Transmissible Diseases are responsible for a large fraction of the illness-related deaths in the world. This fractions is higher in developing countries and for infants and children. Unfortunately, these diseases do not focus an equally large fraction of medical research. Little has been done so far to use novel approaches from the nanomedicine toolbox to create novel strategies to address and counter transmissible diseases. What has been done, though, has been rather promising. In this talk, I will review some of these approaches and present new results recently achieved in my group on this topic. Strategies to develop drugs for major transmissible diseases will be discussed.

Treatment of brain metastases from primary breast cancer is a critical clinical challenge. Approximately 30% of all breast cancer patients have central nervous system (CNS) metastases, based on autopsies. This rate is thought to be increasing as a function of the aging population, better treatment of non-CNS disease, and improved imaging techniques. Drug uptake into the brain is limited by numerous factors, such as the blood-brain barrier (BBB). More than 98% of small molecule drugs (< 400-500 Da) and nearly all large-molecule therapeutics do not cross the BBB. Lapatinib is a small molecule therapy targeted at the over-expression of HER2+ in breast cancer. However, lapatinib currently shows low efficacy in the treatment of CNS disease due to inadequate delivery across the BBB.
In this study, we investigate a macrophage-based “Trojan horse” light-triggered delivery of lapatinib to treat brain metastases. Macrophages, originating as blood monocytes, can infiltrate brain metastases through an intact blood brain barrier and can be readily loaded with nanoparticles. Here, nanoshells are functionalized with double stranded DNA, which serves as a protective host carrier for lapatinib delivery. It is important to be able to remotely trigger the release of the therapeutic cargo once the macrophages have relocated to the site of the metastases. Drug release is triggered using low levels (1-2 W/cm²) of continuous wave near-infrared light, which induces the dehybridization of the double helix and subsequently releases lapatinib. The optimal DNA sequence is determined by circular dichroism and loading is confirmed with surface enhanced Raman scattering. Loading capacity and release are quantified with LC-MS. Intracellular drug release will be demonstrated and the effects of near-infrared triggered lapatinib release on neighboring HER2+ brain-seeking breast cancer cells will be investigated.

Photoactivated theranostics combining phototherapies (such as photothermal, photodynamic, or photo-triggered chemo/gene therapy) with real-time photodiagnostics (such as bioluminescence, fluorescence, optical or photoacoustic imaging), have been actively pursued due to the spatiotemporal selectivity and specificity for disease destruction, and the advantages of optical imaging including real-time, non-ionizing radiation, high spatial and temporal resolution. Gold nanocrystals (GNCs) with attractive optical properties have been widely employed in photoactivated theranostics.
GNCs with various sizes, shapes, and geometries (such as nanoshells, nanorods, nanocages, nanostars, etc.) have shown great potential to tune their localized surface plasmon resonance (LSPR) to the near-infrared (NIR) region. For example, the LSPR of gold nanoshells and nanocages can be red-shifted by tuning the core/cavity diameter and shell thickness. Gold nanorods, nanoprisms, and nanoplates have red-shifted LSPR as a result of increased length or edge size. Many uniquely shaped GNCs have thus been used as contrast agents for photoacoustic imaging (PAI) and as photothermal conversion agents (PTCAs) for photothermal therapy (PTT).
Plasmonic coupling effect is very helpful to improve the performance of GNCs in PAI and PTT. Recently, we designed and prepared a novel gold bellflower (GBF) platform with multiple-branched petals in a liquid/liquid/gas triphase interface system for PAI guided PTT. Upon NIR laser irradiation, the GBFs, with strong NIR absorption, showed very strong PA response and ultrahigh photothermal conversion efficiency (eta;, ~74%). The excellent performance in PAI and PTT is mainly attributed to the unique features of the GBFs: i) multiple-branched petals with enhanced local electromagnetic field; ii) long narrow gaps between adjacent petals that induce strong plasmonic coupling effect; and iii) bell-shaped nanostructure that can effectively amplify the acoustic signals during the acoustic propagation. Besides notable PTT and excellent PAI effect, the NIR-absorbing GBFs may also find applications in NIR light-triggered drug delivery, catalysis, surface enhanced Raman scattering (SERS), stealth, antireflection, IR sensor, telecommunications, and the like.
This study provides a novel concept of a multiphase interface reaction that can potentially be applied to investigate other chemical reactions existing at multiphase interfaces and guide facile preparation of hierarchical micro- and nano-structures.

Multifunctional plasmonic nanostructures that serve as exogenous image contrast and therapeutic agents are highly attractive for locoregional cancer therapy with minimal systemic toxicity due to intense surface enhanced Raman scattering (SERS) and their photothermal efficiency. It has been demonstrated that core-satellite nanostructures can serve as ultra-bright SERS probes for bioimaging applications due to their remarkable SERS activity considering the electromagnetic hotspots formed between the core and satellites and between the satellites. However, most of the reported core-satellites nanostructures involve complex synthesis procedures such as self-assembly using bifunctional molecular linkers or biomolecules such as DNA. Such procedures either severely limit the stability of the assemblies in physiological fluids or make them prohibitively complex for their translation into pre-clinical and clinical applications. In this work, we introduce a novel class of plasmonic superstructures comprised of a shape-controlled Au nanostructure such as a nanorod or nanosphere as core and spherical Au nanosatellites, synthesized using a biopolymer (poly-L-histidine) coating as a nanoreactor. The optical properties of plasmonic superstructures such as LSPR wavelength, SERS activity and photothermal efficiency could be finely tuned by controlling the shape of the core and size and density of the Au nanosatellites decorating the core. Therefore, the photothermal effect and SERS performance of the superstructures could be integrated or decoupled by a rational choice of the size and shape of the core. This precise control over the properties enables the superstructures to serve as multifunctional (imaging+therapy) theranostic agents or non-invasive SERS contrast agents.

Photothermal ablation (PTA) is an emerging technique that uses near-infrared (NIR) laser light-generated heat to destroy tumor cells. Multifunctional hollow gold nanospheres (HAuNS) have been developed to mediate simultaneous PTA and local delivery of chemoterapeutic agents and siRNA, resulting in enhanced antitumor activity. Various targeting ligands including peptides, apatamers, and antibodies have been conjugated to HAuNS to achieve increased tumor delivery of HAuNS. Successful active targeting of HAuNS to the tumors requires overcoming many biological barriers, including extravazation from tumor vasculature and dispersion of nanoparticles from perivascular area. I will discuss different approaches that we have used to enhance tumor delivery of gold nanoparticles. I will also present our studies towards the application of HAuNS in image-guided antitumor therapy using photoacoustic imaging, MRI, and nuclear imaging techniques.

Gold nanoaggregates assembled from individual gold nanoparticles have been studied in ex vivo materials applications for their plasmonic properties, including surface enhanced Raman scattering. These properties of nanoaggregates could be useful in vivo for diagnostic, therapeutic or imaging modalities, but current synthetic methods generally lead to ill-defined nanoaggregates or required sophisticated polymer additives. Additionally current methods rely heavily on organic solvents to produce aggregates, rendering them poorly biocompatible, and the aggregates are difficult to handle due to reactive surfaces. We have developed a straightforward and modular synthetic method to create and cap gold nanoaggregates to enable their use in aqueous, organic and biological environments. Individual nanoparticles 5-20 nm in size can be controllably assembled to produce aggregates ranging in size from 15 - 100 nm. The properties of the nanoparticles aggregates are controlled by selection of capping agent. Capping with a phenyl or PEG layer renders the resulting stable aggregates organically soluble or biocompatible, respectively. Fluorescent labels are also readily introduced using this strategy and efficient and non-toxic uptake into cells was observed. These aggregates hold great promise for in vivo imaging and potentially drug delivery.

We developed non-nanotoxic proteinticle/gold core/shell nanoparticle (PGCS-NP) for targeted cancer therapy. Unlike synthetic nanoparticles, proteinticles are biological nanoparticles, i.e. nano-scale protein particles (e.g. viral capsid) that are self-assembled inside cells with constant structure and surface topology. Proteinticles can be engineered to present cancer targeting peptides on their surface through genetic modification of the N- or C-terminus region of the protein constituent. Both hexa-tyrosine with high reduction potential and affibody peptides with specific affinity for human epidermal growth factor receptor I (EGFR) were presented on the surface of hepatitis B virus capsid, and the Au-ion reduction formed PGCS-NP (40 nm) that is dotted with many small gold NPs (1-3 nm). PGCS-NP shows great photothermal activity and excellent affinity for EGFR-expressing tumor cells. When IV-injected into tumor-bearing mice, PGCS-NP effectively reached the EGFR-expressing tumor cells and caused severe tumor cell necrosis and significant reduction in tumor size upon NIR laser irradiation. Unlike gold NPs causing in vivo toxicity problems, PGCS-NP don't caused any histological lesions in major organs of mice, indicating that it is a safe and potent agent for photothermal therapy of cancer.

It has been proven that magnetic concentration of drug laden magnetic nanoparticles increases the efficiency of treatment by 2 fold. In these techniques, particles can be concentrated by the presence of a magnetic source that delivers a very high magnetic field and a strong magnetic field gradient. Such a source causes even 150 nm nanoparticles to form chains and aggregations in low Reynold&’s number conditions, which exceeds the diameter of the tumor pores and prevents particles from entering cancer cells.
In this work, we have developed theoretical, numerical and experimental frameworks to apply dynamic magnetic fields to the concentration and de-agglomeration of drug-laden magnetic nanoparticles. We have added the effect of Brownian motion to the dynamic model to be able to accurately describe particle suspensions less than 1um in size. As proof of principle, collagen hydrogels with porosities consistent with tumor cells are fabricated and used as matrices to test the diffusion of magnetic nanoparticles with and without advanced field functions. This work enables the magnetic nanoparticles to both be concentrated at the tumor site and remain non-aggregated allowing them to diffuse into the intercellular matrix and deliver drug throughout the site of the tumor.

Supramolecular architectures constructed through a self-assembly of block copolymers receive much interest due to their various morphologies. Among them, polymersomes have recently highlighted as a focused material because of interests in the study of their formation mechanisms, morphology tuning and also their potential applications as delivery carriers of bioactive materials. Among various interactions utilized in constructing polymersomes, a metal coordination bond is attractive because the ligand exchange reaction can make the membrane properties of polymersomes change within water, which allows controlled release of loaded materials in the polymersomes. Here we would like to report spontaneous polymersome formation triggered by metal coordination bond in aqueous medium.
The supramolecular assembly was formed through the reaction of an anticancer agent, DACHPt, with a Y-shaped block copolymer of w-cholesteroyl-poly(L-glutamic acid) and two-armed poly(ethylene glycol) (PEGasus-PLGA-Chole) via coordination bond between platinum of the DACHPt and carboxylic acid groups of the PLGA segment. The analysis of TEM images and the loading capacity of hydrophilic polymers within the assembly affirmed the assembly to be vesicle with size of 100 nm, thus named Metallosomes. Circular dichroism spectrum measurements revealed that the PLGA segment forms an a-helix structure within the Metallosomes, which suggested a secondary-structure formation of metallo-complexed PLGA segment may drive the self-assembly to form vesicular structure. The Metallosomes can load water-soluble fluorescent macromolecules into their inner aqueous space. Responding to chloride ion corresponding to the physiological NaCl concentration, the DACHPt was released in continuous manner from the metallosomes by the ligand exchange reaction, followed by release of the loaded macromolecules. The Metallosomes showed a prolonged blood circulation property and eventually selectively accumulated into tumor tissues in mice through the EPR effect due to the 100 nm size. Ultimately, fluorescent imaging of the tumor was successfully demonstrated along with an appreciable antitumor activity by DACHPt retained in the vesicular wall of the metallosomes.
The simple construction of the Metallosomes in water without use of organic solvents allows encapsulation of fragile bioactive compounds, which positions the Metallosomes as a promising platform for novel nanodevices capable of simultaneous diagnosis and treatment.

Particulate Au nanostructures such as one dimensional nanorods (1D NRs) and three dimensional nanospheres (3D NSs) have shown impressive potential as contrast agents for biological imaging due to their highly effective plasmonic interaction with light. However, there are some points to consider for each Au nanostructure. Although 1D NRs show a broad optical window of resonant wavelength across the near-infrared region, an alignment of light direction and polarization are necessary due to the anisotropic shape of the 1D NRs. The opposite is the case with 3D NSs. In order to achieve complementary optical properties of both 1D NRs and 3D NSs, we propose two-dimensional nanodisks (2D NDs) and top-down physical synthesis, in which Au is vacuum deposited onto a nanoimprinted polymer template. Nanoimprint lithography (NIL) used to define the diameter of the 2D NDs offered distinct benefits of nanoscale resolution, reliable pattern generation, and high throughput. In addition, by using Si nanocone array as a NIL mold and adjusting the time of O₂ plasma etch following the NIL, it was possible to vary the diameters of 20-nm-thick Au NDs from 65 nm to 165 nm to match their dimensional resonances for excitation wavelengths from visible to near-infrared. As well as the resonant wavelength, electromagnetic simulations demonstrated that the relative ratio of optical absorption and scattering in a total extinction of the Au ND could be also controlled by adjusting its diameter and thickness. More efficient light absorption and scattering by the 2D NDs compared to the 1D NRs and 3D NSs were successfully demonstrated by imaging the three kinds of Au nanostructures with two-photon excitation fluorescence microscopy and optical coherence tomography. Considering the present results, we expect the proposed 2D NDs to be used as highly sensitive and reliable contrast agents for in-vivo and in-vitro imaging.

