Lanthanide Inorganic Nanoparticles Enhance Semiconducting Polymer Nanoparticles Afterglow Luminescence for In Vivo Afterglow/Magnetic Resonance Imaging.

Dual/multimodal imaging strategies are increasingly recognized for their potential to provide comprehensive diagnostic insights in cancer imaging by harnessing complementary data. This study presents an innovative probe that capitalizes on the synergistic benefits of afterglow luminescence and magnetic resonance imaging (MRI), effectively eliminating autofluorescence interference and delivering a superior signal-to-noise ratio. Additionally, it facilitates deep tissue penetration and enables noninvasive imaging. Despite the advantages, only a limited number of probes have demonstrated the capability to simultaneously enhance afterglow luminescence and achieve high-resolution MRI and afterglow imaging. Herein, we introduce a cutting-edge imaging platform based on semiconducting polymer nanoparticles (PFODBT) integrated with NaYF4@NaGdF4 (Y@Gd@PFO-SPNs), which can directly amplify afterglow luminescence and generate MRI and afterglow signals in tumor tissues. The proposed mechanism involves lanthanide nanoparticles producing singlet oxygen (1O2) upon white light irradiation, which subsequently oxidizes PFODBT, thereby intensifying afterglow luminescence. This innovative platform paves the way for the development of high signal-to-background ratio imaging modalities, promising noninvasive diagnostics for cancer.

CD9 shapes glucocorticoid sensitivity in pediatric B-cell precursor acute lymphoblastic leukemia.

Resistance to glucocorticoids (GCs), the common agents for remission induction in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), poses a significant therapeutic hurdle. Therefore, dissecting the mechanisms shaping GC resistance could lead to new treatment modalities. Here, we showed that CD9- BCP-ALL cells were preferentially resistant to prednisone and dexamethasone over other standard cytotoxic agents. Concordantly, we identified significantly more poor responders to the prednisone prephase among BCP-ALL patients with a CD9- phenotype, especially for those with adverse presenting features including older age, higher white cell count and BCR-ABL1. Furthermore, gain- and loss-of-function experiments dictated a definitive functional linkage between CD9 expression and GC susceptibility, as demonstrated by the reversal and acquisition of relative GC resistance in CD9low and CD9high BCP-ALL cells, respectively. Despite physical binding to the GC receptor NR3C1, CD9 did not alter its expression, phosphorylation or nuclear translocation but potentiated the induction of GC-responsive genes in GCresistant cells. Importantly, the MEK inhibitor trametinib exhibited higher synergy with GCs against CD9- than CD9+ lymphoblasts to reverse drug resistance in vitro and in vivo. Collectively, our results elucidate a previously unrecognized regulatory function of CD9 in GC sensitivity, and inform new strategies for management of children with resistant BCP-ALL.

Rational Design of NIR-II G-Quadruplex Fluorescent Probes for Accurate In Vivo Tumor Metastasis Imaging.

Accurate in vivo imaging of G-quadruplexes (G4) is critical for understanding the emergence and progression of G4-associated diseases like cancer. However, existing in vivo G4 fluorescent probes primarily operate within the near-infrared region (NIR-I), which limits their application accuracy due to the short emission wavelength. The transition to second near-infrared (NIR-II) fluorescent imaging has been of significant interest, as it offers reduced autofluorescence and deeper tissue penetration, thereby facilitating more accurate in vivo imaging. Nonetheless, the advancement of NIR-II G4 probes has been impeded by the absence of effective probe design strategies. Herein, through a "step-by-step" rational design approach, we have successfully developed NIRG-2, the first small-molecule fluorescent probe with NIR-II emission tailored for in vivo G4 detection. Molecular docking calculations reveal that NIRG-2 forms stable hydrogen bonds and strong π-π interactions with G4 structures, which effectively inhibit twisted intramolecular charge transfer (TICT) and, thereby, selectively illuminate G4 structures. Due to its NIR-II emission (940 nm), large Stokes shift (90 nm), and high selectivity, NIRG-2 offers up to 47-fold fluorescence enhancement and a tissue imaging depth of 5 mm for in vivo G4 detection, significantly outperforming existing G4 probes. Utilizing NIRG-2, we have, for the first time, achieved high-contrast visualization of tumor metastasis through lymph nodes and precise tumor resection. Furthermore, NIRG-2 proves to be highly effective and reliable in evaluating surgical and drug treatment efficacy in cancer lymphatic metastasis models. We are optimistic that this study not only provides a crucial molecular tool for an in-depth understanding of G4-related diseases in vivo but also marks a promising strategy for the development of clinical NIR-II G4-activated probes.

