Comparative Characterization of Different Molecular Formats of Bispecific Antibodies Targeting EGFR and PD-L1.

We generated two IgG1-like bispecific antibodies (BsAbs) with different molecular formats, symmetrical DVD-Ig and asymmetrical knob-in-hole (KIH), targeting the same antigens, EGFR and PD-L1 (designated as anti-EGFR/PD-L1). We performed the physiochemical and biological characterization of these two formats of anti-EGFR/PD-L1 BsAbs and compared some key quality attributes and biological activities of these two formats of BsAbs. Physiochemical binding characterization data demonstrated that both formats bound EGFR and PD-L1. However, the binding affinity of the KIH format was weaker than the DVD-Ig format in Biacore binding assays. In contrast, both DVD-Ig and KIH BsAbs had similar ELISA and cell surface binding activities, comparable to mAbs. Triple-negative breast cancer (TNBC) cells and a xenograft model were used to test the potency of BsAbs and other biological activities. Results showed that anti-EGFR/PD-L1 BsAbs exhibited in vitro and in vivo antitumor proliferation activity, but there was a difference in the potencies of the respective BsAb formats (DVD-Ig and KIH) when different cells or assays were used. This study provides evidence that the potency of the BsAbs targeting the same antigens can be affected by the respective molecular features, and selection of appropriate cell lines and assays is critically important for the assay development and potency testing of BsAbs.

Examining the role of nickel and NiTi nanoparticles promoting inflammation and angiogenesis.

Nickel titanium (NiTi, or Nitinol) alloy is used in several biomedical applications, including cardiac, peripheral vascular, and fallopian tube stents. There are significant biocompatibility issues of metallic implants to nickel ions and nano-/micro-sized alloy particles. Our laboratories have recently shown that microscale CoCr wear particles from metal-on-metal hips crosslink with the innate immune signaling Toll-like receptor 4 (TLR4), prompting downstream signaling that results in interleukin (IL)-1ß and IL-8 gene expression. In vivo, NiTi alloy can also generate wear particles on the nanoscale (NP) that have thus far not been studied for their potential to induce inflammation and angiogenesis that can, in turn, contribute to implant (e.g. stent) failure. Earlier studies by others demonstrated that nickel could induce contact hypersensitivity by crosslinking the human, but not the mouse, TLR4. In the present work, it is demonstrated that NiCl2 ions and NiTi nanoparticles induce pro-inflammatory and pro-angiogenic cytokine/chemokine expression in human endothelial and monocyte cell lines in vitro. These observations prompt concerns about potential mechanisms for stent failure. The data here showed a direct correlation between intracellular uptake of Ni2+ and generation of reactive oxygen species. To determine a role for nickel and NiTi nanoparticles in inducing angiogenesis in vivo, 1-cm silicone angioreactors were implanted subcutaneously into athymic (T-cell-deficient) nude mice. The angioreactors contained Matrigel (a gelatinous protein mixture that resembles extracellular matrix) in addition to one of the following: PBS (negative control), VEGF/FGF-2 (positive control), NiCl2, or NiTi NP. The implantation of angioreactors represents a potential tool for quantification of angiogenic potentials of medical device-derived particles and ions in vivo. By this approach, NiTi NP were found to be markedly angiogenic, while Ni2+ was less-so. The angioreactors may provide a powerful tool to examine if debris shed from medical devices may promote untoward biological effects.

Multiphase Drug Distribution and Exchange in Oil-in-Water Nanoemulsion Revealed by High-Resolution 19F qNMR.

