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Junko Yoshida, Qingguo Xu, Nick Boylan, Jung Soo Suk, Rhonda Grebe, Walter Stark, Justin Hanes, Gerard Lutty; Transfection of Human Corneas using Non-Viral Gene Carriers. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1412.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the optimal methods for transfection of human corneas using DNA nanoparticles.
We tested three polymer systems including 1) CK30PEG, 2) CH12K18PEG, and 3) PEI-PEG. These polymers were used to compact plasmid DNA encoding either CMV promoted DsRed or human beta-actin promoted mCherry into DNA nanoparticles (NPs).In an initial experiment, we tested 3 different approaches to delivery CK30PEG NPs encoding DsRed to human corneal tissue. The first was cornea with scleral rim with half of the epithelium peeled off. The second was to trephine out a cornea button of 8.5mm diameter. These two corneal preparations were soaked in 1.5ml Optisol containing 50ug of DNA. The third corneal preparation received intra-stromal injection of 50ug of DNA (20ul) before incubating in 1.5ml Optisol. Each cornea was cut into half; one half was incubated for 48 h at 37oC and the other half was incubated for 6 h at 37oC then 42 h at 4oC before fixation. Expression of DsRed protein within the cornea was confirmed by confocal microscopy. Our second experiment included 4 groups; naked mCherry plasmid DNA and mCherry compacted in CK30PEG, CH12K18PEG, and PEI-PEG. We cut human corneas into quadrants and each was used in the four experimental groups. 10 ul of each NPs or naked DNA (25ug of DNA) was injected into stroma and incubated in Optisol for 48 h at 37oC before fixation/cryopreservation. After cryosectioning, sections were observed by confocal microscopy.
In our initial experiment, we observed DsRed expression in ketratocytes only after intra-stromal injection of DNA NPs followed by incubation at 37oC for 48 h. In no other specimens could we detect any DsRed fluorescence. In our second experiment, we observed mCherry expression in keratocytes with all conditions tested. The most intense mCherry fluorescence was observed when the CK30PEG was used for DNA delivery. Overall, the sectors of cornea that were injected with NPs had more intense fluorescence than corneas injected with naked DNA.
Intra-stromal injection of DNA nanoparticles into human cornea resulted in good transfection of keratocytes. Our study indicates that successful DNA transfection of human keratocytes can be achieved by non-viral compacted DNA nanoparticles, which suggests DNA nanoparticles may offer promising therapies for corneal diseases.
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