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The development of photothermal agents (PTAs) with robust photostability and high photothermal conversion efficiency is of great importance for cancer photothermal therapy. Herein, a novel PTA was created using two-dimensional intermetallic PtSnBi nanoplates (NPs), which demonstrated excellent photostability and biocompatibility with a high photothermal conversion efficiency of ~61% after PEGylation. More importantly, PtSnBi NPs could be employed as photoacoustic imaging contrast agents for tumor visualization due to their strong absorbance in the NIR range. In addition, both in vitro and in vivo experiments confirmed that PtSnBi NPs had a good photothermal efficacy under NIR laser irradiation. Therefore, the remarkable therapeutic characteristics of PtSnBi NPs make them make them a most promising candidate for cancer theranostics.

Immunotherapy is an attractive approach for cancer therapy, while its antitumor efficacy is still limited, especially for non-immunogenic tumors. Nanomedicines can be utilized to convert the non-immunogenic "cold" tumors to immunogenic "hot" tumors via inducing immunogenic cell death (ICD), thereby promoting the antitumor immune response. Some nanomedicines that can produce local heat and reactive oxygen species upon the stimulation of electromagnetic energy are the main candidates for inducing the ICD effect. However, their applications are often restricted due to the poor tissue penetration depths of electromagnetic energy, such as light. By contrast, ultrasound, X-ray, alternating magnetic field, and microwave show excellent tissue penetration depths and thereby can be used for sonodynamic therapy, radiotherapy, magnetic hyperthermia therapy, and microwave ablation therapy, all of which can effectively induce ICD. Herein, the combination of deep-tissue electromagnetic energy with nanomedicines for inducing ICD and cancer immunotherapy are summarized. In particular, the designs of nanomedicines to amplify ICD effect in the presence of deep-tissue electromagnetic energy and sensitize tumors to various immunotherapies will be discussed. At the end of this review, a brief conclusion and discussion of current challenges and further perspectives in this subfield are provided. Submitted/Accepted version This study was financially supported by the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, the Science and Technology Commission of Shanghai Municipality (22ZR1401100, 22490760700, 20DZ2254900), and Fundamental Research Funds for the Central Universities (2232021A-05).

Chemiluminescence imaging is imperative for diagnostics and imaging due to its intrinsically high sensitivity. To improve in vivo detection of biomarkers, chemiluminophores that simultaneously possess near-infrared (NIR) emission and modular structures amenable to construction of activatable probes are highly desired; however, these are rare. Herein, we report two chemiluminophores with record long NIR emission (>750 nm) via integration of dicyanomethylene-4H-benzothiopyran or dicyanomethylene-4H-benzoselenopyran with dioxetane unit. Caging of the chemiluminophores with different cleavable moieties produces NIR chemiluminescence probes (NCPs) that only produce signals upon reaction with reactive oxygen species or enzymes, for example, β-galactosidase, with a tissue-penetration depth of up to 2 cm. Thus, this study provides NIR chemiluminescence molecular scaffolds applicable for in vivo turn-on imaging of versatile biomarkers in deep tissues. Ministry of Education (MOE) Nanyang Technological University This work was supported by Nanyang Technological University (Start-up Grant: M4081627) and Singapore Ministry of Education Academic Research Fund Tier 1 (2019-T1-002-045,RG125/19) and Tier 2 (MOE2018-T2-2-042)

