ROS-responsive Upconversion Nano-drug Delivery Systems For Targeted Triplenegative Breast Cancer Therapy

Triple negative breast cancer(TNBC)is a special subtype of breast cancer,which is known as the traits of early metastasis,high invasiveness,high recurrence rate,high mortality and poor prognosis.The conventional clinical treatments(radiotherapy,chemotherapy,surgery)for TNBC have a lot of disadvantages,such as limited therapeutic effects and serious side effects.Photodynamic therapy(PDT)is a new type of non-invasive tumor treatment.Its principle is to use photosensitizers,which can produce reactive oxygen species(ROS)with visible light irradiation to kill cancer cells,and PDT can also induce immunogenic cell death(ICD),which can activate systemic anti-tumor immune responses.However,traditional PDT is limited by poor tissue penetration of visible light,lack of tumor targeting of photosensitizers,and immunosuppression of tumor microenvironment,so it is ineffective in deep tumor treatment and has limited clinical applications.Upconversion nanoparticles(UCNPs),as a novel inorganic nanomaterial,can emit visible light with higher energy under the excitation of near-infrared light(NIR)to overcome the defect of poor tissue penetration of traditional light sources.UCNPs can also carry drugs to achieve targeted delivery,so as to solve the deficiencies of photosensitizer’s poor targeting and low bioavailability.However,due to the multidrug resistance and immunosuppressive microenvironment of tumors,a single treatment cannot completely inhibit the TNBC’s development.In recent years,the combination therapy of chemotherapy and PDT has attracted many attentions in preclinical and clinical studies of tumors.On one hand,chemotherapy can enhance the sensitivity of tumors to PDT and realize the synergistic anti-tumor effects of chemotherapy and PDT;on the other hand,some chemotherapeutic drugs and ROS generated by PDT can both induce ICD in tumors,and enhance tumors’immunogenicity,promoting the recognition and clearance of tumors by the immune system.Therefore,the use of upconversion nanomaterials with stimulus-responsive drug delivery technology can achieve tumor-targeted delivery and controlled release of drugs,which is expected to achieve the synergistic anti-tumor effects of deep PDT combined with chemotherapy and improve the effect of ICD-induced anti-tumor immunotherapy.In this study,morphologically intact and uniformly scaled UCNPs with an average particle size of 42.13±1.42 nm were synthesized by high-temperature solvothermal method.UCNPs could emit 540 nm fluorescence under 980 nm NIR excitation,which coincided with the maximum absorption peak of the photosensitizer rose bengal(RB),thus UCNPs could activate RB to produce ROS.Meanwhile,we used sialic acid(SA)as the tumor-targeting ligand and thioketal(TK)as the ROS-responsive linker to synthesize the ROS-responsive polymer SA-PEG-TK-PLGA(SPTP)by esterification and amide reactions.SPTP could self-assemble in water to form micelles.The chemotherapeutic drug doxorubicin(DOX)and photosensitizer RB were applied to prepare the ROS-responsive SA-modified upconversion nano-delivery system(SPTP@UCNP-RB-DOX),using the method of emulsification solvent volatilization and dialysis.The particle size of SPTP@UCNP-RB-DOX was 64.58±2.18 nm,and the drug loading of DOX and RB was4.87±0.24%and 4.36±0.04%,respectively.SPTP@UCNP-RB-DOX could activate RB to produce ROS under NIR irradiation,and excessive ROS would trigger SPTP depolymerization,so that DOX could be quickly released more than 75%at 14 h.The receptor-mediated endocytosis of 4T1 cells was investigated.The results showed that SPTP@UCNP-RB-DOX was more easily taken up by 4T1 cells,because SA modified on the surface of nanoparticles could specifically bind to E-selectin receptor,which overexpressed on the surface of tumor cells.SPTP@UCNP-RB-DOX could increase intracellular levels of ROS under NIR irradiation,which could lead to the quick release of DOX and lysosomal escape,thus effectively killing tumor cells.NIR treated SPTP@UCNP-RB-DOX(50μg/m L)was incubated