C-terminal binding protein (CTBP2) is a novel tumor suppressor targeting the MYC-IRF4 axis in multiple myeloma.

Multiple myeloma (MM) cells are addicted to MYC and its direct transactivation target IRF4 for proliferation and survival. MYC and IRF4 are still considered "undruggable" as the majority of small molecule inhibitors suffers from low potency, suboptimal pharmacokinetic properties and undesirable off-target effects. Indirect inhibition of MYC/IRF4 emerges as a therapeutic vulnerability in MM. Here, we uncover an unappreciated tumor suppressive role of C-terminal Binding Protein 2 (CTBP2) in MM via strong inhibition of the MYC-IRF4 axis. In contrast to epithelial cancers, CTBP2 is frequently downregulated in MM, in association with shortened survival, hyperproliferative features and adverse clinical outcomes. Restoration of CTBP2 exhibited potent anti-tumor effects against MM in vitro and in vivo, with marked repression of MYC-IRF4 network genes. Mechanistically, CTBP2 impeded transcription of MYC and IRF4 by histone H3 lysine 27 deacetylation (H3K27ac), and indirectly via activation of MYC repressor IFIT3. In addition, activation of interferon gene signature by CTBP2 suggested its concomitant immunomodulatory role in MM. Epigenetic studies revealed contribution of polycomb-mediated silencing and DNA methylation to CTBP2 inactivation in MM. Notably, inhibitors of Enhance of zeste homolog 2 (EZH2), histone deacetylase (HDACs) and DNA methyltransferase (DNMTs) currently under evaluation in clinical trials were effective in restoring CTBP2 expression in MM. Our findings indicated that loss of CTBP2 plays an essential role in myelomagenesis and decipher an additional mechanistic link on MYC-IRF4 dysregulation in MM. We envision that identification of novel critical regulators would facilitate the development of selective and effective approaches for treating this MYC/IRF4-addicted malignancy.

Enhancing Human Treg Cell Induction through Engineered Dendritic Cells and Zinc Supplementation.

Regulatory T (Treg) cells hold promise for the ultimate cure of immune-mediated diseases. However, how to effectively restore Treg function in patients remains unknown. Previous reports suggest that activated dendritic cells (DCs) de novo synthesize locally high concentrations of 1,25-dihydroxy vitamin D, i.e., the active vitamin D or 1,25(OH)2D by upregulating the expression of 25-hydroxy vitamin D 1α-hydroxylase. Although 1,25(OH)2D has been shown to induce Treg cells, DC-derived 1,25(OH)2D only serves as a checkpoint to ensure well-balanced immune responses. Our animal studies have shown that 1,25(OH)2D requires high concentrations to generate Treg cells, which can cause severe side effects. In addition, our animal studies have also demonstrated that dendritic cells (DCs) overexpressing the 1α-hydroxylase de novo synthesize the effective Treg-inducing 1,25(OH)2D concentrations without causing the primary side effect of hypercalcemia (i.e., high blood calcium levels). This study furthers our previous animal studies and explores the efficacy of the la-hydroxylase-overexpressing DCs in inducing human CD4+FOXP3+regulatory T (Treg) cells. We discovered that the effective Treg-inducing doses of 1,25(OH)2D were within a range. Additionally, our data corroborated that the 1α-hydroxylase-overexpressing DCs synthesized 1,25(OH)2D within this concentration range in vivo, thus facilitating effective Treg cell induction. Moreover, this study demonstrated that 1α-hydroxylase expression levels were pivotal for DCs to induce Treg cells because physiological 25(OH)D levels were sufficient for the engineered but not parental DCs to enhance Treg cell induction. Interestingly, adding non-toxic zinc concentrations significantly augmented the Treg-inducing capacity of the engineered DCs. Our new findings offer a novel therapeutic avenue for immune-mediated human diseases, such as inflammatory bowel disease, type 1 diabetes, and multiple sclerosis, by integrating zinc with the 1α-hydroxylase-overexpressing DCs.

