高压乳匀法制备中药固体脂质纳米粒

1、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(西安交通大学医学院1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。

2、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&#

3、160; 文献标识码：A  文章编号：1673-4254(2006)05-0541-04PreParation of solid lipid nanoParticles loaded with traditional Chinese medicine by high-pressure homogenizationLI Ying-chao1；DONG Lei2；JIA Ai1；CHANG Xin-ming1；XUE Hui11DePartment of Gastroenterology,First Affiliated Hospital,Medical Colleg

4、e of Xian Jiaotong University,Xian 710061,China;2DePartment of Gastroenterology,Second Affiliated Hospital,Medical College of Xian Jiaotong University,Xian 710004,ChinaAbstract:Objective  To investigate the preParation of solid lipid nanoParticles (SLN) loaded with traditional Chinese medi

5、cines 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. Transm

6、ission 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 chro

7、matography 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±2.6

8、8 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 T

9、ET 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 medicinesSuppo

10、rted by Science and Technology Project of Xian City (GG04133).LI Ying-chao (1974-), PhD, attending physician, specialized in pharmaceutical research of liver fibrosis, Tel: E-mail: l_Corresponding author: DONG Lei, medical professor, Tel: 29368, E-mail: donglei4488  

11、   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 delivery1. ComPared with other Particulate carriers SLN has several advantages for drug

12、delivery such as its good biocomPatibility2, biodegradability3, high bioavailability4, 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 homogenization5. Nevertheless, only a few investigati

13、ons 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 s

14、ilibinin (or silybin, SIL), isosilibinin, silidianin and silychristin. SIL, which constitutes 60%-70% of the silymarin mixture, has been identified as the major active component6. Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid extracted from the traditional Chinese medicinal herb Radix stepha

15、nia tetrandrae. SIL and TET possess wide spectrums of pharmacological activities7891011. 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 i

16、ntravenous 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

17、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

18、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. Gly

19、cerin (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 diss

20、olved 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 or

21、iginal 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 precisel

22、y 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 wi

23、th 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 m) and zeta potential analyzer (Zetasize

24、r 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.Entrapmen

25、t 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

26、40 , and detection wavelength of 288 nm.    The control solutions (0.050, 0.161, 1.605, 14.19, 28.38, 56.75, 113.50 g/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

27、 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%/t

28、heoretical 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 acco

29、rding 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 m) was used with mobile

30、 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 acco

31、rding 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.    Stati

32、stical 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   &

33、#160;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 diamete

34、r 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

35、 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=0.999

36、9) in the concentration range of  0.050-113.50 g/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

37、    The linear calibration curve of TET was obtained in the range of 1.25-25 g/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

38、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)

39、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 possibi

40、lity of controlled drug release and drug targeting, increased drug stability, absence of carrier biotoxicity, and large scale production and sterilization12.    High-pressure homogenization has emerged as a reliable and powerful technique for SLN preParation12. In the present stu

41、dy, 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

42、 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 Particl

43、e 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 mate

44、rials 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

45、 investigated. The shape of SLN may significantly differ from a sphere. Lipids tend to crystallize in the platelet form12,13. 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 rece

46、nt years12,14.    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 disorders7,8. TET has been clinically used to treat a

47、rthritis, silicosis and hypertension. In recent years, TET was reported to reduce liver fibrosis and portal hypertension10. 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 aft

48、er 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

49、system for traditional Chinese medicines for liver protection and combating liver fibrosis due to its incomParable advantages over other drug delivery systems,.    REFERENCES    1Müller RH, Mader K, Gohla S. Solid lipid nanoParticles (SLN) for controlled

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