Computer-controlled microcirculatory support system for endothelial cell culture and shearing

Endothelial cells (ECs) lining the inner lumen of blood vessels are continuously subjected to hemodynamic shear stress, which is known to modify EC morphology and biological activity. This paper describes a self-contained microcirculatory EC culture system that efficiently studies such effects of shear stress on EC alignment and elongation in vitro. The culture system is composed of elastomeric microfluidic cell shearing chambers interfaced with computer-controlled movement of piezoelectric pins on a refreshable Braille display. The flow rate is varied by design of channels that allow for movement of different volumes of fluid per variable-speed pump stroke. The integrated microfluidic valving and pumping system allowed primary EC seeding and differential shearing in multiple compartments to be performed on a single chip. The microfluidic flows caused ECs to align and elongate significantly in the direction of flow according to their exposed levels of shear stress. This microfluidic system overcomes the small flow rates and the inefficiencies of previously described microfluidic and macroscopic systems respectively to conveniently perform parallel studies of EC response to shear stress.     

Elucidating the Influences of Size,Surface Chemistry,and Dynamic Flow on Cellular Association of Nanoparticles Made by Polymerization‐Induced Self‐Assembly

The size and surface chemistry of nanoparticles dictate their interactions with biological systems. However, it remains unclear how these key physicochemical properties affect the cellular association of nanoparticles under dynamic flow conditions encountered in human vascular networks. Here, the facile synthesis of novel fluorescent nanoparticles with tunable sizes and surface chemistries and their association with primary human umbilical vein endothelial cells (HUVECs) is reported. First, a one‐pot polymerization‐induced self‐assembly (PISA) methodology is developed to covalently incorporate a commercially available fluorescent dye into the nanoparticle core and tune nanoparticle size and surface chemistry. To characterize cellular association under flow, HUVECs are cultured onto the surface of a synthetic microvascular network embedded in a microfluidic device (SynVivo, INC). Interestingly, increasing the size of carboxylic acid–functionalized nanoparticles leads to higher cellular association under static conditions but lower cellular association under flow conditions, whereas increasing the size of tertiary amine–decorated nanoparticles results in a higher level of cellular association, under both static and flow conditions. These findings provide new insights into the interactions between polymeric nanomaterials and endothelial cells. Altogether, this work establishes innovative methods for the facile synthesis and biological characterization of polymeric nanomaterials for various potential applications.     

On-chip evaluation of shear stress effect on cytotoxicity of mesoporous silica nanoparticles

In this work, nanotoxicity in the bloodstream was modeled, and the cytotoxicity of sub-50 nm mesoporous silica nanoparticles to human endothelial cells was investigated under microfluidic flow conditions. Compared to traditional in vitro cytotoxicity assays performed under static conditions, unmodified mesoporous silica nanoparticles show higher and shear stress-dependent toxicity to endothelial cells under flow conditions. Interestingly, even under flow conditions, highly organo-modified mesoporous silica nanoparticles show no significant toxicity to endothelial cells. This paper clearly demonstrates that shear stress is an important factor to be considered in in vitro nanotoxicology assessments and provides a simple device for pursuing this consideration.     

Laser-induced damage and recovery of plasmonically targeted human endothelial cells

Laser-induced techniques that employ the surface plasmon resonances of nanoparticles have recently been introduced as an effective therapeutic tool for destroying tumor cells. Here, we adopt a low-intensity laser-induced technique to manipulate the damage and repair of a vital category of noncancerous cells, human endothelial cells. Endothelial cells construct the interior of blood vessels and play a pivotal role in angiogenesis. The degree of damage and repair of the cells is shown to be influenced by laser illumination in the presence of gold nanoparticles of different morphologies, which either target the cellular membrane or are endocytosed. A pronounced influence of the plasmonic nanoparticle laser treatment on the expression of critical angiogenic genes is shown. Our results show that plasmon-mediated mild laser treatment, combined with specific targeting of cellular membranes, enables new routes for controlling cell permeability and gene regulation in endothelial cells.     

