Semiconducting Polymer Dots for Dual-Wavelength Differential Background-Suppressed Photoacoustic Imaging.

Photoacoustic imaging (PAI) can sensitively detect regions and substances with strong optical absorption, which means that diseased tissue can be imaged with high contrast in the presence of surrounding healthy tissue through the photoacoustic effect. However, its signal intensity and resolution may be limited by background signals generated by endogenous chromophores such as melanin and hemoglobin. A feasible method for practical application of this so-called background-suppressed PAI is still lacking. In this work, a dual-wavelength differential background noise-suppressed photoacoustic tomography is developed based on organic semiconducting polymer dots (Pdots). The Pdots have a strong absorption peak at 945 nm, and then the absorption decreases sharply with the increase of wavelength, and the absorption intensity drops to only about a quarter of the original value at 1050 nm. The present system significantly suppresses the strong background noise of blood through dual-wavelength differential PAI, enabling precise monitoring of the distribution information of theranostic agents in diseased tissues. The signal-to-noise ratio of the theranostic agent distribution map is increased by about 20 dB. This work provides a platform for real-time and accurate monitoring of tumors and drugs, which helps avoid damage to healthy tissue during treatment and has clinical significance in cancer treatment.

Metallopolymer strategy to explore hypoxic active narrow-bandgap photosensitizers for effective cancer photodynamic therapy.

Practical photodynamic therapy calls for high-performance, less O2-dependent, long-wavelength-light-activated photosensitizers to suit the hypoxic tumor microenvironment. Iridium-based photosensitizers exhibit excellent photocatalytic performance, but the in vivo applications are hindered by conventional O2-dependent Type-II photochemistry and poor absorption. Here we show a general metallopolymerization strategy for engineering iridium complexes exhibiting Type-I photochemistry and enhancing absorption intensity in the blue to near-infrared region. Reactive oxygen species generation of metallopolymer Ir-P1, where the iridium atom is covalently coupled to the polymer backbone, is over 80 times higher than that of its mother polymer without iridium under 680 nm irradiation. This strategy also works effectively when the iridium atom is directly included (Ir-P2) in the polymer backbones, exhibiting wide generality. The metallopolymer nanoparticles exhibiting efficient O2•- generation are conjugated with integrin αvß3 binding cRGD to achieve targeted photodynamic therapy.

Site-Specific Modification of Single Domain Antibodies by Enzyme-Immobilized Magnetic Beads.

Nanobodies as imaging agents and drug conjugates have shown great potential for cancer diagnostics and therapeutics. However, site-specific modification of a nanobody with microbial transglutaminase (mTGase) encounters problems in protein separation and purification. Here, we describe a facile yet reliable strategy of immobilizing mTGase onto magnetic beads for site-specific nanobody modification. The mTGase immobilized on magnetic beads (MB-mTGase) exhibits catalytic activity nearly equivalent to that of the free mTGase, with good reusability and universality. Magnetic separation simplifies the protein purification step and reduces the loss of nanobody bioconjugates more effectively than size exclusion chromatography. Using MB-mTGase, we demonstrate site-specific conjugation of nanobodies with fluorescent dyes and polyethylene glycol molecules, enabling targeted immunofluorescence imaging and improved circulation dynamics and tumor accumulation in vivo. The combined advantages of MB-mTGase method, including high conjugation efficiency, quick purification, less protein loss, and recycling use, are promising for site-specific nanobody functionalization and biomedical applications.

Near-Infrared II Semiconducting Polymer Dots: Chain Packing Modulation and High-Contrast Vascular Imaging in Deep Tissues.

