Hydrophilic Cationic Star Homopolymers Based on a Novel Diethanol

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Hydrophilic Cationic Star Homopolymers Based on a Novel DiethanolN-Methylamine Dimethacrylate Cross-Linker for siRNA Transfection: Synthesis, Characterization, and Evaluation Kyriaki S. Pafiti,† Nikolaos P. Mastroyiannopoulos,‡ Leonidas A. Phylactou,‡ and Costas S. Patrickios*,† † ‡

Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus Department of Molecular Genetics, Function and Therapy, The Cyprus Institute of Neurology and Genetics, P.O. Box 23462, 1683 Nicosia, Cyprus ABSTRACT:

Four cationic hydrophilic star homopolymers based on the novel hydrophilic, positively ionizable cross-linker bis(methacryloyloxyethyl)methylamine (BMEMA) were synthesized using sequential group transfer polymerization (GTP) and were, subsequently, evaluated for their ability to deliver siRNA to mouse myoblast cells. The nominal degrees of polymerization (DP) of the arms were varied from 10 to 50. For the polymerizations, 2-(dimethylamino)ethyl methacrylate (DMAEMA) was employed as the hydrophilic, positively ionizable monomer. For comparison, four linear DMAEMA homopolymers were also synthesized, whose nominal DPs were the same as those of the arms of the stars. The numbers of arms of the star homopolymers were determined using gel permeation chromatography with static light scattering detection, and found to range from 7 to 19, whereas the hydrodynamic diameters of the star homopolymers in aqueous solution were measured using dynamic light scattering and found to increase with the arm DP from 13 to 26 nm. The presence of the hydrophilic BMEMA cross-linker enabled the solubility of all star homopolymers in pure water. The cloud points of the star homopolymers in aqueous solution increased with the arm DP from 23 to 29 °C, while the cloud points of the linear homopolymers were found to decrease with their DP, from 42 to 32 °C. The effective pK values of the DMAEMA units were in the range of 6.9 to 7.3 for the star homopolymers, whereas they ranged between 7.3 and 7.4 for the linear homopolymers. Subsequently, all star and linear homopolymers were evaluated for their ability to deliver siRNA to the C2C12 mouse myoblast cell line, expressing the reporter enhanced green fluorescent protein (EGFP). All star homopolymers and the largest linear homopolymer presented significant EGFP suppression, whereas the smaller linear homopolymers were much less efficient. For all star homopolymers and the largest linear homopolymer both the EGFP suppression and the cell toxicity increased with polymer loading. The siRNAspecific EGFP suppression, calculated by subtracting the effect of cell toxicity on EGFP suppression, slightly increased with star polymer loading for the two smaller stars, whereas it presented a shallow maximum and a decrease for the other two stars. Moreover, the siRNA-specific EGFP suppression also increased slightly with the DP of the arms of the DMAEMA star homopolymers. Overall, the EGFP suppression efficiencies with the present star homopolymers were at levels comparable to that of the commercially available transfection reagent Lipofectamine.

’ INTRODUCTION Small interfering RNA (siRNA) is a new promising tool for silencing target genes,1,2 and holds great promise for therapeutic applications in the area of cancer and other diseases such as muscular dystrophies, influenza, arthritis, osteoporosis, Alzheimer’s disease, and asthma.3-7 The greatest limitation for using r 2011 American Chemical Society

siRNA in gene therapy is the delivery of siRNA to the target cells. Direct introduction of naked siRNA in to the cells is limited by its Received: November 23, 2010 Revised: February 1, 2011 Published: March 17, 2011 1468

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Biomacromolecules low permeability through the cell membranes, necessitating large siRNA loadings and being applicable only with certain tissues. Nowadays, effective transfer of siRNA in to the cells is usually accomplished using deactivated viruses.8-10 However, the high cost and potential pathogenicity and immunogenicity of viral carriers has prompted the use of nonviral vehicles based on synthetic cationic polymers.11 Many examples of synthetic cationic polymers for use in in vitro and in vivo siRNA transfection can be found in the literature. The most prominent example is polyethylenimine (PEI),12-23 which has been used either in the linear or in the hyperbranched form. Furthermore, PEI has been used in different compositions, including the pure homopolymer,12 the modified homopolymer,13,14 and its copolymers with poly(ethylene glycol) (PEG),15-17 with some receptors, such as the folate,16,17 or with hyaluronic acid.18 Other examples of cationic polymers used are poly(lysine),24,25 poly(histidine),24 chitosan,26-29 and amine-based dendrimers.30-32 Moreover, block copolymers33-35 comprising at least one positively charged block based on monomer repeating units with tertiary (such as poly(2-(dimethylamino)ethyl methacrylate), polyDMAEMA) or quaternized nitrogen have also been used as siRNA carriers. Biodegradable cationic polymers have been used as well.36-38 In particular, Liu et al.36 used a novel synthetic polymeric carrier material composed of a cationic oligomer of PEI, a hydrophilic PEG segment, and a biodegradable lipid-based cross-linker. Lee et al.37 used linear PEI derivatives which were interconnected via biodegradable disulfide bonds, while Zhu et al.38 used cationic micelles of the ABA triblock copolymer poly(DMAEMAb-(ε-caprolactone)-b-DMAEMA) (PDMAEMA-PCL-PDMAEMA), with PCL being the biodegradable block. Finally, cationic, selfassembling nanoparticles based on oligocyclodextrins, carrying transferrin receptors, and sterically stabilized by PEG, have been developed by Davis to effect the first targeted delivery of siRNA in humans.39 To the best of our knowledge, from all published literature reports on synthetic polymer siRNA vehicles, only in one case40 was the delivery of siRNA effected using a cationic star polymer. In particular, that was a four-armed, double-hydrophilic, cationicnonionic, star block copolymer of 3-(N,N-dimethylamino)propyl acrylamide (cationic hydrophilic), and N,N-dimethylacrylamide (nonionic hydrophilic) synthesized by photoliving radical polymerization using 1,2,4,5-tetrakis(N,N-diethyldithiocarbamylmethyl)benzene as iniferter. For the efficient delivery of siRNA to the target cells, synthetic cationic polymer vehicles should present some specific structural characteristics, most notably a moderate density of positive charge and water solubility.41 The former defines both the binding efficiency of the polymers with siRNA and the ease of release of the siRNA from the complex in the endosome, while the latter is necessary for keeping an appropriate particle size of the complex. To satisfy these requirements, we synthesized hydrophilic, cationic star-shaped polymers consisting of arms comprising tertiary amine monomer repeating units of DMAEMA (to obtain the positive charge) that were interconnected with the novel hydrophilic amine-containing cross-linker bis(methacryloyloxyethyl)methylamine (BMEMA), which further increases the polymer solubility and introduces extra positive charge. The synthesis of the star homopolymers was accomplished using the “living” polymerization technique group transfer polymerization (GTP),42-46 with sequential monomer and cross-linker additions. The star homopolymers were characterized in solution (organic and aqueous media) and were,

