Aminated Linear and Star-Shape Poly(glycerol methacrylate)s

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Aminated Linear and Star-Shape Poly(glycerol methacrylate)s: Synthesis and Self-Assembling Properties Hui Gao,†,‡ Mahmoud Elsabahy,‡ Elisabeth V. Giger,§ Dekun Li,‡ Robert E. Prud’homme,| and Jean-Christophe Leroux*,‡,§ School of Chemistry and Chemical Engineering, Tianjin University of Technology, Tianjin, China, 300384, Canada Research Chair in Drug Delivery, Faculty of Pharmacy, and Department of Chemistry, University of Montreal, Montreal (QC), Canada, H3C 3J7, and Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zu¨rich, 8093 Zu¨rich, Switzerland Received October 31, 2009; Revised Manuscript Received February 19, 2010

Over the past 10 years, polyglycerols and their structurally related analogs have received considerable attention in the biomedical field. Poly(glycidyl methacrylate) (PGMA) is a versatile polymer because its pendant epoxide groups can be opened with different functional groups to generate poly(glycerol methacrylate)s (PGOHMA) derivatives. In this work, linear and star-shape PGMAs were synthesized by atom transfer radical polymerization and then functionalized with four different amines by ring-opening addition. This resulted in the formation of polyglycerol-like polymers having both hydroxyl and amine moieties and different water-solubility. The waterinsoluble polymers could form pH-sensitive nanoassemblies, while the soluble derivatives efficiently complexed a short strand polynucleotide. The aminated polyglycerol interacted more avidly with the oligonucleotide than the control poly(ethyleneimine), and high transfection efficacy could be obtained with the linear derivative. Such polymers could find practical applications for the delivery of drugs and nucleic acids.

1. Introduction Nucleic acid delivery is an attractive approach for the treatment of numerous diseases, including cancer and viral infections.1,2 However, the clinical use of nucleic acid-based drugs is still largely hampered by their inability to reach their site of action in sufficient amount. Complexation with oppositely charged molecules such as cationic lipids and polymers (e.g., polyethylenimine (PEI)3 and poly(amidoamine)4,5) has been employed to improve cellular uptake and resistance to nucleases. Likewise, pH-sensitive nanocarriers (e.g., pH-sensitive liposomes) have been exploited to enhance the transfection efficiencies of various nucleic acids.2,6 Although the complexes formed with cationic polymers and lipids are considered to be the most promising carriers for nucleic acid delivery, excess positive charges are usually required for high transfection efficiency, which is usually associated with cellular toxicity.7 In addition, neutral complexes (or complexes with low +/- charge ratios) usually have poor cellular uptake and limited stability.8 Therefore, there is an ultimate need for new materials with good stability, low toxicity, and high transfection capacity. Poly(glycidyl methacrylate) (PGMA)9 is a versatile polymer because its pendant epoxide groups can be opened with several functional groups to generate polyglycerol methacrylate (PGOHMA) derivatives.10 Postpolymerization modification in this fashion is attracting increasing interest owing to the possibility of conveniently preparing many functional polymers, which are possibly inaccessible by direct polymerization of the functional monomer.11 In addition, owing to their polyol structure, they offer multiple attachment sites to ligands and * To whom correspondence should be addressed. Phone: (+41) 44 633 7310. Fax: (+41) 44 633 1314. E-mail: [email protected]. † Tianjin University of Technology. ‡ Faculty of Pharmacy, University of Montreal. § ETH Zu¨rich. | Department of Chemistry, University of Montreal.

drugs.12 Such characteristics have been exploited for drug targeting applications13 and surface modification.14 Recently, polyglycerols have been derivatized by different functional groups and molecules to produce polymers with unique properties. For example, alkylated dendritic, branched, and star-shape polyglycerols can H-bond with various substrates and thus be used to dissolve hydrophilic compounds in apolar media.15–17 Sulfated dendritic polyglycerols have demonstrated promising anti-inflammatory properties.18 Aminated dendritic polyglycerols have been investigated for their ability to complex plasmid DNA through electrostatic interactions.19 In vitro, these polymers were shown to be safe and capable of transfecting cells.20 In this study, we have synthesized and characterized aminated polyglycerols from linear and star-shape PGMA. Depending on their structure, some aminated PGOHMA were found to exhibit pHdependent solubility, while others interacted efficiently with cells to deliver an antisense oligonucleotide (AON).

