Bimodality Biomimetic Nanomolecular Probe For Photothermal Combined Sonodynamic And Synergistic Immunotherapy

PART Ⅰ PREPARATION,CHARACTERIZATION AND BASIC PERFORMANCE INVESTIGATION OF BIOMIMETIC NANOMOLECULAR PROBES MODIFIED BY BREAST CANCER CELL MEMBRANEObjectiveTo prepare a kind of biomimetic nano-molecular probe(CHINPs)with hematoporphyrin monomethyl ether/superparamagnetic iron oxide coated by breast cancer cell membrane,investigate its characterization,photothermal properties and reactive oxygen production performance.Methods1.Preparation and characterization of nanoparticles The cell membrane of breast cancer 4T1 was extracted by chemical lysis and repeated freeze-thaw method,and then biemulsification combined with extrusion method was used to prepare the biomimetic nano-molecular probes(CHINPs)of polylactic acid glycolic acid copolymer(PLGA)coated with CCM and loaded with hemoporphyrin-monomethyl ether(HMME)and superparamagnetic iron oxide(SPIO).Its morphology was observed under optical microscope and its structure was analyzed by transmission electron microscope(TEM).The particle size and potential were detected by Malvin particle size analyzer.The contents of HMME and SPIO in the nanoparticles were determined by ultraviolet spectrophotometry and inductively coupled plasma mass spectrometry(ICP-MS),and the encapsulation rate and drug loading were calculated.Protein gel electrophoresis(SDS-PAGE)was used to detect the proteins carried by the nanoparticles,and the membrane coating effect was verified.2.Basic properties of nanoparticles The photothermal properties and photothermal stability of different concentrations of CHINPs were tested under the excitation of 808 nm laser;The Singlet Oxygen Sensor Green(SOSG)probe was used to verify the performance of reactive oxygen species(ROS)generation by nanoparticles.The cellular ROS production capacity of nanoparticles was determined by using 2,7-dichlorodi-hydrofluorescein diacetate(DCFH-DA).Results1.Nanoparticle characterization CHINPs were prepared by extrusion method and double emulsification method and were light brown.Nanoparticle size was uniform and morphology was regular under light microscope and CLSM,and nanoparticles were spherical "core-shell structure" under TEM.The particle sizes of CCM,HINPs and CHINPs were 337.8 ± 6.4 nm,198.9 ± 1.4 nm and 228.1 ± 6.2 nm,respectively,and the Zeta potential were-20.5 ± 0.4 m V,-10.7 ± 1.3 m V and-19.3 ±0.6 m V,respectively.The encapsulation rate and drug loading of hemoporphyrin monomethyl ether were 82.12% and 3.16% respectively.The enveloping rate of superparamagnetic iron oxide was 89.21% and the drug loading was1.83%.SDS-PAGE results showed that the protein profile of CHINPS was almost the same as that of CCM and 4T1 cell lysates.2.Basic properties of nanoparticles CHINPs has good photothermal conversion performance and stability under 808 nm laser excitation;The SOSG probe showed that CHINPs could produce ROS under ultrasonic irradiation.The DCFH-DA probe detected that a large amount of ROS was produced by ultrasonic irradiation after CHINPs and cells were incubated.ConclusionIn this study,the bionic breast cancer cell membrane nano-molecular probe(CHINPs)was successfully prepared.The molecular probe has good photothermal and reactive oxygen generation properties.PART Ⅱ EVALUATION OF HOMOLOGOUS TARGETING AND PHOTOACOUSTIC/ MAGNETIC RESONANCE BIMODAL IMAGING OF BIOMIMETIC NANOMOLECULAR PROBESObjective To observe the ability of CHINPS targeting homologous breast cancer 4T1 cells in vitro,and to investigate its PAI and MRI effects in vitro.The in situ breast cancer model of Babl/c mice was established and employed to investiagate the targeting effect of CHINPs to homologous tumors in vivo,PAI and MRI in vivo,and to evaluate the imaging ability of CHINPs as a bimodal contrast agent in vivo.Methods 1.In vitro homologous targeting and photoacoustic and magnetic resonance imaging of CHINPs Non-targeted HINPs and targeted CHINPs were co-incubated with 4T1 breast cancer cells for 1h,2h,3h and 4h,respectively,and the phagocytosis of 4T1 cells was observed under laser confocal microscopy and flow cytometry.At the same time,non-targeted HINPs and targeted CHINPs were incubated with other tumor cells(MDA-MB-231 cells,MG63 cells,B16F10 cells)and macrophage RAW264.7 for 4h,respectively,and the phagocytosis of CHINPs by different tumor cells and escaped macrophages was observed by laser confocal microscopy and flow cytometry.The different concentrations of CHINPs were placed into the gel hole model to observe the enhanced photoacoustic imaging effect at the excitation wavelength of 725 nm.Similar CHINPs were loaded inot 2 ml EP tubes and scaned under magnetic resonance imaging system.The acquired signal intensities were quantitatively analyzed.2.In vivo homologous targeting and photoacoustic and magnetic resonance imaging of CHINPs 4T1 orthotopic murine breast-cancer model was established.In vivo fluorescence imaging was performed in tumor-bearing