Preparation And Properties Of Glycyrrhizic Acid Modified Poria Cocos Polysaccharide Carbon Dots Loaded Methotrexate Soluble Microneedles

Rheumatoid Arthritis（RA）is an autoimmune disease characterized by erosive arthritis.MTX is one of the most frequently recommended anti-rheumatic drugs.It can treat RA alone or in combination with other drugs,and the combination of two or more anti-RA drugs can improve the treatment efficiency compared with the use of a single drug.GA is an active ingredient with anti-inflammatory effect in traditional Chinese medicine licorice.It can be used as a multifunctional drug carrier to enhance the activity of drugs,and can improve the water solubility of MTX and increase the loading of MTX in the system.Therefore,it can be combined with MTX.CDs,as a new nanocarrier,can combine two or more drugs,so that the drugs have the physical properties and fluorescence properties of the carrier CDs.Moreover,according to the fluorescence characteristics of CDs,the process of drugs in vivo can be tracked to improve the bioavailability of drugs,increase cycle time,reduce toxicity and control drug delivery.On the other hand,the most commonly used method of MTX in clinical practice is oral,but this can cause great side effects.MNs are a new type of transdermal drug delivery technology,which is safe,effective and painless.It can replace traditional oral administration by transdermal administration.Therefore,using CDs as nanocarriers,GA and MTX are combined to construct a nano-drug delivery system,and MNs dosage form is made to achieve transdermal administration,which provides a new feasible solution for the treatment of RA.Objective:In this study,PCP-CDs were synthesized by hydrothermal method.The anti-inflammatory drug GA was modified to CDs by amide reaction,and then the anti-RA drug MTX was loaded by intermolecular force to prepare a nano-drug delivery system with fluorescence and double anti-inflammatory effects,high biocompatibility and low toxicity.Finally,HA was used as the matrix material to prepare soluble microneedles for transdermal administration to treat RA.Methods:（1）CDs derived from poria cocos polysaccharide were synthesized by hydrothermal method.GA-CDs were prepared by amide reaction between the active-NH₂ on the surface of CDs and the-COOH on GA.The GA-CDs system was characterized and evaluated by transmission electron microscopy,ultraviolet-visible spectroscopy,fluorescence spectroscopy,infrared spectroscopy,differential scanning calorimetry and nuclear magnetic resonance hydrogen spectroscopy.（2）To establish a HPLC method for the detection and analysis of MTX in vitro.（3）GA-CDs@MTX nano drug delivery system was prepared by loading anti-RA drug MTX with GA-CDs through intermolecular forces.It was characterized and evaluated by transmission electron microscopy,ultraviolet-visible spectroscopy,fluorescence spectroscopy,infrared spectroscopy,differential scanning calorimeter and nuclear magnetic resonance spectroscopy,and the in vitro release characteristics of nanoparticles were investigated.（4）LPS was used to induce RA-related cells-RAW264.7 cells to construct a cellular inflammatory model,and FAC was used to induce rats to construct a RA rat model.The anti-inflammatory effect of GA-CDs@MTX nano-drug delivery system was investigated by animal experiments and cell experiments.（5）Using HA as matrix material,GA-CDs@MTX nanoparticles were prepared into soluble microneedle formulations,and their morphology and characteristics of puncture skin were investigated.The anti-inflammatory effect of the prepared microneedles was evaluated by FAC-RA rats.Results:（1）Transmission electron microscopy results showed that CDs and GA-CDs were spherical monodisperse particles with uniform size.The average particle diameter of CDs was 7.20±2.25 nm,and the average particle diameter of GA-CDs was 11.40±3.41nm.The results of ultraviolet spectroscopy,infrared spectroscopy,fluorescence spectroscopy,differential scanning calorimetry and nuclear magnetic resonance showed that GA was successfully modified on CDs,and the grafting rate of GA was 50.48±0.56%.The results of cell experiments showed that the prepared CDs and GA-CDs had low cytotoxicity.（2）After investigation,the standard curve of HPLC method for MTX in vitro detection had good linear relationship and specificity.The RSD values of intra-day and inter-day precision were less than 3.5.The recovery rate was in the range of 98.37-101.97%.The relative standard deviation of the corresponding peak area within 7 days of storage at 4°C was 2.56%.The precision,stability and recovery rate were good,which met the requirements of in vitro analysis.（3）The results of ultraviolet-visible spectroscopy,infrared spectroscopy,fluorescence spectroscopy and nuclear magnetic resonance spectroscopy showed that MTX was successfully loaded onto GA-CDs.After optimization,the nanosystem had high drug loading capacity（39.48±0.79%）and good in vitro release behavior.The final cumulative release rate was 96.22±1.47%in the release medium with p H 5.0,and the final cumulative release rate was 61.55±2.55%in the release medium with p H 7.4.Compared with MTX group,GA-CDs@MTX group showed stronger inhibitory effect on LPS-induced inflammatory model cells（*P<0.05）,and significantly reduced the level of pro-inflammatory cytokines secreted by cells.The results of in vivo animal experiments showed that the toe swelling rate,toe score and plasma pro-inflammatory cytokine levels in the GA-CDs@MTX group were lower than those in the MTX group（*P<0.05）.（5）After the GA-CDs@MTX nanosystem was prepared into microneedles,the drug loading was 70.05±0.72μg.The pyramid-shaped microneedles successfully penetrated the skin to achieve transdermal drug delivery,and the transdermal release effect was good.The cumulative release rate of the drug within30 min was as high as 60.53±2.84%,and the final cumulative release rate was about96.25±2.79%.Compared with the oral MTX group and the oral GA-CDs@MTX group,the efficacy of GA-CDs@MTX and MTX in RA rats was significantly improved after the preparation of microinjection formulations.The toe swelling rate,toe score and plasma pro-inflammatory cytokine levels in the GA-CDs@MTX MNs group were lower than those in the MTX MNs group（*P<0.05）.Conclusion:In this study,GA-CDs@MTX nano-drug delivery system with fluorescence and dual anti-inflammatory effects was successfully prepared.The nanoparticles have good water solubility,small particle size and stable imaging in cells.The results of in vivo animal experiments and in vitro cell experiments showed that the GA-CDs@MTX nano-drug delivery system was more effective than MTX.After the synthesized nanoparticles were prepared into microinjections,they could penetrate the skin and achieve transdermal administration.Using FAC-induced RA rats as a model,the efficacy of GA-CDs@MTX microneedles was stronger than that of oral MTX and MTX microneedles.After transdermal administration of GA-CDs@MTX MNs,GA and MTX worked together to reduce the inflammatory response of RA and alleviate the foot swelling of rats.