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Aim: To characterize the pharmacokinetics of deferoxamine-conjugated nanoparticles (DFO-NPs), a novel nanochelator for removing excess iron. Materials & methods: The pharmacokinetics of DFO-NPs were evaluated in Sprague–Dawley rats at three doses (3.3, 10 and 30 μmol/kg) after intravenous and subcutaneous administration. Results: DFO-NPs exhibited a biphasic concentration-time profile after intravenous administration with a short terminal half-life (2.0–3.2 h), dose-dependent clearance (0.111–0.179 l/h/kg), minimal tissue distribution and exclusive renal excretion with a possible saturable reabsorption mechanism. DFO-NPs after subcutaneous administration exhibited absorption-rate-limited kinetics with a prolonged half-life (5.7–10.1 h) and favorable bioavailability (47–107%). Conclusion: DFO-NPs exhibit nonlinear pharmacokinetics with increasing dose, and subcutaneous administration substantially improves drug exposure, thereby making it a clinically viable administration route for iron chelation.

Non-invasive methods for the in vivo detection of hallmarks of Alzheimer’s disease can facilitate the study of the progression of the disease in mouse models and may enable its earlier diagnosis in humans. Here we show that the zwitterionic heptamethine fluorophore ZW800–1C, which has peak excitation and emission wavelengths in the near-infrared optical window, binds in vivo and at high contrast to amyloid-β deposits and to neurofibrillary tangles, and allows for the microscopic imaging of amyloid-β and tau aggregates through the intact skull of mice. In transgenic mouse models of Alzheimer’s disease, we compare the performance of ZW800–1C with that of the two spectrally similar heptamethine fluorophores ZW800–1A and indocyanine green, and show that ZW800–1C undergoes a longer fluorescence-lifetime shift when bound to amyloid-β and tau aggregates than when circulating in blood vessels. ZW800–1C may prove advantageous for tracking the proteinic aggregates in rodent models of amyloid-β and tau pathologies.

Background Due to the deep tissue penetration and reduced scattering, NIR-II fluorescence imaging is advantageous over conventional visible and NIR-I fluorescence imaging for the detection of bone growth, metabolism, metastasis, and other bone-related diseases. Methods Bone-targeted heptamethine cyanine fluorophores were synthesized by substituting the meso-carbon with a sulfur atom, resulting in a bathochromic shift and increased fluorescence intensity. The physicochemical, optical, and thermal stability of newly synthesized bone-targeted NIR fluorophores was performed in aqueous solvents. Calcium binding, bone-specific targeting, biodistribution, pharmacokinetics, and 2D and 3D NIR imaging were performed in animal models. Results The newly synthesized S-substituted heptamethine fluorophores demonstrated a high affinity for hydroxyapatite and calcium phosphate, which improved bone-specific targeting with signal-background ratios > 3.5. Particularly, P800SO3-PEG showed minimum nonspecific uptake, and most unbound molecules were excreted into the urinary bladder. Histological analyses demonstrated that P800SO3-PEG remained stable in the bone for over two weeks and was incorporated into bone matrices. Interestingly, the flexible thiol ethylene glycol linker on P800SO3-PEG induced a promising photothermal effect upon NIR laser irradiation, demonstrating potential theranostic imaging. Conclusions P800SO3-PEG shows a high affinity for bone tissues, deeper tissue imaging capabilities, minimum nonspecific uptake in the major organs, and photothermal effect upon laser irradiation, making it optimal for bone-targeted theranostic imaging.

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.

Triple-negative breast cancer (TNBC) patients are considered intractable, as this disease has few effective treatments and a very poor prognosis even in its early stages. Here, intratumoral therapy with resveratrol (Res), which has anticancer and metastasis inhibitory effects, was proposed for the effective treatment of TNBC. An injectable Res-loaded click-crosslinked hyaluronic acid (Res-Cx-HA) hydrogel was designed and intratumorally injected to generate a Res-Cx-HA depot inside the tumor. The Res-Cx-HA formulation exhibited good injectability into the tumor tissue, quick depot formation inside the tumor, and the depot remained inside the injected tumor for extended periods.

