siRNA-Chitosan Complexes in Poly(lactic-co-glycolic acid

Jun 11, 2013 - Kozono , D.; Yasui , M.; King , L. S.; Agre , P. Aquaporin water channels: atomic structure molecular dynamics meet clinical medicine J...
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siRNA-Chitosan Complexes in Poly(lactic-co-glycolic acid) Nanoparticles for the Silencing of Aquaporin‑1 in Cancer Cells Cinzia Stigliano,† Santosh Aryal,† Marco Donato de Tullio,‡ Grazia Paola Nicchia,§ Giuseppe Pascazio,‡ Maria Svelto,§ and Paolo Decuzzi*,†,∥

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Department of Translational Imaging and Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, Texas 77030, United States ‡ Department of Mechanics, Mathematics and Management and Centre of Excellence for Computational Mechanics (CEMeC), Politecnico di Bari, 70125 Bari, Italy § Department of Bioscience, Biotechnology and Pharmacological Sciences and Centre of Excellence in Comparative Genomics (CEGBA), University of Bari, 70125 Bari, Italy ∥ Department of Experimental and Clinical Medicine, University of Magna Graecia, Catanzaro, 88100, Italy ABSTRACT: A large number of studies document the strong expression of aquaporin-1 (AQP1) in tumor microvessels and correlate this aberrant expression with higher metastatic potential and aggressiveness of the malignancy. Although small animal experiments have shown that the modulation of AQP1 expression can halt angiogenesis and induce tumor regression, effective and safe strategies for the tissue specific inhibition of AQP1 are still missing. Here, small interference RNAchitosan complexes encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are proposed for the intracellular delivery of siRNA molecules against AQP1. These NPs are coated with poly(vinyl alcohol) (PVA), to improve stability under physiological conditions, and demonstrate a diameter of 160 nm. The partial neutralization of the negatively charged siRNA molecules with the cationic chitosan enhances the loading by 5-fold, as compared to that of the free siRNA molecules, and allows one to modulate the release kinetics in the pH-dependent manner. At pH = 7.4, mimicking the conditions found in the systemic circulation, only the 40% of siRNA is released at 24 h post incubation; whereas at pH = 5.0, recreating the cell endosomal environment, all siRNA molecules are released in about 3 h. These NPs show no cytotoxicity on HeLa cells up to 72 h of incubation. In the same cells, transfected to overexpress AQP1, a silencing efficiency of 70% is achieved at 24 h post treatment with siRNA-loaded NPs. Confocal microscopy analysis of NP uptake demonstrates that siRNA molecules accumulate perinuclearly and in the nucleus. Given the stability, preferential release behavior, and well-known biocompatibility properties of PLGA nanostructures, these siRNA-loaded NPs hold potential for the efficient and safe in vivo silencing of AQPs via systemic administration. KEYWORDS: siRNA, polymeric nanoparticles, aquaporins, tumor therapy

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INTRODUCTION Aquaporins (AQPs) are small membrane channels regulating the bidirectional transport of water molecules across the cell membrane, as driven by osmotic and hydrostatic gradients.1,2 They are physiologically expressed in the epithelia and endothelia of several organs, including the kidneys, exocrine glands, eye, skin, and brain, where water exchange has to be finely modulated.3 Recently, AQPs have been associated with multiple disorders such as brain edema,4 glaucoma,5 and tumor progression.6 An aberrant expression of AQPs has been documented in several tumors: AQP1,7,8 AQP4,8,9 and AQP910 are overexpressed in gliomas; AQP3 is strongly present in human squamous cell carcinomas;11 and together with AQP1, it is also expressed in lung adenocarcinoma,12,13 colorectal tumor,14 and other malignancies.6 Clinical data correlate the overexpression of AQPs with higher metastatic potential and aggressiveness, 6 typically associated with increased cell mobility.6,15 AQP1 has been reported in several © 2013 American Chemical Society

studies for its abnormal expression specifically in tumor endothelial cells,15,16 and it has been demonstrated that AQP1 deletion in knockout mice greatly impairs tumor growth and angiogenesis.15,17,18 The proposed mechanism is based on the ability of the polarized AQPs in the leading edge to enhance cell migration by water influx during lamellipodial extension.6,19 This would suggest that the downregulation of AQPs in different subcellular districts of a malignant mass could alleviate the progression and, possibly, induce the regression of the disease. The identification of AQP inhibitors is impaired by the intrinsic refractoriness of these molecules to drug discovery. The vast majority of compounds so far selected are heavy Received: Revised: Accepted: Published: 3186

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Figure 1. Schematic illustration of the fabrication process for PLGA nanoparticles incorporating siRNA-chitosan complexes. A water-in-oil-in-water solvent evaporation technique is used for the synthesis of the nanostructures. W and O represent the water phase and oil phase, respectively.

