Low doses of zeolitic imidazolate framework-8 nanoparticles alter the actin organization and contractility of vascular smooth muscle cells

Highlights

• Cell biophysical change is overlooked in evaluation of hazardous materials.

• Interactions of low-dose MOF nanoparticles with VSMCs are studied.

• AFM and dSTORM are used to characterize single VSMC biophysical changes.

• Low-dose MOF nanoparticles cause cell morphology and actin cytoskeleton changes.

• Biophysical changes lead to loss of cell contractility and cardiovascular risks.

Abstract

Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles have emerged as a promising platform for drug delivery and controlled release. Considering most ZIF-8 nanoparticle drug carriers are designed to be administered intravenously, and thus would directly contact vascular smooth muscle cells (VSMCs) in many circumstances, the potential interactions of ZIF-8 nanoparticles with VSMCs require investigation. Here, the effects of low doses of ZIF-8 nanoparticles on VSMC morphology, actin organization, and contractility are investigated. Two nanoscale imaging tools, atomic force microscopy, and direct stochastic optical reconstruction microscopy, show that even at the concentrations (12.5 and 25 µg/ml) that were deemed “safe” by conventional biochemical cell assays (MTT and LDH assays), ZIF-8 nanoparticles can still cause changes in cell morphology and actin cytoskeleton organization at the cell apical and basal surfaces. These cytoskeletal structural changes impair the contractility function of VSMCs in response to Angiotensin II, a classic vasoconstrictor. Based on intracellular zinc and actin polymerization assays, we conclude that the increased intracellular Zn²⁺ concentration due to the uptake and dissociation of ZIF-8 nanoparticles could cause the actin cytoskeleton dis-organization, as the elevated Zn²⁺ directly disrupts the actin assembly process, leading to altered actin organization such as branches and networks. Since the VSMC phenotype change and loss of contractility are fundamental to the development of atherosclerosis and related cardiovascular diseases, it is worth noting that these low doses of ZIF-8 nanoparticles administered intravenously could still be a safety concern in terms of cardiovascular risks. Moving forward, it is imperative to re-consider the “safe” nanoparticle dosages determined by biochemical cell assays alone, and take into account the impact of these nanoparticles on the biophysical characteristics of VSMCs, including changes in the actin cytoskeleton and cell morphology.

Introduction

Metal-organic frameworks (MOFs), a class of crystalline hybrid materials consisting of metal ions or clusters coordinated to organic ligands, have received increased interest in the field of nanomedicine.(Liu et al., 2019, Wu and Yang, 2017, Wang et al., 2020, Lu et al., 2018, Zhang et al., 2018, Wan et al., 2020) In particular, zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, as a subclass of MOFs, have emerged as a promising platform for drug delivery and controlled release, thanks to their mild encapsulation conditions,(Wang et al., 2017, Tadepalli et al., 2018, Liang et al., 2015, Luzuriaga et al., 2019) excellent drug loading and stabilization capacity,(Wang et al., 2018, Feng et al., 2019, Doonan et al., 2017, Sun et al., 2020, Welch et al., 2018) and pH-responsive drug release under slightly acidic environments.(Chen et al., 2018, Zheng et al., 2016) So far, ZIF-8 nanoparticles have been used to encapsulate and deliver small anticancer drugs such as doxorubicin and tirapazamine (Zheng et al., 2016, Zhang et al., 2018), peptides and proteins such as insulin and glucose oxidase for diabetes (Chen et al., 2018, Wang et al., 2018), and nucleic acids for gene therapy (Li et al., 2019, Alsaiari et al., 2018). It is widely accepted that ZIF-8 nanoparticles have relatively low cytotoxicity and good biocompatibility. As with other nanoparticles, the typical procedure to determine the “safe dose” of ZIF-8 nanoparticles is through biochemical cell viability assays (e.g., MTT assay). Typically, cells are incubated with a series of concentrations of ZIF-8 nanoparticles for a short period of time (4–48 h), if more than 90% of the cells remain viable, the concentration is considered “safe” (Chen et al., 2018, Zheng et al., 2016; Tamames-Tabar et al., 2014). However, recent studies in the nanotoxicology field indicate that some metal-based nanoparticles can induce abnormalities in the actin cytoskeleton and cell morphology, at lower concentrations than those which show changes in cell viability. For instance, Liang and Parak’s study showed that gold nanoparticles altered the actin organization and cell morphology at lower nanoparticle concentrations, than those that led to the onset of the reduced cell viability (Ma et al., 2017). Another pioneering work by Setyawati, Leong and co-authors demonstrated that titanium dioxide nanoparticles can induce morphological changes and increased mobility for colorectal cancer cells at the “safe” concentrations pre-determined from cell viability assays (Setyawati et al., 2018). Considering the ZIF-8 nanoparticles are metal-containing, and the increased interest in their applications for nanomedicine, we began to ask whether ZIF-8 nanoparticles at “safe” concentrations can alter cell morphology, which could be an overlooked adverse effect of ZIF-8 nanoparticles.

