Preparation And Evaluation Of Tetrodotoxin-PLGA Microspheres

As a highly toxic biological toxin,tetrodotoxin（TTX）was discovered in puffer fish from Tetrodidae in 1909.Studies have shown that TTX can cause sodium ion channel block,thus blocking sodium ion inflow,making nerve signals unable to be transmitted,resulting in sensory nerve disability,quadriplegia,dyspnea and even death of animals.However,with the deepening of subsequent studies,it is found that TTX has good analgesic,local anesthesia,detoxification,anti-tumor and other effects at low dose.Among them,there are a mass of extensive and in-depth studies related to analgesia.In clinical practice,clinical studies on the combination of TTX and other analgesics have been carried out,with good efficacy.Microspheres,a new type of drug carrier,are spherical particles with micron size.When it is released,it has obvious sustained and controlled release characteristics.In recent years,the studies on microspheres have been deepened,and more and more diversified types of microspheres have been developed.The preparation process is quite mature,and the characteristics of microspheres such as morphology and release curve can be accurately controlled by various methods.Microspheres have become an important carrier for long-term sustained and controlled release preparations.In the microsphere preparation material,Biodegradable materials have been widely valued since they were discovered.Due to its good biocompatibility,controllable biodegradability,environmental friendliness and controllable cost,it has been widely developed.Among them,polylactic acid glycolic acid copolymer（PLGA）is an outstanding representative in recent years,which has been recognized by the United States,China,the European Union and other countries.It is completely degraded in human body to produce carbon dioxide and water that are harm Less to human body,and has easy-to-control drug release rate and characteristics.It is a mature and reliable microsphere preparation material.Therefore,PLGA microspheres loaded with TTX were designed and prepared in this study.The research contents are mainly divided into the following parts:First of all,the analytical methods of TTX in different environments were established,and the reversed-phase ion-pair chromatography,hydrophilic interaction chromatography,ELISA kit and colloidal gold kit were comprehensively investigated.Finally,the reversed-phase ion-pair chromatography suitable for various environments was determined as the best solution.The linearity in the range of 1-20μg/m L accords with the determination requirement.The linear regression equation is A=22216c+591.82（R²=0.9999）.Methodological verification results show that the accuracy and precision of this method are good,and RSD is less than 2.0%.The stability of TTX aqueous solution at 37℃and 100rpm oscillation was investigated by this method.The results showed that TTX aqueous solution was extremely unstable under this condition,and the degradation rate in 28 days was about 74.96±2.93%,which provided data support for subsequent research.Secondly,the formulation of TTX-PLGA microspheres was screened and characterized.The optimum formulation of microspheres was selected by single factor method.The optimum formulation of microspheres was 0.5%PVA external water phase containing 5%Na Cl,TTX formic acid aqueous solution as internal water phase containing100μg TTX,dichloromethane phase with 100 mg/m L PLGA as oil phase,the volume ratio of internal water phase to external water phase=0.1:2.5,the stirring rate of colostrum was8000 rpm,the volume ratio of colostrum to external water phase=2.6:20,and the stirring rate of double emulsion was 900 rpm.The entrapment efficiency was 88.56±0.86%,drug loading was 0.036±0.001%,particle size was 111±4.00μm,Span value was 0.172.Scanning electron microscope（SEM）showed that the microspheres had a round surface,and the release characteristics in vitro were fitted to a first-order release model.The equation was ln（100-）=161.53367+0.05061,and the correlation coefficient was 0.9628,and the release rate of microspheres was close to 90%at 14 days,and the concentration at 21 days was lower than the lower limit of the method.Thirdly,the analgesic pharmacodynamics of TTX-PLGA microspheres were investigated.The maximum safe dose of TTX aqueous solution in Balb/c mice was investigated,and the subsequent experiments were carried out under this dose.The model of sciatic nerve block was established.incubation period of thermal withdrawal latency（TWL）was taken as the index.The changes of TWL incubation period in mice and rats under the action of various preparations were studied by hot plate method.The results of hot plate method in mice group showed that there was no statistical difference in TWL between the front and back claws of mice in normal saline group and basic group.Morphine aqueous solution group began to take effect at about 0.17 h.TWL in front paw was significantly different from