Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.

Role of Common Cell Culture Media Supplements on Citrate-Stabilized Gold Nanoparticle Protein Corona Formation, Aggregation State, and the Consequent Impact on Cellular Uptake.

Sodium citrate-stabilized gold nanoparticles (AuNPs) are destabilized when dispersed in cell culture media (CCMs). This may promote their aggregation and subsequent sedimentation, or under the proper conditions, their interaction with dispersed proteins can lead to the formation of a NP-stabilizing protein corona. CCMs are ionic solutions that contain growth substances which are typically supplemented, in addition to serum, with different substances such as dyes, antioxidants, and antibiotics. In this study, the impact of phenol red, penicillin-streptomycin, l-glutamine, and ß-mercaptoethanol on the formation of the NP-protein corona in CCMs was investigated. Similar protein coronas were obtained except in the presence of antibiotics. Under these conditions, the protein corona took more time to be formed, and its density and composition were altered, as indicated by UV-vis spectroscopy, Z potential, dynamic light scattering, and liquid chromatography-mass spectrometry analyses. As a consequence of these modifications, a significantly different AuNP cellular uptake was measured, showing that NP uptake increased as did the NP aggregate formation. AuNP uptake studies performed in the presence of clathrin- and caveolin-mediated endocytosis inhibitors showed that neither clathrin receptors nor lipid rafts were significantly involved in the internalization mechanism. These results suggest that in these conditions, NP aggregation is the main mechanism responsible for their cellular uptake.

Pathways Related to NLRP3 Inflammasome Activation Induced by Gold Nanorods.

The widespread and increasing use of engineered nanomaterials (ENM) increases the risk of human exposure, generating concern that ENM may provoke adverse health effects. In this respect, their physicochemical characteristics are critical. The immune system may respond to ENM through inflammatory reactions. The NLRP3 inflammasome responds to a wide range of ENM, and its activation is associated with various inflammatory diseases. Recently, anisotropic ENM have become of increasing interest, but knowledge of their effects on the immune system is still limited. The objective of the study was to compare the effects of gold ENM of different shapes on NLRP3 inflammasome activation and related signalling pathways. Differentiated THP-1 cells (wildtype, ASC- or NLRP3-deficient), were exposed to PEGylated gold nanorods, nanostars, and nanospheres, and, thus, also different surface chemistries, to assess NLRP3 inflammasome activation. Next, the exposed cells were subjected to gene expression analysis. Nanorods, but not nanostars or nanospheres, showed NLRP3 inflammasome activation. ASC- or NLRP3-deficient cells did not show this effect. Gene Set Enrichment Analysis revealed that gold nanorod-induced NLRP3 inflammasome activation was accompanied by downregulated sterol/cholesterol biosynthesis, oxidative phosphorylation, and purinergic receptor signalling. At the level of individual genes, downregulation of Paraoxonase-2, a protein that controls oxidative stress, was most notable. In conclusion, the shape and surface chemistry of gold nanoparticles determine NLRP3 inflammasome activation. Future studies should include particle uptake and intracellular localization.

Microfluidic In Vitro Platform for (Nano)Safety and (Nano)Drug Efficiency Screening.

Microfluidic technology is a valuable tool for realizing more in vitro models capturing cellular and organ level responses for rapid and animal-free risk assessment of new chemicals and drugs. Microfluidic cell-based devices allow high-throughput screening and flexible automation while lowering costs and reagent consumption due to their miniaturization. There is a growing need for faster and animal-free approaches for drug development and safety assessment of chemicals (Registration, Evaluation, Authorisation and Restriction of Chemical Substances, REACH). The work presented describes a microfluidic platform for in vivo-like in vitro cell cultivation. It is equipped with a wafer-based silicon chip including integrated electrodes and a microcavity. A proof-of-concept using different relevant cell models shows its suitability for label-free assessment of cytotoxic effects. A miniaturized microscope within each module monitors cell morphology and proliferation. Electrodes integrated in the microfluidic channels allow the noninvasive monitoring of barrier integrity followed by a label-free assessment of cytotoxic effects. Each microfluidic cell cultivation module can be operated individually or be interconnected in a flexible way. The interconnection of the different modules aims at simulation of the whole-body exposure and response and can contribute to the replacement of animal testing in risk assessment studies in compliance with the 3Rs to replace, reduce, and refine animal experiments.

Formation of the Protein Corona: The Interface between Nanoparticles and the Immune System.

