Laser-assembled conductive 3D nanozyme film-based nitrocellulose sensor for real-time detection of H2O2 released from cancer cells.

In this work, a nanostructured conductive film possessing nanozyme features was straightforwardly produced via laser-assembling and integrated into complete nitrocellulose sensors; the cellulosic substrate allows to host live cells, while the nanostructured film nanozyme activity ensures the enzyme-free real-time detection of hydrogen peroxide (H2O2) released by the sames. In detail, a highly exfoliated reduced graphene oxide 3D film decorated with naked platinum nanocubes was produced using a CO2-laser plotter via the simultaneous reduction and patterning of graphene oxide and platinum cations; the nanostructured film was integrated into a nitrocellulose substrate and the complete sensor was manufactured using an affordable semi-automatic printing approach. The linear range for the direct H2O2 determination was 0.5-80 µM (R2 = 0.9943), with a limit of detection of 0.2 µM. Live cell measurements were achieved by placing the sensor in the culture medium, ensuring their adhesion on the sensors' surface; two cell lines were used as non-tumorigenic (Vero cells) and tumorigenic (SKBR3 cells) models, respectively. Real-time detection of H2O2 released by cells upon stimulation with phorbol ester was carried out; the nitrocellulose sensor returned on-site and real-time quantitative information on the H2O2 released proving useful sensitivity and selectivity, allowing to distinguish tumorigenic cells. The proposed strategy allows low-cost in-series semi-automatic production of paper-based point-of-care devices using simple benchtop instrumentation, paving the way for the easy and affordable monitoring of the cytopathology state of cancer cells.

Surface Modified ß-Ti-18Mo-6Nb-5Ta (wt%) Alloy for Bone Implant Applications: Composite Characterization and Cytocompatibility Assessment.

Commercially available titanium alloys such as Ti-6Al-4V are established in clinical use as load-bearing bone implant materials. However, concerns about the toxic effects of vanadium and aluminum have prompted the development of Al- and V-free ß-Ti alloys. Herein, a new alloy composed of non-toxic elements, namely Ti-18Mo-6Nb-5Ta (wt%), has been fabricated by arc melting. The resulting single ß-phase alloy shows improved mechanical properties (Young's modulus and hardness) and similar corrosion behavior in simulated body fluid when compared with commercial Ti-6Al-4V. To increase the cell proliferation capability of the new biomaterial, the surface of Ti-18Mo-6Nb-5Ta was modified by electrodepositing calcium phosphate (CaP) ceramic layers. Coatings with a Ca/P ratio of 1.47 were obtained at pulse current densities, -jc, of 1.8-8.2 mA/cm2, followed by 48 h of NaOH post-treatment. The thickness of the coatings has been measured by scanning electron microscopy from an ion beam cut, resulting in an average thickness of about 5 µm. Finally, cytocompatibility and cell adhesion have been evaluated using the osteosarcoma cell line Saos-2, demonstrating good biocompatibility and enhanced cell proliferation on the CaP-modified Ti-18Mo-6Nb-5Ta material compared with the bare alloy, even outperforming their CaP-modified Ti-6-Al-4V counterparts.

Supercritical CO2 Synthesis of Porous Metalloporphyrin Frameworks: Application in Photodynamic Therapy.

A series of porous metalloporphyrin frameworks prepared from the 5,10,15,20-tetra(4-pyridyl)porphyrin (H2TPyP) linker and four metal complexes, M(hfac)2 M = Cu(II), Zn(II), Co(II), and Ni(II) (hfac: 1,1,1,5,5,5-hexafluoroacetylacetonate), were obtained using supercritical CO2 (scCO2) as a solvent. All the materials, named generically as [M-TPyP] n , formed porous metal-organic frameworks (MOFs), with surface areas of ∼450 m2 g-1. All MOFs were formed through the coordination of the metal to the exocyclic pyridine moieties in the porphyrin linker. For Cu(II), Zn(II), and Co(II), incomplete metal coordination of the inner pyrrole ring throughout the structure was observed, giving place to MOFs with substitutional defects and leading to a certain level of disorder and limited crystallinity. These samples, prepared using scCO2, were precipitated as nano- to micrometric powders. Separately, a layering technique from a mixture of organic solvents was used to crystallize high-quality crystals of the Co(II) based MOF, obtained with the formula [{Co(hfac)2}2H2TPyP] n . The crystal structure of this MOF was elucidated by single-crystal synchrotron X-ray diffraction. The Zn(II)-based MOF was selected as a potential photodynamic therapy drug in the SKBR-3 tumoral cell line showing outstanding performance. This MOF resulted to be nontoxic, but after 15 min of irradiation at 630 nm, using either 1 or 5 µM concentration of the product, almost 70% of tumor cells died after 72 h.

