Culture-Independent Multiplexed Detection of Drug-Resistant Bacteria Using Surface-Enhanced Raman Scattering.

The rapid and accurate detection of bacteria resistance to ß-lactam antibiotics is critical to inform optimal treatment and prevent overprescription of potent antibiotics. Here, we present a fast, culture-independent method for the detection of extended-spectrum ß-lactamases (ESBLs) using surface-enhanced Raman scattering (SERS). The method uses Raman probes that release sulfur-based Raman active molecules in the presence of ß-lactamases. The released thiol molecules can be captured by gold nanoparticles, leading to amplified Raman signals. A broad-spectrum cephalosporin probe R1G and an ESBL-specific probe R3G are designed to enable duplex detection of bacteria expressing broad-spectrum ß-lactamases or ESBLs with a detection limit of 103 cfu/mL in 1 h incubation. Combined with a portable Raman microscope, our culturing-free SERS assay has reduced screening time to 1.5 h without compromising sensitivity and specificity.

Bioluminogenic Probe for Rapid, Ultrasensitive Detection of ß-Lactam-Resistant Bacteria.

Increasingly difficult-to-treat infections by antibiotic-resistant bacteria have become a major public health challenge. Rapid detection of common resistance mechanisms before empiric antibiotic usage is essential for optimizing therapeutic outcomes and containing further spread of resistance to antibiotics among other bacteria. Herein, we present a bioluminogenic probe, D-Bluco, for rapid detection of ß-lactamase activity in viable pathogenic bacteria. D-Bluco is a pro-luciferin caged by a ß-lactamase-responsive cephalosporin structure and further conjugated with a dabcyl quencher. The caging and quenching significantly decreased the initial background emission and increased the signal-to-background ratio by more than 1200-fold. D-Bluco was shown to detect a broad range of ß-lactamases at the femtomolar level. An ultrasensitive RAPID bioluminescence assay using D-Bluco can detect 102 to 103 colony forming unit per milliliter (cfu/mL) of ß-lactamase-producing Enterobacterales in urine samples within 30 min. The high sensitivity and rapid detection make the assay attractive for the use of point-of-care diagnostics for lactam-resistant pathogens.

Highly Excretable Gold Supraclusters for Translatable In Vivo Raman Imaging of Tumors.

Raman spectroscopy provides excellent specificity for in vivo preclinical imaging through a readout of fingerprint-like spectra. To achieve sufficient sensitivity for in vivo Raman imaging, metallic gold nanoparticles larger than 10 nm were employed to amplify Raman signals via surface-enhanced Raman scattering (SERS). However, the inability to excrete such large gold nanoparticles has restricted the translation of Raman imaging. Here we present Raman-active metallic gold supraclusters that are biodegradable and excretable as nanoclusters. Although the small size of the gold nanocluster building blocks compromises the electromagnetic field enhancement effect, the supraclusters exhibit bright and prominent Raman scattering comparable to that of large gold nanoparticle-based SERS nanotags due to high loading of NIR-resonant Raman dyes and much suppressed fluorescence background by metallic supraclusters. The bright Raman scattering of the supraclusters was pH-responsive, and we successfully performed in vivo Raman imaging of acidic tumors in mice. Furthermore, in contrast to large gold nanoparticles that remain in the liver and spleen over 4 months, the supraclusters dissociated into small nanoclusters, and 73% of the administered dose to mice was excreted during the same period. The highly excretable Raman supraclusters demonstrated here offer great potential for clinical applications of in vivo Raman imaging.

A TLR7-nanoparticle adjuvant promotes a broad immune response against heterologous strains of influenza and SARS-CoV-2.

The ideal vaccine against viruses such as influenza and SARS-CoV-2 must provide a robust, durable and broad immune protection against multiple viral variants. However, antibody responses to current vaccines often lack robust cross-reactivity. Here we describe a polymeric Toll-like receptor 7 agonist nanoparticle (TLR7-NP) adjuvant, which enhances lymph node targeting, and leads to persistent activation of immune cells and broad immune responses. When mixed with alum-adsorbed antigens, this TLR7-NP adjuvant elicits cross-reactive antibodies for both dominant and subdominant epitopes and antigen-specific CD8+ T-cell responses in mice. This TLR7-NP-adjuvanted influenza subunit vaccine successfully protects mice against viral challenge of a different strain. This strategy also enhances the antibody response to a SARS-CoV-2 subunit vaccine against multiple viral variants that have emerged. Moreover, this TLR7-NP augments antigen-specific responses in human tonsil organoids. Overall, we describe a nanoparticle adjuvant to improve immune responses to viral antigens, with promising implications for developing broadly protective vaccines.