High‐Throughput Continuous Flow Production of Nanoscale Liposomes by Microfluidic Vertical Flow Focusing

Liposomes represent a leading class of nanoparticles for drug delivery. While a variety of techniques for liposome synthesis have been reported that take advantage of microfluidic flow elements to achieve precise control over the size and polydispersity of nanoscale liposomes, with important implications for nanomedicine applications, these methods suffer from extremely limited throughput, making them impractical for large‐scale nanoparticle synthesis. High aspect ratio microfluidic vertical flow focusing is investigated here as a new approach to overcoming the throughput limits of established microfluidic nanoparticle synthesis techniques. Here the vertical flow focusing technique is utilized to generate populations of small, unilamellar, and nearly monodisperse liposomal nanoparticles with exceptionally high production rates and remarkable sample homogeneity. By leveraging this platform, liposomes with modal diameters ranging from 80 to 200 nm are prepared at production rates as high as 1.6 mg min⁻¹ in a simple flow‐through process.     

Quantitative assessment of the comparative nanoparticle-uptake efficiency of a range of cell lines

The mechanism(s) of nanoparticle-cell interactions are still not understood. At present there is little knowledge of the relevant length- and timescales for nanoparticle intracellular entry and localization within cells, or the cell-specificity of nanoparticle uptake and localisation. Here, the effect of particle size on the in-vitro intracellular uptake of model fluorescent carboxyl-modified polystyrene nanoparticles is investigated in various cell lines. A range of micro- and nanoparticles of defined sizes (40 nm to 2 μm) are incubated with a series of cell types, including HeLa and A549 epithelial cells, 1321N1 astrocytes, HCMEC D3 endothelial cells, and murine RAW 264.7 macrophages. Techniques such as confocal microscopy and flow cytometry are used to study particle uptake and subcellular localisation, making significant efforts to ensure reproducibility in a semiquantitative approach. The results indicate that internalization of (nano)particles is highly size-dependent for all cell lines studied, and the kinetics of uptake for the same type of nanoparticle varies in the different cell types. Interestingly, even cells not specialized for phagocytosis are able to internalize the larger nanoparticles. Intracellular uptake of all sizes of particles is observed to be highest in RAW 264.7 cells (a specialized phagocytic cell line) and the lowest in the HeLa cells. These results suggest that (nano)particle uptake might not follow commonly defined size limits for uptake processes, and highlight the variability of uptake kinetics for the same material in different cell types. These conclusions have important implications for the assessment of the safety of nanomaterials and for the potential biomedical applications of nanoparticles.     

Zebrafish Embryos Allow Prediction of Nanoparticle Circulation Times in Mice and Facilitate Quantification of Nanoparticle–Cell Interactions

The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real‐time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.     

Amorphous Silica Nanoparticles Promote Monocyte Adhesion to Human Endothelial Cells: Size‐Dependent Effect

There is evidence that nanoparticles can induce endothelial dysfunction. Here, the effect of monodisperse amorphous silica nanoparticles (SiO₂‐NPs) of different diameters on endothelial cells function is examined. Human endothelial cell line (EA.hy926) or primary human pulmonary artery endothelial cells (hPAEC) are seeded in inserts introduced or not above triple cell co‐cultures (pneumocytes, macrophages, and mast cells). Endothelial cells are incubated with SiO₂‐NPs at non‐cytotoxic concentrations for 12 h. A significant increase (up to 2‐fold) in human monocytes adhesion to endothelial cells is observed for 18 and 54 nm particles. Exposure to SiO₂‐NPs induces protein expression of adhesion molecules (ICAM‐1 and VCAM‐1) as well as significant up‐regulation in mRNA expression of ICAM‐1 in both endothelial cell types. Experiments performed with fluorescent‐labelled monodisperse amorphous SiO₂‐NPs of similar size evidence nanoparticle uptake into the cytoplasm of endothelial cells. It is concluded that exposure of human endothelial cells to amorphous silica nanoparticles enhances their adhesive properties. This process is modified by the size of the nanoparticle and the presence of other co‐cultured cells.     

