Heptamethine Cyanine-Loaded Nanomaterials for Cancer Immuno-Photothermal/Photodynamic Therapy: A Review

The development of strategies capable of eliminating metastasized cancer cells and preventing tumor recurrence is an exciting and extremely important area of research. In this regard, therapeutic approaches that explore the synergies between nanomaterial-mediated phototherapies and immunostimulants/immune checkpoint inhibitors have been yielding remarkable results in pre-clinical cancer models. These nanomaterials can accumulate in tumors and trigger, after irradiation of the primary tumor with near infrared light, a localized temperature increase and/or reactive oxygen species. These effects caused damage in cancer cells at the primary site and can also (i) relieve tumor hypoxia, (ii) release tumor-associated antigens and danger-associated molecular patterns, and (iii) induced a pro-inflammatory response. Such events will then synergize with the activity of immunostimulants and immune checkpoint inhibitors, paving the way for strong T cell responses against metastasized cancer cells and the creation of immune memory. Among the different nanomaterials aimed for cancer immuno-phototherapy, those incorporating near infrared-absorbing heptamethine cyanines (Indocyanine Green, IR775, IR780, IR797, IR820) have been showing promising results due to their multifunctionality, safety, and straightforward formulation. In this review, combined approaches based on phototherapies mediated by heptamethine cyanine-loaded nanomaterials and immunostimulants/immune checkpoint inhibitor actions are analyzed, focusing on their ability to modulate the action of the different immune system cells, eliminate metastasized cancer cells, and prevent tumor recurrence.


Introduction
Cancer survival is, in many cases, a mirage due to metastization processes and tumor relapse [1]. This harsh reality is inherently correlated with the inadequacy of classical treatments (e.g., surgery, chemotherapy, radiotherapy) to completely eliminate metastasized cancer cells and to trigger the creation of immune memory [1,2]. To tackle these problems, researchers and clinicians have focused on developing strategies that can re-engage the immune system in the fight against local and metastasized cancer cells [1,3]. In this regard, nanomaterial-mediated immuno-phototherapy has been yielding remarkable results in preclinical models [4][5][6].
This promising therapeutic modality explores the nanomaterials' physicochemical features for enabling tumor uptake, as well as their optical properties, which strongly influence the phototherapeutic outcome [7][8][9]. Upon irradiation of the primary tumor with light, the primary tumor-homed nanomaterials can absorb its energy, producing heat (photothermal therapy (PTT)) and/or reactive oxygen species (ROS; photodynamic therapy (PDT)) [10,11]. In brief, the photoresponsive agent absorbs light energy and is In this review, the application of HC-loaded nanomaterials in cancer immuno-PTT/PDT is analyzed. In Section 2, a general overview of this therapeutic approach is given. Sections 3 and 4 analyze the application of ICG-loaded nanomaterials and prototypic HC-loaded nanostructures in cancer immuno-PTT/PDT. Finally, an outlook of the state of the art and future directions is provided (Section 5). In this review, the application of HC-loaded nanomaterials in cancer immuno-PTT/PDT is analyzed. In Section 2, a general overview of this therapeutic approach is given. Sections 3 and 4 analyze the application of ICG-loaded nanomaterials and proto-typic HC-loaded nanostructures in cancer immuno-PTT/PDT. Finally, an outlook of the state of the art and future directions is provided (Section 5).

Overview of Nanomaterial-Mediated Immuno-PTT/PDT
The photothermal and photodynamic effects mediated by HC-loaded nanomaterials can trigger a series of events that are crucial for potentiating the antitumoral immune responses [31,33,35]. For this reason, the immuno-PTT/PDT potential of these nanomaterials is being investigated for metastatic cancer treatment [32,37,73,74]. In general, this therapeutic approach starts with the intravenous administration of the nanomaterials [75,76]. Once in circulation, these nanomaterials must avoid interaction with blood components (e.g., albumin, red blood cells), uptake by liver/spleen, and rapid clearance by the kidneys [10,77,78]. This will likely increase the probability of these nanomaterials to extravasate to the tumor zone by taking advantage of abnormal static and dynamic pores occurring in the tumor vasculature [68,79,80]. The ability of nanomaterials to avoid off-target accumulation/clearance and to accumulate in the tumor zone is strongly influenced by their physicochemical properties (e.g., size, surface charge, corona composition). The impact of these features on nanomaterial biodistribution has been extensively reviewed by our and other research groups [10,[81][82][83][84].

