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Sustained delivery of siRNAs targeting viral infection by cell-degradable multilayered polyelectrolyte films
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Edited by Harvey J. Alter, National Institutes of Health, Bethesda, MD, and approved September 12, 2008
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↵¶F.M. and I.N. and J.O. and T.F.B. contributed equally to this work. (received for review January 9, 2008)

Abstract
Gene silencing by RNA interference (RNAi) has been shown to represent a recently discovered approach for the treatment of human diseases, including viral infection. A major limitation for the success of therapeutic strategies based on RNAi has been the delivery and shortlasting action of synthetic RNA. Multilayered polyelectrolyte films (MPFs), consisting of alternate layer-by-layer deposition of polycations and polyanions, have been shown to represent an original approach for the efficient delivery of DNA and proteins to target cells. Using hepatitis C virus infection (HCV) as a model, we demonstrate that siRNAs targeting the viral genome are efficiently delivered by MPFs. This delivery method resulted in a marked, dose-dependent, specific, and sustained inhibition of HCV replication and infection in hepatocyte-derived cells. Comparative analysis demonstrated that delivery of siRNAs by MPFs was more sustained and durable than siRNA delivery by standard methods, including electroporation or liposomes. The antiviral effect of siRNA-MPFs was reversed by a hyaluronidase inhibitor, suggesting that active degradation of MPFs by cellular enzymes is required for siRNA delivery. In conclusion, our results demonstrate that cell-degradable MPFs represent an efficient and simple approach for sustained siRNA delivery targeting viral infection. Moreover, this MPF-based delivery system may represent a promising previously undescribed perspective for the use of RNAi as a therapeutic strategy for human diseases.
Small or short interfering RNAs (siRNA) mediate the degradation of complementary mRNA, which results in a specific silencing of gene expression. RNA interference (RNAi) has become one of the most widely used gene silencing techniques (1). This approach induces gene silencing at a posttranscriptional level and is more reliable, efficient, and specific than previous antisense methods. Furthermore, RNAi is a very attractive option for degrading viral RNA and may represent a promising approach for the treatment of viral infection (2). However, the efficiency, safety, and long-term duration of siRNA delivery remains the major challenge to its clinical application (2).
The layer-by-layer adsorption technique has been shown to represent a promising method for the delivery of bioactive molecules. This technique is a general and versatile tool for the controlled build-up of multilayered functionalized surface coatings on a large variety of surface types (3). Self-assembled multilayer architectures can be built by using macromolecules or materials as different as polyelectrolytes, proteins, inorganic complexes, clay platelets, or colloidal particles, all adsorbed from aqueous solutions or suspensions. Although complex multilayered architectures were shown to deliver a variety of molecules including drugs (4–8) and DNA (9–12), it is unknown whether this technique is feasible for the sustained delivery of siRNAs.
In this study, we aimed to assess the potential of multilayered polyelectrolyte films (MPFs) for delivering siRNA to human tissues, a key issue to overcome the hurdles for the preclinical and clinical application of siRNA-based therapeutic strategies. To test the feasibility of this strategy, we choose to target hepatitis C virus (HCV) infection as a model. HCV infection is a major global health problem (13). The large majority of infected individuals fail to clear the virus and are progressing to chronic infection, including liver cirrhosis and hepatocellular carcinoma. A preventive vaccine is not available, and current therapies based on IFN-α and ribavirin are characterized by limited efficacy, high costs, and substantial side effects (14). Complementary novel antiviral treatment strategies are thus urgently needed (14).
HCV is a positive-stranded RNA virus containing a genome of a single-stranded uncapped linear RNA (15). It carries a long ORF, flanked at the 5′- and 3′-ends by short, highly structured nontranslated regions (NTRs). The 5′-NTR contains an internal ribosome entry site (IRES) required for translation of the HCV genome (15). This sequence is highly conserved within the viral genome (15) and represents an ideal target for RNAi (16, 17). Using a highly efficient HCV cell culture system (18, 19), we demonstrate that cell-degradable MPFs represent an efficient approach for the sustained delivery of siRNA targeting viral infection.
