Polyelectrolyte Vectors for Gene Delivery ... - ACS Publications

Bioconjugate Chem. 1999, 10, 993−1004

993

Polyelectrolyte Vectors for Gene Delivery: Influence of Cationic Polymer on Biophysical Properties of Complexes Formed with DNA Margreet A. Wolfert, Philip R. Dash, Ola Nazarova,† David Oupicky,† Leonard W. Seymour,* Sarah Smart, Jiri Strohalm,† and Karel Ulbrich† CRC Institute for Cancer Studies, University of Birmingham, B15 2TA, U.K., and Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague 6, The Czech Republic. Received March 2, 1999; Revised Manuscript Received August 13, 1999

Cationic polymer/DNA complexes are widely used for gene delivery, although the influence of the cationic polymer on the biophysical properties of the resulting complex is poorly understood. Here, several series of cationic polymers have been used to evaluate the influence of structural parameters on properties of DNA complexes. Parameters studied included the length of side chain, charge type (primary versus tertiary and quaternary), polymer molecular weight, and charge spacing along the polymer backbone. Cationic polymers with short side chains (such as polyvinylamine) formed small complexes, resistant to destabilization by polyanions, with low surface charge, limited transfection activity, and efficient intranuclear transcription. Conversely, cationic polymers with long side chains (e.g., poly[methacryloyl-Gly-Gly-NH-(CH2)6-NH2)] showed inefficient complex formation, high positive surface charge, and better transfection activity. The effects of molecular weight varied between polymers, for example, low molecular weight poly(L-lysine) produced relatively small complexes, whereas low molecular weight poly[2-(trimethylammonio)ethyl methacrylate chloride] produced large aggregates. Polymers containing quaternary ammonium groups showed efficient complex formation but poor transfection. Finally, spreading charges widely on the polymer structure inhibited their ability to condense DNA. In summary, to achieve small, stable complexes, the use of cationic polymers with short side chains bearing primary amino groups is suggested.

INTRODUCTION

The successful application of gene therapy can lead in principle to significant improvements in the treatment of several diseases (1). However, studies to date have highlighted the difficulty of achieving efficient transfection of target cells and tissues, both in vitro and in vivo. Indeed, inadequate transgene expression currently comprises a major factor limiting therapeutic application of sophisticated molecular treatment strategies (2, 3). Most existing gene delivery vectors are relatively inefficient when applied in vivo, requiring administration directly to the target tissue to attain useful levels of therapeutic gene expression (4). For useful deployment of gene therapy protocols, there is an urgent need for development of more efficient and biocompatible vectors (5). Ideally, these should ideally be capable of remote targeting and efficient transfection of specific cells and tissues following intravenous injection (6). We are developing novel vectors for gene delivery based on self-assembly of DNA with synthetic polymers. Although the final vectors are expected to contain several functional groups, the core of the complex will be formed by binding and condensation of DNA expression plasmids by cationic polymers, producing discrete polyelectrolyte particles (7, 8). There are several biophysical requirements of such polyelectrolyte particles. Chief among these are requirements that the particles should be small, to facilitate good biodistribution and extravasation proper* To whom correspondence should be addressed at the University of Birmingham. Phone: 44-121-414-3289. Fax: 44121-414-3263. E-mail: [email protected]. † Institute of Macromolecular Chemistry.

ties (9), and also be capable of undergoing efficient transcription following arrival in the nucleus of the target cell (10). In addition, resistance of complexes to destabilization by serum proteins is desirable, since there is increasing evidence that the rapid clearance of such polyelectrolyte complexes from the circulation follows their interaction with serum proteins (11). The principles of particle formation following cooperative binding of polycations to DNA have been widely studied, although several details of the process remain to be elucidated (12-16). Most of the work performed so far on formation of interpolyelectrolyte complexes has made use of the simple cationic polymer poly(L-lysine) (pLL), and there has been very little systematic study of the influence of polycation structure on self-assembly with DNA and the properties of the resulting complexes. In this study, we have examined a range of synthetic linear cationic polymers, selected to permit systematic evaluation of the influence of individual parameters on the biophysical properties of complexes formed by selfassembly with DNA. Specific molecular parameters selected for study are the distance of cationic charge from polymer backbone (length of side chain), order of cationic charge, influence of charge density along the polymer backbone, and polycation molecular weight. The ability of the cationic polymers to bind and condense DNA into particles has been assessed using gel electrophoresis and fluorescence inhibition assays. Physical properties of the particles formed have been characterized using atomic force microscopy (AFM)1 and ζ-potential (a measurement of charge at the surface of the particle). Their stability has been assessed using a polyanion-disruption assay, and finally, their biological properties have been assessed

10.1021/bc990025r CCC: $18.00 © 1999 American Chemical Society Published on Web 10/23/1999

994 Bioconjugate Chem., Vol. 10, No. 6, 1999

Wolfert et al.

by monitoring transfection of cells in vitro and measuring transcriptional activity directly following microinjection into the nucleus of Xenopus oocytes. In this way, we aim to identify molecular parameters of cationic polymers influencing properties of the complexes formed with DNA, providing information that should allow us to begin the design and development of polyelectrolyte complexes with properties selected for specific applications. EXPERIMENTAL PROCEDURES

DNA Purchase and Preparation. A circular 6 kb expression vector containing a CMV promoter-driven β-galactosidase reporter and ampicillin resistance was used for the atomic force microscopy, electrophoresis, and all studies of biological properties. This was prepared by growth in Escherichia coli DH5R and purified by standard technology. Following the final ethanol precipitation, the purity of the DNA was checked by agarose gel electrophoresis. The ethidium bromide fluorescence quenching and ζ-potential assays were performed using calf thymus DNA (Sigma Chemical Co.), with average molecular size determined to be 8 kb. For calculation of the N-to-P ratio of polyelectrolyte complexes, a mass per charge of 325 Da was used for DNA. Cationic Polymers Studied. The polymers described below were purchased or synthesized for systematic examination of the influence of individual parameters on the properties of complexes formed by self-assembly with DNA. Chemical structures of all cationic polymers examined are shown in Figure 1. (1) Influence of Length of Side Chain Bearing Cationic Residue. Several poly-(methacrylate)-based homopolymers with differing side chains bearing primary amino groups were synthesized as described below. These were poly[methacryloyl-Gly-Gly-NH-(CH2)6-NH2] hydrochloride (pMADGHDA.HCl), mass per primary amino group, 334.6 Da; poly[methacryloyl-Gly-NH-(CH2)2-NH2] hydrochloride (pMAGEDA.HCl), mass per primary amino group, 221.6 Da, and poly[methacryloyl-NH-(CH2)2-NH2] hydrochloride (pMAEDA.HCl), mass per primary amino group, 164.5 Da, together with poly(L-lysine) hydrobromide (pLL.HBr), mass per primary amino group, 209 Da; poly(allylamine) hydrochloride (pAA.HCl), mass per primary amino group, 57 Da; and poly(vinylamine) hydrochloride (pVA.HCl), mass per primary amino group, 79 Da. They were allowed to form complexes with DNA, using cationic polymer preparations of roughly similar molecular weight distribution in each case, and properties of the resulting complexes were determined and compared as described below. (2) Primary, Tertiary, and Quaternary Amino Groups. The influence of order of cationic charge on the properties of complexes formed with DNA was examined. The tertiary amino group-containing polymer poly(2-dimethylaminoethyl methacrylamide) (in free base form, pD1 Abbreviations: HPMA, (2-hydroxypropyl)methacrylamide; pMADGHDA.HCl, poly[methacryloyl-Gly-Gly-NH-(CH2)6-NH2] hydrochloride; pMAGEDA.HCl, poly[methacryloyl-Gly-NH(CH2)2-NH2] hydrochloride; pMAEDA.HCl, poly[methacryloylNH-(CH2)2-NH2] hydrochloride; pLL.HBr, poly(L-lysine) hydrobromide; pAA.HCl, poly(allylamine) hydrochloride; pVA.HCl, poly(vinylamine) hydrochloride; pDMAEMam, poly(2-dimethylaminoethyl methacrylamide); pTMAEM.Cl, poly[2-(trimethylammonio)ethyl methacrylate chloride]; pBTMAIPM.I2, poly[1,3bis-(trimethylammonio)isopropyl methacrylate iodide]; DCC, N,N′-dicyclohexylcarbodiimide; AIBN, azobisisobutyronitrile; IVA, 4,4′-azobis(4-cyanovaleric acid); DMSO, dimethyl sulfoxide; DMF, N,N-dimethyl formamide; BOC, di-tert-butyl dicarbonate; pVFA, poly(N-vinylformamide).

