Quaternary Ammonium Polysaccharides for Gene

Bioconjugate Chem. 2005, 16, 1196−1203

1196

Quaternary Ammonium Polysaccharides for Gene Delivery Ira Yudovin-Farber,† Chava Yanay,‡ Tony Azzam,† Michal Linial,‡ and Abraham J. Domb*,†,§ Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O.B. 12065 Jerusalem 91120, Israel, Department of Biological Chemistry-Life Science, The Hebrew University of Jerusalem, Jerusalem 91905, Israel, and Alex Grass Center for Drug Design and Synthesis, The Hebrew University of Jerusalem. Received March 8, 2005; Revised Manuscript Received July 27, 2005

Cationic polysaccharides were synthesized by conjugation of various monoquaternary (MQ) ammonium oligoamines to oxidized dextran by reductive amination and tested for gene transfection. Polycations of dextran grafted with MQ ammonium oligoamines of two to four amino groups were investigated for their ability to cause pCMV-GFP encoding for green fluorescence protein and β-Gal encoding for β-galactosidase protein transfection on EPC and HEK-293 cell lines. These polycations were expected to strongly complex DNA due to increased surface cationic charge of the carrier, which may result in a higher transfection yield. However, the transfection yields were much lower compared to the parent vector, dextran-spermine conjugate, which was highly effective both in vitro and in vivo.

INTRODUCTION

The basic concept of gene therapy is that disease can be treated by transfer of genetic material into specific cells of a patient to supplement defective genes responsible for disease development (1). The main problem of gene therapy is gene delivery to gain high expression with maximal safety to the patient. A number of techniques have been developed for DNA delivery: direct introduction of transgene using cell electroporation, microinjection of DNA, and incorporation of the gene by viruses (2) and synthetic vectors (3). Viral vectors including retroviruses, adenoviruses, and adeno-associated viruses impress by their high efficiency in introducing their genetic material into host cells (4), although immunogenicity, inflammatory effects, and safety concerns restrict their usefulness. The advantages of nonviral vectors are that they do not integrate into chromosome, can introduce DNA into nondividing cells, do not possess infective risk, and are significantly less expensive and easy to handle than viral vectors (5). The major disadvantage of nonviral systems is their low transfection efficiency. The nonviral delivery vectors should overcome intracellular barriers, such as endosomes and nuclear membranes (6). Polycations are a leading class of nonviral gene delivery systems due to their versatility in molecular weight, polymer type, polymer-DNA ratio, molecular architecture, and the ability to introduce target-specific moieties. Polycations and negatively charged nucleic acids can spontaneously form nanocomplexes by electrostatic interaction. Polycation reduces the electrostatic repulsion between DNA and the cell membrane by neutralizing the DNA negative charge and also protects it from enzymatic digestion by nucleases in serum and extracellular fluids. Cationic materials that are commonly used in gene * To whom correspondence should be addressed. Phone: 9722-6757573. Fax: 972-2-6758959. E-mail: [email protected]. † Department of Medicinal Chemistry and Natural Products. ‡ Department of Biological Chemistry-Life Science. § Alex Grass Center for Drug Design and Synthesis.

delivery include poly(L-lysine) (7), cationic liposomes (8), polyethyleneimine (9), dendrimers (9), poly(L-histidine) (10), polyvinylpyridine, and cationic polysaccharides (11, 12). Polycations that are used as gene delivery systems are polyamines that contain primary, secondary, or tertiary amines (11). Under physiological conditions, polyamines become cationic and condense DNA into compact structures. In our previous publications, we reported on a new type of biodegradable polycation based on grafted oligoamine residues on natural polysaccharides (13). From a large number of oligoamine conjugates, the dextran-spermine conjugate was found to be the most active in transfecting a wide range of cell lines and plasmids in vitro and in vivo (14). This high transfection of dextran-spermine may be attributed to the spermine residues and architecture of the polysaccharide, which apparently play a significant role in cell transfection (14, 15). The effectiveness of the carrier is related to the ability of the polycation to condense DNA that is dependent on the structure of the polymer, the distance of cationic charge from the backbone, charge spacing along the polymer backbone, order of charged amine, and molecular weight. One way to achieve improved uptake into cells is by increasing the surface charge of complexes (16). This modification is expected to result in higher affinity to negatively charged cell membrane constituents and therefore to a higher rate of uptake. The charge of the carrier can also influence the size of the polymer-DNA complex (polyplex) that plays a significant role in the endosomal uptake (17), the cytoplasmatic transport and the migration through the nucleoporecomplexes which mediate the bidirectional transport between cytoplasm and nucleus. This report focuses on the synthesis of various oligoamines bearing a quaternary ammonium group conjugated to dextrans (Scheme 2) and investigates their transfection capacity in vitro. Monoquaternary (MQ) ammonium oligoamine-dextran conjugates were prepared and tested for their gene transfection efficiency on EPC and HEK-293 cell lines. These agents showed

