This PDF file contains the front matter associated with SPIE Proceedings Volume 10079 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Near infrared photoimmunotherapy (NIR-PIT) is a new type of molecularly-targeted photo-therapy based on conjugating a near infrared silica-phthalocyanine dye, IR700, to a monoclonal antibody (MAb) targeting target-specific cell-surface molecules. When exposed to NIR light, the conjugate rapidly induces a highly-selective cell death only in receptor-positive, MAb-IR700-bound cells. Current immunotherapies for cancer seek to modulate the balance among different immune cell populations, thereby promoting anti-tumor immune responses. However, because these are systemic therapies, they often cause treatment-limiting autoimmune adverse effects. It would be ideal to manipulate the balance between suppressor and effector cells within the tumor without disturbing homeostasis elsewhere in the body. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are well-known immune-suppressor cells that play a key role in tumor immuno-evasion and have been the target of systemic immunotherapies. We used CD25-targeted NIR-PIT to selectively deplete Tregs, thus activating CD8+ T and NK cells and restoring local anti-tumor immunity. This not only resulted in regression of the treated tumor but also induced responses in separate untreated tumors of the same cell-line derivation. We conclude that CD25-targeted NIR-PIT causes spatially selective depletion of Tregs, thereby providing an alternative approach to cancer immunotherapy that can treat not only local tumors but also distant metastatic tumors.

The in vivo detection of rare circulating cells using non invasive fluorescence imaging would provide a key tool to study migration of eg. tumoral or immunological cells. Fluorescence detection is however currently limited by a lack of contrast between the small emission of isolated, fast circulating cells and the strong autofluorescence background of the surrounding tissues. We present the development of near infrared emitting quantum dots (NIR-QDs) with long fluorescence lifetime for sensitive time-gated in vivo imaging of circulating cells. These QDs are composed of low toxicity ZnCuInSe/ZnS materials and made biocompatible using a novel multidentate imidazole zwitterionic block copolymer, ensuring their long term intracellular stability. Cells of interest can thus be labeled ex vivo with QDs, injected intravenously and imaged in the near infrared range. Excitation using a pulsed laser coupled to time-gated detection enables the efficient rejection of short lifetime (≈ ns) autofluorescence background and detection of long lifetime (≈ 150 ns) fluorescence from QD-labeled cells. We demonstrate efficient in vivo imaging of single fast-flowing cells, which opens opportunities for future biological studies. [1] M. Tasso et al, “Sulfobetaine-Vinylimidazole block copolymers: a robust quantum dot surface chemistry expanding bioimaging’s horizons”, ACS Nano, 9(11), 2015 [2] S. Bouccara et al, “Time-gated cell imaging using long lifetime near-infrared-emitting quantum dots for autofluorescence rejection”, J Biomed Optc, 19(5), 2014

Short wavelength infrared (SWIR) part of the spectrum offers several distinct advantages for biomedical imaging. In this report we focus on improving research capabilities for generating broadband continuum in spectral region from 1000 to 1900 nm and utilizing it for nonlinear Raman spectroscopy

Plasmonic gold nanoshell (GNS) probe penetrates into tumors for deep imaging, enables superior photoacoustic contrast. Glypican-3 (GPC3) specific peptide (Kd = 71 nM) conjugated gold nanoshell (λabs=770nm) was used to detect HCC xenograft tumors in mice with photoacoustic imaging. This targeting probe demonstrated tumor uptake after 1 hr and cleared in 12 hrs. Images at a mean (±SD) depth of 9.7±1.4 mm from 0 to 2.1 cm beneath the skin revealed increased PA signal from tumors. Highest tumor uptake and tumor to normal tissue ratio occurred at 2 hrs post injection (T/B = 4.45±0.22, n = 8). Molecular targeting GNS showed potential as a simple, effective and rapid technique for noninvasive in vivo monitoring HCC tumor growth and GPC3 expression.

