A novel 3D intestine barrier model to study the immune response upon exposure to microplastics.

The plausibility of human exposure to microplastics has increased within the last years. Microplastics have been found in different food types including seafood, salt, sugar and beverages. So far, human health effects of microplastics after ingestion are unknown. Herein, we designed a novel, three-dimensional in vitro intestinal model consisting of the human intestinal epithelial cell lines Caco-2 and HT29-MTX-E12 as well as human blood monocyte-derived macrophages and dendritic cells that is suitable to assess the possible effects of ingested microplastics. Relevant microplastic particles (in the order of 50-500 µm), including polymers representing tire wear and polyolefins, which represent major sources of microplastic in the EU, were compared to other polymer classes and an inorganic microparticle, healing earth, which is intended for human consumption. Microplastic particles were exposed at concentrations of 823.5-1380.0 µg/cm2 to the model using a dry powder insufflator system to aerosolize the particles directly on the intestinal model's surface. Cytotoxicity was investigated after 6, 24 and 48 h of exposure via measuring the release of lactate dehydrogenase. Inflammatory end points including the cytokines IL-8, TNFα and IL-1ß as well as changes of the barrier integrity after exposure were additionally monitored. We demonstrated that all of the microplastics and the healing earth particles did not cause any significant cytotoxicity or release of (pro-)inflammatory cytokines and did not change the barrier integrity of the co-culture at any of the time points investigated.

An In Vitro Lung System to Assess the Proinflammatory Hazard of Carbon Nanotube Aerosols.

In vitro three-dimensional (3D) lung cell models have been thoroughly investigated in recent years and provide a reliable tool to assess the hazard associated with nanomaterials (NMs) released into the air. In this study, a 3D lung co-culture model was optimized to assess the hazard potential of multiwalled carbon nanotubes (MWCNTs), which is known to provoke inflammation and fibrosis, critical adverse outcomes linked to acute and prolonged NM exposure. The lung co-cultures were exposed to MWCNTs at the air-liquid interface (ALI) using the VITROCELL® Cloud system while considering realistic occupational exposure doses. The co-culture model was composed of three human cell lines: alveolar epithelial cells (A549), fibroblasts (MRC-5), and macrophages (differentiated THP-1). The model was exposed to two types of MWCNTs (Mitsui-7 and Nanocyl) at different concentrations (2-10 µg/cm2) to assess the proinflammatory as well as the profibrotic responses after acute (24 h, one exposure) and prolonged (96 h, repeated exposures) exposure cycles. The results showed that acute or prolonged exposure to different concentrations of the tested MWCNTs did not induce cytotoxicity or apparent profibrotic response; however, suggested the onset of proinflammatory response.

Glyco-functionalized dinuclear rhenium(i) complexes for cell imaging.

The design, synthesis and photophysical characterization of four new luminescent glycosylated luminophores based on dinuclear rhenium complexes, namely Glyco-Re, are described. The derivatives have the general formula [Re2(µ-Cl)2(CO)6(µ-pydz-R)] (R-pydz = functionalized 1,2-pyridazine), where a sugar residue (R) is covalently bound to the pyridazine ligand in the ß position. Different synthetic pathways have been investigated including the so-called neo-glycorandomization procedure, affording stereoselectively glyco-conjugates containing glucose and maltose in a ß anomeric configuration. A multivalent dinuclear rhenium glycodendron bearing three glucose units is also synthesized. All the Glyco-Re conjugates are comprehensively characterized and their photophysical properties and cellular internalization experiments on human cervical adenocarcinoma (HeLa) cells are reported. The results show that such Glyco-Re complexes display interesting bio-imaging properties, i.e. high cell permeability, organelle selectivity, low cytotoxicity and fast internalization. These findings make the presented Glyco-Re derivatives efficient phosphorescent probes suitable for cell imaging application.

Biodistribution of single and aggregated gold nanoparticles exposed to the human lung epithelial tissue barrier at the air-liquid interface.

