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Carbonic anhydrase (CA) was previously proposed as a green alternative for biomineralization of carbon dioxide (CO2). However, enzyme’s fragile nature when in synthetic environment significantly limits such industrial application. Herein, we hypothesized that CA immobilization onto flexible and hydrated “bridges” that ensure proton-transfer at their interfaces leads to improved activity and kinetic behavior and potentially increases enzyme’s feasibility for industrial implementation. Our hypothesis was formulated considering that water plays a key role in the CO2 hydration process and acts as both the reactant as well as the rate-limiting step of the CO2 capture and transformation process. To demonstrate our hypothesis, two types of user-synthesized organic metallic frameworks [metal–organic frameworks (MOFs), one hydrophilic and one hydrophobic] were considered as model supports and their surface characteristics (i.e., charge, shape, curvature, size, etc.) and influence on the immobilized enzyme’s behavior were evaluated. Morphology, crystallinity and particle size, and surface area of the model supports were determined by scanning electron microscopy, dynamic light scattering, and nitrogen adsorption/desorption measurements, respectively. Enzyme activity, kinetics, and stability at the supports interfaces were determined using spectroscopical analyses. Analysis showed that enzyme functionality is dependent on the support used in the immobilization process, with the enzyme immobilized onto the hydrophilic support retaining 72% activity of the free CA, when compared with that immobilized onto the hydrophobic one that only retained about 28% activity. Both CA–MOF conjugates showed good storage stability relative to the free enzyme in solution, with CA immobilized at the hydrophilic support also revealing increased thermal stability and retention of almost all original enzyme activity even after heating treatment at 70 °C. In contrast, free CA lost almost half of its original activity when subject to the same conditions. This present work suggests that MOFs tunable hydration conditions allow high enzyme activity and stability retention. Such results are expected to impact CO2 storage and transformation strategies based on CA and potentially increase user-integration of enzyme-based green technologies in mitigating global warming.

Background Montmorillonite is a type of nanoclay that originates from the clay fraction of the soil and is incorporated into polymers to form nanocomposites with enhanced mechanical strength, barrier, and flammability properties used for food packaging, automotive, and medical devices. However, with implementation in such consumer applications, the interaction of montmorillonite-based composites or derived byproducts with biological systems needs to be investigated. Methods Herein we examined the potential of Cloisite Na+ (pristine) and Cloisite 30B (organically modified montmorillonite nanoclay) and their thermally degraded byproducts’ to induce toxicity in model human lung epithelial cells. The experimental set-up mimicked biological exposure in manufacturing and disposal areas and employed cellular treatments with occupationally relevant doses of nanoclays previously characterized using spectroscopical and microscopical approaches. For nanoclay-cellular interactions and for cellular analyses respectively, biosensorial-based analytical platforms were used, with induced cellular changes being confirmed via live cell counts, viability assays, and cell imaging. Results Our analysis of byproducts’ chemical and physical properties revealed both structural and functional changes. Real-time high throughput analyses of exposed cellular systems confirmed that nanoclay induced significant toxic effects, with Cloisite 30B showing time-dependent decreases in live cell count and cellular viability relative to control and pristine nanoclay, respectively. Byproducts produced less toxic effects; all treatments caused alterations in the cell morphology upon exposure. Conclusions Our morphological, behavioral, and viability cellular changes show that nanoclays have the potential to produce toxic effects when used both in manufacturing or disposal environments. General significance The reported toxicological mechanisms prove the extensibility of a biosensorial-based platform for cellular behavior analysis upon treatment with a variety of nanomaterials.

Nanoclays' functionalization with organic modifiers increases their individual barrier properties, thermal stability, and mechanical properties and allows for ease of implementation in food packaging materials or medical devices. Previous reports have shown that, while organic modifiers integration between the layered mineral silicates leads to nanoclays with different degrees of hydrophobicity that become easily miscible in polymers, they could also pose possible effects at inhalation or ingestion routes of exposure. Through a systematic analysis of three organically modified and one pristine nanoclay, we aimed to relate for the first time the physical and chemical characteristics, determined via microscopical and spectroscopical techniques, with the potential of these nanoclays to induce deleterious effects in in vitro cellular systems, i.e. in immortalized and primary human lung epithelial cell lines. To derive information on how functionalization could lead to toxicological profiles throughout nanoclays' life cycle, both as-received and thermally degraded nanoclays were evaluated. Our analysis showed that the organic modifiers chemical composition influenced both the physical and chemical characteristics of the nanoclays as well as their toxicity. Overall, when cells were exposed to nanoclays with organic modifiers containing bioreactive groups, they displayed lower cellular numbers as well more elongated cellular morphologies relative to the pristine nanoclay and the nanoclay containing a modifier with long carbon chains. Additionally, thermal degradation caused loss of the organic modifiers as well as changes in size and shape of the nanoclays, which led to changes in toxicity upon exposure to our model cellular systems. Our study provides insight into the synergistic effects of chemical composition, size, and shape of the nanoclays and their toxicological profiles in conditions that mimic exposure in manufacturing and disposal environments, respectively, and can help aid in safe-by-design manufacturing of nanoclays with user-controlled functionalization and lower toxicity levels when food packaging applications are considered.

Organomodified nanoclays (ONCs) are increasingly used as filler materials to improve nanocomposite strength, wettability, flammability, and durability. However, pulmonary risks associated with exposure along their chemical lifecycle are unknown. This study's objective was to compare pre- and post-incinerated forms of uncoated and organomodified nanoclays for potential pulmonary inflammation, toxicity, and systemic blood response. Mice were exposed via aspiration to low (30 μg) and high (300 μg) doses of preincinerated uncoated montmorillonite nanoclay (CloisNa), ONC (Clois30B), their respective incinerated forms (I-CloisNa and I-Clois30B), and crystalline silica (CS). Lung and blood tissues were collected at days 1, 7, and 28 to compare toxicity and inflammation indices. Well-dispersed CloisNa caused a robust inflammatory response characterized by neutrophils, macrophages, and particle-laden granulomas. Alternatively, Clois30B, I-Clois30B, and CS high-dose exposures elicited a low grade, persistent inflammatory response. High-dose Clois30B exposure exhibited moderate increases in lung damage markers and a delayed macrophage recruitment cytokine signature peaking at day 7 followed by a fibrotic tissue signature at day 28, similar to CloisNa. I-CloisNa exhibited acute, transient inflammation with quick recovery. Conversely, high-dose I-Clois30B caused a weak initial inflammatory signal but showed comparable pro-inflammatory signaling to CS at day 28. The data demonstrate that ONC pulmonary toxicity and inflammatory potential relies on coating presence and incineration status in that coated and incinerated nanoclay exhibited less inflammation and granuloma formation than pristine montmorillonite. High doses of both pre- and post-incinerated ONC, with different surface morphologies, may harbor potential pulmonary health hazards over long-term occupational exposures.