Recent Advances in Fluorescent Probes for Cancer Biomarker Detection.

Many important biological species have been identified as cancer biomarkers and are gradually becoming reliable targets for early diagnosis and late therapeutic evaluation of cancer. However, accurate quantitative detection of cancer biomarkers remains challenging due to the complexity of biological systems and the diversity of cancer development. Fluorescent probes have been extensively utilized for identifying biological substances due to their notable benefits of being non-invasive, quickly responsive, highly sensitive and selective, allowing real-time visualization, and easily modifiable. This review critiques fluorescent probes used for detecting and imaging cancer biomarkers over the last five years. Focuses are made on the design strategies of small-molecule and nano-sized fluorescent probes, the construction methods of fluorescence sensing and imaging platforms, and their further applications in detection of multiple biomarkers, including enzymes, reactive oxygen species, reactive sulfur species, and microenvironments. This review aims to guide the design and development of excellent cancer diagnostic fluorescent probes, and promote the broad application of fluorescence analysis in early cancer diagnosis.

Near-Infrared Light-Triggered Lysosome-Targetable Carbon Dots for Photothermal Therapy of Cancer.

Photothermal therapy (PTT) has inherent advantages in the treatment of hypoxic tumors due to its optically controlled selectivity on tumor ablation and oxygen-independent nature. The subcellular organelle-targeting capability and photothermal conversion efficiency (PCE) at near-infrared (NIR) wavelength are the key parameters in the assessment of the photothermal agent (PTA). Here, we report that carbon dots (CDs) prepared by the hydrothermal treatment of coronene derivatives show a high PCE of 54.7% at 808 nm, which can be attributed to the narrow band gap and the presence of amounts of continuous energy bands on CDs. Moreover, the vibrations in the layered graphite structures of the CDs also increase the rate of nonradiative transition and thus enhance the PCE. Furthermore, the CDs also possess excellent photostability, biocompatibility, and cell penetration capability and could mainly accumulate in the lysosomes. These experiment results have proved that the CDs are suitable as an efficient NIR light-triggered PTA for efficient PTT against cancer.

Fabrication of small-structure red-emissive fluorescent probes for plasma membrane enables quantification of nuclear to cytoplasmic ratio in live cells and tissues.

Nuclear to cytoplasmic ratio is one of the vital parameters in diagnosis of cancer by means of hematoxylin-eosin (HE) stained histopathology. However, HE histopathology dependent on mechanical tissue slice damages biosamples and exhibits insufficient accuracy. Herein, we rationally prepared two small-molecule plasma membrane fluorescent probes with red-emitting fluorescence for visualizing plasma membrane in living cells and tissues. Their fluorescence intensities are strongly affected by environmental viscosity, which enables the exclusive imaging of plasma membrane in high fidelity. The probes can visualize plasma membrane in SiHa and rat blood red cells. Particularly, the probes are able to visualize T-tubule (transverse tubule) in skeletal muscle tissues successfully, suggesting their ability to image plasma membrane in tissues. In cooperation with Hoechst 33342, the nuclear to cytoplasmic ratio was successfully qualified in live cells and tissues. We believe these probes may have potential applications in facilitating the study on histopathology and the related areas.

Bright Aggregation-Induced Emission Nanoparticles for Two-Photon Imaging and Localized Compound Therapy of Cancers.

Photodynamic therapy (PDT), a noninvasive therapeutic strategy for cancer treatment, which always suffers from the low reactive oxygen species (ROS) yield of traditional organic dyes. Herein, we present lipid-encapsulated aggregation-induced emission nanoparticles (AIE NPs) that have a high quantum yield (23%) and a maximum two-photon absorption (TPA) cross-section of 560 GM irradiated by near-infrared light (800 nm). The AIE NPs can serve as imaging agents for spatiotemporal imaging of tumor tissues with a penetration depth up to 505 µm on mice melanoma model. Importantly, the AIE NPs can simultaneously generate singlet oxygen (1O2) and highly toxic hydroxyl radicals (•OH) upon irradiation with 800 nm irradiation for photodynamic tumor ablation. In addition, the AIE NPs can be effectively cleared from the mouse body after the imaging and therapy. This study provides a strategy to develop theranostic agents for cancer image-guided PDT with high brightness, superior photostability, and high biosafety.

Visualizing semipermeability of the cell membrane using a pH-responsive ratiometric AIEgen.

