Colloidally Stable Small Unilamellar Stearyl Amine Lipoplexes for Effective BMP-9 Gene Delivery to Stem Cells for Osteogenic Differentiation
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The biocompatibility of cationic liposomes has led to their clinical translation in gene delivery and their application apart from cancer to cardiovascular diseases, osteoporosis, metabolic diseases, and more. We have prepared PEGylated stearyl amine (pegSA) lipoplexes meticulously considering the physicochemical properties and formulation parameters to prepare single unilamellar vesicles (SUV) of < 100 nm size which retain their SUV nature upon complexation with pDNA rather than the conventional lipoplexes which show multilamellar nature. The developed PEGylated SA lipoplexes (pegSA lipoplexes) showed a lower N/P ratio (1.5) for BMP-9 gene complexation while maintaining the SUV character with a unique shape (square and triangular lipoplexes). Colloidal and pDNA complexation stability in the presence of electrolytes and serum indicates the suitability for intravenous administration for delivery of lipoplexes to bone marrow mesenchymal stem cells through sinusoidal vessels in bone marrow. Moreover, lower charge density of lipoplexes and low oxidative stress led to lower toxicity of lipoplexes to the C2C12 cells, NIH 3T3 cells, and erythrocytes. Transfection studies showed efficient gene delivery to C2C12 cells inducing osteogenic differentiation through BMP-9 expression as shown by enhanced calcium deposition in vitro, proving the potential of lipoplexes for bone regeneration. In vivo acute toxicity studies further demonstrated safety of the developed lipoplexes. Developed pegSA lipoplexes show potential for further in vivo preclinical evaluation to establish the proof of concept.
KEY WORDSlipoplexes liposomes micelle gene delivery cationic lipids PEGylation
The authors are thankful to the University Grants Commission, Government of India for financial assistance. The authors also thank Dr. Vikram Sarabhai Central Facility, The Maharaja Sayajirao University of Baroda, Vadodara, for experimental support in cell line studies. The authors acknowledge the support of Dr. Sachin Naik and Dr. Subhas Bhowmick (Sun Pharma Advance Research Center Ltd., Vadodara) for cryoTEM studies.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
- 1.Balazs DA, Godbey W. Liposomes for use in gene delivery. J Drug Deliv. 2011;2011:1–12.Google Scholar
- 9.Yoshihara E, Nakae T. Cytolytic activity of liposomes containing stearylamine. Biochim Biophys Acta Biomembr. 1986;854(1):93–101.Google Scholar
- 12.Silva AL, Alexandrino F, Verissimo LM, Agnez-Lima LF, Egito LC, de Oliveira AG, et al. Physical factors affecting plasmid DNA compaction in stearylamine-containing nanoemulsions intended for gene delivery. Pharmaceuticals (Basel). 2012;5(6):643–54.Google Scholar
- 15.Casals E, Soler M, Gallardo M, Estelrich J. Electrophoretic behavior of stearylamine-containing liposomes. Langmuir. 1998;14(26):7522–6.Google Scholar
- 16.Van’t Hag L, Gras SL, Conn CE, Drummond CJ. Lyotropic liquid crystal engineering moving beyond binary compositional space—ordered nanostructured amphiphile self-assembly materials by design. Chem Soc Rev. 2017;46(10):2705–31.Google Scholar
- 20.Patil S, Bhatt P, Lalani R, Amrutiya J, Vhora I, Kolte A, et al. Low molecular weight chitosan–protamine conjugate for siRNA delivery with enhanced stability and transfection efficiency. RSC Adv. 2016;6(112):110951–63.Google Scholar
- 24.Yewale C, Baradia D, Patil S, Bhatt P, Amrutiya J, Gandhi R, et al. Docetaxel loaded immunonanoparticles delivery in EGFR overexpressed breast carcinoma cells. J Drug Deliv Sci Tec. 2018;45:334–45.Google Scholar
- 27.Israelachvili J. The science and applications of emulsions—an overview. Colloids Surf A Physicochem Eng Asp. 1994;91:1–8.Google Scholar
- 28.Dos Santos N, Allen C, Doppen A-M, Anantha M, Cox KAK, Gallagher RC, et al. Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding. Biochim Biophys Acta Biomembr. 2007;1768(6):1367–77.Google Scholar
- 29.Tandia B-M, Vandenbranden M, Wattiez R, Lakhdar Z, Ruysschaert J-M, Elouahabi A. Identification of human plasma proteins that bind to cationic lipid/DNA complex and analysis of their effects on transfection efficiency: implications for intravenous gene transfer. Mol Ther. 2003;8(2):264–73.PubMedGoogle Scholar
- 32.Radler JO, Koltover I, Salditt T, Safinya CR. Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes. Science (New York, NY). 1997;275(5301):810–4.Google Scholar
- 34.de la Torre LG, Rosada RS, Trombone AP, Frantz FG, Coelho-Castelo AA, Silva CL, et al. The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes. Colloids Surf B: Biointerfaces. 2009;73(2):175–84.PubMedGoogle Scholar
- 38.Carmona-Ribeiro AM, Ortis F, Schumacher RI, Armelin MCS. Interactions between cationic vesicles and cultured mammalian cells. Langmuir. 1997;13(8):2215–8.Google Scholar
- 41.Lonez C, Lensink MF, Vandenbranden M, Ruysschaert J-M. Cationic lipids activate cellular cascades. Which receptors are involved? Biochim Biophys Acta Gen Subj. 2009;1790(6):425–30.Google Scholar
- 43.Ciani L, Ristori S, Salvati A, Calamai L, Martini G. DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery: zeta potential measurements and electron spin resonance spectra. Biochim Biophys Acta Biomembr. 2004;1664(1):70–9.Google Scholar
- 45.Szoka FC, Xu Y, Zelphati O. How are nucleic acids released in cells from cationic lipid-nucleic acid complexes? J Liposome Res. 1996;6(3):567–87.Google Scholar