| Proliferative vitreoretinopathy(PVR)is one of the serious eye diseases causing blinding. Although refinements in surgical techniques and equipment have improved the success rate of surgery to repair retinal detachment in recent years, recurrence due to reproliferation is not uncommon and remains the leading cause of failure of retinal reattachment surgery.Drug prevention has become a hotspot in PVR research.Topical instillation of drugs has little therapeutic effect on posterior structures in the eye, and systemically administered drugs penetrate poorly into the eye due to the blood–retinal barrier.Intravitreal drug injection is used to achieve high efficacy rates,but multiple injections may be associated with the risk of cataract, vitreous hemorrhage, retinal detachment, or endophthalmitis et. al.Curcumin, a polyphenolic compound derived from dietary spice turmeric, possesses diverse pharmacologic effects including anti-inflammatory, antioxidant, antiproliferative and antiangiogenic activities. So curcumin for proliferative diseases such as PVR has broad application prospects.Despite its efficacy and safety, the clinical application of curcumin has been limited by its low bioavailability.The reasons for reduced bioavailability of any agent within the body are low intrinsic activity, poor absorption, high rate of metabolism, inactivity of metabolic products and/or rapid elimination and clearance from the body. On the basis of these factors, to improve the intraocular bioavailability of CUR, firstly we prepare biodegradable scleral plugs containing curcumin and evaluate the drug release in vitro and in vivo.And we evaluate the biocompatibility or adverse effects secondary to the implant.Finally we made rabbit PVR model by injecting autologous blood into vitreous space and evaluated the the inhibition effect of biodegradable scleral plugs containing curcumin on the proliferative vitreoretinopathy.The final purpose of the study was to esplore the better way of administration on application of curcumin prevention from PVR. Part Ⅰ The preparationg of biodegradable scleral plugs containing different dose curcumin and the study on the release characteristics of the curcumin in vitro.Objective: To prepare biodegradable scleral plugs containing different dose curcumin and to characterize the release characteristics of the curcumin in vitro.Methods: All polymers and curcumin were available in powder form with an average particle size of approximately 100 um, as determined by a sieve shaker.To fabricate the biodegradable plugs, PLGA were premixed with the drugs. Drug/polymer ratios were 1:5.The mixture was then compression molded to form a sclera plug of about 1.0mm in diameter. The compressed plugs with the mold were then placed in an oven for sintering. The sintering time used was 30 min in order to attain an isothermal sintering of the materials. The plugs were placed in glass test tubes with 1.5 m L of BSS buffer. All tubes were incubated at 37℃. At every 24-hour interval,the dissolution medium was collected for subsequent concentration analyses. Fresh BSS buffer(1.5 m L) was then added for the next 24-hour period and this procedure was repeated for 14 days. The curcumin concentrations in buffer for the elution studies were determined by a high-performance liquid chromatography(HPLC) assay.Results: Preparation into biodegradable sclera plugs containing 0.5 mg, 1.0 mg, 1.5 mg three doses of curcumin, about 1mm in diameter, about 5mm long. The measured result suggested biodegradable sclera plugs containing 0.5 mg, 1.0 mg, 1.5 mg three doses of curcumin persistently release concentrations Of curcumin in vitro for 14 days. All three drugs had maximal release during the initial 24 h,and the drug release stably dropped to a lower amount since the second day.The release concentrations of plugs containing 1.5mg curcumin had been well above the best inhibitory of RPE concentrations(15ug/ml) for 14 days, concentrations of plugs containing 1.0mg curcumin had been well above the best inhibitory of RPE concentrations(15ug/ml) for 12 days.Conclusion: This part of the study has explored the alternative of using biodegradable polymers PLGA as different dose curcumin plugs for a long-term drug release.And plugs containing 1.0mg, 1.5mg curcumin persistently release concentration of curcumin in vitro above 15ug/ml for some time. Part Ⅱ The study on intraocular biocompatibility and toxicity of biodegradable sclera plug containing curcumin in adult rabbitObjective: To evaluate the biocompatibility and toxicity of biodegradable sclera plug containing curcumin and to evaluate the security of the drug release way.Method: 44 rabbits were randomly divided into control group and 0.5mg group, 1.0mg group, 1.5mg group,11 right eyes of each group were was inserted biodegradable sclera plugs containing different dose curcumin and trans-scleral fixed without suture after antiseptic procedure. For control group eyes, sclerotomy only was created with 18-gauge needle without implation of plugs. At last all experiment eyes conjunctiva was then sutured with 8-0 absorbable suture to cover the implantation or sclerotomy site. The plugs biocompatibility and potential tissue toxicity of 8 rabbits among each group were evaluated by slit-lamp biomicroscope, indirect