Article pubs.acs.org/molecularpharmaceutics
Biodegradable Film for the Targeted Delivery of siRNA-Loaded Nanoparticles to Vaginal Immune Cells Jijin Gu, Sidi Yang, and Emmanuel A. Ho* Laboratory for Drug Delivery and Biomaterials, College of Pharmacy, Faculty of Health Sciences, University of Manitoba, 750 McDermot Avenue, Winnipeg, Manitoba Canada, R3E 0T5 S Supporting Information *
ABSTRACT: The goal of this study was to develop and characterize a novel intravaginal film platform for targeted delivery of small interfering RNA (siRNA)-loaded nanoparticles (NP) to dendritic cells as a potential gene therapy for the prevention of sexually transmitted human immunodeficiency virus (HIV) infection. Poly(ethylene glycol) (PEG)-functionalized poly(D, L-lactic-co-glycolic acid) (PLGA)/polyethylenimine (PEI)/siRNA NP (siRNA-NP) were fabricated using a modified emulsionsolvent evaporation method and characterized for particle size, zeta potential, encapsulation efficiency (EE), and siRNA release. siRNA-NP were decorated with anti-HLA-DR antibody (siRNA-NP-Ab) for targeting delivery to HLA-DR+ dendritic cells (DCs) and homogeneously dispersed in a biodegradable film consisting of poly vinyl alcohol (PVA) and λ-carrageenan. The siRNA-NP-Ab-loaded film (siRNA-NP-Ab-film) was transparent, displayed suitable physicomechanical properties, and was noncytotoxic. Targeting activity was evaluated in a mucosal coculture model consisting of a vaginal epithelial monolayer (VK2/E6E7 cells) and differentiated KG-1 cells (HLA-DR+ DCs). siRNA-NP-Ab were rapidly released from the film and were able to penetrate the epithelial layer to be taken up by differentiated KG-1 cells. siRNA-NP-Ab demonstrated higher targeting activity and significantly higher knockdown of synaptosome-associated 23-kDa protein (SNAP-23) mRNA and protein when compared to siRNA-NP without antibody conjugation. Overall, these data suggest that our novel siRNA-NP-Ab-film may be a promising platform for preventing HIV infection within the female genital tract. KEYWORDS: intravaginal delivery, microbicides, polymeric film, SNAP-23, mucosal coculture model
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INTRODUCTION Intravaginal drug delivery is a promising route for both local and systemic drug absorption attributed to its large surface area, rich blood supply, accessibility, and avoidance of hepatic first-pass elimination.1,2 However, successful delivery of drugs via the vagina remains a challenge, primarily due to poor drug retention as a result of “wash-out” and poor drug absorption across the vaginal epithelium, which often requires multiple daily doses to achieve the desired therapeutic effect.3 Acceptability of intravaginal drug administration by women and doctors is increasing, particularly because it is effective for the prevention of sexually transmitted infections including human immunodeficiency virus (HIV) and because the vagina is an excellent route for the delivery of nucleic acids, peptides/proteins, and other therapeutically important macromolecules.2 Recently, there has been an increasing interest in using small interfering RNA (siRNA) to knockdown host and/or viral factors as a strategy to suppress the viral life cycle resulting in reduced viral entry into cells or the establishment of productive infection.4,5 As a result, development of siRNA-based vaginal microbicides is a promising approach for the treatment and prevention of sexually transmitted infections. However, delivery of siRNA across the vaginal mucosa is challenging because the siRNA molecules need to penetrate the cervicovaginal mucus layer, © XXXX American Chemical Society
