Poly(triazolyl methacrylate) glycopolymers as potential targeted unimolecular nanocarriers

© The Royal Society of Chemistry 2019.Synthetic glycopolymers are increasingly investigated as multivalent ligands for a range of biological and biomedical applications. This study indicates that glycopolymers with a fine-tuned balance between hydrophilic sugar pendant units and relatively hydrophobic polymer backbones can act as single-chain targeted nanocarriers for low molecular weight hydrophobic molecules. Non-covalent complexes formed from poly(triazolyl methacrylate) glycopolymers and low molecular weight hydrophobic guest molecules were characterised through a range of analytical techniques-DLS, SLS, TDA, fluorescence spectroscopy, surface tension analysis-and molecular dynamics (MD) modelling simulations provided further information on the macromolecular characteristics of these single chain complexes. Finally, we show that these nanocarriers can be utilised to deliver a hydrophobic guest molecule, Nile red, to both soluble and surface-immobilised concanavalin A (Con A) and peanut agglutinin (PNA) model lectins with high specificity, showing the potential of non-covalent complexation with specific glycopolymers in targeted guest-molecule delivery.Peer reviewedFinal Published versio

Mechanism of mucosal permeability enhancement of CriticalSorb® (Solutol® HS15) investigated In Vitro in cell cultures.

Purpose CriticalSorb™, with the principal component Solutol® HS15, is a novel mucosal drug delivery system demonstrated to improve the bioavailability of selected biotherapeutics. The intention of this study is to elucidate mechanism(s) responsible for the enhancement of trans-mucosal absorption of biological drugs by Solutol® HS15. Methods Micelle size and CMC of Solutol® HS15 were determined in biologically relevant media. Polarised airway Calu-3 cell layers were used to measure the permeability of a panel of biological drugs, and to assess changes in TEER, tight junction and F-actin morphology. The rate of cell endocytosis was measured in vitro in the presence of Solutol® HS15 using a membrane probe, FM 2–10. Results This work initially confirms surfactant-like behaviour of Solutol® HS15 in aqueous media, while subsequent experiments demonstrate that the effect of Solutol® HS15 on epithelial tight junctions is different from a ‘classical’ tight junction opening agent and illustrate the effect of Solutol® HS15 on the cell membrane (endocytosis rate) and F-actin cytoskeleton. Conclusion Solutol® HS15 is the principle component of CriticalSorb™ that has shown an enhancement in permeability of medium sized biological drugs across epithelia. This study suggests that its mechanism of action arises primarily from effects on the cell membrane and consequent impacts on the cell cytoskeleton in terms of actin organisation and tight junction opening

Structural and binding characterization of the LacdiNAc-specific adhesin (LabA; HopD) exodomain from Helicobacter pylori

Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 ​Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20–49%), LabA shares an L-shaped fold with SabA and BabA. The ‘head’ region contains a 4 ​+ ​3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain (‘crown’ region), the ‘J99-type’ (insertion ~31 ​amino acids), and the H. pylori ‘26695 type’ (insertion ~46 ​amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding

Targeted PEG-poly(glutamic acid) complexes for inhalation protein delivery to the lung

Pulmonary delivery is increasingly seen as an attractive, non-invasive route for the delivery of forthcoming protein therapeutics. In this context, here we describe protein complexes with a new ‘complexing excipient’ - vitamin B12-targeted poly(ethylene glycol)-block-poly(glutamic acid) copolymers. These form complexes in sub-200 nm size with a model protein, suitable for cellular targeting and intracellular delivery. Initially we confirmed expression of vitamin B12-internalization receptor (CD320) by Calu-3 cells of the in vitro lung epithelial model used, and demonstrated enhanced B12 receptor-mediated cellular internalization of B12-targeted complexes, relative to non-targeted counterparts or protein alone. To develop an inhalation formulation, the protein complexes were spray dried adopting a standard protocol into powders with aerodynamic diameter within the suitable range for lower airway deposition. The cellular internalization of targeted complexes from dry powders applied directly to Calu-3 model was found to be 2–3 fold higher compared to non-targeted complexes. The copolymer complexes show no complement activation, and in vivo lung tolerance studies demonstrated that repeated administration of formulated dry powders over a 3 week period in healthy BALB/c mice induced no significant toxicity or indications of lung inflammation, as assessed by cell population count and quantification of IL-1β, IL-6, and TNF-α pro-inflammatory markers. Importantly, the in vivo data appear to suggest that B12-targeted polymer complexes administered as dry powder enhance lung retention of their protein payload, relative to protein alone and non-targeted counterparts. Taken together, our data illustrate the potential developability of novel B12-targeted poly(ethylene glycol)-poly(glutamic acid) copolymers as excipients suitable to be formulated into a dry powder product for the inhalation delivery of proteins, with no significant lung toxicity, and with enhanced protein retention at their in vivo target tissue

