Chip-based size-selective sorting of biological cells using high frequency acoustic excitation

This work presents the size-selective sorting of single biological cells using the assembly process known as templated assembly by selective removal (TASR). We have demonstrated experimentally, for the first time, the selective placement and sorting of single SF9 cells (clonal isolate derived from Spodoptera frugiperda (Fall Armyworm) IPLB-Sf21-AE cells) into patterned hemispherical sites on rigid assembly templates using TASR. Nearly 100% of the assembly sites on the template were filled with matching cells (with assembly density as high as 900 sites per mm(2)) within short time spans of 3 minutes. 3-D reconstruction of cell profiles and volume analysis of cells trapped inside assembly sites demonstrates that only those cells that match the assembly site precisely (within 0.5 μm) in size are assembled on the template. The assembly conditions are also compatible with the extension of TASR to mammalian cells. TASR-based size-selective structuring and sorting of biological systems represents a valuable tool with potential for implementation in biological applications such as cell sorting for medical research or diagnostics, templating for artificial tissue replication, or isolation of single cells for the study of biological or mechanical behavior.     

Tandem Molecular Self‐Assembly in Liver Cancer Cells

We herein describe the tandem molecular self‐assembly of a peptide derivative ( 1 ) that is controlled by a combination of enzymatic and chemical reactions. In phosphate‐buffered saline (PBS), compound 1 self‐assembles first into nanoparticles by phosphatase and then into nanofibers by glutathione. Liver cancer cells exhibit higher concentrations of both phosphatase and GSH than normal cells. Therefore, the tandem self‐assembly of 1 also occurs in the liver cancer cell lines HepG2 and QGY7703; compound 1 first forms nanoparticles around the cells and then forms nanofibers inside the cells. Owing to this self‐assembly mechanism, compound 1 exhibits large ratios for cellular uptake and inhibition of cell viability between liver cancer cells and normal liver cells. We envision that using both extracellular and intracellular reactions to trigger tandem molecular self‐assembly could lead to the development of supramolecular nanomaterials with improved performance in cancer diagnostics and therapy.     

Construction of Organelle-Like Architecture by Dynamic DNA Assembly in Living Cells

The design of controllable dynamic systems is vital for the construction of organelle-like architectures in living cells, but has proven difficult due to the lack of control over defined topological transformation of self-assembled structures. Herein, we report a DNA based dynamic assembly system that achieves lysosomal acidic microenvironment specifically inducing topological transformation from nanoparticles to organelle-like hydrogel architecture in living cells. Designer DNA nanoparticles are constructed from double-stranded DNA with cytosine-rich stick ends (C-monomer) and are internalized into cells through lysosomal pathway. The lysosomal acidic microenvironment can activate the assembly of DNA monomers, inducing transformation from nanoparticles to micro-sized organelle-like hydrogel which could further escape into cytoplasm. We show how the hydrogel regulates cellular behaviors: cytoskeleton is deformed, cell tentacles are significantly shortened, and cell migration is promoted.     

Regulating the Rate of Molecular Self‐Assembly for Targeting Cancer Cells

Besides tight and specific ligand–receptor interactions, the rate regulation of the formation of molecular assemblies is one of fundamental features of cells. But the latter receives little exploration for developing anticancer therapeutics. Here we show that a simple molecular design of the substrates of phosphatases—tailoring the number of phosphates on peptidic substrates—is able to regulate the rate of molecular self‐assembly of the enzyme reaction product. Such a rate regulation allows selective inhibition of osteosarcoma cells over hepatocytes, which promises to target cancer cells in a specific organ. Moreover, our result reveals that the direct measurement of the rate of the self‐assembly in a cell‐based assay provides precise assessment of the cell targeting capability of self‐assembly. This work, as the first report establishing rate regulation of a multiple‐step process to inhibit cells selectively, illustrates a fundamentally new approach for controlling the fate of cells.     

Frame‐Guided Assembly of Vesicles with Programmed Geometry and Dimensions

In molecular self‐assembly molecules form organized structures or patterns. The control of the self‐assembly process is an important and challenging topic. Inspired by the cytoskeletal‐membrane protein lipid bilayer system that determines the shape of eukaryotic cells, we developed a frame‐guided assembly process as a general strategy to prepare heterovesicles with programmed geometry and dimensions. This method offers greater control over self‐assembly which may benefit the understanding of the formation mechanism as well as the functions of the cell membrane.     