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subsequently, evaluated in terms of their ability to transfer siRNA to mouse myoblast (C2C12) cells expressing the enhanced green fluorescent protein (EGFP) as the reporter gene. For comparison, linear DMAEMA homopolymers with the same degrees of polymerization (DPs) as the arm DPs of the stars homopolymers were also synthesized, characterized, and evaluated for their siRNA transfection efficiency.

’ MATERIALS AND METHODS Materials. DMAEMA (99%), 1-methoxy-1-trimethylsiloxy-2-methyl propene (MTS, 95%), tetrabutylammonium hydroxide (40% in water), benzoic acid (g99.5%), N-methyldiethanolamine (g99%), methacryloyl chloride (90%), triethylamine (g99%), basic alumina, calcium hydride (CaH2, 90-95%), 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH, 95%), tris(hydroxymethyl)aminomethane (Tris, 99.9%), Tris-HCl (for molecular biology), and potassium metal were all purchased from Aldrich, Germany. Sodium metal was purchased from Fluka, Germany. Deuterated chloroform (CDCl3), NaCl, and β-mercaptoethanol (99%) were purchased from Merck, Germany. Tetrahydrofuran (THF, 99.8%, both HPLC and reagent grade) was purchased from Scharlau, Spain. EGFP-siRNA (for the silencing of the EGFP gene) was purchased from Ambion, U.K., Lipofectamine RNAiMAX (for siRNA transfection) and Lipofectamine2000 (for DNA transfection) were purchased from Invitrogen, U.K. C2C12 cell lines were purchased from ECACC, Salisbury, Wiltshire, U.K. Dulbecco’s phosphate buffered saline (D-PBS) without calcium chloride and magnesium chloride, Dulbecco’s modified Eagle’s medium (DMEM), OPTIMEM (reduced-serum medium (1), liquid), fetal bovine serum (FBS), glutamine (glutaMAX), penicillin-streptomycin, trypsin (0.25% (1) with EDTA 4Na), geneticin-selective antibiotic, and trypan blue were purchased from Gibco, U.K., and were used for the cell culture experiments. Finally, glycerol (99.5%) was purchased from BDH England, tween (ultrapure) was purchased from USB Corporation U.S.A., and complete EDTA-free protease inhibitor was purchased from Roche, Germany. Synthesis of Bis(methacryloyloxyethyl)methylamine (BMEMA). The synthesis of the novel hydrophilic cross-linker bis(methacryloyloxyethyl)methylamine (BMEMA) was accomplished by the esterification reaction of N-methyldiethanolamine with methacryloyl chloride in the presence of triethylamine in freshly distilled, absolute THF at 0 °C (Figure 1). In particular, 9.5 mL (9.8 g, 0.0826 mol) of Nmethyldiethanolamine was diluted in 140 mL of THF and the mixture was cooled down to 0 °C. Subsequently, 20 mL (21.6 g, 0.206 mol) of methacryloyl chloride was added dropwise and the mixture was stirred for 1 h. Afterward, the reaction mixture was filtered to remove the salt of triethylamine hydrochloride, which was formed and, subsequently, passed through a basic alumina column to remove the excess of methacryloyl chloride/methacrylic acid, while the solvent was evaporated off using a rotary evaporator. For further purification, the crosslinker was distilled over CaH2 under dynamic vacuum at 108 °C. The chemical structure of the BMEMA cross-linker was confirmed using 1H and 13C NMR spectroscopies. Methods. In GTP, all reagents and the solvent should be free of protonic impurities. Thus, the DMAEMA monomer and the BMEMA cross-linker were passed through basic alumina columns, which retained all acidic impurities and were, subsequently, stirred over CaH2 (in the presence of added free radical inhibitor DPPH to prevent undesired thermal polymerization) to neutralize the last traces of moisture and were, finally, freshly distilled just prior to use. The polymerization catalyst, tetrabutylammonium bibenzoate (TBABB), was synthesized by the reaction of tetrabutylammonium hydroxide and benzoic acid, as described by Dicker et al.,44 while the polymerization solvent, THF, was dried by being refluxed for 3 days over a potassium/sodium amalgam. For the polymerizations, all glassware was dried overnight at 120 °C and 1469

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Biomacromolecules assembled hot under dynamic vacuum prior to use. The chemical structures and the names of the initiator, the catalyst, the monomer, and the cross-linker used for the synthesis of the star homopolymers are shown in Figure 2. Synthesis of the Star and the Linear Homopolymers. In the present work, four star homopolymers and four linear homopolymers were synthesized at different monomer to initiator molar ratios using the “living” polymerization technique GTP. A typical synthetic procedure for the synthesis of the “arm-first” star homopolymer DMAEMA10-star is detailed below: To a 100 mL round-bottom flask, which was preloaded with a small amount of TBABB and 16.8 mL of freshly distilled THF, 0.30 mL of MTS initiator (0.26 g, 1.50 mmol) was added. The roundbottom flask was kept under an inert nitrogen atmosphere, while the polymerization mixture was under stirring during the whole procedure. Then, 2.50 mL of DMAEMA monomer (2.33 g, 14.8 mmol) was added dropwise, producing a polymerization exotherm (21-32 °C), which abated within 5 min, and subsequently, 1.4 mL of BMEMA cross-linker (1.53 g, 6.00 mmol) was added rapidly, which produced an exotherm (28-30 °C). After the additions of monomer and cross-linker, samples were extracted from the polymerization mixture and were analyzed using gel permeation chromatography (GPC) and 1H NMR spectroscopy. Finally, the thus-produced star homopolymer was recovered by precipitation in n-hexane and was dried under vacuum at room temperature for 72 h. The synthesis of the linear homopolymers was accomplished following exactly the same procedure as for the stars, as described above, but without the addition of cross-linker.

