Ligand Regulation Strategy to Modulate ROS Nature in a Rhodamine-Iridium(III) Hybrid System for Phototherapy.

The efficacy of photodynamic therapy (PDT) is highly dependent on the photosensitizer features. The reactive oxygen species (ROS) generated by photosensitizers is proven to be associated with immunotherapy by triggering immunogenic cell death (ICD) as well. In this work, we establish a rhodamine-iridium(III) hybrid model functioning as a photosensitizer to comprehensively understand its performance and potential applications in photodynamic immunotherapy. Especially, the correlation between the ROS generation efficiency and the energy level of the Ir(III)-based excited state (T1'), modulated by the cyclometalating (C∧N) ligand, is systematically investigated and correlated. We prove that in addition to the direct population of the rhodamine triplet state (T1) formed through the intersystem crossing process with the assistance of a heavy Ir(III) metal center, the fine-tuned T1' state could act as a relay to provide an additional pathway for promoting the cascade energy transfer process that leads to enhanced ROS generation ability. Moreover, type I ROS can be effectively produced by introducing sulfur-containing thiophene units in C∧N ligands, providing a stronger M1 macrophage-activation efficiency under hypoxia to evoke in vivo antitumor immunity. Overall, our work provides a fundamental guideline for the molecular design and exploration of advanced transition-metal-based photosensitizers for biomedical applications.

Controlling NIR-II emitting gold organic/inorganic nanohybrids with tunable morphology and surface PEG density for dynamic visualization of vascular dysfunction.

Luminescent Au nanoparticles (AuNPs) and their organic/inorganic nanohybrids are of interest due to their favorable properties and promising biomedical applications. However, most existing AuNP-based hybrid nanostructures cannot satisfy high efficiency in synthesis, deep tissue penetration, and long blood circulation simultaneously, thus cannot be employed in dynamic monitoring of biomedical applications. In this paper, using Pluronic F127 as a template, we report a robust approach for one-pot synthesis of AuNP-based organic/inorganic nanohybrids (AuNHs) with bright luminescence in the second near-infrared (NIR-II) window, tunable shape, and controllable surface polyethylene glycol (PEG) density. The nanohybrids could be controlled from a necklace-like shape with a dense brush PEG configuration to a spherical structure with a brush PEG coating, which greatly impacts the in vivo biological behavior. Compared to spherical AuNHs, the necklace-shaped AuNHs present a higher quantum yield and longer blood circulation, which are superior to most of the individual AuNPs. With these outstanding features, the necklace-shaped AuNHs could achieve real-time, dynamic visualization of vascular dysfunction, capable of directing the precise administration of thrombolytics (a medicine for the breakdown of blood clots). These findings could provide a powerful guide for designing novel NIR-II nanoprobes toward in vivo dynamic information visualization.

Carbon dots-mediated synthesis of gold nanodendrites with extended absorption into NIR-II window for in vivo photothermal therapy.

BACKGROUND: Photothermal therapy (PTT) in the second near-infrared (NIR-II) window has attracted extensive attention due to the benefits in high maximum permissible exposure and penetration depth. Current photothermal agents generally show a broadband absorption accompanied by a gradual attenuation of absorption in the NIR-II window, leading to poor effect of PTT. It remains a great challenge to gain photothermal agents with strong and characteristic absorption in NIR-II regions. To overcome this problem, based on carbon dots (CDs)-mediated growth strategy, we proposed a simple and feasible approach to prepare plasmonic gold nanodendrites (AuNDs) with NIR-II absorption to enhance the therapeutic effect of PTT. RESULTS: By rationally regulating the size and branch length of AuNDs, the AuNDs exhibited a broadband absorption from 300 to 1350 nm, with two characteristic absorption peaks located at 1077 and 1265 nm. The AuNDs demonstrated desired optical photothermal conversion efficiency (38.0%), which was further applied in NIR-II photoacoustic imaging (PAI) and PTT in human colon cancer cells (HCT 116)-tumor-bearing mice model. The tumor cells could be effectively eliminated in vivo under 1064 nm laser irradiation by the guidance of PAI. CONCLUSIONS: We reported a simple but powerful synthetic method to obtain the unique AuNDs with strong and characteristic absorption peaks in the NIR-II window. This study provides a promising solution to tuning the growth of nanoparticles for bioimaging and phototherapy in the NIR-II window.