2. Experimental Section 2.1. Materials and Techniques. A 20-mer phosphorothioate AON (5′-GTTCTCGCTGGTGAGTTTCA) and its 5′-fluorescein-labeled derivative were supplied by Medicorp Inc. (Montreal, QC, Canada). 5′-TCTCCCAGCGTGCGCCAT AON (Bcl-2 AON) that targets the initiation codon of Bcl-2 mRNA21 was obtained from Alpha DNA (Montreal, QC, Canada). Linear polyethylenimine (PEI; MW 25000) and glycidyl methacrylate (GMA) were purchased from Polysciences Inc. (Oakville, ON, Canada). CellTiter 96 AQueous One Solution Cell Proliferation Assay was supplied by Promega Corporation (Madison, WI). RPMI 1640, sodium pyruvate, nonessential amino acids, fetal bovine serum, and penicillin/streptomycin were purchased from Invitrogen (Paisley, U.K.). All other reagents were obtained from Sigma Aldrich (Oakville, ON, Canada) and used as received, except for tetrahydrofuran (THF), which was dried through PureSolv columns (Innovative Technologies, Newburyport, MA). IR spectra were recorded on a Nicolet FTIR 5 DXB spectrometer (Thermo Scientifc, Waltham, MA) using KBr pellets. 1H NMR spectra

10.1021/bm901241k  2010 American Chemical Society Published on Web 03/04/2010

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Scheme 1. Synthesis of PGMA and Amino PGOHMA

were recorded on an ARX 400 spectrometer (400 MHz, Bruker, Freemont, CA). Samples were either prepared in deuterated chloroform or deuterated water. Cross-polarization/magic-angle spinning (CP/MAS) 13 C NMR experiments were carried out at room temperature on a Bruker AV600WB spectrometer, using a standard 4 mm CPMAS probe. Spin rate was set to 14 kHz, contact time to 1.5 ms, repetition time to 5 s. Gel permeation chromatography (GPC) measurements were performed in THF for PGMA and in 0.4 mM sodium-acetate buffer (pH 5.1)/ acetonitrile (80:20) for amino PGOHMA, using a Viscotek TDAmax system (Viscotek, Houston, TX) equipped with a differential refractive index detector and a low angle light scattering detector. Adequate molecular weight separation was achieved using two ViscoGEL columns (GMHHRM with THF as mobile phase, GMPWXL with buffer as mobile phase) in series at a flow rate of 1.0 mL/min and a temperature of 35 °C. Calibration curves were obtained with near monodisperse polystyrene and poly(ethylene oxide) standards for PGMA and amino PGOHMA, respectively. Elemental analysis was used to determine the molar content of amino groups per gram of polymer. The measurements were conducted in an oxidative atmosphere at 1021 °C using a thermal conductivity probe (Fisons Instruments EA 1108 C, H, N, S Elemental Analyzer, Beverly, MA). The mean hydrodynamic diameter, size distribution, and scattering intensity of the micelles were determined at a temperature of 25 °C on a Malvern Zetasizer NS (Malvern, Worcestershire, U.K.) equipped with a backscattered light detector operating at 173°. The CONTIN program was used to extract size distributions from the autocorrelation functions. Zeta potential measurements were carried out in the standard capillary electrophoresis cell of the same Malvern Zetasizer. Three measurements, each consisting of 20 runs, were performed on each sample. The Smoluchowski model was used to calculate zeta potential values. 2.2. Synthesis of Amino PGOHMA. PGMA was synthesized according to a method reported previously.9 Briefly, glycidyl methacrylate was polymerized in THF using the corresponding atom transfer radical polymerization initiator to yield linear (L_PGMA), 4-arm (S4_PGMA), and 8-arm (S8_PGMA) products. The linear PGMA was then modified with four kinds of aliphatic amines, N-butylmethylamine (B), propylamine (P), N-methylpropylamine (M), and N,N,N′-trimethylethylenediamine (T), by ring-opening of the epoxide groups to obtain L-B, L-P, L-M, and L-T (Scheme 1). PGMA was dissolved in acetonitrile at a concentration of 12.5 g/L. Then, excess amine was added (amine/epoxy group 2:1 molar ratio) to ensure the completion of the reaction. The mixture was refluxed at 90 °C overnight under argon. In addition, the feed ratio of amine/epoxy group was set at 1:1 and 1:0.5 to achieve lower amination degree. After refluxing at 90 °C overnight, distilled water (ratio of water/initial epoxy group in PGMA was 2:1) was added and refluxed at 90 °C for another 12 h. The solution was cooled down, and the products were dialyzed (Spectra/Por RC, cutoff 15000) either against water (watersoluble L-T) or against ethanol (water-insoluble polymers, L-B, L-P, L-M) for 48 h. They were then freeze-dried or rotary evaporated to obtain the pure products (yields ∼90-95%). Then the same procedure was used, S4_PGMA and S8_PGMA were modified with T to yield S4T and S8T, respectively (yields ∼90-95%).

