Current status of ATRP-based materials for gene therapy

Reactive and Functional Polymers 147 (2020) 104453

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Review

Current status of ATRP-based materials for gene therapy Sayeny de Ávila Gonçalves, Roniérik Pioli Vieira

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T

Department of Bioprocesses and Materials Engineering, School of Chemical Engineering, University of Campinas, 13083-852 Albert Einstein av. Campinas, São Paulo, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: ATRP Gene therapy Polymeric gene vectors Well-defined polymers

Gene therapy is an auspicious alternative to treat diseases. However, the design of efficient vectors remains as a challenge due to the innumerous intracellular and extracellular barriers that should be faced during the gene delivery process. Among some types of carries, polymeric gene vectors have gained increasingly attention. Aiming to improve the polymeric vectors' performance, several strategies have been applied such as diversification of the monomers, synthesis routes, polymers architecture, addition of specific targeting units, shielding domains, and inorganic nanoparticles. Besides, the use of controlled polymerization in the synthesis of these carries have led to improvements, especially ATRP, a very robust and versatile technique. Therefore, the aims of this review are summarizing the recent advances in gene vectors produced through ATRP; propose a division according to the main gene carries characteristics and strategies used to improve their performance; and also provide a critical analysis of the current and future perspectives on the use of ATRP in the synthesis of gene vectors.

1. Introduction In recent years, the development of biotechnology and engineering has promoted conditions for new approaches to diagnostic and treat diseases. Among several novel alternatives, gene therapy is one of the most promising. This technique emerged in 1963 and consists in insert nucleic acids, DNA or RNA, into cells to replace or repair or regulate abnormal genes [1–4]. In general, it is possible to deliver the genes into the cell directly using a naked gene. However, this is not an efficient method because of the rapid enzymatic degradation of the nucleic acids and low delivery rate due to the negative charge of both, nucleic acids and membrane cells, being necessary to use a gene carrier or vector [2,4–6]. The gene carriers can be classified as viral and non-viral vectors. Fig. 1 provides a schematic overview of applying nanocarriers for gene delivery. Initially, the researches focused mainly on viral vectors because of their high transfection efficiency and specificity [2,4,5,7,8]. However, the virus vectors have many safety issues such as the activation of immunologic responses, the possible genes mutation, besides the difficult to scale up the production. Hence, the non-viral delivery systems have been increasingly gained prominence mainly for their low immune response, ease of synthesis, and unrestricted plasmid size. Nonviral vectors include lipoplexes (DNA complexed with cationic lipids), polyplexes (nucleic acid complexes with polycations), nanoparticles, and encapsulated DNA in degradable polymer matrices. Regardless the

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kind, all gene vector should guarantee not only delivery with high gene expression in the target cells, but also be biocompatible, non-toxic, stable against enzymatic degradation and in the presence of serum proteins do not trigger any inflammatory response, and facilitate cellular uptake [4,9–12]. Among all alternatives for gene carriers, polymeric vectors have received increasing attention due to their versatility, ease of modification and structure manipulation, low immunogenicity and pathogenicity, and their potential applications in a broad variety of gene-mediated therapies [3,5,6,11]. Even these great characteristics, the polymeric vectors also present poor delivery efficiency and significant cytotoxicity, which makes the clinical use of these materials remains as a challenge. Several strategies have been exploited to improve it, for example, the new highly branched poly(β-amino ester)s synthesis [14–16]. Now researchers have already found that characteristics such as chemical composition, molecular weight, structural architecture, dispersity, surface functionality, charge ratio and size of the polymernucleic acid complex are directly related with the performance of the polymeric gene vectors [4,5,12,17,18]. The relatively recent advances in polymer engineering made possible to design and produce polymers with well-defined properties by controlled radical polymerization techniques (CRPs) [1,5]. CRPs, especially atom-transfer radical polymerization (ATRP) and reversibleaddition fragmentation chain-transfer polymerization (RAFT)

Corresponding author. E-mail address: [email protected] (R.P. Vieira).

https://doi.org/10.1016/j.reactfunctpolym.2019.104453 Received 2 October 2019; Received in revised form 12 December 2019; Accepted 13 December 2019 Available online 14 December 2019 1381-5148/ © 2019 Elsevier B.V. All rights reserved.

Reactive and Functional Polymers 147 (2020) 104453

S.d.Á. Gonçalves and R.P. Vieira

Fig. 1. Polymeric nanocarriers (in orange) for gene delivery [13]. Reproduced with permission from Royal Society of Chemistry Copyright 2015.

ATRP technique is based on a reversible electrochemical reaction from a dormant specie (Pn-X), usually a halogen derivative, to a transition metal salt (Mtm+/L). This process leads to the generation of propagating radicals (Pn*) and the metal complex in the higher oxidation state (X-Mtm+1/L). The radical species formed may then undergo addition to one or more vinyl monomer units. Due to the reversibility of the reaction, the radicals can also react with the oxidized metal complexes, X-Mtm+1/L, reestablishing dormant species and the transition metal complex (Mtm/L) in the lower oxidation state. The rapid chemical equilibrium between a very small concentration of propagating radicals and a much larger concentration of dormant species suppress bimolecular termination and chain transfer reactions, and promotes an effective process control allowing the production of polymers with a narrow molecular weight distributions [19,22–24]. Fig. 2 presents the general mechanism of traditional ATRP. It is well-known that traditional ATRP requires high concentration of activator (transition metal complexes), since the persistent radical effect causes the loss of catalyst in the process (it oxidizes) [25–27]. Several works were developed with the aim of drastically reducing the amount of catalyst through its regeneration. The main variations of the traditional process are provided by addition of chemical reducing agents in activator (re)generation by electron transfer (ARGET-ATRP)

processes, have been frequently applied for the synthesis of well-defined, functionalized polymers that meet the requirements for a successful gene delivery system. ATRP is one of the most robust CRP techniques that allows the controlled synthesis of polymers using several monomers. The polymers narrow molecular weight distribution will be predetermined by the concentration ratio of monomer, initiator and catalyst. In addition, this technique is especially attractive because of it does not require critical experimental conditions; it is not sensible to impurities; most of ATRP catalyst systems and initiators are commercially available or can be easily synthesized; and facility of incorporation a specific functionality site in the chain, allowing the construction of block, alternate and grafted copolymers. The materials produced have structures ranging from purely homopolymers to extremely complex hybrid materials, and depending on the macromolecular architecture obtained, they indicated characteristics quite favorable to the above application. To the best of our knowledge, there is no review that presents the current status of ATRPbased materials for application in gene therapy jointly to a critical analysis of synthesis strategies and challenges regarding copper toxicity. Therefore, considering the importance and great potential application of ATRP for the development and improvements in the design of gene vectors, this review summarizes the recent advances in gene vectors produced through ATRP. The aim of this paper is not only to provide to readers an overview of the materials synthesized by ATRP, but also to propose a subdivision of these materials in order to direct the readers to the main synthesis routes. In addition, this review provides a critical analysis of the strategies to improve gene vectors performance, challenges and future perspectives for the expansion of applications in this area. 2. Atom-transfer radical polymerization Atom-Transfer Radical Polymerization (ATRP) has been demonstrated to be a versatile and robust technique to synthesize well-defined polymers. Through this technique, it is possible to produce polymers with low dispersity, controllable molecular weight (MW), as well as complex (co)polymers with several architectures, organic/inorganic hybrid materials, and multifunctional properties [19–21].

Fig. 2. General mechanism of traditional ATRP [24]. Reproduced with permission from Elsevier. Copyright 2012. 2

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nucleic acid is attributed to the presence of amine groups in these materials that can bind to the phosphate groups of the nucleic acids [60,62,63]. However, most of them have low transfection efficiency and/or significant toxicity [2,9]. Due to its high transfection capability, PEI of molecular weight of 25 kDa is one of the most successful and widely studied polymeric gene vector. It is considered the gold standard and has become the reference to which other polymers, projected and synthesized, are often compared with. In contrast, PEI also shows high cytotoxicity and its synthesis for systematic studies is not simple. Therefore, actually, several studies have been given special attention to PDMAEMA. This polymer can be easily prepared and manipulated by radical polymerization techniques and its gene delivery systems present buffering capacity, high transfection efficiency (90% of the branched PEI) and less cytotoxicity than PEI (25 kDa) [1,2,6,9,40]. The use of cationic polymer vectors is a paradox because in order to obtain a high degree of transfection the carries must possess high density of charges and high molecular weight, but these same characteristics lead to high toxicity due to the interaction of the charges with the cellular components and possible inhibition or alteration of normal cellular processes. A high concentration of amino groups is important to the acid nucleic compaction but it is also an important factor leading to high cytotoxicity. PEI is mainly composed of secondary amino groups while PDMAEMA has only tertiary amines [2,9,50,55,64]. Aiming to face these challenges one of the strategies for improve the polycations performance is to design vectors with varied architectures. Block copolymers, grafted copolymers, hyperbranched, star-shaped polymers, and mult-knot polymers can be produced through ATRP and have shown good results [5,17,65]. Mathew et al. [52] used traditional ATRP followed by DE-ATRP to produce a hyperbranched polymeric system with a linear pDMAEMA block and hyperbranches of polyethylene glycol methyl ether methacrylate (PEGMEMA) and ethylene dimethacrylate (EGDMA). The authors designed two different types of polymers, one with a high content of PEG (lpD-b-P/E 1) and another one with a lower concentration of PEG (lpD-b-P/E 2). pDMAEMA was synthesized through traditional ATRP. In sequence, the linear pDMAEMA was used as a macroinitiator for DE-ATRP of PEGMEMA and EGDMA to obtain a hyperbranched PEG-based polymer (Fig. 3). In this work, DE-ATRP was performed with a modification from the original DE-ATRP technique. A reducing agent, ascorbic acid, was used to facilitate the controlled growth of the polymer chain without gelation. Better control of polymeric chain was possible due to the production of active Cu1+ species, while Cu2+ remaining in the system. Characteristics such as DNA binding capacity, cytotoxicity, and transfection efficiency were evaluated and compared with commercially gene carriers. The assays with the polyplexes showed that both gene vectors could form stable particles that were not degraded by DNAse action and stayed stable for two weeks even in the serum presence. The rise in the degree of PEGylation led to a decrease in the zeta potential and an increase in the polyplexes size. In fibroblasts and in ADSCs, the metabolism of the cells was higher in the presence of both gene vectors produced than in the presence of the commercial transfection agents. Besides, the vectors did not affect the cells reproduction. The polymeric gene vector with a lower concentration of PEG was more toxic than that vector with a higher concentration of PEG, but lower cytotoxic than the commercial gene vectors. Both gene vectors developed in this work maintained their transfection efficiency even at a high concentration of pDNA, which was not noted for commercial transfection agents. This polymeric system can prove favorable for sustained gene therapy applications where long-term and a high DNA dose is required [52,66]. Song et al. [57] prepared two gene delivery vectors by ATRP: hydrophilic poly(vinyl pyrrolidone)-graft-poly[2-(N,N-dimethylamino) ethyl methacrylate] (PVP-g-PDMAEMA) and amphiphilic poly(vinyl pyrrolidone)-graft poly[2-(N,N-dimethylamino) ethyl methacrylate]-

