Nanotechnology-based non-viral vectors for gene delivery in cardiovascular diseases.

Gene therapy is a technique that rectifies defective or abnormal genes by introducing exogenous genes into target cells to cure the disease. Although gene therapy has gained some accomplishment for the diagnosis and therapy of inherited or acquired cardiovascular diseases, how to efficiently and specifically deliver targeted genes to the lesion sites without being cleared by the blood system remains challenging. Based on nanotechnology development, the non-viral vectors provide a promising strategy for overcoming the difficulties in gene therapy. At present, according to the physicochemical properties, nanotechnology-based non-viral vectors include polymers, liposomes, lipid nanoparticles, and inorganic nanoparticles. Non-viral vectors have an advantage in safety, efficiency, and easy production, possessing potential clinical application value when compared with viral vectors. Therefore, we summarized recent research progress of gene therapy for cardiovascular diseases based on commonly used non-viral vectors, hopefully providing guidance and orientation for future relevant research.

Engineered Extracellular Vesicles Expressing Siglec-10 Camouflaged AIE Photosensitizer to Reprogram Macrophages to Active M1 Phenotype and Present Tumor-Associated Antigens for Photodynamic Immunotherapy.

Cancer immunotherapy has attracted considerable attention due to its advantages of persistence, targeting, and ability to kill tumor cells. However, the efficacy of tumor immunotherapy in practical applications is limited by tumor heterogeneity and complex tumor immunosuppressive microenvironments in which abundant of M2 macrophages and immune checkpoints (ICs) are present. Herein, two type-I aggregation-induced emission (AIE)-active photosensitizers with various reactive oxygen species (ROS)-generating efficiencies are designed and synthesized. Engineered extracellular vesicles (EVs) that express ICs Siglec-10 are first obtained from 4T1 tumor cells. The engineered EVs are then fused with the AIE photosensitizer-loaded lipidic nanosystem to form SEx@Fc-NPs. The ROS generated by the inner type-I AIE photosensitizer of the SEx@Fc-NPs through photodynamic therapy (PDT) can convert M2 macrophages into M1 macrophages to improve tumor immunosuppressive microenvironment. The outer EV-antigens that carry 4T1 tumor-associated antigens directly stimulate dendritic cells maturation to activate different types of tumor-specific T cells in overcoming tumor heterogeneity. In addition, blocking Siglec-10 reversed macrophage exhaustion for enhanced antitumor ability. This study presents that a combination of PDT, immune checkpoints, and EV-antigens can greatly improve the efficiency of tumor immunotherapy and is expected to serve as an emerging strategy to improve tumor immunosuppressive microenvironment and overcome immune escape.

Aggregation-Induced-Emission Photosensitizer-Loaded Nano-Superartificial Dendritic Cells with Directly Presenting Tumor Antigens and Reversed Immunosuppression for Photodynamically Boosted Immunotherapy.

The success of tumor immunotherapy highlights the potential of harnessing immune system to fight cancer. Activating both native T cells and exhausted T cells is a critical step for generating effective antitumor immunity, which is determined based on the efficient presentation of tumor antigens and co-stimulatory signals by antigen-presenting cells, as well as immunosuppressive reversal. However, strategies for achieving an efficient antigen presentation process and improving the immunosuppressive microenvironment remain unresolved. Here, aggregation-induced-emission (AIE) photosensitizer-loaded nano-superartificial dendritic cells (saDC@Fs-NPs) are developed by coating superartificial dendritic cells membranes from genetically engineered 4T1 tumor cells onto nanoaggregates of AIE photosensitizers. The outer cell membranes of saDC@Fs-NPs are derived from recombinant lentivirus-infected 4T1 tumor cells in which peptide-major histocompatibility complex class I, CD86, and anti-LAG3 antibody are simultaneously anchored. These saDC@Fs-NPs could directly stimulate T-cell activation and reverse T-cell exhaustion for cancer immunotherapy. The inner AIE-active photosensitizers induce immunogenic cell death to activate dendritic cells and enhance T lymphocyte infiltration by photodynamic therapy, promoting the transformation of "cold tumors" into "hot tumors," which further boosts immunotherapy efficiency. This work presents a powerful photoactive and artificial antigen-presenting platform for activating both native T cells and exhausted T cells, as well as facilitating tumor photodynamic immunotherapy.

