B Y T

TRNG I HC DC H NI

HI OAN

NGHIN CU TO LIPOSOME

RESVERATROL KCH C NANO

BNG PHNG PHP

BC HI PHA O

KHA LUN TT NGHIP DC S

H NI - 2014

B Y T

TRNG I HC DC H NI

HI OAN

NGHIN CU TO LIPOSOME

RESVERATROL KCH C NANO

BNG PHNG PHP

BC HI PHA O

KHA LUN TT NGHIP DC S

Ngi hng dn:

TS. Nguyn Th Lp

Ni thc hin:

B mn Ho Sinh

B mn bo ch

H NI 2014

LI CM N

hon thnh c kho lun tt nghip ny, u tin em xin by t lng

bit n su sc nht n:

TS. Nguyn Th Lp

Ngi thy tn tnh hng dn v gip em trong sut qu trnh thc

hin ti.

Em xin chn thnh cm n cc thy, c gio, cc ch k thut vin trong b

mn Ho sinh to iu kin thun li v gip em trong qu trnh thc

hin ti.

Em cng xin by t lng bit n chn thnh ti cc thy, c gio v cc anh

ch k thut vin trong b mn Bo ch nhit tnh gip em hon thnh

kho lun tt nghip ny.

Cui cng em xin gi li cm n ti gia nh, bn b, nhng ngi lun

bn ng vin, gip em trong sut qu trnh hc tp v thc hin ti

tt nghip ny.

H Ni, ngy 20 thng 4 nm 2014

Sinh vin

Hi oan

i

MC LC

DANH MC VIT TT ................................................................................................................... iii

DANH MC CC BNG ................................................................................................................ iv

DANH MC CC HNH V, TH ............................................................................................. v

T VN ................................................................................................................................... 1

CHNG 1. TNG QUAN .............................................................................................................. 3

1.1. Tng quan v liposome .......................................................................................................... 3

1.1.1. Khi nim .................................................................................................................... 3

1.1.2. Thnh phn.................................................................................................................. 3

1.1.3. Phn loi ..................................................................................................................... 4

1.1.4. Phng php bo ch .................................................................................................. 4

1.2. Tng quan v resveratrol ....................................................................................................... 8

1.2.1. Cng thc ha hc, tnh cht l ha. ........................................................................... 8

1.2.2. Ngun cha resveratrol ............................................................................................... 9

1.2.3. Tc dng ..................................................................................................................... 9

1.2.4. Sinh kh dng ........................................................................................................... 10

1.2.5. Cc bin php ci thin sinh kh dng ca resveratrol ............................................. 10

1.2.6. Tng quan cc nghin cu bo ch liposome resveratrol ......................................... 11

CHNG 2. I TNG V PHNG PHP NGHIN CU ............................................... 14

2.1. Nguyn vt liu, thit b ....................................................................................................... 14

2.1.1. Nguyn vt liu ......................................................................................................... 14

2.1.2. Thit b ...................................................................................................................... 14

2.2. Ni dung nghin cu ............................................................................................................ 15

2.2.1. Kho st t l mol cc thnh phn trong cng thc bo ch liposome RES .............. 15

ii

2.2.2. nh gi tiu phn liposome RES to thnh qua cc thng s ................................. 15

2.3. Phng php thc nghim ................................................................................................... 15

2.3.1. Phng php ch to liposome RES ......................................................................... 15

2.3.2. nh lng RES ........................................................................................................ 17

2.3.3. nh gi tiu phn liposome resveratrol to thnh ................................................... 18

2.3.3. Phng php x l s liu ......................................................................................... 19

CHNG 3. THC NGHIM, KT QU V BN LUN ........................................................ 20

3.1. KT QA ............................................................................................................................ 20

3.1.1. Thm nh phng php nh lng RES ................................................................ 20

3.1.2. Kho st nh hng ca t l mol cc thnh phn trong cng thc n KTTP, PDI v

hiu sut liposome ha .............................................................................................. 22

3.1.3. nh gi liposome to thnh ..................................................................................... 27

3.2. NHN XT, BN LUN ................................................................................................... 28

3.2.1. V phng php bo ch ........................................................................................... 28

3.2.2. V phng php nh lng RES ............................................................................. 29

3.2.3. nh hng ca cc yu t cng thc v quy trnh bo ch ti tnh cht h liposome

to thnh .................................................................................................................... 30

KT LUN, KIN NGH ............................................................................................................... 34

KT LUN ................................................................................................................................. 34

KIN NGH ................................................................................................................................ 34

TI LIU THAM KHO ................................................................................................................ 35

PH LC ......................................................................................................................................... 40

iii

DANH MC VIT TT

Chol

COX 1

COX 2

DMPC

DPPC

DQA-PEG2000-

DSPE

DSPC

EE

LDL

LUV

MLV

PBS

PDI

PEG

PL

RES

SPC

SUV

Tc

VLDL

Cholesterol

Cyclooxygenase 1

Cyclooxygenase 2

Dimyrisitoyl phosphatidyl cholin

Dipalmoyl phosphatidyl cholin

Dequalinium polyethylen glycol-

distearoylphosphatidylethanolamin

Distearoyl phosphatidyl cholin

Encapsulation efficiency (hiu sut liposome ha)

Low-density lipoprotein (lipoprotein t trng thp)

Large unilamellar vesicle (liposome mt lp loi nh)

Multilamellar vesicle (liposome nhiu lp)

Phosphate buffered saline m phosphate

Polydispersity index (ch s a phn tn)

Polyethylen glycol

Phospholipid

Resveratrol

Soy phosphatidylcholin

Small unilamellar vesicle (liposome mt lp loi nh)

Nhit chuyn pha

Very-low-density lipoprotein (lipoprotein t trng rt

thp)

