| Objectives: Type I diabetes mellitus(T1DM)is a chronic autoimmune disease with an unknown pathogenesis.In T1 DM,damage to pancreatic β cells may result in an absolute lack of insulin secretion.A large number of studies have shown that the combination of oxidative stress and inflammatory cytokines lead to serious damage to pancreatic cells,resulting in hyperglycemia to exacerbate.Currently,the most common strategy for T1 DM treatment is directly injecting exogenous insulin to regulate hyperglycemia levels.However,multiplie injections lead to poor patient compliance,and injection may occur after injection.The novel therapies,including pancreatic transplantation,and islet cell transplantation,can be used to provide insulin.However,the lack of donors,and immune rejection limit the clinical application.Therefore,it is of great importance to develop new strategies to address these problems.The aim of this study is to regulate blood glucose levels by the combination of insulin loaded microneedles(INS-MN)and curcumin nancocrystals(CUR-NCs).The INS-MN directly delivery INS to the system through transdermal administration,while CUR-NCs are administered orally as a model drug for assistance treatment of T1 DM.Finally,curcumin is used to assist INS treatment to improve its effect and explore whether it can alleviate or even stop the progression of T1 DM.Methods: In this study,methacrylic anhydride(MA)was used to modified gelatin(Gel)to form gelatin-methacrylic anhydride(Gel-MA).The success of the modified was characterized by UV cross-linking and hydrogen nuclear magnetic resonance spectroscopy(HNMR).Gel-MA was used to prepare INS-MN by micromolding method,and assess the influence of centrifugal temperature,different backing layer materials,Gel-MA concentration,drying temperature and other conditions on the microneedle formability and appearance during the preparation.Visual method,optical microscope,scanning electron microscope for morphological examination of the microneedles;the mechanical strength is evaluated by measuring the force required for the microneedle to produce a similar displacement under different UV irradiation time.The penetration of the microneedle into the skin and the depth of penetration were evaluated by the skin recovery test,trypan blue staining and H&E staining.The distribution of FITC-labeled INS in the microneedle tips was examined using a laser confocal microscope.In order to alleviate pancreatic injury and exert a better blood glucose stabilization effect,this study further prepared CUR-NCs.Poloxamer 188(F68)was used as a stabilizer to prepare CUR-NCs.,and some of the factors affecting CUR-NCs preparation were investigated,including surfactant type,probe-sonication amplitude and time,and the mass ratio of F68 to curcumin.The particle size,polydispersity index(PDI)and zeta potential of CUR-NCs were measured by Malvern particle sizer with DLS measurements.Their apparent morphology and dispersion were determined by TEM,the drug content was detected by UV-Vis spectrophotometry,the crystal form and structure change of CUR-NCs were evaluated by X-ray diffractometer and FTIR respectively.The cytotoxicity of CUR-NCs and INS-MN were detected in the concentration range of0-20 μg/m L by CCK-8 method.At the end,a T1 DM mouse model induced by streptozotocin(STZ)was used to assess the effect of CUR-NCs and INS-MN combinations on fasting blood glucose,body weight,and clinical symptoms over a40-day period.The glucose tolerance test was performed by intraperitoneal injection after 35 days of treatment to assess the impact of treatment on peritoneal glucose tolerance in mice.H&E staining was used to assess the histopathological transition,and the drug’s safety was assessed after the treatment was completed.Results: In this study,methacrylic anhydride was successfully modified onto gelatin.Under UV light(365 nm)irradiation,the crossing effect was obvious,and the modification rate was up to 63% calculated by the HNMR.Under direct observation and optical microscope,the obtained INS-MN was found to be cone-shaped,neatly arranged,and consistent with the mold.It had good molding,smooth backing layer,and no bubble,breakage,etc.Observed by scanning electron microscope,the morphology of the microneedles is consistent with the results of the optical microscope.The surface of the needles forms a rough network structure,which indirectly indicates the successful grafting of methacrylic anhydride.The mechanical properties experiments show that the microneedles are formed more densely as the irradiation time increases.It will improve the mechanical strength of the microneedle.The results of skin recovery test,trypan blue staining and H&E staining showed that the formed INS-MN could be successfully and effectively inserted into the skin with almost no damage to the skin with the insertion depth of 300 μm.The results of laser confocal microscope showed that FITC-labeled insulin was distributed throughout the microneedle.The distribution of FITC-labeled insulin at 500 μm in the front microneedle was more uniform,which can reduce the drug waste compared to its distribution in the entire microneedle.CUR-NCs was successfully prepared in this study which is an orange-yellow suspension.It had a particle size was 219.83±14.94 nm and a PDI of 0.23±0.02 in DSL studies.Under the transmission electron microscope,a rod-like structure of relatively uniform size with diameter of247.86±63.28 nm,no adhesion,and better dispersion was observed.The result determined by UV-Vis spectrophotometry showed that the actual content of curcumin in CUR-NCs was 46.55±0.68%.XRD results show that there are curcumin crystal peaks at the diffraction angles of 14.121,25.774,and 26.788,respectively.The results detected by FTIR showed that compared the active pharmaceutical ingredients of curcumin to their physical mixture;there were characteristic peaks carbon-carbon double bond and carbon-oxygen double bond of curcumin in CUR-NCs,demonstrating that the chemical structure of curcumin has not been changed.The cytotoxicity results showed that the cell survival rates of CUR-NCs and INS-MN were higher than 85.5% and 88.70% respectively in the concentration range of 0-20μg/m L,demonstrating that both have good safety.T1 DM mouse model was successfully established induced by STZ multiple times.The group receiving CUR-NCs and INS-MN had the strongest therapeutic effect after 40 days of continuous administration.The blood glucose levels were improved,while their body weight increased.For the other treatment groups,the blood glucose of the mice was also decreased to varying degrees,and after the intra-peritoneal injection of glucose,the blood glucose of the mice in the treatment groups was lowered more quickly.The results of H&E staining showed that,except for the liver,the other tissues of mice in each group had no obvious damage,while in the treatment group,especially the curcumin and insulin combined treatment group,it only had little liver damage response.Conclusion: This research successfully prepared CUR-NCs and INS-MN based on the experimental findings.Its combinations had strong in vivo therapeutic effects in the STZ-induced T1 DM mouse model,which could lead to new T1DM treatment designs and approaches. |
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