"Zero" Intrinsic Fluorescence Sensing-Platforms Enable Ultrasensitive Whole Blood Diagnosis and In Vivo Imaging.

Abnormal physiological processes and diseases can lead to content or activity fluctuations of biocomponents in organelles and whole blood. However, precise monitoring of these abnormalities remains extremely challenging due to the insufficient sensitivity and accuracy of available fluorescence probes, which can be attributed to the background fluorescence arising from two sources, 1) biocomponent autofluorescence (BCAF) and 2) probe intrinsic fluorescence (PIF). To overcome these obstacles, we have re-engineered far-red to NIR II rhodol derivatives that possess weak BCAF interference. And a series of "zero" PIF sensing-platforms were created by systematically regulating the open-loop/spirocyclic forms. Leveraging these advancements, we devised various ultra-sensitive NIR indicators, achieving substantial fluorescence boosts (190 to 1300-fold). Among these indicators, 8-LAP demonstrated accurate tracking and quantifying of leucine aminopeptidase (LAP) in whole blood at various stages of tumor metastasis. Furthermore, coupling 8-LAP with an endoplasmic reticulum-targeting element enabled the detection of ERAP1 activity in HCT116 cells with p53 abnormalities. This delicate design of eliminating PIF provides insights into enhancing the sensitivity and accuracy of existing fluorescence probes toward the detection and imaging of biocomponents in abnormal physiological processes and diseases.

Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing.

BACKGROUND: CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. RESULTS: We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. CONCLUSIONS: Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

Leveraging CRISPR-Cas9 for Accurate Detection of AAV-Neutralizing Antibodies: The AAV-HDR Method.

The effectiveness of adeno-associated virus (AAV)-based gene therapy is frequently constrained by the presence of AAV-neutralizing antibodies (NAbs). Existing detection techniques have shown inconsistencies across laboratories and cellular dependencies, challenging their universal applicability. Here, we redefine the NAb titer concept to represent the capability to neutralize a specific number of AAV virions per milliliter of serum. We present the AAV-homology-directed repair (HDR) assay, which harnesses the CRISPR-Cas9 system, offering a precise and sensitive means of detecting AAV NAbs. This assay employs a promoterless AAV HDR vector for integration into electroporated cells, facilitating the stable expression of a quantifiable fluorescent reporter and subsequent NAb titer assessment. Comparative evaluations indicated that the AAV-HDR method outperforms the traditional AAV overexpression (AAV-OE) assay regarding sensitivity and consistency. Crucially, it produced consistent outcomes across various cell lines, suggesting its potential as a universal standard for NAb titer measurement. We further confirmed the validity of the AAV-HDR titration approach by juxtaposing it with the established NT50 assay. Notably, the AAV-HDR method correlated robustly with both the AAV-OE assay and NT50 NAb titer values, and it exhibited heightened efficacy in identifying low-titer antibodies compared with the NT50 method. Given its ability to address AAV NAb detection challenges, the AAV-HDR assay holds promise for refining therapeutic strategies in gene therapy, particularly in tailoring AAV doses to neutralize preexisting NAbs.

Chemical Approaches to Optimize the Properties of Organic Fluorophores for Imaging and Sensing.

