A Tumor Environment‐Activated Photosensitized Biomimetic Nanoplatform for Precise Photodynamic Immunotherapy of Colon Cancer

Abstract Aggressive nature of colon cancer and current imprecise therapeutic scenarios simulate the development of precise and effective treatment strategies. To achieve this, a tumor environment‐activated photosensitized biomimetic nanoplatform (PEG2000‐SiNcTI‐Ph/CpG‐ZIF‐8@CM) is fabricated by encapsulating metal‐organic framework loaded with developed photosensitizer PEG2000‐SiNcTI‐Ph and immunoadjuvant CpG oligodeoxynucleotide within fusion cell membrane expressing programmed death protein 1 (PD‐1) and cluster of differentiation 47 (CD47). By stumbling across, systematic evaluation, and deciphering with quantum chemical calculations, a unique attribute of tumor environment (low pH plus high concentrations of adenosine 5′‐triphosphate (ATP))‐activated photodynamic effect sensitized by long‐wavelength photons is validated for PEG2000‐SiNcTI‐Ph/CpG‐ZIF‐8@CM, advancing the precision of cancer therapy. Moreover, PEG2000‐SiNcTI‐Ph/CpG‐ZIF‐8@CM evades immune surveillance to target CT26 colon tumors in mice mediated by CD47/signal regulatory proteins α (SIRPα) interaction and PD‐1/programmed death ligand 1 (PD‐L1) interaction, respectively. Tumor environment‐activated photodynamic therapy realized by PEG2000‐SiNcTI‐Ph/CpG‐ZIF‐8@CM induces immunogenic cell death (ICD) to elicit anti‐tumor immune response, which is empowered by enhanced dendritic cells (DC) uptake of CpG and PD‐L1 blockade contributed by the nanoplatform. The photodynamic immunotherapy efficiently combats primary and distant CT26 tumors, and additionally generates immune memory to inhibit tumor recurrence and metastasis. The nanoplatform developed here provides insights for the development of precise cancer therapeutic strategies.


Introduction
Colon cancer is a malignant tumor with high incidence and mortality worldwide, which seriously affects human health. [1]ifferent therapeutic modalities, such as surgery, radiotherapy, chemotherapy, etc., have been exploited to elicit a certain antitumor effect.7] Photodynamic therapy (PDT) has been proved to be a non-invasive therapeutic modality with high efficacy for various tumors in clinical applications. [8]During the PDT process, photosensitizers (PSs) generate cytotoxic reactive oxygen species (ROS) under laser irradiation to directly induce tumor cell apoptosis or necrosis. [9]Regretfully, most of PDT developed so far realized the tumor-specific treatment only by elaborately manipulating the direction of laser irradiation toward the tumor, proposing a high demand for the focusing of laser irradiation and the necessity to pinpoint the exact location of tumors in advance, otherwise damages to normal tissues were inevitable due to the uncontrollable diffusion of PSs, severely hindering the clinical applications of PDT.Therefore, tumor environment-activated PDT, of which the photodynamic therapeutic process can only be initiated under tumorspecific conditions, appears to be a more reliable and robust scenario to advance the accuracy of PDT, matching the concept of precise cancer therapy.As far as we know, now there are some nanoplatforms reported could achieve tumor environmentactivated PDT. [10,11]However, a few characteristics not suitable for robust in vivo applications in these work essentially need to be improved.For one thing, the utilization of PSs sensitive to shortwavelength visible photons (such as, chlorin e6, 635 nm; methylene blue, 635 nm) [12,13] would result in the limited tissue penetration depth due to strong photon scattering in tissues. [14]For another thing, the implementation of tumor environment-activated PDT in some nanoplatform required elaborate control of the molar ratio and distance between PSs and the quencher, greatly limiting the robust application. [15]In this regard, it is imperative to develop an easily-handling tumor environment-activated PDT platform with long-wavelength photon sensitization (e.g., 750-850 nm, a window more transparent to tissues [16] ).
Besides the direct damage, PDT-caused killing of tumor cells can further release tumor-associated antigens (TAAs), [17] inducing immunogenic cell death (ICD) to trigger anti-tumor immune response, which is expected to boost the cancer therapy.Recently, significant efforts have been devoted to introducing immune adjuvants to amplify anti-tumor immune response. [18,19]CpG oligodeoxynucleotide is a synthetic short DNA fragment that can trigger innate immunity and induce cytokine secretion by activating dendritic cells (DCs). [20,21]Nevertheless, the inferior pharmacokinetics of CpG (e.g., non-specific diffusion and low cell uptake due to the anionic property) plus the easy degradation and clearance within blood attenuated the function of CpG as an immune adjuvant to the greatest extent.Moreover, anti-tumor immune response is seriously weakened by the immunosuppressive mechanism within the tumor. [22]For instance, programmed death-ligand 1 (PD-L1), an immune checkpoint expressed on tumor cells, especially for colon tumor cells, [23] can bind to programmed death protein 1 (PD-1) on T lymphocytes and then shut down the immune recognition.Though approved by Food and Drug Administration (FDA) for the blockade of PD-L1 to advance immune response, the clinical application of the anti-PD-L1 antibody is impeded by the high cost and inferior pharmacokinetics (e.g., clearance by the innate immune system). [24]Therefore, a platform, which can improve in vivo pharmacokinetics of economical alternatives for immune checkpoint PD-L1 blockade and CpG, appears to be the best scenario to empower photodynamic immunotherapy of colon cancer.
Zeolitic imidazolate framework-8 (ZIF-8), a kind of metalorganic framework, has been gradually developed into the most representative delivery nanocarrier owing to its unique characteristics of superior biocompatibility, [21] selective biodegradability toward tumor environment (i.e., high concentrations of adenosine 5′-triphosphate (ATP) [25,26] plus low pH [27] ), and versatile surface modification. [28,29]Although ZIF-8 can protect the loaded agents from physiological assaults [30] (e.g., endogenous enzymes and high concentration of salts) and enhance the cell uptake of loaded agents, [31] as exogenous NPs, they are easily surveilled by the body's immune system for elimination, thus limiting their drug delivery efficiency in vivo.][34][35][36] Specifically, the self-recognition protein cluster of differentiation 47 (CD47) expressed on the surface of red blood cell (RBC) membrane (RM) can activate the CD47/signal regulatory proteins  (SIRP) pathway to escape macrophage clearance to realize long blood circulation, enlightening us that this property can be utilized to improve the in vivo pharmacokinetics of administrated agents. [37]As such, for the purpose of efficient tumor treatment based on the photodynamic immunotherapy, the cell membrane-based biomimetic concept also motivates us to explore a reliable and easily-obtained source of membrane mimicking T cell membrane, which demonstrates to be an economical alternative of the PD-L1 antibody by simple culturing and self-proliferation of parental cells, [38] to fabricate the delivery nanoplatform.This would endow the delivery nanoplatform not only with the capacity of restoration of antitumor immune response, but also with the tumor targeting ability, both of which would advance the anti-tumor effect.
