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B Y T
TRNG I HC DC H NI
HONG TH MINH
NGHIN CU PHNG PHP
Y QUA MNG TRONG LM NH
V NG NHT HA LIPOSOM
RESVERATROL
KHA LUN TT NGHIP DC S
H NI 2014
B Y T
TRNG I HC DC H NI
HONG TH MINH
NGHIN CU PHNG PHP
Y QUA MNG TRONG LM NH
V NG NHT HA LIPOSOM
RESVERATROL
KHA LUN TT NGHIP DC S
Ngi hng dn:
TS. Nguyn Th Lp
Ni thc hin:
1. B mn Ho Sinh
2. B mn Bo Ch
H NI 2014
LI CM N
Li u tin ti xin by t lng bit n su sc ti:
T.S. Nguyn Th Lp.
Ngi thy tn tnh hng dn v gip ti trong qu trnh thc hin
ti ny.
Ti xin chn thnh cm n cc thy, c gio, cc ch k thut vin B
mn Ha sinh gip v to iu kin thun li cho ti hon thnh ti.
Ti cng xin gi li cm n chn thnh ti cc thy, c gio, cc anh, ch
k thut vin B mn Bo ch nhit tnh gip ti trong thi gian thc
hin kha lun.
Cui cng ti xin gi li cm n ti gia nh, bn b nhng ngi lun
bn ng vin, gip ti c th hon thnh ti tt nghip ca mnh.
H Ni, ngy 13/05/2014
Sinh vin
Hong Th Minh.
MC LC
DANH MC CC K HIU, CH VIT TT
DANH MC CC BNG BIU
DANH MC CC HNH V V TH
T VN ................................................................................................... 1
Chng I. TNG QUAN .................................................................................. 3
1.1. Tng quan v liposom ................................................................................ 3
1.1.1. Khi nim ............................................................................................. 3
1.1.2. Phn loi ............................................................................................... 3
1.1.3. Thnh phn liposom ............................................................................. 5
1.1.4. Cc phng php ch to liposom ....................................................... 6
1.1.5. Cc phng php ng nht ha kch thc liposom ......................... 7
1.1.5.1. Phng php siu m ..................................................................... 7
1.1.5.2. Phng php y qua mng ........................................................... 9
1.2. Tng quan v trans - Resveratrol ............................................................ 12
1.2.1. Cng thc ha hc ............................................................................. 12
1.2.2. c tnh l ha ................................................................................... 12
1.2.3. Dc ng hc ................................................................................... 13
1.3. Tng quan v liposom RSV ..................................................................... 14
CHNG II: I TNG V PHNG PHP NGHIN CU ............. 17
2.1. i tng nghin cu, nguyn vt liu v phng tin nghin cu ........ 17
2.2. Ni dung nghin cu ................................................................................ 18
2.3. Phng php nghin cu .......................................................................... 18
2.3.1. Phng php bo ch liposom RSV ................................................. 18
2.3.1.1. To liposom th ........................................................................... 19
2.3.1.2. Lm nh kch thc v ng nht ha liposom RSV .................. 19
2.3.1.3. Tinh ch liposom RSV ................................................................. 20
2.3.2. Phng php nh gi mt s c tnh ca liposom RSV................. 23
2.3.2.1. Phng php nh gi hnh thc, hnh thi, kch thc v phn b
KTTP liposom ........................................................................................... 23
2.3.2.2. Phng php nh lng RSV ..................................................... 23
2.3.2.3. nh gi hiu sut liposom ha ................................................... 24
Chng III. THC NGHIM, KT QU, BN LUN .............................. 26
3.1. Kt qu ..................................................................................................... 26
3.1.1. Thm nh phng php nh lng RSV ......................................... 26
3.1.2. Xy dng quy trnh y qua mng ..................................................... 28
3.1.3. Kho st nh hng ca cc iu kin siu m ti cc c tnh ca
liposom ......................................................................................................... 32
3.1.4. So snh hai phng php lm nh kch thc tiu phn: siu m v
y qua mng ............................................................................................... 35
3.2. Bn lun .................................................................................................... 36
KT LUN V KIN NGH......................................................................... 40
TI LIU THAM KHO
Ph lc
DANH MC CC K HIU, CH VIT TT
Chol Cholesterol
DVN Dc in Vit Nam
DPPC Dipalmitoyl phosphatidylcholin
DPPG Dipalmitoyl phosphatidylglycerol
DSC Phn tch nhit vi sai (Differential scanning calorimetry)
DSPC Disteraroyl phosphatidylcholin
KTTP Kch thc tiu phn
LUV Large unilamellar vesicle (liposom to mt lp)
MLV Multilamellar vesicle (liposom nhiu lp)
N/D Nc/ du
PDI Ch s a phn tn (Polydispersity index)
PEG Poly(ethylenglycol)
PL Phospholipid
RES Reticuloendothelial system (h li ni m)
RSV Resveratrol
SPC Phosphatidylcholine u nnh (Soy phosphatidylcholine)
SUV Small unilamellar vesicle (liposom nh mt lp)
Tc Nhit chuyn pha gel - tinh th lng
TEM Knh hin vi in t truyn qua (Transmission electron
microscope)
TKHH Tinh khit ha ha hc
DANH MC CC BNG BIU
STT Tn bng Trang
1 Bng 1.1. Phn loi liposom theo s lp PL v kch thc 4
2 Bng 2.1. Nguyn vt liu 17
3 Bng 2.2. Lng nguyn liu ch to liposom RSV 19
4 Bng 3.1. Gi tr hp th quang ca cc mu dung dch
RSV chun bc sng 308,5 nm 26
5 Bng 3.2. Kt qu o lp li ca phng php nh lng
bng k thut o quang ph UV VIS 28
6 Bng 3.3. Kch thc, phn b KTTP liposom thu c sau
cc ln y qua mng 400 nm 29
7 Bng 3.4. Kch thc, phn b KTTP v hiu sut liposom
ha ca liposom thu c sau khi y qua mng 80 nm 31
8 Bng 3.5. nh hng ca iu kin siu m ti kch thc,
phn b KTTP 33
9 Bng 3.6. nh hng ca iu kin siu m ti hiu sut
liposom ha RSV 34
10
Bng 3.7. Kch thc, phn b KTTP, hiu sut liposom ha
ca liposom c lm nh KTTP bng siu m v y qua
mng
35
DANH MC CC HNH V V TH
STT Tn hnh Trang
1 Hnh 1.1. Cu trc liposom 3
2 Hnh 1.2. C ch hnh thnh liposom theo phng php bc
hi pha o 7
3 Hnh 1.3. C ch gim KTTP liposom bng phng php y
qua mng 10
4 Hnh 2.1. M hnh thm tch 20
5 Hnh 2.2. S tm tt cc giai on ca quy trnh bo ch
liposom RSV 22
6
Hnh 3.1. th biu din mi tng quan gia hp th
quang v nng RSV trong mi trng m PBS v Triton
1% bc sng 308,5 nm
27
7 Hnh 3.2. th bin thin PDI theo s ln y qua mng
400 nm 30
8 Hnh 3.3. th bin thin PDI theo s ln y qua mng
80 nm 31
9 Hnh 3.4. th kch thc trung bnh ca h qua cc ln y
qua mng 80 nm 32
10 Hnh 3.5. Hnh nh chp TEM ca mu liposom lm nh kch
thc bng phng php siu m (A) v y qua mng (B) 36
1
T VN
Resveratrol l mt polyphenol c tm thy t nht 72 loi thc vt,
c bit trong nho, u tng v nhiu loi thc phm khc. Resveratrol c
phn lp ln u t r cy ct kh c (Polygonum cuspidatum), mt loi thc
vt dng trong y hc c truyn phng ng. T nhng nm 1990 n nay
c rt nhiu nghin cu v resveratrol, trn cc m hnh th nghim
resveratrol c cho l c nhiu tc dng c li cho sc khe nh chng
vim, chng oxy ha, bo v tim mch...[5]. Mc d resveratrol c cho l
c tim nng ci thin sc khe v phng nga bnh mn tnh ngi [36]
nhng do cc d liu lm sng trn ngi cha y nn hin nay
resveratrol vn ch c dng di dng thc phm chc nng theo ng
ung. Trn thc t vic s dng resveratrol dng t do gp nhiu hn ch do
resveratrol km n nh di tc ng ca nh sng, nhit [40], [41]. Thm
vo resveratrol t do sau khi hp thu b chuyn ha nhanh v nhiu gan
lm cho sinh kh dng ng ung rt thp [42]. khc phc cc nhc
im ca dng t do, hin nay nhiu h mang thuc mi c nghin cu,
pht trin v ng dng cho resveratrol, trong c liposom [33]. Liposom
khng ch bo v resveratrol khi tc ng bt li ca mi trng, lm tng
n nh m cn gp phn ci thin sinh kh dng ng ung ca
resveratrol. Nhiu phng php khc nhau c s dng bo ch
liposom resveratrol, tuy nhin cc hn dch liposom to thnh c kch thc
ln vi khong phn b kch thc rng. V vy vic lm nh v ng nht
ha kch thc l bt buc trong quy trnh bo ch liposom resveratrol.
gp phn ng dng liposom lm cht mang va tng n nh va
tng sinh kh dng ng ung cho resveratrol chng ti thc hin ti:
2
Nghin cu phng php y qua mng trong lm nh v ng nht
ha liposom resveratrol nhm mc tiu:
- Nghin cu ng dng phng php y qua mng lm nh v ng nht
ha kch thc liposom resveratrol.
