Novel Polymerizable Surfactants with pH-Sensitive ... - ACS Publications

Bioconjugate Chem. 2007, 18, 2169–2177

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Novel Polymerizable Surfactants with pH-Sensitive Amphiphilicity and Cell Membrane Disruption for Efficient siRNA Delivery Xu-Li Wang, Sergej Ramusovic, Thanh Nguyen, and Zheng-Rong Lu* Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84108. Received July 27, 2007; Revised Manuscript Received September 4, 2007

Small interfering RNA (siRNA) is a promising new therapeutic modality that can specifically silence diseaserelated genes. The main challenge for successful clinical development of therapeutic siRNA is the lack of efficient delivery systems. In this study, we have designed and synthesized a small library of novel multifunctional siRNA carriers, polymerizable surfactants with pH-sensitive amphiphilicity based on the hypothesis that pH-sensitive amphiphilicity and environmentally sensitive siRNA release can result in efficient siRNA delivery. The polymerizable surfactants comprise a protonatable amino head group, two cysteine residues, and two lipophilic tails. The surfactants demonstrated pH-sensitive amphiphilic hemolytic activity or cell membrane disruption with rat red blood cells. Most of the surfactants resulted in low hemolysis at pH 7.4 and high hemolysis at reduced pH (6.5 and 5.4). The pH-sensitive cell membrane disruption can facilitate endosomal–lysosomal escape of siRNA delivery systems at the endosomal–lysosomal pH. The surfactants formed compact nanoparticles (160–260 nm) with siRNA at N/P ratios of 8 and 10 via charge complexation with the amino head group, lipophilic condensation, and autoxidative polymerization of dithiols. The siRNA complexes with the surfactants demonstrated low cytotoxicity. The cellular siRNA delivery efficiency and RNAi activity of the surfactants correlated well with their pH-sensitive amphiphilic cell membrane disruption. The surfactants mediated 40–88% silencing of luciferase expression with 100 nM siRNA and 35–75% with 20 nM siRNA in U87-luc cells. Some of the surfactants resulted in similar or higher gene silencing efficiency than TransFast. EHCO with no hemolytic activity at pH 7.4 and 6.5 and high hemolytic activity at pH 5.4 resulted in the best siRNA delivery efficiency. The polymerizable surfactants with pH-sensitive amphiphilicity are promising for efficient siRNA delivery.

INTRODUCTION RNA interference (RNAi) is a natural biological process involving gene silencing or regulation with small interfering RNA (siRNA) (1, 2). siRNA is a double-stranded RNA molecule (dsRNA) with approximately 20 to 25 nucleotides. The function of siRNA involves specific incorporation into the RNA-induced silencing complex (RISC) and guiding the RNAi machinery to destroy a target mRNA containing complementary sequences (3). It has been demonstrated that siRNA is effective to silence specific genes in mammalian cells and nonhuman primates. It is believed that siRNA can target every gene in the human genome and has unlimited potential to treat human disease with RNAi (4–9). However, the challenge for successful clinical applications of RNAi remains the in ViVo delivery of therapeutic siRNAs into target cells with high efficiency. Systemic in ViVo siRNA delivery is a complicated process, which involves protecting siRNA from enzymatic degradation, preventing rapid elimination from the body, and facilitating specific tissue and cell uptake, endosomal–lysosomal escape, and siRNA release in cytoplasm. Although various systems have been reported for siRNA delivery, most of these delivery systems are developed based on the concept for the delivery of plasmid DNA, using cationic lipids (10, 11) or polymers (12–14). These cationic materials form stable nanoparticles with anionic siRNA via charge complexation and protect siRNA from degradation. These siRNA delivery systems are either toxic or inefficient for specific siRNA delivery (15). Innovative approaches are needed to design and develop safe and efficient * Correspondence to Dr. Zheng-Rong Lu, 421 Wakara Way, Suite 318, Salt Lake City, UT 84108. Phone: 801 587-9450. Fax: 801 5853614. E-mail: [email protected].