with 4T1 cells for 24 h,resulting in about 52.9%cell death.At the same time,both DOX and ROS could induce tumor ICD and increase the exposure levels of damage-associated molecular patterns(DAMPs).In addition,SPTP@UCNP-RB-DOX showed brilliant permeability in the multicellular cancer spheroids(MCSs)and significantly promoted cell apoptosis after NIR irradiation,showing a strong inhibition on MCSs’growth.The TNBC murine model was established by injecting 4T1 cells into the breast pad of mice,and the bio-distribution of nanoparticles in vivo was investigated.The results showed that SPTP@UCNP-RB-DOX could escape the retention of reticuloendothelial system after intravenous injection,and quickly accumulate in tumor sites with good tumor targeting ability.After tumor-bearing mice treated with SPTP@UCNP-RB-DOX and NIR irradiation,the growths of tumors were significantly inhibited,large areas of necrosis and apoptosis appeared in tumor tissues,tumor metastases were reduced in lung tissues,and the expressions of Ki67 were significantly down-regulated,and the survival periods of mice were prolonged.These results indicated that this therapeutic strategy had the best antitumor effects and could effectively inhibit spontaneous pulmonary metastasis of TNBC.At the same time,SPTP@UCNP-RB-DOX with NIR treatment could effectively induce tumor ICD,improve the exposure levels of DAMPs,promote the infiltration of CD8~+T cells in tumor tissues,and effectively activate the systemic anti-tumor immune responses.During the treatment period,the weights of the mice remained stable.The liver and kidney functions of mice were normal,and no obvious cardiotoxicity was caused,indicating that SPTP@UCNP-RB-DOX had a good safety in vivo.Tumor recurrence and metastasis are the main reasons for the high mortality rate of TNBC.Although the SPTP@UCNP-RB-DOX could achieve systemic anti-tumor immune responses induced by NIR-controlled PDT combined with chemotherapy,recent studies have shown that the efficacy of PDT combined with chemotherapy is limited.It is not sufficient to eliminate tumor residual tissues and inhibit metastasis,because tumor ICD is often immunosuppressed by tumor microenvironment.When tumors undergo ICD,a large amount of adenosine triphosphate(ATP)is released to recruit dendritic cells(DCs)and promote their maturation.However,ATP is quickly degraded to adenosine(ADO)by the overexpressed extracellular nucleotide enzyme CD73 in the tumor microenvironment,which activates the inhibitory pathway of immune cells.Therefore,the combination of PDT with chemotherapy and anti-CD73 antibody can block the immunosuppression induced by ADO and strengthen the specific anti-tumor immune responses activated by ICD.In recent years,inspired by biological systems in nature,nanoparticles have been functionalized by cell membranes to make them have good bionic properties,which have got a lot of attentions.Among them,the unique functional membrane proteins of cancer cell membranes enable them to have the unique abilities of immune system evasion and homologous tumor targeting.Thus,the nanoparticles functionalized with cancer cell membranes can have the abilities of long circulation and tumor targeted delivery in vivo.In view of the deficiency of SPTP@UCNP-RB-DOX in the first part of the study,the drug delivery strategy of covalent bonding was adopted in this study.Firstly,the ROS-responsive prodrug PEG-TK-DOX(PTD)was synthesized by chemical bonding reactions.Subsequently,the photosensitizer RB and ROS-responsive prodrug PTD were directly grafted onto the surface of UCNPs by amide reaction and addition reaction respectively to prepare the ROS-responsive co-bonded RB and DOX upconversion nanoparticles(UCNP-RB/PTD),with the particle size of 48.85±4.30 nm,and the drug loading of RB and DOX were 2.03±0.21%and 2.43±0.14%,respectively.With NIR irradiation,UCNP-RB/PTD showed a ROS-responsive drug release behavior,and the cumulative release rate of DOX reached more than 60%at 12 h.Further,4T1 cell membranes were ultrasonicated with UCNP-RB/PTD to fabricate the cancer membranes functionalized