Figure 1. Structure of synthetic polymers used in this study. For simplicity, primary cationic monomers are shown as free bases. Panels a-c represent simple monomers as follows: a, pVA; b, pAA; c, pLL. Panels d-j show the side chains based on the methacrylate backbone shown, as follows: d, pHPMA; e, pMAEDA; f, pMAGEDA; g, pMADGHDA; h, pDMAEMam; i, pTMAEM.Cl; j, pBTMAIP.I2. Systematic names, together with full details of molecular weight averages, counterions, etc., are given in the text.

MAEMam, mass per amino group 157 Da) was synthesized and properties of its DNA complexes were compared with those of the quaternized ester poly[2-(trimethylammonio)ethyl methacrylate chloride] (pTMAEM.Cl, mass per charge 208 Da) and also with those formed by polymers bearing primary amino groups, mentioned above. (3) Influence of Charge Spacing along the Polymer Chain. Charge spacing was varied by synthesising statistical copolymers containing N-(2-hydroxypropyl)methacrylamide (HPMA) and 2-(trimethylammonio)ethyl methacrylate chloride (TMAEM.Cl), with TMAEM.Cl present at monomer ratios of 5, 15, 50, 75, and 100 mol %, masses per charge 2925, 1019, 351, 255, and 208 Da, respectively. In addition, poly[1,3-bis-(trimethylammonio)isopropyl methacrylate iodide] (pBTMAIPM.I2), which bears two cationic groups per monomer, was examined. It had a mass per charge of 249 Da. (4) Influence of Molecular Weight or Degree of Polymerization. Several families of polymers were compared to evaluate the influence of molecular weight on properties of complexes formed with DNA. pLL.HBr was purchased with weight average molecular masses, 4, 10, 24, 54, and 224 kDa (manufacturer’s specifications). Several polymers were synthesized (described below), including pTMAEM.Cl, weight average molecular masses, 5, 8, 21, 34, and 413 kDa; pMADGHDA.HCl, weight average molecular masses, 38, 74, and 330 kDa; pMAGEDA.HCl, weight average molecular masses, 29, 82, and 323 kDa; and pMAEDA.HCl, weight average molecular masses 19

Properties of Cationic Polymer/DNA Complexes

and 42 kDa. Degrees of polymerization (DPn) are given in Table 1. Chemicals. 1-Amino-2-propanol, methacryloyl chloride (freshly distilled), glycine, glycylglycine, 4-nitrophenol, N,N′-dicyclohexylcarbodiimide (DCC), azobisisobutyronitrile (AIBN), 4,4′-azobis(4-cyanovaleric acid) (IVA), 3-mercaptopropionic acid, cysteamine, cysteamine hydrochloride, N,N-dimethyl ethylenediamine, 1,2-diaminoethane, di-tert-butyl dicarbonate, N-BOC-1,6-diaminohexane hydrochloride, 4-(dimethylamino)pyridine, tetramethylammonium chloride, dimethyl sulfoxide (DMSO), N,N-dimethyl formamide (DMF), trifluoracetic acid, and triethylamine were from Fluka A.G., Switzerland. NVinylformamide was from Aldrich, Germany, and poly(L-aspartic acid, sodium salt) and pLL.HBr preparations were from Sigma, U.K. Solvents (all used after distillation) and other chemicals were of analytical grade quality, Lachema Brno, Czech Republic. Synthesis of Cationic Polymers. Cationic monomers and polymers were synthesized as described below. In each case, the composition of the monomers was checked by elemental analysis prior to polymerization and found to be within 0.5% of the theoretical values. In most cases, 1H NMR was used as an additional technique to characterize the monomers and some of the polymers. In addition, the polymer products were routinely characterized by GPC for weight and number average molecular weights. The polydispersity of most of the polycations was about 1.8. Poly[N′-{2-[(tert-butyloxycarbonyl) amino]ethyl}methacryl amide] [MA-NH-(CH2)2-NH-BOC] was prepared as follows. A solution of di-tert-butyl dicarbonate (2.45 g, 0.011 mol) in dioxane (30 mL) was added dropwise to a solution of 1,2-diamino-ethane (5.25 g, 0.087 mol) in dioxane (30 mL). The mixture was stirred for 22 h at room temperature. The dioxane was evaporated in vacuo and the crude product triturated with 50 mL of water. The insoluble bis-substituted diamine was removed by filtration. The filtrate was extracted three times with methylene chloride (150 mL), the organic layer was separated and the solution was dried over Na2SO4. Methylene chloride was evaporated, and the yield of oily BOC-NH-(CH2)2-NH2 was 1.29 g (72%). Contamination of the product with free diamine was checked by TLC, but none could be determined. The oily product was used for the synthesis of MA-Gly-NH-(CH2)2-NH-BOC and MA-NH-(CH2)2-NH-BOC without further purification. N-BOC-1,2-diamino-ethane (1 g, 0.00625 mol) and triethylamine (5.5 mL) were dissolved in 50 mL of freshly distilled chloroform. The mixture was cooled to -5 °C and methacryloyl chloride (0.68 g, 0.0065 mol) was added dropwise under stirring within 3 h. Reaction mixture was extracted four times with 20 mL of water and then a chloroform layer was dried with Na2SO4. Chloroform was evaporated, and after trituration in diethyl ether, the yellow crude product was obtained. This product was purified by recrystallization from the benzene-hexane mixture and characterized by elemental analysis and 1H NMR (CDCl3). Yield was 1.2 g (81%). Melting point: 7376 °C. The monomer and initiator of polymerization (AIBN) were dissolved in methanol to form a solution containing 7-30 wt % of monomer and 0.1-2 wt % of AIBN related to polymerization mixture. The solution was introduced into an ampule and bubbled through with nitrogen. The ampule was sealed, and polymerization was carried out at 50 or 60 °C for 24 h. The polymer was precipitated into diethyl ether and reprecipitated from methanol into diethyl ether. The precipitated polymer

