key: cord-0885618-i9bucuma
authors: Natarajan, Pavithra; Tomich, John M.
title: Understanding the influence of experimental factors on bio-interactions of nanoparticles: Towards improving correlation between in vitro and in vivo studies
date: 2020-09-21
journal: Arch Biochem Biophys
DOI: 10.1016/j.abb.2020.108592
sha: d001d53a3afe527d2b2bb7bcc078679e54e90cd9
doc_id: 885618
cord_uid: i9bucuma

Bionanotechnology has developed rapidly over the past two decades, owing to the extensive and versatile, functionalities and applicability of nanoparticles (NPs). Fifty-one nanomedicines have been approved by FDA since 1995, out of the many NPs based formulations developed to date. The general conformation of NPs consists of a core with ligands coating their surface, that stabilizes them and provides them with added functionalities. The physicochemical properties, especially the surface composition of NPs influence their bio-interactions to a large extent. This review discusses recent studies that help understand the nano-bio interactions of iron oxide and gold NPs with different surface compositions. We discuss the influence of the experimental factors on the outcome of the studies and, thus, the importance of standardization in the field of nanotechnology. Recent studies suggest that with careful selection of experimental parameters, it is possible to improve the positive correlation between in vitro and in vivo studies. This provides a fundamental understanding of the NPs which helps in assessing their potential toxic side effects and may aid in manipulating them further to improve their biocompatibility and biosafety.

The term nanotechnology was coined by Prof. Norio Taniguchi in 1974 and is defined as the science, engineering and technology conducted at the nanoscale i.e. 1 to 100 nm. The nanoscale materials generally referred to as nanoparticles (NPs) are highly desirable because of their small size, optical properties, high surface area to volume ratio and their multifunctional nature. Bionanotechnology comprises research at the interface of nanotechnology and biology 2 that has established a niche in biomedical sciences. Liposomes 3-6 , peptide-based [7] [8] [9] and synthetic polymer-based [10] [11] [12] , three-dimensional macromolecular assemblies and nanocages [13] [14] [15] are examples of hollow/porous core NPs. Solid core NPs may be composed of inorganic metals such as iron oxide, gold, silver, platinum, silicon, quantum dots, titanium dioxide, gadolinium, selenium, copper oxide, zinc oxide or metallic hybrids, or organic carbon nanoparticles. The surfaces of inorganic NPs are generally modified with synthetic or naturally occurring polymers and/or monomers which may be of biological origin such as peptides, proteins, carbohydrates, lipids, DNA, RNA, PNA, aptamers, hybrid bio-synthetic molecules and others. These relatively flexible capping ligands improve the stability, biocompatibility and functionalize the NPs for various applications or for further modifications. Fig. 1 , depicts the various components and configurations of nanoparticle-bioconjugates.

Drugs that have poor pharmacodynamics can be delivered using NPs that may overcome these shortcomings by improving their half-lives, stabilities and bioavailabilities. 17 However, their use is not limited to drug delivery systems (DDS). Their other applications include use as optical imaging agents and analytical probes/biosensors, thus making them suitable theranostics agents. [18] [19] [20] [21] Fifty-one nanomedicines have been approved since 1995 by FDA for clinical use J o u r n a l P r e -p r o o f with ~77 products in clinical trials as of 2016. 22 Owing to their potential, nanomaterials are being utilized in the recent fight against SARS-CoV-2. 23, 24 Gold nanoparticles based immunoassays have been developed that enable rapid detection of SARS-CoV-2 infected asymptomatic patients or individuals showing mild symptoms. 25, 26 An mRNA vaccine which went into Phase 1 clinical trial in March 2020, codes for the prefusion stabilized spike protein of SARS-Cov-2 and it is encapsulated in lipid nanoparticles which serve as effective delivery agents . 27 The focus of this review is on gold and iron oxide NPs which are the top 2 inorganic NPs in clinical trials (Fig. 1D) . Iron oxide NPs are the only metal-containing NPs that have received approval to date for clinical use and most of them are MRI contrast agents. 22 Gold nanoparticles (AuNPs) exhibit plasmon resonance which can be followed using UV-Vis spectrophotometric detection assays 28, 29 , surface-enhanced Raman spectroscopy (SERS) 30 and confocal/ luminescence microscopy. 31, 32 The magnetic iron oxide nanoparticles (FeONPs), also commonly called superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents for magnetic resonance imaging (MRI) 33, 34 , for bio-detection such as tracking the implanted stem cells in vitro 35 , in binding assays and hyperthermia 36, 37 and magnetic field guided drug delivery 21 in cancer treatment. Besides, the electron dense gold and iron NPs are used widely in electron microscopy analyses. Au-Magnetite composites used in (SERS) analyses improve the intracellular signal intensity essential to studying interactions of NPs with biomolecules. 30 Delivery systems must be non-toxic by themselves, should not be cleared quickly from the body and trigger adverse immunological responses. It, therefore, becomes vital to understand J o u r n a l P r e -p r o o f their interactions at a molecular level, to determine how suitable they are for delivery and determine the applications for which they are best suited. The review is divided into four sections which discuss the (I) synthesis and functionalizing of NPs, (II) the discrepancies observed between the effects of NPs in vitro and in vivo, followed by a detailed review of (III) in vitro and (IV) in vivo studies of gold and iron oxide NPs, which demonstrate the need to carefully consider experimental factors to improve the correlation between in vitro and in vivo studies.

