A Degradable Polyethylenimine Derivative with Low Toxicity for Highly

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Bioconjugate Chem. 2003, 14, 934−940

A Degradable Polyethylenimine Derivative with Low Toxicity for Highly Efficient Gene Delivery M. Laird Forrest, James T. Koerber, and Daniel W. Pack* Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801. Received February 5, 2003; Revised Manuscript Received May 2, 2003

Routine clinical implementation of human gene therapy awaits safe and efficient gene delivery methods. Polymeric vectors hold promise due to the availability of diverse chemistries, potentially providing targeting, low immunogenicity, nontoxicity, and robustness, but lack sufficient gene delivery efficiency. We have synthesized a versatile group of degradable polycations, through addition of 800-Da polyethylenimine (PEI) to small diacrylate cross-linkers. The degradable polymers reported here are similar in structure, size (14-30 kDa), and DNA-binding properties to commercially available 25kDa PEI, but mediate gene expression two- to 16-fold more efficiently and are essentially nontoxic. These easily synthesized polymers are some of the most efficient polymeric vectors reported to date and provide a versatile platform for investigation of the effects of polymer structure and degradation rate on gene delivery efficiency.

INTRODUCTION

The need for safe and efficient methods for gene delivery remains a critical stumbling block to the routine clinical implementation of human gene therapy (1). While recombinant viruses are the most efficient gene delivery vectors currently available, polymeric vectors have several advantages that make them a promising alternative. Polyplexes, comprising an electrostatic complex of cationic polymer(s) and plasmid DNA, hold potential for improved safety, easier production and purification, relatively large gene-carrying capacity, and flexibility of design. Despite their promise, none of the existing polymers is generally acceptable for human gene therapy, primarily due to lack of efficiency. One of the most successful and widely studied gene delivery polymers reported to date is polyethylenimine (PEI), an off-the-shelf polycation containing a high density of primary, secondary, and tertiary amines, used previously as a chelator and flocculation agent (2). Due to its relatively high gene delivery efficiency and ready availability, branched, 25-kDa PEI has become a benchmark to which other polymers, especially newly designed and synthesized materials, are often compared. However, several groups have reported that PEI is cytotoxic in many cell lines; at PEI concentrations used in typical transfection protocols, cell metabolic activity may be reduced by 40-90% (3, 4). PEI toxicity appears to decrease with decreasing polymer size. For example, Gosselin et al. found transfection with 25-kDa PEI can reduce cells’ adherence by 50-90% compared to a linear, low-molecular-weight, 800-Da PEI (5). However, gene transfer efficiency was reduced 30- to 50-fold with 800Da PEI compared to the more commonly used 25-kDa PEI. Lack of toxicity should be a major consideration in the design of any new gene delivery material. To generate * Correspondence should be addressed to this author at Department of Chemical and Biomolecular Engineering, Box C-3, 600 S. Mathews Ave., Urbana, IL 61801. Phone: (217) 2442816, Fax: (217) 333-5052, e-mail: [email protected].

biocompatible gene delivery polymers, several degradable polycations have been reported recently. For example, several cationic polyesters have been studied (6-10) including a linear, biodegradable mimic of polylysine (6, 8) and a hyperbranched, poly(amino ester) (9), both of which exhibit negligible toxicity in comparison to the nondegradable 25-kDa PEI. A second class of degradable polycations, generated by the addition of diamines to diacrylates, has been reported by Lynn et al. (11, 12). Their strategy provides a facile route to a diversity of cationic polymer chemistries. Using a combinatorial synthesis strategy, they found polymers that exhibit 4-8fold higher gene transfer activity than 25-kDa PEI. In addition, Pichon et al. (13) and Gosselin et al. (5) each reported gene delivery polymers comprising low-molecular-weight polycations (polylysine and PEI, respectively) cross-linked via reversible disulfide bonds. Cross-linked PEI mediated gene expression 2-fold less efficiently than the standard 25-kDa PEI, but exhibited less toxicity to cells in culture. It is expected that these polymers will decompose in the reducing environment of the cytoplasm to their low-molecular-weight constituents, which are known to exhibit reduced cytotoxicity (4). Finally, Ahn et al. (14) reported the synthesis of copolymers of lowmolecular-weight PEI and difunctional poly(ethylene glycol) (PEG) succinimidyl succinate. The resulting polymers consist of short PEI and PEG (Mw ) 2000) segments cross-linked through ester linkages. These polymers showed significantly lower toxicity than 25-kDa PEI and three times more efficient transfection than the lowmolecular-weight PEI starting material. However, the transfection efficiency was not compared directly to 25kDa PEI. Many questions remain about how to design biodegradable gene delivery polymers. Little is known about where in the cell the degradation of these polymers takes place. Further, one can only hypothesize about where degradation would be most beneficial. For example, degradation in endocytic vesicles may aid plasmid release into the cytoplasm, while keeping the plasmid in the polyplex until the vehicle reaches the nucleus may protect the DNA from enzymatic degradation. Alternatively,