In vivo bioluminescence imaging of granzyme B activity in tumor response to cancer immunotherapy.

Cancer immunotherapy has revolutionized the treatment of cancer, but only a small subset of patients benefits from this new treatment regime. Imaging tools are useful for early detection of tumor response to immunotherapy and probing the dynamic and complex immune system. Here, we report a bioluminescence probe (GBLI-2) for non-invasive, real-time, longitudinal imaging of granzyme B activity in tumors receiving immune checkpoint inhibitors. GBLI-2 is made of the mouse granzyme B tetrapeptide IEFD substrate conjugated to D-luciferin through a self-immolative group. GBLI-2 was evaluated for imaging the dynamics of the granzyme B activity and predicting therapeutic efficacy in a syngeneic mouse model of CT26 murine colorectal carcinoma. The GBLI-2 signal correlated with the change in the population of PD-1- and granzyme B-expressing CD8+ T cells in tumors.

Real-time optical oximetry during FLASH radiotherapy using a phosphorescent nanoprobe.

The rapid depletion of oxygen during irradiation at ultra-high dose rate calls for tissue oximeters capable of high temporal resolution. This study demonstrates a water-soluble phosphorescent nanoprobe and fiber-coupled instrument, which together are used to measure the kinetics of oxygen depletion at 200 Hz during irradiation of in vitro solutions.

Multiparameter Longitudinal Imaging of Immune Cell Activity in Chimeric Antigen Receptor T Cell and Checkpoint Blockade Therapies.

Longitudinal multimodal imaging presents unique opportunities for noninvasive surveillance and prediction of treatment response to cancer immunotherapy. In this work we first designed a novel granzyme B activated self-assembly small molecule, G-SNAT, for the assessment of cytotoxic T lymphocyte mediated cancer cell killing. G-SNAT was found to specifically detect the activity of granzyme B within the cytotoxic granules of activated T cells and engaged cancer cells in vitro. In lymphoma tumor-bearing mice, the retention of cyanine 5 labeled G-SNAT-Cy5 correlated to CAR T cell mediated granzyme B exocytosis and tumor eradication. In colorectal tumor-bearing transgenic mice with hematopoietic cells expressing firefly luciferase, longitudinal bioluminescence and fluorescence imaging revealed that after combination treatment of anti-PD-1 and anti-CTLA-4, the dynamics of immune cell trafficking, tumor infiltration, and cytotoxic activity predicted the therapeutic outcome before tumor shrinkage was evident. These results support further development of G-SNAT for imaging early immune response to checkpoint blockade and CAR T-cell therapy in patients and highlight the utility of multimodality imaging for improved mechanistic insights into cancer immunotherapy.

Reversibly Photoswitching Upconversion Nanoparticles for Super-Sensitive Photoacoustic Molecular Imaging.

Photoacoustic (PA) imaging uses light excitation to generate the acoustic signal for detection and improves tissue penetration depth and spatial resolution in the clinically relevant depth of living subjects. However, strong background signals from blood and pigments have significantly compromised the sensitivity of PA imaging with exogenous contrast agents. Here we report a nanoparticle-based probe design that uses light to reversibly modulate the PA emission to enable photoacoustic photoswitching imaging (PAPSI) in living mice. Such a nanoprobe is built with upconverting nanocrystals and photoswitchable small molecules and can be switched on by NIR light through upconversion to UV energy. Reversibly photoswitching of the nanoprobe reliably removed strong tissue background, increased the contrast-to-noise ratio, and thus improved imaging sensitivity. We have shown that PAPSI can image 0.05 nM of the nanoprobe in hemoglobin solutions and 104 labeled cancer cells after implantation in living mice using a commercial PA imager.

Evaluation of a procaspase-3 activator with hydroxyurea or temozolomide against high-grade meningioma in cell culture and canine cancer patients.