10.1021/bc050066p CCC: $30.25 © 2005 American Chemical Society Published on Web 08/27/2005

Bioconjugate Chem., Vol. 16, No. 5, 2005 1197

Quaternary Ammonium Polycations for Gene Therapy Scheme 1. Synthesis of MQ Ammonium Spermine

Scheme 2. Grafting of Spermine Residues on Oxidized Dextran

moderate toxicity when tested on human embryonal kidney HEK-293 cells. MATERIALS AND METHODS

Materials. Dextran of an average molecular weight of 40 kDa, 1,4-butane diamine and 1,2-ethylenediamine, potassium periodate (KIO4), sodium borohydride (NaBH4), di-tert-butyl dicarbonate, and diisopropylethylamine (DIEA) were obtained from Sigma-Aldrich Israel, Rehovot. Spermine and ethyl trifluoroacetate were obtained from Fluka Chemie (Buchs, Switzerland). All solvents and reagents were of analytical grade and were used as received. Tetrahydrofuran (THF) was dried by distillation over sodium/benzophenone.

A sage-metering pump model-365 (Orion, NJ) was used for slow and reproducible addition of reactants. Average molecular weights of polycations were estimated by a GPC-Spectra Physics instrument (Darmstadt, Germany) consisting of a pump, column (Shodex KB-803), and refractive index (RI) detector using pullulan standards (PSS, Mainz, Germany) with molecular weights between 5 800 and 212 000. Eluents used were 0.05 M NaNO3 for the uncharged polymers and 5% (w/v) NaH2PO4 in 3% (v/v) acetonitrile (pH 4) for the cationic conjugates (18). The degree of conjugation was estimated by elemental microanalysis of nitrogen (%N) using a Perkin-Elmer 2400/II CHN analyzer. 1H NMR spectra (D2O, CDCl3 or DMSO-d6) were obtained on a Varian 300-MHz spec-

