Synthesis and in Vitro Characterization of an ABC Triblock Copolymer

Bioconjugate Chem. 2007, 18, 736−745

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Synthesis and in Vitro Characterization of an ABC Triblock Copolymer for siRNA Delivery Tatiana Segura†,‡ and Jeffrey A. Hubbell*,† Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Institute of Bioengineering and Institute of Chemical Sciences and Engineering, CH-1015, Lausanne, Switzerland, and University of California, Los Angeles, Chemical and Biomolecular Engineering Department, Los Angeles, California. Received September 11, 2006; Revised Manuscript Received December 18, 2006

The ability to specifically down-regulate gene expression using the RNAi pathway in mammalian cells has tremendous potential in therapy and in basic science. However, delivery systems capable of efficient and biocompatible delivery of siRNA to target cells are not yet satisfactory. Here, we report the synthesis and in vitro characterization of ABC triblock copolymers that self-assemble with siRNA based on electrostatics and with each other by hydrophobic interactions. The ABC triblock copolymer is based on poly(ethylene glycol) (PEG), poly(propylene sulfide) (PPS), and a positively charged peptide (PEG-PPS-peptide). The diblock copolymer PEG45-PPS5,10 was synthesized using anionic polymerization of propylene sulfide upon a PEG macroinitiator, and the peptide domain was coupled to the PPS terminus using a disulfide exchange reaction with an N-terminal cysteine residue on the peptide. The peptides were designed to interact electrostatically with siRNA, selecting the TAT peptide domain of HIV (RKKRRQRRR) and an oligolysine (Lys9). The resulting triblock copolymers were able to self-assemble with siRNA as demonstrated by dynamic light scattering and gel electrophoresis. Complex size was found to be dependent on the amount of polymer used (charge ratio) and the length of the hydrophobic PPS block, achieving sizes ranging from 171 nm to 601 nm. Cell internalization and gene expression downregulation studies showed that the triblock copolymers are able to transport siRNA inside the cell and mediate gene expression down-regulation, with the amount of internalization and gene transfer affected by charge ratio, PPS length, and the presence of serum. The proposed triblock was able to mediate gene expression down-regulation of GAPDH, achieving up to 90.5% ( 0.02% down-regulation.

INTRODUCTION In 2001, Elhashir and co-workers (1) demonstrated that incorporation of double-stranded, 21-nucleotide-long pieces of RNA results in activation of the RNA interference (RNAi) pathway and the specific down-regulation of genes with sequences that are homologous to the double-stranded RNA. This RNAi pathway activation did not result in the activation of the interferon response, which was seen with longer doublestranded RNAs (1, 2). They called the double-stranded RNA small interfering RNA or siRNA. Since this finding, the idea of using siRNAs as a genetic therapeutic and as a basic science tool has been investigated by many groups (3). In the area of basic science, decreasing the expression level of a gene within a cell has the power to reveal or confirm the roles of specific components of signaling pathways and can lead to a mechanistic understanding of cell behavior, disease pathogenesis, and drug action (4, 5). Therapeutic strategies using siRNA are also under development and have recently shown promise to reduce of tumor growth (6, 7), reduce neovascularization in the eye (8), reduce viral infection (9), and reduce postoperative adhesions (10). However, the lack of an efficient delivery system that can efficiently target and deliver the siRNA to the desired cell population is limiting the full therapeutic potential of this approach. * Corresponding author. Jeffrey A. Hubbell, EPFL SV-LMRP, Station 15, CH-1015 Lausanne, Switzerland, Phone +41 21 693 9681, Fax + 41 21 693 9665, E-mail [email protected]. † Institute of Bioengineering and Institute of Chemical Sciences and Engineering. ‡ University of California, Los Angeles.

Nonviral siRNA delivery suffers from many of the same limitations as other gene delivery approaches, including packaging efficiency, colloidal stability, targeted internalization, endosomal escape, and vector decomplexation, for which a delivery system has yet to be found that is as efficient as viral vectors. Nevertheless, current strategies for siRNA delivery employ the same delivery vectors as used for nonviral gene delivery such as cationic polymers (7, 11, 12), lipids (13, 14), and peptides (15), which self-assemble with siRNA electrostatically. In the field of drug delivery, there is extensive interest in amphiphilic block copolymers that can self-assemble in aqueous environments into stable supermolecular structures. A variety of such structures can be generated, such as micellar and vesicular assemblies, both of which can be important for pharmacological applications. Extensive investigation has been conducted in poly(ethylene glycol) (PEG)-containing block copolymers, such as copolymers with poly(propylene glycol) (16-18), and poly(ethylene) (19). In our laboratory, a novel class of amphiphilic triblock copolymers (ABA) composed of hydrophilic PEG (A) and hydrophobic poly(propylene sulfide) (PPS) (B) polymer domains has been synthesized using mild polymerization conditions that do not require extremely anhydrous conditions and allow for incorporation of biological molecules such as peptides (20). When these triblock copolymers contain a substantial hydrophobic portion (as determined by PPS block length), they have been found to self-assemble into vesicular polymersomes of ca. 100 nm size that have a watery core, which could be used for encapsulating drugs (21). Other supermolecular structures, such as spherical and cylindrical micelles, have also been observed, being obtained by