BACKGROUND: High-grade meningioma is an aggressive type of brain cancer that is often recalcitrant to surgery and radiotherapy, leading to poor overall survival. Currently, there are no FDA-approved drugs for meningioma, highlighting the need for new therapeutic options, but development is challenging due to the lack of predictive preclinical models. METHODS: To leverage the known overexpression of procaspase-3 in meningioma, PAC-1, a blood-brain barrier penetrant procaspase-3 activator, was evaluated for its ability to induce apoptosis in meningioma cells. To enhance the effects of PAC-1, combinations with either hydroxyurea or temozolomide were explored in cell culture. Both combinations were further investigated in small groups of canine meningioma patients and assessed by MRI, and the novel apoptosis tracer, [18F]C-SNAT4, was evaluated in patients treated with PAC-1 + HU. RESULTS: In meningioma cell lines in culture, PAC-1 + HU are synergistic while PAC-1 + TMZ show additive-to-synergistic effects. In canine meningioma patients, PAC-1 + HU led to stabilization of disease and no change in apoptosis within the tumor, whereas PAC-1 + TMZ reduced tumor burden in all three canine patients treated. CONCLUSIONS: Our results suggest PAC-1 + TMZ as a potentially efficacious combination for the treatment of human meningioma, and also demonstrate the utility of including pet dogs with meningioma as a means to assess anticancer strategies for this common brain tumor.

A dual-caged resorufin probe for rapid screening of infections resistant to lactam antibiotics.

The alarming increase of antimicrobial resistance urges rapid diagnosis and pathogen specific infection management. This work reports a rapid screening assay for pathogenic bacteria resistant to lactam antibiotics. We designed a fluorogenic N-cephalosporin caged 3,7-diesterphenoxazine probe CDA that requires sequential activations to become fluorescent resorufin. A series of studies with recombinant ß-lactamases and clinically prevalent pathogens including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens demonstrated that CDA possessed superior sensitivity in reporting the activity of ß-lactamases including cephalosporinases and carbapenemases. After a simple filtration, lactam-resistant bacteria in urine samples could be detected at 103 colony-forming units per milliliter within 2 hours.

Engineering of magnetic nanoparticles as magnetic particle imaging tracers.

Magnetic particle imaging (MPI) has recently emerged as a promising non-invasive imaging technique because of its signal linearly propotional to the tracer mass, ability to generate positive contrast, low tissue background, unlimited tissue penetration depth, and lack of ionizing radiation. The sensitivity and resolution of MPI are highly dependent on the properties of magnetic nanoparticles (MNPs), and extensive research efforts have been focused on the design and synthesis of tracers. This review examines parameters that dictate the performance of MNPs, including size, shape, composition, surface property, crystallinity, the surrounding environment, and aggregation state to provide guidance for engineering MPI tracers with better performance. Finally, we discuss applications of MPI imaging and its challenges and perspectives in clinical translation.

Visualizing the dynamics of tuberculosis pathology using molecular imaging.

Nearly 140 years after Robert Koch discovered Mycobacterium tuberculosis, tuberculosis (TB) remains a global threat and a deadly human pathogen. M. tuberculosis is notable for complex host-pathogen interactions that lead to poorly understood disease states ranging from latent infection to active disease. Additionally, multiple pathologies with a distinct local milieu (bacterial burden, antibiotic exposure, and host response) can coexist simultaneously within the same subject and change independently over time. Current tools cannot optimally measure these distinct pathologies or the spatiotemporal changes. Next-generation molecular imaging affords unparalleled opportunities to visualize infection by providing holistic, 3D spatial characterization and noninvasive, temporal monitoring within the same subject. This rapidly evolving technology could powerfully augment TB research by advancing fundamental knowledge and accelerating the development of novel diagnostics, biomarkers, and therapeutics.

[18F]-C-SNAT4: an improved caspase-3-sensitive nanoaggregation PET tracer for imaging of tumor responses to chemo- and immunotherapies.

