TRNG I HC HOA HC T NHIN HQG HCM

KHOA HA HC
B MN HA L

Nguyn Th Kim Dung

Nguyn Trung Hi Thnh
Mai Hu Thoi

ti:
NANO VNG V NG DNG NANO
VNG DNG QUE TR BNH UNG TH
BNG PHNG PHP QUANG NHIT

Seminar Ha hc Nano

Thnh ph H Ch Minh, thng 11 nm 2014

TRNG I HC HOA HC T NHIN HQG HCM

KHOA HA HC
B MN HA L

Nguyn Th Kim Dung

Nguyn Trung Hi Thnh
Mai Hu Thoi

ti:
THANH NANO VNG
NG DNG TR BNH UNG TH BNG
PHNG PHP QUANG NHIT
GVHD: Nguyn Th Phng Phong
Seminar Ha hc Nano

Thnh ph H Ch Minh, thng 11 nm 2014

H KHTN Khoa ha BM. Ha L

M u
Bc qua th k XXI, cht lng cuc sng con ngi ngy cng c nng cao. Tuy
nhin bn cnh con ngi khng trnh khi vic phi i mt vi nhiu vn v
sc khe. Bnh ung th ngy cng tr nn ph bin hn v nguy him hn. Theo
thng k ca t chc Y t th gii (WHO) v b Y t Vit Nam th Vit Nam c t l
mc bnh ung th cao trn th gii. Nguyn nhn chnh l do ngi dn tip xc hng
ngy mi trng b nhim v thc phm c cha cht bo qun c hi. T l ngi t
vong v cn bnh ny rt cao. Trong khong mt thp k tr li y, cc nh khoa hc
cho thy nhiu phng php iu tr bnh ung th c hiu qu cao, trong vic ng
dng ht nano vng vo vic tm, nhn bit v tiu dit t bo ung th ang dn cho
nhng kt qu rt kh quan. Cc tnh cht ca ht nano vng hnh cu c s dng
trong vic cho hnh nh ca t bo ung th bng cch s dng knh hin vi ng tiu v
knh hin vi trng ti n gin. gn y liu php quang nhit s dng cc thuc tnh
hp th cu khng th lin hp thanh nano vng (nanorods) v ht nano vng hnh cu
c chng minh tiu dit chn lc t bo ung th khng nh hng n cc t
bo khe mnh. Trong khun kh bi vit ny, chng ti xin php phng php iu
ch thanh (que) nano vng, ng dng phng php quang nhit tr trong vic iu tr
bnh ung th.

Trang 3

H KHTN Khoa ha BM. Ha L

Mc lc
M u .......................................................................................................................... 3
Danh mc hnh nh ........................................................................................................ 5
1. Tng quan ............................................................................................................... 6
1.1

S lc v bnh ung th ....................................................................................... 6

1.2

Ht nano v s ra i ca cng ngh nano............................................................ 7

1.3

ng dng nano vng iu tr bnh ung th. .......................................................... 8

1.4

Cc phng php iu ch ht nano vng ............................................................. 9

2. Thc nghim iu ch nano vng dng thanh ng dng iu tr ung th................ 12

2.1

Ha cht thit b .............................................................................................. 12

2.2

Tng hp thanh nano vng ................................................................................. 13

2.3

Chun b khng th anti-EGFR mang thanh nano vng (anti-EGFR/Au) ............ 13

2.4

Th nghim ln t bo bnh thng v t bo bnh ca h lin hp anti-

EGFR/Au..................................................................................................................... 14
2.5

B mt plasmon hp th v tn x nh sng ca thanh nano vng ln t bo. ..... 14

2.6

Tin hnh tr liu quang nhit. ............................................................................ 14

3. Kt qu v nhn xt ............................................................................................... 15
3.1

Kt qu tng hp nano vng dng thanh ............................................................. 15

3.2

Kt qu tr liu quang nhit ................................................................................ 18

4. Kt lun................................................................................................................. 21
Ti liu tham kho ....................................................................................................... 23

Trang 4

H KHTN Khoa ha BM. Ha L

Danh mc hnh nh
Hnh 1: T l mt s loi ung th thng gp trn ton th gii (tnh n nm 2010) .... 6
Hnh 2: nh tn x ca cc t bo khng ung th (a) v cc t bo ung th (b), (c) sau
khi vi khng th EGFR lien hp nano vng. .............................................................. 9
Hnh 3: Cc loi ht nano vng tng ng vi cc hnh dng khc nhau ...................... 10
Hnh 4: A) Quang ph hp thu plasmon b mt ca cc thanh nano vng vi nhng
bc sng khc nhau. B) nh TEM ca cc thanh nano .............................................. 15
Hnh 5: nh tn x ca cc t bo kho st. Ct tri HaCaT, ct gia HSC, ct phi
HOC. A) tn x ca ht nano vng. B) tn x ca thanh nano vng. .......................... 16
Hnh 6: Ph pht x t cc t bo n c o bng my quanh ph hp thu. A) antiEGFR gn trn ht nano hnh cu. B) anti-EGFR gn trn thanh nano. ........................ 17
Hnh 7: Liu php quang nhit c chn lc vi cc t bo ung th nhng mc nng
lng khc nhau. Ct tri HaCaT, ct gia HSC, ct phi HOC. ........................ 19