In this work, a tumor environment-activated photosensitized biomimetic nanoplatform (PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM) was fabricated through co-loading our developed molecular PS PEG 2000 -SiNcTI-Ph and immunoadjuvant CpG into ZIF-8 NPs, followed by encapsulation within the fusion cell membranes (CM) consisting of cell membrane overexpressing PD-1 (HM) obtained from genetically engineered HEK293T cells (HEK293T-PD-1 cells) and RM, [39] which could exert precise and effective photodynamic immunotherapy of CT26 colon cancer in mice sensitized by long-wavelength photons.Camouflaged by CM, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM could evade immune surveillance by activating the CD47/SIRP pathway to target CT26 tumor cells mediated by PD-1/PD-L1 interaction.Dormancy of the photodynamic effect of PEG 2000 -SiNcTI-Ph can be reversed to activate PDT once the nanoplatform was specifically degraded in response to tumor environment (i.e., low pH plus high concentrations of ATP).The PDT-triggered ICD of CT26 tumor cells combined with enhanced DC uptake of CpG to promote maturation of DCs contributed by the nanoplatform delivery elicited anti-tumor immune response, which was empowered by immune checkpoint PD-L1 blockade by CM.As a result, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM realized precise and efficient photodynamic immunotherapy to combat CT26 tumors and prevent their recurrence and metastasis (Scheme 1).The strategy we developed here would advance the development of precise cancer therapy.

Preparation and Characterization of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM
Phthalocyanine derivatives as highly efficient PS have been applied to many clinical PDT treatment of tumors. [40]Although their maximum absorption red-shifted by ≈100 nm compared with those of phthalocyanines, naphthalocyanine derivatives as PS showed limited application in PDT because of their poor solubility and strong aggregation. [41]In our previous work, Scheme 1. Schematic illustration of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM for precise photodynamic immunotherapy of CT26 colon cancer.PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM was synthesized by embedding our developed molecular PS PEG 2000 -SiNcTI-Ph and immunoadjuvant CpG into the porous organic framework ZIF-8 and finally encapsulating within the CM consisting of HM and RM.When the nanocomposite was intravenously injected into www.advancedscience.comalkyl chains substituted naphthalocyanine tetraimides (SiNcTI-N and SiNcTI-Br) with excellent stability and slight aggregation were reported as cathode interlayer materials in organic solar cells, however they displayed little aqueous solubility, making their PDT usage impossible. [42]To increase their aqueous solubility, we modified the compounds with different hydrophilic groups (i.e., SiNcTI-NO, SiNcTI-TFSI, SiNcTI-2N, and SiNcTI-2I) (Figure S1, Supporting Information), but frustratedly they still possessed limited aqueous solubility.Delightedly, when four isopropylphenyl groups and two methoxy polyethylene glycols chains (i.e., mPEG 1000 , mPEG 1500 , or mPEG 2000 ) were introduced to naphthalocyanine tetraimides simultaneously, sufficient aqueous solubility was achieved.Compared with PEG 1000 -SiNcTI-Ph and PEG 1500 -SiNcTI-Ph, PEG 2000 -SiNcTI-Ph (Figure 1A  Next, PEG 2000 -SiNcTI-Ph and CpG were co-embedded within ZIF-8 in one step during the self-assembly of the metalorganic framework, with the loading efficiency up to 46.6% for PEG 2000 -SiNcTI-Ph.The obtained PEG 2000 -SiNcTI-Ph/CpG-ZIF-8 nanocomposite could be well dispersed in PBS, guaranteeing the premise for in vitro and in vivo applications (Figure 1D).In addition, after encapsulation, the content of CpG in centrifugal supernatant decreased (Figure S12, Supporting Information) and agarose gel electrophoresis directly confirmed the success of CpG loading (Figure S13, Supporting Information).The observation by scanning electron microscopy (SEM) illustrated that PEG 2000 -SiNcTI-Ph/CpG-ZIF-8 demonstrated the spherical structure with an average size of ≈150 nm (Figure S14, Supporting Information).Meanwhile, X-ray diffraction (XRD) measurement revealed no significant difference in the crystal structure and crystallinity among ZIF-8, PEG 2000 -SiNcTI-Ph-ZIF-8, and PEG 2000 -SiNcTI-Ph/CpG-ZIF-8, indicating that the embedding of PEG 2000 -SiNcTI-Ph and CpG barely affected the formation of ZIF-8 (Figure S15, Supporting Information).
Eventually, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8 was encapsulated by the CM consisting of HM and RM.To obtain a reliable alternative for immune checkpoint PD-L1 blockade to advance anti-tumor immune response, we transfected PD-1 plasmifd (C-GFPSpark tag) into HEK293T cells.Fluorescence imaging (Figure 1E) and western blot (WB) analyses (Figure S16, Supporting Information) confirmed the successful transfection of the plasmid and expression of PD-1 on HEK293T cells.Likewise, flow cytometry detection verified the existence of typical markers PD-1 (35%) and CD47 (18%) proteins expressed on HEK293T-PD-1 cells and RBC, respectively (Figure 1F).HM and RM were then extracted from HEK293T-PD-1 cells and RBC, respectively, and co-extruded with PEG 2000 -SiNcTI-Ph/CpG-ZIF-8 to obtain PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM. Dynamic light scattering (DLS) results showed a slight increase in the particle size of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8 after CM encapsulation (Figure S17, Supporting Information).The shift of the zeta potential of the nanocomposites from positive to negative charge also indicated the successful encapsulation by CM (Figure 1G).Worth mentioning, the decrease of zeta potential of PEG 2000 -SiNcTI-Ph-ZIF-8 after embedding with CpG was because of the anionic attribute of CpG.And the corresponding morphological observation by transmission electron micrographs (TEM) directly revealed the thickness of the outer membrane of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM was ≈10 nm (Figure 1H).Furthermore, as can be noted from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results, HM retained most of the proteins of HEK293T-PD-1 cells and RM retained major proteins of RBCs.Similarly, it can be observed that some proteins which might exist in the cytoplasm disappeared from the cell membrane compared to the cells.As expected, major proteins on HM and RM were successfully reserved on PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM (Figure 1I).Among these proteins, the functional proteins PD-1 and CD47 expressed on HEK293T-PD-1 and RBC, respectively, were crucial for achieving immune checkpoint PD-L1 blockade/tumor targeting [33] and evasion of macrophage phagocytosis. [32]WB results exhibited that PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM well preserved PD-1 and CD47 proteins from parental cells, laying down the foundation for empowering the photodynamic immunotherapy (Figure 1J).In addition, there was no obvious variation in the particle size of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM in PBS over the course of 8 days, indicating the superior stability of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM under physiological environment (Figure 1K).

