Abstract

   Near‐infrared (NIR) activated photosensitizers based on heavy‐atom‐free
   have great advantages in photoimmunotherapy, yet the tumor
   microenvironment often restricts their efficacy. To address this, a
   NIR‐activated heavy‐atom‐free photosensitizer (named Cy‐BF) is
   developed. Cy‐BF is then encapsulated with phospholipids and platelet
   exosome vesicles to create platelet exosomes vesicles biomimetic and
   Cy‐BF loaded hybrid liposomes (named CHL) Characterized by high
   phototoxicity, low dark toxicity, and enhanced tumor targeting, CHL
   demonstrates aggregation‐induced broadening of absorption spectra and
   NIR (760 nm laser) activates photothermal therapy and type I
   photodynamic therapy. The CHL‐mediated phototherapy induces
   mitochondrial damage and immunogenic cell death in tumor cells,
   decreases lactate production, and alters the tumor microenvironment by
   reducing regulatory T cells and increasing CD8^+ T cells. To mitigate T
   cell inhibition by excess lactate, a combination therapy is introduced
   using lithium carbonate, which repurposes lactate as an energy source
   for CD8^+ T cells, thereby enhancing the effectiveness of CHL‐mediated
   photoimmunotherapy. This combination approach represents a novel
   strategy for reversing lactate metabolism in both tumor cells and T
   cells, paving the way for future clinical applications in
   photoimmunotherapy.

   Keywords: heavy‐atom‐free photosensitizer, lithium carbonate, NIR
   activated type I/II PDT, photoimmunotherapy, T lymphocytes and cancer
   cells metabolism
     __________________________________________________________________

   This work develops a cyanine dye named Cy‐BF and encapsulates it with
   phospholipids and platelet exosome vesicles to create CHL.
   Characterized by high phototoxicity, low dark toxicity, and enhanced
   tumor targeting, CHL demonstrates aggregation‐induced broadening of
   absorption spectra and NIR‐activated photothermal therapy and type I
   photodynamic therapy. This CHL‐mediated phototherapy induces
   immunogenic cell death in tumor cells, decreases lactate production,
   and alters the tumor microenvironment by reducing Tregs and increasing
   CD8+ T cells. Then a combination therapy using lithium carbonate (LC),
   which repurposes lactate as an energy source for CD8+ T cells, is used
   for enhancing CHL‐mediated photoimmunotherapy. This combination
   approach represents a novel strategy for reversing lactate metabolism
   in both tumor cells and T cells.

   graphic file with name ADVS-12-2416143-g010.jpg

1. Introduction

   Cancer remains a pervasive and deadly disease worldwide, presenting
   significant challenges to public health due to its high prevalence and
   mortality rates.^[ [40]^1 ^] Phototherapy, as a non‐invasive treatment
   method, has shown immense potential in the field of tumor treatment.^[
   [41]^2 ^] Notable for its efficiency, selectivity, and low incidence of
   drug resistance, phototherapy is quickly becoming a focal point of
   research. This approach primarily encompasses photothermal therapy
   (PTT) and photodynamic therapy (PDT).^[ [42]^3 ^] During these
   treatments, photosensitizers (PSs) are activated by specific laser
   wavelengths to either generate heat or produce reactive oxygen species
   (ROS), both of which are effective in destroying tumor cells and
   tissues.^[ [43]^4 ^] The integration of phototherapy with
   immunotherapy, which leverages various immunological techniques to
   prompt the immune system to detect and eliminate tumor cells, offers a
   potent treatment for both primary and metastatic tumors.^[ [44]^5 ^]
   This strategy, known as photoimmunotherapy (PIT), utilizes the targeted
   destruction of primary tumors through light exposure while
   simultaneously triggering immune responses by promoting immunogenic
   cancer cell death.^[ [45]^6 ^] Therefore, by combining these therapies,
   PIT provides a novel anti‐cancer strategy that not only enhances the
   effectiveness of each treatment but also addresses their individual
   limitations.

   Traditional photosensitizers typically use heavy metals because they
   can generate singlet oxygen (^1O[2]) or other reactive oxygen species
   through effective electron and energy transfer mechanisms.^[ [46]^7 ^]
   Heavy‐atom‐free photosensitizers (HAF PSs) are those that exclude heavy
   metals like platinum, iridium, ruthenium, etc.^[ [47]^8 ^] Many HAF PSs
   are based on organic compounds and can degrade in the environment,
   reducing environmental pollution.^[ [48]^8 , [49]^9 ^] The benefits of
   using HAF PSs include their low toxicity in the absence of light,
   prolonged triplet lifetimes, strong photostability, and relatively low
   cost.^[ [50]^10 ^] Thus, there is a pressing need to develop new
   anti‐tumor immunotherapy systems utilizing HAF PSs.

