Study Of Cell And Cell Component-Based Drug Delivery Systems For The Diagnosis And Treatment Of Melanoma

1.BackgroundMalignant melanoma is one of the most aggressive skin malignancies,and its main treatments include surgery,chemotherapy,immunotherapy,and targeted therapy.Chemotherapy drugs are easily eliminated in the body,and have low specificity and tumor targeting,which can cause severe toxicity to normal tissues and organs.Therefore,it is crucial to develop drug delivery systems（DDSs）that can efficiently deliver drugs to tumors,prolong drug blood circulation time,and minimize side effects.In the recent years,the research and development of materials science,nanotechnology,cell engineering,genetic recombination and other technologies has greatly enriched the choice of DDSs.Among them,the tumor cell membrane coated nanoparticles can achieve tumor targeting,controlled drug release,and immune activation.The process of cell membrane extraction and coating is complex with low coating rate,so it is a better choice to use live cells as a drug delivery platform.Taking advantage of the tumor chemotactic capacity of neutrophils（PMNs）to carry nanoparticles and drugs to tumor sites is an effective drug delivery strategy.As a type of immunotherapy,tumor vaccines aim to reactivate the immune system to recognize and eliminate tumor cells,and to generate immune memory to prevent cancer recurrence.Tumor cell vaccines use intact cells or cell lysates.Autologous cancer cells also have patient-specific antigens,which can induce tumor-specific immune responses.Combining tumor vaccines with anti-tumor drugs can directly kill tumors,efficiently activate the immune system,remove remaining cancer cells,and reduce tumor recurrence.Based on previous studies,this study designed three DDSs based on cells and cellular components.The first DDS was to extract B16F10 cell membranes to encapsulate hollow copper sulfide（HCu SNPs）,which was loaded with photothermal agent indocyanine green（ICG）and chemotherapeutic drug doxorubicin（DOX）（ID-HCu SNP@B16F10）,the second DDS extracts PMN to be combined with ABTS-modified Fe C（Fe C@ABTS）.These two DDSs utilize the homologous targeting ability of cancer cells and chemotaxis of PMN,respectively.They can increase the accumulation of drugs in the tumor site and prolong the retention time;the third DDS treats B16F10 cells with liquid nitrogen to eliminate their tumorigenicity,retain the complete cell structure and tumor antigens.DOX can be loaded in their nucleus,which can realize chemotherapy and immune activation.In this study,the three DDSs were characterized respectively,to explore their anti-tumor and imaging capabilities,and to summarize their advantages and disadvantages,so as to provide a research basis and basis for the design of drug delivery platforms in the future.2.Research Contents and Methods2.1 Melanoma cell membrane coated HCu SNPs for homologous targeting,photoacoustic imaging and photothermal chemotherapyThe HCu SNPs were synthesized,the particle size and zeta potential were measured,and the morphology was observed by transmission electron microscope（TEM）.B16F10 cell membrane was extracted,and its purity was characterized by western blot（WB）.ID-HCu SNP@B16F10 was synthesized.TEM and WB were used to characterize whether the cell membrane was successfully coated.The ability to load and release DOX and ICG,and the photothermal properties of nanoparticles were determined.The cytotoxicity of nanoparticles and anti-tumor ability induced by photothermal effect in vitro were studied;the mouse melanoma model was constructed to explore the in vivo photoacoustic imaging,anti-tumor ability and biocompatibility of ID-HCu SNP@B16F10.2.2 PMN combined Fe C@ABTS mediated magnetic resonance imaging（MRI）and melanoma treatmentFe C@ABTS was synthesized,and its particle size and zeta potential were measured.PMN was isolated from mouse peritoneal cavity and combined with Fe C@ABTS.PMN-Fe C@ABTS was characterized by Diff-Quik staining and TEM.The photothermal ability and MR imaging ability of nanoparticles was studied,and the reaction mechanism of PMN catalyzing H₂O₂ and ABTS was explored.The cytotoxicity and the anti-tumor ability induced by photothermal effect of PMN-Fe C@ABTS in vitro were studied;the mouse melanoma model was constructed to study the in vivo MR imaging ability of PMN-Fe C@ABTS.For the in vivo anti-tumor ability,the body weight and tumor volume of the mice were monitored after treatment;the organs were reserved for H&E staining,and the blood was tested for blood routine and liver and kidney function-related indicators to study the in vivo biocompatibility.2.3 Homologous cancer cell vaccine mediated melanoma therapy and preventionLNF cells were obtained by liquid nitrogen treatment,and the cell structure and morphology were observed by Diff-Quik staining,scanning electron microscopy,and cytoskeleton fluorescent staining;immunofluorescence staining and WB were used to characterize the expression of gp100;CCK-8 was used to detect the proliferation ability of LNF cells;the DOX loading and releasing ability of LNF cells were also studied.Mouse bone marrow-derived dendritic cells（BMDCs）were extracted to detect the ability of activating antigen-presenting cells in vitro.The in vivo antitumor ability of LNF-DOX with or without the immune adjuvant R848 was tested in the animal model of melanoma.Tumor volume and mouse body weight were monitored,and immune activation was detected by flow cytometry and ELISA.The ability of LNF cells combined with R848 to inhibit tumorigenesis of the B16F10 cells in vivo was tested,tumor volume and mouse body weight were monitored,and immune activation was detected at different time points.3.Results3.1 Melanoma cell membrane coated HCu SNPs for homologous