DANH DAU VI HAT ALBUMIN VOI DONG VI PHONG XA YTTRIUM-90
NGUYEN THI THU, NGUYEN THI KHANH GIANG, BUI VAN CU^NG, VO THI CAM HOA, DANG HO H 6 N G QUANG, NGUYEN DANG KHOA,
MAI PHUdC THO, NGUYEN THANH BINH, DUONG VAN DONG Vi?n Nghien cuu hgt nhdn. Da Lgt, Vi?t Nam
Abstract: This paper presents the process of the radiolabeling of microaggregated albumm particles with radionuclide Yttrium-90 using the directed method. The albumin microsphere kit was prepared in sodium phosphate buffer. The original solution includes 2 mg albumin particle and 0.5 mg stannous chloride dihydrate. The albumin particles size was ranged from 5 jim to 30 ]xm. The mixture was washed three times with phosphate buffer saline, pH 7.2 by centrifugation and suspended in 0.5 M sodium acetate buffer, pH 6. Yttiium-90 in 1.0 M acetic acid was collected from ^^Sr/^^Y generator. The labeling of the particles with ^Y (185 MBq) was performed at pH 5.5 in acetate buffer with agitating for 60 mm at room temperature. The labeled albumin suspensions were centrifuged at 3,000 rpm for 15 min. Labeling yields was calculated using centrifligation, filtration and compared with paper chromatography, which is developed in the Tris Acetic EDTA. In this system, the unbound of Y-90 migrates to an Rf of 0.9-1.0 and the radiolabeled albumin particles remains at the point of origin (Rf=0). The size of Y-albumin particles was compared with the albumin particles in the original solution to be sure that they didn't change during the labeling treatment.
The radiolabeling yields were more than 80%. The labeled compound was dialysis in phosphate buffer.
The radiochemical purity was 98%. The ^°Y- albumin is an Ideal radiopharmaceutical for potential use in malignant cancer treatment as brachytherapy.
Tir khoa: 90Y- albumin microspheres, radiopharmaceuticals, brachytherapy
I. MO DAU
Ung thu la mpt nhom cae bSnh lien quan din viec phan chia t^ bao mpt each vo to chiic. hien nay, thuoc ddc tri chua co, moi phuong phdp phau tri, xa tri lieu hay hoa tri lieu cUng chi giiip dap iing mot phdn trong dieu tri. Do v?ly, viec nghien ciiu tim ra thuoc dieu tri ung thu dang la van de cdp bach. Hien nay, viec nghien ciiu cac phan tti sinh hpc danh dau vdi dong vi phong xa, da md ddu cho hudng dilu tri ung thu mdi, dd la xa tri dp sdt vdi uu diem tieu diet cdc te bdo bi ung thu vd khong gdy anh hudng tdi cac ti bdo Idnh xung quanh, day la hudng dieu tri dang dupe dp dung ngdy cang nhilu, vd hiia h?n nhung hieu qua dieu tri ro r6t. ^"Y la dong vi phdng xa co quang ch?y ti-ong ti bao dai nhat (11 mm tuong img khoing 600 ta bdo), phdt tia beta (P) vdi nang lupng cao 2308 KeV, do dd ^Y Id d6ng vi phong xa co kha nang tieu diet te bao ung thu noi troi hon so vdi cae d6ng vi phdng xa khac, dac biet la cac khoi u rdn. Hien nay tien thi gidi, cac vi hat albumin da duoc dilu chi va sd dung trong y hpc, vi du nhu kit ^""Tc-MAA, Tc-nanocoll dung dl chyp xa hinh chdn doan phii, tiiy xuong va cac nhilm trimg, ^**Y-albumin dupe nghien curu dung trong xa tri ^P sdt. Trong bao cdo nay chiing tdi trinh bay ket qua nghien ciiu cdc dilu kien tdi uu dl danh ddu vi hat albumin vdi ddng vi phdng xa ^°Y, dong thdi kilm tra chdt lupng hpp chdt danh ddu dat chat lupng sii dung cho y hpc.