Bioconjugate Chem., Vol. 10, No. 6, 1999 995

was filtered off, washed with diethyl ether, and dried in vacuo. Molecular weight of polymers was controlled by varying the monomer concentration, the initiator concentration, or the temperature. BOC protecting group was removed by dissolution of the polymer in a solution of dry hydrochloric acid in methanol (15 wt % of HCl in methanol and 20 wt % of polymer in solution) and stirring for 1 h. The deprotected polymer was diluted with methanol, evaporated in vacuo to remove the excess of hydrochloric acid, and precipitated into diethyl ether. The precipitated polymer was filtered off, washed with diethyl ether and dried in vacuo. The quantitative removal of the protecting BOC groups was confirmed by the absence of the BOC signal (1.4 ppm) in the 1H NMR spectrum of the polymer. Poly[N′-{2-[(tert-butyloxycarbonyl)amino]ethyl}methacryloylglycinamide] [MA-Gly-NH-(CH2)2-NH-BOC] was prepared as follows: BOC-NH-(CH2)2-NH2 (1.1 g, 0.0068 mol) was dissolved in 2 mL of DMSO. MA-GlyONp (1.6 g, 0.0061 mol) was dissolved in 3.5 mL of DMSO and added dropwise to the solution of amine. Reaction mixture was stirred for 2 h at room temperature. DMSO was evaporated in vacuo (bp 55 °C) and an oily residue was dissolved in 50 mL of chloroform. An organic layer was extracted three times with 20 mL of distilled water. Organic layer was dried over Na2SO4. Chloroform was evaporated, and MA-Gly-NH-(CH2)2-NH-BOC was crystallized from ethyl acetate. Melting point: 136-38 °C. Polymerization and deprotection of the amino group were carried out according to the polymerization of MA-NH(CH2)2-NH-BOC with subsequent deprotection of the BOC-containing polymer and NMR check of the BOC group removal. Poly[N′-{(6-tert-butyloxycarbonyl)amino]hexyl}methacryloylglycylglycin amide] [MA-GlyGly-NH-(CH2)6NH-BOC] was prepared as follows. MA-GlyGly-ONp (3 g, 0.0093 mol) and N-BOC-1,6-diamino-hexane hydrochloride were dissolved in 15 mL of DMF. Triethylamine (1.3 mL) was added in three portions, and reaction mixture was stirred 24 h at room temperature. The precipitated triethylamine hydrochloride was filtered off, and the filtrate was evaporated in vacuo to dryness. A crude product was dissolved in chloroform and extracted three times with 15 mL of water. An organic layer was dried with Na2SO4 and evaporated in vacuo. The product was crystallized from a chloroform-diethyl ether mixture. Melting point: 122-124 °C. Yield was 1.98 g (54%). Polymerization and deprotection of the amino group were carried out according to the polymerization of MA-NH(CH2)2-NH-BOC with subsequent deprotection of the BOC-containing polymer and NMR check of the BOC group removal. DMAEMam was prepared as follows. N,N-Dimethyl ethylenediamine (8.22 g, 0.093 mol) was diluted with 30 mL of dichlormethane, and the solution was cooled to -15 °C. The solution of methacryloyl chloride (5.0 g, 0.047 mol) in dichloromethane (5 mL) was added dropwise under stirring within 2 h. The precipitate (N,N-dimethylethylenediamine hydrochloride) was filtered off, and dichlormethane was evaporated from the reaction mixture. The product was purified by rectification at reduced pressure. Polymerization of DMAEMam was carried out according to the polymerization of MA-NH-(CH2)2-NHBOC. Briefly, a polymerization mixture consisting of 13 wt % methanol solution containing 0.6 wt % AIBN was introduced into an ampule and bubbled through with nitrogen. The ampule was sealed and polymerization was carried out at 60 °C for 20 h. A mixture of acetone and

pBTMAIPM.I2 pHPMA25pTMAEM75 pHPMA50pTMAEM50 pHPMA85pTMAEM15 pHPMA95pTMAEM5

pDMAEMam pTMAEM.Cl

pMADGHDA.HCl

pMAGEDA.HCl

pMAEDA.HCl

pAA.HCl pLL.HBr

cationic polymer

37 40 12

41 000

35 000

38 101 760 960 19 48 114 258 1072 112 255 133 370 1457 115 220 997 348 24 41 102 164 1986 374 63

13 000

3000 (Mn) 8 000 (Mn) 60 000 (Mn) 54 700 4000 10 000 24 000 54 000 224 000 18 500 41 900 29 400 82 000 322 800 38 400 73 700 333 500 54 700 5000 (Mn) 8470 21 240 34 150 413 000 93 100 16 000

nd 0.75 nd 1.10 1.28 ( 0.05 0.73 ( 0.15 0.68 ( 0.18 0.85 ( 0.05 1.10 ( 0.05 9 0.95 nd 2.23 1.10 nd 2.1 2.1 >4.0 nd 0.75 0.73 0.80 0.78 1.08 2.2 >5.0 >5.0 >5.0

nd 13.2 ( 1.3 nd 13.6 ( 1.4 22.1 ( 1.0 19.9 ( 0.4 12.5 ( 0.2 22.5 ( 2.5 20.9 ( 0.9 34.2 ( 3.5 16.6 ( 0.6 29.8 ( 4.1 25.9 ( 7.2 27.9 ( 2.6 nd nd 26 ( 5.2 85.8 ( 13 nd 21.2 ( 0.3 23.9 ( 0.4 24.2 ( 1.1 23.9 ( 0.8 25.8 ( 2.4 41.6 ( 2.5 84.8 ( 8.1 95.8 ( 1.2 100 ( 0

nd

nd

nd

55.0 ( 5.0 (3.2) nd 402 ( 54.4 (23.4) 204 ( 79.1 (11.9) 46 ( 5.4 (2.7) 57 ( 3.2 (3.3) 58 ( 1.6 (3.4) 55 ( 3.5 (3.2) 55 ( 4.5 (3.2) 17.0 ( 7.2 (1.0) 14.1 ( 1.1 nd 7.5 ( 1.7 (0.4) nd nd nd nd not applicable nd nd nd 26.9 ( 1.7 (1.6) nd 19.9 ( 0.7 (1.2) nd

no particles

no particles

83 ( 25 (45)

nd aggregated no images possible 78 ( 9 (38) 25 ( 5 (19) 73 ( 13 (49) 88 ( 20 (54) 145 ( 42 (80) 210 ( 90 (105) 129 ( 25 (53) 91 ( 20 (42) 100 ( 10 (45) 106 ( 23 (44) 74 ( 18 (43) 75 ( 19 (35) 76 ( 16 (37) 73 ( 9 (36) 78 ( 16 (42) nd 363 ( 93 (137) 80 ( 25 (44) 94 ( 28 (43) 88 ( 18 (41) 86 ( 17 (51) 79 ( 14 (45)

13 ( 3

31 ( 2

17 ( 5

nd -2 ( 1 -1 ( 0 -1 ( 0 16 ( 0 nd 36 ( 1 36 ( 1 33 ( 1 -1 ( 0 14 ( 3 nd 1(2 nd 42 ( 2 36 ( 1 27 ( 1 -15 ( 1 31 ( 2 nd 29 ( 1 30 ( 2 20 ( 2 41 ( 1 nd

(1.73 ( 1.40) × 103

(1.01 ( 1.38) × 104

(9.45 ( 8.53) × 102

(6.9 ( 2.6) × 101 (1.3 ( 1.6) × 101 (2.12 ( 0.14) × 102 (1.81 ( 1.15) × 104 0 ( 175 63 (3.39 ( 1.41) × 104 (3.61 ( 0.52) × 105 (3.16 ( 0.11) × 105 (1.21 ( 1.69) × 104 (9.37 ( 3.62) × 104 nd (2.69 ( 0.21) × 104 (3.68 ( 3.00) × 105 nd (1.83 ( 0.47) × 105 nd (4.67 ( 0.77) × 104 (4.01 ( 0.56) × 102 nd (5.63 ( 3.61) × 102 (7.23 ( 3.78) × 102 (9.61 ( 4.54) × 102 (8.85 ( 1.39) × 102 (1.16 ( 0.32) × 103

nd

30

20

59 61 63 nd 77 nd 63 nd nd 25 nd 71 46 nd nd 40 3 22 14

nd nd 64 63 64

AFM average X-Y molecular diameter (nm) and Intranuclear weight average degree of RFI50 poly(L-aspartic acid) (µg/mL) and (aspartic acid/ (diameter of equivalent ζ-potential transfection 293 injection (oocytes) (Mw, unless polymerization IF50 cation molar ratio) sphere, nm) (mV) n ) 5 cells (l.u./µg) n ) 3 (% of free DNA control) indicated) (DPn) min F (%) (N-to-P ratio)

