Synthesis and Gene Transfection Efficacies of PEI− Cholesterol-Based

PEI is also one of the main focus systems for gene delivery in many research groups .... The lipopolymer and DNA were complexed in a volume of 200 µL...
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Bioconjugate Chem. 2008, 19, 1640–1651

Synthesis and Gene Transfection Efficacies of PEI-Cholesterol-Based Lipopolymers Avinash Bajaj,† Paturu Kondaiah,‡ and Santanu Bhattacharya*,†,§ Department of Organic Chemistry, and Department of Molecular Reproduction, Development, and Genetics, Indian Institute of Science, Bangalore 560 012, India, and Chemical Biology Unit of JNCASR, Bangalore 560 064, India. Received October 14, 2007; Revised Manuscript Received March 18, 2008

Nine lipopolymers based on low molecular weight polyethyleneimines (PEI) and cholesterol via an ether linkage between the polymer amine and the cholesterol backbone have been synthesized. Different percentage of cholesterol moieties have been grafted on three types of PEI of molecular weights 800, 1200, and 2000. These lipopolymers were studied for gene transfection activities in HeLa cells. All the lipopolymers were first optimized for enhanced transfection efficacies as coliposomes with DOPE. All lipopolymers are better transfecting agents and highly serum compatible than commercially available PEI-25KDa. Transfection efficacies and serum compatibility of lipopolymers were found to be dependent upon the MW of PEI used for lipopolymer synthesis and percentage of cholesterol grafting on lipopolymers. Cell viability assay showed that PEI-25KDa is highly toxic as compared to all the lipopolymers. Lipopolyplexes were characterized by transmission electron microscopy, which showed the presence of spherical aggregates.

INTRODUCTION Gene Therapy (1), which involves the transfer of a desired gene to a target cell, is a new hope for various disorders like cystic fibrosis (2, 3), diabetes, cancer (4, 5), AIDS (6), and so forth (7-9). The first major challenge of the use of gene therapy for curing disorders is to deliver the desired gene efficiently with the least toxicity and low immunogenicity. A second major challenge is to transfer such genes to the specific cells (10-13). Viruses are very potent carriers for delivery of the gene, but due to toxicity, strong immunogenicity, and its limitation to carry less DNA, various researchers are trying to develop different kinds of nonviral vectors for efficient gene delivery (14-17). Among nonviral vectors, cationic lipids (18, 19), polymers (20), and lipopolymers (21) are a major focus for researchers to develop new vectors for efficient gene delivery. Various kinds of cationic lipids have been developed and investigated for their gene transfection activity (22-25). Over the past few years, we have been investigating the aggregation, transfection, and mechanistic studies of various lipid-based formulations (26-32). Among polymers, polyethylenimine (PEI1) (33), polylysine, polyornithine (34, 35), and so forth have been examined for gene delivery (36). Behr and co-workers demonstrated for the first time the transfection efficacy of PEI (33). PEI is also one of the main focus systems for gene delivery in many research groups because of its early endosomal release of DNA, which prevents the DNA from degradation in late endosomes (37). This is because of the different pKa values of the primary, secondary, and tertiary amines in it, which give the PEI a good buffer capacity that does not allow it to lower the pH of early endosomes, which is suitable for various enzymes to act (37). * Corresponding author. E-mail: [email protected]. Phone: (91)-80-2293-2664. Fax: (91)-80-2360-0529. † Department of Organic Chemistry, Indian Institute of Science. ‡ Development, and Genetics, Indian Institute of Science. § Chemical Biology Unit of JNCASR. 1 Abbreviations: PEI, polyethylenimine; DOPE, 1,2-dioleoyl-L-Rglycero-3-phosphatidylethanolamine; FACS, fluorescence-activated cell sorting.

