Bioconjugate Chem. 2005, 16, 1204−1208
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Membrane Activity and Transfection Ability of Amphipathic Polycations as a Function of Alkyl Group Size Darren H. Wakefield,† Jason J. Klein,† Jon A. Wolff,‡ and David B. Rozema*,† Mirus Bio Corporation, 505 S. Rosa Road, Madison, Wisconsin 53711, and Waisman Center, Department of Pediatrics and Medical Genetics, University of WisconsinsMadison, Madison, Wisconsin 53705 . Received March 8, 2005; Revised Manuscript Received July 15, 2005
Cationic membrane disruptive peptides such as melittin would appear to have attributes necessary for DNA delivery: DNA binding via electrostatic interactions and membrane lysis to enable cytoplasmic delivery. However, the relatively small overall charge of membrane disruptive peptides results in weak interactions with DNA. As a model of cationic membrane disruptive peptides, amphiphilic polyvinyl ethers were synthesized. The number of positively charged groups incorporated into these polymers is substantially greater than membrane-active peptides, which enables these polymers to form stable complexes with DNA. By varying the length of the hydrophobic groups incorporated into the polymer from one to four carbons, the dependence of membrane activity on side chain length was established. The ability of these polymers to transfect DNA in tissue culture was tested, and it was found that transfection efficiency is dependent upon the membrane disruptive activity of the polymer. Comparison of melittin and synthetic polymers suggests that transfection and toxicity appear to be dependent upon their affinity for DNA. This demonstration of relationships among membrane lysis, transfection, DNA binding, and polymer side-chain composition establishes a new class of transfection reagents and may guide in the design of polymers and formulations that will enable efficient in vivo transfection.
INTRODUCTION
To protect DNA from nucleases and facilitate gene delivery, DNA is complexed with a cationic agent, either a cationic lipid, lipoplexes, or a cationic polymer, polyplexes (1). Either “plex” enters the cell via endocytosis and is contained in intracellular vesicles that mature into degradative lysosomes. To escape to the cytoplasm and ultimately gain entry to the nucleus, some form of endosomolytic activity must be incorporated into the delivery system. We report in this communication the creation of polymers that simultaneously possess DNA complexation and membrane-disruptive properties. Use of these polymers greatly simplifies formulation of polyplexes and improves biologically relevant delivery of DNA to cells. Membrane-disruptive polycations in polyplexes is a relatively uncommon strategy to promote endosomolysis. All membrane-lytic peptides and polymers are amphipathic in character, i.e., they contain both hydrophilic and hydrophobic functional groups. The most studied membrane-disruptive polymers are negatively charged peptides that form amphiphilic helices capable of membrane insertion. Formation of the helices is pH-dependent and occurs in the acidic environment of the mature endosome or lysosome. Examples of these types of peptides are those derived from viral coat proteins, such as the influenza hemagglutinin-like peptide GALA (2, 3). Synthetic polymers polyethyl and polypropyl acrylic acids also have pH-dependent membrane disruptive (4, 5) and transfection (6) activities. In addition to membrane-active anionic peptides, there are also membrane disruptive cationic peptides, such as melittin (GIGAILKVLATGLPTLISWIKNKRKQ) and the * Corresponding author e-mail:
[email protected]. † Mirus Bio Corporation. ‡ University of WisconsinsMadison.
