Electrospun gelatin matrices with bioactive pDNA polyplexes

Journal Pre-proof Electrospun gelatin matrices with bioactive pDNA polyplexes

Porntipa Pankongadisak, Ekeni Tsekoura, Orawan Suwantong, Hasan Uludağ PII:

S0141-8130(19)39959-3

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.252

Reference:

BIOMAC 14557

To appear in:

International Journal of Biological Macromolecules

Received date:

3 December 2019

Revised date:

22 January 2020

Accepted date:

24 January 2020

Please cite this article as: P. Pankongadisak, E. Tsekoura, O. Suwantong, et al., Electrospun gelatin matrices with bioactive pDNA polyplexes, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.01.252

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© 2020 Published by Elsevier.

Journal Pre-proof

Electrospun Gelatin Matrices with Bioactive pDNA Polyplexes

Porntipa Pankongadisak,a Ekeni Tsekoura b, Orawan Suwantong a,c,*

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and Hasan Uludağb,*

School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai, Thailand

b

Department of Chemical & Material Engineering, Faculty of Engineering, University of Alberta,

Muang, Chiang Rai, Thailand

*: corresponding authors at

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Center of Chemical Innovation for Sustainability (CIS), Mae Fah Luang University, Tasud,

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c

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2-021 RTF, Edmonton, Alberta, Canada.

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a

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E-mail: [email protected]; Fax: +1 780 492 2881; Tel: +1 780 492 0344 E-mail:[email protected]; Fax: +66 5391 6776; Tel: +66 5391 6787

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Journal Pre-proof ABSTRACT Recent advances in electrospinning are yielding intricate scaffolds for use in regenerative medicine. To explore the possibility of creating bioactive scaffolds with functional gene expression systems, electrospun gelatin mats bearing plasmid DNA (pDNA) polyplexes are explored. The pDNA is first condensed with a lipid-modified polyethylenimine (PEI) to create polyplexes including a poly(aspartic acid) (pAsp) additive, and subsequently electrospun after mixing the polyplexes in gelatin solution. The pDNA polyplexes, 82 nm in size with -potential of +20 mV,

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are uniformly entrapped in mats with fiber diameter ranging between ~150 and ~350 nm. The

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additive complexes with pAsp display a significantly higher transfection activity in solution, which was also retained after entrapment in electrospun mats, based on GFP expression to human

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myoblast (C2C12) and mouse osteoblast cells (MC3T3-E1). Electrospinning of gelatin with

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polyethylene glycol improves the transfection efficiency, due to increased pDNA entrapment

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(~71%). To further validate gene-activated mats, a pDNA encoding BMP-2 shows robust alkaline phosphatase (ALP) induction in C2C12 and MC3T3-E1 cells as a marker of osteogenic

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differentiation. We conclude that creating gelatin fiber mats with bioactive pDNA polyplexes was

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feasible and such mats could aid in regenerative repair of a wide range of tissues.

Keywords: Electrospun fiber mats,

gelatin, polyethylene glycol, pDNA polyplexes, Bone

morphogenetic protein, Gene delivery

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Journal Pre-proof 1. Introduction Gene therapy is an interventional approach that deploy a genetic material in a specific target tissue in an effort to express a desired protein and prolong the action of the protein to cure or halt the disease [1]. To utilize the full potential of gene therapy technology, suitable gene carriers are required to facilitate intracellular delivery of genetic materials (polynucleotides). The delivery can be implemented with viral and non-viral carriers, which act in different ways to deliver the genetic material to cell nucleus, which is the site of desired transcription process [2]. Ideal properties of

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gene carriers are the ability to deliver a gene to a specific cell type, allow transgene expression at a

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high level and long duration to obtain the therapeutic effect, while avoiding the host immune response, and displaying no adverse effects [3]. Although viral vectors provide the most of efficient

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delivery, but they have severe limitations due to induction of severe immune response, residual

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virulence, and potential risk of secondary carcinogenesis [4-6]. Non-viral gene carriers facilitate a

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complex formation between DNA-based expression systems (typically plasmid DNA, pDNA) and cationic lipids/polymers (lipoplexes/polyplexes, respectively) by electrostatic interactions. They are

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generally recognized as safe due to their low immunogenicity and reduced risks of genetic mutation

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[7]. However, gene delivery via injection of DNA complexes in liquid form to tissues can lead to low gene transfection efficiency, toxicity to untargeted cells, or loss of DNA complexes due to rapid transport away from the site and degradation [8]. Incorporating DNA complexes into biomaterials for administration into a host can enhance and extend the transgene expression, and avoid the risks of immune response to the vectors and possibly degradation of expression systems [9]. Electrospun mats are being explored as highly effective biomaterial scaffolds in regenerative tissue repair. Electrospinning create a fibrous mat with controlled fiber size in nanometer to micrometer range using high electrostatic fields [10] and effectively manipulate the surface area and porosity of mats to create optimal conditions for biomedical devices and tissue engineering scaffolds. The electrospun mats can control cellular invasion and new tissue induction, and

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Journal Pre-proof moreover can deliver bioactive agents such as growth factors to control tissue induction and repair [11]. The relatively benign fabrication parameters employed during electrospinning (e.g., room temperature, use of biocompatible buffers, etc.) is an additional advantage to incorporate biological factors in a bioactive state. Delivering gene expression systems, rather than the protein growth factors, may be more advantageous since gene delivery may allow continues production of bioactive factors in situ. Some studies have reported on electrospun nanofiber mats as gene delivery vehicles [12-16]. The encapsulation of pDNA complexes into the mats can protect the genetic

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materials against extracellular degradation and release a continuing supply of pDNA to target cells

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[12,17]. Different types of biomaterials can be applied with the electrospinning process including natural and synthetic polymers, ceramics, and their composites [18]. Although the electrospun

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blends of natural and synthetic polymers could improve the mechanical properties and

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biocompatibility for tissue engineering, synthetic polymers typically degrade slowly and their

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prolonged presence inside the body may cause increased inflammatory reactions [19]. Gelatin (Gel) is one of the most extensively used biomaterial in regenerative medicine due to its abundance, low

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cost, biocompatibility, biodegradability and cell adhesive properties similar to that of collagen. In

