重组双功能水蛭素PLGA微球的制备及其特性_英文_

复旦学报(医学版)Fudan Univ J Med Sci2006J an.,33(1)重组双功能水蛭素PL GA 微球的制备及其特性郁　琴　贺进田　莫　炜　张艳玲　王龙生　吴婴君　宋后燕(复旦大学上海医学院生物化学与分子生物学系-分子医学教育部重点室验室　上海　200032)【摘要】　目的　①建立R G D 2Hirudin 微球制备工艺;②建立缓释微球制剂中药物活性、含量的测定方法;③分析微球制备工艺中的各个关键参数对微球质量的各个指标的影响情况。

方法　选择聚乙烯醇(PVA )作为助乳剂,聚乳酸聚羟基乙酸(PL GA )作为微球的生物可降解载体,二氯甲烷作为有机溶剂,溶剂蒸发温度为室温,运用溶剂挥发法制备W/O/W 的双乳微球制剂。

在微球的制备工艺研究中,考察内水相中水油比例对微球粒径以及收率的影响,助乳剂PVA 浓度对微球外观以及微球的包封率和收率的影响,超声次数对微球成球率以及药物活性的影响,挥发搅拌速度对微球的成球率以及外观的影响,不同黏度的PL GA 对微球的外观以及分散度的影响,NaCl 浓度对微球的包封率、收率以及载药量的影响等。

结果　利用颗粒粒径系统(PSS )对所制备微球样品的分析得出,微球平均粒径81.38μm ,适合作进一步体内研究。

几批微球的载药量和收率都很高,分别为83.92%～96.3%和79.93%～95.05%;包封率为23.95%～65.13%。

其中NaCl 浓度、PVA 浓度对微球包封率的影响比较明显。

结论　微球的稳定性实验表明,微球释放率稳定,并且释放的重组双功能水蛭素(RG D 2Hiru 2din )活性稳定性保持良好,体外释放实验得出其具有缓慢释放的效果,为进一步做动物体内实验打下了基础。

