Gene Transfer Efficacies of Novel Cationic Amphiphiles with Alanine

Mukthavaram Rajesh, Joyeeta Sen, Marepally Srujan, Koushik Mukherjee, Bojja Sreedhar ... Mikhail A. Maslov , Tatyana O. Kabilova , Ivan A. Petukhov , ...
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Bioconjugate Chem. 2005, 16, 903−912

903

Gene Transfer Efficacies of Novel Cationic Amphiphiles with Alanine, β-Alanine, and Serine Headgroups: A Structure-Activity Investigation† Joyeeta Sen and Arabinda Chaudhuri* Division of Lipid Science and Technology, Indian Institute of Chemical Technology, Hyderabad-500 007, India . Received February 16, 2005; Revised Manuscript Received June 3, 2005

Herein, we report on the relative in vitro efficacies of nine novel nonglycerol based cationic amphiphiles with increasing hydrophobic tails and the amino acids serine, alanine and β-alanine as the headgroup functionalities (lipids 1-9, Scheme 1) in transfecting multiple cultured cells including CHO, COS-1, MCF-7, and HepG2. The gene transfer efficiencies of lipids 1-9 were evaluated using the reporter gene assays in all the four cell lines and the whole cell histochemical X-gal staining assays in representative CHO cells. In CHO, HepG2, and MCF-7 cells, cationic lipids with alanine (4-6) and β-alanine (7-9) headgroups were found to be remarkably more transfection efficient than their serine headgroup counterparts (1-3). Most notably, in CHO, HepG2, and MCF-7 cells, in combination with cholesterol as auxiliary lipid, the transfection efficiencies of the cationic lipids with alanine and β-alanine headgroups and myristyl and palmityl tails (lipids 4, 5, 7 and 8) were significantly higher (2-3-fold) than that of LipofectAmine-2000, a widely used commercially available liposomal tranfection vectors. Surprisingly, in COS-1 cells, although cationic lipids with β-alanine headgroups (7-9) were strikingly transfection efficient (3-4-fold more efficacious than LipofectAmine-2000), the gene transfer properties of both their structural isomers (4-6) and their serine headgroup counterparts (1-3) were adversely affected. In summary, the present structure-activity investigation demonstrate that high gene delivery efficacies of cationic amphiphiles containing alanine or β-alanine headgroups can get seriously compromised by substituting the alanine or β-alanine with serine presumably due to the enhanced sensitivity of DNA associated with such serine-head-containing cationic lipids.

INTRODUCTION

Design of safe and efficacious therapeutic gene carriers still remains a critical challenge in gene therapy, the modality to combat myriads of inherited diseases, dreadful viral infections, and cancer. The gene transfer reagents currently in use can be broadly classified into two categories: viral and nonviral. Recombinant viral vectors, although remarkably efficient, suffer from numerous biosafety-related disadvantages (1-3). Viral vectors are capable of producing potentially replication-competent virus through recombination events with host genome and generating inflammatory and immunogenic responses against their structural components (4-8). In addition, viral vectors have a low insert-size limit for the therapeutic genes they can pack inside. Consequently, an increasing number of investigations are being reported on the development of safe and efficacious nonviral alternatives including cationic amphiphiles also known as cationic transfection lipids (9-18), cationic polymers (19, 20), dendrimers (21-23), etc. Because of their least immunogenic nature, robust manufacture, ability to deliver large pieces of DNA, and ease of handling and preparation techniques, cationic amphiphiles are gaining increasing popularity as the alternative nonviral transfection vectors of choice for delivering genes into our body cells (9-18, 24-52). The molecular architectures of cationic amphiphiles consist of a positively charged water-loving (hydrophilic) * Corresponding author. Tel: 91-40-27193201. Fax: 91-4027160757. E-mail: [email protected]. † IICT Communication No. 041003.

polar headgroup region and a nonpolar hydrophobic tail region (usually consisting of either two long aliphatic hydrocarbon chains or a cholesterol skeleton) often tethered together via a linker functionality such as ether, ester, amide, amidine group, etc. Systematic structureactivity investigations are being pursued toward understanding how variations in each of these three lipid structural components influence the gene transfer efficacies of cationic amphiphiles continue to be an intensely pursued area of research in nonviral gene delivery (12, 16, 27, 31, 34, 38, 40, 41, 47-52). As part of our ongoing structure-activity program in the area of cationic lipidmediated gene delivery (12, 16, 17, 47-52), in the present investigation, we report on the relative in vitro efficacies of nine novel nonglycerol-based cationic amphiphiles with increasing hydrophobic tails and the amino acids serine, alanine and β-alanine as the headgroup functionalities (lipids 1-9, Scheme 1) in transfecting multiple cultured cells including CHO, COS-1, MCF-7, and HepG2. As delineated below, the present structure-activity investigation demonstrates that high gene delivery efficacies of cationic amphiphiles containing alanine or β-alanine headgroups can get seriously compromised by substituting the alanine or β-alanine with serine presumably due to the enhanced sensitivity of DNA associated with such serine-head-containing cationic lipids. The transfection efficacy profiles of the presently described cationic lipids once again reiterate the proposition that gene transfer efficacies of cationic amphiphiles can be significantly modulated by minor structural variations in both the polar headgroup and hydrophobic tail regions of the lipid.