This review also presents recent in vitro and in vivo studies that assess the biosafety/toxicity of NPs and the influence of surface ligands on nano-bio interactions such as uptake and immune response. We will emphasize the importance of standardization in nanotechnology with a focus on the experimental parameters since they have a significant impact on the outcome of studies.

Standardization is essential to make valid comparisons between studies and to prevent redundancy in research which help develop the field of nanomedicine. 38, 39 J o u r n a l P r e -p r o o f

The basic principle of NPs syntheses is to promote nucleation of the monomeric element (e.g. lipids for liposomes and metal ions for inorganic metal NPs), facilitating their assembly in a controlled manner to form stable and well-structured entities with narrow size distributions.

Multiple routes and techniques used in NP syntheses have been established that are broadly categorized as chemical, physical and biological. Most chemical and biological methods use facile synthesis techniques that are easily controlled and reproducible, yet low in cost and scalable. 40 Functionalization of NPs has proved essential as they affect stability in the presence of salts and prevent aggregation over time, thereby increasing their shelf-life. They may also have other purposes including-promoting cellular uptake, co-functionalization to promote the delivery of drugs and nucleic acids, use in biochemical assays serving as binding partners, and provide additional functionalities to the delivery system. There are a wide range of biocompatible molecules used to functionalize the NPs for use in nanomedicine which have been divided into 5 major categories in this review. ( Ligand exchange by direct substitution of surface ligands is one of the commonly used method for functionalizing NPs. 48 AuNPs form gold-thiol bonds facilitating exchange of smaller ligands such as citrate molecules with larger molecules by direct binding of ligands to NPs via Au-S bond formation (Fig 1C.) . 42 Biodegradable polymers are widely used as surface coatings, as they are easy to synthesize, widely studied, allow for precise chemical binding of molecules or can be modified with functional groups to bind other molecules using facile chemistries like EDC-NHS 56 and disulfide conjugations 19 . They have been recognized to increase the circulation time of the nanoparticles by preventing opsonization by phagocytes in vivo. Therefore, a wide range of FDA-approved nanoparticles and in vivo devices are coated with one or more of the abovementioned polymers. 57 A recent report also suggests that PEG-like polymers may not be as inert as currently believed. Their oxidative degradation in vivo can lead to detrimental effects on the cell membrane and affect signal transduction pathways. 58 Therefore, recent emphasis has been on J o u r n a l P r e -p r o o f the use of natural or synthetic biocompatible surface coatings which display minimal adverse effects.

Lipid amphiphiles comprised of one acyl chain generally form micelles while those with two acyl chains assemble into bilayer-like membrane vesicles called liposomes. Commonly, lipid formulations yield self-assembled structures that are greater than 100 nm. The first liposomal formulation to be approved by the FDA was Doxil in 1995, subsequently 9 additional liposomal formulations with active ingredients (AIs) have been approved. 59 Single chained lipid amphiphiles such as lysophosphatidylcholine and two-chained DOPC, POPC 60 , as well as cholesterol and/or their mixtures have been incorporated into liposomes 3 , polymeric liposomes 6 (polymer modified lipid components) and to functionalize inorganic core NPs.