3
Chng I. TNG QUAN
1.1. Tng quan v liposom
1.1.1. Khi nim
Liposom l dng c bit ca vi nang (microcapsules) v siu vi nang
(nanocapsules), bao gm mt v phospholipid kp c u thn nc hng ra
ngoi, gm mt hay nhiu lp ng trc bao bc ngn nc gia hoc ngn
cch bi cc ngn nc, c kch thc thay i t hng chc n hng ngn
nanomet [1].
Hnh 1.1. Cu trc liposom
1.1.2. Phn loi
C rt nhiu cch khc nhau phn loi liposom.
Da trn s lp phospholipid v kch thc liposomc th chia liposom
thnh liposom mt lp, liposom nhiu lp v liposom nhiu ngn [4], [11],
[27].
Dc cht thn
nc
Dc cht thn
lipid Lp
Phospholipid kp
Cm bin
Lp bo v
4
Bng 1.1. Phn loi liposom theo s lp PL v kch thc
Loi liposom K hiu ng knh
(nm)
S lp
PL
Lip
oso
m
m
t l
p Liposom mt lp loi nh SUV 20 - 100 1
Liposom mt lp loi ln LUV 200 1000 1
Liposom mt lp khng l GUV >1000 1
Lip
oso
m
nhi
u l
p
Liposom nhiu lp loi nh OLV 100 - 1000 Khong
5
Liposom nhiu lp loi ln MLV >500 5 20
Liposom nhiu ngn MVL >1000 a nhn
Da trn thnh phn v ng dng ca liposom [2], [4], [11], [16], [35]
chia liposom thnh:
(1) Liposom quy c (conventional liposomes - CL): thng ch to t t
nhin hoc phospholipid tch in m kt hp vi cholesterol, v khng c
bin i thnh phn v hoc b mt.
Khi bin i b mt hoc thnh phn v liposom thu c cc loi
liposom mi c cc c tnh mong mun nh:
(2) Liposom nhy cm nhit (thermal sensitive liposomes): thng c
bo ch t cc phospholipid c nhit chuyn pha gel tinh th lng (Tc) cao
hn nhit c th mt cht (nh DPPG, DPPC). Dc cht c gii phng
khi liposom nhit > Tc [7].
(3) Liposom nhy cm pH (pH sensitive liposomes): cu to t cc thnh
phn nhy cm vi pH thp thng l cc phospholipid anion nh
5
phosphatidylethanolamine (PE) [12], di tc ng ca pH acid ni bo
hoc khi u dc cht c gii phng.
(4) Liposom cation: iu ch t phospholipid cationic thch hp vn
chuyn gen, khng bn v c liu cao [17], [34].
(5) Liposom tun hon di hay liposom n (long circulating liposomes hoc
Stealth liposomes): liposom c bao b mt bng cc phn t thn nc ph
bin nht l PEG, nh cu trc khng gian cng knh nn hn ch hin tng
opsonin ha v s bt gi ca cc t bo bch cu do ko di c thi
gian tun hon v d nh hng thuc ti ch hn [17], [20].
(6) Liposom vn chuyn ch ng: trn b mt c gn cc ligand - c
nhn din bi cc receptor c trng ti m/c quan ch. Cc ligand thng
s dng hin nay l khng th (ch yu l khng th n dng lp IgG), folat,
tranferin. Loi liposom ny c ng dng vn chuyn ch ng thuc ti
ch [2].
Ngoi ra cn c th phn loi liposom theo phng php bo ch.
1.1.3. Thnh phn liposom
Liposom hin nay thng c ch to t hai thnh phn c bn l
phospholipid v cholesterol.
Phospholipid c th c ngun gc t nhin: PC (phosphatidyl cholin), PG
(phosphatidylglycerol)... hoc tng hp: DSPC (disteraroyl
phosphatidylcholin), DPPC (dipalmitoyl phosphatidylcholin)... [16]. Mt
trong nhng c tnh quan trng c nh hng ln ti s hnh thnh v n
nh ca liposom l nhit chuyn pha Tc ca phospholipid. di nhit
chuyn pha phospholipid tn ti trng thi gel hay rn sp xp c trt
t, khi nhit tng trn Tc phospholipid chuyn sang trng thi tinh th lng
lm tng tnh thm ca mng v c th gy r r dc cht trong qu trnh bo
6
qun. Tc ca cc PL rt khc nhau (t - 20C ti 90C) ph thuc vo di
v bn cht (bo ha hay khng bo ha) ca chui acid bo [34].
Cholesterol c xem l va ca lp mng kp do c tnh v cu trc
phn t v tan cholesterol c th lp vo cc khong trng gia cc phn
t PL gi cho cu trc mng bn vng hn [16]. Khi trong thnh phn khng
c cholesterol, cc phospholipid c xu hng b ko ra khi mng PL kp do
tng tc vi cc protein huyt tng nh albumin, transferin, macroglobulin
lm cho liposom km n nh.
Ngoi hai thnh phn chnh k trn c th phi hp thm cc t dc khc
vi mc ch a chc nng ha b mt liposom nh: gn PEG (liposom tun
hon di), gn khng th, trasferin, folat (liposom vn chuyn ch ng)....
hoc chng oxy ha nh - tocopherol...
1.1.4. Cc phng php ch to liposom
C nhiu phng php ch to liposom, da trn cch thc phn tn
ngi ta chia cc phng php bo ch liposom thnh ba loi ln l: (i) cc
phng php phn tn c hc: thng dng nht hin nay l hydrat ha mng
mng lipid, (ii) cc phng php phn tn nh dung mi: tim ethanol, tim
ether, bc hi pha o v (iii) cc phng php loi b cht din hot [4],
[11], [16].
Trong phng php bc hi pha o liposom c to thnh qua qu trnh
trung gian to micel o. Ban u PL, chol v cc thnh phn tan trong du
c ho tan vo mt dung mi hu c thch hp (thng dng diethylether
hoc isopropyl ether hoc hn hp isopropyl ether v cloroform). Cho thm
dung dch nc ri tc dng bng siu m to nh tng mn N/D. Cc
micel o c to thnh c cu trc gm mt lp PL n lp bao quanh mt
nhn nc. Bc hi dung mi hu c t t di p sut gim chuyn cc
micel o sang trng thi gel nht. Khi n im ti hn ca qu trnh ny
7
trng thi gel b b gy, cc micel o b ph v trong mi trng d
phospholipid s ti sp xp thnh cu trc lipid kp v to thnh liposom.
Phng php ny thch hp vi nhiu loi phospholipid, to ra liposom c
th tch khoang nc ln, ph hp bao gi c cc phn t nh, ln v c
cc i phn t. Nhc im chnh ca phng php ny l dc cht b tc
ng bi nhit v siu m c th nh hng ti bn (v d: ph v ADN
hoc gy bin tnh protein), c s phi nhim vi dung mi hu c [4].
H nh 1.2. C ch h nh thnh liposom theo phng php bc hi pha o
1.1.5. Cc phng php ng nht ha kch thc liposom
Cc phng php bo ch liposom thng dng hin nay khng to c
liposom c kch thc v khong phn b kch thc tiu phn ti u v vy
vic lm gim v ng nht ha kch thc tiu phn l mt yu cu bt buc.