Fluorescence imaging in the second near-infrared (NIR-II) window has attracted considerable interest in investigations of vascular structure and angiogenesis, providing valuable information for the precise diagnosis of early stage diseases. However, it remains challenging to image small blood vessels in deep tissues because of the strong photon scattering and low fluorescence brightness of the fluorophores. Here, we describe our combined efforts in both fluorescent probe design and image algorithm development for high-contrast vascular imaging in deep turbid tissues such as mouse and rat brains with intact skull. First, we use a polymer blending strategy to modulate the chain packing behavior of the large, rigid, NIR-II semiconducting polymers to produce compact and bright polymer dots (Pdots), a prerequisite for in vivo fluorescence imaging of small blood vessels. We further developed a robust Hessian matrix method to enhance the image contrast of vascular structures, particularly the small and weakly fluorescent vessels. The enhanced vascular images obtained in whole-body mouse imaging exhibit more than an order of magnitude improvement in the signal-to-background ratio (SBR) as compared to the original images. Taking advantage of the bright Pdots and Hessian matrix method, we finally performed through-skull NIR-II fluorescence imaging and obtained a high-contrast cerebral vasculature in both mouse and rat models bearing brain tumors. This study in Pdot probe development and imaging algorithm enhancement provides a promising approach for NIR-II fluorescence vascular imaging of deep turbid tissues.

Self-Immolative Photosensitizers for Self-Reported Cancer Phototheranostics.

Photosensitizers to precise target and change fluorescence upon light illumination could accurately self-report where and when the photosensitizers work, enabling us to visualize the therapeutic process and precisely regulate treatment outcomes, which is the unremitting pursuit of precision and personalized medicine. Here, we report self-immolative photosensitizers by adopting a strategy of light-manipulated oxidative cleavage of C═C bonds that can generate a burst of reactive oxygen species, to cleave to release self-reported red-emitting products and trigger nonapoptotic cell oncosis. Strong electron-withdrawing groups are found to effectively suppress the C═C bond cleavage and phototoxicity via studying the structure-activity relationship, allowing us to elaborate NG1-NG5 that could temporarily inactivate the photosensitizer and quench the fluorescence by different glutathione (GSH)-responsive groups. Thereinto, NG2 with 2-cyano-4-nitrobenzene-1-sulfonyl group displays excellent GSH responsiveness than the other four. Surprisingly, NG2 shows better reactivity with GSH in weakly acidic condition, which inspires the application in weakly acidic tumor microenvironment where GSH elevates. To this end, we further synthesize NG-cRGD by anchoring integrin αvß3 binding cyclic pentapeptide (cRGD) for tumor targeting. In A549 xenografted tumor mice, NG-cRGD successfully deprotects to restore near-infrared fluorescence because of elevated GSH in tumor site, which is subsequently cleaved upon light irradiation releasing red-emitting products to report photosensitizer working, while effectively ablating tumors via triggered oncosis. The advanced self-immolative organic photosensitizer may accelerate the development of self-reported phototheranostics in future precision oncology.

Near Infrared-Absorbing Nanoparticle-Mediated Endovascular Photothermal Precision Embolization of Tumor Feeding Vessels for Starvation Treatment.

Photothermal therapy has attracted enormous attention as an efficient treatment modality in cancer ablation but still encounters a major bottleneck due to the limited penetration depth of light inside tissues. To overcome the challenge of deep tissue penetration, we present a strategy of endovascular photothermal precision embolization (EPPE), which employs an endovascular optical fiber to induce local embolization only in the entrance of feeding vessels through photothermal heating for the purpose of fully blocking the blood supply of the whole tumor. In EPPE, we apply a highly efficient and biocompatible photothermal agent, i.e., near-infrared (NIR)-light-absorbing diketopyrrolopyrrole-dithiophene-based nanoparticle, which exhibits a high cell-killing efficacy at a concentration of 200 µg/mL using 808 nm laser irradiation of 0.5 W/cm2 within 5 min in both 2D cell culture and a 3D tumor spheroid model. We verify the feasibility of EPPE in an ex vivo organ-structured recellularized liver model and further confirm the in vivo efficacy of the photothermal treatment in a rat liver model. The photothermal treatment combined with the embolization effect holds promise to serve as an effective starvation therapy to treat tumors of varying sizes and locations.

Development and Characterization of Nanobody-Derived CD47 Theranostic Pairs in Solid Tumors.