Figure 2. Physicochemical characterization of siRNA-loaded NPs. (A) Size distribution from DLS analysis showing the mean size ± SD and PdI. (B) SEM image showing the size and morfology of dried samples. The siRNA-loaded NPs have a spherical shape with no appreciable sign of aggregates. The scale bar for the inset is 100 nm. (C) Surface zeta potential distribution demonstrating the uniformity of the sample population. Mean ± SD is presented in the upper corner of the panel. (D) Effect of the incubation in PBS buffer at 37 °C on the stability of the NPs. Size distribution and PdI of the NPs are determined by DLS analysis performed at each time point.

physiologically present in the vessels of multiple organs.22,23 Delivery systems are needed for preserving the therapeutic integrity and supporting the safe systemic administration of the siRNA molecules.24,25 A variety of nanoparticle-based delivery systems have been investigated for the effective in vivo delivery of siRNAs. These include lipid-based and polymer-based nanoparticles (NPs) where the polynucleotides are encapsulated within the hydrophilic core or adsorbed on the cationic surface. Cationic liposomes, stabilized by PEGylation for improved pharmacokinetics, have successfully been used to deliver siRNA.26 However, several studies have documented possible toxicity associated to the use of cationic liposomes manifested in the form of cell contraction, mitotic inhibition, formation of aggregates in blood, and the tendency to induce inflammatory response.27−31 Some cationic lipid formulations have also been

metals, quaternary ammonium salts, and inorganic salts, which are all characterized by high toxicity.20 Recently, a DNA vaccine has been developed to block AQP1 demonstrating antiangiogenic potential in melanoma, colon, and bladder tumors.21 Indeed, the clinical translation of any DNA vaccine-based therapy is still under careful scrutiny. Small interference RNA molecules (siRNAs) against AQPs have also been developed and hold potential in the modulation of their in vivo expression. Along this line, recently, primary tumor reduction in melanoma bearing mice has been demonstrated by the intratumor injection of siRNA-lipofectamine complexes against AQP1.18 However, despite the potential of RNA interference techniques, the in vivo use of siRNAs remains a challenge for the fragile nature of the molecule, which can be rapidly degraded by endoand exonucleases upon direct intravascular injection, and for possible off-target toxicity, particularly for AQP1 that is also 3187

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shown in vitro to elicit inadvertent gene expression32 and enhance the immune response to siRNA.33,34 In polymeric complexes, the negatively charged siRNA molecules could be easily attached by surface-absorption on cationic polymers and surfactants35,36 or entrapped in the NP hydrophilic core prepared by double emulsion solvent evaporation methods.37,38 Alternatively, polycation complexes (polyplexes) can be generated by neutralizing the negative charge of the siRNA with cationic polymers through electrostatic self-assembly.39,40 Although a variety of highly efficient polycations, such as PEI, have been proposed, their high toxicity is still a major concern.41 An attractive polymer for RNAi application is the poly(lactide-co-glycolide) (PLGA), approved by the Food and Drug Administration (FDA). Nanoparticles composed of PLGA are promising because for their high colloidal stability in biological fluid, facile cellular uptake by endocytosis, low toxicity in vitro and in vivo, and controlled release of their cargo.38 In this work, PLGA nanoparticles (NPs) have been developed for the delivery of siRNA molecules against AQP1. The NPs, obtained by the double emulsion solvent evaporation method, comprise a hydrophilic core with the active agent, a PLGA shell, and a PVA coating to increase the colloidal stability. Prior to particle assembly, siRNA molecules are reacted with chitosan, a naturally derived polycation widely used in gene delivery, to form an almost neutral siRNA− chitosan complex. This leads to higher loading, better stability, and controlled release of the siRNA over time. The physicochemical properties of the NPs are characterized by using various characterization techniques. The loading efficiency and release kinetics are analyzed under different physiological conditions. The silencing efficiency is studied in vitro on transfected HeLa cells.

