- Email: [email protected]
Graphene-based Nano-Carrier modifications for gene delivery applications
Accepted Manuscript Graphene-based Nano-Carrier modifications for gene delivery applications Rana Imani, Fatemeh Mohabatpour, Fatemeh Mostafavi PII:
S0008-6223(18)30827-3
DOI:
10.1016/j.carbon.2018.09.019
Reference:
CARBON 13447
To appear in:
Carbon
Received Date: 22 May 2018 Revised Date:
28 August 2018
Accepted Date: 3 September 2018
Please cite this article as: R. Imani, F. Mohabatpour, F. Mostafavi, Graphene-based Nano-Carrier modifications for gene delivery applications, Carbon (2018), doi: 10.1016/j.carbon.2018.09.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Graphene-based Nano-carrier Modifications for Gene Delivery
2
Applications
RI PT
3
Rana Imani a*, Fatemeh Mohabatpour a,b, Fatemeh Mostafavi a
4
6
a
Department of Biomedical Engineering, Amirkabir University of Technology, Tehran 15875/4413, Iran.
b
Division of Biomedical Engineering, University of Saskatchewan, Saskatoon, Canada.
SC
5
M AN U
7
8
9
TE D
10
EP
11
12 13
*
14
Technology, Tehran 15875/4413, Iran. Email: [email protected] Phone: +98 21-64545676, Fax: +98 21-
15
66468186
17 18 19
AC C
16
Corresponding authors: Rana Imani, Department of Biomedical Engineering, Amirkabir University of
ACCEPTED MANUSCRIPT
1 2
Graphical abstract
6 7 8 9 10 11 12 13
EP
5
AC C
4
TE D
M AN U
SC
RI PT
3
ACCEPTED MANUSCRIPT
Abstract
2
Gene therapy, a therapeutic approach based on nucleotide delivery to the targeted cells, serves as
3
a hot spot research. Regardless of recent developments in various nucleotide-based therapeutics,
4
the success of gene therapy in clinical steps is still limited due to less-efficient delivery
5
mechanisms toward the targeted tissue or cells. In recent decades, nanotechnology has provided
6
a remarkable breakthrough in developing safe and efficient gene carriers.
7
Graphene and its derivatives, as sheet-like carbonic nanomaterials, have been progressively
8
utilized in various biomedical fields. Currently, applications of graphene-based platforms extend
9
to gene delivery as nano-carrier. Considering the unique structure and chemistry of graphitic
10
nanoparticles, they provide high capacity for the loading of genes. To improve biocompatibility,
11
biostability, cellular uptake and increase in gene loading capacity, the surfaces of graphene and
12
its derivatives have been modified with various polymers or ligands. This article presents a
13
comprehensive overview of the current status of graphene family nanoplatforms for gene
14
delivery applications with a focus on the main graphene characteristics as gene nano-carriers. We
15
further highlight the various types of modifications and functionalization of graphene, suitable
16
for nucleotides loading and delivery. We finally address the future perspectives and challenging
17
issues of graphene-based gene therapy for prospect clinical applications.
20 21 22 23 24
SC
M AN U
TE D
EP
19
AC C
18
RI PT
1
ACCEPTED MANUSCRIPT
1
Contents
2 3
1-
Introduction to Gene Therapy .......................................................................................................... 5
4
2-
Graphene as a Versatile Nano-material ............................................................................................ 7 2-1- Different Types of Graphene as Nano-carriers .............................................................................. 8
6
Graphene ......................................................................................................................................... 9
7
Graphene oxide (GO)....................................................................................................................... 9
8
Reduced graphene oxide (rGO) ........................................................................................................ 9
9
Nano-graphene oxide (NGO) ........................................................................................................... 9
10
Graphene quantum dots (GQDs) .................................................................................................... 10
11
Graphene nanoribbons (GNRs) ...................................................................................................... 10
12
2-2- Therapeutic Nucleotides Delivered by Graphitic Materials ......................................................... 10
13
Plasmid DNA (pDNA) ................................................................................................................... 11
14
Antisense Oligonucleotides (AONs)............................................................................................... 11
15
Small interfering RNA (siRNA) ..................................................................................................... 11
16
Short hairpin RNA (shRNA) .......................................................................................................... 11
17
MicroRNA (miRNA) ..................................................................................................................... 12
SC
M AN U
3-
TE D
18
RI PT
5
Nano-carrier Design Criteria for Efficient Gene Delivery ............................................................... 12 3-1- Surface charge............................................................................................................................ 12
20
3-2-Size ............................................................................................................................................. 14
21
3-3- Shape ......................................................................................................................................... 15
22
3-4-Surface Chemistry ....................................................................................................................... 15
23
4-
EP
19
Graphene Modifications as Gene Delivery Vehicle ........................................................................ 16 4-1- Graphene modifiers .................................................................................................................... 22
25
Polyethylenimine (PEI) .................................................................................................................. 22
26
Polyethylene glycol (PEG) ............................................................................................................. 28
27
Dendrimers .................................................................................................................................... 30
28
poly-L-lysine (PLL) ....................................................................................................................... 30
29
Chitosan ........................................................................................................................................ 30
30
Poly(sodium 4-styrenesulfonates) (PSS)......................................................................................... 31
31
Poly(2-dimethyl aminoethyl methacrylate) (PDMAEMA) .............................................................. 32
AC C
24
ACCEPTED MANUSCRIPT
1
Peptides ......................................................................................................................................... 32
2
5-
Multifunctional Graphene Nano-carriers ........................................................................................ 34
3
6-
Dual Gene/Drug Delivery by Graphene-based Nano-carriers .......................................................... 45
4
7-
Conclusions and perspectives ......................................................................................................... 48 Conflict of Interest............................................................................................................................. 50
6
References............................................................................................................................................. 50
RI PT
5
7
9
SC
8
1- Introduction to Gene Therapy
Gene therapy, as a hot spot research, is a therapeutic approach that has attracted scientists and
11
scientific communities for decades. This approach comprises the delivery of nucleotide-based
12
therapeutic agents into the targeted cells to correct the genetic functions in the cell nucleus or
13
cytoplasm [1]. Since the 1990s, the advent of gene therapy started a huge revolution in the
14
history of biomedical sciences and was introduced as an effective strategy to fight genetic
15
disorders. After three decades, today, CRISPR-based genome editing, as emerging concepts in
16
gene therapy, remarkably increases promises of treatment of genetic-based diseases [2].
17
Regardless of recent progress in understanding the molecular biology of genetically-based
18
diseases and various developing nucleotide-based therapeutics, efficient delivery of the nucleic
19
acids into the cells remains a major challenge, which has seriously limited gene therapy success,
20
particularly in clinical trial steps. Therefore, the success of the gene therapy approach is mainly
21
dependent on the development of a safe and efficient gene carrier [3].
22
In recent years, the emergence of nanomedicine and the application of nanotechnology in
23
medicine, has greatly changed the face of gene therapy, where amalgamating the nanotechnology
24
concepts and non-viral gene therapy approach has provided a breakthrough in biomedicine [4].
25
Nanotechnology provides an essential improvement in gene carrier design to have a desirable
26
carrier, which could overcome both extra-cellular and intracellular obstacles. Despite decades of
27
progress in nanoparticle design and synthesis, currently, the US Food and Drug Administration
28
(FDA) has not yet approved any nanoparticle-based formulation for gene therapy application [5].
AC C
EP
TE D
M AN U
10
ACCEPTED MANUSCRIPT
Consequently, designing and developing optimum multifunctional nanoparticles still remains an
2
opportunity for researchers.
3
Growing interest in graphene families, carbon-based nanoparticles, gave the possibility to apply
4
this class of nanomaterials in medical applications, as has been discussed in several reviews [6-
5
9]. In particular, graphene-based nanoplatforms attract tremendous attention in the delivery of
6
various therapeutic agents such as drugs and genes [10-12], whereas these platforms have been
7
suggested as an effective cargo delivery system for cancer therapy [13]. Looking at the trend of
8
publications from 2004 to 2017, which have utilized graphene and its derivatives as gene
9
delivery carrier confirms the success of graphene-based carrier in in-vitro and in-vivo gene
10
therapy applications. However, a few literature reviews have specifically addressed different
11
aspects and challenges of graphene nanoplatforms for gene delivery purposes [14, 15].
12
In this review, we present a comprehensive overview of the graphitic nanomaterials as gene
13
delivery vehicles over the past seven years (Fig.1). We briefly discuss the properties of graphene
14
families’ nanoparticles as well as nano-carrier design criteria with a special focus on graphene
15
modification approaches. We aimed to highlight biomaterial-based strategies in detail in order to
16
design and modify the graphitic nano-carrier suitable for overcoming extra and intracellular
17
barriers to have efficient gene delivery. In addition, more advanced multifunctionalized graphene
18
nanoplatforms, which have attracted much more attention in recent studies, are addressed more
19
specifically in dual delivery (e.g. gene and drug) applications.
AC C
EP
TE D
M AN U
SC
RI PT
1
20
ACCEPTED MANUSCRIPT
1
Figure 1. Number of publications on graphene application as gene delivery platform from 2010-2018(April). Source
2
ISI Web of Science (search keywords: Graphene, gene delivery).
3
2- Graphene as a Versatile Nano-material In October 2004, K. Novoselov and A. Geim shocked the physics world with their discovery of
5
the youngest allotrope of graphite, graphene sheets [16]. Graphene, the basic structure of
6
graphite, is a sheet of two dimensional (2D) single or few layers of sp2 ranged carbon atoms [10,
7
17]. It is a fundamental constructive part for other graphite-based materials with various
8
geometry [18] (Fig 2). Graphitic-based nanostructures mainly include spherical structures (zero-
9
D, e.g. fullerenes), rolled structures (2D, e.g. carbon nanotubes (CNTs)), or layered structures
10
(3D , e.g. graphite) [4]. However, graphene and its derivatives usually address the sheet-like
11
structure of carbon-based nanomaterials. The graphene sheets can be synthesized by either of
12
two approaches: bottom-up or top-down methods. For example, chemical vapor deposition
13
(CVD) serves as a bottom–up method and chemical exfoliation or the mechanical scotch tape
14
method are categorized in the top-down methods [16, 19].
M AN U
SC
RI PT
4
AC C
EP
16
TE D
15
17
ACCEPTED MANUSCRIPT
Figure 2. Graphenic-derived nanoparticles. Graphene is a 2D sheet like carbon-based material. It could be found in
2
the form of zero dimensions (fullerenes), one dimension (carbon nanotubes) and three dimensions (graphite) form.
3
Reprinted with permission Ref. [18]. Copyright (2013) Elsevier.
4
Graphene and its derivatives are interesting for scientists from disparate fields due to their
5
remarkable physical and chemical properties, such as high young's modulus, great thermal and
6
electrical conductivity, huge specific area and biocompatibility [8]. The application fields of
7
graphene and its derivatives are ranging from electronic fields such as photoconductive materials
8
[20], energy technology [21], optoelectronic devices [22], sensors [23] and composite materials
9
[24] to biomedical applications, such as medical imaging/bio-sensing [8, 25, 26], tissue
10
engineering [27], drug delivery [25, 26], cancer therapy [27, 28], antibacterial materials[17] and
11
gene delivery [29].
12
Graphitic materials differ in their structural features, such as number of layers, defect density,
13
surface chemistry and layer size and composition. This interestingly resulted in a wide variety of
14
tunable properties, which are considerable in biomedical applications.
15
Since graphene arrival in 2004, many researches have been conducted on its structure and
16
properties, but there was no discussion on the use of its medical applications until 2008, which is
17
a starting point in graphitic material storming in the field of biomedicine. Nowadays, the range
18
of biomedical applications of graphene and its derivatives is enormous, actually bigger than non-
19
medical applications [9]. However, for most bio-applications, the graphitic materials need to be
20
functionalized with biocompatible modifiers (e.g. polymers) to improve specific properties or
21
reduce toxicity. Among various biomedical applications of graphenic materials, more frequently
22
reported ones, which have attracted ever-increasing interest, are in the field of drug delivery [12],
23
gene delivery [14], biosensing and bioimaging [30] as well as a more recent one, tissue
24
engineering and regenerative medicine [7, 31].
25
2-1- Different Types of Graphene as Nano-carriers
26
Graphene family nanomaterials including graphene, graphene oxide (GO), reduced graphene
27
oxide (rGO), nano-graphene oxide (NGO), graphene quantum dots (GQDs) and graphene
28
nanoribbon (GNR) have been widely used in biomedical applications. However, among the
29
graphene derivatives, GO and rGO have been much more frequently utilized as nano-carriers for
30
intracellular delivery of various cargoes, such as drugs and genes.
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
Graphene
2
Graphene is a two dimensional (2D) structure composed of mono or multi-layers of sp2
3
hybridized carbon atoms. Pristine graphene is extremely hydrophobic and exhibits poor
4
dispersibility and stability in aqueous solutions because of the strong interactions among
5
graphene sheets and the high tendency to aggregate. Biomolecules can be loaded onto the
6
conjugated carbon network of graphene through π- π interaction [11]. Since it is quite hard to
7
synthesize the single layer graphene without defects and also due to its poor stability in solutions,
8
multi-layer graphene and GO are broadly used for biomedical purposes[10].
9
Graphene oxide (GO)
SC
RI PT
1
GO sheets, which are obtained by chemical oxidation of graphite under extremely oxidized
11
conditions, are structurally different from the graphene, mainly in the richness of these oxygen
12
containing groups in the GO sheets surface [11]. In fact, the ionization of carboxyl and hydroxyl
13
groups and the electrostatic repulsion between them results in the formation of stable GO
14
colloids in different polar solvents [32]. A wide variety of bio-functionalization of GO can be
15
performed via chemical interaction between oxygen containing groups of GO and various
16
biomolecules [11]. In addition, GO native functional groups might have undergone chemical
17
reaction to form the other non-oxygenated functional groups (e.g. NH2).
18
Reduced graphene oxide (rGO)
19
rGO is produced by treatment of GO under reducing conditions. Reduction of GO into rGO
20
renovates its electrical conductivity, declines oxygen content and surface charge and enhances
21
hydrophobicity [33]. In addition, the oxygen containing functional groups of GO are entirely or
22
partially removed during the reduction process, which can introduce holes and defects in the rGO
23
structure. The reduction process makes graphitic sheets less hydrophobic compared to the bare
24
graphene and its basal plane reactivity is also lower than GO [10].
25
Nano-graphene oxide (NGO)
26
The term nano-GO is used for defining GO with a small lateral size, characteristically smaller
27
than 100 nm and usually lower than 20 nm, whereas the size of regular GO in both dimensions is
28
greater than 100 nm [34]. In fact, NGO resembles zero-dimensional (0D) material with a higher
29
degree of oxygenation in contrast to conventional GO, which is 2D. Thus, NGO is considered as
AC C
EP
TE D
M AN U
10
ACCEPTED MANUSCRIPT
a decent candidate for biomedical applications due to the fact that its small size provides the
2
ability to facilitate cell internalization and increase dispersion stability [33].
3
Graphene quantum dots (GQDs)
4
GQDs are a type of quantum dots obtained by cutting 2D graphene sheets into 0D small dots,
5
which are regularly shaped and their sizes are from several to 100 nm, and have the properties of
6
both graphene and carbon dots (CDs)[34-36]. This conversion results in opening a band gap in
7
GQDs due to quantum confinement and presents powerful photoluminescence, which does not
8
exist in 2D graphene [37]. GQDs are structurally similar to GO and are composed of the main
9
sp2 bonded carbon network, enriched with oxygen containing functional groups on the edges
10
[11]. Due to their superior properties such as lower toxicity, better biocompatibility and higher
11
resistance to photo-bleaching and blinking, GQDs are considered to be a promising replacement
12
for semiconductor quantum dots, which are toxic [36, 38, 39].
13 14
Graphene nanoribbons (GNRs) GNRs are produced by cutting a 2D graphene sheet into a 1D narrow strip with a specific edge
15
structure and width. Typically, the GNR width and aspect ratio are less than 10 nm and higher
16
than 10, respectively; in fact, GNRs can be considered as unrolled CNTs [34, 40]. Their band
17
gaps are contingent upon their width and as a result, their properties can be changed from semi-
18
metallic to semi-conductive by decreasing the width [34]. rGO nanoribbons (rGONRs) exhibit
19
higher cytotoxicity and genotoxicity compared to rGO sheets [41].
20
2-2- Therapeutic Nucleotides Delivered by Graphitic Materials
21
Graphitic nano-carriers are able to deliver various types of therapeutic nucleotides in order to
22
modulate the gene expression profile of cells. Two major manners exist through which the
23
genetics of the targeted cells can be modulated. In the first method, DNA is delivered into the
24
cells to provide a transcriptionally competent copy of a defective or missed gene and makes up
25
for deficiency of a protein. While in the second approach, therapeutic nucleic acids consisting of
26
Antisense Oligonucleotides (AONs), Small interfering RNA (siRNA), Short hairpin RNA
27
(shRNA) and MicroRNA (miRNA) are employed to inhibit or knockdown the expression of
28
defective genes [42].
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
Plasmid DNA (pDNA)
2
Plasmids, known as double stranded DNA, are mostly annular and comprised of the transgene
3
encoding for a particular protein. pDNA range in size from a few hundred to several thousand
4
basepairs (bp). After internalizing into the cells and being uptaken by the nucleus, this genetic
5
material commences, producing the encoded protein through transcription and translation
6
processes using cellular machines. Besides the transgene, pDNA is composed of other
7
modulatory elements including the promoter and enhancer sequences as well as splicing and
8
polyadenylation sites which have a profound role in gene expression [43, 44].
9
Antisense Oligonucleotides (AONs)
SC
RI PT
1
AONs, which contain 18-21 nucleotides, are delivered as single-stranded deoxyribonucleotides
11
and must detect their complementary mRNA sequence, hybridize with the target mRNA and, as
12
a result, induce gene knockdown [43]. Mechanisms through which AONs function are steric
13
hindrance and target degradation. Steric hindrance, known as non-degradative pathway,
14
influence the attachment of trans-splicing modulatory agents to pre-mRNA and thereby regulate
15
splice models, while the target degradation mechanism can be achieved by the action of RNase H
16
enzyme cleaving mRNA in the RNA-DNA hybrid [45, 46].
17
Small interfering RNA (siRNA)
18
Small interfering RNAs (siRNAs) or short interfering RNAs are double-stranded RNA
19
molecules, composed of 20-25 bp in length, that interfere with the translation process through the
20
RNA interference (RNAi) pathway and degradation of complementary mRNA strands, and thus
21
inhibit gene expression [47]. This function occurs through the gene-silencing mechanism, in
22
which dicer protein chops siRNA into shorter segments among which there is a fragment with
23
affinity for the RNA-induced Silencing Complex (RISC). After attaching to RISC, the passenger
24
strand
25
to the corresponding mRNA leading to further degradation of the mRNA and gene silencing [1,
26
48].
27
Short hairpin RNA (shRNA)
28
shRNA is a RNA sequence which has gene silencing ability through the RNAi mechanism and
29
comprises stems (25-29 bases) and a loop site (4-23 nucleotides). shRNAs are transformed into
AC C
EP
TE D
M AN U
10
of
siRNA
is
cleaved
and
the
guide
strand
is
directed
ACCEPTED MANUSCRIPT
siRNAs by the dicer protein, which eliminates the loop part, and afterwards, siRNAs incorporate
2
to RISC and cleave target mRNA [43, 49].
3
MicroRNA (miRNA)
4
MicroRNA, a single-stranded RNA molecule with 20-24 nucleotides, is considered as a non-
5
coding RNA, which regulates some cellular behaviors including differentiation, proliferation and
6
survival. In fact, these functions are achieved by attaching to target mRNAs and then inhibiting
7
the translational process or cleaving corresponding mRNAs [43, 50].
SC
8
RI PT
1
3- Nano-carrier Design Criteria for Efficient Gene Delivery
For efficient gene delivery, a carrier should be designed with regard to the cargo type as well as
10
barriers, whereas the delivered cargo may be faced with in delivery routes. Firstly, the nano-
11
carrier designed to deliver the genetic materials should be able to interact with nucleotides in
12
order to efficiently load the cargoes. For successful transfection, nano-carriers should be able to
13
protect the genetic materials during transport, overcome the extracellular and intracellular
14
barriers and deliver the functional form of genetic materials into the target cells [51]. Several
15
physiochemical parameters, such as surface charge, size, shape and surface chemistry are
16
principal to be considered in order to design an effective nano-carrier system to that end. By
17
manipulation of these factors, the fate of nano-carriers inside the body can be altered, and
18
consequently the final success of gene therapy would be affected [52]. Herein, the main
19
characteristics, which are needed to be considered in designing the graphitic nano-carriers, are
20
briefly summarized.
21
3-1- Surface charge
22
Surface charge plays a prominent role in the gene/carrier complex formation, cellular uptake
23
mechanism, cytotoxicity and stability of nano-carriers [53]. Genetic materials, such as DNA and
24
RNA have negative charge due to the presence of phosphate groups in their backbone.
25
Consequently, they cannot cross cellular membranes by themselves and need carrier systems,
26
which can form complexes with them, compromise the electrostatic repulsion between them and
27
the negatively charged cell membrane and facilitate their internalization into the cells [51]. One
28
of the simple approaches to bind the genetic materials to carriers is based on electrostatic
29
interactions between positively charged nano-carriers and nucleotides [54]. Both charge polarity
AC C
EP
TE D
M AN U
9
ACCEPTED MANUSCRIPT
and charge density have a crucial influence on the cellular uptake of nanoparticles as well as
2
their cytotoxicity. Generally, positive surface charges juxtaposed with neutral or negative
3
charges have been demonstrated to enhance the cellular uptake of nanoparticles due to
4
electrostatic attraction with the cell membrane that is favorable for adhesion to the cell surface in
5
both phagocytes and non-phagocytic cell lines [55, 56]. Also, carriers with a higher positive
6
charge density have been shown to be up taken at a higher level and in the most fastest rate [53,
7
55]. Additionally, the surface charge of nano-carriers is able to determine the mechanism of
8
cellular endocytosis. Positively charged carriers use the clathrin-mediated endocytosis pathway
9
in order to internalize into the cells, while carriers with negative surface charge could be taken
10
up through other pathways, such as the caveolae-mediated pathway [57, 58]. However,
11
nanoparticles with positive charge have shown more cytotoxic effects compared to negatively or
12
neutral charged nanoparticles [55]. In fact, positive charge of the nanoparticle can increase Ca2+
13
influx into cells and thereby inhibit their proliferation, induce reconstruction of lipid bilayers and
14
fluidity [59]. Furthermore, the stability of nano-carrier systems significantly relies on their
15
surface charge, such that nanoparticles with a higher surface charge can exhibit more resistance
16
to form aggregations mostly due to their strong self-repulsive effect [52].
17
It is noteworthy that the surface charge of the carriers would be affected by gene loading. Due to
18
amine (N)/phosphate (P) electrostatic interaction between the carrier and nucleotides, the surface
19
charge of the complex would be partially neutralized [60]. The carrier/DNA ratio (known as N:P
20
ratio) usually dictates the formed complex charge, while it raises to more positive values by
21
adding N:P ratio. In a lower N:P ratio, the formed complex may be unable to effectively interact
22
with the cell membrane [61]. Therefore, to evaluate the charge effect on gene transfection, the
23
net charge of carrier/gene complexes should be considered.
24
GO sheets contain epoxide (-O-) and hydroxyl (-OH) groups on their basal planes, whereas
25
hydroxyl and carboxyl (-COOH) groups exist at their edges [62]. Therefore, GO with
26
hydrophilic terminates and more hydrophobic basal planes can be considered as an amphiphilic
27
sheet [63]. The presence of terminal carboxyl groups can afford stability in colloidal dispersions
28
and negative charge on the surface, such that the pH has an influential impact on this surface
29
charge. For instance, high pH can upgrade the de-protonation of the carboxyl groups and
30
therefore GO would become more charged [63]. Also, the uncharged groups existing in the basal
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
plane, due to their polarity, provide surface reactions, such as weak interactions and hydrogen
2
bonding. Furthermore, hydrophobic and free surface π electrons exist on the basal planes, which
3
are able to form π- π interactions for non-covalent interactions. By reducing GO into rGO, it
4
becomes a less hydrophilic molecule with a lower oxygen content and surface charge [10].
5
3-2-Size
6
The size of nano-carriers is a crucial factor, which can considerably influence colloidal stability,
7
cellular uptake, transfection efficiency and residence in circulation and clearance [52, 53]. It has
8
been observed that nanoparticles are taken up more efficiently as their size is declined in in-vitro
9
[57]. Nanoparticles with a size around 20 nm are able to diffuse into the cells in an endocytosis
10
pathway-independent manner. On the other hand, Rejman et al. demonstrated that particles
11
smaller than 200 nm in diameter were internalized through the clathrin-mediated pathway,
12
whereas, particles with a diameter larger that 200nm but less than 1µm entered into the cells
13
mostly via the caveole-mediated pathway [64]. In addition, a longer circulation time can be
14
provided by administration of nanoparticles with a size smaller than 200 nm in in-vivo. In fact,
15
such nanoparticles revealed a lower clearance and a better delivery to target tissues instead of
16
others, such as lungs, liver and the spleen. However, particles larger than 200 nm were removed
17
expeditiously via liver, spleen and the lungs [57]. In the case of GO, size also has a critical
18
impact on its amphiphilicity, so that smaller sheets are more hydrophilic due to having a higher
19
edge-to-area ratio. In addition, a more colloidal stability can be made by smaller GO sheets as a
20
result of a higher charge density [65]. In addition to size, the layer number is another prominent
21
parameter that should be considered in the design of graphene family nano-carriers, since the
22
specific surface area and bending stiffness are determined based on it [66]. By decreasing the
23
number of layers to one or more, the loading capacity of the cargo would be effectively
24
increased.
25
Similar to the charge, the size of the carrier can be strongly affected by gene loading. The
26
effective electrostatic interaction of the carrier and nucleotides will result in gene
27
condensation/compaction. After gene loading, the size of the formed carrier/gene complexes
28
usually shows an increase [67]. The degree of condensation remarkably affects the amount of
29
size variation. However, the type and modification of the cationic carrier, the N:P ratio and also
30
the protocol of complex formation can influence the complex size. For instance, by increasing
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
N:P ratio during complexation, the complex size enlargement shows a decreasing trend, which is
2
originated from more condensation of the loaded nucleotides [68]. Considering the importance of
3
carrier size and charge in gene transfection efficacy, as discussed, N:P ratio during complexation
4
should be accurately adjusted before transfection, so that the resultant complex charge/size still
5
remains in a proper range and does not disturb gene transfection [69].
6
3-3- Shape
7
The shape of nano-carriers, particularly in their transportation across biological barriers and also
8
inside the cells, is of great importance [57]. Nanoparticles with various shapes, such as spheres,
9
rods, discs, tubes and cubes reveal different uptake behavior, so that for nanoparticles with a
10
diameter larger than 100nm, rods, spheres, cylinders and cubes have higher uptake, respectively.
11
Spherical-shaped nanoparticles smaller than 100 nm are taken up more efficiently in contrast to
12
rod-shaped ones, whereas a lower cell uptake occurring as the aspect ratio of nanorods becomes
13
larger. Despite the few studies conducted on non-spherical nano-carriers, some researches have
14
demonstrated that the interaction of such nano-carriers with cells can be more complicated [59].
15
In addition, the shape of nanoparticles governs the efficiency of gene transfection. In the case of
16
polycation modified silica nanoparticles, chiral nanorods with a length of 300 nm were
17
ascertained as the most efficient carrier in comparison to nanoparticles, whose shapes were
18
different, including nanospheres with a diameter of 100 nm, hollow nanospheres with a diameter
19
of 100 nm, chiral nanorods with a length of 200nm and ordinary nanorods with a length of 300
20
nm. This is while hollow nanospheres juxtaposed with solid nanoparticles reveal better gene
21
transfection [70]. The graphitic materials resemble a sheet-like structure with various thicknesses
22
almost near a few nanometers, and lateral size around several nanometers to a few micrometers.
23
This sheet-like geometry could provide a sharp edge to cross the cell membrane [71].
24
3-4-Surface Chemistry
25
It is well established that the surface chemistry of nano-carriers can effectively modulate their
26
biocompatibility, circulation in the body, and particularly their interaction with the cells. It has
27
been shown that the blood half-life of nano-carriers is contingent upon their surface
28
hydrophobicity, so the more hydrophobic the surface, the more opsonin proteins absorbed on the
29
surface and the shorter the circulation time [55]. Furthermore, nano-carriers with different types
30
of surface chemistry can be internalized by the cells through various mechanisms. Nanoparticles
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
with a hydrophobic surface internalize more expeditiously than those with a hydrophilic surface
2
due to the hydrophobic nature of the cell membrane [57]. A wide variety of surface modification
3
has been used in order to improve biocompatibility, circulation time, cellular uptake and thereby
4
transfection efficiency of nano-carriers [52, 59]. Surface chemistry is variable for each member
5
of the graphene family. As aforementioned, GO has amphiphilic properties with a water contact
6
angle of 40-45°; whereas graphene is hydrophobic with a water contact angle of about 90° [66].
7
The surface chemical modification of the graphitic materials is a pre-requisite for their
8
application as gene nano-carrier. In what follows, various kinds of methods used for
9
modification of graphene-based materials are presented.
SC
4- Graphene Modifications as Gene Delivery Vehicle
M AN U
10
RI PT
1
Graphene has poor dispersion and stability in aqueous solutions due to its high hydrophobicity
12
and is bereft of oxygen containing hydrophilic groups, which are crucial for water dispersion.
13
Therefore, GO has been easily produced through derivatization of graphene, a covalent
14
functionalization method, in which oxygenated groups, such as carboxyl, epoxy and hydroxyl are
15
generated on the graphene, in order to increase its water solubility [72]. However, GO has the
16
tendency to aggregate in physiological solutions in the presence of salts and proteins as a result
17
of the shielding of electrostatic charges, and also the formation of non-specific interactions with
18
proteins [18]. To enhance physiological stability, cellular uptake and transfection efficiency, two
19
main methods, including covalent and non-covalent modifications by different modifiers have
20
been employed [73]. Non-covalent functionalization is preferred when the electrical conductivity
21
and large surface area of graphene is required, while covalent modification methods are applied
22
when the modified graphene is needed to have stability and strong mechanical properties. Studies
23
have shown that DNA/RNA condensation can be provided with both modification methods,
24
which makes them suitable for intracellular gene delivery applications [74]. This leads to a series
25
of studies by various research groups for the exploration of graphene-based materials in gene
26
delivery as summarized in Table 1.
