It is well-known that pluripotent human embryonic stem cells (hPSC) can differentiate into any cell type. Recently, we reported that hPSC colonies enter stasis when immersed in an extremely soft hydrogel comprising hydroxyl-functional block copolymer worms (I. Canton, N. J. Warren, A. Chahal, K. Amps, A. Wood, R. Weightman, E. Wang, H. Moore and S. P. Armes, ACS Centr. Sci., 2016, 2, 65-74). The gel modulus and chemical structure of this synthetic hydrogel are similar to that of natural mucins, which are implicated in the mechanism of diapause for mammalian embryos. Does stasis induction occur merely because of the very soft nature of such hydrogels or does chemical functionality also play a role? Herein, we address this key question by designing a new hydrogel of comparable softness in which the PGMA stabilizer chains are replaced with non-hydroxylated poly(ethylene glycol) [PEG]. Immunolabeling studies confirm that hPSC colonies immersed in such PEG-based hydrogels do not enter stasis but instead proliferate (and differentiate if no adhesion substrate is present). However, pluripotency is retained if an appropriate adhesion substrate is provided. Thus, the chemical functionality of the hydrogel clearly plays a decisive role in the stasis induction mechanism.

A set of poly(amide triazoles) (PT) was synthesized by azide-alkyne cycloaddition reactions from N,N-Bis[2-(propargylamido)ethyl]-N-[2-(tert-butoxycarbonylamino)ethyl] amine and 1,6-diazido-1,6-dideoxy-u-mannitol derivatives, having the secondary hydroxyl groups protected by removable acetal and/or acetyl groups. Either copper-catalyzed (CuAAC) as well as the classical thermal cycloaddition reactions were used to afford in good yields and medium molecular weight-linear PT featuring either 1,4- or 1,4/1,5-disubstituted triazole rings along the polymer chains, respectively. Full structural characterization of all PT was accomplished and comparatively studied. Protecting group removal allowed for modulating the flexibility and polarity of cationic polytriazoles (CP), entitling for structure-activity relationship insight of their self-assembling capabilities in the presence of nucleic acids. Hence, CP and DNA were formulated at different nitrogen-to-phosphate (N/P) ratios and the resulting polyplexes were characterized by Dynamic Light Scattering (DIS) and Transmission Electron Microscopy (TEM). All CP were able to condense DNA into positively-charged nanometric-sized and narrowpolydispersed particles that could mediated transfection into human embryonic stem cells (hESC) with negligible toxicity.

Dengue fever-mimicking pH-responsive framboidal triblock copolymer vesicles enable delivery of a nucleic acid payload to the nuclei of triple-negative breast cancer cells.

Human pluripotent stem cells (hPSCs; both embryonic and induced pluripotent) rapidly proliferate in adherent culture to maintain their undifferentiated state. However, for mammals exhibiting delayed gestation (diapause), mucin-coated embryos can remain dormant for days or months in utero, with their constituent PSCs remaining pluripotent under these conditions. Here we report cellular stasis for both hPSC colonies and preimplantation embryos immersed in a wholly synthetic thermoresponsive gel comprising poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) [PGMA55-PHPMA135] diblock copolymer worms. This hydroxyl-rich mucin-mimicking nonadherent 3D gel maintained PSC viability and pluripotency in the quiescent G0 state without passaging for at least 14 days. Similarly, gel-coated human embryos remain in a state of suspended animation (diapause) for up to 8 days. The discovery of a cryptic cell arrest mechanism for both hPSCs and embryos suggests an important connection between the cellular mechanisms that evoke embryonic diapause and pluripotency. Moreover, such synthetic worm gels offer considerable utility for the short-term (weeks) storage of either pluripotent stem cells or human embryos without cryopreservation.

Glycopolymer-based nanostructures are invaluable tools to both study biological phenomena and to design future targeted drug delivery systems. Polymerization-induced self-assembly, especially RAFT aqueous dispersion polymerization is a unique method to prepare such polymer nanostructures, as it enables the preparation of very-well-defined morphologies at very high concentrations. Here we describe the implementation of PISA to the synthesis of galactosylated spheres, wormlike micelles and vesicles, and the preliminary results of cell toxicity, cell uptake, and cargo delivering capacity of galactose-decorated vesicles.