Tumor Environment-Activated Photodynamic Effect of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM
Unexpectedly, we found that the photodynamic feature of PEG 2000 -SiNcTI-Ph vanished after being embedded into ZIF-8 framework (Figure 2A).This phenomenon enlightened us if dormancy of the photodynamic effect of PEG 2000 -SiNcTI-Ph could be reversed once the release of PEG 2000 -SiNcTI-Ph was triggered after the degradation of ZIF-8 framework within the tumor due to its sensitivity to tumor-specific stresses of low pH and high concentrations of ATP, [43][44][45] a tumor environment-activated photodynamic effect would be achieved to realize precise cancer therapy.And following evaluations were performed to verify the assumption.First, the morphology of PEG 2000 -SiNcTI-Ph/CpG-CT26 tumor-bearing mice, they would target the tumor due to CD47/SIRP interaction-mediated prolonged circulation and PD-1/PD-L1 interactionmediated endocytosis.Afterward, the photodynamic effect of PEG 2000 -SiNcTI-Ph was restored in acidic and ATP-rich tumor environment.Under 808 nm laser irradiation, PEG 2000 -SiNcTI-Ph generated sufficient ROS to kill tumor cells and released TAAs to stimulate immune response, which would be empowered by immune checkpoint PD-L1 blockade and CpG from PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM.As a result, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM realized precise and efficient photodynamic immunotherapy to combat CT26 tumors and prevent their recurrence and metastasis.ZIF-8@CM after incubation in PBS at different pH or with different concentrations of ATP showed that PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM was selectively degraded in acidic or ATP-enriched conditions (Figure 2B,C), which contributed to the gradual release of PEG 2000 -SiNcTI-Ph, eventually reaching as high as >80% after 24 h, in a striking contrast to the negligible release within conditions of pH = 7.4 or 0 mM ATP (Figure S18, Supporting Information).Meantime, when PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM was incubated in PBS with low pH (5.5) plus high concentrations of ATP, its particle size decreased over time, further indicating that PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM was degraded gradually (Figure S19, Supporting Information).Excitingly, a recovery of ROS generation ability was noticed after the release of PEG 2000 -SiNcTI-Ph under 808 nm laser irradiation.As expected, compared with the action of low pH or high concentrations of ATP alone, under their simultaneous stimuli mimicking tumor environment, ZIF-8 degraded the fastest to release the most PEG 2000 -SiNcTI-Ph, thus demonstrating the most significant recovery of ROS generation (Figure 2D; Figures S20-S24, Supporting Information), which was in stark contrast to the feeble photodynamic effect of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM within conditions of pH = 7.4 plus 0 mM ATP approaching normal physiological environment, laying down the foundation for tumor environment-activated PDT beneficial for the precise treatment of colon cancer.
Next, the ROS generation ability of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM at the cellular level was evaluated to determine their tumor environment-activated photodynamic efficacy in vitro.The intracellular uptake and distribution showed that the red fluorescence from Cy5-labeled PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM was highly co-localized with the green fluorescence from Lyso Tracker or Mito Tracker, indicating that the nanocomposites could be effectively engulfed by CT26 tumor cells and accumulated within lysosome and mitochondria, where acids and ATP were enriched, respectively (Figure 2E). [25,26]With above evidence supported, then, we evaluated the ROS generation using DCFH-DA probe at the cellular level by confocal laser scanning microscopy (CLSM).As shown in Figure 2F, CT26 tumor cells incubated with PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM exhibited much brighter green fluorescence under 808 nm laser irradiation compared with those incubated with normal human lung epithelial cells BEAS-2B, which should be attributed to that the higher concentrations of ATP [43] plus lower pH [44,45] within CT26 tumor cells than BEAS-2B normal cells promoted the release of PEG 2000 -SiNcTI-Ph to recover the photodynamic effect.
In terms of above observed phenomenon, we suspected that PEG 2000 -SiNcTI-Ph would form an aggregated state after being loaded within ZIF-8, which hindered the ability of PEG 2000 -SiNcTI-Ph to generate ROS.Then, we deciphered the underlying mechanism by which PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM demonstrated tumor environment-activated photodynamic effect through quantum chemical calculations.First, we compared the electron paramagnetic resonance (EPR) signals of PEG 2000 -SiNcTI-Ph under liquid (dispersed state) and solid (aggregated state) conditions (Figure S25, Supporting Information).The results showed the PEG 2000 -SiNcTI-Ph molecules were more effective in generating ROS when they were in the dispersed state.