An oil-in-water (o/w) nanoemulsion (NE), composed of oil globules, stabilized by a surfactant, and dispersed in an aqueous phase, is increasingly developed in complex drug formulation. Kinetically stable NEs are used to formulate hydrophobic drugs and typically provide higher dosage strengths and better content uniformity. However, little is known accurately about drug distribution in its multiphase solution, especially for the possible drug presence in the surfactant (s) phase, the interface layer between the dispersed oil (o) and the continuous water (w) phases. Here, high-resolution 19F quantitative NMR spectroscopy was applied directly and noninvasively on an o/w NE drug product containing difluprednate (DFPN). The well-resolved 19F peaks of DFPN depended on the shielding molecules in each phase, which revealed mass-balanced DFPN distribution in multiple phases of (w), (s), and (o) of NE globules at a quantity of 1.8 ± 0.1, 35 ± 2, and 59 ± 3% per labeled content, respectively. Furthermore, the dilution-dependent 19F peak line broadening and shift suggested a millisecond dynamic exchange between the NE and the less-noticed smaller but thermodynamically stable microemulsion (ME) globules in NE solution. The high-resolution NMR result revealed that the drug availability could be quickly achieved using an o/w NE formulation because of the drug multiphase distribution and the ME-assisted fast drug exchange among globules.

Impact of Surface Chemistry of Ultrasmall Superparamagnetic Iron Oxide Nanoparticles on Protein Corona Formation and Endothelial Cell Uptake, Toxicity, and Barrier Function.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) have been investigated for biomedical applications, including novel contrast agents, magnetic tracers for tumor imaging, targeted drug delivery vehicles, and magneto-mechanical actuators for hyperthermia and thrombolysis. Despite significant progress, recent clinical reports have raised concerns regarding USPION safety related to endothelial cell dysfunction; however, there is limited information on factors contributing to these clinical responses. The influence of USPION surface chemistry on nanoparticle interactions with proteins may impact endothelial cell function leading to adverse responses. Therefore, the goal of this study was to assess the effects of carboxyl-functionalized USPION (CU) or amine-functionalized USPION (AU) (approximately 30 nm diameter) on biological responses in human coronary artery endothelial cells. Increased protein adsorption was observed for AU compared with CU after exposure to serum proteins. Exposure to CU, but not AU, resulted in a concentration-dependent decrease in cell viability and perinuclear accumulation inside cytoplasmic vesicles. Internalization of CU was correlated with endothelial cell functional changes under non-cytotoxic conditions, as evidenced by a marked decreased expression of endothelial-specific adhesion proteins (eg, vascular endothelial-cadherin and platelet endothelial cell adhesion molecule-1) and increased endothelial permeability. Evaluation of downstream signaling indicated endothelial permeability is associated with actin cytoskeleton remodeling, possibly elicited by intracellular events involving reactive oxygen species, calcium ions, and the nanoparticle cellular uptake pathway. This study demonstrated that USPION surface chemistry significantly impacts protein adsorption and endothelial cell uptake, viability, and barrier function. This information will advance the current toxicological profile of USPION and improve development, safety assessment, and clinical outcomes of USPION-enabled medical products.

Chemical Characterization and Non-targeted Analysis of Medical Device Extracts: A Review of Current Approaches, Gaps, and Emerging Practices.

The developers of medical devices evaluate the biocompatibility of their device prior to FDA's review and subsequent introduction to the market. Chemical characterization, described in ISO 10993-18:2020, can generate information for toxicological risk assessment and is an alternative approach for addressing some biocompatibility end points (e.g., systemic toxicity, genotoxicity, carcinogenicity, reproductive/developmental toxicity) that can reduce the time and cost of testing and the need for animal testing. Additionally, chemical characterization can be used to determine whether modifications to the materials and manufacturing processes alter the chemistry of a patient-contacting device to an extent that could impact device safety. Extractables testing is one approach to chemical characterization that employs combinations of non-targeted analysis, non-targeted screening, and/or targeted analysis to establish the identities and quantities of the various chemical constituents that can be released from a device. Due to the difficulty in obtaining a priori information on all the constituents in finished devices, information generation strategies in the form of analytical chemistry testing are often used. Identified and quantified extractables are then assessed using toxicological risk assessment approaches to determine if reported quantities are sufficiently low to overcome the need for further chemical analysis, biological evaluation of select end points, or risk control. For extractables studies to be useful as a screening tool, comprehensive and reliable non-targeted methods are needed. Although non-targeted methods have been adopted by many laboratories, they are laboratory-specific and require expensive analytical instruments and advanced technical expertise to perform. In this Perspective, we describe the elements of extractables studies and provide an overview of the current practices, identified gaps, and emerging practices that may be adopted on a wider scale in the future. This Perspective is outlined according to the steps of an extractables study: information gathering, extraction, extract sample processing, system selection, qualification, quantification, and identification.