Next, the stability of the siRNA-NPs was tested under physiological conditions. NPs were incubated in PBS buffer (pH 7.4) at 37 °C, and the size and PdI variation was measured over a period of 6 days by DLS analysis. As shown in Figure 2D, over a period of 6 days of stability test in PBS at 37 °C, NPs maintain their hydrodynamic size within the range of 150−190 nm, demonstrating no significant aggregation. In addition, no significant change in PdI was observed, supporting evidence of particle stability in physiological conditions. siRNA-Chitosan Complexes and siRNA Loading in NPs. The materials used in the preparation of the siRNA-NPs are mostly negatively charged at different degrees, except the positively charged chitosan. The PLGA-COOH and siRNA show a negative zeta potential, −25 mV and −18 mV, respectively. The PVA also exhibits a slightly negative surface. Therefore to form stabile NPs, the siRNA molecules were partially neutralized by forming complexes with chitosan. This is a cationic polyelectrolyte, with a zeta potential of 13.8 mV, which can efficiently form stable complexes with the negatively charged siRNA. First, the proper conditions for obtaining a nearly neutral siRNA-chitosan complex were identified by mixing the two components in different ratios. As shown in Figure 3A, chitosan and siRNA were mixed at different N:P

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RESULTS AND DISCUSSION Synthesis and Characterization of siRNA-Loaded PLGA Nanoparticles. siRNA-loaded nanoparticles (NPs) were synthesized by an adapted double-emulsion (w/o/w) solvent evaporation technique as shown in Figure 1. First, siRNA-chitosan complexes were formed in aqueous solution with the objective of enhancing the loading of the siRNA molecules. Then, an aqueous core containing the siRNAchitosan complexes was coated with carboxyl-terminated PLGA (oil phase) followed by the stabilization with hydrated PVA (water phase). The synthesis protocol was optimized to obtain homogeneous and well-dispersed spherical NPs. Note that, as detailed in the sequel, the partial neutralization of the siRNA molecules with the positively charged chitosan has been instrumental in enhancing the NP loading efficiency. The resulting NPs were characterized for their physicochemical properties, including the size, polydispersity index (PdI), morphology, and surface electrostatic charge. As demonstrated in Figure 2A by dynamic light scattering (DLS) analysis, the NPs exhibit an uniform size of 160 ± 10.4 nm with a narrow PdI of 0.180 ± 0.053, thus suggesting the formation of monodisperse particles. The NP morphology was examined via scanning electron microscopy (SEM), and the resulting micrographs (Figure 2B) show monodisperse, spherical structures of 100 nm in size with no sign of bulk aggregation. The NP surface zeta potential was determined to be −25 ± 6.3 mV (Figure 2C). This negative charge confirms that the siRNA-chitosan complexes were engulfed in the NP hydrophilic core and fully covered by the PLGA/PVA composite shell.

Figure 3. (A) Zeta potential of the siRNA-chitosan complexes formed by mixing 25 nM siRNA with chitosan at different molar ratios of amine nitrogen to nucleic acid phosphate (N:P ratio). From left to right, increasing the quantity of chitosan, the complex charge reduces. The ratio 8:1 was chosen for NP synthesis (mean ± SD; n = 3). (B) The histogram compares the siRNA loading in different PLGA NP formulations, with and without chitosan (CH) (mean ± SD; n = 6; *p = 0.05).

molar ratios, defined as the ratio between the amino groups in the chitosan (N) and the phosphate groups (P) in the siRNA. By increasing the N:P ratio from 3:1 to 15:1, the zeta potential of the complex was modulated. At the 8:1 ratio, the siRNAchitosan complex exhibits an almost neutral surface electrostatic charge with a zeta potential of solely 2.73 ± 2.8 mV. Based on the charge interplay during the formation of the emulsion, the almost neutral droplet of siRNA-chitosan complex is expected 3188

