Publications

Biofabrication of three-dimensional (3D) cultures through the 3D Bioprinting technique opens new perspectives and applications of cell-laden hydrogels. However, to continue with the progress, new BioInks with specific properties must be carefully designed. In this study, we report the synthesis and 3D Bioprinting of an electroconductive BioInk made of gelatin/fibrinogen hydrogel, C2C12 mouse myoblast and 5% w/w of conductive poly (3,4-ethylenedioxythiophene) nanoparticles (PEDOT NPs). The influence of PEDOT NPs, incorporated in the cellladen BioInk, not only showed a positive effect in cells viability, differentiation and myotube functionalities, also allowed the printed constructs to behaved as BioCapacitors. Such devices were able to electrochemically store a significant amount of energy (0.5 mF/cm2), enough to self-stimulate as BioActuator, with typical contractions ranging from 27 to 38 mu N, during nearly 50 min. The biofabrication of 3D constructs with the proposed electroconductive BioInk could lead to new devices for tissue engineering, biohybrid robotics or bioelectronics.

JTD Keywords: 3d bioprinting, Animal, Animals, Bioactuator, Bioactuators, Biocapacitor, Biofabrication, Bioprinting, Biosensing techniques, C2c12 myoblasts, Cells, Chemistry, Electric conductivity, Electroconductive, Electroconductive bioink, Ethylenedioxythiophenes, Genetic procedures, Hydrogel, Hydrogels, Mice, Mouse, Pedot nps, Pedot nps,3d bioprinting,electroconductive bioink,bioactuator,biocapacito, Poly (3,4-ethylenedioxythiophene) nanoparticle, Printing, three-dimensional, Procedures, Skeletal-muscle,cytotoxicity,polymer, Synthesis (chemical), Three dimensional printing, Tissue engineering, Tissue scaffolds

Dental implant-associated infection is a clinical challenge which poses a significant healthcare and socio-economic burden. To overcome this issue, developing antimicrobial surfaces, including antimicrobial peptide coatings, has gained great attention. Different physical and chemical routes have been used to obtain these biofunctional coatings, which in turn might have a direct influence on their bioactivity and functionality. In this study, we present a silane-based, fast, and efficient chemoselective conjugation of antimicrobial peptides (Cys-GL13K) to coat titanium implant surfaces. Comprehensive surface analysis was performed to confirm the surface functionalization of as-prepared and mechanically challenged coatings. The antibacterial potency of the evaluated surfaces was confirmed against both Streptococcus gordonii and Streptococcus mutans, the primary colonizers and pathogens of dental surfaces, as demonstrated by reduced bacteria viability. Additionally, human dental pulp stem cells demonstrated long-term viability when cultured on Cys-GL13K-grafted titanium surfaces. Cell functionality and antimicrobial capability against multi-species need to be studied further; however, our results confirmed that the proposed chemistry for chemoselective peptide anchoring is a valid alternative to traditional site-unspecific anchoring methods and offers opportunities to modify varying biomaterial surfaces to form potent bioactive coatings with multiple functionalities to prevent infection.

JTD Keywords: biocompatibility, cytotoxicity, delivery, dental implants, prevention, release, stability, surface coating, titanium, zirconia, Antimicrobial peptide, Biocompatibility, Dental implants, Peri-implantitis, Surface coating, Titanium

Alzheimer's disease is characterized by a combination of several neuropathological hallmarks, such as extracellular aggregates of beta amyloid (Aβ). Numerous alternatives have been studied for inhibiting Aβ aggregation but, at this time, there are no effective treatments available. Here, we developed the tri-component nanohybrid system AuNPs@POM@PEG based on gold nanoparticles (AuNPs) covered with polyoxometalates (POMs) and polyethylene glycol (PEG). In this work, AuNPs@POM@PEG demonstrated the inhibition of the formation of amyloid fibrils, showing a 75% decrease in Aβ aggregation in vitro. As it is a potential candidate for the treatment of Alzheimer's disease, we evaluated the cytotoxicity of AuNPs@POM@PEG and its ability to cross the blood-brain barrier (BBB). We achieved a stable nanosystem that is non-cytotoxic below 2.5 nM to human neurovascular cells. The brain permeability of AuNPs@POM@PEG was analyzed in an in vitro microphysiological model of the BBB (BBB-on-a-chip), containing 3D human neurovascular cell co-cultures and microfluidics. The results show that AuNPs@POM@PEG was able to cross the brain endothelial barrier in the chip and demonstrated that POM does not affect the barrier integrity, giving the green light to further studies into this system as a nanotherapeutic.