PEIs of different molecular weights ranging 0.6-800 kDa have been examined in gene transfection studies (37). PEIs of higher molecular weights (70-800 kDa) were found to be better transfecting agents for in Vitro studies, whereas low MW PEIs have been known to show better gene delivery activity with intravenous administration. Cytotoxicities of PEI increase with increase in molecular weight. However, low MW PEIs are found to be less cytotoxic, although they are not effective as gene delivery agents. To further improve their transfection potential, PEI had been derivatized with various ligands including sugars (10), amino acids (36), poly(ethylene glycol) (37), long hydrocarbon chains (38), and even cholesterol (39). Hydrophobic anchors are essential for gene delivery vehicles because of increased cellular uptake and enhanced DNA stability against the bloodstream (37). Klibanov et al. had shown the effect of various chemical modifications of nitrogen atoms on the efficiency of PEIs as synthetic vectors for the delivery of plasmid DNA (38). One of the major problems associated with the nonviral vectors for clinical application is their inefficiency in serum conditions (40-43). It has already been mentioned that a hydrophobic part is essential to enhance the stability of the lipoplex in the bloodstream and gene transfer, as the cellular uptake would be increased by favorable hydrophobic groups of gene carrier and cell membrane. Various kinds of long-chain alkyl and cholesteryl groups have been attached to PEI to enhance the efficiency of gene transfer. Modification of PEI of Mw 1800 with cholesterol at the primary amine has been shown to dramatically increase the transfection efficacy of PEI (44). Modification of secondary amines in PEI with cholesterol moieties was also shown to furnish better transfection activities (45). Kim and co-workers have shown the DNA delivery abilities of modified linear PEI (MW 25 kDa) with cholesterol (46). Park et al. studied the transfection properties of cholesterol and myristate-conjugated PEI (39). Recently, dexamethasoneconjugated low molecular weight PEI was reported for gene transfection efficacies as well (47). We have previously reported the aggregation and transfection properties of different types of cationic lipids (48-52). We also

10.1021/bc700381v CCC: $40.75  2008 American Chemical Society Published on Web 07/11/2008

PEI-Cholesterol Lipopolymers

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Figure 1. General structure of the PEI-cholesterol-based lipopolymers used for the study.

probed the interaction of cholesterol in membranes with cationic and phospholipids (53, 54). Cholesterol accordingly was used in the design of various cationic lipids and lipopolymers due to its natural occurrence, which make the vectors biocompatible. It has already been reported from this laboratory that the nature of the linkage between the hydrophobic cholesterol backbone and the headgroup plays an important role in regulating the transfection activities of cationic lipids (55-59). Thus, the cholesterol lipids possessing ether-based linkages between the cationic headgroup and steroid skeleton significantly enhanced transfection activities as compared to its ester- or urethane-based counterparts. Recently, we also showed the serum-compatible gene transfection activities of cholesterol-based gemini lipids differing in the length and nature of the spacer between the headgroups (42, 43). Although few PEI-cholesterol conjugates have been reported (39, 45, 46), the PEI-lipid conjugates based on etherlinked cholesterol units have never been described and examined for transfection activity. Accordingly, we have synthesized nine lipopolymers with variable extent of cholesterol units to three different low molecular weight PEIs (Mw ) 800 (P8), Mn ) 1200 (P12), and Mw ) 2000 (P20)) via ether-based links. We also characterized such cholesterol-PEI-based lipopolymers (Figure 1) and finally investigated their transfection efficiencies.

Scheme 1a

EXPERIMENTAL PROCEDURES Materials and Methods. All reagents, solvents, and chemicals used in this study were of the highest purity available. The solvents were dried prior to use. Column chromatography was performed using 60-120 mesh silica gel. NMR spectra were recorded using Jeol JNM λ-300 (300 MHz for 1H) spectrometer. The chemical shifts (δ) are reported in ppm downfield from the internal standard: TMS, for 1H NMR. Mass spectra were recorded on a MicroMass ESI-TOF spectrometer or ESI-MS (HP1100LC-MSD). Infrared (IR) spectra were recorded on a Jasco FT-IR 410 spectrometer using KBr pellets or neat. Synthesis (Scheme 1). Cholest-5-en-3β-tosylate (2). To an ice-cooled solution of cholesterol (1) (5.0 g, 0.013 mmol) in dry pyridine (5 mL) and dry chloroform (5 mL), p-tosyl chloride (3.7 g, 0.02 mmol) was added. A catalytic amount of DMAP was also added. The reaction mixture was then allowed to stir at 0 °C for 6 h, chloroform (35 mL) was added, and the resulting reaction mixture was washed with 1 N HCl (2 × 50 mL), water (50 mL), and brine (50 mL); the organic layer was separated and dried over anhydrous Na2SO4. Chloroform from this solution was evaporated to leave a residue. From the residue, cholest5-en-3β-tosylate (2) was recrystallized using chloroform and methanol. Yield: White solid, 6.49 g., 0.012 mmol, 92.8%. m.p:

