Poly(ethyleneimine) functionalized carbon nanotubes as efficient nano-vector for transfecting mesenchymal stem cells

Accepted Manuscript Title: Poly(ethyleneimine) functionalized carbon nanotubes as efficient nano-vector for transfecting mesenchymal stem cells Author: Hanieh Moradian Hamidreza Fasehee Hamid Keshvari Shahab Faghihi PII: DOI: Reference:

S0927-7765(14)00349-X http://dx.doi.org/doi:10.1016/j.colsurfb.2014.06.056 COLSUB 6502

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

2-3-2014 1-6-2014 24-6-2014

Please cite this article as: H. Moradian, H. Fasehee, H. Keshvari, S.F. [email protected] Poly(ethyleneimine) functionalized carbon nanotubes as efficient nano-vector for transfecting mesenchymal stem cells, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.06.056 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.

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Poly(ethyleneimine) functionalized carbon nanotubes as efficient nano-vector for transfecting mesenchymal stem cells

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Hanieh Moradiana,b*, Hamidreza Faseheea*, Hamid Keshvarib, Shahab Faghihia,**

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Faculty of Biomedical Engineering, Amirkabir University of Technology, Tehran 15875/4413, Iran

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Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 14965/161, Iran

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* Equal contributions **Corresponding author: Shahab Faghihi, Tissue Engineering and Biomaterials Division, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 14155-6343 Iran E-mail: [email protected]; [email protected] Phone: +98 21 44580461, Fax: +98 21 44580386 1    Page 1 of 38

Abstract

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For gene and drug delivery applications, carbon nanotubes (CNTs) have to be functionalized in

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order to become compatible with aqueous media and bind with genetic materials. In this study,

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combination of polyethyleneimine (PEI) grafted multi-walled carbon nanotubes (PEI-g-MWCNTs)

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and chitosan substrate is used as an efficient gene delivery system for transfection of hard-to-

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transfect bone marrow mesenchymal stem cells (BMSCs) with enhanced green fluorescent protein

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(EGFP) gene.  Fourier transform infrared (FT-IR) spectra, dynamic light scattering (DLS) analysis

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and zeta potential measurements are used to characterize binding of PEI, particle size distribution

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and colloidal stability of the functionalized CNTs, respectively. DNA binding affinity, cellular

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uptake, transfection efficiency and possible cytotoxicity are also tested by agarose gel

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electrophoresis, flow cytometry, cytochemisty and MTT assay. The results demonstrate that

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cytotoxic effect of PEI-g-MWCNTs is negligible under optimal transfection condition. In

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consistency with high cellular uptake (> 82%), PEI-g-MWCNTs give higher delivery of EGFP

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into the BMSCs which results in a more sustained expression of the model gene (EGFP) in short-

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term culture. These results suggest that PEI-g-MWCNTs in corporation with chitosan substrates

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would be a promising delivery system for BMSCs, a cell type with relevancy in the regenerative

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medicine and clinical applications.

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Keywords: Mesenchymal stem cells; Multi-walled carbon nanotubes; Gene delivery system;

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Polyethyleneimine; Transfection efficiency.

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1. Introduction Gene therapy is the insertion of genes into an individual's cells and tissues in order to replace

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deficient genes, modulate gene expression or introduce new functions [1, 2]. Gene therapy is

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concerned with genetically inherited diseases while interfering with gene expression to treat the

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acquired diseases [3, 4]. A wide variety of viral systems has been extensively used as gene

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delivery vectors [5, 6]. Even thought viral vectors have proved to be very efficient in term of level

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of gene expression, their clinical applications are limited as viruses provoke immune responses [7,

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8]. Moreover, a long-term expression of the delivered gene which is obtained using viruses is not

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required as in most cases like regenerative medicine and tissue engineering, a transient signaling is

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normally needed [9, 10]. As an alternative strategy, a range of non-viral synthetic vectors has

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emerged based on lipoplex and polyplex forming carriers [11-13]. Despite the advantages of non-

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viral systems over their viral counterparts, some of them cause destabilization of the cell

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membrane and lead to pronounced cytotoxicity and mostly exhibit low gene delivery efficiency

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especially when dealing with primary cells [14]. Bone marrow-derived mesenchymal stem cells

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(BMSCs) are multipotent cells that have the ability to differentiate into multiple lineages,

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including the osteogenic [15-17], chondrogenic [18, 19], myogenic [20, 21] and adipogenic [22,

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23]. They have remarkable potential for related tissues construction and regeneration [24, 25].

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Furthermore, they are relatively easy to isolate, manipulate and culture while presenting significant

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expansion capability [24]. These characteristics make them very attractive candidates for in vitro

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gene delivery studies. However, MSCs have been shown to be very difficult to transfect by non-

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viral methods [26, 27]. Hoelters et al. transfected human MSC (hMSC) with three different

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liposome-based transfection reagents and a plasmid containing the sequence for enhanced green

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fluorescent protein (EGFP) [28]. On the contrary, Hamm et al. were unable to transfect human

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MSCs using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), Effectene™ or Lipofectamine

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Plus™[26]. In general, the literature reveals that transfection levels attained with liposome-based

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vectors in MSCs are low. Also, due to their capacity to interact with biological membranes, these

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carriers are often cytotoxic [29], which constitutes a limiting factor for application of liposomes in

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gene delivery. Ahn et al. reported transfection up to 10% when pDNA/PEI polyplexes were

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delivered into rat bone marrow derived MSCs [30]. A good transfection efficiency was achieved at

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an N/P ratio of 16, which was comparable with that obtained with Lipofectamine™. However, at

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this N/P ratio, polyplexes presented a high cytotoxicity. In another study, human adipose tissue-

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derived MSCs were employed to study the gene transfer properties of pDNA/PEI polyplexes [31].