To simplify the computational model, we used the monomer and dimer models to represent the unassembled and aggregated states of PEG 2000 -SiNcTI-Ph, respectively (Figure 2G).Overall, based on the distribution of highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs), both the singlet and triplet states showed a typical  → * local excitation character along the horizontal axis of molecular skeleton partly mixed with a  → * charge transfer character along their vertical axes (Figure 2H,I).Interestingly, the timedependent density functional theory (TDDFT) calculations suggested that the two lowest singlet excited states (S 1 and S 2 ) of the PEG 2000 -SiNcTI-Ph molecule possessed almost the same (degenerate) energy levels, indicating the natural advantage of such a molecular configuration having twice as many channels of photosensitization.Due to the strong spin-orbit coupling (SOC) interactions between the singlet (S) and triplet (T) excited states, the intersystem crossing (ISC) process between S and T became stronger and they mainly occurred through S1/T2 and S2/T1 channels with significantly large SOC values of ≈3 cm −1 , which benefits enhancing the reactivity and opening the ROS channel (Figure 2J; Figure S26, Supporting Information).In the dimer model, the ISC process between S and T was extremely attenuated.Although a large SOC value for the S3/T5 channel existed, the large energy gap between T5 and T1-T4 prevented the possible internal conversion process.Therefore, the aggregated molecules of the triplet state were unable to generate ROS sensitized by long-wavelength photons, or the ability to generate ROS was greatly weakened (Figure 2J; Figure S27, Supporting Information).Collectively, these findings suggested the rational design of the molecular structure of PSs and ingenious modulation of their delivery state are essential for the realization of tumor environment-activated photodynamic effect.
that treated with Cy5-labeled PEG 2000 -SiNcTI-Ph/CpG-ZIF-8 owing to the PD-1/PD-L1-mediated endocytosis, beneficial for tumor environment-activated PDT (Figure 3A).After the nanocomposite entered the tumor cell and responded the abundant acids plus rich ATP to release PEG 2000 -SiNcTI-Ph, the recovery of strong ROS generation capacity would possess an outstanding potential to kill tumor cells.Under 808 nm laser irradiation, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-based tumor environment-activated PDT could significantly induce more CT26 tumor cell death than the PEG 2000 -SiNcTI-Ph-or PEG 2000 -SiNcTI-Ph/CpG-ZIF-8-based PDT under the same applied concentration of PEG 2000 -SiNcTI-Ph (Figure 3B).Above-verified different ROS generation ability of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM under laser irradiation within tumor and normal cells encouraged us to evaluate the selective killing effect of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM toward tumor cells under laser irrdiation, of which the attribute would lower unnecessary damages toward normal cells to the greatest extent, advancing the precision of cancer therapy.The results of CT26 tumor and BEAS-2B normal cell viabilities evaluated by CCK-8 assay showed that under laser irradiation, PEG 2000 -SiNcTI-Ph inhibited the two cell proliferation by ≈50%, while PEG 2000 -SiNcTI-Ph/CpG-ZIF-8 and PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM exhibited the negligible inhibition on BEAS-2B normal cells, in a striking contrast to the inhibition rates of 60% and 71% against CT26 tumor cells, respectively (Figure 3C).Besides, the generated ROS could also cause changes in mitochondrial membrane potential (MMP) to induce cell death. [46]The variation from red fluorescence of JC-1 aggregates to green fluorescence of JC-1 monomers indicated that PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-based tumor environment-activated PDT led to the most obvious MMP variation, which would promote CT26 tumor cell death.For BEAS-2B normal cells, PEG 2000 -SiNcTI-Ph caused cellular mitochondrial damage under laser irradiation evidenced by the appearance of green fluorescence.However, the damage was avoided by loading PEG 2000 -SiNcTI-Ph into ZIF-8 as indicated by no green fluorecence appearing within BEAS-2B normal cells treated with PEG 2000 -SiNcTI-Ph/CpG-ZIF-8 or PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM under laser irradiaiton (Figure 3D).In addition, as expected, the live/dead staining assay visually revealed that a significant amount of red fluorescence from propidium iodide (PI)-stained dead cells was observed for CT26 tumor cells treated with PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM under laser irradiation.Whereas, there was only a small amount of cell death happening for BEAS-2B normal cells treated with PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM under laser irradiation, which suggested that PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM selectively caused damage to tumor cells and demonstrated negligible toxicity to normal cells under laser irradiation, consistent with above CCK-8 and MMP evalulation assays, validating the tumor environment-activated PDT realized by PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM (Figure 3E).
PDT could induce ICD [47] to release damage-associated molecular patterns (DAMPs), such as calreticulin (CRT), high mobility group protein 1 (HMGB1), and heat shock protein (HSP70), to maturate DC for the activation of immune response, which would advance the anti-tumor effect.The WB analysis demon-strated that under laser irradiation for CT26 tumor cells, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM induced more expression/release of CRT, HMGB1, and HSP70 (Figure S29, Supporting Information).Subsequently, a co-culturing system of splenic immune cells and supernatants of CT26 tumor cells after different treatments was constructed to evaluate how the immune system reacted to ICD.The flow cytometry analysis showed that the percentages of CD80/CD86 double-positive DC was the highest for PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-based tumor environment-activated PDT, reaching as high as ≈49.8%.It was worth mentioning that the percentages of CD80/CD86 double-positive DC in the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM group was 27.5%, whereas CpG alone cannot elicit considerable non-specific immunity (only ≈10.1%) due to the electro-repulsive force between negatively charged cell membrane and anionic CpG, illustrating the rationality of our design to load CpG to enhance its DC uptake to promote the maturation of DCs for boosting the tumor-associated immune response (Figure 3F).Furthermore, these mature DCs would then present tumorassociated antigens to native T cells, which would differentiate into cytotoxic CD8 + T cells to elicit immune response for the anti-tumor purpose. [48]As a result, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-based tumor environment-activated PDT led to the highest activation of CD8 + T cells (Figure S30, Supporting Information).Correspondingly, we investigated the cytokine secretion levels by enzyme-linked immunosorbent assay (ELISA).The results showed that the levels of pro-inflammatory cytokines (Figure 3G), such as tumor necrosis factor- (TNF-), interferon- (IFN-), and interleukin-6 (IL-6), were significantly increased, while the secreted level of the anti-inflammatory cytokine (Figure 3H) interleukin-10 (IL-10) was significantly reduced when PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-based tumor environment-activated PDT was applied rather than PEG 2000 -SiNcTI-Ph-based treatment or CpG treatment alone.These results suggested that PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-based tumor environment-activated PDT could effectively activate DCs and induce robust immune response by combining non-specific and specific immunities.