   Although phototherapy can activate anti‐tumor immunity, tumor cell
   metabolic reprogramming to shape a specific tumor microenvironment can
   inhibit the PIT effect.^[ [51]^11 ^] Lactate (LA), a primary byproduct
   of tumor glycolysis, is produced by tumor cells at rates up to 40 times
   higher than normal cells, with concentrations reaching 40 µmol g^−1
   within the tumor environment.^[ [52]^12 ^] Tumor‐infiltrating
   lymphocytes (TILs) are submerged in this lactate‐rich fluid, where LA
   transport into the cytoplasm of TILs can inhibit T cell function and
   activation.^[ [53]^13 ^] Additionally, lactate undermines immunotherapy
   effectiveness by aiding tumor DNA repair and increasing the presence of
   tumor‐associated macrophages (TAMs), myeloid‐derived suppressor cells
   (MDSCs), and regulatory T cells (Tregs).^[ [54]^14 ^] Recent studies
   have shown that lithium carbonate (LC, an emotion stabilizer) that
   reduces lactate levels can reverse lactate‐mediated CD8^+ T cell immune
   suppression.^[ [55]^12 ^] LC inhibits lysosomal acidification and
   facilitates the movement of the monocarboxylate transporter 1 (MCT1) to
   the mitochondrial membrane, enabling lactate to be used as an energy
   source for CD8^+ T cells. This innovative approach converts a waste
   product into a valuable resource, enhancing CD8^+ T cell activity and
   overcoming lactate‐induced immune suppression. Consequently,
   integrating PIT with LC to manipulate tumor lactate metabolism could
   enhance the immune environment within tumors while repurposing lactate
   as an energy source for T cells, offering a promising avenue for
   effective tumor immunotherapy. This novel strategy for modulating
   lactate metabolism in cancer treatment is still underexplored in
   existing literature.

   Here, we have prepared a novel near‐infrared (NIR) activated HAF named
   Cy‐BF through molecular engineering. This photosensitizer was
   encapsulated with phospholipids (DSPE‐PEG2000) and platelet exosome
   vesicles (PEV) to create aggregated nanoparticles, referred to as CHL.
   Following administration, CHL was activated by 760 nm NIR laser
   irradiation for phototherapy. CHL possesses several advantageous
   features:1) Due to the absence of heavy atoms, CHL exhibits high
   phototoxicity with minimal dark toxicity; 2) CHL demonstrates
   aggregation‐induced broadening of absorption spectra, facilitating NIR
   PDT; 3) CHL combines Type I PDT capabilities to overcome tumor hypoxia;
   4) Its aggregation‐induced fluorescence quenching enhances the
   conversion of light energy into heat and ROS and 5) platelet exosome
   vesicles (PEV) coating provide effective tumor targeting ability.
   Compared to other biomimetic carriers such as red blood cell exosomes
   (REV) and tumor‐derived exosomes (TEV), PEV has better advantages.^[
   [56]^15 ^] Since PEV originates from blood, their biological safety is
   better than TEV. In addition, the PEV surface contains P‐selectin
   protein targeting tumors, which has excellent tumor‐targeting
   ability.^[ [57]^5c ^] Furthermore, the platelet content in the blood is
   high and easy to obtain, making it one of the best candidates as a drug
   carrier. NIR laser is less absorbed by biological tissues within the
   wavelength range of 700–1700 nm, allowing for deeper penetration of
   tissues.^[ [58]^16 ^] CHL solves the problems such as laser penetration
   depth and oxygen dependence of traditional photosensitizers. During
   treatment with CHL, mice also received oral lithium carbonate (LC)
   therapy, which transformed lactate, a byproduct of tumor metabolism,
   into a valuable energy source for CD8^+ T cells, thus enhancing the
   efficacy of CHL‐mediated photoimmunotherapy. This approach not only
   caused mitochondrial damage and immunogenic cell death in tumor cells
   but also altered the tumor microenvironment by reducing Tregs and
   increasing CD8^+ T cells. Furthermore, this combination therapy
   effectively suppressed distant tumor growth by targeting primary tumors
   and maintained its efficacy even when exogenous lactate was
   administered to tumor‐bearing mice. This combination therapy reversed
   lactate metabolism in both tumor cells and T cells for the first time,
   providing an innovative approach for future clinical photoimmunotherapy
   (Scheme [59]1 ).

Scheme 1.

   Scheme 1
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   Schematic illustration of Biomimetic heavy‐atom‐free photosensitizers
   collaborate with lithium carbonate reverses T lymphocytes and cancer
   cells metabolism to enhance near‐infrared activated photoimmunotherapy.
   A) The preparation process of CHL and combination therapy
   administration mode. B) Photophysical mechanism and advantages of the
   CHL. C) The in vivo mechanism of CHL combined with LC in anti‐tumor
   treatment. In untreated tumor tissue, the production of LA by tumor
   cells through glycolysis inhibits the mitochondrial function of CD8^+ T
   cells, thereby suppressing the production of granzyme and cytokines in
   T cells. After combined treatment with CHL and LC, on the one hand,
   CHL‐mediated PDT and PTT induced tumor cell death and mitochondrial
   damage, reducing their LA production. On the other hand, LC reversed
   the immunosuppressive effect of LA on CD8^+ T cells and instead
   enhanced the killing ability of CD8^+T cells. This dual strategy
   ultimately leads to a systemic anti‐tumor immune response.