Cc k thut c th s dng l: siu m, y qua mng, vi ha lng, ct p lc
Franch, ng - chy [11]. Trong hai k thut c s dng rng ri nht
hin nay l siu m v y qua mng [19].
1.1.5.1. Phng php siu m
Siu m l mt trong nhng phng php u tin, n gin bo ch
SUV. C th dng k thut siu m u d hoc b siu m.
8
Siu m u d: que siu m c t ngp trc tip vo hn dch
liposom. Nng lng siu m a vo hn dch liposom l rt ln v c nguy
c gy qu nhit cc b v vy cn lm lnh mu trong qu trnh siu m.
Ngoi ra kim loi (titan) gii phng t u que gy nhim bn ch phm. Siu
m lin tc trong 1 gi khong trn 5% lipid b de - este ha [4].
Siu m b: mu siu m c ng trong dng c thch hp v t vo
b siu m, vic kim sot nhit trong k thut ny d dng hn so vi siu
m u d. Mu liposom c th c bo v trong l tit trng hoc di
dng kh tr [4].
Kch thc trung bnh v s phn b kch thc ca h liposom sau siu
m ph thuc vo nhiu yu t nh thnh phn v nng lipid, th tch dch
siu m, nhit , mc nng lng, thi gian v thao tc ca ngi siu m
[11]. Trong nghin cu ca Yamaguchi v cng s hn dch liposom ch to
t l - alpha - dilauroyl phosphatidylcholin v c lm nh kch thc bng
siu m u d cc nng lng v tn s khc nhau (43 - 480 kHz). Kch
thc trung bnh ca h gim nhanh hn khi siu m tn s thp. So snh
hiu qu lm gim kch thc ca cc mu siu m s dng cng mt tn s
v tng nng lng th siu m trong thi gian ngn vi thit b c nng lng
ln tt hn l siu m trong thi gian di vi thit b c nng lng nh [44].
Giuseppe Maulucci v cng s tin hnh kho st nh hng ca thi gian
siu m ti kch thc v phn b kch thc liposom bo ch t
dimyristoylphosphatidylcholine (DMPC). Cc mu liposom sau khi bo ch
c lm nh kch thc bng siu m u d vi tn s 20 kHz, nng lng
150 W, nhp pht xung 0,4 s trong cc khong thi gian khc nhau. Kt qu
cho thy kch thc ca h gim khi tng thi gian siu m. Nu thi gian
ngn nng lng siu m khng phn ct cc MLV, OLV n kch
thc ti u, nhng nu siu m qu lu mng lipid c nguy c b thoi ha
9
do sinh ra cc gc t do. Thi gian siu m ti u c xc nh ty theo kch
thc liposom yu cu ph hp vi mc ch ng dng ca liposom [24].
Lapinski Monique v cng s bo ch liposom t hn hp 1,2 - dioleoylsn
- glycero - 3 - phosphocholine (DOPC) v 1 - oleoyl - 2 - [12 - [(7 - nitro - 2 -
1,3 - benzoxadiazol - 4 - yl)amino]dodecanoyl] - sn - glycero - 3 -
phosphocholine (18:1 - 12:0 NBD - PC) c hoc khng c chol. Hai mu
liposom th c x l bng siu m b trong 20 pht kt qu cho thy kch
thc trung bnh ca mu liposom c chol cao hn so vi mu khng c chol
(28 2nm so vi 64 3nm), nh vy chol c nh hng ti kch thc ca
liposom khi siu m [19].
Hn ch chnh ca siu m l: nhit lng ln sinh ra trong qu trnh siu
m v tp kim loi t u que c th lm tng thy phn v oxy ha
phospholipid cng nh nh hng ti bn ca dc cht c liposom
ha; liposom to thnh c khong phn b kch thc tng i rng (cc
MLV tn ti ng thi vi SUV) v khng c s ng nht v kch thc v
phn b kch thc gia cc l m [4], [11], [19].
1.1.5.2. Phng php y qua mng
Phng php ny c p dng ln u trong ch to liposom bi Sozka
v cng s t nm 1980 cho ti nay vn l mt trong nhng phng php bo
ch LUV hiu qu [28]. y qua mng c nhiu u im nh: p dng c
vi nhiu loi lipid v hn hp lipid; d thc hin; thng t gy mt lipid,
khng xut hin dng lysophosphatidylcholin trong mu, khng a tp vo
mu, gim nguy c thoi ha lipid; to c liposom c kch thc mong
mun v ng nht; lp li v kch thc v phn b kch thc cao gia
cc l m, c th thc hin trn quy m cng nghip [6], [28], [19], [31], [38].
C ch: Nn hn dch liposom MLV qua mng lc c kch thc l lc
xc nh thu c liposom c kch thc mong mun. Khi b nn qua l
10
lc hnh tr cc liposom c ng knh nh hn l lc s d dng i qua
mng lc. Liposom kch thc ln hn ng knh l lc, mt s s bin
dng i qua l lc , mt s do tc ng ca lc trt b bc lp v ngoi
v ti to li liposom n, t lp vi kch thc nh hn [1], [28].
H nh 1.3. C ch gim KTTP liposom bng phng php y qua mng
Khi nn liposom qua mng lc s xy ra qu trnh ph v v ti to mng
cc thnh phn gn vo liposom c th thot ra ngoi v vy qu trnh y
qua mng c thc hin trong mi trng cha nng thuc cui cng v
lng dc cht t do ch c loi i sau khi to liposom hon tt [28].
Qu trnh y qua mng chu nh hng ca mt s yu t nh: nhit ,
p sut, mng lc (loi mng, kch thc l mng lc), thnh phn ca
liposom.
Nayer v cng s [30] nghin cu bo ch liposom t DSPC v DSPC/
chol t l mol 55:45, gim kch thc tiu phn bng cch y qua mng lc
kch thc l lc 100 nm. Cc MLV c to ra ch t DSPC khng th y
qua mng nhit thp hn 55 C (nhit chuyn pha Tc ca DSPC) k c
khi dng p sut cao 800 lb/in2 (1 lb/in2 = 6895 Pa), nhng khi nng nhit
ln trn Tc tc n nhanh v khng ph thuc nhit na. Khi trong
thnh phn liposom c mt chol th nhit < Tc tc n rt chm
11
(0,06 ml/pht 40 C) cn nhit 65 C tc n cao gp 200 ln tc
40 C. Cc PL bo ha khc cng cho kt qu tng t. Tm li khi y
nhit thp hn Tc tc n rt chm hoc khng n c c th do
mng lipid kp trng thi gel c nht cao v km linh ng [10]. V vy
nhit n nn c thc hin trn nhit chuyn pha ca lipid. S c
mt ca chol lm tng rt nh tc n nhit < Tc nhng lm hn ch
tc n nhit > Tc iu ny c lin quan ti kh nng lm gim
nht ca mng nhit < Tc v lm tng nht ca mng nhit > Tc
ca chol [28].
Hn dch liposom nu c nng ethanol t 10 - 20% (th tch/th tch)
qu trnh y qua mng s d dng hn. Ethanol to iu kin cho lipid i qua
l lc nh lm gim lc cn, tng tc n v cc liposom to thnh c
xu hng phn b kch thc ng nht hn xung quanh ng knh l lc.
Hn na ethanol l mt trong s t dung mi hu c c php s dng trong
sn xut dc phm v c th c loi b khi sn phm cui nh lc gel,
thm tch hoc lc tip tuyn [28].
Kch thc tiu phn liposom gim khi tng p sut n tuy nhin phn
b kch thc tiu phn li khng ph thuc vo p sut n cng nh c
tnh ca lipid to mng liposom [14].
Mng lc thng dng l loi mng polycarbonat bn v tr v mt ha
hc [28]. ng knh l mng lc v s ln y nh hng quyt nh ti
kch thc v phn b kch thc liposom. Khi y qua mng c ng knh
l mng khong 100 nm thu c liposom c kch thc 100 nm, y qua
mng c ng knh l lc ln hn 100 nm thng c liposom kch thc
nh hn ng knh l lc cn y qua mng c ng knh l lc nh hn
100 nm th kch thc liposom thu c thng ln hn ng knh l lc
[25]. Thng thng khi n p MLV qua mng c ng knh l lc 100 nm
12
kch thc liposom gim qua mi ln y v sau khong 10 ln thu c
liposom c kch thc tng i ng nht [28].
1.2. Tng quan v trans - Resveratrol
1.2.1. Cng thc ha hc
Trans - RSV l mt polyphenol nhm stilben c ngun gc thc vt [5].