However, in most case, immune response for the anti-tumor effect would be severely attenuated due to the self-protection mechanism of tumor cells against activated T cells, e.g., PD-1/PD-L1 interaction.Then, we further evaluated the ability of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM for immune checkpoint PD-L1 blockade to empower anti-tumor immune response.For direct comparison, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM N , which was encapsulated by the fusion cell membrane consisting of un-transfected HEK293T cell membranes and RM, was fabricated and applied along with PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM for incubation with CT26 cells, respectively, followed by the addition of the interleukin-2 (IL-2)-activated T cells (cytotoxic T cells).As shown in Figure 3I, compared with the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM N , the CT26 cell viability with PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM incubation decreased significantly (P = 0.0162), indicating that immune checkpoint PD-L1 blockade by PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM restored the cytotoxic activity of T cells to advance the anti-tumor effect.Due to the aggressive nature of CT26 tumors, the primary and distant tumor growth in the PBS group was rapid.Intriguingly, the tumor growth in the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8+L group was more inhibited than that in the PEG 2000 -SiNcTI-Ph-ZIF-8+L group, which was attributed to the CpG-induced nonspecific immunity.Likewise, without laser irradiation, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM also exhibited slight tumor suppression induced by non-specific immunity resulting from the nanoplatform delivery of CpG.Importantly, a more obvious tumor-inhibiting effect was observed from the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group (Figure 4G; Figure S39, Supporting Information).In addition, excised primary tumors in the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group were the smallest after 20 days of treatment, down to 67.  4I).It was worth noting that the volume of distant tumors in the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group was also significantly suppressed, which should be contributed by the combination of anti-tumor immune response elicited by tumor environment-activated PDT (this also got validated in the following part), immune checkpoint PD-L1 blockade, and CpGenhanced immunity as characterized above (Figure 4H,J; Figure S40, Supporting Information).Moreover, during the whole treatment period, no significant body weight loss was observed in all groups, indicating that the nanoplatforms demonstrated negligible side effects (Figure S41, Supporting Information).
To further confirm the therapeutic effect, H&E staining (Figure 4K), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Figure 4L), and Ki-67 immunohistochemical staining (Figure 4M) were used for histological examination of tumors.H&E staining showed the severest cell death with irregular cell shapes and nuclei atrophy in the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group compared with other groups.Supported by TUNEL staining and Ki-67 different treatments.Scale bar, 275 μm.F) Flow cytometric analysis of the expression levels of mature DC markers CD80/CD86 (Q2 quadrant represents the percentage ratio of mature DC) in splenic immune cells co-cultured with supernatants of CT26 cells after different treatments.G,H) Secretion levels of immunoregulatory cytokines, including TNF-, IFN-, IL-6 (G), and IL-10 (H) in splenic immune cells co-incubated with supernatants of CT26 cells after different treatments.I) Rescue of PD-L1-mediated T cell exhaustion by PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM in co-culturing tumor cells and IL-2-activated T cells.Statistical analysis was calculated via one-way ANOVA.* p <0.05, ** p <0.01, *** p <0.001, and **** p <0.0001."ns" represents no significant difference.Data are presented as mean ± S.D. (n = 3 biologically independent experiments per group).immunohistochemical staining, the severest cell death of tumor cells and the least cell proliferation were observed in the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group.All these results indicated that tumor environment-activated PDT, the tumor nonspecific immunity induced by CpG, immune checkpoint PD-L1 blockade, and prolonged blood circulation plus tumor targeting contributed by the CM coating together led to the satisfactory anti-tumor effect.

PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-Based Tumor Environment-Activated PDT Reshaped the Immune Environment to Advance the Anti-Tumor Effect and Inhibit Tumor Metastasis and Recurrence
PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-based tumor environment-activated PDT was supposed to induce ICD for anti-tumor immune response in vivo, which should reshape the immune environment beneficial for the anti-tumor outcome.
To decipher this, serum samples, spleens, and tumor tissues of above mice from different groups were collected and analyzed.First, the levels of CD80 and CD86 were profiled to evaluate the maturation of DCs.As shown in Figure 5A, the proportions of mature DCs within spleens from the PEG 2000 -SiNcTI-Ph+L, PEG 2000 -SiNcTI-Ph-ZIF-8+L, and PEG 2000 -SiNcTI-Ph/CpG-ZIF-8+L groups increased from 14.5% to 17.9%, while that from the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group reached 20.4%.Mature DCs would engulf DAMPs generated by tumor environment-activated PDT-induced ICD and present them to T cells for activation.Supported by immunofluorescence staining (Figure 5B), all laser irradiation groups showed different extents of exposure of CRT as reflected by the red fluorescence, with the strongest fluorescence appearing for the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group.As a result, analysis of activated T cell populations (i.e., CD4 + /CD8 + T cells) in spleens also revealed that the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group demonstrated the highest percentage of activated T cells, approximating more than twofold higher than the PBS group (Figure 5C).Correspondingly, the infiltration of CD4 + and CD8 + T cells within primary tumors was also significantly enhanced for the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group, guaranteeing the premise of immunotherapy of colon cancer (Figure 5D).As another main reason for the immunosuppressive attribute, the dominated M2 type of macrophage within the tumor severely affects the immunotherapeutic outcome. [49]Intriguingly, it was found that the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L treatment effectively repolarized M2-type macrophages to M1-type macrophages (Figure 5E).Meanwhile, flow cytometry analysis also confirmed that PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group effectively induced repolarization of M2 macrophage to M1 phenotype [50] (Figure S42, Supporting Information).Afterward, the secretion of cytokines in serum from differ-ent treatment groups were evaluated by ELISA.Consistently, the concentrations of cytokines positively regulating immune response (i.e., TNF- and IFN-) increased for all mice with different material treatments compared with those treated with PBS, with the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L treatment still demonstrating the strongest promotion of generation of TNF- and IFN-, leading to approximately sevenfold and approximately threefold enhancements, respectively (Figure 5F).In sum, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-based tumor environment-activated PDT elicited PD-L1 blockade-empowered anti-tumor immune response to further advance the combat of primary and distant CT26 tumors.
