ABSTRACT
Micropipette aspiration (MPA) is one of the gold standards for quantifying biological samples' mechanical properties, which are crucial from the cell membrane scale to the multicellular tissue. However, relying on the manipulation of individual home-made glass pipettes, MPA suffers from low throughput and no automation. Here, we introduce the sliding insert micropipette aspiration method, which permits parallelization and automation, thanks to the insertion of tubular pipettes, obtained by photolithography, within microfluidic channels. We show its application both at the lipid bilayer level, by probing vesicles to measure membrane bending and stretching moduli, and at the tissue level by quantifying the viscoelasticity of 3D cell aggregates. This approach opens the way to high-throughput, quantitative mechanical testing of many types of biological samples, from vesicles and individual cells to cell aggregates and explants, under dynamic physico-chemical stimuli.
ABSTRACT
HYPOTHESIS: There is a lack of understanding of the interplay between the copolymer composition profile and thermal transition observed in aqueous solutions of N-isopropyl acrylamide (NIPAM) copolymers, as well as the correlation between this transition and the formation and structure of copolymer self-assemblies. EXPERIMENTS: For this purpose, we investigated the response of five copolymers with the same molar mass and chemical composition, but with different composition profile in aqueous solution against temperature. Using complementary analytical techniques, we probed structural properties at different length scales, from the molecular scale with Nuclear Magnetic Resonance (NMR) to the colloidal scale with Dynamic Light Scattering (DLS) and Small Angle Neutron Scattering (SANS). FINDINGS: NMR and SANS investigations strengthen each other and allow a clear picture of the change of copolymer solubility and related copolymer self-assembly as a function of temperature. At the molecular scale, dehydrating NIPAM units drag N,N-dimethyl acrylamide (DMA) moieties with them in a gradual collapse of the copolymer chain; this induces a morphological transition of the self-assemblies from star-like nanostructures to crew-cut micelles. Interestingly, the transition spans a temperature range which depends on the monomer distribution profile in the copolymer chain, with the asymmetric triblock copolymer specimen revealing the broadest one. We show that the broad morphological transitions associated with gradient copolymers can be mimicked and even surpassed by the use of stepwise gradient (asymmetric) copolymers, which can be more easily and reproducibly synthesized than linear gradient copolymers.
ABSTRACT
The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.
Subject(s)
Extracellular Vesicles , Cryoelectron Microscopy , Extracellular Vesicles/chemistry , Microscopy, Atomic Force/methods , Lipopolysaccharides , Lipoproteins/analysisABSTRACT
In recent years, lipid cubic nanoparticles have emerged as promising nanocarriers for drug delivery, due to the several advantages they exhibit with respect to other lipid systems. Here, we report on lipid cubic nanoparticles stabilized by PNIPAM-based amphiphilic block copolymers, specifically, poly(N, N-dimethylacrylamide)-block-poly(N-isopropylacrylamide) (PDMA-b-PNIPAM), as a new class of drug delivery systems (DDS). In vitro studies on the internalization efficiency of the DDS towards two types of human cancer cells (colon HCT-116 and bladder T24 cells), carried out employing a set of sensitive techniques (confocal laser scanning microscopy (CLSM), flow cytometry, scanning electron microscopy (SEM), fluorescence spectroscopy), highlight a prominent role of PDMA-b-PNIPAM stabilizer in enhancing the uptake of cubosomes, compared to the standard Pluronic F127-based formulations. The drug delivery potential of cubosomes, tested by encapsulating a chemotherapeutic drug, camptothecin (CPT), and conducting cytotoxicity studies against 2D plated cells and 3D spheroids, confirm that PDMA-b-PNIPAM-stabilized cubosomes improve the efficacy of treatment with CPT. The origin of this effect lies in the higher lipophilicity of the stabilizer, as we confirm by studying the interaction between the cubosomes and biomimetic membranes of lipid vesicles with Small Angle X-Ray Scattering (SAXS) and CLSM experiments. These results corroborate our fundamental understanding of the interaction between cubosomes and cells, and on the role of polymer to formulate lipid cubic nanoparticles as DDS.
