| To reduce acute pain in patients with osteoarthritis during the onset of disease and improve long-term outcomes,we designed a two-pronged approach to prepare an injectable double-layer microspheres loaded with "Nonsteroidal anti-inflammatory drugs-macrophage polarizing factor" by double emulsification.The morphological characterization,structure confirmation,in vitro release behavior,in vitro cytotoxicity,macrophage polarization characterization,in vivo pharmacodynamics,and in vivo safety of the prepared bilayer microsphere preparations were investigated.The clinical and industrial transformation provides a preliminary theoretical basis and an effective method for tissue regeneration under inflammatory response.The specific research contents are as follows:(1)The preparation,characterization and release properties of bilayer microspheres co-loaded with two drugs were investigatedDouble-layer microspheres co-loaded with parecoxib and IL-4 were successfully prepared by double emulsification method.Using XRD,FTIR and DSC methods,it was verified that the two drugs were successfully encapsulated in the inner and outer carriers,IL-4 was encapsulated in the inner layer of the double-layer microspheres,and most of the parecoxib was encapsulated by the outer carrier.The encapsulation rate of the outer drug PXB of the double-layer microspheres was(91.31±1.2)%,the drug loading rate was(9.4±0.95)%,while the encapsulation efficiency of the inner drug IL-4 was(69.41±3.17)%,and the drug loading rate was(4.45±0.22)%.The higher encapsulation rate will be beneficial to achieve long-term release of one-time administration in vivo pharmacodynamics experiments.In the in vitro release experiment,the inner layer protein was rapidly released in a slightly acidic release medium,and the cumulative release rate exceeded 50%in 2 days,while the release in the physiological environment was slow,reaching close to 50%after 17 days.The complete release time of PXB in the release system of pH 6.8 was 9 days,while the cumulative release rate of PXB in the release system of pH 7.4 on the 9th day was less than 60%.The release behaviors of the two drugs in different release systems are different.The rapid release of PXB helps to regulate the inflammatory environment in the joint cavity and contribute to the long-term slow release of inner layer protein drugs.(2)In vitro experiments of bilayer microspheres safety evaluation of bilayer microspheres and verification of the polarization effect of IL-4 on macrophagesRAW264.7 and rat chondrocytes were selected as the research objects to conduct a preliminary evaluation on the safety of double-layer microspheres in vitro.Cell viability was concentration-dependent.Both cells showed good viability when incubated in blank bilayer microspheres at a concentration of 62.5 μg/mL and lower concentration,and showed a tendency to inhibit growth with increasing concentrations.The cell viability of RAW264.7 was reduced by 13.0%and 13.3%after 24 hours and 48 hours of culture in a slightly acidic environment,while that of rat chondrocytes was reduced by 15.4%and 19.2%,respectively.Flow cytometry results demonstrated that IL-4 promotes the transformation of macrophages to the M2 type and reduces their conversion to the M1 type inflammatory phenotype,helping to repair damaged articular cartilage.(3)In vivo pharmacodynamic verification and safety evaluation of bilayer microspheresBy delivering "non-steroidal anti-inflammatory drug-macrophage polarizing factor" bilayer microspheres into the degenerative joint cavity of rats at the 4th week of induced arthritis,after 6 weeks of treatment,it is different from those requiring frequent dosing.Compared with the control group,the width of the joint space of the rats in the preparation group has a tendency to increase,and the joint surface is more smooth and complete,and no obvious osteophyte is observed.The beam density is close to the sham side.Histopathological results showed that the drug-loaded microspheres effectively reduced synovial inflammation.Immunohistochemical and quantitative results showed that the microsphere preparation significantly enhanced the expression of COL2,Aggrecan and IL-10,which are the key cartilage repair factors of M2 macrophages. |
