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Near-infrared (NIR) light possesses many suitable optophysical properties for medical imaging including low autofluorescence, deep tissue penetration, and minimal light scattering, which together allow for high-resolution imaging of biological tissue. NIR imaging has proven to be a noninvasive and effective real-time imaging methodology that provides a high signal-to-background ratio compared to other potential optical imaging modalities. In response to this, the use of NIR imaging has been extensively explored in the field of immunotherapy. To date, NIR fluorescence imaging has successfully offered reliable monitoring of the localization, dynamics, and function of immune responses, which are vital in assessing not only the efficacy but also the safety of treatments to design immunotherapies optimally. This review aims to provide an overview of the current research on NIR imaging of the immune response. We expect that the use of NIR imaging will expand further in response to the recent success in cancer immunotherapy. We will also offer our insights on how this technology will meet rapidly growing expectations in the future.

Surface-enhanced Raman scattering (SERS) enables rapid detection of single molecules with high specificity. However, quantitative and sensitive SERS analysis has been a challenge due to the lack of reliable SERS-active materials. In this study, we developed a quantitative SERS-based immunoassay using enzyme-guided Ag growth on Raman labeling compound (RLC)-immobilized gold nanoparticle (Au NP)-assembled silica NPs (SiO2@Au-RLC@Ag). The enzyme amplified Ag+ reduction as well as Ag growth on the RLC-immobilized Au NP-assembled silica NPs (SiO2@Au-RLC), which resulted in a significant increase in SERS signal. In the presence of target antigens such as immunoglobulinG (IgG) or prostate-specific antigen (PSA), Ab1-Antigen-Ab2 immune complex with alkaline phosphatase triggered an enzyme- catalyzed reaction to convert 2-phospho-L-ascorbic acid (2-phospho-L-AA) to ascorbic acid (AA). As produced AA reduced Ag+ to Ag, forming an Ag hot spot on the surface of SiO2@Au-RLC, which enhanced the SERS signal of SiO2@Au-RLC@Ag in a solution with a target antigen concentration. The plasmonic immunoassay for IgG detection showed a high linearity of SERS intensity in the range of 0.6 to 9.0 ng/mL with a detection limit (LOD) of 0.09 ng/mL, while an LOD of 0.006 ng/mL was obtained for PSA. The results indicate that the sensitivity of our novel SERS-based immunoassay is higher than that of conventional enzyme-based colorimetric immunoassays.

Iron chelators have been widely used to remove excess toxic iron from patients with secondary iron overload. However, small molecule-based iron chelators can cause adverse side effects such as infection, gastrointestinal bleeding, kidney failure, and liver fibrosis. Here we report renal clearable nanochelators for iron overload disorders. First, after a singledose intravenous injection, the nanochelator shows favorable pharmacokinetic properties, such as kidney-specific biodistribution and rapid renal excretion (>80% injected dose in 4 h), compared to native deferoxamine (DFO). Second, subcutaneous (SC) administration of nanochelators improves pharmacodynamics, as evidenced by a 7-fold increase in efficiency of urinary iron excretion compared to intravenous injection. Third, daily SC injections of the nanochelator for 5 days to iron overload mice and rats decrease iron levels in serum and liver. Furthermore, the nanochelator significantly reduces kidney damage caused by iron overload without demonstrating DFO’s own nephrotoxicity. This renal clearable nanochelator provides enhanced efficacy and safety., The build-up of iron in the body can have serious consequences; current treatment therapies suffer from adverse side effects and toxicity. Here, the authors developed renal clearable nanochelators with improved pharmacodynamics and demonstrated their efficacy and safety in iron overload animal models.

Background A new approach targeting aeroallergen sensing in the early events of mucosal immunity could have greater benefit. The CSF1-CSF1R pathway has a critical role in trafficking allergens to regional lymph nodes through activating dendritic cells. Intervention in this pathway could prevent allergen sensitization and subsequent Th2 allergic inflammation. Objective To examine the therapeutic effectiveness of CSF1 and CSF1R inhibition for blocking the dendritic cell function of sensing aeroallergens. Methods We adopted a model of chronic asthma induced by a panel of three naturally occurring allergens and novel delivery system of CSF1R inhibitor encapsulated nanoprobe. Results Selective depletion of CSF1 in airway epithelial cells abolished the production of allergen-reactive IgE, resulting in prevention of new asthma development as well as reversal of established allergic lung inflammation. CDPL-GW nanoprobe containing GW2580, a selective CSF1R inhibitor, showed favorable pharmacokinetics for inhalational treatment and intranasal insufflation delivery of CDPL-GW nanoprobe ameliorated asthma pathologies including allergen-specific serum IgE production, allergic lung and airway inflammation and airway hyper-responsiveness (AHR) with minimal pulmonary adverse reaction. Conclusion The inhibition of the CSF1-CSF1R signaling pathway effectively suppresses sensitization to aeroallergens and consequent allergic lung inflammation in a murine model of chronic asthma. CSF1R inhibition is a promising new target for the treatment of allergic asthma.

BACKGROUND: Advances in tissue engineering and regenerative medicine over the last three decades have made great progress in the development of diagnostic and therapeutic methodologies for damaged tissues. However, regenerative medicine is still not the first line of treatment for patients due to limited understanding of the tissue regeneration process. Therefore, it is prerequisite to develop molecular imaging strategies combined with appropriate contrast agents to validate the therapeutic progress of damaged tissues. METHODS: The goal of this review is to discuss the progress in the development of near-infrared (NIR) contrast agents and their biomedical applications for labeling cells and scaffolds, as well as monitoring the treatment progress of native tissue in living organisms. We also discuss the design consideration of NIR contrast agents for tissue engineering and regenerative medicine in terms of their physicochemical and optical properties. RESULTS: The use of NIR imaging system and targeted contrast agents can provide high-resolution and high sensitivity imaging to track/monitor the in vivo fate of administered cells, the degradation rate of implanted scaffolds, and the tissue growth and integration of surrounding cells during the therapeutic period. CONCLUSION: NIR fluorescence imaging techniques combined with targeted contrast agents can play a significant role in regenerative medicine by monitoring the therapeutic efficacy of implanted cells and scaffolds which would enhance the development of cell therapies and promote their successful clinical translations.

