Evaluation of a Cationic Poly(β-hydroxyalkanoate) as a Plasmid DNA

EGEN, Inc., 601 Genome Way, Suite 3100, Huntsville, Alabama 35806, and Department of Chemistry, University of Alabama in Huntsville, 301 Sparkman Driv...
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Biomacromolecules 2009, 10, 1715–1719

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Evaluation of a Cationic Poly(β-hydroxyalkanoate) as a Plasmid DNA Delivery System Jeff Sparks† and Carmen Scholz*,‡ EGEN, Inc., 601 Genome Way, Suite 3100, Huntsville, Alabama 35806, and Department of Chemistry, University of Alabama in Huntsville, 301 Sparkman Drive, Huntsville, Alabama 35899 Received January 15, 2009; Revised Manuscript Received April 28, 2009

Poly(β-hydroxyalkanoates) (PHAs) are biodegradable polymers produced by a wide range of bacteria. The structures of these polymers may be tuned by controlling the available carbon source composition, but the range of functional groups accessible in this manner is limited to those that the organism is able to metabolize. Much effort has been made to chemically modify the side chains of these polymers to achieve new materials with new applications. We have previously reported the synthesis of the first cationic PHA, poly(β-hydroxyoctanoate)-co-(β-hydroxy11-(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON). Here, we report the use of this polymer as a plasmid DNA delivery system. PHON was found to bind and condense the DNA into positively charged particles smaller than 200 nm. In this manner, PHON was shown to protect plasmid DNA from nuclease degradation for up to 30 min. In addition, treatment of mammalian cells in vitro with PHON/DNA complexes resulted in luciferase expression as the result of the delivery of the encoded gene.

1. Introduction The advent of recombinant DNA technology in the early 1970s has led to the treatment of human disease through methods beyond traditional small molecules drugs. One of the most promising is the transfer of nucleic acid-based drugs into human tissue.1,2 To date, the most clinically explored method of gene transfer is that employing modified viruses. However, major concerns about safety in humans still remain, including acute immune response to the viral coat, which have resulted in significant effort toward alternative delivery methods.3 Although nonviral systems tend to avoid some of the negative properties of viral systems such as acute toxicity, chromosomal integration, and oncogenesis, they are often significantly less efficient in gene transfer. Over the past two decades, the field of nonviral gene delivery has developed as a means to overcome these obstacles through the utilization of materials with a wide array of useful properties. Nonviral delivery systems have been composed of cationic lipids,4-6 polypeptides,7-9 polyethyleneimine,10-12 polysaccharides,13-15 and polyesters.16-18 We have previously reported the synthesis of PHON, which is a modified member of the poly(β-hydroxyalkanoate) (PHA) family of polymers.19 These materials are produced by a wide range of microorganisms as a form of energy storage.20,21 PHON was designed to possess several properties seen in a variety of polymers used in applications requiring interaction with nucleic acids. First, polymers in the PHA family are known to be biocompatible and degrade with little toxicity in biological systems.22-24 Second, PHON was designed to possess properties seen in polymeric transfection reagents, including protonable amine groups that are well-known to bind electrostatically to nucleic acids.25 Third, the combination of these properties makes PHON a likely candidate as a transfection agent, facilitating the delivery of plasmid DNA into mammalian cells resulting in expression of the gene. These properties were evaluated in a series of * To whom correspondence should be addressed. Tel.: +1-256-824-6188. Fax: +1-256-824-6349. E-mail: [email protected]. † EGEN, Inc. ‡ University of Alabama in Huntsville.

experiments used to characterize polymers for gene delivery, including complexation and condensation of plasmid DNA, protection from nuclease degradation, and ultimate transfection of mammalian cells in vitro.

