ABSTRACT
Conjugation of biomolecules on the surface of nanoparticles (NPs) to achieve active targeting is widely investigated within the scientific community. However, while a basic framework of the physicochemical processes underpinning bionanoparticle recognition is now emerging, the precise evaluation of the interactions between engineered NPs and biological targets remains underdeveloped. Here, we show how the adaptation of a method currently used to evaluate molecular ligand-receptor interactions by quartz crystal microbalance (QCM) can be used to obtain concrete insights into interactions between different NP architectures and assemblies of receptors. Using a model bionanoparticle grafted with oriented apolipoprotein E (ApoE) fragments, we examine key aspects of bionanoparticle engineering for effective interactions with target receptors. We show that the QCM technique can be used to rapidly measure construct-receptor interactions across biologically relevant exchange times. We contrast random adsorption of the ligand at the surface of the NPs, resulting in no measurable interaction with target receptors, to grafted oriented constructs, which are strongly recognized even at lower graft densities. The effects of other basic parameters impacting the interaction such as ligand graft density, receptor immobilization density, and linker length were also efficiently evaluated with this technique. Dramatic changes in interaction outcomes with subtle alterations in these parameters highlight the general importance of measuring the interactions between engineered NPs and target receptors ex situ early on in the construct development process for the rational design of bionanoparticles.
ABSTRACT
Nanoparticles-based drug delivery systems have attracted significant attention in biomedical fields because they can deliver loaded cargoes to the target site in a controlled manner. However, tremendous challenges must still be overcome to reach the expected targeting and therapeutic efficacy in vivo. These challenges mainly arise because the interaction between nanoparticles and biological systems is complex and dynamic and is influenced by the physicochemical properties of the nanoparticles and the heterogeneity of biological systems. Importantly, once the nanoparticles are injected into the blood, a protein corona will inevitably form on the surface. The protein corona creates a new biological identity which plays a vital role in mediating the bio-nano interaction and determining the ultimate results. Thus, it is essential to understand how the protein corona affects the delivery journey of nanoparticles in vivo and what we can do to exploit the protein corona for better delivery efficiency. In this review, we first summarize the fundamental impact of the protein corona on the delivery journey of nanoparticles. Next, we emphasize the strategies that have been developed for tailoring and exploiting the protein corona to improve the transportation behavior of nanoparticles in vivo. Finally, we highlight what we need to do as a next step towards better understanding and exploitation of the protein corona. We hope these insights into the "Yin and Yang" effect of the protein corona will have profound implications for understanding the role of the protein corona in a wide range of nanoparticles.
ABSTRACT
The progress achieved over the last three decades in the field of bioconjugation has enabled the preparation of sophisticated nanomaterial-biomolecule conjugates, referred to herein as bionanoconstructs, for a multitude of applications including biosensing, diagnostics, and therapeutics. However, the development of bionanoconstructs for the active targeting of cells and cellular compartments, both in vitro and in vivo, is challenged by the lack of understanding of the mechanisms governing nanoscale recognition. In this review, we highlight fundamental obstacles in designing a successful bionanoconstruct, considering findings in the field of bionanointeractions. We argue that the biological recognition of bionanoconstructs is modulated not only by their molecular composition but also by the collective architecture presented upon their surface, and we discuss fundamental aspects of this surface architecture that are central to successful recognition, such as the mode of biomolecule conjugation and nanomaterial passivation. We also emphasize the need for thorough characterization of engineered bionanoconstructs and highlight the significance of population heterogeneity, which too presents a significant challenge in the interpretation of in vitro and in vivo results. Consideration of such issues together will better define the arena in which bioconjugation, in the future, will deliver functional and clinically relevant bionanoconstructs.
Subject(s)
Biological Products , NanostructuresABSTRACT
Endocytosis, as one of the main ways for nanostructures enter cells, is affected by several aspects, and shape is an especially critical aspect during the endocytosis of nanostructures. However, it has remained challenging to capture the dynamic internalization behaviors of rod-shaped nanostructures while also probing the mechanical aspects of the internalization. Here, using the atomic force microscopy-based force tracing technique, transmission electron microscopy, and molecular dynamic simulation, we mapped the detailed internalization behaviors of rod-shaped nanostructures with different aspect ratios at the single-particle level. We found that the gold nanorod is endocytosed in a noncontinuous and force-rebound rotation manner, herein named "intermittent rotation". The force tracing test indicated that the internalization force (â¼81 pN, â¼108 pN, and â¼157 pN) and time (â¼0.56 s, â¼0.66 s, and â¼1.14 s for a 12.10 nm × 11.96 nm gold nanosphere and 26.15 nm × 13.05 nm and 48.71 nm × 12.45 nm gold nanorods, respectively) are positively correlated with the aspect ratios. However, internalization speed is negatively correlated with internalization time, irrespective of the aspect ratio. Further, the energy analysis suggested that intermittent rotation from the horizontal to vertical direction can reduce energy dissipation during the internalization process. Thus, to overcome the energy barrier of internalization, the number and angle of rotation increases with aspect ratios. Our findings provide critical missing evidence of rod-shaped nanostructure's internalization, which is essential for fundamentally understanding the internalization mechanism in living cells.