Near-infrared (NIR) fluorescence imaging provides a safe and cost-efficient method for immediate data acquisition and visualization of tissues, with technical advantages including minimal autofluorescence, reduced photon absorption, and low scattering in tissue. In this review, we introduce recent advances in NIR fluorescence imaging systems for thoracic surgery that improve the identification of vital tissues and facilitate the resection of tumorous tissues. When coupled with appropriate NIR fluorophores, NIR fluorescence imaging may transform current intraoperative thoracic surgery methods by enhancing the precision of surgical procedures and augmenting postoperative outcomes through improvements in diagnostic accuracy and reductions in the remission rate.

Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancers, which is associated with metastatic potential and poor prognosis in cancer patients. Therefore, development of EGFR-targeted sensitive imaging probes has been a challenge in tumor targeting, image-guided cancer surgery, patient-selective anti-EGFR therapy, and efficient targeted therapies. Methods: We synthesized a zwitterionic near-infrared fluorophore (ATTO655)-conjugated epidermal growth factor (EGF) as a novel activatable molecular probe. Fluorescence OFF/ON property and EGFR-targeting specificity of EGF-ATTO655 as well as its utility in real-time near-infrared (NIR) fluorescence imaging of EGFR-positive cancers were evaluated using in vitro and in vivo studies. Results: When conjugated to EGF, the fluorescence of ATTO655 quenched efficiently by photo-induced electron transfer (PET) mechanism between the conjugated dyes and nearby amino acid quenchers (tryptophan/tyrosine residues), which was stably maintained at physiological pH and in the presence of serum for at least 17 h. The fluorescence of EGF-ATTO655 turned on by receptor-mediated endocytosis and subsequent disintegration of EGF in EGFR-positive A431 cancer cells, thereby enabling specific and real-time fluorescence imaging of EGFR-positive cancer cells. Consequently, EGFR-positive tumors could be clearly visualized 3 h post-injection with a significantly high tumor-to-background ratio (TBR = 6.37). Conclusion: This PET mechanism-based OFF/ON type of EGF probe showed great potential for rapid, real-time, and target-cell-specific imaging of EGFR-overexpressing cancers in vitro and in vivo.

Nanoparticles (NPs) play a key role in nanomedicine in multimodal imaging, drug delivery and targeted therapy of human diseases. Consequently, due to the attractive properties of NPs including high stability, high payload, multifunctionality, design flexibility, and efficient delivery to target tissues, nanomedicine employs various types of NPs to enhance targeting and treatment efficacy. In this review, we primarily focus on light-responsive materials, such as fluorophores, photosensitizers, semiconducting polymers, carbon structures, gold particles, quantum dots, and upconversion crystals, for their biomedical applications. Armed with these nanomaterials, NPs represent a growing potential in biophotonic imaging (luminescence, photoacoustic, surface enhanced Raman scattering, and optical coherence tomography) as well as targeted therapy (photodynamic therapy, photothermal therapy, and light-responsive drug release).

Biophotonic imaging has revolutionized the operation room by providing surgeons intraoperative image-guidance to diagnose tumors more efficiently and to resect tumors with real-time image navigation. Among many medical imaging modalities, near-infrared (NIR) light is ideal for image-guided surgery because it penetrates relatively deeply into living tissue, while nuclear imaging provides quantitative and unlimited depth information. It is therefore ideal to develop an integrated imaging system by combining NIR fluorescence and gamma-positron imaging to provide surgeons with highly sensitive and quantitative detection of diseases, such as cancer, in real-time without changing the look of the surgical field. The focus of this review is to provide recent progress in intraoperative biophotonic imaging systems, NIR fluorescence imaging and intraoperative nuclear imaging devices, and their future perspectives for image-guided interventions.

Fluorescence imaging of cancers using continuous wave (CW) detection of receptor targeted probes offer poor sensitivity and specificity due to background autofluorescence and non-specific probe accumulation. Here we show that fluorescence lifetime (FLT) imaging can significantly improve tumor contrast using epidermal growth factor receptor (EGFR) and programed death ligand 1 (PD-L1) targeted probes in a preclinical model of human breast cancer. Our results suggest that these probes have significantly longer FLTs in tumors than in normal tissue and the FLT enhancement is receptor dependent. We also show potential for simultaneous quantification of EGFR and PD-L1 using in vivo FLT multiplexing.

Cell‐based tissue engineering strategies have been widely established. However, the contributions of the transplanted cells within the tissue‐engineered scaffolds to the process of tissue regeneration remain poorly understood. Near‐infrared (NIR) fluorescence imaging systems have great potential to non‐invasively monitor the transplanted cell‐based tissue constructs. In this study, labeling mesenchymal stem cells (MSCs) using a lipophilic pentamethine indocyanine (CTNF127, emission at 700 nm) as a NIR fluorophore was optimized, and the CTNF127‐labeled MSCs (NIR‐MSCs) were printed embedding in gelatin methacryloyl bioink. The NIR‐MSCs‐loaded bioink showed excellent printability. In addition, NIR‐MSCs in the 3D constructs showed high cell viability and signal stability for an extended period in vitro. Finally, we were able to non‐invasively monitor the NIR‐MSCs in constructs after implantation in a rat calvarial bone defect model, and the transplanted cells contributed to tissue formation without specific staining. This NIR‐based imaging system for non‐invasive cell monitoring in vivo could play an active role in validating the cell fate in cell‐based tissue engineering applications.

While iron is a nutrient metal, iron overload can result in multiple organ failures. Iron chelators, such as deferoxamine, are commonly used to ameliorate iron overload conditions. However, their uses are limited due to poor pharmacokinetics and adverse effects. Many novel chelator formulations have been developed to overcome these drawbacks. In this review, we have discussed various nanochelators, including linear and branched polymers, dendrimers, polyrotaxane, micelles, nanogels, polymeric nanoparticles and liposomes. Although these research efforts have mainly been focused on nanochelators with longer half-lives, prolonged residence of polymers in the body could raise potential safety issues. We also discussed recent advances in nanochelation technologies, including mechanism-based, long-acting nanochelators.

