The In Vivo Evaluation Of Ropivacaine Loaded Poly (Lactide-co-glycolide) Micropheres In Mice

Background and objective:Despite the current scientific research in the basic and clinical have made a great progress,there is still a larger challenge in the management of pain.Local anesthetics and its compounds have been used to reduce and eliminate the pain in various stages.Injection of local anesthetics is the most site-directed and effective analgesic modality for the management of postoperative pain.However,clinical application of water-soluble local anesthetics can not achieve a long period analgesia by a single injection,the duration of the current water-soluble local anesthetics action are not more than 12 hours after administration.Current clinical metholds to prolong analgesia include multiple injections of short acting local anesthetic solutions but are time-consuming and demand expensive equipment and monitoring.Application of local anesthetics via a catheter can produce neural blockade for days to weeks,however,catheters may become infected,blocked or migrate.This lack of efficient treatment for postoperative analgesia highlights the need for new therapeutic principles in this area.Nearly thirty years,the drug delivery release system of PLGA(lactide-co-glycolide) microspheres is a hot research in pharmacy.With the drug delivery technology,long-acting local anesthetics sustained-release microspheres have become possible.Sustained-release local anesthetics would be made well,and a single dose will achieve a long term analgesia, so that it can reduce administration frequency,and because of its slowly but steady releasing,it has low plasma concentration and little fluctuations in bodys,reduces the toxicity of local anesthetics.PLGA is a biodegradable synthetic polymer materials,have the stable quality and good biocompatibility,and the inter-products are lactic acid,the final products are water and carbon dioxide,which are non-toxic and without stimulation.The past decades they have been widely used as a control system of microspheres matrix material,particularly in the field of implantable,some agents have been approved by FDA and used in clinical.PLGA can be completely degraded in a month or more, particularly suitable for the local anesthetics,which require long term used.In abroad study,it had a lot of reports in lidocaine and bupivacaine controlled release microspheres study,and found that the sustained-release and long term analgesic effect can be achieved,but has no report in the application of ropivacaine in the control release field.Hydrochloride ropivacaine(Rop) is a new type local anesthetics,compare to bupivacaine,it had the low potential of central nervous and cardiovascular toxicity,and had high degree of separation feeling-blockade exercise,is widely used in postoperative analgesia and painless delivery,etc.In the preliminary studies,that showed the ropivacaine loading poly(lactide-co-glycolide) micropheres (Rop-PLGA-MS) can be successfully synthesized,and the release experiment in vitro found that it can be a stable release of 80%ropivacaine in one week.However, only in vivo release experiment is safely and reliable.In our study,we will study the slow-release and long term analgesic effect of Rop-PLGA-MS in mice,and also study its biocompatibility. Method:1.In vivo release study of Rop-PLGA-MS:60 Kunming mice,body mass for 20-22g.Every mouse were buried with Rop-PLGA-MS 50mg subcutaneously,and randomly divided into 12 groups(n=5),according to the time interval set points,such as 1h,2h,3h,6h,12h,24h,48h,72h,96h,120h,144h,168h.At the corresponding time point,the corresponding groups of mice were sacrificed,removed the implants (Rop-PLGA-MS) completely from them and drying completely.Then through the procedure as add 2ml acetonitrile in→stirring in CL-2-type thermostatic magnetic (37 degrees,40rpm) for 12 hours→ultrasonic dissolved 30s→remove 50ul and add 1ml methanol in→centrifugal for 5min.The liquid samples were detected by high-performance liquid chromatography(HPLC) determination,calculation of the cumulative release rate(Q,%).2.Pharmacodynamic Study of Rop-PLGA-MS in mice:One hundre sixty-five Kunming mice(body mass for 20-22g) were randomly divided into A,B,C three grounds(N=55),The drug sides were implanted with Rop-PLGA-MS,the mixtures of Rop and PLGA-MS(Rop/PLGA-MS) and injected with 0.5%ropivacaine(25mg/Kg) nearby the sciatic nerves respectively.And the control sides were all implanted with the placebo microspheres(PLGA-MS),each side implanted volume was 400mg/Kg^-1.Each ground was randomly divided