Ru(ii) and Ir(iii) phenanthroline-based photosensitisers bearing o-carborane: PDT agents with boron carriers for potential BNCT.

Four novel transition metal-carborane photosensitisers were prepared by Sonogashira cross-coupling of 1-(4-ethynylbenzyl)-2-methyl-o-carborane (A-CB) with halogenated Ru(ii)- or Ir(iii)-phenanthroline complexes. The resulting boron-rich complexes with one (RuCB and IrCB) or two carborane cages (RuCB2 and IrCB2) were spectroscopically characterised, and their photophysical properties investigated. RuCB displayed the most attractive photophysical properties in solution (λem 635 nm, τT 2.53 µs, and φp 20.4%). Nanosecond time-resolved transient absorption studies were used to explore the 3MLCT nature of the triplet excited states, and the highest singlet oxygen quantum yields (ΦΔ) were obtained for the mono-carborane-phenanthroline complexes (RuCB: 52% and IrCB: 25%). None of the complexes produce dark toxicity in SKBR-3 cells after incubation under photodynamic therapy (PDT) conditions. Remarkably, mono-carboranes RuCB and IrCB were the best internalised by the SKBR-3 cells, demonstrating the first examples of tris-bidentate transition metal-carborane complexes acting as triplet photosensitisers for PDT with a high photoactivity; RuCB or IrCB killed ∼50% of SKBR-3 cells at 10 µM after irradiation. Therefore, the high-boron content and the photoactive properties of these photosensitisers make them potential candidates as dual anti-cancer agents for PDT and Boron Neutron Capture Therapy (BNCT).

Hybrid Cyclobutane/Proline-Containing Peptidomimetics: The Conformational Constraint Influences Their Cell-Penetration Ability.

A new family of hybrid ß,γ-peptidomimetics consisting of a repetitive unit formed by a chiral cyclobutane-containing trans-ß-amino acid plus a Nα-functionalized trans-γ-amino-l-proline joined in alternation were synthesized and evaluated as cell penetrating peptides (CPP). They lack toxicity on the human tumoral cell line HeLa, with an almost negligible cell uptake. The dodecapeptide showed a substantial microbicidal activity on Leishmania parasites at 50 µM but with a modest intracellular accumulation. Their previously published γ,γ-homologues, with a cyclobutane γ-amino acid, showed a well-defined secondary structure with an average inter-guanidinium distance of 8-10 Å, a higher leishmanicidal activity as well as a significant intracellular accumulation. The presence of a very rigid cyclobutane ß-amino acid in the peptide backbone precludes the acquisition of a defined conformation suitable for their cell uptake ability. Our results unveiled the preorganized charge-display as a relevant parameter, additional to the separation among the charged groups as previously described. The data herein reinforce the relevance of these descriptors in the design of CPPs with improved properties.

Biocompatibility and Electrical Stimulation of Skeletal and Smooth Muscle Cells Cultured on Piezoelectric Nanogenerators.

