Calcium Based Non-viral Gene Delivery: An Overview of Methodology and Applications

  • Kianoush Khosravi-Darani Mail National Nutrition and Food Technology Research Institute, Shahid Beheshti Medical University, Tehran, Iran.
  • Mohamaad Reza Mozafari Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia.
  • Ladan Rashidi Department of Food & Agriculture Research, Institute of Standard and Industrial Research of Iran, Karaj, Iran.
  • Mehrdad Mohammadi National Nutrition and Food Technology Research Institute, Shahid Beheshti Medical University, Tehran, Iran.
Calcium phosphate, nanoparticles, gene transfer techniques, transfection


Application of therapeutic gene transfer in the treatment of genetic diseases is a notable progress but there are some disadvantages and limitations in it. The process of overcoming these barriers is a drastic change in gene delivery. Recently, calcium phosphate nanoparticles alone, or in combination with viral and nonviral vectors, were found to have a positive effect on gene transfer especially when incorporated in the colloidal particulate systems. This review elaborates on various successful methods of using calcium phosphate nanoparticles in gene delivery, which are considered an advancing approach to gene delivery.


Wyrozumska P, Stebelska K, Grzybek M, Sikorski AF. Synthetic vectors for genetic drug delivery. In: Mozafari MR, editors. Nanocarrier Technologies: Frontiers of Nanotherapy. The Netherlands: Springer; 2006. p. 139-75.

Romano G, Pacilio C, Giordano A. Gene transfer technology in therapy: current applications and future goals. Stem Cells 1999;17(4):191-202.

Tomanin R, Scarpa M. Why do we need new gene therapy viral vectors? Characteristics, limitations and future perspectives of viral vector transduction. Curr Gene Ther 2004;4(4):357-72.

Khosravi-Darani K, Sadeghi-Zadeh M. Comparison of nonviral systems for gene delivery vehicles. Tabib-e-Shargh J 2005;7(2):149-58. [in Persian]

Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973;52(2):456-67.

Xu H, Miller J, Liang BT. High-efficiency gene transfer into cardiac myocytes. Nucleic Acids Res 1992;20(23):6425-6.

Yang YW, Yang JC. Calcium phosphate as a gene carrier: electron microscopy. Biomaterials 1997;18(3):213-7.

Shen H, Tan J, Saltzman WM. Surface-mediated gene transfer from nanocomposites of controlled texture. Nat Mater2004;3(8):569-74.

Truong-Le VL, Walsh SM, Schweibert E, Mao HQ, Guggino WB, August JT, Leong KW. Gene transfer by DNA-gelatin nanospheres. Arch Biochem Biophys 1999;361(1):47-56.

Urabe M, Kume A, Tobita K, Ozawa K. DNA/calcium phosphate precipitates mixed with medium are stable and maintain high transfection efficiency. Anal Biochem 2000;278(1):91-2.

Kulkarni VI, Shenoy VS, Dodiya SS, Rajyaguru TH, Murthy RR. Role of calcium in gene delivery. Expert Opin Drug Deliv 2006;3(2):235-45.

Frey A, Neutra MR, Robey FA. Peptomer aluminum oxide nanoparticle conjugates as systemic and mucosal vaccinecandidates: synthesis and characterization of a conjugate derived from the C4 domain of HIV-1MN gp120. Bioconjug Chem 1997;8(3):424-33.

Kneuer C, Sameti M, Bakowsky U, Schiestel T, Schirra H, Schmidt H, et al. A nonviral DNA delivery system based onmsurface modified silica-nanoparticles can efficiently transfect cells in vitro. Bioconjug Chem 2000;11(6):926-32.

Zhang S, Gonsalves KE. Preparation and characterization of thermally stable nanohydroxyapatite. J Mater Sci Mater Med 1997;8(1):25-8.

Cheng FY, Su CH, Yang YS, Yeh CS, Tsai CY, Wu CL, et al. Characterization of aqueous dispersions of Fe3O4 nanoparticles and their biomedical and their biomedical applications. Biomaterials 2005;26(7):729-38.

Kakizawa Y, Miyata K, Furukawa S, Kataoka K. Sizecontrolled formation of a calcium phosphate-based organicinorganic hybrid vector for gene delivery using poly(ethylene glycol)-block-poly(aspartic acid). Adv Mater 2004;16(8):699-702.

Madry H, Reszka R, Bohlender J, Wagner J. Efficacy of cationic liposome-mediated gene transfer to mesangial cells in vitro and in vivo. J Mol Med 2001;79(4):184-9.

Schmidt HT, Gray BL, Wingert PA, Ostafin AE. Assembly of aqueous-cored calcium phosphate nanoparticles for drug delivery. Chem Mat 2004;16:4942-7.

