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BỘ Y TẾ TRƯỜNG ĐẠI HỌC DƯỢC HÀ NỘI

Bước Đầu Nghiên Cứu Tạo Liposom Doxorubicin Gắn PEG

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B Y TTRNG I HC DC H NINGUYN VN THNGBC U NGHIN CU TO LIPOSOM DOXORUBICIN GN PEGKHA LUN TT NGHIP DC SH NI - 2013B Y TTRNG I HC DC H NINGUYN VN THNGBC U NGHIN CU TO LIPOSOM DOXORUBICIN GN PEGKHA LUN TT NGHIP DC SNgi hng dn: 1. TS Nguyn Th Lp2. TS. H Phng ThNi thc hin: 1. B mn Ha sinh2. B mn Bo ch3. Vin Cng ngh sinh hc H NI - 2013LI CM NLi u tin, ti xin gi li cm n chn thnh nht ti:TS. Nguyn Th LpCtntnhhngdn,chbovgiptitrongsutqutrnhthchin ti ny. Nhng lc gp kh khn, c lun ng vin ti v ch cho ti hng gii quyt vn . Trong khong thi gian hc tp, nghin cu di s hng dn ca c, ti hc c rt nhiu iu b ch v thc s l nhng hnh trang qu bu gip ti thm vng bc trn con ng i sp ti ca mnh.TicngxinbytlngbitnsusctiTS.HPhngTh, phng Nano Y Sinh,vinKhoahcvtliu.Cchotinhngli khuyn b ch, tn tnh gip ti trong qu trnh nghin cu. TixincmnPGS.TS.LQuangHun,ThS.LThThyDngcngcc cnbcngtctiphngCngnghtbongvtVincngnghsinhhc,nhng ngi nhit tnh gip ti c th hon thnh kha lun.Ti xin cm n cc thy c v cc anh ch k thut vin B mn Ha sinh, Bo ch, DclcTrngihcDcHNi,BmnHaphntch,KhoahahcTrng i hc Khoa hc t nhin, i hc Quc gia H Ni to iu kin v gip ti trong qu trnh nghin cu. Ti xin gi li cm n ti b m ti. Chnh b m l ngun ng lc ln nht gip ti hon thnh c kha lun ny.Tixingilicmntinhngngibn,cbitlbnHTrngTon,ch Nguyn Th Bch Trn st cnh bn ti, h tr, ng vin ti rt nhiu trong qu trnh thc hin kha lun.SV Nguyn Vn ThngMC LCDANH MC CC K HIU, CC CH VIT TTDANH MC CC BNGDANH MC CC HNH V, THT VN 1CHNG I. TNG QUAN31.1. i cng v liposom31.1.1. Khi nim31.1.2. Thnh phn cu to ca liposom31.1.3. Phn loi41.1.4. Cc phng php to liposom51.2. Doxorubicin v liposom doxorubicin61.2.1. Doxorubicin61.2.2. Liposom doxorubicin81.3. S phn ca liposom sau khi tim tnh mch91.4. Nhng n lc ci thin thi gian tun hon trong mu ca liposom111.5. PEG v mt s dn cht PEG-lipid ng dng trong ch to liposom121.6. C ch PEG lm tng thi gian tun hon ca liposom trong mu141.7. Cc phng php to liposom gn PEG161.7.1. Gn PEG trong qu trnh to liposom (phng php truyn thng, pre-modification)161.7.2. Gn PEG sau qu trnh to liposom (phng php post-modification)171.8. ng dng ca liposom gn PEG181.8.1. ng dng trong iu tr ung th181.8.2. ng dng trong iu tr mt s bnh l khc 181.8.3. ng dng trong chn on bnh191.8.4. Kt hp ti u ha h mang thuc liposom19CHNG II. I TNG V PHNG PHP NGHIN CU202.1. Nguyn liu, ha cht, thit b v ng vt th nghim202.1.1. Nguyn liu, ha cht202.1.2. Thit b202.1.3. ng vt th nghim212.2. Ni dung nghin cu212.3. Phng php nghin cu212.3.1. Phng php to liposom doxorubicin gn PEG v liposom doxorubicin khng gn PEG212.3.2. Phng php nh gi liposom to thnh232.3.2.1. Phng php nh gi kch thc tiu phn v phn b kch thc tiu phn 232.3.2.2. Phng php xc nh th zeta242.3.2.3. Phng php nh lng doxorubicin242.3.2.4. Xc nh hiu sut liposom ha doxorubicin252.3.3. Phng php nghin cu nh hng ca gn PEG ln b mt liposom ti thi gian tun hon trong mu ca liposom doxorubicin 252.3.3.1. Chun b cc iu kin tin hnh th nghim252.3.3.2. Tim chut v ly mu262.3.3.3. Phng php chit doxorubicin ton phn trong huyt tng262.3.3.4. Phng php nh lng doxorubicin trong huyt tng282.3.3.5. Tnh gi tr AUC0-24h ca cc dng thuc ca doxorubicin292.3.4. Phng php x l s liu29CHNG III. KT QU V BN LUN303.1. Kt qu303.1.1. Ch to liposom doxorubicin gn PEG303.1.2. nh gi liposom gn PEG to thnh303.1.2.1. Kch thc tiu phn v phn b kch thc tiu phn 303.1.2.2. Th zeta323.1.2.3. Hiu sut liposom ha doxorubicin 323.1.3. Bc u tm hiu nh hng ca gn PEG ln b mt liposom ti thi gian tun hon trong mu ca liposom 353.1.3.1. Kt qu xy dng ng chun doxorubicin trong huyt tng353.1.3.2. Kho st nng doxorubicin trong huyt tng363.2. Bn lun393.2.1. V quy trnh to liposom doxorubicin gn PEG393.2.1.1. La chn nguyn liu v cng thc393.2.1.2. Quy trnh ch to liposom doxorubicin gn PEG v cc ch tiu cht lng ca ch phm to thnh 393.2.2. V quy trnh chit doxorubicin trong huyt tng423.2.3.VnhhngcagnPEGlnbmtliposomtithigiantunhon ca liposom trong mu42KT LUN V KIN NGH44TI LIU THAM KHOPH LCDANH MC CC K HIU, CC CH VIT TTVit tt T/ cm t y ADN Acid deoxyribonucleicAUC Area under the curve (Din tch di ng cong)Chol Cholesterol DLS Dynamic Light Scattering (Tn x nh sng ng) DOX DoxorubicinDMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholinDSPC 1,2-distearoyl-sn-glycero-3-phosphocholinDSPE 1,2-distearoyl-sn-glycero-3-phosphoethanolaminDSPE-PEG-NH21,2-distearoyl-sn-glycero-3-phosphoethanolamin-N-[amino(polyethylen glycol)]ECG Electro-Cardio Gram (in tm )MPEG Monomethoxy polyethylen glycolmPEG-DSPE 1,2-distearoyl-sn-glycero-3-phosphoethanolamin-N-[methoxy(polyethylen glycol)]MPS Mononuclear Phagocytic System (H thc bo n nhn)PDI Polydispersity Index (Ch s a phn tn)PEG Polyethylen glycolPHEPC Partiallyhydrogenatedeggphosphatidylcholine (Phosphatidyl cholin trng hydro ha mt phn)T1/2Thi gian bn thiTMPEG Tresylat monomethoxy polyethylen glycoltt/tt Th tch/th tchUS-FDA Cc Qun l Dc phm v Thc phm Hoa KDANH MC CC BNGBng 1.1. Phn loi liposom theo kch thc v s lp4Bng 2.1. Nguyn liu to liposom gn PEG v liposom khng gn PEG21Bng 3.1. Kch thc tiu phn v phn b kch thc tiu phn ca cc mu liposom gn PEG v liposom khng gn PEG31Bng 3.2. ThzetavdichuyncaccmuliposomgnPEGvliposomkhng gn PEG32Bng 3.3. S ph thuc ca hp th vo nng ca doxorubicin hydroclorid trong mi trng NaCl 0,9% v Triton X-100 1% (tt/tt).33Bng 3.4. HiusutliposomhadoxorubicincamuliposomgnPEGvliposom khng gn PEG34Bng 3.5. Sphthuccamthunhquangvonngdungdchdoxorubicin hydroclorid35Bng 3.6. Nng tng ng doxorubicin trong huyt tng chut nht ti cc thi im 5 pht, 4 gi, 24 gi sau khi tim tnh mch ui 37Bng 3.7. Gi tr AUC0-24h ca cc dng thuc doxorubicin trn chut nht (tim tnh mch ui, liu 10 mg/kg) 38DANH MC CC HNH V, THHnh 1.1. Cng thc cu to ca doxorubicin6Hnh 1.2. Cc giai on sinh l trong s thi tr ca liposom9Hnh 1.3. Cng thc cu to ca polyethylen glycol12Hnh 1.4. Mt s dn cht lipid ca polyethylen glycol13Hnh 1.5. C ch PEG ko di thi gian tun hon ca liposom trong mu 15Hnh 1.6. Cc phng php to liposom gn PEG16Hnh 2.1. Quy trnh chit doxorubicin ton phn t huyt tng27Hnh 3.1. ththhinsphthuccahpthvonngdoxorubicin hydroclorid trong mi trng NaCl 0,9% v Triton X-100 1%33Hnh 3.2. thbiudintngquangiamthunhquangvnng doxorubicin hydroclorid36Hnh 3.3. Nng tng ng doxorubicin trong huyt tng chut nhtti thi im 5 pht, 4 gi, 24 gi sau