Nanogenerators are interesting for biomedical applications, with a great potential for electrical stimulation of excitable cells. Piezoelectric ZnO nanosheets present unique properties for tissue engineering. In this study, nanogenerator arrays based on ZnO nanosheets are fabricated on transparent coverslips to analyse the biocompatibility and the electromechanical interaction with two types of muscle cells, smooth and skeletal. Both cell types adhere, proliferate and differentiate on the ZnO nanogenerators. Interestingly, the amount of Zn ions released over time from the nanogenerators does not interfere with cell viability and does not trigger the associated inflammatory response, which is not triggered by the nanogenerators themselves either. The local electric field generated by the electromechanical nanogenerator-cell interaction stimulates smooth muscle cells by increasing cytosolic calcium ions, whereas no stimulation effect is observed on skeletal muscle cells. The random orientation of the ZnO nanogenerators, avoiding an overall action potential aligned along the muscle fibre, is hypothesised to be the cause of the cell-type dependent response. This demonstrates the need of optimizing the nanogenerator morphology, orientation and distribution according to the potential biomedical use. Thus, this study demonstrates the cell-scale stimulation triggered by biocompatible piezoelectric nanogenerators without using an external source on smooth muscle cells, although it remarks the cell type-dependent response.

Chiral Cyclobutane-Containing Cell-Penetrating Peptides as Selective Vectors for Anti-Leishmania Drug Delivery Systems.

Two series of new hybrid γ/γ-peptides, γ-CC and γ-CT, formed by (1S,2R)-3-amino-2,2,dimethylcyclobutane-1-carboxylic acid joined in alternation to a Nα-functionalized cis- or trans-γ-amino-l-proline derivative, respectively, have been synthesized and evaluated as cell penetrating peptides (CPP) and as selective vectors for anti-Leishmania drug delivery systems (DDS). They lacked cytotoxicity on the tumoral human cell line HeLa with a moderate cell-uptake on these cells. In contrast, both γ-CC and γ-CT tetradecamers were microbicidal on the protozoan parasite Leishmania beyond 25 µM, with significant intracellular accumulation. They were conjugated to fluorescent doxorubicin (Dox) as a standard drug showing toxicity beyond 1 µM, while free Dox was not toxic. Intracellular accumulation was 2.5 higher than with Dox-TAT conjugate (TAT = transactivator of transcription, taken as a standard CPP). The conformational structure of the conjugates was approached both by circular dichroism spectroscopy and molecular dynamics simulations. Altogether, computational calculations predict that the drug-γ-peptide conjugates adopt conformations that bury the Dox moiety into a cavity of the folded peptide, while the positively charged guanidinium groups face the solvent. The favorable charge/hydrophobicity balance in these CPP improves the solubility of Dox in aqueous media, as well as translocation across cell membranes, making them promising candidates for DDS.

Cell Internalization in Fluidic Culture Conditions Is Improved When Microparticles Are Specifically Targeted to the Human Epidermal Growth Factor Receptor 2 (HER2).

PURPOSE: To determine if the specific targeting of microparticles improves their internalization by cells under fluidic conditions. METHODS: Two isogenic breast epithelial cell lines, one overexpressing the Human Epidermal Growth Factor Receptor 2 (HER2) oncogene (D492HER2) and highly tumorigenic and the other expressing HER2 at much lower levels and non-tumorigenic (D492), were cultured in the presence of polystyrene microparticles of 1 µm in diameter, biofunctionalized with either a specific anti-HER2 antibody or a non-specific secondary antibody. Mono- and cocultures of both cell lines in static and fluidic conditions were performed, and the cells with internalized microparticles were scored. RESULTS: Globally, the D492 cell line showed a higher endocytic capacity than the D492HER2 cell line. Microparticles that were functionalized with the anti-HER2 antibody were internalized by a higher percentage of cells than microparticles functionalized with the non-specific secondary antibody. Although internalization was reduced in fluidic culture conditions in comparison with static conditions, the increase in the internalization of microparticles biofunctionalized with the anti-HER2 antibody was higher for the cell line overexpressing HER2. CONCLUSION: The biofunctionalization of microparticles with a specific targeting molecule remarkably increases their internalization by cells in fluidic culture conditions (simulating the blood stream). This result emphasizes the importance of targeting for future in vivo delivery of drugs and bioactive molecules through microparticles.

Permanently hydrophilic, piezoelectric PVDF nanofibrous scaffolds promoting unaided electromechanical stimulation on osteoblasts.