Biocompatible micropatterning of two different cell types

The spatial arrangement of individual cell types can now be routinely controlled using soft-lithography-based micropatterning of complementary cell-adhesive and cell-resistant patterns. However, the application of these tools in tissue engineering to recreate tissue complexity in vitro has been hampered by the challenge of finding noncytotoxic procedures for converting complementary cell-resistant regions that define the arrangement of the first cell type into cell-adhesive regions to allow for the attachment of other cell types. A polyelectrolyte assembly approach is presented here for the first time, which allows for this noncytotoxic conversion and, thus, micropatterning of two different cell types, for example, endothelial cells and fibroblasts, on biodegradable substrates. The flexibility of this approach is further demonstrated by inducing organized capillary formation by endothelial cells on micropatterned lines followed by subsequent assembly of fibroblasts.     

Biomaterials and biofunctionality in layered macromolecular assemblies

Recent research in the field of LbL assembly is summarized and categorized as fabrication, sensing, drug release/delivery, and cell technology. Special emphasis is given to topics such as cell-membrane-mimic assembly, fabrication of free-standing biomolecular structures including protein microtubes, detection of DNA adducts and reactive metabolites, DNA hybridization analysis, sensing of toxic and bio-active chemicals, entrapment of proteins and DNA, biocomponent carriers with barcode encoding, release and delivery of DNA plasmids, multiagent delivery, smart defense capsules and oxidation-resistant films, vector introduction to cells, patterned cell culturing and microfluidic microreactors, stem cell differentiation, cellular uptake and degradation, and control of cellular apoptosis.     

Transmembrane Signaling of T Lymphocytes by Ligand-Induced Receptor Complex Assembly

T lymphocytes (T cells) are the central cell type initiating all immune responses. They are able to recognize other cells in the body that have been invaded by foreign living or nonliving matter. In such cells, foreign peptides generated by intracellular breakdown are complexed with molecules of the major histocompatibility complex (MHC) specially designed for peptide binding. Peptide-loaded MHC molecules appear on the surface of these cells and alert the immune system. The molecular complex which T cells use for recognition of peptide-loaded MHC molecules is among the most sophisticated and versatile receptor systems in biology. It consists of specific and nonspecific transmembrane components which assemble to a functional signal transduction unit as the result of ligand binding. Correct assembly leads to activation and relocation of enzymes including membrane-associated, tyrosin-specific protein kinases and phosphatases. Transmembrane signaling in T cells depends on the correct assembly and cooperation among multiple molecular components. This may be related to a multitude of different cellular responses of T cells at different stages of differentiation, all elicited through the T cell receptor complex.     

Development of porous sintered-ceramic separators for application in a LiAl/LiClKCl/FeS battery

The procedure for fabrication of porous sintered-ceramic separators and the technical feasibility of using such separators in LiAl/molten LiClKCl/FeS battery cells have been investigated. Processing techniques have been developed to fabricate ∼1.5–2.5 mm thick, ∼35–60% porous, flat, sintered Y₂O₂ and MgO separator plates with sufficient strength to allow handling prior to and during cell assembly. These separators performed successfully in laboratory-scale cells for up to ∼2000 h and 283 cycles, indicating that the concept of a sintered separator, is viable for LiAl/FeS batteries. The particularly attractive features of these separators are potentially low cost, prefabricated form that allows easy cell assembly, and small pore size (average diameter 0.5–1.0 μm) which provides good particle retention. The test results from the sintered-separator cells indicate that Y₂O₂ is probably unsuitable for long-term performance in LiAl/FeS cells because of its reaction with the positive active material. This is in agreement with the recently reported data on cells with Y₂O₂ felt and powder separators. Sintered MgO separators, however, showed good chemical and mechanical stability in the cell environment.     

Tubule Nanoclay‐Organic Heterostructures for Biomedical Applications

Natural halloysite nanotubes (HNTs) show unique hollow structure, high aspect ratio and adsorption ability, good biocompatibility, and low toxicity, which allow for various biomedical applications in the diagnosis and treatment of diseases. Here, advances in self‐assembly of halloysite for cell capturing and bacterial proliferation, coating on biological surfaces and related drug delivery, bone regeneration, bioscaffolds, and cell labeling are summarized. The in vivo toxicity of these clay nanotubes is discussed. Halloysite allows for 10–20% drug loading and can extend the delivery time to 10–100 h. These drug‐loaded nanotubes are doped into the polymer scaffolds to release the loaded drugs. The rough surfaces fabricated by self‐assembly of the clay nanotubes enhance the interactions with tumor cells, and the cell capture efficacy is significantly improved. Since halloysite has no toxicity toward microorganisms, the bacteria composed within these nanotubes can be explored in oil/water emulsion for petroleum spilling bioremediation. Coating of living cells with halloysite can control the cell growth and is not harmful to their viability. Quantum dots immobilized on halloysite were employed for cell labeling and imaging. The concluding academic results combined with the abundant availability of these natural nanotubes promise halloysite applications in personal healthcare and environmental remediation.     