khi tim tnh mch ui37T VN Ktkhicphthinvonhngnm1960biBangham,liposomc nghincurngrivngdnglmhmangnhiuthuckhcnhau.uimca liposom l tnh tng hp sinh hc v kh nng mang c c cc thuc tan trong nc v khngtantrongnckhcnhaunhthuciutrungth,cckhngsinh,khngsinh chng nm, glucocorticoidTuy nhin, mt trong nhng nhc im ln nht ca liposom khisdngngtimchnhlbthitrnhanhchngkhivngtunhondostm btcacctbothuchthcbonnhn(MPS)[13],[30].Mttrongnhngbin phphiuqukhcphcnhcimnylgnlnbmtliposomccpolymethn nc,phbinlpolyethylen glycol (PEG) vi khi lng phn t t 1000 n 5000 Da [11],[13].StngthigiantunhoncaliposomgnPEGmanglinhiulich:gim srrthuctrongtunhon,gimcctcdngkhngmongmunca thuc, lm tng c hi thot mch vo khong gian bo ca liposom, tng s tch ly ca cc thuc chng ung th ti khi u v tng hiu qu iu tr [23],[30],[43],[55].Nhngnmgny,nhiunghincuctinhnhtrngihcDc HNinhmnhgikhnngngdngliposomlmchtmangthuc.Trong, liposom doxorubicin c nghin cu nhiu nht v t c nhiu kt qu ng khch l.Ccnghincubcuaracquytrnhsnxutthuctimliposom doxorubicinvihiusutliposomhadoxorubicinlnhn80%.Tuynhin,huhtcc nghincumichdnglivicchtoliposomtnguynliulphospholipidv cholesterol,chacnghincunosdngccdnchtpolymelmtngthigian tunhoncaliposomdoxorubicin.Vimongmuntoraccliposomdoxorubicinc kh nng tun hon ko di, gp phn ci thin tnh hiu qu ca h mang thuc liposom, chngtitinhnhti:Bcunghincutoliposomdoxorubicingn PEG. ti gm c hai mc tiu chnh: 1. Ch to v nh gi mt s ch tiu cht lng ca hn dch liposom doxorubicin gn PEG.2.BcunghincunhhngcagnPEGlnbmtliposomtithigian tun hon trong mu ca liposom doxorubicin. CHNG I. TNG QUAN1.1. i cng v liposom1.1.1. Khi nimLiposomlcctinhhnhcuccutogmlpvphospholipidbaobcly nhnncgia,ckchthctrongkhongtvichcnanometnhngchc micromet [3],[18].1.1.2. Thnh phn cu to ca liposomHuhtcchmangthucliposomcsdnghinnayucutobicc phospholipid v cholesterol.Phospholipidcdngchtoliposomcthcngungctnhin(nh phosphatidylcholin,phosphatidylserin..)hoctnghp(nhdimyrisitoylphosphatidyl cholin (DMPC), distearoyl phosphatidyl cholin (DSPC)[3]. Mt thng s quan trng c trng cho phospholipid cn phi quan tm trong ch to liposom chnh l nhit chuyn pha. Nhit ny ph thuc ch yu vo di v bo ha ca chui acyl. Nhn chung, chui acyl di hn v bo ha hn th nhit chuyn pha s cao hn [53].Phosphatidylcholinlphospholipidcsdngnhiunhttoliposom.Cc phosphatidyl cholin c ngun gc t nhin nh t lng trng hoc t u nnh thng cha bo ha, nhit chuyn pha thp nn lp lipid kp to thnh c tnh thm cao, km n nh. Trong khi , cc phospholipid tng hp cha cc mch acyl di, bo ha s to ra cu trc lp kp chc v kh thm hn [8],[46].Phosphatidylcholinthngcsdngphihpvicclipidkhc,cbitl cholesterolcithinnnhinvitrovin vivo ca liposom. Cholesterol c vai tr iu chnh lng ca mng phospholipid, gp phn lm tng tnh bn vng ca liposom, gimtnhthmcamnglipidviccchttan,btkphospholipidtoliposom bo ha hay cha bo ha [53].Ngoi hai thnh phn trn, liposom c th cha thm cc polyme thn nc tng thi gian tun hon, cc yu t hng ch: khng th, acid folic, transferrin hoc cc cht chng oxy ha nh alfa-tocoferol1.1.3. Phn loiC nhiu cch phn loi liposom khc nhau. Da vo kch thc v s lp, liposom c th c chia thnh 3 loi chnh: liposom n lp, liposom a lp v liposom nhiu ti. (Bng 1.1)Bng 1.1. Phn loi liposom theo kch thc v s lp.Loi liposom K hiuKch thc (nm)S lp lipid kp1. Liposom n lpUV 20 - 1000 1Liposom n lp nh SUV 20 - 100 1Liposom n lp ln LUV >100 1Liposom n lp khng l GUV > 1000 12. Liposom a lpLiposom a lp nh OLV 100 - 1000 Khong 5Liposom a lp ln MLV > 500 5 253. Liposom nhiu ti MV > 1000 a nhnDa vo thnh phn v ng dng, liposom c th c phn chia thnh cc loi: (1) Liposom truyn thng (conventional liposomes): liposom c to thnh t cc phospholipid khc nhau, cholesterol v c th c cc lipid khc nhng khng c bt k s thay i no trn b mt liposom [20].(2)LiposomnhycmvipH(pH-sensitiveliposomes):s dng cc phospholipid nhycmpH nh dioleyl phosphatidyl ethanolamin, ng dng tng gii phng thuc nhng vng c pH acid nh nh cc khi u tin pht, cc v tr vim [10].(3)Liposomintchdng(cationicliposomes):liposomcchacclipidtch in dng, thng c s dng cho h mang ADN hoc ng dng hng ch [49].(4) Liposom tun hon ko di (Long-circulating liposomes): L cc liposom c phlnbmtcc polymetr, tng thch sinh hc to thnh mt lp bo v bn ngoi liposom,kodithigiantunhoncaliposomnh:poly[N-(2-hydroxypropyl) methacrylamid],poly-N-vinylpyrrolidon,polyvinylalcolvphbinnhtvnl polyethylen glycol [49]. (5)Liposomhngchhayliposommindch(immunoliposomes):lliposom cgnthmccligandtrnbmt,ccligandnyckhnngnhnbit,gnvot bo ch to iu kin cho liposom tch ly ti ch [49]. 1.1.4. Cc phng php to liposom Cnhiuquytrnhkhcnhauchtoliposomnhngtuchungliutriqua cc giai on sau: Giai on hydrat ha hnh thnh liposomgiaionny,phngphpcsdng ph bin nht to liposom quy m phng th nghim l phng php hydrat ha mng lipid mng. Phospholipid v cc thnh phnkhctovliposomchatanvodungmihuc.Dungmicbchi dipsutgimtrongbnhctquaychnkhngtothnhlpmngmngtrnthnh bnh. Lp mng mng c hydrat ha bng mt dung dch nc nhit trn nhit chuyn pha ca phospholipid to thnh cc liposom [3],[8].Ngoira,mtsphngphpkhccthsdngtoliposombaogm: phng php bc hi pha o, phng php siu m, tim ethanol, tim ether, loi b cc cht din hot[3],[8].Giai on iu chnh kch thc liposomLiposomsaukhitothnhtgiaionhydrathathngckchthcln,km ngnht.khcphciunyngitacthsdngmtsphngphplm gimkchthctiuphnnh: siu m, ng chy, vi dng chy, ng nht ha di p sut cao, y qua mng...[8].Giai on liposom ha thuc (ti thuc vo khoang nc ca liposom)Giai on ny c th tin hnh ng thi vi giai on hydrat ha to liposom hoc tin hnh sau khi liposom to thnh. Giai on loi thuc t do (khng c liposom ha)Nhiu thuc thn du c i lc cao vi lp kp v c gi hon ton vo liposom. Tuynhin,vinhiuhpchtkhc,hiusutliposomhathngnhhn100%.Phn thuc khng c liposom ha c th gy ra cc tc dng khng mong mun vt gii hn chophphoclmmtnnhvmtvtlcaliposom.loibchng,cths dng cc k thut nh: thm tch, siu li tm, sc k lc gel hoc trao i ion[8].1.2. Doxorubicin v liposom doxorubicin1.2.1. DoxorubicinDoxorubicin l mt khng sinh nhm anthracyclin c dng ph bin trong ha tr liu ung th, c cng thc cu to nh hnh 1.1.Hnh 1.1. Cng thc cu to ca doxorubicin [48]Tnh cht: Tinh th hoc bt v nh hnh mu cam, khng mi. Tan trong nc, dung dch natri clorid 0,9%, methanol; thc t khng tan trong cloroform, ether v cc dung mi hu c khc [48].Dc ng hc: Sau khi tim tnh mch, doxorubicin nhanh chng thi tr khi mu v phn b vo mbaogmphi,gan,tim,l lch v thn. Doxorubicin b chuyn ha nhanh ti gan to thnhccdnchtchuynha,baogmcdoxorubicinollmtchtcnhottnh. Khong 40 50% lng doxorubicin bt u c thi tr qua mt trong vng 