A Versatile Nanovaccine Enhancement Strategy Based on Suction-Inspired Physical Therapy.

Vaccine technology is effective in preventing and treating diseases, including cancers and viruses. The efficiency of vaccines can be improved by increasing the dosage and frequency of injections, but it would bring an extra burden to people. Therefore, it is necessary to develop vaccine-boosting techniques with negligible side effects. Herein, we reported a cupping-inspired noninvasive suction therapy that could enhance the efficacy of cancer/SARS-CoV-2 nanovaccines. Negative pressure caused mechanical immunogenic cell death and released endogenous adjuvants. This created a subcutaneous niche that would recruit and activate antigen-presenting cells. Based on this universal central mechanism, suction therapy was successfully applied in a variety of nanovaccine models, which include prophylactic/therapeutic tumor nanovaccine, photothermal therapy induced in situ tumor nanovaccine, and SARS-CoV-2 nanovaccine. As a well-established physical therapy method, suction therapy may usher in an era of noninvasive and high-safety auxiliary strategies when combined with vaccines.

Facile Synthesis of High Molecular Weight Poly(ethylene glycol)-b-poly(amino acid)s by Relay Polymerization.

Poly(amino acid)s (PAAs) are one kind of favorable biopolymer that can be used as a drug or gene carrier. However, conventional ring-opening polymerization of PAAs is slow and needs a strict anhydrous environment with an anhydrous reagent as well as the product without enough high molecular weight (Mn), which limits the expanding of PAAs' application. Herein, we took BLG-NCA as the monomer to quickly synthesize one kind of high Mn amphiphilic copolymer, poly(ethylene glycol)-b-poly(γ-benzyl-l-glutamic acid) (PEG-PBLG), by relay polymerization with a simple one-pot method within 3 h in mild conditions (open air, moisture insensitive). In the polymerization process, ring-opening polymerization-induced self-assembly in sodium bicarbonate aqueous solution first occurred to obtain low Mn PEG-PBLG seeds without purification. Then γ-benzyl-l-glutamate N-carboxyanhydride (BLG-NCA) dichloromethane solution was added into PEG-PBLG seeds directly and stirred vigorously to form am emulsion; during this process, the amphiphilic PEG-PBLG seeds will anchor on the interface of DCM and water to ensure the concentration of α-helix rigid PBLG in DCM to maintain the following relay polymerization. Then, high Mn PEG-PBLG was obtained in mild conditions in one pot. We found that the α-helix rigid structure was essential for relay polymerization by studying the synthetic speed of amphiphilic copolymer with different secondary structures. MOE simulation results showed that PBLG and BLG-NCA tended to form a double hydrogen bond, which was beneficial to relay polymerization because of higher local concentrations that can produce more double hydrogen bonds. Our strategy can quickly obtain high Mn PEG-PBLG (224.9 KDa) within 3 h from PEG-NH2 and BLG-NCA in one pot and did not need an extra initiator. After deprotection, the poly(ethylene glycol)-b-poly(l-glutamate acid) (PEG-PGA) with high Mn as a second product can be used as an excellent antitumor drug carrier. The high Mn PEG-PGA can achieve an encapsulation rate of 86.7% and a drug loading rate of 47.3%, which is twice that of the low Mn PEG-PGA. As a result, the synthesis of PEG-PBLG by relay polymerization simplified the process of PEG-PAA polymerization and increased the Mn. In addition, this method opened a way to obtain other kinds of high Mn PEG-PBLG values in the future.

Co-delivery of dimeric camptothecin and chlorin e6 via polypeptide-based micelles for chemo-photodynamic synergistic therapy.

