Carboxylesterase Activatable Molecular Probe for Personalized Treatment Guidance by Analyte-Induced Molecular Transformation.

Accurate visualization of tumor microenvironment is of great significance for personalized medicine. Here, we develop a near-infrared (NIR) fluorescence/photoacoustic (FL/PA) dual-mode molecular probe (denoted as NIR-CE) for distinguishing tumors based on carboxylesterase (CE) level by an analyte-induced molecular transformation (AIMT) strategy. The recognition moiety for CE activity is the acetyl unit of NIR-CE, generating the pre-product, NIR-CE-OH, which undergoes spontaneous hydrogen atom exchange between the nitrogen atoms in the indole group and the phenol hydroxyl group, eventually transforming into NIR-CE-H. In cellular experiments and in vivo blind studies, the human hepatoma cells and tumors with high level of CE were successfully distinguished by both NIR FL and PA imaging. Our findings provide a new molecular imaging strategy for personalized treatment guidance.

NIR-II Fluorescence Sensor Based on Steric Hindrance Regulated Molecular Packing for In Vivo Epilepsy Visualization.

Fluorescence sensing is crucial to study biological processes and diagnose diseases, especially in the second near-infrared (NIR-II) window with reduces background signals. However, it's still a great challenge to construct "off-on" sensors when the sensing wavelength extends into the second near-infrared (NIR-II) region to obtain higher imaging contrast, mainly due to the difficult synthesis of spectral overlapped quencher. Here, we present a new fluorescence quenching strategy, which utilizes steric hindrance quenchers (SHQ) to tune the molecular packing state of fluorophores and suppress the emission signal. Density functional theory (DFT) calculations further reveal that large SHQ can competitively packing with fluorophores and prevent their self-aggregation. Based on this quenching mechanism, a novel activatable "off-on" sensing method is achieved via bio-analyte responsive invalidation of SHQ, namely Steric Hindrance Invalidation geNerated Emission (SHINE) strategy. As a proof of concept, the ClO- sensitive SHQ lead to the bright NIR-II signal release in epileptic mouse hippocampus under the skull and high photon scattering brain tissue, providing the real-time visualization of ClO- generation process in living epileptic mice.

Comprehensively Optimizing Fenton Reaction Factors for Antitumor Chemodynamic Therapy by Charge-Reversal Theranostics.

Chemodynamic therapy (CDT) is a highly tumor-specific treatment, while its efficacy is compromised by the intratumoral Fenton reaction efficiency, which is determined by the following reaction factors, including the availability of Fenton ions (e.g., Fe2+), the amount of H2O2, and the degree of acidity. Synchronous optimization of these factors is a big challenge for efficient CDT. Herein, a strategy of comprehensively optimizing Fenton reaction factors was developed for traceable multistage augmented CDT by charge-reversal theranostics. The customized pH-responsive poly(ethylene)glycol-poly(ß-amino esters) (PEG-PAE) micelle (PM) was prepared as the carrier. Glucose oxidase (GOx), Fe2+, and pH-responsive second near-infrared (NIR-II) LET-1052 probe were coloaded by PM to obtain the final theranostics. The activity of metastable Fe2+ remained by the unsaturated coordination with PEG-PAE. Then tumor accumulation and exposure of Fe2+ were achieved by charge-reversal cationization of PEG-PAE, which was further enhanced by a GOx catalysis-triggered pH decrease. Together with the abundant H2O2 generation and pH decrease through GOx catalysis, the limiting factors of the Fenton reaction were comprehensively optimized, achieving the enhanced CDT both in vitro and in vivo. These findings provide a strategy for comprehensively optimizing intratumoral Fenton reaction factors to overcome the intrinsic drawbacks of current CDT.

Activatable NIR-II Photothermal Lipid Nanoparticles for Improved Messenger RNA Delivery.

Endosomal escape remains a central issue limiting the high protein expression of mRNA therapeutics. Here, we present second near-infrared (NIR-II) lipid nanoparticles (LNPs) containing pH activatable NIR-II dye conjugated lipid (Cy-lipid) for potentiating mRNA delivery efficiency via a stimulus-responsive photothermal-promoted endosomal escape delivery (SPEED) strategy. In acidic endosomal microenvironment, Cy-lipid is protonated and turns on NIR-II absorption for light-to-heat transduction mediated by 1064 nm laser irradiation. Then, the heat-promoted LNPs morphology change triggers rapid escape of NIR-II LNPs from the endosome, allowing about 3-fold enhancement of enhanced green fluorescent protein (eGFP) encoding mRNA translation capacity compared to the NIR-II light free group. In addition, the bioluminescence intensity induced by delivered luciferase encoding mRNA in the mouse liver region shows positive correlation with incremental radiation dose, indicating the validity of the SPEED strategy.