In clinical chemotherapy, some basic drugs cannot enter the hydrophobic cell membrane because of ionization in the acidic tumor microenvironment, a phenomenon known as ion trapping. In this study, we developed a method to visualize this ion trapping phenomenon by utilizing a pH-responsive ratiometric AIEgen, dihydro berberine (dhBBR). By observing the intracellular fluorescence of dhBBR, we found that non-ionized dhBBR can enter cells more easily than ionized forms, which is in accordance with the concept of ion trapping. In addition, dhBBR shows superior anti-photobleaching ability to Curcumin thanks to its AIE properties. These results suggest that dhBBR can serve as a bioprobe for ion trapping.

In Vitro Light-Up Visualization of a Subunit-Specific Enzyme by an AIE Probe via Restriction of Single Molecular Motion.

Enzymes contain several subunits to maintain different biological functions. However, it remains a great challenge for specific discrimination of one subunit over another. Toward this end, the fluorescent probe TPEMA is now presented for highly specific detection of the B subunit of cytosolic creatine (CK) kinase isoenzyme (CK-B). Owing to its aggregation-induced emission property, TPEMA shows highly boosted emission toward CK-B with a fast response time and very low interference from other analytes, including the M subunit of CK (CK-M). With the aid of a Job plot assay, ITC assay and molecular dynamics simulation, it was directly confirmed that the remarkably enhanced fluorescence of TPEMA in the presence of CK-B results from the restriction of single molecular motion in the cavity. Selective wash-free fluorescence imaging of CK-B in macrophages under different treatments was successfully demonstrated.

Phage-Guided Targeting, Discriminative Imaging, and Synergistic Killing of Bacteria by AIE Bioconjugates.

New agents with particular specificity toward targeted bacteria and superefficacy in antibacterial activity are urgently needed in facing the crisis of worldwide antibiotic resistance. Herein, a novel strategy by equipping bacteriophage (PAP) with photodynamic inactivation (PDI)-active AIEgens (luminogens with aggregation-induced emission property) was presented to generate a type of AIE-PAP bioconjugate with superior capability for both targeted imaging and synergistic killing of certain species of bacteria. The targeting ability inherited from the bacteriophage enabled the bioconjugates to specifically recognize the host bacteria with preserved infection activity of phage itself. Meanwhile, the AIE characteristic empowered them a monitoring functionality, and the real-time tracking of their interactions with targets was therefore realized via convenient fluorescence imaging. More importantly, the PDI-active AIEgens could serve as powerful in situ photosensitizers producing high-efficiency reactive oxygen species (ROS) under white light irradiation. As a result, selective targeting and synergistic killing of both antibiotic-sensitive and multi-drug-resistant (MDR) bacteria were successfully achieved in in vitro and in vivo antibacterial tests with excellent biocompatibility. This novel AIE-phage integrated strategy would diversify the existing pool of antibacterial agents and inspire the development of promising drug candidates in the future.

Cancer cell discrimination and dynamic viability monitoring through wash-free bioimaging using AIEgens.

Cancer cell discrimination and cellular viability monitoring are closely related to human health. A universal and convenient fluorescence system with a dual function of wide-spectrum cancer cell discrimination and dynamic cellular viability monitoring is desperately needed, and is still extremely challenging. Herein we present a series of aggregation-induced emission luminogens (AIEgens) (denoted as IVP) which can allow accurate discrimination between cancer and normal cells and dynamic monitoring of cellular viability through mitochondria-nucleolus migration. By regulating the lengths and positions of alkyl chains in IVP molecules, we systematically studied the discrimination behavior of these AIEgens between cancer cells and normal cells and further investigated how they can migrate between the mitochondria and nucleolus based on the change of mitochondrial membrane potential (ΔΨ m). Using IVP-02 as a model molecule, wash-free bioimaging, excellent two-photon properties, and low cytotoxicity were demonstrated. This present work proves that these designed IVP AIEgens show great potential for cancer identification and metastasis monitoring, as well as activity evaluation and screening of drugs.

Functionalized Acrylonitriles with Aggregation-Induced Emission: Structure Tuning by Simple Reaction-Condition Variation, Efficient Red Emission, and Two-Photon Bioimaging.

Acrylonitriles with aggregation-induced emission (AIE) characteristics have been found to show promising applications in two-photon biomedical imaging. Generally, elaborate synthetic efforts are required to achieve different acrylonitriles with distinct functionalities. In this work, we first reported the synthesis of two different group-functionalized AIE-active acrylonitriles (TPAT-AN-XF and 2TPAT-AN) obtained simply by mixing the same reactants at different temperatures using a facile and transition metal-free synthetic method. These two AIE luminogens (AIEgens) exhibit unique properties such as bright red emission in the solid state, large Stokes shift, and large two-photon absorption cross section. Water-soluble nanoparticles (NPs) of 2TPAT-AN were prepared by a nanoprecipitation method. In vitro imaging data show that 2TPAT-AN NPs can selectively stain lysosome in live cells. Besides one-photon imaging, remarkable two-photon imaging of live tumor tissues can be achieved with high resolution and deep tissue penetration. 2TPAT-AN NPs show high biocompatibility and are successfully utilized in in vivo long-term imaging of mouse tumors with a high signal-to-noise ratio. Thus, the present work is anticipated to shed light on the preparation of a library of AIE-active functionalized acrylonitriles with intriguing properties for biomedical applications.