Non-invasive activation of intratumoural gene editing for improved adoptive T-cell therapy in solid tumours.

Adoptive T-cell therapy against solid tumours is limited by the apoptosis resistance mechanisms of tumour cells and by the extracellular, immunosuppressive tumour microenvironment. Here we report a temperature-sensitive genome-editing nanodevice that can deliver a Cas9 editor with an external trigger which can be used to edit the genome of tumour cells to reduce resistance to apoptosis and modulate the tumour microenvironment via a mild heating trigger. After local or systemic delivery of Cas9, mild heating is induced by non-invasive near-infrared (NIR) light or focused ultrasound (FUS) to activate Cas9, which initiates simultaneous genome editing of HSP70 (HSPA1A) and BAG3 in tumour cells. This disrupts the apoptotic resistance machinery of the tumour cells against adoptive T cells. At the same time, an NIR- or FUS-induced mild thermal effect reshapes the extracellular tumour microenvironment by disrupting the physical barriers and immune suppression. This facilitates the infiltration of adoptive T cells and enhances their therapeutic activity. Mild thermal Cas9 delivery is demonstrated in different murine tumour models which mimic a range of clinical indications, including a tumour model based on humanized patient-derived xenografts. As a result, the non-invasive thermal delivery of Cas9 significantly enhances the therapeutic efficacies of tumour-infiltrating lymphocytes and chimeric antigen receptor T and shows potential for clinical application.

Detection of Kidney Dysfunction through In Vivo Magnetic Resonance Imaging with Renal-Clearable Gadolinium Nanoprobes.

Kidney dysfunction is a clinical syndrome that can subsequently result in lethal kidney failure. The exploration of emerging bioimaging contrast agents with translational potential is highly challenging for a feasible diagnosis of kidney dysfunction. Herein, a class of renal-clearable gadolinium nanoparticles (Gd@PEG NPs) with an ultrasmall size of ∼5 nm, good monodispersity, and T1 relaxivity are synthesized using mesoporous silica nanoparticles as the template. Assisted by such renal-clearable Gd@PEG NPs, the diagnosis of kidney dysfunction in a mice model with a damaged kidney has been achieved through in vivo noninvasive magnetic resonance imaging. As a result, this work paves the way to synthesize monodispersible ultrasmall Gd contrast agents, facilitating the exploration of translational strategies for an in vivo analysis of kidney dysfunction.

Promoted NIR-II Fluorescence by Heteroatom-Inserted Rigid-Planar Cores for Monitoring Cell Therapy of Acute Lung Injury.

Fluorophores with emission in the second near-infrared (NIR-II) window have displayed salient advantages for biomedical applications. However, exploration of new luminogens with high NIR-II fluorescent brightness is still challenging. Herein, based on the "ring-fusion" strategy, a series of heteroatom-inserted rigid-planar cores is proposed to achieve the bathochromic NIR-II fluorophores with aggregation-induced emission (AIE) performance. Interestingly, one of the representative fluorophores, 4,4'-(5,5'-([1,2,5]thiadiazolo[3,4-i]dithieno[2,3-a:3',2'-c]phenazine-8,12-diyl)bis(4-octylthiophene-5,2-diyl))bis(N,N-diphenylaniline) (TTQiT), enjoys a maximum emission beyond 1100 nm because of the efficiently narrowed energy bandgap by electron-rich sulfur-atom-inserted core, which is verified by theoretical calculation. Taking advantage of the bright NIR-II emission of TTQiT nanoparticles, the desirable in vivo NIR-II imaging with high signal-to-background ratios is successfully performed and a long-term stem cell tracking in the detection of acute lung injury is further realized. Therefore, it is anticipated that this work will provide a promising molecular engineering strategy to enrich the scope of NIR-II fluorophores for catering to diverse demands in biomedical applications.

