AB034. In vivo laser-mediated retinal ganglion cell optoporation using Kv1.1 conjugated gold nanoparticles

Background:There is a current void in efficient,cell-specific,retinal drug delivery systems,thus developing a safe,effective,selective drug delivery system would open novel therapeutic avenues.We previously demonstrated that femtosecond(fs)laser irradiation can transfect DNA plasmids into cultured cells in the presence of gold nanoparticles(AuNPs).These AuNPs locally amplify laser energy at a submicron range creating transient pores allowing exogenous genetic material or cell impermeable dyes to enter the cell.Here,we sought out to selectively optoporate retinal cells in vivo with functionalized AuNPs and a 800 nm femtosecond(fs)laser.Methods:The cell-surface Kv1.1 voltage-gated channel was chosen to selectively target retinal ganglion cells(RGC)in the rat retina.Citrate-capped spherical 100 nm AuNPs functionalized with orthopyridyl-disulfide-poly(ethylene glycol)(5 kDa)-N-hydroxysuccinimide(OPSS-PEG-NHS)conjugated to a Kv1.1 monoclonal antibody were injected intravitreally in Sprague Dawley rats 3 hours prior to irradiation,concomitantly to a FITC-dextran dye to detect optoporation.The eyes of anesthetized rats were placed in the beam path of a laser system consisting of an 800 nm,100 fs laser and a Heidelberg Spectralis HRA ophthalmoscope for fundus visualization.The rat retina was irradiated at powers ranging from 20-750 mW,the eyes fixed in 4%paraformaldehyde,dissected,rinsed,mounted and imaged by confocal microscopy.Results:Our novel laser system coupled to a Heidelberg ophthalmoscope allowed for a clear visualisation of the rat ocular fundus.A timecourse of AuNP intravitreal injections revealed that optimal nanoparticle dispersion on the retinal surface occurred at 3 hours post injection.Following Kv1.1-AuNP and FITC-dextran intravitreal injection and incubation,irradiation at 120-750 mW resulted in FITC uptake by retinal cells.Conclusions:Since living biological tissues absorb energy very weakly at 800 nm,this non-invasive tool may provide a safe,cost effective clinically relevant approach to selectively target retinal cells and limit complications associated with surgical interventions,and potential biological hazards associated with viral-based gene therapy.

Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation

Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications,such as CAR T cell-based therapies.mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral-or DNA transposon-based approaches for the generation of adoptive T cells.However,limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells.In this study,we demonstrate that gold nanoparticle-mediated vapor nanobubble(VNB)photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells.Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration,while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%.Importantly,compared to electroporation,which is the reference technology for nonviral transfection of T cells,a fivefold increase in the number of transfected viable Jurkat T cells was observed.Altogether,our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells,with promising potential for the future engineering of cells in therapeutic and fundamental research applications.

Optical delivery of multiple opsin-encoding genes leads to targeted expression and white-light activation

In photodegenerative diseases such as retinitis pigmentosa(RP)and age-related macular degeneration(AMD),progressive loss of vision occurs as a result of degeneration of the periphery of the retina and the macula,respectively.Current optogenetic stimulation-based approaches to vision restoration offer the advantages of cellular specificity,high resolution,and minimal invasiveness over electrode arrays;however,the clinical translation of optogenetic activation suffers from the lack of a method for the delivery of opsins into spatially targeted regions of a retina that has degenerated.Non-targeted opsin delivery through viral or non-viral methods to non-photodegenerated retinal areas will perturb these already functioning retinal regions.Furthermore,viral methods are subject to limitations on the delivery of large plasmids,such as fusion constructs of multiple spectrally separated opsins(e.g.,channelrhodopsin-2(ChR2),chimeric opsin variants(C1V1),ReaChR),which can provide higher photo-excitability than can a single narrow-band opsin under ambient light conditions.Here,we report the ultrafast near-infrared laser-based spatially targeted transfection of single and multiple opsins and present a comparison with the opsin expression distribution achieved using another non-viral,but non-targeted,transfection method,lipofection.Functional evaluation of cells transfected with multiple opsins using the laser method revealed a significantly higher white-light-induced photocurrent than in cells expressing a single opsin(ChR2).The laser-assisted targeted delivery of multiple opsin-encoding genes to the peripheral retina/macula is ideal for sensitizing retinal areas that have degenerated,thus paving the way toward the restoration of lost vision in RP/AMD patients.