Marina I. Giannotti

Influence of cholesterol on the phase transition of lipid bilayers: a temperature-controlled force spectroscopy study.

Cholesterol (Chol) plays the essential function of regulating the physical properties of the cell membrane by controlling the lipid organization and phase behavior and, thus, managing the membrane fluidity and its mechanical strength. Here, we explore the model system DPPC:Chol by means of temperature-controlled atomic force microscopy (AFM) imaging and AFM-based force spectroscopy (AFM-FS) to assess the influence of Chol on the membrane ordering and stability. We analyze the system in a representative range of compositions up to 50 mol % Chol studying the phase evolution upon temperature increase (from room temperature to temperatures high above the T(m) of the DPPC bilayer) and the corresponding (nano)mechanical stability. By this means, we correlate the mechanical behavior and composition with the lateral order of each phase present in the bilayers. We prove that low Chol contents lead to a phase-segregated system, whereas high contents of Chol can give a homogeneous bilayer. In both cases, Chol enhances the mechanical stability of the membrane, and an extraordinarily stable system is observed for equimolar fractions (50 mol % Chol). In addition, even when no thermal transition is detected by the traditional bulk analysis techniques for liposomes with high Chol content (40 and 50 mol %), we demonstrate that temperature-controlled AFM-FS is capable of identifying a thermal transition for the supported lipid bilayers. Finally, our results validate the AFM-FS technique as an ideal platform to differentiate phase coexistence and transitions in lipid bilayers and bridge the gap between the results obtained by traditional methods for bulk analysis, the theoretical predictions, and the behavior of these systems at the nanoscale.

pH-Responsive Polysaccharide-Based Polyelectrolyte Complexes As Nanocarriers for Lysosomal Delivery of Therapeutic Proteins

Nanopharmaceutics composed of a carrier and a protein have the potential to improve the activity of therapeutical proteins. Therapy for lysosomal diseases is limited by the lack of effective protein delivery systems that allow the controlled release of specific proteins to the lysosomes. Here we address this problem by developing functional polyelectrolyte-based nanoparticles able to promote acidic pH-triggered release of the loaded protein. Trimethyl chitosan (TMC) was synthesized and allowed to form polyelectrolyte complexes (PECs) with the lysosomal enzyme α-GAL through self-assembly and ionotropic gelation, with average particle size <200 nm, polydispersity index (PDI) <0.2, ζ potential of ∼ 20 mV, and a protein loading efficiency close to 65%. These polyelectrolyte nanoparticles were stable and active under physiological conditions and able to release the enzyme at acidic pH, as demonstrated by in situ atomic force microscopy (AFM). These nanoparticles were further functionalized with Atto 647N for single-particle characterization and tracking their cellular uptake and fate using high-resolution fluorescence microscopy. In contrast with their precursor, TMC, PECs were efficiently internalized by human endothelial cells and mostly accumulated in lysosomal compartments. The superior physicochemical characteristics of the TMC/α-GAL PECs together with their excellent cellular uptake properties indicate their enormous potential as advanced protein delivery systems for the treatment of lysosomal storage diseases.

Enhanced cell-material interactions through the biofunctionalization of polymeric surfaces with engineered peptides

Research on surface modification of polymeric materials to guide the cellular activity in biomaterials designed for tissue engineering applications has mostly focused on the use of natural extracellular matrix (ECM) proteins and short peptides, such as RGD. However, the use of engineered proteins can gather the advantages of these strategies and avoid the main drawbacks. In this study, recombinant engineered proteins called elastin-like recombinamers (ELRs) have been used to functionalize poly(lactic) acid (PLA) model surfaces. The structure of the ELRs has been designed to include the integrin ligand RGDS and the cross-linking module VPGKG. Surface functionalization has been characterized and optimized by means of ELISA and atomic force microscopy (AFM). The results suggest that ELR functionalization creates a nonfouling canvas able to restrict unspecific adsorption of proteins. Moreover, AFM analysis reveals the conformation and disposition of ELRs on the surface. Biological performance of PLA surfaces functionalized with ELRs has been studied and compared with the use of short peptides. Cell response has been assessed for different functionalization conditions in the presence and absence of the bovine serum albumin (BSA) protein, which could interfere with the surface-cell interaction by adsorbing on the interface. Studies have shown that ELRs are able to elicit higher rates of cell attachment, stronger cell anchorages and faster levels of proliferation than peptides. This work has demonstrated that the use of engineered proteins is a more efficient strategy to guide the cellular activity than the use of short peptides, because they not only allow for better cell attachment and proliferation, but also can provide more complex properties such as the creation of nonfouling surfaces.