Trang 5

H KHTN Khoa ha BM. Ha L

1.
1.1

Tng quan
S lc v bnh ung th

Ung th l tn gi chung ca mt nhm cc bnh lin quan n vic phn chia t bo

mt cch v t chc, cc t bo ny c kh nng xm ln cc m bng cch pht trin
trc tip vo cc m ln cn hoc di chuyn n cc m khc bng cch i vo mu
hoc h bch huyt.
Nguyn nhn gy ung th l s sai hng ca DNA, to nn cc t bin cc gen thit
yu iu khin qu trnh phn bo cng nh cc c ch quan trng khc. Mt hoc
nhiu t bin c tch ly li s gy ra s tng sinh khng kim sot v to thnh cc
khi u. Khi u l mt khi m bt thng, c th c tnh, tc ung th hoc lnh tnh, tc
khng ung th.
Theo thng k ca T chc nghin cu ung th th gii (International Agency for
Research on Cancer, IARC) th nm 2002, trn ton th gii c tnh c khong 10,9
triu ngi mi mc bnh ung th, t l ung th ngy cng tng do tc th ha v
s nhim mi trng.

Hnh 1: T l mt s loi ung th thng gp trn ton th gii (tnh n nm 2010) [1]

Theo thng tin ca B Y t Vit Nam v T chc Y t th gii (WHO), hng nm Vit
Nam c khong 150.000 n 200.000 ngi mc bnh ung th mi v c khong 50%
Trang 6

H KHTN Khoa ha BM. Ha L

t vong v cn bnh ny, l nguyn nhn gy t vong cao th 2 ca quc gia. Theo
thng k n nm 2010, c nc c t 240.000 n 250.000 ngi mc bnh ung th,
ch yu l ung th phi, d dy, i trng, ung th v, c t cung, v.v Nguyn nhn
gy bnh th c nhiu, song ch yu l do ngun nc nhim v ngun thc phm c
cha cht bo qun c hi.
Vic iu tr ung th l rt quan trng trong chng trnh Phng chng ung th mi
quc gia. Mun nng cao cht lng iu tr, khng ch hon chnh v k thut ca mi
phng php, thit b, m cn phi c kinh nghim, kin thc; chn on tht chnh
xc, xy dng phc iu tr cho mi bnh nhn mt cch hp l nht, thng thng
phi phi hp lm vic trong mt tp th cc thy thuc chuyn khoa. C 2 nhm
phng php iu tr bnh ung th thng dng.
-

Cc phng php iu tr ti ch: bao gm phu thut v tia x. C kh

nng iu tr trit khi bnh cn giai on sm, cc t bo ung th ch khu tr ti

ch hoc ti vng. Nu cc khi u di cn xa, chng ta vn c th dng phng php
ny tr tm thi hoc kt hp vi nhng phng php khc.
-

Cc phng php iu tr ton thn. Bao gm iu tr ho cht (dng thuc

chng ung th), iu tr ni tit (dng ni tit t hoc dng khng ni tit t), iu tr
min dch (lm tng sc khng ca c th dit t bo ung th). Cc phng php
ny c tc dng trn phm vi ton c th, v vy ch thng c p dng iu tr khi
bnh c tnh cht ton thn hoc lan rng.
1.2

Ht nano v s ra i ca cng ngh nano.

Tin t Nano c s dng trong ti liu khoa hc u tin vo nm 1908, khi

Lohmann s dng n ch cc sinh vt rt nh c ng knh 200nm. Vo nm 1974,
Tanigushi s dng thut ng cng ngh nano ch s lin kt cc vt liu cho k thut
chnh xc trong tng lai. Hin ti trong khoa hc, tin t nano c biu th vi kch
thc 1 phn t mt.
T chc Nanotechnology Initiative (NNI) trc thuc chnh ph M nh ngha cng
ngh nano l bt c th g lin quan n cc cu trc c kch thc nh hn 100nm.

Trang 7

H KHTN Khoa ha BM. Ha L

Sau , nh ngha ny c loi b bi cc nghin cu lin quan khc tp trung vo
cc thit b vi lng v cc vt liu ang c tin hnh vi m m.
Sau ny cng ngh nano c nh ngha c th hn l khoa hc, k thut v thao
tc lin quan ti cc h thng c kch thc nano, cc h thng ny thc hin
nhim v in, c, sinh, ha hoc tnh ton c bit. Nn tng ca cng ngh ny l
hin tng cc cu trc, thit b v h thng c tnh cht v chc nng mi khi kch
thc siu nh. Cu trc c bn ca cng ngh nano bao gm cc ht hay tinh th
nano, lp nano v ng nano. Cc cu trc nano ny khc nhau ch chng c to
thnh nh th no v cc nguyn t, phn t ca chng c sp xp ra sao.
Cng ngh nano l mt ngnh khoa hc pht trin nhanh trong nhng nm gn y vi
vic to ra nhng vt liu v thit b c kch thc nano mt. Cng ngh nano tp trung
vo cc lnh vc nh vt l, sinh hc, k thut, ha hc, khoa hc my tnh v cc ngnh
khc. T , vt liu nano c nghin cu rng ri v c to ra tr thnh i tng
nghin cu ca cng ngh nano. Trong s , ht nano ang ng mt vai tr rt quan
trng trong lnh vc cng ngh nano v n mang n mt k nguyn mi trong lnh
vc nano thuc v n lm thay i nn tng ca vic chn on, iu tr v phng
nga bnh da trn c tnh ca n.
Mt trong nhng vt liu nano c to ra v c ng dng rng ri chnh l nano
vng.
1.3

ng dng nano vng iu tr bnh ung th.