The interaction of inorganic nanoparticles and many biological fluids often withstands the formation of a Protein Corona enveloping the nanoparticle. This Protein Corona provides the biological identity to the nanoparticle that the immune system will detect. The formation of this Protein Corona depends not only on the composition of the nanoparticle, its size, shape, surface state and exposure time, but also on the type of media, nanoparticle to protein ratio and the presence of ions and other molecular species that interfere in the interaction between proteins and nanoparticles. This has important implications on immune safety, biocompatibility and the use of nanoparticles in medicine.

Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species.

The interaction of a living organism with external foreign agents is a central issue for its survival and adaptation to the environment. Nanosafety should be considered within this perspective, and it should be examined that how different organisms interact with engineered nanomaterials (NM) by either mounting a defensive response or by physiologically adapting to them. Herein, the interaction of NM with one of the major biological systems deputed to recognition of and response to foreign challenges, i.e., the immune system, is specifically addressed. The main focus is innate immunity, the only type of immunity in plants, invertebrates, and lower vertebrates, and that coexists with adaptive immunity in higher vertebrates. Because of their presence in the majority of eukaryotic living organisms, innate immune responses can be viewed in a comparative context. In the majority of cases, the interaction of NM with living organisms results in innate immune reactions that eliminate the possible danger with mechanisms that do not lead to damage. While in some cases such interaction may lead to pathological consequences, in some other cases beneficial effects can be identified.

Mechanomodulation of Lipid Membranes by Weakly Aggregating Silver Nanoparticles.

Silver nanoparticles (AgNPs) have wide-ranging applications, including as additives in consumer products and in medical diagnostics and therapy. Therefore, understanding how AgNPs interact with biological systems is important for ascertaining any potential health risks due to the likelihood of high levels of human exposure. Besides any severe, acute effects, it is desirable to understand more subtle interactions that could lead to milder, chronic health impacts. Nanoparticles are small enough to be able to enter biological cells and interfere with their internal biochemistry. The initial contact between the nanoparticle and cell is at the plasma membrane. To gain fundamental mechanistic insight into AgNP-membrane interactions, we investigate these phenomena in minimal model systems using a wide range of biophysical approaches applied to lipid vesicles. We find a strong dependence on the medium composition, where colloidally stable AgNPs in a glucose buffer have a negligible effect on the membrane. However, at physiological salt concentrations, the AgNPs start to weakly aggregate and sporadic but significant membrane perturbation events are observed. Under these latter conditions, transient poration and structural remodeling of some vesicle membranes are observed. We observe that the fluidity of giant vesicle membranes universally decreases by an average of 16% across all vesicles. However, we observe a small population of vesicles that display a significant change in their mechanical properties with lower bending rigidity and higher membrane tension. Therefore, we argue that the isolated occurrences of membrane perturbation by AgNPs are due to low-probability mechanomodulation by AgNP aggregation at the membrane.

Dynamic Equilibrium in the Cetyltrimethylammonium Bromide-Au Nanoparticle Bilayer, and the Consequent Impact on the Formation of the Nanoparticle Protein Corona.

Nanoparticles in ionic solutions are usually surrounded by stabilizing molecules that avoid aggregation and determine their surface properties, which strongly influence their behavior. The present work aims to shed light on the static vs dynamic nature of the cetyltrimethylammonium bromide (CTAB) bilayer on gold nanoparticles and to understand its effects on nanoparticle evolution in biological systems. A systematic study of the CTAB bilayer of Au nanorods and nanospheres was carried out, exploring the role of excess free surfactant in solution on the surface properties of nanoparticles and their colloidal stability. The results indicated the presence of a CTAB bilayer in which the external layer was in rapid dynamic equilibrium with the free surfactant in solution. The internal surfactant layer of the gold nanospheres was also found to be in dynamic equilibrium. Conversely, the gold nanorods had a permanent internal layer. Consequently, the CTAB-nanoparticle dispersions always contained free CTAB in excess to maintain the colloidal stability of the NPs. In contrast, decreasing the free CTAB concentration resulted in nanoparticle aggregation. The impact of the dynamic equilibrium on the exposure of particles to biological fluids and on the formation of the nanoparticle protein corona was studied, revealing the different fates of the nanoparticles, which depended on the amount of free CTAB in solution.

Size-Dependent Protein-Nanoparticle Interactions in Citrate-Stabilized Gold Nanoparticles: The Emergence of the Protein Corona.

