IL12-based phototherapeutic nanoparticles through remodeling tumor-associated macrophages combined with immunogenic tumor cell death for synergistic cancer immunotherapy

Abstract

Various cancer therapeutic strategies have been designed for targeting tumor-associated macrophages (TAMs), but TAM reprogramming-based monotherapy is often clinically hindered, likely due to the lack of a coordinated platform to initiate T cell-mediated immunity. Herein, we fabricated reactive oxygen species (ROS)-responsive human serum albumin (HSA)-based nanoparticles (PEG/IL12-IA NPs) consisting of indocyanine green (ICG), arginine (Arg), and interleukin 12 (IL12). Upon laser irradiation, the nanoparticles were found to be able to dissociate, thus facilitating the release of IL12. On the one hand, the phototherapy effects produced by ICG could induce immunogenic cell death (ICD) of tumor cells, activate dendritic cells (DCs) and initiate T cell-mediated immunity. On the other hand, the ROS responsively released IL12 from the PEG/IL12-IA NPs could reprogramme the immunosuppressive M2-TAMs to M1-TAMs, which could further convert Arg to nitric oxygen (NO), thus alleviating immunosuppression and enhancing antitumor immunity. Based on this combined therapeutic effect, the nanoparticles inhibited the tumor growth, prolonged the survival time, delayed lung metastasis, and ultimately improved the antitumor immuno-therapeutic efficiency in 4T1-bearing mice. Taken together, remodeling TAMs combined with phototherapy-induced ICD via the PEG/IL12-IA NPs is a promising cancer immunotherapy strategy for boosting both innate and adaptive antitumor immunity.

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1 Introduction

Despite the prominent clinical efficacy of cancer immunotherapy, achieving broad efficacy still remains a major challenge owing to the immunosuppressive tumor microenvironment (TME) in solid tumors.^1–3 In an immunosuppressive TME, tumor-associated macrophages (TAMs) surrounding tumor cells account for 30%–50% of the total tumor weight and mostly express the M2 phenotype to promote tumor occurrence and growth, severely counteracting the therapeutic efficacy of immunotherapy.^4–6 Macrophages have high plasticity, and thus, modulating the phenotype and function of TAMs has become one of the principal strategies in current cancer immunotherapy.^7–9

Interleukin 12 (IL12), as a potent pro-inflammatory cytokine, typically induces the conversion of tumor-supportive M2 phenotype to tumor-suppressive M1 phenotype macrophages and is also known to enhance the infiltration of antitumor immune cells including CD8⁺ T cells.^10–13 In clinical studies, IL12 has gained wide attention as an antitumor cytokine for treating cancer in many malignancies. However, the systemic administration of IL12 may cause unpredictable proinflammatory toxicity risk. Additionally, the limited clinical efficacy of IL12 can be attributed to its inefficient delivery to tumor sites.^14,15 Therefore, a controllable strategy to maximize the content of IL12 inside the tumor site is urgently required for robust antitumor responses with minimal systemic toxicity. Recently, in preclinical studies, nanomedicine-associated IL12 delivery systems, ranging from recombinant proteins to genes encapsulated in nanoparticles, have demonstrated remarkable antitumor effects with reduced toxicity.^16–18 Although IL12-based cancer immunotherapy via re-educating M2-TAMs to M1-TAMs was shown to produce efficient antitumor effects, in most preclinical trials, relatively modest inhibition of tumor growth was observed with monotherapy of TAM reprogramming due to the lack of a coordinated platform to initiate T cell-mediated immunity.^19,20