Selectively light-up hydrogen peroxide in hypoxic cancer cells with a novel fluorescent probe.

A novel fluorescent turn-on probe (HCyHP) was developed in a simple two-step synthesis for monitoring of exogenous and endogenous H2O2 levels in biological samples and hypoxic cancer diagnosis.

Single Near-Infrared Emissive Polymer Nanoparticles as Versatile Phototheranostics.

Attaining consistently high performance of diagnostic and therapeutic functions in one single nanoplatform is of great significance for nanomedicine. This study demonstrates the use of donor-acceptor (D-A) structured polymer (TBT) to develop a smart "all-five-in-one" theranostic that conveniently integrates fluorescence/photoacoustic/thermal imaging and photodynamic/photothermal therapy into single nanoparticle. The prepared nanoparticles (TBTPNPs) exhibit near-infrared emission, high water solubility, excellent light resistance, good pH stability, and negligible toxicity. Additionally, the TBTPNPs exhibit an excellent singlet oxygen (1O2) quantum yield (40%) and high photothermal conversion efficiency (37.1%) under single-laser irradiation (635 nm). Apart from their two phototherapeutic modalities, fluorescence, photoacoustic signals, and thermal imaging in vivo can be simultaneously achieved because of their enhanced permeability and retention effects. This work demonstrates that the prepared TBTPNPs are "all-five-in-one" phototheranostic agents that can exhibit properties to satisfy the "one-fits-all" requirement for future phototheranostic applications. Thus, the prepared TBTPNPs can provide fundamental insights into the development of PNP-based nanoagents for cancer therapy.

Water-Soluble Polythiophene for Two-Photon Excitation Fluorescence Imaging and Photodynamic Therapy of Cancer.

Positively charged water-soluble polythiophene (PT0) that could self-assemble into nanoparticles in pure water solution was designed and synthesized. PT0 exhibited high photostabilities and pH stabilities, excellent biocompatibility, strong 1O2 generation capability, and large two-photon absorption cross sections. Moreover, we showed that the fluorescence of PT0 was unaffected by the interference of biomolecules and metal ions. As an example application, PT0 was demonstrated to be capable of simultaneous cell imaging and photodynamic therapy under either one-photon or two-photon excitation modes.

Versatile Polymer Nanoparticles as Two-Photon-Triggered Photosensitizers for Simultaneous Cellular, Deep-Tissue Imaging, and Photodynamic Therapy.

Clinical applications of current photodynamic therapy (PDT) photosensitizers (PSs) are often limited by their absorption in the UV-vis range that possesses limited tissue penetration ability, leading to ineffective therapeutic response for deep-seated tumors. Alternatively, two-photon excited PS (TPE-PS) using NIR light triggered is one the most promising candidates for PDT improvement. Herein, multimodal polymer nanoparticles (PNPs) from polythiophene derivative as two-photon fluorescence imaging as well as two-photon-excited PDT agent are developed. The prepared PNPs exhibit excellent water dispersibility, high photostability and pH stability, strong fluorescence brightness, and low dark toxicity. More importantly, the PNPs also possess other outstanding features including: (1) the high 1 O2 quantum yield; (2) the strong two-photon-induced fluorescence and efficient 1 O2 generation; (3) the specific accumulation in lysosomes of HeLa cells; and (4) the imaging detection depth up to 2100 µm in the mock tissue under two-photon. The multifunctional PNPs are promising candidates as TPE-PDT agent for simultaneous cellular, deep-tissue imaging, and highly efficient in vivo PDT of cancer.

Polymer nanoparticles with high photothermal conversion efficiency as robust photoacoustic and thermal theranostics.

Synthesis of photothermal agents with absorption in the near-infrared (NIR) region and featuring excellent photostability, high photothermal conversion efficiency, and good biocompatibility is necessary for the application of photothermal therapy (PTT). In this work, a low band gap thiophene-benzene-diketopyrrolopyrrole (TBD)-based polymer was synthesized and used to fabricate TBD polymer nanoparticles (TBDPNPs) through a one-step nanoprecipitation method. The obtained near-infrared-absorbing TBDPNPs presented good water dispersibility, high photothermal stability, and low toxicity. Significantly, the TBDPNPs exhibited an ultrahigh photothermal conversion efficiency of approximately 68.1% under 671 nm laser irradiation. In addition, photoacoustic (PA) imaging, with high spatial resolution and deep tissue penetration, showed that the TBDPNPs targeted tumor sites through the enhanced permeability and retention effect. Therefore, the robust TBDPNPs with a photothermal conversion efficiency of 68.1% can serve as an excellent therapeutic agent for PA-imaging-guided PTT.