siRNA delivery systems. New siRNA delivery systems should be safe and have a comprehensive multifunctionality to address the complexity of in ViVo delivery of therapeutic siRNA. One of the challenges for efficient siRNA delivery is the escape of siRNA delivery systems from the endosomal–lysosomal compartments and siRNA release in cytoplasm after the systems are internalized through endocytosis. We hypothesize that the incorporation of pH-sensitive amphiphilicity and environmentally sensitive siRNA release will result in efficient siRNA delivery systems that facilitate endosomal–lysosomal escape and cytoplasmic siRNA release. Numerous amphiphilic materials present in nature are able to disrupt cell membranes (16–18). The pH-sensitive amphiphilicity of an siRNA carrier will allow the carrier to change its amphiphilic structure at endosomal–lysosomal pH (5.0–6.0), resulting in disruption of endosomal–lysosomal membranes and escape of siRNA delivery systems. It has been shown that the incorporation of degradable disulfide bonds into cationic polymeric delivery systems promotes the release of nucleic acids in the reductive environment of cytosol (19–21). According to the hypothesis, we have designed polymerizable surfactants containing dithiol groups, protonatable amines with different pKa’s and lipophilic units as novel multifunctional carriers for siRNA delivery. The surfactants can readily form compact nanoparticles with siRNA via charge– charge complexation and hydrophobic interaction. The dithiol groups in the surfactants in the nanoparticles can be polymerized by forming disulfide bonds via autoxidation to further stabilize siRNA nanoparticles. The pH-sensitive amphiphilicity of the carriers can be tuned by modifying the structures of the protonatable amines and lipophilic groups to allow the nanoparticles escaping from endosomal–lysosomal compartments via amphiphilic membrane disruption. The disulfide bonds will be

10.1021/bc700285q CCC: $37.00  2007 American Chemical Society Published on Web 10/17/2007

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reduced in the cytoplasm to facilitate the dissociation of nanoparticles and the release of siRNA. We report here the synthesis and evaluation of a small library of polymerizable surfactants with pH-sensitive amphiphilicity for efficient siRNA delivery. The surfactants were synthesized using combinatorial solid-phase chemistry. The pH-sensitive amphiphilic cell membrane disruption of the surfactants was investigated using hemolysis assay of rat red blood cells at different pH values. The cellular siRNA delivery efficiency of the novel surfactants was evaluated with an antiluciferase siRNA in U87-luc cells with constitutive firefly luciferase expression.

EXPERIMENTAL SECTION Materials. 2-Chlorotrityl chloride resin, N-fluorenylmethoxycarbonyl-N-im-trityl-L-histidine, N-fluorenylmethoxycarbonylS-trityl-L-cysteine, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), N-hydroxybenzotriazole (HOBt), and 2-acetyldimedone (Dde-OH) were purchased from EMD Biosciences (San Diego, CA). Ethylenediamine, pentaethylenehexamine, spermine, triethylenetetraamine, N,N-diisopropylethylamine (DIPEA), methyl acrylate, hydrazine, 4-dithiothreitol (DTT), piperidine, trifluoroacetic acid (TFA), hyperbranched PEI (Mw ) 25 KDa), N-(2,3-dioleoyloxy-1propyl)trimethylammonium methyl sulfate (DOTAP), bovine serum albumin (BSA), and 2,5-diphenyl-3-(4,5-dimethyl-2thiazolyl)tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Anhydrous dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and dichloromethane (DCM) were purchased from Acros (Pittsburgh, PA). ISOLUTE column reservoirs (Charlottesville, VA) were used for the solidphase synthesis. The amines were purified by distillation under reduced pressure before they were used for synthesis. All other materials and solvents were used without additional purification. 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) was purchased from Pierce Inc. (Rockford, IL). TransFast that is formulated with N,N-[bis-(2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl] ammonium iodide and dioleoyl phosphatidylethanolamine (DOPE) was purchased from Promega. siRNA targeting firefly luciferase was purchased from Dharmacon (Chicago, IL). The sequence of antisense is 5′-UCGAAGUACUCAGCGUAAGdTdT-3′ and that of sense is 3′-dTdTAGCUUCAUGAGUCGCAUUC-5′. Synthesis of Polymerizable Surfactants. The polymerizable surfactants were synthesized by solid-phase chemistry. The synthetic procedure of (1-aminoethyl)imino-bis[N-(oleicyl-cysteinyl-histinyl-1-aminoethyl)propionamide] (EHCO) is described as the representative procedure for the synthesis of the library. 2-Chlorotrityl chloride resin (300 mg) was extensively washed with anhydrous DCM. A mixture of ethylenediamine (1.0 mL, excess) and DIPEA (64 mg) in DCM was added to the resin, and the suspension was shaken for 2 h. The solvent was drained, and the resin was washed with DCM and MeOH. The resin was further shaken with 10 mL DCM/MeOH/DIPEA (17/2/1, v/v/v) for 20 min. The resin was then mixed with methyl acrylate (50 mL, excess) in 10 mL DMF to introduce methyl carboxylate via Michael addition. The reaction was carried out in a rotary evaporator at 50 °C with continuous rotating. A solution of 1,2ethylenediamine (50 mL, excess) in 10 mL DMF was then mixed with the resin methyl carboxylate. The mixture was rotated in a rotary evaporator at 50 °C for 5 days. The resin containing primary amines was transferred into an ISOLUTE column, mixed with a solution of activated N-R-Fmoc-N-imtrityl-L-histidine (2.0 g, excess) with TBTU/HOBt/DIPEA (excess) in DMF and shaken for 2 h. The resin was subjected to a washing cycle, and the Fmoc protecting group was removed with 20% piperidine in DMF (20 min × 3) to give a resin containing histidine residues. Cysteine residues were similarly