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Several synthetic biodegradable polymers are being investigated for nucleic acid delivery, such as

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the polyesters polylactic acid (PLA) and poly (lactic-co-glycolic acid) (PLGA) [32-34]. Vectors of

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this kind have the advantage of being eliminated after pDNA release, in the form of nontoxic

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degradation products. Gwak et al. developed PLGA nanospheres as vehicles for gene delivery to

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human cord blood-derived MSCs [35]. Uchimura et al. combined colloidal gold nanoparticles to

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DNA/Jet-PEITM complexes in an attempt to enhance their uptake by human MSCs, having in

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mind their application in cell array-based analyses and in regenerative medicine [36]. A 2.5-fold

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increase in gene transfection was obtained, as compared to the control without gold nanoparticles.

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[37].

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Even though much effort has been made in developing nonviral gene vector systems, the

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traditional nonviral vectors generally cause destabilization of the cell membrane leading to a

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pronounced cytotoxicity in order to achieve effective delivery of DNA. Therefore, the lack of a

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widely accepted gene vector coupled with difficulty in transfection of primary cells is a significant

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hurdle in the advancement of gene delivery systems. Recent findings in nanoscience and

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nanotechnology have revealed that carbon nanotubes (CNTs) can be used as a versatile platform 4    Page 4 of 38

for a variety of biomedical applications, including gene delivery [38]. Owing to unique chemical,

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physical and biological characteristics such as high surface area, biocompatibility, low

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cytotoxicity, and ability to cross the cell membrane [39-41], carbon nanotubes (CNTs) have

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received considerable interest in the biomedical applications such as drug [42, 43] and gene

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delivery [44, 45], scaffolds for tissue engineering [46], biosensing and diagnostics [47]. Many

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studies have reported the intracellular transporting of biomolecules by CNTbased carrying

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materials [48-50]. The first work of utilization of carbon nanotubes as a novel gene delivery vector

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system was reported by Bianco et al [51]. It is shown that the uptake of pristine CNTs into

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mammalian cells is poor, which results in little transport efficiency. However, the succeeding

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works indicated that the transport efficiency can be improved by covalent or non-covalent surface

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modifications of the CNTs [45, 52-54]. Skandani et al. [55] simulated the interaction of single-

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walled CNTs (SWCNTs) with a lipid bilayer and observed that the lower chirality SWCNTs

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exhibit significant adhesion with the membrane. Raffa et al. [56] reported that the nanotube length

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can clearly influence the cellular uptake while the shorter multiwalled CNTs (MWCNTs) were

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easier to be internalized than the longer ones. Besides the transportation efficiency, the cellular

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toxicity of the CNT-based delivery vectors was another predominant characteristic that should be

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considered. During the past decade, many works have examined in vitro toxicity of CNTs [57].

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The CNTs exhibit some degree of cytotoxicity; however, the cytotoxicity is dependent on the route

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of preparation and their surface functionalization. Some studies demonstrated that exposure of

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mammalian cells (human epidermal keratinocytes [58], macrophages, human A549 lung cells lines

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[59], etc.) to pristine CNTs results in oxidative stress in cells as well as induction of cellular

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apoptosis and necrosis which causes depletion of total antioxidant reserves and loss of cell

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viability [60]. In contrast to pristine CNTs, the functionalized CNTs showed improved

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biocompatibility [61]. Different types of functionalized CNTs, for example, phenyl-SO3H-

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functionalized SWCNTs [62], polyethylene glycol-modified MWCNTs [63], RNA-modified

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SWCNTs, protein-functionalized CNTs [64], etc., have been investigated in various laboratories

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resulting in no significant cell damage. On the other hand, the cytotoxicity seems to be also

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dependent on the physical properties of CNTs such as size and morphology. For example, Magrez

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et al. [65] and Tian et al. [66] have shown that the SWCNTs are more toxic than MWCNTs. In

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general, the physical properties and surface functionalization of CNTs are the key factors that

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determine the transportation efficiency and cytotoxicity of CNT-based carrying materials.

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Furthermore, in the study of Liu et al multi-walled CNTs (MWCNTs) of different length were

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functionalized with chitosan–folic acid nanoparticles (CS–FA NPs) using ionotropic gelation

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process. This vector was used to transfect MCF-7 and Hela cells with GFP gene. The nanotube

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length showed a compromise influence on the transfection and cytotoxicity properties of

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MWCNTs. In the study of Behnam et al, single-walled carbon nanotubes (SWNTs) were

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functionalized by non-covalent binding of hydrophobic moieties, which were covalently linked to

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polyethyleneimines. Their results showed that PEI-functionalized SWCNTs exhibited a good

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stability and dispersibility in biological media.

