Helen Y. Fan

Graphene-Based

We demonstrated a novel graphene Q-switched erbium-doped fiber laser with wide pulse-repetition-rate range. The Q-switched fiber laser is constructed with a 37-cm linear cavity formed by two fiber Bragg gratings, a section of highly doped erbium fiber, and a graphene saturable absorber. The laser has a low pump threshold of 16.9 mW, a wide range of repetition rate from 31.7 to 236.3 kHz, and minimum pulse duration of 206 ns. The short-cavity laser structure enables laser action free of self-mode-locking effects.

Q

The effective charge of membrane-active molecules such as the fungicidal lipopeptide surfactin (SF) is a crucial property governing solubility, membrane partitioning, and membrane permeability. We present zeta potential measurements of liposomes to measure the effective charge as well as membrane partitioning of SF by utilizing what we call an equi-activity analysis of several series of samples with different lipid concentrations. We observe an effective charge of -1.0 for SF at pH8.5 and insignificantly lower at pH7.4, illustrating that the effective charge may deviate strongly from the nominal value (-2 for 1 Asp, 1 Glu). The apparent partition coefficient decreases from roughly 100 to 20/mM with increasing membrane content of SF in agreement with the literature. Finally, by comparing zeta potentials measured soon after the addition of peptide to liposomes with those measured after a heat treatment to induce transmembrane equilibration of SF, we quantified the asymmetry of partitioning between the outer and inner leaflets. At very low concentration, SF binds exclusively to the outer leaflet. The onset of partial translocation to the inner leaflet occurs at about 5mol-% SF in the membrane. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

-Switched Erbium-Doped Fiber Laser With Wide Pulse-Repetition-Rate Range

Most drug delivery systems have been developed for efficient delivery to tumor sites via targeting and on-demand strategies, but the carriers rarely execute synergistic therapeutic actions. In this work, C8, a cationic, pH-triggered anticancer peptide, is developed by incorporating histidine-mediated pH-sensitivity, amphipathic helix, and amino acid pairing self-assembly design. We designed C8 to function as a pH-responsive nanostructure whose cytotoxicity can be switched on and off by its self-assembly: Noncytotoxic β-sheet fibers at high pH with neutral histidines, and positively charged monomers with membrane lytic activity at low pH. The selective activity of C8, tested for three different cancer cell lines and two noncancerous cell lines, is shown. Based on liposome leakage assays and multiscale computer simulations, its physical mechanisms of pore-forming action and selectivity are proposed, which originate from differences in the lipid composition of the cellular membrane and changes in hydrogen bonding. C8 is then investigated for its potential as a drug carrier. C8 forms a nanocomplex with ellipticine, a nonselective model anticancer drug. It selectively targets cancer cells in a pH-responsive manner, demonstrating enhanced efficacy and selectivity. This study provides a novel powerful strategy for the design and development of multifunctional self-assembling peptides for therapeutic and drug delivery applications.

Utilizing zeta potential measurements to study the effective charge, membrane partitioning, and membrane permeation of the lipopeptide surfactin.

We present the application of pressure perturbation calorimetry (PPC) as a new method for the volumetric characterization of the micelle formation of surfactants. The evaluation is realized by a global fit of PPC curves at different surfactant concentration ranging, if possible, from below to far above the CMC. It is based on the knowledge of the temperature dependence of the CMC, which can for example be characterized by isothermal titration calorimetry. We demonstrate the new approach for decyl-β-maltopyranoside (DM). It shows a strong volume increase upon micelle formation of 16 ± 2.5 mL/mol (+4%) at 25 °C, and changes with temperature by -0.1 mL/(mol K). The apparent molar expansivity (E(S)) decreases upon micelle formation from 0.44 to 0.31 mL/(mol K) at 25 °C. Surprisingly, the temperature dependence of the expansivity of DM in solution (as compared with that of maltose) does not agree with the principal behavior described for polar (E(S)(T) decreasing) and hydrophobic (E(S)(T) increasing) solutes or moieties before. The results are discussed in terms of changes in hydration of the molecules and internal packing of the micelles and compared with the volumetric effects of transitions of proteins, DNA, lipids, and polymers.