10.1021/bc060284y CCC: $37.00 © 2007 American Chemical Society Published on Web 03/15/2007

ABC Triblock Copolymer for SiRNA Delivery

Bioconjugate Chem., Vol. 18, No. 3, 2007 737

modifying the length of the hydrophobic and hydrophilic polymer domains (22). The presence of PEG in the surface of these supermolecular structures also makes them ideal for biological applications, given that their surface would be relatively protein-resistant. Here, we report the synthesis and in vitro characterization of ABC triblock copolymers that self-assembles with siRNA based on electrostatics and with each other by hydrophobic interactions. The ABC triblock copolymer is based on PEG (A), PPS (B), and a peptide (C). The peptide was designed to interact electrostatically with siRNA: two peptide sequences were investigated, namely, the HIV-1 TAT sequence (CGGWRKKRRQRRR, the terminal C being used for block conjugation, the W for detection at 280 nm, and the intervening GG being used as a linker) or a Lys9 sequence (K9, CGGWKKKKKKKKK, likewise, the C, W, and GG residues being used for block conjugation, detection, and linking). The proposed triblock copolymer was able to form condensed structures with siRNA and mediate gene down-regulation of GAPDH, achieving up to 90.5% ( 0.02% down-regulation.

MATERIALS AND METHODS Materials. Peptides were purchased from NeoMPS (Strasburg, France) at >95% purity by HPLC. All synthesis reagents were purchased from Fluka (Buchs, Switzerland). siGlo (siLamin A/C-Cy3) was purchased from Perbio (Lausanne, Switzerland). KDarlet GAPDH enzymatic activity assay, siRNA GAPDH, siRNA NEG were purchased from Ambion (Austin, TX). All other reagents were purchased from VWR (Dietikon, Switzerland) unless otherwise noted. PEG-Allyl Ether Synthesis. PEG45 monomethyl ether (PEG45) was modified with allyl bromide as previously described (20) with modifications. In short, PEG45 (20 g, 10 mmol) was dissolved in tetrahydrofuran (THF, 300 mL) under inert atmosphere and heated to reflux for 3 to 4 h in a Soxhlet apparatus filled with molecular sieves. The solution was allowed to cool to the touch, and 2.5 equiv of NaH (0.6 g, 25 mmol) was added. The solution was stirred and allowed to react for 15 min before 1.4 mL (3.46 g, 40 mmol) of allyl bromide was added to the solution under stirring and allowed to react for 24 h. The reaction mixture was passed through a filter to remove the NaBr salt formed during the reaction. The volume of the solution was reduced to about 20 mL by evaporation and precipitated two times in diethyl ether. A white solid polymer was obtained (16.68 g, 83.4% yield, conversion 100%). 1H NMR (CDCl3): δ 3.56-3.7 (broad, PEG polymer protons), 3.41 (s, 3H, -OCH3), 4.01-4.04 (dd, 2H, -CH2OCH2CHdCH2), 5.15-5.30 (m, 2H, -CH2OCH2CHdCH2), 5.85-5.98 (m, 1H, -CH2OCH2CHdCH2). PEG-Thioacetate Synthesis. PEG-allyl ether was modified with thioacetic acid to form PEG-thioacetate as previously described (20) with modifications. PEG-allyl ether (8 g, 4 mmol) was introduced to a Schlenck tube in toluene (64 mL). The toluene was degassed with sonication for 20 min and bubbling with argon gas for 30 min. The PEG/toluene solution was further degassed with pump evacuation and filling the Sclenck tube with argon. This procedure was repeated five times. The reaction mixture was then warmed to 60 °C, and the first of five AIBN aliquots were added (1.3 g × 5, 0.8 mmol × 5), followed by the first of five thioacetic acid aliquots (0.456 mL × 5, 6.4 mmol × 5). The five aliquots were added over an 8-10 h period. The reaction was allowed to continue for 18 h at 60 °C and an additional 24 h at room temperature. The reaction mixture was mixed with activated DOWEX 1/8-100 (Cl) resin and incubated for 1 h. After filtration, the volume of the solution was reduced to about 20 mL by evaporation and then precipitated three times with diethyl ether. Dichloromethane

Figure 1. Synthetic scheme of poly(ethylene glycol)-poly(propylene sulfide)-peptide (PEG-PPS-peptide). PEG-thioacetate 1 is activated by sodium methoxylate to form a sulfide that can initiate the anionic polymerization of propylene sulfide 2 to form the AB block copolymer PEG-PPS 3. 2,2-Dipyridine dithione 4 was used as the end-capping agent to generate the AB diblock copolymer terminated with a mixed disulfide 5. The peptides TAT (CGWRKKRRQRRR) and K9 (CGGWKKKKKKKKK) reacted with 5 via a disulfide exchange reaction to generate the ABC triblock copolymers shown, 6 and 7. The formation of the triblock copolymer was monitored by recording the absorbance at 342 nm, which corresponded to the release of pyridine thione, 8 (graphical insert).

