Impact of Protein Corona Formation on the Thermoresponsive Behavior of Acrylamide-Based Nanogels.

The ability to fine-tune the volume phase transition temperature (VPTT) of thermoresponsive nanoparticles is essential to their successful application in drug delivery. The rational design of these materials is limited by our understanding of the impact that nanoparticle-protein interactions have on their thermoresponsive behavior. In this work, we demonstrate how the formation of protein corona impacts the transition temperature values of acrylamide-based nanogels and their reversibility characteristics, in the presence of lysozyme, given its relevance for the ocular and intranasal administration route. Nanogels were synthesized with N-isopropylacrylamide or N-n-propylacrylamide as backbone monomers, methylenebis(acrylamide) (2.5-20 molar %) as a cross-linker, and functionalized with negatively charged monomers 2-acrylamido-2-methylpropanesulfonic acid, N-acryloyl-l-proline, or acrylic acid; characterization showed comparable particle diameter (c.a.10 nm), but formulation-dependent thermoresponsive properties, in the range 28-54 °C. Lysozyme was shown to form a complex with the negatively charged nanogels, lowering their VPTT values; the hydrophilic nature of the charged comonomer controlled the drop in VPTT upon complex formation, while matrix rigidity only had a small, yet significant effect. The cross-linker content was found to play a major role in determining the reversibility of the temperature-dependent transition of the complexes, with only 20 molar % cross-linked-nanogels displaying a fully reversible transition. These results demonstrate the importance of evaluating protein corona formation in the development of drug delivery systems based on thermoresponsive nanoparticles.

The Role of Crosslinker Content of Positively Charged NIPAM Nanogels on the In Vivo Toxicity in Zebrafish.

Polymeric nanogels as drug delivery systems offer great advantages, such as high encapsulation capacity and easily tailored formulations; however, data on biocompatibility are still limited. We synthesized N-isopropylacrylamide nanogels, with crosslinker content between 5 and 20 mol%, functionalized with different positively charged co-monomers, and investigated the in vivo toxicity in zebrafish. Our results show that the chemical structure of the basic unit impacts the toxicity profile depending on the degree of ionization and hydrogen bonding capability. When the degree of crosslinking of the polymer was altered, from 5 mol% to 20 mol%, the distribution of the positively charged monomer 2-tert-butylaminoethyl methacrylate was significantly altered, leading to higher surface charges for the more rigid nanogels (20 mol% crosslinker), which resulted in >80% survival rate (48 h, up to 0.5 mg/mL), while the more flexible polymers (5 mol% crosslinker) led to 0% survival rate (48 h, up to 0.5 mg/mL). These data show the importance of tailoring both chemical composition and rigidity of the formulation to minimize toxicity and demonstrate that using surface charge data to guide the design of nanogels for drug delivery may be insufficient.

Protein-Nanoparticle Interactions Govern the Interfacial Behavior of Polymeric Nanogels: Study of Protein Corona Formation at the Air/Water Interface.

Biomedical applications of nanoparticles require a fundamental understanding of their interactions and behavior with biological interfaces. Protein corona formation can alter the morphology and properties of nanomaterials, and knowledge of the interfacial behavior of the complexes, using in situ analytical techniques, will impact the development of nanocarriers to maximize uptake and permeability at cellular interfaces. In this study we evaluate the interactions of acrylamide-based nanogels, with neutral, positive, and negative charges, with serum-abundant proteins albumin, fibrinogen, and immunoglobulin G. The formation of a protein corona complex between positively charged nanoparticles and albumin is characterized by dynamic light scattering, circular dichroism, and surface tensiometry; we use neutron reflectometry to resolve the complex structure at the air/water interface and demonstrate the effect of increased protein concentration on the interface. Surface tensiometry data suggest that the structure of the proteins can impact the interfacial properties of the complex formed. These results contribute to the understanding of the factors that influence the bio-nano interface, which will help to design nanomaterials with improved properties for applications in drug delivery.

