characterization-biological-absorption-spectra-spanning-visible-to

Characterization of Biological Absorption Spectra Spanning the Visible to the Short-Wave Infrared

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. ...

Washington University in St. Louis

Fluorescence anisotropy in the near-infrared (NIR) spectral range is challenging because of the lack of appropriate NIR fluorescent labels. We have evaluated polymethine fluorescent dyes to identify a leading candidate for NIR anisotropy applications. The NIR dye LS601 demonstrated low fluorescence anisotropy values (r) as a result of its relatively long fluorescent lifetime 1.3 ns. The r value of LS601 unbound and coupled to biological macromolecules was found to have a sufficient dynamic range from 0.24 to 0.37, demonstrating the feasibility of fluorescence anisotropy in the NIR. The viability of fluorescence anisotropy using a NIR label was demonstrated by characterization of dye-protein conjugates. These results open the door to a number of applications in drug discovery, fluorescence anisotropy imaging and contrast agent development.

Defining a polymethine dye for fluorescence anisotropy applications in the near-infrared spectral range.

Nanothermometers composed from a gold nanorod core, temperature sensitive linker and fluorescent dye are reported. The nanothermometers have low fluorescence due to a self-quenching mechanism at temperatures below 50 °C and become highly fluorescence above 70 °C.

Design of irreversible optical nanothermometers for thermal ablations.

We present the rationale, synthesis and evaluation of the first activatable fluorescent probe that utilizes fluorescence lifetime change for detection of nitric oxide. The new probe DAP-LT1 features a near-infrared polymethine skeleton with a diaminobenzene functionality incorporated into the meso-position. The probe is partially quenched, and upon reaction with nitric oxide shows an increase in the fluorescence lifetime from 1.08 ns to 1.24 ns.

Synthesis of nitric oxide probes with fluorescence lifetime sensitivity.

Memory T lymphocytes are commonly viewed as a major barrier for long-term survival of organ allografts and are thought to accelerate rejection responses due to their rapid infiltration into allografts, low threshold for activation, and ability to produce inflammatory mediators. Because memory T cells are usually associated with rejection, preclinical protocols have been developed to target this population in transplant recipients. Here, using a murine model, we found that costimulatory blockade-mediated lung allograft acceptance depended on the rapid infiltration of the graft by central memory CD8+ T cells (CD44(hi)CD62L(hi)CCR7+). Chemokine receptor signaling and alloantigen recognition were required for trafficking of these memory T cells to lung allografts. Intravital 2-photon imaging revealed that CCR7 expression on CD8+ T cells was critical for formation of stable synapses with antigen-presenting cells, resulting in IFN-γ production, which induced NO and downregulated alloimmune responses. Thus, we describe a critical role for CD8+ central memory T cells in lung allograft acceptance and highlight the need for tailored approaches for tolerance induction in the lung.

Central memory CD8+ T lymphocytes mediate lung allograft acceptance.

Current imaging modalities lack the ability to quickly assess and classify nerve injury for predicting favourable versus unfavourable healing outcomes, which could minimize episodes of chronic pain and loss of function by allowing for early intervention. Thus, the development of a technique to noninvasively assess peripheral nerve damage is of critical importance. While the development of nerve specific near infrared (NIR) molecular probes capable of such diagnostics constitutes our long term goal, initial studies to identify a NIR dye for constructing such a probe are required. We have evaluated the properties of a novel highly hydrophilic and functionalizable polymethine dye, and its more hydrophobic analogue indocyanine green, within the sciatic nerve of rats following intra-nerve injection. The reporting ability of both dyes at critical depths for nerve imaging, the importance of hydrophilicity on dye transport through nervous tissue, and their toxicity - or lack thereof - to the neural environment have been evaluated. The results suggest that the novel NIR dye is an appropriate fluorescent reporter for use in designing nerve-specific optical molecular probes for non-invasive diagnosis and classification of nerve injury.

A NIR Dye for Development of Peripheral Nerve Targeted Probes.

We have developed a novel design of optical nanothermometers that can measure the surrounding temperature in the range of 20-85 °C. The nanothermometers comprise two organic fluorophores encapsulated in a crosslinked polymethacrylate nanoshell. The role of the nanocapsule shell around the fluorophores is to form a well-defined and stable microenvironment to prevent other factors besides temperature from affecting the dyes' fluorescence. The two fluorophores feature different temperature-dependent emission profiles; a fluorophore with relatively insensitive fluorescence (rhodamine 640) serves as a reference whereas a sensitive fluorophore (indocyanine green) serves as a sensor. The sensitivity of the nanothermometers depends on the type of nanocapsule-forming lipid and is affected by the phase transition temperature. Both the fluorescence intensity and the fluorescence lifetime can be utilized to measure the temperature.

Design of fluorescent nanocapsules as ratiometric nanothermometers.