Subject(s)
Nanostructures , Nanotubes , Endocytosis , Gold/chemistry , Microscopy, Electron, Transmission , Nanostructures/chemistry , Nanotubes/chemistryABSTRACT
Automatized approaches for nanoparticle synthesis and characterization represent a great asset to their applicability in the biomedical field by improving reproducibility and standardization, which help to meet the selection criteria of regulatory authorities. The scaled-up production of nanoparticles with carefully defined characteristics, including intrinsic morphological features, and minimal intra-batch, batch-to-batch, and operator variability, is an urgent requirement to elevate nanotechnology towards more trustable biological and technological applications. In this work, microfluidic approaches were employed to achieve fast mixing and good reproducibility in synthesizing a variety of gold nanostructures. The microfluidic setup allowed exploiting spatial resolution to investigate the growth evolution of the complex nanoarchitectures. By physically isolating intermediate reaction fractions, we performed an advanced characterization of the shape properties during their growth, not possible with routine characterization methods. Employing an in-house developed method to assign a specific identity to shapes, we followed the particle growth/deformation process and identified key reaction parameters for more precise control of the generated morphologies. Besides, this investigation led to the optimization of a one-pot multi-size and multi-shape synthesis of a variety of gold nanoparticles. In summary, we describe an optimized platform for highly controlled synthesis and a novel approach for the mechanistic study of shape-evolving nanomaterials.
Subject(s)
Metal Nanoparticles , Nanostructures , Gold/chemistry , Metal Nanoparticles/chemistry , Microfluidics , Nanostructures/chemistry , Reproducibility of ResultsABSTRACT
Recent observations suggest a role for complex nanoscale particulate shape in the regulation of specific immune-related cellular and in vivo processes. We suspect that cellular recognition of nanostructure architecture could involve nonmolecular inputs, including cellular transduction of nanoscale spatially resolved stresses induced by complex shape. Here, we report nanoscale shape-dependent control of the cellular epigenome. Interpretation of ChIP-Seq sequencing suggests that differential marking of H3K27me3 may be linked to sensory and synapse-recognition of nanoscale forces induced by complex shape. The observations raise significant questions on the role of particle-shape-induced immune regulation and memory, with potential consequences in both causes and treatment of immune-related disease.
ABSTRACT
Silica nanoparticles (SiNP) trigger a range of innate immune responses in relevant essential organs, such as the liver and the lungs. Inflammatory reactions, including NLRP3 inflammasome activation, have been linked to particulate materials; however, the molecular mechanisms and key actors remain elusive. Although many receptors, including several scavenger receptors, were suggested to participate in SiNP cellular uptake, mechanistic evidence of their role on innate immunity is lacking. Here we present an atomic force microscopy-based approach to physico-mechanically map the specific interaction occurring between nanoparticles and scavenger receptor A1 (SRA1) in vitro on living lung epithelial cells. We find that SiNP recognition by SRA1 on human macrophages plays a key role in mediating NLRP3 inflammasome activation, and we identify cellular mechanical changes as clear indicators of inflammasome activation in human macrophages, greatly advancing our knowledge on the interplay among nanomaterials and innate immunity.
Subject(s)
Inflammasomes , Nanoparticles , Humans , NLR Family, Pyrin Domain-Containing 3 Protein , Macrophages/metabolism , Immunity, Innate , Silicon Dioxide/metabolismABSTRACT
Since it is now possible to make, in a controlled fashion, an almost unlimited variety of nanostructure shapes, it is of increasing interest to understand the forms of biological control that nanoscale shape allows. However, a priori rational investigation of such a vast universe of shapes appears to present intractable fundamental and practical challenges. This has limited the useful systematic investigation of their biological interactions and the development of innovative nanoscale shape-dependent therapies. Here, we introduce a concept of biologically relevant inductive nanoscale shape discovery and evaluation that is ideally suited to, and will ultimately become, a vehicle for machine learning discovery. Combining the reproducibility and tunability of microfluidic flow nanochemistry syntheses, quantitative computational shape analysis, and iterative feedback from biological responses in vitro and in vivo, we show that these challenges can be mastered, allowing shape biology to be explored within accepted scientific and biomedical research paradigms. Early applications identify significant forms of shape-induced biological and adjuvant-like immunological control.
