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Protein corona impact on nanoparticle-cell interactions: Toward an energy-based model ofendocytosis
Abstract
Upon incubation of nanoparticles in biological uids, a new layer called as protein corona is formed on their surface affecting the interactions between nanoparticles and targeted cell during the endocytosis process. In the present study, a mathematical model based on the diffusion of membrane mobile receptors is proposed. Opposing the endocytosis proceeding, membrane bending and tension energies are named as resistant energy. Also, binding energy and free-energy associated with the congurational entropy are called as promoter energy. Utilizing this model, endocytosis of gold nanoparticle (GNP) is simulated to explore the biological media effect. The results reveal that there exists a nanoparticle size of 60nm at which, the endocytosis time is minimum. It has been illustrated that, although for sufficiently small particles of diameter 30nm, membrane tension has a negligible contribution (< 10%) in the resistant energy, it noticeably increases the endocytosis processing time for large particles. Therefore, we report several parametric studies to provide a better insight into the effects of biological media on the ingestion of nanoparticles. Through a detailed analysis of the engulfment of the nanoparticles, it is shown that the nanoparticle radius corresponding to the quickest possible ingestion time is affected in the presence of corona. Moreover, it is found that the formation of this layer can not only affect the endocytosis time but also can lead to incomplete engulfment by decreasing the ligand density on the nanoparticle surface. Use of the proposed model can play a signicant role in advancing the design of nanoparticles in the targeted drug delivery applications.
... Differences in the structure of proteins constituting the PC causes differential cellular binding, even for NPs with identical PC. PC also impacts interactions between NPs and targeted cells during the endocytosis process [89]. Even subtle changes in the protein composition affect the level of NP uptake [90]. ...
... It is generally clear that NP internalization by cells happens predominantly by active transport rather than by low-yielding passive transport, such as pinocytic clathrin-mediated pathways that mediate cellular uptake of NPs, which end up in lysosomes and endosomes [92]. Also, receptor-mediated endocytosis (RME), another kind of active transport, is an effective route through which NPs are ingested into the cell [89]. This mechanism offers specificity, associated with the receptor-ligand interaction between NPs and the cell membrane. ...
... The efficiency of endocytosis is dependent on various factors including NP size [93,94], surface properties [95], and shape [96][97][98]. Moreover, alongside these primary factors, the biological media, under forming PC on the NP, can significantly impact the efficiency of the endocytosis process [89]. ...
Proteins are known to play important roles in the biosynthesis of metallic nanoparticles (NPs), which are biological substitutes for conventionally used chemical capping and stabilizing agents. When a pristine nanoparticle comes in contact with a biological media or system, a bimolecular layer is formed on the surface of the nanoparticle and is primarily composed of proteins. The role of proteins in the biosynthesis and further uptake, translocation, and bio-recognition of nanoparticles is documented in the literature. But, a complete understanding has not been achieved concerning the mechanism for protein-mediated nanoparticle biosynthesis and the role proteins play in the interaction and recognition of nanoparticles, aiding its uptake and assimilation into the biological system. This review critically evaluates the knowledge and gaps in the protein-mediated biosynthesis of nanoparticles. In particular, we review the role of proteins in multiple facets of metallic nanoparticle biosynthesis, the interaction of proteins with metallic nanoparticles for recognition and interaction with cells, and the toxic potential of protein-nanoparticle complexes when presented to the cell.
... The intracellular milieu contains compounds for cell growth, proliferation, differentiation and death that are distributed throughout the cytoplasm, nucleus, mitochondria, endoplasmic reticulum and Golgi complex [4]. Endocytosis opens a window of opportunity for communication of materials, energy and information between the inside and outside of cells; such a process provides the essential link for life and physiological activities [5,6]. ...
... Formation of a protein corona increased the time required for endocytosis. In addition, the presence of a protein corona significantly reduced ligand density on the Au nanostructure surface, resulting in defective engulfment [4]. Surface alteration is not always a negative factor for cellular uptake of nanoparticles. ...
... In addition to the microenvironment, another parameter that should be taken into account when performing in vivo investigations is protein adsorption on the surface of the nanoparticles within the circulation. The adsorbed serum proteins in this protein corona may adversely influence how nanoparticles enter cells, such as the time required for internalization [4,283]. ...
Humans are exposed to nanoscopical nanobiovectors (e.g. coronavirus SARS-CoV-2) as well as abiotic metal/carbon-based nanomaterials that enter cells serendipitously or intentionally. Understanding the interactions of cell membranes with these abiotic and biotic nanostructures will facilitate scientists to design better functional nanomaterials for biomedical applications. Such knowledge will also provide important clues for the control of viral infections and the treatment of virus-induced infectious diseases. In the present review, the mechanisms of endocytosis are reviewed in the context of how nanomaterials are uptaken into cells. This is followed by a detailed discussion of the attributes of man-made nanomaterials (e.g. size, shape, surface functional groups and elasticity) that affect endocytosis, as well as the different human cell types that participate in the endocytosis of nanomaterials. Readers are then introduced to the concept of viruses as nature-derived nanoparticles. The mechanisms in which different classes of viruses interact with various cell types to gain entry into the human body are reviewed with examples published over the last five years. These basic tenets will enable the avid reader to design advanced drug delivery and gene transfer nanoplatforms that harness the knowledge acquired from endocytosis to improve their biomedical efficacy. The review winds up with a discussion on the hurdles to be addressed in mimicking the natural mechanisms of endocytosis in nanomaterials design.
... Efficient delivery of drugs by MNP-coated nanocomposites requires a thorough knowledge of the proteic behavior of the drug molecule [247][248][249]. Pharmacokinetic behavior is of utmost importance since it can lead to better drug administration and by this, to more efficient disease treatment and management [250,251]. ...
Magnetic nanoparticles (MNPs) have evolved tremendously during recent years, in part due to the rapid expansion of nanotechnology and to their active magnetic core with a high surface-to-volume ratio, while their surface functionalization opened the door to a plethora of drug, gene and bioactive molecule immobilization. Taming the high reactivity of the magnetic core was achieved by various functionalization techniques, producing MNPs tailored for the diagnosis and treatment of cardiovascular or neurological disease, tumors and cancer. Superparamagnetic iron oxide nanoparticles (SPIONs) are established at the core of drug-delivery systems and could act as efficient agents for MFH (magnetic fluid hyperthermia). Depending on the functionalization molecule and intrinsic morphological features, MNPs now cover a broad scope which the current review aims to overview. Considering the exponential expansion of the field, the current review will be limited to roughly the past three years.
... Once nanoparticles are injected into the systemic circulation, nanoparticles encounter serum components, such as proteins, resulting in the formation of a protein corona on the surface. The formation of a protein corona is critical for the design of efficient and safe nanoparticles for tissue-targeting, nanomedicines, and other applications, so research related to the protein corona is a subject of great interest [136][137][138][139][140][141][142][143][144][145][146]. Although protein corona formation on a nanoparticle surface may adversely affect targeting [147], controlling them can also be applied to achieve more effective targeting [51,54,55,[148][149][150][151][152][153][154]. ...
