Eye drop delivery of nano-polymeric micelle formulated genes with cornea-specific promoters.
BACKGROUND
This study evaluates the eye drop delivery of genes with cornea-specific promoters, i.e., keratin 12 (K12) and keratocan (Kera3.2) promoters, by non-ionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM) to mouse and rabbit eyes, and investigates the underlying mechanisms.


METHODS
Three PM-formulated plasmids (pCMV-Lac Z, pK12-Lac Z and pKera3.2-Lac Z) containing the Lac Z gene for beta-galactosidase (beta-Gal) whose expression was driven by the promoter of either the cytomegalovirus early gene, the keratin 12 gene or the keratocan gene, were characterized by critical micelle concentration (CMC), dynamic light scattering (DLS), and atomic force microscopy (AFM). Transgene expression in ocular tissue after gene delivery was analyzed by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) color staining, 1,2-dioxetane beta-Gal enzymatic activity measurement, and real-time polymerase chain reaction (PCR) analysis. The delivery mechanisms of plasmid-PM on mouse and rabbit corneas were evaluated by EDTA and RGD (arginine-glycine-aspartic acid) peptide.


RESULTS
The sizes of the three plasmid-PM complexes were around 150-200 nm with unimodal distribution. Enhanced stability was found for three plasmid-PM formulations after DNase I treatment. After six doses of eye drop delivery of pK12-Lac Z-PM three times a day, beta-Gal activity was significantly increased in both mouse and rabbit corneas. Stroma-specific Lac Z expression was only found in pKera3.2-Lac Z-PM-treated animals with pretreatment by 5 mM EDTA, an opener of junctions. Lac Z gene expression in both pK12-Lac Z-PM and pKera3.2-Lac Z-PM delivery groups was decreased by RGD peptide pretreatment.


CONCLUSIONS
Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may involve endocytosis and particle size dependent paracellular transport.
Tong
Yaw-Chong
YC
College of Pharmacy, Taipei Medical University, Taipei, Taiwan.
Chang
Shwu-Fen
SF
Liu
Chia-Yang
CY
Kao
Winston W-Y
WW
Huang
Chong Heng
CH
Liaw
Jiahorng
J
D000818:Animals; D003315:Cornea / Q000378:metabolism*; D003316:Corneal Diseases / Q000628:therapy; D016503:Drug Delivery Systems / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors; D053551:Keratin-12 / Q000008:administration & dosage / Q000235:genetics; D051379:Mice; D008823:Micelles; D053758:Nanoparticles / Q000737:chemistry / Q000627:therapeutic use*; D009883:Ophthalmic Solutions / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000627:therapeutic use; D011401:Promoter Regions, Genetic; D011409:Propylene Glycols / Q000627:therapeutic use; D011509:Proteoglycans / Q000008:administration & dosage / Q000235:genetics; D011817:Rabbits; D019076:Transgenes
C490948:KERA protein, human; D053551:Keratin-12; D008823:Micelles; D009883:Ophthalmic Solutions; C116176:PEO-PPO-PEO; D011409:Propylene Glycols; D011509:Proteoglycans; D011092:Polyethylene Glycols