Molecular Insight into the Ligand–IgG Interactions for 4-Mercaptoethyl-pyridine Based Hydrophobic Charge-Induction Chromatography

Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification. In the present work, the molecular simulation methods were used to investigate the interactions between MEP ligand and Fc fragment of IgG (Fc-A). Six ligands with different structures of spacer arm were studied with molecular docking and dynamics simulation at neutral and acidic pH. The binding modes and the interaction energies were analyzed. The results indicated that all ligands tested could bind into the selected pocket on the C_H2 domain of Fc-A at neutral pH. The pyridine ring on the top of MEP ligands acts as a major role to provide the hydrophobic association and hydrogen bond for the ligand–IgG binding; meanwhile, the sulfone group on the spacer arm might form the additional hydrogen bond and enhance the binding of ligand onto the surface of IgG. The replacements of thioether sulfur atom on the spacer arm with either nitrogen or oxygen atom seem to have little influence on the binding. The influences of pH on the ligand–IgG interactions were also studied with the molecular dynamics simulation. It was found that MEP ligands would departed from the surface of Fc-A at low pH due to the electrostatic repulsion. The ligands with a sulfone group on the spacer arm would weaken the electrostatic repulsion and need more acidic conditions for the departing of ligand. The molecular simulation results were in agreement with some experimental observations, which would be useful to elucidate the molecular mechanism of HCIC and design a novel ligand to improve the efficiency of antibody separation

Subcellular Fate of a Fluorescent Cholesterol-Poly(ethylene glycol) Conjugate: An Excellent Plasma Membrane Imaging Reagent

Cholesterol-containing molecules or nanoparticles play a significant role in achieving favorable plasma membrane imaging and efficient cellular uptake of drugs by the excellent membrane anchoring capability of the cholesterol moiety. By linking cholesterol to a water-soluble component (such as poly­(ethylene glycol), PEG), the resulting cholesterol-PEG conjugate can form micelles in aqueous solution through self-assembly, and such a micellar structure represents an important drug delivery vehicle in which hydrophobic drugs can be encapsulated. However, the understanding of the subcellular fate and cytotoxicity of cholesterol-PEG conjugates themselves remains elusive. Herein, by using cholesterol-PEG2000-fluorescein isothiocyanate (Chol-PEG-FITC) as a model system, we found that the Chol-PEG-FITC molecules could attach to the plasma membranes of mammalian cells within 10 min and such a firm membrane attachment could last at least 1 h, displaying excellent plasma membrane staining performance that surpassed that of commonly used commercial membrane dyes such as DiD and CellMask. Besides, we systematically studied the endocytosis pathway and intracellular distribution of Chol-PEG-FITC and found that the cell surface adsorption and endocytosis processes of Chol-PEG-FITC molecules were lipid-raft-dependent. After internalization, the Chol-PEG-FITC molecules gradually reached many organelles with membrane structures. At 5 h, they were mainly distributed in lysosomes and the Golgi apparatus, with some in the endoplasmic reticulum (ER) and very few in the mitochondrion. At 12 h, the Chol-PEG-FITC molecules mostly aggregated in the Golgi apparatus and ER close to the nucleus. Finally, we demonstrated that Chol-PEG-FITC was toxic to mammalian cells only at concentrations above 50 μM. In summary, Chol-PEG-FITC can be a promising plasma membrane imaging reagent to avoid the fast cellular internalization and quick membrane detachment problems faced by commercial membrane dyes. We believe that the investigation of the dynamic subcellular fate of Chol-PEG-FITC can provide important knowledge to facilitate the use of cholesterol–PEG conjugates in fields such as cell surface engineering and drug delivery

Permeabilization-Tolerant Plasma Membrane Imaging Reagent Based on Amine-Rich Glycol Chitosan Derivatives