[28,29], a current in electrochemically mediated (eATRP) [30], an external radical initiator in initiators for continuous activator (re)generation (ICAR-ATRP) [31], zero valent metals in supplemental activators and reducing agents (SARA-ATRP) [32]. In addition, some of the more recent works deal with the process using photochemical reduction (photoATRP) [33,34], or organic activated photo-catalysts (O-ATRP), also known as metal-free ATRP [35], which presents an enormous potential for the production of non-toxic materials, which would allow the expansion of applications in the gene therapy and other medical field. Recently, one of the most used techniques to tailor the chemical and physical properties of the polymers is the modification on its surfaces. Several researches have been employed surface initiated ATRP (SIATRP) to produce gene vectors with the desired properties. In SI-ATRP, the initiator is introduced on the surface, so the polymers chains will grow in a controlled manner from this solid substrate, generating a material with different properties [23]. It is possible to combine this synthesis strategy with the above-mentioned ATRP variations. Nevertheless, it appears that most published research uses the traditional ATRP form, which has as its main disadvantage the large amount of metal catalyst dispersed in the resulting material. Deactivation enhanced atom-transfer radical polymerization (DEATRP) was developed in 2007 aiming to produce high branched polymers avoiding gel formation. After that, the technique was successfully employed to produce multi knot structures for gene delivery. In general, the DE-ATRP consists in allows the growth of the independent and complex hyperbranched molecule, but at the same time avoiding the crosslink between them. It can be achieved by manipulating the reaction equilibrium. The increase in the concentration of Cu(II) relative to Cu(I) leads the reaction to the deactivated state. Hence, there is a slow growth of polymeric chain due to the fact that fewer monomer units are added to an active center and the onset of gelation is prevented until the system has achieved high levels of conversion [11,36,37]. 3. Polymeric vectors for gene therapy In order to standardize and facilitate the materials comparative analysis, −in this work, the vectors were divided in three major groups according to the presence of some compounds into their structure. Hybrids are those that are composed of polymers and some inorganic component such as metals or oxides as part of the material structure; Biohybrids are characterized by the presence of a biomolecule, natural or synthetic, in their composition, such as polysaccharides or peptides; and at least all polymers that do not have inorganic compound or biomolecules in their structure were classified simply as polycations. It is worth mentioning that all polymeric vectors for gene delivery should be positively charged, not just those that were categorized as polycation. The positive nature of the polymer in a specific environment is a prerequisite for it may be employed in gene therapy because this cationic behavior is what enables the complexation of the nucleic acids. Some of the main strategies used by the scientists to face some challenges in use polymeric gene vector are also described in a specific section. Table 1 displays briefly the latest works developed dealing with ATRP-based materials for gene therapy. 3.1. Polycations Polycations, polymers that are charged positively, can spontaneously interact with anionic nucleic acids and compact them into nanocomplexes through electrostatic interaction [2,9]. Consequently, the cationic polymer vectors are able to reduce the electrostatic repulsion between the gene and the cell surfaces, protect it from enzymatic degradation and facilitate transfection [9]. Several kinds of cationic or functional polymers can be employed as vectors for gene therapy such as poly(L-lysine) (PLL), polyethylenimine (PEI), poly(2-N,N-dimethylaminoethyl methacrylate) (PDMAEMA) and copolymers, polyamidoamines. In general, the ability to compact the 3

Redox Stimulli responsive Stimulli responsive/ targeted/PEGylated

Stimulli responsive PEGylated

Stimulli responsive

Redox Stimulli responsive Redox Stimuli responsitive Redox and pH Stimuli responsive/targeted/ PEGylated pH Stimuli responsive/ PEGylated Redox Stimuli responsive/PEGylated PEGylated

Hybrid

Polycation Polycation

Biohybrid

Polycation

4

Star shaped

Hyperbranched

–

PEGylated

Block copolymer - micelles

Randon Coil and micelles

Stimuli responsive

–

PEGylated

Stimuli responsive

Polycation

Polycation

Hybrid

Star shapped core: poly (arylene oxindole)and arms: PDMAEMA and PDEGMA Multilayer star-shapped core: Au NPs and arms: poly (DAMA-HEMA)

Brush-shaped

PEGylated

Diblock copolymers - micells

Triblock terpolymer

Biohybrid/ Hybrid Polycation

Polycation

Biohybrid

Biohybrid/ Hybrid Polycation

Hybrid

Redox Stimulli responsive/targeted –

Grafted

Redox Stimuli responsive –

Star shapped core: CD and arms: Block copolymer Grafted

Hybrid

Hybrid

Stimuli Responsive/ targeted Zwitterion

Block copolymer

Brush-shaped

Block copolymer

Au-p(DAMA-HEMA)n

PVP-g-PDMAEMA PVP-g-PDMAEMA-bPMMA P(DMAEMA-ran-DEGMA) PDMAEMA-bPDEGMA

CE-PCL-a-(PDMAEMA-co-PPEGMA)

CNC-graft-PPEGEEMA/PDMAEMA@Au

PHB-b-PDMAEMA

Gal-PEEP-a-PCL-ss-PDMAEMA

pDMAEMA-b-PEGMEMA/EDGMA

(DOTA-Gd)7-CD-(PDMA)

Au-g-PDMAEMA

CD-SS-PDMAEMA

CD-PDMA-PMPD

P(PEGMEMA-co-PEGMA-Gal)-bPDMAEMA P(OEGMA)-SS-P(GMA-TEPA),

(PEI-PEGMA)n-ss-PCL-PEI

PCD-acetal-PGEA

PEG-PEI-PCL-SS-PCL-PEI-PEG PEG-PEIPCL-SS-PCL-PEI-PEG-T7

Penta-block copolymers/ Micelles

Brush-shaped

PCL-SS-PDMA

PDMAEMA-PEEDEPE

AMY-PDMEMA

POSS-PDMAEMA POSS-PDMAEMAPPEGMA POSS-PDMAEMA-PPEGMACAGW POSS-PDMAEMA-PPEGMA-CAGTAT-NLS P(GMA-PEGEEMA) P(GMAPEGEEMA) PD-E 8%PEG

POSS-(SS-PDMAEMA)8s

(Co)Polymer

Diblock copolymers - micells

Multiknot

Dendritic

Comb-shapped Cyclized knot

Star-shaped with comb-like structure

Star 8 -arms

Architecture

Polycation

Biohybrid

Polycation

Polycation

Polycation

Polycation

Hybrid

Main characteristics

Group

Table 1 Main works dealing with ATRP-based materials for gene therapy.

EA.hy926 (Epithelial)

p-DNA delivery in endothelial cells

293 T HT-1080

A549

pDNA delivery

siRNA delivery (anti-c-Myc siRNA)

HeLa

HEK293 HepG3

HEK293 HepG2

Fibroblasts and in ADSCs HeLa HepG2

HeLa HepG2

HepG2 and COS8

HepG2 and COS7

HeLa, HEK293 T, HepG2, PC-12 COS-7

MDA-MB-231 HeLa EAT (in vivo) HeLa HepG2

HeLa 293 T MCF-7

HeLa 293 T KB CAL27 MCF-7

RDEB keratinocytes

Codelivery: doxorubicin and pDNA pDNA delivery

Codelivery: doxorubicin and DNA Codelivery: paclitaxel and pDNA (Bcl-2) pDNA delivery

Theragnostic p-DNA delivery Theragnostic p-DNA delivery p-DNA delivery

Theragnostic p-DNA delivery p-DNA delivery

Neuron-Targeted

Codelivery of siRNA (plk1) and DOX Hepatoma cells-targeting

Codelivery of pORFhTRAIL (p-DNA) and DOX - cancer breast treatment pDNA siRNA

Drug and p-DNA delivery

Theragnostic p-DNA delivery p-DNA (COL7A1 gene)

HEK293 and C6 Neu 7 Astrocytes B104 SHSY-5Y PC12 HEK293 and HepG2

HepG2 and COS7

p-DNA delivery

p-DNA delivery Protein delivery

Cell line

Application

250 nm

200.2 nm and 169.7 nm 160 nm and 100 nm 100–200 nm

~137 nm

200–250 nm

150–200 nm

lpD-b-P/E 1: 200-250 nm lpD-b-P/E 2: 50-80 nm < 200 nm

~102 nm

100–200

300 nm*

< 50 nm

< 50 nm

160 nm

~100 nm

[59]

[58]

[57]

[56]

[55]

[54]

[53]

[52]

[51]

[50]

[49]

[48]

[47]

[46]

[45]

[44]

[43]

