Abstract
Efficient delivery of nucleic acids into cells is a promising technique to modulate cellular gene expression for therapeutic and research applications. Cationic lipid-based liposomes represent one of the most intensively studied and employed nonviral vectors. They are positively charged at physiological pH and spontaneously self-assemble with polyanionic nucleic acids forming nanoscaled complexes named lipoplexes. Here, we draft a simple protocol for the development, characterization, optimization, and screening of liposomal formulations for in vitro gene delivery. In particular, we report as a practical example a quick method to formulate and extrude nanometer-sized unilamellar cationic vesicles composed of DOTAP as cationic lipid and DOPE as zwitterionic helper lipid at 1:1 molar ratio. The physico-chemical characterization of liposomes and lipoplexes involves the measurement of mean diameter and overall surface charge using Dynamic Light Scattering (DLS) and Laser Doppler Microelectrophoresis. The outlined transfection procedure takes into account several experimental parameters affecting the in vitro performance of gene delivery systems, paying special attention to the charge ratio (CR). Gene delivery effectiveness is evaluated both in terms of transfection efficiency and cytotoxicity of the vector to find the optimal transfection conditions. Importantly, the proposed protocol can be easily shifted to different types of nonviral vectors.
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References
Candiani G, Pezzoli D, Cabras M, Ristori S, Pellegrini C, Kajaste-Rudnitski A, Vicenzi E, Sala C, Zanda M (2008) A dimerizable cationic lipid with potential for gene delivery. J Gene Med 10:637–645
Candiani G, Frigerio M, Viani F, Verpelli C, Sala C, Chiamenti L, Zaffaroni N, Folini M, Sani M, Panzeri W, Zanda M (2007) Dimerizable redox-sensitive triazine-based cationic lipids for in vitro gene delivery. ChemMedChem 2:292–296
Pezzoli D, Candiani G, Cabras S, Giordano C, Daniele F, Cigada A (2008) Liposomes versus micelles as gene delivery vectors. Biomed Pharmacother 62:493
Candiani G, Pezzoli D, Ciani L, Chiesa R, Ristori S (2010) Bioreducible liposomes for gene delivery: from the formulation to the mechanism of action. PLoS One 5:e13430
Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987) Lipofection –- a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A 84:7413–7417
Ciani L, Ristori S, Calamai L, Martini G (2004) DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery: zeta potential measurements and electron spin resonance spectra. Bba-Biomembranes 1664:70–79
Hui SW, Langner M, Zhao YL, Ross P, Hurley E, Chan K (1996) The role of helper lipids in cationic liposome-mediated gene transfer. Biophys J 71:590–599
Ristori S, Ciani L, Candiani G, Battistini C, Frati A, Grillo I, In M (2012) Complexing a small interfering RNA with divalent cationic surfactants. Soft Matter 8:749–756
van Gaal EVB, van Eijk R, Oosting RS, Kok RJ, Hennink WE, Crommelin DJA, Mastrobattista E (2011) How to screen non-viral gene delivery systems in vitro? J Control Release 154:218–232
Pezzoli D, Olimpieri F, Malloggi C, Bertini S, Volonterio A, Candiani G (2012) Chitosan-graft-branched polyethylenimine copolymers: influence of degree of grafting on transfection behavior. PLoS One 7:e34711
Macdonald RC, Macdonald RI, Menco BPM, Takeshita K, Subbarao NK, Hu LR (1991) Small-volume extrusion apparatus for preparation of large unilamellar vesicles. Biochim Biophys Acta 1061:297–303
Acknowledgments
This work was partly supported by the Politecnico di Milano, 5xmille Junior, “SURGES” Project and by the Italian Ministry for Education, University and Research (MIUR)—FIRB Futuro in Ricerca 2008, Grant RBFR08XH0H (to Dr. Candiani, both), as well as by the Italian Ministry of Health Grant Giovani Ricercatori 2009, GR-2009-1471693 (to Dr. Kajaste-Rudnitski).
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Pezzoli, D., Kajaste-Rudnitski, A., Chiesa, R., Candiani, G. (2013). Lipid-Based Nanoparticles as Nonviral Gene Delivery Vectors. In: Bergese, P., Hamad-Schifferli, K. (eds) Nanomaterial Interfaces in Biology. Methods in Molecular Biology, vol 1025. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-462-3_21
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DOI: https://doi.org/10.1007/978-1-62703-462-3_21
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Publisher Name: Humana Press, Totowa, NJ
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