Preparation Of Curcumol Nanogel And Evaluation Of Their Antitumor Activity

Background:Breast cancer is one of the most common malignant tumors in women,and it is the leading cause of cancer death in women worldwide.Distant recurrence and metastasis are the main reasons for the death of breast cancer patients.Chemotherapy plays an indispensable role in the clinical treatment of breast cancer,which can effectively inhibit distant recurrence and metastasis of breast cancer.However,the toxic side effects of traditional chemotherapy drugs seriously affect the quality of life and treatment effect of patients,which is not conducive to long-term prognosis.The Chinese herbal extract curcumol(CUR)has the ability to inhibit breast cancer cell metastasis and has a good safety.However,its poor water solubility limits its clinical application.Objective:A temperature/reduction dual-responsive nanogel was prepared to achieve targeted delivery of curcumin and improve the anti-tumor and metastasis inhibition effects,aiming to provide new ideas for the treatment of breast cancer prone to recurrence and metastasis,and to provide theoretical support for the clinical treatment of breast cancer with curcumol.Methods:(1)Preparation and characterization of H-SS-P nanogels.Firstly,HA-SS-PNIPAAm copolymer was synthesized by a two-step amide reaction,and its structure was verified by Nuclear Magnetic Resonance(~1H-NMR).Subsequently,H-SS-P blank nanogels were prepared by direct heating method and the appearance of the solution was observed.The lower critical solution temperature(LCST)and critical micelle concentration(CMC)of the copolymers were determined by UV visible spectrophotometry and pyrene fluorescence probe method,respectively.(2)Preparation and characterization of H-SS-P@CUR.H-SS-P@CUR drug-loaded nanogels were prepared by dialysis method.High Performance Liquid Chromatography(HPLC),Dynamic Light Scattering(DLS)and Transmission Electron Microscope(TEM)were used to determine the drug loading and encapsulation efficiency,particle size distribution,zeta potential and particle morphology of H-SS-P@CUR.The in vitro drug release behavior of H-SS-P@CUR was evaluated by dialysis method.(3)Evaluation of in vitro anti-tumor activity of H-SS-P@CUR.Using 4T1 mouse breast cancer cells and L929 mouse fibroblasts as cell models,the effects of H-SS-P@CUR in inhibiting tumor cell growth and metastasis in vitro were investigated by MTT cytotoxicity assay,cell uptake assay,cell scratch healing,Transwell migration and invasion assay,respectively.(4)Evaluation of in vivo anti-tumor activity of H-SS-P@CUR.A 4T1 mammary tumor-bearing mouse model was established to investigate the biodistribution and tumor targeting,anti-tumor effect and safety of the nanogel in mice,as well as the inhibitory effect on lung metastasis of breast cancer in mice.Results:(1)The HA-SS-PNIPAAm copolymer was successfully synthesized by a two-step amidation reaction,and its chemical structure was verified by ~1H-NMR.When the H-SS-P copolymer solution was heated above LCST,the solution changed from colorless transparent to light blue emulsion,indicating the formation of nanogel self-assembly.The low CMC value of this nanogel facilitates its stable existence during in vivo circulation.(2)The H-SS-P@CUR drug-loaded nanogel was successfully prepared by dialysis,with an encapsulation rate of about 80%and a maximum drug loading of 24.45%.TEM and DLS results showed that H-SS-P@CUR had a uniform particle size,a spherical appearance,and a zeta potential of about-20 m V with good stability.In vitro drug release studies showed that the cumulative release of H-SS-P@CUR at high GSH concentration was significantly higher than that at low GSH concentration within 24h,which verified the reduction-sensitive drug release property of H-SS-P@CUR.(3)The anti-tumor activity of H-SS-P@CUR was evaluated by in vitro cytological assays.The results of MTT cytotoxicity assay showed that the blank nanogel had good safety and biocompatibility,and the toxicity of H-SS-P@CUR in 4T1 cells was higher than that of free CUR,while the toxicity to L929 cells was always weaker than that of free CUR.The results of cellular uptake assay showed that 4T1 cells had stronger uptake ability of drug-loaded nanogel,but its uptake effect was significantly reduced after blocked by free HA,which verified that the nanogel played an anti-tumor role by relying on HA and CD44 receptor mediated endocytosis on the surface of tumor cells.The results of cell scratch healing,Transwell migration and invasion assays showed that H-SS-P@CUR inhibited 4T1 cell migration and invasion more effectively than free CUR.(4)The anti-tumor activity of H-SS-P@CUR was evaluated by in vivo zoological experiments.The in vivo imaging results of small animals showed that the fluorescence intensity of the drug-loaded nanogel group was significantly higher than that of the free group and HA pretreatment group at the tumor site in mice,which verified the active targeting of nanogel at the animal level.The in vivo safety and pharmacodynamic results showed that the free CUR and H-SS-P@CUR had good internal safety,and the mice in the H-SS-P@CUR treatment group had the smallest tumor volume and the lowest number of pulmonary metastatic nodules,indicating that H-SS-P@CUR had good inhibitory effects on tumor growth and lung metastasis.Conclusion:In this project,a temperature sensitive copolymers was prepared by grafting and copolymerizing of PNIPAAm with HA as the backbone and cystamine dihydrochloride as the crosslinking agent.Using CUR as a model drug,a temperature/reduction dual responsive drug loaded nanogel H-SS-P@CUR was successfully prepared using the temperature-sensitive property of PNIPAAm and the hydrophobic interaction with CUR.H-SS-P@CUR had a strong drug loading ability and exhibits GSH-triggered drug release behavior.Both free CUR and H-SS-P@CUR exhibited good anti-tumor and metastasis inhibition effects in vitro and in vivo.In addition,nanogels can rely on passive targeting with enhanced permeability and retention effect(EPR)and active targeting mediated by CD44 receptor to achieve drug enrichment at tumor sites and improve anti-tumor efficacy.Therefore,CUR encapsulated in nanogels has more significant anti-tumor activity and anti-tumor metastasis advantages,which can further improve the clinical treatment effect of breast cancer.