Overcoming Breast Tumor Stromal Barrier To Enhance Drug Delivery And Antitumor Immunity

OBJECTIVEMost solid tumors have complex biological barriers,which are mainly composed of abnormal blood vessels,lymphatic vessels,and dense extracellular matrix（ECM）.The tumor matrix barrier hinders the penetration of nano-drugs into the deep tumor tissue,making it difficult for nano-drugs to maximize their anti-tumor effects,and hinder immune cells from exerting their anti-tumor effects.Enhancing the infiltration of drugs and immune cells into tumors has become a non-negligible element in tumor therapy.Based on the above background,this thesis applies three different（chemical,biological,and physical）strategies to promote the penetration of nanomedicines in tumors and simultaneously promote the infiltration of immune cells into tumors to enhance antitumor efficacy.Part 1.Biomimetic liposomes with surface bound elastase for enhanced chemo-immunotherapyMETHODSThe membrane protein of triple-negative breast cancer cell line 4T1was extracted by kit,the membrane protein chimeric liposome LMP was prepared using thin-film dispersion method,and the NE-LMP was developed by direct incubation and binding of elastase through electrostatic adsorption.The characterization of NE-LMP was carried out in terms of appearance,particle size,morphology,characteristic protein and stability,and the binding and release of NE was characterized by micro BCA.The binding of NE on LMP was investigated by electrical competition experiment.In vitro ECM model was constructed to investigate the behavior and diffusion coefficient of NE-LMP penetrating the ECM;a fluorescent multicellular 3D tumor sphere was constructed to investigate the ability of NE-LMP to degrade the ECM and penetrate deeply.The in vivo biodistribution and tumor targeting ability of NE-LMP in TNBC orthotopic tumor-bearing mice were firstly investigated;then the effect of NE-LMP on targeting and degrading tumor stroma was investigated by tissue fluorescence staining and scanning electron microscopy.The anti-tumor effect and safety evaluation of NE-LMP were also investigated;the effect of NE-LMP on enhancing drug and immune cell tumor penetration and the anti-tumor efficacy of combined chemotherapy/immunotherapy were investigated in TNBC-bearing mice.RESULTSMembrane proteins were successfully embedded into liposomes to prepare LMPs and incubated with NE to construct NE-LMPs.The mean size of NE-LMP is about 130 nm.Under the electron microscope,NE-LMP is round-shape,with protein-like particle dots on the surface and a haze-like structure around the particle.Coomassie brilliant blue staining and Western blot results showed that LMP had a protein profile similar to that of membrane proteins,and CD47 was retained on LMP.NE-LMP showed good stability when stored at 4°C,and the particle size,zeta potential and PDI did not change significantly within 4 weeks of storage.The binding rate of NE on LMP is as high as 95%,the loading capacity of NE is about 2.4%.The release of NE from NE-LMP is slow,only 2%of NE is released in 48h.A dose-response relationship was observed for the degradation of p NA by NE-LMP.The activity of free NE is limited whenα-antitrypsin was added,while NE-LMP can effectively tolerate the effect ofα-antitrypsin,and the inhibition rate is less than 30%.The results of the electrical competition experiment showed that the binding of NE on LMP was mainly through electrostatic binding,and the binding could be competitively replaced by other substances with stronger charge.NE-LMP can degrade and penetrate ECM-mimicking gel,and the diffusion coefficient is significantly higher than that of free NE plus LMP group,and the serum environment does not affect the binding of NE and LMP and the activity of NE-LMP.NE-LMP does not affect the viability of normal cells at high dose.3D tumor heterospheroids became scattered after incubation with NE-LMP,and a cavity appeared in the center of the spheroid.Fluorescence microscopy showed that the tumor spheres shrank after incubation with NE-LMP,4T1 and 3T3 cells were separated and the recovery rate of the spheroid was slow.The results of confocal scanning showed that the fluorescence distribution of LMP in the NE-LMP-treated 3D tumorspheres was significantly higher than that of the free NE-treated 3D tumorspheres,and the penetrating ability to the tumor spheres of the nanoparticles