Partial Magneto-Endosomalysis for Cytosolic Delivery of Antibodies.

Modulation of protein functions and interactions is the most direct and effective means to intervene in cellular processes and pathogenesis. The majority of the critical intracellular signaling pathways, however, are considered undruggable using small molecules. In this regard, antibodies are superior in structural and functional diversity and are significantly easier to raise compared to the screening of small molecules. Despite these advantages, the uses of antibodies in live cells (either as an imaging agent or as a therapeutic compound) are substantially undermined, only acting on extracellular targets. The inability of targeting intracellular proteins is because of a fundamental issue: antibodies enter cells through endocytosis where the vast majority are trapped in endosomes for degradation. Here, we report a nanoparticle self-assembly strategy enabling antibody endosomal escape. We demonstrate the intracellular bioavailability of antibodies and the preserved binding specificity to their cytosolic targets. This technology is simple and opens exciting opportunities for live-cell imaging, therapeutics development, and cell engineering.

Quantum dots and mouse strain influence house dust mite-induced allergic airway disease.

Quantum dot nanoparticles (QDs) are engineered nanomaterials (ENMs) that have utility in many industries due to unique optical properties not available in small molecules or bulk materials. QD-induced acute lung inflammation and toxicity in rodent models raise concerns about potential human health risks. Recent studies have also shown that some ENMs can exacerbate allergic airway disease (AAD). In this study, C57BL/6J and A/J mice were exposed to saline, house dust mite (HDM), or a combination of HDM and QDs on day 1 of the sensitization protocol. Mice were then challenged on days 8, 9 and 10 with HDM or saline only. Significant differences in cellular and molecular markers of AAD induced by both HDM and HDM + QD were observed between C57BL/6J and A/J mice. Among A/J mice, HDM + QD co-exposure, but not HDM exposure alone, significantly increased levels of bronchoalveolar lavage fluid (BALF). IL-33 compared to saline controls. BALF total protein levels in both mouse strains were also only significantly increased by HDM + QD co-exposure. In addition, A/J mice had significantly more lung type 2 innate lymphoid cells (ILC2s) cells than C57BL/6J mice. A/J lung ILC2s were inversely correlated with lung glutathione and MHC-IIhigh resident macrophages, and positively correlated with MHC-IIlow resident macrophages. The results from this study suggest that 1) QDs influence HDM-induced AAD by potentiating and/or enhancing select cytokine production; 2) that genetic background modulates the impact of QDs on HDM sensitization; and 3) that potential ILC2 contributions to HDM induced AAD are also likely to be modulated by genetic background.

Membrane-Penetrating Carbon Quantum Dots for Imaging Nucleic Acid Structures in Live Organisms.

The dynamics of DNA and RNA structures in live cells are important for understanding cell behaviors, such as transcription activity, protein expression, cell apoptosis, and hereditary disease, but are challenging to monitor in live organisms in real time. The difficulty is largely due to the lack of photostable imaging probes that can distinguish between DNA and RNA, and more importantly, are capable of crossing multiple membrane barriers ranging from the cell/organelle to the tissue/organ level. We report the discovery of a cationic carbon quantum dot (cQD) probe that emits spectrally distinguishable fluorescence upon binding with double-stranded DNA and single-stranded RNA in live cells, thereby enabling real-time monitoring of DNA and RNA localization and motion. A surprising finding is that the probe can penetrate through various types of biological barriers in vitro and in vivo. Combined with standard and super-resolution microscopy, photostable cQDs allow time-lapse imaging of chromatin and nucleoli during cell division and Caenorhabditis elegans (C. elegans) growth.

Quantum dot induced acute changes in lung mechanics are mouse strain dependent.