was used to redissolve the precipitate. A slightly pale yellow solid was obtained (5.26 g, 65.7% yield, conversion 90%). 1H NMR (CDCl3): δ 1.81-1.90 (q, 2H, -OCH2CH2CH2S-), 2.35 (s, 3H, -SCOCH3), 2.92-2.97 (t, 2H, -OCH2CH2CH2S-), 3.39-3.89 (broad, PEG chain protons), 3.41 (s, 3H, -OCH3). PEG-PPS Diblock Copolymer Synthesis. The polymer PEG45-PPSx-pyridyldithione was synthesized using anionic polymerization of propylene sulfide (PS) with 2,2-dipyridine dithione as the end-capping agent to form PEG-PPS-pyridyldithione (Figure 1). PEG-thioacetic acid (1 g, 0.5 mmol) was dissolved in dry THF (23 mL). Sodium methoxylate (NaMeOH, 1.1 mL, 0.55 mmol) was added to the solution and allowed to react for 30 min. Propylene sulfide (0.394 mL, 5 mmol or 0.196 mL, 2.5 mmol) was added via syringe and allowed to react for 1.5 h. Dipyridine dithione (0.44 mg, 2 mmol) was dissolved in THF and added to the reaction mixture via syringe. The progress of the reaction can be easily followed by the appearance of an intense yellow color. After filtration and solvent evaporation, the product was precipitated in diethyl ether. A pale yellow solid was obtained (336.2 mg, 33.6% yield for n ) 5; and 284 mg, 28.41% yield for n ) 10). 1H NMR

738 Bioconjugate Chem., Vol. 18, No. 3, 2007

(CDCl3): δ 1.35-1.45 (d, CH3 in PPS chain), 1.81-1.90 (broad q, 2H, -OCH2CH2CH2S), 3.6-3.7 (broad PEG chain protons), 7.8-7.83 (m, 1H pyridine group). The degree of polymerization of the PPS block was determined by taking the ratio of PEG protons to PPS protons, resulting in polymers with n ) 5.06 and n ) 10.71. The polydispersity index (PDI) of the polymers was determined by GPC with PDI ) 1.15 and PDI ) 1.26, respectively. PEG-PPS-Peptide Triblock Polymer Synthesis. The peptides Ac-CGGWRKKRRQRRR-NH2 (TAT) and Ac-CGGWKKKKKKKKK-NH2 (K9) were bound to the PEG45-PPSpyridyldithione polymers using a disulfide exchange reaction between the disulfide end-capping PEG-PPS and the cysteine group of the peptide. The course of the reaction was monitored following the release of 2-pyridinethione at 342 nm (see inset in Figure 1). Upon dilution in D2O at 12 mg/mL, the solution turned cloudy, indicating the formation of aggregates. Furthermore, upon dilution in CDCl3 at 12 mg/mL, the solution again turned cloudy with a green color indicating the formation of aggregates and the strong amphiphilic nature of the molecule. 1H NMR in water: δ 1.35-1.45 (broad d, CH in PPS chain), 3 1.81-1.90 (broad peak no q visible and signal integration reduced, 2H, -OCH2CH2CH2S), 3.6-3.7 (broad PEG chain protons unchanged). Protons from the peptide were not observed. 1H NMR in CDCl : δ 1.35-1.45 (broad no d visible, CH in 3 3 PPS chain), 1.81-1.90 (q, 2H, -OCH2CH2CH2S), 3.6-3.7 (broad PEG chain protons), 7.08-7.13 and 7.2-7.24 (t, tryptophan amino acid residue), and 7.5-7.54 and 7.6-7.64 (d, tryptophan amino acid residue). Fluorescence Reduction Assay. The ability of PEG-PPSpeptide to form complexes with siRNA was tested using an ethidium bromide (EtBr) fluorescence reduction assay. siRNA (15 pmol, 100 µL) was mixed with EtBr, and the resulting fluorescence emission was measured using a fluorescence microplate reader (Tecan, Ma¨nnedorf, Switzerland). The reduction of fluorescence emission was measured as triblock copolymer was added to generate the indicated charge ratio. Dynamic Light Scattering Analysis. The ability of PEGPPS-peptide alone or in solution with siRNA to form nanosized aggregates was studied using dynamic light scattering (DLS) in a nanozetasizer (Malvern, U.K.). Size measurements of triblock copolymers alone were measured dissolving the individual polymers in Tris-buffer saline (TBS) at a 10 mg/mL and using the standard settings in the instrument. For PEG-PPSpeptide/siRNA size measurements, complexes were formed by adding the triblock copolymer solution (150 µL) to an siRNA solution (150 pmol, in 150 µL TBS) at different charge ratios. 100 µL of the solution was used to measure size, and the other was dissolved in MiliQ water to 1000 µL and used to measure ζ-potential of the complexes. Cellular Internalization in Vitro. Cell internalization studies were assessed via fluorescence microscopy (Zeiss, Feldbach, Switzerland) and fluorescence-activated cell sorting (FACS). HeLa cells (40 000 cells/well, 24-well plate) were seeded the day before adding complexes with siRNA labeled with Cy3 (microscopy) or Cy5 (FACS). The day of transfection, siRNA/ polymer complexes (25 pmol siRNA) were formed at charge ratios of (6 for the microscopy studies and (3 or (6 for the FACS studies, were added to the cells, and were incubated with the cells for either 24 or 4 h for the microscopy experiments and FACS experiments, respectively. After incubation, the cells were washed three times with PBS and either visualized using a fluorescence microscope or trypsinized, centrifuged, and resuspended in 1% FBS in PBS and analyzed via FACS. The cells were not fixed. Untreated cells were used to determine the native background of the cells and to adjust the gates. The live cell gate was adjusted so that 10 000 events were read.