Overview of Nanomaterial-Mediated Immuno-PTT/PDT
The photothermal and photodynamic effects mediated by HC-loaded nanomater can trigger a series of events that are crucial for potentiating the antitumoral immune sponses [31,33,35]. For this reason, the immuno-PTT/PDT potential of these nanomater is being investigated for metastatic cancer treatment [32,37,73,74].
In general, this therapeutic approach starts with the intravenous administration the nanomaterials [75,76]. Once in circulation, these nanomaterials must avoid interact with blood components (e.g., albumin, red blood cells), uptake by liver/spleen, and ra clearance by the kidneys [10,77,78]. This will likely increase the probability of these na materials to extravasate to the tumor zone by taking advantage of abnormal static dynamic pores occurring in the tumor vasculature [68,79,80]. The ability of nanomater to avoid off-target accumulation/clearance and to accumulate in the tumor zone strongly influenced by their physicochemical properties (e.g., size, surface charge, cor composition). The impact of these features on nanomaterial biodistribution has been tensively reviewed by our and other research groups [10,[81][82][83][84].
Afterward, the primary tumor (i.e., the original tumor) is irradiated with NIR lig and the nanomaterials accumulated in this zone produce a localized temperature incre and/or ROS [32,71,85]. Such effects can damage cancer cells and may be sufficiently str to ablate the primary tumor [32,86,87]. As importantly, the nanomaterials' phototh mal/photodynamic effects can also (i) relieve tumor hypoxia [31,32], (ii) release TAAs DAMPs (e.g., exposure of calreticulin (CRT) on cancer cells' membrane, release ATP high mobility group box 1 protein (HMGB1)) [33,34], and (iii) induced a pro-inflammat response ( Figure 2) [35,36].  Schematic representation of the different events occurring during the immuno-PTT/PDT mediated by HC-loaded nanoparticles. In this process, the nanoparticles are generally administered intravenously. The immunostimulants and the ICIs can be administered in conjugation with the nanoparticles or at a later time point. After nanoparticle administration, the primary tumor is irradiated with NIR light. The nanoparticles' photothermal/photodynamic effects can per se induced damage in the primary tumor and can also trigger (i) TAAs/DAMPs release, (ii) hypoxia relief, and (iii) a pro-inflammatory response. The released TAAs can then be processed, leading to DC maturation. DC maturation is also aided by the DAMPs and by the immunostimulants. Afterward, the ICIs abolish the immuno-suppression mediated by CTLA-4, IDO1, and PD-1/PD-L1. These events contribute to the amelioration of the CTL/T reg cells ratio in the diseased sites, paving the way for the elimination of the primary (local effect) and the metastases/distant tumors (abscopal effect). During this process, memory T cells are also established, which have a crucial role in preventing tumor recurrence.
For instance, Zhao and co-workers demonstrated that PTT/PDT generated by ICGincorporating polymeric nanostructures induced CRT exposure and HMGB1 and ATP release, leading to about 2.20-fold higher dendritic cell (DC) maturation (when compared to the non-irradiated nanostructures and the control group) [32]. Moreover, the photothermal heating produced by these nanostructures also improved tumor oxygenation. This contributed to augment the tumor levels of M1-polarized (pro-inflammatory/antitumoral) tumor-associated macrophages (TAMs) by 4.30-fold and to reduce the levels of M2-polarized (anti-inflammatory/protumoral) TAMs by 1.70-fold ( Figure 3). Tumor hypoxia relief can also be attained or improved through the inclusion of oxygen-generating elements in the nanoformulations (e.g., CeO 2 nanoparticles [88], MnO 2 nanoparticles [34], catalase [89]). Tan and co-workers demonstrated that the PTT mediated by cationic lipidic nanoparticles incorporating IR780 could induced the release of TAAs and HMGB1 as well as the exposure of CRT, leading to enhanced DC maturation [33].