Acceptor engineering of small-molecule fluorophores for NIR-II fluorescence and photoacoustic imaging.

Fluorescence imaging in the second near-infrared window (NIR-II) has been an emerging technique in diverse in vivo applications with high sensitivity/resolution and deep tissue penetration. To date, the design principle of the reported NIR-II organic fluorophores has heavily relied on benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (BBTD) as a strong electron acceptor. Here, we report the rational design and synthesis of a NIR-II fluorescent molecule with the rarely used [1,2,5]thiadiazolo[3,4-f]benzotriazole (TBZ) core to replace BBTD as the electron acceptor. Thanks to the weaker electron deficiency of the TBZ core than BBTD, the newly yielded NIR-II molecule (BTB) based nanoparticles have a higher mass extinction coefficient and quantum yield in water. In contrast, the nanoparticle suspension of its counterpart with BBTD as the core is nearly nonemissive. The NIR-II BTB nanoparticles allow video-rate fluorescence imaging for vasculature imaging in ears, hindlimbs, and the brain of the mouse. Additionally, its large absorptivity in the NIR-I region also promotes bioimaging using photoacoustic microscopy (PAM) and tomography (PAT). Upon surface conjugation with the Arg-Gly-Asp (RGD) peptide, the functionalized nanoparticles ensured targeted detection of integrin-overexpressed tumors through both imaging modalities in two- and three-dimensional views. Thus, our approach to engineering acceptors of organic fluorophores offers a promising molecular design strategy to afford new NIR-II fluorophores for versatile biomedical imaging applications.

Type I macrophage activator photosensitizer against hypoxic tumors.

Photodynamic immunotherapy has emerged as a promising strategy to treat cancer. However, the hypoxic nature of most solid tumors and notoriously immunosuppressive tumor microenvironment could greatly compromise the efficacy of photodynamic immunotherapy. To address this challenge, we rationally synthesized a type I photosensitizer of TPA-DCR nanoparticles (NPs) with aggregation-enhanced reactive oxygen species generation via an oxygen-independent pathway. We demonstrated that the free radicals produced by TPA-DCR NPs could reprogram M0 and M2 macrophages into an anti-tumor state, which is not restricted by the hypoxic conditions. The activated M1 macrophages could further induce the immunogenic cell death of cancer cells by secreting pro-inflammatory cytokines and phagocytosis. In addition, in vivo anti-tumor experiments revealed that the TPA-DCR NPs could further trigger tumor immune response by re-educating tumor-associated macrophages toward M1 phenotype and promoting T cell infiltration. Overall, this work demonstrates the design of type I organic photosensitizers and mechanistic investigation of their superior anti-tumor efficacy. The results will benefit the exploration of advanced strategies to regulate the tumor microenvironment for effective photodynamic immunotherapy against hypoxic tumors.

Recent Advances in AIEgen-Based Photodynamic Therapy and Immunotherapy.

Cancer, one of the leading causes of death, has seriously threatened public health. However, there is still a lack of effective treatments. Nowadays, photodynamic therapy (PDT), relying on photosensitizers to trigger the generation of reactive oxygen species (ROS) for killing cancer cells, has been emerging as a noninvasive anti-cancer strategy. To enhance the overall anti-cancer efficacy of PDT, various approaches including molecular design and combination with other therapeutic techniques have been proposed and implemented. Especially, photodynamic immunotherapy that can effectively evoke the body's immune response has attracted much attention. Recently, a class of photosensitizers with aggregation-induced emission (AIE) character have shown unique promises, taking advantage of their profound fluorescence and ROS-generating ability in the aggregation state. Despite the promising results demonstrated by several groups, the associated studies are few and the mechanism of such AIEgen-based photodynamic immunotherapy has not been fully understood. This review discusses the recent advances in the AIEgen-based enhanced PDT with a special focus on the AIE photosensitizers for photodynamic immunotherapy, aiming to inspire more opportunities for in-depth investigation of the working principles in this emerging anti-cancer approach.