INTRODUCTION: Concerns have been raised regarding occupational exposure to engineered nanomaterials (ENMs). Potential impacts on lung function from inhalation exposures are of concern as the lung is a sensitive ENM target in animals. Epidemiological data suggest that occupational exposure to ENMs may impact respiratory and cardiovascular health. Quantum dots (QDs) are ENMs with outstanding semiconductor and fluorescent properties with uses in biomedicine and electronics. QDs are known to induce inflammation and cytotoxicity in rodents and high dose exposures impact lung function 2 weeks after exposure. However, effects of mouse strain and the temporality of QD effects on lung function at more occupationally relevant doses have not been well-established. OBJECTIVE: We evaluated the impact of QD exposure on respiratory mechanics in C57BL/6J and A/J mice. Previous work found a greater initial inflammatory response to QD exposure in A/J mice compared to C57BL/6J mice. Thus, we hypothesized that A/J mice would be more sensitive to QD-induced effects on lung mechanics. METHODS: C57BL/6J and A/J mice were exposed to 6 µg/kg Cd equivalents of amphiphilic polymer-coated Cd/Se core, ZnS shell QDs via oropharyngeal aspiration. Lung mechanics were measured using forced oscillation, and inflammation was characterized by neutrophils and cytokines in bronchoalveolar lavage fluid. RESULTS: Both strains showed signs of QD-induced acute lung inflammation. However, lung mechanics were impacted by QD exposure in A/J mice only. CONCLUSIONS: Our findings suggest that susceptibility to QDs and similar ENM-induced changes in lung function may depend at least in part on genetic background.

Synthesis of hybrid magneto-plasmonic nanoparticles with potential use in photoacoustic detection of circulating tumor cells.

This article describes a novel synthetic route to obtain hybrid nanostructures that combine the plasmonic properties of gold nanorods with the magnetic properties of iron oxide nanoparticles in a robust silica nanostructure. The silica matrix enhances the physico-chemical stability of the nanostructure and preserves its magneto-plasmonic properties by avoiding the interface between gold and iron oxide. In addition, the magneto-plasmonic features of the nanohybrids can be tuned due to the independent synthesis of each component. The magnetic and plasmonic properties of these nanostructures can potentially enhance the photoacoustic detection of circulating tumor cells. Graphical abstract Schematic presentation of a hybrid magneto-plasmonic nanoparticle with an Au@Fe3O4@SiO2 core-satellite-shell arrangement. The magnetic and plasmonic responses of this kind of nanostructure enable magnetic trapping and photoacoustic detection of circulating tumor cells.

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.

Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.

Nanoparticle probes enable implementation of advanced on-surface assay formats, but impose often underappreciated size-associated constraints, in particular on assay kinetics and sensitivity. The present study highlights substantially slower diffusion-limited assay kinetics due to the rapid development of a nanoprobe depletion layer next to the surface, which static incubation and mixing of bulk solution employed in conventional assay setups often fail to disrupt. In contrast, cyclic solution draining and replenishing yields reaction-limited assay kinetics irrespective of the probe size. Using common surface bioassays, enzyme-linked immunosorbent assays and immunofluorescence, this study shows that this conceptually distinct approach effectively "erases" size-dependent diffusion constraints, providing a straightforward route to rapid on-surface bioassays employing bulky probes and procedures involving multiple labeling cycles, such as multicycle single-cell molecular profiling. For proof-of-concept, the study demonstrates that the assay time can be shortened from hours to minutes with the same probe concentration and, at a typical incubation time, comparable target labeling can be achieved with up to eight times lower nanoprobe concentration. The findings are expected to enable realization of novel assay formats and stimulate development of rapid on-surface bioassays with nanoparticle probes.

Functional Photoacoustic Imaging of Gastric Acid Secretion Using pH-Responsive Polyaniline Nanoprobes.

A stomach functional imaging technique based on photoacoustics achieves noninvasive gastric acid secretory assessment utilizing pH-responsive polyaniline nanoprobes. A testing protocol mimicking clinical practice is established using a mouse model. After imaging, the nanoprobes are excreted outside the body without inducing systematic toxicity. Further optimization and translation of this technology can help alleviate patients' suffering and side effects.

Direct Characterization of Polymer Encapsulated CdSe/CdS/ZnS Quantum Dots.

