Nanoparticulate And Self-assembled Nanoparticles Cross-linking Hydrogel Delivery Systems For Tacrolimus To Treat Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a chronic inflammatory autoimmune disease with synovitis and symmetry,destructive joints disease.The microenvironment of inflammation sites is similar to that of tumors,with the properties of the enhanced permeability and retention(EPR).Liposome and nanoparticle are the widely used carriers to target delivery for RA therapy.In this thesis,the novel liposomes composed of egg yolk phosphatidyl cholines(EPC),soybean lecithin(SPC),cholesterol(Chol)and tocopherol polyethylene glycol 1000 succinate(TPGS),and the novel self-assembled nanoparticles composed of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer(Soluplus)were developed to load tacrolimus(FK506)and the formulations,characteristics,in vitro release and stability,and the therapeutic efficiency against the adjuvant-induced arthritis(AIA)rats were thoroughly investigated.Local delivery of sustained release formulations offers the high localdrug concentration,while minimizes systemic toxicity and decreases the dosing frequency.Sustained without burst-release in the local joint sites are pivotal to the RA treatment.Here,to explore the formulation and potential application of self-assembled nanoparticles cross-linking thermosensitive hydrogel in RA,a novel injectable in situ gel-forming system composed of Soluplus and FK506 was developed.The sol-gel transition temperature(Tsol-gel),gelation time,rheological behaviors,in vitro release,in vivo gelation and retention,and therapeutic efficacy against AIA of Soluplus in situ gels were compared with the traditional poloxamer 407(Kolliphor P407).Liposomal FK506(FK506-Lip)was prepared by the thin-film dispersion-ultrasonic method.The optimal FK506-Lip with the(EPC:SPC):Chol:TPGS molar ratio of 55:15:30,the drug to lipid mass ratio of 1:10,and the EPC:SPC mass ratio of 90:10 was obtained,which showed the encapsulation efficiency of 99.1%±0.1%,the mean volume particle size of 33.1nm±0.1nm and the zeta potential of-27.7mV±2.7mV.To investigate the dilution stability of FK506-Lip post intravenous bolus injection,the minic dilution by various volumes of pH7.4 phosphate buffer solution(PBS)was conducted.The results showed that the leakage of FK506 was only 2.6% when FK506-Lip was diluted 30 fold,which indicated the probable stability in vivo.The in vitro release of FK506 from liposomes was compared in the pH5.0 and pH7.4 PBS containing0.5%(w/v)polysorbate 80(Tween80).The sustained release of FK506 in both media and a little faster in acidic medium were achieved,showing the cumulatively released amount of 47.8%±3.7% and 41.4%±4.5%FK506 for 48 h,respectively.FK506 loaded self-assembled nanoparticles(FK506-SNPs)were prepared by ethanol injection method.The optimal FK506-SNPs with the drug to Soluplus mass ratio of 3:80,and the Soluplus concentration of 80mg/ml was obtained,which showed the encapsulation efficiency of97.6%±0.5%,the mean volume particle size of 88.3 nm±1.3 nm.The dilution stability of FK506-SNPs post intravenous bolus injection was also investigated like liposomes.The results showed that the leakage of FK506 was only 7.2% when FK506-SNPs was diluted 27 fold,which indicated the probable stability in vivo.The method of FK506 vitro released from nanoparticles was same as liposomes.The sustained release of FK506 in the pH5.0 and pH7.4 PBS containing 0.5% Tween80 and a little faster in acidic medium were achieved,showing the cumulatively released amount of 54.0%±3.9% and 47.9%±2.2% FK506 for 48 h,respectively.The gelation properties of Soluplus were compared with the traditional poloxamer 407(Kolliphor P407).The Tsol-gel of Soluplus was dropped by the addition of salts,elevation of pH and ionic strength,exhibiting more sensitive to PBS than 0.9% NaCl.The injectableSoluplus in situ gels were obtained with the concentratons from 10% to20% in phosphate buffer(50mM,pH 7.4),showing the Tsol-gel of33.6-36.6°C and gelation time within 60 s.In comparision,Soluplus gelling system showed the lower viscosity in sol state,higher gelation temperature and stronger gel strength than P407 in situ gels,resulting in longer retention in the local injection site and sustained release FK506 up to 7d in vivo.The therapeutic efficacy against AIA of obtained prepared FK506 formulations were evaluated by paw edema and body weight.And the therapeutic efficacy of two administrations was also compared.During the treatment from d7 to d10,two nanoparticulate delivery systems effectively inhibit paw edmea in rats,but the appetite of rats was affected and weight loss.While,weight began to grow from d11,the paw edema in group of FK506-Lip rebound to the final d21 of 111%±19%,FK506-SNPs was 84%± 7%.Subcutaneous injection only once on d7 with 10%,20% Soluplus and 20% P407 loaded FK506 thermosensitive hydrogels also shown alleviate paw edema,the weight of rats were grown slowly,which is difference from FK506-Lip and FK506-SNPs.The paw edema of AIA animals was continuously decreased for 10 d post local injection of 20% Soluplus loaded FK506.Although both 10% Soluplus and 20% P407 loaded FK506 controlled the therapy for 4d,the edema extent of Soluplus was less than that of P407.Until the end of experiment,the paw edema of 10%,20% Soluplus and 20% P407 loaded FK506 were83%±7%,83%±10% and 100±7%.