Article

Bidirectional Regulation of Cell Mechanical Motion via a Gold Nanorods-Acoustic Streaming System

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Abstract

Cell mechanical motion is a key physiological process that relies on the dynamics of actin filaments. Herein, a localized shear-force system based on gigahertz acoustic streaming (AS) is proposed, which can simultaneously realize intracellular delivery and cellular mechanical regulation. The results demonstrate that gold nanorods (AuNRs) can be delivered into the cytoplasm and even the nuclei of cancer and normal cells within a few minutes by AS stimulation. The delivery efficiency of AS stimulation is four times higher than that of endocytosis. Moreover, AS can effectively promote cytoskeleton assembly, regulate cell stiffness and change cell morphology. Since the inhibitory effect of AuNRs on cytoskeleton assembly, this AuNRs-AS system is able to inhibit or promote cell mechanical motion in a controlled manner by regulating the mechanical properties of cells. The bidirectional regulation of cell motion is further verified via scratch experiments, in which AuNRs-treated cells recover their motion ability through AS stimulation. In particular, the results of AuNRs-AS mechanical regulation on cell are related to the intrinsic properties of cell lines, revealing to more obvious effects on the cells with higher motor capacities. In summary, this acoustic technology has shown superiorities in controllable cell-motion manipulation, indicating its potential in building a multifunctional, integrated cytomechanics regulation platform.

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... Zhao et al. (Zhao et al., 2021) developed a rapid drug screening method by changing the permeability of leukemia cells (THP-1), and increased the speed of drug screening by eight times. He et al. (He et al., 2022) have found that that acoustic treatment significantly facilitated intracellular delivery into both the cytoplasm and the nucleus, as shown in Figure 4A, with much higher efficiency compared to endocytosis. Additionally, they discovered that the acoustic treatment induced changes in the mechanical properties of both normal and cancer cells, leading to improvements in cytoskeleton integrity and cell stiffness. ...
... However, these effects were found to be Acoustic-induced intracellular delivery of gold nanorods (AuNRs) into the cytoplasm and even the nuclei of cancer and normal cells. Reproduced with permission from (He et al., 2022). Copyright 2022, American Chemical Society. ...
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Most cancer-related deaths come from metastasis. It was recently discovered that nanoparticles could inhibit cancer cell migration. While most researchers focus on single-cell migration, the effect of nanoparticle treatment on collective cell migration has not been explored. Collective migration occurs commonly in many types of cancer metastasis, where a group of cancer cells move together, which requires the contractility of the cytoskeleton filaments and the connection of neighboring cells by the cell junction proteins. Here, we demonstrate gold nanorods (AuNRs) and the introduction of near-infrared light could inhibit the cancer cell collective migration by altering the actin filaments and cell junctions with significantly triggered phosphorylation changes of essential proteins, using mass spectrometry-based phosphoproteomics. Further observation using super-resolution stochastic optical reconstruction microscopy (STORM) showed the actin cytoskeleton filament bundles were disturbed, which is difficult to differentiate under a normal fluorescence microscope. The decreased expression level of N-Cadherin junctions and morphological changes of tight junction protein zonula occludens 2 (ZO-2) were also observed. All these results indicate possible functions of the AuNRs treatments in regulating and remodeling the actin filaments and cell junction proteins, which contribute to decreasing cancer cell collective migration.
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Mesenchymal stem cell (MSC) differentiation can be manipulated by nanotopographic interface providing unique strategy to engineering stem cell therapy and circumvent complex cellular reprogramming. However, our understanding of the nanotopographic-mechanosensitive properties of MSCs and the underlying biophysical linkage of the nanotopography-engineered stem cell to directed commitment, remains elusive. Here, we show that osteogenic differentiation of human MSCs (hMSCs) can be largely promoted using our nanoengineered topographic glass substrates in the absence of dexamethasone, a key exogenous factor for osteogenesis induction. We demonstrate that hMSCs sense and respond to surface nanotopography, through modulation of adhesion, cytoskeleton tension and nuclear activation of TAZ (transcriptional coactivator with PDZ-binding motif), a transcriptional modulator of hMSCs. Our findings demonstrate the potential of nanotopographic surfaces as non-invasive tools to advance cell-based therapies for bone engineering and highlight the origin of biophysical response of hMSC to nanotopography.
