| Chronic osteomyelitis has been considered to be the second largest disease following cancer in the medical profession. Conventional treatments include complete debridement and continuous high-dose antibiotic treatment. But as a result of long course of treatment, long-term and large doses of antibiotics increase the happening of resistance, makes the recurrence of chronic osteomyelitis high. Vancomycin hydrochloride is the first generation of peptide antibiotics. It can inhibit the formation of peptides and phospholipids of bacterial cell walls and interfere with the synthesis of cell wall, thus it can inhibiting bacterial growth and reproduction. It has no cross resistance with other antibiotics, and it rarely produce resistant strains. It is effective to most of the gram-positive bacteria, especially staphylococcus aureus. Although its specific role in bacterial cell wall peptidoglycan, but obvious nephrotoxicity, ototoxicity and neuromuscular blocking effects appear in the process of early applications. Patients with chronic osteomyelitis often has poor immunity, easily leading to acute kidney injury and chronic kidney disease. This hampers the treatment of chronic osteomyelitis, and puts forward a new research topic to clinical pharmacy worker.Liposome is a potential sustained release system that has the ability to encapsulate water-soluble drug. Since the lipid bilayer of liposomes are composed of phospholipids and cholesterol, which is similar to the components of the cell membrane, and is easily fused with the infected bacteria, therefore liposomes are considered to be a non-toxic and biodegradable sustained release drug delivery systems. Through dropping method with the theory of layer by layer electrostatic deposition, chitosan is wrapped in the surface of liposome, and the Zeta potential was burden from negetive gradually into positive. Because of the electrostatic effect, it can interact with the negatively charged cell membrane. It can open the close connection between the epithelial cells, and promote macromolecular substances through the epithelial tissue of transshipment, thus it increases the permeability of hydrophilic macromolecular cells bypass. So it can promote the absorption of hydrophilic macromolecular drugs.In this topic, firstly, injection soybean lecithin and cholesterol as carrier materials, VANH-Lip was prepared by the modified reverse phase evaporation method. Then c-VANH-Lip was prepared by the dropping method, the physical/chemical properties, characteristics of drug release in vitro and preliminary safety evaluation of VANH-Lip and c-VANH-Lip were studied. Finally, Vancomycin hydrochloride solution as a control, the two liposomes in vivo pharmacokinetics and tissue distribution were studied to evaluate the sustained release and targeting effect in mice by targeting and pharmacokinetic parameters.1. Preparation and physical/chemical properties of VANH-LipThe method of high-performance liquid chromatography (HPLC) was established to determine the drug concentration of VANH, and a methodological study was carried out. Ultrafiltration centrifugation method was applied to determine the encapsulation efficiency of VANH-Lip, and a methodological study was also carried out. VANH-Lip was prepared by the modified reverse phase evaporation method. The optimal prescription and preparation process was determined by single factor and orthogonal experimental design with the index of particle size and encapsulation efficiency. The morphology of VANH-Lip was observed by transmission electric microscope (TEM). Particle size and zeta potential were determined by laser light scattering using a Delsa Nano C Particle Analyzer. And drug loading(DL), encapsulation efficiency(EE), pH, and other physical/chemical properties were investigated to verify the feasibility of prescription composition and preparation processes.The results of methodology indicated that the analytical method to determine the content of VANH has high specificity, accuracy and precision. Ultrafiltration centrifugation method was applied to determine the encapsulation efficiency of VANH-Lip, and the method is energy saving, it has high recovery, good reproducibility. VANH-Lip was prepared by the optimal prescription and technology. The morphological appearance observed under transmission electron microscopy (sem) is kind of spherical, uniform size distribution.The average particle size was 188.4±5.22nm, the average Zeta potential was-10.1±4.40 mV, the EE was 40.78±1.04%, the DL was 2.55±0.065%, the average pH value was 5.96±0.066. It conform to the requirements of the injection. The reproducibility test results show that the prescription composition is reasonable, and the preparation technology is feasible.2. Preparation and physical/chemical properties of c-VANH-Lipc-VANH-Lip was prepared by the dropping method. The optimal prescription and preparation process was determined by investigating the effect of molecular weight, concentration and liposome/chitosan to Zeta potential. The morphology of