AC C
EP
TE D
11
27 28
Table 1: Graphene-based nanoplatform’s modifications with respect to gene delivery (or gene/drug co-delivery)
29
systems
ACCEPTED MANUSCRIPT
Modifier(s)
Bonding
Nucleotide-
Study
Cell
Co-
Graphene
/modification
based cargo
model
line/anima
loaded
Derivative
mode
model
drug
HeLa
-
GO
PEG
Non-covalent
MB
In vitro
GO
PEI
Non-covalent
pEGFP
In vitro
GO
PEI
Covalent
Luciferase
In vitro
reporter gene GO
PEI
Covalent
pEGFP-N1
CS
Covalent
pRL-CMV
In vitro
In vitro
-
[76]
HeLa
-
[77]
-
[78]
CPT
[79]
DOX
[80]
HeLa
HepG2 HeLa
M AN U
(Renilla
[75]
HeLa
SC
GO
Ref
PC-3
Luciferase reporter gene
RI PT
Type of
luciferase)
PEI
Covalent
GNR
PEI(25KD)
Non-covalent
GO
bPEI (10 KD)/
PEI(covalent)
NLS peptide
Peptide (Non-
(PV7)
covalent)
PEG-FA/ Py
PEG-
GO
Bcl2-siRNA
In vitro
HeLa
LNA-m-MB
In vitro
HeLa
pEGFP-C1
In vitro
HeLa
pDNA
hTERT siRNA
TE D
GO
[81] -
[82]
293 T
In vitro
Hela
-
[83]
In vitro
Hela
-
[84]
-
[85]
-
[60]
-
[86]
FA(covalent) Py(Non-
covalent) rGO
PEG(amine
EP
terminated 6-
Covalent
FAM–
ssRNA
branched , 10 KD)
PEI (25KD)
Covalent
AC C
GO
PEG(10 KD)
GO
In vitro
pEGFP
MDA-MB435s HeLa
bPEI(1.8 KD)PEG(5KD)
(si Plk1)
Stat3 siRNA Covalent
In vitro
mouse
In vivo
malignant cell line B16
GO
PEI (60 kDa)
Covalent
pEGFP
In vitro
H293T U2Os
In vivo
zebrafish embryos
ACCEPTED MANUSCRIPT
GO
PEI/
Non-covalent
miR-21 siRNA
In vitro
PSS
MCF-7/ADR
ADR
cells (an ADR
(adrim
resistant breast
ycin)
[87]
cancer cell line) PEI
Non-covalent
FAM-siRNA
In vitro
Covalent
pEGFP
In vitro
mPEG-SPA Au GO
CS (10KD) PSS
bPEI
Covalent
bPEI-PEG
FA-
Covalent
Non-covalent
trimethyl chitosan (FTMC) PAMAM
GO
lactosylated chitosan
Covalent
EP
dendrimer
GFP-siRNA
pEGFP
pDNA
pDNA
TE D
conjugated
G/GO
Covalent
In vitro
HEK293
-
[90]
Drosophila S2
-
[91]
-
[92]
DOX
[93]
pEGFP
-
[94]
FAM-pDNA
DOX
[95]
-
[96]
Dox
[97]
In vitro
In vitro
In vitro
In vitro
AC C PEG
Covalent
chitosan oligosaccharid e (FACO)
Covalent
PC-3 and NIH/3T3 Hela and A549 cells
HeLa and
In vitro
human hepatic carcinoma cells (QGY7703)
CpG ODNs
PEI
FA-conjugated
HeLa
MG-63 cells
e (LCO)
GO
[89]
cancer
oligosaccharid
GO
DOX
SC
Covalent
bPEG
GO
A549 lung
In vivo
M AN U
LPEI (25 kD) bPEI(25 KD)
rGO
[88]
prostate
nanoparticle
GO
-
cancer C42b
Fe
GO
HL-60
RI PT
GO
In
RAW264.7
vitro/ in
cells
vivo MDR1 siRNA
In vitro
MCF-7
ACCEPTED MANUSCRIPT
GCN
bPEI
Covalent
pDNA
In vitro
PC-3
via a PEG
and NIH/3T3
spacer
cells
oxygen plasma
-
pGFP
In vitro
etching
NIH-3T3 and
-
[92]
-
[98]
NG97 cell
RI PT
rGO
lines
GO
PEI (25kDa)
Non- covalent
mRNAs of
In vitro
4reprogramming
Myc, and Klf4)
GO
Covalent
-
-
Covalent
mPEG mPEG/PDMA EMA thiolated PEG (5KDa) or
Covalent
EP
GO
siRNAs against
-
[100]
-
[101]
-
[102]
-
[103]
neuro-2a
-
[74]
lung cancer
-
[104]
-
[105]
-
[106]
fibroblasts
In vitro
M AN U
GO
bPEI
Human breast
CXCR4
carcinoma cell
(siCXCR4)
line MDA-
FAM-labeled
In vitro
MB-231 mouse or
single-stranded
human neural
siRNAs
stem cells
siRNA
TE D
GO
[99]
SC
Oct4, Sox2, c-
-
derived
transcription factors (RNAs for
adipose tissue-
pDNA
In vitro
HeLa–Luc cells
In vitro
PC-3 Raw 264.7
thiolated bPEI (1.8KDa) PEI/ PPI/
covalent
AC C
GO
pDNA
In vitro
PAMAM
GO
PAMAM
Covalent
anti-miRNA-21
dendrimer /
In vitro
cells
In vitro
malignant
PEG
GO
PEI-PEG
Covalent
plasmid-based Stat3 siRNA
melanoma B16 cells
GO
PEI/PEG/FA
Covalent
si-Stat3
in vitro
tumor hepatocellular
ACCEPTED MANUSCRIPT
carcinoma GO
PEG
Covalent
shRNA
In vitro
-
Cy5
In vitro
HepG2
DOX
[107]
chitosanaconitic
(CS-Aco) GO
-
peptide nucleic
RI PT
anhydride
MCF-7,
-
[108]
-
[61]
MDA-MB-
acid (PNA) probes
231,
MDA-MB-
SC
435
HeLa
GO
LPEI (1.8
Covalent
angiogenic gene
In vitro
H9c2
HUVECs
GO
PDMAEMA
Covalent
M AN U
kDa)
VEGF pro-
pDNA
In vitro
COS7 HepG2
CPT
[109]
GO
PEI (25 kDa)
covalent
pDNA
In vitro
HeLa
-
[91]
-
FAM
In vitro
Raw 264.7
-
[110]
EPI
[111]
-
[112]
-
[29]
28immunoco mpetent rats
Gold nanoparticle cGO
-
TE D
In vivo
PAMAM-
Covalent
AC C
cGO
EP
oligonucleotide
dendrimer and
cells
dT30 (FAMdT30) miRNA (Let-
In vitro
7g)
gadolinium
human glioblastoma
In vivo
(U87) cells
In vitro
murine
(Gd)
GO
PLL
Covalent
Unmethylated cytosine-guanine
Raw264.7
(CpG)
macrophages
dinucleotide motifs cGO-
R8 peptide
Covalent
-
In vitro
L929
ACCEPTED MANUSCRIPT
fibroblast PL-PEG/ R8
rGO
Non covalent
peptide
In vitro
MCF-7
-
[113]
In vitro
RAW264.7
-
[114]
(cd-siRNA)
CS
GO
Cell death siRNA
Non-covalent
CpG ODNs
PAMAM
GO
Covalent
MMP-9 shRNA
In vitro
plasmid PEI (non-
peptide
covalent)
conjugated-
Peptide
PEI
(covalent)
PLL
PLL(covalent)
SDGR peptide
Peptide (covalent)
GO
PEG/
PEG
FA
(covalent)
oxidized
-
-
In vitro In vivo
VEGF-siRNA
DOX
[115]
PC12 cells
-
[116]
-
[117]
-
[118]
-
[119]
-
[120]
C57BL/6 mice
In vitro
HeLa HUVEC
In vivo
ICR mice
HDAC1
In vitro
MIA PaCa-2
K-Ras siRNAs
In vivo
Athymic nude
TE D
FA (Covalent)
pDNA
MCF-7 cells
SC
GO
Neurotensin
M AN U
rGO
RI PT
cells
a double stranded
In vitro
mice
HEK 293T
DNA
GNR
(dsDNA)
Four different
rGO
AC C
CPPs
Non-covalent
EP
GO
PEI
PEG
PEI (noncovalent)
pGL3
In vitro
splice correction oligonucleotides (SCO) siRNA pGFP
In vitro
11 different cell
[121]
lines
PEG (covalent)
1
Abbreviations: G: graphene, GO: graphene oxide, rGO, reduced graphene oxide, GNR: graphene nanoribbon, cGO:
2
carboxylated graphene oxide, GCN: graphene and carbon nanotube nanocomposite, PEG: polyethylene glycol,
3
mPEG: methylated PEG, SCM-PEG: succinimidyl carboxymethyl PEG, PL- PEG: phospholipid-PEG, PEI:
4
polyethylene imine, LPEI: low molecular weight PEI, bPEI: branched PEI, CS: chitosan, PSS: poly(sodium 4-
5
styrenesulfonates), PAMAM: Oleic acid polyamidoamine, PDMAEMA: poly(2-dimethyl aminoethyl methacrylate),
ACCEPTED MANUSCRIPT
PPI: polypropylenimine, PLL: poly-L-lysine, NLS: nuclear localization signal, FA: folic acid, R8: octaarginine,
2
Py:1-pyrenemethylamine hydrochloride, FAM: fluorescent activated molecule, CPT: 10-hydroxycamptothecin,
3
DOX: doxorubicin, EPI: Epirubicin, MB: Molecular beacon, siRNA: small interference RNA, pDNA: plasmid
4
DNA, ODNs: oligodeoxynucleotides, shRNA: short hairpin RNA, miRNA: microRNA, LNA-m-MB: locked
5
nucleic acid modified molecular beacon,
6
4-1- Graphene modifiers
7
Since the packing of genetic materials into carriers is performed through electrostatic interaction
8
among negatively charged genes and positively charged carriers and the graphene family
9
materials are intrinsically non-cationic, modification of graphene derivatives by various
10
modifiers is required for providing a cationic surface having a better interaction with anionic
11
genes. So far, several modifiers, most of which are listed below, have been utilized to that end:
12
Polyethylenimine (PEI)
13
PEI, a synthetic cationic polymer, has been well-known as one of the most effective materials for
14
gene delivery vehicles. Suitable features of PEI, such as its capability to strongly bind to
15
negatively charged nucleic acids through electrostatic interactions and condensation of the genes
16
onto the surface of the vehicle, which are provided by its high nitrogen atom concentration,
17
could enhance cellular uptake. It also shows the “proton sponge” effect, which can lead to
18
escaping from endosomal/ lysosomal pathways after internalization [61, 76]. However, high
19
molecular weight branched PEI (HMW bPEI) exhibited high levels of cytotoxicity and
20
transfection efficiency juxtaposed with low molecular weight ones (LMW bPEI) [77].
21
Chen and coworkers synthesized PEI-GO conjugates by chemical grafting of PEI 25 kDa to
22
carboxylated GO (GO-COOH) [78]. They observed that GO and GO-COOH expeditiously
23
formed aggregation in the simulated physiological solution (PBS), while PEI-grafted GO was
24
hydrolytically stable due to the enhanced solubility and stability provided with PEI conjugation.
25
Moreover, PEI-GO revealed a high buffering capacity, which facilitated the endosomal escape of
26
pDNAs and also provided a comparably higher luciferase expression in HeLa cells compared to
27
PEI 25 kDa. In addition, the grafted PEI to GO showed a significantly lower (about 50%)
28
cytotoxic effect, as carrier compared to the naked PEI.
29
Feng et al. investigated the effect of PEI molecular weight on gene transfection ability as well as
30
cytotoxicity of PEI-functionalized GO nano-carriers. Two different molecular weights of PEI
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
(1.2 and 10 kDa) were introduced to GO through electrostatic (non-covalent) interactions [76].
2
Based on the AFM analysis, a significant increase in the average thickness of GO nanosheets
3
after incubation with PEI (from 1 nm to 3–4 nm) indicated that the electrostatic interactions
4
provided a successful immobilization. GO-PEI-10k showed significantly decreased cytotoxicity
5
in HeLa cells compared to bare PEI-10k, which was relatively toxic to the cells. Not only did the
6
complexation of GO with both PEI-1.2k and PEI-10k reveal no increase in the cytotoxicity of
7
GO, but it also showed rather higher cell viability at the higher concentrations of GO,
8
presumably because of the enhanced stability of GO in physiological solutions (Fig. 3).
9
Enhancing zeta potential to positive values (+30-50 mv) via PEI immobilization promoted the
10
loading of EGFP plasmid via the layer-by-layer (LBL) assembly mechanism. GO-PEI-1.2k
11
showed a higher EGFP expression compared to PEI-1.2k with ineffective transfection, whereas
12
EGFP expression observed in GO-PEI-10k was tantamount to bare PEI-10k (Fig. 3, d-g).
AC C
EP
TE D
M AN U
SC
RI PT
1
1
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
2
Figure 3. (a), (b), (c), Viability of HeLa cells treated with different concentrations of GO, GO-PEI and PEI with two
3
different molecular weights of 1.2 and 10 kDa for PEI, (d), (e), (f), (g), Fluorescent images of HeLa cells treated
4
with GO-PEI-pDNA and PEI-pDNA complexes at various nitrogen/phosphate (N/P) ratios and two different
5
molecular weights of PEI. Reprinterd from Ref. [76] with permission from The Royal Society of Chemistry.
ACCEPTED MANUSCRIPT
In another study, branched PEI (bPEI) was suggested to functionalize GO as gene delivery
2
carrier [77]. Two molecular weights of bPEI (1.8 and 25 kDa) were chemically conjugated to
3
GO with three various ratios of bPEI to GO (22, 8 and 5). The formulation prepared by a simple
4
mixture of bPEI and GO (physical immobilization) showed considerable aggregation, while the
5
chemically conjugated pPEI revealed no aggregation in water, PBS, and 10 % serum-containing
6
media. Conjugation of GO with bPEI under sonication reduced the size of GO from 500-600 nm
7
to 100-200 nm and increased the thickness of GO from 0.6-1.3 nm to 6-8 nm. The impact of
8
increasing the conjugation degree on the size and thickness was not considerable. Gene
9
transfection ability of HeLa and PC-3 cells was increased with increasing conjugation ratios of
10
bPEI to GO, where bPEI-GO with a higher conjugation ratio showed remarkable transfection
11
even at lower N:P ratio, which was comparable to bare bPEI 25KDa.
12
GNR grafted by PEI (PEI-g-GNR) through electrostatic assembly was studied by Dong and
13
coworkers as a nano-carrier of locked nucleic acid modified molecular bacon (LNA-m-MB)
14
probes for the detection of miRNA [81]. PEI-g-GNR provided good protection of the LNA-m-
15
MB probes against single-stranded DNA binding protein interaction or enzymatic degradation. It
16
was also more efficient in the transfection of HeLa cells juxtaposed with PEI (25kDa) and PEI-
17
grafted multi wall carbon nanotubes (PEI-g-MWCNTs) due to the larger surface area of GNR for
18
conjugation of PEI, and subsequently a stronger possible proton sponge effect.
19
In another study, the effect of GO nanosheet size on PEI conjugation and consequent DNA
20
loading was studied. Chemically grafted PEI to NGO with a size of 10-20nm was assessed in
21
order to obtain higher capacity for PEI loading [86]. The PEI-g-NGO revealed three times higher
22
enhanced green fluorescent protein plasmid (pEGFP) condensation capacity than PEI-g-GO.
23
Transfection with PEI-g-NGO was found to be more effective compared to PEI-g-GO in both
24
H293T and U2Os cell lines with similar cytotoxicity (about 90% viabilities). In addition, the
25
pEGFP transfection study of zebrafish embryos showed efficacy of 90% and 80% for PEI-g-
26
NGO and PEI-g-GO, respectively, which were much higher than naked DNA injection (~30%),
27
as an established transfection technique.
28
Choi et al. utilized PEI- functionalized GO via electrostatic interaction for induced pluripotent
29
stem cell (iPSC) production through mRNA delivery to adipose derived fibroblasts [99]. PEI
30
physical immobilization changed the surface charge of nano-carriers from -34.66 mV to +26.68
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
mV, suitable to binding of negatively charged mRNA. Treatment of human dermal fibroblasts
2
(hDFs) with GO-PEI-mRNA did not show significant cytotoxicity or expression of innate
3
immune response genes, while those with GO, PEI, GO-mRNA or PEI-mRNA significantly
4
dwindled cell viability and increased innate immune response gene expression. hDFs treated by
5
GO-PEI-mRNA also showed very efficient transfection and subsequent reprogramming (around
6
50%), where the derived iPSC expressed pluripotency genes.
7
Another platform, based on the graphene/hydrogel nanocomposite, has been tested by Paul et al.
8
for in-situ delivery of the angiogenic gene for myocardial therapy [61]. The nanocomposite was
9
formulated by incorporating the DNAVEGF - loaded GO, which was synthesized through chemical
10
binding of PEI (1.8 kDa) to carboxylated GO into methacrylated gelatin (GelMA) hydrogel,
11
followed by photocrosslinking under UV exposure (Fig. 4, a). GO had a negative charge (-
12
37mV), while PEI-functionalized GO was positively charged (+43 mV), which was reduced after
13
binding with the negatively charged plasmid DNA depending on N/P ratios. In vitro studies
14
showed that PEI-functionalized GO, gradually released from GelMA hydrogel, did not
15
significantly induce cytotoxic effects on H9c2 cell lines and revealed a rapid overexpression of
16
VEGF in the initial 4 days. In addition, in-vivo studies, which were performed by
17
intramyocardial injection of nanocomposite hydrogels in the peri-infarct areas of a rat model,
18
showed enhancement in myocardial capillary density, a decrease in the scar area of infarcted
19
hearts after treatment with nanocomposite hydrogels compared to untreated groups (Fig.4, b-d).
AC C
EP
TE D
M AN U
SC
RI PT
1
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
1 2
Figure 4. (a) Synthesis of GG’ hydrogel, (b,c) the smaller scar size and (d) higher cardiac functions in the group
3
treated with injectable hydrogel. Reprinted with permission from Ref. [61]. Copyright (2014) American Chemical
4
Society.
5
Covalently grafted PEI (25 KDa) onto GO nanosheets was utilized to encapsulate Au
6
nanoparticles (GO-AuNPs) or Au nanorods (GO-AuNRs) through electrostatic self-assembly in
7
order to improve biocompatibility and gene transfection ability of Au-based nano-carriers [67].
8
The size and surface charge of AuNPs respectively increased from 62.5 nm to 113.8 and from
9
6.6 mV to 36.9 mV after encapsulation with GO-PEI nanosheets, which were ascribed to proper
ACCEPTED MANUSCRIPT
self-assembly of GO and PEI on the Au surface. Although, the GO-PEI sheets formed aggregates
2
with an average size of 500-600 nm via electrostatic interactions between pGFP and GO-PEI,
3
which made the cellular internalization difficult and decreased the transfection efficiency, GO-
4
PEI-AuNPs/pDNA created no obvious aggregates conceivably due to the smaller contact area
5
between GO-PEI-AuNPs spheres compared to GO-PEI sheets. Encapsulation by GO
6
significantly declined the rather cytotoxic effects of AuNPs and AuNRs to HeLa cells and
7
enhanced their transfection ability.
TE D
M AN U
SC
RI PT
1
8
Figure 5: The method of synthesizing GO-PEI-AuNPs and GO-PEI and the proposed mechanism for DNA delivery
10
via these vectors. Reprinted with permission from [67]. Copyright (2013) American Chemical Society.
AC C
11
EP
9
12
Polyethylene glycol (PEG)
13
PEG is a biocompatible amphiphilic polymer, which could improve aqueous solubility and
14
stability in physiological solutions, reduce toxicity, prolong nanomaterials’ blood circulation
15
time through hindering protein adsorption and decrease immunogenicity and antigenicity [83,
16
84]. PEG conjugation (termed PEGylation) has been commonly utilized in order to improve
17
graphene-based nano-carrier functions.
ACCEPTED MANUSCRIPT
A pioneering work, conducted by Lu et al. investigated a covalently attached PEG to NGO
2
carrier to deliver molecular beacon (MB), as a model oligonucleotide, into HeLa cells to detect
3
target mRNA. The steric hindrance effect of the attached PEG remarkably hindered DNase I
4
from binding to MB, and consequently protected MB from enzymatic cleavage. Moreover,
5
PEGylated NGO was less cytotoxic even at a concentration of 100mg/ml and target DNA can be
6
successfully hybridized with MB, which was adsorbed on the NGO surface, and then released
7
from NGO [75].
8
PEGylated rGO (PEG-rGO) was developed by Zhang et al. for loading and delivery of single
9
stranded RNA (ssRNA) by covalent conjugation of PEG to GO and reduction of resultant PEG-
10
GO into PEG-rGO [84]. PEGylation and subsequent reduction had no effect on the size, but an
11
increase of thickness from 1 nm (GO) to 2-3 nm (PEG-rGO) was observed. Furthermore, a
12
higher amount of ssRNA was adsorbed on PEG-rGO in comparison to PEG-GO because of the
13
more noticeable affinity of ssRNA to PEG-rGO. Cytotoxicity studies revealed no toxic effect of
14
GO, PEG-GO and PEG-rGO to HeLa cells, whereas little toxicity was exhibited in rGO. Thus,
15
not only did PEG-modification improve the biocompatibility of rGO, but also significantly
16
enhanced the stability of rGO in biological buffer solutions.
AC C
EP
TE D
M AN U
SC
RI PT
1
17 18
Fig. 6 (a) Viability of HeLa cells 24h after treatment with various concentrations of PEG-GO and PEG-rGO
19
Reprinted with permission from Ref. [84]. Copyright (2013) Royal Society of Chemistry.
ACCEPTED MANUSCRIPT
Dendrimers
2
Dendrimers are highly branched polymers with a spherical three dimensional shape and build up
3
from a central core molecule, which is symmetrically surrounded by branched repetitive units.
4
These structures have a nanoscale size with a high density of positive charges due to the presence
5
of amino groups on the surface [74, 122]. Poly (amidoamine)(PAMAM) dendrimer, poly-L-
6
lysine (PLL) dendrimer, poly (prophylenimine) (PPI) dendrimer, poly(etherimine) (PETIM)
7
dendrimer are some used dendrimers, which have been employed as gene carriers [122], among
8
which PAMAM dendrimers have been evaluated as graphene modifier [115]. PAMAM
9
dendrimers, which are molecularly uniform with a unique shape and size have the intrinsic
10
capability for condensation and efficient transportation of DNA into cells owing to the fact that
11
amine groups on the surface of PAMAM dendrimers can electrostatically interact with phosphate
12
groups of DNA at physiological pH. Moreover, the genotoxic and cytotoxic effects, which are
13
exhibited by these PAMAM dendrimers, are relatively low in comparison to PEI since PAMAM,
14
unlike PEI, is a biodegradable polymeric structure in which a backbone of polymer chains with
15
peptide bonds exist [94].
16
poly-L-lysine (PLL)
17
PLL is a cationic polypeptide, which is highly positively charged at a physiological pH. J. Sun et
18
al. evaluated grafted PLL-grafted GO to intracellular delivery of immunostimulatory CpG
19
oligodeoxynucleotides (ODNs) [112]. They utilized two GO platforms, polydispersed GO and
20
uniform NGO to covalent immobilization of PLL. PLL modification provided a large number of
21
amino groups, which electrostatically interact with negatively charged CpG ODNs and facilitated
22
the ODNs loading. NGO-PLL-CpG conjugates revealed better biocompatibility and higher
23
cellular internalization in murine Raw264.7 macrophages juxtaposed with polydispersed GO-
24
PLL-CpG. In addition, NGO-PLL-CpG exhibited comparably higher immunomodulatory effects
25
than NGO-azide-CpG, which can be due to the adverse effect of azide, as a modifier, on cellular
26
uptake and bioactivity.
27
Chitosan
28
Chitosan (CS) or poly-D-glucosamine, a linear cationic polysaccharide derived from chitin, is
29
considered to be an appropriate candidate as modifier for gene and drug delivery because of its
30
properties, such as good biocompatibility and biodegradability compared to other cationic
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
polymers like PEI, improvement of the stability of nano-carriers in aqueous solutions and the
2
ability of surface charge adjustment for electrostatically interacting with negatively charged
3
nucleic acids [114].
4
Chitosan-modified GO was successfully utilized to deliver nucleotide based immunotherapeutic
5
agents. Zhang et al. developed GO-CS nano-composites through the self-assembly of GO and CS
6
via electrostatic interactions for delivery of CpG oligodeoxynucleotides (ODNs) (Fig. 7)[114].
7
The resultant GO-CS /ODNs nanocomplexes were stable under acidic conditions resembling the
8
endosomal pH, and revealed good potential of CS for buffering the endosome environment. The
9
biocompatible nature of CS resulted in decreases of GO cytotoxicity, and also enhanced cellular
10
uptake, compared to free CpG ODNs. The more efficient CpG ODNs delivery mediated by CS-
11
functionalized formulation induced a higher amount of cytokines secretion, juxtaposed to
12
GO/CpG ODNs, which indicated CS potential to improve GO function as gene nano-carrier.
AC C
13
EP
TE D
M AN U
SC
RI PT
1
14
Figure 7.Schematic illustration of preparation and application of GO-CS nano-composite as carrier for intracellular
15
delivery of CpG ODNs. Reprinted with permission from Ref. [114]. Copyright (2017) Elsevier.
16 17
Poly(sodium 4-styrenesulfonates) (PSS)
18
PSS is a water soluble polymer, which is non-toxic and could improve the dispersion of
19
graphene-based materials in aqueous solutions and prevent them from agglomeration. Since PSS
ACCEPTED MANUSCRIPT
is a negatively charged polyelectrolyte, it could participate in layer by layer assembly structures
2
via electrostatic interactions. Moreover, the pH sensitive nature of this polymer makes it a
3
suitable choice for releasing the payloads, which are incorporated into the composite in response
4
to the pH change in the environment [123, 124]. For gene delivery applications, this polymer was
5
usually employed with other cationic modifiers, such as PEI to make a complex structure, which
6
could provide enough hydrocolloidal stability as well as gene interaction potential [87].
8
Poly(2-dimethyl aminoethyl methacrylate) (PDMAEMA)
SC
7
RI PT
1
PDMAEMA, a synthetic water soluble polycationic polymer, is composed of side chains with
10
tertiary amine groups and can make a complex with pDNA through electrostatic interactions.
11
PDMAEMA has been utilized as transfection agent, while exhibiting a high level of
12
cytotoxicity, which is mainly contingent upon the length of its side chains [109, 125-127]. This
13
polymer is able to non-covalently immobilize onto GO nanosheets through π-π interactions and
14
produce a nanostructure with zwitterionic property, due to the presence of amine, phenol
15
hydroxyl and carboxyl groups [109, 128]. A biocleavable PDMAEMA–GO formulation
16
synthesized by the disulfide (S-S) mediated grafting method was introduced by X. Yang and his
17
colleagues as very efficient nano-carriers to transfect plasmid pRL-CMV. It is considered as the
18
reporter gene in COS7 and HepG2 cell lines, with a pH-sensitive gene releasing manner [109].
TE D
M AN U
9
Peptides
20
Peptides have been used for targeted gene/drug delivery and cell uptake since they are small-
21
sized and biocompatible molecules with cell-penetrating ability and can be chemically
22
synthesized and modified through a relatively easy manner [129]. The peptide could be selected
23
as a nano-carrier modifier with various aims, such as increasing cell penetration, cellular
24
targeting, stimulating endosomal escape and intracellular nuclear targeting [130]. In the last
25
decade, among various peptides utilized for gene delivery applications, cell penetrating peptides
26
(CPPs) have attracted considerable attention due to their ability to intracellularly deliver large
27
therapeutic molecules such as genes. In 2015, we introduced a novel CPP-conjugated GO (GOP),
28
as a gene nano-carrier aimed to improve the transfection efficiency of the GO nano-sheet by R8
29
chemical conjugation, while improving GO biocompatibility [29]. The effect of conjugation ratio
30
(R8 to GO) on the nano-carrier characteristics, such as particle size and morphology, surface
AC C
EP
19
ACCEPTED MANUSCRIPT
charge, hydrocolloid stability and plasmid condensation ability was comprehensively
2
investigated. The results (Fig. 8) demonstrated that the designed R8–GO nano-carrier with a
3
peptide molar ratio of 1µmol per mg of GO (GOP1) was selected as the most efficient and
4
biocompatible gene delivery vehicle having desirable properties as well as efficient transfection
5
ability compared to JetPEI® as a commercial reagent.
TE D
M AN U
SC
RI PT
1
Figure 8. The HEK293 cell line treated with the GOP1–pEGFP complex after 48 h: (a) transmission image, (b)
8
fluorescent image and (c) merged image. Quantification of transfection ability after 48 h of cell transfection in
9
the presence and absence of FBS (d). *p-value<0.005 compared to transfectionability in the absence of FBS
10
Reprinted with permission from Ref. [29]. Copyright (2015) Royal Society of Chemistry.
AC C
11
EP
6 7
12
In another study, Dowaidar et al. combined GO with two different kinds of CPP, PepFect14
13
(PF14) and PepFect221 (PF221), for gene delivery of three oligonucleotide (ON) models,
14
including plasmid (pGL3), splice correction oligonucleotides (SCO) and siRNA [120]. The
15
CPPs were sequentially added to ONs/GO complexes and physically loaded on the complexes.
16
The result demonstrated that GO-ONs-CPPs offered several magnitude enhancements of cellular
17
uptake and transfection ability (1.5-25 folds) than CPPs-ONs (Fig. 9).
RI PT
ACCEPTED MANUSCRIPT
Figure 9. Confocal microscopy images for the cell transfection of PF14-SCO without a) and with b) graphene
3
oxide (GO-SCO-PF14). PF14 were labeled with Alxa568-705SCO. Reprinted with permission from Ref [120].
4
Copyright (2017) Elsevier.
SC
1 2
5
Recent progresses in peptide synthesis provide the opportunity to design multipurpose peptides,
7
which are suitable for various biomedical applications. Chimeric peptides are one of the best
8
candidates to be utilized in designing gene delivery carriers. For instance, Ghafari and his
9
coworkers introduced and evaluated peptide-modified GQD with two different (green and red)
10
emission colors for gene delivery and nuclear targeting applications as well as cellular tracking
11
[131]. GQDs are physically conjugated with MPG-2H1 chimeric peptide, which was composed
12
of three different motifs. The selected motifs were responsible for the three specific
13
performances aimed to overcome the biological barriers against gene transfer (DNA packaging,
14
endosomal escape and cellular nuclear targeting). The chimeric peptide-modified GQDs
15
provided efficient cell tracking and showed enhanced internalization of the luciferase plasmid
16
into HEK 293 T cells.