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Significance Metastasis is the primary cause of cancer-related deaths. Current clinical treatments for antimetastasis, however, are not effective. This work aims to develop a strategy to inhibit cancer cell migration using gold nanorods (AuNRs) with systematic understanding of the mechanism. The ability of targeting AuNRs to cancer cell surface integrins and the introduction of NIR light to generate a mild plasmonic photothermal effect caused broad regulation on cytoskeletal proteins, thus impairing cancer cell migration. This strategy provides a potential application for controlling cancer metastasis.
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Sensitive and specific fluorescence imaging-guided photothermal therapy (PTT) with high-efficiency is of essential importance and is still a challenge for nanotheranostics. To address these issues, we developed activatable ultrasmall gold nanorods (AUGNRs) to realize “off-on” switched fluorescence imaging-guided efficient PTT. Herein, the GNRs with an ultrasmall small size (∼4 nm) were set as the PTT platform due to their distinct absorption-dominant characteristics, generating an enhanced photothermal conversion efficiency. A near infrared (NIR) dye, Cy5, was conjugated to the surface of the ultrasmall GNRs for fluorescence imaging. Due to the strong localized surface plasmon resonance (LSPR), the fluorescence of Cy5 could be remarkably quenched by the GNRs and show an “off” state under normal conditions. As the AUGNRs are internalized by tumor cells, their ability of fluorescence imaging would be activated by glutathione (GSH) for the reducing action of GSH. Given the higher intracellular GSH concentration in tumor cells, a highly selective intracellular fluorescence imaging pattern was provided by the AUGNRs. As a result, the obtained AUGNRs revealed a uniformly rod-like structure with an aspect ratio of ∼4 and showed an enhanced photothermal conversion efficiency. The in vitro cellular uptake study indicated that the AUGNRs can efficiently enter the tumor cells. It has been demonstrated by in vitro Cy5 release profiles that the AUGNRs could achieve a triggered Cy5 release in response to GSH. The MTT assay and calcein AM/PI co-staining demonstrated that the cancer cells could be effectively killed when exposed to a NIR laser. Our work presents great potential for activated fluorescence imaging-guided PTT with high specificity and efficiency, as a promising method for future clinical cancer diagnostics and treatment.
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Efficient delivery of genes and therapeutic agents to the interior of the cell is critical for modern biotechnology. Herein, a new type of chemical-free cell poration method— hypersonic poration—is developed to improve the cellular uptake, especially the nucleus uptake. The hypersound (≈GHz) is generated by a designed piezoelectric nano-electromechanical resonator, which directly induces normal/shear stress and “molecular bombardment” effects on the bilayer membranes, and creates reversible temporal nanopores improving the membrane permeability. Both theory analysis and cellular uptake experiments of exogenous compounds prove the high delivery efficiency of hypersonic poration. Since target molecules in cells are accumulated with the treatment, the delivered amount can be controlled by tuning the treatment time. Furthermore, owing to the intrinsic miniature of the resonator, localized drug delivery at a confined spatial location and tunable arrays of the resonators that are compatible with multiwell plate can be achieved. The hypersonic poration method shows great delivery efficacy combined with advantage of scalability, tunable throughput, and simplification in operation and provides a potentially powerful strategy in the field of molecule delivery, cell transfection, and gene therapy.
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Mechanical stress is pervasive in egress routes of malignancy, yet the intrinsic effects of force on tumour cells remain poorly understood. Here, we demonstrate that frictional force characteristic of flow in the lymphatics stimulates YAP1 to drive cancer cell migration; whereas intensities of fluid wall shear stress (WSS) typical of venous or arterial flow inhibit taxis. YAP1, but not TAZ, is strictly required for WSS-enhanced cell movement, as blockade of YAP1, TEAD1-4 or the YAP1–TEAD interaction reduces cellular velocity to levels observed without flow. Silencing of TEAD phenocopies loss of YAP1, implicating transcriptional transactivation function in mediating force-enhanced cell migration. WSS dictates expression of a network of YAP1 effectors with executive roles in invasion, chemotaxis and adhesion downstream of the ROCK–LIMK–cofilin signalling axis. Altogether, these data implicate YAP1 as a fluid mechanosensor that functions to regulate genes that promote metastasis.