A reverse phase evaporation method that involves exchanging the existing surface ligands with lipids in an organic solvent followed by transfer to an aqueous solvent, is commonly used for lipid membrane assembly on NPs. This technique has been employed in the synthesis of hybrid lipid bilayer coatings on NPs where inner and outer layer have different compositions. 6, 60 Another common technique involves adsorption of liposomes 4 , on the NPs where the charged head moieties interact with the surface and encapsulate the NPs within liposomes. 5 However, lipid membranes often have low stability in solution due to fusion, leading to increases in the particle size. 61 This can be remedied by increasing the surface charges that promote repulsion between particles or by incorporating spacers such as PEG that sterically hinder particle association. These methods improve colloidal stability. utilized to functionalize NPs with peptides (Fig 1C.) . Although amines bind to the gold surfaces, the strength of Au-N (~4 kJ/mol) bond is much weaker than the Au-thiol bond (137 kJ/mol) that is commonly used to bind cysteine containing peptides under appropriate conditions. 49, 55, 62 Cell penetrating peptides like HIV-1 derived TAT peptide 63 promote the uptake of molecules or complexes that cannot penetrate the cell membrane efficiently by themselves. They are therefore used to co-functionalize the surface of nanoparticles and are widely explored for delivery of nanoparticles in radiation therapy 63 

Specific proteins can be used to functionalize NPs for targeted delivery or to serve as binding partners in assays. Abraxane® is an FDA-approved chemotherapeutic drug that consists of nanoparticle albumin bound (nab)-paclitaxel. Albumin is an abundant serum protein used as surface coating for NPs as it improves bioavailability, has low immunogenicity and good biocompatibility. 72, 73 Nab-paclitaxel and its variations comprise a major percentage of the protein based nanomedicines in clinical trials (Fig. 1D) . This success has fostered the use of albumin as a surface coating for additional NPs delivery systems. [74] [75] [76] Antibodies/immunoglobulins are widely used due to their high specificity in detecting and binding to specific antigens and have been successfully employed for disease treatments as antibody drug conjugates (ADCs), four of which are commercially available. 77 Since protein structure defines function, any structural alterations due to temperature transitions or pH, limit the chemistries available for attachment to NPs. General strategies for binding antibodies and proteins to inorganic surfaces therefore include covalent binding to a modified surface 78, 79, 80 or by physical adsorption promoted by electrostatic interactions. 79 The orientation of the antibody is more important for its functioning than its coverage on the surface and hence orienting covalent binding strategies are more widely employed. 79 J o u r n a l P r e -p r o o f Finetti et al. 80 used "click" chemistry to immobilize anti CD-63 and anti-rabbit-IgG on the surface of AuNPs. Thus, using the benefits of click chemistry, antibodies immobilized NPs can be produced for a wide range of applications. Antibody immobilized AuNPs are also widely used in immunostaining for analysis using electron microscopy, and plasmon resonance mediated confocal imaging. 31, 81, 82 Antibodies tagged with fluorescence molecule on AuNPs allows for dual imaging, reducing cost and time.

NPs are commonly coated with nucleic acids such as DNA, dsRNA, ssRNA, siRNA, mRNA, and microRNA, as they facilitate the delivery of the nucleic acids into cells or for use in binding assays. DNA grafted polymers such as poly(acrylic acid) embedded DNA are also used for functionalizing nanoparticles as they facilitate polyvalent DNA nanostructure formation. 83 A common strategy for functionalizing NPs with nucleic acids is to utilize the electrostatic interactions between the negatively charged nucleic acids and cationic NPs which mediates their adsorption to NPs. 84, 85 This does not require extensive modification of the nucleic acids. 86 Recently nucleic acids have also been identified as templates that control and facilitate inorganic NPs synthesis. 87 Aptamers that bind with high affinity and specificity to proteins and peptides are commonly conjugated to AuNPs and FeONPs for detection of molecules using colorimetric binding assays 88, 89 and magnetic isolation 90 , respectively. J o u r n a l P r e -p r o o f 