Colorectal and prostate cancers are major causes of cancer-related death, with early detection key to increased survival. However, as symptoms occur during advanced stages and current diagnostic methods have limitations, there is a need for new fluorescent probes that remain bright, are biocompatible and can be targeted. Conjugated polymer nanoparticles have shown great promise in biological imaging due to their unique optical properties. We have synthesised small, bright, photo-stable CN-PPV, nanoparticles encapsulated with poloxamer polymer and a thin silica shell. By incubating the CN-PPV silica shelled cross-linked (SSCL) nanoparticles in mammalian (HeLa) cells; we were able to show that cellular uptake occurred. Uptake was also shown by incubating the nanoparticles in RWPE-1, WPE1-NB26 and WPE1- NA22 prostate cancer cell lines. Finally, HEK cells were used to show the particles had limited cytotoxicity.

Nanodiamonds contain stable fluorescent emitters and hence can be used for molecular fluorescence imaging and precision sensing of electromagnetic fields. The physical properties of these emitters together with their low reported cytotoxicity make them attractive for biological imaging applications. The controlled application of nanodiamonds for cellular imaging requires detailed understanding of surface chemistry, size ranges and aggregation, as these can all influence cellular interactions. We compared these characteristics for graphitic and oxidized nanodiamonds. Oxidation is generally used for surface functionalization, and was optimized by Thermogravimetric Analysis, achieved by 445±5°C heating in air for 5 hours, then confirmed via Raman and Infrared spectroscopies. Size ranges and aggregation were assessed using Atomic Force Microscopy and Dynamic Light Scattering. Biocompatibility in breast cancer cell lines was measured using a proliferation assay. Heating at 445±5°C reduced the Raman signal of graphitic carbon (1575 cm^-1) as compared to that of diamond (1332 cm^-1) from 0.31±0.07 Raman intensity units to 0.07±0.04. This temperature was substantially below the onset of major mass loss (observed at 535±1°C) and therefore achieved cost efficiency, convenience and high yield. Graphitic and oxidized nanodiamonds formed aggregates in water, with a mean particle size of 192±4nm and 166±2nm at a concentration of 66μg/mL. We then applied the graphitic and oxidized nanodiamonds to cells in culture at 1μg/mL and found no significant change in the proliferation rate (-5±2% and -1±3% respectively). Nanodiamonds may therefore be suitable for development as a novel transformative tool in the life sciences.

Publisher's Note: This paper, originally published on 21, February, 2017, was withdrawn per author request.

Recent studies showed that the excitation spectral window lying between 1.6 and 1.8 μm is optimal for in-depth three-photon microscopy of intact tissues due to the reduced scattering and absorption in this wavelength range. Hence, millimeter penetration depth imaging in a living mouse brain has been demonstrated, demonstrating a major potential for neurosciences. Further improvements of this approach, towards much higher imaging frame rates (up to 15-20 s/frame in previous achievements) requires the development of advanced molecular optical probes specifically designed for three-photon excited fluorescence in the 1.6 -1.8 μm spectral range. In order to achieve large three-photon brightness at 1700 nm, novel molecular-based fluorescent nanoparticles which combine strong absorption in the green-yellow region, remarkable stability and photostability in aqueous and biological conditions have been designed using a bottom-up route. Due to the multipolar nature of the dedicated dyes subunits, these nanoparticles show large nonlinear absorption in the NIR region. These new dyes have been experimentally characterized through the measurement of their three-photon action cross-section, fluorescence spectra and lifetimes using a monolithically integrated high repetition rate all-fiber femtosecond laser based on soliton self-frequency shift providing 9 nJ, 75 fs pulses at 1700 nm. The main result is that their brightness could be several orders of magnitude larger than the one of Texas Red in the 1700 nm excitation window. Ongoing experiments involving the use of these new dyes for in vivo cerebral angiography on a mouse model will be presented and the route towards three-photon endomicroscopy will be discussed.

