高压乳匀法制备中药固体脂质纳米粒
作者：厉英超 1；董蕾 2；贾皑 1；苌新明 1；薛挥 1
(西
1
安交通大学医学院 第一附属医院消化内科，陕西 西安
710061；2 第二附属医院消化内科，陕西 西安 710004)
摘要：目的
采用高压乳匀法将中药有效成分包载于固体脂质纳米粒
(SLN)，并研究制备的纳米粒的主要性质。方法
选择水飞蓟宾(SIL)和
汉防己甲素(TET)为模型药物，采用高压乳匀法将其分别包载于 SLN。在电
镜下观察其形态，以粒度分析仪和 Zeta 电位分析仪测定其粒径和 Zeta 电
位，用葡聚糖凝胶柱层析法和 HPLC 测定其包封率和载药量，还观察了 SLN
的稳定性。结果
高压乳匀法制备的 SIL-SLN 呈球状，形态规则，平均
粒径为(157±8) nm，Zeta 电位为(-35.36±2.68)mV ，包封率为 95.64%，
载药量为 4.63%；TET-SLN 呈片状存在，不规则，粒径较小，平均粒径为
(47±3) nm，Zeta 电位为(-32.99±2.54)mV，包封率为 97.82%，载药量为
4.76%。SIL-SLN 和 TET-SLN 有较高稳定性。结论
高压乳匀法适于制备
包载中药的 SLN。
关键词：水飞蓟宾；汉防己甲素；固体脂质纳米粒；高压乳匀法；中药现
代化
中图分类号：R944
文献标识码：A
文章编号：
1673-4254(2006)05-0541-04
PreParation of solid lipid nanoParticles loaded with traditional
Chinese medicine by high-pressure homogenization
LI Ying-chao1；DONG Lei2；JIA Ai1；CHANG Xin-ming1；XUE Hui1
1
DePartment of Gastroenterology,First Affiliated Hospital,Medical
College of Xi’an Jiaotong University,Xi’an
710061,China;2DePartment of Gastroenterology,Second Affiliated
Hospital,Medical College of Xi’an Jiaotong University,Xi’an
710004,China
Abstract:Objective
To investigate the preParation of solid lipid
nanoParticles (SLN) loaded with traditional Chinese medicines by
high-pressure homogenization,and study the physicochemical
characteristics of the Particles produced by this
method.Methods
The model traditional Chinese
medicines,silibinin (SIL) and tetrandrine (TET), were incorporated
into SLN seParately by high-pressure homogenization. Transmission
electron microscope was employed to study the shape of the Particles.
Particle characterization system and zeta potential analyzer were
used to study the diameter and zeta potential of SLN in the suspension.
The entrapment efficiency and drug loading were determined with the
sephadex gel chromatography and high-performance liquid
chromatography. The stability of SLN was also studied.
Results
The SIL-SLNs prePared by high-pressure homogenization
were spherical and regular. The mean diameter and zeta potential of
SIL-SLN in distilled water were 157±8 nm and -35.36±2.68 mV,
respectively. The entrapment efficiency was 95.64%, and the drug
loading was 4.63%. The TET-SLN was platelet-shaped, irregular and
smaller. The mean diameter and zeta potential of TET-SLN were 47±3
nm and -32.99±2.54 mV, respectively, with drug loading of 4.76%,
and up to 97.82% of TET was incorporated. SIL-SLN and TET-SLN had
good stability. Conclusion High-pressure homogenization is feasible
for preParing SLN loaded with traditional Chinese medicines.
Key words: silibinin; tetrandrine; solid lipid nanoParticles;
high-pressure homogeni zation; traditional Chinese medicines
Supported by Science and Technology Project of Xi’an City (GG04133).
LI Ying-chao (1974-), PhD, attending physician, specialized in
pharmaceutical research of liver fibrosis, Tel: 029-85324001,
E-mail: [email protected]
Corresponding author: DONG Lei, medical professor, Tel:
029-87276936- 29368, E-mail: [email protected]
Solid lipid nanoParticles (SLNs) are Particles made from
solid lipids with a mean diameter of approximately 50 to 1000 nm to
serve as an alternative colloidal carrier system for controlled drug
delivery[1]. ComPared with other Particulate carriers SLN has
several advantages for drug delivery such as its good
biocomPatibility[2], biodegradability[3], high bioavailability[4],
and effects targeting the liver and spleen. In recent years, markedly
increasing studies on SLN have been reported, especially with the
method of high-pressure homogenization[5]. Nevertheless, only a few
investigations have been conducted in regard with the incorporation
of effective components of traditional Chinese medicines into SLN.