Overexpression of CD47 is frequently observed in various types of human malignancies, inhibiting myeloid-mediated elimination of tumor cells and affecting the prognosis of cancer patients. By mapping biomarker expression, immuno-positron emission tomography has been increasingly used for patient screening and response monitoring. By immunization alpacas with recombinant human CD47, we prepared a CD47-targeting nanobody C2 and developed [68Ga]Ga-NOTA-C2, followed by an exploration of the diagnostic value in CD47-expressing tumor models including gastric-cancer patient-derived xenograft models. By fusing C2 to an albumin binding domain (ABD), we synthesized ABDC2, which had increased in vivo half-life and improved targeting properties. We further labeled ABDC2 with 68Ga/89Zr/177Lu to develop radionuclide theranostic pairs and evaluated the pharmacokinetics and theranostic efficacies of the agents in cell- and patient-derived models. Both C2 and ABDC2 specifically reacted with human CD47 with a high K D value of 23.50 and 84.57 pM, respectively. [68Ga]Ga-NOTA-C2 was developed with high radiochemical purity (99 >%, n = 4) and visualized CD47 expression in the tumors. In comparison to the rapid renal clearance and short half-life of [68Ga]Ga-NOTA-C2, both [68Ga]Ga-NOTA-ABDC2 and [89Zr]Zr-DFO-ABDC2 showed prolonged circulation and increased tumor uptake, with the highest uptake of [89Zr]Zr-DFO-ABDC2 occurring at 72 h post-injection. Moreover, [177Lu]Lu-DOTA-ABDC2 radioimmunotherapy suppressed the tumor growth but was associated with toxicity, warranting further optimization of the treatment schedules. Taken together, we reported a series of nanobody-derived CD47-targeted agents, of which [68Ga]Ga-NOTA-C2 and [89Zr]Zr-DFO-ABDC2 are readily translatable. Optimization and translation of CD47-targeted theranostic pair may provide new prospects for CD47-targeted management of solid tumors.

Near-Infrared Optical Transducer for Dynamic Imaging of Cerebrospinal Fluid Glucose in Brain Tumor.

Aberrant cerebral glucose metabolism is related to many brain diseases, especially brain tumor. However, it remains challenging to measure the dynamic changes in cerebral glucose. Here, we developed a near-infrared (NIR) optical transducer to sensitively monitor the glucose variations in cerebrospinal fluid in vivo. The transducer consists of an oxygen-sensitive nanoparticle combined with glucose oxidase (GOx), yielding highly sensitive NIR phosphorescence in response to blood glucose change. We demonstrated long-term continuous glucose monitoring by using the NIR transducer. After subcutaneous implantation, the glucose transducer provides a strong luminescence signal that can continuously monitor blood glucose fluctuations for weeks. By using the NIR emission of the transducer, we further observed abnormal dynamic changes in cerebrospinal fluid glucose and quantitatively assessed cerebral glucose uptake rates in transgenic mice bearing brain tumors. This study provides a promising method for the diagnosis of various metabolic diseases with altered glucose metabolism.

In Vivo Imaging of Exosomes Labeled with NIR-II Polymer Dots in Liver-Injured Mice.

Mesenchymal stem cell-derived exosomes (MSC-Exos) are emerging as a promising platform for treating various intractable diseases and organ injuries. Monitoring their migration, homing, and therapeutic capability in vivo is essential to develop exosome-based theranostics. Here, we designed fluorescent semiconductor polymer dots (Pdots) in the second near-infrared window (NIR-II) for bright labeling and tracking of MSC-Exos. Glucose-coated Pdots (Pdots-Glu) were able to label MSC-Exos without changing their biological properties. The NIR-II fluorescent Pdots allow for high labeling brightness and long-term in vivo tracking of MSC-Exos. We investigated the biodistributions and therapeutic functions of these labeled MSC-Exos in liver-resected mice. In vivo and ex vivo imaging demonstrated that the Pdot-labeled MSC-Exos injected via the tail vein mainly accumulated in the residual liver tissue. In terms of the therapeutic effect, MSC-Exos may accelerate postoperative liver function recovery by inhibiting inflammatory responses, promoting cell proliferation, and resisting apoptosis. Our results indicated that MSC-Exos therapeutic systems hold promising applications in liver regenerative medicine.

Biomimetic semiconducting polymer dots for highly specific NIR-II fluorescence imaging of glioma.