a Reaction conditions: (i) p-TsCl/Py/CHCl3, 0 °C, 6 h; (ii) ethylene glycol, 1,4-dioxane, 4 h, reflux; (iii) p-TsCl/Py/CHCl3, 0 °C, 6 h.

133 °C. lit m.p: 132-133 °C (55). IR (neat) (cm-1): 2949, 2867, 1598, 1495, 1467, 1366, 1188, and 1174. 1H NMR (CDCl3, 300 MHz): δ 0.65 (s, 3H), 0.84-2.29 (m, 41H), 2.44 (s, 3H), 4.32 (m, 1H), 5.30 (d, 1H, J ) 4.5 Hz), 7.31-7.34 (d, 2H, J ) 8.1 Hz), 7.78 -7.81 (d, 2H, J ) 8.1 Hz). Cholest-5-en-3β-oxyethane (3). Chlolest-5-en-3β-tosylate (2) (3.5 g, 6.5 mmol) was taken in anhydrous dioxane. To this, dry ethylene glycol (10 g, 0.16 mol) was added and the mixture was refluxed under nitrogen for 4 h. The solution was cooled and solvent was removed under vacuum. A white residue was obtained which was dissolved in chloroform (50 mL) and washed with water. The organic layer was separated; washed with NaHCO3 (50 mL), water (50 mL), and brine (50 mL); and dried over anhydrous Na2SO4. Finally, from this solvent was removed in Vacuo, and the product cholest-5-en-3β-oxyethane (3) was purified by column chromatography over silica gel using a mixture of hexane and ethyl acetate. Yield: white solid, 2.3 g,

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Scheme 2

Table 1. Lipopolymers Synthesized and Percentage of Cholesterol Grafting on Lipopolymers and the Optimized Lipopolymer/DOPE Ratios

lipopolymer

PEIsMW

∆Apei

∆Ac

P8C1 P8C2 P8C3 P12C1 P12C2 P12C3 P20C1 P20C2 P20C3

800 800 800 1200 1200 1200 2000 2000 2000

5.82 8.99 15.71 7.72 8.04 15.18 5.25 8.09 17.17

3 3 3 3 3 3 3 3 3

% cholesterol grafting 104.7a 57.22 29.18 88.10 66.32 30.34 123a 65.68 26.36

optimized lipopolymer. DOPE 1:1 4:1 2:1 1:1 2:1 1:4 1:1 1:0 1:0

a More then 100% cholesterol grafting indicates that the primary amines in PEI get alkylated with two cholesteryl moieties.

