Targeted Liposome-based Combined Chemotherapy-Biotherapy-Hyperthermia For Rheumatoid Arthritis Treatment

Objective:A drug delivery system named FA-Lip(GNRs-ODNs/DEX),which integrates targeting,thermal-chemotherapy and biological therapy was designed and constructed,and its anti-rheumatic activity in vitro and in vivo were evaluated.Methods:Gold nanorods were prepared by classic seed growth methods.GNRs-ODNs were prepared through the electrostatic interaction of GNRs and ODNs and were characterized using agarose gel electrophoresis and transmission electron microscopy.Using liposomes(Lips)as drug carriers,the FA-Lip(GNRs-ODNs/DEX)loaded with Dexamethasone(DEX)and GNRs-ODNs was prepared via thin film dispersion method.HPLC and ICP-MS were used to determine the contents of DEX and GNRs in the liposomes,and the thermal response of FA-Lip(GNRs-ODNs/DEX)under 808 nm laser irradiation.was investigated.Finally,zetasizer was used to analyze the particle size and zeta potential of FA-Lip(GNRs-ODNs/DEX),and its morphology was observed using transmission electron microscopy.After FA-Lip(GNRs-ODNs/DEX)was successfully prepared,CCK-8 method was used to detect the cytotoxicity of FA-Lip(GNRs-ODNs/DEX)in RAW264.7 cells.LPS was used to establish inflammatory cell models for in vitro anti-inflammatory study.The in vitro anti-inflammatory activity was evaluated by detecting the levels of inflammatory factors TNF-α and IL-6 in the cell supernatant with an ELISA kit and the NO content in the cell supernatant with NO detection kit.Fluorescent microscopy and flow cytometry were used to investigate the qualitative and quantitative uptake of FA-Lip(GNRs-ODNs/DEX)by inflammatory cells.The mouse model of arthritis was constructed by subcutaneous injection of complete Freund’s adjuvant.It is administered via tail vein injection.During the treatment,body weight,paw thickness and clinical arthritis index of the mice were measured and calculated every two days.Anti-inflammatory activity of FA-Lip(GNRs-ODNs/DEX)in vivo was also evaluated by detecting the levels of inflammatory factors TNF-α and IL-6 in blood using ELISA kits at the end of treatment,the hind limbs of the mice were collected to prepare pathological tissue sections.Finally,in vivo imaging technology was used to investigate the distribution of FA-Lip(GNRs-ODNs/DEX)in mice.Results:GNRs were successfully synthesized,GNRs-ODNs were prepared by combining GNRs with ODNs through electrostatic adsorption.The binding of GNRs and ODNs was confirmed by agarose gel electrophoresis.Transmission electron microscopy showed that the appearance of gold nanorods did not change after combining with ODNs,and the dispersibility of GNRs-ODNs was good.After a series of prescription and preparation process optimization,FA-Lip(GNRs-ODNs/DEX)with appropriate particle size and zeta potential was prepared,with an average particle size of 82.00 ± 0.99 nm,PDI of 0.249 ± 0.004,and zeta potential of-20.83 ± 0.23 mV.The drug loading and encapsulation efficiency of DEX were 5.56±0.08%and 97.87±0.07%,respectively.The concentration and encapsulation efficiency of gold nanorods were 82.42±0.05 pM and 35.63± 1.95%,respectively.Thermal response experiment showed that FA-Lip(GNRs-ODNs/DEX)had a good light-to-heat conversion ability.Through the transmission electron microscopy showed that FA-Lip(GNRs-ODNs/DEX)were liposomes with spherical shape and uniform distribution,and the particle size was within the target range.The cytotoxicity of FA-Lip(GNRs-ODNs/DEX)was determined by CCK-8 method,results showed that the cytotoxicity of Dexamethasone was significantly reduced after being loaded by liposomes.Cellular uptake experiment showed that FA-Lip(GNRs-ODNs/DEX)could be taken up quickly and efficiently by inflamed cells.FA-Lip(GNRs-ODNs/DEX)could significantly inhibit the secretion of TNF-α,IL-6 and NO and achieve the best anti-inflammatory effect under 808 nm laser irradiation.Animal experiment results showed that FA-Lip(GNRs-ODNs/DEX)treatment did not reduce the body weight of arthritis mice and could significantly reduce paw thickness,clinical score and the levels of inflammatory factors(TNF-andαIL-6)in serum.Histopathological section study showed that FA-Lip(GNRs-ODNs/DEX)treated group had less inflammatory cells infiltration in the joint cavity,and smoother cartilage surface,which was similar to that of normal mice in Blank group.In vivo imaging experiment showed that FA-Lip(GNRs-ODNs/DEX)had increased accumulation at the inflamed joints;and had not been fully eliminated at 48 hours.Conclusion:Under 808 nm laser irradiation,FA-Lip(GNRs-ODNs/DEX)had significantly better anti-inflammatory efficacy in vitro and in vivo via the combined thermal-chemo-biological therapy.