overcome the cervicovaginal epithelial barrier, avoid rapid nuclease degradation, and be taken up by target cervicovaginal lymphocytes.6 There is currently a lack of studies evaluating the intravaginal delivery of siRNA and, consequently, a lack of strategies for overcoming the obstacles mentioned above. Nanosized drug carriers such as nanoparticles (NP) have been investigated for in vitro/in vivo siRNA delivery to overcome typical drug delivery challenges such as physicochemical stability, low cellular uptake, rapid cellular and tissue clearance, the need for multiple dosing, and the potential induction of an immunogenic response.7,8 Mucus-penetrating NP may provide advantageous distribution of microbicides throughout the reproductive tract, including the highly folded epithelial surfaces of the vagina.9,10 Strategies that offer controlled local delivery of siRNA play a significant role in improving gene therapy outcomes.6 Despite the fact that the female genital tract is a suitable route for local and systemic drug administration, the major obstacle with vaginal delivery is patient compliance. Many of the currently Received: January 24, 2015 Revised: June 17, 2015 Accepted: June 22, 2015
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DOI: 10.1021/acs.molpharmaceut.5b00073 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Molecular Pharmaceutics available vaginal dosage forms, such as gels and creams, suffer from leakage, messiness, and low residence time, resulting in poor patient compliance.11,12 Vaginal films have several advantages over conventional intravaginal dosage forms, which include portability, convenience in application, prolonged retention time, easy storage, and improved stability of drug.13,14 Therefore, polymeric films are increasingly being investigated as a promising approach for vaginal drug delivery. Vaginal films can be designed for immediate or controlled release by optimizing the polymeric composition of the film, or combining thin film technology with other drug delivery strategies.15,16 Hence, biodegradable and biocompatible films would be an excellent platform for the vaginal delivery of siRNAencapsulated NP to improve localized transfection efficiency. Retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. The concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. In eukaryotic cells, biological membrane fusion is generally executed by fusogenic soluble N-ethylmaleimidesensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs).17 Synaptosome-associated 23-kDa protein (SNAP-23) is a SNARE protein located on the plasma membrane, and it is involved with the exocytosis of secretory vesicles. Studies have shown that the absence of SNAP-23 expression on host cells results in defects with HIV-1 particle production. This defect correlates with a deficiency in Gag-membrane localization, suggesting a role for SNARE proteins in Gag accumulation at the plasma membrane.17 RNA interference (RNAi) is increasingly being utilized for the specific targeting and down-regulation of disease-causing genes, including those involved in viral infections such as HIV. Hence, using specific siRNA against SNAP-23 will disrupt the host SNARE machinery, resulting in a significant reduction in HIV-1 virus particle production. In this study, we developed and characterized a novel biodegradable film formulation for the active targeted delivery of siRNA-loaded NP (siRNA-NP) to HLA-DR+ immune cells of the vaginal mucosa. NP were manufactured from polyethylene glycol-poly(D, L-lactide-co-glycolide) (PLGA-PEG) and polyethylenimine (PEI)/siRNA complexes, and functionalized with anti-HLA-DR antibody (siRNA-NP-Ab). We investigated the physicochemical properties of the siRNA-NPAb and siRNA-NP-Ab-loaded film (siRNA-NP-Ab-film) and determined its impact on the viability of epithelial and immune cells. Most importantly, we evaluated the ability of siRNA-NP-Ab released from film to penetrate epithelial cells and specifically target delivery into HLA-DR+ immune cells (HIV target cells) using a vaginal mucosal coculture model (Figure 1). SNAP-23 knockdown efficiency was also determined at both mRNA and protein levels.
Figure 1. Schematic representation of anti-HLA-DR antibody conjugated PLGA-PEG/PEI/siRNA NP (siRNA-NP-Ab) formulated into a biodegradable film for targeting siRNA delivery into vaginal HLA-DR+ dendritic cells. siRNA-NP-Ab were homogeneously dispersed in a biodegradable film consisting of poly vinyl alcohol (PVA) and λ-carrageenan. Upon administration, the film will disintegrate within the vaginal lumen allowing siRNA-NP-Ab to penetrate across the vaginal mucosa and target delivery to HLA-DR+ dendritic cells.