2. Materials and Methods 2.1. Materials. Undecylenic acid, sodium octanoate, and all salts necessary for bacterial fermentation, m-chloroperbenzoic acid (mCPBA), diethanolamine (DEA), and all solvents were purchased from Thermo-Fisher (Waltham, MA). Plasmid (pCMV encoding luciferase (pCMV-luc; 7040 bp), DNase I, Express-In (low molecular weight cross-linked linear PEI),12 and African green monkey kidney cells (COS-1) were generous gifts from EGEN, Inc. (Huntsville, AL). Agarose, tris-acetate-EDTA (TAE) and acetate buffers, dextran sulfate, and ethylenediamine tetraacetic acid (EDTA) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Mediatech, Inc. (Herndon, VA). Bicinchoninic acid (BCA) assay was purchased from Pierce (Rockford, IL). Lysis buffer (Calbiochem Protease Inhibitor cocktail, set I) was purchased from Calbiochem (San Diego, CA). 2.2. PHON Synthesis. PHON was synthesized as reported previously.19 Briefly, Pseudomonas putida GPo1 (ATCC 29347) was grown in 1 L batch cultures on modified E* media (per 1 L: 1.1 g (NH4)2HPO4, 5.8 g K2HPO4, 3.7 g KH2PO4, 10 mL of a 100 mM MgSO4 solution, 1 mL of a µ element solution: per 1 L of 1 M HCl solution, 2.78 g FeSO4 · 7H2O, 1.98 g MnCl2 · 4H2O, 2.40 g CoSO4 · 6H2O, 1.67 g CaCl2 · 2H2O, 0.17 g CuCl2 · 2H2O, 0.29 g ZnSO4 · 7H2O), 5 mM sodium octanoate, and 5 mM undecylenic acid as carbon sources. After 48 h, the cells were collected by centrifugation and freeze-dried. The resulting polymer, poly(β-hydroxyoctanoate)-co-(β-hydroxyundecenoate), PHOU, was extracted by refluxing with methylene chloride, the cell debris filtered off, and collected by precipitation in cold methanol. The side chain-terminal vinyl groups of PHOU were converted to epoxide groups by treatment with a 2-fold molar excess of mCPBA in methylene chloride to give the epoxidized polymer, poly(β-hydroxyoctanoate-co10-epoxyundecenoate) (PHOE). The epoxide groups were modified by the addition of DEA in THF. The final product, PHON, was purified by forming the HCl salts and dialyzing against pure water to remove