Monitoring tumor growth with a novel NIR-II photoacoustic probe.

In this chapter, we designed and synthesized a series of thiadiazoloquinoxaline (TQ)-based semiconducting polymers (SPs) with a broad absorption covering from NIR-I to NIR-II regions. Theoretical calculation suggests that the BTD-TQE with ester-substituted TQ-acceptor shows a large dihedral angle and narrow adiabatic energy as well as low radiative decay, resulting in higher reorganization energy for efficient photoinduced nonradiative decay (PNRD). As a result, the obtained BDT-TQE SP-cored nanoparticles, a NIR-II PA probe, exhibit a highest NIR-II photothermal conversion efficiency (61.6%) and achieved PA tracking of in situ hepatic tumor growth for 20 days. Herein, we propose a strategy to construct an effective NIR-II photoacoustic reagent through the enhanced PNRD effect of twisted intramolecular charge transfer (TICT), thereby extending the application of NIR-II PA reagents in in vivo bioimaging.

NIR-II Fluorescent Brightness Promoted by "Ring Fusion" for the Detection of Intestinal Inflammation.

Fluorophores with emission in the second near-infrared window (NIR-II) have displayed salient advantages for biomedical applications. However, the common strategy of reducing the energy bandgap of fluorophores so as to achieve red-shifted wavelengths always leads to compromised fluorescent brightness. Herein, we propose a molecular design concept of "ring-fusion" to modify the acceptor of AIEgen that can extend the luminous wavelength from NIR-I to NIR-II. The fused-acceptor-containing fluorophore yielded, TTQP, has an enhanced absorption coefficient with a higher brightness in nanoparticle formation compared to its NIR-I emissive counterpart (TTQ-DP) with a non-fused acceptor. Theoretical calculation further confirms that the ring fusion can efficiently promote the rigidity and planarity of the electron-deficient core, leading to a lower reorganization energy and nonradiative decay. The TTQP NPs yielded thus allow sensitive NIR-II fluorescence imaging of vasculature and intestinal inflammation in mice models. Therefore, we anticipate that our work will provide a promising molecular-engineering strategy to enrich the library and broaden the application scope of NIR-II fluorophores.

Targeted NIR-II emissive nanoprobes for tumor detection in mice and rabbits.

The functional NIR-II emissive nanoprobe loaded with AIEgen (cRGD-TTB NPs) achieved a high quantum yield (10.32%) and a high signal-to-background (S/B) ratio of 7.7 when employed for the visualization of large tumors (∼600 mm3) in rabbit models for the first time. This work will aid in the investigation of tumor targeting effect of therapeutic agents in large animal models.

Photoacoustic Force-Guided Precise and Fast Delivery of Nanomedicine with Boosted Therapeutic Efficacy.

Precise and efficient delivery of nanomedicine to the target site has remained as a major roadblock in advanced cancer treatment. Here, a novel photoacoustic force (PAF)-guided nanotherapeutic system is reported based on a near-infrared (NIR)-absorbing semiconducting polymer (SP), showing significantly improved tumor accumulation and deep tissue penetration for enhanced phototherapeutic efficacy. The accumulation of nanoparticles in 4T1 tumor-bearing mice induced by the PAF strategy displays a fivefold enhancement in comparison with that of the traditional passive targeting pathway, in a significantly shortened time (45 min vs 24 h) with an enhanced penetration depth in tumors. Additionally, a tumor-bearing mouse model is rationally designed to unveil the mechanism, indicating that the nanoparticles enter solid tumors through enhanced transportation across blood vessel barriers via both inter-endothelial gaps and active trans-endothelial pathways. This process is specifically driven by PAF generated from the nanoparticles under NIR laser irradiation. The study thus demonstrates a new nanotherapeutic strategy with low dose, enhanced delivery efficiency in tumor, and boosted therapeutic efficacy, opening new doors for designing novel nanocarriers.