U.S.A.). λ-carrageenan was purchased from American Custom Chemicals Corp (San Diego, CA, U.S.A.). Ethyl acetate (AR grade) was purchased from Thermo Fisher Scientific Inc. (Toronto, ON, Canada). rhIL-4, rhGM-CSF and rhTNF-α were obtained from R&D Systems (Minneapolis, MN, U.S.A.). 4′-6-Diamidino2-phenylindole (DAPI) and Annexin V-FITC/7-AAD apoptosis kit were purchased from Life Technologies, Inc. (Burlington, ON, Canada). Anti-HLA-DR antibody was obtained from Biolegend (San Diego, CA, U.S.A.). Trypsin-EDTA, penicillin/ streptomycin solution and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Burlington, ON, Canada). cDNA amplification kits for rapid amplification of cDNA ends was obtained from Clontech Laboratories, Inc. (Mountain View, CA, U.S.A.). Double distilled water (ddH2O) was purified using a Millipore Simplicity System, which was used for all the experiments. Cell Lines and siRNA. All cell lines used in this study were obtained from ATCC. The vaginal epithelial cell line, VK2/ E6E7, was maintained in ATCC complete growth medium (Keratinocyte-Serum Free medium with 0.1 ng/mL human recombinant epidermal growth factor (EGF), 0.05 mg/mL bovine pituitary extract, additional calcium chloride (44.1 mg/L), and 1% penicillin-streptomycin) under conditions of 5% CO2 at 37 °C. The KG-1 immune cell line was maintained in ATCCformulated Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 20% FBS and 1% penicillin-streptomycin under conditions of 5% CO2 at 37 °C. All siRNA sequences were chemically synthesized by Ambion-Invitrogen and purified by HPLC. siRNA against SNAP-23 (siSNAP-23) consisted of the following sense strand
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MATERIALS AND METHODS Materials. HOOC-PEG (2k)-b-PLGA (50/50) (10 k), (PLGA-PEG, Mn: 12000; PI: 1.6) was custom synthesized by Advanced Polymer Materials, Inc. (Montreal, QC, Canada). PLGA (MW 15 kDa, acid terminated), branched polyethylenimine (PEI, MW 25 kDa, Aldrich), poly vinyl alcohol (PVA, 98−99% hydrolyzed; MW 31 000−50 000) and ionomycin were obtained from Sigma-Aldrich (Oakville, ON, Canada). 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) were purchased from G-Biosciences (Geno Technology Inc., Maryland Heights, MO, B
DOI: 10.1021/acs.molpharmaceut.5b00073 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Preparation of siRNA-NP-Film and siRNA-NP-Ab-Film. The polymeric film was prepared by a solvent casting and solvent evaporation method.14 Briefly, PVA and λ-carrageenan were completely dissolved in 10 mL of ddH2O at 80 °C and mixed intermittently. Subsequently, 600 μL of glycerin and 300 μL of PEG400 were then added to the solution, followed by the addition of siRNA-NP or siRNA-NP-Ab. The polymeric dispersion was vortexed to achieve a homogneous mixture and then sonicated for 30 min without heat (using sonication bath) to obtain an aqueous dispersion and to remove entrapped air bubbles. The polymeric solution (4.0 mL) was then uniformly spread onto an evaporation dish (30.0 cm2) and dried in an oven at 37 °C for 20 ± 4 h or in a freeze-dryer (0.05 mbar, −47 °C) overnight. Characterization of the Film. Polymeric films were evaluated for various physical and mechanical parameters including thickness, weight, and disintegration time, and they were visually inspected for appearance and transparency. The thickness of each film was measured at five different locations (center and four corners) using a digital caliper, and the mean value was calculated. All measurements were performed in triplicate. For gross qualitative evaluation of film disintegration, 50 mg of film was immersed in 3 mL of ddH2O in a 10 mL vial at 37 °C. The vial was shaken at 100 rpm on an orbital shaker, and the rate of disintegration was visually recorded. Film disintegration was defined as the time for the intact film structure to completely disappear into solution. Surface morphology of film was captured under a Scanning Auger Microprobe (SAM, JEOL JAMP 9500F). The dried film samples were cut into small pieces and attached to slab surfaces with double-sided adhesive tapes. KG-1 Cell Differentiation. KG-1 cells can be differentiated rapidly