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 (siGLO^Green), which was complexed to PEI (N/P = 12), adsorbed on (PLL/PGA)₁₀ films at one, three, or five layers (1.34 μg of siGLO^Green/layer) and further coated with 1.5 (HA-CHI/HA) layers. The resulting architectures were the following: (PLL/PGA)₁₀-(PEI-siGLO^Green-HA-CHI/HA)_n, where n = 1 [1 layer(1L) PEI-siGLO^Green], 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.

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Fig. 1.

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 (siGLO^Green) 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 siGLO^Green 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 siGLO^Green 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.

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Fig. 2.

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.

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Fig. 3.

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 (siGLO^Green) 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 siGLO^Green RNA and siGLO^Green 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.