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A sensitive assay method was developed to determine fentanyl, an opiate agonist, in rat plasma by gas chromatography with nitrogen–phosphorus detection. For the pretreatment of plasma samples, sodium hydroxide was added to denature protein and n-butyl chloride was used to extract fentanyl. The calibration curve was linear within the concentration range 0.5 to 50 ng/ml (r=0.9997). The limit of detection was 0.1 ng/ml, and 0.5 ng/ml could be quantified with acceptable precision. Furthermore, fentanyl could be determined in only 200 μl of rat plasma. The method has been successfully applied to an intramuscular pharmacokinetic study at a dose of 10 μg/kg. Therefore, the current method is a valuable analytical tool for investigating the pharmacokinetics of fentanyl at low clinical doses.

The discovery of small molecule ligands targeted to the surface of live pathogenic bacteria would enable an entirely new class of antibiotics. We report the development and validation of a microarray-based high-throughput screening platform for bacteria that exploits 300 μm diameter chemical spots in a 1 in. × 3 in. nanolayered glass slide format. Using 24 model compounds and 4 different bacterial strains, we optimized the screening technology, including fluorophore-based optical deconvolution for automated scoring of affinity and cyan–magenta–yellow–key (CMYK) color-coding for scoring of both affinity and specificity. The latter provides a lossless, one-dimensional view of multidimensional data. By linking in silico analysis with cell binding affinity and specificity, we could also begin to identify the physicochemical factors that affect ligand performance. The technology we describe could form the foundation for developing new classes of antibiotics.

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.

Stem cell engineering, the manipulation and control of cells, harnesses tremendous potential for diagnosis and therapy of disease; however, it is still challenging to impart multifunctionalization onto stem cells to achieve both. Here we describe a mesenchymal stem cell (MSC)-based multifunctional platform to target orthotopic glioblastoma by integrating the tumor targeted delivery of mesenchymal stem cells and the multimodal imaging advantage of mesoporous silica nanoparticles (MSNs). Rapid cellular uptake, long retention time and stability of particles exemplify the potential that the combination of MSNs and MSCs has as a stem cell-based multifunctional platform. Using such a platform, we verified tumor-targeted delivery of MSCs by in vivo multimodal imaging in an orthotopic U87MG glioblastoma model, displaying higher tumor uptake than particles without MSCs. As a proof-of-concept, this MSC platform opens a new vision for multifunctional applications of cell products by combining the superiority of stem cells and nanoparticles for actively targeted delivery.

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 (≈ 1,000 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.

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.

Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release ‘messenger’ or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelial ‘apoptotic bodies’, as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50–120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after hyperoxia exposure. These circulating EVs also activated systemic macrophages other than the alveolar ones. Collectively, the results show that hyperoxia-induced, lung epithelial cell-derived and caspase-3 enriched EVs activate macrophages and mediate the inflammatory lung responses involved in lung injury.

We describe a rapid and facile method for surface functionalization and ligand patterning of glass slides based on microwave-assisted synthesis and a microarraying robot. Our optimized reaction enables surface modification 42-times faster than conventional techniques and includes a carboxylated self-assembled monolayer, polyethylene glycol linkers of varying length, and stable amide bonds to small molecule, peptide, or protein ligands to be screened for binding to living cells. We also describe customized slide racks that permit functionalization of 100 slides at a time to produce a cost-efficient, highly reproducible batch process. Ligand spots can be positioned on the glass slides precisely using a microarraying robot, and spot size adjusted for any desired application. Using this system, we demonstrate live cell binding to a variety of ligands and optimize PEG linker length. Taken together, the technology we describe should enable high-throughput screening of disease-specific ligands that bind to living cells.