BACKGROUND: The integration of photodynamic therapy with a chemical drug-delivery system has displayed great potential in enhancing anticancer therapy. However, the solubility and non-specific biodistribution of both chemotherapeutic agents and photosensitizers continue to pose challenges that hinder their clinical applications. METHOD: A polypeptide-based nanoscale drug delivery system was fabricated to address the prementioned issues. An amphiphilic polymer was formed by conjugating the photosensitizer chlorin e6 (Ce6) onto a polypeptide poly-(L-lysine)-b-polyphenylalanine (PKF) for encapsulating the model drug dimeric camptothecin (DCPT), and the nanoparticles (PCD) with high drug loading efficiency were further modified with acid-sensitive polyethylene glycol (PEG) to yield the drug delivery sytem (PPCD). RESULTS: The DCPT and Ce6 encapsulation efficiency were analyzed as 99% and 73.5%, respectively. In phosphate-buffered saline (PBS) solution at a pH of 7.4, the PEG shell improved the stability of micelles and shielded their positive charge while in the acidic tumor microenvironment, the pH-sensitive PEG layer was removed to expose the cationic nanoparticles, thus facilitating the cellular uptake of PPCD micelles. Benefiting from the enhanced cellular internalization, the amount of intracellular reactive oxygen species (ROS) treated with PCD and PPCD micelles were obviously increased. Furthermore, the enhanced anti-cancer efficacy prompted by PPCD micelles was validated through cellular and animal study. CONCLUSION: This study presents a promising method to promote the solubility and biodistribution of both chemotherapeutic agent and photosensitizer, thereby facilitating the further application of chemo-photodynamic cancer therapy.

Biodegradable covalent organic frameworks achieving tumor micro-environment responsive drug release and antitumor treatment.

The emergence of nanocarriers has greatly improved the therapeutic efficacy of chemotherapeutic drugs. As emerging nanocarriers, covalent organic frameworks (COFs) have been increasingly used in biomedicine in recent years. However, due to their inherent chemical stability, existing COF nanocarriers hardly undergo in vivo degradation, which brings potential safety hazards to further applications. In this work, we introduce the azo bond into COFs. When the nanocarrier enters the cell, ˙OH generated by the coordinated Fe response to the H2O2 in the cell will break the azo bond and cause the degradation of the framework structure, accelerating the release of internally loaded DOX to effectively realize tumor treatment. We verified the degradation ability of the materials by constructing model compounds, in vitro drug release, MTT assay and antitumor experiments. Compared with the control groups, the degradable COF accelerates the release of DOX and shows a stronger killing effect on 4T1 cells. Serum biochemical analysis and H&E sections of organs show good biocompatibility for both COFs and degradation products. This work provides a new idea for the design of biodegradable COFs in vivo, and greatly explores the potential application of COF materials in the biomedical field.

Gene-guided OX40L anchoring to tumor cells for synergetic tumor "self-killing" immunotherapy.

The low objective response rates and severe side effects largely limit the clinical outcomes of immune checkpoint blockade (ICB) therapy. Here, a tumor "self-killing" therapy based on gene-guided OX40L anchoring to tumor cell membrane was reported to boost ICB therapy. We developed a highly efficient delivery system HA/PEI-KT (HKT) to co-deliver the OX40L plasmids and unmethylated CG-enriched oligodeoxynucleotide (CpG). On the one hand, CpG induced the expression of OX40 on T cells within tumors. On the other hand, OX40L plasmids achieved the OX40L anchoring on the tumor cell membrane to next promote T cells responses via OX40/OX40L axis. Such synergistic tumor "self-killing" strategy finally turned "cold" tumors to "hot", to sensitize tumors to programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) blockade therapy, and promoted an immune-mediated tumor regression in both B16F10 and 4T1 tumor models, with prevention of tumor recurrence and metastasis. To avoid the side effects, the gene-guided OX40L anchoring and PD-L1 silencing was proposed to replace the existing antibody therapy, which showed negligible toxicity in vivo. Our work provided a new possibility for tumor "self-killing" immunotherapy to treated various solid tumors.

Linear-like polypeptide-based micelle with pH-sensitive detachable PEG to deliver dimeric camptothecin for cancer therapy.

Nano drug delivery systems have made significant progress in delivering anticancer drugs camptothecin (CPT). However, many challenges for CPT delivery remain, including low drug loading efficiency, premature drug leakage, and poor cellular internalization. Herein, we report a novel dual-sensitive polypeptide-based micelle with remarkably high drug loading of CPT for cancer therapy. This self-assembled micelle possesses the following essential components for CPT: (1) pH-sensitive PEG (OHC-PEG-CHO) for prolonging blood circulation and allowing biocompatibility by shielding the cationic micelles, which can be detached under the tumor acidic microenvironment and facilitates the cellular uptake; (2) polypeptide polylysine-polyphenylalanine (PKF) synthesized via ring-opening polymerization for micelle formation and CPT analogue loading; (3) dimeric CPT (DCPT) with redox-sensitive linker for increasing CPT loading and ensuring drug release at tumor sites. Interestingly, the linear-like morphology of PEG-PKF/DCPT micelles was able to enhance their cellular internalization when compared with the spherical blank PKF micelles. Also, the anticancer efficacy of DCPT against lung cancer cells was significantly improved by the micelle formation. In conclusion, this work provides a promising strategy facilitating the safety and effective application of CPT in cancer therapy.

