Low molecular weight oligochitosans for non-viral retinal gene therapy.
Ultrapure oligochitosans have recently been evaluated as a promising tool for corneal gene therapy; however, there are no reports regarding the potential use of this polymer in other ocular tissues. We have prepared and characterized at pH 7.1 oligochitosan/pCMS-EGFP polyplexes to evaluate the transfection efficiency in rat retinas after subretinal and intravitreal administration. Polyplexes were characterized in terms of shape, size, surface charge, DNA condensation, and transfection efficiency in HEK-293 and ARPE-19 culture cells. Polyplexes were positively charged, around 10 mV, and size oscillated between 256.5 ± 56 and 67.3 ± 0.44 nm, depending on the nitrogenous/phosphate ratio. Polyplexes efficiently protected the plasmid against enzymatic digestion. A drastic increase in transfection efficiency was observed when pH slightly decreased from 7.4 to 7.1 in both HEK-293 (from 19.1% to 51.5%) and ARPE-19 (from 2.0% to 36.5%) cells (data normalized to Lipofectamine™ 2000). In rat retinas, subretinal administrations transfected cells mainly in the RPE layer, whereas intravitreal injections transfected cells in the inner nuclear and plexiform layers of the retina and mainly in the ganglion cell layer. This study establishes the base for future treatments of genetic retinal disorders with low molecular weight oligochitosan polyplexes.
NanoBioCel Group, University of Basque Country, Vitoria, Spain; Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain.
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Puras
NanoBioCel Group, University of Basque Country, Vitoria, Spain; Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain.
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Zarate
NanoBioCel Group, University of Basque Country, Vitoria, Spain; Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain.
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Aceves
NanoBioCel Group, University of Basque Country, Vitoria, Spain; Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain.
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Murua
Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain; Neuroprothesis and Neuroengineering Research Group, Miguel Hernández University, Spain.
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Díaz
Laboratory of Experimental Ophthalmology, Faculty of Medicine, University of Murcia, Regional Campus of International Excellence "Campus Mare Nostrum", Murcia, Spain.
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Avilés-Triguero
Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain; Neuroprothesis and Neuroengineering Research Group, Miguel Hernández University, Spain.
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Fernández
NanoBioCel Group, University of Basque Country, Vitoria, Spain; Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain. Electronic address: joseluis.pedraz@ehu.es.
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Pedraz
D000818:Animals; D002460:Cell Line; D002686:Chitin / Q000008:administration & dosage / Q000031:analogs & derivatives*; D048271:Chitosan; D004247:DNA / Q000008:administration & dosage*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008970:Molecular Weight; D009844:Oligosaccharides; D010316:Particle Size; D011108:Polymers / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D009844:Oligosaccharides; D011108:Polymers; C493484:oligochitosan; D002686:Chitin; D004247:DNA; D048271:Chitosan