Table 1. Physical and Biological Properties of Complexes Formed by Self-Assembly of DNA with Cationic Polymers

996 Bioconjugate Chem., Vol. 10, No. 6, 1999 Wolfert et al.


diethyl ether was used for deprotection and reprecipitation of the polymer. pTMAEM.Cl was prepared as follows. TMAEM.Cl was prepared by quaternization of 75 g dimethylaminoethyl methacrylate with gaseous methyl chloride in 200 mL of acetone in the presence of 5 mL of N,N-dimethylformamide at room temperature. The crystalline precipitate was isolated by filtration, washed three times with acetone, and dried in vacuo. Melting point: 174 °C. Polymerization was carried out as described elsewhere (17). pBTMAIPM.I2 was prepared by radical polymerization of 1,3-bis-(trimethylammonio)isopropyl methacrylate iodide (10 wt %) in DMF using IVA (0.3%) as initiator. Polymerization was carried out at 70 °C for 40 h in a sealed ampule under nitrogen atmosphere. The polymer was precipitated into dry acetone-diethyl ether mixture, washed with dry acetone, and dried in a vacuum. pVA.HCl was prepared by alkaline hydrolysis of poly(N-vinylformamide) (pVFA). pVFA was prepared by radical precipitation polymerization of 4 g of N-vinylformamide dissolved in methanol, using 0.38 g of initiator (AIBN) and 2-mercaptoethanol as a chain transfer agent for regulation of molecular weight. Polymerization was carried out at 60 °C for 24 h. Precipitated polymer was washed with methanol, dissolved in 28 mL of NaOH (1.7 M), and hydrolyzed at 80 °C for 6 h. The polymer solution was acidified by concentrated HCl solution. Precipitated pVA.HCl was washed with methanol, acetone, and dried in vacuo. Synthesis of statistical copolymers of TMAEM.Cl with HPMA. HPMA was prepared by the reaction of methacryloyl chloride with 1-amino-2-propanol, as described earlier (18). Copolymers were prepared by radical copolymerization of TMAEM.Cl with HPMA in methanol using AIBN as initiator. Monomers in various molar ratio (25-95% HPMA) were dissolved in methanol to obtain 20 wt % solution. Concentration of AIBN was 0.6 wt %, polymerization temperature 60 °C, and polymerization time 24 h. Polymerization was conducted in sealed ampules under nitrogen atmosphere. Copolymers were isolated by precipitation into diethyl ether-acetone and purified on GPC (Sephadex LH-20, methanol). The composition of copolymers was calculated using data of elemental analysis (chlorine content). The molecular weight distribution (weight average molecular weight Mw and polydispersity Mw/Mn) of cationic polymers was determined either by FPLC on Sepharose 12 using 0.25 M sodium acetate buffer, pH 6.6, containing 0.5 M NaCl and 0.028 M tetramethylammonium chloride, or using Catsec 300 column (Micra Scientific) with an aqueous solution of NaNO3 (0.2 M, adjusted to pH 2 using trifluoroacetic acid) as mobile phase. The columns were calibrated with fractionated low-polydispersity polyHPMA standards with molecular weights in a range 18000-300000 g/mol. The molecular weights of polyHPMA standards were determined by static laser light scattering. Weight average of molecular weight (Mw) of selected polycations was determined using light scattering (Fica 40). Formation of Cationic Polymer/DNA Complexes. The conditions of complex formation were standardized to permit comparison of different properties. DNA was dissolved in water at 20 µg/mL, and then cationic polymers were added in a small volume of concentrated (5 mg/mL) solution in water. Samples were mixed very gently and then allowed to stand for at least 1 h at room temperature before more thorough mixing. To permit comparison of different cationic polymers, containing


amino groups with different pKa values, complexes were formed at defined molar ratios of highly charged amino groups to DNA phosphate (N-to-P ratios), and no correction was made for different levels of protonation. Inhibition of Ethidium Bromide/DNA Fluorescence by Cationic Polymers. Decreased ethidium bromide-DNA fluorescence can be used to indicate condensation of the DNA (13). The influence of N-to-P ratio on DNA condensation by each polymer was determined by loss of ethidium bromide fluorescence in aqueous solution using a fluorimeter (λex ) 366 nm, λem ) 590 nm). DNA (20 µg/mL) was incubated in water in the presence of ethidium bromide (400 ng/mL) before sequential addition of small volumes of the cationic polymer (5 mg/ mL), each addition accounting for increased N-to-P ratio of 0.1. The parameters deduced for each interaction were the N-to-P ratio of polymer causing 50% fall in measured fluorescence (IF50) and the lowest fluorescence level achieved at any N-to-P ratio (Min F), expressed as a % the fluorescence of equivalent free DNA. Ethidium Bromide/DNA Fluorescence Restoration by Poly(L-aspartic acid). Polyelectrolyte complexes containing 20 µg/mL DNA were formed at an N-to-P ratio of 2 in the presence of ethidium bromide (400 ng/mL). Poly(L-aspartic acid) was then added in sequential aliquot portions in water. After each addition, solutions were mixed and ethidium bromide/DNA fluorescence was monitored as above. Poly(L-aspartic acid) was added in small portions until fluorescence was restored to 50% of its original value (the RFI50 concentration; Figure 3). Agarose Gel Electrophoresis. Polymer/DNA complexes in water were electrophoresed on agarose gel (0.8% w/v) (BRL) for 45 min at 90 V. Ethidium bromide was included in the gel to show the location of DNA using a UV transilluminator. Atomic Force Microscopy (AFM). Samples containing complexes in water (1-2 µL) with final DNA concentration of 20 µg/mL were deposited onto the center of a freshly split untreated mica disk (Agar Scientific). Following adsorption for 1-2 min at room temperature, excess fluid was taken off by absorption onto filter paper. The mica surface was dried at room temperature before imaging using an AFM-2, part of the NanoScope II system (Digital Instruments). A 200 µm long Si3N4 cantilever with a spring constant of 0.12 N/m and a D scanner head (AFM 351) with 12 µm scan range were used. The image mode was set to constant force mode, hence the images obtained are height images of the sample surface. The scanning speed varied between 2.48 and 8.68 Hz. Analysis of ζ-Potential. The surface charge of the polymer/DNA complexes was assessed by using a Zetamaster system (Malvern Instruments). The system was routinely calibrated using a -55 mV standard. Experimental samples (5 mL) contained a final calf thymus DNA concentration of 20 µg/mL and were measured 5 times for 30 s at 1000 Hz with zero field correction. Microinjection of DNA Complexes into Xenopus oocytes. Reporter gene activity was determined following injection of polymer/DNA complexes directly into the nucleus of Xenopus oocytes. Mature female frogs were killed by benzocaine overdose, and oocytes were individually examined in a Petri dish containing Modified Barth’s Solution (MBS, pH 7.6). Only oocytes at developmental stage IV were used, and complexes were microinjected using a Drummond Auto Oocyte Injector (Drummond Scientific Company). The injection volume was 13.8 nL/ oocyte and DNA concentration 22 pg/nL (either free DNA


Wolfert et al.