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parameter (= 1, exponential curve; > 1, S-shaped curve; < 1, parabolic curve). For the experimental data presented here, the best interpolating parameters take the values a = 0.7 and tscale = 65.96 h for pH = 7.4 (R2 = 0.9900 and SSR = 27.78) and a = 0.7 and tscale = 0.2441 h for pH = 5.0 (R2 = 0.9953 and SSR = 41.17). Using this model, 90% of the siRNA would be released at pH = 7.4 only after about 9 days. As the experimental data of Figure 4 suggest, there is a significant difference in the characteristic time scale of siRNA release under the two different physiological conditions, which is directly quantified through the tscale parameter in the Weibull formula. At pH = 7.4, the siRNA release is mostly governed by the Fickian diffusion of the molecules out of the NP hydrophilic core, through the pores of the PLGA/PVA shell, and eventually in the surrounding environment. This can also be confirmed by using the Ritger−Peppas release model42 which, upon fitting, returns a release exponent of 0.5745 [R2 = 0.9842 and SSR = 0.9843]. Note that a pure Fickian diffusion would be associated with a release exponent of 0.5, thus confirming that in neutral pH the siRNA release is mostly driven by passive diffusion through the pores of the polymeric shell. On the other hand, the rapid release observed at pH = 5.0 is most likely associated with the hydrolysis of the PLGA/PVA shell under acid conditions that would progressively increase the NP porosity with consequent rapid release of its payload.43 NP Uptake in Tumor Cells. NPs loaded with fluorescently labeled siRNAthe siGlo Red DY-547were incubated with HeLa cells for 24 h. After that, cells were fixed, and their nuclei were stained with DAPI. As shown in Figure 5A, lateral, front, and top views show the fluorescent siRNA molecules accumulating within the cell and localizing either perinuclearly or inside the nucleus. To better characterize the NP contribution to the intracellular delivery of siRNA molecules, the internalization efficiency of three different systems were compared, namely, siRNA-NPs, siRNA-lipofectamine, and siRNA-chitosan complexes. The resulting fluorescent microscopy images are shown in Figure 5B. As expected, the commercially available system designed for in vitro cell transfectionsiRNA-lipofectaminedelivered the siRNA molecules with high efficiency, as demonstrated by the several red dots accumulating within the cells. Differently, the siRNAchitosan complex at the N:P ratio of 8:1 did not deliver the siRNA into the cells, possibly due to the unfolding of the complex in physiological solution. This is due to the fact that the chitosan/siRNA complexes do not form stable nanoparticles under physiologically relevant conditions, as confirmed by DLS analysis. Notably, the siRNA-NPs and the siRNAlipofectamine complexes presented comparable delivering efficiency. As it can be seen from the fluorescent confocal images acquired over multiple z-planes (Figure 5A), the fluorescent due to siGlo Red DY-547 localize within the nucleus, suggesting that these NPs are capable of delivering siRNA into the nucleus. The same internalization experiments were performed also at earlier time points at 2, 4, and 6 h (Figure 5C). The siGlo is delivered inside the cell already at 2 h post incubation, demonstrating the high transfection efficiency of our NPs. In Vitro Gene Silencing by siRNA-NPs. To evaluate the gene silencing efficacy of siRNA-NPs, HeLa cells were transfected to transiently express the AQP1 gene. These cells were incubated in 12-multiwell plates with NPs carrying 20 pmol of siRNAs against AQP1. NPs loaded with scrambled siRNA were used separately as control experiments. The

to be more effective in improving the NP stability and siRNA encapsulation as compared to the case of free, negatively charged siRNA molecules.38 An N:P ratio of 8:1 was chosen for the synthesis of the siRNA-NPs. The quantification of loaded siRNA was performed by an adapted aqueous phase extraction technique, where the siRNA molecule signal was further amplified by RiboGreen RNA reagent. As shown in Figure 3B, the siRNA-NPs with a siRNAchitosan complex provided a 24.2 ± 6.72 pmol of siRNA in one milligram of nanoparticles, whereas the direct loading of free siRNA molecules returned a value of only 5.11 ± 0.83 pmol. The partial complexation of the siRNA molecules with chitosan increased the overall loading by 5-fold, as compared to the direct loading of free siRNAs. siRNA Release Kinetics under Different Physiological Conditions. The release of siRNA molecules from the siRNANPs was studied over time at pH 7.4 and 5.0. These two different ionic strengths were selected to mimic the physiological conditions that NPs would found in the circulation (pH = 7.4) and in the acidic environment of the cell endosomes (pH = 5.0), respectively. The percentage of siRNA released over time is shown in Figure 4. For a circulating

Figure 4. In vitro release kinetics of siRNA molecules from NPs. Measurements were performed at 37 °C and at pH 5.0 (circle) and pH 7.4 (triangle). Experimental (symbols) and fitted curves (Weibull model) of cumulative percentage amount of drug released versus time. The siRNA release in the first two hours is shown in the inset. The siRNA release was sustained for over 48 h in neutral pH with over 50% of siRNA molecules being still available after 24 h. Values are presented as the mean ± SE (n = 3).

