Effect of Helper Lipids on Stability and Transfection Activity of Lyophilized Lipoplex Formulations of Antisense and DOTAP Nanoliposomes

Survivin, an inhibitor of apoptosis protein is highly expressed in most cancers and considered as an attractive target for cancer antisense therapy. To vectorize antisense molecules, cationic nanoliposomes are generally used; however, their complexes are too instable, during shelf-life and upon exposure to blood components and extracellular matrix, to be used in-vivo. The present study aimed to develop fresh and lyophilized formulations of antisense/DOTAP nanoliposomes with different helper lipids and compare their shelflife and biologic stabilities and their transfection activities in tumor cell lines. DOTAP nanoliposomes in combination with different helper lipids were prepared in HEPES buffer (20 mM, pH=7.4) by thin-layer hydration followed by thermobarrel extrusion and PTFE membrane filter sterilization. Nanoliposomes were characterized regarding their particle size distribution, final lipid recovery and physical stability. Following antisense loading by direct addition through electrostatic attraction, the degree of complexation was determined by ethidium bromide displacement assay. To stabilize the formulations, they were freezedried with 10% sucrose. The potential of the lyophilized and fresh formulations of FITC-labeled antisense to transfect different cell lines (SK-BR-3, MCF-7) was studied by flow cytometry. Fresh nanoliposomes of different formulations had a size in range of 50100 nm. The degree of complex formation with antisense was determined almost 70-80% (N/P ~ 2) which decreased as incubated with either PBS or complete medium and heparin sulfate. Their average sizes significantly changed after preservation for few days at 4 °C. Lyophilization process compromised the particle size distribution and antisense loading efficiency of different formulations except for DOTAP/DOPE (1:1 mole ratio) which did not change significantly. Both fresh and lyophilized formulations exhibited the highest transfection activity at comparable levels especially in SK-BR-3 cells. As a conclusion, lyophilization process could promote stability and preserve transfection activity of the antisense complexes of DOTAP/DOPE (1:1) nanoliposomes.

[1]  Kazuo Maruyama,et al.  Intracellular targeting delivery of liposomal drugs to solid tumors based on EPR effects. , 2011, Advanced drug delivery reviews.

[2]  Y. Maitani,et al.  Effect of sugars on storage stability of lyophilized liposome/DNA complexes with high transfection efficiency. , 2008, International journal of pharmaceutics.

[3]  N. Zaffaroni,et al.  Targeting survivin in cancer therapy , 2008, Expert opinion on therapeutic targets.

[4]  D. Burgess,et al.  DNA-based therapeutics and DNA delivery systems: A comprehensive review , 2005, The AAPS Journal.

[5]  J. Hughes,et al.  Effect of Lyophilization and Freeze-thawing on the Stability of siRNA-liposome Complexes , 2008, AAPS PharmSciTech.

[6]  H. Moghimi,et al.  Modeling cytoplasmic release of encapsulated oligonucleotides from cationic liposomes. , 2007, International journal of pharmaceutics.

[7]  H. Moghimi,et al.  Preparation of oligodeoxynucleotide encapsulated cationic liposomes and release study with models of cellular membranes , 2007 .

[8]  S. Fukuda,et al.  Survivin, a cancer target with an emerging role in normal adult tissues , 2006, Molecular Cancer Therapeutics.

[9]  W. Hinrichs,et al.  The choice of a suitable oligosaccharide to prevent aggregation of PEGylated nanoparticles during freeze thawing and freeze drying. , 2006, International journal of pharmaceutics.

[10]  S. Hart.  Lipid carriers for gene therapy. , 2005, Current drug delivery.

[11]  H. Frijlink,et al.  Inulin is a promising cryo- and lyoprotectant for PEGylated lipoplexes. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[12]  S. Yoshioka,et al.  Effect of freezing rate on physical stability of lyophilized cationic liposomes. , 2005, Chemical & pharmaceutical bulletin.

[13]  Shubiao Zhang,et al.  Cationic compounds used in lipoplexes and polyplexes for gene delivery. , 2004, Journal of controlled release : official journal of the Controlled Release Society.

[14]  M. Kumar,et al.  Survivin expression induced by doxorubicin in cholangiocarcinoma. , 2004, World journal of gastroenterology.

[15]  A. El-Aneed,et al.  An overview of current delivery systems in cancer gene therapy. , 2004, Journal of controlled release : official journal of the Controlled Release Society.

[16]  E. Winden.  Freeze-drying of liposomes: theory and practice. , 2003 .

[17]  E. C. van Winden.  Freeze-drying of liposomes: theory and practice. , 2003, Methods in enzymology.

[18]  V. Vlassov,et al.  Cationic lipid-DNA complexes-lipoplexes-for gene transfer and therapy. , 2002, Bioelectrochemistry.

[19]  I. S. Blagbrough,et al.  Rapid and sensitive ethidium bromide fluorescence quenching assay of polyamine conjugate-DNA interactions for the analysis of lipoplex formation in gene therapy. , 2000, Journal of pharmaceutical and biomedical analysis.

[20]  C. Nastruzzi,et al.  Effect of DNA complexation and freeze-drying on the physicochemical characteristics of cationic liposomes. , 2000, Antisense & nucleic acid drug development.