Thermoplastic Polyurethane:Polythiophene Nanomembranes for Biomedical and Biotechnological Applications

Nanomembranes have been prepared by spin-coating mixtures of a polythiophene (P3TMA) derivative and thermoplastic polyurethane (TPU) using 20:80, 40:60, and 60:40 TPU:P3TMA weight ratios. After structural, topographical, electrochemical, and thermal characterization, properties typically related with biomedical applications have been investigated: swelling, resistance to both hydrolytic and enzymatic degradation, biocompatibility, and adsorption of type I collagen, which is an extra cellular matrix protein that binds fibronectin favoring cell adhesion processes. The swelling ability and the hydrolytic and enzymatic degradability of TPU:P3TMA membranes increases with the concentration of P3TMA. Moreover, the degradation of the blends is considerably promoted by the presence of enzymes in the hydrolytic medium, TPU:P3TMA blends behaving as biodegradable materials. On the other hand, TPU:P3TMA nanomembranes behave as bioactive platforms stimulating cell adhesion and, especially, cell viability. Type I collagen adsorption largely depends on the substrate employed to support the nanomembrane, whereas it is practically independent of the chemical nature of the polymeric material used to fabricate the nanomembrane. However, detailed microscopy study of the morphology and topography of adsorbed collagen evidence the formation of different organizations, which range from fibrils to pseudoregular honeycomb networks depending on the composition of the nanomembrane that is in contact with the protein. Scaffolds made of electroactive TPU:P3TMA nanomembranes are potential candidates for tissue engineering biomedical applications.

Bioactive nanomembranes of semiconductor polythiophene and thermoplastic polyurethane: thermal, nanostructural and nanomechanical properties

Free-standing and supported nanomembranes have been prepared by spin-coating mixtures of a semiconducting polythiophene (P3TMA) derivative and thermoplastic polyurethane (TPU). Thermal studies of TPU:P3TMA blends with 60 : 40, 50 : 50, 40 : 60 and 20 : 80 weight ratios indicate a partial miscibility of the two components. Analysis of the glass transition temperatures allowed us to identify the highest miscibility for the blend with a 40 : 60 weight ratio, this composition being used to prepare both self-standing and supported nanomembranes. The thickness of ultra-thin films made with the 40 : 60 blend ranged from 11 to 93 nm, while the average roughness was 16.3 ± 0.8 nm. In these films the P3TMA-rich phase forms granules, which are dispersed throughout the rest of the film. Quantitative nanomechanical mapping has been used to determine the Youngs modulus value by applying the Derjanguin–Muller–Toporov (DMT) contact mechanics model and the adhesion force of ultra-thin films. The modulus depends on the thickness of the films, values determined for the thicker (80–140 nm)/thinner (10–40 nm) regions of TPU, P3TMA and blend samples being 25/35 MPa, 3.5/12 GPa and 0.9/1.7 GPa, respectively. In contrast the adhesion force is homogeneous through the whole surface of the TPU and P3TMA films (average values: 7.2 and 5.0 nN, respectively), whereas for the blend it depends on the phase distribution. Thus, the adhesion force is higher for the TPU-rich domains than for the P3TMA-rich domains. Finally, the utility of the nanomembranes for tissue engineering applications has been proved by cellular proliferation assays. Results show that the blend is more active as a cellular matrix than each of the two individual polymers.

Hierarchical Assemblies of Gold Nanoparticles at the Surface of a Film Formed by a Bridged Silsesquioxane Containing Pendant Dodecyl Chains

Hierarchical aggregates of gold nanoparticles (NPs) on different length scales were in situ generated at the surface of a bridged silsesquioxane during the process of film formation by polycondensation and solvent evaporation. A precursor of a bridged silsesquioxane based on the reaction product of (glycidoxypropyl)trimethoxysilane (2 mol) with dodecylamine (1 mol) was hydrolytically condensed in a THF solution at room temperature in the presence of formic acid, water, and variable amounts of dodecanethiol-stabilized gold NPs (average diameter of 2 nm). The initial compatibility of the precursor with gold NPs was achieved by the presence of dodecyl chains in both components. Phase separation of gold NPs accompanied by partitioning to the air-polymer interface took place driven by the polycondensation reaction and solvent evaporation. A hierarchical organization of gold NPs in the structures generated at the air-polymer interface was observed. Small body-centered cubic (bcc) crystals of about 20 nm diameter were formed in the first step, in which the 2 nm gold NPs kept their individuality (high-resolution transmission electron microscopy, field emission scanning electron microscopy, and small-angle X-ray diffraction). In the second step, bcc crystals aggregated, forming compact micrometer-sized spherical particles. Under particular evaporation rates a third step of the self-assembly process was observed where micrometer-sized particles formed fractal structures. Increasing the initial concentration of gold NPs in the formulation led to more compact fractal structures in agreement with theoretical simulations. The surface percolation of NPs in fractal structures can be the basis of useful applications.