Pharmaceutics 2022, 14, 1015 5 of 32 damage in the primary tumor and can also trigger (i) TAAs/DAMPs release, (ii) hypoxia relief, and (iii) a pro-inflammatory response. The released TAAs can then be processed, leading to DC maturation. DC maturation is also aided by the DAMPs and by the immunostimulants. Afterward, the ICIs abolish the immuno-suppression mediated by CTLA-4, IDO1, and PD-1/PD-L1. These events contribute to the amelioration of the CTL/Treg cells ratio in the diseased sites, paving the way for the elimination of the primary (local effect) and the metastases/distant tumors (abscopal effect). During this process, memory T cells are also established, which have a crucial role in preventing tumor recurrence.
For instance, Zhao and co-workers demonstrated that PTT/PDT generated by ICGincorporating polymeric nanostructures induced CRT exposure and HMGB1 and ATP release, leading to about 2.20-fold higher dendritic cell (DC) maturation (when compared to the non-irradiated nanostructures and the control group) [32]. Moreover, the photothermal heating produced by these nanostructures also improved tumor oxygenation. This contributed to augment the tumor levels of M1-polarized (pro-inflammatory/antitumoral) tumor-associated macrophages (TAMs) by 4.30-fold and to reduce the levels of M2-polarized (anti-inflammatory/protumoral) TAMs by 1.70-fold ( Figure 3). Tumor hypoxia relief can also be attained or improved through the inclusion of oxygen-generating elements in the nanoformulations (e.g., CeO2 nanoparticles [88], MnO2 nanoparticles [34], catalase [89]). Tan and co-workers demonstrated that the PTT mediated by cationic lipidic nanoparticles incorporating IR780 could induced the release of TAAs and HMGB1 as well as the exposure of CRT, leading to enhanced DC maturation [33].  These nanomaterials' photothermal/photodynamic effects can also trigger the release of pro-inflammatory cytokines and chemokines [33,90], which are crucial in the recruitment/activation of immune cells and can also enhance the outcome of ICI-based therapies [12,[91][92][93][94].
To further improve DC maturation, immunostimulants can be combined with the HC-loaded nanoparticles. In this regard, CpG oligodeoxynucleotides (CpG ODNs; TLR-9 agonist), due to their hydrophilicity, can be co-administered with the nanoparticles (i.e., non-encapsulated) or incorporated in the hydrophilic shell of the HC-loaded nanoparticles [95][96][97][98]. In turn, hydrophobic immunostimulants such as R837 (Imiquimod; TLR-7 agonist) have been encapsulated in the HC-loaded nanostructures due to their hydrophobicity [69,99]. For example, Chen et al. demonstrated that the ability of the PTT mediated by ICG-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles to improve DC maturation could be further boosted by 1.24 times by including R837 in this nanoformulation [38].
Subsequently, mature DCs (mDCs) can migrate into the lymph nodes and then prime T cells for the TAA [100,101]. Despite these events, the immunosuppressive actions mediated by CTLA-4, IDO1, and PD-1/PD-L1 can still abrogate the T cells' action on the primary and secondary tumors [102][103][104][105]. To overcome this bottleneck, ICIs have also been combined with nanomaterial-mediated PTT/PDT [38,75]. In this regard, anti-CTLA-4, anti-PD-1, and anti-PD-L1 antibodies (Ab) are often intravenously co-administered with the nanomaterials (i.e., non-encapsulated) for performing the blockade of these receptors [38,73]. In turn, the IDO1 inhibitors, such as NLG919 or Epacadostat, due to their hydrophobic character, have been-loaded into nanomaterials' core/reservoirs [85,106]. In general, the combination of ICIs' action with nanomaterial-mediated PTT/PDT can starkly augment the cytotoxic T lymphocytes (CTLs) populations in the tumoral sites and diminish the populations of regulatory T cells (T reg cells; immunosuppressive cells) [75,85,107], enabling the elimination of the primary tumor and abscopal effects on the secondary tumors (reviewed in Sections 3 and 4). The combined effects arising from nanomaterial-mediated PTT/PDT and ICIs can also greatly increase the levels of memory T cells [37,38,75], which have a crucial role in decreasing the likelihood of tumor recurrence (reviewed in Sections 3 and 4).