Surface modifications of highly monodisperse citrate-stabilized gold nanoparticles (AuNPs) with sizes ranging from 3.5 to 150 nm after their exposure to cell culture media supplemented with fetal bovine serum were studied and characterized by the combined use of UV-vis spectroscopy, dynamic light scattering, and zeta potential measurements. In all the tested AuNPs, a dynamic process of protein adsorption was observed, evolving toward the formation of an irreversible hard protein coating known as Protein Corona. Interestingly, the thickness and density of this protein coating were strongly dependent on the particle size, making it possible to identify different transition regimes as the size of the particles increased: (i) NP-protein complexes (or incomplete corona), (ii) the formation of a near-single dense protein corona layer, and (iii) the formation of a multilayer corona. In addition, the different temporal patterns in the evolution of the protein coating came about more quickly for small particles than for the larger ones, further revealing the significant role that size plays in the kinetics of this process. Since the biological identity of the NPs is ultimately determined by the protein corona and different NP-biological interactions take place at different time scales, these results are relevant to biological and toxicological studies.

Quantifying the Sensitivity of Multipolar (Dipolar, Quadrupolar, and Octapolar) Surface Plasmon Resonances in Silver Nanoparticles: The Effect of Size, Composition, and Surface Coating.

The effect of composition, size, and surface coating on the sensitivity of localized multipolar surface plasmon resonances has been spectroscopically investigated in high-quality silver colloidal solutions with precisely controlled sizes from 10 to 220 nm and well-defined surface chemistry. Surface plasmon resonance modes have been intensively characterized, identifying the size-dependence of dipolar, quadrupolar, and octapolar modes. Modifications of the NP's surface chemistry revealed the higher sensitivity of large sizes, long molecules, thiol groups, and low-order resonance modes. We also extend this study to gold nanoparticles, aiming to compare the sensitivity of both materials, quantifying the higher sensitivity of silver.

Little adjustments significantly improve the Turkevich synthesis of gold nanoparticles.

In this report, we show how the classical and widely used Turkevich synthesis can be improved significantly by simple adjustments. The gold nanoparticles (AuNPs) produced with the optimized protocol have a much narrower size distribution (5-8% standard deviation), and their diameters can be reproduced with unrivaled little variation (<3%). Moreover, large volumes of these particles can be produced in one synthesis; we routinely synthesize 1000 mL of ∼3.5 nM AuNPs. The key features of the improved protocol are the control of the pH by using a citrate buffer instead of a citrate solution as the reducing agent or stabilizer and optimized mixing of reagents. Further, the shape uniformity of the particles can be improved by addition of 0.02 mM EDTA. While the proposed protocol is as straightforward as the original Turkevich protocol, it is more tolerant against variations in precursor concentration.

Long-Term Intracellular Tracking of Label-Free Nanoparticles in Live Cells and Tissues with Confocal Microscopy.

The label-free imaging of inorganic nanoparticles (NPs) using confocal laser scanning microscopy (CLSM) provides a powerful and versatile tool for studying interactions between NPs and biological systems. Without the need for exogenous labels or markers, it simply benefits from the differential scattering of visible photons between biomaterials and inorganic NPs. Validation experiments conducted on fixed and living cells in real-time, as well as mouse tissue sections following parenteral administration of NPs. Additionally, by incorporating reporter fluorophores and utilizing both reflectance and fluorescence imaging modalities, the method enables high-resolution multiplex imaging of cellular structures and NPs. Different sizes and concentrations of Au NPs are tested as for Ag, Fe3O4, and CeO2 NPs, all with biological interest. Overall, the comprehensive study of NP imaging by confocal microscopy in reflectance mode provides valuable insights and tools for researchers interested in monitoring the nano-bio interactions.

Effect of the spacer structure on the stability of gold nanoparticles functionalized with monodentate thiolated poly(ethylene glycol) ligands.

Poly(ethylene glycol)- (PEG-) based ligands are well-established for the stabilization of nanoparticles in aqueous solution and are especially interesting for applications in medicine and biotechnology because they are known to improve the pharmacokinetic properties of nanomaterials. In this study, we prepared gold nanoparticles (AuNPs) with ligand shells of different monodentate poly(ethylene glycol)-thiol (PEG-SH) ligands. These ligands differed only in the segment connecting the thiol group with the PEG moiety (Mw ≈ 2000 g/mol) through an ester bond, the spacer. All ligands were synthesized by straightforward esterification. Specifically, we used PEG ligands with a long (C10, PEGMUA) or short (C2, PEGMPA) alkylene spacer or a phenylene (PEGMPAA) spacer. The influence of the spacer on the stability of gold nanoparticle-PEG conjugates (AuNP@PEG) was tested by cyanide etching experiments, electrolyte-induced aggregation, and competitive ligand displacement with dithiothreitol (DTT). In the presence of 100 mM cyanide, AuNPs stabilized with PEGMPA or PEGMPAA were completely dissolved by oxidative etching within a few minutes, whereas AuNPs stabilized with PEGMUA needed more than 20 h to be completely etched. By complementary experiments, we deduced a simplified description for the etching process that takes into account the role of excess ligand. In the presence of free ligand, significantly fewer AuNPs are etched, suggesting a competition of etching and ligand binding to AuNPs. We also compared the stabilizing effect of PEGMUA with that of a bidentate PEG-thiol ligand (PEGLIP) and found a reversed stability against cyanide etching and DTT displacement, in agreement with previously reported observations. Our results clearly demonstrate the strong impact of the spacer structure on conjugate stability and provide valuable information for the rational design of more complex AuNP@PEG conjugates, which are of much interest in the context of biotechnology and medical applications.