Nguyn tc c bn trong vic iu tr ung th chnh l ngn chn s phn

chia tip tc ca cc t bo v tin hnh ph hy chng. Kh khn trong vic ny

chnh l vic pht hin sm s bt thng ca cc t bo lc chng mi hnh thnh tng
nhm nh. Mt khc vic ph hy chng cng rt kh khn khng ph hy nhng t
bo bnh thng khc.
Nm 2005, mt nhm cc nh khoa hc M cng b rng, cc ht nano
vng c th dng pht hin sm ung th mt cch d dng. Nguyn nhn l do cc t
bo ung th c mt loi protein gi l biu b tng trng trung tm (Epidermal
Growth Factor Receptor EFGR) bm trn b mt cn cc t bo bnh thng th
Trang 8

H KHTN Khoa ha BM. Ha L

khng c. Khi nh km cc khng th ca EFGR vo cc ht nano vng ri tim vo c
th, cc khng th ca EFGR khi gp cc t bo ung th s bm cht ln b mt ca
chng. V vy ch no c t bo ung th th c cc ht vng, cn nhng t bo bnh
thng th khng. Do tnh tn x mnh ca cc ht nano vng, khi chiu chm nh sang
trng v quan st k di knh hin vi, chng ta c th thy nhng chm sng pht ra t
cc ht nano. T xc nh c v tr ca cc t bo ung th trong c th.
Thng 3 nm 2009, cc nh khoa hc M tip tc cng b cch dng ht nano
vng tm t bo ung th tiu dit. Nguyn nhn l khi chiu tia hng ngoi vo, cng
nh i vi nhiu loi ht khc, ht nano vng hp th tia hng ngoi v nng ln.
Nhng i vi mi bc sng ca tia hng ngoi c mt kch thc (ng knh) ph
hp ca ht nano vng, c hin tng hp th cng hng xy ra: Ht nano vng rt
nng. Li dng hin tng ny, ngi ta ch to ht nano vng hnh cu rng (v tia
hng ngoi ch lm nng lp mt ngoi) ng knh c 30-50 nanomet. Khi nh cc
khng th vo cc ht nano vng ny ri cho vo c th, chng ch tp trung bm dnh
vo nhng t bo ung th. Chiu tia hng ngoi c bc sng thch hp vo, do hp th
cng hng, cc ht nano vng rt nng lm tiu dit t bo ung th, cn cc t bo
lnh xung quanh nng ln khng ng k. Ngi ta thy rng ch cn lm nng t bo
ung th cao hn nhit bnh thng ca c th khong 6-7 oC l c th tiu dit c
m khng nh hng n cc t bo bnh thng.

Hnh 2: nh tn x ca cc t bo khng ung th (a) v cc t bo ung th (b), (c) sau khi vi khng th EGFR lien
hp nano vng. [3]

1.4

Cc phng php iu ch ht nano vng

Vng c k hiu ha hc Au, s hiu nguyn t 79, l kim loi chuyn tip mm,

do, d un, d dt mng, mu vng ng v chiu sng. Vng khng phn ng vi hu

Trang 9

H KHTN Khoa ha BM. Ha L

ht cc ha cht, nhng li tan trong nc cng toan to thnh Chloroauric acid
(HAuCl4) l tin thn ca Au trong nhiu ng dng. Vng c dng qung hoc ht
trong hoc cc m bi tch.
Ty thuc vo kch thc, hnh dng v tnh cht vt l m c nhiu loi ht
nano vng c pht minh. Nano vng c nghin cu sm nht c dng hnh cu,
tip theo l dng hnh que (nanorod), dng v bc (nanoshell) , dng khung
(nanocage) v mt loi ht nano vng khc c to ra nh c tnh tn x tt trn b
mt ca n l ht nano SERS.

Hnh 3: Cc loi ht nano vng tng ng vi cc hnh dng khc nhau

Trong hai thp k qua, s pht trin khng ngng cng ngh nano to ra cc ht nano
vng c hnh dng v kch thc khc nhau di s kim sot tt, chnh xc cao.
Di y, chng ti xin gii thiu mt s phng php tng hp ht nano vng tiu
biu.
-

Phng php kh ha hc:

L phng php s dng tc nhn ha hc kh ion kim loi Au3+(trong dung

dch mui vng HAuCl 4) thnh Au. y cn c gi l phng php ha t do tc
nhn ha hc c s dng dng lng nh acid citric, vitamin C, natri bohidrua
(NaBH4), etanol, etilen glycol, glycerin, natri citrat, poly sodium acrylate (PSA).
Sau khi ha tan mui vng HAuCl4, cho tc nhn kh vo ng thi khuy nhanh
dung dch. Cc ion Au 3+ s b kh thnh nguyn t Au, cho n khi dung dch tr nn
bo ha th dn xut hin kt ta cc ht vng c kch thc nm; nu khuy mnh s
to ra cc ht c kch thc ng u hn. Thng thng ngi ta cho cht n nh vo
ngn s kt dnh ca cc ht nano vng.

