Antitumor Immunity Mediated by Photodynamic Therapy Using Injectable Chitosan Hydrogels for Intratumoral and Sustained Drug Delivery

Abstract

Photodynamic therapy (PDT) is an anticancer therapy with proven efficacy; however, its application is often limited by prolonged skin photosensitivity and solubility issues associated with the phototherapeutic agents. Injectable hydrogels which can effectively provide intratumoral delivery of photosensitizers with sustained release are attracting increased interest for photodynamic cancer therapies. However, most of the hydrogels for PDT applications are based on systems with high complexity, and often, preclinical validation is not provided. Herein, we provide a simple and reliable pH-sensitive hydrogel formulation that presents appropriate rheological properties for intratumoral injection. For this, Temoporfin (m-THPC), which is one of the most potent clinical photosensitizers, was chemically modified to introduce functional groups that act as cross-linkers in the formation of chitosan-based hydrogels. The introduction of -COOH groups resulted in a water-soluble derivative, named PS2, that was the most promising candidate. Although PS2 was not internalized by the target cells, its extracellular activation caused effective damage to the cancer cells, which was likely mediated by lipid peroxidation. The injection of the hydrogel containing PS2 in the tumors was monitored by high-frequency ultrasounds and in vivo fluorescence imaging which confirmed the sustained release of PS2 for at least 72 h. Following local administration, light exposure was conducted one (single irradiation protocol) or three (multiple irradiation protocols) times. The latter delivered the best therapeutic outcomes, which included complete tumor regression and systemic anticancer immune responses. Immunological memory was induced as ∼75% of the mice cured with our strategy rejected a second rechallenge with live cancer cells. Additionally, the failure of PDT to treat immunocompromised mice bearing tumors reinforces the relevance of the host immune system. Finally, our strategy promotes anticancer immune responses that lead to the abscopal protection against distant metastases.

Figure 1

Schematic illustration of covalently cross-linked CS-based hydrogel photodynamic therapy against cancer. (A) Molecular structure of m-THPC and its tetrafunctionalized derivatives, PS1, 2, and 3. (B) Schematic representation of the hydrogel formation process and application for PDT.

Figure 2

Preparation and characterization of CS-based hydrogels containing m-THPC derivatives. (A) From viscous solution of CS (right) to cross-linked hydrogel loading PS2; (B) macroscopical evaluation of the self-healing properties of the PS1-PEG-CS hydrogel during 1 h after its physical disruption; (C) injectable properties of the PS2-PEG-CS hydrogel; (D) storage (G′) and loss (G′) moduli of the hydrogel formulations containing PS1, 2, or 3 as a function of angular frequency at a fixed strain of 5% (n = 3); (E) self-healing properties of all hydrogel formulations demonstrated by continuous step strain measurements (5% strain → 300% strain → 5% strain → 600% strain → 5% strain) (n = 3); and (F) pH-dependent release of PS1, 2, and 3 from their hydrogel scaffold at pH of 5.0, 6.5, and 7.5 (n = 2).

Figure 3

Dark toxicity and phototoxicity of m-THPC and the tetrafunctionalized derivatives PS1, 2, and 3. Dark toxicity evaluated in (A) B16F10 and (B) CT26 cancer cells; (C) phototoxicity of m-THPC and tetrafunctionalized derivatives PS1, 2, and 3 incubated with B16F10 melanoma cells for 24 h followed by illumination at 660 nm with a LD of 0.4 J/cm²; (D) phototoxicity of m-THPC and tetrafunctionalized derivatives PS1, 2, and 3 incubated with B16F10 melanoma cells for 24 h followed by illumination at 660 nm with a LD of 2 J/cm²; and (E) phototoxicity of m-THPC and tetrafunctionalized derivatives PS1, 2, and 3 incubated with B16F10 melanoma cells or (F) with CT26 colon carcinoma cells for 24 h followed by illumination at 660 nm with a LD of 4 J/cm²; ***p < 0.001 vsm-THPC.

