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Nanofiber-mediated controlled release of siRNA complexes for long term gene-silencing applications
Biomaterials 32 (2011) 5915e5923
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Nanofiber-mediated controlled release of siRNA complexes for long term gene-silencing applications Pim-on Rujitanaroj a, Yu-Cai Wang b, Jun Wang b, Sing Yian Chew a, * a b
Division of Chemical and Biomolecular Engineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, 637459 Singapore, Singapore Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 March 2011 Accepted 23 April 2011 Available online 18 May 2011
Nanofiber scaffold-mediated delivery of small-interfering RNA (siRNA) holds great potential in regenerative medicine by providing biomimicking topographical signals and enhanced gene silencing effects to seeded cells. While the delivery of naked siRNA was demonstrated previously using poly (e-caprolactone) (PCL) nanofibers, the resulting siRNA release kinetics and gene knockdown efficiencies were sub-optimal. In this study, we investigated the feasibility of encapsulating siRNA and transfection reagent (TKO) complexes within nanofibers comprising of a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP, diameter w 400 nm). Sustained release of bioactive naked siRNA and siRNA/TKO complexes were obtained for at least 28 days. By copolymerizing EEP with caprolactone, siRNA release was significantly enhanced (total siRNA that was released by day 49 was w 89.3e97.2% as compared to previously reported 3% by plain PCL nanofiber delivery). Using GAPDH as the model protein, bioactivity analyses by supernatant transfection revealed the partial retention of bioactivity of naked siRNA and siRNA/TKO complexes for at least 30 days. In particular, GAPDH siRNA/TKO supernatant alone induced significant gene silencing (w40%), indicating the feasibility of co-encapsulating siRNA and transfection reagent within a single scaffold construct for sustained delivery. Direct culture of cells on siRNA incorporated scaffolds for scaffold-mediated gene transfection revealed significant gene knockdown even in the absence of transfection reagent (21.3% knockdown efficiency by scaffolds incorporating naked siRNA only). By encapsulating siRNA/TKO complexes, more significant gene knockdown was obtained (30.9% knockdown efficiency as compared to previously reported 18% by plain PCL scaffold-mediated transfection). Taken together, the results demonstrated the feasibility of co-encapsulating siRNA-transfection reagent complexes within a single nanofiber construct for sustained siRNA delivery and enhanced gene knockdown efficiency. The study also highlights the potential of PCLEEP as a platform for tailoring siRNA release kinetics for long-term gene silencing applications. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: RNA interference Scaffold-mediated reverse transfection Tissue engineering Electrospinning Gene knockdown
1. Introduction RNA interference (RNAi) using small-interfering RNA (siRNA) has found useful applications in biomedical research, ranging from the treatment of cancer and genetic disorders to the understanding of basic cell biology and cell signaling pathways [1e3]. The use of siRNA in tissue engineering, however, remains limited. As opposed to the typical approach of enhancing the expressions of biochemical signals for tissue regeneration, RNAi adopts the opposite by silencing targeted genes. This method is attractive particularly in
* Corresponding author. Tel.: þ65 6316 8812; fax: þ65 6794 7553. E-mail address: [email protected] (S.Y. Chew). 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.04.065
areas where regeneration of tissues is often hindered by inhibitory factors such as in the case of the central nervous system. Since tissue regeneration often requires prolonged time periods, to effectively translate RNAi technology into regenerative medicine, sustained availability of siRNA may be required. Thus far, the use of siRNA is often in the form of microsphere-encapsulation or nanoparticle complexation involving liposomes [4e6], cationic polymers [7e9], and peptide conjugation [8,10e13]. Although good transfection efficiencies and desired cellular responses have been reported, the silencing effects are often transient. Scaffoldmediated delivery of siRNA may serve as a potential alternative due to the possibility of allowing a sustained availability of siRNA at the site of injury. The enhanced transfection efficiencies associated with scaffold-mediated reverse transfection is a further advantage [14e16]. Additionally, it is possible to impart topographical [17,18]
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and matrix compliance signals [19] through proper scaffold designs for a synergistic control over cell fate. Nanofiber scaffolds serve as ideal tissue constructs due to their close resemblance of the natural extracellular matrix (ECM) in terms of architecture and size scale. Fibrous topographical cues can direct cellular response [17,18,20]. Compared to 2D substrates, nanofiber constructs offer more three-dimensional microenvironment to seeded cells and induce more physiologically relevant cellular phenotypes [21]. Derived from the versatile electrospinning technique, these fibers can be easily functionalized with biochemical signals to enhance tissue regeneration [22e25]. The feasibility of encapsulating siRNA via electrospinning was demonstrated previously using poly(e-caprolactone) (PCL) with poly(ethylene glycol) (PEG) as the porogen [16]. Although the presence of PEG allowed control over siRNA release kinetics, cell viability was compromised. Furthermore, while enhanced cellular uptake of siRNA was observed by scaffold-mediated reverse transfection, gene silencing efficiency was low (18e26%) and required the supplementation of transfection reagent for more substantial gene knockdown (65e76%). This requirement of a separate addition of transfection reagent inevitably reduced the versatility of the siRNA-encapsulated nanofibers for long-term gene silencing applications. In this study, we explore the possibility of using a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP) for the controlled delivery of siRNA. The feasibility of incorporating transfection reagent and siRNA complexes within a single nanofibrous construct to enhance gene knockdown efficiency was also explored. PCLEEP is a copolymer with hydrophilic properties, low cytotoxicity and good biocompatibility [22,26,27]. Its degradation rate and associated drug release profiles may be tailored by altering copolymer ratio and molecular weight.
2. Materials and methods Ethyl ethylene phosphate (EEP) was purchased from Sinopharm Chemical Reagent Co., Ltd., China. Negative siRNA, DEPC-treated PBS (pH 7.4) and DEPCtreated TE buffer (pH 8.0) were purchased from 1st Base, Singapore. SilencerÒ GAPDH siRNA was purchased from Ambion, USA. TransIT-TKO (TKO) reagent was obtained from Mirusbio, USA. RiboGreenÒ reagent Quanti-ITTM RiboGreen, TRIzolÒ reagent, SYBRÒ Green II RNA Gel Stain, DAPI and Oregon GreenÒ Phalloidin 488 were purchased from Invitrogen, USA. Dulbecco’s Modified Eagle’s Medium (DMEM) and bovine calf serum (BCS) were obtained from Hyclone, USA. Penicillin-streptomycin (10,000 U/mL) was purchased from Gibco, Invitrogen, USA. RQ1 RNase-free DNase, SensiscriptÒ RT kit and iQ SYBR Green Supermix were purchased from Promega, USA, Qiagen Germany and Bio-rad, USA, respectively. WST-1 assay reagent was purchased from Roche, Germany. 2,2,2-trifluoroethanol (TFE, 99.0), tetrahydrofuran (THF, 99.9), chloroform (99.9), 10% Formalin and Triton X-100 were obtained from SigmaeAldrich, USA. All chemicals were used as received without any further purification.
Loading efficiency ¼
2.2. Electrospinning of siRNA-encapsulated PCLEEP nanofibers PCLEEP was dissolved in TFE to obtain a 10% w/v polymer solution. siRNA was reconstituted in RNase-free water to obtain a stock solution of 50 mM concentration. To obtain naked siRNA-encapsulated fibers (denoted as siRNA), 15 mL of siRNA stock solution was mixed with 35 mL of TE buffer. Thereafter, the mixture was added into 500 mL of PCLEEP solution (PCLEEP40k or PCLEEP25k) for electrospinning. To obtain nanofibers encapsulating siRNA and transfection reagent, TKO, complexes (denoted as siRNA/TKO), 5 mL of TE buffer was first added into 30 mL of TranIT-TKO reagent for 10 min. Following that, 15 mL of siRNA stock solution was added and incubated for another 10 min. The resulting 50 mL of siRNA/TKO complex solution was finally added into 500 mL of PCLEEP40k solution for electrospinning. Negative siRNA was used for electrospinning optimization and siRNA release kinetics studies. For in vitro gene knockdown evaluation, GAPDH siRNA was chosen as the model siRNA. A flow rate of 1.5 ml/h (New Era Pump) was adopted for all electrospinning processes. High DC voltage (GAMMA high voltage research, USA) was applied to the polymer mixture at a positive voltage of 10e12 kV. A stationary negatively charged aluminum foil (3e4 kV) was used as the collector. The distance between the polymer supply and the collector was 10e12 cm. All electrospinning parameters were set after initial optimization studies to obtain fibers with uniform diameters. 2.3. Evaluation of scaffold morphology SiRNA- and siRNA/TKO-encapsulated PCLEEP nanofiber meshes were sputter coated with platinum (Pt) and evaluated by scanning electron microscopy (SEM) (JOEL, JSM-6390LA, Japan). The average fiber diameters were determined using Image J (NIH, USA) by measuring 100 fibers per sample. The results were finally presented as mean standard error (SE) of mean. 2.4. Analysis of siRNA release kinetics The release kinetics studies were conducted under dynamic conditions. Scaffolds encapsulating negative siRNA (average weight ¼ 75 5 mg, n ¼ 3) were incubated in 5 mL of PBS at 37 C, 70e90 rpm (Sartorius CertomatÒ R, Germany). At fixed time points, 2 mL of supernatant was retrieved and replenished with 2 mL of fresh PBS. To quantify the amounts of siRNA that was released within the first day, early time points of 0, 2, 4, 6 and 24 h were chosen. The initial burst release of siRNA at 0 h, was quantified by measuring siRNA concentration after initial immersion in PBS. The concentration of siRNA within the supernatants was then determined using RiboGreenÒ assay following manufacturer’s protocol. Fluorescence intensity measurement was carried out using a microplate reader (TecanÒ, Infinite 200, Austria). After the release study, all remaining scaffolds were dried and kept at 20 C until further evaluation. The experimental loading efficiency of siRNA was evaluated based on previous protocols [16]. Briefly, siRNA- and siRNA/TKO-encapsulated nanofibrous scaffolds (n ¼ 3) were cut into halves and dissolved in 1 mL of chloroform each for higher extraction efficacy. Thereafter, 200 mL of TE buffer was added and the aqueous phase was collected. This process was repeated three times. The amount of siRNA within the extracted aqueous solution was finally determined using RiboGreenÒ assay. The efficiency of extracting siRNA from PCLEEP solution was also accounted for by doping PCLEEP solutions with known amounts of siRNA or siRNA/TKO mixtures. Thereafter, the polymer/siRNA or polymer/siRNA/TKO mixtures were placed under vacuum overnight at room temperature to remove all solvents. Next, dry polymer/ siRNA or polymer/siRNA/TKO constructs (n ¼ 3) were cut into halves and dissolved in 1 mL of chloroform each. siRNA was finally extracted and quantified as described above to obtain extraction efficiencies. The experimental loading efficiencies were finally computed using the following equation:
Total amount of siRNA released þ Extracted amount of siRNA after accounting for extraction efficiency 100% Original siRNA theoritical loading
2.1. Poly (e-caprolactone-co-ethyl ethylene phosphate), PCLEEP
2.5. Analysis of structural integrity of nanofiber encapsulated siRNA
PCLEEP copolymer with 1% ethyl ethylene phosphate (EEP) was synthesized through bulk ring-opening polymerization of e-caprolactone and EEP according to previous protocols [28,29]. From our preliminary results, PCLEEP copolymer with more than 1% EEP may be too soft for electrospinning. Therefore, 1% EEP was chosen for the preparation of the nanofibrous scaffolds used in this study. Briefly, e-caprolactone and EEP were mixed and polymerized at 120 C for 48 h with Sn(Oct)2 as the catalyst. Thereafter, the resulting polymer was dissolved in dichloromethane and precipitated in cold ethyl ether/methanol (10/1 v/v), filtrated and dried under vacuum. The precipitated polymer was further purified by dissolving in THF and precipitated in water at room temperature. Thereafter, the mixture was filtrated and freeze-dried to produce the final product. PCLEEP of molecular weight 40,000 and 25,000 (denoted as PCLEEP40k and PCLEEP25k respectively) were used in this study.