[42]

[11]

[41]

[40] [37]

[39]

[38]

Reference

(continued on next page)

consecutive SI-ATRP

ATRP core first consecutive ATRP core first

ROP, ATRP, CuAAC click reaction ATRP

ROP, ATRP, CuAAC click reactions, polymer reaction Traditional ATRP and transesterification ATRP and RAFT

ATRP and click chemistry reaction ATRP and DE-ATRP

ATRP

ATRP

ATRP

Random polymerization and ATRP RAFT and ATRP

ROP and ATRP

ROP and ATRP

ROP, Michael Addition and ATRP

80–120 nm

< 200 nm

ATRP and ROP

70 to 200 nm

in situ DE-ATRP

ATRP

100–150 nm 100 and 320 nm

ATRP and ROP DE-ATRP

Consecutive traditional ATRP and click chemistry

Traditional ATRP

Synthesis technique

200 to 300 nm 90 and 130 nm

101.0–112.1 nm

100–150 nm

Nanoparticle size

S.d.Á. Gonçalves and R.P. Vieira

Reactive and Functional Polymers 147 (2020) 104453

Reactive and Functional Polymers 147 (2020) 104453

[61] ATRP and ROP

[60] ATRP

block-poly(methylmethacrylate) (PVP-g-PDMAEMA-b-PMMA). Hydrophilic PVP-g-PDMAEMA was able to form random coils in water solution and PVP-g-PDMAEMA-b-PMMA was self-assembled into micelles with the PMMA as hydrophobic blocks. Both polycations presented higher buffering capability than PEI (25 kDa). Between them, there was no significant difference, which means that the incorporation of MMA does not affect their buffering capacity. PVP-g-PDMAEMA/pDNA polyplexes showed a lower zeta potential. It can be associated with the shielding effect of the micelle structure. At all N/P ratios tested, PVP-g-PDMAEMA-b-PMMA compacted pDNA into smaller particles than PVP-g-PDMAEMA. The hydrophobic parts can compress the nanoparticles in aqueous solution. The two polymeric vectors could effectively condense pDNA, but the micellar structure could condense more effectively and into a more stable particle. PVP-g-PDMAEMA/pDNA exhibited similar cytotoxicity to PEI (25 kDa) while PVP-g-PDMAEMA-b-PMMA/pDNA had slightly higher cytotoxicity. The decrease in the cell viability of PVP-gPDMAEMA-b-PMMA/pDNA might be the presence of the hydrophobic MMA segments. On the other hand, PVP-g-PDMAEMA-b-PMMA exhibited greater transfection efficiency compared to PVP-g-PDMAEMA at all N/P ratios. Besides, both gene vectors had better a transfection performance than PEI (25 kDa) in 293 T cells. In the presence of serum, the developed polymeric vectors showed more resistance to the protein absorption. Probably the PVP can shield the positive charges by steric effect, while PEI with excess positive charges can combine with more negative charged BSA [57]. Mendrek et al. [58] designed star-shape gene vectors. The authors evaluated how the arms structure and composition could influence the performance of the polymeric vector in gene delivery. For that, they produced stars with random copolymer arms and stars with block copolymer arms. The arm compositions of both kinds varied from 8% to 40% of DEGMA monomer. In both cases, the procedure adopted was the “core first” method. The core was composed by poly(arylene oxindole) (PArOx) containing bromoester groups that were used as macroinitiator of the ATRP of DMAEMA and DEGMA. The star with random copolymer arms, P(DMAEMA-ran-DEGMA), was prepared using the one-pot ATRP method. Stars with block arms of DMAEMA and DEGMA were obtained in a two-pot method. First, PDMAEMA was polymerized into the core and the other monomer DEGMA was introduced into the ATRP polymerization system after the purification of PDMAEMA stars. All polymerizations were executed to low monomer conversions aiming to avoid radical star-star coupling reactions. The star polymeric vectors studied were pH-sensitive in aqueous solutions, being positively charged in physiological condition (pH ∼ 7.4). Regardless of DEGMA content and arm architecture, the values of zeta potential decreased with the rise of pH. Both star copolymers could condense pDNA, however, no compact nanoparticles were observed. It can be a consequence of the presence of uncharged DEGMA in the structures. Random arms star copolymer could bind pDNA more effectively, which suggests a better binding of pDNA when amine groups are located next to each other. All polymeric vectors obtained showed less cytotoxicity than branched PEI at all N/P ratios investigated. Comparing with the PDMAEMA stars, the inclusion of DEGMA into the structures reduced the cytotoxicity. In addition, the star random polyplexes exhibited slightly less toxicity than those composed of block star copolymers. The greatest results for gene expression were also achieved for random copolymer stars with the highest DEGMA concentration (30 to 40 mol%.). Lastly, the authors could infer that the introduction of DEGMA into the star arms was the most important reason for improvements in cell viability and transfection efficiency [58]. The neutral, water-soluble, and biocompatible poly(N-hydroxyethylacrylamide) (HEAA) was polymerized by traditional ATRP to be the backbone of the polymeric gene vector. In sequence, the PHEAA backbone was grafted with 3,30-diaminodipropylamine (DPA). The final PHEAA-DPA produced contained primary amine and tertiary

Block copolymer – Polycation

p(HEMA)-b-p(lys)

pDNA delivery

NIH 3 T3

20 and 30 nm of diameter < 200 nm HepG2 and COS7 pDNA delivery Brush-shaped – Polycation

PHEAA100-DPA PHEAA200-DPA

Architecture Main characteristics Group

Table 1 (continued)

(Co)Polymer

Application

Cell line

Nanoparticle size

Synthesis technique

Reference

S.d.Á. Gonçalves and R.P. Vieira

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Fig. 3. Schematic diagram of the polymeric system synthetized with a linear pDMAEMA block and hyperbranches of PEGMEMA and EGDMA (A and B) and polyplex formation (C) [52]. Reproduced with permission from Elsevier. Copyright 2012.

ratio of 12:1 and 10:1, respectively. In the end, the scientists agreed that PHEAA200-DPA is more appropriate for gene therapy application [60]. Johnson et al. [61] synthesized a block copolymer based on poly(2hydroxyethyl methacrylate) and L-lysine (p(lys)) (Fig. 4). A series of p (HEMA)-b-p(lys) with the size of PHEMA fixed in 40 and the length of p (lys) varying in 40, 80, 120, and 150 were synthesized by ROP and ATRP. An ended functionalized Phthalimido End-Capped p(HEMA) was firstly produced by ATRP. In sequence, p(HEMA)40-NH2 was used as the macroinitiator for the ROP of Z-lys NCA for the synthesis of p (HEMA)40-b-p(lys)n copolymers. All the copolymers could condense the pDNA and form polyplexes with size from 150 to 200 nm at specified N/P ratios. The polyplexes were tested in NIH 3 T3 cells and exhibited significant lower cytotoxicity than PEI (25 kDa). However, the transfection efficiency was slightly lower than PEI (25 kDa). The designed gene carrier taking advantage of the plasmid DNA (pDNA) binding ability of p(lys), biodegradability and biocompatibility of p (HEMA). Yang et al. [40] using the combination of traditional ATRP and ring opening polymerization (ROP) techniques designed two comb-shaped polymers: a poly(glycidyl methacrylate) (PGMA) backbone with PGMA side chains and a PGMA backbone with poly(poly(ethylene glycol)ethyl

amine and the ratio is 2:1. Seeking a better knowledge about how the amine groups could influence the performance of a polymeric vector in gene therapy, the authors analyzed two polymers PHEAA100-DPA and PHEAA200-DPA [60]. PHEAA200-DPA presented more efficiency in condensing pDNA, slightly smaller particles formation, and higher zeta potentials than PHEAA100-DPA. Surely, all these results depend on several factors, but in this case, a great part of them was fixed and then, the result could be attributed to the higher concentration of charged amino groups in PHEAA200-DPA. The buffering capacity of the polymers was similar but lower than that of PEI (25 kDa) and higher than PLL's. PEI has higher charge concentration while has no buffering capability at physiological pH because all primary amino groups with a high pKa are protonated. The cytotoxicity of the gene carriers was measured in COS-7 cells and HepG-2 cells. In both cell lines, PHEAA100-DPA showed lower cytotoxicity than PHEAA200-DPA -DPA, at the same weight ratio, due to the higher positive density of PHEAA200-DPA. Until a weight ratio of vector/DNA of 16:1 the polyplexes produced exhibited lower cytotoxicity than PEI (25 kDa). In relation to the transfection efficiency, PHEAA200-DPA/pDNA presented better results than PHEAA100-DPA/ pDNA. The transfection efficiency of PHEAA200-DPA/pDNA was better than that of PEI (25 kDa) in COS-7 cells and HepG-2 cells at a weight 6

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Fig. 4. Synthesis procedures of p(HEMA)-b-p(lys) copolymers by combining ATRP of HEMA with ROP of Z-lys-NCA [61]. Reproduced with permission from John Wiley & Sons Inc. Copyright 2014.