Cng thc phn t: C14H12O3.
Trng lng phn t: 228,25.
pKa1 = 9.07 (phenol); pKa2 = 10.06 (phenol); pKa3 = 11.56 (phenol).
Tn gi khc: 3,4,5 Trihydroxystilbene [13].
1.2.2. c tnh l ha
- Bt mu trng.
- tan: RSV tan trong du, cng tan trong ethanol 50 mg/ml ( 200 mM)
v trong dimethylsulphoxid (DMSO) 16 mg/ml ( 70 mM) , rt kh tan trong
nc ( 3 mg/100 ml nc) [5].
- Trong methanol RSV c mt cc i hp th ti 305,3 nm [29].
- Trans - RSV l cht nhy cm vi nh sng theo Vian v cng s 80
90% trans - RSV trong dung dch chuyn thnh dng cis khi tip xc vi
nh sng trong mt gi [41], tng t Trela v Waterhouse cng bo co
rng c ti 90,6% trans - RSV chuyn sang dng cis khi chiu trans -
RSV tinh khit di chm tia 366 nm trong hai gi [40].
13
1.2.3. Dc ng hc
Theo Walle v cng s khong 70% tng liu trans - RSV c hp thu
sau khi ung, vi liu tim tnh mch RSV c chuyn sang dng lin hp
sulphat trong vng 30 pht. Sn phm chuyn ha ca RSV khi dng ng
ung gm c dng lin hp sulphat v lin hp glucuronat. Ch mt lng rt
nh RSV khng b chuyn ha (< 5 ng/ml 22 nM) c pht hin trong mu
sau khi dng liu 25 mg ng ung. Nh vy mc d hp thu kh tt nhng
sinh kh dng ng ung ca RSV rt thp do mt lng ln RSV nhanh
chng b chuyn ha bi enzym gan. RSV c thi tr ch yu thn, ngoi
ra cn thi tr qua phn. Trong nghin cu ny tng lng RSV thi tr qua
thn v qua phn sau khi dng liu ung l 71 98%, trong thi tr qua
thn khong 53 85% v qua phn khong 3,3 35% [42].
1.2.4. Tc dng dc l
Mc d c hng ngn cc nghin cu v RSV c thc hin trn cc
m hnh ng vt nhng s lng cc th nghim lm sng trn ngi l rt t
i. T kt qu cc nghin cu thy rng RSV c th c mt s tc dng sinh
hc c li nh: bo v h tim mch [23], phng chng ung th [3], chng tiu
ng [26], [37]... Cc c tnh sinh hc ca RSV c lin quan ti tc dng
chng oxy ha v chng vim ca RSV thng qua c ch qu trnh oxy ha
lipoprotein t trng thp - LDL ngi cng nh c ch enzym
cyclooxygenase 2 v cm ng enzym nitric oxid synthetase. Tc dng
phng nga ung th trn in vivo ca RSV cn c cho l nh kh nng c
ch quinone reductase 2 do iu ha biu hin ca cc cht chng oxy ha
ni bo v cc enzym gii c nng cao sc khng ca t bo vi stress
oxy ha. Ngoi ra RSV cng lm tng hot ng ca SIRT gip nng cao kh
nng khng vi stress ca t bo [5]. Nhn chung cc nghin cu ng h
14
lun im trans - RSV c tim nng ci thin sc khe v phng nga cc
bnh mn tnh ngi [36].
1.3. Tng quan v liposom RSV
Mc d cc kt qu tin lm sng y ha hn vic p dng iu tr ca
RSV cn nhiu hn ch do c tnh khng n nh v sinh kh dng ng
ung thp. RSV thng c s dng di ngy vi mc ch chnh l phng
nga bnh, ng dng ph bin nht ca n l ng ung. Vic tng liu
ung tng tc dng l khng c hiu qu ci thin v sinh kh dng v tim
n kh nng gy hi do cc c tnh c ghi nhn liu cao ca RSV, thm
na l chi ph s rt cao [33]. Cc dng bo ch c in nh: vin nn, vin
nang khng khc phc c cc nhc im ca RSV. t c p ng
ti u th RSV cn c phn phi ti v tr tc dng ca n mt tc v
nng ph hp, va pht huy ti a hiu qu iu tr va gim thiu c
tnh, iu ny i hi phi pht trin c h vn chuyn thuc hiu qu [33].
Hin nay c nhiu nghin cu ng dng cc h a thuc mi cho RSV.
Cyclodextrin c th lm tng tan v n nh ca ca RSV [21]. Cc tiu
phn nano lipid rn gip tng n nh vt l ng thi kim sot gii
phng RSV [39]. Tng t cc vi cu chitosan c ph lp poly(d,l - lactide
- co - glycolide) bo v RSV khi tc ng ca nh sng, lm tng n nh
cng nh kim sot gii phng RSV in vitro [32]. Trong s cc h vn chuyn
thuc hin nay liposom rt c quan tm pht trin lm cht mang cho RSV
bi cc u im nh:
Bo v dc cht:
Isailovi v cng s [15] tin hnh phn tch nhit vi sai (DSC -
Differential scanning calorimetry) vi mu liposom khng cha RSV v
liposom RSV c ch to t SPC v chol. Kt qu phn tch c s xut hin
peak nng chy ca RSV trn th nhit ca mu liposom RSV cho thy
15
RSV c gn vo liposom v c s tng tc gia RSV vi cc thnh
phn lipid cu trc mng trong qu trnh hnh thnh mng. Nh vy RSV nm
xen k trong lp mng lipid kp nh gim tip xc vi mi trng bn
ngoi, trnh hin tng thoi ha gy ra bi nh sng v nhit . Theo
Coimbra M. v cng s khi chiu tia UV vo hn dch liposom RSV c 70%
dng trans cn tn ti sau 16 pht, cn nu dng dng dung dch t do ch c
10% trans - RSV cn tn ti [9].
Tng sinh kh dng:
Liposom l h mang thuc c kh nng gii phng t t RSV trnh s bt
gi v chuyn ha RSV nhanh chng gan nh tng sinh kh dng ca
RSV, gim liu dng, gim chi ph.
Trn m hnh chut thc nghim c tim mng bng doxorubicin liu
4mg/kg/ngy, so snh kh nng bo v tim mch khi cc tc dng ph gy ra
bi doxorubicin khi dng liposom RSV (ch to t HSPC) vi RSV t do liu
4 8 mg/kg/ngy ng ung. Kt qu cho thy liposom RSV lm tng nng
RSV trong mu cng nh tng tc dng bo v tim mch trn chut so vi
RSV t do [29].
Wang. Y v cng s [43] tin hnh bo ch liposom RSV t lecithin :
chol : RSV t l mol l 3:1,5:1 bng phng php hydrat ha mng phim. Hn
dch liposom th c lm nh kch thc bng cch siu m trong 20 pht
ri y qua mng kch thc l mng lc 80 nm. Liposom thu c c kch
thc t 146 585 nm, hiu sut liposom ha khong 73,54%. Th nghim
tc dng bo v ca liposom RSV bo ch theo quy trnh trn v RSV trong
dch chit ct kh c vi t bo thn kinh trn m hnh chut b gy bnh
Parkinson vi liu ung 20 mg/kg/ngy trong thi gian 14 ngy . Kt qu cho
thy c hai dng u c tc dng gim ng k cc hnh vi bt thng, gim
s mt t bo v cht theo chu trnh ca t bo nhn xm, gim lng oxy
16
hot ng v ci thin kh nng chng oxy ha ca cc m nhn xm.
Trong liposom RSV c tc dng r rt hn so vi RSV dng t do.
Julijana Kristl v cng s tin hnh bo ch liposom RSV t hn hp
SPC (129 mol), dicetyl phosphat (36 mol), cholesterol (26 mol) v lm
nh kch thc tiu phn bng cch y qua mng polycarbonat kch thc l
lc 100 nm. Cc mu liposom thu c c hiu sut liposom ha kh cao
> 70%, kch thc phn b tng i ng nht gn kch thc l lc vi PDI
khong 0,2. Th nghim in vivo so snh nh hng ca liposom RSV vi
RSV t do cho thy ch RSV c gn vo liposom c tc dng bo v t bo
khi tc hi ca cc gc t do. Liposom RSV cn c kh nng kim sot gii
phng RSV, bo v t bo khi c tnh do nng cao (100 M) ca RSV,
kch thch hot ng chuyn ha v chng oxy ha ca t bo qua ngn
chn cc nh hng bt li ca stress [18].