Integrating Flexible Electrochemical Sensor into Microfluidic Chip for Simulating and Monitoring Vascular Mechanotransduction

As an interface between the blood flow and vessel wall, endothelial cells (ECs) are exposed to hemodynamic forces, and the biochemical molecules released from ECs–blood flow interaction are important determinants of vascular homeostasis. Versatile microfluidic chips have been designed to simulate the biological and physiological parameters of the human vascular system, but in situ and real‐time monitoring of the mechanical force–triggered signals during vascular mechanotransduction still remains a significant challenge. Here, such challenge is fulfilled for the first time, by preparation of a flexible and stretchable electrochemical sensor and its incorporation into a microfluidic vascular chip. This allows simulating of in vivo physiological and biomechanical parameters of blood vessels, and simultaneously monitoring the mechanically induced biochemical signals in real time. Specifically, the cyclic circumferential stretch that is actually exerted on endothelium but is hard to reproduce in vitro is successfully recapitulated, and nitric oxide signals under normal blood pressure, as well as reactive oxygen species signals under hypertensive states, are well documented. Here, the first integration of a flexible electrochemical sensor into a microfluidic chip is reported, therefore paving a way to evaluate in vitro organs by built‐in flexible sensors.     

Microfluidic platform for controlled synthesis of polymeric nanoparticles

A central challenge in the development of drug-encapsulated polymeric nanoparticles is the inability to control the mixing processes required for their synthesis resulting in variable nanoparticle physicochemical properties. Nanoparticles may be developed by mixing and nanoprecipitation of polymers and drugs dissolved in organic solvents with nonsolvents. We used rapid and tunable mixing through hydrodynamic flow focusing in microfluidic channels to control nanoprecipitation of poly(lactic- co-glycolic acid)- b-poly(ethylene glycol) diblock copolymers as a model polymeric biomaterial for drug delivery. We demonstrate that by varying (1) flow rates, (2) polymer composition, and (3) polymer concentration we can optimize the size, improve polydispersity, and control drug loading and release of the resulting nanoparticles. This work suggests that microfluidics may find applications for the development and optimization of polymeric nanoparticles in the newly emerging field of nanomedicine.     

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer‐related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time‐consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h^?1 in contrast to a flow rate of 1 mL h^?1 standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.     

Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition

This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.     

Erythrocyte–Platelet Hybrid Membrane Coating for Enhanced Nanoparticle Functionalization

Cell‐membrane‐coated nanoparticles have recently been studied extensively for their biological compatibility, retention of cellular properties, and adaptability to a variety of therapeutic and imaging applications. This class of nanoparticles, which has been fabricated with a variety of cell membrane coatings, including those derived from red blood cells (RBCs), platelets, white blood cells, cancer cells, and bacteria, exhibit properties that are characteristic of the source cell. In this study, a new type of biological coating is created by fusing membrane material from two different cells, providing a facile method for further enhancing nanoparticle functionality. As a proof of concept, the development of dual‐membrane‐coated nanoparticles from the fused RBC membrane and platelet membrane is demonstrated. The resulting particles, termed RBC–platelet hybrid membrane‐coated nanoparticles ([RBC‐P]NPs), are thoroughly characterized, and it is shown that they carry properties of both source cells. Further, the [RBC‐P]NP platform exhibits long circulation and suitability for further in vivo exploration. The reported strategy opens the door for the creation of biocompatible, custom‐tailored biomimetic nanoparticles with varying hybrid functionalities, which may be used to overcome the limitations of current nanoparticle‐based therapeutic and imaging platforms.     