Some heptamethine cyanine dyes accumulate in solid tumors in vivo and persist there for several days. The reasons why they accumulate and persist in tumors were incompletely defined, but explanations based on uptake into cancer cells via organic anion transporting polypeptides (OATPs) have been widely discussed. All cyanine-based “tumor-seeking dyes” have a chloride centrally placed on the heptamethine bridge (a “meso-chloride”). We were intrigued and perplexed by the correlation between this particular functional group and tumor uptake, so the following study was designed. It features four dyes (1-Cl, 1-Ph, 5-Cl, and 5-Ph) with complementary properties. Dye 1-Cl is otherwise known as MHI-148, and 1-Ph is a close analog wherein the meso-chloride has been replaced by a phenyl group. Data presented here shows that both 1-Cl and 1-Ph form noncovalent adducts with albumin, but only 1-Cl can form a covalent one. Both dyes 5-Cl and 5-Ph have a methylene (CH(2)) unit replaced by a dimethylammonium functionality (N(+)Me(2)). Data presented here shows that both these dyes 5 do not form tight noncovalent adducts with albumin, and only 5-Cl can form a covalent one (though much more slowly than 1-Cl). In tissue culture experiments, uptake of dyes 1 is more impacted by the albumin in the media than by the pan-OATP uptake inhibitor (BSP) that has been used to connect uptake of tumor-seeking dyes in vivo with the OATPs. Uptake of 1-Cl in media containing fluorescein-labeled albumin gave a high degree of colocalization of intracellular fluorescence. No evidence was found for the involvement of OATPs in uptake of the dyes into cells in media containing albumin. In an in vivo tumor model, only the two dyes that can form albumin adducts (1-Cl and 5-Cl) gave intratumor fluorescence that persisted long enough to be clearly discerned over the background (~4 h); this fluorescence was still observed at 48 h. Tumors could be imaged with a higher contrast if 5-Cl is used instead of 1-Cl, because 5-Cl is cleared more rapidly from healthy tissues. Overall, the evidence is consistent with in vitro and in vivo results and indicates that the two dyes in the test series that accumulate in tumors and persist there (1-Cl and 5-Cl, true tumor-seeking dyes) do so as covalent albumin adducts trapped in tumor tissue via uptake by some cancer cells and via the enhanced permeability and retention (EPR) effect.

The second near-infrared window (NIR-II) beyond 1000 nm has attracted attention for optical contrast imaging in small animals. We sought to assess whether commercially available NIR-II dyes could be easily formulated for this purpose. 13 hydrophobic NIR-II dyes were purchased and screened by formulating them in simple solubilizing agents with established use in humans: propylene glycol, Cremaphor EL, Kolliphor HS15 (HS15), Tween 80, and cyclodextrin. Based on the absorption at 1064 nm (matching the Nd:YAG laser output commonly used in photoacoustic imaging), three of the dyes were further assessed at varying dye and surfactant concentrations. Of these, benzo indole butyl diphenylaminocyclopentene heptamethine (BIBDAH) tetrafluoroborate in HS15 generally showed the most favorable NIR-II character. 1 mg mL−1 BIBDAH in 25% HS15 exhibited a single absorption peak at 1030 nm with a calculated intensity greater than 100, which was relatively stable for weeks in storage. Following intravenous administration to mice, determination of BIBDAH pharmacokinetics was possible by absorption measurements of sampled plasma, revealing a circulating half-life of about one hour. Most of the dye was taken up by the liver. BIBDAH was used in vitro and in vivo as a photoacoustic contrast imaging agent and its accumulation could be detected in subcutaneous tumors in mice. BIBDAH was used for fluorescence imaging of blood vessels in mice, including in the brain (through intact skull), and dye clearance from blood to the liver was visualized. Taken together, this study confirms that accessible, strongly-absorbing dye can readily be formulated for injection by simply dissolving them in biocompatible surfactants and used for high-contrast preclinical optical imaging in the second NIR window.