Nanomaterial-mediated low-temperature photothermal therapy via heat shock protein inhibition.

With the continuous development of nanobiotechnology in recent years, combining photothermal materials with nanotechnology for tumor photothermal therapy (PTT) has drawn many attentions nanomedicine research. Although nanomaterial-mediated PTT is more specific and targeted than traditional treatment modalities, hyperthermia can also damage normal cells. Therefore, researchers have proposed the concept of low-temperature PTT, in which the expression of heat shock proteins (HSPs) is inhibited. In this article, the research strategies proposed in recent years based on the inhibition of HSPs expression to achieve low-temperature PTT was reviewed. Folowing this, the synthesis, properties, and applications of these nanomaterials were introduced. In addition, we also summarized the problems of nanomaterial-mediated low-temperature PTT at this stage and provided an outlook on future research directions.

Ratiometric imaging of butyrylcholinesterase activity in mice with nonalcoholic fatty liver using an AIE-based fluorescent probe.

Butyrylcholinesterase (BChE) is an essential human biomarker which is related to liver and neurodegenerative diseases. It is of great significance to develop a fluorescent probe that can image BChE in vitro and in vivo. Unfortunately, most fluorescent probes that are based on a single change in fluorescence intensity are susceptible to environmental interference. Therefore, we reported an easily available ratiometric fluorescent probe, TB-BChE, with aggregation-induced emission (AIE) characteristics for ratiometric imaging of BChE. TB-BChE demonstrated excellent sensitivity (LOD = 39.24 ng mL-1) and specificity for BChE. Moreover, we have successfully studied the ratiometric imaging of TB-BChE to BChE in a nonalcoholic fatty liver disease model. These results indicated that TB-BChE is expected to become a powerful analysis tool for butyrylcholinesterase research in basic medicine and clinical applications.

Intelligent photothermal dendritic cells restart the cancer immunity cycle through enhanced immunogenic cell death.

Dendritic cells (DCs) play a pivotal role in initiating antigen-specific tumor immunity. However, the abnormal function of DCs owing to the immunosuppressive tumor microenvironment (TME) and the insufficient number of tumor infiltrating DCs could promote immune tolerance and tumor immune escape. Thus, there is great potential to employ DCs to induce efficient antitumor immunity. In this paper, we developed intelligent DCs (iDCs), which consist of nanoparticles loaded with photothermal agents (IR-797) and coated with a mature DC membrane. The DC cell membrane on the surface of iDCs preserves the ability to present antigens and prime T cells. The iDCs can also enter the lymph node and stimulate T cells. The activated T cells reduced the expression of heat shock proteins (HSPs) in tumor cells, rendering them more sensitive to heat stress. Subsequently, we used mild photothermal therapy (42-45 °C) to induce immunogenic cell death and contribute to a synergistic antitumor effect. iDCs as a refined and precise system in combination with DC-based immunotherapy and thermal therapy can be stored long-term and on a large scale, so they can be applied in many patients.

A Biomimetic Aggregation-Induced Emission Photosensitizer with Antigen-Presenting and Hitchhiking Function for Lipid Droplet Targeted Photodynamic Immunotherapy.

Photodynamic therapy (PDT) is a promising alternative approach for effective cancer treatment that is associated with an antitumor immune response. However, immunosuppression of the tumor microenvironment limits the immune response induced by PDT. Stimulation and proliferation of T cells is a critical step for generating immune responses and depends on the efficient presentation of tumor antigens and co-stimulatory molecules by antigen-presenting cells (APCs). Here, biomimetic aggregation-induced emission (AIE) photosensitizers with antigen-presenting and hitchhiking abilities (DC@AIEdots) are developed by coating dendritic cell (DC) membranes on the nanoaggregates of the AIEgens. Notably, the inner AIE molecules can selectively accumulate in lipid droplets of tumor cells, and the outer cell membrane can facilitate the hitchhiking of DC@AIEdots onto the endogenous T cells and enhance the tumor delivery efficiency by about 1.6 times. Furthermore, DC@AIEdots can stimulate the in vivo proliferation and activation of T cells and trigger the immune system. The potential applications of therapeutic agents targeting lipid droplets for immunotherapy are indicated and a new hitchhiking approach for drug delivery is provided. Lastly, the study presents a photoactive and artificial antigen-presenting platform for effective T cell stimulation and cancer photodynamic immunotherapy.