Light Assisted Molecular Immobilization (LAMI) results in spatially oriented and localized covalent coupling of biomolecules onto thiol reactive surfaces. LAMI is possible due to the conserved spatial proximity between aromatic residues and disulfide bridges in proteins. When aromatic residues are excited with UV light (275-295nm), disulphide bridges are disrupted and the formed thiol groups covalently bind to surfaces. Immobilization hereby reported is achieved in a microfabrication stage coupled to a fs-laser, through one- or multi-photon excitation. The fundamental 840nm output is tripled to 280nm and focused onto the sample, leading to one-photon excitation and molecular immobilization. The sample rests on a xyz-stage with micrometer step resolution and is illuminated according to a pattern uploaded to the software controlling the stage and the shutter. Molecules are immobilized according to such pattern, with micrometer spatial resolution. Spatial masks inserted in the light path lead to light diffraction patterns used to immobilize biomolecules with submicrometer spatial resolution. Light diffraction patterns are imaged by an inbuilt microscope. Two-photon microscopy and imaging of the fluorescent microbeads is shown. Immobilization of proteins, e.g. C-reactive protein, and of an engineered molecular beacon has been successfully achieved. The beacon was coupled to a peptide containing a disulfide bridge neighboring a tryptophan residue, being this way possible to immobilize the beacon on a surface using one-photon LAMI. This technology is being implemented in the creation of point-of-care biosensors aiming at the detection of cancer and cardiovascular disease markers.

A quick, cost-effective method for detection of drugs of abuse in biological fluids would be of great value in healthcare, law enforcement, and home testing applications. The alarming rise in narcotics abuse has led to considerable focus on developing potent and versatile analytical tools that can address this societal problem. While laboratory testing plays a key role in the current detection of drug misuse and the evaluation of patients with drug induced intoxication, these typically require expensive reagents and trained personnel, and may take hours to complete. Thus, a significant unmet need is to engineer a facile method that can rapidly detect drugs with little sample preparation, especially the bound fraction that is typically dominant in the blood stream. Here we report an approach that combines the exquisite sensitivity of surface enhanced Raman spectroscopy (SERS) and a facile protein tethering mechanism to reliably detect four different classes of drugs, barbiturate, benzodiazepine, amphetamine and benzoylecgonine. The proposed approach harnesses the reliable and specific attachment of proteins to both drugs and nanoparticle to facilitate the enhancement of spectral markers that are sensitive to the presence of the drugs. In conjunction with chemometric tools, we have shown the ability to quantify these drugs lower than levels achievable by existing clinical immunoassays. Through molecular docking simulations, we also probe the mechanistic underpinnings of the protein tethering approach, opening the door to detection of a broad class of narcotics in biological fluids within a few minutes as well as for groundwater analysis and toxin detection.

The fluorescent tracer agent 3,6-diamino-2,5-bisN-[(1R)-1-carboxy-2-hydroxyethyl]carbamoylpyrazine, designated MB-102, is cleared from the body solely by the kidneys. A prototype noninvasive fluorescence detection device has been developed for monitoring transdermal fluorescence after bolus intravenous injection of MB-102 in order to measure kidney function. A mathematical model of the detected fluorescence signal was created for evaluation of observed variations in agent kinetics across body locations and for analysis of candidate instrument geometries. The model comprises pharmacokinetics of agent distribution within body compartments, local diffusion of the agent within the skin, Monte Carlo photon transport through tissue, and ray tracing of the instrument optics. Data from eight human subjects with normal renal function and a range of skin colors shows good agreement with simulated data. Body site dependence of equilibration kinetics was explored using the model to find the local vasculature-to-interstitial diffusion time constant, blood volume fraction, and interstitial volume fraction. Finally, candidate instrument geometries were evaluated using the model. While an increase in source-detector separation was found to increase sensitivity to tissue optical properties, it reduced the relative intensity of the background signal with minimal effect on the measured equilibration kinetics.

Transcutaneous measurement of Glomerular Filtration Rate (tGFR) is now frequently used in preclinical in vivo animal studies. tGFR allows consecutive measurements on the same animal, including multiple measurements on a daily basis. A description of the measurement device and its many applications, along with examples from the recent literature will be given. We will highlight the fields of interest in which the system is used and give an overview about its performance versus endogenous and other exogenous methods of GFR measurement. A special focus will be put on the precision of tGFR compared to standard measurements employed in the research setting.