Subject(s)
Acrylic Resins , Nanoparticles , Humans , Scattering, Small Angle , X-Ray Diffraction , Nanoparticles/chemistry , Polymers , Lipids/chemistryABSTRACT
Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.
Subject(s)
Liposomes , Nanoparticles , Humans , Liposomes/chemistry , Nanoparticles/chemistry , Proteins , HeLa Cells , Silicon Dioxide/chemistryABSTRACT
The design of cellular functions in synthetic systems, inspired by the internal partitioning of living cells, is a constantly growing research field that is paving the way to a large number of new remarkable applications. Several hierarchies of internal compartments like polymersomes, liposomes, and membranes are used to control the transport, release, and chemistry of encapsulated species. However, the experimental characterization and the comprehension of glycolipid mesostructures are far from being fully addressed. Lipid A is indeed a glycolipid and the endotoxic part of Gram-negative bacterial lipopolysaccharide; it is the moiety that is recognized by the eukaryotic receptors giving rise to the modulation of innate immunity. Herein we propose, for the first time, a combined approach based on hybrid Particle-Field (hPF) Molecular Dynamics (MD) simulations and Small Angle X-Ray Scattering (SAXS) experiments to gain a molecular picture of the complex supramolecular structures of lipopolysaccharide (LPS) and lipid A at low hydration levels. The mutual support of data from simulations and experiments allowed the unprecedented discovery of the presence of a nano-compartmentalized phase composed of liposomes of variable size and shape which can be used in synthetic biological applications.
Subject(s)
Lipopolysaccharides , Liposomes , Lipopolysaccharides/chemistry , Lipid A , Scattering, Small Angle , X-Ray Diffraction , Bacteria , GlycolipidsABSTRACT
Although promising for biomedicine, the clinical translation of inorganic nanoparticles (NPs) is limited by low biocompatibility and stability in biological fluids. A common strategy to circumvent this drawback consists in disguising the active inorganic core with a lipid bilayer coating, reminiscent of the structure of the cell membrane to redefine the chemical and biological identity of NPs. While recent reports introduced membrane-coating procedures for NPs, a robust and accessible method to quantify the integrity of the bilayer coverage is not yet available. To fill this gap, we prepared SiO2 nanoparticles (SiO2NPs) with different membrane coverage degrees and monitored their interaction with AuNPs by combining microscopic, scattering, and optical techniques. The membrane-coating on SiO2NPs induces spontaneous clustering of AuNPs, whose extent depends on the coating integrity. Remarkably, we discovered a linear correlation between the membrane coverage and a spectral descriptor for the AuNPs' plasmonic resonance, spanning a wide range of coating yields. These results provide a fast and cost-effective assay to monitor the compatibilization of NPs with biological environments, essential for bench tests and scale-up. In addition, we introduce a robust and scalable method to prepare SiO2NPs/AuNPs hybrids through spontaneous self-assembly, with a high-fidelity structural control mediated by a lipid bilayer.
