Eliminating the Animal Species Constraints in Antibody Selection for Multicolor Immunoassays

Molecular profiling on the single-cell level helps unveil the mystery of gene expression patterns in individual cells at subcellular resolution, enabling discovery of small but meaningful variations that are often overlooked at the population level. Similar to other immunoassays, the most common and economical protocols are developed by combining primary antibodies (1′Abs) and fluorophore-labeled secondary antibodies (2′Abs). The selection of 1′ and 2′ Abs, however, has been limited by the availability of animal species, consequently resulting in low multiplexing capability. Here we report the development of preassembled Ab pairs using 1′Abs all from the same animal species. We show that multiple molecular targets can be simultaneously labeled without cross reactivity. This simple and general self-assembly technology eliminates the animal species constraints in multicolor immunoassays, offering exciting new opportunities for a wide range of biomedical and clinical applications

Gradient Coating of Polydopamine via CDR

Surfaces with gradient properties are of central importance for a number of chemical and biological processes. Here, we report rapid generation of a polydopamine (PDA) gradient on hydrophobic surfaces by a simple, low cost, and general technology, cyclic draining-replenishing (CDR). Due to the unique surface chemistry of PDA, it enables continuous and precise control of surface wettability and subsequent deposition of organic and inorganic compounds. Using kanamycin as a model compound, we show that the gradient PDA membrane potentially can be used to prepare minimum inhibitory concentration (MIC) test strips for quantifying resistance of antimicrobial agents from microorganisms. Because CDR is experimentally simple, scalable, fast, and does not require specialized reagents or instruments, we envision this platform can be easily adopted to create a variety of functional surfaces

Solid-Phase Bioconjugation of Heterobifunctional Adaptors for Versatile Assembly of Bispecific Targeting Ligands

High-throughput generation of bispecific molecules promises to expedite the discovery of new molecular therapeutics and guide engineering of novel multifunctional constructs. However, high synthesis complexity and cost have hampered the discovery of bispecific molecules in drug development and biomedical research. Herein we describe a simple solid-phase bioconjugation procedure for preparation of Protein A­(G,L)-PEG-Streptavidin heterobifunctional adaptors (with 1:1:1 stoichiometry), which enable self-assembly of unmodified antibodies and biotinylated molecules into bispecific targeting ligands in a versatile mix-and-use manner. Utility of such adaptors is demonstrated by assembly of anti-CD3 and anti-Her2 antibodies into bispecific CD3xHer2 targeting ligands, which efficiently drive T-cell-mediated lysis of Her2-positive cancer cells. In comparison to bioconjugation in solution, the solid-phase procedure described here offers precise stoichiometry control, ease of purification, and high yield of functional conjugates. Simplicity and versatility should prove this methodology instrumental for preparation of bispecific ligands, as well as for high-throughput screening of bispecific combinations, before proceeding to synthesis of lead candidates via recombinant engineering or chemical cross-linking

Lipid Stabilized Solid Drug Nanoparticles for Targeted Chemotherapy

Nanoparticle-based chemotherapeutics have gained widespread interest in medicine due to their tunable pharmacokinetics and pharmacodynamics. Various drug delivery vehicles have been developed including polymer, liposome nanoparticles, and some of them have already made clinical impacts. Despite these advances, drug payload of these formulations is limited (typically <10%). Here, we report a general and scalable approach to prepare lipid-coated solid drug nanoparticles by combining flash nanoprecipitation and extrusion technique, which enables optimization of individual steps separately and flexibility in selection of nanoparticle surface functionalities. Using methotrexate as a model drug, the nanoparticles significantly outperformed free drug in tumor growth suppression

Stably Doped Conducting Polymer Nanoshells by Surface Initiated Polymerization

Despite broad applications ranging from electronics to biomedical sensing and imaging, a long-standing problem of conducting polymers is the poor resistance to dedoping, which directly affects their signature electrical and optical properties. This problem is particularly significant for biomedical uses because of fast leaching of dopant ions in physiological environments. Here, we describe a new approach to engineer multimodal core–shell nanoparticles with a stably doped conductive polymer shell in biological environments. It was achieved by making a densely packed polymer brush rather than changing its molecular structure. Polyaniline (PANI) was used as a model compound due to its concentrated near-infrared (NIR) absorption. It was grafted onto a magnetic nanoparticle via a polydopamine intermediate layer. Remarkably, at pH 7 its conductivity is ca. 2000× higher than conventional PANI nanoshells. Similarly, its NIR absorption is enhanced by 2 orders of magnitude, ideal for photothermal imaging and therapy. Another surprising finding is its nonfouling property, even outperforming polyethylene glycol. This platform technology is also expected to open exciting opportunities in engineering stable conductive materials for electronics, imaging, and sensing