lysineanalogueofGALA(7)(KALA: WEAKLAKALAKALAKHLAKALAKALKACEA) that have been studied for their ability to transfect DNA. However, the interaction between DNA and small polycations such as melittin and KALA (both with +5 net charge) are relatively weak compared to larger polycations, resulting in the formation of particles that dissociate in the presence of physiological salt (8, 9). Therefore, it is not surprising that recent studies using cationic membrane-disruptive peptides as transfection reagents covalently attach the peptides to larger polycations such as poly-L-lysine and polyethyleneimine (10-12). To combine DNA binding ability and membrane disruptive activity into the same polymer, we synthesized polycations that mimic the amphipathic nature of membrane-active peptides, but with charge sufficient for the formation of stable DNA complexes. Since the hydrophobic/ hydrophilic ratio required to attain membrane lysis has, in contrast to polyanions (4, 5), not been considered for polycations, we systematically varied the size of the hydrophobic groups. EXPERIMENTAL SECTION
Materials. 2-Vinyloxyethylphthalimide was synthesized according to literature procedure from phthalimide and 2-chloroethyl vinyl ether (13). 2-Chloroethyl vinyl ether, phthalimide, ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, and boron trifluoride diethyl etherate (BF3‚OEt2) were purchased from Aldrich. Methyl vinyl ether was purchased from Matheson. Melittin was synthesized by an Applied Biosystems 433A peptide synthesizer using standard peptide synthesis methods. Synthesis of Polyvinyl Ethers. 2-Vinyloxyethylphthalimide (1 g, 4.6 mmol) and methyl vinyl ether (0.267 g, 4.6 mmol), ethyl vinyl ether (0.332 g, 4.6 mmol), propyl vinyl ether (0.396 g, 4.6 mmol), or butyl vinyl ether
10.1021/bc050067h CCC: $30.25 © 2005 American Chemical Society Published on Web 08/31/2005
Bioconjugate Chem., Vol. 16, No. 5, 2005 1205
Amphipathic Polycations
(0.460 g, 4.6 mmol) were dissolved in 25 mL of anhydrous dichloromethane. These solutions were then brought to -78 °C, BF3‚OEt2 (0.065 g, 0.46 mmol) was added, and the reaction was allowed to proceed for 3 h at -78 °C. The polymerization was then stopped by the addition of 50/50 mixture of ammonium hydroxide in methanol. The solvents were then removed by rotary evaporation. The polymer was then dissolved in 30 mL of 1,4-dioxane/ methanol (2/1). To this solution was added hydrazine (0.147 g, 46 mmol), and the mixture was heated to reflux for 3 h. The solvents were then removed by rotary evaporation, and the resulting solid was then brought up in 20 mL of 0.5M HCl and refluxed for 15 min, diluted with 20 mL distilled water, and refluxed for additional 1 h. This solution was then neutralized with NaOH, cooled to room temperature, transferred to 3500 molecular weight cutoff cellulose tubing, and dialyzed for 24 h (2 × 20L) against distilled water and lyophilized. Separation of Polymers by Size Exclusion Chromatography. Lyophilized polymer was dissolved in water. A 5 mL aliquot was placed onto a 2.5 × 20 cm column packed with Sephacryl S-200. The column was eluted with 25 mM (NH4)2CO3 5% ethanol, pH 9. Ten microliter fractions were collected after the dead volume (25 mL). Fractions were then assayed by trinitrobenzenesulfonic acid for amine content. The eluted polymer was pooled into four equal fractions and lyophilized. Fractions were submitted for carbon, nitrogen, and hydrogen elemental analysis at Galbraith Laboratories (Knoxville, TN). Due the hydroscopic nature of the polymers, only the carbon and nitrogen data were used to determine monomer incorporation ratios. The fourth fractions showed the most consistent incorporation ratios and were therefore used in subsequent assays. DNA Labeling. Covalent labeling of plasmid DNA with fluorophores was performed using tetramethylrhodamine (TMR) LabelIT reagent (Mirus Corp., Madison, WI) as previously described (14). LabelIT reagents are nitrogen mustard alkylating reagents that react primarily with guanine bases (15). Briefly, plasmid DNA and a solution of LabelIT reagent in methyl sulfoxide (100 mg/mL) were mixed in 1 mL of 10 mM HEPES, pH 7.5, at a LabelIT/DNA ratio of 5:1 (w/w). The reaction mixture was incubated for 1 h at 37 °C. Labeled DNA was then precipitated three times in 70% ethanol with 0.2 MNaCl. DNA Condensation Assay. The collapse of TMRlabeled DNA was assessed using a quantitative assay based on condensation-induced quenching of a fluorophore covalently attached to DNA (14). Briefly, TMRDNA (10 µg) was mixed with various quantities of polyvinyl ethers or melittin in 0.5 mL of 10 mM HEPES, pH 7.5. Rhodamine fluorescence of the samples was measured using a Varian spectrofluorometer (excitation wavelength (λex) of 546 nm; emission wavelength (λem) of 576 nm) at room temperature. A graph of fluorescence as a function of added polymer was composed of two regions: a condensation phase where each addition of polymer resulted in a linear decrease in fluorescence, and the condensed phase where the labeled DNA was fully condensed. The concentration of polymer required for condensation was calculated by fitting of the steepest portion of the condensation phase and condensed phase to linear equations and determining the intercept of the two lines. A summary of data is presented in Table 2. Titration curves are presented as supplementary data. Determination of Polymer Size. The molecular weights of the polymers were then determined by GPC using Eprogen Inc. CATSEC100, CATSEC300, and CATSEC1000 columns in series. The running buffer was 200
Table 1. Composition and Size of Amphipathic Polyvinyl Ethers
polymer
formula from EA
mw per charge by EAa
mw per charge by DNA (ratio of two charge density determinations)
C5N C7N C6N C7N
147 165 153 164 680
154 (1.0) 147 (0.9) 153 (1.0) 166 (0.8) 891 (0.8)
methyl ethyl propyl butyl mellitin
a Molecular weight of polyvinyl ethers per charge is calculated as amine free base. Melittin’s molecular weight is calculated using a trifluoroacetate counterion.