Results and Discussion
Construction of MPFs, Incorporation of PEI-siRNA Complexes, and Stability of the Films.
To assess siRNA delivery by MPFs, we used a recently described antiviral siRNA (siHCV331) targeting the 5′NTR of HCV RNA (17). The build-up of MPFs containing PEI-siRNA was first monitored by quartz crystal microbalance (QCM). To enhance uptake, siRNAs were complexed to linear polyethylenimine (PEI) at a nitrogen/phosphate (N/P) ratio of 12. This complexation neutralizes the siRNA negative charge, and the resulting PEI-siRNAs complexes become positively charged. A precursor film composed of alternating layers of polylysine (PLL) and polyglutamic acid (PGA) was built to provide a surface suitable for the adsorption of PEI–siRNA layers, each adsorption step of PEI–siRNA complexes being followed by the deposition of 1.5 [hyaluronan (HA)-chitosan (CHI)/HA] layers. The evolution of the thickness of the MPF with the number of layers (plotted in Fig. 1 A and B) was estimated from the frequency shifts -Δf/ν (Δf being the resonance frequency shift and ν representing the overtone number) according to the Sauerbrey equation, assuming that the density of the materials deposited was 1.1 g·cm−3. The film thickness increased monotonically to yield a precursor film ≈150 nm after the deposition of 10 PLL/PGA bilayers (Fig. 1A). The adsorption of PEI-siRNAs complexes led to an increase in thickness of ≈10 nm, which was slightly reduced after the following rinsing and HA adsorption steps. Because HA molecules do not exchange PEI-DNA complexes (11), decrease in the signal is likely because of the desorption of a small fraction of preadsorbed PEI-siRNA complexes. The buildup process, further pursued by the addition of 1.5 (HA-CHI/HA) layers and (PEI-siRNA-HA-CHI/HA)4 layers, was demonstrated by the increase in film thickness (Fig. 1B). An atomic force microscopy (AFM) image of the surface topography of the resulting film is shown in Fig. 1C. Next, we investigated the amounts of siRNAs, effectively incorporated in the MPFs, as well as the stability of the resulting films. For this purpose, we used 1.34 μg of a fluorescent 6-FAM-labeled siRNA (siGLOGreen), which was complexed to PEI (N/P = 12), adsorbed on (PLL/PGA)10 films at one, three, or five layers (1.34 μg of siGLOGreen/layer) and further coated with 1.5 (HA-CHI/HA) layers. The resulting architectures were the following: (PLL/PGA)10-(PEI-siGLOGreen-HA-CHI/HA)n, where n = 1 [1 layer(1L) PEI-siGLOGreen], 3 (3L), or 5 (5L). After incubation of the resulting MPFs in cell culture medium at 37°C, siRNA release in the medium was evaluated by measuring the intensity of the fluorescence. In parallel, the siRNA content in the architectures was quantified after solubilization of the MPFs by using 5 M NaCl (20) with subsequent quantitation of embedded siRNA. As shown in Fig. 1D, a large amount of the available, positively charged PEI-siRNA complexes in the deposition solution, adsorbs on the negatively ending (PLL/PGA)10 architecture. In fact, PEI-siRNA adsorption ratios achieved 94% ± 5% (≈1.26 μg of 1.34 μg siRNA was embedded in the film) for the 1L PEI-siRNA construction, 74% ±3% for the 3L films (2.92 μg of 4.02 μg siRNA), and 83% ± 2% for the 5L films (5.55 μg of 6.7 μg siRNA). The amounts of siRNA released from the architectures in the presence of culture medium were small and similar for 1L, 3L, and 5L MPFs with time over a 12-day period of incubation (Fig. 1D), confirming that siRNA remains stably embedded in the film, despite the following rinsing steps and subsequent coating with 1.5 (HA-CHI/HA) layers. This finding suggests that passive diffusion or spontaneous release of siRNA from the films is limited and is restricted to the outer siRNA layer.