BACKGROUND: The lung represents the primary entry route for airborne particles into the human body. Most studies addressed possible adverse effects using single (nano)particles, but aerosolic nanoparticles (NPs) tend to aggregate and form structures of several hundreds nm in diameter, changing the physico-chemical properties and interaction with cells. Our aim was to investigate how aggregation might affect the biodistribution; cellular uptake and translocation over time of aerosolized NPs at the air-blood barrier interface using a multicellular lung system. RESULTS: Model gold nanoparticles (AuNPs) were engineered and well characterized to compare single NPs with aggregated NPs with hydrodynamic diameter of 32 and 106 nm, respectively. Exposures were performed by aerosolization of the particles onto the air-liquid interface of a three dimensional (3D) lung model. Particle deposition, cellular uptake and translocation kinetics of single and aggregated AuNPs were determined for various concentrations, (30, 60, 150 and 300 ng/cm2) and time points (4, 24 and 48 h) using transmission electron microscopy and inductively coupled plasma optical emission spectroscopy. No apparent harmful effect for single and aggregated AuNPs was observed by lactate dehydrogenase assay, nor pro-inflammation response by tumor necrosis factor α assessment. The cell layer integrity was also not impaired. The bio-distribution revealed that majority of the AuNPs, single or aggregated, were inside the cells, and only a minor fraction, less than 5%, was found on the basolateral side. No significant difference was observed in the translocation rate. However, aggregated AuNPs showed a significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h. CONCLUSIONS: Our studies revealed that aggregated AuNPs showed significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h, but the uptake rate was similar at later time points. In addition, aggregation did not affect translocation rate across the lung barrier model since similar translocation rates were observed for single as well as aggregated AuNPs.

Breakable Hybrid Organosilica Nanocapsules for Protein Delivery.

The direct delivery of specific proteins to live cells promises a tremendous impact for biological and medical applications, from therapeutics to genetic engineering. However, the process mostly involves tedious techniques and often requires extensive alteration of the protein itself. Herein we report a straightforward approach to encapsulate native proteins by using breakable organosilica matrices that disintegrate upon exposure to a chemical stimulus. The biomolecule-containing capsules were tested for the intracellular delivery of highly cytotoxic proteins into C6 glioma cells. We demonstrate that the shell is broken, the release of the active proteins occurs, and therefore our hybrid architecture is a promising strategy to deliver fragile biomacromolecules into living organisms.

Combined Delivery of Temozolomide and Anti-miR221 PNA Using Mesoporous Silica Nanoparticles Induces Apoptosis in Resistant Glioma Cells.

Mesoporous silica nanoparticles (MSNPs), 100 nm in size, incorporating a Cy5 fluorophore within the silica framework, are synthesized and loaded with the anti-cancer drug temozolomide (TMZ), used in the treatment of gliomas. The surface of the particles is then decorated, using electrostatic interactions, with a polyarginine-peptide nucleic acid (R8-PNA) conjugate targeting the miR221 microRNA. The multi-functional nanosystem thus obtained is rapidly internalized into glioma C6 or T98G cells. The anti-miR activity of the PNA is retained, as confirmed by reverse transcription polymerase chain reaction (RT-PCR) measurements and induction of apoptosis is observed in temozolomide-resistant cell lines. The TMZ-loaded MSNPs show an enhanced pro-apoptotic effect, and the combined effect of TMZ and R8-PNA in the MSNPs shows the most effective induction of apoptosis (70.9% of apoptotic cells) thus far achieved in the temozolomide-resistant T98G cell line.

Gold nanoparticles explore cells: cellular uptake and their use as intracellular probes.

Understanding uptake of nanomaterials by cells and their use for intracellular sensing is important for studying their interaction and toxicology as well as for obtaining new biological insight. Here, we investigate cellular uptake and intracellular dynamics of gold nanoparticles and demonstrate their use in reporting chemical information from the endocytotic pathway and cytoplasm. The intracellular gold nanoparticles serve as probes for surface-enhanced Raman spectroscopy (SERS) allowing for biochemical characterisation of their local environment. In particular, in this work we compare intracellular SERS using non-functionalised and functionalised nanoparticles in their ability to segregate different but closely related cell phenotypes. The results indicate that functionalised gold nanoparticles are more efficient in distinguishing between different types of cells. Our studies pave the way for understanding the uptake of gold nanoparticles and their utilisation for SERS to give rise to a greater biochemical understanding in cell-based therapies.

