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. 2010 Nov 16;19(2):372–380. doi: 10.1038/mt.2010.242

Poly(oligo--arginine) With Internal Disulfide Linkages as a Cytoplasm-sensitive Carrier for siRNA Delivery

Young-Wook Won 1, Sun-Mi Yoon 1, Kyung-Mi Lee 2, Yong-Hee Kim 1
PMCID: PMC3034849  PMID: 21081902

Abstract

Small interfering RNA (siRNA) has emerged as a therapeutic strategy for various diseases due to its target-specific gene silencing; however, its relatively high molecular weight, negative charge, and low stability hamper in vitro and in vivo applications. Approaches to overcome those drawbacks have relied on nonviral siRNA carriers based on cationic polymers or peptides. Nevertheless, cationic polymer-based siRNA carriers have yet to resolve intrinsic problems such as cytotoxicity and immunogenicity. An environment-sensitive carrier was recently proposed to enhance siRNA bioactivity and to reduce the carrier safety issues. Only a few studies, however, have shown cytoplasm-sensitive dissociation of the polyplex. In the present study, we clearly demonstrated decondensation of siRNA/poly(oligo--arginine) polyplex in the cytoplasm in response to intracellular glutathione (GSH) and the enhanced bioactivity of siRNA against VEGF (siVEGF) used as a model both in vitro and in an animal model. Reducible poly(oligo--arginine) (rPOA) rapidly dissociated in the cytoplasm, resulting in fast siRNA release to its target location while maintaining siRNA bioactivity both in vitro and in vivo.

Introduction

Small interfering RNA (siRNA) is a well-established approach to specifically inhibit gene expression both in vitro and in vivo.1 Among siRNA technologies, synthetic siRNA-based methodologies are currently being used not only for functional genetic analysis but also to develop highly target-specific therapeutics.2 Despite the potency of siRNA technologies, the relatively high molecular weight, negative charge characteristic, and instability of siRNA require a specialized delivery vehicle to localize siRNA to the cytoplasm.3 Attempts to overcome those obstacles have exposed various carriers such as liposomes, nanoparticles, and/or cationic polymers.4 Cationic polymers are the most established approaches to siRNA delivery, but several intrinsic problems remain, including cytotoxicity and immunogenicity. Recent studies have focused on developing environment-responsive delivery vehicles for siRNA delivery in order to minimize toxicity and enhance the siRNA activity.5

Studies of environment-sensitive polymeric gene carriers have reported a wide variety of bioreducible cationic polymers. Cationic polymers connected by internal disulfide linkages have high potential for siRNA delivery due to low toxicity, high transfection efficiency, and rapid siRNA release in the cytoplasm.6,7,8 Various polymers containing disulfide bonds are reduced in cytoplasmic environments and upon exposure to reducing agents, both of which facilitate to release siRNA due to polyplex dissociation.9,10 Bioreducible polymers have unique features that lead to effective siRNA localization in the cytoplasm. Thus, cationic polymers with internal disulfide bonds have the potential to provide cytoplasm-selective controlled siRNA release and improved stability in extracellular spaces.

Protein transduction domains (PTDs) have shown high transduction ability for various cargoes such as proteins and particles. In particular, -9-arginine, the most well-known PTD, has shown higher transduction efficiency than those of other PTDs. Recent studies demonstrated usefulness of PTDs for gene delivery because they can form a polyplex through electrostatic interactions between positively charged PTDs and negatively charged nucleic acids. These interactions may enhance stability and cellular uptake, improving bioactivity of DNA or siRNA.11 In a previous study, we developed a -9-arginine PTD-based reducible poly(oligo--arginine) (rPOA) by introducing internal disulfide linkages via spontaneous oxidation of the cysteine sulfhydryl group.12 rPOA showed considerable DNA transfection efficiency with low cytotoxicity compared to polyethylenimines (PEI) both in vitro and in vivo. The major mechanistic difference between DNA and siRNA is the target location in the cells. DNA must be released from the polymers upon cellular uptake and relocate into the nucleus, whereas siRNA functions in the cytoplasm.6 This locational difference may restrict cationic polymers to either DNA or siRNA applications.

The object of the present study was to demonstrate whether rPOA could deliver siRNA with rapid polyplex dissociation in order to facilitate siRNA release in the cytoplasm in response to intracellular glutathione (GSH). To characterize the polyplex, we examined condensation ability, siRNA stability in serum and the biochemical properties of the polyplex. In addition, we clearly revealed cytoplasm-sensitive dissociation and bioactivity of the siRNA/rPOA polyplex using siRNA against VEGF (siVEGF) as a model both in vitro and in a mouse tumor model.

