Anchoring Microbubbles on Cerebrovascular Endothelium as a New Strategy Enabling Low-Energy Ultrasound-Assisted Delivery of Varisized Agents Across Blood-Brain Barrier.

The protective blood-brain barrier (BBB) prevents most therapeutic agents from entering the brain. Currently, focused ultrasound (FUS) is mostly employed to create microbubbles that induce a cavitation effect to open the BBB. However, microbubbles pass quickly through brain microvessels, substantially limiting the cavitation effect. Here, we constructed a novel perfluoropropane-loaded microbubble, termed ApoER-Pep-MB, which possessed a siloxane bonds-crosslinked surface to increase the microbubble stability against turbulence in blood circulation and was decorated with binding peptide for apolipoprotein E receptor (ApoER-Pep). The microbubble with tailor-made micron size (2 µm) and negative surface charge (-30 mV) performed ApoER-mediated binding rather than internalization into brain capillary endothelial cells. Consequently, the microbubble accumulated on the brain microvessels, based on which even a low-energy ultrasound with less safety risk than FUS, herein diagnostic ultrasound (DUS), could create a strong cavitation effect to open the BBB. Evans Blue and immunofluorescence staining studies demonstrated that the DUS-triggered cavitation effect not only temporarily opened the BBB for 2 h but also caused negligible damage to the brain tissue. Therefore, various agents, ranging from small molecules to nanoscale objects, can be efficiently delivered to target regions of the brain, offering tremendous opportunities for the treatment of brain diseases.

Nanodrug Augmenting Antitumor Immunity for Enhanced TNBC Therapy via Pyroptosis and cGAS-STING Activation.

Pyroptosis is a proinflammatory form of programmed cell death that results in the release of cellular contents and activation of immune responses. However, GSDME (a pyroptosis-executed protein) is suppressed in many cancers. Herein, we constructed a nanoliposome (GM@LR) for codelivering the GSDME-expressing plasmid and manganese carbonyl (MnCO) into TNBC cells. MnCO generated Mn2+ and carbon monoxide (CO) in the presence of H2O2. The CO-activated caspase-3, which cleaved the expressed GSDME, converting apoptosis to pyroptosis in 4T1 cells. In addition, Mn2+ promoted maturation of dendritic cells (DCs) by the activation of STING signaling pathway. The increased proportion of intratumoral mature DCs brought about massive infiltration of cytotoxic lymphocytes, leading to a robust immune response. Besides, Mn2+ could be applied for magnetic resonance imaging (MRI)-guided metastasis detection. Taken together, our study showed that GM@LR nanodrug could effectively inhibit tumor growth via pyroptosis and STING activation combined immunotherapy.

Multifunctional nanodrug performs sonodynamic therapy and inhibits TGF-ß to boost immune response against colorectal cancer and liver metastasis.

Liver metastasis is the leading cause of death in colorectal cancer. Immunotherapy using immune checkpoint blockade (ICB) is ineffective due to its immunological cold tumor nature. Herein, we prepared a nanodrug (NCG) encapsulating the transforming growth factor-ß receptor inhibitor galunisertib (Gal) and the sonosensitizer chlorin e6 (Ce6), which was aimed to turn this type of cold tumor into a hot one to promote the ICB-based immunotherapy against it. After delivery to the tumor, NCG under ultrasonic irradiation generated reactive oxygen species causing tumor immunogenic cell death and releasing immunostimulatory signals such as calreticulin and HMGB1, which increased tumor immunogenicity and activated the innate T lymphocyte immune response. Moreover, NCG responded to the acidic microenvironment and released Gal, inhibiting phosphorylation and inducing immunosuppressive Smad2/3 signaling. Consequently, the differentiation of MDSCs was inhibited, M1-like polarization of tumor-associated macrophages was induced, and the immunosuppressive barrier of tumor-associated fibroblasts was destroyed to increase the infiltration of effector T cells, which reversed the immunosuppression of the tumor microenvironment and improved the therapeutic efficacy of anti-PD-L1 antibodies. Notably, in the liver metastasis mouse model, combination therapy using NCG (+) and aPD-L1 inhibited the growth of colon cancer liver metastasis, manifesting potential in treating this popular yet intractable malignancy. STATEMENT OF SIGNIFICANCE: Only a limited number of patients with colorectal cancer and liver metastasis can benefit from immune checkpoint blockade therapy, as most of them are microsatellite stable, immunologically cold tumors. Interestingly, there is compelling evidence that sonodynamic therapy (SDT) can convert immunosuppressed cold tumors into hot ones, trigger tumor immunogenic cell death non-invasively, and boost cytotoxic T cells infiltration. However, its therapeutic efficacy is constrained by the abundance of transforming growth factor-ß (TGF-ß) cytokines in the tumor microenvironment. Here, we reported a TGF-ß-targeted inhibitory nanodrug that improved SDT in colon cancer and liver metastasis, reversed the immunosuppressive tumor microenvironment and boosted the immune response to anti-PD-L1 therapy in this cancer. It demonstrated the potential to cure this prevalent but incurable malignancy.