7 ngy; ch khong 5% c thi tr qua nc tiu trong vng 5 ngy [48]. Ch nh: Doxorubicincdngphbintrongiutrnhiuloiungth,baogmbnh Hodgkin, u lympho dng khng Hodgkin, bnh bch cu cp, sarcoma xng v m mm, u nguyn bo thn kinh, u c tnh bng quang, v, phi, bung trng v d dy [48].Tc dng khng mong mun: Mt s tc dng khng mong mun ph bin ca doxorubicin l bun nn, nn, rng tc, c ch ty xng, vim ming, vim thc qun c th tin trin n lot, c tnh trn tim, tn thng thot mch dn n hoi t m [48]. Trong cc tc dng ny, c tnh trn timlnghimtrngnht.ctnhtrntimcadoxorubicinchiathnh2loi:cptnh, thng thy l ri lon chc nng tim thong quabiu hin bng s thay i ECG c th hiphc,lonnhptimvmunlccbnhctimkhnghiphc,phthucvoliu, dnnsuytimsunghuyt.Tltvonglkhong60%nhngbnhnhncnhng biuhincactnhmun.Thiimkhiphtcactnhmunthngxyrakhi liu tch ly khong 550 mg/m2 doxorubicin [48].1.2.2. Liposom doxorubicinNhcimcadoxorubicintdolthtchphnbln(khong25L/kg)vb thi tr nhanh chng khi vng tun hon dn n nng thuc khi u thp m li gy ctnhccmbnhthng.Sdng liposomlm h mang thuc doxorubicin s lm thayicsphnbm,cbitlgimtchlytimvtcthitrca doxorubicin, t lm tng tc dng v gim c tnh ca thuc [20].Doxorubicin c th c liposom ha vi hiu sut cao nh s dng k thut chnh lch pH hoc chnh lch nng amoni sulfat. Nguyn tc chung l pH bn trong mng liposomlpHacid(45,5),pHbnngoiciuchnhvkhong7,4,khi doxorubicindngphnt mi trng bn ngoi s khuch tn vo bn trong sau mt thi gian thch hp [38].Hin nay trn th trng c nhiu ch phm liposom doxorubicin, trong 2 ch phm ni bt l Myocet v Doxil. Myocet l ch phm liposom truyn thng c ch totphosphatidylcholintrngvcholesterol(55:45)[18]vsdngkthutchnh lch pH ti thuc [16]. Ch phm c ng lm 3 l ring bit: 1 l cha doxorubicin dng bt kh, 1 l cha hn dch liposom cha ti thuc trong m citric v l th 3 cha dung dch natri carbonat. Thuc s c ti vo liposom ngay trc khi s dng [30]. Th nghimlmsngphaIIIcaMyocetchothytcdngchngungthtngtnhng gim ng k c tnh trn tim so vi doxorubicin t do [18].Doxillchphmliposomdoxorubicintunhonkodi,viccthnhphn phosphatidylcholinunnhhydroha(56,2%),cholesterol(38,3%),DSPE-PEG2000(5,3%)v-tocoferol(0,2%).ScmtcaPEGlmtngngkthigiantunhon trong mu ca liposom (2,5 gi vi Myocet v 55 gi vi Doxil) v tng t Myocet, c tnh an ton ca Doxil cng c ci thin hn hn so vi doxorubicin dng thuc t do[18]. 1.3. S phn ca liposom sau khi tim tnh mch Sau khi tim tnh mch, liposom tip xc vi cc protein huyt tng v cc t bo mu. Rt nhiu trong s tng tc v c th lm mt n nh lp lipid kp ca liposom hoc khi pht cc qu trnh sinh hc dn ti gia tng gii phng thuc v/hoc dn ti thi tr liposom bi h thc bo n nhn (MPS) (hnh 1.2).Hnh 1.2. Cc giai on sinh l trong s thi tr ca liposom [46]utin,liposomcthtngtcvicclipoproteintrongmu.Tythucvo thnh phn ca liposom, lipoprotein c th tn cng v thoi ha liposom thng qua loi b ccphntphospholipidtcutrcmnglipidkp.Tngtcnydnnsmtn nhcalpmng kp v gii phng cc phn t thuc bn trong liposom vo mu (hnh 1.2, A) [17],[30],[31],[46].Liposom cng c th tng tc vi cc protein huyt tng. Cc protein khi hp ph lnbmtcaliposomcthlmtngtnhthmcamng,nhhngnslugi thuc bn trong liposom; hoc to iu kin MPS nhn bit v thc bo liposom (hnh 1.2,B).Ccproteinnycgilccopsonin,vqutrnhhpphchiuhoc khngchiucachnglnbmtliposomcgilopsoninha[31].Mtscc proteinckhnngopsoninhacxcnh l cc globulin min dch, fibronectin, 2-glycoprotein,proteinCphnngv2-macroglobulin,ccthnhphncabth (C3b, iC3b) [30],[41]. Bncnhccopsonin,tronghuyttngcngtnting thi cc thnh phn c chsthcboccliposomnhalbuminvIgA,chngcgilccdysopsonin.S cn bng gia opsonin ha v dyopsonin ha quyt nh s thi tr ca liposom [30].H thc bo n nhn (MPS) ng vai tr quan trng trong s thi tr ca liposom khivngtunhon.MPSbaogmccithcbotronggan(tboKupffer),lch, phi,rut,da,tyxngvccbchcumonotrongmu.Cctbonycchamt lotccreceptor,chiuchoccglycoprotein,lipidvcconFccaglobulinmin dch.Ccopsoninsaukhihpphlnbmtliposomcthgnviccreceptortrn mng cc t bo ca MPS v dn n s thc bo liposom[52].Liposom cng c th b thi tr khi mu thng qua s tng tc trc tip vi cc t bo thc bo. Cc nghin cu in vitro cho thy s tm bt liposom bi cc i thc bo c th xy ra khi khng c protein huyt tng [29]. Tuy nhin, s gn ca protein ln b mtliposomvnngmtvaitrquantrngtrongs tng tc ca liposom vi h thc bo n nhn [46].Vtquacchngrotrn,ccliposomtunhontrongmusctchly chtheocchthnghocchng[10].Vicchchng,liposomcgn thm cc yu t hng ch v c tch ly ch nh i lc ca cc yu t ny vi ch tc dng. Liposom cng c th tch ly th ng ti khi u rn hoc cc v tr vim nh kh nng thot mch qua cc k h ca thnh mch cc v tr ny v c lu gi li do gim sdnlucahbchhuyttikhiu.Hintngnycgilhiungtngtnh thmvkhnnglugi[49].Tuynhin,stchlycaliposomtikhiulmtqu trnhdinratngichm[24],trongkhisthitrcaccliposomtruynthng khi vng tun hon li qu nhanh c th t c iu ny. V vy, tng thi gian tun honlmtyucutracthtiptcphttrinliposomnhmthmangthuc hiu qu.1.4. Nhng n lc ci thin thi gian tun hon trong mu ca liposomSaukhinhnracstmbtcaMPSlnguynnhnchnhdnnsthanh thi nhanh chng ca liposom ra khi mu tun hon, nhiu nghin cu c tin hnh ci thin thi gian tun hon ca liposom.Mt trong s nhng n lc u tin tng thi gian tun hon ca liposom l thay ilngcalpkpbngcchsdngcc phospholipid c nhit chuyn pha cao vcholesteroltoliposom.Ccliposomctotphospholipidboha nh DSPC c i lc thp hn vi cc opsonin, n nh trong mu hn v thi gian tun hon ko di hnsoviliposomchtotccphospholipidchabohanhphosphatidylcholin trng [6],[20],[46]. Vic iu chnh kch thc tiu phn v in tch ca liposom cng lm gim s tm bt bi MPS. Nhn chung, cc liposom c kch thc ln hn s b thi tr khi mu nhanh hn cc liposom c kch thc nh [42]. Cc liposom khng tch in s c thi gian tun hon di hn so vi liposom tch in m hoc dng [30].Mt bc ngot quan trng din ra vo nhng nm 1987-1988 l pht hin s c mt ca cc lipid t nhin (nh gangliosid GM1 [12] v phosphatidylinositol [25]) trn b mtliposomcthlmtngngkthigiantunhontrongmucaliposom.Tuy nhin, kh khn v kh nng thng mi ha ca cc liposom cha gangliosid GM1 dn nnhucutmkimnguynliuthaythrhn,antonvcchpnhntrnlm sng. Cng vi s ng dng lm tng thi gian tun hon ca cc protein, peptid, cc dn chtpolymecchngminhlchiuqukodithi gian tun hon ca liposom vttrisovigangliosidvphosphatidylinositol[13],[16].Chonnay,ccdncht polymecngdngrngrichtoccliposomtunhonkodi,liposom min dch. Trong , dn cht polyethylen glycol (PEG) c nghin cu nhiu v s dng ph bin nht.1.5. PEG v mt s dn cht PEG-lipid ng dng trong ch to liposomPEGlmtpolymeckhilngphntt50030000Da,ccutrcthng hocphnnhnhctothnhdoslinktcaccphntethylenoxidvinhau bng cc lin kt ha hc (hnh 1.3).Hnh 1.3. Cng thc cu to ca polyethylen glycol [44]PEGcnhngctnhcthnil c mt khng hai, cho php n c s dng rng ri trong lnh vc dc phm: (1) Khng c c tnh, khng sinh min dch, khng c tnh khng nguyn [30],[44],[52],[56].