The integrity of siRNA encapsulated within PCLEEP fibers was tested using a 1% agarose gel with SYBRÒ Green II. The positions of the resulting bands were compared with fresh naked siRNA as the positive control. To evaluate the integrity of siRNA in the supernatant, 0.07g of siRNA-encapsulated scaffolds were cut into small pieces of 2e5 mm in size and immersed in 2 mL of PBS in a microcentrifuge tube. 100 mL of the supernatant was taken out and replaced with fresh PBS at each time point. Following that, 18 mL of each supernatant was loaded for gel electrophoresis. To test the integrity of siRNA that remained within the fibers after controlled release of 49 days, scaffolds were washed and dried under vacuum overnight at room temperature. Thereafter, they were dissolved in 1 mL of chloroform followed by extraction with 200 mL of TE buffer. Finally, 18 mL of the extracted solutions were used for gel electrophoresis.
P.-o. Rujitanaroj et al. / Biomaterials 32 (2011) 5915e5923 2.6. Bioactivity analysis of nanofiber encapsulated siRNA 2.6.1. Supernatant transfection NIH 3T3 cells (P15-P30) were cultured in complete medium comprising of DMEM supplemented with 10% BCS and 1% penicillin-streptomycin. Cells were maintained in a humidified incubator at 37 C with 5% CO2. For transfection, cells were kept within 15 passages to ensure similar cellular activity. GAPDH siRNA- and GAPDH siRNA/TKO-encapsulated PCLEEP40k scaffolds (n ¼ 3) with an average weight of 70 mg were sterilized under UV for 1 h. Following that, the scaffolds were placed in 24-well culture plates and immersed in 2 mL of DMEM. The plates were kept in a humidified incubator at 37 C with 5% CO2. At days 5, 10, 15 and 30, 500 mL of siRNA supernatant was removed from each well and replenished with 500 mL of fresh DMEM. One day before transfection, 3T3 cells were plated at a density of 1.0 105 cells/well in 24-well plates with 500 mL of complete medium. For transfection using GAPDH siRNA supernatant, 2.0 mL of TKO reagent was mixed with 150 mL of supernatant for each well. For transfection using GAPDH siRNA/TKO supernatant, 150 mL of supernatant was added directly into each well without further TKO supplementation. The positive control comprised of cells subjected to 20 nM of GAPDH siRNA with 2.0 mL of TKO. After the addition of siRNA, DMEM was added to each well to obtain a final volume of 300 mL per well during transfection. Twenty four hours later, 300 mL of fresh complete medium was added to each well. The negative control comprised of cells that were not subjected to any treatment. Forty-eight hours after transfection, 3 wells of cells were pooled for each sample and GAPDH mRNA expression levels were evaluated by real-time PCR. The entire transfection experiment was repeated 3 times. 2.6.2. Scaffold-mediated transfection GAPDH siRNA and GAPDH siRNA/TKO-encapsulated PCLEEP40k scaffolds (n ¼ 3) with an average weight of 70 mg were cut to fit the wells of 24-well plates and sterilized under UV for 1 h. To evaluate cellular attachment onto nanofibers, 3T3 cells were seeded at 1.0 104 cells/well in 24-well plate on plain PCLEEP fibers. Glass cover slips were used as the 2D controls. Twenty-four hours after cell seeding, cells were fixed with 10% formalin for 30 min at room temperature, permeabilized in 0.3% Triton X-100 for 20 min and stained with DAPI (1:1000 dilution) and Oregon GreenÒ Phalloidin 488 (1:500 dilution) for 30 min. Finally, cells were observed under a confocal microscope (Zeiss, LSM 710 Meta Laser Scanning Confocal Microscope, Germany). For transfection, 3T3 cells were seeded onto the scaffolds at 1.0 105 cells/well in 500 mL of complete medium. In the case where soluble TKO was supplemented (denoted as T-GAPDH siRNA or T-GAPDH siRNA/TKO for scaffolds encapsulating GAPDH siRNA and GAPDH siRNA/TKO respectively), 2.5 mL of TKO was added into each well 24 h following cell seeding. For all samples, half of the medium was replaced with fresh complete medium 48 h after transfection. Ninety-six hours after initial cell seeding, cells were lysed for real-time PCR analysis. For each experimental group, 3 wells of cells were pooled to obtain sufficient RNA. The positive control comprised of cells subjected to 20 nM of GAPDH siRNA with 2.0 mL of TKO. Cells cultured on tissue culture polystyrene without any treatment were used as the negative control. The entire transfection experiment was repeated 3 times.
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positive and negative controls). The total cell number was computed for by multiplying cell density (cells per unit area, which was obtained by DAPI staining and fluorescent imaging of 10 fields per sample at 20 magnification) with the total scaffold area for that sample. 2.9. Statistical analyses All quantitative values were expressed as a mean standard deviation, unless otherwise stated. Statistical comparisons for fiber diameter, cell viability and silencing efficiency, were performed using one-way ANOVA and Tukey post-hoc tests after verifying equal variances. The student’s t-test was used for statistical comparisons involving 2 samples. P < 0.05 was considered statistically significant.
3. Results 3.1. Morphology of siRNA-encapsulated nanofibers SiRNA-encapsulated PCLEEP fibers were successfully fabricated by electrospinning as indicated in Fig. 1. Bead-free fibers with similar diameters were obtained. The average diameters of GAPDH siRNA- and GAPDH siRNA/TKO-encapsulated nanofibers were 408 21 nm and 398 18 nm, respectively. 3.2. Release kinetics of siRNA from PCLEEP nanofibers Sustained release of siRNA was detected for up to 28 days after initial burst releases upon immersion into PBS (Fig. 2). The
2.7. Evaluation of gene silencing by real-time RT-PCR RNA was isolated using TRIzolÒ reagent. RQ1 RNase-free DNase was added to the isolated RNA to improve RNA quality. Reverse transcription was carried out using SensiscriptÒ RT kit according to manufacturer’s protocol. GAPDH mRNA expression levels were determined by real-time PCR using iQ SYBR Green Supermix in an iCycler iQ5 real-time PCR detection system (Bio-rad, USA), with b-actin as the housekeeping gene. Primer sequences for GAPDH gene were: forward 50 -TCAACAGCAACTCCCACTCTTCCA-30 and reverse 50 -ACCCTGTTGCTGTAGCCGTATTCA-30 with a product size of 115 bp. Primer sequences for b-actin were: forward 50 TGTGATGGTGGGAATGGGTCAGAA-30 and reverse 50 -TGTGGTGCCAGATCTTCTCCATGT-30 with a product size of 140 bp. Our preliminary studies showed that these two primers had similar amplification efficiency under the parameters used. Therefore, the DDCT method was used for fold change analysis. The real-time PCR cycling condition used was: 3 min at 95 C, 40 cycles at 95 C for 15 s, followed by 59 C for 30 s. All results were normalized with respect to the negative control. 2.8. Cell viability analyses To evaluate cell viability under supernatant transfection and scaffold-based transfection settings, cells were cultured at 2.0 104 cells/well (in 96 well plates) and 1.0 105 cells/well (in 24 well plates) respectively. Each sample comprised of 3 repeats. The same transfection procedure was implemented with proportionally reduced amounts of siRNA and TKO where applicable. Forty-eight hours after transfection, cells were incubated in WST-1 reagent (1:10 dilution) for 2 h at 37 C. Finally, the relative absorbance (A450-A620) was measured using a microplate reader (Bio-rad, Benchmark PlusÔ, Japan). For scaffold-base transfection, the absorbance values were normalized by the total cell number for each sample in order to account for possible differences due to variations in cell culture substrates (nanofiber samples vs. tissue culture plates in
Fig. 1. SEM images of a) GAPDH siRNA-encapsulated and b) GAPDH siRNA/TKOencapsulated PCLEEP nanofibers illustrating uniform and bead-free fibers.