10.1021/bc0500443 CCC: $30.25 © 2005 American Chemical Society Published on Web 07/01/2005

904 Bioconjugate Chem., Vol. 16, No. 4, 2005

Sen and Chaudhuri

Scheme 1

EXPERIMENTAL PROCEDURES

General Procedures and Materials. FABMS data were acquired by the liquid secondary ion mass spectrometry (LSIMS) technique using m-nitrobenzyl alcohol as the matrix. LSIMS analysis was performed in the scan range 100-1000 amu at the rate of 3 scans/s. 1H NMR spectra were recorded on a Varian FT 200 MHz, AV 300 MHz, or Varian Unity 400 MHz. 1-Bromotetradecane, 1-bromohexadecane, 1-bromooctadecane, n-tetradecylamine, n-hexadecylamine, and n-octadecylamine were procured from Lancaster (Morecambe, England). The progress of the reactions was monitored by thin-layer chromatography on 0.25 mm silica gel plates. Column chromatography was performed with silica gel (Acme Synthetic Chemicals, India, finer than 200 and 60-120 mesh). p-CMV-SPORT-β-gal plasmid was a generous gift from Dr. Nalam Madhusudhana Rao. Lipofectamine was purchased from Invitrogen life technologies. Cell culture media, fetal bovine serum, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), poly(ethylene glycol) 8000, o-nitrophenyl-β-D-galactopyranoside, and cholesterol were purchased from Sigma, St. Louis, MO. NP-40, antibiotics, and agarose were purchased from Himedia, India. Unless otherwise stated, all the other reagents purchased from local commercial suppliers were of analytical grades and were used without further purification. COS-1 (SV 40 transformed African green monkey kidney cells), CHO (Chinese hamster ovary), HepG2 (human hepatocarcinoma), and MCF-7 (human breast adenocarcinoma) cell lines were procured from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were grown at 37 °C in Dulbecco’s modified Eagle’s

medium (DMEM)1 with 10% FBS in a humidified atomosphere containing 5% CO2/95% air. Purity of the final lipids 1-9 were determined by Analytical HPLC (Shimadzu Model LC10A) using a PARTISIL 5 ODS-3 WCS analytical column (4.6 × 250 mm, Whatman Inc., Clifton, NJ) in two different mobile phases. One solvent system (A) was methanol:water:trifluoroacetic acid in the ratio 75:25:0.05 (v/v) for 15 min with a flow rate of 0.8 mL/ min. The other solvent system (B) was methanol:acetonitrile:water:trifluoroacetic acid in the ratio 65:10:25:0.05 for 15 min with a flow rate of 0.8 mL/min. Peaks were detected by UV absorption at 219 nm. All the target lipids (1-9) showed more than 95% purity. Typical retention times in mobile phase A were: 3.56 min (lipid 1); 3.58 mins (lipid 2); 3.60 min (lipid 3), 3.59 mins (lipid 4); 3.63 min (lipid 5); 3.65 min (lipid 6); 3.57 min (lipid 7); 3.61 min (lipid 8); 3.64 min (lipid 9). Synthesis. Lipids 1-9 were synthesized essentially applying the same synthetic steps as depicted schematically in Scheme 1. As a representative experimental detail, synthesis of lipid 2 is described below. For all the other lipids, only TLC, 1H NMR, and mass spectral data are provided below. Due to extensive line broadening in 1 H NMR spectra of the final deprotected lipids (particularly in the region δ/ppm ) 3-5), all the final lipids were characterized by LSIMS. However, 1H NMR spectra of the BOC-protected immediate precursors for all the final 1 Abbreviations used: BOC, tert-butyloxycarbonyl; Chol, cholesterol; DCC, dicyclohexylcarbodiimide; DCM, dichloromethane; DMAP, 4-(N,N-dimethylamino)pyridine; DMEM, Dulbecco’s Modified Eagle’s Medium; DMF, N,N-dimethylformamide; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; FBS, fetal bovine serum; ONPG, o-nitrophenyl-β-D-galactopyranoside; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid.

Transfection Lipids with Amino Acid Heads

lipids 1-9 (provided below) were in complete agreement with their assigned molecular structures. Synthesis of N,N-Di-n-hexadecyl-N-methyl-N-[N′(L-serinyl)aminoethyl]ammonium Chloride (lipid 2). Step i (Scheme 1, Part A). Synthesis of N,N-Di-nhexadecyl-N-[N-(NR-BOC-L-serinyl)aminoethyl]amine. Solid N-hydroxysuccinimide (0.08 g, 0.70 mmol) and DCC (0.16 g, 0.80 mmol) were added sequentially to an ice cold and stirred solution of N-tert-butyloxycarbonyl-L-serine (0.14 g, 0.70 mmol, prepared from L-serine and di-tert-butyl dicarbonate as described previously (53)) in dry DCM/ dry DMF (9:1, v/v). After five minutes, N-(2-aminoethyl)N,N-di-n-hexadecylamine (0.35 g, 0.70 mmol, a readily available precursor in our laboratory prepared as described previously (48)) and DMAP (catalytic) dissolved in dry DCM were added to the reaction mixture. The resulting solution was stirred at room temperature for 6 h, solid DCU was filtered, and the solvent from the filtrate was evaporated. The residue was taken up in ethyl acetate (100 mL) and washed sequentially with icecold 1 N HCl (3 × 100 mL), saturated sodium bicarbonate (3 × 100 mL), and water (3 × 100 mL). The organic layer was dried over anhydrous sodium sulfate and filtered and the solvent from the filtrate removed by rotary evaporation. The residue upon column chromatographic purification with 60-120 mesh silica gel using 3-4% methanoldichloromethane (v/v) as eluent afforded 0.34 g (yield 71%) of the pure N,N-di-n-hexadecyl-N-[N-(NR-BOC-Lserinyl)aminoethyl]amine intermediate (Rf ) 0.45, 10% methanol-dichloromethane, v/v). 1H NMR (200 MHz, CDCl3): δ/ppm ) 0.9 [t, 6H, CH3(CH2)14]; 1.2-1.4 [m, 56H, (CH2)14]; 1.4-1.5 [s, 9H, COOC(CH3) 3]; 2.5 [t, 4H, N(CH2CH2)2]; 2.65 [t, 2H, NCH2CH2NHCO]; 3.3 [m, 2H, NCH2CH2NHCO]; 3.55 [dd, 1H, CH2NHCOCH(CHHOH)NHBOC]; 3.9 [dd, 1H, CH2NHCOCH(CHHOH)NHBOC]; 4.05 [m, 1H, CH2NHCOCH(CHHOH)NHBOC]; 5.5 (m, 1H, Boc-NH); 6.8 [m, 1H, CH2CH2NHCO]. Step ii (Scheme 1, Part A). Synthesis of N,N-Di-nhexadecyl-N-[N(NR-BOC-L-serinyl)aminoethyl]-N-methylammonium Iodide. The intermediate obtained in step i above was dissolved in 3 mL of dichloromethane/ methanol (2:1, v/v), and 3 mL of methyl iodide was added. The solution was stirred at room-temperature overnight. Solvent was removed on a rotary evaporator. The residue upon column chromatographic purification with 60-120 mesh size silica gel and 5-6% methanol in dichloromethane (v/v) as eluent afforded 0.22 g (54% yield) of pure N,N-di-n-hexadecyl-N-[N-(NR-BOC-L-serinyl)aminoethyl]-N-methylammonium iodide intermediate (Rf ) 0.3, 10% methanol in dichloromethane, v/v). 1H NMR (200 MHz, CDCl3): δ/ppm ) 0.9 [t, 6H, CH3(CH2)13]; 1.2-1.4 [m, 52H, CH3(CH2)13]; 1.4-1.5 [s, 9H, COOC(CH3)3]; 1.61.8 [m, 4H, N+(CH2CH2)2]; 3.05 [s, 3H, N+CH3]; 3.2-3.3 [m,4H, N+(CH2CH2)2]; 3.5 [m,2H, N+CH2CH2NHCO]; 3.6-3.75 [m, 3H, N+CH2CH2NHCO; dd, 1H, CH2NHCOCH(CHHOH)NHBOC]; 3.9 [dd, 1H, CH2NHCOCH(CHHOH)NHBOC]; 4.15 [m, 1H, CH2NHCOCH(CHHOH)NHBOC]; 6.0 [m, 1H, BOCNH]; 8.1 [m, 1H, CH2CH2NHCO]. Steps iii and iv (Scheme 1, Part A). Synthesis of N,NDi-n-hexadecyl-N-methyl-N-[N′-(L-serinyl)aminoethyl]ammonium Chloride (lipid 2). The intermediate obtained above in step ii (0.22 g) was dissolved in dry DCM (2 mL), and TFA (2 mL) was added at 0 °C. The resulting solution was stirred at room-temperature overnight to ensure complete deprotection. Excess TFA was removed by flushing with nitrogen to give the title compound as a trifluoroacetate salt. Column chromatographic purification using 60-120 mesh size silica gel and 12-14% (v/v)

