Preparation,Pharmacodynamics And Safety Evaluaton Of Triamcinolone Acetonide Chitosan Liposomes Eye Drops

As a leading cause of vision impairment of the retina,macular edema(ME)has one of the highest clinical demands for treatment.However,the treatment of macular edema mainly relies on invasive treatment methods such as grating laser photocoagulation,intravitreal injection,and retrobulbar/peribulbar injection.These treatments are not only accompanied by complications such as postoperative blind spots,intravitreal hemorrhage,endophthalmitis,but also have low patient compliance.Therefore,there is an urgent problem to be solved is looking for a safe and effective treatment for macular edema.Eye drop is an ideal and optional route of administration,which is simple,easy to store and has few side effects.However,the bioavailability of traditional eye drops is low,only 1-10%or less of the drugs reach the anterior segment of the eye,and no or less of them can be delivered to the posterior segment of the eye due to eye clearance mechanisms such as tear flushing and rapid drainage of nasolacrimal duct,as well as the presence of various ocular barriers.In recent years,the application of nano-delivery systems such as liposomes,nanoparticles and nano-emulsions in ophthalmology has attracted wide attention,and has become the hope of promoting the effective delivery of drugs to the posterior segment of the eye.Previous studies have confirmed that liposome is more suitable for ophthalmology because of its good corneal penetration,strong biocompatibility,non-toxicity,sustained release and long half-life.However,due to the low encapsulation efficiency and low bioavailability of common liposomes,it is difficult to deliver the effective drug concentration to the posterior segment of the eye.Therefore,this study seeks to find a new preparation process and formulation to overcome the problems of low encapsulation efficiency and low bioavailability of common liposomes.Calcium acetate gradient method,as one of the active drug loading methods,generates calcium acetate concentration gradient through the transmembrane movement of calcium acetate(the internal concentration is higher than the external concentration),so that a large number of protons are transported from the inside of the liposome to the outside,forming a pH gradient,which makes the weak acidic drugs in the aqueous phase of the liposomes spontaneously aggregate into the inside of the liposomes,thus greatly improving the encapsulation efficiency of the drugs.Therefore,this experiment attempts to use calcium acetate gradient method to prepare liposomes in order to overcome the low encapsulation rate of common liposomes.Chitosan(CH),as a natural biological material with cationic properties,has unique ocular surface adhesion and permeability enhancement,and has been increasingly applied in the field of ophthalmology.Studies have shown that the positive charge on the surface of chitosan combines with the negative charge in the ocular surface mucin layer to enhance the intermolecular force.Therefore,chitosan was chosen as surface modification in this experiment to increase corneal permeability and ocular adhesion,prolong drug residence time on the ocular surface,and improve drug bioavailability.Triamcinolone acetonide(TA),as a long-acting glucocorticoid drug,is widely used in ophthalmology.It has the characteristics of anti-inflammation,anti-angiogenesis,anti-apoptosis and neuroprotection,can also alleviate the immune response of cells,stabilize the aqueous humor and blood-retinal barrier,prevent the metaplasia of fibroblasts and inhibit the proliferation of epithelial cells.A large number of studies have shown that TA can improve vision and reduce inflammation by treating diabetic retinopathy,diffuse macular edema caused by retinal vein occlusion,macular edema after cataract surgery and macular edema caused by different types of non-infectious uveitis.However,due to its strong hydrophobicity and low solubility,triamcinolone acetonide has poor oral absorption and low bioavailability.Therefore,the clinical application of triamcinolone acetonide can only be achieved by intravitreal injection and retrobulbar/peribulbar injection,which greatly limits its application in ophthalmology.The aim of this study is to establish and evaluate a novel and efficient delivery system for the posterior segment of the eye,which can deliver drugs to the posterior segment of the eye by eye-dropping and play a role.Therefore,triamcinolone acetonide chitosan-coated liposomes(TA-CHL)eye drops were