Adsorption of soft NIPAM nanogels at hydrophobic and hydrophilic interfaces: Conformation of the interfacial layers determined by neutron reflectivity.

The application of stimuli-responsive microgels and nanogels in drug delivery, catalysis, sensing, and coatings is restricted currently by the limited understanding of the factors influencing their adsorption dynamics and structural changes at interfaces. We have used neutron reflectivity to resolve, on the Ångström scale, the structure of 5% crosslinked N-isopropylacrylamide nanogels at both hydrophobic and hydrophilic interfaces in situ, as a function of temperature and bulk nanogel concentration. Our results show that the higher flexibility given by the low crosslinker content allows for a more ordered structure and packing. The adsorption of the thermoresponsive nanogels is primarily driven by temperature, more specifically its proximity to its volume phase transition temperature, while concentration plays a secondary role. Hydrophobic interactions drive the conformation of the first layer at the interface, which plays a key role in influencing the overall nanogel structure. The mobility of the first layer at the air-water interface as opposed to the interfacial confinement at the solid (SiC8)-liquid interface, results in a different conformation, a more compact and less deformed packing structure, which ultimately drives the structure of the subsequent layers. The evidence for the different structural conformations determined by the degree of hydrophobicity of the interface provides new knowledge, which is essential for the development of further applications. The key role of hydrophobic interactions in driving adsorption and interfacial behavior was also confirmed by fluid AFM experiments which visualized adherence of the nanogels to SiC8 modified surfaces.

Influence of surface chemistry and morphology of nanoparticles on protein corona formation.

Nanomaterials offer promising solutions as drug delivery systems and imaging agents in response to the demand for better therapeutics and diagnostics. However, the limited understanding of the interaction between nanoparticles and biological entities is currently hampering the development of new systems and their applications in clinical settings. Proteins and lipids in biological fluids are known to complex with nanoparticles to form a "biomolecular corona". This has been shown to affect particles' morphology and behavior in biological systems and their interactions with cells. Hence, understanding how nanomaterials' physicochemical properties affect the formation and composition of this biocorona is a crucial step. This work evaluates existing literature on how morphology (size and shape), and surface chemistry (charge and hydrophobicity) of nanoparticles influence the formation of protein corona. The latest evidence suggest that although surface charge promotes the interaction with proteins and lipids, surface chemistry plays a leading role in determining the affinity of the nanoparticle for biomolecules and, ultimately, the composition of the corona. More recently the study of additional nanoparticles' properties like shape and surface chirality have demonstrated a significant effect on protein corona architecture, providing new tools to tailor biomolecular corona formation. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier.

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

Systematic Analysis of the Relative Abundance of Polymers Occurring as Microplastics in Freshwaters and Estuaries.

Despite growing interest in the environmental impact of microplastics, a standardized characterization method is not available. We carried out a systematic analysis of reliable global data detailing the relative abundance of polymers in freshwaters and estuaries. The polymers were identified according to seven main categories: polyethylene terephthalate, polyethylene, polyvinyl chloride, polypropylene, polystyrene, polyurethane and a final category of miscellaneous plastic. The results show that microplastics comprised of polyvinyl chloride and polyurethane are significantly less abundant than would be expected based on global production, possibly due to their use. This has implications for models of microplastic release into the environment based on production and fate. When analysed by matrix (water, sediment or biota) distinct profiles were obtained for each category. Polyethylene, polypropylene and polystyrene were more abundant in sediment than in biota, while miscellaneous plastics was more frequent in biota. The data suggest that environmental sorting of microplastic particles, influenced by physical, chemical and biological processes, may play a key role in environmental impact, although partitioning among matrices based on density was not realized. The distinct profile of microplastics in biota raises an important question regarding potential selectivity in uptake by organisms, highlighting the priority for more and better-informed laboratory exposure studies.

