Drug Delivery and Translational Research

, Volume 9, Issue 1, pp 106–122 | Cite as

Cationic cholesterol derivative efficiently delivers the genes: in silico and in vitro studies

  • Jasmin Monpara
  • Divya Velga
  • Tripti Verma
  • Sanjay Gupta
  • Pradeep VaviaEmail author
Original Article


The aims of the research work were to synthesize ethyl(cholesteryl carbamoyl)-l-arginate (ECCA), an arginine-conjugated cholesterol derivative, and to evaluate its application as a gene delivery vector. The interactions of ECCA with DNA duplex were studied using molecular dynamics (MD) simulations. It was found that the guanidine group of ECCA could interact with the phosphate group of DNA through ionic interactions as well as hydrogen bonds. The structure of DNA was stable throughout the simulation time. Liposomes were formulated using ECCA and soya phosphatidylcholine (SPC) by a thin-film hydration method. They had the particle size of ~ 150 nm and the zeta potential of + 51 mV. To ensure the efficient binding of DNA to the liposomes, the ratio of DNA to ECCA was optimized using gel retardation assay. Further, serum stability, haemolysis and cytotoxicity studies were carried out to determine the stability and safety of the lipoplexes. Circular dichroism spectroscopy was used to determine the interaction of DNA and cationic liposomes. Cellular uptake pathway was determined by studying the uptake of coumarin-loaded lipoplexes at 4 °C and in the presence of uptake inhibitors, i.e. genistein, chlorpromazine and methyl-β-cyclodextrin. Transfection studies were carried out to evaluate the transfection efficacy of the ECCA-loaded lipoplexes. The binding of DNA and lipoplexes was found to be stable in the presence of serum, and no degradation of DNA was observed. The lipoplexes showed low haemolysis and cytotoxicity. The uptake of coumarin-loaded liposomes was decreased up to ~ 20% in the presence of clathrin- and caveola-mediated uptake inhibitors, indicating a role of both the pathways in the uptake of the inhibitors. Satisfactory transfection efficiency was obtained compared to Lipofectamine®. Thus, cationic cholesterol derivative is a useful tool for gene delivery vector.


Cationic cholesterol derivative Non-viral vector Gene delivery Uptake pathway MD simulation 



Authors are extremely thankful to Dr. Dhanashree Jagtap, National Institute for Research in Reproductive Health (NIRRH), India, for helping with the CD spectrometry study. Ms. Tripti Verma contributed to the manuscript by carrying out the serum stability study.

Funding information

The authors are thankful to the University Grant Commission (UGC), Government of India, for the financial assistance and AICTE-NAFETIC for providing facilities to perform the experimental work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13346_2018_571_MOESM1_ESM.mpeg (175.1 mb)
ESM 1 (MPEG 179251 kb)