Magnetic resonance imaging (MRI) is of one the most powerful imaging techniques in medical diagnostics. It can offer high spatial resolution while having unique soft tissue contrast. Unlike any other imaging tools such as X-ray imaging, MRI does not provide radiation and damage to the tissues. However, without the use of contrast agent, there is a lack of sensitivity and MRI cannot provide all the crucial details for accurate diagnosis. To overcome this challenge, contrast agents play an important role by changing the longitudinal (T1) and the transverse (T2) relaxation times of water protons.
Based on potential applications of gadolinium oxide (Gd2O3) nanoparticles, here we report synthesis of anisotropic, monodisperse and size-tunable gadolinium oxide nanostructures, capable of being applied in-vivo as enhanced T1 MRI contrast agents. Synthetic parameters, such as surfactant ratio and reaction time can be tailored to attain monodispersed nanocrystals with narrow size distribution. Additionally, as synthesized nanosized nanoparticles must undergo a surface modification in order to render the material water soluble and functional for any biological application. In this work, transferring organic phase nanoparticles into aqueous phase was done using three different polymers.
The MRI properties studies were fulfilled by varying different parameters, such as polymer molecular weight, hydrophilic surface coating and nanoparticle core size. The longitudinal relaxivity of PAMPS_LA coated gadolinium (Gd) nanoparticles resulted approximately 20 times higher than clinically approved Gd contrast agents. The polymer-coated gadolinium oxide nanoparticles are biocompatible based on our in vitro cytotoxicity study in which showed no significant effect on cell viability up to a concentration of 600 ?M of gadolinium. Owing to the large enhancement in relaxivity, we were able to use the nanoparticles as in vitro cellular MR labels; these showed high cellular uptake while increasing the signal intensity on the T1-weighted image which is very promising for cellular labelling. This study demonstrates that water-soluble biocompatible gadolinium oxide nanostructures are potential candidates as enhanced MRI contrast agents suitable for non-specific cell labeling, which makes the visualization of labeled cells in-vivo possible.

Since Tang and Cohen proposed the possibility of creating Electromagnetic fields with chirality larger than that of circularly polarized light much effort has been done to understand and utilize this phenomenon. In the context of Plamsonics it is shown that, due to the enhancement of the electric field, it is possible to create chiral evanescent fields with much higher chirality than that of circularly polarized light. Such enhanced chiral fields are highly desirable for multiple applications such as enantiomer sensing, enhanced light-matter interaction, etc. in the case of enhanced enantiomer sensing there are two major problems associated with the Plasmonic structures proposed so far. First, most of the optical activity regarding the chiral biomolecules take place in UV or higher frequency part of the visible spectrum. However, majority of the prototypical chiral Plasmonic antennas show a resonance at much higher wavelengths. Gammadions, helical antennas and G shaped wheels are some of the typical chiral antennas researchers have used. The problem of the Plasmonic resonances occurring far from the desired frequencies stems from the difficulty of fabricating small enough antennas while preserving their geometrical chirality. Second, due to the mainly dipolar nature of these resonances positive and negative evanescent chiral fields surround the antenna. This makes specific enhancement of one particular handedness of an enantiomer elusive. Here, we propose that using a cluster-based approach to creating the chiral hot spots can alleviate these problems. We numerically demonstrate that through a cluster of achiral Plasmonic nano antennas arranged in a chiral geometry one can create localized evanescent super chiral fields with a specific non-changing sign. Moreover, due to the feasibility of shifting the Plasmonic resonance of the individual components of the cluster to Blue or even Ultraviolet part of the spectrum, enhanced chiral light matter interaction in this range of the spectrum can be achieved.

Gold based nanoparticles with multifunctional properties have found increasing medical applications such as medical diagnostics, imaging, therapies, etc. One of the biggest challenges is the creation of the ability to enable the nanoparticles with both biocompatibility and bio-intervention capability for effective detection of biomolecular activities in bioassay or diagnostic processes. Recently, we have successfully synthesized magnetic nanoalloys such as NiFe and NiCo and use them as cores to create core@shell nanoparticles (e.g., NiFe@Au) with controllable size and composition by hydrothermal method, which could function as biocompatible and magnetically-separable nanoprobes in aqueous solutions. In addition to describing the structural characterization results, this report will discuss preliminary findings in an investigation of the bioconjugation, especially DNA or aptamer functionalized NiFe@Au nanoprobes, for the creation of interparticle “hot-spot” to enable surface enhanced Raman scattering (SERS) detection. The results have demonstrated the viability of the core-shell magnetic nanoparticles for SERS detection in aqueous solutions. Implications of these results to new strategies in medical diagnostics and imaging will also be discussed.

Currently there is pronounced curiosity in the development of nanoparticles that combine plasmonic assets with magnetic properties to achieve the ability of using the particles as a vehicle for simultaneous plasmonic monitoring and magnetic operations. These dual functional probes hold great promise as sensing probes for diagnostics and environmental applications such as immunomagnetic/chemo-magnetic separation under plasmonic imaging, dual mode imaging (MRI and plasmonic) and SERS sensing. Au has traditionally been included in fcc FePt as a dopant to lower the annealing temperature required for the L1₀ fct phase transition, however no significant work has been done to fully characterize their potential in magneto-plasmonic applications. With this in mind, we report the combination of magnetic and plasmonic components in single phase Fe_xPt_100-x-yAu_y ternary NPs, which are used as a new material for dual functional magnetic/plasmonic applications. In this work, we synthesized Fe_xPt_100-x-yAu_y spherical NPs which reveals the potential for dual application of plasmonic imaging and magnetic separation. Magnetic and plasmonic properties are found to be dependent on the final composition of the particles which were carefully studied in terms of the effect of shape, size and composition. Finally by carefully tailoring the various parameters we tried to optimize the best conditions for getting the desired nanoparticles. Our research leads to a deeper understanding of the critical role of physical and chemical properties (precise control of size, shape and composition) and their correlation to the usage of particles in biomedical applications.

Monolayer-protected gold nanoparticles have been shown to embed in the hydrophobic core of lipid bilayers in a manner that resembles the behavior of transmembrane proteins [1]. This fusion capability of the nanoparticles makes them a promising candidate for drug delivery and biological sensing applications. In recent work, we have established that stochastic lipid tail protrusions are responsible for inducing fusion of these nanoparticles with lipid bilayers [2]. Enhancing fusion probability would hence require both an understanding of factors that govern lipid protrusions and tuning the properties of ligands that coat these nanoparticles. Moreover, understanding the mechanisms that promote lipid protrusions would also provide physical insight into more fundamental biological processes such as vesicle fusion.
Using atomistic molecular dynamics simulations, we show that the relative thermodynamic likelihood of protrusion events is insensitive to lipid composition in single-component bilayers. Instead, the packing arrangement of surrounding lipids presents a steric barrier that kinetically limits the incidence of protrusion events. Through membrane surface analysis, we demonstrate that spontaneous protrusion events are facilitated by the presence of geometrical defects in the bilayer. We then utilize this physical understanding to tailor the nanoparticles' protective ligands in a manner that promotes fusogenic behavior. Though the ligand monolayer in recent works has mainly been composed of a binary mixture involving purely hydrophobic and anionic end-functionalized linear alkanethiols to match experimental work, we use simulations to identify new candidate ligands with higher fusion probability.
[1] R. C. Van Lehn et al., "Effect of particle diameter and surface composition on the spontaneous fusion of monolayer-protected gold nanoparticles with lipid bilayers", Nano Letters 13 (9), 4060-4067 (2013).
[2] R. C. Van Lehn et al., "Lipid tail protrusions mediate the insertion of nanoparticles into model cell membranes", Nature Communications, Accepted (2014).

As interests in the application of noble metal nanoparticles, especially gold nanoparticles, in medical theranostics rapidly expand in many cutting-edge areas, there is an increasing need for the development of effective strategies for the fabrication of nanomaterials and the detection of the biomolecules. One important strategy is to establish an effective coupling of the unique optical properties of gold-based nanoparticles and the functional properties for an enhanced detection or visualization. This report describes recent findings of an investigation using gold-based nanoparticles to construct a microfluidic platform for the detection of proteins or DNA in the biomolecular recognition process. In addition to establishing an effective protocol for building a well-defined gold-based nanoparticle array on a substrate, one important emphasis is the understanding of molecular and biomolecular interactions at the interfaces of the array in a solution. Atomic force microscopy, surface enhanced Raman scattering, and confocal Raman microscopy are discussed for the characterization and the detection of biomolecular activities such as protein recognition and DNA binding. Results involving gold nanoparticles and magnetic nanoparticles coated or decorated with gold shells will be discussed, along with their implication to biomedical imaging.

Leukemia is a disease caused by the malignant transformation of immature lymphoid cell, which has the ability to reproduce and form a clone of the parent cell; these cells are found blocked in a differentiation point. The leukemic cells from most patients with B-lineage acute lymphoblastic leukemia (ALL) appears to originate from normal B-lymphocyte precursors who co-express antigens such as CD10⁺ and CD19⁺ on their cell surfaces during the immatureness stages; while the mature cells don&’t express the CD10+. In this work, we studied CD10⁺ and CD19⁺ markers by using functionalized gold nanostars (NS) and Surface Enhanced Raman Spectroscopy (SERS). The NS were synthesized by wet chemical treatment (chemical reactions in solution). The nanomaterial&’s characterization was made through UV-Vis spectroscopy within the range from 400 to 900 nm with one nm of resolution. The morphology and size of the NS were determined by Transmission Electron Microscopy. The Raw spectra were recorded with a Reninshaw inVia Raman system, with a 785 nm laser source and we used 15 s of exposition time over the sample. All spectra were recorded in the range from 550 to 1700 cm^-1 and we recollected 50 Raman spectra per sample by mapping with 4 µm of distance between each recorded spectra. The NS were coated by pegylation with OPSS-PEG-NHS and mPEG-SH (CREATIVE PEGWORKS); the antibodies anti-CD10 and anti-CD19 (ABCAM) by chemical attachment of their heavy chains. The study was made with leukemic B lymphoblast from the SUP-B15 cell line (ATCC CRL-1929), the cells were cultured at 37°C in Iscove&’s modified Dulbecco&’s medium supplied with 4mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 0.05 mM 2-mercaptoethanol, 80%; fetal bovine serum, 20%. The NS functionalized were incubated with the B lymphoblast for 2 hours. The adhesion of the NS to the cell surface was confirmed by TEM. Three different treatments (cells, cells with nanoparticles and functionalized NS with cells) were analyzed by Raman spectroscopy. The ratios of some band intensities were analyzed, and some of them resulted relevant and corresponded to proteins, phospholipids, amino acids and polysaccharides. It showed important differences between CD10⁺ and C19⁺ markers in the peaks 632, 1003, 1047, 1110, 1231, 1452 and 1585 cm^-1. Characteristic patterns were observed when the principal components analysis were applied to the entire spectrum block (CD10 and CD19), and moreover, was possible to identify the presence of SERS in both groups. And thus generating new fields of study to find alternative therapeutic techniques to assist the medical community for the treatment of leukemia.

Engineering the plasmonic properties of noble metal nanostructures with facile control over their size, shape and materials combination holds great promise for cancer nanomedicine. Here, we demonstrate multifunctional plasmonic sphere-in-star (SIS) nanostructures with built-in gold core and internal Raman reporter (gold sphere-in-star) for simultaneous high-resolution Raman bioimaging and photothermal ablation therapy. Raman reporter encoded between the gold core and the gold star shell was subjected to enhanced electromagnetic field due to their plasmon hybridization, resulting in ultra-bright probes for non-invasive high resolution Raman bioimaging. The star geometry of the SIS nanostructures enables their strong absorption of light in the near infrared therapeutic window that allows to deploy them not only for Raman bioimaging but also for photothermal ablation therapy and image-guided therapy applications. In vitro targeting SIS nanostructures to SKBR3 breast cancer cells via biofunctionalization with the ErbB2-receptor targeting antibodies is demonstrated to significantly improve the cellular uptake of the nanostructures. Owing to their compact size (~40 nm), cell-specific uptake at minimally invasive dosages and enabling simultaneous bioimaging and photothermal therapy, SIS nanostructures can be ideal candidates for medical nanotheranostics such as spectrum-guided therapy applications and paves the way to design novel plasmonic-engineered nanomedicine with ideal optical properties with desired multifunctionality.

Different geometrical and physico-chemical configurations of iron oxide nanocubes (NCs) have been proposed as either probes for magnetic resonance imaging (MRI) with superior relaxometric properties or efficient heat generators for hyperthermia and tissue ablation. Here, individual and clustered NCs, with a characteristic edge length ranging from 11 to 28 nm, are synthesized via thermal decomposition methods with various combinations of iron precursors, surfactants, and solvents. Clustered NCs exhibit better magnetic properties than individual NCs, for both MRI relaxivity and specific absorption rate (SAR). In particular, 20 nm clustered NCs possess the highest transversal relaxivity, with a r₂ of 680.3 ± 60.3 mM^-1s^-1 at 1.41T, and magnetic heating efficiency, with a SAR of 113.8 ± 21.6 W/g at 512 KHz and 10 kA/m. As compared to individual NCs, the 20 nm clustered NCs manifest a higher magnetization saturation (86.6 vs 21.9 emu/g), a larger Fe³⁺/ Fe²⁺ ratio (34 vs 3%), and a more homogeneous crystalline structure, with one sole crystalline direction [311]. Wrapping NCs in serum albumin increases significantly their colloidal stability and returns an average hydrodynamic diameter of ~ 100 nm which is preserved up to 7 days in a physiological solution. In vitro viability tests on J-774 murine macrophages and U87-MG glioblastoma multiforme tumor cells demonstrate no significant toxicity up to NC concentrations of 100 ppm. This study suggests that 20 nm, albumin coated, clustered iron oxide NCs can simultaneously behave as superior MRI contrast agents and efficient heat generators for potential cancer theranosis.