Results

Condensation and stability tests

The electrostatic interactions between negatively charged siRNA and positively charged polymer drive siRNA condensation, resulting in polyplex formation. In order to confirm the ability of rPOA to condense siRNA, siRNA and rPOA were mixed in a ratio-dependent manner before a gel retardation assay. The siRNA condensation by rPOA retarded siRNA migration in an agarose gel as shown in Figure 1a (left). At and above the charge ratio of 5, the siRNA was completely retarded, suggesting fully condensed siRNA and showing that rPOA effectively condensed the siRNA. The siRNA condensation via rPOA interaction was further examined in the presence of 2-mercaptoethanol to reduce the internal disulfide bonds of rPOA, because rPOA fragmentation may weaken the electrostatic interactions. Figure 1a (right) shows no siRNA retardation at a ratio of 7.5 and complete siRNA condensation at a ratio of 10, demonstrating that reduced rPOA does not efficiently condense siRNA compared to the condensation associated with oxidized rPOA.

Figure 1.

Figure 1

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Polyplex formation of siRNA/rPOA, and siRNA stability in serum. (a) siRNA was condensed by rPOA ratio-dependently with or without reducing agent. siVEGF was mixed with various ratios of rPOA; thereafter, siRNA/rPOA polyplexes were incubated in the absence or presence of 40% 2-mercaptoethanol (2-ME). (b) Naked siRNA and siRNA/rPOA polyplexes were incubated in 90% mouse serum for 3, 5, and 10 minutes. For the heparin competition assay, the siRNA/rPOA polyplex prepared at a ratio of 5 was further incubated for 1 hour in the presence of heparin. The ratios represent a charge ratio of rPOA to siRNA. rPOA, poly(oligo--arginine); siRNA, small interfering RNA.

Instability is a major problem in siRNA applications because siRNA degrades in serum within only a few minutes. Therefore, in vitro or in vivo tests require improved siRNA stability. Polyplex formation may help to overcome the instability problem by protecting siRNA against serum. To test whether rPOA protects siRNA in serum, naked siRNA and siRNA/rPOA polyplexes were incubated in 90% mouse serum for 3, 5, and 10 minutes. As shown in Figure 1b, naked siRNA rapidly degraded in serum, whereas rPOA prolonged siRNA stability in serum at and above a siRNA/rPOA ratio of 5. The heparin competition assay further confirmed that polyplex formation stabilized and protected siRNA in the serum (Figure 1b). The siRNA bands appeared at the same position as the control bands after dissociation of the siRNA/rPOA polyplexes. These results indicate that rPOA can protect siRNA against degradation in serum under the test conditions.

Characterization of siRNA/rPOA polyplexes

Biophysical properties of polyplexes, such as the mean diameter, zeta potential, morphology, size distribution, and homogeneity, may influence internalization, intracellular tracking of siRNA and transfection efficiency. To characterize the siRNA/rPOA polyplexes, the sizes and zeta potentials were measured using a dynamic light scattering, and the morphological characteristics and homogeneities were observed using an atomic force microscope. The average polyplex mean diameter and zeta potential were 90.3 ± 2.5 nm with polydispersity index of 0.171 ± 0.012 and 36.7 ± 6.4 mV, respectively. Moreover, siRNA/rPOA polyplexes were well dispersed with a narrow size distribution and homogeneity, as shown in Figure 2a,b. We hypothesized that upon rPOA exposure to reducing environments, internal disulfide bond reduction weakens the binding affinity between siRNA and rPOA as shown in Figure 1a because rPOA reverses back to 9-arginine. In order to test the hypothesis, time-dependent changes in polyplex size were observed in the presence or absence of dithiothreitol (DTT), used to mimic a reducing environment similar to that of the cytoplasm. Polyplex size reached a steady state within 30 minutes, with no significant changes up to ~50 minutes incubation, before adding the reducing agent (Figure 3). After 5 minutes of incubation with DTT, the polyplex size increased rapidly with a high-polydispersity index value (>0.5) for 30 minutes followed by a slow increase throughout the measurement period. On the other hand, the polyplex without reducing agent maintained its size, in the range of 70–100 nm, with a low-polydispersity index value.

Figure 2.

