Investigation On The Treatment Of Spinal Cord Injury By Using Hydrogel Microneedles/CD-MOF Loaded With Methylprednisolone Sodium Succinate For Transdural And Sustained Release Therapy

【Background】Spinal cord injury(SCI),with high mortality rate,high disability rate and poor prognosis,is a global clinical problem with no effective treatment available.For individuals,SCI is undoubtedly catastrophic and the primary damage is unavoidable while the secondary damage can be effectively controlled.At present,the main research direction of SCI focuses on microglia and astrocytes,and the most commonly used treatment is early surgery decompression combined with drug therapy.Due to the time window limitation of SCI treatment,there are problems such as single route of administration,high incidence of complications and difficulty in maintaining concentration.At present,a series of emerging technologies have provided some options,but effective transmembrane drug delivery and sustained release programs are still to be solved.Combining with the existing technologies,this study designed a hydrogel microneedles(MNs)drug delivery system loaded with cyclodextrin metal-organic frameworks(CD-MOF)for local transmembrane and drug controlled release,and verified its feasibility through in vitro and in vivo experiments,with a view to being applied to the transdural sustained release delivery of drugs after SCI decompression surgery.【Objectives】1.Construct and characterize the physical properties of CD-MOF nanoparticles(CDMOF@MPSS)loaded with methylprednisolone sodium succinate(MPSS).2.Design and verify a multifunctional drug delivery system that can cross the dura mater and release slowly,and synthesize hyaluronic acid methacrylate(HAMA)microneedles(MNs)loaded with CD-MOF@MPSS of suitable size(MNs@CD-MOF@MPSS),verify its mechanical properties,and evaluate the release effect of the drug delivery system on MPSS.3.Verify in vivo and in vitro transmembrane effect,sustained effect and biocompatibility of HAMA hydrogel microneedles loaded with CD-MOF drug delivery system(MNs@CD-MOF@MPSS),and research its therapeutic effect on SCI.【Methods】1.The CD-MOF was synthesized by a hydrothermal method combined with a vapor diffusion method,and CD-MOF@MPSS was prepared by an immersion method.The physical appearance,scanning electron microscope(SEM)images,transmission electron microscope(TEM)images,X-ray diffraction(XRD),Fourier transform infrared spectroscopy(FTIR),ultraviolet spectroscopy(UV),nitrogen adsorption-desorption isotherms and specific surface area,thermogravimetric analysis(TGA),zeta potential,and encapsulation efficiency were used to confirm the successful loading of MPSS onto CDMOF.The biocompatibility of CD-MOF@MPSS was verified by a cell toxicity test.2.Based on the above,a versatile drug delivery system that can cross the dura mater and sustain release was further designed.The solution required for preparing the HAMA hydrogel microneedles was formulated,and HAMA hydrogel microneedles and MNs@CDMOF@MPSS were prepared using a polydimethylsiloxane(PDMS)mold.The mechanical characteristics of MNs@CD-MOF@MPSS were analyzed through SEM,TEM,rheological properties,microneedle mechanical testing,and swelling experiments.At the same time,the sustained release effect of MPSS under different p H values was analyzed to verify the sustained release effect of MNs@CD-MOF@MPSS.3.To verify the transmembrane effect,sustained release effect,and biocompatibility of the MNs@CD-MOF@MPSS dosing system in vivo and in vitro,MNs@CD-MOF@MPSS were combined with the rat skin stratum corneum(simulated dura mater)which was fixed and tightly adhered to the top of the culture medium with the microneedle tip facing outward(non-puncturing)or inward(puncturing).Immunofluorescence(IF)staining and flow cytometry(FCM)were used to determine the transmembrane effect in vitro;MNs@CD-MOF@MPSS and lipopolysaccharide(LPS)treated BV2 cells were cocultured and IF staining and enzyme-linked immunosorbent assay(ELISA)were used to determine the anti-inflammatory effect in vitro.SCI rat model was established and divided into sham operation group,SCI group,SCI+CD-MOF@MPSS group,outward(nonpuncturing)SCI+MNs@CD-MOF@MPSS group and inward(puncturing)SCI+MNs@CD-MOF@MPSS group.In vivo sustained release effect and transmembrane effect of MNs@CD-MOF@MPSS were observed by in vivo imaging;behavior tests were used to analyze the motor function of rats in different groups;hematoxylin-eosin staining(H&E),IF staining,ELISA,and Western blot(WB)were used to analyze the effect of MNs@CDMOF@MPSS on reducing neuroinflammation in vivo.