Understanding the mechanism of protamine in solid lipid nanoparticle-based lipofection: the importance of the entry pathway.

The aim of our study was to evaluate the effect of protamine on the transfection capacity of solid lipid nanoparticles (SLNs) by correlating it to the internalization mechanisms and intracellular trafficking of the vectors. Vectors were prepared with SLN, DNA, and protamine. ARPE-19 and HEK-293 cells were used for the evaluation of the formulations. Protamine induced a 6-fold increase in the transfection of SLNs in retinal cells due to the presence of nuclear localization signals (NLS), its protection capacity, and a shift in the internalization mechanism from caveolae/raft-mediated to clathrin-mediated endocytosis. However, protamine produced an almost complete inhibition of transfection in HEK-293 cells. In spite of the high DNA condensation capacity of protamine and its content in NLS, this does not always lead to an improvement in cell transfection since it may impair some of the limiting steps of the transfection processes.

[1]  F. Barry,et al.  Enhanced lipoplex‐mediated gene expression in mesenchymal stem cells using reiterated nuclear localization sequence peptides , 2010, The journal of gene medicine.

[2]  M. Conese,et al.  Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[3]  Y. Barenholz,et al.  Relationships between chemical composition, physical properties and transfection efficiency of polysaccharide-spermine conjugates. , 2006, Biomaterials.

[4]  N. M. Rao,et al.  Cationic lipid-mediated nucleic acid delivery: beyond being cationic. , 2010, Chemistry and physics of lipids.

[5]  Shengrong Guo,et al.  Synthesis and characterization of stearyl protamine and investigation of their complexes with DNA for gene delivery. , 2009, Colloids and surfaces. B, Biointerfaces.

[6]  Yoshio Okahata,et al.  Characterization of Protamine as a Transfection Accelerator for Gene Delivery , 2006 .

[7]  Hadas Keren,et al.  Effect of peptides bearing nuclear localization signals on therapeutic ultrasound mediated gene delivery , 2008, Journal of Gene Medicine.

[8]  S. Futaki,et al.  Octaarginine-modified liposomes: enhanced cellular uptake and controlled intracellular trafficking. , 2008, International journal of pharmaceutics.

[9]  Kelly Shintani,et al.  Review and update: current treatment trends for patients with retinitis pigmentosa. , 2009, Optometry.

[10]  B. Ruozi,et al.  Nuclear localization of cationic solid lipid nanoparticles containing Protamine as transfection promoter. , 2010, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[11]  H. Harashima,et al.  Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microinjection: The use of a nuclear transfer score , 2005, FEBS letters.

[12]  Ernst Wagner,et al.  The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[13]  J. Pedraz,et al.  Solid lipid nanoparticles for retinal gene therapy: transfection and intracellular trafficking in RPE cells. , 2008, International journal of pharmaceutics.

[14]  B. Röder,et al.  Lipid nanoparticles for skin penetration enhancement-correlation to drug localization within the particle matrix as determined by fluorescence and parelectric spectroscopy. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[15]  Gaurav Sahay,et al.  Endocytosis of nanomedicines. , 2010, Journal of controlled release : official journal of the Controlled Release Society.

[16]  E. Giralt,et al.  A proline-rich peptide improves cell transfection of solid lipid nanoparticle-based non-viral vectors. , 2009, Journal of controlled release : official journal of the Controlled Release Society.

[17]  A. Urtti,et al.  Long-Lasting Secretion of Transgene Product from Differentiated and Filter-Grown Retinal Pigment Epithelial Cells After Nonviral Gene Transfer , 2005, Current eye research.

[18]  Hui Peng,et al.  Interaction of DNA/nuclear protein/polycation and the terplexes for gene delivery , 2010, Nanotechnology.

[19]  S. Simões,et al.  Association of albumin or protamine to lipoplexes: enhancement of transfection and resistance to serum , 2004, The journal of gene medicine.

[20]  Vadim Zinchuk,et al.  Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images , 2008, Current protocols in cell biology.

[21]  K. Biegeleisen.  The probable structure of the protamine-DNA complex. , 2006, Journal of theoretical biology.

[22]  H. Okada,et al.  Enhancement of gene transfection into human dendritic cells using cationic PLGA nanospheres with a synthesized nuclear localization signal. , 2009, International journal of pharmaceutics.

[23]  T. Restle,et al.  Recent Developments in Peptide-Based Nucleic Acid Delivery , 2008, International journal of molecular sciences.

[24]  D. Hoekstra,et al.  Cationic lipids, lipoplexes and intracellular delivery of genes. , 2006, Journal of controlled release : official journal of the Controlled Release Society.

[25]  C. Di Giorgio,et al.  Synthesis of acridine-nuclear localization signal (NLS) conjugates and evaluation of their impact on lipoplex and polyplex-based transfection. , 2005, European journal of medicinal chemistry.

[26]  A. Griffioen,et al.  Targeted gene-delivery strategies for angiostatic cancer treatment. , 2007, Trends in molecular medicine.

[27]  H. Cohen,et al.  Nanoparticles for gene delivery to retinal pigment epithelial cells. , 2005, Molecular vision.

[28]  P. Dario,et al.  Evaluation of cationic liposomes composed of an amino acid-based lipid for neuronal transfection. , 2010, Nanomedicine : nanotechnology, biology, and medicine.

[29]  J. Pedraz,et al.  Solid lipid nanoparticles as potential tools for gene therapy: in vivo protein expression after intravenous administration. , 2010, International journal of pharmaceutics.

[30]  M. Tabrizian,et al.  Cell line-dependent internalization pathways and intracellular trafficking determine transfection efficiency of nanoparticle vectors. , 2008, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[31]  Shubiao Zhang,et al.  The biological routes of gene delivery mediated by lipid-based non-viral vectors , 2009, Expert opinion on drug delivery.

[32]  J. Barichello,et al.  Complexation of siRNA and pDNA with cationic liposomes: the important aspects in lipoplex preparation. , 2010, Methods in molecular biology.

[33]  J. Davies.  Gene and cell therapy for cystic fibrosis. , 2006, Paediatric respiratory reviews.

[34]  J. Pedraz,et al.  Solid lipid nanoparticles: formulation factors affecting cell transfection capacity. , 2007, International journal of pharmaceutics.

[35]  J. Pedraz,et al.  Short- and long-term stability study of lyophilized solid lipid nanoparticles for gene therapy. , 2009, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[36]  G. Giammona,et al.  Novel cationic solid-lipid nanoparticles as non-viral vectors for gene delivery , 2007, Journal of drug targeting.

[37]  M. Lamfers,et al.  Evolving gene therapy approaches for osteosarcoma using viral vectors: review. , 2007, Bone.