5.3 mmol, 85%. m.p: 99-100 °C. lit mp: 97-98 °C (60). IR (neat) (cm-1): 3438, 2936, 2868, 1466, and 1376. 1H NMR (CDCl3, 300 MHz): δ 0.67 (s, 3H), 0.85-2.35 (m, 41H), 3.20 (m, 1H), 3.57-3.60 (t, 2H), 3.72 (t, 2H), 5.34 (d, 1H). ESIMS: 453 (M + Na+). Cholest-5-en-3β-oxyethane tosylate (4). To an ice-cooled solution of cholest-5-en-3β-oxyethan-2-ol (3) (2.2 g, 5.1 mmol) in dry pyridine (5 mL) and dry chloroform (5 mL), p-tosyl chloride (1.5 g, 7.86 mmol) was added. The reaction mixture was allowed to stir at 0 °C for 6 h, chloroform (40 mL) was added, and then the reaction mixture was washed with 1 N HCl (2 × 50 mL), water (50 mL), and brine (50 mL). Finally, the organic layer was separated and dried over anhydrous Na2SO4. Solvent was removed using a rotary evaporator, and the product cholest-5-en-3β-oxyethane tosylate (4) was purified by column chromatography over silica gel using a mixture of hexane and ethyl acetate. Yield: 2.68 g, 7.04 mmol, 90.0%. IR (neat) (cm-1): 2935, 2886, 1598, 1465, 1360, 1189, and 1177. 1H NMR (CDCl3, 300 MHz): δ 0.67 (s, 3H), 0.85-2.25 (m, 41H), 2.44 (s, 3H), 3.06-3.13 (m, 1H), 3.63-3.66 (t, 2H), 4.13-4.16 (t, 2H), 5.31 (d, 1H), 7.32-7.35 (d, 2H), 7.79-7.82 (d, 2H). ESIMS: 607 (M + Na+).

Lipopolymer Synthesis (Scheme 2). Cholest-5-en-3β-oxyethane-tosylate (4) (170 mg, 0.29 mmol) was taken in 3 mL of dry toluene. Desired amounts of PEI solutions (2-8 equiv) from the stock solution of PEI in dry MeOH were added to reaction mixture. The reaction mixture was refluxed, the reaction was monitored by TLC, and reflux was continued until the disappearance of cholest-5-en-3β-oxyethane-tosylate (4). The reaction mixture was evaporated, and 30 mL of chloroform was added to the crude mixture. The chloroform layer was washed with 5 mL of 1 N NaOH, water (5 mL), and brine solution (5 mL). The organic layer was passed through dry K2CO3 and evaporated to get lipopolymer. All the lipopolymers were characterized by 1 H NMR. Transmission Electron Microscopy. Freshly prepared aqueous suspensions of lipopolymer-DNA complexes were examined under transmission electron microscopy with negative staining using 1% uranyl acetate. A 10 µL sample of the suspension was loaded onto Formvar-coated 400 mesh copper grids and allowed to remain for 1 min. Excess fluid was wicked off the grids by touching their edges to filter paper, and 10 µL of 1% uranyl acetate was applied on the same grid after which the excess stain was similarly wicked off. The grid was airdried for 30 min, and the specimens were observed under TEM (JEOL 200-CX) operating at an acceleration voltage of 120 keV. Micrographs were recorded at a magnification of 5000-20 000×. Plasmid DNA. pEGFP-c3 (Clontech, USA), which encodes for an enhanced green fluorescence protein (GFP) under a CMV promoter, was amplified in Escherichia coli (DH5R) and purified using Qiagen Midi Prep Plasmid Purification protocol (Qiagen, Germany). Purity of the plasmid was checked by electrophoresis on 1.0% agarose gel. Concentration of the DNA was estimated spectroscopically by measuring the absorption at 260 nm and confirmed by gel electrophoresis. The plasmid preparations showing a value of OD260/OD280 > 1.8 were used. Cell Culture. Cells (HeLa) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS) in T25 culture flasks (Nunc, Denmark) and were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Cells were regularly passaged

PEI-Cholesterol Lipopolymers

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Figure 2. Representive 1H NMR (300 MHz) of cholest-3β-oxyethan-tosylate.

Figure 3. Representive 1H NMR (300 MHz) of one lipopolymer molecule.