Organic fluorophores are indispensable tools in cells, tissue and in vivo imaging, and have enabled much progress in the wide range of biological and biomedical fields. However, many available dyes suffer from insufficient performances, such as short absorption and emission wavelength, low brightness, poor stability, small Stokes shift, and unsuitable permeability, restricting their application in advanced imaging technology and complex imaging. Over the past two decades, many efforts have been made to improve these performances of fluorophores. Starting with the luminescence principle of fluorophores, this review clarifies the mechanisms of the insufficient performance for traditional fluorophores to a certain extent, systematically summarizes the modified approaches of optimizing properties, highlights the typical applications of the improved fluorophores in imaging and sensing, and indicates existing problems and challenges in this area. This progress not only proves the significance of improving fluorophores properties, but also provide a theoretical guidance for the development of high-performance fluorophores.

The design strategies for CRISPR-based biosensing: Target recognition, signal conversion, and signal amplification.

Rapid, sensitive and selective biosensing is highly important for analyzing biological targets and dynamic physiological processes in cells and living organisms. As an emerging tool, clustered regularly interspaced short palindromic repeats (CRISPR) system is featured with excellent complementary-dependent cleavage and efficient trans-cleavage ability. These merits enable CRISPR system to improve the specificity, sensitivity, and speed for molecular detection. Herein, the structures and functions of several CRISPR proteins for biosensing are summarized in depth. Moreover, the strategies of target recognition, signal conversion, and signal amplification for CRISPR-based biosensing were highlighted from the perspective of biosensor design principles. The state-of-art applications and recent advances of CRISPR system are then outlined, with emphasis on their fluorescent, electrochemical, colorimetric, and applications in POCT technology. Finally, the current challenges and future prospects of this frontier research area are discussed.

Enhancing lipid peroxidation via radical chain transfer reaction for MRI guided and effective cancer therapy in mice.

Lipid peroxidation (LPO), the process of membrane lipid oxidation, is a potential new form of cell death for cancer treatment. However, the radical chain reaction involved in LPO is comprised of the initiation, propagation (the slowest step), and termination stages, limiting its effectiveness in vivo. To address this limitation, we introduce the radical chain transfer reaction into the LPO process to target the propagation step and overcome the sluggish rate of lipid peroxidation, thereby promoting endogenous lipid peroxidation and enhancing therapeutic outcomes. Firstly, radical chain transfer agent (CTA-1)/Fe nanoparticles (CTA-Fe NPs-1) was synthesized. Notably, CTA-1 convert low activity peroxyl radicals (ROO·) into high activity alkoxyl radicals (RO·), creating the cycle of free radical oxidation and increasing the propagation of lipid peroxidation. Additionally, CTA-1/Fe ions enhance reactive oxygen species (ROS) generation, consume glutathione (GSH), and thereby inactivate GPX-4, promoting the initiation stage and reducing termination of free radical reaction. CTA-Fe NPs-1 induce a higher level of peroxidation of polyunsaturated fatty acids in lipid membranes, leading to highly effective treatment in cancer cells. In addition, CTA-Fe NPs-1 could be enriched in tumors inducing potent tumor inhibition and exhibit activatable T1-MRI contrast of magnetic resonance imaging (MRI). In summary, CTA-Fe NPs-1 can enhance intracellular lipid peroxidation by accelerating initiation, propagation, and inhibiting termination step, promoting the cycle of free radical reaction, resulting in effective anticancer outcomes in tumor-bearing mice.

Superoxide Anion-Mediated Afterglow Mechanism-Based Water-Soluble Zwitterion Dye Achieving Renal-Failure Mice Detection.

