Synthesis of star-branched poly(vinyl alcohol) and ice recrystallization inhibition activity

Antifreeze proteins are potent inhibitors of ice crystal growth (recrystallization), which is a highly desirable property for cryopreservation and other low temperature applications. It has emerged that relatively simple polymers based on poly(vinyl alcohol) can mimic this activity, but the link between architecture and activity is not known. Here, a trifunctional xanthate was designed and synthesized to prepare star-branched poly(vinyl alcohols) by RAFT/Xanthate mediated polymerization, and their ice growth inhibition activity probed for the first time. The trifunctional agent design affords the formation of well-defined star polymers, with no evidence of star-star linking, even at high conversions, and narrow molecular weight dispersity. It is observed that three-arm stars have identical activity to two-armed (i.e. linear) equivalents, suggesting that the total hydrodynamic size of the polymer (diameter three-arm ~ two-arm) rather than total valence of the functional groups is the key descriptor of activit

Mechanisms of the drug penetration enhancer propylene glycol interacting with skin lipid membranes

Very few drugs have the necessary physicochemical properties to cross the skin’s main permeability barrier, the stratum corneum (SC), in sufficient amounts. Propylene glycol (PG) is a chemical penetration enhancer that could be included in topical formulations in order to overcome the barrier properties of the skin and facilitate the transport of drugs across it. Experiments have demonstrated that PG increases the mobility and disorder of SC lipids and may extract cholesterol from the SC, but little is known about the molecular mechanisms of drug permeation enhancement by PG. In this work, we have performed molecular dynamics (MD) simulations to investigate the molecular-level effects of PG on the structure and properties of model SC lipid bilayers. The model bilayers were simulated in the presence of PG concentrations over the range of 0–100% w/w PG, using both an all-atom and a united atom force field. PG was found to localize in the hydrophilic headgroup regions at the bilayer interface, to occupy the lipid–water hydrogen-bonding sites, and to slightly increase lipid tail disorder in a concentration-dependent manner. We showed with MD simulation that PG enhances the permeation of small molecules such as water by interacting with the bilayer interface; the results of our study may be used to guide the design of formulations for transdermal drug delivery with enhanced skin permeation, as well as topical formulations and cosmetic products

Ethanol induces the formation of water-permeable defects in model bilayers of skin lipids

We show that ethanol can induce the formation of water-permeable defects in model membranes of skin, providing a fresh perspective on ethanol as a membrane modulator. We rationalise our findings in terms of the chemical nature of ethanol, i.e., a combination of its hydrogen bonding propensity and amphiphilic character

Complete structure of an epithelial keratin dimer: implications for intermediate filament assembly

Keratins are cytoskeletal proteins that hierarchically arrange into filaments, starting with the dimer sub-unit. They are integral to the structural support of cells, in skin, hair and nails. In skin, keratin is thought to play a critical role in conferring the barrier properties and elasticity of skin. In general, the keratin dimer is broadly described by a tri-domain structure: a head, a central rod and a tail. As yet, no atomistic-scale picture of the entire dimer structure exists; this information is pivotal for establishing molecular-level connections between structure and function in intermediate filament proteins. The roles of the head and tail domains in facilitating keratin filament assembly and function remain as open questions. To address these, we report results of molecular dynamics simulations of the entire epithelial human K1/K10 keratin dimer. Our findings comprise: (1) the first three-dimensional structural models of the complete dimer unit, comprising of the head, rod and tail domains; (2) new insights into the chirality of the rod-domain twist gained from analysis of the full domain structure; (3) evidence for tri-subdomain partitioning in the head and tail domains; and, (4) identification of the residue characteristics that mediate non-covalent contact between the chains in the dimer. Our findings are immediately applicable to other epithelial keratins, such as K8/K18 and K5/K14, and to intermediate filament proteins in general

Impact of oxetane incorporation on the structure and stability of alpha-helical peptides

Peptide-based drugs combine advantages of larger biological therapeutics with those of small molecule drugs, but they generally display poor permeability and metabolic stability. Recently, we introduced a new type of peptide bond isostere, in which the backbone carbonyl is replaced with a 3-amino oxetane heterocycle, into short linear peptides with the aim of improving their therapeutic potential. In this study, we have explored the impact of oxetane modification on α-helical peptides to establish whether or not this modification is tolerated in this biologically important structural motif. The oxetane modification was introduced at two positions in a well-characterised helical peptide sequence, and circular dichroism and NMR spectroscopy were used to measure the resulting secondary structure content under different experimental conditions. Our data demonstrated that introduction of an oxetane into the peptide backbone results in a significant loss of helicity, regardless of where in the sequence the modification is placed. The molecular determinants of this destabilisation were then explored using steered molecular dynamics simulations, a computational method analogous to single molecule spectroscopy. Our simulations indicated that oxetane modification introduces a kink in the helical axis, alters the dihedral angles of residues up to three positions away from the modification, and disrupts the (i, i + 4) hydrogen bonding pattern characteristic of α-helices in favour of new, short-range hydrogen bonds. The detailed structural understanding provided in this work can direct future design of chemically modified peptides

Synthesis and functionalization of azetidine‐containing small macrocyclic peptides

