Real-time monitoring the staged interactions between cationic surfactants and a phospholipid bilayer membrane.

The cationic surfactant-lipid interaction directs the development of novel types of nanodrugs or nanocarriers. The membrane action of cationic surfactants also has a wide range of applications. In this work, combining a photo-voltage transient method with the traditional dynamic giant unilamellar vesicle (GUV) leakage assay and molecular dynamics (MD) simulations, we monitored the molecular actions of a representative cationic surfactant, tetradecyl trimethyl ammonium bromide (TTAB), in a wide concentration range (i.e., 0.5 µM-10 mM), on a phospholipid bilayer membrane in real time. With low concentrations (e.g., ≤10 µM), TTAB performed a three-stage acting process, including the structural-disturbance-dominated, adsorption-dominated, and dynamic equilibrium stages. At higher concentrations (e.g., ≥100 µM), this process was accelerated to two stages. Furthermore, TTAB induced deformation and even rupture of the membrane, due to the asymmetric disturbance of surfactant molecules on the two leaflets of a bilayer. All these disturbances induced membrane permeabilization, and the times at which these transitions occurred are given. This work provides information on time and molecular mechanism during the membrane actions of cationic surfactants, and provides a simple and real-time method in studying the dynamic processes at the membrane interface.

One-pot synthesis of silicon based nanoparticles with incorporated phthalocyanine for long-term bioimaging and photo-dynamic therapy of tumors.

Combining the merits of delivery vectors with drug molecules is one of the key directions for development of efficient cancer monitoring and treatment techniques. In this work, a novel type of silicon based composite nanoparticles (NPs) with incorporated hydrophobic phthalocyanine molecules (Pc) was synthesized via a facile one-pot method. The as-synthesized Pc@Si NPs, with a small size of 4.2 ± 0.8 nm, have excellent dispersibility in water and good biocompatibility with cells, in addition to favorable photoluminescence and robust photostability even in cells. Moreover, the Pc@Si NPs show significant in vitro cancer cell killing and in vivo tumor inhibiting abilities upon near-infrared light exposure, due to the photodynamic therapy (PDT) effect of Pc. This work develops an efficient fluorescent PDT drug carrier; moreover, the facile one-pot synthesis strategy may be used generally to prepare silicon-based composite NPs incorporated with diverse hydrophobic drugs/diagnostic molecules for a wide range of biomedical applications.

Reduced graphene oxide directed self-assembly of phospholipid monolayers in liquid and gel phases.

The response of cell membranes to the local physical environment significantly determines many biological processes and the practical applications of biomaterials. A better understanding of the dynamic assembly and environmental response of lipid membranes can help understand these processes and design novel nanomaterials for biomedical applications. The present work demonstrates the directed assembly of lipid monolayers, in both liquid and gel phases, on the surface of a monolayered reduced graphene oxide (rGO). The results from atomic force microscopy indicate that the hydrophobic aromatic plane and the defect holes due to reduction of GO sheets, along with the phase state and planar surface pressure of lipids, corporately determine the morphology and lateral structure of the assembled lipid monolayers. The DOPC molecules, in liquid phase, probably spread over the rGO surface with their tails associating closely with the hydrophobic aromatic plane, and accumulate to form circles of high area surrounding the defect holes on rGO sheets. However, the DPPC molecules, in gel phase, prefer to form a layer of continuous membrane covering the whole rGO sheet including defect holes. The strong association between rGO sheets and lipid tails further influences the melting behavior of lipids. This work reveals a dramatic effect of the local structure and surface property of rGO sheets on the substrate-directed assembly and subsequent phase behavior of the supported lipid membranes.

Assembly-Induced Membrane Selectivity of Artificial Model Peptides through Entropy-Enthalpy Competition.