A prototype medical device for trans-cutaneous monitoring of kidney function has been developed, validated, and used in a clinical trial on 16 healthy subjects having a wide range of skin color types. The fluorescent tracer agent MB-102 was administered intravenously as a bolus that was varied between 0.5 and 4 μmole/kg subject weight. The tracer agent was tracked as a function of time in plasma by blood sampling and trans-cutaneously at four body sites (sternum, forehead, arm, and side) simultaneously. Excitation was performed with a very low level of amplitude-modulated LED light at 450 nm (<50 μW/cm²), and fluorescence emission was synchronously detected at 570 nm. With adjustment of detection gain between subjects, no skin color dependence was observed of the signal-to-noise ratio (SNR) of the transcutaneous measurements. The primary source of measurement noise appeared to be subject motion, likely due to variations in blood content at the skin measurement site. A typical two-compartment pharmacokinetic dependence was observed with equilibration of the fluorescent agent between the vascular space into which it was injected and the extracellular space into which it subsequently diffused. Variation of this equilibration time was observed across body sites, with the sternum providing the shortest and most consistent equilibration. After equilibration, the terminal fluorescence time dependence at the sternum site was found to be highly correlated with tracer agent concentration time dependence sampled from the blood plasma.

Silica nanoparticles have been increasingly used in developing bioanalytical, biomedical and in many other applications. Silica nanoparticles can easily be synthesized and with the advent of wide availability of modified TEOS reactive analogues only the researcher imagination is the limit of preparing silica nanoparticles that contain different molecules that are either copolymerized inside of the silica nanoparticle or chemically attached (bonded) to the silica nanoparticle surface. Relatively non-porous silica nanoparticles can contain copolymerized dyes for the creation of bright fluorescence labels while the surface of these silica nanoparticles can be bonded with reactive moieties that are suitable for covalently labeling the molecule of interest. Also the surface bonded moieties can serve other purposes, e.g., molecular recognition either on a non-fluorescent or fluorescent silica nanoparticle. As far as the fluorescent nanoparticles development concerns near-infrared (NIR) absorbing carbocyanine dyes have been increasingly used as they can be useful for developing bioanalytical, biomedical methods and in many other applications. Carbocyanines are preferred as they are relatively easy to synthesize and can be designed to achieve particular spectroscopic properties. For example either copolymerized or surface bound dyes can contain appropriate functional moieties absorption and fluorescence properties change when it is complexed to metal ions, to detect pH changes, bind to biological molecules, etc. Fluorescence intensity of carbocyanines significantly increases by enclosing several dye molecules in a single silica nanoparticle due to shielding however self quenching may become a problem at high dye concentrations in confined spaces. Large Stokes’ shift dyes can significantly decrease this problem. This can be achieved by substituting meso position halogens in the NIR fluorescent carbocyanines with a linker containing amino moiety which can also serve as linker to covalently attach the dye molecule during the nanoparticle synthesis. This presentation discusses facile synthesis and applications of silica nanoparticles containing copolymerized fluorophores and/or surface bound moieties. Applications include silica nanoparticles containing several dye molecules as bright labels in immunochemical uses, cell imaging and forensic applications for latent blood detection. This latter application was developed using leuco fluorescein copolymerized silica nanoparticles. This synthesis proved that copolymerized dyes can be further modified after the dye containing silica nanoparticle was formed. Surface bound moiety examples will be given for capillary electrochromatography using amino acid-bonded silica nanoparticles as pseudostationary phases as chiral selectors.