Figure 2. Influence of side-chain length on structure of complexes formed with DNA, viewed by AFM. Polymer/DNA complexes were formed at an N-to-P ratio of 2.0. The images show conjugates formed using (i) pAA.HCl (54 700 Mw; DPn 960), (ii) pMAEDA.HCl (41 900 Mw; DPn 255), (iii) pMAGEDA.HCl (82 000 Mw; DPn 370), and (iv) pMADGHDA.HCl (73 700 Mw; DPn 220). The height of the AFM image is represented by a graded black-white scale, with white indicating a height of 30 nm above the mica surface. The x and y dimensions are scaled as shown. The images are typical of the results of multiple evaluations performed on separate days.

or as polymer/DNA complexes). Oocytes were maintained in MBS for a further 48 h in small Petri dishes at 18-20 °C (individually grouped for specific studies), at which time healthy looking oocytes were pooled together (10 oocytes/mL) in lysis solution (100 mM potassium phosphate, 0.2% vol/vol Triton X-100, and 1 mM dithithreitol, pH 7.8). Oocytes were lysed by pipetting them up and down, and samples were centrifuged at 13 000 rpm (MicroCentaur, 10 min). The clear supernatant (20 µL) was then assayed for β-galactosidase activity by Galactolight, as described below. Efficiency of Transfection in Vitro. Polymer/DNA complexes were prepared in water at an N-to-P ratio of 2 and with a DNA concentration of 20 µg/mL. Complexes were added to 293 cells and incubated for 4 h at a final DNA concentration of 2.6 µg/mL in serum-free medium containing chloroquine (100 µM). Cells were washed after exposure and reincubated in the presence of serum (10%). After 48 h, medium was removed from the culture and the cells were washed once with PBS (100 µL). Aliquots of lysis buffer (100 mM potassium phosphate, pH 7.8, and 0.2% Triton X-100) (100 µL) were added, and the plates were left for 40 min at room temperature until the lysis was complete. After centrifugation, the cell lysates were assayed for gene expression and total protein content (see below). Evaluation of Gene Expression. Total β-galactosidase activity was quantified using the chemiluminescent reporter assay system Galacto-Light Plus (Tropix, Cam-

Figure 3. Restoration of ethidium bromide/DNA fluorescence by addition of poly(L-aspartic acid) to preformed polymer/DNA complexes. Complexes were formed at an N-to-P ratio of 2.0, in the presence of ethidium bromide (400 ng/mL), and allowed to stabilize for 2 h, and then poly(L-aspartic acid) was added in small portions, with fluorescence determined at λex 366 and λem 590 nm. The behavior of DNA complexes formed with pVA.HCl [60 000 Mw, DPn 760 (b)], pAA.HCl [54 000 Mw, DPn 960 (O)], pMAEDA,HCl [41 900 Mw, DPn 255 (9)], and pMAGEDA.HCl [82 000 Mw, DPn 370 (0)] is shown.


bridge, U.K.). Cell lysates were diluted 1:1 with lysis buffer containing 2 mM dithiothreitol. After centrifugation, 50 µL of the supernatant was then added to 200 µL of chemiluminescent substrate solution (Galacton-Plus in 100 mM sodium phosphate, pH 8.0, and 1 mM magnesium chloride). After incubation for 1 h, 300 µL of luminescence accelerator reagent was added, and the sample was assayed on a monolight luminometer (Lumat LB9501, Berthold) by counting emitted light units for 10 s. Transfection was quantified as β-galactosidase activity measured per µg of protein (light units/µg). The total protein content was measured in the absence of dithiothreitol using the bicinchonninic acid (BCA) assay for protein. RESULTS

Influence of Length of Polycation Side Chain on Properties of Complexes Formed with DNA. Different distance of cationic charge from the polymer backbone had an important influence on the properties of complexes formed with DNA. Polymers with charges close to the backbone (e.g., pVA.HCl) showed generally efficient condensation, with relatively low values of IF50 and Min F (Table 1). Conversely, polymers with long lateral side chains (e.g., pMADGHDA.HCl) showed poor condensation. It is important throughout these studies comparing different side chains to consider the influence of DPn, since polymers based on small monomers have more charges per polymer of the same molecular weight compared with polymers based on larger monomers. However, even bearing this in mind, it is clear that polymers with long side chains tend to be less efficient at condensing DNA than polymers with short side chains (Table 1). Nonetheless, the influence of side-chain length is not simple as some polymers with intermediate length side chains, such as pLL.HBr, also achieved efficient DNA condensation judged by inhibition of ethidium bromide/DNA fluorescence. AFM was used to measure sizes of polymer/DNA complexes. However, pVA.HCl/DNA complexes tended to flocculate in water, particularly with lower molecular weight pVA.HCl, and no AFM images were possible. pAA.HCl and cationic polymers with side chains of intermediate length produced discrete, well-defined complexes with DNA, typical diameter about 100 nm. DNA complexes produced with cationic polymers with long side chains (pMADGHDA.HCl) were often loose with structural irregularities, and there was still DNA protruding from the complexes even several hours following initial mixing of pMADGHDA.HCl with DNA (Figure 2). The influence of side-chain length was also evident when complexes were subject to interaction with pLAA, and restoration of ethidium bromide/DNA fluorescence was monitored. Although complexes based on all other polymers showed fluorescence restoration (RFI 50) at concentrations of pLAA less than 60 µg/mL (a molar ratio of aspartic acid to cation of 3.5), those based on polymers with shortest side chains (pVA.HCl and pAA.HCl) showed much greater resistance to pLAA, pVA.HCl achieving RFI50 values even over 400 µg/mL (aspartic acid/cation ratios over 20; Table 1 and Figure 3). This does not appear to be strongly influenced by the DPn and may reflect high stability of DNA complexes formed with cationic polymers such as pAA.HCl and pVA.HCl. When the surface charge of complexes was monitored (using N-to-P ratio of 2.0), DNA complexes formed with pVA.HCl or pAA.HCl were found to have ζ-potentials of approximately zero (Table 1), perhaps contributing to the


tendency of pVA.HCl complexes to flocculate in water. Comparing complexes formed with medium molecular mass (40-80 kDa) cationic polymers, most of the cationic polymers with medium or long pendent side chains produced positive ζ-potential values, for example, pMADGHDA.HCl, 36 mV; pLL.HBr, 36 mV; pTMAEM.Cl, 9 mV. Although it is intuitively attractive to consider that longer side chains may result in a surface layer of nonneutralized positive charges, any such proposal is compromised by the low value of ζ-potential (1 mV) produced by pMAGEDA.HCl and the higher value (14 mV) produced by the shorter side chain pMAEDA.HCl. DNA complexes formed using the short side-chain PVA.HCl gave no significant spontaneous transfection when applied to 293 cells in vitro, and pAA.HCl also produced only slight increases compared with DNA alone. Complexes based on all the other nonquaternized cationic homopolymers examined produced higher levels of gene expression, and this may relate to the known positive ζ-potential of the latter complexes, possibly mediating greater membrane adsorption. Complexes formed using pMADGHDA.HCl, known from AFM studies to be incompletely condensed, also mediated high levels of transgene expression, similar to the levels achieved using high molecular weight pLL.HBr (Table 1). Following direct inoculation into Xenopus oocytes, complexes based on most homopolymers produced levels of gene expression comparable to that following injection of free DNA. pAA.HCl and pVA.HCl complexes showed over 60% expression, suggesting that despite their high stability to polyanions they are still suitable for intranuclear transcription. The only indication of the influence of side-chain length was from the long side-chain pMADGHDA.HCl polymer, which gave relatively low gene expression (25%) (Table 1). Influence of Charge Strength on Properties of Complexes Formed with DNA. Comparison of similar molecular weight poly(methacrylamide)-based polymers containing primary (pMAEDA.HCl), tertiary (pDMAEMam), or quaternary (pTMAEM.Cl) amino groups permitted examination of the influence of these different types of charge on properties of complexes formed with DNA. Quaternary charged pTMAEM.Cl was relatively efficient at DNA condensation, achieving low IF50 values (Table 1). In contrast, the tertiary amino-based pDMAEMam was very poor at DNA condensation, and failed to decrease ethidium bromide/DNA fluorescence to 50% of the starting value even at N-to-P ratios of 4.0. This poor ability of pDMAEMam to form complexes with DNA was probably due to its presence in free base form, leading to lower levels of protonation in water than with the other cationic polymers which were salts. When poly(L-aspartic acid) was added to complexes to restore fluorescence of ethidium bromide/DNA, complexes based on pTMAEM.Cl/DNA required greater levels of poly(L-aspartic acid) than DNA complexes formed using methacrylate polymers containing primary amino groups (Table 1). AFM showed no obvious differences between DNA complexes formed using methacrylate-based polymers of different order of charge, with all polymers capable of producing discrete complexes of about 100 nm diameter. Similarly, there were no trends in ζ-potential determined for the complexes, except that DNA complexes formed with the tertiary amino-containing pDMAEMam displayed negative values of ζ-potential (Table 1). Spontaneous transfection of 293 cells in vitro could be achieved to levels of about 105 l.u./µg protein by DNA