In vitro studies are often indicators of potential outcomes in animal studies and provide mechanistic information at the cellular level. They allow researchers to explore the effect of different doses, chemicals at relatively lower cost and reduced time. They also allow for probing POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; DOPE, Dioleoylphosphatidylethanolamine; OQLCS, octadecyl-quaternized lysine modified chitosan; TAT, Transactivator of transcription; CPP, Cell penetrating peptide; ScFvEGFR, short chain variable, anti-EFGR the underlying mechanisms leading to toxicity, immunogenicity, metabolic changes and analyzing gene expression profiles. These cell culture studies reduce the number and cost of animals required to statistically assess the effect of NPs. 91 NPs on the other hand encounter a very complex environment in vivo which cannot be mimicked accurately in vitro. And therefore, there are obvious discrepancies due to these inherent differences between in vitro and in vivo environments. Khlebtsov et al. 92 have examined the lack of correlation between in vitro and in vivo behavior of NPs. They emphasize on the need for systematization of data obtained from various studies on NPs, to gain a fundamental understanding of factors affecting their biointeractions.

The inconsistencies observed between their effects in vitro and in vivo is also due to differences in experimental factors. [93] [94] [95] [96] [97] For example, one basic consideration is to use the cell lines/ primary cell types for in vitro studies that belong to the same species that is being investigated in vivo. Surprisingly, this is overlooked often. 93 

There are hundreds of reports on the toxicity of NPs in vitro 'or' in vivo but very few recent studies have compared their effects in vitro 'and' in vivo. Table 2 summarizes the studies belonging to latter group. 

Recent reviews by Foroozandeh et al. 100 and Behzadi et al. 101 discussed the effect of nanoparticle physicochemical properties such as size, shape, surface composition on their uptake and intracellular trafficking. Unfortunately, few articles discuss the effect of experimental parameters on cellular uptake. In the following section the importance of carefully selecting cell lines, determining effect of dosage, time and media type in understanding NP interactions will be J o u r n a l P r e -p r o o f discussed. We also review recent studies that explore cellular uptake routes, immune responses and toxicity induced by AuNPs and FeONPs with different surface compositions.

A. Influence of various experimental parameters

Cell lines used to study NPs are commonly selected based on availability; they should be chosen based on the applications of NPs and the expected in vivo exposure. 102 Several studies have shown that nanoparticle uptake and toxicity profiles vary between cell lines, cell sub-types and to some extent between species. [102] [103] [104] [105] [106] The uptake of NPs is also dependent on cell-specific functions. 107 Although immortalized cell lines are easier to maintain, readily accessible and widely studied, they differ from cells in vivo due to repeated in vitro manipulations and the initial immortalization itself. 102 Joris et al. 102 facilitates co-culturing of multiple cell types simultaneously, to evaluate the effect of NPs treatment on the crosstalk between the cell types or to study transcytosis. [110] [111] [112] Three-D cell cultures that make use of a scaffold increase the surface area of exposure, while only ~50% area is available in a 2D cell culture. The MD1-MB231 breast cancer cells in 2D culture, in comparison to their 3D counterpart, had increased viability and showed a lesser change in the cytoplasmic actin network that plays a major role in intracellular processes. 103

Thus, the toxicity of the NPs could be underestimated by testing their effect in just 2D cell cultures and immortalized cell lines. In vivo, NPs and drugs have a tendency to accumulate in the liver generally, which clears foreign materials and thus, the liver is an important tissue to consider for studying NPs. The sandwich hepatocyte culture model uses primary hepatocytes, grown between two layers of collagen that keeps them competent and polarized with functional J o u r n a l P r e -p r o o f bile networks and helps to assess the hepatotoxicity of drugs and NPs accurately. 113 While 3-D cultures mimic the in vivo environment more closely not all labs have transitioned to this approach. Traditional 2D cultures still predominate in the current literature.