Table 2. Characterization of Amphipathic Polyvinyl Ethers polymer
molecular weight of polymera (charge)
[NaCl]* (M)b
methyl ethyl propyl butyl melittin
8500 (57) 2500 (15) 4200 (27) 5000 (30) 3275 (5)
0.6 0.4 0.5 0.5 0.1
a Molecular weight based on free bases, number of charges per polymer in parentheses. b [NaCl]*,the concentration of NaCl required to displace polycation from plasmid DNA.
mM NaCl and 10% MeOH. Polyvinylpyridine standards (1.5, 6.5 12, 21, 47, 65, 78, 115, 287, and 1,260 kD) were used as the calibration curve. To detect the polyvinyl ethers, they were labeled with Cy3 with Cy3-N-hydroxysuccinimide (Amersham), which was detected at 555 nm. The log of the molecular weight of the polyvinylpyridine standards were linear with respect to retention time, and from this relationship we were able to estimate the molecular at any retention time. From this relationship, we calculated the molecular weight Mw of the polymers according to the equation Mw) ∑tAtMt/∑tMt where At is the absorbance measured at time t and Mt is the molecular weight based upon standards that elutes at that time. Polycation-pDNA Stability Assay. To determine the relative strength of the interactions between the peptides and polycations, the concentration of salt required to displace a polycation from TMR-labeled DNA was determined. TMR-DNA (10 µg) was condensed at N:P ratio of 2:1, as determined by condensation assays, in 10 mM HEPES, pH 7.5. To this solution were added increasing concentrations of NaCl from a 5 M stock. The fluorescence of the samples was then measured (λex) 546 nm λem) 576 nm). A graph of fluorescence as a function of [NaCl] was a composed of two regions: a lag phase where increasing salt resulted in little or no increase in fluorescence and a decondensation phase where each addition of salt resulted in a linear increase in fluorescence. The concentration of NaCl required for decondensation was calculated by fitting of the lag phase and decondensed phase to a linear equation and determining the intercept of the two lines. Particle Sizing. To a solution of 5 mM HEPES pH 7.5, 150 mM NaCl, and 10 µg/mL plasmid pCIluc was added polyvinyl ether to 20 µg/mL or polyethyleneimine to 10 µg/mL. After 30 min, the size of the particles was measured by light scattering at 532 nm using a Brookhaven Instruments Corporation, ZetaPlus Particle Sizer, I90. Liposome Lysis. Egg phosphatidylcholine (10 mg) was hydrated with 1 mL of buffer containing 100 mM carboxyfluorescein (CF) and 10 mM HEPES pH 7.5.
1206 Bioconjugate Chem., Vol. 16, No. 5, 2005 Scheme Ethers
1.