10.1021/bc034014g CCC: $25.00 © 2003 American Chemical Society Published on Web 08/26/2003

Degradable PEI Derivative for Gene Delivery

release of the plasmid from the polyplex may facilitate transport into the nucleus, for example through the nuclear pores, in which case degradation of the polymer in the cytoplasm may be desirable. Further complicating matters, little is known about how the polymer structure, including the size, cross-linking density, branching, amine density, etc., affects degradation kinetics and ultimately gene delivery efficiency. To address these issues, we sought to develop a system that is flexible, allowing the polymer structure to be varied easily, while maintaining high gene-delivery efficiency. The polymer reported here, a biodegradable analogue of 25-kDa PEI, was produced by addition of amino groups on 800-Da PEI to diacrylates of varying spacer length following the strategy reported by Lynn et al. (11, 12). The resulting polymers are highly branched, 14-30 kDa polycations. We show that these polymers are capable of gene delivery activity two- to 16-fold greater than nondegradable, 25-kDa PEI. Importantly, these polymers show negligible toxicity in the cell lines studied to date. MATERIALS AND METHODS

Cells and Plasmid DNA. The MDA-MB-231 human breast carcinoma cell line was purchased from the American Type Culture Collection, and the C2C12 murine myoblast cell line was a gift from Prof. Stephen Kaufman (University of Illinois, Urbana, IL). All cell lines were maintained according to their respective ATCC protocols, at 37 °C and 5% CO2, but were adapted from fetal bovine serum to heat-inactivated horse serum. The 5.3-kilobase pair expression vector, pGL3 (Promega, Madison, WI), containing the luciferase gene driven by the SV40 promoter and enhancer, was grown in DH5R E. coli (Gibco BRL, Rockville, MD) and purified using a commercial plasmid purification kit (Bio-Rad, Hercules, CA). Plasmids were further purified by ethanol precipitation; the ratio of absorbances at 260 and 280 nm was 1.8 or greater. General Polymer Cross-Linking Procedure. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification unless noted otherwise. Cross-linking was performed following the procedure of Lynn et al. (11, 12). One gram of PEI (branched, 800 Da) was transferred to a 1-oz scintillation vial and dissolved in 3 mL of freshly distilled methylene chloride (417 mM solution). An equimolar amount of diacrylate linker (417 mM) was added dropwise, and the vial was sealed with a solvent-resistant cap. The reaction was carried out at 45 °C, with shaking, for 6 h. The polymer was then precipitated with hexanes, lyophilized (Labconco 18L Freeze-Dry System, Kansas City, MO), and stored at -80 °C. Synthesis of Polymer 1. Polymer 1 was prepared according to the procedure above using 1,3-butanediol diacrylate as the cross-linker. 1H NMR (400 MHz, D2O): δ ) 3.95 (br m, 2 H, NHCH2CH2COOCH2, ester linker), 3.88 (br m, 2 H, NHCH2CH2COOCH(CH3), ester linker), 3.5-3.35 (br m, 2 H, OHCH2CH2, hydrolyzed ester), 3.32.5 (br m, 49 H, [CH2CH2N]x[CH2CH2NH]y[CH2CH2NH2]z, PEI ethylenes, 2.4 (br m, 2 H, NHCH2CH2COOCH2, ester linker), 2.33 (br m, 2 H, NHCH2CH2COOCH2, ester linker), 1.48 (br m, 2 H, COOCH2CH2, ester linker), 1.37 (t, 1 H, CH2CH2CH(CH3)OH, hydrolyzed ester), 1.2 (t, 3 H, CH2CH2CH(CH3)OCOCH2CH2NH, ester linker). Elemental anal. (mass fraction): (C: 0.409, H: 0.0853, N: 0.1784). Synthesis of Polymer 2. Polymer 2 was prepared according to the procedure above using 1,6-hexanediol