Cell culture media composition varies depending upon the requirements of each cell line. observed a similar effect where AuNPs pre-exposed to protein poor medium had a higher tendency to aggregate than in protein rich medium. Interestingly, 15 nm AuNPs exerted more adverse effects on cells in RPMI in comparison to DMEM. Hence, while designing and implementing studies, we should consider the choice of cell culture media which is crucial. 117 Another non-trivial factor to be considered is the method by which NPs are administered as documented by Moore et al. 107 When poly(vinylpyrrolidone) (PVP) coated AuNPs were administered as a concentrated bolus directly to J774A.1 mouse macrophages, the protein corona formation was 2-fold higher than AuNPs pre-mixed with media. The macrophages also phagocytosed more AuNPs administered as a concentrated dose in comparison to the pre-mixed

AuNPs. This study emphasizes how a minor detail such as the initial administration of NPs can affect the outcome of the study. Thus, to be able to compare studies between research groups, we should consider every minor detail and develop a robust analytical method. Due to a lack of standardized/universal methods of testing NPs, it is difficult to compare and obtain a better understanding of NPs bio-interactions.

The Hence, more studies are needed to assess the effect of repeated exposure to NPs at prescribed intervals. 91 Gokduman et al. 91 Cell viability tests are widely used to assess the toxicity of NPs. This typically involves a single dose of NPs followed by short-term evaluations of viability. Whereas, in vivo studies focus on studying the systemic effects and accumulation of NPs. Therefore, there is an apparent disconnect between most in vitro and in vivo studies. 120 Reactive oxygen species produced by cells in response to NPs is a potent early marker for nanoparticle toxicity. 94 119 Oxidative stress exerted by NPs may be inevitable in some cases and can be ameliorated by the naturally occurring antioxidants 94 or by supplementation with antioxidants such as thymoquinone to reduce these effects. 121 Feng et al. 99 observed that cationic PEI coated FeONPs were endocytosed in high numbers compared to PEG-FeONPs and were more toxic to cells as they dramatically reduced cell viability in a concentration dependent manner. Increased ROS generation that disrupted the cell cycle by arresting cells in G2-phase cell cycle, led to apoptosis. Genotoxicity induced by the PEI-FeONPs was observed to be an indirect effect and not due to direct interaction with the DNA. 99 In contrast 60 nm 'naked' FeONPs intercalated with DNA base pairs in primary lymphocytes and generated high levels of ROS that reduced the cell viability. 121 

Cells may use an active, energy dependent endocytic pathway or energy independent passive diffusion to internalize NPs. Table 4 summarizes the uptake pathways used by nanoparticles with different surface chemistries in various cell types. Endocytosis is broadly classified as -Clathrin mediated endocytosis (CME), caveolae mediated endocytosis (CvME), macropinocytosis and clathrin and caveolae independent endocytosis. Phagocytosis is a type of endocytic pathway which is only employed by immune cells such as macrophages, neutrophils and dendritic cells. 127 Cargo is transported intracellularly in endocytic vesicles formed by cell membrane invaginations. Endocytic vesicles can be classified based on the protein markers on the vesicle membrane associated with the endocytic pathway, further influencing the cargo's intracellular sorting. (Fig. 4 ) CME and macropinocytosis promote the fusion of endocytic vesicles with the highly acidic lysosomes (~ pH 5) that can cause degradation of the functionalizing ligands and NPs themselves. While the cargo transported in the caveosomes, enter the Golgi and endoplasmic reticulum, bypassing the lysosomes. CvME also favor transcytosis like in the case of Nab-paclitaxel. [127] [128] [129] [130] Some oncology and viral medications such as trastuzumab emtansine

(T-DMI) 131 and chloroquine 132 , respectively, target the endocytosis pathways. Hence, studying the mechanism of uptake is important for the fundamental understanding of nano-bio interactions and drug delivery.

Chemical inhibitors of endocytosis are commonly used to study the endocytic uptake pathways. Some inhibitors may have a generalized inhibitory effect while some are relatively J o u r n a l P r e -p r o o f more specific. Methyl-β-cyclodextrin although commonly used as an inhibitor of CvME, it can also inhibit cholesterol dependent clathrin and caveolin independent pathways. 133 Similarly, dynasore may inhibit dynamin independent endocytic pathways as well. 134 Therefore, the chemical inhibitors should be selected wisely and the results should be interpreted appropriately.