NP (pH = 7.4), the release rate is extremely low within the first few hours, being of just 20% at 4 h post incubation. After 2 days, only about 50% of the loaded siRNA is released. Conversely, within a cell endosome (pH = 5.0), all of the siRNA molecules are released within the first 3 h post incubation. These different release kinetics are indeed associated to different release mechanisms. The classical empirical model of Weibull can be used for fitting the release profiles.42 Thus, the ratio m between the released siRNA and the originally loaded siRNA is given as m=

⎡ ⎛ t − tlag ⎞a ⎤ M = 1 − exp⎢ −⎜ ⎟⎥ ⎢⎣ ⎝ tscale ⎠ ⎥⎦ M∞

(1)

where t is the time; tlag is the lag time before the onset of release, which is zero in the present cases; tscale is the characteristic time scale for the process; a is the shape 3189

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Figure 5. (A) Internalization of siGlo-Red encapsulated NPs in HeLa cells. Images were captured along multiple z-planes with fluorescent confocal microscopy after 24 h of incubation. In red, the siGlo Red DY-547 (557 nm/570 nm) delivered by the NPs. Cell nuclei were counterstained with DAPI (blue). The scale bar is 5 μm. (B) The confocal images showed the differential cellular uptake of siGlo-Red delivered by PLGA NPs, lipofectamine, chitosan (CH), and without carrier. (C) Internalization of siGlo-Red encapsulated NPs at different time points (2, 4, and 6 h post incubation; scale bar: 10 μm).

efficacy of two different siRNA sequences against the AQP1 was tested. Both sequences were designed to be specific for mouse and human AQP1 genes that exhibit an identity of 87% by BLAST alignment results (data not shown). We have chosen

a 50% mixture of the two to improve the silencing efficacy and try to mimic the physiological RNAi machinery that uses multiple sequences to destroy in different position the complementary mRNA,44 as already demonstrated in previous 3190

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work.18 The AQP1 expression knockdown efficiency was analyzed after 24 h by Western blot. As shown in Figure 6,

Figure 7. (A) Viability of the HeLa cells incubated with different concentrations of NPs loaded with CTRL siRNA or AQP1 siRNA, at 24, 48, and 72 h post incubation. (B) ELISA assay for detecting the secretion of TNF-α and IL-6 by J774.A1 murine macrophages after 2 and 24 h of NPs incubation (*p < 0.005, n = 6).

Figure 6. Immunoblot analysis of AQP1 expression in HeLa cells was performed to test the efficacy of AQP1 siRNA-loaded NPs. A scrambled siRNA (CTRL siRNA) was used to make NPs as control. The same quantity of siRNA (20 pmol) complexed with lipofectamine and chitosan (Ch) was tested respectively as positive and negative controls. Actin was revealed in parallel for each sample and used as a nontargeted internal control housekeeping gene for the densitometry analysis. The histogram reports the densitometric analysis of the protein bands, calculated as the AQP1/actin expression ratio. The histogram shows the analysis of five independent experiments performed 24 h after transfection with the siRNA (*p < 0.005 vs CTRL siRNA, n = 5).

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CONCLUSIONS siRNA-loaded NPs have been synthesized for silencing the AQP1 genes in tumor cells. The NP comprises of a siRNAchitosan complex encapsulated into a multilayer shell of PLGA and PVA. These nanostructures have shown higher stability under physiological conditions with an average diameter of 160 nm. The quasi-neutral electrostatic charge of the hydrophilic siRNA-chitosan complex supports the overall stability of the NPs and controls the siRNA release kinetics. The siRNAloaded NPs have reduced the expression of AQP1 proteins in HeLa cells by 70%, presenting a silencing performance comparable with the results obtained by using transfection reagents, such as lipofectamine, optimized for in vitro use. Differently from other systems proposed for siRNA delivery, such as cationic liposomes and polymeric nanoparticles, the present siRNA-loaded NPs did not induce any cytotoxicity in HeLa cells up to 72 h post incubation, and any secretion of proinflammatory cytokines (IL-6 and TNF-α) from J774.A1 murine macrophages. These results, together with the wellknown biodegradability and biocompatibility of PLGA-based nanoparticles, make these siRNA-loaded NPs interesting candidates for the in vivo, tissue specific silencing of AQPs in cancer treatment.