When self-assembly meets biology: luminescent platinum complexes for imaging applications.

Luminescent platinum complexes have attractive chemical and photophysical properties such as high stability, emission in the visible region, high emission quantum yields and long excited state lifetimes. However the absorption spectrum of the compounds in the UV region, preventing their excitation in the harmless visible/red region, as well as the strong quenching of the luminescent triplet state, caused by dioxygen in water and biological fluids, reduces their possible applications for imaging. Therefore a possible solution to these drawbacks is to take advantage of the high tendency of such square planar compounds to self-assemble in supramolecular structures. The assemblies can be considered new chemical species with enhanced and tunable properties. Furthermore the assembly and disassembly process can be explored as a tool to obtain dynamic labels that can be applied in biomedicine. The change in color, the turn on and off of luminescence but also of the reactivity, the protection from quenching and environmental degradation are some of the attractive properties connected to the aggregation of the complexes.

Multifunctional inorganic nanocontainers for DNA and drug delivery into living cells.

The design and synthesis of multifunctional nanomaterials could lead to applications relevant for biomedicine. Manufacturing porous particles to make them able to carry bioactive molecules into living cells represents a substantial goal towards the development of powerful tools for nanomedicine. This work describes a first example of using zeolite-L crystals as multifunctional nanocontainers to simultaneously deliver DNA oligonucleotides and organic molecules into living cells. Multifunctional zeolite-L was prepared by filling the pore system with guest molecules, whilst DNA was adsorbed electrostatically on their surface. The release kinetics of DNA and of the guest molecules into living cells was studied to prove the multiple-drug-delivery ability of the system. The localization of all the components in different cellular compartments was followed. The presented system may be a prototype for the development of novel nanoparticles for drug delivery and gene therapy.

A 3D multi-cellular tissue model of the human omentum to study the formation of ovarian cancer metastasis.

Reliable and predictive experimental models are urgently needed to study metastatic mechanisms of ovarian cancer cells in the omentum. Although models for ovarian cancer cell adhesion and invasion were previously investigated, the lack of certain omental cell types, which influence the metastatic behavior of cancer cells, limits the application of these tissue models. Here, we describe a 3D multi-cellular human omentum tissue model, which considers the spatial arrangement of five omental cell types. Reproducible tissue models were fabricated combining permeable cell culture inserts and bioprinting technology to mimic metastatic processes of immortalized and patient-derived ovarian cancer cells. The implementation of an endothelial barrier further allowed studying the interaction between cancer and endothelial cells during hematogenous dissemination and the impact of chemotherapeutic drugs. This proof-of-concept study may serve as a platform for patient-specific investigations in personalized oncology in the future.

Cellular lasers for cell imaging and biosensing.