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incorporated by reacting the resin with N-R-Fmoc-S-trityl-Lcysteine (2.0 g, excess) and TBTU/HOBt/DIPEA (excess) in DMF, followed by removal of Fmoc protecting groups. Finally, oleicyl groups were incorporated by reacting the resin with oleic acid (2 g) in the presence of TBTU/HOBt/DIPEA in DMF for 2 h. The quality of each coupling reaction involving primary amino groups was monitored with the Kaiser test. The resin in each reaction cycle was extensively washed with DMF, MeOH, and DCM and dried under reduced pressure before proceeding to the next reaction. The final resin was then suspended in a solution of TFA/H2O/EDT/TIBS (94/2.5/2.5/1) and shaken for 3 h at room temperature. The solution was collected and concentrated under reduced pressure. The residue was washed with cold diethyl ether (40 mL × 5) and dried. The final product EHCO was purified by preparative HPLC equipped with a ZORBAX PrepHT C-18 column using an Agilent 1100 series purification system. Product fractions were collected and lyophilized. The purity of the final product was verified by analytic HPLC. The structure of the compound was analyzed by 1H NMR spectroscopy using a Varian Mercury 400 (Palo Alto, CA) and matrix-assisted laser desorption–ionization (MALDI) timeof-flight (TOF) mass spectrometry. Hemolysis Assay. The surfactants (16.7 µM), DOTAP (16.7 µM), and Triton X-100 (1%, w/v) were dissolved in phosphate buffered saline (PBS) with pH adjusted to 7.4, 6.5, or 5.4 as stock solutions. Rat erythrocytes (RBC) were suspended in prechilled PBS at a concentration of 2 % (w/v) RBC at appropriate pH values and then seeded (100 µL) into a 96-well plate. The sample solutions (100 µL) were added to each well of the RBC suspensions and incubated for 1 h at 37 °C. The absorbance of the supernatant from each sample was measured at 550 nm using a microplate reader to determine the released hemoglobin. The relative hemolysis efficiency was calculated as the percentage of released hemoglobin by the surfactants or DOTAP to that by Triton X-100. Formation and Size Measurement of Surfactant/siRNA Nanoparticles. Surfactant/siRNA nanoparticulate complexes were prepared by mixing siRNA solution with an equal volume of surfactant solutions at predetermined N/P ratios, followed by 30 min incubation. The size of the nanoparticles was analyzed using a Brookhaven Instruments BI-200SM system equipped with a 5 mW helium neon laser with a wavelength output of 633 nm. The effective diameter and population distribution were computed from the diffusion coefficient. Measurements were made at 25 °C at an angle of 90°, and each sample was analyzed in triplicate. Autoxidation of the Polymerizable Surfactants. The polymerizable surfactants were diluted from stock solutions (N2protected, 2 mg/mL) to an initial thiol concentration of 150 µM in Tris buffer (10 mM pH 8.0). The autoxidation of the dithiols in the surfactants was performed with the working solutions (400 µL) in the absence or presence of 10 µg siRNA (N/P ) 10). For each predetermined time point, an aliquot was taken and mixed with an equal volume of Ellman’s solution (2 mM DTNB in 50 mM NaOAc solution), and the free thiol concentration was determined by Ellman assay using UV–vis spectrophotometry (Cary-300 Bio). Polymerizable Surfactant Mediated in Vitro Gene Silence with siRNA. U87-luc cells with constitutive expression of firefly luciferase were maintained in minimal essential medium (ATCC) containing 10% FBS, G418 (300 µg/mL), streptomycin (100 µg/mL), and penicillin (100 units/mL). U87Luc cells were seeded 24 h prior to transfection into a 96-well plate at a density of 5000 cells/well. At the time of siRNA transfection, the medium in each well was replaced with fresh serum-free medium. Antiluciferase siRNA was complexed with the polymerizable surfactants, TransFast, or DOTAP and in-