The era of the aging society has arrived, and this is accompanied by an increase in the absolute numbers of patients with neurological disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Such neurological disorders are serious costly diseases that have a significant impact on society, both globally and socially. Gene therapy has great promise for the treatment of neurological disorders, but only a few gene therapy drugs are currently available. Delivery to the brain is the biggest hurdle in developing new drugs for the central nervous system (CNS) diseases and this is especially true in the case of gene delivery. Nanotechnologies such as viral and non-viral vectors allow efficient brain-targeted gene delivery systems to be created. The purpose of this review is to provide a comprehensive review of the current status of the development of successful drug delivery to the CNS for the treatment of CNS-related disorders especially by gene therapy. We mainly address three aspects of this situation: (1) blood-brain barrier (BBB) functions; (2) adeno-associated viral (AAV) vectors, currently the most advanced gene delivery vector; (3) non-viral brain targeting by non-invasive methods.
... Shadmani and workers at cellular level disclosed the how impact of corona formation leads to receptor mediated endocytosis of gold nanoparticles. The optimal value based on mathematical model for corona particle (bare diameter 40 nm vs. 60 nm corona particle) and ligand density ~1500 μm − 2 over 100 nm of gold nanoparticle are accounted to reduce endocytosis time via balance approach of membrane-tension energy and ligand receptor interaction density indicating the pivotal role in silico modeling of NC design [94]. ...
Background
In past few decades, the research on engineered nanocarriers (NCs) has gained significant attention in cancer therapy due to selective delivery of drug molecules on the diseased cells thereby preventing unwanted uptake into healthy cells to cause toxicity.
Scope of review
The applicability of enhanced permeability and retention (EPR) effect for the delivery of nanomedicines in cancer therapy has gained limited success due to poor accessibility of the drugs to the target cells where non-specific payload delivery to the off target region lack substantial reward over the conventional therapeutic systems.
Major conclusions
In spite of the fact, nanomedicines fabricated from the biocompatible nanocarriers have reduced targeting potential for meaningful clinical benefits. However, over expression of receptors on the tumor cells provides opportunity to design functional nanomedicine to bind substantially and deliver therapeutics to the cells or tissues of interest by alleviating the bio-toxicity and unwanted effects. This critique will give insight into the over expressed receptor in various tumor and targeting potential of functional nanomedicine as new therapeutic avenues for effective treatment.
General significance
This review shortly shed light on EPR-based drug targeting using nanomedicinal strategies, their limitation, and advances in therapeutic targeting to the tumor cells.
... Shadmani and workers at cellular level disclosed the how impact of corona formation leads to receptor mediated endocytosis of gold nanoparticles. The optimal value based on mathematical model for corona particle (bare diameter 40 nm vs. 60 nm corona particle) and ligand density ~1500 μm − 2 over 100 nm of gold nanoparticle are accounted to reduce endocytosis time via balance approach of membrane-tension energy and ligand receptor interaction density indicating the pivotal role in silico modeling of NC design [94]. ...
Abstract
Background
In past few decades, the research on engineered nanocarriers (NCs) has gained significant attention in cancer therapy due to selective delivery of drug molecules on the diseased cells thereby preventing unwanted uptake into healthy cells to cause toxicity.
Scope of review
The applicability of enhanced permeability and retention (EPR) effect for the delivery of nanomedicines in cancer therapy has gained limited success due to poor accessibility of the drugs to the target cells where non-specific payload delivery to the off target region lack substantial reward over the conventional therapeutic systems.
Major conclusions
In spite of the fact, nanomedicines fabricated from the biocompatible nanocarriers have reduced targeting potential for meaningful clinical benefits. However, over expression of receptors on the tumor cells provides opportunity to design functional nanomedicine to bind substantially and deliver therapeutics to the cells or tissues of interest by alleviating the bio-toxicity and unwanted effects. This critique will give insight into the over expressed receptor in various tumor and targeting potential of functional nanomedicine as new therapeutic avenues for effective treatment.
General significance
This review shortly shed light on EPR-based drug targeting using nanomedicinal strategies, their limitation, and advances in therapeutic targeting to the tumor cells.
Keywords
Cancer,Tumor metastasis, Nanomedicines, Drug delivery, Receptor targeting,Theranostics.
A real-time and molecule-level monitoring of the interfacial dynamic interactions between molecules and a cell membrane is of vital importance. Herein, taking TAT, one of the most representative cell penetrating peptides, as an example, a photo-voltage transient technique and a dynamic giant bistratal vesicle (GBV) leakage method were combined with the traditional giant unilamellar vesicle (GUV) leakage assays, to provide a molecule-level understanding of the dynamic membrane interaction process performed in a low ionic strength and neutral pH condition. The photo-voltage test based on supported phospholipid bilayers showed a quick disturbance (<1 min) followed by a continuous reconstruction of the membrane by peptides, leading to a slight destruction (at TAT concentrations lower than 1 μg mL⁻¹, i.e., 0.64 μM) or strong damage (e.g. at 10 μg mL⁻¹, i.e., 6.4 μM) of the bilayer structure. The GUV/GBV leakage assays further demonstrated the TAT-induced membrane deformation and transmembrane diffusion of dyes, which occurred in an immediate, linear, and TAT-concentration dependent manner. Moreover, the flux of dye across the substrate-immobilized membranes was approximately three times of that across the substrate-free ones. This work gives information on time and molecular mechanism of the TAT-membrane interactions, demonstrates the different permeabilizing effects of TAT on immobilized and free membranes. Overall, it provides useful strategies to investigate the nano-bio interfacial interactions in a simple, global and real-time way.
Drug delivery to tumors suffers from poor solubility, specificity, diffusion though the tumor micro-environment and non-optimal interactions with components of the extra-cellular matrix and cell surface receptors. Nanoparticles and drug-polymer complexes address many of these problems. However, large size exasperates the problem of slow diffusion through the tumor. Three-dimensional tumor spheroids are good models to evaluate approaches to mitigate these difficulties and aid in design strategies to improve the delivery of drugs to treat cancer effectively. Diffusion of drug carriers is highly dependent on cell uptake rate parameters (association/dissociation) and temperature. Hyperthermia increases molecular transport and is known to act synergistically with chemotherapy to improve treatment. This study presents a new inverse estimation approach based on Bayesian probability for estimating nanoparticle cell uptake rates from experiments. The parameters were combined with a finite element computational model of nanoparticle transport under hyperthermia conditions to explore its effect on tumor porosity, diffusion and particle binding (association and dissociation) at cell surfaces. Carboxy-PEG-Silane (cPEGSi) nanoparticles showed higher cell uptake compared to Methoxy- PEG- Silane (mPEGSi) nanoparticles. Simulations were consistent with experimental results from Skov-3 ovarian cancer spheroids. Amorphous silica (cPEGSi) nanoparticles (58 nm) concentrated at the periphery of the tumor spheroids at 37 °C but mild hyperthermia (43 °C) increased nanoparticle penetration. Thus, hyperthermia may enhance cancer treatment by improving blood delivery to tumors, enhancing extravasation and penetration into tumors, trigger release of drug from the carrier at the tumor site and possible lead to synergistic anti-cancer activity with the drug.