2. Results and Discussion

2.1. Preparation and Characterization of Cy‐BF and CHL

   First, we designed a novel small molecule‐based cyanine boron fluoride
   derivative photosensitizer named Cy‐BF. The synthesis route, molecular
   structure, High‐Resolution Mass Spectrometry (HRMS), and ^1H Nuclear
   Magnetic Resonance (NMR) spectrum of Cy‐BF were displayed in Scheme
   [61]S1 (Supporting Information), Figure [62]1A and Figures [63]S1 and
   [64]S2 (Supporting Information) respectively, confirming the successful
   synthesis of these compounds. The standard curve of 660 nm Cy‐BF is
   shown in Figure [65]S3 (Supporting Information). To understand the
   detailed mechanism of triplet state formation, we performed theoretical
   calculations on the Cy‐BF structures using time‐dependent density
   functional theory (TD‐DFT). As illustrated in Figure [66]1B, the
   highest occupied molecular orbital (HOMO) of Cy‐BF spans the entire
   molecular framework, while the lowest unoccupied molecular orbital
   (LUMO) is predominantly localized in the central acceptor region. The
   triplet state (T1−S0) energy gaps for Cy‐BF were calculated to be
   1.599 eV, which were higher than those required to excite ^3O[2] for
   ^1O[2] (0.977 eV) via energy transfer.^[ [67]^17 ^] Similarly, the
   ΔE[S1−T1] value was 0.7905 eV, which is too large to facilitate
   intersystem crossing (ISC). However, the ΔE[S1−T2] was significantly
   reduced to 0.4079 eV, suggesting a viable ISC pathway from the S1 to
   the T2 state (Figure [68]1C), thereby enhancing ROS generation. The
   energy gap (Δ[EST]) between the first excited singlet state (S1) and
   the first excited triplet state (T1) of dimeric Cy‐BF (Figure [69]S4,
   0.6222 eV) was smaller than that of the monomer (0.7905 eV), and the Tn
   (n > 1) energy levels of the dimer are significantly decreased. With
   the increase of polymerization degree, the energy level difference
   between the excited states is gradually approaching, which provides
   more feasible pathways for inter‐system transition from singlet to
   triplet states, thereby facilitating inter‐system transition and
   improving the efficiency of singlet oxygen production.^[ [70]^18 ^]
   Subsequently, Cy‐BF (referred to as CL) was encapsulated with
   DSPE‐PEG2000, and the platelet exosome vesicles (PEV) were physically
   extruded onto the surface of CL to form CHL. Fluorescence spectroscopy
   showed that Cy‐BF in chloroform displayed strong fluorescence under 660
   or 760 nm light, but this fluorescence was significantly quenched in
   the CHL format (Figure [71]1D; Figure [72]S5, Supporting Information).
   The quenching of fluorescence indicates that upon aggregation, Cy‐BF
   efficiently converts light energy into heat and demonstrates robust ISC
   capabilities, enhancing the effectiveness of PDT. As shown in
   Figure [73]1E, at the same concentration, while the absorption peak of
   CHL at the same concentration was lower than that of Cy‐BF, there was a
   noticeable broadening of the absorption spectrum and a double peak
   occurrence. This could be likely due to the M‐aggregation of Cy‐BF in
   CHL.^[ [74]^19 ^] The absence of a red or blue shift in the CHL
   spectrum indicates that it does not exhibit either H or
   J‐aggregation.^[ [75]^20 ^] This also significantly increased the
   near‐infrared (NIR) absorption capacity of Cy‐BF, facilitating its
   activation by NIR laser for both PDT. The molar extinction coefficients
   of Cy‐BF and CHL at 760 nm were 1.25 × 10^3 L mol^−1 cm^−1 and
   1.40 × 10^4 L mol^−1 cm^−1, respectively, indicating that CHL has
   improved 760 nm light‐harvesting ability. TEM images of CL, PEV, and
   CHL have been shown in Figures [76]1F,G and [77]S6 (Supporting
   Information). There was an obvious membrane structure on the surface of
   CHL, with a particle size ≈10 nm higher than CL, and a zeta potential
   like PEV (Figure [78]1H), confirming that PEV has effectively enveloped
   the surface of CL. The drug loading efficiency of Cy‐BF in CHL is to be
   37.4 ± 5.2% through absorption spectroscopy and standard curves. CHL
   demonstrated good dimensional stability (Figure [79]1I) and
   photostability (Figure [80]1J), and the surface contains platelet
   membrane intrinsic proteins CD41 and P‐selectin (Figure [81]1K).
   P‐selectin plays a critical role in targeting the CD44 receptor on
   tumor surfaces.^[ [82]^21 ^] Compared to CL, CHL exhibited better
   tumor‐targeting ability (Figure [83]S7, Supporting Information). In
   summary, we have successfully synthesized CHL, which has excellent NIR
   absorption, tumor targeting, and photostability, laying the foundation
   for subsequent biological experiments.