Summary Design of tissue-specific contrast agents to delineate tumors from background tissues is a major unmet clinical need for ultimate surgical interventions. Bioconjugation of fluorophore(s) to a ligand has been mainly used to target overexpressed receptors on tumors. However, the size of the final targeted ligand can be large, >20 kDa, and cannot readily cross the microvasculature to meet the specific tissue, resulting in low targetability with a high background. Here, we report a small and hydrophilic phenoxazine with high targetability and retention to pancreatic neuroendocrine tumor. This bioengineered fluorophore permits sensitive detection of ultrasmall (, Graphical Abstract, Highlights • Bioengineered near-infrared fluorescent small molecules for PNET imaging • Highlight tumors within a minute after a single bolus injection with improved retention • Sensitive and specific detection of ultrasmall ectopic tumors • Intraoperative image-guided navigation provides a surgical margin, Histology; Chemistry; Cancer

Steady-state visual evoked potentials (SSVEPs) have been extensively utilized to develop brain&ndash, computer interfaces (BCIs) due to the advantages of robustness, large number of commands, high classification accuracies, and information transfer rates (ITRs). However, the use of several simultaneous flickering stimuli often causes high levels of user discomfort, tiredness, annoyingness, and fatigue. Here we propose to design a stimuli-responsive hybrid speller by using electroencephalography (EEG) and video-based eye-tracking to increase user comfortability levels when presented with large numbers of simultaneously flickering stimuli. Interestingly, a canonical correlation analysis (CCA)-based framework was useful to identify target frequency with a 1 s duration of flickering signal. Our proposed BCI-speller uses only six frequencies to classify forty-eight targets, thus achieve greatly increased ITR, whereas basic SSVEP BCI-spellers use an equal number of frequencies to the number of targets. Using this speller, we obtained an average classification accuracy of 90.35 &plusmn, 3.597% with an average ITR of 184.06 &plusmn, 12.761 bits per minute in a cued-spelling task and an ITR of 190.73 &plusmn, 17.849 bits per minute in a free-spelling task. Consequently, our proposed speller is superior to the other spellers in terms of targets classified, classification accuracy, and ITR, while producing less fatigue, annoyingness, tiredness and discomfort. Together, our proposed hybrid eye tracking and SSVEP BCI-based system will ultimately enable a truly high-speed communication channel.

Assessing lymph node (LN) status during tumor resection is fundamental for the staging of colorectal cancer. Current guidelines require a minimum of 12 LNs to be harvested during resection and ultra-staging regional lymph nodes by sentinel lymph node (SLN) assessment is being extensively investigated. The current study presents novel near-infrared (NIR) fluorescent dyes for simultaneous pan lymph node (PanLN; regional) and SLN mapping. PanLN-Forte was intravenously injected in mice and assessed for accumulation in regional LNs. SLN800 was injected intradermally in mice, after which the collection and retention of fluorescence in SLNs were measured using indocyanine green (ICG) and its precursor, SLN700, as references. LNs in the cervical, inguinal, jejunal, iliac, and thoracic basins could clearly be distinguished after a low dose intravenous injection of PanLN-Forte. Background fluorescence was significantly lower compared to the parent compound ZW800-3A (p < 0.001). SLN700 and SLN800 specifically targeted SLNs with fluorescence being retained over 40-fold longer than the current clinically used agent ICG. Using SLN700 and SLN800, absolute fluorescence in SLN was at least 10 times higher than ICG in second-tier nodes, even at 1 hour post-injection. Histologically, the fluorescent signal localized in the LN medulla (PanLN-Forte) or sinus entry (SLN700/SLN800). PanLN-Forte and SLN800 appear to be optimal for real-time NIR fluorescence imaging of regional and SLNs, respectively.

Early diagnosis and monitoring of disease progress are of significant importance in the effective treatment of rheumatoid arthritis (RA), because the continuing inflammation can lead to irreversible joint damage and systemic complications. However, applying imaging modalities for the prognosis of RA remains challenging, because no tissue‐specific guidelines are available to monitor the progressive course of RA. In this study, fluorometric imaging of RA is reported using bioengineered targeted agents of the blood vessel, bone, and cartilage in combination with the customized optical fluorescence imaging system. Separate but simultaneous tissue‐specific images of synovitis, cartilage destruction, and bone resorption are obtained from a mouse model of RA, which allows quantification of the prognosis of diseases at each stage. Thus, the fluorometric imaging of RA by using tissue‐specific contrast agents plays a key role in the systemic treatment of RA by monitoring structural damage and disease progression., Near‐infrared fluorescence imaging is a fast, inexpensive, nonionizing, yet accurate and quantitative method for the early diagnosis and prognosis of rheumatoid arthritis. Combined with targeted contrast agents, separate but simultaneous tissue‐specific imaging of synovitis, cartilage destruction, and bone resorption can be obtained. This strategy allows quantitative biomedical imaging of articular tissue in response to the progressive course of rheumatoid arthritis.

Background: Tumor-associated macrophages (TAMs) are one of the most abundant immune cell types in solid tumors and implicated in tumor progression. Toll-like receptor 4 (TLR4) is expressed in TAMs and plays a key role in immune surveillance and tumor progression. Therefore, molecular imaging of TLR4 has potential not only for detection of TAM-enriched progressing tumors, but also evaluation of TLR4 expression in tumor microenvironment. Methods: Here, we report that near-infrared (NIR) fluorescence imaging can provide a real-time imaging of a syngeneic model of murine hepatocellular carcinoma using targeted strategy against TLR4. We conjugated a zwitterionic NIR fluorophore ZW800-1C with minimal nonspecific tissue interactions to anti-TLR4 antibody and observed its targetability. The bioconjugates showed high affinity to murine macrophages in cell culture and in vivo . Results: Interestingly, we observed predominant NIR signals in the tumor site, which persisted for more than 48 h after single intravenous administration of the bioconjugate. Conclusions: This result suggests that TLR4 targeting combined with NIR fluorescence imaging is a useful tool for cancer imaging. This imaging strategy could be used to detect cancerous tissue with the increased TAM content and evaluate the status of TLR4 signaling in solid tumors, ultimately impacting on the diagnostic and prognostic imaging of human cancers.

