Preparation And Evaluation Of Baicalin Nanocrystal In Situ Gel For Ophthalmic Use

Objective To improve the solubility and dissolution rate of insoluble baicalin and toprolong the retention time of pharmaceuticals with better treatment effect after ophthalmicadministration, baicalin nanocrystal-loaded in situ eyegel was prepared and evaluated by invitro and in vivo methods. The present study aims to ensure the quality controllability,stability, safty and effectiveness of the obtained preparation, and also lay a solid foundationfor research and development of new products.Method1Optimazation of formulations for in situ eyegel. Baicalin nanocrystal suspensions wereobtained by co-precipitation technique and nano-emulsion of Wolfberry seed oil by ultrasonictechnique. The solutions of gelling materials were prepared by cold method or hot method.Tgelwas measured by “magnetic bar method”, and viscosity values measured by viscometer.Using Tgel, flowability, viscosity and gel ability as index, the major gelling materials werescreened for temperature-sensitive, pH-sensitive and ion-sensitive in situ eyegel formulationsrespectively. The influence of adhesive materials, other additives, drugs and their carriers ongelling process of all3types of in situ eyegels were tested by rheometer, and finally the bestformulation for ophthalmic administration was optimized.2Preparation and evaluation of baicalin nanocrystal-loaded in situ eyegel. The baicalinnanocrystal-loaded in situ eyegel was prepared, appearance and microstructure of the gel wasobserved. Its water-holding capacity and gel strength were tested. The rheological propertiesof the samples under non-physiological or physiological conditions were measured by arheometer. The methodology of HPLC analysis of baicalin was validated for baicalin assay and in vitro dissolution test by dialysis bag method. Through franz diffusion cell methodtested the corneal permeability of the preparations. The stimulation of eyegel to rabbit eyeswas evaluated by single and multiple dosing experiments, and the retention time of eyegel inrabbit eyes by photographing method under UV light. The experimental uveitis in rats wereestablished and used to evaluated the effectiveness of the eyegel in vivo. Further more, thepreliminary stability of eyegel was investigated under4℃and room temperature.Result1The obtained baicalin nanocrystals showed as irregular granules with a mean particlessize of258.6±21nm(PI=0.153±0.014) and of500.8±34nm(PI=0.391±0.018) aftercentrifugation. And nano-emulsion of Wolfberry seed oil has a narrow particle size of190±22nm (PI=0.147±0.013) with opalescent appearance.The results for formulation screen of temperature-sensitive in situ eyegel were as follows:20%P407and0.5%P188as the major gelling material, the addition of adhesive material ofMC or chitosan increased the Tgelof the system while HPMC or sodium alginate no suchinfluence. Tgeldecreased but viscosity values increased after sodium chloride or mannitoladded. Tgelincreased but viscosity values has no change after LBP added. Both Tgelandviscosity values increased after baicalin nanocrystal or nano-emulsion of Wolfberry seed oiladded. The addition of other additives showed no remarkable influence on Tgeland viscosity.The results for formulation screen of pH-sensitive in situ eyegel were as follows:0.4%carbomer as the major gelling material,0.2%HPMC as adhesive additive, the addition ofsodium chloride, baicalin nanocrystal, LBP or nano-emulsion of Wolfberry seed oil caused nogelation of the system under physiological conditions. The addition of LBP or nano-emulsionof Wolfberry seed oil slightly increased the viscosity of the system.The results for formulation screen of ion-sensitive in situ eyegel were as follows:0.5%gellan gum as the major gelling material,0.2%sodium alginate as adhesive additive, theaddition of mannitol, benzalkonium, LBP, sophoridine or baicalin nanocrystal showed no remarkable influence on viscosity of the system, the addition of sodium chloride ornano-emulsion of Wolfberry seed oil has an increasing effect on viscosity of the system.The optimized formulation was turned out to be ion-sensitive in situ gel as follows:0.5%gellan gum as the major gelling material,0.2%sodium alginate as adhesive additive,5%mannitol as osmotic agent and0.02%benzalkonium as bacteriostatic agent, and1%baicalinnanocrystal as the drug.2The eyegel without baicalin was transparent and semisolid gel after mixed with artificialtear solution, slightly yellow solution was obtained with the addition of baicalin nanocrystaland turned into gel after mixed with artificial tear solution. The eyegel showed as packed andtwisted bulk under TEM. After gelling under physiological conditions, the obtained gel had awater-holding capacity of over85%after centrifuagation for30min, with a solving time of52±2.1min. The rheological test indicated that shear stress-shear rate curve was linear undernonphysiological conditions, and the viscosity values became diluted with shear rateincreasing under physiological conditions.HPLC detection wavelength was280nm for baicalin assay. The negative sample showedno interference. The precision, reproducibility, stability and recovery of methodology were allmeet the requirements.The in vitro profile showed a sustained-release characteristic with a completely drugrelease after10h. Nanocrystal in situ gel corneal transmittance was higher than baicalinsolution. During retention time experiment, no fluorescence were tested at22min for eyedropgroup and yellow fluorescence can stilled be investigated at35min for eyegel group.No irritation was investigated after single or multiple dosing and the pathological sectionof cornea after multiple dosing showed no damage. The protein concentration in aqueoushumor at4h and8h for eyegel group were both lower than eyedrop group.The particle size of nanocystals showed no remarkable change with slightly fluctuation ofPI values during storage under4℃and room temperature. Conclusion The optimized formulation of baicalin nanocrystal-loaded in-situ eyegel can begelled rapidly under physiological conditions and showed a prolonged retention time onocular surface and sustained release profile. The preparation has acceptable preliminarystability and showed no irritation after ophthalmic administration, with betteranti-inflammatory effect compared with general eyedrops.