Studies On The Pharmacotherapy Of Diabetic Keratopathy By Targeting Trigeminal Ganglion

ObjectiveTo describe a novel curcumin containing a nanomicelle formulation using a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol(PVCL-PVA-PEG)graft copolymer,and this nanomicelle curcumin intranasal delivery to correct pathophysiological conditions in TG to promote corneal epithelial/nerve wound healing in streptozotocin-induced diabetic mice,then,to reveal the mechanisms that intranasal administration treatment promotes corneal epithelial/nerve wound healing(from the pharmacokinetic viewpoint)in mice,and verify the existence of the nasal mucosa epithelium-trigeminal ganglion-corneal nerve transporting pathway.Methods1.Nanomicelle curcumin ophthalmic solution was prepared by a solvent evaporation-hydration method and its mean diameter,encapsulation efficiency and stability were detected,respectively.The antioxident activity was perfomed by FRAP.The cytotoxicity and cellular uptake of the nanomicelle were tested by in vitro cellular experiments.The in vivo ocular irritation tests were investigated by using eye comprehensive score.The eye local inflammatory models were set up by instilling arachidonic acid solution in rabbit eyes to evaluate the anti-inflammatory activity of nanomicelle.2.STZ-induced diabetic mice model with corneal epithelium abrasion was established.Ocular topical and/or intranasal nanomicelle curcumin treatments were performed,and treatment efficacy,including corneal epithelial wound healing,corneal nerve rescovery,oxidative stress in corneal epithelium and TG neuron,inflammatory cytokine expression in cornea and TG neuron,expressions of major intracellular free radical scavengers in corneal epithelium and TG neuron,and neurotrophic factor expression in cornea and TG neuron were evaluated.3.Ocular topical or intranasal administration of nanomicelle coumarin-6 was performed in mice,and tissue distribution after administration(0.25,1,2,4,6,8,and 10h)was analyzed.Fluoro-Gold was used as a retrograde tracer to identify corneal and nasal neurons in the trigeminal ganglion(TG).Pharmacokinetic profiles after ocular topical or intranasal administration were explored in detail.Results1.Nanomicelle curcumin ophthalmic solution were successfully prepared by using PVCL-PVA-PEG with the mean diameter and encapsulation efficiency of 50.1 ± 1.0nm and 99.37 ± 0.76%.The remaining drugs of nanomicelle curcumin with 18:1 mass ratio were 98.13 ± 0.97% after 12 weeks.The antioxidant activity of curcumin was improved after the encapsulation of nanomicelle.The in vitro cellular experiments showed that the cell viability were 86.89% as the concentration of 500μg/ml after 48 h cell incubation,and there were no cytotoxicity found after 1h cell incubation with the nanomicelle curcumin’s concentration of 4.5mg/ml.To determine the ocular tolerance in rabbits’ eyes,the values of the clinical scores were 0~2 at different timepoints in all three groups.The curcumin’s concentration of 4.5mg/ml exhibited powerful anti-inflammatory efficiency as the anti-inflammatory activity results displayed.2.Intranasal nanomicelle curcumin treatment promoted corneal epithelial wound healing and recovery of corneal sensation.Enhanced accumulation of reactive oxygen species,reduced free radical scavengers,increased mRNA expressions of inflammatory cytokines,and decreased mRNA expressions of neurotrophic factors in the cornea and TG neuron were observed in diabetic mice with corneal epithelium abrasions.Intranasal nanomicelle curcumin treatment effectively recovered these pathophysiological conditions,especially that of the TG neuron,and an strengthened recovery was observed with ocular topical combined with intranasal treatment.3.The mean diameter and encapsulation efficiency were 51.1 ± 0.9nm and 98.85 ± 0.93%.Coumarin-6 levels in the TG were significantly higher in intranasal administration groups than in topical administration groups,and the difference was statistically significant(P<0.05)at all time points except for 10 h.Interestingly,in cornea,coumarin-6 was detected after intranasal administration,indicating the existence of a nasal mucosa epithelium-trigeminal ganglion-corneal nerve transporting pathway.For intranasal administration groups,it was also interestingly found that coumarin-6 levels in the TG were much higher than that in the brain,suggesting that the TG was a target tissue after the intranasal administration of nanomicelle coumarin-6.These levels also indicated the safety of brain tissue after intranasal administration.Using Fluoro-Gold tract tracing techniques,coumarin-6 was detected in TG neurons after either ocular topical or intranasal administration of nanomicelle coumarin-6,indicating the high colocalization of corneal and nasal neurons in the TG,which verified the existence of a nasal mucosa epithelium-trigeminal ganglion-corneal nerve transporting pathway.ConclusionCurcumin was successfully encapsulated in the PVCL-PVA-PEG nanomicelles using a simple and environmentally friendly process,and this novel nanomicelle formulation is a potential vehicle for curcumin delivery in Ophthalmology;Intranasal nanomicelle curcumin treatment effectively corrected TG neurons,and these helped promote diabetic corneal epithelial/nerve wound healing.This novel treatment might be a promising strengthened therapy for diabetic keratopathy;Intranasal nanomicelles could effectively target TG neurons and transport to cornea through the nasal mucosa epithelium-trigeminal ganglion-corneal nerve transporting pathway.Thus,intranasal administration treatment might be a promising therapy for ocular diseases of corneal nerves,such as diabetic keratopathy,and can potentially enrich the knowledge of pathway targeting.