Owing to the prevalence of rotator cuff (RC) injuries and suboptimal healing outcome, rapid and functional regeneration of the tendon-bone interface (TBI) after RC repair continues to be a major clinical challenge. Given the essential role of the RC in shoulder movement, the engineering of biomimetic multi-tissue constructs presents an opportunity for complex TBI reconstruction after RC repair. Here, we propose a gradient cell-laden multi-tissue construct combined with compositional gradient TBI-specific bioinks via 3D cell-printing technology.

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.

The ability of brain tissue to regenerate is limited; therefore, brain diseases (i.e., trauma, stroke, tumors) often lead to irreversible motor and cognitive impairments. Therapeutic interventions using various types of injectable biomaterials have been investigated to promote endogenous neural differentiation. Despite promising results in pre-clinical studies, the translation of regenerative medicine to the clinic has many challenges due to the lack of reliable imaging systems to achieve accurate evaluation of the treatment efficacy. Methods: In this study, we developed a dual-channel fluorescence imaging technique to simultaneously monitor tissue ingrowth and scaffold disintegration. Enzymatically crosslinked gelatin-hyaluronic acid hydrogel was labeled with 800 nm fluorophore, ZW800-3a, while the regenerated tissue was highlighted with 700 nm brain-specific contrast agent, Ox1. Results: Using the multichannel fluorescence imaging system, tissue growth and degradation of the NIR hydrogel were simultaneously imaged in the brain of mice. Images were further analyzed and reconstructed to show both visual and quantitative information of each stage of a therapeutic period. Conclusion: Dual-channel in vivo imaging systems can provide highly accurate visual and quantitative information of the brain tissue ingrowth for the evaluation of the therapeutic effect of NIR hydrogel through a simple and fast operating procedure.

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.

In this study, we developed injectable intratympanic hyaluronic acid (HA) depots for the treatment of hearing loss. We prepared an injectable click-crosslinking formulation by modifying HA with tetrazine (HA-TET) and trans-cyclooctene (HA-TCO), which crosslinked to form an HA depot (Cx-HA). Preparation of the click-crosslinking HA formulation was facile, and Cx-HA depot formation was reproducible. Additionally, the Cx-HA hydrogel was significantly stiffer than HA hydrogel. To monitor the degradation pattern of hydrogels, we mixed a zwitterionic near-infrared (NIR) fluorophore (e.g., ZW800-1C) in the click-crosslinking HA formulation. Then, HA-TET and HA-TCO solutions containing ZW800-1C were loaded separately into the compartments of a dual-barrel syringe for intratympanic injection. The Cx-HA depots formed quickly, and an extended residence time in the tympanic cavity was confirmed by performing NIR fluorescence imaging. We have successfully prepared an injectable click-crosslinking HA formulation that has promise as an intratympanic drug depot.

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.

Here, we propose a zwitterionic near-infrared (NIR) fluorophore-tryptophan (Trp) conjugate with a cleavable linker as a minimal-sized versatile platform (MP) for the preparation of peptide ligand-based off-on type molecular probes. The zwitterionic NIR fluorophore in MP undergoes fluorescence quenching via a photoinduced electron transfer mechanism when in close proximity to tryptophan, and nonspecific binding with serum proteins is minimized by the zwitterionicity of the fluorophore. The linker can be cleaved inside cancer cells in response to tumor-associated stimuli. As a proof-of-concept experiment, ATTO655 was covalently linked with Trp via a diarginine linker to form an MP. A cyclic peptide consisting of Arg-Gly-Asp-d-Phe-Lys (cRGD) was used as a cancer-targeting ligand and was conjugated to the MP to form cRGD-MP. The NIR fluorescence of cRGD-MP could be selectively turned on inside the target cancer cells, thereby enabling specific fluorescence imaging of integrin αvβ3-overexpressing cancer cells in vitro and in vivo.

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.

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.

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

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).

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.

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.