Targeted biodegradable nanoparticles for drug delivery to smooth muscle cells

Targeted delivery of therapeutic agents to prevent smooth muscle cell (SMC) proliferation is important in averting restenosis (a narrowing of blood vessels). Since platelet derived growth factor (PDGF) receptors are over-expressed in proliferating SMCs after injury from cardiovascular interventions, such as angioplasty and stent implantation, our hypothesis is that conjugation of PDGF-BB (platelet-derived growth factor BB (homodimer)) peptides to biodegradable poly (D,L-lactic-co-glycolide) (PLGA) nanoparticles (NPs) would exhibit an increased uptake of these NPs by proliferating SMCs. In this study, poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles containing dexamethasone were formulated and conjugated with PDGF-BB peptides. These NPs were stable, biocompatible, and exhibited a sustained drug release over 14 days. Various particle uptake studies using HASMCs (human aortic smooth muscle cells) demonstrated that PDGF-BB peptide-conjugated nanoparticles significantly increased cellular uptake and decreased proliferation of HASMCs compared to control nanoparticles (without conjugation of PDGF-BB peptides). These NPs were internalized primarily by clathrin-mediated endocytosis and macropinocytosis. Our in vitro results suggest that PDGF-BB peptide-conjugated NPs could represent as an effective targeted, sustained therapeutic delivery system to reduce restenosis and neointimal hyperplasia.     

Endothelial cell culture model for replication of physiological profiles of pressure, flow, stretch, and shear stress in vitro

The phenotype and function of vascular cells in vivo are influenced by complex mechanical signals generated by pulsatile hemodynamic loading. Physiologically relevant in vitro studies of vascular cells therefore require realistic environments where in vivo mechanical loading conditions can be accurately reproduced. To accomplish a realistic in vivo-like loading environment, we designed and fabricated an Endothelial Cell Culture Model (ECCM) to generate physiological pressure, stretch, and shear stress profiles associated with normal and pathological cardiac flow states. Cells within this system were cultured on a stretchable, thin (～500 μm) planar membrane within a rectangular flow channel and subject to constant fluid flow. Under pressure, the thin planar membrane assumed a concave shape, representing a segment of the blood vessel wall. Pulsatility was introduced using a programmable pneumatically controlled collapsible chamber. Human aortic endothelial cells (HAECs) were cultured within this system under normal conditions and compared to HAECs cultured under static and "flow only" (13 dyn/cm(2)) control conditions using microscopy. Cells cultured within the ECCM were larger than both controls and assumed an ellipsoidal shape. In contrast to static control control cells, ECCM-cultured cells exhibited alignment of cytoskeletal actin filaments and high and continuous expression levels of β-catenin indicating an in vivo-like phenotype. In conclusion, design, fabrication, testing, and validation of the ECCM for culture of ECs under realistic pressure, flow, strain, and shear loading seen in normal and pathological conditions was accomplished. The ECCM therefore is an enabling technology that allows for study of ECs under physiologically relevant biomechanical loading conditions in vitro.     

Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.     

Cellular Interactions of Liposomes and PISA Nanoparticles during Human Blood Flow in a Microvascular Network

A key concept in nanomedicine is encapsulating therapeutic or diagnostic agents inside nanoparticles to prolong blood circulation time and to enhance interactions with targeted cells. During circulation and depending on the selected application (e.g., cancer drug delivery or immune modulators), nanoparticles are required to possess low or high interactions with cells in human blood and blood vessels to minimize side effects or maximize delivery efficiency. However, analysis of cellular interactions in blood vessels is challenging and is not yet realized due to the diverse components of human blood and hemodynamic flow in blood vessels. Here, the first comprehensive method to analyze cellular interactions of both synthetic and commercially available nanoparticles under human blood flow conditions in a microvascular network is developed. Importantly, this method allows to unravel the complex interplay of size, charge, and type of nanoparticles on their cellular associations under the dynamic flow of human blood. This method offers a unique platform to study complex interactions of any type of nanoparticles in human blood flow conditions and serves as a useful guideline for the rational design of liposomes and polymer nanoparticles for diverse applications in nanomedicine.     

Uptake of silica-coated nanoparticles by HeLa cells

Nanoparticles have seen wide applications in cellular research and development. One major issue that is unclear is the uptake of nanoparticles by cells. In this study, we have investigated the uptake of silica-coated nanoparticles by HeLa cells, employing rhoadime 6G isothiocyanate (RITC)-doped nanoparticles as a synchronous fluorescent signal indicator. These nanoparticles were synthesized with reverse microemulsion. A few factors, such as nanoparticle concentration, incubation time and temperature, and serum and inhibitors in culture medium were assessed on the nanoparticle's cellular uptake. The experimental results demonstrated that uptake was maximum after a 6 h incubation and was higher at 37 degrees C than that at 4 degrees C. Nanoparticle uptake depended on the nanoparticle concentration and was inhibited by hyperosmolarity, K+ depletion. In addition, serum in culture medium decreased the cellular uptake of nanoparticles. The results indicated that the uptake of silica-coated nanoparticles by HeLa cells was a concentration-, time-, and energy-dependent endocytic process. Silica-coated nanoparticles could be transported into HeLa cells in part through adsorptive endocytosis and in part through fluid-phase endocytosis.     