significant molecular weight increase. The remaining vinyl groups were end-functionalized with 1,3-diaminopropane through Michael Addition [11]. The polymeric multi-knot gene vectors showed superior results for transfection, and expression, besides lower cytotoxicity when compared with PEI (25 kDa) and Lipofectamine®2000. These improvements in the results are attributed to the innovative architecture and degradability of the polymer, which favors, at the same time, the DNA condensation and the DNA release [11]. Seeking a solution for drug resistance in cancer patients during chemotherapeutics, researchers developed a co-delivery system of paclitaxel and nuclear receptor Nur77 [54]. According to the authors, Bcl2 protein family is the main factor for the drug resistance due to the blockage of the apoptosis signal in the cancer cell, and it is overexpressed in > 50% of cancer patients. The polymeric vector designed to achieve this objective was an amphiphilic poly[(R)-3-hydroxybutyrate]-poly(2-(dimethylamino)ethyl methacrylate) (PHB-bPDMAEMA). PHB is a natural biodegradable polyester that due to its high hydrophobicity is able to form stable micelles [54].Through transesterification reaction it was produced three PHB with different degree of hydroxylation: low molecular weight mono-hydroxylated PHB (PHB01), di-hydroxylated PHB (PHB02), and tri-hydroxylated PHB (PHB03). In sequence, the hydroxyl end groups were bromoesterified to produce a bromo-terminated PHB macroinitiator for ATRP reaction. Thereby, ATRP of PDMAEMA was conducted in a traditional path. Compounds with a high degree of hydroxylation generated cationic copolymers with higher amount of PDMAEMA branches (Fig. 5). PHB-PDMAEMA could bind pDNA into stable nanoparticles of 150–200 nm of diameter, at N/P ratio above 10. All polymeric systems showed lower cytotoxicity than PEI (25 kDa). This result is attributed to the presence of the biocompatible PHB that may reduce cationic charge density of PDMAEMA improving the cell viability. Under complete serum or reduced serum conditions and at its optimal N/P ratios, only PHB01-DMAEMA showed a comparable or better transfection efficiency than PEI (25 kDa). The co-delivery of Nur77 and paclitaxel fully reached its objective (Fig. 6). Nur77 could effectively accumulate in the mitochondria where it might interact with Bcl-2 changing its function and PTX could effectively inhibit the growth of the drug-resistant cancer cells [54].

ether methacrylate) (PPEGEEMA) side chains. In both cases, the branches were attached to the main chain through hydrolyzable ester bonds. In sequence, the branches of each kind of polymer were functionalized with ethanolamine (EA). Besides a PGMA chain was only functionalized with EA to be used for comparisons. Briefly, ROP was used to convert the epoxy groups of PGMA into initiation sites, while the side chains were polymerized by ATRP. Finally, ROP was again applied to functionalize the polymer side chains with EA. In cases when ATRP is realized from a multifunctional backbone with a high local concentration of initiation sites, the radical-radical linkage of the propagating chains can lead to a gel formation. It can be avoided by controlling the concentration of initiation sites, which was made in this work by adjusting the feed ratio between the ring opening agent - bromoisobutyric acid (BIBA) and epoxy units. All the polymeric gene carriers synthesized showed, at different N/P ratios, lower cytotoxicity than PEI (25 kDa). However, among them, there were no significant difference in cytotoxicity. At the N/P ratios of above 10, all PGEAs and PGEAPEGs could condense pDNA into nanoparticles of 200 to 300 nm in diameters. At N/P ratio of 10, only the functionalized PGMA-EA polymer, that did not have a comb-shape, exhibited lower transfection efficiency than PEI (25 kDa). The best transfection results were reached for polyplexes containing PGEEMA branches. The species containing a higher amount of PGEEMA in its branches had a better buffering capacity, but the lower DNA condensation capability [40]. A research group has been worked in improvements in DE-ATRP technique to produce knot structure for gene delivery. In earlier researches, they find out that a single non-degradable knot gene vector interacts differently with p-DNA exhibiting superior transfection profile and lower toxicity [36]. Thenceforward Aied et al. [11] projected a multi-knot polymeric structure with multi vinyl functional groups made from in situ DE-ATRP of two starting monomers: dimethyl amino ethyl methacrylate (DMAEMA), that supply the cationic unit, and propenoyloxy ethyl disulfanyl ethyl propenoate (PEEDEPE), that acts as knot and connecting points. Besides, PEEDEPE links the chain points by disulfide bonds that in cells environment may be easily broken and then the multi-knot polymers will be degraded into smaller molecules, (Mw = 5 kDa), allowing the release of DNA and avoiding the toxicity of high molecular weight polymers. Aiming to evaluate the performance of the multi-knot gene carrier they used it to correct the collagen type VII-null skin cells of Recessive Dystrophic Epideromylis Bullosa (RDEB) [11]. During the DE-ATRP reaction, the polymer chains exhibit a linearlike growth until to reach high monomer conversion the single knot molecules start to combine into multi-knot molecules, along with a

3.2. Hybrids Polymeric hybrid vectors are those that contain in their structure, besides the polymer, some inorganic component predominantly metals, metal sulfides, oxides, semiconductors, and quantum dots (QD). These materials are of interest to applications as gene vectors due to their 7

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Fig. 5. General synthesis procedure of PHB01-b-PDMAEMA cationic copolymer: (i) transesterification of natural PHB with hexanol, (ii) bromoesterification, and (iii) ATRP reaction of DMAEMA monomer, at the presence of HMTETA and CuBr [54]. Reproduced with permission from John Wiley & Sons Inc. Copyright 2017.

Lately, vectors containing inorganic nanoparticles have been widely investigated for applications in theranostic due to their optical, magnetic, and electrical properties. In this approach, there is the union between the diagnosis, monitoring and the treatment of the disease

large surface area, inherent properties such as magnetic and optical, non-immunogenicity, resistance to degradation under physiological conditions, and relative ease of functionalizing them through surface modification [13,67,68].

Fig. 6. Illustration of PTX delivery in the hydrophobic domain of PHB-PDMAEMA micelles; in addition their complex formation with Nur77/ΔDBD plasmid to render PHB-PDMAEMA@polyplex [54]. Reproduced with permission from John Wiley & Sons Inc. Copyright 2017. 8

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Fig. 7. Schematic diagram of biocleavable PDMAEMA–graphene oxide sheets synthesis by ATRP [49]. Republished with permission from Royal Society of Chemistry. Copyright 2014.

(CD) surface aiming its application in theranostics. Basically, the gene vector synthesis had three steps: preparation luminescent CDs by onestep microwave pyrolysis method; synthesis of CD-Br initiator through carbodiimide-assisted conjugation; and ATRP of poly- [2-(dimethylamino) ethyl methacrylate]-b-poly[N-(3-(methacryloylamino)propyl)N,N-dimethyl-N-(3 sulfopropyl) (PDMAEMA-b-PMPDSAH) on the surface of the CD-Br initiator. The final polymeric gene vector was denoted as CD-PDMA-PMPD. In CD-PDMA-PMPD's structure CD cores had the role of multicolor cell imaging agent; the cationic PDMAEMA acted as a DNA condensing agent; the zwitterionic PMPDSAH was the shell that protected the polyplex from undesired interactions with serum components [69]. It was analyzed three polymers: one with no shell component, just the CD core and PDMA; and another two that had CD core, PDMA with fixed length in 80 and PMPD zwitterions shield varying their length to 20 and 40, respectively. All polymeric vectors could condense the DNA into stable spheres, however, the presence of the PDMA decreased their ability to condense the DNA and their zeta potential. The vector containing PMPDSAH showed lower protein adsorption and superior hemocompatibility compared to PEI (25 kDa) and to the hybrid that did not have this shield. Zwitterionic PMPD modified particles exhibited less cytotoxicity than non-zwitterionic modified particles and PEI (25 kDa). The transfection assay showed that, at their optimum complexing ratios, the three polymeric vectors analyzed presented higher transfection efficiency than PEI (25 kDa). Besides, an increased serum concentration leads to a dramatic decrease in PEI (25 kDa) transfection efficiency, while the transfection capability of CD-PDMA-PMPD was maintained. The carbon dots in the transfected cells exhibited excitation dependent fluorescent emissions. Finally, the polymeric gene

using only a complex for that [2,13,67,68]. Adjusting characteristics such as composition, geometry, and structure it is possible to manipulate the optical, magnetic, and electrical properties of inorganic particles, which makes them a very interesting tunable detection modality [67]. Yang, Zhao, and Xu [49] produced a gene vector composed by a Graphene Oxide (GO) backbone and disulfide-linked PDMAEMA side chains with different lengths (Fig. 7). The first step was to create the ATRP initiator sites containing disulfide bonds onto the GO surface. Then, PDMAEMA was polymerized by traditional ATRP on de the GO–SS–Br sides. The polymeric vector produced was tested for both, drug and gene delivery. It was evaluated five hybrid structures, that varying the length of PDMAEMA which was made by controlling the ATRP time [49]. All the polymeric gene carries were able to condense the pDNA in small and stable particles. However, this property was dependent on the N/P and PDMEMA size chain. At high N/P ratios, all vectors condensed pDNA into nanoparticles with diameter of about 300 nm. The cytotoxicity of the vectors showed a rising trend to increase of PDMAEMA length. On the other hand, the transfection capability reached better results in vectors that had a longer PDMEMA chain. When compared with PEI (25kDA) and PDMAEMA homopolymer, at their optimum N/P rates, 10 and 15 respectively, all the vectors showed higher transfection efficiency and lower toxicity. In relation to the drug delivery capability, due to the conjugated structure of the graphene basal plane, SS–GPD could effectively attach and absorb aromatic, hydrophobic drugs, such as Camptothecin (CPT), to destroy cancer cells. The behavior of the gene vectors in the serum environment was not evaluated [49]. Cheng et al. [69] designed a zwitterion polymer in a carbon dot 9