Subject(s)
Metal Nanoparticles , Nanoparticles , Lipid Bilayers/chemistry , Metal Nanoparticles/chemistry , Gold/chemistry , Silicon Dioxide/chemistry , Biomimetics , Nanoparticles/chemistryABSTRACT
The design of drug delivery systems (DDS) for the encapsulation of therapeutic agents and the controlled release to the target site of the disease is one of the main goals of nanomedicine. Although already explored in an extensive number of studies over the years, lipid assemblies, and particularly liposomes, are still considered the most promising and interesting candidates as DDS due to their biocompatibility and structural similarity with plasma membranes. Lately, this research area has been extended to include more complex lipid assemblies, such as cubosomes. Cubosomes are an emerging structural platform for the delivery of molecules with pharmaceutical interest, such as drugs, bioactives and contrast agents. Here we report on the application of a thermo-responsive copolymer poly(N,N-dimethylacrylamide)-block-poly(N-isopropylacrylamide) (PDMA-b-PNIPAM), as a thermoresponsive stabilizer of lipid-based nanoparticles for drug-delivery. First, we assessed the affinity of PDMA-b-PNIPAM towards supported and free-standing bilayers; then, we explored the colloidal and thermoresponsive properties of cubic self-assembled DDS composed of glycerol-monooleate (GMO), where PDMA-b-PNIPAM replaces the conventional stabilizer Pluronic F127 (PEOx-PPOy-PEOx), normally used for cubosomes. We prepared dispersions of cubic lipid nanoparticles with two PDMA-b-PNIPAM block copolymers of different molar mass. The colloidal properties were then assessed and compared to those exhibited by standard lipid cubic dispersions stabilized by Pluronic F-127, combining a series of experimental techniques (Quartz Crystal Microbalance with Dissipation monitoring, Dynamic Light Scattering, Small-Angle X-rays Scattering, Cryo-Transmission Electron Microscopy). Interestingly, PDMA-b-PNIPAM stabilized cubosomes display additional benefits with respect to those stabilized by Pluronic, thanks to the combination of a "sponge " effect for the controlled release of encapsulated molecules and an increased affinity towards lipid bilayer membranes, which is a promising feature to maximize fusion with the target-cellular site.
Subject(s)
Liposomes , Nanoparticles , Delayed-Action Preparations , Nanoparticles/chemistry , Drug Delivery Systems , Polymers , Poloxamer/chemistry , ExcipientsABSTRACT
In the past decades, events occurring at the nano-bio interface (i.e., where engineered nanoparticles (NPs) meet biological interfaces such as biomembranes) have been intensively investigated, to address the cytotoxicity of nanomaterials and boost their clinical translation. In this field, lamellar synthetic model membranes have been instrumental to disentangle non-specific interactions between NPs and planar biological interfaces. Much less is known on nano-biointeractions occurring at highly curved biological interfaces, such as cubic membranes. These non-lamellar architectures play a crucial -but far from understood-role in several biological processes and occur in cells as a defence mechanism against bacterial and viral pathologies, including coronaviruses infections. Despite its relevance, the interaction of cubic membranes with nano-sized objects (such as viral pathogens, biological macromolecules and synthetic NPs) remains largely unexplored to date. Here, we address the interaction of model lipid cubic phase membranes with two prototypical classes of NPs for Nanomedicine, i.e., gold (AuNPs) and silver NPs (AgNPs). To this purpose, we challenged lipid cubic phase membranes, either in the form of dispersed nanoparticles (i.e., cubosomes) or solid-supported layers of nanometric thickness, with citrate-stabilized AuNPs and AgNPs and monitored the interaction combining bulk techniques (UV-visible spectroscopy, Light and Synchrotron Small-Angle X-ray Scattering) with surface methods (Quartz Crystal Microbalance and Confocal Laser Scanning Microscopy). We show that the composition of the metal core of NPs (i.e., Au vs Ag) modulates their adsorption and self-assembly at cubic interfaces, leading to an extensive membrane-induced clustering of AuNPs, while only to a mild adsorption of isolated AgNPs. Such differences mirror opposite effects at the membrane level, where AuNPs induce lipid extraction followed by a fast disruption of the cubic assembly, while AgNPs do not affect the membrane morphology. Finally, we propose an interaction mechanism accounting for the different behaviour of AuNPs and AgNPs at the cubic interface, highlighting a prominent role of NPs' composition and surface chemistry in the overall interaction mechanism.