Glioma with very short medium survival time consists of 80% of primary malignant types of brain tumors. The unique microenvironment such as the existence of the blood-brain barrier (BBB) makes the glioma theranostics exhibit low sensitivity in diagnosis, a poor prognosis and low treatment efficacy. Therefore, the development of multifunctional nanoplatform that can cross BBB and target the glioma is essential for the high-sensitivity detection and ablation of cancer cells. In this study, C6 cell membrane-coated conjugated polymer dots (Pdots-C6) were constructed for targeted glioma tumor detection. As a new kind of biomimetic and biocompatible nanoprobes, Pdots-C6 preserve the complex biological functions of natural cell membranes while possessing physicochemical properties for NIR-II fluorescence imaging of glioma. After encapsulating C6 cell membrane on the surface of conjugated Pdots, Pdots-C6 exhibited the most favorable specific targeting capabilities in vitro and in vivo. In particular, this pilot study demonstrates that biomimetic nanoparticles offer a potential tool to enhance specific targeting to the brain, hence improving glioma tumor detection accuracy.

Multicolor Photoacoustic Volumetric Imaging of Subcellular Structures.

Photoacoustic imaging (PAI) has been widely used in multiscale and multicontrast imaging of biological structures and functions. Optical resolution photoacoustic microscopy (OR-PAM), an emerging submodality of PAI, features high lateral resolution and rich optical contrast, indicating great potential in visualizing cellular and subcellular structures. However, three-dimensional (3D) imaging of subcellular structures using OR-PAM has remained a challenge due to the limited axial resolution. In this study, we propose a multicolor 3D photoacoustic microscopy with high lateral/axial resolutions of 0.42/2 and 0.5/2.5 µm at 532 and 780 nm excitation, respectively. Owing to the significantly increased axial resolution, we could visualize the volumetric subcellular structures of melanoma cells using intrinsic contrast. In addition, we carried out multicolor imaging of labeled microtubules/clathrin-coated pits (CCP) and microtubules/mitochondria, respectively, with one scanning by using two different excitation wavelengths. The internal connections between different subcellular structures are revealed by quantitatively comparing the spatial distributions of microtubules/CCP and microtubules/mitochondria in a single cell. Current results suggest that the proposed OR-PAM may serve as an efficient tool for subcellular and cytophysiological studies.

Light-Controlled Precise Delivery of NIR-Responsive Semiconducting Polymer Nanoparticles with Promoted Vascular Permeability.

The endothelial barrier plays an essential role in health and disease by protecting organs from toxins while allowing nutrients to access the circulation. However, it is the major obstacle that limits the delivery of therapeutic drugs to the diseased tissue. Here, it is reported for the first time that near-infrared (NIR) laser pulses can transiently promote the delivery of semiconducting polymer nanoparticles passing the vascular barrier via photoacoustic-effect-induced accumulation, only by the aid of pulse laser irradiation. This strategy enables selective and substantial accumulation of the NIR-absorbing nanoparticles inside specific tissues, implying the discovery of an unprecedented approach for light-controlled nanoparticle delivery. Especially, the nanoparticle delivery in solid tumors by 10-min laser scanning is approximately six times higher than that of the enhanced permeability and retention (EPR) effect in 24 h under current experimental conditions. Further results confirm that this strategy facilitates substantial accumulation of nanoparticles in the mouse brain with intact skull. This approach thus opens a new door for tissue-specific delivery of nanomaterials with an unprecedented level of efficiency and precision.

A biodegradable nano-photosensitizer with photoactivatable singlet oxygen generation for synergistic phototherapy.