Nanoengineered CAR-T Biohybrids for Solid Tumor Immunotherapy with Microenvironment Photothermal-Remodeling Strategy.

Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable clinical success in eradicating hematologic malignancies. However, hostile microenvironment in solid tumors severely prevents CAR-T cells migrating, infiltrating, and killing. Herein, a nanoengineered CAR-T strategy is reported for enhancing solid tumor therapy through bioorthogonal conjugation with a nano-photosensitizer (indocyanine green nanoparticles, INPs) as a microenvironment modulator. INPs engineered CAR-T biohybrids (CT-INPs) not only retain the original activities and functions of CAR-T cells, but it is further armed with fluorescent tracing and microenvironment remodeling abilities. Irradiated with laser, CT-INPs demonstrate that mild photothermal intervention destroys the extracellular matrix, expanded blood vessels, loosened compact tissue, and stimulated chemokine secretion without damping CAR-T cell activities. Those regulations induce an immune-favorable tumor microenvironment for recruitment and infiltration of CT-INPs. CT-INPs triggered photothermal effects collapse the physical and immunological barriers of solid tumor, and robustly boosted CAR-T immunotherapy. Therefore, CAR-T biohybrids provide reliable treatment strategy for solid tumor immunotherapy via microenvironment reconstruction.

In situ poly I:C released from living cell drug nanocarriers for macrophage-mediated antitumor immunotherapy.

Immunotherapy is one of the most promising approaches to inhibit tumor growth and metastasis by activating host immune functions. However, the arising problems such as low immune response caused by complex tumor microenvironment and extremely systemic immune storm still limit the clinical applications of immunotherapy. Here, we construct Poly I: C-encapsulated poly (lactic-co-glycolic acid) nanoparticles (PLP NPs) with a slow release profile. A biomimetic system (MPLP), which loads PLP NPs on the surface of bone marrow-derived macrophage (BMDM) via the maleimide-thiol conjugation, is synthesized to effectively deliver PLP, control drug release and activate the tumor-specific immune response in situ. The results show that PLP NPs loading does not affect the activity and function of BMDM. Then, BMDM acts as a living cell drug vehicle and promotes the accumulation of PLP NPs in tumors, where Poly I: C is released from PLP NPs and reprograms BMDM into tumoricidal M1 macrophage. Furthermore, MPLP triggers potent antitumor immune responses in vivo and effectively inhibits local and metastatic tumors without causing adverse pathological immune reactions. This study offers an inspiration to facilitate clinical translation through the delivery of drugs by living immune cells for future anticancer therapy.

Natural-Killer-Cell-Inspired Nanorobots with Aggregation-Induced Emission Characteristics for Near-Infrared-II Fluorescence-Guided Glioma Theranostics.

Nature has always inspired robotic designs and concepts. It is conceivable that biomimic nanorobots will soon play a prominent role in medicine. The "Terminator" in the science fiction film is a cybernetic organism with living tissue over a metal endoskeleton, which inspired us to develop natural-killer-cell-mimic nanorobots with aggregation-induced emission (AIE) characteristics (NK@AIEdots) by coating a natural kill cell membrane on an AIE-active polymeric endoskeleton, PBPTV, a highly bright NIR-II AIE-active conjugated polymer. Owing to the AIE and soft-matter characteristics of PBPTV, as-prepared NK@AIEdots maintained a superior NIR-II brightness (quantum yield ∼7.9% in water) and good biocompatibility. Besides, they can serve as a tight junction (TJ) modulator to trigger an intracellular signaling cascade, causing TJ disruption and actin cytoskeleton reorganization to form an intercellular "green channel" to help them to cross the blood-brain barrier (BBB) silently. Furthermore, they can initiatively accumulate in glioblastoma cells in the complex brain matrix for high-contrast and through-skull tumor imaging. The tumor growth was also greatly inhibited by these NK@AIEdots under the NIR light illumination. As far as we know, the quantum yield of PBPTV is the highest among the existing NIR-II luminescent conjugated polymers. Besides, the NK-cell biomimetic nanorobots showed great potential for BBB-crossing active delivery.