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methanol-chloroform as eluent followed by chloride ion exchange chromatography (using amberlyst A-26 chloride ion-exchange resin) afforded 102 mg (57% yield) of pure lipid 2 (Rf ) 0.3-0.4, 15% methanol in chloroform, v/v). LSIMS (lipid 2): m/z: 611 [M + 1+] (calcd forC38H80O2N3, 35%). Lipids 1, 3-9 were synthesized following the same four-step protocols as described above for the synthesis lipid 2 using the appropriate starting mixed amine (I, Scheme 1) and N-BOC-protected appropriate amino acids shown in Scheme 1. The TLC characteristics, 1H NMR spectral data for the immediate precursor of the remaining final lipids, and the LSIMS data for the remaining final lipids are provided below. Lipid 1. 1H NMR (200 MHz, CDCl3) of N,N-di-ntetradecyl-N-[N-(NR-BOC-L-serinyl)aminoethyl]-N-methylammonium iodide (immediate precursor of lipid 1, Rf ) 0.3, 10% methanol in dichloromethane, v/v): δ/ppm ) 0.9 [t, 6H, CH3(CH2)11]; 1.2-1.4 [m, 44H, CH3(CH2)11]; 1.4-1.5 [s, 9H, COOC(CH3)3]; 1.6-1.8 [m, 4H, N+(CH2CH2)2]; 3.05 [s, 3H, N+CH3]; 3.2-3.3 [m,4H, N+(CH2CH2)2]; 3.5 [m,2H, N+CH2CH2NHCO]; 3.6-3.75 [m, 3H, N+CH2CH2NHCO; dd, 1H, CH2NHCOCH(CHHOH)NHBOC]; 3.9[dd, 1H, CH2NHCOCH(CHHOH)NHBOC]; 4.15 [m, 1H, CH2NHCOCH(CHHOH)NHBOC]; 6.0 [m, 1H, BOCNH]; 8.1 [m, 1H, CH2CH2NHCO]. LSIMS (lipid 1): m/z: 555 [M + 1+] (calcd forC34H72O2N3, 100%). Lipid 3. 1H NMR (200 MHz, CDCl3) of N,N-di-noctadecyl-N-[N-(NR-BOC-L-serinyl)aminoethyl]-N-methylammonium iodide (immediate precursor of lipid 3, Rf ) 0.3, 10% methanol in dichloromethane, v/v). δ/ppm ) 0.9 [t, 6H, CH3(CH2)15]; 1.2-1.4 [m, 60H, CH3(CH2)15]; 1.4-1.5 [s, 9H, COOC(CH3)3]; 1.6-1.8 [m, 4H, N+(CH2CH2)2]; 3.05 [s, 3H, N+CH3]; 3.2-3.3 [m,4H, N+(CH2CH2)2]; 3.5 [m,2H, N+CH2CH2NHCO]; 3.6-3.75 [m, 3H, N+CH2CH2NHCO; dd, 1H, CH2NHCOCH(CHHOH)NHBOC]; 3.9[dd, 1H, CH2NHCOCH(CHHOH)NHBOC]; 4.15 [m, 1H, CH2NHCOCH(CHHOH)NHBOC]; 6.0 [m, 1H, BOCNH]; 8.1 [m, 1H, CH2CH2NHCO]. LSIMS (lipid 3): m/z: 667 [M + 1+] (calcd forC42H88O2N3, 100%). Lipid 4. 1H NMR (200 MHz, CDCl3) of N-[N-(NR-BOCL-alanyl)aminoethyl]-N,N-di-n-tetradecyl-N-methylammonium iodide (immediate precursor of lipid 4, Rf ) 0.5, 10% methanol in dichloromethane, v/v). δ/ppm ) 0.9 [t, 6H, CH3(CH2)13]; 1.2-1.4 [m, 44H, (CH2)11; 3H, NHCOCH(CH3)NHBoc]; 1.4-1.5 [s, 9H, COOC(CH3)3]; 1.6-1.7 [m, 4H, N+(CH2CH2)2]; 3.2 [s, 3H, N+CH3]; 3.3 [m,4H, N+(CH2CH2)2]; 3.7-3.9 [bs, 4H, N+CH2CH2NHCO]; 4.05 [m, 1H, NHCOCH(CH3)NHBOC]; 5.2 [m, 1H, BOC NH]; 8.2 [m, 1H, CH2CH2NHCO]. LSIMS (lipid 4): m/z: 539 [M + 1+] (calcd forC34H72ON3, 100%). Lipid 5. 1H NMR (200 MHz, CDCl3): N,N-di-nhexadecyl-N-[N-(NR-BOC-L-alanyl)aminoethyl]-N-methylammonium iodide (immediate precursor of lipid 5, Rf ) 0.5, 10% methanol in dichloromethane, v/v). δ/ppm ) 0.9 [t, 6H, CH3(CH2)13]; 1.2-1.4 [m, 52H, (CH2)13; 3H, NHCOCH(CH3)NHBOC]; 1.4-1.5 [s, 9H, COOC(CH3)3]; 1.6-1.7 [m, 4H, N+(CH2CH2)2]; 3.2 [s, 3H, N+CH3]; 3.3 [m, 4H, N+(CH2CH2)2]; 3.7-3.9 [bs, 4H, N+CH2CH2NHCO]; 4.05 [m, 1H, NHCOCH(CH3)NHBOC]; 5.2 [m, 1H, BOC NH]; 8.2 [m, 1H, CH2CH2NHCO]. LSIMS (lipid 5): m/z: 595 [M + 1+] (calcd forC38H80N3O, 100%). Lipid 6. 1H NMR (200 MHz, CDCl3) of N-[N-(NR-BOCL-alanyl]aminoethyl]-N,N-di-n-octadecyl-N-methylammonium iodide (immediate precursor of lipid 6, Rf ) 0.5, 10% methanol in dichloromethane, v/v). δ/ppm ) 0.9 [t, 6H, CH3(CH2)13]; 1.2-1.4 [m, 60H, (CH2)15; 3H, NHCOCH(CH3)NHBOC]; 1.4-1.5 [s, 9H, COOC(CH3)3]; 1.6-1.7 [m, 4H, N+(CH2CH2)2]; 3.2 [s, 3H, N+CH3]; 3.3 [m, 4H,