18
TE D
EP 5- Multifunctional Graphene Nano-carriers
AC C
17
M AN U
6
19
Multifunctional nano-carriers can synchronously exhibit various functions including drug/gene
20
delivery, optical imaging, induction of apoptosis in cancer cells, increase in biocompatibility,
21
gene transfection and condensation ability as well as stability and solubility within physiological
22
solutions. There exist various advantages of using multifunctional carriers, including the ability
23
of having specific beneficial effects synergically, which can be obtained by less frequent
24
administration of therapeutic carriers, enabling researchers to have more control on vehicles and
25
also achieving more information regarding nano-carriers’ fate in the body [132]. Considering the
ACCEPTED MANUSCRIPT
unique chemical structure of graphene family nanoparticles, they showed a great potential to
2
insert various functionalities in order to achieve a multipurpose nano-platform for cargo delivery
3
(Fig. 10). Among the graphene family derivatives, GO could provide a suitable platform to
4
immobilize the modifier ligands/molecules on the basal surface as well as the edges of nano-
5
sheets via physical or chemical interactions (Fig. 11, up). Beside π-π interactions, which are
6
mostly utilized to introduce hydrophobic functionalities, carboxyl groups on the basal plane or
7
edges of GO sheets provide more reactive sites for electrostatic or chemical immobilization of
8
hydrophilic functionalities (Fig. 11, down) [133]. However, the other oxygen containing
9
functional groups are utilized for GO modification as well.
AC C
EP
TE D
M AN U
SC
RI PT
1
AC C
1
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
2
Figure 10. Schematic representation of modification of a multipurpose graphene-based platform for nucleotide based
3
cargo delivery
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 11: Up) A representation of structure and main functional groups of GO. Reprinted with permission from
3
Ref. [29]. Copyright (2015) Royal Society of Chemistry. Down) An illustration of common routes for covalent
4
and non-covalent carboxyl-mediated GO modifications. a: covalent PEG (branched or linear) conjugation for
5
increased bio-distribution water solubility, b: conjugation of amine-contained synthetic or natural polymers
6
(PEI, chitosan), c: targeting ligands such as folic acid, d: antibodies immobilization for cell targeting or
7
biosensor designing, e: fluorescence marker labeling for cell tracking and imaging, f: conjugation of amine-
8
decorated nano-particles (e.g., Fe2O3), g: proteins and peptide immobolization, electrostatically immobilization
9
of h: natural or synthetic cationic polymers, i: positively charged nano-particles, j: cationic ligands as mediator
10
for loading negatively charged biomolecules, e.g. nucleic acids DNAs, RNAs. Reprinted with permission from
11
Ref. [133]. Copyright (2015) Springer.
AC C
EP
1 2
12 13
Several studies have been dually reported on the functionalization of graphene-based nano-
14
carriers with PEG and PEI polymers [60, 85, 91, 92, 96, 105, 134] or with other modifiers, such
ACCEPTED MANUSCRIPT
as folic acid (FA)[106], chitosan[107] and mPEG-SPA[88]. In 2013, Feng et al. produced
2
covalently co-conjugated PEI (25 kDa) and PEG (10 kDa) nano-GO (Fig. 12), which exhibited
3
superior stability in salts and serum, excellent gene transfection efficiency and decreased cell
4
toxicity with respect to free PEI or GO-PEI [85]. The instability of GO-PEI in the serum, which
5
is mainly ascribed to the protein adsorption on GO via hydrophobic and electrostatic interactions,
6
were extensively hindered by PEG coating. Increased cellular uptake of synthesized nano-
7
carriers, intracellular delivery of siRNA and also gene silencing, in terms of down-regulating the
8
target gene (Polo-like kinase 1) were observed by the applied dual functionalization. In another
9
study by Yin et al., they investigated the antitumor activities of the signal transducer and
10
activator of transcription 3 (Stat3) delivered by PEG and PEI dual functionalized-GO after
11
intratumoral administration in mouse malignant melanoma and then infrared region irradiation.
12
The zeta potential of nanosheets increased from -49.08 mV to +43.13 mV after PEI conjugation,
13
whereas this amount decreased to +21.02 mV after PEGylation and also to +7.53 mV after the
14
formation of GO–PEI–PEG and plasmid Stat3 siRNA complexes. Their results indicated that due
15
to the positive charges, GO-PEI-PEG completely condensed the plasmid Stat3 siRNA, and had
16
no cytotoxic effect even at a concentration of 350 mgL-1 and transfected B16 cells with an
17
efficiency of above 60%. In addition, the GO–PEI–PEG/si-Stat3 complexes significantly
18
diminished tumor size in comparison to PBS, GO, PEI-GO and GO/si-Scramble when injected
19
intratumorally in mouse malignant melanoma. Examinations performed 30 days after injection
20
demonstrated that GO–PEI–PEG did not produce any toxic effects on liver and kidney organs
21
[60].
AC C
EP
TE D
M AN U
SC
RI PT
1
22
ACCEPTED MANUSCRIPT
1
Figure 12. Schematic of NGO-PEG-PEI preparation as a dual-functionalized formulation [85].
2
In another study, PEI and PEG dual-functionalized rGO (RGPP) was evaluated as a gene
4
delivery carrier to deliver gene plasmids and siRNA in 11 different cell lines, compared to a
5
commercial polyalkyleneimine cationic transfection reagent (TR) [121]. RGPP exhibited
6
remarkable delivery ability for both the FAM-labeled siRNA model and GFP plasmid into
7
HepG2 cells and GFP plasmids than TR. In addition, the modified GO nano-carrier interestingly
8
provided about 47.1% transfection efficiency in rabbit articular chondrocytes. However, it
9
provided acceptable GFP transfection of about 27%, 37.2% and 26.0% in A549 cell lines, EMT6
10
cells and LO2 cells, respectively. The results of the study concluded that RGPP nano-carrier
11
should be optimized for different cell lines.
12
Modified GO based carriers also showed the potential to be utilized in the genome editing
13
approach based on CRISPR/Cas 9 technology. Yue et al. utilized GO-based nano-carrier for
14
CRISPR/Cas9 complexes delivery in the genome editing approach [135]. In this study, PEI/PEG
15
dually functionalized GO was electrostatically loaded by high molecular weight Cas9/single-
16
guide RNA (sgRNA) complexes, where Cas9 maintains endonuclease activity equivalent to that
17
of its free state. GFP expressing a human AGS cell line, treated by GO-mediated Cas9/sgRNA,
18
showed 39% gene disruption with good cytocompatibility and non-significant cell toxicity.
19
PEG modifier alone, not with PEI, was also used dually along with other molecules, including
20
FA and Pyrenemethylamine hydrochloride (PyNH2) [83] and with PDMAEMA [102]. Yang et
21
al. conjugated amino-PEG (NH2-PEG-NH2) to FA and linked NH2-PEG-FA covalently to GO to
22
obtain GO-PEG-FA (Fig. 13), in which enhancement of the biocompatibility and solubility and
23
also decrease in toxicity of GO provided by PEG modification were combined with the tumor
24
targeting function of FA. Human telomerase reverse transcriptase (hTERT) was loaded onto the
25
surface of nano-carrier through π-π stacking with the assistance of PyNH2, which was non-
26
covalently coupled onto the GO-PEG-FA without cytotoxic effects. The GO-PEG-FA-PyNH2
27
showed a good dispersibility in blood. It was found that FAM-labeled DNA was delivered
28
efficiently into HeLa cells by both GO-PEG-FA-PyNH2 and GO-PEG -PyNH2 (FA free) with a
29
faster manner of targeting in GO-PEG-FA-PyNH2 [83].
30
AC C
EP
TE D
M AN U
SC
RI PT
3
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
1 2
Figure 13. Schematic illustration of GO-PEG-FA-PyNH2 synthesis for siRNA delivery into cells. Reprinted with
3
permission from Ref. [83]. Copyright (2012) Royal Society of Chemistry.
4
A combination of photothermal therapy and gene delivery has been reported as a combinatorial
6
strategy against cancer treatment. Graphene and its derivatives are one of the best candidates for
7
photothermal therapy. For instance, FA- grafted PEG (PEG-FA) was assessed to modified GO in
8
order to co-deliver HDAC1 and K-Ras siRNAs to target pancreatic cancer cells (Fig.14, a)[118].
9
They combined dual gene silencing effects with NIR light thermotherapy and tried to enhance
10
treatment effects. The FA-PEG served as a ligand with a potential of providing hydrocolloid
11
stability and cancerous cell targeting ability, simultaneously. To achieve better transfection
12
ability, the GO-PEG-FA was further non-covalently modified with a cationic polymer, poly-
13
allylamine hydrochloride (PAH). Therefore, the PEG-FA/PAH modified-GO provided a
14
multipurpose nano-carrier to co-deliver the two siRNAs. The results of this study demonstrated
15
that synergistically utilizing the gene silencing potential and NIR light thermotherapy capability
16
provided remarkable anticancer efficacy, consequently inhibiting in vivo tumor volume growth
17
by >80% (Fig. 14, b-d).
AC C
EP
TE D
5
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 14. a) Schematic representation of the FA/PEG/GO synthesis and subsequently gene loading. Folic acid (FA)
3
was conjugated with NH2-mPEG-NH2 to form FA/PEG-NH2. Subsequently, FA/GO nanosheets were prepared by
4
conjugating the amine-functionalized FA/PEG-NH2 to increase water solubility and biocompatibility. For siRNA
5
delivery, PEGylated or FA/PEGylated GO were functionalized with positive polymer PAH to form positively
6
charged GO/PEG/PAH or GO/PEG/FA/PAH which were able to deliver siRNA by electrostatic interaction.
7
Antitumor activities of GO-based nanoformulations in a MIA PaCa-2 xenograft animal model. (b) Representative
8
tumor tissue images of mice treated with (1) PBS, (2) FA/GO/scramble siRNA, (3) FA/GO with NIR light, (4)
9
FA/GO/(H+K) siRNA or (5) FA/GO/(H+K) siRNA with NIR light. Mice treated with FA/GO/(H+K) siRNA with
10
NIR light in the last group exhibited the smallest tumor size. (c) Tumor volume measuring over time and (d) tumor
11
weights of mice treated with the same nanoformulations as in (b), respectively. Relative tumor volume was defined
12
as (V-V0)/V0, where V and V0 indicate the tumor volume on a particular day and day 0, respectively [118].
14
EP
AC C
13
TE D
1 2
15
Peptides have also been used with other modifiers including PEI [82, 116], PLL [117], PEG
16
[136] and phospholipid-based amphiphilic polymer (PL-PEG) [113]. Functionalization of GO-
17
PEI was performed with NLS peptides in order to carry out nuclear targeting and localization
18
[82] and in another study, with neurotensin (NT, a neurospecifc peptide) mainly due to
ACCEPTED MANUSCRIPT
enhancement of the targeting property and biocompatibility and decrease in the cell toxicity
2
effect of PEI [116]. Also, GO–PLL was covalently modified with RGD peptides, which were
3
able to target the tumor through interaction with the integrin avb3, which is overexpressed on the
4
cell membrane of cancer cells [117]. rGO-PL-PEG was non-covalently functionalized with
5
CPPs, which could provide buffering capacity and elevate resistance to the acidic endolysosomal
6
environment and, as a result, improve siRNA transfection ability [113]. Ren et al. covalently
7
modified GO with low molecular weight PEI, in order to dwindle the cytotoxicity of PEI 25 kDa
8
and then PV7 was non-covalently incorporated into GO-PEI through three different routes,
9
including after the complexation of GO-PEI/DNA, simultaneously with the GO-PEI and DNA,
10
and before treatment of cells with GO-PEI/DNA. The incorporation of PV7 into GO-PEI 10 kDa
11
reduced the cytotoxicity and also its post-addition and simultaneous addition to the composite
12
enhanced gene expression efficiency, whereas prior-addition revealed slight changes juxtaposed
13
to GO-PEI/DNA [82]. In our recent study, we non-covalently functionalized rGO with
14
phospholipid-PEG (PL-PEG), which was more hydrocolloidally stable compared to rGO and
15
afterwards, the PL-PEG-rGO was non-covalently modified with R8 CPP (Fig. 15) [113]. The
16
observed good colloidal stability and dispersibility of double functionalized nanosheets in
17
aqueous solutions were the results of steric hindrance, provided by hydrophilic chains of PEG,
18
and the repulsion among positively charged amino groups of PEG. MCF-7 cells, which were
19
treated with rGON-PLPEG-R8, revealed superior viability and proliferation juxtaposed to rGO
20
nanosheets. The results indicated the successful condensation of siRNA on the surface of
21
nanosheets, the complete protection of siRNA from enzymatic degradation and the high
22
transfection efficiency of 82 ± 5.1%.
SC
M AN U
TE D
EP
AC C
23
RI PT
1
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
1 2
Figure 15. a) A schematic illustration of rGO based nano-carriers preparation via dual functionalization with PL-
3
PEG and R8 to deliver siRNA into the cancer cell. b) The functionalized nano-carriers successfully internalized the
4
FITC-siRNA into the cells juxtaposed to HiPerfect®, a commercialized siRNA transfection reagent. Reprinted with
5
permission from Ref. [113]. Copyright (2016) Elsevier.
6 7
In a study by Ren et al., dual functionalized GO were synthesized based on the PLL and peptide
8
(Arg-Gly-Asp-Ser, SDGR) covalent immobilization in order to actively target tumors and deliver
ACCEPTED MANUSCRIPT
anti-VEGF siRNA [117]. PLL and SDGR were utilized to improve the water solubility/loading
2
capacity and targeting ability to tumors, respectively. The results of RT-qPCR and ELISA assays
3
showed that the VEGF expressions in the level of mRNA and protein were remarkably down-
4
regulated by 40.86% and 51.71%, respectively. In addition, the in-vivo experiment on the S180
5
tumor-bearing mice model revealed that intravenously injected VEGF-siRNA at a dose of 0.3 mg
6
kg-1 (once every other day) provided the tumor inhibitory rate of about 51.74% (Fig. 16).
EP
TE D
M AN U
SC
RI PT
1
Figure 16. The in vivo evaluation of anti-tumoral effect of GPR/VEGF-siRNA. (a) Image of tumors, (b) tumor
9
weight and (c) tumor inhibitory rate. Reprinted with permission from Ref.[117]. Copyright (2017) Royal Society of
10 11
AC C
7 8
Chemistry.
12
PAMAM dendrimers have also been employed together with other molecules consisting of PEG
13
[104] and oleic acid [94] for dual functionalization of graphene-based materials as gene carriers.
14
Liu et al. developed graphene-oleate-PAMAM hybrids by chemical adsorption of oleci acid on
15
graphene and then covalent functionalization of graphene-oleic acid with PAMAM. Oleic acid is
16
able to improve gene transfection efficiency, as a result of its strong affinity to the
17
cytomembrane and ability to further the destabilization of the membrane and also provide
ACCEPTED MANUSCRIPT
terminal carboxyl groups for covalent bonding with PAMAM through amidation reaction.
2
Hybrids became positively charged in aqueous solution after modification with PAMAM, which
3
made them appropriate for immobilization of genetic materials. In addition, graphene-oleate-
4
PAMAM hybrids were stably dispersible in aqueous media and showed dose-dependent and cell
5
type-dependent cytotoxicity. Treatment of HeLa cells with the hybrids diminished the viability
6
of HeLa cells as the concentrations of hybrids increased, but the viability was still above 80%
7
after incubation by 100µg.mL-1 of hybrids for 24h. The GFP transfection efficiency of graphene-
8
oleate-PAMAM hybrids was 18.3% in HeLa cells at the mass ratio of 4:1, while it was 9.9% in
9
MG-63 cells at that of 2:1 [94].
SC
RI PT
1
11
M AN U
10
6- Dual Gene/Drug Delivery by Graphene-based Nano-carriers The nano-carriers potential for the delivery of gene and drug could simultaneously provide much
13
stronger approaches to fighting diseases. Graphene and its derivatives are good candidates for
14
co-delivery of nucleotide based therapeutics along with chemical based drugs due to their huge
15
surface area and unique conjugated structure [12]. The strong bonding between carbon-carbon
16
atoms, the aromatic structure, presence of free π electrons as well as reactive sites on the
17
graphene basal plane, make graphene suitable for the simultaneous load of hydrophilic and
18
hydrophobic biomolecules via chemical or physical interactions. For instance, co-delivery of
19
anticancer drugs and genes to the cancer cells has been utilized in cancer therapy applications.
20
However, there are few studies on the application of graphene-based nano-carrier for co-delivery
21
or sequential delivery of chemotherapeutic drugs and genes. Results reported by various studies
22
revealed remarkable superposed efficient therapy compared to merely gene or drug delivery
23
protocols alone.
24
One of the common and effective anticancer drugs is doxorubicin (DOX), which has been used
25
in cancer therapy over the past 30 years [137]. CS-functionalized GO was also utilized to co-
26
deliver platinum complexes drugs, including cisplatin, carboplatin, oxaliplatin as well as
27
nucleotides [138]. It usually undergoes various biological actions such as binding to DNA-
28
associated enzymes and intercalating with DNA base pairs. DOX was commonly delivered by
29
graphene-based nano-carrier along with the anticancer nucleotides. Zhang et al. [80] developed
AC C
EP
TE D
12
ACCEPTED MANUSCRIPT
a PEI-grafted GO nano-carrier for co-delivery of Bcl2-targeted siRNA and DOX. PEI-GO shows
2
considerably lower cytotoxicity and substantially higher efficacy of siRNA, at optimal N/P ratio
3
and then PEI of 25 K. The result showed that sequential delivery of siRNA and DOX by the PEI-
4
GO nano-carrier led to enhanced anticancer activity. In another study, folate conjugated
5
trimethyl chitosan-functionalized GO (FGNCs) was introduced as a capable candidate for the
6
delivery of both anticancer drugs and genes [93]. This complex nanoparticle was used as a
7
targeted delivery vector for DOX and pDNA delivery to Hela and A549 cells. The higher uptake
8
level in Hela cells with Folate-receptor was evidence for the targeting ability of FGNCs. Cao et
9
al. [97]
RI PT
1
SC
synthesized a functionalized-GO with FA-conjugated CS oligosaccharide (FACO)
including quadruple ammonium groups. GO-FACO was used for sequential delivery of MDR1
11
siRNA and DOX. The results presented higher cytotoxicity against DOX- treated MCF-7 cells
12
and also rather than DOX/ GO-FACO treated cells. In another study, the PEG/PEI/CS-Aco
13
complex was successfully utilized for the delivery of DOX and shRNA to the human hepatic cell
14
line [107]. PEI/ chitosan/PSS/Fe nanoparticles complicated multi-functionalized GO (CMG)
15
were introduced by Wang et al. for the delivery of pEGFP and DOX to A549 lung cancer and
16
C42b prostate cancer cell lines [89]. The DOX loaded nano-carriers (DOX–CMGs) release DOX
17
faster at acidic pH, and are more effective in killing A549 lung cancer cells than free DOX.
18
These results demonstrated the multifunctional capability of CMGs for targeted cancer
19
chemotherapy, gene therapy, and MR imaging. CS-functionalized GO was also utilized to co-
20
deliver platinum complex drugs including cisplatin, carboplatin, oxaliplatin as well as
21
nucleotides [138].
22
Zhi et al. [87] prepared a nano-complex composed of PEI, poly(sodium 4-styrenesulfonates), GO
23
and termed PPG (PEI/PSS/GO) to carry Adriamycin (ADR) along with miR-21 targeted siRNA.
24
First, ADR was loaded onto the PPG surface by physical mixing, and then anti-miR-21 was
25
loaded by electrical absorption (anti-miR-21/PPG/ADR). The superior therapeutic efficacy of
26
this nano-carrier could be exhibited by gene silencing of miR-21 and better accumulation of
27
ADR in tumor cells. PAMAM dendrimer-grafted gadolinium-functionalized GO (Gd-NGO) was
28
introduced as a nano-carrier to co-deliver drugs and specific gene-targeting agents to cancer cells
29
[111]. The positively charged surface of this vector was able to absorb the anti-cancer drug
30
epirubicin (EPI) and interaction with Let-7g miRNA. Also, Gd-NGO can be used as MRI-
AC C
EP
TE D
M AN U
10
ACCEPTED MANUSCRIPT
detectable to identify the tumor place. Therefore, Gd-NGO could be a potential vector for both
2
drug and gene therapy and molecular imaging diagnosis.
3
In another study, a PAMAM dendrimer-functionalized GO (GO-PAMAM) was utilized to
4
simultaneously deliver MMP-9 shRNA plasmid and DOX in order to enhance the efficacy of
5
anti-breast cancer treatment [115]. The PAMAM-functionalized GO showed the pH dependent
6
DOX release rate. In addition, compared to PEI-25k, this nano-carrier interestingly provided
7
more transfer ability as well as transfection efficiency in the presence of serum proteins. It
8
demonstrated that PAMAM could provide superior stability against protein adsorption as well as
9
a high amine covered surface to load much more shRNA. Co-delivery assay results demonstrated
10
that the cell survival rate of GO-PAMAM/DOX/MMP-9 treated MCF-7 breast cancer cells was
11
lower than DOX or MMP-9 shRNA treated once, which confirmed the significant potential of
12
breast cancer via the co-delivery approach.
13
Combining photothermal therapy and effective gene therapy have shown promising in cancer
14
treatment due to the enhanced combined therapeutic effects [139]. Graphene and its derivatives
15
can also be used in photothermal treatment of cancer because of their unique features like NIR
16
absorbance, huge specific surface area and functional groups [140]. One of the recent works in
17
this field was conducted by Cheng et al. [88]. They used the PEGylated PEI-grafted graphene/Au
18
complex to deliver siRNA to HL-60 cells. Moreover, this complex displayed photothermal
19
response to enhance the gene transfection efficacy. NGO-PEG-PEI is another composite
20
exhibiting remarkably enhanced pDNA transfection efficiencies under NIR laser irradiation [85].
21
In another study, Kim et al. [92] used the PEG–BPEI–rGO nano-composite to deliver plasmid
22
DNA in PC-3 and NIH/3T3 cells. In addition, this carrier demonstrated enhanced pDNA
23
transfection efficiencies under NIR irradiation that showed the ability of using NGO-PEG-PEI as
24
a light-responsive gene carrier.
25
Taken together, the co-delivery of drugs and nucleotide-based therapeutics by graphene-based
26
nano-carriers would have wide prospects for treatment of the target diseases, particularly cancer
27
[13].
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
1
7- Conclusions and perspectives
2
To date, graphene-based nanoplatform applications in biomedicine, particularly gene and drug
4
delivery, have progressively been the focus of interest. The sheet-like structure of the graphenic
5
nanoparticles, which can provide ultrahigh surface area, as well as a purely carbon composition,
6
offer a remarkable advantage to load cargoes with high efficiency compared with other nano-
7
carriers. In addition, relatively easy synthesis and the possibility of chemical modification of
8
such a carbon-based platform, for instance oxidation or reduction, makes it a suitable candidate
9
to design multifunctional nano-carriers. However, regarding the nucleotides negative charge, the
10
most possible interaction of genes with the graphene nanosheet would be based on the
11
electrostatic ones, which are limited by the relatively negative charge of graphene. Therefore, a
12
key design feature of such a nano-carrier is the cationic charge induction via cationic polymer
13
immobilization or cationic functional group insertion on the surface (e.g. amine). As seen in
14
Table1, cationic polymers, synthetic or natural, have been utilized as modifier to provide
15
electrostatic interaction with nucleotide based cargo. However, these kinds of modifiers could
16
also provide better cell membrane interactions, and consequently more efficient cell entrance.
17
One of the challenging issues in extra-cellular gene delivery with the power of nanoparticles is
18
undesired interaction with proteins, which are available in cell culture media or blood, and result
19
in nano-carrier instability and consequently precipitation before reaching the targeted cells. This
20
is because the large surface area of graphitic nanosheets and surface charge density are very
21
suseptible to agglomeration in such media, particularly after in-vivo administration.
22
Consequently, minimizing these nonspecific interactions and improving hemocompatibility via
23
surface functionalization with long chain hydrophilic modifiers, which could provide steric
24
hindrance seems essential to developing such carriers in clinical applications. Although
25
PEGylation are commonly utilized for this purpose, and results of various studies showed the
26
effectiveness of PEGylation on biostability as well as biocompatibility, additional investigations
27
on more efficient modifiers, which could provide biostability without interference in cell
28
entrance potential seems a more helpful strategy.
29
Unlike chemical drugs, for nucleotide-based cargoes, the intracellular delivery route even toward
30
the nucleous is another restrictive issue, where non-efficient endosomal escape results in such
31
severe cargo damages, and the damaged cargo obviously could not show therapeutic effects. In
AC C
EP
TE D
M AN U
SC
RI PT
3
ACCEPTED MANUSCRIPT
the nano-carrier design step, considering the intracellular obstacles and providing possible
2
solutions are very important. Although some cationic modifiers like PEI or CPPs could stimulate
3
endosomal escape, the co-delivery of some lysosomotropic agents (e.g. chloroquin, NH4Cl,
4
methylamine) into the cells could likely aid to overcome this challenge. However, the
5
cytotoxicity aspect of such agents should be considered and controlled.
6
From the synthesis and modification point of view, controlling the size of nanosheets and
7
immobolized ligands’ density per particle are challenging issues, which both effect the
8
nanoparticle biodistribution and cell entrance mechanisms. In addition, nucleotide loading
9
capacity is a function of the size and surface modification density. Non-uniformity usually
10
originated from various synthesis methods or a diverse level of oxidaton/reduction during further
11
functionalization of graphene. For the translation of graphene-based gene delivery systems to
12
clinical applications, developing manufacturing protocols, which could be scaled up based on the
13
laboratory studies, is an unignorable step.
14
Regarding the limited cell source (primary or cell line) having been utilized to evaluate the
15
potential of graphene-based nanomaterial as gene delivery vectors, one of the other challenges
16
ahead may be to confirm whether their successful performance is dependent on the treated cell’s
17
type or they could be considered as a general platform to transfect all cells. In addition, since in
18
various reviewed reports different eventual treatment protocols (i.e. transfection in presence or
19
absence of serum, different incubation times, pre/post treatment by some reagents) have been
20
used, there is a need for further investigation to find a clear relationship between the applied
21
treatments and the transfection efficacy in order to standardize the protocols.
22
Although numerous studies of graphene based nanoplatforms as gene delivery systems are
23
currently in progress by diverse research groups, only a few studies assessed the fate of
24
functionalized graphene-based particles through in-vivo gene delivery. Like other nanoparticles,
25
biocompatibility and biodegradation of graphitic nanoparticles are still challenging issues.
26
However, the results of studies demostrated that biocompatibility is a dose-dependent manner
27
and different functionalization could impact its improvement. Biodegradation and metabolization
28
of these kinds of particles are also under investigation. Although the overall findings of studies
29
have almost consistently confirmed the safety of the modified graphitic nano-carrier at suitable
30
doses, regarding a few in-vivo assessments in their gene delivery applications, these challenges
31
still await more investigations in the future before clinical trial attempts.
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
Under the light of such supplementary studies, it will create hope for the gene therapiast to open
2
up promising tools for efficent and safe gene delivery to the targeted cells and tissues.
3
Conflict of Interest
4
The authors declare there is no conflct of interest.
5
References
[5]
[6]
[7]
[8] [9]
[10] [11] [12]
[13] [14] [15] [16] [17]
SC
M AN U
[4]
TE D
[2] [3]
D. Scherman, A. Rousseau, P. Bigey, and V. Escriou, "Genetic pharmacology: progresses in siRNA delivery and therapeutic applications," Gene therapy, vol. 24, pp. 151-156, 2017. D. Cyranoski, "CRISPR gene-editing tested in a person for the first time," Nature, vol. 539, 2016. H. Yin, R. L. Kanasty, A. A. Eltoukhy, A. J. Vegas, J. R. Dorkin, and D. G. Anderson, "Non-viral vectors for gene-based therapy," Nature Reviews Genetics, vol. 15, p. 541, 2014. V. Wagner, A. Dullaart, A.-K. Bock, and A. Zweck, "The emerging nanomedicine landscape," Nature biotechnology, vol. 24, p. 1211, 2006. J. K. Wong, R. Mohseni, A. A. Hamidieh, R. E. MacLaren, N. Habib, and A. M. Seifalian, "Will nanotechnology bring new hope for gene delivery?," Trends in biotechnology, vol. 35, pp. 434451, 2017. M. Nurunnabi, K. Parvez, M. Nafiujjaman, V. Revuri, H. A. Khan, X. Feng, et al., "Bioapplication of graphene oxide derivatives: drug/gene delivery, imaging, polymeric modification, toxicology, therapeutics and challenges," Rsc Advances, vol. 5, pp. 42141-42161, 2015. H. Zhao, R. Ding, X. Zhao, Y. Li, L. Qu, H. Pei, et al., "Graphene-based nanomaterials for drug and/or gene delivery, bioimaging, and tissue engineering," Drug discovery today, vol. 22, pp. 1302-1317, 2017. L. Feng and Z. Liu, "Graphene in biomedicine: opportunities and challenges," Nanomedicine, vol. 6, pp. 317-324, 2011. G. Reina, J. M. González-Domínguez, A. Criado, E. Vázquez, A. Bianco, and M. Prato, "Promises, facts and challenges for graphene in biomedical applications," Chemical Society Reviews, vol. 46, pp. 4400-4416, 2017. S. Goenka, V. Sant, and S. Sant, "Graphene-based nanomaterials for drug delivery and tissue engineering," Journal of Controlled Release, vol. 173, pp. 75-88, 2014. H. Dong, C. Dong, T. Ren, Y. Li, and D. Shi, "Surface-engineered graphene-based nanomaterials for drug delivery," Journal of biomedical nanotechnology, vol. 10, pp. 2086-2106, 2014. G. Shim, M.-G. Kim, J. Y. Park, and Y.-K. Oh, "Graphene-based nanosheets for delivery of chemotherapeutics and biological drugs," Advanced drug delivery reviews, vol. 105, pp. 205-227, 2016. M. Nejabat, F. Charbgoo, and M. Ramezani, "Graphene as multifunctional delivery platform in cancer therapy," Journal of Biomedical Materials Research Part A, vol. 105, pp. 2355-2367, 2017. M. Vincent, I. de Lázaro, and K. Kostarelos, "Graphene materials as 2D non-viral gene transfer vector platforms," Gene therapy, vol. 24, p. 123, 2017. F. Yin, B. Gu, Y. Lin, N. Panwar, S. C. Tjin, J. Qu, et al., "Functionalized 2D nanomaterials for gene delivery applications," Coordination Chemistry Reviews, vol. 347, pp. 77-97, 2017. K. Novoselov and A. Geim, "The rise of graphene," Nat. Mater, vol. 6, pp. 183-191, 2007. A. M. Pinto, I. C. Gonçalves, and F. D. Magalhães, "Graphene-based materials biocompatibility: a review," Colloids and Surfaces B: Biointerfaces, vol. 111, pp. 188-202, 2013.