into mature dendritic cells (mKG-1) when cultured in serum-free medium at a concentration of 2 × 105 cells/mL for 1−3 days in a humidified incubator at 37 °C and 5% CO2 with rhGM-CSF (100 ng/mL), rhTNF-α (10 ng/mL), and 200 ng/mL ionomycin.21 During differentiation into mature DCs, the cells exhibit de novo cell-surface expression of CD83 and intracellular synthesis of IL-10, and up-regulation of HLA-DR cell surface expression.21 After differentiation, the mKG-1 cells were used for NP uptake and targeting studies. mKG-1 cells were identified according to the following method: cultured cells were washed, resuspended in cold PBS at a density of 1.5 × 105 cells/mL and incubated with monoclonal antibody FITC-labeled antiCD83, PE-conjugated anti-HLA-DR or Alexa647-conjugated anti-IL-10 or appropriate isotypic controls for 30 min. Stained cells were analyzed for three-color immunofluorescence with a flow cytometer (FACSCalibur, BD, U.S.A.). In Vitro Cytotoxicity Studies. For cytotoxicity studies, VK2/E6E7 and mKG-1 cells were seeded in a 96-well plate at 2 × 104 cells per 100 μL per well, respectively. After 24 h of incubation (37 °C, 5% CO2), the cell culture medium was replaced with 100 μL positive and negative control medium and various treatment groups. Cell viability was determined using the MTS assay (CellTiter 96 A-Queous One Solution Cell Proliferation Assay, Promega), following the manufacturer’s instructions. Levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) in supernatant of each well were evaluated using enzyme-linked immunosorbent assay kits (ELISA) obtained from R&D Systems. Treatment groups included siRNA-NP, siRNA-NP-Ab, blank film, siRNA-NP-film, and siRNA-NP-Ab-film. Various NPs and films were diluted in cell culture medium to achieve an appropriate concentration
5′-CCAACAGAGAUCGUAUUGAtt-3′ and antisense strand 5′-UCAAUACGAUCUCUGUUGGtg-3′. Nontargeting scrambled siRNA was used as a negative control (siNC) with a sense strand consisting of 5′-UUCUCCGAACGUGUCACGUTT-3′ and an antisense strand of 5′-ACGUGACACGUUCGGAGAATT-3′. Fluorescent siRNA used in this study were labeled at the 5′-end of the sense strand with carboxyfluorescein (FAM) (FAM-siRNA) or cyanine dye (Cy3) (Cy3-siRNA). Preparation of siRNA-NP and siRNA-NP-Ab. siRNA-NP was formulated by a double-emulsion solvent evaporation method previously described with some modifications.18−20 In brief, siRNA was complexed to PEI (N/P ratio 3:1) in TE buffer and incubated at room temperature for 15 min with intermittent vortexing. This aqueous solution (100 μL) was then added dropwise to ethyl acetate (500 μL) solution of PLGA-PEGCOOH (10 mg/mL) to form the first emulsion with 10 s sonication on ice using a probe sonicator. This mixture was then added dropwise to 4.0 mL of 0.5% (w/v) PVA solution and sonicated to form a double emulsion. The final emulsion was poured into a vial and stirred for 4 h to evaporate the ethyl acetate before centrifuging at 12 000g for 15 min at 4 °C. The particles were washed twice and resuspended in 1 mL ddH2O, rapidly frozen, and lyophilized. Prior to antibody conjugation, the carboxyl groups on the siRNA-NP surface (from PEG-COOH) were activated by EDC (1.92 mg/mL) and sulfo-NHS (1.08 mg/mL) coupling reaction in 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.0) for 1 h. The activated siRNA-NP were collected by centrifugation (12 000g, 15 min), resuspended in pH 7.4 PBS, and reacted with 20 μL of anti-HLA-DR antibody (0.5 mg/mL) for 2 h, with stirring at room temperature. The siRNA-NP conjugated to antibody were collected by centrifugation (12 000g, 15 min), washed three times with DEPC-treated water to remove free antibody, and resuspended in ddH2O. Characterization of siRNA-NP and siRNA-NP-Ab. Particle size and zeta potential of the NP were measured by dynamic light scattering (DLS) using the ZetaPALS (Brookhaven). Transmission electron microscopy (TEM) was utilized to investigate the morphology of the NP after negative staining with 2% (w/v) phosphotungstic acid solution. The encapsulation efficiency (EE) of siRNA loaded in the NP was calculated as EE (%) = (the total amount of siRNA − the amount