In most cases, tumor metastasis and recurrence will result in the ultimate failure of cancer therapy.Promoted by the positive modulation of immune environment as characterized above, we wondered whether PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-based tumor environment-activated PDT can induce immune memory to inhibit tumor metastasis and recurrence.To evaluate this, CT26 bilateral tumor mice were randomly divided into two groups, which were intravenously injected with PBS and PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM (denoted as the PBS group and PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group; "L" represented laser irradiation.),respectively, where primary tumors were irradiated with an 808 nm laser for 5 min at 24 h postinjection, followed by re-challenging the mice with CT26 tumor cells through tail vein after 20 days' treatment to simulate tumor metastasis and recurrence (Figure 5G).Ten days later, the mice were dissected and their lungs were photographed and observed.The results showed that lung tissues of mice in the PBS group exhibited obvious pulmonary nodules, which was in a striking contrast to the effect of the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group on the inhibition of pulmonary nodule formation (Figure 5H).H&E staining (Figure 5I) further evidenced new metastatic nodules in the lungs of mice in the PBS group, while no obvious tumor metastasis to lung was observed in the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group.According to immunofluorescence analysis, higher intratumoral infiltration of the central memory CD44 + CD62L + T cells and CD4 + /CD8 + T cells were observed in the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group compared with the PBS group (Figure 5J; Figure S43, Supporting Information).This indicated that CD44 + /CD62L + memory T cells induced by immune response could rapidly transform into tumor antigen-specific CD4 + and CD8 + T cells, respectively, once they were attacked by homologous tumor cells again to achieve long-term protection, thus effectively inhibiting tumor metastasis and recurrence.
Cell Lines and Animals: All the cell lines used in the study were obtained from Procell Life Science & Technology Co., Ltd (Wuhan, China).CT26 cells and the three cell lines (HEK293T, BEAS-2B, and RAW264.7 cells) were cultured in RPMI 1640 and DMEM medium, respectively, with 10% FBS and 1% penicillin-streptomycin in a humidified atmosphere at 37 °C containing 5% CO 2.
C57BL/6 mice (male, 5 weeks) were obtained from Gempharmatech Co., Ltd (Jiangsu, China).All mice were raised in the Animal Science Laboratory Center of Nanchang University with a photoperiod of 12 h light/12 h dark, humidity of 40%−68%, and temperature of 22 °C ± 1 °C.All animal experiments were grouped randomly and investigators were blinded to the group allocation during data collection and/or analysis.All animal studies were conducted according to the guidelines approved by Institutional Animal Care and Use Committee (IACUC) of Nanchang University (Nanchang, China, approval number: NCULAE-20231128009).
Synthesis of PEG 2000 -SiNcTI-Ph: PEG 2000 -SiNcTI-Ph was synthesized according to the synthesis route shown in Figure S2 (Supporting Information).Compound 1-8 were synthesized with the reported methods. [51]ynthesis of Compound 9: A mixture of compound 8 (1 g, 2.49 mmol), sodium methylate (NaOCH 3 , 100 mg, 1.85 mmol), and ammonia (NH 3 , 7 M in methanol (CH 3 OH), 24.9 mmol) were stirred at reflux in CH 3 OH (30 mL) for 1 h.After cooling to room temperature, the mixture was poured into 30 mL of water.The white precipitate was filtered, recrystallized, and dried in a vacuum oven.And compound 9 (692 mg, 65.2%) was obtained as a yellow solid for the next step without further purification. 1 Synthesis of Compound 10: Compound 9 (500 mg, 0.018 mol) and 10 mL of quinoline were heated to 210 °C in a flask under argon (Ar 2 ), and then silicon tetrachloride (SiCl 4 , 101 mg, 0.60 mmol) was added dropwise.The mixture was stirred for 30 min, the temperature was lowered to 145 °C, then 1 mL of water was added to the reaction mixture and stirred for 1 h.After cooling to room temperature, 60 mL of CH 3 OH was added.The as-obtained mixture was filtered and purified through a silica gel column with an eluent of dichloromethane (CH 2 Cl 2 ).And compound 10 (120 mg, 30.0%) was obtained as a dark green solid. 1  Synthesis of compound PEG 2000 -SiNcTI-Ph: Compound 10 (100 mg, 0.059 mol) and PEG 2000 (480 mg, 0.24 mol) were dissolved in 10 mL of 1,2-dichlorobenzene.The mixture was refluxed under Ar 2 atmosphere for 8 h.After cooling to room temperature, the mixture was purified through a silica gel column (eluent, CH 3 OH:CH 2 Cl 2 = 1:100).PEG 2000 -SiNcTI-Ph was obtained (230 mg, 68%) as a dark green solid. 1  hydroxide (NaOH) solution and stirring for 30 min away from light.The DCFH solution (10 μm, 100 μL) was then added into PEG 2000 -SiNcTI-Ph solution (10 μg mL −1 , 1 mL) and mixed well.Then, the spectra of the mixed solutions were measured by a fluorescence spectrometer.Subsequently, an 808 nm laser with an optical density of 0.3 W cm −2 was irradiated for 10 s and the spectrum of the mixed solution was measured.This experiment was performed in parallel for six times and the peak intensity was observed at 525 nm.
For DPBF detection, the DPBF solution (1 mm, 100 μL) was added into PEG 2000 -SiNcTI-Ph solution (30 μg mL −1 , 1 mL) and mixed well.Then, the spectrum of the mixed solution was measured by a UV-vis spectrophotometer.Similarly, the 808 nm laser with a density of 0.3 W cm −2 was irradiated for 10 s and the spectrum of the mixed solution was measured.This experiment was performed in parallel for six times and the peak intensity was observed at 410 nm.Preparation of HM, RM, and PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM: For extraction of HM, HEK293T-PD-1 cells were first obtained by transfecting PD-1 plasmid (C-GFPSpark tag) into HEK293T cells and then centrifuged for 3 min at 300 g and rinsed three times with PBS.The cells were then resuspended in cell membrane isolation buffer (0.25 mM sucrose, 1 mM EDTA, 20 mM HEPES-NaOH, and protease inhibitor; pH = 7.4) and crushed with an ultrasonic pulverizer for 20 s.The supernatant was then collected and centrifuged again at 10 000 g for 20 min to discard the pellet.The cell membrane was collected by re-centrifugation at 30 000 g for 60 min, which were finally freeze-dried and stored in at −80 °C.The above process was always performed at 4 °C.
RM was extracted according to the reported method. [32]First, the obtained RBCs from mice were resuspended in 0.25 × PBS for 2 h to induce RBCs rupture.Then, RM was collected by centrifugation at 14 000 g for 30 min.The above procedure was repeated until the RM solution was colorless.All above experimental operations were carried out at 4 °C.