complexes formed using the methacrylate based primary amino polymer pMAEDA.HCl, and also by the tertiary amino pDMAEMam. These levels were comparable with those achieved using pLL.HBr. Complexes based on quaternary pTMAEM.Cl, however, showed much lower levels of spontaneous transfection, achieving maximal levels of 103 l.u./µg protein. This was only marginally increased compared with the intrinsic transfection activity of free DNA. Following direct intranuclear injection in Xenopus oocytes, complexes based on primary aminobearing pMAEDA.HCl showed transgene expression at a level of 77% that of the equivalent dose of free DNA. pDMAEMam/DNA complexes achieved 71%, and pTMAEM.Cl/DNA complexes 40%. However, the 2-fold decreased intranuclear activity of the quaternary pTMAEM.Cl-based complexes is not sufficient to explain their very low levels of spontaneous cell transfection, suggesting that cellular transfection by pTMAEM.Cl/ DNA complexes may be subject to particular problems of gaining access to the cytoplasm or nucleus (Table 1). Influence of Charge Spacing along Polymer Backbone on Properties of Complexes Formed with DNA. A series of random copolymers of TMAEM.Cl and HPMA permitted examination of the influence of charge spacing on the properties of complexes formed with DNA. To permit comparison, all copolymers were used at standard concentrations of charge, which for some materials (particularly the 5% TMAEM.Cl copolymer) necessitated use of very high total concentrations of copolymer. Copolymers containing 50% pHPMA were unable to mediate 50% inhibition of ethidium bromide/DNA fluorescence at any N-to-P ratio examined (Figure 4a). Interestingly, agarose gel electrophoresis showed that all random copolymers, including the material containing 95% HPMA (where each positively charged TMAEM.Cl group is surrounded by an average 19 noncharged HPMA groups) was capable of binding DNA and mediating a gelshift assay (Figure 4b). At N-to-P ratio of 1.0, there was still some electrophoretic mobility of DNA condensed with the 5% TMAEM.Cl copolymer, although at N-to-P ratio of 2.0, this had disappeared. In addition, fluorescence of DNA complexes retained in the wells was lower using polymers with greater TMAEM.Cl content, corresponding with data in Figure 4a, and suggesting formation of tighter complexes. AFM analysis showed that the higher charge density cationic polymers was capable of forming discrete complexes, while copolymers containing 50% or less TMAEM.Cl produced images of extended structures, probably representing DNA/cationic polymer complexes which are not subject to charge neutralization-driven particle self-assembly (Figure 5). Measurement of ζ-potential also showed that every copolymer was capable of binding DNA, with all ζ-potentials measured in the range 10-50 mV, compared with the ζ-potential of free DNA (-80 mV, approximately). Although all copolymer complexes showed some activity in spontaneous transfection of 293 cells, levels were very low compared with pLL.HBr/DNA complexes, similar to the level achieved using complexes formed using pTMAEM.Cl homopolymer. After direct intranuclear injection to Xenopus oocytes, copolymer/DNA complexes showed low levels of gene expression (14-30% of DNA control), although again this is comparable with the activity of pTMAEM.Cl/DNA complexes (Table 1). pBTMAIPM.I2 is a polymer bearing two quaternary positive charges per methacrylate-based monomer, and hence the charge density of the polymer is relatively high. Properties of the complexes formed between pBTMAIPM.I2 and DNA were very similar to pTMAEM/DNA

Wolfert et al.

Figure 4. Assessment of DNA condensation by TMAEM.Clco-HPMA random copolymers. (a) Fluorescence of ethidium bromide allowed to integrate into the DNA before addition of the cationic polymers, as described in the text. The lines represent sequential addition of copolymers of different composition: 100 mol % TMAEM.Cl (Mw 21 240; DPn 102; b), 75 mol % TMAEM.Cl (Mw 16 000; DPn 63; O), 50 mol % TMAEM.Cl (Mw 13 000, DPn 37; 9), 15 mol % TMAEM.Cl (Mw 41 000, DPn 40; 0), 5 mol % TMAEM.Cl (Mw 35 000, DPn 12, 3). (b) Agarose gel shift assay. Complexes were formed by addition of precise amounts of TMAEM.Cl-co-HPMA random copolymers to DNA. After 2 h, samples were analyzed by agarose gel electrophoresis, with ethidium bromide present in the gel to permit visualization of DNA. Samples applied are as follows: lanes 1 and 7, free DNA; lanes 2 and 8, 5 mol % TMAEM.Cl (Mw 35 000); lanes 3 and 9, 15 mol % TMAEM.Cl (Mw 41 000); lanes 4 and 10, 50 mol % TMAEM.Cl (Mw 13 000); lanes 5 and 11, 75 mol % TMAEM.Cl (Mw 16 000); lanes 6 and 12, 100 moly TMAEM.Cl (Mw 34 150). Complexes were formed at an N-to-P ratio of 1.0 (lanes 2-6) or 2.0 (lanes 8-12). The gel was photographed using a UV transilluminator, and the arrow marks the position of free DNA running into the gel.

complexes, showing similar condensation parameters and poor ability to transfect cells despite positive surface charge. This may reflect the poor transcriptional ability of pBTMAIPM.I2/DNA complexes observed following intranuclear injection, similar to that observed for complexes based on high molecular weight pTMAEM.Cl.



Figure 5. Influence of charge spacing in TMAEM.Cl-co-HPMA random copolymers on complexes formed with DNA, visualized by AFM. Complexes were formed at an N-to-P ratio of 2.0. The images show conjugates formed using (i) 100 mol % TMAEM.Cl (Mw 21 240), (ii) 75 mol % TMAEM.Cl (Mw 16 000), (iii) 50 mol % TMAEM.Cl (Mw 13 000), and (iv) 15 mol % TMAEM.Cl (Mw 41 000). The height of the AFM image is represented by a graded black-white scale, with white indicating a height of 30 nm above the mica surface. The x and y dimensions are scaled as shown. The images are typical of the results of multiple evaluations performed on separate days.