siRNA mediated knockdown of proteins, essential to specific endocytic routes on the other hand is less ambiguous than chemical inhibitors. 135 In some cases other endocytic pathways may be upregulated to compensate for inhibition of one pathway. Although the net uptake of NPs may seemingly be unaffected, one should not discount changes in the uptake mechanism. 71, 135, 136 Endocytosis of NPs is time dependent. 137 He et al. 138 observed that although the uptake of cationic CALRRRRRRRR (R8) peptide functionalized AuNPs was slower in comparison to the hydrophobic CALNNPFVYLI (PFV) peptide coated AuNPs, in the initial one hour, their net uptake was higher at the end of 12 h of incubation. IEC-18 epithelial cells also seemed to use different endocytosis pathways to internalize peptide bilayer coated FeONPs in a time dependent manner. 71 The surface composition plays a crucial role since they may also help in endosomal escape as observed for highly cationic NPs. 70, 139 Different cell types may use different endocytic pathways for the uptake of the same NPs 140 and a single cell type may use multiple pathways for the uptake of NPs. 71 Srijampa et al. 141 identified that monocytes and macrophages generally studied for their phagocytic response may also use other endocytosis pathways alongside phagocytosis for NPs uptake. B.End endothelial cells internalized more of the negatively charged FeONPs in comparison to epithelial cells, using CvME, which was enhanced in the endothelial cells since they overexpressed the caveolin-1 J o u r n a l P r e -p r o o f

NPs can elicit an immune response by interfering and interacting with intracellular signaling pathways directly or indirectly via the reactive oxygen and nitrogen species produced.

For example, the transition metals on the surface of NPs or in SPIONs generate ROS as described previously, which triggers a pro-inflammatory response. 147 

The NPs bio-interactions in the complex in vivo environment are dependent on their physicochemical properties, contributing to their translocation to the different organs and tissues and ultimate clearance. 97, 156 Therefore, it is vital to discern the relationship between the NPs and the interactions with endogenous molecules that influence their biodistribution. In this section, The route of administration has an obvious role to play on the tissue distribution which is generally chosen based on the end application of NPs. However, i.v. injections are used more commonly since they can provide a near instantaneous response and is suitable for delivery of materials that cannot be absorbed efficiently or that can undergo proteolytic or pH disruption.

Another major advantage of i.v. injections is the increased bioavailability of drugs. 157 The animal model selected for a particular study may influence the administration route. 158 Intramuscular delivery in mice is generally not recommended as their muscles are small, making it difficult to get reproducible results. 159 The genetic background of animals will also show variations in NPs interactions due to differences in their response to foreign molecules. The C57BL/6 and the BALB/c mice, for example, fundamentally exhibit different immune responses that could affect J o u r n a l P r e -p r o o f their adaptive immunity. C57Bl/6 and BALB/c are prototypical, Th1 and Th2 type mouse strains, respectively, and therefore can have an altered response to NPs. 160 When NPs are administered, they have to cross various hurdles before they reach the There has been an increase in studies exploring intradermal delivery using microneedles as it is minimally invasive. 172 Dur et al. 173 

In vitro studies can guide one in explaining the effects of NPs in vivo. For instance, ROS and RNS generated in response to NPs activates the cells and induces secretion of cytokines/chemokines. (Fig. 4) This leads further to the infiltration of immune cells, which may cause tissue necrosis or induce apoptosis of cells causing organ damage. Thus, the immune response to the NPs can lead to a cascade of events that induces toxicity. In vivo, toxicity is determined by assessing ultrastructural changes in the tissues (Fig.3) , comparing cytokine levels and other molecular markers in serum and analyzing blood cell counts (hematology) 171 injection. 168 PEI-FeONPs at 5 and 2.5 mg/kg doses were highly toxic to mice leading to death, but a dose of 1.5 mg/kg was well tolerated. 99 J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f This may also lead to metabolic changes. Reactive nitrate species (RNS) produced mainly by immune cells such as macrophages and neutrophils along with the ROS are considered to be indicators of cellular activation. Altogether, the intracellular changes may cause cellular toxicity (iv) and cause an immune response by inducing changes in cytokine and chemokines secretion (v). NPs can also be exocytosed in vesicles called exosomes which may be inherently targeted to different tissues. Therefore, a cascade of events determines the bioavailability, clearance, toxicity profile and thus, the net effect of NPs. 

Analyzing nanomaterial bioconjugates: a review of current and emerging purification and characterization techniques

Functionalizing nanoparticles with biological molecules: developing chemistries that facilitate nanotechnology

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Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-beta Conformation

Cationic Lipid-Coated Gold Nanoparticles as Efficient and Non-Cytotoxic Intracellular siRNA Delivery Vehicles

Polymeric Liposomes-Coated Superparamagnetic Iron Oxide Nanoparticles as Contrast Agent for Targeted Magnetic Resonance Imaging of Cancer Cells

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MRI Guided Magnetochemotherapy with High-Magnetic-Moment Iron Oxide Nanoparticles for Cancer Theranostics

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Synthesis and characterization of superparamagnetic iron oxide nanoparticles as calcium-responsive MRI contrast agents

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Poly(ethylene glycol)-lipid conjugates regulate the calciuminduced fusion of liposomes composed of phosphatidylethanolamine and phosphatidylserine

Silicon Quantum Dots Metal-Enhanced Photoluminescence by Gold Nanoparticles in Colloidal Ensembles: Effect of Surface Coating

Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer

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Penetratin Peptide-Functionalized Gold Nanostars: Enhanced BBB Permeability and NIR Photothermal Treatment of Alzheimer's Disease Using Ultralow Irradiance

Biocompatible Peptide-Coated Ultrasmall Superparamagnetic Iron Oxide Nanoparticles for In Vivo Contrast-Enhanced Magnetic Resonance Imaging

A Study of the Cellular Uptake of Magnetic Branched Amphiphilic Peptide Capsules

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Bovine Serum Albumin (BSA) coated iron oxide magnetic nanoparticles as biocompatible carriers for curcumin-anticancer drug

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Acquired Resistance to Antibody-Drug Conjugates

Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

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A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy

Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer

DNA-Grafted Poly(acrylic acid) for One-Step DNA Functionalization of Iron Oxide Nanoparticles

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Nucleic acid and nucleotide-mediated synthesis of inorganic nanoparticles

Gold Nanoparticle-Aptamer-Based LSPR Sensing of Ochratoxin A at a Widened Detection Range by Double Calibration Curve Method

Aptamer-modified gold nanoparticles for colorimetric determination of platelet-derived growth factors and their receptors

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Biodistribution and toxicity of engineered gold nanoparticles: a review of in vitro and in vivo studies

Gold nanoparticles: Distribution, bioaccumulation and toxicity. In vitro and in vivo studies

Assessing cell-nanoparticle interactions by high content imaging of biocompatible iron oxide nanoparticles as potential contrast agents for magnetic resonance imaging

Natural tripeptide capped pH-sensitive gold nanoparticles for efficacious doxorubicin delivery both in vitro and in vivo

Effect of Surface Chemistry and Associated Protein Corona on the Long-Term Biodegradation of Iron Oxide Nanoparticles In Vivo

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Uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes and coatings

Cellular uptake of nanoparticles: journey inside the cell

Parametrizing the exposure of superparamagnetic iron oxide nanoparticles in cell cultures at different in vitro environments

Effects of Cell Culture Media on the Dynamic Formation of Protein-Nanoparticle Complexes and Influence on the Cellular Response

Functionalized Gold Nanoparticles as Biosensors for Monitoring Cellular Uptake and Localization in Normal and Tumor Prostatic Cells

Selection of potential iron oxide nanoparticles for breast cancer treatment based on in vitro cytotoxicity and cellular uptake

Nanoparticle administration method in cell culture alters particle-cell interaction

Evaluating the effect of assay preparation on the uptake of gold nanoparticles by RAW264.7 cells

Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues

Gold quantum dots impair the tumorigenic potential of glioma stem-like cells via beta-catenin downregulation in vitro

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Sandwich-Cultured Hepatocytes as a Tool to Study Drug Disposition and Drug-Induced Liver Injury

Is it time to reinvent basic cell culture medium?

Protein Corona Composition of Superparamagnetic Iron Oxide Nanoparticles with Various Physico-Chemical Properties and Coatings

Effect of cell media on polymer coated superparamagnetic iron oxide nanoparticles (SPIONs): Colloidal stability, cytotoxicity, and cellular uptake studies

How Entanglement of Different Physicochemical Properties Complicates the Prediction of in Vitro and in Vivo Interactions of Gold Nanoparticles

Intracellular Accumulation of Gold Nanoparticles Leads to Inhibition of Macropinocytosis to Reduce the Endoplasmic Reticulum Stress