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Multifunctional drug delivery and combined multimodal therapy strategies are very promising in tumor theranostic applications. In this work, a simple and versatile nanoplatform based on biologically inspired polydopamine capped gold nanorods (GNR-PDA) is developed. Dopamine, a well-known neurotransmitter associated with many neuronal disorders, can undergo self-polymerization on the surface of GNRs to form a stable PDA shell. Its unique molecular adsorption property, as well as its high chemical stability and biocompatibility, facilitate GNR-PDA as an ideal candidate for drug delivery. Methylene blue (MB) and doxorubicin (DOX) are directly adsorbed on GNR-PDA via electrostatic and/or π-π stacking interactions, forming GNR-PDA-MB and GNR-PDA-DOX nanocomposites, respectively. The GNR-PDA-MB can generate reactive oxygen species (ROS, from MB) or hyperthermia (from GNR-PDA) with high efficiency under deep-red/NIR laser irradiation, while the GNR-PDA-DOX exhibits light-enhanced drug release under NIR laser irradiation. The combined dual-modal light-mediated therapy, by using GNR-PDA-MB [photodynamic/photothermal therapy (PDT/PTT)] and GNR-PDA-DOX (Chemo/PTT) is carried out and shows remarkable cancer cell killing efficiency in vitro and significant suppression of tumor growth in vivo, which are much more distinct than any single-modal therapy strategy. Our work illustrates that GNR-PDA could be a promising nanoplatform for multifunctional drug delivery and multimodal tumor theranostics in the future.
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Background: Cell stiffness is a crucial mechanical property that is closely related to cell motility. AFM is the most prevalent method used to determine cell stiffness by the quantitative parameter designated as Young's modulus. Young's modulus is regarded as a biomarker of cell motility, especially in estimating the metastasis of cancer cells, because in recent years, it has been repeatedly shown that cancerous cells are softer than their benign counterparts. However, some conflicting evidence has shown that cells with higher motility are sometimes stiffer than their counterparts. Thus, the correlation between cell stiffness and motility remains a matter of debate. Scope of review: In this review, we first summarize the reports on correlations between cell motility and stiffness determined by AFM and then discuss the major determinants of AFM-determined cell stiffness with a focus on the cytoskeleton, nuclear stiffness and methodological issues. Last, we propose a possible correlation between cell stiffness and motility and the possible explanations for the conflicting evidence. Major conclusions: The AFM-determined Young's modulus is greatly affected by the characteristics of the cytoskeleton, as well as the procedures and parameters used in detection. Young's modulus is a reliable biomarker for the characterization of metastasis; however, reliability is questioned in the evaluation of pharmacologically or genetically modified motility. General significance: This review provides an overview of the current understanding of the correlation between AFM-determined cell stiffness and motility, the determinants of this detecting method, as well as clues to optimize detecting parameters.
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ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvβ3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn2+ concentration prior to cell death. The response of the cells within the population to intracellular Zn2+ is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies.
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We study the correlation between cytoskeleton organization and stiffness of three epithelial breast cancer cells lines with different degree of malignancy: MCF-10A (healthy), MCF-7 (tumorigenic/non-invasive) and MDA-MB-231 (tumorigenic/invasive). Peak-force modulation atomic force microscopy is used for high-resolution topography and stiffness imaging of actin filaments within living cells. In healthy cells, local stiffness is maximum where filamentous actin is organized as well-aligned stress fibers, resulting in apparent Young's modulus values up to one order of magnitude larger than in regions where these structures are not observed. But these organized actin fibers are barely observed in tumorigenic cells. We further investigate cytoskeleton conformation in the three cell lines by immunofluorescence confocal microscopy. The combination of both techniques determines that actin stress fibers are present at apical regions of healthy cells, while in tumorigenic cells they appear only at basal regions, where they cannot contribute to stiffness as probed by atomic force microscopy. These results substantiate that actin stress fibers provide a dominant contribution to stiffness in healthy cells, while the elasticity of tumorigenic cells appears not predominantly determined by these structures. We also discuss the effects of the high-frequency indentations inherent to peak-force atomic force microscopy for the identification of mechanical cancer biomarkers. Whereas conventional low loading rate indentations (1 Hz) result in slightly differentiated average stiffness for each cell line, in high-frequency measurements (250 Hz) healthy cells are clearly discernible from both tumorigenic cells with an enhanced stiffness ratio; however, the two cancerous cell lines result undistinguishable.