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directly decreased the vector positive charge and hence it condenses capacity. At the same N/P ratio, the cytotoxicity of CNC-based vectors exhibited an upward trend with the increase of the PDMAEMA chain sizes. Besides, the vectors that had PPEGEEMA in its structure showed lower cytotoxicity than its non-PEGylated counterparts. All the CNCbased gene carriers showed lower cytotoxicity than PDMAEMA homopolymer probably due to the presence of CNC that is biocompatible. The presence of Au NPs slight improved the cell viability in HEK293 at low N/P ratios. It can be explained by the reduction in vector charge caused by Au NPs. The transfection efficiency was also depended on the PDMAEMA chain length. Those vectors that had larger PDMAEMA chain presented a superior gene transfection result. The importance of the PPEGEEMA was evident because high cell viability enables the cell expression even at a relatively high concentration of cationic vectors. Au NPs diminished the transfection efficiency, once again for occupying the PDMAEMA amine groups, however, this decrease was not significant considering that all gene vectors produced presented better transfection efficiency than PEI (25 kDa) and PDMAEMA homopolymer at their optimum N/P ratios. Au NPs could really act as a contrast agent for computed tomography. Finally, it could be concluded that PDMAEMA brushes interacted well with the genes, while the biocompatible PPEGEEMA brushes induced shielding effect and reduced the cytotoxicity as the biocompatible CNC [55]. Li et al. [51] developed a bioconjugated Janus-type star copolymers to provide gene delivery and magnetic resonance imaging contrast enhancement. The polymeric vector had a gadolinium (Gd3+) functionalized β-cyclodextrin (CD) core and PDMA arms. β-cyclodextrin has an asymmetrical structure. On one side 14 α-bromopropionate that were used as ATRP initiator for PDMA arms polymerization and on the other site 7 azide reactive sites that were covalently conjugated to DOTA-Gd complex by click chemistry, where DTA is where 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid. At similar N/P ratio, (DOTA-Gd)7-CD-(PDMA)14 showed a higher capability to compact the DNA in stable particles than PDMA homopolymer. The transfection efficiency assay was carried out with two expressing genes: green fluorescent protein (GFP) and luciferase (Luc). For GFP-expressing pDNA at an N/P ratio of 8 showed transfection comparable to that of branched PEI (25 kDa) at an N/P ratio of 10. Increasing the N/P ratio to 16, the transfection efficiency was reduced, probably due to the rise in cytotoxicity of the gene carriers at elevated concentrations. Using luciferase expressing pDNA as the reporter gene, branched PEI (25 kDa) had better transfection efficiency in all N/P ratios. (DOTA-Gd)7-CD-(PDMA11)14 reached a transfections efficiency 18% lower than branched PEI (25 kDa), which is considered acceptable. The cell viability was higher than 80% and had no obvious trend for cytotoxicity [51]. Wang et al. [41] prepared and analyzed the properties of a series of dendritic cationic gene vectors composed by amylopectin backbones and poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) side chains with different lengths. Furthermore, the resultant amylopectingraft-PDMAEMA vectors were functionalized with gold nanoparticles (Au NPs) aiming applications in theragnostic. The dendritic structure is interesting because it provides a high density of reactive spots that can be manipulated by CRPs, such as ATRP. For the synthesis of the polymeric gene vectors, the first step was the activation of the amylose hydroxyl groups to produce the initiators for subsequent ATRP. In sequence, PDMAEMA were polymerized with different lengths by varying the time of ATRP. The produced polymers condensed the pDNA in small particles successfully. The particle size depended on the N/P ratio, however, at N/P of 20 and above, all the dendritic cationic polymers compacted the pDNA into stable nanoparticles of 100 to 150 nm of diameter. All the dendritic vectors exhibit lower toxicity than PDMAEMA at the same N/ P ratio. At N/P ratio of 10, PEI has more transfection efficiency, but at ratios from 15 to 30, all the amylopectin-PMAEMA dendritic vectors had equivalent or higher transfection efficiency than PEI (25 kDa). The

vectors designed have the potential to be used as a multifunctional vector with the ability of bioimaging [69]. Yan et al. [50] reported the preparation of hybrid gene vectors composed by gold nanorods (Au NRs) grafted with PDMAEMA aiming their application as contrast agents for computed tomography and gene delivery. The ATRP initiator was immobilized onto Au NR surface. After that, a traditional ATRP was conducted to obtain the nanohybrids structures with different PDMAEMA chain size on the Au NR surface. The length of the PDMAEMA brush was controlled by the amount of monomer used and reaction time. Four different gene vectors were analyzed. All four nanohybrids were able to condense pDNA into nanoparticles. The particle size of all nanoplexes decreased with the increase in the N/P ratio. Besides, the particle size also decreased with the rise of the PDMAEMA length chain. These both results are a consequence of the higher electrostatic interaction provided by bigger PDMAEMA chain size and higher N/P ratio. Following the expected tendency, the cytotoxicity increased in vectors with larger PDMAEMA chains. Even so, all the gene carriers were less toxic than PEI (25 kDa) and PDMEMA at all N/P ratios. At N/P ratio of 10 and above, most of the Au-gPDMAEMA exhibited better transfection capability when compared with PEI (25 kDa) and PDMEMA. Moreover, the vectors presented a great performance as a contrast agent for computed tomography. Lastly, the scientists concluded that Au-g-PD nanohybrids at N/P ratios of 10 and 15 are recommended to be gene carriers for their great performance [50]. 3.3. Biohybrids gene vectors Biohybrids materials are those that contain in their structure a biopolymer, a polymer produced by living organisms, and a synthetic polymer. In general, the biopolymer is a protein or polysaccharide, and its main characteristic is biocompatibility and non-immunogenicity [5,7]. Protein-based biopolymers are highly stable, biodegradable carriers with unique abilities such as gelation, emulsify and bind to water. Fibronectin, collagen, and elastin are some of the most commonly used protein-based biopolymers for the production of gene vectors [5,70]. Similar to protein-based vectors, polysaccharide-based biopolymers also exhibit high degrees of biocompatibility and biodegradability, and are highly susceptible to chemical modification for specific purposes, owing to their various functional groups including hydroxyl, carboxyl, and amino groups. Currently, chitosan is the most studied polysaccharide to compose gene vectors. It is a linear binary hetero-polysaccharide derived from the alkaline de-N-acetylation of chitin. In addition, it is the only cationic natural polymer and this polyelectrolyte nature allows strong electrostatic interactions with anionic nucleic acids [7,71]. Hu et al. [55] suggested the use of spindly cellulose nanocrystals (CNCs) branched with PPEGEEMA and PDMAEMA and functionalized with gold for theranostic applications. The authors used two controllable polymerization techniques to graft the CNCs superficies, RAFT and ATRP. First, the PPEGEEMA brushes were polymerized via RAFT, and in sequence, PDMAEMA was produced by traditional ATRP. Gold nanoparticles were conjugated into PDMAEMA chain to be the contrast agent for computed tomography (Fig. 8). ATRP time determined the PDMAEMA chain size, being produced three vectors with distinct PDMAEMA length chain. The performance of the gene carriers was compared with the non-PEGylated counterparts and PDMAEMA homopolymer. All polymeric gene vectors designed could bind pDNA into nanoparticles with the size of 200–250 nm at N/P ratio of 15. As expected, the PEGylated vectors showed a lower capacity to condense the DNA because of the PPEGEEMA that decreases the positive copolymer charge. The tertiary-amine groups of PDMAEMA are the sites to provide positive charge and also to fix Au NPs. Then, the presence of the Au NPs 10

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Fig. 8. Schematic diagram for the synthesis of heterogeneous polymer brush-coated cellulose nanocrystals and resultant pDNA delivery processes [55]. Republished with permission from Royal Society of Chemistry. Copyright 2016.

desirable characteristics, scientists have been used several strategies. Some of them are described below.

transfection efficiency increased with the rise of the length of the PDMAEMA chains. The presence of gold nanoparticles did not have a significant influence on any of the parameters evaluated. In the other hand, the results showed that the Au NPs functionalized nanoplexes had potential application as a contrast agent for computerized tomography of cancer cells in vivo during the process of gene therapy without causing any damage to the patient [41].

4.1. Stimuli-responsive gene vectors Stimuli-responsive polymeric vectors represent an important alternative to optimize the delivery of nucleic acids and drugs. In this kind of system, the vectors are active; they react to changes in the environment undergoing chemical or physical transformations that result in the protection or in the release of the material to be delivered [10,76]. Several types of molecules, with different compositions and architectures, can be produced and manipulated to generate polymeric vectors with the desired stimuli-responsive property [74,77,78]. Oftentimes the diseased cells produce biological signals that are used as the stimulus source for stimuli-responsive polymeric gene vectors. In this case, the stimuli are internal and it can be pH conditions, redox potentials, enzymatic activations, and thermal gradients [79,80]. However, recently scientists developed polymeric vectors that can respond to artificial triggers, such as ultrasound, light, magnetic fields, and electrical fields [77,81,82]. In many cases, the scientists use as strategy the combination of stimuli as triggers and also multiple responses at the same time. The polyplex response can be a change in conformation, solubility, modification in hydrophilic/hydrophobic characteristic or release of the gene or/and drug. According to Ganta et al., stimuli responsive carriers are especially important when the

4. Strategies to improve polymer gene vectors performance Gene delivery is a multifactorial issue and during the process of delivery, numerous extracellular and intracellular barriers must be faced for an optimum gene transfection, mainly in vivo [72]. The biological barriers are numerous and they vary from their site of application to their target, including those that depends on the method of administration, systematic barriers in blood circulation, and cellular barriers. In blood, the polymeric gene vectors will face enzymatic degradation, opsonization, and undesirable interactions with blood components. The carriers, that are usually are unstable colloids in physiological conditions, must remain stable, with no protein absorption and aggregation. Besides, they must transfect the specific cell type rather than all others present. Finally, the gene vectors must overcome cellular such as cell entrance, endosomal escape and nuclear uptake [47,72–75]. All these requirements should be combined to low cytotoxicity. Aiming to produce an optimum gene vector that has all the 11

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Fig. 9. Synthetic processes of polyhedral oligomeric silsesquioxane based gene vectors via ATRP [38]. Reprinted with permission from American Chemical Society. Copyright 2014.