A critical limitation of bioorthogonal click chemistry for in vivo applications has been its low reaction efficiency due to the pharmacokinetic barriers, such as blood distribution, circulation, and elimination in living organisms. To identify key factors that dominate the efficiency of click chemistry, here a rational design of near-infrared fluorophores containing tetrazine as a click moiety is proposed. Using trans-cyclooctene-modified cells in live mice, it is found that the in vivo click chemistry can be improved by subtle changes in lipophilicity and surface charges of intravenously administered moieties. By controlling pharmacokinetics, biodistribution, and clearance of click moieties, it is proved that the chemical structure dominates the fate of in vivo click ligation.

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.

Stem cell-based tissue engineering has the potential to use as an alternative for autologous tissue grafts; however, the contribution of the scaffold degradation along with the transplanted stem cells to in vivo tissue regeneration remains poorly understood. Near-infrared (NIR) fluorescence imaging has great potential to monitor implants while avoiding autofluorescence from the adjacent host tissue. To utilize NIR imaging for in vivo monitoring of scaffold degradation and cell tracking, we synthesized 800-nm emitting NIR-conjugated PCL-ran-PLLA-ran-PGA (ZW-PCLG) copolymers with three different degradation rates and labeled 700-nm emitting lipophilic pentamethine (CTNF127) on the human placental stem cells (CT-PSCs). The 3D bioprinted hybrid constructs containing the CT-PSC-laden hydrogel together with the ZW-PCLG scaffolds demonstrate that NIR fluorescent imaging enables tracking of in vivo scaffold degradation and stem cell fate for bone regeneration in a rat calvarial bone defect model. This NIR-based monitoring system can be effectively utilized to study cell-based tissue engineering applications.

Significance: Photobiomodulation is a well-established therapeutic modality. However, the mechanism of action is poorly understood, due to lack of research in the causal relationship between the near-infrared (NIR) light irradiation and its specific biological effects, hindering broader applications of this technology. Aim: Since biological chromophores typically show several absorption peaks, we determined whether specific effects of photobiomodulation are induced with a combination of two wavelengths at a certain range of irradiance only, rather than a single wavelength of NIR light. Approach: In order to analyze a wide array of combinations of multispectral NIR light at various irradiances efficiently, we developed a new optical platform equipped with two distinct wavelengths of NIR lasers by high-throughput multiple dosing for single-cell live imaging. Two wavelengths of 1064 and 1270 nm were selected based on their photobiomodulatory effects reported in the literature. Results: A specific combination of wavelengths at low irradiances (250 to 400 mW/cm2 for 1064 nm and 55 to 65 mW/cm2 for 1270 nm) modulates mitochondrial retrograde signaling, including intracellular calcium and reactive oxygen species in T cells. The time-dependent density functional theory computation of binding of nitric oxide (NO) to cytochrome c oxidase indicates that the illumination with NIR light could result in the NO release, which might be involved in these changes. Conclusions: This optical platform is a powerful tool to study causal relationship between a specific parameter of NIR light and its biological effects. Such a platform is useful for a further mechanistic study on not only photobiomodulation but also other modalities in photomedicine.

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.

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.

The screening of living cells using high-throughput microarrays is technically challenging. Great care must be taken in the chemical presentation of potential ligands and the number of collisions that cells make with them. To overcome these issues, we have developed a glass slide-based microarray system to discover small molecule ligands that preferentially bind to one cell type over another, including when the cells differ by only a single receptor. Chemical spots of 300 ± 10 µm in diameter are conjugated covalently to glass slides using an arraying robot, and novel near-infrared fluorophores with peak emission at 700 nm and 800 nm are used to label two different cell types. By carefully optimizing incubation conditions, including cell density, motion, kinetics, detection, etc. we demonstrate that cell-ligand binding occurs, and that the number of cells bound per chemical spot correlates with ligand affinity and specificity. This screening system lays the foundation for high-throughput discovery of novel ligands to the cell surface.