1198 Bioconjugate Chem., Vol. 16, No. 5, 2005

trometer in 5-mm outside diameter tubes. D2O/CDCl3/ DMSO-d6 containing tetramethylsilane served as solvent and shift reference. Fourier transform (FT) IR spectra were recorded on a Perkin-Elmer, 2000 FTIR. Purified plasmid (pCMV-GFP and pCMV-β-Gal) were from Qiagen kit, Germany. Fetal calf serum (FCS), 4 mM glutamine, penicillin, streptomycin, and amphotericin B were from Beit Haemek, Israel. Alamarblue reagent was from Biosorce, Israel. Cell transfection with pCMV-GFP was analyzed by fluorescence microscope instrument using model Axiovert 35, Zeiss, Jena, Germany. Methods. Synthesis of tri-tert-Butyl Carbonyl Spermine (tri-BOC Spermine). Spermine was selectively protected on one of the primary amino functional groups by reaction with ethyl trifluoroacetate. Spermine (21.96 mmol, 4.4 g) was dissolved in 250 mL of absolute methanol, and the solution was cooled to -78 °C, then an equimolar (21.96 mmol, 3.1 g) amount of ethyl trifluoroacetate was added during 1 h at -78 °C under nitrogen atmosphere. Stirring was continued for 1 h at 0 °C to complete the reaction. The remaining three amino functional groups were BOC protected with di-tert-butyl dicarbonate (4.0 equiv, 87.84 mmol, 19.1 g) that was added, and the mixture was stirred at 0 °C for 1 h under nitrogen atmosphere and then overnight at room temperature. The trifluoroacetamide protecting group was then cleaved by adding 30 mL of 25% ammonia solution (pH of the mixtrure was 11), and stirring was continued for 16 h at room temperature under nitrogen atmosphere. The resulting tri-BOC spermine was purified from diBOC spermine by chromatography over silica gel (dichloromethane (DCM):MeOH:NH4OH 70:10:1 to 50:10:1 v/v/ v). Average yield: 50%. 1 H NMR (DMSO, ppm): 1.4 (s, 27H, BOC hydrogens), 1.6 (m, 4H, NH(BOC)(CH2)3N(BOC)CH2CH2CH2CH2N(BOC)(CH2)3NH2), 1.72 (m, 4H, NH(BOC)CH2CH2CH2N(BOC)(CH2)4 N(BOC)CH2CH2CH2NH2), 2.6 (m, 2H, NH(BOC)(CH2)3N(BOC)(CH2)4N(BOC)CH2CH2CH2NH2), 3.1 (m, 10H, NH(BOC)CH2CH2CH2N(BOC) CH2CH2CH2CH2N(BOC)CH2CH2CH2NH2). Synthesis of MQ Ammonium Spermine (MQ-Spermine). Tri-BOC spermine (2.0 mmol, 1.0 g) was dissolved in 25 mL of anhydrous THF. The solution was cooled to 0 °C, then diisopropylethylamine (5.0 equiv, 10.0 mmol, 1.75 mL) and dimethyl sulfate (4.0 equiv, 8.0 mmol, 0.75 mL) dissolved in minimum amount of THF were added to the flask. The mixture was stirred for 24 h at room temperature under nitrogen atmosphere. Diisopropylethylammonium salt was discarded by filtration. The filtrate was evaporated under reduced pressure. The crude was dissolved in a mixture of trifluoroacetic acid (TFA) and DCM (TFA:DCM 10:90 v/v) for 2 h at room temperature to deprotect three amino functional groups. DCM and TFA were evaporated under reduced pressure, and the residue was redissolved in double deionized water (DDW) and purified from sulfate by Dowex-1 anion-exchange resin (chloride form). The fractions were picked and freeze dried. Average yield: 90%. 1H NMR (D O, ppm, tetrahydrochloride form): 1.62 2 (m, 4H, H3N(CH2)3NH2CH2CH2CH2CH2NH2(CH2)3N(CH3)3), 1.9 (m, 2H, H3NCH2CH2CH2NH2(CH2)4 NH2(CH2)3N(CH3)3), 2.08 (m, 2H, H3N(CH2)3NH2(CH2)4- NH2CH2CH2CH2N(CH3)3), 3.0 (m, 12H, H3NCH2CH2CH2NH2CH2CH2CH2CH2NH2-CH2CH2CH2N(CH3)3), 3.28 (m, 9H, H3N(CH2)3NH2(CH2)4NH2CH2CH2CH2N(CH3)3). Elemental Analysis: %N ) 13.36, %Cl ) 34. Synthesis of Dextran-MQ-Spermine Conjugate. Oxidized polysaccharide (1.0 g, 6.25 mmol of aldehyde groups) (19) was dissolved in 100 mL of DDW, and the

Yudovin-Farber et al.