Background We hypothesized that two independent wavelengths of near-infrared (NIR) fluorescent light could be used to identify bile ducts and hepatic arteries simultaneously, and intraoperatively. Materials and Methods Three different combinations of 700 and 800 nm fluorescent contrast agents specific for bile ducts and arteries were injected into N = 10 35-kg female Yorkshire pigs intravenously. Combination 1 (C-1) was methylene blue (MB) for arterial imaging and indocyanine green (ICG) for bile duct imaging. Combination 2 (C-2) was ICG for arterial imaging and MB for bile duct imaging. Combination 3 (C-3) was a newly developed, zwitterionic NIR fluorophore ZW800-1 for arterial imaging and MB for bile duct imaging. Open and minimally invasive surgeries were imaged using the fluorescence-assisted resection and exploration (FLARE) surgical imaging system and minimally invasive FLARE (m-FLARE) imaging systems, respectively. Results Although the desired bile duct and arterial anatomy could be imaged with contrast-to-background ratios (CBRs) ≥ 6 using all three combinations, each one differed significantly in terms of repetition and prolonged imaging. ICG injection resulted in high CBR of the liver and common bile duct (CBD) and prolonged imaging time (120 min) of the CBD (C-1). However, because ICG also resulted in high background of liver and CBD relative to arteries, ICG produced a lower arterial CBR (C-2) at some time points. C-3 provided the best overall performance, although C-2, which is clinically available, did enable effective laparoscopy. Conclusions We demonstrate that dual-channel NIR fluorescence imaging provides simultaneous, real-time, and high resolution identification of bile ducts and hepatic arteries during biliary tract surgery.

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.

Fluorescence lifetime imaging (FLi) could potentially improve exogenous near-infrared (NIR) fluorescence imaging, because it offers the capability of discriminating a signal of interest from background, provides real-time monitoring of a chemical environment, and permits the use of several different fluorescent dyes having the same emission wavelength. We present a high-power, LED-based, NIR light source for the clinical translation of wide-field (larger than 5 cm in diameter) FLi at frequencies up to 35 MHz. Lifetime imaging of indocyanine green (ICG), IRDye 800-CW, and 3,3(')-diethylthiatricarbocyanine iodide (DTTCI) was performed over a large field of view (10 cm by 7.5 cm) using the LED light source. For comparison, a laser diode light source was employed as a gold standard. Experiments were performed both on the bench by diluting the fluorescent dyes in various chemical environments in Eppendorf tubes, and in vivo by injecting the fluorescent dyes mixed in Matrigel subcutaneously into CD-1 mice. Last, measured fluorescence lifetimes obtained using the LED and the laser diode sources were compared with those obtained using a state-of-the-art time-domain imaging system and with those previously described in the literature. On average, lifetime values obtained using the LED and the laser diode light sources were consistent, exhibiting a mean difference of 3% from the expected values and a coefficient of variation of 12%. Taken together, our study offers an alternative to laser diodes for clinical translation of FLi and explores the use of relatively low frequency modulation for in vivo imaging.

Nanoparticles (NPs) have the potential to revolutionize drug delivery, however, administering them to the human body without the need for intravenous injection remains a major challenge. In this study, a series of near-infrared (NIR) fluorescent NPs were systematically varied in chemical composition, shape, size, and surface charge, and their biodistribution and elimination were quantified in rat models after lung instillation. We demonstrate that NPs with hydrodynamic diameter (HD) less than ≈ 34 nm and a non-cationic surface charge translocate rapidly from lung to mediastinal lymph nodes. NPs of HD < 6 nm can traffic rapidly from the lungs to lymph nodes and the bloodstream, and then be subsequently cleared by the kidneys. We discuss the importance of these findings to drug delivery, air pollution, and carcinogenesis.