Construction of MPFs, incorporation of PEI-siRNA complexes and stability of the architectures. (A and B) Evolution of the thickness of MPFs was estimated step by step from the frequency shifts −Δf/ν (Δf, the resonance frequency shift; ν, the overtone number) with QCM after successive injections of polyelectrolytes (PLL, PGA, HA, or CHI) and PEI-siRNA complexes. PEI-siRNA complexes were deposited on the PGA10 layer (A) and then followed by injection of HA-CHI/HA and (PEI-siRNA-HA-CHI/HA)4 (B). All polyelectrolytes were dissolved in Hepes 20 mM/NaCl 0.15 M solutions at 1 mg/ml (pH 7.4) and adsorbed for 5 min. PEI-siRNA complexes (1.34 μg of siRNAs per layer, N/P 12, in 1.5 ml of NaCl 0.15 M, pH 7.4) were adsorbed during 15 min. (C) AFM of the surface topography of the resulting MPF. AFM was performed as described in Materials and Methods. (D) PEI-siRNA incorporation ratios in MPFs and film stability. The (PLL/PGA)10-(PEI-siRNA-HA-CHI/HA)n films (n = 1, 3, or 5) were built up by using 1.34 μg per layer of fluorescent 6-FAM-labeled siRNA (siGLOGreen) complexed to PEI (N/P = 12), and incubated in complete cell culture medium (DMEM + 10% FCS) at 37°C in 5% CO2 for 2–12 days. The siGLOGreen amount, which remained stably incorporated in the MPFs, was quantified by determining the fluorescence because of the release of 6-FAM-labeled siRNA complexes by complete dissolution of the architectures after treatment with 5 M NaCl for 10 min. In parallel, the siGLOGreen amount, spontaneously released (REL-siRNA) into the cell culture medium, was quantified by measuring the intensity of the fluorescence. The siRNA amounts were calculated by using a standard curve established in the same medium conditions. Means ± SD of three independent experiments are shown. REL, Release.
Efficient Inhibition of HCV Replication and Infection by Antiviral siRNAs Delivered by MPFs.
To assess the effects of siHCV-charged MPFs on HCV replication we used (PLL/PGA)10 films on which PEI-siHCV331 complexes were absorbed at one, three, or five layers and further coated with 1.5 (HA-CHI/HA) layers as described above. For the study of HCV replication and infection, we used a highly efficient HCV tissue culture system based on chimeric viral genomes containing a luciferase reporter (Jc1-Luc) (18). Huh7.5 cells containing replicating HCV Jc1-Luc RNA (18) were incubated with PEI-siRNA-charged MPFs and inhibition of HCV replication was evaluated by expression of the luciferase reporter (18). As shown in Fig. 2A, HCV Jc1-Luc replication was not affected when the cells were incubated with nonfunctionalized MPFs, MPFs functionalized by the nontargeting control siRNAs (siCTRL) or MPFs containing PEI without siRNA. These data clearly rule out that PEI embedded within the MPFs or the MPFs themselves had an effect on replication. In contrast, when Huh7.5 cells were incubated with MPFs in which siHCV RNAs were incorporated at one (1L), three (3L), or five (5L) layers, a marked and sustained decrease in HCV Jc1-Luc replication was observed (Fig. 2A). The MPF-siHCV construction most potently inhibiting HCV replication was the architecture containing siHCV complexes adsorbed at five layers (5L) suggesting that even the most deeply embedded siRNAs are efficiently available for delivery into target cells. The number of layers was not always directly proportional to the magnitude of inhibition of viral replication. The reason for this observation is most likely related to the MPF-layer-to-target-cell ratio resulting in different ratios between the amount of siRNAs delivered by cell-degradable MPFs and viral target RNAs at different time points. Furthermore, the kinetics of viral replication has been shown to depend on cell number and confluency (18). Differences in viral kinetics may alter siRNA-HCV RNA interaction, resulting in quantitatively different effects of delivered siRNA at different time points.
