Multifunctional folate receptor-targeting and pH-responsive nanocarriers loaded with methotrexate for treatment of rheumatoid arthritis

International Journal of Nanomedicine Multifunctional folate receptor-targeting and ph- responsive nanocarriers loaded with methotrexate for treatment of rheumatoid arthritis Jinlong Zhao 1 Menghui Zhao 1 changhui Yu 1 Xueyan Zhang 1 Jiaxin liu 1 Xinwei cheng 0 robert J lee 0 1 Fengying sun 1 lesheng Teng 1 Youxin li 1 0 college of Pharmacy, Ohio state University , columbus, Oh, Usa , USA 1 s chool of l ife s ciences, Jilin University , changchun, china Rheumatoid arthritis (RA) is an autoimmune disease characterized by progressive cartilage and bone destruction. Activated macrophages that overexpress folic acid (FA) receptors play an important role in RA, due to their abundance in inflamed synovial membrane and joints. In an effort to deliver drugs to the inflamed tissues, multifunctional FA receptor-targeting and pH-responsive nanocarriers were developed. They were composed of lipids, polyethylene glycol (PEG)-poly(lactic-co-glycolic acid) (PLGA) forming a hydrophilic shell, FA around the hydrophilic shell as a targeting ligand, and poly(cyclohexane-1,4-diylacetone dimethylene ketal) (PCADK) and PLGA as a hydrophobic core. PCADK also acts as a pH-responsive material. Methotrexate (Mtx) was encapsulated in the nanoparticles, which exhibited pH-responsive release in vitro. Cellular uptake and cytotoxicity experiments revealed that FA-PEG-PLGA/ PCADK-lipid nanoparticles loaded with Mtx (FA-PPLNPs) exhibited superior cellular uptake and higher cytotoxicity to activated macrophages than PPLNPs/Mtx. The therapeutic effect of FA-PPLNPs/Mtx in RA was confirmed in an adjuvant-induced arthritis rat model. These results suggest that the multifunctional folate receptor-targeting and pH-responsive nanocarriers are promising for the treatment of RA. - Scheme 1 structure and mechanism of cellular uptake of Fa-PPlNPs/Mtx. Abbreviations: Fa-PPlNPs/Mtx, methotrexate-loaded folic acid–polyethylene glycol–poly(lactic- co-glycolic acid)–poly(cyclohexane-1,4-diylacetone dimethylene ketal)–lipid nanoparticles; PcaDK, poly(cyclohexane-1,4-diylacetone dimethylene ketal); Peg, polyethylene glycol; Plga, poly(lactic-co-glycolic acid). Materials and methods Materials Mtx was purchased from Yuanye Biotechnology (Shanghai, China). PLGA was purchased from Boehringer Ingelheim Pharma (Ingelheim am Rhein, Germany). DMEM was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lipopolysaccharide (LPS) and polyvinyl alcohol (87%–89% 6736 hydrolyzed, molecular weight [MW] 13,000–23,000 Da) were purchased from Sigma-Aldrich (St Louis, MO, USA). fetal bovine serum (FBS) and RAW264.7 cells were obtained from Procell Biological Technology (Wuhan, China). 4′,6diamidino-2-phenylindole (DAPI) was obtained from Beyotime Institute of Biotechnology (Haimen, China). Rhodamine B was obtained from Sinopharm Chemical Reagent (Shanghai, China). Acetonitrile was purchased from Thermo Fisher Scientific. MTT was obtained from Yuanye Biotechnology. Enzymelinked immunosorbent assay (ELISA) kits for TNFα and IL6 were obtained from Elabscience Biotechnology (Wuhan, China). Complete Freund’s adjuvant was obtained from Chondrex (Redmond, WA, USA). All chemicals used were of analytical grade and used without further purification. Preparation of lPNPs PCADK and FA-PEG-PLGA were synthesized as described previously.35 Mtx-loaded FA-PPLNPs (FA-PPLNPs/Mtx) were prepared using modified emulsion–solvent evaporation. Briefly, PLGA, folate-PLGA-PEG, PCADK, and lipid were dissolved in 4 mL dichloromethane acetone solvent (1:1, v:v). Then, 5 mg Mtx was dissolved in the organic phase under sonication. The obtained organic phase containing Mtx was added slowly to 8 mL of 1% (w:v) polyvinyl alcohol (MW 30,000–70,000 Da, Sigma-Aldrich), and emulsified for 3 minutes by sonication in an ice bath at 100 W. The resulting emulsion was then added to 15 mL deionized water and evaporated under magnetic stirring at room temperature for 4 hours. The LPNPs obtained were pelleted by centrifugation (18,000 rpm, 15 minutes, 4°C) and washed three times with 30 mL deionized water. Rhodamine B-loaded LPNPs for cell-uptake studies were prepared by the same method, except for replacing Mtx with rhodamine B. The resulting LPNPs were lyophilized for 24 hours and kept at 4°C until use. In the formulation design, polymer:lipid (w:w), PLGA:PCADK (w:w), and PLGA:FA-PEG-PLGA (w:w) were optimized to obtain the smallest particle size. characterization of lPNPs The size and polydispersity index (PDI) of LPNPs were examined at 25°C by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 from Malvern Instruments (Malvern, UK). Prior to measurement, LPNPs were reconstituted in deionized water at a concentration of 1 mg/mL and sonicated for 30 seconds. Drug loading (DL) was determined by high-performance liquid chromatography (HPLC). Briefly, 2 mg LPNPs were dissolved in 200 μL dichloromethane. Then, 1 mL phosphate buffer solution (PBS) (0.1 M, pH 7.4) was added to extract the encapsulate (...truncated)