Silymarin is a purified extract from the milk thistle
Silybum marianum (L.) Gaertn, which is composed of a mixture of 4
isomeric flavonolignans, namely silibinin (or silybin, SIL),
isosilibinin, silidianin and silychristin. SIL, which constitutes
60%-70% of the silymarin mixture, has been identified as the major
active component[6]. Tetrandrine (TET) is a bisbenzylisoquinoline
alkaloid extracted from the traditional Chinese medicinal herb Radix
stephania tetrandrae. SIL and TET possess wide spectrums of
pharmacological activities[7][8][9][10][11]. These two effective
components of the traditional Chinese medicines have high
lipophilicity and are excellent candidates for SLN encapsulation.
By using this drug delivery system, a high bioavailability and an
intravenous administration are possible. In the present study,
SIL-SLN and TET- SLN were prePared by high-pressure homogenization,
and the physicochemical characteristics of the Particles produced
by this method were analyzed.
MATERIALS AND METHODS
Drugs and reagents
SIL (95%) was purchased from Panjin Green Biological
Development Co. Ltd., China. TET (98%) was purchased from Shanchuan
Biological Co. Ltd., Xi'an. Cholesterin (obtained from Zhengxiang
Chemical Research Institute, Shanghai) and stearic acid (Tianda
Chemical Industry Ltd., Tianjin) were used seParately as the lipid
materials of SLN. Soybean lecithin was obtained from Auboxing Co.
Ltd., Beijing. Sephadex gel-50 was purchased from Tianjin Chemical
Industry Ltd. Methanol (HPLC grade) and absolute alcohol was
supplied by Xi'an Chemical Industry Ltd. Glycerin (Amoy Glycerin
Industry Ltd.) was used as a coemulsifier in water phase.
PreParation of SIL-SLN
SIL (75 mg), cholesterin (1.5 g) and soybean lecithin (1.0
g) were weighed precisely with electronic balance (BP-121S,
sartorius Ltd., Germany) and dissolved in 10 ml absolute alcohol in
water bath at 70 ℃. An aqueous phase was prePared by dissolving 45
ml glycerin in 75 ml distilled water. The resultant organic solution
was rapidly injected into the stirred aqueous phase (80 ℃). The
resulting suspension was stirred continuously at 80 ℃ for 2 h. The
original SIL-SLN suspension was then loaded into a high-pressure
homogenizer (15M-8BA, APV, UK, 5 cycles at 50 MPa) and the samples
were kept at 4 ℃.
PreParation of TET-SLN
TET (75 mg), stearic acid (1.5 g) and soybean lecithin (1.0
g) were weighed precisely and prePared into TET-SLN suspension
according to the method described above.
Transmission electron microscopy
The morphology of SIL-SLN and TET-SLN was examined with
transmission electron microscope (H-600, Hitachi, JaPan). The
samples were stained with 2% (m/V) phosphotungstic acid for 30 s and
placed on copper grids with films for viewing.
Mean diameter and zeta potential
Particle characterization system (Mastersizer 2000,
Malvern Instruments, UK, 20 nm-2000 μm) and zeta potential analyzer
(Zetasizer Nano, Malvern Instruments, UK) were used to study the
diameter and zeta potential of SLN in distilled water. Three samples
of SIL-SLN/TET- SLN were prePared according to the previously
described method and each sample was measured 3 times to calculate
the mean diameter and zeta potential.Entrapment efficiency (EE) and
drug loading (DL) of SIL-SLN.
Chromatographic condition: The chromatographic column of
Planetsil C18 (4.6 mm×15 cm) was used with mobile phase of
methanol/0.1mol/L phosphate buffer (35/65, V/V, pH 3.0), flow rate
of 1.0 ml/min, column temperature of 40 ℃, and detection wavelength
of 288 nm.
The control solutions (0.050, 0.161, 1.605, 14.19, 28.38,
56.75, 113.50 μg/ml) was prePared by dissolving precisely weighed
SIL in the mobile phase. The amount of SIL entering the receptor
comPartment was determined with high-performance liquid
chromatogram (HPLC, LC-2010, Shimadzu, JaPan). The integral
calculus of the chromatographic peak area (A) was recorded as the
Y axis, and the concentration of SIL (C) as the X axis. Drug recovery
was calculated from the following equation:Drug recovery=measured
drug weight in SLN×100%/theoretical drug weight loaded in the
system.
The SIL-SLN suspension was seParated by Sephadex gel-50
column chromatography. The concentrations of SIL in the suspension
(n1) and free drug (n2) were assayed by HPLC after dilution with
methanol. EE and DL could be calculated according to the following
equations: EE%=(n1-n2)/n1×100%, DL=Wdrug loaded in system/Wlipid matrix×100%.
EE and DL of TET-SLN
Chromatographic condition: The chromatographic column of
Spherisorb ODS C18 (250 mm×4.6 mm, 5 μm) was used with mobile phase
of methanol/ether/ ethylamine (volume proportion of 100:1:0.05) and
flow rate of 1.0 ml/min at room temperature and detection wavelength
of
282 nm.