EP
[1]
AC C
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
RI PT
1
ACCEPTED MANUSCRIPT
[23] [24] [25] [26] [27] [28]
[29]
[30] [31]
[32] [33] [34]
[35]
[36] [37] [38]
RI PT
[22]
SC
[21]
M AN U
[20]
TE D
[19]
J. Liu, L. Cui, and D. Losic, "Graphene and graphene oxide as new nanocarriers for drug delivery applications," Acta biomaterialia, vol. 9, pp. 9243-9257, 2013. M. Jahan, Q. Bao, J.-X. Yang, and K. P. Loh, "Structure-directing role of graphene in the synthesis of metal− organic framework nanowire," Journal of the American Chemical Society, vol. 132, pp. 14487-14495, 2010. M. Freitag, T. Low, F. Xia, and P. Avouris, "Photoconductivity of biased graphene," Nature Photonics, vol. 7, p. 53, 2013. C. Liu, Z. Yu, D. Neff, A. Zhamu, and B. Z. Jang, "Graphene-based supercapacitor with an ultrahigh energy density," Nano letters, vol. 10, pp. 4863-4868, 2010. Q. Bao and K. P. Loh, "Graphene photonics, plasmonics, and broadband optoelectronic devices," ACS nano, vol. 6, pp. 3677-3694, 2012. Y. Shao, J. Wang, H. Wu, J. Liu, I. A. Aksay, and Y. Lin, "Graphene based electrochemical sensors and biosensors: a review," Electroanalysis, vol. 22, pp. 1027-1036, 2010. J. R. Potts, D. R. Dreyer, C. W. Bielawski, and R. S. Ruoff, "Graphene-based polymer nanocomposites," Polymer, vol. 52, pp. 5-25, 2011. M. Pumera, "Graphene in biosensing," Materials today, vol. 14, pp. 308-315, 2011. Y. Wang, Y. Shao, D. W. Matson, J. Li, and Y. Lin, "Nitrogen-doped graphene and its application in electrochemical biosensing," ACS nano, vol. 4, pp. 1790-1798, 2010. H. Shen, L. Zhang, M. Liu, and Z. Zhang, "Biomedical applications of graphene," Theranostics, vol. 2, pp. 283-294, 2012. A. R. Maity, A. Chakraborty, A. Mondal, and N. R. Jana, "Carbohydrate coated, folate functionalized colloidal graphene as a nanocarrier for both hydrophobic and hydrophilic drugs," Nanoscale, vol. 6, pp. 2752-2758, 2014. R. Imani, S. H. Emami, and S. Faghihi, "Synthesis and characterization of an octaarginine functionalized graphene oxide nano-carrier for gene delivery applications," Physical Chemistry Chemical Physics, vol. 17, pp. 6328-6339, 2015. T. Kuila, S. Bose, P. Khanra, A. K. Mishra, N. H. Kim, and J. H. Lee, "Recent advances in graphenebased biosensors," Biosensors and Bioelectronics, vol. 26, pp. 4637-4648, 2011. N. Ren, J. Li, J. Qiu, M. Yan, H. Liu, D. Ji, et al., "Growth and accelerated differentiation of mesenchymal stem cells on graphene-oxide-coated titanate with dexamethasone on surface of titanium implants," Dental Materials, vol. 33, pp. 525-535, 2017. D. Li, M. B. Müller, S. Gilje, R. B. Kaner, and G. G. Wallace, "Processable aqueous dispersions of graphene nanosheets," Nature nanotechnology, vol. 3, pp. 101-105, 2008. V. C. Sanchez, A. Jachak, R. H. Hurt, and A. B. Kane, "Biological interactions of graphene-family nanomaterials–an interdisciplinary review," Chemical research in toxicology, vol. 25, p. 15, 2012. L. Chen, Y. Hernandez, X. Feng, and K. Müllen, "From nanographene and graphene nanoribbons to graphene sheets: chemical synthesis," Angewandte Chemie International Edition, vol. 51, pp. 7640-7654, 2012. R. Sekiya, Y. Uemura, H. Naito, K. Naka, and T. Haino, "Chemical functionalisation and photoluminescence of graphene quantum dots," Chemistry–A European Journal, vol. 22, pp. 8198-8206, 2016. H. Sun, L. Wu, W. Wei, and X. Qu, "Recent advances in graphene quantum dots for sensing," Materials Today, vol. 16, pp. 433-442, 2013. L. Li, G. Wu, G. Yang, J. Peng, J. Zhao, and J.-J. Zhu, "Focusing on luminescent graphene quantum dots: current status and future perspectives," Nanoscale, vol. 5, pp. 4015-4039, 2013. H. Dong, W. Dai, H. Ju, H. Lu, S. Wang, L. Xu, et al., "Multifunctional PLA-PEG-grafted Graphene Quantum Dots for Intracellular MicroRNA Imaging and Combined Specific Gene-targeting Agents Delivery for Improved Therapeutics," ACS Appl. Mater. Interfaces, vol. 7, pp. 11015-11023, 2015.
EP
[18]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
ACCEPTED MANUSCRIPT
[44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56]
[57] [58]
[59]
[60]
RI PT
[43]
SC
[42]
M AN U
[41]
TE D
[40]
S. Zhou, H. Xu, W. Gan, and Q. Yuan, "Graphene quantum dots: recent progress in preparation and fluorescence sensing applications," RSC Advances, vol. 6, pp. 110775-110788, 2016. R. S. Duryat, "Graphene nanoribbons (GNRs) for future interconnect," in IOP Conference Series: Materials Science and Engineering, 2016, p. 012018. O. Akhavan, E. Ghaderi, H. Emamy, and F. Akhavan, "Genotoxicity of graphene nanoribbons in human mesenchymal stem cells," Carbon, vol. 54, pp. 419-431, 2013. M. K. Riley and W. Vermerris, "Recent Advances in Nanomaterials for Gene Delivery—A Review," Nanomaterials, vol. 7, p. 94, 2017. M. Elsabahy, A. Nazarali, and M. Foldvari, "Non-viral nucleic acid delivery: key challenges and future directions," Current drug delivery, vol. 8, pp. 235-244, 2011. C. Scholz and E. Wagner, "Therapeutic plasmid DNA versus siRNA delivery: common and different tasks for synthetic carriers," Journal of controlled release, vol. 161, pp. 554-565, 2012. P. M. Moreno and A. P. Pêgo, "Therapeutic antisense oligonucleotides against cancer: hurdling to the clinic," Frontiers in chemistry, vol. 2, 2014. R. W. Collin and A. Garanto, "Applications of antisense oligonucleotides for the treatment of inherited retinal diseases," Current opinion in ophthalmology, vol. 28, pp. 260-266, 2017. W. Guo, W. Chen, W. Yu, W. Huang, and W. Deng, "Small interfering RNA-based molecular therapy of cancers," Chinese journal of cancer, vol. 32, p. 488, 2013. K. Tatiparti, S. Sau, S. K. Kashaw, and A. K. Iyer, "siRNA Delivery Strategies: A Comprehensive Review of Recent Developments," Nanomaterials, vol. 7, p. 77, 2017. J. J. Rossi, "Expression strategies for short hairpin RNA interference triggers," Human gene therapy, vol. 19, pp. 313-317, 2008. R. Rupaimoole and F. J. Slack, "MicroRNA therapeutics: towards a new era for the management of cancer and other diseases," Nature Reviews Drug Discovery, vol. 16, pp. 203-222, 2017. X. J. Loh, T.-C. Lee, Q. Dou, and G. R. Deen, "Utilising inorganic nanocarriers for gene delivery," Biomaterials science, vol. 4, pp. 70-86, 2016. J. M. Ageitos, J.-A. Chuah, and K. Numata, "Design Considerations for Properties of Nanocarriers on Disposition and Efficiency of Drug and Gene Delivery," 2016. A. F. Adler and K. W. Leong, "Emerging links between surface nanotechnology and endocytosis: impact on nonviral gene delivery," Nano Today, vol. 5, pp. 553-569, 2010. S. Jin and K. Ye, "Nanoparticle-Mediated Drug Delivery and Gene Therapy," Biotechnology progress, vol. 23, pp. 32-41, 2007. R. Misra, M. Upadhyay, and S. Mohanty, "Design considerations for chemotherapeutic drug nanocarriers," Pharmaceutica Analytica Acta, vol. 2014, 2014. K. Kettler, K. Veltman, D. van de Meent, A. van Wezel, and A. J. Hendriks, "Cellular uptake of nanoparticles as determined by particle properties, experimental conditions, and cell type," Environmental toxicology and chemistry, vol. 33, pp. 481-492, 2014. R. Agarwal and K. Roy, "Intracellular delivery of polymeric nanocarriers: a matter of size, shape, charge, elasticity and surface composition," Therapeutic delivery, vol. 4, pp. 705-723, 2013. V. Mundra and R. I. Mahato, "Design of nanocarriers for efficient cellular uptake and endosomal release of small molecule and nucleic acid drugs: learning from virus," Frontiers of Chemical Science and Engineering, vol. 8, pp. 387-404, 2014. A. Albanese, P. S. Tang, and W. C. Chan, "The effect of nanoparticle size, shape, and surface chemistry on biological systems," Annual review of biomedical engineering, vol. 14, pp. 1-16, 2012. D. Yin, Y. Li, H. Lin, B. Guo, Y. Du, X. Li, et al., "Functional graphene oxide as a plasmid-based Stat3 siRNA carrier inhibits mouse malignant melanoma growth in vivo," Nanotechnology, vol. 24, p. 105102, 2013.
EP
[39]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
ACCEPTED MANUSCRIPT
[66]
[67]
[68]
[69]
[70]
[71]
[72] [73] [74]
[75] [76] [77]
RI PT
[65]
SC
[64]
M AN U
[63]
TE D
[62]
A. Paul, A. Hasan, H. A. Kindi, A. K. Gaharwar, V. T. Rao, M. Nikkhah, et al., "Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair," ACS nano, vol. 8, pp. 8050-8062, 2014. M.-j. Li, C.-m. Liu, Y.-b. Xie, H.-b. Cao, H. Zhao, and Y. Zhang, "The evolution of surface charge on graphene oxide during the reduction and its application in electroanalysis," Carbon, vol. 66, pp. 302-311, 2014. J. Kim, L. J. Cote, F. Kim, W. Yuan, K. R. Shull, and J. Huang, "Graphene Oxide Sheets at Interfaces," Journal of the American Chemical Society, vol. 132, pp. 8180-8186, 2010/06/16 2010. J. Rejman, V. Oberle, I. S. Zuhorn, and D. Hoekstra, "Size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis," Biochemical Journal, vol. 377, pp. 159-169, 2004. J. Kim, L. J. Cote, and J. Huang, "Two dimensional soft material: new faces of graphene oxide," Accounts of Chemical Research, vol. 45, pp. 1356-1364, 2012. V. C. Sanchez, A. Jachak, R. H. Hurt, and A. B. Kane, "Biological interactions of graphene-family nanomaterials: an interdisciplinary review," Chemical research in toxicology, vol. 25, pp. 15-34, 2011. C. Xu, D. Yang, L. Mei, B. Lu, L. Chen, Q. Li, et al., "Encapsulating gold nanoparticles or nanorods in graphene oxide shells as a novel gene vector," ACS applied materials & interfaces, vol. 5, pp. 2715-2724, 2013. H. Tian, W. Xiong, J. Wei, Y. Wang, X. Chen, X. Jing, et al., "Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG," Biomaterials, vol. 28, pp. 2899-2907, 2007. M. Ogris, P. Steinlein, M. Kursa, K. Mechtler, R. Kircheis, and E. Wagner, "The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells," Gene therapy, vol. 5, p. 1425, 1998. X. Lin, N. Zhao, P. Yan, H. Hu, and F.-J. Xu, "The shape and size effects of polycation functionalized silica nanoparticles on gene transfection," Acta biomaterialia, vol. 11, pp. 381392, 2015. Y. Li, H. Yuan, A. von dem Bussche, M. Creighton, R. H. Hurt, A. B. Kane, et al., "Graphene microsheets enter cells through spontaneous membrane penetration at edge asperities and corner sites," Proceedings of the National Academy of Sciences, vol. 110, pp. 12295-12300, 2013. K. P. Loh, Q. Bao, P. K. Ang, and J. Yang, "The chemistry of graphene," Journal of Materials Chemistry, vol. 20, pp. 2277-2289, 2010. Y. Pan, N. G. Sahoo, and L. Li, "The application of graphene oxide in drug delivery," Expert opinion on drug delivery, vol. 9, pp. 1365-1376, 2012. M. Teimouri, A. H. Nia, K. Abnous, H. Eshghi, and M. Ramezani, "Graphene oxide–cationic polymer conjugates: Synthesis and application as gene delivery vectors," Plasmid, vol. 84, pp. 51-60, 2016. C.-H. Lu, C.-L. Zhu, J. Li, J.-J. Liu, X. Chen, and H.-H. Yang, "Using graphene to protect DNA from cleavage during cellular delivery," Chemical Communications, vol. 46, pp. 3116-3118, 2010. L. Feng, S. Zhang, and Z. Liu, "Graphene based gene transfection," Nanoscale, vol. 3, pp. 12521257, 2011. H. Kim, R. Namgung, K. Singha, I.-K. Oh, and W. J. Kim, "Graphene oxide–polyethylenimine nanoconstruct as a gene delivery vector and bioimaging tool," Bioconjugate chemistry, vol. 22, pp. 2558-2567, 2011.
EP
[61]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
ACCEPTED MANUSCRIPT
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90] [91]
[92] [93]
RI PT
[82]
SC
[81]
M AN U
[80]
TE D
[79]
B. Chen, M. Liu, L. Zhang, J. Huang, J. Yao, and Z. Zhang, "Polyethylenimine-functionalized graphene oxide as an efficient gene delivery vector," Journal of Materials Chemistry, vol. 21, pp. 7736-7741, 2011. H. Bao, Y. Pan, Y. Ping, N. G. Sahoo, T. Wu, L. Li, et al., "Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery," Small, vol. 7, pp. 1569-78, Jun 6 2011. L. Zhang, Z. Lu, Q. Zhao, J. Huang, H. Shen, and Z. Zhang, "Enhanced chemotherapy efficacy by sequential delivery of siRNA and anticancer drugs using PEI-grafted graphene oxide," Small, vol. 7, pp. 460-464, 2011. H. Dong, L. Ding, F. Yan, H. Ji, and H. Ju, "The use of polyethylenimine-grafted graphene nanoribbon for cellular delivery of locked nucleic acid modified molecular beacon for recognition of microRNA," Biomaterials, vol. 32, pp. 3875-3882, 2011. T. Ren, L. Li, X. Cai, H. Dong, S. Liu, and Y. Li, "Engineered polyethylenimine/graphene oxide nanocomposite for nuclear localized gene delivery," Polymer Chemistry, vol. 3, pp. 2561-2569, 2012. X. Yang, G. Niu, X. Cao, Y. Wen, R. Xiang, H. Duan, et al., "The preparation of functionalized graphene oxide for targeted intracellular delivery of siRNA," Journal of Materials Chemistry, vol. 22, pp. 6649-6654, 2012. L. Zhang, Z. Wang, Z. Lu, H. Shen, J. Huang, Q. Zhao, et al., "PEGylated reduced graphene oxide as a superior ssRNA delivery system," Journal of Materials Chemistry B, vol. 1, pp. 749-755, 2013. L. Feng, X. Yang, X. Shi, X. Tan, R. Peng, J. Wang, et al., "Polyethylene glycol and polyethylenimine dual-functionalized nano-graphene oxide for photothermally enhanced gene delivery," Small, vol. 9, pp. 1989-1997, 2013. X. Zhou, F. Laroche, G. E. Lamers, V. Torraca, P. Voskamp, T. Lu, et al., "Ultra-small graphene oxide functionalized with polyethylenimine (PEI) for very efficient gene delivery in cell and zebrafish embryos," Nano Research, vol. 5, pp. 703-709, 2012. F. Zhi, H. Dong, X. Jia, W. Guo, H. Lu, Y. Yang, et al., "Functionalized graphene oxide mediated adriamycin delivery and miR-21 gene silencing to overcome tumor multidrug resistance in vitro," PloS one, vol. 8, p. e60034, 2013. F.-F. Cheng, W. Chen, L.-H. Hu, G. Chen, H.-T. Miao, C. Li, et al., "Highly dispersible PEGylated graphene/Au composites as gene delivery vector and potential cancer therapeutic agent," Journal of Materials Chemistry B, vol. 1, pp. 4956-4962, 2013. C. Wang, S. Ravi, U. S. Garapati, M. Das, M. Howell, J. Mallela, et al., "Multifunctional chitosan magnetic-graphene (CMG) nanoparticles: a theranostic platform for tumor-targeted co-delivery of drugs, genes and MRI contrast agents," Journal of Materials Chemistry B, vol. 1, pp. 43964405, 2013. S. K. Tripathi, R. Goyal, K. C. Gupta, and P. Kumar, "Functionalized graphene oxide mediated nucleic acid delivery," Carbon, vol. 51, pp. 224-235, 2013. J. Zhang, L. Feng, X. Tan, X. Shi, L. Xu, Z. Liu, et al., "Dual-polymer-functionalized nanoscale graphene oxide as a highly effective gene transfection agent for insect cells with cell-typedependent cellular uptake mechanisms," Particle & Particle Systems Characterization, vol. 30, pp. 794-803, 2013. H. Kim and W. J. Kim, "Photothermally controlled gene delivery by reduced graphene oxide– polyethylenimine nanocomposite," Small, vol. 10, pp. 117-126, 2014. H. Hu, C. Tang, and C. Yin, "Folate conjugated trimethyl chitosan/graphene oxide nanocomplexes as potential carriers for drug and gene delivery," Materials Letters, vol. 125, pp. 82-85, 2014.
EP
[78]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
ACCEPTED MANUSCRIPT
[99]
[100]
[101]
[102]
[103]
[104]
[105]
[106]
[107]
[108]
[109]
RI PT
[98]
SC
[97]
M AN U
[96]
TE D
[95]
X. Liu, D. Ma, H. Tang, L. Tan, Q. Xie, Y. Zhang, et al., "Polyamidoamine dendrimer and oleic acidfunctionalized graphene as biocompatible and efficient gene delivery vectors," ACS applied materials & interfaces, vol. 6, pp. 8173-8183, 2014. X. Cao, S. Zheng, S. Zhang, Y. Wang, X. Yang, H. Duan, et al., "Functionalized Graphene Oxide with Hepatocyte Targeting as Anti-Tumor Drug and Gene Intracellular Transporters," Journal of Nanoscience and Nanotechnology, vol. 15, pp. 2052-2059, 2015. Y. Tao, E. Ju, J. Ren, and X. Qu, "Immunostimulatory oligonucleotides-loaded cationic graphene oxide with photothermally enhanced immunogenicity for photothermal/immune cancer therapy," Biomaterials, vol. 35, pp. 9963-9971, 2014. X. Cao, F. Feng, Y. Wang, X. Yang, H. Duan, and Y. Chen, "Folic acid-conjugated graphene oxide as a transporter of chemotherapeutic drug and siRNA for reversal of cancer drug resistance," Journal of nanoparticle research, vol. 15, p. 1965, 2013. L. Hollanda, A. Lobo, M. Lancellotti, E. Berni, E. Corat, and H. Zanin, "Graphene and carbon nanotube nanocomposite for gene transfection," Materials Science and Engineering: C, vol. 39, pp. 288-298, 2014. H. Y. Choi, T.-J. Lee, G.-M. Yang, J. Oh, J. Won, J. Han, et al., "Efficient mRNA delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells," Journal of Controlled Release, vol. 235, pp. 222-235, 2016. Y.-P. Huang, C.-M. Hung, Y.-C. Hsu, C.-Y. Zhong, W.-R. Wang, C.-C. Chang, et al., "Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by PolyethylenimineFunctionalized Graphene Oxide," Nanoscale research letters, vol. 11, pp. 1-8, 2016. M. Y. Kim, F. L. Do Won Hwang, Y. Choi, J. W. Byun, D. Kim, J.-E. Kim, et al., "Detection of intrabrain cytoplasmic 1 (BC1) long noncoding RNA using graphene oxide-fluorescence beacon detector," Scientific reports, vol. 6, 2016. Y. Sun, J. Zhou, Q. Cheng, D. Lin, Q. Jiang, A. Dong, et al., "Fabrication of mPEGylated graphene oxide/poly (2-dimethyl aminoethyl methacrylate) nanohybrids and their primary application for small interfering RNA delivery," Journal of Applied Polymer Science, vol. 133, 2016. H. Kim, J. Kim, M. Lee, H. C. Choi, and W. J. Kim, "Stimuli-Regulated Enzymatically Degradable Smart Graphene-Oxide-Polymer Nanocarrier Facilitating Photothermal Gene Delivery," Advanced healthcare materials, 2016. F. Wang, B. Zhang, L. Zhou, Y. Shi, Z. Li, Y. Xia, et al., "Imaging dendrimer-grafted graphene oxide mediated anti-miR-21 delivery with an activatable luciferase reporter," ACS applied materials & interfaces, vol. 8, pp. 9014-9021, 2016. D. Yin, Y. Li, B. Guo, Z. Liu, Y. Xu, X. Wang, et al., "Plasmid-Based Stat3 siRNA Delivered by Functional Graphene Oxide Suppresses Mouse Malignant Melanoma Cell Growth," Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, vol. 23, pp. 229-236, 2016. C. Wang, X. Wang, T. Lu, F. Liu, B. Guo, N. Wen, et al., "Multi-functionalized graphene oxide complex as a plasmid delivery system for targeting hepatocellular carcinoma therapy," RSC Advances, vol. 6, pp. 22461-22468, 2016. Y. He, L. Zhang, Z. Chen, Y. Liang, Y. Zhang, Y. Bai, et al., "Enhanced chemotherapy efficacy by codelivery of shABCG2 and doxorubicin with a pH-responsive charge-reversible layered graphene oxide nanocomplex," Journal of Materials Chemistry B, vol. 3, pp. 6462-6472, 2015. S.-R. Ryoo, J. Lee, J. Yeo, H.-K. Na, Y.-K. Kim, H. Jang, et al., "Quantitative and multiplexed microRNA sensing in living cells based on peptide nucleic acid and nano graphene oxide (PANGO)," ACS nano, vol. 7, pp. 5882-5891, 2013. X. Yang, N. Zhao, and F.-J. Xu, "Biocleavable graphene oxide based-nanohybrids synthesized via ATRP for gene/drug delivery," Nanoscale, vol. 6, pp. 6141-6150, 2014.
EP
[94]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
ACCEPTED MANUSCRIPT
[114]
[115]
[116]
[117]
[118]
[119]
[120]
[121]
[122] [123]
[124]
[125]
RI PT
SC
[113]
M AN U
[112]
TE D
[111]
C.-J. Hsieh, Y.-C. Chen, P.-Y. Hsieh, S.-R. Liu, S.-P. Wu, Y.-Z. Hsieh, et al., "Graphene oxide based nanocarrier combined with a pH-sensitive tracer: a vehicle for concurrent pH sensing and pHresponsive oligonucleotide delivery," ACS applied materials & interfaces, vol. 7, pp. 1146711475, 2015. H.-W. Yang, C.-Y. Huang, C.-W. Lin, H.-L. Liu, C.-W. Huang, S.-S. Liao, et al., "Gadoliniumfunctionalized nanographene oxide for combined drug and microRNA delivery and magnetic resonance imaging," Biomaterials, vol. 35, pp. 6534-6542, 2014. J. Sun, J. Chao, J. Huang, M. Yin, H. Zhang, C. Peng, et al., "Uniform small graphene oxide as an efficient cellular nanocarrier for immunostimulatory CpG oligonucleotides," ACS applied materials & interfaces, vol. 6, pp. 7926-7932, 2014. R. Imani, W. Shao, S. Taherkhani, S. H. Emami, S. Prakash, and S. Faghihi, "Dual-functionalized graphene oxide for enhanced siRNA delivery to breast cancer cells," Colloids and Surfaces B: Biointerfaces, vol. 147, pp. 315-325, 2016. H. Zhang, T. Yan, S. Xu, S. Feng, D. Huang, M. Fujita, et al., "Graphene oxide-chitosan nanocomposites for intracellular delivery of immunostimulatory CpG oligodeoxynucleotides," Materials Science and Engineering: C, vol. 73, pp. 144-151, 2017. Y. Gu, Y. Guo, C. Wang, J. Xu, J. Wu, T. B. Kirk, et al., "A polyamidoamne dendrimer functionalized graphene oxide for DOX and MMP-9 shRNA plasmid co-delivery," Materials Science and Engineering: C, vol. 70, pp. 572-585, 2017. T. Y. Hsieh, W. C. Huang, Y. D. Kang, C. Y. Chu, W. L. Liao, Y. Y. Chen, et al., "NeurotensinConjugated Reduced Graphene Oxide with Multi-Stage Near-Infrared-Triggered Synergic Targeted Neuron Gene Transfection In Vitro and In Vivo for Neurodegenerative Disease Therapy," Advanced healthcare materials, vol. 5, pp. 3016-3026, 2016. L. Ren, Y. Zhang, C. Cui, Y. Bi, and X. Ge, "Functionalized graphene oxide for anti-VEGF siRNA delivery: preparation, characterization and evaluation in vitro and in vivo," RSC Advances, vol. 7, pp. 20553-20566, 2017. F. Yin, K. Hu, Y. Chen, M. Yu, D. Wang, Q. Wang, et al., "SiRNA Delivery with PEGylated Graphene Oxide Nanosheets for Combined Photothermal and Genetherapy for Pancreatic Cancer," Theranostics, vol. 7, p. 1133, 2017. H.-C. C. Foreman, G. Lalwani, J. Kalra, L. T. Krug, and B. Sitharaman, "Gene delivery to mammalian cells using a graphene nanoribbon platform," Journal of Materials Chemistry B, vol. 5, pp. 2347-2354, 2017. M. Dowaidar, H. N. Abdelhamid, M. Hällbrink, X. Zou, and Ü. Langel, "Graphene oxide nanosheets in complex with cell penetrating peptides for oligonucleotides delivery," Biochimica et Biophysica Acta (BBA)-General Subjects, vol. 1861, pp. 2334-2341, 2017. L. Wu, J. Xie, T. Li, Z. Mai, L. Wang, X. Wang, et al., "Gene delivery ability of polyethylenimine and polyethylene glycol dual-functionalized nanographene oxide in 11 different cell lines," Royal Society open science, vol. 4, p. 170822, 2017. J. Yang, Q. Zhang, H. Chang, and Y. Cheng, "Surface-engineered dendrimers in gene delivery," Chemical reviews, vol. 115, pp. 5274-5300, 2015. X. Sun, W. Wang, T. Wu, H. Qiu, X. Wang, and J. Gao, "Grafting of graphene oxide with poly (sodium 4-styrenesulfonate) by atom transfer radical polymerization," Materials Chemistry and Physics, vol. 138, pp. 434-439, 2013. H.-K. Jeong, M. H. Jin, K. H. An, and Y. H. Lee, "Structural stability and variable dielectric constant in poly sodium 4-styrensulfonate intercalated graphite oxide," The Journal of Physical Chemistry C, vol. 113, pp. 13060-13064, 2009. R. A. Jones, M. H. Poniris, and M. R. Wilson, "pDMAEMA is internalised by endocytosis but does not physically disrupt endosomes," Journal of controlled release, vol. 96, pp. 379-391, 2004.
EP
[110]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
ACCEPTED MANUSCRIPT
42 43
[131]
[132]
[133]
[134]
[135] [136]
[137]
[138] [139]
[140]
RI PT
[130]
SC
[129]
M AN U
[128]
TE D
[127]
S. Agarwal, Y. Zhang, S. Maji, and A. Greiner, "PDMAEMA based gene delivery materials," Materials Today, vol. 15, pp. 388-393, 2012. Y.-Z. You, D. S. Manickam, Q.-H. Zhou, and D. Oupický, "Reducible poly (2-dimethylaminoethyl methacrylate): synthesis, cytotoxicity, and gene delivery activity," Journal of Controlled Release, vol. 122, pp. 217-225, 2007. K. Hu, D. D. Kulkarni, I. Choi, and V. V. Tsukruk, "Graphene-polymer nanocomposites for structural and functional applications," Progress in Polymer Science, vol. 39, pp. 1934-1972, 2014. J. Zong, S. L. Cobb, and N. R. Cameron, "Peptide-functionalized gold nanoparticles: versatile biomaterials for diagnostic and therapeutic applications," Biomaterials Science, vol. 5, pp. 872886, 2017. V. P. Torchilin, "Cell penetrating peptide-modified pharmaceutical nanocarriers for intracellular drug and gene delivery," Peptide Science, vol. 90, pp. 604-610, 2008. S. M. Ghafary, M. Nikkhah, S. Hatamie, and S. Hosseinkhani, "Simultaneous Gene Delivery and Tracking through Preparation of Photo-Luminescent Nanoparticles Based on Graphene Quantum Dots and Chimeric Peptides," Scientific reports, vol. 7, p. 9552, 2017. R. Guo, L. Zhang, H. Qian, R. Li, X. Jiang, and B. Liu, "Multifunctional nanocarriers for cell imaging, drug delivery, and near-IR photothermal therapy," Langmuir, vol. 26, pp. 5428-5434, 2010. R. Imani, S. H. Emami, and S. Faghihi, "Nano-graphene oxide carboxylation for efficient bioconjugation applications: a quantitative optimization approach," Journal of Nanoparticle Research, vol. 17, p. 88, February 13 2015. H. Kim, J. Kim, M. Lee, H. C. Choi, and W. J. Kim, "Stimuli-Regulated Enzymatically Degradable Smart Graphene-Oxide-Polymer Nanocarrier Facilitating Photothermal Gene Delivery," Advanced healthcare materials, vol. 5, pp. 1918-1930, 2016. H. Yue, X. Zhou, M. Cheng, and D. Xing, "Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing," Nanoscale, 2018. R. Imani, S. Prakash, H. Vali, and S. Faghihi, "Polyethylene glycol and octaarginine n dualfunctionalized nano-graphene oxide: An optimization for efficient nucleic acids delivery," Biomaterials Science, 2018. O. Tacar, P. Sriamornsak, and C. R. Dass, "Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems," Journal of Pharmacy and Pharmacology, vol. 65, pp. 157-170, 2013. H. Bao, Y. Pan, Y. Ping, N. G. Sahoo, T. Wu, L. Li, et al., "Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery," Small, vol. 7, pp. 1569-1578, 2011. Y.-W. Chen, Y.-L. Su, S.-H. Hu, and S.-Y. Chen, "Functionalized graphene nanocomposites for enhancing photothermal therapy in tumor treatment," Advanced drug delivery reviews, vol. 105, pp. 190-204, 2016. M. Guo, J. Huang, Y. Deng, H. Shen, Y. Ma, M. Zhang, et al., "pH-Responsive Cyanine-Grafted Graphene Oxide for Fluorescence Resonance Energy Transfer-Enhanced Photothermal Therapy," Advanced Functional Materials, vol. 25, pp. 59-67, 2015.