Figure 2

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Size distribution and morphological characteristic of siVEGF/rPOA polyplexes. (a) Polyplexes were prepared with 3.5 µg siVEGF at a charge ratio of 5 in 500 µl PBS. After 30 minutes of incubation, the sizes were measured at 37 °C, and the size distributions were expressed based on the intensity. The data represents the average ± SD of three independent experiments. (b) The polyplexes were prepared with 2.5 µg siVEGF at a ratio of 5 in 300 µl PBS, thereafter 10 µl of the polyplexes were dropped onto the mica. An AFM picture was taken in the close contact mode. AFM, atomic force microscope; rPOA, poly(oligo--arginine); siRNA, small interfering RNA.

Figure 3.

Figure 3

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Sizes and PDI changes in siVEGF/rPOA polyplexes in the absence or presence of DTT. The polyplex preparation and size measurements were same as those in Figure 2a. After 50 minutes, 10 mmol/l DTT or equivalent volume of PBS were added to the samples. The changes in size were observed up to 120 minutes. DTT, dithiothreitol; PBS, phosphate-buffered saline; rPOA, poly(oligo--arginine).

In vitro transfection studies

The transfection efficiency of rPOA in squamous cell carcinoma (SCC) cells was determined by measuring luciferase activity using a luminometer. As shown in Figure 4a,b, luciferase expression by rPOA was compared with PEI and poly--arginine (PLR) at varying ratios and doses. rPOA showed a transgene expression level similar to that of PEI and higher than that of PLR; however, the luciferase activities of rPOA, PEI, and PLR were independent of the ratio. When plasmid luciferase (pLuc) was transfected in different doses at a constant cationic polymer ratio, the activities increased slightly with increasing DNA dose in rPOA, PEI, and PLR, and rPOA showed transfection efficiency almost equal to that of PEI. The polyplex cytotoxicities were concurrently determined to be <10% for pLuc/rPOA, ~20% for pLuc/PEI and ~15% for pLuc/PLR at ratios of 2 and 4 (Figure 4c).

Figure 4.

Figure 4

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In vitro transfection efficiency. (a) Ratio-dependent and (b) dose-dependent transfection efficiencies of pLuc/rPOA, pLuc/PEI, and pLuc/PLR in SCC cells. The polyplexes were prepared with a fixed DNA concentration of 2 µg/well at ratios of 2 and 4 or at a constant ratio of 2 with DNA doses of 1 and 2 µg. (c) Cytotoxicities of rPOA, PEI, and PLR polyplexes. Polyplexes were prepared with 2 µg pLuc at ratios of 2 and 4. After 48 hours of incubation, the luciferase activity and cytotoxicity were measured using a luminometer and an MTT assay, respectively. The data represent the average ± SD of three independent experiments with four replicates. DTT, dithiothreitol; PBS, phosphate-buffered saline; PLR, poly--arginine; PEI, polyethylenimines; pLuc, plasmid luciferase; rPOA, poly(oligo--arginine); SCC, squamous cell carcinoma.

Intracellular siRNA delivery

When nonviral carriers deliver siRNA, siRNA has to be localized in the cytoplasm. Thus, siRNA internalization with and without rPOA was observed to occur in a time-dependent manner using a confocal laser scanning microscopy (CLSM). To show intracellular siRNA delivery, fluorescein isothiocyanate-labeled siLuc (luciferase-targeted siRNA) was condensed and transfected using rPOA. As shown in Figure 5, siRNA internalized within 30 minutes, mainly located at the cell boundaries. After an additional 30 minutes, siRNA was localized mostly in the cytoplasm. The spread continued to 120 minutes post-transfection, when no naked siRNA was observed in the cells (fixed cell). The internalization of siRNA/rPOA polyplex in live cells was further visualized by live CLSM because cell fixation may influence the internalization of polyplex. As shown in Figure 5 (live cell), siRNA was uniformly distributed in the cytoplasm within 2 hours, indicating that the internalization of siRNA/rPOA polyplex was not artificially occurred by the cell fixation. The siRNA internalization by rPOA was rapid and well dispersed in the cytoplasm, providing direct evidence of effective siRNA delivery by rPOA to the cytoplasm.

Figure 5.

Figure 5

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Internalization of siRNA by rPOA. FITC-labeled siLuc was condensed and transfected to SCC cells using rPOA. Nuclei were counter-stained with DAPI (blue); FITC-labeled siRNA (green); optical images of cells (DIC, gray). After 30, 60, and 120 minutes post-transfection, cells were washed, fixed, and mounted for confocal microscope analysis. For live cell analysis, the FITC-siLuc/rPOA polyplex was transfected to the cells, and cells were washed after 2 hours of incubation prior to the live CLSM. FITC, fluorescein isothiocyanate; PEI, polyethylenimines; pLuc, plasmid luciferase; rPOA, poly(oligo--arginine); SCC, squamous cell carcinoma; siRNA, small interfering RNA.