In Situ Reprogramming of Tumors for Activating the OX40/OX40 Ligand Checkpoint Pathway and Boosting Antitumor Immunity.

OX40 (CD134, TNFRSF4) is a member of the tumor necrosis factor receptor superfamily that can be activated by its cognate ligand OX40L (CD252, TNFSF4) and functions as a pair of T cell costimulatory molecules. The interaction between OX40 and OX40L (OX40/OX40L) plays a critical role in regulating antitumor immunity, including promoting effector T cells expansion and survival, blocking natural regulatory T cells (Treg) activity, and antagonizing inducible Treg generation. However, current OX40 agonists including anti-OX40 monoclonal antibodies (aOX40) have serious side effects after systemic administration, which limits their clinical success and application. Herein, we propose a strategy to reprogram tumor cells into OX40L-expressing "artificial" antigen-presenting cells (APCs) by OX40L plasmid-loaded nanoparticles for boosting antitumor immunity in situ. A novel gene transfection carrier was prepared by a modular hierarchical assembly method, which could efficiently transfect various tumor cells and express OX40L proteins on their surface. These surface-decorated OX40L proteins were proved to stimulate T cell proliferation in vitro while stimulating strong antitumor immune responses in vivo. Importantly, this in situ reprogramming strategy did not induce any toxicity as observed in aOX40 treatment, thus providing a novel method for immune checkpoint stimulator application.

Etching Bulk Covalent Organic Frameworks into Nanoparticles of Uniform and Controllable Size by the Molecular Exchange Etching Method for Sonodynamic and Immune Combination Antitumor Therapy.

To improve the therapeutic effect of sonodynamic therapy (SDT), more effective and stable sonosensitizers and therapeutic strategies are still required. A covalent organic framework (COF) sonosensitizer is developed by using a new nanoscale COF preparation strategy. This strategy uses molecular etching based on the imine exchange reaction to etch the bulk COF into nanoparticles and has universal applicability to imine-bond-based COF. The regular COF structure can prevent the loss of sonodynamic performance caused by the aggregation of porphyrin molecules and improve the chemical stability of the porphyrin unit. In addition, the coordination of Fe3+ to COF endows the nanoparticle with chemodynamic therapy performance and glutathione consumption ability. The combination of enhanced SDT and α-PD-L1 antibody achieves a good antitumor effect. The innovative nanoscale COF sonosensitizer preparation strategy provides a new avenue for clinical antitumor therapy.

Novel Cocktail Therapy Based on a Nanocarrier with an Efficient Transcytosis Property Reverses the Dynamically Deteriorating Tumor Microenvironment for Enhanced Immunotherapy.

The immune checkpoint blockade (ICB) faces a low response rate in clinical cancer treatment. Chemotherapy could enhance the response rate of the ICB, but patients would suffer from side effects. The off-target toxicity could be reduced by loading the chemotherapeutic agent through nanocarriers. Therefore, we developed a polymeric carrier for doxorubicin (DOX) loading to form DOX nanoparticles (DOX NPs), which were spatiotemporally responsive to the tumor microenvironment (TME). DOX NPs had an efficient transcytosis property for deep tumor infiltration and sustained drug release ability. Unfortunately, a binary therapy of DOX NPs and ICB induces tumor adaptive resistance and causes dynamic deterioration of the TME. We propose for the first time that TGF-ß1 is a major cause of tumor adaptive resistance and developed an immune cocktail therapy containing DOX NPs, ICB, and TGF-ß1 gene silencing nanoparticles. This therapy successfully overcame tumor adaptive resistance by reversing the immunosuppressive TME and achieved enhanced tumor treatment efficiency.

Reactive oxygen species activated by mitochondria-specific camptothecin prodrug for enhanced chemotherapy.