Recent advances in bioimaging and nanomedicine have permitted the exploitation of molecular optical imaging in image-guided surgery; however, the parameters mediating optimum performance of contrast agents are not yet precisely determined. To develop ideal contrast agents for image-guided surgery, we need to consider the following criteria: (1) excitation and emission wavelengths in the near-infrared (NIR) window, (2) optimized optical characteristics for high in vivo performance, (3) overcoming or harnessing biodistribution and clearance, and (4) reducing nonspecific uptake. The design considerations should be focused on optimizing the optical and physicochemical property criteria. Biodistribution and clearance should first be considered because they mediate the fate of a contrast agent in the body such as how long after intravenous injection a contrast agent reaches the peak signal-to-background ratio (SBR) and how long the signal lasts (retention). ©

【Bioimaging has advanced the field of nanomedicine, drug delivery, and tissue engineering by directly visualizing the dynamic mechanism of diagnostic agents or therapeutic drugs in the body. In particular, wide-field, planar, near-infrared (NIR) fluorescence imaging has the potential to revolutionize human surgery by providing real-time image guidance to surgeons for target tissues to be resected and vital tissues to be preserved. In this review, we introduce the principles of NIR fluorescence imaging and analyze currently available NIR fluorescence imaging systems with special focus on optical source and packaging. We also introduce the evolution of the FLARE intraoperative imaging technology as an example for image-guided surgery.】

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.

When designing drug carriers, the drug-to-carrier ratio is an important consideration, because the use of high quantities of carriers can result in toxicity as a consequence of poor metabolism and elimination of the carriers. However, these issues would be of less concern if both the drug and carrier had therapeutic effects. (-)-Epigallocatechin-3-O-gallate (EGCG), a major ingredient of green tea, has been shown, for example, to possess anticancer effects, anti-HIV effects, neuroprotective effects and DNA-protective effects. Here, we show that sequential self-assembly of the EGCG derivative with anticancer proteins leads to the formation of stable micellar nanocomplexes, which have greater anticancer effects in vitro and in vivo than the free protein. The micellar nanocomplex is obtained by complexation of oligomerized EGCG with the anticancer protein Herceptin to form the core, followed by complexation of poly(ethylene glycol)-EGCG to form the shell. When injected into mice, the Herceptin-loaded micellar nanocomplex demonstrates better tumour selectivity and growth reduction, as well as longer blood half-life, than free Herceptin.

Nerve preservation is an important issue during most surgery because accidental transection or injury results in significant morbidity, including numbness, pain, weakness, or paralysis. Currently, nerves are still identified only by gross appearance and anatomical location during surgery, without intraoperative image guidance. Near-infrared (NIR) fluorescent light, in the wavelength range of 650-900 nm, has the potential to provide high-resolution, high-sensitivity, and real-time avoidance of nerve damage, but only if nerve-specific NIR fluorophores can be developed. In this study, we evaluated a series of Oxazine derivatives to highlight various peripheral nerve structures in small and large animals. Among the targeted fluorophores, Oxazine 4 has peak emission near into the NIR, which provided nerve-targeted signal in the brachial plexus and sciatic nerve for up to 12 h after a single intravenous injection. In addition, recurrent laryngeal nerves were successfully identified and highlighted in real time in swine, which could be preserved during the course of thyroid resection. Although optical properties of these agents are not yet optimal, chemical structure analysis provides a basis for improving these prototype nerve-specific NIR fluorophores even further.