mice at different time points(pre,1h,3h,6h,24 h and 48h)afer injecting DIR labeled HINPs or CHINPs through the tail vein.Then fluorescence signal values in the tumor region of mice were measured by imaging system software to evaluate the tumor targeting performance of CHINPs.At the same time,24 h after the injection of nanoparticles,the mice were sacrificed,and the tumor and important organs(heart,liver,spleen,lung,kidney)were taken for in vitro fluorescence imaging,and the tissue distribution of CHINPs was further quantitatively analyzed by ICP-MS.HINPs or CHINPs were injected through the tail vein.Photoacoustic imaging and T2-weighted magnetic resonance imaging were performed at different time points(pre,1 h,3 h,6 h,24 h and 48 h),and signal intensity was quantitatively analyzed.Results 1.In vitro homologous targeting and photoacoustic and magnetic resonance imaging of CHINPs In the CHINPs group,a large number of nanoparticles gathered around breast cancer 4T1 cells with high fluorescence intensity,while in other tumor cells(MDA-MB-231 cells,MG63 cells,and B16F10 cells),there were fewer nanoparticles with low fluorescence intensity.In the non-targeted HINPs group,only a few nanoparticles clustered around all tumor cells,suggesting that CHINPs could target homologous 4T1 cells.There were a large number of non-targeted HINPs and a few targeted CHINPs around macrophage RAW264.7,suggesting that CHINPs could escape macrophage phagocytosis after cell membrane modification.In vitro PAI and MRI results showed that CHINPs nanoparticles could enhance photoacoustic imaging,and the photoacoustic signal increased with the increase of their concentration.The T2-weighted MRI showed negative enhancement,the signal intensity decreased with the increase of its concentration,and the relaxation rate gradually increased with the increase of its concentration.2.In vivo homologous targeting and photoacoustic and magnetic resonance imaging of CHINPs In vivo fluorescence imaging of tumor-bearing mice showed that the targeted nanoparticle CHINPS group distributed in the tumor region,and the fluorescence signal of the tumor site in this group was significantly higher than that of the non-targeted nano-particle HINPS group,and the value of the fluorescence signal increased gradually after injection,peaked at 24 h and then decreased gradually.Invitro fluorescence imaging and ICP-MS further confirmed that CHINPS can target tumor regions.In the tumor-bearing mice,the photoacoustic signals in the tumor region gradually increased after the injection of nano-nanoparticles CHINPS through the tail vein,and then slowly decreased after the peak at 24 hours,while the T2-weighted magnetic resonance signals were on the opposite side,which first decreased to the lowest value and then gradually increased,showing a negative enhancement.Conclusion The prepared cell membrane-coated nanomolecular probe CHINPs can actively target homologous breast cancer cells and has good photoacoustic and T2-weighted magnetic resonance imaging performance in vitro.CHINPs can effectively concentrate at tumor sites and can be used as an ideal contrast agent for PAI/MRI bimodal imaging.PART Ⅲ PHOTOTHERMAL/ SONODYNAMIC EFFECT AND SYNERGISTIC THERAPY WITH PD-1 ANTIBODY OF BIONIC NANOMOLECULAR PROBESObjective To observe the photothermal/sonodynamic effect of CHINPs and synergistic therapy with PD-1 antibody for breast cancer,and analyze the immune mechanism in vivo to evaluate the biosecurity of CHINPs in vitro and in vivo.Methods 1.In vitro cytotoxicity and combined photothermal/ sonodynamic antitumor of CHINPs The cytotoxicity of CHINPs nanomolecular probe on breast cancer 4T1 cells was evaluated by CCK-8 assay.Under the irradiation of 808 nm laser and low intensity focused ultrasound,the killing effect of combined photothermal and sonodynamic therapy on breast cancer 4T1 cells was evaluated by flow cytometry and laser confocal microscopy.2.In vivo CHINPs for photothermal/ sonodynamic therapy combined with PD-1 antitumor therapy Bilateral in-situ tumor model of BALB/c mice was established(right: primary tumor,left: artificial distant metastasis tumor).When the volume of primary tumor was about 50-60 mm3,tumor-bearing mice were randomly divided into 9 groups(n=6),including: control group;Laser+US group;alone CHINPs group;anti-PD-1 group;CHINPs+laser group(PTT group);CHINPs+US group(SDT group);HINPs+laser+US group(non-targeted PTT/SDT group);CHINPs+laser+US group(targeted PTT/SDT group);CHINPs+laser+US+anti-PD-1 group(targeted PTT/SDT/anti-PD-1 group).After injection of 200μl PBS,HINPs or CHINPs via tail vein for 24 hours,the tumor was irradiated by laser or/or ultrasound.The body weight and tumor growth of mice in each group were observed.The maximum diameter and smallest diameter of tumor were measured every 2 days,and the mice were weighed for 16 days.At the same time,one of the tumor-bearing mice in each group was randomly selected to be killed 72 hours after treatment and the tumors were removed