Cellular delivery of antibodies: effective targeted subcellular imaging and new therapeutic tool

It is already more than a century since the pioneering work of the Nobel Laureate Ehrlich gave birth to the side chain theory1, which helped to define antibodies and their ability to target specific biological sites. However, the use of antibodies is still restricted to the extracellular space due to the lack of a suitable delivery vehicle for the efficient transport of antibodies into live cells without inducing toxicity. In this work, we report the efficient encapsulation and delivery of antibodies into live cells with no significant loss of cell viability or any deleterious affect on the 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. These polymersomes can successfully deliver relatively high antibody payloads within live cells. Once inside the cells, we demonstrate that these antibodies can target their epitope by immune-labelling of cytoskeleton, Golgi, and transcription factor proteins in live cells. We also demonstrate that this effective antibody delivery mechanism can be used to control specific subcellular events, as well as modulate cell activity and pro-inflammatory process

Antimicrobial graft copolymer gels

In view of the growing worldwide rise in microbial resistance, there is considerable interest in designing new antimicrobial copolymers. The aim of the current study was to investigate the relationship between antimicrobial activity and copolymer composition/ architecture to gain a better understanding of their mechanism of action. Specifically, the antibacterial activity of several copolymers based on 2- (methacryloyloxy)ethyl phosphorylcholine [MPC] and 2-hydroxypropyl methacrylate (HPMA) toward Staphylococcus aureus was examined. Both block and graft copolymers were synthesized using either atom transfer radical polymerization or reversible addition−fragmentation chain transfer polymerization and characterized via 1H NMR, gel permeation chromatography, rheology, and surface tensiometry. Antimicrobial activity was assessed using a range of well-known assays, including direct contact, live/dead staining, and the release of lactate dehydrogenase (LDH), while transmission electron microscopy was used to study the morphology of the bacteria before and after the addition of various copolymers. As expected, PMPC homopolymer was biocompatible but possessed no discernible antimicrobial activity. PMPC-based graft copolymers comprising PHPMA side chains (i.e. PMPC-g-PHPMA) significantly reduced both bacterial growth and viability. In contrast, a PMPC−PHPMA diblock copolymer comprising a PMPC stabilizer block and a hydrophobic core-forming PHPMA block did not exhibit any antimicrobial activity, although it did form a biocompatible worm gel. Surface tensiometry studies and LDH release assays suggest that the PMPC-g-PHPMA graft copolymer exhibits surfactantlike activity. Thus, the observed antimicrobial activity is likely to be the result of the weakly hydrophobic PHPMA chains penetrating (and hence rupturing) the bacterial membrane

Evidence of ratchet effect in nanowires of a conducting polymer

Ratchet effect, observed in many systems starting from living organism to artificially designed device, is a manifestation of motion in asymmetric potential. Here we report results of a conductivity study of Polypyrrole nanowires, which have been prepared by a simple method to generate a variation of doping concentration along the length. This variation gives rise to an asymmetric potential profile that hinders the symmetry of the hopping process of charges and hence the value of measured resistance of these nanowires become sensitive to the direction of current flow. The asymmetry in resistance was found to increase with decreasing nanowire diameter and increasing temperature. The observed phenomena could be explained with the assumption that the spatial extension of localized state involved in hopping process reduces as the doping concentration reduces along the length of the nanowires.Comment: Revtex, two column, 4 pages, 10 figure

A Critical Appraisal of RAFT-Mediated Polymerization-Induced Self-Assembly

Recently, polymerization-induced self-assembly (PISA) has become widely recognized as a robust and efficient route to produce block copolymer nanoparticles of controlled size, morphology, and surface chemistry. Several reviews of this field have been published since 2012, but a substantial number of new papers have been published in the last three years. In this Perspective, we provide a critical appraisal of the various advantages offered by this approach, while also pointing out some of its current drawbacks. Promising future research directions as well as remaining technical challenges and unresolved problems are briefly highlighted

RAFT Aqueous Dispersion Polymerization of N -(2-(Methacryloyloxy)ethyl)pyrrolidone: A Convenient Low Viscosity Route to High Molecular Weight Water-Soluble Copolymers