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Unpredictable and uncontrollable protein adsorption on nanoparticles remains a considerable challenge to achieving effective application of nanotechnologies within biological environments. Nevertheless, engineered nanoparticles offer unprecedented functionality and control in probing and altering biological systems. In this review, we highlight recent advances in harnessing the “protein corona” formed on nanoparticles as a handle to tune functional properties of the protein-nanoparticle complex. Towards this end, we first review nanoparticle properties that influence protein adsorption and design strategies to facilitate selective corona formation, with the corresponding characterization techniques. We next focus on literature detailing corona-mediated functionalities, including stealth to avoid recognition and sequestration while in circulation, targeting of predetermined in vivo locations, and controlled activation once localized to the intended biological compartment. We conclude with a discussion of biocompatibility outcomes for these protein-nanoparticle complexes applied in vivo. While formation of the nanoparticle-corona complex may impede our control over its use for the projected nanobiotechnology application, it concurrently presents an opportunity to create improved protein-nanoparticle architectures by exploiting natural or guiding selective protein adsorption to the nanoparticle surface.
Nanotoxicity to fetal brains after maternal oral exposures during pregnancy is often in question because nanoparticles have to cross multiple biological barriers such as intestinal barrier, maternal blood placental barrier (BPB) and fetal blood brain barrier (BBB). Here, we investigated this seemingly impossible passage for ZrO2 nanoparticles (ZrO2 NPs) from maternal body to fetal brains using a pregnant mouse model. After three oral exposures to pregnant mice at late pregnancy (GD16, 17, 18), ZrO2 NPs were able to accumulate in fetal brains at GD19 via crossing the well-developed maternal BPB and fetal BBB. Moreover, ZrO2 NPs crossed the mature biological barriers with increasing the expression levels of caveolae, clathrin and arf6 proteins as well as decreasing the expression levels of the tight junction proteins claudin-5, occludin and ZO-1 in placenta and fetal brain. From this investigation, we speculated that the main mechanisms for such translocation were receptor-mediated endocytosis transcellular pathway and breakthrough of tight junctions paracellular pathway in mature maternal BPB and fetal BBB. These findings have important implications for other nanoparticles exposures during pregnancy and provide crucial information to safeguard fetal development from contamination of widely used nanoproducts.
One of the crucial issues in the pharmacological field is developing new drug delivery systems. The main concern is to develop new methods for improving the drug delivery efficiencies such as low disruptions, precise control of the target of delivery and drug sustainability. Nowadays, there are many various methods for drug delivery systems. Carbon-based nanocarriers are a new efficient tool for translocating drug into the defined area or cells inside the body. These nanocarriers can be functionalized with proteins, peptides and used to transport their freight to cells or defined areas. Since functionalized carbon-based nanocarriers show low toxicity and high biocompatibility, they are used in many nanobiotechnology fields. In this study, different shapes of nanocarrier are investigated, and the suitable magnetic field, which is applied using MRI for the delivery of the nanocarrier, is proposed. In this research, based on the force required to cross the membrane and MD simulations, the optimal magnetic field profile is designed. This optimal magnetic force field is derived from the mathematical model of the system and magnetic particle dynamics inside the nanocarrier. The results of this paper illustrate the effects of the nanocarrier’s shapes on the percentage of success in crossing the membrane and the optimal required magnetic field.
Biophysical-factor-dependent cellular uptake of nanoparticles (NPs) through receptor-diffusion-mediated endocytosis bears significance in pathology, cellular immunity and drug-delivery systems. Advanced nanotechnology of NP synthesis provides methods for modifying NP surface with different ligand distributions. However, no report discusses effects of ligand distribution on NP surface on receptor-diffusion-mediated cellular uptake. In this article, we used a statistical dynamics model of receptor-diffusion-mediated endocytosis to examine ligand-distribution-dependent cellular uptake dynamics by considering that ligand–receptor complexes drive engulfing to overcome resistance to membrane deformation and changes in configuration entropy of receptors. Results showed that cellular internalization of NPs strongly depended on ligand distribution and that cellular-uptake efficiency of NPs was high when ligand distribution was within a range around uniform distribution. This feature of endocytosis ensures robust infection ability of viruses to enter host cells. Interestingly, results also indicated that optimal ligand distribution associated with highest cellular-uptake efficiency slightly depends on distribution pattern of ligands and density of receptors, and the optimal distribution becomes uniform when receptor density is sufficiently large. Position of initial contact point is also a factor affecting dynamic wrapping. This study explains why most enveloped viruses present almost homogeneous ligand distribution and is useful in designing controlled-release drug-delivery systems.
After administration of nanoparticle (NP) into biological fluids, an NP–protein complex is formed, which represents the “true identity” of NP in our body. Hence, protein–NP interaction should be carefully investigated to predict and control the fate of NPs or drug-loaded NPs, including systemic circulation, biodistribution, and bioavailability. In this review, we mainly focus on the formation of protein corona and its potential applications in pharmaceutical sciences such as prediction modeling based on NP-adsorbed proteins, usage of active proteins for modifying NP to achieve toxicity reduction, circulation time enhancement, and targeting effect. Validated correlative models for NP biological responses mainly based on protein corona fingerprints of NPs are more highly accurate than the models solely set up from NP properties. Based on these models, effectiveness as well as the toxicity of NPs can be predicted without in vivo tests, while novel cell receptors could be identified from prominent proteins which play important key roles in the models. The ungoverned protein adsorption onto NPs may have generally negative effects such as rapid clearance from the bloodstream, hindrance of targeting capacity, and induction of toxicity. In contrast, controlling protein adsorption by modifying NPs with diverse functional proteins or tailoring appropriate NPs which favor selective endogenous peptides and proteins will bring promising therapeutic benefits in drug delivery and targeted cancer treatment.
A theory of the elasticity of lipid bilayers is proposed. Three types of strain, i. e. stretching, tilt and curvature, are distinguished and the associated stresses are identified. It is argued that in the case of vesicles (= closed bilayer films) the only elasticity controlling nonspherical shapes is that of curvature. Euler-Lagrange equations are derived for the shape in magnetic fields and under excess outside pressure. It is shown that magnetic fields can deform spherical vesicles into ellipsoids of revolution. Under excess outside pressure the spherical shape becomes unstable at a certain threshold pressure. Both effects can be influenced by a spontaneous curvature of the bilayer. Some possible experiments to determine the elastic properties are also discussed.
In this work we systematically investigate the pathways of the interaction between elastic vesicles and lipid membranes with the aid of computer simulation techniques. Different vesicle responses to the vesicle–membrane adhesion, including vesicle fusion, vesicle hemi-fusion, vesicle adhesion, vesicle endocytosis and vesicle rupture, are observed from our simulations. We also investigate how the pathways of vesicle–membrane interaction depend on the adhesion strength, and the membrane and vesicle properties.
Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.
When nanoparticles (NP) enter the body they come into contact with body fluids containing proteins which can adsorb to their surface. These proteins may influence the NP interactions with the biological vicinity, eventually determining their biological fate inside the body.Adsorption of the most abundantly binding proteins was studied after an in vitro 24 hr incubation of monodisperse, negatively charged 5, 15 and 80 nm gold spheres (AuNP) in mouse serum by a two-step analysis: proteomic protein identification and quantitative protein biochemistry. The adsorbed proteins were separated from non-adsorbed proteins by centrifugation and gel electrophoresis and identified using a MALDI-TOF-MS-Proteomics-Analyzer. Quantitative analysis of proteins in gel bands by protein densitometry, required the focus on predominantly binding serum proteins.Numerous proteins adsorbed to the AuNP depending on their size, e.g. apolipoproteins or complement C3. The qualitative and quantitative amount of adsorbed proteins differed between 5, 15 and 80 nm AuNP. Band intensities of adsorbed proteins decreased with increasing AuNP sizes based not only on their mass but also on their surface area.Summarizing, the AuNP surface is covered with serum proteins containing transport and immune related proteins among others. Hence, protein binding depends on the size, surface area and curvature of the AuNP.