Magneto-Optical Nanoparticles for Cyclic Magnetomotive Photoacoustic Imaging

Photoacoustic imaging has emerged as a highly promising tool to visualize molecular events with deep tissue penetration. Like most other modalities, however, image contrast under <i>in vivo</i> conditions is far from optimal due to background signals from tissue. Using iron oxide–gold core–shell nanoparticles, we have previously demonstrated the concept of magnetomotive photoacoustic (mmPA) imaging, which is capable of dramatically reducing the influence of background signals and producing high-contrast molecular images. Here, we report two significant advances toward clinical translation of this technology. First, we introduce a new class of compact, uniform, magneto-optically coupled core–shell nanoparticles, prepared through localized copolymerization of polypyrrole (PPy) on an iron oxide nanoparticle surface. The resulting iron oxide–PPy nanoparticles feature high colloidal stability and solve the photoinstability and small-scale synthesis problems previously encountered by the gold coating approach. In parallel, we have developed a new generation of mmPA featuring cyclic magnetic motion and ultrasound speckle tracking (USST), whose imaging capture frame rate is several hundred times faster than the photoacoustic speckle tracking (PAST) method we demonstrated previously. These advances enable robust artifact elimination caused by physiologic motions and demonstrate the application of the mmPA technology for <i>in vivo</i> sensitive tumor imaging

Cross-Platform Cancer Cell Identification Using Telomerase-Specific Spherical Nucleic Acids

Distinguishing tumor cells from normal cells holds the key to precision diagnosis and effective intervention of cancers. The fundamental difficulties, however, are the heterogeneity of tumor cells and the lack of truly specific and ideally universal cancer biomarkers. Here, we report a concept of tumor cell detection, bypassing the specific genotypic and phenotypic features of different tumor cell types and directly going toward the hallmark of cancer, uncontrollable growth. Combining spherical nucleic acids (SNAs) with exquisitely engineered molecular beacons (SNA beacons, dubbed SNAB technology) is capable of identifying tumor cells from normal cells based on the molecular phenotype of telomerase activity, largely bypassing the heterogeneity problem of cancers. Owing to the cell-entry capability of SNAs, the SNAB probe readily achieves tumor cell detection across multiple platforms, ranging from solution-based assay, to single cell imaging and <i>in vivo</i> solid tumor imaging (unlike PCR that is restricted to cell lysates). We envision the SNAB technology will impact cancer diagnosis, therapeutic response assessment, and image-guided surgery

<i>In Vitro</i> Toxicity Assessment of Amphiphillic Polymer-Coated CdSe/ZnS Quantum Dots in Two Human Liver Cell Models

Semiconductor quantum dots (Qdots) are a promising new technology with benefits in the areas of medical diagnostics and therapeutics. Qdots generally consist of a semiconductor core, capping shell, and surface coating. The semiconductor core of Qdots is often composed of group II and VI metals (<i>e.g.</i>, Cd, Se, Te, Hg) that are known to have toxic properties. Various surface coatings have been shown to stabilize Qdots and thus shield cells from the toxic properties of their core elements. In this study, HepG2 cells and primary human liver (PHL) cells were chosen as <i>in vitro</i> tissue culture models of human liver to examine the possible adverse effects of tri-<i>n</i>-octylphosphine oxide, poly(maleic anhydride-<i>alt</i>-1-tetradecene) copolymer (TOPO-PMAT)-coated CdSe/ZnS Qdots (TOPO-PMAT Qdots). The TOPO-PMAT coating is desirable for increasing aqueous solubility and ease of conjugation to targeting moieties (<i>e.g.</i>, aptamers and peptides). HepG2 cells avidly incorporated these TOPO-PMAT Qdots into subcellular vesicles. However, PHL cells did not efficiently take up TOPO-PMAT Qdots, but nonparenchymal cells did (especially Kupffer cells). No acute toxicity or morphological changes were noted in either system at the exposure levels used (up to 40 nM). However, cellular stress markers and pro-inflammatory cytokines/chemokines were increased in the PHL cell cultures, suggesting that TOPO-PMAT Qdots are not likely to cause acute cytotoxicity in the liver but may elicit inflammation/hepatitis, demonstrating the importance of relevant preclinical safety models. Thus, further <i>in vivo</i> studies are warranted to ensure that TOPO-PMAT-coated Qdots used in biomedical applications do not induce inflammatory responses as a consequence of hepatic uptake