EP
[126]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
S0008-6223(18)30827-3
DOI:
10.1016/j.carbon.2018.09.019
Reference:
CARBON 13447
To appear in:
Carbon
Received Date: 22 May 2018 Revised Date:
28 August 2018
Accepted Date: 3 September 2018
Please cite this article as: R. Imani, F. Mohabatpour, F. Mostafavi, Graphene-based Nano-Carrier modifications for gene delivery applications, Carbon (2018), doi: 10.1016/j.carbon.2018.09.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Graphene-based Nano-carrier Modifications for Gene Delivery
2
Applications
RI PT
3
Rana Imani a*, Fatemeh Mohabatpour a,b, Fatemeh Mostafavi a
4
6
a
Department of Biomedical Engineering, Amirkabir University of Technology, Tehran 15875/4413, Iran.
b
Division of Biomedical Engineering, University of Saskatchewan, Saskatoon, Canada.
SC
5
M AN U
7
8
9
TE D
10
EP
11
12 13
*
14
Technology, Tehran 15875/4413, Iran. Email: [email protected] Phone: +98 21-64545676, Fax: +98 21-
15
66468186
17 18 19
AC C
16
Corresponding authors: Rana Imani, Department of Biomedical Engineering, Amirkabir University of
ACCEPTED MANUSCRIPT
1 2
Graphical abstract
6 7 8 9 10 11 12 13
EP
5
AC C
4
TE D
M AN U
SC
RI PT
3
ACCEPTED MANUSCRIPT
Abstract
2
Gene therapy, a therapeutic approach based on nucleotide delivery to the targeted cells, serves as
3
a hot spot research. Regardless of recent developments in various nucleotide-based therapeutics,
4
the success of gene therapy in clinical steps is still limited due to less-efficient delivery
5
mechanisms toward the targeted tissue or cells. In recent decades, nanotechnology has provided
6
a remarkable breakthrough in developing safe and efficient gene carriers.
7
Graphene and its derivatives, as sheet-like carbonic nanomaterials, have been progressively
8
utilized in various biomedical fields. Currently, applications of graphene-based platforms extend
9
to gene delivery as nano-carrier. Considering the unique structure and chemistry of graphitic
10
nanoparticles, they provide high capacity for the loading of genes. To improve biocompatibility,
11
biostability, cellular uptake and increase in gene loading capacity, the surfaces of graphene and
12
its derivatives have been modified with various polymers or ligands. This article presents a
13
comprehensive overview of the current status of graphene family nanoplatforms for gene
14
delivery applications with a focus on the main graphene characteristics as gene nano-carriers. We
15
further highlight the various types of modifications and functionalization of graphene, suitable
16
for nucleotides loading and delivery. We finally address the future perspectives and challenging
17
issues of graphene-based gene therapy for prospect clinical applications.
20 21 22 23 24
SC
M AN U
TE D
EP
19
AC C
18
RI PT
1
ACCEPTED MANUSCRIPT
1
Contents
2 3
1-
Introduction to Gene Therapy .......................................................................................................... 5
4
2-
Graphene as a Versatile Nano-material ............................................................................................ 7 2-1- Different Types of Graphene as Nano-carriers .............................................................................. 8
6
Graphene ......................................................................................................................................... 9
7
Graphene oxide (GO)....................................................................................................................... 9
8
Reduced graphene oxide (rGO) ........................................................................................................ 9
9
Nano-graphene oxide (NGO) ........................................................................................................... 9
10
Graphene quantum dots (GQDs) .................................................................................................... 10
11
Graphene nanoribbons (GNRs) ...................................................................................................... 10
12
2-2- Therapeutic Nucleotides Delivered by Graphitic Materials ......................................................... 10
13
Plasmid DNA (pDNA) ................................................................................................................... 11
14
Antisense Oligonucleotides (AONs)............................................................................................... 11
15
Small interfering RNA (siRNA) ..................................................................................................... 11
16
Short hairpin RNA (shRNA) .......................................................................................................... 11
17
MicroRNA (miRNA) ..................................................................................................................... 12
SC
M AN U
3-
TE D
18
RI PT
5
Nano-carrier Design Criteria for Efficient Gene Delivery ............................................................... 12 3-1- Surface charge............................................................................................................................ 12
20
3-2-Size ............................................................................................................................................. 14
21
3-3- Shape ......................................................................................................................................... 15
22
3-4-Surface Chemistry ....................................................................................................................... 15
23
4-
EP
19
Graphene Modifications as Gene Delivery Vehicle ........................................................................ 16 4-1- Graphene modifiers .................................................................................................................... 22
25
Polyethylenimine (PEI) .................................................................................................................. 22
26
Polyethylene glycol (PEG) ............................................................................................................. 28
27
Dendrimers .................................................................................................................................... 30
28
poly-L-lysine (PLL) ....................................................................................................................... 30
29
Chitosan ........................................................................................................................................ 30
30
Poly(sodium 4-styrenesulfonates) (PSS)......................................................................................... 31
31
Poly(2-dimethyl aminoethyl methacrylate) (PDMAEMA) .............................................................. 32
AC C
24
ACCEPTED MANUSCRIPT
1
Peptides ......................................................................................................................................... 32
2
5-
Multifunctional Graphene Nano-carriers ........................................................................................ 34
3
6-
Dual Gene/Drug Delivery by Graphene-based Nano-carriers .......................................................... 45
4
7-
Conclusions and perspectives ......................................................................................................... 48 Conflict of Interest............................................................................................................................. 50
6
References............................................................................................................................................. 50
RI PT
5
7
9
SC
8
1- Introduction to Gene Therapy
Gene therapy, as a hot spot research, is a therapeutic approach that has attracted scientists and
11
scientific communities for decades. This approach comprises the delivery of nucleotide-based
12
therapeutic agents into the targeted cells to correct the genetic functions in the cell nucleus or
13
cytoplasm [1]. Since the 1990s, the advent of gene therapy started a huge revolution in the
14
history of biomedical sciences and was introduced as an effective strategy to fight genetic
15
disorders. After three decades, today, CRISPR-based genome editing, as emerging concepts in
16
gene therapy, remarkably increases promises of treatment of genetic-based diseases [2].
17
Regardless of recent progress in understanding the molecular biology of genetically-based
18
diseases and various developing nucleotide-based therapeutics, efficient delivery of the nucleic
19
acids into the cells remains a major challenge, which has seriously limited gene therapy success,
20
particularly in clinical trial steps. Therefore, the success of the gene therapy approach is mainly
21
dependent on the development of a safe and efficient gene carrier [3].
22
In recent years, the emergence of nanomedicine and the application of nanotechnology in
23
medicine, has greatly changed the face of gene therapy, where amalgamating the nanotechnology
24
concepts and non-viral gene therapy approach has provided a breakthrough in biomedicine [4].
25
Nanotechnology provides an essential improvement in gene carrier design to have a desirable
26
carrier, which could overcome both extra-cellular and intracellular obstacles. Despite decades of
27
progress in nanoparticle design and synthesis, currently, the US Food and Drug Administration
28
(FDA) has not yet approved any nanoparticle-based formulation for gene therapy application [5].
AC C
EP
TE D
M AN U
10
ACCEPTED MANUSCRIPT
Consequently, designing and developing optimum multifunctional nanoparticles still remains an
2
opportunity for researchers.
3
Growing interest in graphene families, carbon-based nanoparticles, gave the possibility to apply
4
this class of nanomaterials in medical applications, as has been discussed in several reviews [6-
5
9]. In particular, graphene-based nanoplatforms attract tremendous attention in the delivery of
6
various therapeutic agents such as drugs and genes [10-12], whereas these platforms have been
7
suggested as an effective cargo delivery system for cancer therapy [13]. Looking at the trend of
8
publications from 2004 to 2017, which have utilized graphene and its derivatives as gene
9
delivery carrier confirms the success of graphene-based carrier in in-vitro and in-vivo gene
10
therapy applications. However, a few literature reviews have specifically addressed different
11
aspects and challenges of graphene nanoplatforms for gene delivery purposes [14, 15].
12
In this review, we present a comprehensive overview of the graphitic nanomaterials as gene
13
delivery vehicles over the past seven years (Fig.1). We briefly discuss the properties of graphene
14
families’ nanoparticles as well as nano-carrier design criteria with a special focus on graphene
15
modification approaches. We aimed to highlight biomaterial-based strategies in detail in order to
16
design and modify the graphitic nano-carrier suitable for overcoming extra and intracellular
17
barriers to have efficient gene delivery. In addition, more advanced multifunctionalized graphene
18
nanoplatforms, which have attracted much more attention in recent studies, are addressed more
19
specifically in dual delivery (e.g. gene and drug) applications.
AC C
EP
TE D
M AN U
SC
RI PT
1
20
ACCEPTED MANUSCRIPT
1
Figure 1. Number of publications on graphene application as gene delivery platform from 2010-2018(April). Source
2
ISI Web of Science (search keywords: Graphene, gene delivery).
3
2- Graphene as a Versatile Nano-material In October 2004, K. Novoselov and A. Geim shocked the physics world with their discovery of
5
the youngest allotrope of graphite, graphene sheets [16]. Graphene, the basic structure of
6
graphite, is a sheet of two dimensional (2D) single or few layers of sp2 ranged carbon atoms [10,
7
17]. It is a fundamental constructive part for other graphite-based materials with various
8
geometry [18] (Fig 2). Graphitic-based nanostructures mainly include spherical structures (zero-
9
D, e.g. fullerenes), rolled structures (2D, e.g. carbon nanotubes (CNTs)), or layered structures
10
(3D , e.g. graphite) [4]. However, graphene and its derivatives usually address the sheet-like
11
structure of carbon-based nanomaterials. The graphene sheets can be synthesized by either of
12
two approaches: bottom-up or top-down methods. For example, chemical vapor deposition
13
(CVD) serves as a bottom–up method and chemical exfoliation or the mechanical scotch tape
14
method are categorized in the top-down methods [16, 19].
M AN U
SC
RI PT
4
AC C
EP
16
TE D
15
17
ACCEPTED MANUSCRIPT
Figure 2. Graphenic-derived nanoparticles. Graphene is a 2D sheet like carbon-based material. It could be found in
2
the form of zero dimensions (fullerenes), one dimension (carbon nanotubes) and three dimensions (graphite) form.
3
Reprinted with permission Ref. [18]. Copyright (2013) Elsevier.
4
Graphene and its derivatives are interesting for scientists from disparate fields due to their
5
remarkable physical and chemical properties, such as high young's modulus, great thermal and
6
electrical conductivity, huge specific area and biocompatibility [8]. The application fields of
7
graphene and its derivatives are ranging from electronic fields such as photoconductive materials
8
[20], energy technology [21], optoelectronic devices [22], sensors [23] and composite materials
9
[24] to biomedical applications, such as medical imaging/bio-sensing [8, 25, 26], tissue
10
engineering [27], drug delivery [25, 26], cancer therapy [27, 28], antibacterial materials[17] and
11
gene delivery [29].
12
Graphitic materials differ in their structural features, such as number of layers, defect density,
13
surface chemistry and layer size and composition. This interestingly resulted in a wide variety of
14
tunable properties, which are considerable in biomedical applications.
15
Since graphene arrival in 2004, many researches have been conducted on its structure and
16
properties, but there was no discussion on the use of its medical applications until 2008, which is
17
a starting point in graphitic material storming in the field of biomedicine. Nowadays, the range
18
of biomedical applications of graphene and its derivatives is enormous, actually bigger than non-
19
medical applications [9]. However, for most bio-applications, the graphitic materials need to be
20
functionalized with biocompatible modifiers (e.g. polymers) to improve specific properties or
21
reduce toxicity. Among various biomedical applications of graphenic materials, more frequently
22
reported ones, which have attracted ever-increasing interest, are in the field of drug delivery [12],
23
gene delivery [14], biosensing and bioimaging [30] as well as a more recent one, tissue
24
engineering and regenerative medicine [7, 31].
25
2-1- Different Types of Graphene as Nano-carriers
26
Graphene family nanomaterials including graphene, graphene oxide (GO), reduced graphene
27
oxide (rGO), nano-graphene oxide (NGO), graphene quantum dots (GQDs) and graphene
28
nanoribbon (GNR) have been widely used in biomedical applications. However, among the
29
graphene derivatives, GO and rGO have been much more frequently utilized as nano-carriers for
30
intracellular delivery of various cargoes, such as drugs and genes.
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
Graphene
2
Graphene is a two dimensional (2D) structure composed of mono or multi-layers of sp2
3
hybridized carbon atoms. Pristine graphene is extremely hydrophobic and exhibits poor
4
dispersibility and stability in aqueous solutions because of the strong interactions among
5
graphene sheets and the high tendency to aggregate. Biomolecules can be loaded onto the
6
conjugated carbon network of graphene through π- π interaction [11]. Since it is quite hard to
7
synthesize the single layer graphene without defects and also due to its poor stability in solutions,
8
multi-layer graphene and GO are broadly used for biomedical purposes[10].
9
Graphene oxide (GO)
SC
RI PT
1
GO sheets, which are obtained by chemical oxidation of graphite under extremely oxidized
11
conditions, are structurally different from the graphene, mainly in the richness of these oxygen
12
containing groups in the GO sheets surface [11]. In fact, the ionization of carboxyl and hydroxyl
13
groups and the electrostatic repulsion between them results in the formation of stable GO
14
colloids in different polar solvents [32]. A wide variety of bio-functionalization of GO can be
15
performed via chemical interaction between oxygen containing groups of GO and various
16
biomolecules [11]. In addition, GO native functional groups might have undergone chemical
17
reaction to form the other non-oxygenated functional groups (e.g. NH2).
18
Reduced graphene oxide (rGO)
19
rGO is produced by treatment of GO under reducing conditions. Reduction of GO into rGO
20
renovates its electrical conductivity, declines oxygen content and surface charge and enhances
21
hydrophobicity [33]. In addition, the oxygen containing functional groups of GO are entirely or
22
partially removed during the reduction process, which can introduce holes and defects in the rGO
23
structure. The reduction process makes graphitic sheets less hydrophobic compared to the bare
24
graphene and its basal plane reactivity is also lower than GO [10].
25
Nano-graphene oxide (NGO)
26
The term nano-GO is used for defining GO with a small lateral size, characteristically smaller
27
than 100 nm and usually lower than 20 nm, whereas the size of regular GO in both dimensions is
28
greater than 100 nm [34]. In fact, NGO resembles zero-dimensional (0D) material with a higher
29
degree of oxygenation in contrast to conventional GO, which is 2D. Thus, NGO is considered as
AC C
EP
TE D
M AN U
10
ACCEPTED MANUSCRIPT
a decent candidate for biomedical applications due to the fact that its small size provides the
2
ability to facilitate cell internalization and increase dispersion stability [33].
3
Graphene quantum dots (GQDs)
4
GQDs are a type of quantum dots obtained by cutting 2D graphene sheets into 0D small dots,
5
which are regularly shaped and their sizes are from several to 100 nm, and have the properties of
6
both graphene and carbon dots (CDs)[34-36]. This conversion results in opening a band gap in
7
GQDs due to quantum confinement and presents powerful photoluminescence, which does not
8
exist in 2D graphene [37]. GQDs are structurally similar to GO and are composed of the main
9
sp2 bonded carbon network, enriched with oxygen containing functional groups on the edges
10
[11]. Due to their superior properties such as lower toxicity, better biocompatibility and higher
11
resistance to photo-bleaching and blinking, GQDs are considered to be a promising replacement
12
for semiconductor quantum dots, which are toxic [36, 38, 39].
13 14
Graphene nanoribbons (GNRs) GNRs are produced by cutting a 2D graphene sheet into a 1D narrow strip with a specific edge
15
structure and width. Typically, the GNR width and aspect ratio are less than 10 nm and higher
16
than 10, respectively; in fact, GNRs can be considered as unrolled CNTs [34, 40]. Their band
17
gaps are contingent upon their width and as a result, their properties can be changed from semi-
18
metallic to semi-conductive by decreasing the width [34]. rGO nanoribbons (rGONRs) exhibit
19
higher cytotoxicity and genotoxicity compared to rGO sheets [41].
20
2-2- Therapeutic Nucleotides Delivered by Graphitic Materials
21
Graphitic nano-carriers are able to deliver various types of therapeutic nucleotides in order to
22
modulate the gene expression profile of cells. Two major manners exist through which the
23
genetics of the targeted cells can be modulated. In the first method, DNA is delivered into the
24
cells to provide a transcriptionally competent copy of a defective or missed gene and makes up
25
for deficiency of a protein. While in the second approach, therapeutic nucleic acids consisting of
26
Antisense Oligonucleotides (AONs), Small interfering RNA (siRNA), Short hairpin RNA
27
(shRNA) and MicroRNA (miRNA) are employed to inhibit or knockdown the expression of
28
defective genes [42].
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
Plasmid DNA (pDNA)
2
Plasmids, known as double stranded DNA, are mostly annular and comprised of the transgene
3
encoding for a particular protein. pDNA range in size from a few hundred to several thousand
4
basepairs (bp). After internalizing into the cells and being uptaken by the nucleus, this genetic
5
material commences, producing the encoded protein through transcription and translation
6
processes using cellular machines. Besides the transgene, pDNA is composed of other
7
modulatory elements including the promoter and enhancer sequences as well as splicing and
8
polyadenylation sites which have a profound role in gene expression [43, 44].
9
Antisense Oligonucleotides (AONs)
SC
RI PT
1
AONs, which contain 18-21 nucleotides, are delivered as single-stranded deoxyribonucleotides
11
and must detect their complementary mRNA sequence, hybridize with the target mRNA and, as
12
a result, induce gene knockdown [43]. Mechanisms through which AONs function are steric
13
hindrance and target degradation. Steric hindrance, known as non-degradative pathway,
14
influence the attachment of trans-splicing modulatory agents to pre-mRNA and thereby regulate
15
splice models, while the target degradation mechanism can be achieved by the action of RNase H
16
enzyme cleaving mRNA in the RNA-DNA hybrid [45, 46].
17
Small interfering RNA (siRNA)
18
Small interfering RNAs (siRNAs) or short interfering RNAs are double-stranded RNA
19
molecules, composed of 20-25 bp in length, that interfere with the translation process through the
20
RNA interference (RNAi) pathway and degradation of complementary mRNA strands, and thus
21
inhibit gene expression [47]. This function occurs through the gene-silencing mechanism, in
22
which dicer protein chops siRNA into shorter segments among which there is a fragment with
23
affinity for the RNA-induced Silencing Complex (RISC). After attaching to RISC, the passenger
24
strand
25
to the corresponding mRNA leading to further degradation of the mRNA and gene silencing [1,
26
48].
27
Short hairpin RNA (shRNA)
28
shRNA is a RNA sequence which has gene silencing ability through the RNAi mechanism and
29
comprises stems (25-29 bases) and a loop site (4-23 nucleotides). shRNAs are transformed into
AC C
EP
TE D
M AN U
10
of
siRNA
is
cleaved
and
the
guide
strand
is
directed
ACCEPTED MANUSCRIPT
siRNAs by the dicer protein, which eliminates the loop part, and afterwards, siRNAs incorporate
2
to RISC and cleave target mRNA [43, 49].
3
MicroRNA (miRNA)
4
MicroRNA, a single-stranded RNA molecule with 20-24 nucleotides, is considered as a non-
5
coding RNA, which regulates some cellular behaviors including differentiation, proliferation and
6
survival. In fact, these functions are achieved by attaching to target mRNAs and then inhibiting
7
the translational process or cleaving corresponding mRNAs [43, 50].
SC
8
RI PT
1
3- Nano-carrier Design Criteria for Efficient Gene Delivery
For efficient gene delivery, a carrier should be designed with regard to the cargo type as well as
10
barriers, whereas the delivered cargo may be faced with in delivery routes. Firstly, the nano-
11
carrier designed to deliver the genetic materials should be able to interact with nucleotides in
12
order to efficiently load the cargoes. For successful transfection, nano-carriers should be able to
13
protect the genetic materials during transport, overcome the extracellular and intracellular
14
barriers and deliver the functional form of genetic materials into the target cells [51]. Several
15
physiochemical parameters, such as surface charge, size, shape and surface chemistry are
16
principal to be considered in order to design an effective nano-carrier system to that end. By
17
manipulation of these factors, the fate of nano-carriers inside the body can be altered, and
18
consequently the final success of gene therapy would be affected [52]. Herein, the main
19
characteristics, which are needed to be considered in designing the graphitic nano-carriers, are
20
briefly summarized.
21
3-1- Surface charge
22
Surface charge plays a prominent role in the gene/carrier complex formation, cellular uptake
23
mechanism, cytotoxicity and stability of nano-carriers [53]. Genetic materials, such as DNA and
24
RNA have negative charge due to the presence of phosphate groups in their backbone.
25
Consequently, they cannot cross cellular membranes by themselves and need carrier systems,
26
which can form complexes with them, compromise the electrostatic repulsion between them and
27
the negatively charged cell membrane and facilitate their internalization into the cells [51]. One
28
of the simple approaches to bind the genetic materials to carriers is based on electrostatic
29
interactions between positively charged nano-carriers and nucleotides [54]. Both charge polarity
AC C
EP
TE D
M AN U
9
ACCEPTED MANUSCRIPT
and charge density have a crucial influence on the cellular uptake of nanoparticles as well as
2
their cytotoxicity. Generally, positive surface charges juxtaposed with neutral or negative
3
charges have been demonstrated to enhance the cellular uptake of nanoparticles due to
4
electrostatic attraction with the cell membrane that is favorable for adhesion to the cell surface in
5
both phagocytes and non-phagocytic cell lines [55, 56]. Also, carriers with a higher positive
6
charge density have been shown to be up taken at a higher level and in the most fastest rate [53,
7
55]. Additionally, the surface charge of nano-carriers is able to determine the mechanism of
8
cellular endocytosis. Positively charged carriers use the clathrin-mediated endocytosis pathway
9
in order to internalize into the cells, while carriers with negative surface charge could be taken
10
up through other pathways, such as the caveolae-mediated pathway [57, 58]. However,
11
nanoparticles with positive charge have shown more cytotoxic effects compared to negatively or
12
neutral charged nanoparticles [55]. In fact, positive charge of the nanoparticle can increase Ca2+
13
influx into cells and thereby inhibit their proliferation, induce reconstruction of lipid bilayers and
14
fluidity [59]. Furthermore, the stability of nano-carrier systems significantly relies on their
15
surface charge, such that nanoparticles with a higher surface charge can exhibit more resistance
16
to form aggregations mostly due to their strong self-repulsive effect [52].
17
It is noteworthy that the surface charge of the carriers would be affected by gene loading. Due to
18
amine (N)/phosphate (P) electrostatic interaction between the carrier and nucleotides, the surface
19
charge of the complex would be partially neutralized [60]. The carrier/DNA ratio (known as N:P
20
ratio) usually dictates the formed complex charge, while it raises to more positive values by
21
adding N:P ratio. In a lower N:P ratio, the formed complex may be unable to effectively interact
22
with the cell membrane [61]. Therefore, to evaluate the charge effect on gene transfection, the
23
net charge of carrier/gene complexes should be considered.
24
GO sheets contain epoxide (-O-) and hydroxyl (-OH) groups on their basal planes, whereas
25
hydroxyl and carboxyl (-COOH) groups exist at their edges [62]. Therefore, GO with
26
hydrophilic terminates and more hydrophobic basal planes can be considered as an amphiphilic
27
sheet [63]. The presence of terminal carboxyl groups can afford stability in colloidal dispersions
28
and negative charge on the surface, such that the pH has an influential impact on this surface
29
charge. For instance, high pH can upgrade the de-protonation of the carboxyl groups and
30
therefore GO would become more charged [63]. Also, the uncharged groups existing in the basal
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
plane, due to their polarity, provide surface reactions, such as weak interactions and hydrogen
2
bonding. Furthermore, hydrophobic and free surface π electrons exist on the basal planes, which
3
are able to form π- π interactions for non-covalent interactions. By reducing GO into rGO, it
4
becomes a less hydrophilic molecule with a lower oxygen content and surface charge [10].
5
3-2-Size
6
The size of nano-carriers is a crucial factor, which can considerably influence colloidal stability,
7
cellular uptake, transfection efficiency and residence in circulation and clearance [52, 53]. It has
8
been observed that nanoparticles are taken up more efficiently as their size is declined in in-vitro
9
[57]. Nanoparticles with a size around 20 nm are able to diffuse into the cells in an endocytosis
10
pathway-independent manner. On the other hand, Rejman et al. demonstrated that particles
11
smaller than 200 nm in diameter were internalized through the clathrin-mediated pathway,
12
whereas, particles with a diameter larger that 200nm but less than 1µm entered into the cells
13
mostly via the caveole-mediated pathway [64]. In addition, a longer circulation time can be
14
provided by administration of nanoparticles with a size smaller than 200 nm in in-vivo. In fact,
15
such nanoparticles revealed a lower clearance and a better delivery to target tissues instead of
16
others, such as lungs, liver and the spleen. However, particles larger than 200 nm were removed
17
expeditiously via liver, spleen and the lungs [57]. In the case of GO, size also has a critical
18
impact on its amphiphilicity, so that smaller sheets are more hydrophilic due to having a higher
19
edge-to-area ratio. In addition, a more colloidal stability can be made by smaller GO sheets as a
20
result of a higher charge density [65]. In addition to size, the layer number is another prominent
21
parameter that should be considered in the design of graphene family nano-carriers, since the
22
specific surface area and bending stiffness are determined based on it [66]. By decreasing the
23
number of layers to one or more, the loading capacity of the cargo would be effectively
24
increased.
25
Similar to the charge, the size of the carrier can be strongly affected by gene loading. The
26
effective electrostatic interaction of the carrier and nucleotides will result in gene
27
condensation/compaction. After gene loading, the size of the formed carrier/gene complexes
28
usually shows an increase [67]. The degree of condensation remarkably affects the amount of
29
size variation. However, the type and modification of the cationic carrier, the N:P ratio and also
30
the protocol of complex formation can influence the complex size. For instance, by increasing
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
N:P ratio during complexation, the complex size enlargement shows a decreasing trend, which is
2
originated from more condensation of the loaded nucleotides [68]. Considering the importance of
3
carrier size and charge in gene transfection efficacy, as discussed, N:P ratio during complexation
4
should be accurately adjusted before transfection, so that the resultant complex charge/size still
5
remains in a proper range and does not disturb gene transfection [69].
6
3-3- Shape
7
The shape of nano-carriers, particularly in their transportation across biological barriers and also
8
inside the cells, is of great importance [57]. Nanoparticles with various shapes, such as spheres,
9
rods, discs, tubes and cubes reveal different uptake behavior, so that for nanoparticles with a
10
diameter larger than 100nm, rods, spheres, cylinders and cubes have higher uptake, respectively.
11
Spherical-shaped nanoparticles smaller than 100 nm are taken up more efficiently in contrast to
12
rod-shaped ones, whereas a lower cell uptake occurring as the aspect ratio of nanorods becomes
13
larger. Despite the few studies conducted on non-spherical nano-carriers, some researches have
14
demonstrated that the interaction of such nano-carriers with cells can be more complicated [59].
15
In addition, the shape of nanoparticles governs the efficiency of gene transfection. In the case of
16
polycation modified silica nanoparticles, chiral nanorods with a length of 300 nm were
17
ascertained as the most efficient carrier in comparison to nanoparticles, whose shapes were
18
different, including nanospheres with a diameter of 100 nm, hollow nanospheres with a diameter
19
of 100 nm, chiral nanorods with a length of 200nm and ordinary nanorods with a length of 300
20
nm. This is while hollow nanospheres juxtaposed with solid nanoparticles reveal better gene
21
transfection [70]. The graphitic materials resemble a sheet-like structure with various thicknesses
22
almost near a few nanometers, and lateral size around several nanometers to a few micrometers.
23
This sheet-like geometry could provide a sharp edge to cross the cell membrane [71].
24
3-4-Surface Chemistry
25
It is well established that the surface chemistry of nano-carriers can effectively modulate their
26
biocompatibility, circulation in the body, and particularly their interaction with the cells. It has
27
been shown that the blood half-life of nano-carriers is contingent upon their surface
28
hydrophobicity, so the more hydrophobic the surface, the more opsonin proteins absorbed on the
29
surface and the shorter the circulation time [55]. Furthermore, nano-carriers with different types
30
of surface chemistry can be internalized by the cells through various mechanisms. Nanoparticles
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
with a hydrophobic surface internalize more expeditiously than those with a hydrophilic surface
2
due to the hydrophobic nature of the cell membrane [57]. A wide variety of surface modification
3
has been used in order to improve biocompatibility, circulation time, cellular uptake and thereby
4
transfection efficiency of nano-carriers [52, 59]. Surface chemistry is variable for each member
5
of the graphene family. As aforementioned, GO has amphiphilic properties with a water contact
6
angle of 40-45°; whereas graphene is hydrophobic with a water contact angle of about 90° [66].
7
The surface chemical modification of the graphitic materials is a pre-requisite for their
8
application as gene nano-carrier. In what follows, various kinds of methods used for
9
modification of graphene-based materials are presented.
SC
4- Graphene Modifications as Gene Delivery Vehicle
M AN U
10
RI PT
1
Graphene has poor dispersion and stability in aqueous solutions due to its high hydrophobicity
12
and is bereft of oxygen containing hydrophilic groups, which are crucial for water dispersion.
13
Therefore, GO has been easily produced through derivatization of graphene, a covalent
14
functionalization method, in which oxygenated groups, such as carboxyl, epoxy and hydroxyl are
15
generated on the graphene, in order to increase its water solubility [72]. However, GO has the
16
tendency to aggregate in physiological solutions in the presence of salts and proteins as a result
17
of the shielding of electrostatic charges, and also the formation of non-specific interactions with
18
proteins [18]. To enhance physiological stability, cellular uptake and transfection efficiency, two
19
main methods, including covalent and non-covalent modifications by different modifiers have
20
been employed [73]. Non-covalent functionalization is preferred when the electrical conductivity
21
and large surface area of graphene is required, while covalent modification methods are applied
22
when the modified graphene is needed to have stability and strong mechanical properties. Studies
23
have shown that DNA/RNA condensation can be provided with both modification methods,
24
which makes them suitable for intracellular gene delivery applications [74]. This leads to a series
25
of studies by various research groups for the exploration of graphene-based materials in gene
26
delivery as summarized in Table 1.