Recently, photodynamic therapy (PDT) and photothermal therapy (PTT), as the two most common phototherapies, have attracted much attention because they can trigger immunogenic cell death (ICD) of tumor cells for activating dendritic cells (DCs) and promoting the activation, proliferation and differentiation of T cells.^21,22 However, due to the weak immunogenicity of some cancers, phototherapy-based immunotherapy is not sufficient to induce a robust antitumor response.^23,24 Our previous research works demonstrated that nano-enabled phototherapy combined with immunotherapy, including small molecular immunomodulators and immune checkpoint inhibitors, could produce an antitumor immune response and improve the antitumor effect substantially.^25–27 Here, we hypothesized that the combination of phototherapy with TAM re-education can achieve a synergistic antitumor immunotherapeutic effect, which could induce ICD of tumor cells, activate DC, trigger a T cell-mediated antitumor immune response, re-educate M2-to-M1 TAMs, and ultimately boost both the innate and adaptive immune response. However, there are still crucial issues to be addressed regarding the construction of a versatile, coordinated nanoplatform to dually promote TAM reprogramming and the phototherapy-based ICD of tumor cells.

Herein, we fabricated reactive oxygen species (ROS)-responsive human serum albumin (HSA)-based nanoparticles consisting of indocyanine green (ICG), arginine (Arg), and IL12 (Scheme 1). Briefly, HSA, ICG and Arg were first constructed into IA NPs using the desolvation method.²⁸ With an ROS-responsive thioketal containing cross-linker (TK), IL12 was cross-linked into IA NPs with further modification by poly(ethylene glycol) (PEG) to obtain PEG/IL12-IA NPs as the final nanoparticles. Upon arrival at the tumor, the PEG/IL12-IA NPs generated ROS under the first wave of near-infrared (NIR) laser irradiation, together with a relatively high concentration of ROS at the tumor site, triggered the dissociation of the nanoparticles, thus facilitating IL12 release. Subsequently, phototherapy can induce ICD of tumor cells for promoting DC maturation and CD8⁺ T cell activation. The released IL12 could repolarize TAMs from the M2 to M1 phenotype for restraining the immunosuppressive TME. Moreover, Arg in the nanoparticles was expected to be converted to nitric oxygen (NO) by M1 macrophages, which provided the potential to further induce apoptosis in tumor cells.^29,30 This strategy exhibits great potential to design functional nanoplatforms to sustainably promote antitumor T cell immunity as well as facilitate macrophage reprogramming, and ultimately synergistically boost both the innate and adaptive antitumor immunity.

Scheme 1 Schematic of the therapeutic mechanism of the PEG/IL12-IA NPs prepared using Figdraw.

2 Results and discussion

2.1 Fabrication and characterization of the PEG/IL12-IA NPs

In this study, HSA was chosen as the carrier, which has the ability to non-covalently bind with ICG and Arg based on their structure comprising anionic, lipophilic and aromatic characteristics. It has been found that ICG can be adsorbed on HSA by hydrophobic binding and Arg can be adsorbed on HSA through cation–π interactions, respectively.^31–34 As schematically illustrated in Scheme 1, ICG, Arg, HSA and IL12 could be assembled into IL12-IA NPs using the desolvation method and stabilized by an ROS-sensitive TK cross-linker. After the further conjugation of PEG to the surface of the IL12-IA NPs through TK linkers, the PEG/IL12-IA NPs were obtained. As expected, all the prepared nanoparticles had a relatively uniform spherical shape (Fig. 1A). The average hydrodynamic diameters of the ICG NPs, IA NPs, and PEG/IL12-IA NPs were 153.3 ± 0.8 nm, 131.7 ± 1.0 nm, and 160.1 ± 4.0 nm in aqueous solution, respectively (Table S1). The zeta potential of all the prepared nanoparticles is shown in Fig. 1B. Considering their stability, no obvious changes in the size and zeta potential of the PEG/IL12-IA NPs were found in 10% fetal bovine serum (FBS) during 72 h (Fig. 1C).

Fig. 1 Characterization of the PEG/IL12-IA NPs. (A and B) Size distribution and TEM images of the nanoparticles and zeta potential of the nanoparticles (scale bar: 200 nm). (C) Changes in zeta potential and size of the PEG/IL12-IA NPs in 10% FBS for 72 h (n = 3). (D and E) Changes in size and TEM images of the PEG/IL12-IA NPs after treatment with H₂O₂ and laser irradiation (n = 3) (scale bar: 200 nm). (F and G) IL12 and ICG release from the nanoparticles in PBS at pH 7.4 under different conditions (n = 3). (H and I) In vitro ROS production and photothermal effect of the nanoparticles (n = 3).