of siRNA in supernatant)/total amount of siRNA × 100. The amount of free siRNA recovered in the NP wash solution obtained during the formulation step was quantified using the Quant-iT Picogreen reagent. siRNA loading in NP was determined by subtracting the amount of siRNA recovered in the wash solutions from initial amount of siRNA added. In vitro release studies were carried out using Cy3-siRNA-NP and Cy3-siRNA-NP-Ab. Briefly, NP (5.0 mg) were suspended in 1.0 mL of release medium (PBS, pH 7.4) in RNase-free microcentrifuge tubes, and incubated at 37 °C with gentle shaking (100 rpm). At predetermined time intervals, the NP suspension was centrifuged at 12 000g, 4 °C for 10 min, and 100 μL of the supernatant was withdrawn for quantification of siRNA and replaced with an equivolume of fresh release medium. The concentration of released Cy3-siRNA in the supernatant was determined using a microplate reader (BioTek Synergy) with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The percentage of released siRNA was normalized to the total amount of siRNA encapsulated. Samples were taken and analyzed in triplicates for each time point. C
DOI: 10.1021/acs.molpharmaceut.5b00073 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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siNC. mKG-1 cells were harvested 24, 48, or 72 h posttreatment and centrifuged at 600 rpm for 5 min at 4 °C, following removal of the incubation medium. Quantitative real-time polymerase chain reaction (qPCR) was used to determine the down-regulation of SNAP-23 mRNA level. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene for normalization of results. The comparative CT (ΔΔCT) method was used to quantitate relative SNAP-23 mRNA expression level, comparing treated samples to nontreated controls.22,23 To demonstrate that siRNA-mediated SNAP-23 gene silencing can engage the RNAi machinery, 5′-rapid amplification of cDNA ends (RACE) PCR assay was performed to characterize the mRNA cleavage products using SMARTer RACE cDNA amplification kit (Clontech) according to the manufacturer’s protocol. Briefly, mKG-1 cells were treated with different siRNA formulations using the mucosal coculture model. After transfection, total RNA was extracted from mKG-1 cells and subjected to 5′-RACE to identify the specific cleavage products. cDNA was synthesized from 1 μg of total RNA and 5′-CDS primer A for the 5′ RACE. RACE PCR was performed with the cDNA, SNAP-23 gene specific primer and universal primer mixture. PCR products were run on a 2% agarose gel for 40 min at 120 V and visualized using SYBR Safe gel stain (Life Technologies). For analysis of SNAP-23 protein expression, mKG-1 cells were analyzed by flow cytometry after stained with FITC-labeled SNAP-23 antibody. The mKG-1 cells stained with FITC-labeled nonspecific antibody were used as isotype stain control. Cells were gated to evaluate 10 000 viable cells per experiment. The mean fluorescence intensity of fluorescence-positive cell population was determined. The extent of SNAP-23 silencing with different formulations was expressed relative to the cells treated with the scrambled siRNA of the corresponding formulations. Cells were also imaged using a fluorescence microscope (Nikon TE2000). Impact of the various siRNA formulations on cell viability was also determined using the apoptosis kit with Annexin V FITC/ 7-aminoactinomycin D (7-AAD) according to the manufacturer’s instructions (Life Technologies). Briefly, cell populations were washed twice with PBS and then resuspended in 100 μL of 1 × binding buffer at a concentration of 1 × 106 cells/mL. Afterward, the resuspended cells were stained with 5 μL Annexin V FITC and 1 μL 7-AAD for 15 min at room temperature in the dark, and then 400 μL of binding buffer was added. The stained samples were analyzed immediately by flow cytometry. For each sample, the fluorescence of 1 × 104 events was measured. Every experiment was performed in triplicate. Statistical Analysis. Data were analyzed by a two-way ANOVA test with P < 0.05 considered statistically significant. All data were shown as means ± SD of at least N ≥ 3 unless otherwise indicated.