Hemin activation abrogates Mycoplasma hyorhinis replication in chronically infected prostate cancer cells via heme oxygenase-1 induction.

Mycoplasma hyorhinis (M. hyorhinis) lacks a cell wall and resists multiple antibiotics. We describe here the striking > 90% inhibitory effect of hemin, a natural inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1), on M. hyorhinis replication in chronically infected LNCaP prostate cancer cells. The role of HO-1 in interrupting M. hyorhinis replication was confirmed by HO-1-specific siRNA suppression of hemin-induced HO-1 protein expression, which increased intracellular M. hyorhinis DNA levels in LNCaP cells. Proteomic analysis and transmission electron microscopy of hemin-treated cells confirmed the complete absence of M. hyorhinis proteins and intact microorganisms, respectively, strongly supporting these findings. Our study is the first to our knowledge suggesting therapeutic potential for activated HO-1 in cellular innate responses against mycoplasma infection.

Crossover between anti- and pro-oxidant activities of different manganese oxide nanoparticles and their biological implications.

Manganese oxide nanoparticles (MnOx NPs) have been suggested to possess several enzyme-like activities. However, studies often used either color change or fluorescence to determine the catalytic activity. Despite the simplicity and sensitivity of these probes, these methods may give distracting artifacts or not reflect the catalytic activities in biological systems. To address this issue, herein, we used electron spin resonance (ESR) spectroscopy, a technique proven effective in identifying and quantifying the free radicals produced/scavenged in nanomaterial-catalyzed reactions, to systematically evaluate the catalytic activities of three MnOx NPs (MnO2, Mn2O3, and Mn3O4 NPs) towards biologically relevant antioxidants (ascorbate and glutathione (GSH)) and reactive oxygen species (ROS) (hydrogen peroxide (H2O2), superoxide anion, and hydroxyl radical). We found that all three MnOx NPs possess both pro- and anti-oxidant activities, including oxidase-, catalase-, and superoxide dismutase (SOD)-like activities but without peroxidase-like or hydroxyl radical scavenging activity. In addition, there are differences among these MnOx NPs in their catalytic activities towards different reactions. Mn2O3 shows the strongest ascorbate oxidation activity, followed by MnO2 and Mn3O4, while Mn3O4 shows the strongest oxidation efficiency towards GSH compared to Mn2O3 and MnO2. In the catalyzed decomposition of H2O2, MnO2 NPs show higher efficiency in the generation of molecular oxygen than Mn2O3 or Mn3O4. Cellular studies indicate that all three MnOx NPs induced concentration-dependent decreases in the cell viability, with Mn3O4 > Mn3O2 > MnO2. At lower concentrations (<100 µM), consistent with the enzyme-like activities detected in solution, all three NPs significantly decreased H2O2-induced cytotoxicity in Caco-2 cells. Our study determined the multi-enzymatic activities of MnOx NPs and exhibited differences among MnOx NPs of different valences in their enzyme-like activities and their biological implications; these results provide valuable information for safe and efficient applications of MnOx NPs as ROS-scavenging biomedical nanomaterials.

Coexistence of oil droplets and lipid vesicles in propofol drug products.