Release of PEG 2000 -SiNcTI-Ph: To study pH-and ATP-responsive release of PEG 2000 -SiNcTI-Ph, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM was dispersed in PBS (pH 7.4 or 5.5) and/or incubated with different concentrations of ATP (0, 2, and 5 mm), respectively.At different time periods, the supernatant was obtained by centrifugation and the absorbance at 790 nm was measured with a UV-vis spectrophotometer.The released profiles of PEG 2000 -SiNcTI-Ph at each time point were calculated according to the standard curves.
Flow Cytometry: To evaluate the expression of PD-1 protein on HEK293-PD-1 cells and CD47 protein on RBC, HEK293-PD-1 cells and RBCs were washed with PBS for three times, and then incubated with FITCanti-PD-1 and FITC-anti-CD47 for 30 min, respectively, and finally washed with PBS for three times for flow cytometry analysis.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis: Samples were added to 10% SDS-PAGE gels and separation was initiated under 120 V.After the separation, incubate the gel for staining by adding it to the coomassie blue staining solution.After that, it was washed with coomassie blue staining destaining solution.Finally, wash the gel with water.
Western Blotting: Samples were added to 10% SDS-PAGE gels for separation, followed by being transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 5% skimmed milk for 1 h at room temperature.After incubation with the corresponding primary antibodies, the membrane was washed three times with TBST for 10 min and then incubated with the secondary antibody for 1 h at room temperature.The membrane was washed three times with TBST and then stained with enhanced chemiluminescent (ECL) detection reagent.Proteins were observed using a digital gel image analysis.
Lysosomal/Mitochondrial Localization Experiment: CT26 cells were seeded into the cell culture dishes for 24 h of incubation at 37 °C.Afterward, fresh medium containing Cy5-labeled PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM was added for another 2 h of incubation.After washing the cells with PBS for three times, they were stained with Lyso Tracker/Mito Tracker for 20 min at 37 °C.Finally, the cells were washed with PBS for three times and then imaged by CLSM.
Intracellular ROS Generation: CT26 cells or BEAS-2B cells were seeded into the cell culture dishes for 24 h of incubation at 37 °C.Fresh medium containing PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM (PEG 2000 -SiNcTI-Ph equivalent dose: 30 μg mL −1 ) was added for another 4 h of incubation.After washing the cells with PBS for three times, they were incubated with 10 μm DCFH-DA for 20 min at 37 °C in the dark.After washing, the cells were irradiated with an 808 nm laser (1 W cm −2 ) for 15 min, followed by CLSM imaging.Conditions: excitation wavelength: 488 nm; emission filter: 500-550 nm.
Quantum Chemical Calculations: The ground-state (S 0 ) geometries of monomer and aggregation dimer models were optimized using density functional theory (DFT) at the B3LYP-D3 (BJ) [52][53][54] /6-31G (d) level.Both the singlet and triplet excitation energies were calculated at the time-dependent (TD) DFT/6-31G (d) level with the polarizablecontinuum model in water, with three selected B3LYP, M062X, and B97XD functionals.And the TDDFT calculations suggested that the (TD)-B3LYP/6-31G (d) level was proved to reasonably reproduce the experimental measurement and was employed in this work (Table S1, Supporting Information).The corresponding HOMOs and LUMOs for ground states, as well as the hole and electron distribution for various excited states, were plotted and rendered using variational mode decomposition (VMD) code. [55]All the DFT and TDDFT calculations were performed using the Gaussian 16 program. [56]The SOC elements between singlet and triplet states were evaluated using the linear-response (LR) TDDFT method employing Casida-type wave functions and the Breit-Pauli (BP) spin-orbit Hamiltonian with an effective charge approximation by PySOC code. [57]ndocytosis Experiments: CT26 cells or RAW264.7 cells were seeded into 24-well plates with a density of 1 × 10 5 cells well −1 for 24 h of incubation.Different samples were added for another 2 h of incubation.Then, the medium was removed and washed with PBS for three times, followed by staining with Hoechst 33 342 for 15 min.After that, the samples were washed with PBS and imaged by a fluorescence microscope to investigate the cell endocytosis of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8 or PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM.
Cell Viability Test: CT26 cells or BEAS-2B cells were seeded into 96-well plates with a density of 5 × 10 3 cells well −1 for 12 h of incubation.Fresh medium containing different materials (equivalent contents of PEG 2000 -SiNcTI-Ph in different materials) was added for another 12 h of incubation and the medium was removed and washed with PBS for three times.Afterward, the cells were irradiated with an 808 nm laser (1 W cm −2 ) for 2 min.Next, the old culture medium was replaced with serum-free medium containing 10% CCK8 for 2 h of incubation at 37 °C.The absorbance at 450 nm was measured by a microplate reader.
Cellular Mitochondrial Damage by JC-1 Assays: CT26 cells or BEAS-2B cells were seeded into 12-well plates for 12 h of incubation.Fresh medium containing different materials (equivalent dose of PEG 2000 -SiNcTI-Ph: 30 μg mL −1 ) was added for 4 h of incubation.After that, the cells were irradiated with an 808 nm laser (1 W cm −2 ) for 15 min.Afterward, the medium was removed and the cells were stained with 10 μg mL −1 of JC-1 for 20 min at 37 °C.Finally, the cells were washed with PBS for three times and then imaged by a fluorescence microscope.
Live/Dead Cell Staining: CT26 cells or BEAS-2B cells were seeded into 24-well plates at a density of 1 × 10 5 cells well −1 for 24 h of incubation.Afterward, fresh medium containing different materials (equivalent dose of PEG 2000 -SiNcTI-Ph: 30 μg mL −1 ) was added for 4 h of incubation.Subsequently, the cells were washed and irradiated with an 808 nm laser (1 W cm −2 ) for 15 min.After that, the cells were incubated at 37 °C for another 4 h, then successively stained with Calcein-AM and PI.Subsequently, the cells were washed with PBS for three times and observed by a fluorescence microscope.
Analysis of DAMPs: CT26 cells were seeded into 6-well plates for 24 h.Fresh culture media containing PBS or PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM (equivalent dose of PEG 2000 -SiNcTI-Ph: 30 μg mL −1 ) was then added and incubated for 12 h.Subsequently, the cells were washed and irradiated with an 808 nm laser (1 W cm −2 ) for 15 min.Finally, we took apoptotic CT26 cells for WB analysis of CRT and cell supernatant for WB analysis of HMGB-1 and HSP-70.