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One of the most relevant chemical modifications to create carboxylic acid groups on the multi-

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walled carbon nanotubes (MWCNTs) is oxidation which [67, 68] provides opportunity to

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functionalize MWCNTs for conjugation of surfactant polymers [69]. At the same time, oxidation

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damages nanotubes, resulting in structural defects, shortening of tubes, accumulation of

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carbonaceous impurities, and loss of small diameter nanotubes [67, 68]. To oxidize nanotubes

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efficiently and preventing significant material loss, oxidation conditions should be selected

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carefully. Nitric acid has been the mostly utilized agent for oxidation of carbon nanotubes [68]. It

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can be used solely in boiling temperature or in combination with concentrated sulfuric acid [70]. In 6    Page 6 of 38

order to maximize the loading of nucleic acids onto the surface of carboxylated carbon nanotubes,

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a highly cationic-charge density polymer such as polyethyleneimine (PEI) has been used [69, 71].

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Across the literature the branched 25 kDa PEI polymer is favored as a gene transfer agent [30].

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The delivery of genes to MSCs as well as PEI-modified CNT carriers are well-documented

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separately, however; the application of PEI-modified CNTs as non-viral gene delivery vectors for

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MSCs is not reported. Thus, in this study, a PEI-grafted MWCNT in combination with chitosan

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substrate as a nanocarrier system for gene delivery is developed and its transfection efficiency in

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bone marrow mesenchymal stem cells (BMSCs) is evaluated using plasmid DNA encoding

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enhanced green fluorescent protein (pEGFP) as an exogenous reporter gene. The PEI binding,

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particle size distribution, and colloidal stability of the functionalized CNTs were analyzed by

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Fourier transform infrared (FT-IR) spectra, dynamic light scattering (DLS), and zeta potential,

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respectively. DNA binding affinity, cellular uptake, transfection efficiency and possible

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cytotoxicity were also tested by agarose gel electrophoresis, flow cytometry, cytochemisty and

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MTT assay.

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2. Experimental Methods

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2.1. Materials

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PEI (bPEI-25K), Chitosan (medium molecular weight), Hyaluronan (HA), Multi-walled carbon

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nanotubes, fluorescein-5-isothiocyanate (FITC), EDC and sulfo-NHS were obtained from Sigma-

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Aldrich; plasmid DNA encoding enhanced green fluorescent protein (pEGFP) were purchased

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from Invitrogen (USA), A large amount of these plasmids were prepared using a Qiagen Plasmid

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Maxi Kit (Qiagen, Germany), Fetal bovine serum (FBS), alpha-MEM media, opti-MEM serum-

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free media, PBS buffer, Trypsin/EDTA and penicillin-streptomycin were purchased from GIBCO 7    Page 7 of 38

(USA); MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay kit was

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obtained from Roche (Germany).

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2.2. Preparation of functionalized CNTs

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The MWCNTs were firstly treated with concentrated HNO3/H2SO4 solution. In a typical

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procedure, 50 mg of MWCNTs were suspended in 40 mL of a 3:1 mixture of concentrated H2SO4

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(98wt%) /HNO3(16M) and transferred into a round-bottom flask equipped with a magnetic stirring

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bar and a reflux condenser. The flask was immersed in an oil bath at 120 °C. The mixture was

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refluxed for 2 h and cooled down to room temperature. The resultant suspension was filtered to

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collect oxidized-MWCNTs (ox-MWCNTs) on a 100-nm-pore membrane filter and washed with

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deionized water until the filtrate was neutral followed by overnight drying in vacuum at 4oC. The

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consequential MWCNTs–COOH were coated with PEI, following the EDC (1-ethyl-3-(3-

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dimethylaminopropyl) carbodiimide hydrochloride) chemical activation of the COOH groups that

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yields a PEI/MWCNT complex. The ratio of PEI to MWCNTs used for functionalization process

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was from 0.1-0.6. Subsequently, the complex was dispersed in 100mM MES buffer at

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concentration of 1 mg/ml using an ultrasonic bath for 1 h, at room temperature. The suspension

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was then centrifuged at 22,000 g for 30 min to remove bulky ox-MWCNTs and impurities whereas

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the excess PEI was removed through filtration in Millipore Microcon 50,000 molecular weight cut-

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off spin filter. The obtained PEI-grafted-MWCNT (PEI-g-MWCNT) solution was stored at 4oC for

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further experiments. Figure 1 represents these procedures schematically.  

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2.3. Characterization of PEI-g-MWCNT

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2.3.1. Dispersability of PEI-g-MWCNT

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To determine the best sample in terms of “solubility” a dispersion of functionalized nanotubes

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with different surfactant ratio was diluted with deionized water in 10 mL glass vials to yield series

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of concentrations ranging from 0.01 to 3.5 mg/mL. Each vial was sonicated by an ultrasonic bath

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for 10 min. After standing for 3 h, the dispersions were analyzed for the presence of visible

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particles. The highest concentration of functionalized nanotubes with no visible particles is

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reported as the solubility value for each sample.

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2.3.2. FT-IR analysis of functionalized CNTs

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spectrometer (Madison, WI) for the sample which was selected from the solubility test. 2.3.3. Dynamic light scattering and zeta potential measurements

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Fourier transform infrared (FT-IR) spectra recorded on Nicolet 400 Fourier transform infrared

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The mean particle size of the ox-MWCNT and PEI-g-MWCNT solutions was determined by

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dynamic light scattering using photon correlation spectroscopy. The measurements were

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performed using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) equipped with a

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helium-neon laser at 25°C and a scattering angle of 173°. Additionally, the zeta potential of the

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same solutions was measured with the same instrument at 25°C by electrophoretic mobility.