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excess reagents. The structure of polycationic PHON was verified by 1 H, COSY, and HSQC NMR spectroscopy and the molecular weight was determined by gel permeation chromatography (GPC). 2.3. PHON-DNA Binding and Condensation. The capacity for PHON to interact with and condense plasmid DNA was examined using agarose gel electrophoresis. pCMV-luc plasmid DNA was used for all PHON-DNA experiments. Based on a PHON Mn of 20 kDa and an average of 46 diethanolamine-terminated side chains per polymer chain, as well as an anionic phosphorus density of DNA of 330 Da/P, PHON-DNA solutions were made at a range of N/P ratios from 1:1 to 50:1. This calculation is based on 100% conversion of side chainterminal epoxide groups to the DEA-terminated derivative, however, it does not consider the ∼15% DEA impurity inherent in the PHON synthesis.19 As such, the N/P ratios, which only take into account the amine groups from PHON and not those from DEA, are likely slightly lower than reported. It is anticipated that this low-level impurity had little effect on the conclusions drawn from the subsequent experiments due to the range of N/P ratios tested. DEA was included as a negative control and a commercial PEI-based transfection reagent containing cationic amine groups, Express-In, was included as a positive control. Express-In is a modified form of PEI and was deemed suitable for use as a positive control for these studies. Acetate buffer (25 mM, pH 5.0) and plasmid (dissolved in pure water) were added to 1.5 mL centrifuge tubes. PHON (dissolved in DMSO) was then added and the mixtures vortexed for 5 s. The solutions were incubated at room temperature for 10 min to allow PHON/DNA complexes to form. To each solution of 20 µL, 2 µL loading dye (10× glycerol loading buffer) was added and each solution was added to single lanes of a 1% agarose gel prepared in 1× TAE buffer. The experiment was run in 1× TAE buffer at 80 V and 50 mA for 45 min. The gel was then stained with ethidium bromide solution and viewed using an AlphaImager 2200 MultiImage light cabinet (Alpha Innotech, San Leandro, CA). 2.4. PHON-DNA Complex Particle Size and Zeta Potential. To measure the size of PHON/DNA complexes at a range of N/P ratios, PHON/DNA complexes were prepared as above (section 2.3), diluted with 0.5 M acetate buffer and 1.5 mL pure water and filtered through a 0.8 µm nylon syringe filter into individual polystyrene cuvettes. Particle size was determined by dynamic light scattering using a Brookhaven Instruments 90 Plus Particle Size Analyzer (Holtsville, NY). Particle size was measured in a series of five runs at an angle of 90° and at 25 °C for each solution. Zeta potential analysis was performed during one cycle of 30 individual measurements at 25 °C for each solution. Both particle size and zeta potential experiments were performed under default instrument settings recommended by the manufacturer. 2.5. DNase protection. The capacity for PHON to condense plasmid DNA and protect it from DNase I degradation was examined using agarose gel electrophoresis. PHON/DNA complexes were incubated in the presence of DNase I at 37 °C for a range of time periods. Both treated (DNase I) and untreated (DNase I-free) experiments were performed concurrently. DEA was included as a negative control and Express-In was included as a positive control. Twenty five mM acetate buffer (pH 5.0) and plasmid (dissolved in deionized water) were added to 1.5 mL centrifuge tubes. PHON at a concentration of 1.5 g/L was then added and the mixtures vortexed for 5 s. The solutions were incubated at room temperature for 10 min to allow PHON/DNA complexes to form. One unit DNase (1 U/µL) was added to each solution and incubated for the appropriate time at 37 °C. Immediately following incubation, all samples were treated with 5 µL of 100 mM EDTA for 10 min to inactivate the DNase I. A total of 10 µL of a 5 mg/mL dextran sulfate solution was added to each tube, and the tubes were incubated for 2 h at room temperature to allow complete dissociation of PHON from DNA. To each solution of 20 µL, 2 µL loading dye was added and each solution was added to single lanes of a 1% agarose gel. The experiment was run in 1× TAE buffer at 80 V and 160 mA for 45 min. The gel was then stained with ethidium bromide and viewed as noted above.

Sparks and Scholz

Figure 1. Structure of poly(β-hydroxyoctanoate)-co-(β-hydroxy-11(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON; m ) 60, n ) 46, x ) 3, y ) 4).

2.6. COS-1 Transfection. PHON was examined for potential transfection ability with COS-1 cells (ATCC CRL-1650). PHON/DNA complexes were formed as above (section 2.3) at N/P ratios of 10:1, 20:1, and 50:1, containing 100 ng of DNA. To each 20 µL solution, 80 µL serum-free DMEM was added. Serum-free media was used to prevent aggregation of PHON/DNA complexes with serum proteins. A total of 100 µL of each solution was added to single wells of a 96well plate containing COS-1 cells, which had been plated previously at a density of 10000 cells per well and grown to 80% confluency. The cells were incubated in the presence of PHON/DNA complexes for 3 h, after which the media was replaced with 200 µL DMEM medium containing 10% FBS. The cells were grown for 60 h then measured for luciferase expression. Cell lysates were prepared by adding 100 µL lysis buffer to each well. Luciferase activity was determined using the Promega luciferase assay system with an Orion Microplate Luminometer (Zylux Corp., Huntsville, AL). The total protein concentrations in the cell lysates were determined using a BCA assay.