Trang 10

H KHTN Khoa ha BM. Ha L

Ty thuc vo tng loi ha cht v tc nhn kh tng ng khc nhau m to ra
cc ht nano vng vi cht lng, kch thc v hnh dng khc nhau. Do , khi iu
ch vng bng phng php ha hc cn la chn ha cht s dng vi nng thch
hp, cht n nh, to ra sn phm theo yu cu.
-

Phng php vt l

Dng tc nhn in t hay sng in t c nng lng cao nh tia , tia t ngoi,
tia laser kh ion kim loi thnh kim loi. y l phng php bottom-up.
Vi phng php chiu x gamma- Co-60 v dng chitosan va lm cht kh va
lm cht n nh v ang c nghin cu ph bin to ra cc ht nano vng c dng
hnh cu kch thc khong 16-25 nm v ng nht cao. Ngoi ra, nano vng cn
c to thnh trong micelle khng ion (u phn cc khng b ion ha) nh poly
oxyetylen, isooctyl eter (thng c bit l Triton X-100 hay TX-100) m khng cn
tc nhn kh no khc v n cng ng vai tr l cht n nh.
Phng php ny n gin, tin hnh nhit thng, sn phm c tinh khit
cao v kh nng ch to ra cc ht nano c kch thc nh hn so vi phng php kh
ha hc.
-

Phng php sinh hc

Phng php kh sinh hc dng tc nhn kh l vi khun, vi nm, vi rtkh ion

kim loi.
Nhiu loi vi nm v vi khun c s dng nh Bacillus subtilis, Bacillis
Licheniformic, khun Lactobacillus (khun acid lactic), nm Verticillium to ra ht
nano vng c kch thc t 20-50 nm, khun Actinomycete tng hp ht nano vng c
kch thc 7-12 nm
Ngoi ra trn th gii ngi ta cn s dng cc loi nm mc, to v cy trng nh
c linh lng, l cy rau mi, v cy qu ch to nano vng.
Phng php kh sinh hc n gin, thn thin vi mi trng, c th to ra s
lng ln cc ht tuy nhin thi gian di thng trn 3 ngy.

Trang 11

H KHTN Khoa ha BM. Ha L

-

Phng php vi nh

S dng dung dch cha cc micellce rt sch, n nh v mt nhit ng bao gm

pha nc v pha du to thnh h vi nh. Trong h vi nh, nhng git nc c kch
thc nano c to bi nhng du a nc ca cht hot ng b mt trong khi ui
k nc c solvat ha bi pha du. Nc trong cc micelle o ng vai tr nh cc
trung tm phn ng rt nh cho cc phn ng kh ion vng to thnh cc ht vng.
Dung dch cha mui vng c ha trn vi cht kh nh sodium bis (2
ethylhexyl) sulfosuccinate (ATO) trong dung mi alcan lng. Tc nhn kh s thc y
qu trnh kh ca ion kim loi vng to thnh ht vng kim loi. S va chm gia c
micelle gy nn s tranh dnh li, dn n s pht trin ht trong micelle cho n khi
t kch thc ti a c quyt nh bi t l khi lng nc/cht hot ng b mt.
Theo thi gian phn ng xy ra cc ht nano vng c chit t micelle bi ly tm, ra
dung mi loi cht hot ng b mt tha.
-

Phng php s dng nhit vi sng

S dng tc nhn kh ha hc di tc dng nhit ca l vi sng kh ion vng,

qu trnh kh din ra nhanh.Cc cht kh thng dng l cc hp cht polyol nh etilen
glycol, glycerin, ncPhng php ny to ra cc ht nano vi kch thc ng u
v nh hn so vi nhng phng php khc, v khi nhit c cung cp cho ton b h
thng nh nhau.u im ca phng php l thi gian kh nhanh, thit b n gin, d
s dng. Tuy nhin, phi s dng thm cht kh v cht n nh trnh lm ht vng
keo t tr li.
2.
2.1

Thc nghim iu ch nano vng dng thanh ng dng iu tr ung th

Ha cht thit b
-

Auric acid - HAuO2 0.0005M (Sigma)

Cetyltrimethylammonium bromide (CTBA) C19H42BrN 0.2M (Sigma)

Sodium borohydride NaBH4 0.01M

Benzyldimethylhexadecylammonium chloride (BDAC) 0.15M

Ascorbic acid 0.788M

N-(2-hydroxyethyl)piperazineN-2-ethanesulfonic acid (HEPES)

Trang 12

H KHTN Khoa ha BM. Ha L

2.2

Poly(styrenesulfonate) (PSS, MW=18 000, Polysciences Inc.)