Introduction Macleaya cordata (Willd) R. Br. (Papaveraceae family) is a well-known traditional Chinese medicine used to treat muscle pain, inflamed wounds, and bee bites. Benzo[c]phenanthridine alkaloids are the main active ingredients in M. cordata. In this work, sanguinarine and chelerythrine were efficiently extracted and purified by ultrahigh-pressure extraction (UHPE) technique and pH-zone-refining counter-current chromatography (PZRCCC) from M. cordata. Objective To develop an efficient UHPE method followed by an efficient separation technique using PZRCCC for benzo[c]phenanthridine alkaloids from the study plant species, and to evaluate the study samples for anti-breast cancer activity. Methodology The optimal extraction conditions were optimised as extraction pressure 200 MPa, extraction solvent 95% ethanol, solid-liquid ratio 1:30 (g/mL) and extraction time 2 min. A two-phase n-hexane/ethyl acetate/i-propanol/water (1:3:1.5:4.5, v/v) solvent system was optimised with 10 mmol triethylamine in the upper phase and 10 mmol trifluoroacetic acid in lower phase in PZRCCC. The sample loading was optimised as 2.50 g. Moreover, the samples were evaluated for anti-breast cancer activity later on. Results The 2.50 g sample loading yielded 0.45 g of sanguinarine and 0.59 g chelerythrine in one-step separation using PZRCCC. The anti-breast cancer activities of sanguinarine and chelerythrine were found stronger than positive control (vincristine 5.04 μg/mL) with half-maximal inhibitory concentration values of 0.96 and 3.00 μg/mL, respectively. Conclusion This study showed that the established methods were efficient in extraction (UHPE) and separation (PZRCCC) of the sanguinarine and chelerythrine from M. cordata.

This study reports the development of iron-chelated semiconducting polycomplex nanoparticles (SPFeN) for photoacoustic (PA) imaging-guided photothermal ferrotherapy of cancer. The hybrid polymeric nanoagent comprises a ferroptosis initiator (Fe3+ ) and an amphiphilic semiconducting polycomplex (SPC ) serving as both the photothermal nanotransducer and iron ion chelator. By virtue of poly(ethylene glycol) (PEG) grafting and its small size, SPFeN accumulates in the tumor of living mice after systemic administration, which can be monitored by PA imaging. In the acidic tumor microenvironment, SPFeN generates hydroxyl radicals, leading to ferroptosis; meanwhile, under NIR laser irradiation, it generates localized heat to not only accelerate the Fenton reaction but also implement photothermal therapy. Such a combined photothermal ferrotherapeutic effect of SPFeN leads to minimized dosage of iron compared to previous studies and effectively inhibits the tumor growth in living mice, which is not possible for the controls. Ministry of Education (MOE) Nanyang Technological University K.P. thanks Nanyang Technological University (Start-Up grant: M4081627) and Singapore Ministry of Education Academic Research Fund Tier 1 (2017-T1-002-134 RG147/17 & 2019-T1-002-045 RG125/19) and Academic Research Fund Tier 2 (MOE2018-T2-2-042) for the financial support.

Noninvasive regulation of CRISPR/Cas9 gene editing is conducive to understanding of gene function and development of gene therapy; however, it remains challenging. Herein, a photolabile semiconducting polymer nanotransducer (pSPN) is synthesized to act as the gene vector to deliver CRISPR/Cas9 plasmids into cells and also as the photoregulator to remotely activate gene editing. pSPN comprises a 1 O2 -generating backbone grafted with polyethylenimine brushes through 1 O2 -cleavable linkers. NIR photoirradiation spontaneously triggers the cleavage of gene vectors from pSPN, resulting in the release of CRISPR/Cas9 plasmids and subsequently initiating gene editing. This system affords 15- and 1.8-fold enhancement in repaired gene expression relative to the nonirradiated controls in living cells and mice, respectively. As this approach does not require any specific modifications on biomolecular components, pSPN represents the first generic nanotransducer for in vivo regulation of CRISPR/Cas9 gene editing.

Despite the great promises of sonodynamic therapy (SDT) in combination cancer therapy, its clinical applications are hindered by the "always-on" pharmacological activities of therapeutic agents and the lack of efficient sonosensitizers. Herein, the development of semiconducting polymers as efficient sonosensitizers and further development of sono-immunotherapeutic nanobodies (SPNAb ) for activatable cancer sono-immunotherapy are reported. Conjugation of anti-CTLA-4 antibodies onto the polymer nanoparticles through a 1 O2 -cleavable linker affords SPNAb with relatively low CTLA-4 binding affinity. Upon sono-irradiation, SPNAb generates 1 O2 not only to elicit a sonodynamic effect to induce immunogenic cell death, but also to release anti-CTLA-4 antibodies and trigger in situ checkpoint blockade. Such a synergistic therapeutic action mediated by SPNAb modulates the tumoricidal function of T-cell immunity by promoting the proliferation of cytotoxic T lymphocytes and depleting immunosuppressive regulatory T cells, resulting in effective tumor regression, metastasis inhibition, durable immunological memory, and prevention of relapse. Therefore, this study represents a proof-of-concept sonodynamic strategy using semiconducting polymers for precise spatiotemporal control over immunotherapy. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) K.P. thanks Singapore Ministry of Education, Academic Research Fund Tier 1 (2019-T1-002-045, RG125/19, RT05/20), Academic Research Fund Tier 2 (MOE2018-T2-2-042; MOE-T2EP30220-0010), and A*STAR SERC AME Programmatic Fund (SERC A18A8b0059) for the financial support.

