MSC-based PDT/PTT Nanoparticle Delivery System For Melanoma Metastasis Therapy

Fast clearance from the systemic circulation and poor penetrating avascular tumor tissues are two big challenges for targeted drug delivery systems.Prolonged blood circulation and effective penetration are achieved by reducing the interactions with blood components and the reticuloendothelial system?RES?and enhancement of tumor accumulation.Mesenchymal stem cell?MSC?is a stromal cell that can actively homing to tumor cells and has low immunogenicity.It also has the advantages of easy culture and expansion in vitro.MSC could be a potential biomimetic drug delivery vehicle for tumor-targeted therapy.Photodynamic therapy?PDT?and photothermal therapy?PTT?,which are non-invasive,less toxic and side effects,no multidrug resistance,are new methods for selective treatment of malignant lesions in recent years.In this paper,polydopamine conjugated with chlorin e6?PDA-Ce6?nanoparticle as photoactive units and photothermal conversion units was prepared.The nanoparticles were loaded into MSC by endocytosis to construct the biological tumor-targeting system?MSC-PDA-Ce6?.Due to the receptors on the surface of MSC,MSC-PDA-Ce6 would be attracted by the chemokines or cytokines secreted in the tumor microenvironment and finally migrate and penetrate the depth of the tumor.In this process,nanoparticles released via exocytosis of MSC can transfer between cells and widespread distribution in tumor tissues.After being stimulated by local light,PDT/PTT nanoparticles can exert photodynamic and photothermal effects to kill tumor cells and realize the self-clearing of MSC.MSCs have inherent homing/targeting capacity.PDA nanoparticles were synthesized by the solution oxidation method,and Ce6was conjugated by forming an amide bond between PDA and Ce6.It was confirmed that Ce6 successfully bound to the surface of PDA by ultraviolet and fluorescence scanning.TEM showed that PDA-Ce6 was round and uniformly distributed.The particle size of PDA-Ce6 is 161.2±3.2nm,Zeta potential is-35.3±0.5mV,and the drug loading and encapsulation efficiency of Ce6 was 36.28±4.79%and 62.0±5.20%,respectively.The amide bond between PDA-Ce6 was stable in pH 4.0-7.4.The temperature and ROS generation were monitored to demonstrate that PDA-Ce6maintained excellent photothermal conversion effect and photodynamic performance.After incubating MSC and PDA for 4 hours,PDA-Ce6 can be successfully taken up by MSC and widely distributed in the cytoplasm.SEM and co-localization experiments proved that the nanoparticles were distributed in MSC lysosomes.Trypan blue and trypsin methods were applied to determine the membrane-associated particles,and about 10%were on the cell membrane and nearly 90%were in the cytoplasm.MTT test results showed that PDA-Ce6 had low dark toxicity to MSC.At the same time,the toxicity of the PDT/PTT combination therapy was the maximum,with an IC₅₀0 value about 0.247±0.004?g/mL,which was significantly lower than that of the PDT and PTT single laser irradiation?0.618±0.004?g/mL and 0.763±0.016?g/mL?.Then it was proved that MSC can still maintain migration ability in vitro with different drug loading?0.5-10?g/mL?.In order to investigate the release of MSC-PDA-Ce6,we analyzed the form of Ce6 in the cell culture medium?CM?and cell lysate of the drug-loaded MSC,and found that the drug was in the form of PDA-Ce6 particles.The exocytosis kinetic curve was plotted.The results showed that as the culture time prolonged,the level of Ce6 in the MSC cells decreased,and the content of Ce6 in the culture medium increased.The cumulative release rate of the drug after 3 days was about 60%.There were still particles in the cell.To evaluate the anti-tumor effect of MSC-PDA-Ce6 in vitro,we used flow cytometry to detect the survival rate of tumor cells.The results showed that the survival rate of the cancer cells receiving the combinatory PDT/PTT was significantly reduced compared with those single PDT or PTT.Even at a low drug loading?0.1?g/mL?,MSC-PDA-Ce6 caused 75% tumor cell death.Subsequently,we evaluated the survival of total cells in the co-culture system by using the MTT method.The photocytotoxicity was dose-dependent.In the co-culture system,high-loading MSC-PDA-Ce6 subjected to a single PDT treatment can induce the death of B16-F10 cells.The effect of combined PDT/PTT treatment is more significant,about 80%of MSC and tumor cells can be killed.The cell-to-cell delivery process of the drug was further explored.The particle-to-cell transfer phenomenon was observed in CLSM,and the penetration of MSC-PDA-Ce6 into the tumor sphere was tracked in the 3D tumor spheroids.Finally,we evaluated the in vivo distribution of MSC-PDA-Ce6 and its antitumor effect in a mouse model of melanoma lung metastasis.The Ce6 fluorescence signal in the tumor area reached the maximum 24 hours after the tail vein infusion of the MSC-PDA-Ce6 system.In antitumor experiments,after three intravenous injections and three PDT/PTT treatments,compared with the single treatment of PDT or PTT,PDA-Ce6?PDT+PTT?combined significantly inhibited tumor growth,significantly reduced the number of black metastatic nodules and lung weight,significantly prolonged the survival time of mice,and there was no significant difference between MSC and control group.MSC did not affect tumor growth.In summary,targeted delivery of PDT/PTT particles by MSC tumors can exert significant anti-tumor effects in vivo and in vitro.