High‐contrast Noninvasive Imaging of Kidney Clearance Kinetics Enabled by Renal Clearable Nanofluorophores

Noninvasive imaging of kidney clearance kinetics (KCK) of renal clearable probes is key to studying unilateral kidney function diseases, but such imaging is highly challenging to achieve with in vivo fluorescence. While this long‐standing challenge is often attributed to the limited light penetration depth, we found that rapid and persistent accumulation of conventional dyes in the skin “shadowed” real fluorescence signals from the kidneys and prevented noninvasive imaging of KCK, which, however, can be addressed with renal clearable nanofluorophores. By integrating near infrared emission with efficient renal clearance and ultralow background interference, the nanofluorophores can increase kidney‐contrast enhancement and imaging‐time window by approximately 50‐ and 1000‐fold over conventional dyes, and significantly minimize deviation between noninvasive and invasive KCK, laying down a foundation for translating in vivo fluorescence imaging in preclinical noninvasive kidney function assessments.     

A Renal-Clearable Macromolecular Reporter for Near-Infrared Fluorescence Imaging of Bladder Cancer

Bladder cancer (BC) is a prevalent disease with high morbidity and mortality; however, in vivo optical imaging of BC remains challenging because of the lack of cancer-specific optical agents with high renal clearance. Herein, a macromolecular reporter (CyP1) was synthesized for real-time near-infrared fluorescence (NIRF) imaging and urinalysis of BC in living mice. Because of the high renal clearance (ca. 94 % of the injection dosage at 24 h post-injection) and its cancer biomarker (APN=aminopeptidase N) specificity, CyP1 can be efficiently transported to the bladder and specially turn on its NIRF signal to report the detection of BC in living mice. Moreover, CyP1 can be used for optical urinalysis, permitting the ex vivo tracking of tumor progression for therapeutic evaluation and easy translation of CyP2 as an in vitro diagnostic assay. This study not only provides new opportunities for non-invasive diagnosis of BC, but also reveals useful guidelines for the development of molecular reporters for the detection of bladder diseases.     

Cartilage‐Specific Near‐Infrared Fluorophores for Biomedical Imaging

A novel class of near‐infrared fluorescent contrast agents was developed. These agents target cartilage with high specificity and this property is inherent to the chemical structure of the fluorophore. After a single low‐dose intravenous injection and a clearance time of approximately 4 h, these agents bind to all three major types of cartilage (hyaline, elastic, and fibrocartilage) and perform equally well across species. Analysis of the chemical structure similarities revealed a potential pharmacophore for cartilage targeting. Our results lay the foundation for future improvements in tissue engineering, joint surgery, and cartilage‐specific drug development.     

Near‐Infrared Light and pH‐Responsive Polypyrrole@Polyacrylic acid/Fluorescent Mesoporous Silica Nanoparticles for Imaging and Chemo‐Photothermal Cancer Therapy

We have rationally designed a new theranostic agent by coating near‐infrared (NIR) light‐absorbing polypyrrole (PPY) with poly(acrylic acid) (PAA), in which PAA acts as a nanoreactor and template, followed by growing small fluorescent silica nanoparticles (fSiO₂ NPs) inside the PAA networks, resulting in the formation of polypyrrole@polyacrylic acid/fluorescent mesoporous silica (PPY@PAA/fmSiO₂) core–shell NPs. Meanwhile, DOX‐loaded PPY@PAA/fmSiO₂ NPs as pH and NIR dual‐sensitive drug delivery vehicles were employed for fluorescence imaging and chemo‐photothermal synergetic therapy in vitro and in vivo. The results demonstrate that the PPY@PAA/fmSiO₂ NPs show high in vivo tumor uptake by the enhanced permeability and retention (EPR) effect after intravenous injection as revealed by in vivo fluorescence imaging, which is very helpful for visualizing the location of the tumor. Moreover, the obtained NPs inhibit tumor growth (95.6 % of tumors were eliminated) because of the combination of chemo‐photothermal therapy, which offers a synergistically improved therapeutic outcome compared with the use of either therapy alone. Therefore, the present study provides new insights into developing NIR and pH‐stimuli responsive PPY‐based multifunctional platform for cancer theranostics.     