【关键词】　重组双功能水蛭素;　生物可降解材料;　溶剂挥发法;　包封率;　控释制剂;　微球【中图分类号】　R 945 【文献标识码】　APreparation and characterization of poly (lactic 2co 2glycolic acid)microspheres containing RG D 2hirudinYU Qin ,H E J IN 2tian ,MO Wei ,ZHAN G Yan 2ling ,WAN G Long 2sheng ,WU Y ing 2jun ,SON G Hou 2yan(Key L aboratory of Molecular Medicine ,Minist ry of Education 2Departmant of B iochemist ry and MolecularB iology ,S hanghai Medical College Fudan Universit y S hanghai 200032,China )【Abstract 】　Purpose The main purposes of our research were to :1.set up t he met hod of t he R G D 2Hirudin microsphere preparation ;2.set up t he met hod to test t he activity and t he content of t he medi 2cine contained in t he microsphere ;3.analyse t he key factors on t he quality of the microsphere prepara 2tion.　Methods Co 2poly lactic acid glycolic acid (PL GA )microsphere was prepared by a modified sol 2vent evaporation met hod by a double emulsion wit h t he use of polyvinylalcohol (PVA )as emulsifica 2tion ;PL GA was used as biodegradable material and dichloromet hane as organic solvent.The influence of formulation factors including the W 1/O on microsphere diameter distribution and yield coefficient ;PVA concentration on microsphere appearance ,encapsulation and yield coefficient ;ultrasound on spherulization average and medicine activity ;stirring speed on spherulization average and microsphere appearance ;PL GA on microsphere appearance and microsphere dispersity ;concentration of NaCl on encap sulation efficiency ,yield coefficient and medicine content etc were st udied.　R esults The size of all t he fabricated microsphere was measured according to the several factors t hat affect t he particle size.The average diameter was 81.38μm ,which is good for furt her research.The medicine content and t he percent yield of all t he microsphere was high ,which ranged from 83.92%-96.3%and79.93%-95.05%respectively.The encapsulation efficiency was about 23.95%-65.13%.We foundt hat t he concentration of t he NaCl and PVA were t he very important factors to t he encapsulation effi 2ciency.Physiological activity of R G D 2Hirudin containing in t he microsphere and the release rate of the microsphere were controlled.Furt hermore ,t he release rate was stable.　Conclusions The physio 271复旦学报(医学版)　2006年1月,33(1)　logic activity of R G D2Hirudin released from t he microspheres was stable.PL GA2R G D2Hirudin micro2 spheres were controlled released by t he i n vit ro st udies.Therefore,the i n vivo experiment was well grounded.【K ey w ords】　R G D2Hirudin;　biogradable material;　solvent evaporation met hod;　encapsulation efficiency;　controlled released preparation;　microsphereIntroductionHirudin was originally isolated fro m leeches, and it is now available in recombinant forms. R GD2Hirudin,an analogue of t he Hirudin,exhib2 it s two main f unctions.It is t he mo st potent spe2 cific inhibitor of t hrombin and is used mainly in t he management of t hrombus,such as in t he treat ment and p revention of various forms of acute coronary syndromes.It is also t he mo st potent specific in2 hibitor of agglomeration of t hrombocytes.Micro sp here was one of t he multiparticulate delivery systems and was p repared to obtain pro2 longed or cont rolled drug delivery.It is understood t hat micro sp heres will be able to improve bioavail2 ability or stability and to target drug to specific sites.Micro sp heres can also offer advantages like limiting fluct uatio n wit hin t herapeutic range,re2 ducing side effect s,decreasing do sing frequency and improving patient compliance.We prepared co2poly lactic acid/glycolic acid (PL GA)micro sp heres containing water2soluble p roteins,R GD2Hirudin to achieve a cont rolled drug release p rofile suitable via injection subcuta2 neously.Firstly,we investigated some formulation variables to obtain sp herical particles,and t hen an2 alyzed t he yield of production,particle size dist ri2 bution,encap sulation efficiency,surface properties and i n vit ro release rate f rom microsp heres.The influences of formulation variables on t he micro2 sp here p roperties were examined,t he microsp here formulations suitable to achieve our goal were de2 termined as well.Materials and methodsPL GA,wit h a75∶25ratio of lactic to glycolic acid,0.3-0.75dL/g of viscosity and130000D of a weight2average molecular weight(MW)were used for t he microsp here preparation.Polyvinylal2 cohol(PVA)wit h average M r′s77000-79000, 88%hydrolyzed,was p urchased f rom Aldrich Chemical Co.and ot her materials and reagent s used were all of p harmaceutical grade.Microsphere preparationPL GA microsp heres were prepared by a modi2 fied solvent evaporation met hod by double emul2 sion.Briefly,2.0mg of R GD2Hirudin dissolved in distilled,deionized water or0.5%～2.0%W/V PVA(W1p hase,250μL)was poured into200～300mg PL GA dissolved in2.5-3.0mL of dichlo2 romet hane in a test t ube to create t he original e2 mulsion.The entire mixt ure was mixed for1min by vortex at maximum speed(Vortex Genie,Sci2 entifin Inc.),or probe sonicated(J Y922I)at out2 p ut80w for40sec to form t he original inner emul2 sion(W1/O).The emulsion was poured,under vigorous mixing using a magnetic bar into a350 mL glass beaker containing75mL of an aqueo us PVA solution(0.5%-2%W/V).The