Peptide design and drug development offer a promising solution for combating serious diseases or infections. In this study, using an AI-human negotiation approach, we have designed a class of minimal model peptides against tuberculosis (TB), among which K7W6 exhibits potent efficacy attributed to its assembly-induced function. Comprising lysine and tryptophan with an amphiphilic α-helical structure, the K7W6 sequence exhibits robust activity against various infectious bacteria causing TB (including clinically isolated and drug-resistant strains) both in vitro and in vivo. Moreover, it synergistically enhances the effectiveness of the first-line antibiotic rifampicin while displaying low potential for inducing drug resistance and minimal toxicity toward mammalian cells. Biophysical experiments and simulations elucidate that K7W6's exceptional performance can be ascribed to its highly selective and efficient membrane permeabilization activity induced by its distinctive self-assembly behavior. Additionally, these assemblies regulate the interplay between enthalpy and entropy during K7W6-membrane interaction, leading to the peptide's two-step mechanism of membrane interaction. These findings provide valuable insights into rational design principles for developing advanced peptide-based drugs while uncovering the functional role played by assembly.

Heterogeneous Structural Disturbance of Cell Membrane by Peptides with Modulated Hydrophobic Properties.

Extensive effort has been devoted to developing new clinical therapies based on membrane-active peptides (MAPs). Previous models on the membrane action mechanisms of these peptides mostly focused on the MAP−membrane interactions in a local region, while the influence of the spatial heterogeneity of the MAP distribution on the membrane was much ignored. Herein, three types of natural peptide variants, AS4-1, AS4-5, and AS4-9, with similar amphiphilic α-helical structures but distinct hydrophobic degrees (AS4-1 < AS4-5 < AS4-9) and net charges (+9 vs. +7 vs. +5), were used to interact with a mixed phosphatidylcholine (PC) and phosphatidylglycerol (PG) membrane. A combination of giant unilamellar vesicle (GUV) leakage assays, atomic force microscopy (AFM) characterizations, and molecular dynamics (MD) simulations demonstrated the coexistence of multiple action mechanisms of peptides on a membrane, probably due to the spatially heterogeneous distribution of peptides on the membrane surface. Specifically, the most hydrophobic peptide (i.e., AS4-9) had the strongest membrane binding, perturbation, and permeabilization effects, leading to the formation of large peptide−lipid aggregates (10 ± 5 nm in height and 150 ± 50 nm in size), as well as continuous fragments and ridges on the supported membrane surface. The AS4-5 peptides, with a half-hydrophilic and half-hydrophobic structure, induced membrane lysis in addition to reconstruction. The most hydrophilic peptide AS4-1 only exhibited unstable binding on the supported membrane surface. These results demonstrate the heterogeneous structural disturbance of model cell membranes by amphiphilic α-helical peptides, which could be significantly strengthened by increasing the degree of hydrophobicity and/or local number density of peptides. This work provides support for the modulation of the membrane activity of MAPs by adjusting their hydrophobicity and local concentration.

Real-time monitoring the interfacial dynamic processes at model cell membranes: Taking cell penetrating peptide TAT as an example.

A real-time and molecule-level monitoring of the interfacial dynamic interactions between molecules and a cell membrane is of vital importance. Herein, taking TAT, one of the most representative cell penetrating peptides, as an example, a photo-voltage transient technique and a dynamic giant bistratal vesicle (GBV) leakage method were combined with the traditional giant unilamellar vesicle (GUV) leakage assays, to provide a molecule-level understanding of the dynamic membrane interaction process performed in a low ionic strength and neutral pH condition. The photo-voltage test based on supported phospholipid bilayers showed a quick disturbance (<1 min) followed by a continuous reconstruction of the membrane by peptides, leading to a slight destruction (at TAT concentrations lower than 1 µg mL-1, i.e., 0.64 µM) or strong damage (e.g. at 10 µg mL-1, i.e., 6.4 µM) of the bilayer structure. The GUV/GBV leakage assays further demonstrated the TAT-induced membrane deformation and transmembrane diffusion of dyes, which occurred in an immediate, linear, and TAT-concentration dependent manner. Moreover, the flux of dye across the substrate-immobilized membranes was approximately three times of that across the substrate-free ones. This work gives information on time and molecular mechanism of the TAT-membrane interactions, demonstrates the different permeabilizing effects of TAT on immobilized and free membranes. Overall, it provides useful strategies to investigate the nano-bio interfacial interactions in a simple, global and real-time way.

