Small interfering (si)RNA mediated inhibition of oncogenes or viral genes may offer great opportunities for the treatment of several diseases such as hepatocellular carcinoma and viral hepatitis. However, the development of siRNAs as therapeutic agents strongly depends on the availability of safe and effective intracellular delivery systems. Poly(β-amino esters) (PbAEs) are, in contrast to many other cationic polymers evaluated in siRNA delivery, biodegradable into smaller, nontoxic molecules.
Methods and Results
We show for the first time that PbAE : siRNA complexes, containing 1,4-butanediol (PbAE1) or 1,6-hexanediol (PbAE2) diacrylate-based polymers, induced efficient gene silencing in both hepatoma cells and primary hepatocytes without causing significant cytotoxicity. Furthermore, carriers that slowly release the siRNA into the cytoplasm and hence induce a prolonged gene silencing are of major clinical interest, especially in fast dividing tumour cells. Therefore, we also studied the duration of gene silencing in the hepatoma cells and found that it was maintained for at least 5 days after siRNA delivery with PbAE2, the polymer with the slowest degradation kinetics.
Hepatocellular carcinoma (HCC) is worldwide one of the most prevalent human cancers, with approximately 600 000 new cases diagnosed annually and almost as many deaths 1. For the vast majority of HCC cases, no effective therapy is available. Hence, new treatment approaches for HCC are urgently needed 2. The major cause of HCC is chronic hepatitis B (HBV) and hepatitis C virus (HCV) infection. The former can be prevented by vaccination and also different treatments are currently available, for example, with nucleo(s)(t)ide analogues 3. By contrast, there is no vaccine available against HCV infection and all HCV treatments so far rely on the antiviral activity of pegylated interferon (IFN)-α that is administered alone or in combination with ribavirin 4. Unfortunately, only a part of the HCV patients clear the virus during therapy and currently no alternative treatment exists for nonresponders 5. Clearly, more effective treatments are needed against HCV and HCC. Therefore, the selective inhibition of highly active genes involved in liver oncogenesis 6 or HCV genes involved in viral replication 7–14 via RNA interference (RNAi) may offer great improvement in the treatment of HCC and HCV infections.
RNAi is a naturally occurring post-transcriptional sequence-specific gene silencing mechanism that is initiated by double-stranded RNAs (dsRNAs) that are homologous in sequence to the target mRNA 15. These dsRNAs are enzymatically processed into 21–22 nucleotide small interfering RNAs (siRNAs) by the RNase III-like cellular enzyme Dicer 16. The generated siRNAs are subsequently incorporated into a silencing complex called RNA-induced silencing complex that scans and cleaves mRNA in a sequence-specific manner 17.
Therapeutic RNAi applications mainly depend on the availability of safe delivery systems that cause a sufficient cellular delivery of siRNA. DNA molecules encoding short hairpin RNAs (shRNAs) that are subsequently processed by Dicer into siRNAs, are often used to demonstrate the therapeutic potential of RNAi. Both viral and nonviral carriers have been used to deliver these DNAs. However, viral delivery of DNA encoding shRNA is not feasible for therapeutic applications due to safety and production concerns. Nonviral carriers provide no suitable alternative because they lack the ability to efficiently deliver DNA into the nucleus 18. Additionally, it was suggested that HCV has evolved mechanisms to inhibit Dicer-dependent cleavage of longer dsRNAs like shRNAs 19, 20. Therefore, to avoid the necessity of nuclear uptake involved with DNA delivery and safety concerns involved with viral carriers, the most desirable approach appears to be the cytoplasmic delivery of synthetic siRNAs by means of nonviral carriers. To date, nonviral carriers for delivering synthetic siRNA are mostly based on transfection with cationic lipids 21–25. Cationic polymers, such as polyethylenimine (PEI) and chitosan, have also been tested as delivery agents 26–29 but they are often less efficient 30 and more cytotoxic 31.