2.3. Preparation of Nanoparticles and pH-Sensitivity. Nanoparticles were prepared by nanoprecipitation from amino PGOHMA, which is insoluble at neutral pH (L-B, L-P, L-M). In this procedure, the nanoparticles were formed by adding the polymer solution dropwisely to a nonsolvent.22,23 Briefly, 5 mL of the polymer solution (2 mg/mL) in acetone was added dropwise to 10 mL of water under magnetic stirring. The suspension was dialyzed (Spectra/Por RC, cutoff 25000) against water overnight to remove acetone. The size of the nanoparticles and distribution width index (DWI) were determined by dynamic light scattering (DLS). The resulting nanoparticles were then titrated with 0.1 M HCl. The dissociation point, defined as the pH corresponding to the lowest absorbance, was monitored spectrophotometrically at 400 nm. Then, the polymer solution was titrated with 0.1 M NaOH to check the reversibility of the process. 2.4. Atomic Force Microscopy (AFM). Thin films were prepared by adding polymer suspension/solution (0.01 g/L) onto freshly cleaved mica followed by air drying at room temperature. A Nanoscope Multimode AFM (Digital Instruments, Santa Barbara, CA), operated in tapping mode, was used to capture images at ambient conditions. 2.5. Gel Electrophoresis. L-T, S4T, S8T, and PEI were used to form complexes with AON. Polyplexes were prepared in 10 mM Tris, pH 7.4, at nitrogen-to-phosphate (N/P) ratios varying from 0.5 to 7 using an AON solution spiked with fluorescein-labeled AON (25 mol %). Samples were incubated for 15 min, mixed with glycerol, and loaded onto a 15% precasted polyacrylamide gel (Bio-Rad, Mississauga, ON, Canada). Following migration, the AON was visualized by UV irradiation using a ChemiImager 5500 imaging system (Alpha Innotech Corp., San Leandro, CA). 2.6. Preparation and Characterization of T-PGOHMA/AON Complexes. Complexes were prepared by first dissolving T-PGOHMA (0.05 and 0.5 mg/mL) and AON (0.05 mg/mL) separately in 10 mM Tris buffer (pH 7.4). The polymer solution was gradually added to the AON solution to obtain N/P ratios of 0.5-3 and then incubated at room temperature for 45 min. Both the stock polymer and the AON solutions were passed through 0.22 µm nylon filters. To evaluate stability of the amino PGOHMA/AON complexes, a polyanion competition assay was performed as reported previously.24,25 Nanocomplexes were prepared in 10 mM Tris, pH 7.4, at an N/P ratio of 2 (final AON concentration of 2.75 µg/mL) and incubated at room temperature for 30 min in the presence of ethidium bromide (EtBr; 1 eq per base pair). Increasing amounts of low molecular weight heparin (sodium salt from porcine intestinal mucosa, MW of ∼3000 g/mol) were then added and the complexes were incubated for an additional 75 min. The fluorescence of free EtBr (Fi), AON/EtBr (Fo), and EtBr/ complex was recorded after addition of heparin (Fh) on a Safire plate reader (Tecan, Medford, MA; λex ) 523 nm, λem ) 587 nm). Relative fluorescence intensities were calculated from the following equation:

relative fluorescence )

(Fh - Fi) × 100 (Fo - Fi)

(1)

An increase in relative fluorescence is indicative of micelle destabilization.