Figure 1.

   Figure 1
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   A) The chemical structural formula of Cy‐BF. B) HOMO‐LUMO distribution
   determined by DFT calculations for Cy‐BF. C) Energy levels for
   simplified monomeric and dimeric Cy‐BF at the optimized molecular
   geometries. D) Photoluminescence (PL) spectroscopy and E) absorption
   spectra of Cy‐BF in CHCl[3] solution compared to CHL in PBS solution,
   using an excitation wavelength of 760 nm. F) TEM image of CL and G)
   CHL, including a magnified view of a particle. H) Particle diameter and
   Zeta potential measurements for PEV, CL, and CHL. Data are shown as the
   mean ± SD (n = 3). I) Stability of CHL in various solutions. J)
   Absorption spectra of CHL before and after exposure to 760 nm laser
   irradiation at 0.5W/cm^2 for varying durations. K) Western blot (WB)
   analysis of key proteins in different formulations (I: Actin, II:
   P‐selectin, III: CD41) at a Cy‐BF concentration of 10 µg mL^−1.

2.2. Photothermal Ability, ROS Generation, and In Vitro Anticancer Ability of
CHL

   Next, we measured the photothermal performance of CHL, as shown in
   Figure [85]2A–C. As the concentration increases, CHL heats up faster
   under near‐infrared light. CHL has stable photothermal cycling ability
   and high photothermal conversion efficiency. Electron spin resonance
   (ESR) spectroscopy was used to detect ROS production in Cy‐BF and CHL.
   As shown in Figure [86]2D–F, both CHL and Cy‐BF can produce ^1O[2], OH,
   and •O[2] ^−, with CHL producing more ROS than Cy‐BF at equivalent
   concentrations. We detected the production of ROS by CHL at different
   laser exposure times using different ROS indicators (as shown in
   Figure [87]2G–I; Figure [88]S8, Supporting Information). These
   experimental results suggested that CHL can perform both type I and
   type II PDT simultaneously under 760 nm laser irradiation. According to
   previous studies, photosensitizers facilitating type I PDT are
   particularly effective in treating hypoxic tumor environments,
   highlighting the potential of CHL as a promising HAF photosensitizer.
   In addition, CHL can perform NIR‐activated PDT, which is crucial for
   advanced photosensitizers. Due to the issue of laser penetration
   ability, traditional visible light excited photosensitizers cannot
   penetrate deep tissues, which is not conducive to clinical
   translation.^[ [89]^22 ^] The development of NIR‐excited
   photosensitizers has always been a hot topic in the field of
   phototherapy research.^[ [90]^16 , [91]^23 ^]

Figure 2.

   Figure 2
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   A) Heating profile of CHL containing different concentrations of Cy‐BF,
   PBS, and chloroform solution containing 200 ug mL^−1 Cy‐BF under 760 nm
   laser irradiation (0.5W/cm^2) for 3 min. Data are shown as the mean ±
   SD (n = 3). B) Photothermal cycling curves of different formulations.
   C) Calculation of the time constant for heat transfer using a linear
   regression of the CHL cooling profile. D) Production of ^1O[2], E) •OH
   and F) •O[2] ^− by the CHL or CHL+NIR (0.5 W cm^−2, 3 min) was
   determined by ESR. G) Fluorescence spectra of SOSG, H) HPF, and I) DCF
   after CHL plus 760 nm laser irradiation (0.5 W cm^−2) for different
   times. J) Fluorescence images and flow cytometry analysis of
   intracellular ROS treated with different components (NIR: 760 nm laser,
   0.5 W cm^−2, 10 min). Scale bars: 20 µm.