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diacrylate as the cross-linker. 1H NMR (400 MHz, D2O): δ ) 4.1-3.9 (br m, 2 H, NHCH2CH2COOCH2, ester linker), 3.55-3.35 (br m, 2 H, OHCH2CH2, hydrolyzed ester), 3.3-2.5 (br m, 90 H, [CH2CH2N]x[CH2CH2NH]y[CH2CH2NH2]z, PEI ethylenes, 2.45 (br m, 2 H, NHCH2CH2COOCH2, ester linker), 2.35 (br m, 2 H, NHCH2CH2COOCH2, ester linker), 1.2 (br m, 2 H, COOCH2CH2, ester linker), 1.1 (br m, 2 H, -COOCH2CH2- ester). Elemental anal. (mass fraction): (C: 0.412, H: 0.0986, N: 0.1754). Polymer Degradation Studies. Polymers were dried in vacuo overnight. Polymer (150.0 mg) was dissolved in 1.0 mL of D2O. The solutions were then adjusted to pH 5.0 or 7.0 using deuterated buffer salts and incubated at 37 °C for specified times from 0 to 60 h. 1H NMR spectra were obtained on a Varian Unity 500 MHz spectrometer with a 5-mm probe. The fraction of esters was determined by comparing δ 4.0-3.8 (ester linker [see synthesis]) and δ 3.3-2.5 ([CH2CH2N]x[CH2CH2NH]y[CH2CH2NH2]z, PEI) peaks. Complex Formation and Transfection. DNApolymer complexes (polyplexes) were prepared in 20 mM PIPES, 150 mM NaCl (pH 7.3) by addition of 150 µL of polymer to an equal volume of 3 µg of plasmid to achieve the desired polymer to DNA ratio. Polyplexes were then incubated at 4 °C for 15 min. Cells were cultured in DMEM supplemented according to ATCC protocols and plated in six-well plates at 1 × 105 cells/well 24 h prior to transfection. Immediately before transfection, the growth medium was replaced with serum-free medium, and 100 µL of polyplexes (1 µg plasmid/well) was added to each well. Transfection medium was replaced with growth medium 4 h post transfection. Luciferase expression was quantified 24 h later using a Promega luciferase assay system. Luciferase activity was measured in relative light units (RLU) using a Lumat LB 9507 luminometer (Berthold, GmbH, Germany) and converted to luciferase concentration by comparing to recombinant luciferase standards (Promega). Results were normalized to total cell protein as determined using a Bio-Rad Protein Assay Kit. All samples were run in triplicate and on two or more separate occasions. Cytotoxicity Determination. Cytotoxicity was characterized as a decrease in metabolic rate measured using the XTT assay (15). Cells were plated in 96-well plates at an initial density of 50 000 cells per well in 100 µL of growth medium for 24 h. Afterward, the growth medium was replaced with fresh, serum-free medium containing the polymer of interest. Cells were incubated with polymers for 4 h, and the medium was replaced with complete growth medium for 24 h. Fresh XTT (1 mg/mL) and coenzyme Q0 (80 µg/mL) stock were prepared each day in PBS and filter sterilized (0.22-µm syringe filter). Both components were diluted in PBS (0.5 µg/µL XTT and 0.04 µg/µL coenzyme Q0), and 10-µL aliquots were added to each well. The samples were incubated for 4 h at 37 °C, and the absorbance was read at 450 nm relative to blank wells prepared without cells. The cytotoxicity of polymer degradation products was measured by first incubating the degradable polymers for 60 h (PBS, 37 °C) before applying the polymers to the cells. Viscosity Measurements. Polymers were dissolved in PBS to concentrations of 150 mg/mL to 20 mg/mL, and pH was adjusted to 7.3. Solution viscosity, η, was determined with an AR1000-N Rheometer with a 6-cm, 0.29° plate (TA Instruments, New Castle, DE) at 25 °C. The intrinsic viscosity, ηint, was determined by ηint ) limcf0 [ln(η/η0)/c], where η0 is solvent viscosity and c is polymer concentration. The molecular weight of cross-

