Construction Of Nanoparticles-in-microspheres Composite Carriers And Their Study In Rheumatoid Arthritis Therapy

Rheumatoid arthritis(RA)is a chronic autoimmune disease that seriously affects the life quality of patients,and even causes disability or death.Although its etiology remains obscure,inflammatory cells,inflammatory cytokines,and cell signaling pathways are considered as important aspects leading to RA.In the RA pathogenesis,a large number of inflammatory cells including macrophages infiltrate synovium and promote the release of pro-inflammatory cytokines.Meanwhile,multiple intracellular signaling pathways are involved in cytokines signal transduction,and accelerate the process of RA.The complicated pathogenesis of RA leads to numerous targets,and it is difficult for single-drug therapy to achieve ideal efficacy.Combination therapy can act on different targets of RA to produce better therapeutic effect than single-drug therapy.Myeloid leukemia gene-1(Mcl-1)siRNA can specifically silence the expression of Mcl-1 gene,induce macrophage apoptosis to inhibit the process of RA.Tofacitinib(Tof)blocks the signal transduction of many inflammatory cytokines by inhibiting the Janus kinase/signal transducer and activator of transcription signaling pathway.In this study,we combined Mcl-1 siRNA and Tof to improve the efficacy to act on inflammatory macrophages and signaling pathways in the pathological process of RA to improve the efficacy.However,there are some obstacles to deliver siRNA and Tof effectively.First,siRNA is highly hydrophilic and polyanionic,which makes it difficult to enter the cell interior.Second,siRNA is susceptible to degradation by serum nucleases.Meanwhile,Tof is absorbed and eliminated rapidly in vivo,which leads to frequent use of them,and thus reducing medication compliance.Finally,the non-specific distribution of siRNA and Tof lead to reduced efficacy and increased systemic side effects.Therefore,the development of a reasonable drug delivery system is essential for the treatment of RA.Nano-carriers can carry drugs to the inflammatory site passively,enhance efficacy and reduce side effects,but they often lead to faster drug release behavior;polymer microspheres as long-acting sustained-release carriers can reduce the appearance of drug peaks and troughs in the blood and improve the compliance of patients,but they are not targeted.In this study,we combined nano-carriers with micro-carriers to develop a compound-scale preparation that has both the targeting function of nanoparticles and the sustained release function of microspheres.According to the different characteristics of siRNA and Tof,we designed corresponding nanocarriers.In addition,PLGA and PCADK were used as microsphere carriers to encapsulate the two kinds of nanoparticles or a combination of both,forming a nano-micro composite drug delivery system.The composite carrier was evaluted in vivo and in vitro.This research is divided into the following three parts:1.Construction and evaluation of siRNA loaded nanoparticles-in-microspheresIn this part,cationic chitosan(CS)nanoparticles were prepared to encapsulate negatively charged siRNA.According to the characteristic that HA can bind to CD44 receptor overexpressed on the macrophages surface,HCNPs-siRNA was formed by modifying HA on the surface of CS nanoparticles.Taking the particle size as a screening index,the amount of HA,CS and the cross-linking agent ammonium tripolyphosphate(TPP)was adjusted to obtain the optimal prescription of nanoparticles.The optimized nanoparticles had a particle size of 113.6 ± 2.2 nm,a PDI of 0.154 ±0.013,and an encapsulation efficiency of 90.02 ± 3.02%.Agarose gel electrophoresis showed that HCNPs can protect siRNA against degradation for more than 24 h.Cell uptake and cytotoxicity experiments showed that HCNPs-siRNA had enhanced celluptake and cell proliferation inhibition to LPS-activated macrophages through HAmediated endocytosis.Subsequently,we used PLGA and PCADK as polymer carrier matrix to encapsulate HCNPs-siRNA for preparing nanoparticles-in-microspheres drug delivery system(Ni MPs-siRNA).Meanwhile,conventional microspheres