Accurate identification of sentinel lymph nodes (SNL) and complete tumor removal in cancer patients is of critical importance and leads to improved patient recovery and survival. Gamma probes along with near infrared (NIR) probes are the gold standard in SNL identification but have failed to reduce false negative rates (FNR) to the desirable 5% value. By developing a high-resolution Positron Emission Tomography (PET) detector atop the patient in coincidence with a partially populated detector ring placed underneath the patient bed with

Background Breast cancer–related lymphedema affects more than 400,000 survivors in the United States. In 2009, lymphatic microsurgical preventive healing approach (LYMPHA) was first described as a surgical technique to prevent lymphedema by bypassing divided arm lymphatics into adjacent veins at the time of an axillary lymph node dissection. We describe the first animal model of LYMPHA. Methods In Yorkshire pigs, each distal hind limb lymphatic system was cannulated and injected with a different fluorophore (human serum albumin–conjugated indocyanine green or Evans Blue). Fluorescence-assisted resection and exploration imaging system was used to map the respective lymphangiosomes to the groin. Baseline lymphatic clearance of each hind limb lymphangiosome was obtained by measuring the fluorescence of each dye from centrally obtained blood samples. A lymphadenectomy versus lymphadenectomy with LYMPHA was then performed. The injections were then repeated to obtain clearance rates that were compared against baseline values. Results Human serum albumin–conjugated indocyanine green and Evans Blue allowed for precise lymphatic mapping of each respective hind limb using fluorescence-assisted resection and exploration imaging. Lymphatic clearance from the distal hind limb dropped 68% when comparing baseline clearance versus after a groin lymphadenectomy. In comparison, lymphatic clearance dropped only 21% when comparing baseline clearance versus a lymphadenectomy with LYMPHA. Conclusions We describe the first animal model for LYMPHA, which will enable future studies to further evaluate the efficacy and potential limitations of this technique. Of equal importance, we demonstrate the power of optical imaging to provide real-time lymphatic clearance rates for each hind limb.

Apoptosis is a key factor in unstable plaques. The aim of this study is to evaluate the utility of visualizing atherosclerotic plaques with radiolabeled duramycin and Annexin V. ApoE−/− mice were fed with a high-fat diet to develop atherosclerosis, C57 mice as a control. Using a routine conjugation protocol, highly pure [99mTc]duramycin and [99mTc]Annexin V were obtained, which were applied for in vitro cell assays of apoptosis and in vivo imaging of atherosclerotic plaques in the animal model. Oil Red O staining, TUNEL, hematoxylin-eosin (HE), and CD68 immunostaining were used to evaluate the deposition of lipids and presence of apoptotic macrophages in the lesions where focal intensity positively correlated with the uptake of both tracers. [99mTc]duramycin and [99mTc]Annexin V with a high radiochemical purity (97.13 ± 1.52 and 94.94 ± 0.65 %, respectively) and a well stability at room temperature were used. Apoptotic cells binding activity to [99mTc]duramycin (Kd, 6.92 nM and Bmax, 56.04 mol/1019 cells) was significantly greater than [99mTc]Annexin V (Kd, 12.63 nM and Bmax, 31.55 mol/1019 cells). Compared with [99mTc]Annexin V, [99mTc]duramycin bound avidly to atherosclerotic lesions with a higher plaque-to-background ratio (P/B was 8.23 ± 0.91 and 5.45 ± 0.48 at 20 weeks, 15.02 ± 0.23 and 12.14 ± 0.22 at 30 weeks). No plaques were found in C57 control mice. Furthermore, Oil Red O staining showed lipid deposition areas were significantly increased in ApoE−/− mice at 20 and 30 weeks, and TUNEL and CD68 staining confirmed that the focal uptake of both tracers contained abundant apoptotic macrophages. This stable, fast clearing, and highly specific [99mTc]duramycin, therefore, can be useful for the quantification of vulnerable atherosclerotic plaques.

Miniaturized microfluidic biosensors have recently been advanced for portable point-of-care diagnostics by integrating lab-on-a-chip technology and electrochemical analysis. However, the design of a small, integrated, and reliable biosensor for multiple and simultaneous electrochemical analyses in a single device remains a challenge. Here, we present a simultaneous microfluidic electrochemical biosensing system to detect multiple biomarkers of pulmonary hypertension diseases in a single device. The miniaturized biosensor, which is composed of five chambers, is precisely and individually controlled using in-house-built pneumatic microvalves to manipulate the flow pathway. Each chamber is connected to an electrochemical sensor designed to detect four different biomarkers plus a reference control. Our design allows for loading of multiple reagents for simultaneous analyses. On the basis of the developed microfluidic electrochemical sensor system, we successfully detected four well-defined pulmonary hypertension-associated biomarkers, namely, fibrinogen, adiponectin, low-density lipoprotein, and 8-isoprostane. This novel approach offers a new platform for a rapid, miniaturized, and sensitive diagnostic sensor in a single device for various human diseases.

Cell-based therapies hold great potential to treat a wide range of human diseases, yet the mechanisms responsible for cell migration and homing are not fully understood. Emerging molecular imaging technology enables in vivo tracking of transplanted cells and their therapeutic efficacy, which together will improve the clinical outcome of cell-based therapy. Particularly, optical imaging provides highly sensitive, safe (non-radioactive), cost-effective, and fast solutions for real-time cellular trafficking compared to other conventional molecular imaging modalities. This review provides a comprehensive overview of current advances in optical imaging for cell-based therapy and tissue engineering. We discuss different types of fluorescent probes and their labeling methods with a special focus on cardiovascular disease, cancer immunotherapy, and tissue regeneration. In addition, advantages and limitations of optical imaging-based cell tracking strategies along with the future perspectives to translate this imaging technique for a clinical realm are discussed.

Despite advances in diagnostic and therapeutic technology of human diseases, cancer remains among the leading causes of morbidity and mortality worldwide. The development of molecular imaging has made it possible to diagnose and treat cancer at early stages, which increases the likelihood of survival. Nuclear medicine has played a key role in diagnosis and staging of human malignancy. However, most imaging technology can only be used in the preoperative diagnosis stage, and these methods are time consuming and often expose patients to a high amount of radiation. Combined with appropriate contrast agents, fluorescence molecular imaging is an easy-to-use imaging tool that can be implemented for intraoperative cancer surgery to delineate tumor margins. In particular, near-infrared fluorescence is useful for image-guided cancer surgery because of the relatively high tissue penetration, low tissue absorption and scattering, and reduced autofluorescence. In this review, the basic principles of fluoresce...