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linked polymers was determined using the following relationship: ηint ) KMa, where M is the molecular weight and K and a are Mark-Houwink parameters determined from PEI standards of known molecular weight. Gel Retardation and Dye Exclusion Assays. Appropriate amounts of each polymer, in 10 µL of 20 mM PIPES, 150 mM NaCl (pH 7.3), were added to an equal volume of DNA solution (500 ng/10 µL) to achieve the desired polymer to DNA ratio. Polyplexes were incubated at 4 °C for 15 min, after which 10 µL was loaded and run on a 0.75% agarose gel (70 V, 1 h). Gels were visualized with Vista Green staining (Molecular Probes, Eugene, OR). For intercalating dye exclusion assays, polyplexes were prepared as above, diluted in PBS to a concentration of 1 µg DNA/mL, and incubated at 37 °C for 24 h. Ethidium bromide (1 µM) was added and the fluorescence intensity measured (λexcitation ) 500 nm, λemission ) 600 nm). Dynamic Light Scattering (DLS) Size Determination. Polyplexes were prepared as in the above transfection procedure. Polyplexes were then diluted with PBS to a final concentration of 1 µg DNA/mL. Polyplex size determination was performed on a DynaPro-MS800 (Protein Solutions, Lakewood, NJ) with Dynamics v6.3 software. The effect of polymer degradation on polyplexes was examined by incubating the complexes at 37 °C and performing light scattering analyses at specified time intervals. Samples were run in triplicate. Transmission Electron Microscopy. Polyplexes were prepared as described above in the transfection procedure. Samples were placed on a carbon-coated grid and negatively stained with uranyl acetate. Micrographs were obtained using a Hitachi H600 transmission electron microscope, and 15-nm gold particles were used as size standards.

Forrest et al.

Figure 1. Synthesis of degradable PEI derivatives. (A) 800Da PEI is reacted with diacrylates (1,3-butanediol diacrylate shown) to generate the ester-cross-linked polymers. (B) The acrylate groups can react with either primary or secondary amines, resulting in a highly branched structure.

RESULTS

Synthesis of Degradable Polyethylenimines. The biodegradable polyethylenimines, 1 and 2, were synthesized by cross-linking 800-Da PEI using the diacrylate cross-linkers 1,3-butanediol diacrylate and 1,6-hexanediol diacrylate, respectively (Figure 1). The cross-linking was confirmed by observing extensive ester bond formation in the final product using 1H NMR. Although it is not possible to determine from NMR whether the observed cross-linking was inter- or intramolecular, the increased viscosity of 1 and 2, relative to the starting material, can be directly correlated to molecular weight using MarkHouwink theory. We determined the intrinsic viscosity of the cross-linked polymer with a cone-and-plate rheometer and, based on PEI standards, found that polymer 1 had a molecular weight of ∼14 kDa. Polymer 2 was found to have a molecular weight of ∼30 kDa. Given the starting material molecular weight of 800 Da, extensive intermolecular cross-linking apparently occurred in the formation of 1 and 2. Previous reports have indicated that PEI with molecular weight between 10 and 70 kDa are generally efficient for gene delivery (4, 16-18). Characterization of Degradable Polyethylenimines. High molecular weight PEI forms very compact particles with DNA through electrostatic attraction (19). The formation of these polyplexes can be observed as a reduction of mobility of the DNA in gel electrophoresis. We mixed DNA with increasing amounts of polymer to determine the ability of 1 and 2 to form polyplexes with DNA. Polymers 1 and 2 both completely retarded the DNA migration at a ratio of ∼1:3 (w:w) (Figure 2), indicating the formation of charge-neutral complexes.

