| Rheumatoid arthritis(RA)is a systemic autoimmune disease,patients with RA would suffer great pain and lower quality of life if they do not receive timely and effective treatment.Methotrexate(MTX),the first line antirheumatic drug,has been applied to clinical practice for several decades.However,long-tearm and high-dose usage always cause severe tolerance and toxic side effects.Therefore,how to deliver MTX to affected joints selectively become an urgent problem to be solved.The activated macrophages are abundant in affected joints,which can secrete a great deal of inflammatory factors and thus accentuate further inflammation.Folate receptor ?(FR?)is always overexpressed on the surface of activated macrophages.And therefore,FR? becomes a promising target for RA therapies.Lipid polymer hybrid nanoparticles(LPNs)as novel nanocarriers combine the advantages of polymeric nanoparticles and liposomes.LPNs could accumulate at affected joints due to extravasation through leaky vasculature and subsequent inflammatory cell-mediated sequestration(ELVIS).Thus,LPNs which could target to FR? overexpressed on activated macrophages could be a promising candidate for RA treatment.The properties of nanoparticles mainly depend on their component material.Polyketal is a kind of novel hydrophobic polymer,it can degrade into neutral products and it possess favorable acid sensitivity.Polyketal-based nanoparticles can be stable in neutral environment of bloodstream.And once in acid environment of endosome through uptake by cells,polyketal-based nanoparticles will degrade and release their payload.Our main work could be summarized as the following four parts:1.Synthesis and characterization of polyketalIn this section,two kinds of polyketal PCADK and PK3 were synthesized.Compared with PCADK,PK3 has more significant acid sensitivity.The structure of PCADK and PK3 were determined by NMR and FTIR.Through XPRD and DSC,a portion of PCADK and PK3 were identified to be crystals.The analysis results of GPC showed that the weight-average molecular weight of PCADK and PK3 were 6463 Da and 3844 Da,respectively.2.Establishment of MTX in vitro analytical methodsIn this section,a HPLC method was developed to determine MTX.And the method was assessed to be specific,accurate with good precision.3.Preparation and evaluation of PCADK-based LPNs decorated with folate.In this section,nanoparticles were prepared with PCADK,Folate-PEG-PLGA and Egg PC.The ratio of polymer to lipid(polymer/lipid),PLGA to PCADK(PLGA/PCADK)and Folate-PEG-PLGA to PLGA(Folate-PEG-PLGA/PLGA)were optimized to obtain nanoparticles loaded with MTX.Through analyzing the particle size and drug loading of MTX,polymer/lipid(6:4),PLGA/PCADK(5:5),Folate-PEG-PLGA/PLGA(10:0)were selected as the optimized formulation.The particle size,PDI and drug loading of optimized nanoparticles were 133.6±5.1 nm,0.14±0.021 and 2.35±0.003%,respectively.SEM images indicated that FA-PPLNs were smooth and spherical without adhesion.The hemolysis assay results showed that hemolysis rate of FA-PPLNs was lower than 5%,which indicated FA-PPLNs possessed good biocompatibility.The in vitro release studies revealed that MTX release rate from the optimized nanoparticles in PBS(p H 5.0)was faster than that in PBS(p H 7.4).The result showed that the optimized nanoparticles possess acid sensitivity.In cellular experiments,RAW 264.7 cells were activated with 1000 ng/m L LPS for 48 hours.Cellular uptake studies and cytotoxicity test revealed the uptake efficiency and cytotoxicity of FA-PPLNs was significantly higher than PPLNs.In vivo therapeutic effects on adjuvant-induced arthritis(AIA)rats showed that the clinical score and paw thickness of the rats treated with FA-PPLNs were 0.6 and 6.18 mm,which were obviously lower than PPLNs and MTX solution.The histological evaluation by X-ray and pathological section suggested that FA-PPLNs could decrease bone and cartilage destruction.Levels of TNF-?and IL-6 in serum of the rats treated with FA-PPLNs were 85.6 pg/m L and 177.2 pg/m L,which were significant lower than that of the rats treated with PPLNs or MTX solution.These results demonstrated that FA-PPLNs had good therapeutic effect on AIA.4.Preparation and evaluation of PK3-based LPNs decorated with folate and Sta-R8.In this section,nanoparticles were prepared by PK3,Folate-PEG-PLGA,Egg PC and Sta-R8.The ratio of polymer to Folate-PEG-PLGA to PK3(Folate-PEG-PLGA/PK3)and Sta-R8 content were optimized.The particle size,PDI,zeta potential and drug loading of the optimized nanoparticles were 136.6±3.5 nm,0.16±0.024,12.0±2.91 m V and 2.55±0.11%,respectively.SEM images indicated the optimized nanoparticles possess smooth and spherical shape without adhesion.The in vitro release behavior of the optimized nanoparticles in PBS(p H 7.4)and PBS(p H 5.0)revealed that they have favorable acid sensitivity.The results of hemolysis assay suggested the optimized nanoparticles had excellent biocompatibility.Cellular uptake studies and cytotoxicity test revealed that comparison with the nanoparticles decorated with Sta-8(Sta-R8-PPLNs)or folate(FA-PPLNs),Sta-R8-FA-PPLNs could be internalized more efficiently and with greater cytotoxicity.In vivo therapeutic effects on adjuvant-induced arthritis(AIA)rats showed that the clinical score and paw thickness of the rats treated with Sta-R8-FA-PPLNs were significantly lower than that treated with Sta-R8-PPLNs or FA-PPLNs.The histopathologic results indicated Sta-R8-FA-PPLNs could reduce bone and cartilage destruction significantly.The serum concentration of TNF-?,IL-6 and IL-1? from the rats treated with Sta-R8-FA-PPLNs were 240.9,87.5 and 90.8 pg/m L,respectively,which were lower than Sta-R8-PPLNs and FA-PPLNs.In summary,our prepared polyketal-based nano drug carriers possessed great acid sensitivity and the folate on their surface could promote uptake efficiency by activated macrophages.And they showed good therapeutic effect on AIA rats.These results suggested that the polyketal-based nano drug carriers were promising for the treatment of RA. |
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