ABSTRACT
In recent years, many efforts have been devoted to investigating the interaction of nanoparticles (NPs) with lipid biomimetic interfaces, both from a fundamental perspective aimed at understanding relevant phenomena occurring at the nanobio interface and from an application standpoint for the design of novel lipid-nanoparticle hybrid materials. In this area, recent reports have revealed that citrate-capped gold nanoparticles (AuNPs) spontaneously associate with synthetic phospholipid liposomes and, in some cases, self-assemble on the lipid bilayer. However, the mechanistic and kinetic aspects of this phenomenon are not yet completely understood. In this study, we address the kinetics of interaction of citrate-capped AuNP with lipid vesicles of different rigidities (gel-phase rigid membranes on one side and liquid-crystalline-phase soft membranes on the other). The formation of AuNP-lipid vesicle hybrids was monitored over different time and length scales, combining experiments and simulation. The very first AuNP-membrane contact was addressed through molecular dynamics simulations, while the structure, morphology, and physicochemical features of the final colloidal objects were studied through UV-visible spectroscopy, small-angle X-ray scattering, dynamic light scattering, and cryogenic electron microscopy. Our results highlight that the physical state of the membrane triggers a series of events at the colloidal length scale, which regulate the final morphology of the AuNP-lipid vesicle adducts. For lipid vesicles with soft membranes, the hybrids appear as single vesicles decorated by AuNPs, while more rigid membranes lead to flocculation with AuNPs acting as bridges between vesicles. Overall, these results contribute to a mechanistic understanding of the adhesion or self-assembly of AuNPs onto biomimetic membranes, which is relevant for phenomena occurring at the nano-bio interfaces and provide design principles to control the morphology of lipid vesicle-inorganic NP hybrid systems.
ABSTRACT
The mechanical response of lipid membranes to nanoscale deformations is of fundamental importance for understanding how these interfaces behave in multiple biological processes; in particular, the nanoscale mechanics of non-lamellar membranes represents a largely unexplored research field. Among these mesophases, inverse bicontinuous cubic phase QII membranes have been found to spontaneously occur in stressed or virally infected cells and to play a role in fundamental processes, such as cell fusion and food digestion. We herein report on the fabrication of thin ( Ì´150 nm) supported QII cubic phase lipid films (SQIIFs) and on their characterization via multiple techniques including Small Angle X-Ray Scattering (SAXS), Ellipsometry and Atomic Force Microscopy (AFM). Moreover, we present the first nanomechanical characterization of a cubic phase lipid membrane, through AFM-based Force Spectroscopy (AFM-FS). Our analysis reveals that the mechanical response of these architectures is strictly related to their topology and structure. The observed properties are strikingly similar to those of macroscopic 3D printed cubic structures when subjected to compression tests in material science; suggesting that this behaviour depends on the 3D organisation, rather than on the length-scale of the architecture. We also show for the first time that AFM-FS can be used for characterizing the structure of non-lamellar mesophases, obtaining lattice parameters in agreement with SAXS data. In contrast to classical rheological studies, which can only probe bulk cubic phase solutions, our AFM-FS analysis allows probing the response of cubic membranes to deformations occurring at length and force scales similar to those found in biological interactions.
Subject(s)
Lipids , Mechanical Phenomena , Microscopy, Atomic Force , Scattering, Small Angle , X-Ray DiffractionABSTRACT
The bioactivity, biological fate and cytotoxicity of nanomaterials when they come into contact with living organisms are determined by their interaction with biomacromolecules and biological barriers. In this context, the role of symmetry/shape anisotropy of both the nanomaterials and biological interfaces in their mutual interaction, is a relatively unaddressed issue. Here, we study the interaction of gold nanoparticles (NPs) of different shapes (nanospheres and nanorods) with biomimetic membranes of different morphology, i.e. flat membranes (2D symmetry, representative of the most common plasma membrane geometry), and cubic membranes (3D symmetry, representative of non-lamellar membranes, found in Nature under certain biological conditions). For this purpose we used an ensemble of complementary structural techniques, including Neutron Reflectometry, Grazing Incidence Small-Angle Neutron Scattering, on a nanometer lengthscale and Confocal Laser Scanning Microscopy on a micrometer length scale. We found that the structural stability of the membrane towards NPs is dependent on the topological characteristic of the lipid assembly and of the NPs, where a higher symmetry gave higher stability. In addition, Confocal Laser Scanning Microscopy analyses highlighted that NPs interact with cubic and lamellar phases according to two distinct mechanisms, related to the different structures of the lipid assemblies. This study for the first time systematically addresses the role of NPs shape in the interaction with lipid assemblies with different symmetry. The results will contribute to improve the fundamental knowledge on lipid interfaces and will provide new insights on the biological function of phase transitions as a response strategy to the exposure of NPs.
