Modulation of Intracellular Oxygen Pressure by Dual‐Drug Nanoparticles to Enhance Photodynamic Therapy

Oxygen plays an essential role in the photodynamic therapy (PDT) of cancer. However, hypoxia inside tumors severely attenuates the therapeutic effect of PDT. To address this issue, a novel strategy is reported for cutting off the oxygen consumption pathway by using sub‐50 nm dual‐drug nanoparticles (NPs) to attenuate the hypoxia‐induced resistance to PDT and to enhance PDT efficiency. Specifically, dual‐drug NPs that encapsulate photosensitizer (PS) verteporfin (VER) and oxygen‐regulator atovaquone (ATO) with sub‐50 nm diameters can penetrate deep into the interior regions of tumors and effectively deliver dual‐drug into tumor tissues. Then, ATO released from NPs efficiently reduce in advance cellular oxygen consumption by inhibition of mitochondria respiratory chain and further heighten VER to generate greater amounts of ¹O₂ in hypoxic tumor. As a result, accompanied with the upregulated oxygen content in tumor cells and laser irradiation, the dual‐drug NPs exhibit powerful and overall antitumor PDT effects both in vitro and in vivo, and even tumor elimination. This study presents a potential appealing clinical strategy in photodynamic eradication of tumors.     

In Situ Formed Fibrin Scaffold with Cyclophosphamide to Synergize with Immune Checkpoint Blockade for Inhibition of Cancer Recurrence after Surgery

Unsatisfied cytoreductive surgery predicts worse clinical outcomes. Previous studies have found that cyclophosphamide (CTX) is a rhythmic immune modulator that can target suppressive regulatory immune cells and meanwhile enhance effector cells. Here, a therapeutic scaffold is engineered based on a fibrin hydrogel to codeliver CTX and anti‐PD‐L1 antibody (aPDL1) for the prevention of cancer recurrence postsurgery. It is demonstrated that the sequential release of CTX and aPDL1 from the fibrin hydrogel can lead to selective depletion of regulatory T cells (Treg) in the residual tumor, which would then synergize the immune checkpoint blockade therapy. The therapeutic benefit is demonstrated in an orthotopic breast tumor and an orthotopic ovarian tumor model after incomplete resection of primary tumors. In this work, the strategy provides a clinically valuable option for preventing cancer recurrence postsurgery.     

Persistent Luminescence Immune Hydrogel for Photodynamic-Immunotherapy of Tumors In Vivo

Persistent luminescence material (PLM)-based photodynamic therapy (PDT) has shown tremendous promise in tumor elimination via avoiding continuous external light illumination. In addition, the tumor-associated antigens produced by PDT can trigger systemic antitumor immune responses, but only exhibit a limited immunotherapy effect. Herein, a persistent luminescence immune hydrogel is developed via a “turning solid into gel” strategy by introducing a PLM and an immunoadjuvant (R837) into an alginate-Ca²⁺ hydrogel for rechargeable photodynamic-immunotherapy of tumors, for the first time. The designed PLM-R837-ALG hydrogel exhibits the intact persistent luminescence of the PLM, 100% of utilization efficiency of the hydrophobic precursors, good biocompatibility and syringeability, and can be easily injected into tumors to serve as an internal light source for efficiently activating photosensitizers to induce a sustained PDT effect. Moreover, the loaded R837 can significantly amplify the immunogenicity of tumor-associated antigens originating from PL sensitized PDT, thereby leading to a powerful immune response to suppress tumors in vivo. The proposed PL-based photodynamic-immunotherapy provides a novel combined tumor treatment paradigm.     

Fluorination-Induced Multi-Enhancement of a Nano-Photosensitizer for Deep Photodynamic Therapy against Hypoxia Tumor

Organic dyes hold great promise for application in photodynamic therapy (PDT). However, they currently face challenges such as inadequate photodynamic activity, limited tumor penetration, and constraints imposed by tumor hypoxia. Here, a facile and efficient strategy is presented for multi-enhanced PDT through the fluorination of a squarylium indocyanine dye-based photosensitizer (FCy). The amphiphilic FCy features perfluorooctane and PEG-biotin moieties conjugated to a squarylium indocyanine core. In aqueous environments, FCy spontaneously self-assembles into stable nano-sized photosensitizers (FCy NPs), demonstrating a high oxygen loading ability attributable to the presence of perfluoroalkyl groups. Consequently, the aggregation of squarylium indocyanine dyes remarkably boosts the photodynamic effect, yielding a 15-fold improvement in singlet oxygen quantum yield. Owing to the perfluoroalkyl group, FCy NPs exhibit increased endoplasmic reticulum (ER)- accumulating abilities, which further induce ER stress upon laser irradiation and enhance the PDT effect. Furthermore, the superior deep tumor penetration ability of FCy NPs is confirmed through both in vitro and in vivo studies. With efficient oxygen supply to the deep tumor regions, FCy NPs demonstrate potent imaging-guided PDT against hypoxia tumors. The study substantiates the enhanced ER-accumulating ability of the perfluoroalkyl group and presents a facile fluorination strategy for the multi-enhancement of photosensitizers.     