loaded with siRNA(CMPs-siRNA)were prepared as control group.The particle size of Ni MPs-siRNA was17.63 ± 0.68 ?m,and the encapsulation efficiency was 87.09 ± 3.91%.Compared with CMPs,siRNA was dispersed inside Ni MPs more uniformly detected by laser scanning confocal microscopy.In vitro release experiments showed that,compared with CMPs,Ni MPs reduced siRNA burst release.Cytotoxicity experiments proved that the released supernatant from Ni MPs exhibited a strong cytotoxic effect.Western blot results showed that Ni MPs released supernatant reduced the expression of Mcl-1 protein in macrophages,which proved that the biological activity of siRNA could be protected by Ni MPs-siRNA.The pharmacokinetic experiments were carried out in SD rats,compared with HCNPs-siRNA group,the blood drug concentration of Ni MPs-siRNA group was more stable.The bio-distribution experiment indicated that Ni MPs increased siRNA accumulation in inflamed joints.In vivo pharmacodynamic experiments proved that Ni MPs-siRNA alleviated inflammation and inhibited the process of RA.2.Construction and evaluation of siRNA loaded nanoparticles-in-microspheresIn this part,the acid-sensitive carrier polyketal(PCADK)was selected to encapsulate Tof for preparing nanoparticles(LPNPs-Tof).Egg PC was added to adjust the particle size,and CS was added to protect the nanoparticles.Taking the particle size and drug loading as screening indexs,the optimal prescription of nanoparticles was obtained.The optimized nanoparticles were spherical and uniformly dispersed.The particle size and drug loading were 153.0 ± 2.2 nm and 8.21 ± 0.24%,respectively.In vitro release experiments indicated that LPNPs-Tof was acid-sensitive.Subsequently,LPNPs-Tof were encapsulated into PLGA and PCADK to prepare nanoparticles-in-microspheres composite drug delivery system(Ni MPs-Tof).Meanwhile,conventional microspheres loaded with Tof(CMPs-Tof)was prepared as a control.Ni MPs-Tof had a particle size of 23.1 ± 0.77 ?m and a drug loading of 1.36± 0.16%.In vitro release results showed Ni MPs-Tof had a reduced burst compared to CMPs-Tof,and exhibited a sustained-release behavior for 16 days.SEM observation proved the integrity of the nanoparticles released from Ni MPs.In vivo distribution studies showed that Ni MPs-Tof increased accumulation in inflammation sites.Pharmacodynamic experiments in AIA rats proved that Ni MPs-siRNA reduced inflammatory cells and the expression of pro-inflammatory cytokines,thereby inhibiting the process of RA.3.Construction and evaluation of nanoparticles-in-microspheres co-loaded with siRNA and TofIn this part,two nanoparticles HCNPs-siRNA and LPNPs-Tof were coencapsulated into PLGA and PCADK to form nanoparticles-in-microspheres: Ni MPs(siRNA+Tof).Meanwhile,conventional microspheres loaded with siRNA and Tof was prepared as a control,named as CMPs(siRNA+Tof).The siRNA encapsulation efficiency in Ni MPs(siRNA+Tof)was 92.70 ± 3.62%,and the drug loading of Tof was1.32 ± 0.17%.The in vitro release results showed that,compared with CMPs(siRNA+Tof),Ni MPs(siRNA+Tof)reduced the burst release of siRNA and Tof,and prolonged siRNA release.Pharmacodynamic results showed that Ni MPs(siRNA+Tof)exhibited a more obvious inhibitory effect on RA rats,compared with single-loaded Ni MPssiRNA or Ni MPs-Tof,which confirmed the synergistic effect of siRNA and Tof.In addition,compared with nanoparticles and conventional microspheres co-loaded with siRNA and Tof,Ni MPs(siRNA+Tof)also showed a stronger inhibitory effect on RA,demonstrated the superiority of the composite microspheres over conventional microspheres as well as nanoparticles.Results of H&E staining of viscera showed that Ni MPs(siRNA+Tof)is safe,and after its treatment,the organ damage caused by RA was restored.In conclusion,Ni MPs(siRNA+Tof)combined the dual advantages of nanoparticles and microspheres,which not only reduced the frequency of administration,but also achieved effective drug delivery.It is a promising delivery system for the treatment of RA.Meanwhile,the synergistic effect of siRNA and Tof enhanced the therapeutic effect.This study presents a new idea for combination therapy of RA.