Poly(β-amino esters) (PbAEs) are polyamines that are synthesized by Michael addition of either primary amines or bis (secondary amines) to diacrylate esters (Figure 1a) 32. PbAEs are fully biodegradable via hydrolysis of their backbone esters to yield small molecular weight bis(β-amino acid) and diol products, which, along with the parent polymer, are significantly less toxic than many other polycations, such as PEI and poly(L-lysine) 33. Anderson et al.34 identified a library of PbAEs that transfect pDNA in COS-7 cells as good as or even better than PEI. Additionally, several of the synthesized PbAEs were shown to be effective for in vivo pDNA delivery 35–37. Therefore, in the present study, different PbAEs were synthesized and, for the first time, their capacity to deliver siRNA and to evoke a biological siRNA effect was tested. Two PbAEs that successfully delivered the siRNA in the cells were identified: PbAE1 (polymer 1; Figure 1b) and PbAE2 (polymer 2; Figure 1c). These were obtained by conjugation, respectively, 1,4-butanediol diacrylate (PbAE1) and 1,6-hexanediol diacrylate (PbAE2), to 4,4′-trimethylenedipiperidine. Both polymers showed a strong gene silencing effect in both human hepatoma cells and primary rat hepatocytes. Furthermore, in contrast to PbAE1, PbAE2 was able to induce a prolonged gene silencing effect in the hepatoma cells.
Materials and methods
4,4′-Trimethylenedipiperidine, CH2Cl2, tetrahydrofurane (THF), triethylamine, insulin, glucagon, kanamycin monosulfate, streptomycin sulfate and ampicillin sodium salt were purchased from Sigma-Aldrich (Bornem, Belgium). Hydrocortisone hemisuccinate came from Upjohn (Windsor, UK). 1,4-Butanediol diacrylate and 1,6-hexanediol diacrylate were purchased from Alfa Aesar Organics (Karlsruhe, Germany). SiRNA against firefly (Photinus pyralis) luciferase (pGL3), Alexa488-labelled pGL3 siRNA, negative control siRNA and jetSI™-ENDO were purchased from Eurogentec (Seraing, Belgium). All siRNAs were purchased in their annealed form, dissolved in RNase free water at a concentration of 20 µ M, aliquoted and stored at − 80 °C. Dulbecco's modified Eagle's medium (DMEM), OptiMEM, L-glutamine (L-Gln), heat-inactivated fetal bovine serum (FBS), G418 (geneticine) and penicillin/streptomycin (P/S) were obtained from Invitrogen (Merelbeke, Belgium).
Synthesis of PbAE1 and PbAE2
All glassware was flame-dried under vacuum before use. 37.8 mmol 1,4-butanediol diacrylate (in the case of PbAE1) or 1,6-hexanediol diacrylate (in the case of PbAE2) and 37.8 mmol 4,4′-trimethylenedipiperidine were separately dissolved in 50 ml of CH2Cl2. The 4,4′-trimethylenedipiperidine solution was added dropwise to the 1,4-butanediol diacrylate (or 1,6-hexanediol diacrylate) solution under vigorous stirring. The reaction mixture was placed in an oil bath at 50 °C and the polymerization was allowed to proceed over 48 h under a nitrogen atmosphere. After cooling to room temperature, the reaction product was precipitated in diethyl ether saturated with HCl. The precipitate was filtered and thoroughly washed with diethyl ether. A white powder was obtained after overnight drying under vacuum. The molecular weight of the polymers was determined by size exclusion chromatography using a gel permeation chromatography system system equipped with two PLgel 5 micron MIXED-D, 300 × 7.5 mm columns (Polymer Laboratories, St-Katelijne-Waver, Belgium). THF/0.1 M triethylamine was used as mobile phase at a flow rate of 1 ml/min. The molecular weight of the polymers was calculated relative to polystyrene standards. 1H-NMR (300 mHz) spectra were recorded on a Varian Mercury 300 spectrometer (Varian Inc., Palo, Alto, CA, USA) in CDCl3 as solvent. The obtained chemical shifts δ were 4.12 (4H), 2.87 (4H), 2.52 (4H), 1.97 (4H), 2.63 (8H), 1.16 (12H) and 4.06 (4H), 2.91 (4H), 2.71 (4H), 2.55 (4H), 2.01 (4H), 1.65 (8H), 1.37 (4H), 1.22 (12H) for PbAE1 and PbAE2, respectively.
Preparation of siRNA complexes
The synthesized PbAEs were dissolved in acetate buffer (100 mM, pH 5.4) at different concentrations depending on the desired nitrogen to phosphate (N : P) ratio (10 : 1, 20 : 1 and 30 : 1) of the PbAE : siRNA complexes and prior to use filtered through a 0.22 µm membrane syringe filter. The PbAE : siRNA complexes were formed by adding an equal volume of PbAE solution to 0.5 µ M siRNA, followed by vigorously mixing. The resulting PbAE : siRNA complexes were incubated at room temperature for at least 30 min before addition to the cells.