Evaluation of DNA interaction, genotoxicity and oxidative stress induced by iron oxide nanoparticles both in vitro and in vivo: attenuation by thymoquinone

Cytotoxicity and Cell Cycle Effects of Bare and Poly(vinyl alcohol)-Coated Iron Oxide Nanoparticles in Mouse Fibroblasts

Bare surface of gold nanoparticle induces inflammation through unfolding of plasma fibrinogen

Functionalized gold nanoparticles for topical delivery of methotrexate for the possible treatment of psoriasis

Endocytosis of nanomedicines

Physical Principles of Nanoparticle Cellular Endocytosis

Uptake mechanisms of non-viral gene delivery

Uptake mechanism of metabolic-targeted gold nanoparticles

Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles

Dynasore -not just a dynamin inhibitor

Caveolin-1 and CDC42 mediated endocytosis of silica-coated iron oxide nanoparticles in HeLa cells

Increased cellular uptake of peptide-modified PEGylated gold nanoparticles. Biochemical and Biophysical Research Communications

Mechanisms for cellular uptake of nanosized clinical MRI contrast agents

Effects of Gold Nanoparticles with Different Surface Charges on Cellular Internalization and Cytokine Responses in Monocytes

Differential internalization of brick shaped iron oxide nanoparticles by endothelial cells

Interactions at the cell membrane and pathways of internalization of nano-sized materials for nanomedicine

The gene transfection and endocytic uptake pathways mediated by PEGylated PEI-entrapped gold nanoparticles

Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis

Mechanisms of Nanoparticle-Induced Oxidative Stress and Toxicity

Iron-mediated lipid peroxidation and lipid raft disruption in low-dose silica-induced macrophage cytokine production. Free Radical Biology and Medicine

Gold Nanoparticles as a Vaccine Platform: Influence of Size and Shape on Immunological Responses in Vitro and in Vivo

Gold nanoparticles as an adjuvant: Influence of size, shape, and technique of combination with CpG on antibody production

Iron Oxide Nanoparticles-Based Vaccine Delivery for Cancer Treatment

Physical characterization and in vivo organ distribution of coated iron oxide nanoparticles

Administration of Substances to Laboratory Animals: Routes of Administration and Factors to Consider

Routes of Administration

Innate immune response in Th1-and Th2-dominant mouse strains

Evaluating the potential of gold, silver, and silica nanoparticles to saturate mononuclear phagocytic system tissues under repeat dosing conditions

Effect of surface-modified superparamagnetic iron oxide nanoparticles (SPIONS) on mast cell infiltration: An acute in vivo study

Effect of removing Kupffer cells on nanoparticle tumor delivery

Biodistribution, Clearance And Morphological Alterations Of Intravenously Administered Iron Oxide Nanoparticles In Male Wistar Rats

PEG-copolymer-coated iron oxide nanoparticles that avoid the reticuloendothelial system and act as kidney MRI contrast agents

Biodegradable Gold Nanoclusters with Improved Excretion Due to pH-Triggered Hydrophobic-to-Hydrophilic Transition

Effect of Gold Nanoparticle Size on Their Properties as Contrast Agents for Computed Tomography

Comparisons of the biodistribution and toxicological examinations after repeated intravenous administration of silver and gold nanoparticles in mice

Effects of core size and PEG coating layer of iron oxide nanoparticles on the distribution and metabolism in mice

Intradermal delivery of vaccine nanoparticles using hollow microneedle array generates enhanced and balanced immune response

Conjugation of a peptide autoantigen to gold nanoparticles for intradermally administered antigen specific immunotherapy

The toxicity and distribution of iron oxide-zinc oxide core-shell nanoparticles in C57BL/6 mice after repeated subcutaneous administration

Accumulation of biosynthesized gold nanoparticles and its impact on various organs of Sprague Dawley rats: a systematic study

Nanoparticle-based vaccines: opportunities and limitations

The Physiological Sources of, Clinical Significance of, and Laboratory-Testing Methods for Determining Enzyme Levels. Labmedicine

Size-and cell type-dependent cellular uptake, cytotoxicity and in vivo distribution of gold nanoparticles

Biodistribution, pharmacokinetics, and toxicity of dendrimer-coated iron oxide nanoparticles in BALB/c mice

This manuscript is contribution number 20-333-J from the Kansas Agricultural Experiment