Objective Mitochondrial dysfunction, oxidative stress and nucleus pulposus (NP) cell apoptosis are important contributors to the development and pathogenesis of intervertebral disc degeneration (IDD). Here, we comprehensively evaluated the effects of mitochondrial dynamics, mitophagic flux and Nrf2 signalling on the mitochondrial quality control, ROS production and NP cell survival in in vitro and ex vivo compression models of IDD and explored the effects of the mitochondria‐targeted anti‐oxidant MitoQ and its mechanism. Material and methods Human NP cells were exposed to mechanical compression to mimic pathological conditions. Results Compression promoted oxidative stress, mitochondrial dysfunction and NP cell apoptosis. Mechanistically, compression disrupted the mitochondrial fission/fusion balance, inducing fatal fission. Concomitantly, PINK1/Parkin‐mediated mitophagy was activated, whereas mitophagic flux was blocked. Nrf2 anti‐oxidant pathway was insufficiently activated. These caused the damaged mitochondria accumulation and persistent oxidative damage. Moreover, MitoQ restored the mitochondrial dynamics balance, alleviated the impairment of mitophagosome‐lysosome fusion and lysosomal function and enhanced the Nrf2 activity. Consequently, damaged mitochondria were eliminated, redox balance was improved, and cell survival increased. Additionally, MitoQ alleviated IDD in an ex vivo rat compression model. Conclusions These findings suggest that comodulation of mitochondrial dynamics, mitophagic flux and Nrf2 signalling alleviates sustained mitochondrial dysfunction and oxidative stress and represents a promising therapeutic strategy for IDD; furthermore, our results provide evidence that MitoQ might serve as an effective therapeutic agent for this disorder.

Tumor immunometabolism contributes substantially to tumor proliferation and immune cell activity, and thus plays a crucial role in the efficacy of cancer immunotherapy. Modulation of immunometabolism to boost cancer immunotherapy is mostly based on small-molecule inhibitors, which often encounter the issues of off-target adverse effects, drug resistance, and unsustainable response. In contrast, enzymatic therapeutics can potentially bypass these limitations but has been less exploited. Herein, an organic polymer nanoenzyme (SPNK) with near-infrared (NIR) photoactivatable immunotherapeutic effects is reported for photodynamic immunometabolic therapy. SPNK is composed of a semiconducting polymer core conjugated with kynureninase (KYNase) via PEGylated singlet oxygen (1 O2 ) cleavable linker. Upon NIR photoirradiation, SPNK generates 1 O2 not only to exert photodynamic effect to induce the immunogenic cell death of cancer, but also to unleash KYNase and trigger its activity to degrade the immunosuppressive kynurenine (Kyn). Such a combinational effect mediated by SPNK promotes the proliferation and infiltration of effector T cells, enhances systemic antitumor T cell immunity, and ultimately permits inhibition of both primary and distant tumors in living mice. Therefore, this study provides a promising photodynamic approach toward remotely controlled enzymatic immunomodulation for improved anticancer therapy. Ministry of Education (MOE) Nanyang Technological University K.P. thanks Nanyang Technological University (Start-up Grant No. NTU-SUG: M4081627.120) and the Singapore Ministry of Education, Academic Research Fund Tier 1 (2019-T1-002-045; 2018-T1-001-173) and Academic Research Fund Tier 2 (MOE2018-T2-2-042) for the financial support.

