Nghiên cứu phương pháp đẩy qua màng trong làm nhỏ và đồng nhất hóa liposom reveratrol

BỘ Y TẾ
TRƯỜNG ĐẠI HỌC DƯỢC HÀ NỘI


HOÀNG THỊ MINH

NGHIÊN CỨU PHƯƠNG PHÁP
ĐẨY QUA MÀNG TRONG LÀM NHỎ
VÀ ĐỒNG NHẤT HÓA LIPOSOM
RESVERATROL

KHÓA LUẬN TỐT NGHIỆP DƯỢC SĨ

HÀ NỘI  2014

BỘ Y TẾ
TRƯỜNG ĐẠI HỌC DƯỢC HÀ NỘI

HOÀNG THỊ MINH

NGHIÊN CỨU PHƯƠNG PHÁP
ĐẨY QUA MÀNG TRONG LÀM NHỎ
VÀ ĐỒNG NHẤT HÓA LIPOSOM
RESVERATROL

KHÓA LUẬN TỐT NGHIỆP DƯỢC SĨ

Người hướng dẫn:
TS. Nguyn Th Lp
Nơi thực hiện:
1. B 

2. B 

HÀ NỘI  2014
LI C
L c ti:
T.S. Nguyn Th Lp.
i thng d c hin
 
 k thu
 u kin thun l 
i li c
k thu   i gian thc
hin.
Cui li c
     t nghip c

 13/05/2014

 Minh.


MỤC LỤC
DANH MU, CH VIT TT
DANH MNG BIU
DANH M  TH
T V 1
NG QUAN 3
1.1. Tng quan v liposom 3
m 3
1.1.2. i 3

1.1.3.  phn liposom 5
 to liposom 6
ng nhc liposom 7
 7
 9
1.2. Tng quan v trans - Resveratrol 12
c 12
 12
1.2.3. ng hc 13
1.3. Tng quan v liposom RSV 14
U 17
t liu 17
2.2. Nu 18
u 18
 liposom RSV 18
2.3.1.1. T 19
 ng nh 19
2.3.1.3. Tinh ch liposom RSV 20
t s a liposom RSV 23

KTTP liposom 23
ng RSV 23
u su 24
C NGHIM, KT QUN 26
3.1. Kt qu 26
3.1.1. Thng RSV 26
 28
3.1.3. Khng cu kia
liposom 32
nh c ti

 35
n 36
KT LUN NGH 40
U THAM KHO
Ph lc


DANH MỤC CÁC KÝ HIỆU, CHỮ VIẾT TẮT
Chol
Cholesterol

n Vit Nam
DPPC
Dipalmitoyl phosphatidylcholin
DPPG
Dipalmitoyl phosphatidylglycerol
DSC
t vi sai (Differential scanning calorimetry)
DSPC
Disteraroyl phosphatidylcholin
KTTP
c ti
LUV
Large unilamellar vesicle (liposom to mt lp)
MLV
Multilamellar vesicle (liposom nhiu lp)
N/D
c/ du
PDI
Ch s index)

PEG
Poly(ethylenglycol)
PL
Phospholipid
RES
Reticuloendothelial system (h i n
RSV
Resveratrol
SPC

SUV
Small unilamellar vesicle (liposom nh mt lp)
Tc
Nhit chuyn pha gel - tinh th lng
TEM
   n t truyn qua (Transmission electron
microscope)
TKHH
Tinh khic



DANH MỤC CÁC BẢNG BIỂU
STT
ng
Trang
1
Bng 1.1. i liposom theo s lc
4
2

Bt liu
17
3
Bu ch to liposom RSV
19
4
Bng 3.1.    hp th quang c  u dung dch
RSV chun  
26
5
Bng 3.2. Kt qu  lp li cng
bng k thu UV  VIS
28
6
Bng 3.3.  KTTP liposom c sau

29
7
B u sut liposom
a liposom 
31
8
Bng 3.5. ng ca u kin c,
 KTTP
33
9
Bng 3.6.  ng ca u kin   i hiu sut
liposom 
34
10