ICG-Loaded Nanomaterials in Cancer Immuno-PTT/PDT
ICG-loaded nanomaterials are among the most explored for cancer-immuno-PTT/ PDT [107][108][109][110]. The FDA-approval status of ICG for angiography is certainly a key contributor to this phenomenon. The ICG-loaded nanomaterials can be used for theragnostic applications since these can produce a photothermal/photodynamic effect upon NIR laser irradiation as well as emit fluorescence [111,112].
In recent work, Huang et al. verified that the PTT mediated by ICG-loaded Poly(ethylene glycol) functionalized (PEGylated) liposomes could ablate the primary tumor and enrich the CTL/T reg cells ratio in the secondary tumors by 3.30-fold (when compared to the control) [113]. However, such effect was not able to impact the growth of the secondary tumors, which was attributed to the high expression of PD-1 and mucin domain-containing protein 3 (TIM-3) by the secondary tumor-homed CTLs. By combining the nanomaterials' PTT with PD-1 and TIM-3 blockade (using anti-PD-1 and anti-TIM-3 Abs), secondary tumor regression was attained.
In fact, the combination of ICG-loaded nanomaterials' PTT/PDT capacity with immunostimulants and/or ICIs can pave the way to a remarkable therapeutic outcome [38,39,85,108]. For instance, Liu's team prepared hyaluronic acid (HA)-coated metal organic frameworks (MOF)-loaded with ICG and R837 for application in cancer immuno-PTT [108]. The combined photothermal and immunostimulatory effects mediated by this nanosystem boosted the levels of mDCs in the lymph nodes to ≈55%, being 1.40 times greater than those attained after the sole application of nanomaterials' PTT (ICG-loaded MOF plus NIR light) and nanomaterials' immunostimulant delivery (ICG and R837-loaded MOF) [108]. Due to this reason, the nanomaterial-mediated PTT and R837 delivery induced 1.50 times higher CTL infiltration, thus leading to the greatest reduction in primary and distant tumor growth ( Figure 4). This treatment also prompted the highest levels of memory T cells, being the only therapeutic regimen that diminished the growth of the reinoculated tumors.
Pharmaceutics 2022, 14, 1015 7 of 32 CTL infiltration, thus leading to the greatest reduction in primary and distant tumor growth ( Figure 4). This treatment also prompted the highest levels of memory T cells, being the only therapeutic regimen that diminished the growth of the reinoculated tumors. In another study, Chen et al., explored the therapeutic capacity of nanoparticle-mediated PTT and R837 delivery (PLGA nanoparticles-loaded with ICG and R837 plus NIR light) followed by CTLA-4 blockade (systemic administration of anti-CTLA-4 Ab after the PTT) [38]. This combined treatment induced a remarkable effect since it could eliminate the primary and secondary tumors as well as prevent the establishment of metastases. A key contributor to this outcome was the combined treatment's ability to improve the CTLs/Treg cell ratio in the malignant tissue. In fact, the nanoparticle-mediated PTT and R837 delivery plus CTLA-4 blockade prompted a 1.40, 15.40, 10.20, and 5.60-fold higher CTLs/Treg cell ratio than nanomaterials' R837 delivery plus CTLA-4 blockade, nanomaterials' PTT, nanomaterials' R837 delivery, and CTLA-4 Ab administration, respectively. On other hand, the combined treatment also prompted the highest levels of effector memory T In another study, Chen et al., explored the therapeutic capacity of nanoparticlemediated PTT and R837 delivery (PLGA nanoparticles-loaded with ICG and R837 plus NIR light) followed by CTLA-4 blockade (systemic administration of anti-CTLA-4 Ab after the PTT) [38]. This combined treatment