Characterization of the Star and the Linear Homopolymers in Organic Solvents. Solutions of all the star homopolymers were characterized using GPC with refractive index (RI) detection (GPC-RI; 1% w/w in THF), GPC with dual RI and static light scattering (SLS) detection (GPC-SLS; 2% w/w in THF), and 1H NMR spectroscopy (3% w/w in CDCl3) to determine their size and confirm their constitution. The linear homopolymers were characterized using only GPC-RI and 1H NMR spectroscopy. Gel Permeation Chromatography (GPC) with RI Detection. All the star homopolymers and their precursors as well as the linear homopolymers were characterized in terms of their molecular weights (MWs) and polydispersity indices (PDIs) using GPC-RI. GPC-RI was performed on a Polymer Laboratories system equipped with a Waters 515 isocratic pump, an ERC-7515A Polymer Laboratories RI detector and a PL Mixed “D” column. The eluent was THF, pumped at 1 mL min-1.

Figure 1. Synthetic route followed for the preparation of the BMEMA cross-linker.

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The MW calibration was performed using linear poly(methyl methacrylate) (PMMA) standards supplied by Polymer Laboratories, which provided rather accurate MWs for the linear homopolymers and the linear precursors to the star homopolymers, but only qualitative estimations for the MWs of the star homopolymers. GPC with Dual RI and Static Light Scattering (SLS) Detection. The absolute weight-average MWs, Mw, of the star homopolymers were determined using GPC with dual, SLS and RI, detection. A BIMwA Brookhaven SLS spectrophotometer, equipped with a 30 mW red diode laser operating at 673 nm, was used for the scattering measurements. Scattered intensities were measured at seven different angles: 35, 50, 75, 90, 105, 130, and 145°. The BIMwA spectrophotometer was connected with a PL-Mixed “D” column from Polymer Laboratories. The eluent was THF, delivered at 1 mL min-1 using an isocratic LC 1120 HPLC pump also from Polymer Laboratories. The system also included a PL-RI 800 Polymer Laboratories RI detector connected right after the SLS detector. All the samples were dissolved in THF and were filtered through 0.45 μm PTFE syringe filters. The RI increment (dn/dc) of the star homopolymers was determined using an ABBE refractometer. Proton Nuclear Magnetic Resonance ( 1H NMR) Spectroscopy. The 1 H NMR spectra of all the star homopolymers and their linear precursors as well as those of the linear homopolymers in deuterated chloroform (CDCl3) were recorded using a 300 MHz Avance Bruker NMR spectrometer equipped with an Ultrashield magnet. The DPs of the DMAEMA units were calculated from the NMR spectra as the ratio of the normalized area of the peak due to the two oxymethylene protons of polyDMAEMA at 4.1 ppm divided by the normalized area of the peak due to the three methoxy protons of the MTS initiator at 3.6 ppm.

Characterization of the Star and the Linear Homopolymers in Aqueous Media. Aqueous solutions of all the star and all the linear homopolymers (1% w/w in distilled water) were characterized in terms of their cloud points and effective pK values using turbidimetry and hydrogen ion titration, respectively, whereas the star homopolymers were also characterized in terms of their hydrodynamic sizes in water using DLS. Dynamic Light Scattering (DLS). A 90 Plus Brookhaven DLS spectrophotometer equipped with a BI-9000 correlator and a 30 mW red diode laser operating at 673 nm was used for the DLS measurements at an angle of 90° and at room temperature to determine the hydrodynamic diameters of the star homopolymers in aqueous solution (1% w/w). Five 2 min runs were performed for each sample, and the data were averaged. The data were processed using multimodal size distribution (MSD) analysis based on non-negatively constrained least-squares (NNCLS). Prior to the DLS experiments, the solutions of the star homopolymers were filtered through 0.45 μm PTFE syringe filters and left still for at least 1 h so that any air bubbles could escape. Turbidimetry. A single beam Lambda 10 Perkin-Elmer UV-vis spectrometer was used for the turbidity measurements. The polymer aqueous solution (1% w/w) was placed in a 10 mm path-length quartz cuvette containing a small magnetic stirring bar, set in motion with the aid of a miniature magnetic stirrer. A small temperature probe was immersed in the upper part of the solution which was heated from 15 to

Figure 2. Chemical structures and names of the main reagents used for the star homopolymer synthesis. 1470


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Figure 3. 1H and 13C NMR spectra of the novel BMEMA cross-linker. 65 °C. The optical density at 500 nm and the temperature were simultaneously monitored using software TempLab (version 1.56) along with UVWinLab (version 2.7). The cloud point was taken as the temperature where the first large increase in optical density occurred. Hydrogen Ion Titration. Aqueous polymer solutions (1% w/w) were titrated between pH 2 and 12 using a standard 0.5 M NaOH solution under continuous stirring. The pH was measured using a Corning PS30 portable pH meter. The solution was visually observed at all times during titration, and the pH range where any increased turbidity occurred was noted. The effective pK values were taken as the pH at 50% ionization.

Evaluation of the Star and the Linear Homopolymers as Transfection Reagents. After their characterization in organic and aqueous media, star and linear homopolymers were complexed with EGFPsiRNA and evaluated for their ability to deliver this siRNA to C2C12 mouse myoblast cells which express stably the EGFP gene. Preparation of the Polymer/siRNA Complex. A total of 30 mg of each star or linear homopolymer was dissolved in 10 mL of pure, deionized, and sterile water, thus, resulting in a 3 mg mL-1 polymer solution. The polymer/siRNA complex was prepared by mixing 1 μL of 50 μM siRNA (0.665 μg μL-1) solution in OPTIMEM with the appropriate volume of polymer solution in OPTIMEM, followed by incubation at room temperature for 25 min in the dark. The amount of polymer used was varied between 15 and 100 μg, corresponding to volumes of polymer solution from 5 to 33 μL and N/P ratios between 45 and 303. Each volume of polymer solution was completed to 100 μL by adding OPTIMEM. As a control, a LipofectamineRNAiMAX/siRNA complex containing 5 μL of LipofectamineRNAiMAX (5% v/v in OPTIMEM) and 1 μL of siRNA solution (0.02 μg μL-1 in OPTIMEM) was also prepared as described above. Preparation of Myoblast Cells Expressing the EGFP Genes. C2C12 mouse myoblast cells were plated onto 6-well plates containing 2 mL of DMEM medium (DMEM supplemented with 10% v/v fetal bovine serum (FBS), 2% v/v glutamine, and 1% v/v penicillin-streptomycin). Cells were grown to 90% confluence before being transfected with 10 μg NIT EGFP plasmid DNA (40 mg L-1 in OPTIMEM) using 10 μL of Lipofectamine2000 (40 mg L-1 in OPTIMEM). At 6 h after transfection, cells were rinsed with PBS before replenishing with fresh DMEM medium. Cells were detached by adding 100 μL trypsin EDTA to each well until the cells were detached (ca. 2 min after the addition of trypsin) from the monolayer. A total of 2 mL of DMEM medium was then added to each well to deactivate the trypsin activity and resuspend the cells into the medium. Volumes of 20, 30, and 50 μL were taken from this suspension and placed onto 100 mm plates containing fresh DMEM medium. Untransfected cells were also plated onto 100 mm plates. After 24 h, the medium was changed to DMEM medium containing geneticin (G418) at a final concentration of 400 μg mL-1. Cells were then allowed to grow for 6-10 days, and colonies expressing EGFP were selected under a fluorescent microscope. Cloning discs (3 mm) were soaked in