Figure 2. Agarose gel electrophoresis of polymer/DNA complexes. (A) Polymer 1 and (B) polymer 2 (polymer:DNA, w:w): lane 1, 0.1:1; lane 2, 0.15:1; lane 3, 0.2:1; lane 4, 0.25:1; lane 5, 0.3:1; lane 6, 0.35:1; lane 7, 0.4:1; lane 8, 0.45:1.

The same ratio, 1:3 w:w, of 25-kDa PEI/DNA was found to completely retard migration of plasmid (data not shown). Therefore, amidation of PEI by the cross-linker does not appear to affect its ability to condense DNA. For efficient endocytosis and transfection to occur, the complexes must be small and compact (20). Dynamic light scattering showed that 1 and 2 formed small particles, 30 to 80 nm in diameter, over the ratio of polymer:DNA ratios examined (Figure 3A). Complexes formed in the same way using commercially available 25-kDa PEI were similar in size. The sizes of the particles were confirmed by transmission electron microscopy (TEM) using 15-nm gold particles as size standards (Figure 3B), and the complexes appeared to be compact particles. Previous reports have indicated that polyplexes in this size range are efficiently endocytosed by cells (2, 19, 21-23). Degradation of Polyethylenimine Derivatives. The degradation of gene delivery polymers is an important concern for design of safe and efficient gene therapy vehicles. The commonly used 25-kDa PEI is known to be cytotoxic in many cell lines. Further, nondegradable PEI may accumulate in vivo since there is no degradation or excretion pathway, increasing its potential cytotoxicity (4). The ester bonds in 1 and 2 are susceptible to hydrolysis at physiological conditions to form the diol

Degradable PEI Derivative for Gene Delivery

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Figure 5. Ethidium bromide exclusion from polymer/DNA complexes. White bars, 25-kDa PEI; gray bars, polymer 1; black bars, polymer 2. Fraction DNA intercalated is calculated as (F - F0)/(FDNA - F0). F, fluorescence from solution containing ethidium bromide and the complexes; F0, fluorescence from solution of ethidium bromide only; and FDNA, fluorescence from solution of DNA and ethidium bromide (N ) 3, error bars represent standard deviation).

Figure 3. (A) Dynamic light scattering of polymer/DNA polyplexes. White bars, 25-kDa PEI; gray bars, polymer 1; black bars, polymer 2. (N ) 3, error bars represent standard deviation). (B) Transmission electron microscopy of polymer/DNA complexes.

Figure 4. Degradation of polyethylenimine derivatives. O, polymer 1, pH 7.0; 0, polymer 1, pH 5.0; b, polymer 2, pH 7.0; 9, polymer 2, pH 5.0. The fraction of esters remaining was determined using 1H NMR by integrating ester peaks (COOH, linker) and normalizing to the polyethylenimine backbone ([CH2CH2N]x[CH2CH2NH]y[CH2CH2NH2]z).

linker and amino acid. Using NMR, we investigated the hydrolysis of the ester bond in the degradable polyethylenimines. Polymer 1 had a half-life of 4 h, and 2 had a half-life of 30 h, based on the remaining fraction of ester bonds (Figure 4). Degradation rates remained relatively constant between near-neutral, cytoplasmic conditions and endolysosomal conditions, pH 5.0. A further question was whether polymer degradation could weaken the complex, exposing the condensed DNA to cellular degradation mechanisms. Well-condensed DNA is inaccessible to intercalating dyes, such as ethidium bromide, which have a very high binding affinity for DNA (24). We found that after 24 h of incubation in PBS at 37 °C, 1 and 2 prevented ethidium bromide intercalation, suggesting that the partially degraded polymers are still capable of complexing with and protecting the plasmid (Figure 5). DLS studies also showed that degradable polyplexes remain compact for at least 6 h after formation. DNA was condensed with 1 and 2, and DLS measurements were taken at 0, 6, and 24 h postforma-