beaker was placed on a magnetic stirrer and beat up at t he speed of500-600r/min for1min to form t he final emulsion[(W1/O)/W2].The resulting double e2 mulsion was poured into225mL of distilled,dei2 onized water wit h t he whole300ml of solution containing0.5%-5%W/V NaCl and beat up at t he speed of300-350r/min for3-4h at room temperat ure until most of t he dichloromet hane e2 vaporated,resulting in precipitation of t he dis2 solved PL GA and subsequently t he formation of t he micro sp heres.The microsp heres were t hen col2 lected by cent rif ugation,washed wit h distilled,de2 ionized water,and finally vacuum2dried(AlpahⅠ2 6).This met hod p roduced a free2flowing powder t hat could be easily handled and stored at-20℃before use.All micro sp heres formulations were prepared in t riplicate.Microsperes dried at room temperat ure were81　郁琴,等.重组双功能水蛭素PL GA微球的制备及其特性t hen weighed and t he yield of micro sp here prepara2 tion was calculated using t he formula:Percentage yield:　=The amount of microspheres obtained(g)The theoretical amount(g)×100%　Encap sulation efficiency:=The amount of microsp heres obtained (g)×drug efficiency(%)The t heoretical amount of t he drug(g)×100%　Drug loading:　=Drug amount of t he microsp here (g)The amount of t he microsp here(g)×100% Morphology of the microspheresThe surface morp hology of t he micro sp heres was observed by scanning elect ron micro scopy (SEM,S2520).Dry microsp heres were mounted onto metal st ubs using double2sided adhesive tape and sp utter coated for1min wit h a50∶50gold/ platinum mixt ure.Size distributionThe diameter of microsp heres and dimension of t he scale bar were measured on micrograp hs wit h1500×magnificatio n SEM pict ure.And t he size dist ribution analysed wit h t he met hod of Parti2 cle Sizing Systems(PSS,Santa Barbara,CA., U SA).The mean microsp here volume in microns was reported f rom at least t hree micrograp hs each containing1000-55000microsp heres.E ncapsulation eff iciency and percentage yieldThe percentage yield of t he microsp here was investigated comparing t he amount of micro sp heres to t heoretical calculation.The ent rap ment efficien2 cy of R GD2Hirudin in t he microsp heres was deter2 mined by comparing t he starting amount of R GD2 Hirudin wit h t he quantity co ntained by t he micro2 sp heres after t he solvent evaporation met hod.En2 cap sulated R GD2Hirudin was determined by dissol2 ving15mg of microsp heres crushed and powdered by using a mortar in t he mixt ure of0.5mL dichlo2 romet hane and0.2mL0.1mol/L hydrochloric acid,and t he drug R GD2Hirudin will ext racted to t he layer part of hydrochloric acid.The R GD2Hir2 udin concentration was determined by BCA p rotein quantitative analysis met hod and absorbed at570 nm in a microplate reader(Bio2Rad model500, U SA).Calibration curves were used for t he deter2 mination of t he amount s dissolved.Physiologic stability of RG D2HirudinR GD2Hirudin in an amount of excess of it s solubility was added to15mL of dissolution medi2um in a20mL bottle maintained at(37±5)℃and shaken in a constant temperat ure water bat h for48 h.At app ropriate times aliquot s of1mL were taken f rom t he dissolution medium,filtered and t hen diluted to100mL.Drug content of t he sam2 ples were analyzed wit h t he BCA2100protein quan2 titative analysis met hod and were assayed wit h t he microplate reader(Bio2Rad model500,U SA)at50 nm.Calibration curves were used for t he determi2 nation of t he amount s dissolved.I n vit ro releasing studiesPL GA microsp heres(50mg)were suspended in4mL0.033mol/L p hosp hate buffer,p H7.2;0.02%Tween80was added to p revent t he micro2 sp heres f rom clumping.Releasing st udies were conducted in a rotating10mL polypropylene bottle at(37±0.5)℃and250r/min in an air incubating shaker(SCS224shaker).The samples were col2 lected periodically,cent rif uged for10min at2000 r/min,and analyzed for t he released R GD2Hirudin wit h t he BCA protein quantitative analysis met hod after suitable dilution and t he absorbance was mo2 nitored at570nm.R esultsDifferent factors affecting the preparation of microsphereChoise of t he PV A concent rationPVA,as t he emulsification,affected t he sur2 face morp hology and t he encap sulatio n efficiency of t he microsp here.We chose t hree different concen2 t ration of PVA:0.5%,2%,4%(W/V)respec2 tively.The scanning elect ron micrograp hs showed us t hat when t he concent ration of PVA increased, t he dispersivity of t he microsp heres went bad (Fig1).And we also found t hat t he PVA concen2 t ration affected t he percentage yield and t he encap2 sulation efficiency(Tab1).When PV A concentration increased,dispersivity of microsphere went bad,especially with the concentra291复旦学报(医学版)　2006年1月,33(1)　tion of 4%(W/V ),the microspheressuperposed.Fig 1　A ffection of PVAconcentrationto surface morphologyT ab 1　A ffection of PVA concentration to percentage yield and the encapsulation eff iciency of microspherePVA concentration[W/V )%]Encapsulation efficiency (%)Percentage yield (%)0.539.8782.782.065.1395.05Tab 1showed us t hat ,when PVA concentra 2tion was 2%(W/V ),t hepercentage yield and t he encap sulation efficiency of t he microsp heres werebot h higher t hat t ho se of 0.5%(W/V ).A f f ection of ult