ophthalmoscope, ERG on postoperation 1d, 7d, 14 d, 21 d and 28d; additionally, 2 eyeballs of each group were removed for the examination of light microscope and transmission electron microscope on 14 d, 28 d and 60 d.Result: At each detect point after implantation of plugs,intraocular pressure was no statistical difference between each groups. At post experiment 1d, 7d, there were different numbers of experimental eyes appeared different degree of conjunctival reaction between groups. But there was no significant difference between groups, and the rest of the checkpoint without conjunctival reaction. At slit-lamp biomicroscopy, the cornea of each experiment eye was transparent and no obvious anterior chamber reaction was found. Major complications such as infection, vitreous hemorrhage, or retinal detachment at sclerotomy site were not observed throughout the experiment. The sclerotomy wound healed after total degradation of the scleral implant without leakage and local necrosis. Finally the sclerotomy wound site was replaced by dense fibrosis-like tissue. The a-wave and b-wave amplitudes of ERG were no significant difference between groups at all detect points. The retinal tissue structure examined by light microscope and transmission electron microscope were normal on all stages.Conclusion: This part of the study showed good biocompatibility without significant inflammation or infection after the PLGA biodegradable sclera plugs containing curcumin implantation, and the scleral plugs degraded finally without any sclerotomy dehiscence or leakage. The animal model showed good biocompatibility without significant inflammation or infection, and after 1 to 2 months the scleral plugs degraded finally without second surgery.The intraocular tissue toxicity was found in the process of the whole experiment. In this study, the experimental results suggested that biodegradableplugs containing curcumin may be a novel type of curcumin treatment PVR. Part Ⅲ The study on the pharmacokinetics of curcumin from the biodegradable plugs in vitreous of rabbit and for to provide a reference for clinical applicationObjective: To evaluate the release characteristics of curcumin from the biodegradable plugs and to Provide a reference basis for clinical application.Methods: Twenty-two healthy and mature New Zealand albino rabbits were randomly divided into standard and quality control group(3 rabbits, 6 eyes) and experimental group(16 rabbits), The scleral plug containing curcumin was inserted and trans-scleral fixed without suture at superior-temporal quadrant 3 mm from the right eye limbus of experimental rabbits. After implantation, intra-vitreal tapping was performed at superior-nasal quadrant with 23-gauge needle on day 1, 7, 14, 21, 28. About 0.1 m L of vitreal fluid was aspirated each time and simultaneous scleral indentation was routinely performed to prevent vitreal hemorrhage. The vitreous samples were assayed by HPLC.Results: The chromatographic peak of curcumin and endogenous substance were separated well with the lasting time 4.3 min. The extract recovery of curcumin was 90%, and the relative standard difference(RSD) of intra-day and inter-day was low. The calibration curve of curcumin presented linear through 2 to 128 mg/m L with a regression formation as follow: y=32572x+46109(r= 0.999). Vitreous curcumin levels of all sclera plugs were measured at in implanted eyes with HPLC assay. Vitreous levels of all three drugs were measured over 4 weeks in implanted eyes with HPLC assay.The intravitreal curcumin concentration of sclera plugs containing 1.5mg curcumin group peaked at day 7(45.71±5.61ug/m L), and dropped to 9.51±1.03ug/m L at day 28. The intravitreal curcumin concentration of sclera plugs containing 1.0mg curcumin group also peaked at day 7(24.18±2.14ug/m L),and dropped gradually to 7.23±0.83ug/m L at day 28. The intravitreal curcumin levels of sclera plugs containing 1.5mg curcumin group were above 15ug/ml at 1d, 7d, 14 d, 21 d check point; and the intravitreal curcumin levels of sclera plugs containing 1.0mg curcumin group were above 15ug/ml at 1d, 7d, 14 d check point.Conclusion: Biodegradable scleral plugs containing curcumin release curcumin into vitreous after implantion into ocular at lease for 4 weeks. The intravitreal curcumin concentration had been above 15ug/ml for some time. Biodegradable scleral plugs containing curcumin can be used as a novel method curcumin inhibition of PVR. Part Ⅳ Establishment of proliferative vitreoretinopathy in rabbit eye and measurement of relative factorsObjective: To choose a high successul rate, effctive and economical establishment of proliferative vitreoretinopathy in rabbit eye in the process of drug prevention and treatment of PVR. At the same time, to detect relative factors of PVR with the method of antibody chip.Methods: Sixteen healthy and mature New Zealand albino rabbits were randomly divided into control and model group.The right eye of each rabbit was for experiment.For all experiment rabbits, a 4mm sclera incision with the paralle limbus through the pars plana to the center of the vitreous after limbus by 3mm, 0.2ml vitreal tapping was performed at the same sclera incision with 2ml syringe after an 8-0 absorbable suture of incision. 