Antibody-fluorophore conjugates have high potential for the specific fluorescence detection of target cancer cells in vitro and in vivo. However, the antibody-fluorophore conjugates described to date are inappropriate for real-time imaging of target cells because removal of unbound antibody is required to reduce background fluorescence before quantifiable analysis by microscopy. In addition, clinical applications of the conjugates have been limited by persistent background retention due to their long systemic circulation and nonspecific uptake. Here we report fast and real-time near-infrared fluorescence imaging of target cancer cells using an antigen-responsive molecular "on-off" sensor: the fluorescence of trastuzumab-ATTO680 conjugate is dark (i.e., turned off) in the extracellular region, while it becomes highly fluorescent (i.e., turned on) upon binding to the target antigen HER2 on cancer cell surface. This molecular switch enables fast and real-time imaging of target cancer cells in vitro and in vivo.

Prostate-specific membrane antigen (PSMA) can serve as a molecular cell surface target for the detection and treatment of prostate cancer. Near-infrared (NIR) fluorescence imaging enables highly sensitive, rapid, and non-radioactive imaging of PSMA, though specific targeting still remains a challenge because no optimized contrast agents exist.

An optical-imaging instrument that integrates multispectral imaging for the detection of fluorescence in the first and second near-infrared windows aids the surgical resection of liver tumours in patients.

Background: Iatrogenic or traumatic ureteral injuries are life-threatening but difficult to diagnose early. Ureteral visualization is essential for both the prevention and diagnosis of iatrogenic or traumatic ureter injuries. In the present study, we evaluated the feasibility of near-infrared (NIR) with ZW800-1C as a diagnostic tool of iatrogenic or traumatic ureteral injury in addition to ureter visualization, compared to methylene blue. Methods: With mice model, we compared the image quality of ZW800-1C with methylene blue for ureter visualization. We also made ureter perforation, obstruction, crushing injury, and transection model with mice and evaluated the feasibility of ZW800-1C for diagnostic tool for ureteral injuries. Results: We could confirm the ureter in the ZW800-1C images in maximally 30 minutes after injection, and the ureter was visible until NIR imaging concluded at 180 minutes after injection. However, methylene blue failed to provide clear ureter imaging during the same period. ZW800-1C imaging successfully visualized ureters subjected to obstruction, transection, perforation, and crush injuries, although urinary leakage was not visible by eye. Conclusions: Our results indicate ZW800-1C is better suited for ureter visualization than methylene blue and that ZW800-1C has considerable potential for the early diagnosis of various ureteral injuries.

Site-specific imaging agents play a key role in tumor targeting, but only a few agents are currently available for inflammation targeting. Since the P2X7 receptor (P2X7R) is a promising molecular target for inflammation, we evaluated the potential value of the (18)F-labeled tracer (18)F-PTTP (5-{[2-Chloro-3-(trifluoromethyl)phenyl]carbonyl}-1-pyrimidin-2-yl-4,5,6,7-tetrahydro-1H-[1,2,3]triazolo[4,5-c]pyridin) for targeting P2X7Rs and thus differentiating inflammation from tumors. Methods: The radioligand (18)F-PTTP was achieved by a 1-step (18)F-trifluoromethylation reaction. The binding affinity of the ligand for P2X7R and its stability were evaluated in vitro. Blood pharmacokinetics tests and biodistribution studies were performed in vivo. Dynamic (18)F-PTTP small-animal PET/CT imaging was performed for 60 min on A549 tumor–bearing mice and inflammation-model mice for targeting differentiation. Results:(18)F-PTTP was afforded with decay-corrected radiochemical yields of 2.5%–7.0%, specific activity of 296–370 MBq/μmol, and radiochemical purity over 95%. (18)F-PTTP showed excellent stability in 0.9% NaCl and 0.1% bovine serum albumin, good affinity to RAW264.7 cells, and rapid blood clearance in mice. In inflammation-model mice, uptake of (18)F-PTTP peaked at 5 min after injection and kept at an imageable level till 30 min, whereas no significant radioactivity uptake was found in tumor grafts till 1 h after injection. The specificity of (18)F-PTTP was verified by blocking studies and histologic analysis. Conclusion: The current study provides compelling data that (18)F-PTTP is a novel radioligand targeting P2X7R and has potential to screen new drugs, quantify peripheral inflammation, and distinguish inflammation from certain solid tumors.

Nanomaterials have revolutionized cancer imaging, diagnosis, and treatment. Multifunctional nanoparticles in particular have been designed for targeted cancer therapy by modulating their physicochemical properties to be delivered to the target and activated by internal and/or external stimuli. This review will focus on the fundamental “chemical” design considerations of stimuli-responsive nanosystems to achieve favorable tumor targeting beyond biological barriers and, furthermore, enhance targeted cancer therapy. In addition, we will summarize innovative smart nanosystems responsive to external stimuli (e.g., light, magnetic field, ultrasound, and electric field) and internal stimuli in the tumor microenvironment (e.g., pH, enzyme, redox potential, and oxidative stress).