Design and Characterization of a Multifunctional pH‐Triggered Peptide C8 for Selective Anticancer Activity

Volumetric parameters have long been used to elucidate the phenomena governing the stability of protein structures, ligand binding, or transitions in macromolecular or colloidal systems. In spite of much success, many problems remain controversial. For example, hydrophobic groups have been discussed to condense adjacent water to a volume lower than that of bulk water, causing a negative contribution to the volume change of unfolding. However, expansivity data were interpreted in terms of a structure-making effect that expands the water interacting with the solute. We have studied volume and expansivity effects of transfer of alkyl chains into micelles by pressure perturbation calorimetry and isothermal titration calorimetry. For a series of alkyl maltosides and glucosides, the methylene group contribution to expansivity was obtained as 5 uL/(mol K) in a micelle (mimicking bulk hydrocarbon) but 27 uL/(mol K) in water (20 °C). The latter value is virtually independent of temperature and similar to that obtained from hydrophobic amino acids. Methylene contributions of micellization are about -60 J/(mol K) to heat capacity and 2.7 mL/mol to volume. Our data oppose the widely accepted assumption that water-exposed hydrophobic groups yield a negative contribution to expansivity at low temperature that would imply a structure-making, water-expanding effect.

Volume and Expansivity Changes of Micelle Formation Measured by Pressure Perturbation Calorimetry

Doxorubicin (DOX) and other anti-cancer drugs are often formulated using nanoparticles for passive or active targeting and reducing detrimental side effects. Anionic polymers have been shown to effectively facilitate loading of cationic DOX hydrochloride into nanoparticles with high efficiency. One powerful method to study DOX loading into anionic polymeric nanoparticles has been isothermal titration calorimetry (ITC), but the curves are complex and were previously interpreted in a largely qualitative manner only. Here we present detailed quantitative modelling of such ITC data, corroborated by zeta potential measurements and dynamic light scattering. The model takes into account 3 coupled equilibria. First, DOX self-associates in solution to dimers and larger aggregates. This effect is modelled in terms of the stepwise aggregation model. Second, DOX binds with a 1:1 stoichiometry to the carboxylic acids in the polymer at low salt. At about 33% saturation, the nanoparticles collapse in size and the enthalpy of further binding becomes less exothermic. Third, free DOX also stacks onto polymer-bound DOX. This stacking effect is very weak and hardly detected by ITC. It is, however, revealed by a positive zeta potential. The present work demonstrates the power of combining ITC with light scattering and zeta potential measurements for studying the thermodynamics of drug loading into polymeric nanoparticles.

Effect of hydrophobic interactions on volume and thermal expansivity as derived from micelle formation.

The human G protein-coupled receptor M2 muscarinic receptor has been functionally reconstituted in its tetrameric state into mixed lipid bilayers (Redka et al. 2013). This is achieved by first solubilizing the receptor in mixed detergent micelles composed of digitonin and sodium cholate, then reconstituting it into vesicles composed of phosphatidylcholine, phosphatidylserine and cholesterol, followed by detergent removal. To understand how the individual detergent and lipid components used in this empirical protocol contribute to the stability and activity of the receptor, we used isothermal titration calorimetry (ITC) to study the self-assembly of the mixed surfactant system; differential scanning calorimetry and pressure perturbation calorimetry to probe the phase behavior of the membrane; ITC, fluorescence (time-resolved) leakage assays and dynamic light scattering to characterize detergent-lipid interactions. The results suggest ideal mixing between digitonin and sodium cholate in the formation of mixed micelles that stabilize the receptor. Differences in membrane-partitioning behavior between the two detergents and the presence of a significant fraction of gel phase at the temperature used in the protocol contribute to non-equilibration of the detergents in the bilayer. The insights gained from this biophysical approach will aid in the mechanistic selection of detergents and conditions that influence function, oligomeric state, orientation, and accessibility of membrane proteins in future studies.

Coupled equilibria of a self-associating drug loaded into polymeric nanoparticles

It has long been observed that the net volume change upon protein unfolding is very small. The loss of structural voids and hydration of otherwise shielded charged groups from unfolding contribute negatively to volume change, while confusion persists regarding whether the exposure of hydrophobic groups has a condensing effect or a structure-making effect on water. The development of pressure perturbation calorimetry in recent years has made it possible to determine expansivity directly with great precision. Data obtained for proteins were interpreted in terms of a structure-making effect that expands the water interacting with the solute. We have studied volume and expansivity effects of transfer of alkyl chains into micelles by pressure perturbation calorimetry and isothermal titration calorimetry. The contribution of methylene groups to expansivity upon hydration is positive and independent of temperature, similar to that obtained with hydrophobic amino acids. Our data oppose the widely accepted assumption that water-exposed hydrophobic groups yield a negative contribution to expansivity at low temperatures that would imply a structure-making, water-expanding effect. It is of worth to note that group contributions should be obtained by subtracting molar expansivities, not coefficients of expansion.