induced a remarkable effect since it could eliminate the primary and secondary tumors as well as prevent the establishment of metastases. A key contributor to this outcome was the combined treatment's ability to improve the CTLs/T reg cell ratio in the malignant tissue. In fact, the nanoparticle-mediated PTT and R837 delivery plus CTLA-4 blockade prompted a 1.40, 15.40, 10.20, and 5.60-fold higher CTLs/T reg cell ratio than nanomaterials' R837 delivery plus CTLA-4 blockade, nanomaterials' PTT, nanomaterials' R837 delivery, and CTLA-4 Ab administration, respectively. On other hand, the combined treatment also prompted the highest levels of effector memory T cells (T EM ),  Liu and co-workers prepared PEGylated nanoparticles containing ICG and Epacadostat for cancer immuno-PTT/PDT [85]. The events triggered by the nanoparticles' photothermal/photodynamic effects could improve DC maturation by up to 2.50-fold (the levels of mDCs in the tumor-draining lymph node (TDLN) reached 16% after nanomaterialmediated PTT/PDT, contrasting with the 6.4% attained when non-irradiated nanoparticles were used). The nanomaterials' PTT/PDT combined with IDO1 inhibition (performed by Epacadostat) was able to induced the elimination of the primary tumor and slow the growth of the secondary tumor. By adding PD-L1 blockers to this therapy (PEGylated nanoparticles containing ICG and Epacadostat + NIR light + Anti-PD-L1 Ab), the primary tumor was also eliminated, but the secondary tumor experienced a stronger delay in its growth. Such events were correlated with a higher CTL infiltration and higher amelioration of the CTL/Treg cells ratio in the secondary tumors after the nanomaterials' PTT/PDT combined with IDO1 inhibition and PD-L1 blockade. In other work, Lam's team demonstrated that the application of two treatment cycles composed of R837-loaded PEGylated ICG-based nanoparticles plus NIR light plus anti-PD-1 Ab administration could lead to the elimination of both primary and secondary tumors [39].
The immuno-PTT/PDT capability of other ICG-loaded nanomaterials is summarized in Tables 1 and 2. Liu and co-workers prepared PEGylated nanoparticles containing ICG and Epacadostat for cancer immuno-PTT/PDT [85]. The events triggered by the nanoparticles' photothermal/photodynamic effects could improve DC maturation by up to 2.50-fold (the levels of mDCs in the tumor-draining lymph node (TDLN) reached 16% after nanomaterialmediated PTT/PDT, contrasting with the 6.4% attained when non-irradiated nanoparticles were used). The nanomaterials' PTT/PDT combined with IDO1 inhibition (performed by Epacadostat) was able to induced the elimination of the primary tumor and slow the growth of the secondary tumor. By adding PD-L1 blockers to this therapy (PEGylated nanoparticles containing ICG and Epacadostat + NIR light + Anti-PD-L1 Ab), the primary tumor was also eliminated, but the secondary tumor experienced a stronger delay in its growth. Such events were correlated with a higher CTL infiltration and higher amelioration of the CTL/T reg cells ratio in the secondary tumors after the nanomaterials' PTT/PDT combined with IDO1 inhibition and PD-L1 blockade. In other work, Lam's team demonstrated that the application of two treatment cycles composed of R837-loaded PEGylated ICG-based nanoparticles plus NIR light plus anti-PD-1 Ab administration could lead to the elimination of both primary and secondary tumors [39].
The immuno-PTT/PDT capability of other ICG-loaded nanomaterials is summarized in Tables 1 and 2. Table 1. Outcome generated by the immuno-PTT/PDT mediated by ICG-based nanomaterials in the levels of mDCs and T cells.