Background The aim of this study was to determine whether the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB), a dye already approved by the U.S. Food and Drug Administration for other indications, could be exploited for real-time, intra-operative identification of the ureters. Methods The optical properties of MB were quantified in vitro. Open surgery and laparoscopic NIR fluorescence imaging systems were employed. Yorkshire pigs were injected intravenously with 0.1-mg/kg MB ( n = 8), 10-mg furosemide followed by 0.1-mg/kg MB ( n = 6), or 0.5-mg/kg MB ( n = 6). The contrast-to-background ratio (CBR) of the kidney and ureters, and the MB concentration in the urine, were quantified. Results Peak MB absorbance, emission, and intensity in urine occurred at 668 nm, 688 nm, and 20 μmol/L, respectively. After intravenous injection, doses as low as 0.1-mg/kg MB provided prolonged imaging of the ureters, and a dose of 0.5 mg/kg provided statistically significant improvement of CBR. The preinjection of furosemide increased urine volume but did not improve CBR. Laparoscopic identification of the ureter using MB NIR fluorescence was demonstrated. Conclusion Ureteral imaging using MB NIR fluorescence provides sensitive, real-time, intra-operative identification of the ureters during open and laparoscopic surgeries.

Neuroendocrine tumors of the pancreas, such as insulinoma, are difficult to localize, and complete resection is essential for cure. Our hypothesis is that a near-infrared (NIR) fluorophore exhibiting uptake in insulinoma could provide high-sensitivity detection intraoperatively.The optical properties of methylene blue (MB) were measured in vitro in 100% serum at 37 degrees C and in vivo after tissue uptake. MB was injected as a rapid intravenous bolus at doses ranging from 0.25 to 2 mg/kg into wildtype rats and pigs, and into insulinoma-bearing transgenic mice. The FLARE imaging system was used to acquire color video and NIR fluorescence images simultaneously and in real-time. The signal-to-background ratios (SBR) of tissues and tumors were quantified using FLARE software.When appropriately diluted, MB exhibits moderate NIR fluorescence emission peaking at 688 nm. At dosesor =1 mg/kg, certain normal tissues, such as pancreas, accumulate MB and remain NIR fluorescent for up to 1 h with an SBRor = 1.6. MB spectral properties are maintained after uptake into tissue. Interestingly, insulinoma exhibits even higher MB signal than normal pancreas, resulting in insulinoma-to-pancreas ratios of 3.7 and insulinoma-to-muscle ratios of 16.2. MB permitted high-sensitivity, real-time localization of primary, multicentric, and metastatic insulinoma and permitted differentiation among tumor, normal pancreas, and other abdominal structures.A single intravenous injection of a clinically available, commonly used NIR fluorophore provides prolonged intraoperative localization of normal pancreas and insulinoma using invisible NIR fluorescent light.

A significant portion of the field of nanomedicine is predicated on being able to target nanoparticles to sites of disease. However, in vivo biodistribution and clearance of nanoparticles are poorly understood. In this study, a novel formulation of near-infrared fluorescent InAs(ZnS) quantum dots was synthesized and coated with a systematically increasing chain length of PEG. We found that varying PEG chain length resulted in major changes in organ/tissue-selective biodistribution and clearance from the body.

PI3K is important in the regulation of growth, proliferation, and survival of tumor cells. We show that class 1A PI3K is also critical in the tumor microenvironment by regulating the integrity of the tumor vasculature. Using Tie2Cre-mediated deletion of the PI3K regulatory subunits (p85alpha, p55alpha, p50alpha, and p85beta), we generated mice with endothelial cell-specific loss of class 1A PI3K. Complete loss of all subunits caused acute embryonic lethality at E11.5 due to hemorrhaging, whereas retention of a single p85alpha allele yielded viable mice that survived to adulthood. These heterozygous mice exhibited no vascular defects until challenged with a pathological insult, such as tumor cells or high levels of VEGF. Under these pathological conditions, heterozygous mice exhibited localized vascular abnormalities, including vessel leakage and the inability to maintain large vessels, which caused a deceleration of tumorigenesis. Furthermore, we show that a PI3K inhibitor can mimic the effects of class 1A PI3K loss, which suggests that targeting class 1A PI3K may be a promising therapy for blocking tumor angiogenesis.

