Nanoparticles for gene delivery to retinal pigment epithelial cells.
OBJECTIVE
To evaluate the safety and potential use of poly(lactic) acid (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as vectors for gene transfer to RPE cells.


METHODS
Experiments were conducted with primary bovine RPE cells and with the ARPE-19 human RPE cell line. Rhodamine loaded NPs were used to study factors influencing the internalization process by the various RPE cells: concentrations of NPs, duration of contact time, stage of cell culture and ambient temperature. The extent of NPs internalization was evaluated by fluorescence and phase microscopy. Potential NP toxicity was measured by the trypan blue exclusion dye test and the MTT method. Green fluorescent protein (GFP) plasmid or red nuclear fluorescent protein (RNFP) plasmid were sequestered in NPs. The ability ot these "loaded" NPs to generate gene transfection and protein expression in RPE cells was assessed both in vivo and in vitro by fluorescence and confocal microscopy.


RESULTS
The extent of NP internalization in cultured cells increases with their concentration reaching a plateau at 1 mg/ml and a contact time of up to 6 h. Temperature and culture stage did not influence the in vitro internalization process. No toxic effects on RPE cells could be detected when these were incubated with up to 4 mg/ml of NPs. In human and bovine RPE cells incubated with GFP loaded NPs, cytoplasmic green fluorescence was observed in 14+/-1.65% of the cultured cells. Incubation with RNFP loaded NPs yielded a nuclear red fluorescence in 18.9+/-1.6% of the cells. These percentage levels of expression initially detected after 48 h of incubation remained unchanged during the following 8 additional days in culture. No significant differences in the extent of cytoplasm or nuclear fluorescence expression were observed between bovine or human RPE cultured cells. In vivo, a preferential RNFP expression within the RPE cell layer was detected after intra vitreous injection of RNFP plasmid loaded NPs.


CONCLUSIONS
The ability of PLGA NPs to sequester plasmids, their nontoxic characteristics, and rapid internalization enables gene transfer and expression in RPE cells. These findings may be of potential use when designing future gene therapy strategies for ocular diseases of the posterior segment.
Bejjani
Riad Antoine
RA
French National Institute of Health and Medical Research, Paris, France.
BenEzra
David
D
Cohen
Hagit
H
Rieger
Jutta
J
Andrieu
Charlotte
C
Jeanny
Jean-Claude
JC
Gollomb
Gershon
G
Behar-Cohen
Francine F
FF
D000818:Animals; D002417:Cattle; D002452:Cell Count; D002460:Cell Line; D002470:Cell Survival; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008863:Microspheres; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids; D011098:Polyglactin 910; D012235:Rhodamines / Q000378:metabolism; D014162:Transfection; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011098:Polyglactin 910; D012235:Rhodamines; D000097573:Red Fluorescent Protein; C026188:rhodamine 6G