Explore the vast range of titles available.

Polymerization reactions conducted inside cells must be compatible with the complex intracellular environment, which contains numerous molecules and functional groups that could potentially prevent or quench polymerization reactions. Here we report a strategy for directly synthesizing unnatural polymers in cells through free radical photopolymerization using a number of biocompatible acrylic and methacrylic monomers. This offers a platform to manipulate, track and control cellular behaviour by the in cellulo generation of macromolecules that have the ability to alter cellular motility, label cells by the generation of fluorescent polymers for long-term tracking studies, as well as generate a variety of nanostructures within cells. It is remarkable that free radical polymerization chemistry can take place within such complex cellular environments. This demonstration opens up a multitude of new possibilities for how chemists can modulate cellular function and behaviour and for understanding cellular behaviour in response to the generation of free radicals. A strategy for directly synthesizing unnatural polymers in cells through radical polymerization has now been developed. This approach provides a platform to manipulate, track and control cellular behaviour by the in cellulo generation of macromolecules and a variety of nanostructures.

The capacity to culture stem cells in a controllable, robust and scalable manner is necessary in order to develop successful strategies for the generation of cellular and tissue platforms for drug screening, toxicity testing, tissue engineering and regenerative medicine. Creating substrates that support the expansion, maintenance or directional differentiation of stem cells would greatly aid these efforts. Optimally, the substrates used should be chemically defined and synthetically scalable, allowing growth under defined, serum-free culture conditions. To achieve this, the chemical and physical attributes of the substrates should mimic the natural tissue environment and allow control of their biological properties. Herein, recent advances in the development of materials to study/manipulate stem cells, both in vitro and in vivo, are described with a focus on the novelty of the substrates’ properties, and on application of substrates to direct stem cells. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

The cellular plasma membrane plays a fundamental role in biological processes, including cell growth, signaling and transport. The labelling of the plasma membrane with targeted fluorescent probes offers a convenient and non-invasive way to image the morphological changes and dynamics of a membrane in real-time and, despite many examples of fluorescent plasma membrane probes, a “universal targeting/anchoring moiety” is still required. In this study, a small number of stearic acid-based probes labelled with 6-carboxyfluorescein was designed and fabricated via solid-phase synthesis in which variations in both charge and hydrophobicity were explored. To ease the synthesis process, a gram-scale synthesis of the Fmoc-Lys(6-carboxyfluoresein diacetate)-OH building block was developed, allowing the discovery of optimal probes that carried a positively charged amino group and a stearic acid tail that exhibited intense plasma membrane brightness and robust retention.

Cathepsin D is a protease that is an effector in the immune response of macrophages, yet to date, only a limited number of probes have been developed for its detection. Herein, we report a water soluble, highly sensitive, pH insensitive fluorescent probe for the detection of Cathepsin D activity that provides a strong OFF/ON signal upon activation and with bright emission at 515 nm. The probe was synthesised using a combination of solid and solution-phase chemistries, with probe optimisation to increase its water solubility and activation kinetics by addition of a long PEG chain (5 kDa) at the C -terminus. A BODIPY fluorophore allowed detection of Cathepsin D across a wide pH range, important as the protease is active both at the low pH found in lysosomes and also in higher pH phagolysosomes, and in the cytosol. The probe was successfully used to detect Cathepsin D activity in macrophages challenged by exposure to bacteria. Cathepsin D (CatD) is an aspartic acid protease involved in the immune response of macrophages to bacterial infection, and the development of pH insensitive CatD probes is vital in order to detect its activity across multiple cellular components. Here, the authors develop a water soluble and pH insensitive FRET-based fluorescent probe for the sensitive detection of CatD.

The in situ detection of caspase-3 activity has applications in the imaging and monitoring of multiple pathologies, notably cancer. A series of cell penetrating FRET-based fluorogenic substrates were designed and synthesised for the detection of caspase-3 in live cells. A variety of modifications of the classical caspase-3 and caspase-7 substrate sequence Asp-Glu-Val-Asp were carried out in order to increase caspase-3 affinity and eliminate caspase-7 cross-reactivity. To allow cellular uptake and good solubility, the substrates were conjugated to a cationic peptoid. The most selective fluorogenic substrate 27, FAM-Ahx-Asp-Leu-Pro-Asp-Lys(MR)-Ahx, conjugated to the cell penetrating peptoid at the C-terminus, was able to detect and quantify caspase-3 activity in apoptotic cells without cross-reactivity by caspase-7.

The capacity to culture stem cells in a controllable, robust and scalable manner is necessary in order to develop successful strategies for the generation of cellular and tissue platforms for drug screening, toxicity testing, tissue engineering and regenerative medicine. Creating substrates that support the expansion, maintenance or directional differentiation of stem cells would greatly aid these efforts. Optimally, the substrates used should be chemically defined and synthetically scalable, allowing growth under defined, serum-free culture conditions. To achieve this, the chemical and physical attributes of the substrates should mimic the natural tissue environment and allow control of their biological properties. Herein, recent advances in the development of materials to study/manipulate stem cells, both in vitro and in vivo, are described with a focus on the novelty of the substrates' properties, and on application of substrates to direct stem cells. This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.