906 Bioconjugate Chem., Vol. 16, No. 4, 2005

N+(CH2CH2)2]; 3.7-3.9 [bs, 4H, N+CH2CH2NHCO]; 4.05 [m, 1H, NHCOCH(CH3)NHBOC]; 5.2 [m, 1H, BOC NH]; 8.2 [m, 1H, CH2CH2NHCO]. LSIMS (lipid 6): m/z: 651 [M + 1+] (calcd forC42H88ON3, 100%). Lipid 7. 1H NMR (200 MHz, CDCl3) of N-[N-(NR-BOCL-β-alanyl)]aminoethyl]-N,N-di-n-tetradecyl-N-methylammonium iodide (immediate precursor of lipid 7, Rf ) 0.5, 10% methanol in dichloromethane, v/v). δ/ppm ) 0.9 [t, 6H, CH3(CH2)11]; 1.2-1.4 [m, 44H, CH3(CH2)11]; 1.41.5 [s, 9H, COOC(CH3)3]; 1.7 [m, 4H, N+(CH2CH2)2]; 2.45 [t, 2H, COCH2CH2NHBOC]; 3.3 [s, 3H, N+CH3]; 3.4-3.5 [bs, 6H, N+(CH2CH2)2; N+CH2CH2NH]; 3.7 [bs, 4H, N+CH2CH2NHCOCH2CH2NHBOC]; 5.3 [m, 1H, BOCNH]; 8.1 [m, 1H, CH2CH2NHCO]. LSIMS (lipid 7): m/z: 539 [M + 1+] (calcd forC34H72ON3, 100%). Lipid 8. 1H NMR (200 MHz, CDCl3) of N-[N-(NR-BOCL-β-alanyl)]aminoethyl]-N,N-di-n-hexadecyl-N-methylammonium iodide (immediate precursor of lipid 8, Rf ) 0.5, 10% methanol in dichloromethane, v/v). δ/ppm ) 0.9 [t, 6H, CH3(CH2)13]; 1.2-1.4 [m, 52H, CH3(CH2)13]; 1.41.5 [s, 9H, COOC(CH3)3]; 1.7 [m, 4H, N+(CH2CH2)2]; 2.45 [t, 2H, COCH2CH2NHBOC]; 3.3 [s, 3H, N+CH3]; 3.4-3.5 [bs, 6H, N+(CH2CH2)2; N+CH2CH2NH]; 3.7 [bs, 4H, N+CH2CH2NHCOCH2CH2NHBOC]; 5.3 [m, 1H, BOCNH]; 8.1 [m, 1H, CH2CH2NHCO]. LSIMS (lipid 8): m/z: 595 [M+] (calcd forC38H80ON3, 100%). Lipid 9. 1H NMR (200 MHz, CDCl3) of N-[N-(NR-BOCL-β-alanyl)]aminoethyl]-N,N-di-n-octadecyl-N-methylammonium iodide (immediate precursor of lipid 9, Rf ) 0.5, 10% methanol in dichloromethane, v/v). δ/ppm ) 0.9 [t, 6H, CH3(CH2)15]; 1.2-1.4 [m, 60H, CH3(CH2)15]; 1.41.5 [s, 9H, COOC(CH3)3]; 1.7 [m, 4H, N+(CH2CH2)2]; 2.45 [t, 2H, COCH2CH2NHBOC]; 3.3 [s, 3H, N+CH3]; 3.4-3.5 [bs,6H, N+(CH2CH2)2; N+CH2CH2NH]; 3.7 [bs, 4H, N+CH2CH2NHCOCH2CH2NHBOC]; 5.3 [m, 1H, BOCNH]; 8.1 [m, 1H, CH2CH2NHCO]. LSIMS (lipid 9): m/z: 651 [M + 1+] (calcd forC42H88ON3, 100%). Cell Culture. COS-1 (SV 40 transformed African green monkey kidney cells), CHO (Chinese hamster ovary), HepG2 (human hepatocarcinoma), and MCF-7 (human breast adenocarcinoma cell) cell lines were procured from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, 50 µg/mL penicillin, 50 µg/mL streptomycin, and 20 µg/mL kanamycin in a humidified atmosphere containing 5% CO2. Preparation of Liposomes. The cationic lipid and the colipid (Chol) in 1:1 mole ratio were dissolved in a mixture of chloroform and methanol (3:1, v/v) in a glass vial. The solvent was removed with a flow of moisture free nitrogen gas, and the dried lipid film was then kept under high vacuum for 8 h. A 5 mL amount of sterile deionized water was added to the vacuum-dried lipid film, and the mixture was allowed to swell overnight. The vial was then vortexed for 2-3 min at room temperature and occasionally sonicated in a bath sonicator to produce multilamellar vesicles (MLVs). MLVs were then sonicated in an ice bath (usually 45-60 s) until clarity using a Branson 450 sonifier at 100% duty cycle and 25 W output power. The resulting clear aqueous liposomes were used in forming lipoplexes. Plasmid DNA. pCMV-SPORT-β-gal plasmid was a generous gift from Dr. Nalam Madhusudhana Rao (Centre for Cellular and Molecular Biology, Hyderabad, India) and was amplified in DH5R strain of Escherichia coli, isolated by alkaline lysis procedure, and finally purified by PEG-8000 precipitation as described previously (54).

