ABSTRACT
Crotamine is a basic, 42-residue polypeptide from snake venom that has been shown to possess cell-penetrating properties. Here we describe the preparation, purification, biochemical and biophysical analysis of venom-derived, recombinant, chemically synthesized, and fluorescent-labeled crotamine. We also describe the formation and characterization of crotamine-DNA and crotamine-RNA nanoparticles; and the delivery of these nanoparticles into cells and animals. Crotamine forms nanoparticles with a variety of DNA and RNA molecules, and crotamine-plasmid DNA nanoparticles are selectively delivered into actively proliferating cells in culture or in living organisms such as mice, Plasmodium, and worms. As such, these nanoparticles could form the basis for a nucleic acid drug-delivery system. We also describe here the design and characterization of crotamine-functionalized gold nanoparticles, and the delivery of these nanoparticles into cells. We also evaluated the viability of using the combination of crotamine with silica nanoparticles in animal models, aiming to provide slow delivery, and to decrease the crotamine doses needed for the biological effects. In addition, the efficacy of administering crotamine orally was also demonstrated.
Subject(s)
Antineoplastic Agents/administration & dosage , Cell-Penetrating Peptides/administration & dosage , Crotalid Venoms/administration & dosage , Melanoma, Experimental/drug therapy , Administration, Oral , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Crotalid Venoms/chemistry , Crotalid Venoms/pharmacology , DNA/metabolism , Fluorescent Dyes/chemistry , Mice , Nanoparticles , RNA/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Activated proximal tubular epithelial cells (PTECs) play a crucial role in progressive tubulo-interstitial fibrosis in native and transplanted kidneys. Targeting PTECs by non-viral delivery vectors might be useful to influence the expression of important genes and/or proteins in order to slow down renal function loss. However, no clinical therapies that specifically target PTECs are available at present. We earlier showed that a cationic cell penetrating peptide isolated from South American rattlesnake venom, named crotamine, recognizes cell surface heparan sulfate proteoglycans and accumulates in cells. In healthy mice, crotamine accumulates mainly in kidneys after intraperitoneal (ip) injection. Herein we demonstrate for the first time, the overall safety of acute or long-term treatment with daily ip administrated crotamine for kidneys functions. Accumulation of ip injected crotamine in the kidney brush border zone of PTECs, and its presence inside these cells were observed. In addition, significant lower in vitro crotamine binding, uptake and reporter gene transport and expression could be observed in syndecan-1 deficient HK-2 PTECs compared to wild-type cells, indicating that the absence of syndecan-1 impairs crotamine uptake into PTECs. Taken together, our present data show the safety of in vivo long-term treatment with crotamine, and its preferential uptake into PTECs, which are especially rich in HSPGs such as syndecan-1. In addition to the demonstrated in vitro gene delivery mediated by crotamine in HK-2 cells, the potential applicability of crotamine as prototypic non-viral (gene) delivery nanocarrier to modulate PTEC gene and/or protein expression was confirmed.
