[Preparation and Characterization of Clopidogrel Bisulfate Liposomes].

OBJECTIVE: To prepare encapsulated clopidogrel bisulfate (CLP) liposomes so as to deal with the poor water solubility of CLP, and to provide the experimental basis for the development of CLP formulations for intravascular injection. METHODS: CLP-loaded liposomes were prepared using thin film hydration/sonication method and pH gradient active drug loading technology. Then, the morphology, particle size, encapsulation efficiency, drug loading capacity, Zeta potentials and in vitro release behavior were characterized. Bilateral renal arteries of Sprague-Dawley (SD) rats were clamped with micro-artery clamps to establish the model of renal ischemia-reperfusion injury (IRI) in male SD rats. The study aimed to preliminarily investigate the therapeutic effect of CLP-loaded liposome pretreatment on renal IRI in rats. RESULTS: It was found that the optimal formulation and preparation technology of CLP liposomes were as follows: the CLP-to-phospholipid weight ratio of 1∶10, phospholipid-to-cholesterol ratio of 6∶1, octadecylamine-to-CLP ratio of 1.2∶1, PEG 400-to-CLP ratio of 1∶1, and incubation at 50 ℃ for 40 min. Then, following ultrasonication of 100 W efficiency at 5-second intervals for 20 times, CLP loading was conducted using 5 mL of 0.1 mol/L citric acid buffer at pH 3.0. Liposome samples were prepared with the film dispersion method, and the pH value was adjusted to 7.5 through pH gradient active drug loading technology. The CLP-loaded liposomes obtained in this way had a rounded shape, good dispersity, an average particle size of (134.13±2.60) nm, polydispersity index (PDI) of 0.25±0.02, and a Zeta potential of (2.12±0.23) mV. The encapsulation efficiency was found to be (98.66±0.14)%, and the drug loading capacity was (7.47±0.01)%. The in vitro release results showed that 66.24% of CLP was released cumulatively within 72 h. Preliminary efficacy experiments showed that animals pretreated with CLP-loaded liposomes had lower serum levels of blood urea nitrogen and creatinine compared to the levels of IRI model rats without any pretreatment. CONCLUSION: CLP-loaded liposomes were successfully prepared, which might provide the experimental foundation for the future development of CLP formulations for injection.

[Characterization of thermal denaturation process of proteinase K by spectrometry].

The effect of different temperatures on the activity and conformational changes of proteinase K was studied. Methods Proteinase K was treated with different temperatures, then denatured natural substrate casein was used to assay enzyme activity, steady-state and time-resolved fluorescence spectroscopy was used to study tertiary structure, and circular dichroism was used to study secondary structure. Results show with the temperature rising from 25 to 65 degrees C, the enzyme activity and half-life of proteinase K dropped, maximum emission wavelength red shifted from 335 to 354 nm with fluorescence intensity decreasing. Synchronous fluorescence intensity of tryptophan residues decreased and that of tyrosine residues increased. Fluorescence lifetime of tryptophan residues reduced from 4. 427 1 to 4. 032 4 ns and the fraction of alpha-helix dropped. It was concluded that it is simple and accurate to use steady-state/time-resolved fluorescence spectroscopy and circular dichroism to investigate thermal stability of proteinase K. Thermal denaturation of proteinase K followed a three-state process. Fluorescence intensity of proteinase K was affected by fluorescence resonance energy transfer from tyrosine to tryptophan residues. The alpha-helix was the main structure to maintain conformational stability of enzyme active site of proteinase K.

Release test of alliin/alliinase double-layer tablet by HPLC-Allicin determination.

A simple, precise and accurate method was developed and validated for the determination of allicin release from alliin/alliinase double-layer tablets. According to Appendix XC II of Chinese Pharmacopoeia 2010 edition Volume II, a small glass-method was adopted at the rotational speed of 100 r/min using 100 mL phosphate buffer (pH 6.8) as release medium. The release amount was determined by HPLC with a C18 column (250 mm×4.6 mm, 5 µm) using the mobile phase consisting of methanol -0.4% carboxylic acid (65:35) at a flow rate of 1 mL/min and UV detection at 242 nm. The current method demonstrates good linearity over the range 4.052-405.2 µg/mL (r2=0.9999) with an average recovery of 105.5%(RSD=1.25%). The accumulative release of alliin/alliinase double-layer tablets had good homogeneity for within- and between-batches. The method established is simple, accurate and repeatable for the determination of allicin release from alliin/alliinase double-layer tablets.

[Preparation of a hyaluronic acid modified bovine serum albumin nanoparticle and its anti-tumor effect].