【Results】1.After mixing CD-MOF and MPSS in a mass ratio of 1:1,the encapsulation efficiency of CD-MOF@MPSS was 60.49% at a reaction time of 12 hours.CD-MOF@MPSS obtained under these conditions was subjected to subsequent physical characterization.SEM and TEM showed that CD-MOF had a flake-like structure,a porous surface,and no significant change in shape after loading MPSS.The diameter of CD-MOF and CD-MOF@MPSS was around 100 nm.XRD,FTIR,UV,and zeta potential confirmed the successful loading of MPSS onto CD-MOF.TGA demonstrated that CD-MOF@MPSS had good decomposition resistance,and cell counting kit-8(CCK-8)experiments confirmed the biocompatibility of CD-MOF@MPSS.2.Hydrogel microneedles with appropriate sizes were designed based on the thickness of the rat dura mater,with a needle tip spacing of 470 μm,a bottom diameter of 170 μm,and a height of 300 μm.Rheological characterization showed that HAMA hydrogel microneedles maintained good stability after loading CD-MOF@MPSS.Swelling tests demonstrated that HAMA hydrogel microneedles could swell and dissolve slowly.Mechanical tests showed that MNs@CD-MOF@MPSS had sufficient mechanical strength to puncture the dura mater.The sustained release curve showed that the release amount of MPSS from CDMOF@MPSS or MNs@CD-MOF@MPSS was higher at lower p H and decreased with the increase of p H.Moreover,MNs@CD-MOF@MPSS had a release time of over 4 days,and the total release amount exceeded 80%.Therefore,the versatile drug delivery system MNs@CD-MOF@MPSS was successfully constructed.3.To further verify the biological effects of MNs@CD-MOF@MPSS,in vitro experiments showed that the microneedle tip facing outward(non-puncturing)MNs@CD-MOF@MPSS was almost completely removed during the first wash,while the microneedle tip facing inward(puncturing)MNs@CD-MOF@MPSS adhered to the stratum corneum,quickly swelled and dissolved slowly,suggesting that MNs could effectively penetrate the spinal cord,thus avoiding the absorption of MPSS by surrounding tissues and achieving targeted drug delivery.FCM and IF results showed that inward MNs@CD-MOF@MPSS could effectively cross the membrane in vitro,and its antiinflammatory effect was mainly aimed at M1 phenotype of microglia cells and could effectively inhibit the nucleic acid binding oligomerization domain pattern receptor protein3(NLRP3)inflammasome.in vivo imaging verified that loading with HAMA hydrogel microneedles delayed the release of CD-MOF@MPSS,and the inward(puncturing)group with MNs@CD-MOF@MPSS could effectively improve the motor dysfunction after SCI.【Results】The ratio of microglial cells M2/M1 of rats after MNs@CD-MOF@MPSS treatment was significantly increased,suggesting that the drug delivery system played an antiinflammatory effect,and WB and ELISA results showed that it could effectively inhibit downstream inflammatory factors.H&E and IF staining at day 28 suggested that MNs@CDMOF@MPSS could effectively inhibit astrocyte scar formation in the later stage,thus creating conditions for SCI recovery.【Conclusions】This study successfully synthesized CD-MOF nanoparticles and loaded them with MPSS to form CD-MOF@MPSS.A multifunctional drug delivery system,MNs@CDMOF@MPSS,was further constructed by designing HAMA hydrogel microneedles suitable for the size of the rat injury site dura mater.The changes in nanoparticle size before and after drug loading and microneedles size were analyzed by SEM and TEM.The ability of CDMOF to effectively load MPSS was verified by XRD,FTIR,UV,nitrogen adsorptiondesorption curve,specific surface area,TGA,and Zeta potential.Cell toxicity experiments showed that CD-MOF@MPSS had good biocompatibility.Rheological tests,swelling experiments,and mechanical testing further demonstrated that the designed HAMA hydrogel microneedles had good mechanical effects.The designed multifunctional drug delivery system could dissolve slowly in phosphate buffer solution(PBS)and provided a theoretical basis for future applications.The MPSS sustained release curve suggested that HAMA hydrogel microneedles mediated CD-MOF@MPSS could prolong the release time and effectively improve the utilization rate of MPSS.In vitro results showed that MNs@CDMOF@MPSS could effectively penetrate the rat skin stratum corneum,providing a basis for subsequent transdural delivery in vivo.Its specific mechanism was to improve the inflammatory microenvironment after SCI by inhibiting the pro-inflammatory response of BV2 cells treated with LPS,down-regulating NLRP3-positive inflammasome and cascade pro-inflammatory cytokines.In vivo experiments were consistent with in vitro experiments,and in vivo imaging experiments showed that MNs@CD-MOF@MPSS could effectively penetrate the dura mater and continuously release CD-MOF@MPSS to achieve transdural sustained release,effectively utilizing MPSS.Behavioral assessments further confirmed its ability to effectively improve motor dysfunction after SCI,and improve the Basso,Beattie& Bresnahan(BBB)score of SCI rats.Its specific mechanism relied mainly on inhibiting the polarization of M1 phenotype,down-regulating pro-inflammatory cytokines,and inhibiting the formation of neural glial scars caused by GFAP-positive astrocytes.Therefore,we believe that the HAMA hydrogel microneedles-loaded CD-MOF multifunctional drug delivery system(MNs@CD-MOF@MPSS)can achieve transdural sustained release of MPSS to treat SCI and may have broad prospects in the clinical application of spinal cord diseases.