Polymer‐Coated Radioluminescent Nanoparticles for Quantitative Imaging of Drug Delivery

Some theranostic nanoparticle (NP) drug delivery systems are capable of measuring drug release rates in situ. This can provide quantitative information regarding drug biodistribution, and drug dose that is delivered to cells or tissues. Here, X‐ray excited optical luminescent (XEOL) NPs coated with poly(glycolide)‐poly(ethylene glycol) (XGP) are used measure the amount of drug released into cells. The photoactive drug protoporphyrin IX (PpIX) is loaded into XGP and is able to attenuate the XEOL NP emission. Measuring an increase in XEOL intensity as PpIX is released enables the measurement of drug release into glioblastoma cells (GBM). Biodistribution studies in a BALB/c mouse GBM intracranial xenograft model show significant XGP accumulation at the site of the GBM xenograft within the brain, and not in adjacent healthy brain tissues. There is no uptake of XGP in the heart or kidneys, the primary organs associated with drug and gadolinium ion toxicity. NP toxicity is tested with U‐138MG GBM in vitro, and NPs show low cytotoxicity at concentrations of 100 μg/mL. In vivo dose escalation studies in BALB/c mice show no adverse effects at doses up to 75 mg/kg. These theranostic NPs offer an approach to quantitatively measure drug release into cells.     

siRNA Nanoparticle Suppresses Drug-Resistant Gene and Prolongs Survival in an Orthotopic Glioblastoma Xenograft Mouse Model

Temozolomide (TMZ) is the standard-of-care chemotherapy drug for treating glioblastomas (GBMs), the most aggressive cancer that affects people of all ages. However, its therapeutic efficacy is limited by the drug-resistance mediated by a DNA repair protein, O⁶-methylguanine-DNA methyltransferase (MGMT), which eliminates the TMZ-induced DNA lesions. Here, the development of an iron oxide nanoparticle (NP) system for targeted delivery of small interfering RNAs to suppress the TMZ-resistance gene (MGMT) is reported. The NPs are able to overcome biological barriers, bind specifically to tumor cells, and reduce MGMT expression in tumors of mice bearing orthotopic GBM serially passaged patient-derived xenografts. The treatment with sequential administration of this NP and TMZ result in increased apoptosis of GBM stem-like cells, reduced tumor growth, and significantly prolonged survival as compared to mice treated with TMZ alone. This study introduces an approach that holds great promise to improve the outcomes of GBM patients.     

光动力疗法对Louis肺癌鼠的杀伤及免疫效应

应用光动力疗法对 30只接种Louis肺癌瘤株的昆明小鼠作杀伤实验研究。对实验组与对照组荷瘤鼠抑瘤曲线、抑瘤率及免疫学指标进行检测。结果表明 ,两组肿瘤生长曲线存在明显差异 (P<0 0 5 )。两组肿瘤生长体积和肿瘤重量均存在明显差异 (P <0 0 1)。实验组瘤体积抑制率为 5 2 94 % ,瘤重量抑制率为 37 2 4 %。各免疫指标与对照组差异有显著性。说明光动力疗法对肿瘤细胞具有杀伤和抑制生长作用 ,并且对荷瘤小鼠的免疫功能有调节作用     

Two-Photon Absorption Induced Cancer Immunotherapy Using Covalent Organic Frameworks