In vitro VEGF silencing

Cytoplasm-sensitive siRNA delivery ability of rPOA was confirmed by measuring vascular endothelial growth factor (VEGF) secretion level after the transfection of siRNA for VEGF in the absence or presence of DL-buthionine-[S,R]-sulfoximine (BSO), which inhibits GSH production in the cytosol. VEGF silencing was first tested in SCC cells by measuring secreted VEGF using an ELISA kit. As shown in Figure 6a, VEGF secretions reduced in the siVEGF/PEI, siVEGF/PLR, and siVEGF/rPOA treatment groups by ~20%, ~5%, and ~50%, respectively, whereas those treated with scVEGF/rPOA (scrambled VEGF) showed a ~10% reduction. The VEGF and fibroblast growth factor mRNA levels were further assessed by quantitative reverse transcriptase-PCR as shown in Figure 6b. VEGF mRNA levels were reduced by ~70% in the siVEGF/rPOA and siVEGF/Lipofectamine treatment groups, whereas fibroblast growth factor mRNA levels maintained 90–95%. On the other hand, scVEGF treatment showed no silencing of VEGF and fibroblast growth factor mRNA in both rPOA and Lipofectamine groups. PEI and PLR were omitted in quantitative reverse transcriptase-PCR analysis because of their poor effects on VEGF silencing as shown in Figure 6a. The difference in VEGF mRNA and protein expression levels may be originated from various factors affecting the mRNA-protein correlation because the correlation between mRNA level and protein abundance is modest. Strikingly, siVEGF/rPOA reduced the VEGF secretion by 20% in the presence of BSO, whereas siVEGF/PEI and siVEGF/PLR showed no change, regardless of the BSO presence (Figure 6c). These results indicate that GSH plays a crucial role in the controlled release of siRNA in the cytoplasm. Cell viability measurements confirmed that the siVEGF/rPOA polyplex was nontoxic to cells, and that the reduction of VEGF secretion was not originated from the siVEGF/rPOA cytotoxicity (Figure 6d).

Figure 6.

Figure 6

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In vitro VEGF silencing. (a) The polyplexes prepared with 100 pmol siVEGF at a ratio of 5 were transfected into SCC cells for 48 hours. VEGF secreted from cells was quantified using an ELISA kit (*P < 0.001 versus control). (b) Relative mRNA levels of VEGF and FGF to GAPDH were assessed by qRT-PCR (*P < 0.001). (c) VEGF silencing was measured at the same transfection conditions as in a in the absence or presence of 10 mmol/l BSO (*P < 0.001). (d) At the same time, cell viability was assayed using MTT. The data represent the average ± SD of three independent experiments with four replicates. BSO, DL-buthionine-[S,R]-sulfoximine; FGF, fibroblast growth factor; qRT-PCR, quantitative reverse transcriptase-PCR; rPOA, poly(oligo--arginine); SCC, squamous cell carcinoma; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

Intracellular rPOA reducibility

Cytoplasm-sensitive reductions of the disulfide linkages may facilitate the siRNA release by dissociating siRNA/rPOA polyplexes. Cy5.5-rPOA tagged by an amide bond and fluorescein isothiocyanate-labeled siRNA were used to visualize the intracellular trafficking of rPOA and siRNA using CLSM. To show the effects of GSH production on polyplex dissociation, the fluorescent-labeled polyplexes were transfected into SCC cells in the absence or presence of BSO. As shown in Figure 7, intact siRNA/rPOA polyplexes and rapidly released siRNA were observed at the boundaries of cells in the first 30 minutes post-transfection in both groups. The polyplexes rapidly dissociated at 2 hours of incubation, and at 4 hours post-transfection, there was no colocalization in the absence of BSO (Figure 7, left), whereas siRNA and rPOA remained stable in the cytoplasm as intact polyplexes in the presence of BSO (Figure 7, right). These results indicate that GSH plays a crucial role to dissociate siRNA/rPOA polyplexes in the cytoplasm, thus facilitating the siRNA release in the proper location.

Figure 7.

Figure 7

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Intracellular reducibility of siRNA/rPOA polyplexes. Equal volumes of plain medium or plain medium-containing BSO were added to SCC cells 6 hours prior to the transfection at a final BSO concentration of 10 mmol/l. The samples were prepared by mixing 1 µg FITC-siRNA (green) and Cy5.5-rPOA (red) at a ratio of 5; nuclei were counter-stained with DAPI (blue); cell morphologies were shown in DIC images (gray). After 0.5, 2, and 4 hours post-transfection, cells were washed, fixed, and mounted for confocal microscope analysis. BSO, DL-buthionine-[S,R]-sulfoximine; FITC, fluorescein isothiocyanate; rPOA, poly(oligo--arginine); SCC, squamous cell carcinoma; siRNA, small interfering RNA.

