Construction Of Sinomenine-loaded Targeted And PH-responsive Nanoparticles And Their Study In Rheumatoid Arthritis Therapy

The main symptoms of rheumatoid arthritis(RA)are joint inflammation and pain,which brings great inconvenience to the life and work of patients.Sinomenine(SIN)has been used more and more frequently in the treatment of RA in recent years.It has the pharmacological effects of both non-steroidal anti-inflammatory drugs and antirheumatic drugs,which can reduce pain and inflammation,slow down the disease process through immunosuppression.However,SIN is not selective,its systemic distribution reduces the therapeutic effect of SIN for RA therapy and the bioavailability of the drug,meanwhile increases the toxicity to non-lesional sites.In addition,SIN is sensitive to light,heat,and alkali,and the instability of its easy decomposition limits its application.Therefore,the development of a nano-delivery system that can safely and effectively deliver SIN to the RA site to enhance its local drug concentration is crucial for the treatment of RA.Lipid polymer hybrid nanoparticles have good biocompatibility,biostability and high drug-carrying capacity.Macrophages play an important role in the pathophysiological response of RA.Activated macrophages overexpressed CD44 receptor on the surface,which can be used as an effective target for the treatment of RA.Hyaluronic acid(HA)can specifically bind to the CD44 receptor,and the surfacemodified HA nanoparticles can be actively delivered to macrophages at the site of RA inflammation.In this paper,sinomenine-loaded p H-responsive nanoparticles(SIN-PPNPs)were prepared with poly(D,L-lactic-co-glycolic acid)(PLGA),poly(cyclohexane-1,4-diyl acetone dimethylene ketal)(PCADK)and egg phosphatidylcholine(egg PC),which had acid-sensitive properties to control drug release and improved the instability of SIN.Afterwards,(2,3-Dioleoyloxy-propyl)-trimethylammonium(DOTAP)was added to electrostatically adsorb the negatively charged HA,so that the final obtained sinomenine-loaded HA-targeted p H-responsive nanoparticles(SIN-PPNPs-HA)had the ability of active delivery to activated macrophages,which could improve instability and in vivo distribution of SIN,and increase the effective drug concentration of SIN at RA lesions for the treatment of RA.The specific content includes the following parts:1.Establishment of in vitro analysis method of SINIn this chapter,a high-performance liquid chromatography analysis method for the in vitro detection of SIN was established.After detection,the maximum UV absorption peak of SIN was at 261 nm.It has been verified that the intra-day and inter-day relative standard deviations and recoveries of SIN meet the standards.It has been verified that the method has good specificity,precision and accuracy.The linear relationship of the standard curve of SIN between 1.00 and 500.00 μg/m L concentration was stable and good,in line with in vitro analysis standards.2.Preparation,characterization and in vitro evaluation of sinomenine-loaded HAtargeted and p H-responsive nanoparticlesSIN-PPNPs and SIN-PPNPs-HA were prepared by an improved emulsification solvent evaporation method,and the formulations were screened by particle size,polydispersity index,zeta potential,encapsulation efficiency and drug loading.The particle sizes of SIN-PPNPs and SIN-PPNPs-HA under the obtained optimal formulations were 117.3 ± 2.7 nm and 161.2 ± 3.7 nm,the polydispersity coefficients were 0.113 ± 0.023 and 0.216 ± 0.011,and the zeta potentials were-4.14 ± 0.07 m V and-6.56 ± 1.11 m V,the encapsulation efficiencies were 91.01 ± 0.27% and 88.10 ±0.07%,and the drug loadings were 11.60 ± 0.33% and 10.22 ± 0.33%,respectively.The morphology of SIN-PPNPs and SIN-PPNPs-HA was smooth and round without adhesion.The stability results showed that SIN-PPNPs and SIN-PPNPs-HA could exist stably for 48 h during storage,indoor placement and in vivo delivery.The release results showed that the acidic environment would accelerate the release of SIN from SINPPNPs and SIN-PPNPs-HA,and the cumulative release of SIN reached about 93% at48 h.3.Biological effects evaluation of sinomenine-loaded HA-targeted and p Hresponsive nanoparticlesCell viability experiments showed that blank nanoparticles with a concentration below 1000 μg/m L were relatively safe,and the survival rate of activated macrophages incubated with 200 μg/m L SIN-PPNPs-HA was 38.65%,which was significantly lower than that of SIN(***p < 0.001)and SIN-PPNPs(##p < 0.01).The results of cellular uptake efficiency experiments showed that the uptake of SIN-PPNPs-HA by activated macrophages was more than 1.5 times that of SIN-PPNPs,and it was verified that this better uptake was mediated by HA through HA competition experiments.The results of in vitro tissue imaging showed that the carrier PPNPs-HA could increase the distribution of the drug in vivo and enhance the aggregation of the drug at the RA site.The results of pharmacodynamic experiments in rats showed that the average clinical score of RA in the SIN-PPNPs-HA group was 1.33,which was significantly lower than that in the SIN group(***p < 0.001).The average paw thickness was about 7.22 mm,the swelling degree was lighter than the SIN group(9.05 mm).And the pathological changes of the joint tissue also proved that SIN-PPNPs-HA could more effectively relieve RA in rats.In addition,the plasma levels of TNF-α,IL-6,and IL-1β in the SINPPNPs-HA group decreased while the IL-10 level increased(**p < 0.01),indicating that SIN-PPNPs-HA effectively reduced the degree of inflammation in RA.In conclusion,in order to increase the effective concentration of SIN at the RA site and improve its in vivo stability and bioavailability,the HA-targeted p H-responsive nanoparticles were designed and prepared for SIN delivery.It could improve the systemic distribution of SIN,increase its accumulation in RA joints,and have a bright future in the treatment of RA.