Camptothecin (CPT) has attracted much attention due to its potent antitumor activities. However, the undesirable physicochemical properties, including poor water-solubility, unstable lactone ring and severe adverse effects limit its further application. In this study, two water-soluble prodrugs, CPT-lysine (CPTK) and CPT-arginine (CPTR), were designed and synthesized by conjugating lysine or arginine with CPT, improving its solubility, pharmacokinetic properties and tumor penetration. Importantly, the introduction of arginine into CPTR contributed to the mitochondria-specific delivery, which increased mitochondrial reactive oxygen species (ROS) generation, induced mitochondria dysfunction and enhanced cell apoptosis and in vivo anti-cancer effect. This strategy is believed to hold great potential for organelle-specific synergistic anti-tumor therapy.

Synthetic Helical Polypeptide as a Gene Transfection Enhancer.

The relatively low transfection efficiency limits further application of polymeric gene carriers. It is imperative to exploit a universal and simple strategy to enhance the gene transfection efficiency of polymeric gene carriers. Herein, we prepared a cationic polypeptide poly(γ-aminoethylthiopropyl-l-glutamate) (PALG-MEA, termed PM) with a stable α-helical conformation, which can significantly improve the gene transfection efficiency of cationic polymers. PM can be integrated into polymeric gene delivery systems noncovalently through electrostatic interactions. With the assistance of PM, polymeric gene delivery systems exhibited excellent cellular uptake and endosomal escape, thereby enhancing transfection efficiency. The transfection enhancement effect of PM was applicable to a variety of cationic polymers such as polyethylenimine (PEI), poly-l-lysine (PLL), and polyamidoamine (PAMAM). The ternary gene delivery system PM/pshVEGF/PEI exhibited an excellent antitumor effect against the B16F10 tumor model. Moreover, we demonstrated that PM could also enhance the delivery of gene editing systems (sgRNA-Cas9 plasmids). This work provides a facile and effective strategy for constructing polymeric gene delivery systems with a high transfection efficiency.

Combination of epigenetic regulation with gene therapy-mediated immune checkpoint blockade induces anti-tumour effects and immune response in vivo.

Immunotherapy has become a powerful cancer treatment, but only a small fraction of patients have achieved durable benefits due to the immune escape mechanism. In this study, epigenetic regulation is combined with gene therapy-mediated immune checkpoint blockade to relieve this immune escape mechanism. PPD (i.e., mPEG-b-PLG/PEI-RT3/DNA) is developed to mediate plasmid-encoding shPD-L1 delivery by introducing multiple interactions (i.e., electrostatic, hydrogen bonding, and hydrophobic interactions) and polyproline II (PPII)-helix conformation, which downregulates PD-L1 expression on tumour cells to relieve the immunosuppression of T cells. Zebularine (abbreviated as Zeb), a DNA methyltransferase inhibitor (DNMTi), is used for the epigenetic regulation of the tumour immune microenvironment, thus inducing DC maturation and MHC I molecule expression to enhance antigen presentation. PPD plus Zeb combination therapy initiates a systemic anti-tumour immune response and effectively prevents tumour relapse and metastasis by generating durable immune memory. This strategy provides a scheme for tumour treatment and the inhibition of relapse and metastasis.

Prodrug-Based Versatile Nanomedicine with Simultaneous Physical and Physiological Tumor Penetration for Enhanced Cancer Chemo-Immunotherapy.

Chemo-immunotherapy combination effect remains to be a great challenge due to the poor tumor penetration of therapeutic agents that resulted from condensed extracellular matrix (ECM), T cell-related immune escape, and thus the potential recurrence. Herein, a helix self-assembly camptothecin (CPT) prodrug with simultaneous physical and physiological tumor penetration was constructed to realize effective chemo-immunotherapy. Specifically, CPT was modified with arginine to self-assemble into nanofibers to physically improve tumor penetration. Two plasmids, pshPD-L1 and pSpam1 for expressing small hairpin RNA PD-L1 and hyaluronidase, respectively, were loaded to down-regulate tumor surface PD-L1 expression for converting anergic state of T cells into the tumor-reactive T cells and produce hyaluronidase to physiologically degrade ECM for further enhanced tumor penetration. Moreover, the degraded ECM could also increase immune cells' infiltration into tumor sites, which may exert a synergistic antitumor immunity combined with immune checkpoint inhibition. Such a nanomedicine could cause significant inhibition of primary, distant tumors, and effective prevention of tumor recurrence.

Cationic Flexible Organic Framework for Combination of Photodynamic Therapy and Genetic Immunotherapy Against Tumors.