trypsin, placed on top of the colonies, and then removed carefully to separate wells containing DMEM medium using tweezers, washed with ethanol, and sterilized over a flame between colony picking. siRNA Transfection. EGFP-expressing mouse myoblast (C2C12-E) cells were placed in 12-well plates at a density 100000 cells/well and cultured under standard conditions (37 °C, 5% CO2) in DMEM medium. For the transfection experiments, cells were seeded in 12-well plates in DMEM for 24 h to reach 40-60% confluency. On the day of the transfection, cells were rinsed with PBS and 0.8 mL of DMEM without penicillin, and streptomycin was added. Cells were transfected with the siRNA/polymer complex (200 μL) and incubated at 37 °C for 6 h. Afterward, the medium was replaced with 2 mL of fresh DMEM, and the cells were further cultured for 24 h. Cells were then rinsed with PBS to remove dead cells, and the living cells were detached from the plate using a trypsin solution. Subsequently, the extraction of the proteins from cells was accomplished using 200 μL of Lysis Buffer (10 mM TrisHCl, pH 7.6, 150 mM NaCl, 10% v/v glycerol, 1% v/v Tween, 10 mM βmercaptoethanol, 0.04% v/v protease inhibitor). The amount of EGFP was measured using a PBS-380 mini Fluorometer detecting at 509 nm (extinction coefficient, ε = 61000 cm-1 M-1), with an excitation wavelength of 488 nm. Determination of Cell Viability. The effect of the presence of polymer on the viability of the cells was investigated. To this end, 10 μL of the cells (∼900 cells μL-1) were mixed gently with 10 μL of Trypan Blue. Then, 10 μL of the resulting mixture was put in a Bio-Rad iMark Microplate Reader and the number of viable cells was counted. The toxicity of the polymer to the cells was calculated as one minus the ratio of the number of viable cells in the transfected well (Tr.) divided by the number of viable cells in the untransfected well (Untr.): ! number of viable cellsTr: 100 %toxicity ¼ 1 number of viable cellsUntr: From the experimental data, the EGFP suppression and the siRNAspecific EGFP suppression were also determined. The EGFP suppression was calculated as one minus the ratio of the EGFP (EP) concentration (in ng mL-1) from the transfected cells (EPTr.) divided by the EGFP concentration from the untransfected cells (EPUntr.): ! EPTr: %EGFP suppression ¼ 1 - Untr: 100 EP Finally, the siRNA-specific EGFP suppression was calculated as the EGFP suppression multiplied by the cell viability, with the latter being equal to one minus the cell toxicity.

%siRNA-specific EGFP suppression ¼ ½EGFP suppression viability 100 1471


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Figure 4. Synthetic procedure followed for the preparation of the star homo polymer DMAEMA20-star.

Figure 6. GPC-RI traces of the linear homopolymers. Figure 5. GPC-RI traces of the star homopolymers and their linear precursors.

’ RESULTS Bis(methacryloyloxyethyl)methylamine (BMEMA) CrossLinker. The novel hydrophilic cross-linker was successfully

synthesized through an esterification reaction between Nmethyldiethanolamine and methacryloyl chloride. The overall yield of the reaction, after purification, was 59%. Figure 3 shows the 1H and the 13C NMR spectra of the purified BMEMA crosslinker. Star and Linear Homopolymers. In the present work, four star homopolymers with nominal arm DPs of 10, 20, 35, and 50 and four linear homopolymers with the same nominal DPs were synthesized using GTP. For all the polymerizations, TBABB was used as catalyst, MTS was used as initiator, and THF was used as solvent. The positively ionizable hydrophilic DMAEMA was used as monomer and the novel, hydrophilic BMEMA was used as cross-linker. Figure 4 illustrates the synthetic procedure followed for the synthesis of DMAEMA20-star. In the figure, the DMAEMA units are colored in light blue and the BMEMA core is painted in dark red, while the asterisks at the tip of the chains of the linear precursors and around the core of the star polymer denote the active polymerization sites. As is shown in the figure, the polymerization was accomplished in two steps. In the first step, the synthesis of the linear precursors (arms of the star) took place by the polymerization of the DMAEMA monomer in the presence of a monofunctional GTP initiator. Subsequently, the arms were interlinked at one end in the same pot via the polymerization of BMEMA, which led to the formation of an “arm-first” star homopolymer. The molar ratio of the cross-linker to the initiator was 4 : 1 for all star homopolymers. Molecular Weights of the Star and the Linear Homopolymers. The successful formation of the star and the linear homopolymers was confirmed by GPC-RI. The GPC-RI traces of the star and the linear homopolymers are presented in Figures 5 and 6, respectively. Tables 1 and 2 list the values of