In situ functionalization and PEO coating of iron oxide nanocrystals using seeded emulsion polymerization.

Herein we demonstrate that seeded emulsion polymerization is a powerful tool to produce multiply functionalized PEO coated iron oxide nanocrystals. Advantageously, by simple addition of functional surfactants, functional monomers, or functional polymerizable linkers-solely or in combinations thereof-during the seeded emulsion polymerization process, a broad range of in situ functionalized polymer-coated iron oxide nanocrystals were obtained. This was demonstrated by purposeful modulation of the zeta potential of encapsulated iron oxide nanocrystals and conjugation of a dyestuff. Successful functionalization was unequivocally proven by TXRF. Furthermore, the spatial position of the functional groups can be controlled by choosing the appropriate spacers. In conclusion, this methodology is highly amenable for combinatorial strategies and will spur rapid expedited synthesis and purposeful optimization of a broad scope of nanocrystals.

Sculpting Windows onto AuAg Hollow Cubic Nanocrystals.

Using surfactants in the galvanic replacement reaction (GRR) offers a versatile approach to modulating hollow metal nanocrystal (NC) morphology and composition. Among the various surfactants available, quaternary ammonium cationic surfactants are commonly utilised. However, understanding how they precisely influence morphological features, such as the size and void distribution, is still limited. In this study, we aim to uncover how adding different surfactants-CTAB, CTAC, CTApTS, and PVP-can fine-tune the morphological characteristics of AuAg hollow NCs synthesised via GRR at room temperature. Our findings reveal that the halide counterion in the surfactant significantly controls void formation within the hollow structure. When halogenated surfactants, such as CTAB or CTAC, are employed, multichambered opened nanoboxes are formed. In contrast, with non-halogenated CTApTS, single-walled closed nanoboxes with irregularly thick walls form. Furthermore, when PVP, a polymer surfactant, is utilised, changes in concentration lead to the production of well-defined single-walled closed nanoboxes. These observations highlight the role of surfactants in tailoring the morphology of hollow NCs synthesised through GRR.

The development of highly dense highly protected surfactant ionizable lipid RNA loaded nanoparticles.

The long quest for efficient drug administration has been looking for a universal carrier that can precisely transport traditional drugs, new genomic and proteic therapeutic agents. Today, researchers have found conditions to overcome the two main drug delivery dilemmas. On the one side, the versatility of the vehicle to efficiently load, protect and transport the drug and then release it at the target place. On the other hand, the questions related to the degree of PEGylation which are needed to avoid nanoparticle (NP) aggregation and opsonization while preventing cellular uptake. The development of different kinds of lipidic drug delivery vehicles and particles has resulted in the development of ionizable lipid nanoparticles (iLNPs), which can overcome most of the typical drug delivery problems. Proof of their success is the late approval and massive administration as the prophylactic vaccine for SARS-CoV-2. These ILNPs are built by electrostatic aggregation of surfactants, the therapeutic agent, and lipids that self-segregate from an aqueous solution, forming nanoparticles stabilized with lipid polymers, such as PEG. These vehicles overcome previous limitations such as low loading and high toxicity, likely thanks to low charge at the working pH and reduced size, and their entry into the cells via endocytosis rather than membrane perforation or fusion, always associated with higher toxicity. We herein revise their primary features, synthetic methods to prepare and characterize them, pharmacokinetic (administration, distribution, metabolization and excretion) aspects, and biodistribution and fate. Owing to their advantages, iLNPs are potential drug delivery systems to improve the management of various diseases and widely available for clinical use.

Asymmetrical Plasmon Distribution in Hybrid AuAg Hollow/Solid Coded Nanotubes.