generation of energy by the cells occurs mainly through glycolysis rather than oxidative phosphorylation, which leads to the accumulation of lactic acid into the cellular environment. Generally, there are two approaches for pH-responsive systems: polymers that have ionizable groups and polymers that have acid-labile linkages. Polymers that contain ionizable functional groups can be protonated or deprotonated depending on the pH medium in which they are inserted. Weak bases and acids such as carboxylic acids, phosphoric acid, and amines, respectively, exhibit the ability to change the ionization depending on the pH [10,78,81,84]. Cationic polymers, such as PEI and PDMAEMA, remains with some free tertiary amines even after complexation. Into the endosome and lysosome, they became protonated and buffer the acidic pH making the complex swell through the increase of osmotic pressure, which is known as proton sponge effect. Cationic polymers that induce the proton sponge effect, may ease endosomal membrane rupture, improve the DNA from the endosome to the cytoplasm, and enhance the gene transfection efficiency [60,85–87]. However, the discussion about the real contribution of this phenomenon to the great performance of some cationic gene is still ongoing. The copolymerization of monomers with distinct hydrophobicity and hydrophilicity features originate amphiphilic complexes capable of self-assemble themselves into micelles according to the medium pH. This ability can improve polyplex serum stability and adsorptive endocytosis [88,89]. This kind of system has been widely investigated for the co-delivery of drugs and genes and it has been presenting good results [42,54,56,57].

stimulus which the vector is sensitive are unique to a pathology [77]. Undeniably, the stimuli response technologies that have been more widely used in ATRP based materials for gene therapy are pH and redox responsiveness. Surely, the other approaches are very promising alternatives to improvements in gene therapy. However, for ATRP-based polymers these technologies have not been applied yet and therefore will not be addressed in this work. Redox-responsive polymers are designed taking into account the difference in glutathione (GSH) concentration in the oxidative extracellular medium (2–20 μM) and reducing intracellular environment (1–10 mM). Besides this normal variation, in tumor cells due to its proliferation and metabolism, the amount of GSH is about 10–7 times higher than in normal cells. GSH is a linear tripeptide containing a thiol group that has as the main function the defense of the body against the increase of free radicals. Polymeric gene vectors that have disulfide linkage (SeS) can maintain itself stable in blood and extracellular environment, but they degrade in the intracellular environment because of the interaction of SeS bond and thiol group of GSH. The strategy to introduce SeS bonds in gene vectors may not only facilitate the gene/ drug release but in some cases may also decrease the cytotoxicity of the carrier. Some copolymers are constructed to be degraded into the cytoplasm by GSH action into smaller parts less aggressive to the cells [10,77,81,83]. Different organs, tissues, organelles, and other cellular components may have great differences in pH, which makes this parameter suitable stimulus. Cancer cells present an acid environment. Due to the fast reproduction of tumor cells, there is lack of oxygen supply then the 12

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caprolactone) (PCL) and PDMA (Fig. 10). The length of PCL was fixed in 80 units while the length of the PDMA was 55, 115, and 155 monomers units. ROP was used to prepare the ATRP initiator and PDMA polymerization was carried out by ATRP. These two kinds of polymers were chosen because of PDMA has relatively low toxicity and high buffer capacity in gene delivery and PCL is degradable and hydrophobic, being widely used in anticancer drug encapsulation. In a reducer condition the produced polymers, due to hydrophobic interaction, were able to self-assemble into micelles with size ranging from 70 to 200 nm and positive surface charges from +24 to +37 mV. In general, the PCL-SS-PDMA block copolymers exhibited high DNA condensation and cellular uptake efficiency. The tertiary nitrogen present in PDMA confers to polymer the ability to protonate and de-protonate according to the pH condition. Hence, the vector presents a high acid-resistance in the range of pH 5–7 which makes possible the escape of the polyplex from endosome before releasing the encapsulated gene or drug into the cytoplasm. The gene transfection efficiency of PCL-SSPDMA/p-DNA complex showed relatively low transfection efficiency in 293 T and HeLa cells, compared to PEI (25 kDa). However, the transfection efficacy was about ten times higher than PEI (25 kDa) transfection in human oral carcinoma cell lines (KB and CAL-27 cells) [42]. Zhang et al. [44] synthesized a series of brush-shaped and pH-sensitive gene vectors in a host-guest system. The delivery system had a supramolecular approach with two modules with specific functions. Basically, the host module was composed by a brush shaped and pHsensitive polymer that contained poly(β-cyclodextrin) (PCC) as the backbone and acetal bond-linked PGEA as the arms. It was expected that this host condensed the DNA into stable nanoplexes in the neutral extracellular environment and to release quickly the DNA in the intracellular acidic environment. The guest modules tested were adamantyl terminated polyethylene glycol (Ad-PEG-OH) and adamantlyfolate terminated PEG (Ad-PEG-FA). The guest modules should increase the gene delivery serum stability and cell-targeting capacity. The host module was produced using a combination of ATRP and ROP. First, the PCD macroinitiator for ATRP with pH-sensitive acetal bonds was prepared. In sequence, through traditional ATRP technique, it was produced PCD-acetal-PGMA brush-shaped polymers composed by PCD backbone and acetal-linked PGMA arms. Finally, PCD-acetal-PGEAs were prepared by ROP of the PCD-acetal-PGMAs with excess of

The insertion of acid-labile linkages into the polymer structure is another approach to produce pH-responsive polymers. Vectors with this characteristic are stable at physiological pH but at acidic pH they have its bonds cleaved. Thus, it is possible to enhance the serum stability of the polyplex and facilitate the nucleic acid release. Commonly, the acidlabile linkages more used are acetal, imine, hydrazone orthoester, and ketal acid bonds. ATRP-based materials with these characteristics mostly use acetal bonds. A star-shaped biodegradable gene carrier was prepared via traditional ATRP from a biocompatible core polyhedral oligomeric silsesquioxane (POSS) and DMAEMA. The molecule of POSS has a threedimensional structure of a silica cube with eight organic sites that were functionalized and then synthetized a macro-ATRP agent (POSS-(SSBr)8) with eight disulfide-linked initiation spots. Thenceforth, the PDMAEMA arms, with different molecular weights, were polymerized (POSS-(SS-PDMAEMA)8s), as can be seen in Fig. 9. Controlling the reaction time, it was possible to obtain vectors with arms of different PDMAEMA length. It was investigated the relation among the size of the arms and the cytotoxicity, transfection efficiency and DNA condensation capability. The reaction was stopped in 20, 30, and 40 min, when was obtained arms with 15, 20, and 24 monomer repeat units per arm respectively [38]. All the vectors tested presented an excellent DNA condensation capability. The nanoplexes obtained was stable at 100–150 nm, which favor their cellular uptake. Regarding to the cytotoxicity, it was noticed that all POSS-(SS-PDMAEMA)8s, and also the control POSS(PDMAEMA)8s, that did not contain the disulfide linkage being nondegradable, showed much lower cytotoxicity than PEI (25 kDa). As expected, the cytotoxicity increased with the rise in arm length. The controlled vector exhibited arm length similar to the bigger arms POSS(SS-PDMAEMA)8s however its cytotoxicity was higher, proving that the inclusion of the disulfide bonds was effective in the degradability and hence in the toxicity reduction of the gene vector. Among the all the gene vectors synthetized, the one with intermediate length size, 20 monomer repeat units per arm displayed the best transfection efficiency at the N/P ratio of 15 [38]. Aiming the codelivery of pDNA and an anticancer drug, doxorubicin (DOX), Li et al. [42] prepared, by a combination of ROP and ATRP, diblock copolymers linked by disulfide-bonds from poly(3-

Fig. 10. Synthetic route of PCL-SS-PDMA diblock copolymer [42]. Republished with permission from Royal Society of Chemistry. Copyright 2014. 13

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that ligand-assisted active targeting or size-dependent localization strategies can be used. Assays in vivo exhibited good results with great inhibition of tumor growth by modulation of the gene expression and no significant toxicity. Wei et al. [47] proposed the development of an innovative welldefined polymeric gene carrier that at the same time addressed three problems faced by gene vectors in an in vivo environment: specific cell target, stability in physiological conditions, and efficient intracellular delivery. According to the authors, the key to this is the reducible double-head agent composed by both a reversible addition−fragmentation chain transfer (RAFT) agent and an atom transfer radical polymerization (ATRP) initiator connected by a disulfide bond. Thought this double-head agent it is possible to design block copolymers based on diverse hydrophilic and hydrophobic monomers with cleavable links in the block junctions for several applications. It was the first research that combines controlled living radical polymerization techniques, ATRP and RAFT, to produce a degradable block copolymer. Usually, these kinds of polymerizations produce copolymers that have their blocks linked by non-degradable CeC bonds. Other works that produce degradable polymeric vectors, most of the times, use the combination of ATRP or RAFT and ROP, but this approach is limited to cycle monomers. The final structure produced contained PGMA backbone with pendant oligoamine side chains, that is linked by a disulfide bond to a P(OEMA) that contains a Tet 1 targeting peptide conjugated, being called Tet1-P(OEGMA)15-SS-P(GMA-TEPA)50. Briefly, double-head agent, CPADB-SS-iBuBr, was synthesized by N,N′ dicyclohexylcarbodiimide coupling between the RAFT chain transfer agent (CTA) 4 cyanopentanoic acid dithiobenzoate (CPADB)7 and 2-hydroxyethyl-2′ (bromoisobutyryl)ethyl disulfide initiator (OHSS-iBuBr)8 with 4- (dimethylamino)pyridine as a catalyst. In adittion, CPADB-SS-iBuBr was used as a RAFT CTA to polymerize both OEGMA and HPMA. The hydrophilic OEGMA and HPMA blocks then acted as macroinitiators for ATRP of GMA. To avoid the possibility of competition between RAFT-ATRP, GMA was polymerized with short reaction time because its polymerization has quicker kinetics in ATRP than in RAFT. The P(GMA)’s epoxy groups were functionalized with TEPA by Michael addition. Finally, Tet1 was conjugated by Michael addition to the terminal free thiols that were produced during TEPA functionalization. It was expected that in the complex Tet1-P(OEGMA)15-SS-P (GMA-TEPA)50/DNA., PGMA-TEPA acted as the electrostatic core; P (OEGMA) should act as the shell protecting the complex from serum interaction; TEPA should facilitate the endosomal escape because it has protonated amines in its structure that enhance the proton sponge effect; and the degradable SeS linkage should be broken by the action of the glutathiones (GSH) present in cytosol, leading to detachment of hydrophobic P(OEGMA) shell and release of DNA [47]. Not only the final polymeric vector with all components, Tet1-P (OEGMA)15-SS-P(GMA-TEPA)50, was evaluated, but also all the intermediates: nonreducible, nontargeted complexes; nontargeted, reducible complexes; nonreducible targeted complexes. All polymeric vectors were able to compact the DNA in small particles stable in a saline solution. The transfection efficiency was evaluated in four cell types: PC12, HeLa, HEK293 T, HepG2. PC-12 has a neuron-like phenotype that includes increased binding of the Tet1 peptide. The results showed that the presence of the Tet1 in the vector improved the transfection in PC12 when compared with the vector without Tet1. Among all the copolymers tested, polyplexes that contained both targeting ligand and releasable shielding coronas reached transfection efficiency equivalent to their homopolycations. Tet1-P(OEGMA)15-SS-P(GMA-TEPA)50 exhibiting the highest transfection. The gene vectors' cytotoxicity was not evaluated [47].

