Design And Evaluation Of Loaded Nepafenac Nanostructured Lipid Carriers Into Dual Sensitive Hydrogel For Ocular Drug Delivery

The eye is a relatively independent organ and there are many protection mechanisms.Traditional eye drops account for about 90%of ophthalmic medications for front-of-the-eye diseases but the corneal bioavailability of drugs delivered via eye drops is less than 10%,much being absorbed systemically via conjunctiva and nasolacrimal ducts.These protection mechanisms bring a lot of difficulties for the treatment of eye diseases.Based on the characteristic of eyes,we develop a novel drug delivery system by loading nanostructured lipid carrier incorporating nepafenac into the hydrogel.This drug delivery system can increase the permeation of ocular drugs through the different ocular structures and the precorneal residence time of the formulation.In this work,a stimuli-responsive three-dimensional cross-linked hydrogel system containing carboxymethyl chitosan(CMC)and poloxamer composed of a poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide)(PEO-PPO-PEO)block copolymer was constructed,and its aqueous solution was found to undergo a reversible sol-gel transition upon a temperature and/or pH change at a very low concentration.The hydrogel was synthesized via a cross-linking reaction using glutaraldehyde(GA)as the cross-linking agent.The structure of the hydrogel was characterized by fourier-transform infrared spectroscopy(FTIR),X-ray diffraction(XRD),nuclear magnetic resonance(NMR)and scanning electron microscope(SEM)studies and the swelling behaviour was studied in different buffered solutions.The results obtained indicated that cross-linked F127-CMC underwent discontinuous phase transition in different temperature and pH solutions.The hydrogel at 35℃ and pH 7.4 had largest swelling ratio.In addition,the cytotoxicity and in vitro release was studied at different pH values and temperature.The results of a CCK-8(Cell Counting Kit-8)assay showed that the hydrogel and its physical mixture solution were not cytotoxic to human corneal epithelial cells at a low concentration.A sustained release of NP from the hydrogel can be obtained and the release rate was found to be maximum at 35℃ and pH 7.4.Nepafenac(NP)was chosen as a model molecule and its properties were studied,such as solubility and physicochemical property.A HPLC analytical method was developed.The nepafenac incorporated nanostructured lipid carrier(NP-NLC)was developed using meltemulsification method,by using Glyceryl Behenate and Miglyol 812 as the oil phase,and soybean lecithin and Cremophor EL as the emulsifiers.Based on the results of single factor investigation,a central composite design plus response surface method was applied to optimize the formulation of NP-NLC.The effects of nepafenac concentration,liquid lipid concentration,and Cremophor EL concentration on the particle size,particle distribution and encapsulation efficiency(EE)were investigated and the morphology of NP-NLC,the crystallinity of nepafenac in NLC,and stability were also evaluated.The results showed that the optimized formulation of NP-NLC is as follows:nepafenac 10 mg;Compritol 888 ATO 110 mg;Miglyol 812N 90 mg;soy lecithin 113 mg;Cremophor EL 187 mg.The average particle size was 92.4 nm,distribution coefficient is 0.25 and the EE of 78.0%.Drugs are amorphous in lipids.Stability test results show that the NP-NLC was stable for around 72 hours in 25℃.In vitro release results show that the release of NP-NLC is 12 hours,with a bi-phase release characteristic.Nepafenac was loaded into the hydrogel sample using the swelling-loading technique.New Zealand albino rabbits were chosen as a model animal.The enhanced transcorneal penetration of the NP-NLC-Gel was evaluated using isolated rabbit corneas,with a significant increase of 1.9-fold in the apparent permeability coefficient compared to that of the NP eye drops(p<0.05).Precorneal retention assessed by fluorescence imaging technology in vivo showed that NP-NLC-Gel had a longer retention time(30 min)on the corneal surface compared with the nepafenac eye drops(5 min).The results might suggest that the hydrogel has good adhesiveness and could prolong the retention time.The human corneal epithelial cells(HCEC)were used for the cellular study of NP-NLCGel.The results of CCK-8 cytotoxicity studies also suggested that the blank NLC,blank NLCGel,NP-NLC and NP-NLC-Gel were biocompatible with no significant cytotoxicity observed in the human corneal epithelial cells(HCECs).The fluorescent Rhodamine B was incorporated into NLC to mimic the cellular behavior of NP-NLC.The results showed that the NLC can be effectively absorbed by corneal epithelial cells,mainly by clathrin-mediated endocytosis.Meanwhile,the cell uptake was dependent on energy.The in vivo ocular tolerance study indicated that there was no difference in irritation between the nepafenac eye drops and NPNLC-Gel.The concentration of nepafenac in the aqueous humor and lacrimal fluid was determined using HPLC.Lacrimal fluid results showed that the area under the curve(AUC0-∞)and average residence time(MRT0-∞)of NP-NLC-Gel were significantly higher than the group of eye drops,showing a 2.2-fold and 2.0-fold increase,respectively.Aqueous humor results showed that the AUC0-∞ and Cmax of NP-NLC-Gel were significantly higher than the group of eye drops,showing a 6.9-fold and 11.4-fold increase,respectively.Based on the results above,the NP-NLC-Gel drug delivery system can prolong the precorneal retention time and enhance the cornea permeation rate.The novel formulation demonstrates the high potential for ophthalmic drug delivery to overcome constrains of the conventional eye drops.