An Activatable Polymeric Reporter for Near‐Infrared Fluorescent and Photoacoustic Imaging of Invasive Cancer

Discriminative detection of invasive and noninvasive breast cancers is crucial for their effective treatment and prognosis. However, activatable probes able to do so in vivo are rare. Herein, we report an activatable polymeric reporter (P‐Dex) that specifically turns on near‐infrared (NIR) fluorescent and photoacoustic (PA) signals in response to the urokinase‐type plasminogen activator (uPA) overexpressed in invasive breast cancer. P‐Dex has a renal‐clearable dextran backbone that is linked with a NIR dye caged with an uPA‐cleavable peptide substrate. Such a molecular design allows P‐Dex to passively target tumors, activate NIR fluorescence and PA signals to effectively distinguish invasive MDA‐MB‐231 breast cancer from noninvasive MCF‐7 breast cancer, and ultimately undergo renal clearance to minimize the toxicity potential. Thus, this polymeric reporter holds great promise for the early detection of malignant breast cancer.     

A Multifunctional Nanomicelle for Real‐Time Targeted Imaging and Precise Near‐Infrared Cancer Therapy

Simultaneous targeted cancer imaging, therapy and real‐time therapeutic monitoring can prevent over‐ or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near‐infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH‐activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer‐specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real‐time imaging. Upon NIR irradiation, R16FP‐mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.     

Phosphonated Near‐Infrared Fluorophores for Biomedical Imaging of Bone

The conventional method for creating targeted contrast agents is to conjugate separate targeting and fluorophore domains. A new strategy is based on the incorporation of targeting moieties into the non‐delocalized structure of pentamethine and heptamethine indocyanines. Using the known affinity of phosphonates for bone minerals in a model system, two families of bifunctional molecules that target bone without requiring a traditional bisphosphonate are synthesized. With peak fluorescence emissions at approximately 700 or 800 nm, these molecules can be used for fluorescence‐assisted resection and exploration (FLARE) dual‐channel imaging. Longitudinal FLARE studies in mice demonstrate that phosphonated near‐infrared fluorophores remain stable in bone for over five weeks, and histological analysis confirms their incorporation into the bone matrix. Taken together, a new strategy for creating ultra‐compact, targeted near‐infrared fluorophores for various bioimaging applications is described.     

Noninvasive Staging of Kidney Dysfunction Enabled by Renal‐Clearable Luminescent Gold Nanoparticles

As a “silent killer”, kidney disease is often hardly detected at an early stage but can cause lethal kidney failure later on. Thus, a preclinical imaging technique that can readily differentiate between the stages of kidney dysfunction is highly desired for improving our fundamental understanding of kidney disease progression. Herein, we report that in vivo fluorescence imaging, enabled by renal‐clearable near‐infrared‐emitting gold nanoparticles, can noninvasively detect kidney dysfunction, report on the dysfunctional stages, and even reveal adaptive function in a mouse model of unilateral obstructive nephropathy, which cannot be diagnosed with routine kidney function markers. These results demonstrate that low‐cost fluorescence kidney functional imaging is highly sensitive and useful for the longitudinal, noninvasive monitoring of kidney dysfunction progression in preclinical research.     

Renal‐clearable Molecular Semiconductor for Second Near‐Infrared Fluorescence Imaging of Kidney Dysfunction

Real‐time imaging of kidney function is important to assess the nephrotoxicity of drugs and monitor the progression of renal diseases; however, it remains challenging because of the lack of optical agents with high renal clearance and high signal‐to‐background ratio (SBR). Herein, a second near‐infrared (NIR‐II) fluorescent molecular semiconductor (CDIR2) is synthesized for real‐time imaging of kidney dysfunction in living mice. CDIR2 not only has a high renal clearance efficiency (≈90 % injection dosage at 24 h post‐injection), but also solely undergoes glomerular filtration into urine without being reabsorbed and secreted in renal tubules. Such a unidirectional renal clearance pathway of CDIR2 permits real‐time monitoring of kidney dysfunction in living mice upon nephrotoxic exposure. Thus, this study not only introduces a molecular renal probe but also provides useful molecular guidelines to increase the renal clearance efficiency of NIR‐II fluorescent agents.     