into eleven subsets(n=5) by the time point(5min,15min,30min,1h,2h,3h,4h,10h,18h,30h,48h) after administration.The efficacy of the drug was determined by measure the sensory and motor nerve blockade by the YLS-6B hot plate and 4-point scale.And measure the blood drug concentration by the high-performance liquid chromatography(HPLC) in corresponding time point.3.Biocompatibility study of Rop-PLGA-MS:10 Kunming mice(body mass 20-22g) were randomly divided into A,B groups(n=5),A group mice exposed to the side of lower extremity surgical incision of the sciatic nerve,perineural sutured after injecting saline 0.1ml;group B mice surgery incision exposed the sciatic nerve,and its surrounding is implanted with Rop-PLGA-MS and then sutured.Control study by group A,review the Inflammatory reaction,exudation and healing time in operative incision with group A and group B.One week later,compare the development situation with two groups.Two weeks later,observation the pathological changes in major organs and local tissue nearby the sciatic nerve under light microscope.4.Statistical analysis:Experimental data and charts are used statistical software SPSS13.0,experimental parameters separately using paired T test,One-Way ANOVA and Wilcoxon mean rank test,P＜0.05 represent a statistically significant difference between the experimental data.Result:1.The results of in vivo release study:Rop-PLGA-MS have a significant controlled-release effect in mice,obvious burst effect can be seen in the first hour,and the most ropivacaine release stability during 3-48h.6-24h is the peak hours of release,and the drug release tends to slowly after 48h.The accumulative release rate of drugs within 48 hours is 66.74±4.33%,91.83±1.31%Rop were released within 168hours,and the estimated half-life time is 42.26hrs.2.The results of Pharmacodynamic Study:(1) The results of nerve blockade:①the results of sensory blockade:Rop-PLGA-MS have significantly increased the time to reaction by using the YLS-6B hot plate at 3h post-implanted in A group mice(P= 0.000),10-18h reach the peak.The average during of the thermal nociceptive block from Rop-PLGA-MS was at least 30 hours.Group A and B mice were non-thermal escape response from 5min to 2h after drug administration P=0.000,and restore in 4h.The PLGA-MS did not change the animals' response P＞0.05.②the results of motor blockade:Rop-PLGA-MS showed slight levels of motor blockade during 4-18h post-implanted,and start restore in 3Oh.By 48h the motor function was normal.Rop and Rop/PLGA-MS caused severe motor blockade during 5min-2h,and restore in 4h.PLGA-MS did not cause any impairment in motor function.(2) The results of blood drug concentration:The micro-ropivacaine were measured in plasma of Ground A mice in 3h,and reach peak(Cmax) slowly in 10h and 18h time point,were129.80±22.59.70ng/ml and 120.20±23.35ng/ml,it hadn't detected after 48h,drug stay about 30 hours.In 5min,the higher concentration of ropivacaine in the plasma can be measured in Ground B and Ground C mice and set up rapidly,the Cmax were 1559.00±253.39ng/ml and 1470.80±113.77ng/ml respectively in 30min,then rapidly decline,after 4 hours there is nothing to detect.3.The results of the biocompatibility study:Two groups of mice have no local swelling and marked exudative reaction in the operative incision;the development situation have no statistical differences in two groups mice during one week.After two weeks,in the pathological findings:the mouse heart,brain,lung,liver,kidney and spleen were no clear organizational structure anomalies and a variety of histopathological changes;the muscle and the connective tissue around the Rop-PLGA-MS implanted side only see a slight inflammatory reaction,no infection, tissue necrosis and fibrosis,and macrophage phagocytosis can detected around the organizational structure.In the control group of mice can also see that mild local tissue inflammatory response,but not peripheral macrophage phagocytosis.Conclusion:We study Rop-PLGA-MS in vivo release,efficacy and biocompatibility in mice,we reached the following conclusions:1.Rop-PLGA-MS can release slowly and steady in mice,but more quickly than In vitro.2.Rop-PLGA-MS have an effective extension of ropivacaine limitation in mice,and low plasma concentration,decreased the toxicity of local anesthetics.3.Rop-PLGA-MS in mice has a good biocompatibility.In short,through this experimental study,we can see that Rop-PLGA-MS can not only successfully synthesized,but also has a good role in drug delivery and good biocompatibility,has a study value in clinical pain research in future.