Efficient and precise delivery of microRNA by photoacoustic force generated from semiconducting polymer-based nanocarriers.

One major challenge in miRNA-based therapy is to explore facile delivery strategies, which can facilitate the efficient and precise accumulation of intrinsically instable microRNAs (miRNAs) at targeted tumor sites. To address this critical issue, for the first time we demonstrate that a near-infrared (NIR) pulse laser can guide efficient delivery of miRNAs mediated by a NIR-absorbing and photoacoustic active semiconducting polymer (SP) nanocarrier, which can generate photoacoustic radiation force to intravascularly overcome the endothelial barriers. Importantly, we demonstrate an ultrafast delivery of miRNA (miR-7) to tumor tissues under the irradiation of pulse laser in 20 min, showing a 5-fold boosted efficiency in comparison to the traditional passive targeting strategy. The delivered miR-7 acts as a sensitizer of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and synergizes with TRAIL-inducing compound (TIC), leading to sustained TRAIL upregulation for effective tumor suppression in mice. As such, our results indicate that the NIR-absorbing semiconducting polymer-mediated nanocarrier platform can significantly enhance the targeted delivery efficiency of therapeutic miRNAs to tumors, resulting in potent tumor growth inhibition.

Self-assembled AIEgen nanoparticles for multiscale NIR-II vascular imaging.

In the recent decades, fluorogens with aggregation-induced emission (AIEgens) have been intensively explored in biomedical applications. One main strategy to bring these hydrophobic AIEgens into the aqueous biological environment is to encapsulate them in nanoparticles with functionalized polymeric matrices. However, exploration of reliable strategies that can afford AIE nanoparticles with uniform size and stable loading efficiency with minimized variation still remains a challenge. Here, we rationally designed amphiphilic AIEgens, constructed by a hydrophobic donor-acceptor-donor (D-A-D) core and hydrophilic polyethylene glycol (PEG) chain. The afforded amphiphilic AIEgens can self-assemble into uniform nanoparticles with average sizes of ~35 nm, showing an emission maximum beyond 1000 nm and quantum yields (QYs) above 10%. We then used the bright AIE nanoparticles for multiscale intravital vascular fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) in mouse and rabbit models with a high-resolution of ~38 µm and a penetration depth of ~1 cm. As such, our results demonstrate an efficient self-assembly strategy to construct advanced AIE nanoparticles for angiography.

Acceptor Engineering for Optimized ROS Generation Facilitates Reprogramming Macrophages to M1 Phenotype in Photodynamic Immunotherapy.

Reprogramming tumor-associated macrophages to an antitumor M1 phenotype by photodynamic therapy is a promising strategy to overcome the immunosuppression of tumor microenvironment for boosted immunotherapy. However, it remains unclear how the reactive oxygen species (ROS) generated from type I and II mechanisms, relate to the macrophage polarization efficacy. Herein, we design and synthesize three donor-acceptor structured photosensitizers with varied ROS-generating efficiencies. Surprisingly, we discovered that the extracellular ROS generated from type I mechanism are mainly responsible for reprogramming the macrophages from a pro-tumor type (M2) to an anti-tumor state (M1). In vivo experiments prove that the photosensitizer can trigger photodynamic immunotherapy for effective suppression of the tumor growth, while the therapeutic outcome is abolished with depleted macrophages. Overall, our strategy highlights the designing guideline of macrophage-activatable photosensitizers.

An Ester-Substituted Semiconducting Polymer with Efficient Nonradiative Decay Enhances NIR-II Photoacoustic Performance for Monitoring of Tumor Growth.

