Amino-functionalized Polystyrene Nanoparticles Research Articles

Engineered Nanoparticles as Potential Therapeutics for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignancy associated with poor prognosis. Particularly, older patients suffer greatly from the standard chemotherapy and have a 5‐year survival of only 4%. Hence, new therapeutic agents with higher specificity and lower general cytotoxicity are urgently required.We have discovered that positively charged amino‐functionalized polystyrene nanoparticles (NP‐PS+) induce accumulation of acidic vesicular organelles with elevated pH and impaired processing of procathepsin B leading to mTOR inhibition, activation of autophagy, and induction of caspase‐dependent apoptosis in leukemia cells, but not in normal human macrophages. The antileukemic effect of NP‐PS+ was also preserved in vivo, where NP‐PS+ inhibited proliferation and induced apoptosis in leukemia xenografts grown on chick chorioallantoic membranes.Similar to polystyrene particles, amino‐functionalized gold nanoparticles (NP‐Au+) exhibited selective cytotoxicity towards AML cell lines as well as primary patient‐derived AML cells. Thus, NP‐Au+ particles induced cell death in primary human leukemia cells and reduced their colony‐forming potential, whereas normal hematopoietic cells remained unaffected by the treatment with NP‐Au+. NP‐Au+ targeted specifically the oxidative mitochondrial respiration, which is, different to normal hematopoietic cells, the main source of energy production in AML blasts and leukemic stem cells. Different to NP‐Au+, conventional chemotherapeutics such ascytarabine act in a cell cycle‐dependent manner and target only on proliferating AML blasts but not on quiescent leukemic stem cells. In agreement with the in vitro data, NP‐Au+ exhibited antileukemic efficacy against primary human AML xenografted into mice applied either as monotherapy or as a cytarabine combination regimen in the absence of detectable adverse events.Thus, this engineered nanomaterial that targets particularly resistant quiescent leukemic stem cells holds great promise as a novel nanotherapeutic for the treatment of acute myeloid leukemia independent of its cytogenetic profile.

Single and combined toxicity of amino-functionalized polystyrene nanoparticles with potassium dichromate and copper sulfate on brine shrimp Artemia franciscana larvae.

The increasing use and disposal of plastics has become a persistent problem in the marine environment, calling for studies that refer to realistic scenarios to understand their effects on biota. Particularly, the understanding about the effects of co-exposure with nanoplastic particles and metals on aquatic organisms is still limited. The present work aimed to investigate the acute toxicity of amino-functionalized polystyrene nanoparticles (PS-NH2; 50 nm) as proxy for nanoplastics on brine shrimp Artemia franciscana larvae under different culture conditions and at different stages of development, as well as the combined effect with two reference toxicants - potassium dichromate (K2Cr2O7) and copper sulfate (CuSO4). Nauplii (instar II or III larval stages) were exposed to different concentrations of PS-NH2 (0.005 to 5 μg mL-1) for up to 48 h, with or without agitation in order to mimic a more realistic environmental scenario. Larval mobility and PS-NH2 accumulation were monitored under microscopy. PS-NH2 alone showed toxicity only at the highest concentration tested (5 μg mL-1) regardless the incubation method used (61.2 + 3.1% and 65.0 + 4.5% with and without agitation, respectively). Moreover, instar III stage was the most sensitive to PS-NH2 exposure (38.2% immobility in 24 h of exposure; 5 μg mL-1). Evidence of PS-NH2 retention in the gastrointestinal tract in a concentration- and time-dependent manner was also obtained. Mixtures of PS-NH2 (0.005 and 5 μg mL-1) with different concentrations of K2Cr2O7 increased the immobilization rate of the larvae after 48 h of exposure, when compared to the K2Cr2O7 alone. Similar results were observed for CuSO4 in the co-exposure conditions at different concentrations. However, exposing nauplii to a mixture of PS-NH2 (0.005 μg mL-1) and CuSO4 decreased immobilization rate, in comparison to the group exposed to CuSO4 alone. The present work highlights the potential risk posed by nanoplastics to zooplanktonic species through their interaction with other toxicants.