Synthesis
of
Amphipathic
Wakefield et al. Polyvinyl
Liposomes were then extruded through 100-nm pore polycarbonate filters (Nucleopore, Pleasanton, CA). Unentrapped CF was removed by size exclusion chromatography using Sepharose 4B-200 eluting with 10 mM HEPES, pH 8, 0.1 M NaCl. A 200 µL aliquot of the CFloaded liposomes was added to 1.8 mL of isotonic buffer. Fluorescence (λex ) 488, λem ) 540) was measured 30 min after addition of 0.25 µg of polymers or melittin to vesicle suspensions. At the end of each experiment, vesicles were disrupted by the addition of 40 µL of a 1% Triton X-100 solution to determine maximal lysis. Transfection. Hepa-1clc7 cells (mouse hepatoma) were cultured in 1 mL of Dubelco’s modified Eagle Media containing 10% fetal bovine serum. Amphipathic polyvinyl ethers, melittin, or PEI was added to plasmid DNA at a 2:1 wt:wt ratio, which corresponds to approximately a 4:1 N:P ratio (10 µg/mL, pCIluc; prepared according to published procedure (16)) in 0.5 mL of 150 mM NaCl and 5 mM HEPES, pH 7.5. Formulated DNA (1 µg) was added to the cells, and 48 h later the confluency of the cells was estimated. All samples, except melittin, had levels of confluency comparable to cells grown in the absence of polymer. The cells were harvested and assayed for luciferase expression as previously reported (17). The amount of transfection is average transfection for two separate wells of cells. Cell Toxicity Assay. After 48 h of incubation with DNA-polycation complexes according to transfection protocol, 500 µg of MTT in PBS solution (5 mg/mL) was added to the culture for 1 h. The media were then aspirated, and the cells were washed with 1 mL of PBS. The cells were then dissolved by addition of 1 mL of 10% SDS in 10 mM HCl. After rocking the samples overnight at room temperature, 300 µL of solution was transferred into 96-well plates and the absorbance of the solution at 410 nm was measured using a Molecular Devices SpectraMax Plus (Sunnyvale, CA). Absorbances were normalized to cells grown in the absence of transfection complexes. Each experiment was average of six experiments. RESULTS AND DISCUSSION
Amphiphilic polyvinyl ethers were synthesized from amino and hydrophobic vinyl ether monomers. Amino groups were incorporated into polyvinyl ethers using an amine-protected phthalimido vinyl ether (13). Alkyl groups were incorporated using methyl, ethyl, propyl, or butyl alkyl vinyl ethers (R ) CH3, CH2CH3, CH2CH2CH3, or CH2 CH2CH3 in Scheme 1). After removal of the phthalimido groups, the polymers were dialyzed to remove salts, monomers, and oligomers. The polymers were further purified using size exclusion chromatography to remove unreacted monomers and oligomers. The incorporation of monomers into the polymers was measured by elemental analysis, which determines the ratio of carbon to nitrogen and thereby the ratio of amine to alkyl groups in the polymer (Table 1). By elemental analysis and DNA condensation (vide infra), we determined that the smaller size exclusion fractions had amine
Figure 1. Transfection and lysis as function of side chain composition. Transfection ability (bars, see Table 3) and liposome lysis activity of polymers as a function of polymer side chain length.
to alkyl group incorporation ratios that were more consistent with each other in the series and with the feed ratios. For this reason, only comparisons between these fractions are presented in this report. In addition to measuring amine incorporation ratios by elemental analysis, the charge density, and consequently the amine content, of the polymer was measured by determining the amount of polymer required to condense fluorophore-labeled DNA (Table 1) (14). As one would expect, the DNA condensing ability of a polycation correlates with the number of positively charged functional groups. For comparison, the charge density of the amphipathic peptide melittin was also measured. Note that, in low salt, melittin’s charge density as measured by its ability to condense DNA does correlate with its charge. The molecular weights, and thereby the number of amines, of the polymers were measured using size exclusion columns. The smallest polymer, the ethyl vinyl ether-derived polymer, had an average charge of +15, which is well above melittin and KALA. More importantly for transfection is that these polymers bind DNA in the presence of physiological concentrations of salt, which roughly corresponds to 150 mM NaCl. To establish the relative stabilities of the complexes between the polycations and DNA, we determined the amount of salt required to induce release of DNA from the complex ([NaCl]* in Table 2). Using fluorophore-labeled DNA condensed at a 2:1 amine-to-phosphate ratio, we measured the amount of NaCl to induce DNA release. For the oligocation melittin, the DNA complex completely dissociates below physiological salt concentrations, ca. 70 mM NaCl. In contrast, the amphiphilic polyvinyl ethers are displaced at much higher concentrations of salt (>400 mM). The membrane-lytic abilities of the amphipathic polyvinyl ethers were measured by analyzing their ability to lyse carboxyfluorescein-loaded liposomes. Similar to data obtained by others studying membrane-lytic polyanions (4, 5), the abilities of the polyvinyl ethers to lyse liposomes are indeed dependent on the size of the hydrophobic chain. The polymer containing butyl groups is the most lytic followed by the propyl and ethyl in order of lytic ability (Figure 1, circles). The butyl-containing polymer possesses lytic activity comparable to melittin (80% lysis with 0.25 µg of melittin). The methyl-derived polymer was completely inactive for lysis.