3. Results and Discussion For a polymer to serve as an effective plasmid DNA delivery agent, it must interact with the nucleic acid. Among nonviral delivery systems, this is most often accomplished through electrostatic interactions.4-18 PHON was synthesized to contain accessible protonable tertiary amines, which, at physiological pH, would provide a polycationic nature to the polymer (Figure 1). Polymers of this type have been shown to electrostatically bind and condense polyanionic nucleic acids such as plasmid DNA.25-27 The majority of polyamines such as poly(L-lysine) investigated as DNA-complexing agents or transfection vectors contain amines at the termini of short, conformationally flexible side chains.28,29 Other polyesters containing amines such as poly(β-amino esters) and poly(4-hydroxy-L-proline esters) possess cationic amines within the polymer main chain.27,30 3.1. PHON/Plasmid DNA Complex Formation. The ability of PHON to complex with plasmid DNA was examined using an agarose gel mobility shift assay. Hence, the immobilization of DNA on an agarose gel in the presence of increasing concentrations of polycationic polymer can be used as an assay to determine the point at which complete DNA charge neutralization is achieved.31,32 The tertiary amines of PHON were estimated to have pKas of 8.0-8.5 (SPARC v3.1) based on theoretical calculations. Other β-amino ester polymers also contain tertiary amines in the main polymer chains but are estimated to have lower pKas in the range of 4-8.27 This difference in pKa is presumed to be the result of three hydroxyl groups in close proximity to the tertiary amine, which are present in PHON but not poly(β-amino esters). Both structures contain

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Figure 2. Agarose gel mobility shift assay of plasmid DNA with PHON. Lane 1, plasmid only; lane 2, 1:1; lane 3, 3:1; lane 4, 5:1; lane 5, 8:1; lane 6, 10:1; lane 7, 13:1; lane 8, 25:1; lane 9, 50:1; lane 10, DEA (40K:1); lane 11, Express-In (20:1).

cationic amines at a physiological pH of 7.4. In the case of PHON, solutions containing plasmid DNA were prepared at a range of N/P ratios in 25 mM acetate buffer at a pH of 5.0 to ensure full protonation of all amines. The resulting PHON/DNA complexes remained soluble when diluted in electrophoresis running buffer (TAE, pH 7.2). Figure 2 shows the migration of plasmid DNA on an agarose gel in the presence of increasing concentrations of PHON. The N/P ratio can determine the degree of DNA complexation and overall charge density of the complex, and can also influence transfection efficiency and cytotoxicity. It was therefore important to evaluate PHON/DNA complexes at a range of N/P ratios. Polymer/DNA complexes were prepared by adding a DMSO solution of PHON to an acetate-buffered solution of DNA and vortexed. The solutions were immediately miscible and remained so upon dilution in running buffer. As depicted in Figure 2, retardation of DNA migration began at an N/P ratio of 1:1 (lane 2), as evidenced by plasmid DNA bands that parallel those in the plasmid-only control (lane 1). Full retardation was achieved at 5:1 (lane 4) as evidenced by the absence of free plasmid bands. Complexes above 5:1 showed complete DNA binding and condensation (lanes 5-9) in that PHON completely retarded plasmid DNA mobility in these wells. PHON required a slightly higher N/P ratio for complete DNA complexation compared to poly(β-amino esters), which also only contain tertiary amines as cationic species, which completely retarded DNA migration at N/P ratios as low as 2:1.27,33,34 This effect, however, was strongly dependent on the specific structures surrounding the cationic amines for a series of similar polymers. In an effort to confirm that complexation in this case was due to protonated amines covalently bound to PHON side chains, the amine reactant which was added to the terminal epoxide, DEA, was added to DNA alone and failed to retard DNA (lane 10; Figure 2). A commercial PEI-based transfection reagent, Express-In, which is known to bind and condense plasmid DNA was also included as a positive control at the manufacturers suggested N/P ratio of 20:1 and was observed to retard DNA migration completely (lane 11; Figure 2). This data suggests that the ability to complex and retard DNA migration in an agarose gel requires the large-scale connectivity of a polymer and not merely the presence of excess cations. Although a small amount of DNA remains visible in the wells after ethidium bromide staining, suggesting PHON may not bind DNA as tightly as PEI, the absence of any traces of DNA that escaped from the PHON wells is readily apparent. It is also clear that the small amount of DEA impurity carried over from the PHON functionalization reaction does not contribute to the DNA-binding observed in PHON-containing wells. Thus, PHON bound and condensed DNA, and fully neutralized the negative charge of the DNA between N/P ratios of 3:1 and 5:1.