Background Currently, only x-ray fluoroscopy is available for visualization of the extrahepatic bile ducts intraoperatively. We hypothesized that with an appropriate fluorophore and imaging system, invisible near-infrared (NIR) light could be used for image-guided procedures on the extrahepatic bile ducts. Methods We quantified the performance of three 800 nm NIR fluorophores, differing primarily in their degree of hydrophilicity, for real-time imaging of the extrahepatic bile ducts in rats and pigs: IR-786, indocyanine green (ICG), and the carboxylic form of IRDye™ 800CW (CW800-CA). The signal-to-background ratio (SBR) of the common bile duct relative to liver and pancreas was measured as a function of the dose of contrast agent, injection site, and kinetics using an intraoperative NIR fluorescence imaging system described previously. Bile samples were examined by high performance liquid chromatography tandem mass spectrometry (HPLC/MS) to determine the chemical form of fluorophores in bile. Results Non-sulfonated (IR-786) and di-sulfonated (ICG) NIR fluorophores had poor efficiency and kinetics of excretion into bile. Tetra-sulfonated CW800-CA, however, provided sensitive, specific, and real-time visualization of the extrahepatic bile ducts after a single low-dose given either intraportally or intravenously via systemic vein. A SBR ≥2 provided sensitive assessment of extrahepatic bile duct anatomy and function for over 30 min post-injection, including the detection of millimeter-sized, radiolucent inclusions in pigs. CW800-CA remained intact chemically after secretion into bile. Conclusion The combination of invisible NIR light and an IV injection of CW800-CA provides prolonged, real-time visualization of the extrahepatic bile duct, without ionizing radiation and without changing the look of the operative field.

Near-infrared (NIR) fluorescence has the potential to provide surgeons with real-time intraoperative image-guidance. Increasing the signal-to-background ratio of fluorescent agents involves delivering a controllable excitation fluence rate of proper wavelength and/or using complementary imaging techniques such as FLIM. In this study we describe a low-cost linear driver circuit capable of driving Light Emitting Diodes (LEDs) from DC to 35 MHz, at high power, and which permit fluorescence CW and lifetime measurements. The electronic circuit Gerber files described in this article and the list of components are available online at www.frangionilab.org.

We have developed a versatile nanoparticle construct using a compact cysteine coating on a CdSe(ZnCdS) core(shell) nanocrystal (QD-Cys) that is biologically compatible, exceptionally compact, highly fluorescent, and easily functionalized. The small hydrodynamic diameter of QD-Cys ( approximately 6 nm) allows for renal clearance of these nanoparticles in rat models. Moreover, the ability to directly conjugate to QD-Cys opens up the possibility of functionalized nanocrystals for in vivo targeted imaging, in which small targeting molecules can be appended to QD-Cys, and unbound QDs can be rapidly cleared to achieve high signal/noise ratios and to reduce background toxicity.

Invisible near-infrared light is safe and it penetrates relatively deeply through tissue and blood without altering the surgical field. Our hypothesis was that near-infrared fluorescence imaging would enable visualization of the ureteral anatomy and flow intraoperatively and in real time.CW800-CA (LI-COR, Lincoln, Nebraska), the carboxylic acid form of near-infrared fluorophore IRDye 800CW, was injected intravenously, and its renal clearance kinetics and imaging performance were quantified in 350 gm rats and 35 kg pigs. High performance liquid chromatography and electrospray time-of-flight mass spectrometry were used to characterize CW800-CA metabolism in urine. The clinically available near-infrared fluorophore indocyanine green was also used via retrograde injection into the ureter. Using the 2 near-infrared fluorophores the ureters were imaged under the conditions of steady state, intraluminal foreign bodies and injury.In rat models the highest signal-to-background ratio for visualization occurred after intravenous injection of 7.5 microg/kg CW800-CA with values of 4.0 or greater and 2.3 or greater at 10 and 30 minutes, respectively. In pig models 7.5 microg/kg CW800-CA clearly visualized the normal ureter and intraluminal foreign bodies as small as 2.5 mm in diameter. Retrograde injection of 10 microM indocyanine green also permitted the detection of normal ureter and pinpointed urine leakage caused by injury. Electrospray time-of-flight mass spectrometry, and absorbance and fluorescence spectral analysis confirmed that the fluorescent material in urine was chemically identical to CW800-CA.A convenient intravenous injection of CW800-CA or direct injection of indocyanine green permits high sensitivity visualization of the ureters under steady state and abnormal conditions using invisible light.