Yu D, Wong J, Matsuda Y, Fox JL, Higuchi WI, Otsuka M. Self-setting hydroxyapatite cement: a novel skeletal drugdelivery system for antibiotics. J Pharm Sci 1992;81(6):529-31.

Morçöl T, Nagappan P, Nerenbaum L, Mitchell A, Bell SJ. Calcium phosphate-PEG-insulin-casein (CAPIC) particles as oral delivery systems for insulin. Int J Pharm 2004;277(1-2):91-7.

Matsumoto T, Okazaki M, Inoue M, Yamaguchi S, Kusunose T, Toyonaga T, et al. Hydroxyapatite particles as a controlled release carrier of protein. Biomaterials 2004;25(17):3807-12.

Joosten U, Joist A, Frebel T, Brandt B, Diederichs S, von Eiff C. Evaluation of an in situ setting injectable calcium phosphate as a new carrier material for gentamicin in the treatment of chronic osteomyelitis: studies in vitro and in vivo. Biomaterials 2004;25(18):4287-95.

Paul W, Sharma CP. Development of porous spherical hydroxyapatite granules: application towards protein delivery. J Mater Sci Mater Med 1999;10(7):383-8.

Tolou H. Administration of oligonucleotides to cultured cells by calcium phosphate precipitation method. Anal Biochem 1993;215(1):156-8.

Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 1987;7(8):2745-52.

Jordan M, Wurm F. Transfection of adherent and suspended cells by calcium phosphate. Methods 2004;33(2):136-43.

Orrantia E, Chang PL. Intracellular distribution of DNA internalized through calcium phosphate precipitation. Exp Cell Res 1990;190(2):170-4.

Seelos C. A critical parameter determining the aging of DNAcalcium- phosphate precipitates. Anal Biochem 1997;245(1):109-11.

Zhang Y, Kohler N, Zhang M. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake. Biomaterials 2002;23(7):1553-61.

Roy I, Mitra S, Maitra A, Mozumdar S. Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery. Int J Pharm 2003;250(1):25-33.

Lopata MA, Cleveland DW, Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferasegene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res 1984;12(14):5707-17.

Frost E, Williams J. Mapping temperature-sensitive and hostrange mutations of adenovirus type 5 by marker rescue. Virology 1978;91(1):39-50.

Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Press; 2001.

Jordan M, Schallhorn A, Wurm FM. Transfecting mammalian cells: optimization of critical parameters affecting calciumphosphate precipitate formation. Nucleic Acids Res 1996;24(4):596-601.

Zhdanov RI, Bogdaneko EV, Zarubina TV, Dominiva SI, Krivstov GG, Borisenko AS, et al. New lipid and glycolipidbased nanosystems for targeted gene delivery: cholenims, glycoclipids and chitosan. In: Mozafari MR, editor. Nanomaterials and Nanosystems for Biomedical Applications. Netherlands: Springer; 2007. p. 27-53.

Wilson SP, Smith LA. Addition of glycerol during DNA exposure enhances calcium phosphate transfection. Anal Biochem 1997;246(1):148-50.

Mozafari MR, Reed CJ, Rostron C, Kocum C, Piskin E. Construction of stable anionic liposome-plasmid particles using the heating method: a preliminary investigation. Cell Mol Biol Lett 2002;7(3):923-7.

O'Mahoney JV, Adams TE. Optimization of experimental variables influencing reporter gene expression in hepatoma cells following calcium phosphate transfection. DNA Cell Biol 1994;13(12):1227-32.

Wilson SP, Liu F, Wilson PR, Housley PR. Optimization of calcium phosphate transfection for bovine chromaffin cells: relationship to calcium phosphate precipitate formation. Anal Biochem 1995;226(2):212-20.

Luo D, Saltzman WM. Synthetic DNA delivery systems. Nat Biotechnol 2000;18(1):33-7.

Chowdhury EH, Kunou M, Nagaoka M, Kundu AK, Hoshiba T, Akaike T. High-efficiency gene delivery for expression in mammalian cells by nanoprecipitates of Ca-Mg phosphate. Gene 2004;341:77-82.

Kakizawa Y, Kataoka K. Block copolymer self-assembly into monodispersive nanoparticles with hybrid core of antisense DNA and calcium phosphate. Langmuir 2002;18:4539-43.

Strain AJ, Wyllie AH. The uptake and stability of simianvirus- 40 DNA after calcium phosphate transfection of CV-1 cells. Biochem J 1984;218(2):475-82.

Welzel T, Radtke I, Meyer-Zaika W, Heumann R, Epple M. Transfection of cells with custom-made calcium phosphate nanoparticles coated with DNA. J Materials Chem 2004;14(14):2213-17.