Subject(s)
Nanostructures , Reproducibility of Results , Nanostructures/chemistry , Microfluidics , Machine Learning , ImmunomodulationABSTRACT
Liposomes, especially cationic liposomes, are the most common and well-investigated nanocarriers for biomedical applications, such as drug and gene delivery. Like other types of nanomaterials, once liposomes are incubated in a biological milieu, their surface can be immediately cloaked by biological components to form a protein corona, which confers a new 'biological identity' and modulates downstream interactions with cells. However, it remains unclear how the protein corona affects the transportation mechanism after liposomes interact with cells. Here, we employed home-made aggregation-induced-emission-visualized nanoliposomes TR4@Lipo as a model to investigate transportation with or without the protein corona by optical imaging techniques. The results show that the protein corona can change the cellular transportation mechanism of TR4@Lipo from energy-independent membrane fusion to energy-dependent endocytosis. The protein corona also modulates the intracellular distribution of loaded cargoes. This knowledge furthers our understanding of bio-nano interactions and is important for the efficient use of cationic liposomes.
ABSTRACT
Despite the high level of interest in bio-nano interactions, detailed intracellular mechanisms that govern nanoscale recognition and signalling still need to be unravelled. Magnetic nanoparticles (NPs) are valuable tools for elucidating complex intracellular bio-nano interactions. Using magnetic NPs, it is possible to isolate cell compartments that the particles interact with during intracellular trafficking. Studies at the subcellular scale rely heavily on optical microscopy; therefore, combining the advantages of magnetic recovery with excellent imaging properties to allow intracellular NP tracking is of utmost interest for the nanoscience field. However, it is a challenge to prepare highly magnetic NPs with a suitable fluorescence for the fluorescence imaging techniques typically used for biological studies. Here we present the synthesis of biocompatible multifunctional superparamagnetic multicore NPs with a bright fluorescent silica shell. The incorporation of an organic fluorophore in the silica surrounding the magnetic multicore was optimised to enable the particles to be tracked with the most common imaging techniques. To prevent dye loss resulting from silica dissolution in biological environments, which would reduce the time that the particles could be tracked, we added a thin dense encapsulating silica layer to the NPs which is highly stable in biological media. The synthesised multifunctional nanoparticles were evaluated in cell uptake experiments in which their intracellular location could be clearly identified using fluorescence imaging microscopy, even after 3 days. The magnetic properties of the iron oxide core enabled both efficient recovery of the NPs from the intracellular environment and the extraction of cell compartments involved in their intracellular trafficking. Thus, the NPs reported here provide a promising tool for the study of the processes regulating bio-nano interactions.
Subject(s)
Multifunctional Nanoparticles , Nanoparticles , Fluorescent Dyes , Magnetic Iron Oxide Nanoparticles , Silicon DioxideABSTRACT
X-ray-based analytics are routinely applied in many fields, including physics, chemistry, materials science, and engineering. The full potential of such techniques in the life sciences and medicine, however, has not yet been fully exploited. We highlight current and upcoming advances in this direction. We describe different X-ray-based methodologies (including those performed at synchrotron light sources and X-ray free-electron lasers) and their potentials for application to investigate the nano-bio interface. The discussion is predominantly guided by asking how such methods could better help to understand and to improve nanoparticle-based drug delivery, though the concepts also apply to nano-bio interactions in general. We discuss current limitations and how they might be overcome, particularly for future use in vivo.
Subject(s)
Nanoparticles , Synchrotrons , Lasers , Radiography , X-RaysABSTRACT
Advances in nanofabrication methods have enabled the tailoring of new strategies towards the controlled production of nanoparticles with attractive applications in healthcare. In many cases, their characterisation remains a big challenge, particularly for small-sized functional nanoparticles of 5 nm diameter or smaller, where current particle sizing techniques struggle to provide the required sensitivity and accuracy. There is a clear need for the development of new reliable characterisation approaches for the physico-chemical characterisation of nanoparticles with significant accuracy, particularly for the analysis of the particles in the presence of complex biological fluids. Herein, we show that the Differential Centrifugal Sedimentation can be utilised as a high-precision tool for the reliable characterisation of functional nanoparticles of different materials. We report a method to correlate the sedimentation shift with the polymer and biomolecule adsorption on the nanoparticle surface, validating the developed core-shell model. We also highlight its limit when measuring nanoparticles of smaller size and the need to use several complementary methods when characterising nanoparticle corona complexes.