In vivo tests

VEGF has angiogenic property, which is necessary for the tumor growth; therefore, the in vitro findings were further extended to in vivo study using a subcutaneous tumor model. Figure 8a,b shows tumor suppression in the siVEGF/rPOA-treated group compared with those of the other groups, and a less increase in the body weight was observed in siVEGF/rPOA group in comparison with phosphate-buffered saline (PBS) (Figure 8c). Although the body weight difference between PBS and siVEGF/rPOA-treated groups was around 2 g, there was not a statistically significant difference between two values (P = 0.459). The tumor volumes reached ~5,500, ~4,500, ~3,000, and ~1,000 mm3 at 17 days postinjection in PBS, scVEGF/rPOA, naked siRNA, and siVEGF/rPOA groups, respectively. In addition, intratumoral VEGF expression levels measured 2 days after the second administration demonstrated that the siVEGF/rPOA group showed approximately twofold decrease in VEGF expression compared with those of the naked siVEGF or scVEGF/rPOA groups as shown in Figure 8d. This result indicates that the suppression in tumor growth was resulted from VEGF silencing, because tumor growth is associated with VEGF-induced angiogenesis.

Figure 8.

Figure 8

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Antitumor effects of siVEGF/rPOA polyplexes in vivo. The polyplexes were directly injected into the tumor twice per week at a dose of 3.5 µg siVEGF/mouse. (a) The tumor volumes were measured (*P < 0.05, **P < 0.01 versus naked siVEGF; #P < 0.001 versus PBS), (b) images of the tumors were taken at 3, 14, and 17 days, and (c) body weights were measured twice a week. The data represent the average ± SE of five mice. (d) The tumors were dissected, homogenized, and lysed to quantify the intracellular VEGF levels, and the samples were collected 2 days after the second administration (*P < 0.001 versus naked siVEGF). The data represent the average ± SD of three mice. rPOA, poly(oligo--arginine); PBS, phosphate-buffered saline; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

Discussion

The localization of siRNA to the cytoplasm is a critical step in effective siRNA-mediated gene silencing because the target mRNA degradation occurs in the cytoplasm. Nonviral vectors, therefore, must deliver synthetic siRNA to the cytoplasm. Attempts to carry siRNA into the cytoplasm have focused on developing environment-sensitive nonviral vectors in which siRNA polyplexes are stable in extracellular spaces and rapidly dissociate in intracellular environments. In particular, polymeric vectors containing internal disulfide bonds have been shown to provide great advances in terms of stable polyplex formation and cytoplasm-sensitive siRNA release. Disulfide bonds spontaneously formed by the oxidation of two sulfhydryl groups (-SH) are relatively stable in the extracellular space and easily reversed under reductive intracellular environments.13

Branched PEI (25 kd) was chosen as a comparison for gene silencing efficiency analysis with 9-arginine-based reducible peptide, rPOA. PEI is the most frequently used nonviral carrier for gene delivery in vitro as well as in vivo due to its high transfection efficiency through the proton-buffering effect.14 Despite the effectiveness of branched PEI, high molecular weight PEI leads to severe cytotoxicity. Low-molecular weight PEI (~10 kd), on the other hand, is less toxic but has poor transfection efficiency. High molecular weight PEI composed of repeating units with low-molecular weight PEI (~10 kd) can efficiently deliver genes with low toxicity.15 Thus, we hypothesized that 9-arginine-based reducible peptide connected by disulfide linkages would be cleaved in the reductive environment of the cytoplasm, thereby facilitating siRNA release in the cytoplasm with less toxicity.

Although several studies have reported reducible nonviral vectors to be useful for siRNA delivery, only a few studies have shown intracellular reduction. To our knowledge, this is the first paper that clearly shows cytoplasm-sensitive dissociation of the polyplex in response to intracellular GSH. GSH level is expected as to be a major factor effecting reduction of internal disulfide bonds. GSH levels in normal body fluids and extracellular spaces remain low, whereas intracellular GSH is high.16 The different GSH levels provide a relatively high-redox potential that stabilizes disulfide bonds in extracellular spaces and highly reducing environments inside cells. The high-redox potential gradient between extracellular and intracellular environments provides the ability to dissociate rapidly, resulting in fast siRNA release from reducible peptides including rPOA.17 Therefore, the dissociation rate of siRNA/rPOA is expected to decline when intracellular GSH concentration decreases due to blocked glutamyl-S-transferase upon exposure to BSO.6,7,12,18 The polyplexes remained intact nano-sized aggregates with negligible siRNA/rPOA dissociation after internalization in the presence of BSO (Figure 7). Disassembly of intact polyplexes and good rPOA dispersion in the cytoplasm under the normal conditions provides direct evidence for the facilitation of cytoplasm-sensitive siRNA release.

