Construction Of 5-fluorouracil-cinnamaldehyde Prodrug Nano-delivery System And Its Anti-tumor Effect In Vivo And In Vitro

Chemotherapy is one of the main clinical treatments for cancer at present.However,traditional chemotherapeutic drugs have limited efficacy due to a series of problems such as strong side effects,low bioavailability and no targeting to tumor sites.Nanodrug delivery system can accurately enrich drugs in tumor tissues,improve the blood circulation cycle of the drugs and increase bioavailability.However,the preparation of nano-drug delivery system is complicated,the drug loading rate is low,the difference between batches is large,and the carrier may have toxicity or immunogenicity.Therefore,carrier-free drug delivery system based on chemotherapeutic drugs has received a lot of research in recent years.Carrier-free nanomedicine systems can be obtained without synthesis or through a simple preparation process,usually by means of free drug self-assembly or the preparation of certain chemotherapeutic drugs into prodrugs and then through prodrug self-assembly.In this paper,we designed a carrier-free nano-delivery system for prodrug based on chemotherapy drugs of 5-fluorouracil(5FU)and cinnamaldehyde(CA).5-Fluorouracil(5FU)is an anti-metabolic drug,which has anti-tumor effect by interfering with DNA or RNA synthesis,Cinnamaldehyde(CA)can promote the apoptosis of tumor cells by triggering the production of reactive oxygen species(ROS)and the release of cytochrome C in cells.Considering that these two drugs have different anti-cancer mechanisms,we designed a small molecule drug conjugate of 5FU and CA,then prepared it into nanoparticles by self-assembly,which is expected to produce synergistic anti-tumor effects.First,small molecule prodrug 5FUCA was synthesized by simple synthesis and then self-assembled into 5FU-CA NPs.A series of experiments were conducted to explore the anti-tumor effects of 5FU-CA NPs in vivo and in vitro.The main contents are as follows:(1)First,cinnamaldehyde derivative(CA-OH)was synthesized by acetal reaction and 5-fluorouracil 1-acetic acid(5FU-COOH)was synthesized according to the method described in literature.Then,the target conjugate of 5FU-CA was obtained by esterification reaction of the two intermediates.5FU-CA nanoparticles(5FU-CA NPs)were self-assembled into carrier-free nanoparticles by emulsification-solvent evaporation method.(2)The morphology,particle size distribution,physiological stability and acid response of5FU-CA NPs were observed by scanning electron microscopy and DLS.The results show that the size of 5FU-CA NPs is similar and it is spherical.It showed good stability under simulated physiological environment.Because the acetal bond is an acid-responsive bond,the acetal bond of 5FU-CA NPs can be rapidly degraded in acidic environment.Dialysis method was used to evaluate the drug release of prodrug particles.The results showed that the nanoparticle had good performance in releasing 5FU-COOH and CA in the simulated tumor microenvironment.Under the condition of p H 5.0,the cumulative release rate of 5FU-COOH and CA could reach55% and 52% within 24 hours.(3)The ability of Hep G2 cells to take up 5FU-CA NPs was evaluated by laser confocal microscopy and flow cytometry.The results showed that 5FU-CA NPs could be effectively ingested through endocytosis.The killing effect of 5FU-CA NPs on tumor cells or tissues was investigated by in vitro cytotoxicity assay,apoptosis assay and Hep G2 multicellular spherical inhibition assay.The results showed that nanoparticles can also achieve effective killing effect on cancer cells at low dose.(4)A mouse tumor model(H22 cells)was established to investigate the anti-tumor effect of nanoparticles in vivo.The results showed that 5FU-CA NPs had a higher tumor suppressive effect than 5FU-COOH and CA mixed free drug(5FU-COOH+CA)at the same dose.In vivo systemic toxicity assay and pathological section results showed that 5FU-CA NPs had little toxicity to normal organs of mice.In summary,this nanoparticle of 5FU-CA NPs can maintain stability and good biocompatibility during in blood circulation and can target in tumor tissue sites through EPR effect,then improve the anti-tumor effect by using dual drug synergistic effect.Therefore,this method of obtaining a good carrier-free drug delivery system by rationally connecting drugs with different mechanisms of action can be extended to the study of other chemotherapeutic drugs.