Afterglow materials-based biological imaging has promising application prospects, due to negligible background. However, currently available afterglow materials mainly include inorganic materials as well as some organic nanoparticles, which are difficult to translate to the clinic, resulting from non-negligible metabolic toxicity and even leakage risk of inorganic heavy metals. Although building small organic molecules could solve such obstacles, organic small molecules with afterglow ability are extremely scarce, especially with a sufficient renal metabolic capacity. To address these issues, herein, we designed water-soluble zwitterion Cy5-NF with renal metabolic capacity and afterglow luminescence, which relied on an intramolecular cascade reaction between superoxide anion (O2•-, instead of 1O2) and Cy5-NF to release afterglow luminescence. Of note, compared with different reference contrast agents, zwitterion Cy5-NF not only had excellent afterglow properties but also had a rapid renal metabolism rate (half-life period, t1/2, around 10 min) and good biocompatibility. Unlike prior afterglow nanosystems possessing a large size, for the first time, zwitterion Cy5-NF has achieved the construction of water-soluble renal metabolic afterglow contrast agents, which showed higher sensitivity and signal-to-background ratio in afterglow imaging than fluorescence imaging for the kidney. Moreover, zwitterion Cy5-NF had a longer kidney retention time in renal-failure mice (t1/2 more than 15 min). More importantly, zwitterion Cy5-NF can be metabolized very quickly even in severe renal-failure mice (t1/2 around 25-30 min), which greatly improved biosecurity. Therefore, we are optimistic that the O2•--mediated afterglow mechanism-based water-soluble zwitterion Cy5-NF is very promising for clinical application, especially rapid detection of kidney failure.

Enhancing cord blood stem cell-derived NK cell growth and differentiation through hyperosmosis.

BACKGROUND: Natural killer (NK) cells hold great promise in treating diverse hematopoietic and solid tumors. Despite their availability from peripheral blood and cord blood, stem cell-derived NK cells offer an 'off-the-shelf' solution. Hematopoietic stem and progenitor cells (HSPCs) derived from cord blood pose no risk to the newborn or mother and are virtually ideal sources for NK cell differentiation. METHODS: We developed a modified protocol to differentiate HSPCs to NK cells under serum-free conditions using defined factors. The HSPC-derived NK (HSC-NK) cells could be expanded in a K562 feeder cell-dependent manner. Furthermore, using lentivirus transduction, chimeric antigen receptor (CAR)-modified HSPCs could be differentiated into NK cells, leading to the establishment of CAR-NK cells. RESULTS: The efficiency of NK cell differentiation from HSPCs was increased through the simple modulation of osmotic pressure by the addition of sodium chloride or glucose. Furthermore, the hyperosmosis-primed HSC-NK cells exhibited enhanced proliferation capacity and maintained normal functional characteristics, including transcriptome and antitumor efficacy. The optimized protocol yielded approximately 1.8 million NK cells from a single CD34-positive cell within a 28-day cycle, which signifies more than a ten-fold increase in efficiency relative to the conventional methods. This optimized protocol was also suitable for generating CAR-NK cells with high yields compared to standard conditions. CONCLUSIONS: The results of this study establish high osmotic pressure as a simple yet powerful adjustment that significantly enhances the efficiency and functionality of HSC-NK cells, including CAR-NK cells. This optimized protocol could lead to cost-effective, high-yield NK cell therapies, potentially revolutionizing cancer immunotherapy strategies.

Noninvasive Imaging of Tumor Glycolysis and Chemotherapeutic Resistance via De Novo Design of Molecular Afterglow Scaffold.