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Fig. 2. Release kinetics of naked siRNA and siRNA/TKO complexes from PCLEEP25k and PCLEEP40k nanofibers as observed in PBS at 37 C.
encapsulation of naked siRNA within PCLEEP40K fibers resulted in a significantly higher burst release than PCLEEP25k fibers. The complexation of siRNA with TKO, however, significantly reduced the initial burst release (48.5 4% vs 12.3 1% at time ¼ 0 for PCLEEP40k siRNA scaffolds and PCLEEP40k siRNA/TKO scaffolds respectively, p < 0.05). Concurrently, the rate and amount of siRNA/ TKO complexes that was released were also lower as compared to naked siRNA (total siRNA amount that was released by day 49 was 97.2 1% vs 89.3 2% for PCLEEP40k siRNA scaffolds and PCLEEP40k siRNA/TKO scaffolds respectively, p < 0.05). TKO complexation did not alter the loading efficiency of siRNA significantly. The experimental loading efficiencies for PCLEEP40k siRNA scaffolds and PCLEEP40k siRNA/TKO scaffolds were 62.2 1.5 and 59.3 1.8% respectively.
remained at least partially bioactive throughout the period of sustained release. Furthermore, GAPDH siRNA/TKO supernatant exhibited gene silencing without further supplementation of transfection reagent, suggesting the feasibility of co-encapsulating transfection reagent within the nanofibers for sustained gene silencing applications. While cell viability was slightly lower in the presence of TKO as compared to the negative control (Fig. 4b), the difference was not statistically significant. Similarly, no apparent difference was observed between GAPDH siRNA supernatant, GAPDH siRNA/TKO supernatant and the positive control at all time points.
3.3. Structural integrity of nanofiber encapsulated siRNA Fig. 3 shows the structural integrity of siRNA that was recovered from siRNA and siRNA/TKO supernatants at various time points. The molecular weight of the samples matched that of fresh siRNA indicating that the siRNA remained structurally intact throughout the period of sustained release. The lower siRNA band intensity of siRNA/TKO samples corresponded with the lower siRNA release amounts as observed from the release profiles in Fig. 2. At day 49, a significant amount of intact siRNA/TKO remained entrapped within the nanofibers (Fig. 3c). 3.4. Bioactivity analysis of nanofiber encapsulated siRNA 3.4.1. Supernatant transfection As shown in Fig. 4a, silencing efficiencies of 58% and 40% were obtained using GAPDH siRNA and GAPDH siRNA/TKO supernatants respectively. The effects were significantly different as compared to the positive and negative controls, indicating that the siRNA
Fig. 3. Structural integrity of siRNA in polymer nanofibers as evaluated by 1.0% agarose gel electrophoresis. Gel electrosphoresis results of siRNA supernatants from a) PCLEEP siRNA fibers, b) PCLEEP siRNA/TKO fibers of various time points. Lane 1: dsRNA marker, Lane 2: naked siRNA and Lane 3-8: siRNA supernatants collected at 7, 14, 21, 28, 35 and 49 days respectively. c) siRNA extracted from nanofiber scaffolds after 49 days of release study. Lane 1: dsRNA marker, Lane 2: naked siRNA, Lane 3: siRNA within PCLEEP siRNA nanofibers, and Lane 4: siRNA within PCLEEP siRNA/TKO nanofibers.
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Fig. 4. GAPDH siRNA supernatant transfection in 3T3 cells, illustrating retention of siRNA bioactivity throughout period of sustained release. a) GAPDH mRNA levels in transfected 3T3 cells, and b) cell viability of transfected 3T3 cells at 96h. T-GAPDH siRNA: GAPDH siRNA supernatants precomplexed with TKO reagent during transfection. GAPDH siRNA/TKO: GAPDH siRNA/TKO supernatants only. POS: positive control of cells on culture plate subjected to 20 nM GAPDH siRNA transfection. NEG: negative control of cells without treatment. * # $ , , , and % indicate p < 0.05 as compared to the positive control. ** indicates p < 0.05. n ¼ 3, mean SE.
3.4.2. Scaffold-mediated transfection Plain PCLEEP scaffolds supported cell attachment as illustrated in Fig. 5. While cell spreading and stress fiber formation were more prominent on the 2D controls, cells on nanofibers adopted morphological changes that were typical of cells seeded on 3D matrices. In particular, cells on nanofibers adopted more spinal-like
shape and possessed stress fibers that were located at the periphery of the cells [30]. Cells also responded to the underlying nanofiber topography with cellular protrusions (White arrows). As shown in Fig. 6a, 3T3 cells seeded directly on GAPDH siRNA and GAPDH siRNA/TKO scaffolds showed significant gene silencing as compared to untreated cells (negative control). This significant
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efficiency on both scaffolds (T-GAPDH siRNA vs T-GAPDH siRNA/ TKO, p > 0.05) Fig. 6b shows the viability of cells during scaffold-based transfection. All cells remained viable indicating that the scaffolds were cytocompatible. However, the presence of TKO significantly decreased cell viability. Cells seeded directly on the GAPDH siRNA/ TKO scaffolds appeared to show lower cell viability as compared to GAPDH siRNA scaffolds. When more TKO was added into the medium (i.e., T-GAPDH siRNA and T-GAPDH siRNA/TKO), cell viability was further reduced.
4. Discussion
Fig. 5. Confocal fluorescent microscopy images of 3T3 cells on a) glass cover slip and b) electrospun PCLEEP nanofibrous scaffold. Blue: cell nuclei; green: actin cytoskeleton. Scale bar ¼ 20 mm (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
gene knockdown was consistently observed in cells that were treated with (p < 0.05) and without (p < 0.05) the additional bolus supplementation of transfection reagent. The results clearly demonstrate the efficacy of nanofiber scaffold-mediated transfection. In the absence of TKO supplementation, a significantly higher GAPDH silencing efficiency was observed in cells that were cultured directly on GAPDH siRNA/TKO fibers as compared to GAPDH siRNA fibers (30.9% vs 21.3% respectively, p < 0.05). While the delivery of siRNA-TKO complex enhanced gene silencing efficiency as compared to naked siRNA delivery, further addition of TKO abrogated this difference, resulting in similar gene knockdown
Encapsulating biomolecules within electrospun fibers is a feasible and convenient way to produce biofunctional scaffolds that are capable of providing topographical signals and the sustained delivery of drugs [31e33] to seeded cells. Polymer fibers provide physical protection to the encapsulated biomolecules, preserve their bioactivity over prolonged time periods and enable localized release at target sites. Although we have demonstrated scaffold-mediated delivery of siRNA using PCL [16], the release of siRNA from plain PCL fibers was low even after 7 weeks in vitro, resulting in a cumulative release of only 3%. Therefore, in this study, siRNA-encapsulated PCLEEP fibers were fabricated to improve the siRNA release kinetics and scaffold-mediated gene silencing efficiency. SiRNA-encapsulated PCLEEP fibers were successfully fabricated by electrospinning and the average diameters of the samples were not significantly different. The similarity in fiber size despite the addition of TKO was likely due to the low volume used as compared to PCLEEP. This allowed the slight changes in drug composition to be tolerated by the optimized electrospinning parameters. Fiber size affects cell fate [34,35]. Therefore, maintaining similar fiber diameters throughout this study helped eliminate fiber size effect on cellular response. By encapsulating siRNA within PCLEEP fibers, a sustained release of bioactive siRNA was achieved for at least 28 days (Fig. 2). The initial burst releases are likely attributed to siRNA that was located on the surface of the scaffolds, since uniform PCLEEP-siRNA mixtures were electrospun. PCLEEP40k fibers showed significantly higher burst release as compared to PCLEEP25k fibers. This is likely due to the enhanced polymer hydrophilicity with increasing amounts of phosphate groups as PCLEEP molecular weight increased. Given the higher amount of siRNA released, PCLEEP40k was chosen for further analyses involving siRNA/TKO complex encapsulation. The rate and amount of siRNA/TKO complexes that was released were lower as compared to naked siRNA, likely due to the increase in particle size as a result of TKO complexation. As compared to using PCL, the experimental loading efficiency of siRNA within PCLEEP was similar. However, the total amount of siRNA that was being released by day 49 was significantly higher in PCLEEP. Taken together, the results demonstrated the possibility of control over siRNA release through changes in EEP content and polymer molecular weight. Further changes in copolymerization ratio and polymer molecular weight will, therefore, allow specific tailoring of siRNA release kinetics for desired applications. As shown in Fig. 3, a small amount of siRNA remained entrapped within siRNA/TKO fibers even after 49 days. Coupled with the observation that less than 8% mass loss was detected after incubating PCLEEP nanofibers under similar in vitro conditions for 3 months [31], it is likely that the encapsulated siRNA was released by diffusion and the remaining entrapped molecules can be released only upon matrix degradation. In the case of naked siRNA encapsulation, no visible band was observed within the detection limits
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Fig. 6. Silencing of GAPDH gene expression in cells seeded directly on siRNA-encapsulated PCLEEP 40k scaffolds. a) GAPDH mRNA expression levels in transfected 3T3 cells and b) cell viability of transfected 3T3 cells at 96h. T-GAPDH siRNA, T-GAPDH siRNA/TKO: cells seeded on PCLEEP siRNA and PCLEEP siRNA/TKO nanofibers with additional TKO supplementation. GAPDH siRNA, GAPDH siRNA/TKO: cells seeded on PCLEEP siRNA and PCLEEP siRNA/TKO nanofibers without TKO supplementation. POS: positive control of cells on culture plate subjected to 20 nM GAPDH siRNA transfection. NEG: negative control of cells without treatment. * indicates p < 0.05 as compared to the negative control, # indicates p < 0.05 as compared to the positive control, $ and % indicate p < 0.05. n ¼ 3, mean SE.
of SYBRÒ Green II, likely due to the higher release rate of naked siRNA. The supernatant transfection study was carried out to evaluate the bioactivity of the encapsulated siRNA, in the absence of other extracellular signaling factors such as scaffold topography, compliance and surface chemistry differences, which may affect cellular response [15,16,36] and gene silencing efficiency. When cells were treated with siRNA (precomplexed with TKO) and siRN/ TKO supernatants, significant gene knockdown was observed at all time points. This showed that the nanofibrous scaffolds provided efficient protection for the encapsulated siRNA for at least 30 days.
