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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.

共同研究チームは、近赤外線レーザーを照射することで卵巣がん細胞を手術中に可視化する新しい蛍光色素を開発しました。卵巣がん治療において手術で病巣を完全に切除することは、治療成績を向上させるための最も重要な因子です。近年、がんや組織をはっきりと標識することができる蛍光ガイド手術は、腫瘍の局在を明らかにすることで取り残しを減らし、外科的な腫瘍切除成績を向上させる技術として注目を集めています。今回本研究グループは、近赤外領域に蛍光を持つスクアライン色素の化学構造や電荷に修飾を加えることで、高輝度で安定した卵巣がん指向性のある蛍光色素OCTL14の開発に成功しました。OCTL14を卵巣がん腹膜播種マウスモデルに投与したところ、投与24時間後まで1ミリ以下の微小な腫瘍組織を検出し、その蛍光ガイド下での手術では肉眼だけに比べて多数の腫瘍の除去が可能でした。OCTL14は低分子蛍光色素分子であり、比較的簡単、安価に大量合成も可能であり広い臨床応用にも適しています。この蛍光物質が将来的に認可され、手術に応用されると、産婦人科医による卵巣がんの手術成績を向上させ、女性患者の予後を改善する画期的な新技術となることが期待されます。, The residual tumor after surgery is the most significant prognostic factor of patients with epithelial ovarian cancer. Near-infrared (NIR) fluorescence-guided surgery is actively utilized for tumor localization and complete resection during surgery. However, ......

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.

The use of chemoattractants to promote endogenous stem cell-based in situ tissue regeneration has recently garnered much attention. This study is the first to assess the endogenous stem cell migration using a newly discovered substance P (SP) analog (SP1) by molecular dynamics simulations as an efficient chemoattractant. Further, a novel strategy based on electrostatic interaction using cationic chitosan (Ch) and anionic hyaluronic acid (HA) to prepare an SP1-loaded injectable C/H formulation without SP1 loss is developed. The formulation quickly forms an SP1-loaded C/H hydrogel in situ through in vivo injection. The newly discovered SP1 is found to possess human mesenchymal stromal cells (hMSCs) migration-inducing ability that is approximately two to three times higher than that of the existing SP. The designed VEGF-mimicking peptide (VP) chemically reacts with the hydrogel (C/H-VP) to sustain the release of VP, thus inducing vasculogenic differentiation of the hMSCs that migrate toward the C/H-VP hydrogel. Similarly, in animal experiments, SP1 attracts a large number of hMSCs toward the C/H-VP hydrogel, after which VP induces vasculogenic differentiation. Collectively, these findings indicate that SP1-loaded C/H-VP hydrogels are a promising strategy to facilitate endogenous stem cell-based in situ tissue regeneration.

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.

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.

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). ©

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.

The physicochemical properties of nanomaterials play a key role in tissue-specific targeting by reducing nonspecific background uptake as well as controlling biodistribution and clearance. Due to the strong influence of surface chemistry, chemical modulation of bioinert exosomes with chargeable and traceable small molecule fluorophores has a significant effect on the targeting, stability, and toxicity of the final conjugates. In this study, charge-variable exosomes are designed by conjugating their surface proteins with near-infrared fluorophores to control the in vivo fate of exosomes. Interestingly, zwitterionic fluorophore-labeled exosomes show rapid renal clearance with minimum to none nonspecific tissue uptake, whereas anionic exosomes are excreted through the hepatobiliary route with high uptake in the liver. The biodistribution and pharmacokinetics of exosome conjugates are comparable to their corresponding free fluorophores, demonstrating that the surface characteristics govern the fate of final conjugates in the living organism. Such unique surface properties of chemically modulated exosomes are confirmed in the lymphatic system after intradermal administration, which results in distinctive kinetic profiles in the secondary lymphoid tissues. This finding can subsequently serve as the foundation for developing tissue-specific exosome-based therapeutics.

