Preparation And Release Profiles In Vitro Of Ropivacaine-dexamethasone Acetate-loaded Poly(Lactide-co-glycolide) Microspheres

Research background and purposeHuman have been unremittingly endeavoring to reveal the mystery of pain though several thousand years,especially the past two to three decades the application of advanced scientific technology to carry out basic and clinical study we had made great achievements,so that we can gradually evaluate the pain by biological point of view.9th IASP academic conference of pain put forward formally,"some of the pain itself is a disease" and medical personnel can relieve or ease patients' pain that is the "life-saving,humanitarian".Pain seriously affects the quality of mankind life which is not only a medical problem in the scope of a world,but also is one of the major health problems in our country.Local anesthetics as an effective drug in treatment pain had been widely used in clinic,but its action time only maintains 6-12hours,it can not meet the need of intractable pain control and treatment,such as post-operative pain, advanced cancer pain and trigeminal neuralgia.Over the years,people had used various ways to prolong analgesia time of local anesthetics,but the results can be not ideal.Micro-ball technology is a new technology which had been applied to the field of pharmaceutical preparations nearly 40 years.Micro-ball technology can use natural or synthetic polymers as a drug carrier in order to achieve the slow- release of drugs and extend the duration of drugs.In recent years,with the development of micro-ball technology,study direction of long-acting local anesthetics had shifted to various forms of preparation local anesthetics microspheres.Local anesthetics had been made slow-release or controlled-release formulations to extend the duration of single-dose analgesic effect,reduce the number of drug delivery and reduce its plasma concentration fluctuations and drug toxicity.PLA and Poly(lactic-co-glycolide)(PLGA) are polymer which polymerize by the different ratio of glycolide monomer and lactide monomer and are developed with a non-toxic,no stimulation the last 10 years.PLGA could be completely biodegradable polymers with good biocompatibility and human adaptability.In organisms,the ultimately degradable products of PLGA are lactic acid which can completely degrade to carbon dioxide and water ultimately,and all these degradable products are discharged out of organism.So PLGA is a kind of safe biodegradable material and can not cause obvious inflammatory response,immune response and cell toxicity response.PLGA has its own characteristics,i.e.completely biodegradation in vivo and can be discharged out of organism to avoid secondary victimization for patients. PLGA is a kind of good biocompatibility and biodegradable carrier material. Simultaneously PLGA as a carrier,the release rate of encapsulated drugs is controlled by changing the molecular weight of PLGA and the proportion of glycolide and lactide.PLGA microspheres have been used in anti-cancer drugs,antibiotics, treatment of eye diseases,gene therapy and diabetes treatment and many other studies. However,there is no sophisticated formulations of local anesthetics in clinic.The PLA and PLGA microspheres were used in the study of local anesthetics which has been about thirty years.Early in 1981,Wakiyama N prepared the PLA microspheres containing several local anesthetics.In 1994,Le Corre et al studied preparation and properties of bupivacaine PLA and PLGA microspheres.These had proved that the method of preparation sustained-release microspheres is feasible by using biodegradable materials containing local anesthetics and sustained-release microspheres can significantly extend the release time of local anesthetics.Nineties, Joanne PhD,Christiane DVM et al scholars studied pharmacokinetics and pharmacodynamic characteristics of local anesthetics slow-release microspheres by different animals as study subjects.Results showed that local anesthetics slow-release microspheres can significantly extend analgesic effects time of the single dose of local anesthetics and reduce drug dosage and the volatility of drug concentration in vivo.Ropivacaine as the first new type of pure L-lactam precursor and long-term of local anesthetics.Structure,nature and metabolic path ways of ropivacaine are similar with bupivacaine,but the central nervous system and cardiovascular system toxicity of ropivacaine are lower and the role time is longer than bupivacaine.A more prominent characteristic of ropivacaine are the high separation of feeling-motor nerve block,low drug concentration produces almost exclusively sensory nerve block, non-progressive motor nerve block which can make patients quickly restore the possibility of exercise.Ropivacaine is more suitable for treatment various pain.The experimental group in the pre-work preparated successfully ropivacaine poly(lactide-co-glycolide) microspheres and confirmed that its release time in vitro maintained 8 days.The local analgesia experiments by New Zealand rabbits and mice found that ropivacaine poly(lactide-co-glycolide) microspheres group produced more prolonged analgesia time than ropivacaine injection group(about 10 times).Joanne C in l996 and Christiane DVM,Drager et al in 1998 added dexamethasone acetate into local anesthetics microspheres in order to further extent the duration of analgesic effects on the original foundation.In 2003,Dan J.Kopacz found that peak plasma concentration and peak time of Bup-Dex-PLGA-MS are less than Bup-PLGA-MS,as well as duration of analgesia are longer in the body by the same dose(drug loading) of the Bup-Dex-PLGA-MS and Bup-PLGA-MS in intercostal nerve block in human trials.For these reasons,the effect of dexamethasone acetate in Rop-PLGA-MS had aroused our great interest.we had adopted a fully biodegradable polymer materials PLGA as the carrier material and a new type of long-acting local anesthetics ropivacaine and dexamethasone acetate as model drugs for