The Study Of Sustained-releasing Endostar PLGA Microspheres Combined Calcium Phosphate Cement In The Treatment Of Osteosarcoma

It was confirmed by many studies at home and abroad that the solid tumor could not develop if there were not new endothelial cells to supplement and the formation of neogenesis blood capillary was the cause of the amplification and metastasis of tumors. Rh-Endostatin was discovered by Folkman et al at 1997 and it is the fragment of non-collegenous dormain which is at the C-terminal of extracellular matrix(ECM) collagen protein XVIII. It exerts a negative effect on the angiogenesis of tumor. And it is a specific kind of angiogenic inhibitor. The experiment in vitro indicated that it can inhibit the proliferation and migration of vascular endothelial cells effectively and inhibit the angiogenesis of carcinoma. So the nutrition supply of tumor cells was obstructed and the proliferation and migration of tumor were inhibited. Therefore rh-Endostatin is featured in broad-spectrum, high-efficiency and no resistance to prevent neoplasm of tumor blood vessels. Calcium phosphates possess good biocompatibility and osteoconductivity as the filling materials for repairing bone defects. When solidified, CPC produces strong compressive strength without releasing much heat and has good plasticity, biocompatibility, osteoconductivity and degradability in vivo. Currently, CPC is the only material available capable of spontaneous solidification and inducing bone regeneration, and has been widely used in dental and orthopedic surgeries. The mild condition required for solidification and the porous structure of CPC suggest great potential of CPC as a drug carrier for sustained drug release in vivo to maintain sufficient local drug concentration and minimize potential systemic adverse effects of the drugs, especially the agents for tumor therapy.Endostar is a neotype self-designed rh-Endostatin and it is produced by using escherichia coli as expression vectors. Endostar raise the stability of the protein efficiently and the antiangiogenenic effect, and make the purification of protein easy. As shown in these researchs, the inhibitory effect of Endostar to new vessels is greater two times than Endostatin at least. PhaseⅢclinical trials confirm that the efficacy of Endostar combined with NP regimen in treating advanced non-small-cell lung cancer(NSCLC) is raised significantly and the treatment prolong the live time and improve the quality of life. At present in home and abroad many researchers are attempting to use Endostar combined chemotherapy to treatment many kinds of advanced malignant tumour. Calcium phosphates possess good biocompatibility and osteoconductivity as the filling materials for repairing bone defects. When solidified, CPC produces strong compressive strength without releasing much heat and has good plasticity, biocompatibility, osteoconductivity and degradability in vivo. Currently, CPC is the only material available capable of spontaneous solidification and inducing bone regeneration, and has been widely used in dental and orthopedic surgeries. The mild condition required for solidification and the porous structure of CPC suggest great potential of CPC as a drug carrier for sustained drug release in vivo to maintain sufficient local drug concentration and minimize potential systemic adverse effects of the drugs, especially the agents for tumor therapy.At present, the research of the pharmaceutical dosage form of Endostar is very seldom and there is no report about sustained-release dosage form of Endostar. Because Endostar is easy to be degradated and inactivated and has a very short half-life in vivo, it is required to inject many times and the promising prospects in utilization are restricted. In this study we design to prepare controlled release of Endostar encapsulated within poly(D,L-lactide-co-glycolide)(PLGA).The lasting time of the drug is 4 weeks and the quality of drug and the compliance of patients are raised. The delayed release microspheres are used to combinate with calcium phosphate cement (CPC), then the complex is used to fill the defect after the focal cleaning of bone tumor. Because the diameter of Osteoblast is about 100～150um, in this study the particle diameter of these microspheres are controlled about 100～150um. After delayed release the cellules are profit to osteoblast to enter and newly formed bone tissue to deposite.This study is divided into 4 parts. The first part begun with the preparation of PLGA microspheres by W/O/O phase separation method and observed the appearance and the size and distribution of PLGA microspher. Single-factor experiment was designed to investigate the influential factors of the size and encapsulation efficiency of microspheres. The influential factors include stirring rate when preparing double emulsion, polyvinyl alcohol (PVA) volume, PLGA concentration, the volume of inner water phase. Adjusting all kinds of influential factors, the encapsulation efficiency of microspheres are usually high(more than 45%). In vitro test confirmed that PLGA microspheres for Endostar delivery systems prepared by the solvent evaporation with W/O/O phase separation method have better functions of sustained releasing Endostar, moreover, their stability and reproducibility are favorable, and encapsulation efficiency are high, which could provide the local sustained releasing Endostar.The second part of this study researched on the properties of CPC combined with PLGA microspheres for Endostar. We studied the properties of CPC combined with different content of PLGA microspheres, including the setting time, compressive strength, microstructural development and the interval porosity. The result showed that high content of PLGA microspheres(10%) affected the properties of CPC that had the longer setting time and lower compressive strength. On the other hand, the properties of CPC combined with low content of PLGA microspheres(5% and 8%) were not influenced.The third part of this study researched on the release characteristics in vitro of the PLGA microspheres for Endostar combined with CPC and the biological activities of Endostar delayed release from CPC. The experimental result showed that the delayed release of Endostar from CPC is like as the delayed release of Endostar from PLGA, their stability and reproducibility are favorable, and it could meet the need of the local controlled releasing.The last part of this study is the in vivo research of the PLGA microspheres for Endostar combined with CPC on tumor. Implanted the PLGA microspheres for Endostar combined with CPC and MTX CPC into osteosarcoma model of the nude mouse. It has shown that the PLGA microspheres for Endostar combined with CPC has the stronger ability of inhibiting the growth and inducing the apoptosis of the of the tumor cell.Conclusion:Endostar can be encapsulated in microspheres to yield continuous release when using biodegradable polymers PLGA as carrier material, and this technique will have a favorable perspective in the near future. The properties of CPC combined with low content of PLGA microspheres(5% and 8%) were not influenced negatively. The delayed release of Endostar from CPC is like as the delayed release of Endostar from PLGA, their stability and reproducibility are favorable, and it could meet the need of the local controlled releasing. The compressive strength of three groups CPC after Endostar delayed release from them did not cut down, and the porosity of CPC (8% and 10%) raised obviously. In summary, the composite cement serves as an ideal drug carrier and Endostar released from the composites retains good bioactivity. The composite cement may find potential clinical application in bone defect reconstruction with sustained release of therapeutic agents to improve the therapeutic effect, simplify the drug administration and minimize the adverse effects of the drugs.All aboved experimental result suggested that the PLGA microspheres for Endostar combined with CPC could be used for local treatment of malignant bone tumor. And this research has certain instructive function to the clinical application of drug-loaded CPC.The new ideas of this study: 1,Discuss the feasibility of CPC on the spinal tumor.2,Compounded the PLGA microspheres for Endostar combined with CPC for the first time and carried out the basic research.