Bng 3.7 KTTP, hiu sut liposom 
ca liposom   KTTP b    y qua

35

DANH MỤC CÁC HÌNH VẼ VÀ ĐỒ THỊ
STT

Trang
1
liposom
3
2
1.2 liposom c
o
7
3
1.3 gim KTTP liposom by

10
4

20
5
 n c
liposom RSV
22
6
 th biu din m hp th
 

1%  
27
7
 th bi theo s l
400 nm
30
8
 th bi l
80 nm
31
9
 th a h y

32
10
nh chp TEM ca mu liposom  
thc b
36
1

ĐẶT VẤN ĐỀ
y  c vt,
c bic phc
p lu t r  (Polygonum cuspidatum), mt loi thc
vc c truyT nh
 t nhi  u v resveratrol,      nghim
resveratrol      ng  i cho sc khe  ng
o v tim mch [5]. M
i thin sc kha bnh m i [36]
    li         n nay

resveratrol vn ch i dng thc phm chng
uc t vic s dng resveratrol dng t do gp nhiu hn ch do
ng c [40], [41]. 
 do sau khi hp thu b chuyu  gan
 dng ung rt thp [42] khc phc
m ca dng t do, hin nay nhiu h mang thuc mu,
 ng dng cho  liposom [33]. Liposom
 bo v resveratrol khng bt li cng, 
      n ci thin sinh kh dng ng ung ca
resveratrol. Nhi      c s d   
liposom resveratrol, n dch liposom tc
ln vi kho c rng. Vy vi ng nht
t bu liposom resveratrol.
 n ng dng liposom t mang v nh va
 dng ng ung  c hi  
2

“Nghiên cứu phương pháp đẩy qua màng trong làm nhỏ và đồng nhất
hóa liposom resveratrol” nhm m
- u ng d  ng nht
c liposom resveratrol.

3

Chương I. TỔNG QUAN
1.1. Tổng quan về liposom
1.1.1. Khái niệm
Liposom   c bit c       
(nanocapsules), bao gm mt v ng ra
m mt hay nhiu lng trc bao bc  gia ho

i t 
nanomet [1].

Hình 1.1. Cấu trúc liposom
1.1.2. Phân loại
t nhi i liposom.
D lc liposom chia liposom
liposom mt lp, liposom nhiu lliposom nhi],
[27].

Dược chất thân
nước
Dược chất thân
lipid
Lớp
Phospholipid kép
Cảm biến
Lớp bảo vệ
4

Bi liposom theo s lc
Loi liposom
u

(nm)
S lp
PL
Liposom
mt lp
Liposom mt lp loi nh

SUV
20 - 100
1
Liposom mt lp loi ln
LUV
200  1000
1
Liposom mt lp khng l
GUV
>1000
1
Liposom
nhiu lp
Liposom nhiu lp loi nh
OLV
100 - 1000
Khong
5
Liposom nhiu lp loi ln
MLV
>500
5  20
Liposom nhi
MVL
>1000

D ng dng ca liposom [2], [4], [11], [16], [35]
chia liposom 
(1) Liposom quy ước (conventional liposomes - CL): thng ch to t t
t hp vi cholesterol, c

bin v hoc b mt.
Khi bi i b mt ho  n v liposom    i
liposom m
(2) Liposom nhạy cảm nhiệt độ (thermal  c
 t t chuyn pha gel  tinh th lng (Tc) cao
  mc chc gi
khi liposom  nhi > Tc [7].
(3) Liposom nhạy cảm pH (pH  sensitive liposomes): cu to t 
phn nhy cm vi pH th      
5

phosphatidylethanolamine (PE) [12ng ca pH acid  n
hoc khc chc gi
(4) Liposom cation u ch t phosphol     vn
chuyc  liu cao [17], [34].
(5) Liposom tuần hoàn dài hay liposom ẩn (long  circulating liposomes hoc
Stealth liposomes): liposom c bao b mt b c ph
bin nh cng kn ch hing
 bt gi c ch cu c thi
gian tu ng thuc t [17], [20].
(6) Liposom vận chuyển chủ động mt   - c
nhn din bng
s dng hi (ch y p IgG), folat,
tranferin. Loi liposom c ng d vn chuyn ch ng thuc ti
 [2].
 i liposom .
1.1.3. Thành phần liposom
Liposom hi   c ch to t     n 
.
Phospholipid  n gc t  cholin), PG