The facile hydrothermal synthesis of polyethyleneimine (PEI)-coated iron oxide (Fe3O4) nanoparticles (NPs) doped with Gd(OH)3 (Fe3O4-Gd(OH)3-PEI NPs) for dual mode T1- and T2-weighted magnetic resonance (MR) imaging applications is reported. In this approach, Fe3O4-Gd(OH)3-PEI NPs are synthesized via a hydrothermal method in the presence of branched PEI and Gd(III) ions. The PEI coating onto the particle surfaces enables further modification of poly(ethylene glycol) (PEG) in order to render the particles with good water dispersibility and improved biocompatibility. The formed Fe3O4-Gd(OH)3-PEI-PEG NPs have a Gd/Fe molar ratio of 0.25:1 and a mean particle size of 14.4 nm and display a relatively high r2 (151.37 × 10−3m−1 s−1) and r1 (5.63 × 10−3m−1 s−1) relaxivity, affording their uses as a unique contrast agent for T1- and T2-weighted MR imaging of rat livers after mesenteric vein injection of the particles and the mouse liver after intravenous injection of the particles, respectively. The developed Fe3O4-Gd(OH)3-PEI-PEG NPs may hold great promise to be used as a contrast agent for dual mode T1- and T2-weighted self-confirmation MR imaging of different biological systems.

A unique dendrimer-assisted approach is reported to create Fe3O4/Au nanocomposite particles (NCPs) for targeted dual mode computed tomography/magnetic resonance (CT/MR) imaging of tumors. In this approach, preformed Fe3O4 nanoparticles (NPs) are assembled with multilayers of poly(γ-glutamic acid) (PGA)/poly(L-lysine)/PGA/folic acid (FA)-modified dendrimer-entrapped gold nanoparticles via a layer-by-layer self-assembly technique. The interlayers are crosslinked via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chemistry, the assembled Au core NPs are then used as seed particles for subsequent seed-mediated growth of Au shells via iterative Au salt reduction process, and subsequent acetylation of the remaining amines of dendrimers leads to the formation of Fe3O4/Au(n.)Ac-FA NCPs with a tunable molar ratio of Au/Fe3O4. It is shown that the Fe3O4/Au(n.)Ac-FA NCPs at an optimized Au/Fe3O4 molar ratio of 2.02 display a relatively high R2 relaxivity (92.67 × 10(-3) M(-1) s(-1)) and good X-ray attenuation property, and are cytocompatible and hemocompatible in the given concentration range. Importantly, with the FA-mediated targeting, the Fe3O4/Au(n.)Ac-FA NCPs are able to be specifically uptaken by cancer cells overexpressing FA receptors, and be used as an efficient nanoprobe for targeted dual mode CT/MR imaging of a xenografted tumor model. With the versatile dendrimer chemistry, the developed Fe3O4/Au NCPs may be differently functionalized, thereby providing a unique platform for diagnosis and therapy of different biological systems.

This article reports the recent advances in hydrothermally synthesized iron oxide nanoparticles (Fe3O4 NPs) used for magnetic resonance (MR) imaging applications. The hydrothermally synthesized Fe3O4 NPs have a controllable size, narrow size distribution, and tunable magnetic properties. For biomedical imaging applications, the surface-functionalized Fe3O4 NPs with good water dispersibility, colloid stability, and relatively high r 2 relaxivity can be synthesized using modified hydrothermal routes, and can be further surface functionalized with targeting ligands, enabling effective tumor MR imaging with high specificity via receptor-mediated active targeting pathway. What is more, the hydrothermal route can also be used to synthesize Fe3O4@Au composite nanoparticles (CNPs) for dual-mode MR and computed tomo­graphy (CT) imaging applications. Some of the key developments in this area of research will be introduced in detail.

Inhibition of protein biosynthesis is a promising strategy to develop new therapeutic modalities for cancers; however, noninvasive precise regulation of this cellular event in living systems has been rarely reported. In this study, a semiconducting polymer nanoblockader (SPNB ) is developed that can inhibit intracellular protein synthesis upon near-infrared (NIR) photoactivation to synergize with photodynamic therapy (PDT) for metastasis-inhibited cancer therapy. SPNB is self-assembled from an amphiphilic semiconducting polymer which is grafted with poly(ethylene glycol) conjugated with a protein biosynthesis blockader through a singlet oxygen (1 O2 ) cleavable linker. Such a designed molecular structure not only enables generation of 1 O2 under NIR photoirradiation for PDT, but also permits photoactivation of blockaders to terminate protein translation. Thereby, SPNB exerts a synergistic action to afford an enhanced therapeutic efficacy in tumor ablation. More importantly, SPNB -mediated photoactivation of protein synthesis inhibition precisely and remotely downregulates the expression levels of metastasis-related proteins in tumor tissues, eventually contributing to the complete inhibition of lung metastasis. This study thus proposes a photoactivatable protherapeutic design for metastasis-inhibited cancer therapy.