X-ray photons generate photoelectrons and Auger electrons, which cause ionization of water and formation of reactive free radicals (mostly hydroxyl radicals). The free radicals diffuse through chain reactions in cells, and damage DNA in mitochondria and nuclei by extracting hydrogen atoms from ribose sugars, leading to cleavage of polynucleotide backbone. A challenge of X-ray radiation therapy is that high dose X-ray can damage normal cells and cause side effects due to its low tumor selectivity. Nanoparticles of gold, platinum and bismuth are proposed to enhance radiation therapy, but the measured effect of nanoparticles is negligible. This is likely due to the fact that these nanoparticles are attached on cell membrane, and X-ray generated free radicals have to diffuse into vicinity of DNA to cause damage. This abstract describes the use of a nanoparticle-based method to enhance X-ray radiation therapy by delivering radio-sensitizing gold nanoparticles in cancer cells. The nanoparticles are modified with cationic polyelectrolytes to allow internalization into cells. Upon X-ray irradiation of nanoparticles, more photoelectrons and Auger electrons are generated to cause water ionization, leading to formation of free radicals that damage DNA of cancer cells. The enhanced DNA damage is confirmed with HaloChip assay, the expression of DNA repair protein (γ-H2AX) and flow cytometry assay. X-ray dose required for DNA damage and cell killing is reduced by putting gold nanoparticles inside cancer cells. More cells can be killed due to combined effect of X-ray radiation and nanoparticles.

Recently, we reported our advancements with tantalum oxide nanoparticles for potential use as next generation of x-ray contrast media.^a Desirable properties of these materials that require further study are reduced viscosity and osmolality of concentrated particle solutions, and reduction of the total amount of Ta retained after administration. Here, we report on a family of new zwitterionic (carboxybetaine) siloxane polymer nanoparticle coatings, some of which showed dramatic reduction in viscosity of highly concentrated solutions of tantalum oxide particles (>250mgTa/mL), with exceedingly low tissue retention of injected tantalum. Subtle differences in the chemistry/coating structure were found to have a profound impact on both physico-chemical properties and excretion profiles.
^aBonitatibus, P., et al. ACS Nano2012, 6, 6650-6658.

Photothermal effect of polymer-coated magnetite Fe3O4 nanoparticle was investigated via near-infrared (NIR) laser at 785 nm for cancer therapy. For clinical applications, the tissue-depth dependence of the photothermal power was studied using various simulated body issues. These included Agar gel with cow blood. Three different NIR laser wavelengths were used in order to determine the critical penetration depths for hyperthermic ablation of cancer cells. The photothermal heating curves were obtained and cytotoxicity experiments were carried out for various Fe3O4 nanoparticles. The power and concentration dependences of different Fe3O4 nanoparticles were established from the heating curves. Also discussed is the operating photothermal mechanism of the Fe3O4 nanoparticles.

Hollow hydroxyapatite nanospheres are widely used in areas of orthopedic, dental surgery, drug delivery applications because of its unique properties related to good biocompatibility and excellent adsorption properties. The HA nanospheres are more effective in fighting infection by loading them with Silver nanoparticles. Hollow HA nanospheres have large specific surface area, good flowability and easily tune the size of the spheres. Several methods have been developed to prepare HA nanospheres with dimensions ranging from nano to micron size for use in sustained release drug delivery systems. In this study the spherical porous HA nanoparticles are fabricated by the modified SBF solution method using carbon nanosphere as template. The SBF solution method is particularly attractive because of its wide spread use, controlled morphology and the particles are synthesized in human body environment. As derived HA nanospheres were further decorated with Ag nanodots using AgNO₃ precursor. Hollow Ag-CaP nanospheres were obtained by removing carbon template at 300°C. XRD results confirm the presence of nanocrystalline CaP decorated with Ag. FTIR result shows distinct peak of PO₄^-2, CO₃^-2, -OH confirming presence of CaP in the sample. SEM result show spherical Ag decorated CaP nanospheres in the size range of 400 nm to 1 µm. Protein loading in Ag-CaP was assessed by cytorchrome C absorption. A high loading of 32.5 µg/mgNS was obtained for CaP nanosphere. The protein release study shows a steady release of 6.1 µgm/Day from High loading and discharge of protein is attributed to highly porous CaP surface. These results are superior to earlier studies on drug release from oxide nanoparticles. Our results indicate that Ag-CaP nanospheres can effectively be used for drug delivery application.

With the rising incidence of cancer, many forms of therapy have been developed to impede progression of cancer. Surgery, chemo- and radiation therapy continue to be the main treatments for cancer with limited success rates. Use of nanoparticles for anti-cancer drug delivery is a rapidly growing field and has received approval for clinical use. Unlike radiation/chemotherapy, drug-loaded liposomes can be specifically targeted to attack cancer cells. Advances in nanotechnology have permitted new possibilities for theranostics, which are defined as the combination of therapy and imaging within a single platform. We have been working to optimize the construction of theranostic liposomes loaded with gold nanoparticles (GNP) and methylene blue (MB). Encapsulating GNP and MB in targeted liposomes would allow for multimodal imaging and treatment of breast cancer. Folic acid can be conjugated to the drug loaded liposomes to target the overexpression of folate receptors on breast cancer cells. MB can be excited by light of appropriate wavelength to produce reactive oxygen species through photodynamic therapy, an emerging treatment modality that is used in the clinic. MB can also be used as an imaging agent for fluorescence-based optical imaging. Metallic nanoparticles like GNP are also used to enhance the contrast in computed tomography (CT). GNP can also absorb light at certain wavelengths, convert it into heat, and cause irreversible damage to the targeted cancer cells and this is referred to as photothermal therapy. We have been experimenting with several variables and tweaking our protocol to allow for optimal encapsulation in the targeted liposomes. We have also used imaging techniques such as confocal microscopy to test the specific uptake of these nano-constructs by breast cancer cells. Preliminary results on synthesis, optimization and characterization of these theranostic liposomes as well as imaging and treatment results against breast cancer cells will be presented.

Magnetic nanoparticles (NPs) are expected to be used in a wide variety of biomedical applications such as magnetic cell separation, drug delivery etc. However, many researchers have used large magnetic beads combined with fluorescence molecule to trace the NPs. The large size of NPs creates steric hindrance, which makes it difficult to interact with biological cells of smaller size while fluorescence molecules lose their activity with time. To overcome these problems, we synthesized FeCo@Ag magnetic core plasmonic shell NPs. FeCo has very high saturation magnetization and show response to external magnetic field while plasmonic Ag shell enables the optical tracing using plasmon scattering. The synthesis of FeCo@Ag is done by polyol one-pot synthesis method. The resulting NPs were characterized by TEM, UV-vis, XRD and SQUID magnetometer. The surfaces of as-synthesized NPs were modified with poly-L-lysine which contains thiol and lactose moieties (PLL-SH-Lac). The PLL-SH-Lac modified FeCo@Ag NPs were then attached on HepG2 cells which have lactose receptors. The PLL-SH-Lac FeCo@Ag NPs-cell attachment is carried out by incubation of HepG2 cells with PLL-SH-Lac FeCo@Ag NPs. The attachment was confirmed by dark field microscopy and confocal laser scanning microscopy. These NPs also possess strong potential to be used for the magnetic-cell separation of these cancerous cell lines from the normal cell lines.

Introduction: In dentistry, a successful root canal treatment depends on the complete removal of contaminants and on the adequate sealing. The most common sealants endodontic cements are based on Zinc Oxide-Eugenol whose antimicrobial effect is reduced, but there are other antimicrobial agents successfully used in different biomedical applications that have not been explored in the endodontic field. Objective: To obtain an antimicrobial root canal sealer adding micro or nanoparticles for the inhibition of Candida albicans and Enterococcus faecalis cultures and being noncytotoxic. Methods: The Fourier Transformed Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), or Transmission Electron Microscopy (TEM) characterization was performed to the antimicrobial microparticles (triclosan, iodoform, terbinafine and chitosan) and to silver nanoparticles. The Zinc Oxide-Eugenol (ZOE) root canal sealer were mixed with the micro or nanoparticles. The prepared experimental groups were: Group-1 (ZOE+terbinafine); Group-2 (ZOE+triclosan); Group-3 (ZOE+, iodoform); Group-4 (ZOE+ chitosan) and Group-5 (ZOE+AgNPs). Each group was evaluated: the antimicrobial activity on Candida albicans and Enterococcus faecalis cultures by the diffusion test (n=4); the cytotoxicity was evaluated on 3T3-NIH fibroblast cultures by the MTT test (n=8). The assays were repeated by triplicate and One-Way ANOVA (p<0.05) was applied to the results. Results: The functional groups of the antimicrobial particles analyzed by FT-IR resulted according to previous reports. The morphology of the microparticles (SEM), showed irregular particles with particle size in: terbinafine (5-10 mu;m ), triclosan (15-50 µm), iodoform(10-40 µm), chitosan (15-50 µm); while the TEM observations indicated that the silver nanoparticles are spherical in shape and 10-25 nm in size. The antimicrobial assay showed different length in the inhibition zone (mm) of each ZOE cement when modified with different particles; the higher ihibition of Candida albicans was in the group of ZOE+chitosan (p=0.0019), while for Enterococcus faecalis the higher inhibition zone was in the group ZOE+triclosan (p<0.05). The cytotxicity assay (p=0.001) in the group of ZOE with terbinafine inidicating less viability cell (20%), while all other goups shown no cytotoxic effec to the fibroblast cell line.
Conclusions: The root canal sealer added with chitosan microparticles presented the higher inhibition of Candida albicans, and the sealer cement with triclosan microparticles better inhibited Enterococcus faecalis cultures. Most of the evaluated groups results with no cytotoxic, so that, the experimental root canal sealer with triclosan, iodoform, chitosan or silver nanoparticles could be used in endodontic treatments in order to avoid the post-treatment infections. Acknowledgements: DGAPA-UNAM: PAPIIT-TA200414 and PAPIME-PE200214. For technical support to: Dra. Genoveva-Hernández Padroacute;n, Dra. Marina Vega and Mtro. Gerardo Fonseca.

During implant surgeries, antibacterial agents are needed to prevent bacterial infections, which can cause the formation of biofilms between implanted materials and tissue. Silver nanoparticles, which release silver ions, have been considered to be a promising antibacterial agent because of their broad antibacterial activity. Mussel adhesive proteins (MAPs) derived from marine mussels are bioadhesives that show strong adhesion and coating ability on various surfaces even in wet environment. Here, we proposed a novel surface-independent antibacterial coating biomaterial by incorporating the strong adhesion ability of recombinant mussel adhesive protein into a silver-binding peptide. This sticky recombinant fusion protein enabled the efficient coating on target surface and the easy generation of silver nanoparticles on the coated-surface. The synthesized silver nanoparticles showed excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria, and also revealed good cytocompatibility with mammalian cells. Moreover, the silver nanoparticles were fully synthesized on various surfaces including metal, plastic, and glass by a simple, surface-independent coating manner, and they were also successfully synthesized on a nanofiber surface fabricated by electrospinning of the fusion protein. Thus, this facile surface-independent silver nanoparticle-generating antibacterial coating biomaterial has great potential to be used for the prevention of bacterial infection in diverse biomedical fields.

Functionalized silver nanoparticles coated with graphene nanostructures were synthesized by laser irradiation of benzene-silver colloid. The functionalization of this Ag-Graphene nanocomposite with polyethylene glycol provides stabilization and higher solubility in aqueous solution. The Ag-Graphene nanocomposite was characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscope (FE-SEM) coupled with energy dispersive spectrometry (EDS), and Fourier transform IR spectroscopy (FT-IR). The antibacterial activity of Ag-Graphene was tested using Pseudomonas aeruginosa and Staphylococcus aureus as model strains of Gram-negative and Gram-positive bacteria, respectively. The Ag-Graphene nanocomposite solution exhibits strong antibacterial activity against both types of bacteria, as tested using Kirby-Bauer assay disk diffusion and Minimum Inhibitory Concentration (MIC) assay. By applying an external electric bias, a decrease in the bacterial inhibition time (less than 6 hrs) can be attained. The success of this method allows us to propose a mechanism which describes the bactericide effect in terms the ion transport in the solution when exposed to the bacteria. Taking advantage of the high biocompatibility of graphene and the antibactericidal activity of silver, the Ag-Graphene nanocomposite is a nontoxic biomaterial suitable for numerous applications, such as self-sterile textiles, biomedical devices, coatings, and cosmetics.