that in basic group.TWL in back paw increased to more than 60 s,reached its peak at 0.5 h,and TWL in front and back paw increased to more than60 s,but lasted for a short time.The effect began to decline at 2-4 h,and recovered to the same level as that in basic group at 8 h.TWL of front paw increased from 15.32±8.49 s to59.43±0.84 s,and TWL of back paw increased from 21.30±8.85 s to 55.48±9.03 s,which indicated that TTX aqueous solution had significant inhibitory effect on the pain induced by hot plate in mice,but the analgesic time was also short,and the effect began to weaken after 2-4 h.The analgesic effect of TTX aqueous solution increased from 15.32±8.49 s to 59.43±0.84 s and 21.30±8.85 s to 55.48±9.03 s.At 8h,it recovered to the basic level.After administration of TTX-PLGA microspheres,there was no significant difference between TWL of front paw and TWL of base,TWL of the posterior paw increased to 53.21±8.59 s at 2 h,and remained at a high level from 2 h to 14 days,which was significantly different from that in basic group.TWL of the posterior paw recovered to the level of the basic group at 17 days,indicating that the drug began to take effect at 2 h and the action time was as long as 14 days,indicating that TTX-PLGA microspheres mainly exerted local analgesic effect at the administration site of the posterior paw,and it has a significant effect of inhibiting heat pain in mice during this period.In the rat sciatic nerve block model,the changes of TWL measured by hot plate method in different groups were consistent with those of mice,with the significant difference.There was no significant difference between TWL and basal level in saline group and blank microsphere group.Morphine aqueous solution reached its peak at 0.5 h,and there were significant differences in TWL between front paw and back paw and basic group.The TTX aqueous solution also reached its peak at 0.5 h.Compared with the basic group,the TWL of front paw and back paw was significantly different,TWL in front paw of TTX-PLGA microsphere group showed no significant difference with basic group in the whole time period,but TWL in back paw began to show significant difference with basic group at 0.5h,and remained at a high level from 0.5 h to 14 days.TWL in back paw recovered to basic group at 17 days,indicating that TTX-PLGA microsphere mainly plays local analgesic effect at the administration site of back paw.It can obviously prolong the analgesic effect of TTX.Finally,a method for the determination of TTX in biological samples was established,and the pharmacokinetics of TTX aqueous solution and TTX-PLGA microspheres in rats were investigated.The detection method used is LC-MS/MS,Adopt ESI positive ion source,Tolbutamide was selected as internal standard,The detection ion pairs of TTX and tolbutamide are 320.1→301.9（m/z）and 271→171.7（m/z）,respectively.The content determination method has good specificity,and there was no interference between endogenous substances and internal standard substances in rat plasma.The linear regression equation was Y=0.008338X-0.001664,R²=0.9991.The lower limit of quantification（LLOQ）was 1 ng/m L.The method had good accuracy and precision,good matrix effect and extraction recovery.It could be used as a method for the detection of TTX in rat plasma.The drug-time curves of TTX aqueous solution at tail vein and sciatic nerve and TTX microspheres at sciatic nerve were measured by this detection method,and the pharmacokinetic parameters were calculated.The results show that the half-life of TTX aqueous solution injected into tail vein was about 19.76±6.58 h,The half-life of TTX was 30.38±12.93 h,and the half-life of TTX-PLGA microspheres was390.70±137.79 h after injection into sciatic nerve.The concentration of TTX in TTX microspheres was lower within 21 days after administration,which was higher than the detection limit but lower or close to the lowest quantitative limit.Comparing the AUC _0-∞of TTX aqueous solution（61.24±12.95 ng/m L*h）and microsphere group（641.70±169.18 ng/m L*h）at sciatic nerve,it was found that the AUC_0-∞of microsphere group was10 times that of TTX aqueous solution,but the actual dosage was 40 times that of TTX aqueous solution.Considering the local analgesic effect of TTX-PLGA microspheres,it can be preliminarily judged that TTX released by TTX-PLGA microspheres is concentrated at the local administration site.The comprehensive results showed that there was no systemic toxicity when TTX-PLGA microspheres exerted its efficacy,and the concentration in whole blood remained within the safe dose range all the time.In a word,this study preliminarily proved that the prepared TTX-PLGA microspheres have good sustained-release characteristics,and pharmacodynamic experiments proved that it effectively prolonged the action time of TTX and played a good long-term analgesic effect in ensuring its safe concentration range.It has certain guiding significance for the development and expansion of long-acting analgesics.