Photoacoustic (PA) imaging in the second near‐infrared (NIR‐II) window (1000–1700 nm) holds great promise for deep‐tissue diagnosis due to the reduced light scattering and minimized tissue absorption; however, exploration of such a noninvasive imaging technique is greatly constrained by the lack of biodegradable NIR‐II absorbing agents. Herein, the first series of metabolizable NIR‐II PA agents are reported based on semiconducting polymer nanoparticles (SPNs). Such completely organic nanoagents consist of π‐conjugated yet oxidizable optical polymer as PA generator and hydrolyzable amphiphilic polymer as particle matrix to provide water solubility. The obtained SPNs are readily degraded by myeloperoxidase and lipase abundant in phagocytes, transforming from nonfluorescent nanoparticles (30 nm) into NIR fluorescent ultrasmall metabolites (≈1 nm). As such, these nanoagents can be effectively cleared out via both hepatobiliary and renal excretions after systematic administration, leaving no toxicity to living mice. Particularly these nanoagents possess high photothermal conversion efficiencies and emit bright PA signals at 1064 nm, enabling sensitive NIR‐II PA imaging of both subcutaneous tumor and deep brain vasculature through intact skull in living animals at a low systematic dosage. This study thus provides a generalized molecular design toward organic metabolizable semiconducting materials for biophotonic applications in NIR‐II window. MOE (Min. of Education, S’pore) Accepted version

Cancer immunotherapy have recently gained much attention in the search of cancer cure due to its ability to “train” the immune system in seeking out and removing residual tumor cells. However, relying on cancer immunotherapy alone may fail to ablate primary tumors. Phototherapy is a promising modality that offers an elegant solution to eradicate tumors through the simple application of light irradiation and meanwhile triggers immune responses by immunogenic cell death to enhance antitumor immunity. Uniting phototherapy with cancer immunotherapy have been found to achieved synergistic outcomes, promote cancer regression and even attain immunologic memory. This review summarizes the recent studies on utilizing phototherapy and immunotherapy combinatorial approach to treat cancer. A variety of nanoplatforms have been investigated in combination with immunoadjuvants and immune checkpoint blockades such as CTLA4 and PD-L1 pathways. Higher levels of proinflammatory cytokines, improved migration of dendritic cells and an increased ratio of tumor-infiltrating cytotoxic T cells against regulatory T cells are revealed in many studies, offering a glimpse into the mechanistic principles. Future advancements targeting different cancer checkpoints that could offer better immunosuppression coupled with other phototherapeutic strategies remains to be explored in accomplishing complete elimination of cancer. MOE (Min. of Education, S’pore) Accepted version

The fact that cancer metastasis is the main cause of death for most cancer patients necessitates the development of imaging tools for sensitive detection of metastases. Although optical imaging has high temporospatial resolution, tissue autofluorescence compromises the sensitivity for in vivo imaging of cancer metastasis. Herein, the synthesis of a series of photosensitizer-incorporated poly(p-phenylenevinylene)-based semiconducting copolymers and their utility as near-infrared (NIR) afterglow imaging nanoagents that emit light after cessation of light irradiation are reported. As compared with nondoped nanoparticles, the nanoparticles derived from the photosensitizer-incorporated copolymers have red-shifted NIR luminescence and amplified afterglow signals, allowing the detection of tiny peritoneal metastatic tumors almost invisible to naked eye. Moreover, the intrinsic oxygen-sensitive nature of afterglow makes those nanoagents potentially useful for in vivo imaging of oxygen levels. Thus, this study introduces a generation of light-excitation-free background-minimized optical imaging agents for the sensitive detection of diseased tissues in vivo. MOE (Min. of Education, S’pore) Accepted version