Inhibition of HCV replication by siRNA delivered by MPFs, PEI, liposomes (LT), and EP. (A) Delivery of siRNA by MPFs. siRNAs, targeting the 5′ UTR of the HCV genome (siHCV) or nontargeting control siRNAs (siCTRL) were complexed to PEI and incorporated in the following MPF architectures: (PLL/PGA)10-(PEI-siRNA-HA-CHI/HA)n, with n = 1 (1 PEI-siRNA layer, 1L), n = 3 (3L), or n = 5 (5L) layers. Huh7.5 cells, electroporated with HCV Jc1-Luc RNA, were incubated with MPFs, MPFs-PEI and PEI-complexed siHCV- (blue) or siCTRL (gray)-charged MPFs. In all experiments shown in Fig. 2, electroporated cells containing replicating HCV RNA were pooled before seeding onto 24-well plates to ensure that in all experiments comparing control and HCV siRNAs, HCV replication, and protein expression started at the same level. HCV replication was quantified by luciferase reporter expression as described in Materials and Methods. For each time point, means ± SD of three independent experiments are shown. The threshold for a positive signal in luciferase reporter protein expression was 3.1 × 103 RLU/μg protein. (B) Delivery in solution of siRNA by PEI. In side-by-side experiments, Huh7.5 cells, electroporated with HCV Jc1-Luc RNA, were incubated with PEI-siRNA complexes containing the same amounts of siRNA as MPFs. Increasing amounts of required complexes to deliver the same amount of siRNA (siHCV × 3 and siCTRL × 3) present in MPFs with 3L resulted in cell death on day 9 indicated by +. (C) Delivery of siRNA by liposomes. Liposome-siRNA complexes containing siHCV or siCTRL RNAs (same amounts as in A) were added to cells before EP of cells with HCV Jc1-Luc RNA. (D) Delivery of siRNA by co-EP. Huh7.5 cells were coelectroporated with HCV Jc1-Luc RNA and siHCV or siCTRL by using the same siRNA amounts as incorporated into the MPFs. (E) Comparative analysis of inhibition of HCV replication by different delivery approaches. Change of replication on days 2, 7, and 12 after EP of HCV Jc1-luc RNA is depicted as change in reporter gene expression (normalized for protein content) in the presence of siHCV RNA delivered by MPFs, PEI, EP, LT subtracted by the change in reporter gene expression in the presence of delivery of control siRNA. (F) Inhibition of HCV infection by HCV siRNA delivered by MPFs. Huh7.5 cells were incubated with the siHCV- or siCTRL-charged MPFs. 24 h later, the cells were incubated with cell culture derived HCV (Jc1-Luc strain). Forty-eight hours after inoculation viral infection was assessed by quantitation of intracellular HCV RNA by using RT-PCR (18). The detection limit for HCV RNA was 2,000 copies/assay. siHCV ×1/×3/×5, siRNA corresponding to the same amount of siRNA incorporated into MPFs with one, three, and five layers (1.34, 4.02, and 6.70 μg of siRNA, respectively).