solution was slowly added over several hours to a solution containing 6.25 mmol MQ-spermine dissolved in 50 mL of borate buffer (0.1 M, pH 11). The mixture was stirred at room temperature for 24 h. NaBH4 (1.0 g, 4.0 equimolar) was added to reduce the formed imine bonds and stirring was continued for 48 h at room temperature. The reduction was repeated with an additional portion of NaBH4 (1 g), and stirring was continued for additional 24 h. The resulting solution was poured into a dialysis membrane (3 500 cutoff, Membrane Filtration Products, Inc., San Antonio, TX) and dialyzed against DDW at 4 °C for 3 days. The dialysate was lyophilized to dryness. Average yield: 50% (w/w). 1 H NMR (D2O, ppm): 1.45 (m, 4H, MQ-spermine hydrogens), 1.63 (m, 4H, MQ-spermine hydrogens), 2.64 (m, 10H, MQ-spermine hydrogens), 3.0 (m, 9H, methyl hydrogens of MQ-spermine), 3.0 (m, 2H, MQ-spermine hydrogens), 3.30-4.45 (m, glucose hydrogens), and 5.01 (m, 1H, anomeric hydrogen). Synthesis of BOC-1,2-ethylenediamine. Solution of ditert-butyl dicarbonate (2.45 g, 0.011 mol) in dioxane (30 mL) was added over a period of 3 h to a solution of 1,2ethylenediamine (5.25 g, 0.087 mol) in 30 mL of dioxane. The mixture was allowed to stir for 24 h at room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in 50 mL of DDW, and the insoluble bis-substituted product was collected by filtration. The white filtrate that contained monoprotected 1,2-ethylenediamine was extracted with DCM (3 × 50 mL), dried over anhydrous MgSO4, filtered, and evaporated to dryness. The crude yellow oil was further dried under vacuum over solid NaOH pellets. Average yield: 90%. 1 H NMR (CDCl3, ppm): 1.4 (s, 9H, COOC(CH3)3), 2.8 (t, 2H, -CH2-NH2), 3.15 (m, 2H, BOC-NH-CH2-) and 5.02 (br, 1H, NH-BOC). Synthesis of MQ Ammonium 1,2-Ethylenediamine (MQ1,2-Ethylenediamine). BOC-1,2-ethylenediamine (1.0 g, 6.25 mmol) was dissolved in 5 mL of anhydrous dimethylformamide (DMF), then 3.7 mL of anhydrous tributylamine (TBA) was added to the flask. The mixture was cooled to 0 °C, and 3.3 g of methyl iodide (3.5 equimolar) was added. Stirring was continued overnight at room temperature under nitrogen atmosphere. Solvent and excess TBA were removed under reduced pressure and the resulting product was incubated for 2 h in 5 mL of TFA:DCM (10:90 v/v) at room temperature to deprotect the amino groups. The product was precipitated in 100 mL of diethyl ether, and the yellow precipitate was further dried under vacuum over solid NaOH pellets. The yellow precipitate was dissolved in DDW (5 mL) and purified from iodide by Dowex-1 anion-exchange resin (chloride form). The fractions were picked and freeze dried. Average yield: 80%. 1 H NMR (DMSO, ppm, dichloride form): 3.140 (s, 9H, -N(CH3)3), 3.282 (t, 2H, -CH2-NH3), 3.481 (t, 2H, -CH2-N(CH3)3), and 7.942 (br, 3H, -CH2NH3). Synthesis of Dextran-MQ-1,2-Ethylenediamine Conjugate. Oxidized dextran (100.0 mg, 0.625 mmol) was dissolved in 10 mL of DDW, and the solution was slowly added over several hours to a solution containing 0.625 mmol of MQ ammonium-1,2-ethylenediamine (1.0 equimolar) dissolved in 5 mL of borate buffer (0.1 M, pH 11). The mixture was stirred at room temperature for 24 h. NaBH4 powder (100.0 mg, 4 equimolar) was added to reduce the imine bonds to amines, and the mixture was further stirred for 48 h under the same conditions. The reduction was repeated with an additional portion of NaBH4 (100.0 mg), and stirring was continued for ad-