Immunofluorescence staining is a crucial tool for studying the structure and behavior of intracellular proteins and organelles. During the staining process, the permeabilization treatment is usually required to enhance the penetration of a fluorescent antibody into the cells. However, since most of the membrane imaging dyes as well as the membrane lipids will detach from the cell surface after permeabilization, membrane labeling using these dyes is not compatible with immunofluorescence staining. Herein, by linking cholesterol-polyethylene glycol (PEG-Chol) and fluorescein isothiocyanate (FITC) with the amine-rich glycol chitosan (GC), we prepared a multifunctional polymeric construct, GC-PEG Chol-FITC, and realized permeabilization-tolerant plasma membrane imaging. Owing to the presence of abundant amine groups in the labeling reagent and the membrane proteins/lipids, the addition of paraformaldehyde in the fixation step induces the amine-cross-linking between the labeling reagents and the membrane proteins/lipids, thus preventing the detachment of fluorophores from the cell surface after permeabilization. Besides, the large molecular weight effect of the imaging reagent may also account for its antipermeabilization property. Furthermore, by combining immunofluorescence staining with the plasma membrane labeling by GC-PEG Chol-FITC, we simultaneously imaged the plasma membrane and cytoskeletons, and clearly observed metaphase cells and binucleated cells. The concept of using amine-rich polymeric dyes for plasma membrane imaging will inspire the development of more permeabilization-resistant membrane labeling dyes with better performance, which can realize simultaneous membrane and intracellular protein imaging and facilitate the future studies of membrane–intracellular protein interactions

Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy

Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan–10% PEG2000 cholesterol–10% biotin (abbreviated as “GC-Chol-Biotin”), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period

Qualitative and Quantitative Analyses of the Molecular-Level Interaction between Memantine and Model Cell Membranes

Sum frequency generation (SFG) vibrational spectroscopy was employed to study the interaction between memantine (a water-soluble drug for treating Alzheimer’s disease) and lipid bilayers (including zwitterionic PC and negatively charged PG lipid bilayers) at the molecular level in real time and <i>in situ</i>. SFG results revealed how the memantine affected these lipid bilayers in terms of the lipid dynamics, average tilt angle (θ), as well as angle distribution width (σ). It was found that memantine could adsorb onto the zwitterionic PC surface but did not affect the flip-flop rate of the PC bilayer even in the presence of 5.0 mM memantine, indicating the negligible interaction between memantine and the PC bilayer. However, for the negatively charged PG bilayer, it was found that the outer PG leaflet could be significantly destroyed by memantine at a relatively low memantine concentration (1.0 mM), while the inner PG leaflet remained intact. Besides, the θ and σ of CD₃ groups in the outer PG lipid leaflet were calculated to be ∼82.0° and ∼19.5° after adding 5 mM memantine, respectively, indicating that these CD₃ groups were prone to lie down at the membrane surface (versus the surface normal) with the addition of 5 mM memantine while nearly standing up without the addition of drug molecules. These monolayer- and molecular-level results could hardly be obtained by other techniques. To the best of our knowledge, this is the first experimental attempt to quantify the drug-induced orientational changes of lipid molecules within a lipid bilayer. The present work provided an in-depth understanding on the interaction between memantine and model cell membranes, which will potentially benefit the development of new drugs for neurodegenerative diseases involving drug–membrane interaction

Highly Sensitive and Selective Detection of Dopamine Using One-Pot Synthesized Highly Photoluminescent Silicon Nanoparticles

A simple and highly efficient method for dopamine (DA) detection using water-soluble silicon nanoparticles (SiNPs) was reported. The SiNPs with a high quantum yield of 23.6% were synthesized by using a one-pot microwave-assisted method. The fluorescence quenching capability of a variety of molecules on the synthesized SiNPs has been tested; only DA molecules were found to be able to quench the fluorescence of these SiNPs effectively. Therefore, such a quenching effect can be used to selectively detect DA. All other molecules tested have little interference with the dopamine detection, including ascorbic acid, which commonly exists in cells and can possibly affect the dopamine detection. The ratio of the fluorescence intensity difference between the quenched and unquenched cases versus the fluorescence intensity without quenching (Δ<i>I</i>/<i>I</i>) was observed to be linearly proportional to the DA analyte concentration in the range from 0.005 to 10.0 μM, with a detection limit of 0.3 nM (<i>S</i>/<i>N</i> = 3). To the best of our knowledge, this is the lowest limit for DA detection reported so far. The mechanism of fluorescence quenching is attributed to the energy transfer from the SiNPs to the oxidized dopamine molecules through Förster resonance energy transfer. The reported method of SiNP synthesis is very simple and cheap, making the above sensitive and selective DA detection approach using SiNPs practical for many applications