Subject(s)
Gold , Metal Nanoparticles , Anisotropy , Lipids , Scattering, Small AngleABSTRACT
The mechanical properties of biogenic membranous compartments are thought to be relevant in numerous biological processes; however, their quantitative measurement remains challenging for most of the already available force spectroscopy (FS)-based techniques. In particular, the debate on the mechanics of lipid nanovesicles and on the interpretation of their mechanical response to an applied force is still open. This is mostly due to the current lack of a unified model being able to describe the mechanical response of both gel and fluid phase lipid vesicles and to disentangle the contributions of membrane rigidity and luminal pressure. In this framework, we herein propose a simple model in which the interplay of membrane rigidity and luminal pressure to the overall vesicle stiffness is described as a series of springs; this approach allows estimating these two contributions for both gel and fluid phase liposomes. Atomic force microscopy-based FS, performed on both vesicles and supported lipid bilayers, is exploited for obtaining all the parameters involved in the model. Moreover, the use of coarse-grained full-scale molecular dynamics simulations allowed for better understanding of the differences in the mechanical responses of gel and fluid phase bilayers and supported the experimental findings. The results suggest that the pressure contribution is similar among all the probed vesicle types; however, it plays a dominant role in the mechanical response of lipid nanovesicles presenting a fluid phase membrane, while its contribution becomes comparable to the one of membrane rigidity in nanovesicles with a gel phase lipid membrane. The results presented herein offer a simple way to quantify two of the most important parameters in vesicle nanomechanics (membrane rigidity and internal pressurization), and as such represent a first step toward a currently unavailable, unified model for the mechanical response of gel and fluid phase lipid nanovesicles.
Subject(s)
Biological Phenomena , Lipid Bilayers , Liposomes , Mechanical Phenomena , Microscopy, Atomic ForceABSTRACT
Citrate capping is one of the most common strategies to achieve the colloidal stability of Au nanoparticles (NPs) with diameters ranging from a few to hundreds of nanometers. Citrate-capped Au nanoparticles (CNPs) represent a step of the synthesis of Au NPs with specific functionalities, as CNPs can be further functionalized via ligand-exchange reactions, leading to the replacement of citrate with other organic ligands. In vitro, CNPs are also used to address the fundamental aspects of NP-membrane interactions, as they can directly interact with cells or model cell membranes. Their affinity for the bilayer is again mediated by the exchange of citrate with lipid molecules. Here, we propose a new computational model of CNPs compatible with the coarse grained Martini force field. The model, which we develop and validate through an extensive comparison with new all-atom molecular dynamics (MD) simulations and UV-vis and Fourier transform infrared spectroscopy data, is aimed at the MD simulation of the interaction between citrate-capped NPs and model phosphatidylcholine lipid membranes. As a test application we show that, during the interaction between a single CNP and a flat planar 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, the citrate coating is spontaneously replaced by lipids on the surface of Au NPs, while the NP size and shape determine the final structural configuration of the NP-bilayer complex.