Polymerizable Surfactants for siRNA Delivery

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Scheme 1. General Structure of Polymerizable Surfactants

cubated for 30 min before use. Complexes were incubated with the cells for 4 h at 37 °C. The medium was then replaced with 100 µL of fresh complete medium, and cells were incubated for an additional 44 h. The cells were then washed with prewarmed PBS, treated with 200 µL cell lysis buffer, and subjected to a freeze–thaw cycle. Cellular debris was removed by centrifugation at 14 000 g for 5 min. The luciferase activity in the cell lysate (20 µL) was measured using a luciferase assay kit (100 µL luciferase assay buffer) on a luminometer for 10 s (Lumat 9605, EG&G Wallac). The gene silencing efficiency was normalized against the luciferase expression of untreated cells. Cytotoxicity of Surfactant/siRNA Complexes. U87-luc cells were incubated with siRNA complexes of different carriers according to the protocol described above. MTT in PBS (5 mg/ mL, 25 µL) was added to each well after incubation, and cells were incubated for another 2 h. After cell culture medium was removed, 200 µL DMSO was added to each well, and cells were incubated at 37 °C for 5 min. The optical absorption was measured at 570 nm using a microplate reader (model 550, BioRad Laboratory. Hercules, CA). The relative cell viability was calculated with the equation ([Abs]sample – [Abs]blank)/([Abs]control – [Abs]blank) × 100%.

RESULTS AND DISCUSSION Design and Synthesis of Polymerizable and pH-Sensitive Amphiphilic Surfactants. The polymerizable surfactants with pH-sensitive amphiphilicity are designed based on the hypothesis that pH-sensitive amphiphilicity can facilitate the escape of siRNA delivery systems from endosomal–lysosomal compartments and environmentally sensitive disulfide crosslinking can stabilize the siRNA delivery system in the plasma and facilitate the release of siRNA in cytoplasm for efficient siRNA delivery and effective RNAi. The general structure of the designed polymerizable surfactants is shown in Scheme 1. Each surfactant is composed of three key components in its