Immune checkpoint blockade therapy is revolutionizing the traditional treatment model of multiple tumor types, but remains ineffective for a large subset of patients. Photodynamic therapy (PDT) has been shown to induce cancer cell death and provoke an immune response, and may represent a potential strategy to synergize with immune checkpoint blockade therapy. However, the limited tissue penetration of exciting light for conventional PDT largely hinders its application in the clinic and its further combination with immunotherapy. Here, a serrated packing covalent organic framework (COF), COF-606, with excellent two-photon absorption (2PA) property and photostability, largely avoids aggregation-caused quenching, therefore offering high reactive oxygen species (ROS) generation efficiency; it is used as a 2PA photosensitizer for PDT in deep tumor tissue. COF-606 induced PDT is shown to be efficient in inducing immunogenic cell death, provoking an immune response and normalizing the immunosuppressive status for the first time. This makes it possible to combine 2PA induced PDT using COF with programmed cell death protein 1 immune checkpoint blockade therapy. Such combination leads to strong abscopal tumor-inhibiting efficiency and long-lasting immune memory effects, standing as a promising combinatorial therapeutic strategy for cancer treatment.     

5-氨基酮戊酸光动力治疗小鼠皮肤乳头状瘤的实验研究

目的:研究5-氨基酮戊酸光动力学疗法(ALA-PDT)对小鼠皮肤乳头状瘤的治疗作用。方法:二阶段使用二甲基苯蒽/巴豆油诱发小鼠皮肤产生乳头状瘤,在肿瘤表面局部应用不同剂量的5-氨基酮戊酸溶液(10%、20%、30%),3小时后用波长为632.8nm的氦氖激光照射肿瘤10min,照射剂量为90J/cm2。辐照后,每天观察肿瘤变化情况,每周测量肿瘤体积三次,计算相对肿瘤增殖率T/C(%)。记录肿瘤消失(瘤体脱落)数及肿瘤复发情况。并设单用激光、单用药物、阴性对照及阳性对照组。结果:ALA低、中、高三个剂量光动力治疗组和阳性对照组均可使肿瘤显著缩小,到治疗后第14天,相对肿瘤增殖率分别为4.8%、3.4%、0。ALA高剂量光动力治疗组小鼠瘤体全部脱落,继续观察四周未见复发。药物对照组、激光对照组对肿瘤无作用。结论:ALA-PDT对小鼠皮肤乳头状瘤生长有显著的抑制作用,其抑制作用与ALA剂量明显相关。     

FAP-Targeted Photodynamic Therapy Mediated by Ferritin Nanoparticles Elicits an Immune Response against Cancer Cells and Cancer Associated Fibroblasts

Cancer-associated fibroblasts (CAFs) are present in many types of tumors and play a pivotal role in tumor progression and immunosuppression. Fibroblast-activation protein (FAP), which is overexpressed on CAFs, has been indicated as a universal tumor target. However, FAP expression is not restricted to tumors, and systemic treatment against FAP often causes severe side effects. To solve this problem, a photodynamic therapy (PDT) approach is developed based on ZnF₁₆Pc-loaded and FAP-specific single chain variable fragment (scFv)-conjugated apoferritin nanoparticles, or αFAP-Z@FRT. αFAP-Z@FRT PDT efficiently eradicates CAFs in tumors without inducing systemic toxicity. When tested in murine 4T1 models, the treatment elicits anti-cancer immunity, causing suppression of both primary and distant tumors, that is, the abscopal effect. Treatment efficacy is enhanced when αFAP-Z@FRT PDT is used in combination with anti-PD1 antibodies. Interestingly, it is found that the PDT treatment not only elicits a cellular immunity against cancer cells, but also stimulates an anti-CAFs immunity. This is supported by an adoptive cell transfer study, where T cells taken from 4T1-tumor-bearing animals treated with αFAP PDT retard the growth of A549 tumors established on nude mice. Overall, this approach is unique for permitting site-specific eradication of CAFs and inducing a broad spectrum anti-cancer immunity.     

Ferric Ion-Driven Chlorin E6 Nanoparticles: Simple and Effective Multimodal Theranostics

Theranostics integrating therapeutic power and imaging exhibit great prospects in precision medicine. It is a big challenge to develop simple and selective theranostic systems for potential clinical translation. Herein the Fe (III) driven assembly of chlorin E6 (Ce6) is reported to form multifunctional nanoparticles. The resulting Fe-Ce6 NPs possess high loading content of photosensitizer Ce6, red-shifted absorption, good colloidal stability, and photostability, and robust photothermal performance. After cellular internalization, Fe-Ce6 NPs can respond to intracellularly enriched glutathione to release Ce6, thus priming photodynamic therapy (PDT) effect, fluorescence, and afterglow luminescence. In vitro and in vivo results prove that Fe-Ce6 NPs remarkably enhance the tumor regression through photothermal therapy and in situ PDT. This study provides a facile and efficient method to construct versatile nanotheranostics for tumor treatment.     

