Fluorescent Sensor Arrays Can Predict and Quantify the Composition of Multicomponent Bacterial Samples

Fast and reliable identification of infectious disease agents is among the most important challenges for the healthcare system. The discrimination of individual components of mixed infections represents a particularly difficult task. In the current study we further expand the functionality of a ratiometric sensor array technology based on small-molecule environmentally-sensitive organic dyes, which can be successfully applied for the analysis of mixed bacterial samples. Using pattern recognition methods and data from pure bacterial species, we demonstrate that this approach can be used to quantify the composition of mixtures, as well as to predict their components with the accuracy of ~80% without the need to acquire additional reference data. The described approach significantly expands the functionality of sensor arrays and provides important insights into data processing for the analysis of other complex samples

Multimodal Imaging Nanoparticles Derived from Hyaluronic Acid for Integrated Preoperative and Intraoperative Cancer Imaging

Surgical resection remains the most promising treatment strategy for many types of cancer. Residual malignant tissue after surgery, a consequence in part due to positive margins, contributes to high mortality and disease recurrence. In this study, multimodal contrast agents for integrated preoperative magnetic resonance imaging (MRI) and intraoperative fluorescence image-guided surgery (FIGS) are developed. Self-assembled multimodal imaging nanoparticles (SAMINs) were developed as a mixed micelle formulation using amphiphilic HA polymers functionalized with either GdDTPA for T1 contrast-enhanced MRI or Cy7.5, a near infrared fluorophore. To evaluate the relationship between MR and fluorescence signal from SAMINs, we employed simulated surgical phantoms that are routinely used to evaluate the depth at which near infrared (NIR) imaging agents can be detected by FIGS. Finally, imaging agent efficacy was evaluated in a human breast tumor xenograft model in nude mice, which demonstrated contrast in both fluorescence and magnetic resonance imaging

Near Infrared Fluorescent Nanoparticles Derived from Hyaluronic Acid Improve Tumor Contrast for Image-Guided Surgery.

Tumor tissue that remains undetected at the primary surgical site can cause tumor recurrence, repeat surgery, and treatment strategy alterations that impose a significant patient and healthcare burden. Intraoperative near infrared fluorescence (NIRF) imaging is one potential method to identify remaining tumor by visualization of NIR fluorophores that are preferentially localized to the tumor. This requires development of fluorophores that consistently identify tumor tissue in different patients and tumor types. In this study we examined a panel of NIRF contrast agents consisting of polymeric nanoparticle (NP) formulations derived from hyaluronic acid (HA), with either physically entrapped indocyanine green (ICG) or covalently conjugated Cy7.5. Using orthotopic human breast cancer MDA-MB-231 xenografts in nude mice we identified two lead formulations. One, NanoICGPBA, with physicochemically entrapped ICG, showed 2.3-fold greater tumor contrast than ICG alone at 24 h (p \u3c 0.01), and another, NanoCy7.5100-H, with covalently conjugated Cy7.5, showed 74-fold greater tumor contrast than Cy7.5 alone at 24 h (p \u3c 0.0001). These two lead formulations were then tested in immune competent BALB/c mice bearing orthotopic 4T1 breast cancer tumors. NanoICGPBA showed 2.2-fold greater contrast than ICG alone (p \u3c 0.0001), and NanoCy7.5100-H showed 14.8-fold greater contrast than Cy7.5 alone (p \u3c 0.0001). Furthermore, both NanoICGPBA and NanoCy7.5100-H provided strong tumor enhancement using image-guided surgery in mice bearing 4T1 tumors. These studies demonstrate the efficacy of a panel of HA-derived NPs in delineating tumors in vivo, and identifies promising formulations that can be used for future in vivo tumor removal efficacy studies

Combination of an Integrin-Targeting NIR Tracer and an Ultrasensitive Spectroscopic Device for Intraoperative Detection of Head and Neck Tumor Margins and Metastatic Lymph Nodes

Despite major advances in targeted drug therapy and radiation therapy, surgery remains the most effective treatment for most solid tumors. The single most important predictor of patient survival is a complete surgical resection of the primary tumor, draining lymph nodes, and metastatic lesions. Presently, however, 20%–30% of patients with head and neck cancer who undergo surgery still leave the operating room without complete resection because of missed lesions. Thus, major opportunities exist to develop advanced imaging tracers and intraoperative instrumentation that would allow surgeons to visualize microscopic tumors during surgery. The cell adhesion molecule integrin αvβ3 is specifically expressed by tumor neovasculature and invading tumor cells, but not by quiescent vessels or normal cells. Here we report the combined use of an integrin-targeting near-infrared tracer (RGD-IRDye800CW) and a handheld spectroscopic device, an integrated point spectroscopy with wide-field imaging system, for highly sensitive detection of integrin overexpression on infiltrating cancer cells. By using an orthotopic head and neck cancer animal model, we show that this tracer–device combination allows intraoperative detection of not only invasive tumor margins but also metastatic lymph nodes. Correlated histological analysis further reveals that microscopic clusters of 50–100 tumor cells can be detected intraoperatively with high sensitivity and specificity, raising new possibilities in guiding surgical resection of microscopic tumors and metastatic lymph nodes

