Domain-Targeted Membrane Partitioning of Specific Proteins with DNA Nanodevices.

The cell membrane exhibits a remarkable complexity of lipids and proteins that dynamically segregate into distinct domains to coordinate various cellular functions. The ability to manipulate the partitioning of specific membrane proteins without involving genetic modification is essential for decoding various cellular processes but highly challenging. In this work, by conjugating cholesterols or tocopherols at the three bottom vertices of the DNA tetrahedron, we develop two sets of nanodevices for the selective targeting of lipid-order (Lo) and lipid-disorder (Ld) domains on the live cell membrane. By incorporation of protein-recognition ligands, such as aptamers or antibodies, through toehold-mediated strand displacement, these DNA nanodevices enable dynamic translocation of target proteins between these two domains. We first used PTK7 as a protein model and demonstrated, for the first time, that the accumulation of PTK7 to the Lo domains could promote tumor cell migration, while sequestering it in the Ld domains would inhibit the movement of the cells. Next, based on their modular nature, these DNA nanodevices were extended to regulate the process of T cell activation through manipulating the translocation of CD45 between the Lo and the Ld domains. Thus, our work is expected to provide deep insight into the study of membrane structure and molecular interactions within diverse cell signaling processes.

Unsaturation effects on lipid transmembrane asymmetry.

Within cell plasma membranes, unsaturated lipids are asymmetrically distributed over the inner and outer leaflets, offering an attractive local structural feature. However, the mechanism to keep lipid transmembrane asymmetry and the closely related transmembrane movement (flip-flop) for unsaturated lipids remain poorly understood. Here, we applied sum frequency generation vibrational spectroscopy to investigate this lipid transmembrane asymmetry upon mimicking the cell membrane homeostatic processes. On the one hand, unsaturated lipids were found to hinder the flip-flop process and preserve lipid transmembrane asymmetry in model cell membranes, owing to the steric hindrance caused by their bent tails. On the other hand, local unsaturated lipids in the mixed unsaturated/saturated lipid bilayer were conducive to the formation of the local asymmetry. Therefore, lipid unsaturation can be recognized as an intrinsic key factor to form and maintain lipid transmembrane asymmetry in cell membranes.

Re-Examining Interaction between Antimicrobial Peptide Aurein 1.2 and Model Cell Membranes via SFG.

Aurein 1.2 (Aur), a highly efficient 13-residue antimicrobial peptide (AMP) with a broad-spectrum antibiotic activity originally derived from the Australian frog skin secretions, can nonspecifically disrupt bacterial membranes. To deeply understand the molecular-level detail of the antimicrobial mechanism, here, we artificially established comparative experimental models to investigate the interfacial interaction process between Aur and negatively charged model cell membranes via sum frequency generation vibrational spectroscopy. Sequencing the vibrational signals of phenyl, C-H, and amide groups from Aur has characteristically helped us differentiate between the initial adsorption and subsequent insertion steps upon mutual interaction between Aur and the charged lipids. The phenyl group at the terminal phenylalanine residue can act as an anchor in the adsorption process. The time-dependent signal intensity of α-helices showed a sharp rise once the Aur molecules came into contact with the negatively charged lipids, indicating that the adsorption process was ongoing. Insertion of Aur into the charged lipids then offered the detectable interfacial C-H signals from Aur. The achiral and chiral amide I signals suggest that Aur had formed ß-folding-like aggregates after interacting with the charged lipids, along with the subsequent descending α-helical amide I signals. The above-mentioned experimental results provide the molecular-level detail on how the Aur molecules interact with the cell membranes, and such a mechanism study can offer the necessary support for the AMP design and later application.

Exploring Interfacial Hydrolysis of Artificial Neutral Lipid Monolayer and Bilayer Catalyzed by Phospholipase C.