Figure 4

Cellular uptake, phototoxicity, and self-aggregation in aqueous medium of m-THPC and the tetrafunctionalized derivatives PS1, 2, and 3. (A) Cell uptake of m-THPC and derivatives PS1, 2, and 3, after 24 h of incubation with B16F10 cancer cells. Bars indicate the mean ± SEM of three independent experiments; *** vsm-THPC. (B) Phototoxicity of m-THPC and tetrafunctionalized derivatives PS1, 2, and 3 in B16F10 melanoma or (C) in CT26 colon carcinoma cells by using an extracellular PDT protocol with a LD of 15 J/cm². Each point indicates the mean ± SEM of three independent experiments; ***p < 0.001, **p < 0.01, and *p < 0.05 vsm-THPC. (D–F) Representative absorption spectra of PS1, PS2, and PS3 recorded in pure DMSO and in PBS (0.4% DMSO).

Figure 5

m-THPC and PS2 activated extracellularly induced significant lipid peroxidation. (A) Quantitative analysis of lipid peroxidation, obtained by the 590/510 fluorescence ratio, on B16F10 cancer cells after extracellular PDT with PS2 (40 μM). Bars indicate the mean ± SEM of triplicates from one representative experiment with B16F10 cells, ***p < 0.001 vs untreated cells incubated with the BODIPY. (B) Oxidation of the BODIPY 581/591 C11 was evaluated by flow cytometry in B16F10 cancer cells after extracellular PDT with PS2 (20 and 40 μM) and m-THPC (20 and 40 μM). Cumene hydroperoxide (40 μM) was included as positive control. Results are expressed as the mean ± SEM of three independent experiments, ***p < 0.001 and **p < 0.01 vs untreated cells incubated with the BODIPY. (C) Representative images of lipid peroxidation on B16F10 cancer cells caused by photoactivated PS2 (40 μM). Reduced (red) and oxidized (green) forms of the BODIPY 581/591 probe were obtained via confocal microscopy; scale bar = 20 μm; cumene hydroperoxide (10 μM) was used as a positive control.

Figure 6

Time-dependent in vivo fluorescence images of PS2-PEG-CS and free PS2. (A) Indicated formulations (0.08 mg PS2/mouse) were directly injected in CT26 tumors (Balb/c mice) or in (B) B16F10 tumors (C57BL/6J mice). At various time points, in vivo fluorescence images of the whole body were taken. Each group contained three mice with the exception of the control group that only included one animal. (C,D) Quantitative analysis of the obtained fluorescence images. The background signal detected before PS2 injection was subtracted from each signal obtained at varied time points. Each point represents the mean ± SEM of three mice; ***p < 0.001, **p < 0.01, and *p < 0.05 PS2-PBSvsPS2-PEG-CS.

Figure 7

PDT after local administration of PS2 for the treatment of colon cancer. (A) Schematic illustration of the tumor treatment process against CT26 tumors. (B) Kaplan–Meier survival analysis of male Balb/c mice bearing CT26 tumors treated with the single irradiation PDT protocol using the PS2-PEG-CS or PS2-PBS formulations. Each treatment group contains 6–15 mice (DLI = 24 h, drug dose 0.07 mg, LD = 20 J/cm², and λ = 660 nm); ***p < 0.001, **p < 0.01 vs control. (C) Tumor volume represented as mean ± SEM 8 days post-PDT. (D) Kaplan–Meier survival analysis mice comparing the single and multiple irradiation PDT protocol using the PS2-PEG-CS or PS2-PBS formulations. Each treatment group contains six mice (DLI = 24, 48, and 72 h, drug dose = 0.04 or 0.07 mg, LD = 20 J/cm², and λ = 660 nm). ***p < 0.001, **p < 0.01 vs control. (E) Tumor volume represented as mean ± SEM 8 days post-PDT. (F) Representative images of Balb/c mice treated with single and multiple irradiation PDT protocols using the PS2-PEG-CS or PS2-PBS formulations.