Core-shell nanoclusters have recently received considerable attention owing to their physical and chemical properties that are strongly dependent on the structure of the core, shell, and interface. The core-shell magnetic nanoclusters are of special interests since the heterogeneous nanostructures offer opportunities for developing devices and cluster-assembled materials with new functions for magnetic recording, bio, and medical applications. In fact, superparamagnetic nanoparticles with suitable biocompatible coatings have important implications in biology, biotechnology, and other biomedical disciplines¹.
Here, we report the synthesis and characterization of CoFe2O4/MnFe2O4 coated Au NPs that were prepared by reducing a gold (III) salt in the presence of respective magnetic NPs. Co and Mn NPs were prepared using standard co-precipitation methods, respectively. An Au layer was subsequently added using seed-mediated approach. Such Core-shell nanoclusters were characterized by Ultraviolet-visible (UV-vis) absorption spectroscopy, X-ray diffraction (XRD), Transmission electron microscopy (TEM) and High Resolution-Transmission electron microscopy (HRTEM). Line mapping EDS was employed for elemental composition of core and shell. Saturation magnetization of such clusters was also determined to be 78 emu/gm using Vibrating sample magnetometer. Such Au coated magnetic NPs were transferred into aqueous solution and then bio-functionalized with doxorubicin for drug release studies. The presence of Au shell on the magnetic core makes it possible to functionalize the NPs with dox molecules by exploiting Au-N chemistry². FTIR studies confirm the binding of dox onto the surface of nanoclusters. The biocompatible studies of these nanoclusters were investigated using MTT assay on L6 cell lines. They also exhibit very high T2 relaxivitiy, hence can be evaluated as an efficient contrast agent in MRI. Dox functionalized nanoclusters have proved to be effectively cytotoxic against Hep-2 Larynx carcinoma cell lines leaving normal and healthy cells. This is in good agreement with Wu et al³, who have synthesized highly biocompatible nanoclusters for Cancer Therapeutics.
References:
Sadighian S, Rostamizadeh K, Hosseini-Monfared H, Hamidi M. Colloids Surf B Biointerfaces. 2014 Mar 12;117C:406-413.
Sibnath Kayal and Raju Vijayaraghavan Ramanujan, J. Nanosci. Nanotechnol. 10, 1-13, 2010.
Ya-Na Wu, Dong-Hwang Chen, Xian-Yu Shi, Chiao-Ching Lian, Ting-Yu Wang, Chen-Sheng Yeh, Kyle R. Ratinac, Pall Thordarson, Filip Braet, Dar-Bin Shieh, Nanomedicine: Nanotechnology, Biology, and Medicine, 7 (2011) 420-427

A low-cost and direct green synthetic strategy was developed for the preparation of polymer composite spheres composed of silver nanoparticles (AgNPs) decorated on the polymer colloids in a raspberry-like fashion. The polymer colloids were employed as both the reductant and the substrate support. The environmentally benign reaction system consists of AgNO₃, polymer spheres and ethanol solvent, without any additional reducing agent or stabilizers. The obtained composite spheres are surprisingly stable. The size range of the formed AgNPs is from several to tens of nanometers, dependent on the reaction time, AgNO₃ concentration, and the amount of polymer spheres. A four-step mechanism was proposed for the formation of AgNPs-coated polymer spheres emphasizing the role of ethanol to saturate the external surface of the polymer spheres firstly. These composites exhibit an excellent antibacterial activity, dependent on both the concentration of the composite spheres and the size of AgNPs.

The temperature increase inside mesoporous silica nanoparticles that is induced by encapsulated smaller gold nanocrystals when exposed to light and by encapsulated superparamagnetic nanocrystals in an oscillating magnetic field is effective in actuating thermosensitive nanovalves for biomedical drug delivery applications. The release mechanism is caused by local internal heat, and not a bulk temperature increase.
The internal temperature is measured using a crystalline optical nanothermometer. The detection mechanism is based on the temperature-dependent intensity ratio of two luminescence bands in the upconversion emission spectrum of NaYF₄:Yb³⁺, Er³⁺. A facile stepwise phase transfer method to embed both a nanoheater and a nanothermometer in 100 nm mesoporous silica nanoparticles is developed. The magnetically induced heating inside the dual-core nanoparticles varies with different experimental conditions, including the magnetic field induction power, the exposure time to the magnetic field, and the magnetic nanocrystal size. The temperature increase of the immediate nano environment around the magnetic nanocrystals is monitored continuously during the magnetic oscillating field exposure. The interior of the nanoparticles becomes much hotter than the macroscopic solution and cools to the temperature of the ambient fluid on a time scale of seconds after the magnetic field is turned off.
A thermally sensitive gatekeeper attached to the mesoporous nanoparticle functions as a unique drug delivery system. A mesoporous silica framework surrounding a zinc-doped iron oxide nanocrystal was surface modified with pseudorotaxanes. Upon application of an oscillating field, internal heating causes the molecular machines to disassemble and allows the cargos (drugs) to be released. When breast cancer cells (MDA-MB-435) were treated with doxorubicin-loaded particles and exposed to an oscillating field, cell death was induced.
Light-operated nanovalves that control the pore openings of mesoporous silica nanoparticles containing gold nanoparticles cores were also synthesized. The nanoparticles consist of 20 nm gold cores inside ~150 nm mesoporous silica spherical shells. The nanovalves are comprised of cucurbit[6]uril rings encircling stalks that are attached to the ~2 nm pore openings. Plasmonic heating of the gold core raises the local temperature and decreases the ring-stalk binding constant, thereby unblocking the pore and releasing the cargo molecules that were preloaded inside. Bulk heating of the suspended particles to 60 ⁰C is required to release the cargo, but no bulk temperature change was observed in the plasmonic heating release experiment. These light- or magnetically-stimulated, thermally activated mechanized nanoparticles demonstrate new systems with potential utility for non-invasive, externally controlled, on-command drug delivery with cancer-killing properties.

We demonstrate the use of laser-triggered release of DNA from gold nanorods (NRs) to switch on and off blood clotting. The NRs can be excited by laser irradiation at their longitudinal surface plasmon resonance (SPR), which heats the NRs and releases their payloads. Because the SPR can be tuned throughout the NIR with NR size and shape, selective release of multiple species is possible. We utilize this property of NRs to release a DNA aptamer and its complement in a mutually exclusive manner to control the activity of thrombin, the central protein in the blood coagulation cascade. One NR is loaded with the thrombin binding aptamer (TBA), so triggering of its release by one excitation wavelength inhibits thrombin and stops blood clotting. Another NR is loaded with its complement, and triggering its release by excitation at a second wavelength inhibits the TBA and restores blood clotting. In order to obtain high enough DNA loading on the NR that can be used for controlling blood coagulation, we use protein coronas because they can hold large quantities of small molecules and DNA at a capacity higher than what is achievable by covalent attachment strategies. We characterize the loading of the protein corona and its release properties to enable optimization of DNA and drug loading.

Transdermal delivery is an attractive method for drug/protein delivery, even though the stratum corneum is a major barrier of their translocation into the skin. To achieve protein delivery through the stratum corneum, we examined thermal ablation of the stratum corneum by photothermal effect of gold nanorods.
We first casted gold nanorods, acting as a heating device in response to near-infrared light irradiation, onto the skin surface. After applying an aqueous solution of ovalbumin to the skin, the skin was irradiated by near-infrared laser light. Irradiation of the skin with a continuous-wave laser increased the skin temperature and enhanced the permeability of ovalbumin through the stratum corneum. Inflammation cells were also observed to migrate to the heated area of the skin and HSP70 was induced. In contrast, skin irradiation with a pulsed-laser enhanced the permeability of the stratum corneum without causing an increase in skin temperature and inflammation. The pulsed-laser irradiation intensively heats the gold nanorods, but immediately change the rod shape to spherical form, resulting in reduction of the photothermal effects. Such transient photothermal effects in a localized area could enhance the permeability of ovalbumin through the stratum corneum by selectively ablating the stratum corneum.
Thus, the physiological response of skin was dependent on the type of laser light used. It will be an important foundation on which to develop transdermal protein delivery and vaccination combined with external stimuli.

Breast cancer is the second leading cause of cancer related deaths in women. The majority of those deaths are due to triple negative breast cancer (TNBC), for which all current targeted therapies are rendered useless. Consequently, we have been exploring the use of silver nanoparticles (AgNPs) for the treatment of TNBC. AgNPs have shown anti-cancer efficacy in vitro and in vivo against leukemia, glioblastoma, and colon cancer. In addition, AgNPs also possess anti-bacterial and anti-inflammatory properties which are currently being exploited in the clinic as wound dressings and as coatings for neurosurgical shunts. We utilized transmission electron microscopy, dynamic light scattering, and nanoparticle tracking analysis to aid in the characterization of our lyophilized AgNPs stabilized with polyvinylpyrrolidone (PVP) as the capping agent. Using this combinatorial analysis, we showed our AgNPs to be approximately 108 nm in size with a zeta potential of -36 mV which confers coloidal stability. To assess the cytotoxic properties of our AgNPs, viability was determined via MTT conversion, and short term proliferation was assessed via BrdU incorporation. Using three TNBC cell lines and three non-tumorigenic cell lines, we have demonstrated a 10 fold decrease in viability for TNBC cells treated with various concentrations of AgNPs (1-100 mu;g/ml) compared to non-tumorigenic breast cells. Significantly decreased short term proliferation was also shown compared to luminal A breast cancer cells and non-tumorigenic breast cells. AgNPs inhibit migration of TNBC cells while the growth of non-tumorigenic breast cells are uninhibited as demonstrated via a scratch assays. This data indicates that AgNPs may be beneficial for preventing local recurrence after breast conserving surgery. Our AgNPs also demonstrated minimal cytotoxicity against cells derived from the liver, kidney, and monocyte lineages. In addition to selective cytotoxicity against TNBC cell lines, synergy between AgNPs and ionizing radiation was observed at low concentrations of AgNPs (1 µg/ml) via clonogenic assays in two different TNBC cell lines. We have demonstrated that AgNPs deplete glutathione, a glutamate-cysteine-glycine tripeptide necessary for mitigating damage induced by cancer drugs, radiation, and toxins. Supplementation with glutathione prior to AgNP exposure significantly decreased the damage induced by AgNPs. However, the depletion of glutathione with buthionine sulfoximine largely increased the cytotoxicity induced by AgNPs. AgNPs also induce DNA double strand breaks which were additively increased when combined with ionizing radiation. An in vivo study showed that AgNPs slow TNBC xenograft growth and enhance radiotherapy. Our current study indicates that AgNPs have significant anti-cancer activity toward TNBC cells in vitro and in vivo, and point to potential vulnerabilities in TNBC cells that could be exploited for the development of new therapeutic agents.

Gene therapy, which uses exogenous genes to perform therapeutic functions, offers highly promising treatments for cancer. The therapeutic genes can be delivered to replace defective ones which cause diseases or silence the expression of mutated genes. Although viral vectors have been proven efficient and stable delivery vehicles, safety concerns over viral vectors make non-viral vectors preferable delivery vehicles. Nanotechnology now offers unprecedented opportunities to explore potential non-viral vectors. A variety of nanoparticle (NP) -based gene delivery vehicles have emerged, using cationic polymers such as polyethyleneimine and chitosan. Inorganic NPs such as gold NPs and silica NPs have also shown promises for gene delivery due to their good biocompatibility, facile functionalization and capability for targeted delivery and controlled release. Meanwhile, integrating multiple nanocomponents into a single nanostructure can provide both diagnostic and therapeutic functions for cancer. In the present study, we synthesized hybrid NPs with an Au-Ag bimetallic core and a folic acid-chitosan shell (Au-Ag@CS-FA), which not only give targeting and imaging functions but also possess good DNA binding ability. The synthesis of Au-Ag NPs was performed through successive layer-by-layer deposition of Au and Ag precursors with FA-conjugated CS molecules acting as a reductant and also as a structure-directing agent for forming highly branched Au-Ag NPs. Tests showed that sufficient FA molecules were conjugated to Au-Ag NPs, which would provide strong targeting ability for folate receptor overexpressed cancer cells. Highly branched Au-Ag core could provide high surface-enhanced Raman scattering, which would be used for cancer cell sensing and imaging. The cationic polyelectrolyte nature of the CS-FA polymer shell provided strong electrostatic interaction with negatively charged DNA and protected it from nuclease degradation. The plasmid DNA encoding enhanced green fluorescent protein with a CMV promoter (pEGFP) was used as report gene. Nanoparticle/pEGFP complexes with different molar ratios were prepared to investigate the effect of incubation time and pH values on the binding ability of nanoparticles with pEGFP. Results showed Au-Ag@CS-FA NPs were stable and had good biocompatibility and strong DNA binding ability in the acidic environment.

The natural defenses of biological systems for exogenous oligonucleotides, such as synthetic antisense DNA and siRNA, present many challenges for the delivery of nucleic acids in an efficient, non-toxic, and non-immunogenic fashion. Indeed, because nucleic acids are negatively charged and prone to enzymatic degradation, researchers have historically relied on transfection agents such as cationic polymers, liposomes, and modified viruses to facilitate cellular entry and protect delivered biomolecules from degradation. However, each of these platforms is subject to several drawbacks, which include toxicity at high concentrations, the requirement of specialty nucleic acids to enhance stability, and severe immunogenicity.
Spherical nucleic acid (SNA) gold nanoparticle conjugates (inorganic nanoparticle cores functionalized with a spherical shell of densely organized, highly oriented nucleic acids) pose one possible solution for circumventing these problems in the context of both antisense and RNAi pathways. Remarkably, these highly negatively charged SNA structures do not require cationic transfection agents or additional particle surface modifications and naturally enter all cell lines tested to date (over 50, including primary cells). Further work has shown the cellular uptake of these particles to be dependent upon DNA surface density: higher densities lead to higher levels of particle uptake. The high-density polyvalent nucleic acid surface layer is believed to recruit scavenger receptors from the cells that facilitate endocytosis. Moreover, the ion cloud associated with the high-density oligonucleotide shell, combined with steric inhibition at the surface of the particles, inhibits enzymatic nucleic acid degradation and activation of the enzymes that trigger the innate immune response of certain cells. In this talk, methods to synthesize such structures and novel applications that take advantage of the interesting properties unique to spherical and other forms of three-dimensional nucleic acids will be described.

One of the important trends of next-generation nanomedicine is theranostics that is defined by the combination of therapeutics and diagnostics on a single platform. Magnetic nanoparticles are among one of the most essential platforms for targeted imaging, therapy, and simultaneous monitoring of therapeutic efficacy. In this talk, I will discuss magnetic nanoparticles as a core platform material for theranostics and add a variety of functionalities such as drug, targeting moiety, and gene to enhance their performance. Their unique utilization in highly accurate dual-modal MR imaging, therapeutic hyperthermia of cancer cells, controlled drug release, gene delivery, and molecular level cell signaling and cell fate control will be discussed.

