Drug Loading Performance And Corneal Injury Repair Mechanism Of Bovine Serum Albumin Porous Film

Objective:Corneal chemical burns are common ocular emergencies.If effective treatment is not carried out in time,they may cause corneal damage,such as corneal cell degeneration and corneal neovascularization,which lead to irreversible eye tissue damage and even permanent visual impairment.Eye drops are the most common method for treating corneal chemical burn.However,due to the limitation of the ocular barrier,the retention time of eye drops on the cornea is very short(<5 min),resulting in a low bioavailability of drugs,and the effective absorption of eye drops on the cornea is less than 5%.As an alternative to eye drops,drugs loaded therapeutic corneal contact lenses can adhere to corneal tissue for a long time and release drugs,significantly improving the retention time and bioavailability of drugs,thus it receives an increasing attention.Preparation of eye drug delivery systems with high drug loading,long drug release time and low eye irritation is currently the focus of research on corneal contact lenses.Based on this,in this study,we used bovine serum albumin porous films as the lens substrate for corneal contact lenses to explore its drug loading property,studied the repair effect of drug loaded bovine serum albumin porous films on corneal alkali burn,and evaluated the application potential of bovine serum albumin porous films as the therapeutic corneal contact lens.Method:1.Bovine serum albumin(BSA)molecules self-assembled in a phosphate-ethanol solution(60 vol.%,p H 7.4)at 65℃to obtain a BSA nanofibers dispersion,which formed BSA porous film through a simple reverse dialysis method.2.Soaked in a silver nitrate solution,the BSA porous films adsorbed Ag~+into the structure of BSA porous films through physical adsorption such as electrostatic force,and utilized the reducibility of some functional groups in the BSA structure to reduce Ag~+to Ag nanoparticles,and the Ag nanoparticles loaded BSA/Ag porous films were obtained;Then,hyaluronic acid(HA)molecules are connected to the BSA/Ag porous films through an EDC/NHS assisted chemical crosslinking process to obtain the BSA/Ag/HA porous films.The physical and chemical properties(microstructure,water content,hydrophilicity and transparency),antibacterial properties and biocompatibility(blood compatibility and cytotoxicity)of BSA/Ag/HA porous films were studied;Established an animal model of corneal alkali burn in mice to explore the potential therapeutic effect of BSA/Ag/HA porous films on corneal alkali burn in mice;Mouse corneal was stained with fluorescein sodium to observe the repair status of mouse corneal epithelium;Evaluated corneal opacity;Measured corneal sensitivity by using a corneal sensitivity meter;The growth of corneal neovascularization in mice was observed by using FITC-glucan perfusion method;Determined the content of VEGF and IL-1βin mouse corneal by ELISA.The pathological changes of the mouse corneal were detected by staining the mouse corneal tissue;The main organs of mice were sectioned and stained to confirm the biological safety of the porous film.3.The flavonoid anti-inflammatory drug kaempferol(KAE)and BSA were co dissolved in a phosphate-ethanol solution(60 vol.%,p H 7.4)by using a"one pot"method.Through BSA fibrosis self-assembly induced by ethanol and reverse dialysis,we obtained the KAE loaded BSA/KAE porous films.Studied the physical and chemical properties and in vivo/in vitro biocompatibility of BSA/KAE porous films;Established a mouse corneal alkali burn model to explore the repair effect and mechanism of BSA/KAE porous films on mouse corneal alkali burns and to evaluate the application potential of BSA/KAE porous films as the therapeutic corneal contact lenses.Result:1.The microstructure of BSA nanofibers is wormlike nanofibers with a diameter of approximately 10.0 nm,and the microstructure of BSA porous films assembled from nanofibers is a three-dimensional porous structure.2.The BSA/Ag/HA porous film had a high water content(>84.9%),good hydrophilicity(water contact angle<73.0°),high transparency(the transmittance of BSA/Ag/HA porous films with different HA content at 600 nm>76.0%)and antibacterial properties,as well as good blood compatibility(hemolysis rate<5.0%)and cell compatibility(cell viability>110.3%after 72 h co culture).The experimental results of mouse corneal alkali burn animal models showed that BSA/Ag/HA porous films could accelerate corneal healing by promoting corneal epithelial regeneration,lessening corneal opacity and neovascularization,as well as reducing inflammatory reactions.Ag nanoparticles endowed BSA porous films with antibacterial properties.HA accelerated endogenous repair by regulating cell function.BSA porous films prolonged the retention time of Ag nanoparticles and HA on the corneal surface,and the three worked together to promote corneal repair.3.In order to avoid the potential biological toxicity of Ag nanoparticles(e.g.,the accumulation of metal ions led to biological toxicity),the loading of effective components of traditional Chinese medicine on BSA porous films was studied.BSA/KAE porous films were prepared by using kaempferol as a model drug.The results of in vitro experiments showed that the drug loading capacity of BSA/KAE porous films improved with the increase of the initial amount of KAE,and the drug loading capacity reached 83.3 mg/g when the amount of KAE was 250.0 mg;BSA/KAE porous films had a high transparency(transmittance of 17.0μm BSA/KAE porous films at 600 nm>87.1%)and high water content(>91.4%),high blood compatibility(hemolysis<5.0%),good cell compatibility(cell viability>94.9%after 72h co culture),and high antioxidant capacity.KAE was continuously released from BSA/KAE porous films(release time>340 h),which greatly extended the retention time of KAE on the corneal.The experimental results of a mouse corneal alkali burn animal model showed that BSA/KAE porous films retained the anti-inflammatory and antioxidant properties of KAE,and could accelerate corneal repair by promoting corneal re epithelialization,reducing corneal turbidity,inhibiting neovascularization,and reducing inflammation.BSA/KAE porous films extended the retention time of KAE on the corneal surface,which improved the bioavailability of KAE,so then promoted corneal repair.Conclusion:BSA porous films can prolong the retention time of drugs such as HA and KAE on the corneal while maintain the nature and structure of drugs,thereby drugs can accelerate the repair of alkali burned corneas.The preparation method of BSA porous films is simple,chemical modification methods of BSA porous films are diverse,and they are no biologically toxic.It is expected to use BSA porous films as a substrate material for new therapeutic corneal contact lenses.