Figure 8

PDT after local administration of PS2 for the treatment of melanoma tumors. (A) Schematic illustration of the tumor treatment process against B16F10 tumors. (B) Kaplan–Meier survival of female C57BL/6J mice bearing B16F10 tumors after the single irradiation PDT protocol using the PS2-PEG-CS hydrogel. Each treatment group consists of six mice (DLI = 24 h, drug dose = 0.08 mg, LD = 36 J/cm², and λ = 660 nm). ***p < 0.001 vs control. (C) Tumor volume represented as mean ± SEM 8 days post-PDT. (D) Kaplan–Meier survival comparing the single and multiple irradiation PDT protocols using the PS2-PEG-CS or PS2-PBS formulations. Each treatment group contains six mice (DLI = 24, 48, and 72 h, drug dose = 0.1, LD = 40 J/cm², and λ = 660 nm). **p < 0.01, *p < 0.05 vs control. (E) Tumor volume represented as mean ± SEM 8 days post-PDT. (F) Representative images of C57BL/6J mice treated with single and multiple irradiation PDT protocols using the PS2-PEG-CS or PS2-PBS formulations.

Figure 9

PDT with local administration of PS2-PEG-CS induces systemic antitumor immunity. (A) Tumor protection observed following tumor rechallenge (with CT26 cells) of male BALB/c mice that remained CT26 tumor-free for 60 days after PDT using the PS2-PEG-CS hydrogel (drug dose = 0.07 mg, DLI = 24 h, LD = 20 J/cm²). ***p < 0.001 vs control. (B) Cross-tumor protection observed following tumor rechallenge (with 4T1 cells) of male BALB/c mice that were previously cured, from CT26 tumors, with single or multiple PDT using the PS2-PEG-CS hydrogel (drug dose = 0.07 mg, DLI = 24 or 24, 48, and 72 h, LD = 20 J/cm²); **p < 0.01 PS2-PEG-CS 3× light vs control, *p < 0.05 PS2-PEG-CS 3× light vsPS2-PEG-CS 1× light. (C) Survival curves of male Balb/c vs male Balb/c nude mice bearing CT26 tumors after treatment with the multiple irradiation PDT protocol using the PS2-PEG-CS formulation (DLI = 24, 48, and 72 h, drug dose = 0.04 mg, LD = 20 J/cm², and λ = 660 nm). Each treatment group contains five mice. *p < 0.05 vs control and vsPS2-PEG-CS 3× light Nu/Nu. (D) Post-PDT images of nude Balb/c and WT Balb/c mice undergoing the multiple irradiation protocol using the PS2-PEG-CS formulation (DLI = 24, 48, and 72 h, drug dose = 0.04 mg, LD = 20 J/cm², and λ = 660 nm).

Figure 10

Abscopal effects of PDT with PS2-PEG-CS and PS2-PBS against distant metastasis. (A) Schematic representation of the experiment of the abscopal effects of PDT with PS2-PEG-CS against distant metastasis. (B) Kaplan–Meier survival analysis of male Balb/c mice bearing one CT26 tumor in each flank. The primary tumor was submitted to the PDT multiple irradiation protocol for using the PS2-PEG-CS or PS2-PBS formulations (DLI = 24, 48, and 72 h, drug dose = 0.07 mg, LD = 20 J/cm², and λ = 660 nm). Each treatment group contains eight mice ***p < 0.001 vs control and **p < 0.01 vsPS2-PBS 3× light. (C) Growth kinetics of secondary tumors which were measured during the time interval where no mouse reached the humane end point. Each treatment group contains eight mice (DLI = 24, 48, and 72 h, drug dose = 0.07 mg, LD = 20 J/cm², and λ = 660 nm).