Propofol is intravenously administered oil-in-water emulsion stabilized by egg lecithin phospholipids indicated for the induction and maintenance of general anesthesia or sedation. It is generally assumed to be structurally homogenous as characterized by commonly used dynamic light scattering technique and laser diffraction. However, the excessive amount of egg lecithin phospholipids added to the propofol formulation may, presumably, give rise to additional formation of lipid vesicles (i.e., vesicular structures consisting of a phospholipid bilayer). In this study, we investigate the use of high-resolution cryogenic transmission electron microscopy (cryo-TEM) in morphological characterization of four commercially available propofol drug products. The TEM result, for the first time, reveals that all propofol drug products contain lipid vesicles and oil droplet-lipid vesicle aggregated structures, in addition to oil droplets. Statistical analysis shows the size and ratio of the lipid vesicles varies across four different products. To evaluate the impact of such morphological differences on active pharmaceutical ingredient (API)'s distribution, we separate the lipid vesicle phase from other constituents via ultracentrifuge fractionation and determine the amount of propofol (2,6-diisopropylphenol) using high performance liquid chromatography (HPLC). The results indicate that a nearly negligible amount of API (i.e., NMT 0.25% of labeled content) is present in the lipid vesicles and is thus primarily distributed in the oil phase. As oil droplets are the primary drug carriers and their globule size are similar, the findings of various lipid vesicle composition and sizes among different propofol products do not affect their clinical outcomes.

Influences of simulated gastrointestinal environment on physicochemical properties of gold nanoparticles and their implications on intestinal epithelial permeability.

Gold nanoparticles (Au NPs) hold great promise in food, industrial and biomedical applications due to their unique physicochemical properties. However, influences of the gastrointestinal tract (GIT), a likely route for Au NPs administration, on the physicochemical properties of Au NPs has been rarely evaluated. Here, we investigated the influence of GIT fluids on the physicochemical properties of Au NPs (5, 50, and 100 nm) and their implications on intestinal epithelial permeability in vitro. Au NPs aggregated in fasted gastric fluids and generated hydroxyl radicals in the presence of H2O2. Cell studies showed that GIT fluids incubation of Au NPs affected the cellular uptake of Au NPs but did not induce cytotoxicity or disturb the intestinal epithelial permeability.

Chitosan-Lysozyme Conjugates for Enzyme-Triggered Hydrogel Degradation in Tissue Engineering Applications.

Tuning hydrogel degradation enables effective and successful tissue regeneration by modulating cellular behaviors and matrix formation. In this work, we develop a novel degradable hydrogel scaffold on the basis of a unique enzyme-substrate complex by photocrosslinking. Chitosan and lysozyme are chemically modified with methacrylate moieties to be tethered in hydrogels, and in the presence of riboflavin initiator, these hydrogels are cured by blue light irradiation. The incorporation of lysozyme to chitosan hydrogels accelerates the degradation rate of the crosslinked hydrogels in a dose-dependent manner, as evidenced by an increase in pore size and interconnectivity through cryogenic scanning electron microscopy over time. Those noncytotoxic materials significantly enhance cellular proliferation and migration, which contribute to osteogenic differentiation of encapsulated mesenchymal stem cells in vitro and bone formation in mouse calvarial defects. These findings suggest a promising strategy to modulate the degradation behavior of hydrogels for use in tissue engineering.

Platinum nanoparticles: an avenue for enhancing the release of nitric oxide from S-nitroso-N-acetylpenicillamine and S-nitrosoglutathione.

Nitric oxide (NO) is an endogenous bioregulator with established roles in diverse fields. The difficulty in the modulation of NO release is still a significant obstacle to achieving successful clinical applications. We report herein our initial work using electron spin resonance (ESR) spectroscopy to detect NO generated from S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) donors catalyzed by platinum nanoparticles (Pt NPs, 3 nm) under physiological conditions. With ESR spectroscopy coupled with spin trapping and spin labeling techniques, we identified that Pt NPs can significantly promote the generation of NO from SNAP and GSNO under physiological conditions. A classic NO colorimetric detection kit was also employed to verify that Pt NPs truly triggered the release of NO from its donors. Pt NPs can act as promising delivery vehicles for on-demand NO delivery based on time and dosage. These results, along with the detection of the resulting disulfide product, were confirmed with mass spectrometry. In addition, cellular experiments provided a convincing demonstration that the triggered release of NO from its donors by Pt NPs is efficient in killing human cancer cells in vitro. The catalytic mechanism was elucidated by X-ray photo-electron spectroscopy (XPS) and ultra-high performance liquid chromatography/high-resolution mass spectrometry (UHPLC-HRMS), which suggested that Pt-S bond formation occurs in the solution of Pt NPs and NO donors. Identification of Pt NPs capable of generating NO from S-nitrosothiols (RSNOs) is an important step in harnessing NO for investigations into its clinical applications and therapies.