Surface engineering advances of semiconductor quantum dots (QDs) have enabled their application to molecular labeling, disease diagnostics and tumor imaging. For biological applications, hydrophobic core/shell QDs are transferred into aqueous solutions through the incorporation of water-solubility imparting moieties, typically achieved via direct exchange of the native surface passivating ligands or indirectly through the adsorption of polymers. Although polymeric encapsulation has gained wide acceptance, there are few reports addressing the characterization of the adsorbed polymers and existing theoretical analyses are typically based on simple geometric models. In this work, we experimentally characterize and quantify water-soluble QDs prepared by adsorption of amphiphilic poly(maleic anhydride-alt-1-tetradecene) (PMAT, MW~9000) onto commercially available CdSe/CdS/ZnS (CdSe/CdS/ZnS-PMAT). Using x-ray photoelectron spectroscopy (XPS) we determined that ~15 PMAT molecules are adsorbed onto each QD and sum frequency generation (SFG) vibrational spectra was utilized to investigate the mechanism of interaction between PMAT molecules and the QD surface. Importantly, when employed together, these techniques constitute a platform with which to investigate any polymer-nanoparticle complex in general.

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.

Cross-Platform DNA Encoding for Single-Cell Imaging of Gene Expression.

Integration of imaging data across different molecular target types can provide in-depth insight into cell physiology and pathology, but remains challenging owing to poor compatibility between target-type-specific labeling methods. We show that cross-platform imaging analysis can be readily achieved through DNA encoding of molecular targets, which translates the molecular identity of various target types into a uniform in situ array of ssDNA tags for subsequent labeling with complementary imaging probes. The concept was demonstrated through multiplexed imaging of mRNAs and their corresponding proteins with multicolor quantum dots. The results reveal heterogeneity of cell transfection with siRNA and outline disparity in RNA interference (RNAi) kinetics at the level of both the mRNA and the encoded protein.

Susceptibility to quantum dot induced lung inflammation differs widely among the Collaborative Cross founder mouse strains.

Quantum dots (QDs) are engineered semiconductor nanoparticles with unique physicochemical properties that make them potentially useful in clinical, research and industrial settings. However, a growing body of evidence indicates that like other engineered nanomaterials, QDs have the potential to be respiratory hazards, especially in the context of the manufacture of QDs and products containing them, as well as exposures to consumers using these products. The overall goal of this study was to investigate the role of mouse strain in determining susceptibility to QD-induced pulmonary inflammation and toxicity. Male mice from 8 genetically diverse inbred strains (the Collaborative Cross founder strains) were exposed to CdSe-ZnS core-shell QDs stabilized with an amphiphilic polymer. QD treatment resulted in significant increases in the percentage of neutrophils and levels of cytokines present in bronchoalveolar lavage fluid (BALF) obtained from NOD/ShiLtJ and NZO/HlLtJ mice relative to their saline (Sal) treated controls. Cadmium measurements in lung tissue indicated strain-dependent differences in disposition of QDs in the lung. Total glutathione levels in lung tissue were significantly correlated with percent neutrophils in BALF as well as with lung tissue Cd levels. Our findings indicate that QD-induced acute lung inflammation is mouse strain dependent, that it is heritable, and that the choice of mouse strain is an important consideration in planning QD toxicity studies. These data also suggest that formal genetic analyses using additional strains or recombinant inbred strains from these mice could be useful for discovering potential QD-induced inflammation susceptibility loci.

Nanoparticle counting: towards accurate determination of the molar concentration.