preparation local anesthetics slow-release microspheres.we studied preparation and preparation process of Rop-Dex-PLGA-MS and orthogonal experimental design to draw up a more optimized preparation process and preparation parameters.Through observation Rop-Dex-PLGA-MS in vitro release characteristics,we studied its in vitro release drug model and whether dexamethasone acetate played a role in ropivacaine release in vitro from microspheres.Methods:1.We took surface morphology,particle size,drug entrapment efficiency,drug loading and drug burst as observed indicators and PLGA as carrier materials and ropivacaine and dexamethasone acetate as parceled drugs,as well as we studied single-factor of W₁/O/W₂ double emulsion methods-solvent evaporation method of preparation microspheres.Single factor included the organic phase PLGA concentration,continuous phase/organic phase volume ratio,internal aqueous phase volume and continuous phase with polyvinyl alcohol concentration.Then we selected the three factors which had significantly impacted characteristics of the microspheres and used the orthogonal experimental design(L₉(3³) three factors and three levels) to select the best preparation method.Rop-Dex-PLGA-MS and Rop-PLGA-MS were prepared by the best preparation method.2.In vitro release studies of Rop-Dex-PLGA-MS were carried out.we took sway column method and phosphate buffered salts(pH=7.4) as release medium.50mg Rop-Dex-PLGA-MS were dispersed in 10mL release medium in a covered test tube at(37±0.5)℃with 100rpm speed of oscillation.Respectively,at 0.5,6,12,24h and thereafter once a day,by regular centrifugal,sampling and exhausting all the release medium in test tube,the microspheres were separated from the release medium,then added the same temperature and fresh release medium 10mL into the test tube. Content of ropivacaine and dexamethasone acetate in samples was determinated by RP-HPLC.The cumulative release of drug was calculated.Release drug curve in vitro of Rop-Dex-PLGA-MS was fitted by Origin6.0 to explore its possible mechanism of drug release.3.We comparised ropivacaine release characteristics in vitro of Rop-Dex-PLGA-MS and Rop-PLGA-MS and studied where dexamethasone acetate extended duration of ropivacaine release in vitro from microspheres.4.Statistical analysis:all measurement data denotes((?)±s).Three groups factors of the organic phase PLGA solution concentration,continuous phase/organic phase volume ratio,continuous phase PVA solution concentration can impact the characteristics of microspheres,different variables in each group compared by one-way ANOVA.Orthogonal design information used analysis of variance and multiple comparisons.All data were treated by statistical software SPSS 13.0 or Origin6.0,P≤0.05 for significant difference.Results:1.Single-factor study found that internal aqueous phase volume,organic phase PLGA concentration and continuous phase/organic phase volume ratio significantly impacted particle size,drug loading and encapsulation efficiency,burst release of the microspheres.These three factors had been done by a three-level orthogonal design to optimization of the preparation program.The best method for the preparation was internal aqueous phase volume 0.55mL,organic phase PLGA concentration 10%(w/ v) and continuous phase/organic phase volume ratio 50.Rop-Dex-PLGA-MS which were prepared by the optimum method have smooth surface,spherical shape,uniform particle size,distribution of more than 85%between 20-90μm,average particle size 50.93±1.73μm,ropivacaine drug loading(7.60±0.08)%,encapsulation efficiency (74.10±1.42)%,dexamethasone acetate drug loading(1.54±0.02)%,encapsulation efficiency(60.16±1.55)%.W₁/O/W₂ double emulsion-solvent evaporation method can prepare very ideal Rop-Dex-PLGA-MS.2.Rop-Dex-PLGA-MS can have a significant role in sustained-release and sustain drug release for up to 14 days.Its process of drug release in vitro accorded with diffusion-corrosion model and showed a typical S-shaped three-stage model. namely:a small burst,short-term slow release and a quick release.Drug of microspheres released faster in the initial 6 hours,ropivacaine and dexamethasone acetate cumulative release rate were respectively 15.26%and 7.73%.Next drug release rate was relatively stable,after 4d the drug release speed was accelerated.The cumulative release rate of ropivacaine and dexamethasone acetate rates were 95.37% and 96.55%within 14 days respectively.3.The release curves in vitro of ropivacaine in Rop-Dex-PLGA-MS and Rop-PLGA-MS were similar which had shown that dexamethasone acetate had no extended duration of ropivacaine release in vitro from microspheres.Conclusion:We study Rop-Dex-PLGA-MS preparation method and that the major influencing factors impacted on the particle size,surface morphology,drug loading, encapsulation efficiency and burst behavior of microspheres by using orthogonal test to optimize preparation process,as well as we studied its in vitro release characteristics.we reached the following conclusions:1.we can prepare ideal Rop-Dex-PLGA-MS by W₁/O/W₂ double emulsion-solvent evaporation method under certain influencing conditions with high drug loading and encapsulation efficiency,small size and distribution uniform,appearance of the smooth and whole round,and the preparation methods had good repeatability.2.Rop-Dex-PLGA-MS can significantly extend the drug release time and sustaine drug release for up to 14 days.Its process of drug release in vitro showed a typical S-shaped three-stage model.3.The release curves in vitro of ropivacaine in Rop-Dex-PLGA-MS and Rop-PLGA-MS were similar which had shown that dexamethasone acetate has no extended duration of ropivacaine release in vitro from microspheres.The test provided local anesthetics slow-release formations for further research and development and post-agents pharmacokinetics,pharmacodynamics research with platform.And it may provid treatment pain in clinic with an even better system of long-acting sustained-release local anesthetics.