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addition, Gel contains the arginine-glycine-aspartic acid motif that can improve the binding of host cells and facilitate integration with the extracellular matrix (ECM), consequently enhancing the biological behavior compared to other synthetic polymers that lack cell recognition motifs [20]. Although Gel can dissolve in water at temperatures over 40 ℃ [21], it has not been possible to fabricate electrospun mats from aqueous gelatin solutions at room temperature due to nonvolatilized water that leads to gel formation during the electrospinning process. 2,2,2trifluoroethanol (TFE) or phosphate buffer saline (PBS)/ethanol (EtOH) mixtures have been used to dissolve Gel for electrospinning at room temperature [22,23]. Among the non-viral gene carriers, polyethylenimine (PEI) and its derivatives have been widely used to deliver expression systems for gene therapy [24]. In a previous study, a function carrier was prepared from low molecular weight PEI by grafting the lipid linoleic acid (LA) onto

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Journal Pre-proof the PEI [25]. The pDNA complexes prepared with the lipophilic PEI displayed improved dissociation as compared to native PEI and were less toxic with the bone narrow derived mesenchymal stem cells (BM-MSC) and umbilical cord blood derived mesenchymal stem cells (UCB-MSC) as compared to commercial transfection reagents, ultimately leading to improved transfection efficiencies. Moreover, the incorporation of hyaluronic acid (HA) into the pDNA polyplexes increased the transfection efficiency in BM-MSC and UCB-MSC under optimal conditions [25].

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These distinct materials approaches are amalgamated in this study to explore the feasibility

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of creating electrospun Gel mats bearing bioactive pDNA complexes for the first time. By using lipid-modified PEI polymers to condense pDNA into polyplexes [25], the polyplexes were

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electrospun embedded in a Gel solution to create mats. The polyplexes incorporated the anionic

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poly(aspartic acid) (pAsp) as an additive to improve the transfection efficiency. Polyethylene glycol

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(PEG) was additionally mixed with Gel solution to fabricate create Gel-PEG mats, which improved the transfection efficiency. The morphological appearance, fiber diameter, pDNA encapsulation

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efficiency, size of pDNA polyplexes, in vitro transfection efficiency, and ALP induction by the

system.

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designed mats were investigated to assess the potential of this system to act as a gene delivery

2. Materials and methods 2.1 Materials

The gelatin Type A (300 Bloom; porcine skin) and ALP substrate p-nitrophenyl-phosphate (pNPP) were purchased from Sigma-Aldrich (St Louis, MO), while PEG (Mn = 20 kDa) was obtained from Fluka (Buchs, Switzerland). pAsp was purchased from Alamanda Polymers (Huntsville, Alabama, US) and TFE (>99.0%) used as solvent for gelatin was from Alfa Aesar (Cambridge, UK). The nuclease free water, Hank’s Balanced Salt Solution (HBSS), tissue culture mediums DMEM and F12, SybrTM Green I and antibiotic solutions Penicillin and Streptomycin

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Journal Pre-proof were from Life Technologies (ThermoFisher). The control gWIZ plasmid (no protein expression) and Green Fluorescent Protein (GFP)-expressing gWIZ-GFP plasmid were from Aldevron (Fargo, ND). The BMP-2 expression plasmid (pBMP-2) was prepared in-house as described in [26]. The polymer used for pDNA condensation was described in [25] and obtained from RJH Biosciences (Edmonton, AB) under the trade name ALL-Fect. The 20X PBS buffer used for dissolving Gel for electrospinning had the composition of 0.05 M KCl, 0.035 M KH2PO4, 2.73 M NaCl and 0.2 M

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Na2HPO4. Cy3 used for pDNA labeling was from Mirus (Madison, WI).

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2.2 Preparation of Additive Polyplexes

The pDNA polyplexes with the pAsp additive was prepared using a similar approach as

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Bahadur et al.,[25] where HA was replaced with pAsp for polyplex formation. Briefly, 0.2 µg µL-1

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of both pDNA and pAsp solutions were prepared in nuclease free water from 0.4 µg µL-1 stock

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solutions. 20 µL of pDNA was mixed with 10 µL of pAsp to give a weight ratio of pAsp/pDNA = 0.5. The ALL-Fect solution (1 mg mL-1; 40 µL) was added to the pDNA/pAsp solution to form the

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polyplexes at ALL-Fect/pDNA ratio of 10. These complexes were added in DMEM serum-free

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media to give a total volume of 100 μL, after which the solutions were vortexed rapidly for 3-5 s and incubated for 30 min at room temperature before addition to the cells or used for electrospinning.

2.3 Fabrication of Electrospun Mats Encapsulated with pDNA Polyplexes Gel powder was dissolved in 20X PBS/EtOH mixture (volume ratio: 2:3, respectively) to obtain a concentration of 10 wt% according to the method of Erencia et al.[23] and stirred at room temperature until Gel was dissolved completely (designated as Gel(PBS:EtOH)). Gel powder was also prepared in pure TFE (designated as Gel(TFE)). To fabricate Gel/PEG solutions, 10 wt% PEG solution was prepared by dissolving the PEG pellets in water and mixed with Gel(TFE) at volume

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Journal Pre-proof ratios of 100:0, 70:30, 50:50, and 30:70 (designated as Gel(TFE), Gel-PEG70, Gel-PEG50 and GelPEG30, respectively). Before the electrospinning process, 100 µL of complexes (typically bearing 4 µg pDNA) was dispersed in 300 µL of Gel solutions (without and with PEG) at a volume ratio 1:3 (polyplexes:Gel solution) and stirred for one min to disperse the polyplexes in base electrospinning solutions. Such a short mixing time was used in order to avoid any possible dissociation or aggregation of the particles in the high concentration Gel solution. 400 µL of the solution was

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transferred into a 1-mL plastic syringe fitted with a 20-gauge needle. The spinneret was injected

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using an AL-4000 programmable syringe-pump at a flow rate of 0.35 mL/h onto a collector plate covered with an aluminum foil. The distance between the tip of the needle and the collector was 10

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cm, and 19 kV voltage was applied between the needle and the collector plate. The collected mats

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were dried under a biological safety cabinet for 30 min to evaporate any solvent remaining. We did

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not systematically explore any residual solvent remaining in the system after this relatively short drying time. Since no adverse effects of the mats was seen on cell viability in subsequent

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experiments, and we wanted to avoid any undesirable changes to polyplexes upon incubation, we

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used the mats after such a short incubation time.