(2) Tan tt trong nc v nhiu dung mi hu c [30],[44],[56].(4) Chui c t l hydrat ha cao v linh hot [9],[50].(5) C sn trn th trng vi cc mc gi tng i thp vi nhiu loi khi lng phn t v cu trc khc nhau.(6) C th gn hoc hp ph d dng ln cc h mang thuc.(7) c US-FDA chp thun cho s dng trn ngi.ckhnnggnvomngliposom,PEGhocccdnchtthnnccan clinktvimtphnthndu,phbinnhtlphosphatidylethanolamine(DSPE) tothnhphchpPEG-DSPE(hnh1.4).KhilngphntcaPEGtrongphchp ctht750n20000Da.Tuynhin,ccnghincuchothykhnngkodithi gian tun hon l tt nht khi khi lng phn t ca PEG t 1000 n 5000 Da [11],[13],[33].MtsccdnchtlipidkhccaPEGcngchothytngthigiantunhonl PEG-Cholesterol,PEG-distearoylglycerol,PEG-oxycarbonyl-3-amino-1,2-propanediol distearoyleste [11].Hnh 1.4. Mt s dn cht lipid ca polyethylen glycol [21],[36]1.6. C ch PEG lm tng thi gian tun hon ca liposom trong muGnPEGlnbmtliposomcchngminhlcnhiuuim,trong quan trng nht l ko di thi gian tun hon trong mu ca liposom, to iu kin t cnng thuc cao ti v tr khi u, ci thin hot tnh chng ung th. Tuy nhin, c ch lm bn vng v tng thi gian tun hon liposom ca PEG vn cn nhiu im cha r rng. C ch c cp nhiu v chp nhn rng ri l PEG lm gim s tng tc ca liposom vi cc protein, gim s tm bt liposom ca MPS.Lchtthndugiabmtlipidcaliposomvproteinto iu kin protein hpphlnbmttiuphn[52].KhignPEGlnbmtliposom,lponcc hnh thnh do i lc cao ca PEG vi cc phn t nc s lm cho b mt liposom tr nn thnnc,lmgimlchtthndu,do,lmstngtccaliposomviprotein huyttng(hnh1.5A).ngthi,scmtcaPEGtrnbmtliposomcntora mtlcykhnggian.Lcynytnglnkhicctiuphnkhclignliposomdo lppolymebnnli(hnh1.5B).Lcykhnggiangpmtphnquantrnglm hnchstngtcgiaccliposomvinhaucngnhgialiposomviccphnt protein v cc t bo khc [37].StngthigiantunhoncaliposomgnPEGcncthcgiithchda trn nh hng ca PEG ti s kt t ca cc tiu phn liposom. Lc y khng gian gia cc liposom gn PEG lm gim s kt t ca chng, gip duy tr cc liposom trng thi ring r vi ng knh nh (khong 100 nm)- kch thc ti u ko di thi gian tun hon[9],[19].Ahlvcngs(1997)nghincumiquanhgiaskttca liposominvitrovithigiantunhoninvivocachng[7].Ktquchothycmi lin quan mt thit gia gia xu hng kt t ca liposom v thi gian tun hon. S kt t ca cc liposom trong huyt tng/ huyt thanh c quan st trn knh hin vi v c tnginhlngcl275,185v15tngngviccchphmliposomch chaDSPC,chaDSPC/Chol(2:1)vDSPC/DSPE-PEG2000(9:1).Thigiantun hon,cphnnhquaAUCtrungbnhchothyliposomgnPEGcthigiantun hon ko di hn hn (AUC l 1,16 0,05) so vi hai ch phm liposom khng gn PEG. (AUC l 0,04 0,02, 0,[35]34 0,04 ln lt vi liposom DSPC v DSPC/Chol).ABHnh 1.5. C ch PEG ko di thi gian tun hon ca liposom trong mu(A): Gim hin tng opsonin ha [16] (B): Tng tc y khng gian lm gim kt t tiu phnMtiuthvlmcdtnhthnncvlcykhnggianctorabi PEGchnhlcchkodithigiantunhoncaliposomnhngkhigndextran, mtpolymetantttrongncvctnhthnnccaolnbmtliposomlikhng chng minh c kh nng ko di thi gian tun hon. iu ny c th lin quan n c tnhlinhng(flexibility)(squaytdocaccnvpolymexungquanhcclinkt ca chng) ca PEG. S linh ng ny khin lp polyme kh thm v to thnh mt lp o baobcvbovtonbliposom,trongkhiccpolymekmlinhngnhdextran khng to ra c s bo v ton din trn b mt liposom [50].1.7. Cc phng php to liposom gn PEGPEG c th c gn vo liposom trong hoc sau qu trnh to liposom. 1.7.1.GnPEGtrongqutrnhtoliposom(phngphptruynthng,pre-modification)Viphngphpny,dnchtlipidcaPEG,vdmPEG-DSPE,cngcclipid khcchatantrongmtphadungmihucvtinhnhhydrathatoliposom theo cc phng php trnh by mc 1.4. PEG s phn b c 2 bn mng lipid kp ca liposom to thnh (hnh 1.6A).Hnh 1.6. Cc phng php to liposom gn PEG [35]1.7.2. Gn PEG sau qu trnh to liposom (phng php post-modification)C2kthutchnhgnPEGlnbmtccliposomtothnhttrc(pre-formed liposomes). K thut th nht da trn c s to lin kt ng ha tr ca PEG hot ha vi nhm phn ng trn b mt ca liposom (hnh 1.6C). K thut ny c xut bi Senior v cng s (1991) [47]. Monomethoxy-poly (ethylene glycol) (M-PEG) c hot ha ti nhmhydroxyltdovitresylclorid.0,5mlliposomnlp(cha20% phosphatidylethanolamin)ctrnvingthtchtresylatmonomethoxypolyethylen glycol (TMPEG) trong dung dch m phosphat (pH 8,5). Sau khi 2 gi 22oC, TMPEG khng phn ng (khng gn ln liposom) c loi i bng sc k lc gel (Sepharose 4B). Cc liposom gn PEG to thnh c thi gian thi gian bn thi di hn liposom khng gn PEG (thi gian bn thi ca liposom gn PEG l 10 gi, liposom khng gn PEG l 7 gi). MtkthutkhccthsdnggnPEGlnbmtccliposomlkthut post-insertion(hnh1.6B).Cscakthutnylkhnngdichuyncaphnt lipid t pha lipid ny sang pha lipid khc trong mi trng nc. nng trn nng micell ti hn, mPEG-DSPE cng nh mt s dn cht lipid khc ca PEG hnh thnh cc micell.KhihndchmicellmPEG-DSPEvihndchliposomiukinnhitv thi gian thch hp, mPEG-DSPE s c chuyn t micell sang cc liposom. K thut cthchinutintrongnghincucaUstervcngs(1996)[51].iung ngc nhin l s chuyn pha ny din ra ngay khi ng thi liposom v micell nhit thp hn nhiu so vi nhit chuyn pha (20oC so vi 58oC)v trong thi gian rt ngn (5 pht). Sau khi qu trnh gn hon tt, mPEG-DSPE tn ti dng monome v c loi bng cch s dng sc k lc gel. 1.8. ng dng ca liposom gn PEG1.8.1. ng dng trong iu tr ung th: Doxil l ch phm liposom gn PEG u tin c US-FDA ph duyt cho iu tr lmsng.MtchphmkhccaliposomdoxorubicingnPEGcmttrnthtrng hinnaylLipo-Dox(TTYBiopharmCompanyLtd,TaipeiTaiwan).Chphm ny s dng DSPC, mt phospholipid bo ha c nhit chuyn pha cao (55oC) thay v s dng phosphatidylcholin u nnh hydro ha nh trng hp ca Doxil . Trong th nghim lmsngphaI,Lipo-Doxtcthigiantunhonkodilnti65gi[18]. Nhvy,cDoxilvLipo-Dox,nhngchphmliposomgnPEGuchothythi gian tun hon vt tri so vi liposom truyn thng nh Myocet.LiposomgnPEGcngangcnghincungdngvicchotchtchng ung th khc nh cisplatin, camptothecin[18],[53].1.8.2. ng dng trong iu tr mt s bnh l khcDohiungtngtnhthmvkhnng lu gi, cc liposom tun hon ko di s c kh nng tch ly th ng khng ch v tr cc khi u m cn cc v tr vim, nhim khun.ChnhvldonymliposomgnPEGcngcngdngtrongiutrcc bnh l vim hoc nhim khun. Bakkervcngs(1993)nghincutcdngcaliposomgnPEG(vitl molPEG-DSPE:PHEPC:Chol=0,15:1,85:1)chagentamicinvceftazidimtrn2m hnhchutcngvimphitridoKlebsiellapneumonianhycmcaovkhng gentamicin/ceftazidim.Viphc liunbtulc24gisaukhicyvi khun cho thyhiuquiutrvttricadngthuckhngsinhtrong liposom gn PEG so vi khngsinhtdo:tngtlsngstcachutnhimkhuncngnhtngtc dng dit khun cc m phi nhim khun [14]. 