In-situ-sprayed therapeutic hydrogel for oxygen-actuated Janus regulation of postsurgical tumor recurrence/metastasis and wound healing

Abstract

Surgery is the mainstay of treatment modality for malignant melanoma. However, the deteriorative hypoxic microenvironment after surgery is recognized as a stemming cause for tumor recurrence/metastasis and delayed wound healing. Here we design and construct a sprayable therapeutic hydrogel (HIL@Z/P/H) encapsulating tumor-targeted nanodrug and photosynthetic cyanobacteria (PCC 7942) to prevent tumor recurrence/metastasis while promote wound healing. In a postsurgical B16F10 melanoma model in female mice, the nanodrug can disrupt cellular redox homeostasis via the photodynamic therapy-induced cascade reactions within tumor cells. Besides, the photosynthetically generated O₂ by PCC 7942 can not only potentiate the oxidative stress-triggered cell death to prevent local recurrence of residual tumor cells, but also block the signaling pathway of hypoxia-inducible factor 1α to inhibit their distant metastasis. Furthermore, the long-lasting O₂ supply and PCC 7942-secreted extracellular vesicles can jointly promote angiogenesis and accelerate the wound healing process. Taken together, the developed HIL@Z/P/H capable of preventing tumor recurrence/metastasis while promoting wound healing shows great application potential for postsurgical cancer therapy.

Fig. 1. Schematic illustration of sprayable HIL@Z/P/H for efficiently preventing tumor recurrence/metastasis and simultaneously promoting wound healing during the postsurgical cancer treatment.

a Preparation of HIL@Z nanodrug. b Schematic showing the in situ formation and action mechanism of sprayed HIL@Z/P/H containing HIL@Z nanodrug and PCC 7942 within the postsurgical wound bed. Under Red laser irradiation, HIL@Z/P/H produces abundant O₂ through photosynthesis and effectively relieved the hypoxia microenvironment. In tumor cells, the intracellular cascade reactions induced by HIL@Z nanodrug generate plentiful reactive species (ROS, NO and RNS) and lower the GSH level, accompanied by significant HIF-1α downregulation with the aid of O₂, resulting in effective inhibition of residual tumor recurrence/metastasis. Within the postsurgical wound, the excessively generated O₂ and PCC 7942-secreted EVs accelerate the wound healing process by downregulating HIF-1α expression and upregulating VEGF level.

Fig. 2. Characterization of nanoparticles.

a SEM, b TEM and c element mapping images of HIL@Z. d Size distribution, e zeta potential patterns, f UV-vis spectra, g FT-IR spectra, and h XRD patterns of different nanoparticles. i The cumulative release profiles of ICG from HIL@Z in PBS with different pH values. j Time-dependent absorbance change of DPBF co-incubated with HIL@Z at 410 nm under NIR irradiation. k NO production by HIL@Z with and without NIR irradiation. l Fluorescence spectrum of ONOO^- characterized by DHR. The results in a, b were representative of three independent experiments. Data in e, i, k were presented as mean ± SD, n = 3 independent samples. Source data are provided as a Source Data file.

Fig. 3. Characterization and photosynthetic O₂-producing capacity of sprayable HIL@Z/P/H.

a Photographs of different hydrogels before and after gelation. b SEM images showing the microstructures of HIL@Z/P/H with different magnifications (inset: photograph of the lyophilized HIL@Z/P/H). c Element mapping images of HIL@Z/P/H. d Fluorescence images of HIL@Z/P/H. e Released dissolved O₂ during the storage of HIL@Z/P/H at different days. f Light-triggered cyclic O₂ production of HIL@Z/P/H. The results in b, d were representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 4. In vitro cytophagocytosis and cellular oxygenation.

a Fluorescence images of B16F10 cells treated with different nanoparticles. b Flow cytometry analysis of Rhm B signal and c corresponding mean fluorescence intensities (MFI) in B16F10 cells treated with different nanoparticles. Fluorescence images of B16F10 cells incubated with HIL@Z for 2.5 h (d) and 4 h (f). Blue fluorescence represents the nucleus, red fluorescence represents Rhm B and green fluorescence represents Lyso-Tracker. e and g are the line scan profiles of the fluorescence intensities at the white arrows in d and f, respectively. h Fluorescence images of Ru(dpp)₃Cl₂-stained B16F10 cells after different treatments and i their corresponding fluorescence intensities. j Western blotting (WB) analysis of HIF-1α/MMP-9 protein expressions in B16F10 cells after different treatments. k Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of HIF-1α mRNA expression in B16F10 cells after different treatments. The results in a, d, f were representative of three independent experiments. Data in c, i, k were presented as mean ± SD, n = 3 biologically independent samples. P values were calculated via multiple comparisons one-way ANOVA method t-test. Source data are provided as a Source Data file.