Linear and Star-Shape Poly(glycerol methacrylate)s


2.7. Assessment of the Antisense Activity. Prostate cancer (PC-3) cells were plated in a six-well plate (1.5 × 105 cells/well). After 24 h, the medium was replaced by Opti-Mem I reduced serum medium. The cells were incubated for 5 h at 37 °C with the PEI, L-T, and S8T at N/P ratios of 2 and 4. Then, the cells were washed with complete RPMI medium (RPMI supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 1% sodium pyruvate and penicillin (100 U/mL)/streptomycin (100 µg/mL). After total incubation time of 72 h, cells were washed with phosphate buffered saline (PBS), and extracted in 200 µL lysis buffer consisting of Tris-HCl, pH 8.0 (0.01 M), NaCl (0.14 M), Triton X-100 (1% v/v), aprotinin (0.1 U/mL), and phenylmethylsulfonyl fluoride (0.5 mM) The cell lysates were incubated for 1 h at 4 °C, and centrifuged at 8000 g for 10 min at 4 °C. Samples protein contents were determined with the BCA protein assay kit using BSA standards (Pierce, Rockford, IL). The remaining samples were electrophoresed through a 15% (w/v) poly(acrylamide) gel and the resolved proteins were transferred to a poly(vinylidene fluoride) membrane by electrotransfer. Bcl-2 expression was quantified by the use of an anti-Bcl-2 monoclonal antibody (Medicorp, Montreal, QC, Canada). To confirm equal loading, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantified using an anti-GAPDH monoclonal antibody (Advanced Immunochemical Inc., Long Beach, CA). 2.8. Evaluation of Cellular Toxicity by MTS Assay. In vitro cytotoxicity was determined by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS). PC-3 cells were maintained in complete RPMI medium. Cells were seeded in 96-well plates at a density of 8000 cells/well in 100 µL complete RPMI medium and cultured for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. The medium was then replaced by 100 µL/well fresh medium containing different concentrations of L-8 and PEI ranging from 1 to 800 µg/mL. After a 48 h incubation period, cells were rinsed twice with PBS and fed with 100 µL per well of fresh medium plus 20 µL CellTiter 96 AQueous One Reagent containing the tetrazolium compound MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. After incubation for about 3 h, the absorbance was measured at 490 nm using a Tecan Infinite M200 plate reader (Tecan, Ma¨nnedorf, Switzerland). Cell viability was calculated according to the equation: cell viability(%) ) (OD490 sample/OD490 control) × 100

(2)

where OD490 sample represents the optical density of the wells treated with polymers and OD490 control represents the wells treated with growth medium only.

3. Results and Discussion Three PGMA having a linear (L), 4-arm (S4), or 8-arm (S8) backbone and an average molecular weight (Mn) of about 10 k Table 1. Molecular Weights of PGMA polymer

Mn (kDa)

Mw/Mn

L_PGMA S4_PGMA S8_PGMA

9.9 9.8 10.2

1.37 1.16 1.19

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Figure 1. FTIR spectra of PGMA (black line) and L-P (gray line; L-P is shown as a typical example).

were synthesized (Table 1). The linear PGMA was functionalized with four different amines added in excess, namely, N-butylmethylamine (B), propylamine (P), N-methylpropylamine (M), and N,N,N′-trimethylethylenediamine (T; Table 2) by ring-opening addition (Scheme 1). This synthetic route is generally used for modification of microparticles or resin in suspension.26,27 Here, it was applied to modify the epoxy group in solution by choosing a proper reaction medium and to improve the yield of modification. The polymers were characterized by FT-IR, 1H NMR, and cross-polarization/magic-angle spinning (CP/MAS) 13C NMR spectroscopy, elemental analysis, and pH-titration (Table 2, Figures 1-3). Only L-T (with two tertiary amines) exhibited good aqueous solubility at near neutral pH values, the others were only soluble at acidic pH. Therefore, the 4- and 8-arm PGMA were derivatized only with T (Table 2, S4T and S8T). FTIR and NMR spectroscopy confirmed the transformation from epoxide to -OH and -NH addition. The amination conversion ranged between 88-98% for L-B, L-P and L-M, and 72-81% for L-T, S4T, and S8T, respectively (Table 2). The steric hindrance of T caused a lower yield of amination. The aminated PGOHMA had an apparent pKa comprised between 7.1 and 8.3, which was influenced by the chemical structure and the density of the amino groups28 in the polymer. In the case of L-T, two more polymers with lower degree of amination (25 and 40%) were synthesized (data not shown). Surprisingly, these polymers were insoluble in water and several organic solvents, and adequate characterizations could not be performed. Intermolecular/ intramolecular cross-linking side reactions could probably occurred during hydrolysis, which was evidenced by IR spectroscopy (data not shown). Interestingly, pH-dependent nanoassemblies could be obtained from the water-insoluble polymers L-B, L-P, and L-M by nanoprecipitation.22 Figure 4A is an AFM picture of typical L-B particles. The mean diameter was 63 ( 4 nm with a mean height