Magnetic nanoparticles (MNPs) have a major role as contrast agent in diagnostic imaging and therapeutic monitoring. In order to research on MNP exposition, degradation and elimination of those nano composites as well as the consequences of the MNP exposition in relation with social economic relevant diseases (cancer, infectious diseases), the comprehensive characterization of magnetic and structural properties is of high importance. Within this contribution, the magnetic characterization of theranostic relevant MNPs is introduced. Applying a vibrating sample magnetometer (VSM), it is found, that the nanocomposites show superparamagnetic behavior and the recorded data confirm iron oxide cores (magnetite/maghemite). Employing Raman spectroscopy, the typical fingerprint information of magnetite is detected. By increasing the laser power, the transition to maghemite and hematite due to the oxidation of the magnetic core is illustrated. Moreover, IR spectroscopy is applied to characterize the coating material e.g. starch or other biocompatible polymers. To determine the stability of MNPs as well as the MNP’s elimination under physiological conditions, different buffer systems were tested i.e. simulated body fluid (SBF) and artificial lysosomal fluid (ALF). The investigated MNPs are stable in SBF; thus, the stability in blood after injection of the contrast agent is guaranteed. Finally, the storage in ALF leads to a complete decomposition of the MNPs, which reflects the conditions in lysosomes and guarantee for a fast MNP elimination. Acknowledgement: We thank the Federal Ministry of Education and Research (BMBF), Germany as well as the Project Management Jülich (PTJ), Germany for funding the research project NanoBEL (03XP0003F).

The porous carbon structures (sorbent) with a density of 1.4 g/cm³ are of most interest compared with existing porous carbon structures. This interest can be explained the technology of synthesis such structures. This technology allows to change the pore size in the sorbent leaving a constant concentration of the particles. Investigation of mechanical properties of sorbents allows choosing the synthesis conditions of the sorbent and its parameters so that it was possible to filter the heavy particles moving at high speed. Therefore, the aim of this work is to study the mechanical strength of the porous carbon materials with a density of 1.4 g/cm³ with different pore sizes. The influence of pore size and form of the sorbent on the adsorption capacity were investigated.

Perspective materials in adsorption medicine are the composite carbon nanostructures based on carbon nanotubes and graphene because of their unique mechanical properties and because of their ability to attach other types of atoms. The ability to control the pore size in synthesis process is an important feature of this material. The deformation of nanotubes and graphene in the longitudinal direction of the graphene sheet will occur during the filtration of microorganisms by the composite. Investigation the deformation of the composite under tension along the graphene sheet is carried out for the first time in this work by molecular mechanical method based on potential of DFT.

Hepatocellular carcinoma (HCC) has been the third most common cause of cancer-related death worldwide. Glypican-3 (GPC3) is a heparin sulfate proteoglycan linked to the cell membrane by a glycosyl-phosphatidylinositol anchor (GPI) and is expressed by 75% of all hepatocellular carcinomas but undetectable in healthy liver tissue or liver with focal lesions. What’s more, GPC3 has been gradually applied in clinical applications as a specific indicator for the early detection and prognosis of HCC. As GPC3 can also regulate many pathways in HCC pathogenesis including Wnt, Hh and Yap signaling, it has been shown that GPC3 knockdown can inhibit HCC growth, reinforcing the important roles of GPC3 in HCC development. For HCC early detection, we designed a peptide targeting GPC3 that allows to establish a fluorescent dyes-labeled probe. Firstly, according to the structure of the GPC3 antibody GC33 and the positive peptide reported in the literature, we generated a peptide consisting of twelve amino acids named 12P that may bind to GPC3 with tight binding ability and specificity. In vitro testing, we utilized FCM and laser confocal microscopy to verify its specificity of targeting to the high expression cells of GPC3. What’s more, we linked 12P with a near infrared dye to verify its in vivo targeting ability. All results indicated that 12P possessed potent binding capacity which could be used as a targeting module in GPC3 detection probe.

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, including proliferation, development, metabolism, immunological response, tumorigenesis, and viral infection. The detection of miRNAs is imperative for gaining a better understanding of the functions of these biomolecules and has great potential for the early diagnosis of human disease as well as the discovery of new drugs through the use of miRNAs as targets. In this article, we develop a highly sensitive, and specific miRNA assay based on the two-stage isothermal amplification reactions and molecular beacon. The two-stage isothermal amplification reactions involves two templates and two-stage amplification reactions under isothermal conditions. The first template enables the amplification of miRNA, and the second template enables the conversion of miRNA to the reporter oligonucleotide(Y). Importantly, different miRNAs can be converted to the same Y seperately, which can hybridize with the same set of molecular beacon to generate fluorescent signals. This assay is highly sensitive and specific with a detection limit of 1 fM and can even discriminate single-nucleotide differences. Moreover, in combination with the specific templates, this method can be applied for multiplex miRNA assay by simply using the same molecular beacon. This method has potential to become a promising miRNA quantification method in biomedical research and clinical diagnosis.