Hin nay trn th trng liposom RSV c thng mi ha di dng
thc phm chc nng.
17
CHNG II: I TNG V PHNG PHP NGHIN CU
2.1. i tng nghin cu, nguyn vt liu v phng tin nghin cu
i tng nghin cu: Liposom Resveratrol.
Nguyn vt liu
Bng 2.1. Nguyn vt liu
STT Tn nguyn liu Ngun gc Tiu chun
1 Trans - Resveratrol
DSM Nutritional
Products Ltd - Thy
S
NSX
2 Cholesterol Merch - c NSX
3 Phosphatidylcholin u nnh
(Soy phosphatidylcholine)
Avanti polar Lipids -
M NSX
4 Diethyl ether Trung Quc TKHH
5 Dinatri hydrophosphat Trung Quc TKHH
6 Kali dihydrophosphat Trung Quc TKHH
7 Natri clorid Trung Quc TKHH
8 Kali clorid Trung Quc TKHH
9 Nc ct hai ln Vit Nam DVN IV
10 Triton X 100 MP Biomedicals
M NSX
Phng tin nghin cu
- H thng ct quay Rovapor R - 210 (Buchi c), bnh cu 29/32 GG 17,
dung tch 500 ml.
- H thng thit b phn tch kch thc Zetasizer nano ZS90 (Malvern
Anh).
- Thit b liposome extruder cm tay (M).
18
- My siu m LabsonicM.
- My o quang Spectro UV VIS Double, Labomed, Inc. (M).
- My o pH Inolab (c).
- Knh hin vi in t truyn qua (TEM) JEOL 1010 (Nht Bn).
- Mng thm tch kch thc l xp 12000 14000 Da (Sepetrum - M).
- Cn phn tch, t sy, t lnh, b siu m, ni un cch thy, cc dng c
thy tinh....
2.2. Ni dung nghin cu
- ng dng phng php y qua mng trong lm nh v ng nht ha
liposom RSV bo ch bng phng php bc hi pha o.
Kho st nh hng ca s ln y qua mng n kch thc v phn b
KTTP liposom, hiu sut liposom ha.
- Kho st nh hng ca mt s iu kin siu m (thi gian, nhp pht
xung) ti kch thc, phn b KTTP ca liposom, hiu sut liposom ha.
- So snh liposom thu c sau khi ng nht ha bng phng php y
qua mng v phng php siu m v hnh thi, kch thc, phn b kch
thc, hiu sut liposom ha.
2.3. Phng php nghin cu
2.3.1. Phng php bo ch liposom RSV
Nghin cu la chn phng php bc hi pha o bo ch liposom
RSV c tham kho nghin cu trc [22] v c iu chnh ph hp vi
iu kin thc nghim.
Lng cc thnh phn nguyn liu dng cho ch to liposom RSV c
trnh by bng 2.2:
19
Bng 2.2. Lng nguyn liu ch to liposom RSV
1 SPC 400 mg
2 Cholesterol 40 mg
3 Resveratrol 10 mg
4 Diethyl ether 30 ml
5 m PBS pH 7,4 10 ml
2.3.1.1. To liposom th
Cn cc thnh phn SPC, chol, RSV theo cng thc bng 2.2 vo cc c
m, thm 30 ml diethyl ether ha tan hon ton. Thm 10 ml dung dch
m PBS vo dung dch trn thu c h phn tn hai pha. Tin hnh siu m
u d trong thi gian khong 5 pht, kim sot nhit khong 10C n khi
c nh tng mn N/D.
Chuyn ton b nh tng vo bnh cu ca h thng ct quay. Ct quay
di p sut gim loi dung mi hu c, duy tr nhit 40C, tc quay
100 vng/pht, iu chnh p sut ether bay hi t t trnh to bt. Sau
khong 10 pht ht chn khng ether bay hi ht h chuyn sang trng thi
gel nht. Tip tc ct quay, sau khong 15 pht trng thi gel b ph v hnh
thnh liposom. Ct thm 1 gi na loi hon ton dung mi hu c thu
c hn dch liposom th.
2.3.1.2. Lm nh kch thc v ng nht ha liposom RSV
Nghin cu ny s dng hai phng php lm nh kch thc l siu
m v y qua mng
Phng php y qua mng
S dng thit b y lm nh kch thc liposom cm tay th tch 1 ml.
Hn dch liposom c m nhit 45C trc khi y. u tin y qua
mng polycarbonat kch thc l mng lc 400 nm, sau y tip qua mng
20
80 nm. Thay i s ln y mi loi mng v kho st nh hng ca s ln
y ti cc c tnh ca liposom RSV.
Phng php siu m
Hn dch liposom th sau khi bo ch bng phng php bc hi pha o
c cho vo l thy tinh, th tch mu liposom khong 10 ml. S dng thit
b siu m LabsonicM, cng 100% lm gim kch thc tiu phn.
Que siu m c t ngp vo khong 2/3 mu. Kho st nh hng ca
nhp pht xung (lin tc hay gin on) v thi gian siu m ti kch thc
tiu phn, phn b KTTP, hiu sut liposom ha.
2.3.1.3. Tinh ch liposom RSV
Do iu kin thc nghim khng c sc k lc gel hoc h thng lc tip
tuyn, nghin cu ny s dng phng php thm tch loi RSV t do ra
khi hn dch liposom.
H nh 2.1. M h nh thm tch
Tin hnh: Ht hn dch liposom sau khi lm nh v ng nht ha kch
thc bng y qua mng hoc siu m cho vo ti thm tch (12000 14000
Bt u thm
tch
Trng thi cn
bng
Mng thm
tch
Dung mi
Dch cn
thm tch
21
Da) c kp cht hai u. Mi trng thm tch l m PBS pH 7,4 (thnh
phn 137 mM NaCl; 2,6 mM KC1; 6,4 mM Na2HPO4; 1,4 mM KH2PO4 [38])
c th tch gp t nht 100 ln th tch dch liposom trong ti thm tch. Tng
thi gian thm tch l 8 gi, c mi 2 gi thay m mi mt ln, duy tr nhit
2 - 8 C, trnh nh sng.
Sau khi thm tch liposom c ng l thy tinh, bc giy bc trnh nh
sng v bo qun nhit 2 - 8C.
Quy trnh bo ch liposom RSV bng phng php bc hi pha o c
tm tt trong s sau:
22
H nh 2.2. S tm tt cc giai on ca quy tr nh bo ch liposom RSV
Ha tan trong diethyl ether
Siu m to nh tng N/D
Ct quay loi dung mi hu c
Hn dch liposome th
Lm nh kch thc tiu phn
Thm tch loi RSV t do
ng l thy tinh, bo qun 2-8C
Nhit : 40C
Tc : 100 vng/pht
SPC, chol, RSV
m PBS pH 7,4 Thi gian: 5 pht
Nhit : 10C
Nn
Siu m hoc y
qua mng
23
2.3.2. Phng php nh gi mt s c tnh ca liposom RSV
2.3.2.1. Phng php nh gi hnh thc, hnh thi, kch thc v phn b
KTTP liposom
- V hnh thc: Bng cm quan, liposom RSV sau khi thm tch phi l hn
dch mu trng c, khng c cc tiu phn kch thc ln c th quan st
bng mt thng, c th lng sau mt thi gian bo quan nhng phi nhanh
chng phn tn ng nht tr li sau khi lc.
- V hnh thi liposom: S dng knh hin vi in t truyn qua (TEM) vi
k thut nhum soi m bn quan st hnh thi bn ngoi ca liposom.
Ngoi ra k thut ny cn cho bit kch thc v phn b KTTP liposom.
- nh gi KTTP, phn b KTTP bng phng php tn x nh sng ng
(DLS) s dng thit b Zetasizer ZS 90: Mu liposom c pha long khong
100 ln bng dung dch m PBS pH 7,4 lc qua mng cellulose acetat
0,2 m, chn cc thng s ph hp v tin hnh o, thit b s cho ra gi tr
kch thc, ch s a phn tn PDI, phn b kch thc theo phn trm th
tch ca h.