ObjectiveIntraoperative imaging of intravascular thrombi is limited by the inability of visible light to penetrate thick-walled vessels. Near-infrared (NIR) light has relatively high tissue penetration and low autofluorescence and scatter, offering significant advantages. We hypothesized that the development of 700-nm NIR fluorophores for platelet labeling, in conjunction with existing 800-nm NIR fluorophores, would permit simultaneous and separable quantitation of intravascular thrombi and measurement of the antiplatelet effect of drugs.MethodsWe synthesized a series of lipophilic, cationic, polymethine indocyanine dyes (MHI-86, 94, 106, and 114) that emit at approximately 700 nm. Platelet uptake was optimized in vitro and the bioactivity and blood half-life of labeled platelets was characterized in vitro and in vivo. FeCl3-induced injury of the femoral arteries and intravascular thrombus formation was performed in 35-kg Yorkshire pigs. A combination of 700-nm and 800-nm NIR fluorophore-labeled platelets was used in conjunction with the fluorescence-assisted resection and exploration imaging system to image and quantify the antiplatelet effect of cilostazol and acetylsalicylic acid.ResultsMHI-114 was incorporated at nearly 4.1 × 106 molecules per platelet without affecting platelet function. When infused into pigs, the signal-to-background ratio of MHI-114-labeled platelets exhibited a blood half-life of 16.4 ± 2.2 (mean ± SEM; n = 3) minute and generated a signal-to-background ratio of 2.5 ± 0.5 (mean ± SEM; n = 3) at the site of thrombi. Using dual-NIR-labeled platelet populations, cilostazol and acetylsalicylic acid were found to cause a reduction in platelet incorporation into thrombi of 51 ± 2% and 10 ± 1% (mean ± SEM; n = 3), respectively, relative to vehicle-only treated control thrombi.ConclusionsNew platelet-avid 700-nm NIR fluorophores permit simultaneous two-wavelength NIR fluorescence imaging and quantitation of intravascular thrombi in intact vessels approaching the size of humans and can be used to study the antiplatelet effect of drugs.Clinical RelevanceOccult thrombus formation during surgery leads to significant morbidity and mortality. We describe the synthesis of a novel family of 700-nm near-infrared fluorophores that concentrate into platelets and render them highly fluorescent while maintaining bioactivity. Using these platelets and a near-infrared imaging system, we can detect thrombi in thick-walled vessels intraoperatively with high signal-to-background ratio ≈ 2.5. The optical properties and blood half-life of these 700-nm fluorophores complement a previously described 800-nm platelet-avid fluorophore and enable the assessment of antiplatelet interventions intraoperatively.

The completion of surgical resection is a key prognostic factor in brain tumor treatment. This requires surgeons to identify residual tumors in theater as well as to margin the proximity of the tumor to adjacent normal tissue. Subjective assessments, such as texture palpation or visual tissue differences, are commonly used by oncology surgeons during resection to differentiate cancer lesions from normal tissue, which can potentially result in either an incomplete tumor resection, or accidental removal of normal tissue. Moreover, malignant brain tumors are even more difficult to distinguish from normal brain tissue, and resecting noncancerous tissue may create neurological defects after surgery. To optimize the resection margin in brain tumors, a variety of intraoperative guidance techniques are developed, such as neuronavigation, magnetic resonance imaging, ultrasound, Raman spectroscopy, and optical fluorescence imaging. When combined with appropriate contrast agents, optical fluorescence imaging can provide the neurosurgeon real-time image guidance to improve resection completeness and to decrease surgical complications.

Inorganic/organic hybrid nanoparticles are potentially useful in biomedicine, but to avoid non-specific background fluorescence and long-term toxicity, they need to be cleared from the body within a reasonable timescale1. Previously, we have shown that rigid spherical nanoparticles such as quantum dots can be cleared by the kidneys if they have a hydrodynamic diameter of approximately 5.5 nm and a zwitterionic surface charge2. Here, we show that quantum dots functionalized with high-affinity small-molecule ligands that target tumours can also be cleared by the kidneys if their hydrodynamic diameter is less than this value, which sets an upper limit of 5–10 ligands per quantum dot for renal clearance. Animal models of prostate cancer and melanoma show receptor-specific imaging and renal clearance within 4 h post-injection. This study suggests a set of design rules for the clinical translation of targeted nanoparticles that can be eliminated through the kidneys., National Science Foundation (U.S.) (NSF-0070319), National Institutes of Health (U.S.) (NIH GM68762), National Institutes of Health (U.S.) (NIH grant no. R33-EB-000673), National Institutes of Health (U.S.) ( NIH grant no. R01-CA-115296), National Institutes of Health (U.S.) (MIT-Harvard NanoMedical Consortium (1U54-CA119349, a Center of Cancer Nanotechnology Excellence)), Bank of America, Medical Foundation, inc. (Charles A. King Trust Postdoctoral Research Fellowship Program), cancer