Positron emission tomography (PET) imaging of apoptosis can noninvasively detect cell death in vivo and assist in monitoring tumor response to treatment in patients. While extensive efforts have been devoted to addressing this important need, no apoptosis PET imaging agents have yet been approved for clinical use. This study reports an improved 18F-labeled caspase-sensitive nanoaggregation tracer ([18F]-C-SNAT4) for PET imaging of tumor response to chemo- and immunotherapies in preclinical mouse models. METHODS: We rationally designed and synthesized a new PET tracer [18F]-C-SNAT4 to detect cell death both in vitro and in vivo. In vitro radiotracer uptake studies were performed on drug-sensitive and -resistant NSCLC cell lines (NCI-H460 and NCI-H1299, respectively) treated with cisplatin at different doses. In vivo therapy response monitoring by [18F]-C-SNAT4 PET imaging was evaluated with two treatment modalities-chemotherapy and immunotherapy in two tumor xenografts in mice. Radiotracer uptake in the tumors was validated ex vivo using γ-counting and cleaved caspase-3 immunofluorescence. RESULTS: This [18F]-C-SNAT4 PET tracer was facilely synthesized and displayed improved serum stability profiles. [18F]-C-SNAT4 cellular update was elevated in NCI-H460 cells in a time- and dose-dependent manner, which correlated well with cell death. A significant increase in [18F]-C-SNAT4 uptake was measured in NCI-H460 tumor xenografts in mice. In contrast, a rapid clearance of [18F]-C-SNAT4 was observed in drug-resistant NCI-H1299 in vitro and in tumor xenografts. Moreover, in BALB/C mice bearing murine colon cancer CT26 tumor xenografts receiving checkpoint inhibitors, [18F]-C-SNAT4 showed its ability for monitoring immunotherapy-induced apoptosis and reporting treatment-responding mice from non-responding. CONCLUSION: The uptake of [18F]-C-SNAT4 in tumors received chemotherapy and immunotherapy is positively correlated with the tumor apoptotic level and the treatment efficacy. [18F]-C-SNAT4 PET imaging can monitor tumor response to two different treatment modalities and predict the therapeutic efficacy in preclinical mouse models.

In Vivo Imaging of Methionine Aminopeptidase II for Prostate Cancer Risk Stratification.

Prostate cancer is one of the most common malignancies worldwide, yet limited tools exist for prognostic risk stratification of the disease. Identification of new biomarkers representing intrinsic features of malignant transformation and development of prognostic imaging technologies are critical for improving treatment decisions and patient survival. In this study, we analyzed radical prostatectomy specimens from 422 patients with localized disease to define the expression pattern of methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that has been identified as a druggable target in cancer. MetAP2 was highly expressed in 54% of low-grade and 59% of high-grade cancers. Elevated levels of MetAP2 at diagnosis were associated with shorter time to recurrence. Controlled self-assembly of a synthetic small molecule enabled design of the first MetAP2-activated PET imaging tracer for monitoring MetAP2 activity in vivo. The nanoparticles assembled upon MetAP2 activation were imaged in single prostate cancer cells with post-click fluorescence labeling. The fluorine-18-labeled tracers successfully differentiated MetAP2 activity in both MetAP2-knockdown and inhibitor-treated human prostate cancer xenografts by micro-PET/CT scanning. This highly sensitive imaging technology may provide a new tool for noninvasive early-risk stratification of prostate cancer and monitoring the therapeutic effect of MetAP2 inhibitors as anticancer drugs. SIGNIFICANCE: This study defines MetAP2 as an early-risk stratifier for molecular imaging of aggressive prostate cancer and describes a MetAP2-activated self-assembly small-molecule PET tracer for imaging MetAP2 activity in vivo.

Mitochondrial copper depletion suppresses triple-negative breast cancer in mice.

Depletion of mitochondrial copper, which shifts metabolism from respiration to glycolysis and reduces energy production, is known to be effective against cancer types that depend on oxidative phosphorylation. However, existing copper chelators are too toxic or ineffective for cancer treatment. Here we develop a safe, mitochondria-targeted, copper-depleting nanoparticle (CDN) and test it against triple-negative breast cancer (TNBC). We show that CDNs decrease oxygen consumption and oxidative phosphorylation, cause a metabolic switch to glycolysis and reduce ATP production in TNBC cells. This energy deficiency, together with compromised mitochondrial membrane potential and elevated oxidative stress, results in apoptosis. CDNs should be less toxic than existing copper chelators because they favorably deprive copper in the mitochondria in cancer cells instead of systemic depletion. Indeed, we demonstrate low toxicity of CDNs in healthy mice. In three mouse models of TNBC, CDN administration inhibits tumor growth and substantially improves survival. The efficacy and safety of CDNs suggest the potential clinical relevance of this approach.

Different PEG-PLGA Matrices Influence In Vivo Optical/Photoacoustic Imaging Performance and Biodistribution of NIR-Emitting π-Conjugated Polymer Contrast Agents.