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Gold nanomaterials are intensively studied for applications in disease detection, diagnosis and therapeutics, and this has motivated considerable research to determine their interaction with biomolecules, cells and cell behaviors. However, few studies look at how nanomaterials alter the extracellular matrix (ECM) and cell-ECM interactions. Nanomaterials in the body would interact with the entire cellular environment, and it is imperative to account for this when studying the impact of nanomaterials on living systems. Furthermore, recent evidence finds that migration rates of cells in 2D can be affected by nanomaterials and uptake of the nanomaterials is not necessary to exert an effect. In this study, three-dimensional nested type I collagen matrices were utilized as a model ECM to study how gold nanorods affect the migration of MDA-MB-231 human breast cancer cells. Spontaneous cell migration through collagen containing gold nanorods was found to increase with increasing concentrations of gold nanorods, independent of intracellular uptake of the nanorods. Gold nanorods in the collagen matrix were found to alter collagen mechanical properties and structure, molecular diffusion, cellular adhesion, cell morphology, mode of migration and protease expression. Correlation between decreased cellular adhesion and rounded cell morphology and locomotion in nanorod-containing collagen suggests the induction of an amoeboid-like migratory phenotype.
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Viscoelastic and other physical properties of cancerous cells are particularly important since cells interact with the extracellular matrix and other cells constantly during malignant proliferation, adhesion, invasion and metastasis process. Atomic force microscope (AFM) has an unparalleled advantage in the measurement of viscoelastic properties of living cells. In this paper, a stress relaxation test using atomic force microscopy was conducted to obtain viscoelastic characteristics of lung cancer cells. The experimental data obtained were well fitted with a special theoretical model which is appropriate to samples with infinite thickness, such as cells. This theoretical model takes into account the thin thickness of the measured sample and the substrate effect generated by a relatively larger indention of AFM probe can be avoided. Two different non-small cell lung cancer cell lines with varying metastatic potential show distinct stress relaxation characteristics. The metastatic NCI-H1299 cells, which are originally isolated from a patient's lymph node metastases, appeared a lower viscoelastic response compared to the non-metastatic A549 tumorous cells. When cancerous cells release from the primary tumor site, intravasate into lymphatic or blood circulation, and squeeze through a variety of cell gaps to transfer other where, they become easily deformed and thus show lower viscoelastic properties. The emerging insight into these viscoelastic properties may promote the understanding of the underlying mechanism for cancer metastatic and invasive progress.
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Nanoparticle vehicles may improve the delivery of contrast agents and therapeutics to diseased tissues, but their rational design is currently impeded by a lack of robust technologies to characterize their in vivo behavior in real-time. This study demonstrates that fluorescent-labeled gold nanoparticles can be optimized for in vivo detection, perform pharmacokinetic analysis of nanoparticle designs, analyze tumor extravasation, and clearance kinetics in tumor-bearing animals. This optical imaging approach is non-invasive and high-throughput. Interestingly, these fluorescent gold nanoparticles can be used for multispectral imaging to compare several nanoparticle designs simultaneously within the same animal and eliminates the host-dependent variabilities across measured data. Together these results describe a novel platform for evaluating the performance of tumor-targeting nanoparticles, and provide new insights for the design of future nanotherapeutics.
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The hydrodynamic radius, rh, of low molar mass polyethylene glycol, MPEG = (200 to 1000) g·mol−1, in a homologous series of primary alcohols, acetone, and toluene has been determined from viscosity measurements. The viscosity data have been collected using a fast one-point method as well as a more generally used multipoint method. The results for both approaches are in good agreement. For a given average molar mass of PEG, rh is the largest in acetone, methanol, and toluene and shows a decrease with the chain length of the alcohol. For the solvents studied, rh shows an increase with MPEG that can be described adequately by the two-parameter Mark–Houwink equation for MPEG = (400 to 1000) g·mol−1. In the range T = (298.2 to 323.2) K, the influence of the temperature is not significant.
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Cell volume perturbation initiates a wide array of intracellular signalling cascades, leading to protective and adaptive events and, in most cases, activation of volume-regulatory osmolyte transport, water loss, and hence restoration of cell volume and cellular function. Cell volume is challenged not only under physiological conditions, e.g. following accumulation of nutrients, during epithelial absorption/secretion processes, following hormonal/autocrine stimulation, and during induction of apoptosis, but also under pathophysiological conditions, e.g. hypoxia, ischaemia and hyponatremia/hypernatremia. On the other hand, it has recently become clear that an increase or reduction in cell volume can also serve as a specific signal in the regulation of physiological processes such as transepithelial transport, cell migration, proliferation and death. Although the mechanisms by which cell volume perturbations are sensed are still far from clear, significant progress has been made with respect to the nature of the sensors, transducers and effectors that convert a change in cell volume into a physiological response. In the present review, we summarize recent major developments in the field, and emphasize the relationship between cell volume regulation and organism physiology/pathophysiology.