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2.3.4. DNA binding assay

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PEI-g-MWCNT/pDNA (C=250µg/ml) complexes were prepared by mixing dilutions of PEI-g-

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MWCNT consist of 2, 1, 1/2, 1/4, 1/6, 1/8, 1/10, 1/20, 1/100 and 1/200 to reach different N:P

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ratios (where N=number of primary amines in the PEI; P=number of phosphate groups in the

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pDNA backbone). Phosphate buffered saline (PBS) solution was used to prepare the solutions.

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Complex solutions were gently vortexed and allowed to incubate for 30 min at room temperature

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prior to experiment. The binding of DNA to PEI-g-MWCNT was assessed by agarose gel electrophoresis. Each PEI-

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g-MWCNT/pDNA dispersion with a desired N:P ratio was mixed with 6×loading buffer

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(bromophenol blue/xylene cyanol) and loaded on a 1%(w/v) agarose gel in tris-acetate-EDTA

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(TAE) buffer. The amount of pDNA loaded into each well was 2.5 µg in total volume of 10 µL.

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The electrophoresis was performed under 100 V for 45 min. Ethidium bromide was used for

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pDNA staining and their bands were visualized under UV.

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2.4. In vitro gene transfection

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2.4.1. BMSCs isolation and expansion

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Rat bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from long bones of 8

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week-old male Wistar rats according to a standard protocol [41]. Following euthanasia by

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pentobarbital 20% (v/v), the femora were aseptically excised, cleaned of soft tissue, and washed in

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PBS. Subsequently, marrow was flushed out from the tibiae and femora with 5 ml of α-Minimum

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Essential Medium (α-MEM) using a 23-Gauge needle and syringe. The cells were centrifuged (600

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g, 5 min), suspended in fresh medium containing 10% heat-inactivated FBS, 100 U/ml penicillin

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and 100 µg/ml of streptomycin and seeded in 25 cm2 flasks. Flasks were incubated at 37 °C in 5%

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CO2 and 90% humidity. After removal of non-adherent cells and medium exchange at day 3,

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colonies became compact and cells were detached with 0.25% trypsin/EDTA at day 7. Following

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cultures were passaged at 5–7 day intervals and expanded to passage 5 for further experiments.

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2.4.2. Preparation of chitosan -modified substrates

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To improve transfection efficiency, chitosan substrates were prepared as previously described

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by Hsu et al.[72] Briefly, chitosan was dissolved in 1% acetic acid by gentle stirring for 12 h at

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room temperature. The solution (1% chitosan) was filtered and then coated on coverslip glass (100

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ml of solution on each 15 mm-diameter glass) and air-dried for 2 days. The chitosan membranes

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formed on the coverslip glass were immersed in 0.5N NaOH solution for about 5 min, and washed

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extensively by distilled water. The chitosan membranes were air-dried for further transfection

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experiments. These samples (1.5 cm in diameter) fit the size of each well in a 24-well plate.

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2.4.3. BMSCs transfection by PEI-g-MWCNT/pDNA complexes

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Plasmid DNA encoding enhanced green fluorescent protein (pEGFP) was used as an exogenous

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reporter gene to assess transfection efficiency. Cells were cultured until 65–70% confluency and

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seeded at a density of 15×104 cells per well in adherent 24 well plates 24 h prior to transfection.

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Media was removed an hour before transfection and the monolayer of cells was washed with 1 ml

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of PBS per well. Subsequently, 500 μl of OptiMEM serum-free media was added to each well and

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incubated at 37 °C for one hour. PEI-g-MWCNT/pDNA complexes were produced in OptiMEM at

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varying doses (1 μg, 2 μg, 4 μg) of pDNA and incubated at room temperature for 30 min.

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Transfection medium (100 μl) were then added to the cells and supernatant was removed after

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approximately 4 h followed by PBS wash. Complete culture media was added and the cells were

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incubated at 37 °C to allow expression of the transgene. Additionally, HEK293 cells were cultured

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in 24-well culture plate and transfected in the same manner as control. Non-transfected cells and

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cells transfected with naked pDNA were used as negative controls. The transfection efficiency of

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PEI-g-MWCNT/pDNA was directly analyzed by imaging under a fluorescence microscope

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(Nikon, Germany).

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2.4.4. Cytotoxicity assay The cytotoxicity of free PEI-g-MWCNT and PEI-g-MWCNT/pDNA dispersion was evaluated

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on rat BMSCs 24 and 60 h post transfection using MTT assay. BMSCs were seeded in a 96-well

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plate at an initial density of 5000 cells/well. After overnight incubation, PEI-g-MWCNT as well as

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PEI-g-MWCNT/pDNA complex were added to the cells at concentrations ranging from 0-100

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µg/ml in PBS buffer (pH 7.4). Subsequently 10 μl of MTT reagent (5 mg/ml) was added to each

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well in 90 μl of media and incubated for an additional 4 h at 37 °C to detect the metabolically

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active cells in each well. The medium was then removed and replaced by 100 µL DMSO to

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dissolve the formazan crystals formed as a result of MTT cleavage by viable cells. Absorbance

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was measured at 570 nm by Titertek multiscan Eliza reader (Labsystems multiscan, Rodon,

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Netherlands) with a reference with non-transfected cells serving as a 100% viability control. Cell

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viability (%) was estimated according to the following equation: Abs (transfected)/Abs (control)

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×100.