Quaternary Ammonium Polycations for Gene Therapy

ditional 24 h under the same conditions. Then, the reaction mixture was dialyzed (3500 cutoff, Membrane Filtration Products, Inc., San Antonio, TX) against DDW at 4 °C for 3 days. The dialysate was lyophilized to dryness. Average yield: 35% w/w. 1 H NMR (D2O, ppm): 2.9 (m, 2H, methylene hydrogens), 3.3 (s, 9H, methyl hydrogens), 3.3 (m, 2H, methylene hydrogens), 3.3-4.45 (m, glucose hydrogens) and 5.01 (m, 1H, anomeric hydrogen). Synthesis of BOC-1,4-Butane Diamine. Solution of ditert-butyl dicarbonate (2.45 g, 0.011 mol) in dioxane (30 mL) was added over a period of 3 h to a solution of 1,4butane diamine (7.67 g, 0.087 mol) in 30 mL of dioxane. The mixture was stirred for 24 h at room temperature, and the solvent was evaporated. The residue was dissolved in 50 mL of DDW, and the insoluble bissubstituted product was collected by filtration. Contained monoprotected 1,4-butane diamine filtrate was extracted with DCM (3 × 50 mL), dried over anhydrous MgSO4, filtered, and evaporated to dryness. The crude was dried under vacuum over solid NaOH pellets. Average yield: 90%. 1 H NMR (CDCl3, ppm): 1.4 (m, 9H, COOC(CH3)3), 1.45 (m, 4H, BOC-NH-CH2-(CH2)2-CH2-NH2), 2.6 (t, 2H, -CH2-NH2), 3.07 (m, 2H, BOC-NH-CH2-), and 4.8 (br, 1H, NH-BOC). Synthesis of MQ Ammonium-1,4-Butane Diamine (MQ-1,4-Butane Diamine). BOC-1,4-butane diamine (1.0 g, 5.31 mmol) was dissolved in 5 mL of anhydrous dimethylformamide (DMF), then 3.2 mL of anhydrous tributylamine (TBA) was added to the flask. The mixture was cooled to 0 °C, and 2.8 g of methyl iodide (3.5 equimolar) was added. Solution was stirred overnight at room temperature under nitrogen atmosphere. Solvent and excess TBA were removed under reduced pressure, and the resulting product was incubated for 2 h in 5 mL of TFA:DCM (10:90 v/v) at room temperature. The product was precipitated in 100 mL of diethyl ether, and the precipitate was further dried under vacuum over solid NaOH pellets. Average yield: 80%. 1 H NMR (D2O, ppm, dichloride form): 1.8 (m, 2H, -CH2-CH2-NH3), 1.9 (m, 2H, -CH2-CH2-N(CH3)3), 3.09 (m, 2H, -CH2-NH3), 3.15 (s, 9H, -N(CH3)3), and 3.4 (t, 2H, -CH2-N(CH3)3). Synthesis of Dextran-MQ-1,4-Butane Diamine Conjugate. Oxidized dextran (100.0 mg, 0.625 mmol) was dissolved in 10 mL of DDW, and the solution was slowly added over several hours to a solution containing 0.625 mmol of MQ ammonium 1,4-butane diamine (1.0 equimolar) dissolved in 5 mL of borate buffer (0.1 M, pH 11). The mixture was stirred at room temperature for 24 h. NaBH4 (100.0 mg, 4 equimolar) was added to reduce the imine bonds to amines, and the mixture was further stirred for 48 h under the same conditions. The reduction was repeated with an additional portion of NaBH4 (100.0 mg), and stirring was continued for additional 24 h under the same conditions. Then, the reaction mixture was dialyzed (3500 cutoff, Membrane Filtration Products, Inc., San Antonio, TX) against DDW at 4 °C for 3 days. The dialysate was lyophilized to dryness. Average yield: 35% w/w. 1 H NMR (D2O, ppm): 1.41 (m, 2H, -CH2-CH2-NHglucose unit), 1.7 (m, 2H, -CH2-CH2-N(CH3)3), 2.5 (m, 2H, -CH2-NH-glucose unit), 3.25 (m, 2H, -CH2N(CH3)3), 3.3 (m, 9H, -N(CH3)3), 3.3-4.45 (m, glucose hydrogens), and 5.01 (m, 1H, anomeric hydrogen). Preparation of Plasmid DNAs. Plasmid DNAs used in the transfection experiments were pCMV-GFP encoding for green fluorescence protein and pCMV-β-Gal encoding