OBJECTIVE: To prepare hyaluronic acid modified bovine serum albumin nanoparticle, investigate the modification extent, the drug entrapment property and its anti-tumor effect. METHODS: Bovine serum albumin nanoparticles were prepared by desolvation method, then use hyaluronic acid to modify the surface of bovine serum albumin nanoparticle. The decrement of reactive amino groups on the surface of bovine serum albumin nanoparticles was applied to evaluate the modification extent of hyaluronic acid. Single factor was used to study the effects of several influencing factors in preparation on the modification extent of hyaluronic acid modified bovine serum albumin nanoparticles. The influence of pH on drug entrapment ratio as well as the drug entrapment property were studied. The inhibition rate of nanoparticle on HepG2 was evaluated with MTT asasay. RESULTS: The prepared hyaluronic acid modified bovine serum albumin nanoparticle had a average particle size of 396 nm, and Zeta potential of - 19.7 mV, the deceased ratio of surface reactive amino groups was 34.28%; hyaluronic acid modified mitoxantrone bovine serum albumin nanoparticles had a property of drug load ratio 11.13%, drug entrapment ratio 94.64% and 398 nm average particle size, Zeta potential - 17.9 mV, with a distinguished delayed drug release effect. A higher inhibition rate was observed for hyaluronic acid modified bovine serum albumin nanoparticle than that of mitoxantrone solution (P < 0.05). CONCLUSION: The preparation technique is stable which could be used to prepare hyaluronic acid modified bovine serum albumin nanoparticle, and the prepared nanoparticle has a higer anti-tumor effect than that of mitoxantrone solution.

Immunotargeting of collagenase on thrombus.

In this study, we aimed to develop a thrombus-targeting delivery system of collagenase bound to a monoclonal antibody, and to investigate the thrombolysis of an immune-conjugate in vitro and in vivo as well as the targeting effect. We prepared the immunizing conjugation of collagenase by the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI) method. In order to conjugate collagenase and a monoclonal antibody, bovine serum albumin was used as a linker, increasing the number of collagenase molecules carried and keeping collagenase and the monoclonal antibody active. In vitro thrombolysis experiments showed that collagenase had a strong dissolving effect on collagen-embolus within 24 hours. We established a rabbit pulmonary embolism model to investigate the thrombolysis effect of collagenase and collagenase immunizing conjugation in vivo. Our results revealed a significant difference between collagenase and collagenase immunizing conjugation (P < 0.05). We also established a rabbit ear edge vein model to investigate the active target of collagenase immunizing conjugation. We found that collagenase immunizing conjugation had active targets, and had a strong ability to dissolve organized thrombi. In conclusion, the thrombus-targeting delivery system of collagenase we developed has active targeting effects on thrombi.

[Preparation and quality evaluation of intravenous tanshinone II (A) emulsion].

OBJECTIVE: To prepare and evaluate an intravenous emulsion of tanshinone II(A). METHOD: Soybean phospholipid mixing with poloxamer 188 was used as emulsifier. Oleic acid and glycerol were used as co-emulsifier and isoosmotic adjusting agent, respectively. The coarse emulsion was first prepared and following homogenization was carried out for the coarse emulsion by using a high pressure homogenizer. RESULT: The average diameter of the prepared tanshinone II (A) emulsion was 211 nm with a zeta potential of -32. 1 mV. There had no changes of diameter, zeta potential, pH value, content and physical appearance for the tanshinone II (A) emulsion stored at 25 degrees C away from light during one year. CONCLUSION: The physicochemical properties of the prepared tanshinone II (A) emulsion was stable, which could meet the requirements of intravenous administration.

[Determination of ivermectin in pig serum by high performance liquid chromatography].

OBJECTIVE: To establish a high performance liquid chromatographic method for the determination of micro-amount of ivermectin in pig serum. METHODS: Ivermectin in pig serum was extracted with ethyl acetate after the serum protein was precipitated with 0.05% metaphosphoric acid-methanol in the ratio of 7:3 (V/V). Then the sample was centrifuged at 4000 r/min for 5 min and the supernate was evaporated to dryness with rotary vacuum evaporator. The residue was dissolved with 0.20 ml of methanol as the sample solution for HPLC analysis. HPLC column used was a phenomenex C18 (5 microm, 250 mm x 4.6 mm) with a same type of guard column. Mobile phase consisted of methanol and water in the ratio of 90:10 (V/V) and the flow rate was 1.0 ml/min. The detection wavelength was 245 nm. RESULTS: The linear range of the method was found to be 0.010-20 mg/L and its detection limit was 0.010 mg/L. The relative standard deviation of the method was 0.78%-3.82% and the recoveries varied from 94.0% to 100.0%, with an average recovery of 97.3%. CONCLUSION: This method is simple, reproducible, accurate and suitable to the determination of micro-amount of ivermectin in pig serum.