Morphological control at the nanoscale paves the way to fabricate nanostructures with desired plasmonic properties. In this study, we discuss the nanoengineering of plasmon resonances in 1D hollow nanostructures of two different AuAg nanotubes, including completely hollow nanotubes and hybrid nanotubes with solid Ag and hollow AuAg segments. Spatially resolved plasmon mapping by electron energy loss spectroscopy (EELS) revealed the presence of high order resonator-like modes and localized surface plasmon resonance (LSPR) modes in both nanotubes. The experimental findings accurately correlated with the boundary element method (BEM) simulations. Both experiments and simulations revealed that the plasmon resonances are intensely present inside the nanotubes due to plasmon hybridization. Based on the experimental and simulated results, we show that the novel hybrid AuAg nanotubes possess two significant coexisting features: (i) LSPRs are distinctively generated from the hollow and solid parts of the hybrid AuAg nanotube, which creates a way to control a broad range of plasmon resonances with one single nanostructure, and (ii) the periodicity of the high-order modes are disrupted due to the plasmon hybridization by the interaction of solid and hollow parts, resulting in an asymmetrical plasmon distribution in 1D nanostructures. The asymmetry could be modulated/engineered to control the coded plasmonic nanotubes.

Facet-engineered TiO2 drives photocatalytic activity and stability of supported noble metal clusters during H2 evolution.

Metal clusters supported on TiO2 are widely used in many photocatalytic applications, including pollution control and production of solar fuels. Besides high photoactivity, stability during the photoreaction is another essential quality of high-performance photocatalysts, however systematic studies on this attribute are absent for metal clusters supported on TiO2. Here we have studied, both experimentally and with first-principles simulation methods, the stability of Pt, Pd and Au clusters prepared by ball milling on nanoshaped anatase nanoparticles preferentially exposing {001} (plates) and {101} (bipyramids) facets during the photogeneration of hydrogen. It is found that Pt/TiO2 exhibits superior stability than Pd/TiO2 and Au/TiO2, and that {001} facet-based photocatalysts always are more stable than their {101} analogous regardless of the considered metal species. The loss of stability associated with cluster sintering, which is facilitated by the transfer of photoexcited carriers from the metal species to the neighbouring Ti and O atoms, most significantly and detrimentally affects the H2-evolution photoactivity.

Nanoceria as Safe Contrast Agents for X-ray CT Imaging.

Cerium oxide nanoparticles (CeO2NPs) have exceptional catalytic properties, rendering them highly effective in removing excessive reactive oxygen species (ROS) from biological environments, which is crucial in safeguarding these environments against radiation-induced damage. Additionally, the Ce atom's high Z number makes it an ideal candidate for utilisation as an X-ray imaging contrast agent. We herein show how the injection of albumin-stabilised 5 nm CeO2NPs into mice revealed substantial enhancement in X-ray contrast, reaching up to a tenfold increase at significantly lower concentrations than commercial or other proposed contrast agents. Remarkably, these NPs exhibited prolonged residence time within the target organs. Thus, upon injection into the tail vein, they exhibited efficient uptake by the liver and spleen, with 85% of the injected dose (%ID) recovered after 7 days. In the case of intratumoral administration, 99% ID of CeO2NPs remained within the tumour throughout the 7-day observation period, allowing for observation of disease dynamics. Mass spectrometry (ICP-MS) elemental analysis confirmed X-ray CT imaging observations.

Exploiting endocytosis for transfection of mRNA for cytoplasmatic delivery using cationic gold nanoparticles.

Introduction: Gene therapy holds promise to cure various diseases at the fundamental level. For that, efficient carriers are needed for successful gene delivery. Synthetic 'non-viral' vectors, as cationic polymers, are quickly gaining popularity as efficient vectors for transmitting genes. However, they suffer from high toxicity associated with the permeation and poration of the cell membrane. This toxic aspect can be eliminated by nanoconjugation. Still, results suggest that optimising the oligonucleotide complexation, ultimately determined by the size and charge of the nanovector, is not the only barrier to efficient gene delivery. Methods: We herein develop a comprehensive nanovector catalogue comprising different sizes of Au NPs functionalized with two different cationic molecules and further loaded with mRNA for its delivery inside the cell. Results and Discussion: Tested nanovectors showed safe and sustained transfection efficiencies over 7 days, where 50 nm Au NPs displayed the highest transfection rates. Remarkably, protein expression was increased when nanovector transfection was performed combined with chloroquine. Cytotoxicity and risk assessment demonstrated that nanovectors are safe, ascribed to lesser cellular damage due to their internalization and delivery via endocytosis. Obtained results may pave the way to design advanced and efficient gene therapies for safely transferring oligonucleotides.