NGHIEN C t r u TAC DUNG CUA BANG NANO BAC LEN QUA TRINH DIEU TRI VET THUONG BONG
TrSn Thi Ngoc Dung'. Nguyin Hoai Chau', Dao Trong Hien', Nguyin Thiiy Phuong', Ngo Quoc Bun', Nguyen Gia Tien^
Vien Cong nghe moi truang; Vien Bong quoc gia Email: ttndzung(5),gmail.com
Tom tat:
Ngodi khd ndng khir triing duac biit din tir Idu ra, nano bgc vdi ndng luong bi mat Idm cdn cd khd ndng diiu hda qua trinh chira tri vit thuong bdng cdch phdt triin cdc cytokin chuyin hd tra diiu tri hoac irc chi cdc cytokin chuyen hd trty viim, cho phep rut ngdn thdi gian diiu tri, ddng thdi lam cho vit thuong sau diiu tri dgt gid tri thdm my cao, khdng di Igi di chung. Bdo cdo trinh bdy mdt sd kit qud icng dung bdng nano bgc diiu tri vit thuong bdng. Bdng nano bgc duoc chi tgo bdng cdch ngdm vdi khdng dit vdo dung dich nano bgc cd ndng dp 500 mg/l vd kich thudc hgt trung binh 20 -25 nm. Dung dich nano bgc duac diiu chi bdng phuong phdp dung dich nude sic dung borohydrid lam chdt khic vd chitozan la chdt dn dinh. Tdc dung diiu tri vit bdng cua bdng nano bgc tu chi tgo da dupe nghiin cicu song song vdi bdng sunfadiazin duac tdm vdi mudi AgNO}. Nghiin cuu duac thuc hien tren chupt thi nghiem vd trin nguai Kit qud thu duoc cho thdy tdn thuong bdng duac diiu tri bdng bdng nano bgc cho kit qud tdt han ddng ki so vdi bdng sunfadiazin vdi thdi gian phuc hdi nhanh hon vd khdng di Igi seo ldi nhu trong trudng hpp sic dung bdng sunfadiazin.
Abstract:
Silver was known as a strongest antimicrobial element in the nature, moreover in the nanosized form it can modulate the wound healing process by developing pro-healing or inhibiting pro-inflammatory cytokines, resulting in accelerated wound recovery and improved cosmetic appearence. This report represents some results of using nanosilver-coated wound dressings for therapeutic treatment of burns. The dressings were manufactured by immersing non-woven fabric material into a nanosilver solution of concentration of 500 mg/l with an average size of 20 - 25 nm, which was produced by aqueous molecular solution method. The healing effect of the tested dressing was studied through the comparison with silver sulfadiazine-coated one. The experimental data obtained showed that nanosilver- coated dressing heals burns with accelerated healing and without scars, much better than silver sulfadiazine-coated dressing.
l.DATVANDE
Dieu tri vit thuong la mgt qua trinh dien biin phiic tap va da tir lau luon la dli tugng nghien cuu cua cac nha y hgc. Muc tieu culi cimg cua dilu tri tin thuong la su phuc hli nhanh va khong de lai di chimg. Qua trinh dilu tri diin ra thong qua mot loat cac su c6 chIng cheo len nhau nhu keo ty, viem nhiem, tang truang va tai tao mo [1]. D I cho qua'trinh phuc hoi ton thuong co the dien ra mgt each hoan hao, mgt s6 hgp chit hoa hoc dac biet - goi la cac chat kich thich phan bao (cytokin) - xuit hien dl giri tin hieu din cac tl bao [2,3]. Co thi
Hgi nghj Khoa hgc ky niem 35 ndm Vien Khoa hgc vd Cong nghe Viet Nam - Hd Not 10/2010
dan ra o day mgt so chat cytokin dien hinh thuong gap trong dieu tri vet thuong nhu: TGF-P (yeu to tang truong bien doi - transforming growth factor), VEGF (yeu to tang truang noi mac huyet quan - vascular endothelial growth factor), interleukin IL-6, IL-10, interferon IFN-y, v.v... Cac chat nay co vai tro quan trgng trong cac qua trinh khoi mao, duy tri va dieu tiet cac phan ling xay ra sau khi ton thuong xuat hien. Chinh cac chat nay la nguyen nhan lam cho tinh trang vet thuong co the xau di, hinh thanh seo loi, hoac xuat hien cac phan ling viem