Figure 6. Gene delivery activity of degradable PEI derivatives. Luciferase expression in (A) MDA-MB-231 (†, p < 0.05; ‡, p < 0.02). (B) Luciferase expression in C2C12 cells transfected with polymer/DNA polyplexes (*, p < 0.005; //, p < 0.00005). White bars, 25-kDa PEI; gray bars, polymer 1; black bars, polymer 2. Luciferase expression was normalized by total cellular protein (N ) 6, error bars represent standard deviation).

tion. Polyplexes containing 1 and 2 increased in size by 25 and 50%, respectively, but remained smaller than 80 nm up to 6 h after being formed. This was similar to the control, 25-kDa PEI, which increased in size by 40% at 6 h. Beyond 6 h, complex aggregation and precipitation made accurate DLS measurements impossible. Cell Transfection Studies. We investigated the efficacy of the cross-linked polyethylenimine polymers in gene delivery to human breast carcinoma cells, MDAMB-231, and murine myoblasts, C2C12. Both cell lines were transfected in vitro with 1 µg of plasmid DNA complexed with various polymers. Gene transfection efficiency was measured as luciferase enzyme activity and normalized to total cell protein (Figure 6). The starting material, 800-Da PEI, induced no measurable gene expression over the range of polymer:DNA ratios investigated (data not shown). These data are in agreement with results from other groups that have reported a lack of transfection of various cell lines using 30 vs ∼4 h) and was more toxic to the PEI-sensitive C2C12 cell line. And of course, nondegradable 25-kDa PEI is the most toxic. The reduced toxicity of 1 and 2 relative to 25-kDa PEI may partially explain the higher levels of gene transfer mediated with degradable PEI. Reduced toxicity cannot be the only factor responsible for increased gene transfer, however. MDA-MB-231 cells are resistant to all three polymers, yet polymers 1 and 2 are ∼10-fold better transfection agents than 25-kDa PEI in these cells. In addition, polymer 2 is more effective at gene transfer in C2C12 cells than polymer 1, yet it is more toxic (polymer 2 reduced the metabolic activity of the cells twice as much as 1 at 15 µg/mL). This comparison is complicated, however, by the different molecular weights of 1 and 2. Several studies have shown that there is a correlation between increased gene transfer activity and increased molecular weight of PEI in a similar range of polymer sizes (4, 18).

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The mechanism of cytotoxicity of PEI is poorly understood. One hypothesis is that PEI aggregates on the cell surface and impairs important membrane functions (4, 30). Other possibilities are that PEI, free or in complexes with DNA, interferes with critical intracellular processes. We have shown reduced cytotoxicity even with polymers 1 and 2 that do not degrade significantly until long after most complexes have been endocytosed. Thus, our data support the importance of intra- rather than extracellular effects of PEI on the health of target cells. Polymer degradation may also enhance transfection by facilitating the “unpackaging” of the DNA-polymer complex. Tight binding of the polymer to the plasmid is expected to hinder binding of proteins required for initiation of gene expression. In fact, several studies have found that reducing the polymer/DNA binding strength by reducing the number of positive charges (31), conjugation of PEG chains (32), or decreasing the polymer molecular weight (4, 17) leads to increased gene expression. We may expect that the degraded polymer, consisting of 800-Da PEI segments, may similarly allow more facile transcription of the plasmid. In conclusion, we have shown that polymers comprising 800-Da PEI cross-linked with diacrylates exhibit low toxicity and can be more than 1 order of magnitude more efficient gene transfer agents than commercially available 25-kDa PEI. Low molecular weight PEI, in general, shows low cytotoxicity. In addition, the smaller polymers are hypothesized to allow easier transcription of the packaged plasmid DNA. However, small PEI polymers are known to be poor transfection agents. The degradable PEI reported here appears to provide the beneficial functions of 25-kDa PEI, but upon degradation we believe the polymer allows for more efficient transcription and interferes less with normal cell functions. Future studies will examine the effects of the polymer structure and degradation kinetics on specific barriers including cell uptake, escape from endocytic vesicles, and unpackaging. ACKNOWLEDGMENT