ethanolamine. Vectors with these compositions and morphology, varying only the brush lengths, were analyzed. Besides, their (Ad-PEGOH) and (Ad-PEG-FA) conjugated counter partners were also analyzed. The results showed that PCD-acetal-PGEA polymer was able to condense the pDNA into stable nanosized particles in the neutral extracellular environment and to liberate the DNA in the acidic cellular condition. The conjugation of the two guest modules provided better serum stability and cell-targeting capability. The gene vector produced showed lower cytotoxicity and higher transfection efficiency than PEI (25 kDa). The FA-target polyplexes presented the greatest results for cellular uptake, pDNA transfection and siRNA silencing efficiency in folate-receptor positive cells [44]. Hao et al. [56] investigated the production and performance for drug and gene codelivery of a new acid-responsible and fluorescent cationic block copolymer composed by of poly(3-caprolactone) (PCL), poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) and poly [poly(ethylene glycol)methyl ether methacrylate] (PPEGMA). A fluorescent coumarin derivative (CE) was also part of the vector structure, and its presence aimed to provide a fluorescence detection. The final vector was CE-PCL-a-(PDMAEMA-co-PPEGMA). The functionalized copolymer was synthetized by a combination of ROP, ATRP, and CuAAC “click” reaction. Briefly, ROP was used to produce the CE-PCL part. The acetal and azide-containing homopolymer, CE-PCL-a-N3, was obtained by the reaction of 2-chloroethyl vinyl ether, followed by the azidation reaction with NaN3. In an independent reaction, copolymer alkynylPDMAEMA-co-PPEGMA was synthetized by the ATRP of DMAEMA and PEGMA monomers using propargyl 2-bromoisobutyrate as the initiator. Through a CuAAC “click” reaction, alkynyl-PDMAEMA-co-PPEGMA and CE-PCL-a-N3 were linked by the acetal bond, generating the final polymeric vector CE-PCL-a-(PDMAEMA-co-PPEGMA). The polymeric vector could self-assemble into stable micelles to encapsulate hydrophobic DOX in the core, and condense pDNA in the positively charged PDMAEMA. The acetal linkage was effectively cleaved in the acid intracellular environment, resulting in the disruption of the micelle structure and the release of DOX and DNA. The cytotoxicity of the micelles increased with rising concentrations of the copolymer due to the higher concentration of free positively charged amino groups in PDMAEMA. This parameter was not compared to other polymeric vectors for co-delivery. The micelles maintained the drug activity to effectively inhibit the growth of cancer. At N/P ratio of 15, it was possible to internalize the complexes and express the gene in HeLa cells. The fluorescence of the complex was investigated under irradiation of 365 nm UV light, and a considerable blue fluorescence could be observed. From three consecutive SI-ATRPs in Au nanoparticles, Kim et al. [59] produced a multi-shelled cationic corona of poly(2-(dimethylamino) ethyl methacrylate-2-hydroxyethyl methacrylate) (DAMAHEMA) for the delivery of siRNA in A549 tumor cells. The Au nanoparticles were initiated with a disulfide, and then the polymeric layers were linked by disulfide bonds that could be cleaved in reducing conditions of cytoplasm, releasing the genetic material. The amount of siRNA loaded in the vector could be controlled by varying the number of shells. The system containing more layers exhibited more interaction with siRNA and hence lower release and more protection from nucleases. The increase in the number of layers leads to the rise in the capacity to load siRNA, and the size of the nanoparticles also increased. However, when the siRNA was complexed into the structure the size of the nanoparticles did not show significant change, staying in about 250 nm. Therefore, the gene vector created could load a great amount of siRNA without compromise size and colloidal stability. The high surface charge and the dendritic architecture of the three DAMA-HEMA layers enhanced, at the same time, the interaction with the membrane cell and cellular uptake. The gene therapy systems were tested in relation to the bioaccumulation in liver, lungs, heart, spleen, and kidneys. The results showed that the nanoparticles presented a significant accumulation in the liver. To overcome this issue the authors suggested

4.2. Use of pegylated polymeric segments The introduction of molecules characterized by their hydrophilic nature such as poly(ethylene glycol) (PEG), hyaluronic acid (HA), and 14

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the gene vector has a better transfection efficiency in HepG2 than in HeLa due to the interaction between Gal and asialoglycoprotein receptors on the surface of HepG2 cells. Finally, the authors concluded that the gene delivery system has great potential to be used in hepatoma-targeting gene delivery [46,92]. Scientists designed a transfection agent to delivery in the central nervous system cells directly a protein - glial derived neurotrophic factor (GDNF) - aiming improvements in therapeutic intervention for neurological diseases such as Parkinson's disease. The research group applied DE-ATRP, via a simple one-pot reaction, to produce a vector with knot structure made from EGDMA, DMAEMA, and PEGMEMA. The one containing 8% PEG with a molecular weight of 30 kDa showed a generally higher transfection efficiency profile with lower cytotoxicity than the others and because of it was used in this study. The transfection capability and cell viability analysis were analyzed in five central nervous system cells: Neu 7, Astrocytes, B104, SHSY-5Y, and PC12. In summary, PD-E 8% PEG showed a comparable transfection efficiency to PEI (25 kDa), but with evidently lower cytotoxicity, which gives to this polymeric vector a great potential for neuronal gene therapy applications [37].

zwitterions like carboxybetaine (PCB) and poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) has been widely used as strategy to enhance performance of the polymeric gene vector because it avoid undesirable non-specific interactions with blood or cellular components [1,10,66,89]. The presence of hydrophilic groups on the cationic polyplexes surfaces has been shown to reduce their interactions with anionic proteins like glucosaminoglycans (GAGs) and serum proteins, which diminishes polyplex unpackaging and/or rapid elimination by the immune system [1,10,66]. Hence, there are an increase in the stability of the polyplexes in physiological environment and a decrease of their immunogenicity. PEG is most explored “stealth” polymer in gene therapy due to its well-known safety uses in humans and its classification as Generally Regarded as Safe (GRAS) by the U.S. Food and Drug Administration Agency (FDA) [66]. However, the PEGylation of polymeric vectors also presents drawbacks because it strongly inhibits cellular uptake and endosomal escape, which negatively affects the performance of the delivery system [90]. In addition, there is a limit for the amount PEG that can be used for that the clinical application would not be compromised. These factors stimulate investigations using other hydrophilic groups into the polymeric structure and manipulations on the PEGylated vectors such as in their length, density or linking PEG by degradable bonds [1,10,42,66,90,91]. ATRP technique is an important tool that allows the synthesis of cationic polymers containing hydrophilic groups of varying chain lengths, architectures and molecular weights helping to identify an optimum design for the gene delivery and expression. Nehate et al. [45] aiming to improve the efficiency of chemotherapy developed a redox-sensitive copolymer from ATRP. The gene vector structure was diblock copolymer composed by poly[poly(ethylene glycol)methacrylate]-s-spolycaprolactone ((PEI-PEGMA)n-ss-PCL-PEI). The multiple PEG chains in polymer protect the nanoparticles, meanwhile, short PEI chains provided positive charge over their surface for siRNA complexation. For the synthesis, the first step was the production of the macroinitiator, Br-ss-PCL-OH by ROP. In sequence [(PEGMA)nss-PCL-OH] diblock copolymer was producing through ATRP using the macroinitiator previously produced. In a N2 environment, the CuBr/ PMDETA catalytic system lead to the ATRP of PEGMA, generating an amphiphilic diblock copolymer with a controlled hydrophobic and hydrophilic ratio in the polymer chain. The system delivered simultaneously doxorubicin, an anti-cancer drug, and polo like kinase I siRNA (plk1 siRNA), as silencing agent for the endogenous enzyme plk1 that is overexpressed in tumor cells, into MDA-MB-231 and HeLa cells. The co-delivery system was evaluated regarding the hemolysis, protein adsorption, coagulation, transfection efficiency, and MTT cytotoxicity assay. The results showed that the codelivery polyplex was stable at physiological conditions, biocompatible and hemocompatible in vitro and in vivo, even after consecutive doses administration. Besides, it was able to self-assembling in spherical nanoparticles, ensuring the load of the drug and siRNA complexation. The results in vitro presented 50–70% knockdown of plk1 mRNA expression and in vivo assays exhibited the reduction of about 29% in tumor growth in relation to the control group [45]. Hao et al. [46] elaborated a series of galactosamine (Gal)-conjugated brush-type cationic copolymer P(PEGMEMA-co-PEGMA-Gal)-bPDMAEMA prepared by a combination of ATRP in two-steps and polymer reaction. In the first moment, ATRP initiator was synthesized through a random polymerization of ether methacrylate (PEGMEMA) and poly(ethylene glycol) methacrylate (PEGMA). Then, it was produced by ATRP the P(PEGMEMA-co-PEGMA)-b-PDMAEMA by the polymerization of the monomer 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA). Lastly, the pendant hydroxyl groups were functionalized with galactosamine using N,N′ carbonyldiimidazole. As expect the DNA condensation capability of the gene carriers was dependent on the N/P ratio. The analysis of in vitro cytotoxicity demonstrated that the developed non-viral gene delivery system has lower cytotoxicity than PEI (25 kDa). Besides, transfection capability evaluation showed that

