Integration of AIEgens into covalent organic frameworks for pyroptosis and ferroptosis primed cancer immunotherapy

Immunogenic programmed cell death, such as pyroptosis and ferroptosis, efficiently induces an acute inflammatory response and boosts antitumor immunity. However, the exploration of dual-inducers, particularly nonmetallic inducers, capable of triggering both pyroptosis and ferroptosis remains limited. Here we show the construction of a covalent organic framework (COF-919) from planar and twisted AIEgen-based motifs as a dual-inducer of pyroptosis and ferroptosis for efficient antitumor immunity. Mechanistic studies reveal that COF-919 displays stronger near-infrared light absorption, lower band energy, and longer lifetime to favor the generation of reactive oxygen species (ROS) and photothermal conversion, triggering pyroptosis. Because of its good ROS production capability, it upregulates intracellular lipid peroxidation, leading to glutathione depletion, low expression of glutathione peroxidase 4, and induction of ferroptosis. Additionally, the induction of pyroptosis and ferroptosis by COF-919 effectively inhibits tumor metastasis and recurrence, resulting in over 90% tumor growth inhibition and cure rates exceeding 80%.

(AIEgens) into COF skeletons to elicit a strong inflammatory response and boost antitumor immunity.Nanoscale COFs are crystalline organic porous materials and have attracted much attention in the field of cancer phototherapy owing to their excellent light absorption, high porosity, photostability, and good biocompatibility [23][24][25][26][27] .Currently, the major cell death mode caused by COF-mediated phototherapy is apoptosis.However, others and we showed the feasibility of decorating metal-containing inducers onto the skeleton or into the pores of COFs to trigger pyroptosis or ferroptosis [28][29][30] .However, it remains challenging to create non-metallic COF-based pyroptosis or ferroptosis inducers due to the stringent requirements of ROS generation efficiency.Traditional COF-based photosensitizers (PSs) suffer from strong undesirable aggregation-caused quenching (ACQ) effects.This leads to no or weak emission and limited capability to generate reactive oxygen species (ROS), ultimately failing to trigger pyroptosis or ferroptosis.In this work, a pyroptosis and ferroptosis dual-inducer, called COF-919, was rationally designed by integrating planar and twisted motifs with AIE characteristics 31,32 into the COF skeleton.Such a kind of planar plus twisted AIE COF has several advantages over the existing COF-based photosensitizers.First, in comparison to traditional COFs with all planar or twisted motifs (e.g., COF-909 and COF-818), this planar plus twisted AIE COF (COF-919) displayed better nearinfrared (NIR) light absorption, lower band energy and a longer lifetime to contribute good phototherapeutic effect, which is favorable for inducing pyroptosis.Second, benefiting from its good ROS production capability, intracellular lipid peroxidation (LPO) is also upregulated, which leads to glutathione depletion, low glutathione peroxidase 4 (GPX4) expression, and the induction of a GPX4-related ferroptosis process.Third, the superb photothermal therapy (PTT) performance of COF-919 helps improve the pyroptosis and ferroptosis process.Finally, the high porosity of AIE COFs not only effectively avoids the undesirable ACQ and poor photosensitization effects but also facilitates the ROS diffusion process 33,34 .These advantages make COF-919 favorable for inducing pyroptosis and ferroptosis simultaneously (Fig. 1).
In this work, COF-919-mediated photodynamic and photothermal combination therapy elicits GPX4-related ferroptosis and gasdermin E (GSDME)-dependent pyroptosis, inducing an acute inflammatory response and boosting cancer immunotherapy.Specifically, GSDME is cleaved by overexpressed cleaved Caspase-3 to produce N-terminal domains, which further assemble into transmembrane pores and swell with large bubbles.During this process, the release of abundant cell contents, such as high mobility group protein B1 (HMGB1), lactic dehydrogenase (LDH), and adenosine triphosphate (ATP), induces a strong immune response.On the other hand, the successful induction of ferroptosis by COF-919 is revealed by the overexpression of representative pyroptosis indicators, a decreased GSH/GSSG ratio, an increased cellular iron level, and a shriveled mitochondrial morphology after treatment with COF-919.As a result, COF-919 induces pyroptosis and ferroptosis, promoting the proportion of T cell infiltration in the microenvironment while reducing the proportion of regulatory T cells (Tregs) and immunosuppressive myeloid-derived suppressor cells (MDSCs) in the macroenvironment.This indicates the significant potential of COF-919 in the induction of pyroptosis and ferroptosis.Importantly, COF-919 treatment synergistically improves the response rate of immune checkpoint blockade therapy by αPD-1, and the combination of COF-919 + αPD-1 demonstrates a favorable synergistic effect in inhibiting tumor metastasis and recurrence, resulting in over 90% tumor growth inhibition and cure rates exceeding 80%.