Polymersomes have the potential to encapsulate and deliver chemotherapeutic drugs into tumor cells, reducing off-target toxicity that often compromises anticancer treatment. Here, we assess the ability of the pH-sensitive poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)- poly 2-(diisopropylamino)ethyl methacrylate (PDPA) polymersomes to encapsulate chemotherapeutic agents for effective combinational anticancer therapy. Polymersome uptake and ability to deliver encapsulated drugs into healthy normal oral cells and oral head and neck squamous cell carcinoma (HNSCC) cells was measured in two and three-dimensional culture systems. PMPC-PDPA polymersomes were more rapidly internalized by HNSCC cells compared to normal oral cells. Polymersome cellular uptake was found to be mediated by class B scavenger receptors. We also observed that these receptors are more highly expressed by cancer cells compared to normal oral cells, enabling polymersome-mediated targeting. Doxorubicin and paclitaxel were encapsulated into pH-sensitive PMPC-PDPA polymersomes with high efficiencies either in isolation or as a dual-load for both singular and combinational delivery. In monolayer culture, only a short exposure to drug-loaded polymersomes was required to elicit a strong cytotoxic effect. When delivered to three-dimensional tumor models, PMPC-PDPA polymersomes were able to penetrate deep into the center of the spheroid resulting in extensive cell damage when loaded with both singular and dual-loaded chemotherapeutics. PMPC-PDPA polymersomes offer a novel system for the effective delivery of chemotherapeutics for the treatment of HNSCC. Moreover, the preferential internalization of PMPC polymersomes by exploiting elevated scavenger receptor expression on cancer cells opens up the opportunity to target polymersomes to tumors.

Therapeutic DeliveryVol. 5, No. 2 EditorialGalactosylated block copolymers: a versatile nano-based tool for effective intracellular drug delivery?Irene Canton, Burcin Ustbas & Steven P ArmesIrene Canton* Author for correspondenceDepartment of Biomedical Sciences, University of Sheffield, Sheffield, South Yorkshire, S10 2TN, UK. , Burcin UstbasDepartment of Biomedical Sciences, University of Sheffield, Sheffield, South Yorkshire, S10 2TN, UK & Steven P ArmesDepartment of Chemistry, University of Sheffield, Sheffield, South Yorkshire, S3 7HF, UKPublished Online:31 Jan 2014https://doi.org/10.4155/tde.13.141AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: block copolymerscell targetingdrug deliverygalactosegalectinslectinsnanoparticlessugar ligandsReferences1 Avila-Olias M, Pegoraro C, Battaglia G, Canton I. 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The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

A new cysteine-based methacrylic monomer (CysMA) was conveniently synthesized via selective thia-Michael addition of a commercially available methacrylate-acrylate precursor in aqueous solution without recourse to protecting group chemistry. Poly(cysteine methacrylate) (PCysMA) brushes were grown from the surface of silicon wafers by atom-transfer radical polymerization. Brush thicknesses of ca. 27 nm were achieved within 270 min at 20 °C. Each CysMA residue comprises a primary amine and a carboxylic acid. Surface zeta potential and atomic force microscopy (AFM) studies of the pH-responsive PCysMA brushes confirm that they are highly extended either below pH 2 or above pH 9.5, since they possess either cationic or anionic character, respectively. At intermediate pH, PCysMA brushes are zwitterionic. At physiological pH, they exhibit excellent resistance to biofouling and negligible cytotoxicity. PCysMA brushes undergo photodegradation: AFM topographical imaging indicates significant mass loss from the brush layer, while XPS studies confirm that exposure to UV radiation produces surface aldehyde sites that can be subsequently derivatized with amines. UV exposure using a photomask yielded sharp, well-defined micropatterned PCysMA brushes functionalized with aldehyde groups that enable conjugation to green fluorescent protein (GFP). Nanopatterned PCysMA brushes were obtained using interference lithography, and confocal microscopy again confirmed the selective conjugation of GFP. Finally, PCysMA undergoes complex base-catalyzed degradation in alkaline solution, leading to the elimination of several small molecules. However, good long-term chemical stability was observed when PCysMA brushes were immersed in aqueous solution at physiological pH.

Diblock copolymer vesicles are tagged with pH-responsive Nile Blue-based labels and used as a new type of pH-responsive colorimetric/fluorescent biosensor for far-red and near-infrared imaging of live cells. The diblock copolymer vesicles described herein are based on poly(2-(methacryloyloxy)ethyl phosphorylcholine-block-2-(diisopropylamino)ethyl methacrylate) [PMPC-PDPA]: the biomimetic PMPC block is known to facilitate rapid cell uptake for a wide range of cell lines, while the PDPA block constitutes the pH-responsive component that enables facile vesicle self-assembly in aqueous solution. These biocompatible vesicles can be utilized to detect interstitial hypoxic/acidic regions in a tumor model via a pH-dependent colorimetric shift. In addition, they are also useful for selective intracellular staining of lysosomes and early endosomes via subtle changes in fluorescence emission. Such nanoparticles combine efficient cellular uptake with a pH-responsive Nile Blue dye label to produce a highly versatile dual capability probe. This is in marked contrast to small molecule dyes, which are usually poorly uptaken by cells, frequently exhibit cytotoxicity, and are characterized by intracellular distributions invariably dictated by their hydrophilic/hydrophobic balance.