Photodynamic therapy is a new type of anti-tumor therapy with excellent therapeutic effects and minor side effects. The key factor for photodynamic therapy is highly efficient loading and protection of photosensitizers. Covalent organic framework is a new type of organic porous material with rich sources and has huge development potential in the loading of photosensitizers. However, the π-π interaction between the rigid monomers inevitably causes aggregation and quenching between photosensitizers, which in turn affects the rate of reactive oxygen production. Here, newly designed cationic flexible organic framework nanoparticles (PEI-Por NPs) are synthesized via one-step method with PEI25K and meso-tetra(p-formylphenyl)porphyrin under microwave irradiation. The structure of the flexible organic framework can effectively inhibit the aggregation and quenching of porphyrin. In addition, PEI-Por NPs had excellent gene transfection ability both in vitro and in vivo. Excellent antitumor effect can be achieved by combining PEI-Por NPs' photodynamic therapy capacity and PEI-Por NPs-mediated PD-L1 gene silencing with the guidance of fluorescence imaging and photoacoustic imaging. This cationic flexible organic framework material combines the advantages of flexible building units and rigid monomers, which provides a basis for the development of nano-photosensitizers and excellent gene carriers, and has great potential for clinical application.

Synthesis of Copolymers Polyethyleneimine-co-Polyphenylalanine as Gene and Drug Codelivery Carrier.

In this study, a series of hyperbranched copolymers polyethyleneimine-co-polyphenylalanine (PEI-co-PPhe) are synthesized by ring-opening polymerization with phenylalanine-N-carboxyanhydride as monomer and PEI-25k as initiator, using as a gene and drug codelivery carrier. Among them, PEI-co-PPhe (1:170) is selected out from transfection efficiency and cytotoxicity tests. Then, doxorubicin-cis-aconitic anhydride (CAD) and BCl2-shRNA (as a therapeutic gene) are coloaded into the PEI-co-PPhe carrier to form PEI-co-PPhe/Bcl2-shRNA/CAD complexes as a codeliver system. When the mass ratio of PEI-co-PPhe:Bcl2-shRNA:CAD is 5:1:1, the codeliver system has the most obvious synergistic therapeutic effect against B16F10 cells. Confirmed by confocal laser scanning microscope and flow cytometry, compared with drug and gene alone, the codeliver complexes can be endocytosed into B16F10 cells efficiently. As a result, the appropriate length of PPhe grafted on PEI will improve the gene transfer efficiency and decrease cytotoxicity, as well as effective codelivery of gene and drug into cancer cells to be a promising codelivery carrier for cancer therapy.

An immune cocktail therapy to realize multiple boosting of the cancer-immunity cycle by combination of drug/gene delivery nanoparticles.

Immune checkpoint blockade therapy (ICT) has shown potential in the treatment of multiple tumors, but suffers poor response rate in clinic. We found that even combining ICT with chemotherapy, which was wildly used in clinical trials, failed to achieve satisfactory tumor inhibition in the B16F10 model. Thus, we further constructed a previously unexplored immune cocktail therapy and realized multiple boosting of the cancer-immunity cycle. Cocktail therapy consisted of two kinds of tumor microenvironment-responsive drug and gene delivery nanoparticles to achieve specific delivery of doxorubicin and codelivery of plasmids expressed small hairpin RNA of PD-L1 (pshPD-L1) and hyaluronidase (pSpam1) in the tumor area. Experimental evidences proved that any component in the cocktail therapy was indispensable, and the cocktail therapy exhibited excellent antitumor effects against different types of tumors. The cocktail therapy presented here offers a searching strategy for more synergistic units with ICT and is meaningful for developing more efficient antitumor immunotherapy.

Highly Enhanced Antitumor Immunity by a Three-Barreled Strategy of the l-Arginine-Promoted Nanovaccine and Gene-Mediated PD-L1 Blockade.