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2.4.5. Cellular uptake of PEI-g-MWCNT/pDNA by flow cytometry

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To quantitatively assess PEI-g-MWCNT/pDNA uptake by BMSCs, PEI-g-MWCNT was first

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labeled with fluorescein isothiocyanate (FITC) by mixing 11.2 mg of FITC with 360 mg of PEI-g-

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MWCNT, followed by stirring the mixture overnight at room temperature. Subsequently, FITC-

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labeled nanoparticles were incubated with BMSCs in 6-well plates (in 2ml serum free basic

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medium/well) for 4 h. After removing the PEI-g-MWCNT/pDNA containing media, the cells were

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washed with HBSS, trypsinized, centrifuged, and suspended in HBSS with 3.7% formalin for flow

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cytometric analysis (λex = 488 nm). The instrument threshold for the negative control sample (i.e.,

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untreated BMSCs) was setup at ~ 1% level. The percentage of cells exhibiting FITC-fluorescence 12    Page 12 of 38

beyond this threshold value was calculated as a function of nanoparticle concentration in the

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medium.

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2.5. Statistical analysis

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Each experiment was performed at least in triplicate. All results are summarized as means ±

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standard deviation and statistical differences were determined by analysis of variance followed by

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Student’s t-test. Results were considered statistically significant when p<0.05.

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3. Results

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3.1. Characterization of PEI-g-MWCNT

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The solubility is defined as the maximum weight percent of nanotubes that can be dispersed in a

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solvent with no particle visible for at least 2 h. Figure 2(A) reports the solubilities of PEI-g-

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MWCNTs in water with different weight fraction of PEI to MWCNTs. The solubility of mixture

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enhanced rapidly at first as the ratio of the PEI to MWCNTs was increased. Functionalization of

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nanotubes with greater amounts of PEI (>0.3 (w/w)) gave smaller increases of solubility. Figure

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2(B) shows a PEI-g-MWCNT dispersion with optimum ratio of PEI to MWCNT (0.3 (w/w)) after

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3 months.

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The FT-IR was employed to characterize the formation of oxidized MWCNTs and PEI-g-

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MWCNTs with optimum surfactant to nanotube ratio. As shown in figure 3, spectrum of the

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oxidized MWCNTs (curve a) and PEI-g-MWCNTs (curve b) presented the characteristic peaks

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which indicated the successful oxidation of MWCNTs by acid treatment process and ideal

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assembly of the PEI-g-MWCNTs, respectively. In curve a, spectra of oxidized MWCNTs shows

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four major peaks, located at 3450, 2370, 1740 and 1635 cm−1. The broad peak at 3450 cm−1 refers 13    Page 13 of 38

to the O-H stretch from carboxyl groups (O=C−OH and C−OH), while the peak at 2370 cm−1 can

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be associated with the O−H stretch from strongly hydrogen-bonded −COOH [73, 74]. The peak at

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1740 cm−1 is associated with the stretch mode of carboxylic groups indicating that carboxylic

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groups are formed due to the oxidation of carbon atoms on the surfaces of the MWCNTs by nitric

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and sulfuric acids [75, 76]. The peak at 1635 cm−1 can be associated with the stretching of the

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carbon nanotubes backbone [73]. In addition, peak assigned to the C–O stretching corresponds to

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the oxidized MWCNTs at the 1165 cm−1 could be clearly observed in curve a [77]. The peaks at

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2845 and 2920 cm-1 correspond to the H−C stretch modes of H−C=O in the carboxyl group. In

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curve b, the strong and broad absorption peak located at 3450 cm-1 could be attributed to the N−H

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bond of PEI and −OH groups in PEI and MWCNTs, a −NH2 bending peak at 1559 cm-1 and a

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C−N stretching vibration at 1460 cm-1 were also detected [78].

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Dynamic light scattering (DLS) measurement was carried out to determine the size distribution

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of the formed complexes (Figure. 4(A)). Conjugation of hydrophobic chains of PEI to MWCNTs

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resulted in a significant increase (p<0.05) in complex mean diameter. In general, the size of the

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complexes increased with the length of the hydrophobic chain from 70.89 to 110.1 nm. The

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polydispersity indices (PDI) varied among oxidized (0.224) and PEI-g-MWCNT (0.179) showing

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that the heterogeneity of PEI-g-MWCNT was lower as revealed by the sharpness of the

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corresponding peak in figure 4(A).

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The surface modification and colloidal stability of MWCNTs were also assessed by zeta

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potential measurements (Figure 4(B)). The surface potential of the acid-treated MWCNTs (-40.7

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mV) became positive (39.3 mV) after modification with PEI. The zeta potential changes reflect the

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successful surface modification of MWCNTs suggesting that the surface potentials of MWCNTs

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can be manipulated through PEI-mediated reactions. 14    Page 14 of 38

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3.2. Optimization of DNA binding The potential of functionalized MWCNTs to neutralize, bind and condense plasmid DNA was

2

studied by agarose gel electrophoresis, due to its importance for efficient gene delivery. The

4

plasmid used for this study was the pEGFP that encodes the enhanced green fluorescent protein.