Nanoparticle-based diagnosis-therapy integrative systems represent an emerging approach to cancer treatment. However, the diagnostic sensitivity, treatment efficacy, and bioavailability of nanoparticles as well as the heterogeneity and drug resistance of tumours pose tremendous challenges for clinical implementation. Herein we report on the fabrication of tumor pH-sensitive nanoparticle/polymer hybrids composed of self-assembled noble metal (such as Pt and Au) or metal oxide nanoparticles and pH-responsive polymeric ligands. These nanohybrids can readily target tumors via surface-charge switching triggered by the acidic tumor microenvironment, and are further disassembled into a highly active state in acidic subcellular compartments that “turn on” their imaging and therapeutic activity. We successfully visualized small tumors implanted in mice via unique pH-responsive imaging, demonstrating early-stage diagnosis of tumors without using any targeting agents. Furthermore tumor pH-triggered disassembly of these nanohybrids enabled pH-dependent cancer therapy. In particular, we demonstrate the superior therapeutic efficacy of these nanohybrids in highly heterogeneous drug-resistant tumors, showing a great potential for clinical applications.
References
D. Ling et al. J. Am. Chem. Soc., 2014, 136, 5647-5655
D. Ling et al. Nature Materials2014, 13, 122-124
D. Ling et al. Nano Today 2014, in press

A family of small (~3nm) water-soluble gold nanoparticles (AuNP) based on triethylene glycol ligands and containing interfacial "clickable" reaction moieties was synthesized. These include AuNPs that incorporate clickable interfacial maleimide, an azide or a strained alkyne, and a substituted triphenylphosphine. The Maleimide-AuNP can undergo an interfacial Michael addition reaction, the azide- or strained alkyne-AuNP can participate in interfacial strain-promoted alkyne-azide cycloaddition (I-SPAAC) reaction with the appropriate reactive partner, whereas the substituted triphenylphosphine-AuNP can undergo a Staudinger ligation. Each of these clickable nanoparticles were completely characterized and the number of interfacial clickable functionalities was estimated with good precision using thermogravimetric analysis, multinuclear NMR spectroscopy, transmission electron microscopy and X-ray photoelectron spectroscopy. The reactivity of the clickable AuNP was then tested and monitored using model molecules. Each approach leads to covalently modified AuNP, with high efficiency and high yields, and thus represents a convenient and desirable way to introduce biomolecules, drugs and contrast agents onto the AuNP surface. The modified-AuNPs created through this technology were finally tested in vivo showing promising results. In this presentation aspects of the synthesis, characterization and reactivity of the clickable-AuNP and results of their use in imaging and bioconjugation will be presented.

A variety of organic and inorganic materials have been utilized to generate nanoparticles
for drug delivery applications, including polymeric nanoparticles, dendrimers, nanoshells,
liposomes, nucleic acid based nanoparticles, magnetic nanoparticles, and virus
nanoparticles. Most commonly used systems are polymeric nanoparticles and liposomes.
Controlled release polymer technology impacts every branch of medicine. Polymeric
nanoparticles can deliver drugs in the optimum dosage over time, thus increasing the
efficacy of the drug, maximizing patient compliance and enhancing the ability to use
highly toxic, poorly soluble, or relatively unstable drugs, and can also be used to codeliver
two or more drugs for combination therapy. The surface engineering of these
nanoparticles may yield them “stealth” to prolong their residence in blood and the
functionalization of these particles with targeting ligands can differentially target their
delivery or uptake by a subset of cells, further increasing their specificity and efficacy.
The successful clinical translation of therapeutic nanoparticles requires optimization of
many distinct parameters including: variation in the composition of the carrier system,
drug loading efficiency, surface hydrophilicity, surface charge, particle size, density of
possible ligands for targeting, etc., resulting in potential variables for optimization which
is impractical to achieve using a low throughput approach. Combinatorial approaches
precisely engineer nanoparticles and screen multiple nanoparticle characteristics
simultaneously with the goal of identifying formulations with the desired physical and
biochemical properties for each specific application. This review demonstrates our efforts
in the design and optimization of polymeric nanoparticles for medical applications, which
formed the foundation for the clinical translation of the first-in-human targeted and
controlled-release nanoparticles, BIND-014 and SEL-068.

Computed tomography (CT) is an important clinical diagnostic imaging modality which enables three-dimensional anatomical imaging at high spatial resolution, but requires the administration of an X-ray contrast agent to distinguish tissues with similar or low X-ray attenuation. Over the last decade, gold nanoparticles (Au NPs) have gained attention as a potential X-ray contrast agent due to exhibiting a high X-ray attenuation, low cytotoxicity, and facile synthesis and surface functionalization enabling colloidal stability and targeted delivery. Potential applications of Au NPs in diagnostic X-ray imaging include blood pool imaging, passive targeting, and active targeting, where actively targeted Au NPs could enable molecular imaging capabilities with CT. The current state of knowledge for Au NP X-ray contrast agents will be reviewed within a paradigm of key structure-property-function relationships in order to provide guidance in the design of Au NP contrast agents. Au NPs must be designed to meet the necessary functional requirements for a contrast agent in a particular clinical or preclinical application. These functional requirements include delivery to the site of interest, non-toxicity during delivery and clearance, targeting or localization at the site of interest (e.g., blood, tumor, microcalcification, etc.), and contrast-enhancement for the site of interest compared to surrounding tissues. Importantly, design is achieved by strategically controlling structural characteristics (e.g., composition, mass concentration, size, shape, and surface functionalization) for optimized properties and functional performance. Examples from the literature will be used to highlight current design tradeoffs that exist between different functional requirements. Novel applications will also be introduced, including targeted labeling of breast microcalcifications for improved sensitivity and specificity during mammographic screening of breast cancer, and spectral (color) CT using multiple contrast agents.

Nanomaterials (NMs) have attracted much attention as probes for tumor imaging due to their unique physical properties as well as the ease of surface functionalization. Radionuclide imaging has an important role in cancer diagnosis due to its unique advantages like high sensitivity and the ability to conduct quantitative analysis of the whole-body images. We directly doped ⁶⁴Cu into quantum dots (QDs) via cation exchange reaction. These self-illuminated ⁶⁴Cu doped QDs show greatly enhanced Cerenkov resonance energy transfer efficiency and has been successfully applied for in vivo luminescence and PET (positron emission tomography) imaging. We also integrated ⁶⁴Cu onto polyethylene glycol stablized Au NMs via chemical reduction. Our ⁶⁴Cu integrated NMs are proved to be radiochemically stable and can provide the accurate distribution of Au NMs via non-invasive PET imaging. Further study on ⁶⁴Cu integrated arginine-glycine-aspartic acid (RGD) peptide modified Au nanorods (NRs) showes these NRs not only have high tumor targeting ability but also could be successfully used for PET image guided photothermal therapy. These intrinsically radioactive NMs are emerging probes for theranostic applications.

Single imaging tools (MRI, CT, ultrasound, optical imaging, etc.) have limitations that sensitivity, resolution, tissue penetration, etc.. So, various types of multimodal nanoplatforms have been developed to come over the shortages of single modality. Here, we present that a multimodal imaging nanoprobe emitting radiation, SERS and Fluorescence signal (SERF-AuNSs) simultaneously. We used DTTC NIR dye which simultaneously irradiates fluorescence and SERS signal, and star-shaped gold nanoparticles which have the strong plasmonic extinction in NIR region and rough and sharp morphology called as ‘hot spot&’, which can afford to generate SERS (The enhancement factor is almost 10⁸). AuNSs were synthesized by PVP method efficiently, and the functionalities corresponding to a application were invested to surface and the inside of AuNSs. The shapes and physical properties of nanoparticles at each step were characterized by UV-Vis spectroscopy, TEM, ELS, and their optical properties including SERS and fluorescence were analysed by photoluminescence (PL) spectroscopy and confocal Raman spectroscopy (Figure 1a, 1b and 1c). For the triplmodal imaging, RSF-AuNSs were labelled with ¹²³I radionuclide and they were injected into mice through tail vein. We successfully obtained multimodal images using IVIS for NIR fluorescence imaging and SPECT/CT for radioimaging (Figure 1d and 1e) Furthermore, RSF-AuNSs were applied to the active tumor targeting and multimodal imaging by conjugating cyclic RGD.

Gold nanostructures provide a multifucntional platform for an array of applications in biomedicine, including cancer diagnosis and treatment. In this talk, I will discuss some new developments in engineering the properties of gold nanostructures for biomedial applications. Examples include 1) the development of a method for precisely controlling the sizes from 5 to 150 nm for gold nanoparticles with a truly spherical shape; 2) the development of a method for incorporating radioactive Au-198 into the lattice of gold nanostructures for both radioactive and Cerenkov luminescence imaging; and 3) the development of various methods for controlling the shape, structure, and plasmonic properties of gold nanostructures for photoacoustic imaging and photothermal cancer treatment. I will also discuss some future directions and issues in pushing this class of nanomaterials toward clinical applications.

Fabrication of nanoparticles to sustain higher electromagnetic field enhancement for surface enhanced Raman scattering (SERS) spectroscopy has become a field of interest mainly in analytical and biomedical-related research areas. SERS relies on the highly localized confinement of the electromagnetic energy within the narrow gap between plasmonic nanoparticles or at the sharp edges or tips of isolated nanoparticles. Based on this fact, many attempts in designing assembled nanostructures for improved SERS properties can be found in the literature.
Herein, we report the development of water-based, physiologically stable, regiospecific core-satellite nano-assemblies as excellent SERS probes. In this system, gold nanostars with multiple sharp tips, which produce SERS enhancement factors equal to 10⁹, were used as the core particle onto which spherical satellite nanoparticles were assembled. The assembled structures are here envisioned to yield a high SERS enhancement per nanoparticle due the possibility of converting each tip into a SERS hot spot. Our assembly scheme involves nanoparticles capped with small, rigid SERS active organic molecules, and a conjugation linker chemistry that creates narrow gaps between the nanoparticles via covalent linking. The assembled nanostructures are capped with polyethylene glycol to provide stability in physiological conditions. Transmission electron micrographs show the core-satellite structures with 1-2 nm interparticle gap with a preferential assembly of the spheres at the tips of the nanostars, which are confirmed by changes in the SERS spectra. Taken together, these results demonstrate a great potential for our core-satellite nanoparticle assembly particularly as substrates in SERS-based bio imaging and bioanalytical applications.

Computed tomography (CT) is an important tool in clinical diagnostic imaging enabling three-dimensional anatomic imaging at high spatial resolution with short scan times. However, X-ray attenuation differences in physiological fluids and soft tissues are relatively small, requiring the use of contrast agents to achieve sufficient imaging contrast. Recent advances in energy-sensitive X-ray detectors have made spectral (color) CT commercially feasible by unmixing the energy-dependent attenuation profile of different materials and will potentially enable molecular imaging in CT. In order to leverage these capabilities for diagnostic imaging, we are developing a spectral library of nanoparticle contrast agents with K-shell absorption edges spaced at least 10 keV apart. The objective of this study was to demonstrate the ability of spectral CT to simultaneously detect up to three different contrast agents and unmix their signals to create color images. Gadolinium oxide (Gd), hafnium oxide (Hf) and gold (Au) were chosen due to exhibiting exhibiting K-edges spaced 10-20 keV apart. Core-shell nanoparticles of each composition were synthesized by various methods to have a core diameter of 15-20 nm and were coated with a silica shell 2-4 nm in thickness to create a common platform for surface functionalization. The contrast agents were imaged in a soft tissue equivalent phantom using both source-side and detector-side methods for spectral CT imaging. The source-side approach utilized monochromatic synchrotron radiation at the Argonne National Laboratory which, while not clinically applicable, served as a gold standard due to providing the highest spectral resolution. The detector-side approach utilized a photon-counting detector in an advanced preclinical system. The nanoparticles designed for this study have broad appplications in biomedical imaging due to their modular assembly, potential for enabling multi-modal detection, and surface functionalization with biomolecules (e.g., antibodies, peptides or enzymes) for active targeting.

We recently discovered ‘porphysomes&’, the first all-organic nanoparticles with intrinsic multimodal photonic properties. They are self-assembled from porphyrin-lipid building blocks to form liposome-like bilayer vesicle (~100 nm diameter). The very high porphyrin packing density (>80,000 per particle) results in both ‘super&’-absorption and structure-dependent ‘super&’-quenching, which, in turn, converts light energy to heat with extremely high efficiency, giving them ideal photothermal and photoacoustic properties that are unprecedented for organic nanoparticles. Upon porphysome nanostructure dissociation, fluorescence of free porphyrins is restored to enable low background fluorescence imaging. In addition, metal ions (e.g., radioactive copper-64) can be directly incorporated into the porphyrin building blocks of the preformed porphysomes thus unlocking their potential for PET, MRI and radiation therapy. As a result of their organic nature, porphysomes were biodegradable in vivo and induced minimal acute toxicity in mice with high intravenous doses. In a similar manner to liposomes, porphysomes can be easily scaled up via commercial extrusion techniques and the large aqueous core of porphysomes could be passively or actively loaded with drugs, opening up a new avenue for image-guided drug delivery. By changing the way porphyrin-lipid assembles, we developed ultra small high-density lipoprotein (HDL)-like porphyrin nanoparticles (<20nm), trimodal (US/photoacoustic/fluorescence) porphyrin shell microbubbles (~2um), confocal microscopy-controlled porphyrin microreactors (~100um), and hybrid porphyrin-gold nanoparticles, expanding the purview of porphyrin nanophotonics. Compared with classical “all-in-one” nanoparticles containing many functional modules, the simple yet “one-for-all” nature of porphysomes represents a novel approach to the design of multifunctional nanoparticle and confers high potential for clinical translation.