AFM-Based Force-Clamp Monitors Lipid Bilayer Failure Kinetics

The lipid bilayer rupture phenomenon is here explored by means of atomic force microscopy (AFM)-based force clamp, for the first time to our knowledge, to evaluate how lipid membranes respond when compressed under an external constant force, in the range of nanonewtons. Using this method, we were able to directly quantify the kinetics of the membrane rupture event and the associated energy barriers, for both single supported bilayers and multibilayers, in contradistinction to the classic studies performed at constant velocity. Moreover, the affected area of the membrane during the rupture process was calculated using an elastic deformation model. The elucidated information not only contributes to a better understanding of such relevant process, but also proves the suitability of AFM-based force clamp to study model structures as lipid bilayers. These findings on the kinetics of lipid bilayers rupture could be extended and applied to the study of other molecular thin films. Furthermore, systems of higher complexity such as models mimicking cell membranes could be studied by means of AFM-based force-clamp technique.

Electronic, electric and electrochemical properties of bioactive nanomembranes made of polythiophene:thermoplastic polyurethane

The electronic, electric and electrochemical response of nanomembranes prepared by using spin-coating mixtures of a semiconducting polythiophene derivative (P3TMA) and thermoplastic polyurethane (TPU) has been exhaustively examined by UV-vis spectroscopy, conductive AFM, current/voltage measurements and cyclic voltammetry. TPU:P3TMA nanomembranes were reported to be good substrates for applications related to tissue engineering, acting as a cellular matrix for cell adhesion and proliferation. Both TPU:P3TMA and P3TMA nanomembranes show semiconductor behavior with very similar band gap energy (2.35 and 2.32 eV, respectively), which has been attributed to the influence of the fabrication process on the π-conjugation length and packing interactions of P3TMA chains. This behavior is in opposition to the observations in THF solution, which indicates that the band gap energy of P3TMA is clearly lower than that of the mixture, independently of the concentration. The current and conductivity values determined for the nanomembranes, which range from 0.43 to 1.85 pA and from 2.23 × 10−5 to 5.19 × 10−6 S cm−1, respectively, evidence inhomogeneity in the P3TMA-rich domains. This has been associated with the irregular distribution of the doped chains and the presence of insulating TPU chains. The voltammetric response of TPU:P3TMA and P3TMA nanomembranes is similar in terms of ability to store charge and electrochemical stability. Overall results indicate that TPU:P3TMA nanomembranes are potential candidates for the fabrication of bioactive substrates able to promote cell regeneration through electrical or electrochemical stimulation.

Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information.

Morphological and Nanomechanical Behavior of Supported Lipid Bilayers on Addition of Cationic Surfactants

The addition of surfactants to lipid bilayers is important for the modulation of lipid bilayer properties (e.g., in protein reconstitution and development of nonviral gene delivery vehicles) and to provide insight on the properties of natural biomembranes. In this work, the thermal behavior, organization, and nanomechanical stability of model cationic lipid-surfactant bilayers have been investigated. Two different cationic surfactants, hexadecyltrimethylammonium bromide (CTAB) and a novel derivative of the amino acid serine (Ser16TFAc), have been added (up to 50 mol %) to both liposomes and supported lipid bilayers (SLBs) composed by the zwitterionic phospholipid DPPC. The thermal phase behavior of mixed liposomes has been probed by differential scanning calorimetry (DSC), and the morphology and nanomechanical properties of mixed SLBs by atomic force microscopy-based force spectroscopy (AFM-FS). Although DSC thermograms show different results for the two mixed liposomes, when both are deposited on mica substrates similar trends on the morphology and the mechanical response of the lipid-surfactant bilayers are observed. DSC thermograms indicate microdomain formation in both systems, but while CTAB decreases the degree of organization on the liposome bilayer, Ser16TFAc ultimately induces the opposite effect. Regarding the AFM-FS studies, they show that microphase segregation occurs for these systems and that the effect is dependent on the surfactant content. In both SLB systems, different microdomains characterized by their height and breakthrough force Fb are formed. The molecular organization and composition is critically discussed in the light of our experimental results and literature data on similar lipid-surfactant systems.