targeting,photoacoustic imaging and photothermal chemotherapy3.1.1 Syntheses and characterization of HCu SNPs and HCu SNP@B16F10HCu SNPs are spherical with mesoporous structure,and the extracted B16F10cell membrane has high purity.HCu SNPs have a strong ability to load DOX or ICG,and the drug release of ID-HCu SNP@B16F10 is laser-induced.3.1.2.Photothermal effect of synthetic nanoparticlesHCu SNPs and ID-HCu SNP@B16F10 have ideal photothermal ability,and ICG can improve the photothermal ability of ID-HCu SNP@B16F10.In addition,HCu SNPs and HCu SNP@B16F10 have low cytotoxicity,and ID-HCu SNP@B16F10has excellent anti-tumor ability induced by photothermal effect.3.1.3 ID-HCu SNP@B16F10 for in vivo photothermal therapyIn vivo experimental results show that ID-HCu SNP@B16F10 has tumor targeting ability,and its accumulation in the tumor is about twice that of ID-HCu SNP.Moreover,it can be used for PA imaging of melanoma.In addition,ID-HCu SNP@B16F10+laser can effectively reduce tumor size,and the results of body weight monitoring and H&E staining showed good biocompatibility.3.2 PMN combined Fe C@ABTS mediated MR imaging and melanoma treatment3.2.1 Synthesis and characterization of PMN-Fe C@ABTSFe C@ABTS was successfully synthesized with a zeta potential of-6.33 m V and a particle size of about 280 nm.PMN-Fe C@ABTS was successfully synthesized,and some Fe C@ABTS nanoparticles were endocytosed by PMN.3.2.2.The mechanism of PMN catalyzing the reaction of H₂O₂ and ABTSPMN can catalyze the reaction between ABTS and H₂O₂,and the reaction product is the same as that of HRP.After inhibiting MPO in PMN,the catalytic phenomenon disappeared,indicating that MPO is the key catalytic enzyme in the reaction.3.2.3 Photothermal properties of synthetic nanoparticlesFe C has ideal photothermal performance;H₂O₂ can increase the maximum temperature of PMN-Fe C@ABTS,and the phenomenon disappears after suppressing MPO.Fe C has low cytotoxicity,and PMN-Fe C@ABTS has a certain ability to kill cancer cells,which may be due to the presence of PMN in the system.PMN-Fe C@ABTS has excellent anti-tumor ability induced by photothermal effect,and H₂O₂can enhance this ability.3.2.4.MR imaging ability of synthetic nanoparticlesIn vitro MR imaging capability showed that the relaxation of Fe C nanoparticles was 70.864 m M^-1s^-1,which was similar to that of contrast agents in clinical application.In vivo T₂-MRI results indicated that PMN could enhance the accumulation and retention time of Fe C@ABTS in tumor sites.3.2.5 In vivo anti-tumor ability of PMN-Fe C@ABTSIn vivo experimental results show that PMN-Fe C@ABTS can effectively target melanoma,and its accumulation in tumor is about 2.5 times that of Fe C@ABTS.PMN-Fe C@ABTS+laser can reduce tumor volume effectively.The results of body weight monitoring,H&E staining,blood routine examination and liver and kidney function test in mice showed that all the treatments had good biocompatibility.3.3 Homologous cancer cell vaccine mediated melanoma therapy and prevention3.3.1 Synthesis and characterization of LNF cellsLNF cells retained intact cell structure and gp100 expression,and the cell diameter was smaller than that of B16F10.In addition,DOX was successfully loaded into the nucleus of LNF cells and released slowly within 3 h at 37℃.3.3.2.The ability to promote the maturation of DCs in vitroLNF cells with or without R848 can promote the maturation of BMDCs and increase the proportion of CD11c⁺CD80⁺CD86⁺cells.In addition,the concentration of IL-10,IL-12 and TNF-αin cell culture supernatant was also higher than that in negative control group.3.3.3 Anti-tumor ability of LNF-DOXBoth cell and animal experiments proved that the anti-tumor ability of DOX was not significantly affected after loading into the nucleus of LNF cells.In addition,after co-administration with R848,the anti-tumor ability of LNF-DOX in vivo was improved.In addition,LNF-DOX/R848 also increased the proportion of CD4⁺T cells and CD8⁺T cells in the spleen and tumor microenvironment of mice,and the serum IL-12 content was also significantly increased.3.3.4 In vivo prophylactic efficiency of LNF cellsAfter immunizing mice by injecting LNF/R848 for 4 times,the tumor volume was significantly smaller than that of PBS and R848 groups.After tumor bearing,the proportion of CD4⁺T cells and CD8⁺T cells in spleen and tumor microenvironment and the content of IL-12 in serum were significantly increased.4.ConclusionIn this study,three cell-based DDSs were designed,and the characterization,in vivo and in vitro anti-tumor ability were tested.Among them,ID-HCu SNP@B16F10has good tumor targeting ability and can be used for melanoma PA imaging.Moreover,the combined chemotherapy and photothermal therapy can effectively eradicate tumor cells.However,due to the complexity and low production rate of cell membrane extraction,and HCu SNPs is potentially toxic to the body,thus,it is not the best choice for DDS.PMN-Fe C@ABTS overcomes the above shortcomings and uses PMN’s targeting ability to increase the accumulation at the tumor site.Moreover,the T₂ MR imaging and photothermal ability of Fe C integrate the diagnosis and treatment of melanoma.However,mouse peritoneal PMN does not guarantee a sufficient number for each experiment and is therefore not an ideal DDS for universal application.LNF cells are simple to prepare and have complete cell structure,which can be used to deliver DOX for chemotherapy.Moreover,it has a strong ability to activate dendritic cells,which can activate T cells in vivo to further enhance the anti-tumor effect and thus,can also be used as tumor vaccine.Therefore,among the three DDSs designed in this paper,LNF cells are relatively ideal drug delivery platforms,although it still has disadvantages such as the type of drugs can be loaded are limited.