khong kiem soat dugc [3-5]. Chang ban trong cac trucmg hgp seo loi hoac seo sieu dinh duong nguofi ta phat hien thay thanh phan cytokin TGF-P gia tang dang ke, trong khi o cac ton thuong bao thai khong de lai seo, thi yeu to nay giam ban. Nhu vay, neu tim dugc mot tac nhan nao d6 c6 kha nang lie che yeu to TGF-P thi chiing ta co the kiem soat dugc qua trinh phuc hoi vet thuong. Interleukin IL-6 la mgt cytokin ho trg phan ling viem (pro-inflamatory), dugc biet din nhu la mgt tac nhan kich thich qua trinh phat trien tran lan cua cac nguyen bao sgi [6]. Chang ban, trong ton thuong bao thai, ham lugng IL-6 thuong khong nhieu, nhung neu chiing dugc dua them tir ngoai vao thi ton thuong se de lai seo. Nhu vay c6 the n6i sir suy giam lugng IL-6 trong ton thuong bao thai cung la dieu kien can thiet de phuc boi nhanh ton thuong khong de lai di chiing. Trong khi do IL-10 lai la mgt cytokin khang viem (anti- inflamatory), giir vai tro quan trgng trong viec dieu tiet su hien dien cua cac dai thuc bao va bach cau trung tinh tren ton thuong viem, cu the la lic che cac cytokin ho trg viem tren ton thuong [7]. Mgt trong nhung tac dung dac biet cua cytokin IL-10 la kha nang tie che qua trinh tong hgp cac cytokin ho trg viem trong d6 co IL-6 [8]. Interferon (IFN-y) do cac te bao bach huyet T v^ dai thuc bao san sinh ra giii vai tro quan trgng trong viec tai tao mo cua vet thuong. Khi ham lugng IFN-y trong vet thuong tang len, thi qua trinh san sinh colagen bi cham lai trong khi lugng men phan giai collagen tang, dan den su suy giam toe do co lai cua vet thuong [9]. Noi each khac, khi lugng IFN-y tang len, thi toe do hinh thanh lop colagen tren vet thuong bi cham lai, tao dieu kien thuan Igi cho viec ngan chan nguy co tao seo loi.
Cac hat nano bac da dugc biet nhu mgt phuong tien khir triing dac biet bieu qua. Nhung ngoai dac tinh do ra nano bac con the bien kha nang tac dung len cac cytokin nham ho trg qua trinh phuc hoi vet thuong. Nhu chiing ta biet, truoc khi c6 cong nghe nano, trong dieu tri benh nguoi ta thuong sir dung bac duoi dang hgp chat (thi du sunfadiazin chiia muoi bac), do co dgc tinh nen khong the tranh khoi cac phan ling phu. Cong nghe nano da tao ra cac hat nano kim loai bac co kha nang giai phong ion bac theo y muon, nen loai trir dugc hau het cac phan ling phu. Cac cong trinh nghien ciiu ve kha nang dieu tri vet thuong cua nano bac hien khong nhieu va co che tac dung cua no len vet thuong ciing chua dugc sang to hoan toan. Tuy nhien, ket qua nghien ciiu gan day cua cac nha khoa hoc thuoc truong Dai hgc Hong Kong [10] da cho thay tac dung cua nano bac thiic day qua trinh dieu tri vet thuong va ban che viec de lai di chung sau dieu tri. Nhom tac gia nay cho thay nano bac co the lam giam miic do viem cua vet thuong bang each dieu chinh sir xuat hien cua cac cytokin khac nhau.
Mgt so cong trinh nghien ciiu da tim each danh gia mgt each dinh lugng tac dung ciia m6i cytokin len kit qua dieu tri vit thuong [1-9], dong thai tim kiem cac tac nhan co kha nang tac dgng len hieu qua boat dgng cua cac cytokin trong dieu tri vet thuong. Cac cong bo nay da chi ra rang nano bac co kha nang dieu bien cac cytokin trong qua trinh dieu tri vet thuong, dac biet la vit thuong bong. D I nghien ciiu vin dl nay nguoi ta sir dung phuong phap PCR-thai gian thuc (Real time - polymerase chain reaction), ma qua do sir hien dien cua moi cytokin dugc danh gia theo miic mRNA do dugc cua no. Hinh 1 cho thay miic mRNA cua cac cytokin biin dli trong qua trinh phuc hoi vet thuong phu thuoc vao tac nhan xir ly dugc ap dung [10].