Peripheral nerve injury evokes a complex cascade of chemical reactions including generation of molecular radicals. Conversely, the reactions within nerve induced by stress are difficult to directly detect or measure to establish causality. Monitoring these reactions in vivo would enable deeper understanding of the nature of the injury and healing processes. Here, we utilized near-infrared fluorescence molecular probes delivered via intra-neural injection technique to enable live, in vivo imaging of tissue response associated with nerve injury and stress. These initially quenched fluorescent probes featured specific sensitivity to hydroxyl radicals and become fluorescent upon encountering reactive oxygen species (ROS). Intraneurally delivered probes demonstrated rapid activation in injured rat sciatic nerve but minimal activation in normal, uninjured nerve. In addition, these probes reported activation within sciatic nerves of living rats after a stress caused by a pinprick stimulus to the abdomen. This imaging approach was more sensitive to detecting changes within nerves due to the induced stress than other techniques to evaluate cellular and molecular changes. Specifically, neither histological analysis of the sciatic nerves, nor the expression of pain and stress associated genes in dorsal root ganglia could provide statistically significant differences between the control and stressed groups. Overall, the results demonstrate a novel imaging approach to measure ROS in addition to the impact of ROS within nerve in live animals.

Imaging of radicals following injury or acute stress in peripheral nerves with activatable fluorescent probes.

Rapid detection of multifocal cancer without the use of complex imaging schemes will improve treatment outcomes. In this study, dynamic fluorescence imaging was used to harness differences in the perfusion kinetics of near-infrared (NIR) fluorescent dyes to visualize structural characteristics of different tissues. Using the hydrophobic nontumor-selective NIR dye cypate, and the hydrophilic dye LS288, a high tumor-to-background contrast was achieved, allowing the delineation of diverse tissue types while maintaining short imaging times. By clustering tissue types with similar perfusion properties, the dynamic fluorescence imaging method identified secondary tumor locations when only the primary tumor position was known, with a respective sensitivity and specificity of 0.97 and 0.75 for cypate, and 0.85 and 0.81 for LS288. Histological analysis suggests that the vasculature in the connective tissue that directly surrounds the tumor was a major factor for tumor identification through perfusion imaging. Although the hydrophobic dye showed higher specificity than the hydrophilic probe, use of other dyes with different physical and biological properties could further improve the accuracy of the dynamic imaging platform to identify multifocal tumors for potential use in real-time intraoperative procedures.

Perfusion-based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near-infrared molecular probes.

Several biologically active bivalent Pd and Pt complexes with two structurally similar cyanoxime ligands abbreviated as H(DECO): 2-oximino-2-cyano-N,N'-diethylacetamide, and H(PyrCO): 2-oximino-2-cyan-N-pyrrolidine acetamide were synthesized and characterized using spectroscopic methods, thermal analysis and X-ray crystallography. Structures revealed planar cis-geometry of studied complexes. Freshly obtained Pt(DECO), Pd(DECO), Pt(PyrCO) and Pd(PyrCO) complexes were used in for in vitro cytotoxicity assays using two different etiology human cancer cell lines HeLa and WiDr cells. Investigated compounds showed cytotoxicity levels at or above cisplatin. Pt(DECO) was also tested in vivo in healthy C57BL/6 mice. The complex was administered at three different dosage (0, 7.5, 15 mg/kg, i.p. once/week), over a total period of 8 weeks. No changes were observed in the animal weight in the treated mice compared to the control dextrose-treated group. The levels of erythrocytes, leukocytes, and hemoglobin were within the normal level suggesting low myelotoxicity. Negligible cardiotoxicity was observed from the histological evaluation of the hearts from the treated animals. Results from the tail nerve conduction velocity (NCV) and nerve histomorphometry suggested no impact of Pt(DECO) on peripheral nerves. The complex, however, induced certain hepatotoxicity and lead to the elevation of IL-6, a pro-inflammatory cytokine. Overall, Pt(DECO)2 showed minimal in vivo toxicity, thus presenting a promising candidate for future testing in animal models of cancer.

New in vitro highly cytotoxic platinum and palladium cyanoximates with minimal side effects in vivo.

This work demonstrates the application of hyaluronan-conjugated nitrogen-doped carbon quantum dots (HA-nCQDs) for bioimaging of tumor cells and illustrates their potential use as carriers in targeted drug delivery. Quantum dots are challenging to deliver with specificity, which hinders their application. To facilitate targeted internalization by cancer cells, hyaluronic acid, a natural ligand of CD44 receptors, was covalently grafted on nCQDs. The HA-nCQD conjugate was synthesized by carbodiimide coupling of the amine moieties on nCQDs and the carboxylic acids on HA chains. Conjugated HA-nCQD retained sufficient fluorescence, although with 30% lower quantum efficiency than the original nCQDs. Confocal microscopy showed enhanced internalization of HA-nCQDs, facilitated by CD44 receptors. To demonstrate the specificity of HA-nCQDs toward human tumor cells, patient-derived breast cancer tissue with high-CD44 expression was implanted in adult mice. The tumors were allowed to grow up to 200-250 mm prior to the injection of HA-nCQDs. With either local or systemic injection, we achieved a high level of tumor specificity judged by a strong signal-to-noise ratio between the tumor and the surrounding tissue . Overall, the results show that HA-nCQDs can be used for imaging of CD44-specific tumors in preclinical models of human cancer and potentially used as carriers for targeted drug delivery into CD44-rich cells.