ABSTRACT
Polyethylene glycol grafting has played a central role in preparing the surfaces of nano-probes for biological interaction, to extend blood circulation times and to modulate protein recognition and cellular uptake. However, the role of PEG graft dynamics and conformation in determining surface recognition processes is poorly understood primarily due to the absence of a microscopic picture of the surface presentation of the polymer. Here a detailed NMR analysis reveals three types of dynamic ethylene glycol units on PEG-grafted SiO2 nanoparticles (NPs) of the type commonly evaluated as long-circulating theranostic nano-probes; a narrow fraction with fast dynamics associated with the chain ends; a broadened fraction spectrally overlapped with the former arising from those parts of the chain experiencing some dynamic restriction; and a fraction too broad to be observed in the spectrum arising from units closer to the surface/graft which undergo slow motion on the NMR timescale. We demonstrate that ethylene glycol units transition between fractions as a function of temperature, core size, PEG chain length and surface coverage and demonstrate how this distribution affects colloidal stability and protein uptake. The implications of the findings for biological application of grafted nanoparticles are discussed in the context of accepted models for surface ligand conformation.
Subject(s)
Nanoparticles , Silicon Dioxide , Polyethylene Glycols , Polymers , Protein Binding , Surface PropertiesABSTRACT
Nanoscale objects are processed by living organisms using highly evolved and sophisticated endogenous cellular networks, specifically designed to manage objects of this size. While these processes potentially allow nanostructures unique access to and control over key biological machineries, they are also highly protected by cell or host defence mechanisms at all levels. A thorough understanding of bionanoscale recognition events, including the molecules involved in the cell recognition machinery, the nature of information transferred during recognition processes and the coupled downstream cellular processing, would allow us to achieve a qualitatively novel form of biological control and advanced therapeutics. Here we discuss evolving fundamental microscopic and mechanistic understanding of biological nanoscale recognition. We consider the interface between a nanostructure and a target cell membrane, outlining the categories of nanostructure properties that are recognized, and the associated nanoscale signal transduction and cellular programming mechanisms that constitute biological recognition.
Subject(s)
Cell Membrane/genetics , Nanostructures/chemistry , Nanotechnology/trends , Cell Membrane/chemistry , Humans , Signal Transduction/geneticsABSTRACT
The field of nanomedicine has the potential to be a game-changer in global health, with possible applications in prevention, diagnostics, and therapeutics. However, despite extensive research focus and funding, the forecasted explosion of novel nanomedicines is yet to materialize. We believe that clinical translation is ultimately hampered by a lack of understanding of how nanoparticles really interact with biological systems. When placed in a biological environment, nanoparticles adsorb a biomolecular layer that defines their biological identity. The challenge for bionanoscience is therefore to understand the evolution of the interactions of the nanoparticle-biomolecules complex as the nanoparticle is trafficked through the intracellular environment. However, to progress on this route, scientists face major challenges associated with isolation of specific intracellular compartments for analysis, complicated by the diversity of trafficking events happening simultaneously and the lack of synchronization between individual events. In this perspective article, we reflect on how magnetic nanoparticles can help to tackle some of these challenges as part of an overall workflow and act as a useful platform to investigate the bionano interactions within the cell that contribute to this nanoscale decision making. We discuss both established and emerging techniques for the magnetic extraction of nanoparticles and how they can potentially be used as tools to study the intracellular journey of nanomaterials inside the cell, and their potential to probe nanoscale decision-making events. We outline the inherent limitations of these techniques when investigating particular bio-nano interactions along with proposed strategies to improve both specificity and resolution. We conclude by describing how the integration of magnetic nanoparticle recovery with sophisticated analysis at the single-particle level could be applied to resolve key questions for this field in the future.
ABSTRACT
The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.
