Chemo-photothermal Therapy Promoting Immunogenic Cell Death Based on NIR-II Light-responsive Drug Delivery Systems

Background: Several recent studies have well demonstrated that the chemotherapy or near-infrared-II (NIR-II) photothermal therapy (PTT) can induce immunogenic cell death (ICD). However, single treatment based on the independent chemotherapy or PTT to induce ICD may require high dose of drug, high laser power, or high temperature, which limits their clinical application. We hypothesize that combination of chemotherapy and NIR-II PTT possesses great promise to overcome respective limitations. This manuscript describes the development of polyethylene glycol (PEG) modified hollow Cu x S nanoparticles (NPs) for synergistic chemo-photothermal therapy to effectively promote ICD. Results: Hollow structure Cu x S-PEG NPs were prepared under mild condition by using Cu 2 O NPs as sacrificial templates. Cu x S loaded with doxorubicin (Dox) as NDDSs were characterized for hydrate particle size and surface charge. The morphology, photothermal effect, drug loading/releasing abilities, synergistic chemo-photothermal therapy, and ICD from synergistic therapy of Cu x S-PEG NPs have been investigated. The in vitro outcomes of ICD and chemo-photothermal therapy were assessed in EMT-6 cells. In vivo therapeutic studies and immunoreaction were performed in EMT-6 bearing mice where therapeutic outcomes were assessed by tumor volume, immunohistochemical staining, and expression of CD8 + cytotoxic T-lymphocytes. The Cu x S-PEG NPs with hollow structure show high drug loading capacity (~255 μg Dox per mg of Cu x S NPs) and stimuli-responsive drug release triggered by NIR-II laser irradiation. The chemo-photothermal strategy more effectively induces ICD than that of the single treatment, accompanying with the release of adenosine triphosphate, pre-apoptotic calreticulin, and high mobility group box-1. Finally, the synergistic chemo-photothermal therapy based on the Dox/Cu x S-PEG NPs promotes CD8 + cytotoxic T-lymphocytes infiltration into tumors and achieves ~98.5% tumor elimination. Conclusion: Therefore, our study emphasizes that the great potentials of Cu x S-PEG NPs can be used as NIR-II light-responsive NDDSs for the applications of biomedicine and immunotherapy.