Exploring the activities of ruthenium nanomaterials as reactive oxygen species scavengers.

Research on noble metal nanoparticles (NPs) able to scavenge reactive oxygen species (ROS) has undergone a tremendous growth recently. However, the interactions between ruthenium nanoparticles (Ru NPs) and ROS have never been systematically explored thus far. This research focused on the decomposition of hydrogen peroxide (H2O2), scavenging of hydroxyl radicals (•OH), superoxide radical (O2•-), singlet oxygen (1O2), 2,2'-azino-bis(3-ethylbenzenothiazoline- 6-sulfonic acid ion (ABTS•+), and 1,1-diphenyl-2-picrylhydrazyl radical (•DPPH) in the presence of commercial Ru NPs using the electron spin resonance technique. In vitro cell studies demonstrated that Ru NPs have excellent biocompatibility and exert a cytoprotective effect against oxidative stress. These findings may spark fresh enthusiasm for the applications of Ru NPs under relevant physiologically conditions.

Nanosilver-PMMA composite coating optimized to provide robust antibacterial efficacy while minimizing human bone marrow stromal cell toxicity.

Porous PMMA is a versatile biomaterial with good biocompatibility but high susceptibility to bacterial colonization, which we mitigated by utilizing immobilized antimicrobial silver nanoparticles (AgNPs). A uniform porous thin film was deposited onto silicon wafers by simultaneously ablating PMMA and silver (Ag) using pulsed laser deposition (PLD) optimized for minimal human cell toxicity and antibacterial efficacy. PMMA without Ag became heavily colonized by E. coli in simulated dynamic conditions, while Ag-containing samples prevented all colonization. ICP-MS analysis demonstrated that the amount of leached Ag after 24h under simulated in vivo conditions (with serum media at 37°C and 5% CO2) increased in proportion to film thickness (and total silver content). 10,000, 14,000, and 20,000 laser pulse-deposited films released 0.76, 1.05, and 1.67µg/mL Ag, respectively, after 24h. Human bone marrow stromal cells (hBMSCs) grown directly on 10,000-pulse films (0.76µg/mL Ag released) for 24-h exhibited no cytotoxicity. Exposure to the remaining films produced cytotoxicity, necrosis, and apoptosis detected using flow cytometry. Examining both leachates and direct cell contact allowed us to develop an in vitro cytotoxicity test method and optimize a novel device material and coating to be nontoxic and bactericidal during both potential initial implantation and external use.

Biological evaluation of ultrananocrystalline and nanocrystalline diamond coatings.

Nanostructured biomaterials have been investigated for achieving desirable tissue-material interactions in medical implants. Ultrananocrystalline diamond (UNCD) and nanocrystalline diamond (NCD) coatings are the two most studied classes of synthetic diamond coatings; these materials are grown using chemical vapor deposition and are classified based on their nanostructure, grain size, and sp3 content. UNCD and NCD are mechanically robust, chemically inert, biocompatible, and wear resistant, making them ideal implant coatings. UNCD and NCD have been recently investigated for ophthalmic, cardiovascular, dental, and orthopaedic device applications. The aim of this study was (a) to evaluate the in vitro biocompatibility of UNCD and NCD coatings and (b) to determine if variations in surface topography and sp3 content affect cellular response. Diamond coatings with various nanoscale topographies (grain sizes 5-400 nm) were deposited on silicon substrates using microwave plasma chemical vapor deposition. Scanning electron microscopy and atomic force microscopy revealed uniform coatings with different scales of surface topography; Raman spectroscopy confirmed the presence of carbon bonding typical of diamond coatings. Cell viability, proliferation, and morphology responses of human bone marrow-derived mesenchymal stem cells (hBMSCs) to UNCD and NCD surfaces were evaluated. The hBMSCs on UNCD and NCD coatings exhibited similar cell viability, proliferation, and morphology as those on the control material, tissue culture polystyrene. No significant differences in cellular response were observed on UNCD and NCD coatings with different nanoscale topographies. Our data shows that both UNCD and NCD coatings demonstrate in vitro biocompatibility irrespective of surface topography.

