Design And The Evaluation Of The Co-assemble System Of Pheophorbide A And Diphenylalanine Peptide For The Treatment Of Colon Cancer

Objective:According to the latest statistics in China,the national incidence rate of colorectal cancer is 28.20 per 100,000 people,ranking third among all cancers.The mortality rate was 13.61 per 100,000,ranking fifth among all cancers.Although with the breakthrough of diagnosis and treatment technology,the survival of patients has been significantly prolonged by various treatments including chemotherapy,there are still many problems such as low efficiency and poor targeting of traditional chemotherapy.Therefore,it is of great significance to develop new therapeutic methods and research and development of new drug delivery carriers to realize targeted delivery of drugs while increasing sensitization and efficacy.Although nanocarriers loaded with chemotherapy drugs can achieve tumor targeted delivery and sensitization effect of chemotherapy drugs,there are still many problems in practical application of nanocarriers,such as toxic and side effects caused by poor biocompatibility.Therefore,how to construct an appropriate nanodrug delivery system is a major challenge.In recent years,self-assembly systems based on small molecule chemotherapeutic drugs have attracted attention,and their advantages such as simple molecular structure and easy preparation have become a research hotspot in the field of drug delivery.Among them,short peptide nanomaterials have become a hot research topic because of their ability to form a variety of nanostructures,such as spherical nanomicelles and tubular nanofibers,and their good biocompatibility.Phenylalanine dipeptide(PHE),which has good biocompatibility,can also be combined with other small chemotherapeutic drugs to form nanomedicine in aqueous solution.Photodynamic therapy(PDT)is a therapy that destroys tumor tissue oxygen by photoactivated nanoparticles to form singlet oxygen.At present,despite the rapid progress of photodynamic therapy,the clinical transformation of the therapy has encountered some problems.First,for deep visceral tumors such as colon cancer,percutaneous light excitation of nanoparticles is inefficient.Secondly,the low delivery efficiency of most photosensitizers leads to poor enrichment of photosensitizers in tumor sites,resulting in poor efficacy of PDT.At present,there is a technology that has been developed to excite nanoparticles for PDT therapy by direct irradiate tumor lesions in the intestine assisted by colonoscopy,which improves the excitation efficiency of nanoparticles.Therefore,how to construct an appropriate nano-carrier to target the delivery of photosensitizer to the tumor site for PDT has become the key to improve the efficacy of PDT on colon cancer.In conclusion,considering the limitations of conventional therapies,especially chemotherapy,we explored the application of photodynamic therapy in colon cancer treatment.In order to better promote the clinical transformation of PDT,we selected the photosensitizer pheophorbide a A(PPA),which has been deeply studied as the research drug.By using diphenylalanine Peptide and PPA co-assembly to form nanoparticles,we solved the problem of poor biocompatibility of PPA,improved the peripheral circulation time of PPA and the enrichment content of PPA in tumor,and then enhanced the anti-tumor efficacy and anti-tumor immune activation effect of PPA.Methods:1.PPA and PHE self-assemble nanoparticles(PPA@PHE)were prepared by successively adding corresponding compounds through agitation method.Subsequently,the particle size and potential were characterized by particle size analyzer to preliminarily verify the successful synthesis of the particle.Transmission electron microscopy(TEM)was used to confirm the successful synthesis of the particles and characterize their basic morphology.2.We simulated the binding force and stability of PPA and phenylalanine dipeptide in water phase through molecular docking and molecular dynamics,and preliminarily simulated and discussed their interaction mode.Subsequent daily monitoring of PPA@PHE particle size changes in vitro confirmed the stability of PPA@PHE.3.We verified the presence of characteristic peaks of PPA in PPA@PHE nanoparticles by detecting full-wavelength excitation and emission pops of PPA@PHE.Subsequently,the uptake of PPA@PHE in CT26 cells was detected by confocal and flow cytometry in vitro,and the killing of CT26 cells by PPA@PHE was detected by CCK-8assay to verify the killing ability of the nanoparticles on colorectal cancer cell lines.After the tumor ball model of CT26 colon cancer cells was constructed in vitro,the tumor ball penetration experiment was conducted to verify the infiltration of nanoparticles in the tumor center,so as to preliminarily confirm the antitumor activity of PPA@PHE nanoparticles.4.Balb/C mice loaded with CT26 colorectal cancer cells were constructed,and the organ distribution and peripheral circulation of PPA@PHE in tumor-bearing mice were detected to illustrate the general situation of nanoparticles in vivo,and to verify that PPA@PHE has a good circulation ability and can be passively enriched in tumor sites.5.Balb/C mice loaded with CT26 colorectal cancer cells were constructed to conduct pharmacodynamics experiments.The efficacy of PPA@PHE was explained by detecting tumor volume,tumor quality,tumor burden and other indicators of the mice.6.HE staining was used to detect the pathological changes and necrosis of heart,liver,spleen,lung,kidney and tumor tissues of mice in each group after treatment,and combined with the safety indexes such as mouse body weight,blood routine and liver and kidney function,to demonstrate the safety of the preparation in vivo.7.The contents of CD4~+and CD8~+T cells in tumor tissues of each group were qualitatively detected by immunofluorescence staining.The changes of inflammatory factors in tumor tissue were detected by Elisa assay.8.After the spleen and tumor tissues of mice receiving different treatments were dispersed into single-cell suspensions,the infiltration of DC cells,CD8~+T cells and Treg in tumor tissues was detected to explain the