Photophysical properties of COF-818 and COF-919
The phototherapy efficacy of COF-based PSs is dictated by their specific photophysical properties, including light absorption, steadystate fluorescence, lifetime, and band energy (Fig. 3a) 35 .To study these photophysical properties, a series of spectroscopic experiments were carried out.Ultraviolet/visible diffuse reflectance spectroscopy was conducted to investigate the light absorption properties of these AIE COFs.A clear red-shift was observed in the spectrum of COF-919 in comparison with that of COF-818, demonstrating the better coefficient absorption of COF-919 (Fig. 3b, Supplementary Figs.9-11).Then, both steady-state and time-resolved photoluminescence (PL) spectra were obtained to study the chargetransfer properties of these COFs.As depicted in Fig. 3c, d, compared with COF-818, COF-919 showed decreased steady-state PL and a longer lifetime, which unveiled faster electron-hole separation and a lower charge recombination rate upon photoexcitation, critical for improving the PDT performance (Fig. 3c, d).Furthermore, the band energy properties, including the band gap and valence band, were also studied by ultraviolet photoelectron spectroscopy, where COF-919 possessed a lower valence band and band gap compared to that of COF-818 (Fig. 3e-h).These results, combined with the calculation results of COF-818 and COF-919, where COF-919 also lowers band energy (Fig. 3i), unambiguously reveal the excellent light absorption of COF-919.

In vitro investigation of the photothermal and photodynamic therapy of COF-818 and COF-919
Considering the excellent photophysical properties of COF-919, we then studied its potential to act as a photosensitizer for photothermal and photodynamic therapy (Fig. 4a).As shown in Fig. 4b, both COF-818 and COF-919 can generate heat under 808 nm laser irradiation, and COF-919 displayed better photothermal conversion capability, owing to its better photophysical properties (Fig. 4c, d).The excellent photothermal conversion of COF-919 was further confirmed by experiments conducted in vitro and in vivo (Fig. 4e, f).Subsequently, the ROS generation ability of these AIE COFs was evaluated using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as a ROS detection probe 36 .The results showed that after adding DCFH-DA to the COF samples, no obvious fluorescence intensity change was observed in either the control or the COF-818 sample.However, a drastic enhancement in fluorescence intensity was observed in the COF-919 sample under the same conditions, demonstrating its excellent ROS generation capability and outlining the superiority of COF-919 for phototherapy (Fig. 4g-i).Then, nanoscale COF samples were successfully generated by ultrasound treatment in PBS.The sample size determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM) was ~200 nm, which is suitable for in vivo experiments.(Fig. 4d).As displayed in Supplementary Fig. 12, after 12 h of coculture, 4T1 cells showed a clear red fluorescence signal, indicating that these nanoscale COFs could be effortlessly engulfed by 4T1 cells.We then evaluated the cytotoxicity of COF-818 and COF-919 by using an MTT assay.After treatment with COF solutions with different concentrations, over 95% of the 4T1 cells survived, demonstrating the good biocompatibility of COF-818 and COF-919.In contrast, after laser irradiation, less than 25% of the 4T1 cells survived after treatment with COF-919 (Supplementary Figs.13-15), unveiling the higher photo cytotoxicity of COF-919.

