PH-responsive PCL/PEG-naringenin Nanofiber Membrane Used In The Treatment Of Osteoarthritis Research

OBJECTIVE: To investigate the pH-responsive mPEG-COOH(mPEG-COOH,Methoxy-PEG-Carboxymethy)and Nar in the mildly acidic environment of OA(osteoarthritis,osteoarthritis).Naringenin is synthesized into PEG-Nar through an esterification reaction,the linking compound is mixed with the material PCL(polycaprolactone,polycaprolactone),and an electrospinning technique is used to produce a PCL with pH response function.PEG-Nar nanofiber membrane.Study PCL/PEG-Nar nanofiber membrane in vivo and in vitro for osteoarthritis treatment,verify its pH response,and develop pH-responsive PCL/PEG-Nar nanofiber membrane DDS(drug delivery system drug delivery system)as a treatment platform For OA treatment.Part I: Effect of PCL/Nar nanofiber membrane on IL-1β(recombinant rat IL-1β,interleukin 1β)-induced osteoarthritis in vitro.(1)First is the synthesis of PCL/Nar nanofiber membrane.PCL nanofiber membranes and PCL/Nar nanofiber membranes were prepared by mixing PCL and Nar in different proportions using electrostatic spinning technology.(2)Characterization of nanofiber membrane materials.Biocompatibility using SEM(3)nanofiber membranes.The biocompatibility of nanofiber membranes was detected using CCK-8 and Calcein-AM/PI staining.(3)Biocompatibility of nanofiber membrane.(Scanning electron microscope,scanning electron microscope)Observe the three-dimensional structure of the fiber.4)Use q RT-PCR to explore the anti-inflammatory ability of nanofiber membranes in vitro.QRT-PCR was used to analyze the expression of chondrocyte-related genes MMP3,MMP13,IL-1,IL-6 and cartilage-related gene COL2a1.Extend the foundation for subsequent research on pH-responsive drug delivery sustained-release systems.(1)First,the synthesis and characterization of PEG-Nar complex.mPEG-COOH and Nar were synthesized by the esterification reaction using EDC/DMAP as a catalyst.The chemical composition and structure of PEG-Nar were verified using Fourier infrared and ultraviolet spectrophotometry.(2)Fabrication of PCL/PEG-Nar nanofiber membrane.After the synthesis of Nar,PCL/PEG-Nar nanofiber membranes were prepared by mixing PCL and PEG-Nar in different ratios and using electrostatic spinning technology.(3)Biocompatibility detection of PCL/PEG-Nar nanofiber membrane.Using CCK-8,4 wt% was screened as the optimal amount of PEG-Nar complex.The ratio of PCL,PEG,and Nar was calculated according to the calculated structural formula,and according to the ratio of these three,PCL(4)nanofiber membranes were fabricated using electrostatic spinning technology.First,in vitro drug release was performed to verify the pH response of PCL/PEG-Nar nanofiber membranes.Scanning electron microscopy,Fourier infrared,mechanics,and contact angle were used to detect nanometer(5)in vitro experiments.The experiments were divided into 5 groups: control group(chondrocytes);OA group(spinal cells + IL-1β);PCL/PEG nanofiber membrane group(chondrocytes + IL-1β).),PCL/PEG/Nar nanofiber membrane group(spinal cell+ IL-1β);PCL/PEG-Nar nanofiber membrane group(experimental group,chondrocyte + IL-1β),and then chondrocytes were implanted into nanofiber After 24 hours,IL-1β was added to induce microorganisms.The biocompatibility of the nanofiber membrane was tested using CCK-8,calpain-AM/PI staining and scanning electron microscopy,and the bone marrow in vitro was explored by q RT-PCR.Cell inflammation(6)In vivo experiments.Osteoarthritis model after anterior cruciate ligament resection in SD rats was used for in vivo animal experiments and animal experiments.Expression of related genes MMP3,MMP13,IL-1,IL-6 and cartilage-related gene COL2a1.There are 6 groups in total: the fiber membrane group.After the model of osteoarthritis was successfully established,the results of the treatment were sampled at the fourth week after implantation of the nanofiber membrane.Gross evaluation of joint tissue.Samples were embedded in sections,fixed with H & E and saffron O-fast green staining and histologically scored to verify the in vivo OA treatment of the PCL/PEG-Nar nanofiber membrane group.Experimental results: In the first part,we successfully produced nano-DDS of PCL/Nar nanofiber membranes,transformed the material characterization,and explored the biocompatibility and flame-retardant treatment effect of nanofiber membranes.In this study,we have successfully produced PCL/Nar nanofiber membranes through electrospinning technology.SEM results show that PCL/Nar nanofiber membranes have a smaller fiber diameter and a 3D structure.PCL/Nar nanofiber membrane has good biocompatibility to capillary cells.With the intervention of IL-1β,PCL/Nar nanofiber membrane can effectively promote the adhesion and proliferation of chondrocytes and exhibit low cytotoxicity.And