Dynamic Covalent Hydrogel as a Single-dose Vaccine Adjuvant for Sustained Antigen Release and Significantly Elevated Humoral Immunity.

Vaccine is the most important way for fighting against infection diseases. However, multiple injections and unsatisfied immune responses are the main obstacles for current vaccine application. Herein, a dynamic covalent hydrogel (DCH) was used as a single-dose vaccine adjuvant for eliciting robust and sustained humoral immunity. By adjusting the mass ratio of the DCH gel, we successfully realized 10 to 30 days constant release of the loaded recombinant protein antigens, and proved that sustained release of antigens could significantly improve the vaccine efficacy. When loading SARS-CoV-2 RBD (Wuhan and Omicron BA.1 strains) antigens into this DCH gel, an over 32,000 times and 8,000 times improvement was observed in antigen-specific antibody titers compared to conventional Aluminum adjuvanted vaccines. The universality of this DCH gel adjuvant was confirmed in a Nipah G antigen test as well as a H1N1 influenza virus antigen test, with much improved protection of C57BL/6 mice against H1N1 virus infection than conventional Aluminum adjuvanted vaccines. This sustainably released, single-dose DCH gel adjuvant provides a new promising option for designing next-generation infection vaccines. This article is protected by copyright. All rights reserved.

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.

miR-548c-3p targets TRIM22 to attenuate the Peg-IFN-α therapeutic efficacy in HBeAg-positive patients with chronic hepatitis B.

Chronic hepatitis B (CHB) patients treated with interferon shows encouraging results. However, its clinical efficacy is limited by significant individual differences in treatment responses. We identified an interferon-inducible effector, TRIM22, as the likely causal target of such differential responses. We found that TRIM22 was highly expressed in interferon-responsive patients and negatively correlated with HBV DNA and HBeAg serum levels. Stable cells overexpressing TRIM22 carried significantly less HBsAg, HBeAg, and HBV DNA, and cells with knocked-down TRIM22 by shRNA displayed higher levels of these markers than controls. Integrated bioinformatics analysis and subsequent experiments revealed that TRIM22 overexpression significantly increased the supernatant levels of IL-1ß and IL-8, two important cytokines of NOD2/NF-κB pathway involved in interferon-induced antiviral activities. We identified three candidate microRNAs binding to 3'UTR of TRIM22 at various locations through typical imperfect paring using the TargetScan program. MiR-548c-3p appeared to be highly expressed, while the TRIM22 level was low in the suboptimal response group of CHB patients. The Luciferase reporter assay revealed an interaction between miR-548c-3p and the 3'UTR of TRIM22, leading to a controlled suppression of TRIM22 endogenous expression. This resulted in interferon's substantially weakened therapeutic efficacy, as indicated by the elevation of the serum levels of HBsAg, HBeAg and HBV DNA in miR-548c-3p-transfected HepAD38 cells. Our study demonstrated that a particular miR-548c-3p is the key negative regulator of TRIM22 in CHB patients with a weak response to interferon treatment, providing a novel marker and target in interferon-α therapy evaluation.

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.

Functionalized PAMAM constructed nanosystems for biomacromolecule delivery.

Polyamidoamines (PAMAMs) are a class of dendrimer with monodispersity and controlled topology, which can deliver biologically active macromolecules (e.g., genes and proteins) to specific regions with high efficiency and minimum side effects. In detail, PAMAMs can be functionalized easily by core modification or surface amendment to encapsulate a wide range of biomacromolecules. Besides, self-assembled, cross-linked and hybrid PAMAMs with customized therapeutic purposes are developed as delivery vehicles, which makes PAMAMs promising for biomacromolecule therapy. In this review, we comprehensively summarize the application of PAMAMs in biomacromolecule delivery from the synthesis of functionalized PAMAM carriers to the development of PAMAM-based drug delivery systems. The underlying strategies for PAMAM functionalization and assembly are first systematically discussed, and then the current applications of PAMAMs for biomacromolecule delivery are reviewed. Finally, a brief perspective on the further applications of PAMAMs concludes, aiming to provide insights into developing PAMAM-based biomacromolecule delivery systems.

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.

Cationic polymer synergizing with a disulfide-containing enhancer achieved efficient nucleic acid and protein delivery.

To improve the efficiency of nucleic acid and protein delivery by cationic polymers, there is a trade-off between increasing the positive charge density of cationic polymers and decreasing cytotoxicity. In this work, a strategy to introduce multiple interactions between the cell membrane and a delivery system based on cationic polymers was proposed. A novel delivery system consisting of PEI1.8k and an enhancer (LA-RT) was fabricated. The introduction of LA-RT contributed to multiple interactions between the delivery system and the cell membrane including electrostatic interactions, hydrogen bonding, hydrophobic interaction, and dynamic sulfur exchange reactions, which enabled efficient intracellular delivery of nucleic acids and proteins. For nucleic acid delivery, plasmid DNA and mRNA were loaded to realize CRISPR/Cas 9 gene editing in vivo and protein expression in vivo, respectively. For protein delivery, the delivery system carrying OVA protein and CpG formed a nano-vaccine, which induced enhanced humoral and cellular immunity in vivo. In addition, the delivery system based on PEI1.8k revealed negligible cytotoxicity. This work provided a novel strategy to prepare efficient delivery systems based on cationic polymers via the introduction of a multifunctional enhancer.

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.