Versatile Prodrug Nanoparticles for Acid‐Triggered Precise Imaging and Organelle‐Specific Combination Cancer Therapy

Integration of chemotherapy with photodynamic therapy (PDT) has been emerging as a novel strategy for treatment of triple negative breast cancer (TNBC). However, the clinical translation of this approach is hindered by the unwanted dark toxicity due to the “always‐on” model and low tumor specificity of currently approved photosensitizer (PS). Here, the design of a multifunctional prodrug nanoparticle (NP) is described for precise imaging and organelle‐specific combination cancer therapy. The prodrug NP is composed of a newly synthesized oxaliplatin prodrug, hexadecyl‐oxaliplatin‐trimethyleneamine (HOT), an acid‐activatable PS, derivative of Chlorin e6 (AC), and functionalized with a targeting ligand iRGD for tumor homing and penetration. HOT displays much higher antitumor efficiency than oxaliplatin by simultaneously inducing mitochondria depolarizing and DNA cross‐linking. AC is specifically activated in the orthotopic or metastatic TNBC tumor for fluorescence imaging and PDT, while it remains inert in blood circulation to minimize the dark toxicity. Under the guide of acid‐activatable fluorescence imaging, PDT and chemotherapy can be synergistically performed for highly efficient regression of TNBC. Taken together, this versatile prodrug nanoplatform could achieve tumor‐specific imaging and organelle‐specific combination therapy, which can provide an alternative option for cancer theranostic.     

Metformin Mediated PD-L1 Downregulation in Combination with Photodynamic-Immunotherapy for Treatment of Breast Cancer

Intelligent nanomaterials open up new avenues for realizing safer and more effective combination immunotherapy. Herein, a kind of simple enzymatically cleavable self-delivery nanoparticles (MA-pepA-Ce6 NPs) is developed by conjugating acidic-sensitive small-molecule programmed cell death ligand 1 (PD-L1) inhibitor (Metformin, MET) with photosensitizer (chlorin e6, Ce6) through matrix metalloproteinase-2 (MMP-2) cleavable peptide (GPLGVRGDK, pepA). Noticeably, these self-delivery peptide-based NPs can circumvent the controversial biosafety facing nanomaterials. Moreover, MA-pepA-Ce6 NPs are degraded by overexpressed MMP-2 in tumor microenvironment (TME) and expose the VRGDK-Ce6. The exposed VRGDK-Ce6 shows superior targeting ability towards integrin α_vβ₃ receptor, ensuring sufficient accumulation and laser-activated robust antitumor immune effects. Remarkably, the released MET in tumor microenvironment hampers the PD-L1 expression and augments the antitumor immune response elicited by photodynamics therapy (PDT), thus significantly improving therapeutic outcomes. Overall, this study offers a potential appealing paradigm of synergistic PDT-triggered immunotherapy by revealing MET-mediated PD-L1 downregulation to achieve tumor eradication.     

A Near-Infrared Light-Responsive ROS Cascade Nanoplatform for Synergistic Therapy Potentiating Antitumor Immune Responses

Tumor occurrence is closely related to the unlimited proliferation and the evasion of the immune surveillance. However, it remains a challenge to kill tumor cells and simultaneously activate antitumor immunity upon spatially localized external stimuli. Herein, a robust tumor synergistic therapeutic nanoplatform is designed in combination with dual photosensitizers-loaded upconversion nanoparticles (UCNPs) and ferric-tannic acid (FeTA) nanocomplex. Dual photosensitizers-loaded UCNPs can induce photodynamic therapy (PDT) effect by generation of cytotoxic reactive oxygen species (ROS) on demand under near-infrared (NIR) light irradiation. FeTA can robustly respond to acidic tumor microenvironment to produce Fe²⁺ and subsequently induce chemodynamic therapy (CDT) by reacting with H₂O₂ in the tumor microenvironment. More importantly, the CDT/PDT synergy can not only exhibit significant antitumor ability but also induce ROS cascade to evoke immunogenic cell death. It increases tumor immunogenicity and promotes immune cell infiltration at tumor sites allowing further introduction of systemic immunotherapy responsiveness to inhibit the primary and distant tumor growth. This study provides a potential tumor microenvironment-responsive nanoplatform for imaging-guided diagnosis and combined CDT/PDT with improved immunotherapy responses and an external NIR-light control of photoactivation.     