Figure 3. Particle size and zeta potential measurements for PHON/ DNA complexes at increasing N/P ratios [9 particle size (nm), --- zeta potential (mV); n ) 3; error bars indicate standard deviation].

3.2. PHON-DNA Complex Particle Size and Zeta Potential. Gel mobility shift assays are useful in determining the extent to which polycations interact with DNA, but to be useful transfection reagents and deliver DNA to the interior of the cell, they must also be able to self-assemble plasmid into complexes small enough to enter the cell through endocytosis. For most cell types, this limit is roughly 200 nm or less.35 The ability of PHON to condense plasmid DNA into small particles was investigated by forming complexes at a range of N/P ratios, then measuring the size of the polymer/DNA particles using light scattering. In addition, the net surface charge of the resulting particles was measured in the form of zeta potential. PHON/DNA particles used for these studies were formed as described for the agarose gel experiments to make valid comparisons. Figure 3 shows the average effective diameter of the complexes as well as the zeta potential. In fully condensed form above an N/P ratio of 5:1, PHON/ DNA complexes had effective diameters ranging from 183 to 291 nm. These results are consistent with the data obtained from agarose gel electrophoresis experiments above. Below an N/P ratio of 5:1, however, a mixture of particle sizes was observed, ranging from 400 to 750 nm, suggesting a heterogeneous solution of noncondensed DNA, partially condensed DNA, and fully condensed DNA, due to the lack of sufficient concentrations of PHON. Most notably, at an N/P ratio of 1:1, particles were on the order of near-micrometer size. In this case, measured particle size increased within the time frame of the measurements, which suggests strong particle aggregation. This behavior is presumed to be due to lack of charge repulsion by charge neutralized particles. Particles at higher N/P ratios, which were found to have relatively large net positive charges, effectively resisted aggregation, which can be seen in relatively uniform particle sizes (smaller standard error, error bars, Figure 3) and smaller effective diameters. A sharp transition is observed in particle size from an N/P ratio of 1:1 to an N/P ratio of 5:1, clearly indicating the complete complexation of DNA and effective polymer/DNA complex repulsion. Interestingly, zeta potential measurements suggested DNA charge neutralization occurred at an N/P ratio of roughly 1:1. DNA is compacted into small positively charged particles at relatively low PHON concentrations and net surface charge seems to level after reaching a certain maximum value near an N/P ratio of 5:1. This behavior is consistent with that observed for a structurally similar poly(β-amino esters).27 However, the N/P ratio at charge neutralization (1:1) is in contrast to the ratio suggested by the agarose gel electrophoresis experiments of 3:1 to 5:1. Similar poly(L-ornithine) cationic polymers, which

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Figure 4. Evaluation of the ability of PHON to protect plasmid DNA from DNase I degradation. PHON (N/P ) 20:1; lanes 2-5 and 8-11), Express-In (lanes 6 and 12), and plasmid DNA only (lanes 1 and 7) were examined with (left pane) and without (right pane) DNase I at 1, 15, 30, and 45 min.