Weak T cell responses and immune checkpoints within tumors could be two key factors for limiting antitumor efficacy in the field of cancer immunotherapy. Thus, the combined strategy of tumor vaccines and immune checkpoint blockade has been widely studied and expected to boost antitumor immune responses. Herein, we first developed a two-barreled strategy to combine the nanovaccine with a gene-mediated PD-L1 blockade. On the one hand, polyethyleneimine (PEI) worked as a vaccine carrier to codeliver the antigen ovalbumin (OVA) and the adjuvant unmethylated cytosine-phosphate-guanine (CpG) to formulate the PEI/OVA/CpG nanovaccine through electrostatic binding, which realized both dendritic cell activation and antigen cross-presentation enhancement. On the other hand, the PD-L1 silence gene was loaded by PEI to form PEI/pshPD-L1 complexes, which were further in situ shielded by aldehyde-modified polyethylene glycol (OHC-PEG-CHO) via pH-responsive Schiff base bonds. The formed pshPD-L1@NPs could decrease PD-L1 expression on the tumor cells. However, such a combined two-barreled strategy improved feebly for tumor inhibition in comparison with monotherapy, exhibiting the antagonistic effect, which might be due to the limited T cell response enhancement in the tumor microenvironment. To solve this problem, we have further developed a three-barreled strategy to combine oral administration of l-arginine, which worked as an amplifier to induce robust T cell response enhancement, without causing the upregulation of other negative immune regulators. Superior antitumor behavior and tumor rechallenge protection were realized by the three-barreled strategy in B16F10-OVA (B16-OVA)-bearing mice. The unique three-barreled strategy we developed might offer a novel clinical therapeutic treatment.

Poly(l-glutamic acid)-Based Zwitterionic Polymer in a Charge Conversional Shielding System for Gene Therapy of Malignant Tumors.

Recently, pH-sensitive polymers have received extensive attention in tumor therapy. However, the rapid response to pH changes is the key to achieving efficient treatment. Here, a novel shielding system with a rapidly pH-responsive polymer (PAMT) is synthesized by click reaction between poly(γ-allyl-l-glutamate) and thioglycolic acid or 2-(Boc-amino)ethanethiol. The zwitterionic biodegradable polymer PAMT, which is negatively charged at physiological pH, can be used to shield positively charged nanoparticles. PAMT is electrostatically attached to the surface of the positively charged PEI/pDNA complex to form a ternary complex. The zwitterionic PAMT-shielded complex exhibits rapid charge conversion when the pH decreases from 7.4 to 6.8. For the in vivo tumor inhibition experiment, PAMT/PEI/shVEGF injected intravenously shows a more significant inhibitory effect on tumor growth. The excellent results are mainly attributed to introduction of the zwitterionic copolymer PAMT, which can shield the positively charged PEI/shVEGF complex in physiological conditions, while the surface potential of the shielded complexes changes to a positive charge in the acidic tumor environment.

Helix Self-Assembly Behavior of Amino Acid-Modified Camptothecin Prodrugs and Its Antitumor Effect.

For effective antitumor treatment, it is important to increase the water solubility of hydrophobic antitumor drugs and improve their cell absorption efficiency and nuclear transmission capacity. Here, we use endogenous hydrophilic arginine to modify camptothecin (CPT) to increase its water solubility. Surprisingly, the modified CPT can self-assemble into helical nanofibers through intermolecular π-π stacking and hydrophilic-hydrophobic interactions. Prodrug-based nanofibers were better endocytosed into the nucleus than their nonassembled CPT. Moreover, in vivo, such nanofibers had a longer blood circulation time and a better ability to accumulate in the tumor site. Further, we found that the cationic nanofibers can be combined with the anionic cisplatin-polyglutamic acid through electrostatic interaction to achieve a combined antitumor effect. This provides a new idea for achieving more effective cancer chemotherapy effects.

Porphyrin-based covalent organic framework nanoparticles for photoacoustic imaging-guided photodynamic and photothermal combination cancer therapy.

Combination of photodynamic therapy (PDT) and photothermal therapy (PTT) generally requires different components to build a composite irradiated with different excitation lights. One component photoactive agent for enhanced combination of PDT and PTT under the excitation of a single wavelength light source is more urgent in tumor phototherapy via adjusting spatial arrangement of photoactive units. Herein, porphyrin-based covalent organic framework nanoparticles (COF-366 NPs) were synthesized to control the orderly spatial arrangement of the photoactive building units and firstly used for antitumor therapy in vivo. COF-366 NPs provide the simultaneous therapy of PDT and PTT under a single wavelength light source with the monitoring of photoacoustic (PA) imaging, which makes the operation simpler and more convenient. COF-366 NPs had achieved good phototherapy effect even in the face of large tumors. The prepared multifunctional COF-366 NPs open up a new avenue to phototherapeutic materials and expand the application range of covalent organic framework.