2.4 Characterization of Electrospun Mats with pDNA polyplexes The morphology of electrospun mats prepared with and without polyplexes was observed by a Sigma 300VP Field Emission SEM (ZEISS, Germany) with an acceleration voltage of 15 kV. The fiber mats, collected on an aluminum foil, were typically cut into 1 cm x 1 cm pieces and coated with a conductive film by sputtering gold using a Nanotech SEMprep2 sputter coater for 1.5 min. The average fiber diameters were measured from the SEM images using SemAfore 5.21 software. The mats bearing pDNA polyplexes were also observed by a Morgagni 268D TEM with Gatan Digital Camera (Philips/FEI, US). The mats were electrospun directly onto 400 mesh copper grids under the spinneret for 2-3 min prior to observation.

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Journal Pre-proof To verify the presence of pDNA polyplexes in nanofibers, the mats were prepared as described before except the pDNA polyplexes used in electrospinning were prepared using Cy3labeled pDNA (labeling procedure using gWIZ plasmid was described in [25]). The mats were collected on glass slides, closed with cover slips and then observed under oil plan apochromat lenses by a Laser Scanning Confocal Microscope (LSM710, Carl Zeiss Ag, Oberkochen, Germany). The encapsulation efficiency of pDNA in electrospun mats was determined by quantification of DNA content of the mats using SYBRTM Green I dye. Briefly, gWIZ-GFP plasmid

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was electrospun into mats, which were dissolved in water to achieve an estimated concentration of

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2.5 µg mL-1. The samples were then added to the SYBRTM Green I dye solution and the fluorescence was measured at EX: 497 and EM: 520 nm with a Fluoroskan Ascent FL microplate

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reader (Thermo Fisher Scientific; Waltham, MA). A calibration curve with known concentrations of

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pDNA was constructed to convert the readings from the mats into pDNA concentration. The

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percentage of encapsulation efficiency was calculated as follows: 100% × (actual amount of pDNA in mats) / (DNA originally electrospun).

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The particles size and surface charge of the polyplexes were investigated by Zetasizer Nano

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ZS system (Malvern Instruments, UK) with dynamic light scattering and zeta potential measurements, respectively. Freshly prepared polyplexes (i.e., pAsp/pDNA = 0.5, and polymer/pDNA = 10) were diluted 10-fold with ddH2O for measurements at room temperature. The polyplexes released from the mats were also evaluated for particles size and surface charge by dissolving the mats with ddH2O and then measuring the particles at room temperature.

2.5 Cell Culture Two cell types, human myoblast C2C12 cells and mouse osteoblast MC3T3-E1 cells most frequently used for assessment of bioactivity studies in bone tissue engineering applications [27,28], were used to investigate the bioactivity of electrospun pDNAs. Both cells were cultured in Dulbecco’s Modified Eagle Medium:F12 medium (DMEM:F12 = 1:1) containing 10% Fetal

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Journal Pre-proof Bovine Serum (FBS), 0.1% GlutaMax-1, 0.1% MEM NEAA, Penicillin (100 U mL-1) and Streptomycin (100 g mL-1). Cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37℃. When the cells in a tissue culture flask reached ~75% confluence, they were subcultured (1:10) for passage or seeded in multiwell plates at a given concentration for specific experiments (see below).

2.6 Transfection Efficiency of Electrospun Mats Encapsulated with pDNA Polyplexes

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The transfection activities of polyplex loaded electrospun mats were investigated in C2C12

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and MC3T3-E1 cells by fluorescence microscopy and flow cytometry using a gWIZ-GFP plasmid expressing the reporter GFP gene. All sample preparation and handling in these experiments were

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carried out in a sterile biological safety hood. Prior to transfection studies, C2C12 and MC3T3-E1

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cells were seed in 48 well-plates at 10,000 cells/well (0.5 mL medium/well), and then incubated at a

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humidified atmosphere of 95% air and 5% CO2 at 37℃ for 20 h. The free polyplex samples (i.e., unspun samples) were directly added to each well at pDNA concentrations of 0.25, 0.5, and 0.75 µg

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mL-1. For transfections with polyplexes in mats, the fiber mats were first dissolved with serum free

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media (DMEM/F12) and a pre-determined volume of these solutions were added into each well to give final pDNA concentrations of 0.25, 0.5 and 0.75 µg mL-1 (based on SybrTM Green I estimated pDNA content in mats). After 24 h of incubation, the cells that expressed GFP were directly observed with an Olympus FSX100 Fluorescence Microscope. After 48 h of incubation, the cells were washed with HBSS, trypsinized and fixed with 150 µL of 3.7% formaldehyde. The transfection efficiency was quantified based on GFP positive population and mean fluorescence intensity of the cells using a BD AccuriTM C6 Plus flow cytometer with BD CSampleTM Plus software.

2.7 BMP-2 Bioactivity by Alkaline Phosphatase (ALP) Assay

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Journal Pre-proof To analyze the osteogenic differentiation of cells, cellular ALP activity was determined by pNPP hydrolysis using a kinetic ALP assay. Different electrospun Gel(PBS/EtOH), Gel(TFE) and Gel-PEG mats were used to encapsulate the gWIZ-GFP and pBMP-2 polyplexes. All sample preparations were carried out under sterile conditions. Prior to addition of samples to cells, C2C12 and MC3T3-E1 cells were seeded in 48 well-plates at 30,000 cells/well (0.5 mL medium/well). After incubation for 20 h, the samples were directly added to each well in the same manner as described in the transfection procedure. The ALP assay was performed after the incubation of the

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cells for 7 days. To perform the ALP assay, the cells were washed with HBSS solution and lysed

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with 200 μL of ALP buffer (0.5 M 2-amino-2-methylpropan-1-ol and 0.1% (v/v) Triton X-100; pH 10.5). After 2 h, 200 μL of 1.0 mg mL-1 ALP substrate p-NPP was added to 200 μL of the cell

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lysate, and the rate of change in the optical density was measured at 405 nm by a BioTek ELx800

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Universal Microplate Reader with intervals of 90 s for seven cycles. Untreated cells served as the

(mAbs/min).

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2.8 Statistical Analysis

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negative control. The kinetic ALP activity was expressed as the change of optical density per min

Experimental results were represented as the means ± standard deviation (SD), and the data had n ≥ 3 independent experiments. Statistical analyses were performed using a one-way analysis of variance (ANOVA), followed by Tukey’s post hoc tests in SPSS (IBM SPSS, USA). The level statistical significance used to compare different treatment groups was set at p<0.05.