Mn, Mp, and PDI of all star and linear homopolymers calculated from the GPC-RI traces. The tables also show the DPs of the arms of the stars and the DPs of the linears as measured using GPC-RI and 1H NMR spectroscopy. In Figure 6, the chromatograms of the linear homopolymers were successively shifted to lower elution volumes as the DPs were increased. In Figure 5, the chromatograms of the star homopolymers were shifted to lower elution volumes compared to those of their linear precursors, consistent with the interconnection of the precursors via the cross-linker molecules to larger polymeric entities. With the exception of DMAEMA10-star, the GPC traces of the star homopolymers exhibited two peaks, one due to the star homopolymer and the second due to a fraction of unattached arm. Incomplete incorporation of the linear homopolymers into the stars may be attributed to increased solution viscosity, lower chain mobility, and possible chain termination. The effect of unattached arms on transfection efficiency is thoroughly investigated by the independent preparation of the pure linear arms and the evaluation of their own efficiency as transfection reagents. Another possible concern with the present star homopolymers is the fact that they do not possess a precise number of arms. Instead, there is a distribution in the number of arms, which is a result of the random cross-linking procedure, characterized by the interconnection of a larger number of arms, limited mainly by steric hindrance. Our approach here was to prepare a number of star homopolymers with this facile strategy, evaluate their transfection efficiency, and, if necessary, synthesize in a subsequent study the best samples using a more precise method to extract more accurate structure-property relationships. It is noteworthy that the relatively large number of arms in these star homopolymers secures a high density of DMAEMA groups which would favor complexation with siRNA. Absolute Molecular Weights of the Star Homopolymers. All star homopolymers were characterized in terms of their absolute MWs, Mw, in THF using GPC-SLS. The relevant chromatograms are plotted in Figure 7, in which the unattached arms are invisible due to the much weaker light scattering from 1472


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Table 1. MWs, PDIs, and Experimental Arm DPs of the Star Homopolymers and Their Linear Precursors GPC results no. 1 2

3

4

polymer theoretical formula

theor. MW

Mp

1HNMR results

Mn

PDI

arm DP 11

DMAEMA10

1670

2820

1770

1.35

DMAEMA10-star

-----

26100

15200

1.69

DMAEMA20

3240

4710

4090

1.14

DMAEMA20-star

-----

34100

23800

1.47

4880

4270

1.04

DMAEMA35

5600

12800

10900

1.14

DMAEMA35-star

-----

72100

57400

1.29

DMAEMA50

7960

13500 16300

11600 13200

1.06 1.18

DMAEMA50-star

-----

157000

112000

1.41

17100

12700

1.07

arm DP 19 17

25

37.5 30

69

75 75

83

Table 2. MWs, PDIs, and Experimental DPs of the Linear Homopolymers 1

GPC results

H NMR results

no


theor. MW

Mp

Mn

PDI

1

DMAEMA10-linear

1670

1800

1240

2

DMAEMA20-linear

3240

4280

3610

3

DMAEMA35-linear

5600

10500

4

DMAEMA50-linear

7960

34100

Figure 7. GPC-SLS traces of the star homopolymers.

the smaller entities, that is, the free arms compared to the multiarmed stars. The absolute Mw values and the calculated numbers of arms are listed in Table 3. The absolute Mw values increased monotonically with the arm DP, from 51000 to 269000 g mol-1. The numbers of arms of the star homopolymers were calculated by first subtracting from the absolute Mw values of the star the contribution from the BMEMA cross-linker and dividing the result by the Mn value of the corresponding arms previously determined by GPC-RI. The calculated numbers of arms were found to be in the range from 7 to 19. Aqueous Solution Properties of the Star and the Linear Homopolymers. The cloud points and the effective pK values in water of the star and the linear homopolymers were characterized using turbidimetry and hydrogen ion titration, respectively. The star homopolymers were also characterized in terms of their hydrodynamic diameters using DLS. The results for the star and the linear homopolymers are presented in Tables 4 and 5, respectively.

DP

DP

1.30

7

13.6

1.17

22

37.5

9320

1.11

59

26200

1.19

166

Hydrodynamic Diameters. The hydrodynamic diameters of all star homopolymers were determined in pure water (pH ≈ 9.4) but also in aqueous solutions containing 0.07 M HCl and 1 M NaCl at pH 3. Table 4 presents the experimental hydrodynamic diameters in both above-mentioned aqueous media and also the theoretically maximum possible diameters. The theoretically maximum possible diameters were calculated as twice the value of the arm contour length which was estimated by multiplying the contribution of one vinyl monomer repeating unit of 0.252 nm47 times the total number of vinyl repeating units per arm. The last number was taken as the sum of the arm DP (from GPC) plus 8, where 8 reflects the fact that each of the four BMEMA units per arm bears two vinyl groups. In the size distribution histograms, all the star homopolymers presented 2-4 peaks (Table 4), with that of the smallest size corresponding to unattached linear precursors and the others to star polymer and star polymer aggregates. The maximum of the most populated peak in each sample appears in bold in Table 4. In pure water, the most populated hydrodynamic diameters for DMAEMA10-star and DMAEMA20-star corresponded to polymer aggregates, while the most populated hydrodynamic diameters for the other two stars, DMAEMA35-star and DMAEMA50-star, corresponded to that expected for star homopolymers. In the acidic (pH 3) aqueous solution with 1 M NaCl, the most populated peaks of all the star homopolymers corresponded to star polymers. With the exception of DMAEMA10-star, the experimental hydrodynamic diameters which corresponded to star polymers were lower than the maximum possible value because the latter was calculated for fully stretched arms. The higher value of the experimental hydrodynamic diameter of DMAEMA10-star in acidic salt buffer than the theoretical upper limit may be due to star-star coupling during the synthesis of this star polymer arising from the weak steric hindrance (very short arms). 1473


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Table 3. Absolute MWs and Number of Arms of all the Star Homopolymers, as Determined by GPC-SLS

a

core weight fractiona

Mcore w

core Mstar w -Mw

51300

0.34

17400

33900

1770

19.2

60600

0.20

12100

48500

4090

11.9

DMAEMA35-star

87700

0.13

11400

76300

10900

7.0

DMAEMA50-star

269000

0.09

24200

244800

13200

18.5

no.

polymer formula

1

DMAEMA10-star

2

DMAEMA20-star

3 4

Mw of star from SLS

Mn of arm by GPC

number of arms

(DPtheor.BMEMA MWBMEMA)/(DPtheor.DMAEMA MWDMAEMA þ DPtheor.BMEMA MWBMEMA).