The field of nanotechnology holds great promise for the diagnosis and treatment of human disease. However, the size and charge of most nanoparticles preclude their efficient clearance from the body as intact nanoparticles. Without such clearance or their biodegradation into biologically benign components, toxicity is potentially amplified and radiological imaging is hindered. Using intravenously administered quantum dots in rodents as a model system, we have precisely defined the requirements for renal filtration and urinary excretion of inorganic, metal-containing nanoparticles. Zwitterionic or neutral organic coatings prevented adsorption of serum proteins, which otherwise increased hydrodynamic diameter by >15 nm and prevented renal excretion. A final hydrodynamic diameter

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

The typical method for creating targeted contrast agents requires covalent conjugation of separate targeting and fluorophore domains. In this study, we demonstrate that it is possible to create near-infrared (NIR) fluorophores with different tissue specificities driven by their inherent chemical structures. Thus, a single compact molecule performs both targeting and imaging. We use this strategy to solve a major problem in head and neck surgery: the identification and preservation of parathyroid and thyroid glands. We synthesized 700-nm and 800-nm halogenated fluorophores that show high uptake into these glands after a single intravenous (IV) injection of 0.06 mg kg(-1) in a pig. By using a dual-channel NIR imaging system, we observed-in real time and with high sensitivity-the unambiguous distinction of parathyroid and thyroid glands simultaneously in the context of blood and surrounding soft tissue. This novel technology lays a foundation for performing head and neck surgery with increased precision and efficiency along with potentially lower morbidity, and it provides a general strategy for developing targeted NIR fluorophores.

Direct electrochemical (EC) monitoring in a cell culture medium without electron transporter as called mediator is attractive topic in vitro organoid based on chip with frequently and long-time monitoring since it can avoid to its disadvantage as stability, toxicity. Here, direct monitoring with nonmediator is demonstrated based on impedance spectroscopy under the culture medium in order to overcome the limitation of mediator. The applicability of EC monitoring is shown by detecting alpha-1-anti trypsin (A1AT) which is known as biomarkers for cardiac damage and is widely chosen in organoid cardiac cell-based chip. The validity of presented EC monitoring is proved by observing signal processing and transduction in medium, mediator, medium–mediator complex. After the observation of electron behavior, A1AT as target analyte is immobilized on the electrode and detected using antibody–antigen interaction. As a result, the result indicates limit of detection is 10 ng mL−1 and linearity for the 10–1000 ng mL−1 range, with a sensitivity of 3980 nF (log [g mL])−1 retaining specificity. This EC monitoring is based on label-free and reagentless detection, will pave the way to use for continuous and simple monitoring of in vitro organoid platform.

We present theoretical and experimental methods for in vivo fluorescence lifetime multiplexing and validate the approach using implanted and organ targeted fluorophores in mice.

Biodegradable scaffolds could revolutionize tissue engineering and regenerative medicine; however, in vivo matrix degradation and tissue ingrowth processes are not fully understood. Currently a large number of samples and animals are required to track biodegradation of implanted scaffolds, and such nonconsecutive single-time-point information from various batches result in inaccurate conclusions. To overcome this limitation, we developed functional biodegradable scaffolds by employing invisible near-infrared fluorescence and followed their degradation behaviors in vitro and in vivo. Using optical fluorescence imaging, the degradation could be quantified in real-time, while tissue ingrowth was tracked by measuring vascularization using magnetic resonance imaging in the same animal over a month. Moreover, we optimized the in vitro process of enzyme-based biodegradation to predict implanted scaffold behaviors in vivo, which was closely related to the site of inoculation. This combined multimodal imaging will benefit tissue engineers by saving time, reducing animal numbers, and offering more accurate conclusions.

The signal-to-background ratio (SBR) is the key determinant of sensitivity, detectability and linearity in optical imaging. As signal strength is often constrained by fundamental limits, background reduction becomes an important approach for improving the SBR. We recently reported that a zwitterionic near-infrared (NIR) fluorophore, ZW800-1, exhibits low background. Here we show that this fluorophore provides a much-improved SBR when targeted to cancer cells or proteins by conjugation with a cyclic RGD peptide, fibrinogen or antibodies. ZW800-1 outperforms the commercially available NIR fluorophores IRDye800-CW and Cy5.5 in vitro for immunocytometry, histopathology and immunoblotting and in vivo for image-guided surgery. In tumor model systems, a tumor-to-background ratio of 17.2 is achieved at 4 h after injection of ZW800-1 conjugated to cRGD compared to ratios of 5.1 with IRDye800-CW and 2.7 with Cy5.5. Our results suggest that introducing zwitterionic properties into targeted fluorophores may be a general strategy for improving the SBR in diagnostic and therapeutic applications.

