A Porphyrin Nanomaterial for Photoimmunotherapy for Treatment of Melanoma

Abstract

The incidence of melanoma, the third most common skin cancer, has been on the rise in recent years. In addition, it has a high mortality rate due to its high aggressiveness. Phototherapy, as a promising treatment method, can effectively kill tumor cells, but it is incapable of the treatment of tumor metastasis. Herein, a nanomaterial (TPC@OVA NPs) is developed for phototherapy in conjunction with immunotherapy against melanoma. TPC, as a derivative of porphyrin, is used as a photosensitizer with excellent biosafety and photostability. After assembly with ovalbumin (OVA), TPC@OVA NPs with vaccine properties is formed, which can not only ablate the primary tumor but also induce immunogenic cell death (ICD). In addition, DC cells can be stimulated to mature by exogenous OVA, enhancing the immune response against tumors by further activating T lymphocytes. Combined with immune checkpoint inhibitor aPD-1, the immune microenvironment is reshaped, and the increased activity of immunotherapy are validated. This work highlights the potential of combining phototherapy and immunotherapy against metastasis.

Keywords: combination therapy; immunogenic cell death; melanoma; phototherapy; tumor vaccine.

Scheme 1

Schematic diagram of TPC@OVA NPs photoimmunotherapy for treatment of melanoma.

Figure 1

Characterization of TPC@OVA NPs. A) Particle size and PDI of TPC@OVA NPs with different proportions of OVA: TPC. B) Size of TPC@OVA NPs in deionized water. C) TEM image of TPC@OVA NPs. D) SDS‐PAGE analysis of OVA and TPC@OVA NPs. E) The UV–vis spectra of TPC and TPC@OVA NPs. F) The fluorescence spectra of TPC and TPC@OVA NPs. G) FTIR spectra of TPC@OVA NPs and OVA. H) Particle size and PDI change curves of TPC@OVA NPs after treatment with Urea, SDS, and Triton X‐100, respectively. I) Variations of hydrodynamic diameter and PDI of TPC@OVA NPs in PBS solution containing 10% FBS for a week. Bars express SD (n = 3).

Figure 2

Photothermal and photodynamic performance of TPC@OVA NPs. A) The temperature changes of water, OVA, and TPC@OVA NPs under illumination. B) Warming curves of different concentrations of TPC@OVA NPs after illumination. C) Temperature rise curves of TPC@OVA NPs irradiated at different power densities. D) Heat‐up and cool‐down curves of TPC@OVA NPs. E) A linear relationship between negative logarithm of temperature change and cooling time. F) Temperature changes of TPC@OVA NPs during 5 cycles of heating and cooling. Changes in UV absorption values of TPC@OVA NPs G) and ICG H) before and after illumination (inset: before illumination on the left and after illumination on the right). I) UV absorption curves at 417 nm of DPBF after different treatments (L:685 nm, 0.2 W cm⁻²).

Figure 3

Phototherapy performance of TPC@OVA NPs in cells. A) Fluorescent pictures of B16‐OVA cells treated with TPC@OVA NPs for 0.5, 2, and 4 h. Scale bar, 20 µm. B) The fluorescence of ROS in cells after different treatments. Scale bar, 50 µm. C) Quantitative fluorescence of B16‐OVA cells after incubation with TPC@OVA NPs for 0.5, 2, and 4 h by FCM. Cytoactive of B16‐OVA cells treated with OVA D) and TPC@OVA NPs E). (F) Fluorescence images of live and dead cells co‐stained B16‐OVA cells treated with different conditions. Scale bar, 100 µm. G) The analysis of apoptotic and necrotic B16‐OVA cells after various treatments. L: 685 nm, 0.6 W  cm⁻². Bars express SD (n = 3).

Figure 4

In vitro immune response. CLSM images of CRT A) and HMGB1 B) exposure in B16‐OVA cells after various treatments. Scale bar, 20 µm. C) The content of ATP in each group supernatant. Immuno‐inflammatory cytokines secretion of TNF‐α D), IFN‐γ E), and IL‐6 F) in different groups detected by ELISA. G) The quantification of DC2.4 cells maturation after different treatments. H) Analysis of DC2.4 cells maturation by FCM after incubation with different cellular supernatants. Means ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001 and ****p <0.0001.

Figure 5

The antitumor effect in vivo. A) In vivo fluorescence images of tumors after intravenous injection of TPC@OVA NPs for different times. B) Fluorescent images of major organs 24 h after injection. C) Infrared thermography of tumor sites during irradiation (685 nm laser, 0.6 W cm⁻²) in mice 24 h after intravenous injection with TPC@OVA NPs (6 mg kg⁻¹). D) Temperature detection values of tumor sites in mice during 10 min of illumination. E) The timeline of the mice receiving various treatments. Volume of in situ F) and distal I) tumors in each group of mice. The weights G) and photos H) of primary tumors in different groups. The weights J) and photos K) of distant tumors in each group. H&E L) and TUNEL M) staining of excised tumor sections of each group. Scale bar: 100 µm. N) Ki67 staining of slices of excised tumors after various treatments. Scale bar: 50 µm.*P < 0.05, **P < 0.01, and ***P < 0.001, ****p <0.0001.

Figure 6

In vivo immunostimulatory ability. FCM analysis and the corresponding quantitative result of maturated DCs A, C) in TDLN in different groups. FCM analysis and the corresponding quantitative result of CD8⁺ T cells B, D) and MDSCs E, G) in tumors of different groups. Corresponding quantitative analysis of CD8⁺ T cells within spleen F) and TDLN H) in different groups. Cytokine levels of TNF‐α I), IFN‐γ J), and IL‐6 K) after different treatments. Immunofluorescence staining results of CRT L), HMGB‐1 M), CD8 T cells N), and Treg cells O) expression in tumor tissues of different groups. Scale bar: 50 µm. *P < 0.05, **P < 0.01, and ***P < 0.001, ****p <0.0001.