When treated with naked siRNA only, no gene silencing was observed in 3T3 cells. Therefore, GAPDH siRNA supernatants were precomplexed with TKO prior to addition into cell culture medium. In contrast, gene silencing using supernatants from siRNA/TKO fibers resulted in a silencing efficiency of w40% even without further supplementation of transfection reagent. This suggested the possibility of co-encapsulating transfection reagent and siRNA within a single scaffold for sustained gene silencing applications. While our cellular assays indicated the retention of siRNA bioactivity, the extent of bioactivity remains unknown. However, compared to the positive control of 20 nM siRNA, the lower
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silencing efficiency of the supernatants implied that the amounts of bioactive siRNA that was present were likely less than 20 nM. In the scaffold-mediated gene knockdown study, the observed gene silencing in the absence of transfection reagent was likely due to the localized concentration of siRNA. Such cellular behavior in response to localized availability of nucleic acids was also observed previously [16,36,37]. Although lower amounts of siRNA were released from GAPDH siRNA/TKO scaffolds, GAPDH silencing efficiency was significantly higher than GAPDH siRNA scaffolds (w31% vs w21% in the latter). This gene knockdown efficiency was also greater than that achieved by PCL nanofiber delivery (18% in plain PCL and up to 26% with PEG inclusion) [16]. This indicated the need of a transfection reagent to facilitate cellular uptake of siRNA and the feasibility of co-encapsulating the transfection reagent with siRNA within a single scaffold construct for dual biomolecule delivery. Transfection reagent induces slight cytotoxicity [16,38,39]. This was also observed in the supernatant transfection study (Fig. 4), although the changes in cell viability were not statistically significant. However, nanofiber scaffolds appeared to sensitize cells towards TKO, resulting in significant changes in cell viability. While the exact reasons behind the observation remain to be elucidated, it is noteworthy that microenvironmental differences, such as topography, surface chemistry, three-dimensionality and compliance of matrices, existed between supernatant and scaffold-mediated transfection studies and could contribute to cell phenotypic changes. Although decreased cell viability was observed as compared to GAPDH siRNA scaffolds, significant number of cells remained viable on GAPDH siRNA/TKO scaffolds and cell viability was still higher as compared to additional supplementation of TKO. Further TKO supplementation could enhance gene silencing efficiency (Fig. 6a). This means that TKO reagent was also able to complex with siRNA that was released into the culture medium. Similar to supernatant transfection, an overall lower silencing efficiency was observed as compared to the positive control, likely due to the lower amounts of bioactive siRNA that was present. Comparing the results between supernatant transfection and scaffold-mediated transfection, transfection using bolus delivery of naked siRNA in the former resulted in no gene knockdown. However, scaffold-mediated transfection enabled gene silencing even in the absence of TKO. In the presence of TKO, a silencing efficiency of w 40% was obtained for GAPDH siRNA/TKO samples in both transfection studies. Similarly, a silencing efficiency of w 60% was observed in T-GAPDH siRNA samples. The results suggested that the presence of transfection reagent, either co-encapsulated within nanofibers or supplemented additionally, appeared to dominate over any effects of localized concentration of biochemicals and topography on gene knockdown. GAPDH is crucial in cell metabolic pathways and is required for long-term cell viability and survival [40,41]. As a result, gene knockdown study by scaffold-mediated approach was restricted to 96 h after initial cell seeding. Further studies focusing on other cellular targets that are less critical to cell survival, e.g. ECM deposition, are currently ongoing to evaluate the efficacy of siRNA nanofibers for long-term gene silencing applications. 5. Conclusion This study demonstrates the feasibility of delivering siRNA and transfection reagent complexes within a single nanofiber construct for enhanced gene silencing efficiency in scaffold-mediated transfection. Co-encapsulation of siRNA and TKO within PCLEEP fibers resulted in a sustained release of bioactive siRNA for at least 28 days. The copolymerization of EEP with caprolactone enhanced siRNA delivery rate and gene knockdown efficiency as compared to
PCL. Such biofunctional nanofiber constructs may provide synergistic topographical and biochemical signals for regulating cellular behaviors by RNA interference. Acknowledgements The partial funding support from AStar BMRC grant (07/1/22/19/ 519), AStar BMRC SSCC grant (09/16), AcRF Tier One grant (RG36/ 07) and the National Basic Research Program of China (973 Program, 2010CB934000) are acknowledged. P. Rujitanaroj expresses her gratitude to Ms. HQ Cao for her help with initial studies on the nanofibrous scaffold-mediated siRNA delivery. References [1] Suggate EL, Ahmed Z, Read ML, Eaton-Charnock K, Douglas MR, Gonzalez AM, et al. Optimisation of siRNA-mediated RhoA silencing in neuronal cultures. Mol Cell Neurosci 2009;40(4):451e62. [2] Veiseh O, Kievit FM, Fang C, Mu N, Jana S, Leung MC, et al. Chlorotoxin bound magnetic nanovector tailored for cancer cell targeting, imaging, and siRNA delivery. Biomaterials 2010;31(31):8032e42. [3] Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, Heimerikx M, et al. A large-scale RNAi screen in human cells identifies new components of the p53 p.thway. Nature 2004;428(6981):431e7. [4] Colonna C, Conti B, Genta I, Alpar OH. Non-viral dried powders for respiratory gene delivery prepared by cationic and chitosan loaded liposomes. Int J Pharm 2008;364(1):108e18. [5] Sioud M, Sorensen DR. Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun 2003;312(4):1220e5. [6] Chen JL, Wang H, Gao JQ, Chen HL, Liang WQ. Liposomes modified with polycation used for gene delivery: preparation, characterization and transfection in vitro. Int J Pharm 2007;343(1e2):255e61. [7] Howard KA, Rahbek UL, Liu XD, Damgaard CK, Glud SZ, Andersen MO, et al. RNA interference in vitro and in vivo using a chitosan/siRNA nanoparticle system. Mol Ther 2006;14(4):476e84. [8] Trabulo S, Resina S, Simoes S, Lebleu B, de Lima MCP. A non-covalent strategy combining cationic lipids and CPPs to enhance the delivery of splice correcting oligonucleotides. J Control Release 2010;145(2):149e58. [9] Zhang SB, Zhao B, Jiang HM, Wang B, Ma BC. Cationic lipids and polymers mediated vectors for delivery of siRNA. J Control Release 2007;123(1):1e10. [10] Davidson TJ, Harel S, Arboleda VA, Prunell GF, Shelanski ML, Greene LA, et al. Highly efficient small interfering RNA delivery to primary mammalian neurons induces microRNA-like effects before mRNA degradation. J Neurosci 2004;24(45):10040e6. [11] Lundberg P, El-Andaloussi S, Sutlu T, Johansson H, Langel U. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides. Faseb J 2007; 21(11):2664e71. [12] Crombez L, Aldrian-Herrada G, Konate K, Nguyen QN, McMaster GK, Brasseur R, et al. A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells. Mol Ther 2009;17(1):95e103. [13] Veldhoen S, Laufer SD, Trampe A, Restle T. Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect. Nucl Acids Res 2006;34(22): 6561e73. [14] Bengali Z, Pannier AK, Segura T, Anderson BC, Jang JH, Mustoe TA, et al. Gene delivery through cell culture substrate adsorbed DNA complexes. Biotechnol Bioeng 2005;90(3):290e302. [15] Jang JH, Bengali Z, Houchin TL, Shea LD. Surface adsorption of DNA to tissue engineering scaffolds for efficient gene delivery. J Biomed Mater Res Part A 2006;77A(1):50e8. [16] Cao HQ, Jiang X, Chai C, Chew SY. RNA interference by nanofiber-based siRNA delivery system. J Control Release 2010;144(2):203e12. [17] Chew SY, Mi R, Hoke A, Leong KW. The effect of the alignment of electrospun fibrous scaffolds on schwann cell maturation. Biomaterials 2008;29(6):653e61. [18] Lim SH, Liu XY, Song HJ, Yarema KJ, Mao HQ. The effect of nanofiber-guided cell alignment on the preferential differentiation of neural stem cells. Biomaterials 2010;31(34):9031e9. [19] Nathan AS, Baker BM, Nerurkar NL, Mauck RL. Mechano-topographic modulation of stem cell nuclear shape on nanofibrous scaffolds. Acta Biomater 2011;7(1):57e66. [20] Cao HQ, McHugh K, Chew SY, Anderson JM. The topographical effect of electrospun nanofibrous scaffolds on the in vivo and in vitro foreign body reaction. J Biomed Mater Res Part A 2010;3:1151e9. [21] Delgado-Rivera R, Harris SL, Ahmed I, Babu AN, Patel RP, Ayres V, et al. Increased FGF-2 secretion and ability to support neurite outgrowth by astrocytes cultured on polyamide nanofibrillar matrices. Matrix Biol 2009; 28(3):137e47. [22] Chew SY, Mi RF, Hoke A, Leong KW. Aligned protein-polymer composite fibers enhance nerve regeneration: a potential tissue-engineering platform. Adv Funct Mater 2007;17(8):1288e96.
P.-o. Rujitanaroj et al. / Biomaterials 32 (2011) 5915e5923 [23] Il Cho Y, Choi JS, Jeong SY, Yoo HS. Nerve growth factor (NGF)-conjugated electrospun nanostructures with topographical cues for neuronal differentiation of mesenchymal stem cells. Acta Biomater 2010;6(12):4725e33. [24] Cao HQ, Liu T, Chew SY. The application of nanofibrous scaffolds in neural tissue engineering. Adv Drug Del Rev 2009;61(12):1055e64. [25] Jiang X, Lim SH, Mao HQ, Chew SY. Current applications and future perspectives of artificial nerve conduits. Exp Neurol 2010;223(1):86e101. [26] Chua KN, Lim WS, Zhang PC, Lu HF, Wen J, Ramakrishna S, et al. Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold. Biomaterials 2005;26(15):2537e47. [27] Wen J, Kim GJA, Leong KW. Poly(D, Llactide-co-ethyl ethylene phosphate)s as new drug carriers. J Control Release 2003;92(1e2):39e48. [28] Xiao CS, Wang YC, Du JZ, Chen XS, Wang J. Kinetics and mechanism of 2ethoxy-2-oxo-1,3,2-dioxaphospholane polymerization initiated by stannous octoate. Macromolecules 2006;39(20):6825e31. [29] Wang YC, Li Y, Yang XZ, Yuan YY, Yan LF, Wang J. Tunable thermosensitivity of biodegradable polymer micelles of poly (epsilon-caprolactone) and polyphosphoester block bopolymers. Macromolecules 2009;42(8):3026e32. [30] Hakkinen KM, Harunaga JS, Doyle AD, Yamada KM. Direct comparisons of the morphology, migration, cell adhesions, and actin cytoskeleton of fibroblasts in four different three-dimensional extracellular matrices. Tissue Eng Part A 2011;17(5e6):713e24. [31] Chew SY, Wen J, Yim EKF, Leong KW. Sustained release of proteins from electrospun biodegradable fibers. Biomacromolecules 2005;6(4):2017e24. [32] Luu YK, Kim K, Hsiao BS, Chu B, Hadjiargyrou M. Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers. J Control Release 2003;89(2):341e53.