Ch s Zaverage (n v o d.nm: s nanomet ng knh tiu phn) phn
nh KTTP trung bnh ca mu. Ch s PDI c gi tr thay i t 0 1, i
din cho s phn b ca KTTP trong hn dch, PDI cng gn 0 h cng ng
nht v KTTP. Khi PDI nh c th dng Zaverage nh gi kch thc gia
cc mu liposom khc nhau, tuy nhin nu PDI ln (> 0,5) h rt khng ng
nht ch s Zaverage khng c ngha khi da vo v tr cc peak trn th
phn b KTTP so snh kch thc gia cc mu.
2.3.2.2. Phng php nh lng RSV
Nghin cu la chn phng php o quang nh lng RSV
- Pha long Triton X100 trong nc t l 1 Triton X100 : 99 nc (th
tch/th tch) thu c Triton 1%.
24
- Pha Triton 9/1: ly 9 th tch Triton 1% pha vi 1 th tch dung dch m
PBS.
- Pha dung dch RSV gc : Pha 0,01 g RSV trong 100 ml Triton 9/1 c
dung dch gc c nng 100 g/ml.
- Mu trng: l dung dch Triton 9/1.
- Qut ph tm cc i hp th ca RSV trong mi trng m PBS Triton -
pH 7,4.
Qut ph dung dch RSV gc trong khong bc sng 200 nm 800 nm
tm cc i hp th ca RSV trong mi trng Triton m PBS.
- nh lng RSV trong hn dch liposom
Ht chnh xc 1 ml dch liposom trc hoc sau khi thm tch loi RSV t
do thm Triton 1% va 10 ml, lc u, trong ni un cch thy 40C
thnh thong lc u qu trnh ph mng xy ra hon ton. Sau khong hai
gi ht chnh xc 1 ml dch sau khi ph mng, thm Triton 9/1 nh mc va
25 ml, lc u em o quang cc i hp th ca RSV thu c gi tr
hp th quang A.
2.3.2.3. nh gi hiu sut liposom ha
nh lng RSV trong hn dch liposom trc v sau khi thm tch loi
RSV t do theo phng php nu mc 2.3.2.2.
Hiu sut liposom ha (EE) c tnh bng t l % gia nng RSV
trong hn dch liposom sau khi thm tch loi RSV t do vi nng RSV
trong hn dch liposom trc khi thm tch theo cng thc (1).
EE% =
(1)
Trong :
CE l nng RSV tronng hn dch liposom sau khi thm tch loi RSV t do
(g/ml).
CT l nng RSV trong hn dch liposme trc khi thm tch (g/ml).
25
Sau khi ph mng v em o quang, nu gi AE l hp th quang ca
hn dch liposom sau khi thm tch v AT l hp th quang ca hn dch
liposom trc khi thm tch th cng thc (1) c th bin i thnh:
EE% =
(2)
26
Chng III. THC NGHIM, KT QU, BN LUN
3.1. Kt qu
3.1.1. Thm nh phng php nh lng RSV
Sau khi qut ph pht hin trans - RSV c mt cc i hp th ti bc
sng 308,5 nm trong mi trng Triton 1% v m PBS pH 7,4.
Kho st nh hng ca t dc
Ti bc sng 308,5 nm SPC v chol khng hp th quang nh vy hai t
dc ny khng nh hng ti phng php nh lng.
Kho st tnh tuyn tnh
Chun b mt dy 6 dung dch chun c nng cht chun RSV ln lt
l 2, 3, 4, 5, 6, 7 g/ml t dung dch RSV gc bng cch ht chnh xc ln
lt 0,5; 0,75; 1; 1,25; 1,5; 1,75 ml dung dch gc pha long bng hn hp
Triton 9/1 va 25 ml. Lc u em o quang bc sng cc i thu c
cc gi tr hp th AS. S dng chng trnh Excel ca b phn mm
Microsolf Office dng ng chun th hin s tng quan gia hp th
quang v nng RSV trong dung dch. Kt qu c trnh by bng 3.1 v
hnh 3.1.
Bng 3.1. Gi tr hp th quang ca cc mu dung dch RSV chun
bc sng 308,5 nm
Nng RSV
(g/ml) 2 3 4 5 6 7
Ga tr hp
th quang 0,236 0,341 0,452 0,562 0,664 0,775
Cc gi tr hp th quang l kt qu trung bnh ca ba ln o.
27
H nh 3.1. th biu din mi tng quan gia hp th quang v nng
RSV trong mi trng m PBS v Triton 1% bc sng 308,5 nm
Nhn xt: T th hnh 3.1 ta thy trong khong nng RSV kho
st (2 - 7 g/ml), hp th quang ph thuc tuyn tnh vo nng RSV.
ng chun thu c dng ng thng c phng trnh hi quy tuyn tnh
l y = 0,1078x + 0,0198 vi h s tng quan R2 = 0,9999 rt gn 1. Nh vy
c th s dng phng php o quang bc sng 308,5 nm vi khong
nng trn nh lng RSV trong liposom.
Kho st lp li ca phng php
Ht chnh xc 5 ml dung dch RSV gc pha long bng hn hp Triton
9/1, nh mc 150 ml. o hp th quang ca dung dch ny ti bc
sng cc i 6 thi im khc nhau trong ngy (mi thi im cch nhau
khong 1 gi) trn cng mt thit b v trong cng mt phng th nghim.
Mi gi tr hp th quang c o ba ln ly gi tr trung bnh. Tnh
lch chun tng i RSD ca dy gi tr thu c, nu RSD < 2% th c th
kt lun phng php c lp li cao. Kt qu kho st lp li c trnh
by bng 3.2.
y = 0,1078x + 0,0198
R = 0,9999
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
0 1 2 3 4 5 6 7 8
hp th quang
Nng RSV g/ml
28
Bng 3.2. Kt qu o lp li ca phng php nh lng bng k thut
o quang ph UV VIS
STT Ga tr hp th
quang
1 0,375
2 0,372
3 0,376
4 0,377
5 0,380
6 0,381
Atb 0,377
SD 0,003312
RSD% 0,88
Nhn xt: lch chun tng i ca gi tr hp th quang l RSD =
0,88% < 2% php o c lp li tt.
Phng php nh lng RSV bng k thut o quang khng b nh
hng bi t dc SPC, chol, c lp li cao v c tnh tuyn tnh nn c
th p dng nh lng RSV v nh gi hiu sut liposom ha RSV.
3.1.2. Xy dng quy trnh y qua mng
Hn dch liposom trc tin c y qua mng c kch thc l lc
400 nm, kho st s ln y khc nhau: 7, 9, 11, 13. Ly mu liposom sau cc
ln y khc nhau thm tch loi RSV t do nh gi kch thc v
phn b KTTP bng thit b Zetasizer ZS 90.
Kt qu nh gi kch thc v phn b KTTP liposom sau khi y qua
mng 400 nm c trnh by bng 3.3:
29
Bng 3.3. Kch thc, phn b KTTP liposom thu c sau cc ln
y qua mng 400 nm
S
ln
y
PDI Zaverage ca
h (d.nm) peak
KTTP trung
bnh mi peak
theo ng knh
(d.nm)
% theo th
tch
rng
peak
(dnm)
0 0,535 970,9
1 1000 37,1 269
2 143,86 2,6 31,88
3 5502 60,3 620,8
7 0,256 259,9
1 150,72 7,8 29,12
2 364,8 15,3 164,5
3 4456 76,9 1115,6
9 0,197 251,9
1 95,4 27,5 26,78
2 373,6 72,5 165,64
3 0 0 0
11 0,197 246,9
1 134,02 32,6 35,8
2 414,2 67,4 180,18
3 0 0 0
13 0,140 224,4
1 248 100 102,28
2 0 0 0
3 0 0 0
S bin i PDI ca h qua cc ln y c th hin r hn hnh 3.2
30
H nh 3.2. th bin thin PDI theo s ln y qua mng 400 nm
Nhn xt: Hn dch liposom trc khi y qua mng c kch thc ln v
phn b kch thc tiu phn rng (PDI ln). Sau khi y qua mng 400 nm
kch thc trung bnh v ch s PDI ca h gim dn theo s ln y chng t
h tr nn ng nht hn. Sau 7 ln y PDI gim mnh nht, gim ti 50%
(t 0,535 xung cn 0,256). Cc ln y tip theo PDI gim trung bnh 20
30%. S lng peak v t l phn trm theo th tch ca cc tiu phn kch
thc ln hng nghn nm gim v t l phn trm theo th tch ca cc tiu
phn kch thc nh tng ln qua cc ln y v n ln y th 13 thu c
h n phn tn vi 1 peak.