Formulation
Immuno Therapy Agent

Mg and ICG-loaded PES NPs -ICG
Mg and ICG-loaded PES NPs + Laser induced two times higher mDC levels than Mg and ICG-loaded PES NPs, and PES (in the primary tumor); Mg and ICG-loaded PES NPs + Laser induced 2.27 times higher mDC levels than the control (in the primary tumor); Mg and ICG-loaded PES NPs + Laser induced about two times higher mDC levels than Mg and ICG-loaded PES NPs, PES, and the control (in lymph nodes). [32] Mg and ICG-loaded PES NPs + Laser induced about 2.72 times higher CTLs levels than Mg and ICG-loaded PES NPs, PES, and the control (in the secondary tumor).

Mg and ICG-loaded PES NPs -ICG
Mg and ICG-loaded PES NPs + Laser caused tumor regression while the other treatment groups only caused tumor growth reduction; Mg and ICG-loaded PES NPs + Laser caused a great secondary tumor growth reduction compared to the other treatment groups; The number of metastatic nodules after Mg and ICG-loaded PES NPs + Laser treatment strongly decreases compared to control (3.39 vs. 41.53). [32]

ICG-loaded COF coated with ovalbumin
Anti-PD-L1 Ab (non-loaded) ICG; COF ICG-loaded COF coated with ovalbumin + Laser, with and without Anti-PD-L1 Ab both caused primary tumor eradication; ICG-loaded COF coated with ovalbumin + Laser + Anti-PD-L1 Ab caused secondary tumor eradication while the other treatment groups only caused tumor growth reduction. [73] Metastases after ICG-loaded COF coated with ovalbumin + Laser + Anti-PD-L1 Ab do not occur in mice after tumor reinoculation.

ICG-loaded lipid-PLGA NPs decorated with FimH
FimH ICG ICG-loaded lipid-PLGA NPs decorated with FimH + Laser caused primary tumor eradication while the other treatment groups only caused tumor growth reduction.
[115] Metastases after ICG-loaded lipid-PLGA NPs decorated with FimH + Laser treatment do not occur in mice after tumor reinoculation.

CpG ODNs-loaded ICG functionalized MOF CpG ODNs ICG
CpG-loaded ICG functionalized MOF + Laser caused primary tumor eradication while the other treatment groups only caused tumor growth reduction.

