Study On Characteristics Of Enhanced PLGA Nanoparticles Uptake In Corneal Epithelial Cells

Age-related macular degeneration,diabetic macular edema,cataract,glaucoma and uveitis are the main causes of visual impairment.The presence of unique anatomical and physiological barriers of the eye,such as the tear barrier,corneal barrier,scleral barrier,blood-aqueous humor,and blood-retinal barrier,offers many challenges to developing effective retinal drug delivery systems.Polymer nanoparticles as ocular drug delivery carriers have the advantages of improving drug solubility,increasing corneal permeability,reducing adverse reactions,controllable morphology and surface modification.By coating hydrophilic substances on the outer layer of the polymer nanoparticles,a speed-limiting barrier can be created for drug molecules to release from the polymer matrix,regulate hydration and control drug release,prolong drug retention time in the tear film,thereby prolonging the release of the drug.In this study,PLGA nanoparticles were modified with oligosaccharides,and the physicochemical properties and drug release in vitro were investigated.The aqueous humor pharmacokinetics were investigated by microdialysis.The transport mechanism of nanoparticles in corneal epithelial cells was studied.The main contents and results are as follows:1.Triamcinolone acetonide PLGA nanoparticles were prepared by a modified emulsification-solvent evaporation method.The average particles sizes of PLGA nanoparticles,2-HP-β-CD/PLGA nanoparticles,trehalose/PLGA nanoparticles and chitosan oligosaccharide/PLGA nanoparticles were（170.8±71.0）nm,（149.4±52.4）nm,（135.6±52.5）nm,（160.7±66.5）nm,respectively.The particle sizes were mainly distributed within 200nm,reaching the maximum size requirements of delivering to the posterior segment of the eye.PDI values were all within 0.2,indicating that the nanoscale system was stable.The zeta potential were-（18.90±2.76）mV,-（12.42±4.51）mV,-（15.28±3.05）mV and-（9.90±3.96）mV,respectively.TEM results showed that the particles were evenly dispersed with uniform size.The results of surface tension were all meet the requirement of ocular formulation.The results of differential scanning calorimetry（DSC）,infrared spectroscopy and X-ray diffraction showed that triamcinolone acetonide were interacted with nanoparticles and existed in amorphous state.2.In the evaluation of corneal permeability in vitro,the cumulative penetration of 2-HP-β-CD/PLGA nanoparticles for 8 hours reached 38.44±5.77μg?（88）^-2,which was significantly higher（P<0.01）than that of PLGA nanoparticles 7.98±3.24μg?（88）^-2,drug solution 4.81±0.58μg?（88）^-2,trehalose/PLGA nanoparticles 8.91±2.00μg?（88）^-2 and chitosan oligosaccharide/PLGA nanoparticles 9.40±1.66μg?（88）^-2.The value of₍（6）of the 2-HP-β-CD/PLGA nanoparticles was higher（P<0.01）,by 5.08folds,4.07 folds,3.89 folds and 8.24 folds,respectively,compared with the₍（6）of the PLGA nanoparticles,trehalose/PLGA nanoparticles,chitosan oligosaccharide/PLGA nanoparticles and drug solution.From the results above,2-HP-β-CD/PLGA nanoparticles have better corneal permeability,and drug can quickly distributed from nanoparticles to the corneal epithelium and form a drug reservoir,thus maintaining the driving force of drug diffusion into aqueous humor and increasing the drug concentration across cornea,thereby improving drug bioavailability.Dialysis bag method were used to predict the release behavior of nanoparticles in vivo.The results showed that the 12h cumulative release of 2-HP-β-CD/PLGA nanoparticles reached95.64%,which was higher,by 1.69 folds,4.07 folds,1.89 folds and 2.0 folds,respectively,compared with the₍（6）for the PLGA nanoparticles,trehalose/PLGA nanoparticles and chitosan oligosaccharide/PLGA nanoparticles,with statistical difference（P<0.05）.3.The pharmacokinetic parameters of PLGA nanoparticles,2-HP-β-CD/PLGA nanoparticles and drug solution in rabbit aqueous humor showed that the peak concentration of 2-HP-β-CD/PLGA nanoparticles was 23.2 and 36.8times higher than that of PLGA nanoparticles and drug solution,respectively（P<0.01）.The AUC of 2-HP-β-CD/PLGA nanoparticles and PLGA nanoparticles in aqueous humor were 44.93 times and 2.20 times higher than that of drug solution group（P<0.01）.The results showed that2-HP-β-CD/PLGA nanoparticles help to form a drug reservoir in front of the cornea,and uneasy to be washed away by tears,which could sustain the drug release for a period of time and maintain effective drug concentration in aqueous humor.4.The cytotoxicity and transport mechanism of nanoparticles were studied by immortalized human corneal epithelial cells（HCE-T）.CCK8assay showed that PLGA nanoparticles and 2-HP-β-CD/PLGA nanoparticles used on HCE-T cells did not show obvious toxic effects.The uptake results showed that the uptake of nanoparticles increased with uptake time within 4h,and the uptake of 2-HP-β-CD/PLGA nanoparticles by HCE-T was significantly higher than PLGA nanoparticles,that is 2-HP-β-CD/PLGA nanoparticles were easier to penetrate HCE-T cells.The uptake mechanism of nanoparticles showed that 2-HP-β-CD/PLGA nanoparticles uptake by HCE-T cells were mainly through active transport,which was mainly transported into cells through micropinocytosis,this may be due to the fact that the size of nanoparticles larger than 150nm triggered the endocytosis transport process mediated by micropinocytosis.At the same time,endocytosis mediated by clathrin,caveolin/lipid rafts structure also affected the uptake of nanoparticles.Only 24.0%of PLGA nanoparticles were transported into HCE-T cells via active transport,which was significantly lower than2-HP-β-CD/PLGA nanoparticles,indicating that PLGA nanoparticles may enter HCE-T cells mainly through passive transport.In this study,the physicochemical characterization,cornea penetration,drug release mechanism,rabbit aqueous humor pharmacokinetics and cellular transport mechanism of nanoparticles were studied.The results indicated that2-HP-β-CD/PLGA nanoparticles can further enhance aqueous solubility of a water-insoluble drug without affecting the lipophilicity of the drug molecule.2-HP-β-CD act as drug carriers by keeping the drug molecules in solution and delivering them to the surface of the eye where they partition into the eye,improving the drug concentration on the surface of the eyeball so as to improve drug bioavailability.