Simultaneous Quantification of Antioxidants Paraxanthine and Caffeine in Human Saliva by Electrochemical Sensing for CYP1A2 Phenotyping.

The enzyme CYP1A2 is responsible for the metabolism of numerous antioxidants in the body, including caffeine, which is transformed into paraxanthine, its main primary metabolite. Both molecules are known for their antioxidant and pro-oxidant characteristics, and the paraxanthine-to-caffeine molar ratio is a widely accepted metric for CYP1A2 phenotyping, to optimize dose-response effects in individual patients. We developed a simple, cheap and fast electrochemical based method for the simultaneous quantification of paraxanthine and caffeine in human saliva, by differential pulse voltammetry, using an anodically pretreated glassy carbon electrode. Cyclic voltammetry experiments revealed for the first time that the oxidation of paraxanthine is diffusion controlled with an irreversible peak at ca. +1.24 V (vs. Ag/AgCl) in a 0.1 M H2SO4 solution, and that the mechanism occurs via the transfer of two electrons and two protons. The simultaneous quantification of paraxanthine and caffeine was demonstrated in 0.1 M H2SO4 and spiked human saliva samples. In the latter case, limits of detection of 2.89 µM for paraxanthine and 5.80 µM for caffeine were obtained, respectively. The sensor is reliable, providing a relative standard deviation within 7% (n = 6). Potential applicability of the sensing platform was demonstrated by running a small scale trial on five healthy volunteers, with simultaneous quantification by differential pulse voltammetry (DPV) of paraxanthine and caffeine in saliva samples collected at 1, 3 and 6 h postdose administration. The results were validated by ultra-high pressure liquid chromatography and shown to have a high correlation factor (r = 0.994).

Influence of Buffers, Ionic Strength, and pH on the Volume Phase Transition Behavior of Acrylamide-Based Nanogels.

The use of covalently crosslinked nanogels for applications in biology and medicine is dependent on their properties and characteristics, which often change because of the biological media involved. Understanding the role of salts, ionic strength and pH in altering specific properties is key to progress in this area. We studied the effect of both chemical structure and media environment on the thermoresponsive behavior of nanogels. A small library of methylenebisacrylamide (MBA) crosslinked nanogels were prepared using N-isopropylacrylamide (NIPAM) or N-n-propylacrylamide (NPAM), in combination with functional monomers N-hydroxyethylacrylamide (HEAM) and N-acryloyl-l-proline (APrOH). The thermoresponsive properties of nanogels were evaluated in phosphate buffer, tris-acetate buffer and Ringer HEPES, with varying concentrations and ionic strengths. The presence of ions facilitates the phase separation of nanogels, and this "salting-out" effect strongly depends on the electrolyte concentration as well as the specificity of individual anions, e.g., their positions in the Hofmeister series. A subtle change in the chemical structure of the side chain of the monomer from NIPAM to NPAM leads to a reduction of the volume phase transition temperature (VPTT) value by ~10 °C. The addition of hydrophilic comonomers such as HEAM, on the other hand, causes a ~20 °C shift in VPTT to higher values. The data highlight the significant role played by the chemical structure of the monomers used, with hydrophobicity and rigidity closely interlinked in determining thermoresponsive behavior. Furthermore, the volume phase transition temperature (VPTT) of nanogels copolymerized with ionizable APrOH comonomer can be tailored by changes in the pH of buffer solutions. This temperature-controlled phase transition is driven by intricate interplay involving the entropy of mixing, electrostatic interactions, conformational transitions, and structural rigidity. These results highlight the importance of understanding the physiochemical properties and behavior of covalently crosslinked nanogels in a biological environment prior to their applications in life-science, such as temperature/pH-triggered drug delivery systems.

Fluorescent Imprinted Nanoparticles for the Effective Monitoring of Irinotecan in Human Plasma.