Table 4. Hydrodynamic Diameters, Cloud Points, Effective pK Values, and Precipitation pHs of the Star Homopolymers hydrodynamic diameters (nm) experimental in water

a

polymer theoretical

Pure water,

Acidic salt buffer,

Theoretical

clouds points

clouds points

effective

precip.

no.

formula

pH = 9.4

pH = 3

upper limita

(°C; dissolution pH)

(°C; pH = 8)

pK

pH

1

DMAEMA10-star

9.0, 32.1, 141.0, 944.0

12.8, 278.0

9.7

23.1

43.5

6.86

9.19

2

DMAEMA20-star

133.8, 479.7

14.7, 378.5, 453.4

16.8

23.5

50.0

7.14

9.41

3

DMAEMA35-star

8.2, 21.1

21.5, 346.0

39.1

27.5

55.5

7.28

9.82

4

DMAEMA50-star

11.2, 26.5

5.5, 26.2, 108.8

46.2

29.3

56.0

7.33

9.38

For the calculation of the theoretically maximum diameter, the arm DP from the GPC results was used.

Table 5. Cloud Points and Effective pK Values of the Linear Homopolymers no.


cloud points °C

effective pK

1

DMAEMA10-linear

41.9

7.33

2

DMAEMA20-linear

38.7

7.37

3

DMAEMA35-linear

35.2

7.37

4

DMAEMA50-linear

31.7

7.45

Cloud Points. The cloud points of all star homopolymers were determined in pure water (at their dissolution pH of 8.5-9.5) and in an aqueous buffer of pH = 8. The cloud points of the linear homopolymers were determined in pure water. The results are shown in Tables 4 and 5. All values of cloud points for the star homopolymers increased monotonically with the arm length as a result of better steric stabilization of the star polymer in water by longer arms.48 In pure water, the cloud points increased from 23.1 to 29.3 °C as the nominal arm DP increased from 10 to 50, as expected for DMAEMA-based star homopolymers,49a whereas in the buffer solution of pH = 8 the increase was from 43.5 and 56.0 °C. In the buffer solution, the cloud points were approximately 20 °C higher than those in pure water due to the partial ionization of the DMAEMA and the BMEMA units at pH 8. The cloud points of the linear homopolymers in pure water were found to decrease with their DP from 41.9 to 31.7 °C. The cloud points of all the linear homopolymers were higher compared with those of the star homopolymers due to their increased watersolubility arising from their very low MW. Furthermore, the cloud points of the linear homopolymers followed an inverse MW-dependence, again due to their very small size (highest solubility for the smallest oligomer). Effective pKs. The effective pK values of the star and the linear homopolymers were calculated as the pH at 50% ionization. The pKs of the star homopolymers are listed in Table 4 and increased with the arm DP from 6.86 to 7.33, in agreement with previous investigations on DMAEMA-based linear homopolymers50 and star homopolymers.49a The effective pK values for the linear

homopolymers, presented in Table 5, were found to increase with the DP within a narrower range, from 7.33 to 7.45 (and close to the experimental error in the pH measurement of ca. 0.1 pH units). pH of Precipitation. The pH of precipitation of the star homopolymers (Table 4) slightly increased with their arm DP due to increased steric stabilization by longer arms.48 The linear homopolymers did not precipitate at all within the studied pH range (2-12) at room temperature. Transfection Performance. After their organic and aqueous solution characterization, all star homopolymers were evaluated for their ability to deliver siRNA to mouse myoblast (C2C12) cells and down-regulate endogenous EGFP gene expression. The evaluation of the transfection efficiency of the star homopolymers involved three indicators: the EGFP suppression, the siRNA-specific EGFP suppression, and the cell toxicity. These indicators were studied against star polymer loading. Transfections were also performed using linear homopolymers with the same DPs as the arm DPs of the star homopolymers. Finally, for comparison, transfections were performed using the commercially available transfection reagent Lipofectamine as well. Figure 8 presents the transfection results using the star homopolymers, while Figure 9 presents the results using the linear homopolymers. The graphs plot the EGFP suppression, the siRNA-specific EGFP suppression, and the cell toxicity against the amount of polymer used in the transfection. The polymer amount was varied from 30 to 100 μg (N/P ratios from 91 to 303) for DMAEMA10-star and from 15 to 80 μg (N/P ratios from 45 to 243) for DMAEMA20-star. For DMAEMA35star and DMAEMA50-star, a lower polymer amount range, between 15 and 60 μg (N/P ratios from 45 to 182), was employed due to the increased cytotoxicity of these star homopolymers. The amounts of DMAEMA10-linear and DMAEMA20linear were varied from 15 to 100 μg (N/P ratios from 45 to 303), while those of DMAEMA35-linear and DMAEMA50-linear ranged between 15 and 60 μg (N/P ratios from 45 to 182). Form Figure 8 for all star homopolymers, EGFP suppression and cell toxicity increased with the amount of star polymer used. 1474


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Figure 8. Dependence of EGFP suppression, siRNA-specific EGFP suppression, and cell toxicity on the amount of polymer at a constant amount of siRNA for all star homopolymers. The range of polymer amounts from 15 to 100 μg corresponds to an N/P ratio range from 45 to 303.

The siRNA-specific EGFP suppression, which also takes into account the effect of star polymer toxicity on the cells, presented a slight increase with polymer loading for the star homopolymers with arm DPs 10 and 20, while for DMAEMA35-star it presented a clear maximum. The siRNA-specific EGFP suppression with DMAEMA50-star decreased with polymer loading due to its high values of toxicity. From Figure 9, the EGFP suppression and the siRNA-specific EGFP suppression of DMAEMA10-linear and DMAEMA20linear were fairly independent of polymer loading, while those of DMAEMA35-linear presented a slight increase. The EGFP suppression and cell toxicity of DMAEMA50-linear clearly increased with the polymer loading, while its siRNA-specific EGFP suppression slightly increased. With the exception of DMAEMA50-linear, the cell toxicity of the linear homopolymers remained at very low levels. The low transfection efficiency of the linear homopolymers is in agreement with one example given in

the literature37 where a linear DMAEMA homopolymer with a MW of 20000 g mol-1 was used. It is clear from Figure 8 that the transfection efficiency (the EGFP suppression and the siRNA-specific EGFP suppression) of all the star homopolymers was comparable with that of the commercially available, and expensive, reagent Lipofectamine. Moreover, Figure 9 indicated that DMAEMA50-linear was the only linear homopolymer exhibiting substantial transfection efficiency, accompanied, however, with increased cell toxicity. Figures 10 and 11 present the dependence of EGFP suppression, siRNA-specific EGFP suppression, and cell toxicity using 30 μg of polymer and 1 μg of siRNA (N/P ratio of 90) on the arm DP of the stars and on the DP of the linear homopolymers, respectively. Both for the star and the linear homopolymers, the EGFP suppression and the siRNA-specific EGFP suppression increased with the DP. The toxicity of the star homopolymers increased with the arm DP (up to 20%), while that of the linear 1475


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Figure 9. Dependence of EGFP suppression, siRNA-specific EGFP suppression, and cell toxicity on the amount of polymer at a constant amount of siRNA for all linear homopolymers. The range of polymer amounts from 15 to 100 μg corresponds to an N/P ratio range from 45 to 303.

homopolymers remained at very low levels, below 5%. Because the DP of the arms of the stars and the DP of the linears were proportional to the MWs of each group of polymers, the DP x-axis can be thought of as a MW axis. Thus, Figures 10 and 11 present, at the same time, the qualitative dependence of these three indicators on the MW of the star and the linear homopolymers.