According to our assumption in Scheme 1, the PEG/IL12-IA NPs would be susceptible to ROS because of the presence of the TK cross-linker. To test this assumption, dynamic light scattering measurements (DLS) and transmission electron microscopy (TEM) were applied to study the PEG/IL12-IA NPs after H₂O₂ incubation with or without laser irradiation (808 nm, 1 W cm⁻², 1 min). As expected, the size distribution and the polydispersity of the PEG/IL12-IA NPs drastically increased after H₂O₂ incubation due to the disassembly of the PEG/IL12-IA NPs, implying the superior ROS-sensitivity of the nanoparticles (Fig. 1D). More interestingly, by TEM observation after H₂O₂ incubation with laser irradiation, it was found that the majority of the disassembled PEG/IL12-IA NPs had a smaller size than that treated with H₂O₂ only (Fig. 1E); these results suggested the complete disassembly of the PEG/IL12-IA NPs and release of albumin-based particles, which have a size of 10–20 nm in solution.^35,36 Given that IL12 was encapsulated in the PEG/IL12-IA NPs by the TK linker, the ROS-responsive release of IL12 from the PEG/IL12-IA NPs was determined by ELISA. IL12 was quickly released from the NPs in the presence of H₂O₂. Specifically, after about 3 h, approximately 20% of IL12 was released (Fig. 1F). However, in the absence of H₂O₂, almost none of the IL12 was released. Notably, in the presence of H₂O₂ with laser irradiation, IL12 was released more rapidly and presented 42.5% of IL12 in 96 h, demonstrating the necessity to amplify the ROS signal in the TME using laser pre-irradiation for the PEG/IL12-IA NPs. Meanwhile, the release of ICG was also much higher in the presence of H₂O₂ with laser irradiation than that in its absence (Fig. 1G).

It is well known that ICG is a potent photosensitizer for cancer phototherapy.^37–39 In this study, the in vitro PDT and PTT performance of the nanoparticles under 808 nm laser irradiation was evaluated. Firstly, ROS generation was detected using 1,3-diphenylisobenzofuran (DPBF) as a probe. The absorbance of DPBF had a noticeable time-dependent decrease, suggesting the efficient ROS generation induced by the ICG-loaded nanoparticles upon laser irradiation (Fig. 1H). According to Fig. 1I, under laser irradiation, a rapid temperature increase was observed in the ICG-loaded nanoparticles. Taken together, the PEG/IL12-IA NPs could be used as a phototherapeutic agent for PDT and PTT of cancer.

2.2 In vitro evaluation of the ICD effect

To start with, the dark cytotoxicity of all the formulations on 4T1 tumor cells was determined using the CCK-8 assay. Free ICG and all the prepared nanoparticles without laser irradiation showed no obvious cytotoxicity in 4T1 cells (Fig. S1). Then, flow cytometry (FCM) was employed to evaluate the cellular uptake of all the formulations in 4T1 cells. Stronger ICG fluorescence intensity was observed in all the nanoparticle-treated cells compared with that of the free ICG-treated ones at the designed time points (Fig. 2A). In all the nanoparticle-treated cells, the ICG fluorescence intensity increased with the prolongation of the incubation time, indicating the time-dependent cellular uptake of nanoparticles. Additionally, compared with the ICG NPs, the IA NPs showed stronger ICG fluorescence intensity in the treated cells, which is probably due to the arginine encapsulated in the nanoparticles. It has been reported that arginine-rich nanoparticles can enhance cell uptake and are widely used as drug delivery carriers.^40,41 Similar to the IA NPs, PEG/IL12-IA NP also showed increased cellular uptake compared with the ICG NPs. In this study, the PEG/IL12-IA NPs subjected to 808 nm laser pre-irradiation (1 W cm⁻², 1 min) before addition to the cells were denoted as the PEG/IL12-IA NPs^L. As expected, the cells treated with the PEG/IL12-IA NPs^L exhibited a higher ICG fluorescence intensity than that treated with the PEG/IL12-IA NPs, which is possibly due to the dissociation of the PEG/IL12-IA NPs and the acceleration of ICG release from the nanoparticles upon laser irradiation. The 4T1-tumor cell apoptosis level under laser irradiation after different treatments is shown in Fig. 2B. All the nanoparticle groups showed the stronger induction of apoptotic cell death compared with the free ICG group, which was consistent with the result of the cellular uptake study, indicating that all nanoparticle-mediated phototherapies have a stronger killing effect on 4T1 tumor cells.