Fluorescent, imprinted nanosized polymers for the detection of irinotecan have been synthesised using a napthalimide polymerisable derivative (2-allyl-6-[2-(aminoethyl)-amino] napthalimide) as functional monomer. The imprinted polymers contain ethylene glycol dimethacrylate (EGDMA) as a cross-linker and were prepared by high dilution radical polymerisation in dimethylsulphoxide (DMSO). The material was able to rebind irinotecan up to 18 nmol/mg with good specificity. Fluorescence emission at 525 nm (excitation at 448 nm) was quenched by increasing concentrations of irinotecan via a static mechanism and also in analytically useful environments as mixtures of human plasma and organic solvents. This allowed the direct detection of irinotecan (in the 10 nM-30 µM range) in human plasma treated with acetonitrile; the limit of detection (LOD) was 9.4 nM, with within-run variability of 10% and day-to-day variability of 13%.

Towards point of care systems for the therapeutic drug monitoring of imatinib.

Therapeutic drug monitoring is used in the clinical setting in the optimisation of dosages to overcome inter-patient pharmacokinetic variability, increasing efficacy whilst reducing toxicity. Imatinib is a tyrosine kinase inhibitor, displaying large variations in plasma concentrations that impact therapeutic success. As a result, imatinib has been the focus in the development of innovative techniques, aimed at its quantification in plasma. Liquid chromatography coupled with tandem mass spectrometry is currently the gold standard; however, cost and availability of the equipment limit its wider application in clinical settings. Recent advances in the field have shown Raman spectroscopy and electrochemistry to be key techniques for the development of promising analytical tools. This article reviews the latest advances towards less costly, more portable solutions that can be used at the point of care. Graphical abstract.

Microbial Degradation of Plastic in Aqueous Solutions Demonstrated by CO2 Evolution and Quantification.

The environmental accumulation of plastics worldwide is a consequence of the durability of the material. Alternative polymers, marketed as biodegradable, present a potential solution to mitigate their ecological damage. However, understanding of biodegradability has been hindered by a lack of reproducible testing methods. We developed a novel method to evaluate the biodegradability of plastic samples based on the monitoring of bacterial respiration in aqueous media via the quantification of CO2 produced, where the only carbon source available is from the polymer. Rhodococcus rhodochrous and Alcanivorax borkumensis were used as model organisms for soil and marine systems, respectively. Our results demonstrate that this approach is reproducible and can be used with a variety of plastics, allowing comparison of the relative biodegradability of the different materials. In the case of low-density polyethylene, the study demonstrated a clear correlation between the molecular weight of the sample and CO2 released, taken as a measure of biodegradability.

Dietary Antioxidants in Coffee Leaves: Impact of Botanical Origin and Maturity on Chlorogenic Acids and Xanthones.

Natural polyphenols are important dietary antioxidants that significantly benefit human health. Coffee and tea have been shown to largely contribute to the dietary intake of these antioxidants in several populations. More recently, the use of coffee leaves to produce tea has become a potential commercial target, therefore prompting studies on the quantification of polyphenols in coffee leaves. In this study a variety of coffee leaf species, at different development stages, were analyzed using ultra-high pressure liquid chromatography. The results demonstrate that both the botanical origin of the samples and their maturity influence significantly the concentration of the antioxidants; for total chlorogenic acids a two-fold difference was found between different species and up to a three-fold variation was observed between young and mature leaves. Furthermore, the range of concentrations of chlorogenic acids in young leaves (35.7-80.8 mg/g of dry matter) were found to be comparable to the one reported for green coffee beans. The results provide important data from which potential new commercial products can be developed.

Covalently Crosslinked Nanogels: An NMR Study of the Effect of Monomer Reactivity on Composition and Structure.