siRNA-NPs against AQP1 reduced the protein expression to approximately 70% of the control, obtained by using a scrambled siRNA (CTRL siRNA) loaded NPs. As a positive control, the same cells were treated under the same conditions with siRNA-lipofectamine complexes. The lipofectamine complex, carrying 20 pmol of AQP1 siRNA, reduced by about 80% the AQP1 expression as compared to the scrambled siRNA. Furthermore, 20 pmol of siRNAs were complexed with chitosan at the molar N:P ratio of 8:1. As expected based on the results of the internalization assay, these siRNA-chitosan complexes did not exhibit any significant silencing effect. Cytotoxicity and Cytokines Secretion Analysis for the siRNA-NPs. The in vitro cytotoxicity of the siRNA-NPs was estimated by performing a XTT test on HeLa cells. NPs encapsulating the two different siRNAs, the scrambled siRNA and the AQP1 siRNA, were incubated at various doses with HeLa cells. As shown in Figure 7A, the NPs did not show any significant cytotoxic effect over a wide range of concentrations (from 0.5 to 4 mg/mL), at 24, 48, and 72 h post incubation. To test whether the siRNA/chitosan loaded NPs could possibly induce immune activation, the secretion of the pro-inflammatory cytokines, TNF-α and IL-6, was quantified via ELISA. NPs were incubated with J774.A1 murine macrophages, and the cytokine levels were measured at 2 and 24 h post incubation. As observed in Figure 7B, the NPs did not induce any significant pro-inflammatory reaction, the TNF-α and IL-6 levels being much smaller than the positive control (LPS stimulation).

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MATERIALS AND METHODS Materials. All chemicals were purchased from Sigma− Aldrich unless otherwise noted: PLGA (lactide:glycolide = 50:50, MW = 38 000−54 000 Da); poly(vinyl alcohol) (PVA, 99% MW = 30 000−70 000); glycol chitosan (degree of polymerization ≥ 400). All siRNAs sequences were purchased through Dharmacon: siRNA AQP1 target sequences used were: AQP1 siRNA (1) (5′-GGGUGGAGAUGAAGCCCAAUU-3′), AQP1 siRNA (2) (5′-UGGAGAUGAAGCCCAAAUAUU-3′). A scrambled siRNA (5′-UGGAGAAGGCCAACUAGGGUU3′) was used as a negative control (CTRL siRNA). The selected siRNA sequences were submitted to a BLAST search to avoid the targeting of other homologous. siGlo Red DY-547 (557 nm (λ ex) /570 nm (λ em)) was used as a fluorescent siRNA for visual indication of delivery. Quant-iT RiboGreen

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Scientific). The samples were dialyzed in the dialysis minicups, in a large volume of PBS pH 7.4 and pH 5.0, and incubated at 37 °C under stirring at 100 rpm. At calculated time point, three dialysis mini-cups (N = 3) were taken, and the residual siRNA in the NPs was determined as described in aforementioned section; the release amount of siRNA was calculated accordingly. Mathematical Modeling of the siRNA Release Profiles. Two mathematical models are considered to interpolate the experimental data derived for the siRNA release: the Weibull experimental distribution and the Ritger−Peppas power-law model.42 For the Weibull experimental distribution, the ratio of released siRNA m follows the expression:

