Jin-Woo Kim

Golden carbon nanotubes as multimodal photoacoustic and photothermal high-contrast molecular agents

Carbon nanotubes have shown promise as contrast agents for photoacoustic and photothermal imaging of tumours and infections because they offer high resolution and allow deep tissue imaging. However, in vivo applications have been limited by the relatively low absorption displayed by nanotubes at near-infrared wavelengths and concerns over toxicity. Here, we show that gold-plated carbon nanotubes-termed golden carbon nanotubes-can be used as photoacoustic and photothermal contrast agents with enhanced near-infrared contrast ( approximately 10(2)-fold) for targeting lymphatic vessels in mice using extremely low laser fluence levels of a few mJ cm(-2). Antibody-conjugated golden carbon nanotubes were used to map the lymphatic endothelial receptor, and preliminary in vitro viability tests show golden carbon nanotubes have minimal toxicity. This new nanomaterial could be an effective alternative to existing nanoparticles and fluorescent labels for non-invasive targeted imaging of molecular structures in vivo.

In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells.

The spread of cancer cells between organs, a process known as metastasis, is the cause of most cancer deaths. Detecting circulating tumour cells -- a common marker for the development of metastasis -- is difficult because ex vivo methods are not sensitive enough owing to limited blood sample volume and in vivo diagnosis is time-consuming as large volumes of blood must be analysed. Here, we show a way to magnetically capture circulating tumour cells in the bloodstream of mice followed by rapid photoacoustic detection. Magnetic nanoparticles, which were functionalized to target a receptor commonly found in breast cancer cells, bound and captured circulating tumour cells under a magnet. To improve detection sensitivity and specificity, gold-plated carbon nanotubes conjugated with folic acid were used as a second contrast agent for photoacoustic imaging. By integrating in vivo multiplex targeting, magnetic enrichment, signal amplification and multicolour recognition, our approach allows circulating tumour cells to be concentrated from a large volume of blood in the vessels of tumour-bearing mice, and this could have potential for the early diagnosis of cancer and the prevention of metastasis in humans.

Photoacoustic flow cytometry: principle and application for real-time detection of circulating single nanoparticles, pathogens, and contrast dyes in vivo

The goal of this work is to develop in vivo photoacoustic (PA) flow cytometry (PAFC) for time-resolved detection of circulating absorbing objects, either without labeling or with nanoparticles as PA labels. This study represents the first attempt, to our knowledge, to demonstrate the capability of PAFC with tunable near-infrared (NIR) pulse lasers for real-time monitoring of gold nanorods, Staphylococcus aureus and Escherichia coli labeled with carbon nanotubes (CNTs), and contrast dye Lymphazurin in the microvessels of mouse and rat ears and mesenteries. PAFC shows the unprecedented threshold sensitivity in vivo as one gold nanoparticle in the irradiated volume and as one bacterium in the background of 10(8) of normal blood cells. The CNTs are demonstrated to serve as excellent new NIR high-PA contrast agents. Fast Lymphazurin diffusion in live tissue is observed with rapid blue coloring of a whole animal body. The enhancement of the thermal and acoustic effects is obtained with clustered, multilayer, and exploded nanoparticles. This novel combination of PA microscopy/spectroscopy and flow cytometry may be considered as a new powerful tool in biological research with the potential of quick translation to humans, providing ultrasensitive diagnostics of pathogens (e.g., bacteria, viruses, fungi, protozoa, parasites, helminthes), metastatic, infected, inflamed, stem, and dendritic cells, and pharmacokinetics of drug, liposomes, and nanoparticles in deep vessels (with focused transducers) among other potential applications.

In vivo fiber‐based multicolor photoacoustic detection and photothermal purging of metastasis in sentinel lymph nodes targeted by nanoparticles

This report introduces a novel diagnostic and therapeutic platform for in vivo non-invasive detection and treatment of metastases in sentinel lymph nodes (SLNs) at single cell level using an integrated system of multicolor photoacoustic (PA) lymph flow cytometry, PA lymphography, absorption image cytometry, and photothermal (PT) therapy. A melanoma-bearing mouse model was used to demonstrate the capability of this platform for real-time lymphatic mapping, counting of disseminated tumor cells (DTCs) in prenodal lymphatics, and detecting metastasis in SLNs and its purging. The detection and ablation of non-pigmented breast cancer cells in SLNs was achieved by labeling them with nanoparticles. The association between DTC count and SLN metastasis progression supports lymphatic DTCs as a novel prognostic marker of metastasis. The fiber-based portable PA device may replace the conventional SLN(s) excision and histology-based staging. The earliest detection of DTCs in the lymphatic vessels before the establishment of nodal metastasis may prevent metastasis by well-timed ablation of DTCs.

Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics

Various nanoparticles have raised significant interest over the past decades for their unique physical and optical properties and biological utilities. Here we summarize the vast applications of advanced nanoparticles with a focus on carbon nanotube (CNT)-based or CNT-catalyzed contrast agents for photoacoustic (PA) imaging, cytometry and theranostics applications based on the photothermal (PT) effect. We briefly review the safety and potential toxicity of the PA/PT contrast nanoagents, while showing how the physical properties as well as multiple biological coatings change their toxicity profiles and contrasts. We provide general guidelines needed for the validation of a new molecular imaging agent in living subjects, and exemplify these guidelines with single-walled CNTs targeted to α(v) β(3) , an integrin associated with tumor angiogenesis, and golden carbon nanotubes targeted to LYVE-1, endothelial lymphatic receptors. An extensive review of the potential applications of advanced contrast agents is provided, including imaging of static targets such as tumor angiogenesis receptors, in vivo cytometry of dynamic targets such as circulating tumor cells and nanoparticles in blood, lymph, bones and plants, methods to enhance the PA and PT effects with transient and stationary bubble conjugates, PT/PA Raman imaging and multispectral histology. Finally, theranostic applications are reviewed, including the nanophotothermolysis of individual tumor cells and bacteria with clustered nanoparticles, nanothrombolysis of blood clots, detection and purging metastasis in sentinel lymph nodes, spectral hole burning and multiplex therapy with ultrasharp rainbow nanoparticles.

Nanotechnology-based molecular photoacoustic and photothermal flow cytometry platform for in-vivo detection and killing of circulating cancer stem cells.

In-vivo multicolor photoacoustic (PA) flow cytometry for ultrasensitive molecular detection of the CD44+ circulating tumor cells (CTCs) is demonstrated on a mouse model of human breast cancer. Targeting of CTCs with stem-like phenotype, which are naturally shed from parent tumors, was performed with functionalized gold and magnetic nanoparticles. Results in vivo were verified in vitro with a multifunctional microscope, which integrates PA, photothermal (PT), fluorescent and transmission modules. Magnet-induced clustering of magnetic nanoparticles in individual cells significantly amplified PT and PA signals. The novel noninvasive platform, which integrates multispectral PA detection and PT therapy with a potential for multiplex targeting of many cancer biomarkers using multicolor nanoparticles, may prospectively solve grand challenges in cancer research for diagnosis and purging of undetectable yet tumor-initiating cells in circulation before they form metastasis.

Photothermal nanodrugs: potential of TNF-gold nanospheres for cancer theranostics

Nanotechnology has been extensively explored for drug delivery. Here, we introduce the concept of a nanodrug based on synergy of photothermally-activated physical and biological effects in nanoparticle-drug conjugates. To prove this concept, we utilized tumor necrosis factor-alpha coated gold nanospheres (Au-TNF) heated by laser pulses. To enhance photothermal efficiency in near-infrared window of tissue transparency we explored slightly ellipsoidal nanoparticles, its clustering, and laser-induced nonlinear dynamic phenomena leading to amplification and spectral sharpening of photothermal and photoacoustic resonances red-shifted relatively to linear plasmonic resonances. Using a murine carcinoma model, we demonstrated higher therapy efficacy of Au-TNF conjugates compared to laser and Au-TNF alone or laser with TNF-free gold nanospheres. The photothermal activation of low toxicity Au-TNF conjugates, which are in phase II trials in humans, with a laser approved for medical applications opens new avenues in the development of clinically relevant nanodrugs with synergistic antitumor theranostic action.