structure, including a protonatable amino head group, dual cysteine residues, and lipophilic tails. The protonatable amino head group comprises primary, secondary, tertiary, and aromatic amino groups of various pKa’s, which is designed to complex with siRNA and alter the pH-sensitive amphiphilicity of the surfactants. Lipophilic tails are designed to introduce lipophilicity in the surfactants and to condense the nanoparticulate siRNA complexes though hydrophobic interactions. Cysteine residues are designed to further stabilize nanoparticles through polymerization of the surfactants by forming disulfide bonds via autoxidation of thiols. The disulfide bonds are relatively stable in the plasma during the delivery process and can be reduced in the reductive cytosolic environment to facilitate siRNA release. The pH-sensitive amphiphilicity of the surfactants can be tuned by varying the composition of amino groups of different pKa’s and structures of the lipophilic groups. Different polyamines, including ethylenediamine, triethylenetetraamine, pentaethylenehexamine, and spermine, and histidine have been chosen to build the protonatable head groups. Fatty acids of different chain lengths and structures, including lauric acid, stearic acid, and unsaturated oleic acid, have been used as the hydrophobic units. The small library of surfactants with different combinations of protonatable head groups and lipophilic tails were synthesized using resin-supported solidphase chemistry. Scheme 2 shows the synthetic procedure of EHCO. The structures of the polymerizable surfactants and their abbreviated names, according to their head group, peptide linkage, and tail chains (Scheme 1), are shown in Scheme 3. For triethylenetetraamine, pentaethylenehexamine, and spermine, their primary and secondary amines were selectively protected by 2-acetyldimedone (Dde-OH) and tert-butyloxycarbonyl (Boc) groups, respectively, as described in our previous publication . The polymerizable surfactants with high purity were obtained after purification with preparative HPLC and characterized by 1 H NMR and mass spectrometry. The 1H NMR spectrum of EHCO is shown in Figure 1 as a representative example. The


Wang et al.

Scheme 2. Synthetic Procedure of EHCO

Scheme 3. Chemical Structures and Abbreviated Names of Different Polymerizable Surfactants with pH-Sensitive Amphiphilicity

calculated mass and measured mass of the polymerizable surfactants are summarized in Table 1. No evidence of oligomers or cyclic byproducts was observed in the mass spectra of the compounds. Over 98% of thiol groups remained in a reduced state as determined by Ellman’s assay. TGCO with glycine replacing histidine was prepared to study the impact of imidazole groups on the pH-sensitive amphiphilicity and cell membrane disruption of the surfactants. pH-Sensitive Hemolysis. Amphiphilic cell membrane disruption is a common phenomenon in nature. Numerous amphiphilic materials have been found to have a fusogenic property of mediating material transport across cell membranes (22–24). Amphiphilic peptide sequences are used by viruses, the highly efficient gene delivery systems, in their membrane surface

protein to facilitate cell entry or endosomal–lysosomal escape (25–27). Several amphiphilic lipids, e.g., DOPE, have been used to enhance the fusion of nucleic acid lipoplexes with cellular membrane for gene delivery (28, 29). However, these lipids may not have the ability of selective amphiphilic membrane disruption at the endosomal–lysosomal pH. It is critical for siRNA delivery if siRNA delivery systems have low amphiphilicity at the physiological pH and high amphiphilicity at the endosomal–lysosomal pH, which will only cause selective endosomal– lysosomal membrane disruption. The polymerizable surfactants may not have strong amphiphilicity before the amino head groups are completely protonated at neutral pH. The amphiphilicity of the surfactants may increase with increasing protonation degrees of the head groups at reduced pH.



Figure 1. 1H NMR Spectra of EHCO. Table 1. Measured Mass of the Polymerizable Surfactants by Mass Spectroscopy entry

MFC abbr

formula

Mw (calcd) m/e

Mw (found) [M + 1]+

1 2 3 4 5 6 7 8 9 10

EHCL EHCO EHCSt THCL THCO THCSt TGCO PHCL PHCO SHCO

C54H96N14O8S2 C66H116N14O8S2 C66H120N14O8S2 C58H106N16O8S2 C70H126N16O8S2 C70H130N16O8S2 C62H118N12O8S2 C62H116N18O8S2 C74H136N18O8S2 C74H134N16O8S2

