Directional Conjugation Of Trop2 Antibody To Black Phosphorus Nanosheets For Precise Phototherapy In Orthotopic Gastric Carcinoma

BACKGROUND:Gastric cancer is a common and aggressive malignant throughout the world with limited treatment options and low patient survival rates.Gastric cancer is a heterogeneous disease with an extremely unique pathogenesis and active carcinogenic pathways.As researchers gain a better understanding of the molecular pathways of gastric carcinogenesis,targeted therapy is a promising approach for tumor treatment.However,drug targets on cell membranes usually face problems such as target mutations or drug resistance,which seriously impair the therapeutic efficacy of targeted therapies.Therefore,the search for new and less mutated targets will serve as an alternative therapeutic strategy to address drug resistance in tumor cells.Human trophoblast cell surface glycoprotein（TROP2）,highly specific expression in various human epithelial cancers including GC,is a promising target for antibody-drug conjugates（ADC）.Black phosphorus（BP）is a novel two-dimensional material with high drug loading,good biocompatibility and major photothermal therapy/photodynamic therapy（PTT/PDT）properties,which is a promising nanomaterial for future cancer treatment.Therefore,the combination of TROP2 antibody for tumor targeting and phototherapy agents could exhibit synergetic antitumor activity.Phototherapy agent of black phosphorus nanosheets（BP）characterized with excellent biocompatibility are covalently modified with TROP2 Ig G antibodies via heterobifunctional linker of polyethylene glycol（PEG）.The linker connected to BP is prepared by esterification reaction.Then the TROP2 antibody was directionally conjugated to BP via Schiff base reaction between aldehyde group from oxidized TROP2 antibody and amino group of PEG,and without influence on the tumor targeting ability of TROP2 antibody.The TROP2-funcationalzied BP can significantly increase the endocytosis of BP in TROP2-positive GC cells exhibiting a reinforced antitumor activity under near infrared（NIR）irradiation.More importantly,a murine orthotopic GC model demonstrates that TROP2 antibody modification can significantly promote the accumulation of BP at tumor tissues and strengthen antitumoral activity of phototherapy.Directional conjugation of TROP2 antibody to BP facilitates the BP with superior stability,tumor targeting ability and excellent anti-tumor activity under NIR irradiation without systemic toxicity in an orthotopic TROP2-positve GC model.OBJECTIVE:This project focuses on the investigation of black phosphorus nanosheets modified by TROP2 antibody as a novel drug delivery system,which has the ability to precisely target gastric tumors and has excellent photothermal therapeutic effect at the tumor tissue after irradiation by exogenous near-infrared light.Both in vivo and in vitro experiments exhibited significant anti-tumor activity.During the treatment,the TROP2-modified black phosphorus nanosheets also had uncommon biocompatibility and did not cause damage to the major organs of the treated mice.In summary,TROP2 antibody-modified black scale nanosheets as a novel drug delivery system provide a new option for targeted therapy of gastric cancer.METHODS:1.Using the Purpald experiment,a gradual fading of the purple color of the mixed solution of BP_Land TROP2 antibody was observed,which proved the successful preparation of BP_TROP2.The results of Fourier transform infrared spectra and thermogravimetric analysis also proved the successful preparation of BP_TROP2.The BP_{Ig G}of the control group was prepared according to the same principle as above.2.The nanoparticles BP_TROP2,BP were characterized by measuring the average particle size as well as by observing the structure.3.The in vitro cellular uptake of BP_TROP2was probed by fluorescence microscopy and flow cytometry using DAPI staining technique on gastric cancer cell MFC.4.Detection of BP_TROP2combined with NIR irradiation-mediated intracellular ROS release using a fluorescent probe.5.The cytotoxicity of BP_TROP2combined with NIR light irradiation on HGC27 cells and MFC cells was measured by MTT kit,and the absorbance at 570 nm was measured by enzyme standard analyzer to quantify the results.6.Live and dead cells are detected using live/dead staining kits and observed under a fluorescent microscope.7.The murine orthotopic GC model was established,and BP_TROP2and BP_{Ig G}were injected into the tail vein and combined with NIR light treatment.To detect the in vivo anti-tumor performance of BP_TROP2and each treatment group in the murine orthotopic GC model.8.All mice were euthanized at the end of the experiment and the treatment of the drug and the biocompatibility of the drug were assessed using H&E staining technique.RESULTS:1.Using thermogravimetric analysis experiments,it was observed that the inflection points of mass change during heating were different for BP_TROP2and BP and BP_L,and the weight loss steps of the three materials were not consistent,which proved the successful preparation of BP_TROP2.2.The size distribution of BP_TROP2was characterized by dynamic light scattering and transmission electron microscopy,and the average diameter of BP_TROP2was 98.7 nm,and the negative potential of BP_TROP2surface was favorable for its long time circulation in vivo without degradation.The experiments using NIR light irradiation on BP_TROP2demonstrated that BP was also modified by TROP2 functionalization with excellent photothermal effect.3.The fluorescence intensity of MFC cells co-incubated with Cy5.5-labeled BP_TROP2increased significantly after 4 h.The fluorescence decreased significantly when the cells were pretreated with free TROP2 antibody.The results suggest that the targeted coupling of TROP2 antibody to BP plays an important role in the enhanced BP uptake by the cells.4.TROP2 antibody modification of BP enhances photodynamic therapy by increasing the release of ROS after NIR irradiation exposure.5.The results of MTT assay showed that both free TROP2 antibody and BP_TROP2exhibited anti-tumor activity against TROP2-positive MFC cells.Under NIR light irradiation,BP_TROP2showed the strongest antitumor activity and BPIg G showed a parallel activity independent of TROP2expression.6.Cell live/dead staining confirmed that BP_TROP2combined with NIR light irradiation had stronger activity in killing the TROP2-positive gastric cancer cell line MFC compared to all other groups.7.Monitor bioluminescence intensity to determine tumor growth.（1）BP_TROP2exhibited moderate anti-tumor effects in vivo compared to PBS or BP_{Ig G}treatment.（2）BP_{Ig G}+NIR has more tumor growth inhibitory activity compared to BP_{Ig G}and BP_TROP2.（3）BP_TROP2+NIR exhibited the strongest antitumor activity.8.The development of tumor growth was detected by IHC staining,and tumor cells and Ki67-positive cells were found to be significantly reduced in the BP_TROP2+NIR group.According to H&E staining,no significant damage was observed in the major organs such as heart,liver,spleen,lung and kidney of the treated mice in each group,indicating that the combined BP_TROP2+NIR treatment has good biocompatibility.In summary,in the present work,the TROP2 antibody was attached to the black scale nanosheets by bifunctional PEG to make the black phosphorus have the function of precisely targeting TROP2 tumors.In vivo experiments as well as in vitro experiments demonstrated the photothermal therapeutic effect and precise targeting ability of BPTROP2,while possessing good biocompatibility without systemic toxicity to users.BPTROP2 may become a new option for clinical targeted treatment of gastric cancer.