Photodynamic therapy (PDT) is a promising method for cancer therapy and also may initiate unexpected damages to normal cells and tissues. Herein, we develop a near-infrared (NIR) light-activatable nanophotosensitizer, which shows negligible phototoxicity before photoactivation to improve the specificity of PDT. The nanophotosensitizer is prepared by indocyanine green carboxylic (ICG), Chlorin e6 (Ce6), and biodegradable poly (lactic acid) (PLA) and poly (lactic-co-glycolic acid) (PLGA), and all these materials have been approved by the Food and Drug Administration. Initially the phototoxicity of Ce6 is effectively inhibited by ICG through fluorescence resonance energy transfer (FRET). Upon 808 nm laser activation, ICG generate hyperthermia for photothermal therapy (PTT) and simultaneously is degraded due to the inherently poor photostability. The FRET is disrupted and followed by the recovery of phototoxicity of Ce6 for PDT. We investigated the photoactivation and the resulting phototherapy by cellular assays and mouse models, which indicate a superior synergistic treatment effect and selective PDT activated by near-infrared 808 nm light. This study presents a promising strategy for activatable and synergistic phototherapy with minimal damage to normal tissues.

Reversible Ratiometric NADH Sensing Using Semiconducting Polymer Dots.

Reduced nicotinamide adenine dinucleotide (NADH) is a key coenzyme in living cells due to its role as an electron carrier in redox reactions, and its concentration is an important indicator of cell metabolic state. Abnormal NADH levels are associated with age-related metabolic diseases and neurodegenerative disorders, creating a demand for a simple, rapid analytical method for point-of-care NADH sensing. Here we develop a series of NADH-sensitive semiconducting polymer dots (Pdots) as nanoprobes for NADH measurement, and test their performance in vitro and in vivo. NADH sensing is based on electron transfer from semiconducting polymer chains in the Pdot to NADH upon UV excitation, quenching Pdot fluorescence emission. In polyfluorene-based Pdots, this mechanism resulted in an on-off NADH sensor; in DPA-CNPPV Pdots, UV excitation resulted in NADH-sensitive emission at two wavelengths, enabling ratiometric detection. Ratiometric NADH detection using DPA-CNPPV Pdots exhibits high sensitivity (3.1 µM limit of detection), excellent selectivity versus other analytes, reversibility, and a fast response (less than 5 s). We demonstrate applications of the ratiometric NADH-sensing Pdots including smartphone-based NADH imaging for point-of-care use.

NIR-II Fluorescence Imaging Reveals Bone Marrow Retention of Small Polymer Nanoparticles.

The concept that systemically administered nanoparticles are highly accumulated into the liver, spleen and kidney is a central paradigm in the field of nanomedicine. Here, we report that bone is an important organ for retention of small polymer nanoparticles using in vivo fluorescence imaging in the second near-infrared (NIR-II) window. We prepared different sized polymer nanoparticles with both visible and NIR-II fluorescence. NIR-II imaging reveals that the behavior of nanoparticle distribution in bone was largely dependent on the particle size. Small polymer nanoparticles of ∼15 nm diameter showed fast accumulation and long-term retention in bone, while the nanoparticles larger than ∼25 nm were dominantly distributed in liver. Confocal microscopy of bone sections indicated that the nanoparticles were largely distributed in the endothelial cells of sinusoidal vessels in bone marrow. The study provides promising opportunities for bone imaging and treatment of bone-related disease.

Thermosensitive Polymer Dot Nanocomposites for Trimodal Computed Tomography/Photoacoustic/Fluorescence Imaging-Guided Synergistic Chemo-Photothermal Therapy.

Precision delivery of theranostic agents to the tumor site is essential to improve their diagnostic and therapeutic efficacy and concurrently minimize adverse effects during treatment. In this study, a novel concept of near-infrared (NIR) light activation of conjugated polymer dots (Pdots) at thermosensitive hydrogel nanostructures is introduced for multimodal imaging-guided synergistic chemo-photothermal therapy. Interestingly, owing to the attractive photothermal conversion efficiency of Pdots, the Pdots@hydrogel as theranostic agents is able to undergo a controllable softening or melting state under the irradiation of NIR laser, resulting in light-triggered drug release in a controlled way and concurrently hydrogel degradation. Besides, the novel Pdots@hydrogel nanoplatform can serve as the theranostic agent for enhanced trimodal photoacoustic (PA)/computed tomography (CT)/fluorescence (FL) imaging-guided synergistic chemo-photothermal therapy of tumors. More importantly, the constructed intelligent nanocomposite Pdots@hydrogel exhibits excellent biodegradability, strong NIR absorption, bright PA/CT/FL signals, and superior tumor ablation effect. Therefore, the concept of a light-controlled multifunctional Pdots@hydrogel that integrates multiple diagnostic/therapeutic modalities into one nanoplatform can potentially be applied as a smart nanotheranostic agent to various perspectives of personalized nanomedicine.