1.8.3. ng dng trong chn on bnhLiposom gn PEG cng c s dng lm h vn chuyn cc cht nh du phng x,ngdngtrongchnonbnhvimcchkhutrvtrcaccchtphngxv lmgimctnhcachng.Mtschtphngxcsdngnh 67Ga, 111In, 99mTc [49].Trong nghin cu lm sng, k thut chp sciti s dng liposom gn PEG mang 99mTc cho thy nhy v c hiu cao (94% v 89%) 35 bnh nhn nghi ng l c bnh l vim hoc nhim khun [32]. Nhiu nghin cu khc cng cho thy s dng cc liposom gn PEG mang cht phng x l mt phng php hiu qu v thun tin chp sciti,gpphnchnontnhtrngvimhocnhimkhun.Tuynhin, nhng phn ng khng mong mun ngn cn vic s dng cc ch phm ny trn ngi v cn c thm cc nghin cu khc ng dng vo thc tin.1.8.4. Kt hp ti u ha h mang thuc liposomKt hp cc nguyn liu khc nhau trong ch to liposom t c thi gian tun honkodi,khnnghngchvgiiphngthuchpltichlmtxuhng trongnghincu,phttrinliposomhinnay.KazuoMaruyama(2011)gntrasferrin lnbmtPEG-liposomchaoxaliplatin.Cctransferrin-PEG-liposomvackhnng tun hon ko di, duy tr thuc trong khoang gian bo, va c kh nng phn phi chng vobotngcatboungththngquatrunggianreceptortransferrin.Dovy, transferrin-PEG-liposom ci thin tnh an ton v hiu qu ca thuc [40]. CHNG II. I TNG V PHNG PHP NGHIN CU2.1. Nguyn liu, ha cht, thit b v ng vt th nghim2.1.1. Nguyn liu, ha cht: Nguyn liu: -Phospholipidlng trng hydro ha c tiu chun ha theo nghin cu trc [4]. - Cholesterol (Merck, c), DSPE-PEG2000-NH2 (Laysanbio, M)- Doxorubicin hydroclorid (Merck, c)Ha cht:- Cloroform (Trung Quc), isopropanol, amonisulfat (Merck, c)- Triton X-100 (MP Biomedicals, M)- NaCl, KCl, Na2HPO4, KH2PO4, BaCl2, HCl (Trung Quc)V mt s ha cht khc t tiu chun dng cho phn tch.2.1.2. Thit b- My ct quay BUCHI R-210, Thy S. - My khuy t IKA C-MAG HS4, c- My o quang ph UV-VIS U1900 HITACHI, Nht Bn. - Ti thm tch, MWCO: 12000 14000 Da, Spectrum Labs M- My Emulsiflex C5, Canada (Vin Cng ngh sinh hc)- My hunh quang Shimadzu RF1501, Nht Bn (B mn Ha phn tch, Khoa Ha hc i hc Khoa hc t nhin, i hc Quc gia H Ni).- My ly tm lnh Hermle Z326K, c.- My o kch thc tiu phn v o th zeta Zetasizer ZS90 (Anh)- ng heparin ha, bm kim tim 1ml (Vit Nam)-Cnphntch,cnkthut,myopH,tsy,nihptittrng,tlnh,mnglc PTFE 0,2m, mng polycarbonat kch thc l 0,4; 0,08 m2.1.3. ng vt th nghimChut nht trng dng Swiss, BALC 57/6, ging c do Vin v sinh dch t Trung ng cung cp. 2.2. Ni dung nghin cu-ChtoliposomdoxorubicingnPEGvsosnhviliposomdoxorubicinkhnggn PEG v mt s ch tiu: + Kch thc tiu phn, phn b kch thc tiu phn+ Th zeta+ Hiu sut ti thuc- Bc u nghin cu nh hng ca gn PEG ln b mt liposom ti thi gian tun hon caliposomdoxorubicintrongmuthngquavickhostnngdoxorubicintimt sthiimsaukhitimccdngthuckhcnhaucadoxorubicin:dngthuctdo, dng liposom khng gn PEG v liposom gn PEG.2.3. Phng php nghin cu2.3.1.PhngphptoliposomdoxorubicingnPEGvliposomdoxorubicinkhng gn PEGBng 2.1. Nguyn liu to liposom gn v liposom khng gn PEGSTT Tn nguyn liu Liposom gn PEG Liposom khng gn PEG1. Phospholipid trng231,25 mg 250 mg2. Cholesterol 62,82 mg 62,82 mg3. DSPE-PEG2000-NH274,26 mg 0HaimuliposomdoxorubicinkhnggnPEG(liposomdoxorubicin)vliposom doxorubicin gn PEG (PEG-liposom doxorubicin) vi cc thnh phn nguyn liu ban u nh trong bng 2.1 c ch to vi cng mt quy trnh. Quy trnh ch to c xy dng da trn s tham kho cc nghin cu trc [1],[2],[15] v c mt vi thay i ph hp vi iu kin thc t, c th gm cc bc nh sau: Bc 1: Hydrat ha mng lipid trong dung dch amoni sulfat to liposom nhiu lpTo lp lipid mng: Ha tan phospholipid trng hydro ha, cholesterol v DSPE-PEG2000-NH2(nuc)vo15mlcloroform.Tonbdungdchcchovobnhcu dung tch 1000 ml, tin hnh ct quay chn khng 50oC,tc quay t mc 5. Sau khilpmngmnglipidctothnh,tiptc ct quay thm 3 gi lm bay hi ht dung mi hu c. Hydrat ha mng mng lipid bng 25 ml dung dch amoni sulfat nng 250 mM, 50oC,tcquaymc5.Qutrnhhydrathacthchin2gimbolp mng lipid phn tn hon ton vo dung mi phn cc v to thnh cc liposom nhiu lp. Bc2: Lmnhvngnhthakchthctiuphnbngphngphpyqua mng.Liposom nhiu lp to thnh bc trn c y ln lt qua mng polycarbonat vi kch thc l 0,40 m 8 ln v 0,08 m 10 ln. Thit b y qua mng Emulsiflex C5 v mu liposom c lm nng nhit 50oC trc khi tin hnh5 10 pht. p sut y qua mng khong 174 psi. mu liposom sau khi y qua mng n nh trong 1 gi nhit phng. Liposomcboquntrongtlnhnhit 4oC tip tc cc giai on tip theo. Bc 3: Hnh thnh chnh lch nng amoni sulfat Chnhlchnngamonisulfatctothnhbngcchsdngphngphp thmtchtrongmitrngmPBSpH7,4(NaCl8,0g;KCl0,2g;Na2HPO4.12H2O 2,9g;KH2PO40,2gphatrong1Lncct)loimuiamonisulfatmitrng bn ngoiliposom.Thtchdungdchmlntnhtgp100lnthtchdchthmtch. Dung dch m c thay mi sau 2 gi, 7 gi v qu trnh thm tch kt thc sau 24 gi. Trong qu trnh ny, mu c trnh nh sng bng cch bc giy nhm. Bc 4: Liposom ha doxorubicinTrckhitinhnh,hndchliposomclmnngnhit50oCtrong10 pht.Tinhnhrcnhmtlngdoxorubicinthchhpvohndchliposomt nng2mg/mltrongiukinkhuyt.mutrong60phtnhit50oC.Trong qu trnh , mu c bc giy nhm trnh nh sng. Mt phn dch liposom c gi li xc nh nng doxorubicin ban u. Bc 5: Loi doxorubicin khng c liposom haDoxorubicin khng c liposom ha c loi bng phng php thm tch tng t nh bc 3. Dung dch m PBS pH7,4 c thay vo lc 3 gi, 7 gi v qu trnh kt thc sau 24 gi. 2.3.2. Phng php nh gi liposom to thnh: 2.3.2.1. Phng php nh gi kch thc tiu phn v phn b kch thc tiu phnNguyn tc: Kch thc tiu phn c nh gi bng k thut tn x nh sng nng ng (DLS).Khichiuchmtiasngvohndchliposomckchthckhcnhausthu c mc tn x khc nhau ca chm sng ra. Da vo mc tn x ca chm sng ra, ta c th tnh c kch thc ca liposom [27].Tinhnh:Sdngthitbokchthctiuphnvphnbkchthctiuphn ZetasizerZS90.Dchliposomcphalong200lnbngncct2lnlcqua mnglc0,2mvchovocuvetsaochochiucaophndch khong 1 cm. Cho cuvet vo bung o v tin hnh chy my, kt qu thu c hin th trn mn hnh. 2.3.2.2. Phng php xc nh th zeta: Nguyn tc: Th zeta c xc nh gin tip da trn hin tng in di, tc da trn s dichuyntngicahtmanginsovichtlngmncphntnditc dng ca in trng [26].Tin hnh: Pha long mu liposom 200 ln bng nc ct 2 ln lc qua mng lc 0,2 m. Bmttvocuvetsaochokhngcbtkh.Chocuvetvobungocathitb zetasizer ZS90 v tin hnh o. Kt qu hin th trn mn hnh. 2.3.2.3. Phng php nh lng doxorubicinNguyn tc: Ph v mng liposom bng cht din hot Triton X-100, tin hnh nh lng doxorubicinbngphngphpoquang.Nngcadoxorubicinctnhdavo ng chun xy dng trc . Tin hnh: * Xy dng ng chun doxorubicinDydungdchchuncphanhsau:Cnchnhxc25mgdoxorubicin hydroclorid,hatantrongdungdchNaCl0,9%vchovobnhnhmc25ml.Thm nc n vch c nng 1 mg/ml. T dung dch ny pha long bng NaCl 0,9% cccdungdchcnngdoxorubicinhydrocloridlnltl0,1;0,15;0,2;0,25; 0,3; 0,35 v 0,4 mg/ml. Ht chnh xc 1 ml cc dung dch trn v thm vo 9 ml dung dch TritonX-1001%phatrongncct2ln(tt/tt).Lckvemohpthtibc