Phospholipase C (PLC) represents an important type of enzymes with the feature of hydrolyzing phospholipids at the position of the glycerophosphate bond, among which PLC extracted from Bacillus cereus (BC-PLC) has been extensively studied owing to its similarity to hitherto poorly characterized mammalian analogues. This study focuses on investigating the interfacial hydrolysis mechanism of phosphatidylcholine (PC) monolayer and bilayer membranes catalyzed by BC-PLC using sum frequency generation vibrational spectroscopy (SFG-VS) and laser scanning confocal microscopy (LSCM). We found that, upon interfacial hydrolysis, BC-PLC was adsorbed onto the lipid interface and catalyzed the lipolysis with no net orientation, as evidenced by the silent amide I band, indicating that ordered PLC alignment was not a prerequisite for the enzyme activity, which is very different from what we have reported for phospholipase A1 (PLA1) and phospholipase A2 (PLA2) [Kai, S. Phys. Chem. Chem. Phys. 2018, 20(1), 63-67; Wang, F. Langmuir 2019, 35(39), 12831-12838; Zhang, F. Langmuir 2020, 36(11), 2946-2953]. For the PC monolayer, one of the two hydrolysates, phosphocholine, desorbed from the interface into the aqueous phase, while the other one, diacylglycerol (DG), stayed well packed with high order at the interface. For the PC bilayer, phosphocholine dispersed into the aqueous phase too, similar to the monolayer case; however, DG, presumably formed clusters with the unreacted lipid substrates and desorbed from the interface. With respect to both the monolayer and bilayer cases, mechanistic schematics were presented to illustrate the different interfacial hydrolysis processes. Therefore, this model experimental study in vitro provides significant molecular-level insights and contributes necessary knowledge to reveal the lipolysis kinetics with respect to PLC and lipid membranes with monolayer and bilayer structures.

Interaction between Antimicrobial Peptide CM15 and a Model Cell Membrane Affected by CM15 Terminal Amidation and the Membrane Phase State.

Antimicrobial peptides (AMPs) have been proposed as an effective class of antimicrobial agents against microorganisms. In this work, the interaction between an antimicrobial peptide, CM15, and a negatively charged phospholipid bilayer, DPPG, was studied via sum frequency generation (SFG) vibrational spectroscopy. Two structurally correlated characteristic variables were introduced to reveal the interaction mechanism/efficiency, i.e. C-terminal amidation and temperature variation (∼20 °C, room temperature, and ∼35 °C, close to human body temperature). Experimental results indicated that owing to the increased positive charge, C-terminal amidation resulted in rapid adsorption onto the bilayer surface and efficient disruption of the outer layer, exhibiting less ordered insertion orientation. The elevated temperature (from ∼20 °C to ∼35 °C) promoted the penetration of both the outer and inner leaflets by the peptides and finally led to the disruption of the whole bilayer owing to the enhanced fluidity of the bilayer. From the perspective of the interaction mechanism, this experimental study provides two practical cues to understand the disruption process of the negatively charged model biomembranes, which can lay the structural foundation for designing and developing high-efficiency antimicrobial peptides.

Polar Solvents Induce Sum Frequency Generation Activity for Multiwalled Carbon Nanotubes.

Regarding methods of process and use of carbon nanotubes (CNTs), solvents are generally employed to disperse or dissolve CNTs as a pretreatment or intermediate process step. This naturally imposes an essential issue on how CNTs and solvents interact with each other, which seems trivial, comparatively inconsequential, and might often be overlooked from the perspective of engineering scenarios. However, as a matter of fact, it is indeed a fascinating and significant topic. In this article, to investigate the interfacial properties of multiwalled CNTs (MWCNTs) exposed to widely utilized solvents, we applied sum frequency generation vibrational spectroscopy (SFG-VS) to probe solvent-wetted MWCNTs and proved that polar solvents can substantially alter the interfacial optical property of MWCNTs. First, the interfacial optical phonon vibrational modes were detected when MWCNTs were wetted by polar solvents, i.e., water and dimethylformamide (DMF), while such modes were inactive when the solvents were nonpolar, i.e., decalin and air. Second, the interfacial optical phonon vibration frequency displayed distinct dependence on surface defects of MWCNTs. Combining theoretical analysis with experimental verification, a valid conjecture with respect to surface phonon vibration activity for MWCNTs was proposed. This phenomenon of polar solvent-induced SFG activity may have the potential to find applications in optical detection and environmental sensing in the near future.

Sum Frequency Generation of Interfacial Lipid Monolayers Shows Polarization Dependence on Experimental Geometries.