tumor immunity of mice after treatment.9.The interaction between PPA@PHE nanoparticles and protein was predicted by molecular docking assay.Results:1.Firstly,PPA@PHE nanoparticles were prepared by reverse solvent precipitation method using PPA and phenylalanine dipeptide solution,with a particle size of 70.419±5.519 nm and PDI of 0.125±0.007.The particle potential was negative,and the potential was-16.1±2.81 m V.The results of particle stability showed that PPA@PHE nanoparticles had good stability in vitro,and the particle size of PPA@PHE nanoparticles did not change significantly on day 7 compared with day 0.2.Molecular docking results showed that PPA and phenylalanine dipeptide were stable in aqueous solution.The overall energy of PPA and phenylalanine dipeptide was stable within 50 nanoseconds of their binding,with no clear dispersion,and the binding index was-3.116 kcal/mol.3.By scanning the excitation and emission spectra of PPA@PHE nanoparticles and pure PPA solution,the results show that PPA@PHE nanoparticles have the characteristic excitation and emission peaks of PPA,which does not affect the photoactivation activity of PPA.4.Uptake of PPA@PHE nanoparticles in mouse colon cancer CT26 cells was detected.Confocal results showed that CT26 cells had a higher uptake of PPA@PHE nanoparticles compared with free PPA.Fluorescence quantitative results showed that PPA@PHE nanoparticles were incubated with cells for 2 h,4 h and 8 h,and the uptake of nanoparticles by cells increased gradually,and the uptake of PPA@PHE nanoparticles by cells was higher than that of free PPA at the same time point.Flow cytometry results confirmed that more cells ingested PPA@PHE nanoparticles than free PPA.CCK-8results showed that PPA@PHE nanoparticles showed better cytotoxicity against CT26cells than free PPA solution,while phenylalanine dipeptide did not kill tumor cells.5.A 3D tumor sphere model was constructed to detect the tumor permeability of PPA@PHE nanoparticles and free PPA.The results showed that PPA@PHE nanoparticles penetrated into the tumor sphere at 50μm,100μm and 150μm from the tip of the tumor,while free PPA only penetrated into the outer layer of the tumor sphere.6.The pharmacokinetics of PPA@PHE nanoparticles and free PPA solution in peripheral blood were further detected,and the results showed that compared with free PPA,PPA@PHE nanoparticles had better circulation characteristics.7.The tissue distribution of PPA@PHE nanoparticles and free PPA in vivo was further detected at 3,6,9,12 and 24 hours.The results showed that both PPA@PHE nanoparticles and free PPA were enriched in the liver within 24 hours.The difference was that compared with free PPA,PPA@PHE nanoparticles were enriched in tumor significantly more than free PPA group.8.Bal B/C mice with murine colon cancer(CT26)were used as animal models to study the pharmacodynamics in vivo.The results showed that treatment with PBS or phenylalanine dipeptide in mice did not produce tumor inhibition.Tumor growth in mice continued rapidly,with tumor volume reaching 1200 mm~3 in the PBS group and1100mm~3 in the phenylalanine dipeptide group at day 12 of administration.Statistical analysis of tumor growth curves showed no significant difference between the two groups.Free PPA solution showed certain anti-tumor ability.On the 12th day after administration,the tumor size of the PPA solution group was maintained at 600mm~3,and the tumor inhibition rate was 46.50%.The PPA@PHE co-assembled nanoparticles group showed the best antitumor activity,the tumor volume remained below 300 mm~3 and the tumor inhibition rate was 82.09%after 12 days of administration.9.Subsequently,the safety of each treatment group was further evaluated.Compared with mice treated with PBS alone,no significant weight loss occurred in each group after three treatments.After treatment,there was no significant difference in organ index between the two groups and PBS group.The important organs of the treated mice such as heart,liver,spleen,lung and kidney were observed after HE staining,and the results showed no obvious damage.No significant difference in blood biochemical indexes such as liver and kidney function.These results confirmed the safety of PPA@PHE nanoparticles.10.Immunofluorescence results showed that the content of CD8~+T cells increased significantly after the tumor was treated with PPA@PHE nanoparticles,indicating that PPA@PHE nanoparticles have a good anti-tumor cell immune stimulation effect.Elisa results showed that the concentration of TNF-αin tumor tissues treated with PPA@PHE nanoparticles was 1.66,2.70 and 2.96 times higher than that in free PPA group,phenylalanine dipeptide group and PBS group,respectively.The concentration of IL-6was 1.83,3.82 and 3.36 times higher than that of free PPA group,phenylalanine dipeptide group and PBS group,respectively.These results suggest that PPA@PHE nanoparticles can induce inflammatory response in tumor tissue and improve tumor microenvironment.11.CD8~+T lymphocytes and DC cells in tumor and spleen of mice treated by each group were detected,memory T cells and Regulatory cells(Treg).The results showed that the PPA@PHE group had the highest concentration of CD8~+T lymphocytes,DC cells,and memory T cells,showing strong anti-tumor immunity after the treatment of PPA@PHE nanoparticles.Meanwhile,the number of Treg cells in the peripheral and tumor cells in the PPA@PHE group was the lowest,indicating that the immunosuppressive microenvironment of tumor was alleviated after the treatment with PPA@PHE nanoparticles.12.Through molecular docking assay,we found that PPA@PHE nanoparticles may interact with glutathione S-transferase(GST).Conclusions:We found that PPA and phenylalanine dipeptide can self-assemble to form nanoparticles by reverse solvent precipitation method with good stability.Compared with free PPA solution,PPA@PHE nanoparticles have better tumor cell uptake,tumor cell killing and tumor penetration ability.More importantly,PPA@PHE nanoparticles owned good biocompatibility,good peripheral circulation,more enrichment content in tumor tissues than free PPA solution,and can show better anti-tumor activity in vivo.