We elucidate, from thermodynamic arguments, the governing factors of receptor-mediated endocytosis of nanoparticles (NPs). We show that the endocytic energetics specifies a minimal particle size and a minimal ligand density below which endocytosis is not possible. Due to the entropic penalty involved in ligand-receptor binding, endocytosis may occur with a large fraction of ligands unbound with receptors. Our analyses suggest that the endocytic time depends interrelatedly on the particle size and ligand density. There exists an optimal condition at which the endocytic time minimizes. These findings may provide valuable guidance to the rational designs of NP-based biomarkers and anticancer bioagents.
We investigated the penetration and thermotherapy efficiency of different surface coated gold nanorods (Au NRs) in multicellular tumor spheroids. The current data show that negatively charged Au NRs, other than positively charged Au NRs, can penetrate deep into the tumor spheroids and achieve a significant thermal therapeutic benefit.
We theoretically study the elastic deformation of a fluid membrane induced by an adhering spherical colloidal particle within the framework of a Helfrich energy. Based on a full optimization of the membrane shape we find a continuous binding and a discontinuous envelopment transition, the latter displaying a potentially substantial energy barrier. A small-gradient approximation permits membrane shape and complex energy to be calculated analytically. While this only leads to a good representation of the complex geometry for very small degrees of wrapping, it still gives the correct phase boundaries in the regime of low tension.
Nanostructures of different sizes, shapes and material properties have many applications in biomedical imaging, clinical diagnostics and therapeutics. In spite of what has been achieved so far, a complete understanding of how cells interact with nanostructures of well-defined sizes, at the molecular level, remains poorly understood. Here we show that gold and silver nanoparticles coated with antibodies can regulate the process of membrane receptor internalization. The binding and activation of membrane receptors and subsequent protein expression strongly depend on nanoparticle size. Although all nanoparticles within the 2-100 nm size range were found to alter signalling processes essential for basic cell functions (including cell death), 40- and 50-nm nanoparticles demonstrated the greatest effect. These results show that nanoparticles should no longer be viewed as simple carriers for biomedical applications, but can also play an active role in mediating biological effects. The findings presented here may assist in the design of nanoscale delivery and therapeutic systems and provide insights into nanotoxicity.
The highly effectiveness and robustness of receptor-mediated viral invasion of living cells shed lights on the biomimetic design of nanoparticle(NP)-based therapeutics. Through thermodynamic analysis, we elucidate that the mechanisms governing both the endocytic time of a single NP and the cellular uptake can be unified into a general energy-balance framework of NP-membrane adhesion and membrane deformation. Yet the NP-membrane adhesion strength is a globally variable quantity that effectively regulates the NP uptake rate. Our analysis shows that the uptake rate interrelatedly depends on the particle size and ligand density, in contrast to the widely reported size effect. Our model predicts that the optimal radius of NPs for maximal uptake rate falls in the range of 25-30 nm, and optimally several tens of ligands should be coated onto NPs. These findings are supported by both recent experiments and typical viral structures, and serve as fundamental principles for the rational design of NP-based nanomedicine.
A spatio-temporal mapping of the uptake of silica (SiO(2)) nanoparticles of different sizes by lung epithelial cells has been obtained. Based on high control of nanoparticle dispersion in cell media and cell exposure, one obtains reproducible and quantitative time-resolved data using a combination of flow cytometry, fluorescence and electron microscopies. We are thereby able to give a rather detailed account of the journey of SiO(2) nanoparticles from the early events of uptake to their final sub-cellular localization.
In order to better understand the physical basis of the biological activity of nanoparticles (NPs) in nanomedicine applications and under conditions of environmental exposure, we performed an array of photophysical measurements to quantify the interaction of model gold NPs having a wide range of NP diameters with common blood proteins. In particular, absorbance, fluorescence quenching, circular dichroism, dynamic light scattering, and electron microscopy measurements were performed on surface-functionalized water-soluble gold NPs having a diameter range from 5 to 100 nm in the presence of common human blood proteins: albumin, fibrinogen, gamma-globulin, histone, and insulin. We find that the gold NPs strongly associate with these essential blood proteins where the binding constant, K, as well as the degree of cooperativity of particle--protein binding (Hill constant, n), depends on particle size and the native protein structure. We also find tentative evidence that the model proteins undergo conformational change upon association with the NPs and that the thickness of the adsorbed protein layer (bare NP diameter <50 nm) progressively increases with NP size, effects that have potential general importance for understanding NP aggregation in biological media and the interaction of NP with biological materials broadly.
Rapid growth in nanotechnology is increasing the likelihood of engineered nanomaterials coming into contact with humans and the environment. Nanoparticles interacting with proteins, membranes, cells, DNA and organelles establish a series of nanoparticle/biological interfaces that depend on colloidal forces as well as dynamic biophysicochemical interactions. These interactions lead to the formation of protein coronas, particle wrapping, intracellular uptake and biocatalytic processes that could have biocompatible or bioadverse outcomes. For their part, the biomolecules may induce phase transformations, free energy releases, restructuring and dissolution at the nanomaterial surface. Probing these various interfaces allows the development of predictive relationships between structure and activity that are determined by nanomaterial properties such as size, shape, surface chemistry, roughness and surface coatings. This knowledge is important from the perspective of safe use of nanomaterials.
The unique properties of synthetic nanostructures promise a diverse set of applications as carriers for drug delivery, which are advantageous in terms of biocompatibility, pharmacokinetics, targeting and controlled drug release. Historically, more traditional drug delivery systems have focused on spherical carriers. However, there is a growing interest in pursuing non-spherical carriers, such as elongated or filamentous morphologies, now available due to novel formulation strategies. Unique physiochemical properties of these supramolecular structures offer distinct advantages as drug delivery systems. In particular, results of recent studies in cell cultures and lab animals indicate that rational design of carriers of a given geometry (size and shape) offers an unprecedented control of their longevity in circulation and targeting to selected cellular and subcellular locations. This article reviews drug delivery aspects of non-spherical drug delivery systems, including material selection and formulation, drug loading and release, biocompatibility, circulation behavior, targeting and subcellular addressing.
As materials are produced at smaller scales, the properties that make them especially useful for biological applications such as drug delivery, imaging or sensing applications also render them potentially harmful. There has been a reasonable amount of work addressing the interactions of biological fluids at material surfaces that demonstrates the high affinity of protein for particle surfaces and some looking at the role of particle surface chemistry in cellular associations, but mechanisms have been too little addressed outside the context of intended, specific interactions. Here, using cultured endothelium as a model for vascular transport, we demonstrate that the capacity of nanoparticle surfaces to adsorb protein is indicative of their tendency to associate with cells. Quantification of adsorbed protein shows that high binding nanoparticles are maximally coated in seconds to minutes, indicating that proteins on particle surfaces can mediate cell association over much longer time scales. We also remove many of the most abundant proteins from culture media which alters the profile of adsorbed proteins on nanoparticles but does not affect the level of cell association. We therefore conclude that cellular association is not dependent on the identity of adsorbed proteins and therefore unlikely to require specific binding to any particular cellular receptors.