In Situ Synthesis of an Aptamer‐Based Polyvalent Antibody Mimic on the Cell Surface for Enhanced Interactions between Immune and Cancer Cells

An ability to promote therapeutic immune cells to recognize cancer cells is important for the success of cell‐based cancer immunotherapy. We present a synthetic method for functionalizing the surface of natural killer (NK) cells with a supramolecular aptamer‐based polyvalent antibody mimic (PAM). The PAM is synthesized on the cell surface through nucleic acid assembly and hybridization. The data show that PAM has superiority over its monovalent counterpart in powering NKs to bind to cancer cells, and that PAM‐engineered NK cells exhibit the capability of killing cancer cells more effectively. Notably, aptamers can, in principle, be discovered against any cell receptors; moreover, the aptamers can be replaced by any other ligands when developing a PAM. Thus, this work has successfully demonstrated a technology platform for promoting interactions between immune and cancer cells.     

Tailored Combinatorial Microcompartments through the Self‐Organization of Microobjects: Assembly,Characterization, and Cell Studies

A new concept enables the generation of cell microenvironments by microobject assembly at an water/air interface. As the orientation of 30 μm sized polymer cubes and their capillary force assembly are controlled by the surface wettability, which in turn can be modulated by coating the initially exposed surfaces with gold and self‐assembled monolayers, unique niches in closely packed arrays of cubes with vertex up orientation can be realized. The random assembly of distinctly different cubes, prefunctionalized or surface‐structured exclusively on their top surface, facilitates the parallel generation of different microenvironments in a combinatorial manner, which paves the way to future systematic structure–property relationship studies with cells.     

Tailored Combinatorial Microcompartments through the Self‐Organization of Microobjects: Assembly,Characterization, and Cell Studies

A new concept enables the generation of cell microenvironments by microobject assembly at an water/air interface. As the orientation of 30 μm sized polymer cubes and their capillary force assembly are controlled by the surface wettability, which in turn can be modulated by coating the initially exposed surfaces with gold and self‐assembled monolayers, unique niches in closely packed arrays of cubes with vertex up orientation can be realized. The random assembly of distinctly different cubes, prefunctionalized or surface‐structured exclusively on their top surface, facilitates the parallel generation of different microenvironments in a combinatorial manner, which paves the way to future systematic structure–property relationship studies with cells.     

Assembly of Biologically Functional Structures by Nucleic Acid Templating: Implementation of a Strategy to Overcome Inhibition by Template Excess

Delivery of therapeutic molecules to pathogenic cells is often hampered by unintended toxicity to normal cells. In principle, this problem can be circumvented if the therapeutic effector molecule is split into two inactive components, and only assembled on or within the target cell itself. Such an in situ process can be realized by exploiting target-specific molecules as templates to direct proximity-enhanced assembly. Modified nucleic acids carrying inert precursor fragments can be designed to co-hybridize on a target-specific template nucleic acid, such that the enforced proximity accelerates assembly of a functional molecule for antibody recognition. We demonstrate the in vitro feasibility of this adaptation of nucleic acid-templated synthesis (NATS) using oligonucleotides bearing modified peptides (“haplomers”), for templated assembly of a mimotope recognized by the therapeutic antibody trastuzumab. Enforced proximity promotes mimotope assembly via traceless native chemical ligation. Nevertheless, titration of participating haplomers through template excess is a potential limitation of trimolecular NATS. In order to overcome this problem, we devised a strategy where haplomer hybridization can only occur in the presence of target, without being subject to titration effects. This generalizable NATS modification may find future applications in enabling directed targeting of pathological cells.     

In Situ Synthesis of an Aptamer-Based Polyvalent Antibody Mimic on the Cell Surface for Enhanced Interactions between Immune and Cancer Cells

An ability to promote therapeutic immune cells to recognize cancer cells is important for the success of cell-based cancer immunotherapy. We present a synthetic method for functionalizing the surface of natural killer (NK) cells with a supramolecular aptamer-based polyvalent antibody mimic (PAM). The PAM is synthesized on the cell surface through nucleic acid assembly and hybridization. The data show that PAM has superiority over its monovalent counterpart in powering NKs to bind to cancer cells, and that PAM-engineered NK cells exhibit the capability of killing cancer cells more effectively. Notably, aptamers can, in principle, be discovered against any cell receptors; moreover, the aptamers can be replaced by any other ligands when developing a PAM. Thus, this work has successfully demonstrated a technology platform for promoting interactions between immune and cancer cells.     