3. Results and Discussion 3.1 Characterization of Electrospun Mats Encapsulated with pDNA Polyplexes 3.1.1 Morphology of Electrospun Mats. The morphology and average fiber diameters of electrospun mats are shown in Figure 1 and Table 1 (see also Figure 1S in Supporting Information), respectively. After initial studies to optimize Gel concentration, all mats were electrospun from

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Journal Pre-proof 10% Gel as the base polymer. Higher Gel concentrations resulted in uneven extrusion during the process whereas mats from lower concentration lacked integrity during handling. The three different types of electrospun mats were studied: (i) Gel(PBS:EtOH) mats prepared from gelatin in PBS:EtOH solvent, (ii) Gel(TFE) mats prepared from gelatin in TFE solvent, and (iii) Gel-PEG mats prepared from the mixture of 10% Gel in TFE and 10% of PEG in water. Each type of mats is prepared with and without pDNA polyplexes. Gel in PBS:EtOH was initially employed for electrospinning by Erencia et al. [23]; Gel in pure EtOH does not dissolve and minimal amount of

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PBS needs to be added to break down the Gel networks with Na+ and K+. The Gel solution

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containing low salts concentration of PBS (<10X) was not suitable for electrospinning because of large amount of beads in fiber mats.

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The electrospun Gel(PBS:EtOH) mats without pDNA polyplexes was smooth and fine, with

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an average fiber diameter of 491.1 ± 112.4 nm (Figure 1A). This was in line with fiber diameters

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(300-400 nm) independently reported from 12% Gel(PBS/EtOH) solution [23]. For electrospun Gel(PBS:EtOH) mats with pDNA polyplexes, there was no apparent difference during the

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electrospinning process but the average diameter of the fibers was decreased to 345.3 ± 126.0 nm

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based on SEM analysis. The fibers also contained beads with the average diameters of 513.1 ± 129.9 nm (Figure 1B). The dispersion of pDNA polyplexes that have cationic gene carrier in polymer solutions can increase the charge density of the polymer solutions, making it more conductive. This result may be attributed to the higher charge density formation resulting in an increase in force exerted on the jet during electrospinning, which reduces the fiber diameters and/or form the beads in the fibers [29]. In the case of Gel(TFE), the electrospun mats without pDNA polyplexes showed smooth fibers (Figure 1C), with the average fiber diameters of 1118.1 ± 513.3 nm, a value significant larger than the fiber size for the Gel(PBS:EtOH). This phenomenon may be due to the differences in the conductivity of the Gel solution. Generally, the viscosity and surface tension significantly change upon salt addition from PBS into a polymer solution. The Gel(PBS:EtOH) solution containing high

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Journal Pre-proof amount of salts will have stronger charge density and elongation forces on the surface of the ejected jests during spinning, leading to decreased diameters of fibers [30]. In contrast, the electrospun Gel(TFE) mats electrospun with pDNA polyplexes exhibited a continuous randomly arranged fiber network with the average fiber diameters of 169.2 ± 32.3 nm (Figure 1D). The decrease in fiber diameters from the addition of pDNA polyplexes was expected to due to the accumulation of a higher charge density from cationic polymer leading to a significant increase in jet elongation in electrospinning. However, incorporation of pDNA polyplexes during electrospinning of Gel(TFE)

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mats did not cause ‘neading’ in the fibers, while the bead formation was obtained in the electrospun

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Gel(PBS:EtOH) mats. The Gel(TFE) solution might display the optimal conductivity, viscosity, surface tension and vapor pressure that favor bead-free fibers [31]. Zhang et al. reported that the

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electrospun mats from 10% Gel(TFE) solution was bead-free and randomly arrayed fibers with

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fiber diameter of 200-300 nm (with electric field of 10 kV/13 cm and flow rate of 0.8 mL/h) [32].

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However, other operating parameters can influence the fiber diameter [33] and can explain larger diameters observed by Zhang et al [32].

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PEG blending into Gel solutions (typically at a volume ratio of 50:50) was additionally

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explored for electrospinning. Gel/PEG mats were previously fabricated [34] using modified PEGs and Gel as an ‘additive’. As shown in Figure 1E, the electrospun Gel-PEG50 mats without pDNA polyplexes showed poor quality fibers containing many beads in fibers. The average fiber diameter in our hands were 91.8 ± 23.1 nm with bead sizes of 483.2 ± 157.2 nm. Similarly, the pDNA polyplexes encapsulated in electrospun Gel-PEG50 mats showed beads in fibers (Figure 1F) and had an average fiber diameter of 146.9 ± 50.2 nm and bead sizes of 667.3 ± 402.2 nm. These are smaller size fibers as compared to the mats in Mollet et al. (680 ± 230 nm) [34], but the size difference in understandable considering the differences in spinning concentration, solvent (1,1,1,3,3,3-hexafluoro-2-propanol) and voltage gradient (12 kV/12 cm) in the latter study. Comparing the Gel(TFE) and Gel-PEG50 electrospinnings, it was observed that the incorporation of the PEG in the Gel solution, beads in fibers were obtained. This was most likely due to

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Journal Pre-proof insufficient resistance of the polymer solution against the stretching forces under the voltage gradient due to low viscosity and high surface tension of electrospinning solution [29]. The electrospun mats from Gel are expected to have a high surface area to volume ratio, but other processes, for example using a microfluidic bubbling system via a T-junction [35], were also reported to create gelatin scaffolds with homogenous and high surface area to volume ratio, allowing enhanced cell adhesion, viability, and migration through the interconnected porous network. The latter process, however, is sensitive to gas pressure and excess pressure was observed

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to result in loss of near-monodisperse bubble generation [36].

3.1.2 Confirmation of pDNA Polyplexes Loaded into Nanofiber Mats. The fact that beaded

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structures were seen only for polyplex electrospun Gel(PBS:EtOH) and Gel-PEG50 mats suggested

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the entrapment of polyplex inside the fiber structures. To confirm and investigate the distribution of

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pDNA polyplexes in the fiber mats, Cy3-labeled pDNA polyplexes were electrospun under similar conditions and confocal microscopy was used to confirm the presence of pDNA in mats. Strong red

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signal of Cy3-pDNA was readily detected on the surface of all fiber mats (Figure 2). Furthermore,

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Z-stack image for each mats (shown in Figure 3S, Supporting Information) confirmed that the fabricated mats successfully entrapped the polyplexes, which were uniformly distribution throughout the mats.