Pyroptosis-inducing capability of COF-818 and COF-919
Based on previous studies, the release of a large amount of ROS caused by PDT may trigger acute inflammation and elicit pyroptosis, which is characterized by the formation of large bubbles on the membrane (Fig. 5a) [37][38][39][40] .The gasdermin family proteins have been proven to be crucial for pyroptotic cell death 41,42 .The classical pyroptosis process is usually activated by Caspase-1, where the protein gasdermin D (GSDMD) is cleaved to release GSDMD-N domains and form large bubbles [43][44][45] .Recently, a gasdermin family protein-dependent pyroptosis process was reported by Shao et al., which can be activated by Caspase-1/3/4/5/11 [46][47][48][49] .To investigate whether these COFs could trigger pyroptosis, optical confocal microscopy was used to monitor morphological changes in situ.As shown in Fig. 5f, 4T1 cells treated with COF-919 + Laser generated bubbles, and obvious bubbles were formed with 660 nm plus 808 nm laser irradiation, outlining the importance of dual-wavelength laser irradiation.The occurrence of pyroptosis was further confirmed by western blotting.Although both COF-818 and COF-919 could cause cleaved Caspase-3 overexpression, GSDME-N was only overexpressed in COF-919-treated 4T1 cells (Fig. 5e).This revealed that pyroptosis was successfully induced by COF-919.In contrast, COF-818 only triggered apoptosis.This was further evidenced by the quantitative evaluation of the representative pyroptosis parameters, such as ATP, LDH, and HMGB1.A notable increase in ATP and LDH concentrations was found in COF-919-treated 4T1 cells under 660 and/or 808 nm laser irradiation (Fig. 5b, c).In contrast, no distinct change in ATP or LDH concertation was observed in other groups, including phosphate buffer solution (PBS), laser, and COF-818.Similarly, a sharp decrease in the concertation of HMGB1 was detected in only COF-919 treated 4T1 cells under 660 and/or 808 nm laser irradiation (Fig. 5d).These results clearly revealed the good pyroptosis-inducing efficacy of COF-919.

Ferroptosis-inducing capability of COF-818 and COF-919
Generally, generations of sufficient ROS in lipids will result in intracellular lipid peroxidation, GSH depletion, and low expression of GPX4 to trigger another immunogenic programmed cell death mode, called ferroptosis, and PTT can facilitate ferroptosis efficacy [50][51][52] .We hypothesized that the excellent PDT and PTT performance of COF-919 may elicit ferroptosis (Fig. 6a).To test our hypothesis, the expression of three representative ferroptosis indicators, including GPX4, transferrin receptor (TFRC), solute carrier family 7 and member 11 (xCT), was assessed by western blot.As seen in Fig. 6b, Supplementary Fig. 16, a clear reduction in the expression of GPX4 and xCT was observed in 4T1 cells treated with COF-919 + 660 nm, COF-919 + 808 nm, and COF-919 + 660 + 808 nm groups.Among them, COF-919 + 660 + 808 nm treatment resulted in the lowest expression, outlining the importance of PDT/PTT combination therapy.Similarly, TFRC, another ferroptosis indicator, was also found to be obviously overexpressed after treatment with COF-919 + 660 + 808 nm.These results, combined with the drastic enhancement in cellular iron levels and decreased GSH/GSSG ratio, indicated the occurrence of strong ferroptosis caused by COF-919 + 660 + 808 nm (Fig. 6c, d).The high ferroptosis-inducing performance of COF-919 was further confirmed by detecting the lipid ROS levels, where the 4T1 cells treated with COF-919 + 660 + 808 nm displayed the strongest green fluorescence (Fig. 6e).Moreover, the TEM results also indicated that compared with normal ones, the morphology mitochondria treated with that of COF-919 + 660 + 808 nm was notably shriveled (Fig. 6f).These results clearly demonstrated the successful elicitation of ferroptosis by COF-919-mediated PDT and PTT.