5

Agarose gel electrophoresis assay revealed that binding of plasmid DNA to functionalized

6

MWCNTs inhibits ethidium bromide intercalation [79], as the pDNA is in a condensed form. The

7

level of binding can thereby be assessed by the measurement of the non-bounded DNA (Figure. 5

8

lanes 5–10). The DNA binding capacity of dispersed PEI-g-MWCNTs can be estimated by

9

reference to the lowest concentration of nanotubes that demonstrates detectable DNA binding (lane

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5 in Figure. 5). Based on these results, a dilution of 1/4, which can package complete pDNA was

11

selected for further experiments.

12

3.3. In vitro gene transfection

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In order to determine the effectiveness of functionalized MWCNTs in transfecting mammalian

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cells, two different cell lines (i.e. BMSC and HEK293) were transfected in monolayer culture with

15

different amounts of pEGFP (2, 4 and 6 µg/ml) complexed by optimum ratio of PEI-g-MWCNTs.

16

Subsequently, gene delivery was qualitatively studied 60 h post-transfection by morphological

17

analysis and expression of EGFP using optical and fluorescence microscopy, respectively (Figure

18

6). The fluorescent green color of the cultures is indicative of successful transfection of PEI-g-

19

MWCNTs/pDNA and EGFP expression. The highest EGFP expression was observed in both cell

20

lines which transfected with 4 µg/ml of pDNA. Furthermore, results show that by increasing the

21

amount of pDNA (6 µg/ml), the transfection efficiency was decreased possibly because of

22

cytotoxic effect of MWCNTs in higher doses. No fluorescent green color was observed for control

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groups and cells that were transfected with complexes containing 2 µg of pDNA/ml. Furthermore,

2

the transfection efficiency of cells transfected on chitosan substrate was more than cells which

3

transfected on untreated culture plate.

4

3.4. BMSCs viability assay

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The MTT assay was performed to evaluate viability of BMSCs 24 and 60 h post transfection.

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by incubation of cells with PEI-g-MWCNTs and PEI-g-MWCNTs/pDNA at concentrations

7

ranging from 0-100 µg/ml. As shown in figure 7, there was no statistically significant difference in

8

the cell viability of BMSCs treated with PEI-g-MWCNTs/pDNA complexes compared to the

9

controls at concentrations of 0-37.5 μg/ml (p>0.05) after 24 h. However, PEI-g-MWCNTs/pDNA

10

start to exhibit cytotoxicity at concentrations of 50 μg/ml and higher. Since free PEI-g-MWCNTs

11

were cytotoxic at all concentrations, it can be concluded that cytotoxicity of PEI-g-

12

MWCNT/pDNA was considerably lower than free PEI-g-MWCNT to bone marrow mesenchymal

13

stem cells. Furthermore, the cell viability of BMSCs treated with PEI-g-MWCNTs/pDNA

14

complexes at concentrations of 0-5 μg/ml was not significantly different compared to the controls

15

60 h after the transfection (p>0.05).

16

3.5. Cellular uptake of PEI-g-MWCNT/pDNA

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To determine the uptake of PEI-g-MWCNT/pDNA complex by BMSCs, FITC-labeled PEI-g-

17 18

MWCNTs were incubated with cells and examined by flow cytometry. To evaluate the propensity

19

of PEI-g-MWCNT/pDNA to be internalized, the intracellular fluorescence values were determined

20

by measuring non-transfected cells fluorescent (Figure 8. A,C). It can be seen that non-transfected

21

cells were unable to exhibit FITC-fluorescent (RN1=0.08%, figure. 8A). Additionally, almost all

22

cells (89.17 %) displayed PEI-g-MWCNT/pDNA uptake after 4 h incubation (Figure. 8B). These 16    Page 16 of 38

experiments provide quantitative data, which indicate an effective uptake of PEI-g-

2

MWCNTs/pDNA by the bone marrow mesenchymal stem cells.

3

4. Discussion

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Recently, due to the limitations associated with protein delivery [80, 81], gene therapy has

5

alternatively been applied to provide sustained protein production by transfected cells. Very little

6

work has been reported using functionalized-MWCNTs as the BMSCs transfection agent. In this

7

study, we developed and evaluated the use of PEI grafted MWCNT as a gene delivery vector for

8

expression of a model gene (EGFP) in primary BMSCs. Even though, MWCNTs have been

9

utilized in the past for gene delivery, their application has been usually evaluated in immortal cells

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that are not clinically useful [45]. On the other hand, BMSCs are multipotent cells that have the

11

ability to differentiate into multiple lineages and have remarkable potential for related tissues

12

construction and regeneration [24, 25]. In view of the fact that there is no ideal vector for effective

13

gene transfer in BMSCs, a delivery system using PEI-g-MWCNT in corporation with chitosan

14

substrates was successfully optimized for BMSC transfection in monolayer cultures.