Biomimetic functional scaffolds for tissue engineering should fulfil specific requirements concerning structural, bio-chemical and electro-mechanical characteristics, depending on the tissue that they are designed to resemble. In bone tissue engineering, piezoelectric materials based on poly(vinylidene fluoride) (PVDF) are on the forefront, due to their inherent ability to generate surface charges under minor mechanical deformations. Nevertheless, PVDF's high hydrophobicity hinders sufficient cell attachment and expansion, which are essential in building biomimetic scaffolds. In this study, PVDF nanofibrous scaffolds were fabricated by electrospinning to achieve high piezoelectricity, which was compared with drop-cast membranes, as it was confirmed by XRD and FTIR measurements. Oxygen plasma treatment of the PVDF surface rendered it hydrophilic, and surface characterization revealed a long-term stability. XPS analysis and contact angle measurements confirmed an unparalleled two-year stability of hydrophilicity. Osteoblast cell culture on the permanently hydrophilic PVDF scaffolds demonstrated better cell spreading over the non-treated ones, as well as integration into the scaffold as indicated by SEM cross-sections. Intracellular calcium imaging confirmed a higher cell activation on the piezoelectric electrospun nanofibrous scaffolds. Combining these findings, and taking advantage of the self-stimulation of the cells due to their attachment on the piezoelectric PVDF nanofibers, a 3D tissue-like functional self-sustainable scaffold for bone tissue engineering was fabricated.

Membrane reorganization after photochemical internalization to release transferrin-biofunctionalized polystyrene microparticles.

Therapeutic drug carriers can drive their cargo to their target cells. However, an obstacle is usually the entrapment of the drug inside the endolysosomal compartment, which physically impedes its actuation by the impossibility of reaching its molecular site of action. To overcome this hurdle, photochemical internalization (PCI) has been proposed, but the extent of PCI-induced membrane disruption and its capability to allow the release of microparticles is unknown. The aim of the present study was to determine if PCI allows the release of microparticles from the endolysosomal compartment to the cytosol and to analyze at the ultrastructural level the effect of PCI on the membrane surrounding the particles. Confocal microscope allowed us to detect that endolysosomal membranes suffered some disruption after PCI, evidenced by the diffusion of soluble transferrin from the endolysosomes to the cytosol and by a decrease of LAMP1-microparticles co-localization. Transmission electron microscopy (TEM) showed a decrease in the number of well-defined membranes around microparticles after PCI, and scanning TEM combined with energy dispersive x-ray revealed an increase in the width of endolysosomal membranes after treatment. These results suggest that endolysosomal membranes suffered an ultrastructure alteration after PCI, enough to liberate soluble transferrin but not the entire microparticles.

Biodegradable FeMnSi Sputter-Coated Macroporous Polypropylene Membranes for the Sustained Release of Drugs.

Pure Fe and FeMnSi thin films were sputtered on macroporous polypropylene (PP) membranes with the aim to obtain biocompatible, biodegradable and, eventually, magnetically-steerable platforms. Room-temperature ferromagnetic response was observed in both Fe- and FeMnSi-coated membranes. Good cell viability was observed in both cases by means of cytotoxicity studies, though the FeMnSi-coated membranes showed higher biodegradability than the Fe-coated ones. Various strategies to functionalize the porous platforms with transferrin-Alexa Fluor 488 (Tf-AF488) molecules were tested to determine an optimal balance between the functionalization yield and the cargo release. The distribution of Tf-AF488 within the FeMnSi-coated PP membranes, as well as its release and uptake by cells, was studied by confocal laser scanning microscopy. A homogeneous distribution of the drug within the membrane skeleton and its sustained release was achieved after three consecutive impregnations followed by the addition of a layer made of gelatin and maltodextrin, which prevented exceedingly fast release. The here-prepared organic-inorganic macroporous membranes could find applications as fixed or magnetically-steerable drug delivery platforms.

Study of Galfenol direct cytotoxicity and remote microactuation in cells.