The beautifully orchestrated regulation of cell shape and volume are central themes in cell biology and physiology. Though it is less well recognized, cell surface area regulation also constitutes a distinct task for cells. Maintaining an appropriate surface area is no automatic side effect of volume regulation or shape change. The issue of surface area regulation (SAR) would be moot if all cells resembled mammalian erythrocytes in being constrained to change shape and volume using existing surface membrane. But these enucleate cells are anomalies, possessing no endomembrane. Most cells use endomembrane to continually rework their plasma membrane, even while maintaining a given size or shape. This membrane traffic is intensively studied, generally with the emphasis on targeting and turnover of proteins and delivery of vesicle contents. But surface area (SA) homeostasis, including the controlled increase or decrease of SA, is another of the outcomes of trafficking. Our principal aims, then, are to highlight SAR as a discrete cellular task and to survey evidence for the idea that membrane tension is central to the task. Cells cannot directly "measure" their volume or SA, yet must regulate both. We posit that a homeostatic relationship exists between plasma membrane tension and plasma membrane area, which implies that cells detect and respond to deviations around a membrane tension set point. Maintenance of membrane strength during membrane turnover, a seldom-addressed aspect of SA dynamics, we examine in the context of SAR. SAR occurs in both animal and plant cells. The review shows the latter to be a continuing source of groundbreaking work on tension-sensitive SAR, but is principally slanted to animal cells.
0. Abbreviations 106
1. Introduction: overview of forces in biology 108
1.1 Subtleties of biological forces and interactions 108
1.2 Specific and non-specific forces and interactions 113
1.3 van der Waals (VDW) forces 114
1.4 Electrostatic and ’double-layer‘ forces (DLVO theory) 122
1.4.1 Electrostatic and double-layer interactions at very small separation 126
1.5 Hydration and hydrophobic forces (structural forces in water) 131
1.6 Steric, bridging and depletion forces (polymer-mediated and tethering forces) 137
1.7 Thermal fluctuation forces: entropic protrusion and undulation forces 142
1.8 Comparison of the magnitudes of the major non-specific forces 146
1.9 Bio-recognition 146
1.10 Equilibrium and non-equilibrium forces and interactions 150
1.10.1 Multiple bonds in parallel 153
1.10.2 Multiple bonds in series 155
2. Experimental techniques for measuring forces between biological molecules and surfaces 156
2.1 Different force-measuring techniques 156
2.2 Measuring forces between surfaces 161
2.3 Measuring force–distance functions, F(D) 161
2.4 Relating the forces between different geometries: the ‘Derjaguin Approximation’ 162
2.5 Adhesion forces and energies 164
2.5.1 An example of the application of adhesion mechanics of biological adhesion 166
2.6 Measuring forces between macroscopic surfaces: the surface forces apparatus (SFA) 167
2.7 The atomic force microscope (AFM) and microfiber cantilever (MC) techniques 173
2.8 Micropipette aspiration (MPA) and the bioforce probe (BFP) 177
2.9 Osmotic stress (OS) and osmotic pressure (OP) techniques 179
2.10 Optical trapping and the optical tweezers (OT) 181
2.11 Other optical microscopy techniques: TIRM and RICM 184
2.12 Shear flow detachment (SFD) measurements 187
2.13 Cell locomotion on elastically deformable substrates 189
3. Measurements of equilibrium (time-independent) interactions 191
3.1 Long-range VDW and electrostatic forces (the two DVLO forces) between biosurfaces 191
3.2 Repulsive short-range steric–hydration forces 197
3.3 Adhesion forces due to VDW forces and electrostatic complementarity 200
3.4 Attractive forces between surfaces due to hydrophobic interactions: membrane adhesion and fusion 209
3.4.1 Hydrophobic interactions at the nano- and sub-molecular levels 211
3.4.2 Hydrophobic interactions and membrane fusion 212
3.5 Attractive depletion forces 213
3.6 Solvation (hydration) forces in water: forces associated with water structure 215
3.7 Forces between ‘soft-supported’ membranes and proteins 218
3.8 Equilibrium energies between biological surfaces 219
4. Non-equilibrium and time-dependent interactions: sequential events that evolve in space and time 221
4.1 Equilibrium and non-equilibrium time-dependent interactions 221
4.2 Adhesion energy hysteresis 223
4.3 Dynamic forces between biomolecules and biomolecular aggregates 226
4.3.1 Strengths of isolated, noncovalent bonds 227
4.3.2 The strengths of isolated bonds depend on the activation energy for unbinding 229
4.4 Simulations of forced chemical transformations 232
4.5 Forced extensions of biological macromolecules 235
4.6 Force-induced versus thermally induced chemical transformations 239
4.7 The rupture of bonds in series and in parallel 242
4.7.1 Bonds in series 242
4.7.2 Bonds in parallel 244
4.8 Dynamic interactions between membrane surfaces 246
4.8.1 Lateral mobility on membrane surfaces 246
4.8.2 Intersurface forces depend on the rate of approach and separation 249
4.9 Concluding remarks 253
5. Acknowledgements 255
6. References 255
While the intermolecular forces between biological molecules are no different from those that arise between any other types of molecules, a ‘biological interaction’ is usually very different from a simple chemical reaction or physical change of a system. This is due in part to the higher complexity of biological macromolecules and systems that typically exhibit a hierarchy of self-assembling structures ranging in size from proteins to membranes and cells, to tissues and organs, and finally to whole organisms. Moreover, interactions do not occur in a linear, stepwise fashion, but involve competing interactions, branching pathways, feedback loops, and regulatory mechanisms.
Using coarse-grained molecular dynamics simulations, we systematically investigate the receptor-mediated endocytosis of elastic nanoparticles (NPs) with different sizes, ranging from 25 nm to 100 nm, and shapes, including sphere-like, oblate-like and prolate-like. Simulation results provide clear evidence that the membrane wrapping efficiency of NPs during endocytosis is a result of competition between receptor diffusion kinetics and thermodynamic driving force. The receptor diffusion kinetics refer to the kinetics of receptor recruitment that are affected by the contact edge length between the NP and membrane. The thermodynamic driving force represents the amount of required free energy to drive NPs into a cell. Under volume constraint of elastic NP, the soft spherical NPs are found to have similar contact edge lengths as rigid ones and to less efficiently be fully wrapped due to their elastic deformation. Moreover, the difference in wrapping efficiency between soft and rigid spherical NPs increases with their sizes, due to the increment of their elastic energy change. Furthermore, because of its prominent large contact edge length, the oblate ellipsoid is found to be the least sensitive geometry to the variation in NP's elasticity among the spherical, prolate and oblate shapes during the membrane wrapping. In addition, simulation results indicate that conflicting experimental observations on the efficiency of cellular uptake of elastic NPs could be caused by their different mechanical properties. Our simulations provide a detailed mechanistic understanding about the influence of NPs' size, shape and elasticity on their membrane wrapping efficiency, which serves as a rational guidance for the design of NP-based drug carriers.