Table 2. Characterization of Amino PGOHMA name

elemental N (%)a

amination conversion (%)a

theoretical Mn (kDa)b

Mn (kDa)c

Mw/Mnc

pKa,appd

dissociation pHe

L-B L-P L-M L-T S4T S8T PGOHMA

5.61 6.26 6.26 9.85 9.38 9.20 0

90 88 98 81 73 72 0

14.7 13.0 14.9 13.9 12.4 14.6 11.0

19.4 13.6 19.3 18.4 15.1 12.2 17.9

1.10 1.12 1.22 1.30 1.24 1.12 1.52

7.1 8.3 7.8 7.4 7.3 7.3 N/A

6.6 8.0 7.5 N/A N/A N/A N/A

Note: a Elemental analysis. b Calculated from Mn of PGMA and amination conversion. c GPC. d pH titration. e DLS; PGOHMA is the linear polymer. Amine used for modification. B: N-butylmethylamine; P: propylamine; M: N-methylpropylamine; T: N,N,N′-trimethylethylenediamine. N/A: not available.

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Figure 2. 1H NMR spectra of L_PGMA (I) and L-P (II) in CDCl3 (L-P is shown as a typical example).

Figure 3. CP/MAS

13

C NMR spectra of L_PGMA (I) and L-P (II) (L-P is shown as a typical example).

Figure 4. AFM images of L-B nanoparticles prepared by nanoprecipitation at pH 7 (A) and 3.6 (B) and AON complexes of S8T at N/P ratios of 1 (C) and 3 (D).

of 10 ( 2 nm. The diameter is comparable to that obtained by DLS (69 nm). The lower height could be due to flattening of the soft particles on the mica surface during the drying process.29 The particles formed by nanoprecipitation was titrated by 0.1 M HCl, which led to the ionization of the amino group, the dissociation of the assembly, and the complete dissolution of the polymer. This was evidenced by the decrease in absorbance at 400 nm concurrent with dissociation of the nanoparticles. Indeed, no particles were observed by AFM when the pH of

the suspension was adjusted to pH 3.6 (Figure 4B). The dissociation pH is shown in Table 2. A polymer such as L-B, with a dissociation pH of 6.6, could for instance be useful for the pH-triggered endosomal release of drugs.30,31 The nanoparticle formation was, however, nonreversible. When the pH of the acidic solution was increased by NaOH titration, large aggregates were observed (data not shown). The water-soluble pH-sensitive polymers (i.e., T-derivatives) were then examined for their ability to complex a fluorescently



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Figure 5. Gel electrophoresis of polymer/AON complexes prepared at N/P ratios of 0-7 using L-T (A), S4T (B), S8T (C), and PEI (D). As a negative control PGOHMA (E) was also tested and added at concentrations corresponding to equivalent N/P ratios.

labeled phosphorothioate AON, which inhibits the synthesis of the antiapoptotic protein Bcl-2. Complex formation was examined by gel electrophoresis at N/P ratios of 0.5-7, and compared to unmodified PGOHMA (negative control) and poly(ethylenimine) (PEI; positive control; Figure 5). In the absence of amino groups, PGOHMA was unable to complex the AON, at least in an effective fashion. The incorporation of the labeled-AON in the L-T, S4T, S8T, and PEI complexes was evidenced by fluorescence self-quenching of the AON. The fact that complex formation occurred at a lower N/P ratio for aminated PGOHMA versus PEI could be attributed to the stabilization of the assembly through H-bonds originating from the hydroxyl groups.32,33 Indeed, if electrostatic interaction was the sole mechanism of complex formation, one would have expected linear PEI to condensate AON at a lower N/P ratio than aminated PGOHMA given its higher pKa value.34,35 Even though secondary amine groups of PEI can also form H-bonds with AON, the additional H-bonding interactions provided by the hydroxyl groups located in the neighbor atoms of amine groups of PGOHMA derivatives may further stabilize the complexes by enhanced cooperative interactions.36 The zeta potential of the complexes of PGMA derivatives changed from a negative to a positive value at an N/P ratio of 2. In the case of PEI, this shift occurred at an N/P ratio of 3 (Figure 6). Likewise, the Z-average diameters of the nanocomplexes and their relative scattering intensity (RSI) increased with the N/P ratios, and reached their maximum value (ca. 200 nm for particle size) at an N/P ratio of 2 and 3, respectively (Figure

Figure 6. Effects of N/P ratio on zeta potential. Mean ( standard deviation (SD; n ) 3).