Dulbeccos modification of Eagles medium (DMEM, Cellpro)

Blue trypan 0.4% (Sigma)

Knh hin vi o ngc (Olympus IX70)

My ly tm

Cng mt s thit b v ha cht ph bin khc.

Tng hp nano vng hnh que

Cc nano vng hnh que c tng hp theo phng php tng trng ht qua

trung gian (seed-mediated growth method) vi mt s thay i. u tin dung dch

gm 0.0005 M acid auric trong 0.2M CTAB c kh nhit phng bi NaBH4
lnh mang cc ht nano nh ( < 5nm) nh dung dch keo. 100 mL dung dch tng
trng c iu ch bng cch kh 0.001 M acid auric trong dung dch cha 0.2 M
CTAB, 0.15 M BDAC bng 0.004 M Ag+ v 70L acid ascobic 0.0788M. 8 L dung
dch ht a vo dung dch tng trng v cc thanh nano vng thu c sau vi gi.
Hiu sut phn ng s cao hn nu thi gian phn ng lu hn. Thanh nano vng vi
cc t l kch thc khc nhau thu c bng cch thay i nng Ag+ [4].
2.3

Chun b khng th anti-EGFR mang nano vng hnh que (anti-EGFR/Au)

Cc nano vng hnh que va tng hp trn nn CTAB c tch in dng sau

ly tm 14000 vng/pht loi CTAB. Cc b mt tch in dng ca que nano

c thay i thnh mt b mt tch in m bng cch cho cc que nano vo dung dch
polymer dn PSS - Poly(styrenesulfonate). PSS thm vo dung dch c tch ra bng
cch ly tm dung dich cha cc thanh nano 8000 vng/pht, cc ht c phn tn
trong dung dch HEPES pH=7,4. Cc que nano PSS trng trn vi dung dch khng
th sau c pha long trong HEPES cng mt b m v cho php phn ng trong
20 pht. Cc khng th lin kt vi cc que nano PSS bi s tng tc ca hp ph vt
l (hp ph tnh in), phng php tng t nh c s dng to h lin hp khi
ht hnh cu. Bi s tng tc ca hp ph vt l (hp ph tnh in). Cc que nano
lin hp vi khng th n dng anti - EGFR c ly tm v phn tn vo m

Trang 13

H KHTN Khoa ha BM. Ha L

photphat (pH=7.4) to thnh mt dung dch gc vi mt quang khong 0.5 800
nm. Hp cht anti - EGFR/Au n nh nhit 4 oC trong vi ngy [4].
2.4

Th nghim gn t bo bnh thng v t bo bnh ln h lin hp anti-

EGFR/Au.
T bo biu m lnh tnh HaCaT (t bo sng), dng t bo c tnh HOC 313 th
h 8 v HSC 3 (t bo biu m sng b ung th) c nui cy trong DMEM c 5%
huyt thanh thai b (FBS) 37 C di 5% CO2. Cc t bo c phn ct bi enzym
trypsin v phn tn ln mt knh thy tinh 18 mm trong 12 tm nui cy, thi gian tng
trng tng trng 3 ngy. Cc lp v c bc li bng colagen to iu kin cho t
bo pht trin ti u. Cc t bo c ly ra t mi trng nui cy, ra sch vi m
PSB, sau ngm vo dung dch cha anti-EGFR/Au trong 30 pht nhit phng.
Sau khi ngm trong dung dch cha thanh nano vng, lp t bo c ra bng m
PSB, c nh vi paraformandehide, trng li vi glycerol, v c gn vo vi lp v
khc [4].
2.5

B mt plasmon hp th v tn x nh sng ca nano vng dng que ln t

bo.
Cc hnh nh tn x nh sng c ghi li bng cch s dng mt knh hin vi o
ngc Olympus IX70 vi trng ti t cao tng (U-DCW, 0,9 -1,2). S dng nh sng
trng t n vonfram, iu chnh thnh 1 tia rt hp n phn trn ca mu. Du iris
100/1.35 (UPLANAPO) c s dng hp thu cc nh sng tn x t mu. Hnh nh
thu c bng cch s dng tm phim (Kodak E100 VS). Ph pht x ca cc ht nano
vng trn cc t bo n c o bng vi quang ph k SEE1100 vi phng i 20x
cho khu vng tp trung ng knh 8m. nh sng tn x t cc t bo c th b qua do
s hp th tng cng v tn x t cc ht nano vng [4].
2.6

Tin hnh tr liu quang nhit.