Dual‐Targeting Heparin‐Based Nanoparticles that Re‐Assemble in Blood for Glioma Therapy through Both Anti‐Proliferation and Anti‐Angiogenesis

The efficient and specific delivery of nanoparticles (NPs) to brain tumors is crucial for successful glioma treatment. Heparin‐based polymers decorated with two peptides self‐assemble into multi‐functional NPs that specifically target glioma cells. These NPs re‐self‐assemble to a smaller size in blood, which is beneficial for in‐vivo brain drug delivery. The hydrodynamic size of one type of these NPs is 63 ± 11 nm under blood‐mimic conditions (10% fetal bovine serum), but it is 164 ± 16 nm in water. Additionally their zeta potential is more neutral in the blood‐mimic conditions. Transmission electron microscopy reveals the morphology of the spherical NPs. In vitro experiments demonstrate that the NPs exhibit a high cellular uptake and the ability to efficiently discourage proliferation, endothelial‐lined vessels, and vasculogenic mimicry. In vivo studies demonstrate that the NPs can by‐pass the normal blood–brain and blood–(brain tumor) barriers and specifically accumulate in glioma tissues; moreover, they present an excellent anti‐glioma effect in subcutaneous/intracranial glioma‐bearing mice. Their superiority is due to their appropriate size in blood and the synergic effect arising from their targeting of two different receptors. The data suggests that these NPs are ideal for anti‐glioma therapy.     

Cationic Lipids-Mediated Dual-Targeting of Both Dendritic Cells and Tumor Cells for Potent Cancer Immunotherapy

Impaired antigen presentation either in dendritic cells (DCs) or tumor cells impedes the triggering of antitumor immunity or tumor cell killing, resulting in failures of multiple types of cancer immunotherapy. Herein, the strategy of using dual-targeting nanomedicines to simultaneously improve the presentation of tumor antigens by both DCs and tumor cells is proposed. It is shown that tuning of surface charge of nanoparticles (NPs) by incorporating different amounts of cationic lipids alters the in vivo NP tissue accumulation and cellular targeting profiles. NPs with moderately positive surface charge (≈20 mV) achieve efficient accumulation in tumors and lymph nodes and dual-targeting to both DCs and tumor cells. As a proof-of-concept demonstration, siRNA against YTH N6−methyladenosine RNA binding protein 1 (YTHDF1) is delivered by the dual-targeting NPs to inhibit excessive antigen degradation in both DCs and tumor cells. For DCs, YTHDF1 downregulation promotes tumor antigen cross-presentation and cross-priming of antigen-specific T cells. For tumor cells, it enhances the presentation of endogenous tumor antigens and hence improves both the recognition and killing of tumor cells by primed antigen-specific T cells. The dual-targeting nanomedicines generate efficient antitumor immunity.     

Covalent Organic Framework‐Supported Molecularly Dispersed Near‐Infrared Dyes Boost Immunogenic Phototherapy against Tumors

Photodynamic therapy (PDT) mediated by near‐infrared (NIR) dyes is a promising cancer treatment modality; however, its use is limited by significant challenges, such as hypoxic tumor microenvironments and self‐quenching of photosensitizers. These challenges hamper its utility in inducing immunogenic cell death (ICD) and triggering potent systemic antitumor immune responses. This study demonstrates that molecular dispersion of NIR dyes in nanocarriers can significantly enhance their ability to produce reactive oxygen species and potentiate synergistic PDT and photothermal therapy against tumors. Specifically, NIR dye indocyanine green (ICG) can be spontaneously adsorbed to covalent organic frameworks (COFs) via π–π conjugations to prevent intermolecular stacking interactions. Then, ICG‐loaded COFs are ultrasonically exfoliated and coated with polydopamine (PDA) to construct a new phototherapeutic agent ICG@COF‐1@PDA with enhanced efficacy. In conjunction with ICG@COF‐1@PDA, a single round of NIR laser irradiation can induce obvious ICD, elicit antitumor immunity in colorectal cancer, and yield 62.9% inhibition of untreated distant tumors. ICG@COF‐1@PDA also exhibits notable phototherapeutic efficacy against 4T1 murine breast to lung metastasis, a spontaneous metastasis mode for triple‐negative breast cancers (TNBCs). Overall, this study reveals a novel nanodelivery system for molecular dispersion of NIR dyes, which may present new therapeutic opportunities against primary and metastatic tumors.     