Hgi nghi Khoa hgc vd Cdng ngh? hgt nhdn todn qudc Idn thu IX, Ninh Thudn 18-19/8/2011 393
II. PHU'ONG PHAP NGHIEN CI^tT 1. Nguyen li^u, hoa chat
Cac boa chdt diu Id nhung hoa chat tinh khiet dupe sii dung trong nghien eihi vl sinh hpc phdn tir va danh ddu phong x?. '^'Y dupe tdch tu nguon '^Sr (Vien Nghien ciru hat nhan), djt cdc tieu chudn boat dp phdng xa, dp sach hat nhdn, dp sach hoa phong xa. Vi hat albumin dupe dieu che tit human serum albumin (HSA) tai Vien Nghien ciiu hat nhan, dang hot dong kh6, kich tiiudc 5-30 pm. Cdc loai hda chdt khac nhu NaH2PO4.2H20, KH2PO4, KCI, SnCl2.2H20, EDTA, Tris, NaOH 2M, HCi 2M, acid acetic, NaCI, acid ascorbic dimg dl pha cac loai dem deu dupe mua tii hang Sigma bode Merk.
2. Phtrcmg phap danh dau
Phucmg phdp nay dupe dua ra bdi nhom nghien ciiu Naoyuki Watanabe va cpng su (Nhat Ban). Diing 5 mg albumin (kieh thudc 5-30 pm) ly tdm va nia 3 Idn trong dem PBS pH 7,2, sau dd cho dem acetat 0,1 M, pH 6 vdo trong Idn ly tam sau cung (thdi gian ly tam 5 phiit, toe do 3.000 rpm). De ddnh ddu vdi '''Y, cho 1,8 ml dung djch albumin vao cac chai danh ddu, them vao do 200 jil (5 mCi) dong vj phong xa '°Y, lac tron manh tien may Idc trong thdi gian 60 phiit, nhi?t dp phong. Sau khi xong phdn iing, phdn tich kilm tra bieu sudt danh dau.
3. Cac phircnig phap kiem tra d$ sach hoa phong x^ va tinh hn djnh cua san phdm ddnh dau
Phuong phdp sdc ky lap mdng: Gidy sic ky ITLC (Instant Thin Layer Chromatography) kich thudc 1x10 cm, chdm 5 ^il mdu len gidy ITLC tai vi tri em thii 2, m6i mau chdm 2 bdng, khi gidy khd, dat bang giay vao binh chiia sdn hon hpp dem TAE (0,04 M Tris acetat, 0,002 M ethylendiamininetetraacetic acid), dd^ ndp kin, thdi gian sdc ky Id 3 phiit, khi dung moi di chuyen den centimet thii 10, Idy bang gidy ra, de khd rdi cdt thanh tiing bang nho, moi bdng rpng 1 cm vuong goc vdi chieu di ehuyen ciia dung moi. Dat timg bang giay nay vdo cdc dng nghiem, do dem boat dp phong xa tien mdy do.
Phuong phdp sdc ky gidy: Cdc budc th\rc hien tuong tu nhu phuong phdp sac ky ldp mong ITLC nhung sii dung loai giay sac ky Whatman No. 1 kich thudc 1,5 x 17 cm. Thdi gian sac ky Idu hon khoang 60 phiit, dung moi sdc ky Id dem TAE. Khi dung moi di chuyen den centimet thii 15, lay bdng gidy ra de khd rdi cdt thanh timg bang nhd rpng 1 cm. Sau do do dem boat dp phdng xa tien may do.
Kiem tia tinh 6n djnh sdn pham theo thdi gian: San pham danh ddu phong xa dupe bdo quan d dieu ki?n 2-8''C, chtra chdt bdo quan va khong cd chdt bao quan. Sau cdc khodng thdi gian 1, 2, 3, 4, 5, 6, 7 ngay, mdi ngay lay sdn pham ra, de ve nhiet dp phong, phdn tich dp sach hod phdng xa va hieu suat danh ddu bang phuong phdp sdc ky.
Kiem tra dp dn dinh tiong buyet thanh va tiong NaCl 0,9%: Chia san pham ra cdc 6ng nghiem. tl d 37°C tion^ thdi gian 6 ngdy. Sau moi ngay Idy ra do boat dp phong xa, tinh ti le ho?t dp phan gan va phan t\r do.
III. KET QUA NGHIEN ClTU
1. Ket qua khao sat anh hudng pH ISn qua tiinh danh dau:
™Y dung dl ddnh ddu vdi albumin cd ndng dp phdng xa 16 mCi/ml. Qua trinh ddnh dau '"Y- albumin th\rc hi§n vdi cac pH khdc nhau 2; 4; 4,7; 5; 5,5; 7 vdi thdi gian phan ung la 60 phiit d nhi?t dp phdng (22-25°C).
