Cell Penetrating Peptide Transfection and Genetic Engineering of Stem Cells

Grant Winners

Dean

Abstract

Research into micro- and nano-biological processes has spiked in the last few decades due to technological advances and new methods for visualizing molecules and events within cells. These developments have led to scientific breakthroughs such as the ability to grow cells directly from living patients in a laboratory setting (termed “primary” cell culture), observe and affect cellular processes with nanoscopic agents such as luminescent semiconducting quantum dot (QD) nanoparticles, as well engineer cells to treat disease factors by means of cellular transfection. Efficient cell transfection can also facilitate the genetic engineering of medically helpful “nurse” cells from primary cells that act as bio-reactors, producing strategically engineered molecules for the treatment of various cells and tissue types in the body. In order to be successful therapeutic agents in live patients, the cells must have high survival rates following transfection. Cell penetrating peptides (CPPs) aid in cargo delivery into cells with minimal damage, maintaining high cell viability while being superlative cell transfection mediators. Understanding which CPP sequences and combinations can be used to best aide in therapeutic delivery to primary cells can be facilitated by using luminescent QD nanoparticles, for visualization and therapeutic packaging. The utility of knowing how to safely and effectively transfect primary cells and transform them into “nurse” cells to fight disease and promote healing will advance medicine one step closer towards being truly personalized. The goals of this work are to develop novel technical insights into the transfection of primary human cells using CPPs loaded onto QD delivery vectors, as well as to demonstrate the proof of principle for the subsequent generation of “nurse” cell bio-reactors from primary adult human mesenchymal stem cells (hMSCs). In order to accomplish these goals, first, the best performing CPPs for the transfection of hMSCs will be determined from a selection of known CPP sequences attached to QDs for cargo packing and intracellular tracking. Next, hMSC transfection with CPP-QDs will be optimized by systematic combinations of the best performing CPP-QDs. Finally, the optimum CPP-QD platforms will be used to genetically engineer the hMSCs by delivering a plasmid gene for a red fluorescent protein to be cellularly excreted, as a model of a therapeutic molecule to be built and distributed by the “nurse” cell. Cell transfection efficiency, gene expression, and overall cell health will be monitored by fluorescence and light microscopy at regular time intervals. Success will be evaluated numerically by relative fluorescence intensities expressed fluorescent protein, as well as by the number of fluorescence events per cell versus controls.