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[33] Kenawy ER, Bowlin GL, Mansfield K, Layman J, Simpson DG, Sanders EH, et al. Release of tetracycline hydrochloride from electrospun poly(ethylene-covinylacetate), poly(lactic acid), and a blend. J Control Release 2002;81(1e2): 57e64. [34] Christopherson GT, Song H, Mao HQ. The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials 2009;30(4):556e64. [35] Bashur CA, Shaffer RD, Dahlgren LA, Guelcher SA, Goldstein AS. Effect of fiber diameter and alignment of electrospun polyurethane meshes on mesenchymal progenitor cells. Tissue Eng Part A 2009;15(9):2435e45. [36] Pannier AK, Wieland JA, Shea LD. Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes. Acta Biomater 2008;4(1):26e39. [37] Zhang XQ, Tang HH, Hoshi R, De Laporte L, Qiu HJ, Xu XY, et al. Sustained transgene expression via citric acid-based polyester elastomers. Biomaterials 2009;30(13):2632e41. [38] Ghonaim HM, Li S, Blagbrough IS. N-1, N-12-Diacyl Spermines: SAR studies on non-viral lipopolyamine vectors for plasmid DNA and siRNA formulation. Pharm Res 2010;27(1):17e29. [39] Andersen MO, Howard KA, Paludan SR, Besenbacher F, Kjems J. Delivery of siRNA from lyophilized polymeric surfaces. Biomaterials 2008;29(4):506e12. [40] Hara MR, Agrawal N, Kim SF, Cascio MB, Fujimuro M, Ozeki Y, et al. S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat Cell Biol 2005;7(7):U640e665. [41] Tristan C, Shahani N, Sedlak TW, Sawa A. The diverse functions of GAPDH: views from different subcellular compartments. Cell Signal 2011;23(2): 317e23.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Nanofiber-mediated controlled release of siRNA complexes for long term gene-silencing applications Pim-on Rujitanaroj a, Yu-Cai Wang b, Jun Wang b, Sing Yian Chew a, * a b
Division of Chemical and Biomolecular Engineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, 637459 Singapore, Singapore Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 March 2011 Accepted 23 April 2011 Available online 18 May 2011
Nanofiber scaffold-mediated delivery of small-interfering RNA (siRNA) holds great potential in regenerative medicine by providing biomimicking topographical signals and enhanced gene silencing effects to seeded cells. While the delivery of naked siRNA was demonstrated previously using poly (e-caprolactone) (PCL) nanofibers, the resulting siRNA release kinetics and gene knockdown efficiencies were sub-optimal. In this study, we investigated the feasibility of encapsulating siRNA and transfection reagent (TKO) complexes within nanofibers comprising of a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP, diameter w 400 nm). Sustained release of bioactive naked siRNA and siRNA/TKO complexes were obtained for at least 28 days. By copolymerizing EEP with caprolactone, siRNA release was significantly enhanced (total siRNA that was released by day 49 was w 89.3e97.2% as compared to previously reported 3% by plain PCL nanofiber delivery). Using GAPDH as the model protein, bioactivity analyses by supernatant transfection revealed the partial retention of bioactivity of naked siRNA and siRNA/TKO complexes for at least 30 days. In particular, GAPDH siRNA/TKO supernatant alone induced significant gene silencing (w40%), indicating the feasibility of co-encapsulating siRNA and transfection reagent within a single scaffold construct for sustained delivery. Direct culture of cells on siRNA incorporated scaffolds for scaffold-mediated gene transfection revealed significant gene knockdown even in the absence of transfection reagent (21.3% knockdown efficiency by scaffolds incorporating naked siRNA only). By encapsulating siRNA/TKO complexes, more significant gene knockdown was obtained (30.9% knockdown efficiency as compared to previously reported 18% by plain PCL scaffold-mediated transfection). Taken together, the results demonstrated the feasibility of co-encapsulating siRNA-transfection reagent complexes within a single nanofiber construct for sustained siRNA delivery and enhanced gene knockdown efficiency. The study also highlights the potential of PCLEEP as a platform for tailoring siRNA release kinetics for long-term gene silencing applications. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: RNA interference Scaffold-mediated reverse transfection Tissue engineering Electrospinning Gene knockdown
1. Introduction RNA interference (RNAi) using small-interfering RNA (siRNA) has found useful applications in biomedical research, ranging from the treatment of cancer and genetic disorders to the understanding of basic cell biology and cell signaling pathways [1e3]. The use of siRNA in tissue engineering, however, remains limited. As opposed to the typical approach of enhancing the expressions of biochemical signals for tissue regeneration, RNAi adopts the opposite by silencing targeted genes. This method is attractive particularly in
* Corresponding author. Tel.: þ65 6316 8812; fax: þ65 6794 7553. E-mail address: [email protected] (S.Y. Chew). 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.04.065
areas where regeneration of tissues is often hindered by inhibitory factors such as in the case of the central nervous system. Since tissue regeneration often requires prolonged time periods, to effectively translate RNAi technology into regenerative medicine, sustained availability of siRNA may be required. Thus far, the use of siRNA is often in the form of microsphere-encapsulation or nanoparticle complexation involving liposomes [4e6], cationic polymers [7e9], and peptide conjugation [8,10e13]. Although good transfection efficiencies and desired cellular responses have been reported, the silencing effects are often transient. Scaffoldmediated delivery of siRNA may serve as a potential alternative due to the possibility of allowing a sustained availability of siRNA at the site of injury. The enhanced transfection efficiencies associated with scaffold-mediated reverse transfection is a further advantage [14e16]. Additionally, it is possible to impart topographical [17,18]
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and matrix compliance signals [19] through proper scaffold designs for a synergistic control over cell fate. Nanofiber scaffolds serve as ideal tissue constructs due to their close resemblance of the natural extracellular matrix (ECM) in terms of architecture and size scale. Fibrous topographical cues can direct cellular response [17,18,20]. Compared to 2D substrates, nanofiber constructs offer more three-dimensional microenvironment to seeded cells and induce more physiologically relevant cellular phenotypes [21]. Derived from the versatile electrospinning technique, these fibers can be easily functionalized with biochemical signals to enhance tissue regeneration [22e25]. The feasibility of encapsulating siRNA via electrospinning was demonstrated previously using poly(e-caprolactone) (PCL) with poly(ethylene glycol) (PEG) as the porogen [16]. Although the presence of PEG allowed control over siRNA release kinetics, cell viability was compromised. Furthermore, while enhanced cellular uptake of siRNA was observed by scaffold-mediated reverse transfection, gene silencing efficiency was low (18e26%) and required the supplementation of transfection reagent for more substantial gene knockdown (65e76%). This requirement of a separate addition of transfection reagent inevitably reduced the versatility of the siRNA-encapsulated nanofibers for long-term gene silencing applications. In this study, we explore the possibility of using a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP) for the controlled delivery of siRNA. The feasibility of incorporating transfection reagent and siRNA complexes within a single nanofibrous construct to enhance gene knockdown efficiency was also explored. PCLEEP is a copolymer with hydrophilic properties, low cytotoxicity and good biocompatibility [22,26,27]. Its degradation rate and associated drug release profiles may be tailored by altering copolymer ratio and molecular weight.
2. Materials and methods Ethyl ethylene phosphate (EEP) was purchased from Sinopharm Chemical Reagent Co., Ltd., China. Negative siRNA, DEPC-treated PBS (pH 7.4) and DEPCtreated TE buffer (pH 8.0) were purchased from 1st Base, Singapore. SilencerÒ GAPDH siRNA was purchased from Ambion, USA. TransIT-TKO (TKO) reagent was obtained from Mirusbio, USA. RiboGreenÒ reagent Quanti-ITTM RiboGreen, TRIzolÒ reagent, SYBRÒ Green II RNA Gel Stain, DAPI and Oregon GreenÒ Phalloidin 488 were purchased from Invitrogen, USA. Dulbecco’s Modified Eagle’s Medium (DMEM) and bovine calf serum (BCS) were obtained from Hyclone, USA. Penicillin-streptomycin (10,000 U/mL) was purchased from Gibco, Invitrogen, USA. RQ1 RNase-free DNase, SensiscriptÒ RT kit and iQ SYBR Green Supermix were purchased from Promega, USA, Qiagen Germany and Bio-rad, USA, respectively. WST-1 assay reagent was purchased from Roche, Germany. 2,2,2-trifluoroethanol (TFE, 99.0), tetrahydrofuran (THF, 99.9), chloroform (99.9), 10% Formalin and Triton X-100 were obtained from SigmaeAldrich, USA. All chemicals were used as received without any further purification.
