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.

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.

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.

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.

[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.

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.

Targeting MMP-14 for dual PET and fluorescence imaging of glioma in preclinical models.

PURPOSE: There is a clinical need for agents that target glioma cells for non-invasive and intraoperative imaging to guide therapeutic intervention and improve the prognosis of glioma. Matrix metalloproteinase (MMP)-14 is overexpressed in glioma with negligible expression in normal brain, presenting MMP-14 as an attractive biomarker for imaging glioma. In this study, we designed a peptide probe containing a near-infrared fluorescence (NIRF) dye/quencher pair, a positron emission tomography (PET) radionuclide, and a moiety with high affinity to MMP-14. This novel substrate-binding peptide allows dual modality imaging of glioma only after cleavage by MMP-14 to activate the quenched NIRF signal, enhancing probe specificity and imaging contrast. METHODS: MMP-14 expression and activity in human glioma tissues and cells were measured in vitro by immunofluorescence and gel zymography. Cleavage of the novel substrate and substrate-binding peptides by glioma cells in vitro and glioma xenograft tumors in vivo was determined by NIRF imaging. Biodistribution of the radiolabeled MMP-14-binding peptide or substrate-binding peptide was determined in mice bearing orthotopic patient-derived xenograft (PDX) glioma tumors by PET imaging. RESULTS: Glioma cells with MMP-14 activity showed activation and retention of NIRF signal from the cleaved peptides. Resected mouse brains with PDX glioma tumors showed tumor-to-background NIRF ratios of 7.6-11.1 at 4 h after i.v. injection of the peptides. PET/CT images showed localization of activity in orthotopic PDX tumors after i.v. injection of 68Ga-binding peptide or 64Cu-substrate-binding peptide; uptake of the radiolabeled peptides in tumors was significantly reduced (p < 0.05) by blocking with the non-labeled-binding peptide. PET and NIRF signals correlated linearly in the orthotopic PDX tumors. Immunohistochemistry showed co-localization of MMP-14 expression and NIRF signal in the resected tumors. CONCLUSIONS: The novel MMP-14 substrate-binding peptide enabled PET/NIRF imaging of glioma models in mice, warranting future image-guided resection studies with the probe in preclinical glioma models.

Pre-targeted Imaging of Protease Activity through In†Situ Assembly of Nanoparticles.

The pre-targeted imaging of enzyme activity has not been reported, likely owing to the lack of a mechanism to retain the injected substrate in the first step for subsequent labeling. Herein, we report the use of two bioorthogonal reactions-the condensation reaction of aromatic nitriles and aminothiols and the inverse-electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO)-to develop a novel strategy for pre-targeted imaging of the activity of proteases. The substrate probe (TCO-C-SNAT4) can be selectively activated by an enzyme target (e.g. caspase-3/7), which triggers macrocyclization and subsequent in situ self-assembly into nanoaggregates retained at the target site. The tetrazine-imaging tag conjugate labels TCO in the nanoaggregates to generate selective signal retention for imaging in vitro, in cells, and in mice. Owing to the decoupling of enzyme activation and imaging tag immobilization, TCO-C-SNAT4 can be repeatedly injected to generate and accumulate more TCO-nanoaggregates for click labeling.

Exploring the Condensation Reaction between Aromatic Nitriles and Amino Thiols To Optimize In†Situ Nanoparticle Formation for the Imaging of Proteases and Glycosidases in Cells.

The condensation reaction between 6-hydroxy-2-cyanobenzothiazole (CBT) and cysteine has been shown for various applications such as site-specific protein labelling and in vivo cancer imaging. This report further expands the substrate scope of this reaction by varying the substituents on aromatic nitriles and amino thiols and testing their reactivity and ability to form nanoparticles for cell imaging. The structure-activity relationship study leads to the identification of the minimum structural requirement for the macrocyclization and assembly process in forming nanoparticles. One of the scaffolds made of 2-pyrimidinecarbonitrile and cysteine joined by a benzyl linker was applied to design fluorescent probes for imaging caspase-3/7 and ß-galactosidase activity in live cells. These results demonstrate the generality of this system for imaging hydrolytic enzymes.

[18F]-SuPAR: A Radiofluorinated Probe for Noninvasive Imaging of DNA Damage-Dependent Poly(ADP-ribose) Polymerase Activity.

Poly(ADP ribose) polymerase (PARP) enzymes generate poly(ADP ribose) post-translational modifications on target proteins for an array of functions centering on DNA and cell stress. PARP isoforms 1 and 2 are critically charged with the surveillance of DNA integrity and are the first line guardians of the genome against DNA breaks. Here we present a novel probe ([18F]-SuPAR) for noninvasive imaging of PARP-1/2 activity using positron emission tomography (PET). [18F]-SuPAR is a radiofluorinated nicotinamide adenine dinucleotide (NAD) analog that can be recognized by PARP-1/2 and incorporated into the long branched polymers of poly(ADP ribose) (PAR). The measurement of PARP-1/2 activity was supported by a reduction of radiotracer uptake in vivo following PARP-1/2 inhibition with talazoparib treatment, a potent PARP inhibitor recently approved by FDA for treatment of breast cancer, as well as ex vivo colocalization of radiotracer analog and poly(ADP ribose). With [18F]-SuPAR, we were able to map the dose- and time-dependent activation of PARP-1/2 following radiation therapy in breast and cervical cancer xenograft mouse models. Tumor response to therapy was determined by [18F]-SuPAR PET within 8 h of administration of a single dose of radiation equivalent to one round of stereotactic ablative radiotherapy.