1132.70 1296.85 1300.89 1218.78 1382.94 1386.97 1222.86 1304.87 1469.02 1439.00

1133.65 1297.90 1301.87 1219.79 1383.94 1387.97 1223.86 1305.87 1470.94 1439.97

The pH-sensitive amphiphilic cell membrane disruption of the polymerizable surfactants was evaluated by hemolysis assay with rat red blood cells at different pHs. Figure 2 shows the hemolytic activity of the compounds at pHs 7.4, 6.5, and 5.4 in PBS buffer. The positive control Triton X-100 (1%, w/v) resulted in complete hemolysis. Little hemolysis was shown with the buffer and DOTAP in all cases. The polymerizable surfactants showed various pH-dependent hemolytic activities. Generally, all surfactants had lower hemolytic activity at pH 7.4 than at pHs 6.5 and 5.4. At pH 7.4, EHCL and EHCO resulted in negligible hemolysis, while the rest of the surfactants showed moderate hemolytic activities with 10–35% hemolysis. At pH 6.5, hemolytic activity of the surfactants increased except for EHCO, which still showed negligible hemolysis. At pH 5.4, all surfactants exhibited high hemolysis, and the surfactants except EHCL resulted in 50–80% hemolysis. The pH-sensitive hemolysis of the surfactants is clearly governed by their structural characteristics. For the surfactants containing histidine residues and the same lipophilic tails, the hemolytic activity increased with increasing number of protonatable amino groups in the head group at pHs 7.4 and 6.5. More protonatable amino groups could result in more charges in the head groups and higher amphiphilicity for the surfactants at neutral pH. For the EHC and THC series, the unsaturated oleicyl group resulted in less hemolysis than the saturated lipophilic tails in the same series at pHs 7.4 and 6.5 except for EHTL at pH 7.4. The comparison between THCO and TGCO suggests that the histidine residues are critical for pH-sensitive

Figure 2. Hemolytic activity of polymerizable surfactants (8.3 µM) and DOTAP (8.3 µM) at pHs of 7.4, 6.5, and 5.4, respectively. Triton X-100 (1%, w/v) and PBS were used as controls.

amphiphilicity of the surfactants. Overall, the pH-sensitive hemolysis, disruption of membrane of red blood cells, is caused by the pH-sensitive amphiphilicity of the surfactants derived from the combination of protonatable amino head groups and lipophilic tails. When more amino groups are protonated, the surfactants become more amphiphilic and result in more significant hemolysis. The protonation and pH-sensitive amphiphilicity may be governed by the overall pKa of the head groups of the surfactants. As we have shown here, they can be tuned by using amino groups with different pKa’s. The pH-sensitive amphiphilicity of the surfactants is critical for efficient cellular siRNA delivery. It is known that pH in the late endosomal compartment is in the range 5.0–6.0 and pH in the lysosomal compartment is 5.0–5.5. Therefore, the pHresponsive amphiphilicity and membrane disruption of the polymerizable surfactants is an ideal feature to trigger endosomal–lysosomal membrane destabilization. Low amphiphilicity of the surfactants at physiological pH will minimize nonspecific cell membrane disruption and nonspecific tissue uptake of siRNA delivery systems. To this end, EHCO with no hemolytic activity at pHs 7.4 and 6.5 and high membrane disruption activity at pH 5.4 could be a suitable carrier for siRNA delivery.


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Figure 4. Autoxidation profile of EHCO in the absence or presence of siRNA based on thiol concentration measured by Ellman’s assay.

Figure 3. Particle size of the siRNA complexes with the polymerizable surfactants: (a) size of EHCO/siRNA complexes at different N/P ratios; (b) sizes of siRNA complexes with the polymerizable surfactants at N/P ratios of 8 and 10.