sng480nmvimutrngcphat1mlNaCl0,9%v9mlTritonX-1001%.S dng phn mm Excel v th th hin tng quan gia nng dc cht (CS) v hp th (DS), xc nh phng trnh hi quy v h s tng quan R. * nh lng doxorubicin trong h liposomMu th: ly chnh xc 125 l dch liposom, pha long bng NaCl 0.9% thnh 1 ml. Thm vo 9 ml Triton X-100 1%. Lc mnh. un cch thy hn hp 60oC trong 4 pht, thnhthonglcgiiphngthuchonton.hnhpvnhitphngritin hnh o quang bc sng 480 nm. Mu th c lm 3 ln v ly kt qu trung bnh. Mutrng:lychnhxc1mlNaCl0,9%,thmvo9mlTritonX1001%.Lc mnh.2.3.2.4. Xc nh hiu sut liposom ha doxorubicin Tin hnh: nh lng doxorubicin trong h liposom trc (Ct) v sau (Cs) khi thm tch loidoxorubicintdo(khngcliposomha)theophngphpnutrongmc 2.3.2.3. Hiu sut liposom ha doxorubicin c xc nh theo cng thc (1): (1)Trong:Ct vCslnltlnngdoxorubicin trc v sau khi thm tch loi doxorubicin t do. 2.3.3.PhngphpnghincunhhngcagnPEGlnbmtliposomti thi gian tun hon trong mu ca liposom doxorubicin2.3.3.1. Chun b cc iu kin tin hnh th nghim* ng vt th nghim: Chut nht trng c a v phng th nghim (b mn Dc lc) nui 5 ngy cho nnh.Trckhitimthuc1ngy,chochutnhniquamxcnhcnnng cachut.Chnnhngcthckhilngt2228g,chia ngu nhin thnh 4 nhm: nhmT(nhmtrng,timncmuisinhl),nhmF(timdoxorubicindngtdo), nhm C (tim liposom doxorubicin), nhm S (tim PEG-liposom doxorubicin). * Thuc tim: - Thuc tim doxorubicin dng t do: ha tan doxorubicin hydroclorid trong NaCl 0,9% v khuncnngkhong2mg/ml.LcquamngPTFE0,2m,bmtphndch lc u. Phn dch sau c ng vo l hp tit khun trong ni hp 100oC trong 60 pht.Trnuvhtmt phn dch ra bng bm tim v khun tin hnh nh lng nng doxorubicin theo phng php m t trong mc 2.3.2.3.-ThuctimliposomdoxorubicinvPEG-liposomdoxorubicin:lcquamngPTFE0,2m,bmtphndchlcu.Lcudchthucvmtphncngclyra nh lng nng doxorubicin.-Cuicng,phalongccdngthuctimtrn bng dung dch NaCl 0,9% v khun t nng 1 mg/ml. 2.3.3.2. Tim chut v ly mu 4 nhm chut li c chia ra thnh cc nhm nh (3 con/nhm nh), tng ng vi cc thi im s ly mu sau khi tim: 5 pht, 4 gi v 24 gi. Chut c tim tnh mch uincmuisinhl(nhmT)hocdoxorubicin(ccdngtngng)viliu10 mg/kgtrongthtchtim10ml/kg.Sauccthiim5pht,4gi,24gi,muchut clythcmtvongheparinha.ngheparinhacynplivlnnh giahailngbntayva ln a xung nh nhng mu trn u vi heparin trong ng. 2.3.3.3. Phng php chit doxorubicin ton phn trong huyt tngNguyn tc: S dng isopropanol acid ha (0,075N HCl trong isopropanol 90% (tt/tt)) chit doxorubicin ton phn t huyt tng [25],[28].Tin hnh: Quy trnh chit c m t trong hnh 2.1.Hnh 2.1. Quy trnh chit doxorubicin ton phn t huyt tng2.3.3.4. Phng php nh lng doxorubicin trong huyt tngNguyn tc: nh lng doxorubicin bng phng php o quang ph hunh quang. Nng doxorubicin c xc nh t mt hunh quang ca dch chit doxorubicin v ng chun xy dng t cc dung dch doxorubicin bit trc nng . Do trong huyt tng tn ti c cc dn cht chuyn ha ca doxorubicin m phng php o quang ph hunh quangkhngphnbitcnnnngxcnhclnngtngng doxorubicin (doxorubicin equivalents) [28].Tin hnh: * Xy dng ng chun doxorubicin: - Pha cc dung dch chun: Cn chnh xc 25 mg doxorubicin, ha tan bng NaCl 0,9% ri chovobnhnhmc25ml,bsungNaCl0,9%nvchcdungdch doxorubicin hydroclorid nng 1 mg/ml. Ht chnh xc 0,5 ml cho vo bnh nh mc 10 ml, thm NaCl 0,9% n vch c dung dch 50 g/ml. Sau pha long bng NaCl 0,9% c cc dung dch chun c nng 0,25; 0,5; 1,0; 5; 10 g/ ml. Ht 100 l cc dungdchchun,thmvo2,4mlisopropanolacidha,lcu.Tinhnhomt hunh quang bc sng kch thch 471 nm, bc sng pht x 554 nm. -Mutrng: ht 100 l dung dch NaCl 0,9%, thm vo 2,4 ml isopropanol acid ha, lc u. * o hunh quang cc mu chit doxorubicin t huyt tng: Ccngnghimboqun-20oCclyravvnhitphng.Lcu ng nghim 1-2 pht. Tin hnh o mt hunh quang cc mu chit nhm F,C,S bc sng kch thch 471 nm, bc sng pht x 554 nm vi mu trng l dch chit t nhm T. Trong trng hp gi tr hunh quang nm ngoi ng chun, mu chit c pha long vi isopropanol acid ha v mu trng cng c pha long vi t l tng t. 2.3.3.5. Tnh gi tr AUC0-24h ca cc dng thuc ca doxorubicinGitrdintchdingcong0n24gi(AUC0-24h)caccdngthuc: doxorubicin t do, liposom doxorubicin v PEG-liposom doxorubicin c tnh t nng tng ng doxorubicin trung bnh trong huyt tng theo quy tc hnh thang. Cng thc tnh c th nh sau: AUC0-24h = 0,5 x [(C0,083 + C4) x (4 0,083) + (C4 + C24) x 20] (2)Trong : AUC0-24h l gi tr din tch di ng cong t 0 24 gi.C0,083, C4, C24 l nng tng ng doxorubicin trung bnh ti thi im 5 pht, 4 gi v 24 gi. 2.3.4. Phng php x l s liu- Cc s liu c x l bng phn mm Microsoft Office Excel 2007 tnh gi tr trung bnh v lch chun SD. - S dng Students T-Test nh gi s khc nhau gia cc kt qu. S khc bit c ngha thng k khi p < 0,05.CHNG III. THC NGHIM, KT QU V BN LUN3.1. Kt qu3.1.1. Ch to liposom doxorubicin gn PEGLiposomgnPEGctothnhtccnguynliuPLlngtrnghydro ha, cholesterol v DSPE-PEG-NH2 vi t l mol tng ng 1,85:1:0,15. Sau khi ha tan hon ton cc nguyn liu vo cloroform v s dng my ct quay chn khng bc hi cloroformnhit50oC,tcquaymc5,lplipidtothnhtngimng. Hydrat ha bng dung dch amoni sulfat thu c hn dch liposom c mu trng c, hi vng, ng nht. Cc liposom to thnh sau c lm nh kch thc v ng nht ha bng cch y nhiu ln qua cc mng c kch thc gim dn. Hn dch liposom thu c sau qu trnh ny v cm quan thy trong hn. Liposom khng gn PEG cng c to ra viquytrnhtngtvgnnhkhngcskhcbitviliposomgnPEGkhinhn bng mt thng. Sau khi doxorubicin c ti vo liposom, hn dch liposom c mu , phn tn tt, khng b kt ta hay vn cc. 3.1.2. nh gi liposom gn PEG to thnh 3.1.2.1. Kch thc tiu phn v s phn b kch thc tiu phn Kchthctiuphntrungbnhvphnbkchthctiuphncaccliposom to thnh c xc nh bng thit b Zetasizer ZS90 nh trnh by mc 2.3.2.1. Kt qu c th hin trong bng 3.1 v ph lc 1.Bng3.1.Kchthctiuphnvphnbkchthctiuphncaccmu liposom gn PEG v liposom khng gn PEGMu liposom PDIKch thc trung bnh theo ng knh (nm)Phn b KTTP theo th tch (%) rng pic (nm)Trc khi ti thucLiposom 0,074 108,9 100 34,98PEG-liposom 0,081 100,6 100 33,12Liposom sau khi ti thucLiposom doxorubicin0,110 135,8 100 51,53PEG-liposom doxorubicin0,179 124,9 100 70,18Nhn xt: -Viliposomtrckhitithuc:Khngcskhcbitngkgialiposomgnv khnggnPEGsaukhisdngphngphpyquamnglmnhkchthcv ng nht ha tiu phn. Ch s a phn tn (PDI) tng i thp (PDI 30mV)chothykhnngnnhcahlkhcao.Th zeta ca liposom gn PEG rt thp, - 10,1 mV trc khi ti thuc v - 8,88 mV sau khi ti thuc. So vi liposom khng gnPEG,liposomgnPEGdichuynchmhnvgitrtuyticathzetado cng gim.3.1.2.3. Hiu sut liposom ha doxorubicin3.1.2.3.1. Kt qu xy dng ng chun doxorubicin hydrocloridDungdchchundoxorubicinhydroclorid(DOX)cphatrongNaCl0,9%vi cc nng 0,1; 0,15; 0,2; 0,25; 0,3; 0,35; 0,4 