β-Galactosidase. Plasmid DNAs were separately amplified in a XL-blue Escherichia coli and isolated using Qiagen Maxi kit-25 (Qiagen kit, Germany). Briefly, the grown bacteria were harvested and lysed in a 1% aqueous solution of sodium dodecyl sulfate (SDS) and 0.1% RNase A solution in NaOH (pH 8.0). The lysate was neutralized by the addition of 3 M potassium acetate (pH 5.5). After separation of the insoluble portion by centrifuging at 14 000 g for 30 min, the lysate was applied to an anionexchange resin (Qiagentip, Germany), followed by high salt washing (buffer containing 1 M NaCl) to remove traces of RNAs and denatured proteins. The plasmid DNA was eluted with an elution buffer containing 1.25 M NaCl at pH 8.5, desalted, and precipitated by 2-propanol. The precipitated plasmid DNA was centrifuged at 14 000 rpm for 10 min at 4 °C, and washed twice with 70% ethanol-water. After centrifugation (14 000 rpm, 2 min, 4 °C), the resulting plasmid DNA was air dried and dissolved in a small volume of 10 mM Tris-Cl pH 7.5. The purity of plasmid DNA was checked routinely, and the absorbance ratio of 260 nm to 280 nm ranged from 1.8 to 2.0. In vitro Transfection. In vitro transfection procedure was based on the previous publications (20). Human embryonal kidney (HEK-293) cells were used in the transfection experiments. Purified plasmid per well of transfected cells (0.5 µg) was mixed with the polycation at various weight-mixing ratios ranging from 3 to 9 w/w (polycation/ DNA). The polycation/DNA complex mixtures were diluted to a final volume of 200 µL with Dulbecco’s modified Eagle’s Medium (DMEM) and allowed to stand at room temperature for 40 min. Twenty-four-well plates coated with polyethyleneimine were seeded 24 h before transfection at 7 × 104 cells per well. Cultures were washed with preheated (37 °C) phosphate-buffered saline (PBS), and the solution of the complexes was added to the wells and cultured at 37 °C in 95% air/5% CO2. Four hours after transfection, cell medium was replaced with DMEM containing 10% fetal calf serum (FCS), 4 mM glutamine, penicillin, streptomycin, and amphotericin B. Cells were further cultured for 48-72 h. At this time, transfection efficiency and toxicity were assayed. For determination of the transfection efficiency, cultures were washed gently with warm PBS and then subjected to lysis and colorimetric β-galactosidase quantitative determination, measuring the amount of 2-nitrophenol formed in situ from cleavage of 2-nitrophenyl-B-D-galactopyranoside (ONPG) by β-galactosidase. Briefly: 300 µL of the reaction solution consisting of 1 mM MgCl2, 45 mM β-mercaptoEtOH, 73 mM Na2HPO4, 16 mM NaH2PO4, 0.1% SDS, and 0.88 mg/mL ONPG were applied onto each well, and the cells were then incubated at 37 °C for 30 min until a yellow color developed. Reaction was stopped with 500 µL of 1 M solution Na2CO3. The amount of 2-nitrophenol formed was determined by measuring optical density at 420 nm. Cells transfected with pCMV-GFP were analyzed for gene expression using fluorescence microscope instrument. For the assessment of the number of vital cells in each well, Alamarblue method was used, in which the reduction of Alamarblue from the oxidized indigo blue, nonfluorescing state to the reduced fluorescent pink state is indicative of the proportion of living cells in each well. Alamarblue reagent was diluted to 5% in growing media and applied to the culture for 4 h, after which the OD at 570 nm was measured. Percent toxicity was calculated as 100 - OD570SAMPLE/ OD570CTRL × 100, where the control group was not



Figure 1. (A) 1H NMR spectrum of the MQ-spermine. (B) 1H NMR spectrum of the compound #C.

subjected to any treatment but underwent the identical culturing and washing procedure. RESULTS AND DISCUSSION

Chemistry. In the present study, we have prepared quaternorized conjugates and investigated their transfection efficiency and cytotoxicity. Oligoamine derivatives having a quaternary ammonium salt in one end and a primary amine group in the other end were synthesized as illustrated in Scheme 1. Tri-BOC spermine derivative

was prepared in several steps using TFA and BOC orthogonal protecting groups. Ethyl trifluoroacetate was added at -78 °C to obtain the predominantly monotrifluoroacetamide derivative, followed by reaction with excess of di-tert-butyl dicarbonate to obtain the fully mixed protected derivative. The trifluoroacetamide protecting groups were removed by concentrated ammonia, yielding the tri-BOC spermine derivative. The tri-BOC spermine was then treated with dimethyl sulfate or methyl iodide to obtain the tri-BOC monoquaternary


Quaternary Ammonium Polycations for Gene Therapy

Table 1. Structures of Oligoamines Used in the Preparation of Dextran-Oligoamine Polycations

Table 2. Chemical Characterization of Dextran-Oligoamine Conjuagatesa code

oligoamine

%Nb

primary aminec

A

MQ-spermine:spermine 5:95 mole ratio MQ-spermine:spermine 10:90 mole ratio MQ-spermine:spermine 20:80 mole ratio MQ-spermine:spermine 5:95 mole ratio MQ-spermine:spermine 10:90 mole ratio spermine 100% MQ-spermine 100% MQ-1,2-ethylenediamine 100% MQ-1,4-butane diamine