To address the longevity and efficiency of the MPF approach, we performed a side-by-side comparison of the efficiency of MPFs in delivering siRNA and three siRNA transfection methods including PEI (Fig. 2B), cationic lipids (Fig. 2C), and electroporation (EP) (Fig. 2D). EP, PEI, and lipid transfection (LT) protocols were optimized by using various conditions, and the best conditions were used for side-by-side analysis. To ensure that, in all experiments, comparing control and HCV siRNAs HCV replication started at the same level, electroporated cells containing replicating HCV RNA were pooled before seeding onto 24-well plates. Whereas the effects of LT, PEI, and EP peaked on days 2–5 after transfection and fell off on day 7 (Fig. 2 B–E), delivery of siRNA by MPFs resulted in a sustained inhibition of HCV replication over a time period of 12 days (Fig. 2 A and E). siRNA delivery by PEI in solution showed similar potency as EP during early time points (Fig. 2B). However, the amounts of PEI required for the delivery of siRNAs amounts equivalent to amounts present in the architectures containing three layers (Fig. 2B, experiments marked with a cross) or five layers (data not shown) was characterized by marked toxicity resulting in cell death. The toxicity of this approach was clearly related to PEI, because PEI without siRNA resulted in comparable toxicity and similar amounts of siCTRL or HCV RNA did not exhibit any toxicity in other delivery approaches (Fig. 2). In contrast, when PEI had been embedded into the architectures without siRNA, no unspecific effect on viral replication (Fig. 2A) or cell toxicity (data not shown) was observed. A comparative analysis of the effect on viral replication of the delivery methods is shown in Fig. 2E. This side-by-side analysis clearly demonstrates the superiority of MPFs for long-term and sustained siRNA delivery in comparison to established techniques.
Using the experimental conditions outlined in Fig. 2, the level of inhibition by siRNA delivered by MPFs appeared to be comparable to the level of inhibition by BILN2061 (21), an HCV protease inhibitor [supporting information (SI) Fig. S1]. In these experiments, BILN2061 was less potent than in previous studies by using HCV genotype 1 replicons (21), because BILN2061 is less active against genotype 2a protease (21).
The potential use of MPFs carrying siRNA for the treatment of HCV infection requires not only an efficient inhibition of viral replication but also inhibition of HCV infection. To address this question, we performed infection experiments in which Huh7.5 cells were seeded on MPFs containing HCV siRNA and subsequently inoculated with cell-culture-derived infectious virions (HCVcc) containing a luciferase reporter [Jc1-Luc (18)]. As shown in Fig. 2F, siRNAs delivered by MPFs markedly inhibited infection of Huh7.5 cells. Inhibition of infection depended on siRNA amount because the 5L architecture was the most potent architecture in inhibiting infection (Fig. 2F, Fig. S2). In contrast, HCV infection was not affected when the cells were incubated with nonfunctionalized MPFs (Fig. 2F, Fig. S2), MPFs functionalized by the nontargeting control siRNAs-PEI complexes (siCTRL; Fig. 2F, Fig. S2) or MPFs containing PEI without siRNA (data not shown). These data clearly rule out that PEI embedded within the MPFs or the MPFs themselves affect viral infection.
siRNA Delivery Requires Degradation of MPFs by Cellular Hyaluronidase.
At least three mechanisms, or a combination of them, could account for siRNA delivery by MPFs: these include passive diffusion or spontaneous release of siRNA from MPFs, passive degradation, or swelling of MPFs or active degradation of MPFs by cellular enzymes with subsequent uptake of embedded siRNAs. As shown in Fig. 1D, spontaneous release or passive diffusion of siRNA over a time period of 12 days was limited to marginal amounts, suggesting that this mechanism does not play a major role for the observed sustained delivery of siRNA complexes. The marginal amounts of released siRNA complexes were without functional impact, as shown by an absent effect on viral replication after transfection of Huh7.5 cells with supernatants of MPFs (Fig. S3A). Next, we considered whether siRNA delivery was because of passive degradation or swelling. To evaluate this possibility, we added cell culture medium at the end of the build-up of a 5L film. After 24 h of contact of the film with the medium, no QCM-D signal change was detected (data not shown). This findings suggests that passive degradation or swelling does not play an important role for delivery of siRNA complexes.