AC C
EP
TE D
11
27 28
Table 1: Graphene-based nanoplatform’s modifications with respect to gene delivery (or gene/drug co-delivery)
29
systems
ACCEPTED MANUSCRIPT
Modifier(s)
Bonding
Nucleotide-
Study
Cell
Co-
Graphene
/modification
based cargo
model
line/anima
loaded
Derivative
mode
model
drug
HeLa
-
GO
PEG
Non-covalent
MB
In vitro
GO
PEI
Non-covalent
pEGFP
In vitro
GO
PEI
Covalent
Luciferase
In vitro
reporter gene GO
PEI
Covalent
pEGFP-N1
CS
Covalent
pRL-CMV
In vitro
In vitro
-
[76]
HeLa
-
[77]
-
[78]
CPT
[79]
DOX
[80]
HeLa
HepG2 HeLa
M AN U
(Renilla
[75]
HeLa
SC
GO
Ref
PC-3
Luciferase reporter gene
RI PT
Type of
luciferase)
PEI
Covalent
GNR
PEI(25KD)
Non-covalent
GO
bPEI (10 KD)/
PEI(covalent)
NLS peptide
Peptide (Non-
(PV7)
covalent)
PEG-FA/ Py
PEG-
GO
Bcl2-siRNA
In vitro
HeLa
LNA-m-MB
In vitro
HeLa
pEGFP-C1
In vitro
HeLa
pDNA
hTERT siRNA
TE D
GO
[81] -
[82]
293 T
In vitro
Hela
-
[83]
In vitro
Hela
-
[84]
-
[85]
-
[60]
-
[86]
FA(covalent) Py(Non-
covalent) rGO
PEG(amine
EP
terminated 6-
Covalent
FAM–
ssRNA
branched , 10 KD)
PEI (25KD)
Covalent
AC C
GO
PEG(10 KD)
GO
In vitro
pEGFP
MDA-MB435s HeLa
bPEI(1.8 KD)PEG(5KD)
(si Plk1)
Stat3 siRNA Covalent
In vitro
mouse
In vivo
malignant cell line B16
GO
PEI (60 kDa)
Covalent
pEGFP
In vitro
H293T U2Os
In vivo
zebrafish embryos
ACCEPTED MANUSCRIPT
GO
PEI/
Non-covalent
miR-21 siRNA
In vitro
PSS
MCF-7/ADR
ADR
cells (an ADR
(adrim
resistant breast
ycin)
[87]
cancer cell line) PEI
Non-covalent
FAM-siRNA
In vitro
Covalent
pEGFP
In vitro
mPEG-SPA Au GO
CS (10KD) PSS
bPEI
Covalent
bPEI-PEG
FA-
Covalent
Non-covalent
trimethyl chitosan (FTMC) PAMAM
GO
lactosylated chitosan
Covalent
EP
dendrimer
GFP-siRNA
pEGFP
pDNA
pDNA
TE D
conjugated
G/GO
Covalent
In vitro
HEK293
-
[90]
Drosophila S2
-
[91]
-
[92]
DOX
[93]
pEGFP
-
[94]
FAM-pDNA
DOX
[95]
-
[96]
Dox
[97]
In vitro
In vitro
In vitro
In vitro
AC C PEG
Covalent
chitosan oligosaccharid e (FACO)
Covalent
PC-3 and NIH/3T3 Hela and A549 cells
HeLa and
In vitro
human hepatic carcinoma cells (QGY7703)
CpG ODNs
PEI
FA-conjugated
HeLa
MG-63 cells
e (LCO)
GO
[89]
cancer
oligosaccharid
GO
DOX
SC
Covalent
bPEG
GO
A549 lung
In vivo
M AN U
LPEI (25 kD) bPEI(25 KD)
rGO
[88]
prostate
nanoparticle
GO
-
cancer C42b
Fe
GO
HL-60
RI PT
GO
In
RAW264.7
vitro/ in
cells
vivo MDR1 siRNA
In vitro
MCF-7
ACCEPTED MANUSCRIPT
GCN
bPEI
Covalent
pDNA
In vitro
PC-3
via a PEG
and NIH/3T3
spacer
cells
oxygen plasma
-
pGFP
In vitro
etching
NIH-3T3 and
-
[92]
-
[98]
NG97 cell
RI PT
rGO
lines
GO
PEI (25kDa)
Non- covalent
mRNAs of
In vitro
4reprogramming
Myc, and Klf4)
GO
Covalent
-
-
Covalent
mPEG mPEG/PDMA EMA thiolated PEG (5KDa) or
Covalent
EP
GO
siRNAs against
-
[100]
-
[101]
-
[102]
-
[103]
neuro-2a
-
[74]
lung cancer
-
[104]
-
[105]
-
[106]
fibroblasts
In vitro
M AN U
GO
bPEI
Human breast
CXCR4
carcinoma cell
(siCXCR4)
line MDA-
FAM-labeled
In vitro
MB-231 mouse or
single-stranded
human neural
siRNAs
stem cells
siRNA
TE D
GO
[99]
SC
Oct4, Sox2, c-
-
derived
transcription factors (RNAs for
adipose tissue-
pDNA
In vitro
HeLa–Luc cells
In vitro
PC-3 Raw 264.7
thiolated bPEI (1.8KDa) PEI/ PPI/
covalent
AC C
GO
pDNA
In vitro
PAMAM
GO
PAMAM
Covalent
anti-miRNA-21
dendrimer /
In vitro
cells
In vitro
malignant
PEG
GO
PEI-PEG
Covalent
plasmid-based Stat3 siRNA
melanoma B16 cells
GO
PEI/PEG/FA
Covalent
si-Stat3
in vitro
tumor hepatocellular
ACCEPTED MANUSCRIPT
carcinoma GO
PEG
Covalent
shRNA
In vitro
-
Cy5
In vitro
HepG2
DOX
[107]
chitosanaconitic
(CS-Aco) GO
-
peptide nucleic
RI PT
anhydride
MCF-7,
-
[108]
-
[61]
MDA-MB-
acid (PNA) probes
231,
MDA-MB-
SC
435
HeLa
GO
LPEI (1.8
Covalent
angiogenic gene
In vitro
H9c2
HUVECs
GO
PDMAEMA
Covalent
M AN U
kDa)
VEGF pro-
pDNA
In vitro
COS7 HepG2
CPT
[109]
GO
PEI (25 kDa)
covalent
pDNA
In vitro
HeLa
-
[91]
-
FAM
In vitro
Raw 264.7
-
[110]
EPI
[111]
-
[112]
-
[29]
28immunoco mpetent rats
Gold nanoparticle cGO
-
TE D
In vivo
PAMAM-
Covalent
AC C
cGO
EP
oligonucleotide
dendrimer and
cells
dT30 (FAMdT30) miRNA (Let-
In vitro
7g)
gadolinium
human glioblastoma
In vivo
(U87) cells
In vitro
murine
(Gd)
GO
PLL
Covalent
Unmethylated cytosine-guanine
Raw264.7
(CpG)
macrophages
dinucleotide motifs cGO-
R8 peptide
Covalent
-
In vitro
L929
ACCEPTED MANUSCRIPT
fibroblast PL-PEG/ R8
rGO
Non covalent
peptide
In vitro
MCF-7
-
[113]
In vitro
RAW264.7
-
[114]
(cd-siRNA)
CS
GO
Cell death siRNA
Non-covalent
CpG ODNs
PAMAM
GO
Covalent
MMP-9 shRNA
In vitro
plasmid PEI (non-
peptide
covalent)
conjugated-
Peptide
PEI
(covalent)
PLL
PLL(covalent)
SDGR peptide
Peptide (covalent)
GO
PEG/
PEG
FA
(covalent)
oxidized
-
-
In vitro In vivo
VEGF-siRNA
DOX
[115]
PC12 cells
-
[116]
-
[117]
-
[118]
-
[119]
-
[120]
C57BL/6 mice
In vitro
HeLa HUVEC
In vivo
ICR mice
HDAC1
In vitro
MIA PaCa-2
K-Ras siRNAs
In vivo
Athymic nude
TE D
FA (Covalent)
pDNA
MCF-7 cells
SC
GO
Neurotensin
M AN U
rGO
RI PT
cells
a double stranded
In vitro
mice
HEK 293T
DNA
GNR
(dsDNA)
Four different
rGO
AC C
CPPs
Non-covalent
EP
GO
PEI
PEG
PEI (noncovalent)
pGL3
In vitro
splice correction oligonucleotides (SCO) siRNA pGFP
In vitro
11 different cell
[121]
lines
PEG (covalent)
1
Abbreviations: G: graphene, GO: graphene oxide, rGO, reduced graphene oxide, GNR: graphene nanoribbon, cGO:
2
carboxylated graphene oxide, GCN: graphene and carbon nanotube nanocomposite, PEG: polyethylene glycol,
3
mPEG: methylated PEG, SCM-PEG: succinimidyl carboxymethyl PEG, PL- PEG: phospholipid-PEG, PEI:
4
polyethylene imine, LPEI: low molecular weight PEI, bPEI: branched PEI, CS: chitosan, PSS: poly(sodium 4-
5
styrenesulfonates), PAMAM: Oleic acid polyamidoamine, PDMAEMA: poly(2-dimethyl aminoethyl methacrylate),
ACCEPTED MANUSCRIPT
PPI: polypropylenimine, PLL: poly-L-lysine, NLS: nuclear localization signal, FA: folic acid, R8: octaarginine,
2
Py:1-pyrenemethylamine hydrochloride, FAM: fluorescent activated molecule, CPT: 10-hydroxycamptothecin,
3
DOX: doxorubicin, EPI: Epirubicin, MB: Molecular beacon, siRNA: small interference RNA, pDNA: plasmid
4
DNA, ODNs: oligodeoxynucleotides, shRNA: short hairpin RNA, miRNA: microRNA, LNA-m-MB: locked
5
nucleic acid modified molecular beacon,
6
4-1- Graphene modifiers
7
Since the packing of genetic materials into carriers is performed through electrostatic interaction
8
among negatively charged genes and positively charged carriers and the graphene family
9
materials are intrinsically non-cationic, modification of graphene derivatives by various
10
modifiers is required for providing a cationic surface having a better interaction with anionic
11
genes. So far, several modifiers, most of which are listed below, have been utilized to that end:
12
Polyethylenimine (PEI)
13
PEI, a synthetic cationic polymer, has been well-known as one of the most effective materials for
14
gene delivery vehicles. Suitable features of PEI, such as its capability to strongly bind to
15
negatively charged nucleic acids through electrostatic interactions and condensation of the genes
16
onto the surface of the vehicle, which are provided by its high nitrogen atom concentration,
17
could enhance cellular uptake. It also shows the “proton sponge” effect, which can lead to
18
escaping from endosomal/ lysosomal pathways after internalization [61, 76]. However, high
19
molecular weight branched PEI (HMW bPEI) exhibited high levels of cytotoxicity and
20
transfection efficiency juxtaposed with low molecular weight ones (LMW bPEI) [77].
21
Chen and coworkers synthesized PEI-GO conjugates by chemical grafting of PEI 25 kDa to
22
carboxylated GO (GO-COOH) [78]. They observed that GO and GO-COOH expeditiously
23
formed aggregation in the simulated physiological solution (PBS), while PEI-grafted GO was
24
hydrolytically stable due to the enhanced solubility and stability provided with PEI conjugation.
25
Moreover, PEI-GO revealed a high buffering capacity, which facilitated the endosomal escape of
26
pDNAs and also provided a comparably higher luciferase expression in HeLa cells compared to
27
PEI 25 kDa. In addition, the grafted PEI to GO showed a significantly lower (about 50%)
28
cytotoxic effect, as carrier compared to the naked PEI.
29
Feng et al. investigated the effect of PEI molecular weight on gene transfection ability as well as
30
cytotoxicity of PEI-functionalized GO nano-carriers. Two different molecular weights of PEI
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
(1.2 and 10 kDa) were introduced to GO through electrostatic (non-covalent) interactions [76].
2
Based on the AFM analysis, a significant increase in the average thickness of GO nanosheets
3
after incubation with PEI (from 1 nm to 3–4 nm) indicated that the electrostatic interactions
4
provided a successful immobilization. GO-PEI-10k showed significantly decreased cytotoxicity
5
in HeLa cells compared to bare PEI-10k, which was relatively toxic to the cells. Not only did the
6
complexation of GO with both PEI-1.2k and PEI-10k reveal no increase in the cytotoxicity of
7
GO, but it also showed rather higher cell viability at the higher concentrations of GO,
8
presumably because of the enhanced stability of GO in physiological solutions (Fig. 3).
9
Enhancing zeta potential to positive values (+30-50 mv) via PEI immobilization promoted the
10
loading of EGFP plasmid via the layer-by-layer (LBL) assembly mechanism. GO-PEI-1.2k
11
showed a higher EGFP expression compared to PEI-1.2k with ineffective transfection, whereas
12
EGFP expression observed in GO-PEI-10k was tantamount to bare PEI-10k (Fig. 3, d-g).
AC C
EP
TE D
M AN U
SC
RI PT
1
1
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
2
Figure 3. (a), (b), (c), Viability of HeLa cells treated with different concentrations of GO, GO-PEI and PEI with two
3
different molecular weights of 1.2 and 10 kDa for PEI, (d), (e), (f), (g), Fluorescent images of HeLa cells treated
4
with GO-PEI-pDNA and PEI-pDNA complexes at various nitrogen/phosphate (N/P) ratios and two different
5
molecular weights of PEI. Reprinterd from Ref. [76] with permission from The Royal Society of Chemistry.
ACCEPTED MANUSCRIPT
In another study, branched PEI (bPEI) was suggested to functionalize GO as gene delivery
2
carrier [77]. Two molecular weights of bPEI (1.8 and 25 kDa) were chemically conjugated to
3
GO with three various ratios of bPEI to GO (22, 8 and 5). The formulation prepared by a simple
4
mixture of bPEI and GO (physical immobilization) showed considerable aggregation, while the
5
chemically conjugated pPEI revealed no aggregation in water, PBS, and 10 % serum-containing
6
media. Conjugation of GO with bPEI under sonication reduced the size of GO from 500-600 nm
7
to 100-200 nm and increased the thickness of GO from 0.6-1.3 nm to 6-8 nm. The impact of
8
increasing the conjugation degree on the size and thickness was not considerable. Gene
9
transfection ability of HeLa and PC-3 cells was increased with increasing conjugation ratios of
10
bPEI to GO, where bPEI-GO with a higher conjugation ratio showed remarkable transfection
11
even at lower N:P ratio, which was comparable to bare bPEI 25KDa.
12
GNR grafted by PEI (PEI-g-GNR) through electrostatic assembly was studied by Dong and
13
coworkers as a nano-carrier of locked nucleic acid modified molecular bacon (LNA-m-MB)
14
probes for the detection of miRNA [81]. PEI-g-GNR provided good protection of the LNA-m-
15
MB probes against single-stranded DNA binding protein interaction or enzymatic degradation. It
16
was also more efficient in the transfection of HeLa cells juxtaposed with PEI (25kDa) and PEI-
17
grafted multi wall carbon nanotubes (PEI-g-MWCNTs) due to the larger surface area of GNR for
18
conjugation of PEI, and subsequently a stronger possible proton sponge effect.
19
In another study, the effect of GO nanosheet size on PEI conjugation and consequent DNA
20
loading was studied. Chemically grafted PEI to NGO with a size of 10-20nm was assessed in
21
order to obtain higher capacity for PEI loading [86]. The PEI-g-NGO revealed three times higher
22
enhanced green fluorescent protein plasmid (pEGFP) condensation capacity than PEI-g-GO.
23
Transfection with PEI-g-NGO was found to be more effective compared to PEI-g-GO in both
24
H293T and U2Os cell lines with similar cytotoxicity (about 90% viabilities). In addition, the
25
pEGFP transfection study of zebrafish embryos showed efficacy of 90% and 80% for PEI-g-
26
NGO and PEI-g-GO, respectively, which were much higher than naked DNA injection (~30%),
27
as an established transfection technique.
28
Choi et al. utilized PEI- functionalized GO via electrostatic interaction for induced pluripotent
29
stem cell (iPSC) production through mRNA delivery to adipose derived fibroblasts [99]. PEI
30
physical immobilization changed the surface charge of nano-carriers from -34.66 mV to +26.68
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
mV, suitable to binding of negatively charged mRNA. Treatment of human dermal fibroblasts
2
(hDFs) with GO-PEI-mRNA did not show significant cytotoxicity or expression of innate
3
immune response genes, while those with GO, PEI, GO-mRNA or PEI-mRNA significantly
4
dwindled cell viability and increased innate immune response gene expression. hDFs treated by
5
GO-PEI-mRNA also showed very efficient transfection and subsequent reprogramming (around
6
50%), where the derived iPSC expressed pluripotency genes.
7
Another platform, based on the graphene/hydrogel nanocomposite, has been tested by Paul et al.
8
for in-situ delivery of the angiogenic gene for myocardial therapy [61]. The nanocomposite was
9
formulated by incorporating the DNAVEGF - loaded GO, which was synthesized through chemical
10
binding of PEI (1.8 kDa) to carboxylated GO into methacrylated gelatin (GelMA) hydrogel,
11
followed by photocrosslinking under UV exposure (Fig. 4, a). GO had a negative charge (-
12
37mV), while PEI-functionalized GO was positively charged (+43 mV), which was reduced after
13
binding with the negatively charged plasmid DNA depending on N/P ratios. In vitro studies
14
showed that PEI-functionalized GO, gradually released from GelMA hydrogel, did not
15
significantly induce cytotoxic effects on H9c2 cell lines and revealed a rapid overexpression of
16
VEGF in the initial 4 days. In addition, in-vivo studies, which were performed by
17
intramyocardial injection of nanocomposite hydrogels in the peri-infarct areas of a rat model,
18
showed enhancement in myocardial capillary density, a decrease in the scar area of infarcted
19
hearts after treatment with nanocomposite hydrogels compared to untreated groups (Fig.4, b-d).
AC C
EP
TE D
M AN U
SC
RI PT
1
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
1 2
Figure 4. (a) Synthesis of GG’ hydrogel, (b,c) the smaller scar size and (d) higher cardiac functions in the group
3
treated with injectable hydrogel. Reprinted with permission from Ref. [61]. Copyright (2014) American Chemical
4
Society.
5
Covalently grafted PEI (25 KDa) onto GO nanosheets was utilized to encapsulate Au
6
nanoparticles (GO-AuNPs) or Au nanorods (GO-AuNRs) through electrostatic self-assembly in
7
order to improve biocompatibility and gene transfection ability of Au-based nano-carriers [67].
8
The size and surface charge of AuNPs respectively increased from 62.5 nm to 113.8 and from
9
6.6 mV to 36.9 mV after encapsulation with GO-PEI nanosheets, which were ascribed to proper
ACCEPTED MANUSCRIPT
self-assembly of GO and PEI on the Au surface. Although, the GO-PEI sheets formed aggregates
2
with an average size of 500-600 nm via electrostatic interactions between pGFP and GO-PEI,
3
which made the cellular internalization difficult and decreased the transfection efficiency, GO-
4
PEI-AuNPs/pDNA created no obvious aggregates conceivably due to the smaller contact area
5
between GO-PEI-AuNPs spheres compared to GO-PEI sheets. Encapsulation by GO
6
significantly declined the rather cytotoxic effects of AuNPs and AuNRs to HeLa cells and
7
enhanced their transfection ability.
TE D
M AN U
SC
RI PT
1
8
Figure 5: The method of synthesizing GO-PEI-AuNPs and GO-PEI and the proposed mechanism for DNA delivery
10
via these vectors. Reprinted with permission from [67]. Copyright (2013) American Chemical Society.
AC C
11
EP
9
12
Polyethylene glycol (PEG)
13
PEG is a biocompatible amphiphilic polymer, which could improve aqueous solubility and
14
stability in physiological solutions, reduce toxicity, prolong nanomaterials’ blood circulation
15
time through hindering protein adsorption and decrease immunogenicity and antigenicity [83,
16
84]. PEG conjugation (termed PEGylation) has been commonly utilized in order to improve
17
graphene-based nano-carrier functions.
ACCEPTED MANUSCRIPT
A pioneering work, conducted by Lu et al. investigated a covalently attached PEG to NGO
2
carrier to deliver molecular beacon (MB), as a model oligonucleotide, into HeLa cells to detect
3
target mRNA. The steric hindrance effect of the attached PEG remarkably hindered DNase I
4
from binding to MB, and consequently protected MB from enzymatic cleavage. Moreover,
5
PEGylated NGO was less cytotoxic even at a concentration of 100mg/ml and target DNA can be
6
successfully hybridized with MB, which was adsorbed on the NGO surface, and then released
7
from NGO [75].
8
PEGylated rGO (PEG-rGO) was developed by Zhang et al. for loading and delivery of single
9
stranded RNA (ssRNA) by covalent conjugation of PEG to GO and reduction of resultant PEG-
10
GO into PEG-rGO [84]. PEGylation and subsequent reduction had no effect on the size, but an
11
increase of thickness from 1 nm (GO) to 2-3 nm (PEG-rGO) was observed. Furthermore, a
12
higher amount of ssRNA was adsorbed on PEG-rGO in comparison to PEG-GO because of the
13
more noticeable affinity of ssRNA to PEG-rGO. Cytotoxicity studies revealed no toxic effect of
14
GO, PEG-GO and PEG-rGO to HeLa cells, whereas little toxicity was exhibited in rGO. Thus,
15
not only did PEG-modification improve the biocompatibility of rGO, but also significantly
16
enhanced the stability of rGO in biological buffer solutions.
AC C
EP
TE D
M AN U
SC
RI PT
1
17 18
Fig. 6 (a) Viability of HeLa cells 24h after treatment with various concentrations of PEG-GO and PEG-rGO
19
Reprinted with permission from Ref. [84]. Copyright (2013) Royal Society of Chemistry.
ACCEPTED MANUSCRIPT
Dendrimers
2
Dendrimers are highly branched polymers with a spherical three dimensional shape and build up
3
from a central core molecule, which is symmetrically surrounded by branched repetitive units.
4
These structures have a nanoscale size with a high density of positive charges due to the presence
5
of amino groups on the surface [74, 122]. Poly (amidoamine)(PAMAM) dendrimer, poly-L-
6
lysine (PLL) dendrimer, poly (prophylenimine) (PPI) dendrimer, poly(etherimine) (PETIM)
7
dendrimer are some used dendrimers, which have been employed as gene carriers [122], among
8
which PAMAM dendrimers have been evaluated as graphene modifier [115]. PAMAM
9
dendrimers, which are molecularly uniform with a unique shape and size have the intrinsic
10
capability for condensation and efficient transportation of DNA into cells owing to the fact that
11
amine groups on the surface of PAMAM dendrimers can electrostatically interact with phosphate
12
groups of DNA at physiological pH. Moreover, the genotoxic and cytotoxic effects, which are
13
exhibited by these PAMAM dendrimers, are relatively low in comparison to PEI since PAMAM,
14
unlike PEI, is a biodegradable polymeric structure in which a backbone of polymer chains with
15
peptide bonds exist [94].
16
poly-L-lysine (PLL)
17
PLL is a cationic polypeptide, which is highly positively charged at a physiological pH. J. Sun et
18
al. evaluated grafted PLL-grafted GO to intracellular delivery of immunostimulatory CpG
19
oligodeoxynucleotides (ODNs) [112]. They utilized two GO platforms, polydispersed GO and
20
uniform NGO to covalent immobilization of PLL. PLL modification provided a large number of
21
amino groups, which electrostatically interact with negatively charged CpG ODNs and facilitated
22
the ODNs loading. NGO-PLL-CpG conjugates revealed better biocompatibility and higher
23
cellular internalization in murine Raw264.7 macrophages juxtaposed with polydispersed GO-
24
PLL-CpG. In addition, NGO-PLL-CpG exhibited comparably higher immunomodulatory effects
25
than NGO-azide-CpG, which can be due to the adverse effect of azide, as a modifier, on cellular
26
uptake and bioactivity.
27
Chitosan
28
Chitosan (CS) or poly-D-glucosamine, a linear cationic polysaccharide derived from chitin, is
29
considered to be an appropriate candidate as modifier for gene and drug delivery because of its
30
properties, such as good biocompatibility and biodegradability compared to other cationic
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
polymers like PEI, improvement of the stability of nano-carriers in aqueous solutions and the
2
ability of surface charge adjustment for electrostatically interacting with negatively charged
3
nucleic acids [114].
4
Chitosan-modified GO was successfully utilized to deliver nucleotide based immunotherapeutic
5
agents. Zhang et al. developed GO-CS nano-composites through the self-assembly of GO and CS
6
via electrostatic interactions for delivery of CpG oligodeoxynucleotides (ODNs) (Fig. 7)[114].
7
The resultant GO-CS /ODNs nanocomplexes were stable under acidic conditions resembling the
8
endosomal pH, and revealed good potential of CS for buffering the endosome environment. The
9
biocompatible nature of CS resulted in decreases of GO cytotoxicity, and also enhanced cellular
10
uptake, compared to free CpG ODNs. The more efficient CpG ODNs delivery mediated by CS-
11
functionalized formulation induced a higher amount of cytokines secretion, juxtaposed to
12
GO/CpG ODNs, which indicated CS potential to improve GO function as gene nano-carrier.
AC C
13
EP
TE D
M AN U
SC
RI PT
1
14
Figure 7.Schematic illustration of preparation and application of GO-CS nano-composite as carrier for intracellular
15
delivery of CpG ODNs. Reprinted with permission from Ref. [114]. Copyright (2017) Elsevier.
16 17
Poly(sodium 4-styrenesulfonates) (PSS)
18
PSS is a water soluble polymer, which is non-toxic and could improve the dispersion of
19
graphene-based materials in aqueous solutions and prevent them from agglomeration. Since PSS
ACCEPTED MANUSCRIPT
is a negatively charged polyelectrolyte, it could participate in layer by layer assembly structures
2
via electrostatic interactions. Moreover, the pH sensitive nature of this polymer makes it a
3
suitable choice for releasing the payloads, which are incorporated into the composite in response
4
to the pH change in the environment [123, 124]. For gene delivery applications, this polymer was
5
usually employed with other cationic modifiers, such as PEI to make a complex structure, which
6
could provide enough hydrocolloidal stability as well as gene interaction potential [87].
8
Poly(2-dimethyl aminoethyl methacrylate) (PDMAEMA)
SC
7
RI PT
1
PDMAEMA, a synthetic water soluble polycationic polymer, is composed of side chains with
10
tertiary amine groups and can make a complex with pDNA through electrostatic interactions.
11
PDMAEMA has been utilized as transfection agent, while exhibiting a high level of
12
cytotoxicity, which is mainly contingent upon the length of its side chains [109, 125-127]. This
13
polymer is able to non-covalently immobilize onto GO nanosheets through π-π interactions and
14
produce a nanostructure with zwitterionic property, due to the presence of amine, phenol
15
hydroxyl and carboxyl groups [109, 128]. A biocleavable PDMAEMA–GO formulation
16
synthesized by the disulfide (S-S) mediated grafting method was introduced by X. Yang and his
17
colleagues as very efficient nano-carriers to transfect plasmid pRL-CMV. It is considered as the
18
reporter gene in COS7 and HepG2 cell lines, with a pH-sensitive gene releasing manner [109].
TE D
M AN U
9
Peptides
20
Peptides have been used for targeted gene/drug delivery and cell uptake since they are small-
21
sized and biocompatible molecules with cell-penetrating ability and can be chemically
22
synthesized and modified through a relatively easy manner [129]. The peptide could be selected
23
as a nano-carrier modifier with various aims, such as increasing cell penetration, cellular
24
targeting, stimulating endosomal escape and intracellular nuclear targeting [130]. In the last
25
decade, among various peptides utilized for gene delivery applications, cell penetrating peptides
26
(CPPs) have attracted considerable attention due to their ability to intracellularly deliver large
27
therapeutic molecules such as genes. In 2015, we introduced a novel CPP-conjugated GO (GOP),
28
as a gene nano-carrier aimed to improve the transfection efficiency of the GO nano-sheet by R8
29
chemical conjugation, while improving GO biocompatibility [29]. The effect of conjugation ratio
30
(R8 to GO) on the nano-carrier characteristics, such as particle size and morphology, surface
AC C
EP
19
ACCEPTED MANUSCRIPT
charge, hydrocolloid stability and plasmid condensation ability was comprehensively
2
investigated. The results (Fig. 8) demonstrated that the designed R8–GO nano-carrier with a
3
peptide molar ratio of 1µmol per mg of GO (GOP1) was selected as the most efficient and
4
biocompatible gene delivery vehicle having desirable properties as well as efficient transfection
5
ability compared to JetPEI® as a commercial reagent.
TE D
M AN U
SC
RI PT
1
Figure 8. The HEK293 cell line treated with the GOP1–pEGFP complex after 48 h: (a) transmission image, (b)
8
fluorescent image and (c) merged image. Quantification of transfection ability after 48 h of cell transfection in
9
the presence and absence of FBS (d). *p-value<0.005 compared to transfectionability in the absence of FBS
10
Reprinted with permission from Ref. [29]. Copyright (2015) Royal Society of Chemistry.
AC C
11
EP
6 7
12
In another study, Dowaidar et al. combined GO with two different kinds of CPP, PepFect14
13
(PF14) and PepFect221 (PF221), for gene delivery of three oligonucleotide (ON) models,
14
including plasmid (pGL3), splice correction oligonucleotides (SCO) and siRNA [120]. The
15
CPPs were sequentially added to ONs/GO complexes and physically loaded on the complexes.
16
The result demonstrated that GO-ONs-CPPs offered several magnitude enhancements of cellular
17
uptake and transfection ability (1.5-25 folds) than CPPs-ONs (Fig. 9).
RI PT
ACCEPTED MANUSCRIPT
Figure 9. Confocal microscopy images for the cell transfection of PF14-SCO without a) and with b) graphene
3
oxide (GO-SCO-PF14). PF14 were labeled with Alxa568-705SCO. Reprinted with permission from Ref [120].
4
Copyright (2017) Elsevier.
SC
1 2
5
Recent progresses in peptide synthesis provide the opportunity to design multipurpose peptides,
7
which are suitable for various biomedical applications. Chimeric peptides are one of the best
8
candidates to be utilized in designing gene delivery carriers. For instance, Ghafari and his
9
coworkers introduced and evaluated peptide-modified GQD with two different (green and red)
10
emission colors for gene delivery and nuclear targeting applications as well as cellular tracking
11
[131]. GQDs are physically conjugated with MPG-2H1 chimeric peptide, which was composed
12
of three different motifs. The selected motifs were responsible for the three specific
13
performances aimed to overcome the biological barriers against gene transfer (DNA packaging,
14
endosomal escape and cellular nuclear targeting). The chimeric peptide-modified GQDs
15
provided efficient cell tracking and showed enhanced internalization of the luciferase plasmid
16
into HEK 293 T cells.
