Adam Blanazs

Self‐Assembled Block Copolymer Aggregates: From Micelles to Vesicles and their Biological Applications

The ability of amphiphilic block copolymers to self-assemble in selective solvents has been widely studied in academia and utilized for various commercial products. The self-assembled polymer vesicle is at the forefront of this nanotechnological revolution with seemingly endless possible uses, ranging from biomedical to nanometer-scale enzymatic reactors. This review is focused on the inherent advantages in using polymer vesicles over their small molecule lipid counterparts and the potential applications in biology for both drug delivery and synthetic cellular reactors.

Aqueous Dispersion Polymerization: A New Paradigm for in Situ Block Copolymer Self-Assembly in Concentrated Solution

Reversible addition-fragmentation chain transfer polymerization has been utilized to polymerize 2-hydroxypropyl methacrylate (HPMA) using a water-soluble macromolecular chain transfer agent based on poly(2-(methacryloyloxy)ethylphosphorylcholine) (PMPC). A detailed phase diagram has been elucidated for this aqueous dispersion polymerization formulation that reliably predicts the precise block compositions associated with well-defined particle morphologies (i.e., pure phases). Unlike the ad hoc approaches described in the literature, this strategy enables the facile, efficient, and reproducible preparation of diblock copolymer spheres, worms, or vesicles directly in concentrated aqueous solution. Chain extension of the highly hydrated zwitterionic PMPC block with HPMA in water at 70 °C produces a hydrophobic poly(2-hydroxypropyl methacrylate) (PHPMA) block, which drives in situ self-assembly to form well-defined diblock copolymer spheres, worms, or vesicles. The final particle morphology obtained at full monomer conversion is dictated by (i) the target degree of polymerization of the PHPMA block and (ii) the total solids concentration at which the HPMA polymerization is conducted. Moreover, if the targeted diblock copolymer composition corresponds to vesicle phase space at full monomer conversion, the in situ particle morphology evolves from spheres to worms to vesicles during the in situ polymerization of HPMA. In the case of PMPC(25)-PHPMA(400) particles, this systematic approach allows the direct, reproducible, and highly efficient preparation of either block copolymer vesicles at up to 25% solids or well-defined worms at 16-25% solids in aqueous solution.

Sterilizable Gels from Thermoresponsive Block Copolymer Worms

Biocompatible hydrogels have many applications, ranging from contact lenses to tissue engineering scaffolds. In most cases, rigorous sterilization is essential. Herein we show that a biocompatible diblock copolymer forms wormlike micelles via polymerization-induced self-assembly in aqueous solution. At a copolymer concentration of 10.0 w/w %, interworm entanglements lead to the formation of a free-standing physical hydrogel at 21 °C. Gel dissolution occurs on cooling to 4 °C due to an unusual worm-to-sphere order-order transition, as confirmed by rheology, electron microscopy, variable temperature (1)H NMR spectroscopy, and scattering studies. Moreover, this thermo-reversible behavior allows the facile preparation of sterile gels, since ultrafiltration of the diblock copolymer nanoparticles in their low-viscosity spherical form at 4 °C efficiently removes micrometer-sized bacteria; regelation occurs at 21 °C as the copolymer chains regain their wormlike morphology. Biocompatibility tests indicate good cell viabilities for these worm gels, which suggest potential biomedical applications.

Controlling Cellular Uptake by Surface Chemistry, Size, and Surface Topology at the Nanoscale

Cell cytosol and the different subcellular organelles house the most important biochemical processes that control cell functions. Effective delivery of bioactive agents within cells is expected to have an enormous impact on both gene therapy and the future development of new therapeutic and/or diagnostic strategies based on single-cell-bioactive-agent interactions. Herein a biomimetic nanovector is reported that is able to enter cells, escape from the complex endocytic pathway, and efficiently deliver actives within clinically relevant cells without perturbing their metabolic activity. This nanovector is based on the pH-controlled self-assembly of amphiphilic copolymers into nanometer-sized vesicles (or polymersomes). The cellular-uptake kinetics can be regulated by controlling the surface chemistry, the polymersome size, and the polymersome surface topology. The latter is controlled by the extent of polymer-polymer phase separation within the external envelope of the polymersome.

Anionic polyelectrolyte-stabilized nanoparticles via RAFT aqueous dispersion polymerization.