iv

DANH MC CC BNG

Bng Tn bng Trang

1 tan ca RES trong cc dung mi khc nhau 8

2 Nguyn vt liu 14

3

hp th ca dung dch RES trong mi trng

m PBS pH 7.4 v Triton 1% ti cc thi im sau

khi pha

20

4 S ph thuc ca hp th vo nng RES

trong mi trng m PBS pH 7.4 v Triton 1% 21

5 Thnh phn cng thc bo ch cc mu kho st

t l mol RES/SPC 22

6 nh hng ca t l RES/SPC n kch thc, phn

b kch thc tiu phn liposome 23

7 nh hng ca t l RES/SPC n hiu sut

liposome ha 23

8 Thnh phn CTBC cc mu kho st t l mol

Chol/SPC 25

9 nh hng ca t l chol/SPC n kch thc, phn

b kch thc tiu phn liposome 25

10 nh hng ca t l chol:SPC n hiu sut

liposome ha 26

v

DANH MC CC HNH V, TH

Hnh Tn hnh Trang

1 Cu to liposome 3

2 C ch hnh thnh liposome trong phng php bc

hi pha o 5

3 Cng thc ha hc ca resveratrol 8

4 Mt s ch phm liposome RES trn th trng 13

5 Quy trnh bo ch liposome resveratrol 15

6 th s ph thuc ca hp th vo nng

RES trong mi trng m PBS pH 7.4 v Triton 1%

21

7 th biu din hiu sut liposome ha cc mu

liposome kho st t l RES/SPC 24

8 th biu din hiu sut liposome ha cc mu

liposome kho st t l chol/SPC 26

9 Hnh nh chp TEM ca liposome sau khi siu m 27

1

T VN

Resveratrol (RES) l mt polyphenol khng c t t nhin c tng hp

bi nhiu loi cy nh: nho (Vitis spp.), du (Vaccinium spp.), u tng

(Arachis spp.), ct kh c (Polygonum cuspidatum), c tc dng nh hng

ro chng li nm, virus, vi khun v tc ng ca tia UV [32]. c pht

hin t nm 1939. Tuy nhin, n u nhng nm 90 ca th k 20, RES mi

c nghin cu v tc dng bo v tim mch trong nghch l ca ngi

Php (French Paradox) [24]. T , RES c quan tm su sc hn vi cc

nghin cu v cc tc dng c li nh bo v thn kinh, chng oxy ha,

chng vim, chng ung th, chng lo ha [9]. Mc d RES c nhiu tc

dng in vitro, tuy nhin cng nh nhiu polyphenol t nhin khc, RES c

sinh kh dng b hn ch bi ha tan km trong dch sinh hc, cng nh

nhanh chng b chuyn ha v thi tr [38]. Do vy, cn c cht mang thch

hp c c hiu qu iu tr vi liu dng thp. Liposome l mt cht

mang u vit cho mc ch a thuc vo c th, v tnh thch ng sinh hc,

phn hy sinh hc hon ton v c th mang c dc cht thn du v dc

cht thn nc. Hin nay, trn th gii c mt s cng trnh nghin cu

cng nh sn xut ch phm liposome RES. Tuy nhin, Vit Nam cha c

cng trnh khoa hc c cng b v ng dng liposome lm cht mang

RES, m mi ch nghin cu tng hp RES bng phng php ha hc, hay

phn lp t cy ct kh c [3].

Do , chng ti thc hin nghin cu ti: Nghin cu to liposome

resveratrol kch c nano bng phng php bc hi pha o nhm mc

ch b sung thm thng tin v liposome RES, v bc u bo ch c

liposome RES trong iu kin ha cht cng nh thit b ti Vit Nam.

Mc tiu nghin cu:

2

- Kho st c t l mol RES : SPC v chol : SPC trong cng thc bo ch

liposome resveratrol bng phng php bc hi pha o.

- nh gi c tiu phn liposome RES to thnh v mt s thng s:

Kch thc tiu phn, ch s a phn tn v hiu sut liposome ha.

3

CHNG 1. TNG QUAN

1.1. Tng quan v liposome

1.1.1. Khi nim

Liposome l dng c bit ca microcapsule v nanocapsule (hnh 1), gm

mt nhn nc gia v bao ngoi l mt hay nhiu lp phospholipid ng

trc, c kch thc thay i t hng chc n hng ngn nanomet (0,03 10

m) [1], [6].

Hnh 1. Cu to liposome

1.1.2. Thnh phn

- Thnh phn ch yu: phospholipid (PL)

Phospholipid l nhng phn t lng tnh va thn du va thn nc, cu

to t khung phn t glycerol gn vi cc nhm phn cc v cc acid bo.

Cc PL c kh nng t ng vng to liposome khi chng c trn ln

vi mi trng nc. u phn cc s hng ra mi trng nc, ui khng

phn cc quay vo vi nhau, to ra lp lipid kp [17].

- Cholesterol (chol) v dn cht c thm vo PL lm tng bn vng

v gim tnh thm ca liposome, nng ti a chol c th kt hp trong

Gn PEG bo v Khng th

Dc cht thn nc kt tinh

trong pha nc Lp lipid kp Dc cht thn du

trong mng kp

4

mng PL kp l 66 %mol i vi mng phosphatidyl cholin, 51 %mol i vi

mng phosphatidyl ethanolamine [21].

Khi b sung chol vo lp mng kp ca liposome u phn cc ca chol s

nm gn u phn cc ca PL, trong khi ui k nc c xp song song vi

cc chui acid bo ca phn t PL. Do , chol c vai tr c nh c hc lm

cho ui acid bo tr nn bt ng, cc phn t PL kh c th chuyn dch

c, t lm tng n nh ca mng, gim tnh thm ca mng v tng

sc chu ng ca liposome trc s tc ng ca p sut thm thu.

1.1.3. Phn loi

Da vo kch thc v s lp PL (hnh 3)

- Liposome mt lp: v ch c mt lp PL, ty theo kch thc chia lm hai

loi:

+ Loi nh (SUV) c ng knh khong 20 50 nm.

+ Loi ln (LUV) c ng knh 200 1000 nm.

- Liposome nhiu lp (MLV): cu to gm nhiu lp PL v nhiu ngn nc

ng trc, c ng knh t 400 - 3500 nm.[1], [6]

1.1.4. Phng php bo ch

Phng php Bangham (phng php hydrat ha mng film)

Ho PL v cc thnh phn to v liposome vo dung mi hu c

(chloroform, methanol). Bc hi dung mi di p sut gim trong bnh ct

quay, PL s to thnh mng mng bm ln thnh bnh ct. Nhit tin hnh

xp x nhit chuyn pha ca PL. Thm dung dch nc c h m, va cho

va lc PL hydrat ho to thnh liposome. Phng php ny thu c

liposome nhiu lp, c kch thc khng ng nht [1], [6], [18], [27].

Phng php Batzri v Korn (phng php tim/ ha tan ethanol)

Ha tan PL v cc thnh phn to mng vo ethanol. Bm nhanh dung dch

ny vo dung dch KCl 0,1 - 0,2 M (hoc mt dung dch khc tng ng, v

5

d MgSO4), do thay i dung mi s to thnh cc liposome. Siu lc loi

ethanol v tinh ch liposome. Phng php ny to ra liposome c kch thc

khng u [1], [6], [31].

Phng php bc hi pha o

Phng php ny da trn s hnh thnh micel o khi siu m hn hp

pha nc cha dc cht thn nc vi pha du ha tan cc cht thn du.

Bc hi dung mi hu c t t hnh thnh gel nht. Ti im ti hn,

trng thi gel b ph v, hnh thnh mng kp bao quanh cc micel o cn

li, to thnh liposome (hnh 4). T l pha nc: dung mi hu c l 1/3 vi

diethylether v 1/6 i vi hn hp dung mi isopropylether v chloroform

[7].

Phng php ny cho hiu sut to liposome cao, p dng c vi nhiu

loi PL, nhng to ra liposome kch thc ln, dc cht chu tc ng ca

nhit v siu m, c th nh hng n bn [1], [6].

Hnh 2. C ch hnh thnh liposome trong phng php bc hi pha o [6]

Ngoi ra cn nhiu phng php khc nh: Phng php ha tan v thm

tch cht din hot, phng php pha long polyol, phng php ng kh,

phng php vi knh[27].

6

lm nh kch thc tiu phn liposome to thnh, ngi ta thng s

dng cc phng php nh: Phng php y qua mng, phng php siu

m, phng php ng chy [22].

Phng php y qua mng

y qua mng l mt trong nhng phng php ph bin nht c dng

to liposome mt lp v kim sot kch thc ca chng. Trong phng

php ny, hn dch liposome c y qua mt mng polycarbonate to ra

liposome c kch thc gn vi kch thc l lc ca mng. Phng php ny

hn ch vic phi loi tp cht trong sn phm to thnh. u im ln nht l

kch thc v phn b kch thc thy phn (KTTP) c lp li cao gia

cc l m sn xut [28].