Sen and Chaudhuri

The purity of plasmid was checked by A260/A280 ratio (around 1.9) and 1% agarose gel electrophoresis. Transfection of Cells. Cells were seeded at a density of 15000 (for COS-1) and 20000 cells (for CHO, HepG2, and MCF-7) per well in a 96-well plate 18-24 h before the transfection. A 0.3 µg amount of plasmid DNA was complexed with varying amounts of lipids (0.09-8.1 nmol) in plain DMEM medium (total volume made up to 100 µL) for 30 min. The charge ratios were varied from 0.1:1 to 9:1 (() over these ranges of the lipids. Immediately prior to transfection, cells plated in the 96well plate were washed with PBS (2 × 100 µL) followed by the addition of lipoplexes. After 3h of incubation, 100µL of DMEM with 20% FBS was added to the cells. The medium was changed to 10% complete medium after 24 h and the reporter gene activity was estimated after 48 h. The cells were washed twice with PBS (100 µL each) and lysed in 50 µL of lysis buffer [0.25 M Tris-HCl (pH 8.0) and 0.5% NP40]. Care was taken to ensure complete lysis. The β-galactosidase activity per well was estimated by adding 50 µL of 2X-substrate solution [1.33 mg/mL of ONPG, 0.2 M sodium phosphate (pH 7.3) and 2 mM magnesium chloride] to the lysate in a 96-well plate. Absorbance of the product o-nitrophenol at 405 nm was converted to β-galactosidase units by using a calibration curve constructed using pure commercial β-galactosidase enzyme. Each transfection experiment was repeated three times on three different days. The transfection values reported were average values from three replicate transfection plates assayed on three different days. The values of β-galactosidase units in replicate plates assayed on the same day varied by less than 20%. The day to day variation in transfection efficiency values for identically treated transfection plates was mostly within 2-3-fold and was dependent on the cell density and condition of the cells. Toxicity Assay. Cytotoxicities of lipids 1-9 were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay as described earlier (37). The cytotoxicity assay was performed in 96-well plates by maintaining the same ratio of number of cells to amount of cationic lipid, as used in the transfection experiments. MTT was added 3 h after addition of cationic lipid to the cells. Results were expressed as percent viability ) [A540(treated cells) background/A540(untreated cells) - background] × 100. Zeta Potential (ζ) and Size Measurements. The sizes and the surface charges (zeta potentials) of liposomes and lipoplexes were measured by photon correlation spectroscopy and electrophoretic mobility on a Zeta sizer 3000HSA (Malvern, UK). The sizes were measured in deionized water with a sample refractive index of 1.59 and a viscosity of 0.89. The system was calibrated by using the 200 ( 5 nm polystyrene polymer (Duke Scientific Corp., Palo Alto, CA). The diameters of liposomes and lipoplexes were calculated by using the automatic mode. The zeta potential was measured using the following parameters: viscosity, 0.89 cP; dielectric constant, 79; temperature, 25 °C; F(Ka), 1.50 (Smoluchowski); maximum voltage of the current, V. Measurements were done 10 times with the zero field correction. The potentials were calculated by using the Smoluchowski approximation. DNase 1 Sensitivity Assays. Briefly, in a typical assay, 1.5 nmol of DNA (500 ng) was complexed with lipid using the indicated lipid:DNA charge ratios in 10 mM HEPES buffer (pH 7.4) in a volume of 40 µL and the mixture incubated at room temperature for 30 min on a rotary shaker. Subsequently, the complexes were treated

Transfection Lipids with Amino Acid Heads

Bioconjugate Chem., Vol. 16, No. 4, 2005 907

with DNase I (at a final concentration of 1 ng/1.5 nmol of DNA) in the presence of 10 mM MgCl2 and incubated for 20 min at 37 °C. The reactions were then halted by adding EDTA (to a final concentration of 50 mM) and incubated at 60 °C for 10 min in a water bath. The aqueous layer was washed with 50 µL of phenol:chloroform mixture (1:1, v/v) and centrifuged at 10 000 g for 5 min. The aqueous supernatants were separated, loaded (15 µL) on a 1% agarose gel, and electrophoresed at 100 V for 2 h. The bands were visualized with ethidium bromide staining. X-Gal Staining. Cells expressing β-galactosidase were histochemically stained with the substrate 5-bromo-4chloro-3-indolyl-β-D-galactopyranoside (X-gal) as described previously (47). Briefly, 48 h after transfection with lipoplexes in 96-well plates, the cells were washed two times (2 × 100 µL) with phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and fixed with 0.5% glutaraldehyde in PBS (225 µL). After 15 min incubation at room temperature, the cells were washed again with PBS three times (3 × 250 µL) and subsequently were stained with 1.0 mg/mL X-gal in PBS containing 5.0 mM K3[Fe(CN)6], and 5.0 mM K4[Fe(CN)6] and 1 mM MgSO4 for 2-4 h at 37 °C. Blue colored cells were identified by light microscope (Leica, Germany). RESULTS