JetSI™-ENDO : siRNA complexes were prepared as described by the manufacturer. Briefly, the jetSI™-ENDO solution was diluted into OptiMEM and vigorously vortexed. After incubation at room temperature for 10 min, the jetSI™-ENDO mixture was added all at once to an equal volume of a 0.5 µ M siRNA solution, immediately vortexed for 10 s and incubated at room temperature for 15 min before addition to the cells.
Characterization of siRNA complexes
The average particle size and the zeta potential of the different siRNA complexes were measured by photon correlation spectroscopy (Autosizer 4700; Malvern, Worcestershire, UK) and particle electrophoresis (Zetasizer 2000; Malvern), respectively. Before measurement, the jetSI™-ENDO : siRNA and PbAE : siRNA complexes were diluted two-fold in 20 mM Hepes buffer (pH 7.4) and 0.1 M acetate buffer (pH 5.4), respectively. The control jetSI™-ENDO : siRNA complexes had a size of 221 ± 5 nm and a zeta potential of 23 ± 4 mV. The results for the PbAE : siRNA complexes are summarized in Table 1.
Table 1. The particle size and zeta potential of the different PbAE : siRNA complexes
Particle size (nm)
Zeta potential (mV)
As indicated, PbAE : siRNA complexes were made at three different nitrogen to phosphate (N : P) ratios. Mean values with their corresponding standard deviations are shown (n = 5).
PbAE1 : siRNA
N : P 10 : 1
326 ± 14
15 ± 3
N : P 20 : 1
315 ± 6
20 ± 2
N : P 30 : 1
380 ± 9
31 ± 1
PbAE2 : siRNA
N : P 10 : 1
517 ± 29
23 ± 3
N : P 20 : 1
457 ± 24
13 ± 3
N : P 30 : 1
374 ± 23
18 ± 3
Native polyacrylamide gelelectrophoresis (PAGE) was used to study the complexation state of siRNA in the jetSI™-ENDO : siRNA and PbAE : siRNA complexes. Loading buffer containing 10% glycerol was added to the siRNA complexes, containing 0.3 µg of siRNA, and these samples were consequently loaded on a gel that contained 20% polyacrylamide in TBE buffer (89 mM Tris·borate pH 8.3 and 2 mM ethylenediaminetetracetic acid). The PAGE gel was subjected to electrophoresis at 100 V for 2 h and the siRNA was visualized by ultraviolet (UV) transillumination using 1 : 10 000 diluted SYBR Green II RNA stain (Molecular Probes, Merelbeke, Belgium) prior to photography.
Production of recombinant adenoviruses
For the generation of recombinant adenovirus the AdEasy Adenoviral Vector system (Stratagene, La Jolla, CA, USA) was employed. The firefly luciferase open reading frame was cloned in the pShuttle-cytomegalovirus (CMV) vector and was constitutively expressed under control of the CMV promoter. Subsequently, recombinant, replication-deficient adenoviruses expressing luciferase (Adluc) were generated following the standard protocol as described by He et al.38.
Gene silencing in primary hepatocytes
As human primary hepatocytes are difficult to obtain because of their increasing use in transplantation, rat primary hepatocytes were used in this study. These hepatocytes were isolated from outbred adult male Sprague-Dawley rats (200–250 g; Iffa Credo, L'Arbresle, France), with free access to food and water, as described previously 39. Cell integrity was tested by trypan blue exclusion.
For gene silencing experiments, rat hepatocytes were cultured (37 °C, 5% CO2, 100% humidity) as a monolayer in 24-well plates at a density of 6 × 104 cells per cm2 in DMEM containing 0.5 U/ml insulin, 7 ng/ml glucagon, 10% FBS and 1% antibiotic mix [sodium ampicillin (10 µg/ml), kanamycin monosulfate (50 µg/ml), benzyl penicillin (7.3 IU/ml) and streptomycin sulfate (50 µg/ml)] 40. After 4 h, the medium was renewed with the same medium as described above but supplemented with 7.5 µg/ml hydrocortisone hemisuccinate and incubated overnight. Prior to transfection with the siRNA complexes, the hepatocytes were transduced with replication-deficient adenoviruses expressing firefly luciferase at a multiplicity of infection of 50. Subsequently, 30 min after addition of the adenoviral vectors, the PbAE : siRNA or jetSI™-ENDO : siRNA complexes, containing 25 pmol siRNA, were added to the cells and incubated for 4 h at a final concentration of 50 nM in serum free medium. The remaining viruses and siRNA complexes were removed and replaced by 500 µl culture medium. Thirty hours later, cells were lysed with 80 µl passive lysis buffer (PLB; Promega, Leiden, The Netherlands) under vigorous shaking. Luciferase activity was determined with the Promega luciferase assay kit according to the manufacturer's instructions and expressed in relative light units (RLU). Briefly, 100 µl substrate was added to 20 µl sample and after a 2-s delay, the luminescence was measured during 10 swith the GloMax™ 96 plate luminometer (Promega) with injector. To correct for the amount of cells per well, the protein concentration was determined with the BCA kit (Pierce, Rockford, IL, USA). Two hundred microlitres of mastermix, containing 50 parts reagent A to 1 part B, was mixed with 20 µl of cell lysate, incubated at 37 °C for 30 min and measured on a Wallac Victor2 absorbance plate reader (PerkinElmer Life Sciences, Boston, MA, USA) at 590 nm. For each carrier, the RLU value per mg protein of the anti-luciferase siRNA transfected cells was compared with the RLU value per mg protein of the mock siRNA transfected cells and expressed as luciferase expression level (%).