RAFT solution polymerization of N-(2-(methacryoyloxy)ethyl)pyrrolidone (NMEP) in ethanol at 70 °C was conducted to produce a series of PNMEP homopolymers with mean degrees of polymerization (DP) varying from 31 to 467. Turbidimetry was used to assess their inverse temperature solubility behavior in dilute aqueous solution, with an LCST of approximately 55 °C being observed in the high molecular weight limit. Then a poly(glycerol monomethacylate) (PGMA) macro-CTA with a mean DP of 63 was chain-extended with NMEP using a RAFT aqueous dispersion polymerization formulation at 70 °C. The target PNMEP DP was systematically varied from 100 up to 6000 to generate a series of PGMA63–PNMEPx diblock copolymers. High conversions (≥92%) could be achieved when targeting up to x = 5000. GPC analysis confirmed high blocking efficiencies and a linear evolution in Mn with increasing PNMEP DP. A gradual increase in Mw/Mn was also observed when targeting higher DPs. However, this problem could be minimized (Mw/Mn < 1.50) by utilizing a higher purity grade of NMEP (98% vs 96%). This suggests that the broader molecular weight distributions observed at higher DPs are simply the result of a dimethacrylate impurity causing light branching, rather than an intrinsic side reaction such as chain transfer to polymer. Kinetic studies confirmed that the RAFT aqueous dispersion polymerization of NMEP was approximately four times faster than the RAFT solution polymerization of NMEP in ethanol when targeting the same DP in each case. This is perhaps surprising because both 1H NMR and SAXS studies indicate that the core-forming PNMEP chains remain relatively solvated at 70 °C in the latter formulation. Moreover, dissolution of the initial PGMA63–PNMEPx particles occurs on cooling from 70 to 20 °C as the PNMEP block passes through its LCST. Hence this RAFT aqueous dispersion polymerization formulation offers an efficient route to a high molecular weight water-soluble polymer in a rather convenient low-viscosity form. Finally, the relatively expensive PGMA macro-CTA was replaced with a poly(methacrylic acid) (PMAA) macro-CTA. High conversions were also achieved for PMAA85–PNMEPx diblock copolymers prepared via RAFT aqueous dispersion polymerization for x ≤ 4000. Again, better control was achieved when using the 98% purity NMEP monomer in such syntheses

Phosphonic Acid-Functionalized Diblock Copolymer Nano-Objects via Polymerization-Induced Self-Assembly: Synthesis, Characterization, and Occlusion into Calcite Crystals

Dialkylphosphonate-functionalized and phosphonic acid-functionalized macromolecular chain transfer agents (macro-CTAs) were utilized for the reversible addition–fragmentation chain transfer (RAFT) dispersion polymerization of benzyl methacrylate (BzMA) at 20% w/w solids in methanol at 64 °C. Spherical, worm-like and vesicular nano-objects could all be generated through systematic variation of the mean degree of polymerization of the core-forming PBzMA block when using relatively short macro-CTAs. Construction of detailed phase diagrams is essential for the reproducible targeting of pure copolymer morphologies, where these were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). For nano-objects prepared using the phosphonic acid-based macro-CTA, transfer from methanol dispersion to water leads to the development of anionic surface charge as a result of ionization of the stabilizer chains, but this does not adversely affect the copolymer morphology. Given the well-known strong affinity of phosphonic acid for calcium ions, selected nano-objects were evaluated for their in-situ occlusion within growing CaCO3 crystals. Scanning electron microscopy (SEM) studies provide convincing evidence for the occlusion of both worm-like and vesicular phosphonic acid-based nano-objects and hence the production of a series of interesting new organic-inorganic nanocomposites

RAFT dispersion polymerization of glycidyl methacrylate for the synthesis of epoxy-functional block copolymer nanoparticles in mineral oil

Epoxy-functional poly(stearyl methacrylate)-poly(glycidyl methacrylate) (PSMA-PGlyMA) diblock copolymer nanoparticles are synthesized via reversible addition–fragmentation chain transfer (RAFT) dispersion polymerization of glycidyl methacrylate (GlyMA) in mineral oil at 70 °C. This efficient polymerization-induced self-assembly (PISA) formulation yields well-defined spheres of tunable diameter as confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies. 1H NMR spectroscopy and gel permeation chromatography (GPC) studies indicate that such non-polar dispersions exhibit greater stability during their long-term storage at 20 °C compared to related epoxy-functional nanoparticles prepared via RAFT aqueous emulsion polymerization. Model epoxy-amine ring-opening reactions using N-methylaniline demonstrate the potential for post-polymerization functionalization of such spherical nanoparticles

Block Copolymer Nanoparticles Prepared via Polymerization-Induced Self-Assembly Provide Excellent Boundary Lubrication Performance for Next-Generation Ultralow-Viscosity Automotive Engine Oils