Segura and Hubbell

The positive cell gate was defined as having less than 0.5% positive cells for untreated cells (a 95% confidence interval). Cell Proliferation Assay. The toxicity of the triblock copolymer/siRNA complexes was assessed using the MTT assay (Promega Corp, Madison, Wisconsin). HeLa cells (7000 cells/ well) were plated on a 96-well plate the day before they were exposed to the triblock copolymers/siRNA complexes. To determine the effect of complex concentration on toxicity, different siRNA concentrations were delivered with complexes at a charge ratio of (6. Complexes were incubated with the cells for 24 h, then removed, and new media added. The MTT solution (20 µL) was added to each well and incubated for 2 h. Absorbance readings were measured at 490 nm. Untreated cells were used as the 100% cell proliferation control and siRNA complexed with Lipofectamine 2000 (LF2000, Invitrogen, Basel, Switzerland) was used as a comparative control using a 20 pmol siRNA/µL LF2000. GAPDH Gene Expression Down-Regulation. The efficacy of the delivery strategy was studies using a GAPDH enzyme activity assay (Ambion, Austin, Texas). HeLa cells (7000 cells/ well) were plated in 96-well plates the day before transfection and transfected with 10 pmol siRNA at a charge ratio of (6. The transfections were done both in the presence and absence of FBS (0% or 10%) and in the presence and absence of chloroquine (Sigma, Lyon, France, 100 µM). Before transfection, the cell culture medium was replaced with transfection medium. For complexation, siRNA (30 pmol, 60 µL OptimMem) was mixed with the triblock copolymer or unmodified peptide (60 µL, TBS) and mixed by vortexing for 10 s. The complex was allowed to form undisturbed for 10 min. The extent of gene expression down-regulation was assessed 48 h posttransfection. LF2000 complexes were formed using a 1 µL of LF2000 to 20 pmol of siRNA. To determine if the transfection procedure affected GAPDH expression, a nontargeting sequence of siRNA was used (siNEG). GAPDH activity was only significantly reduced when LF2000 was used as the delivery vector for siNEG. The remaining GAPDH activity was measured as described by the manufacturer using the kinetic fluorescence increase method over 5 min and was calculated according to the following equation: % remaining expression ) ∆fluorescence,GAPDH/∆fluorescence,siNEG, where ∆fluorescence ) fluorescence1min fluorescence5min for either transfected (GAPDH), mock transfected (siNEG), or untransfected cells. Untransfected cells are cells that are exposed to serum or serum-free media for the 4 h of transfection and then replaced with cMEM. For LF2000, the control is a mock transfection using siNEG to determine the level of down-regulation that occurs due to the transfection procedure. Statistics. All statistical analyses were performed using the computer program Prism (GraphPad, San Diego, CA). Experiments were statistically analyzed using a one-way analysis of variance using the Tukey test, which compares all pairs of columns, using a 95% confidence interval. When only two groups were compared, the Student t test was used as indicated in the figure legends.

RESULTS Polymer Synthesis and Characterization. The ABC triblock copolymer PEG-PPS-peptide was synthesized using anionic polymerization of propylene sulfide upon the terminus of an activated PEG-thiol under an inert atmosphere, followed by peptide grafting using a disulfide exchange reaction (see Figure 1 and Material and Methods). The peptide coupling reaction was monitored using the release of 2-pyridine dithione at 342 nm, which demonstrated that the reaction reached a plateau after 2 h (see inset in Figure 1). After each step of the synthesis and purification, the intermediate product was characterized with


Figure 2. Relative fluorescence intensity of siRNA-EtBr complexes with PEG-PPSn-TAT (A) and PEG-PPSn-K9 (B) at different charge ratios. The florescence intensity decreases as more polymer is added until a plateau is reached, indicating that particles have formed. The dotted line indicates the charge ratio at which complex migration in agarose gel electrophoresis is stopped. 1H