rasonic t reatment ti mesUlt rasonic t reat ment affected t he emulsifica 2tion.When t he ult rasonic t reat ment times in 2creased ,t he glomeration rate of t he micro sp heres deseased (Fig 2,3).A f f ection of sti rri ng s peedDuring t he stage of glomeration ,we chose two different stirring speeds as 600r/min and 800r/min (Fig 4,5).Fig 2　A ffection of ultrasonic treatment times toglomeration rateKeeping t he ot her ultrasonic factors t he same (ultrasonic worktime 1s ,ult rasonic interval time 1s ,power 80W )Fig 3　A ffection of ultrasonic treatment times to formeremulsion activityWe set t he activity of t he former emulsion as 100%,t he activity of t he former emulsion under t he 20,40,80ultrasonic treat menttimes is 88.89%,94.45%,and 22.22%respectively■Former emulsion wit h different ultrasonic treat ment times□Former emulsion wit hout ultrasonic treat mentFig 4　Affection of stirring speed to glomeration rateWit h t he 600r/min ,t he glomeration rate washigher t hat t hat wit h 800r/minA f f ection of PL GA t y peWe chose the two PL G A with the different vis 2cosity ,one was 75∶25with the viscosity of 0.082-0.098dL/g ,the other was 50∶50with the viscosity of 0.154-0.186dL/g.When the viscosity increased ,2　郁琴,等.重组双功能水蛭素PL GA 微球的制备及其特性the surface of the microsphere became less smooth (Fig 6),and the dispersivity of the microsphere were differ 2ent (Fig 7).Fig 5　A ffection of stirring sp eed to su rface morphology (SEM)The microspher of picture B was smoot her t han t hat of pictureAFig 6　A ffection of P LG A viscosity to su rface morphology (SEM)S ingle microsphere in picture B was less smooth than that in pictureAFig 7　A ffection of P LG A dispersivity to microsph ere viscositivityWit h t he lower viscosity of t he PL GA ,t he microspheresin picture A had better dispersivityN aCl concentrationWit h t he change of t he NaCl concent ration in W 2,t he drug loading (Fig 8),encap sulation effi 2ciency (Fig 9)and t he percentage field (Fig 10)changed.Fig 8　Affection of N aCl concentration to drug loadingDrug loading was between 82.93%and 96.3%under t he different NaClconcentrationFig 9　A ffection of N aCl concentration toencapsulation eff iciencyEncapsulation efficiency was between 23.95%and 65.13%under t he different NaClconcentrationFig 10　Affection of N aCl concentration to percentage f ieldPercentage field was between 79.93%and 95.05%under t he different NaCl concent ration.Size distributionAll fabricated microspheres prepared according to the several factors were between 4.98μm and 192.78μm in diameter and the average sizes for the 55000micro sp heres formulations wit h t he particle sizing system analysis met hod was 81.38μm.Fig 5showed us t he volume percent of t he microparticles p repared.The size dist ribution was good for our f urt her st udies i n vi vo .Physiologic stability of RG D 2HirudinThe st udy showed us t hat t he p ure R GD 2Hir 2udin was basically stable during t he period time of1mont h.Thus ,it is possible to co mpare t he p ureR GD 2Hirudin and t he micorsp here containingR GD 2Hirudin.Fig 12showed us t hat t he concern 212复旦学报(医学版)　2006年1月,33(1)　t ration of t he R GD 2Hirudin compared to t he total drug was between 3.75%and 9.36%..Fig 11　P article sizingdistributionFig 12　physiologic stabilityThe physiologic stability of t he R GD 2Hirudin was comparatively good wit h t he daily release rate between 3.75%and 9.36%I n vit ro release studies of microspheresThe PL GA microsp heres were cont rolled re 2leased slowly and t he Fig 13showed us t hat t he av 2erage amount of released drug f rom t he micro 2sp heres was 98.16%at t he 30t h paring to t he p hysiologic activity of t he p ure drug ,t he re 2lease rate of t he micro sp heres were cont rolled re 2leased and t he release rate was stable.Thus ,it could be easily used in t he clinical medicine to con 2t rol t he drug concent ration in t heblood.Fig 13　I n vit ro release studies of microspheresThe average amount of released drug from t he microspheres was 98.16%at t he 30th dayDiscussion and ConclusionsPL GA micro sp heres were prepared successf ul 2ly using modified solvent evaporation met hod usingdo uble emulsion.The type of PL GA ,concentra 2tion of NaCl and PVA ,stirring speed and stirring time were t he important factors to o btain sp herical particles wit h smoot h surface.The percentageyields of p reparation ,t he drug loading and t he en 2cap sulation efficiency was different wit h t he differ 2ent factors especially t he concent ration of NaCl in W 2.PVA ,emulsion of t he preparation ,affected t he dispersivity of t he micro sp heres.When t he concent ration of t he PVA increased ,t he dispersivi 2ty of t he microsp heres become conglutination ;while t he concentration become low ,t he percent 2age yield and t he encap sulation efficiency de 2creased.So we chose t he 2%(W/V )as t he best concent ration.The first stirring stage was t he glomeration stage.If t he stirring speed was too low ,t here was no enough power to form t he mi 2cro sp here ;but if t he stirring speed was too high ,t here were too many broken microsp heres.Finally we cho se t he 600r/min ×1min ,and t he glomera 2tion of t he micro sp here was perfect.And 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