(phosphatidylglycerol) hoc tng hp: DSPC (disteraroyl
phosphatidylcholin), DPPC (dipalmitoyl phosphatidylcholin) [16]. Mt
trong nhng ng ln ti s  n
nh ca liposom  chuyn pha Tc ca phospholipid.  i nhit
 chuyn pha phospholipid tn ti  tr p xt
t, khi nhi pid chuyn sang tr lng
m c  c cho
6

qun. Tc c -  thu 
n cha chu [34].
a l c
   lng trng gi
t PL gi cho cn v[16]
pid ng b 
obulin
liposom nh.
  phi h
vi ma ch mt liposom n PEG (liposom tun
, trasferin, folat (liposom vn chuyn ch ng)
hoc ch - tocopherol
1.1.4. Các phương pháp chế tạo liposom
     to liposom, d     
 liposom i l (i) c
c: t hi
mng lipid, (ii) c 
ether, b(iii) ci b cht din hot [4],
[11], [16].
o liposom c t
trung gian tn tan trong du


hoc isopropyl ether hoc hn h
dung dc rng b t
c tm mt lp bao quanh mt
c. B t t gi chuy
o sang trm ti hn c
7

tr    b    o b      
phospholipid s p xliposom.
p vi nhiu loi phospholipid, to ra liposom 
th c lp  c  nh, l
c cht b 
ng bi nhi ng t bn (:  ADN
ho i nhim v [4].

1.2 liposom o
1.1.5. Các phương pháp đồng nhất hóa kích thước liposom
 liposom hin nay c
liposom  c tiy
ving nhc tiu bt buc.
 thu s dng, cc
 - chy [11].  thuc s dng rt
hi [19].
1.1.5.1. Phương pháp siêu âm
t trong nh 
  thu hoc b .
8

 Siêu âm đầu dò     t ngp trc ti  n dch

liposomn dch liposom t l
t cc b y cnh m 
i (titan) gi y nhim bn ch ph
c trong 1 gi kho de - ].
 Siêu âm bể: mng trong dng c 
b c ki trong k thu 
  u liposom   c bo v trong l tii
].
  c ca h liposom 
 thu nhiu yu t   lipid, th ch
, mng, th
[11]. u ca Yamaguchi ng s hn dch liposom ch to
t l - alpha - dilauroyl phosphatidylcholin  c bng
   n s  - 
a h gi tn s th
hiu qu c c dng t tn s
i gian ngn vi thit b ng
ln ti thit b ng nh [44].
Giuseppe Maulucci ng s ng ca thi gian
         c liposom   t
dimyristoylphosphatidylcholine (u liposom 
 c bi tn s 20 kHz, ng
150 W, nhs ng tht qu
cho thc ca h giu thi gian
ng              
c t 
9

c t do. Th
c liposom    p vi m  ng dng ca liposom [24].

Lapinski ng s   liposom t hn hp 1,2 - dioleoylsn
- glycero - 3 -  - oleoyl - 2 - [12 - [(7 - nitro - 2 -
1,3 - benzoxadiazol - 4 - yl)amino]dodecanoyl] - sn - glycero - 3 -
phosphocholine (18:1 - 12:0 NBD -        u
liposom c x  t qu cho th
a mu liposom i m
 3nm), y ng tc ca
liposom kh[19].
Hn ch cng l
  p kim loi t           
   ng t  bn c c ch c liposom
liposom t  i r 
MLV tn tng thi v  ng nht v 
 c gi [4], [11], [19].
1.1.5.2. Phương pháp đẩy qua màng
ng lu trong ch to liposom bi Sozka
 cng s t  cho ti nay vt trong nh
ch LUV hiu qu [28]c
vi nhiu lon hp lipid; d thc hinmt lipid,
xut hin dng lysophosphatidylcholin trong mu, 
mu, gi   ; t c liposom  c mong
mung nh lp li v  c cao gia
 thc hip [6], [28], [19], [31], [38].
n dch liposom c l lc
 c liposom c mong mun. Khi b 
10

l    liposom       lc s d   
 c. Liposom  c l   nh l lc, mt s s bin
d  lc , mt s ng ca lt b p v 

o li liposom p vi c nh [28].

1.3 gim KTTP liposom b
liposom c s x 
 y
c thc hing cha n thuc cu
c cht t do ch c lo khi to liposom t [28].
u ng ca mt s yu t ,
   c (lo   c l  c)  n ca
liposom.
ng s [30]  liposom t 
chol t l mol 55:45, gic tic
c l lc to ra ch t  y
 nhi tht chuyn pha Tc ca DSPC) k c
t cao 800 lb/in
2
(1 lb/in
2

      thuc nhi  na. Khi trong
 n liposom      nhi  < Tc t   t chm
11

(0,06 ml/  nhi  p 200 ln tc 
 40 t qu y 
nhi th t chm ho do
 tr nhng [10y
nhi c thc hin   chuyn pha ca lipid. S 
mt ct nh t  nhi n ch
t  nhi i kh 

