Systematic investigation of alkyl sulfonate initiators for the cationic ring-opening polymerization of 2-oxazolines revealing optimal combinations of monomers and initiators

A

Conformational properties of biocompatible poly(2-ethyl-2-oxazoline)s in phosphate buffered saline

Inspired by the increasing popularity of poly(2-ethyl-2-oxazoline) (PEtOx) for biomedical applications, this study reports the complete and thorough solution analysis of the homologous series of biocompatible PEtOx samples in a very broad range of molecular weights ranging from 11.2 x 10(3) g mol(-1) up to 260 x 10(3) g mol(-1). The main focus of the research was on the determination of the conformational properties of PEtOx macromolecules at a temperature of 37 degrees C in phosphate buffered saline (PBS) simulating the parameters of physiological media. The polymers were studied in PBS solutions by analytical ultracentrifugation, dynamic light scattering (DLS), translational diffusion, and intrinsic viscosity measurements in a temperature range from 15 degrees C up to 72 degrees C. The complete set of Kuhn-Mark-Houwink-Sakurada scaling relationships revealed linear trends over the whole range of the studied molar masses, while the determined scaling indices at 37 degrees C correspond to the coil conformation in a thermodynamically good solvent ([eta] = 0.045 x M-0.62, s(0) = 0.010 x M-0.46 and D-0 = 1750 x M-0.54). Based on the intrinsic viscosity values (most sensitive characteristic to the size variations of polymer coils, [eta] similar to r(3)), it was demonstrated that PEtOx macromolecules in PBS solutions undergo a transition from swollen polymer coils with gradual deterioration of thermodynamic quality of solutions within the temperature range of 15-45 degrees C, reaching theta-conditions at 55 degrees C with further precipitation at 62-72 degrees C. Also, to the best of our knowledge, the conformational parameters (equilibrium rigidity/the Kuhn segment length and the diameter of the polymer chain) of PEtOx macromolecules were evaluated under physiological conditions for the first time and constitute A = 1.8 +/- 0.3 nm and d = 0.7 +/- 0.4 nm. These equilibrium rigidity values classify PEtOx as a flexible macromolecule with rigidity similar to that of poly(ethylene glycol). For the first time, we were able to demonstrate a direct influence of thermosensitivity on the rigidity of the biocompatible polymer: PEtOx. The Kuhn segment length is undoubtedly decreasing when approaching the LCST

Uniform Linear Poly(ethylenimine) via Poly(oxazoline)s - Synthesis, Mechanism of Toxicity and the Role of the Free Polymer during Transfection

Linear poly(ethylenimine) is the current “gold standard” gene transfection agent. It is synthesised via the cationic ring opening polymerisation of 2-ethyl-2-oxazoline followed by the acid catalysed hydrolysis to the hydrochloride salt. However the synthesis of the parent polymer produces a broad distribution of molecular weights. Ideally the polymer should have a narrow molecular weight distribution - it should be “uniform”. By exploring the possible side reactions it is possible to minimise them and optimise the polymerisation of 2-oxazolines. By using a non-interfering solvent (chlorobenzene) in place of acetonitrile at a low temperature (40-45°C) the majority of side-reactions cease. Increasing the substitution on the side-chain increased steric hindrance of both the β and the 2-positions, but increased crystallinity complicated the polymerisation. When an extremely nucleofugic counterion (triflate) was used the propagation rate increased dramatically, despite no apparent change in the equilibrium between the ionic propagating species and inactive ester. The nature of propagation appears to have changed. The l-PEI derived from uniform poly(oxazoline)s together with a broad 22 kDa l-PEI simulating the commercial polymer “in-vivo jetPEITM” and a range of poly(l-lysine)s were tested on lung adenocarcinoma cells (A549) revealing a strong correlation between the molecular weight of the polymer and the degree of cell membrane disruption via LDH release. Mitochondrial activity assays carried out with short (4 hr) incubation times did not correlate with LDH release except in the case of immediate cellular destruction. In fact values much greater then 100% viability were typically observed, indicating that mitochondrial activity had actually increased. However when incubation time was increased to 24 or 48 hrs the increase in mitochondrial activity was less pronounced and the correlation with cell membrane damage was restored. This suggests that damaged cells were increasing their activity to maintain homeostatis, which obfuscates cellular toxicity in the normal MTT protocol. With the destruction of cell membranes strongly implicated in the toxicity of l-PEI and PLL (and assumidly other polycations) it was hypothesised that the mechanism of cell membrane destruction was acid catalysed hydrolysis of the ester bonds of phospholipids. This would lead to changes in cell membrane curvature resulting in the opening of pores/ holes in the cell surface and, eventually would simulate apoptotic cell death despite a necrotic origin since apoptotic “squibs” are formed by hydrolysis of phospholipid esters by an enzyme. Experiments on DOPC liposomes incubated with l-PEI revealed the concentration of DOPC falling over time with MOPC forming transiently before further hydrolysis occurred. The rate of hydrolysis is implied to be related to the MW of the polymer. Finally the observations of hydrolysis lead to a thought experiment and a new hypothesis of the underlying mechanism of polymeric transfection.Imperial Users Onl