Core size determination and structural characterization of intravenous iron complexes by cryogenic transmission electron microscopy.

Understanding physicochemical properties of intravenous (IV) iron drug products is essential to ensure the manufacturing process is consistent and streamlined. The history of physicochemical characterization of IV iron complex formulations stretches over several decades, with disparities in iron core size and particle morphology as the major source of debate. One of the main reasons for this controversy is room temperature sample preparation artifacts, which affect accurate determination of size, shape and agglomeration/aggregation of nanoscale iron particles. The present study is first to report the ultra-fine iron core structures of four IV iron complex formulations, sodium ferric gluconate, iron sucrose, low molecular weight iron dextran and ferumoxytol, using a cryogenic transmission electron microscopy (cryo-TEM) preservation technique, as opposed to the conventional room temperature (RT-TEM) technique. Our results show that room temperature preparation causes nanoparticle aggregation and deformation, while cryo-TEM preserves IV iron colloidal suspension in their native frozen-hydrated and undiluted state. In contrast to the current consensus in literature, all four IV iron colloids exhibit a similar morphology of their iron oxide cores with a spherical shape, narrow size distribution and an average size of 2nm. Moreover, out of the four tested formulations, ferumoxytol exhibits a cluster-like community of several iron carbohydrate particles which likely accounts for its large hydrodynamic size of 25nm, measured with dynamic light scattering. Our findings outline a suitable method for identifying colloidal nanoparticle core size in the native state, which is increasingly important for manufacturing and design control of complex drug formulations, such as IV iron drug products.

Contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by the cytokinesis-block micronucleus assay.

Extensive human exposure to food- and cosmetics-related consumer products containing nanosilver is of public concern because of the lack of information about their safety. Genotoxicity is an important endpoint for the safety and health hazard assessment of regulated products including nanomaterials. The in vitro cytokinesis-block micronucleus (CBMN) assay is a very useful test for predictive genotoxicity testing. Recently, we have reported the genotoxicity of 20 nm nanosilver in human liver HepG2 and colon Caco2 cells evaluated using the CBMN assay. The objective of our present study was three-fold: (i) to evaluate if HepG2 and Caco2 cells are valuable in vitro models for rapid genotoxicity screening of nanosilver; (ii) to test the hypothesis that the nanoparticle size and cell types are critical determinants of its genotoxicity; and (iii) to determine if ionic silver contributes to the nanosilver genotoxicity. With these objectives in mind, we evaluated the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge, obtained from the same commercial source, under the same experimental conditions and the same genotoxic CBMN endpoint used for the previously tested 20 nm silver. The ionic silver (silver acetate) was also evaluated under the same conditions. Results of our study show that up to the concentrations tested in these cell types, the smaller (20 nm) nanosilver induces micronucleus formation in both the cell types but the larger (50 nm) nanosilver and the ionic silver provide a much weaker response compared with controls under the same conditions.

Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells.

Exposure to nanosilver found in food- and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food- and cosmetics-related chemicals including nanosilver.

Silver Nanoparticle-Induced Autophagic-Lysosomal Disruption and NLRP3-Inflammasome Activation in HepG2 Cells Is Size-Dependent.