Covalently crosslinked nanogels are widely explored as drug delivery systems and sensors. Radical polymerization provides a simple, inexpensive, and broadly applicable approach for their preparation, although the random nature of the reaction requires careful study of the final chemical composition. We demonstrate how the different reactivities of the monomers influence the total degree of incorporation into the polymer matrix and the role played by the experimental parameters in maximizing polymerization efficiency. Nanogels based on N-isopropylacrylamide, N-n-propylacrylamide, and acrylamide crosslinked with N,N'-methylenebisacrylamide were included in this study, in combination with functional monomers N-acryloyl-l-proline, 2-acrylamido-2-methyl-1-propanesulfonic acid, and 4-vinyl-1H-imidazole. Total monomer concentration and initiator quantities are determining parameters for maximizing monomer conversions and chemical yields. The results show that the introduction of functional monomers, changes in the chemical structure of the polymerizable unit, and the addition of templating molecules can all have an effect on the polymerization kinetics. This can significantly impact the final composition of the matrices and their chemical structure, which in turn influence the morphology and properties of the nanogels.

Prediction of self-assembly of adenosine analogues in solution: a computational approach validated by isothermal titration calorimetry.

The recent discovery of the role of adenosine-analogues as neuroprotectants and cognitive enhancers has sparked interest in these molecules as new therapeutic drugs. Understanding the behavior of these molecules in solution and predicting their ability to self-assemble will accelerate new discoveries. We propose a computational approach based on density functional theory, a polarizable continuum solvation description of the aqueous environment, and an efficient search procedure to probe the potential energy surface, to determine the structure and thermodynamic stability of molecular clusters of adenosine analogues in solution, using caffeine as a model. The method was validated as a tool for the prediction of the impact of small structural variations on self-assembly using paraxanthine. The computational results were supported by isothermal titration calorimetry experiments. The thermodynamic parameters enabled the quantification of the actual percentage of dimer present in solution as a function of concentration. The data suggest that both caffeine and paraxanthine are present at concentrations comparable to the ones found in biological samples.

Interactions of NIPAM nanogels with model lipid multi-bilayers: A neutron reflectivity study.

In dermal drug delivery, the influence of the chemical structure of the carriers on their penetration mechanisms is not yet fully understood. This is a key requirement in order to design highly efficient delivery systems. In this study, neutron reflectivity is used to provide insights into the interactions between thermoresponsive N-isopropylacrylamide based nanogels, cross-linked with 10%, 20% and 30% N,N'-methylenebisacrylamide, and skin lipid multi-bilayers models. Ceramide lipid multi-bilayers and ceramide/cholesterol/behenic acid mixed lipid multi-bilayers were used for this work. The results indicated that in both multi-bilayers the lipids were depleted by the nanogels mainly through hydrophobic interactions. The ability of nanogels to associate with skin lipids to form water-dispersible complexes was found to be a function of the percentage cross-linker. An enhanced depletion of lipids was further observed in the presence of benzyl alcohol, a well-known skin penetration enhancer.

Biocompatible Phenylboronic-Acid-Capped ZnS Nanocrystals Designed As Caps in Mesoporous Silica Hybrid Materials for on-Demand pH-Triggered Release In Cancer Cells.

Biocompatible ZnS-based nanocrystals capped with 4-mercaptophenylboronic acid (ZnS@B) have been size-designed as excellent pH-responsive gatekeepers on mesoporous silica nanoparticles (MSNs), which encapsulate fluorophore safranin O (S2-Saf) or anticancer drug epirubicin hydrochloride (S2-Epi) for delivery applications in cancer cells. In this novel hybrid system, the gate mechanism consists of reversible pH-sensitive boronate ester moieties linking the nanocrystals directly to the alcohol groups from silica surface scaffold, avoiding tedious intermediate functionalization steps. The ∼3 nm size of the ZnS@B nanocrystals was tailored to allow efficient sealing of the pore voids and achieve a "zero premature cargo release" at neutral pH (7.4). The system selectively released the cargo in acidic conditions (pH 5.4 and 3.0) because of the hydrolysis of the boronate esters, which unblocked the pore voids. Delivery of the cargo by off-on cycles was demonstrated by changes in pH from 7.4 to 3.0, showing its potential pH-switching behavior. Cellular uptake of these nanocarriers within human cervix adenocarcinoma (HeLa) cells was achieved and the controlled release of the chemotherapeutic drug epirubicin was shown to occur within the endogenous endosomal/lysosomal acidified cancer cell microenvironment and further diffused into the cytosol. Cytotoxicity tests done on the mesoporous support without cargo and covalently linked with ZnS@B nanocrystals as caps were negative, suggesting that the proposed system is biocompatible and can be considered as a very promising drug nanocarrier.