ICG and poly I:C (c) -loaded liposomes (d)
poly I:C ICG ICG and poly I:C-loaded liposomes + Laser. and ICG-loaded liposomes + Laser caused primary tumor regression while the other treatment groups do not reduce tumor growth.
[121] Metastases after ICG and poly I:C-loaded liposomes + Laser treatment strongly decrease compared to control in mice after tumor reinoculation.

Prototypic HC-Loaded Nanomaterials in Cancer Immuno-PTT/PDT
Nanoparticles containing prototypic HC also hold great potential for application in cancer immuno-PTT/PDT due to their improved optical properties (reviewed in detail in [10,17,68]). Among these, IR780-loaded nanomaterials have been the most applied, followed by IR820-loaded nanostructures.
As described in Section 2, the events triggered by the nanomaterial-mediated PTT/PDT can per se support the development of antitumoral immunological responses. In this regard, Borrathybay and co-workers verified that the photothermal/photodynamic effects generated by IR780-loaded PEG-Poly(caprolactone) (PCL) nanoparticles trigger the release of DAMPs (ATP, HMGB1, CRT), leading to a 1.50-and 2-fold greater DCs' maturation and CTLs' infiltration when compared to the control, respectively [122]. These effects paved the way for a slight decrease in the primary tumors' growth and reduction of the occurrence of lung metastases.
The inclusion of immunostimulants and/or ICIs in the nanomaterials' phototherapies is crucial to further boost the therapeutic outcome [37,65,71,75,106]. For instance, Ou and co-workers prepared PEGylated Glucocorticoid-induced Cancer Necrosis Factor Receptor (GITR)-functionalized PLGA nanoparticles incorporating IR780 and Imatinib (diminishes immunosuppression mediated by T reg cells [123]) for application in cancer immuno-PTT/PDT [71]. The irradiation of these nanoparticles with NIR light stimulated the release of TAAs and HMBG1. This could augment the intratumoral levels of matured DCs to about 52%, being 2.40 times greater than those attained in the control group [71]. Moreover, the IR780 and Imatinib-loaded nanoparticles combined with NIR light also reduced the intratumoral T reg cells' levels by 3.40-fold. Such events mediated by the nanomaterials' immuno-PTT/PDT led to complete tumor elimination. In another work, Qian et al., developed PEG-PCL micelles-loaded with NLG919 and IR780 for application in cancer immuno-PTT [106]. By combining the IDO1 inhibitory capacity of NLG919 with the local hyperthermia produced by the micelles upon NIR laser irradiation, this treatment could ablate the primary tumor and strongly diminish the growth of the secondary tumors ( Figure 6). Moreover, this combined approach also decreased the establishment of lung metastases. This outcome was correlated with the ability of the micelle-mediated immuno-PTT to greatly improve the CTLs/T reg cells ratio. In fact, the micelles' immuno-PTT prompted a 7-and 33 times higher CTLs/T reg cells ratio than the micelles' immunotherapy (NLG919 and IR780-loaded micelles) and micelles' PTT (IR780-loaded micelles plus NIR light), respectively. Therefore, the micelles' immunotherapy and micelles' PTT were only capable of reducing the growth of the primary and secondary tumors. In another work, Luan team prepared HA-coated IR820-loaded MOFs and Mannancoated R837 and 1-Methyl-D-tryptophan (1MT; IDO1 inhibitor)-loaded MOFs for cancer immuno-PTT. The photothermal effect mediated by the IR820-loaded MOFs strongly stimulated DAMPs and TAAs release [37]. Such effect could improve DCs' maturation levels from 17.6 to 33.2%. By combining the PTT capacity of IR820-loaded MOFs with the immunomodulating capacity of R837 and 1MT-loaded MOFs, the levels of mDCs could be further improved to about 42%. In vivo, the combined treatment (IR820-loaded MOFs + NIR light + R837 and 1MT-loaded MOFs) prompted the greatest enrichment in the CTL/Treg cells ratio. Such events contributed to the regression of the primary tumor and almost inhibited the growth of the secondary tumor. This combined treatment could also abolish the establishment of metastases upon reinoculation of the cancer cells, an effect attributed to the more pronounced presence of memory T cells.
Luan's team explored the phototherapeutic potential of IR820-1MT conjugate nanoparticles in combination with Anti-PD-L1 Ab [75]. Pairing the double-ICI strategy with the PTT (IR820-1MT + NIR light + Anti-PD-L1 Ab) yielded the best therapeutic outcome (the strongest reduction in the growth of the primary and secondary tumors), due to In another work, Luan team prepared HA-coated IR820-loaded MOFs and Mannancoated R837 and 1-Methyl-D-tryptophan (1MT; IDO1 inhibitor)-loaded MOFs for cancer immuno-PTT. The photothermal effect mediated by the IR820-loaded MOFs strongly stimulated DAMPs and TAAs release [37]. Such effect could improve DCs' maturation levels from 17.6 to 33.2%. By combining the PTT capacity of IR820-loaded MOFs with the immunomodulating capacity of R837 and 1MT-loaded MOFs, the levels of mDCs could be further improved to about 42%. In vivo, the combined treatment (IR820-loaded MOFs + NIR light + R837 and 1MT-loaded MOFs) prompted the greatest enrichment in the CTL/T reg cells ratio. Such events contributed to the regression of the primary tumor and almost inhibited the growth of the secondary tumor. This combined treatment could also abolish the establishment of metastases upon reinoculation of the cancer cells, an effect attributed to the more pronounced presence of memory T cells.
Luan's team explored the phototherapeutic potential of IR820-1MT conjugate nanoparticles in combination with Anti-PD-L1 Ab [75]. Pairing the double-ICI strategy with the PTT (IR820-1MT + NIR light + Anti-PD-L1 Ab) yielded the best therapeutic outcome (the strongest reduction in the growth of the primary and secondary tumors), due to greater DCs' maturation, CTLs' infiltration, and CTL/T reg ratio improvement. This approach also prompted the highest levels of T EM cells, and therefore the establishment of lung metastases did not occur after tumor reinoculation (Figure 7). greater DCs' maturation, CTLs' infiltration, and CTL/Treg ratio improvement. This approach also prompted the highest levels of TEM cells, and therefore the establishment of lung metastases did not occur after tumor reinoculation (Figure 7).   [75]. Copyright 2019 American Chemical Society. NS: normal saline; αPD-L1: anti-PD-L1 Ab; IR820: IR820 + Laser irradiation; IR820-1MT: IR820-1MT conjugate nanoparticles + Laser irradiation. ** p < 0.01, and *** p < 0.001. The blue arrows represent metastatic nodules.
The immuno-PTT/PDT potential of other prototypic HC-loaded nanomaterials is summarized in Tables 3 and 4. Table 3. Outcome generated by the immuno-PTT/PDT mediated by prototypic HC-based nanomaterials in the levels of mDCs and T cells.