Silver nanoparticles (AgNPs) are incorporated into medical and consumer products to exploit their excellent antimicrobial properties; however, potential mechanisms of toxicity of AgNPs in mammalian cells are not fully understood. The objective of this study was to determine the mechanism of size- and concentration-dependent cytotoxicity of AgNPs in human liver-derived hepatoma (HepG2) cells. Mechanisms of toxicity were explored at subcytotoxic concentrations (≤10 µg/ml AgNPs) and autophagy induction, lysosomal activity, inflammasome-dependent caspase-1 activation, and apoptosis were examined. Using enhanced dark-field light microscopy, hyperspectral imaging, electron microscopy, and energy dispersive X-ray spectroscopy, AgNPs were shown to rapidly accumulate in cytoplasmic vesicles for up to 24 h and 10-nm AgNPs exhibited the highest uptake and accumulation. Autophagy and enhanced lysosomal activity were induced at noncytotoxic concentrations (1 µg/ml; primary particle size:10 > 50 >100 nm), whereas increased caspase-3 activity (associated with apoptosis) was observed at cytotoxic concentrations (10, 25, and 50 µg/ml). Subcytotoxic concentrations of AgNPs enhanced expression of LC3B, a pro-autophagic protein, and CHOP, an apoptosis inducing ER-stress protein, and activation of NLRP3-inflammasome (caspase-1, IL-1ß). Disrupting the autophagy-lysosomal pathway through chloroquine or ATG5-siRNA exacerbated AgNPs-induced caspase-1 activation and lactate dehydrogenase release, suggesting that NLRP3-inflammasome plays an important role in AgNPs-induced cytotoxicity. Overall, 10-nm AgNPs showed the highest cellular responses compared with 50- and 100-nm AgNPs based on equal mass dosimetry. The results indicate the potential of vesicle-engulfed 10-nm AgNPs to induce cytotoxicity by a mechanism involving perturbations in the autophagy-lysosomal system and inflammasome activation.

Platinum nanoparticles inhibit antioxidant effects of vitamin C via ascorbate oxidase-mimetic activity.

The development and application of nanomaterials as consumer products including food, drugs, and cosmetics are rapidly expanding. However, interactions between these novel materials and other chemical components of consumer products have not been thoroughly studied. Here, by using electron spin resonance techniques, we compared the effects of Au, Ag, and Pt nanoparticles (NPs) on the antioxidant activity of vitamin C (sodium l-ascorbate, NaA). Chemical studies showed that Pt NPs exhibit ascorbate oxidase-mimetic activity, thereby oxidizing NaA but Au and Ag NPs do not. This ascorbate oxidase-mimetic activity of Pt NPs results in a dramatic loss of antioxidant activity of NaA for scavenging hydroxyl radicals and superoxide radicals. A further study suggested that the ascorbate oxidase-mimetic activity of Pt NPs is critically dependent on the particle size. Finally, in vitro cell studies demonstrated that Pt NPs with ascorbate oxidase-mimetic activity inhibit the cytoprotective effect of NaA on cells challenged by oxidative stress. Our findings provide a better understanding of enzyme-mimicking NP interactions with naturally-occurring antioxidants and should guide future applications.

Nanoporous Aluminum Oxide Membranes Coated with Atomic Layer Deposition-Grown Titanium Dioxide for Biomedical Applications: An In Vitro Evaluation.

The surface topographies of nanoporous anodic aluminum oxide (AAO) and titanium dioxide (TiO2) membranes have been shown to modulate cell response in orthopedic and skin wound repair applications. In this study, we: (1) demonstrate an improved atomic layer deposition (ALD) method for coating the porous structures of 20, 100, and 200 nm pore diameter AAO with nanometer-thick layers of TiO2 and (2) evaluate the effects of uncoated AAO and TiO2-coated AAO on cellular responses. The TiO2 coatings were deposited on the AAO membranes without compromising the openings of the nanoscale pores. The 20 nm TiO2-coated membranes showed the highest amount of initial protein adsorption via the micro bicinchoninic acid (micro-BCA) assay; all of the TiO2-coated membranes showed slightly higher protein adsorption than the uncoated control materials. Cell viability, proliferation, and inflammatory responses on the TiO2-coated AAO membranes showed no adverse outcomes. For all of the tested surfaces, normal increases in proliferation (DNA content) of L929 fibroblasts were observed over from 4 hours to 72 hours. No increases in TNF-alpha production were seen in RAW 264.7 macrophages grown on TiO2-coated AAO membranes compared to uncoated AAO membranes and tissue culture polystyrene (TCPS) surfaces. Both uncoated AAO membranes and TiO2-coated AAO membranes showed no significant effects on cell growth and inflammatory responses. The results suggest that TiO2-coated AAO may serve as a reasonable prototype material for the development of nanostructured wound repair devices and orthopedic implants.