Carboxymethyl Cellulose-coated Tacrolimus Non-spherical Microcrystals For Mouse Dry Eye Treatment

ObjectiveDry eye（DE）is a highly prevalent and multifactorial ocular surface disease which affects the life quality and results in low working efficiency.The management and treatment methods of DE are diverse,while topical administration represents the easiest and least invasive route to deliver drugs to the ocular surface.Because of the low bioavailability of traditional eye drops,the development of a novel formulation containing both anti-inflammatory drugs and artificial tears is expected to improve the efficiency of DE treatment.This study aimed to synthesize carboxymethyl cellulose-coated tacrolimus microcrystals,endowing the complex microcrystals with dual capability of anti-inflammatory and lubricate functions as well as prolonging their retention time on the ocular surface,hence providing a better option for the management of DE.MethodsFirstly,non-spherical tacrolimus microcrystals（TAC MCs）were synthesized by precipitation.Different antisolvent condition were compared for their effects on distribution and morphology of TAC MCs.These TAC MCs were modified by alternate deposition of poly（allylamine hydrochloride）（PAH）and carboxymethyl cellulose（CMC）by layer-by-layer assembly（Lb L）subsequently to obtain CMC-coated TAC MCs（TAC-（PAH/CMC）₃）.The physiochemical properties of TAC MCs and TAC-（PAH/CMC）₃ were characterized by scanning electron microscopy（SEM）,zeta-potential measurements and X-ray diffraction analysis（XRD）.The resultant formulations were evaluated in vivo in a mouse DE model induced by an intelligently controlled environmental system.Compared to the conventional eye drops,all ten experimental and control groups were set.The experiment lasted 28days and evaluation were carried on 0,14th and 28th days.The therapeutic effects were explored in three levels:clinical manifestations,tissues morphological structure and the relative expression of representative inflammatory mediators,Interleukin-1β（IL-1β）and matrix metalloproteinase-9（MMP-9）,in m RNA level.ResultsTAC MCs are hexagonal and lamellar.Using pure water or 1%poly（vinyl alcohol）aqueous solution as antisolvent agent,the distribution of TAC MCs are aggregated or homogeneous.The average length,width and thickness of TAC MCs are 8.90±0.35μm,3.89±0.19μm and 0.48±0.02μm,respectively.The average thickness of TAC-（PAH/CMC）₃ increased to 0.54±0.02μm and the difference is statistically significant.The zeta-potential of TAC MCs charges-18.27mV,and charge reversal happens in the Lb L progress accompanied by PAH and CMC deposition.Before and after crosslinking,the zeta-potential of TAC-（PAH/CMC）₃ are-21.41 mV and-13.16 mV respectively.XRD patterns demonstrated that precipitation method did not alter the native crystallinity of TAC.Compared with commercially available tacrolimus eye drops and the TAC MCs counterpart,TAC-（PAH/CMC）₃ exhibited superior therapeutic performance with reduced drug instillation frequency.After treatment for 14 days,the volume of tear production in the TAC-（PAH/CMC）₃BID and TAC-（PAH/CMC）₃QD groups increased the most（0.52mm）.The corneal fluorescein staining score was lowest in the TAC-（PAH/CMC）₃BID group（1.00±0.33）.The corneal epithelium integrity and microvilli structure in the TAC-（PAH/CMC）₃BID,TAC-（PAH/CMC）₃QD and Talymus^?+Refresh Plus^?groups were as normal as the Normal group,however,the TAC-（PAH/CMC）₃BID and TAC-（PAH/CMC）₃QD groups displayed reduced administration frequency.From the perspective of the protective effect on conjunctival goblet cells,the TAC-（PAH/CMC）₃BID group was superior to the TAC-（PAH/CMC）₃QD group.The TAC-（PAH/CMC）₃BID group decreased the relative m RNA expression of IL-1βand MMP-9,which was consistent with other groups containing tacrolimus.ConclusionIn this study,TAC non-spherical MCs were synthesized using precipitation method,and the novel formulation TAC-（PAH/CMC）₃ was obtained by Lb L.The therapeutic efficiency was found to be TAC-（PAH/CMC）₃>TAC MCs>commercial Talymus^?eye drops.The superior therapeutic performance and reduced instillation frequency of TAC-（PAH/CMC）₃ stem from synergistic effects of the non-spherical geometry,the lubricant and mucoadhesive CMC,as well as the anti-inflammatory TAC.In conclusion,the as-synthesized TAC-（PAH/CMC）₃ non-spherical composite microcrystals is expected to confer improved patient compliance and reduced medical costs,which would open a new avenue for the management of DE.