In Situ Transformable Nanoplatforms with Supramolecular Cross-Linking Triggered Complementary Function for Enhanced Cancer Photodynamic Therapy.

In vivo cross-linking of nanoparticles is widely used to increase accumulation of therapeutic agents at tumor site for enhanced therapy. However, the components in nanoplatforms usually only play for one role and are independent of each other, unable to amplify their biofunctions. Herein, a complementary functioning tumor microenvironment triggered, supramolecular coordination-induced nanoparticle cross-linking strategy is constructed for enhanced photodynamic therapy. Manganese oxide (MnOx ) and polyhydroxy photosensitizer hypericin (Hyp) are coated and loaded onto lanthanide-doped upconversion nanoparticles (UCNPs) to form transformable UCNP@MnOx -Hyp. In CT26 mouse colon cancer cells and xenograft tumors, UCNP@MnOx -Hyp is reduced by glutathione and H2 O2 , releasing Mn2+ and Hyp for in situ cross-linking to transform to UCNP@Mn2+ -Hyp. Compared to the simple photosensitizer-loaded UCNP@PEI-Hyp, the Mn2+ -Hyp coordination redshifts absorbance of Hyp and improves the energy transfer efficiency from UCNPs to Hyp (5.6-fold). In turn, the supramolecular coordination-induced UCNPs cross-linking exhibits enhanced luminescence recovery and increased intracellular accumulation of both UCNPs and Hyp, thus enhancing the photodynamic therapy efficacy both at cellular level (2.1-fold) and in vivo, realizing the function amplification of each component after responsive transformation and offering a new avenue for enhanced cancer therapy.

An Integrated Polymeric mRNA Vaccine without Inflammation Side Effects for Cellular Immunity Mediated Cancer Therapy.

Among the few available mRNA delivery vehicles, lipid nanoparticles (LNPs) are the most clinically advanced but they require cumbersome four components and suffer from inflammation-related side effects that should be minimized for safety. Yet, a certain level of proinflammatory responses and innate immune activation are required to evoke T-cell immunity for mRNA cancer vaccination. To address these issues and develop potent yet low-inflammatory mRNA cancer vaccine vectors, a series of alternating copolymers "PHTA" featured with ortho-hydroxy tertiary amine (HTA) repeating units for mRNA delivery is synthesized, which can play triple roles of condensing mRNA, enhancing the polymeric nanoparticle (PNP) stability, and prolonging circulation time. Unlike LNPs exhibiting high levels of inflammation, the PHTA-based PNPs show negligible inflammatory side effects in vivo. Importantly, the top candidate PHTA-C18 enables successful mRNA cancer vaccine delivery in vivo and leads to a robust CD8+ T cell mediated antitumor cellular immunity. Such PHTA-based integrated PNP provides a potential approach for establishing mRNA cancer vaccines with good inflammatory safety profiles.

A LRET Nanoplatform Consisting of Lanthanide and Amorphous Manganese Oxide for NIR-II Luminescence Lifetime Imaging of Tumor Redox Status.

Designing luminescence lifetime sensors in the second near-infrared (NIR-II) window is a great challenge due to the difficult structural construction. Here, we report a tumor redox responsive and easily synthesized material, amorphous manganese oxide (MnOx ) with indirect band gap of 1.02 eV, as an energy acceptor to build a luminescence resonance energy transfer (LRET) toolbox for universally regulating NIR-I to NIR-II luminescence lifetimes of lanthanide nanoparticles, in which energy transfer is based on matched energy gap instead of conventional overlapped spectra. We further utilize ytterbium (Yb3+ )-doped YbNP@MnOx as an NIR-II luminescence lifetime sensor to realize in vitro quantitative redox visualization with relative errors under 5 % in samples covered with mouse skin. Furthermore, HepG2 cells and tumors with high redox state have been accurately distinguished by NIR-II luminescence lifetime imaging. The quantified intracellular and intratumor glutathione (GSH) levels are highly consistent with the commercial kit results, illustrating the reliable redox visualization ability in biological tissue.