Fig. 5. In vitro evaluation of the anticancer effect of HIL@Z/P/H on B16F10 cells.

a The relative cell viabilities of HUVECs and B16F10 cells co-cultured with different concentrations of HIL@Z/P/H. b The relative cell viabilities of B16F10 cells after different treatments. c Fluorescence images of live/dead staining of B16F10 cells in different groups. d Flow cytometry analysis of the B16F10 cell apoptosis in different groups. e Population of early apoptotic, apoptotic, and necrotic B16F10 cells. f Fluorescence images showing intracellular ROS, NO, and RNS detection in B16F10 cells. g, j Flow cytometric assay and corresponding MFI of B16F10 cells stained with DCFH-DA (ROS fluorescent probe) after different treatments. h, k Flow cytometric assay and corresponding MFI of B16F10 cells stained with DAF-FM DA (NO fluorescent probe) after different treatments. i, l Flow cytometric assay and corresponding MFI of B16F10 cells stained with DHR (ONOO^– fluorescent probe) after different treatments. The results in c, f were representative of three independent experiments. Data in a, b, j–l were presented as mean ± SD, n = 3 biologically independent samples. P values were calculated via multiple comparisons one-way ANOVA method t-test. Source data are provided as a Source Data file.

Fig. 6. In vivo antitumor performance of HIL@Z/P/H on an incomplete melanoma resection model.

a Schematic illustration of HIL@Z/P/H for inhibiting tumor recurrence in an incomplete melanoma resection model. b Photographs of tumor/wound sites in different groups during the 14-day treatment period. c Photographs of the excised tumors after different treatments on day 14. Changes of d tumor volume, e tumor weight, f mouse survival rate, g H&E, TUNEL, and Ki67 and HIF-1α stained tumor slices and h–j their quantification analysis in different groups. k Photographs showing metastatic nodules (red circles) in lung tissues. Treatments: (1) Control, (2) Red+NIR, (3) HIL@Z/P/H (4) HL@Z/P/H+NIR, (5) HI@Z/P/H+NIR, (6) HIL@Z/P/H+Red, (7) HIL@Z/P/H+NIR, (8) HIL@Z/P/H+Red+NIR. The results in g, k were representative of three independent mice. Data in d–f, h–j were presented as mean ± SD, n = 3 biologically independent mice in h–j, n = 5 biologically independent mice in d–f. P values were calculated via multiple comparisons one-way ANOVA method t-test. Source data are provided as a Source Data file.

Fig. 7. In vitro proangiogenic performance of PCC 7942.

a Illustration showing the wound-healing promotion of P/H. b TEM images of PCC 7942-secreted EVs. c Hydrated particle size distribution of PCC 7942-secreted EVs. d, e Representative images and quantification of HUVEC migration. f–h Representative images of HUVECs’ tube formation and quantification of tudes and nodes. The results in b were representative of three independent experiments. Data in e, g, h were presented as mean ± SD, n = 3 biologically independent samples. P values were calculated via multiple comparisons one-way ANOVA method t-test. Source data are provided as a Source Data file.

Fig. 8. In vivo promotion effect of HIL@Z/P/H on wound healing.

a Schematic illustration of HIL@Z/P/H for promoting wound healing in a mouse model with full-thickness skin defect. b Photographs of skin wounds, c traces of unhealed wounds, and (d) quantitative analysis of the wound areas in different groups during the treatment period. e Complete wound closure times in different groups. f H&E and h Masson staining of the wounds in different groups on day 12. Quantification of the g epithelial thickness and i collagen deposition in different groups on day 12. Data in d, e, g, i were presented as mean ± SD, n = 3 biologically independent mice in d, g, i, n = 5 biologically independent mice in e. P values were calculated via multiple comparisons one-way ANOVA method t-test. Source data are provided as a Source Data file.

Fig. 9. Immunohistological and quantitative analysis of wounds after 12 days of treatment.

a–d HIF-1α, VEGF, CD31 and α-SMA staining of wound tissues after different treatments. e–h Quantitative analysis of HIF-1α-positive area, VEGF-positive area, blood vessels and α-SMA-positive area in regenerated dermis after different treatments. Data in e–h were presented as mean ± SD, n = 3 biologically independent mice. P values were calculated via multiple comparisons one-way ANOVA method t-test. Source data are provided as a Source Data file.