were also increased.Biodistribution results showed that,the liver was still the organ with the highest fluorescence intensity,NE-LMP showed significant targeting ability to the tumor site and the fluorescence intensity was significantly stronger than that of LP without membrane protein modification.There was no significant inhibitory effect of NE-LMP on tumor growth.In the internal tumor stroma,we found that a large number of fibers were broken by scanning electron microscopy for the NE-LMP group.Extensive exposure of tumor cells.EVG staining of tumor tissue,collagen I immunohistochemistry and elastin immunohistochemistry showed that NE-LMP had a significant degradation effect on tumor stromal components.In terms of safety concern,NE-LMP showed high biocompatibility for the serum tests and H&E staining of major organ tissues.Fluorescent scanning of tumor tissues showed that NE-LMP treatment significantly enhanced the distribution and penetration of the chemotherapeutic drug doxorubicin at the tumor site.The anti-tumor results of NE-LMP combined with chemotherapy showed that NE-LMP significantly enhanced the anti-tumor effects of free paclitaxel and nab-paclitaxel,prolonging the survival of tumor-bearing mice.The combined treatment strategy also showed an inhibitory effect on lung metastasis.NE-LMP also enhanced the therapeutic effect of immune checkpoint PD-1inhibitor by increasing the infiltration of CD8⁺T cells in tumor,and decreasing the number of Treg.NE-LMP combined with PD-1 inhibitor and chemotherapeutic drug showed superior anti-tumor effect,and the tumor volume was further decreased,with prolonged survival.During the combinational treatment period,the no significant side effects or lung metastasis were observed.CONCLUSIONSNE-LMP successfully mimics the structure and function of NE-Exo for effective degradation of ECM components while being resistant toα1-antitrypsin.NE-LMP can target the tumor site and break the dense tumor stroma barrier,thus enhancing the antitumor effect of chemotherapeutic drugs and immunotherapy without affecting normal tissues.The development and successful application of NE-LMP in preclinical experiments provide a strategy for the future biomimetic nanomedicine for the enhancement of tumor chemotherapy and immunotherapy.Part 2.Bacterial outer membrane vesicles-based therapeutic platform eradicates triple-negative breast tumor by combinational photodynamic/chemo-/immunotherapyMETHODSEscherichia coli DH5αwas cultured,and bacterial outer membrane vesicles（OMVs）were extracted by differential ultracentrifugation.OMVs were characterized by particle size,morphology,protein and toxoid content.The preliminary anti-tumor effect and safety of different doses of OMVs were tested on 4T1 tumor-bearing mice model.Serum biochemical indexes and immuno-inflammatory indexes were monitored,and liver and kidney tissue sections were observed by H&E staining.Different doses of OMVs@M were prepared by loading OMVs into Raw264.7 macrophages by incubation.OMVs@M was observed by transmission electron microscopy and fluorescent staining,and the protein profile of OMVs@M was characterized by Coomassie brilliant blue staining.The tumor targeting ability of OMVs@M was investigated in orthotopic 4T1 breast cancer mice models,and the anti-tumor effects and safety of different doses of OMVs@M were further investigated.Ce6/DOX-OMVs@M was prepared by preloading photosensitizer Ce6 and chemotherapeutic drug doxorubicin DOX into OMVs by co-incubation and then encapsulating them into Raw264.7 macrophages.Transmission electron microscopy was used to investigate the release of OMVs from Ce6/DOX-OMVs@M before and after laser on.The drug loading and releasing profile of Ce6/DOX-OMVs were investigated by UV and fluorescence spectrophotometry.The effect of Ce6/DOX-OMVs@M on cell viability was investigated.The in vitro anti-tumor and reactive oxygen species generation abilities were investigated.The antitumor effect and safety of Ce6/DOX-OMVs@M were also investigated in tumor-bearing mice.The anti-tumor mechanism of OMVs was explored in vitro and in vivo in regards to their ability to shift macrophage polarization and to activate the apoptotic pathway in tumor cells.RESULTSThe particle size distribution of OMVs determined by NTA showed that the particle diameter ranged from 35 to 200 nm,and the peak particle size was 76 nm.The OMVs under TEM showed