nht c nhi  nht c nhi > Tc
ca chol [28].
Hn dch liposom n ethanol t 10 - 20% (th th 
 s d Ethanol tu ki
l lc nh m lc c liposom t
 ng nh lc.
t trong s  dng trong
sn xuc ph c loi b khi sn phm cui nh lc gel,
thc lc tip tuyn [28].
c tiliposom gi
b c ti thuc
a lipid tliposom [14].
 v m
hc [28].   ly ng quynh ti
 c liposomy 
l c liposom y qua
 lc lc liposom c
nh  ly  lc nh 
c liposom ng l lc
[25] lc 100 nm
12

 c liposom gim qua mi l y   ng 10 l  c
liposom ng nht [28].
1.2. Tổng quan về trans - Resveratrol
1.2.1. Công thức hóa học
Trans - t [5].

: C
14

H
12
O
3
.
Tr: 228,25.
pKa
1
= 9.07 (phenol); pKa
2
= 10.06 (phenol); pKa
3
= 11.56 (phenol).


,5  Trihydroxystilbene [13].
1.2.2. Đặc tính lý hóa
- Bng.
-  tan: RSV tan trong d 50 mg200 mM)
70 mM) , r
3 mg/c) [5].
- t ci hp th ti 305,3 nm [29].
- Trans - t nhy cm vng s 80 
90% trans - RSV trong dung dng cis khi tii
t gi [41] Waterhouse 
r i 90,6% trans -  n sang dng cis khi chiu trans -
RSV tinh khi [40].
13

1.2.3. Dược động học

Theo cng s khong 70% tng liu trans - RSV c hp thu
sau khi ung, vi lich RSV c chuyn sang dp
n phm chuyng
ung gm c dp glucuronat. Ch mng rt
nh  chuy (< 5 ng/ml  
ng uy mp thu t
sinh kh dng ung ca RSV rt thp do mng ln RSV nhanh
 chuyc thi tr ch yu  thn, 
i tr ng RSV thi tr qua
thu u 98%i tr qua
thn khong 53  85% ng 3,3  35% [42].
1.2.4. Tác dụng dược lý
Mu v c thc hi
ng v  nghi
i. T kt qu u thy r t s ng sinh
ho v h tim mch [23]], chng tiu
ng [26], [37] c cng
chc ch 
lipoprotein t trng thp - LDL     c ch enzym
cyclooxygenase  2 v m       ng
in vivo c kh c
ch quinu hin ct ch
n  a t i stress
ng c
 i stress ca t u ng h
14

lum trans - i thin sc kh
b ngi [36].
1.3. Tổng quan về liposom RSV


 

 
 
 
 [33]



,  [33].

Cyclodextrin [21].  
               
[39].  - lactide
- co - glycoli , 
  in vitro [32]. 
liposom   cho RSV

 
    [15]          -
Differential scanning calorimet   liposom    
liposom 
 liposom R
15

liposom 


              Theo

Coimbra M. liposom %
t 
10% trans -  [9].
 
Liposom  
 
RSV,  .


liposom 
4  liposom 

 [29].
[43]  liposom R
chol : RSV 1 b
h liposom 
Liposom 
 liposom %. Tm
liposom RSV RSV trong
           



16

               
liposom RSV 
Julijana Kristl  liposom 
mol), dmol), mol) 


    liposom     liposom   
> 70%
   in vivo     liposom  
liposom 
Liposom 
 (100  

 [18].
liposom RSV 
.

17

CHƯƠNG II: ĐỐI TƯỢNG VÀ PHƯƠNG PHÁP NGHIÊN CỨU
2.1. Đối tượng nghiên cứu, nguyên vật liệu và phương tiện nghiên cứu
 u: Liposom Resveratrol.
 t liu
Bt liu
STT
u
Ngun gc
n
1
Trans - Resveratrol
DSM Nutritional
Products Ltd - Thy

NSX
2
Cholesterol

Merch - c
NSX
3

(Soy phosphatidylcholine)
Avanti polar Lipids -
M
NSX
4
Diethyl ether
Trung Quc
TKHH
5
Dinatri hydrophosphat
Trung Quc
TKHH
6
Kali dihydrophosphat
Trung Quc
TKHH
7
Natri clorid
Trung Quc
TKHH
8
Kali clorid
Trung Quc
TKHH
9
c ct hai ln

Vit Nam

10
Triton X  100
MP Biomedicals 
M
NSX

 u
- H thng ct quay Rovapor R - 210 (Buchi  u 29/32 GG 17,
 ml.
- H thng thit b    c Zetasizer nano ZS90 (Malvern 
Anh).
- Thit b liposome extruder cm tay (M).