José M. Rodrigo‐Muñoz

Metal-organic frameworks (MOFs), network structures wherein metal ions or clusters link organic ligands into porous materials, are being actively researched as nanoscale drug delivery devices as they offer tunable structures with high cargo loading that can easily be further functionalized for targeting and enhanced physiological stability. The excellent biocompatibility of Zr has meant that its MOFs are among the most studied to date, in particular the archetypal Zr terephthalate UiO-66. In contrast, the isoreticular analog linked by fumarate (Zr-fum) has received little attention, despite the endogenous linker being part of the Krebs cycle. Herein, we report a comprehensive study of Zr-fum in the context of drug delivery. Reducing particle size is shown to increase uptake by cancer cells while reducing internalization by macrophages, immune system cells that remove foreign objects from the bloodstream. Zr-fum is compatible with defect loading of the drug dichloroacetate (DCA) as well as surface modification during synthesis, through coordination modulation and postsynthetically. DCA-loaded, PEGylated Zr-fum shows selective in vitro cytotoxicity toward HeLa and MCF-7 cancer cells, likely as a consequence of its enhanced caveolae-mediated endocytosis compared to uncoated precursors, and it is well tolerated by HEK293 kidney cells, J774 macrophages, and human peripheral blood lymphocytes. Compared to UiO-66, Zr-fum is more efficient at transporting the drug mimic calcein into HeLa cells, and DCA-loaded, PEGylated Zr-fum is more effective at reducing HeLa and MCF-7 cell proliferation than the analogous UiO-66 sample. In vitro examination of immune system response shows that Zr-fum samples induce less reactive oxygen species than UiO-66 analogs, possibly as a consequence of the linker being endogenous, and do not activate the C3 and C4 complement cascade pathways, suggesting that Zr-fum can avoid phagocytic activation. The results show that Zr-fum is an attractive alternative to UiO-66 for nanoscale drug delivery, and that a wide range of in vitro experiments is available to greatly inform the design of drug delivery systems prior to early stage animal studies.

The high drug-loading and excellent biocompatibilities of metal-organic frameworks (MOFs) have led to their application as drug-delivery systems (DDSs). Nanoparticle surface chemistry dominates both biostability and dispersion of DDSs while governing their interactions with biological systems, cellular and/or tissue targeting, and cellular internalization, leading to a requirement for versatile and reproducible surface functionalization protocols. Herein, we explore not only the effect of introducing different surface functionalities to the biocompatible Zr-MOF UiO-66 but also the efficacy of three surface modification protocols: (i) direct attachment of biomolecules [folic acid (FA) and biotin (Biot)] introduced as modulators for UiO-66 synthesis, (ii) our previously reported "click-modulation" approach to covalently attach polymers [poly(ethylene glycol) (PEG), poly-l-lactide, and poly-N-isopropylacrylamide] to the surface of UiO-66 through click chemistry, and (iii) surface ligand exchange to postsynthetically coordinate FA, Biot, and heparin to UiO-66. The innovative use of a small molecule with metabolic anticancer activity, dichloroacetate (DCA), as a modulator during synthesis is described, and it is found to be compatible with all three protocols, yielding surface-coated, DCA-loaded (10-20 w/w %) nano-MOFs (70-170 nm). External surface modification generally enhances the stability and colloidal dispersion of UiO-66. Cellular internalization routes and efficiencies of UiO-66 by HeLa cervical cancer cells can be tuned by surface chemistry, and anticancer cytotoxicity of DCA-loaded MOFs correlates with the endocytosis efficiency and mechanisms. The MOFs with the most promising coatings (FA, PEG, poly-l-lactide, and poly-N-isopropylacrylamide) were extensively tested for selectivity of anticancer cytotoxicity against MCF-7 breast cancer cells and HEK293 healthy kidney cells as well as for cell proliferation and reactive oxygen species production against J774 macrophages and peripheral blood lymphocytes isolated from the blood of human donors. DCA-loaded, FA-modified UiO-66 selectively kills cancer cells without harming healthy ones or provoking immune system response in vitro, suggesting a significant targeting effect and great potential in anticancer drug delivery. The results provide mechanistic insight into the design and functionalization of MOFs for drug delivery and underline the availability of various in vitro techniques to potentially minimize early-stage in vivo animal studies following the three Rs: reduction, refinement, and replacement.