Dng n lasers Ti-sapphire pht ra tia c bc sng 800 nm. Bc sng ny nm

vng NIR m ti cc m c s hp th thp. N cng trng vi cc dy hp ph

theo chiu dc ca cc nano vng dng que. Cc lp t bo ngm vo dung dch lin
hp nano, ra sch vi m PBS. V sau tip xc vi nh sng laser vi cng
Trang 14

H KHTN Khoa ha BM. Ha L

khc nhau. Cc laser 800 nm c tp trung ti vng c ng knh 1 mm trn
mu. Nhiu khu vc trn vng kho st c tip xc vi nh sng laser vi mt
khc nhau cho mi 4 pht v sau nhum vi Blue trypan 0.4% trong 10 pht kim
tra kh nng di chuyn ca t bo. Cc t bo b nhum xanh trong khi cc t bo sng
vn bnh thng. Sau khi c nhum mu c chp nh vi phng i 10x [4].
3.
3.1

Kt qu v nhn xt
Kt qu tng hp nano vng dng que

Hnh 4: A) Quang ph hp thu plasmon b mt ca cc thanh nano vng vi nhng bc sng khc nhau. B) nh
TEM ca cc thanh nano

Ht nano vng hnh cu hp th nh sng trong vng kh kin khong 520 nm. S
hp th plasmon trn b mt ca cc que nano vng c 2 phn: mt phn c bc sng
di mnh do s dao ng theo chiu dc ca cc electron v mt di c bc sng ngn
yu xung quanh 520 nm do s dao ng ngang ca cc electron. Hnh 4A cho thy s
hp th quang hc ca cc que nano vng vi t l hnh dng khc nhau thu c bng
cch thay i nng Ag+ trong qu trnh pht trin. Cc dy hp th theo chiu dc
thay i t vng nh sng kh kin n vng gn hng ngoi cho cc hnh dng quetng
ln. Trong nghin cu, t l 3,9 c la chn do s hp ph trng vi s pht x ti
thiu ca t bo m ca con ngi. Cc dy hp ph ca que nano cng trng vi bc
sng laser 800 nm. Hnh nh TEM ca cc thanh nano vng vi t l 3,9 c th hin
trong hnh 1B [4].

Trang 15

H KHTN Khoa ha BM. Ha L

Hnh 5: nh tn x ca cc t bo kho st. Ct tri HaCaT, ct gia HSC, ct phi HOC. A) tn x ca ht

nano vng. B) tn x ca thanh nano vng.

Trong knh hin vi trng ti, mt chm nh sng trnng hp c s dng vi

phng i 100x, cc ht nano khc nhau s tn x nh sng khc nhau, ty thuc vo
kch thc v hnh dng ca chng so snh hnh nh tn x ca anti-EGFR trn ht
nano vng hnh cu (ng knh 35 nm) sau khi vi t bo cng c th hin hnh
5A. Do cc b mt plasmon tn s dao trong trong vng kh kin, nn s hp ph v
pht x iu trong vng nh sng kh kin. Cc ht nano vng hnh cu tn x nh sng
t xanh l cy n vng. Hnh 5B cho thy hnh nh tn x ca anti EGFR gn trn
que nano vng (t l 3,9) sau khi lin kt vi cc t bo c tnh. Cc nano vng dng que
tn x mnh m nh sng t cam ti do s dao ng theo chiu dc ca b mt
plasmon trong khu vc gn hng ngoi. C 2 anti-EGFR gn trn nano vng hnh cu v
nano vng dng que u tng tc vi 2 loi t bo c tnh v gip phn bit cc t bo
ung th v t bo thng. Cc t bo bnh thng khng c nhn dng do tng tc
c hiu vi cc ht nano vng [4].
nh lng cc ht nao vng tng tc vi mi loi t bo, ph pht x t cc
t bo n c o bng my quang ph hp thu. Hnh 6A cho thy ph pht x trung
bnh ca anti EGFR gn trn nano vng hnh cu trn 20 t bo ring l khc nhau ca
tng loi, hnh 6B cho thy quang ph pht x trung bnh ca anti EGFR gn trn
Trang 16

H KHTN Khoa ha BM. Ha L

nano vng dng que, trn 20 t bo ring l khc nhau ca tng loi. Trong c 2 trng
hp ht nano vng hnh cu v nano vng dng que, hm lng vng trn cc t bo c
tnh nhiu gp 2 ln so vi cc t bo thng. Vic xc nh hm lng ny khng nht
thit tng ng vi s khc bit v nng ca EGFR gia t bo c tnh v t bo
thng v cc t bo c tnh c th lin kt vi cc ht nano vng do tng tc khng
c hiu

Hnh 6: Ph pht x t cc t bo n c o bng my quanh ph hp thu. A) anti-EGFR gn trn ht nano

hnh cu. B) anti-EGFR gn trn nano vng dng que.

. So vi cc ht nano hnh cu, hm lng ca cc nano vng dng que cao hn rt

nhiu trn c t bo c tnh v t bo thng, ch y do tng tc gia PSS trn b mt
ca que nano v cc protein trn b mt t bo. Mc d ph pht x y cc que
nano vng khng hon ton c ghi nhn y do hn ch ca thit b 1000 nm, c
th thy rng v tr pht x theo chiu dc c o mt cch chnh xc v s dch
chuyn sang vng nh sng so vi s pht x ca que nano vng t do c cng kch
thc (xem Hnh 1A, ng cong mu cam). S dch chuyn sang vng nh sng c
th l do hai nguyn nhn. Mt l do s thay i ch s khc x trn b mt ht nano
gy ra bi s lin hp vi cc anti EGFR vi cc EGFR trn b mt t bo. Hai l kt
qu s tng tc ca cc ht nano trn b mt t bo. S tng tc trn plasmon ca
Trang 17