Once in biological fluids, the surface of nanoparticles (NPs) is rapidly covered with a layer of biomolecules (i.e., “protein corona”) whose composition strongly determines its biological identity, regulating interactions with biological entities including cells and the immune system, and consequently direct biological fate and pharmacokinetics of nanoparticles. We recently introduced the concept of “personalized protein corona” which refers to the formation of the different biological identity of the exact same type of NPs after being exposed to extract plasmas from individuals who have various type of diseases. As different diseases have distinct metabolome profile and metabolomes can interact with proteins, it is legitimate to hypothesize that metabolome profiles in plasma may have a capacity to, at least partially, drive the formation of personalized protein corona. To test this hypothesis, we employed a multi-scale approach composed of coarse-grained (CG) and all atom (AA) molecular dynamics (MD) simulations to probe the role of glucose and cholesterol (model metabolomes in diabetes and hypercholesterolemia patients) on the interaction of fibrinogen protein and polystyrene NP. Our results revealed that the glucose and cholesterol had the capacity to induce substantial changes on the binding site of fibrinogen to the surface of NPs. More specifically, the simulation results demonstrated that increasing the metabolite amount could change profiles of fibrinogen adsorption and replacement, what is known as Vroman effect, on the NP surface. In addition, we also found out that metabolites can substantially determine the immune triggering potency of the fibrinogen-NP complex. Our proof-of-concept outcomes further emphasize the need for the development of patient-specific NPs in a disease type-specific manner for high yielding and safe clinical applications.
Significance
Most previous work on endocytosis (the way cells ingest particles) has focused on engulfment of spherical targets. However, this is rarely relevant outside the laboratory, where cells must engulf a huge variety of target shapes, from capped cylinders (like many bacteria) to hourglasses (such as dividing cells). Here, we map this problem to the well-studied physics problem of the freezing of water. This allows us to study a wide range of different, biologically relevant shapes. In particular, for the first time, to our knowledge, it allows us to examine the orientation dependence of phagocytosis, to explain the shape dependence of phagocytosis and receptor-meditated endocytosis, and to explain why nonspherical targets often engulf faster when the most highly-curved tip is engulfed first.
The interaction at nanobio is a critical issue in designing safe nanomaterials for biomedical applications. Recent studies have reported that it is nanoparticle-protein corona rather than bare nanoparticle that determines the nanoparticle-cell interactions, including endocytic pathway and biological responses. Here, we demonstrate the effects of protein corona on cellular uptake of different sized gold nanoparticles in different cell lines. The experimental results show that protein corona significantly decreases the internalization of Au NPs in a particle size- and cell type-dependent manner. Protein corona exhibits much more significant inhibition on the uptake of large-sized Au NPs by phagocytic cell than that of small-sized Au NPs by nonphagocytic cell. The endocytosis experiment indicates that different endocytic pathways might be responsible for the differential roles of protein corona in the interaction of different sized Au NPs with different cell lines. Our findings can provide useful information for rational design of nanomaterials in biomedical application.
This review article focuses on the physiochemical mechanisms underlying nanoparticle uptake into cells. When nanoparticles are in close vicinity to a cell, the interactions between the nanoparticles and the cell membrane generate forces from different origins. This leads to the membrane wrapping of the nanoparticles followed by cellular uptake. This article discusses how the kinetics, energetics, and forces are related to these interactions and dependent on the size, shape, and stiffness of nanoparticles, the biomechanical properties of the cell membrane, as well as the local environment of the cells. The discussed fundamental principles of the physiochemical causes for nanoparticle-cell interaction may guide new studies of nanoparticle endocytosis and lead to better strategies to design nanoparticle-based approaches for biomedical applications.
The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRζ chain expressed as a chimeric protein (TTζ and Tζζ), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tζζ fused to green fluorescent protein (ZAP-70 GFP and Tζζ–GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRζ chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTζ was indicated using mutant ZAP-70 GFPs and a truncated ζ chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tζζ– GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRζ–ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.
Using two independent methods, incorporation of radioactive amino-acid and quantitative immunoblotting, we have determined that the rate of synthesis of each of the Semliki Forest virus (SFV) proteins in infected baby hamster kidney (BHK) cells is 1.2 X 10(5) copies/cell/min. Given the absolute surface areas of the endoplasmic reticulum and Golgi complex presented in the companion paper (Griffiths, G., G. Warren, P. Quinn , O. Mathieu - Costello , and A. Hoppeler , 1984, J. Cell Biol. 98:2133-2141), and the approximate time spent in these organelles during their passage to the plasma membrane (Green J., G. Griffiths, D. Louvard , P. Quinn , and G. Warren 1981, J. Mol. Biol. 152:663-698), the mean density of each viral protein in these organelles can be calculated to be 90 and 750 molecules/micron 2 membrane, respectively. In contrast, we have determined that the density of total endogenous integral membrane proteins in these organelles is approximately 30,000 molecules/micron 2 so that the spike proteins constitute only 0.28 and 2.3% of total membrane protein in the endoplasmic reticulum and Golgi, respectively. Quantitative immunoblotting was used to give direct estimates of the concentrations of one of the viral membrane protein precursors (E1) in subcellular fractions; these agreed closely with the calculated values. The data are discussed with respect to the sorting of transported proteins from those endogenous to the intracellular membranes.
Nanoparticles have recently been widely applied in biomedicine. Understanding how nanoparticles interact with biosystems (especially cells) is crucial for their further biomedical application. On page 1055, H.-m. Ding and Y.-q. Ma summarize the current progress on theoretical and computational investigations of nanoparticle-biomembrane interactions in cellular delivery. The parameters that determine the cellular uptake and subsequent intracellular trafficking of nanoparticles are discussed in detail.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Despite being of vital importance to the immune system, the mechanism by which cells engulf relatively large solid particles during phagocytosis is still poorly understood. From movies of neutrophil phagocytosis of polystyrene beads, we measure the fractional engulfment as a function of time and demonstrate that phagocytosis occurs in two distinct stages. During the first stage, engulfment is relatively slow and progressively slows down as phagocytosis proceeds. However, at approximately half-engulfment, the rate of engulfment increases dramatically, with complete engulfment attained soon afterwards. By studying simple mathematical models of phagocytosis, we suggest that the first stage is due to a passive mechanism, determined by receptor diffusion and capture, whereas the second stage is more actively controlled, perhaps with receptors being driven toward the site of engulfment. We then consider a more advanced model that includes signaling and captures both stages of engulfment. This model predicts that there is an optimum ligand density for quick engulfment. Further, we show how this model explains why nonspherical particles engulf quickest when presented tip-first. Our findings suggest that active regulation may be a later evolutionary innovation, allowing fast and robust engulfment even for large particles.