A hierarchical tumor-targeting strategy for eliciting potent antitumor immunity against triple negative breast cancer.

Triple negative breast cancer (TNBC) as a highly aggressive and metastatic malignancy lacks targeting therapies nowadays. Moreover, although immune checkpoint blockade (ICB) is known to trigger anti-tumor immune response, most TNBC falls into the immunologically "cold" category unsuitable for ICB therapy due to insufficient lymphocyte infiltration. Herein, we develop a hierarchical targeting strategy for preparing a core-shell-structural nanodrug to concurrently block the programmed death ligand 1 (PD-L1) and deliver a stimulator of interferon gene (STING) agonist into tumor-infiltrating antigen-presenting cells (APCs). The nanodrug complexed the interferon stimulatory DNA (ISD) for STING activation in its core, conjugated PD-L1 antibody (aPD-L1) on its shell through a matrix metalloproteinase-2 (MMP-2) substrate peptide, and incorporated "hidden" mannose in its sublayer. Through aPD-L1-mediated active targeting of tumor cells and tumor-infiltrating APCs, the nanodrug efficiently accumulated in tumor sites. Then, the PD-L1-conjugating peptide was cleaved by tumor-enriched MMP-2, leaving aPD-L1 on target cells for ICB while exposing mannose to mediate targeted delivery of ISD into tumor-infiltrating dendritic cells (DCs) and tumor-associated macrophages (TAMs). Activating the STING signaling in DCs and TAMs not only stimulated the APCs maturation to prime anti-tumor immunity but also induced their chemokine secretion to promote tumor infiltration of anti-tumor effector T cells, thus sensitizing TNBC to the ICB therapy. Consequently, a potent antitumor immunity was evoked to effectively inhibit the tumor growth and metastasis in mice bearing orthotopic 4T1 breast cancer, showing the great potential in treating immunologically "cold" tumors.

Nanodrug regulates lactic acid metabolism to reprogram the immunosuppressive tumor microenvironment for enhanced cancer immunotherapy.

A majority of cancers fail to respond to immunotherapy due to the immunosuppressive tumor microenvironment (TME), and metabolic regulation of the TME has been a promising strategy to improve immunotherapy. Lactate is a key metabolic player in tumor immune response since its excess secretion aggravates tumor immune escape by favoring the polarization of tumor-associated macrophages (TAMs) to an immunosuppressive phenotype meanwhile impeding the tumor infiltration of the cytotoxic T lymphocyte. Here, we proposed a metabolic reprogramming mechanism to ameliorate tumor immunosuppression by using lonidamine and syrosingopine incorporated liposomes (L@S/L) to regulate lactate production and efflux. Concretely, lonidamine reduced lactate production by affecting the glycolytic metabolic pathway while syrosingopine decreased lactate efflux by inhibiting the key protein expression of the lactate transporter MCT-4. Consequently, both the drugs synergistically normalize the pH of the TME to overcome the tumor immunosuppressive microenvironment. In vivo studies demonstrated that the decreased extracellular lactate preferentially polarized TAMs to the M1 phenotype, simultaneously increased the proportion of NK cells and reduced the number of Treg cells. These results validated an efficient tumor immunotherapy in the breast cancer model. This new strategy of lactic acid metabolism regulation is proposed to operate in concert with immune modulation in the TME, which shows great potential for immunotherapy of immunologically "cold" tumors.