NMR to confirm that the correct product was achieved and that there were not remaining impurities. At the concentrations required for 1H NMR analysis, however, the triblock copolymer formed aggregates, presumably micelles, both in an aqueous environment (D2O) or in deuterated chloroform, due to its strong amphiphilic nature. 1H NMR of the triblock thus contained broad peaks that were difficult to integrate. On the basis of the characterization performed, the polymers PEG45-PPS5-peptide and PEG45-PPS10-peptide were obtained, where the subscripted numbers indicate the degree of polymerization of the respective blocks. EtBr Exclusion Assay. The ability of the triblock polymer to form condensed structures with siRNA was studied using an EtBr dye exclusion assay (Figure 2). All polymers were able to exclude EtBr from the siRNA as more polymer was added (higher charge ratios), indicating that condensed particles were forming. Both the TAT and K9 peptides when used alone resulted in the highest condensation, reaching up to an 80% decrease in fluorescence intensity. Triblock copolymers based on TAT (Figure 2A) showed little dependence on the PPS degree of polymerization in their ability to form condensed particles with siRNA, with the maximum fluorescence decrease being reached at a charge ratio of 3, showing a 50% decrease. Triblock copolymers based on K9 (Figure 2B), on the other hand, showed more of an effect of PPS length, with PPS5 resulting in more condensed structures than PPS10. The maximum fluorescence decrease was also 50%. Size and ζ-Potential Characterization. In the absence of siRNA, the triblock polymers PEG-PPS5-TAT, PEG-PPS10TAT, PEG-PPS5-K9, and PEG-PPS10-K9 each formed


aggregates in solution at a concentration of 10 mg/mL, with sizes of 11.3 ( 0.4, 16.0 ( 0.3, 13.1 ( 0.1, and 16.0 ( 0.6 nm, respectively (Figure 3A,D). These measurements are consistent with a nanostructure morphology of spherical micelles. There was a statistical significance between the sizes of PEG-PPS5-peptide and PEG-PPS10-peptide aggregates for both TAT and K9 peptides (p < 0.001), indicating that the degree of polymerization of the PPS domain affects the overall size of the particles formed. Furthermore, for triblock copolymers with PPS degree of polymerization of 5, there was a statistical difference of the size of aggregates formed between TAT and K9 peptides (p < 0.01), indicating that the character of the peptide also affects the size of the resulting micellar aggregates. Size and surface charge of the aggregates formed between siRNA and PEG-PPS5-TAT, PEG-PPS10-TAT, PEGPPS5-K9, and PEG-PPS10-K9, as well as the TAT and K9 peptides not associated to the block copolymer, were measured using DLS and ζ-potential (Figure 3). Complex size was found to be dependent on the amount of polymer used (charge ratio) and the length of the hydrophobic domain (PPS degree of polymerization). For PEG-PPS5-TAT triblock polymers, the size of the particles formed decreased as the amount of polymer increased, with sizes ranging from 199.0 ( 79.3 to 160.2 ( 1.5 nm (Figure 3B, open triangles). However, for the triblock polymers where the PPS unit was longer (PPS10), the trend was reversed, such that increasing the amount of polymer from a charge ratio of (3 to (24 increased the size of the particles, with particle size ranging from 222.2 ( 0.8 to 601.2 ( 72.8 nm and 182.4 ( 6.5 to 312.3 ( 42.7 nm for PEG-PPS10TAT (Figure 3B, closed triangles) and PEG-PPS10-K9 (Figure 3E, closed triangles), respectively. ζ-Potential measurement for particles formed with TAT-containing polymers increased from ∼-20 mV to ∼0 mV (Figure 3C) and for K9-containing polymers from ∼0 mV and to ∼+10 mV (Figure 3F) as charge ratio was increased from (3 to (24. PEG-PPS-Peptide/siRNA Internalization Studies. HeLa cell internalization of PEG-PPS-peptide complexes with siRNA was assessed by FACS and confirmed with fluorescence microscopy. FACS data were analyzed by determining the number of cells in the positive gate and the total mean fluorescence intensity (MFI) of the cell population (Figures 4 and 5). Cellular internalization of PEG-PPS-peptide complexes was greatly affected by the presence of serum as well as the length of the PPS domain and the charge ratio of the complex. For the TAT-containing polymers, maximal internalization in the presence of serum was only 38.2 ( 0.1%, whereas in the absence of serum, 99.9 ( 0.1% of the cells had internalized the polymer complex with siRNA (Figure 4A compared to C). Polymers formed with the K9 peptide were also sensitive to the presence of serum (Figure 5A compared to C); however, polymer structure could somehow overcome this difference. For example, 93.3 ( 1.6% of the cells treated with PEG-PPS10-K9 at a charge ratio (6 were positive for internalization in the presence of serum, compared with 99.0 ( 0.3% in the absence of serum. For these polymers, charge ratio was important: the PEG-PPS10-K9 polymer at a charge ratio of (3 were internalized by only 63.4 ( 3.1% and 94.7 ( 0.6% of the cells in the presence and absence of serum, respectively, compared to the above-mentioned 93.3% and 99.00% for (6 (p < 0.001). In contrast, internalization by TATcontaining polymers was not affected by the charge ratio used. The mean fluorescence intensity (MFI) of the cells was also affected by the presence of serum, the length of the PPS domain, and the charge ratio of the complexes (Figures 4 and 5, panels B,D). In general, longer PPS domains and higher charge ratio achieved more particle internalization (higher MFI) than shorter