The possibility to produce laser action involving biomaterials, in particular (single) biological cells, has fostered the development of cellular lasers as a novel approach in biophotonics. In this respect, cells that are engineered to carry gain medium (e.g., fluorescent dyes or proteins) are placed inside an optical cavity (i.e., typically a sandwich of highly reflective mirrors), allowing the generation of stimulated emission upon sufficient optical pumping. In another scenario, micron-sized optical resonators supporting whispering-gallery mode (WGM) or semiconductor-based laser probes can be internalized by the cells and support light amplification. This review summarizes the recent advances in the fields of biolasers and cellular lasers, and most importantly, highlights their potential applications in the fields of in vitro and in vivo cell imaging and analysis. They include biosensing (e.g., in vitro detection of sodium chloride (NaCl) concentration), cancer cell imaging, laser-emission-based microscope, cell tracking, cell distinction study, and tissue contraction monitoring in zebrafish. Lastly, several fundamental issues in developing cellular lasers including laser probe fabrication, biocompatibility of the system, and alteration of local refractive index of optical cavities due to protein absorption or probe aggregation are described. Cellular lasers are foreseen as a promising tool to study numerous biological and biophysical phenomena. STATEMENT OF SIGNIFICANCE: Biolasers are generation of laser involving biological materials. Biomaterials, including single cells, can be engineered to incorporate laser probes or fluorescent proteins or fluorophores, and the resulting light emission can be coupled to optical resonator, allowing generation of cellular laser emission upon optical pumping. Unlike fluorescence, this stimulated emission is very sensitive and is capable of detecting small alterations in the optical property of the cells and their environment. In this review, recent development and applications of cellular lasers in the fields of in vitro and in vivo cell imaging, cell tracking, biosensing, and cell/tissue analysis are highlighted. Several challenges in developing cellular lasers including probe fabrication and biocompatibility as well as alteration of cellular environment are explained.

A Near-Infrared Mechanically Switchable Elastomeric Film as a Dynamic Cell Culture Substrate.

Commercial static cell culture substrates can usually not change their physical properties over time, resulting in a limited representation of the variation in biomechanical cues in vivo. To overcome this limitation, approaches incorporating gold nanoparticles to act as transducers to external stimuli have been employed. In this work, gold nanorods were embedded in an elastomeric matrix and used as photothermal transducers to fabricate biocompatible light-responsive substrates. The nanocomposite films analysed by lock-in thermography and nanoindentation show a homogeneous heat distribution and a greater stiffness when irradiated with NIR light. After irradiation, the initial stiffness values were recovered. In vitro experiments performed during NIR irradiation with NIH-3T3 fibroblasts demonstrated that these films were biocompatible and cells remained viable. Cells cultured on the light stiffened nanocomposite exhibited a greater proliferation rate and stronger focal adhesion clustering, indicating increased cell-surface binding strength.

Substrate stiffness reduces particle uptake by epithelial cells and macrophages in a size-dependent manner through mechanoregulation.

Cells continuously exert forces on their environment and respond to changes in mechanical forces by altering their behaviour. Many pathologies such as cancer and fibrosis are hallmarked by dysregulation in the extracellular matrix, driving aberrant behaviour through mechanotransduction pathways. We demonstrate that substrate stiffness can be used to regulate cellular endocytosis of particles in a size-dependent fashion. Culture of A549 epithelial cells and J774A.1 macrophages on polystyrene/glass (stiff) and polydimethylsiloxane (soft) substrates indicated that particle uptake is increased up to six times for A549 and two times for macrophages when cells are grown in softer environments. Furthermore, we altered surface characteristics through the attachment of submicron-sized particles as a method to locally engineer substrate stiffness and topography to investigate the biomechanical changes which occurred within adherent epithelial cells, i.e. characterization of A549 cell spreading and focal adhesion maturation. Consequently, decreasing substrate rigidity and particle-based topography led to a reduction of focal adhesion size. Moreover, expression levels of Yes-associated protein were found to correlate with the degree of particle endocytosis. A thorough appreciation of the mechanical cues may lead to improved solutions to optimize nanomedicine approaches for treatment of cancer and other diseases with abnormal mechanosignalling.

Intracellular gold nanoparticles influence light scattering and facilitate amplified spontaneous emission generation.

Generation of amplified stimulated emission inside mammalian cells has paved the way for a novel bioimaging and cell sensing approach. Single cells carrying gain media (e.g., fluorescent molecules) are placed inside an optical cavity, allowing the production of intracellular laser emission upon sufficient optical pumping. Here, we investigate the possibility to trigger another amplified emission phenomenon (i.e., amplified spontaneous emission or ASE) inside two different cell types, namely macrophage and epithelial cells from different species and tissues, in the presence of a poorly reflecting cavity. Furthermore, the resulting ASE properties can be enhanced by introducing plasmonic nanoparticles. The presence of gold nanoparticles (AuNPs) in rhodamine 6G-labeled A549 epithelial cells results in higher intensity and lowered ASE threshold in comparison to cells without nanoparticles, due to the effect of plasmonic field enhancement. An increase in intracellular concentration of AuNPs in rhodamine 6G-labeled macrophages is, however, responsible for the twofold increase in the ASE threshold and a reduction in the ASE intensity, dominantly due to a suppressed in and out-coupling of light at high nanoparticle concentrations.