Gene silencing in human hepatoma cells
The human hepatoma cell lines HuH-7 and HuH-7_eGFPLuc, stably expressing firefly luciferase, were cultured (37 °C and 5% CO2) in DMEM : F12 supplemented with 2 mML-Gln, 10% heat-inactivated FBS and 100 U/ml P/S.
HuH-7_eGFPLuc cells stably expressing eGFP-Luciferase were generated by transfecting HuH-7 cells with the vector pEGFPLuc (Clontech, Palo Alto, CA, USA) as previously described 41.
For gene silencing experiments, HuH-7_eGFPLuc cells were seeded into 24-well plates at a density of 5 × 104 cells per cm2 and allowed to attach overnight. Cells were washed with PBS and to each well the siRNA complexes, containing 25 pmol of siRNA, were added at a final concentration of 50 nM in serum free medium. After 4 h, the remaining complexes were removed and replaced by 500 µl of culture medium. Thirty hours later, cells were lysed with 80 µl of CCLR buffer (Promega), and luciferase activity and protein concentration were determined as described above.
For the long-term gene silencing experiments, cells were seeded in 24-well plates at a density of 5 × 104 cells per cm2 and allowed to attach overnight. All transfection experiments were performed as described above, but in duplex. Twenty-four hours later, one sample was lysed (day 1) whereas the parallel sample was trypsinized and 1 : 5 diluted into four 24-well plates. These were incubated at 37 °C and cells were lysed at days 2, 3, 4 or 5. After collection of all samples, luciferase activity and protein concentration were determined as described above. To correct for the dilution of the cells after trypsinization, the RLU value per mg protein of the anti-luciferase transfected cells was compared with similarly treated cells, transfected with mock siRNA and expressed as luciferase expression level (%).
The influence of the siRNA complexes on the cell viability was determined using the CellTiter-Glo Assay (Promega) and the tetrazolium salt-based colorimetric MTT assay (EZ4U; Biomedica, Vienna, Austria). 5 × 104 cells per cm2 were seeded into 96-well plates and allowed to adhere. After 24 h, cells were washed with PBS and incubated either with the PbAE : siRNA complexes, the jetSI™-ENDO : siRNA complexes or free carrier. After 4 h, the remaining siRNA complexes or carriers were removed from the cells and replaced by culture medium. For the CellTiter-Glo Assay, the 96-well plate was incubated at 37 °C for 24 h, subsequently incubated at room temperature for 30 min and 100 µl of CellTiter-Glo reagent was added to each well. After shaking the plate for 2 min and a 10-min incubation at room temperature, the luminescence was measured on a GloMax 96 luminometer with 1 s of integration time. In the case of the EZ4U assay, medium was removed after 24 h and 20 µl substrate, along with 180 µl culture medium, was added to each well. After 4 h incubation at 37 °C reduced formazan was measured at 450 nm and 630 nm on a Wallac Victor2 absorbance plate reader.