3.1.3 Determination of pDNA Encapsulation Efficiency. To further quantitate the extent of pDNA encapsulation during electrospinning process, pDNA encapsulation efficiency was determined in mats. Different mats containing the pDNA polyplexes were assessed for percentage of encapsulation efficiency by using 4 and 8 µg of pDNA during the electrospinning process. All mats were dissolved in water for this analysis; the dissolution was rapid and complete in <1 min with no differences among different fiber types. This was indicative of no undesirable crosslinking reaction during the fabrication process that might have prevented dissolution of the mats. The rapid

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Journal Pre-proof dissolution also facilitated analysis since no polyplexes were presumably retained in the mats, unlike the situation where the mats do not readily dissolve (e.g., with collagen) and extra digestion steps are need to be included to extract the entrapped pDNA (e.g., collagenase digestion) and lead to possible errors in analysis. As shown in Figure 3A, the encapsulation efficiency for electrospun pDNA polyplexes-loaded Gel-PEG50 fiber mats was in the range of 65-71%, which was significantly (p<0.05) higher than those of pDNA polyplexes in Gel(PBS:EtOH) mats (34 - 41%) and Gel(TFE) mats (34 - 51%). This phenomenon may be attributed to the choice of solvents and

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the the presence of PEG. For examples, high amount of salts and EtOH in the 2:3 PBS(20X):EtOH

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solvent can cause pDNA damage that may affect the encapsulation of pDNA polyplexes in the mats [37,38]. Our results are in line with a previous report has reported increased encapsulation

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efficiency with PEG-modified thiolated Gel nanoparticles, that was attributed to steric hindrance

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caused by PEG addition on the surface of nanoparticles to prevent DNA damage from the

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surrounding [39]. Comparing the encapsulation efficiency between the 4 µg vs. 8 µg processes, the encapsulation efficiency did not vary too much between the 2 amounts, but better encapsulation

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with PEG-bearing mats.

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efficiency was evident with Gel(TFE) mats for the 8 µg amount, reaching to high levels observed

3.1.4 Particle Size and Charge Analysis The particle size and surface charge of polyplexes are major parameters to determine the cellular uptake efficiency and their delivery efficiency in vivo [40,41]. The particle size and surface charge of free pDNA polyplexes and pDNA polyplexes released from the electrospun mats were analyzed (Figure 3B). The size of free pDNA was found to be 473.0 ± 33.5 nm, while the pDNA polyplexes had a hydrodynamic size of 82.5 ± 0.5 nm. The free polyplexes were additionally investigated with the TEM, which yielded average particle sizes of ~83 nm in line with hydrodynamic size measurements (Figure 2S, Supporting Information). These are ideal sizes for particles to undergo cellular internalization since larger particles were shown to be less optimal for uptake [42,43]. The particle size of pDNA polyplexes released from

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Journal Pre-proof mats were in the range of 65.5 ± 15.5 to 80.4 ± 17.0 nm, similar in size compared to the free (i.e., un-spun) polyplexes. It appears that the applied voltage gradient during the electrospinning process did not compromise the integrity of the particles. It is in principle possible that the polyplexes might have dissociated during the electrospinning and they might have re-assembled during dissolution process, but this is unlikely to yield similar size particles with the presence of excess gelatin and PEG in medium. The optimum surface charges for polyplexes should minimize non-specific clearance of

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polyplexes and prevent their loss to undesirable surfaces. The cellular uptake of polyplexes via

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receptor-mediated of endocytosis pathway is facilitated by electrostatic interactions between anionic cell membranes and cationic polyplexes [44,45]. Our results showed that the -potential of the free

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pDNA were initially negative (-13.4 ± 0.1 mV) and it became positively-charged at +20.0 ± 0.5 mV

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after polyplex formation with the polymer carrier ALL-Fect. The surface charge of pDNA

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polyplexes released from the mats were in the range of +6.2 ± 0.2 to +21.1 ± 6.3 mV. It appeared that the polyplexes in 20X PBS mats retained their original -potential while the other polyplexes

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displayed reduced -potential. Gelatin may have incorporated (or coated) into the polyplexes,

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reducing the -potential. Our past studies [46] indicated that gelatin coating of polyplexes results in better stability of the particles, in terms of better dissociation after prolonged incubation and better preservation of cell uptake. The -potential was also reduced with gelatin coating (by ~50%), which was in line with the current observations, without compromising the transfection efficiency of polyplexes. Nevertheless, all polyplexes retained a net positive charge in mats that should facilitate interactions with the cell surface membranes upon release from the mats.

3.2 In Vitro Transfection Efficiency To evaluate the ‘bioactivity’ of electrospun polyplexes, transfection experiments were carried out using GFP as a reporter gene in C2C12 (Figure 4A and B) and MC3T3 (Figure 5A and B) cells. Figure 4A and 5A show GFP transfection observed by fluorescent microscopy after 24 h of 15

Journal Pre-proof transfection. A significant difference in transfection efficiency was observed between the pDNA polyplexes with and without pAsp additive. This was the case for both free polyplexes as well as that polyplexes encapsulated into electrospun Gel(PBS:EtOH) mats. At equivalent concentrations (0.25, 0.5 and 0.75 µg mL-1 pDNA), transfection efficiencies from the mats were significantly less. To quantitate GFP expression, the samples were analyzed by flow cytrometry 48 h posttransfection. Some GFP expression was evident in polyplexes without the pAsp additive (~10% GFP-positive cells), but a large increase was seen in flow cytometry analysis upon formulating the

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complexes with the additive (>90% GFP-positive cells) (Figure 4B). Increasing the total pDNA

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concentrations from 0.25 to 0.75 µg mL-1 increased the extent of transfection with pAsp additive complexes, but no effect was seen in the absence of the additive. With polyplexes in electrospun

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mats, GFP-positive population of C2C12 cells without pAsp was ~6%, ~17%, and ~31% for 0.25,

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0.50 and 0.75 µg mL-1, respectively, while the mats with pAsp additive had ~31%, ~50%, and

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~38% GFP-positive population, respectively. While appearing comparable, the mean values for both types of polyplexes were significantly less than free (un-electrospun) polyplexes. The

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transfection efficiencies of polyplexes in mats were additionally compared using ALL-Fect/pDNA

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ratios of 10, 15 and 20 (at pAsp/pDNA ratio of 0.5) for mat fabrication; ALL-Fect/pDNA ratios of 10 gave the highest transfection efficiency (Figure 4S, Supporting Information) and increasing the ratio further did not help in transfection. Therefore, this optimal composition was used in our subsequent experiments.

