High-throughput mechanical phenotyping and transcriptomics of single cells.

The molecular system regulating cellular mechanical properties remains unexplored at single-cell resolution mainly due to a limited ability to combine mechanophenotyping with unbiased transcriptional screening. Here, we describe an electroporation-based lipid-bilayer assay for cell surface tension and transcriptomics (ELASTomics), a method in which oligonucleotide-labelled macromolecules are imported into cells via nanopore electroporation to assess the mechanical state of the cell surface and are enumerated by sequencing. ELASTomics can be readily integrated with existing single-cell sequencing approaches and enables the joint study of cell surface mechanics and underlying transcriptional regulation at an unprecedented resolution. We validate ELASTomics via analysis of cancer cell lines from various malignancies and show that the method can accurately identify cell types and assess cell surface tension. ELASTomics enables exploration of the relationships between cell surface tension, surface proteins, and transcripts along cell lineages differentiating from the haematopoietic progenitor cells of mice. We study the surface mechanics of cellular senescence and demonstrate that RRAD regulates cell surface tension in senescent TIG-1 cells. ELASTomics provides a unique opportunity to profile the mechanical and molecular phenotypes of single cells and can dissect the interplay among these in a range of biological contexts.

Versatile Mitogenic and Differentiation-Inducible Layer Formation by Underwater Adhesive Polypeptides.

Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation-inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin-like growth factor with a key amino acid (3,4-dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid-phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long-lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.

Controlled quercetin release from high-capacity-loading hyperbranched polyglycerol-functionalized graphene oxide.

PURPOSE: An efficient drug-delivery system was prepared based on graphene oxide using a facile and one-step strategy for controlling the release of anticancer drugs. METHODS: Fabrication of single-layer graphene oxide (GO) sheets was carried out by both modified and improved Hummers method. Biocompatible hyperbranched polyglycerol (HPG) was grafted on the surface of GO through the ring-opening hyperbranched polymerization of glycidol. Various ratios of GO and glycidol were used for polymer grafting. An anticancer drug, quercetin (Qu), was loaded into modified GO via noncovalent interactions. RESULTS: Polymer grafting on the surface of GO sheets was confirmed by results obtained from Fourier-transform infrared and Raman spectroscopy, thermogravimetric analysis, energy-dispersive X-ray and X-ray spectroscopy, scanning electron microscopy, and atomic force microscopy. It was revealed that polymerization increased d-spacing between the basal planes. In addition, as a hydrophilic polymer, HPG improved the stability and dispersion of GO sheets in biological solutions and endowed extra drug-loading capacity for the sheets. The effect of hyperbranched structure on drug loading and release was investigated by comparing drug loading and release for HPG-modified GO and linear PPO-modified GO. Our experiments indicated high drug-loading capacity (up to 185%), and excellent encapsulation efficiency (up to 93%) for HPG-GO compared to linear PO-grafted GO. The release profile of Qu under various pH levels exhibited controlled and sustained drug release without an initial burst effect for HPG-GO, suggesting that an acidic solution could facilitate drug release. HPG-GO did not show any cytotoxicity on the MCF7 cell line in different concentrations during 72 hours' incubation. Uptake and entrance of HPG-GO into the cells were verified by determining the intracellular amount of Qu by high-performance liquid chromatography. CONCLUSION: A combination of the unique properties of GO and the biodegradable polymer polyglycerol revealed high drug-loading capacity, pH-dependent drug release, and cytocompatibility with HPG-GO, thus introducing it as a promising nanocarrier for anticancer drug delivery.

Wash-free and selective imaging of epithelial cell adhesion molecule (EpCAM) expressing cells with fluorogenic peptide ligands.

Detection of the cells expressing an epithelial cell adhesion molecule (EpCAM) is a crucial step to identify circulating tumor cells (CTCs) from blood. To detect the EpCAM, we here designed and synthesized a series of fluorogenic peptides. Specifically, we functionalized an EpCAM-binding peptide, Ep114, by replacing its amino acids to an aminophenylalanine that was modified with environmentally sensitive 7-nitro-2,1,3-benzoxadiazole (NBD-amPhe). Among six synthesized peptides, we have found that two peptides, Q4X and V6X (X represents NBD-amPhe), retain the Ep114's binding ability and specifically mark EpCAM-expressing cells by just adding these peptides to the cultivation medium. Our wash-free, fluorogenic peptide ligands would boost the development of next generation devices for CTC diagnoses.

Radiation grafting of N-vinylcaprolactam onto nano and macrogels of chitosan: Synthesis and characterization.

The aim of this study was to synthesize chitosan hydrogels, in macro- and nano-size, grafted with N-vinylcaprolactam (NVCL) using gamma radiation, and evaluate their potential application as a drug delivery system, using 5-fluorouracil (5-FU) as a model drug. The effect of dose and monomer concentration in the grafting process was studied, and the materials were characterized by FTIR, TGA, DLS, SEM and AFM. Higher grafting percentages were observed for the nanogels system. Although both the grafted macro- and nanogels, (net-CS)-g-NVCL, showed a response to pH (4.75) and temperature (31-33°C), the nanogels showed a better swelling response to both stimuli because of their higher surface area. Both systems were able to load 5-FU in small amounts (2-3.5mgg-1) and the release was sustained for more than 12h, showing that the modified macro and nanogels can be a potential alternative for the administration of drugs.

Novel microarrays for simultaneous serodiagnosis of multiple antiviral antibodies.

We developed an automated diagnostic system for the detection of virus-specific immunoglobulin Gs (IgGs) that was based on a microarray platform. We compared efficacies of our automated system with conventional enzyme immunoassays (EIAs). Viruses were immobilized to microarrays using a radical cross-linking reaction that was induced by photo-irradiation. A new photoreactive polymer containing perfluorophenyl azide (PFPA) and poly(ethylene glycol) methacrylate was prepared and coated on plates. Inactivated measles, rubella, mumps, Varicella-Zoster and recombinant Epstein-Barr viruse antigen were added to coated plates, and irradiated with ultraviolet light to facilitate immobilization. Virus-specific IgGs in healthy human sera were assayed using these prepared microarrays and the results obtained compared with those from conventional EIAs. We observed high correlation (0.79-0.96) in the results between the automated microarray technique and EIAs. The microarray-based assay was more rapid, involved less reagents and sample, and was easier to conduct compared with conventional EIA techniques. The automated microarray system was further improved by introducing reagent storage reservoirs inside the chamber, thereby conserving the use of expensive reagents and antibodies. We considered the microarray format to be suitable for rapid and multiple serological diagnoses of viral diseases that could be developed further for clinical applications.