H KHTN Khoa ha BM. Ha L

nano vng dng que cng c quan st trn b mt ng carbon nano v trong dung
dch. S pht x ca thanh nano vng trn t bo bnh thng HaCat v t bo c tnh
HSC v HOC l qu rng a ra mt gi tr chnh xc v hu ch cho cc bc sng
cc i cho mc ch chun on. iu ny khng ging nh cc kt qu t c trn
cc ht hnh cu, di bc sng cc i l khc nhau cho cc t bo c tnh v khng c
tnh cung cp mt k thut chun on ung th n gin [4].
3.2

Kt qu tr liu quang nhit

Cc anti EGFR gn trn ht nano vng hnh cu ng knh 35nm. ng vai tr

hp thu quang nhit hiu qu v chn lc ph hy cc t bo ung th bng tia laser

ion argon m khng nh hng n cc t bo bnh thng xung quanh. Cc bc sng
laser ti 514 nm chng ln b mt hp th cc ht nano vng hnh cu, trong c mt
tia hp thu cc i 520 nm.
Bng cch lin hp vi cc anti EGFR nhm nh du cc phn t EGFR, cc t
bo c tnh c th b ph hy vi nng lng t hn na nng lng cn thit tiu dit
cc t bo bnh thng do s lin hp ca EGFR trn cc t bo c tnh. Tuy nhin, ti
bc sng ny, s thm nhp m ca nh sng l rt thp (di 500 m). Trong khi
iu kin ny c th c li cho cc tn thng trn b mt, iu tr ung th trong c
th mong mun s thm nhp su hn vo cc m. Vng quang ph gn hng ngoi cho
s thm nhp ti a do tn x tng i thp hn v hp th t bn trong m.
T vic hp th nh sng ca cc kim loi qu c th c iu chnh bng cch
thay i hnh dng hoc kch thc nano, liu php quang nhit hp th c th c
pht trin. Hnh dng khc ca cc ht nano vng nh phn nhnh, ng gic, lng knh
ln c b mt hp th plasmon khu vc gn hng ngoi. Nano dng que l ng c
vin tt nht cho ng dng ny do kh nng kim sot chnh xc, ti a hp th ca cc
bc sng cn thit bng cch thay i cc t l hnh dng, c th c thc hin bng
cch thay i nng Ag+ trong qu trnh tng hp nano vng. Tuy nhin, cc tnh
nng khc, nh lin hp vi cc dng nano khc v cc phn t ca h khng th nn
c nghin cu nhiu hn trong tng lai [4].

Trang 18

H KHTN Khoa ha BM. Ha L

Hnh 7: Liu php quang nhit c chn lc vi cc t bo ung th nhng mc nng lng khc nhau. Ct tri
HaCaT, ct gia HSC, ct phi HOC.

Sau khi khng th lin hp anti EGFR gn ln nano vng dng que trong 30
pht, cc t bo c tip xc vi bc x lasers cc cng khc nhau 40, 80, 120,
160, v 200 mW vi mt im trng tm ca 1 mm ng knh cho mi 4 pht. Cc t
bo ny sau c nhum vi blue trypan kim sot s n nh ca liu php
quang nhit. Hnh 7 cho thy nh ca mu chiu x mc nng lng laser khc nhau.
Tip xc vi tia laser 800 nm v trn 160 mW ( 20W/cm 2) gy ra s ph hy
cc t bo bnh thng HaCaT, c kim tra bi kh nng di chuyn vi blue Trypan
Trang 19

H KHTN Khoa ha BM. Ha L

(Hnh 7, ct bn tri). T bo cht c th hin l mt im mu xanh trong khu vc
trn ph hp vi kch thc iu laser. Cc t bo bn ngoi im laser l tn ti, do
khng b nh hng bi blue trypan. iu ny cng ch ra rng anti EGFR gn trn
thanh nano vng khng nh hng n t bo. Gim nng lng laser n 120 mW, s
lng t bo b nhum bi blue trypan gim v tr laser. Ti nng lng ny ch c
trung tm im laser cha nng lng cao gy ra s ph hy t bo. Mt nng
lng cnh ca im laser khng tn thng t bo, nn cc t bo cht b
nhum mu xanh tr nn nh hn [4].
Hnh 7 ct gia cho thy rng cc t bo c tnh HSC b tn thng quang nhit ti
cng ca tia laser thp hn. T bo cht xy ra trong vng kim sot ca tia laser
sau khi tip xc vi cc tia laser trn 80 mW ( tng ng 10W/cm2) ngng nng lng
cho t bo cht ca c t bo HSC l khong mt na l cn thit gy cht cc t bo
bnh thng HaCaT. Mu xanh m hin th trong hnh nh bn ngoi im laser t bo
HSC, l do cc t bo b cht do tip xc ko di ca cc t bo trong dung dch m
bn ngoi mi trng t bo.
Cc t bo c tnh HOC cng b ph hy quang nhit ti trn 80 mW, trong khi
khng c t bo cht quan st cng thp hn ( hnh 3 ct bn phi). Nng lng
tiu dit t bo c tnh bng mt na nng lng cn tiu dit t bo bnh thng
do s tc ng ca EGFR trn cc t bo ung th v s lng cao hn ca cc khng th
lin hp anti EGFR gn trn thanh nano vng hp th nh sng v chuyn i chng
thnh nhit b mt t bo. T quang ph hp th ca 3 loi t bo trong hnh 2, c th
thy rng s lng thanh nano vng trn hai loi t bo c tnh cao hn so vi cc t bo
bnh thng 2 ln. Cc que nano vng hp thu nng lng quang ph v chuyn chng
thnh nhit cung cp vo mng t bo c tnh lm cho chng cht mc nng lng
thp hn so vi t bo bnh thng.
Cc kt qu trn cho thy rng que nano vng lin hp vi cc khng th c th
c s dng nh mt tc nhn quang nhit chon lc v hiu qu cho liu php cha tr
bnh ung th bng cch s dng nng lng thp, v hi tia laser hng ngoi. v vy
bit thm trong ng dng c th, l mong rng cc khi u s c ph hy chn lc
Trang 20