Sl lieu din ra tren hinh la va lb cho thiy nano bac lic chi manh me cytokin ho trg viem IL-6, ding thai kich thich cytokin khang viem IL-10, dan din qua trinh phuc hIi vit thuong hoan thien ban va khong dl lai seo. Nhu da noi a tren, cytokin tham gia vao qua trinh xo boa mo va hinh thanh seo 16i trong qua trinh vit thuong phuc hIi. Hinh Ic cho thiy su co mat cua nano bac tren vit thuong bong da gift gia tri mRNA cua TGF-P a mirc thap hon dang kl so voi truong hgp xir ly vit thuong bing sunfadiazin bac. Tren hinh ve co the cle dang nhan ra ring, dli voi cac cytokin dugc khao sat 6f tren, su co mat cua sunfadiazin deu lam cho qua trinh phuc hIi vit thuong bong xiu di, tham chi xiu ban ca truang hgp vet thuong phuc hoi tu nhien (khong xu ly). Bao cao nay trinh bay mgt sl kl qua nghien ciin taC dung cua bang nano bac len qua trinh phuc hoi vet thuong bong tren tho thi nghiem va tren benh nhan.
1 3 5 7 10 15 20 25 30
Th^i gian dieu tri, ngay
1 3 5 7 10 15 20 25 30
Thcri gian di6u trj, ngay
3 5 7 10 I.S 20 25 30
Thcri gian dieu trj, ngay
Hinh I: Biin diiu cua cdc cytokin trong qud trinh phuc hdi vit thuong bdng phu thudc vdo tdc nhdn xit ly duoc dp dung [10]: muc mRNA ciia IL-6 (a), IL-10 (b) vd TGF-^ (c).
Ghi chu: Du&ng cong "Nano Ag": vet thuang bong dugc phu bdng chica nano bgc; du&ng cong "SSD": vet thuong bong dugc phu bdng chua sunfadiazin bgc; du&ng cong "Khong xic ly": vet thuang tu phuc hoi.
2. PHAN THLTC NGHIEM 2.1 Hoa chat, vat lieu va doi tuong nghien cuu
AgNOs, NaBH4, NaOH, axit acetic va chitozan deu thuoc loai p.a. Vai cottong khong det dugc sir dung de co dinh nano bac. Doi tugng nghien cmx la bang nano bac dieu tri vit thuong tu che, bang nano bac Anson Trung Quoc va kem sunfadiazin \% bac do Cong ty Dugc pham Trung uong Hue san xuat. Thi nghiem dugc tien hanh tren dgng vat thi nghiem (chugt va tho) va tren benh nhan bong. Cac chung vi khuan Pseudomonas aeruginosa. Staphylococcus aureus vd Acindobader baumannii dugc phan lap tu cac ton thuong bong tren benh nhan.
2.2 Phuong phap nghien cuu
Cac hat nano bac dugc dieu che bang phuang phap dung dich nuac [II], trong do phan ling hinh thanh nano bac dugc thuc hien trong dung dich nuac muoi nitrat bac vai chitozan lam chit In dinh.
Dung dich khir NaBILt dugc nho gigt tir tu vao hon hgp phan ung trong dilu kien khuiy manh liet tai nhiet do phong va pH 5-6 cho tod khi dung dich chuyen dan sang mau nau vang.
Bang dilu tri vet thuang chiia nano bac dugc che tao nhu sau: cac manh vai khong det c6 kich CO 30x40 cm sau khi dugc tiet trung 15 phiit tai 150 C dugc ngam trong dung dich nano bac voi nIng do 500 mg/L, khuiy lien tuc 1 gia tai nhiet do phong, sau do cho sieu am rli siy kho o 50*^C trong 24 gia. Khoi lugng dung dich nano bac tieu hao cho qua trinh cl dinh nano bac len vai bang la
1 L/m^. Hoat luc cua bang nano bac khao sat bang each do duong kinh vong vo khuin tren dTa thach trong moi truang Muller- Hinton [12]. Lieu dgc cap ciia nano bac dugc khao sat tren chugt nhit tring vod trgng lugng trung binh 20g/con dugc chia thanh 5 16, moi 16 12 con. Cac 16 dugc cho uIng
Hgi nghj Khoa hgc ky niem 35 ndm Vien Khoa hgc vd Cong nghe Viet Nam - Hd Noi 10/2010
dung dich nano bac (5000 mg/L) vod lieu khoi diem la 0,3mL/10g/24gia va tang thi tich uIng len gap doi sau moi 16 cho den miic ma da day chugt co the chiia dugc.
