Smart Assembled Human Serum Albumin Nanodrug Delivery System For Breast Cancer Treatment By Chemo-photothermal Therapy

Breast cancer,a highly aggressive and metastatic malignancy,is the most common diagnostic cancer among women and is one of the leading causes of premature death among women worldwide.Studies have shown that due to low selectivity and non-targeting,single chemotherapy drugs,such as doxorubicin（DOX）,are difficult to achieve complete elimination of tumors.In addition,multidrug resistance（MDR）will occur after long-term use of chemotherapy drugs.Photothermal therapy（PTT）,as a non-invasive treatment,shows low toxicity and high selectivity,which provides a new strategy for the treatment of breast cancer.However,single PTT for photothermal tumor ablation relies on hyperthermia（>65℃）.Excessive fever will cause overheating damage to the surrounding normal tissues and reduce the patient’s compliance.At the same time,excessive fever will produce heat shock phenomenon,thus affecting the efficacy of PTT.Although mild PTT can effectively overcome the deficiency of hyperthermia,it cannot be used for tumor monotherapy because of the reduced tumor photothermal ablation efficacy.Therefore,the combination of mild PTT and chemotherapy for anti-tumor therapy has become a new option.The key to the realization of combination therapy is the design of drug delivery system.In recent years,the commonly used nano-drug delivery systems include but are not limited to liposomes,micelles,dendrimers,etc.Among them,albumin-based nano-drug delivery system has been widely used in the delivery of anti-tumor drugs.However,the stability of traditional albumin nanoparticles depends on the use of crosslinking agents（such as glutaraldehyde）,which may cause complications.Therefore,how to prepare albumin nanoparticles with high stability while avoiding the use of crosslinking agents is the key to the application of albumin nanodrug delivery system in combination therapy.In this study,human serum albumin（HSA）with high biocompatibility,high biodegradability,no antigenicity and rich functional groups was used as carrier.The stable and reduction sensitive HSA NPs were prepared by reduction self-assembly method without crosslinking agent addition,just utilizing the abundant disulfide bonds and free sulfhydryl groups in HSA structure.The average hydrodynamic diameter of HSA NPs was 126.89±1.66 nm and the average zeta potential was-27.34±0.65 m V.Doxorubicin（DOX）and ICG were co-loaded into HSA NPs to obtain albumin loaded nanoparticles（DI@HSA NPs）.The average hydrodynamic diameter of DI@HSA NPs was 122.79±0.51 nm,the average zeta potential was-27.66±0.77 m V and PDI was 0.26±0.06.Transmission electron microscopy（TEM）showed that DI@HSA NPs was approximately spherical with uniform size.When the feed ratio was 1:1,the drug loading rate of DOX and ICG determined by UV spectrophotometry and fluorescence spectrophotometry were5.39%and 5.34%,respectively,and the encapsulation efficiency was 97.06%and 96.7%,respectively.In aqueous medium,the temperature of DI@HSA NPs increased by about26℃under the irradiation of near infrared（NIR）laser,which was equivalent to the photothermal conversion efficiency of free ICG in vitro.The results of release kinetics showed that DI@HSA NPs exhibited a redox-responsive behaviors.The cumulative release of DOX was more than 60%after being treated with 10 m M GSH for 8 h.Compared with free ICG,DI@HSA NPs can significantly enhance the stability of aqueous solution and physical conditions.4T1 cells were used as model cells.The cytotoxicity of 4T1 cells was significantly enhanced by DI@HSA NPs mediated chemo-photothermal synergism（combination therapy index,CI=0.72）.Compared with free ICG group and free DOX group,the IC₅₀of DI@HSA NPs group decreased by 2.8 and 2.6 folds,respectively.The results of cell uptake showed that the uptake of DI@HSA NPs by 4T1 cells was positively correlated with the incubation time.Laser confocal microscopy exhibited that the uptake of DI@HSA NPs with laser irradiation group was significantly higher than that of without laser irradiation group.Compared with ICG@HSA NPs plus laser illumination group and DI@HSA NPs without laser irradiation group,flow cytometry results showed that DI@HSA NPs plus laser illumination significantly increased the apoptosis/necrosis ratio of 4T1 cells.4T1 orthotopic breast cancer model was established.In vivo biodistribution assay,DI@HSA NPs showed exhibited enhanced tumor-accumulation and tumor-retention capabilities.After 48 h of intravenous injection,the fluorescence signal intensity of ICG in DI@HSA NPs group was 1.9 folds higher than that of free ICG.After irradiation with NIR laser,the results of real-time infrared thermography showed that,compared with free ICG and PBS,the tumor temperature of DI@HSA NPs group increased slowly and persistently 48.1℃and increased by 13.1℃,which is conducive to achieving mild tumor photothermal ablation.Compared with the normal saline group,the growth inhibition rates of free ICG,free DOX,ICG@HSA NPs,DI@HSA NPs,DI@HSA NPs and DI@HSA NPs were 33.16%,26.44%,54.2%,40.83%and 97.41%,respectively.The lung metastasis inhibition rate of DI@HSA NPs group was up to 94.91%.These results indicate that DI@HSA NPs mediated chemo-photothermal therapy can significantly inhibited the growth and metastasis of breast cancer.Compared with other groups,the expression of Ki67 in DI@HSA NPs group was significantly decreased,suggested the proliferation of tumor cells was decreased;the expression of Caspase-3 was increased,suggested the apoptosis of tumor cells was increased.The results of immunofluorescence staining showed that DI@HSA NPs significantly enhanced the infiltration of CD4⁺and CD8⁺T cells in tumor,and significant immune activation ability.This study shows that the combination of albumin based redox-responsive nanodrug delivery system（DI@HSA NPs）can significantly inhibit the growth and metastasis of breast cancer,and activate the immune system,providing a potential strategy for the future therapy of breast cancer.