Chemistry. Lipids 1-9 were prepared by conventional DCC coupling of the common precursor I (a readily available hydrophobic mixed primary-tertiary amine in our laboratory prepared as described previously (48)) and the appropriate BOC-protected amino acids (Scheme 1). The resulting intermediates were then subjected to quaternization with excess methyl iodide and BOCdeprotection with trifluoroacetic acid. The BOC-deprotected products afforded pure target lipids upon chloride ion exchange chromatography over Amberlyst-26 (Scheme 1). Minor variation in lipid structures were ensured by designing lipid pairs varying either only in structurally isomeric headgroup functionalities (e.g. lipid pairs 4 and 7, 5 and 8, and 6 and 9) or by designing lipid pairs whose sole structural difference is substitution of a hydrogen atom in the headgroup side chain of one lipid by a hydroxyl functionality in the other (e.g. lipid pairs 1 and 4, 2 and 5, and 3 and 6). Structures of all the synthetic intermediates and the final lipids shown in Scheme 1 were confirmed by 1H NMR and LSIMS. Transfection Biology. The in vitro gene transfer efficacies of lipids 1-9 were first measured by the reporter gene expression assay using equimolar cholesterol as colipid (cholesterol was found to be a more efficacious colipid than the other commonly used colipid dioleoyllphosphatidyl ethanolamine, DOPE, data not shown) and pCMV-SPORT-β-gal plasmid as the reporter gene across the lipid:DNA charge ratios 9:1-0.3:1. Figures 1-4 summarize the relative in vitro gene delivery efficacies of lipids 1-9 in transfecting CHO, HepG2, MCF7, and COS-1 cells, respectively. In CHO, HepG2, and MCF-7 cells, cationic lipids with alanine and β-alanine headgroups were, in general, found to be more transfection efficient than LipofectAmine-2000 (by about 2-fold in CHO and MCF-7 cells), a commercially available popular liposomal transfection kit (Figures 1-3, Parts B and C). Except in MCF-7 cells at 1:1 lipid:DNA charge ratio and in COS-1 cells, the sterylated lipids with alanine (lipid 6) and β-alanine (lipid 9) headgroups were found to be less efficient than their myristyl and palmityl

Figure 1. In vitro transfection efficiencies in CHO cells of lipids 1, 2, 3 (A); lipids 4, 5, 6 (B); and lipids 7, 8, 9 (C) using cholesterol as colipid (at lipid:cholesterol mole ratio of 1:1). Units of β-galactosidase activity were plotted against the varying lipid to DNA (() charge ratios. The transfection efficiencies of the lipids were compared to that of commercially available LipofectAmine 2000. Transfection experiments were performed as described in the text. The transfection values shown are average of duplicate experiments performed on the same day.

analogues (lipids 4 and 5, and 7 and 8, respectively, Figures 1-4, parts B and C). However, unlike the previously reported high transfection efficacies of cationic lipids containing hydroxyalkyl headgroups (31, 40, 41, 49, 50, 52), the cationic lipids with serine headgroups (lipids 1-3) in spite of possessing hydroxymethyl headgroup functionalities were found to be less efficacious than their alanine (lipids 4-6) and β-alanine (lipids 7-9) analogues in transfecting all the four cells (Figures 1-4). Unlike the comparable gene transfer efficacies of the cationic lipids with structurally isomeric alanine (lipids 4-6) and β-alanine headgroups (lipids 7-9) in CHO, HepG2, and MCF-7 cells (Figures 1-3, parts B and C), lipids 7-9 with β-alanine headgroups showed significantly higher transfection properties in COS-1 cells than its alanine analogues (Figure 4, parts B and C). In particular, at 3:1 lipid:DNA charge ratio, lipid 9 was found to be strikingly more efficient (>10-fold) than its alanine counterpart (lipid 6) in transfecting COS-1 cells (Figure 4, parts B and C). The β-alanine lipid with a palmityl chain (8) were 3-4-fold more transfection efficient than LipofectAmine-2000 at lipid:DNA charge ratios of 3:1 (Figure 4, part C). The general transfection profiles for lipids 1-9 found in the above-mentioned reporter gene assays (Figures 1-4) were further confirmed by the whole cell histochemical X-gal staining assay. Representative X-gal staining patterns for CHO

908 Bioconjugate Chem., Vol. 16, No. 4, 2005

Figure 2. In vitro transfection efficiencies in HepG2 cells of lipids 1, 2, 3 (A); lipids 4, 5, 6 (B); and lipids 7, 8, 9 (C) using cholesterol as colipid (at lipid:cholesterol mole ratio of 1:1). Units of β-galactosidase activity were plotted against the varying lipid to DNA (() charge ratios. The transfection efficiencies of the lipids were compared to that of commercially available LipofectAmine 2000. Transfection experiments were performed as described in the text. The transfection values shown are average of duplicate experiments performed on the same day.