Fig. 2 In vitro evaluation of the ICD effect of the PEG/IL12-IA NPs against 4T1 cells. (A) Cellular uptake of all formulations in 4T1 cells determined by FCM (n = 3). (B) Apoptosis rate of 4T1 cells under laser irradiation after different treatments (n = 3). (C) Evaluation of CRT exposure on 4T1 cells upon various treatments by CLSM (scale bar: 20 μm). (D and E) Quantification of HMGB1 release and intracellular ATP determined using ELISA assay (n = 3). (F and G) BMDC maturation (CD80⁺CD86⁺ in CD11c⁺ cells) after different treatments determined by FCM (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001).

Phototherapy can induce ICD of tumor cells and promote the surface expression of calcium reticulin (CRT), the release of high-mobility group box1 (HMGB1) and adenosine triphosphate (ATP). The confocal laser scanning microscopy (CLSM) images showed that free ICG moderately induced CRT expression on the surface of 4T1 cells (Fig. 2C). However, all the nanoparticle formulations significantly promoted CRT expression. Furthermore, compared with the free ICG group, all the nanoparticle groups triggered apparent HMGB1 release and decreased intracellular ATP secretion from the treated 4T1 cells (Fig. 2D and E), respectively. Next, the ICD-induced maturation of bone marrow-derived dendritic cells (BMDCs) was evaluated through the analysis of the expression of CD80 and CD86 on BMDCs co-incubated with different formulation-pretreated 4T1 cells (Fig. 2F and G), respectively. In accordance with the ICD effect induced by all the nanoparticle treatments, compared to the free ICG group, all the nanoparticle groups exhibited a higher proportion of mature BMDCs. Therefore, these results confirmed that all the ICG nanoparticle-mediated phototherapies could induce ICD of tumor cells and elicit the maturation of BMDCs in vitro.

2.3 In vitro evaluation of repolarization of M2-to-M1 macrophages and 4T1 cell-killing effect

The immune stimulatory IL12 can induce the M2-to-M1 repolarization of TAMs. Therefore, next we investigated the polarity of macrophages after the free IL12, PEG/IL12-IA NP and PEG/IL12-IA NP^L treatments. The specific phenotypic biomarker expression and cytokine secretion in the treated bone marrow-derived macrophages (BMDMs) were assayed using FCM and enzyme-linked immunosorbent assay (ELISA), respectively. In comparison with PBS, the free IL12, PEG/IL12-IA NP and PEG/IL12-IA NP^L treatment groups significantly increased the percentage of M1 macrophages (CD86⁺F4/80⁺) and simultaneously decreased the percentage of M2 macrophages (CD206⁺F4/80⁺), proving the role played by IL12 to repolarize M2 macrophages to the M1 phenotype, which is consistent with previously reported studies (Fig. 3A–C).^11,12 Furthermore, the proportion of CD86⁺F4/80⁺ macrophages in the PEG/IL12-IA NP^L group was remarkably higher than that in the free IL12 and PEG/IL12-IA NPs groups. Additionally, the secretion of both TNF-α and IL-6 was significantly enhanced in macrophages treated with the PEG/IL12-IA NPs^L compared with the other groups (Fig. 3D and E), respectively.