Bare carbon electrodes as simple and efficient sensors for the quantification of caffeine in commercial beverages.

Food quality control is a mandatory task in the food industry and relies on the availability of simple, cost-effective and stable sensing platforms. In the present work, the applicability of bare glassy carbon electrodes for routine analysis of food samples was evaluated as a valid alternative to chromatographic techniques, using caffeine as test analyte. A number of experimental parameters were optimized and a differential pulse voltammetry was applied for quantification experiments. The detection limit was found to be 2 × 10-5 M (3σ criterion) and repeatability was evaluated by the relative standard deviation of 4.5%. The influence of sugars, and compounds structurally related to caffeine on the current response of caffeine was evaluated and found to have no significant influence on the electrode performance. The suitability of bare carbon electrodes for routine analysis was successfully demonstrated by quantifying caffeine content in seven commercially available drinks and the results were validated using a standard ultra-high performance liquid chromatography method. This work demonstrates that bare glassy carbon electrodes are a simple, reliable and cost-effective platform for rapid analysis of targets such as caffeine in commercial products and they represent therefore a competitive alternative to the existing analytical methodologies for routine food analysis.

Interaction of thermal responsive NIPAM nanogels with model lipid monolayers at the air-water interface.

Understanding the interaction of nanoparticles (NP) with ceramide lipids is important in developing strategies to overcome the formidable obstacle that is skin. This paper presents studies of interactions between N-isopropylacrylamide nanogels, crosslinked with 30% N,N'-methylenebisacrylamide, and model ceramide lipid monolayers at the air-water interface as a function of temperature. In the case of the mixed ceramide/cholesterol/behenic acid monolayer, the interaction of nanogels with the ceramide was strongly mediated by the fatty acids. This interaction between nanogels and monolayer components is dominated by hydrophobic-hydrophobic binding. The data show the important intermediary role of the fatty acid in facilitating transmembrane transport. For a pure ceramide lipid monolayer, the neutron reflectivity (NR), Brewster angle microscopy (BAM) and surface pressure results showed a lipid-nanogel complex formation and the subsequent depletion/solubilisation of the lipids from the interface when the area per molecule for the lipid was increased from 42 to 44 Å2.

Adsorption versus aggregation of NIPAM nanogels: new insight into their behaviour at the air/water interface as a function of concentration.

We have used neutron reflectivity (NR) measurements in combination with dynamic light scattering (DLS), surface tension and ellipsometry, to study the adsorption behaviour at the air/water interface of N-isopropylacrylamide-based nanogels as a function of concentration. The data provide clear evidence that the nanogels are adsorbed at the interface in a strongly deformed shape and forming a multi-layer where the thickness increases with nanogel concentration in the bulk. The combination of surface characterisation techniques and bulk studies indicate that interfacial film formation is preferred over bulk aggregation. This observation at the air/water interface supports the Derjaguin prediction, that a sphere's interaction with a plane (the thick adsorbed nanogel layer at interface) is much larger than nanogel-nanogel (sphere-sphere) association in the bulk. These findings, in particular the changes in conformations and the thick layer adsorption at the interface as a function of concentration, impact significantly on a number of applications for which nanogels are currently being investigated. These results contribute to the understanding of the behaviour of soft colloids at the interfaces.