Nanoparticles as potential therapeutics for acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is associated with an unfavourable prognosis. Hence, new therapeutic agents with higher specificity and lower general cytotoxicity are urgently required. We synthesized biocompatible engineered nanoparticles and used various physico-chemical methods for their characterization. Cell viability was assessed by analysis of activities of mitochondrial dehydrogenases and colony-formation assay. Intracellular targets were identified by Western immunoblotting, fluorescent microscopy, flow cytometry, and various functional tests. We have previously shown that amino-functionalized polystyrene nanoparticles (PS-NH2) induce autophagy in leukemia cells and are selectively cytotoxic to leukemia cells compared to human macrophages and PBMC. We created now biocompatible amino-functionalized nanoparticles with an inert metallic gold core, Au-NH2, which were selectively highly cytotoxic towards different AML cell lines and PBMC from acute myeloid leukemia patients, but not to PBMC from healthy donors. We show that in leukemia cells, Au-NH2 treatment induces BID cleavage to tBID, increases mitochondrial superoxide levels, inhibits activity of complexes I, III, IV of the mitochondrial electron transport chain, and induces loss of the mitochondrial membrane potential as well as cytochrome c release from the mitochondrial intermembrane space and caspase 3 activation. Likewise, Au-NH2 treatment induces a rapid decrease in mTOR pathway activity, induces lysosomal rupture in THP-1 and HL-60 leukemia cells but not so in macrophages and PBMC. Thus, this engineered material holds great promise as a novel nanotherapeutic for treatment of acute myeloid leukemia independent of cytogenetic profile.

Metabolomic method to detect a metabolite corona on amino-functionalized polystyrene nanoparticles

Protein coronas on nanoparticles (NPs) affect their physicochemical properties, cellular uptake, and toxicity, and have been described extensively. To date, studies of the occurrence of small molecule (metabolite) coronas are limited. We sought to determine whether a metabolite corona forms on NPs, using high-sensitivity metabolomics combined with a model system for freshwater ecotoxicology (Daphnia magna feeding on Chlorella vulgaris). Using amino-functionalized polystyrene NPs (NH2-pNPs), we showed the impact of this material on Daphnia feeding to provide a rationale for the detailed molecular investigations. We then employed a targeted LC-MS/MS approach for sodium dodecyl sulfate (SDS) as an analog to signaling molecules known to occur in our freshwater model system and optimized a corona extraction method for this representative metabolite. Next, we performed an untargeted discovery-based metabolomics study – using high-sensitivity nanoelectrospray direct infusion mass spectrometry (DIMS) – to enable an unbiased assessment of the metabolite corona of NH2-pNPs in the freshwater model system. Our results demonstrate that SDS was successfully recovered from NH2-pNPs, confirming that the extraction protocol was fit-for-purpose. Untargeted DIMS metabolomics reproducibly detected 100 s of small molecule peaks extracted from NH2-pNPs exposed to conditioned media from the D. magna–C. vulgaris model system. Attempts to annotate these extracted metabolites, including by using van Krevelen and Kendrick Mass Defect plots, indicate a diverse range of metabolites that were not clustered into any particular class. Overall we demonstrate the existence of an ecologically relevant metabolite corona on the surface of NPs through application of a high-sensitivity, untargeted mass spectrometry metabolomics workflow.

A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death

The temporal context of cell death decisions remains generally hidden in ensemble measurements with endpoint readouts. Here, we describe a method to extract event times from fluorescence time traces of cell death-related markers in automated live-cell imaging on single-cell arrays (LISCA) using epithelial A549 lung and Huh7 liver cancer cells as a model system. In pairwise marker combinations, we assess the chronological sequence and delay times of the events lysosomal membrane permeabilization, mitochondrial outer membrane permeabilization and oxidative burst after exposure to 58 nm amino-functionalized polystyrene nanoparticles (PS-NH2 nanoparticles). From two-dimensional event-time scatter plots we infer a lysosomal signal pathway at a low dose of nanoparticles (25 µg mL−1) for both cell lines, while at a higher dose (100 µg mL−1) a mitochondrial pathway coexists in A549 cells, but not in Huh7. In general, event-time correlations provide detailed insights into heterogeneity and interdependencies in signal transmission pathways.

Inhibition of Mitochondrial Respiration by Amino‐Functionalized Gold Nanoparticles in Acute Myeloid Leukemia Cells