Core cross-linked poly(stearyl methacrylate)–poly(benzyl methacrylate)–poly(ethylene glycol dimethacrylate) [S31–B200–E20] triblock copolymer nanoparticles were synthesized directly in an industrial mineral oil via polymerization-induced self-assembly (PISA). Gel permeation chromatography analysis of the S31–B200 diblock copolymer precursor chains indicated a well-controlled reversible addition–fragmentation chain transfer dispersion polymerization, while transmission electron microscopy, dynamic light-scattering (DLS), and small-angle X-ray scattering studies indicated the formation of well-defined spheres. Moreover, DLS studies performed in THF, which is a common solvent for the S and B blocks, confirmed successful covalent stabilization because well-defined solvent-swollen spheres were obtained under such conditions. Tribology experiments using a mini-traction machine (MTM) indicated that 0.50% w/w dispersions of S31–B200–E20 spheres dramatically reduce the friction coefficient of base oil within the boundary lubrication regime. Given their efficient and straightforward PISA synthesis at high solids, such nanoparticles offer new opportunities for the formulation of next-generation ultralow-viscosity automotive engine oils

Determining the Effective Density and Stabilizer Layer Thickness of Sterically Stabilized Nanoparticles.

A series of model sterically stabilized diblock copolymer nanoparticles has been designed to aid the development of analytical protocols in order to determine two key parameters: the effective particle density and the steric stabilizer layer thickness. The former parameter is essential for high resolution particle size analysis based on analytical (ultra)centrifugation techniques (e.g., disk centrifuge photosedimentometry, DCP), whereas the latter parameter is of fundamental importance in determining the effectiveness of steric stabilization as a colloid stability mechanism. The diblock copolymer nanoparticles were prepared via polymerization-induced self-assembly (PISA) using RAFT aqueous emulsion polymerization: this approach affords relatively narrow particle size distributions and enables the mean particle diameter and the stabilizer layer thickness to be adjusted independently via systematic variation of the mean degree of polymerization of the hydrophobic and hydrophilic blocks, respectively. The hydrophobic core-forming block was poly(2,2,2-trifluoroethyl methacrylate) [PTFEMA], which was selected for its relatively high density. The hydrophilic stabilizer block was poly(glycerol monomethacrylate) [PGMA], which is a well-known non-ionic polymer that remains water-soluble over a wide range of temperatures. Four series of PGMA x -PTFEMA y nanoparticles were prepared (x = 28, 43, 63, and 98, y = 100-1400) and characterized via transmission electron microscopy (TEM), dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS). It was found that the degree of polymerization of both the PGMA stabilizer and core-forming PTFEMA had a strong influence on the mean particle diameter, which ranged from 20 to 250 nm. Furthermore, SAXS was used to determine radii of gyration of 1.46 to 2.69 nm for the solvated PGMA stabilizer blocks. Thus, the mean effective density of these sterically stabilized particles was calculated and determined to lie between 1.19 g cm(-3) for the smaller particles and 1.41 g cm(-3) for the larger particles; these values are significantly lower than the solid-state density of PTFEMA (1.47 g cm(-3)). Since analytical centrifugation requires the density difference between the particles and the aqueous phase, determining the effective particle density is clearly vital for obtaining reliable particle size distributions. Furthermore, selected DCP data were recalculated by taking into account the inherent density distribution superimposed on the particle size distribution. Consequently, the true particle size distributions were found to be somewhat narrower than those calculated using an erroneous single density value, with smaller particles being particularly sensitive to this artifact

Synthesis of well-defined epoxy-functional spherical nanoparticles by RAFT aqueous emulsion polymerization

The environmentally-friendly synthesis of epoxy-functional spherical nanoparticles has been achieved using polymerization-induced self-assembly (PISA) in aqueous solution. Firstly, a non-ionic hydrophilic stabilizer block, poly(glycerol monomethacrylate) (PGMA), was prepared by reversible addition–fragmentation chain transfer (RAFT) solution polymerization in ethanol. This water-soluble precursor was subsequently chain-extended via RAFT aqueous emulsion polymerization of glycidyl methacrylate (GlyMA) at 50 °C and neutral pH to ensure maximum retention of the epoxy functionality. PISA leads to the formation of well-defined PGMA-PGlyMA spherical diblock copolymer nanoparticles at up to 35% w/w solids and 1H NMR spectroscopy studies indicated that virtually all of the epoxy groups survive such relatively mild conditions. DMF GPC studies confirmed that relatively low dispersities (Mw/Mn </p