Influence of Cationic Polymer Molecular Weight or DPn on Properties of Complexes Formed with DNA. Self-assembly of cationic polymers with DNA, measured by inhibition of ethidium bromide/DNA fluorescence, showed molecular weight (or DPn) effects that varied from polymer to polymer. The simplest methacrylate-based polymer, pMAEDA.HCl, showed much better DNA condensation using a higher molecular weight preparation, achieving lower values of IF50 and Min F. Similar results were obtained using the longer side chain pMAGEDA.HCl. With the quaternary pTMAEM.Cl, no real effects of molecular weight were seen, and all polymer fractions showed similar condensation abilities. In contrast, pLL.HBr showed clear effects of molecular mass, with medium molecular mass pLL (24 kDa) mediating more efficient condensation of DNA than high or low molecular mass preparations. Sizes of complexes formed by self-assembly with DNA, measured by AFM, showed molecular weight influences that varied between polymers. There were no reproducible effects for the polymer pMAEDA.HCl, although pMAGEDA.HCl and pMADGHDA.HCl both seemed to give better defined complexes at higher molecular weight, with lower molecular weight preparations yielding more extended structures. pTMAEM.Cl showed a clear effect of molecular weight, with complexes based on low molecular weight polymer showing a larger size, probably due to aggregation. pLL.HBr also showed a clear molecular weight effect that was effectively the opposite, namely significantly smaller complexes formed using

lower molecular weight polycation. This phenomenon has been observed before (7). ζ-Potential of complexes formed between DNA and polycations showed no trends with molecular weight for any of the cationic polymers examined except pLL.HBr. In the case of pLL.HBr, low molecular weight polycations yielded lower values of ζ-potential, with pLL.HBr 4 kDa showing a relatively low positive value (16 mV). Smaller preparations (e.g. pLL.HBr 1 kDa) showed further decreased ζ-potential, perhaps reflecting incomplete condensation, but also a tendency to flocculate. Following microinjection of complexes into the nucleus of Xenopus oocytes, complexes based on high molecular weight pTMAEM.Cl showed much lower levels of gene expression than even those based on midrange pTMAEM.Cl. This may reflect relatively high stability of these complexes and protection of DNA from polymerase enzymes, or could reflect other effects, possibly toxicological, of this high molecular weight polycation. Complexes based on pLL.HBr show no such effects of molecular weight, with even complexes based on very high molecular weight pLL.HBr showing roughly the same levels of transcription as all other pLL.HBr/DNA complexes. This was in sharp contrast to the levels of gene expression achieved by simple transfection, where the use of complexes based on high molecular weight pLL.HBr achieved much greater levels of gene expression (Figure 6). The other polymers examined also showed the same trend in transfection, with complexes based on larger polymers mediating higher levels of transgene expres-


Figure 6. Influence of molecular weight of cationic polymers on transfection activity against 293 cells. (a) Influence of molecular weight of pLL.HBr, while panel b shows the influence of molecular weight of pTMAEM.Cl. Complexes were formed, cells were transfected, and expression of the β-galactosidase gene delivered was assayed as described in the text.

sion, although the effect was not always statistically significant. DISCUSSION

Several techniques have been employed in this study to characterize the biological and physical properties of complexes formed spontaneously between cationic polymers and DNA. Some of the techniques are still under development, however, and results must be interpreted cautiously. For example, it is not clear whether loss of ethidium bromide/DNA fluorescence upon complex formation (13, 19) results from expulsion or quenching of the fluorophore, and in the case of expulsion, whether the motive force is physical compression or electrostatic repulsion of the ethidium bromide from the complex. Subject to these qualifications, the techniques employed have yielded considerable information on the interaction of cationic polymers with DNA. Considering first the influence of side-chain length, polymers studied with long side chains (e.g., pMADGHDA.HCl) were incapable of efficient complex formation with DNA. The resulting complexes maintained considerable residual fluorescence and high ζ-potential and showed loops of

Wolfert et al.

nonincorporated DNA protruding from the structure when viewed by AFM. They did show considerable transfection activity, however, despite poor gene expression following direct intranuclear injection. At the other extreme were complexes formed using pVA.HCl and pAA.HCl, both with very short side chains. These polymers were efficient at complex formation, yielding complexes that displayed little residual fluorescence and no appreciable surface charge. They appeared to form complexes efficiently with DNA, showing remarkable stability to subsequent polyanion-mediated disruption. Efficient charge neutralization may also explain their lack of surface charge, which probably underlies their tendency to aggregate. Their low transfection activity may also reflect their low surface charge, but their ability to undergo efficient intranuclear transcription is perhaps surprising in light of their stability to polyanions. The ability of cells to transcribe DNA bound even relatively firmly to cationic polymers may increase the opportunities for design of complexes combining good extracellular stability with efficient transcription following arrival within the nucleus (20). The next parameter studied in depth was the influence of cationic charge strength, using primary, tertiary, or quaternary amino groups, on the properties of complexes formed with DNA. Quaternary amino groups (pTMAEM.Cl) seemed to form complexes relatively efficiently with DNA, perhaps reflecting a stronger bond than with primary amino groups. Perhaps the most striking observation was the very poor transfection activity associated with the quaternarized polycation, pTMAEM.Cl, being 10-100-fold less active than complexes based on most other cationic polymers. It did undergo reasonably efficient intranuclear transcription, however, (except at high molecular weights) and the reason for its poor transfection activity may involve relatively poor access to the nucleus. Complexes formed using cationic polymers with pKa values in the endosomal range [e.g. poly(ethylene imine), Starburst dendrimers] are thought to mediate selective membrane activity on the endosome, perhaps via the so-called proton sponge mechanism (21), and this leads to extremely high transfectional activity. Hence, it seems possible that the complete absence of pH responsiveness in the quaternized polycation may restrict its ability to enter the cytoplasm/nucleus of target cells. The tertiary amine-containing pDMAEMam was less effective at DNA condensation in water, probably reflecting low protonation of its free base form, and complexes displayed a low ζ-potential (-15 mV). However, AFM showed that discrete complexes were formed and there was efficient transcription following their injection into the nuclei of Xenopus oocytes. These complexes also demonstrated reasonably high levels of transfection of 293 cells in vitro, compatible with the suggestions of good transfection activity associated with polymers with pKa in the endosomal range (21, outlined above). It should be noted that the pDMAEMam polymer is based on a methacrylamide backbone, while most of the other polymers are methacrylate-based; however, this difference is thought unlikely to affect properties of the complexes formed. Indeed, the corresponding methacrylate-based polymer has been studied in great detail (22), and high molecular weight forms are reported to have particularly good activity as in vitro transfection agents (23). The influence of the density of positive charge along the cationic polymer on the properties of complexes formed with DNA was examined by comparing pTMAEM.Cl with pBTMAIPM.I2, which bears two quater-