Purpose: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/ 64 Cu dual-labeled cyclic RGD peptide. Methods: The integrin αvβ3 binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data. Results: The dual-labeled probe 64 Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026). Conclusion: The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models.

The imaging of sentinel lymph nodes (SLNs), the first defense against primary tumor metastasis, has been considered as an important strategy for noninvasive tracking tumor metastasis in clinics. In this study, we report the development and application of mesoporous silica-based triple-modal nanoprobes that integrate multiple functional moieties to facilitate near-infrared optical, magnetic resonance (MR) and positron emission tomography (PET) imaging. After embedding near-infrared dye ZW800, the nanoprobe was labeled with T(1) contrast agent Gd(3+) and radionuclide (64)Cu through chelating reactions. High stability and long intracellular retention time of the nanoprobes was confirmed by in vitro characterization, which facilitate long-term in vivo imaging. Longitudinal multimodal imaging was subsequently achieved to visualize tumor draining SLNs up to 3 weeks in a 4T1 tumor metastatic model. Obvious differences in uptake rate, amount of particles, and contrast between metastatic and contra-lateral sentinel lymph nodes were observed. These findings provide very helpful guidance for the design of robust multifunctional nanomaterials in SLNs' mapping and tumor metastasis diagnosis.

Anastomotic complications such as stenosis and leakage in the gastrointestinal (GI) tract can cause high patient morbidity and mortality. To identify the potential preconditions of these complications intraoperatively, we explored the use of two 700 nm near-infrared (NIR) fluorophores administered intraluminally: (1) chlorella, an over-the-counter herbal supplement containing high concentrations of chlorophyll, and (2) methylene blue (MB). In parallel, we administered the 800 nm NIR fluorophore indocyanine green (ICG) intravenously to assess vascular function. Dual-channel, real-time intraoperative imaging and quantitation of the contrast to background ratio (CBR) were performed under normal conditions or after anastomosis or leakage of the stomach and intestines in 35 kg Yorkshire pigs using the Fluorescence-Assisted Resection and Exploration (FLARE) imaging system. Luminal integrity could be assessed with relatively high sensitivity with either chlorella or MB, although chlorella provided significantly higher CBR. ICG angiography provided assessment of blood perfusion of normal, ischemic, and anastomotic areas of the GI tract. Used simultaneously, 700 nm (chlorella or MB) and 800 nm (ICG) NIR fluorescence permitted independent assessment of luminal integrity and vascular perfusion of the GI tract intraoperatively and in real time. This technology has the potential to identify critical complications, such as anastomotic leakage, intraoperatively, when correction is still possible.

Supramolecular structures composed of inorganic nanoparticles and DNA strands can efficiently target tumours and then be disassembled for ease of elimination from the body.

The field of biomedical optics has matured rapidly over the last decade and is poised to make a significant impact on patient care. In particular, wide-field (typically > 5 cm), planar, near-infrared (NIR) fluorescence imaging has the potential to revolutionize human surgery by providing real-time image guidance to surgeons for tissue that needs to be resected, such as tumors, and tissue that needs to be avoided, such as blood vessels and nerves. However, to become a clinical reality, optimized imaging systems and NIR fluorescent contrast agents will be needed. In this review, we introduce the principles of NIR fluorescence imaging, analyze existing NIR fluorescence imaging systems, and discuss the key parameters that guide contrast agent development. We also introduce the complexities surrounding clinical translation using our experience with the Fluorescence-Assisted Resection and Exploration (FLARE™) imaging system as an example. Finally, we introduce state-of-the-art optical imaging techniques that might someday improve image-guided surgery even further.