Primary hepatocytes or HuH-7 cells were seeded on sterile glass bottom culture dishes (MatTek Corporation, Ashland, MA, USA) at a density of 5 × 104 cells per cm2 and allowed to attach overnight. PbAE : siRNA and jetSI™-ENDO : siRNA complexes, containing Alexa488-labelled siRNA, were prepared and added to the cells as described above. The distribution of the fluorescence in the cells was visualized using a Nikon C1si confocal laser scanning module attached to a motorized Nikon TE2000-E inverted microscope (Nikon Benelux, Brussels, Belgium). A nonconfocal diascopic DIC (differential interference contrast) image was collected simultaneously with the confocal images. For lysosome staining, cells were incubated for 60 min with 1 : 15 000 diluted Lysotracker Red (Molecular Probes) prior to image recording. Images were captured with a Nikon Plan Apochromat 60 × oil immersion objective lens (numerical aperture of 1.4) using the 488 nm and 639 nm line from an Ar-ion and a diode laser for the excitation of the Alexa488-siRNA and Lysotracker Red, respectively.
Results and Discussion
Synthesis and characterization of PbAEs
Different PbAEs were synthesized (Figure 1a) and the structures of the most efficient PbAEs for siRNA delivery are shown in Figure 1b and 1c. Polymer 1 (PbAE1; Figure 1b) and polymer 2 (PbAE2; Figure 1c) were obtained by reaction between 4,4′-trimethylenedipiperidine and, respectively, 1,4-butanediol diacrylate and 1,6-hexanediol diacrylate. The structure of PbAE1 and PbAE2 was verified by 1H NMR spectroscopy and the integration of the peaks yielded values as expected for the proposed chemical structures (see Materials and Methods). As these PbAEs were not soluble at a neutral or basic pH, they were dissolved in acetate buffer with pH 5.4. The average molecular weight of the polymers was determined by size exclusion chromatography and found to be 18 kDa for PbAE1 and 22 kDa for PbAE2.
Characterization and cytotoxicity of the PbAE : siRNA complexes
The ability of PbAEs to complex siRNA was tested via polyacrylamide gel electrophoresis (PAGE) (Figure 2). As no bands were present at the position of free siRNA, both PbAE1 and PbAE2 were able to bind siRNA. In the case of PbAE2 N : P ratio 20 : 1 and 30 : 1, no fluorescence signal was visible in the lanes, which indicates that no free siRNA is present in these complexes. However, the lanes containing the PbAE1 : siRNA complexes showed, independent of the N : P ratio, a siRNA band just beneath the slots. Similarly, PbAE2 : siRNA complexes with a N : P ratio 10 : 1 also displayed some traces of siRNA at this position. This may indicate the presence of partially complexed siRNA that is able to enter the gel, but that migrates slower than the free siRNA. Alternatively, the electric field may have caused a dissociation of these PbAE : siRNA complexes, generating free siRNA that entered the gel at a later time point than the free siRNA present in the first lane. Nevertheless, these observations indicate that PbAE2 forms tighter complexes with siRNA than PbAE1.
Besides the ability to bind siRNA, we also determined the size and zeta potential of the PbAE : siRNA complexes (Table 1). PbAE1 : siRNA complexes were, except at N : P ratio 30 : 1, smaller than PbAE2 : siRNA complexes and their zeta potential clearly increased as a function of the N : P ratio. By contrast, the zeta potential of PbAE2 : siRNA complexes was more or less independent of the N : P ratio. In general, both PbAEs were able to form relatively small siRNA complexes with a positive surface charge, a feature known to facilitate cellular binding and uptake.
Before performing siRNA transfection experiments, the cytotoxicity of PbAEs (data not shown) and PbAE : siRNA complexes (Figure 3) was assessed by quantifying the intracellular ATP levels after transfection. Measuring ATP levels is a sensitive marker of cell viability. Indeed, within minutes after a loss of membrane integrity, cells lose the ability to synthesize ATP. Additionally, endogenous ATPases destroy any remaining ATP. We found that the free PbAEs and PbAE : siRNA complexes only slightly reduced cell viability, with a maximal cytotoxicity of approximately 20% in the case of PbAE2 : siRNA N : P 30 : 1. JetSI™-ENDO : siRNA complexes, which are commonly used for in vitro siRNA delivery and which are claimed to lack cytotoxicity, did indeed not decrease cell viability. Furthermore, we also performed an MTT-based cell viability test (EZ4U assay), a test that relies on the ability of live but not dead cells to reduce MTT into a colorimetric formazan product by active mitochondrial dehydrogenases. In this approach, none of the free polymers or complexes showed significant cytotoxicity (data not shown), which can be explained by the fact that this test only detects severe cytotoxicity effects, in contrast to the ATP-based cytotoxicity test.
