Organic & Biomolecular Chemistry Dynamic Article Links Cite this: Org. Biomol. Chem., 2011, 9, 4600 www.rsc.org/obc PAPER Syntheses, Transfection Efficacy and Cell Toxicity Properties of Novel Cholesterol-based Gemini Lipids having Hydroxyethyl Head group† Joydeep Biswas, a Santosh K. Mishra, a Paturu Kondaiah b and Santanu Bhattacharya* a,c Received 27th October 2010, Accepted 26th January 2011 DOI: 10.1039/c0ob00940g We have synthesized five new cholesterol based gemini cationic lipids possessing hydroxyethyl (-CH 2 CH 2 OH) function on each head group, which differ in the length of the polymethylene spacer chain. These gemini lipids are important for gene delivery processes as they possess pre-optimized molecular features, e.g., cholesterol backbone, ether linkage and a variable spacer chain between both the headgroups of the gemini lipids. Cationic liposomes were prepared from each of these lipids individually and as a mixture of individual cationic gemini lipid and 1,2-dioleoyl phosphatidylethanolamine (DOPE). Each gemini lipid based formulation induced better transfection activity than that of their monomeric counterpart. One such gemini lipid with a -(CH 2 ) 12 - spacer, HG-12, showed dramatic increase in the mean fluorescence intensity due to the expression of green-fluorescence protein (GFP) in the presence of 10% FBS compared to the conditions where there was no serum. Other gemini lipids retained their gene transfection efficiency without any marked decrease in the presence of serum. The only exception was seen with the gemini with a -(CH 2 ) 3 - spacer, HG-3, which on gene transfection in the presence of 10% FBS lost ~70% of its transfection efficiency. Overall the gemini lipid with a -(CH 2 ) 5 - spacer, HG-5, showed the highest transfection activity at N/P (lipid/DNA) ratio of 0.5 and lipid : DOPE molar ratio of 2. Upon comparison of the relevant parameters, e.g., %-transfected cells, the amount of DNA transfected to each cell and %-cell viability all together against Lipofectamine 2000, one of the best commercial transfecting agents, the optimized lipid formulation based on DOPE/HG-5 was found to be comparable. In terms of its ability to induce gene-transfer in the presence of serum and shelf-life DOPE/HG-5 liposome was found to be superior to its commercial counterpart. Confocal imaging analysis confirmed that in the presence of 10% serum using a Lipid : DOPE of 1 : 4 and N/P charge ratio of 0.75 with 1.2 mg DNA per well, HG-5 is better than Lipofectamine 2000. Introduction Cholesterol 1–6 and glycerol based 7–11 cationic lipids have been extensively used for gene delivery across a number of mam- malian cells. 12 Some of these lipids with natural trans- fection enhancers e.g., 1,2-dioleoyl-L-a-glycero-3-phosphatidyl- ethanolamine (DOPE) express synergistically higher level of trans- fection in many cases, although a few lipids have shown efficient gene transfer activity without any helper lipids. 13 Molecular level structural variations in cationic lipids influence both biophysical a Department of Organic Chemistry, Indian Institute of Science, Bangalore, 560 012, India. E-mail: sb@orgchem.iisc.ernet.in; Fax: (91)-80-2360–0529; Tel: (91)-80-2293–2664 b Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560 012, India c Chemical Biology Unit of JNCASR, Bangalore, 560 064, India. E-mail: sb@ orgchem.iisc.ernet.in; Fax: (91)-80-2360–0529; Tel: (91)-80-2293–2664 † Electronic supplementary information (ESI) available. See DOI: 10.1039/c0ob00940g characteristics of liposomes and their transfection efficiency. Reports describing variations in the headgroup structures, 4,5 hydrophobic moieties, 6 linker types, 13–16 linker lengths 17 and linker polarity 18 as well as balance between the head group and tail group have appeared in the literature. 3 A significant improvement in the transfection efficiency of cationic cholesterol based cytofectins was shown when the cationic center is linked to the steroid backbone via an ether type of linkage as opposed to their ester or urethane based counterparts. 13,14 High transfection efficacies of non-glycerol-based simple monocationic transfection lipids with hydroxyethyl (-CH 2 CH 2 OH) head groups were reported by Banerjee and coworkers. 19 The presence of hydroxyl function in the headgroup region of such lipids contributes favorably towards liposomal gene delivery. 20 These authors proposed a role of hydrogen-bonding interactions between the lipid headgroups and the cell surface of the biological membranes for the improvement in the transfection efficiency. Park et al. utilized OH group terminated dendrimers to avoid cytotoxicity and aggregation problems for polyamido amine based cytofectins. 21 Mahato et al. 4600 | Org. Biomol. Chem., 2011, 9, 4600–4613 This journal is © The Royal Society of Chemistry 2011 Downloaded by Indian Institute of Technology Kharagpur on 01 March 2013 Published on 03 May 2011 on http://pubs.rsc.org | doi:10.1039/C0OB00940G View Article Online / Journal Homepage / Table of Contents for this issue