ABSTRACT
Salcaprozate sodium (SNAC) and sodium caprate (C10) are the two leading intestinal permeation enhancers (PEs) in oral peptide formulations in clinical trials. There is debate over their mechanism of action on intestinal epithelia. The aims were: (i) to compare their effects on the barrier function by measuring transepithelial electrical resistance (TEER), permeability of FITC-4000 (FD4) across Caco-2 monolayers, and on immunohistochemistry of tight junction (TJ)-associated proteins; and (ii) to compare cellular parameters using conventional end-point cytotoxicity assays and quantitative high content analysis (HCA) of multiple sub-lethal parameters in Caco-2 cells. C10 (8.5 mM) reversibly reduced TEER and increased FD4 permeability across monolayers, whereas SNAC had no effects on either parameter except at cytotoxic concentrations. C10 exposure induced reorganization of three TJ proteins, whereas SNAC only affected claudin-5 localization. High concentrations of C10 and SNAC were required to cause end-point toxicology changes in vitro. SNAC was less potent than C10 at inducing lysosomal and nuclear changes and plasma membrane perturbation. In parallel, HCA revealed that both agents displayed detergent-like features that reflect initial membrane fluidization followed by changes in intracellular parameters. In conclusion, FD4 permeability increases in monolayers in response to C10 were in the range of concentrations that altered end-point cytotoxicity and HCA parameters. For SNAC, while HCA parameters were also altered in a similar overall pattern as C10, they did not lead to increased paracellular flux. These assays show that both agents are primarily surfactants, but C10 has additional TJ-opening effects. While these in vitro assays illucidate their epithelial mechanism of action, clinical experience suggests that they over-estimate their toxicology in the dynamic intestinal environment.
Subject(s)
Caprylates/chemistry , Decanoic Acids/chemistry , Intestinal Absorption/drug effects , Intestinal Mucosa/metabolism , Intestines/drug effects , Permeability/drug effects , Caco-2 Cells , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Electric Impedance , Humans , Tight Junctions/drug effects , Tight Junctions/metabolismABSTRACT
Here we present a blood-brain barrier (BBB) model that enables high-resolution imaging of nanoparticle (NP) interactions with endothelial cells and the capture of rare NP translocation events. The enabling technology is an ultrathin silicon nitride (SiN) membrane (0.5 µm pore size, 20% porosity, 400 nm thickness) integrated into a dual-chamber platform that facilitates imaging at low working distances (â¼50 µm). The platform, the µSiM-BBB (microfluidic silicon membrane-BBB), features human brain endothelial cells and primary astrocytes grown on opposite sides of the membrane. The human brain endothelial cells form tight junctions on the ultrathin membranes and exhibit a significantly higher resistance to FITC-dextran diffusion than commercial membranes. The enhanced optical properties of the SiN membrane allow high-resolution live-cell imaging of three types of NPs, namely, 40 nm PS-COOH, 100 nm PS-COOH, and apolipoprotein E-conjugated 100 nm SiO2, interacting with the BBB. Despite the excellent barrier properties of the endothelial layer, we are able to document rare NP translocation events of NPs localized to lysosomal compartments of astrocytes on the "brain side" of the device. Although the translocation is always low, our data suggest that size and targeting ligand are important parameters for NP translocation across the BBB. As a platform that enables the detection of rare transmission across tight BBB layers, the µSiM-BBB is an important tool for the design of nanoparticle-based delivery of drugs to the central nervous system.
Subject(s)
Blood-Brain Barrier/metabolism , Models, Biological , Nanoparticles/metabolism , Optical Imaging , Silicon/metabolism , Biological Transport , Blood-Brain Barrier/chemistry , Cell Line , Coculture Techniques , Humans , Nanoparticles/chemistry , Particle Size , Silicon/chemistry , Surface PropertiesABSTRACT
Ultrasmall nanoparticles are attracting an increasing interest for a variety of biomedical applications, from therapeutic targeting to imaging, in virtue of the peculiar behavior shown in vivo (i.e., efficient renal clearance, low liver accumulation, etc.). In evaluating their potential to overcome some of the challenges that larger particles have faced, it is important to understand their mechanisms of interaction with the cell membrane in relation to the biological environment and their tendency to transiently interact with biomolecules. In this work, the mechanism of cellular uptake across a range of serum concentrations is investigated using 2 nm gold nanoparticles with different surface chemistries as a model. The data suggest that despite their ultrasmall size, for these nanoparticles, internalization occurs via energy-dependent processes, and the surface chemistry could play a key role in determining the modality of the transient protein interaction, especially in conditions close to the in vivo scenario (large excess of the protein content). These aspects might be exploited to define novel targeting strategies.