Segura and Hubbell

Figure 3. Mean diameter of micelles forming in 1% w/v solutions of PEG-PPSn-TAT (A) and PEG-PPSn-K9 (D) in the absence of siRNA. Size and ζ-potential measurements as a function of charge ratio for TAT peptide-based polymers (B,C) and K9-based polymers (E,F). The siRNA complexes with copolymers are much larger than the copolymer micelles that form without siRNA. Statistical analyses were done using the Student t test. The symbol *** indicates statistical significance at a level of p < 0.001.

PPS domains or lower charge ratio. Interestingly, the MFI was also affected by the type of peptide used, with the polymers formed with the K9 peptide (Figure 5B,D) being less adversely affected by serum than the polymers formed with the TAT peptide (Figure 4B,D). In the presence of serum, the maximum MFI observed with PEG-PPS10-K9 was 12.4 ( 0.1 (at a charge ratio of (6), compared to 22.4 ( 1.6 in the absence of serum (Figure 5, B vs D). The effect for the polymers formed with the TAT peptide was stronger: in the presence of serum, the maximum MFI observed with PEG-PPS10-K9 was 4.7 ( 0.01 (at a charge ratio of (6), compared to 263.3 ( 63.8 in the absence of serum (Figure 4, B vs D). Fluorescence microscopy of complexes formed with Cy3-labeled siRNA corroborate the data found in FACS analysis. Figures 4E and 5E show fluorescence images for internalization of PEG-PPS5-TAT and PEG-PPS5-K9 complexes, respectively, which show substantially more internalization than those complexes formed with TAT or K9 (Figures 4F and 5F). Comparisons with LF2000 were also made, and in all cases evaluated at the same siRNA dose (as in Figures 4 and 5),

complex uptake was less with the polymer vectors than with the cationic lipid vector. Toxicity of PEG-PPS-Peptide/siRNA Complexes. A cellular proliferation assay was used as an indirect measure of the toxicity caused by exposure to the triblock copolymer/siRNA complexes (Figure 6). None of the concentrations used for the different triblock copolymer/siRNA transfections resulted in a statistically significant reduction of cell proliferation, indicating that no toxicity occurred as a result of treatment with the complexes. In contrast, LF2000/siRNA complexes resulted in significant toxicity at concentrations of more than 10 pmol/ well. Gene Expression Down-Regulation. The ability of PEGPPS-peptide/siRNA complexes to mediate gene expression down-regulation of GAPDH enzyme in vitro in the presence and absence of serum was investigated at 20 pmol/well siRNA (Figure 7). In the presence of serum, significant down-regulation was observed with PEG-PPS10-K9 (p < 0.01), but not with any other triblock polymer. In the absence of serum, however, significant down-regulation was observed for all triblock



Figure 4. HeLa cell internalization of siRNA-Cy5 complexes with TAT peptide-based polymers over a 4 h period in the presence of 10% serum (A,B) and 0% serum (C,D). The bars in A and C represent the percent of cells in the positive gate, and B and D represent the mean fluorescent intensity of the cells. Open bars represent cells treated at a charge ratio of (3, and filled bars at (6. Fluorescence micrographs of complexes with PEG-PPS5-TAT (E) and TAT peptide (F) are shown. Statistical analyses were done using multiple comparisons and the Tukey analysis of variance. The symbols *, **, and *** indicate statistical significance at levels of p < 0.05, p < 0.01, and p < 0.001, respectively, for the comparison indicated. Symbols placed directly on top of the bar indicate a significant difference between the TAT peptide and the all-TAT peptide-based polymers.

polymers, with PEG-PPS5-TAT and PEG-PPS10-TAT achieving 53 ( 0.01 and 56 ( 0.1 GAPDH gene expression downregulation, respectively. PEG-PPS5-TAT and PEG-PPS10TAT were statistically more able to mediate gene expression down-regulation than the TAT peptide alone (p < 0.01), achieving more than 50% down-regulation with the triblock copolymers compared to only 30% with the TAT peptide alone. Interestingly, the K9 peptide-containing polymers in general were not as able to mediate gene expression down-regulation as the TAT polymers, achieving at most a 34 ( 0.1% GAPDH gene expression down-regulation for PEG-PPS5-K9 at a dose of 20 pmol/well. To determine the effect of siRNA complex concentration on transfection efficiency, transfections were done using 10, 20, or 100 pmol/well of siRNA at a charge ratio of (6 in the absence of serum (Figure 8). It was found that an increase in complex concentration from 10 to 20 pmol/affected GAPDH down-regulation of all complexes tested, including those with

LF2000 (p < 0.05). However, an increase from 20 to 100 pmol/ well only resulted in enhanced GAPDH down-regulation for the K9 peptide-containing polymers (at least p < 0.01). Results for transfection with the PEG-PPS5-K9 polymer were particularly satisfactory, the level of gene expression downregulation reaching 90.5%, statistically higher than that achieved with LF2000 at its highest acceptable concentration, 20 pmol/ well (p < 0.01). Transfection at 100 pmol/well with LF2000 was not possible, due to the toxicity of the vector.