contain side chain-terminal amines in close proximity to other polar groups (-COOH), show similar DNA charge neutralization at N/P ratios of 0.8:1.36 In the same study, poly(L-lysine) showed the same behavior at 1.2:1. This is in contrast to reported PEIbased cationic polymers which possess high densities of protonable amines. Such N/P ratios are reported based on full theoretical amine protonation, although at such a high density (every third atom), it is unlikely to be complete.37 This leads to an inflated estimation of cation density, and as a result, N/P ratio at the point of charge neutralization. It is more likely, therefore, that the amount of PHON required for DNA charge neutralization is within the reasonable range of those reported for other types of cationic nitrogen-containing polymers that also bind DNA. 3.3. DNase Protection. The ability for a polymer to protect DNA from nuclease degradation is vital for its use as a gene delivery agent. DNase I protection assays may be used to demonstrate stable complex formation between polymer and DNA. DNase I is a potent plasmid DNA nuclease and readily cleaves unprotected DNA. To test the ability of PHON to prevent such degradation of complexed plasmid DNA, and in addition to verify the physical association of PHON with plasmid DNA, an experiment was performed at an N/P ratio of 20:1 to ensure full complexation of plasmid DNA in the samples. The PHON/DNA complexes were incubated in the presence of DNase I at 37 °C for a range of time periods. Both DNase I-treated and untreated experiments were performed for comparison. Dextran sulfate was used to displace PHON from plasmid DNA in order for the remaining intact nucleic acids to move through the gel. PHON was able to protect the plasmid from degradation for up to 30 min at 37 °C and pH 5.0, based on the appearance of intact plasmid DNA in lanes 2-4 (Figure 4). In contrast, uncomplexed plasmid DNA is completely degraded after only 1 min (lane 1). As observed in lane 5, after 30 min of exposure to DNase I, no bands for either the open circular or supercoiled DNA are visible. This compares favorably with PEI-based systems, which protected plasmid DNA for up to 15 min (PEI-PEG)38 and 45 min (Express-In control). When PHON/DNA complexes are incubated without DNase I (lanes 7-11), for up to 45 min, no degradation is observed, as indicated by strong bands for both open circular and supercoiled plasmid DNA. The relatively weak intensities of the intact bands in the treated lanes (lanes 2-4) compared to the untreated lanes

Figure 5. PHON transfection of COS-1 cells with pCMV-luciferase. Luciferase transfection was measured in relative light units (RLU) and viability of cells after transfection was determined by total protein, both as a function of N/P ratio. PO ) plasmid only (n ) 3; error bars indicate standard deviation).

(lanes 7-11) could be due to fractional degradation of DNA. The presence of a significant fraction of DNA remaining in the wells could suggest PHON binds plasmid DNA very well, and attempts to dissociate the two components, even after several hours, is difficult. The exact nature of the degradation process is unclear. It appears that the majority of the exposure of plasmid DNA to nuclease, and the subsequent degradation, is not a result of degradation of the main chain polyester. Incubation of the untreated sample for 45 min (lane 11) appears to result in no major increase in naked DNA compared to the samples, which were incubated for shorter times (lanes 8-10), as judged by the intensity of the DNA bands. This suggests that PHON protects plasmid DNA from nuclease degradation for extended periods of time compared to naked DNA. 3.4. COS-1 Transfection. PHON was also tested for its ability to transfect mammalian cells in vitro. PHON formulations containing luciferase plasmid were added to African green monkey kidney cells (COS-1) and examined for expression as a function of N/P ratio. At N/P ratios of 10:1 and 50:1 PHON produced little expression of luciferase when compared to naked plasmid. However, the 20:1 ratio is near optimal and shows relatively high expression of luciferase (Figure 5). Although all three samples contain fully condensed, cationic complexes, the intermediate polymer content (20:1) appears to have the