Phng php siu m

Siu m l mt trong nhng phng php ph bin bo ch liposome t h

phn tn PL trong pha nc, c p dng t khi bt u nghin cu v

liposome. Siu m c th coi l phng php ph bin nht c p dng

bo ch SUV. C th dng siu m b hoc siu m cm tay lm nh kch

thc tiu phn, p lc gy ra bi nng lng siu m ph v cc liposome

nhiu lp kch thc ln thnh cc liposome kch thc nh hn, c th l

liposome mt lp hoc liposome nhiu lp kch thc nh [40]. Kch thc

liposome to thnh ph thuc vo thi gian v cng siu m [17], khng

ph thuc vo chiu di chui hydrocarbon ca cc PL. Theo nghin cu ca

Yamaguchi T. v cng s (2009), siu m vi tn s thp lm gim KTTP

nhanh hn so vi siu m tn s cao. Cng tn s v tng nng lng, siu m

vi cng mnh hn trong thi gian ngn cho hiu qu cao hn siu m

cng yu trong thi gian di [40]. u im chnh ca phng php siu

m l tit kim thi gian.. Tuy nhin c th gy qu nhit cc b [17]. Hn

7

na, liposome to thnh c khong phn b kch thc tng i rng (cc

MLV tn ti ng thi vi SUV), khng c s ng nht gia cc l m [7].

- Siu m cm tay: que siu m c t ngp trc tip vo hn dch

liposome. Nng lng siu m a vo 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.

- 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 cm tay. Mu liposome c th c bo v trong l tit trng hoc di

dng kh tr [7].

Phng php ng chy

ng - chy l qu trnh lm nh kch thc tiu phn liposome (thng l

MLV) thnh cc tiu phn liposome c s lp t hn (nh LUV) bng chu k

lm lnh hn dch liposome nhit thp, ri tip tc lm chy. Thng s

dng lp i lp li nhiu ln chu k ng chy ni trn. Kch thc trung

bnh v s lp ca liposom gim dn khi s chu k tng ln [33].

8

1.2. Tng quan v resveratrol

1.2.1. Cng thc ha hc, tnh cht l ha.

Hnh 3. Cng thc ha hc ca trans-resveratrol

Tn khoa hc: Trans-3,5,4-Trihydroxystilbene; hay 3,4,5-Stilbenetriol;

hay (E)-5-(p-hydroxystyryl) resorcinol; hay (E)-5-(4-hydroxystyryl) benzene-

1,3-diol

CTPT: C14H12O3 PTL: 228,4 g/mol

Tnh cht l ha

Bt mu trng hay vng nht, tan rt t trong nc, d tan trong ethanol.

RES tn ti di hai dng ng phn cis- v trans-. Trong , dng trans c

tc dng dc l mnh hn. Dng trans c th chuyn thnh dng cis khi tip

xc vi tia UV, nhit . Theo Trela v Waterhouse, khi phi nhim trong

vng 120 pht vi tia UV bc sng 366 nm, 90.6 % trans - RES b chuyn

thnh dng cis [36].

Bng 1. tan ca RES trong cc dung mi khc nhau [41]

nh tnh: bng sc k lp mng

Bn mng silicagel GF 254, hot ha 100oC trong 1h

H dung mi: n-hexane/ ethyl acetat (2/1)

Thuc th hin mu: dung dch FeCl3 5%

So snh Rf vi Rf ca cht chun tin hnh trong cng iu kin [3].

Dung mi tan

Nc 0.03 g/l

DMSO 16 g/l

Ethanol 50 g/l

9

nh lng: Bng phng php o quang ph hp th ca RES ti max

[14].

1.2.2. Ngun cha resveratrol

c cp ln u tin trong mt bi bo ca Nht Bn vo nm 1939

bi Michio Takaoka, RES c phn lp t cy Veratrum grandiflorum [35].

Mt trong s cc ngun giu RES nht l r cy ct kh c (Polygonum

cuspidatum), v qu nho (Vitis vinifera), du (Vaccinium spp.), u tng

(Arachis spp.), thng, da, ca cao[32].

1.2.3. Tc dng

Cc nghin cu in vitro v nghin cu trn ng vt thc nghim cho thy

RES c cc tc dng nh:

Tc dng chng oxy ha

- RES c ch qu trnh peroxide ha LDL.

- RES lm gim qu trnh oxy ha xc tc bi ng bng cch to phc

chelat vi ng.[19]

- RES c kh nng dn gc t do mnh hn c vitamine C v E [34].

Tc dng chng ung th

RES c tc dng trn nhiu loi ung th ngi, nh ung th da (dng ti

ch hoc ng ung), ung th v, ung th d dy v rut kt, ung th thc

qun, ung th tuyn tin lit, ung th gan, ung th nguyn bo thn kinh, ung

th ty, sarcoma dng si v leukemia [8].

Tc dng trn bnh tiu ng [20].

Tc dng ca RES trn tim mch

- Tc dng chng kt tp tiu cu.

- Tc dng gin mch.[19]

- RES c ch s oxy ha LDL [19], [23].

Tc dng trn qu trnh vim v min dch

10

RES c ch hot ng ca COX v do c tc dng chng vim [23].

iu bin chuyn ha lipid v lipoprotein [19].

V mt s tc dng khc.

1.2.4. Sinh kh dng

RES c hp thu t nht l 70 % i vi liu 25 mg ng ung. Tuy

nhin, sinh kh dng ca RES l gn bng 0, nng nh trong huyt tng

ca RES v cc cht chuyn ha l 419 90 ng/ml (khong 2 M), thi gian

bn thi ca RES v ton b cc cht chuyn ha ca n khong 9,2 h. Tuy

vy, ch mt lng nh RES khng b chuyn ha c tm thy trong huyt

tng. C ba con ng chuyn ha RES trong c th l: Lin hp sulfate,

lin hp vi acid glucuronic v hydro ha lin kt i (c th do h vi sinh vt

ng rut), trong yu t chnh hn ch sinh kh dng ca RES c th do

b chuyn ha theo con ng lin hp sulfate [38]. Ngoi ra, hp thu v

chuyn ha RES cn khc nhau gia cc c th, ph thuc vo chc nng gan

v hot ng ca h vi khun ng rut [30].

1.2.5. Cc bin php ci thin sinh kh dng ca resveratrol

Do kh nng ha tan km trong nc ca RES, nn trong nhiu th

nghim, RES c ha tan trong cc dung mi hu c khc nhau (DMSO,

acetone, ethanol) l khng thch hp vn chuyn thuc. Ngoi ra, RES c

thi gian bn thi ngn, km n nh. Do vy, vic n nh RES l cn thit

bo tn tc dng sinh hc v tc dng dc l ca n v ci thin SKD.

Pht trin cc h a thuc n v tr ch v bo v RES trong qu trnh vn

chuyn trong c th l rt quan trng v i hi nhiu k nng. Cng ngh

nano pht trin dn n s pht trin ca h vn chuyn nano, c kh nng

ko di thi gian bn thi, bo v dc cht khi qu trnh oxy ha, kim sot

gii phng v c th vn chuyn RES n m ch, c th k n nh: nh

tng nano, nano polymer, liposome, ht nano lipid rn, cyclodextrin

11

Nano polymer

Cc polymer c cu trc mt u thn nc v mt ui k nc c kh

nng ti thuc thn du bng cch t tp hp to thnh cu trc nano hnh cu

vi mt lp v thn nc v mt li k nc. S hp thu cc phn t polymer

theo c ch nhp bo, do , c th ci thin qu trnh xm nhp thuc vo t

bo v cho hiu qu iu tr cao hn dng t do [30].