Universal Cell Surface Imaging for Mammalian, Fungal, and Bacterial Cells

Because of the distinct surface structures of different cells (mammalian cells, fungi, and bacteria), surface labeling for these cells requires a variety of fluorescent dyes. Besides, fluorescent dyes (especially the commercial ones) for staining Gram-negative bacterial cell walls are still lacking. Herein, a conformation-adjustable glycol chitosan (GC) derivative (GC-PEG cholesterol-FITC) with “all-in-one” property was developed to realize universal imaging for plasma membranes of mammalian cells (via hydrophobic interaction) and cell walls of fungal and bacterial cells (via electrostatic interaction). By comparing the different staining behaviors of GC-PEG cholesterol-FITC and three other analogs (GC-PEG-FITC, GC-FITC, and cholesterol-PEG-FITC), we have elucidated the different roles the hydrophobic and electrostatic interactions play in the staining performance of these different cells. Such a simple, noncytotoxic, economic, and universal cell surface staining reagent will be very useful for investigating cell surface-related biological events and advancing cell surface engineering of various types of cells

Shape-Dependent Radiosensitization Effect of Gold Nanostructures in Cancer Radiotherapy: Comparison of Gold Nanoparticles, Nanospikes, and Nanorods

The shape effect of gold (Au) nanomaterials on the efficiency of cancer radiotherapy has not been fully elucidated. To address this issue, Au nanomaterials with different shapes but similar average size (∼50 nm) including spherical gold nanoparticles (GNPs), gold nanospikes (GNSs), and gold nanorods (GNRs) were synthesized and functionalized with poly­(ethylene glycol) (PEG) molecules. Although all of these Au nanostructures were coated with the same PEG molecules, their cellular uptake behavior differed significantly. The GNPs showed the highest cellular responses as compared to the GNSs and the GNRs (based on the same gold mass) after incubation with KB cancer cells for 24 h. The cellular uptake in cells increased in the order of GNPs > GNSs > GNRs. Our comparative studies indicated that all of these PEGylated Au nanostructures could induce enhanced cancer cell-killing rates more or less upon X-ray irradiation. The sensitization enhancement ratios (SERs) calculated by a multitarget single-hit model were 1.62, 1.37, and 1.21 corresponding to the treatments of GNPs, GNSs, and GNRs, respectively, demonstrating that the GNPs showed a higher anticancer efficiency than both GNSs and GNRs upon X-ray irradiation. Almost the same values were obtained by dividing the SERs of the three types of Au nanomaterials by their corresponding cellular uptake amounts, indicating that the higher SER of GNPs was due to their much higher cellular uptake efficiency. The above results indicated that the radiation enhancement effects were determined by the amount of the internalized gold atoms. Therefore, to achieve a strong radiosensitization effect in cancer radiotherapy, it is necessary to use Au-based nanomaterials with a high cellular internalization. Further studies on the radiosensitization mechanisms demonstrated that ROS generation and cell cycle redistribution induced by Au nanostructures played essential roles in enhancing radiosensitization. Taken together, our results indicated that the shape of Au-based nanomaterials had a significant influence on cancer radiotherapy. The present work may provide important guidance for the design and use of Au nanostructures in cancer radiotherapy

A Water-Soluble, Green-Light Triggered, and Photo-Calibrated Nitric Oxide Donor for Biological Applications

Nitric oxide (NO) is a versatile endogenous molecule, involved in various physiological processes and implicated in the progression of many pathological conditions. Therefore, NO donors are valuable tools in NO related basic and applied applications. The traditional spontaneous NO donors are limited in scenarios where flux, localization, and dose of NO could be monitored. This has promoted the development of novel NO donors, whose NO release is not only under control, but also self-calibrated. Herein, we reported a phototriggered and photocalibrated NO donor (<b>NOD565</b>) with an N-nitroso group on a rhodamine dye. <b>NOD565</b> is nonfluorescent and could release NO efficiently upon irradiation by green light. A bright rhodamine dye is generated as a side-product and its fluorescence can be used to monitor the NO release. The potentials of <b>NOD565</b> in practical applications are showcased in in vitro studies, e.g., platelet aggregation inhibition and fungi growth suppression