Rapid diagnostics are needed to detect dangerous human viruses that are either geographically endemic or introduced through potential bioterror attacks. Some human viruses, including dengue fever virus, have multiple serotypes. Infection with one dengue serotype does not confer immunity against the other serotypes, and in fact, secondary infections from different serotypes of dengue are correlated with severe and even fatal symptoms. Current technology in rapid diagnostic devices does not distinguish among viral serotypes, and this gap in technology allows for potentially avoidable epidemics. Thus, the ability to rapidly identify viral serotypes can serve as an early warning to alert public health officials. Dengue is a positive sense RNA flavivirus that is estimated to affect 390 million people each year. Symptoms can range from flu-like symptoms to fatal dengue shock syndrome. To aid in predicting the occurrence of patients with severe dengue symptoms, we have developed a point-of-care (POC) device that can distinguish among different serotypes of dengue. When coupled with a mobile phone reader, the device provides real-time geographic monitoring capabilities. Our POC device applies lateral flow chromatography technology, using anti-dengue NS1 antibodies and gold nanoparticles. To increase sensitivity of the device, we compared three types of surface chemistries to optimize antibody conjugation to the nanoparticle surface: thiol binding, electrostatic, and commercial covalent (proprietary) binding. Conjugation via thiol and electrostatic binding led to higher limits of detection in lateral flow tests than commercial covalent binding, as analyzed by ImageJ software. For thiol conjugation, antibodies were conjugated to gold nanoparticles via heterbifunctional molecule comprised of a dithiol moiety on one end to bind to the gold nanoparticle surface, and a hydrazide on the other end to bind to the Fc region of antibodies. Antibody-conjugated nanoparticles were characterized by UV Vis, DLS, gel electrophoresis, and ultimately, lateral flow tests. Lateral flow tests were validated against samples of a dengue marker, dengue NS1 protein, which is typically circulating in the blood of infected patients in early stages of dengue. Samples of dengue NS1 protein were developed in the supernatants of mammalian Vero cells that were individually infected with dengue serotypes 1-4. This POC device is affordable, and does not require special storage conditions, handling of special reagents, or trained personnel for use in the field.

Medical countermeasures surveillance and reporting during and after a public health emergency event require sensitive and specific detection/diagnostic methods and devices. We are designing, building, and testing a rapid, multiplexed, mobile phone-enabled diagnostic device to detect dengue virus and Ebola virus, and yellow fever virus. The goal is to deliver a device that will permit screening for multiple pathogen markers without the need for refrigeration, specialized training, specialized equipment or chemicals. Mobile phone technology is used to analyze the lateral flow data, quantify the results, and upload the results for real time epidemiology.
The device is based on lateral flow chromatography, an established technology. Current multiplexing permits assay for up to eight pathogen markers concurrently using one hundred microliters of sample. Monoclonal antibodies have been screened using flow cytometry and lateral flow chromatography to define functional pairs when conjugated to gold nanoparticles and bound to nitrocellulose paper. Nanoparticle surface chemistries are being evaluated to identify low cost approaches to prepare conjugated nanoparticles. A mobile phone app has been coded to record the image of the multiplexed diagnostic, correct the image for user photography errors, quantify the signal intensities, and upload data to a server, with GIS.
A prototype device that detects and distinguishes the four serotypes of dengue virus, dengue IgG/IgM, Ebola glycoprotein, and yellow fever NS1protein has been built and tested. Initial specificity and sensitivity tests using laboratory proteins and human patient serum samples are favorable. A multiplexed rapid lateral flow diagnostic for field use detects Category A pathogens and uploads data for real time epidemiology.

Biomolecule-nanoparticles conjugates is a topic of intense and growing interest for extending the applications of nanomaterials in biomedicine. Despite the recent advances, the biomedical applications of these materials are still limited, among other factors, by the low efficiency of functionalization, low stability and high toxicity. Overcoming these obstacles requires a complete understanding of the interactions between nanomaterials and biomolecules. Here, we present the development of jacalin-conjugated gold nanoparticles (AuNPs/jacalin) for leukemia cells detection via impedimetric measurements. Jacalin is a lectin that may specifically recognize a tumor-associated disaccharide that is overexpressed in most types of human cancers. AuNPs were synthesized in presence of poly(amido amine) generation 4 (PAMAM G4) and conjugated with a jacalin targeted with the fluorescein isothiocyanate (FITC) Typical AuNPs/Jacalin conjugates are shown in figure 1a. The AuNPs/jacalin formation is driven by an entropic process with good affinity, as revealed by isothermal titration calorimetry and quenching fluorescence measurements. Moreover, in vitro tests revealed that the AuNPs/jacalin-FITC complexes presented higher affinity against K 562 leukemia cells compared to normal ones (figure 1b). The nanoconjugates were successfully immobilized on specific electrodes for impedimetric detection of THP-1 leukemia cells, as shown in figure 2. Our findings are relevant for extending the understanding of the interactions between nanomaterials and biomolecules besides of their applications in biomedicine, especially for cancer cells detection.

Detection of rare cancer cells in human blood has important implications for detecting metastasis at early stage. However, it is very difficult to capture and detect these cells because of the background blood cells that are billions time higher in concentration than the cancer cells. We report a simple and highly sensitive detection method based on the dual magnetic-optical properties of iron oxide-gold core-shell nanoparticles. Anisotropic iron oxide-gold core-shell nanoparticles were prepared using a seed-mediated growth method. The hybrid nanoparticles were coated with QSY21 Raman reporters and linked with antibodies against epithelial cell adhesion molecules and human epidermal growth factor receptors to form magnetic and surface enhanced Raman scattering (SERS)-active nanoprobes targeting breast cancer. Using the dual functional nanoprobes in conjunction with a capillary-based flow system, we demonstrated that breast cancer cells spiked in human whole blood could be captured with high efficiency (about 90%) via magnetic separation, followed by on-line SERS detection with the sensitivity down to 1-2 cells/mL blood. The use of magnetic-metal nanoparticles bridged the gap of current rare cell enrichment and detection methods, making a huge step forward in developing a simple, rapid, quantitative and ultrasensitive technique for the detection of circulating tumor cells in blood of cancer patients.

Fluorescent imaging in the second near-infrared window (NIR-II, 1.0~1.4 mu;m) is appealing due to minimal autofluorescence and negligible tissue scattering in this region, affording maximal penetration depth for deep tissue imaging with high feature fidelity. Simulations and modeling studies suggested that fluorophores with emission in the 1000-1320 nm NIR-II region could significantly improve signal-to-noise ratio compared to those emitting at 650-950 nm (NIR-I). Recent efforts have been devoted to identifying NIR-II emitting agents for in vivo imaging applications. Quantum dots (QDs) such as PbSe, PbS, and CdHgTe with NIR emission have been successfully developed. However, the highly toxic nature of Pb, Cd and Hg is of concern for in vivo applications. Therefore, highly biocompatible NIR-II fluorescent probes that do not contain Cd, Pd or Hg will facilitate biological imaging in this beneficial spectral region. Herein, we first reported a new type of NIR QDs, Ag₂S QDs, with emission in the NIR-II region.
1) Highly selective in vitro targeting and imaging of different cell lines were achieved using biocompatible NIR-II Ag₂S QDs with different targeting ligand.
2) In vivo imaging of early-stage tumor in mice with Ag₂S QDs was also achieved. Video-rate dynamic contrast-enhanced imaging revealed deep inner organs and tumor in mice.
3) In vivo real-time visualization of lymphatic structures, blood flow, and angiogenesis mediated by a subcutaneous xenograft 4T1 mammary tumor utilizing Ag₂S QDs.
4) PEGylated-Ag₂S QDs are mainly accumulated in the reticuloendothelial system (RES) including liver and spleen after intravenous administration and can be gradually cleared, mostly by fecal excretion, without appreciable toxicity to the treated mice over a period of 2 months as evidenced by blood biochemistry, hematological analysis and histological examinations.
1. Du, Y.; Xu, B.; Fu, T.; Cai, M.; Li, F.; Zhang, Y.; Wang, Q. J. Am. Chem. Soc. 2010, 132, 1470-1471.
2. Zhang, Y.; Zhang, Y.; Hong, G.; Chen, G.; Li, F.; Dai, H.; Wang, Q. ACS Nano2012, 6, 3695-3702.
3. Hong, G.; Robinson, J. T.; Zhang, Y.; Diao, S.; Antaris, A. L.; Wang, Q.; Dai, H. Angew. Chem. Int. Ed. 2012, 51, 9818-9821.
4. Li, C.; Zhang, Y.; Wang, M.; Zhang, Y.; Chen, G.; Li, L.; Wu, D.; Wang, Q. Biomaterials2014, 35, 393-400.
5. Zhang, Y.; Zhang, Y.; Hong, G.; He, W.; Zhou, K.; Yang, K.; Li, F.; Chen, G.; Liu, Z.; Dai, H.; Wang, Q. Biomaterials2013, 34, 3639-3646.
6. Chen, G.; Tian, F.; Zhang, Y.; Li, C.; Wang, Q. Adv. Funct. Mater. 2014, 24, 2481-2488.

Depletion of intracellular antioxidants is linked to major cytotoxic events and cellular disorders such as oxidative stress and multiple sclerosis. In addition to medical diagnosis, determining the concentration of antioxidants in foodstuffs, food preservatives and cosmetics has proved to be very vital. Gold nanoclusters (Au-NCs) have a core size below 2 nm and contain several of metal atoms. They have interesting photophysical properties, are readily functionalized, and are safe to use in various biomedical applications. Herein, a simple and quantitative spectroscopic method based on gold nanoclusters (Au-NCs) is developed to detect and image antioxidants such as ascorbic acid (AA). The sensing mechanism is based on the fact that antioxidants can protect the fluorescence of Au-NCs against quenching by highly reactive oxygen species (hROS). Our method shows great accuracy when employed to detect the total antioxidant capacity (TAC) in commercial fruit juice. Moreover, confocal fluorescence microscopy images of HeLa cells show that this approach can be successfully used to image antioxidant levels in living cells. Finally, the potential application of this “light-on” detection method in multiple logic gate fabrication was discussed using the fluorescence intensity of Au-NCs as output.

Inexpensive sensors verifying quickly the presence of multiple analytes within a small drop would bring drastic improvements for medical diagnosis, e.g. in discriminating SARS from normal flu patients. Common approaches for parallel analyte detection in small liquid samples couple specific receptor molecules to spectrally encoded markers (e.g. SERS barcode, luminex) limiting the number of independent targets in a parallel assay to a few dozen. A (potentially) larger number of targets are possible on micro-spot arrays (e.g. DNA microarrays, multiplexed ELISA) where the type of target is encoded in the position of the corresponding spot. However, current multiplexed detection schemes are too complex, slow and/or expensive for routine use ‘in the field&’. We show a new approach to detect multiple analytes simultaneously in a microfluidic flow cell using randomly deposited gold nanorods. Random deposition allows an inexpensive, simple and high-throughput sensor fabrication. Each nanorod responds with a spectral shift of its plasmon resonance specifically to one target, acting effectively as a ‘nano-SPR&’ device. Using four distinct proteins as targets, we demonstrate the feasibility of the concept, sensitivity down to nanomolar concentrations, the reactivation and reuse of the sensor over several cycles, and estimate the potential for up-scaling the concept to hundreds or thousands of targets. Our technique has the potential to simplify multiplexed detection and reduce the costs of each sensor to negligible dimensions, especially if combined with advanced nanofabrication methods like nano-stamping or optical trapping. Such inexpensive sensor platform could be used, for example, to discriminate influenza subtypes in a doctor&’s office. Even a simplified home version is imaginable provided the readout machine is produced with costs compatible to consumer budgets. Besides medical uses, applications for inexpensive multiplexed sensors are abundant, for example identifying plant diseases, monitoring food composition, quality and safety, screening for explosives, drugs or environmental hazards.

Monitoring protein markers in easily accessible bodily fluids (e.g., blood, urine, ascites) is an important diagnostic and prognostic tool. We herein report a rapid and sensitive platform for protein detection, based on star-shaped Au nanoparticles. Noble metallic nanoparticles (e.g., Au, Ag) have been used as a sensing element for localized surface plasmon resonance (LSPR), as the resonance characteristic changes upon the binding of target molecules. LSPR assays offer 1) high kinetics as the reaction occurs in bulk solution; 2) facile readout from visual color changes using simple instrumentation; and 3) no need for precise temperature control. We reasoned that the LSPR sensitivity could be further enhanced by morphing conventional spherical particles into star-shaped ones; such a structure is expected to generate strongly localized electromagnetic fields (hot spots) for more pronounced spectral changes upon molecular binding. Finite-difference time-domain modeling revealed that Au nanostars have >100-fold larger spectral changes than Au nanospheres. We further validated this prediction by synthesizing 70-nm Au nanostars and applying them to a biotin-avidin model system. As an example of clinical application, we used the Au nanostar platform to detect kidney injury molecule-1 (KIM-1), a novel protein marker for renal proximal tubule injury. We show that Au nanostar-based LSPR allowed for fast (<30 min) and simplified target detection of KIM-1.

Gold nanoparticles have brilliant optical plasmonic properties that make them attractive for biomedical applications in diagnostics, imaging, and therapeutics. Targeting gold nanoparticles to cells is desirable to improve the specificiity in sensing, imaging, and photothermal therapy. In this talk I will discuss how we make and surface-modify our gold nanoparticles, and quantify specificity. The presence of gold nanoparticles in the extracellular matrix surrounding cells influences cellular activity; two possible mechanisms will be discussed.