ABSTRACT
Hybrid materials composed of superparamagnetic iron oxide nanoparticles (SPIONs) and lipid self-assemblies possess considerable applicative potential in the biomedical field, specifically, for drug/nutrient delivery. Recently, we showed that SPIONs-doped lipid cubic liquid crystals undergo a cubic-to-hexagonal phase transition under the action of temperature or of an alternating magnetic field (AMF). This transition triggers the release of drugs embedded in the lipid scaffold or in the water channels. In this contribution, we address this phenomenon in depth, to fully elucidate the structural details and optimize the design of hybrid multifunctional carriers for drug delivery. Combining small-angle X-ray scattering (SAXS) with a magnetic characterization, we find that, in bulk lipid cubic phases, the cubic-to-hexagonal transition determines the magnetic response of SPIONs. We then extend the investigation from bulk liquid-crystalline phases to colloidal dispersions, i.e., to lipid/SPIONs nanoparticles with cubic internal structure ("magnetocubosomes"). Through Synchrotron SAXS, we monitor the structural response of magnetocubosomes while exposed to an AMF: the magnetic energy, converted into heat by SPIONs, activates the cubic-to-hexagonal transition, and can thus be used as a remote stimulus to spike drug release "on-demand". In addition, we show that the AMF-induced phase transition in magnetocubosomes steers the realignment of SPIONs into linear string assemblies and connect this effect with the change in their magnetic properties, observed at the bulk level. Finally, we assess the internalization ability and cytotoxicity of magnetocubosomes in vitro on HT29 adenocarcinoma cancer cells, in order to test the applicability of these smart carriers in drug delivery applications.
Subject(s)
Drug Delivery Systems , Magnetic Iron Oxide Nanoparticles/chemistry , Adenocarcinoma/drug therapy , Antineoplastic Agents/administration & dosage , Drug Liberation , HT29 Cells , Humans , Phase Transition , Scattering, Small Angle , X-Ray DiffractionABSTRACT
In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.
ABSTRACT
[This corrects the article DOI: 10.3389/fbioe.2019.00452.].
ABSTRACT
Nanosized lipid vesicles are ubiquitous in living systems (e.g. cellular compartments or extracellular vesicles, EVs) and in formulations for nanomedicine (e.g. liposomes for RNA vaccine formulations). The mechanical properties of such vesicles are crucial in several physicochemical and biological processes, ranging from cellular uptake to stability in aerosols. However, their accurate determination remains challenging and requires sophisticated instruments and data analysis. Here we report the first evidence that the surface plasmon resonance (SPR) of citrated gold nanoparticles (AuNPs) adsorbed on synthetic vesicles is finely sensitive to the vesicles' mechanical properties. We then leverage this finding to show that the SPR tracking provides quantitative access to the stiffness of vesicles of synthetic and natural origin, such as EVs. The demonstration of this plasmon-based "stiffness nanoruler" paves the way for developing a facile, cost-effective and high-throughput method to assay the mechanical properties of dispersions of vesicles of nanometric size and unknown composition at a collective level.
Subject(s)
Gold , Metal Nanoparticles , Lipids , Liposomes , Surface Plasmon ResonanceABSTRACT
In the present study, we investigated lipid membrane interactions of silica nanoparticles as carriers for the antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES). In doing so, smooth mesoporous nanoparticles were compared to virus-like mesoporous nanoparticles, characterized by a "spiky" external surface, as well as to nonporous silica nanoparticles. For this, we employed a combination of neutron reflectometry, ellipsometry, dynamic light scattering, and ζ-potential measurements for studies of bacteria-mimicking bilayers formed by palmitoyloleoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol. The results show that nanoparticle topography strongly influences membrane binding and destabilization. We found that virus-like particles are able to destabilize such lipid membranes, whereas the corresponding smooth silica nanoparticles are not. This effect of particle spikes becomes further accentuated after loading of such particles with LL-37. Thus, peptide-loaded virus-like nanoparticles displayed more pronounced membrane disruption than either peptide-loaded smooth nanoparticles or free LL-37. The structural basis of this was clarified by neutron reflectometry, demonstrating that the virus-like nanoparticles induce trans-membrane defects and promote incorporation of LL-37 throughout both bilayer leaflets. The relevance of such effects of particle spikes for bacterial membrane rupture was further demonstrated by confocal microscopy and live/dead assays on Escherichia coli bacteria. Taken together, these findings demonstrate that topography influences the interaction of nanoparticles with bacteria-mimicking lipid bilayers, both in the absence and presence of antimicrobial peptides, as well as with bacteria. The results also identify virus-like mesoporous nanoparticles as being of interest in the design of nanoparticles as delivery systems for antimicrobial peptides.