Cellular uptake of PbAE : siRNA complexes by primary hepatocytes
To tackle viral hepatitis, the siRNA molecules must be delivered to primary hepatocytes. Although numerous mechanical, electrical, and chemical delivery methods have been applied, efficient transfer of siRNAs into primary cells is restricted to only a few cell types 42. To study cellular uptake, one of the first steps in siRNA delivery, we visualized the cellular entry of the PbAE : siRNA complexes in primary hepatocytes via confocal laser scanning microscopy (CLSM). PbAE : siRNA complexes containing Alexa-488 labelled siRNA (green colour in Figure 4) were incubated over 4 h with primary hepatocytes and the lysosomes were visualized using the Lysotracker Red (red colour in Figure 4). Figure 4 a–i shows the outcome of a typical uptake experiment analysed by CLSM. After 4 h, all siRNA complexes gave rise to a punctuated green nonhomogeneous distribution pattern of the siRNA inside the cell, which is indicative of an endocytotic uptake mechanism. This is confirmed by co-localization of the green pattern with the red labelled lysosomes. PbAE : siRNA complexes are thus clearly taken up by the hepatocytes by endocytosis, but they do not show a clear cytoplasmic localization of the siRNA after 4 h and not even at 24 h (data not shown), which is required to evoke a RNAi-mediated inhibitory effect.
Gene silencing in primary rat hepatocytes
The ability of PbAE : siRNA complexes to induce gene silencing of viral genes was tested in primary rat hepatocytes transduced with replication-deficient adenoviruses expressing firefly luciferase. As a control, complexes containing nonspecific siRNA (i.e. siRNA with no known target in the cells) were added to the cells under identical conditions to ascertain that the reduction in gene expression was due to a specific siRNA effect and not to nonspecific effects such as IFN induction. The gene silencing observed 30 h after the adenoviral transduction was independent of the N : P ratio and equalled approximately 55% and 65% for the PbAE1 : siRNA and PbAE2 : siRNA complexes, respectively (Figure 5). Our lipid-based reference siRNA complexes (jetSI™-ENDO : siRNA complexes) were much less efficient and silenced only 30% of the luciferase expression in primary hepatocytes. Additionally, both polymers were also used to target the endogenous connexin (Cx) proteins Cx32 and Cx43, the building stones of gap junctions controlling direct communication between cells. In both cases, a strong gene silencing was observed by western blotting (data not shown).
These results imply that, although not clearly visible by CLSM after 4 h (Figure 4) and not even after 24 h (data not shown), a portion of the siRNA molecules must be released from the endosomes after endocytotic uptake. Because of the recycling of the siRNA after degradation of a target mRNA, it can be expected that very low amounts of siRNA in the cytosol can cause an efficient gene silencing. Indeed, it has been calculated that approximately 300 siRNA molecules per cell are sufficient to reduce luciferase activity with 50% 43. Such small amounts of cytosolic siRNA are most likely not visible via CLSM. Therefore, the absence of visible siRNA in the cytosol of our cells does not exclude that a small fraction is present in the cytosol that is causing the observed gene silencing effect. Because no fusogenic function is present in our polymers and based on the physicochemical properties, two possible mechanisms may mediate endosomal escape of the siRNA. First, PbAE : siRNA complexes may disrupt endosomes via the proton sponge mechanism that is considered to be responsible for the high transfection efficiency of PEI-based DNA complexes. The ‘proton sponge’ nature of PEI is thought to act as a strong buffer that prevents the pH drop inside endosomes. The endosomes try to overcome this high buffer capacity of PEI by pumping in more protons. To maintain charge neutrality, the influx of protons is accompanied by an influx of chloride ions. These ion influxes cause a drastic increase of the ionic strength inside the endosomes, which leads to an osmotic swelling and, finally, a physical rupture of the endosomes, resulting in the escape of PEI based DNA complexes from the endosomes 44. Alternatively, endosomal escape of our siRNA may also occur via an increase of the colloidal osmotic pressure in the endosomes after degradation of the PbAE into low molecular weight fragments, as suggested by Murthy et al.45.