by trypsinization with 0.1% trypsin (EDTA 0.02%, dextrose 0.05%, and trypsin 0.1%) in PBS (pH 7.2). Cytotoxicity. Toxicity of each lipopolymer formulation toward HeLa cells was determined using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide reduction assay following literature procedures (61, 62). Cytotoxicity of the lipopolymer formulations optimal for transfection experiments was determined under conditions exactly the same as transfection conditions. Nearly 12 000 cells/ well were plated in 96 well plates. After 24 h, optimized lipopolymer/DOPE formulations were complexed with 0.2 µg of the DNA at various N/P ratios for 30 min. DNA-lipopolymer complexes were added to the cells in the absence of serum. After 6 h of incubation, lipoplexes were removed and 200 µL

of media with 10% FBS was added. After 42 h, 20 µL of MTT solution was added and the cells were incubated further for 6 h. Blue formazan crystals were seen when checked under microscope. Media was removed and 200 µL of DMSO was added per well. The absorbance was measured using a microtiter plate reader. The % viability was then calculated as [{A590(treated cells) - background]/[A590(untreated cells) - background}] × 100. Transfection Procedure. All transfection experiments were carried out in HeLa Cells in antibiotic-free media unless specified otherwise. In a typical experiment, 24 well plates were seeded with 45 000 cells/well in antibiotic-free media 24 h before transfection such that they were at least ∼70% confluent at the time of transfection. For transfection, lipopolymer

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Figure 4. Transfection efficiency of PEI-25KDa at different polymer/ DNA weight ratios (w/w) using EGFP-c3 plasmid in the presence of different percentages of serum.

Figure 5. Transfection efficiencies of lipopolymers at different lipopolymer/DNA weight ratios (w/w) using EGFP-c3 plasmid in the presence of different percentages of serum using optimized lipopolymer/ DOPE ratio: (a) P8C1; (b) P8C2; (c) P8C3.

formulation and DNA were serially diluted separately in DMEM containing no serum to have the required working stocks. DNA was used at a concentration of 0.8 µg/well unless specified otherwise. The lipopolymer and DNA were complexed in a volume of 200 µL by incubating the desired amount of

Bajaj et al.

lipopolymer formulation and DNA together at room temperature for about 30 min. The lipopolymer concentrations were varied so as to obtain the required lipopolymer/DNA (w/w) charge ratios. After 30 min of complexation, 200 µL of media was added to the complexes. Old medium was removed from the wells, cells were washed with DMEM, and lipopolymer-DNA complexes in 200 µL media were added to the cells. The plates were then incubated for 6 h at 37 °C in a humidified atmosphere containing 5% CO2. At the end of incubation period, medium was removed, and cells were washed with DMEM, and then 500 µL of DMEM containing 10% FBS was added per well. Plates were further incubated for a period of 42 h before checking for the reporter gene expression. GFP expression was examined by fluorescence microscopy and was quantified by flow cytometry analysis. All the experiments were done in duplicate, and results presented are the average of at least two such independent experiments done on two different days. For transfections in the presence of serum, lipopolymer and DNA were separately diluted in serum-free media as already mentioned, and the complexation was done in serum-free media (200 µL) for 30 min. The complex was then diluted to 400 µL with DMEM containing 20% FBS so as to achieve a final serum concentration of 10%. The cells were then incubated with this complex for 6 h. At the end of the incubation period, medium was removed and cells were washed with DMEM, and 500 µL of DMEM containing 10% FBS was added per well. For transfections at 30% and 50% of serum concentrations, complexes were diluted with DMEM containing 60% FBS or with neat FBS, respectively. Flow Cytometry. The reporter gene expression was examined by fluorescence microscopy at regular intervals and was quantified 48 h post-transfection by flow cytometry. The percentages of transfected cells were obtained by determining the statistics of cells fluorescing above the control level where nontransfected cells were used as the control. Approximately 10 000 cells were analyzed to generate the statistical data, which have been presented as the average of at least two independent measurements. For the flow cytometry analysis, ∼48 h posttransfection, old medium was removed from the wells; cells were washed with PBS and trypsinized by adding 100 µL of 0.1% trypsin. To each well, 200 µL of PBS containing 20% FBS was added. Duplicate cultures were pooled and analyzed by flow cytometry immediately using Becton and Dickinson flow cytometer equipped with a fixed laser source at 488 nm. FACS Analysis. FACS data were analyzed by public domain WinMDI software to eliminate data from cell debris (particles smaller than cells), dead cells, and clumps of two or more cells. Subcellular debris and clumps can be distinguished from single cells by size (estimated by the intensity of low angle forward scatter). Dead cells have lower forward-scatter and higher sidescatter than living cells. The FACS scans were configured to display the fluorescence signals only from those particles with a specified set of scatter properties, namely, living single cells. This is called a scatter-gated fluorescence analysis. Therefore, the data from dead cells had been eliminated by gating out brightly fluorescent cells. We have presented the representative FACS scans (as well as scatter plots) for the transfection experiments using lipopolymers and the respective FACS scans (as well as scatter plots) of the untransfected cells in the Supporting Information. We have set up the baseline in FACS scans, so that we observed only 1-3% transfection in the untreated cells as shown by the FACS scans presented in the Supporting Information. Baseline setup for the representative FACS scans has been shown in the Supporting Information.