Photoacoustic agents have been of vital importance for improving the imaging contrast and reliability against self-interference from endogenous substances. Herein, we synthesized a series of thiadiazoloquinoxaline (TQ)-based semiconducting polymers (SPs) with a broad absorption covering from NIR-I to NIR-II regions. Among them, the excited s-BDT-TQE, a repeating unit of SPs, shows a large dihedral angle and narrow adiabatic energy as well as low radiative decay, attributing to its strongly electron-deficient ester-substituted TQ-segment. In addition, its more vigorous molecular motions trigger a higher reorganization energy that further yields an efficient photoinduced nonradiative decay, which has been carefully examined and understood by theoretical calculation. Thus, BDT-TQE SP-cored nanoparticles with twisted intramolecular charge transfer (TICT) feature exhibit a high NIR-II photothermal conversion efficiency (61.6 %) and preferable PA tracking of in situ hepatic tumor growth for more than 20 days. This study highlights a unique strategy for constructing efficient NIR-II photoacoustic agents via TICT-enhanced PNRD effect, advancing their applications for in vivo bioimaging.

Sub-10†nm Aggregation-Induced Emission Quantum Dots Assembled by Microfluidics for Enhanced Tumor Targeting and Reduced Retention in the Liver.

A robust platform is developed to assemble sub-10 nm organic aggregation-induced emission (AIE) particles using four different AIE luminogens (AIEgens) with emissions from green to the second near-infrared window (NIR-II). They are called AIE quantum dots (QDs) to distinguish from typical AIE dots which are larger than 25 nm. Compared with AIE dots that are larger than 25 nm, AIE QDs allow more efficient cellular uptake and imaging without surface modification of any membrane-penetrating peptides or other targeting molecules. NIR-II AIEgens, which have nearly no background fluorescence from organisms, are used to demonstrate that AIE QDs can achieve high contrast at the tumor as small as 80 mm3 and evade the liver more efficiently than AIE dots. AIE QDs hold a good promise for sensitive and precise diagnosis of the latent solid tumor in clinical medicine with much lower off-targeting to the liver than AIE dots.

A Photoinduced Nonadiabatic Decay-Guided Molecular Motor Triggers Effective Photothermal Conversion for Cancer Therapy.

It remains highly challenging to identify small molecule-based photothermal agents with a high photothermal conversion efficiency (PTCE). Herein, we adopt a double bond-based molecular motor concept to develop a new class of small photothermal agents to break the current design bottleneck. As the double-bond is twisted by strong twisted intramolecular charge transfer (TICT) upon irradiation, the excited agents can deactivate non-radiatively through the conical intersection (CI) of internal conversion, which is called photoinduced nonadiabatic decay. Such agents possess a high PTCE of 90.0 %, facilitating low-temperature photothermal therapy in the presence of a heat shock protein 70 inhibitor. In addition, the behavior and mechanism of NIR laser-triggered molecular motions for generating heat through the CI pathway have been further understood through theoretical and experimental evidence, providing a design principle for highly efficient photothermal and photoacoustic agents.

Planar AIEgens with Enhanced Solid-State Luminescence and ROS Generation for Multidrug-Resistant Bacteria Treatment.

Planar luminogens have encountered difficulties in overcoming intrinsic aggregation-caused emission quenching by intermolecular π-π stacking interactions. Although excited-state double-bond reorganization (ESDBR) can guide us on designing planar aggregation-induced emission (AIE) luminogens (AIEgens), its mechanism has yet been elucidated. Major challenges in the field include methods to efficiently restrict ESDBR and enhance AIE performance without using bulky substituents (e.g., tetraphenylethylene and triphenylamine). In this study, we rationally developed fluoro-substituent AIEgens with stronger intermolecular H-bonding interaction for restricted molecular motions and increased crystal density, leading to decreased nonradiative decay rate by one order of magnitude. The adjusted ESDBR properties also show a corresponding response to variation in viscosity. Furthermore, their aggregation-induced reactive oxygen species (ROS) generations have been discovered. The application of such planar AIEgen in treating multidrug-resistant bacteria has been demonstrated in a mouse model. The relationship between ROS generation and distinct E/Z-configurational stacking behaviors have been further understood, providing a design principle for synthesizing planar AIEgen-based photosensitizers.