vesicle-like morphology.Coomassie brilliant blue staining showed that OMVs contained multiple proteins.4×10¹² OMVs particles contained about 0.6 EU lipopolysaccharide（LPS）.High,medium and low doses of OMVs were directly injected into tumor-bearing mice via tail vein,and the survival rates of mice after once injection were 1/6,3/6,and 6/6,respectively.After injection of OMVs,the tumor site turned red and black.Different doses of OMVs inhibited tumor growth while the effect is not statistically significant.After injection of OMVs,serum inflammatory cytokine levels and liver and kidney function indexes all increased transiently.Significant splenocyte proliferation and enlargement of spleen corpuscles were observed.OMVs@M were prepared by loading OMVs into Raw264.7macrophages.OMVs widely existed in cells by transmission electron microscopy and fluorescence confocal microscopy.Coomassie brilliant blue staining proved that OMVs@M had high levels of OMVs protein bands and Raw264.7 cell protein bands at the same time.In vivo biodistribution experiments demonstrated that OMVs@M had significant tumor targeting compared with OMVs,and reduced the distribution of OMVs in the spleen.After injection of OMVs@M,the tumor sites of tumor-bearing mice also turned red and black.The safety of OMVs@M was improved compared with that of free OMVs.The survival rates of mice in the high-dose,medium-dose and low-dose OMVs@M groups were 3/6,6/6,and 6/6,respectively.All doses of OMVs@M inhibited tumor growth,and the spleen tissue of tumor-bearing mice increased and the weight increased.OMVs@M more mildly and consistently increased the levels of inflammatory cytokines in tumor-bearing mice.Ce6 and DOX were loaded into OMVs by co-incubation method to prepare Ce6/DOX-OMVs@M.The drug loadings of Ce6 and DOX were41.92±4.86μg and 31.44±5.21μg in 1×10¹¹ OMVs particles,respectively.DOX and Ce6 were released by 15%and 10%in OMVs for 24 hours,respectively.Photodynamic irradiation could stimulate the release rapidly,and the release amount could reach 80%within a few minutes.Transmission electron microscopy showed that the macrophages were obviously cracked after laser on,and OMVs were released.Free DOX had a greater effect on decreasing the viability of Raw264.7 cells,but the effect could be effectively reduced by loading DOX into OMVs.Ce6 had little effect on the viability of Raw264.7 cells.Ce6/DOX-OMVs@M can generate a large amount of ROS under photodynamic irradiation and exert anti-tumor effects.In vivo studies demonstrated that Ce6/DOX-OMVs@M had a strong combinational anti-tumor effect and the tumor volume was not increased during the treatment,and almost all the bioluminescence of tumor cells disappeared after two times of treatment,and the tumors in most mice were almost eliminated and cannot be surgically excised.TUNEL immunofluorescent staining and Ki67 immunohistochemistry results also showed that Ce6/DOX-OMVs@M massively killed tumor cells and inhibited tumor cell proliferation.Compared with the PBS control group,all treatment group reduced tumor metastasis to the lung.The body weight of some mice in the treatment group decreased during the treatment period but recovered soon after the treatment,indicating that Ce6/DOX-OMVs@M is relatively safe with high biocompatibility.To further explore the anti-tumor mechanism of OMVs.After co-incubating Raw264.7 cells with OMVs,both the morphology and phenotype shift to M1-like macrophage.Immunofluorescence staining of tumors also indicate that there were more M1 macrophages in the OMVs treatment group,suggesting the anti-tumor effect of OMVs may be related to its ability to polarize macrophages.In addition,after co-incubating 4T1tumor cells with OMVs,the expressions of pyroptosis pathway-related proteins such as NLRP3,Caspase-1 and Gasdermin D were significantly increased after the tumor-bearing mice were injected with OMVs.Tumor tissue q RCR and transcriptome sequencing also showed that the expression of multiple pyroptosis-related transcriptomes was up-regulated,indicating that the anti-tumor effect of OMVs was also related to the activation of tumor cell pyroptosis.CONCLUSIONSOMVs have immune-stimulating activity and anti-tumor effects,but the safety concern limited its application.A biomimetic delivery platform was constructed using macrophages to target tumors,and the chemotherapeutic drug DOX and photosensitizer Ce6 were