Purpose: Imaging techniques for highly specific detection of cancer cells in vivo can have applications ranging from preclinical drug discovery studies to clinical cancer diagnosis and surgical therapy. Although fluorescence imaging using cancer-targeted antibodies has shown promise, nonspecific probe accumulation in tissue results in significant background fluorescence, reducing detection sensitivity using traditional intensity–based continuous-wave (CW) fluorescence imaging. Here we demonstrate that fluorescence lifetime (FLT) imaging can provide significant tumor contrast enhancement over CW intensity in preclinical models of human breast cancer. Experimental Design: Mice bearing MDA-MB-231 tumors were injected with anti-EGFR antibody conjugated to the fluorescent dye IRDye 800CW (anti-EGFR-800). Time domain fluorescence imaging was performed in vivo and in situ up to 48 hours after dye injection. Results: Mice injected with anti-EGFR-800 showed a significantly longer FLT (0.7 ± 0.03 ns) compared with the FLT of nonspecific probe uptake in liver (0.63 ± 0.05 ns), providing a dramatic improvement in sensitivity and specificity compared with CW intensity. IgG antibody–conjugated IRDye 800CW did not show an increased FLT compared with normal tissue, suggesting that the FLT increase of anti-EGFR-800 in tumors was associated with receptor expression. Using serial surgery, we show that FLT allows the detection of smaller residual tumors in the surgical bed than possible using CW intensity. Conclusions: Our data suggest that FLT can significantly enhance tumor contrast using fluorescently labeled antibodies, thereby accelerating the efficient clinical application of these probes for margin assessment in image-guided surgery and for highly specific detection of tumor receptors in vivo.

Efficient and timely delivery of vaccine antigens to the secondary lymphoid tissue is crucial to induce protective immune responses by vaccination. However, determining the longitudinal biodistribution of injected vaccines in the body has been a challenge. Here, the near-infrared (NIR) fluorescence imaging is reported that can efficiently enable the trafficking and biodistribution of vaccines in real time. Zwitterionic NIR fluorophores are conjugated on the surface of model vaccines and tracked the fate of bioconjugated vaccines after intradermal administration. Using an NIR fluorescence imaging system, it is possible to obtain time-course imaging of vaccine trafficking through the lymphatics, observing notable uptake in lymph nodes with minimal nonspecific tissue interactions. Flow cytometry analysis confirmed that the uptake in lymph nodes by antigen presenting cells was highly dependent on the hydrodynamic diameter of vaccines. These results demonstrate that the combination of a real-time NIR fluorescence imaging system and zwitterionic fluorophores is a powerful tool to determine the fate of vaccine antigens. Since such non-specific vaccine uptake causes serious adverse reactions, this method is not only useful for optimization of vaccine design, but also for safety evaluation of clinical vaccine candidates.

International audience; Advances in molecular imaging modalities have accelerated the diagnosis and treatment of human diseases. However, tumors less than 1 cm in size still remain difficult to localize by conventional means because of the difficulty in specific targeting/delivery to the tumor site. Furthermore, high nonspecific uptake in the major organs and persistent background retention results in low tumor-to-background ratio. The targeting and therapy of gastrointestinal stromal tumors (GIST) using nonsticky and renal clearable theranostic nanoparticles (a.k.a. H-Dots) are demonstrated. H-Dots not only target GIST for image-guided surgery, but also tailor the fate of anticancer drugs such as imatinib (IM) to the tumor site resulting in efficient treatment of unresectable GIST. In addition, H-Dots can monitor targetability, pharmacokinetics, and drug delivery, while also showing therapeutic efficacy in GIST-bearing xenograft mice following surgical resection. More importantly, IM loaded H-Dots exhibit lower uptake into the immune system, improved tumor selectivity, and increased tumor suppression compared to free IM, which accumulates in the spleen/liver. Precisely designed H-Dots can be used as a promising theranostic nanoplatform that can potentially reduce the side effects of conventional chemotherapies.

The instability of recombinant basic fibroblast growth factor (bFGF) is a major disadvantage for its therapeutic use and means frequent applications to cells or tissues are required for sustained effects. Originating from silkworm hemolymph, 30Kc19α is a cell-penetrating protein that also has protein stabilization properties. Herein, it was investigated whether fusing 30Kc19α to bFGF could enhance the stability and skin penetration properties of bFGF, which may consequently increase its therapeutic efficacy. The fusion of 30Kc19α to bFGF protein increased protein stability, as confirmed by ELISA. 30Kc19α-bFGF also retained the biological activity of bFGF as it facilitated the migration and proliferation of fibroblasts and angiogenesis of endothelial cells. It was discovered that 30Kc19α could improve the transdermal delivery of a small molecular fluorophore through the skin of hairless mice. Importantly, it increased the accumulation of bFGF and further facilitated its translocation into the skin through follicular routes. Finally, when applied to a skin wound model in vivo, 30Kc19α-bFGF penetrated the dermis layer effectively, which promoted cell proliferation, tissue granulation, angiogenesis, and tissue remodeling. Consequently, our findings suggest that 30Kc19α improves the therapeutic functionalities of bFGF, and would be useful as a protein stabilizer and/or a delivery vehicle in therapeutic applications.

One author name and corresponding affiliation of the original version of this article were unfortunately mislabeled.

Near-infrared (NIR) fluorescence light has been widely utilized in clinical imaging by providing surgeons highly specific images of target tissue. The "NIR window" from 650 to 900 nm is especially useful due to several special features such as minimal autofluorescence and absorption of biomolecules in tissue, as well as low light scattering. Compared with visible wavelengths, NIR fluorescence light is invisible, thus allowing highly sensitivity real-time image guidance in human surgery without changing the surgical field. The benefit of using NIR fluorescence light as a clinical imaging technology can be attributed to its molecular fluorescence as an exogenous contrast agent. Indeed, whole body preoperative imaging of single-photon emission computed tomography (SPECT) and positron emission tomography (PET) remains important in diagnostic utility, but they lack the efficacy of innocuous and targeted NIR fluorophores to simultaneously facilitate the real-time delineation of diseased tissue while preserving vital tissues. Admittedly, NIR imaging technology has been slow to enter clinical use mostly due to the late-coming development of truly breakthrough contrast agents for use with current imaging systems. Therefore, clearly defining the physical margins of tumorous tissue remains of paramount importance in bioimaging and targeted therapy. An equally noteworthy yet less researched goal is the ability to outline healthy vital tissues that should be carefully navigated without transection during the intraoperative surgery. Both of these paths require optimizing a gauntlet of design considerations to obtain not only an effective imaging agent in the NIR window but also high molecular brightness, water solubility, biocompatibility, and tissue-specific targetability. The imaging community recognizes three strategic approaches which include (1) passive targeting via the EPR effect, (2) active targeting using the innate overall biodistribution of known molecules, and (3) activatable targeting through an internal stimulus, which turns on fluorescence from an off state. Recent advances in nanomedicine and bioimaging offer much needed promise toward fulfilling these stringent requirements as we develop a successful catalog of targeted contrast agents for illuminating both tumors and vital tissues in the same surgical space by employing spectrally distinct fluorophores in real time. These tissue-specific contrast agents can be versatile arsenals to physicians for real-time intraoperative navigation as well as image-guided targeted therapy. There is a versatile library of tissue-specific fluorophores available in the literature, with many discussed herein, which offers clinicians an array of possibilities that will undoubtedly improve intraoperative success and long-term postoperation prognosis.