DTT or 2-mercaptoethanol is well-known reducing agents that mimic the reductive environment of the cytoplasm. These agents reduce the internal disulfide bonds of rPOA, reverting rPOA back to its low-molecular weight counterparts, 9-arginines.19 Upon exposing rPOA to the reducing agents, the resulting peptides were not sufficient to condense siRNA, even at high charge ratios, suggesting that the reduction of the internal disulfides of rPOA would trigger siRNA release (Figure 1a). Moreover, the increased mean diameter and polydispersity index values obtained from dynamic light scattering measurements under DTT-induced reductive conditions support the environment-responsive dissociation of the siRNA/rPOA polyplex (Figure 3).5 These observations lead us to conclude that intracellular GSH could reduce internal disulfides of rPOA, triggering siRNA release from the polyplex.

A wide variety of growth factors have been reported to induce tumor progress, among which VEGF is the only growth factor consistently found in various angiogenic conditions.2,11 Inhibiting VEGF secretion or activity has been shown to suppress tumor growth by inhibiting angiogenesis in animal tumor models.20,21 Although siRNA is a potent therapeutic for the treatment of various diseases, it loses bioactivity and stability due to serum nucleases or inappropriate delivery vehicles.22 The present study investigated the bioactivities of siRNA/rPOA polyplexes both in vitro and in vivo using siRNA against VEGF to suppress tumor growth. Relative increases in VEGF secretion in the presence of BSO compared to that in the absence of BSO demonstrate that cytoplasm-sensitive dissociation of the polyplex constitutes a promising strategy for the intracellular siRNA delivery.18,23 In addition, VEGF expression in tumors decreased after the injection of siVEGF/rPOA polyplexes into the tumor, validating siRNA bioactivity after the formation of polyplex with rPOA. Despite the therapeutic potency, siVEGF/rPOA polyplex showed a less gain in the body weight in comparison with PBS. This result may be due to one or more factors such as an intrinsic toxicity of siRNA, immunogenicity of rPOA or tumor volume increase. Although the polyplexes were injected locally to minimize the risk of such effects, liver function, immune activation by siRNA and immunogenicity of rPOA upon siRNA/rPOA injection remain to be examined.

In summary, the cytoplasm-sensitive polypeptide, rPOA, was sufficient to condense and stabilize siRNA and rapidly deliver siRNA to the cytoplasm. We demonstrated rapid dissociation of siRNA/rPOA polyplexes in response to GSH in the cytoplasm, which resulted in enhanced target gene silencing. Moreover, the bioactivity of the siRNA/rPOA polyplex was confirmed using siVEGF to suppress tumor growth in an animal model. The cytoplasm-sensitive rPOA constitutes a promising strategy for siRNA delivery in terms of high extracellular stability and rapid siRNA release in intracellular environments.

Materials and Methods

Materials. Peptide Cys-(D-R9)-Cys (CRRRRRRRRRC, Mw 1628) was purchased from Peptron (Daejeon, Korea). PEI (branched form, average molecular weight 25,000) and PLR (average molecular weight >70,000) were obtained from Sigma (St Louis, MO). pLuc (pGL3-promoter, 5,010 bp) and the luciferase assay kit were from Promega (Madison, WI). siRNA was purchased from Samchully Pharmaceuticals (Seoul, Korea) (siVEGF: 5′-AUGUGAAUGCAGACCAAAGAATT-3′, 5′-UUCUUUG GUCUGCAUUCACAUTT-3′ scVEGF: 5′-GAUAGCA AUGACGAAUGCGUATT-3′, 5′-UACGCGAUUCGUCAUUGCUAUC TT-3′).11 Primers for quantitative reverse transcriptase-PCR were purchased from Bioneer (Daejeon, Korea). All other reagents were of analytical grade.