Molecular recognition in chemistry and biology is a kind of host-guest phenomena, which concerns inclusion complexes formed through noncovalently controlled interactions between the host and guest. Possessing well-defined hydrophobic cavities of cyclodextrins (CDs), which can bind various organic, inorganic, and biological molecules to form stable inclusion complexes or supramolecular species, have been employed as excellent hosts in supramolecular chemistry and chiral selectors in separation science and technology. Hence, a great deal of efforts has been devoted to the design and syntheses of novel CD derivatives that display enhanced molecular binding abilities and selectivities for specific substrates.

Invisible near-infrared (NIR) fluorescent light permits high sensitivity, real-time image-guidance during oncologic surgery without changing the look of the surgical field. In this study, we complete pre-clinical development of the technology for sentinel lymph node (SLN) mapping using a large animal model of spontaneous melanoma.Sinclair swine with spontaneous melanoma metastatic to regional lymph nodes were used because of their similarity to human melanoma. Organic lymphatic tracers tested included FDA-approved indocyanine green adsorbed non-covalently to human serum albumin (HSA), and NIR fluorophore CW800 conjugated covalently to HSA (HSA800). The inorganic/organic hybrid tracer tested was type II NIR quantum dots with an anionic coating. Primary tumors received four peri-tumoral injections of each tracer, with a fluorophore dose of 100 pmol to 1 nmol per injection. SLN mapping and image-guided resection were performed in real-time.Each of the 3 lymphatic tracers was injected into n = 4 separate primary melanomas in a total of 6 animals. All 12 injections resulted in identification of the SLN(s) and their associated lymphatic channels within 1 minute in 100% of cases, despite highly pigmented skin and black fur. Hydrodynamic diameter had a profound impact on tracer behavior in vivo.This study completes the pre-clinical development of NIR fluorescence-guided SLN mapping and provides insight into imaging system optimization and tracer choice for future human clinical trials. The technology is likely to eliminate the need for radioactive and colored tracers, permits real-time image guidance throughout the procedure, and assists the pathologist in tissue analysis.

A functional polyrotaxane of a PEI-b-PEG-b-PEI copolymer is synthesized in aqueous solution in a one-pot sequence. To obtain a polyrotaxane with PEG-block-selective inclusion complexes, the solution pH of the polypseudorotaxane is lowered to 4.4 in the presence of 9-anthraldehyde (AN), which triggers the expulsion of the alpha-cyclodextrins (alpha-CDs) from the flank PEI chains. Synthetic strategy of a block-selective polyrotaxane between a PEI-b-PEG-b-PEI copolymer and alpha-cyclodextrins.

A biocleavable polyrotaxane, having a necklace-like structure consisting of many cationic alpha-cyclodextrins (alpha-CDs) and a disulfide-introduced poly(ethylene glycol) (PEG), was synthesized and examined as a nonviral gene carrier. The polyrotaxane formed a stable polyplex having positively charged surface even at low charge ratio. This is likely to be due to structural factors of the polyrotaxane, such as the mobile motion of alpha-CDs in the necklace-like structure. Rapid endosomal escape was observed 90 min after transfection. The positively charged surface and the good buffering capacity are advantageous to show the proton sponge effect. The pDNA decondensation occurred through disulfide cleavage of the polyrotaxane and subsequent supramolecular dissociation of the noncovalent linkages between alpha-CDs and PEG. Transfection of the DMAE-SS-PRX polyplex is independent of the amount of free polycation. Those properties played a key role for delivery of pDNA clusters to the nucleus. Therefore, the polyplex nature and the supramolecular dissociation of the polyrotaxane contributed to the enhanced gene delivery.