Similar trends were confirmed for the MC3T3 cells (Figure 5B); the GFP-positive population for the polyplexes without pAsp was ~21%, ~38%, and 49% for the 0.25, 0.50 and 0.75 µg mL-1 pDNA concentrations, respectively, whereas the GFP-positive population was increased to ~51%, ~60%, and ~63% with the pAsp additive, respectively. The GFP-positive population for the polyplexes without pAsp in electrospun mats was ~6%, 19%, and 34% for the 0.25, 0.50 and 0.75 µg mL-1 pDNA, respectively, while the fiber mats bearing polyplexes with pAsp was ~31%, 46%, and 35%, respectively. While the beneficial effect of pAsp additive was again confirmed (~2-fold

16

Journal Pre-proof difference based on mean GFP fluorescence), the differences were not as pronounced as the C2C12 cells, which displayed ~10-fold increased transfection with pAsp bearing polyplexes. The polyplexes entrapped in fiber mats ultimately led to a similar level of GFP expression in both C2C12 and MC3T3 cells, based on both mean GFP expressed and the percentage of the cell population modified. To improve transfection from mats, we incorporated PEG into the Gel mats and assessed transfection efficiency of gWIZ-GFP polyplexes with pAsp additive in C2C12 cells. It was not

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possible to electrospun intact mats with PEG and 20X PBS as the Gel solvent, so that the base

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solvent used in this series of studies were TFE for the Gel dissolution. Using 0.25, 0.5 and 0.75 µg mL-1 pDNA concentration and fluorescent microscopy (Figure 6A), low levels of transfection

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was again observed with the polyplexes in Gel(TFE), but the transfection efficiency was

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significantly improved at 30, 50 and 70% PEG content, although lower transfection was evident

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at the highest PEG content. The results from the flow cytometry analysis 48 h post-transfection are summarized in Figure 6B. Quantitative flow cytometry also indicated a beneficial effect of

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PEG addition on the transfection efficiency; adding 30% and 50% of PEG increased the

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transfection efficiency and provided mean GFP fluorescence/cell values half of that of free polyplexes (Figure 6B, right). Increasing the PEG content to 70% decreased the mean GFP fluorescence in line with visual observation. The GFP-positive population for polyplexes from electrospun Gel(TFE), Gel-PEG30, Gel-PEG50, and Gel-PEG70 fiber mats was ~3.8%, ~12.2%, ~28.6%, and ~7.5% for the 0.25 µg mL-1, respectively. At 0.50 µg mL-1, the corresponding values were ~9.8%, ~12.5%, ~27.8%, and ~34.5%, and, at 0.75 µg mL-1, the values were ~10.6%, ~11.5%, ~21.9%, and ~17.1%, respectively. These values were generally lower than the un-spun pDNA polyplexes (~21% to 34%) but the differences with PEG bearing mats were relatively minor. Therefore, incorporation of PEG into Gel fiber mats resulted in a significant increase in the transfection efficiency in C2C12 cells and, based on GFP expression efficiency, we considered the optimal formulation to be Gel-PEG50 mats since they showed a transfection

17

Journal Pre-proof ability that was relatively robust with respect to the concentration of pDNA used in medium . Others also reported beneficial effect of PEG in improving gene expression efficiency. For instance, Yang et al. prepared core-sheath fibers with a core loading of pDNA/PEI polyplexes inside a fiber sheath of poly(DL-lactide)-poly(ethylene glycol) (PLA-PEG) as a potential tissue engineering scaffolds. Increased PEG content in fiber mats showed faster release of polyplexes, resulting in higher transfection efficiencies of NIH3T3 fibroblasts after 7 days [47]. In addition to release, PEG might have enhanced the biocompatibility of the polyplexes [48], resulting in

3.3 Bioactivity of Electrospun BMP-2 Polyplexes

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increased transfection efficiency.

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Bone morphogenetic protein-2 (BMP-2) is known to be one of the most potent growth

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factors that induces osteoblast differentiation in both C2C12 [49] and MCT3T-E1 [50] cells.

lP

However, BMP-2 protein easily disperses away from the administered site rather than remain locally in the target tissue. BMP-2 protein must be combined with a scaffold to control its release at

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the target site over an extended period. Among several strategies, [51-53] electrospun nanofiber

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mats are one option to implement gene therapy for osteoinductive growth factors to enhance bone reconstruction. For instance, Kim et al. fabricated BMP-2-loaded polycarpolactone-gelationbiphasic calcium phosphate fibrous scaffolds to obtain higher ALP activity in MC3T3-E1 cells. Additionally, the scaffolds released pDNA encoding for BMP-2 and enhanced cell proliferation and cell adhesion for a better bone growth [14]. To investigate the effects of electrospun mats with pBMP-2 polyplexes on osteogenic differentiation behavior, ALP induction was assessed in C2C12 and MCT3T cells after 7 days of treatment. The results were summarized in Figure 7 at different concentration of pBMP-2. Two groups of electrospun mats including Gel(PBS:EtOH) and GelPEG50 were used for this study. Each type of mat was encapsulated with two different types of polyplexes, namely gWIZ-GFP polyplexes (as negative control) and pBMP-2 polyplexes. For the C2C12 cells (Figure 7A), untreated sample showed low ALP activity on day 7, which was

18

Journal Pre-proof equivalent to the ALP activity in samples treated with gWIZ-GFP complexes (either free or in mats). The free pBMP-2 polyplexes gave significantly high ALP activity as compared to gWIZGFP polyplexes, especially at 0.75 µg mL-1 pDNA concentration. The ALP activity of C2C12 cells treated with pBMP-2 polyplexes in Gel(PBS:EtOH) was not significant (equivalent to gWIZ-GFP polyplexes). However, the pBMP-2 polyplexes loaded in Gel-PEG50 mats gave significantly higher ALP induction, which was even higher than the free polyplexes for the 0.5 and 0.75 µg mL-1 pDNA concentrations.