In vivo therapeutic effect of AIE COFs
To in vivo validate the therapeutic effect of COF-818 and COF-919, a 4T1 tumor-bearing mouse model was set up (Fig. 7a).As shown in Fig. 7b-d, the growth rate of the 4T1 tumors was effectively inhibited after treatment with COF-919 irradiated under 660 and/or 808 nm laser and almost all of the tumors were eliminated after treated with COF-919 under 660 and 808 nm dual-wavelength laser irradiation.This indicated the excellent therapeutic effect of COF-919 + 660 + 808 nm.Then, western blotting and immunohistochemical staining were performed to analyze tumor tissues from each group of animals after euthanasia.The Hematoxylin and eosin (H&E) staining images of the major organs showed no apparent histopathological abnormalities after different treatments (Supplementary Fig. 17).This, combined with no obvious changes in body weight (Fig. 7c), demonstrated the good biocompatibility of these COFs.The immunohistochemical staining results showed a sharp decrease in the proliferation marker Ki67 after treatment with COF-919 + 660 + 808 nm, indicating successful tumor growth inhibition (Fig. 7e, Supplementary Fig. 18).Moreover, after treatment with COF-919 + 660 + 808 nm, granzyme B and CD8 were also overexpressed, demonstrating the clear activation of the immune system (Supplementary Fig. 19).In addition, the cleaved Caspase-3 and 4-Hydroxynonenal (4-HNE) were also overexpressed after treatment with COF-919 + 660 + 808 nm (Fig. 7f, Supplementary Fig. 18).These results, combined with the overexpression of the protein GSDME-N and reduced expression of the GPX4 determined by western blotting after treatment with COF-919 + 660 + 808 nm (Fig. 7g), unveiled the successful occurrence of pyroptosis and ferroptosis within 4T1 tumor tissues.
As immunogenic programmed cell death modes, pyroptosis and ferroptosis have demonstrated their potential to induce an acute inflammatory response to produce antitumor immune activity and boost cancer immunotherapy (Fig. 7h) [53][54][55] .To investigate the influence of COF-919-mediated pyroptosis and ferroptosis on the immune cell response, a flow cytometry system was utilized to analyze both the draining lymph (DLN) and the spleen of each tumor-bearing mouse (Supplementary Figs.20-23).The population of dendritic cells (DCs), a kind of first-line cell that initiates adaptive immune responses 56,57 , was found to be notably increased in DLN, implying the potential of activated immune cells (Fig. 7m).Second, the proportions of CD4 + and CD8 + T cells (both T CM and T EM ) were drastically increased in the DLN and spleen of COF-919 + 660 + 808 nm treated mouse, indicating that COF-919-mediated phototherapy is favorable for promoting T-cell clone expansion (Fig. 7j, k, n, o).This was further evidenced by the immunohistochemical staining, where both CD8 and granzyme B, which play a critical role in cytotoxic T-cell-mediated antitumor immunity [58][59][60] , were overexpressed in tumor tissues treated with COF-919 + 660 + 808 nm (Supplementary Fig. 19).Moreover, the proportions of immunosuppressive cells, including MDSCs and Treg cells, in DLN and spleen of COF-919 + 660 + 808 nm treated mouse were also sharply decreased (Fig. 7i, l, m, p), revealing that the COF-919-mediated phototherapy can relieve the immunosuppressive status and effectively reshape the tumor immune microenvironment for effective immunotherapy.Collectively, these results revealed that the COF-919mediated phototherapy efficiently boosted the T-cell-mediated immune response.

Synergistic effect of COF-919 and αPD-1
Encouraged by the above in vivo therapeutic results, we suspected that COF-919-mediated phototherapy may be capable of improving the immune response rate of checkpoint blockade therapy.Therefore, a 4T1 tumor-bearing dual-flank model was set up with PD-1 served as the immune checkpoint inhibitor (Fig. 8a).As seen in Figs.8b, c, although the primary tumor was inhibited by COF-919 or COF-919 + αPD-1 treatment, the distant tumor could be suppressed only after treatment with COF-919 + αPD-1, demonstrating the great power of the synergistic effect of COF-919 plus αPD-1.Furthermore, the tumor volume of the mice was examined using in vivo bioluminescence imaging every week.The data showed that COF-919 + αPD-1 combination therapy drastically prolonged the survival time of the 4T1 mice, where all of the COF-919 + αPD-1 treated mice survived euthanasia  (Fig. 8d, e).In contrast, the control group showed a 30-day median survival time.To further investigate the potential of inhibiting tumor recurrence, a rechallenge 4T1 mouse model was established (Fig. 8f).