9.08

0.56

9.05

B C D E F G H I

Mnd

P

relative transfection yielde

14.400

3.3

0.2

0.64

13.300

2.7

0.16

9.01

0.58

15.600

3.1

0.13

9.78

0.822

1800

1.7

0.17

9.96

0.66

1100

2.5

0.09

10.73 4.13 5.57 4.13

1.7 0.05 0.02 0.05

1.6 1.17 1.35 1.75

1.46 0 0 0

13.600 7.900 5.700 4.500

a Oligoamine was reacted with oxidized dextran (∼50% dialdehyde) at a 1:1 aldehyde/oligoamine mole ratio. b Found nitrogen content (elemental analysis). c Amount of primary amine (mmol/g) in conjugates determined by the TNBS method. d Average molecular weight (Mn) and polydispersity (P ) Mw/Mn) were determined by GPC. The remarkable difference in molecular weight of the #A and #B and #D and #E conjugates is explained by the use of dextran of 40 and 10 kDa as starting material. e β-Galactosidase quantitative determination (OD). Trasfection yield of the polycations, prepared at 3:1 weight ratio (polymer/DNA), were compared to calcium phosphate used as a positive reference (OD ) 1.307).

ammonium spermine derivative as a sulfate or iodide salt. Deprotection of BOC groups was achieved by treating with trifluoroacetic acid to obtain the MQ-spermine derivative. Figure 1A shows the 1H NMR spectrum obtained from this derivative. The remaining MQ ammonium alkane diamine derivatives were similarly prepared. Oxidized dextrans having similar molecular weight and aldehyde content were reacted under the same conditions with MQ ammonium tetramine and diamine (Table 1) followed by treating with sodium borohydride to obtain the amine conjugate (Scheme 2). MQ-oligoamine derivatives were chemically characterized (Table 2). The degree of substitution with MQ-oligoamine moieties was estimated by microanalysis (%N), primary amine content (TNBS) (13), and 1H NMR spectroscopy. Negligible primary amino group content was obtained for conjugates that were fully grafted with MQ ammonium oligoamines. This was expected by the fact that these oligoamine derivatives are being all grafted and terminated with quaternary ammonium functionalities. Transfection Results. Transfection experiments of grafted oxidized dextran with MQ-spermine, MQ ammonium-1,4-butane diamine, and MQ ammonium-1,2ethylenediamine were performed using EPC cell line in serum-free medium (SFM). Plasmid used for these studies was pCMV-luciferase encoding for luciferase protein.

Luciferase was quantitatively assayed using commercial enzyme-linked immunosorbent assay kits according to standart protocols. Dextran-spermine based polycations were found to be highly effective in transfecting cells in vitro (21) and in vivo (14, 22), therefore dextranspermine was used as a positive reference. Since spermine is clearly the active moiety in the transfection (21), it was plausible that increasing the charge level of the carrier by MQ-spermine conjugation onto dextran (Scheme 2, product #d) would result in enhanced transfection efficiency. However, in comparison with leading derivative dextran-spermine, all quaternary ammonium derivatives resulted in negligible transfection in all tests (Table 2). Possible explanation for this behavior is probably a result of the high cationic nature of the quaternary ammonium moieties, which form a too strong complex with DNA. In other words, quaternary ammonium groups seem to form too strong and stable complexes with DNA to be released, which led to a dramatic decrease in the transfection efficiencies. These results strengthen Margreet hypothesis about quaternary ammonium groups that form complexes efficiently with DNA, reflecting a stronger bond than with primary amino groups (23). We hypothesize that an additional explanation for the reduced transfection yield is the difference in the branching of the polymer. It was shown that dextran-spermine is a branched polymer with some



spermine residues serve as branching agents (13, 23). The degree of branching can be determined according to the following equation