Immunocyte Membrane-Coated Nanoparticles for Cancer Immunotherapy.

Despite the advances in surface bioconjugation of synthetic nanoparticles for targeted drug delivery, simple biological functionalization is still insufficient to replicate complex intercellular interactions naturally. Therefore, these foreign nanoparticles are inevitably exposed to the immune system, which results in phagocytosis by the reticuloendothelial system and thus, loss of their biological significance. Immunocyte membranes play a key role in intercellular interactions, and can protect foreign nanomaterials as a natural barrier. Therefore, biomimetic nanotechnology based on cell membranes has developed rapidly in recent years. This paper summarizes the development of immunocyte membrane-coated nanoparticles in the immunotherapy of tumors. We will introduce several immunocyte membrane-coated nanocarriers and review the challenges to their large-scale preparation and application.

Scaffolds biomimicking macrophages for a glioblastoma NIR-Ib imaging guided photothermal therapeutic strategy by crossing Blood-Brain Barrier.

Glioblastoma (GBM) is one of the most malignant cancers, and Blood-Brain Barrier (BBB) is the main obstacle to diagnose and treat GBM, hence scientists are making great efforts to develop new drugs which can pass BBB and integrate diagnosis and therapeutics together. Here, we designed plasma membrane of macrophage camouflaged DSPE-PEG loaded near-infrared Ib (NIR-Ib) fluorescence dye IR-792 nanoparticles (MDINPs). MDINPs were able to penetrate BBB and selectively accumulate at tumor site, and then could be used as NIR-Ib fluorescence probes for targeted tumor imaging. At the same time, MDINPs could kill tumor cells by photothermal effect. Our results showed that MDINPs-mediated NIR-Ib fluorescence imaging could clearly observe orthotopic GBM, and the NIR-Ib imaging-guided photothermal therapy significantly suppressed the growth of GBM and prolonged the life of mice. This work not only provided a method to mimic the biological function of macrophage, but also provided an integrative strategy for diagnosis and treatment in GBM.

Near-infrared fluorescence imaging for vascular visualization and fungal detection in plants.

We found that heptamethine dye IR-820 showed distinct emission peaks in both the NIR-Ia and NIR-Ib windows. IR-820 yielded images of vascular structures in the NIR-Ib window with unprecedented details. NIR-Ib fluorescence imaging was useful not only for studying plant transpiration, but also for detecting and differentiating fungal pathogens.

Cell-Membrane Immunotherapy Based on Natural Killer Cell Membrane Coated Nanoparticles for the Effective Inhibition of Primary and Abscopal Tumor Growth.

Developing effective immunotherapies with low toxicity and high tumor specificity is the ultimate goal in the battle against cancer. Here, we reported a cell-membrane immunotherapy strategy that was able to eliminate primary tumors and inhibited distant tumors by using natural killer (NK) cell membrane cloaked photosensitizer 4,4',4'',4'''-(porphine-5,10,15,20-tetrayl) tetrakis (benzoic acid) (TCPP)-loaded nanoparticles (NK-NPs). The proteomic profiling of NK cell membranes was performed through shotgun proteomics, and we found that NK cell membranes enabled the NK-NPs to target tumors and could induce or enhance pro-inflammatory M1-macrophages polarization to produce antitumor immunity. The TCPP loaded in NK-NPs could induce cancer cell death through photodynamic therapy and consequently enhanced the antitumor immunity efficiency of the NK cell membranes. The results confirmed that NK-NPs selectively accumulated in the tumor and were able to eliminate primary tumor growth and produce an abscopal effect to inhibit distant tumors. This cell-membrane immunotherapeutic approach offers a strategy for tumor immunotherapy.

Near-infrared fluorescence imaging in the largely unexplored window of 900-1,000 nm.