Management of fluorescent organic/inorganic nanohybrids for biomedical applications in the NIR-II region.

Biomedical fluorescence imaging in the second near-infrared (NIR-II, 100-1700 nm) window provides great potential for visualizing physiological and pathological processes, owing to the reduced tissue absorption, scattering, and autofluorescence. Various types of NIR-II probes have been reported in the past decade. Among them, NIR-II organic/inorganic nanohybrids have attracted widespread attention due to their unique properties by integrating the advantages of both organic and inorganic species. Versatile organic/inorganic nanohybrids provide the possibility of realizing a combination of functions, controllable size, and multiple optical features. This tutorial review summarizes the reported organic and inorganic species in nanohybrids, and their biomedical applications in NIR-II fluorescence and lifetime imaging. Finally, the challenges and outlook of organic/inorganic nanohybrids in biomedical applications are discussed.

A "Self-Checking" pH/Viscosity-Activatable NIR-II Molecule for Real-Time Evaluation of Photothermal Therapy Efficacy.

We present a second near-infrared (NIR-II) self-checking molecule, LET-1052, for acidic tumor microenvironment (TME) turn-on photothermal therapy (PTT), followed by viscosity based therapeutic efficacy evaluation by itself in two independent channels, denoted as "self-checking" strategy. In acidic TME, LET-1052 was protonated and turned on NIR-II absorption for PTT under 1064 nm laser irradiation. Subsequently, PTT-induced cellular death increases intracellular viscosity, which inhibited the intramolecular rotation of LET-1052, resulting in the enhancement of NIR-I fluorescence for real-time evaluation of PTT efficacy. After PTT of tumor-bearing mice for different periods of NIR-II laser irradiation, NIR-I fluorescence in the tumor region showed positive correlation with tumor growth inhibition rate, demonstrating reliable and prompt prediction of PTT efficacy. The strategy may be expanded for instant evaluation of other therapeutic modalities for personalized medicine.

Near-infrared probes for luminescence lifetime imaging.

Biomedical luminescence imaging in the near-infrared (NIR, 700-1700 nm) region has shown great potential in visualizing biological processes and pathological conditions at cellular and animal levels, owing to the reduced tissue absorption and scattering compared to light in the visible (400-700 nm) region. To overcome the background interference and signal attenuation during intensity-based luminescence imaging, lifetime imaging has demonstrated a reliable imaging modality complementary to intensity measurement. Several selective or environment-responsive probes have been successfully developed for luminescence lifetime imaging and multiplex detection. This review summarizes recent advances in the application of luminescence lifetime imaging at cellular and animal levels in NIR-I and NIR-II regions. Finally, the challenges and further directions of luminescence lifetime imaging are also discussed.

Neutrophil-like Cell-Membrane-Coated Nanozyme Therapy for Ischemic Brain Damage and Long-Term Neurological Functional Recovery.

Oxidative stress and a series of excessive inflammatory responses are major obstacles for neurological functional recovery after ischemic stroke. Effective noninvasive anti-inflammatory therapies are urgently needed. However, unsatisfactory therapeutic efficacy of current drugs and inadequate drug delivery to the damaged brain are major problems. Nanozymes with robust anti-inflammatory and antioxidative stress properties possess therapeutic possibility for ischemic stroke. However, insufficiency of nanozyme accumulation in the ischemic brain by noninvasive administration hindered their application. Herein, we report a neutrophil-like cell-membrane-coated mesoporous Prussian blue nanozyme (MPBzyme@NCM) to realize noninvasive active-targeting therapy for ischemic stroke by improving the delivery of a nanozyme to the damaged brain based on the innate connection between inflamed brain microvascular endothelial cells and neutrophils after stroke. The long-term in vivo therapeutic efficacy of MPBzyme@NCM for ischemic stroke was illustrated in detail after being delivered into the damaged brain and uptake by microglia. Moreover, the detailed mechanism of ischemic stroke therapy via MPBzyme@NCM uptake by microglia was further studied, including microglia polarization toward M2, reduced recruitment of neutrophils, decreased apoptosis of neurons, and proliferation of neural stem cells, neuronal precursors, and neurons. This strategy may provide an applicative perspective for nanozyme therapy in brain diseases.