Amphiphilic polymer-coated CdSe/ZnS quantum dots induce pro-inflammatory cytokine expression in mouse lung epithelial cells and macrophages

<p>Quantum dots (Qdots) are semiconductor nanoparticles with size-tunable fluorescence capabilities with diverse applications. Qdots typically contain cadmium or other heavy metals, hence raising concerns of their potential toxicity, especially in occupational settings where inhalation of nanomaterials may increase the risk of lung disease. Accordingly, we assessed the effects of tri-<i>n</i>-octylphosphine oxide, poly(maleic anhydride-<i>alt</i>-1-tetradecene) (TOPO-PMAT) coated CdSe/ZnS Qdots on mouse lung epithelial cells and macrophages. Mouse tracheal epithelial cells (MTEC), grown as organotypic cultures, bone marrow-derived macrophages (BMDM), and primary alveolar macrophages (AM) were derived from C57BL/6J or A/J mice and treated with TOPO-PMAT CdSe/ZnS Qdots (10–160 nM) for up to 24 h. Cadmium analysis showed that Qdots remained in the apical compartment of MTEC cultures, whereas they were avidly internalized by AM and BMDM, which did not differ between strains. In MTEC, Qdots selectively induced expression (mRNA and protein) of neutrophil chemokines CXCL1 and CXCL2 but only low to no detectable levels of other factors assessed. In contrast, 4 h exposure to Qdots markedly increased expression of CXCL1, IL6, IL12, and other pro-inflammatory factors in BMDM. Higher inflammatory response was seen in C57BL/6J than in A/J BMDM. Similar expression responses were observed in AM, although overall levels were less robust than in BMDM. MTEC from A/J mice were more sensitive to Qdot pro-inflammatory effects while macrophages from C57BL/6J mice were more sensitive. These findings suggest that patterns of Qdot-induced pulmonary inflammation are likely to be cell-type specific and genetic background dependent.</p

The Glutathione Synthesis Gene <i>Gclm</i> Modulates Amphiphilic Polymer-Coated CdSe/ZnS Quantum Dot–Induced Lung Inflammation in Mice

<div><p>Quantum dots (QDs) are unique semi-conductor fluorescent nanoparticles with potential uses in a variety of biomedical applications. However, concerns exist regarding their potential toxicity, specifically their capacity to induce oxidative stress and inflammation. In this study we synthesized CdSe/ZnS core/shell QDs with a tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) (TOPO-PMAT) coating and assessed their effects on lung inflammation in mice. Previously published <i>in vitro</i> data demonstrated these TOPO-PMAT QDs cause oxidative stress resulting in increased expression of antioxidant proteins, including heme oxygenase, and the glutathione (GSH) synthesis enzyme glutamate cysteine ligase (GCL). We therefore investigated the effects of these QDs <i>in vivo</i> in mice deficient in GSH synthesis (<i>Gclm</i> +/− and <i>Gclm</i> −/− mice). When mice were exposed via nasal instillation to a TOPO-PMAT QD dose of 6 µg cadmium (Cd) equivalents/kg body weight, neutrophil counts in bronchoalveolar lavage fluid (BALF) increased in both <i>Gclm</i> wild-type (+/+) and <i>Gclm</i> heterozygous (+/−) mice, whereas <i>Gclm</i> null (−/−) mice exhibited no such increase. Levels of the pro-inflammatory cytokines KC and TNFα increased in BALF from <i>Gclm</i> +/+ and +/− mice, but not from <i>Gclm</i> −/− mice. Analysis of lung Cd levels suggested that QDs were cleared more readily from the lungs of <i>Gclm</i> −/− mice. There was no change in matrix metalloproteinase (MMP) activity in any of the mice. However, there was a decrease in whole lung myeloperoxidase (MPO) content in <i>Gclm</i> −/− mice, regardless of treatment, relative to untreated <i>Gclm</i> +/+ mice. We conclude that in mice TOPO-PMAT QDs have <i>in vivo</i> pro-inflammatory properties, and the inflammatory response is dependent on GSH synthesis status. Because there is a common polymorphism in humans that influences GCLM expression, these findings imply that humans with reduced GSH synthesis capabilities may be more susceptible to the pro-inflammatory effects of QDs.</p></div