%branching ) [(spermine (total) spermine (graft))/spermine (total)] × 100 where spermine (total) is determined by %N (microanalysis) and spermine (graft) can be determined by primary amine content (TNBS). In our case, 11% branching spermine moieties were obtained in the compound #F. On the other hand, use of the MQ-spermine reduces the degree of branching and thus a less branched polycation is obtained which affects the transfection yield. More studies will be needed to confirm this hypothesis. The structures of dextran-spermine and dextran-MQ-spermine are given in Scheme 2 (products #c and #d, respectively). Grafting of the oligoamines mixtures to oxidized dextran was the further step of our study. Dextran conjugates of mixtures of intact spermine and MQ-spermine were prepared by reacting oxidized dextran starting from different molecular weights with mixtures of spermine: MQ-spermine at 95:5 to 80:20 mole ratios followed by reduction with NaBH4 (Scheme 2, product #e). Chemical characteristic of these compounds and 1H NMR spectrum of one of them is given in Table 2 (compounds #A-E) and Figure 1B, respectively. The purpose of this step was to evaluate transfection efficiencies of these polymers as a function of MQ ammonium spermine percentages to the total grafted oligoamine. The transfection with these derivatives was performed in SFM applying HEK-293 cells. Plasmids used for these studies were pCMV-GFP encoding for green fluorescence protein as a marker gene and β-Gal encoding for β-galactosidase protein. Stock solutions of DNA and polycations were mixed at various weight ratios, diluted with DMEM, and left at room temperature. After 40 min, complex solutions were added separately to each cell well. After 4 h, SFM was replaced with a fresh medium. Incubation was continued for 48-72 h to complete transfection. Transfection yield was defined by colorimetric β-galactosidase quantitative determination, measuring the amount of 2-nitrophenol formed in situ from cleavage of 2-nitrophenyl-B-D-galactopyranoside by β-galactosidase. In the case of GFP protein, the transfection yield was quantified by visual counting of fluorescent cells in a certain field using a fluorescent microscope (data not shown). Calcium phosphate precipitating technique and dextran-spermine were used as positive reference. MQ ammonium conjugates were prepared and tested for their transfectivity. Each single polymer was tested over a range of polymer/DNA ratios from 3 to 9. Figure 2 demonstrates a series of transfection results, applying HEK-293 cells and β-Gal transfection systems. These conjugates showed low transfection yield even at 5% MQ-spermine content at both low and high molecular weight conjugates (compounds #A and #D). Further increase of weight ratio (polymer/DNA) resulted only in a slight improvement of the transfection yield (Figure 2). These results indicate that conjugation of MQ-spermine and probably other quaternary ammonium derivatives have negative effect on the transfection yield and that spermine remains the most effective as oligoamine graft. Evaluation of the Cytotoxicity. One of the requirements of the polymeric vectors for use in gene therapy is the absence of cytotoxicity. Cytotoxicity evaluation of the tested conjugates was performed by the Alamarblue method (24). Polycation solutions and plasmid DNA were

Figure 2. Transfection efficiencies of β-Gal system in HEK293 cells applying on mixed MQ-spermine/spermine grafted derivatives as vectors (Table 2). Calcium phosphate and dextran-spermine were used as positive reference.

Figure 3. Average cytotoxicity of the tested compounds. Cytotoxicity test was performed using Alamarblue method (24) on HEK-293 cells applying dextran-spermine and mixed MQspermine/spermine derivatives (see Table 2).

mixed at 3:1, 6:1, and 9:1 weight ratios (polymer/DNA). Toxicity of the polyplexes was presented by the number of vital cells in each well. Several conjugates showed a dose-response correlation between the polymer concentration and cell-growth inhibition (compounds #F and #A). Majority of the compounds demonstrated moderate effect on the cell viability or no toxicity at all at 3:1 weight ratio (dextran-spermine/DNA). However, an additional increase in the weight ratio resulted in a raise of cytotoxic properties of the majority of the samples (Figure 3). CONCLUSION

On the basis of the assumption that quaternorization may enhance the DNA-condensing ability of the cationic polysaccharides, various polycations were prepared by reductive amination between MQ ammonium oligoamines and oxidized dextran. The cationic polysaccharides demonstrated low transfection efficiency in EPC and HEK-293 cells. Mixed grafted MQ-spermine/spermine caused loss of transfection yield. This study proves again that the dextran-spermine conjugate, a biodegradable cationic polysaccharide, remains to be a promising gene carrier. Further attempts to improve its performance by quaternorization of the spermine residue resulted in the loss of activity and increased toxicity. ACKNOWLEDGMENT

This study was supported in part by the US-Israel Binational Fund and by the AFIRST, France-Israel initiative.


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Quaternary Ammonium Polysaccharides for Gene

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