Photodynamic therapy (PDT), which utilizes reactive oxygen species to kill cancer cells, has found wide applications in cancer treatment. However, the hypoxic nature of most solid tumors can severely restrict the efficiency of PDT. Meanwhile, the hydrophobicity and limited tumor selectivity of some photosensitizers also reduce their PDT efficacy. Herein, a photosensitizer-Pd@Pt nanosystem (Pd@Pt-PEG-Ce6) is designed for highly efficient PDT by overcoming these limitations. In the nanofabrication, Pd@Pt nanoplates, exhibiting catalase-like activity to decompose H2O2 to generate oxygen, are first modified with bifunctional PEG (SH-PEG-NH2). Then the Pd@Pt-PEG is further covalently conjugated with the photosensitizer chlorin e6 (Ce6) to get Pd@Pt-PEG-Ce6 nanocomposite. The Pd@Pt-PEG-Ce6 exhibits good biocompatibility, long blood circulation half-life, efficient tumor accumulation, and outstanding imaging properties. Both in vitro and in vivo experimental results clearly indicate that Pd@Pt-PEG-Ce6 effectively delivers photosensitizers to cancer cells/tumor sites and triggers the decomposition of endogenous H2O2 to produce oxygen, resulting in a remarkably enhanced PDT efficacy. Moreover, the moderate photothermal effect of Pd@Pt nanoplates also strengthen the PDT of Pd@Pt-PEG-Ce6. Therefore, by integrating the merits of high tumor-specific accumulation, hypoxia modulation function, and mild photothermal effect into a single nanoagent, Pd@Pt-PEG-Ce6 readily acts as an ideal nanotherapeutic platform for enhanced cancer PDT.

Nanostructured materials have drawn a broad attention for their applications in biomedical fields. Ligand-modified nanomaterials can well mimic the dynamic extracellular matrix (ECM) microenvironments to regulate cell functions and fates. Herein, ECM mimetic gold nanoparticles (Au NPs) with tunable surface arginine-glycine-aspartate (RGD) density are designed and synthesized to induce the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). The biomimetic Au NPs with an average size of 40 nm shows good biocompatibility without affecting the cell proliferation in the studied concentration range. The RGD motifs on Au NPs surface facilitate cellular uptake of NPs into monolayer hMSCs through integrin-mediated endocytosis. The biomimetic NPs have a promotive effect on cartilaginous matrix production and marker gene expression in cell pellet culture, especially for the biomimetic Au NPs with high surface RGD density. This study provides a novel strategy for fabricating biomimetic NPs to regulate cell differentiation, which holds great potentials in tissue engineering and biomedical applications.

Development of multifunctional nanoprobes for tumor diagnosis is extremely important in the field of molecular imaging. In this study, the facile synthesis of lactobionic acid (LA)-targeted superparamagnetic iron oxide (Fe3O4) nanoparticles (NPs) with ultrahigh relaxivity for targeted magnetic resonance (MR) imaging of an orthotopic hepatocellular carcinoma (HCC) is reported. Polyethyleneimine (PEI)-stabilized Fe3O4 NPs prepared via a mild reduction route are sequentially coupled with fluorescein isothiocyanate and polyethylene glycol-LA (LA-PEG-COOH) segment, followed by acetylation of the remaining PEI surface amines. The formed LA-targeted Fe3O4 NPs are thoroughly characterized. It is shown that the developed multifunctional LA-targeted Fe3O4 NPs are colloidally stable and water-dispersible, display an ultrahigh r 2 relaxivity (579.89 × 10−3 m −1 s−1) and excellent hemocompatibility and cytocompatibility in the given concentration range, and can target HepG2 cells overexpressing asialoglycoprotein receptors as confirmed by in vitro cellular uptake assay, flow cytometry, and confocal microscopy. Most strikingly, the developed multifunctional LA-targeted Fe3O4 NPs can be used as a nanoprobe for targeted MR imaging of HepG2 cells in vitro and an orthotopic tumor model of HCC in vivo. With the ultrahigh r 2 relaxivity and the versatile PEI amine-mediated conjugation chemistry, a range of different Fe3O4 NP-based nanoprobes may be developed for theranostics of different types of cancer.