’ DISCUSSION In the present work, four hydrophilic star homopolymers of DMAEMA, bearing the novel hydrophilic cross-linker BMEMA, with nominal arm DPs 10, 20, 35 and 50, were successfully synthesized, and characterized in organic and in aqueous media. For comparison, four linear homopolymers of DMAEMA with the same nominal DPs were also synthesized and characterized.

The MWs and PDIs of the star homopolymers and their linear precursors were determined using GPC-RI. The Mns of all the linear precursors were (slightly) higher than their theoretically anticipated values due to partial deactivation of the initiator. The PDIs of the star homopolymers were between 1.3 and 1.7. The determination of the MWs and the PDIs of the linear homopolymers was also performed using GPC-RI. The Mn values of DMAEMA10-linear and DMAEMA20-linear were relatively close to the theoretical values, while those of the two other linear homopolymers were higher than the theoretical values due to greater deactivation of the initiator. From SLS studies, the absolute Mw values of the star homopolymers were found to increase monotonically with their arm DP. From the absolute MWs, the number of arms was measured and found to range from 7 to 19. The greatest number of arms 1476


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Figure 10. Dependence of (a) EGFP suppression, (b) siRNA-specific EGFP suppression, and (c) toxicity on the arm DP of the star homopolymers at a loading of 30 μg of star polymer and 0.665 μg (50 pmol) of siRNA.

Figure 11. Dependence of (a) EGFP suppression, (b) siRNA-specific EGFP suppression, and (c) toxicity on the DP of the linear homopolymers at a loading of 30 μg of linear polymer and 0.665 μg (50 pmol) of siRNA.

(=19) was presented by DMAEMA10-star whose short arms were less capable of preventing star-star coupling. Moreover, the star homopolymers were characterized in aqueous media. Their hydrodynamic diameters in pure water and in acidic aqueous solution (with 1 M NaCl and 0.07 M HCl) were found to correspond to star homopolymers and star polymer aggregates. The cloud points of the stars were found to increase with their MW. This is in agreement with previous studies by our research group,49a where the cloud points of star homopolymers were found to increase, attaining a limiting value at sufficiently high MW. In buffer solution, the cloud points of the star homopolymers were found to be 20 °C higher than in pure water due to the partial ionization of the DMAEMA units under these conditions as compared to their almost uncharged state in pure water. The cloud points of the linear homopolymers were higher compared to those of the star homopolymers, while they followed an inverse MW-dependence. This is in agreement with the MW-dependence of the cloud points of linear homopolymers of 2-hydroxyethyl methacrylate (prepared using a hydrophilic, morpholine-containing initiator) with DPs between 20 and 45 in aqueous solution at pH 6.5.51 All the star homopolymers and DMAEMA50-linear presented considerable EGFP suppression, comparable with that of the commercially available reagent Lipofectamine. The greatest suppression appeared with the highest polymer amounts. This is in agreement with the study of Hoffman et al.33 who observed that the suppression of the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) increased as the loading of their DMAEMA-containing diblock copolymer also increased. These authors attributed this to the formation of more condensed particles at higher polymer loadings, which could be internalized by the cells more efficiently. At this point we should mention that the present study is just the second report38 on the use of starshaped polymers for siRNA transfection.

Increase of transfection efficiency with the polymer loading was also observed using other polycation systems which presented globular structure, similar to that of our star-shaped polymers, including highly branched peptides composed of histidine and lysine,24 PEI-graft-PEG-folate,17 and PAMAM dendrimers.30 Another example was the use of chitosan/siRNA nanoparticles by Liu et al.,28 where they ascribed the increase in gene silencing to the amount of chitosan that did not participate in complex formation, but improved complex stability. Another important indicator studied was the cell toxicity of the star homopolymers. The cell toxicity increased with the polymer amount due to the toxicity of the excess free polymer bound to the cell membrane, resulting in its destabilization and, subsequently, leading to cell death. In their study, Liu et al.28 observed an increase in the toxicity as the amount of chitosan used increased, which is in agreement with our findings. The last and most important indicator studied was the siRNAspecific EGFP suppression. This indicator takes into account not only the suppression of the EGFP signal but also the toxicity during transfection. For star homopolymers DMAEMA10-star and DMAEMA20-star, the siRNA-specific EGFP suppression slightly increased with the polymer loading. DMAEMA35-star presented a maximum with the polymer amount, while DMAEMA50-star exhibited a reduction with the polymer loading. This last trend arises from the large increase in cytotoxicity with the loading of the largest star polymer (Figure 8). Note that the best siRNA-specific suppression afforded by each star homopolymer in Figure 8 was comparable to that measured for the commercially available, and expensive, transfection reagent Lipofectamine (result also plotted in Figure 8), suggesting the good efficiency of our star homopolymers in gene silencing. Although the siRNA-specific EGFP suppression is a very important indicator, combining the two other important indicators of suppression efficiency and cytotoxicity that usually 1477