Mc tiu qu trnh y qua mng 400 nm l gim t l tiu phn kch
thc ln v tng t l tiu phn kch thc nh lm cho h c kch thc nh
hn v phn b KTTP ng nht hn trnh hin tng nt v liposom khi y
qua mng kch thc l lc nh (80 nm). T bng 3.3 v hnh 3.2 ta thy sau
13 ln y qua mng 400nm thu c h n phn tn vi ch s PDI nh
(0,14) nn la chn s ln y l 13 ln cho giai on nghin cu tip theo.
Mu liposom sau khi y 13 ln qua mng c kch thc l lc 400 nm
c y tip qua mng 80 nm, kho st s ln y khc nhau: 5, 7, 9, 11, 13,
15 ln. Ly mu s ln y khc nhau em thm tch loi RSV t do, sau
0
0,1
0,2
0,3
0,4
0,5
0,6
0 7 9 11 13
PD
I
S ln y
31
nh gi nh hng ca s ln y ti kch thc, phn b KTTP v hiu sut
liposom ha. Kt qu c trnh by bng 3.4.
Bng 3.4. Kch thc, phn b KTTP v hiu sut liposom ha ca
liposom thu c sau khi y qua mng 80 nm
S
ln
y
PDI
Zaverage
ca h
(d.nm)
Peak
KTTP trung
bnh mi
peak theo
ng knh
% theo
th tch
rng
peak
(d.nm)
Hiu
sut
(%)
5 0,111 127 1 112,4 100 44,63 95,1
7 0,096 117,9 1 106,5 100 33,58 94,8
9 0,080 113,2 1 102,06 100 30,64 95,18
11 0,062 112,9 1 101,5 100 30,1 95,04
13 0,054 110,8 1 100,94 100 27,78 94,93
15 0,043 109,9 1 100,46 100 26,82 95,06
PDI ca h thu c sau cc ln y qua mng 80 nm nh, h n phn
tn vi 1 peak v rng peak hp. S thay i kch thc trung bnh v ch
s PDI ca h theo s ln y c th hin r hn hnh 3.3 v hnh 3.4.
H nh 3.3. th bin thin PDI theo s ln y qua mng 80 nm
0
0,02
0,04
0,06
0,08
0,1
0,12
5 7 9 11 13 15
PD
I
S ln y
32
H nh 3.4. th kch thc trung b nh ca h qua cc ln
y qua mng 80 nm
Nhn xt: Qua cc ln y qua mng 80 nm PDI ca h gim dn v
khng chng t h cng tr nn ng nht hn. Kch thc trung bnh ca h
gim theo s ln y. T sau ln y th 9 kch thc trung bnh ca h gim
khng ng k, PDI gim dn n nh sau 15 ln y.
T bng 3.4 ta thy hiu sut liposom ha ca cc mu liposom RSV thu
c s ln y khc nhau kh cao 95% v c th thy s ln y qua
mng dng nh khng nh hng ti hiu sut liposom ha.
Qua kho st c th chn s ln y qua mng 80 nm l 15 ln cho cc
nghin cu tip theo.
3.1.3. Kho st nh hng ca cc iu kin siu m ti cc c tnh ca
liposom
Ly cc mu liposom sau cc khong thi gian siu m khc nhau vi
tn s siu m lin tc hay gin on (nhp pht xung 0,6 s), thm tch loi
RSV t do. Tin hnh nh gi kch thc, phn b KTTP, hiu sut liposom
ha theo phng php trnh by mc 2.3.2.
100
105
110
115
120
125
130
5 7 9 11 13 15
Z a
ver
age
(d.n
m)
S ln y
33
- Kt qu nh gi v kch thc v phn b KTTP c trnh by bng
3.5:
Bng 3.5. nh hng ca iu kin siu m ti kch thc, phn b KTTP
iu kin PDI
Zaverage
ca h
(d.nm)
peak
KTTP trung
bnh mi peak
theo ng
knh
% theo
th tch
rng
peak
(d.nm)
Cha siu m 0,535 970,9
1 1000 37,1 269
2 143,86 2,6 31,88
3 5502 60,3 620,8
Siu m lin tc 3
pht 0,31 183,4
1 82,22 60,7 42,7
2 524,6 19,1 264,4
3 5046 20,2 829,4
Siu m lin tc 5
pht 0,302 158,6
1 70,08 87,2 40,38
2 385,6 12,8 144,1
3 0 0 0
Siu m gin
on nhp pht
xung 0,6s 3 pht
0,492 257,4
1 159,5 16,3 60,38
2 1375,6 30,6 449,4
3 5294 53,1 708
Siu m gin
on nhp pht
xung 0,6s 5 pht
0,447 219,6
1 35,3 5,9 5,97
2 132,06 28,2 94,9
3 2968 65,8 1549
Siu m gin
on nhp pht
xung 0,6s 7 pht
0,422 212,8
1 193,96 13,8 125,16
2 3482 63,6 1247
3 40,56 22,6 10,30
Siu m gin
on nhp pht
xung 0,6s 10 pht
0,396 193,82
1 84,56 32,4 41,84
2 439,4 9,2 187,98
3 4892 58,4 904
34
Nhn xt:
- Mu cha siu m c kch thc v ch s a phn tn ln hn mu sau
siu m. Siu m lm gim kch thc v PDI ca h.
- Thi gian siu m cng di kch thc trung bnh v PDI cng gim (t
0,535 trc khi siu m xung 0,302 khi siu m lin tc 5 pht), h tr nn
ng nht hn (t peak hn v/ hoc peak gn hn).
- Vi cng thi gian siu m th siu m lin tc to ra h liposom c kch
thc v PDI nh hn tc l ng nht hn so vi siu m ngt qung.
Kt qu nh gi hiu sut liposom ha RSV ca cc mu siu m iu
kin khc nhau c trnh by bng 3.6:
Bng 3.6. nh hng ca iu kin siu m ti hiu sut liposom ha RSV
iu kin
siu m
Nhp pht xung
Lin tc Gin on (nhp pht xung 0,6 s)
3 pht 5 pht 3 pht 5 pht 7 pht 10 pht
Hiu sut
% 92,2 90,03 95,1 94,8 93,79 92,9
Nhn xt: Thi gian siu m cng di hiu sut liposom ha cng gim.
Vi cng thi gian siu m th siu m lin tc c hiu sut liposom ha thp
hn siu m gin on.
T bng 3.5 v bng 3.6 ta thy khi siu m lin tc kch thc v PDI
gim nhiu hn siu m gin on tuy nhin hiu sut liposom ha li thp
hn. Vi siu m gin on mc d h liposom sau siu m 10 pht c PDI
gim nhiu nht (0,396 so vi 0,535 trc khi siu m) tuy nhin hiu sut
liposom ha li thp nht. m bo hiu sut liposom chn iu kin siu
m gin on nhp pht xung 0,6 s trong thi gian 5 pht.
35
3.1.4. So snh hai phng php lm nh kch thc tiu phn: siu m v
y qua mng
Liposom c bo ch bng phng php bc hi pha o theo quy trnh
mc 2.3.1.1 v c lm nh kch thc tiu phn theo mt trong hai
phng php siu m u d (thi gian 5 pht, nhp pht xung 0,6 s) hoc y
qua mng (y ln lt 13 ln qua mng 400 nm v 15 ln qua mng 80 nm).
nh gi liposom to thnh v kch thc, phn b KTTP, hiu sut liposom
ha v s b hnh thi liposom. Kt qu th hin bng 3.7 v hnh 3.5.
Bng 3.7. Kch thc, phn b KTTP, hiu sut liposom ha ca liposom
c lm nh KTTP bng siu m v y qua mng
Phng php
Kch thc tiu
phn trung bnh
theo ng knh
Ch s a phn
tn (PDI) Hiu sut %
Siu m 219,6 0,447 94,80
y qua mng 109,9 0,043 95,06
Nhn xt: H liposom to thnh theo hai phng php siu m v y qua
mng c hiu sut gn tng t nhau. Tuy nhin phng php y qua mng
cho tiu phn liposom kch thc nh hn 1/2 ln v ch s PDI nh hn rt
nhiu ln, tc l h ng nht hn so vi phng php siu m.
nh gi hnh thi liposom to thnh bng thit b TEM vi k thut
nhum soi m bn. Kt qu thu c th hin hnh 3.5.
36
A B
H nh 3.5. H nh nh chp TEM ca mu liposom lm nh kch thc
bng phng php siu m (A) v y qua mng (B)
Nhn xt: C hai phng php u to c liposom c dng gn cu.