NIR-II pH Sensor with a FRET Adjustable Transition Point for In Situ Dynamic Tumor Microenvironment Visualization.

Monitoring the pH in tumor lesions provides abundant physiological information. However, currently developed pH sensors only achieve sensitive detection in the settled response region around the pH transition point (pHt ). To realize tumor pH monitoring with high sensitivity within a wider response region, reported here are serial pHt adjustable sensors (pTAS) that simply regulate the component ratio of second near-infrared (NIR-II) emission aza-BODIPY (NAB) donor and pH sensitive rhodamine-based pre-acceptor (NRh) in Förster resonance energy transfer system. Combining the pH response regions of pTAS, a twofold widened pH detection range (6.11-7.22) is obtained compared to the pHt settled sensor (6.38-6.94). With an adjustable pHt , in vivo tumor pH increase and decrease processes could be dynamically visualized through dual-channel ratiometric bioimaging within the NIR-II window, with a coefficient of variation under 1 % compared to the standard pH meter.

Molecular Fluorophores for Deep-Tissue Bioimaging.

Fluorescence imaging has made tremendous inroads toward understanding the complexity of biological systems, but in vivo deep-tissue imaging remains a great challenge due to the optical opacity of biological tissue. Recent improvements in laser and detector manufacturing have allowed the expansion of nonlinear and linear fluorescence imaging to the underexplored "tissue-transparent" second near-infrared (NIR-II; 1000-1700 nm) window, opening up new opportunities for optical access deep inside opaque tissue. Molecular fluorophores have historically played a major role in fluorescence bioimaging. It is increasingly important to design new molecular fluorophores to fully unlock the potential of NIR-II imaging techniques. In this outlook, we give an overview of the novel molecular fluorophores developed for deep-tissue bioimaging in the past five years and discuss their pros and cons in applications. Guidelines for designing new molecular fluorophores with the desirable properties are also provided.

NIR-II bioluminescence for in vivo high contrast imaging and in situ ATP-mediated metastases tracing.

Bioluminescence imaging has been widely used in life sciences and biomedical applications. However, conventional bioluminescence imaging usually operates in the visible region, which hampers the high-performance in vivo optical imaging due to the strong tissue absorption and scattering. To address this challenge, here we present bioluminescence probes (BPs) with emission in the second near infrared (NIR-II) region at 1029 nm by employing bioluminescence resonance energy transfer (BRET) and two-step fluorescence resonance energy transfer (FRET) with a specially designed cyanine dye FD-1029. The biocompatible NIR-II-BPs are successfully applied to vessels and lymphatics imaging in mice, which gives ~5 times higher signal-to-noise ratios and ~1.5 times higher spatial resolution than those obtained by NIR-II fluorescence imaging and conventional bioluminescence imaging. Their capability of multiplexed imaging is also well displayed. Taking advantage of the ATP-responding character, the NIR-II-BPs are able to recognize tumor metastasis with a high tumor-to-normal tissue ratio at 83.4.

A Tumor-Microenvironment-Responsive Lanthanide-Cyanine FRET Sensor for NIR-II Luminescence-Lifetime In Situ Imaging of Hepatocellular Carcinoma.

Deep tissue imaging in the second near-infrared (NIR-II) window holds great promise for widespread fundamental research. However, inhomogeneous signal attenuation due to tissue absorption and scattering hampers its application for accurate in vivo biosensing. Here, lifetime-based in situ hepatocellular carcinoma (HCC) detection in NIR-II region is presented using a tumor-microenvironment (peroxynitrite, ONOO- )-responsive lanthanide-cyanine Förster resonance energy transfer (FRET) nanosensor. A specially designed ONOO- -responsive NIR-II dye, MY-1057, is synthesized as the FRET acceptor. Robust lifetime sensing is demonstrated to be independent of tissue penetration depth. Tumor lesions are accurately distinguished from normal tissue due to the recovery lifetime. Magnetic resonance imaging and liver dissection results illustrate the reliability of lifetime-based detection in single and multiple HCC models. Moreover, the ONOO- amount can be calculated according to the standard curve.

Organic NIR-II molecule with long blood half-life for in vivo dynamic vascular imaging.