Remote microactuators are of great interest in biology and medicine as minimally-invasive tools for cellular stimulation. Remote actuation can be achieved by active magnetostrictive transducers which are capable of changing shape in response to external magnetic fields thereby creating controlled displacements. Among the magnetostrictive materials, Galfenol, the multifaceted iron-based smart material, offers high magnetostriction with robust mechanical properties. In order to explore these capabilities for biomedical applications, it is necessary to study the feasibility of material miniaturization in standard fabrication processes as well as evaluate the biocompatibility. Here we develop a technology to fabricate, release, and suspend Galfenol-based microparticles, without affecting the integrity of the material. The morphology, composition and magnetic properties of the material itself are characterized. The direct cytotoxicity of Galfenol is evaluated in vitro using human macrophages, osteoblast and osteosarcoma cells. In addition, cytotoxicity and actuation of Galfenol microparticles in suspension are evaluated using human macrophages. The biological parameters analyzed indicate that Galfenol is not cytotoxic, even after internalization of some of the particles by macrophages. The microparticles were remotely actuated forming intra- and extracellular chains that did not impact the integrity of the cells. The results propose Galfenol as a suitable material to develop remote microactuators for cell biology studies and intracellular applications.

Rapid on-chip apoptosis assay on human carcinoma cells based on annexin-V/quantum dot probes.

Despite all the efforts made over years to study the cancer expression and the metastasis event, there is not a clear understanding of its origins and effective treatment. Therefore, more specialized and rapid techniques are required for studying cell behaviour under different drug-based treatments. Here we present a quantum dot signalling-based cell assay carried out in a segmental microfluidic device that allows studying the effect of anti-cancer drugs in cultured cell lines by monitoring phosphatidylserine translocation that occurs in early apoptosis. The developed platform combines the automatic generation of a drug gradient concentration, allowing exposure of cancer cells to different doses, and the immunolabeling of the apoptotic cells using quantum dot reporters. Thereby a complete cell-based assay for efficient drug screening is performed showing a clear correlation between drug dose and amount of cells undergoing apoptosis.

Effect of Surface Modifications of Ti40Zr10Cu38Pd12 Bulk Metallic Glass and Ti-6Al-4V Alloy on Human Osteoblasts In Vitro Biocompatibility.

The use of biocompatible materials, including bulk metallic glasses (BMGs), for tissue regeneration and transplantation is increasing. The good mechanical and corrosion properties of Ti40Zr10Cu38Pd12 BMG and its previously described biocompatibility makes it a potential candidate for medical applications. However, it is known that surface properties like topography might play an important role in regulating cell adhesion, proliferation and differentiation. Thus, in the present study, Ti40Zr10Cu38Pd12 BMG and Ti6-Al-4V alloy were surface-modified electrochemically (nanomesh) or physically (microscratched) to investigate the effect of material topography on human osteoblasts cells (Saos-2) adhesion, proliferation and differentiation. For comparative purposes, the effect of mirror-like polished surfaces was also studied. Electrochemical treatments led to a highly interconnected hierarchical porous structure rich in oxides, which have been described to improve corrosion resistance, whereas microscratched surfaces showed a groove pattern with parallel trenches. Cell viability was higher than 96% for the three topographies tested and for both alloy compositions. In all cases, cells were able to adhere, proliferate and differentiate on the alloys, hence indicating that surface topography plays a minor role on these processes, although a clear cell orientation was observed on microscratched surfaces. Overall, our results provide further evidence that Ti40Zr10Cu38Pd12 BMG is an excellent candidate, in the present two topographies, for bone repair purposes.

Polysilicon-chromium-gold intracellular chips for multi-functional biomedical applications.

The development of micro- and nanosystems for their use in biomedicine is a continuously growing field. One of the major goals of such platforms is to combine multiple functions in a single entity. However, achieving the design of an efficient and safe micro- or nanoplatform has shown to be strongly influenced by its interaction with the biological systems, where particle features or cell types play a critical role. In this work, the feasibility of using multi-material pSi-Cr-Au intracellular chips (MMICCs) for multifunctional applications by characterizing their interactions with two different cell lines, one tumorigenic and one non-tumorigenic, in terms of biocompatibility, internalization and intracellular fate, has been explored. Moreover, the impact of MMICCs on the induction of an inflammatory response has been assessed by evaluating TNFα, IL1b, IL6, and IL10 human inflammatory cytokines secretion by macrophages. Results show that MMICCs are biocompatible and their internalization efficiency is strongly dependent on the cell type. Finally as a proof-of-concept, MMICCs have been dually functionalized with transferrin and pHrodo™ Red, SE to target cancer cells and detect intracellular pH, respectively. In conclusion, MMICCs can be used as multi-functional devices due to their high biocompatibility, non-inflammatory properties and the ability of developing multiple functions.

Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells.

The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 µm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

Chromosome instability in mouse embryonic stem cells.

Embryonic Stem Cells (ESCs) are expected to show a stable euploid karyotype, but in the last decade (sub)chromosomal aberrations have been systematically described in these cell lines when maintained in vitro. Culture conditions and long-term culture have been traditionally proposed as possible factors involved in the acquisition of chromosomal abnormalities. Thus, we analyzed the chromosome constitution, the undifferentiated state and the functional pluripotency of three different mouse ESCs grown under the same culture conditions. Two cell lines were unstable from early passages, whereas the third one retained its chromosome integrity after long-term culture despite using enzymatic methods for cell disaggregation. Trisomy 8 and 11 were clonally selected in both unstable cell lines, which also showed a higher growth rate than our normal cell line and suffered morphological changes in colony shape with increasing passage number. Regardless of the length of culture or the chromosome instability, all cell lines preserved their differentiation potential. These results confirm that double trisomy 8 and 11 confers a growth advantage to the abnormal cells, but not at the expense of cell differentiation. The presence of chromosome instability, widely related to tumor development and cancer disease, highlights the risk of using pluripotent cells in regenerative medicine.

Detection of circulating cancer cells using electrocatalytic gold nanoparticles.

A rapid cancer cell detection and quantification assay, based on the electrocatalytic properties of gold nanoparticles towards the hydrogen evolution reaction, is described. The selective labeling of cancer cells is performed in suspension, allowing a fast interaction between the gold nanoparticle labels and the target proteins expressed at the cell membrane. The subsequent electrochemical detection is accomplished with small volumes of sample and user-friendly equipment through a simple electrochemical method that generates a fast electrochemical response used for the quantification of nanoparticle-labeled cancer cells. The system establishes a selective cell-detection assay capable of detecting 4 × 10(3) cancer cells in suspension that can be extended to several other cells detection scenarios.

Simple monitoring of cancer cells using nanoparticles.

Here we present a new strategy for a simple and fast detection of cancer circulating cells (CTCs) using nanoparticles. The human colon adenocarcinoma cell line (Caco2) was chosen as a model CTC. Similarly to other adenocarcinomas, colon adenocarcinoma cells have a strong expression of EpCAM, and for this reason this glycoprotein was used as the capture target. We combine the capturing capability of anti-EpCAM functionalized magnetic beads (MBs) and the specific labeling through antibody-modified gold nanoparticles (AuNPs), with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction (HER) detection technique. The fully optimized process was used for the electrochemical detection of Caco2 cells in the presence of monocytes (THP-1), other circulating cells that could interfere in real blood samples. Therefore we obtained a novel and simple in situ-like sensing format that we applied for the rapid quantification of AuNPs-labeled CTCs in the presence of other human cells.

Internalization and cytotoxicity analysis of silicon-based microparticles in macrophages and embryos.

Microchips can be fabricated, using semiconductor technologies, at microscopic level to be introduced into living cells for monitoring of intracellular parameters at a single cell level. As a first step towards intracellular chips development, silicon and polysilicon microparticles of controlled shape and dimensions were fabricated and introduced into human macrophages and mouse embryos by phagocytosis and microinjection, respectively. Microparticles showed to be non-cytotoxic for macrophages and were found to be localized mainly inside early endosomes, in tight association with endosomal membrane, and more rarely in acidic compartments. Embryos with microinjected microparticles developed normally to the blastocyst stage, confirming the non-cytotoxic effect of the particles. In view of these results silicon and polysilicon microparticles can serve as the frame for future intracellular chips development and this technology opens the possibility of real complex devices to be used as sensors or actuators inside living cells.