Fig. 3 PEG/IL12-IA NPs could effectively induce repolarization of M2-to-M1 macrophages in vitro. (A–C) Representative FCM plots and quantitative analysis of BMDM repolarization from M2 (CD206⁺F4/80⁺) to M1 (CD86⁺F4/80⁺) after indicated treatments (n = 3). (D and E) Quantitative analysis of IL-6 and TNF-α secreted by BMDMs with different treatments (n = 3). (F) NO production levels of M2-BMDMs after indicated treatments (n = 3). (G and H) Schematic of 4T1-RAW264.7 Transwell co-culture system, prepared using Figdraw, and percentage of apoptotic 4T1 cells in the lower chamber determined by FCM (n = 3) (^#vs. PBS, ^##p < 0.01, ^###p < 0.001; *p < 0.05, **p < 0.01, ***p < 0.001).

The reported results indicated that M1 macrophages could convert the intracellular Arg into NO with the help of nitric oxide synthase (iNOS).^30,42 Hence, we detected the NO production ability of M1 macrophages repolarized by different IL12 formulations using the Griess reagent kit. Fig. 3F shows that in comparison with the PBS group, free IL12 alone resulted in certain but not significant NO production. As expected, the NO level could be dramatically enhanced by the PEG/IL12-IA NPs and PEG/IL12-IA NPs^L, respectively, revealing that both Arg and IL12 of the PEG/IL12-IA NPs contribute to the NO generation by M1 macrophages. The generated NO as gas therapy has the potential to kill tumor cells, which could combine with the repolarized M1 macrophages to synergistically induce apoptosis in tumor cells.^29,30 Then, the apoptosis level in the 4T1 cells induced by the different IL12 formulation-treated M2 macrophages was examined in the 4T1–M2 TAM Transwell system using flow cytometry (Fig. 3G). As shown in Fig. 3H, the M2 macrophages treated with the PEG/IL12-IA NPs and PEG/IL12-IA NPs^L could significantly induce the apoptosis of 4T1 cells, which was higher than that by the free IL12 alone. Additionally, 35.4% cell apoptosis was induced by the PEG/IL12-IA NPs^L, which was much higher than that by the PEG/IL12-IA NPs (29.6%). Taken together, the PEG/IL12-IA NPs^L could effectively kill tumor cells by promoting M2-to-M1 macrophage repolarization and NO generation.

2.4 In vivo evaluation of the synergistic immunotherapeutic effect

Firstly, the in vivo biodistribution of all the ICG nanoparticles was evaluated through NIR imaging in 4T1-bearing mice, and the results indicated all the ICG nanoparticles could gradually enrich at the tumor sites after i.v. injection (Fig. S2). In addition, the tumor of the mice treated with the PEG/IL12-IA NPs^L had the maximum temperature of ∼59 °C, which is significantly higher than that in the free ICG group (∼43 °C), indicating the potential of the PEG/IL12-IA NPs^L for photothermal cancer therapy (Fig. S3). Next, the 4T1 tumor-bearing mice were randomly divided into 6 groups, as follows: PBS, free ICG, ICG NPs, IA NPs, PEG/IL12-IA NPs and PEG/IL12-IA NPs^L, and i.v. injected with these formulation as described in the Methods section. As shown in Fig. 4A, the free ICG and ICG NPs exhibited rather limited inhibition effects on the tumor growth compared with the PBS group. In contrast, compared with the other groups, the tumor size significantly reduced in the PEG/IL12-IA NP and PEG/IL12-IA NP^L-treated groups, respectively. Hematoxylin and eosin (H&E) staining of the tumor sections determined that the tumor cells in the PEG/IL12-IA NP^L group clearly caused obvious damage and necrosis in the tumor cells (Fig. S4). Furthermore, obviously prolonged survival was observed in the PEG/IL12-IA NP^L-treated groups, with 100% of the mice surviving over 60 days (Fig. 4B). There was not any significant body weight reduction and obvious histological major organ damages caused by the PEG/IL12-IA NPs^L, preliminarily indicating the biocompatibility of the PEG/IL12-IA NPs^L (Fig. 4C and Fig. S5). Notably, the 4T1-bearing mice had a strong metastatic profile, and the lung samples from the mice after different treatments were collected. The number of metastases were significantly reduced by the PEG/IL12-IA NP^L treatment, as verified by the similar results from the lung images and hematoxylin and eosin (H&E) staining of the lung tissues, indicating the considerable immunotherapy effect induced by the PEG/IL12-IA NPs^L (Fig. 4D, E and Fig. S6).

