Studies On Vincristine-loaded Polymer Ultrasound Microbubbles Contrast Agent

In recent years, with the development of preparation and image technology of UMCA, ultrasound image have gradually developed a new technology. Technology of drug positioning release that microbubbles carrying drug or gene are destructed by ultrasound has already been a new non-invasive gene grafting and drug delivery technology, and the main mechanism are cavitation and sonoporation.UMCA usually refers to microbubbles with shell whose diameter is from 2μm to tens of microns, and the thickness of shell is from 1 to 500 nm. Shell materials are mostly albumin, phospholipids, galactose, polymers, etc. The gas in microbubbles are usually CO2, fluorocarbon gas, N2 and air.VCR is an alkaloid which is extracted from the plant catharanthus apocynaceae and is widely used in clinical treatment of neuroblastoma, brain tumors, lung cancers and other solid tumors. VCR belongs to a cytotoxic anticancer drug and the mechanism of action is the binding of tubulin, inhibiting tubulin polymerization and affecting microtubule assembly and spindle fiber formation.In this theme, PLGA/(PLGA-PEG) was used as the inclusion material and NH4HCO3 was used as porogen, VCR-PLGA-PEG-UMCA were prepared through double emulsion-solvent evaporation. To UMCA as a drug carrier, UMCA were broke down at the scheduled tumor region with the energy of ultrasonic cavitation in vitro so that drug were released and penetrating into local tissue to improve the local drug concentration to achieve the purpose of targeted therapy.PartⅠPreformulation studyUV method of VCR was developed for assay. The regression equation was A = 0.0121C-0.0025, r = 0.9998, which was linear over the range of 4.00~20.00μg·mL-1. Its recovery, precision, stability all met the requirement of technology.Shaking flask test was used to determine O/W apparent partition coefficients of VCR in different CH2Cl2/0.050 M PBS (pH3.0, pH4.0, pH5.0, pH6.0) and CH2Cl2/H2O. The P value was 0.855, 1.887, 2.174, 2.224 and 2.264, respectively. It was showed that the lipophilia of VCR was increased with pH value increasing. To improve the encapsulation efficiency, VCR should be distributed into water phase as much as possible, so CH2Cl2/PBS (pH 4.0) should be chosen as oil-water system.PartⅡPreparation of VCR-PLGA-PEG-UMCATaken for size and appearance of microbubbles as the index, some conditions of UMCA preparation were investigated such as the ultrasonic time, emulsifier type, emulsifier concentration, internal phase volume and the NH4HCO3 aqueous concentration. Taken for EE as the index, primary single factor test was applied to choose preparation conditions, which were respectively dosage, different organic solvents, oil-water phase ratio, PLGA/(PLGA-PEG) ratio, PLGA/(PLGA-PEG) concentration, Span80 content, the external phase pH, NaCl concentration in the external phase and CH2Cl2 evaporating method.Single-factor test was utilized to screen out influential factors on VCR-PLGA-PEG-UMCA preparation: The volume ratio of water phase and oil phase, PLGA/(PLGA-PEG) concentration, PLGA/(PLGA-PEG) ratio. Taken for EE as the index, orthogonal design was optimized and the optimized process conditions was that: VCR dosage was 2.0 mg, the volume ratio of oil phase and water phase was 2:0.1, PLGA/(PLGA-PEG) concentration was 0.5%, PLGA and PLGA-PEG ratio was 2:1.Though the best prescription, acquired VCR-PLGA-PEG-UMCA whose appearance was spherical, smooth surface and no adhesion, and the distribution of particle size was even, average particle size was 1.27μm, EE was (37.63±0.61)%, DL was (1.25±0.23)%, and zeta potential was -24.88 mV.Preliminary stability of lyophilized VCR-PLGA-PEG-UMCA showed that there were 60 days for good stability at 4℃. VCR-PLGA-PEG-UMCA was redissoluted with saline. One week later, the solution became undertint and there were some sediments in the bottle bottom. One month later, the solution was delaminated, the upper layer was light white and the underlayer was the majority of precipitation.PartⅢRelease study of VCR-PLGA-PEG-UMCA in vitroUV method of VCR was developed for assay. The regression equation was A = 0.0157 C-0.0007, r = 0.9997, which was linear over the range of 2.00 ~ 20.00μg·mL-1. Its recovery, precision, stability all met the requirement of technology.Compared drug release behavior of UMCA of molecular weight 20 kDa with UMCA of molecular weight 40 kDa in vitro, experiments showed that the release of UMCA of molecular weight 20 kDa was faster and was fundamentally completed about 20 days, however, the release of UMCA of molecular weight 40 kDa was accomplished about 30 days.PEG on drug release behavior of the UMCA had a certain influence in vitro. Drug release from UMCA could be accelerated by adding PEG, possibly because the rate of PLGA degradation was increased and dissolution was enhanced by hydrophilic PEG, so the release rate was speeded up.Study drug release behavior of UMCA with ultrasound irradiation and without ultrasound irradiation in vitro, experimental results showed that UMCA's release characteristics without ultrasound irradiation was consistent with the release of sustained-release preparation. However, the drug release of UMCA with ultrasonic irradiation could rapidly come to 80% in the 60 min. PartⅣPreliminary study of ultrasound imaging of VCR-PLGA-PEG-UMCA in vitro and in vivoUMCA imaging was observed with ultrasound diagnostic apparatus and there was a good ability to increase the ultrasonic signal. Imaging was very clearly in vitro.UMCA imaging was observed with using ultrasound diagnostic apparatus in vivo, which remarkably could be increased to ultrasound imaging effects on heart of rabbits with ultrasound irradiation and was provided with a direct and reliable basis for ultrasonic positioning targeted drug delivery further.PartⅤPreliminary study of pharmacokinetics of VCR-PLGA-PEG-UMCA in rabbitHPLC method of VCR was developed for assay. The regression equation was A = 264.43 C– 58.237, r = 0.9996, which was linear over the range of 0.105 ~ 20.010μg·mL-1. Its recovery, precision, stability all met the requirement of technology.To VCR-aqueous injection as the reference agent, the pharmacokinetic study of VCR-PLGA-PEG-UMCA was performed in rabbits and data was analysized by DAS pharmacokinetic software. The results showed that: VCR-PLGA-PEG-UMCA and VCR-aqueous injection were consistent with two-compartment model (weight factor: 1/C2). The concentration-time curve was very similar. In this paper, PLGA-PEG was used as coating materials and NH4HCO3 was used as the pore material. UMCA were acquired by double emulsion-solvent evaporated method. The drug release was controlled by the corrosion rate of polymer material in vitro, which had a good cotroolled-release effect.In this paper, taken for UMCA size, appearance and EE as index, effects of various factors on the conditions of preparation UMCA were investigated. Method that EE and drug released in vitro were determined by UV was established.Prepared VCR-PLGA-PEG-UMCA had a good effect of imaging in vivo/ vivo. Pharmacokinetics experiments in rabbit in vivo showed that AUC of VCR-PLGA-PEG-UMCA incresaed and there was better bioavailability, which was provided with a foundation to study further UMCA targeted drug delivery.The paper's design, experiment and evaluation in vitro/vivo haven't been found in pertinent literatures.