Cyclodextrin

Cyclodextrin cha mt khoang k nc bn trong v mt b mt thn nc

bn ngoi, c th tng tc vi vi cc phn t khc nhau to thnh dng

phc hp khng cng ha tr. Cyclodextrin c th ci thin n nh v thi

gian bn thi ca RES bng vic bao RES trong khoang k nc [30].

Tiu phn nano lipid rn (SLN)

Tiu phn nano lipid rn c dng hnh cu, bn trong l li lipid, c th ha

tan cc dc cht thn du, b mt c bao quanh bi cc cht hot ng b

mt. SLN c th qua mng nhanh khng gy ra s thay i ng k v hnh

thi, hot ng trao i cht hay chu trnh t bo [30].

Liposome

Trong s cc h vn chuyn nano c ng dng lm cht mang RES,

liposome c ng dng rng ri vn chuyn thuc, da vo gi thuyt l

mng liposome ha vi mng t bo v sau gii phng thnh phn bn

trong vo t bo cht. Liposome l h vn chuyn ti u mang v a

thuc vo t bo v n kt hp vi RES trong mng kp. Liposome cng c

th gii phng RES t t, trnh qu ti cho mng t bo v gy c. T l v

n nh ha hc ca RES trong liposome c nh gi bng HPLC qua

60 ngy bo qun 4oC v khng thy s thay i, cho thy khng c qu

trnh isomer ha trans/cis [30].

1.2.6. Tng quan cc nghin cu bo ch liposome resveratrol

12

Liposome RES c th c bo ch bng nhiu phng php khc nhau.

Trong s , mt s nghin cu s dng phng php hydrat ha mng film

nh nghin cu ca Claudia Bonechi v s dng DPPC v chol vi t l mol

50 : 50 v 25 : 75, dung mi chloroform v ethanol, lm nh kch thc tiu

phn bng cch kt hp cc phng php ng chy, siu m v y qua

mng, cho hiu sut liposome ha thp, khong 50%, KTTP khong 250-

350nm [11]. Theo nghin cu ca Bojana D. Isailovic v cng s, t l

RES/SPC = 1/20 (w/w), PL : chol = 2,3 : 1 (w/w), dng siu m lm nh

KTTP [10]. Tuy nhin phng php ny cho hiu sut liposome ha khng

cao (44,22%), kch thc v phn b KTTP ln (KTTP t 120 n 290 nm,

PDI = 0,47).

Isailovic v cng s cn s dng mt phng php to liposome RES

khc l phng php proliposome, dng siu m lm nh kch thc tiu

phn [10]. Phng php ny cho hiu sut liposome ha khng cao (55,88%),

kch thc v phn b KTTP kh ln (250 nm, PDI 0,25) .

Mt trong nhng phng php cho hiu sut liposome ha cao c ng

dng bo ch liposome RES l phng php bc hi pha o, chng

hn, nghin cu ca Lu X. Y. v cng s (2012), bo ch liposome RES gn

hoc khng gn PEG - 2000, vi thnh phn to mng l phospholipid u

nnh, cho hiu sut liposome ha cao, khong 95 %, kch thc v phn b

KTTP nh (KTTP 100 - 120 nm, PDI khong 0,3) [26].

- Vic gn RES vo liposome em li mt s li ch:

Tng tan ca RES

Bo v RES khi tc ng ca tia UV. Theo nghin cu ca Maria

Coimbra v cng s, khi tip xc vi tia UV, 70 % trans - RES c gn

trong liposome vn cn n nh sau 16 pht, trong khi RES t do ch cn

10 % sau 16 pht tip xc vi tia UV [15].

13

Khi nghin cu trn t bo Hela v Hep G2 ca ngi, Lu X. Y. v cng

s cho thy kh nng c ch s pht trin khi u ca liposome RES

nng 10 80 g/ml mnh hn r rt so vi dng RES t do sau 24 -

48 h [26].

Th nghim trn chut, liposome gn RES gip tng nng RES trong

huyt tng v ci thin tc dng ca RES n h tim mch so vi RES t

do [29].

RES nng 10 M trong liposome lm tng r rt tc dng chng li

qu trnh oxy ha do tia UV-B so vi RES t do. Hn na, liposome RES

cn gip bo v t bo trnh khi cc tc dng c hi ca RES nng

cao [13].

Th nghim hot tnh chng oxy ha ca RES cho thy gn RES vo

liposome lm tng c ngha hot tnh chng oxy ha ca RES [25], [37].

LipoRES c th c gn cc yu t hng ch nh: gn DQA - PEG2000

- DSPE hng ch mc tiu l ti th ca t bo ung th [39].

Mt s ch phm trn th trng

Hnh 4: Mt s ch phm liposome RES trn th trng

14

CHNG 2. I TNG V PHNG PHP NGHIN CU

2.1. Nguyn vt liu, thit b

2.1.1. Nguyn vt liu

Bng 2. 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 phosphatidylcholin-SPC)

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

2.1.2. Thit b

- My ct quay Rovapor R- 210 (Buchi c).

- My siu m Labsonic M.

- My o quang ph Spectro UV-VIS Double, Labomed, Inc. M.

- H thng thit b phn tch kch thc Zetasizer ZS90 (Malvern, Anh).

- Knh hin vi in t truyn qua TEM JEOL 1010 (Nht Bn).

- My o pH Inolab (c).

- Mng thm tch kch thc l xp 12000 14000 Da (Sepetrum - M)

- Bn mng Silicagel GF254,cn phn tch, cn k thut, t sy, t lnh

15

2.2. Ni dung nghin cu

2.2.1. Kho st t l mol cc thnh phn trong cng thc bo ch liposome

RES

2.2.1.1 T l mol RES/SPC

2.2.1.2 T l mol chol/SPC

2.2.2. nh gi tiu phn liposome RES to thnh qua cc thng s

- Hiu sut liposome ha

- Kch thc v phn b kch thc tiu phn

- Hnh thi tiu phn.

2.3. Phng php thc nghim

2.3.1. Phng php ch to liposome RES

Ch to liposome RES theo phng php bc hi pha o [26]

Quy trnh:

Hnh 5. Quy trnh bo ch liposome resveratrol

Ha tan cc thnh phn trong

dung mi hu c (diethyl ether)

Siu m to nh tng N/D

Ct quay loi dung mi hu c

Siu m

Thm tch loi RES t do

Liposome RES

PL,chol,

RES

m PBS pH 7,4

Thi gian: 5 pht Nhit : ~10oC

Thi gian: 1,5h Nhit : 40oC Tc : 100v/p

Thi gian: 5 pht Nhit : ~10oC NPX: 0,6s

Thi gian: 18h Nhit : ~2-8oC

16

Cc bc c th nh sau:

- Bc 1: Ha tan cc thnh phn trong dung mi hu c

Cn cc thnh phn trong cng thc: SPC, chol, RES vo cc c m

Ha tan trong dung mi diethylether

- Bc 2: B sung pha nc, siu m to nh tng nc/du

Thm m PBS pH 7,4 (thnh phn: NaCl 137 mM, KCl 2,7 mM,

Na2HPO4.12H2O 10 mM, KH2PO4 1,8 mM )[42].