Bioconjugate Chem., Vol. 16, No. 5, 2005 1207
Amphipathic Polycations Table 3. Transfection and Toxicity of Amphipathic Polycations in Hepa Cells polycation
ng luciferase per well
% MTTa
methyl vinyl ether ethyl vinyl ether propyl vinyl ether butyl vinyl ether polyethyleneimine melittin
0.068 ( 0.029 0.054 ( 0.028 0.30 ( 0.12 19 ( 14 1.1 ( 0.4 0.015 ( 0.003
94 ( 11 100 ( 3 90 ( 4 94 ( 9 105 ( 5 67 ( 12
a Percent of metabolized MTT relative to cells grown in absence of DNA complexes.
The ability of the amphiphilic polyvinyl ethers to transfect DNA was tested at various (N/P) ratios using a mouse hepatoma cell line. Optimal transfections are observed for the polyvinyl ethers at a 4/1 N/P ratio (Figure 1: bars and Table 3: column 1). Branched PEI, a standard polyplex-based transfection reagent, and melittin were also tested in the assay at a 8:1 and 4:1 N:P ratios, respectively. The ability of the polyvinyl ethers to transfect DNA positively correlated with their membrane lytic activity; the highest levels of transfection were obtained with the polymers containing butyl groups, whereas transfection levels obtained using polymers containing shorter alkyl side chains were progressively lower. These data suggest that transfection activity of the polymers is derived at least in part from membrane activity. Similar results were obtained using the HEK (human epithelial kidney) 293 cell line (see Supporting Information). The sizes of the complexes are important for transfection. Complexes were formulated at physiologically relevant ionic strength (150 mM NaCl), which results in particles that are roughly 900 ( 200 nm in size. Formulation at low salt conditions does result in smaller particles (ca 100 nm); however, these particles are roughly 100 times less effective transfection reagents. This dependence upon size is seen for other transfection reagents (18, 19). The reason for the dependence of transfection upon complex size is a matter of debate; however, the dependence appears to be common to all transfection reagents and is an impediment that needs to be removed for most in vivo delivery applications. One would expect membrane active polymers to have inherent cell toxicity. Cell confluencies, a qualitative measure of cell viability based upon cell coverage on the well surface as judged by microscopy, indicated little or no toxicity at the levels used for transfection. We do observe decreased levels of cell confluency if higher concentrations of butyl-containing polymer were used. To get a more quantitative measure of cell viability, we assayed cell metabolism using methylthiazolyldiphenyltetrazolium bromide (MTT). MTT is reduced by mitochondrial dehydrogenases of metabolically active cells into a water insoluble blue formazan, which may be quantified by absorbance spectroscopy. Testing the transfection complexes we found that there appears to be slight, although only the propyl-containing polymer is statistically significant, toxicity (Table 3). In general, more toxicity was observed for 293 cells (see Supporting Information), which, as expected, correlates with membrane lytic activity. The polycation PEI is, to date, the most effective and studied polyplex-based transfection reagent. However, under the conditions reported in Table 3, the butyl-based polyvinyl ether is 10-fold more effective at DNA delivery. Transfection levels comparable to the butyl polyvinyl ether can be achieved with PEI at higher N:P ratios and if the transfection is performed in serum free conditions.