ROS-responsive upconversion nanoparticles(CM@UCNP-RB/PTD).The surface of CM@UCNP-RB/PTD successfully retained the functional membrane proteins of cancer cells,including CD47,N-cadherin and Ep CAM,which determined by western blotting and immunogold labeling.The cellular uptake behavior of the nanoparticles was investigated by using 4T1 cells and RAW264.7 cells.The results showed that the endocytosis of CM@UCNP-RB/PTD was significantly enhanced in 4T1 cells,indicating its ability to specifically target homologous tumor cells;the uptake of CM@UCNP-RB/PTD in RAW264.7 cells was significantly reduced,indicating its ability to escape macrophages’phagocytosis.The ROS level,drug releasing behavior and cytotoxicity in 4T1 cells were investigated.The results showed that CM@UCNP-RB/PTD(50μg/m L)with NIR irradiation could produce a large amount of ROS and induce DOX release at the cellular level.The released DOX could quickly escape from the lysosomes and enter the nuclei for therapeutic effects,resulting in about 57.6%cell death after 24 h co-incubation.At the same time,CM@UCNP-RB/PTD combined with NIR could significantly improve the exposure levels of DAMPs,induce tumor ICD,and effectively stimulate the maturation of DCs in the co-incubation system,which helped the immune system to recognize and kill tumor cells.The 4T1 tumor-bearing murine model was established by injecting 4T1 cells into the breast pad of mice,and the in vivo bio-distribution,tumor growth,spontaneous lung metastasis and immune activation were evaluated by intravenous injection of CM@UCNP-RB/PTD.The results showed that CM@UCNP-RB/PTD had a concentrated distribution in tumor sites after 2 h’s injection,and the accumulation of nanoparticles in tumors increased with the extension of administration time.In comparison with other treatment groups,CM@UCNP-RB/PTD combined with anti-CD73 antibody and NIR treatment could maximally inhibit tumor growth,with the tumor inhibition rate of about93.4%,and effectively inhibit spontaneous lung metastasis and prolong the survival time of mice.At the same time,this strategy could significantly improve the exposure levels of DAMPs,and improve the proportions of mature DCs in the lymph nodes,from about7.1%in the saline group to about 30.3%,further promote cytotoxic T cells and the effective memory T cells’proliferation and infiltration,significant inhibit the regulatory T cells’differentiation,improve the contents of antitumor cytokines in tumors,spleens,and peripheral blood,which indicated that the tumor microenvironment was relieved and the systemic antitumor immune response was effectively activated.The inhibitory effects of immune surveillance function activated by this strategy on distant tumors and lung metastasis were investigated by constructing a bilateral tumor murine model and lung metastasis murine model.The results showed that CM@UCNP-RB/PTD combined with anti-CD73 antibody and NIR irradiation significantly inhibited the expressions of CD73in distant tumors,and had the best inhibitory effects on the growth of distant tumors,with a growth inhibition rate of about 89.7%,and significantly reduced the number of lung metastases within 15 days after drug administration.In addition,CM@UCNP-RB/PTD was safe in vivo and did not cause cell or tissue damages.In view of the rapid growth and high invasions of TNBC,and lack of effective clinical therapies,this study constructed two kinds of ROS-responsive upconversion nano-drug delivery systems,both of which could achieve targeted delivery and controlled drug release in tumor sites,producing synergistic effects of chemotherapy and PDT under NIR irradiation.In this treatment process,ICD could be induced to improve the tumor immunogenicity,and alleviated the immunosuppression of tumor microenvironment,and effectively activated the systemic anti-tumor immune responses,inhibited tumor metastasis and recurrence,which finally realized the safe and efficient antitumor effects of PDT,chemotherapy and immunotherapy.These novel therapeutic strategies provided new methods and ideas for clinical treatment of TNBC.