Formation of Nanoparticles of the Polymerizable Surfactants with siRNA. The complexation of the polymerizable surfactants with siRNA and formation of nanoparticles of the complexes were investigated by dynamic light scattering. EHCO was first used to study the complexation with siRNA and the impact of N/P ratios on the complexation and formation of nanoparticles. No nanoparticulate formation was detected in either EHCO or siRNA solutions. When EHCO was mixed with siRNA and incubated for 30 min, the formation of nanoparticulate complexes was observed with an N/P ratio as low as 0.5. The particle size changed with N/P ratios of the complexes (Figure 3a). The initial size was approximately 200 nm in diameter at an N/P ratio of 0.5, and the size then increased with increasing N/P ratio up to an N/P ratio of 4. The particle size was as large as about 3 µm at the N/P ratio of 4, possibly due to aggregation of relatively neutral complex particles. The particle size decreased to approximately 240, 200, and 151 nm at the N/P ratios of 6, 8, and 10, respectively. The particle size of siRNA complexes with other polymerizable surfactants was measured at the N/P ratios of 8 and 10 based on the observation with EHCO. As shown in Figure 3b, the average particle sizes of the complexes were in the ranges of 160–260 nm at N/P ratio of 8 and 160–210 nm at N/P ratio of 10. The sizes of most complexes were under the reported cutoff size of 250 nm for efficient cellular uptake (30). The polymerizable surfactants formed relatively compact and small nanoparticles as compared to other reported siRNA delivery systems (21). It is speculated that complexes were formed by charge interaction between the carriers and siRNA. Hydrophobic interaction of lipophilic tails and polymerization of dithiols facilitated the formation of stable compact nanoparticles. The polymerization of the surfactants via oxidation was confirmed by the disappearance of thiols after they were mixed with siRNA, as determined by Ellman’s assay (Figure 4). The autoxidation rate of thiols was faster in the presence of siRNA than that in the absence of siRNA, possibly because the complexation with siRNA facilitated autoxidation. These disulfide linkages could further stabilize the nanopaticulate complexes. Due to the significant difference of thiol/disulfide redox potential between the extracellular space and the intracellular environment, the disulfide bonds would be stable in the

Figure 5. EHCO-mediated luciferase gene silencing efficiency in U87luc cells at different N/P ratios.

extracellular space during the delivery process and then reduced in cytosol, facilitating the disassociation of nanoparticles and the release of siRNA. A number of recent reports have shown that incorporation of disulfide cross-linking in the siRNA/ cationic polymers can stabilize the siRNA nanoparticles and facilitate siRNA release into the reductive cytoplasm, resulting in high transfection efficiency (19–21). Cellular siRNA Delivery. The efficacy of the polymerizable surfactants for cellular siRNA delivery was evaluated using an antiluciferase siRNA in U87-luc cell line with stable expression of firefly luciferase. Commercial transfection agents TransFast and DOTAP were used as controls. The silencing efficiency of luciferase expression mediated by the surfactants was first evaluated with EHCO at different N/P ratios to obtain the best N/P ratios for effective siRNA delivery. High luciferase expression knockdown efficiency was observed with N/P ratios ranging from 8 to 20 for EHCO (Figure 5). The peak silencing efficiency was observed at the N/P ratio of 10 for the surfactants. The silencing efficiency of luciferase expression mediated by the polymerizable surfactants was evaluated at the fixed N/P ratio of 10. Viability of cells incubated with the siRNA complexes was also investigated at the same time by MTT assay to assess the cytotoxicity of the surfactants. As shown in Figure 6a, TransFast resulted in an 89.6 ( 5.6% knockdown of luciferase expression as compared to untreated cells with 100 nM siRNA, but significant cellular cytotoxicity was observed with a cell viability of only 57.6 ( 2.2%. The relatively high gene silencing efficiency of TransFast with 100 nM siRNA could also be the result of low cell viability. DOTAP/siRNA complexes at the same siRNA concentration had higher viability (85.8 ( 1.6%), but resulted in significantly lower luciferase knockdown efficiency (56.7 ( 3.1%). The cells incubated with the siRNA complexes of the polymerizable surfactants had a relatively high viability ranging from 78.6 ( 5.7% to 88.2 ( 1.3%. The luciferase expression knockdown efficiencies varied from 47.8 ( 4.2% to 88.4 ( 3.1%. EHCO resulted in the highest



Figure 6. The polymerizable surfactants mediated luciferase gene silencing efficiency and the viability of cells incubated with MFC/siRNA complexes in U87-Luc cells. Transfast and DOTAP were used as controls. Transfection experiments were performed at siRNA concentration of 100 nM (a) or 20 nM (b).