7). In all cases, the size distributions were narrow, with DWI values of less than 0.2. Figure 4C,D show AFM pictures of S8T/AON complexes prepared at an N/P ratio of 1 and 3, with an average size of 118 ( 27 and 209 ( 32 nm, respectively. The mean height of both aggregates was 11 ( 2 nm. Apparently, these particles flattened out on the substrate due to their soft nature. To evaluate the stability of the formulations, a competition assay was performed by incubating the complexes with heparin, an anionic macromolecule that can typically be found

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Figure 7. Effects of N/P ratio on average diameter (squares), scattering intensity (circles), and DWI (triangles) are shown. (A) L-T, (B) S4T, (C) S8T, and (D) PEI/AON complexes prepared at N/P ratios varying from 0.5 to 3. Mean ( SD (n ) 3).

Figure 8. Destabilization of complexes in presence of heparin.

in blood. For all polymers, the addition of heparin released AON in a concentration-dependent manner (Figure 8). The aminated PGOHMA bound more avidly to the AON than did PEI, which may reflect different interaction modes between the two types of polymers. Stability increased with the degree of branching and was the highest for S8T. This might reflect a better molecular packing of the assembly for branched architecture. These results are also consistent with previously published data obtained with PEI, where it was

shown that branched PEI are more effective condensing agents than their linear counterparts.37,38 The safety of L-T was evaluated by colorimetric MTS assay on PC-3 cells and directly compared to that of linear PEI with similar molecular weight. As shown in Figure S1, L-T exhibited a similar cytotoxicity profile to PEI. The concentrations and incubation times chosen for the transfection assays were therefore kept below that where toxicity occurred. The in vitro antisense activity of the formulations was assessed by measuring the inhibition of Bcl-2 synthesis by the AON (Figure 9). Polyplexes were prepared at N/P ratios of 2 and 4 using L-T, S8T, and PEI. At the concentrations tested, the complexes did not show any cell toxicity. The mismatched AON sequence (5′TCTCCCAGCATGTGCCAT; Alpha DNA) failed to downregulate the Bcl-2 protein (data not shown). L-T exhibited a slightly higher transfection efficacy than PEI at an N/P ratio of 2, which might be attributed to the greater stability of those complexes.39 The activity decreased upon raising the N/P ratio to 4. The formation of larger aggregates at this ratio possibly reduced the cellular uptake of the complexes.33 Unexpectedly, the S8T polyplexes did not show any antisense activity. Although additional experiments will be required to understand the difference in transfection efficacy between the linear and branched polymers, the strong interaction between AON and S8T (see Figure 8), possibly related to the polymer architecture, may partially hinder the intracellular disassembly of the AON which reduces their intracellular bioavailability.40 Indeed, L-T and S8T have similar pKa,app (Table 2) and, therefore, the lower


Figure 9. Bcl-2 gene silencing in PC-3 cells transfected for 5 h with AON (400 nM) complexed to PEI, L-T, and S8T. Control cells were treated with medium alone. Mean ( SD (n ) 3).

efficacy of S8T cannot be attributed to a difference in the ionization state. Similar findings were obtained for oligoethylenimine-grafted polypropylenimine dendrimers. In this case, increased stability of G3 over G2 core derivatives led to a reduced intracellular disassembly of polyplexes and lower transfection efficiency.41 Future experiments will be aimed at addressing the effect of polymer structure on transfection activity in more detail as well as investigating the role of H-bonds.

4. Conclusion In conclusion, different amine functionalities were introduced into linear and star-shape PGMA by addition to epoxy groups. The water-insoluble polymers could form pH-sensitive nanoassemblies, while the soluble derivatives efficiently complexed a short strand polynucleotide. These polyplexes interacted more avidly with the AON than PEI and high transfection efficacy could be obtained with the linear derivative. Acknowledgment. Financial support from the NSERC and the Canada Research Chair program is acknowledged. Prof. J. Claverie, Dr. A. Arnold, and Mrs. K. Fuhrmann are acknowledged for their assistance with the GPC measurements. We also thank Drs. J. Chen and X. H. Wang for their help with AFM measurements. Drs. M. A. Gauthier and J. Chain are thanked for their critical reading of this manuscript. Supporting Information Available. Cytotoxicity data of L-T and PEI. This material is available free of charge via the Internet at http://pubs.acs.org.

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