Poly(β-hydroxyalkanoate) for Plasmid DNA Delivery

necessary balance of size (∼180 nm) and zeta potential (36 mV) to pass the cell membrane and release the plasmid for transport to the nucleus. However, for this amount of polymer, the level of viable cells, based on total protein and compared to plasmidonly wells, decreased to 65%. At 50:1, viability decreases even further to 33% (Figure 5). It should be noted that the introduction of foreign material into cells as in transfection is inherently associated with a certain level of toxicity. Although many commercial transfection reagents show low levels of toxicity, some of the most effective systems at shuttling plasmid into the cells and into the nuclei are designed to do so by destabilizing membranes, specifically the lysosomal membrane. It is likely this behavior results in some level of outer cell membrane destabilization, which is observed in toxicity. In contrast, wells containing 10:1 complexes showed virtually no toxicity and an intermediate level of gene expression. It can be assumed that the optimum balance between toxicity and transfection is between the N/P ratios of 10:1 and 20:1.

4. Conclusions Confirmation of covalent modification of PHOE with amines was achieved through a study of the interaction of the polymer with plasmid DNA. Polycationic behavior was observed for PHON in DNA complexation experiments, as well as charge neutralization and condensation studies. The stability of the interaction of PHON with plasmid DNA was determined to be substantial, as the polymer was able to protect DNA from nuclease degradation for extended periods of time. This data suggested PHON might be suitable as a transfection agent to promote gene expression in mammalian cell culture. Acknowledgment. This work was supported in part by the Alabama Space Grant Consortium and the Partnership for Biotechnology Research. J.S. would like to thank EGEN, Inc. for the use of their facilities for much of this work.

References and Notes (1) (2) (3) (4)

Mulligan, R. Science 1993, 260, 926. Crystal, R. Science 1995, 270, 404. Thomas, C.; Ehrhardt, A.; Kay, M. Nat. ReV. Genet. 2003, 4, 346. Felgner, P.; Gadek, T.; Holm, M.; Roman, R.; Chan, H.; Wenz, M.; Ringold, G.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7413. (5) Ambegia, E.; Ansell, S.; Cullis, P.; Heyes, J.; Palmer, L.; MacLachlan, I. Biochim. Biophys. Acta 2005, 1669, 155. (6) Akinc, A.; Zumbuehl, A.; Goldberg, M.; Leshchiner, E.; Busini, V.; Hossain, N.; Bacallado, S.; Nguyen, D.; Fuller, J.; Alvarez, R.; Borodovsky, A.; Borland, T.; Constein, R.; de Fougerolles, A.; Dorkin, J.; Jayaprakash, K.; Jayaraman, M.; John, M.; Koteliansky, V.; Manoharan, M.; Nechev, L.; Qin, J.; Racie, T.; Raitcheva, D.; Rajeev,

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(7) (8)

(9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38)