   We next assessed the cytotoxic impact of CHL on 4T1 cells. As shown in
   Figures [93]2J,A and [94]S9 (Supporting Information), CHL+NIR treatment
   led to an increase in ROS and altered mitochondrial membrane potential
   in 4T1 cells, compared to PBS+NIR, CHL, and CL+NIR groups. TEM results
   showed a decrease in mitochondrial cristae in tumor cells in the
   CHL+NIR group (Figure [95]S10, Supporting Information), suggesting
   mitochondrial damage induced by CHL+NIR. CHL+NIR treatment also led to
   more cell apoptosis and immunogenic cell death (ICD, Figure [96]3B–D:
   Figures [97]S11–[98]S13, Supporting Information). Immunofluorescence
   staining for ICD markers (HMGB1 and calreticulin, CRT) showed that
   cells treated with CHL and exposed to 760 nm laser radiation displayed
   markedly reduced green fluorescence in the nucleus (indicative of HMGB1
   release) and increased green fluorescence on the cell membrane
   (indicative of elevated CRT expression). The release of HMGB1 and ATP
   from cells in the CHL+NIR group also significantly increased. These
   results suggest that the oxidative stress triggered by CHL+NIR can
   cause ICD of 4T1 cells. CHL+NIR treatment also resulted in decreased
   glutathione (GSH) and lactate (LA) levels in 4T1 cells
   (Figure [99]3E,F). The CCK‐8 assay corroborated the cytotoxic effects
   of CHL+NIR on 4T1 cells (Figure [100]3G). The results of transcriptomic
   sequencing illustrated that differential genes were significantly
   enriched in signaling pathways related to ICD, ROS, and immune
   regulation, further proving the potential of CHL in immunosensitization
   therapy (Figure [101]4A–E). Relative to the PBS+NIR group, CHL+NIR
   treatment upregulated 1059 genes and 2263 transcripts while
   downregulating 2085 genes and 4053 transcripts (Figure [102]S14,
   Supporting Information). For instance, the transcription levels of
   ATF3, HSPA1A, DDIT3, and HSPA8, related to ROS‐induced ER stress, were
   upregulated, likely due to ICD.^[ [103]^24 ^] Elevated transcription
   levels of GADD45G, NOTCH2, BAG3, and DUSP1 linked to apoptosis,^[
   [104]^25 ^] indicated profound apoptosis triggered by CHL+NIR. Further
   KEGG pathway enrichment analysis indicated significant impacts on genes
   associated with ICD and apoptosis due to CHL+NIR treatment. As shown in
   Figure [105]S15 (Supporting Information), under hypoxic conditions, the
   anti‐tumor ability of CL is not affected by oxygen. CL has better
   anti‐tumor effects compared to ICG‐DSPE. This is because Cy‐BF can
   generate hydroxyl radicals through type‐I PDT, which does not consume
   oxygen. Therefore, CL is less oxygen‐dependent compared to traditional
   photosensitizers such as ICG. And there is a hypoxic microenvironment
   in tumor tissue,^[ [106]^26 ^] so Cy‐BF has greater advantages. Under
   no‐laser conditions, CHL showed minimal cytotoxic effects on RAW 264.7
   mouse macrophages and Cy‐BF showed minimal cytotoxic effects on 4T1
   cells (Figures [107]S16 and [108]S17, Supporting Information),
   indicating its low dark toxicity and favorable biocompatibility.

Figure 3.

   Figure 3
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   A) JC‐1 flow cytometry analysis and B) apoptosis detection of 4T1 cells
   treated with different components (NIR: 760 nm laser, 0.5 W cm^−2,
   10 min). C) Expression of HMGB1 and CRT (scale bars: 10 µm) and D)
   HMGB1 release from 4T1 cells following treatment with different
   formulations (NIR: 760 nm laser, 0.5 W cm^−2, 10 min). E) Intracellular
   GSH and F) lactate levels after different treatments. Cy‐BF
   concentration: 100 µg mL^−1. G) The relative cellular viability of 4T1
   cells after various treatments with different Cy‐BF concentrations.
   NIR: 760 nm laser, 0.5 W cm^−2, 10 min. Data are shown as the mean ± SD
   (n = 3). Statistical significance was calculated via one‐way ANOVA with
   Tukey's test: ^*** p < 0.001.

Figure 4.

   Figure 4
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   A) Heatmap illustrating differentially expressed genes after treatment.
   B) The Venn diagram revealed the number of genes transcribed in the
   indicated treatment group. C) Volcano plots illustrating differentially
   expressed genes. D) Results of KEGG pathway enrichment analysis. E)
   Heatmap of gene expressions related to apoptosis, immunity, and
   oxidative stress in 4T1 cells treated with PBS + NIR and CHL + NIR.NIR:
   760 nm laser, 0.5 W cm^−2, 10 min.

   Given that the release of damage‐associated molecular patterns (DAMPs)
   can stimulate dendritic cells (DCs), we assessed DC maturation by
   co‐incubating bone marrow‐derived dendritic cells (BMDCs) with 4T1
   cells treated using different groups in a Transwell system for 24 h
   (Figure [111]5A). The expression of costimulatory molecules CD80 and
   CD86 served as indicators of DC maturity (Figure [112]5B,C). Following
   the release of DAMPs, cancer cells treated with CHL+NIR notably
   enhanced BMDC maturation to 65.2%, a 1.97 fold increase compared to
   those treated with CL+NIR. This maturation was accompanied by a
   significant rise in proinflammatory cytokines such as IL‐12p70, TNF‐α,
   and IL‐6 compared to the control group (Figure [113]5D–F).
   Subsequently, we investigated the effects of LC combined with CHL on
   immune cells in vitro. As shown in Figure [114]5G, previous studies
   have shown that LC‐mediated transfer of MCT1 to the mitochondrial
   membrane allows lactate to enter the mitochondria as an energy source,
   thereby providing energy for CD8^+ T cells.^[ [115]^12 ^] Using
   fluorescence microscopy and western blot analyses (Figure [116]5H,I;
   Figure [117]S18, Supporting Information), we observed that after LC
   treatment, MCT1 protein in CD8^+ T cells predominantly localized to the
   mitochondria, unaffected by CHL or LA treatments. As shown in
   Figure [118]5J, we established an assay based on BMDCs and splenic T
   lymphocytes in vitro. Flow cytometry was used to demonstrate that the
   CHL+NIR+LC group or CHL+NIR+LC+LA group can effectively activate T
   lymphocytes after activating BMDCs. This resulted in a significant
   increase in IL‐2 release and the expression of IFN‐γ and GZMB compared
   to the LA or PBS groups (Figure [119]5K,M–P). When these T lymphocytes
   were co‐cultured with 4T1 tumor cells for 24 h, lactate dehydrogenase
   (LDH) levels in the supernatant confirmed effective tumor cell killing
   by the CHL+NIR+LC and CHL+NIR+LC+LA treatment (Figure [120]5L). It is
   worth noting that LA treatment can significantly reduce T cell
   activation and anti‐tumor factor production, but the introduction of LC
   counteracted this effect, demonstrating that this combination therapy
   successfully overcomes lactate‐induced suppression of T cells.