In recent years, nanomaterials emerged as versatile tools for biomedical applications. Due to their special physicochemical characteristics, many different types of nanomaterials are under investigation for diagnostic as well as therapeutic purposes. We have previously shown the intrinsic cytotoxic activity of amino‐functionalized polystyrene nanoparticles towards acute myeloid leukemia cells. Here, we developed biocompatible gold nanoparticles with amino‐functionalization and investigated their mechanism of cellular toxicity.Acute myeloid leukemia (AML) is characterized by the proliferation and accumulation of mostly undifferentiated myeloid progenitor cells. In comparison to other leukemias, AML is a disease of the elderly with a mean age at diagnosis of 68 years. Due to severe side effects of the standard therapy, elderly AML patients often receive only palliative care, which results in profoundly reduced survival rates in older patients. Even though, a range of molecular changes in AML has already been investigated in detail, only few therapeutic agents have been approved. One possible explanation for the limited efficacy of targeted agents in AML might be the high level of genetic heterogeneity in myeloid leukemia.For a long time, glycolysis has been considered the major pathway of energy production in cancer cells, also known as the Warburg effect. Recently, however, oxidative phosphorylation emerged as a crucial metabolic pathway for the survival and proliferation of leukemia cells. The high dependence of acute myeloid leukemia cells on oxidative phosphorylation suggests the possibility to target the mitochondrial respiratory chain for antileukemic efficacy.Here, we performed high resolution respirometry using an Oroboros oxygraph and show the enhanced respiratory activity of THP‐1 leukemia cells compared to healthy peripheral mononuclear cells. Furthermore, chromogenic complex activity assays employed to study the effect of gold nanoparticles on respiratory complex activities of isolated mitochondria demonstrated that the amino‐functionalized gold nanoparticles, but not the carboxy‐functionalized control particles, inhibit mitochondrial complex activities. We could further show that such direct inhibitory effect on mitochondrial complex activities also impairs mitochondrial respiration in intact THP‐1 cells. Inhibition of mitochondrial respiration further led to depolarization of the mitochondrial membrane, depletion of ATP, and cell death of THP‐1 cells and primary AML blasts.Hence, our findings confirm the high dependence of AML cells on oxidative phosphorylation. On that basis, our amino‐functionalized gold nanoparticles represent novel nanotherapeutics for a broad application in AML therapy, specifically targeting and inhibiting the sensitive energy production pathway in AML cells.Support or Funding InformationThis work was supported by the Volkswagen Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nanoparticle core stability and surface functionalization drive the mTOR signaling pathway in hepatocellular cell lines

Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization and biodegradability. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH2), but not amino- or hydroxyl-functionalized silica particles, trigger cell death in hepatocellular carcinoma Huh7 cells. Importantly, biodegradability of nanoparticles plays a crucial role in regulation of essential cellular processes. Thus, biodegradable silica nanoparticles having the same shape, size and surface functionalization showed opposite cellular effects in comparison with similar polystyrene nanoparticles. At the molecular level, PS-NH2 obstruct and amino-functionalized silica nanoparticles (Si-NH2) activate the mTOR signalling in Huh7 and HepG2 cells. PS-NH2 induced time-dependent lysosomal destabilization associated with damage of the mitochondrial membrane. Solely in PS-NH2-treated cells, permeabilization of lysosomes preceded cell death. Contrary, Si-NH2 nanoparticles enhanced proliferation of HuH7 and HepG2 cells. Our findings demonstrate complex cellular responses to functionalized nanoparticles and suggest that nanoparticles can be used to control activation of mTOR signaling with subsequent influence on proliferation and viability of HuH7 cells. The data provide fundamental knowledge which could help in developing safe and efficient nano-therapeutics.

Carboxyl- and amino-functionalized polystyrene nanoparticles differentially affect the polarization profile of M1 and M2 macrophage subsets

Macrophages are key regulators of innate and adaptive immune responses. Exposure to microenvironmental stimuli determines their polarization into proinflammatory M1 and anti-inflammatory M2 macrophages. M1 exhibit high expression of proinflammatory TNF-α and IL-1β, and M2 promote tissue repair, but likewise support tumor growth and cause immune suppression by expressing IL-10. Thus, the M1/M2 balance critically determines tissue homeostasis. By using carboxyl- (PS-COOH) and amino-functionalized (PS-NH2) polystyrene nanoparticles, the effects of surface decoration on the polarization of human macrophages were investigated. The nanoparticles did not compromise macrophage viability nor did they affect the expression of the M1 markers CD86, NOS2, TNF-α, and IL-1β. By contrast, in M2, both nanoparticles impaired expression of scavenger receptor CD163 and CD200R, and the release of IL-10. PS-NH2 also inhibited phagocytosis of Escherichia coli by both, M1 and M2. PS-COOH did not impair phagocytosis by M2, but increased protein mass in M1 and M2, TGF-β1 release by M1, and ATP levels in M2. Thus, nanoparticles skew the M2 macrophage polarization without affecting M1 markers. Given the critical role of the M1 and M2 polarization for the immunological balance in patients with cancer or chronic inflammation, functionalized nanoparticles might serve as tools for reprogramming the M1/M2 polarization.

Quantification of PEG-maleimide ligands and coupling efficiencies on nanoparticles with Ellman's reagent.

