Study Of Hyaluronic Acid Coated PH Sensitive Co-delivery Nanoparticles For Adjuvant-induced Arthritis Therapy

Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease,characterized swelling,pain,and stiffness in joints,which seriously affects the life quality of RA patients.Myeloid leukemia-1(MCL-1)siRNA can inhibit the progression of RA by inhibiting the expression of MCL-1 in macrophages without toxicity,but it does not rapidly eliminate inflammation and pain.Dexamethasone(Dex),a classic arthritis-inhibiting drug,could quickly relieve pain and inflammation,but its high and frequent dosage caused systemic side effects.Therefore,we hypothesize that the combination of MCL-1 siRNA and Dex can increase the efficacy of treating RA while reducing systemic side effects.Many cells are involved in RA,and activated macrophages could release pro-inflammatory cytokines which significantly contribute to inflammation and joint destruction.Hyaluronic acid(HA)could be used as a targeting moiety of drug delivery systems for RA therapy because it could bind to CD44 overexpressed by activated macrophages.Polyketal,a new type of acid-sensitive polymer,which degrades slowly under neutral or alkaline conditions,and can be rapidly degraded into neutral small molecules in an acidic environment,and thus can be used as an excellent carrier for drug delivery in inflammatory diseases.In this paper,the nanoparticles were prepared from PCADK or PK3 with externally modified hyaluronic acid as a targeting group,encapsulating Dex,siRNA or a combination of both.And the in vivo efficacy of nanoparticles was then evaluated on an adjuvant-induced arthritis rat model.This experiment is divided into the following three parts: 1.Preparation and evaluation of PCADK-based nanoparticles loaded with Dex and modified with HA(HAPNPs/Dex)In this part,nanoparticles were prepared with PCADK,polyethyleneimine(PEI),egg yolk lecithin(egg PC)and modified with HA.The amount of PEI,egg PC and HA were the conditions for formulation optimization,and PCADK 20 mg,PEI 1 mg,egg PC 8 mg,0.1% HA 500 ?L were selected.The particle size,zeta potential and drug loading of optimized nanoparticles were 150.5 ± 2.47 nm,-2.84 ± 0.53 mV and 7.26±0.71%,respectively.Cell uptake and cytotoxicity experiments indicate that HAPNPs/Dex are capable of specifically targeting activated macrophages and could kill activated macrophages without a significant effect on the proliferation of non-activated macrophages.The in vitro release results showed that the release rate of HAPNPs/Dex under acidic conditions was significantly faster than under neutral conditions,indicating that HAPNPs/Dex were acid-sensitive.The in vivo pharmacodynamic experiment showed HAPNPs/Dex could effectively reduce inflammatory cells,impair cartilage destruction,reduce the expression of pro-inflammatory cytokines and inhibit the progression of arthritis.2.Preparation and evaluation of PK3-based nanoparticles loaded with MCL-1 siRNA and modified with HA(HAPKNPs/siRNA)In this part,we first established an in vitro assay method for MCL-1 siRNA,then labeled MCL-1 siRNA with Cy5,and used a fluorescence spectrophotometer to detect the standard curve of Cy5-siRNA in blank nanoparticle solution.The results showed a good linear relationship between Cy5-siRNA's fluorescence intensity and the concentration of Cy5-siRNA.Nanoparticles were prepared with PK3,hydrogenated soybean phospholipids(DOTAP),egg PC and modified with HA.The amount of DOTAP-siRNA,egg PC and HA were the conditions for formulation optimization,and PK3 20 mg,DOTAP-siRNA 1.5 nM,egg PC 6 mg,0.1% HA 200 ?L were selected.The particles size,zeta potential and drug loading of HAPKNPs/siRNA were 115.56 ± 5.44 nm,11.38 ± 0.98 mV and 5.43 ± 0.003%,respectively.Cytotoxicity experiments indicate that HAPKNPs/siRNA are capable of promoting apoptosis of activated macrophages.And the in vitro release experiments showed that HAPKNPs/siRNA released faster in the acidic environment than in the neutral conditions,indicating that HAPKNPs/siRNA were acid-sensitive.The results in vivo on arthritic rat model indicated that HAPKNPs/siRNA could reduce some inflammatory cells and inhibit the progression of arthritis.3.Preparation and evaluation of PK3-based nanoparticles modified with HA and co-loaded with MCL-1 siRNA and Dex(HAPKNPs/Dex+siRNA)In this part,we prepared co-loaded MCL-1 siRNA and Dex nanoparticles using the method in part 2.The particle size and zeta potential of HAPKNPs/Dex+siRNA were 117.07 ± 2.21 nm and 15.53 ± 1.06 mV,respectively.The drug loading and encapsulation efficiency of MCL-1 siRNA in HAPKNPs/Dex+siRNA was 4.34 ± 0.026% and 93.33 ± 5.51%,and the drug loading and encapsulation efficiency of Dex were 5.03 ± 0.13% and 40.55 ± 2.32%.The in vitro release results indicated that HAPKNPs/Dex+siRNA were acid-sensitive.The results of cytotoxicity experiments showed that HAPKNPs/Dex+siRNA had a higher cytotoxicity on activated macrophages compared with the two single-loaded nanoparticles.The results of pharmacodynamic experiments in animals showed that HAPKNPs/Dex+siRNA have a stronger inhibitory effect on RA,compared with the two single-loaded nanoparticles.We next examined the safety of HAPKNPs/Dex+siRNA in cells and rats.The cell viability of 3T3,C2C12 and PC12 were more than 90% after incubation with HAPKNPs/Dex+siRNA for 24 h.And the HAPKNPs/Dex+siRNA were not toxic to heart,liver,spleen,lung and kidney in rats.In summary,acid-sensitive nanoparticles modified with HA could effectively deliver drugs for the treatment of RA.And the combination of MCL-1 siRNA and Dex could better inhibit the progression of RA.This provides a new idea for the targeted therapy and drug combination therapy of RA in the future.