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K.; Sah, D.; Soutschek, J.; Toudjarska, I.; Vornlocher, H.; Zimmerman, T.; Langer, R.; Anderson, D. Nat. Biotechnol. 2008, 26, 561. Cotten, M.; Langle-Roualt, F.; Kirlappos, H.; Wagner, E.; Metchler, K.; Zenke, M.; Beug, H.; Birnistiel, M. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 4033. Konstan, M.; Davis, P.; Wagener, J.; Hilliard, K.; Stern, R.; Milgram, L.; Kowalczyk, T.; Hyatt, S.; Fink, T.; Gedeon, C.; Oette, S.; Payne, J.; Muhammad, O.; Ziady, A.; Moen, R.; Cooper, M. Hum. Gene Ther. 2004, 15, 1255. Itaka, K.; Ohba, S.; Miyata, K.; Kawaguchi, H.; Nakamura, K.; Takato, T.; Chung, U.; Kataoka, K. Mol. Ther. 2007, 15, 1655. Boussif, O.; Lezoualac’h, F.; Zanta, M.; Mergny, M.; Scherman, D.; Demeneix, B.; Behr, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297. Fewell, J.; Matar, M.; Slobodkin, G.; Han, S.; Rice, J.; Hovanes, B.; Lewis, D.; Anwer, K. J. Controlled Release 2005, 109, 288. Matar, M.; Slobodkin, G.; Rea-Ramesy, A.; Brunhoeber, E.; Skoyen, J.; Fewell, J.; Lewis, D.; Anwer, K. J. Biomed. Nanotechnol. 2006, 2, 53. MacLaughlan, F.; Mumper, R.; Wang, J.; Tagliaferri, J.; Gill, I.; Hinchcliffe, M.; Rolland, A. J. Controlled Release 1998, 56, 259. Davis, M.; Pun, S.; Bellocq, N.; Reineke, T.; Popielarski, S.; Mishra, S.; Heidel, J. Curr. Med. Chem. 2004, 11, 179. Jiang, H.; Kwon, J.; Kim, Y.; Kim, E.; Arote, R.; Jeong, H.; Nah, J.; Choi, Y.; Akaike, T.; Cho, M.; Cho, C. Gene Ther. 2007, 14, 1389. Rolland, A.; Mumper, R. AdV. Drug DeliVery ReV. 1998, 30, 151. Arote, R.; Kim, T.; Kim, Y.; Hwang, S.; Jiang, H.; Song, H.; Nah, J.; Cho, M.; Cho, C. Biomaterials 2007, 28, 735. Yamanouchi, D.; Wu, J.; Lazar, A.; Kent, K.; Chu, C.; Liu, B. Biomaterials 2008, 29, 3269. Sparks, J.; Scholz, C. Biomacromolecules 2008, 9, 2091. Findley, R. Appl. EnViron. Microbiol. 1983, 45, 71–78. Steinbuchel, A. Polyhydroxyalkanoic Acids; Macmillan Publishers: NewYork, NY, 1992. Gogelewski, S.; Jovanovich, M.; Perren, S.; Dillon, J.; Hughes, M. Biomed. Mater. Res. 1993, 27, 1135. Chen, G.-Q.; Wu, Q. Biomaterials 2005, 26, 6565. Qu, X.-H.; Wu, Q.; Zhang, K.-Y.; Chen, G. Biomaterials 2006, 27, 3540. De Laporte, L.; Rea, J.; Shea, L. Biomaterials. 2006, 27, 947. Kabanov, V.; Kabanov, A. AdV. Drug DeliVery ReV. 1998, 30, 49. Lynn, D.; Langer, R. J. Am. Chem. Soc. 2000, 122, 10761. Hashida, M.; Takemura, S.; Nishikawa, M.; Takakura, Y. J. Controlled Release 1998, 53, 301. Kwoh, D.; Coffin, C.; Lollo, C.; Jovenal, J.; Banaszczyk, M.; Mullen, P.; Phillips, A.; Amini, A.; Fabrycki, J.; Bartholomew, R.; Brostoff, S.; Carlo, D. Biochim. Biophys. Acta 1999, 1444, 171. Putnam, D.; Langer, R. Macromolecules 1999, 32, 3658. Lim, Y.; Choi, Y.; Park, J. J. Am. Chem. Soc. 1999, 121, 5633. Gonzalez, H.; Hwang, S.; Davis, M. Bioconjugate Chem. 1999, 10, 1068. Lynn, D.; Anderson, D.; Putnam, D.; Langer, R. J. Am. Chem. Soc. 2001, 123, 8155. Akinc, A.; Lynn, D.; Anderson, D.; Langer, R. J. Am. Chem. Soc. 2003, 125, 5316. Zauner, W.; Orgis, M.; Wagner, E. AdV. Drug DeliVery ReV. 1998, 30, 97. Ramsay, E.; Gumbleton, M. J. Drug Target. 2002, 10, 1. Suh, J.; Paik, H.; Hwang, B. Bioorg. Chem. 1994, 22, 318. Gebhart, C.; Sadibhatla, S.; Vinogradov, S.; Lemieux, P.; Alakhov, V.; Kabanov, A. Bioconjugate Chem. 2002, 13, 937.

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