Near-infrared (NIR) fluorescence imaging has relied on fluorophores that emit in the 700-900 nm NIR-Ia or 1,000-1,700 nm NIR-II window for generating deep-tissue images. Up until now, there have been few fluorophores developed for the 900-1,000 nm NIR-Ib window. This is largely because NIR-Ib light is thought to be strongly absorbed by water. Methods: Here we found that six heptamethine dyes had distinct emission peaks in both the NIR-Ia and NIR-Ib window. We tested the performance of these contrast agents by introducing them into the leaves of the common house plant Epipremnum aureum with early stage anthracnose leaf infections from Khaya senegalensis, as well as injecting them into the hind feet of nude mice and tails of tumour-bearing mice in vivo. Results: Heptamethine dyes yielded superior images of leaf venation, anthracnose infection locations, sentinel lymph nodes, brain tumours and subcutaneous tumours in the NIR-Ib window. We found that NIR-Ib images had markedly enhanced signal-to-background ratio because autofluorescence, scattering and light absorption by biological tissues and water were weaker at longer wavelengths. Conclusion: NIR-Ib fluorescence imaging was a powerful method for studying sentinel lymph nodes, tumours, leaf veins and early anthracnose infection locations in plant leaves. The findings challenge our current view of NIR fluorescence imaging and may have important implications for biomedical research and image-guided cancer surgery.

Dye-Anchored MnO Nanoparticles Targeting Tumor and Inducing Enhanced Phototherapy Effect via Mitochondria-Mediated Pathway.

Phototherapy is a promising treatment method for cancer therapy. However, the various factors have greatly restricted phototherapy development, including the poor accumulation of photosensitizer in tumor, hypoxia in solid tumor tissue and systemic phototoxicity. Herein, a mitochondrial-targeted multifunctional dye-anchored manganese oxide nanoparticle (IR808@MnO NP) is developed for enhancing phototherapy of cancer. In this nanoplatform, IR808 as a small molecule dye acts as a tumor targeting ligand to make IR808@MnO NPs with capacity to actively target tumor cells and relocate finally in the mitochondria. Meanwhile, continuous production of oxygen (O2 ) and regulation of pH induced by the high reactivity and specificity of MnO NPs toward mitochondrial endogenous hydrogen peroxide (H2 O2 ) could effectively modulate tumor hypoxia and lessen the tumor subacid environment. Large amounts of reactive oxide species (ROS) are generated during the reaction process between H2 O2 and MnO NPs. Furthermore, under laser irradiation, IR808 in IR808@MnO NPs turns O2 into a highly toxic singlet oxygen (1 O2 ) and generates hyperthermia. The results indicate that IR808@MnO NPs have the high efficiency of specific targeting of tumors, relieving tumor subacid environment, improving the tumor hypoxia environment, and generating large amounts of ROS to kill tumor cells. It is expected to have a wide application in treating cancer.

Hypoxia-triggered single molecule probe for high-contrast NIR II/PA tumor imaging and robust photothermal therapy.

Hypoxia is a common characteristic of solid tumors. This important feature is associated with resistance to radio-chemotherapy, which results in poor prognosis and probability of tumor recurrence. Taking advantage of background-free NIR II fluorescence imaging and deeper-penetrating photoacoustic (PA) imaging, we developed a hypoxia-triggered and nitroreductase (NTR) enzyme-responsive single molecule probe for high-contrast NIR II/PA tumor imaging and hypoxia-activated photothermal therapy (PTT), which will overcome cellular resistance during hypoxia. Methods: The single molecule probe IR1048-MZ was synthesized by conjugating a nitro imidazole group as a specific hypoxia trigger with an IR-1048 dye as a NIR II/PA signal reporter. We investigated the NIR II fluorescence, NIR absorbance and photothermal effect in different hypoxia conditions in vitro, and performed NIR II/PA tumor imaging and hypoxia-activated photothermal therapy in mice. Results: This versatile molecular probe IR1048-MZ not only realized high-contrast tumor visualization with a clear boundary by NIR II fluorescence imaging, but also afforded deep-tissue penetration at the centimeter level by 3D PA imaging. Moreover, after being activated by NTR that is overexpressed in hypoxic tumors, the probe exhibited a significant photothermal effect for curative tumor ablation with no recurrence. Conclusions: We have developed the first hypoxia-triggered and NTR enzyme-responsive single molecule probe for high-contrast NIR II/PA tumor imaging and hypoxia-activated photothermal therapy. By tracing the activity of NTR, IR1048-MZ may be a promising contrast agent and theranostic formulation for other hypoxia-related diseases (such as cancer, inflammation, stroke, and cardiac ischemia).