Siu m lin tc bng my siu m cm tay trong 5 pht, cng 100%

to nh tng mn nc/du.

- Bc 3: Ct quay loi dung mi hu c di p sut gim

Cho nh tng va to thnh vo bnh ct quay

Ct quay thu hi dung mi trong iu kin: nhit 40 oC, p sut - 0.08

mPa, tc quay 100 vng/ pht, trong thi gian 30 pht, thu c

liposome kch thc ln

Tip tc ct quay thm 1 h loi hon ton dung mi ether.

- Bc 4: Lm nh kch thc tiu phn bng siu m

Hn dch liposome to thnh c siu m gin on bng siu m cm

tay, cng 100%, nhp pht xung 0,6, trong iu kin nhit thp, thi

gian 5 pht.

- Bc 5: Thm tch loi RES t do

Hn dch liposome sau khi siu m c cho vo ti thm tch loi

COMW = 12000 14000 Da, kp cht 2 u, sau cho vo m PBS pH

7,4 (th tch m ln hn 100 ln th tch hn dch liposome em thm

tch), thm tch trong 18 h trong iu kin nhit thp (2 8 oC), thay

m sau 2 h, 4 h v tip tc thm tch qua m (lu bc giy nhm

trnh nh sng trong qu trnh thm tch).

17

Liposome sau khi thm tch c cho vo l thy tinh c np y, bo

qun nhit 2 8 oC.

Tin hnh kho st t l mol cc thnh phn trong cng thc

Khi kho st t l mol RES/SPC, gi nguyn t l mol chol/SPC, thay i t

l RES/SPC cc thng s khc thuc quy trnh bo ch khng thay i.

Tng t, khi kho st t l mol chol/SPC, gi nguyn t l mol RES/SPC,

thay i t l chol/SPC cc thng s khc khng thay i.

2.3.2. nh lng RES

nh lng RES bng phng php o quang ph UV.

- Qut ph hp thu ca RES trong mi trng m PBS v Triton tm ra

bc sng hp th cc i ca RES trong mi trng ny (max).

- Kho st mi tng quan gia nng RES v mt quang

Mi trng pha cc dung dch: cha m PBS pH 7,4 v Triton 1%

(t l 1/9) (Triton 9/1)

Ha tan 0,01 g RES trong Triton 9/1. Siu m trong 2 h RES tan hon

ton (nhit khong 30oC). nh mc 100 ml bng Triton 9/1 (thu

c dung dch RES gc, nng 100 g/ml). Pha mt dy gm 6 dung

dch nh lng: Ht chnh xc mt th tch dung dch RES gc nht

nh, nh mc va 25 ml bng Triton 9/1.

o hp th (A) ca dy dung dch RES va pha ti max va qut

c (mi nng o 3 ln, kt qu l gi tr trung bnh ca 3 ln o).

Lp ng chun mi tng quan gia hp th v nng RES.

- nh gi lp li ca phng php

Ph mng liposome ca mt mu liposome RES bng Triton X-100 1%.

Pha long bng Triton 9/1. o hp th quang ca dung dch ti cc thi

im 0h, 1h, 2h, 3h, 4h, 5h sau khi pha (mi thi im o 3 ln, ly gi tr

trung bnh), mu trng l Triton 9/1. Tnh lch chun tng i.

18

Phng php c nh gi l c lp li cao khi RSD < 2%

- nh gi nh hng ca t dc n php nh lng

Kho st hp th quang ca SPC v chol ti bc sng hp th cc i

ca RES trong mi trng m PBS pH 7,4 v Triton 1 %, nh gi nh

hng ca n n php nh lng.

Tin hnh: Bo ch liposome trng (khng cha hot cht) theo quy trnh

bo ch liposome RES, ph mng liposome trng bng Triton X-100 1 %, pha

long bng Triton 9/1, qut ph hp th ca dung dch thu c trong khong

bc sng 200 600 nm, mu trng l Triton 9/1.

2.3.3. nh gi tiu phn liposome resveratrol to thnh

Hnh thi hn dch liposome

Quan st hn dch liposome to thnh v ng nht, mu sc.

Kch thc v phn b kch thc tiu phn (PDI)

nh gi kch thc v phn b kch thc tiu phn bng thit b

Zetasizer ZS90, s dng phng php nhiu x nh sng ng.

Hnh thi tiu phn

S dng knh hin vi in t truyn qua (TEM) ti Vin v sinh dch t

trung ng bng k thut nhum soi m bn, thu c hnh nh b mt cc

tiu phn liposome.

nh gi hiu sut liposome ha

Nguyn tc: Ph v mng liposome bng cht hot ng b mt Triton X-

100, sau pha long v tin hnh o quang.

Hiu sut liposome ha (EE %) c tnh theo cng thc:

=

x 100%

Trong :

Lng RES c liposome ha l lng RES c trong liposome

sau khi thm tch loi RES t do.

19

Tng lng RES l lng RES c trong hn dch liposome trc

khi thm tch.

AS: hp th quang ca mu sau khi thm tch

AT: hp th quang ca mu trc khi thm tch.

Tin hnh:

- Ht chnh xc 1,00 ml liposome trc hoc sau khi thm tch vo bnh

nh mc 10 ml.

- Thm dd Triton 1 %, nh mc va 10 ml.

- 40 oC trong 2 h liposome b ph hon ton (ch bc giy nhm

trnh nh sng trong sut qu trnh ph mng liposome)

- Ht chnh xc 1,00 ml liposome sau khi ph, thm Triton 9/1 n va

25 ml, nh lng bng phng php o quang ph hp th nh sng

bc sng hp th cc i ca RES va qut c (max RES), mu trng l

Triton 9/1.

2.3.3. Phng php x l s liu

- Lp bng thng k s liu theo cc ch s nghin cu.

- S dng phn mm Microsoft Excell tnh gi tr trung bnh v

lch chun SD.

- So snh hai gi tr trung bnh vi mc ngha

| |

Trong :

: gi tr trung bnh ca i lng X.

: gi tr trung bnh ca i lng Y.

n: s ln thc hin ca X; m: s s ln thc hin ca Y.

: lch chun ca X; : lch chun ca Y.

Nu > x hai gi tr trung bnh khc bit c ngha thng k.

Nu < x hai gi tr trung bnh khc bit khng c ngha thng k.

x c tra trong bng phn phi chun sao cho F(x) = 1- /2.

20

CHNG 3. THC NGHIM, KT QU V BN LUN

3.1. KT QA

Kt qu qut ph hp th ca RES trong mi trng m PBS pH 7,4 v

Triton X-100 1 % cho thy bc sng hp th cc i ca RES max = 308,5

nm, do s o hp th ca RES ti 308,5 nm.

3.1.1. Thm nh phng php nh lng RES

3.1.1.1. Kho st lp li ca phng php

o hp th ca cng mt mu th ti cc thi im khc nhau, kt qu

c trnh by trong bng 3.

Bng 3: hp th ca dung dch RES trong mi trng m PBS pH

7,4 v Triton 1 % ti cc thi im sau khi pha

Thi im hp th

0h 0,328

1h 0,329

2h 0,324

3h 0,321

4h 0,326

5h 0,332

Trung bnh 0,327

SD 0,004

RSD % 1,20 %

Kt qu l gi tr trung bnh ca 3 ln o ti cng mt thi im.