The surface modification of nanometer- and micrometer-sized particles and planar substrates with polyethylene glycol (PEG) ligands of varying length is a very common strategy to tune the hydrophilicity and biocompatibility of such materials, minimize unspecific interactions, improve biofunctionalization efficiencies, and enhance blood circulation times. Nevertheless, simple methods for the quantification of PEG ligands are comparatively rare. Here, we present a new concept for the quantification of PEG ligands for maleimide-functionalized PEG molecules and the determination of PEG coupling efficiencies, exploiting the quantitative reaction of maleimide with l-cysteine, and the subsequent determination of the unreacted thiol with the photometric Ellman's test. This is shown for heterobifunctional PEG spacers of varying length and amino-functionalized polystyrene nanoparticles (PS NP) without and with differently charged encoding dyes. The reaction of l-cysteine with the Ellman's reagent was monitored photometrically and with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) to derive the reaction mechanism and to obtain the stoichiometry factor for l-cysteine quantification. Mass balances and quantification of l-cysteine via its sulfur concentration using elemental analysis and inductively coupled plasma mass spectrometry (ICP-MS) confirmed the accuracy and reliability of this approach that can be extended to other surface groups and ligands.

Synthesis, Characterization and Biological Evaluation of Functionalized Polystyrene Particles

In this paper we synthesized a series of carboxylate and amino functionalized polystyrene nanoparticles with controllable size and surface properties by conventional and seed emulsion polymerization methods. They are characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). Additionally the content of carboxylate and amino groups on the surface of the particles are determined accurately by conductometric titration and fluorescamine technique respectively. Afterwards the toxicity of amino modified polystyrene particles was tested in 1321N1 astrocytoman cell line and the results showed the degree of the toxicity is proportional to the amino content of the particles, suggesting the amino group is responsible for the toxicity of the particles. Furthermore those particles were labeled by fluorescent dye and confocal fluorescent microscopy experiment showed that the particles can be well absorbed by cells.

Amino‐functionalized polystyrene nanoparticles activate the NLRP3 inflammasome in human macrophages

Nanoparticles are increasingly used in different biological and biomedical applications. However, their impact on cell functions is not well understood. Here we demonstrate that aminofunctionalized polystyrene nanoparticles (PS‐NH2) of ~100 nm but not carboxy‐functionalized (PS‐COOH) or non‐functionalized (PS) particles of the same size induce NLRP3 inflammasome activation and subsequent release of IL‐1β by human macrophages. PS‐NH2 were found to induce time‐dependent lysosomal destabilization followed by release of lysosomal enzymes, which trigger mitochondrial damage and increased formation of reactive oxygen species (ROS). Accumulation of mitochondria‐derived ROS causes oxidation of thioredoxin and its dissociation from the thioredoxin‐interacting protein (TXNIP). Liberated TXNIP forms a complex with the inflammasome, which promotes recruitment and activation of caspase‐1 followed by processing of pro‐IL‐1β to IL‐ 1β. Treatment of macrophages with the ROS scavenger N‐acetyl‐ L‐cysteine abolishes both, the caspase‐1 activation and the subsequent release of IL‐1β. Using an in vitro knockdown approach, we showed that PS‐NH2 but not PS‐COOH or PS induce activation of specifically the NLRP3 inflammasome. Thus, aminofunctionalized nanoparticles may induce proinflammatory activation of human macrophages, an effect that can be antagonized by radical scavengers. Supported by the DFG.

Amino-Functionalized Polystyrene Nanoparticles Activate the NLRP3 Inflammasome in Human Macrophages

Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH(2)) of ∼100 nm in diameter, but not carboxyl- or nonfunctionalized particles, trigger NLRP3 inflammasome activation and subsequent release of proinflammatory interleukin 1β (IL-1β) by human macrophages. PS-NH(2) induced time-dependent proton accumulation in lysosomes associated with lysosomal destabilization, release of cathepsin B, and damage of the mitochondrial membrane. Accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. Upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (TXNIP). Liberated TXNIP, in turn, interacted with the NLRP3 protein, resulting in a conformational change of the pyrin domain of the NLRP3 protein, as was predicted by molecular modeling. Consequently, this prompted assembly of the NLRP3 inflammasome complex with recruitment and activation of caspase-1, inducing IL-1β release by cleavage of pro-IL-1β. The central role of the NLRP3 inflammasome for cytokine production was confirmed by in vitro knockdown of NLRP3 and of the adaptor protein ASC, confirming that other inflammasomes were not activated by PS-NH(2). The PS-NH(2)-mediated proinflammatory macrophage activation could be antagonized by the radical scavenger N-acetyl-L-cysteine, which prevented mitochondrial damage, caspase-1 activation, and the subsequent release of IL-1β. Our study reveals the molecular mechanism of NLRP3 inflammasome activation by amino-functionalized nanoparticles and suggests a strategy as to how such adverse effects could be antagonized.