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For MC3T3 cells, the untreated cells (Figure 7B) showed low ALP activity, whereas free

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pBMP-2 polyplexes at 0.75 µg mL-1, pBMP-2 polyplexes in Gel(PBS:EtOH) mats at 0.25 µg mL-1, and in Gel-PEG50 mats at 0.25 and 0.5 µg mL-1 mats had significantly increased ALP activity on

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day 7 (p<0.05 vs. untreated cells). The ALP activity in MC3T3-E1 cells treated pBMP-2 polyplexes

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loaded Gel-PEG50 mats at 0.75 µg mL-1 was insignificant, which might be due to some toxicity on

lP

the cells. In general, the ALP induction by the pBMP-2 polyplexes was higher in the C2C12 cells (compared to MCT3T cells) but the observed trends followed a similar pattern where PEG

na

containing mats were superior. Taken together, these results corroborate the results obtained from

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the GFP polyplexes and confirm that biologically active polyplexes could be electrospun into mats for transfection of target tissues. Such mats should provide a novel approach to regenerative medicine efforts by providing a convenient way to deliver polyplexes. Gelatin mats were chosen in this study to demonstrate the proof of principle for electrospinning bioactive polyplexes, since they readily dissolve and release the polyplexes in free form. This makes assessment of bioactivity convenient since un-dissolvable mats require forced extraction or release of entrapped pDNA over an extended time. One can envision electrospinning more resilient mats, for example from collagen or poly(lactide/glycolide) polymers, to create bioactive scaffolds for cell invasion and differentiated due to impregnated polyplexes. It may be also possible to sequester pDNA polyplexes in fiber cores to better control release rates [54]. This will be the scope of future studies in the authors’ lab.

19

Journal Pre-proof Finally, we are aware that other fabrication techniques for tissue engineering scaffold could be alternative to the electrospinning as practiced in this study. We recently reviewed various fabrication technologies to this end [55]; methods to create fibers, such as wet spinning, with and without pressure induction [56], and direct writing techniques [57] may be particularly suitable due to involvement of hydrated states (i.e., compatible with sensitive plasmid expression systems) at low temperatures. Methods involving high temperature and/or reactive species to set the scaffold will not be compatible with the bioactive gene-incorporated scaffolds. The high voltage gradient

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inherent in electrospinning, although not detrimental in our set-up, might not be suitable for more

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fragile pDNA complexes, and protective biomaterials might need to be also spinned along with

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pDNA complexes in that case.

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4. Conclusions

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In this study, pDNA polyplexes loaded Gel or Gel-PEG fiber mats were successfully prepared using an electrospinning process. The pDNA polyplexes loaded in electrospun

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Gel(PBS:EtOH) fiber mats showed nanofibers (~345 nm) and contained small beads, while

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Gel(TFE) mats showed smooth fibers and reduced size (~169 nm). The pDNA polyplex loaded GelPEG50 mats showed more heterogeneous fibers (146.9 ± 50.2 nm) with many large beads in fibers. In vitro tests, Gel fiber mats bearing pDNA polyplexes with pAsp readily transfected C2C12 and MC3T3-E1 cells with a reporter gene, as compared with pDNA polyplexes without pAsp. The incorporation of PEG to Gel fiber mats resulted in a significant increase in the transfection efficiency of fabricated mats, due to increased pDNA encapsulation efficiency. Additionally, the BMP-2 polyplexes loaded in electrospun Gel-PEG fiber mats was able to induce the expected osteogenic ALP activity in both C2C12 and MC3T3-E1 cells. Further studies on pDNA release, cytotoxicity and transfection efficiency of the fiber mats in animal models are needed. The latter will especially be critical to demonstrate that implanted electrospun mats could delivery polyplexes to host cells under physiological conditions and that the appropriate therapeutic responses are

20

Journal Pre-proof secured. We conclude that the polyplexes-loaded electrospun Gel-PEG fiber mats exhibited an excellent gene transfection efficiency, and represent a feasible approach for tissue regeneration and regeneration.

Acknowledgements The authors would like to thank the other members of Uludag Lab for their assistance in this research. We acknowledge the scientific guidance and equipment support by Dr. Cagri Ayranci

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(University of Alberta) in the development of this project. Ms. Porntipa Pankongadisak was

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supported by studentships from the Royal Golden Jubilee PhD scholarship (PHD/0094/2558), Thailand Research Fund (TRF). The financial support of the Canadian Institutes of Health Research

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(CIHR) and Natural Sciences and Engineering Council of Canada (NSERC) are acknowledged. Dr.

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Uludag is the scientific founder and share holder in RJH Biosciences Inc.

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H. Alenezi, M.E. Can, M. Edirisinghe, Experimental and theoretical investigation of the fluid behavior during polymeric fiber formation with and without pressure, Appl. Phys. Rev. 6 (2019) 041401. https://doi.org/10.1063/1.5110965 H. Jo, M. Yoon, M. Gajendiran, K. Kim. Recent strategies in fabrication of gradient hydrogels for tissue engineering applications. Macromol. Biosci. (2019) e1900300.

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Journal Pre-proof LEGENDS Figure 1

SEM images of electrospun Gel(PBS:EtOH) fiber mats without (A) and with pDNA polyplexes (B); Gel(TFE) fiber mats without (C) and with pDNA polyplexes (D); Gel-PEG50 fiber mats without (E) and with polyplexes (F) (scale bar = 2 µm)

Figure 2

Confocal fluorescence microscopy images of electrospun fiber mats with Cy3-pDNA complexes; Gel(PBS:EtOH) (first row), Gel(TFE) (second row) and Gel-PEG50 (third row). All scale bars are 20 µm. The pDNA encapsulation efficiency with 4 and 8 g pDNA loading for the 3

of

Figure 3

ro

different mats (A). Particle size and -potential of free pDNA, free pDNA

Figure 4

-p

polyplexes and pDNA polyplexes electrospun in mats (B). In vitro transfection in C2C12 cells. Fluorescence microscopic images (A) and Flow

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cytometry analysis (B) of transfection efficiency for electrospun Gel(PBS:EtOH)

In vitro transfection in MC3T3-E1 cells. Fluorescence microscopic images (A) and Flow

cytometry

analysis

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Figure 5

lP

loaded with GFP polyplexes with and without pAsp as additive.

(B)

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transfection

efficiency for

electrospun

Figure 6

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Gel(PBS:EtOH) loaded with GFP polyplexes with and without pAsp as additive. In vitro transfection in C2C12 cells. Fluorescence microscopic images (A) and Flow cytometry analysis (B) of electrospun pDNA polyplexes-loaded Gel/PEG blended fiber mats. *p<0.05 compared with GFP-loaded Gel(TFE) mats. Figure 7

ALP activity of electrospun pDNA polyplexes-loaded in Gel(PBS:EtOH) and GelPEG50 fiber mats on (A) C2C12 and (B) MC3T3 cells. *p<0.05 compared with untreated and #p<0.05 compared with BMP-2 polyplexes.

Table 1

Average fiber diameters and bead size of electrospun fiber mats without and with pDNA polyplexes.