DISCUSSION Efficient and safe siRNA-mediated gene silencing could benefit both basic science and therapeutic applications. Working toward these ends, we have developed a novel self-assembling triblock copolymer for siRNA delivery based on a triblock copolymer architecture. We sought to use short cationic peptides to reversibly bind siRNA, here using the HIV-1 TAT peptide


Segura and Hubbell

Figure 5. HeLa cell internalization of siRNA-Cy5 complexes with K9 peptide-based polymers over a 4 h period in the presence of 10% serum (A,B) and 0% serum (C,D). The bars in A and C represent the percent of cells in the positive gate, and B and D represent the mean fluorescent intensity of the cells. Open bars represent cells treated at a charge ratio of (3, and filled bars at (6. Fluorescence micrographs of complexes with PEG-PPS5-K9 (E) and K9 peptide (F) are shown. Statistical analyses were done using multiple comparisons and the Tukey analysis of variance. The symbol *** indicates statistical significance at a level of p < 0.001 for the comparison indicated. Symbols placed directly on top of the bar indicate significant difference between the K9 peptide and the all-K9 peptide-based polymers.

sequence and a synthetic lysine 9-mer, K9. In order to enhance the formation of nanoparticles from these short peptides, we conjugated them to a hydrophobic polymer of controllable length, based on PPS with a degree of polymerization of either 5 or 10. Since a dimer of PPS with the peptide might have had limited solubility, the third block, a hydrophilic polymer also of controllable length, namely, a PEG of degree of polymerization of 45, was utilized. Our group has previously investigated PEG-PPS block copolymers (20, 22) and has determined that this architecture is within a range that will form micelles, as was also demonstrated by measurements of particle size using DLS in this study as well. This concept for nanoparticle design, forming nanoparticles based on assembly of the hydrophobic PPS domains controlled by the attached hydrophilic PEG domains, and binding siRNA based on electrostatic interactions with the cationic peptides TAT and K9 was explored for siRNA delivery. The TAT peptide was investigated as a means to enhance endosomal escape and K9 as a control peptide to allow conclusions regarding endosomal escape enhancements. As

mentioned in the Results section, however, the triblock copolymers made with the TAT peptide did not seem to have any added benefit as to endosomal escape compared to K9-containing polymers; both polymers achieved enhanced gene transfer when choloquine was added to the media. The triblock copolymers, PEG-PPS5-TAT, PEG-PPS10TAT, PEG-PPS5-K9, and PEG-PPS10-K9 were able to selfassemble with siRNA into nanoparticles and mediate siRNA internalization with an efficiency (fraction of cells taking up the siRNA/copolymer complex) of up to 93% in the presence of serum and 100% in the absence of serum. Furthermore, the relative number of complexes internalized (mean fluorescence intensity) was 45% of that of a reference transfection agent, cationic liposomes (LF2000). Given that the copolymers demonstrated no measurable cytotoxicity, whereas the LF2000 demonstrated substantial cytotoxicity, much higher doses of siRNA/copolymer complexes could be applied, which resulted in up to 90.5 ( 0.02% GAPDH expression down-regulation


Figure 6. MTS toxicity assay (using cell proliferation) of (A) TAT peptide-based polymers and (B) K9 peptide-based polymers, using lipofectamine 2000 (LF2000) as a positive control and untreated cells as a negative control.

when PEG-PPS5-K9 was used as the delivery vehicle in the absence of serum. For some formulations, the hydrophobic character of the nanoparticles appeared to affect the internalization of PEGPPS-peptide/siRNA complexes. The length of the hydrophobic domain in PEG-PPS-K9 triblocks affected the amount of PEG-PPS-K9/siRNA nanoparticle internalization, resulting in complexes with longer PPS domains achieving higher rates of internalization. This finding is not likely to be a result of the charge of the particles or their size, given that they were the same for both PEG-PPS5-K9 and PEG-PPS10-K9, suggesting that the hydrophobic domain directly affects internalization (Figure 5A). Limited hydrophobicity has been used in the design of other vectors as well: our results are consistent with findings exploring a dextran-spermine (D-SPM) delivery strategy for DNA, which found that the addition of hydrophobic domains, N-oleyl moieties (ODS), to D-SPM (D-SPM-ODS) enhanced transfection in the presence of serum (23). Taken together, these results suggest that optimizing the hydrophobic nature of the complexes may be another strategy to improve transfection efficiency. An influence of PPS block length was less apparent with the TAT peptide-based copolymer vectors. With PEG-PPS5-K9 and PEG-PPS10-K9 formulations, the charge ratio of the PEG-PPS-peptide/siRNA nanoparticles also affected their internalization, with higher charge ratios ((6) resulting in statistically higher internalization than at lower charge ratios ((3) (p < 0.001 for all comparisons). Differing charge ratios may have resulted in differing levels of condensation of the siRNA within the nanoparticles; increasing copolymer