Fig. 4 Evaluation of in vivo synergistic immunotherapeutic effect by the PEG/IL12-IA NPs. (A–C) Tumor growth, survival rate and body weight change in mice after various treatments under laser irradiation (808 nm, 1 W cm⁻², 5 min) (n = 5). (D and E) Number of metastatic nodules in the lungs (n = 5) and lung images and H&E staining images collected from the mice in the different groups. (F) FCM quantification of CD206⁺F4/80⁺ M2 macrophages in tumors (n = 3). (G) FCM quantification of CD11c⁺CD80⁺CD86⁺ cells in the TDLNs (n = 3). (H and I) FCM quantification of CD3⁺CD8⁺ T cells and CD3⁺CD4⁺ T cells in tumors (n = 3). (J and K) IFN γ and IL10 level in sera from mice in different groups (n = 3) (^#vs. PBS, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; *p < 0.05, **p < 0.01, ***p < 0.001).

Next, to investigate the detailed mechanisms of the synergistic antitumor effects induced by the PEG/IL12-IA NPs^L, the tumors and tumor-draining lymph nodes (TDLNs) were collected from the treated mice to make single-cell suspensions for the detection of FCM. Correspondingly, it was found that compared with the other groups, the PEG/IL12-IA NP^L treatment significantly increased the M2-to-M1 repolarization of TAMs in the tumors by significantly decreasing the number of CD206⁺F4/80⁺ cells, as detected by FCM (Fig. 4F and Fig. S7). Meanwhile, it was shown that the PEG/IL12-IA NP treatment could also decrease the number of CD206⁺F4/80⁺ cells, but significantly less than that elicited by the PEG/IL12-IA NP^L treatment, indicating the necessity of the PEG/IL12-IA NPs with laser pre-irradiation on TAM repolarization in vivo. Then, we evaluated the in vivo maturation of DCs in TDLNs triggered by the nanoparticles. As shown in Fig. 4G and Fig. S8, the proportion of matured DCs was ∼9.27%, ∼8.35%, ∼11.07% and ∼11.97% in the PBS group, free drug group, ICG NP group and IA NP group, respectively, which is much lower than that in the PEG/IL12-IA NP group (∼16.27%) and PEG/IL12-IA NPs^L (∼18.23%). Moreover, the capability of the PEG/IL12-IA NPs^L to improve the infiltration of CD8⁺ T cells and CD4⁺ T cells was investigated next (Fig. 4H, I, Fig. S9 and S10). It was found that both the CD8⁺ T cells and CD4⁺ T cells in the tumors of the PEG/IL12-IA NP-treated mice increased by ∼6-fold compared to the PBS group. As expected, the infiltration of CD8⁺ T cells and CD4⁺ T cells could be further enhanced by the PEG/IL12-IA NPs^L by ∼10-fold than that in the PBS group. Meanwhile, the cytokine levels of IFN-γ and IL10 in the serum samples from the mice after various treatments were evaluated. The PEG/IL12-IA NP^L treatment exhibited the highest level of pro-inflammatory cytokine IFN-γ, and conversely, the lowest level of anti-inflammatory cytokine IL-10, suggesting the effectively activated systemic immune response induced by the PEG/IL12-IA NPs^L (Fig. 4J and K), respectively. The above-mentioned results confirmed that the PEG/IL12-IA NPs^L under laser irradiation could induce ICD of tumor cells, stimulate the maturation of DCs in TDLNs, promote the transformation of M2-to-M1 macrophages and enhance the T cell trafficking to tumors, which work together to further inhibit tumor growth.