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Figure 1. SEM images of electrospun Gel(PBS:EtOH) fiber mats without (A) and with pDNA polyplexes (B); Gel(TFE) fiber mats without (C) and with pDNA polyplexes (D); Gel-PEG50 fiber mats without (E) and with polyplexes (F) (scale bar = 2 µm) Table 1. Average fiber diameters and bead size of electrospun fiber mats without and with pDNA polyplexes. Average fiber diameters (nm) Samples

Without polyplexes Fiber Bead

With polyplexes Fiber Bead

492 ± 112

-

345 ± 126

Gel(TFE) fiber mats

1118 ± 513

-

169 ± 32

-

Gel-PEG50 fiber mats

91.8 ± 23.1

483 ± 157

147 ± 50

667 ± 402

Gel(PBS:EtOH) fiber mats

513 ± 1230

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Journal Pre-proof Bright field

Merge

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Cy3

Figure 2. Confocal fluorescence microscopy images of electrospun fiber mats with Cy3-pDNA complexes; Gel(PBS:EtOH) (first row), Gel(TFE) (second row) and Gel-PEG50 (third row). All scale bars are 20 µm.

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pDNA encapsulation efficiency (%)

A

4 g pDNA in mats 8g pDNA in mats

*

80

* #

60

40

20

50

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EG el -P

-potential (mV) -13.4 ± 0.1 +20.0 ± 0.5

80.4 ± 17.0 70.7 ± 6.4 65.5 ± 15.5

+21.1 ± 6.3 +8.0 ± 1.6 +6.2 ± 0.2

-p

Particles size (nm) 473.0 ± 33.5 82.5 ± 0.5

lP

Free pDNA pDNA polyplexes pDNA polyplexes released from - Gel(PBS:EtOH) fiber mats - Gel(TFE) fiber mats - Gel-PEG50 fiber mats

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pD N A /G

el

Samples

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B

pD N A /G

pD N A /G

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(P

BS :E tO

H

(T FE )

)

0

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Figure 3. The pDNA encapsulation efficiency with 4 and 8 g pDNA loading for the 3 different

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mats (A). Particle size and -potential of free pDNA, free pDNA polyplexes and pDNA polyplexes electrospun in mats (B).

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A

GFP

pAsp/GFP

0.25 µg mL-1 0.25 µg mL-1

0.5 µg mL-1

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0.75 µg mL-1 Gene-loaded Gel fiber mats

600000

400000

0.75 µg/mL

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300000

200000

100000

100 0.25 g mL-1 0.50 g mL-1 0.75 g mL-1

80

60

40

20

ad ed

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Mean GFP fluorescence

500000

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0.25 g mL-1 0.50 g mL-1 0.75 g mL-1

GFP positive C2C12 cells (%)

B

Gene-loaded Gel fiber mats

Polyplexes

lP

Polyplexes

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0.75 µg mL-1

Figure 4. In vitro transfection in C2C12 cells. Fluorescence microscopic images (A) and flow cytometry analysis (B) of transfection efficiency for electrospun Gel(PBS:EtOH) loaded with GFP polyplexes with and without pAsp as additive.

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GFP

A

pAsp/GFP

0.25 µg mL-1 0.25 µg mL-1

0.50 µg mL-1

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0.50 µg mL-1

0.75 µg mL-1 Gene-loaded Gel fiber mats

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B

150000

100000

80

GFP positive MC3T3-E1 cells (%)

0.25 g mL-1 0.50 g mL-1 0.75 g mL-1

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200000

50000

0.25 g mL-1 0.50 g mL-1 0.75 g mL-1

60

40

20

m at s el f

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pA sp /G

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pA sp /G

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m at s

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m at s

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G ad ed

pA sp /G FP

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ib er

m at s

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G

FP -lo G

xe s

0

0

U nt re at ed

Mean GFP fluorescence

Gene-loaded Gel fiber mats

Polyplexes

lP

Polyplexes

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0.75 µg mL-1

Figure 5. In vitro transfection in MC3T3-E1 cells. Fluorescence microscopic images (A) and flow cytometry analysis (B) of transfection efficiency for electrospun Gel(PBS:EtOH) loaded with GFP polyplexes

with

and

without

pAsp

as

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A GFP-loaded Gel(TFE) fiber mats

Polyplexes

GFP-loaded GelPEG30 fiber mats

GFP-loaded GelPEG50 fiber mats

GFP-loaded GelPEG70 fiber mats

0.25 µg mL-1

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0.50 µg mL-1

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lP

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200000 150000

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100000 50000

**

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50

** *

0

40

* **

30

* 20

* *

*

10

at s

G

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G FP

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Mean GFP fluorescence

250000

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0.25 g mL-1 0.50 g mL-1 0.75 g mL-1

60

GFP positive C2C12 cells (%)

300000

Figure 6. In vitro transfection in C2C12 cells. Fluorescence microscopic images (A) and flow cytometry analysis (B) of electrospun pDNA polyplexes-loaded Gel-PEG fiber mats. *p<0.05 compared with GFP-loaded Gel(TFE) mats. 34

0

3

5

6

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BM P-

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m at s

m at s

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d

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BS :E tO

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FP -lo

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G

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ALP activity (mAbs/min)

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xe ad s ed BM 2 p G o Ply el 2pl (P lo ex BS ad es : ed Et O G H el )m (P G BS FP at s :E -lo tO ad H ed BM )m G Pat el 2-P s lo E ad G 50 ed m G at el -P s EG 50 m at s

FP -lo

G

G

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ALP activity (mAbs/min)

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0.25 g mL-1 0.50 g mL-1 0.75 g mL-1 #

*

8

* #

6

*

*

4

*

* #

#

*

* *

Figure 7. ALP activity of electrospun pDNA polyplexes-loaded in Gel(PBS:EtOH) and Gel-

PEG50 fiber mats on (A) C2C12 and (B) MC3T3 cells. *p<0.05 compared with untreated

and #p<0.05 compared with free BMP-2 polyplexes.

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Journal Pre-proof Authors contributions

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Authors contributions were as follows: Conceptualized by PP, ET, OS and HU; Methodology developed by PP, ET and HU; Formal analysis by PP, ET and HU; Funding acquisition by OS and HU; Original draft by PP and ET; Review & editing by HU and OS.

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Journal Pre-proof Graphical abstract

Electrospun Gelatin Matrices with Bioactive pDNA Polyplexes

Porntipa Pankongadisak, Ekeni Tsekoura, Orawan Suwantong

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