Longitudinal tracking of living cells is crucial to understanding the mechanism of action and toxicity of cell-based therapeutics. To quantify the presence of administered cells in the host tissue without sacrifice of animals, labeling of the target cells with a nontoxic and stable contrast agent is a prerequisite. However, such long-term live cell tracking is currently limited by the lack of fluorophores with steady optical and physicochemical properties in the near-infrared (NIR) window. Herein, for the first time, the design of fixable cell-tracking NIR fluorophores (CTNFs) with high optical properties, excellent cell permeation and retention, and high stability against chemical treatments is reported. Efficient cellular labeling and tracking of CTNFs using intraoperative optical fluorescence imaging by following the fate of NIR-labeled cells from the time of injection into animals to ex vivo cellular analysis after resection of the target tissue is demonstrated. Due to the lipophilic cationicity and primary amine docking group, CTNF126 outperforms the other tested fluorophores with rapid diffusion into the cytoplasmic membrane and sequestration inside the lysosomes, which prevents cellular efflux and improves cellular retention. Thus, CTNF126 will be useful to track cells in living organisms for the mechanism of action at the single cell level.

Image-guided surgery using optical imaging requires the availability of large quantities of clinical-grade fluorophores. We describe the cGMP-compatible synthesis of the zwitterionic heptamethine indocyanine near-infrared fluorophore ZW800-1 at the 10 g scale (~1000 patient doses) using facile and efficient solvent purification, and without the need for column chromatography. ZW800-1 has >90% yield at the final step and >99% purity as measured by fluorescence and evaporative light scatter detection. We describe an analytical framework for qualifying impurities, as well as a detailed analysis of counterion identities. Finally, we report the unique in vivo properties of ZW800-1 in large animals approaching the size of humans, thus laying the foundation for rapid clinical translation of these methods. Copyright © 2012 John Wiley & Sons, Ltd.

A longstanding problem in the field of image-guided surgery is the development of ideal near-infrared (NIR) fluorophores. The heptamethine NIR fluorophore indocyanine green (ICG) has been used extensively for image-guided surgery because of clinical availability and safety.[1-3] However, ICG is far from ideal because it exhibits high uptake in the liver, contaminates the gastrointestinal (GI) tract, provides moderate optical properties,[4] is unstable in aqueous media,[3,5] and is unable to conjugate covalently to targeting ligands.[2] Although several classes of novel molecules have been described,[6-13] none to date exhibit simultaneous low background binding, bifunctionality, excellent optical properties, low protein binding, and high serum stability. Although it is intuitive that physicochemical properties, i.e., positive/negative charge density, hydrophilicity/lipophilicity, and charge distribution, will impact in vivo performance, chemical structures that exhibit ideal characteristics have not yet been defined.

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.

A polypseudorotaxane consisting of a linear polyethylenimine with Mn of 22,000 (LPEI22k) and gamma-cyclodextrins (gamma-CDs; LPEI22k/gamma-CD) has been examined as a gene carrier. The polyplex formation with luciferase-encoding plasmid DNA (pDNA), intracellular trafficking of polyplex, cytotoxicity, and transfection efficiency were evaluated by various characteristic methods. LPEI22k/gamma-CD formed a pDNA polyplex at higher N/P ratios than LPEI22k; this suggests that the gamma-CD threading sterically interfered with the polyplex formation. In addition, the zeta potentials of the polyplex significantly decreased due to the reduction in charge density of LPEI22k caused by gamma-CD threading. The cellular uptake of pDNA in the LPEI22k/gamma-CD polyplex was enhanced by free gamma-CDs released from the polyplex that might accelerate the cellular uptake through enhanced membrane affinity. LPEI22k/gamma-CD significantly increased cell viability even at high N/P ratios, and the polyplex showed high transfection efficacy. The low cytotoxicity and high gene expression of LPEI22k/gamma-CD are advantageous to polyplex administration in vivo.

Poly(propylene glycol) (PPG)-grafted dextran was synthesized by conjugating amino-terminated PPG with hydroxyl groups of dextran, and its inclusion complexation property was investigated. The average number of grafted PPG per dextran was changeable in the range of 1.1 to 38.5. The formation of inclusion complexes between the PPG grafts and β-cyclodextrins (β-CDs) and their crystalline structures were characterized by 13 C CP/MAS NMR and X-ray spectroscopies. These hydrogel systems showed a thermall reversible sol-gel transition based on supramolecular assembly and dissociation between host and guest moieties. The results of theological measurements and solgel transition temperature of the hydrogels suggested that the aggregate channel-type crystalline domain was critical to control the transition temperature in terms of initial feed molar ratio of PPG and β-CD and the graft number of PPG constituents. These thermoreversible hydrogel systems showed rapid gelation properties, which may be useful for biomedical application, especially injectable drug deliver systems.

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.