Hyaluronan-Conjugated Carbon Quantum Dots for Bioimaging Use.

Bioconjugates are important next-generation drugs and imaging agents. Assembly of these increasingly complex constructs requires precise control over processing conditions, which is a challenge for conventional manual synthesis. This inadequacy has motivated the pursuit of new approaches for efficient, controlled modification of high-molecular-weight biologics such as proteins, carbohydrates, and nucleic acids. We report a novel, hands-free, semiautomated platform for synthetic manipulation of biomolecules using acoustically responsive microparticles as three-dimensional reaction substrates. The microfluidic reactor incorporates a longitudinal acoustic trap that controls the chemical reactions within a localized acoustic field. Forces generated by this field immobilize the microscale substrates against the continuous flow of participating chemical reagents. Thus, the motion of substrates and reactants is decoupled, enabling exquisite control over multistep reaction conditions and providing high-yield, high-purity products with minimal user input. We demonstrate these capabilities by conjugating clinically relevant antibodies with a small molecule. The on-bead synthesis comprises capture of the antibody, coupling of a fluorescent tag, product purification, and product release. Successful capture and modification of a fluorescently labeled antibody are confirmed via fold increases of 49 and 11 in the green (antibody)- and red (small-molecule dye)-channel median intensities determined using flow cytometry. Antibody conjugates assembled on acoustically responsive, ultrasound-confined microparticles exhibit similar quality and quantity to those prepared manually by a skilled technician.

Antibody Conjugate Assembly on Ultrasound-Confined Microcarrier Particles.

Understanding peripheral nerve micro-anatomy can assist in the development of safe and effective neuromodulation devices. However, current approaches for imaging nerve morphology at the fiber level are either cumbersome, require substantial instrumentation, have a limited volume of view, or are limited in resolution/contrast. We present alternative methods based on MUSE (Microscopy with Ultraviolet Surface Excitation) imaging to investigate peripheral nerve morphology, both in 2D and 3D. For 2D imaging, fixed samples are imaged on a conventional MUSE system either label free (via auto-fluorescence) or after staining with fluorescent dyes. This method provides a simple and rapid technique to visualize myelinated nerve fibers at specific locations along the length of the nerve and perform measurements of fiber morphology (e.g., axon diameter and g-ratio). For 3D imaging, a whole-mount staining and MUSE block-face imaging method is developed that can be used to characterize peripheral nerve micro-anatomy and improve the accuracy of computational models in neuromodulation. Images of rat sciatic and human cadaver tibial nerves are presented, illustrating the applicability of the method in different preclinical models.

Imaging peripheral nerve micro-anatomy with MUSE, 2D and 3D approaches.

Exogenous extracellular matrix (ECM) proteins, such as fibrinogen and the thrombin-polymerized scaffold fibrin, are used in surgical repair of severe nerve injuries to supplement ECM produced via the injury response. Monitoring the dynamic changes of fibrin during nerve regeneration may shed light on the frequent failure of grafts in the repair of long nerve gaps.

Article	Year
Defining a polymethine dye for fluorescence anisotropy applications in the near-infrared spectral range. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 22302715	2012
Design of irreversible optical nanothermometers for thermal ablations. Chemical communications (Cambridge, England)\| PubMed ID: 23223185	2013
Synthesis of nitric oxide probes with fluorescence lifetime sensitivity. Organic & biomolecular chemistry\| PubMed ID: 24166035	2013
Central memory CD8+ T lymphocytes mediate lung allograft acceptance. The Journal of clinical investigation\| PubMed ID: 24569377	2014
A NIR Dye for Development of Peripheral Nerve Targeted Probes. MedChemComm\| PubMed ID: 24575295	2012
Design of fluorescent nanocapsules as ratiometric nanothermometers. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 25044240	2014
Imaging of radicals following injury or acute stress in peripheral nerves with activatable fluorescent probes. Free radical biology & medicine\| PubMed ID: 27693326	2016
Perfusion-based fluorescence imaging method delineates diverse organs and identifies multifocal tumors using generic near-infrared molecular probes. Journal of biophotonics\| PubMed ID: 29206348	2018
New in vitro highly cytotoxic platinum and palladium cyanoximates with minimal side effects in vivo. Journal of inorganic biochemistry\| PubMed ID: 32413634	2020
Hyaluronan-Conjugated Carbon Quantum Dots for Bioimaging Use. ACS applied materials & interfaces\| PubMed ID: 33355448	2021
Antibody Conjugate Assembly on Ultrasound-Confined Microcarrier Particles. ACS biomaterials science & engineering\| PubMed ID: 33449635	2020
Imaging peripheral nerve micro-anatomy with MUSE, 2D and 3D approaches. Scientific reports\| PubMed ID: 35715554	2022
near-infrared fluorescent fibrin highlights growth of nerve during regeneration across a nerve gap. Journal of biomedical optics\| PubMed ID: 36451699	2022

Mikhail Y. Berezin

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