dioleoyl-phosphatidylethanolamine and arginyl-glycyl-aspartic-acid

In recent times, search for potent and highly selective thrombolytic agents with minimal side effects has become a major area of research. The aim of the present study was to develop and characterize target sensitive (TS) liposomes encapsulating streptokinase, a thrombolytic agent. The developed TS liposomes were composed of dioleylphophatidyl ethanolamine (DOPE) and dipalmityl-c(RGDfK) (10:1mol/mol). Dipalmityl-c(RGDfK) was synthesized using typical carbodiimide chemistry using palmitic acid and c(RGDfK), while lysine was used as a spacer. Liposomes were of 100-120nm size. In vitro drug release study showed that nearly 40% drug of the entrapped drug was released in 12h in the PBS (pH 7.4), however on incubation with activated platelet about 90% of drug was released within 45min. The results suggested target sensitivity of the liposomes. Further, targeting potential was confirmed using fluorescent microscopy and flow cytometry. Clot lysis study revealed that TS liposomes could not only reduce the clot lysis time but also increase the extent of clot lysis as compared to non-liposomal streptokinase solution. In conclusion, the present liposomal formulation will target the thrombolytic agent to the activated platelets in the thrombus and hence will improve the therapeutic efficacy of the drug.

Topics: Blood Coagulation; Blood Platelets; Cells, Cultured; Drug Delivery Systems; Fibrinolytic Agents; Flow Cytometry; Humans; Liposomes; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Molecular Structure; Oligopeptides; Palmitic Acids; Phosphatidylethanolamines; Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Streptokinase; Substrate Specificity

The recently developed siRNA oligonucleotides are an attractive alternative to antisense as a therapeutic modality because of their robust, gene selective silencing of drug target protein expression. To achieve therapeutic success, however, several hurdles must be overcome including rapid clearance, nuclease degradation, and inefficient intracellular localization. In this presentation, we discuss design strategies for development of self-assembling nanoscale carriers for neovasculature targeted delivery of siRNA inhibiting tumor or ocular angiogenesis.

Topics: Angiogenesis Inhibitors; Animals; Cells, Cultured; Drug Delivery Systems; Flow Cytometry; Humans; Indicators and Reagents; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Neovascularization, Pathologic; Oligopeptides; Peptides; Phosphatidylethanolamines; Polyethylene Glycols; RNA, Small Interfering

Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cell Line; Cornea; Drug Carriers; Genetic Vectors; Humans; Ligands; Liposomes; Microscopy, Atomic Force; Molecular Sequence Data; Oligopeptides; Peptides; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Rabbits; Receptors, Fibronectin; Recombinant Fusion Proteins; Transfection