Preparation And Characterization Of Lysozyme-PLLA Complex Microparticles

In this study, the drug carries based on PLLA was prepared using solution-enhanced dispersion by supercritical CO₂ (SEDS), and its application in drug delivery for protein drugs was investigated.Firstly, microparticles of poly-L-lactide (PLLA) were prepared with SEDS process by using supercritical carbon dioxide as an anti-solvent. The effects of several key factors on surface morphology, and particle size and particle size distribution were investigated. These factors included temperature (33-39°C), pressure (8-16 MPa), PLLA concentration (0.5-1.5%, w/v), flow rate of organic solution (0.5-1.5 ml·min^-1), mixing acetone into PLLA dichloromethane solution (1:1, v/v) and different molecular weight of PLLA. The results indicated that concentration of the organic solution and the flow rate of the solution played important roles on the properties of products; the mean particles size was decreased with the decreasing of PLLA concentration or flow rate of organic solution; the temperature and pressure had minor effects on the properties of products, however, the mean particles size was decreased slightly with the increasing of CO₂ density, and the agglomeration of products was increased with the increasing of temperature; the PLLA microparticles with MW of 50 kDa and 100 kDa exhibited rather spherical shape, smooth surface, and narrow particle size distribution. Various microparticles with the mean particle size ranging from 0.64μm to 6.64μm, could be prepared by adjusting the operational parameters. Fine microparticles were obtained in the SEDS process with dichloromethane/acetone mixture as solution.Secondly, lysozyme was used as macromolecular drug model, the micronization of lysozyme was performed in SEDS process and both the change in the secondary structure and activity of lysozyme were studied. The results of Fourier-transform infrared (FTIR) spectra and Circular Dichoism (CD) spectra both showed that there was no significant change in the secondary structure of lysozyme after SEDS process, while the activity test indicated that its activity was increased 51.2% after this process;Thirdly, the micronized lysozyme were used to prepare lysozyme-PLLA microparticles, the effect of molecular weight (MW) of PLLA, Polyethylene Glycol (PEG) ratio, PEG MW and drug loading on the properties of lysozyme-PLLA microparticles were studied. The results indicated that with the increasing of drug loading, PEG ratio, or PEG MW, the particle size of lysozyme-PLLA microparticles decreased, and its rate of drug release increased, while with the increasing of PLLA MW, the particle size of lysozyme-PLLA microparticles increased first and then decreased, and its drug-release rate was controlled by both particle size and PLLA MW.Finally, the in vitro degradation of PLLA microparticles was investigated by incubating PLLA microparticles in 37℃pH 6.8 PBS, and the toxicity of PLLA microparticles was measured by MTT assay. It was found that after being incubated for 10 weeks, there were some tiny holes on the surface of PLLA microparticles, the PLLA MW was decreased, the larger was the MW, the bigger was the decreasing scope of MW; the MTT assay demonstrated that the PLLA microparticles had no cytotoxicity, they would be biocompatible.