ROS
As shown in Fig. 8g, h, the naive mice suffered grave tumor recurrence, whereas the mice treated with COF-919 + αPD-1 showed no noticeable tumor recurrence.This indicated that this synergistic strategy of COF-919 + αPD-1 was favorable for restraining tumor recurrence.

Discussion
In summary, a non-metallic AIE COF-based pyroptosis and ferroptosis dual-inducer was constructed by integrating planar and twisted motifs into a COF skeleton.Unlike traditional COFs constructed with solely planar or twisted motifs, this planar plus twisted AIE COF displayed better NIR light absorption, a lower band energy, and a longer lifetime which facilitate ROS generation and photothermal conversion, thus triggering a GSDME-dependent pyroptosis.Furthermore, owing to its exceptional ROS production capacity, COF-919 also upregulates intracellular lipid peroxidation, leading to glutathione depletion, low expression of glutathione peroxidase 4, and induction of a GPX4-related ferroptosis.Notably, COF-919-induced pyroptosis and ferroptosis effectively reshape the TME by promoting T-cell infiltration and alleviating immunosuppression, thereby  boosting T-cell-mediated immune responses that are conducive to inhibiting tumor metastasis and recurrence.This work not only broadened the biomedical applications of AIE COFs but also provided an additional strategy for pyroptosis and ferroptosis dualinducer design, which will benefit the further advancement in the field of cancer immunotherapy.

Synthesis of COF-818 and COF-919
A Pyrex tube was charged with M-TPh (77 mg, 0.12 mmol) or M-Tpy (78.5 mg, 0.12 mmol) and M-TPA (57 mg, 0.12 mmol).Next, 1.5 mL of o-dichlorobenzene and n-butanol were added along with 0.3 mL aqueous acetic acid (6 M).Then, the tube was swiftly sealed by flame and subjected to a 120 °C oven for 3 days.The ensuing products were filtered and activated with dioxane and acetone.These AIE COFs were dried under a dynamic vacuum at 120 °C for 24 h to attain complete activation.

Photothermal properties
Totally, 1.0 mL of COF samples with different concentrations were added and irradiated by an 808 nm NIR laser (Beijing Laserwave Optoelectronics Technology Co., Ltd.).An infrared camera (FOTRIC 225s, China) was utilized to monitor the temperature change in real time.

Ferroptosis assay
4T1 cells (5 × 10 4 ) and COF samples (100 μg mL −1 ) were incubated for 24 h, then irradiated with 660 nm (0.5 W cm −2 , 5 min) and/or 808 nm (1.5 W cm −2 , 5 min) lasers.Then, the cells were used for GSH/GSSG assay.The ratio of GSH/GSSG in cells was measured using the GSH/ GSSG assay kit (Beyotime Biotechnology, S0053) as per the manufacturer's instructions.Total GSH and oxidized GSH were quantified using a luminescence reader.Totally, 5 × 10 4 4T1 cells were collected and washed twice with PBS from each group.After treatment, according to the manufacturer's instructions (E1042, APPLYGEN, Beijing), the supernatant was collected, and the absorbance value of 550 nm was detected by the microplate reader and zeroized with a blank hole.

Measurement of lipid ROS
C11-BODIPY (581/591) dye (D3861, Invitrogen) was employed as a probe to assay the lipid ROS level, which is readily oxidized by lipid ROS, leading to a gradual increase in fluorescent intensity.Cells from each experimental group were washed and incubated in 1 mL of PBS containing 0.5 µM C11-BODIPY at 37 °C for 60 min.The fluorescence signal change was evaluated using confocal microscopy.

Flow cytometry analysis
Single-cell suspensions of splenic and inguinal nodes were prepared by perfusing mice with PBS, followed by staining with various antibodies and analyzing using flow cytometry on FACS caliber (Beckman).The eFluor 506 dye (eBioscience) was utilized to exclude dead cells.The stained cells were run on a flow cytometer (Beckman) using the CytExpert software v. 2.3 (Beckman) and then analyzed by FlowJo software (v. 10, TreeStar).