Cyclic peptides are increasingly important structures in drugs but their development can be impeded by difficulties associated with their synthesis. Here, we introduce the 3-aminoazetidine (3-AAz) subunit as a new turn-inducing element for the efficient synthesis of small head-to-tail cyclic peptides. Greatly improved cyclizations of tetra-, penta- and hexapeptides (28 examples) under standard reaction conditions are achieved by introduction of this element within the linear peptide precursor. Post-cyclization deprotection of the amino acid side chains with strong acid is realized without degradation of the strained four-membered azetidine. An special feature of this chemistry is that further late-stage modification of the resultant macrocyclic peptides can be achieved via the 3-AAz unit. This is done by: (i) chemoselective deprotection and substitution at the azetidine nitrogen, or by (ii) a click-based approach employing a 2-propynyl carbamate on the azetidine nitrogen. In this way, a range of dye and biotin tagged macrocycles are readily produced. Structural insights gained by XRD analysis of a cyclic tetrapeptide indicate that the azetidine ring encourages access to the less stable, all-trans conformation. Moreover, introduction of a 3-AAz into a representative cyclohexapeptide improves stability towards proteases compared to the homodetic macrocycle

Breaching the skin barrier — insights from molecular simulation of model membranes

Breaching the skin's barrier function by design is an important strategy for delivering drugs and vaccines to the body. However, while there are many proposed approaches for reversibly breaching the skin barrier, our understanding of the molecular processes involved is still rudimentary. Molecular simulation offers an unprecedented molecular-level resolution with an ability to reproduce molecular and bulk level properties. We review the basis of the molecular simulation methodology and give applications of relevance to the skin lipid barrier, focusing on permeation of molecules and chemical approaches for breaching the lipid barrier by design. The bulk kinetic model based on Fick's Law describing absorption of a drug through skin has been reconciled with statistical mechanical quantities such as the local excess chemical potential and local diffusion coefficient within the membrane structure. Applications of molecular simulation reviewed include investigations of the structure and dynamics of simple models of skin lipids, calculation of the permeability of molecules in simple model membranes, and mechanisms of action of the penetration enhancers, DMSO, ethanol and oleic acid. The studies reviewed illustrate the power and potential of molecular simulation to yield important physical insights, inform and rationalize experimental studies, and to predict structural changes, and kinetic and thermodynamic quantities

Hydrophobic Biomimetic Nanoparticles drives Size-dependent Remodelling in Asymmetric Bilayers

The interactions between heterogeneous components in a biomimetic bilayer can control its physical properties such as its rigidity, local and bulk curvature and propensity towards phenomena such as membrane fission and fusion. In particular, nanoparticles (NPs) have been subjects of intense interest due to their similar scale to the bilayer width and its ability to affect local membrane structure. Generally, it is understood that hydrophobic components are energetically favoured to adsorb within the hydrophobic interior of a biomimetic bilayer. However, how such NPs interact in the presence of heterogeneous aggregates in the bilayer has been the subject of much debate. To better understand the effects of the integration of nanoscale components on heterogeneous mixed bilayer, we have simulated a series of generic hydrophobic NPs interacting with a phase-separating two-component surfactant bilayer. We find that the hydrophobic NP tends to aggregate at the phase interface, acting as a line tension relaxant i.e. a lineactant on the phase separated interface, which results in a variety of demixing behavior. We demonstrate that depending on the size of the NP, the localized softening of surfactants and the formation of a mixing gradient of surfactants can drive the a cap/bud formation around the NP, as well as the formation of a NP-micelle structure<br /

Comparison of Umbrella Sampling and Steered Molecular Dynamics Methods for Computing Free Energy Profiles of Aromatic Substrates through Phospholipid Bilayers

Understanding the permeation of molecules through lipid membranes is fundamental for predicting the cellular uptake of solutes and drug delivery mechanisms. In molecular simulations the usual approach is to compute the free energy (FE) profile of a molecule across a model lipid bilayer, which can then be used to estimate the permeability of the molecule. Umbrella sampling (US), which involves carrying out a series of biased simulations along a defined reaction coordinate (usually the bilayer normal direction), is a popular method for the computation of such FE profiles. However, US can be challenging to implement because the results are dependent on the strength of the biasing potential and the spacing of windows along the reaction coordinate, which, in practice, are usually optimized by an inefficient trial and error approach. The Steered Molecular Dynamics implementation of the Jarzynski Equality (JE-SMD) has been identified as an alternative to equilibrium sampling methods for measuring the FE change across a reaction coordinate. In the JE-SMD approach, equilibrium FE values are evaluated from the average of rapid non-equilibrium trajectories, thus avoiding the practical issues that come with US. Here, we use three different corrections of the JE-SMD method to calculate the FE change for the translocation of two aromatic substrates, phenylalanine and toluene, across a lipid bilayer, and compare the accuracy and computational efficiency of these approaches to the results obtained using US. We show evidence that when computing the free energy profile, the JE-SMD approach suffers from insufficient sampling convergence of the bilayer environment, and is dependent on the characteristic of the aromatic substrate itself. We deduce that, despite its drawbacks, US remains the more viable approach of the two for computing the FE profile

Size-Dependent Interaction of Nanoparticles with Non-ionic Bilayers

Understanding the mechanism of transit of a nanoparticle (NP) through a biomimetic bilayer has been at the forfront of research for the design of efficient drug-delivery mechanisms, nanotechnology and biomedicine. Establishing a consistent picture of how the transit mechanism depends on the physiochemical property of a NP is critical to understanding what approach may be the most effective for nanomedicine design. In this study, using molecular simulation techniques, we have analyzed the key properties of a NP that may affect the mechanism of transit - the effect of size and hydrophobicity. By using a continuum model of a NP based on the Hamaker potential, we have created NP of tunable hydrophobic properties. The effect of hydrophilic, hydrophobic, and mixed properties of the NP is analyzed against a biomimetic bilayer - we show that this model can illustrate three distinct properties - where the hydrophilic type shows rupture of the bilayer, the hydrophobic type showing a entrapment of the NP around the hydrophobic tailgroups of the bilayer, and the mixed type showing a distinct, direct translocation type mechanism. Increasing the NP size shows different effects for each type of NP, and hence, may provide insight into the design of NPs with these types of mechanisms involved