Dual-Responsive Molecular Probe for Tumor Targeted Imaging and Photodynamic Therapy.

The precision oncology significantly relies on the development of multifunctional agents to integrate tumor targeting, imaging and therapeutics. In this study, a first small-molecule theranostic probe, RhoSSCy is constructed by conjugating 5'-carboxyrhodamines (Rho) and heptamethine cyanine IR765 (Cy) using a reducible disulfide linker and pH tunable amino-group to realize thiols/pH dual sensing. In vitro experiments verify that RhoSSCy is highly sensitive for quantitative analysis and imaging intracellular pH gradient and biothiols. Furthermore, RhoSSCy shows superb tumor targeted dual-modal imaging via near-infrared fluorescence (NIRF) and photoacoustic (PA). Importantly, RhoSSCy also induces strongly reactive oxygen species for tumor photodynamic therapy (PDT) with robust antitumor activity both in vitro and in vivo. Such versatile small-molecule theranostic probe may be promising for tumor targeted imaging and precision therapy.

Dual-modal imaging-guided highly efficient photothermal therapy using heptamethine cyanine-conjugated hyaluronic acid micelles.

Targeted phototherapy and multi-modal imaging can effectively improve the therapeutic efficacy and reduce the side effects of theranostics. Herein, we constructed novel biocompatible cyanine dye IR808-conjugated hyaluronic acid nanoparticles (HAIR NPs) for photothermal therapy (PTT) with near-infrared fluorescence (FL) and photoacoustic (PA) dual-modal imaging. The nanoparticles formed stable nanostructures under aqueous conditions with uniform size distribution. The HAIR NPs were rapidly taken up by the human lung cancer cells A549 via CD44 (the hyaluronic acid receptor on the surface of tumor cells) receptor-mediated endocytosis. Upon laser irradiation, the HAIR NPs enabled good near-infrared fluorescence imaging and photoacoustic imaging in tumor-bearing mice. In addition, the tight nanostructure arising from the covalent link between HA and IR808 could significantly improve the light-thermal conversion efficiency of IR808. Under a low dose of laser power, the HAIR NPs presented more effective photothermal therapy for the suppression of tumor growth than free IR808 in vitro and in vivo. Overall, these results indicate that the HAIR NPs may be an extremely promising nanoplatform in cancer theranostics for targeted PTT under FL and PA dual-modal imaging.

Tumor-targeted small molecule for dual-modal imaging-guided phototherapy upon near-infrared excitation.

Near-infrared (NIR) organic dyes have received increasing attention in recent years as diagnostic and therapeutic agents in the field of tumor research. In this study, IR-822, a near-infrared fluorescence (NIRF) heptamethine cyanine dye, was chosen as a fluorophore due to its high extinction coefficients, and native preferential tumor accumulation property. To enhance its specificity in tumor imaging, N1-(pyridin-4-ylmethyl)ethane-1,2-diamine (PY) was conjugated to IR-822 as a pH-sensing receptor, forming a fluorophore-spacer-receptor molecular probe (IR-PY) that can modulate the fluorescence emission intensity through a fast photoinduced electron-transfer process, which allowed the probe to "switch on" significantly in an acidic tumor microenvironment and which realized enhanced NIRF imaging in vivo. Having a strong NIR absorption at 600-850 nm, this small-molecule IR-PY showed not only high spatial resolution photoacoustic (PA) imaging in mice, but also effective tumor photothermal ablation in vivo. After photothermal therapy (PTT) with IR-PY under NIR 808 nm laser irradiation, the mice exhibited remarkable ablation with no tumor recurrence after treatment. Therefore, a single smart small-molecule probe IR-PY has been designed, synthesized and verified as an "all in one" multifunctional agent, including pH sensitivity, tumor targeting, "switch-on" near-infrared fluorescence imaging, high-spatial-resolution PA imaging and efficient near-infrared photothermal therapy, which is promising for clinical application in NIRF/PA dual-modal imaging-guided cancer diagnosis and treatment.