cells are shown in Figure 5 for lipids 1, 3, 4, 6, 7, and 9 at both lipid:DNA charge ratios of 3:1 and 1:1. Cell Viabilities. MTT-based cell viability assays were performed in representative CHO cells with lipids 1-9 as well as with the commercially available LipofectAmine across the entire range of lipid:DNA charge ratios used in the actual transfection experiments. Percent cell viabilities of lipids 1-9 were found to be high (more than 75%), particularly up to the lipid:DNA charge ratios of 3:1 (Figure 6). However, LipofectAmine was found to be less toxic than the present lipids, particularly at lipid:DNA charge ratios 9:1 and 3:1 (Figure 6). Physicochemical Characterizations of Liposomes and Lipoplexes. Sizes and Zeta Potentials. The nanosizes and the global surface charges of the representative lipoplexes prepared with lipids 1, 3, 4, 6, 7 and 9 were measured using dynamic laser light scattering instrument equipped with zeta-sizing capacity across the lipid:DNA charge ratios of 0.1:1 to 9:1 in the presence of DMEM. Broadly speaking, the nanosizes of lipoplexes prepared using both transfection efficient (e.g. 4 and 7) and transfection-incompetent lipids (e.g. 3 and 6) similarly increased with increasing lipid:DNA charge ratios (up to 3:1) within the range 200-430 nm (data not shown). At the highest lipid:DNA charge ratios of 9:1, all the lipoplexes grew somewhat larger (around 600 nm). Interestingly, in the presence of DMEM, the global

Sen and Chaudhuri

Figure 3. In vitro transfection efficiencies in MCF-7 cells of lipids 1, 2, 3 (A); lipids 4, 5, 6 (B); and lipids 7, 8, 9 (C) using cholesterol as colipid (at lipid:cholesterol mole ratio of 1:1). Units of β-galactosidase activity were plotted against the varying lipid to DNA (() charge ratios. The transfection efficiencies of the lipids were compared to that of commercially available LipofectAmine 2000. Transfection experiments were performed as described in the text. The transfection values shown are average of duplicate experiments performed on the same day.

surface charges of both the inefficient as well as efficient lipoplexes prepared using lipid:DNA charge ratios of 1:1 or 3:1 were found to be either slightly positive (2-5 mV) or close to electroneutrality (Table 1). Gel-Mobility Shifts. Toward understanding the role of lipid:DNA electrostatic binding interactions (if any) in modulating the in vitro transfection efficacies of lipids 1-9, gel-mobility shift assays were performed using representative lipoplexes prepared with lipids 1 and 3 (serine head), 4 and 6 (alanine head), and 7 and 9 (βalanine head) across the lipid:DNA charge ratios of 9:1 to 0.1:1. The band intensities of the free DNA (unassociated or loosely bound with lipoplexes) for all these lipoplexes were found to be remarkably diminished at lipid:DNA charge ratios of 3:1 and 9:1 (Figure 7), indicating that at such high lipid:DNA charge ratios, all the lipids were strongly associated with plasmid DNA. At lower lipid:DNA charge ratios (1:1-0.1:1), the band intensities of free DNA for all the lipoplexes increased significantly, the highest increase being observed with serine-lipids (lipids 1 and 3, Figure 7, parts A-C). DNase I Sensitivities. Toward obtaining insights into the relative accessibilities of the lipoplex associated DNA to DNase I, DNase I sensitivity assays were carried out using lipoplexes made with lipids 1-9 across the entire range of lipid:DNA charge ratios 0.1:1-9:1. After the free DNA digestion by DNase I, the total DNA (both digested

Transfection Lipids with Amino Acid Heads

Bioconjugate Chem., Vol. 16, No. 4, 2005 909

Figure 4. In vitro transfection efficiencies in COS-1 cells of lipids 1, 2, 3 (A); lipids 4, 5, 6 (B); and lipids 7, 8, 9 (C) using cholesterol as colipid (at lipid:cholesterol mole ratio of 1:1). Units of β-galactosidase activity were plotted against the varying lipid to DNA (() charge ratios. The transfection efficiencies of the lipids were compared to that of commercially available LipofectAmine 2000. Transfection experiments were performed as described in the text. The transfection values shown are average of duplicate experiments performed on the same day.

and inaccessible DNA) was separated from the lipid and DNase I (by extracting with organic solvent) and loaded onto a 1% agarose gel. Resulting electrophoretic gel pattens in such DNase I protection experiments for representative lipoplexes prepared with lipids 1 and 3, 4 and 6, and 7 and 9 are shown in Parts A-C of Figure 8. In general, the band intensities of inaccessible and therefore undigested DNA associated with transfection incompetent lipids (1 and 3 in particular) was observed to be significantly less than those associated with transfection efficient lipids (e.g. 4 and 7, Figure 4, Parts A-C). DISCUSSION

As a part of our ongoing structure-activity investigations in cationic lipid mediated gene delivery (12, 16, 17, 47-52), in the present study, we have studied the influence of minor headgroup modifcations in liposomal gene delivery using nine novel cationic amphiphiles 1-9 containing simple aliphatic hydrocarbon chains (myristyl, palmityl, and stearyl) as hydrophobic tails and amino acids alanine, β-alanine and serine as their polar heads. Structurally speaking, lipid series 4-6 and 7-9 are very similar in nature. Except the headgroups of 4-6 being the structural isomers of lipids 7-9, there are no differences between them. The only difference in the headgroup regions of lipids 1-3 and lipids 4-6 is that one hydrogen atom of the side-chain methyl groups of lipids

Figure 5. Histochemical whole cell X-gal staining of CHO cells transfected with lipids 1, 3, 4, 6, 7, and 9 at lipid:DNA charge ratios of 3:1 and 1:1. Cells expressing β-galactosidase were stained with X-gal as described in the text.