reagent, MEM alpha+GlutaMAX medium, RPMI medium, fetal bovine serum (FBS), penicillin/streptomycin, and lipofectamine were obtained from Invitrogen. TACS XTT kit was purchased from Trevigen. X-tremeGENE HP DNA transfection reagent was obtained by Roche. RIPA buffer, proteases inhibitor cocktail, and a chemiluminescence ECL kit were obtained by Thermo Scientific. Rabbit polyclonal anti-AQP1, mouse monoclonal anti-β-actin, goat antirabbit IgG-HRP, and rabbit antimouse IgG-HRP antibodies were purchased from Santa Cruz. The ELISA kit was obtained from eBioscience. Preparation of siRNA-Loaded PLGA Nanoparticles. Nanoparticles were prepared using a water-in-oil-in-water (w/ o/w) double emulsion solvent evaporation technique. Briefly, siRNA was reconstituted in RNase-free water and was complexed with glycol chitosan by incubating at room temperature for 15 min on a rotary shaker. The siRNA (2 nmol) was combined with the chitosan previously dissolved in 1 mM AcOH to ensure a positive charge, at molar ratio of the chitosan amine to the polynucleotide phosphate (N/P ratio) of 8:1 to obtain neutral complexes. Next, siRNA complex was emulsified with 5 mg of PLGA dissolved in 0.5 mL of dichloromethane (DCM) by sonication for 30 s (Sonicator Q55, Qsonica, LLC, Newtown, CT). This first emulsion was added dropwise to 2 mL of a 5% (w/v) aqueous solution of PVA and sonicated again for 90 s to obtain second emulsion. The resulting double emulsion was then poured into 20 mL of a 0.3% (v/v) aqueous PVA solution and then maintained under mechanical stirring overnight at 500 rpm. Next, the nanoparticle suspension was washed three times with RNase-free water by centrifugation (12 000 rpm, 20 min, 4 °C) and then resuspended in water. Size and Surface Charge Characterization. The PLGA NP size measurements were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS apparatus (Malvern, Worcestershire, UK). The NPs were suspended in deionized water at a concentration of 0.1 mg/mL. The zpotential (surface charge) of the complexes siRNA-chitosan at different molar ratios (3:1; 8:1; 15:1) was determined using a Zetasizer, at 25 °C and in deionized water. The Smoluchowski model was used to calculate the zeta potential values. All data represent 10 measurements from one sample followed by triplicate measurement of sample prepared in three different preparations. The nanoparticles were further characterized by their size and surface morphology using scanning electron microscopy (FEI Nova NanoSEM 230) operated at 20 keV. Samples were prepared by drop casting and evaporation technique using silicon wafer as a substrate. Finally, the dried samples were sputter-coated with platinum prior to imaging. siRNA Loading. To calculate loading, 2 mg of siRNA NPs was dissolved in 0.5 mL of chloroform at room temperature for 30 min on a rotary shaker. The siRNA was extracted from the organic phase by adding two volumes of RNase-free water and vortexing vigorously for 1 min followed by centrifuging at 12 000 rpm for 10 min at 4 °C. The siRNA content in the aqueous fraction was analyzed with the Quant-iT RiboGreen reagent according the manufacturer’s instructions. A standard curve correlating fluorescence and siRNA concentration was used to determine the amount of siRNA loaded into the PLGA nanoparticles. In Vitro siRNA Release. Nanoparticles (12 mg) were suspended in 12 mL of phosphate buffer (pH 7.4) and fractioned (300 μL) in disposable dialysis mini-cups (Slide-ALyzer MINI Dialysis Units, 10 000 MWCO by Thermo

m=

⎡ ⎛ t − tlag ⎞a ⎤ M = 1 − exp⎢ −⎜ ⎟⎥ ⎢⎣ ⎝ tscale ⎠ ⎥⎦ M∞

where M∞ is the amount of drug released after an infinite time; M is the amount of drug released after a finite time t; tlag is the lag time from the start of the release process (here tlag = 0; tscale is the characteristic time scale of the release process; and a is a coefficient defying the shape of the curve (= 1: exponential curve; < 1: parabolic curve; > 1: sigmoid (S-shape) curve). For the semiempirical Ritger−Peppas’ power-law equation, the ratio of released siRNA m follows the expression:

M = KRP·t n M∞

(2)