3 Conclusion

In this study, we constructed a versatile, coordinated nanoplatform (PEG/IL12-IA NPs) by cross-linking the pro-inflammatory cytokine IL12 into self-assembled HSA nanoparticles with ICG and Arg, which could promote the reprogramming of TAMs as well as T cell-mediated immune responses. Upon laser irradiation, the nanoparticles were found to be able to dissociate, thus facilitating the release of IL12. On the one hand, the PEG/IL12-IA NPs could cause a phototherapy-induced direct tumor cell-killing effect, activate DCs and initiate T cell-mediated immunity. On the other hand, IL12, the ROS responsively released from the PEG/IL12-IA NPs could reprogramme the immunosuppressive M2-TAMs to M1-TAMs, which further converted Arg to NO, and thus enhanced the antitumor immunity and abated immunosuppression. Based on this combined therapeutic effect, the nanoparticles were able to inhibit tumor growth and lung metastasis in 4T1-bearing mice. Taken together, we disclosed new IL12-based phototherapeutic nanoparticles that can promote antitumor T cell immunity as well as facilitate macrophage reprogramming, ultimately synergistically boosting both the innate and adaptive immune responses, which can be considered a potential clinical option for antitumor immunotherapy.

4 Methods

4.1 Preparation and characterization of nanoparticles

Briefly, 10 mg HSA and 0.5 mg ICG were first dissolved in 1 mL deionized water and the pH calibrated to 8.0 using 0.1M NaOH. Then, 1 mL of ethanol was incorporated and the reaction stirred for 3 h. Finally, the pre-activated TK linker with EDC/NHS was added to chemically cross-link to form ICG NPs and kept for 3 h on stirring. After that, the above-mentioned solution was dialyzed and the obtained nanoparticles were lyophilized for further characterization and treatment. IA NPs were prepared basically the same as the above-mentioned method except that 0.5 mg of L-Arg was premixed with a solution of HSA and ICG. Similarly, the PEG/IL12-IA NPs containing IL12 were prepared. Briefly, 1.5 μg of recombinant mouse IL12 in 15 μL of phosphate-buffered saline (PBS) was added in the reaction solution of IA NPs, as described above. Following this, 1.0 mg of pre-activated PEG-TK (PEG, M_w = 2 kDa) was added for chemical cross-linking to form the PEG/IL12-IA NPs.

A Nanosizer Nano ZS (Brookhaven) was used to determine the size and zeta potential of the nanoparticles. The morphology of the nanoparticles was characterized using TEM (Tecnai F20, EFI, Netherlands). The stability of the nanoparticles was assessed in 10% FBS by monitoring the changes in the particle size and zeta potential at different time points. The content of ICG and IL12 in the nanoparticles was measured using a UV-Vis spectrophotometer (Thermo Fisher Scientific Varioskan, USA) and specific ELISA kit, respectively. In vitro ROS production and temperature elevation of the nanoparticles were evaluated under laser irradiation (808 nm, 1 W cm⁻²).

4.2 In vitro ROS-responsiveness

To investigate the ROS responsiveness of the PEG/IL12-IA NPs, the nanoparticles were irradiated a with laser (808 nm, 1 W cm⁻², 1 min) or without, and then incubated in PBS with or without the addition of H₂O₂ at 37 °C with shaking at 200 rpm for 12 h, followed by TEM observation and DLS assay. The release behavior of ICG or IL12 from the nanoparticles was examined in PBS at pH7.4 with or without laser irradiation (808 nm, 1 W cm⁻², 1 min) and the addition of H₂O₂.

4.3 Cellular uptake

4T1 cells were treated with the free ICG, ICG NPs, IA NPs, PEG/IL12-IA NPs and PEG/IL12-IA NPs^L (ICG: 20 μg mL⁻¹) in RPMI 1640 medium for 4 h and 8 h, respectively. Then, the intracellular fluorescence intensity of ICG in the treated 4T1 cells was detected by FCM and the treated cells were observed using CLSM.

4.4 Apoptosis and ICD effect

4T1 cells (1 × 10⁵ cells per well) in 24-well plates were incubated with the free ICG, ICG NPs, IA NPs, PEG/IL12-IA NPs and PEG/IL12-IA NPs^L (ICG: 20 μg mL⁻¹) for 8 h and then washed with PBS and irradiated with an 808 nm laser (1 W cm⁻², 5 min). After an additional 24 h of incubation, the cells were stained with Annexin V-FITC and PI for apoptosis analysis by FCM.