improve the vitality of cells.The results of CCK-8 and Calcein-AM/PI staining showed that the PCL/Nar(1%)nanofiber membrane showed good ability to promote cell growth and proliferation.PCL/Nar nanofiber membrane has excellent arthritis treatment effect.Compared with PCL nanofiber membrane group,effective chondrocytes of PCL/Nar nanofiber membrane significantly down-regulate antibacterial marker genes MMP3,MMP13,IL-1,IL-6,and up-regulate capillaries.Marker gene COL2a1.In summary,the PCL/Nar nanofiber membrane prepared by electrospinning has good biological properties,is beneficial to the growth of chondrocytes,and has anti-inflammatory effects.Based on the research results in the first part,in the second part,PEG-Nar was successfully synthesized,and the pH-responsive PCL/PEG-Nar nanofiber membrane nano drug-loading system was successfully fabricated using electrostatic spinning technology.First,it was characterized and Explore the effects of OA treatment in vivo and in vitro.First,mPEG-COOH and Nar were synthesized by esterification reaction with PEG-Nar,which was verified by Fourier infrared and ultraviolet absorption spectra.Then,PCL/PEG-Nar nanofiber membrane was successfully synthesized using electrospinning technology.The nanofiber membrane was characterized by drug release,SEM,mechanics,and contact angle experiments.The results showed that the material was stable.The results of in vitro release experiments showed the cumulative release of PCL/PEG-Nar nanofiber membranes at pH = 7.4.It is only 50.28%,and the cumulative release amount is 69.43% at pH = 5.0,which proves that the PCL/PEG-Nar nanofiber membrane releases drugs through the pH response function.The diameter body of PCL/PEG-Nar nanofibers is between 254-885 nm,the average diameter is 503 ± 169 nm,the mechanical properties are Young’s modulus 42.96 ± 6.32 MPa,and the contact angle of the nanofiber membrane is 36.1 ± 2.51 °.Water-based.The results of in vitro experiments show that PCL/PEG-Nar nanofiber membranes are non-toxic and have good biological tolerance.The results of CCK-8,Calcein-AM/PI staining and SEM showed that PCL/PEG-Nar nanofiber membrane added with PEG-Nar at 4wt%showed good biocompatibility,and had a value-added effect on spinal cord cells.In vitro experiments show that PCL/PEG-Nar nanofiber membrane has a good anti-inflammatory effect on capillary cells,and q RT-PCR results show that PCL/PEG-Nar nanofiber membrane and IL-1β-induced plasma model of chondrocyte culture 24 hours per hour Later,the expression of the antibiotic-related gene was down-regulated to about 90%,and the expression of the chest-related gene Col2a1 was up-regulated by 416.67%.PCL/PEG-Nar nanofiber membrane also has good osteoarthritis treatment effect in SD in vivo experiments.In vivo results After the anterior cruciate ligament resection was successfully performed in rats,the nanofiber membrane was implanted into the graft joint cavity.After 4 weeks of internal treatment,PCL/PEG-Nar nanofiber membrane was shown to have a good therapeutic effect on osteoarthritis.The results showed that the PCL /PEG-Nar nanofiber membrane would not trigger normal spinous processes,and the PCL/PEG-Nar nanofiber membrane’s osteoarthritis treatment effect was simply a mixed PCL/PEG/Nar nanofiber membrane for each group.The joints were photographed,scored,histologically stained,and histologically scored.The results showed that the PCL/PEG-Nar nanofiber membrane had the best treatment effect.Experimental evidence: In short,in the first part of this study,we successfully demonstrated the feasibility of making Nar/PCL nanofiber membranes,and successfully demonstrated their good biocompatibility and anti-inflammatory effects.In the second part,a new functional compound nanoparticle drug delivery system was successfully designed.The system not only has a pH stimulus response function,but also can predict the release of a therapeutic dose in the joint cavity according to needs.The characterization of nanofiber membranes was examined from drug release,SEM,in vitro drug release,mechanics,and contact angle experiments.It is proved that nanofiber membrane can slowly release Nar in an acidic microenvironment and has good mechanical properties.By using CCK-8,Calcein-AM/PI staining and SEM experiments,the nanofiber membrane was proved to be non-existent.The q RT-PCR experiment proved that the nanofiber membrane has effective down-regulation of pathological genes MMP3,MMP13,IL-1,and IL-6.Genes also have good biocompatibility and can promote the appreciation of thymocytes.Animal experiment SD rat OA model After 4 weeks of treatment,it was evaluated by gross score,HE staining,safranin 0-fast green.The color results indicate that the nanofiber membrane does not cause an inflammatory response and has a therapeutic effect on osteoarthritis.