18
TE D
EP 5- Multifunctional Graphene Nano-carriers
AC C
17
M AN U
6
19
Multifunctional nano-carriers can synchronously exhibit various functions including drug/gene
20
delivery, optical imaging, induction of apoptosis in cancer cells, increase in biocompatibility,
21
gene transfection and condensation ability as well as stability and solubility within physiological
22
solutions. There exist various advantages of using multifunctional carriers, including the ability
23
of having specific beneficial effects synergically, which can be obtained by less frequent
24
administration of therapeutic carriers, enabling researchers to have more control on vehicles and
25
also achieving more information regarding nano-carriers’ fate in the body [132]. Considering the
ACCEPTED MANUSCRIPT
unique chemical structure of graphene family nanoparticles, they showed a great potential to
2
insert various functionalities in order to achieve a multipurpose nano-platform for cargo delivery
3
(Fig. 10). Among the graphene family derivatives, GO could provide a suitable platform to
4
immobilize the modifier ligands/molecules on the basal surface as well as the edges of nano-
5
sheets via physical or chemical interactions (Fig. 11, up). Beside π-π interactions, which are
6
mostly utilized to introduce hydrophobic functionalities, carboxyl groups on the basal plane or
7
edges of GO sheets provide more reactive sites for electrostatic or chemical immobilization of
8
hydrophilic functionalities (Fig. 11, down) [133]. However, the other oxygen containing
9
functional groups are utilized for GO modification as well.
AC C
EP
TE D
M AN U
SC
RI PT
1
AC C
1
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
2
Figure 10. Schematic representation of modification of a multipurpose graphene-based platform for nucleotide based
3
cargo delivery
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 11: Up) A representation of structure and main functional groups of GO. Reprinted with permission from
3
Ref. [29]. Copyright (2015) Royal Society of Chemistry. Down) An illustration of common routes for covalent
4
and non-covalent carboxyl-mediated GO modifications. a: covalent PEG (branched or linear) conjugation for
5
increased bio-distribution water solubility, b: conjugation of amine-contained synthetic or natural polymers
6
(PEI, chitosan), c: targeting ligands such as folic acid, d: antibodies immobilization for cell targeting or
7
biosensor designing, e: fluorescence marker labeling for cell tracking and imaging, f: conjugation of amine-
8
decorated nano-particles (e.g., Fe2O3), g: proteins and peptide immobolization, electrostatically immobilization
9
of h: natural or synthetic cationic polymers, i: positively charged nano-particles, j: cationic ligands as mediator
10
for loading negatively charged biomolecules, e.g. nucleic acids DNAs, RNAs. Reprinted with permission from
11
Ref. [133]. Copyright (2015) Springer.
AC C
EP
1 2
12 13
Several studies have been dually reported on the functionalization of graphene-based nano-
14
carriers with PEG and PEI polymers [60, 85, 91, 92, 96, 105, 134] or with other modifiers, such
ACCEPTED MANUSCRIPT
as folic acid (FA)[106], chitosan[107] and mPEG-SPA[88]. In 2013, Feng et al. produced
2
covalently co-conjugated PEI (25 kDa) and PEG (10 kDa) nano-GO (Fig. 12), which exhibited
3
superior stability in salts and serum, excellent gene transfection efficiency and decreased cell
4
toxicity with respect to free PEI or GO-PEI [85]. The instability of GO-PEI in the serum, which
5
is mainly ascribed to the protein adsorption on GO via hydrophobic and electrostatic interactions,
6
were extensively hindered by PEG coating. Increased cellular uptake of synthesized nano-
7
carriers, intracellular delivery of siRNA and also gene silencing, in terms of down-regulating the
8
target gene (Polo-like kinase 1) were observed by the applied dual functionalization. In another
9
study by Yin et al., they investigated the antitumor activities of the signal transducer and
10
activator of transcription 3 (Stat3) delivered by PEG and PEI dual functionalized-GO after
11
intratumoral administration in mouse malignant melanoma and then infrared region irradiation.
12
The zeta potential of nanosheets increased from -49.08 mV to +43.13 mV after PEI conjugation,
13
whereas this amount decreased to +21.02 mV after PEGylation and also to +7.53 mV after the
14
formation of GO–PEI–PEG and plasmid Stat3 siRNA complexes. Their results indicated that due
15
to the positive charges, GO-PEI-PEG completely condensed the plasmid Stat3 siRNA, and had
16
no cytotoxic effect even at a concentration of 350 mgL-1 and transfected B16 cells with an
17
efficiency of above 60%. In addition, the GO–PEI–PEG/si-Stat3 complexes significantly
18
diminished tumor size in comparison to PBS, GO, PEI-GO and GO/si-Scramble when injected
19
intratumorally in mouse malignant melanoma. Examinations performed 30 days after injection
20
demonstrated that GO–PEI–PEG did not produce any toxic effects on liver and kidney organs
21
[60].
AC C
EP
TE D
M AN U
SC
RI PT
1
22
ACCEPTED MANUSCRIPT
1
Figure 12. Schematic of NGO-PEG-PEI preparation as a dual-functionalized formulation [85].
2
In another study, PEI and PEG dual-functionalized rGO (RGPP) was evaluated as a gene
4
delivery carrier to deliver gene plasmids and siRNA in 11 different cell lines, compared to a
5
commercial polyalkyleneimine cationic transfection reagent (TR) [121]. RGPP exhibited
6
remarkable delivery ability for both the FAM-labeled siRNA model and GFP plasmid into
7
HepG2 cells and GFP plasmids than TR. In addition, the modified GO nano-carrier interestingly
8
provided about 47.1% transfection efficiency in rabbit articular chondrocytes. However, it
9
provided acceptable GFP transfection of about 27%, 37.2% and 26.0% in A549 cell lines, EMT6
10
cells and LO2 cells, respectively. The results of the study concluded that RGPP nano-carrier
11
should be optimized for different cell lines.
12
Modified GO based carriers also showed the potential to be utilized in the genome editing
13
approach based on CRISPR/Cas 9 technology. Yue et al. utilized GO-based nano-carrier for
14
CRISPR/Cas9 complexes delivery in the genome editing approach [135]. In this study, PEI/PEG
15
dually functionalized GO was electrostatically loaded by high molecular weight Cas9/single-
16
guide RNA (sgRNA) complexes, where Cas9 maintains endonuclease activity equivalent to that
17
of its free state. GFP expressing a human AGS cell line, treated by GO-mediated Cas9/sgRNA,
18
showed 39% gene disruption with good cytocompatibility and non-significant cell toxicity.
19
PEG modifier alone, not with PEI, was also used dually along with other molecules, including
20
FA and Pyrenemethylamine hydrochloride (PyNH2) [83] and with PDMAEMA [102]. Yang et
21
al. conjugated amino-PEG (NH2-PEG-NH2) to FA and linked NH2-PEG-FA covalently to GO to
22
obtain GO-PEG-FA (Fig. 13), in which enhancement of the biocompatibility and solubility and
23
also decrease in toxicity of GO provided by PEG modification were combined with the tumor
24
targeting function of FA. Human telomerase reverse transcriptase (hTERT) was loaded onto the
25
surface of nano-carrier through π-π stacking with the assistance of PyNH2, which was non-
26
covalently coupled onto the GO-PEG-FA without cytotoxic effects. The GO-PEG-FA-PyNH2
27
showed a good dispersibility in blood. It was found that FAM-labeled DNA was delivered
28
efficiently into HeLa cells by both GO-PEG-FA-PyNH2 and GO-PEG -PyNH2 (FA free) with a
29
faster manner of targeting in GO-PEG-FA-PyNH2 [83].
30
AC C
EP
TE D
M AN U
SC
RI PT
3
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
1 2
Figure 13. Schematic illustration of GO-PEG-FA-PyNH2 synthesis for siRNA delivery into cells. Reprinted with
3
permission from Ref. [83]. Copyright (2012) Royal Society of Chemistry.
4
A combination of photothermal therapy and gene delivery has been reported as a combinatorial
6
strategy against cancer treatment. Graphene and its derivatives are one of the best candidates for
7
photothermal therapy. For instance, FA- grafted PEG (PEG-FA) was assessed to modified GO in
8
order to co-deliver HDAC1 and K-Ras siRNAs to target pancreatic cancer cells (Fig.14, a)[118].
9
They combined dual gene silencing effects with NIR light thermotherapy and tried to enhance
10
treatment effects. The FA-PEG served as a ligand with a potential of providing hydrocolloid
11
stability and cancerous cell targeting ability, simultaneously. To achieve better transfection
12
ability, the GO-PEG-FA was further non-covalently modified with a cationic polymer, poly-
13
allylamine hydrochloride (PAH). Therefore, the PEG-FA/PAH modified-GO provided a
14
multipurpose nano-carrier to co-deliver the two siRNAs. The results of this study demonstrated
15
that synergistically utilizing the gene silencing potential and NIR light thermotherapy capability
16
provided remarkable anticancer efficacy, consequently inhibiting in vivo tumor volume growth
17
by >80% (Fig. 14, b-d).
AC C
EP
TE D
5
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 14. a) Schematic representation of the FA/PEG/GO synthesis and subsequently gene loading. Folic acid (FA)
3
was conjugated with NH2-mPEG-NH2 to form FA/PEG-NH2. Subsequently, FA/GO nanosheets were prepared by
4
conjugating the amine-functionalized FA/PEG-NH2 to increase water solubility and biocompatibility. For siRNA
5
delivery, PEGylated or FA/PEGylated GO were functionalized with positive polymer PAH to form positively
6
charged GO/PEG/PAH or GO/PEG/FA/PAH which were able to deliver siRNA by electrostatic interaction.
7
Antitumor activities of GO-based nanoformulations in a MIA PaCa-2 xenograft animal model. (b) Representative
8
tumor tissue images of mice treated with (1) PBS, (2) FA/GO/scramble siRNA, (3) FA/GO with NIR light, (4)
9
FA/GO/(H+K) siRNA or (5) FA/GO/(H+K) siRNA with NIR light. Mice treated with FA/GO/(H+K) siRNA with
10
NIR light in the last group exhibited the smallest tumor size. (c) Tumor volume measuring over time and (d) tumor
11
weights of mice treated with the same nanoformulations as in (b), respectively. Relative tumor volume was defined
12
as (V-V0)/V0, where V and V0 indicate the tumor volume on a particular day and day 0, respectively [118].
14
EP
AC C
13
TE D
1 2
15
Peptides have also been used with other modifiers including PEI [82, 116], PLL [117], PEG
16
[136] and phospholipid-based amphiphilic polymer (PL-PEG) [113]. Functionalization of GO-
17
PEI was performed with NLS peptides in order to carry out nuclear targeting and localization
18
[82] and in another study, with neurotensin (NT, a neurospecifc peptide) mainly due to
ACCEPTED MANUSCRIPT
enhancement of the targeting property and biocompatibility and decrease in the cell toxicity
2
effect of PEI [116]. Also, GO–PLL was covalently modified with RGD peptides, which were
3
able to target the tumor through interaction with the integrin avb3, which is overexpressed on the
4
cell membrane of cancer cells [117]. rGO-PL-PEG was non-covalently functionalized with
5
CPPs, which could provide buffering capacity and elevate resistance to the acidic endolysosomal
6
environment and, as a result, improve siRNA transfection ability [113]. Ren et al. covalently
7
modified GO with low molecular weight PEI, in order to dwindle the cytotoxicity of PEI 25 kDa
8
and then PV7 was non-covalently incorporated into GO-PEI through three different routes,
9
including after the complexation of GO-PEI/DNA, simultaneously with the GO-PEI and DNA,
10
and before treatment of cells with GO-PEI/DNA. The incorporation of PV7 into GO-PEI 10 kDa
11
reduced the cytotoxicity and also its post-addition and simultaneous addition to the composite
12
enhanced gene expression efficiency, whereas prior-addition revealed slight changes juxtaposed
13
to GO-PEI/DNA [82]. In our recent study, we non-covalently functionalized rGO with
14
phospholipid-PEG (PL-PEG), which was more hydrocolloidally stable compared to rGO and
15
afterwards, the PL-PEG-rGO was non-covalently modified with R8 CPP (Fig. 15) [113]. The
16
observed good colloidal stability and dispersibility of double functionalized nanosheets in
17
aqueous solutions were the results of steric hindrance, provided by hydrophilic chains of PEG,
18
and the repulsion among positively charged amino groups of PEG. MCF-7 cells, which were
19
treated with rGON-PLPEG-R8, revealed superior viability and proliferation juxtaposed to rGO
20
nanosheets. The results indicated the successful condensation of siRNA on the surface of
21
nanosheets, the complete protection of siRNA from enzymatic degradation and the high
22
transfection efficiency of 82 ± 5.1%.
SC
M AN U
TE D
EP
AC C
23
RI PT
1
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
1 2
Figure 15. a) A schematic illustration of rGO based nano-carriers preparation via dual functionalization with PL-
3
PEG and R8 to deliver siRNA into the cancer cell. b) The functionalized nano-carriers successfully internalized the
4
FITC-siRNA into the cells juxtaposed to HiPerfect®, a commercialized siRNA transfection reagent. Reprinted with
5
permission from Ref. [113]. Copyright (2016) Elsevier.
6 7
In a study by Ren et al., dual functionalized GO were synthesized based on the PLL and peptide
8
(Arg-Gly-Asp-Ser, SDGR) covalent immobilization in order to actively target tumors and deliver
ACCEPTED MANUSCRIPT
anti-VEGF siRNA [117]. PLL and SDGR were utilized to improve the water solubility/loading
2
capacity and targeting ability to tumors, respectively. The results of RT-qPCR and ELISA assays
3
showed that the VEGF expressions in the level of mRNA and protein were remarkably down-
4
regulated by 40.86% and 51.71%, respectively. In addition, the in-vivo experiment on the S180
5
tumor-bearing mice model revealed that intravenously injected VEGF-siRNA at a dose of 0.3 mg
6
kg-1 (once every other day) provided the tumor inhibitory rate of about 51.74% (Fig. 16).
EP
TE D
M AN U
SC
RI PT
1
Figure 16. The in vivo evaluation of anti-tumoral effect of GPR/VEGF-siRNA. (a) Image of tumors, (b) tumor
9
weight and (c) tumor inhibitory rate. Reprinted with permission from Ref.[117]. Copyright (2017) Royal Society of
10 11
AC C
7 8
Chemistry.
12
PAMAM dendrimers have also been employed together with other molecules consisting of PEG
13
[104] and oleic acid [94] for dual functionalization of graphene-based materials as gene carriers.
14
Liu et al. developed graphene-oleate-PAMAM hybrids by chemical adsorption of oleci acid on
15
graphene and then covalent functionalization of graphene-oleic acid with PAMAM. Oleic acid is
16
able to improve gene transfection efficiency, as a result of its strong affinity to the
17
cytomembrane and ability to further the destabilization of the membrane and also provide
ACCEPTED MANUSCRIPT
terminal carboxyl groups for covalent bonding with PAMAM through amidation reaction.
2
Hybrids became positively charged in aqueous solution after modification with PAMAM, which
3
made them appropriate for immobilization of genetic materials. In addition, graphene-oleate-
4
PAMAM hybrids were stably dispersible in aqueous media and showed dose-dependent and cell
5
type-dependent cytotoxicity. Treatment of HeLa cells with the hybrids diminished the viability
6
of HeLa cells as the concentrations of hybrids increased, but the viability was still above 80%
7
after incubation by 100µg.mL-1 of hybrids for 24h. The GFP transfection efficiency of graphene-
8
oleate-PAMAM hybrids was 18.3% in HeLa cells at the mass ratio of 4:1, while it was 9.9% in
9
MG-63 cells at that of 2:1 [94].
SC
RI PT
1
11
M AN U
10
6- Dual Gene/Drug Delivery by Graphene-based Nano-carriers The nano-carriers potential for the delivery of gene and drug could simultaneously provide much
13
stronger approaches to fighting diseases. Graphene and its derivatives are good candidates for
14
co-delivery of nucleotide based therapeutics along with chemical based drugs due to their huge
15
surface area and unique conjugated structure [12]. The strong bonding between carbon-carbon
16
atoms, the aromatic structure, presence of free π electrons as well as reactive sites on the
17
graphene basal plane, make graphene suitable for the simultaneous load of hydrophilic and
18
hydrophobic biomolecules via chemical or physical interactions. For instance, co-delivery of
19
anticancer drugs and genes to the cancer cells has been utilized in cancer therapy applications.
20
However, there are few studies on the application of graphene-based nano-carrier for co-delivery
21
or sequential delivery of chemotherapeutic drugs and genes. Results reported by various studies
22
revealed remarkable superposed efficient therapy compared to merely gene or drug delivery
23
protocols alone.
24
One of the common and effective anticancer drugs is doxorubicin (DOX), which has been used
25
in cancer therapy over the past 30 years [137]. CS-functionalized GO was also utilized to co-
26
deliver platinum complexes drugs, including cisplatin, carboplatin, oxaliplatin as well as
27
nucleotides [138]. It usually undergoes various biological actions such as binding to DNA-
28
associated enzymes and intercalating with DNA base pairs. DOX was commonly delivered by
29
graphene-based nano-carrier along with the anticancer nucleotides. Zhang et al. [80] developed
AC C
EP
TE D
12
ACCEPTED MANUSCRIPT
a PEI-grafted GO nano-carrier for co-delivery of Bcl2-targeted siRNA and DOX. PEI-GO shows
2
considerably lower cytotoxicity and substantially higher efficacy of siRNA, at optimal N/P ratio
3
and then PEI of 25 K. The result showed that sequential delivery of siRNA and DOX by the PEI-
4
GO nano-carrier led to enhanced anticancer activity. In another study, folate conjugated
5
trimethyl chitosan-functionalized GO (FGNCs) was introduced as a capable candidate for the
6
delivery of both anticancer drugs and genes [93]. This complex nanoparticle was used as a
7
targeted delivery vector for DOX and pDNA delivery to Hela and A549 cells. The higher uptake
8
level in Hela cells with Folate-receptor was evidence for the targeting ability of FGNCs. Cao et
9
al. [97]
RI PT
1
SC
synthesized a functionalized-GO with FA-conjugated CS oligosaccharide (FACO)
including quadruple ammonium groups. GO-FACO was used for sequential delivery of MDR1
11
siRNA and DOX. The results presented higher cytotoxicity against DOX- treated MCF-7 cells
12
and also rather than DOX/ GO-FACO treated cells. In another study, the PEG/PEI/CS-Aco
13
complex was successfully utilized for the delivery of DOX and shRNA to the human hepatic cell
14
line [107]. PEI/ chitosan/PSS/Fe nanoparticles complicated multi-functionalized GO (CMG)
15
were introduced by Wang et al. for the delivery of pEGFP and DOX to A549 lung cancer and
16
C42b prostate cancer cell lines [89]. The DOX loaded nano-carriers (DOX–CMGs) release DOX
17
faster at acidic pH, and are more effective in killing A549 lung cancer cells than free DOX.
18
These results demonstrated the multifunctional capability of CMGs for targeted cancer
19
chemotherapy, gene therapy, and MR imaging. CS-functionalized GO was also utilized to co-
20
deliver platinum complex drugs including cisplatin, carboplatin, oxaliplatin as well as
21
nucleotides [138].
22
Zhi et al. [87] prepared a nano-complex composed of PEI, poly(sodium 4-styrenesulfonates), GO
23
and termed PPG (PEI/PSS/GO) to carry Adriamycin (ADR) along with miR-21 targeted siRNA.
24
First, ADR was loaded onto the PPG surface by physical mixing, and then anti-miR-21 was
25
loaded by electrical absorption (anti-miR-21/PPG/ADR). The superior therapeutic efficacy of
26
this nano-carrier could be exhibited by gene silencing of miR-21 and better accumulation of
27
ADR in tumor cells. PAMAM dendrimer-grafted gadolinium-functionalized GO (Gd-NGO) was
28
introduced as a nano-carrier to co-deliver drugs and specific gene-targeting agents to cancer cells
29
[111]. The positively charged surface of this vector was able to absorb the anti-cancer drug
30
epirubicin (EPI) and interaction with Let-7g miRNA. Also, Gd-NGO can be used as MRI-
AC C
EP
TE D
M AN U
10
ACCEPTED MANUSCRIPT
detectable to identify the tumor place. Therefore, Gd-NGO could be a potential vector for both
2
drug and gene therapy and molecular imaging diagnosis.
3
In another study, a PAMAM dendrimer-functionalized GO (GO-PAMAM) was utilized to
4
simultaneously deliver MMP-9 shRNA plasmid and DOX in order to enhance the efficacy of
5
anti-breast cancer treatment [115]. The PAMAM-functionalized GO showed the pH dependent
6
DOX release rate. In addition, compared to PEI-25k, this nano-carrier interestingly provided
7
more transfer ability as well as transfection efficiency in the presence of serum proteins. It
8
demonstrated that PAMAM could provide superior stability against protein adsorption as well as
9
a high amine covered surface to load much more shRNA. Co-delivery assay results demonstrated
10
that the cell survival rate of GO-PAMAM/DOX/MMP-9 treated MCF-7 breast cancer cells was
11
lower than DOX or MMP-9 shRNA treated once, which confirmed the significant potential of
12
breast cancer via the co-delivery approach.
13
Combining photothermal therapy and effective gene therapy have shown promising in cancer
14
treatment due to the enhanced combined therapeutic effects [139]. Graphene and its derivatives
15
can also be used in photothermal treatment of cancer because of their unique features like NIR
16
absorbance, huge specific surface area and functional groups [140]. One of the recent works in
17
this field was conducted by Cheng et al. [88]. They used the PEGylated PEI-grafted graphene/Au
18
complex to deliver siRNA to HL-60 cells. Moreover, this complex displayed photothermal
19
response to enhance the gene transfection efficacy. NGO-PEG-PEI is another composite
20
exhibiting remarkably enhanced pDNA transfection efficiencies under NIR laser irradiation [85].
21
In another study, Kim et al. [92] used the PEG–BPEI–rGO nano-composite to deliver plasmid
22
DNA in PC-3 and NIH/3T3 cells. In addition, this carrier demonstrated enhanced pDNA
23
transfection efficiencies under NIR irradiation that showed the ability of using NGO-PEG-PEI as
24
a light-responsive gene carrier.
25
Taken together, the co-delivery of drugs and nucleotide-based therapeutics by graphene-based
26
nano-carriers would have wide prospects for treatment of the target diseases, particularly cancer
27
[13].
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
1
7- Conclusions and perspectives
2
To date, graphene-based nanoplatform applications in biomedicine, particularly gene and drug
4
delivery, have progressively been the focus of interest. The sheet-like structure of the graphenic
5
nanoparticles, which can provide ultrahigh surface area, as well as a purely carbon composition,
6
offer a remarkable advantage to load cargoes with high efficiency compared with other nano-
7
carriers. In addition, relatively easy synthesis and the possibility of chemical modification of
8
such a carbon-based platform, for instance oxidation or reduction, makes it a suitable candidate
9
to design multifunctional nano-carriers. However, regarding the nucleotides negative charge, the
10
most possible interaction of genes with the graphene nanosheet would be based on the
11
electrostatic ones, which are limited by the relatively negative charge of graphene. Therefore, a
12
key design feature of such a nano-carrier is the cationic charge induction via cationic polymer
13
immobilization or cationic functional group insertion on the surface (e.g. amine). As seen in
14
Table1, cationic polymers, synthetic or natural, have been utilized as modifier to provide
15
electrostatic interaction with nucleotide based cargo. However, these kinds of modifiers could
16
also provide better cell membrane interactions, and consequently more efficient cell entrance.
17
One of the challenging issues in extra-cellular gene delivery with the power of nanoparticles is
18
undesired interaction with proteins, which are available in cell culture media or blood, and result
19
in nano-carrier instability and consequently precipitation before reaching the targeted cells. This
20
is because the large surface area of graphitic nanosheets and surface charge density are very
21
suseptible to agglomeration in such media, particularly after in-vivo administration.
22
Consequently, minimizing these nonspecific interactions and improving hemocompatibility via
23
surface functionalization with long chain hydrophilic modifiers, which could provide steric
24
hindrance seems essential to developing such carriers in clinical applications. Although
25
PEGylation are commonly utilized for this purpose, and results of various studies showed the
26
effectiveness of PEGylation on biostability as well as biocompatibility, additional investigations
27
on more efficient modifiers, which could provide biostability without interference in cell
28
entrance potential seems a more helpful strategy.
29
Unlike chemical drugs, for nucleotide-based cargoes, the intracellular delivery route even toward
30
the nucleous is another restrictive issue, where non-efficient endosomal escape results in such
31
severe cargo damages, and the damaged cargo obviously could not show therapeutic effects. In
AC C
EP
TE D
M AN U
SC
RI PT
3
ACCEPTED MANUSCRIPT
the nano-carrier design step, considering the intracellular obstacles and providing possible
2
solutions are very important. Although some cationic modifiers like PEI or CPPs could stimulate
3
endosomal escape, the co-delivery of some lysosomotropic agents (e.g. chloroquin, NH4Cl,
4
methylamine) into the cells could likely aid to overcome this challenge. However, the
5
cytotoxicity aspect of such agents should be considered and controlled.
6
From the synthesis and modification point of view, controlling the size of nanosheets and
7
immobolized ligands’ density per particle are challenging issues, which both effect the
8
nanoparticle biodistribution and cell entrance mechanisms. In addition, nucleotide loading
9
capacity is a function of the size and surface modification density. Non-uniformity usually
10
originated from various synthesis methods or a diverse level of oxidaton/reduction during further
11
functionalization of graphene. For the translation of graphene-based gene delivery systems to
12
clinical applications, developing manufacturing protocols, which could be scaled up based on the
13
laboratory studies, is an unignorable step.
14
Regarding the limited cell source (primary or cell line) having been utilized to evaluate the
15
potential of graphene-based nanomaterial as gene delivery vectors, one of the other challenges
16
ahead may be to confirm whether their successful performance is dependent on the treated cell’s
17
type or they could be considered as a general platform to transfect all cells. In addition, since in
18
various reviewed reports different eventual treatment protocols (i.e. transfection in presence or
19
absence of serum, different incubation times, pre/post treatment by some reagents) have been
20
used, there is a need for further investigation to find a clear relationship between the applied
21
treatments and the transfection efficacy in order to standardize the protocols.
22
Although numerous studies of graphene based nanoplatforms as gene delivery systems are
23
currently in progress by diverse research groups, only a few studies assessed the fate of
24
functionalized graphene-based particles through in-vivo gene delivery. Like other nanoparticles,
25
biocompatibility and biodegradation of graphitic nanoparticles are still challenging issues.
26
However, the results of studies demostrated that biocompatibility is a dose-dependent manner
27
and different functionalization could impact its improvement. Biodegradation and metabolization
28
of these kinds of particles are also under investigation. Although the overall findings of studies
29
have almost consistently confirmed the safety of the modified graphitic nano-carrier at suitable
30
doses, regarding a few in-vivo assessments in their gene delivery applications, these challenges
31
still await more investigations in the future before clinical trial attempts.
AC C
EP
TE D
M AN U
SC
RI PT
1
ACCEPTED MANUSCRIPT
Under the light of such supplementary studies, it will create hope for the gene therapiast to open
2
up promising tools for efficent and safe gene delivery to the targeted cells and tissues.
3
Conflict of Interest
4
The authors declare there is no conflct of interest.
5
References
[5]
[6]
[7]
[8] [9]
[10] [11] [12]
[13] [14] [15] [16] [17]
SC
M AN U
[4]
TE D
[2] [3]
D. Scherman, A. Rousseau, P. Bigey, and V. Escriou, "Genetic pharmacology: progresses in siRNA delivery and therapeutic applications," Gene therapy, vol. 24, pp. 151-156, 2017. D. Cyranoski, "CRISPR gene-editing tested in a person for the first time," Nature, vol. 539, 2016. H. Yin, R. L. Kanasty, A. A. Eltoukhy, A. J. Vegas, J. R. Dorkin, and D. G. Anderson, "Non-viral vectors for gene-based therapy," Nature Reviews Genetics, vol. 15, p. 541, 2014. V. Wagner, A. Dullaart, A.-K. Bock, and A. Zweck, "The emerging nanomedicine landscape," Nature biotechnology, vol. 24, p. 1211, 2006. J. K. Wong, R. Mohseni, A. A. Hamidieh, R. E. MacLaren, N. Habib, and A. M. Seifalian, "Will nanotechnology bring new hope for gene delivery?," Trends in biotechnology, vol. 35, pp. 434451, 2017. M. Nurunnabi, K. Parvez, M. Nafiujjaman, V. Revuri, H. A. Khan, X. Feng, et al., "Bioapplication of graphene oxide derivatives: drug/gene delivery, imaging, polymeric modification, toxicology, therapeutics and challenges," Rsc Advances, vol. 5, pp. 42141-42161, 2015. H. Zhao, R. Ding, X. Zhao, Y. Li, L. Qu, H. Pei, et al., "Graphene-based nanomaterials for drug and/or gene delivery, bioimaging, and tissue engineering," Drug discovery today, vol. 22, pp. 1302-1317, 2017. L. Feng and Z. Liu, "Graphene in biomedicine: opportunities and challenges," Nanomedicine, vol. 6, pp. 317-324, 2011. G. Reina, J. M. González-Domínguez, A. Criado, E. Vázquez, A. Bianco, and M. Prato, "Promises, facts and challenges for graphene in biomedical applications," Chemical Society Reviews, vol. 46, pp. 4400-4416, 2017. S. Goenka, V. Sant, and S. Sant, "Graphene-based nanomaterials for drug delivery and tissue engineering," Journal of Controlled Release, vol. 173, pp. 75-88, 2014. H. Dong, C. Dong, T. Ren, Y. Li, and D. Shi, "Surface-engineered graphene-based nanomaterials for drug delivery," Journal of biomedical nanotechnology, vol. 10, pp. 2086-2106, 2014. G. Shim, M.-G. Kim, J. Y. Park, and Y.-K. Oh, "Graphene-based nanosheets for delivery of chemotherapeutics and biological drugs," Advanced drug delivery reviews, vol. 105, pp. 205-227, 2016. M. Nejabat, F. Charbgoo, and M. Ramezani, "Graphene as multifunctional delivery platform in cancer therapy," Journal of Biomedical Materials Research Part A, vol. 105, pp. 2355-2367, 2017. M. Vincent, I. de Lázaro, and K. Kostarelos, "Graphene materials as 2D non-viral gene transfer vector platforms," Gene therapy, vol. 24, p. 123, 2017. F. Yin, B. Gu, Y. Lin, N. Panwar, S. C. Tjin, J. Qu, et al., "Functionalized 2D nanomaterials for gene delivery applications," Coordination Chemistry Reviews, vol. 347, pp. 77-97, 2017. K. Novoselov and A. Geim, "The rise of graphene," Nat. Mater, vol. 6, pp. 183-191, 2007. A. M. Pinto, I. C. Gonçalves, and F. D. Magalhães, "Graphene-based materials biocompatibility: a review," Colloids and Surfaces B: Biointerfaces, vol. 111, pp. 188-202, 2013.