A real-time and in-situ monitoring of the molecular interactions between drug carrier polymers and a phospholipid membrane.

The dynamic interactions between drug carrier molecules and a cell membrane can not be ignored in their clinical use. Here a simple, label-free and non-invasive approach, photo-voltage transient method, combined with the atomic force microscopy, dynamic giant unilamellar vesicle leakage assay and cytotoxicity method, was employed for a real-time monitoring of the interaction process. Two representative polymer molecules, polyoxyethylene (35) lauryl ether (Brij35) and polyvinylpyrrolidone (PVPk30), were taken as examples to interact with a phospholipid bilayer membrane in a low ionic strength and neutral pH condition. Brij35 demonstrated an adsorption-accumulation-permeabilization dominated process under the modulation of polymer concentration in the solution. In contrast, PVPk30 performed a dynamic balance between adsorption-desorption of the molecules and/or permeabilization-resealing of the membrane. Such difference explains the high and low cytotoxicity of them, respectively, in the living cell tests. Briefly, through combining the photo-voltage approach with conventional fluorescent microscopy method, this work demonstrates new ideas on the time and membrane actions of polymer surfactants which should be taken into account for their biomedical applications.

Biophysical Impact of Lipid A Modification Caused by Mobile Colistin Resistance Gene on Bacterial Outer Membranes.

Expression of mobile colistin resistance gene mcr-1 results in the addition of phosphoethanolamine (pEtN) to the lipid A headgroup in the bacterial outer membrane (OM) of Gram-negative bacteria, increasing the resistance to the last-line polymyxins. However, the potential biological consequences of such modification remain unclear. Using coarse-grained molecular simulations with quantitative lipidomics models, we discovered pEtN modification of the lipid A headgroup caused substantial changes to the morphology and physicochemical properties of the OM. Single-lipid level structural and energetic analyses revealed that this modification resulted in lipid A-pEtN adopting an abnormally twisted and slanted conformation with a closer packing state because of strengthened inter-lipid attraction. The consequent accumulation of lipid A-pEtN produced a negative curvature of the OM and altered the membrane's tension, fluidity, and rigidity. Our results provide a key mechanistic connection between mcr-1 expression and biophysical changes in the bacterial OM.

Plasmon-enhanced fluorescence imaging with silicon-based silver chips for protein and nucleic acid assay.

Metal-enhanced fluorescence shows great potential for improving the sensitivity of fluoroscopy, which has been widely used in protein and nucleic acid detection for biosensor and bioassay applications. In comparison with the traditional glass-supported metal nanoparticles (MNPs), the introduction of a silicon substrate has been shown to provide an increased surface-enhanced Raman scattering (SERS) effect due to the coupling between the MNPs and the semiconducting silicon substrate. In this work, we further study the fluorescence-enhanced effect of the silicon-supported silver-island (Ag@Si) plasmonic chips. In particular, we investigate their practical application of improving the traditional immunoassay such as the biotin-streptavidin-based protein assay and the protein-/nucleic acid-labeled cell and tissue samples. The protein assay shows a wavelength-dependent enhancement effect of the Ag@Si chip, with an enhancement factor ranging from 1.2 (at 532 nm) to 57.3 (at 800 nm). Moreover, for the protein- and nucleic acid-labeled cell and tissue samples, the Ag@Si chip provides a fluorescence enhancement factor of 3.0-4.1 (at 800 nm) and a significant improvement in the signal/background ratio for the microscopy images. Such a ready accommodation of the fluorescence-enhanced effect for the immunoassay samples with simple manipulations indicates broad potential for applications of the Ag@Si chip not only in biological studies but also in the clinical field.