Chemotherapeutic resistance poses a significant challenge in cancer treatment, resulting in the reduced efficacy of standard chemotherapeutic agents. Abnormal metabolism, particularly increased anaerobic glycolysis, has been identified as a major contributing factor to chemotherapeutic resistance. To address this issue, noninvasive imaging techniques capable of visualizing tumor glycolysis are crucial. However, the currently available methods (such as PET, MRI, and fluorescence) possess limitations in terms of sensitivity, safety, dynamic imaging capability, and autofluorescence. Here, we present the de novo design of a unique afterglow molecular scaffold based on hemicyanine and rhodamine dyes, which holds promise for low-background optical imaging. In contrast to previous designs, this scaffold exhibits responsive "OFF-ON" afterglow signals through spirocyclization, thus enabling simultaneous control of photodynamic effects and luminescence efficacy. This leads to a larger dynamic range, broader detection range, higher signal enhancement ratio, and higher sensitivity. Furthermore, the integration of multiple functionalities simplifies probe design, eliminates the need for spectral overlap, and enhances reliability. Moreover, we have expanded the applications of this afterglow molecular scaffold by developing various probes for different molecular targets. Notably, we developed a water-soluble pH-responsive afterglow nanoprobe for visualizing glycolysis in living mice. This nanoprobe monitors the effects of glycolytic inhibitors or oxidative phosphorylation inhibitors on tumor glycolysis, providing a valuable tool for evaluating the tumor cell sensitivity to these inhibitors. Therefore, the new afterglow molecular scaffold presents a promising approach for understanding tumor metabolism, monitoring chemotherapeutic resistance, and guiding precision medicine in the future.

Oxidative stress biomarker triggered multiplexed tool for auxiliary diagnosis of atherosclerosis.

Oxidative stress is integral in the development of atherosclerosis, but knowledge of how oxidative stress affects atherosclerosis remains insufficient. Here, we design a multiplexed diagnostic tool that includes two functions (photoacoustic imaging and urinalysis), for assessing intraplaque and urinary malondialdehyde (MDA), a well-recognized end-product of oxidative stress. Molecular design is conducted to develop the first near-infrared MDA-responsive molecule (MRM). Acid-unlocked ratiometric photoacoustic nanoprobe is designed to report intraplaque MDA, enabling it to reflect plaque burden. Furthermore, MRM is tailored for urinary MDA detection with excellent specificity in a blind study. Moreover, we found a significant difference in urinary MDA between healthy adults and atherosclerotic patients (more than 600 participants). Combining these two functions, such a multiplexed diagnostic tool can dynamically report intraplaque and systemic oxidative stress levels during atherosclerosis progression, pneumonia infection, and drug treatment in atherosclerotic mice, which is promising for the auxiliary diagnosis of atherosclerosis.

Vanin-1-Activated Chemiluminescent Probe: Help to Early Diagnosis of Acute Kidney Injury with High Signal-to-Noise Ratio through Urinalysis.

Acute kidney injury (AKI) is a common medical condition with high morbidity and mortality. Although urinalysis provides a noninvasive and convenient diagnostic method for AKI at the molecular level, the low sensitivity of current chemical probes used in urinalysis hinders the time diagnosis of AKI. Herein, we achieved the sensitive and early diagnosis of AKI by the development of a chemiluminescent probe CL-Pa suitable for detection of urinary Vanin-1. Vanin-1 is considered as an early and sensitive biomarker for AKI, while few chemical probes have been applied to for its efficient detection. By virtue of the low autofluorescence interference during urine imaging in the chemiluminescence model, CL-Pa could realize the monitoring of the up-regulated urinary Vanin-1 with a high signal-to-noise ratio (∼588). Importantly, under the help of CL-Pa, the up-regulation of urinary Vanin-1 of cisplatin-induced AKI mice at 12 h post cisplatin injection was detected, which was much earlier than clinical biomarkers (sCr and BUN) and change of kidney histology (48 h post cisplatin injection). Furthermore, using this probe, the fluctuation of urinary Vanin-1 of mice with different degrees of AKI was monitored. This study demonstrated the ability of CL-Pa in sensitively detecting drug-induced AKI through urinalysis and suggested the great potential of CL-Pa for early diagnosis of AKI and evaluate the efficiency of anti-AKI drugs clinically.

FINDER: A Fluidly Confined CRISPR-Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification.

The accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the naturally confined enzymes on fluid cell membranes, a fluidly confined CRISPR-based DNA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR-Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamer-based DNA logic gate for cell recognition, thus a FINDER system capable of accurate, rapid and sensitive cancer cell identification was constructed. The FINDER rapidly identified target cells in only 20 min, and achieved over 80 % recognition efficiency with only 0.1 % of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.