Sum frequency generation (SFG) vibrational spectroscopy has been widely employed to investigate molecular structures of biological surfaces and interfaces including model cell membranes. A variety of lipid monolayers or bilayers serving as model cell membranes and their interactions with many different molecules have been extensively studied using SFG. Here, we conducted an in-depth investigation on polarization-dependent SFG signals collected from interfacial lipid monolayers using different experimental geometries, i.e., the prism geometry (total internal reflection) and the window geometry (external reflection). The different SFG spectral features of interfacial lipid monolayers detected using different experimental geometries are due to the interplay between the varied Fresnel coefficients and second-order nonlinear susceptibility tensor terms of different vibrational modes (i.e., ss and as modes of methyl groups), which were analyzed in detail in this study. Therefore, understanding the interplay between the interfacial Fresnel coefficients and χ((2)) tensors is a prerequisite for correctly understanding the SFG spectral features with respect to different experimental geometries. More importantly, the derived information in this paper should not be limited to the methyl groups with a C3v symmetry; valid extension to interfacial functional groups with different molecular symmetries and even chiral interfaces could be expected.

Calcium-dependent and -independent annexin V binding: distinct molecular behaviours at cell membrane interfaces.

Distinct annexin V binding behaviours in Ca2+-dependent and Ca2+-independent cases were comparatively investigated using sum frequency generation vibrational spectroscopy. It was discovered that binding affected the molecular arrangement of both membrane leaflets, and the initial Ca2+-independent binding went through a transition with annexin V reorientation to a more stable state upon adding Ca2+.

Ultrasmall and photostable nanotheranostic agents based on carbon quantum dots passivated with polyamine-containing organosilane molecules.

In this work, we demonstrate that ultrasmall, photostable and multifunctional carbon quantum dots (or carbon dots, CDs) passivated with polyamine-containing organosilane molecules can realize simultaneous cell imaging and anticancer drug delivery. The presence of abundant surface amine groups makes these CDs be able to covalently link with the anticancer drug, doxorubicin (DOX), with an extremely high drug loading capacity (62.8%), while the surface hydroxyl groups ensure the good water-dispersibility of the CDs-DOX. Besides the use as a drug carrier, the fluorescent CDs also enable the dynamic tracing of the drug release process. When the CDs-DOX complexes were internalized by the human breast cancer cells (MCF-7), DOX could gradually detach from the surface of CDs and enter into the cell nucleus, while the CDs themselves still resided in the cytoplasm. In addition, the in vivo experiments showed that the CDs-DOX complexes exhibited a better tumor inhibition performance than free DOX molecules, which may be ascribed to the prolonged drug accumulation in tumor tissues. Furthermore, the as-synthesized CDs also exhibited negligible cytotoxicity/systemic side effects, and could successfully illuminate mammalian, bacterial and fungal cells, making them good candidates as not only drug delivery vehicles but also universal cell imaging reagents. The present work may have implications for the fabrication of functional carbon-based nanomaterials and foster the development of carbon dots as novel nanotheranostics for various biomedical applications.

Carbon Dot-Based Platform for Simultaneous Bacterial Distinguishment and Antibacterial Applications.

In this work, we prepared quaternized carbon dots (CDs) with simultaneous antibacterial and bacterial differentiation capabilities using a simple carboxyl-amine reaction between lauryl betaine and amine-functionalized CDs. The obtained quaternized CDs have several fascinating properties/abilities: (1) A long fluorescence emission wavelength ensures the exceptional bacterial imaging capability, including the super-resolution imaging ability; (2) the polarity-sensitive fluorescence emission property leads to significantly enhanced fluorescence when the quaternized CDs interact with bacteria; (3) the presence of both hydrophobic hydrocarbon chains and positively charged quaternary ammonium groups makes the CDs selectively attach to Gram-positive bacteria, realizing the bacterial differentiation; (4) excellent antimicrobial activity is seen against Gram-positive bacteria with a minimum inhibitory concentration of 8 µg/mL for Staphylococcus aureus. Besides, the quaternized CDs are highly stable in various aqueous solutions and exhibit negligible cytotoxicity, suggesting that they hold great promise for clinical applications. Compared to the traditional Gram staining method, the selective Gram-positive bacterial imaging achieved by the quaternized CDs provides a much simpler and faster method for bacterial differentiation. In summary, by combining selective Gram-positive bacterial recognition, super-resolution imaging, and exceptional antibacterial activity into a single system, the quaternized CDs represent a novel kind of metal-free nanoparticle-based antibiotics for antibacterial application and a new type of reagent for efficient bacterial differentiation.