The work was supported by awards from the American Heart Association and the National Science Foundation (BES-0120101 and BES-0134163). In addition, acknowledgment is made to the donors of the Petroleum Research Fund, administered by the ACS, for partial support of this research. We thank Amy Balija for her valuable assistance with synthesis and NMR, and also Prof. Steven C. Zimmerman (University of Illinois, Urbana, IL) and Prof. Anthony McHugh (University of Illinois) for the generous use of their equipment and facilities. NOTE ADDED AFTER ASAP POSTING

In Figure 1 of the manuscript posted August 26, 2003, one of the carbonyl groups was missing in the top diacrylate. The corrected version of the manuscript was published September 8, 2003. LITERATURE CITED (1) Verma, I. M., and Somia, N. (1997) Gene therapy promises, problems and prospects. Nature 389, 239-242. (2) Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Poly(ethylenimine) and its role in gene delivery. J. Controlled Release 60, 149-160. (3) Lim, Y.-B., Kim, S.-M., Suh, H., and Park, J.-S. (2002) Biodegradable, endosome disruptive, and cationic networktype polymer as a highly efficient and nontoxic gene delivery carrier. Bioconjugate Chem. 13, 952-957. (4) Fischer, D., Bieber, T., Li, Y., Elsa¨sser, H.-P., and Kissel, T. (1999) A novel nonviral vector for DNA delivery based on

940 Bioconjugate Chem., Vol. 14, No. 5, 2003 low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res. 16, 1273-1279. (5) Gosselin, M. A., Guo, W., and Lee, R. J. (2001) Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine. Bioconjugate Chem. 2001, 989-994. (6) Lim, Y. B., Han, S. O., Kong, H. U., Lee, Y., Park, J. S., Jeong, B., and Kim, S. W. (2000) Biodegradable polyester, poly[R-(4-aminobutyl)-L-glycolic acid], as a nontoxic gene carrier. Pharm. Res. 17, 811-816. (7) Lim, Y.-B., Choi, Y. H., and Park, J.-S. (1999) A selfdestroying polycationic polymer: Biodegradable poly(4-hydroxy-L-proline ester). J. Am. Chem. Soc. 121, 5633-5639. (8) Lim, Y.-B., Kim, C.-H., Kim, K., Kim, S. W., and Park, J.-S. (2000) Development of a safe gene delivery system using biodegradable polymer, poly[R-(4-aminobutyl)-L-glycolic acid]. J. Am. Chem. Soc. 122, 6524-6525. (9) Lim, Y.-B., Kim, S.-M., Lee, Y., Lee, W.-K., Yang, T.-G., Lee, M.-J., Suh, H., and Park, J.-S. (2001) Cationic hyperbranched poly(amino ester): A novel class of DNA condensing molecule with cationic surface, biodegradable three-dimensional structure, and tertiary amine groups in the interior. J. Am. Chem. Soc. 123, 2460-2461. (10) Putnam, D., and Langer, R. (1999) Poly(4-hydroxy-L-proline ester): Low-temperature polycondensation and plasmid DNA complexation. Macromolecules 32, 3658-3662. (11) Lynn, D. M., Anderson, D. G., Putnam, D., and Langer, R. (2001) Accelerated discovery of synthetic transfection vectors: Parallel synthesis and screening of a degradable polymer library. J. Am. Chem. Soc. 123, 8155-8156. (12) Lynn, D. M., and Langer, R. (2000) Degradable poly(βamino esters): synthesis, characterization, and self-assembly with plasmid DNA. J. Am. Chem. Soc. 122, 10761-10768. (13) Pichon, C., LeCam, E., Gue´rin, B., Coulaud, D., Delain, E., and Midoux, P. (2002) Poly[Lys-(AEDTP)]: A cationic polymer that allows dissociation of pDNA/cationic polymer complexes in a reductive medium and enhances polyfection. Bioconjugate Chem. 13, 76-82. (14) Ahn, C.-H., Chae, S. Y., Bae, Y. H., and Kim, S. W. (2002) Biodegradable poly(ethylenimine) for plasmid DNA delivery. J. Controlled Release 80, 273-282. (15) Stevens, M. G., and Olsen, S. (1993) Comparative analysis of using MTT and XTT in colorimetric assays for quantitating bovine neutrophil bactericidal activity. J. Immunol. Methods 157, 225-231. (16) Remy, J.-S., Abdallah, B., Zanta, M. A., Boussif, O., Behr, J.-P., and Demeneix, B. (1998) Gene transfer with lipospermines and polyethylenimines. Adv. Drug Deliv. Rev. 30, 85-95. (17) Bieber, T., and Elsa¨sser, H.-P. (2001) Preparation of a low molecular weight polyethylenimine for efficient cell transfection. Biotechniques 30, 74-81. (18) Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Size matters: molecular weight affects the efficiency of poly(ethylenimine) as gene delivery vehicle. J. Biomed. Mater. Res. 45, 268-275.

