POZylation: a new approach to enhance nanoparticle diffusion through mucosal barriers

The increasing use of nanoparticles in the pharmaceutical industry is generating concomitant interest in developing nanomaterials that can rapidly penetrate into, and permeate through, biological membranes to facilitate drug delivery and improve the bioavailability of active pharmaceutical ingredients. Here, we demonstrate that the permeation of thiolated silica nanoparticles through porcine gastric mucosa can be significantly enhanced by their functionalization with either 5 kDa poly(2-ethyl-2-oxazoline) or poly(ethylene glycol). Nanoparticle diffusion was assessed using two independent techniques; Nanoparticle Tracking Analysis, and fluorescence microscopy. Our results show that poly(2-ethyl-2-oxazoline) and poly(ethylene glycol) have comparable abilities to enhance diffusion of silica nanoparticles in mucin dispersions and through the gastric mucosa. These findings provide a new strategy in the design of nanomedicines, by surface modification or nanoparticle core construction, for enhanced transmucosal drug delivery

Poly(amidoamine)-BSA conjugates synthesised by Michael addition reaction retained enzymatic activity

Polymer-protein conjugates are key to overcome some of the therapeutic protein limitations, including inefficient intracellular delivery. Poly(amidoamine)s are bioresponsive polyelectrolytes, which can form complexes with proteins and promote their delivery into the cytosol of cells. To investigate if conjugation would affect the activity of the protein, two poly(amidoamine)-BSA conjugates were synthesised using a “grafted to” method and Michael addition reaction. Following purification, the conjugates were characterised by electrophoresis, size exclusion chromatography (Mn(C1) = 140.7 kDa ; Mn(C2) = 218.6 kDa) and light scattering (Dh(C1) = 37.5 nm ; Dh(C2) = 75.1 nm). As a result of the conjugation with the cationic polymer, the conjugates had a positive zeta potential (?(C1) = +15.4 mV; ?(C2) = +20.2 mV). TNBS assays demonstrated that 16% to 25% of the protein amine groups were modified and HPLC analysis indicated that the amount of protein in the conjugate was 0.76 mg of BSA/mg of PAA (C1) and 0.43 mg of BSA /mg of PAA (C2). Enzymatic assays indicated the conjugates displayed an esterase activity similar (C1) or reduced ~ 35% (C2) compare to BSA. Altogether the results demonstrated that the conjugation of poly(amidoamine)s to a model protein can lead to the formation of bioconjugates that retain the enzymatic activity of the native protein. Such conjugates could have some application in protein delivery and enzyme engineering for biocatalysis and biosensors

Protein Coating of DNA Nanostructures for Enhanced Stability and Immunocompatibility

Fully addressable DNA nanostructures, especially DNA origami, possess huge potential to serve as inherently biocompatible and versatile molecular platforms. However, their use as delivery vehicles in therapeutics is compromised by their low stability and poor transfection rates. This study shows that DNA origami can be coated by precisely defined one-to-one protein-dendron conjugates to tackle the aforementioned issues. The dendron part of the conjugate serves as a cationic binding domain that attaches to the negatively charged DNA origami surface via electrostatic interactions. The protein is attached to dendron through cysteine-maleimide bond, making the modular approach highly versatile. This work demonstrates the coating using two different proteins: bovine serum albumin (BSA) and class II hydrophobin (HFBI). The results reveal that BSA-coating significantly improves the origami stability against endonucleases (DNase I) and enhances the transfection into human embryonic kidney (HEK293) cells. Importantly, it is observed that BSA-coating attenuates the activation of immune response in mouse primary splenocytes. Serum albumin is the most abundant protein in the blood with a long circulation half-life and has already found clinically approved applications in drug delivery. It is therefore envisioned that the proposed system can open up further opportunities to tune the properties of DNA nanostructures in biological environment, and enable their use in various delivery applications.Peer reviewe

Site-selective protein-modification chemistry for basic biology and drug development.

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.We thank FCT Portugal (FCT Investigator to G.J.L.B.), the EU (Marie-Curie CIG to G.J.L.B. and Marie-Curie IEF to O.B.) and the EPSRC for funding. G.J.L.B. is a Royal Society University Research Fellow.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/nchem.239

Modifiable and Protein-Stabilizing Polymers Prepared Using Controlled Polymerization Techniques

Even after significant advances in polymerization and protein modification chemistries, the majority of polymeric biomaterials focus on poly(ethylene glycol) (PEG)-based monomers. Despite their widespread use, these polymers present concerns due to their non-biodegradable nature, the possibility of toxicity and immunogenicity, and their limited chemical functionality. Therefore, there is significant interest in the rational design of alternative polymers with specific chemical or biological properties. Specifically, approaches that allow for the rapid and divergent synthesis of a large number of biodegradable polymeric materials capable of functionalization would be broadly applicable. This dissertation focuses on novel degradable and modifiable polymers with applications in protein conjugation and stabilization.In Chapter 1, the history of protein-polymer conjugates for therapeutic use is outlined, from PEGylation techniques to next-generation conjugation strategies. Alternative polymer technologies and future directions of the field are also presented. In Chapter 2, the synthesis and biological application of a degradable trehalose glycopolymer is described. The polymer is shown to stabilize the therapeutic protein granulocyte colony stimulating factor (G-CSF) against heat stress. While the polymer was noncytotoxic, its degradation products inhibited cell proliferation at high concentrations.Chapter 3 details the development of poly(caprolactone)-based polyesters for protein stabilization. An alkene-substituted polyester was synthesized and modified using thiol-ene chemistry with thiols containing glucose, lactose, trehalose, PEG, and carboxybetaine units. The relative stabilizing ability of these side-chains toward G-CSF was assessed. Trehalose and carboxybetaine were found to maintain the most protein activity upon exposure to heat stress. We varied the size of these polymers and found a dependence on molecular weight, where longer polymers were more effective protein stabilizers. These materials and their degradation products were cytocompatible, yet exhibited minimal degradation in aqueous conditions. Chapter 4 describes the synthesis of trehalose- and carboxybetaine-funcitonalized polyesters and polycarbonates with tunable degradability, with half-lives from 10 hours to over 4 months. We expect these materials will be useful in the development of novel protein-polymer therapeutics.We also describe the development of novel PEG analogs using ring-opening metathesis polymerization (ROMP). Chapter 5 describes the synthesis of these PEG analogs and subsequent conjugation to the model protein lysozyme. Exploration of post-polymerization modifications to install thiols onto the unsaturated polymer backbone are also described