Prabha S, Zhou WZ, Panyam J, Labhasetwar V. Sizedependency of nanoparticle-mediated gene transfection: studies with fractionated nanoparticles. Int J Pharm 2002;244(1-2):105-15.

Washbourne P, McAllister AK. Techniques for gene transfer into neurons. Curr Opin Neurobiol 2002;12(5):566-73.

Lasic DD. Templeton NS. Liposomes in gene therapy. Adv Drug Deliv Rev 1996; 20: 221-6.

Ishii N, Fukushima J, Kaneko T, Okada E, Tani K, Tanaka SI, et al. Cationic liposomes are a strong adjuvant for a DNA vaccine of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 1997;13(16):1421-8.

Tang MX, Redemann CT, Szoka FC Jr. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjug Chem 1996;7(6):703-14.

Loyter A, Scangos G, Juricek D, Keene D, Ruddle FH. Mechanisms of DNA entry into mammalian cells. II. Phagocytosis of calcium phosphate DNA co-precipitate visualized by electron microscopy. Exp Cell Res 1982;139(1):223-34.

Loyter A, Scangos GA, Ruddle FH. Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc Natl Acad Sci U S A 1982;79(2):422-6.

Lechardeur D, Verkman AS, Lukacs GL. Intracellular routing of plasmid DNA during non-viral gene transfer. Adv Drug Deliv Rev 2005;57(5):755-67.

Sokolova VV, Radtke I, Heumann R, Epple M. Effective transfection of cells with multi-shell calcium phosphate-DNA nanoparticles. Biomaterials 2006;27(16):3147-53.

Bisht S, Bhakta G, Mitra S, Maitra A. pDNA loaded calcium phosphate nanoparticles: highly efficient non-viral vector for gene delivery. Int J Pharm 2005;288(1):157-68.

Paine PL, Moore LC, Horowitz SB. Nuclear envelope permeability. Nature 1975;254(5496):109-14.

Zhu SH, Huang BY, Zhou KC, Huang SP, Liu F, Li YM, et al.bHydroxyapatite nanoparticles as a novel gene carrier. J Nanoparticle Res 2004;6(2):307-11.

Maitra A. Calcium phosphate nanoparticles: secondgeneration nonviral vectors in gene therapy. Expert Rev Mol Diagn 2005;5(6):893-905.

Cullis PR, Chonn A. Recent advances in liposome technologies and their applications for systemic gene delivery. Adv Drug Deliv Rev 1998;30(1-3):73-83.

Fenske DB, MacLachlan I, Cullis PR. Stabilized plasmid-lipid particles: a systemic gene therapy vector. Methods Enzymol 2002;346:36-71.

Alton EW, Geddes DM. Gene therapy for cystic fibrosis: a clinical perspective. Gene Ther 1995;2(2):88-95.

Liu Y, Liggitt D, Zhong W, Tu G, Gaensler K, Debs R. Cationic liposome-mediated intravenous gene delivery. J Biol Chem 1995;270(42):24864-70.

Smith JG, Walzem RL, German JB. Liposomes as agents of DNA transfer. Biochim Biophys Acta 1993;1154(3-4):327-40.

Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem 1995;270(32):18997-9007.

Bonté F, Juliano RL. Interactions of liposomes with serum proteins. Chem Phys Lipids 1986;40(2-4):359-72.

Oku N, Yamazaki Y, Matsuura M, Sugiyama M, Hasegawa M, Nango M. A novel non-viral gene transfer system, polycation liposomes. Adv Drug Deliv Rev 2001;52(3):209-18.

Deol P, Khuller GK. Lung specific stealth liposomes: stability, biodistribution and toxicity of liposomal antitubercular drugs in mice. Biochim Biophys Acta 1997;1334(2-3):161-72.

Lechardeur D, Sohn KJ, Haardt M, Joshi PB, Monck M, Graham RW, et al. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther 1999;6(4):482-97.

Filion MC, Phillips NC. Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells. Biochim Biophys Acta 1997;1329(2):345-56.

Khosravi-Darani K, Mozafari MR. The Second Generation of Anionic Liposome for Gene Therapy. Tabib-e-Shargh 2005;7(1):71-9.

Gao X, Huang L. Potentiation of cationic liposome-mediated gene delivery by polycations. Biochemistry 1996;35(3):1027-36.

Hagstrom JE, Sebestyen MG, Budker V, Ludtke JJ, Fritz JD, Wolff JA. Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation. Biochim Biophys Acta 1996;1284(1):47-55.

Ibáñez M, Gariglio P, Chávez P, Santiago R, Wong C, Baeza I. Spermidine-condensed DNA and cone-shaped lipids improve delivery and expression of exogenous DNA transfer by liposomes. Biochem Cell Biol 1996;74(5):633-43.