Near-Infrared Organic Dye-Based Nanoagent for the Photothermal Therapy of Cancer.

Given their easy structural modification and good biocompatibility advantages, near-infrared (NIR) organic dyes with a large molar extinction coefficient, while a superlow fluorescence quantum yield shows considerable potential application in photothermal therapy (PTT). Herein, a new NIR-absorbing asymmetric cyanine dye, namely, RC, is designed and synthesized via the hybrid of rhodamine and hemicyanine derivatives. RC-BSA nanoparticles (NPs) are fabricated by using the bovine serum albumin (BSA) matrix. The NPs exhibit a strong NIR absorption peak at ∼868 nm and 28.7% photothermal conversion efficiency. Based on these features, RC-BSA NPs exhibit excellent performance in ablating tumor under a 915 nm laser radiation through a PTT mechanism. These NPs show no obvious toxicity to the treated mice. Thus, RC-BSA NPs can used as a new NIR laser-triggered PTT agent in cancer treatment.

Gold nanorod@silica-carbon dots as multifunctional phototheranostics for fluorescence and photoacoustic imaging-guided synergistic photodynamic/photothermal therapy.

Phototheranostics, which is the application of light in the diagnostic imaging and therapy of cancer, has shown great promise for multimodal cancer imaging and effective therapy. Herein, we developed multifunctional gold nanorod@silica-carbon dots (GNR@SiO2-CDs) as a phototheranostic agent by incorporating carbon dots (CDs) with gold nanorods (GNRs), using SiO2 as a scaffold. In GNR@SiO2-CDs, the GNRs act as both photoacoustic (PA) imaging and photothermal therapy (PTT) agents, and the CDs serve as fluorescence (FL) imaging and photodynamic therapy (PDT) agents. The introduction of SiO2 not only improves the chemical stability of the GNRs and CDs in the physiological environment but also prevents the absolute quenching of the fluorescence of the CDs by GNRs. These collective properties make GNR@SiO2-CDs a novel phototheranostic agent, in which high sensitivity and good spatial resolution of FL/PA imaging can be achieved to guide PDT/PTT treatments through i.v. administration. The combination of PDT and PTT proved to be more efficient in killing cancer cells compared to PDT or PTT alone under a low dose of laser irradiation (≤0.5 W cm(-2)). Furthermore, GNR@SiO2-CDs could be cleared out from the body of mice, indicating the low toxicity of this phototheranostic agent. Our work highlights the potential of using GNRs and CDs as novel phototheranostic agents for multifunctional cancer therapies.

Aminobenzofuran-fused rhodamine dyes with deep-red to near-infrared emission for biological applications.

Aminobenzofuran-fused rhodamine dyes (AFR dyes) containing an amino group were constructed by an efficient condensation based on 3-coumaranone derivatives. AFR dyes exhibited significantly improved properties, including deep-red and near-infrared emissions, a large Stokes shift, good photostability, and wide pH stability. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium assay experiments show that these AFR dyes are biocompatible for their low cytotoxicity to both A549 and HeLa cells. Cell imaging data reveal that AFR1, AFR1E, and AFR2 are mainly located in the mitochondria, while AFR3 is a lysosome tracker. As far as we know, NIR AFR3 is the longest fluorescent rhodamine derivative containing the amino group. These amino group-containing AFR dyes hold great potential in fluorogenic detection, biomolecule labeling, and cell imaging.

A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation.

Clinical applications of current photodynamic therapy (PDT) agents are often limited by their low singlet oxygen ((1)O2) quantum yields, as well as by photobleaching and poor biocompatibility. Here we present a new PDT agent based on graphene quantum dots (GQDs) that can produce (1)O2 via a multistate sensitization process, resulting in a quantum yield of ~1.3, the highest reported for PDT agents. The GQDs also exhibit a broad absorption band spanning the UV region and the entire visible region and a strong deep-red emission. Through in vitro and in vivo studies, we demonstrate that GQDs can be used as PDT agents, simultaneously allowing imaging and providing a highly efficient cancer therapy. The present work may lead to a new generation of carbon-based nanomaterial PDT agents with overall performance superior to conventional agents in terms of (1)O2 quantum yield, water dispersibility, photo- and pH-stability, and biocompatibility.