In vivo measurement of blood flow in a micro‐scale stenosis model generated by laser photothermal blood coagulation

Blood flow in a stenosed vessel is one of the most important issues, because it is closely related to the outbreak of circulatory diseases. To overcome the technological limitations encountered in the haemodynamic studies using in vitro stenosis models, the authors induced a stenosed flow model in the extraembryonic vessels of a chicken embryo. Blood was coagulated by laser irradiation to artificially form a stenosis on the designated spot in a straight blood vessel. Owing to photothermal coagulation of red blood cells (RBCs), the blood is denatured and a stable blood coagulum is induced in the vessel. The blood coagulum adheres firmly and stably on the vessel wall without any size variation. It disturbs the on‐coming blood flow significantly. To investigate the haemodynamic characteristics of the blood flow in the stenosed vessel, a micro particle image velocimetry technique was employed using RBCs as tracers to measure the spatial distributions of velocity vectors, streamlines and shear rate. The present simple modelling of in vivo stenosis would be useful for investigating the basic haemodynamic mechanisms underlying circulatory vascular diseases.Inspec keywords: adhesion, biological effects of laser radiation, biomedical optical imaging, blood, blood flow measurement, blood vessels, cellular biophysics, coagulation, diseases, flow visualisation, laser applications in medicine, photothermal effects, shear flowOther keywords: in vivo measurement, blood flow, microscale stenosis model, laser photothermal blood coagulation, stenosed vessel, circulatory diseases, haemodynamics, extraembryonic vessels, chicken embryo, laser irradiation, blood vessel, red blood cells, blood coagulum, adherence, vessel wall, microparticle image velocimetry, shear rate, circulatory vascular diseasesInspec keywords: adhesion, biological effects of laser radiation, biomedical optical imaging, blood, blood flow measurement, blood vessels, cellular biophysics, coagulation, diseases, flow visualisation, laser applications in medicine, photothermal effects, shear flowOther keywords: in vivo measurement, blood flow, microscale stenosis model, laser photothermal blood coagulation, stenosed vessel, circulatory diseases, haemodynamics, extraembryonic vessels, chicken embryo, laser irradiation, blood vessel, red blood cells, blood coagulum, adherence, vessel wall, microparticle image velocimetry, shear rate, circulatory vascular diseases     

Endothelial cell alignment on cyclically-stretched silicone surfaces

Endothelial cells at the interface between the bloodstream and the vessel wall are continuously subjected to mechanical stimulation in vivo, and it widely recognised that such stimulation plays an important role in cardiovascular physiology. Cell deformation is induced by mechanical forces such as cyclic stretch, fluid shear stress, and transmural pressure. Although much of the work in this field has dealt with the effect of fluid shear stress, very little is known about how cyclic forces modulate and alter the morphology of single endothelial cells, and thereafter, how they effect the confluent layer of endothelial cells lining the vessel wall. The aim of this study is to investigate the response of endothelial cells when subjected to substrate deformation of similar magnitude to those experienced in vivo. Human umbilical vein endothelial cells (HUVEC) were cultured on plasma-treated silicone strips and uni-axially cyclically stretched using a custom made mechanical device. Results showed that endothelial cells subject to 10% deformation for as little as 4 h reoriented perpendicular to the stretch direction. In addition, although no integrin coating was applied to the substrate, it was found that plasma-treated silicone provided a cell adhesion substrate comparable to the commonly used collagen type I. Thus the results show that the stretch stimulus alone affects the morphology of endothelial cells. Further studies are required to establish the relative importance of substrate strain vs. fluid flow stimuli.