Three Steps of Fluorescence Resonance Energy Transfer Observation of Complexation of DNA/Drug/Polymeric Micelles on Cornea Transport

Project: A - Government Institutionb - Ministry of Science and Technology

Project Details

Description

Understanding the process of in vitro / in vivo stability of genes/ or drugs core-loaded with polymeric micelles (PM) and their biodistribution in the cellular/tissue uptake of polymeric micelles is critical for developing effective nano PM delivery systems. Using a dynamic molecular orientations within 10 nm distance between donor and acceptor fluorescence molecules, Forster/fluorescence resonance energy transfer (FRET) efficiencies have been enhanced/improved the visibility of the complexes of signaling proteins in specific cellular environments levels. Under our preliminary cornea application of two fluorescence molecules core-loaded with PM, we found increasing FRET from donor molecules to acceptor fluorescence only observed in two fluorescence molecules core-loaded with PM. In addition, FRET intensity increased steady after 150 min on cornea transport. In order to analyze three microenvironment interactions among multi-layers of tissues with genes/or drugs core-loaded with PM, three steps of FRET methods is proposed. By using our previous specific cornea green fluorescence protein (donor) expression in epithelial or stroma layers after a specific cornea promoters plasmids (pK12-eGFP and pKera3.2-eGFP) delivery, the microenvironment interaction among specific cornea layer, gene and PM should be elucidated by two FRET donor-acceptor pairs. Our major objective of this project is to design a cornea nano-sized particle of labeled DsRed2-PM with labeled rhodamined-DNA plasmids, whose expression are driven by epithelial or stroma-specific promoter, as two FRET donor-acceptor pairs with consequent to achieve a timed cornea tissue distribution for observation of gene with PM transfer pathway. The optimization of physicochemical kinetic formation of PM (size/shape), in vitro and in vivo stability/retention, distribution of tissue/cells pharmacokinetic and function expression properties of anti-apoptotic genes in two different tissue layers will be systematically investigated. In year 1, we will synthesize fluorochrome-labeled polymer (PM), DsRed2-PM and core loaded rhodamine-gene for in vitro FRET observation. A time-resolved fluorescence measurement of one donor-acceptor pair for gene core loaded with PM will be evaluated as well as release/ stability of gene from PM. In year 2, we will focus on the assessment of two steps of FRET on penetration and distribution of anti-apoptotic plasmids delivered with DsRed2-PM. The permeation orientation/distance of rhodamine-genes complexes with DeRed2-PM will be measured by one donor-acceptor pair of FRET. Pre-treated with different permeation inhibitors (caveolae-, clathrin-dependent endocytosis, energy dependence etc.) will be evaluated the influence of penetration distance/distribution pathway between both of labeled-genes and PM. In year 3, we will apply the three steps of FRET occur after an eye drop rhodamine-gene/DsRed2-PM delivery with eGFP protein expression at specific epithelial or stroma target cornea in vivo. The pharmacokinetic biodistribution and functional expression of eGFP, Bcl-xL genes with DsRed2-PM in specific cornea animal model will be examined by multi-modality image, real time confocal microscopy, electron microscopy, western blotting, and immunohistochemistry observations. The transport/permeability rate will be also calculated to evaluate the modulation of barrier rates of plasmid with PM’s permeability.
StatusFinished
Effective start/end date8/1/1412/31/15