Hieu sudt ddnh dau^cua ^Y-albumin dupe xac dinh bdng phuang phap sdc ky. Hieu suat ddnh ddu cao tiong miln pH 4,7-6,0 (Hmh 1). Dilu nay cd the gidi tiiich khi pH Idn hon
394 Hdi nghi Khoa hgc vd Cdng nghe hgt nhdn todn qudc idn thur IX, Ninh Thudn 18-19/8/2011 6,0 hieu suat danh ddu gidm, co thi do trong miln pH Idn bon 6 ho^c kilm, ngodi dilm dajw dien cua protein, cac phdii tu albumin dl bi boa tan. Trong miln pH nhd hon 4,5 hieu suat danh ddu thdp bon. Co the la do trong mdi trudng cang acid cdc nhdm chirc boat dpng tren phan tu albumin cang gidm. Tren be m^t cdc bat albumin, cd cdc nhom chiic boat dpng nhu- OH, -SH, -NH, trong miln thich hpp (pH 4,7-6,0) cdc nhom ndy d tr^g thai boat dpng. D ^ acetat co vai tro duy tri pH cho qud trinh danh dau xdy ra.
IfSl^^jerrin: * -, '* v-'F?*'^^
Hinh 1. Anh hudngpH len qud trinh ddnh ddu tfinh 2. Hal albumin 2, Ket qua khao sat anh hudng thoi gian phan irng len hi^u suat ddnh dau
Thdi gian phan iing ciing dupe khao sdt tai nhiet dp phdng. Bang 1 cho thay thdi gian can thiet toi uu de phan ung dat hieu suat cao la khoang 60 phiit.
Bdng 1. Kit qud khdo sdt dnh hudng thdi gian phdn ung len hi?u sudt ddnh ddu Thoi gian (phut)
Hieu suat danh ddu (%)
1 80,5
IS 93,4
30 95,3
60 99,6
90 99,6
120 99,8
150 99,8 Thdi gian it hon cd the khong du cho phan iing xay ra hodn todn. Do vdy thi nghiSm tien hdnh cho phan iing danh ddu hat albumin Id 60 phiit.
3. Ket qua khao sat anh hirdng nhift dp len hieu suat danh dau
Bdng 2 Kit qud khdo sdt dnh hudng nhiet do len hi?u sudt ddnh ddu
Nhiet dou (°C) 24 50 70 90
Hieu suat danh ddu {%)
99,6 99,5 99,3 99,7
Ghi chu Ldc tron tren may Lie tron tren may Ldc tron tren may
Khong lac _ Nhiet dp phong hay nhet dp 90°C, phdn iing gdn ^Y len hat diu cho ket qua hon 99w Do vdy chpn dieu kien phdn iing tai nhipt dp phdng la thich hpp.
•^ Hgi nghi Khoa hgc vd Cdng nghi hgt nhdn todn qudc idn thir IX, Ninh Thudn 18-I9/8/20I1 395 4. Tinh che san pham va so sanh hi|u suat danh dau
Hpp chdt danh ddu dupe thdm tich dl thu dupe sdn phdm tinh khilt. Cho dung dich hat danh ddu vao tiii thdm tich, cot chdt, nhiing vao dem thdm tich TAE (Tris acetic EDTA), khudy lien tuc, thay nude 3 Idn, sau khi thdm tich phiic khong bin duoc tdch ra qua mdng thdm tich. Ket qua thu dupe san phdm ^'^Y-albumin cdn lai hon 80%. Ddy la san phdm tinh khiet dupe dimg de nghien ciiu dn dinh. Quy trinh danh ddu va tinh che dupe so sanh vdi kit MAASOL cua hang Health-Care (Hinh 3 vd 4).
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Hten a t i t danh d i a h a l alliundn voiY-90
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Hinh 3. So sdnh ddnh ddu hgt albuminVNCHNvai kit MAASOL
Hmh 4. Kiem tra hi?u sudt ddnh ddu ciia ov- albumin bangphircmg phdp sac ky gidy Do thi cho thdy quy trinh gan vi hat albumin vd vi hat albumin kit MAASOL vdi ^**Y la giong nhau, hieu sudt ddnh ddu cua '^Y-albumin dat hon 98%.
S. Ket qua kiem tra dp s^ch hoa ph6ng xa cua san pham
Sdn phdm ddnh dau ludn dat dp sach boa phong xa hon 98% (Hinh 5, 6).