JTD Keywords: beta-amyloid, blood-brain barrier organ-on-a-chip, cellular uptake, citrate, cytotoxicity, electrocatalytic reduction, gold nanoparticles, hypothesis, nanorods, polyoxometalates, size, stability, surface, Alzheimers-disease, Blood–brain barrier organ-on-a-chip, Gold nanoparticles, Nanovehicle, Polyoxometalates, Β-amyloid

The lack of predictive models that mimic the blood-brain barrier (BBB) hinders the development of effective drugs for neurodegenerative diseases. Animal models behave differently from humans, are expensive and have ethical constraints. Organ-on-a-chip (OoC) platforms offer several advantages to resembling physiological and pathological conditions in a versatile, reproducible, and animal-free manner. In addition, OoC give us the possibility to incorporate sensors to determine cell culture features such as trans-endothelial electrical resistance (TEER). Here, we developed a BBB-on-a-chip (BBB-oC) platform with a TEER measurement system in close distance to the barrier used for the first time for the evaluation of the permeability performance of targeted gold nanorods for theranostics of Alzheimer's disease. GNR-PEG-Ang2/D1 is a therapeutic nanosystem previously developed by us consisting of gold nanorods (GNR) functionalized with polyethylene glycol (PEG), angiopep-2 peptide (Ang2) to overcome the BBB and the D1 peptide as beta amyloid fibrillation inhibitor, finally obtaining GNR-PEG-Ang2/D1 which showed to be useful for disaggregation of the amyloid in in vitro and in vivo models. In this work, we evaluated its cytotoxicity, permeability, and some indications of its impact on the brain endothelium by employing an animal-free device based on neurovascular human cells.In this work, we fabricated a BBB-oC with human astrocytes, pericytes and endothelial cells and a TEER measuring system (TEER-BBB-oC) integrated at a micrometric distance of the endothelial barrier. The characterization displayed a neurovascular network and the expression of tight junctions in the endothelium. We produced GNR-PEG-Ang2/D1 and determined its non-cytotoxic range (0.05-0.4 nM) for plated cells included in the BBB-oC and confirmed its harmless effect at the highest concentration (0.4 nM) in the microfluidic device. The permeability assays revealed that GNR-PEG-Ang2/D1 cross the BBB and this entry is facilitated by Ang2 peptide. Parallel to the permeability analysis of GNR-PEG-Ang2/D1, an interesting behavior of the TJs expression was observed after its administration probably related to the ligands on the nanoparticle surface.BBB-oC with a novel TEER integrated setup which allow a correct read-out and cell imaging monitoring was proven as a functional and throughput platform to evaluate the brain permeability performance of nanotherapeutics in a physiological environment with human cells, putting forward a viable alternative to animal experimentation.© 2023. The Author(s).

JTD Keywords: alzheimer disease (ad), cell-culture, cytotoxicity, endothelial-cells, gold nanoparticles, microfluidic platform, model, organ-on-a-chip (ooc), peptide, tight junction, trans-endothelial electrical resistance (teer), transport, Alzheimer disease (ad), Blood-brain barrier (bbb), Blood-brain-barrier, Blood–brain barrier (bbb), Gold nanoparticles, Organ-on-a-chip (ooc), Trans-endothelial electrical resistance (teer)

Biohybrid robots, or bio-bots, integrate living and synthetic materials following a synergistic strategy to acquire some of the unique properties of biological organisms, like adaptability or bio-sensing, which are difficult to obtain exclusively using artificial materials. Skeletal muscle is one of the preferred candidates to power bio-bots, enabling a wide variety of movements from walking to swimming. Conductive nanocomposites, like gold nanoparticles or graphene, can provide benefits to muscle cells by improving the scaffolds' mechanical and conductive properties. Here, boron nitride nanotubes (BNNTs), with piezoelectric properties, are integrated in muscle-based bio-bots and an improvement in their force output and motion speed is demonstrated. A full characterization of the BNNTs is provided, and their piezoelectric behavior with piezometer and dynamometer measurements is confirmed. It is hypothesized that the improved performance is a result of an electric field generated by the nanocomposites due to stresses produced by the cells during differentiation. This hypothesis is backed with finite element simulations supporting that this stress can generate a non-zero electric field within the matrix. With this work, it is shown that the integration of nanocomposite into muscle-based bio-bots can improve their performance, paving the way toward stronger and faster bio-hybrid robots.

JTD Keywords: Bio-bots, Biohybrid robots, Biomaterials, Boron nitride nanotubes, Cells, Cytotoxicity, Differentiation, Myoblasts, Skeletal muscle tissue, Skeletal-muscle, Stimulation

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.