Peptides are widely used to direct the growth of metal nanostructures and important for molecular recognition. The molecular mechanism of selective binding to gold surfaces and nanoparticles of various shape will be discussed, as shown by simulations with the INTERFACE force field in consistency with observations by phage display, LEED, binding constants, and shape-selective selective nanocrystal growth. The dominance of soft epitaxial coordination of polarizable atoms in the biological molecules (C, N, O) with epitaxial (hollow) sites on the metal surfaces in competition with water will be illustrated, and its impact on binding differentials between {111}, {100}, and {110} facets. In addition, differences in binding near the edges versus center portions of facets, and overall differences in attraction as a function of nanoparticle size will be explained using accurate simulation tools at the 1 to 100 nm scale.
Additional contributions to ligand binding arise from induced charges in the metal surface, although only significant in the presence of ionic ligands. The influence of such conditions will be highlighted for ionic liquids that are used as solvents and reaction media due to low volatility and stability up to high temperature. A combination of quantum mechanical and classical simulations provides insight into the structure and energetics of the self-assembly of ionic liquids on metal surfaces from single ion pairs to multilayers, using the example of 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIM][ES]) on the crystallographic {111}, {100}, and {110} facets of gold. Adsorption is then controlled by the interplay of soft epitaxy, ionic interactions, induced charges, and steric effects related to the geometry of the cation and anion. These factors lead to characteristic molecular patterns on individual surfaces. Binding energies are similar irrespective of surface coverage and only slightly increase from {111} to {100} and {110} surfaces due to stronger surface corrugation and higher induced charge. The results explain experimental observations on isotropic growth in the absence of shape regulating agents and aid in understanding particle growth. A mechanistic hypothesis for the formation of anisotropic gold nanorods in the presence of silver ions is presented, in which silver retards the growth along {100} and {110} facets through underpotential deposition.

Noble metal nanoparticles have a series of unique optical properties, which make them appealing labels for a broad range of biomedical applications. Noble metal nanoparticles do not blink or bleach, and they offer large resonant optical cross-section. Their precise resonance wavelength depends on the environment, which provides entirely new opportunities for sensing the local refractive index as well as measuring interparticle separations on subdiffraction limit length scales. While the photophysical properties of the nanoparticles are enabling, a meaningful application requires specific surface properties that allows a selective binding of the nanoparticle to the target of interest. Virus nanoparticles are evolutionarily optimized smart nanoparticles that have perfected these functionalities. We describe here a unique class of biomimetic nanoparticles that contains a membrane of defined composition wrapped around a solid metal core. These artificial virus nanoparticles combine the functionality of biological membranes with the unique photophysical properties of noble metal nanoparticles. We demonstrate that these hybrid materials achieve an efficient targeting of sialoadhesin (CD169) expressing dendritic cells under simulated in vivo conditions. The impact of the self-assembled membrane around the metal core on non-specific corona formation is also characterized quantitatively. Upon integration of the monosialoldihexosylganglioside GM3 into the membrane of the nanoparticles, a unique nanoparticle sequestration into peripheral non-lysosomal compartments is triggered. We demonstrate that the characterization of the segregation mechanism is enabled by the unique physical properties provided by the noble metal nanoparticle cores.

Cytotoxic gold nanoparticles (AuNPs) with a diameter of 1.4 nm were functionalized with the peptide (Lys³)-bombesin to specifically target the gastrin-releasing peptide receptor (GRPR), which is strongly expressed in prostate and colon cancer cells. AuNPs with a mixed ligand shell consisting of sodium 3-(diphenylphosphino)-benzenesulfonate (TPPMS) and 4-(diphenylphosphino)benzoic acid (TPPMC) were synthesized; the former to ensure solubility and stability in cell culture media and the latter to couple (Lys³)-bombesin through an amide bond formation. Using GeneBLAzer® reporter cells we demonstrated receptor binding and activation via a fluorescence signal transduction assay for bombesin and bombesin-modified AuNPs whereas bombesin-free TPPMS-stabilized AuNPs showed no receptor binding and activation. The toxicity of bombesin-labeled AuNPs was similar to same sized TPPMS-stabilized AuNPs. Thus bombesin-modified AuNPs may be used for tumor specific cell targeting in cancer diagnosis and therapy.

Gold, silica, and titanium dioxide nanoparticles inhibit vascular endothelial growth factor (VEGF)-driven pathological angiogenesis by binding with VEGF, which promotes pathological responses in age-related macular degeneration, cancers, and diabetic retinopathy. Interestingly, 20 nm-sized nanoparticles demonstrate more potent anti-angiogenic effect on pathological choroidal and retinal neovascularization than their 100 nm-sized counterparts. This phenomenon might be due to differences in the binding affinity between nanoparticles of different diameters and VEGF. We demonstrated that 20 nm-sized nanoparticles bind to VEGF, inhibit in vitro angiogenesis processes, and in vivo pathological neovascularization more efficiently than 100 nm-sized ones. As such, size-dependent biological activities of nanoparticles are determined by nanoparticle interaction with surrounding pathological proteins such as VEGF. Furthermore, corona formation with physiological proteins in biological systems affects therapeutic effects of nanoparticles as well. In particular, protein binding occurs differently according to biological fluids exposed to nanoparticles. In the treatment of pathological choroidal and retinal neovascularization, nanoparticles experience the vitreous after intravitreous injection; whereas, serum after intravenous administration. Interestingly, there are size-dependent differences in the quantity and the quality of proteins bound to nanoparticles which are immersed into the vitreous. Furthermore, nanoparticles bound to proteins maintain size-dependent anti-angiogenic effects related with differential binding affinity with VEGF. In this context, corona formation according to the size of nanoparticles seems to affect the discrepancy in size-dependent in vivo anti-angiogenic effects. Taken together, we suggest that differential protein binding to nanoparticles according to the size govern anti-angiogenic effects of them.

Recently, gold nanoparticles (NPs) protected by a binary mixture of purely hydrophobic and anionic end-functionalized linear alkanethiols were observed to spontaneously penetrate cell membranes via a non-disruptive, non-endocytic mechanism [1]. This non-toxic direct penetration process is highly promising for applications in drug delivery, bioimaging, or biosensing, as NPs can reach the cytosol without being trapped in endosomal compartments or inducing cytosolic leakage. However, to the best of our knowledge, no mechanism has yet emerged to explain the experimental observations limiting the ability to rationally engineer NPs for optimal internalization.
Inspired by these findings, in this work we use detailed atomistic molecular dynamics simulations to show that the same charged, amphiphilic, monolayer-protected NPs can spontaneously and rapidly insert into lipid bilayers, establishing a transmembrane configuration earlier predicted by implicit modeling methods [2]. We first show that the rate-limiting step for initial NP-bilayer insertion is the stochastic protrusion of aliphatic lipid tails into solvent and into contact with hydrophobic material in the amphiphilic NP monolayer. Upon contact with a protrusion, the NP begins to rapidly fuse with the bilayer in order to shield hydrophobic ligands within the bilayer core. Fusion is also facilitated by the “snorkeling” of charged ligands to minimize the exposure of charged end groups to the low dielectric constant aliphatic tail region. Our simulations further suggest that there is a second barrier to fusion associated with the eventual “flipping” of charges across the bilayer in a long-timescale process. We estimate the free energy cost for such charge translocation and show that successful flipping stabilizes a fully embedded transmembrane state. This study thus reveals a novel mechanism for the NP-bilayer fusion that could be prominent in biological systems given the widespread use of NPs in biomedical applications. Furthermore, the non-disruptive, non-endocytic nature of the insertion process suggests that our findings may also explain the direct penetration behavior observed experimentally.
[1] A. Verma et al, “Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles,” Nature Materials 7 (2008).
[2] R. C. Van Lehn et al, “Effect of particle diameter and surface composition on the spontaneous fusion of monolayer-protected gold nanoparticles with lipid bilayers,” Nano Letters 13 (2013).

The introduction of nanoparticles (NP) into biological fluids leads to an adsorption of proteins on the surface of the nanoparticles, forming a protein corona. The consensus is that some classes of proteins bind strongly to the NPs, making up the “hard corona”, while others have a weaker interaction with the surface, forming the “soft corona”. The protein corona is multi-layered and can be used to hold high concentrations of therapeutic payloads on the nanoparticles.
We show that protein coronas on gold nanorods can be loaded with DNA at capacities 5-10-times higher than what is achievable by covalent chemistry. Furthermore, we can manipulate the release rates of DNA payloads from protein coronas by engineering the corona composition or by actively triggering the release of the bound species. We explore strategies to tune the passive release profile, such as the proteins forming the corona, the passivating ligands on the nanoparticles, or the buffer strength of the solution and find that the material properties of the nanoparticles, as well as the molecules in their surroundings have a great impact on the loading and release of payloads from the carriers.
These advances in understanding the protein corona, behaviour and composition can be used for the design of better carriers for drug delivery and disease diagnostics applications.

Advances in nanotechnology have culminated in the development of novel engineered nanomaterials with different physicochemical properties for numerous applications including electronic, chemical, biological and medical fields. Engineered metal nanoparticles (ENPs) are among the most widely studied nanomaterials used in biomedical applications, including biosensors, targeted drug and gene delivery, tracking, and theranostics. AuNPs with diameters between 4-100 nm have been focus of various biomedical applications due to their low toxicity, biocompatibility and tunable surface functionalities. When gold nanoparticles enter into the biological milieu, they interact with various biomolecules, especially proteins, forming a dynamic protein corona on their surface. Formation of the protein corona can significantly affect the faith of the nanoparticle in the living environment. Therefore, the parameters affecting the nanoparticle-biomolecule interactions require special attention for their safer use in biomedical applications. These nonspecific nanoparticle-biomolecule interactions change depending on the particular cell type and the defining parameters of the nanomaterials that interact with the proteins. Up to now, short term (acute) toxicity of gold nanoparticles inside cells has been explored. However; long term effects of in vitro toxicity has not been reported sufficiently. Herein, we developed an in vitro experimental setup to investigate the effect of long term exposure of nanoparticles inside the cells. Here, Turkevich method was used for fabrication of gold nanoparticles (around 12 nm). The size, morphology, and colloidal stability of 12 nm AuNPs were fully characterized with dynamic light scattering (DLS), transmission electron microscopy (TEM), and UV-Vis spectrum. A bathochromic shift was observed in UV-Vis spectra of AuNPs in 5% FBS/DMEM indicating corona formation around AuNPs. According to dose dependent cell viability assay, 8 µg/mL was determined as the (acute) non-toxic dose for long-term accumulation experiments in endothelial cells. After long term accumulation with determined concentration of AuNPs, the uptake of AuNPs was demonstrated with inductively coupled plasma mass spectroscopy (ICP-MS) and silver staining. Specifically, time dependent fluctuation in cellular uptake of AuNPs was observed. Interestingly, cell death was not observed even after 57 days of exposure of AuNPs. We believe that this experimental setup will provide valuable information for understanding of the effects of long-term exposure and accumulation of engineered nanomaterials on the cellular behavior.

One of the most common types of nanoparticles (NPs) studied are citrate-coated Au nanoparticles. Gold nanoparticles (AuNPs) have attracted vast scientific interest due to their chemical stability, unique optical properties and relatively easy synthesis. These NPs have an enormous potential in chemical sensing, biological imaging, drug delivery, and cancer treatment. Therefore, it is important to know their potential toxicity and short- and long-term health impacts. Here we present the effect of AuNPs on human epidermal keratinocytes where we found that the penetration rate for 13 nm AuNPs is 2.5-fold greater than that for 45 nm AuNPs , despite the fact that cells were treated with a 7-fold greater concentration of 45 nm AuNPs, suggesting that AuNP size influences the uptake mechanism. TEM data indicated that both 13 nm and 45 nm AuNPs penetrate keratinocytes and are sequestered in a ring of vacuoles around the nucleus, however no evidence of nuclear or mitochondrial penetration was found. Keratinocyte proliferation rates were found to decrease with increasing AuNP concentration, or for a given concentration, increasing storage time of the particles. The decrease was greater in the case of 45 nm particles compared to 13 nm particles, indicating that large nanoparticles are more toxic, and that limit need to be imposed regarding the stability of these particles.
After 3 days post-AuNP removal, cell numbers in samples treated with AuNPs are similar to control, indicating partial recovery. The recovery rate was faster for the samples exposed to the 13 nm AuNPs compared to the 45 nm ones. Finally, the effect of the AuNPs on the morphology of keratinocytes was investigated. Using confocal microscopy, we observed that after exposure to AuNPs, cells were unable to form a monolayer or to make tight connections; however, no damage to the cells as a function of size was observed. After 7 days of exposure, samples treated with AuNPs revealed broken actin fibers. AuNPs have no effect on the keratinocyte differentiation process as was confirmed by the involucrin expression, a known marker of keratinocyte differentiation. Results of other factors influencing toxicity and recovery, such as the age of the tissue donor or different stress factors imposed on the cells in culture will be discussed.

H₂O₂ is an important reactive oxygen species generated in cells via oxygen metabolism and is involved actively in cell signaling and cell growth. However its uncontrolled over-production can cause detrimental oxidation of biomolecules and lead to aging, cancer and other diseases. In this talk, I will present our new method of developing core/shell Au/MnO nanoparticles as sensitive probe for H₂O₂ detection. We first prepared monodisperse 5 nm AuMn nanoparticles by hydride reduction of manganese acetylacetonate in the presence of 5 nm Au nanoparticles. The alloy was formed via fast Mn diffusion into the Au structure. The AuMn nanoparticles were converted to Au-MnO composite particles via air annealing at 1700C. These Au-MnO particles, especially the core/shell Au/MnO nanoparticles, were active for electrochemical reduction of H₂O₂ with the detection limit reaching 8 nM. The highly sensitive electrochemical sensor based on the Au/MnO nanoparticles was used to monitor H₂O₂ concentration levels released from living cells, from which tumorigenic cells were detected to have higher level of H₂O₂ than the non-tumorigenic ones. The Au/MnO nanoparticle probe can be used for sensitive cancer detection.