300000 250000 200000 150000 100000 50000
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y /
10 12 14 16
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Hinh 5. Dg sgch hda phdng xg ^"Y Hmh 6. Kiem tra dg sgch hda phdng xg ciia
^"Y-albumin bdng phuang phdp sdc ky gidy
6. Ket qua kiem tra do on dinh trong huyet thanh va on dinh theo thdi gian
Ca hai chat danh dau ^"Y-albumin A va B diu cho thay dp sach hoa phong xa luon Idn hon 95% frong su6t thdi gian li vdi huylt thanh ngudi vd u trong nude muoi sinh ly tai nhiet dp 37''C trong v6ng 6 ngay. Thi nghiem chimg to rdng trong nghien cuu invitro, ^°Y-albumin khong bi phdn hiiy, dn dinh tiong ca huylt thanh vd trong nude mudi sinh ly d nhiet dp 37°C vi vdy cho phep sdn phdm Y-albumin co thi 6n dinh trong nghien curu phdn bd sinh hpc tren lam sang (Bang 3).
396 Hdi nghj Khoa hgc vd Cdng nghe hgt nhdn tadn qudc idn thu" IX, Ninh Thudn 18-19/8/2011 Bdng 3. Kit qud kiem tra tinh dn dinh hgp chdt ddnh ddu Y-albumin trong huyit thanh
ngudi vd trong NaCl 0,9%
Ngay On dinh trong huyk thanh (%) On dinh trong NaCl 0,9% (%)
1 99,8 99,8
2 99,0 99,3
3 98,5 98,9
4 97,8 98,2
5 96,6 97,8
e
95,8 96,5 Tinh on dinh theo thdi gian cua sdn phdm dupe theo doi theo thdi gian bao quan.
Trong moi trudng co them axit ascorbic, phiic ben trong vong 7 ngdy. Trong moi truong bao qudn khong co acid ascorbic, phiic khong dn dinh, sau 7 ngdy, d§ sach hoa ph6ng xa, it hon 90% (Hinh 7).
100 -
^ 9S J
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=• 94 1 3 92 - ,S 90 1
a
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1
^ -~—.^__^ C6 acid ascorbic
4 \ ^ ^ ^ " ^ " ^ Khong CO acid 3 s c o r b i c \ ^
2 3 4 3 6 7 Thai gian (ngay) 8
Hinh 7. Kiem tra tinh dn dinh sdn phdm theo thdi gian IV. KET LUAN
Vi hat albumin co the danh dau voi dong vj phong xa ^'^Y trong cac dilu Icien toi uu cua phan img danh dau. Ket qua cho thay phan ung danh ddu dat hieu sudt cao 98 - 99% a Ichoang pH 5,5; thai gian phan img danh dau Id 60 phiit; nhiet dp thich hgp cho phan img ]h nhiet do phong. Sau danh dau, hop chdt danh ddu dugc thdm tich va san phdm thu dugc co do on dinh, do sach hod phong xa cao hon 98%. Hgp chdt danh ddu dugc ki8m tra chat luong theo tieu chudn dugc chdt phong xa. San phdm da dugc kiem tra vd dat yeu cdu ve dp on dinh trong huySt thanh nguoi, San phdm 6n dinh theo thcri gian bdo quan la 7 ngdy trong axit ascorbic, dat cac tieu chuan cua thuoc phong xa cho mijc dich diSu tri trong y hpc.
TAI LIEU THAM K H A O
[I] IAEA-TECDOC-1340, "Manual for reactor produced radioisotopes", IAEA, Vienna, 2003.
[2] Gopal B. Saha, "Fundamentals of Nuclear Pharmacy", Third Edition, Springer, 2000.
[3] Phan Van Duyet, "Phongxay hgc", Nha xudt bdn y hpc - Hd Npi 1979.
[4] Tecchnical Series Number 470, "Therapeutic Radionuclide Generators: "Sr^Y and '^'W/'"Re Generators", IAEA, Vienna, 2009.
[5] Azu Wuike Owunwanne and Samy Sadek, "The Handbook of Radiopharmaceuticals', Chapman & Hall Medical, London, Glasgow, Weinheim. New York, 1995.
[6] Watanabe N, Oriuchi N, Endo K, Inoue T, Tanada S, Murata H and Sasaki Y, "K/riw- 90-labeled human macroaggregated albumin for internal radiotherapy: combined use with DTPA ", Nucl. Med. Biol.; 26:847-851,1999.