Figure 5.

   Figure 5
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   A) Schematic illustration of the Transwell system of the Transwell
   system used to induce maturation of BMDCs in vitro. B) Flow cytometry
   analysis and C) quantitative analysis of mature BMDCs after different
   treatments. The Cy‐BF concentration was 20 µg mL^−1. Data are shown as
   the mean ± SD (n = 3). D) The levels of TNF‐α, E) IL‐6, and F) IL‐12p70
   secreted by matured BMDCs. G) A schematic showing how LC facilitates
   the translocation of MCT1 from the T cell membrane to mitochondria. H)
   Immunofluorescence staining of MCT1 and COX IV in CD8^+ T cells treated
   with indicated formulations. Blue: Nuclei, Green: COX IV and Red: MCT1.
   I) Western blot of MCT1 in the mitochondria of CD8^+ T cells treated
   with indicated formulations. J) A schematic representing specific
   immune activation experimental setups in vitro. K) Detection of IL‐2 in
   supernatant from treated CD8 ^+ T cells by ELISA after LA or/and LC for
   24h. L) Measurement of LDH levels in the supernatant after
   co‐incubation of the splenic T lymphocytes with 4T1 tumor cells for
   24h. M) Representative plots and N) quantification of IFN‐γ in CD8^+ T
   cells after different treatments analyzed by the flow cytometry. O)
   Representative plots and P) quantification of granzyme B (GZMB) in
   CD8^+ T cells after different treatments analyzed by flow cytometry. LA
   and LC were used at a concentration of 10 mM, and Cy‐BF at 100 µg mL^−1
   Data are shown as the mean ± SD (n = 3). Statistical significance was
   calculated via one‐way ANOVA with Tukey's test: ns: Non‐Significant,
   ^*** p < 0.001.