(films: 0−10 mg/mL; NP was administrated at the concentration according to the loading of film: 0.043 nmol siRNA/ 0.15 mg NP/mg film). Cells cultured in medium alone without treatment were used as negative control. Cells treated with 1% (v/v) Triton X-100 were used as positive control for the MTS assay. Nonoxynol-9 (N-9; 200 μg/mL; Spectrum Chemical Corp., New Brunswick, NJ, U.S.A.) diluted in culture medium was used as a positive control for IL-1β ELISA (R&D System Inc., Minneapolis, MN, U.S.A.). Lipopolysaccharide (LPS; 50 μg/mL; from Escherichia coli 0111:B4, Sigma) prepared in culture medium was used as positive control for IL-6 and IL-8 ELISA (R&D System). Cells were incubated for 4, 12, and 24 h at 37 °C and 5% CO2. In Vitro Cellular Uptake of siRNA-NP and siRNA-NP-Ab. To examine the cellular uptake of siRNA-NP and siRNA-NPAb, VK2/E6E7 or mKG-1 cells were seeded in a 24-well plate at a density of 3 × 105 cells per 500 μL per well and incubated for 24 h. The cells were then incubated with FAM-siRNA-NP or FAM-siRNA-NP-Ab formulations at a concentration of 0.15 nmol FAM-siRNA/well (1 mg/mL NP) for 0.5−24 h. At the end of the incubation period, the medium containing NP was removed, and the cells were rinsed three times with icecold PBS before DAPI (5 μg/mL) staining. The fluorescence of the noninternalized NP attached to the plasma membrane was quenched by incubation in 0.4% trypan blue (GIBCO BRL, Invitrogen) for 5 min. After being washed with PBS, fluorescence was directly observed and imaged under a fluorescence microscope (Nikon TE2000). For quantification, the cells were collected at various time points, rinsed with PBS, and analyzed using flow cytometry (FACSCalibur, BD, U.S.A.). For the competitive inhibition assay, VK2/E6E7 and mKG-1 cells were pretreated with excess amounts of free anti-HLA-DR. After 2 h of incubation, the FAM-siRNA-NP with or without antibody conjugation was added to the cells. The fluorescence intensity of each sample was then analyzed as described above. Evaluation of Penetration of NP Using a Vaginal Mucosal Coculture Model. VK2/E6E7 cells were seeded onto a cell culture insert consisting of a polycarbonate membrane (mean pore size 3.0 μm, effective cell growth area of membrane 0.33 cm2, Corning Inc. Life Sci., MA, U.S.A.) at a density of 5 × 104 cells/200 μL medium per well and were cultured until the formation of tight junctions was achieved. Trans-epithelial electrical resistance (TEER) was monitored daily by a volt-ohm meter (Millicell-ERS-2, Millipore, U.S.A.) attached to Endohm-12 chamber electrodes to evaluate the cell monolayer integrity. Cell monolayers with TEER values over 150 Ω were used for the following experiments. Six days after seeding, VK2/E6E7 cell monolayers grown in the transwell chambers were transferred to a 24-well plate containing mKG-1 cells in complete culture medium and then cocultured for another day. Culture medium (300 μL) containing siRNA-NP, siRNA-NP-Ab, siRNA-NP-film, or siRNA-NP-Abfilm was then added into the upper apical chamber of each transwell. siRNA-NP and siRNA-NP-Ab were incorporated with coumarin-6 for fluorescent quantitation of NP and the transport of NP from the apical to basolateral direction was studied. At various time points (4, 12, 24, and 48 h), mKG-1 cells were collected from the basolateral chamber, quenched with 0.4% trypan blue, washed with PBS, and analyzed using a flow cytometer to determine cellular uptake. Silencing Activity of siSNAP-23 in a Vaginal Mucosal Coculture Model. As described earlier, cells were treated with various NP and NP-film formulations containing siSNAP-23 or
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RESULTS Characterization of siRNA-NP and siRNA-NP-Ab. In this study, the mean particle size and zeta potential of the NP formulations used are summarized in Table 1. Blank NP had a mean particle size of 161.0 ± 4.6 nm and a polydispersity index (PDI) of 0.108 ± 0.016. The PDI of NP is smaller than 0.2, indicating narrow size distribution. The zeta potential of blank NP in ddH2O was found to be −32.76 ± 1.01 mV. In the presence of PEI/siRNA complexes, the siRNA-NP had an effective hydrodynamic diameter of 201.4 ± 4.3 nm in ddH2O, with no appreciable increase in zeta potential (−29.65 ± 3.35 mV). D
DOI: 10.1021/acs.molpharmaceut.5b00073 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Molecular Pharmaceutics Table 1. Particle Size, Zeta Potential, and EE (%) of Various NP Formulations parameter
particle size (nm)
PDI
zeta potential (mV)
EE (%)
blank NP siRNA-NP siRNA-NP-Ab
161.0 ± 4.6 201.4 ± 4.3 232.0 ± 3.0
0.108 ± 0.016 0.131 ± 0.009 0.149 ± 0.022
−32.76 ± 1.01 −29.65 ± 3.35 −27.37 ± 2.45
75.89 ± 3.67 70.68 ± 4.89
Data represent means ± SD; N = 5. siRNA-NP = siRNA-encapsulated nanoparticles; siRNA-NP-Ab = antibody conjugated siRNA-encapsulated nanoparticles; EE (%) = siRNA encapsulation efficiency.