BACKGROUND: Eosinophils, a central factor in asthma pathogenesis, have the ability to secrete exosomes. However, the precise role played by exosomes in the biological processes leading up to asthma has not been fully defined. OBJECTIVE: We hypothesized that exosomes released by eosinophils contribute to asthma pathogenesis by activating structural lung cells. METHODS: Eosinophils from asthmatic patients and healthy volunteers were purified from peripheral blood, and exosomes were isolated from eosinophils of asthmatic and healthy individuals. All experiments were performed with eosinophil-derived exosomes from healthy and asthmatic subjects. Epithelial damage was evaluated using primary small airway epithelial cell lines through 2 types of apoptosis assays, that is, flow cytometry and TUNEL assay with confocal microscopy. Additionally, the epithelial repair was analysed by performing wound healing assays with epithelial cells. Functional studies such as proliferation and inhibition-proliferation assays were carried out in primary bronchial smooth muscle cell lines. Also, gene expression analysis of pro-inflammatory molecules was evaluated by real-time PCR on epithelial and muscle cells. Lastly, protein expression of epithelial and muscle cell signalling factors was estimated by Western blot. RESULTS: Asthmatic eosinophil-derived exosomes induced an increase in epithelial cell apoptosis at 24 hour and 48 hour, impeding wound closure. In addition, muscle cell proliferation was increased at 72 hours after exosome addition and was linked with higher phosphorylation of ERK1/2. We also found higher expression of several genes when both cell types were cultured in the presence of exosomes from asthmatics: CCR3 and VEGFA in muscle cells, and CCL26, TNF and POSTN in epithelial cells. Healthy eosinophil-derived exosomes did not exert any effect over these cell types. CONCLUSIONS AND CLINICAL RELEVANCE: Eosinophil-derived exosomes from asthmatic patients participate actively in the development of the pathological features of asthma via structural lung cells.

Abstract Eosinophils are able to secrete exosomes that have an undefined role in asthma pathogenesis. We hypothesized that exosomes released by eosinophils autoregulate and promote eosinophil function. Eosinophils of patients with asthma (n = 58) and healthy volunteers (n = 16) were purified from peripheral blood, and exosomes were isolated and quantified from eosinophils of the asthmatic and healthy populations. Apoptosis, adhesion, adhesion molecules expression, and migration assays were performed with eosinophils in the presence or absence of exosomes from healthy and asthmatic individuals. Reactive oxygen species (ROS) were evaluated by flow cytometry with an intracellular fluorescent probe and nitric oxide (NO) and a colorimetric kit. In addition, exosomal proteins were analyzed by mass spectrometry. Eosinophil-derived exosomes induced an increase in NO and ROS production on eosinophils. Moreover, exosomes could act as a chemotactic factor on eosinophils, and they produced an increase in cell adhesion, giving rise to a specific augmentation of adhesion molecules, such as ICAM-1 and integrin α2. Protein content between exosomes from healthy and asthmatic individuals seems to be similar in both groups. In conclusion, we found that exosomes from the eosinophils of patients with asthma could modify several specific eosinophil functions related to asthma pathogenesis and that they could contribute fundamentally to the development and maintenance of asthma.

Intercellular communication is crucial to the immune system response. In the recent years, the discovery of exosomes has changed the way immune response orchestration was understood. Exosomes are able to operate as independent units that act as mediators in both physiological and pathological conditions. These structures contain proteins, lipidic mediators, and nucleic acids and notoriously include microRNAs (miRNAs). miRNAs are short RNA sequences (around 19-22 nucleotides) with a high phylogenetic conservation and can partially or totally regulate multiple mRNAs, inhibiting protein synthesis. In respiratory diseases such as asthma and allergic sensitization, exosomes released by several cell types and their specific content perform crucial functions in the development and continuation of the pathogenic mechanisms. Released exosomes and miRNAs inside them have been found in different types of clinical samples, such as bronchoalveolar lavage fluids and sputum supernatants, providing new data about the environmental factors and mediators that participate in the inflammatory responses that lead to the exacerbation of asthma. In this review, we summarize our current knowledge of the role of exosomes and miRNAs in asthma and allergic sensitization, paying attention to the functions that both exosomes and miRNAs are described to perform through the literature. We review the effect of exosomes and miRNAs in cells implicated in asthma pathology and the genes and pathways that they modify in them, depicting how their behavior is altered in disease status. We also describe their possible repercussion in asthma diagnosis through their possible role as biomarkers. Therefore, both exosomes and miRNAs can be viewed as potential tools to be added to the arsenal of therapeutics to treat this disease.