The plasmon resonance of metallic nanoparticles makes them attractive for biomedical applications, e.g. for photothermal cancer therapey. Most research on such particles has been focused on silver and gold nanoparticles. Silver, however, exhibits intrinsically stronger plasmonic performance than gold because of its lower losses in the UV and visible spectrum [2]. On the other hand, nanosilver exhibits significant toxicity due to the release of Ag⁺ ions [2]. This prohibits the use of nanosilver in biomedical applications where inert materials are sought [3]. As a result, research in biomedical applications focuses mostly on the plasmonically inferior and rather expensive gold. Here, we synthesize Ag-Au nanoalloys by scalable flame aerosol technology searching for maximum plasmonic performance at minimal toxicity. The addition of Au hinders the Ag surface oxide layer formation drastically minimizing the toxic Ag⁺ ion release in aqueous solutions. This renders the nanoalloys biocompatible while their photothermal plasmonic performance is thrice than that of pure gold.
References
[1] G.A. Sotiriou, S.E. Pratsinis, “Engineering nanosilver as an antibacterial, biosensor and bioimaging material”, Curr. Opinion Chem. Eng.1, 3-10 (2011).
[2] G.A. Sotiriou, A. Meyer, J.T.N. Knijnenburg S. Panke, S.E. Pratsinis, “Quantifying the origin of nanosilver ions and their antibacterial activity”, Langmuir, 28, 15929-15936 (2012).
[3] G.A. Sotiriou, “Biomedical Applications of Multifunctional Plasmonic Nanoparticles”, WIREs Nanomed. Nanobiotechnol.5, 19-30 (2013).

SiO₂ based metal nanoparticle hybrids form an important class of material which finds application in catalysis, sensors and biotechnology. Although several protocols exist for synthesizing these hybrids, the complete understanding of the morphology, composition and distribution of the heterounits in three dimensions is lacking. Conventional imaging techniques like SEM and TEM gives information on the size, external/surface morphology and partially on the shape of the nanostructure, but it can be misleading for detailed understanding of the internal distribution and structural composition of the hybrid with a 3D shape. However, these are important factors governing their functionalities like catalytic activity, stability, sensitivity, plasmonic behavior etc. Also, thermal stability of such hybrids are important to be investigated. Here we present a simple wet chemical route to obtain extremely stable Au nanoparticles (5 - 10 nm) decorated SiO₂ spheres without using any external linkers. We investigated the composition by STEM-EDX and 3D ordering of the heterounits of the hybrid using STEM tomography. It reveals the presence of Au nanoparticles exclusively on the surface of the SiO₂ spheres and not inside the matrix. The same characterization method has been used for understanding the mechanism of formation of the hybrid, intermediate nanostructures resulted in course of reaction. This study reveals that the hybrid formation is mediated by self-assembling of Au nanoparticles due to the presence of oleyl amine and (3-mercaptopropyl) trimethoxysilane (MPTMS) on the Au surface and a formation mechanism of the hybrid is deduced. Thermal stability of the hybrid is investigated as well by electron tomography which reveals a good stability of the structure up to 400^oC beyond which the Au particles starts migrating from the surface of the SiO₂ sphere into the matrix. However, no significant coarsening of the particles are observed. These kind of structural changes can have significant impact on physical properties or their functional behavior related to surface activity. This also implies low mobility of the Au particles on the SiO₂ surface which is advantageous for several applications. The method being general could be used for making similar SiO₂ based hybrid to stabilize metal nanoparticles.

DNA-modified gold nanoparticles (AuNPs) have been successfully used in the field of bioanalysis, diagnosis, drug delivery and material construction. DNA surface coverage correlates directly with the performance of DNA-modified AuNPs. Herein we propose an Exonuclease III (Exo III)-assisted, target-recycling based assay for rapidly quantifying DNA surface coverage on AuNPs. Specially, we introduced two abasic sites into the fluorophore-labeled DNA probe immobilized on AuNPs, employed a perfectly-matched target to form the target/probe duplexes to achieve Exo III-assisted target recycling on the surface through combination of Exo&’s apurinic endonuclease activity and its 3&’-to-5&’ exonuclease activity. We obtained a complete release of the tagged fluorophore on the conjugated DNA from the AuNPs after 80 minutes at room temperature. Our newly developed method gives a high accuracy of DNA surface coverage, which matched well with the DTT-displaced, golden standard method. It is applicable for a high or low surface coverage of DNA strands anchored on surface of gold or other materials with significantly shortened time. By accurate determination of DNA surface coverage, we demonstrated a successful control of reaction time on the AuNP-based assay optimized for a 5-min point-of-care testing.

It&’s been recently found that lipids in the viral membranes play roles far more important than simple scaffolds for carrying proteins. Some phospholipids, especially phosphatidylserine (PS), have been shown to play key roles in infectivity of viruses such as Vaccinia and Ebola. PS is also shown to be a cofactor in capture of HIV-1 by monocytic cells. In addition, there are multiple lines of evidence for the crucial effect of glycosphingolipids (GSLs) such as G_M1 and G_M3 in capture of HIV-1 by dendritic cells. These findings have attracted special attention to the field of lipidomics of viruses, mainly through mass spectrometry (MS). However, despite its qood performance for lipid quantification, MS normally requires lipid extraction and a significant amount of sample to satisfy the sensitivity of the spectrometer&’s detector. Therefore, the need for a simple yet precise method of lipid quantification led us to devise an all-optical approach that has the potential for working with low concentrations of virus particles as is common in many patient-derived samples. We have developed a plasmon-coupling based optical method for the quantification of gold nanoparticle targeted lipids on virus particles. This approach requires only hundreds of virus particles to detect lipids in the outer leaflet of their envelope with a sensitivity of down to 1 mole% lipid. Here, in addition to the basics of our optical method, I&’ll introduce its calibration on liposomes, as known model systems, and its application to quantification of PS and G_M1 lipids on the surface of HIV-1 (with various cellular origins) and Ebola virus-like particles.

This report will describe the functionalization strategies for gold nanocrystals of different shape and size and enhancement of their uptake by cancer cells. Imaging of breast cancer cells with positively-charged nanorods, microrods, and nanowires by optical microscopy, SEM, and TEM will be discussed. The use of small gold nanoparticles as therapeutic platform for the delivery of paclitaxel will also be presented.

Gold nanorods were applicable for Laser-induced desorption and ionization (LDI) mass spectrometry of oligopeptides [1]. As well as the LDI of organic molecules, gold ions (Au_n⁺: n = 1shy;3) were also detectable when they were irradiated by the pulsed-laser light. In this work, the LDI of the gold ions was observed from gold nanorods in a liver tissue section (thickness: 10 micros). Larger amounts of gold nanorods produced stronger mass signals. Imaging mass spectrometry technique was successfully applied for assessing the distribution of gold nanorods. In this case, organic mass-marker molecules were not needed on the nanorod surfaces. Organic molecules in the tissue were not detected and did not inhibit the LDI processes of the gold nanorods in the tissue sections. Aun+ ions could be detected from liver sections of mice in which gold nanorods were intravenously injected. Highly stable chemical natures of gold and the high sensitivity of mass spectrometry served the gold nanorods as a mass marker in a body tissue. The maximum spatial resolution of the technique is about 10 microns, which is sufficient for extracting information about the distribution of gold nanorods in tissues.
1. Y. Nakamura, Y. Tsuru, M. Fujii, Y. Taga, A. Kiya, N. Nakashima, Y. Niidome, Nanoscale,3, 3793 (2011).
2. M. Fujii, N. Nakashima, T. Niidome, Y. Niidome, Chem. Lett.,43, 131 (2014).

Due to the steadily increasing number of elderly people and the resulting rise in age-related diseases such as osteoporosis, there is a high demand for long lasting and biocompatible orthopedic implant materials. In the current focus of research are beta-type titanium alloys such as Ti-40Nb which exhibit superior mechanical properties, e.g. much lower stiffness compared to cp-Ti and Ti-6Al-4V, as well as very low metal release rates. However, naturally passivated alloy surfaces are bioinert and strategies for bioactivation are needed.
The use of human mesenchymal stem cells (hMSC) is of ever growing interest in regenerative medicine. Stem cells are responsive to physical features of the extracellular matrix (ECM), e.g. topography and stiffness, and to chemical features, e.g. molecular composition of the ECM and ligand density. The presence of non-proliferative senescent cells is a problematic issue. It is of upmost significance to carefully control initial cell settling on metallic implant materials by modification of their surfaces. This can be achieved by immobilization of specific ligands, like proteins or bioactive peptides present in the ECM. A promising method to control the spatial density of bioactive peptides and the orientation of the immobilized ligand is a dip-coating technique based on self-assembly of diblock copolymer micelles. Using Au as micelle core, an ordered pattern of Au nanoparticles can be created on various materials and can be used to direct cell adhesion.
For the present study beta-type Ti-40Nb alloy samples with very low elastic modulus were prepared by casting and solution annealing treatment and their microstructural and initial surface states were characterized. A successful method for decoration of fine-polished alloy surfaces with patterns of Au-nanoparticles by means of block-copolymer micelle nanolithography (BCMN) is demonstrated. HR-SEM evaluation revealed the formation of hexagonal patterns with interparticle distances of approximately 68 nm. Auger electron spectroscopy (AES) depth profile analysis indicated the presence of a Ti- and Nb-oxide layer with a thickness of approx. 18 nm. TEM investigations confirmed the presence of this oxide layer which is amorphous. TEM and STEM images revealed nanoparticles on top of this oxide layer, large ones with 6-7 nm diameter and a smaller ones with 2-3 nm. Their diffractograms clearly identified the crystal structure of face centered cubic (fcc) Au. Further biofunctionalization of these nanoparticle patterns with cRGDfK peptides was conducted. The effects of nanotopography and biofunctionalization on hMSC early adhesion and phenotype selection will be discussed [1].
Funding from the DFG (SFB-Transregio 79) and from the EC (FP7/2007-13, grant no. 264635 (BioTiNet-ITN)) is acknowledged.
[1] R. Medda et al., Interface Focus 4: 20130046 (2014).

Silver as antibacterial agent is increasingly used in many applications. To meet the diversity of application types, kinds of silver compounds have been developed to serve this market. The most potent compounds for a rapid silver release are soluble silver salts like silver nitrate or silver acetate. They are often used in cell culture experiments to elucidate the biological effect of silver. In these cases is tacitly assumed that the concentration of free silver ions is the same as that in the added silver salts. This obviously cannot be true because of the presence of a whole set of proteins, biomolecules and inorganic ions like chloride and phosphate in the biological medium. These will react with the silver ions in one or the other way, reducing the concentration of dissolved silver ions, also for engineered silver nanoparticles after their release into the environment. Consequently, there have been attempts to study the biological effect of silver chloride which is the likely precipitation product. However, in all these studies, silver chloride was prepared as precipitated solid, i.e. not in the presence of proteins and therefore not colloidally stable. Here we show that silver chloride is present as dispersed colloidal species if proteins are present (due to the formation of a stabilizing protein corona) and that it exhibits the same toxicity towards eukaryotic cells and bacteria as a dissolved silver salt. Therefore, it is deduced that this is the predominant species in "solutions" of soluble silver salts in biological fluids. We have investigated the behavior of silver ions in biologically relevant concentrations (10 to 100 ppm) in different media, from physiological salt solution over phosphate-buffered saline solution to protein-containing cell culture media. The results show that the initially present silver ions are bound as silver chloride due to the presence of chloride. Only in the absence of chloride, glucose is able to reduce Ag⁺ to Ag⁰. The precipitation of silver phosphate was not observed in any case. We conclude that the predominant silver species in biological media is dispersed nanoscopic silver chloride, surrounded by a protein corona which prevents the growth of the crystals and leads to colloidal stabilization. Therefore, in cell culture experiments where dissolved silver ions are studied in the upper ppm range, in fact the effect of colloidally dispersed silver chloride is observed. We have confirmed this by cell culture experiments (human mesenchymal stem cells; T-cells; monocytes) and bacteria (S. aureus) where the cells were incubated with synthetically prepared silver chloride nanoparticles (diameter ca. 100 nm). These were easily taken up by eukaryotic cells and showed the same toxic effect at the same silver concentration as ionic silver (as silver acetate).

Some reactive organic functionalities linked to alkoxysilane precursors have shown potential contribution serving as reducing and stablilizing agents during the synthesis of novel functional nanomaterials. It has been found that 3-aminopropyltrimethoxysialne (3-APTMS) capped metal ions are converted into respective nanoparticles in the presence additional suitable organic reducing agents like 3-glycidoxypropyltrimethoxy-silane and many others. The choice of such organic reducing agents results into the formation of functional nanomaterials dispersible in both aqueous and non-aqueous media for practical applications. The role of cyclohexanone, tetrahydrofuran hydroperoxide and 3-glycidoxypropyltrimethoxysilane (3-GPTMS) are demonstrated for the synthesis of gold and prussian blue nanoparticles justifying functional ability for the formation of nanocomposite with known materials displaying significant improvement in catalytic ability. The use of 3-GPTMS enable the disperbility of nanomaterials mostly in organic media with limited option for their use in aqueous medium as a function of 3-APTMS/3-GPTMS ratio. However when tetrahydrofuran hydroperoxide is used for the conversion of 3-APTMS capped metal ions into nanoparticles, the process enable the formation of organic -inorganic hybrid that display dramatic improvement in catalytic activity of nanopaterial during practical application as a function 3-APTMS concentrations. An increase in 3-APTMS concentration allow an increase in the catalytic behavior of nanomaterial justifying the functional ability of the nanomaterial in practical application. Such functional ability of gold nanoparticle also enable the nanocomposite formation with Prussian nanoparticles. The present talk will focus on the synthesis of gold and Prussian blue nanoparticle and their use as peroxidase mimetics for practical biomedical application.