Enhanced oligonucleotide delivery to mouse retinal cells usingiontophoresis

[摘要] Purpose: To study the combination of oligodeoxynucleotides (ODNs)intravitreous injection and saline transpalpebral iontophoresis on thedelivery of ODNs to photoreceptors in the newborn rd1/rd1 mice.Methods: Cathodal or anodal transpalpebral iontophoresis (1.43mA/cm2 for 5 min) was applied to eyes of postnatal day 7 (PN7)rd1/rd1 mice immediately before the intravitreous injection of ODNs.The effect of cathodal iontophoresis after ODNs injection was alsoevaluated. The influence of current intensity (0.5, 1.5, and 2.5 mA) wasassayed with cathodal iontophoresis performed prior to ODNs injection.The duration of current-induced facilitation of ODNs delivery tophotoreceptors was evaluated for 6 h following iontophoresis. One groupof control eyes received cathodal iontophoresis prior to theintravitreous injection of phosphate buffered saline (PBS) orhexachlorofluorescein (Hex). The second control group received ODN orHex intravitreous injection without iontophoresis. The penetration offluorescent ODNs in the outer nuclear layer (ONL) was quantified byimage analysis of the ONL fluorescence intensity on cryosectionmicrophotographs. Integrity of ODN was assessed using acrylamide gelmigration after its extraction from the retina of treated mice. Theintegrity of retinal structure, 1 and 24 h after iontophoresis, wasanalyzed using light and electron microscopy.Results: Transpalpebral anodal or cathodal saline iontophoresisenhanced the penetration of ODNs in all retinal layers. Cathodaliontophoresis was more efficient than anodal iontophoresis in enhancingthe tissue penetration of the injected ODN. Photoreceptor delivery ofODN was significantly higher when cathodal saline transpalpebraliontophoresis was applied prior than after the injection. The extent ofenhanced tissue penetration decreased in parallel to the increasedinterval between iontophoresis application and the intravitreousinjection. Current of 1.5 mA was safe and optimal for the delivery ofODNs to the ONL. One hour after iontophoresis followed by injection, ODNextracted from the retina of treated eyes remained intact. Histology andelectron microscopy observations demonstrated that iontophoresis usingthe optimal parameters did not induce any permanent tissue alterationsor structure damage.Conclusions: Saline transpalpebral iontophoresis facilitates thepenetration of injected ODNs in photoreceptors for at least 3 h. Thismethod may be considered for photoreceptor targeted gene therapy.

[学科分类] 生物化学/生物物理