Sterically Shielded Heptamethine Cyanine Dyes for Bioconjugation and High Performance Near‐Infrared Fluorescence Imaging

The near‐infrared window of fluorescent heptamethine cyanine dyes greatly facilitates biological imaging because there is deep penetration of the light and negligible background fluorescence. However, dye instability, aggregation, and poor pharmacokinetics are current drawbacks that limit performance and the scope of possible applications. All these limitations are simultaneously overcome with a new molecular design strategy that produces a charge balanced and sterically shielded fluorochrome. The key design feature is a meso‐aryl group that simultaneously projects two shielding arms directly over each face of a linear heptamethine polyene. Cell and mouse imaging experiments compared a shielded heptamethine cyanine dye (and several peptide and antibody bioconjugates) to benchmark heptamethine dyes and found that the shielded systems possess an unsurpassed combination of photophysical, physiochemical, and biodistribution properties that greatly enhance bioimaging performance.     

Click‐Assembled,Oxygen‐Sensing Nanoconjugates for Depth‐Resolved,Near‐Infrared Imaging in a 3 D Cancer Model

Hypoxia is an important contributing factor to the development of drug‐resistant cancer, yet few nonperturbative tools exist for studying oxygenation in tissues. While progress has been made in the development of chemical probes for optical oxygen mapping, penetration of such molecules into poorly perfused or avascular tumor regions remains problematic. A click‐assembled oxygen‐sensing (CAOS) nanoconjugate is reported and its properties demonstrated in an in vitro 3D spheroid cancer model. The synthesis relies on the sequential click‐based ligation of poly(amidoamine)‐like subunits for rapid assembly. Near‐infrared confocal phosphorescence microscopy was used to demonstrate the ability of the CAOS nanoconjugates to penetrate hundreds of micrometers into spheroids within hours and to show their sensitivity to oxygen changes throughout the nodule. This proof‐of‐concept study demonstrates a modular approach that is readily extensible to a wide variety of oxygen and cellular sensors for depth‐resolved imaging in tissue and tissue models.     

A Renal‐Clearable Duplex Optical Reporter for Real‐Time Imaging of Contrast‐Induced Acute Kidney Injury

Despite its high morbidity and mortality, contrast‐induced acute kidney injury (CIAKI) remains a diagnostic dilemma because it relies on in vitro detection of insensitive late‐stage blood and urinary biomarkers. We report the synthesis of an activatable duplex reporter (ADR) for real‐time in vivo imaging of CIAKI. ADR is equipped with chemiluminescence and near‐infrared fluorescence (NIRF) signaling channels that can be activated by oxidative stress (superoxide anion, O₂^.?) and lysosomal damage (N‐acetyl‐β‐d ‐glucosaminidase, NAG), respectively. By virtue of its high renal clearance efficiency (80 % injected doses after 24 h injection), ADR detects sequential upregulation of O₂^.? and NAG in the kidneys of living mice prior to a significant decrease in glomerular filtration rate (GFR) and tissue damage in the course of CIAKI. ADR outperforms the typical clinical assays and detects CIAKI at least 8 h (NIRF) and up to 16 h (chemiluminescence) earlier.     