T bng trn cho thy phng php c lp li cao vi gi tr RSD = 1,2 %

(< 2 %)

3.1.1.2. Kho st nh hng ca t dc

Qut ph hp th ca dung dch sau khi ph liposome trng v pha long

bng Triton 9/1 cho thy SPC v chol trong mi trng m PBS pH 7,4 v

21

Triton 1 % khng hp th quang ti bc sng 308,5 nm. Do , SPC v chol

khng nh hng n php nh lng RES s dng trong nghin cu ny.

3.1.1.3. Kho st mi tng quan gia hp th v nng resveratrol

Kt qu mi tng quan gia hp th (As) v nng RES (Cs) c

th hin trong bng 4.

Bng 4. S ph thuc ca hp th vo nng RES trong mi trng

m PBS pH 7,4 v Triton 1 %

STT 1 2 3 4 5 6

Th tch

dung dch gc(ml) 0,50 0,75 1,00 1,25 1,50 1,75

Nng (g/ml) 2 3 4 5 6 7

Hnh 6. th s ph thuc ca hp th vo nng RES trong

mi trng m PBS pH 7,4 v Triton 1%

Nhn xt: trong khong nng kho st (2 7 g/ml), hp th ca

dung dch RES ph thuc tuyn tnh vo nng , ng chun thu c c

y = 0.1117x + 0.0015

R = 0.9997

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

Nng (g/ml)

22

dng ng thng, phng trnh hi quy: y = 0,111x + 0,001, vi h s tng

quan R = 0.999 1.

Phng php nh lng RES bng o quang c lp li cao, c tnh

tuyn tnh (trong khong nng kho st: 2 7 g/ml) v khng b nh

hng bi SPC v chol, do vy c th s dng phng php o quang trong

mi trng m PBS pH 7,4 v Triton X-100 1 % ti bc sng 308,5 nm

trong khong nng trn nh lng RES trong mu liposome.

3.1.2. Kho st nh hng ca t l mol cc thnh phn trong cng thc

n KTTP, PDI v hiu sut liposome ha

3.1.2.1. T l mol RES/SPC

Tin hnh bo ch liposome RES theo quy trnh c nu bc 2.3.1,

trong c nh t l mol chol/SPC = 1/5, t l mol RES/SPC ln lt l 1/6;

1/9; 1/12; 1/15. Th tch diethyl ether ha tan cc thnh phn l 30 ml, th

tch m PBS pH 7,4 l 10 ml. Tin hnh nh gi KTTP, phn b KTTP v

hiu sut liposome ha ca cc mu. Kt qu c trnh by trong bng 6,

bng 7, hnh 7.

Bng 5. Thnh phn cng thc bo ch cc mu kho st t l mol

RES/SPC

T l RES/SPC 1/6 1/9 1/12 1/15

mRES (g) 0,01 0,01 0,01 0,01

mSPC (g) 0,2036 0,3054 0,4072 0,5090

mChol (g) 0,0203 0,0305 0,0406 0,0508

23

Bng 6. nh hng ca t l RES/SPC n kch thc, phn b

kch thc tiu phn liposome

RES/SPC PDI

Zaverage

ca h

(d.nm)

Peak

KTTP trung

bnh mi

peak theo

ng knh

(d.nm)

% theo

th tch

rng

peak(dnm)

1/6 0.371 233.2

1 199 16,8 93,48

2 3629 83,2 1446

3 0,000 0,0 0,000

1/9 0,457 256,7

1 155,9 18,4 106,4

2 3306 81,6 1485

3 0,000 0,0 0,000

1/12 0,405 237,9

1 77,4 17,4 31,23

2 348,8 9,9 142,6

3 4922 72,8 888,6

1/15 0,436 251,9

1 177,2 12,9 63,02

2 2602 87,1 889,5

3 0,000 0,0 0,000

Bng 7. nh hng ca t l RES/SPC n hiu sut liposome ha

T l mol

RES/SPC 1/6 1/9 1/12 1/15

EE% SD 81,15 1,29 88,11 0,98 95,66 1,07 94,81 0,91

24

Hnh 7. th biu din hiu sut liposome ha cc mu liposome kho

st t l RES/SPC

(Kt qu mi t l c tnh bng gi tr trung bnh ca 3 ln lm lp li SD)

Nhn xt:

Kt qu thu c t bng 6, 7 v hnh 7 cho thy khi thay i t l

RES/SPC v gi nguyn t l chol/SPC th:

- Cc mu thu c c kch thc tiu phn liposome kh ln, phn b

KTTP rng (PDI ~ 0,4), h km ng nht. C 4 t l u cho hiu sut

liposome ha trn 80 %, trong , 2 mu cho hiu sut trn 90 % (1/12 v

1/15).

- Khi gim t l RES/SPC, hiu sut ti thuc tng ln, t 81,15 % (1/6) n

88,11 % (1/9) v 95,66 % (1/12) (cao hn c ngha thng k so vi mu t l

1/9, p < 0,05), nhng khi gim xung cn 1/15 th hiu sut li gim nh cn

94,81 % (khng c ngha thng k, p> 0,05).

Do , chng ti la chn t l RES/SPC = 1/12 cho hiu sut ti thuc cao

nht, kch thc v PDI kh nh p dng cho cc nghin cu sau.

81.15

88.11

95.66 94.81

0

10

20

30

40

50

60

70

80

90

100

1:6 1:9 1:12 1:15

EE%

T l RES : PL

25

3.1.2.2. T l mol chol/SPC

Bo ch liposome RES theo quy trnh c nu bc 2.3.1, c nh t l

mol RES/SPC = 1/12, t l mol chol/SPC ln lt l 1/2;1/3;1/4;1/5. Th tch

diethyl ether ha tan cc thnh phn l 30 ml, th tch m PBS pH 7,4 l

10 ml. Kt qu c trnh by trong bng 9, 10, hnh 8.

Bng 8. Thnh phn CTBC cc mu kho st t l mol Chol/SPC

T l Chol/SPC 1/2 1/3 1/4 1/5

mRES (g) 0,01 0,01 0,01 0,01

mSPC (g) 0,4072 0,4072 0,4072 0,4072

mChol (g) 0,1016 0,0677 0,0508 0,0406

Bng 9.nh hng ca t l chol/SPC n KT v phn b KTTP liposome

Chol/SPC PDI

Zaverage

ca h

(d.nm)

Peak

KTTP TB

mi peak

theo ng

knh (d.nm)

% theo

th tch

rng

peak(dnm)

1/2 0,397 237,8

1 92,55 26,7 37,16

2 444,8 17,8 198,9

3 5302 55,5 705,1

1/3 0,383 235,6

1 62,77 32,9 24,02

2 417,0 19,1 204,5

3 5103 48,0 843,2

1/4 0,433 234,1

1 83,18 32,2 35,42

2 494 20,3 221,7

3 5269 47,4 718,3

1/5 0,405 237,9

1 77,4 17,4 31,23

2 348,8 9,9 142,6

3 4922 72,8 888,6

26

Bng 10. nh hng ca t l chol/SPC n hiu sut liposome ha

T l mol chol:SPC

1/2 1/2 1/4 1/5

T l mol RES/SPC : chol

tng ng 1/12/6 1/12/4 1/12/3 1/12/2,4

EE% SD 85,85 1,74 89,58 0,71 93,55 0,74 95,66 1,07

(Kt qu mi t l c tnh bng gi tr trung bnh ca 3 ln lm lp li SD)

Hnh 8. th biu din hiu sut liposome ha cc mu liposome kho

st t l chol/SPC

Nhn xt:

- T kt qu trong bng 9, 10 v hnh 8 ta thy, liposome thu c c kch

thc kh ln v khng c s khc bit nhiu gia cc mu, phn b KTTP

cha tht ng u, PDI cn kh ln (khong 0,4), cc mu u c hiu sut

liposome ha cao, t hn 85 n hn 95 %.

