Injectable thermosensitive hydrogel co-loading with ATRi and doxorubicin for the treatment of triple-negative breast cancer

Abstract

Chemotherapy has been the first-line treatment option for cancer. However, acquired chemo-resistance led by DNA damage repair (DDR) of cancer cells and serious side-effects of chemotherapeutic agents are huge hurdles to effectively suppress metastatic tumors. Herein, we developed an injectable thermosensitive hydrogel for localized co-delivery of ATRi-BAY-1895344 (BAY) and doxorubicin (DOX), serving as a localized drug depot to minimize systemic toxicity while ensuring sustained tumor-specific drug release exceeding 4 days. The in vitro cumulative drug release rate of DOX and BAY reached up to 73.9% and 63.3% under pH 6.5 conditions. This study pioneers the synergistic combination of a DNA-damaging agent and Ataxia telangiectasia and RAD3-related (ATR) kinase inhibitor ATRi to disrupt the DDR pathway. The ATRi-mediated inhibition of ATR kinase effectively disrupts the replication stress response by impairing the repair of DOX-induced DNA lesions. This dual mechanism significantly enhances tumor cell vulnerability to chemotherapy, ultimately achieving an 8-fold increase in chemosensitivity compared to monotherapy regimens. In triple-negative breast cancer models, the hydrogel-based DOX + BAY@Gel formulation achieved a tumor inhibition rate of 79.4%, significantly surpassing the 58% observed with free DOX monotherapy. This dual-action strategy overcomes chemo-resistance by disabling DDR compensatory mechanisms and prolongs tumor suppression through controlled drug release. The hydrogel platform represents a functional innovation in localized combination therapy, integrating stimuli-responsive drug delivery with DDR pathway disruption for synergistic efficacy.

Scheme 1. Schematic illustration of DOX + BAY@Gel localized and extended release of DOX and BAY in the treatment of tumor. DOX + BAY@Gel is prepared by cross-linking CS and β-GP before performing thermosensitive gelation in vivo. Then the localized injection of DOX + BAY@Gel reduces the size of the tumor by increasing the concentration of DOX and BAY in the tumor. DOX can trigger DSBs and BAY induces more severe DNA damage by inhibiting the DNA damage repair (DDR).

Fig. 1. Characterization of hydrogel. (a) The blank gel was prepared using different CS to β-GP ratios at 25 °C before undergoing thermosensitive gelation at 37 °C. (b) SEM images of a mixture of CS and β-glycerol phosphate disodium salt pentahydrate. (c) SEM images of blank hydrogel. (d) SEM images of DOX + BAY@Gel. Scale bar = 100 μm. (e) FT-IR spectra of a hydrogel. (f) Rheological analysis of blank hydrogel at 37 °C. (g) Rheological analysis of blank hydrogel at different temperatures (G′ is the storage modulus, G′′ is the loss modulus). (h) The release curve of DOX in DOX + BAY@Gel at pH = 7.4, 6.5 and 5.5. (i) The release curve of BAY in DOX + BAY@Gel at pH = 7.4, 6.5 and 5.5.

Fig. 2. Therapeutic efficacy of DOX + BAY in vitro. (a) CCK-8 assay of co-incubate MCF-10A and 4T1 cells with 0, 0.125, 0.25, 0.5, 0.75, 1 μmol per L DOX for 48 h. (b) CCK-8 assay of co-incubate MCF-10A and 4T1 cells with 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1 μmol per L BAY for 48 h. (c) Different administration of DOX and BAY on 4T1 cells. (d) CCK-8 assay of co-incubate 4T1 cells with 0.5 μmol per L DOX plus 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1 μmol per L BAY for 48 h. (e) Combination index (CI) in 4T1 cells upon treatment with BAY and DOX. The CI was calculated using cell viability data presented in (a, b and d). (f) The statistic of cell apoptosis at 48 h. (g) The analysis of cell apoptosis by flow cytometric at 48 h. Statistical analysis: group DOX + BAY was compared with other groups. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 3. The mechanism of DSBs. (a) Images of DSBs tested by γ-H2AX foci. Scale bar = 20 μm. (b) The corresponding number of γ-H2AX foci in cells. (c) The protein expression level of γ-H2AX. (d) The corresponding protein expression level of the G2/M pathway. (e) Cell cycle analysis by flow cytometric. (f) The statistic of the cell cycle. Statistical analysis: group control was compared with other groups. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4. The antitumor efficacy of DOX + BAY@Gel in vivo. (a) Schematic illustration therapeutic design (b) The volume of tumors in each group. (c) Tumor volume growth curves of mice. (d) The average weight of the tumor from each group. Statistical analysis: group DOX + BAY@Gel was compared with other groups. Data are presented as mean ± SD (***p < 0.001). (e) Body weight curves of mice. (f) The survival curves of mice. (g) Images of H&E, TUNEL, and ki67 staining of tumor slices. Scale bar = 100 μm.

Fig. 5. The safety and biocompatibility of DOX + BAY@Gel in vivo. (a) Images of in vivo degradation of hydrogels (n = 3 mice/group). (b) The statistic of (a). Group 21 was compared with group 0. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). (c) Cell viability of HUVEC cells after indirect treatment with CS/β-GP Gel. (d) The blood routine analysis of BALB/c mice (n = 5 mice/group) after 14 days of different treatment. (e) The pivotal blood biochemical indicators of BALB/c mice (n = 5 mice/group) after 14 days of different treatment. Data are presented as mean ± SD (^nsp > 0.05, *p < 0.05, **p < 0.01). (f) H&E staining of the major organs. Scale bar = 100 μm.