Real-time monitoring of vessel dysfunction is of great significance in preclinical research. Optical bioimaging in the second near-infrared (NIR-II) window provides advantages including high resolution and fast feedback. However, the reported molecular dyes are hampered by limited blood circulation time (~ 5-60 min) and short absorption and emission wavelength, which impede the accurate long-term monitoring. Here, we report a NIR-II molecule (LZ-1105) with absorption and emission beyond 1000 nm. Thanks to the long blood circulation time (half-life of 3.2 h), the fluorophore is used for continuous real-time monitoring of dynamic vascular processes, including ischemic reperfusion in hindlimbs, thrombolysis in carotid artery and opening and recovery of the blood brain barrier (BBB). LZ-1105 provides an approach for researchers to assess vessel dysfunction due to the long excitation and emission wavelength and long-term blood circulation properties.

Real-time monitoring and accurate diagnosis of drug-induced hepatotoxicity in vivo by ratio-fluorescence and photoacoustic imaging of peroxynitrite.

Because of the low tissue penetration depth and poor photostability of organic cyanine dye, in addition to environmental interference, it is a great challenge to monitor the degree of drug-induced hepatotoxicity by the in vivo detection of peroxynitrite (ONOO-). Herein, we fabricated heptamethine cyanine dye (P-cy7)-coordinated upconversion nanoparticles (UCNPs), namely UCY7, as a fluorescent nanoprobe for evaluating drug-induced hepatotoxicity. Due to the luminescence resonance energy transfer (LRET) between UCNPs and the cyanine dye (P-cy7), the irradiation changed from visible light at 660 nm to near infrared (NIR) light at 980 nm; therefore, the issues of poor photostability and severe photobleaching of cyanine dye can be effectively solved. After injecting via the tail vein, the nanoprobes are rapidly concentrated in the liver. Since the level of ONOO- is up-regulated during the drug-induced liver injury, the LRET between UCNPs and P-cy7 is disrupted to release the upconversion luminescence at 656 nm, while the upconversion luminescence at 800 nm remains constant, thus achieving the ratio-fluorescent imaging (RFLI) of ONOO- in the liver to calibrate the influence of the environment. In addition, the reduction in the absorption of nanoprobes in the presence of ONOO- allows for sensitive photoacoustic imaging (PAI). Based on the RFLI and PAI of the liver, the real-time monitoring and accurate diagnosis of different degrees of hepatotoxicity using the model of Acetaminophen (APAP) induction was achieved successfully, providing a new approach for the clinical evaluation of drug-induced hepatotoxicity.

Activatable fluorescence sensors for in vivo bio-detection in the second near-infrared window.

Fluorescence imaging in the second near-infrared (NIR-II, 1000-1700 nm) window has exhibited advantages of high optical resolution at deeper penetration (ca. 5-20 mm) in bio-tissues owing to the reduced photon scattering, absorption and tissue autofluorescence. However, the non-responsive and "always on" sensors lack the ability of selective imaging of lesion areas, leading to the low signal-to-background ratio (SBR) and poor sensitivity during bio-detection. In contrast, activatable sensors show signal variation in fluorescence intensity, spectral wavelength and fluorescence lifetime after responding to the micro-environment stimuli, leading to the high detection sensitivity and reliability in bio-sensing. This minireview summarizes the design and detection ability of recently reported NIR-II activatable sensors. Furthermore, the challenges, opportunities and prospects of NIR-II activatable bio-sensing are also discussed.

J-Aggregates of Cyanine Dye for NIR-II in Vivo Dynamic Vascular Imaging beyond 1500 nm.

Light in the second near-infrared window, especially beyond 1500 nm, shows enhanced tissue transparency for high-resolution in vivo optical bioimaging due to decreased tissue scattering, absorption, and autofluorescence. Despite some inorganic luminescent nanoparticles have been developed to improve the bioimaging around 1500 nm, it is still a great challenge to synthesize organic molecules with the absorption and emission toward this region. Here, we present J-aggregates with 1360 nm absorption and 1370 nm emission formed by self-assembly of amphiphilic cyanine dye FD-1080 and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Molecular dynamics simulations were further employed to illustrate the self-assembly process. Superior spatial resolution and high signal-to-background ratio of J-aggregates were demonstrated for noninvasive brain and hindlimb vasculature bioimaging beyond 1500 nm. The efficacy evaluation of the clinically used hypotensor is successfully achieved by high-resolution in vivo dynamic vascular imaging with J-aggregates.