Covalent Organic Framework for Improving Near‐Infrared Light Induced Fluorescence Imaging through Two‐Photon Induction

Fluorescent materials exhibiting two‐photon induction (TPI) are used for nonlinear optics, bioimaging, and phototherapy. Polymerizations of molecular chromophores to form π‐conjugated structures were hindered by the lack of long‐range ordering in the structure and strong π–π stacking between the chromophores. Reported here is the rational design of a benzothiadiazole‐based covalent organic framework (COF) for promoting TPI and obtaining efficient two‐photon induced fluorescence emissions. Characterization and spectroscopic data revealed that the enhancement in TPI performance is attributed to the donor‐π‐acceptor‐π‐donor configuration and regular intervals of the chromophores, the large π‐conjugation domain, and the long‐range order of COF crystals. The crystalline structure of TPI‐COF attenuates the π–π stacking interactions between the layers, and overcomes aggregation‐caused emission quenching of the chromophores for improving near‐infrared two‐photon induced fluorescence imaging.     

A Near‐Infrared Fluorescence‐Based Optical Thermosensor

A polymeric thermosensor composed of the thermo‐responsive block copolymer Pluronic F127 (PF127) and the near‐infrared (NIR) dye Cy5.5 can simply monitor, image, and analyze temperature changes. The thermoprobe exhibited linear NIR fluorescent emission changes (see figure) over a broad temperature range (0–80 °C).

     

Near‐Infrared Fluorescent Molecular Probe for Sensitive Imaging of Keloid

Early detection of skin diseases is imperative for their effective treatment. However, fluorescence molecular probes that allow this are rare. The first activatable near‐infrared (NIR) fluorescent molecular probe is reported for sensitive imaging of keloid cells, skin cells from abnormal scar fibrous lesions. As keloid cells have high expression levels of fibroblast activation protein‐alpha (FAPα), the probe (FNP1) is designed to have a caged NIR dye and a FAPα‐cleavable peptide substrate linked by a self‐immolative segment. FNP1 can quickly and specifically turn on its fluorescence at 710 nm by 45‐fold in the presence of FAPα, allowing it to effectively recognize keloid cells from normal skin cells. Integration of FNP1 with a simple microneedle‐assisted topical application enables sensitive detection of keloid cells in metabolically‐active human skin tissue with a theoretical limit of detection down to 20 000 cells.     

Synthesis of a Near‐Infrared BODIPY Dye for Bioimaging and Photothermal Therapy

The development of robust photothermal agents for near‐infrared (NIR) imaging is a great challenge. Herein, we report the design and synthesis of a new photothermal agent, based on the aza‐boron‐dipyrromethene framework (azaBDP). This compound possessed excellent photostability and high photothermal‐conversion efficiency (50 %) under NIR laser irradiation. When the photothermal properties of this compound were utilized for tumor inhibition, stable long‐term fluorescence was observed in living animals. Photothermal treatment efficiently suppressed tumor growth, as evidenced by in vitro and in vivo experiments. Furthermore, NIR emission could be detected by using an imaging system and therapeutic self‐monitoring was achieved by using NIR imaging.     

Near‐Infrared‐Light‐Mediated Imaging of Latent Fingerprints based on Molecular Recognition

Photoluminescence is one of the most sensitive techniques for fingerprint detection, but it also suffers from background fluorescence and selectivity at the expense of generality. The method described herein integrates the advantages of near‐infrared‐light‐mediated imaging and molecular recognition. In principle, upconversion nanoparticles (UCNPs) functionalized with a lysozyme‐binding aptamer were used to detect fingerprints through recognizing lysozyme in the fingerprint ridges. UCNPs possess the ability to suppress background fluorescence and make it possible for fingerprint imaging on problematic surfaces. Lysozyme, a universal compound in fingerprints, was chosen as the target, thus simultaneously meeting the selectivity and generality criteria in photoluminescence approaches. Fingerprints on different surfaces and from different people were detected successfully. This strategy was used to detect fingerprints with cocaine powder by using UCNPs functionalized with a cocaine‐binding aptamer.     

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red‐shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red‐shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual‐color, far‐red to near‐infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far‐red to nIR emission maxima up to λ_max=706 nm with different Fluc mutants. This emission is the most red‐shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep‐tissue bioluminescence imaging.