RESULTS Synthesis and Characterization. Nine PEI-cholesterolbased lipopolymers were synthesized using different molecular

PEI-Cholesterol Lipopolymers

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Figure 6. Transfection efficiencies of lipopolymers at different lipopolymer/DNA weight ratios (w/w) using EGFP-c3 plasmid in the presence of different percentages of serum using optimized lipopolymer/ DOPE ratio: (a) P12C1; (b) P12C2; (c) P12C3.

weight polyethylenimines as starting materials (Figure 1, Table 1). Different percentages of cholesteryl units were incorporated covalently on PEI by its reaction with cholest-5-en-3β-oxyethane tosylate (4). First, cholesterol (1) was tosylated using ptoluenesulfonyl chloride in pyridine-chloroform (v/v: 1/1) with a catalytic amount of DMAP for 6 h at 0 °C in 92% yield. Cholesterol tosylate (2) was then subjected to a reaction with ethylene glycol in dioxane under reflux for 4 h to afford cholest5-en-3β-oxyethan-2-ol (3) in 85% yield. Compound 3 was then tosylated with p-toluenesulfonyl chloride in pyridine-chloroform (v/v: 1/1) for 6 h at 0 °C to get 4 in 90% yield. Reaction of the tosylate (4) with PEI of different molecular weights in different mole ratios resulted in the formation of PEI-cholesterol-based lipopolymers. All the lipopolymers were characterized by 1H NMR studies (47). Characterization of Lipopolymers. Figure 2 displays the 1 H NMR of cholest-3β-oxyethan-tosylate (4). The 1H peak at δ of 0.67 is due to the angular methyl group directly linked to cyclohexane and cyclopentane rings of the cholesterol backbone (signal a designated by proton a). The chemical shifts at δ 3.64 and δ 4.14 were attributed to protons d and e, respectively. The

Figure 7. Transfection efficiencies of lipopolymers at different lipopolymer/DNA weight ratios (w/w) using EGFP-c3 plasmid in the presence of different percentages of serum using optimized lipopolymer/ DOPE ratio: (a) P20C1; (b) P20C2; (c) P20C3. 1

H peaks of δ 3.09 and δ 5.3 were associated with the -CHsO- (proton c, signal c) and -CHdC- (proton f, signal f) protons of cholesterol, respectively. Singlets at δ 2.3 (proton b, signal b) and δ 7.3, 7.8 (protons g and h, signal g and h) were due to methyl and aromatic protons of the tosylate group. The ratio of the peak areas was determined to be 3:3:1:2:2:1: 2:2 for signals/protons a:b:c:d:e:f:g:h, confirming the successful synthesis of cholest-3β-oxyethan-tosylate (4). The successful synthesis of lipopolymers was evidenced by 1 H NMR spectra as shown in Figure 3. The 1H NMR of lipopolymers illustrates peaks at δ 0.67, 3.14, and 5.3 because of the presence of protons a, c, and e due to the cholesterol moiety. Various peaks at 0.7-1.2 were also attributed to the presence of cholesterol units with polymer. The peak at 5.38 arose from the proton of dCH- in the cholesterol units (signal e). The peak at δ 0.7 was from the methyl group directly linked to the cyclic hydrocarbon (signal a). The broad peak in the