To determine the prepared nanoparticle-induced ICD of the tumor cells, the extracellular release of HMGB-1, amount of intracellular ATP, and the expression of CRT on the cell surface were examined.

4.5 BMDC maturation

BMDCs were extracted from the femurs and tibias of 4–6 weeks female BALB/c mice and cultured with 20 ng mL⁻¹ GM-CSF for 7 days. The 4T1 tumor cells were treated with all formulations (ICG: 20 μg mL⁻¹) for 8 h, followed by 808 nm laser irradiation (1 W cm⁻², 5 min). Immature BMDCs were co-cultured with the pre-treated 4T1 cells for 24 h and then stained with antibodies to detect CD11c⁺CD80⁺CD86⁺ DCs.

4.6 BMDM repolarization

Similar to BMDCs, BMDMs were harvested and cultured with 20 ng mL⁻¹ M-CSF for 7 days. M2 polarized BMDMs (20 ng mL⁻¹ of IL4) were treated with the free IL12, PEG/IL12-IA NPs, and PEG/IL12-IA NPs^L (IL12: 60 ng mL⁻¹) for 24 h and then stained with antibodies to detect CD206⁺F4/80⁺ and CD86⁺F4/80⁺ cells, respectively. Additionally, the culture supernatants were collected for cytokine determination and NO concentration detection with the ELISA kit and Griess Reagen kit, respectively.

4.7 4T1 cells apoptosis assessed in a transwell system

4T1 cells were co-cultured with and Raw264.7 macrophages in a Transwell system. Briefly, 4T1 cells were seeded in the lower chamber of the Transwell, while RAW264.7 cells were put in the upper chamber and treated with different formulations as the same treatments mentioned above. After co-incubation for 24 h, 4T1 cells were harvested and stained with Annexin V-FITC and PI for apoptosis analysis by FCM.

4.8 In vivo antitumor effect

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and approved by the Animal Ethics Committee of the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences. 4T1-bearing mice (Female BALB/c mice, 6–8 weeks) were established by inoculation on their right flank (1 × 10⁶ cells per mouse). The mice (∼50 mm³) were randomly divided into 6 groups (n = 5) as follows: (1) PBS, (2) free ICG, free L-Arg and free IL12, (3) ICG NPs, (4) IA NPs, (5) PEG/IL12-IA NPs and (6) PEG/IL12-IA NPs^L (ICG 7.5 mg kg⁻¹, IL12 25 μg kg⁻¹, every 2 days for 3 doses). The tumor sites of the mice in group 6 were pre-irradiated with laser irradiation (808 nm, 1 W cm⁻², 1 min) at 2 h post-injection. All groups were treated with laser irradiation (808 nm, 1 W cm⁻², 5 min) at 4 h after administration. The tumor sizes and body weights were measured every two days thereafter, and when the tumor volume became nearly 2000 mm³, the mice were sacrificed. At the time of sacrificing, lung tissues were collected for H&E staining and surface lung tumor nodules were counted.

To analyze the infiltration of immune cells, the treated mice were sacrificed to collect their tumors and tumor-draining lymph nodes for the preparation of single-cell suspensions for FCM analysis. Additionally, cytokines in the blood were determined using ELISA kits.

4.9 Statistical analysis

Data are presented as the mean value ± SD. Statistical significance was calculated by one-way ANOVA with Tukey's post hoc test or log-rank test (GraphPad Prism 5.0 software).

Conflicts of interest

All authors declare no competing interests.

Data availability

The data supporting this article have been included as part of the SI. The supplementary information provides additional experimental section and supporting figures that complement the main findings of the study. See DOI: https://doi.org/10.1039/d5bm00848d.

Acknowledgements

We acknowledge the financial support from the National Natural Science Foundation of China (No. 82172089 and 22178270) and CAMS Innovation Fund for Medical Sciences (2021-I2M-1-058).

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Footnote

† These authors contributed equally.

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