Different types of cells have been employed to investigate the cytotoxicity of ZIF-8 using biochemical cell viability assays. Early studies indicated that ZIF-8 nanoparticles had half maximal effective concentrations (EC50, the concentration causing 50% cell viability) of 45 µg/ml and 100 µg/ml on human breast cancer cells and human cervical cancer cells, respectively (Tamames-Tabar et al., 2014, Zhuang et al., 2014). More recently, a systematic study demonstrated that ZIF-8 microparticles had no significant cytotoxicity up to a threshold value of 30 µg/ml on different cell lines including mouse macrophages, mouse embryo fibroblasts, human bone fibroblasts, human kidney epithelial cells, human keratinocyte epithelial cells, and human breast cancer cells (Hoop et al., 2018). However, it should be noted that the impact of ZIF-8 nanoparticles on vascular cells including vascular smooth muscle cells and endothelial cells are still unclear, even though most ZIF-8 nanoparticles as drug carriers are designed to be administered intravenously and therefore could directly contact cells lining blood vessels. Although considerable effort has been made in studying the effects of metal-based nanoparticles on vascular endothelial cells (i.e., the monolayer covering the lumen of blood vessels)(Cao et al., 2017, Napierska et al., 2009, Setyawati et al., 2018, Setyawati et al., 2013), there is limited information available on whether metal-based nanoparticles would affect the vascular smooth muscle cells underneath the endothelium. Herein, we chose to focus on the impact of ZIF-8 nanoparticles on vascular smooth muscle cells (VSMCs) based on the following justifications: (i) A number of risk factors such as hypertension, diabetes, mechanical damage, smoking, aging, inflammation, cancer, and viral infection could result in endothelial dysfunction and its increased permeability (Hadi et al., 2005, Rajendran et al., 2013, Komarova and Malik, 2010, Wang et al., 2016), thereby directly exposing underlying VSMCs to the nanoparticles; (ii) Accumulating evidence suggests that metal-based nanoparticles could lead to endothelial leakage at endothelial cell junctions (Setyawati et al., 2017, Tee et al., 2019), which could cause the accumulation of nanoparticles in VSMCs; and (iii) Under various stimuli, VSMCs tend to undergo phenotypic change, from contractile to synthetic phenotype, which could initiate atherosclerosis, a main cause of heart attack, stroke, and heart failure (Bennett et al., 2016, Doran et al., 2008, Ren et al., 2019). With these considerations, it is important to evaluate the impact of ZIF-8 nanoparticles on VSMCs before applying them to treat diseases such as cancers and diabetes, in order to ascertain whether or not ZIF-8 nanoparticles could increase the risk of cardiovascular diseases.

In this study, we aim at the effects of low doses of ZIF-8 nanoparticles on VSMCs at the level of cell actin cytoskeleton and cell morphology, both of which are important structural characteristics that determine the main function of VSMCs: regulating vascular tones through the cell contraction (Hong et al., 2014, Sanyour et al., 2018). First, biochemical cell assays were conducted to identify the accepted “safe” concentrations of ZIF-8 nanoparticles. Under these “safe” concentrations, atomic force microscopy (AFM) was performed to investigate the effects of ZIF-8 nanoparticles on VSMC morphology, actin cytoskeleton at the cell apical surfaces, and cell mechanical properties at the single cell level. Meanwhile, direct stochastic optical reconstruction microscopy (dSTORM), a super-resolution fluorescence imaging approach, was utilized to examine the effect of ZIF-8 nanoparticles on VSMC actin organization at the cell basal layers. The results showed that, even at the “safe” concentrations, ZIF-8 nanoparticles caused changes in actin cytoskeleton at the cell apical and basal surfaces, and the cell morphology. These changes further impaired the contractility of VSMCs in response to a classic vasoconstrictor (Angiotensin II). We hypothesized that the increased intracellular zinc concentration due to the uptake and dissociation of ZIF-8 nanoparticles could cause the actin cytoskeleton disorganization, and this hypothesis was tested by an actin polymerization assay and AFM imaging. Overall, the implications of our findings could be important for in vivo applications that would administer ZIF-8 nanoparticles intravenously, as even low doses of ZIF-8 nanoparticles may promote the risks of cardiovascular diseases. Moving forward, it is imperative to re-consider the “safe” nanoparticle dosages that are determined by the biochemical cell assays alone, and take into account the impact of nanoparticles on the actin cytoskeleton and cell morphology, especially for vascular smooth muscle cells.