  1. 1.
    Ju J, Huan M-L, Wan N, Hou Y-L, Ma X-X, Jia Y-Y, et al. Cholesterol derived cationic lipids as potential non-viral gene delivery vectors and their serum compatibility. Bioorg Med Chem Lett. 2016;26(10):2401–7.Google Scholar
  2. 2.
    McCain J. The future of gene therapy. Biotechnol Healthc. 2005;2(3):52–60.Google Scholar
  3. 3.
    Ginn SL, Alexander IE, Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical trials worldwide to 2012—an update. J Gene Med. 2013;15(2):65–77. Scholar
  4. 4.
    U.S. Food & Drug Administration FDA approval brings first gene therapy to the United States. 2017.Google Scholar
  5. 5.
    Marshall E. Gene therapy death prompts review of adenovirus vector. Science. 1999;286(5448):2244–5.Google Scholar
  6. 6.
    Mintzer MA, Simanek EE. Nonviral vectors for gene delivery. Chem Rev. 2009;109(2):259–302. Scholar
  7. 7.
    Niidome T, Huang L. Gene therapy progress and prospects: nonviral vectors. Gene Ther. 2002;9(24):1647–52. Scholar
  8. 8.
    Yin H, Kanasty RL, Eltoukhy AA, Vegas AJ, Dorkin JR, Anderson DG. Non-viral vectors for gene-based therapy. Nat Rev Genet. 2014;15(8):541–55. Scholar
  9. 9.
    Kaneda Y, Tabata Y. Non-viral vectors for cancer therapy. Cancer Sci. 2006;97(5):348–54. Scholar
  10. 10.
    Wasungu L, Hoekstra D. Cationic lipids, lipoplexes and intracellular delivery of genes. J Control Release. 2006;116(2):255–64. Scholar
  11. 11.
    Zhi D, Zhang S, Cui S, Zhao Y, Wang Y, Zhao D. The headgroup evolution of cationic lipids for gene delivery. Bioconjug Chem. 2013;24(4):487–519. Scholar
  12. 12.
    Niculescu-Duvaz D, Heyes J, Springer CJ. Structure-activity relationship in cationic lipid mediated gene transfection. Curr Med Chem. 2003;10(14):1233–61.Google Scholar
  13. 13.
    Sen J, Chaudhuri A. Design, syntheses, and transfection biology of novel non-cholesterol-based guanidinylated cationic lipids. J Med Chem. 2005;48(3):812–20. Scholar
  14. 14.
    Cooper RG, Etheridge CJ, Stewart L, Marshall J, Rudginsky S, Cheng SH, et al. Polyamine analogues of 3β-[N-(N′, N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) as agents for gene delivery. Chem Eur J. 1998;4(1):137–51.Google Scholar
  15. 15.
    Medvedeva DA, Maslov MA, Serikov RN, Morozova NG, Serebrenikova GA, Sheglov DV, et al. Novel cholesterol-based cationic lipids for gene delivery. J Med Chem. 2009;52(21):6558–68. Scholar
  16. 16.
    Oudrhiri N, Vigneron J-P, Peuchmaur M, Leclerc T, Lehn J-M, Lehn P. Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo. Proc Natl Acad Sci. 1997;94(5):1651–6.Google Scholar
  17. 17.
    Ghosh YK, Visweswariah SS, Bhattacharya S. Nature of linkage between the cationic headgroup and cholesteryl skeleton controls gene transfection efficiency. FEBS Lett. 2000;473(3):341–4.Google Scholar
  18. 18.
    Sheng R, Luo T, Li H, Sun J, Wang Z, Cao A. ‘Click’ synthesized sterol-based cationic lipids as gene carriers, and the effect of skeletons and headgroups on gene delivery. Bioorg Med Chem. 2013;21(21):6366–77.Google Scholar
  19. 19.
    Liu Q, Jiang Q-Q, Yi W-J, Zhang J, Zhang X-C, Wu M-B, et al. Novel imidazole-functionalized cyclen cationic lipids: synthesis and application as non-viral gene vectors. Bioorg Med Chem. 2013;21(11):3105–13.Google Scholar
  20. 20.
    Biswas J, Mishra SK, Kondaiah P, Bhattacharya S. Syntheses, transfection efficacy and cell toxicity properties of novel cholesterol-based gemini lipids having hydroxyethyl head group. Org Biomol Chem. 2011;9(12):4600–13.Google Scholar
  21. 21.
    Uchida E, Mizuguchi H, Ishii-Watabe A, Hayakawa T. Comparison of the efficiency and safety of non-viral vector-mediated gene transfer into a wide range of human cells. Biol Pharm Bull. 2002;25(7):891–7.Google Scholar
  22. 22.
    Fitch CA, Platzer G, Okon M, Garcia-Moreno E, McIntosh LP. Arginine: its pKa value revisited. Protein Sci. 2015;24(5):752–61.Google Scholar
  23. 23.
    Liederer BM, Borchardt RT. Enzymes involved in the bioconversion of ester-based prodrugs. J Pharm Sci. 2006;95(6):1177–95. Scholar
  24. 24.
    Zhi D, Zhang S, Zhao Y, Cui S, Wang B, Chen H, et al. In vitro study of carbamate-linked cationic lipid for gene delivery against cervical cancer cells. Adv Mater Phys Chem. 2013;2(04):229.Google Scholar
  25. 25.
    Burk MJ, Allen JG. A mild amide to carbamate transformation. J Org Chem. 1997;62(20):7054–7.Google Scholar
  26. 26.