0

10

20

30

40

50

60

70

80

90

100

1:2 1:3 1:4 1:5

EE%

T l chol:PL

27

- Khi gim t l chol trong mu, hiu sut liposome ha tng kh nhiu. Mu

c t l chol cao nht cho hiu sut liposome ha thp nht (85,85 %). Mu c

t l chol thp nht (1/5) cho hiu sut liposome ha cao nht, cao hn c

ngha so vi mu c t l 1/4 (p < 0,05).

Do , t kt qu nghin cu trn, chng ti la chn cng

thc c t l mol chol/SPC = 1/5 cho hiu sut liposome ha cao nht dng

trong cc nghin cu sau ny.

3.1.3. nh gi liposome to thnh

Hn dch liposome ng nht, mu trng sa, hi nht, mt thi gian c

th b lng cc tiu phn, nhng phn tn ng nht tr li sau khi lc.

- Hnh thi liposome: quan st hnh nh chp TEM cho thy liposome c

hnh cu, phn b kch thc tiu phn khng u, liposome t b ph v, cc

tiu phn liposome khng b kt t (hnh 9) .

Hnh 9. Hnh nh chp TEM ca liposome sau khi siu m

28

3.2. NHN XT, BN LUN

3.2.1. V phng php bo ch

Trong rt nhiu phng php bo ch liposome, phng php bc hi pha

o l mt trong nhng phng php n gin v cho hiu sut cao [26]. V

hn ch ca phng php ny l c th tn d dung mi hu c trong ch

phm to thnh, chng ti nghin cu v tham kho nghin cu ca Ng

Th Bch Phng [4] quyt nh la chn thi gian ct quay l 1,5 h loi

ht dung mi. Chng ti th bo ch liposome RES bng phng php

hydrat ha mng film s dng dung mi ethanol. Tuy nhin, phng php ny

cho hiu sut liposome ha khng cao, ch khong 50 60 %, kt qu ny ph

hp vi mt s nghin cu bo ch liposome bng phng php hydrat ha

mng film [10],[11],[15].

Phng php bo ch s dng trong nghin cu ny cho hiu sut liposome

ha cao hn cc nghin cu khc. So vi phng php hydrat ha, mng film

c s dng trong nghin cu ca Claudia Bonechi v cng s cho hiu sut

liposome ha khong 50 % [11], hay nghin cu ca Bojana D. Isailovic v

cng s ch cho hiu sut ti thuc di 50 % (44,2 %) [10]. Phng php s

dng trong nghin cu ny cho hiu sut cao hn hn, t 80 n 90%, mu

cao nht cho hiu sut 95.66% (bng 7), ngoi ra, c KTTP v PDI ca

liposome to ra trong nghin cu cng thp hn ng k so vi nghin cu

ca Claudia Bonechi v cng s (250 nm so vi 350 nm)[11].

Nu so snh vi nghin cu ca Xin Y. L. v cng s [26], s dng cng

phng php bo ch cng nh phng php lm nh KTTP, th nghin cu

ny cho hiu sut cao hn, tuy nhin, KTTP v PDI l ln hn (hiu sut

95,66% so vi 95%) (KTTP khong 250 nm v PDI khong 0.4 so vi 100

nm, PDI < 0,2). C th do khi tin hnh chng ti ci tin quy trnh, khng

ct quay 20 pht nh trong nghin cu ca Xin Y. L. v cng s [26], m

29

chng ti tin hnh ct quay thm 1 h sau khi ether bay hi ht, v theo

Cortesi R. ether tn d trong ch phm c th gy r r dc cht, ngoi ra

chng ti cn tin hnh lm lnh mu bng nc khi siu m trnh hin

tng qu nhit gy oxy ha RES [16]

V phng php lm nh KTTP, chng ti s dng phng php siu m

trong nghin cu, bi phng php ny n gin, thi gian tin hnh nhanh

v khng cn thit b qu phc tp. Tuy nhin, liposome thu c c kch

thc tiu phn kh ln, phn b KTTP cha ng nht (bng 6, 7), cng nh

nghin cu ch mi tin hnh siu m trong thi gian 5 pht m cha kho st

c thi gian siu m khc nhau. Do vy, cn c thm cc nghin cu tip

theo kho st nh hng ca thi gian siu m n KTTP, PDI v hiu sut

liposome ha cng nh so snh phng php siu m vi cc phng php

lm nh kch thc tiu phn khc nh phng php y qua mng.

3.2.2. V phng php nh lng RES

Hin nay trn th gii cng nh trong nc p dng rng ri cc phng

php nh lng RES hin i, cho chnh xc cao nh: HPLC, LC-MS [2],

[5], [12]. Phng php nh lng bng o hp th quang kh c in v t

c p dng. Tuy nhin, u im ca phng php ny l n gin, cho kt

qu nhanh Theo Camont L. v cng s, nh lng RES bng phng php

o quang cho kt qu khc bit khng ng k so vi dng HPLC [14]. Do

vy, chng ti la chn phng php o quang nh lng RES.

nh lng RES c ti trong liposome, chng ti tin hnh ph mng

bng Triton X-100 1 %, sau pha long o quang. V ph hp th ca

RES trong cc mi trng khc nhau l khc nhau, chng ti tin hnh qut

li ph hp th ca RES trong mi trng Triton 1 % v m PBS pH 7,4,

cho thy max ca RES trong mi trng ny l 308,5 nm (trong nc l 304

nm) [41].

30

3.2.3. nh hng ca cc yu t cng thc v quy trnh bo ch ti

tnh cht h liposome to thnh

3.2.3.1. nh hng ca cng thc bo ch

PL l thnh phn c bn cu to liposome, chol c thm vo ng vai tr

n nh mng. Cc nghin cu trc y s dng cc phng php bo ch

khc nhau vi t l RES/SPC/chol khc nhau. Thng thng, s dng t l

khi lng RES/SPC = 1/20 (tng ng t l mol l 1/6) [10], [12], [13] . T l

khi lng chol/SPC khc nhau ty theo nghin cu. Trong nghin cu ca

Caddeo v cng s (2008) t l chol/SPC = 1/5 tng ng t l mol l 1/2 [13].

Trong nghin cu ca Claudia Bonechi v cng s (2012), bo ch liposome

RES vi t l mol chol/SPC = 1/1 v 1/2 [11]. Nm 2013, Bojana D. v cng

s kho st t l khi lng chol/SPC = 1/2,3 tng ng t l mol l 1/1 [10].

Cc nghin cu trn, s dng ch yu phng php hydrat ha mng film

[10], [11]. Tuy nhin cc nghin cu ny cho hiu sut liposome ha thp.