gene silencing efficiency (88.4 ( 3.1%) with high cell viability (86.7 ( 8.3%) among the polymerizable surfactants. All of the siRNA complexes demonstrated low cytotoxicity at a low siRNA concentration (20 nM), (Figure 6b). Cell viability was high in all cases including Transfast (87.6 ( 4.6%). Under this condition, the polymerizable surfactants except EHCL resulted in higher luciferase silencing efficiency than DOTAP. THCL, THCO, TGCO, PHCO, and SHCO resulted in comparable transfection efficiency to that of TransFast (62.6 ( 6.4%). EHCO (74.5 ( 1.0%) resulted in significantly higher transfection efficiency than TransFast. The cellular siRNA delivery efficiency correlated well to the pH-sensitive hemolytic activity and amphiphilicity of the polymerizable surfactants. EHCO exhibited the highest gene silencing efficiency at both siRNA concentrations. THCO and SHCO with relatively low hemolysis at pHs 7.4 and 6.5 also showed relatively high gene silencing efficiency among the surfactants. Although the polymerizable surfactants have similar gemini lipid tails to other commercial lipid transfection agents, e.g.,

DOTAP, no helper molecule like DOPE or cholesterol is needed to achieve high siRNA delivery efficiency. This is an advantageous feature for the polymerizable surfactants as ready-to-use carriers compared to the commercially available liposome or lipid-based transfection agents. To the best of our knowledge, this is the first example of lipid-containing siRNA carriers achieving high siRNA delivery efficiency without a helper lipid. However, helper molecules are required for commercially available lipid-based siRNA carriers, including TransITsiQUEST and X-tremeGENE, to enhance delivery efficiency (15). Some of helper molecules may introduce cytotoxicity in formulations. It is reported that DOTAP was nontoxic as a siRNA carrier, but the formulation of DOTAP with DOPE resulted in significant toxicity (31). It is speculated that the polymerizable surfactants form compact nanoparticles with siRNA through charge–charge complexation, lipophilic condensation, and oxidative polymerization. The lipophilic chains may facilitate lipophilic condensation rather than forming lipid bilayers, because they are further


apart in the polymerizable surfactants as compared to the gemini lipophilic chains in commercial lipids. The cellular siRNA delivery efficiency clearly correlates to pH-sensitive amphiphilicity and pH-sensitive cell membrane disruption of the surfactants. EHCO with no hemolytic activity at pHs 7.4 and 6.5 and high hemolytic activity at the endosomal–lysosomal pH (5.4) has shown the highest cellular siRNA delivery efficiency among the tested polymerizable surfactants. The polymerizable surfactants may have another advantage in that the surface of their siRNA nanoparticles can be readily modified by reacting the unreacted thiols at the surface for various purposes, including pegylation and incorporation of targeting agents. Further study is ongoing to expand the library of the polymerizable surfactants, to develop targeted siRNA delivery systems with the surfactants and to study the factors, including formation of disulfide bonds and polymerization degree of the surfactants, on siRNA delivery efficiency and the mechanism of cellular uptake and intracellular siRNA release with the novel carriers.

CONCLUSION We have designed and synthesized a small library of novel polymerizable surfactants as multifunctional siRNA carriers based on the hypothesis that pH-sensitive amphiphilicity and environmentally sensitive siRNA release of siRNA delivery systems will result in efficient siRNA delivery and effective RNAi. The polymerizable surfactants showed pH-sensitive amphiphilic hemolysis or cell membrane disruption of rat red blood cells. They formed compact nanoparticles with siRNA. The siRNA delivery and gene silencing efficiency of the surfactants correlated well to their pH-sensitive amphiphilicity. EHCO with low hemolytic activity at the physiological pH and high hemolytic activity at pH 5.4 showed the best gene silencing efficiency in U87-luc cells. The polymerizable surfactants with pH-sensitive amphiphilicity are promising multifunctional carriers for siRNA delivery.

ACKNOWLEDGMENT We thank Mr. Shawn Owen for his technical support in the synthesis of some of the compounds and Dr. Randy Jensen for providing the U87-luc cells.

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