4-6 is substituted by one hydroxyl group in lipids 1-3. Taking the high transfection efficacies of many previously reported cationic lipids containing a hydroxyalkyl headgroup (31, 40, 41) including our own (49, 50, 52) into account, we expected lipids 1-3 to be more transfection efficacious than lipids 4-6. Surprisingly, lipids 1-3 turned out to be the most inefficient among all the lipids 1-9 (Figures 1-5). Findings in MTT-assay-based cell viability assays (Figure 6) ruled out the possibility of varying cell cytotoxicities playing any key role behind the compromised gene transfer efficacies of the serine lipids (1-3). The compromised transfection efficacies of lipids 1-3 in transfecting CHO, HepG2, MCF-7, and COS-1 cells across the entire lipid:DNA charge ratios of 0.1:9:1 (Figures 1-4, parts A) demonstrate that even minor structural substitution in the headgroup region can significantly lower the in vitro gene transfer properties of cationic amphiphiles. Such remarkable influence of minor headgroup modification in cationic lipid-mediated gene delivery is consistent with our recently reported findings on the dramatically superior transfer properties of a cholesterol-based cationic lipid with a β-alanine headgroup to its glycine counterpart (47). Findings in the

910 Bioconjugate Chem., Vol. 16, No. 4, 2005

Sen and Chaudhuri

Table 1. Zeta Potentiala (mV) of Liposomes and Lipoplexes (in DI water and plain DMEM, respectively) lipid:DNA charge ratio (N/P) no.

sample code

1:0

9:1

3:1

1:1

0.3:1

0.1:1

1 2 3 4 5 6

Lipid 1 Lipid 3 Lipid 4 Lipid 6 Lipid 7 Lipid 9

18.6 ( 1.6 17.8 ( 1.2 27.1 ( 1.6 27.7 ( 2.1 20.2 ( 4.7 28.8 ( 3.2

6.4 ( 1.3 6.6 ( 1.0 1.6 ( 2.8 2.9 ( 1.9 11.9 ( 6.8 6.3 ( 1.3

3.2 ( 1.6 0.9 ( 2.1 0.4 ( 1.8 -4.5 ( 2.1 5.9 ( 2.7 2.3 ( 2.0

1.6 ( 2.3 -0.2 ( 3.4 -1.9 ( 4.4 -8.7 ( 3.2 0.3 ( 2.5 1.3 ( 2.4

-32.0 ( 1.6 -29.2 ( 2.4 -15.5 ( 5.6 -22.6 ( 2.8 -33.7 ( 2.0 -30.6 ( 2.4

-30.4 ( 1.7 -33.6 ( 1.8 -25.6 ( 2.9 -30.7 ( 2.4 -28.6 ( 1.9 -27.3 ( 2.2

a ξ Potentials were measured by laser light scattering technique using Zetasizer 3000A (Malvern Instruments, UK). Values shown are the averages obtained from 10 (zeta potential) measurements.

Figure 6. MTT-assay based cellular cytotoxicities of lipids 1-9 and commercially available LipofectAmine against representative CHO Cells. The percent cell viability values shown are average of duplicate experiments performed on the same day.

Figure 7. Electrophoretic gel patterns for lipoplex-associated DNA in gel retardation for lipid 1 and 3 (A), for lipid 4 and 6 (B), and for lipid 7 and 9 (C). The lipid:DNA charge ratios are indicated at the top of each lane. The details of treatment are as described in the text.

Figure 8. Electrophoretic gel patterns for lipoplex-associated DNA in DNase I sensitivity assay for lipids 1 and 3 (A), for lipids 4 and 6 (B), and for lipids 7 and 9 (C). The lipid:DNA charge ratios are indicated at the top of each lane. The details of treatment are as described in the text.

Transfection Lipids with Amino Acid Heads

gel mobility shift assay indicate similar DNA-binding characteristics of lipids 1-9 (Figure 7). However, the electrophoretic gel patterns in DNase I sensitivity assays revealed pronounced sensitivities of DNA associated with lipoplexes of lipids 1-3 compared to DNA associated with lipids 4-9 (Figure 8). Thus, the findings summarized in Figures 7 and 8, taken together, support the notion that enhanced DNase I sensitivity of DNA associated with lipids 1-3 might play some important role behind their relatively poor gene transfer properties. However, the origin of such relatively pronounced DNase I sensitivities of the plasmid DNA associated with serine-lipids 1-3 across the entire lipid:DNA charge ratios of 9:1 to 0.1:1 remains elusive at this point of investigation. The transfection profiles in COS-1 cells (Figure 4) provided the most convincing examples of dramatic headgroup influence in cationic lipid-mediated gene delivery. In COS-1 cells, although lipids 7-9 with β-alanine headgroups were found to be highly transfection efficient (lipid 8 at lipid:DNA charge ratios of 3:1 were 3-4-fold more transfection efficient than LipofectAmine2000), gene transfer properties of lipids 4-6 containing their structurally isomeric alanine headgroups were observed to be significantly less (Figure 4, parts B and C). The strikingly enhanced gene transfection properties of lipid 9 with a β-alanine headgroup and a stearyl tail compared to lipid 6 with the same stearyl tail but a structurally isomeric alanine headgroup at 3:1 lipid:DNA charge ratio in COS-1 cells (Figure 4, parts B and C) demonstrate that in cationic lipid-mediated gene delivery even lipids with structurally isomeric headgroups can exhibit phenomenally contrasting transfection properties. The compromised transfection efficacies of all the present lipids at 9:1 lipid:DNA charge ratios (Figures 1-5) may partly originate from the big lipoplex sizes (>600 nm, data not shown). The global surface charge data summarized in Table 1 rules out the possibility of varying lipoplex surface potentials playing any key role behind the contrasting transfection profiles of the present lipids. In conclusion, the present in-depth structure-activity investigation using nine novel cationic lipids with alanine, β-alanine, and serine headgroups demonstrates that high gene delivery efficacies of cationic amphiphiles containing alanine or β-alanine headgroups can get seriously compromised by substituting the alanine or β-alanine with serine, presumably due to the enhanced sensitivity of DNA associated with such serine-headcontaining cationic lipids. The transfection efficacy profiles of the presently described cationic lipids once again reiterate the proposition that gene transfer efficacies of cationic amphiphiles can be significantly modulated by minor structural variations in both the polar headgroup and hydrophobic tail regions of the lipid. ACKNOWLEDGMENT

Financial support received from the Department of Biotechnology, Government of India (to A.C.) is gratefully acknowledged. J.S. sincerely thanks the University Grant Commission (UGC), Government of India, for her doctoral research fellowship. Supporting Information Available: Reverse phase HPLC chromatograms for the lipids 1-9 in two mobile phases with details of used HPLC parameters. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Verma, I. M., and Somina, M. (1997) Gene TherapyPromises, Problems and Prospects. Nature 389, 239-242.

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