Synergy between nanotechnology and drug delivery has created a multitude of novel drug-delivery systems with great therapeutic potential. However, directing these systems across the biological barriers to the target site has proven difficult. Nanotechnology is looking for inspiration in natural systems that have evolved to overcome such barriers. Here, we review nature-inspired strategies and fundamental features common to successful drug-delivery systems across biological barriers.

Recent advances in polymer science are enabling substantial progress in nanobiotechnology, particularly in the design of new tools for enhanced understanding of cell biology and for smart drug delivery formulations. Herein, a range of novel galactosylated diblock copolymer nano-objects is prepared directly in concentrated aqueous solution via reversible addition-fragmentation chain transfer polymerization using polymerization-induced self-assembly. The resulting nanospheres, worm-like micelles, or vesicles interact in vitro with galectins as judged by a turbidity assay. In addition, galactosylated vesicles are highly biocompatible and allow intracellular delivery of an encapsulated molecular cargo.

Fluorescent microscopy becomes an essential tool for live imaging analysis of complex biological pathways and events as it enables noninvasive real-time/real-space imaging. The design of fluorescent probes to provide dynamic information and long-term tracking of samples without altering physiological and structural integrity is critical in live imaging. In recent years, nanotechnology has produced a new generation of imaging probes with promising applications in live imaging. In particular, we describe the use of pH-sensitive amphiphilic block copolymer PMPC25-PDPA70. This polymer forms biomimetic nanometer-sized vesicles (known as polymersomes) that are readily uptaken by a wide variety of cell types. The pH sensitivity confers much needed endolysomal escape capability without inducing cellular toxicity or stress. Two different characteristic compartments in the polymersomes (hydrophilic core and hydrophobic membrane) allow for encapsulation of different labeling cargoes such as lipidic cell membrane probes, quantum dots, fluorescent dyes, and fluorescent biomolecules such as nucleic acid and protein probes.

Polymeric nanoparticles, specifically polymersomes, are at the leading edge of the rapidly developing field of nanotechnology. However, their use for biological applications is primarily limited by the biocompatibility of the components. Hence, optimization of polymersome synthesis protocols should carefully consider aspects of cellular toxicity. In this work, we investigate the viability of HDF and HeLa cells treated with photo-cross-linked and pH-sensitive polymersomes. We demonstrate how aspects of polymersome preparation conditions such as cross-linking density and UV irradiation time may affect their cytotoxic properties. Additionally, we also study the cellular uptake of our polymersomes into the cell types mentioned.

There is an emerging need both in pharmacology and within the biomedical industry to develop new tools to target intracellular mechanisms. The efficient delivery of functionally active proteins within cells is potentially a powerful research strategy, especially through the use of antibodies. In this work, we report on a nanovector for the efficient encapsulation and delivery of antibodies into live cells with no significant loss of cell viability or any deleterious effect on cell metabolic activity. This delivery system is based on poly[2-(methacryloyloxy)ethyl phosphorylcholine]-block-[2-(diisopropylamino)ethyl methacrylate] (PMPC-PDPA), a pH-sensitive diblock copolymer that self-assembles to form nanometer-sized vesicles, also known as polymersomes, at physiological pH. Polymersomes can successfully deliver relatively high antibody payloads within different types of live cells. We demonstrate that these antibodies can target their respective epitope showing immunolabeling of γ-tubulin, actin, Golgi protein, and the transcription factor NF-κB in live cells. Finally, we demonstrate that intracellular delivery of antibodies can control specific subcellular events, as well as modulate cell activity and proinflammatory processes.

Endocytosis is a fundamental process in which eukaryotic cells internalise molecules and macromolecules via deformation of the membrane and generation of membrane-bound carriers. Functional aspects are not only limited to uptake of nutrients, but also play a primary role in evolutionary conserved processes such as the regulation of plasma membrane protein activity (i.e. signal-transducing receptors, small-molecule transporters and ion channels), cell motility and mitosis. The macromolecular nature of the material transported by endocytosis makes this route one of the most important targets for nanomedicine. Indeed, many nanoparticle formulations have been customised to enter cells through endocytosis and deliver the cargo within the cell. In this critical review, we present an overview of the biology of endocytosis and discuss its implications in cell internalisation of nanoparticles. We discuss how nanoparticle size, shape and surface chemistry can control this process effectively. Finally, we discuss different drug delivery strategies on how to evade lysosomal degradation to promote effective release of the cargo (376 references).