Thermoresponsive hydrogels formed by poly(2-oxazoline) triblock copolymers

Hydrogels are useful materials for drug delivery and tissue engineering. A subset of these are responsive hydrogelators that are liquids until a stimulus triggers their gelation. Poly(2-oxazoline)s with moderately hydrophobic side chains, such as ethyl and propyl, are inherently thermoresponsive exhibiting lower critical solution temperature behavior. However, previous attempts to make thermoresponsive poly(2-oxazoline) block copolymer hydrogelators have all failed. In this work we report the first working thermoresponsive poly(2-oxazoline) hydrogelator based on ABA triblock copolymers with thermoresponsive outer blocks composed of poly(2-n-propyl-2-oxazoline) and a more hydrophilic poly(2-ethyl-2-oxazoline) inner block that can form hydrogels upon heating an aqueous solution. It was found that thermoresponsive hydrogels are only formed if the block size is tuned to adjust the cloud point temperature and, presumably, to allow sufficient flexibility to non-covalently crosslink the flower-like micelles that these materials form. These triblocks copoly(2-oxazoline)s formed relatively soft thermoresponsive hydrogels that could be suitable for drug release and cell culture applications

Synthesis of defined high molar mass poly(2-methyl-2-oxazoline)

In this communication, we report for the first time the synthesis of defined high molar mass poly(2-methyl-2-oxazoline) (PMeOx), a water-soluble polymer with excellent anti-fouling properties. So far, there has been no report on low dispersity (D < 1.2) PMeOx longer than 10 kg mol(-1). Higher molar mass would be beneficial for synthesis of polymer-drug conjugates, excipients as well as for other biomedical applications. We report our attempts to prepare defined high molar mass PMeOx via living cationic ring-opening polymerization (CROP) of its monomer using our optimized method that failed due to extensive chain transfer and chain coupling side reactions. Therefore, we proposed an alternative strategy to high molar mass PMeOx based on acetylation of well-defined linear polyethyleneimine (PEI) prepared by controlled side-chain hydrolysis of defined high molar mass PEtOx. This method allowed us to synthesize a series of low-dispersity PMeOx up to 58 kg mol(-1) (D = 1.07). Considering the biomedical potential of PMeOx, the synthesis of such polymers might open a way to a new class of effective polymer-based therapeutics

Detailed Understanding of Solvent Effects for the Cationic Ring-Opening Polymerization of 2-Ethyl-2-oxazoline

Polymerization of 2-ethyl-2-oxazoline (EtOx) has often been in the spotlight for fundamental studies of poly(2-alkyl/aryl-2-oxazoline)s (PAOx) polymerization, especially initiator screening, solvent screening, and copolymerization trends. In this work, we build on previous observations of solvent effects on the cationic ring-opening polymerization (CROP) of EtOx, with additional experimental observations of previously unreported solvents to expand the explored parameter space. Our objective is to find solvents with the lowest activation energy (Ea) and higher Arrhenius preexponential factor (A), which will allow us to produce narrow molar mass distributions at higher molecular weights, in the least time. To achieve this, we examined the various single factors like Dimroth ET(30) values, the Kamlet-Abraham-Taft (KAT) linear free-energy relationship (LFER) equation(s), and the Catalan LFER equations. Only one of Catalan’s equations sufficiently disentangled dipolarity and polarizability to give a good fit due to contradictory effects. It was found that solvent nucleophilicity, electrophilicity, and polarizability affected the Ea, but not dipolarity. All four factors affected the A. This indicates that the Ea is minimized in solvents that do not solvate ions well (i.e. force ion-pairing), and A was minimized in more dipolar solvents that solvate the polymer chains well. A strongly negative activation entropy (ΔS‡) shows that the propagation reaction is associative. The Catalan LFER allows for the prediction of Ea, A, ΔH‡, and ΔS‡, and the derived kp, across a broad range of solvents