A Simple and Sensitive LC-MS/MS for Quantitation of ICG in Rat Plasma: Application to a Pre-Clinical Pharmacokinetic Study

A selective, sensitive, and rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of ICG in rat plasma. The chromatographic separation was achieved using an ACE excel C18 (3 µm, 50 × 3.0 mm) column, with a mobile phase composition of 0.1% formic acid and 0.1% formic acid in acetonitrile, using a gradient flow at a rate of 0.3 mL/min. The MS was operated at a unit resolution in the multiple reaction monitoring mode, using the precursor ion → product ion combinations of 753.3 → 330.2 m/z (ICG) and 747.45 → 717.50 (Cy7.5 amine) with a run time of 5 min. The assay was linear over a concentration range of 1–1000 ng/mL with a regression coefficient (r2) of 0.998 or better. The inter and intra-batch precision (% relative standard deviation, %RSD) was lower than 13.5%, with accuracy (%Bias) between −10.03% and 11.56%. The ICG was stable under laboratory storage and handling conditions. The validated method was successfully applied to preclinical pharmacokinetic (PK) studies of ICG at a dose of 0.39 mg/kg in rats. PK parameters suggested the highest plasma concentration within 2 min of intravenous dosing with restricted systemic distribution and rapid clearance

Direct Observation of a Photochemical Alkyne–Allene Reaction and of a Twisted and Rehybridized Intramolecular Charge-Transfer State in a Donor–Acceptor Dyad

The excited-state dynamics of an aniline–triazine electron donor–acceptor dyad with an alkyne spacer has been investigated using a combination of ultrafast broadband mid-IR and visible transient absorption and fluorescence spectroscopies. The transient IR data reveal the occurrence of an efficient alkyne to allene isomerization of the spacer with a time constant increasing from a few hundreds of femtoseconds to a few picoseconds with solvent viscosity. This process is faster than the vibrational cooling of the Franck–Condon excited state, indicative of nonequilibrium dynamics. The transient electronic absorption and fluorescence data evidence that this transformation is accompanied by a charge separation between the donor and the acceptor subunits. The allene character of the spacer implies an orthogonal orientation of the donor and acceptor moieties, similar to that proposed for twisted intramolecular charge-transfer states. Such states are often invoked in the excited-state dynamics of donor–acceptor dyads, but their involvement could never be unambiguously evidenced spectroscopically. The alkyne–allene isomerization involves not only a torsional motion but also a bending of the molecule due to the sp to sp2 rehybridization of one of the alkyne carbon atoms. This twisted and rehybridized intramolecular charge transfer (“TRICT”) state decays back to the planar and linear alkyne ground state on a time scale decreasing from a few hundred to ten picoseconds upon going from weakly to highly polar solvents. The different solvent dependencies reveal that the dynamics of the allene buildup are controlled by the structural changes, whereas the decay is limited by the charge recombination step

Ratiometric Fluorescent Sensor Array as a Versatile Tool for Bacterial Pathogen Identification and Analysis

Rapid and reliable identification of pathogenic microorganisms is of great importance for human and animal health. Most conventional approaches are time-consuming and require expensive reagents, sophisticated equipment, trained personnel, and special storage and handling conditions. Sensor arrays based on small molecules offer a chemically stable and cost-effective alternative. Here we present a ratiometric fluorescent sensor array based on the derivatives of 2-(4′-<i>N</i>,<i>N</i>-dimethylamino)-3-hydroxyflavone and investigate its ability to provide a dual-channel ratiometric response. We demonstrate that, by using discriminant analysis of the sensor array responses, it is possible to effectively distinguish between eight bacterial species and recognize their Gram status. Thus, multiple parameters can be derived from the same data set. Moreover, the predictive potential of this sensor array is discussed, and its ability to analyze unknown samples beyond the list of species used for the training matrix is demonstrated. The proposed sensor array and analysis strategies open new avenues for the development of advanced ratiometric sensors for multiparametric analysis

Solvent tuning of photochemistry upon excited-state symmetry breaking

The nature of the electronic excited state of many symmetric multibranched donor–acceptor molecules varies from delocalized/multipolar to localized/dipolar depending on the environment. Solvent-driven localization breaks the symmetry and traps the exciton in one branch. Using a combination of ultrafast spectroscopies, we investigate how such excited-state symmetry breaking affects the photochemical reactivity of quadrupolar and octupolar A–(π-D)2,3 molecules with photoisomerizable A–π–D branches. Excited-state symmetry breaking is identified by monitoring several spectroscopic signatures of the multipolar delocalized exciton, including the S2 ← S1 electronic transition, whose energy reflects interbranch coupling. It occurs in all but nonpolar solvents. In polar media, it is rapidly followed by an alkyne–allene isomerization of the excited branch. In nonpolar solvents, slow and reversible isomerization corresponding to chemically-driven symmetry breaking, is observed. These findings reveal that the photoreactivity of large conjugated molecules can be tuned by controlling the localization of the excitation