Thus, we hypothesized that delivery of siRNA complexes is predominantly mediated by active degradation of MPFs with subsequent uptake of embedded siRNA. MPF degradation could be either mediated by cellular enzymes secreted into the cell culture supernatant or cellular enzymes acting during cell-to-MPF interaction with local enzymatic MPF degradation. To investigate whether siRNA release from MPFs was mediated by cellular enzymes secreted into the tissue culture medium, we incubated MPFs with conditioned cell culture supernatants from Huh7.5 cells cultured for 4 days (Fig. 3A and Fig. S3B). Similar to the results when architectures were incubated with fresh cell culture medium (Fig. 1D), only limited amounts of siRNA were released (Fig. 3A and Fig. S3B). Thus, we hypothesized that the availability of embedded siRNA for cells was based on an active cell-dependent process requiring cell-to-MPF contact with local MPF degradation. To study whether degradation of MPFs by specific cellular enzymes resulted in siRNA release, we incubated the architectures with cellular lysates. Incubation with cell lysates resulted in release of significant amounts of siRNA from the architectures paralleled by a decrease of the siRNA content within the MPFs (Fig. 3A). Although this experimental approach does not completely reflect the cell-to-MPF interaction in living cells, it demonstrates that Huh7.5 cells contain enzymatic activity able to degrade MPFs with subsequent release of siRNA complexes. Indeed, a number of cellular enzymes have been shown to degrade HA (22) and CHI (23), the key components of MPFs. HA is degraded by hyaluronidase, an enzyme with high expression levels in many tissues including hepatocytes. To study whether cellular hyaluronidases were directly involved in MPF degradation, we investigated the impact of a hyaluronidase inhibitor, neomycin trisulfate (24), on inhibition of viral replication induced by MPF-siRNA. As shown in Fig. 3B, siRNA delivery to the cells was markedly inhibited in the presence of neomycin trisulfate (NT), because viral replication in infected cells incubated with specific siRNA-functionalized MPFs was restored to baseline levels in the presence of the inhibitor in a dose-dependent manner. These data demonstrate that siRNA delivery to cells by MPFs appears to be predominantly mediated by local degradation of the architectures by cellular enzymes such as hyaluronidases.
Active degradation of MPFs by cellular enzymes is required for siRNA delivery. (A) Release of siRNA from MPFs incubated with Huh7.5 culture medium or cell lysates. The (PLL/PGA)10-(PEI-siRNA-HA-CHI/HA)n films (n = 1, 3, or 5) were built up by using 1.34 μg per layer of fluorescent 6-FAM-labeled siRNA (siGLOGreen) as described in Fig. 1 and incubated for 72 h with PBS (CTRL), conditioned cell culture medium (MED), or lysates (LYS) from Huh7.5 cells as described in Materials and Methods. REL siGLOGreen RNA and siGLOGreen RNA remaining incorporated in the MPFs (MPF) was quantified as described in Fig. 1D. (B) Inhibition of siRNA delivery and restoration of HCV replication by hyaluronidase inhibitor NT. Huh7.5 cells, electroporated with HCV Jc1-Luc RNA, were incubated with siHCV- or siCTRL-charged MPFs in the presence or absence of 0.1, 0.5 or 1 mM NT. HCV replication was quantified by luciferase reporter expression as described in Fig. 2. The detection limit for luciferase reporter protein expression was 3.1 × 103 RLU/μg of protein. Means ± SD of three independent experiments are shown.
In conclusion, we have demonstrated a functionalization of cell-degradable MPFs with PEI-siRNA complexes targeting viral infection. This functionalization allows an efficient and longlasting sustained delivery of siRNAs to human liver cells resulting in a marked and sustained inhibition of HCV replication. The efficacy observed is most likely the consequence of active enzymatic film degradation during cell-to-MPF contact by cellular enzymes such as hyaluronidase.
Delivery remains a major limitation for therapies based on RNAi, because siRNAs do not cross the mammalian cell membrane unaided and most of the transfection methods used for in vitro studies cannot be used in vivo (2). One possibility is to express siRNA precursors in viral vectors. However, this approach is limited by safety and immunogenicity problems associated with viral vectors (2). Another approach for siRNA delivery is the use of cationic liposomes (25). However, cationic liposome-mediated siRNA delivery cannot provide a controlled longlasting delivery and would probably require administration to be often repeated in single doses. Side-by-side analyses (Fig. 2) demonstrated that MPFs were clearly superior to EP, cationic liposomes, and PEI in achieving long-term siRNA delivery. The reason for the superiority of MPFs in long-term suppression of HCV replication is most likely the longlasting and constant availability of siRNAs embedded in the MPFs. Sustained and longlasting siRNA delivery by MPFs is important for in vivo applications of siRNA and provides a significant advancement compared with other state-of-the-art delivery systems.