Loading efficiency ¼
2.2. Electrospinning of siRNA-encapsulated PCLEEP nanofibers PCLEEP was dissolved in TFE to obtain a 10% w/v polymer solution. siRNA was reconstituted in RNase-free water to obtain a stock solution of 50 mM concentration. To obtain naked siRNA-encapsulated fibers (denoted as siRNA), 15 mL of siRNA stock solution was mixed with 35 mL of TE buffer. Thereafter, the mixture was added into 500 mL of PCLEEP solution (PCLEEP40k or PCLEEP25k) for electrospinning. To obtain nanofibers encapsulating siRNA and transfection reagent, TKO, complexes (denoted as siRNA/TKO), 5 mL of TE buffer was first added into 30 mL of TranIT-TKO reagent for 10 min. Following that, 15 mL of siRNA stock solution was added and incubated for another 10 min. The resulting 50 mL of siRNA/TKO complex solution was finally added into 500 mL of PCLEEP40k solution for electrospinning. Negative siRNA was used for electrospinning optimization and siRNA release kinetics studies. For in vitro gene knockdown evaluation, GAPDH siRNA was chosen as the model siRNA. A flow rate of 1.5 ml/h (New Era Pump) was adopted for all electrospinning processes. High DC voltage (GAMMA high voltage research, USA) was applied to the polymer mixture at a positive voltage of 10e12 kV. A stationary negatively charged aluminum foil (3e4 kV) was used as the collector. The distance between the polymer supply and the collector was 10e12 cm. All electrospinning parameters were set after initial optimization studies to obtain fibers with uniform diameters. 2.3. Evaluation of scaffold morphology SiRNA- and siRNA/TKO-encapsulated PCLEEP nanofiber meshes were sputter coated with platinum (Pt) and evaluated by scanning electron microscopy (SEM) (JOEL, JSM-6390LA, Japan). The average fiber diameters were determined using Image J (NIH, USA) by measuring 100 fibers per sample. The results were finally presented as mean standard error (SE) of mean. 2.4. Analysis of siRNA release kinetics The release kinetics studies were conducted under dynamic conditions. Scaffolds encapsulating negative siRNA (average weight ¼ 75 5 mg, n ¼ 3) were incubated in 5 mL of PBS at 37 C, 70e90 rpm (Sartorius CertomatÒ R, Germany). At fixed time points, 2 mL of supernatant was retrieved and replenished with 2 mL of fresh PBS. To quantify the amounts of siRNA that was released within the first day, early time points of 0, 2, 4, 6 and 24 h were chosen. The initial burst release of siRNA at 0 h, was quantified by measuring siRNA concentration after initial immersion in PBS. The concentration of siRNA within the supernatants was then determined using RiboGreenÒ assay following manufacturer’s protocol. Fluorescence intensity measurement was carried out using a microplate reader (TecanÒ, Infinite 200, Austria). After the release study, all remaining scaffolds were dried and kept at 20 C until further evaluation. The experimental loading efficiency of siRNA was evaluated based on previous protocols [16]. Briefly, siRNA- and siRNA/TKO-encapsulated nanofibrous scaffolds (n ¼ 3) were cut into halves and dissolved in 1 mL of chloroform each for higher extraction efficacy. Thereafter, 200 mL of TE buffer was added and the aqueous phase was collected. This process was repeated three times. The amount of siRNA within the extracted aqueous solution was finally determined using RiboGreenÒ assay. The efficiency of extracting siRNA from PCLEEP solution was also accounted for by doping PCLEEP solutions with known amounts of siRNA or siRNA/TKO mixtures. Thereafter, the polymer/siRNA or polymer/siRNA/TKO mixtures were placed under vacuum overnight at room temperature to remove all solvents. Next, dry polymer/ siRNA or polymer/siRNA/TKO constructs (n ¼ 3) were cut into halves and dissolved in 1 mL of chloroform each. siRNA was finally extracted and quantified as described above to obtain extraction efficiencies. The experimental loading efficiencies were finally computed using the following equation:
Total amount of siRNA released þ Extracted amount of siRNA after accounting for extraction efficiency 100% Original siRNA theoritical loading
2.1. Poly (e-caprolactone-co-ethyl ethylene phosphate), PCLEEP
2.5. Analysis of structural integrity of nanofiber encapsulated siRNA
PCLEEP copolymer with 1% ethyl ethylene phosphate (EEP) was synthesized through bulk ring-opening polymerization of e-caprolactone and EEP according to previous protocols [28,29]. From our preliminary results, PCLEEP copolymer with more than 1% EEP may be too soft for electrospinning. Therefore, 1% EEP was chosen for the preparation of the nanofibrous scaffolds used in this study. Briefly, e-caprolactone and EEP were mixed and polymerized at 120 C for 48 h with Sn(Oct)2 as the catalyst. Thereafter, the resulting polymer was dissolved in dichloromethane and precipitated in cold ethyl ether/methanol (10/1 v/v), filtrated and dried under vacuum. The precipitated polymer was further purified by dissolving in THF and precipitated in water at room temperature. Thereafter, the mixture was filtrated and freeze-dried to produce the final product. PCLEEP of molecular weight 40,000 and 25,000 (denoted as PCLEEP40k and PCLEEP25k respectively) were used in this study.
The integrity of siRNA encapsulated within PCLEEP fibers was tested using a 1% agarose gel with SYBRÒ Green II. The positions of the resulting bands were compared with fresh naked siRNA as the positive control. To evaluate the integrity of siRNA in the supernatant, 0.07g of siRNA-encapsulated scaffolds were cut into small pieces of 2e5 mm in size and immersed in 2 mL of PBS in a microcentrifuge tube. 100 mL of the supernatant was taken out and replaced with fresh PBS at each time point. Following that, 18 mL of each supernatant was loaded for gel electrophoresis. To test the integrity of siRNA that remained within the fibers after controlled release of 49 days, scaffolds were washed and dried under vacuum overnight at room temperature. Thereafter, they were dissolved in 1 mL of chloroform followed by extraction with 200 mL of TE buffer. Finally, 18 mL of the extracted solutions were used for gel electrophoresis.
P.-o. Rujitanaroj et al. / Biomaterials 32 (2011) 5915e5923 2.6. Bioactivity analysis of nanofiber encapsulated siRNA 2.6.1. Supernatant transfection NIH 3T3 cells (P15-P30) were cultured in complete medium comprising of DMEM supplemented with 10% BCS and 1% penicillin-streptomycin. Cells were maintained in a humidified incubator at 37 C with 5% CO2. For transfection, cells were kept within 15 passages to ensure similar cellular activity. GAPDH siRNA- and GAPDH siRNA/TKO-encapsulated PCLEEP40k scaffolds (n ¼ 3) with an average weight of 70 mg were sterilized under UV for 1 h. Following that, the scaffolds were placed in 24-well culture plates and immersed in 2 mL of DMEM. The plates were kept in a humidified incubator at 37 C with 5% CO2. At days 5, 10, 15 and 30, 500 mL of siRNA supernatant was removed from each well and replenished with 500 mL of fresh DMEM. One day before transfection, 3T3 cells were plated at a density of 1.0 105 cells/well in 24-well plates with 500 mL of complete medium. For transfection using GAPDH siRNA supernatant, 2.0 mL of TKO reagent was mixed with 150 mL of supernatant for each well. For transfection using GAPDH siRNA/TKO supernatant, 150 mL of supernatant was added directly into each well without further TKO supplementation. The positive control comprised of cells subjected to 20 nM of GAPDH siRNA with 2.0 mL of TKO. After the addition of siRNA, DMEM was added to each well to obtain a final volume of 300 mL per well during transfection. Twenty four hours later, 300 mL of fresh complete medium was added to each well. The negative control comprised of cells that were not subjected to any treatment. Forty-eight hours after transfection, 3 wells of cells were pooled for each sample and GAPDH mRNA expression levels were evaluated by real-time PCR. The entire transfection experiment was repeated 3 times. 2.6.2. Scaffold-mediated transfection GAPDH siRNA and GAPDH siRNA/TKO-encapsulated PCLEEP40k scaffolds (n ¼ 3) with an average weight of 70 mg were cut to fit the wells of 24-well plates and sterilized under UV for 1 h. To evaluate cellular attachment onto nanofibers, 3T3 cells were seeded at 1.0 104 cells/well in 24-well plate on plain PCLEEP fibers. Glass cover slips were used as the 2D controls. Twenty-four hours after cell seeding, cells were fixed with 10% formalin for 30 min at room temperature, permeabilized in 0.3% Triton X-100 for 20 min and stained with DAPI (1:1000 dilution) and Oregon GreenÒ Phalloidin 488 (1:500 dilution) for 30 min. Finally, cells were observed under a confocal microscope (Zeiss, LSM 710 Meta Laser Scanning Confocal Microscope, Germany). For transfection, 3T3 cells were seeded onto the scaffolds at 1.0 105 cells/well in 500 mL of complete medium. In the case where soluble TKO was supplemented (denoted as T-GAPDH siRNA or T-GAPDH siRNA/TKO for scaffolds encapsulating GAPDH siRNA and GAPDH siRNA/TKO respectively), 2.5 mL of TKO was added into each well 24 h following cell seeding. For all samples, half of the medium was replaced with fresh complete medium 48 h after transfection. Ninety-six hours after initial cell seeding, cells were lysed for real-time PCR analysis. For each experimental group, 3 wells of cells were pooled to obtain sufficient RNA. The positive control comprised of cells subjected to 20 nM of GAPDH siRNA with 2.0 mL of TKO. Cells cultured on tissue culture polystyrene without any treatment were used as the negative control. The entire transfection experiment was repeated 3 times.
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positive and negative controls). The total cell number was computed for by multiplying cell density (cells per unit area, which was obtained by DAPI staining and fluorescent imaging of 10 fields per sample at 20 magnification) with the total scaffold area for that sample. 2.9. Statistical analyses All quantitative values were expressed as a mean standard deviation, unless otherwise stated. Statistical comparisons for fiber diameter, cell viability and silencing efficiency, were performed using one-way ANOVA and Tukey post-hoc tests after verifying equal variances. The student’s t-test was used for statistical comparisons involving 2 samples. P < 0.05 was considered statistically significant.
3. Results 3.1. Morphology of siRNA-encapsulated nanofibers SiRNA-encapsulated PCLEEP fibers were successfully fabricated by electrospinning as indicated in Fig. 1. Bead-free fibers with similar diameters were obtained. The average diameters of GAPDH siRNA- and GAPDH siRNA/TKO-encapsulated nanofibers were 408 21 nm and 398 18 nm, respectively. 3.2. Release kinetics of siRNA from PCLEEP nanofibers Sustained release of siRNA was detected for up to 28 days after initial burst releases upon immersion into PBS (Fig. 2). The
2.7. Evaluation of gene silencing by real-time RT-PCR RNA was isolated using TRIzolÒ reagent. RQ1 RNase-free DNase was added to the isolated RNA to improve RNA quality. Reverse transcription was carried out using SensiscriptÒ RT kit according to manufacturer’s protocol. GAPDH mRNA expression levels were determined by real-time PCR using iQ SYBR Green Supermix in an iCycler iQ5 real-time PCR detection system (Bio-rad, USA), with b-actin as the housekeeping gene. Primer sequences for GAPDH gene were: forward 50 -TCAACAGCAACTCCCACTCTTCCA-30 and reverse 50 -ACCCTGTTGCTGTAGCCGTATTCA-30 with a product size of 115 bp. Primer sequences for b-actin were: forward 50 TGTGATGGTGGGAATGGGTCAGAA-30 and reverse 50 -TGTGGTGCCAGATCTTCTCCATGT-30 with a product size of 140 bp. Our preliminary studies showed that these two primers had similar amplification efficiency under the parameters used. Therefore, the DDCT method was used for fold change analysis. The real-time PCR cycling condition used was: 3 min at 95 C, 40 cycles at 95 C for 15 s, followed by 59 C for 30 s. All results were normalized with respect to the negative control. 2.8. Cell viability analyses To evaluate cell viability under supernatant transfection and scaffold-based transfection settings, cells were cultured at 2.0 104 cells/well (in 96 well plates) and 1.0 105 cells/well (in 24 well plates) respectively. Each sample comprised of 3 repeats. The same transfection procedure was implemented with proportionally reduced amounts of siRNA and TKO where applicable. Forty-eight hours after transfection, cells were incubated in WST-1 reagent (1:10 dilution) for 2 h at 37 C. Finally, the relative absorbance (A450-A620) was measured using a microplate reader (Bio-rad, Benchmark PlusÔ, Japan). For scaffold-base transfection, the absorbance values were normalized by the total cell number for each sample in order to account for possible differences due to variations in cell culture substrates (nanofiber samples vs. tissue culture plates in
Fig. 1. SEM images of a) GAPDH siRNA-encapsulated and b) GAPDH siRNA/TKOencapsulated PCLEEP nanofibers illustrating uniform and bead-free fibers.