Surgical Tumor-Derived Photothermal Nanovaccine for Personalized Cancer Therapy and Prevention.

Recent breakthroughs in cell membrane-fabricated nanovaccine offer innovateive therapeutic options for preventing tumor metastasies and recurrence, yet the treatment of patient-specific solid tumor remained challenging owing to the immunosuppressive tumor microenvironment. Herein, we developed a personalized photothermal nanovaccine based on the surgical tumor-derived cell membranes (CMs) coating resiquimod (R848) loaded mesoporous polydopamine (MPDA) nanoparticles for targeting tumor photothermal immunotherapy and prevention. The fabricated photothermal nanovaccine MPDA-R848@CM (MR@C) demonstrates outstanding imaging-guided photothermal immunotherapy efficacy to eradicate solid tumors under near-IR laser irradiation and further inhibiting metastasis tumors by the resulted antitumor immunities, especially in combination with programmed death-ligand 1 antibody therapy (aPD-L1). Furthermore, from in vivo prophylactic testing results, it is confirmed that the 4T1 cells rechallenge can be prevented 100% in postsurgical tumor model after vaccination of the photothermal nanovaccine. Our work fabricates a personalized photothermal nanovaccine that possesses great potential for tumor-specific treatment and for preventing postoperative tumor recurrence.

GSH-Responsive Metal-Organic Framework for Intratumoral Release of NO and IDO Inhibitor to Enhance Antitumor Immunotherapy.

Immunotherapy brings great benefits for tumor therapy in clinical treatments but encounters the severe challenge of low response rate mainly because of the immunosuppressive tumor microenvironment. Multifunctional nanoplatforms integrating effective drug delivery and medical imaging offer tremendous potential for cancer treatment, which may play a critical role in combinational immunotherapy to overcome the immunosuppressive microenvironment for efficient tumor therapy. Here, a nanodrug (BMS-SNAP-MOF) is prepared using glutathione (GSH)-sensitive metal-organic framework (MOF) to encapsulate an immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) inhibitor BMS-986205, and the nitric oxide (NO) donor s-nitrosothiol groups. The high T1 relaxivity allows magnetic resonance imaging to monitor nanodrug distribution in vivo. After the nanodrug accumulation in tumor tissue via the EPR effect and subsequent internalization into tumor cells, the enriched GSH therein triggers cascade reactions with MOF, which disassembles the nanodrug to rapidly release the IDO-inhibitory BMS-986205 and produces abundant NO. Consequently, the IDO inhibitor and NO synergistically modulate the immunosuppressive tumor microenvironment with increase CD8+ T cells and reduce Treg cells to result in highly effective immunotherapy. In an animal study, treatment using this theranostic nanodrug achieves obvious regressions of both primary and distant 4T1 tumors, highlighting its application potential in advanced tumor immunotherapy.

Multifunctional Nanodrug Mediates Synergistic Photodynamic Therapy and MDSCs-Targeting Immunotherapy of Colon Cancer.