Forrest et al. (19) Dunlap, D. D., Maggi, A., Soria, M. R., and Monaco, L. (1997) Nanoscopic structure of DNA condensed for gene delivery. Nucleic Acids Res. 25, 3095-3101. (20) Liu, G., Molas, M., Grossmann, G. A., Pasumarthy, M., Perales, J. C., Cooper, M. J., and Hanson, R. W. (2001) Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation. J. Biol. Chem. 276, 34379-34387. (21) Boussif, O., Zanta, M. A., and Behr, J.-P. (1996) Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther. 3, 1074-1080. (22) Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J.-P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301. (23) Godbey, W. T., Barry, M. A., Saggau, P., Wu, K. K., and Mikos, A. G. (2000) Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery. J. Biomed. Mater. Res. 51, 321-328. (24) Pack, D. W., Putnam, D., and Langer, R. (1999) Design of imidazole-containing endosomolytic biopolymers for gene delivery. Biotechnol. Bioeng. 67, 217-223. (25) Godbey, W. T., Wu, K. K., and Mikos, A. G. (1997) Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. Proceed. Int. Symp. Control. Relat. Bioact. Mater. 25, 230-231. (26) Protocols by Vector. J. Gene Med. (http://www.wiley.co.uk/ genetherapy/clinical/). Accessed Jan. 3, 2003. (27) Perales, J. C., Ferkol, T., Molas, M., and Hanson, R. W. (1994) An evaluation of receptor-mediated gene transfer using synthetic DNA- ligand complexes. Eur. J. Biochem. 226, 255266. (28) Hwang, S. J., and Davis, M. E. (2001) Cationic polymers for gene delivery: Designs for overcoming barriers to systemic administration. Curr. Opin. Biotechnol. 3, 183191. (29) Pack, D. W. (2001) Gene delivery polymers, in Encyclopedia of Polymer Science and Technology (J. I. Kroschwitz, Ed.) John Wiley & Sons, Inc., New York. (30) Ryser, H. J.-P. (1967) A membrane effect of basic polymers dependent on molecular size. Nature 215, 934936. (31) Erbacher, P., Roche, A. C., Monsigny, M., and Midoux, P. (1997) The reduction of the positive charges of polylysine by partial gluconoylation increases the transfection efficiency of polylysine/DNA complexes. Biochim. Biophys. Acta 1324, 27-36. (32) Banaszczyk, M. G., Lollo, C. P., Kwoh, D. Y., Phillips, A. T., Amini, A., Wu, D. P., Mullen, P. M., Coffin, C. C., Brostoff, S. W., and Carlo, D. J. (1999) Poly-L-lysine-graft-PEG-combtype polycation copolymers for gene delivery. J. Macromol. Sci., Pure Appl. Chem. 36, 1061-1084.

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