Janus Iron Oxides @ Semiconducting Polymer Nanoparticle Tracer for Cell Tracking by Magnetic Particle Imaging.

Iron oxides nanoparticles tailored for magnetic particle imaging (MPI) have been synthesized, and their MPI signal intensity is three-times that of commercial MPI contrast (Ferucarbotran, also called Vivotrax) and seven-times that of MRI contrast (Feraheme) at the same Fe concentration. MPI tailored iron oxide nanoparticles were encapsulated with semiconducting polymers to produce Janus nanoparticles that possessed optical and magnetic properties for MPI and fluorescence imaging. Janus particles were applied to cancer cell labeling and in vivo tracking, and as few as 250 cells were imaged by MPI after implantation, corresponding to an amount of 7.8 ng of Fe. Comparison with MRI and fluorescence imaging further demonstrated the advantages of our Janus particles for MPI-super sensitivity, unlimited tissue penetration, and linear quantitativity.

Real-time Imaging of Mycobacterium tuberculosis, Using a Novel Near-Infrared Fluorescent Substrate.

Slow growth of Mycobacterium tuberculosis, the causative agent of tuberculosis, hinders advancement in all areas of research toward prevention and treatment. Real-time imaging with reporter enzyme fluorescence (REF) that uses custom fluorogenic substrates for bacterial enzymes allows rapid and specific detection of M. tuberculosis in live animals. We have synthesized a novel REF substrate, CNIR800, that carries a near-infrared (NIR) fluorochrome IRDye 800CW, with a quencher connected through the lactam ring that is hydrolyzed by the enzyme BlaC (ß-lactamase) that is naturally expressed by M. tuberculosis. CNIR800 produces long-wavelength emission at 795 nm upon excitation (745 nm) and exhibits significantly improved signal to noise ratios for detection of M. tuberculosis. The detection threshold with CNIR800 is approximately 100 colony-forming units (CFU) in vitro and <1000 CFU in the lungs of mice. Additionally, fluorescence signal from cleaved CNIR800 reaches maximal levels 4-6 hours after administration in live animals, allowing accurate evaluation of antituberculous drug efficacy. Thus, CNIR800 represents an excellent substrate for accurate detection of M. tuberculosis rapidly and specifically in animals, facilitating research toward understanding pathogenic mechanisms, evaluation of therapeutic outcomes, and screening new vaccines.

Point-of-Care Detection of ß-Lactamase in Milk with a Universal Fluorogenic Probe.

The illegal addition of ß-lactamase (Bla) in milk to disguise ß-lactam antibiotics has been a serious issue in the milk industry worldwide. Herein, we report a method for point-of-care detection of Bla based on a probe, Tokyo Green-tethered ß-lactam (CDG-1), as a common substrate of various Blas (Bla A, B...) which can enzymatically convert CDG-1 (low fluorescence) to Tokyo Green (high fluorescence). This approach allows rapid screening of a broad spectrum of Blas in real milk samples within 15 min without any pretreatment. Combined with the immuno-magnetic separation, we achieved sensitive and quantitative detection of Bla (10(-5) U/mL), which provides a universal platform for screening and determining Blas in complex samples with high efficiency and accuracy.

Semiconducting Polymer Nanoparticles with Persistent Near-Infrared Luminescence for In Vivo Optical Imaging.

Materials with persistent luminescence are attractive for in vivo optical imaging since they have a long lifetime that allows the separation of excitation of fluorophores and image acquisition for time-delay imaging, thus eliminating tissue autofluorescence associated with fluorescence imaging. Persistently luminescent nanoparticles have previously been fabricated from toxic rare-earth metals. This work reports that nanoparticles made of the conjugated polymer MEH-PPV can generate luminescence persisting for an hour upon single excitation. A near-infrared dye was encapsulated in the conjugated polymer nanoparticle to successfully generate persistent near-infrared luminescence through resonance energy transfer. This new persistent luminescence nanoparticles have been demonstrated for optical imaging applications in living mice.

Development of novel tumor-targeted theranostic nanoparticles activated by membrane-type matrix metalloproteinases for combined cancer magnetic resonance imaging and therapy.