for H&E,TUNEL and Ki67 staining to evaluate the proliferation and apoptosis of tumor cells and conduct quantitative analysis.3.The immune mechanism of assessment To evaluate the mechanism of CHINPs for photothermal/sonodynamic therapy combined with PD-1 antitumor therapy,each group of tumor-bearing mice was treated 7 days after treatment,the distant tumors were collected and prepared into single-cell suspensions for immunofluorescence antibody staining.T cell types were analyzed by flow cytometry.Meanwhile,blood samples were collected and the secretion of serum cytokines TNF-α,IFN-γ and IL-12 was detected by ELISA.4.Evaluate the biosafety of CHINPs To evaluate the acute and chronic toxicity of CHINPS in healthy Kunming mice,200μl CHINPs were injected into healthy Kunming mice via a tail vein.The mice were sacrificed at 1,7,14 and 28 days after injection.Blood samples were collected for routine blood and blood biochemical examination.The main organs(heart,liver,spleen,lung,kidney and intestine)were taken out for H&E staining and morphological changes were observed.Results 1.In vitro cytotoxicity and combined photothermal/ sonodynamic antitumor of CHINPs There was no significant effect on cell survival rate when CHINPs cells were co-incubated with 4T1 cells alone without the application of ultrasonic irradiation.Under the irradiation of 808 nm laser and low intensity focused ultrasound,breast cancer 4T1 cells could be killed by photothermal effect and sonodynamic generation of reactive oxygen species.Compared with the photothermal therapy group alone and the sonodynamic therapy group alone,the photothermal therapy combined with sonodynamic therapy can significantly enhance the killing effect of 4T1 breast cancer cells,and the combined therapy effect is further enhanced after enveloped by the cell membrane.2.In vivo CHINPs for photothermal/ sonodynamic therapy combined with PD-1 antitumor therapy In the study of in vivo treatment of tumor-bearing mice,there was no statistical difference in tumor volume,tumor inhibition rate and tumor weight between the laser+ultrasound group alone and the CHINPs group alone in both primary and distant tumors compared with the control group,indicating that the laser +ultrasound group alone and the CHINPs group alone could not inhibit tumor growth.However,the growth of primary and metastatic tumors was slightly inhibited in both the photothermal therapy group and the sonodynamic therapy group,indicating that both photothermal therapy and sonodynamic therapy can inhibit tumor growth to a certain extent.However,the growth of primary tumor was completely inhibited in the first 1 week in the targeted nanoparticle CHINPs-mediated photothermal therapy combined with sonodynamic therapy,and the therapeutic effect was better than that in the non-membrane-coated HINPs-mediated photothermal therapy combined with sonodynamic therapy group.However,the primary tumor recurred after 1 week,and the metastases were not significantly inhibited.Only in the targeted nanoparticle CHINPs-mediated photothermal/sonodynamic therapy synergy PD-1 group,the growth of both primary tumor and distant tumor was completely inhibited,indicating that CHINPs-mediated photothermal combined with sonodynamic therapy can cooperate with anti-tumor therapy of PD-1 antibody,which can not only effectively eliminate primary tumor,but also inhibit the growth of metastatic tumor.The results of H&E staining of tumor tissue showed that the morphology of tumor cells in the PD-1 treatment group was destroyed,including nuclear pyropysis,fragmentation and dissolution.TUNEL and Ki67 staining results showed that the cell proliferation index and apoptosis index of the PD-1 treatment group were lower and higher than those of the other groups,with statistical differences(P<0.05).3.The immune mechanism of assessment The distant tumors of tumor-bearing mice in each group were stained by immunofluorescence antibody and analyzed by flow cytometry.There were a large number of activated effector T cells CD8+T cells in the PD-1 treatment group with photothermal/sonodynamic combination,and the proportion was higher than that in the other groups,while the proportion of regulatory T cells Treg was the lowest.The results were statistically significant(P<0.05).ELISA serum analysis showed that the experimental group secreted more cytokines TNF-α,IFN-γ,IL-12,with statistical significance(P<0.05).4.Evaluate the biosafety of CHINPs The results of blood routine and biochemical indexes of healthy Kunming rats in each group were within the normal reference range,and the H&E staining results of main organs did not show significant pathological changes.Conclusion CHINPs has good biocompatibility.Under the irradiation of 808 nm laser and low-intensity focused ultrasound,CHINPs can directly kill primary tumors through photothermal effect and ROS generation,and combine with PD-1 antibody to activate systemic immune response,thus effectively inhibiting the growth of metastatic tumors.