Understanding the role of serum protein in the process of nanoparticle delivery is of great importance in biomedicine. Here, by using dissipative particle dynamics simulations, we systematically investigate the interactions between the nanoparticle-protein corona complex and cell membranes of different types. It is found that the human serum albumin (HSA) will just adsorb onto charged (especially for positively charged) and hydrophobic nanoparticle surface. More importantly, we also provide specific insights into the effect of HSA adsorption on the in vivo transportation of nanoparticle (i.e., immune response and targeted cellular uptake). Our results show that the protein corona can change the interaction modes of hydrophobic nanoparticles and enhance the interaction of charged nanoparticles with macrophage cell membranes, while it may also cause the failure of insertion of hydrophobic nanoparticles and the loss of targeting specificity of charged nanoparticles with cancer cell membranes. These results can help better understand the biological significance of protein corona and may give some useful suggestions on better design of future nanoparticles in drug delivery.
This article provides a brief overview of recent progress in the synthesis and functionalization of magnetic nanoparticles and their applications in the early detection of malignant tumors by magnetic resonance imaging (MRI). The intrinsic low sensitivity of MRI necessitates the use of large quantities of exogenous contrast agents in many imaging studies. Magnetic nanoparticles have recently emerged as highly efficient MRI contrast agents because these nanometer-scale materials can carry high payloads while maintaining the ability to move through physiological systems. Superparamagnetic ferrite nanoparticles (such as iron oxide) provide excellent negative contrast enhancement. Recent refinement of synthetic methodologies has led to ferrite nanoparticles with narrow size distributions and high crystallinity. Target-specific tumor imaging becomes possible through functionalization of ferrite nanoparticles with targeting agents to allow for site-specific accumulation. Nanoparticulate contrast agents capable of positive contrast enhancement have recently been developed in order to overcome the drawbacks of negative contrast enhancement afforded by ferrite nanoparticles. These newly developed magnetic nanoparticles have the potential to enable physicians to diagnose cancer at the earliest stage possible and thus can have an enormous impact on more effective cancer treatment.
Poor penetration of therapeutic drugs into tumor is a major challenge in anti-cancer therapy, especially in solid tumors, leading to reduced therapeutic efficacy in vivo. In the study, we used a new tumor penetrating peptide, CRGDK, to conjugate onto the surface of doxorubicin encapsulated nanoscale micelles. The CRGDK peptide triggered specific binding to neuropilin-1, leading to enhanced cellular uptake and cytotoxicity in vitro and highly accumulation and penetration in the tumors in vivo.
When nanoparticles (NPs) enter a biological fluid (e.g. human plasma (HP)), proteins and other biomolecules adsorb on the surface leading to formation of a rich protein shell, referred to as 'protein corona'. This corona is dynamic in nature and its composition varies over time due to continuous protein association and dissociation events. Understanding the time evolution of the protein corona on the time-scales of particle's lifetime in blood is fundamental to predict its fate in vivo. In this study, we used lipid NPs made the cationic lipid 3β-[N-(N´,N´-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE) that are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DC-Chol-DOPE NPs upon exposure to HP. On time scales between one and sixty minutes, nano-liquid tandem mass spectrometry revealed that the protein corona of DC-Chol-DOPE NPs is mainly constituted of apolipoproteins (Apo A-I, Apo C-II, Apo D and Apo E are the most enriched). Since the total apolipoprotein content is relevant, we exploited the protein corona to target PC3 prostate carcinoma cell line that express high levels of scavenger receptor class B type 1 receptor, which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins and cells. Combining laser scanning confocal microscopy experiments with flow cytometry we demonstrated that DC-Chol-DOPE/HP complexes enter PC3 cells by a receptor-mediated endocytosis mechanism.
When nanoparticles (NPs) are exposed to the biological environment, their surfaces become covered with proteins and biomolecules (e.g. lipids). Here, we report that this protein coating, or corona, reduces the targeting capability of surface engineered NPs by screening the active sites of the targeting ligands.
Nanoparticles have been proposed as carriers for drugs, genes and therapies to treat various diseases. Many strategies have been developed to target nanomaterials to specific or over-expressed receptors in diseased cells, and these typically involve functionalizing the surface of nanoparticles with proteins, antibodies or other biomolecules. Here, we show that the targeting ability of such functionalized nanoparticles may disappear when they are placed in a biological environment. Using transferrin-conjugated nanoparticles, we found that proteins in the media can shield transferrin from binding to both its targeted receptors on cells and soluble transferrin receptors. Although nanoparticles continue to enter cells, the targeting specificity of transferrin is lost. Our results suggest that when nanoparticles are placed in a complex biological environment, interaction with other proteins in the medium and the formation of a protein corona can 'screen' the targeting molecules on the surface of nanoparticles and cause loss of specificity in targeting.
The search for understanding the interactions of nanosized materials with living organisms is leading to the rapid development of key applications, including improved drug delivery by targeting nanoparticles, and resolution of the potential threat of nanotechnological devices to organisms and the environment. Unless they are specifically designed to avoid it, nanoparticles in contact with biological fluids are rapidly covered by a selected group of biomolecules to form a corona that interacts with biological systems. Here we review the basic concept of the nanoparticle corona and its structure and composition, and highlight how the properties of the corona may be linked to its biological impacts. We conclude with a critical assessment of the key problems that need to be resolved in the near future.
Nanoparticles enter cells through active processes, thanks to their capability of interacting with the cellular machinery. The protein layer (corona) that forms on their surface once nanoparticles are in contact with biological fluids, such as the cell serum, mediates the interactions with cells in situ. As a consequence of this, here we show that the same nanomaterial can lead to very different biological outcomes, when exposed to cells in the presence or absence of a preformed corona. In particular, silica nanoparticles exposed to cells in the absence of serum have a stronger adhesion to the cell membrane and higher internalization efficiency, in comparison to what is observed in medium containing serum, when a preformed corona is present on their surface. The different exposure conditions not only affect the uptake levels but also result in differences in the intracellular nanoparticle location and impact on cells. Interestingly, we also show that after only one hour of exposure, a corona of very different nature forms on the nanoparticles exposed to cells in the absence of serum. Evidence suggests that these different outcomes can all be connected to the different adhesion and surface properties in the two conditions.
Unlabelled:
It is generally accepted that the presentation of multiple ligands on a nanoparticle (NP) surface can improve cell targeting; however, little work has been done to determine whether an optimal ligand density exists. We have recently developed a site-specific bioconjugation strategy that allows for distinct control of ligand density on a NP through the combined utilization of expressed protein ligation (EPL) and copper-free click chemistry. This EPL-Click conjugation strategy was applied to create superparamagnetic iron oxide (SPIO) NPs labeled with HER2/neu targeting affibodies at differing ligand densities. It was discovered that an intermediate ligand density provided statistically significant improvements in cell binding in comparison with higher and lower ligand densities. This intermediate optimal ligand density was conserved across NPs with differing hydrodynamic diameters, different HER2/neu targeting ligands and also to cells with lower receptor densities. Additionally, an intermediate optimal ligand density was also evident when NPs were labeled with folic acid.
From the clinical editor:
The authors of this study investigated optimal ligand density with SPIO-based labeling and concluded that intermediate density appears to have the most optimal labeling properties from the standpoint of its T2* shortening effect.