2.3. In Vivo Anti‐Tumor Ability of CHL

   Building on the promising in vitro findings, we further explored the
   therapeutic potential of CHL‐mediated NIR photoimmunotherapy in
   combination with LC in an in vivo setting. BALB/c mice were
   subcutaneously injected with 5 × 10^6 4T1 cells into the right flank to
   form tumors. All animal procedures were performed in accordance with
   the guidelines for Care and Use of Laboratory Animals of the Ministry
   of Health in the People's Republic of PR China and approved by the
   Animal Ethics Committee of Guangxi Medical University (Approval number:
   2023‐KY (0931)). As shown in Figure [122]6A, CHL demonstrated superior
   tumor‐targeting capabilities compared to CL. Compared to the CL + NIR
   group, the CHL+NIR group reached a higher temperature at the tumor site
   after 10 min of illumination (Figure [123]6B). The flowchart of the CHL
   combined with the LC anti‐tumor experiment is shown in Figure [124]6C.
   LC was administered orally to mice every alternate day over a span of
   10 days. CHL was intravenously injected through the tail vein on day 0
   and phototherapy was performed 12 h later. The survival curve, tumor
   volume growth, and tumor weight at day 18 have been shown in
   Figure [125]6D–F. The data clearly indicates that the combination of
   CHL‐mediated phototherapy and LC treatment yielded the most significant
   anti‐tumor effects and enhanced survival rates. Furthermore, the
   lactate content in the tumor was notably lower in this group compared
   to control groups (Figure [126]6G), indicating effective inhibition of
   lactate production in tumor cells by this combination therapy. In vivo
   ROS staining indicated that the ROS levels induced by CHL+NIR or
   CHL+NIR+LC were higher than those induced by CL+NIR+LC, highlighting
   greater photodynamic PDT efficiency with CHL (Figure [127]S19,
   Supporting Information). Histological examination of tumor tissues
   through hematoxylin‐eosin (HE) staining, immunofluorescence staining
   for CRT, HMGB1, and terminal‐deoxynucleotidyl transferase‐mediated nick
   end labeling (TUNEL) revealed extensive necrosis, ICD, and apoptosis in
   tumors treated with CHL+NIR+LC (Figure [128]6H; Figure [129]S20,
   Supporting Information). Additionally, this treatment group exhibited a
   significant reduction in cell proliferation, as evidenced by lower
   Ki‐67 levels, indicating a substantial inhibition of tumor growth. We
   isolated CD8^+ T cells from tumor tissue for western blot (WB) and flow
   cytometry analysis. As shown in Figure [130]6I,J and Figures [131]S21
   and [132]S22 (Supporting Information), the expression of MCT1 in CD8^+
   T cell mitochondria significantly increased after LC treatment,
   aligning with our in vitro, thereby indicating LC‐mediated T cell
   membrane MCT1 transfer. The maturation of DCs in tumor‐draining lymph
   nodes markedly increased in the CHL+NIR+LC group, alongside a rise in
   the number of tumor‐infiltrating CD8^+ T cells and the expression of
   IFN‐γ and GZMB in these cells (Figure [133] 7A–F), demonstrating
   effective activation of T cell‐mediated immunity. In addition, the
   number of Tregs in tumor tissue significantly decreased, thanks to the
   decrease in lactate content in tumor tissue. Moreover, the serum levels
   of key cytokines such as tumor necrosis factor‐alpha (TNF‐α),
   interleukin‐6 (IL‐6), and IFN‐γ were significantly elevated in the
   CHL+NIR+LC group, as measured by enzyme‐linked immunosorbent assay
   (ELISA) (Figure [134]7G), indicating a robust systemic anti‐tumor
   immune response. In addition, we compared the anti‐tumor ability of
   CL+NIR and CHL+NIR (as shown in Figure [135]S23, Supporting
   Information) and found that CHL+NIR has better anti‐tumor ability than
   CL+NIR, possibly due to CHL's better tumor targeting ability. Finally,
   after treatment, no significant liver or kidney toxicity or weight loss
   was observed, confirming the good biocompatibility of the system
   (Figure [136]S24, Supporting Information).

Figure 6.

   Figure 6
   [137]Open in a new tab

   A) Ex vivo fluorescence imaging and corresponding intensity data of
   tumors and organs from 4T1 tumor‐bearing mice at various post‐injection
   intervals of CL or CHL. B) Infrared thermography of tumor tissue after
   different treatments. C) A schematic Diagram outlining the timeline for
   tumor inoculation, nanodrug administration, laser treatment, and
   monitoring of tumor growth. D) Survival curves after treatment. E)
   Tumor volumes were measured every 3 days across all groups. F) Tumor
   weights were recorded for each treatment at the end of the study. G)
   Relative lactate content in 4T1 tumor tissues after different
   treatments. H) H&E, TUNEL, CRT, and Ki‐67 staining of tumor sections
   after the indicated treatments. Scale bars: 20 µm. I) Schematic
   illustration of western blotting analysis of protein expression in
   CD8^+ T cells. J) Western blotting analysis of VDAC1 and MCT1
   expression in CD8^+ T cell mitochondrion. Cy‐BF dose: 10 mg kg^−1. Data
   are shown as the mean ± SD (n = 5). Statistical significance was
   calculated via one‐way ANOVA with Tukey's test: ^*** p < 0.001.

Figure 7.

   Figure 7
   [138]Open in a new tab

   A) Flow cytometry data showing the effects of treatments on DCs
   maturation in lymph nodes, infiltration of CD3^+ CD8^+ T lymphocytes in
   4T1 tumor tissues, and levels of IFN‐γ^+ and GZMB^+ cells among CD8^+ T
   cells, as well as CD25^+ Foxp3^+ Tregs in tumor tumor tissues. B)
   Quantitative analysis of DCs maturation in lymph nodes induced by
   treatments, C) CD3^+ CD8^+ T lymphocytes infiltration in the 4T1 tumor
   tissues, D) IFN‐γ^+ and E) GZMB^+ cells among CD8^+ T cells, and F)
   CD4^+ Foxp3^+ Tregs in tumor tissues. G) Secretion of pro‐inflammatory
   cytokines (TNF‐α, IFN‐γ, and IL‐6) in sera after exposure to different
   treatments. Data are shown as the mean ± SD (n = 5). Statistical
   significance was calculated via one‐way ANOVA with Tukey's test: ^** p
   < 0.01, ^*** p < 0.001.