An ideal tumor treatment is supposed to eliminate the primary tumor and simultaneously trigger the host antitumor immune responses to prevent tumor recurrence and metastasis. Herein, a liposome encapsulating phosphoinositide 3-kinase gamma (PI3Kγ) inhibitor IPI-549 and photosensitizer chlorin e6 (Ce6), denoted by LIC, is prepared for colon cancer treatment. LIC internalized into CT26 cells generates reactive oxygen species (ROS) under laser irradiation to cause immunogenic tumor cell death, during which immunostimulatory signals such as calreticulin are released to further induce T lymphocyte-mediated tumor cell killing. Meanwhile, IPI-549 transported by liposome can inhibit PI3Kγ in the myeloid-derived suppressive cells (MDSCs), resulting in downregulation of arginase 1 (Arg-1) and ROS to promote MDSCs apoptosis and reduce their immunosuppressive activity to CD8+ T cells. LIC-mediated immunogenic photodynamic therapy synergizes with MDSCs-targeting immunotherapy, which significantly inhibits tumor growth via facilitating the dendritic cell maturation and tumor infiltration of CD8+ T cells while decreasing the tumor infiltration of immunosuppressive regulatory T cells, MDSCs, and M2-like tumor-associated macrophages. Moreover, the synergistic therapy increases the number of effector memory T cells (TEM ) in spleen, which suggests a favorable immune memory to prevent tumor recurrence and metastasis. The Ce6 and IPI-549-coloaded multifunctional nanodrug demonstrates high efficacy in colon cancer treatment.

Redox Responsive Metal Organic Framework Nanoparticles Induces Ferroptosis for Cancer Therapy.

Ferroptosis is attracting significant attention due to its effectiveness in tumor treatment. The efficiency to produce toxic lipid peroxides (LPOs) at the tumor site plays a key role in ferroptosis. A hybrid PFP@Fe/Cu-SS metal organic framework (MOF) is synthesized and shown to increase intratumoral LPO content through redox reactions generating ·OH. In addition, glutathione (GSH) depletion through disulfide-thiol exchange leads to the inactivation of glutathione peroxide 4 (GPX4), which results in a further increase in LPO content. This MOF exhibits high inhibitory effect on the growth of xenografted Huh-7 tumors in mice. The coadministration of a ferroptosis inhibitor reduces the antitumor effect of the MOF, leading to a restoration of GPX4 activity and an increase in tumor growth. Moreover, the construction of Cu into mesoporous PFP@Fe/Cu-SS not only allows the MOF to be used as a contrast agent for T1 -weighted magnetic resonance imaging, but also renders its photothermal conversion capacity. Thus, near-infrared irradiation is able to induce photothermal therapy and transform the encapsulated liquid perfluoropentane into microbubbles for ultrasound imaging.

TRAIL-based gene delivery and therapeutic strategies.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), also known as APO2L, belongs to the tumor necrosis factor family. By binding to the death receptor 4 (DR4) or DR5, TRAIL induces apoptosis of tumor cells without causing side toxicity in normal tissues. In recent years TRAIL-based therapy has attracted great attention for its promise of serving as a cancer drug candidate. However, the treatment efficacy of TRAIL protein was under expectation in the clinical trials because of the short half-life and the resistance of cancer cells. TRAIL gene transfection can produce a "bystander effect" of tumor cell killing and provide a potential solution to TRAIL-based cancer therapy. In this review we focus on TRAIL gene therapy and various design strategies of TRAIL DNA delivery including non-viral vectors and cell-based TRAIL therapy. In order to sensitize the tumor cells to TRAIL-induced apoptosis, combination therapy of TRAIL DNA with other drugs by the codelivery methods for yielding a synergistic antitumor efficacy is summarized. The opportunities and challenges of TRAIL-based gene delivery and therapy are discussed.

Antiglioma via regulating oxidative stress and remodeling tumor-associated macrophage using lactoferrin-mediated biomimetic codelivery of simvastatin/fenretinide.

Effective treatment of malignant glioma still remains a formidable challenge due to lack of the effective BBB-permeable drugs and efficient brain delivery methods, and the pharmacotherapy options are very limited. Therefore, to develop an effective therapeutic strategy is a pressing need. In this work, a noncytotoxic drug combination (i.e., simvastatin and fenretinide) was revealed to be potent for treating glioma, which was co-encapsulated into a TPGS-TAT-embedded lactoferrin nanoparticle system for achieving brain-targeted biomimetic delivery via the LRP-1 receptor. It was shown that the lactoferrin nanoparticle repolarized the tumor-associated macrophages from the M2 phenotype to M1 via regulating the STAT6 pathway, as well as induced the ROS-mediated mitochondrial apoptosis by inhibiting the Ras/Raf/p-Erk pathway in the glioma cells. The antiglioma efficacy was further demonstrated in both the subcutaneous and orthotopic glioma models. The repolarization of tumor-associated macrophages not only prompted the ROS generation but also induced the innate immunity (e.g., antitumor cytokine release). This delivery and therapeutic strategy provides a novel modality for the glioma treatment.