Figure 7. siRNA GAPDH down-regulation assay. HeLa cells were transfected with siRNAs at 20 pmol/well encoding for GAPDH enzyme using complexes with TAT peptide-based polymers (A) or K9 peptidebased polymers (B) in the presence of 10% FBS (dark bars) or 0% FBS (medium bars). LF2000 was used as a comparison (white bar). Statistical analyses were done using multiple comparisons and the Tukey analysis of variance. The symbols *, **, and *** indicate statistical significance at levels of p < 0.05, p < 0.01, and p < 0.001, respectively, for the comparison indicated. Symbols placed directly on top of the bar indicate a significant difference between the peptide and the allpeptide-based polymers for the same charge ratio. The symbol † indicates a statistical significance at a level of p < 0.001 for all comparisons with LF2000.

content in the complexes resulted in more condensed particles (Figure 2), suggesting that, if the particles are more compact, they can be internalized at a higher rate. More compact DNA/ polyethyleneimine and DNA/polylysine complexes have been found to internalize at higher rates and achieve higher transfection efficiencies (24, 25). The nature of the cationic peptide used in the copolymer also affected internalization of siRNA and gene expression downregulation. Internalization by PEG-PPS5-K9/siRNA complexes at charge ratios of (3 and (6 was statistically higher than internalization mediated by PEG-PPS5-TAT, with p < 0.05 and p < 0.001, respectively. Likewise, internalization of PEGPPS10-K9/siRNA complexes resulted in statistically more internalization than with PEG-PPS10-TAT, with p < 0.001 for both charge ratios tested. Moreover, PEG-PPS5-TAT and PEG-PPS10-TAT were more affected by serum and less affected by charge ratio than PEG-PPS5-K9 and PEG-PPS10K9. Differences in internalization could be hypothesized to be due to differences in the biological activity of the peptides or differences in surface charge of the complexes. HIV-1 TAT has been reported to be able to translocate across cell membranes independently of receptors and temperature (26). More recently,


Segura and Hubbell

similar levels of gene expression down-regulation as with LF2000. The polymer PEG45-PPS5-K9 was particularly interesting in this regard, achieving 90% gene expression downregulation. Although we were able to achieve significant levels of down-regulation with higher doses of siRNA, it is more desirable to achieve significant levels using lower doses of siRNA. This could be accomplished by more efficient intracellular trafficking of the siRNAs such as by adding fusogenic peptides.

ACKNOWLEDGMENT We would like to acknowledge the Simona Cerritelli for training on the PEG-PPS polymer synthesis and general discussions, and Andre Van der Vlies, Yun Suk Jo, and Conlin O’Neil for assistance with the NMR analysis of the polymer and helpful comments. This project was supported in part by grant F32GM72428 (TS).

LITERATURE CITED

Figure 8. siRNA GAPDH down-regulation assay. HeLa cells were transfected with siRNAs encoding for GAPDH enzyme using increasing concentrations of siRNA (10, 20, and 100 pmol/well) using complexes with TAT peptide-based polymers (A) and K9 peptide-based polymers (B). Measurement of GAPDH activity at 100 pmol/well for LF2000 was not possible due to extreme toxicity. Statistical analyses were done using multiple comparisons and the Tukey analysis of variance. The symbols *, **, and *** indicate statistical significance at levels of p < 0.05, p < 0.01, and p < 0.001, respectively, for the comparison indicated. Symbols placed directly on top of the bar indicate a significant difference between the peptide and the all-peptide-based polymers for the same charge ratio. The symbol † indicates a statistical significance at a level of at least p < 0.05 for PEG-PPS5-K9 with all other conditions.

however, these findings have been disputed, and the mechanism of TAT-mediated internalization has been associated with adsorptive endocytosis (27, 28) and fluid-phase macropinocytosis (29, 30). No specific biological activity has been associated with short polylysine oligomers. Complexes with the K9 peptide are positively charged, whereas complexes with TAT are not. Positively charged complexes have been previously demonstrated to affect internalization of cationic polymer/DNA complexes, with positively charged complexes achieving higher internalization rates and transgene expression (31, 32). In comparison to the TAT or K9 peptides alone, the ABC triblock copolymers were able to achieve more siRNA particle internalization (Figures 4 and 5) and higher gene expression down-regulation levels in the presence and absence of serum (Figures 7 and 8), indicating that the architecture of the triblock positively affects siRNA uptake and trafficking. In terms of comparison with reference materials such as cationic liposomes, the triblock copolymers demonstrated enhanced biocompatibility compared to the commercially available delivery vehicle LF2000. This, in turn, allowed use of higher siRNA doses with the triblock PEG-PPS-peptide copolymers, which achieved

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Synthesis and in Vitro Characterization of an ABC Triblock Copolymer

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