Toxicity of this delivery approach appeared to be low, as indicated by an absent effect of nonfunctionalized MPFs or MPFs loaded with PEI and control siRNA on viral replication (Fig. 2A), viral infection (Fig. 2F), or cell viability (data not shown). In contrast, PEI in solution (Fig. 2B) exhibited a marked dose-dependent negative effect on cell viability. In this study, we demonstrate that in contrast to PEI in solution, PEI embedded in MPFs was characterized by a low or absent toxicity (Fig. 2). This finding represents an important advantage for future in vivo applications (26) of MPF-based delivery systems, because a limiting factor for gene delivery strategies in animal models based on PEI has been toxicity.
Furthermore, our study demonstrates that MPFs can be equipped with different amounts of siRNA, incorporated at different layers and further coated with a variable number of polyelectrolyte bilayers. Because cellular uptake of siRNAs depended on the enzymatic activity of cells during cell-to-MPF contact (Fig. 3), MPFs seem to act as reservoirs that allow a temporal control over availability and cellular uptake of siRNA. Thus, in contrast to other delivery methods, the MPF platform allows a tunable, time-specific and most importantly long-lasting delivery of siRNAs.
With the aim to target distinct tissues, the MPF siRNA delivery approach could take advantage of tissue-specific functionalization, typically achieved by incorporating different bioactive molecules targeting specific human tissues (20). Aiming to target hepatotropic viruses, this could include direct coupling of one of the polyelectrolytes to a molecule specifically targeting the liver such as lactose (25).
Our proof-of-concept study demonstrates that cell-degradable MPFs are an efficient and tunable approach for sustained delivery of siRNAs targeting viral infection. Moreover, this MPF-based delivery system may represent a promising perspective for the use of RNAi as a therapeutic strategy for human diseases.
Materials and Methods
Chemicals.
Poly-(L-lysine) hydrobromide (PLL, 30 kDa), poly(L-glutamic acid) (PGA, 54 kDa), CHI (50 kDa), HA (400 kDa), and neomycin trisulfate were from Sigma; linear polyethylenimine (PEI, 22 kDa) from Polyplus-transfection; BILN2061 from Boehringer Ingelheim; and Lipofectamin 2000 from Invitrogen.
Polyelectrolyte Multilayered Film Preparation.
MPFs were prepared as described (27). Positively charged PEI-siRNA complexes (1.34 μg, N/P = 12, diluted in 300 μl of 0.15 M NaCl, 20 mM Hepes, pH 7.4) were adsorbed for 60 min on architectures ending in polycation (PGA or HA). The buildup was then continued by the addition of 1.5 (HA-CHI/HA) layers.
siRNAs.
siRNAs were provided by Dharmacon. siHCV331 (17), targeting the HCV IRES region, was synthesized as a double-stranded 21-bp-long siRNA, with UU overhangs as described (17). siCTRL is a nontargeting negative control siRNA, siGLOGreen is a 6-FAM-labeled fluorescent siRNA.
PEI-siRNA Complex Preparation.
Complexes of PEI and siRNAs were obtained by diluting siRNAs and PEI separately. An appropriate volume of PEI, diluted to a concentration of 10 mM nitrogen atom in 0.15 M NaCl, 20 mM Hepes, pH 7.4, was added to the siRNA solution (1.34 μg in 100 μl of the same buffer) at an N/P ratio of 12, assuming that 1 μg of siRNA contains 3 nmol anionic phosphate. The solution was incubated for 15 min before incorporation into MPFs.
QCM and AFM.