successfully prepared by using triamcinolone acetonide as model drug,liposome as carrier and chitosan as surface modification.The physical and chemical properties of TA-CHL were investigated by electron microscopy,particle size distribution,surface potential,entrapment efficiency,differential scanning calorimetry,in vitro release and stability.Optical coherence tomography system(OCT),the uptake experiment of corneal epithelial cells(HCEC)and retinal pigment epithelial cells(ARPE-19)and the establishment of diabetic retinopathy rat model were further explored the pharmacodynamic point of formulation.The safety of eyes after topical administration TA-CHL was preliminarily evaluated by HE staining and TUNEL.In this study,the encapsulation efficiency of liposomes was used as the main evaluation index,and the effects of different preparation techniques on the encapsulation efficiency of triamcinolone acetonide were investigated.The preparation methods,selection of organic solvents,incubation temperature,incubation time,probe ultrasound power,calcium acetate concentration,cholesterol-lecithin ratio,drug-lipid ratio and other factors were investigated.The orthogonal design was used to screen the encapsulation efficiency of triamcinolone acetonide.The optimal prescription and preparation process were as follows:calcium acetate gradient method,calcium acetate concentration 120 mmol/1,cholesterol:lecithin ratio 1:8,drug-lipid ratio 1:15,using anhydrous ethanol as organic solvent,incubation temperature 55 ?,incubation 30 min,ultrasonic power 90 w,ultrasonic time 8 min.The TA-CHL preparation prepared under the above optimized conditions is a white colloidal solution with milky light.Under the electron microscope,it presented round cystic particles of uniform size with a rough shell surrounding the vesicle structure,which were well dispersed without adhesion.The particle size was 135.46 ± 4.49 nm,the Zeta potential was 17.98 ±3.21 mv,and the encapsulation rate was 90.66 ± 3.21%.Differential scanning calorimetry results suggest triamcinolone acetonide at 328.27 ? absorption peak disappeared in TA-CHL,that TA was successfully encapsulated in TA-CHL,no longer exist in crystal structure;In vitro release experiments indicated that TA release from TA-CHL was persistent and slow,and about 80%TA release from TA-CHL took 24 h,which had a significant sustained release effect.Stability experiments showed that TA-CHL was stable for 30 days and 60 days at 4 ?.In order to evaluate the delivery efficiency of CHL as a drug carrier to the anterior and posterior segments of eyes from pharmacodynamic point of view,fluorescent coumarin-6(C6-liposomes)and coumarin-6 chitosan-coated liposomes(C6-CHL)were prepared.OCT was used to dynamically observe the distribution of drugs in the anterior and posterior segments of BN rats and relative intensity of fluorescence was compared at different time points.The results showed that the relative fluorescence density of chitosan-modified liposomes was higher than that of normal liposomes at different time points.The experimental results show that the delivery efficiency of CHL is better than that of common liposomes.The uptake of C6-CHL and C6-liposomes by HCEC and ARPE-19 cells after incubation for 5 min,10 min,15 min,30 min and 240 min was observed by fluorescence microscopy.The results showed that the uptake of C6-CHL by HCEC and ARPE-19 cells at different time points was higher than that of C6-liposomes.Eye safety after topical administration of liposomes was preliminarily evaluated using hematoxylin eosin staining(HE)and TdT-mediated dUTP nick and labeling(TUNEL).HE staining showed that there was no difference in cell structure between the conjunctiva and cornea tissue after topical administration TA-CHL,no tissue cell edema and no infiltration of abnormal inflammatory cells.TUNEL results showed that there were no significant tunel-positive cells in the retinal tissue after topical administration TA-CHL.The above results showed that TA-CHL was a novel,safety and no obvious eye irritation and side effects eye drops.This study confirmed that TA-CHL eye drops can effectively deliver triamcinolone acetonide to the posterior segment of the eye and play its role,overcome the problems of low encapsulation rate and low bioavailability of common liposomes,and is a new,efficient and safe eye drops.This study provides a safe and effective new dosage form for the clinical application of triamcinolone acetonide,and is expected to provide new ideas and methods for the treatment of macular edema caused by different causes.