We report the synthesis of anionic sterically stabilized diblock copolymer nanoparticles via polymerization-induced self-assembly using a RAFT aqueous dispersion polymerization formulation. The anionic steric stabilizer is a macromolecular chain-transfer agent (macro-CTA) based on poly(potassium 3-sulfopropyl methacrylate) (PKSPMA), and the hydrophobic core-forming block is based on poly(2-hydroxypropyl methacrylate) (PHPMA). The effect of varying synthesis parameters such as the salt concentration, solids content, relative block composition, and anionic charge density has been studied. In the absence of salt, self-assembly is problematic when using a PKSPMA stabilizer because of lateral repulsion between highly charged anionic chains. However, in the presence of added salt this problem can be overcome by reducing the charge density within the coronal stabilizer layer by either (i) statistically copolymerizing the KSPMA monomer with a nonionic comonomer (2-hydroxyethyl methacrylate, HEMA) or (ii) using a binary mixture of a PKSPMA macro-CTA and a poly(glycerol monomethacrylate) (PGMA) macro-CTA. These diblock copolymer nanoparticles were analyzed by (1)H NMR spectroscopy, gel permeation chromatography (GPC), dynamic light scattering (DLS), transmission electron microscopy (TEM), and aqueous electrophoresis. NMR studies suggest that the HPMA polymerization is complete within 2 h at 70 °C, and DMF GPC analysis confirms that the resulting diblock copolymers have relatively low polydispersities (M(w)/M(n) < 1.30). NMR also suggests a significant degree of hydration for the core-forming PHPMA chains. Depending on the specific reaction conditions, a series of spherical nanoparticles with mean diameters ranging from 50 to 200 nm with tunable anionic surface charge can be prepared. If a binary mixture of anionic and nonionic macro-CTAs is utilized, then it is also possible to access a vesicular morphology.

Efficient synthesis of sterically-stabilized nano-objects via RAFT dispersion polymerization of benzyl methacrylate in alcoholic media.

Synthesis of diblock copolymer nano-objects: alcohol is a good idea! RAFT dispersion polymerization of benzyl methacrylate in alcohol using weak polyelectrolyte-based chain transfer agents allows the facile synthesis of sterically stabilized diblock copolymer nano-objects with very high monomer conversions. Such syntheses are usually problematic when conducted in water due to electrostatic repulsion between highly charged stabilizer chains, which impedes in situ self-assembly. Construction of a detailed phase diagram facilitates reproducible syntheses of well-defined diblock copolymer spheres, worms or vesicles, since it allows mixed phase regions to be avoided. Aqueous electrophoresis studies confirm that these nano-objects can acquire substantial surface charge when transferred to aqueous solution due to ionization (or protonation) of the polyacid (or polybase) stabilizer chains.

Non-cytotoxic polymer vesicles for rapid and efficient intracellular delivery

We have recently achieved efficient cytosolic delivery by using pH-sensitive poly(2-(methacryloyloxy)ethylphosphorylcholine)-co-poly(2-(diisopropylamino)ethylmethacrylate) (PMPC-PDPA) diblock copolymers that self-assemble to form vesicles, known as polymersomes, in aqueous solution. It is particularly noteworthy that these diblock copolymers form stable polymersomes at physiological pH but rapidly dissociate below pH 6 to give molecularly-dissolved copolymer chains (unimers). These PMPC-PDPA polymersomes are used to encapsulate nucleic acids for efficient intracellular delivery. Confocal laser scanning microscopy and fluorescence flow cytometry are used to quantify cellular uptake and to study the kinetics of this process. Finally, we examine how PMPC-PDPA polymersomes affect the viability of primary human cells (human dermal fibroblasts (HDF)), paying particular regard to whether inflammatory responses are triggered.

Nile Blue-Based Nanosized pH Sensors for Simultaneous Far-Red and Near-Infrared Live Bioimaging

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.

Non-spherical morphologies from cross-linked biomimetic diblock copolymers using RAFT aqueous dispersion polymerization

The RAFT synthesis of cross-linked diblock copolymer nanoparticles was explored using an aqueous dispersion polymerisation formulation. This diblock copolymer comprised biomimetic poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) stabilizing chains and a highly cross-linked poly(2-hydroxypropyl methacrylate) (PHPMA) core-forming block. At either zero or relatively low levels of ethylene glycol dimethacrylate (EGDMA) cross-linker, only spherical morphologies were observed. However, using higher levels of EGDMA led to increasing particle anisotropy, with both a worm-like morphology and a novel ‘lumpy rod’ morphology (comprising fused nano-sized aggregates of primary spherical particles) being observed.

How does cross-linking affect the stability of block copolymer vesicles in the presence of surfactant?

Block copolymer vesicles are conveniently prepared directly in water at relatively high solids by polymerization-induced self-assembly using an aqueous dispersion polymerization formulation based on 2-hydroxypropyl methacrylate. However, dynamic light scattering studies clearly demonstrate that addition of small molecule surfactants to such linear copolymer vesicles disrupts the vesicular membrane. This causes rapid vesicle dissolution in the case of ionic surfactants, with nonionic surfactants proving somewhat less destructive. To address this problem, glycidyl methacrylate can be copolymerized with 2-hydroxypropyl methacrylate and the resulting epoxy-functional block copolymer vesicles are readily cross-linked in aqueous solution using cheap commercially available polymeric diamines. Such epoxy-amine chemistry confers exceptional surfactant tolerance on the cross-linked vesicles and also leads to a distinctive change in their morphology, as judged by transmission electron microscopy. Moreover, pendent unreacted amine groups confer cationic character on these cross-linked vesicles and offer further opportunities for functionalization.