with the DLS measurements. The release of siRNA from siRNA-NP and siRNA-NP-Ab (loaded with 1.5 nmol Cy3labeled siRNA) was evaluated in PBS (pH 7.4) for 15 days (Figure 2C). There was no significant difference in siRNA release between siRNA-NP and siRNA-NP-Ab. In the first 4 h, siRNA-NP-Ab had a burst release of ∼8.9% of total loading with ∼35% of siRNA released cumulatively after 15 days. Preparation and Characteristics of the Films. Polymeric films were prepared by a solvent evaporation method with formulation A (Table 2) containing siRNA-NP-Ab (5.0 mg).27
When siRNA-NP were conjugated with antibody, the diameter of the conjugated NP increased to 232.0 ± 3.0 nm. However, no significant effect was observed on the zeta potential, which indicates the presence of negatively charged surfaces. PEI was included in the formulation because studies have shown that preformed PEI/siRNA complexes result in higher encapsulation of siRNA in the final formed siRNA-NP.24 In our studies, we found that prior to antibody conjugation, the EE was 75.89 ± 3.67%, and after conjugation, the EE was 70.68 ± 4.89%. The slight siRNA loss may be due to the release of siRNA from the siRNA-NP during the conjugation process, which involves repeating wash steps, centrifugation, and prolonged stirring. This observation has been reported previously.25 The morphology of siRNA-NP and siRNA-NP-Ab were examined by TEM, and the representative images are shown in Figure 2. The micrograph images revealed that the siRNA-NP
Table 2. Vaginal Film Formulations (10 mL Solution) formulation
PVA (%, m/v)
λ-carrageenan (mg)
glycerin (mL)
PEG400 (mL)
NPs (mg)
A B C D E F G
1 2 5 2 2 2 2
50 50 50 25 100 50 50
0.6 0.6 0.6 0.6 0.6 0.6 0.6
0.3 0.3 0.3 0.3 0.3 0.3 0.3
0 0 0 0 0 50 100
To remove the solvent, two methods of drying the film were evaluated. One method involved the incubation of films at 37 or 60 °C, which resulted in a 3.4 ± 2.6% and 21.2 ± 4.3% loss of siRNA, respectively. The second method evaluated the use of a freeze-dryer, which significantly improved the stability of siRNA with 1.4 ± 0.2% loss of siRNA. These results indicated that siRNA quickly degraded at high temperatures. Furthermore, the appearance of the films using the two drying methods was also compared. When the polymer solution was lyophilized under reduced pressure in a freeze-dryer, an opaque and porous film was produced (Supporting Information, Figure S1A). The conventional heat drying method (37 °C) produced films that were transparent and homogeneous (Supporting Information, Figure S1B). These results suggest that water was quickly released from the frozen polymer solution during the drying process. As a result, the films used for this study were prepared under the conventional heat drying method (37 °C). Films prepared from varying polymeric compositions (Table 2) were evaluated on the basis of physical parameters including thickness, weight, and disintegration time and were visually inspected for appearance and transparency (Table 3). The thickness of the films varied between 33.3 ± 3.56 and 77.3 ± 9.88 μm, and the average weight of the films was between 231.0 ± 18.1 and 342.5 ± 27.1 mg/dish (30.0 cm2). The thickness of the films increased as the polymer concentration increased but did not change significantly when the loading of NP increased. Our results showed that the major contributing factor regulating film appearance (e.g., smoothness and transparency), and disintegration time was related to the amount of PVA and λ-carrageenan in the formulation. The films became more
Figure 2. Surface morphology and release kinetics of siRNA from NP. TEM images (250 000× magnification) of (A) siRNA-NP and (B) siRNA-NP-Ab. (C) Cumulative release of Cy3-labeled siRNA from siRNA-NP and siRNA-NP-Ab in PBS (pH 7.4). Data represent the mean ± SD; N = 5.
