Human serum albumin nanoparticles for efficient delivery of Cu, Zn superoxide dismutase gene.
OBJECTIVE
To assess the potential of human serum albumin nanoparticles (HSA NP) as a nonviral vector for ocular delivery of Cu, Zn superoxide dismutase (SOD1) gene.


METHODS
Cu, Zn superoxide dismutase (SOD1) gene-encapsulated nanoparticles (NP) were developed using human serum albumin (HSA), an endogenous protein, by a desolvation-crosslinking method. The pSOD-loaded HSA NP was evaluated for in vitro release characteristics, stability against DNase I and vitreous humor degradation, cytotoxicity, cellular uptake mechanisms, in vitro transfection efficiency, and in vivo gene expression. In vitro studies employed cultured human retinal pigment epithelial (ARPE-19) cells and in vivo studies employed a mouse model. For cell uptake analysis, fluorescein isothiocyanate (FITC)-labeled human serum albumin (HSA) was used.


RESULTS
Plasmid containing SOD1 gene was encapsulated in HSA by a desolvation-crosslinking method. Gene-loaded HSA NP has a mean size of 120 nm, zeta potential of -44 mV, and plasmid encapsulation efficiency of 84%. At high crosslinking degree, HSA NP sustained the in vitro release of plasmid over 6 days, and stabilized plasmid DNA against DNase I and vitreous humor degradation. No cytotoxicity was observed in ARPE 19 cells treated with blank HSA NP at concentrations up to 5 mg/ml for 96 h. Cellular uptake of HSA NP was via receptor-mediated endocytosis that involves primarily caveolae-pathways. Confocal analysis indicated rapid endo/lysosomal escape of HSA NP. Further, confocal studies indicated that HSA readily enters the cell nucleus. In vitro, pSOD-HSA NP resulted in more than 80% transfection efficiency in ARPE-19 cells, which was 5 fold higher than Lipofectamine. HSA NP-transfected cells exhibited enhanced SOD1 activity that was 5 fold higher than untreated cells, indicating the overexpression of the functional gene. Intravitreal injection of HSA NP to the mouse eye at a dose of 130 ng of plasmid produced detectable level of fusion protein expression at 48 h, compared to non-detectable expression in control animals.


CONCLUSIONS
The HSA NP developed in this study offers a very promising approach for nonviral gene delivery to the retina.
Mo
Yun
Y
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198-5840, USA.
Barnett
Micheal E
ME
Takemoto
Dolores
D
Davidson
Harriet
H
Kompella
Uday B
UB
D000818:Animals; D021941:Caveolae / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002477:Cells / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D053758:Nanoparticles; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism; D010957:Plasmids; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D012709:Serum Albumin / Q000737:chemistry* / Q000493:pharmacokinetics; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism*
D002966:Clathrin; D012709:Serum Albumin; D013482:Superoxide Dismutase