Tac dung cua nano bac len ton thuong bong dugc tien hanh nghien ciiu tren tho va true tiep tren benh nhan bong. Tho tbi nghiem voi so lugng 10 con can nang tir 2kg/con dugc gay bong cap do II tai hai viing tren lung de dap bang dieu tri len d6: vimg A dip bang nano bac tu che va vimg B - bang Anson cua Trung Quoc.
3. KET QUA VA THAO LUAN
Ket qua khao sat lieu dgc cap tinh cua dung dich nano bac (5000mg/L) trinh bay tren bang 1 cho thay tat ca chugt a cac 16 thi nghiem sau 72 gio cho uong nano bac voi cac lieu tang dan deu khoe manh binh thuong, ke ca 16 dugc cho u6ng voi lieu t6i da ma day day chugt co the chiia. Nhu vay, co the khang dinh rang nano bac co doc tinh thap.
Bdng 1: Kit qud nghiin cieu liiu ddc cdp tinh ciia dung dich nano bgc tren chupt nhdt trdng STT
L6 1 L6 2 L6 3 L6 4 L6 5
Liiu sir dung (ml/IOg TLCT) 0,3
0,6 0,9 1,2 1,5
Sd ddng vdt thi nghiem 12
12 12 12 12
Sd chit sau 72 gid 0
0 0 0 0
San pham bang nano bac dieu tri vet thuong da dugc nghien ciiu danh gia boat tinh khir trimg. Bang 2 trinh bay ket qua xac dinh vong khang khuan cua bang nano bac tu che va bang Anson cua Trung Qu6c d6i voi mgt so chimg vi khuan thuang gap trong benh vien, trong do cho thay bang tu che co boat luc khir triing tuong duong bang Anson (p > 0,05).
Bdng 2: Vdng khdng khudn ciia bdng nano bgc ddi vdi mdt sd chiing vi khudn chudn qudc te
Tin vi khudn
P.aeruginosa ATCC 27853 S.aureus ATCC 25923 E.coli ATCC 25922
Dudng kinh vdng khdng khudn, mm Bdng che tgo (n=30)
24,58 + 3,26 24,28 2,15 17,37 2,45
Bdng Anson (n=30) 23,48 5,46 23,29 2,06 19,18 2,87
P
0,157 0,174 0,145 Tren hinh 2 co the thay dien bien tai ch6 vet thuong bong tren tho thi nghiem dugc dieu tri bang bang nano bac dat ket qua t6t. Cac vet bong deu it phu viem, it xung huyet va tiet dich rit it. Tren tat ca ca the tho thi nghiem deu ghi nhan kh6ng thay co hien tugng tho di irng voi bang nano bac. Qua trinh phuc h6i vet thuong bong dieu tri bang bang nano bac tu che va bang Anson deu dien ra thuan Igi, sau 3 tuan dien tich vet thuang da co lai con 1/3.
Hinh 2: Dien biin vit thuang bdng trin thd thi nghiem trong qud trinh diiu tri bdng nano
bgc. Cdc ky hieu: viing A - bdng nano bgc dieu che; viing B - bdng Anson; No-02 - sd thic tu cita tho thi nghiem;
DIO - ngdy dieu tri thu 10
Hinh 3: Diiu tri vit bdng ndng trin cdng tay. a) Viing
A phu bdng nano bgc che tgo; Vung B phu bdng Anson;
Viing C phii bgc -sunfadiazin; b) sau 3 ngdy dieu tri
Hinh 4: Diin biin md hpc ciia vit bdng ndng trin tieu bdn dp trong qud trinh diiu tri bdng nano bgc tu chi (hdng tren) vd kem bgc - sunfadiazin (hdng dudi).