Phng php y qua mng cho liposom c kch thc phn b kh ng u
kch thc khong t 111 130 nm. Phng php siu m to liposom kch
thc phn tn hn, quan st thy c hin tng nt v liposom.
3.2. Bn lun
V phng php nh lng RSV v nh gi hiu sut liposom ha:
Nghin cu la chn phng php o quang ph hp th nh lng RSV,
phng php ny c u im l d thc hin, ph hp vi iu kin thit b
thc nghim. Sau mt s php thm nh thy rng cc t dc SPC, chol
khng hp th quang ti bc sng nh lng 308,5 nm do khng nh
hng ti php o. Mt khc phng php ny c tnh tuyn tnh v lp li
cao. V vy c th ng dng phng php o quang ph hp th nh
lng RSV trong liposom v nh gi hiu sut liposom ha. Mt im cn
lu y l trans - RSV dng t do trong dung dch rt nhy cm vi nh
sng do trong qu trnh ph mng phi c bin php trnh nh sng (bc
giy bc), thao tc o nhanh chng trnh hin tng chuyn dng ng
phn trans - RSV sang dng cis lm nh hng ti kt qu php o.
37
V phng php lm nh v ng nht ha kch thc tiu phn:
Nghin cu ny s dng hai phng php thng dng nht hin nay l
siu m v y qua mng. Trong phng php siu m u d cc tiu phn
cng gn que siu m cng chu tc ng lc siu m vi cng ln hn,
do cc tiu phn c lm nh vi mc khc nhau. V vy h liposom
thu c c khong phn b kch thc rng (bng 3.5). Siu m u d a
mt nng lng ln vo mu, lc phn ct ln c th lm nt v liposom v
lm tng nhit mu siu m c bit l khi siu m lin tc. Nu khng
kim sot tt nhit c th tng trn nhit chuyn pha ca SPC. Hu qu
l lp mng lipid kp trng thi tinh th lng kh nng lu gi RSV km i
v RSV c th b lc siu m y ra khi mng lm gim hiu sut liposom
ha. iu ny c th gii thch cho hin tng gim hiu sut liposom ha
RSV khi tng thi gian siu m v thay i nhp pht xung (bng 3.6). Nghin
cu ny cho thy phng php y qua mng khc phc c cc nhc
im ca siu m. y qua mng cho php d dng kim sot nhit , trong
qu trnh y khng c hin tng qu nhit cc b. Thm na quy trnh y
13 ln qua mng lc th kch thc l lc 400 nm trc khi y 15 ln qua
mng lc tinh kch thc l lc 80 nm gip hn ch hin tng nt v
liposom. Ngoi ra y qua mng khng a tp kim loi vo mu nh siu m
u d (tiu phn titan gii phng t u que siu m). Kt qu nghin cu
cho thy phng php y qua mng to ra h liposom c phn b kch thc
tiu phn ng nht hn phng php siu m u d (PDI = 0,043 vi y
qua mng v PDI = 0,447 vi siu m u d).
Trong mt nghin cu trc Isailovic v cng s [15] tin hnh bo
ch liposom RSV t nguyn liu SPC c hoc khng c chol, t l khi lng
RSV:SPC l 1:20, bng cc phng php hydrat ha mng phim (SPC:chol t
l khi lng 2,3:1) v phng php proliposom (khng dng chol). Hn
38
dch liposom th c lm nh kch thc bng k thut siu m u d (siu
m lin tc trong thi gian 6 pht) hoc y qua mng (y ln lt qua cc
mng 800 nm, 400 nm, 200 nm v 100 nm, mi mng y 11 ln). nh gi
phn b KTTP cho thy phng php y qua mng to ra h liposom ng
nht hn siu m u d (PDI bng 0,22 ca mu liposom y qua mng so
vi 0,47 ca mu liposom siu m u d), kt qu ny ph hp vi nghin
cu ca chng ti. Kch thc trung bnh ca h liposom trong nghin cu
ca Isailovic cao hn nghin cu ca chng ti (290 nm so vi 158,6 nm sau
siu m lin tc). C th l do thnh phn cng thc bo ch bng phng
php hydrat ha mng mng lipid ca Isailovic v cng s c s dng mt t
l chol kh cao. S c mt ca chol trong thnh phn mng PL c xu hng
lm tng kch thc trung bnh ca h liposom [19]. Trong nghin cu ca
Isailovic cc mu liposom lm gim kch thc tiu phn bng y qua mng
cho hiu sut liposom ha cao (92 - 97%) tuy nhin mu liposom gim kch
thc bng siu m ch c hiu sut liposom ha thp (44 - 55%). Theo cc
nh nghin cu ny trong qu trnh siu m c s mt ng k RSV do nh
hng ca lc siu m ln ph v lp mng PL gii phng RSV khi
liposom. Kt qu ny cng ph hp vi kho st chng ti v cc iu kin
siu m (hiu sut liposom ha gim khi tng thi gian siu m v siu m
lin tc).
Hiu sut liposom ha cao khi ng dng phng php y qua mng
trong lm nh v ng nht ha kch thc tiu phn cng c th hin
trong mt nghin cu khc. Caddeo v cng s [8] tin hnh bo ch
liposom RSV t hn hp SPC (129 mol), dicetyl phosphat (36 mol), chol
(26 mol) v RSV (6,5 mol ). Hn dch liposom th c lm nh kch
thc tiu phn bng cch y 21 ln qua mng polycarbonat kch thc l
lc 100 nm. Mu liposom thu c c kch thc 70,7 1,9 nm, PDI 0,237
39
0,031, th Zeta - 38,4 0,6 (mV), hiu sut liposom ha 79,7 3,8%. Kch
thc trung bnh ca h v hiu sut liposom ha trong nghin cu ny thp
hn nghin cu ca chng ti. Tuy nhin cng thc ca Caddeo c u im l
b sung thm thnh phn dicetyl phosphat lm tng kh nng tch in b mt
ca liposom, trnh kt t v lm tng n nh cho liposom.
Nh vy t kt qu ca nghin cu ny v cc nghin cu trc [8],
[15] c th kt lun phng php y qua mng c nhiu u im vt tri so
vi phng php siu m trong vic lm nh v ng nht ha kch thc
liposom RSV. Do c th ng dng quy trnh y qua mng lm nh v
ng nht ha liposom c xy dng trong nghin cu ny cho cc nghin
cu tip theo.
40
KT LUN V KIN NGH
Kt lun
Trong khun kh kha lun tt nghip vi ti Nghin cu phng
php y qua mng trong lm nh v ng nht ha liposom resveratrol,
chng ti thu c nhng kt qu sau:
nghin cu xy dng v p dng phng php y qua mng trong
lm nh v ng nht ha liposom resveratrol. Kt qu cho thy khi y hn
dch liposom th 13 ln qua mng 400 nm ri y tip 15 ln qua mng 80 nm
thu c h liposom c kch thc trung bnh theo ng knh l 109,9 nm v
PDI 0,043.
So snh phng php y qua mng vi phng php siu m u d:
phng php y qua mng to ra h liposom c kch thc trung bnh nh
hn v ng nht hn phng php siu m u d (kch thc trung bnh
109,9 nm, ch s a phn tn 0,043 ca mu y qua mng so vi kch thc
trung bnh 219,6 nm, ch s a phn tn 0,447 ca mu siu m u d).
Kin ngh
- Nghin cu thay th SPC bng nguyn liu phospholipid hydrogen ha,
b sung thm cc cht chng oxy ha v cc thnh phn lm tng kh nng
tch in b mt cho liposom tng n nh cho liposom resveratrol.
- Nghin cu n nh di hn ca liposom resveratrol.
- Nghin cu kh nng gii phng in vitro, in vivo ca liposom resveratrol.
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Ph lc
Cha lm nh KTTP
y qua mng 400 nm - 7 ln
y qua mng 400 nm 9 ln
y qua mng 400 nm 11 ln
y qua mng 400 nm 13 ln
y qua mng 80 nm 5 ln
y qua mng 80 nm 9 ln
y qua mng 80 nm 7 ln
y qua mng 80 nm 13 ln
y qua mng 80 nm 11ln
y qua mng 80 nm 15 ln
Siu m lin tc 3 pht
Siu m lin tc 5 pht
Siu m gin on 3 pht
Siu m gin on 5 pht
Siu m gin on 7 pht
Siu m gin on 10 pht