These requirements are achievable in vitro but put severe limitations on the use of PEI in vivo. As might be predicted given its weak interaction with DNA, melittin performed extremely poorly as a transfection reagent. Poor transfection efficiency was observed for melittin at all N:P ratios with greater toxicity at increased levels of melittin. DNA-melittin complexes dissociate under physiological conditions (Table 3), which we believe is the cause of poor transfection and toxicity. In contrast, the relatively strong interaction between the butyl-containing polyvinyl ether and DNA attenuates its membrane lytic activity and, presumably, its cell toxicity. The butyl-containing polymer with DNA has slight toxicity (Table 3), but polymer alone without DNA causes observable toxicity (75 ( 5% MTT metabolized relative to untreated cells). The inhibition of polymer membrane activity by DNA is also demonstrated in the observation that toxicity is observed for the butyl-containing polymer at higher N:P ratios. For amphiphilic polycation-based transfections, membrane lytic activity may be masked until endocytosis forces the membrane surface to come in close contact with the polymer. Contact with the membrane causes displacement of the polymer from DNA, membrane lysis, and, ultimately, transfection. A similar displacement of cation and DNA is hypothesized to occur in cationic lipid-based transfections (20). The transfection activity of the butyl-containing polyvinyl ether may then be a result of a fine balance between DNA and membrane binding, which implies that modifications of either interaction would greatly affect transfection efficiency. We believe this is the first study to demonstrate a correlation among transfection activity, membrane lytic ability, and polymer side-chain composition for polycations, which creates a new class of transfection reagents and will aid in the design of polymers and formulations that will enable efficient in vivo transfection. ACKNOWLEDGMENT
The authors thank Sean Monahan for synthesis of melittin, Stephanie Bertin for tissue culture work, and So Wong and Vladimir Budker for helpful discussions. Supporting Information Available: Charge density titrations, salt displacement titrations, and 293 cell transfection data. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Felgner, P., et al. (1997) Nomenclature for synthetic gene delivery systems. Hum Gene Ther. 8, 511-2. (2) Li, W., Nicol, F., and Szoka, F. C., Jr. (2004) GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery. Adv. Drug Delivery Rev. 56, 967-85. (3) Plank, C., Oberhauser, K., Mechtler, K., and Koch, C. (1994) The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. J. Biol. Chem. 269, 12918-12924. (4) Tirrell, D. A., Takigawa, D. Y., and Seki, K. (1985) pH sensitization of phospholipid vesicles via complexation with synthetic poly(carboxylic acid)s. Ann. N.Y. Acad. Sci. 446, 237-48. (5) Murthy, N., Robichaud, J. R., Tirrell, D. A., Stayton, P. S., and Hoffman, A. S. (1999) The design and synthesis of polymers for eukaryotic membrane disruption. J. Controlled Release 61, 137-43. (6) Cheung, C. Y., Murthy, N., Stayton, P. S., and Hoffman, A. S. (2001) A pH-sensitive polymer that enhances cationic lipidmediated gene transfer. Bioconjugate Chem. 12, 906-10.
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Wakefield et al. Amino Functions. J. Polym. Sci.: Part A: Polym. Chem. 26, 3361-3374. (14) Trubetskoy, V. S., Slattum, P. M., Hagstrom, J. E., Wolff, J. A., and Budker, V. G. (1999) Quantitative assessment of DNA condensation. Anal. Biochem. 267, 309-13. (15) Slattum, P. S., Loomis, A. G., Machnik, K. J., Watt, M. A., Duzeski, J. L., Budker, V. G., Wolff, J. A., and Hagstrom, J. E. (2003) Efficient in vitro and in vivo expression of covalently modified plasmid DNA. Mol. Ther. 8, 255-63. (16) Danko, I., Williams, P., Herweijer, H., Zhang, G., Latendresse, J. S., Bock, I., and Wolff, J. A. (1997) High expression of naked plasmid DNA in muscles of young rodents. Hum Mol Genet. 6, 1435-43. (17) Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A., and Felgner, P. L. (1990) Direct gene transfer into mouse muscle in vivo. Science 247, 1465-8. (18) Ross, P. C., and Hui, S. W. (1999) Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Ther. 6, 651-9. (19) Ogris, M., Steinlein, P., Kursa, M., Mechtler, K., Kircheis, R., and Wagner, E. (1998) The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther. 5, 1425-33. (20) Xu, Y.; and Szoka, F. C., Jr., (1996) Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 35, 5616-23.
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