To give pH sensitivity to a thermoreversible supramolecular-structured hydrogel system, poly(epsilon-lysine) (PL), as a cationic polymer, was grafted to dextran and used for inclusion complexation with alpha-cyclodextrins (alpha-CDs). The synthesized graft copolymer was characterized by 1H NMR spectroscopy, and the hydrogel formation was confirmed by X-ray diffraction and solid-state 13C NMR analysis. The hydrogelation was induced from a phase-separated structure of hydrated dextrans and hydrophobically aggregated inclusion complexes in buffer solution at pH 10.0. The prepared hydrogels showed thermoreversible gel-sol transitions as well as pH-sensitive phase transitions, which were recorded by the changes in UV/Vis transmittance. A rapid phase transition from gel to sol was observed upon decreasing the pH value to 4.0, which resulted from the dissociation process between the protonated guest polymer and alpha-CDs. The stimuli-responsive physical properties of the hydrogels were improved by modulating the degree of substitution of the grafted PL and the combination with alpha-CDs.

beta-Cyclodextrin-conjugated poly(epsilon-lysine) (beta-CDPL) was synthesized as a novel polymeric host for constructing a smart supramolecular assembling system. Systematic studies on the inclusion complexation between the polymeric host with an alpha- or beta-CD cavity and a model guest molecule provided evidence that dual cooperative interactions, specific host-guest interaction and intermolecular ionic interaction, played a dominant role in leading to a fast aggregation phenomenon. In addition, a rapid phase transition induced by the supramolecular assembly was observed reversibly in response to a small change in pH or temperature.

Nuclear-quadrupole tensors of ${}^{69}\mathrm{Ga}$ and ${}^{71}\mathrm{Ga}$ in high-quality free-standing 0.3-mm-thick GaN single crystals, with the lattice constants $a=0.3205(1)\mathrm{nm}$ and $c=0.5135(1)\mathrm{nm},$ grown by hydride vapor-phase epitaxy, have been precisely determined with NMR rotation patterns measured at room temperature. The quadrupole coupling constants of ${}^{69}\mathrm{Ga}$ and ${}^{71}\mathrm{Ga}$ are $2.72\ifmmode\pm\else\textpm\fi{}0.01$ and $1.72\ifmmode\pm\else\textpm\fi{}0.01\mathrm{MHz},$ respectively. The asymmetry parameter is obtained as zero, explicitly determined by experiment, as expected from the wurtzite crystal structure. The principal z axis of the electric-field gradient is along the crystallographic c axis. The experimental results are compared with theoretical calculations based on the WIEN-97 program and the simple point charge model.

A biocleavable polyrotaxane, having a necklace-like structure consisting of many cationic α-cyclodextrins (α-CDs) and a disulfide-introduced poly(ethylene glycol) (PEG), was synthesized and examined as a nonviral gene carrier. The polyrotaxane formed a stable polyplex having positively charged surface even at low charge ratio. This is likely to be due to structural factors of the polyrotaxane, such as the mobile motion of α-CDs in the necklace-like structure. Rapid endosomal escape was observed 90 min after transfection. The positively charged surface and the good buffering capacity are advantageous to show the proton sponge effect. The pDNA decondensation occurred through disulfide cleavage of the polyrotaxane and subsequent supramolecular dissociation of the noncovalent linkages between α-CDs and PEG. Transfection of the DMAE−SS−PRX polyplex is independent of the amount of free polycation. Those properties played a key role for delivery of pDNA clusters to the nucleus. Therefore, the polyplex nature and...