4.3. Specific targeted gene vectors Non-viral gene delivery systems did not present selectivity of cell surface binding and internalization [93,94]. Owing to address this limitation and improve the cellular uptake, several ligands targeting specific cells or tissues have been conjugated to the polymeric gene carriers. The specific ligand will be recognized by the appropriate receptor on the diseased cell surface and this set will intermediate and facilitate the endocytosis In addition to these improvements, targeting systems may minimize potential adverse effects at other healthy cells and lead to greater therapy results [89,94,95]. The targeting ligands can be proteins (antibodies), nucleic acids, peptides, vitamins, and carbohydrates, which generally bind to the receptor uniquely overexpressed on the plasma membrane of diseased cells [95–97]. One of the major challenges in using this kind of delivery system is to find highly specific ligands that are non-immunogenic. The most widely studied targets are transferrin, folate, epidermal growth factor receptors (EGFRs), and glycoproteins [89,98,99]. Wang et al. [39] proposed a star-shaped pDNA delivery system composed by a POSS core and PDMAEMA-PPEGMA arms functionalized, by click chemistry reaction, with two different kinds of peptides, CAGW, and CAG-TAT-NLS. First, the sites of POSS were initiated with eight-tertiary CeBr, and PDMAEMA-PPEGMA were polymerized through two consecutive ATRP. The authors evaluated how the presence of PPEGMA and of each peptide influenced the gene vector performance. Then, the researchers produced four different gene carriers to deliver pDNA into endothelial cells: POSS-PDMAEMA, POSSPDMAEMA-PPEGMA, POSS-PDMAEMA-PPEGMA-CAGW, and POSSPDMAEMA-PPEGMA-CAG-TAT-NLS. All the structures could form stable micelles in aqueous solution and efficiently compact the pDNA. The gene vector with multifunctional peptides exhibited higher cellular uptake, cell viability, nuclear internalization and transfection efficiency than PEI 25 kDa. Besides, they could promote migration and angiogenesis, being a potential gene delivery system for endothelialization [39]. Feng et al. [43] designed a multifunctional redox-sensitive micelle for the co-delivery of DNA and doxorubicin (DOX) for the treatment of breast cancer. The penta-block copolymer vector, PEG-PEI-PCL-SS-PCLPEI-PEG, was produced in four steps: ROP, acrylate termination, Michel Addition, and ATRP. After that, aiming to improve the cellular uptake and specificity of the vector, the copolymer was conjugated with a T7 that has an affinity with the transferrin receptor - PEG-PEI-PCL-SS-PCLPEI-PEG-T7 (PPPT). This strategy was used because many tumor cells show a high expression of transferrin receptors. A deep investigation about the performance, in vivo and in vitro, of the gene vectors, modified 15

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generating new variations that reduces the copper concentration to the ppm range, such as SARA ATRP, eATRP, ARGET ATRP, ICAR ATRP or more recently the use of organic catalyst instead of copper, O-ATRP [22,24,101,104,105]. Surely, these technologies represent an important path to design and produce better gene vectors. However, to the best of our knowledge, they have not been applied for this purpose yet, which should happen in the near future. Moreover, alternative transition metals, such as iron [106–108], ruthenium and others [25], have been employed to mediate ATRP. Among them, Fe was considered advantageous due to its low cost, wide availability, low toxicity and biocompatibility [107]. In this context, Femediated ATRP when used in aqueous media presents an interesting and powerful alternative for synthesizing well-defined functional materials [109], especially for gene therapy applications. However, studies on Fe-mediated ATRP in aqueous media are still scarce due to the high equilibrium constant values, low catalytic activity, Fe catalyst selectivity, and partial dissociation of the halide ion from the deactivator [108]. Usually, the metal catalyst removal is carried out in difficult and time-consuming procedures. Among the various procedures for recovering transition metals, one may highlight washing or extraction with aqueous precipitants (for example Na2S and NaOH) [110], repeated precipitation in weak solvents that can dissolve the metal complexes [111], passing through the catalyst-containing polymer solution through alumina and silica gel columns [25]. Acid-group ion exchange resins successfully enable the recovery and recycling of transition metals [112]. In addition, numerous industrial-grade absorbents such as activated carbon and various types of clay (mainly kaolinite, montmorillonite, acid clay, and bentonite) have been successfully used to drastically reduce the residual metal concentration [113–115]. Nevertheless, it is noted that small amounts (ppm) of metals may remain in the polymer, which may make their application in gene therapy unfeasible.

or not with T7, was carried out. At a polymer weight ratio/DNA above 15, the produced gene carriers could compact the pDNA into stable nanoparticles with a size lower than 150 nm. The critical micellar concentration value found was very low, which could guarantee that the nanoparticles could maintain their form when they were in vivo diluted conditions, as in the bloodstream. The disulfide bonds of PPPT vector were disassembled in the presence of GSH in cancer cells environment, which lead to a rapid DNA and DOX release from PPPT/DOX/DNA micelles. PPPT/DOX/DNA presented a much higher cytotoxicity than PPPT/DNA and PPPT/DOX in MCF-7 cells. This result may confirm that the redox-sensitive codelivery of DOX and DNA micelles was much more effective when compared with drug or DNA delivery singly. The synergistic effects of DNA and DOX delivery on cell apoptosis enhanced chemotherapeutic efficacy in about 50%. In the absence and in the presence of blood proteins at low polymer/DNA ratios, the transfection efficiency of PEI (25 kDa) was better than that achieved by PPPT. At polymer/DNA ratio of 10 and above, PPPT presented transfection results that exceeded those achieved for PEI. In vivo assay demonstrated that the co-delivery of pDNA and DOX using PPPT vector reduced tumor growth and cellular apoptosis. Moreover, the mice did not show symptoms that demonstrated some health damage such as weight loss and anomalies in the organs. It is attributed to their targeting ability and redox responsiveness, which prevented drug release during circulation in the blood and allowed cancer-specific targeting accumulation [43]. Zhang et al. [53] developed a responsive gene vector to two kinds of stimulus, reduction, thought the disulfide group, (−ss-) and pH, thought the acetal group (−a-). Besides, the triblock terpolymer was also functionalized with galactosamine (Gal) for the targeted co-delivery of anticancer drugs, DOX, and DNA. The synthesis of the innovative gene carrier was made by a combination of ROP, ATRP, a Cu (I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” reaction, and a polymer reaction. Briefly, the role of ATRP was to polymerize the PDMAEMA block. The final gene carrier produced was a poly(ethylethylene phosphate)-a-poly(3-caprolactone)-ss-poly[2-(dimethylamino)ethyl methacrylate] (Gal-PEEP-a-PCL-ss-PDMAEMA). The results showed that the system was able to encapsulate, simultaneously, the DNA and the hydrophobic DOX. Besides, the acetal and disulfide linkages present in the gene vectors were really disrupted in the intracellular environment. Probably, under acid conditions of endosomes, the acetal bonds were degraded. After living the endosomes, the polyplexes had the disulfide bonds cleaved due to the high concentration of GSH, which resulted in the complete release of the DOX and pDNA into the cytosol. At pH 5.0 with 10 mM GSH, the release and the liberation rate of DOX showed an enhancement. The cellular uptake and also the transfection were more effective in HepG2 cells than in HeLa cells because the presence of the Gal moieties that could be recognized by asialoglycoprotein receptor (ASGPR) overexpressing HepG2 cells. Regarding the cytotoxicity, DOX-loaded micelles could inhibit the growth of both cell types, however, it was more effective in HepG2 cells than HeLa cells [53].

6. Conclusion Gene therapy is a very promising alternative to modern medicine, representing the hope of cure for chronic diseases that are still considered incurable. All the efforts are in improve the delivery efficiency, specificity and safety for the patient. Seeking these goals, the scientists have been used several synthesis techniques, often combined, and diverse strategies such as architecture diversification, addition of specific targeting units, shielding domains, and endosomolytic units. These strategies may help to overcome the biological barriers. However, they make the gene carriers increasingly sophisticated, being essential methodologies to produce well-defined reproducible polymers. Controlled polymerization techniques have led to great advances in the development of polymeric gene vectors. Specially ATRP is a very robust and versatile technique that enables the polymeric vectors design with different characteristics and functionalities that are of interest for specific applications. The knowledge acquired in the last decades about the reaction mechanism allowed the creation of the new techniques that face the problem of metal presence in ATRP-based polymers, reducing the amount of metal catalyst used or novel metal-free ATRP systems. These techniques, especially metal-free alternatives, present greater potential to be applied in the synthesis of gene vectors with well-defined properties, without the need to conduct costly purification procedures of synthesized material.

5. Concerns about the toxicity of ARTP-produced gene vectors Despite the great performance of ATRP to design gene vectors due to its robustness, monomer and architecture versatility, and great cost benefit, the necessity of a transition metal catalyst, most of times copper, is a concern especially for the preparation of biomaterials [24,100,101]. Although the effects of metal traces remain in discussion, their presence at least limits the extensive use of ATRP for biomedical applications. Motivated by the increase in of copper nanoparticles (Cu NP) in medicine and industry some authors evaluated the cytotoxicity of Cu NP [102], and copper oxides nanoparticles [103]. Both studies concluded that this species could greatly affect cytotoxicity. The negative effect caused by the presence of the metal onto the ATRP based structures lead to the improvements in ATRP technique,

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 16

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Acknowledgements

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