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region δ 2.0-2.5 was because of the protons of the PEI backbone (signal b, protons b), which also includes the protons of -CH2- from the -CH2sCH2sO- group of the cholesterol unit attached to the PEI backbone by alkylation. Disappearance of the peaks at δ 2.3 and δ 7-8 in 1H NMR of lipopolymers (Figure 3) (signals b, g, and h observed in the case of the 1H NMR spectra of cholest-3β-oxyethan-tosylate (Figure 2)) confirms the substitution of the tosylate group by the amine of PEI. The information provided by the 1H NMR spectrum of lipopolymers proved that the cholesteryl group was successfully grafted onto the PEI main chain. Degree of Cholesterol Grafting in PEI. The grafting degree was obtained from the 1H NMR spectra. The degree of cholesterol grafting has been defined as the relationship of the number of amines alkylated by the cholesterol moiety to the total number of amines on the PEI chain. The signal at 0.67 is due to the protons attached to the -CH3 group directly linked to the hexane and pentane cycles of the cholesterol backbone (signal a, proton a). The broad peak in the region δ 2.0-2.5 is attributed to -CH2- of the PEI backbone as well as the protons of -CH2- from the -CH2-CH2-O- group attached to the cholesterol backbone, which are now attached to PEI backbone by alkylation. The ratio of the protons due to -CH3 at δ 0.67 and -CH2 from the -CH2-CH2-O- group attached to the cholesterol backbone, which are now attached to the PEI backbone by alkylation is 3:2, or 1.5:1. Therefore, the area of the curve in the region δ 2.0-2.5 (∆Apei) also includes the area (∆Ac/1.5) which is due to -CH2 from the -CH2-CH2-Ogroup attached to the cholesterol backbone, which are now attached to the PEI backbone by alkylation. Therefore, the actual area of the PEI backbone would be (∆Apei - ∆Ac/1.5). The percentage of grafting may be estimated as follows: Rg )

(

∆Ac × Npei ∆Ac ∆Apei × Nc 1.5

)

where ∆Ac is the area of the selected peak from the cholesterol backbone, (∆Apei - ∆Ac/1.5) is the area of the selected peak of the PEI backbone, Nc is the number of hydrogen atoms from the selected region of cholesterol backbone, and Npei is the number of hydrogen atoms from the selected region of the PEI backbone. Only suitable protons from the pendant chain and the main chain of the polymers were selected in the calculation. The proton signal selected should not overlap with the signals from other protons. On the basis of the peak area of the signals a and b, Rg for lipopolymers was estimated as given in Table 1. Transfection Efficacy. Optimization of the Lipopolymer/ DOPE Ratio. Incorporation of the helper lipid, DOPE, is known to enhance the transfection efficiencies of many of cationic lipidbased formulations (63). Therefore, we first decided to investigate the optimized lipopolymer/DOPE mole ratios for our newly synthesized lipopolymers. These data were obtained from flow cytometric analysis (64). In order to determine the most effective formulations, transfections were performed with identical amounts of the DNA and lipopolymer upon variation of mole ratios of lipopolymers in DOPE. Optimized lipopolymer/DOPE mole ratios were found to depend upon the percentage of cholesterol grafting and the molecular weight of PEI. The optimized lipopolymer/DOPE ratios for all lipopolymers are given in Table 1. Lipopolymers (P8C1, P12C1, P20C1) with highest cholesterol drafting showed optimized transfection at lipopolymer/DOPE ratio of 1:1. In contrast, lipopolymers P20C2 and P20C3 showed maximum transfection activities in the absence of DOPE. In further studies, we used these optimized lipopolymer/DOPE formulations. All the optimized lipopolymer formulations were examined for transfection studies using

Bajaj et al.

EGFP-c3 plasmid in HeLa cells at different DNA/lipoolymer weight ratios. The transfection experiments were performed in the absence and in the presence of serum. To get further insight, we investigated the transfection activities at higher serum concentrations as well. The transfection experiments have also been performed with the unmodified polymers. We have not observed any transfection activity with these polymers. It is also well-known in the literature that low molecular weight PEI does not show transfection. Kim et al. (45) reported only