0.2ml BSS was injected into control group eyes vitreous space and 0.2ml autologous blood was injected into model group eyes vitreous space. All eyes were examined by slit-lamp biomicroscope, indirect ophthalmoscope and B ultrasonograph at different stages at 1d, 3d, 7d, 14 d, 21 d and 28 d of postoperation and evaluate the PVR grade. 0.2 m L of vitreal fluid was aspirated at 1d, 7d, 14 d, 21 d, 28 d,and then the expression of PVR relative fractors in vitreous specimen were detected by the method of QAL-CYT-2 chip.Results: At each detecting point, there was no PVR occured in control group. Postoperative PVR classification in model group: 5 eyes in stage 0 and 3 eyes in stage1 at 7d; 1 eye in stage 0, 5 eyes in stage 1, 1 eye in stage 2 and 1 eye in stage 3 at 14d; 2 eyes in stage 2, 5 eyes in stage 3 and 1eye in stage 4 at 21d; 2 eyes in stage 3, 4 eyes in stage 4 and 2 eyes in stage 5 at 28 d. At each detecting points, the levels of IL-1α, IL-1β, IL-8, Leptin, MMP-9, NCAM-1, in the model group were significantly higher than those in the control group(P<0.05). The expression of IL-1α, IL-1β, IL-8, TNF-α, MMP-9 gradually increased along with the time progress. The expression of IL-17 A, IL-21 was not detected in control group vitreous at each detecting point. At 14 d and 21 d, the level of IL-17 A was 1.2±0.50pg/ml and 9.2±3.9pg/ml respectively. At 7d and 14 d, the level of IL-21 was 6.0±2.3 and 9.8±3.0 pg/ml respectively.At other detecting time, the expression of IL-17 A and IL-21 was not detected. Model group fluorescence scan showed higher fluorescence intensity or larger area than those of model group at each detect point.The expression of MIP-1β was not found in the model group and the control group.Conclusion: This PVR model established by autologous blood injection into rabbit vitreous with the association of sclera inscion and vitreous extraction is consistent with PVR clinical process.This method of PVR model establishment was found in short time, economical and facility, and a variety of inflammatory factor expression can be detected, which can be used as drug prevention and treatment of PVR research experiments. PartⅤ the Exprimental study on the effect of biodegradable scleral plugs containing different dose curcumin in inhibition of proliferative vitreoretinopathy and related factors in rabbit eyeObjective: To evaluate the inhibition effect of biodegradable scleral plugs containing different dose curcumin on the proliferative vitreoretinopathy and related factors after the implantation of intraocularMethods: 30 healthy and mature New Zealand albino rabbits were randomly divided into model group and treatment group.The right eye of each rabbit was for experimental model group: to inject autologous blood into vitreous space; treantment group: to inject autologous blood into vitreous space and implant biodegradable plugs containing 1.5mg curcumin into ocular. All eyes were examined by slit-lamp biomicroscope, indirect ophthalmoscope and B ultrasonograph at different stages at 1d, 3d, 7d, 14 d, 21 d and 28 d of postoperation and evaluate the PVR grade. 0.2 m L of vitreal fluid was aspirated at 1d, 7d, 14 d, 21 d, 28 d,and then the expression of PVR relative fractors in vitreous specimen were detected by the method of QAL-CYT-2 chip.Results: On each detect points,the PVR degree of treantment group was lower than that of model group respectively, there was significant difference between groups(P < 0.05). On each detect points, there was significant decrease in the levels of IL-1α, IL-1β, IL-8, MMP-9, TNF-α, NCAM-1, leptin in treantment groups in compared with model group.At 14 d, 21 d, the IL-17 A levels of model group was 1.2±0.5pg/ml, 9.2±3.0pg/ml, 6.2±2.1pg/ml respectively. The statistic difference was significant(t=3.148, P=0.004). At 7d, 14 d and the IL-21 levels of model and treantment groups were 6.0±2.2 pg/ml, 3.8±1.3 pg/ml and 10.0±3.0,6.8±1.9pg/ml respectively. The statistic difference was significant(t=3.169, P=0.004; t=3.490, P=0.002). The expression of IL-17 A, IL-21 was not detected in model group and treatment group vitreous at other detect points. At every detecting point, the expression of MIP-1β was not detected both in the model group and in the treantment group.Treament group fluorescence scan showed lower fluorescence intensity or less area than those of model group.Conclusion: The implantation of biodegradable plugs containing 1.5mg curcumin can effectively inhibit the occurrence and development of PVR of rabbit eyes by inhibiting expression of IL-1, IL-8, TNF, MMP-9, NCAM-1 and other cytokines.According to the researches in those five parts above, it’s concluded that biodegradable plugs containing curcumin were prepared using PLGA as carrier material. HPLC assay in vitro and invivo showed that biodegradable plugs released sustained curcumin for at least two weeks. In addition, the animal model showed good biocompatibility without significant inflammation or infection, and the scleral plugs degraded finally without any sclerotomy dehiscence or leakage after 4-8 weeks. The study showed that the biodegradable plugs containing curcumin can effectively restrain the occurrence and development of PVR caused by autologous blood injection into rabbit vitreous. |
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