nary ammonio groups per monomer. Properties of complexes were remarkably similar, suggesting that a high density of positive charges does not adversely affect the interaction with DNA, although neither were any significant benefits observed. The effect of charge dilution was addressed by spreading out the positive charges using random copolymers containing TMAEM.Cl and HPMA as monomers. All copolymers were capable of binding DNA, according to ζ-potential and effects on electrophoretic mobility of the DNA, although copolymers with greater than 50% TMAEM.Cl content were not capable of forming particulate complexes, judged by AFM. Hence, it seems likely that these copolymers cannot drive hydrophobic self-assembly of polymer/DNA complexes even at charge neutrality; instead they form extended structures which can even display greater ethidium bromide/DNA fluorescence than untreated DNA controls (Figure 4a). It seems likely that the effects noted using these copolymers are mediated more by the presence of the hydrophilic HPMA component than directly by the spreading of the positive charges. In terms of biological activity, complexes based on the random copolymers showed low levels of transfection similar to the parent pTMAEM.Cl quaternary homopolymer. However, they also showed particularly poor ability to undergo transcription following intranuclear injection. Although the reasons for this are not clear, it may involve steric protection of the DNA from polymerases by the presence of the hydrophilic polymer. Finally, the influence of cationic polymer molecular weight average on properties of complexes formed with DNA has been examined. With DNA complexes based on pLL.HBr, polycation molecular weight is known to influence several properties (7). For example, complexes based on larger pLL.HBr are significantly bigger, more resistant to disruption by poly(L-aspartic acid), show a higher ζ-potential and are more active in transfecting cells (Figure 6). One explanation may be that, during complex formation, binding of small pLL.HBr to DNA is essentially reversible and leads to formation of thermodynamically stable complexes based on single DNA molecules. Such small complexes are efficiently charge neutralized and can often flocculate. Binding of larger pLL.HBr can be irreversible, however, and often proceeds inefficiently due to the inability of the polyelectrolytes to rearrange themselves. This leads to regions of excess positive charge, cross-linking of DNA molecules, and eventually to formation of complexes which are stable kinetically but thermodynamically locked in an unstable configuration. However, despite the simplicity of this hypothesis, it remains unsubstantiated when the same principles do not seem to govern self-assembly of DNA with other polymers such as pTMAEM.Cl. There were some indications of effects of molecular weight on transfection activity against 293 cells for many of the polymers examined in this study, including pVA.HCl, pLL.HBr, pMAEDA.HCl, pMAGEDA.HCl, and pTMAEM.Cl. With these polymers, DNA complexes formed with higher molecular weight cationic polymer preparations often showed greater expression of reporter genes. This was generally parallel with increased cytotoxicity of preparations based on larger cationic polymers, and it is not clear whether the cationic polymer which is free in solution at these N-to-P ratios is exerting an important effect in either cytotoxicity or transfection. The observations made in this study permit us to begin identifying parameters of cationic polymers that may affect the properties of the complexes they form with DNA. Many people working in this field are searching


for complexes with improved transfectional activity. It is clear from the above that polymer/DNA complexes formed using high molecular weight polymers are generally more transfection active, although the involvement of quaternary charges may lead to less efficient passage through the target cell to the nucleus. In contrast, targeted gene delivery for systemic application requires DNA complexes that are stable in the circulation, small enough to undergo extravasation into target tissues, able to recognize and enter target cells, including translocation into the nucleus, and suitable for efficient transcription within the nucleus of the target cell. One key issue is stability of complexes in the plasma, where they may interact with many different biological components (24-27). This area is the subject of several related studies, and at present, it is uncertain whether all of these requirements can be fulfilled by a simple selfassembling polycation/DNA system. We are therefore presently developing means of postassembly modification of complexes to mediate some of the objectives specified, using the self-assembling system as the core of the final complex (27-29). Chief requirements of the core complex are therefore small size, formation of monodisperse complexes, and the ability to permit efficient transcription within the nucleus of target cells. Consideration of the information generated in this analysis leads us to believe that the most appropriate complexes will be based on cationic polymers with a high charge density, containing nonquaternary amino groups, short side chains, and medium-to-low molecular weight distribution. ACKNOWLEDGMENT

This work was supported in part by the European Biotechnology Program 972334, PECO project Grant IC20CT970005, Grant Agency of the Czech Republic (307/ 96/K226), British Heart Foundation, and the Cancer Research Campaign. LITERATURE CITED (1) Dalgleish, A. G. (1997) Why: Gene therapy? Gene Ther. 4, 629-630. (2) Wright, M. J., Rosenthal, E., Stewart, L., Wightman, L. M. L., Miller, A. D., Latchman, D. S., and Marber, M. S. (1998) Beta-galactosidase staining following intracoronary infusion of cationic liposomes in the in vivo rabbit heart is produced by microinfarction rather than effective gene transfer: a cautionary tale. Gene Ther. 5, 301-308. (3) Bosch, A., McCray, P. B., Walters, K. S., Bodner, M., Jolly, D. J., Van Es, H. H. G., Nakamura, T., Matsumoto, K., and Davidson, B. L. (1998) Effects of keratinocyte and hepatocyte growth factor in vivo: Implications for retrovirus-mediated gene transfer to liver, Hum. Gene Ther. 9, 1747-1754. (4) Kong, H. L., Hecht, D., Song, W., Kovesdi, I., Hackett, N. R., Yayon, A., and Crystal, R. G. (1998) Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secreted form of the extracellular domain of the flt-1 vascular endothelial growth factor receptor. Hum. Gene Ther. 9, 823833. (5) Velu, P. T. (1997) Publication of the European Commission ETD/94/B5-3000/M1/11. (6) Marshall, E. (1995) The trouble with vectors. Science 269, 1052-1053. (7) Wolfert, M. A., and Seymour, L. W. (1996) Atomic force microscopic analysis of the influence of the molecular weight of poly(L)lysine on the size of polyelectrolyte complexes formed with DNA. Gene Ther. 3, 269-273. (8) Wolfert, M. A., Schacht, E. H., Toncheva, V., Ulbrich, K., Nazarova, O., and Seymour, L. W. (1996) Characterisation of vectors for gene therapy formed by self-assembly of DNA with synthetic block copolymers. Hum. Gene Ther. 7, 21232133.

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Wolfert et al. (20) Pollard, H., Remy, J. S., Loussouarn, G., Demolombe, S., Behr, J. P., and Escande, D. (1998) Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells, J. Biol. Chem. 273, 7507-7511. (21) Behr, J. P. (1997) The proton sponge: A trick to enter cells the viruses did not exploit. Chimia 51, 34-36. (22) Cherng, J. Y., VandeWetering, P., Talsma, H., Crommelin, D. J. A., and Hennink, W. E. (1996) Effect of size and serum proteins on transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles. Pharm. Res. 13, 1038-1042. (23) VendeWetering, P., Cherng, J. Y., Talsma, H., Crommelin, D. J. A., and Hennink, W. E. (1998) 2-(Dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents J. Controlled Release 53, 145-153. (24) Dash, P. R., Read, M. L., Barrett, L. B., Wolfert, M. A., and Seymour, L. W. (1999) Protein binding by polyelectrolyte complexes for gene delivery, Gene Ther. 6, 643-650. (25) Oupicky, D., Konac, C., Dash, P., Seymour, L. W., and Ulbrich, K. Effect of albumin and polyanions on the structure of DNA complexes with polycation-containing hydrophilic nonionic blocks. Bioconjugate Chem. 10, 764-772. (26) Ward, C. M., Ogris, M., Howard, K., and Seymour, L. W. Increased stability and prolonged plasma circulation of polyelectrolyte complexes containing DNA and high molecular weight poly(L-lysine) (manuscript in preparation). (27) Ogris, M., Brunner, S., Schuller, S., Kircheis, R., and Wagner, E. (1999) PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 6, 595-605. (28) Dash, P. R., Read, M. L., Fisher, K. D., Wolfert, M. A., Oupicky, D., Subr, V., Strohalm, J., Ulbrich, K., and Seymour, L. W., Decreased binding to proteins and cells of polymeric gene delivery vectors surface-modified with a multivalent hydrophilic polymer (submitted for publication). (29) Fisher, K. D., Ulbrich, K., Strohalm, J., Ward, C. M., Mautner, V., Blakey, D., and Seymour, L. W. (1999) A versatile system for receptor-mediated gene delivery permits increased entry of DNA into target cells, enhanced delivery to the nucleus and elevated rates of transgene expression (submitted for publication).

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