Synthesis of reducible rPOA. rPOA was prepared by dimethyl sulfoxide -mediated oxidative polycondensation as previously described.12 In brief, Cys-(D-R9)-Cys was reacted in PBS (pH 7.4) containing 30% dimethyl sulfoxide at a concentration of 57 mmol/l. After 6 days, the reaction was terminated by adding 5 mmol/l HEPES buffer. Impurities were removed by dialysis (MWCO: 10,000 Da), and the purified peptides were collected and lyophilized using a vacuum-freeze dryer (Freezone 4.5; Labconco, Kansas City, MO).

siRNA condensation. Various amounts of rPOA were added to PBS containing 50 pmol siVEGF, and the samples were incubated for 20 minutes at room temperature and electrophoresed on 0.8% (wt./vol.) agarose gel for 30 minutes at 100 V in 0.5% TBE buffer solution. Ratios were expressed as charge ratios of rPOA to siRNA for all data. For the gel retardation assay in the presence of 2-mercaptoethanol, siRNA/rPOA polyplexes were prepared at a final volume of 6 µl, and the samples were incubated for 30 minutes at room temperature. After incubation, 4 µl 2-mercaptoethanol was added to the mixture to break the disulfide bonds between cysteines. The reaction was performed under gentle stirring for 1 hour at 37 °C. The samples were electrophoresed as described above.

Size and zeta potential determinations. The mean diameters and surface zeta potentials of siRNA/rPOA polyplexes were measured using dynamic light scattering (Zetasizer-Nano ZS; Malvern Instruments, Worcestershire, UK). At a ratio of 5, the mean diameters and zeta potentials were measured three times with triplicates. For polyplex behaviors under reductive environments, DTT was added to samples at a final concentration of 10 mmol/l, and an equivalent volume of PBS was added to the control.

Atomic force microscopy. Atomic force microscope (Nano-R AFM; Pacific Nanotechnology, Santa Clara, CA) was used to visualize the morphological characteristics of siRNA/rPOA polyplexes in the close contact mode in air. The polyplex was prepared with 2.5 µg siRNA at a charge ratio of 5 in 300 µl PBS. After 30 minutes of incubation, 10 µl siRNA/rPOA polyplex was placed on freshly cleaved mica. After 3 minutes, the remaining solution was washed with a drop of water, and the extra water was dried with a gentle stream of nitrogen.

siRNA stability in serum. The siRNA protection ability of rPOA was examined in mouse serum. The polyplexes were prepared at charge ratios of 5 and 7.5 in PBS and incubated at room temperature for 30 minutes. After incubation, mouse serum was added to the polyplexes at a final concentration of 90% (vol./vol.), thereafter samples were further incubated at 37 °C with shaking at 150 r.p.m. The reaction was terminated at predetermined time points. After incubation, heparin was added to the polyplexes (charge ratio 5) at a final heparin to rPOA weight ratio of 300 in the presence of 0.01 mol/l EDTA. After 1 hour of incubation, the mixtures were electrophoresed under the conditions described above.

Cell culture. SCC was obtained from ATCC (Rockville, MD) and cultured in RPMI 1640, 10% fetal bovine serum and penicillin (100 IU/ml)/streptomycin (100 µg/ml). Cells were incubated to 80% confluency at 37 °C with 5% CO2.

In vitro transfection efficiency. SCC cells were seeded onto 12-well plates at a density of 2 × 104 cells/well. After 1 day of incubation, the culture medium was replaced with 1 ml 10% fetal bovine serum-supplemented RPMI 1640 containing pLuc/rPOA, pLuc/PEI, or pLuc/PLR polyplexes. The polyplexes were prepared by mixing pLuc and rPOA, PEI or PLR at a charge ratio of 2 or 4 in RPMI 1640. After 48 hours of cell incubation, cells were washed twice with PBS and treated with 150 µl 1× cell lysis buffer reagent (Promega) for 20 minutes. The cell lysates were scraped, harvested, transferred to 1.5-ml micro tubes, and centrifuged for 3 minutes at 13,000 r.p.m. Luciferase RLU of the cell lysates were measured on a 96-well plate luminometer (Berthold Detection System, Pforzheim, Germany) with 20 seconds integration, and the results were expressed as RLU/mg of cell protein determined by the DC protein assay kit with a bovine serum albumin standard (Bio-Rad Laboratories, Hercules, CA).

In vitro VEGF silencing. SCC cells were seeded onto 12-well plates at a density of 2 × 104 cells/well. After 1 day of incubation, the culture medium was replaced with 1 ml 10% fetal bovine serum-supplemented RPMI 1640 containing siRNA/rPOA, siRNA/PEI, or siRNA/PLR polyplexes. The polyplexes were prepared by mixing 100 pmol siRNA and rPOA, PEI or PLR at a charge ratio of 5 in RPMI 1640. After 48 hours of cell incubation, 500-µl culture medium was collected for ELISA, and MTT was added to the remaining culture medium for the cytotoxicity test. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used to confirm the efficiency of siRNA according to the manufacturer's protocol. The amount of VEGF secreted from the cells was determined using a mouse VEGF ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. For quantitative reverse transcriptase-PCR analysis, total RNA was isolated using RNeasy Mini kit according to manufacturer's protocol (QIAGEN Science, Germantown, MD). VEGF and fibroblast growth factor mRNA levels relative to GAPDH were quantified using ABI7500 (Applied Biosystems, Foster city, CA). VEGF silencing was measured three times with four replicates.