The film buildups were monitored in situ by QCM by using the axial flow chamber QAFC 302 (QCM-D, D300, Q-Sense), as described (27). AFM imaging was performed in contact mode under liquid condition by using Nanoscope IV (Veeco), as described (27).
siRNA-Charged MPF Stability Evaluation.
The (PLL/PGA)10-(PEI-siRNA-HA/CHI/HA)n films were built by using 1.34 μg of fluorescent 6-FAM-labeled siRNA (siGLOGreen) complexed to PEI (N/P = 12) and incubated in 1 ml of DMEM (Invitrogen), cell culture supernatants from Huh7.5 cells cultured for 4 days, or lysates of Huh7.5 cells (prepared by five freeze–thaw cycles and resuspension in PBS containing 100 mg/liter CaCl2, 100 mg/l MgCl2, and 40 units/μl of RNAsin; Promega) at 37°C. Dissolution of the underlying films and fluorescence analysis was performed as described (20).
Analysis of HCV siRNA Delivery on HCV Replication.
HCV RNA was transcribed from the cDNA of HCV isolate Jc1-Luc and Huh7.5 cells (cultured in DMEM/10% FCS; Invitrogen) were electroporated with purified transcribed Jc1-Luc RNA with subsequent seeding in 24-well plates, as described (18, 19). In all experiments, electroporated cells containing replicating HCV RNA were pooled before seeding onto 24-well plates to ensure that in all experiments comparing control and HCV siRNAs, HCV replication, and protein expression started at the same level. For the study of siRNA delivery by MPFs, PEI, and liposomes, Huh7.5 cells, electroporated with Jc1-Luc RNA were transfected 24 h later with equivalent amounts of siRNAs by using PEI or Lipofectamine 2000, according to the manufacturer's instructions. For the study of siRNA delivery by EP, siRNAs and HCV RNA were coelectroporated. BILN2061 (100 or 200 nM) or neomycin trisulfate (0.1–1 mM) was added to the culture medium 6 h after EP. HCV replication was analyzed by luciferase activity as described (18). The detection limit of the luciferase assay was 3,100 relative light units (RLU)/μg of protein corresponding to the mean ± 2 SD of background levels, i.e., luciferase activity of naïve nonreplicating cells (averaging 1.9 ± 0.6 × 103 RLU/μg protein).
Analysis of MPF-siRNA Delivery on HCV Infection.
Huh7.5 cells were transfected by EP with Jc1-Luc HCV RNA and HCVcc produced as described (18). For infection assays, Huh7.5 cells were seeded at a density of 2 × 104 per well in 24-well plates and inoculated 24 h later with Jc1-Luc HCVcc (TCID50 between 103 and 104/ml). Forty-eight hours later, infection was analyzed by quantitation of intracellular HCV RNA by using RT-PCR or luciferase activity (18).
Acknowledgments
We thank M. Parnot for excellent technical assistance; C. Betscha, V. Ball, and B. Senger for help with QCM-D experiments; L. Richert for AFM imaging; and C. Rice (Rockefeller University, New York) for providing Huh7.5 cells. This work was supported by European Union Grants LSHM-CT-2004-503359, ANR-05-CEXC-008, and ANRS-06221; Canceropôle Grand Est-Fond National pour la Santé Grant ACI 2004; and CONECTUS, Louis Pasteur University, Strasbourg.
Footnotes
- **To whom correspondence may be addressed. E-mail: joelle.ogier{at}medecine.u-strasbg.fr or thomas.baumert{at}viro-ulp.u-strasbg.fr
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Author contributions: M.D., F.M., C.S., J.-C.V., J.O., and T.F.B. designed research; M.D., C.A., F.M., I.N., and D.G.R. performed research; R.B. contributed new reagents/analytic tools; M.D., F.M., J.-C.V., J.O., and T.F.B. analyzed data; and M.D. and T.F.B. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0800156105/DCSupplemental.
- © 2008 by The National Academy of Sciences of the USA
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