Statistical analysis
Data are represented as the mean ± standard error of the mean (s.e.m.) or standard deviation (s.d.) as indicated in the figure legends.One-way ANOVA with Tukey's multiple comparisons was used for multiple comparisons when more than two groups were compared, and twotailed Student's t-test was used for two-group comparisons.The survival benefit was determined using a log-rank (Mantel-Cox) test.All statistical tests were performed using GraphPad Prism software v. 8.0 (GraphPad Software) and Excel 2016 software (Microsoft).In all types of statistical analysis, values of P < 0.05 were considered significant.

Fig. 1 |
Fig. 1 | Illustration of the mechanism of COF-919-mediated phototherapy.Planar and twisted motifs were integrated into covalent organic frameworks for pyroptosis and ferroptosis-primed cancer immunotherapy.

Fig. 3 |
Fig. 3 | Characterization of the photophysical properties of AIE COFs. a Illustration of the advantages of COF-919 with planar and twisted monomers.b UV/vis light absorption spectra of AIE COFs.Steady-state (c) and time-resolved (d) photoluminescence spectra of AIE COFs.e, f Band gap of COF-818 and COF-919.g Binding energy of COF-818 and COF-919.h Band structure of COF-818 and COF-919.i DFT calculations for COF-818 and COF-919.Source data are provided as a Source Data file.

Fig. 5 |
Fig. 5 | Characterization of the pyroptosis-eliciting capability of COF-818 and COF-919.a illustration of the mechanism of GSDME-dependent pyroptosis induced by COF-818 and COF-919.Quantitative evaluation of the representative pyroptosis factors, ATP (b) and LDH (c) in 4T1 cells after various treatments; data are presented as mean ± SEM (n = 3 independent samples).d Representative images of HMGB1 immunofluorescence staining in 4T1 cells after treatment with different samples.e Evaluate the expression levels of cleaved Caspase-3 and GSDME-N in 4T1 cells through western blot analysis; Western blot was done thrice independently with similar results.f Representative images of 4T1 cell morphology change after various treatments; the photographs of cell morphology were representative of those generated from three independent samples, each group with similar results; scale bar = 50 μm.Statistical significance was calculated via one-way ANOVA with Tukey's multiple comparisons test.Source data are provided as a Source Data file.

Fig. 6 |
Fig. 6 | Characterization of the ferroptosis eliciting capability of COF-818 and COF-919.a Illustration the mechanism of GPX4-related ferroptosis induced by COF-818 and COF-919.b Western blot results of representative pyroptosis factors, including GPX4, TFRC, and xCT in 4T1 cells treated under different conditions.Quantitative evaluation of the changes in cellular iron concentration (c) and GSH/ GSSG ratio (d), treated under different conditions; data are presented as mean ± SEM (n = 3 independent samples).e Confocal fluorescence images of 4T1 cells, where the green fluorescence was attributed to BODIPY 581/591-C11 staining of lipid peroxidation; the photographs of immunofluorescence staining were representative of those generated from three independent samples with similar results.f Bio-TEM images of mitochondria in normal cells or that of COF-919 treatment; the photographs of TEM were representative of those generated from three independent samples with similar results.Statistical significance was calculated via one-way ANOVA with Tukey's multiple comparisons test.Source data are provided as a Source Data file.

Fig. 7 |
Fig. 7 | Investigation of the in vivo therapeutic effect of COF-919-mediated phototherapy.a Treatment schedule of COF-919-mediated phototherapy.Tumor volumes (b), body weights (c), and tumor images (d) of 4T1 tumor-bearing mice treated under different conditions; data are presented as mean ± SEM (n = 5 independent samples).Quantitative evaluation of Ki67(e) and 4-HNE (f); data are presented as mean ± SEM (n = 5 independent samples).g Western blot analysis was performed to determine the GPX4 and GSDME-N expression levels in tumor tissues;Western blot was done thrice independently with similar results.h Illustration of COF-919-mediated phototherapy to promote DC maturation.Quantitative evaluation of DCs in the DLN (m); T CM (j), and T EM (k) cells in the spleen; T CM (n) and T EM (o) cells in DLN; Tregs cells in the spleen (l) and DLN (p); MDSCs in the spleen (i); data are presented as mean ± SEM (n = 5 independent samples).Statistical significance was calculated via two-way ANOVA (b) and one-way ANOVA with Tukey's multiple comparisons test (e, f, i-p).Source data are provided as a Source Data file.