Background
Recently, several studies have well demonstrated that the chemotherapy or photothermal therapy (PTT) in the second near-infrared (NIR-II) window can induce immunogenic cell death (ICD), accompanying with the release of damage-related molecular patterns (DAMPs), which serve as immunostimulatory "danger" signals to elicit T cell activation [1][2][3][4][5]. However, single treatment based on the independent chemotherapy or PTT to induce ICD may require high dose of drug, high laser power, or high temperature, which limits their clinical application [6,7]. We hypothesize that combination of chemotherapy and NIR-II PTT possesses great promise to overcome respective limitations (i.e. requirement of high laser power, high dose of drug, and less efficient therapeutic efficacy, etc.) and more effectively induces ICD to minimize damage to healthy tissues. Therefore, rationally designing drug delivery systems for chemo-photothermal therapy and improving ICD efficiency is the key to this study.
Compared to the traditional chemotherapy, nanocarrier-based drug delivery systems (NDDSs) have great prospects for precise cancer therapy due to its higher bioavailability, multifunctionality, and low side effects for non-target tissues [8][9][10][11][12][13]. In particular, NIR-II light-responsive NDDSs serve as promising modalities to realize the demand of drug release at the target disease site and combination with other therapeutic modes [14][15][16][17][18]. Light has been widely used as an external stimulus to control drug release because of its unique advantages such as safety, minimal cross-reaction, and spatiotemporal precision [19,20]. In addition, the NIR-II light could penetrate a few inches into tissues deeper than NIR-I or visible light, and displayed no apparent damage to tissues or cells [21,22]. Meanwhile, based on the photothermal conversion agents (PTCAs) as NDDSs, NIR-II light can be converted into heat for PTT, thereafter triggering drug release from the nanocarriers through thermal effect. Moreover, PTT is a complementary approach to enhance chemotherapeutic efficiency. Thus, using PTCAs as NIR-II light-responsive NDDSs can effectively combine chemotherapy and PTT, which has potential to promote tumor ICD.
Various PTCAs such as noble metal, carbon materials, metal oxide and metal sulfide NPs have been developed to generate heat efficiently for NIR light-responsive NDDSs [23][24][25][26][27][28][29]. For example, noble metal nanoparticles (NPs) (gold nanorods, gold nanoparticle agglomerates, platinum NPs) and drugs have been encapsulated into polymeric micelles to create vectors which allow NIR light-triggered drug release [30][31][32][33][34][35]. Carbon materials are also able to absorb NIR light and generate heat, and therefore the high temperature controls drug delivery from vectors containing carbon materials [36]. In addition, nanocarriers containing metal sulfide or metal oxide as PTCAs release cargoes through photothermal conversion under NIR laser irradiation [37,38]. However, the reported NIR light-responsive vectors encapsulating both PTCAs and drugs have some limitations including high cost, relatively low drug loading capacity, premature drug leakage, and complex preparation process, which prevented their further widespread applications. Therefore, development of NIR-II light-responsive NDDSs with high drug loading capacity, low cost, and facile preparation process is demanded.
Among the various NIR-II PTCAs, copper sulfide has an optical absorption band peaking wavelength at NIR-II region caused by the d−d energy band transition of Cu 2+ ions which is independent of the dielectric constant of the surrounding medium [39]. Further, copper sulfide is much less expensive than other NIR-II PTCAs.
Different from embedding PTCAs in nanocarriers, hollow nanostructured copper sulfide which exhibits numerous mesoporous pores and a large specific surface area has great application potential to be used as NIR-II light-responsive NDDSs [40].
Importantly, hollow copper sulfide can be eliminated from the living body by slow dissociation of Cu ion from NPs, indicating low biotoxicity of copper sulfide in vivo [41].
In this work, we develop a NIR-II photo-responsive NDDS mediated hollow nanostructured copper sulfide (Cu x S) with modification of polyethylene glycol-lipoic acid (PEG-LA) for drug delivery, NIR-II PTT, and effectively promoting tumor ICD. Figure 1A, the hollow Cu x S NPs are first prepared through Cu 2 O NPs template under mild condition. Then, drug molecules (doxorubicin) are loaded into hollow Cu x S NPs (Defined as Dox/Cu x S NPs), resulting in a drug a loading factor as high as ~255 μg Dox per mg of Cu x S NPs, and the obtained Dox/Cu x S NPs are subsequently modified with PEG-LA as Dox/Cu x S-PEG NPs. The Dox/Cu x S-PEG NPs exhibit a controllable drug release manner triggered by the NIR-II laser irradiation ( Figure 1B). Moreover, excellent photothermal effect of Dox/Cu x S-PEG NPs has been demonstrated benefiting from strong NIR-II absorption. By synergistic chemo-photothermal therapy, the Dox/Cu x S-PEG NPs can effectively induce ICD, accompanying with the release of DAMPs from dying cancer cells. In addition, we found that DAMPs such as adenosine triphosphate (ATP), pre-apoptotic calreticulin (CRT), and high mobility group box-1 (HMGB-1) in dying cells induced by the chemo-photothermal therapy could simultaneously trigger immune responses ( Figure   1C). Both in vitro and in vivo results well demonstrated that such multifunctional nanoplatforms could achieve excellent efficiency for tumor suppression. This work develops a NIR-II light-responsive NDDS based on a single-component hollow Cu x S NPs for synergistic chemo-photothermal therapy and effectively inducing ICD. Utilizing Cu 2 O nanospheres as Cu x S NPs precursor and template, we report a mild formation of hollow Cu x S NPs through a facile reduction method in aqueous solution.

As illustrated in
The precursor Cu 2 O nanospheres are synthesized through one-step method, in which PVP is used as the stabilizing agent, copper nitrate as the Cu x S NPs precursor, and  of Cu x S crystal, indicating that the obtained hollow NPs is Cu x S. In addition, the powder X-ray diffraction (PXRD) patterns ( Figure S4) of hollow NPs verifies that the all diffraction peaks are assigned to the Cu x S crystal (PDF#41-0959) [42]. Next, the high-resolution X-ray photoelectron spectroscopy (XPS) spectrum of Cu 2p exhibit Cu element in NPs possessed Cu + and Cu 2+ (Figure 2D), and S element is S 2state ( Figure 2E) [43]. Furthermore, the corresponding UV-vis-NIR spectrum ( Figure 2F) of hollow Cu x S NPs showed the optical absorption band peaking wavelength at NIR region (range from NIR-I to NIR-II), which displayed the potential for the NIR-II phototherapy. These results well demonstrated that we successfully achieved hollow Cu x S NPs with absorption at NIR-II region.
To improve the biocompatibility of Dox/Cu x S NPs, PEG-LA due to good biocompatibility and long-term circulation is used to modify the hollow NPs based on the Cu-S bond. The typical TEM image ( Figure