Fluorination Enhances NIR-II Fluorescence of Polymer Dots for Quantitative Brain Tumor Imaging.

Here, we describe a fluorination strategy for semiconducting polymers for the development of highly bright second near-infrared region (NIR-II) probes. Tetrafluorination yielded a fluorescence QY of 3.2 % for the polymer dots (Pdots), over a 3-fold enhancement compared to non-fluorinated counterparts. The fluorescence enhancement was attributable to a nanoscale fluorous effect in the Pdots that maintained the molecular planarity and minimized the structure distortion between the excited state and ground state, thus reducing the nonradiative relaxations. By performing through-skull and through-scalp imaging of the brain vasculature of live mice, we quantitatively analyzed the vascular morphology of transgenic brain tumors in terms of the vessel lengths, vessel branches, and vessel symmetry, which showed statistically significant differences from the wild type animals. The bright NIR-II Pdots obtained through fluorination chemistry provide insightful information for precise diagnosis of the malignancy of the brain tumor.

In vivo dynamic cell tracking with long-wavelength excitable and near-infrared fluorescent polymer dots.

Development of cell-based therapeutic systems has attracted great interest in biomedicine. In vivo cell tracking by fluorescence provides indispensable information for further advancing cell therapy in clinical applications. However, it is still challenging in many cases because of the limited light penetration depth as well as the variations in fluorescent probes, cell lines, and labeling brightness. Here, we designed highly fluorescent polymer dots (Pdots) with far-red-light absorption and near-infrared (NIR) emission for cell tracking. The Pdots consisted of a donor-acceptor polymer blending system where intra-particle energy transfer yielded a narrow-band emission at 800 nm with a high quantum yield of ~0.22. We investigated biocompatibility and cell labeling brightness of the Pdots coated with cell penetrating peptides. Flow cytometry indicated that the cell-labeling brightness of both stem cells and cancer cells increased as much as ~4 orders of magnitude comparing the intensity measurements of labeled cells and controls. Yet, in vivo cell tracking results revealed distinctive fluorescence distribution for the same number of cells that were administered into mice through the tail vein. The stem cells initially accumulated in the lung and remained for seven days, whereas the cancer cells tended to be cleared by the liver in four days. The difference is likely due to the fact that cancer cells are easily attacked by the immune system, whereas stem cells have low immunogenicity. Results obtained herein confirm that NIR-fluorescent Pdots are promising platforms for in vivo cell tracking in small animals.

Utilizing Polymer Micelle to Control Dye J-aggregation and Enhance Its Theranostic Capability.

We utilize polymer micelle to precisely control indocyanine green (ICG) J-aggregation in a fast and highly efficient way. In addition to simple encapsulation, the polymer micelle plays a role as a host template to drive ICG J-aggregation by the synergy of electrostatic and hydrophobic attractions. We further demonstrate that, due to the robust host-guest interaction, the intact of ICG J-aggregate will be secured by the polymer encapsulation during the intracellular and in vivo incubation. These features make this hierarchical assembly between ICG J-aggregate and the micelle polymer a promising biomedicine for cancer phototheranostics. Therefore the complex micelles are further modified by introduction of doxorubicin for chemotherapy and DNA aptamer for tumor targeting, and the final multi-functional micellar medicine shows high therapeutic efficacy for tumor, i.e., the tumor can be completely eliminated with no local reoccurrence and without long-term toxicity or side effects during a 24-day period after the treatment.

Spiro[pyrrol-benzopyran]-based probe with high asymmetry for chiroptical sensing via circular dichroism.

This contribution describes an efficient approach to constructing chiral spiro[pyrrol-benzopyran] (SPP) scaffold with high asymmetry. By combining chiral SPP and the achiral sensing moiety phenothiazine, the target probe P showed unprecedented reaction-based chiroptical sensing towards hypochlorite with switching CD signals.