Palladium Nanocrystals-Engineered Metal–Organic Frameworks for Enhanced Tumor Inhibition by Synergistic Hydrogen/Photodynamic Therapy

Hydrogen therapy, as a star therapeutic modality, has recently acquired much attention in the field of anticancer medicine. Evidence suggests that hydrogen can selectively reduce intratumoral overexpressed hydroxyl radicals (•OH) to break the redox homoeostasis and thereby result in redox stress and cell damage. As a reactive oxygen species-related noninvasive modality, photodynamic therapy (PDT) has been approved for varied tumor treatments clinically. For implementing tumor therapy with enhanced anticancer efficacy and attenuated side effects, here a biocompatible palladium nanocrystals-integrated nanoscale porphyrinic metal–organic framework (NPMOF) is designed to develop a novel combined therapy modality, that is, synergistic hydrogen/photodynamic therapy. The NPMOF is employed simultaneously as the photosensitizer for PDT and as the nanocarrier to support palladium nanocrystals, which is further used as the hydrogen vehicle. The final hydrogen-containing nanosystems exhibit a persistent reductive hydrogen release behavior and considerable light-activated singlet oxygen (¹O₂) generation without mutual interference, contributing to the adequate disturbance of tumor microenvironment redox steady-state for synergistically inducing tumor cell death. Ultimately, by coupling of tumor-selective hydrogen therapy and PDT, the designed nanosystems realize the augmented therapeutic outcome with minimal side effects, providing a safe and efficient tumor treatment for future clinical translation.     

Precise Molecular Engineering of Photosensitizers with Aggregation‐Induced Emission over 800 nm for Photodynamic Therapy

Owing to efficient singlet oxygen (¹O₂) generation in aggregate state, photosensitizers (PSs) with aggregation‐induced emission (AIE) have attracted much research interests in photodynamic therapy (PDT). In addition to high ¹O₂ generation efficiency, strong molar absorption in long‐wavelength range and near‐infrared (NIR) emission are also highly desirable, but difficult to achieve for AIE PSs since the twisted structures in AIE moieties usually lead to absorption and emission in short‐wavelength range. In this contribution, through acceptor engineering, a new AIE PS of TBT is designed to show aggregation‐induced NIR emission centered at 810 nm, broad absorption in the range between 300 and 700 nm with a large molar absorption coefficient and a high ¹O₂ generation efficiency under white light irradiation. Further, donor engineering by attaching two branched flexible chains to TBT yielded TBTC8 , which circumvented the strong intermolecular interactions of TBT in nanoparticles (NPs), yielding TBTC8 NPs with optimized overall performance in ¹O₂ generation, absorption, and emission. Subsequent PDT results in both in vitro and in vivo studies indicate that TBTC8 NPs are promising candidates in practical application.     

Synergy of Immunostimulatory Genetherapy with Immune Checkpoint Blockade Motivates Immune Response to Eliminate Cancer

Normalizing the tumor-induced immune deficiency in the immunosuppressive tumor microenvironment (TME) through increasing the efficient infiltration and activation of antitumoral immunity in TME is the core of promising immunotherapy. Herein, a Cyclo(Arg-Gly-Asp-d -Phe-Lys) (RGD) peptides-modified combinatorial immunotherapy system based on the self-assembly of the nanoparticles named RGD-DMA composed of RGD-PEG-PLA, methoxy poly(ethylene glycol)-poly(lactide) (MPEG-PLA) and 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) is used to codeliver the immunostimulatory chemokine CCL19-encoding plasmid DNA (CCL19 pDNA) and immune checkpoint ligand PD-L1 inhibitor (BMS-1). The RGD-DMA/pCCL19-BMS-1 system not only exhibited significant inhibition of tumor progression but also induced locally high concentrations of immunostimulatory cytokines at tumor sites without causing an obviously systemic inflammatory response. The immunosuppressive TME is efficaciously reshaped by the coadministration of RGD-DMA/pCCL19 and BMS-1, as indicated by the activated T lymphocytes, increased intratumoral-infiltration of mature dendritic cells (DCs), and the repolarization of macrophages from pro-tumoral M2-phenotype toward tumoricidal M1-phenotype. The upregulated PD-L1 expression at tumor sites caused by the increased IFN-γ levels after immunostimulatory gene therapy further demonstrated the synergistic effects of BMS-1 in counteracting the inhibitory role of PD-L1 expression in antitumor immunity. Therefore, the combination of immunostimulating therapy and immune checkpoint inhibitor that synergistically target multiple immune regulatory pathways demonstrates significant potential as a novel immunotherapy approach.