Formulation
Immuno Therapy Agent

Conclusions and Future Outlook
In this review, the recent progress in the application of HC-loaded nanomaterials for cancer immuno-PTT/PDT was analyzed.
Among the HC family, the ICG-loaded nanomaterials have been the most explored for this therapeutic modality, followed by those-loaded with IR780 and then by those incorporating IR820, IR797, and IR775. This trend is concomitant with the usage of these nanostructures in other areas (e.g., standalone PTT/PDT, chemo-PTT/PDT). On the one hand, the FDA-approved status of ICG has fomented the development of nanomaterials containing this NIR dye for cancer-related applications. On the other hand, prototypic HCs such as IR780 and IR820 have superior optical properties when compared to ICG, which has motivated their loading into nanomaterials aimed for cancer therapy. Despite the potential of other prototypic HCs (e.g., Cypate, IR808, IR825), these have not yet been explored for cancer immuno-PTT/PDT. Therefore, in the future, the development of nanoformulations incorporating such prototypic HCs could be interesting to fully unveil their immuno-phototherapeutic potential.
In general, the coordinated action of HC-loaded nanomaterials' photothermal/ photodynamic effects (e.g., inducers of cell death and release of TAAs/DAMPs), immunostimulants (enhancers of DCs' maturation), and ICIs (strong modulators of CTLs and T reg cells) could elicit both local (on the primary tumor) and abscopal (on the secondary tumor/metastases) antitumoral responses. In some few cases, the magnitude of such combined effects led to the complete elimination of the primary tumor and also induced a reduction in the growth of the secondary tumor or even its elimination. These combined immuno-phototherapeutic effects also had an important role in the establishment of immune memory that could prevent/delay tumor's recurrence. Together, these facts depict the potential of HC-loaded nanomaterials for cancer immuno-PTT/PDT.
In order to further amplify the magnitude of HC-loaded nanomaterials' immuno-PTT/PDT, it could be interesting to (i) boost HC-loaded nanomaterials' photothermal/photodynamic capacity, (ii) optimize the delivery regiment of immunostimulants/ICIs, and (iii) incorporate additional therapeutic agents in the combined therapy. Boosting the HC-loaded nanomaterials' photothermal/photodynamic effects will be crucial to enhance the therapeutic outcome in the primary tumor as well as to potentially improve the release of DAMPs/TAAs, which play an important role in the abscopal antitumoral T cell responses. This could be achieved by improving HC-loaded nanomaterials' photostability (in order to sustain the phototherapeutic effects over time) or by incorporating additional NIR responsive agents in the nano-formulations (e.g., gold nanorods, graphene derivatives) [125][126][127].
The events occurring in HC-loaded nanomaterials' immuno-PTT/PDT set the optimal time points for the action of the immunostimulants and ICIs. Initially, the nanomaterials' photothermal/photodynamic effects must occur to trigger TAAs/DAMPs release, which will be crucial for DC maturation. In this way, the immunostimulants' action is best suited after the nanomaterials' PTT/PDT. The same applies to ICIs, whose action is optimal after DC maturation. Therefore, the development of technologies that can perform the sequential delivery of nanomaterials, immunostimulants, and ICIs can potentially pave the way for an improved therapeutic outcome. In this context, hierarchically designed injectable hydrogels, microneedle patches, and scaffolds are promising tri-dimensional matrixes for performing the sequential delivery of these agents [128][129][130][131]. Finally, the inclusion of other hydrophobic agents in the nanomaterials' core/reservoirs (e.g., chemotherapeutic drugs) or hydrophilic agents in the abovementioned tri-dimensional matrices (e.g., antitumoral peptides) can lead to an even greater therapeutic outcome by exploring synergistic interactions among the enrolled agents [9,96,[132][133][134].
Despite the potential of HC-loaded nanomaterials' immuno-PTT/PDT, this therapeutic approach still faces critical challenges before validation in clinical trials can be envisioned. So far, HC-loaded nanomaterials' immuno-PTT/PDT has been mainly applied to treat breast and melanoma tumors and their metastases in mice. This applicability to superfi-cial tumors is highly correlated with the penetration depth limits of NIR light [135,136]. Furthermore, human tumors are also located in deeper zones when compared to their equivalents in mice [137]. In this regard, the use of endoscopes coupled with fiber-type laser to irradiate deeper primary tumors may be an interesting strategy to address the previous limitations at the cost of increasing the procedures' invasiveness [137].
There are also hurdles associated with the nanomaterials' tumor-homing capacity. Classically, nanomaterials have been described to accumulate in the tumor by extravasating through the tumor's leaky vasculature (the so-called enhanced permeability and retention (EPR) effect), hence being designed based on this rationale. However, a review by Wilhelm et al., highlighted that the dose of intravenously administered nanoparticles that reaches the tumor is, in many cases/studies, very low [138]. Recently, other mechanisms involved in nanomaterials' tumor accumulation have been unveiled (e.g., dynamic vents, active transport through endothelial cells) [79,139]. In this way, it is crucial to continue to investigate the mechanisms responsible for nanomaterials' tumor uptake after systemic administration and to optimize the nanoparticles' physicochemical properties accordingly. Moreover, strategies aimed to modulate the tumor vasculature could be a route for mitigating this tumor uptake problem (e.g., vascular permeabilization, normalization, or disruption approaches) [140][141][142][143]. On the other hand, the encapsulation of nanomaterials in macroscale delivery systems (e.g., injectable hydrogels, microneedles) is also appealing [130,131]. These macroscale systems can be used to perform the direct delivery of nanoparticles and ICIs/immunostimulants into the tumor site, possibly avoiding the abovementioned systemic administration issues [131,144].
On the other hand, the efficacy of HC-loaded nanomaterials' immuno-PTT/PDT has not yet been validated in larger animal models. These studies are of utmost importance since they can expose some of the limitations described above. Moreover, long-term studies are also required. Such studies are crucial to establish the safety of this approach since some possible side effects may have a delayed onset (e.g., immune-related adverse events) [145]. Moreover, the outcome of nanomaterial-mediated immuno-PTT/PDT can be, in some cases, highly heterogenous (the same also occurs in the clinic for ICIs) [106,107,110]. In this regard, finding biomarkers that can predict the therapeutic response may also be a path to push the translation of this strategy [146].
Overall, continuing this line of research based on HC-loaded nanomaterials' immuno-PTT/PDT can unlock potent antitumoral T cell responses against local and metastasized cancer cells as well as generate immune memory that prevents tumor's recurrence.