Mizuguchi H, Nakagawa T, Nakanishi M, Imazu S, Nakagawa S, Mayumi T. Efficient gene transfer into mammalian cells using fusogenic liposome. Biochem Biophys Res Commun 1996;218(1):402-7.

Li S, Huang L. In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes. Gene Ther 1997;4(9):891-900.

Perez-Terzic C, Pyle J, Jaconi M, Stehno-Bittel L, Clapham DE. Conformational states of the nuclear pore complex induced by depletion of nuclear Ca2+ stores. Science 1996;273(5283):1875-7.

Zaitsev SV, Haberland A, Otto A, Vorob'ev VI, Haller H, Böttger M. H1 and HMG17 extracted from calf thymus nuclei are efficient DNA carriers in gene transfer. Gene Ther 1997;4(6):586-92.

Lam AM, Cullis PR. Calcium enhances the transfection potency of plasmid DNA-cationic liposome complexes. Biochim Biophys Acta 2000;1463(2):279-90.

Kharakoz DP, Khusainova RS, Gorelov AV, Dawson KA. Stoichiometry of dipalmitoylphosphatidylcholine-DNA interaction in the presence of Ca2+: a temperature-scanning ultrasonic study. FEBS Lett 1999;446(1):27-9.

Lappalainen K, Jääskeläinen I, Syrjänen K, Urtti A, Syrjänen S. Comparison of cell proliferation and toxicity assays using two cationic liposomes. Pharm Res 1994;11(8):1127-31.

Anwer K, Bailey A, Sullivan SM. Targeted gene delivery: a= two-pronged approach. Crit Rev Ther Drug Carrier Syst 2000;17(4):377-424.

Gulati M, Bajad S, Singh S, Ferdous AJ, Singh M. Development of liposomal amphotericin B formulation. J Microencapsul 1998;15(2):137-51.

Tardi PG, Boman NL, Cullis PR. Liposomal doxorubicin. J Drug Target 1996;4(3):129-40.

Perrie Y, Gregoriadis G. Liposome-entrapped plasmid DNA: characterisation studies. Biochim Biophys Acta 2000;1475(2):125-32.

Patil SD, Rhodes DG. Influence of divalent cations on the conformation of phosphorothioate oligodeoxynucleotides: a circular dichroism study. Nucleic Acids Res 2000;28(12):2439-45.

Lakkaraju A, Dubinsky JM, Low WC, Rahman YE. Neurons are protected from excitotoxic death by p53 antisense oligonucleotides delivered in anionic liposomes. J Biol Chem 2001;276(34):32000-7.

Wilson RW, Bloomfield VA. Counterion-induced condesation of deoxyribonucleic acid. a light-scattering study. Biochemistry 1979;18(11):2192-6.

Zareie MH, Mozafari MR, Hasirci V, Piskin E. Scanning tunnelling microscopy investigation of liposome-DNA-Ca2+ complexes. J Liposome Res 1997;7(4):491-502.

Mozafari MR, Zareie MH, Piskin E, Hasirci V. Formation of supramolecular structures by negatively charged liposomes in the presence of nucleic acids and divalent cations. Drug Deliv 1998;5(2):135-41.

Mozafari MR, Hasirci V. Mechanism of calcium ion induced multilamellar vesicle-DNA interaction. J Microencapsul 1998;15(1):55-65.

McManus JJ, Radler JO, Dawson KA. Does calcium turn a zwitterionic lipid cationic? J Phys Chem B 2003a;107(36):9869-75.

McManus JJ, Radler JO, Dawson KO. Phase behavior of DPPC in a DNA-calcium-zwitterionic lipid complex studied by small-angle X-ray scattering. Langmuir 2003b;19(23):9630-7.

Bailey AL, Sullivan SM. Efficient encapsulation of DNA plasmids in small neutral liposomes induced by ethanol and calcium. Biochim Biophys Acta 2000;1468(1-2):239-52.

Patil SD, Rhodes DG, Burgess DJ. Anionic liposomal delivery system for DNA transfection. AAPS J 2004;6(4):e29.

Reston JT, Gould-Fogerite S, Mannino RJ. Potentiation of DNA mediated gene transfer in NIH3T3 cells by activators of protein kinase C. Biochim Biophys Acta 1991;1088(2):270-6.

Eanes ED, Meyer JL. The maturation of crystalline calcium phosphates in aqueous suspensions at physiologic pH. Calcif Tissue Res 1977;23(3):259-69.

How to Cite
Khosravi-Darani K, Mozafari MR, Rashidi L, Mohammadi M. Calcium Based Non-viral Gene Delivery: An Overview of Methodology and Applications. Acta Med Iran. 48(3):133-141.