Nghin cu ca Lu X. Y. v cng s, kho st t l khi lng RES/SPC ln

lt t 1/20; 1/30; 1/40; 1/50 (tng ng t l mol l 1/6; 1/9; 1/12; 1/15). T

l khi lng chol/SPC ln lt l 1/5; 1/7,5; 1/10 (tng ng t l mol t

1/2,5;1/4; 1/5) bng phng php bc hi pha o cho hiu sut liposome ha

cao (95 %), ng thi quy trnh bo ch n gin [26]. Do vy, nghin cu

ny, chng ti tin hnh kho st t l mol RES/SPC t 1/6; 1/9; 1/12; 1/15, t

l chol/SPC l 1/2; 1/2; 1/4 v 1/5 bng phng php bc hi pha o nhm

a ra t l c hiu sut liposome ha ti u ph hp vi phng php nghin

cu.

Khi gim t l RES/SPC, hiu sut liposome ha tng ln, c th gii thch

do RES nm trong mng PL kp, nu t l RES cao vt qu kh nng ti

thuc ca liposome th RES khng c bao ht dn n d tha v tn ti

di dng ha tan hoc kt ta trong mi trng nc v s b loi i khi

31

thm tch, do t l thuc c ti vo liposome so vi tng lng RES s

dng s thp. Khi gim t l RES so vi SPC th lng RES c SPC bao

gi cng s tng n khi ton b RES c bao trong liposome th hiu sut

ny s khng tng na, vic tng SPC khng gip lm tng hiu sut

liposome ha, hn na vic tng SPC trong khi khng gip tng hiu sut

liposome ha cng gy tn km ha cht khi sn xut quy m ln.

Khi gim t l chol/SPC, hiu sut liposome ha tng ng k. Cng thc

c t l chol cao (chol/SPC = 1/2) cho hiu sut liposome ha thp nht.

Nguyn nhn l do c chol v RES cng nm trong mng SPC, m SPC ch

c mt gii hn nht nh, khi t l chol cao, n s cnh tranh v tr trong

mng vi RES v y RES ra khi mng dn n gim hiu sut liposome

ha. Khi t l chol gim, khng gian trong mng c bo v bi SPC tng

ln lm cho lng RES c vo bn trong mng liposome tng, do tng

hiu sut liposome ha. Tuy nhin, chol c vai tr n nh mng, lm tng

cng ca mng, tng sc chu ng ca liposome di p lc thm thu v

bo v liposome khi tng tc vi protein trong dch sinh hc, khi t l chol

qu thp c th gy tch pha v lm chol phn b khng ng u trong

mng. Do vy chng ti ch dng nghin cu t l chol/SPC = 1/5.

3.2.3.2. nh hng ca cc yu t thuc quy trnh bo ch

Trong quy trnh bo ch, giai on to nh tng nc/du rt quan trng.

Khi thm pha nc vo pha dung mi hu c, do khng trn ln vi nc,

nn pha dung mi hu c tn ti tch ring vi pha nc, do cn s dng

nng lng siu m ln to nh tng n nh. Mt s nghin cu trc

dng siu m b to nh tng N/D, nh nghin cu ca Lu X. Y. (siu

m b trong vng 30 pht) [26], chng ti cng th bin php dng b siu

m. Tuy nhin, nng lng khng ln nn khng to thnh nh tng. Do

vy, chng ti dng siu m cm tay v siu m lin tc to c nh

32

tng ng nht. Qu trnh siu m c kt hp lm lnh trnh hin tng

qu nhit c th gy oxy ha SPC v RES.

Khi thay i t l RES/SPC cng nh t l chol/SPC th KTTP v phn b

KTTP thay i khng theo quy lut, c th gii thch do nng lng siu m

phn tn khng ng u gia cc v tr: Cc tiu phn nm cng gn que siu

m th chu tc ng lc cng ln dn n b lm nh mc ln hn

nhng tiu phn nm xa hn, qu trnh siu m chu nh hng ca nhiu yu

t, nh: Qu trnh lc khi siu m, th tch dch khi siu m do , kch

thc tiu phn thu c kh ln, phn b kch thc tiu phn rng v

khng c s khc bit nhiu gia cc mu. Do , rt kh so snh cc mu

vi nhau thng qua KTTP v PDI.

Nhit khi siu m nn c kim sot di nhit chuyn pha ca

lipid, v khi nhit cao hn Tc ca SPC, SPC tn ti dng v nh hnh,

lin kt lng lo. Do , di nng lng siu m cao c th y RES ra khi

mng SPC kp. Ngoi ra, c SPC v RES u d b oxy ha, c bit nhit

cao m qu trnh siu m d gy qu nhit cc b, nn cn lm lnh trong

khi siu m cng nh ch siu m ngt qung.

Trong nghin cu ca Bojana D. I. v cng s s dng li tm loi

RES t do [10]. Phng php ny tuy n gin, d thc hin v c th tnh

hiu sut liposome ha m khng cn ph mng liposome, nhng c th cho

sai s do khi li tm, c th c mt lng RES khng tan kt ta xung y

ng li tm, nn khng loi ht RES khng c liposome ha dn n lm

tng hiu sut so vi hiu sut thc. Nghin cu ca chng ti la chn

phng php thm tch loi RES t do. Phng php ny c p dng

trong nhiu nghin cu, theo c th ha tan v loi b RES t do [12].

33

Ether l dung mi d bay hi, do vy ch cn ct trong khong 30 pht l

ether bay hi ht. Tuy nhin, cn ct thm khong 1 h loi hon ton

dung mi, trnh tn d dung mi gy c v gim n nh ca ch phm.

RES rt d b ging ha bi nh sng, do ton b qu trnh cn trnh nh

sng.

34

KT LUN, KIN NGH

KT LUN

Sau mt thi gian thc hin nghin cu ti b mn Ha sinh v b mn

Bo ch trng i hc Dc H Ni, chng ti thu c cc kt qu

sau:

- to c liposome RES t phospholipid u nnh bng phng php

bc hi pha o c kch thc khong 200 nm, hnh cu, khng b kt t, t b

ph v.

- nh gi nh hng ca t l cc thnh phn trong cng thc n hiu

sut liposome ha RES.

Kt qu cho thy KTTP thu c khong 200 nm, PDI khong 0.4. T l

mol RES/SPC: Chol ph hp nht l 1/12/2,4.

KIN NGH

Do iu kin thi gian cng nh iu kin thc nghim cn hn ch nn

chng ti ch dng li vic chn ra cng thc bo ch liposome RES bng

phng php bc hi pha o. Trong thi gian ti, chng ti ngh tip tc

nghin cu cc vn sau:

- Tip tc kho st nh hng ca cc yu t thuc quy trnh bo ch nh

nhit v thi gian ct quay, thi gian siu m n cc thng s: Hiu

sut liposome ha, KTTP v phn b KTTP.

- Nghin cu so snh liposome to thnh theo phng php siu m vi cc

phng php lm nh KTTP khc.

TI LIU THAM KHO

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PH LC

th phn b kch thc tiu phn

Mu t l RES/SPC = 1/6

Mu t l RES/SPC = 1/9

Mu t l RES/SPC = 1/12

Mu t l RES/SPC = 1/15

Mu t l chol/SPC = 1/2

Mu t l chol/SPC = 1/ 3

Mu t l chol/SPC = 1/4

Mu t l chol/SPC = 1/5