   An in vivo study on anti‐bilateral tumor effects was conducted through
   the systemic administration of various treatment formulations (Figure
   [139]8A). The growth of primary and distant tumors was monitored every
   three days over a period of 15 days. Treatment regimens including free
   CL+NIR+LC, CHL+NIR, and CL+NIR+LC showed a relative delay in tumor
   growth compared to the PBS control group (Figure [140]8B–F).
   Remarkably, the CHL+NIR+LC treatment group significantly hindered the
   growth of both primary and distant tumors, achieving inhibition rates
   of 81.7% and 90.9%, respectively. This group also showed superior
   performance in reducing the tumor weights of both primary and distant
   tumors. In addition, no obvious weight loss of mice was observed after
   various treatments (Figure [141]8G). H&E staining revealed extensive
   nuclear disintegration and significant necrotic areas in distant tumors
   from the CHL+NIR, CL+NIR+LC, and CHL+NIR+LC groups post‐phototherapy,
   whereas no significant cell death was observed in tumors from other
   treatment groups (Figure [142]8H). After treatment with CHL+NIR+LC,
   there was a marked increase in the population of CD8^+ T cells and the
   subsets expressing high levels of IFN‐γ in the distant tumor tissues
   (Figure [143]8I; Figure [144]S25, Supporting Information). ELISA
   analysis revealed that combinatory therapy by CHL+NIR + LC dramatically
   induced sera IFN‐γ and TNF‐α (Figure [145]8J,K). Collectively, these
   results confirm that CHL‐mediated NIR PIT combined with LC effectively
   delays primary tumor growth and induces abscopal effects, thereby
   inhibiting the growth of distant tumors.

Figure 8.

   Figure 8
   [146]Open in a new tab

   A) Schematic illustration of the studies of 4T1 bilateral tumor
   therapy. B) Survival curves after different treatments. C) Evolution of
   the primary tumor volume and D) tumor weight after various treatments.
   E) Development of distant tumor volume and F) tumor weight following
   various treatments. G) Changes in body weight during the treatment
   period. H) H&E and CD8 staining analyses of distant tumor tissues after
   different treatments. I) Quantification of treatment‐induced IFN‐γ^+
   cells among CD8^+ T cells. J) Levels of pro‐inflammatory cytokines
   (TNF‐α and K) IFN‐γ) in serum following different treatments. L)
   Schematic illustration of the studies of 4T1 tumor therapy. M) Lactate
   content in tumor tissue after different treatments. N) Survival curves
   after different treatments. O) Changes in tumor volume and P) tumor
   weight after different treatments. Q) H&E and CD8 staining analyses of
   tumor tissues treated with various treatments. R) Flow cytometry
   analysis and S) Quantification of CD3^+ CD8^+ T lymphocytes
   infiltration in the 4T1 tumor tissues. T) Quantification of
   treatment‐induced IFN‐γ^+ and U) GZMB^+ cells among CD8^+ T cells. Data
   are shown as the mean ± SD (n = 5). Statistical significance was
   calculated via one‐way ANOVA with Tukey's test: ^** p < 0.01, ^*** p
   < 0.001.

   To further validate the effect of LA on this combination therapy, we
   administered exogenous LA via intraperitoneal injection to mice, as
   shown in Figure [147]8L. After intraperitoneal injection of LA, the
   amount of LA in tumor tissue increased (Figure [148]8M). The
   introduction of LA significantly diminished the therapeutic efficacy of
   the CHL+NIR group yet had minimal impact on the efficacy of the
   CHL+NIR+LC group (Figure [149]8N–P). The proportion of CD8^+ T cells,
   GZMB^+ CD8^+, and IFN‐γ^+ CD8^+ T cells increased in the tumor tissues
   of the CHL+NIR+LC+LA group (Figure [150]8Q–U; Figure [151]S26,
   Supporting Information). There was no notable change in the CHL+NIR+LC
   group compared to the first model8. Overall, the addition of LC
   mitigated the suppressive effects of tumor tissue lactate on T cells
   and enhanced the efficacy of CHL‐mediated PIT.

3. Conclusion

   In conclusion, we have developed a novel NIR‐activated HAF
   photosensitizer named Cy‐BF, which was encapsulated with phospholipids
   and PEV to create CHL. CHL exhibits good tumor‐targeting capabilities
   and minimal toxicity in the absence of light. CHL plus NIR laser
   treatment leads to mitochondrial damage, lactate decrease, and
   immunogenic cell death in tumor cells. Additionally, CHL reduced
   regulatory T cells (Tregs) and increased CD8^+ T cells within the tumor
   microenvironment. Then the integration of LC was able to transform
   lactate, allowing CD8^+ T cells to utilize LA as an energy source, thus
   significantly boosting the effectiveness of CHL‐mediated
   photoimmunotherapy. This combination approach markedly inhibits the
   growth of distant tumors by treating primary tumors. Interestingly,
   while the administration of exogenous lactate to tumor‐bearing mice
   impaired phototherapy, it did not modulate the efficacy of the combined
   treatment. This innovative combination therapy, which can alter lactate
   metabolism in both tumor cells and T cells for the first time, holds
   tremendous potential for enhancing future clinical PIT. In the future,
   this combination therapy may increase the sensitivity of immune
   checkpoint inhibitor therapy and radiation immunotherapy. Furthermore,
   it may resolve the issues of hypoxia and T‐cell suppression that occur
   during radiotherapy.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [152]ADVS-12-2416143-s001.docx^ (8.8MB, docx)

Acknowledgements