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Fig. 2. Release kinetics of naked siRNA and siRNA/TKO complexes from PCLEEP25k and PCLEEP40k nanofibers as observed in PBS at 37 C.
encapsulation of naked siRNA within PCLEEP40K fibers resulted in a significantly higher burst release than PCLEEP25k fibers. The complexation of siRNA with TKO, however, significantly reduced the initial burst release (48.5 4% vs 12.3 1% at time ¼ 0 for PCLEEP40k siRNA scaffolds and PCLEEP40k siRNA/TKO scaffolds respectively, p < 0.05). Concurrently, the rate and amount of siRNA/ TKO complexes that was released were also lower as compared to naked siRNA (total siRNA amount that was released by day 49 was 97.2 1% vs 89.3 2% for PCLEEP40k siRNA scaffolds and PCLEEP40k siRNA/TKO scaffolds respectively, p < 0.05). TKO complexation did not alter the loading efficiency of siRNA significantly. The experimental loading efficiencies for PCLEEP40k siRNA scaffolds and PCLEEP40k siRNA/TKO scaffolds were 62.2 1.5 and 59.3 1.8% respectively.
remained at least partially bioactive throughout the period of sustained release. Furthermore, GAPDH siRNA/TKO supernatant exhibited gene silencing without further supplementation of transfection reagent, suggesting the feasibility of co-encapsulating transfection reagent within the nanofibers for sustained gene silencing applications. While cell viability was slightly lower in the presence of TKO as compared to the negative control (Fig. 4b), the difference was not statistically significant. Similarly, no apparent difference was observed between GAPDH siRNA supernatant, GAPDH siRNA/TKO supernatant and the positive control at all time points.
3.3. Structural integrity of nanofiber encapsulated siRNA Fig. 3 shows the structural integrity of siRNA that was recovered from siRNA and siRNA/TKO supernatants at various time points. The molecular weight of the samples matched that of fresh siRNA indicating that the siRNA remained structurally intact throughout the period of sustained release. The lower siRNA band intensity of siRNA/TKO samples corresponded with the lower siRNA release amounts as observed from the release profiles in Fig. 2. At day 49, a significant amount of intact siRNA/TKO remained entrapped within the nanofibers (Fig. 3c). 3.4. Bioactivity analysis of nanofiber encapsulated siRNA 3.4.1. Supernatant transfection As shown in Fig. 4a, silencing efficiencies of 58% and 40% were obtained using GAPDH siRNA and GAPDH siRNA/TKO supernatants respectively. The effects were significantly different as compared to the positive and negative controls, indicating that the siRNA
Fig. 3. Structural integrity of siRNA in polymer nanofibers as evaluated by 1.0% agarose gel electrophoresis. Gel electrosphoresis results of siRNA supernatants from a) PCLEEP siRNA fibers, b) PCLEEP siRNA/TKO fibers of various time points. Lane 1: dsRNA marker, Lane 2: naked siRNA and Lane 3-8: siRNA supernatants collected at 7, 14, 21, 28, 35 and 49 days respectively. c) siRNA extracted from nanofiber scaffolds after 49 days of release study. Lane 1: dsRNA marker, Lane 2: naked siRNA, Lane 3: siRNA within PCLEEP siRNA nanofibers, and Lane 4: siRNA within PCLEEP siRNA/TKO nanofibers.
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Fig. 4. GAPDH siRNA supernatant transfection in 3T3 cells, illustrating retention of siRNA bioactivity throughout period of sustained release. a) GAPDH mRNA levels in transfected 3T3 cells, and b) cell viability of transfected 3T3 cells at 96h. T-GAPDH siRNA: GAPDH siRNA supernatants precomplexed with TKO reagent during transfection. GAPDH siRNA/TKO: GAPDH siRNA/TKO supernatants only. POS: positive control of cells on culture plate subjected to 20 nM GAPDH siRNA transfection. NEG: negative control of cells without treatment. * # $ , , , and % indicate p < 0.05 as compared to the positive control. ** indicates p < 0.05. n ¼ 3, mean SE.
3.4.2. Scaffold-mediated transfection Plain PCLEEP scaffolds supported cell attachment as illustrated in Fig. 5. While cell spreading and stress fiber formation were more prominent on the 2D controls, cells on nanofibers adopted morphological changes that were typical of cells seeded on 3D matrices. In particular, cells on nanofibers adopted more spinal-like
shape and possessed stress fibers that were located at the periphery of the cells [30]. Cells also responded to the underlying nanofiber topography with cellular protrusions (White arrows). As shown in Fig. 6a, 3T3 cells seeded directly on GAPDH siRNA and GAPDH siRNA/TKO scaffolds showed significant gene silencing as compared to untreated cells (negative control). This significant
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efficiency on both scaffolds (T-GAPDH siRNA vs T-GAPDH siRNA/ TKO, p > 0.05) Fig. 6b shows the viability of cells during scaffold-based transfection. All cells remained viable indicating that the scaffolds were cytocompatible. However, the presence of TKO significantly decreased cell viability. Cells seeded directly on the GAPDH siRNA/ TKO scaffolds appeared to show lower cell viability as compared to GAPDH siRNA scaffolds. When more TKO was added into the medium (i.e., T-GAPDH siRNA and T-GAPDH siRNA/TKO), cell viability was further reduced.
4. Discussion
Fig. 5. Confocal fluorescent microscopy images of 3T3 cells on a) glass cover slip and b) electrospun PCLEEP nanofibrous scaffold. Blue: cell nuclei; green: actin cytoskeleton. Scale bar ¼ 20 mm (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
gene knockdown was consistently observed in cells that were treated with (p < 0.05) and without (p < 0.05) the additional bolus supplementation of transfection reagent. The results clearly demonstrate the efficacy of nanofiber scaffold-mediated transfection. In the absence of TKO supplementation, a significantly higher GAPDH silencing efficiency was observed in cells that were cultured directly on GAPDH siRNA/TKO fibers as compared to GAPDH siRNA fibers (30.9% vs 21.3% respectively, p < 0.05). While the delivery of siRNA-TKO complex enhanced gene silencing efficiency as compared to naked siRNA delivery, further addition of TKO abrogated this difference, resulting in similar gene knockdown
Encapsulating biomolecules within electrospun fibers is a feasible and convenient way to produce biofunctional scaffolds that are capable of providing topographical signals and the sustained delivery of drugs [31e33] to seeded cells. Polymer fibers provide physical protection to the encapsulated biomolecules, preserve their bioactivity over prolonged time periods and enable localized release at target sites. Although we have demonstrated scaffold-mediated delivery of siRNA using PCL [16], the release of siRNA from plain PCL fibers was low even after 7 weeks in vitro, resulting in a cumulative release of only 3%. Therefore, in this study, siRNA-encapsulated PCLEEP fibers were fabricated to improve the siRNA release kinetics and scaffold-mediated gene silencing efficiency. SiRNA-encapsulated PCLEEP fibers were successfully fabricated by electrospinning and the average diameters of the samples were not significantly different. The similarity in fiber size despite the addition of TKO was likely due to the low volume used as compared to PCLEEP. This allowed the slight changes in drug composition to be tolerated by the optimized electrospinning parameters. Fiber size affects cell fate [34,35]. Therefore, maintaining similar fiber diameters throughout this study helped eliminate fiber size effect on cellular response. By encapsulating siRNA within PCLEEP fibers, a sustained release of bioactive siRNA was achieved for at least 28 days (Fig. 2). The initial burst releases are likely attributed to siRNA that was located on the surface of the scaffolds, since uniform PCLEEP-siRNA mixtures were electrospun. PCLEEP40k fibers showed significantly higher burst release as compared to PCLEEP25k fibers. This is likely due to the enhanced polymer hydrophilicity with increasing amounts of phosphate groups as PCLEEP molecular weight increased. Given the higher amount of siRNA released, PCLEEP40k was chosen for further analyses involving siRNA/TKO complex encapsulation. The rate and amount of siRNA/TKO complexes that was released were lower as compared to naked siRNA, likely due to the increase in particle size as a result of TKO complexation. As compared to using PCL, the experimental loading efficiency of siRNA within PCLEEP was similar. However, the total amount of siRNA that was being released by day 49 was significantly higher in PCLEEP. Taken together, the results demonstrated the possibility of control over siRNA release through changes in EEP content and polymer molecular weight. Further changes in copolymerization ratio and polymer molecular weight will, therefore, allow specific tailoring of siRNA release kinetics for desired applications. As shown in Fig. 3, a small amount of siRNA remained entrapped within siRNA/TKO fibers even after 49 days. Coupled with the observation that less than 8% mass loss was detected after incubating PCLEEP nanofibers under similar in vitro conditions for 3 months [31], it is likely that the encapsulated siRNA was released by diffusion and the remaining entrapped molecules can be released only upon matrix degradation. In the case of naked siRNA encapsulation, no visible band was observed within the detection limits
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Fig. 6. Silencing of GAPDH gene expression in cells seeded directly on siRNA-encapsulated PCLEEP 40k scaffolds. a) GAPDH mRNA expression levels in transfected 3T3 cells and b) cell viability of transfected 3T3 cells at 96h. T-GAPDH siRNA, T-GAPDH siRNA/TKO: cells seeded on PCLEEP siRNA and PCLEEP siRNA/TKO nanofibers with additional TKO supplementation. GAPDH siRNA, GAPDH siRNA/TKO: cells seeded on PCLEEP siRNA and PCLEEP siRNA/TKO nanofibers without TKO supplementation. POS: positive control of cells on culture plate subjected to 20 nM GAPDH siRNA transfection. NEG: negative control of cells without treatment. * indicates p < 0.05 as compared to the negative control, # indicates p < 0.05 as compared to the positive control, $ and % indicate p < 0.05. n ¼ 3, mean SE.
of SYBRÒ Green II, likely due to the higher release rate of naked siRNA. The supernatant transfection study was carried out to evaluate the bioactivity of the encapsulated siRNA, in the absence of other extracellular signaling factors such as scaffold topography, compliance and surface chemistry differences, which may affect cellular response [15,16,36] and gene silencing efficiency. When cells were treated with siRNA (precomplexed with TKO) and siRN/ TKO supernatants, significant gene knockdown was observed at all time points. This showed that the nanofibrous scaffolds provided efficient protection for the encapsulated siRNA for at least 30 days.