Assembly of three-dimensional polymeric constructs containing cells/biomolecules using carbon dioxide

Using low-pressure carbon dioxide (CO2), we demonstrated a novel and versatile approach to assembling polymeric constructs in the presence of cells and/or biomolecules in an aqueous environment. By regulating the CO2 pressure, the assembly was completed at biologically permissive temperatures with excellent preservation of the original structures. We further demonstrated that mammalian cells can survive the CO2-assisted bioassembly process (37 degrees C, 1.38 MPa, approximately 1 h). Human mesenchymal stem cells from bone marrow (hMSCs) exhibited the same cell morphology and proliferation potential as the untreated control. Mouse embryonic stem cells (mESCs) maintained ES-specific Oct-4 gene expression and differentiation potential after CO2 treatment as well. This method highlights the ability to construct multiple biodegradable polymeric scaffolds with well-defined architecture, on which various types of cells were grown, into a predesigned three-dimensional complex. In addition, protein and DNA bioactivity can be preserved in the context of a CO2-assisted assembly. This CO2-assisted bioassembly method provides for a manufacturing platform that, thus far, has been lacking in the fields of tissue engineering, cell-based biochips, cell therapy, and drug delivery.     

Dimethylarsinic acid targets tubulin in mitotic cells to induce abnormal spindles

Dimethylarsinic acid (DMA) is the most effective inducer of cell‐cycle disruption among the arsenic compounds and their metabolites. The present study was conducted to gain further insight into cell‐cycle disruption induced by DMA. The inhibition of cell proliferation and the mitotic arrest induced by DMA were significant and dose‐dependent in Chinese hamster V79 cells and the two seemed to be closely related. At less than 140 µM the DMA did not inhibit the proliferation of cells, but it significantly induced mitotic arrest. An indirect immunofluorescence assay using anti‐α‐tubulin antibodies revealed that DMA induced the formation of abnormal spindles in the metaphase cells even at 350 µM with 5 h of treatment. At 1.4 mM DMA no metaphase cells could form a definite spindle structure. The spindle figures were similar to those induced by colchicine (125 nM ) or vinblastine (110 nM ), major antimitotic agents. In DMA‐treated interphase cells, the microtubule networks were indistinguishable from those of normal cells. With the tubulin‐assembly assay estimated by turbidity, DMA at less than 200 µM suppressed tubulin assembly in a dose‐dependent manner, whereas at more than 700 µM it enhanced tubulin polymerization remarkably with or without addition of excess guanosine‐5′‐triphosphate. According to the above findings, we discussed the possibility that DMA, a primary metabolite of inorganic arsenic in mammals, is related to arsenic carcinogenicity. Copyright © 2001 John Wiley & Sons, Ltd.     

Supramolecular Polyphenol-DNA Microparticles for In Vivo Adjuvant and Antigen Co-Delivery and Immune Stimulation

DNA-based materials have attracted interest due to the tunable structure and encoded biological functionality of nucleic acids. A simple and general approach to synthesize DNA-based materials with fine control over morphology and bioactivity is important to expand their applications. Here, we report the synthesis of DNA-based particles via the supramolecular assembly of tannic acid (TA) and DNA. Uniform particles with different morphologies are obtained using a variety of DNA building blocks. The particles enable the co-delivery of cytosine-guanine adjuvant sequences and the antigen ovalbumin in model cells. Intramuscular injection of the particles in mice induces antigen-specific antibody production and T cell responses with no apparent toxicity. Protein expression in cells is shown using capsules assembled from TA and plasmid DNA. This work highlights the potential of TA as a universal material for directing the supramolecular assembly of DNA into gene and vaccine delivery platforms.     

DNA‐Edited Ligand Positioning on Red Blood Cells to Enable Optimized T Cell Activation for Adoptive Immunotherapy

Artificial antigen presenting cells (aAPCs) with surface‐anchored T cell activating ligands hold great potential in adoptive immunotherapy. However, it remains challenging to precisely control the ligand positioning on those platforms using conventional bioconjugation chemistry. Utilizing DNA‐assisted bottom‐up self‐assembly, we were able to precisely control both lateral and vertical distributions of T cell activation ligands on red blood cells (RBCs). The clustered lateral positioning of the peptide‐major histocompatibility complex (pMHC) on RBCs with a short vertical distance to the cell membrane is favorable for more effective T cell activation, likely owing to their better mimicry of natural APCs. Such optimized RBC‐based artificial APCs can stimulate T cell proliferation in vivo and effectively inhibit tumor growth with adoptive immunotherapy. DNA technology is thus a unique tool to precisely engineer the cell membrane interface and tune cell–cell interactions, which is promising for applications such as immunotherapy.