Cytotoxicity. To measure cytotoxicity, the polyplexes were transfected as described above. After 48 hours, 500-µl culture medium was removed, and 50-µl MTT solution (5 mg/ml) was added to each well. After 2 hours of incubation, dimethyl sulfoxide replaced the medium before an absorbance reading at 540 nm using a UV/Vis spectrophotometer (SpectraMax M2e; Molecular Devices, Sunnyvale, CA). The relative cell viability was calculated and expressed as percent cell viability relative to the untreated control. The viability was measured three times with four replicates.

Intracellular delivery of siRNA. SCC cells were seeded onto 60-mm dishes at a density of 1 × 106 cells/dish. Cells were incubated for 24 hours before transfection. The siRNA/rPOA polyplexes were prepared as described above. To visualize siRNA, fluorescein isothiocyanate-siLuc was used. After 30, 60, and 120 minutes incubation, cells were washed twice with PBS and fixed with 4% formaldehyde solution for 40 minutes. After fixation, cells were washed twice with PBS and mounted using DAPI-fluoromount G (SouthernBiotech, Birmingham, AL). For live cell analysis, cells were treated with the polyplex for 2 hours and washed before CLSM. The intracellular siRNA delivery was visualized using a multiphoton CLSM system (LSM510 META NLO; Carl Zeiss Jena, Germany; Korea Basic Science Institute, Chuncheon Center, Chuncheon, Korea).

Intracellular reducibility of siRNA/rPOA polyplexes. To visualize the reducibility of rPOA in the cells, rPOA was labeled with Cy5.5 and condensed fluorescein isothiocyanate-siLuc. SCC cells were seeded onto 60-mm dishes at a density of 1 × 106 cells/dish. Cells were incubated in the absence or presence of 10 mmol/l BSO for 6 hours before transfection. The siRNA/rPOA polyplexes were prepared as described above. After 30, 120, and 240 minutes incubation, confocal samples were prepared as described above. The intracellular reducibility of siRNA/rPOA polyplexes was visualized using CLSM.

Tumor model. A subcutaneous tumor model was generated by injecting SCC cells into 6-week-old male nude mice. Before injection, the cells were grown to 80% confluence at 37 °C with 5% CO2, thereafter, the cells were trypsinized and suspended in PBS at a concentration of 1 × 106 cells/ml. The mice were anaesthetized by an intraperitoneal injection of ketamine and xylazine (100 mg/kg), and SCC cells (1 × 105) were subcutaneously injected into the left hind flanks of the mice.

Intratumoral administration of rPOA/siVEGF. Tumor size was measured using a Vernier calipers before injection, and tumor volume was calculated as V = 0.5ab2 using the longest (a) and shortest (b) diameters of the tumor. When the tumor volume reached ~70 mm3, the mice were randomly separated into four groups. For intratumoral administration, polyplexes were prepared by mixing 3.5 µg siVEGF or scVEGF and rPOA at a charge ratio of 5 in 50 µl PBS. Fifty micro liters of siRNA/rPOA or scVEGF/rPOA polyplexes, naked siVEGF or PBS were injected directly into the tumor twice a week for 3 weeks.

In vivo VEGF silencing. Two days after the second administration, tumor tissues were dissected, and the tumor weights were measured. Equal weights of tumor tissues were homogenized using a homogenizer in a lysis buffer, lysed by five freeze-thaw cycles, and centrifuged for 5 minutes at 13,000 r.p.m. The amount of VEGF in the tumor was quantified from the supernatants using a mouse VEGF ELISA kit according to the manufacturer's instructions. The total tissue protein concentration was measured using a DC protein assay kit (Bio-Rad Laboratories) according to the manufacturer's instructions.

Acknowledgments

This work was partially supported by grants from the Korea Science and Engineering Foundation (2010K001247, 2010K001350) funded by the Ministry of Education, Science, and Technology, and KORUS Tech program from Korea Ministry of Knowledge Economy. This work was supported by the research fund of Hanyang University Institute of Aging Society in 2010. K.-M.L. was supported by a grant from KICOS (K20704000007-09A0500-00710).

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