When treated with naked siRNA only, no gene silencing was observed in 3T3 cells. Therefore, GAPDH siRNA supernatants were precomplexed with TKO prior to addition into cell culture medium. In contrast, gene silencing using supernatants from siRNA/TKO fibers resulted in a silencing efficiency of w40% even without further supplementation of transfection reagent. This suggested the possibility of co-encapsulating transfection reagent and siRNA within a single scaffold for sustained gene silencing applications. While our cellular assays indicated the retention of siRNA bioactivity, the extent of bioactivity remains unknown. However, compared to the positive control of 20 nM siRNA, the lower
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silencing efficiency of the supernatants implied that the amounts of bioactive siRNA that was present were likely less than 20 nM. In the scaffold-mediated gene knockdown study, the observed gene silencing in the absence of transfection reagent was likely due to the localized concentration of siRNA. Such cellular behavior in response to localized availability of nucleic acids was also observed previously [16,36,37]. Although lower amounts of siRNA were released from GAPDH siRNA/TKO scaffolds, GAPDH silencing efficiency was significantly higher than GAPDH siRNA scaffolds (w31% vs w21% in the latter). This gene knockdown efficiency was also greater than that achieved by PCL nanofiber delivery (18% in plain PCL and up to 26% with PEG inclusion) [16]. This indicated the need of a transfection reagent to facilitate cellular uptake of siRNA and the feasibility of co-encapsulating the transfection reagent with siRNA within a single scaffold construct for dual biomolecule delivery. Transfection reagent induces slight cytotoxicity [16,38,39]. This was also observed in the supernatant transfection study (Fig. 4), although the changes in cell viability were not statistically significant. However, nanofiber scaffolds appeared to sensitize cells towards TKO, resulting in significant changes in cell viability. While the exact reasons behind the observation remain to be elucidated, it is noteworthy that microenvironmental differences, such as topography, surface chemistry, three-dimensionality and compliance of matrices, existed between supernatant and scaffold-mediated transfection studies and could contribute to cell phenotypic changes. Although decreased cell viability was observed as compared to GAPDH siRNA scaffolds, significant number of cells remained viable on GAPDH siRNA/TKO scaffolds and cell viability was still higher as compared to additional supplementation of TKO. Further TKO supplementation could enhance gene silencing efficiency (Fig. 6a). This means that TKO reagent was also able to complex with siRNA that was released into the culture medium. Similar to supernatant transfection, an overall lower silencing efficiency was observed as compared to the positive control, likely due to the lower amounts of bioactive siRNA that was present. Comparing the results between supernatant transfection and scaffold-mediated transfection, transfection using bolus delivery of naked siRNA in the former resulted in no gene knockdown. However, scaffold-mediated transfection enabled gene silencing even in the absence of TKO. In the presence of TKO, a silencing efficiency of w 40% was obtained for GAPDH siRNA/TKO samples in both transfection studies. Similarly, a silencing efficiency of w 60% was observed in T-GAPDH siRNA samples. The results suggested that the presence of transfection reagent, either co-encapsulated within nanofibers or supplemented additionally, appeared to dominate over any effects of localized concentration of biochemicals and topography on gene knockdown. GAPDH is crucial in cell metabolic pathways and is required for long-term cell viability and survival [40,41]. As a result, gene knockdown study by scaffold-mediated approach was restricted to 96 h after initial cell seeding. Further studies focusing on other cellular targets that are less critical to cell survival, e.g. ECM deposition, are currently ongoing to evaluate the efficacy of siRNA nanofibers for long-term gene silencing applications. 5. Conclusion This study demonstrates the feasibility of delivering siRNA and transfection reagent complexes within a single nanofiber construct for enhanced gene silencing efficiency in scaffold-mediated transfection. Co-encapsulation of siRNA and TKO within PCLEEP fibers resulted in a sustained release of bioactive siRNA for at least 28 days. The copolymerization of EEP with caprolactone enhanced siRNA delivery rate and gene knockdown efficiency as compared to
PCL. Such biofunctional nanofiber constructs may provide synergistic topographical and biochemical signals for regulating cellular behaviors by RNA interference. Acknowledgements The partial funding support from AStar BMRC grant (07/1/22/19/ 519), AStar BMRC SSCC grant (09/16), AcRF Tier One grant (RG36/ 07) and the National Basic Research Program of China (973 Program, 2010CB934000) are acknowledged. P. Rujitanaroj expresses her gratitude to Ms. HQ Cao for her help with initial studies on the nanofibrous scaffold-mediated siRNA delivery. References [1] Suggate EL, Ahmed Z, Read ML, Eaton-Charnock K, Douglas MR, Gonzalez AM, et al. Optimisation of siRNA-mediated RhoA silencing in neuronal cultures. Mol Cell Neurosci 2009;40(4):451e62. [2] Veiseh O, Kievit FM, Fang C, Mu N, Jana S, Leung MC, et al. Chlorotoxin bound magnetic nanovector tailored for cancer cell targeting, imaging, and siRNA delivery. Biomaterials 2010;31(31):8032e42. [3] Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, Heimerikx M, et al. A large-scale RNAi screen in human cells identifies new components of the p53 p.thway. Nature 2004;428(6981):431e7. [4] Colonna C, Conti B, Genta I, Alpar OH. Non-viral dried powders for respiratory gene delivery prepared by cationic and chitosan loaded liposomes. Int J Pharm 2008;364(1):108e18. [5] Sioud M, Sorensen DR. Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun 2003;312(4):1220e5. [6] Chen JL, Wang H, Gao JQ, Chen HL, Liang WQ. Liposomes modified with polycation used for gene delivery: preparation, characterization and transfection in vitro. Int J Pharm 2007;343(1e2):255e61. [7] Howard KA, Rahbek UL, Liu XD, Damgaard CK, Glud SZ, Andersen MO, et al. RNA interference in vitro and in vivo using a chitosan/siRNA nanoparticle system. Mol Ther 2006;14(4):476e84. [8] Trabulo S, Resina S, Simoes S, Lebleu B, de Lima MCP. A non-covalent strategy combining cationic lipids and CPPs to enhance the delivery of splice correcting oligonucleotides. J Control Release 2010;145(2):149e58. [9] Zhang SB, Zhao B, Jiang HM, Wang B, Ma BC. Cationic lipids and polymers mediated vectors for delivery of siRNA. J Control Release 2007;123(1):1e10. [10] Davidson TJ, Harel S, Arboleda VA, Prunell GF, Shelanski ML, Greene LA, et al. Highly efficient small interfering RNA delivery to primary mammalian neurons induces microRNA-like effects before mRNA degradation. J Neurosci 2004;24(45):10040e6. [11] Lundberg P, El-Andaloussi S, Sutlu T, Johansson H, Langel U. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides. Faseb J 2007; 21(11):2664e71. [12] Crombez L, Aldrian-Herrada G, Konate K, Nguyen QN, McMaster GK, Brasseur R, et al. A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells. Mol Ther 2009;17(1):95e103. [13] Veldhoen S, Laufer SD, Trampe A, Restle T. Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect. Nucl Acids Res 2006;34(22): 6561e73. [14] Bengali Z, Pannier AK, Segura T, Anderson BC, Jang JH, Mustoe TA, et al. Gene delivery through cell culture substrate adsorbed DNA complexes. Biotechnol Bioeng 2005;90(3):290e302. [15] Jang JH, Bengali Z, Houchin TL, Shea LD. Surface adsorption of DNA to tissue engineering scaffolds for efficient gene delivery. J Biomed Mater Res Part A 2006;77A(1):50e8. [16] Cao HQ, Jiang X, Chai C, Chew SY. RNA interference by nanofiber-based siRNA delivery system. J Control Release 2010;144(2):203e12. [17] Chew SY, Mi R, Hoke A, Leong KW. The effect of the alignment of electrospun fibrous scaffolds on schwann cell maturation. Biomaterials 2008;29(6):653e61. [18] Lim SH, Liu XY, Song HJ, Yarema KJ, Mao HQ. The effect of nanofiber-guided cell alignment on the preferential differentiation of neural stem cells. Biomaterials 2010;31(34):9031e9. [19] Nathan AS, Baker BM, Nerurkar NL, Mauck RL. Mechano-topographic modulation of stem cell nuclear shape on nanofibrous scaffolds. Acta Biomater 2011;7(1):57e66. [20] Cao HQ, McHugh K, Chew SY, Anderson JM. The topographical effect of electrospun nanofibrous scaffolds on the in vivo and in vitro foreign body reaction. J Biomed Mater Res Part A 2010;3:1151e9. [21] Delgado-Rivera R, Harris SL, Ahmed I, Babu AN, Patel RP, Ayres V, et al. Increased FGF-2 secretion and ability to support neurite outgrowth by astrocytes cultured on polyamide nanofibrillar matrices. Matrix Biol 2009; 28(3):137e47. [22] Chew SY, Mi RF, Hoke A, Leong KW. Aligned protein-polymer composite fibers enhance nerve regeneration: a potential tissue-engineering platform. Adv Funct Mater 2007;17(8):1288e96.
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