Biodegradable scaffolds have been extensively used in the field of tissue engineering and regenerative medicine. However, noninvasive monitoring of in vivo scaffold degradation is still lacking. In order to develop a real-time trafficking technique, a series of meso-brominated near-infrared (NIR) fluorophores were synthesized and conjugated to biodegradable gelatin scaffolds. Since the pentamethine cyanine core is highly lipophilic, the side chain of each fluorophore was modified with either quaternary ammonium salts or sulfonate groups. The physicochemical properties such as lipophilicity and net charge of fluorophores played a key role in the fate of NIR-conjugated scaffolds in vivo after biodegradation. The positively charged fluorophore-conjugated scaffold fragments were found in salivary glands, lymph nodes, and most of the hepatobiliary excretion route. However, halogenated fluorophores intensively accumulated into lymph nodes and the liver. Interestingly, balanced-charged gelatin scaffolds were degraded into urine in a short period of time. These results demonstrate that the noninvasive optical imaging using NIR fluorophores can be useful for the translation of biodegradable scaffolds into the clinic.

Longitudinal monitoring of cell migration, division and differentiation is of paramount importance in cell-based medical treatment. However, currently available optical techniques for tracing cell growth and tissue development are limited in applications due to genetic modification, toxicity and inaccurate detection when utilizing the visible spectrum. We have developed lipophilic near-infrared (NIR) fluorophores with high optical properties and a low background signal that allows longitudinal monitoring of cell proliferation and differentiation. Intracellular labeling efficacy was highly dependent on the physicochemical properties of fluorophores such as lipophilicity, charge, polar surface area and rotational bonds. Among the series of NIR cyanine fluorophores, ESNF 13 showed high solubility in aqueous buffer, high membrane penetration, low cytotoxicity and a long-term signal maintainability with a high signal intensity. This study will guide tissue engineers in designing long-term cell trafficking agents with better physicochemical and optical properties.

Objective Chylothorax resulting from thoracic duct damage is often difficult to identify and repair. We hypothesized that near-infrared fluorescent light could provide sensitive, real-time, high-resolution intraoperative imaging of thoracic duct anatomy and function. Methods In 16 rats, 4 potential near-infrared fluorescent lymphatic tracers were compared in terms of signal strength and imaging time: indocyanine green, the carboxylic acid of IRDye 800CW (LI-COR, Lincoln, Neb), indocyanine green adsorbed to human serum albumin, and IRDye 800CW conjugated covalently to human serum albumin. Optimal agent was validated in 8 pigs approaching human size (n = 6 by open surgery with FLARE imaging system [Beth Israel Deaconess Medical Center, Boston, Mass] and n = 2 by video-assisted thoracoscopic surgery minimally invasive [m-FLARE] imaging system [Beth Israel Deaconess Medical Center]). Lymphatic tracer injection site, dose, and timing were optimized. Results For signal strength, sustained imaging time, and clinical translatability, the best lymphatic tracer was indocyanine green, which is already Food and Drug Administration approved for other indications. In pigs, a simple subcutaneous injection of indocyanine green into lower leg (≥36 μg/kg), provided thoracic duct imaging with onset of about 5 minutes after injection, sustained imaging for at least 60 minutes after injection, and signal-to-background ratio of at least 2. With this technology, normal thoracic duct flow, collateral flow, injury models, and repair models could all be observed under direct visualization. Conclusions Near-infrared fluorescent light could provide sensitive, sustained, real-time imaging of thoracic duct anatomy and function during both open and video-assisted thoracoscopic surgery in animal models.

Because of their large size compared to small molecules and their multifunctionality, nanoparticles (NPs) hold promise as biomedical imaging, diagnostic, and theragnostic agents. However, the key to their success hinges on a detailed understanding of their behavior after administration into the body. NP biodistribution, target binding, and clearance are complex functions of their physicochemical properties in serum, which include hydrodynamic diameter, solubility, stability, shape and flexibility, surface charge, composition, and formulation. Moreover, many materials used to construct NPs have real or potential toxicity or may interfere with other medical tests. In this review, we discuss the design considerations that mediate NP behavior in the body and the fundamental principles that govern clinical translation. By analyzing those nanomaterials that have already received regulatory approval, most of which are actually therapeutic agents, we attempt to predict which types of NPs hold potential as diagnostic agents for biomedical imaging. Finally, using quantum dots as an example, we provide a framework for deciding whether an NP-based agent is the best choice for a particular clinical application.