Many efforts have been made to understand the neural mechanisms of the human brain. However, visualization of human brain processing has been a main challenge in the field. It is still largely unknown how the human brain allocates attention to target objects while excluding unrelated information in a complex visual environment. Using simultaneous electroencephalogram and eye tracking measurements, in this study, we analyzed two brain regions separately to detect the brain wave activity during visual information processing. We observed an activation difference between sensory (P100) and cognitive (P300) processing, and the behavioral response was improved by providing valid cue-target location information. Furthermore, neural processing was evaluated according to the specific area of brain activation and eye movements during cognitive processing. Our results demonstrate the correlation between behavior performance and visual stimuli and suggest an advantage of combined paradigms for efficient visua...

Despite all the advances in multimodal imaging, it remains a significant challenge to acquire both magnetic resonance and nuclear imaging in a single dose because of the enormous difference in sensitivity. Indeed, nuclear imaging is almost 10(6)-fold more sensitive than magnetic resonance imaging (MRI); thus, repeated injections are generally required to obtain sufficient MR signals after nuclear imaging. Here, we show that strategically engineered magnetoferritin nanoprobes can image tumors with high sensitivity and specificity using SPECT and MRI in living mice after a single intravenous injection. The magnetoferritin nanoprobes composed of (125)I radionuclide-conjugated human H-ferritin iron nanocages ((125)I-M-HFn) internalize robustly into cancer cells via a novel tumor-specific HFn-TfR1 pathway. In particular, the endocytic recycling characteristic of TfR1 transporters solves the nuclear signal blocking issue caused by the high dose nanoprobes injected for MRI, thus enabling simultaneous functional and morphological tumor imaging without reliance on multi-injections.

The adrenal glands (AGs) are relatively small yet require definitive identification during their resection, or more commonly their avoidance. To enable image-guided surgery involving the AGs, we have developed novel near-infrared (NIR) fluorophores that target the AGs after a single intravenous injection, which provided dual-NIR image-guided resection or avoidance of the AGs during both open and minimally-invasive surgery.

Passive targeting of large nanoparticles by the enhanced permeability and retention (EPR) effect is a crucial concept for solid tumor targeting in cancer nanomedicine. There is, however, a trade-off between the long-term blood circulation of nanoparticles and their nonspecific background tissue uptake. To define this size-dependent EPR effect, we designed near-infrared fluorophore-conjugated polyethylene glycols (PEG-ZW800s; 1–60 kDa) and evaluated their biodistribution, pharmacokinetics, and renal clearance in tumor-bearing mice. The targeting efficiency of size-variant PEG-ZW800s was investigated in terms of tumor-to-background ratio (TBR). Interestingly, smaller sized PEGs (≤20 kDa, 12 nm) exhibited significant tumor targeting with minimum to no nonspecific uptakes, while larger sized PEGs (>20 kDa, 13 nm) accumulated highly in major organs, including the lungs, liver, and pancreas. Among those tested, 20 kDa PEG-ZW800 exhibited the highest TBR, while excreting unbound molecules to the urinary bladder. This result lays a foundation for engineering tumor-targeted nanoparticles and therapeutics based on the size-dependent EPR effect.

Inflammation and calcification concomitantly drive atherosclerotic plaque progression and rupture and are the compelling targets for identifying plaque vulnerability. However, current imaging modalities for vulnerable atherosclerotic plaques are often limited by inadequate specificity and sensitivity. Here, we show that natural H-ferritin nanocages radiolabeled with technetium-99m (99mTc-HFn) can identify and accurately localize macrophage-rich, atherosclerotic plaques in living mice using combined SPECT and CT. Focal 99mTc-HFn uptake was observed in the atherosclerotic plaques with multiple high-risk features of macrophage infiltration, active calcification, positive remodeling, and necrosis on histology and in early active ongoing lesions with intense macrophage infiltration. The uptake of 99mTc-HFn in plaques enabled quantitative measuring of the dynamic changes of inflammation during plaque progression and anti-inflammation treatment. This strategy lays the foundation of using bioengineered endogenous human ferritin nanocages for the identification of vulnerable and early active plaques as well as potential assessment of anti-inflammation therapy.

Lymphadenectomy is a prerequisite for most malignancies to define the precise staging of cancer, as well as resect the possible metastases completely. While it improves prognosis, lymphadenectomy often causes postoperative edema or bleeding because of unclear surgical margins. In this study, we synthesized near-infrared (NIR) fluorescent nanoprobes with conjugating various mannose moieties on the surface to target macrophages in the lymph node. Armed with these NIR nanoprobes, we demonstrated the feasibility of intraoperative pan lymph nodes (PLN) mapping and real-time optical imaging under the NIR fluorescence imaging system. We found that even single mannose-conjugated ZW800-1 showed specific uptake in lymph nodes within 4 h, and multiple mannose-employed polyrotaxanes highlighted PLN efficiently with low background signals in major organs. This technology can help surgeons perform lymphadenectomy with ease and safety by identifying all regional lymph nodes proficiently after a single intravenous injection of NIR nanoprobes.