EP
[1]
AC C
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
RI PT
1
ACCEPTED MANUSCRIPT
[23] [24] [25] [26] [27] [28]
[29]
[30] [31]
[32] [33] [34]
[35]
[36] [37] [38]
RI PT
[22]
SC
[21]
M AN U
[20]
TE D
[19]
J. Liu, L. Cui, and D. Losic, "Graphene and graphene oxide as new nanocarriers for drug delivery applications," Acta biomaterialia, vol. 9, pp. 9243-9257, 2013. M. Jahan, Q. Bao, J.-X. Yang, and K. P. Loh, "Structure-directing role of graphene in the synthesis of metal− organic framework nanowire," Journal of the American Chemical Society, vol. 132, pp. 14487-14495, 2010. M. Freitag, T. Low, F. Xia, and P. Avouris, "Photoconductivity of biased graphene," Nature Photonics, vol. 7, p. 53, 2013. C. Liu, Z. Yu, D. Neff, A. Zhamu, and B. Z. Jang, "Graphene-based supercapacitor with an ultrahigh energy density," Nano letters, vol. 10, pp. 4863-4868, 2010. Q. Bao and K. P. Loh, "Graphene photonics, plasmonics, and broadband optoelectronic devices," ACS nano, vol. 6, pp. 3677-3694, 2012. Y. Shao, J. Wang, H. Wu, J. Liu, I. A. Aksay, and Y. Lin, "Graphene based electrochemical sensors and biosensors: a review," Electroanalysis, vol. 22, pp. 1027-1036, 2010. J. R. Potts, D. R. Dreyer, C. W. Bielawski, and R. S. Ruoff, "Graphene-based polymer nanocomposites," Polymer, vol. 52, pp. 5-25, 2011. M. Pumera, "Graphene in biosensing," Materials today, vol. 14, pp. 308-315, 2011. Y. Wang, Y. Shao, D. W. Matson, J. Li, and Y. Lin, "Nitrogen-doped graphene and its application in electrochemical biosensing," ACS nano, vol. 4, pp. 1790-1798, 2010. H. Shen, L. Zhang, M. Liu, and Z. Zhang, "Biomedical applications of graphene," Theranostics, vol. 2, pp. 283-294, 2012. A. R. Maity, A. Chakraborty, A. Mondal, and N. R. Jana, "Carbohydrate coated, folate functionalized colloidal graphene as a nanocarrier for both hydrophobic and hydrophilic drugs," Nanoscale, vol. 6, pp. 2752-2758, 2014. R. Imani, S. H. Emami, and S. Faghihi, "Synthesis and characterization of an octaarginine functionalized graphene oxide nano-carrier for gene delivery applications," Physical Chemistry Chemical Physics, vol. 17, pp. 6328-6339, 2015. T. Kuila, S. Bose, P. Khanra, A. K. Mishra, N. H. Kim, and J. H. Lee, "Recent advances in graphenebased biosensors," Biosensors and Bioelectronics, vol. 26, pp. 4637-4648, 2011. N. Ren, J. Li, J. Qiu, M. Yan, H. Liu, D. Ji, et al., "Growth and accelerated differentiation of mesenchymal stem cells on graphene-oxide-coated titanate with dexamethasone on surface of titanium implants," Dental Materials, vol. 33, pp. 525-535, 2017. D. Li, M. B. Müller, S. Gilje, R. B. Kaner, and G. G. Wallace, "Processable aqueous dispersions of graphene nanosheets," Nature nanotechnology, vol. 3, pp. 101-105, 2008. V. C. Sanchez, A. Jachak, R. H. Hurt, and A. B. Kane, "Biological interactions of graphene-family nanomaterials–an interdisciplinary review," Chemical research in toxicology, vol. 25, p. 15, 2012. L. Chen, Y. Hernandez, X. Feng, and K. Müllen, "From nanographene and graphene nanoribbons to graphene sheets: chemical synthesis," Angewandte Chemie International Edition, vol. 51, pp. 7640-7654, 2012. R. Sekiya, Y. Uemura, H. Naito, K. Naka, and T. Haino, "Chemical functionalisation and photoluminescence of graphene quantum dots," Chemistry–A European Journal, vol. 22, pp. 8198-8206, 2016. H. Sun, L. Wu, W. Wei, and X. Qu, "Recent advances in graphene quantum dots for sensing," Materials Today, vol. 16, pp. 433-442, 2013. L. Li, G. Wu, G. Yang, J. Peng, J. Zhao, and J.-J. Zhu, "Focusing on luminescent graphene quantum dots: current status and future perspectives," Nanoscale, vol. 5, pp. 4015-4039, 2013. H. Dong, W. Dai, H. Ju, H. Lu, S. Wang, L. Xu, et al., "Multifunctional PLA-PEG-grafted Graphene Quantum Dots for Intracellular MicroRNA Imaging and Combined Specific Gene-targeting Agents Delivery for Improved Therapeutics," ACS Appl. Mater. Interfaces, vol. 7, pp. 11015-11023, 2015.
EP
[18]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
ACCEPTED MANUSCRIPT
[44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56]
[57] [58]
[59]
[60]
RI PT
[43]
SC
[42]
M AN U
[41]
TE D
[40]
S. Zhou, H. Xu, W. Gan, and Q. Yuan, "Graphene quantum dots: recent progress in preparation and fluorescence sensing applications," RSC Advances, vol. 6, pp. 110775-110788, 2016. R. S. Duryat, "Graphene nanoribbons (GNRs) for future interconnect," in IOP Conference Series: Materials Science and Engineering, 2016, p. 012018. O. Akhavan, E. Ghaderi, H. Emamy, and F. Akhavan, "Genotoxicity of graphene nanoribbons in human mesenchymal stem cells," Carbon, vol. 54, pp. 419-431, 2013. M. K. Riley and W. Vermerris, "Recent Advances in Nanomaterials for Gene Delivery—A Review," Nanomaterials, vol. 7, p. 94, 2017. M. Elsabahy, A. Nazarali, and M. Foldvari, "Non-viral nucleic acid delivery: key challenges and future directions," Current drug delivery, vol. 8, pp. 235-244, 2011. C. Scholz and E. Wagner, "Therapeutic plasmid DNA versus siRNA delivery: common and different tasks for synthetic carriers," Journal of controlled release, vol. 161, pp. 554-565, 2012. P. M. Moreno and A. P. Pêgo, "Therapeutic antisense oligonucleotides against cancer: hurdling to the clinic," Frontiers in chemistry, vol. 2, 2014. R. W. Collin and A. Garanto, "Applications of antisense oligonucleotides for the treatment of inherited retinal diseases," Current opinion in ophthalmology, vol. 28, pp. 260-266, 2017. W. Guo, W. Chen, W. Yu, W. Huang, and W. Deng, "Small interfering RNA-based molecular therapy of cancers," Chinese journal of cancer, vol. 32, p. 488, 2013. K. Tatiparti, S. Sau, S. K. Kashaw, and A. K. Iyer, "siRNA Delivery Strategies: A Comprehensive Review of Recent Developments," Nanomaterials, vol. 7, p. 77, 2017. J. J. Rossi, "Expression strategies for short hairpin RNA interference triggers," Human gene therapy, vol. 19, pp. 313-317, 2008. R. Rupaimoole and F. J. Slack, "MicroRNA therapeutics: towards a new era for the management of cancer and other diseases," Nature Reviews Drug Discovery, vol. 16, pp. 203-222, 2017. X. J. Loh, T.-C. Lee, Q. Dou, and G. R. Deen, "Utilising inorganic nanocarriers for gene delivery," Biomaterials science, vol. 4, pp. 70-86, 2016. J. M. Ageitos, J.-A. Chuah, and K. Numata, "Design Considerations for Properties of Nanocarriers on Disposition and Efficiency of Drug and Gene Delivery," 2016. A. F. Adler and K. W. Leong, "Emerging links between surface nanotechnology and endocytosis: impact on nonviral gene delivery," Nano Today, vol. 5, pp. 553-569, 2010. S. Jin and K. Ye, "Nanoparticle-Mediated Drug Delivery and Gene Therapy," Biotechnology progress, vol. 23, pp. 32-41, 2007. R. Misra, M. Upadhyay, and S. Mohanty, "Design considerations for chemotherapeutic drug nanocarriers," Pharmaceutica Analytica Acta, vol. 2014, 2014. K. Kettler, K. Veltman, D. van de Meent, A. van Wezel, and A. J. Hendriks, "Cellular uptake of nanoparticles as determined by particle properties, experimental conditions, and cell type," Environmental toxicology and chemistry, vol. 33, pp. 481-492, 2014. R. Agarwal and K. Roy, "Intracellular delivery of polymeric nanocarriers: a matter of size, shape, charge, elasticity and surface composition," Therapeutic delivery, vol. 4, pp. 705-723, 2013. V. Mundra and R. I. Mahato, "Design of nanocarriers for efficient cellular uptake and endosomal release of small molecule and nucleic acid drugs: learning from virus," Frontiers of Chemical Science and Engineering, vol. 8, pp. 387-404, 2014. A. Albanese, P. S. Tang, and W. C. Chan, "The effect of nanoparticle size, shape, and surface chemistry on biological systems," Annual review of biomedical engineering, vol. 14, pp. 1-16, 2012. D. Yin, Y. Li, H. Lin, B. Guo, Y. Du, X. Li, et al., "Functional graphene oxide as a plasmid-based Stat3 siRNA carrier inhibits mouse malignant melanoma growth in vivo," Nanotechnology, vol. 24, p. 105102, 2013.
EP
[39]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
ACCEPTED MANUSCRIPT
[66]
[67]
[68]
[69]
[70]
[71]
[72] [73] [74]
[75] [76] [77]
RI PT
[65]
SC
[64]
M AN U
[63]
TE D
[62]
A. Paul, A. Hasan, H. A. Kindi, A. K. Gaharwar, V. T. Rao, M. Nikkhah, et al., "Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair," ACS nano, vol. 8, pp. 8050-8062, 2014. M.-j. Li, C.-m. Liu, Y.-b. Xie, H.-b. Cao, H. Zhao, and Y. Zhang, "The evolution of surface charge on graphene oxide during the reduction and its application in electroanalysis," Carbon, vol. 66, pp. 302-311, 2014. J. Kim, L. J. Cote, F. Kim, W. Yuan, K. R. Shull, and J. Huang, "Graphene Oxide Sheets at Interfaces," Journal of the American Chemical Society, vol. 132, pp. 8180-8186, 2010/06/16 2010. J. Rejman, V. Oberle, I. S. Zuhorn, and D. Hoekstra, "Size-dependent internalization of particles via the pathways of clathrin-and caveolae-mediated endocytosis," Biochemical Journal, vol. 377, pp. 159-169, 2004. J. Kim, L. J. Cote, and J. Huang, "Two dimensional soft material: new faces of graphene oxide," Accounts of Chemical Research, vol. 45, pp. 1356-1364, 2012. V. C. Sanchez, A. Jachak, R. H. Hurt, and A. B. Kane, "Biological interactions of graphene-family nanomaterials: an interdisciplinary review," Chemical research in toxicology, vol. 25, pp. 15-34, 2011. C. Xu, D. Yang, L. Mei, B. Lu, L. Chen, Q. Li, et al., "Encapsulating gold nanoparticles or nanorods in graphene oxide shells as a novel gene vector," ACS applied materials & interfaces, vol. 5, pp. 2715-2724, 2013. H. Tian, W. Xiong, J. Wei, Y. Wang, X. Chen, X. Jing, et al., "Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG," Biomaterials, vol. 28, pp. 2899-2907, 2007. M. Ogris, P. Steinlein, M. Kursa, K. Mechtler, R. Kircheis, and E. Wagner, "The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells," Gene therapy, vol. 5, p. 1425, 1998. X. Lin, N. Zhao, P. Yan, H. Hu, and F.-J. Xu, "The shape and size effects of polycation functionalized silica nanoparticles on gene transfection," Acta biomaterialia, vol. 11, pp. 381392, 2015. Y. Li, H. Yuan, A. von dem Bussche, M. Creighton, R. H. Hurt, A. B. Kane, et al., "Graphene microsheets enter cells through spontaneous membrane penetration at edge asperities and corner sites," Proceedings of the National Academy of Sciences, vol. 110, pp. 12295-12300, 2013. K. P. Loh, Q. Bao, P. K. Ang, and J. Yang, "The chemistry of graphene," Journal of Materials Chemistry, vol. 20, pp. 2277-2289, 2010. Y. Pan, N. G. Sahoo, and L. Li, "The application of graphene oxide in drug delivery," Expert opinion on drug delivery, vol. 9, pp. 1365-1376, 2012. M. Teimouri, A. H. Nia, K. Abnous, H. Eshghi, and M. Ramezani, "Graphene oxide–cationic polymer conjugates: Synthesis and application as gene delivery vectors," Plasmid, vol. 84, pp. 51-60, 2016. C.-H. Lu, C.-L. Zhu, J. Li, J.-J. Liu, X. Chen, and H.-H. Yang, "Using graphene to protect DNA from cleavage during cellular delivery," Chemical Communications, vol. 46, pp. 3116-3118, 2010. L. Feng, S. Zhang, and Z. Liu, "Graphene based gene transfection," Nanoscale, vol. 3, pp. 12521257, 2011. H. Kim, R. Namgung, K. Singha, I.-K. Oh, and W. J. Kim, "Graphene oxide–polyethylenimine nanoconstruct as a gene delivery vector and bioimaging tool," Bioconjugate chemistry, vol. 22, pp. 2558-2567, 2011.
EP
[61]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
ACCEPTED MANUSCRIPT
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90] [91]
[92] [93]
RI PT
[82]
SC
[81]
M AN U
[80]
TE D
[79]
B. Chen, M. Liu, L. Zhang, J. Huang, J. Yao, and Z. Zhang, "Polyethylenimine-functionalized graphene oxide as an efficient gene delivery vector," Journal of Materials Chemistry, vol. 21, pp. 7736-7741, 2011. H. Bao, Y. Pan, Y. Ping, N. G. Sahoo, T. Wu, L. Li, et al., "Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery," Small, vol. 7, pp. 1569-78, Jun 6 2011. L. Zhang, Z. Lu, Q. Zhao, J. Huang, H. Shen, and Z. Zhang, "Enhanced chemotherapy efficacy by sequential delivery of siRNA and anticancer drugs using PEI-grafted graphene oxide," Small, vol. 7, pp. 460-464, 2011. H. Dong, L. Ding, F. Yan, H. Ji, and H. Ju, "The use of polyethylenimine-grafted graphene nanoribbon for cellular delivery of locked nucleic acid modified molecular beacon for recognition of microRNA," Biomaterials, vol. 32, pp. 3875-3882, 2011. T. Ren, L. Li, X. Cai, H. Dong, S. Liu, and Y. Li, "Engineered polyethylenimine/graphene oxide nanocomposite for nuclear localized gene delivery," Polymer Chemistry, vol. 3, pp. 2561-2569, 2012. X. Yang, G. Niu, X. Cao, Y. Wen, R. Xiang, H. Duan, et al., "The preparation of functionalized graphene oxide for targeted intracellular delivery of siRNA," Journal of Materials Chemistry, vol. 22, pp. 6649-6654, 2012. L. Zhang, Z. Wang, Z. Lu, H. Shen, J. Huang, Q. Zhao, et al., "PEGylated reduced graphene oxide as a superior ssRNA delivery system," Journal of Materials Chemistry B, vol. 1, pp. 749-755, 2013. L. Feng, X. Yang, X. Shi, X. Tan, R. Peng, J. Wang, et al., "Polyethylene glycol and polyethylenimine dual-functionalized nano-graphene oxide for photothermally enhanced gene delivery," Small, vol. 9, pp. 1989-1997, 2013. X. Zhou, F. Laroche, G. E. Lamers, V. Torraca, P. Voskamp, T. Lu, et al., "Ultra-small graphene oxide functionalized with polyethylenimine (PEI) for very efficient gene delivery in cell and zebrafish embryos," Nano Research, vol. 5, pp. 703-709, 2012. F. Zhi, H. Dong, X. Jia, W. Guo, H. Lu, Y. Yang, et al., "Functionalized graphene oxide mediated adriamycin delivery and miR-21 gene silencing to overcome tumor multidrug resistance in vitro," PloS one, vol. 8, p. e60034, 2013. F.-F. Cheng, W. Chen, L.-H. Hu, G. Chen, H.-T. Miao, C. Li, et al., "Highly dispersible PEGylated graphene/Au composites as gene delivery vector and potential cancer therapeutic agent," Journal of Materials Chemistry B, vol. 1, pp. 4956-4962, 2013. C. Wang, S. Ravi, U. S. Garapati, M. Das, M. Howell, J. Mallela, et al., "Multifunctional chitosan magnetic-graphene (CMG) nanoparticles: a theranostic platform for tumor-targeted co-delivery of drugs, genes and MRI contrast agents," Journal of Materials Chemistry B, vol. 1, pp. 43964405, 2013. S. K. Tripathi, R. Goyal, K. C. Gupta, and P. Kumar, "Functionalized graphene oxide mediated nucleic acid delivery," Carbon, vol. 51, pp. 224-235, 2013. J. Zhang, L. Feng, X. Tan, X. Shi, L. Xu, Z. Liu, et al., "Dual-polymer-functionalized nanoscale graphene oxide as a highly effective gene transfection agent for insect cells with cell-typedependent cellular uptake mechanisms," Particle & Particle Systems Characterization, vol. 30, pp. 794-803, 2013. H. Kim and W. J. Kim, "Photothermally controlled gene delivery by reduced graphene oxide– polyethylenimine nanocomposite," Small, vol. 10, pp. 117-126, 2014. H. Hu, C. Tang, and C. Yin, "Folate conjugated trimethyl chitosan/graphene oxide nanocomplexes as potential carriers for drug and gene delivery," Materials Letters, vol. 125, pp. 82-85, 2014.
EP
[78]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
ACCEPTED MANUSCRIPT
[99]
[100]
[101]
[102]
[103]
[104]
[105]
[106]
[107]
[108]
[109]
RI PT
[98]
SC
[97]
M AN U
[96]
TE D
[95]
X. Liu, D. Ma, H. Tang, L. Tan, Q. Xie, Y. Zhang, et al., "Polyamidoamine dendrimer and oleic acidfunctionalized graphene as biocompatible and efficient gene delivery vectors," ACS applied materials & interfaces, vol. 6, pp. 8173-8183, 2014. X. Cao, S. Zheng, S. Zhang, Y. Wang, X. Yang, H. Duan, et al., "Functionalized Graphene Oxide with Hepatocyte Targeting as Anti-Tumor Drug and Gene Intracellular Transporters," Journal of Nanoscience and Nanotechnology, vol. 15, pp. 2052-2059, 2015. Y. Tao, E. Ju, J. Ren, and X. Qu, "Immunostimulatory oligonucleotides-loaded cationic graphene oxide with photothermally enhanced immunogenicity for photothermal/immune cancer therapy," Biomaterials, vol. 35, pp. 9963-9971, 2014. X. Cao, F. Feng, Y. Wang, X. Yang, H. Duan, and Y. Chen, "Folic acid-conjugated graphene oxide as a transporter of chemotherapeutic drug and siRNA for reversal of cancer drug resistance," Journal of nanoparticle research, vol. 15, p. 1965, 2013. L. Hollanda, A. Lobo, M. Lancellotti, E. Berni, E. Corat, and H. Zanin, "Graphene and carbon nanotube nanocomposite for gene transfection," Materials Science and Engineering: C, vol. 39, pp. 288-298, 2014. H. Y. Choi, T.-J. Lee, G.-M. Yang, J. Oh, J. Won, J. Han, et al., "Efficient mRNA delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells," Journal of Controlled Release, vol. 235, pp. 222-235, 2016. Y.-P. Huang, C.-M. Hung, Y.-C. Hsu, C.-Y. Zhong, W.-R. Wang, C.-C. Chang, et al., "Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by PolyethylenimineFunctionalized Graphene Oxide," Nanoscale research letters, vol. 11, pp. 1-8, 2016. M. Y. Kim, F. L. Do Won Hwang, Y. Choi, J. W. Byun, D. Kim, J.-E. Kim, et al., "Detection of intrabrain cytoplasmic 1 (BC1) long noncoding RNA using graphene oxide-fluorescence beacon detector," Scientific reports, vol. 6, 2016. Y. Sun, J. Zhou, Q. Cheng, D. Lin, Q. Jiang, A. Dong, et al., "Fabrication of mPEGylated graphene oxide/poly (2-dimethyl aminoethyl methacrylate) nanohybrids and their primary application for small interfering RNA delivery," Journal of Applied Polymer Science, vol. 133, 2016. H. Kim, J. Kim, M. Lee, H. C. Choi, and W. J. Kim, "Stimuli-Regulated Enzymatically Degradable Smart Graphene-Oxide-Polymer Nanocarrier Facilitating Photothermal Gene Delivery," Advanced healthcare materials, 2016. F. Wang, B. Zhang, L. Zhou, Y. Shi, Z. Li, Y. Xia, et al., "Imaging dendrimer-grafted graphene oxide mediated anti-miR-21 delivery with an activatable luciferase reporter," ACS applied materials & interfaces, vol. 8, pp. 9014-9021, 2016. D. Yin, Y. Li, B. Guo, Z. Liu, Y. Xu, X. Wang, et al., "Plasmid-Based Stat3 siRNA Delivered by Functional Graphene Oxide Suppresses Mouse Malignant Melanoma Cell Growth," Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, vol. 23, pp. 229-236, 2016. C. Wang, X. Wang, T. Lu, F. Liu, B. Guo, N. Wen, et al., "Multi-functionalized graphene oxide complex as a plasmid delivery system for targeting hepatocellular carcinoma therapy," RSC Advances, vol. 6, pp. 22461-22468, 2016. Y. He, L. Zhang, Z. Chen, Y. Liang, Y. Zhang, Y. Bai, et al., "Enhanced chemotherapy efficacy by codelivery of shABCG2 and doxorubicin with a pH-responsive charge-reversible layered graphene oxide nanocomplex," Journal of Materials Chemistry B, vol. 3, pp. 6462-6472, 2015. S.-R. Ryoo, J. Lee, J. Yeo, H.-K. Na, Y.-K. Kim, H. Jang, et al., "Quantitative and multiplexed microRNA sensing in living cells based on peptide nucleic acid and nano graphene oxide (PANGO)," ACS nano, vol. 7, pp. 5882-5891, 2013. X. Yang, N. Zhao, and F.-J. Xu, "Biocleavable graphene oxide based-nanohybrids synthesized via ATRP for gene/drug delivery," Nanoscale, vol. 6, pp. 6141-6150, 2014.
EP
[94]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
ACCEPTED MANUSCRIPT
[114]
[115]
[116]
[117]
[118]
[119]
[120]
[121]
[122] [123]
[124]
[125]
RI PT
SC
[113]
M AN U
[112]
TE D
[111]
C.-J. Hsieh, Y.-C. Chen, P.-Y. Hsieh, S.-R. Liu, S.-P. Wu, Y.-Z. Hsieh, et al., "Graphene oxide based nanocarrier combined with a pH-sensitive tracer: a vehicle for concurrent pH sensing and pHresponsive oligonucleotide delivery," ACS applied materials & interfaces, vol. 7, pp. 1146711475, 2015. H.-W. Yang, C.-Y. Huang, C.-W. Lin, H.-L. Liu, C.-W. Huang, S.-S. Liao, et al., "Gadoliniumfunctionalized nanographene oxide for combined drug and microRNA delivery and magnetic resonance imaging," Biomaterials, vol. 35, pp. 6534-6542, 2014. J. Sun, J. Chao, J. Huang, M. Yin, H. Zhang, C. Peng, et al., "Uniform small graphene oxide as an efficient cellular nanocarrier for immunostimulatory CpG oligonucleotides," ACS applied materials & interfaces, vol. 6, pp. 7926-7932, 2014. R. Imani, W. Shao, S. Taherkhani, S. H. Emami, S. Prakash, and S. Faghihi, "Dual-functionalized graphene oxide for enhanced siRNA delivery to breast cancer cells," Colloids and Surfaces B: Biointerfaces, vol. 147, pp. 315-325, 2016. H. Zhang, T. Yan, S. Xu, S. Feng, D. Huang, M. Fujita, et al., "Graphene oxide-chitosan nanocomposites for intracellular delivery of immunostimulatory CpG oligodeoxynucleotides," Materials Science and Engineering: C, vol. 73, pp. 144-151, 2017. Y. Gu, Y. Guo, C. Wang, J. Xu, J. Wu, T. B. Kirk, et al., "A polyamidoamne dendrimer functionalized graphene oxide for DOX and MMP-9 shRNA plasmid co-delivery," Materials Science and Engineering: C, vol. 70, pp. 572-585, 2017. T. Y. Hsieh, W. C. Huang, Y. D. Kang, C. Y. Chu, W. L. Liao, Y. Y. Chen, et al., "NeurotensinConjugated Reduced Graphene Oxide with Multi-Stage Near-Infrared-Triggered Synergic Targeted Neuron Gene Transfection In Vitro and In Vivo for Neurodegenerative Disease Therapy," Advanced healthcare materials, vol. 5, pp. 3016-3026, 2016. L. Ren, Y. Zhang, C. Cui, Y. Bi, and X. Ge, "Functionalized graphene oxide for anti-VEGF siRNA delivery: preparation, characterization and evaluation in vitro and in vivo," RSC Advances, vol. 7, pp. 20553-20566, 2017. F. Yin, K. Hu, Y. Chen, M. Yu, D. Wang, Q. Wang, et al., "SiRNA Delivery with PEGylated Graphene Oxide Nanosheets for Combined Photothermal and Genetherapy for Pancreatic Cancer," Theranostics, vol. 7, p. 1133, 2017. H.-C. C. Foreman, G. Lalwani, J. Kalra, L. T. Krug, and B. Sitharaman, "Gene delivery to mammalian cells using a graphene nanoribbon platform," Journal of Materials Chemistry B, vol. 5, pp. 2347-2354, 2017. M. Dowaidar, H. N. Abdelhamid, M. Hällbrink, X. Zou, and Ü. Langel, "Graphene oxide nanosheets in complex with cell penetrating peptides for oligonucleotides delivery," Biochimica et Biophysica Acta (BBA)-General Subjects, vol. 1861, pp. 2334-2341, 2017. L. Wu, J. Xie, T. Li, Z. Mai, L. Wang, X. Wang, et al., "Gene delivery ability of polyethylenimine and polyethylene glycol dual-functionalized nanographene oxide in 11 different cell lines," Royal Society open science, vol. 4, p. 170822, 2017. J. Yang, Q. Zhang, H. Chang, and Y. Cheng, "Surface-engineered dendrimers in gene delivery," Chemical reviews, vol. 115, pp. 5274-5300, 2015. X. Sun, W. Wang, T. Wu, H. Qiu, X. Wang, and J. Gao, "Grafting of graphene oxide with poly (sodium 4-styrenesulfonate) by atom transfer radical polymerization," Materials Chemistry and Physics, vol. 138, pp. 434-439, 2013. H.-K. Jeong, M. H. Jin, K. H. An, and Y. H. Lee, "Structural stability and variable dielectric constant in poly sodium 4-styrensulfonate intercalated graphite oxide," The Journal of Physical Chemistry C, vol. 113, pp. 13060-13064, 2009. R. A. Jones, M. H. Poniris, and M. R. Wilson, "pDMAEMA is internalised by endocytosis but does not physically disrupt endosomes," Journal of controlled release, vol. 96, pp. 379-391, 2004.
EP
[110]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
ACCEPTED MANUSCRIPT
42 43
[131]
[132]
[133]
[134]
[135] [136]
[137]
[138] [139]
[140]
RI PT
[130]
SC
[129]
M AN U
[128]
TE D
[127]
S. Agarwal, Y. Zhang, S. Maji, and A. Greiner, "PDMAEMA based gene delivery materials," Materials Today, vol. 15, pp. 388-393, 2012. Y.-Z. You, D. S. Manickam, Q.-H. Zhou, and D. Oupický, "Reducible poly (2-dimethylaminoethyl methacrylate): synthesis, cytotoxicity, and gene delivery activity," Journal of Controlled Release, vol. 122, pp. 217-225, 2007. K. Hu, D. D. Kulkarni, I. Choi, and V. V. Tsukruk, "Graphene-polymer nanocomposites for structural and functional applications," Progress in Polymer Science, vol. 39, pp. 1934-1972, 2014. J. Zong, S. L. Cobb, and N. R. Cameron, "Peptide-functionalized gold nanoparticles: versatile biomaterials for diagnostic and therapeutic applications," Biomaterials Science, vol. 5, pp. 872886, 2017. V. P. Torchilin, "Cell penetrating peptide-modified pharmaceutical nanocarriers for intracellular drug and gene delivery," Peptide Science, vol. 90, pp. 604-610, 2008. S. M. Ghafary, M. Nikkhah, S. Hatamie, and S. Hosseinkhani, "Simultaneous Gene Delivery and Tracking through Preparation of Photo-Luminescent Nanoparticles Based on Graphene Quantum Dots and Chimeric Peptides," Scientific reports, vol. 7, p. 9552, 2017. R. Guo, L. Zhang, H. Qian, R. Li, X. Jiang, and B. Liu, "Multifunctional nanocarriers for cell imaging, drug delivery, and near-IR photothermal therapy," Langmuir, vol. 26, pp. 5428-5434, 2010. R. Imani, S. H. Emami, and S. Faghihi, "Nano-graphene oxide carboxylation for efficient bioconjugation applications: a quantitative optimization approach," Journal of Nanoparticle Research, vol. 17, p. 88, February 13 2015. H. Kim, J. Kim, M. Lee, H. C. Choi, and W. J. Kim, "Stimuli-Regulated Enzymatically Degradable Smart Graphene-Oxide-Polymer Nanocarrier Facilitating Photothermal Gene Delivery," Advanced healthcare materials, vol. 5, pp. 1918-1930, 2016. H. Yue, X. Zhou, M. Cheng, and D. Xing, "Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing," Nanoscale, 2018. R. Imani, S. Prakash, H. Vali, and S. Faghihi, "Polyethylene glycol and octaarginine n dualfunctionalized nano-graphene oxide: An optimization for efficient nucleic acids delivery," Biomaterials Science, 2018. O. Tacar, P. Sriamornsak, and C. R. Dass, "Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems," Journal of Pharmacy and Pharmacology, vol. 65, pp. 157-170, 2013. H. Bao, Y. Pan, Y. Ping, N. G. Sahoo, T. Wu, L. Li, et al., "Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery," Small, vol. 7, pp. 1569-1578, 2011. Y.-W. Chen, Y.-L. Su, S.-H. Hu, and S.-Y. Chen, "Functionalized graphene nanocomposites for enhancing photothermal therapy in tumor treatment," Advanced drug delivery reviews, vol. 105, pp. 190-204, 2016. M. Guo, J. Huang, Y. Deng, H. Shen, Y. Ma, M. Zhang, et al., "pH-Responsive Cyanine-Grafted Graphene Oxide for Fluorescence Resonance Energy Transfer-Enhanced Photothermal Therapy," Advanced Functional Materials, vol. 25, pp. 59-67, 2015.
EP
[126]
AC C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41