mg/ml. Pha long 10 ln cc dung dch trn bngTritonX-1001%ritinhnhohpthbcsng480nm.Ktqumi tngquangiahpth(DS-DOX)vnngdungdchDOX(CS-DOX)cth hin trong bng 3.3.Bng 3.3. S ph thuc ca hp th vo nng ca doxorubicin hydroclorid trong mi trng NaCl 0,9% v Triton X-100 1% (tt/tt)CS-DOX (mg/ml)0,01 0,015 0,02 0,025 0,03 0,035 0,04DS-DOX0,201 0,299 0,403 0,507 0,608 0,711 0,824Ch thch:CS-DOX: Nng ca dung dch DOX (mg/ml)DD-DOX: hp th ca dung dch DOX ti bc sng 480nm.Tktqutrn,chngtidngththhinstngquangiahpthv nngDOXtrongmitrngNaCl0,9%vTritonX-1001%.thcbiudin trong hnh 3.1.Hnh 3.1. th th hin s ph thuc ca hp th vo nng doxorubicin hydroclorid trong mi trng NaCl 0,9% v Triton X-100 1%Nhn xt:TthtathytrongnngDOXkhost(t0,010,04mg/ml),hpth phthuctuyntnhvonngDOX.ngchunthucdngngthng,c phng trnh hi quy l y = 20,7x 0,009 vi h s tng quan R = 0,999 1. Do vy, c th s dng phng php o quang bc sng 480 nm vi khong nng trn nh lng DOX. 3.1.2.3.2. Hiu sut liposom ha doxorubicin Doxorubicinctivolinccaliposomnhschnhlchnngamoni sulfat. Doxorubicin t do (khng c liposom ha) c loi i bng phng php thm tch.Nngdoxorubicintonphncamuliposomtrcvsaukhithmtchloi doxorubicintdocxcnhdavophngtrnhhiquythitlpctrongmc 3.1.2.3.1. Hiu sut liposom ha doxorubicin c tnhtheo cng thc (1) mc 2.3.2.4. Kt qu c th hin bng 3.4.Bng 3.4. Hiu sut liposom ha ca mu liposom gn PEG v liposom khng gn PEGMu liposom Trc loi DOX t do Sau loi DOX t do Hiu sut(%) hp thNng (mg/ml) hp thNng (mg/ml)(%)Liposom0,481 1,894 0,430 1,697 89,60PEG-liposom 0,483 1,901 0,397 1,569 82,54Nhn xt:Hiusutliposomhadoxorubicinca hai mu liposom u kh cao. C s chnh lchnhgiahiusutliposomhaca2muliposom:muPEG-liposomchiusut thp hn (82,54% so vi 89,60%).3.1.3.BcutmhiunhhngcagnPEGlnbmtliposomtithigian tun hon trong mu ca liposomThigiantunhontrongmucaliposomcnhgithngquanng doxorubicintonphntronghuyttng.Doxorubicintronghuyttng c chit theo quytrnhmtmc2.3.3.3vcohunhquangbcsngkchthch471nm, bcsngphtx554nm.Tmthunhquangthuc,nngtngng doxorubicincxcnhdavongchunxydngtccdungdchchun doxorubicin bit trc nng . 3.1.3.1. Kt qu xy dng ng chun doxorubicin Phaccdungdchdoxorubicinhydrocloridcnng0,25;0.5;1,0;2,5;5;10 g/ml.100lccdungdchtrncthmvo2,4mldungdchisopropanolacidha. Tinhnhohunhquangvibcsngkchthch 471 nm, bc sng pht x 554 nm. Kt qu thu c c th hin trong bng 3.5.Bng 3.5. S ph thuc ca mt hunh quang vo nng dung dch doxorubicin hydrocloridNng DOX (g/ml)0,01 0,02 0,04 0,1 0,2 0,4Mt hunh quang0,485 1,743 4,273 11,247 24,865 45,171Tccktqutrn,chngtidngththhinstngquangiamt hunh quang v nng DOX. th c biu din trong hnh 3.2.Hnh 3.2. th biu din tng quan gia mt hunh quang v nng doxorubicin hydrocloridNhn xt: th cho thy s tng quan cao gia nng DOX v mt hunh quang. Do vycthsdngphngphpoquangphhunhquangnhlngdoxorubicin trong dch chit. 3.1.3.2. Kho st nng doxorubicin trong huyt tng Nng doxorubicin trong huyt tng chut nht c nh lng v so snh gia 3nhm:nhmtimthucdoxorubicintdo(F-DOX),nhmtimliposomdoxorubicin khnggnPEG(CL-DOX)vnhmtimliposomdoxorubicingn PEG (SSL-DOX) vi liu 10 mg/kg. Kt qu c th hin trong bng 3.6 vhnh 3.3. Bng 3.6. Nng tng ng doxorubicin trong huyt tng chut nht ti cc thi im 5 pht, 4 gi v 24 gi sau khi tim tnh mch uiThi im (gi)Nng tng ng doxorubicin trung bnh (g/ml huyt tng) SDp(gi) F-DOX (1) CL-DOX (2) SSL-DOX (3)0,083 13,79 1,33 133,73 10,98 152,49 14,06p2-1 < 0,05 p3-1 < 0,05p3-2 > 0,054 5,09 1,98 64,24 5,42 112,06 8,41p2-1 < 0,05p3-1 < 0,05p3-2 < 0,0524 - 8,64 0,56 51,61 3,51 p3-2 < 0,05(- : Khng pht hin c.)Hnh 3.3. Nng tng ng doxorubicin trong huyt tng chut nht ti thi im 5 pht, 4 gi, 24 gi sau khi tim tnh mch uiNhn xt: Ti thi im 5 pht, nng tng ng doxorubicin ca dng doxorubicin t do thphnkhong9-10lnsovidngliposom.thiim4gi,nngtng doxorubicincnlikhthpvnthiim24githkhng pht hin c. Nng tng ng doxorubicin ti thi im 5 pht vi 2 ch phm liposom c chnh lch mt cht,tuynhinskhcbitkhngcnghathngk(p>0,05).Schnhlchtngln theothigian.Cth,nngtngngdoxorubicintithiim4gikhidng PEG-liposom doxorubicin cao hn gp khong 2 ln so vi dng liposom khng gn PEG v n 24 gi s chnh lch ny ln ti gn 6 ln. T nng tng ng doxorubicin trong mu, AUC0-24h c tnh theo cng thc (2) nh trnh by mc 2.3.3.5. Kt qu c th hin trong bng 3.7.Bng 3.7. Gi tr AUC0-24h ca cc dng thuc doxorubicin trn chut nht (tim tnh mch ui, liu 10 mg/kg)Dng thuc DOX t do Liposom DOX PEG-liposom DOXAUC0-24h(g.h/ml)87,88 1116,52 2154,82Nhn xt: Gi tr AUC0-24h vi liu tim tnh mch 10 mg/kg ca PEG-liposom doxorubicin l cao nht, gp khong 1,9 ln so vi dng liposom doxorubicin khng gn PEG v khong 24,5 ln so vi dng doxorubicin t do. Nh vy, t cc kt qu trong bng 3.6 v 3.7, c th kt lun s b l gn PEG ln b mt liposom c kh nng lm tng thi gian tun hon ca liposom trong mu.3.2. Bn lun3.2.1. V quy trnh to liposom doxorubicin gn PEG3.2.1.1. La chn nguyn liu v cng thcNghincucthchinlstipniccktqutcvchto liposomdoxorubicintrcvimongmunkhcphcnhcimchnhcaliposom truyn thng l nhanh chng b thi tr khi vng tun hon, to mt bc tin gn hn ng dng liposom vo s dng trong thc tin. Trong qu trnh nghin cu, chng ti c gng vn dng cc ci tin k thut, cng nh la chn quy trnh tin hnh c th to ra liposomdoxorubicinckhnngtunhonkodittnhtdatrniukinhinc. utin,lsdngnguynliulphospholipidlng trng hydro ha thay cho phospholipid lng trng cha c hydro ha. S bo ha cc dy ni i s lm tng nhitchuynphacaphospholipid,tngtnhbnvng,gimtnhthmcalpmng kp. Cng thm vic s dng cholesterol cng ng gp mt phn quan trng tng tnh n nhcamngvtngthigiantunhoninvivocaliposom[20].LiposomgnPEG ctothnhtdnchtPEG-lipidlDSPE-PEG2000-NH2cchngminhlc khnngtngthigiantunhon[57].TlcaDSPE-PEG-NH2chngtilachnl 5% v s mol so vi tng lipid. T l ny cng l t l thch hp t c thi gian tun hon ko di [11],[57].3.2.1.2.QuytrnhchtoliposomdoxorubicingnPEGvccch tiu cht lng ca ch phm to thnhS dng phng php truyn thng to liposom gn PEGPhngphpgnPEGtrongqutrnhtoliposom(phngphptruynthng)l phngphpcsdngphbintrnthgiitoliposomgnPEG.Dodncht PEG-lipidctrncng vi cc nguyn liu to liposom trc khi hydrat ha to thnh mngkp,nnphnPEGsphnbc2bnmnglipidkpcaliposom(hnh1.5). Nh vy, so vi phng php gn PEG sau khi to liposom, phng php ny cn s dng mt lng ln hn dn cht DSPE-PEG t c mt PEG tng ng bn ngoi liposom. Tuy nhin, phng php ny li c nhng u im: t bc thc hin hn, khng cn loi micell sau khi qu trnh gn kt thc (so vi k thut post-insertion), liposom to thnh khng b nh hng bi cc tc nhn phn ng (so vi k thut to lin kt cng ha tr).Clchnhvvymphngphpgntrongqutrnhtoliposomvnlphng php c s dng ph bin trong cc nghin cu to liposom