Gene silencing in hepatoma cells
In contrast to, for example viral HCV RNA, the candidate target genes for siRNA therapeutics in tumour cells are stably and massively expressed. Therefore, we also tested the gene silencing efficiency of the PbAE : siRNA complexes in human hepatoma (HuH-7) cells that stably express the eGFP-luciferase fusion protein (HuH-7_eGFPLuc). In several studies, the targeted gene that needs to be silenced is introduced in the cells via the same carrier as the siRNA 46. In such experiments, an efficient gene silencing is highly expected because the gene and the siRNA are co-delivered to the same cells. Furthermore, it was recently questioned whether simultaneous transfection of an exogenous gene and the siRNA is suitable to quantify RNA interference 47. Additionally, it is currently unknown whether siRNA-mediated knockdown of transiently expressed proteins is an acceptable quantitative surrogate for stably expressed proteins 48. In conclusion, targeting a stably expressed gene can be considered as a model for oncogene silencing and targeting a transient gene as a model for viral gene silencing, if the viral genome is not integrated in the host's genome. Therefore, the stably transfected hepatoma cells used in the present study are the most appropriate model for silencing of genes in tumour cells.
As shown in Figure 6, the extent of gene silencing of the PbAE : siRNA complexes depends on the N : P ratio. The gene silencing increased as a function of the N : P ratio and a gene silencing of approximately 75% was obtained at N : P ratio 30 : 1. PbAE1 : siRNA complexes with higher N : P ratios were not tested due to cytotoxicity concerns. The gene silencing of the PbAE2 : siRNA complexes was also higher at higher N : P ratios but, at the highest N : P ratios, it stagnated at approximately 60%. With the jetSI™-ENDO : siRNA complexes, a gene silencing efficacy of only 20% could be detected.
Prolonged gene silencing in hepatoma cells
A major disadvantage of the current available nonviral carriers for synthetic siRNA delivery is that the period of effective gene silencing is very brief. It has been demonstrated that the rapid restoration of gene expression after delivery of synthetic siRNA is mainly due to dilution of the siRNA during cell division, and not to a rapid intracellular degradation of siRNA by nucleases 49. This implies that prolonged gene silencing after siRNA delivery is of major importance for siRNA delivery to especially tumour cells, which exhibit rapid growth with doubling times in the order of only a few days. Therefore, carriers that slowly release their siRNA in the cytoplasm could be of interest for therapeutic siRNA delivery to tumour cells. The only difference between PbAE1 and PbAE2 is the 4- or 6-carbon linkers situated between the esters in the repeat units, which implies variations in both charge density and hydrophobicity. It was found that these relatively minor changes in polymer structure play an important role in determining the hydrolysis rate of the polymers when incubated in physiologically relevant media. Polyelectrolyte complexes containing PbAE1 and PbAE2 eroded completely in approximately 50 h and 6 days, respectively 50. Therefore, we aimed to analyse whether this difference in degradation rate could also influence the gene silencing kinetics of both PbAE1 : siRNA and PbAE2 : siRNA complexes. As discussed above, obtaining a sustained gene silencing is most challenging in dividing cells due to dilution of the siRNA during cell division. Hence, we monitored gene silencing up to 5 days after siRNA transfection in the HuH-7 hepatoma cells that stably expressed luciferase.
In Figure 7, huge differences in the gene silencing kinetics between both PbAE : siRNA complexes are shown. After siRNA delivery with PbAE1, we observed a gradual recovery of the gene expression (Figure 7a). The rate of recovery inversely correlated with the N : P ratio of these siRNA complexes. Indeed, at an N : P ratio 10 : 1, the luciferase expression started to recover at day 2 and was completely restored at day 5. By contrast, the onset of recovery at N : P ratios 20 : 1 and 30 : 1 occurred later and, at day 5, the luciferase level reached approximately 90% and 60%, respectively. An increase in N : P ratio, and thus in polymer concentration, probably leads to more tight complexes that release their siRNA more slowly. This may explain why PbAE1 : siRNA complexes with a higher N : P ratio can suppress gene expression during longer periods. By sharp contrast to the PbAE1 : siRNA complexes, the gene silencing was almost completely maintained until day 5 when PbAE2 : siRNA complexes with a N : P 20 : 1 and especially 30 : 1 were used. A small recovery in luciferase expression was observed when PbAE2 : siRNA complexes were used at a N : P ratio of 10 : 1. The observation that PbAE2 forms stronger complexes with the siRNA (see above) is probably not the main reason for the more prolonged gene silencing observed with PbAE2. Indeed, at a N : P ratio of 10 : 1, PbAE2 also formed, like PbAE1, ‘weaker’ complexes with siRNA that were still able to cause prolonged gene silencing (Figure 7b).