JTD Keywords: adsorption, cryo-soft x-ray tomography, cytotoxicity, expression, flow cytometry, internalisation, intracellular calcium, magnesium, nano, nanomaterials, nanoparticles, proliferation, protein corona, ultrastructure, Calcium-phosphate nanoparticles, Cryo-soft x-ray tomography, Flow cytometry, Hydroxyapatite, Internalisation, Intracellular calcium, Nanoparticles

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanopar-ticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaf-folds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concen-trations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: antibiotics, antimicrobial activity, behavior, cytotoxicity, delivery, drug, drug delivery, hydroxyapatite nanoparticles, in-vitro, mechanisms, mitochondria, polylactide, release, streptomycin, Antimicrobial activity, Cancer stem-cells, Cytotoxicity, Drug delivery, Hydroxyapatite nanoparticles, Polylactide, Streptomycin

Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.

JTD Keywords: 3d cell-derived matrices, adipose mesenchymal stem cells, collagen matrix, colorectal adenocarcinoma, cytotoxicity assay, deposition, expansion, extracellular microenvironment, extracellular-matrix, fibronectin, growth, macromolecular crowders, microcarriers, scaffolds, tissue, 3d cell-derived matrices, Adipose mesenchymal stem cells, Cytotoxicity assay, Extracellular microenvironment, Macromolecular crowders, Mesenchymal stem-cells, Microcarriers

© 2020 Elsevier Ltd The use of nanoparticles in consumer products is currently on the rise, so it is important to have reliable methods to predict any associated toxicity effects. Traditional in vitro assays fail to mimic true physiological responses of living organisms against nanoparticles whereas murine in vivo models are costly and ethically controversial. For these reasons, this study aimed to evaluate the efficacy of Galleria mellonella as an alternative, non-rodent in vivo model for examining nanoparticle toxicity. Silver, selenium, and functionalized gold nanoparticles were synthesized, and their toxicity was assessed in G. mellonella larvae. The degree of acute toxicity effects caused by each type of NP was efficiently detected by an array of indicators within the larvae: LD50 calculation, hemocyte proliferation, NP distribution, behavioral changes, and histological alterations. G. mellonella larvae are proposed as a nanotoxicological model that can be used as a bridge between in vitro and in vivo murine assays in order to obtain better predictions of NP toxicity.

JTD Keywords: cellular uptake, cytotoxicity, galleria mellonella, gold nanoparticles, hemocytes, nanoparticles, nanotoxicity, non-rodent in vivo model, non-rodent in vivo model, oxidative stress, selenium-compounds, silica nanoparticles, silver nanoparticles, toxicity, toxicity screening, vitro, Galleria mellonella, Hemocytes, In-vivo model, Nanoparticles, Nanotoxicity, Non-rodent in vivo model, Toxicity screening

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non‐toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non‐covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ‐PEG) and a pyrrolidone containing linear derivative (PZ‐PYR) for their ability to intracellularly deliver FITC‐avidin, a model protein. In endothelial cells, PZ‐PYR/protein exhibited both faster internalization and higher uptake levels than PZ‐PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ‐PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ‐PEG/avidin colocalized more profusely with endo-lysosomes, and PZ‐PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ‐PYR-delivered anti‐F‐actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell‐impermeable Bax‐BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ‐PYR, demonstrating endo‐lysosomal escape. These biodegradable PZs were non‐toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

JTD Keywords: cytosolic delivery, cytotoxicity, delivery of apoptotic peptides, endosomal escape, intracellular delivery of antibody, intracellular protein delivery, Cytosolic delivery, Cytotoxicity, Delivery of apoptotic peptides, Endosomal escape, Intracellular delivery of antibody, Intracellular protein delivery, Polyphosphazene polymers

The green synthesis of nanoparticles (NPs) is currently under worldwide investigation as an eco-friendly alternative to traditional routes (NPs): the absence of toxic solvents and catalysts make it suitable in the design of promising nanomaterials for nanomedicine applications. In this work, we used the extracts collected from leaves of two cultivars (Leccino and Carolea) belonging to the species Olea Europaea, to synthesize silver NPs (AgNPs) in different pH conditions and low temperature. NPs underwent full morphological characterization with the aim to define a suitable protocol to obtain a monodispersed population of AgNPs. Afterwards, to validate the reproducibility of the mentioned synthetic procedure, we moved on to another Mediterranean plant, the Laurus Nobilis. Interestingly, the NPs obtained using the two olive cultivars produced NPs with different shape and size, strictly depending on the cultivar selected and pH. Furthermore, the potential ability to inhibit the growth of two woman cancer cells (breast adenocarcinoma cells, MCF-7 and human cervical epithelioid carcinoma, HeLa) were assessed for these AgNPs, as well as their capability to mitigate the bacteria concentration in samples of contaminated well water. Our results showed that toxicity was stronger when MCF-7 and Hela cells were exposed to AgNPs derived from Carolea obtained at pH 7 presenting irregular shape; on the other hand, greater antibacterial effect was revealed using AgNPs obtained at pH 8 (smaller and monodispersed) on well water, enriched with bacteria and coliforms.

JTD Keywords: Green synthesis, Silver nanoparticles, Olea Europaea, Leccino, