Cellular Uptake Of Gold Nanoparticles Research Articles

Identifying factors controlling cellular uptake of gold nanoparticles by machine learning

There is strong interest to improve the therapeutic potential of gold nanoparticles (GNPs) while ensuring their safe development. The utility of GNPs in medicine requires a molecular-level understanding of how GNPs interact with biological systems. Despite considerable research efforts devoted to monitoring the internalisation of GNPs, there is still insufficient understanding of the factors responsible for the variability in GNP uptake in different cell types. Data-driven models are useful for identifying the sources of this variability. Here, we trained multiple machine learning models on 2077 data points for 193 individual nanoparticles from 59 independent studies to predict cellular uptake level of GNPs and compared different algorithms for their efficacies of prediction. The five ensemble learners (Xgboost, random forest, bootstrap aggregation, gradient boosting, light gradient boosting machine) made the best predictions of GNP uptake, accounting for 80–90% of the variance in the test data. The models identified particle size, zeta potential, GNP concentration and exposure duration as the most important drivers of cellular uptake. We expect this proof-of-concept study will foster the more effective use of accumulated cellular uptake data for GNPs and minimise any methodological bias in individual studies that may lead to under- or over-estimation of cellular internalisation rates.

Pre-exposure to titanium or iron oxide nanoparticles suppresses the subsequent cellular uptake of gold nanoparticles

Humans are exposed to a wide variety of natural and engineered nanoparticles (NPs) during their lifetime. However, the effects of pre-exposure to NPs on subsequent uptake of other NPs have not been investigated. In the present study, we investigated the effects of pre-exposure to three NPs (TiO2, Fe2O3, and SiO2 NPs) on the subsequent uptake of gold NPs (AuNPs) by hepatocellular carcinoma cells (HepG2). When HepG2 cells were pre-exposed to TiO2 or Fe2O3 NPs, but not SiO2 NPs for 2 days, their subsequent uptake of AuNPs was inhibited. Such inhibition was also observed in human cervical cancer (HeLa) cells, suggesting that this phenomenon is present in different cell types. The mechanisms underlying the inhibitory effect of NP pre-exposure include altered plasma membrane fluidity due to changes in lipid metabolism and reduced intracellular ATP production due to decreased intracellular oxygen. Despite the inhibitory effects of NP pre-exposure, full recovery was observed after transferring the cells to medium without NPs, even when the pre-exposure time was extended from 2 days to 2 weeks. Overall, the pre-exposure effects observed in the present study should be considered in the biological application and risk evaluation of NPs.

Laser Ablated Albumin Functionalized Spherical Gold Nanoparticles Indicated for Stem Cell Tracking.

Cell tracking in cell-based therapy applications helps distinguish cell participation among paracrine effect, neovascularization, and matrix deposition. This preliminary study examined the cellular uptake of gold nanoparticles (AuNPs), observing cytotoxicity and uptake of different sizes and AuNPs concentrations in Adipose-derived stromal cells (ASCs). ASCs were incubated for 24 h with Laser ablated Albumin functionalized spherical AuNPs (LA-AuNPs), with average sizes of 2 nm and 53 nm in diameter, in four concentrations, 127 µM, 84 µM, 42 µM, and 23 µM. Cytotoxicity was examined by Live/Dead assay, and erythrocyte hemolysis, and the effect on the cytoskeleton was investigated by immunocytochemistry for β-actin. The LA-AuNPs were internalized by the ASCs in a size and concentration-dependent manner. Clusters were observed as dispersed small ones in the cytosol, and as a sizeable perinuclear cluster, without significant harmful effects on the cells for up to 2 weeks. The Live/Dead and hemolysis percentage results complemented the observations that the larger 53 nm LA-AuNPs in the highest concentrated solution significantly lowered cell viability. The demonstrated safety, cellular uptake, and labelling persistency with LA-AuNPs, synthesized without the combination of chemical solutions, support their use for cell tracking in tissue engineering applications.

Glycocalyx Components Detune the Cellular Uptake of Gold Nanoparticles in a Size- and Charge-Dependent Manner.

Functionalized nanoparticles (NPs) are widely used in targeted drug delivery and biomedical imaging due to their penetration into living cells. The outer coating of most cells is a sugar-rich layer of the cellular glycocalyx, presumably playing an important part in any uptake processes. However, the exact role of the cellular glycocalyx in NP uptake is still uncovered. Here, we in situ monitored the cellular uptake of gold NPs─functionalized with positively charged alkaline thiol (TMA)─into adhered cancer cells with or without preliminary glycocalyx digestion. Proteoglycan (PG) components of the glycocalyx were treated by the chondroitinase ABC enzyme. It acts on chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate and slowly on hyaluronate. The uptake measurements of HeLa cells were performed by applying a high-throughput label-free optical biosensor based on resonant waveguide gratings. The positively charged gold NPs were used with different sizes [d = 2.6, 4.2, and 7.0 nm, small (S), medium (M), and large(L), respectively]. Negatively charged citrate-capped tannic acid (CTA, d = 5.5 nm) NPs were also used in control experiments. Real-time biosensor data confirmed the cellular uptake of the functionalized NPs, which was visually proved by transmission electron microscopy. It was found that the enzymatic digestion facilitated the entry of the positively charged S- and M-sized NPs, being more pronounced for the M-sized. Other enzymes digesting different components of the glycocalyx were also employed, and the results were compared. Glycosaminoglycan digesting heparinase III treatment also increased, while glycoprotein and glycolipid modifying neuraminidase decreased the NP uptake by HeLa cells. This suggests that the sialic acid residues increase, while heparan sulfate decreases the uptake of positively charged NPs. Our results raise the hypothesis that cellular uptake of 2-4 nm positively charged NPs is facilitated by glycoprotein and glycolipid components of the glycocalyx but inhibited by PGs.

Vibratome sectioning of tumors to evaluate the interactions between nanoparticles and the tumor microenvironment ex-vivo.

Nanoparticles have been investigated as drug carriers and promising agents for cancer therapy. However, the tumor microenvironment (TME), which is formed by the tumor, is considered a barrier for nanocarriers to enter the internal tumor tissue. Therefore, the evaluation of the biological distribution of nanocarriers in TME can provide useful information on their role in tumor-targeted drug delivery. Although the tumor-bearing mouse model is commonly used to investigate the distribution of nanocarriers in the TME, there is currently a lack of a testing system to predict the distribution of nanocarriers in tumor tissues, especially in patients. This study revealed that the macrophages and dendritic cells (DCs) were more distributed in the peripheral part than the central part of the tumor, which might be an obstacle to the uniform distribution of nanoparticles in the tumor. In addition, the cellular uptake of gold nanoparticles (AuNR and AuNS) in macrophages and DCs cell lines (RAW264.7 and DC1.2) was markedly different from that in the TME. Hence, the study model of the interaction between nanoparticles and macrophages and DCs has an important impact on the accuracy of the results. The vibratome sections of tumor tissues preserved the spatial distribution of immune cells and tumor cells, and had very little effects on their morphologies and activities. More importantly, we found that the distribution of nanocarriers in vibratome sections was similar to that in tumors in vivo. In all, ex vivo analysis using vibratome sections of tumor tissues provides a more convenient and stable method for elucidating the influences of TME on the distribution of nanocarriers.

A Cell-Culture Technique to Encode Glyco-Nanoparticles Selectivity.

Nanoparticles (NPs) embedded with bioactive ligands such as carbohydrates, peptides, and nucleic acid have emerged as a potential tool to target biological processes. Traditional in vitro assays performed under statistic conditions may result in non-specific outcome sometimes, mainly because of the sedimentation and self-assembly nature of NPs. Inverted cell-culture assay allows for flexible and accurate detection of the receptor-mediated uptake and cytotoxicity of NPs. By combining this technique with glyco-gold nanoparticles, cellular internalization and cytotoxicity were investigated. Regioselective glycosylation patterns and shapes of the NPs could tune the receptors' binding affinity, resulting in precise cellular uptake of gold nanoparticles (AuNPs). Two cell lines HepG2 and HeLa were probed with galactosamine-embedded fluorescent AuNPs, revealing significant differences in cytotoxicity and uptake mechanism in upright and invert in vitro cell-culture assay, high-specificity toward uptake, and allowing for a rapid screening and optimization technique.

Effect of the Ligand Binding Strength on the Morphology of Functionalized Gold Nanoparticles.

Functionalized gold nanoparticles are investigated by density functional theory calculations in the context of cancer radiotherapy. Several typical experimental shapes, including nanostars, nanospheres, and nanorods, are modeled by optimizing Au clusters covered by organic monolayers composed of hydrated short-chain polyethylene glycol (PEG) ligands. The PEGylation stabilizes significantly the stellation of decahedral Au54 by deforming significantly its geometry at the spikes. The higher stability of the PEG molecules adsorbed on this stellated nanocluster with respect to the more spherical icosahedral Au55 and truncated octahedral Au79 leads to a larger energy cost to desorb them and thus a weaker propensity for the starred nanoparticle to exchange ligands with the cell membrane, in agreement with experiments. These results open interesting possibilities for advancing our understanding of the cellular uptake of gold nanoparticles.

Size, Surface Functionalization, and Genotoxicity of Gold Nanoparticles In Vitro.

Several studies suggested that gold nanoparticles (NPs) could be genotoxic in vitro and in vivo. However, gold NPs have currently produced present a wide range of sizes and functionalization, which could affect their interactions with the environment or with biological structures and, thus, modify their toxic effects. In this study, we investigated the role of surface charge in determining the genotoxic potential of gold NPs, as measured by the induction of DNA damage (comet assay) and chromosomal damage (micronucleus assay) in human bronchial epithelial BEAS-2B cells. The cellular uptake of gold NPs was assessed by hyperspectral imaging. Two core sizes (~5 nm and ~20 nm) and three functionalizations representing negative (carboxylate), positive (ammonium), and neutral (poly(ethylene glycol); (PEG)ylated) surface charges were examined. Cationic ammonium gold NPs were clearly more cytotoxic than their anionic and neutral counterparts, but genotoxicity was not simply dependent on functionalization or size, since DNA damage was induced by 20-nm ammonium and PEGylated gold NPs, while micronucleus induction was increased by 5-nm ammonium and 20-nm PEGylated gold NPs. The 5-nm carboxylated gold NPs were not genotoxic, and evidence on the genotoxicity of the 20-nm carboxylated gold NPs was restricted to a positive result at the lowest dose in the micronucleus assay. When interpreting the results, it has to be taken into account that cytotoxicity limited the doses available for the ammonium-functionalized gold NPs and that gold NPs have earlier been described to interfere with the comet assay procedure, possibly resulting in a false positive result. In conclusion, our findings show that the cellular uptake and cytotoxicity of gold NPs are clearly enhanced by positive surface charge, but neither functionalization nor size can single-handedly account for the genotoxic effects of the gold NPs.

Cellular Uptake of Gold Nanoparticles and Their Movement in 3D Multicellular Tumor Spheroids: Effect of Molecular Weight and Grafting Density of Poly(2-hydroxyl ethyl acrylate).

It is known that the size of gold nanoparticles (GNPs) is not the only determining factor in the uptake by cells such as cancer cells. The surface functionalization plays a crucial role, in particular the nature of the ligand as well as the molecular weight and the grafting density. Here, poly(2-hydroxy ethyl) acrylate (pHEA) with molecular weights ranging from 10, 20 to 39 g mol-1 via reversible addition-fragmentation chain transfer polymerization is synthesized. These polymers are used directly to coat GNPs with sizes of 20, 40, and 70 nm as the trithiocarbonate functionality can strongly bind to the gold surface. The library of nine GNP is found to be nontoxic against lung carcinoma cells A549 and has negligible albumin protein absorption as determined by quartz crystal microbalance. Laser scanning confocal microscopy and flow cytometry reveal that GNP coated with medium length pHEA displays the highest cellular uptake while the effect of the size is not statistically significant. In contrast, multicellular tumor spheroids, which is a 3D model that simulates the tissue, enable the penetration of GNP coated with the longest pHEA chain while it also appears that smaller GNPs have now a clear advantage.

Improving the Flow Cytometry-based Detection of the Cellular Uptake of Gold Nanoparticles

Due to the considerable amount of applications of gold nanoparticles (AuNPs) in biological systems, there is a great need for an improved methodology to quantitatively measure the uptake of AuNPs in cells. Flow cytometry has the ability to measure intracellular AuNPs by collecting the light scattering from a large population of live cells through efficient single cell analysis. Traditionally, the side scattering setting of the flow cytometer, which is associated with a 488 nm excitation laser (SSC channel), is used to detect nanoparticle uptake. This method is limited as AuNPs do not have the optimized response when excited with this laser. Here, we reported that the use of more red-shifted excitation lasers will greatly enhance the optical signal needed for the flow cytometry-based detection of AuNSs (26 nm in diameter) and AuNRs (67 nm × 33 nm, length × width) uptake in triple negative breast cancer cells (MDA-MB-231).

Mixed Fluorinated/Hydrogenated Self-Assembled Monolayer-Protected Gold Nanoparticles: In Silico and In Vitro Behavior.

Gold nanoparticles (AuNPs) covered with mixtures of immiscible ligands present potentially anisotropic surfaces that can modulate their interactions at complex nano-bio interfaces. Mixed, self-assembled, monolayer (SAM)-protected AuNPs, prepared with incompatible hydrocarbon and fluorocarbon amphiphilic ligands, are used here to probe the molecular basis of surface phase separation and disclose the role of fluorinated ligands on the interaction with lipid model membranes and cells, by integrating in silico and experimental approaches. These results indicate that the presence of fluorinated amphiphilic ligands enhances the membrane binding ability and cellular uptake of gold nanoparticles with respect to those coated only with hydrogenated amphiphilic ligands. For mixed monolayers, computational results suggest that ligand phase separation occurs on the gold surface, and the resulting anisotropy affects the number of contacts and adhesion energies with a membrane bilayer. This reflects in a diverse membrane interaction for NPs with different surface morphologies, as determined by surface plasmon resonance, as well as differential effects on cells, as observed by flow cytometry and confocal microscopy. Overall, limited changes in monolayer features can significantly affect NP surface interfacial properties, which, in turn, affect the interaction of SAM-AuNPs with cellular membranes and subsequent effects on cells.

Safety considerations for nanoparticles in tumor treatment.

Safety considerations for nanoparticles in tumor treatment.

Evaluating cellular uptake of gold nanoparticles in HL-7702 and HepG2 cells for plasmonic photothermal therapy.

A novel methodology is provided to quantitatively measure the gold (Au) mass internalized in a cell for effective implementation of plasmonic photothermal therapy (PPTT). The cellular uptake of 4-mercaptobenzoic acid-labeled Au nanoparticles (NPs) is investigated via Raman mapping and inductively coupled plasma-mass spectrometry and the efficiency of in vitro PPTT is evaluated. The cellular uptake is strongly affected by the size of the Au NPs, concentration of the Au NPs, incubation time and cell type. By optimizing the experimental parameters, the results show that a significant damage is caused to the HepG2 cells and slight harm is caused to the HL-7702 cells during PPTT. This demonstrates a high potential for developing effective photothermal therapy for tumor tissues.

Cellular Uptake of Gold Nanoparticles Triggered by Host-Guest Interactions.

We describe an approach to regulate the cellular uptake of small gold nanoparticles using supramolecular chemistry. The strategy relies on the functionalization of AuNPs with negatively charged pyranines, which largely hamper their penetration in cells. Cellular uptake can be activated in situ through the addition of cationic covalent cages that specifically recognize the fluorescent pyranine dyes and counterbalance the negative charges. The high selectivity and reversibility of the host-guest recognition activates cellular uptake, even in protein-rich biological media, as well as its regulation by rational addition of either cage or pyranine.

Dose enhancement effects of gold nanoparticles specifically targeting RNA in breast cancer cells.

Localization microscopy has shown to be capable of systematic investigations on the arrangement and counting of cellular uptake of gold nanoparticles (GNP) with nanometer resolution. In this article, we show that the application of specially modified RNA targeting gold nanoparticles (“SmartFlares”) can result in ring like shaped GNP arrangements around the cell nucleus. Transmission electron microscopy revealed GNP accumulation in vicinity to the intracellular membrane structures including them of the endoplasmatic reticulum. A quantification of the radio therapeutic dose enhancement as a proof of principle was conducted with γH2AX foci analysis: The application of both—SmartFlares and unmodified GNPs—lead to a significant dose enhancement with a factor of up to 1.2 times the dose deposition compared to non-treated breast cancer cells. This enhancement effect was even more pronounced for SmartFlares. Furthermore, it was shown that a magnetic field of 1 Tesla simultaneously applied during irradiation has no detectable influence on neither the structure nor the dose enhancement dealt by gold nanoparticles.

Cellular processing of gold nanoparticles: CE-ICP-MS evidence for the speciation changes in human cytosol

The cellular uptake of gold nanoparticles (AuNPs) may (or may not) affect their speciation, but information on the chemical forms in which the particles exist in the cell remains obscure. An analytical method based on the use of capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) has been proposed to shed light on the intracellular processing of AuNPs. It was observed that when being introduced into normal cytosol, the conjugates of 10–50 nm AuNPs with albumin evolved in human serum stayed intact. On the contrary, under simulated cancer cytosol conditions, the nanoconjugates underwent decomposition, the rate of which and the resulting metal speciation patterns were strongly influenced by particle size. The new peaks that appeared in ICP-MS electropherograms could be ascribed to nanosized species, as upon ultracentrifugation, they quantitatively precipitated whereas the supernatant showed only trace Au signals. Our present study is the first step to unravel a mystery of the cellular chemistry for metal-based nanomedicines.

Engineering of pectin-reduced gold nanoparticles for targeted delivery of an antiviral drug to macrophages: in vitro and in vivo assessment

In this study, we have attempted one pot green synthesis of non-cytotoxic gold nanoparticles (AuNPs). The formation of pectin (PEC)-reduced and stabilized gold nanoparticles was ascertained by monitoring surface plasmon resonance (SPR) band. The as-synthesized gold nanoparticles demonstrated SPR band at 518 nm, having a spherical shape with a particle size of 13 nm and zeta potential value of − 34.17 mV. The pectin-reduced gold nanoparticles (PEC-AuNPs) showed excellent stability under varying pH and electrolyte conditions. The potential of PEC-AuNPs as a carrier of antiretroviral drug zidovudine (AZT) was explored. In vitro cellular uptake of gold nanoparticles by macrophages was visualized by confocal microscopy and quantified by atomic absorption spectroscopy. The in vivo tissue distribution of PEC-AuNPs was carried out in Wistar rats to evaluate its potential to target HIV reservoir sites. These finding could be further utilized for imaging and cancer therapy using tumor-associated macrophages.

The Effect of shape on Cellular Uptake of Gold Nanoparticles in the forms of Stars, Rods, and Triangles

Gold nanomaterials have attracted considerable interest as vehicles for intracellular drug delivery. In our study, we synthesized three different shapes of methylpolyethylene glycol coated-anisotropic gold nanoparticles: stars, rods, and triangles. The cellular internalization of these nanoparticles by RAW264.7 cells was analyzed, providing a parametric evaluation of the effect of shape. The efficiency of cellular uptake of the gold nanoparticles was found to rank in the following order from lowest to highest: stars, rods, and triangles. The possible mechanisms of cellular uptake for the three types of gold nanoparticles were examined, and it was found that different shapes tended to use the various endocytosis pathways in different proportions. Our study, which has demonstrated that shape can modulate the uptake of nanoparticles into RAW264.7 cells and that triangles were the shape with the most efficient cellular uptake, provides useful guidance toward the design of nanomaterials for drug delivery.