    Huang TL, Szekacs A, Uematsu T, Kuwano E, Parkinson A, Hammock BD. Hydrolysis of carbonates, thiocarbonates, carbamates, and carboxylic esters of alpha-naphthol, beta-naphthol, and p-nitrophenol by human, rat, and mouse liver carboxylesterases. Pharm Res. 1993;10(5):639–48.Google Scholar
  27. 27.
    D’Souza AJ, Topp EM. Release from polymeric prodrugs: linkages and their degradation. J Pharm Sci. 2004;93(8):1962–79. Scholar
  28. 28.
    Wang Z. Schotten-Baumann Reaction. In: Comprehensive organic name reactions and reagents. Hoboken: John Wiley & Sons, Inc.; 2010. p. 2536–9.Google Scholar
  29. 29.
    Zhang Y, Zou H, Chen J-M. Determination of entrapment efficiency of teniposide liposomes by Sephadex G-50 gel minicolumn centrifugation-HPLC. Chin J New Drugs. 2009;16:031.Google Scholar
  30. 30.
    Braun CS, Jas GS, Choosakoonkriang S, Koe GS, Smith JG, Middaugh CR. The structure of DNA within cationic lipid/DNA complexes. Biophys J. 2003;84(2):1114–23.Google Scholar
  31. 31.
    Xiong F, Mi Z, Gu N. Cationic liposomes as gene delivery system: transfection efficiency and new application. Pharmazie. 2011;66(3):158–64.Google Scholar
  32. 32.
    Singh J, Michel D, Chitanda JM, Verrall RE, Badea I. Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles. J Nanobiotechnol. 2012;10(1):7.Google Scholar
  33. 33.
    Patel K, Doddapaneni R, Sekar V, Chowdhury N, Singh M. Combination approach of YSA peptide anchored docetaxel stealth liposomes with oral antifibrotic agent for the treatment of lung cancer. Mol Pharm. 2016;13(6):2049–58.Google Scholar
  34. 34.
    Lee J, Saw PE, Gujrati V, Lee Y, Kim H, Kang S, et al. Mono-arginine cholesterol-based small lipid nanoparticles as a systemic siRNA delivery platform for effective cancer therapy. Theranostics. 2016;6(2):192–203.Google Scholar
  35. 35.
    Aissaoui A, Oudrhiri N, Petit L, Hauchecorne M, Kan E, Sainlos M, et al. Progress in gene delivery by cationic lipids: guanidinium-cholesterol-based systems as an example. Curr Drug Targets. 2002;3(1):1–16.Google Scholar
  36. 36.
    Ciani L, Casini A, Gabbiani C, Ristori S, Messori L, Martini G. DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery studied by circular dichroism and other biophysical techniques. Biophys Chem. 2007;127(3):213–20.Google Scholar
  37. 37.
    Amin K, Dannenfelser RM. In vitro hemolysis: guidance for the pharmaceutical scientist. J Pharm Sci. 2006;95(6):1173–6.Google Scholar
  38. 38.
    Liu Y, Fong S, Debs RJ. Cationic liposome-mediated gene delivery in vivo. Methods Enzymol. 2003;373:536–50.Google Scholar
  39. 39.
    Mochizuki S, Kanegae N, Nishina K, Kamikawa Y, Koiwai K, Masunaga H, et al. The role of the helper lipid dioleoylphosphatidylethanolamine (DOPE) for DNA transfection cooperating with a cationic lipid bearing ethylenediamine. Biochim Biophys Acta Biomembr. 2013;1828(2):412–8.Google Scholar
  40. 40.
    Khalil IA, Kogure K, Akita H, Harashima H. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol Rev. 2006;58(1):32–45.Google Scholar
  41. 41.
    Futaki S, Nakase I, Tadokoro A, Takeuchi T, Jones AT. Arginine-rich peptides and their internalization mechanisms. Biochem Soc Trans. 2007;​35(4):784–787. Scholar
  42. 42.
    Rejman J, Bragonzi A, Conese M. Role of clathrin-and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther. 2005;12(3):468–74.Google Scholar
  43. 43.
    Rejman J, Conese M, Hoekstra D. Gene transfer by means of lipo- and polyplexes: role of clathrin and caveolae-mediated endocytosis. J Liposome Res. 2006;16(3):237–47.Google Scholar
  44. 44.
    Pichon C, Billiet L, Midoux P. Chemical vectors for gene delivery: uptake and intracellular trafficking. Curr Opin Biotechnol. 2010;21(5):640–5.Google Scholar

Copyright information

© Controlled Release Society 2018

Authors and Affiliations

  • Jasmin Monpara
    • 1
  • Divya Velga
    • 2
    • 3
  • Tripti Verma
    • 2
    • 3
  • Sanjay Gupta
    • 2
    • 3
  • Pradeep Vavia
    • 1
    Email author
  1. 1.Department of Pharmaceutical Sciences and Technology, Institute of Chemical TechnologyUniversity Under Section 3 of UGC Act—1956, Elite Status and Center of Excellence—Government of Maharashtra, TEQIP Phase II FundedMumbaiIndia
  2. 2.Gupta Laboratory, Epigenetics and Chromatin Biology Group, Cancer Research Institute, Tata Memorial CentreAdvanced Center for Treatment, Research and Education in Cancer (ACTREC)Navi MumbaiIndia
  3. 3.Homi Bhabha National InstituteMumbaiIndia

Personalised recommendations