The regression equation, percentage recoveries of TET, EE
and DL of TET-SLN were determined and calculated according to the
methods and equations described previously.
Evaluation of stability
SIL-SLN and TET-SLN were stored at 37 ℃ and the Particle
sizes were determined after 7, 45 and 90 days, respectively, to
evaluate their stability.
Statistical analysis
The results were presented as Mean±SD. Statistical
analysis was performed using Student's t test with P<0.05 indicating
significant difference.
RESULTS
Transmission electron microscopy
The electron microscopy micrographs of SLN loaded with
traditional Chinese medicines prePared by high-pressure
homogenization were shown in Fig.1. The SIL-SLN was spherical and
regular (a), and the TET- SLN appeared platelet-shaped, irregular
and smaller (b).
Mean diameter and zeta potential
The mean diameter of SIL-SLN was 157±8 nm, and the zeta
potential was -35.36±2.68 mV in distilled water. The mean diameter
of TET-SLN was 47±3 nm, and the zeta potential -32.99±2.54 mV.
Fig.1 Electron micrographs of the prePared SLN loaded
with traditional Chinese medicines (Original magnification:
×40 000)
a: SIL-SLN; b: TET-SLN
EE and DL of SIL-SLN
The regression equation of SIL was A=3 307.1C+ 9 910.2. The
assay was linear (r=0.9999) in the concentration range
of
0.050-113.50 μg/ml. The percent- age recoveries at high and
low concentrations were 98.99% and 98.49%, respectively, with a mean
of 98.96%. The EE of SIL-SLN was (95.64±1.33)% and the DL was
4.63%±0.21%.
EE and DL of TET-SLN
The linear calibration curve of TET was obtained in the range
of 1.25-25 μg/ml (r=0.9999). The regression equation of TET was A=18
526C-8 342.2. The percentage recoveries ranged from 98.72% to
101.80% (mean 99.46%). Up to (97.82±1.45)% of TET was incorporated
in SLN, and the DL was (4.76±0.26)%.
Stability
Tab.1 shows the data of Particle sizes of SIL-SLN and TET-SLN
after 7, 45 and 90 days of storage at 37 ℃. These two SLN suspensions
showed sufficient long-term stability with only slight Particle
growth (P>0.05) after storage at 37 ℃ for 90 days.
DISCUSSION
SLNs are a colloidal carrier system for controlled drug
delivery, and it is claimed that SLN combines the advantages and
avoids the disadvantages of other colloidal carriers. Its advantages
include the possibility of controlled drug release and drug
targeting, increased drug stability, absence of carrier biotoxicity,
and large scale production and sterilization[12].
High-pressure homogenization has emerged as a reliable and
powerful technique for SLN preParation[12]. In the present study,
this method proved to be feasible for preParing SIL-SLN and TET-SLN,
which are small, steady and highly incorporated. This success
indicates the possibility of incorporating various lipophilic
effective components extracted from the traditional Chinese
medicines in SLN by this method, which make possible high
bioavailability, controlled drug release, drug targeting, decreased
drug toxicities and minimized side effects, and represents a
successful attempt of novel approach to the modernization of
traditional Chinese medicines.
Various factors may influence the Particle size in
high-pressure homogenization, including, for instance,
homogenization pressure, number of cycles, lipids and
emulsifiers/coemulsifiers
used, and operating tempera- ture. In
the present study, the two kinds of SLN were prePared under identical
conditions with almost the same materials except the drugs
incorporated in SLN and the lipid material. The difference of the
lipids (cholesterin and stearic acid) and the interactions between
the drugs and the lipids might enormously contribute to the
differences of Particle sizes. However, the detailed mechanisms
remain to be further investigated. The shape of SLN may significantly
differ from a sphere. Lipids tend to crystallize in the platelet
form[12],[13]. What factors cause the different shapes of SLN? Which
shape is in favor of drug protection and controlled release? These
questions have attracted increasing attention in recent
years[12],[14].
Silymarin is composed mainly of SIL, and the extracts of milk
thistle, which have been empirically used as hePatoprotective agents
from ancient times, is found to produce beneficial effects in several
hePatic disorders[7],[8]. TET has been clinically used to treat
arthritis, silicosis and hypertension. In recent years, TET was
reported to reduce liver fibrosis and portal hypertension[10]. An
increased drug absorbability and a high bioavailability can be
achieved after oral administration of the SLN loaded with SIL or TET.
SLNs are phagocytized by macrophages after intravenous
administration and targeted to the liver and spleen to increase the
drug concentrations in the liver and spleen. Moreover, the lecithin
of SLN can inhibit lipid peroxidation and protect the membrane of
the hePatocytes. Therefore, SLN holds great prospect of an effective
drug delivery system for traditional Chinese medicines for liver
protection and combating liver fibrosis due to its incomParable
advantages over other drug delivery systems,.
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