Multiparametric Assessment of Gold Nanoparticle Cytotoxicity in Cancerous and Healthy Cells: The Role of Size, Shape, and Surface Chemistry.

In recent years, we and others have become interested in evaluating the use of surface-enhanced Raman scattering (SERS) tags for early cancer detection and in designing new approaches to demonstrate the applicability of this spectroscopic technique in the clinic. SERS-based imaging in particular offers ultra sensitivity up to the single molecule, multiplexing capability, and increased photostability and has been shown to outperform fluorescence. However, to employ SERS tags for early cancer detection, it is important to understand their interaction with cells and determine their cytotoxicity. We have been particularly interested for quite some time in determining if and how gold nanostars, which have been demonstrated as outstanding SERS enhancing substrates, can be safely employed in living systems and translated to the clinic. In this study, we carried out a multiparametric in vitro study to look at the cytotoxicity and cellular uptake of gold nanoparticles on human glioblastoma and human dermal fibroblast cell lines. Cytotoxicity was evaluated by incubating cells with three different morphologies of AuNPs, namely nanospheres, nanorods, and nanostars, each having three different surface chemistries (cetyltrimethylammonium bromide (CTAB), poly(ethylene glycol) (PEG), and human serum albumin (HSA)). Our results showed that the surface chemistry of the nanoparticles had predominant effects on cytotoxicity, and the morphology and size of the nanoparticles only slightly affected cell viability. CTAB-coated particles were found to be the most toxic to cells, and PEGylated nanostars were determined to be the least toxic. Caspase-3 assay and LDH assay revealed that cell death occurs via apoptosis for cancerous cells and via necrosis for healthy ones. Cellular uptake studies carried out via TEM showed that the particles retain their shape even at long incubation times, which may be beneficial for in vivo SERS-based disease detection. Overall, this study provides valuable information on gold-nanoparticle-induced cytotoxicity that can be leveraged for the development of safe and effective nanoparticle-based therapeutic and diagnostic systems.

Interaction of gold nanoparticles and nickel(II) sulfate affects dendritic cell maturation

Despite many investigations have focused on the pristine toxicity of gold nanoparticles (GNPs), little is known about the outcome of co-exposure and interaction of GNPs with heavy metals which can possibly detoxify or potentiate them. Here, the combined exposure of nickel (II) sulfate (NiSO4) and GNPs on the maturation response of dendritic cells (DCs) was explored. Exposure to GNPs or NiSO4 separately induced cell activation. When cells were exposed to a mixture of both, however, the observed cell activation pattern indicated a competitive rather than an additive effect of both inducers with levels similar to those induced by NiSO4 alone. Quantification of the GNP uptake by DCs demonstrated a significant decrease in intracellular gold content during co-incubation with NiSO4. An extensive physiochemical characterization was performed to determine the interaction between GNPs and NiSO4 in the complex physiological media using nanoparticle tracking analyses, disc centrifugation, UV–visible spectroscopy, ICP-MS analyses, zeta potential measurements, electron microscopy, and proteomics. Although GNPs and NiSO4 did not directly interact with each other, the presence of NiSO4 in the physiological media resulted in changes in GNPs' charge and their associated protein corona (content and composition), which may contribute to a decreased cellular uptake of GNPs and sustaining the nickel-induced DC maturation. The presented results provide new insights in the interaction of heavy metals and NPs in complex physiological media. Moreover, this study highlights the necessity of mixture toxicology, since these combined exposures are highly relevant for human subjection to NPs and risk assessment of nanomaterials.