The π-conjugated polymer poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b0]-dithiophene)-alt-4,7-(2,1,3-benzothiadiazole)] (PCPDTBT) with deep-red/near-infrared (NIR) absorption and emission has been investigated as a contrast agent for in vivo optical and photoacoustic imaging. PCPDTBT is encapsulated within poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG2kDa -PLGA4kDa or PEG5kDa -PLGA55kDa ) micelles or enveloped by the phospholipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2kDa -DPPE), to investigate the formulation effect on imaging performance, biodistribution, and biocompatibility. Nanoparticles that meet the quality requirements for parenteral administration are generated with similar physicochemical properties. Optical phantom imaging reveals that both PEG-PLGA systems exhibit a 30% higher signal-to-background ratio (SBR) than PEG2kDa -DPPE. This trend cannot be observed in a murine HeLa xenograft model following intravenous administration since dramatic differences in biodistribution are observed. PEG2kDa -PLGA4kDa systems accumulate more rapidly in the liver compared to other formulations and PEG2kDa -DPPE demonstrates a higher tumor localization. Protein content in the "hard" corona differs between formulations (PEG2kDa -DPPE < PEG2kDa -PLGA4kDa < PEG5kDa -PLGA55kDa ), although this observation alone does not explain biodistribution patterns. PEG2kDa -PLGA4kDa systems show the highest photoacoustic amplitude in a phantom, but also a lower signal in the tumor due to differences in biodistribution. This study demonstrates that formulations for conjugated polymer contrast agents can have significant impact on both imaging performance and biodistribution.

A Fluorogenic Trehalose Probe for Tracking Phagocytosed Mycobacterium tuberculosis.

Tuberculosis (TB) disease is a global epidemic caused by the pathogenic Mycobacterium tuberculosis (Mtb). Tools that can track the replication status of viable Mtb cells within macrophages are vital for the elucidation of host-pathogen interactions. Here, we present a cephalosphorinase-dependent green trehalose (CDG-Tre) fluorogenic probe that enables fluorescence labeling of single live Bacille Calmette-Guérin (BCG) cells within macrophages at concentrations as low as 2 µM. CDG-Tre fluoresces upon activation by BlaC, the ß-lactamase uniquely expressed by Mtb, and the fluorescent product is subsequently incorporated within the bacterial cell wall via trehalose metabolic pathway. CDG-Tre showed high selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder. The unique labeling strategy of BCG by CDG-Tre provides a versatile tool for tracking Mtb in both pre- and postphagocytosis and elucidating fundamental physiological and pathological processes related to the mycomembrane.

Reduction Triggered In Situ Polymerization in Living Mice.

"Smart" biomaterials that are responsive to physiological or biochemical stimuli have found many biomedical applications for tissue engineering, therapeutics, and molecular imaging. In this work, we describe in situ polymerization of activatable biorthogonal small molecules in response to a reducing environment change in vivo. We designed a carbohydrate linker- and cyanobenzothiazole-cysteine condensation reaction-based small molecule scaffold that can undergo rapid condensation reaction upon physiochemical changes (such as a reducing environment) to form polymers (pseudopolysaccharide). The fluorescent and photoacoustic properties of a fluorophore-tagged condensation scaffold before and after the transformation have been examined with a dual-modality optical imaging method. These results confirmed the in situ polymerization of this probe after both local and systemic administration in living mice.

Engineered algae: A novel oxygen-generating system for effective treatment of hypoxic cancer.

Microalgae, a naturally present unicellular microorganism, can undergo light photosynthesis and have been used in biofuels, nutrition, etc. Here, we report that engineered live microalgae can be delivered to hypoxic tumor regions to increase local oxygen levels and resensitize resistant cancer cells to both radio- and phototherapies. We demonstrate that the hypoxic environment in tumors is markedly improved by in situ-generated oxygen through microalgae-mediated photosynthesis, resulting in notably radiotherapeutic efficacy. Furthermore, the chlorophyll from microalgae produces reactive oxygen species during laser irradiation, further augmenting the photosensitizing effect and enhancing tumor cell apoptosis. Thus, the sequential combination of oxygen-generating algae system with radio- and phototherapies has the potential to create an innovative treatment strategy to improve the outcome of cancer management. Together, our findings demonstrate a novel approach that leverages the products of photosynthesis for treatment of tumors and provide proof-of-concept evidence for future development of algae-enhanced radio- and photodynamic therapy.