Time-dependent intracellular fate of siRNA delivered via PbAE
The obtain further insight into the intracellular mechanisms that govern the prolonged gene silencing after siRNA delivery via PbAE2, we compared the time-dependent intracellular distribution of PbAE1 : siRNA (N : P 10 : 1) and PbAE2 : siRNA (N : P 30 : 1) complexes. PbAE1 : siRNA (N : P 10 : 1) and PbAE2 : siRNA (N : P 30 : 1) complexes containing Alexa-488 labelled siRNA (Figure 8, shown in green) were added to cultured HuH-7 cells and the intracellular fluorescence was monitored for 5 days. To visualize the lysosomes, we again used Lysotracker Red (Figure 8, shown in red). The inserts in Figure 8 show the intracellular distribution of the labelled siRNA 24 h after transfection. After 24 h, a punctuated pattern, that co-localized with the Lysotracker Red, was observed with both PbAE : siRNA complexes. However, 5 days after transfection, a clear difference in the intracellular siRNA distribution pattern was visible. The intracellular fluorescence of the siRNA after delivery via PbAE1 (N : P 10 : 1) became weaker and the dotted fluorescence signal was replaced by a more diffuse distribution of the fluorescence. By contrast, the intracellular fluorescence of the siRNA after delivery via PbAE2 (N : P 30 : 1) showed a similar intensity and intracellular localization at day 5 (i.e. in vesicles) as it did after 24 h. As stated above, a vesicular localization of the siRNA is not necessarily expected to result in a good RNAi effect because the RNAi machinery is present in the cytosol. Therefore, the siRNA or the siRNA complexes will have to escape from the endosomes.
With the slow degrading polymer (PbAE2), the siRNA remained in vesicles up to day 5 (Figure 8d to 8f), whereas the siRNA signal became more weak and diffuse at day 5 with the fast degrading polymer (PbAE1) (Figure 8a to 8c). If we correlate these observations with the duration of gene silencing (Figure 7), it is tempting to speculate that the endosomal release kinetics of siRNA, and hence the duration of the gene silencing, are driven by the degradation kinetics of the PbAEs. This is in line with the hypothesis that the endosomal escape of the siRNAs or PbAE : siRNA complexes occurs via the colloidal osmotic pressure hypothesis explained above, in which the osmotic pressure in the endosomes is due to the formation of small PbAE degradation products. The prolonged gene silencing with PbAE2 also implies that not all endosomes are ruptured at the same time. Such a continuous rupture of endosomes is very likely to occur because the kinetics of the osmotic pressure increase in the endosomes will also depend on the amount of PbAE in the endosomes and the latter is expected to vary from endosome to endosome. Therefore, the siRNA filled vesicles can be considered as a depot from which siRNA is continuously released in the cytoplasm during a time period that is mainly determined by the degradation kinetics of the PbAE.
Inhibition of highly active genes involved in liver oncogenesis or viral replication via siRNA delivery may offer great opportunities for the treatment of several diseases, such as HCC and HCV infections. Unfortunately, clinical applications of siRNAs are currently limited because no safe and efficient delivery systems are available. In the present study, we show for the first time that biodegradable polymers (PbAEs) are able to induce efficient siRNA-mediated gene silencing of a viral (transduced) gene in primary rat hepatocytes and a stably expressed gene in hepatoma cells without causing significant cytotoxicity. However, besides the development of carriers that efficiently deliver the siRNA, there is also major clinical interest for siRNA carriers that slowly release the siRNA in the cytoplasm and hence induce a prolonged gene silencing effect. We show that, in contrast to PbAE1, PbAE2 is also able to induce a long-term gene silencing in the hepatoma cells (up to 5 days). Interestingly, the prolonged gene silencing of PbAE2-based siRNA complexes correlated with the slower degradation kinetics of PbAE2 and a prolonged vesicular localization of the siRNA. This supports the hypothesis that the release of the PbAE : siRNA complexes or siRNA is based on an increase in colloidal osmotic pressure in the endosomal vesicles after polymer hydrolysis. In this way, the siRNA filled endosomal vesicles act as a siRNA depot from which siRNA is continuously released in the cytoplasm during a time period that is mainly determined by the degradation kinetics of the PbAE. We conclude that both PbAEs, and especially PbAE2, open up new perspectives for the development of efficient biodegradable siRNA carriers suitable for clinical applications.
The HuH-7_eGFPLuc cells were kindly provided by Professor Ernst Wagner, LMU University Munich. Niek Sanders, Tamara Van Haecke, Harry Heimberg and Stefan Bonné are supported by the Fund for Scientific Research—Flanders (FWO). The financial support of this institute is acknowledged with gratitude. This work was supported by grants from Ghent University (BOF), FWO and the European Union (MediTrans).