Purpose The aim of this work was to use an innovative drug delivery system based on engineered blood erythrocytes, erythro-magneto-HA virosome (EMHVs), to transfer a synthetic DNA decoy oligonucleotide, targeting Elk-1 transcription factor inside porcine vascular smooth muscle cells (VSMC) used as a model of vascular pathology. Elk-1 transcription factor is involved in the signalling cascade promoting both cell proliferation and migration. These events are accountable for vascular restenosis after percutaneous transluminal coronary angioplasty. Methods Porcine erythrocytes were engineered by adding superparamagnetic nanoparticles, hemagglutinin (HA) influenza virus glycoprotein, and purposely synthesized and purified Elk-1 decoy. The internalization of EMHVs in VSMC were measured by HPLC and observed by fluorescence confocal microscope. Primary porcine VSMC were isolated by porcine coronaries. The Elk-1 decoy was delivered into the cells either after being introduced into EMHV or bound to PEI (polyethyleneimine). Migratory and proliferative activities were evaluated with specific assays. Results The internalization of oligonucleotide molecules into engineered erythrocyte was measured by HPLC analysis. Confocal microscope imagines showed that EMHVs are able to vehicle the decoy into VSMCs. Moreover, migratory and proliferative assays demonstrated that Elk-1 decoy actually inhibits cell proliferation and migration. Our data also suggested that erythrocyte-based carriers are more efficient than other conventional and more exploited vehicles such as PEI in delivering oligonucleotides inside the cells. As a consequence, with engineered erythrocytes as carriers, a lower dose of Elk-1 decoy, was sufficient to repress cell growth. Conclusions and Perspectives An innovative approach for gene-drug delivery was assessed and characterized. This system is highly efficient thus allowing the use of less drug quantity in comparison to the more traditional delivery systems. This experimental strategy represents a first decisive step for in vivo experiments on pigs and it could be a new milestone in the development of therapeutic strategies for the local application of gene therapy against restenosis after stent implantation.
Engineered magnetic erythrocyte applied for local gene therapy to prevent restenosis.
CECCHETTINI, ANTONELLA;TEDESCHI, LORENA;
2012-01-01
Abstract
Purpose The aim of this work was to use an innovative drug delivery system based on engineered blood erythrocytes, erythro-magneto-HA virosome (EMHVs), to transfer a synthetic DNA decoy oligonucleotide, targeting Elk-1 transcription factor inside porcine vascular smooth muscle cells (VSMC) used as a model of vascular pathology. Elk-1 transcription factor is involved in the signalling cascade promoting both cell proliferation and migration. These events are accountable for vascular restenosis after percutaneous transluminal coronary angioplasty. Methods Porcine erythrocytes were engineered by adding superparamagnetic nanoparticles, hemagglutinin (HA) influenza virus glycoprotein, and purposely synthesized and purified Elk-1 decoy. The internalization of EMHVs in VSMC were measured by HPLC and observed by fluorescence confocal microscope. Primary porcine VSMC were isolated by porcine coronaries. The Elk-1 decoy was delivered into the cells either after being introduced into EMHV or bound to PEI (polyethyleneimine). Migratory and proliferative activities were evaluated with specific assays. Results The internalization of oligonucleotide molecules into engineered erythrocyte was measured by HPLC analysis. Confocal microscope imagines showed that EMHVs are able to vehicle the decoy into VSMCs. Moreover, migratory and proliferative assays demonstrated that Elk-1 decoy actually inhibits cell proliferation and migration. Our data also suggested that erythrocyte-based carriers are more efficient than other conventional and more exploited vehicles such as PEI in delivering oligonucleotides inside the cells. As a consequence, with engineered erythrocytes as carriers, a lower dose of Elk-1 decoy, was sufficient to repress cell growth. Conclusions and Perspectives An innovative approach for gene-drug delivery was assessed and characterized. This system is highly efficient thus allowing the use of less drug quantity in comparison to the more traditional delivery systems. This experimental strategy represents a first decisive step for in vivo experiments on pigs and it could be a new milestone in the development of therapeutic strategies for the local application of gene therapy against restenosis after stent implantation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
