Preparation Of Curcumin-loaded Hybrid Nanoparticles And Its Pharmacodynamics Study

Cancer is one of the most terrible diseases that threaten human life and health.There are many problems in present cancer drugs,such as severe side effects,poor targeting,low bioavailability and so on.Recently,nano drug delivery system has been developed to improve the water solubility,targeting and bioavailability of insoluble drugs,which is a promising strategy to improve therapeutic efficiency.In this paper,we intend to develop a novel hybrid nanoparticle for the delivery of drug by using amphiphilic three-arm copolymer Tri-CL-mPEG and enzyme-targeting four-arm copolymer PET-CL-P.The PEGlation will be used to increase the retention and long circulation time in vivo and the enzymatic peptide will be used to specify the system to overexpress MMP-2 and MMP-9 of tumor cell and cell penetrating.The star-cyclic ester polymer will be employed to enhance the drug loading capacity and the water solubility of nanoparticles will be augmented to improve the drug stability and its systemic circulation time,while the microchannel formation method is to be used for the continuous production of hybrid nanoparticles with narrow and controllable size distributions.Firstly,Tri-CL-mPEG was designed and synthesized with1,2,3-propanetricarboxylic acid and?-caprolactone by ring-opening polymerization,followed by decoration with hydrophilic mPEG.PET-CL-P was prepared with pentaerythritol and?-caprolactone by ring-opening polymerization,subsequently decorated with an enzyme-targeting and cell penetrating peptide ACP-GPLGIAGQr₉-ACP.The copolymers were characterized with ¹H NMR,FT-IR and GPC.Secondly,microchannel formation method was applied to the preparation of hybrid nanoparticles in order to fabricate reproducible and homogeneous nanoparticles.The organic phase was comprised of Tri-CL-mPEG,PET-CL-P and curcumin dissolved in acetone in a certain proportion,meanwhile the water phase was purified water.The organic phase and water phase were injected into the main microchannel and sub-microchannels by precision injection pumps,respectively.The nanoparticles selfassembled due to the local supersaturation of curcumin and copolymers as the two miscible solvents came together and curcumin was encapsulated into the core of the hybrid nanoparticles.The optimum condition obtained by single factor experiments were as follows:the flow rate of water phase was 1.20 mL/min,the flow rate of oil phase was 0.60 mL/min,the concentration of curcumin was 1.00 mg/mL,the material ratio was 1:1 and the ratio of curcumin and materials was 1:15.The average particle size of Cur-P-NPs was in a range of126.0–160.0 nm.The hybrid nanoparticles Cur-P-NPs fabricated under the optimum condition have an average diameter of?146.1±1.940?nm and polydispersity index?PDI?of?0.175±0.014?with a Zeta potential of?10.1±0.300?mV.The drug loading capacity and drug entrapment efficiency of Cur-P-NPs were?5.38±0.316?%and?74.66±0.671?%,respectively.TEM imaging of Cur-P-NPs revealed that the hybrid nanoparticles were nearly spherical in shape with diameter around 150 nm,which was consistent with DLS result?146.0 nm?.XRD results indicated that curcumin was encapsulated into the core of the hybrid nanoparticles instead of adsorbed on the surface of nanoparticles.The result of in vitro release showed that the release rate of Cur-P-NPs increased with the decrease of pH.In vitro stability study demonstrated that hybrid nanoparticles Cur-P-NPs could be stored stably at 4?for 30 days.The particle size and PDI of Cur-P-NPs diluted in five-fold fetal bovine serum did not change significantly within 7 days,suggesting the stability of Cur-P-NPs in serum.Thirdly,fibroblast cell L929 was selected to investigate the cytotoxicity of Blank-NPs and Blank-P-NPs.Enzyme-linked immunosorbent assay?Elisa?kits were used to detect the concentrations of MMP-2 and MMP-9 in the supernatant of tumor cells.Tumor cell for the pharmacodynamics investigation was selected according to the contents of MMP-2 and MMP-9.The in vitro anti proliferation effects of curcumin aqueous solution?Cur-Solution?,amphiphilic hybrid nanoparticles?Cur-NPs?and enzyme targeting hybrid nanoparticles?Cur-P-NPs?were investigated.The MTT assay against L929 cells comfired the biocompatibility of the copolymers in the range of 0.0175–1.4 mg/mL.The concentrations of MMP-2 and MMP-9 of U251 cells were higher than that of A549,MCF-7 and Hela.The results of MTT assay and cell uptake test showed that Cur-P-NPs were internalized by U251 cells more easily when compared with Cur-Solution and Cur-NPs,leading to the highest apoptosis rate of U251 cells.The similar phenomenon was observed by flow cytometry.After 19 days of administration on U251-bearing nude mice,Cur-P-NPs exhibted better inhibitory effect on tumor cells growth,compared with Cur-NPs and Cur-Solution.In a word,this novle curcumin-loaded enzyme targeting hybrid nanoparticles can be fabricated by physical mixture of copolymers and stay stable.In vitro and in vivo anti-tumor activity studies indicated that curcumin-loaded enzyme targeting hybrid nanoparticles increased the bioavailability and anti-tumor effect of curcumin.It is a promising delivery system for clinical application.