The standard view of mechanical adhesive contact is as a competition between a reduction in free energy when surfaces with bonding potential come into contact and an increase in free energy due to elastic deformation that is required to make these surfaces conform. An equilibrium state is defined by an incremental balance between these effects, akin to the Griffith crack growth criterion. In the case of adhesion of biological cells, the molecules that tend to form surface-to-surface bonds are confined to the cell wall but they are mobile within the wall, adding a new phenomenon of direct relevance to adhesive contact. In this article, the process of adhesive contact of an initially curved elastic plate to a flat surface is studied for the case in which the binders that account for adhesion are able to migrate within the plate. This is done by including entropic free energy of the binder distribution in the total free energy of the system. By adopting a constitutive assumption that binders migrate at a speed proportional to the local gradient in chemical potential, the transient growth of an adhesion zone due to binder transport is analyzed. For the case of a plate of very large extent, the problem can be solved in closed form, whereas numerical methods are invoked for the case of a plate of limited extent. Results are presented on the rate of growth of an adhesion zone in terms of system parameters, on the evolution of the distribution of binders and, in the case of a plate of limited extent, on the long-term limiting size of the adhesion zone.
As an emerging applied material, graphene has shown tremendous application potential in many fields, including biomedicine. However, the biological behavior of these nanosheets, especially their interactions with cells, is not well understood. Here, we report our findings about the cell surface adhesion, subcellular locations, and size-dependent uptake mechanisms of protein-coated graphene oxide nanosheets (PCGO). Small nanosheets enter cells mainly through clathrin-mediated endocytosis, and the increase of graphene size enhances phagocytotic uptake of the nanosheets. These findings will facilitate biomedical and toxicologic studies of graphenes and provide fundamental understanding of interactions at the interface of two-dimensional nanostructures and biological systems.
The significant role of protein nanoparticle interactions in nanomedicine and nanotoxicity is emerging recently through the identification of the nanoparticles (NP) protein (biomolecule) corona. The dynamic layer of proteins and/or other biomolecules adsorbed to the nanoparticle surface determines how a NP interacts with living systems and thereby modifies the cellular responses to the NP. Ehrenberg and co-workers used cultured endothelium cells as a model for vascular transport of polystyrene NP with various functional groups, which showed that the capacity of the various NP surfaces to adsorb proteins was indicative of their tendency to associate with cells. The quantification of the adsorbed proteins showed that high-binding NP were maximally coated within seconds to minutes, indicating that proteins on the surface of NP could mediate cell association over much longer time scales. The adsorption or covalent binding of a protein onto a NP's surface can strongly alter the physio-chemical and structural properties of both of them.
In vitro experiments typically measure the uptake of nanoparticles by exposing cells at the bottom of a culture plate to a suspension of nanoparticles, and it is generally assumed that this suspension is well-dispersed. However, nanoparticles can sediment, which means that the concentration of nanoparticles on the cell surface may be higher than the initial bulk concentration, and this could lead to increased uptake by cells. Here, we use upright and inverted cell culture configurations to show that cellular uptake of gold nanoparticles depends on the sedimentation and diffusion velocities of the nanoparticles and is independent of size, shape, density, surface coating and initial concentration of the nanoparticles. Generally, more nanoparticles are taken up in the upright configuration than in the inverted one, and nanoparticles with faster sedimentation rates showed greater differences in uptake between the two configurations. Our results suggest that sedimentation needs to be considered when performing in vitro studies for large and/or heavy nanoparticles.
Unlabelled:
Central to understanding how nanoscale objects interact with living matter is the need for reproducible and verifiable data that can be interpreted with confidence. Likely this will be the basis of durable advances in nanomedicine and nanomedical safety. To develop these fields, there is also considerable interest in advancing the first generation of theoretical models of nanoparticle (NP) uptake into cells, and NP biodistribution in general. Here we present an uptake study comparing the outcomes for free molecular dye and NPs labeled with the same dye. A simple flux-based approach is presented to model NP uptake. We find that the intracellular NP concentration grows linearly in time, and that the uptake is essentially irreversible, with the particles accumulating in lysosomes. A wide range of practical challenges, from labile dye release to NP aggregation and the need to account for cell division, are addressed to ensure that these studies yield meaningful kinetic information.
From the clinical editor:
The authors present an uptake study comparing the outcomes for free molecular dye and NPs labeled with the same dye. A wide range of practical challenges are addressed including labile dye release, NP aggregation and the need to account for cell division with the goal that these studies yield meaningful kinetic information.
In this work, we explore the formation of the protein corona after exposure of metallic Au nanoparticles (NPs), with sizes ranging from 4 to 40 nm, to cell culture media containing 10% of fetal bovine serum. Under in vitro cell culture conditions, zeta potential measurements, UV-vis spectroscopy, dynamic light scattering and transmission electron microscope analysis were used to monitor the time evolution of the inorganic NP-protein corona formation and to characterize the stability of the NPs and their surface state at every stage of the experiment. As expected, the red-shift of the surface plasmon resonance peak, as well as the drop of surface charge and the increase of the hydrodynamic diameter indicated the conjugation of proteins to NPs. Remarkably, an evolution from a loosely attached toward an irreversible attached protein corona over time was observed. Mass spectrometry of the digested protein corona revealed albumin as the most abundant component which suggests an improved biocompatibility.
A thermodynamic model for receptor-mediated endocytosis of ligand-coated nanoparticles (NPs) was discovered by demonstrating the cellular uptake of ligand-coated NPs. The study also investigated the effects of membrane tension and of the surface concentration of NPs on the cellular uptake. Three regimes were identified and separated by two characteristic particle radii, R min and Rmax. The model predicts an optimal surface concentration beyond which the cellular uptake decreases due to the competition of receptors among NPs. During endocytosis, wrapping of NPs may proceed by insertion of lipids from cytoplasm into highly curved regions rather than by laterally pulling the lipids, which effectively lowers the stretching energy. The results showed that the predicted maximal cellular uptake of NPs is valuable for assessing NP toxicity, the efficiency of NP-based bioimaging and biomakers, and the therapeutic efficacy of NP based drug carriers.
A study was conducted to demonstrate the synthesis of monodisperse mesoporous silica nanoparticle (MSN) suspensions and investigate the effect of nanoparticle size on cell uptake. The MSNs were synthesized by adjusting the pH of the reaction medium, leading to a series of MSNs with uniform size ranging from 30-280 nm. The physical properties of the MSNs were characterized with various methods, such as nitrogen adsorption-desorption, transmission electron microscopy (TEM), and X-ray powder diffraction (XRD). The particle size effect on the cell uptake efficiency with human cervical cancer cells and cellular uptake of MSNs of various sizes were investigated by confocal laser scanning microscopy (CLSM). The uptake efficiencies were determined by inductively coupled plasma mass spectrometry (ICP-MS) analysis of total silicon content.
To enable cells to crawl, adhesion receptors such as integrins must bind to extracellular molecules and simultaneously interact with force-generating components of the cytoskeleton. We show here that the binding of extracellular ligand in living cells induces the attachment of beta1 integrins to the retrograde-moving cytoskeleton. Unliganded integrins are not associated with the rearward-moving cytoskeleton: gold particles attached to beta1 integrin by a monoclonal antibody diffuse in the membrane. However, addition of soluble RGD peptide (single-letter amino-acid code) or the use of fibronectin-coated gold particles causes the attachment of integrins to the rearward-moving cytoskeleton. Deletion of the beta1 cytoplasmic tail blocks cytoskeletal attachment. The directed movement of integrins in response to ligand indicates that ligand binding is the critical step in regulating organized receptor movement on the cell surface and the migration of adherent cells.