co-loaded in OMVs as Ce6/DOX-OMVs@M for enhanced antitumor effects and laser-triggered release by combinational photodynamic/chemotherapy/immune therapy.The antitumor activity of OMVs may be related to their induction of macrophage polarization toward M1 antitumor phenotype and activation of pyroptosis-related pathways in tumor cells.Two times of Ce6/DOX-OMVs@M treatment partially eliminating tumors and inhibiting metastasis in mice.The successful application of Ce6/DOX-OMVs@M provided a new strategy for the development of OMVs-based tumor therapy.Part 3.Tin monosulfide（Sn S）nanodots boost antitumor immunity through enhanced sonodynamic therapyMETHODSStannous sulfide nanodots（Sn SNDs）were synthesized by the liquid phase exfoliation method,and Sn SNDs@PEG were prepared by PEG modification on the surface of Sn SNDs.The Sn SNDs@PEG were characterized by particle size,transmission electron microscopy（TEM）,diffuse reflectance spectroscopy,X-ray electron spectroscopy（XPS）,X-ray diffraction（XRD）and Fourier transform infrared transform（FTIR）.The photothermal properties of Sn SNDs@PEG were investigated in vitro by near-infrared NIR₈₀₈ irradiation,and the sonodynamic properties of Sn SNDs@PEG,including ROS generation and GSH depletion,were investigated by probe ultrasound（US）.Cellular experiments were performed to investigate the biocompatibility,cellular uptake,and in vitro sonodynamic therapy（SDT）efficacy of Sn SNDs@PEG.The tumor collagen-mimicking gel was prepared,and the ability of Sn SNDs@PEG to degrade tumor collagen and promote the penetration of Sn SNDs@PEG was investigated in vitro.4T1 triple-negative breast cancer mouse models were constructed to investigate the biodistribution and tumor targeting of Sn SNDs@PEG,and to evaluate the anti-tumor efficacy and enhancement of anti-tumor immunity of Sn SNDs@PEG-based enhanced SDT.RESULTSSn SNDs showed nanodot structure under TEM,and the d-spacing is0.28 nm.The particle size of Sn SNDs@PEG measured by DLS is 32.6±1.8 nm.The calculated band gap of Sn SNDs is 1.18 e V.XPS results showed that Sn SNDs contained Sn and S elements,XRD results showed typical Sn S structure,and FTIR and thermogravimetric analysis confirmed the PEG modification on Sn SNDs.1,3-Diphenylisobenzofuran（DPBF）and methylene blue（MB）assays demonstrated that US-irradiated Sn SNDs can efficiently generate ¹O₂and·OH,respectively.The DTNB indicator demonstrated that US-irradiated Sn SNDs could also effectively deplete GSH,further enhancing the ROS level.Sn SNDs@PEG showed a mild photothermal effect under NIR₈₀₈ irradiation.Sn SNDs@PEG showed high biocompatibility and can be effectively taken up by tumor cells.In the absence of external irradiations,Sn SNDs@PEG is not cytotoxic,but it can effectively increase the level of intracellular ROS and kill tumor cells in the presence of US irradiation.In 4T1 tumor-bearing mouse models,Sn SNDs@PEG showed accumulation at the tumor site after intravenous injection,and the temperature of the tumor site can be effectively and smoothly increased after under NIR irradiation.Significant accumulation of Sn SNDs@PEG and the generation of ROS at the tumor site can be observed in tumor sections,while Masson staining showed a significant reduction of collagen fibers.The therapeutic effect of Sn SNDs@PEG-based strategy on tumor was investigated in 4T1 tumor-bearing mice.The results showed that a single US-irradiated Sn SNDs@PEG treatment significantly inhibited tumor growth,the combination with NIR₈₀₈ further enhanced the anti-tumor efficacy,and two consecutive treatments successfully eliminated tumors without recurrence.The effect of this therapeutic strategy on anti-tumor immunity was further investigated.It was found that this treatment strategy can effectively enhance the infiltration of cytotoxic T cells（CTL）in the spleen and lymph nodes,and significantly increase the level of CTL at the tumor site.CONCLUSIONSSn SNDs have a low band gap,and their efficient ROS generation ability makes them useful as potential sonosensitizers for tumor SDT.In addition,the mild photothermal effect of Sn SNDs@PEG can simultaneously increase the oxygen supply at the tumor site and degrade the tumor matrix,thereby enhancing the penetration of Sn SNDs@PEG at the tumor site and the infiltration of immune cells to enhance SDT and anti-tumor immunity,making it useful as a promising multifunctional tumor therapy nanoplatform.