H KHTN Khoa ha BM. Ha L

mc nng lng laser s khng gy nh hng n c m bnh thng xung quanh l
do hm lng ca c thanh nano vng lin kt chn lc vi cc khi u. Nng lng
ngng tiu dit c t bo ung th tm c l 10 W/cm2 thp hn nng lng cn
thit trong trng hp s dng v nano vng. S khc bit ny l kt qu ca mt ct
hp thu nng lng cao hn, mt hng s i lc cao hn cho cc lin hp ca que nano
vng v khng th, hoc do s tng tc cao hn cho s lin hp gia cc khng th
trn b mt t bo ung th.
c bit, nhiu khi u rn, bao gm c no, bng quang, d dy, v, phi, ni mc t
cung, c t cung, m h, bung trng, thc qun, d dy, tuyn tin lit, thn, tuyn
ty, u nguyn bo xp, v cc t bo ung th biu m, tc ng trn cc t bo cht cht
mc khc nhau. Cc phn t c bit trn mng t bo tng tc chn lc vi cc
nano vng dng que vi hm lng cao hn vi c t bo ung th, cho php quang nhit
chn lc vi tia laser hng ngoi gn cho nhiu loi t bo ung th. S tn x mnh m
ca que nano vng cho php chng tng tc hnh nh tt. Nh vy, nano vng dng
que cung cp mt phng php chun on hnh nh v iu tr lp trong cm bin
sinh hc v iu tr ung th, c th iu chnh s dng trong cc vng nh sng kh
kin v gn hng ngoi.
4.

Kt lun

Nghin cu ny cho thy rng nano vng dng que l mt nhm cc tc nhn tng
phn hnh nh v iu tr kp quang hc hot ng do s hp thu mnh m v tn x
hng ngoi. Bng cch s dng plasmon b mt quang ph cng hng v hnh nh tn
x nh sng, cc t bo c tnh HSC v HOC d dng phn bit vi cc t bo bnh
thng HaCaT do s tc ng chn lc ca cc protein EGFR trn b mt ca t bo c
tnh. Sau khi tip xc vi cc t bo, khng th lin hp anti EGFR gn trn que nano
vng, di tc ng ca bc x lasers c bc sng 800 nm vi cc cng khc nhau
c kho st gy ra s quang nhit hy gia cc t bo c tnh v bnh thng.
Tng s hp th ca cc thanh nano ca t bo c tnh lm gim nng lng cn thit
quang nhit hy cc t bo xung bng mt na so vi nng lng cn thit gy
ra s ph hy ca t bo bnh thng. Que nano vng i din cho mt cht hp ph
Trang 21

H KHTN Khoa ha BM. Ha L

quang hc hot ng tng thch sinh hc v phn tn c kh nng nh hng cho s
quang nhit hy chn lc iu tr bnh ung th.

Trang 22

H KHTN Khoa ha BM. Ha L

Ti liu tham kho
[1] ] http://www.benhvienhyvongmoi.vn/chi-tiet-tin/l-24/t-78 , 2014.
[2] Ivan H. El-Sayed,

Xiaohua Huang,

Mostafa A. El-Sayed. Surface Plasmon

Resonance Scattering and Absorption of anti-EGFR Antibody Conjugated Gold

Nanoparticles in Cancer Diagnostics: Applications in Oral Cancer. Nano Letters Vol 5,
No 5 (2005). 829-834.
[3] Srivalleesha Mallidi, Timothy Larson, Justina Tam, Pratixa P. Joshi,
Karpiouk,

Konstantin

Sokolov

and

Stanislav

Emelianov.

Andrei

Multiwavelength

Photoacoustic Imaging and Plasmon Resonance Coupling of Gold Nanoparticles for

Selective Detection of Cancer. Nano Letters Vol 9, No 8 (2009). 2825-2831.
[4] Xiaohua Huang, Ivan H. El-Sayed, Wei Qian and Mostafa A. El-Sayed. Cancer Cell
Imaging and Photothermal Therapy in the Near-Infrared Region by Using Gold
Nanorods. Jounal of the American Chemical Society(JACS). Vol 128, No 6, (2006).
2115-2120.

Trang 23