Sequence Defined Disulfide-Linked Shuttle for Strongly Enhanced Intracellular Protein Delivery

Intracellular protein transduction technology is opening the door for a promising alternative to gene therapy. Techniques have to address all critical steps, like efficient cell uptake, endolysosomal escape, low toxicity, while maintaining full functional activity of the delivered protein. Here, we present the use of a chemically precise, structure defined three-arm cationic oligomer carrier molecule for protein delivery. This carrier of exact and low molecular weight combines good cellular uptake with efficient endosomal escape and low toxicity. The protein cargo is covalently attached by a bioreversible disulfide linkage. Murine 3T3 fibroblasts could be transduced very efficiently with cargo nlsEGFP, which was tagged with a nuclear localization signal. We could show subcellular delivery of the nlsEGFP to the nucleus, confirming cytosolic delivery and expected subsequent subcellular trafficking. Transfection efficiency was concentration-dependent in a directly linear mode and 20-fold higher in comparison with HIV-TAT-nlsEGFP containing a functional TAT transduction domain. Furthermore, β-galactosidase as a model enzyme cargo, modified with the carrier oligomer, was transduced into neuroblastoma cells in enzymatically active form

The involvement of microtubules and actin filaments in the intracellular transport of non-viral gene delivery system

It is known that two cytoskeleton components, microtubules and actins filaments, are required for efficient endocytosis. The relative importance of these two components in the cellular uptake of 2-(dimethylamino)ethyl methacrylate (DMAEMA)-DNA polyplexes was investigated in this study by applying microtubule depolymerising agent, colchicine, and actin polymerising inhibitor, cytochalasin D, in a cell transfection study. The effect of colchicine on transfection efficiency of polyplexes was found to be a time-dependent phenomenon, whereby the level of gene expression was inhibited at early stage, presumably to the disruption of a transport of vesicles along microtubules by colchicine. As time progressed, the level of gene expression was significantly enhanced relative to the control, possibly due to the failure in transport of vesicles from endosomes to late lysosomes, or due to the breakdown of nuclear membrane when mitosis was arrested at metaphase by colchicine. On the other hand, transfection efficiency was significantly reduced at all time points by cytochalasin D, which is considered to primarily affects invagination of vesicles at the early stage of endocytosis by inhibiting actin polymerisation. Further investigation to identify the endocytotic route of DMAEMA polyplexes was conducted applying clathrin- and caveolae- pathways inhibitors in cell transfection study. The results indicate that DMAEMA polyplexes were internalized primarily through clathrin-mediated pathway, with a minor fraction possibly entering cells via a caveolae-mediated pathway. © 2011 Informa UK, Ltd.link_to_subscribed_fulltex

Folate conjugated phosphorylcholine-based polycations for specific targeting in nucleic acids delivery

Folic acid has been investigated as a targeting ligand for imaging and therapeutic agent for over a decade; however, studies on its use in targeting of nonviral gene or nucleic acids delivery systems are sparse. This study assesses potential application of a new folic acid conjugate with aminomethacrylatephosphoryl-choline based copolymer (DMAEMA-MPCFA) as a targeting gene delivery vector. The folate-conjugated polymers produce colloidally stable polyplexes with a particle size <200nm and demonstrate the ability to protect DNA from enzymatic degradation to a certain extent. In cells that overexpress folate receptors (MCF-7 and KB cultures), the conjugated systems show a folate-specific association and achieved significantly enhanced transfection efficiency, compared to the nonconjugated control, with a dramatically reduced nonspecific cellular association. The transfection enhancement is achieved without a corresponding increase in cellular association, suggesting that an internal cellular trafficking of folate-conjugated system may be altered, resulting in an increased transfection efficacy. In summary, a new folate-conjugated aminomethacrylatephosphorylcholine copolymer is capable of forming colloidal complexes with DNA, modulating their specific cell uptake and improving the level of cell transfection in folate expressing cells. © 2009 Informa UK Ltd.link_to_subscribed_fulltex