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A major obstacle in stem cell transfection and gene therapy is the lack of an efficient vector for

15 16

non-viral transfection. The unique properties of CNTs such as high surface area (theoretically 1300

17

m2/g) have made this nanocarrier more attractive than any other non-viral vectors for biomolecular

18

delivery [82]. However, there are still remaining problems when using carbon nanotubes as a

19

biological carrier. The main problem is their inherent difficulty in handling as they tend to

20

aggregate, thus their application in aqueous media particularly in biological medium is limited [44,

21

69]. Furthermore, the cytotoxic effect of carbon nanotubes is a major obstacle in utilizing them for

22

clinical applications [83]. The CNTs have so many features which could considerably influence

23

their toxicity including: surface charge, agglomeration, concentration, and purity. Even thought, 17    Page 17 of 38

pristine CNTs are not suitable for direct transfer of genes into biological system, it has been shown

2

that functionalization of CNTs with hydrophilic agents or using an optimized concentration can

3

decrease their intrinsic toxicity and allow their applications in biomedical field. Therefore,

4

development of functionalization methods to obtain soluble carbon nanotubes with less cytotoxic

5

effect is primordial. Although the capability of different functionalized CNTs as non-viral vehicle

6

has been previously explored, the surfactant ratio and CNT doses specifically for BMSCs have not

7

been optimized yet [69, 84]. By optimizing the PEI-g-MWCNT/pDNA complex specifically for

8

BMSCs this research would find a greater clinical relevance. Our results demonstrate that the PEI-

9

g-MWCNTs has the property of homogenously dispersing in water, where it has been observed to

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be stable for over 3 months at room temperature, without any solid phase aggregation. This

11

probably relates to the stability of the covalent bond between the PEI polycation and the

12

MWCNTs–COOH, which occurs through an amidic bond. Moreover, solubility increases as the

13

number of polar functional groups on the nanotubes enhances. The oxidation reactions by nitric

14

and sulfuric acids would create many carboxylic acid functional groups on the side walls of CNTs

15

[85-87]. Although several methods have been reported for determination of the “solubility” of

16

carbon nanotubes [88-90] neither the term solubility nor a standard method for its determination

17

has been established yet. Most “solutions” of MWCNTs are in fact dispersions. We found that the

18

ratio of surfactant to MWCNT was also crucial in order to optimize the dispersion [91]. As

19

indicated in the dispersion curves (Fig.2 (A)), MWCNTs were dispersed by conjugating with a

20

range of PEI concentrations. It is evident that by increasing the amount of PEI used for grafting,

21

the amount of dispersed MWCNTs enhances until a maximum is reached where the optimum

22

conditions for dispersion are obtained. Above this optimal concentration of PEI, the yield of

23

dispersed nanotubes is approximately constant. This is likely due to limited amount of MWCNTs

24

being shared between high concentrations of surfactants.

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The length distribution of nanotubes in solution has been measured within hydrodynamic

1

approximations using dynamic light scattering (DLS) [92, 93]. DLS provides information for

3

length distribution whereas the zeta potential measurement provides information for the degree of

4

dispersion. The length of nanotubes is closely related to their degree of dispersion. It is therefore

5

necessary to combine both methods to get comprehensive information in evaluating dispersivity

6

and dispersion stability of nanotubes.

cr

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The FT-IR spectra and electrophoresis results showed that the covalent binding of PEI

8

surfactants to MWCNTs increased the efficient binding of plasmid DNA and enhanced DNA

9

condensation. It is evident that the covalent attachment of cationic surfactants to MWCNTs would

10

be a good method for condensation and binding of DNA to nanotubes. The lowest concentration of

11

nanotubes that demonstrates detectable DNA binding (lane 5, Fig. 5) referred as ‘DNA binding

12

capacity’ of dispersed carbon nanotubes. Vanesa Sanz et al. reported that among different

13

surfactants used for MWCNTs functionalization, the highest binding affinity was obtained for PEI

14

which is being 10 times higher compared to poly(Lys:Phe, 1:1) and 100 times more than PL-PEG-

15

NH2 [84].

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16

On the other hand, in order to enhance the effective transfection of cells, substrate-mediated

17

control of endocytosis has been proposed [72]. As can be seen in figure 7 the cells are successfully

18

transfected both with and without chitosan substrate. However, the transfection efficiency of

19

BMSCs on the chitosan substrates was significantly higher compared to the common cell culture

20

substrates. It is known that the increased transfection efficiency on chitosan substrate is not

21

attributed to higher proliferation rate on these substrates as the expression of surface markers such

22

as CD73, CD90, and CD105 after transfection remains positive (>95). It is therefore apparent that

23

cells remain undifferentiated on chitosan substrates [72]. The intracellular uptake of PEI-g-

24

MWCNTs on chitosan substrates found in this study was comparable to the highest level reported 19    Page 19 of 38

for BMSCs transfected with polycationic agents [94]. This suggests that endocytosis and endocytic

2

phenotype may be altered for BMSCs cultured on chitosan [72]. It is shown that BMSCs migrate

3

very fast and form spheroids on these substrates. It is assumed that the increased endocytosis

4

described here could be associated with the BMSC spheroids formation [95]. It should be noted

5

that there is not enough information about the fate of CNTs in cells. However, Kang et al. (2010)

6

performed a study on the subcellular fate of CNTs and reached to a conclusion that the fate of

7

CNTs in the cells is a size-dependent process. As they reported, 100–200 nm CNTs are mainly

8

distributed in cytoplasm, but 50–100 nm CNTs are localized in the cell nucleus [96]. Other

9

experiments [97] have shown that CNTs can be degraded in the cells by the possible enzyme-

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1

catalyzed oxidative biodegradation process. However, these different studies focused mainly on

11

the degradability of the graphitic lattices of CNTs.