A major drawback with current cancer therapy is the prevalence of unrequired dose-limiting toxicity to non-cancerous tissues and organs, which is further compounded by a limited ability to rapidly and easily monitor drug delivery, pharmacodynamics and therapeutic response. In this report, the design and characterization of novel multifunctional "theranostic" nanoparticles (TNPs) is described for enzyme-specific drug activation at tumor sites and simultaneous in vivo magnetic resonance imaging (MRI) of drug delivery. TNPs are synthesized by conjugation of FDA-approved iron oxide nanoparticles ferumoxytol to an MMP-activatable peptide conjugate of azademethylcolchicine (ICT), creating CLIO-ICTs (TNPs). Significant cell death is observed in TNP-treated MMP-14 positive MMTV-PyMT breast cancer cells in vitro, but not MMP-14 negative fibroblasts or cells treated with ferumoxytol alone. Intravenous administration of TNPs to MMTV-PyMT tumor-bearing mice and subsequent MRI demonstrates significant tumor selective accumulation of the TNP, an observation confirmed by histopathology. Treatment with CLIO-ICTs induces a significant antitumor effect and tumor necrosis, a response not observed with ferumoxytol. Furthermore, no toxicity or cell death is observed in normal tissues following treatment with CLIO-ICTs, ICT, or ferumoxytol. These findings demonstrate proof of concept for a new nanotemplate that integrates tumor specificity, drug delivery and in vivo imaging into a single TNP entity through attachment of enzyme-activated prodrugs onto magnetic nanoparticles. This novel approach holds the potential to significantly improve targeted cancer therapies, and ultimately enable personalized therapy regimens.

Redox-triggered self-assembly of gadolinium-based MRI probes for sensing reducing environment.

Controlled self-assembly of small molecule gadolinium (Gd) complexes into nanoparticles (GdNPs) is emerging as an effective approach to design activatable magnetic resonance imaging (MRI) probes and amplify the r1 relaxivity. Herein, we employ a reduction-controlled macrocyclization reaction and self-assembly to develop a redox activated Gd-based MRI probe for sensing a reducing environment. Upon disulfide reduction at physiological conditions, an acyclic contrast agent 1 containing dual Gd-chelates undergoes intramolecular macrocyclization to form rigid and hydrophobic macrocycles, which subsequently self-assemble into GdNPs, resulting in a ∼60% increase in r1 relaxivity at 0.5 T. Probe 1 has high r1 relaxivity (up to 34.2 mM(-1) s(-1) per molecule at 0.5 T) upon activation, and also shows a high sensitivity and specificity for MR detection of thiol-containing biomolecules.

Comparison of two site-specifically (18)F-labeled affibodies for PET imaging of EGFR positive tumors.

The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules (64)Cu-DOTA-ZEGFR:1907 and (18)F-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with (64)Cu, (18)F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of (18)F-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of (18)F labeled affibody probes (Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with Al(18)F to produce Al(18)F-NOTA-ZEGFR:1907. In a second approach the prosthetic group (18)F-labeled-2-cyanobenzothiazole ((18)F-CBT) was conjugated to Cys-ZEGFR:1907 to produce (18)F-CBT-ZEGFR:1907. Binding affinity and specificity of Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al(18)F-NOTA-ZEGFR:1907 or (18)F-CBT-ZEGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although (18)F-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al(18)F-NOTA-ZEGFR:1907 demonstrated high in vitro and in vivo stability, high tumor uptake, and relative low uptake in most of the normal organs except the liver and kidneys at 3 h after injection. The specificity of both probes for A431 tumors was confirmed by their lower uptake on coinjection of unlabeled affibody. PET studies showed that Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 could clearly identify EGFR positive tumors with good contrast. Two strategies for (18)F-labeling of affibody molecules were successfully developed as two model platforms using NOTA or CBT coupling to affibody molecules that contain an N-terminal cysteine. Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 can be reliably obtained in a relatively short time. Biodistribution and PET studies demonstrated that Al(18)F-NOTA-ZEGFR:1907 is a promising PET probe for imaging EGFR expression in living mice.

Engineering the stereochemistry of cephalosporin for specific detection of pathogenic carbapenemase-expressing bacteria.

Reported herein is the design of fluorogenic probes specific for carbapenem-resistant Enterobacteriaceae (CRE) and they were designed based on stereochemically modified cephalosporin having a 6,7-trans configuration. Through experiments using recombinant ß-lactamase enzymes and live bacterial species, these probes demonstrate the potential for use in the specific detection of carbapenemases, including metallo-ß-lactamases in active bacterial pathogens.

Fluorogenic probes with substitutions at the 2 and 7 positions of cephalosporin are highly BlaC-specific for rapid Mycobacterium tuberculosis detection.

Current methods for the detection of Mycobacterium tuberculosis (Mtb) are either time consuming or require expensive instruments and are thus are not suitable for point-of-care diagnosis. The design, synthesis, and evaluation of fluorogenic probes with high specificity for BlaC, a biomarker expressed by Mtb, are described. The fluorogenic probe CDG-3 is based on cephalosporin with substitutions at the 2 and 7 positions and it demonstrates over 120,000-fold selectivity for BlaC over TEM-1 Bla, the most common ß-lactamase. CDG-3 can detect 10 colony-forming units of the attenuated Mycobacterium bovis strain BCG in human sputum in the presence of high levels of contaminating ß-lactamases expressed by other clinically prevalent bacterial strains. In a trial with 50 clinical samples, CDG-3 detected tuberculosis with 90% sensitivity and 73% specificity relative to Mtb culture within one hour, thus demonstrating its potential as a low-cost point-of-care test for use in resource-limited areas.