In Vivo Biocompatibility Evaluation: To evaluate the biocompatibility of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM (equivalent dose of PEG 2000 -SiNcTI-Ph: 2.5 mg kg −1 body weight) was injected into the mice via tail vein.Mice were sacrificed at 21 days post injection and major organs were collected for H&E staining.Meanwhile, mouse blood was obtained for routine analysis of liver function, kidney function, and other biochemical indicators.
In Vivo Blood Retention and Targeting Capability: To evaluate the blood circulation time of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM, C57BL/6 mice were divided into three groups and administered with PEG 2000 -SiNcTI-Ph, PEG 2000 -SiNcTI-Ph/CpG-ZIF-8, or PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM at a PEG 2000 -SiNcTI-Ph equivalent dose of 2.5 mg kg −1 .At different time points, mouse blood was taken out through the eyeball.The blood samples were then photographed by a fluorescence imaging device.The fluorescence intensity of the blood was analyzed by an image J software to estimate the content of these nanocomposites in blood and calculate their blood half-life time.
Tumor models were generated by injection of CT26 cells (5 × 10 6 cells mouse −1 ) on the right side of each mouse.When the tumor volume reached ≈80 mm 3 , the mice were divided into two groups (n = 3) and injected with ICG-labeled PEG 2000 -SiNcTI-Ph/CpG-ZIF-8, ICG-labeled PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM N , and ICG-labeled PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM (equivalent dose of PEG 2000 -SiNcTI-Ph: 2.5 mg kg −1 body weight) via tail vein, respectively.At different time periods, mice were imaged by a fluorescence imaging system.The fluorescence intensity at the tumor was evaluated by image J software.Finally, the mice were sacrificed at 24 h post injection and their tumors were isolated and photographed by the imaging instrument.
In Vivo PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM-Mediated Tumor Suppression: CT26 cells (5 × 10 6 cells mouse −1 ) were injected subcutaneously on the right side of C57BL/6 mice to generate primary tumors.After the tumor volume reached ≈80 mm 3 , the left side of the same mice was inoculated with the same number of CT26 cells to establish a bilateral tumor model.The mice were randomly divided into six groups (n = 6) (denoted as 1: the PBS group, 2: the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM group, 3: the PEG 2000 -SiNcTI-Ph+L group, 4: the PEG 2000 -SiNcTI-Ph-ZIF-8+L group, 5: the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8+L group, 6: the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group; "L" represented laser irradiation).Different materials were injected into mice via tail vein, followed by irradiation of primary tumors at 24 h post-injection (808 nm, 1 W cm −2 , 5 min).Afterward, mouse body weight and bilateral tumor volume were measured every two days.Tumor volume was calculated as (width 2 × length)/2.In the in vivo efficacy study, tumor growth was tracked until day 20 when the mice were executed and tumor tissue and major organs were then collected for further analysis.And the spleens were made into singlecell suspensions.Cell suspension was then stained with the FITC-antimouse CD80 and PE-anti-mouse CD86 antibodies to detect DCs, and T cells were detected with the FITC-anti-mouse CD4 and PE-anti-mouse CD8 antibodies.
To further evaluate the ability of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM to inhibit tumor metastasis and recurrence, a lung metastasis and tumor recurrence model was established.CT26 cells (5 × 10 6 cells mouse −1 ) were injected subcutaneously on the right side of C57BL/6 mice to generate primary tumors.After the tumor volume reached ≈80 mm 3 , the distant tumors of mice were generated through inoculating the same number of CT26 cells on the left side of the same mice.The mice were randomly divided into two groups (n = 6) (denoted as 1: the PBS group, 2: the PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM+L group; "L" represented laser irradiation).PBS and PEG 2000 -SiNcTI-Ph/CpG-ZIF-8@CM were injected into mice via tail vein, followed by irradiation of primary tumors at 24 h post-injection (808 nm, 1 W cm −2 , 5 min).After 20 days' of treatment, CT26 cells were injected into the mice again via tail vein to establish a lung metastasis and tumor recurrence model.Ten days later, the mice were dissected and their lungs were observed and further stained with H&E for histological analysis.
Statistical Analysis: All experiments were performed at least three times and all data represented mean ± s.d.GraphPad Prism 8 was used to conduct the statistical analysis between the test and control groups using the Student's t-test and one-way ANOVA.* p <0.05, ** p <0.01, *** p <0.001, and **** p <0.0001 represented different statistical significance."ns" stood for no significant difference.
(left picture); Figures S2-S9, Supporting Information) demonstrated the best solubility and well dispersion in aqueous solution, which was chosen for following studies.The maximum absorption of PEG 2000 -SiNcTI-Ph was located at 790 nm (Figure 1A (right picture)), much longer than that of conventional commercial PSs, such as Ce6 (650 nm) and rose Bengal (530 nm).The high-efficiency long-wavelength photon sensitization of PEG 2000 -SiNcTI-Ph was confirmed by the fluorescence increase of 2,7dichlorodihydronflurescein diacetate (DCFH-DA) solution and adsorption decrease of 1,3-diphenylisobenzofuran (DPBF) solution under 808 nm laser irradiation, indicating the generation of ROS (Figure 1B,C; Figures S10 and S11 Supporting Information).
Synthesis of PEG 2000 -SiNcTI-Ph-ZIF-8 and PEG 2000 -SiNcTI-Ph/CpG-ZIF-8: For the preparation of PEG 2000 -SiNcTI-Ph-ZIF-8, first, 40 mg of Zn (NO 3 ) 2 •6H 2 O and 0.77 g of 2-MiM were dissolved in 1 mL and 4 mL of deionized (DI) water, respectively.After that, 8 mg of PEG 2000 -SiNcTI-Ph was added to the Zn (NO 3 ) 2 solution and mixed for 2 min, followed by adding the 2-MiM solution and magnetically stirring for 5 min.The light green product was obtained by centrifugation at 12 000 rpm min −1 for 10 min and washed three times with DI water.For the synthesis of PEG 2000 -SiNcTI-Ph/CpG-ZIF-8, similarly, 8 mg of PEG 2000 -SiNcTI-Ph and 500 μg of CpG was added to above Zn (NO 3 ) 2 •6H 2 O solution and mixed for 2 min, followed by adding the 2-MiM solution and magnetically stirring for 5 min.The light green product was obtained by centrifugation at 12 000 rpm min −1 for 10 min and washed three times with DI water.