| Colorectal cancer(CRC)is a common gastrointestinal cancer worldwide,with high incidence and mortality rates ranking among the top in malignant tumors.Current clinical treatment drugs and methods not only cannot completely eradicate it,but also bring seri-ous adverse reactions to patients.Existing studies have proved that pathogenic bacteria such as fusobacterium nucleatum(F.nucleatum),escherichia coli(E.coli),bacteroides fragilis(B.fragilis)and proteus mirabilis(P.mirabilis)are abundant in colon mucosa,they can promote the formation and progression of tumors,and induce the recurrence of CRC;In addition,due to the special physiological structure of the gastrointestinal tract,drugs are unable to effectively target tumor sites.All of these factors contribute to the significant challenges in treating colorectal cancer.Therefore,a treatment strategy that can effectively deliver drugs to colon tissue with anticancer and antibacterial effects was proposed.The protonated form of hemiprotonic phenanthroline-phenanthroline~+(ph-ph~+)re-ported in the literature is a novel compound with broad-spectrum antibacterial and selec-tive antitumor activity.Additionally,it exhibits high safety with no significant toxic side effects observed in animal studies.Therefore,ph-ph~+has the potential to become an ef-fective treatment for CRC.This study employed egg phosphatidylcholine(EPC)and so-dium deoxycholate(SDC)as carrier materials to prepare drug-carrying nanomicelles(ph-ph~+/SDC/EPC)and carried out in vitro characterization,antibacterial and anti-tumor ac-tivities and in vivo pharmacology of ph-ph~+/SDC/EPC.In this study,ph-ph~+was encapsulated into ph-ph~+/SDC/EPC by thin-film dispersion method.The optimum conditions of each factor were determined by single-factor exper-imental investigation.On this basis,the optimum process for preparing ph-ph~+/SDC/EPC was established by Box-Behnken design-response surface method,namely,the ratio of salt to fat was 0.8(mg/mg),the ratio of drug to fat was 0.2(mg/mg)and the hydration temperature was 40℃.The encapsulation efficiency of ph-ph~+/SDC/EPC obtained by the optimum preparation process was 94.8(±2.3)%,the drug loading was 10%,the particle size was 28±1.34)nm,and the Zeta potential was(-38.50±1.27)m V.The morphol-ogy of ph-ph~+/SDC/EPC was observed by atomic force microscope and transmission electron microscope.The ph-ph~+/SDC/EPC/SDC/EPC was spherical,and its size was consistent with the particle size.Then the critical micelle concentration(CMC)of SDC/EPC blank micelles was determined to be 0.086mg/m L by pyrene fluorescence probe method.After placing ph-ph~+/SDC/EPC at normal atmospheric temperature for 12days,it was found that its particle size and encapsulation efficiency had almost no change,indicating that ph-ph~+/SDC/EPC had good stability.In vitro release experiments were conducted to investigate the drug release characteristics of ph-ph~+/SDC/EPC in simulated gastrointestinal fluids.It was found that the ph-ph~+in ph-ph~+/SDC/EPC exhibited sus-tained release in both simulated gastric and intestinal fluids,providing a foundation for the effective treatment of CRC using ph-ph~+/SDC/EPC.In the in vitro test,the activity of ph-ph~+/SDC/EPC and ph-ph~+against four common CRC-related pathogenic bacteria(F.nucleium,E.coli,B.fragilis and P.mirabilis)was determined firstly.The results showed that ph-ph~+/SDC/EPC had strong antibacterial ef-fect on the four bacteria,which was consistent with the broad-spectrum antibacterial ef-fect of ph-ph~+reported in the literature.After treatment with ph-ph~+/SDC/EPC and ph-ph~+,the morphological changes of Escherichia coli were observed by transmission elec-tron microscopy.The results demonstrated that the cells presented typical characteristics of apoptosis,including cell shrinkage and content degradation.The results showed that ph-ph~+/SDC/EPC and ph-ph~+could inhibit bacterial activity by inducing apoptosis.Then the effect of ph-ph~+/SDC/EPC and ph-ph~+in the range of 0.0025-0.4μmol/m L on the proliferation of human colon cancer cells(HCT-116)was determined by MTT method.The results showed that ph-ph~+/SDC/EPC could significantly inhibit the proliferation of HCT-116 cells in a concentration-dependent manner,and the inhibition of ph-ph~+/SDC/EPC on the proliferation of HCT-116 cells was better than ph-ph~+.It was con-firmed by microscope observation and TUNEL staining that ph-ph~+played an anti-tumor role by inducing cell apoptosis.Finally,it was speculated that the different anti-tumor ability of ph-ph~+and ph-ph~+/SDC/EPC was caused by the different ways of entering cells,therefore the intracellular pathways of ph-ph~+and ph-ph~+/SDC/EPC were investigated.The results showed that ph-ph~+entered the cells through passive diffusion without ATP consumption,while ph-ph~+/SDC/EPC entered the cells through trellisin-mediated path-way with ATP consumption.In the in vivo experiment,the distribution of ph-ph~+/SDC/EPC and ph-ph~+in the gastrointestinal tissue of mice was measured firstly.The experimental results showed that the content of released ph-ph~+from ph-ph~+/SDC/EPC in colon tissue was 3.3 times that of free ph-ph~+.This shows that ph-ph~+can be effectively released from ph-ph~+/SDC/EPC to colorectal tissue.Judging from this,the anti-tumor ability of ph-ph~+/SDC/EPC in colon tissue was stronger than that of free ph-ph~+.In order to ensure the safety of the drug in animals,the changes of blood routine indexes and histopathological sections of mice be-fore and after administration were detected,and these results showed that ph-ph~+/SDC/EPC had a certain safety.Then,the inhibition of ph-ph~+/SDC/EPC on CRC was evaluated by establishing a mouse CRC model.The results showed that ph-ph~+/SDC/EPC significantly reduced the number and volume of mouse tumors,and improved the life cycle and life quality of mice.Through HE staining and TUNEL staining of colon tissue,it was found that ph-ph~+/SDC/EPC could effectively induce CRC cell apoptosis.Besides,the colon tissue fluid was cultured in vitro,and it was found that the bacterial load of the ph-ph~+/SDC/EPC treatment group was the lowest.The results showed that ph-ph~+/SDC/EPC had significant antibacterial effect on facultative anaerobes and anaerobes in the colon of colorectal cancer animals.The degree of colorectal inflammation was measured by HE staining and inflammatory factor measurement.The results demon-strated that the model group and oxaliplatin group had hyperplasia of inflammatory tissue and severe infiltration of inflammatory cells.However,the inflammation symptoms of mice treated with ph-ph~+improved,and the inflammation reaction of colorectal tissue in ph-ph~+/SDC/EPC group was slight.Finally,through the determination of inflammatory factors in colon tissue,it was found that after ph-ph~+/SDC/EPC administration,the con-tent of colorectal inflammatory factor in mice decreased significantly,which suggested that ph-ph~+/SDC/EPC can inhibit inflammatory reaction through anti-cancer and bacteri-ostasis,thus improving the inflammatory symptoms of CRC animals.In conclusion,this study prepared drug-loaded nanomicelles with good performance,whose main function was to effectively release drugs into colorectal tissue.The nanomi-celle not only had the capability to inhibit CRC cell proliferation,but also killed the bac-teria related to CRC.In CRC model mice,drug-loaded nanomicelles played a dual role in antibacterial and anticancer,thus alleviating the inflammatory reactions of CRC,and improving the lifespan of mice.This study provides a new insight and approach for treat-ing CRC. |
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