Innovations in nanotechnology have brought tremendous opportunities for the advancement of many research frontiers, ranging from electronics, photonics, energy, to medicine. To maximize the benefits of nano-scaled materials in different devices and systems, precise control of their concentration is a prerequisite. While concentrations of nanoparticles have been provided in other forms (e.g., mass), accurate determination of molar concentration, arguably the most useful one for chemical reactions and applications, has been a major challenge (especially for nanoparticles smaller than 30 nm). Towards this significant yet chronic problem, a variety of strategies are currently under development. Most of these strategies are applicable to a specialized group of nanoparticles due to their restrictions on the composition and size range of nanoparticles. As research and uses of nanomaterials are being explored in an unprecedented speed, it is necessary to develop universal strategies that are easy to use and are compatible with nanoparticles of different sizes, compositions, and shapes. This review outlines the theories and applications of current strategies to measure nanoparticle molar concentration, discusses the advantages and limitations of these methods, and provides insights into future directions.

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.

An aggregation-induced-emission platform for direct visualization of interfacial dynamic self-assembly.

An in-depth understanding of dynamic interfacial self-assembly processes is essential for a wide range of topics in theoretical physics, materials design, and biomedical research. However, direct monitoring of such processes is hampered by the poor imaging contrast of a thin interfacial layer. We report in situ imaging technology capable of selectively highlighting self-assembly at the phase boundary in real time by employing the unique photophysical properties of aggregation-induced emission. Its application to the study of breath-figure formation, an immensely useful yet poorly understood phenomenon, provided a mechanistic model supported by direct visualization of all main steps and fully corroborated by simulation and theoretical analysis. This platform is expected to advance the understanding of the dynamic phase-transition phenomena, offer insights into interfacial biological processes, and guide development of novel self-assembly technologies.

Emerging applications of conjugated polymers in molecular imaging.

In recent years, conjugated polymers have attracted considerable attention from the imaging community as a new class of contrast agent due to their intriguing structural, chemical, and optical properties. Their size and emission wavelength tunability, brightness, photostability, and low toxicity have been demonstrated in a wide range of in vitro sensing and cellular imaging applications, and have just begun to show impact in in vivo settings. In this Perspective, we summarize recent advances in engineering conjugated polymers as imaging contrast agents, their emerging applications in molecular imaging (referred to as in vivo uses in this paper), as well as our perspectives on future research.

Luminescent quantum dots for molecular toxicology.

Recent developments in nanotechnology have made available a host of new approaches for the improved quantitative detection of biomarkers due to the enhanced sensitivity of nanoparticle-based assays. The majority of molecular toxicology studies revolve around sensitive measurement of cell-death (apoptosis) and cell-health biomarkers present in living cells or formalin-fixed and paraffin embedded (FFPE) tissue samples. In this regard, semi-conductor quantum dots (QDs) which exhibit high brightness, photo-stability and degree of multiplexing, are predicted to have a significant impact on research in molecular toxicology. Due to these superior photophysical properties of QDs as compared to traditional fluorophores and the unsurpassed versatility of QDs as enabling components for new assays, these nanoparticles promise to facilitate new discoveries in molecular toxicology. Indeed, multiplexed QD-based assays have been incorporated into cell imaging, flow cytometry and other homogenized sample-based assays for detecting multiple biomarkers including those associated with cell injury and apoptosis.

Magneto-Endosomalytic Therapy for Cancer.

A remarkably simple yet effective mode of cancer treatment is reported by repurposing clinically approved magnetic nanoparticles (MNPs). Intracellular nanoparticle self-assembly directed by static parallel magnetic fields leads to cell death in targeted tissues while leaving other cells and organs intact. This simple concept opens a new avenue to treat cancer, capitalizing on nanosciences and the nanoparticle (NP) design principles accumulated in the past decades.

Rapid multitarget immunomagnetic separation through programmable DNA linker displacement.

Immunomagnetic separation has become an essential tool for high-throughput and low-cost isolation of biomolecules and cells from heterogeneous samples. However, as magnetic selection is essentially a "black-and-white" assay, its application has been largely restricted to single-target and single-parameter studies. To address this issue, we have developed an immunomagnetic separation technology that can quickly sort multiple targets in high yield and purity using selectively displaceable DNA linkers. We envision that this technology will be readily adopted for experiments requiring high-throughput selection of multiple targets or further adapted for selection of a single target based on multiple surface epitopes.