A novel nanoparticle, magnetic graphene quantum dot (MGQD), was synthesized by hydrothermally cutting graphene oxide-iron oxide sheet for contrast agent in magnetomotive optical coherence tomography (MMOCT) and confocal fluorescence microscopy (CFM). The MGQD has superparamagnetism, which allows the MGQD to be tracked and imaged using MMOCT. The MMOCT can display paramagnetic nanoparticle in vivo and provide an anatomical information with micron scale resolution and long imaging depth in clinic application. Moreover, the MGQD has excitation-depend fluorescence and emits visible fluorescence under the excitation of 360nm light, which allows the MGQD to be used as tracer in CFM. CFM can offer intracellular details due to higher resolution, while CFM is unsuitable for imaging anatomical structure because of the limited view of field. The use of MGQD for cell or tissue tracking realizes the combination of MMOCT and CFM, and gives a more comprehensive diagnosis.

By means of an AMBER/AIREBO hybrid method we investigated indentation of layered graphene/phospholipids composite in which the individual phospholipid molecules arranged between the graphene layers. As a result of calculations it was established that such composite is characterized by negative enthalpy of formation. An armchair carbon nanotube approaching with the speed of 10 m/s to the considered composite was used as an indenter. During the simulation it was found that under the action of indenter upper graphene layer in the composite starts to sag, exerting the pressure on the phospholipids which are located under it. Under the influence of pressure phospholipids begins to move on graphene trying to get away from the indenter. Therefore, by placing the phospholipids under improvised press it is possible to achieve their selective localization on graphene platform. The results of the calculations of the total energy of the studied molecular system showed that the value of energy begins to increase as the tube penetration deep inside the composite, indicating the loss of the structure stability. It was found that the strength of the layered graphene/phospholipids composite will increase with the increase in the number of graphene layers.

A new hybrid mathematical model allowing us to investigate the interaction between the components of the DNA + carbon nanostructure molecular complex on the atomic and molecular levels are developed. Within the developed model we proposed to describe the carbon nanostructures by means of the methods and approaches of atomistic modeling, and to describe the DNA molecule using the methods and approaches of coarse-grained modeling. A coarse-grained structure of DNA is built based on 3-Site-Per-Nucleotide model. The proposed hybrid model has been implemented in the original software complex for molecular modeling KVAZAR using modern IT-solutions. The novelty of the model is concluded to a finding the weight coefficients for the interaction of large particles, simulating DNA, and conventional particle, simulating carbon nanostructure, and also for the intermolecular interactions. On the basis of established regularities for interaction between DNA and carbon nanostructures we will develop the model of the sensor device.

The subject of our scientific interest is the dynamics of the phospholipid molecules into a corrugated graphene sheet. According to our assumption by changing the topology of graphene properly it is possible to find the ways for management of the selective localization of phospholipid molecules to form the desired configuration of these structures. We considered DPPC (dipalmitoylphosphatidylcholine) phospholipids, which are the part of cell membranes and lipoproteins. We investigated the behavior of the phospholipids on the graphene sheet consisting of 1710 atoms with the size of 6.9 nm along the zigzag edge and 6.25 nm along the armchair edge. The numerical experiment was carried out using the original AMBER/AIREBO hybrid method with Lennard-Jones potential to describe the interaction between unbound atoms of different structures. The temperature was maintained at 300 K during the numerical experiment. All numerical experiments were performed using KVAZAR software system. We considered several cases of corrugated graphene with different width and dept of the corrugation. Special attention in our work was paid to the orientation of the phospholipids in the plane of graphene sheet.