DNA‐Linked Nanoparticle Building Blocks for Programmable Matter

Programmable matter is a distributed system of agents that act cooperatively to configure themselves into arbitrary shapes with arbitrary functions. Molecular self-assembled structures containing many nanoparticles are candidates for programmable matter. Programmability implies that system designers are able to control the properties of assembly products. The system should be able to assemble into arbitrary, anisotropic shapes, like an electronic circuit, with the capability of incorporating different materials at specific locations within the structure. Defects or errors should be minimized, and 3D assembly should be possible. In self-assembly, component parts, or building blocks, interact locally to produce a coherent and organized whole. At the molecular level, the interactions are determined by “patches” that react between building blocks. Frequently, the assemblies exhibit collective properties that are distinct from those of their constituent components. These properties often depend upon the shape of the structure. Thus, the difficulty of programmability is really the difficulty of controlling the shape of resulting nanostructures. The ability to program the shape of a final assembly is computationally difficult and subject to frequent errors. Nevertheless, through careful design and implementation of building blocks, desired shapes and properties might be achieved. To maximize programmability (i.e., control), there should be a large number of types of patches available. Otherwise, there is no variety of interactions to assemble complicated shapes. The placement and relative orientation of patches on the surface of the building block should be controlled. Different types of patches should be able to be placed on the same building block to diversify the shapes available. Finally, the chemistry for patch conjugation to the building block should be relatively simple and sustainable, and it should be able to be used with a variety of materials. Because of its unique molecular recognition properties, structural features, and ease of manipulation, beginning with seminal work by Seeman and Chen, DNA has been considered as a promising material to achieve programmable assembly of nanostructures. Nanoparticle (NP) building blocks with different surface functionalities for DNA linkers have been reported. DNA computing verified the programmability of DNA-based nanotechnology and, in fact, demonstrated that DNA self-assembly was computationuniversal. DNA programmability has demonstrated the ability to assemble a variety of shapes 9] and, when NPs are incorporated, to control the position of NPs in linear, 2D, and 3D assemblies, including those based upon origami techniques. 10] Nevertheless, the rational self-assembly of functional structures with arbitrary shapes in all dimensions and at all scales that can incorporate many different NPs into a variety of final geometries remains difficult to attain. Herein we present a strategy to control the number, placement, and relative orientation of DNA linkers on the surface of a colloidal NP building block to maximize its programmability and realize enhanced control over the shape and function of final self-assembled structures. Figure 1 shows a schematic illustration of the assembly sequence to produce the DNA-linked colloidal gold NP building blocks (termed nBLOCKs). A measure of control was achieved by the sequential ligand replacement approach and stiff DNA linkers, which were shorter than the persistence length of double-stranded DNA. In the assembly reaction, the electrostatic repulsions and steric hindrances of DNA molecules influence the layout of DNA on a NP. The net charge of DNA at a pH value above its isoelectric point (i.e., pH 5) is negative, so it would tend to position on a NP to minimize its mutual electrostatic repulsions, in analogy to the valence shell electron pair repulsion model, thus contributing to the molecular geometry. Also, mutual steric hindrance could be another factor to further constrain the overall geometry, particularly using small NPs. In our strategy, a Au NP is functionalized by DNA strand by strand: for example, a NP with one DNA strand is the starting material for the second DNA attachment, a NP with two DNA strands is the starting material for the third DNA attachment, etc. (Figure 1; see Figure S1 in the Supporting Information). With this constraint, the position of DNA attachment would be chosen to minimize the electrostatic and steric interactions with existing DNA on the NP, yielding the optimal arrangement of DNA on a NP with up to sixfold symmetry, that is, linear (one and two DNA strands), T-shaped (three DNA strands), square planar (four DNA strands), square pyramidal (five DNA [*] Prof. J.-W. Kim, Dr. J.-H. Kim Bio/Nano Technology Laboratory Department of Biological and Agricultural Engineering and Institute for Nanoscience and Engineering University of Arkansas, Fayetteville, AR 72701 (USA) E-mail: jwkim@uark.edu

In Vivo Magnetic Enrichment, Photoacoustic Diagnosis, and Photothermal Purging of Infected Blood Using Multifunctional Gold and Magnetic Nanoparticles

Bacterial infections are a primary cause of morbidity and mortality worldwide. Bacteremia is a particular concern owing to the possibility of septic shock and the development of metastatic infections. Treatment of bacteremia is increasingly compromised by the emergence of antibiotic resistant strains, creating an urgent need for alternative therapy. Here, we introduce a method for in vivo photoacoustic (PA) detection and photothermal (PT) eradication of Staphylococcus aureus in tissue and blood. We show that this method could be applicable for label-free diagnosis and treatment of in the bloodstream using intrinsic near-infrared absorption of endogenous carotenoids with nonlinear PA and PT contrast enhancement. To improve sensitivity and specificity for detection of circulating bacteria cells (CBCs), two-color gold and multilayer magnetic nanoparticles with giant amplifications of PA and PT contrasts were functionalized with an antibody cocktail for molecular targeting of S. aureus surface-associated markers such as protein A and lipoprotein. With a murine model, the utility of this approach was demonstrated for ultrasensitive detection of CBCs with threshold sensitivity as low as 0.5 CBCs/mL, in vivo magnetic enrichment of CBCs, PT eradication of CBCs, and real-time monitoring of therapeutic efficacy by CBC counting. Our PA-PT nano-theranostic platform, which integrates in vivo multiplex targeting, magnetic enrichment, signal amplification, multicolor recognition, and feedback control, could be used as a biological tool to gain insights on dissemination pathways of CBCs, infection progression by bacteria re-seeding, and sepsis development and treatment, and could potentially be feasible in humans, especially using bypass schematic.

Design and test of noncrosshybridizing oligonucleotide building blocks for DNA computers and nanostructures

DNA oligonucleotides that anneal to form duplexes in specific, planned configurations are a basic construction material for DNA-based computers and nanotechnology. Unplanned duplex configurations introduce errors in computations and defects in structures, and thus, the sequences must be designed to minimize these effects. A software design tool has been developed that uses thermodynamic models of DNA duplex thermal stability and algorithms from graph theory to select good sets of oligonucleotides. An example set was tested in the laboratory, and the designed sequences formed no unplanned duplexes and had no detectable secondary structure.