Aligned and Oriented Collagen Nanocomposite Fibers as Substrates to Activate Fibroblasts.

Purified collagen possesses weak mechanical properties, hindering its broad application in tissue engineering. Strategies based on manipulating the hydrogel to induce fiber formation or incorporate nanomaterials have been proposed to overcome this issue. Herein, we use a microfluidic device to fabricate, for the first time, collagen hydrogels with aligned and oriented fibers doped with gold nanoparticles and carbon nanotubes. Results based on rheology, atomic force microscopy, and scanning electron microscopy reveal the formation of aligned and oriented collagen fibers possessing greater rigidity and stiffness on the doped hydrogels in comparison with native collagen. The mechanical properties of the hydrogels increased with the nanomaterial loading percentage and the stiffest formulations were those prepared in the presence of carbon nanotubes. We further evaluate the in vitro response of NIH-3T3 fibroblasts to the change in stiffness. The cells were found to be viable on all substrates with directional cell growth observed for the carbon nanotube-doped collagen fibers. No significant differences in the cell area, aspect ratio, and intensification of focal adhesions driven by the increase in stiffness were noted. Nonetheless, fibroblast proliferation and secretion of TGF-ß1 were greater on the hydrogels doped with carbon nanotubes. This nanomaterial-collagen composite provides unique features for cell and tissue substrate applications.

Design of Perfused PTFE Vessel-Like Constructs for In Vitro Applications.

Tissue models mimic the complex 3D structure of human tissues, which allows the study of pathologies and the development of new therapeutic strategies. The introduction of perfusion overcomes the diffusion limitation and enables the formation of larger tissue constructs. Furthermore, it provides the possibility to investigate the effects of hematogenously administered medications. In this study, the applicability of hydrophilic polytetrafluoroethylene (PTFE) membranes as vessel-like constructs for further use in perfused tissue models is evaluated. The presented approach allows the formation of stable and leakproof tubes with a mean diameter of 654.7 µm and a wall thickness of 84.2 µm. A polydimethylsiloxane (PDMS) chip acts as a perfusion bioreactor and provides sterile conditions. As proof of concept, endothelial cells adhere to the tube's wall, express vascular endothelial cadherin (VE-cadherin) between neighboring cells, and resist perfusion at a shear rate of 0.036 N m-2 for 48 h. Furthermore, the endothelial cell layer delays significantly the diffusion of fluorescently labeled molecules into the surrounding collagen matrix and leads to a twofold reduced diffusion velocity. This approach represents a cost-effective alternative to introduce stable vessel-like constructs into tissue models, which allows adapting the surrounding matrix to the tissue properties in vivo.

Particle Stiffness and Surface Topography Determine Macrophage-Mediated Removal of Surface Adsorbed Particles.

Cellular surface recognition and behavior are driven by a host of physical and chemical features which have been exploited to influence particle-cell interactions. Mechanical and topographical cues define the physical milieu which plays an important role in defining a range of cellular activities such as material recognition, adhesion, and migration through cytoskeletal organization and signaling. In order to elucidate the effect of local mechanical and topographical features generated by the adsorption of particles to an underlying surface on primary human monocyte-derived macrophages (MDM), a series of poly(N-isopropylacrylamide) (pNIPAM) particles with differing rigidity are self-assembled to form a defined particle-decorated surface. Assembly of particle-decorated surfaces is facilitated by modification of the underlying glass to possess a positive charge through functionalization using 3-aminopropyltriethoxysilane (APTES) or coating with poly(L-lysine) (PLL). MDMs are noted to preferentially remove particles with higher degrees of crosslinking (stiffer) than those with lower degrees of crosslinking (softer). Alterations to the surface density of particles enabled a greater area of the particle-decorated surface to be cleared. Uniquely, the impact of particle adsorption is evinced to have a direct impact on topographical recognition of the surface, suggesting a novel approach for controllably affecting cell-surface recognition and response.