Biomimetic albumin-modified gold nanorods for photothermo-chemotherapy and macrophage polarization modulation.

Nanotechnology-based photothermal therapy has attracted great attention in the past decade. Nevertheless, photothermal therapy has some inherent drawbacks, such as the uneven heat production and limited laser penetration, often leading to insufficient treatment outcomes. Here, we developed a combination strategy to improve cancer therapy. The biomimetic albumin-modified gold nanorods (AuNRs) were prepared with incorporation of paclitaxel (PTX). This therapeutic system was characterized by several features. First, the albumin modification enhanced the biocompatibility and colloidal stability. Second, the surface-coated albumin promoted cellular uptake via the albumin-binding protein pathway. Third, PTX was incorporated via hydrophobic interaction between PTX and the albumin lipophilic domain. Fourth, the system can be used for combined photothermo-chemotherapy for yielding synergistic effects. The antitumor activity of the system was evaluated both in vitro and in vivo using the HCT116 colon cancer cell and tumor model. The combination therapy was found with an enhanced treatment efficiency and no obvious side effect. Most importantly, the thermal effect was also discovered with the ability to modulate the tumor microenvironments and suppress the macrophages polarization towards the M2 pro-tumor phenotype. It could be a mechanism for photothermal immunotherapy. The combination strategy and the system provide a potential method for cancer therapy.

Targeting death receptors for drug-resistant cancer therapy: Codelivery of pTRAIL and monensin using dual-targeting and stimuli-responsive self-assembling nanocomposites.

Chemoresistance remains a formidable hurdle against cancer therapy. Seeking for novel therapy strategies is an urgent need for those who no longer benefit from chemotherapy. Chemoresistance is usually associated with the dysfunction of intrinsic apoptosis. Targeting extrinsic apoptosis via TRAIL signaling and the death receptors could be a potential solution to treat chemoresistant cancer. A highly biocompatible nano system for codelivery of the TRAIL DNA and the death receptor sensitizer monensin was developed, in which low-molecular-weight PEI (LMW-PEI) was crosslinked by the sulfhydryl cyclodextrin via disulfide bonds, and then bound with DNA, thus forming the bioreducible polyplex cores. In addition, the cyclodextrin also functioned as a carrier for the hydrophobic monensin via host-guest inclusion. Poly-γ-glutamic acid (γ-PGA) was used to modify the polyplex core via charge interaction. The γ-PGA corona can specifically bind with the tumor-associated gamma-glutamyl transpeptidase (GGT) overexpressed on the tumor cells, and achieve tumor-targeting delivery. Moreover, the tumor-homing peptide RGD-modified γ-PGA was also prepared as the surface coating materials for further improving gene delivery efficiency. This gene delivery system was characterized by the dual ligand-targeting, dual stimuli-responsive features. The ligands of RGD and γ-PGA can target the tumor-associated receptors (i.e., integrin and GGT). The conformation of γ-PGA is pH-sensitive, and the tumor acidic micro environments could trigger the detachment of surface-coating γ-PGA. The disulfide crosslinking LMW-PEI is redox-sensitive, and its fast disassembling in the tumor cells could favor the efficient gene delivery. The anti-tumor efficacy was demonstrated both in vitro and in vivo. Moreover, MYC-mediated synthetic lethality could be an important mechanism for overcoming the drug resistance. An important finding of our studies is the demonstration of the in vivo treatment efficacy of TRAIL/monensin, thus providing a potential novel therapeutic strategy for overcoming drug-resistant cancer.