F G
To date, ICD has been well demonstrated in the chemotherapy or photothermal therapy. As shown in schematic illustration (Figure 4A), DAMPs release from the dying cells after treatment with phototherapy. A "find-me" signal is delivered by the released ATP molecules which cause dendritic cells (DCs) to produce cytokine [44,45]. In comparison to the groups of PBS, Cu x S-PEG NPs, Dox, Dox/Cu x S-PEG NPs, Cu x S-PEG NPs plus laser, we observe a highest level of extracellular ATP at 25.3 nM after treatment with the synergistic chemo-photothermal therapy by using a luciferase-based probe ( Figure 4B). Moreover, CRT exposure is a mediator of tumor immunogenicity and potent "eat-me" signal [46]. Flow cytometry measurement reveals that the CRT is most highly expressed after treatment with Dox/Cu x S-PEG NPs under 1064 nm laser irritation, indicating that combination chemotherapy and PTT can more effectively induce the CRT exposure ( Figure 4C). The direct fluorescence microscopy observation ( Figure 4D) and MIF of CRT from fluorescence image ( Figure 4E) further confirm the most significant exposure of CRT after treatment with synergistic chemo-photothermal therapy. To the best of our knowledge, HMGB-1 releasing from cell nucleus in dying cells can trigger inflammation, attract various immune cells and cause DC maturation [47]. In our work, we observe that the treatment of Dox/Cu x S-PEG NPs plus 1064 nm laser also shows a most significant increase of the translocation of HMGB-1 from nuclei to extracellular space ( Figure   4F). The MIF of HMGB-1 in extracellular space from Figure 4F indicates synergistic chemo-photothermal therapy can effectively promote the release of HMGB-1 ( Figure   4G). irradiation with the 1064 nm laser were recorded by the infrared camera ( Figure S14).

Next, in vitro
The temperature at tumor sites with the treatments of Cu x S-PEG NPs plus laser increases to 45.06 o C within 5 min (Figure S15), while the temperature of PBS group is silghtly increased. These results reveal that the enhanced permeability and retention (EPR) effect induced Cu x S-PEG NPs accumulating into the the tumor sites passively.
Next, we investigate the antitumor efficacy of the synergistic chemo-photothermal therapy based on the Cu x S-PEG NPs triggered by NIR-II laser.
As the schematic illustration shown in the Figure 5A Figure   5F). These experimental results indicate that the breast cancer could be effectively inhibited by chemo-photothermal therapy.
Next, we aslo investigated the immune response from ICD after treatments with chemo-photothermal therapy, which can trigger cytotoxic T-lymphocytes and induce anti-tumor immunity [48]. In our experiments, the EMT-6 tumor bearing mice were

Synthesis of Cu x S-PEG NPs
The LA-PEG was prepared according to our previous report [49]. Typically, 5 g mPEG 5k was dissolved in 20 mL dichloromethane. After fully dissolved, lipoic acid

Study of the NIR-II photothermal effect
To explore the photothermal conversion effect of the synthesized Cu x S-PEG NPs To study the intracellular drug delivery of the Dox/Cu x S-PEG NPs, EMT-6 cells were incubated with Dox/Cu x S-PEG NPs dispersion for 0, 1, 2, and 4 h with or without 5 min NIR-II laser irradiation. After the treatments, the cells were measured by the inverted fluorescence microscope for different time point.

In Vitro chemotherapy and chemo-photothermal therapy
To study the chemotherapy and chemo-photothermal effect of the Dox/Cu x S-PEG NPs in cancer cells, EMT-6 cells (3 × 10 3 ) were seeded in 96-well plate and treated with the free Dox, Dox/Cu x S-PEG, and Dox/Cu x S-PEG + laser, respectively. The concentration of free Dox was normalized to be equivalent to the loaded Dox in the Cu x S-PEG NPs. After the treatments, the cell viability of cancer cells was measured by MTT assay.

In vivo chemo-photothermal therapy
When the tumor volumes were about 100 mm 3  Committee.

Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing Interests
The authors have declared that no competing interest exists.