Understanding selectivity of metabolic labelling and click-targeting in multicellular environments as a route to tissue selective drug delivery.

Cancer cells generally exhibit higher metabolic demands relative to that of normal tissue cells. This offers great possibilities to exploit metabolic glycoengineering in combination with bio-orthogonal chemistry reactions to achieve tumour site-targeted therapeutic delivery. This work addresses the selectivity of metabolic glycan labelling in diseased (i.e., cancer) versus normal cells grown in a multicellular environment. Dibenzocylooctyne (DBCO)-bearing acetylated-d-mannosamine (Ac4ManNDBCO) was synthesised to metabolically label three different types of cell lines originating from the human lung tissues: A549 adenocarcinomic alveolar basal epithelial cells, MeT5A non-cancerous mesothelial cells, and MRC5 non-cancerous fibroblasts. These cell lines displayed different labelling sensitivity, which trended with their doubling time in the following order: A549 ≈ MeT5A > MRC5. The higher metabolic labelling efficiency inherently led to a higher extent of specific binding and accumulation of the clickable N3-conjugated gold nanoparticles (N3-AuNps, core diameter = 30 nm) in the DBCO-glycan modified A549 and MeT5A cells, but to a less prominent effect in MRC5 cells. These findings demonstrate that relative rates of cell metabolism can be exploited using metabolic labelling to recruit nanotherapeutics whilst minimising non-specific targeting of surrounding tissues.

From Bioinspired Glue to Medicine: Polydopamine as a Biomedical Material.

Biological structures have emerged through millennia of evolution, and nature has fine-tuned the material properties in order to optimise the structure-function relationship. Following this paradigm, polydopamine (PDA), which was found to be crucial for the adhesion of mussels to wet surfaces, was hence initially introduced as a coating substance to increase the chemical reactivity and surface adhesion properties. Structurally, polydopamine is very similar to melanin, which is a pigment of human skin responsible for the protection of underlying skin layers by efficiently absorbing light with potentially harmful wavelengths. Recent findings have shown the subsequent release of the energy (in the form of heat) upon light excitation, presenting it as an ideal candidate for photothermal applications. Thus, polydopamine can both be used to (i) coat nanoparticle surfaces and to (ii) form capsules and ultra-small (nano)particles/nanocomposites while retaining bulk characteristics (i.e., biocompatibility, stability under UV irradiation, heat conversion, and activity during photoacoustic imaging). Due to the aforementioned properties, polydopamine-based materials have since been tested in adhesive and in energy-related as well as in a range of medical applications such as for tumour ablation, imaging, and drug delivery. In this review, we focus upon how different forms of the material can be synthesised and the use of polydopamine in biological and biomedical applications.

Bioprinting for Human Respiratory and Gastrointestinal In Vitro Models.

Increasing ethical and biological concerns require a paradigm shift toward animal-free testing strategies for drug testing and hazard assessments. To this end, the application of bioprinting technology in the field of biomedicine is driving a rapid progress in tissue engineering. In particular, standardized and reproducible in vitro models produced by three-dimensional (3D) bioprinting technique represent a possible alternative to animal models, enabling in vitro studies relevant to in vivo conditions. The innovative approach of 3D bioprinting allows a spatially controlled deposition of cells and biomaterial in a layer-by-layer fashion providing a platform for engineering reproducible models. However, despite the promising and revolutionizing character of 3D bioprinting technology, standardized protocols providing detailed instructions are lacking. Here, we provide a protocol for the automatized printing of simple alveolar, bronchial, and intestine epithelial cell layers as the basis for more complex respiratory and gastrointestinal tissue models. Such systems will be useful for high-throughput toxicity screening and drug efficacy evaluation.