Engineering of cyanine-based nanoplatform with tunable response toward reactive species for ratiometric NIR-II fluorescent imaging in mice.

High-quality second near-infrared (NIR-II) nanoprobes are of great significance for real-time bioimaging and medical diagnosis. Cyanine is an important class of fluorophores to construct activatable probes; however, there are still significant challenges hindering their biological applications, including weak fluorescence in aqueous solution, instability, and insufficient specificity. Herein, an integrated engineering strategy is conducted to develop the cyanine-based activatable NIR-II nanoplatforms with bright, stable emission and high specificity. Specifically, poly(styrene-co-maleic anhydride) (PSMA) is employed to encapsulate NIR-II fluorescent molecules (IR1048) to render the stable and bright NIR-II nanoparticles (PSMA@IR1048 NPs). By charge-modulated strategy, a series of cyanine-fluorophores are loaded on the surface of PSMA@IR1048 NPs and exhibit tunable response toward reactive species. Combing those two strategies, NIR-II ratiometric fluorescent nanoprobes (RNPs, including RNP1, RNP2, and RNP3) are constructed; among them, RNP2 displays hypochlorous acid (HClO) responsive performance and generates a higher NIR-II fluorescent ratio (FL2/FL1) signal. Such nanoprobe can reliably report the pathological HClO level in models of diabetic liver injury and lower limb ischemia-reperfusion (I/R) injury mice. Our study paves an engineering strategy to construct cyanine-based stable, bright, and specific NIR-II probes for bioimaging.

Rational Design of a Double-Locked Photoacoustic Probe for Precise In Vivo Imaging of Cathepsin B in Atherosclerotic Plaques.

Atherosclerotic plaque rupture is a significant cause of acute cardiovascular events such as heart attack and stroke, triggered by the decomposition of fiber caps induced by cysteine cathepsin. However, the accurate measurement of cathepsin B (CTB) activity in plaques is challenging due to the low specificity and insufficient penetration depth of available atherosclerosis-associated cathepsin fluorescent probes, hampering reliable assessment of plaque vulnerability. To address these limitations, we added both lipophilic alkyl chain and hydrophilic CTB substrate to the hemicyanine scaffold to develop a lipid-unlocked CTB responsive probe (L-CRP) that uses lipids and CTB as two keys to unlock photoacoustic (PA) signals for measuring CTB activity in lipophilic environments. Such properties allow L-CRP for the reliable imaging of specific CTB activities in foam cells and atherosclerotic plaques while keeping in silence toward CTB in lipid-deficient environments, such as M1-type macrophages and LPS-induced inflammatory lesions. Moreover, the activatable PA signals of L-CRP exhibit a deeper tissue penetration ability (>1.0 cm) than current CTB probes based on near-infrared fluorescent imaging (∼0.3 cm), suitable for atherosclerosis imaging in living mice. In atherosclerotic mice, L-CRP dynamically reports intraplaque CTB levels, which is well-correlated with the plaque vulnerability characteristics such as fiber cap thickness, macrophage recruitment, and necrotic core size, thus enabling risk stratification of atherosclerotic mice complicated with pneumonia. Moreover, L-CRP successfully identifies atherosclerotic plaques in excised human artery tissues, promising for auxiliary diagnosis of plaque vulnerability in clinical application.

Design of a near-infrared fluoro-photoacoustic probe for rapid imaging of carboxylesterase in liver injury.

Carboxylesterase (CE) is crucial in metabolizing ester-containing biomolecules and is particularly significant in liver metabolic diseases. Herein, we present the first activatable NIRF/PA dual-mode imaging probe QHD-CE for detection of CE in vitro and in vivo. QHD-CE displays excellent sensitivity and selectivity for CE with a high reaction efficiency (∼90 min). By utilizing QHD-CE, the dynamic changes of CE in drug-induced liver injury and diabetic mice models were monitored.