gn PEG. cng l l do chng ti la chn phng php ny to liposom gn PEG.Kch thc tiu phn v phn b kch thc ca liposom to thnhCi tin hn cc nghin cu trc [1],[2], trong nghin cu ny s dng phng php y qua mng lm gim v ng nht ha kch thc tiu phn. Phng php ny chokchthctiuphnnhvngnhthnsoviphngphpsiummtcgi Nguyn Th Hu [1], Nguyn Vn Lm [2] s dng. Kchthctiuphnlmtyutquantrng,khngchnhhngtithigian tun hon ca liposom trong mu m cn nh hng ti kh nng tch ly ca liposom ti khi u. Nghin cu trc y ch ra liposom gn PEG c kch thc 100 200 nm c thi gian tun hon ko di nht, trong khi cc liposom c kch thc > 200 nm hoc < 70 nm nhanh b thi tr khi vng tun hon hn [39]. Ngoi ra, mc d cc liposom c kch thc khong 400 nm vn c kh nng thot mch vo khi u, nhng t c hiu quttnht,kchthcliposomnndi200nm[43],[45].Chnhvnhngldotrn, chng ti la chn mng y cui cng vi kch thc l lc l 80 nm nhm to ra cc liposomckchthckhong100nm[20].LiposomdoxorubicingnPEGtothnhc kch thc trung bnh 124,9 nm l tng i ph hp t c hiu qu tun hon ko di v c th a thuc ti ch nh hiu ng tng tnh thm v kh nng lu gi. Th zeta ca liposom to thnhTh zeta ca cc liposom khng gn PEG l tng i tt (|Z| > 30 mV) (-32,6 mV v-40,1mVtngngvitrcvsaukhitithuc)vtthnsoviccnghincu trc(-26mV[1],-25,8mVv-25,6mV[5]).Ktqubng3.2cho thy liposom gn PEG to thnh c th zeta nh hn nhiu so vi liposom khng gn PEG. Nguyn nhn c thdoscmtcachuiPEGtrnbmtliposomlmgimsdichuynca liposom trong in trng v do lm gim th zeta [33],[54]. Ngoi ra, theo Kostarelos vcngs(1999),khignccpolymelnbmtliposomlmchomtphngtrtdi chuyn ra xa b mt liposom v do lm gim gi tr tuyt i ca th zeta [34]. McdsgnPEGlmgimgitrtuyticathzetanhngnkhnglm gim in tch b mt ca liposom [54]. Do , th zeta trong trng hp ca liposom gn PEG khng gip ta d on c v s n nh ca h. Hiu sut liposom ha doxorubicinCn c vo kt qu t c ca tc gi Nguyn Th Hu [1], chng ti la chn k thut k thut chnh lch amoni sulfat ti doxorubicin vo liposom gn PEG. y cng lkthutcsdngtrongquytrnhchtocaDoxil[16].Hiusutliposomha doxorubicintc(bng3.4)tngicao(>80%).LiposomgnPEGchohiusut hi thp hn mt cht (82,54% so vi 89,60 % ca liposom khng gn PEG). iu ny c th l gii l do chng ti cha ti u ha c t l ti thuc, cng nh l iu kin tin hnh loi thuc t do. Khi tin hnh thm tch loi thuc t do trong khong thi gian di(24gi)nhitphngthbncnhvicthuctdobloiiththuctrong liposom cng c th b r r, dn n lm gim hiu sut. khc phc iu ny c th s dngthitblctiptuynnhcatcgiNguynVnLm[2]hocsdngphng php sc k lc gel [22] lm gim thi gian tip xc vi nhit cao ca liposom. 3.2.2. V quy trnh chit doxorubicin trong huyt tngTrongqutrnhchitdoxorubicinthuyttng,chngtinhnthycmtvi im cn lu . Th nht v thi gian ly tm mu ton phn ly huyt tng. Khi ly tm 3000 vng/ pht trong 10 pht th s m bo huyt tng tch ra hon ton v d ly hn l 5 pht. Trong nhng trng hp nng doxorubicin cao, huyt tng s c mu tng t dung dch doxorubicin. Th hai, qu trnh tin hnh nn c thc hin 25 27oC v cc ng nghim phi yntknhnchsbayhicaisopropanol.Bclytmloiproteinnntin hnh trong thi gian l 20 pht vi tc quay 4000 vng/ pht. Thi gian ngn hn, v d 10 pht, c th khng tch ht protein v khi bo qun -20oC protein s b ta li. 3.2.3.VnhhngcagnPEGlnbmtliposomtithigiantunhonca liposom trong muQua kho st nng doxorubicin trong huyt tng ca cc dng thuc khc nhau chng ti thy nng tng ng doxorubicin ca doxorubicin t do thp hn nhiu so vi doxorubicin dng liposom ngay t thi im u (5 pht). iu ny phn no phn nh s thay i v c tnh phn b ca thuc: doxorubicin t do sau khi tim tnh mch phn b nhanh chng vo cc t chc, dn n nng doxorubicin trong pha mu thp v lm tngctnhticcmbnhthng.Khcvidoxorubicintdo,khitimcng1liu, dngliposomdoxorubicinchonngtngngdoxorubicintronghuyttngcao hnmtcchcnghac3thiim.Tktquny,cthdonliposom doxorubicindochngtichtockhnngscithincphnnoctnhca doxorubicin. Ktquhnh3.3chothyliposomgnPEGchonngtngng doxorubicintronghuyttngcaonhttrongc3thiimkhost.Mcd cha tch ringcliposomtronghuyttngnhlngnngthuctrongliposomnhng s phn b v chuyn ha nhanh chng ca doxorubicin t do cng l c s chng ti tintngphnlndoxorubicintronghuyttngtntidngtrongliposom.Stin tng ny cng da trn c s cc nghin cu trc [23],[28]. ng thi, trong nghin cucaGabizonvcngs(1994)vichphmliposomdoxorubicingnPEG,tcgi khngphthinclngngknoccdnchtchuynhacadoxorubicin trongtronghuyttngbngkthutscklnghiunngcao[23].Dovy,nng tng ng doxorubicin trong huyt tng ca nhm chut tim liposom doxorubicin gn PEG cao hn c ngha thng k so vi nhm chut tim liposom doxorubicin khng gn PEG ti cc thi im xa (4 gi, 24 gi) phn no chng minh kh nng ca PEG ko di thi gian tun hon trong mu ca liposom. GitrAUC0-24htnhcbng3.7chothyAUC0-24htngkhong1,9ln nhmchuttimliposomdoxorubicingnPEG(AUC0-24h=2154,82g.h/ml)sovi nhmchuttimliposomkhnggnPEG(1116,52g.h/ml).Cncvonng doxorubicin ti thi im 24 gi bng 3.6 chng ti d on s khc bit v gi tr AUC c th s tng ln nu tip tc kho st cc thi im xa hn nh 48 gi, 72 gi. Nhvy,viccktqutc,cth s b kt lun, khi gn PEG ln b mt liposom vi t l 5% theo s mol so vi tng lipid c tc dng ko di thi gian tun hon ca liposom trong mu. KT LUN V KIN NGHKT LUN ti hon thnh c 2 mc tiu ra:1.ChtocliposomdoxorubicingnPEGbngphngphpgnPEGtrong qutrnhtoliposom, s dng phng php y qua mng lm nh v ng nht kch thctiuphnvkthutchnhlchnngamonisulfattithuc.Liposomto thnh c kch thc di 200 nm, hiu sut liposom ha khong 82%. 2. Bc u quan st c kh nng ko di thi gian tun hon liposom trong mu ca gn PEG ln b mt liposom qua vic kho st nng doxorubicin trong mu ti cc thi im 5 pht, 4 gi, 24 gi sau khi tim tnh mch ui chut. Kt qu cho thy nng tngngdoxorubicinticcthiimxanh4gi,24gikhitimliposom doxorubicingnPEGucaohncnghathngksovitimliposomdoxorubicin khng gn PEG. AUC0-24h ca liposom gn PEG cao hn 1,9 ln so vi liposom khng gn PEG.KIN NGHDo thi gian v iu kin tin hnh nghin cu cn hn ch, chng ti mi ch dng li vic to liposom doxorubicin gn PEG v bc u quan st liposom doxorubicin gn PEGckhnngtunhonko di hn liposom doxorubicin khng gn PEG. Chng ti kin ngh trong thi gian ti cn:- Tip tc nghin cu hon thin quy trnh ch to doxorubicin gn PEG.- Nghin cu nh hng ca gn PEG ln b mt liposom ti cc thng s dc ng hc v s tch ly ca liposom doxorubicin ti khi u.- nh gi tc dng ca liposom doxorubicin gn PEG trn khi u ng vt. TI LIU THAM KHOTI LIU TING VIT 1. NguynThHu(2012),Nghincucitinkthuttoliposomdoxorubicint phospholipid lng trng, Kha lun tt nghip dc s i hc, Trng i hc Dc H Ni.2. Nguyn Vn Lm (2012), Nghin cu bo ch thuc tim liposome doxorubicin v nhgitcdngtrnkhiungvt,Lunvnttnghipthcsdchc, Trng i hc Dc H Ni.3. 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