In this work, we chose cartilage acellular matrix (CAM) as a promising antiadhesive material because CAM effectively inhibits the formation of blood vessels, and we used electrospinning to prepare antiadhesive barriers. Additionally, we synthesized N-hydroxysuccinimide (NHS)-poly(caprolactone-co-lactide-co-glycolide)-NHS (MP) copolymers (to tune degradation) as a cross-linking agent for CAM. This is the first report on the development of electrospun cross-linked (Cx) CAM/MP (CA/P) nanofiber (NF) (Cx-CA/P-NF) with a tunable degradation period as an antiadhesive barrier. Compared with the CA/P-NF before cross-linking, the electrospun Cx-CA/P-NF after cross-linking showed different biodegradation. Cx-CA/P-NF significantly inhibited the in vitro attachment and proliferation of human umbilical vein endothelial cells (HUVECs), as confirmed by an MTT assay and scanning electron microscopy images. Cx-CA/P-NFs implanted between a surgically damaged peritoneal wall and cecum gradually degraded in 7 days; this process was monitored by NIR imaging. The in vivo evaluation of the anti–tissue adhesive effect of Cx-CA/P-NFs revealed little adhesion, few blood vessels, and negligible inflammation at 7 days determined by hematoxylin and eosin staining. ED1 staining of Cx-CA/P-NFs showed infiltration of few macrophages because of the inflammatory response to the Cx-CA/P-NF as compared with an untreated injury model. Additionally, Cx-CA/P-NFs significantly suppressed the formation of blood vessels between the peritoneal wall and cecum, according to CD31 staining. Overall, Cx-CA/P-NFs yielded little adhesion, infiltration by macrophages, or formation of blood vessels in a postoperative antiadhesion assay. Thus, it is reasonable to conclude that the Cx-CA/P-NF designed herein successfully works as an antiadhesive barrier with a tunable degradation period. Statement of Significance The cartilage acellular matrix (CAM) can inhibit the formation of fibrous tissue bridges and blood vessels between the tissue at an injured site and the surrounding healthy tissues. However, CAM has not been rigorously investigated as an antiadhesive barrier. In this manuscript, the cross-linked CAM nanofiber (Cx-CA/P-NF) designed herein successfully works as an antiadhesive barrier. Cx-CA/P-NFs yielded little adhesion, infiltration by macrophages, or formation of blood vessels in a postoperative antiadhesion assay. Moreover, we demonstrated the suitable properties of Cx-CA/P-NF such as easy cross-linking by maintaining the antiadhesive properties, controllable biodegradation, and in vivo antiadhesive effect of Cx-CA/P-NF.

Background The injection of botulinum toxin (BTX) to reduce facial wrinkles is one of the most frequently performed plastic surgery procedures. The biocompatible hydrogels are injected with BTX for effective tissue augmentation. However, it is difficult to determine the interval of injection for effective tissue augmentation. Method BTX and hyaluronate (HA) hydrogels were labeled with zwitterionic (ZW) near-infrared (NIR) fluorophores and visualized for 3 weeks after injection to BALB/c nude mice. Results BTX-ZW conjugates and diaminohexane (DAH)-HA-ZW hydrogels were successfully prepared by the conventional EDC/NHS chemistry. Using the NIR fluorescence imaging, we confirmed that approximately 10% of BTX-ZW conjugates and 50% of DAH-HA-ZW hydrogels remained 3 weeks post-injection. Conclusion This bioimaging technique using invisible NIR fluorescence light can be exploited for various biomedical applications.

Objective: Pancreas-related complications are some of the most serious ones in abdominal surgery. The goal of this study was to develop and validate novel near-infrared (NIR) fluorophores that would enable real-time pancreas imaging to avoid the intraoperative pancreatic injury. Design: After initial screening of a large NIR fluorophore library, the performance of 3 selected pancreas-targeted 700 nm NIR fluorophores, T700-H, T700-F, and MB, were quantified in mice, rats, and pigs. Dose ranging using 25 and 100 nmol, and 2.5 µmol of T700-F, and its imaging kinetics over a 4 h period were tested in each species. Three different 800 nm NIR fluorophores were employed for dual-channel FLARE™ imaging in pigs: 2 μmol of ZW800-1 for vessels and kidney, 1 μmol of ZW800-3C for lymph nodes, and 2 μmol of ESNF31 for adrenal glands. Results: T700-F demonstrated the highest signal to background ratio (SBR), with peak SBR at 4 h postinjection in mice. In pigs, T700-F produced an SBR ≥ 2 against muscle, spleen, and lymph nodes for up to 8 h after a single intravenous injection. The combination of T700-F with each 800 nm NIR fluorophore provided simultaneous dual-channel intraoperative imaging of pancreas with surrounding organs in real time. Conclusion: Pancreas-targeted NIR fluorophores combined with the FLARE dual-channel imaging system enable the real-time intraoperative pancreas imaging which helps surgeons perform safer and more curative abdominal surgeries.

Cardiorenal syndrome type 1 causes acute kidney injury but is poorly understood; animal models and diagnostic aids are lacking. Robust noninvasive measurements of glomerular filtration rate are required for injury models and clinical use. Several have been described but are untested in translational models and suffer from biologic interference. We developed a mouse model of cardiorenal syndrome and tested the novel near-infrared fluorophore ZW800-1 to assess renal and cardiac function. We performed murine cardiac arrest and cardiopulmonary resuscitation followed by transthoracic echocardiography, 2 and 24 h later. Transcutaneous fluorescence of ZW800-1 bolus dispersion and clearance was assessed with whole animal imaging and compared with glomerular filtration rate (GFR; inulin clearance), tubular cell death (using unbiased stereology), and serum creatinine. Correlation, Bland-Altman, and polar analyses were used to compare GFR with ZW800-1 clearance. Cardiac arrest and cardiopulmonary resuscitation caused reversible cardiac failure, halving fractional shortening of the left ventricle ( n = 12, P = 0.03). Acute kidney injury resulted with near-zero GFR and sixfold increase in serum creatinine 24 h later ( n = 16, P < 0.01). ZW800-1 biodistribution and clearance were exclusively renal. ZW800-1 t1/2and clearance correlated with GFR ( r = 0.92, n = 31, P < 0.0001). ZW800-1 fluorescence was reduced in cardiac arrest, and cardiopulmonary resuscitation-treated mice compared with sham animals 810 s after injection ( P < 0.01) and bolus time-dispersion curves demonstrated that ZW800-1 fluorescence dispersion correlated with left ventricular function ( r = 0.74, P < 0.01). Cardiac arrest and cardiopulmonary resuscitation lead to experimental cardiorenal syndrome type 1. ZW800-1, a small near-infrared fluorophore being developed for clinical intraoperative imaging, is favorable for evaluating cardiac and renal function noninvasively.

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.