c-VANH-Lip was observed by transmission electric microscope (TEM). Particle size and zeta potential were determined by laser light scattering using a Delsa Nano C Particle Analyzer. And drug loading(DL), encapsulation efficiency(EE), pH, and other physical/chemical properties were investigated to verify the feasibility of prescription composition and preparation processes.The results indicated that c-VANH-Lip was prepared by the dropping method successfully. It is kind of spherical under TEM. After coating with chitosan, the particle size was slightly increased, and the Zeta potential was burden from negative gradually into positive. The average particle size was 220.4±3.56nm, the average Zeta potential was 25.7±1.12 mV, the EE was 32.65±0.586%, the DL was 2.18±0.039%, the average pH value was 4.95±0.082. It conform to the requirements of the injection. The reproducibility test results show that the prescription composition is reasonable, and the preparation technology is feasible.3. Characteristics of drug release in vitro and preliminary safety evaluation of VANH-Lip and c-VANH-LipCharacteristics of drug release in vitro was determined by the dynamic membrane dialysis method. And the release profile was fitting to mathematical model. Then the preliminary safety of VANH-Lip and c-VANH-Lip was evaluated by in vitro hemolysis test.The results showed that in vitro release, compared with VANH-Sol, the drug release curve of two kinds of liposome was similar, and they were in accordance with the Weibull equation. The VANH release from c-VANH-Lip was relatively slow and controlled. Maybe it is because the coating of chitosan reduced the exposure of the lipid, thereby reducing the leakage of drug, and the burst release effect was obviously decreased. And it increases thickness of c-VANH-Lip membrane, delaying the release of drug in liposome. The results of in vitro hemolysis test indicated that VANH-Lip and c-VANH-Lip were safe to intravenous injection without hemolysis during the intravenous injection.4. Pharmacokinetics in vivo and biodistribution studies of VANH-Lip and c-VANH-Lip.The method of high-performance liquid chromatography (HPLC) was established to determine the drug concentration of VANH in plasma and different organs, and a methodological study was carried out. Compared to VANH-Sol, pharmacokinetics in vivo and tissue distribution was investigated after intravenous administration of VANH-Lip and c-VANH-Lip, and targeting parameters was provided to evaluate the targeting propertyof the tissue.The results of methodology indicated that the analytical method has high specificity, accuracy and precision. The results of pharmacokinetics in vivo showed, t1/2β of VANH-Sol, VANH-Lip and c-VANH-Lip were 1.888 h,2.240 h,3.490 h, relatively. Compared to VANH-Sol, 1/2β and MRT of VANH-Lip and c-VANH-Lip was prolonged 0.19 and 0.85 times. Similarly, MRT 0-∞ of VANH-Lip and c-VANH-Lip was prolonged 0.63 and 1.60 times. As to AUC, VANH-Sol, VANH-Lip and c-VANH-Lip were 22.490 mg/1h,44.710 mg/1h,59.908 mg/1h, relatively. AUC of c-VANH-Lip and VANH-Lip were increased 1.66 and 0.34 times bigger than that of VANH-Sol. In another word, VANH was encapsulated into c-VANH-Lip, and the circulation time in the body was prolonged, and the therapeutic effect of VANH was improved. We can reduce the frequency and the dose of administration to achieve the same treatment effect with vancomycin hydrochloride injection. The results of tissue distribution showed, the Re of VANH-Lip in liver and spleen were 2.167 and 1.982; the Re of c-VANH-Lip in liver and spleen were 3.594 and 1.985; the Re of VANH-Lip and c-VANH-Lip in kidney were 0.645 and 0.605, relatively. Compared to VANH-Lip, drug accumulation in kidney was reduced and the side effects could be decreased. It provides the clinical application value for the long-term application of VANH and the treatment of osteomyelitis VANH long-term treatment provides the clinical application value.In a word, c-VANH-Lip was prepared by the dropping method successfully. Preparation process is simple and reliable, reproducible, and the EE is relatively high, the physical/chemical properties was conform to the requirements of the intravenous injection; the results of in vitro release experiments show that, compared to VANH-Lip, the slow-release effect of c-VANH-Lip is stronger; the results of in vitro hemolysis test indicated that VANH-Lip and c-VANH-Lip were safe to intravenous injection without hemolysis during the intravenous injection. The results of pharmacokinetics in vivo showed, compared to VANH-Sol, the circulation time in the body was prolonged, and improved the therapeutic effect of VANH. The results of distribution showed, compared to VANH-Sol, drug accumulation in kidney was reduced and the side effects could be decreased. It provides the clinical application value for the long-term application of VANH and the treatment of osteomyelitis VANH long-term treatment provides the clinical application value. |
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