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The present study showed that the MWCNTs display enhanced water solubility after covalent

12

functionalization with PEI. The solutions of PEI-g-MWCNTs are stable in aqueous solution up to

14

several months. Moreover the PEI-g-MWCNTs/pDNA complexes do not display cytotoxicity at

15

concentrations up to 37.5 μg/ml 24 h and to 5 μg/ml 60 h after transfection while without pDNA

16

complexation, the PEI-modified MWCNTs are cytotoxic. These results suggest that combination

17

use of PEI-g-MWCNTs and chitosan cell-substrate could be an excellent gene delivery system for

18

transfecting some hard-to-transfect cells like BMSCs.

19

5. Conclusion

20

Gene delivery to MSCs as well as the application of PEI-modified CNT carriers is well-

21

documented, separately. However; the application of PEI-modified CNT for delivery of genes to

22

MSCs are not reported. In this work a non-viral system consists of PEI-g-MWCNT and chitosan

23

substrate has been optimized for efficient transfection of BMSCs which is typically regarded as

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20    Page 20 of 38

difficult to transfect cell type. The results show that PEI has been successfully grafted onto the

2

surface of CNTs and increased their stability in aqueous media whilst reduced cytotoxicity of PEI-

3

g-MWCNTs under optimal transfection condition. In consistency with high cellular uptake (>

4

82%), PEI-g-MWCNTs give higher delivery of pDNA into the BMSCs which results in a more

5

sustained expression of the model gene (EGFP). Our findings highlighted the importance of

6

chitosan substrate to enhance the transfection efficacy of BMSCs. We anticipate that the approach

7

could be extended by conjugation of various biomolecules onto functionalized carbon nanotube

8

surfaces which may provide opportunities for applications in biomedical sciences. However, future

9

studies should be directed toward the assessment of this gene delivery system in vivo and to

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develop an efficient non-viral carrier system for regenerative medicine.

11

Acknowledgments

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We gratefully acknowledge the financial support of this work by Iranian Council of Stem Cell

12

Technology (ICST).

14

Disclosures

15

We declare that we have no conflicts of interest.

16

Role of the funding source

17

The sponsor of the study had no role in study design, data collection, data analysis, data

18

interpretation, or writing of the report. The corresponding author had full access to all the data in

19

the study and had final responsibility for the decision to submit for publication.

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6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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Figure Captions Figure 1. Schematic representation of the functionalization process of PEI-g-MWCNTs and their

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interaction with DNA.

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Figure 2. (A) Dispersion of PEI-g-MWCNTs as a function of PEI to MWCNTs ratios. (B)

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Aqueous dispersion of PEI-g-MWCNTs at room temperature (3 (w/w)(PEI/MWCNT) at

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concentration of 3 mg/ml).

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Figure 3. FT-IR spectra of (a) oxidized MWCNTs, and (b) PEI-g-MWCNTs.

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Figure 4. (A)The size distribution of oxidized and PEI grafted MWCNTs assessed by DLS.

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(B) The zeta potential of oxidized and PEI grafted MWCNTs.

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Figure 5. Agarose gel electrophoresis for the PEI-g-MWCNTs presenting the effective binding of

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plasmid DNA with different dilution factors of 2, 1, 1/2, 1/4, 1/6, 1/8, 1/10, 1/20, 1/100 and 1/200,

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lane 1- 10, respectively.

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Figure 6. Optical and fluorescent images of PEI-g-MWCNTs/pDNA transfection in HEK293 (A,

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B) and BMSCs with(C, D) and without (E,F) chitosan substrate. Fluorescent images show EGFP

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expression in both cell lines in monolayer culture 60 h post-transfection using complexes

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containing 4 µg of pDNA/ml.

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Figure 7. The viability of BMSCs transfected with PEI-g-MWCNTs/pDNA. The cell viability of

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BMSCs is shown (A) 24 h and (B) 60 h after transfection with various concentrations of PEI-g-

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MWCNTs. Each bar represents the mean ± standard deviation (n=3) * p<0.05 when compared

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with non-transfected cells.

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Figure 8. Uptake of FITC-labeled PEI-g-MWCNTs/pDNA complexes were evaluated by flow

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cytometry. The intracellular fluorescence values were determined by measuring non-transfected

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cells fluorescent (A). Almost all of the cells (89.17 %) displaying PEI-g-MWCNT/pDNA complex

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uptake after 4 h incubation (B). Population of cells was used to study fluorescent emition (C, D).

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Highlights 

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A non‐viral system consists of PEI‐g‐MWCNT and chitosan substrate is optimized for efficient transfection  of BMSCs. 

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The model gene (EGFP) is successfully and efficiently transfected and expressed in BMSCs 

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The approach may be extended upon replacing the reporter gene with therapeutic genes. 

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The result has enormous implication in genetically directing BMSCs towards a particular lineage for  regenerative medicine.  

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*Graphical Abstract (for review)

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Figure 8

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