where KRP is a constant accounting for the proper features of the polymeric network and the loaded molecule; and n is the diffusional exponent, which provides specific information on the release mechanisms. For n = 0.5, the release occurs by pure Fickian diffusion through the pores of the polymeric network. All interpolating analysis were performed by nonlinear direct fitting through minimization of the sum of squared residuals (SSR). Cell Cultures. HeLa cells from ATCC were maintained in MEM alpha+GlutaMAX medium from Invitrogen, supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen). J774.A1 cells from ATCC were maintained in RPMI medium from Invitrogen, supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen). Cells were incubated at 37 °C in a 5% CO2 atmosphere. Cellular Uptake of Nanoparticles. HeLa cells were seeded into 8-chamber glass cultured slides (Falcon) in complete medium to reach 70% of confluence. The cells were then incubated with PLGA NPs loaded with fluorescent siGlo Red suspended in complete medium at different concentrations. For microscopic observation, after 2, 4, 6, and 24 h of incubation with NPs, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Coverslips were mounted on glass microscope slides with a mounting medium with DAPI for cell nuclei staining. The cellular uptake of nanoparticles was visualized by a laser scanning confocal microscope (Nikon A1 Confocal Imaging System). Images were captured along the z-axis, and the middle panel from the z-stack with focused nuclei was taken to determine intracellular and perinuclear localization of nanoparticles. In Vitro Gene Silencing with siRNA-Encapsulated PLGA Nanoparticles. HeLa cells were seeded at 80% confluence into a 12-well plate and grown overnight. A transient transfection was performed using pTarget mammalian 3192

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Molecular Pharmaceutics expression vector cloned with the murine gene AQP1, using the X-tremeGENE HP DNA transfection reagent (Roche) according to the manufacturer’s instructions. Six hours after transfection, the nanoparticles with AQP1 siRNA or control siRNA were suspended in fresh medium and used to substitute the transfection medium. A sample of 20 pmol siRNA loaded NPs was used for the silencing experiment. siRNA and lipofectamine (Invitrogen) complexes were used as a positive control of siRNA efficacy; 20 pmol of siRNA was incubated with the lipofectamine according to manufacturer’s instructions. To compare the silencing efficacy of NPs and chitosan alone, 20 pmol of siRNAs were complexed with chitosan at ratio N/P of 8:1 and used to transfected the cells. The cells were harvested after 24 h of incubation. Western Blot Analysis. Total protein was extracted from the cells to evaluate the expression of AQP1 protein normalized on the expression of actin protein as control. Briefly, the cells were washed twice with cold PBS and lysate with RIPA buffer (Thermo Scientific) supplemented with the proteases inhibitor cocktail (Thermo Scientific). The lysates were then analyzed by electrophoresis on 10% (w/v) sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels. Blotting was performed using a semidry system. Blots were incubated overnight at 4 °C with primary antibodies diluted 1:500, and binding was detected using HRP-conjugated secondary antibodies diluted 1:15 000. All of the antibodies were from Santa Cruz. Signals were detected via an enhanced chemiluminescence ECL kit (Thermo Scientific) with Li-COR instrument (Bioscience, Lincoln, NE). Cell Viability Measurement. Cell viability was determined using the TACS XTT kit (Trevigen) against HeLa cells incubated with PLGA nanoparticles. Cell were plated in 96-well plates at 10 000 cells per well in complete medium and cultured for 24 h. To determine the NPs effects on cell viability and proliferation, control siRNA or AQP1 siRNA loaded in NPs were resuspended in complete medium at different concentrations and used to replace the culture medium. The cells were further cultured for 24, 48, and 72 h. XTT working solution was added according to the manufacturer’s instructions. The absorbance was measured at 490 nm in a microplate reader (SynergyH4, Bioteck, Winooski, VT), and the data were collected when the absorbance was between 0.8 and 1.2. Cell viability was normalized to that of HeLa cells cultured without nanoparticles. Measurement of Cytokines Secretion by ELISA. J774.A1 murine macrophages were plated in 96-well plates at 20 000 cells per well in complete medium and cultured for 24 h. The day after, the siRNA/chitosan loaded NPs (2 mg/mL) were incubated with the cells. The positive control cell groups were treated with LPS (1 μg/mL) (Sigma Aldrich). To measure the macrophage cytokine levels, cultured medium was harvested 2 and 24 h after incubation. TNF-α and IL-6 levels were measured by ELISA (eBioscience) according to the manufacturer’s instructions.

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The authors would like to thank the reviewers for their valuable comments and suggestions. C.S. acknowledges the “Scuola di Dottorato in Fisiologia e Biotecnologie Cellulari e Molecolari” of the University of Bari for partial support. P.D. acknowledges the program “Messageri della Conoscenza” of the Italian Minister for Education under the Progetto Didattico “Progettazione ottimale di nanoparticelle per applicazioni biomedicali” and The Methodist Hospital Research Institute for partial support.

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