Ghi chii: a,b) Be mat vit bong tru&c dieu tri; c,d) Bi mat vit bong sau 3 ngdy dieu tri; e,g) Be mat vet bong sau 7 ngdy dieu tri
Theo quan diem hien dai ve lien vet thuong, trong cac vit thuang bong va vit thuang man tinh viem co vai tro quyet dinh d6i voi qua trinh phuc h6i vit thuang. Vi vay vai tro dilu tilt cytokin ciia nano bac nham giam thieu viem tren t6n thuang b6ng da dugc chiing t6i nghien ciiu true tiep tren benh nhan bong.
Hinh 3 m6 ta qua trinh dieu tri vit thuong bong tren cing tay cua mgt benh nhan, su dung bang nano bac dilu chi (vung A), bang Anson Trung QuIc (vung B) va kem bac- sunfadiazin (viing C), trong khi hinh 4 the hien diin biin m6 hgc cua vit thuang trong qua trinh dilu tri. Tren be rnat t6n thuong bong n6ng truoc khi dip thulc, cac tl bao viem nim rai rac giua cac dam te bao thoai hoa (hinh 4a,b). Nhung sau 3 ngay dilu tri (hinh 4c,d) cac bach cau nhan da hinh (polymorphonuclear leucocytes) tap hgp lai thanh dam voi cac dai thuc bao voi mat do nhilu ban tren tieu ban bac-sunfadiazin 1%, trong khi tren tieu ban nano bac da rai rac xuat hien cac nguyen bao sgi. Sau 7 ngay, tren tieu ban nano bac da thiy rai rac xuit hien cac tl bao sirng non, trong khi tren tieu ban bac-sunfadiazin con CO nhieu gia mac hoai tir (hinh 4e,g).
Nhilu nghien ciiu truoc day thua nhan ring dilu ti-i bong bing nano bac cho hieu qua cao do nano bac lic cbl vi khuan phat trien, nhung kh6rig giai thich dugc tai sao then gian phuc hIi tin thuang lai nhanh hon so vod bac-sunfadiazin. Dilu nay co thi giai thich nhu sau. Bac duoi dang muli ion co dgc tinh tirong dli cao lam cho vit thuang de bi viem, ti-ong khi cac hat nano bac do
Hgi nghi Khoa hgc ky niem 35 ndm Vien Khoa hgc vd Cong nghe Viet Nam - Hd Noi 10/2010
CO kha nang dieu tiet giai phong tir tu cac ion bac vao dich vet thuong de kich thich cac cytokin h6 trg dieu tri hoac lie che cac cytokin h6 trg viem va gia tang giang boa cac tl bao t6n thuang.
Tac dung d6ng thod cua cac yeu t6 nay cho phep nit ngan thai gian phuc hIi tin thuang va kh6ng de lai seo (hinh 3 va 4). Su dung bac-sunfadiazin de dieu tri bong thuong keo theo phan ling viem va phii ne qua mirc tren vet thuong, dan den tinh trang u tre tuan hoan, lam r6i loan chuyen boa te bao vung lan can. Tu do giai phong ra cac chat chuyen boa trung gian, lam gian mach va tang tinh tham, dan den phii ne xung huyet manh hon, va ket qua la keo dai thod gian phuc bli. Mat khac, trong dich phu ne con co nhiing chat nhu hystamin, bradykinin.. .co kha nang kich thich cac thu the gay cam giac dau [13]. Cac bieu hien nay kh6ng dugc tim thay a t6n thuong bong dugc dilu tri bang nano bac, boi vi nhu da noi o tren, nano bac co kha nang dieu tiet cac cytokin nhu giam yeu t6 IL-6 kich thich viem va yeu t6 TGF-P ho trg hinh thanh seo 16i, tang cuong cad yeu t6 IL-
10 h6 trg khang viem va VEGF h6 trg phat trien cac mao quan noi mac, giiip nit ngan qua trinh phuc h6i t6n thuong.. Cac cytokin noi tren con co kha nang lic che dang ke lugng enzym MMP gay viem manh thuang dugc tim thay rat nhieu tren vet thuong bong ciing nhu cac vet loet kho lanh. Dieu d6 cho thay nano bac co kha nang dieu tri dac biet hieu qua d6i voi cac t6n thuong bong Cling nhu vet loet lau lanh.
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