Study On The Sustained-release System Of PLGA Drug-loaded Microspheres And Its Application In The Repair Of Spinal Cord Injury

Spinal cord injury(SCI)is a kind of traumatic injury with decreased function of sensory and motor neurons,which brings heavy burden to individuals and society.Lack of nerve growth factor(NGF)after spinal cord injury makes it difficult to repair spinal cord injury.However,it is hard for NGF to pass through the blood-brain barrier and it can not be administered directly for a long time.Traditional SCI stents have some problems such as short drug release time or difficult to achieve clinical application.Therefore,the design of a SCI stent which can release NGF for a long time and has potential clinical application value has become an urgent problem.In this study,two kinds of biological stents for the treatment of SCI were designed with PLGA microspheres as the negative carrier of NGF.The main contents of the study are as follows:(1)In this project,firstly,oil-soluble chitosan/gelatin drug-loaded microspheres and water-soluble polylactic acid glycolic acid(PLGA)microspheres were prepared by electrospray.By studying the relationship between the solid content of the chitosan/gelatin blend and the conductivity and viscosity,it is found that the solid content is within the range of 6.310-6.431 mg/100mL,the conductivity and viscosity are appropriate,and microspheres with good morphology can be formed.The diameter is about 450 nm.The main factor affecting the preparation of PLGA microspheres is the concentration of the organic phase.When the concentration of the organic phase of the drug-loaded microspheres is 7 wt%,and the water-oil ratio is 1:200,the microspheres have the best appearance.Through drug release experiments and circular dichroism spectroscopy for drug release analysis,chitosan/gelatin microspheres quickly released NGF within 2 days,with a release rate of about 87%.PLGA microspheres have a sustained drug release time of up to 14 days,with a release rate of about At 60%,both microspheres can maintain the drug activity well.Studies have shown that oil-soluble PLGA microspheres have higher yields and spheroidization rates than water-soluble chitosan/gelatin microspheres.At the same time,they have better drug release properties due to their difficulty in swelling,and can be combined with other biological materials to prepare SCI scaffolds(2)To solve the problem that traditional biological scaffolds are difficult to achieve clinical application,the PLA/NGF-PLGA/CS composite membrane was designed with PLGA microspheres as the carrier of NGF.The compactness of PLA film is used as a sealing layer to prevent the diffusion of drugs and provide certain mechanical support.The fiber uniformity is the best when the concentration is 7 wt%.PLGA microspheres serve as the load-release layer.As the implanted layer,CS membrane can directly contact the injured area and at the same time plant bone marrow mesenchymal stem cells(BMSCs)to repair nerves.The fiber morphology is best when the solution concentration is 6 wt%.The unidirectional tensile test shows that the composite membrane structure has a certain mechanical strength and can be directly applied to the injured area,and at the same time,the space-occupying effect is small,and it does not cause obvious compression and secondary injury to the spinal cord.Through drug release experiments and circular dichroism spectrum analysis of drug release,the results show that the composite film can release drugs for more than two months and maintain good drug activity.Cytotoxicity experiments and cell differentiation experiments show that the composite membrane has no cytotoxicity and can successfully induce PC-12 cells to differentiate into neurons within 5 days.At the same time,animal experiments showed that the application of PLA/NGF-PLGA/CS composite membrane to the injured site significantly improved neuronal regeneration and motor function recovery in rats with spinal cord injury.The addition of BMSCs further promoted the repair of spinal cord injury.These results show that the sustained-release time of the composite film drug can meet the time required for repair and can successfully improve spinal cord injury,and has great prospects in clinical application of SCI.(3)In view of the problem that the composite membrane needs to be surgically implanted and cannot fill the cavity in the ward,we use P407 and P188 as the matrix,blend into SA,and obtain a physical-chemical double cross-linked composite gel through Ca2+cross-linking.The concentration of P407 is 16 wt%and the concentration of P188 is 3 wt%.At this time,the gelation temperature of the composite gel is about 34℃,and the solid content is low.Characterize the rheological properties of the gel through viscosity-temperature curve test,frequency sweep sequence,compression test,and shear thinning test.The viscosity-temperature curve shows that the composite gel has temperature sensitivity.The frequency sweep sequence showed that the hydrogel was formed,and the gel at 37℃ turned into a solid state strength higher than 25℃.The composite gel exhibits shear thinning behavior and can be injected through a conventional syringe,and compression tests show that the gel can maintain a certain shape after being injected into the body.After Ca2+cross-linking,the composite gel gel network structure tightens and the pore size becomes smaller,the release time can reach 14 days,and the release rate is about 48%.The circular dichroism spectrum showed that the activity of NGF was maintained well,and the composite gel showed no cytotoxicity,and successfully induced PC-12 cells to differentiate into neurons within 5 days,which was helpful for nerve repair.The results show that the NGF-PLGA/P407/P188/SA composite gel has good injectability and has a wide range of applications in SCI repair and more medical fields.