were generally spherical in shape and possessed a smooth surface (Figure 2A) even after being functionalized with antibody (Figure 2B). Because TEM measurement requires samples to be air-dried before analysis, particle sizes measured by TEM are always slightly smaller than those from DLS with samples suspended in solution.26 Generally speaking, the particle size directly measured from the TEM images was in good agreement E
DOI: 10.1021/acs.molpharmaceut.5b00073 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
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Molecular Pharmaceutics Table 3. Properties of Vaginal Film Formulations formulation A B C D E F G
thickness (μm) 33.3 56.4 77.3 47.7 69.4 54.6 58.2
± ± ± ± ± ± ±
3.56 5.67 9.88 3.99 3.29 1.49 2.51
weight (mg)
disintegration time
appearance
± ± ± ± ± ± ±
10 min 24 h swelling 20 h >24 h 24 h 24 h
semitransparent semitransparent transparent semitransparent semitransparent semitransparent semitransparent
231.0 271.7 342.5 253.6 301.4 289.3 294.5
18.1 3.32 27.1 11.9 9.28 8.87 4.91
Figure 3. Surface morphology of films (A) blank film, (B) siRNA-NP-Ab-film, (C) blank film, (D) siRNA-film, (E) siRNA-PLGA-NP-Ab-film and (F) siRNA-PLGA-PEG-NP-Ab-film. Images C−F were captured using SAM at 500× magnification; inset image depicts partially enlarged morphology of NPs (2000× magnification).
aggregation on the film surface when compared to siRNA-NP prepared from PEG- PLGA (Figure 3F). Differentiation of KG-1 Cells. After culturing for 3 days in serum-free medium supplemented with rhGM-CSF, TNF-α, and ionomycin, KG-1 cells differentiated completely into mature DCs (mKG-1) with characteristic stellate morphology (Supporting Information, Figure S2A), de novo cell-surface expression of CD83, up-regulation of HLA-DR cell-surface expression, and de novo intracellular synthesis of IL-10 (Supporting Information, Figure S2B). Notably, mKG-1 cells contained a high percentage of HLA-DR (∼99.8%) cell subpopulation after differentiation. The cells retained all phenotypic properties of mature DCs for approximately 3 weeks when serum-free medium supplemented with rhGM-CSF, TNF-α, and ionomycin was exchanged with complete culture medium at day 3. Our observations are similar to those previously reported.21 As expected, there was no detection of HLA-DR receptor expression on the surface of VK2/E6E7 cells, and hence, these cells were used as the control for targeting studies. In Vitro Cytotoxicity Evaluation. The cytotoxicity of the various formulations was evaluated in the presence of VK2/E6E7 and mKG-1 cells in vitro at varying time points. As indicated in Figure 4, the viability of VK2/E6E7 cells was higher than 90%, and none of the treatment groups had any significant impact on cell viability when compared to the negative control group for up to 12 h. After 24 h, there was some slight reduction in cell viability for the film groups at 10 mg/mL. Furthermore, cytotoxicity was observed to be concentrationdependent. In comparison to VK2/E6E7 cells, the mKG-1 cells appear to be more sensitive to siRNA-NP and siRNA-NP-Ab treatment demonstrating a significant reduction in cell viability after 4 h of incubation with the highest concentration.
textured, rigid, and opaque as the ratio of PVA was increased. Furthermore, as the concentration of either PVA or λ-carrageenan was increased, the time for film disintegration increased as well. Changes in PEG400 and glycerin did not have any effect on the rate of film disintegration.14 Films from formulation A formed a gel structure and started to lose its structural integrity within 5 min. Films from formulation B showed steady hydration and kept their structural integrity for the first hour followed by slow disintegration. Films from formulation C maintained their structural integrity for 24 h and only showed swelling with no signs of obvious disintegration. Finally, none of the NP loading dose tested in this study had any effect on film appearance, texture, disintegration, or pliability. However, when the loading dose of NPs was greater than 40 mg, the films would begin to exhibit a cloudy surface. From the various film formulations evaluated, formulation A was the most acceptable based on its physicomechanical properties (e.g.,