Biomacromolecules present opposite dependencies on polymer loadings, to the best of our knowledge, this is not commonly used in the literature. Our group has previously studied this indicator in DNA transfection experiments several years ago,49 where it was called “overall transfection efficiency”. In those experiments, DMAEMA star homopolymers were used again. Those stars presented a clear maximum of the overall DNA transfection efficiency versus polymer loading. Moreover, studying the influence of polymer DP on EGFP suppression (Figures 10a and 11a), we observed that the latter increased with DP (both for the linears and the stars). As already mentioned, the increase of the DP corresponds to the increase of the steric stabilization of the star homopolymers in solution. Thus, the increase of the EGFP suppression can be attributed to more stable star polymers. Our observation is in agreement with the study of Crayson et al.12 who reported an increase in the gene silencing efficiency with the MW of their branched PEIs. Similar observations were also made by Liu et al.28 who documented greater transfection efficiencies as the MW or the degree of deacetylation of chitosan increased due to the formation of more stable and compact chitosan/siRNA nanoparticles. The siRNA-specific EGFP suppression (Figures 10b and 11b) followed the same trend as the EGFP suppression, confirming the great influence of the stabilization and the MW of the star homopolymers on the transfection efficiency. The toxicity of the star homopolymers (Figure 10c) also increased with the arm DP (and overall MW), as already observed in our previous study.49a This was attributed to the destabilization of the cell membranes by larger polymers, which could cause higher cell death rates. Some other examples in the literature, using PEI, also confirm these findings. In particular, Thomas et al.14 showed an increase in cytotoxicity as the MW increased both with linear and deacylated PEI. After realizing the high toxicity of unmodified PEI, Zintchenko et al.13 used succinylated derivatives of branched PEI and they observed up to a 10-fold lower polymer toxicity of these systems in comparison with the unmodified PEI. From these transfection experiments, it is clear that the star homopolymers are better transfection reagents than the corresponding linear homopolymers. For instance, comparing the second star homopolymer of nominal arm DP 20 with the largest linear homopolymer DMAEMA50-linear, having similar MWs, these homopolymers exhibited differences in their efficiency in transferring siRNA and in cell toxicity. In fact, the star polymer seemed to be much less toxic than the linear analogue. The greater transfection efficiency of the star polymers can be attributed to their star-shaped structure which probably contributes to the formation of more compact complexes that are able to go through the cell membrane, while, at the same time, they are sufficiently compact to protect siRNA from protease degradation. Thus, the star homopolymers can be successfully used as novel, efficient, and inexpensive siRNA transfection reagents.

’ CONCLUSIONS In conclusion, in the present work we have developed a new nonviral gene silencing system based on star-shaped DMAEMA homopolymers which can efficiently deliver siRNA in vitro. These star homopolymers were based on the novel hydrophilic BMEMA cross-linker. In total, four star homopolymers with nominal arm DPs 10, 20, 35, and 50 were prepared. The star homopolymers were characterized in organic and aqueous

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media. Our results indicated that these polymers formed aggregates both in pure water and in aqueous acidic solution. The cloud points, the effective pKs, and the precipitation pHs of the star homopolymers increased with the arm DP (increase of the steric stabilization of the star homopolymer). Furthermore, their transfection efficiency was calculated as the siRNA-specific EGFP suppression, which was the pure outcome of EGFP suppression after subtracting the effect of polymer/complex cytotoxicity. The siRNA-specific EGFP suppression of the two smaller stars slightly increased with the star polymer loading, while that of the other two stars with arm DPs 35 and 50 presented a shallow maximum and a decrease, respectively. All star homopolymers presented transfection efficiencies comparable with that of the commercially available reagent Lipofectamine, confirming their potential as useful transfection reagents.

’ AUTHOR INFORMATION Corresponding Author

*Tel.: (þ357) 22 892768. Fax: (þ357) 22 892801. E-mail: [email protected].

’ ACKNOWLEDGMENT We thank the Cyprus Research Promotion Foundation along with the EU Structural and Cohesion Funds for Cyprus for supporting this work in the form of a PENEK2008 Doctoral Research Grant (Project ENISX/0308/048) to K.S.P. We are also grateful to the A. G. Leventis Foundation for a generous donation that enabled the purchase of the NMR spectrometer of the University of Cyprus and a grant awarded to L.A.P. ’ REFERENCES (1) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Nature 1998, 391, 806–811. (2) Zamore, P. D. Science 2002, 296, 1265–1269. (3) Behlke, M. A. Mol. Ther. 2006, 13, 644–670. (4) Charles, X. L.; Parker, A.; Menocal, E.; Xiang, S.; Borodyansky, L.; Fruehauf, J. H. Cell Cycle 2006, 5, 2103–2109. (5) Castanotto, D.; Rossi, J. J. Nature 2009, 457, 426–433. (6) Tiemann, K.; Rossi, J. J. EMBO Mol. Med. 2009, 1, 142–151. (7) Markert, C. D.; Ning, J.; Staley, J. T.; Heinzke, L.; Childers, C. K.; Ferreira, J. A.; Brown, M.; Stoker, A.; Okamura, C.; Childers, M. K. Neuromascul. Disord. 2008, 18, 413–422. (8) Liu, Q.; Muruve, D. A. Gene Ther. 2003, 10, 935–940. (9) Sun, J. Y.; Anand-Jawa, V.; Chatterjee, S.; Wong, K. K. Gene Ther. 2003, 10, 964–976. (10) Carter, B. J.; Samulski, R. J. Int. J. Mol. Med. 2000, 6, 17–27. (11) Heath, W. H.; Senyurt, A. F.; Layman, J.; Long, T. E. Macromol. Chem. Phys. 2007, 208, 1243–1249. (12) Crayson, A. C.; Doody, A. M.; Putnam, D. Pharm. Res. 2006, 23, 1868–1876. (13) Zintchenko, A.; Philipp, A.; Dehshahri, A.; Wagner, E. Bioconjugate Chem. 2008, 19, 1448–1455. (14) Thomas, M.; Lu, J. J.; Ge, Q.; Zhang, C.; Chen, J.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5679–5684. (15) Mao, S.; Neu, M.; Germershaus, O.; Merkel, O.; Sitterberg, J.; Bakowsky, U.; Kissel, T. Bioconjugate Chem. 2006, 17, 1209–1221. (16) Kim, K. H.; Mok, J. H.; Kim, W. S.; Park, T. G. Bioconjugate Chem. 2006, 17, 241–244. (17) Kim, S. H.; Jeong, K. H.; Cho, K. C.; Kim, S. W.; Park, T. G. J. Controlled Release 2005, 104, 223–232. (18) Jiang, G.; Park, K.; Kim, J.; Kim, K. S.; Hahn, S. K. Mol. Pharm. 2009, 6, 727–737. 1478


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Hydrophilic Cationic Star Homopolymers Based on a Novel Diethanol

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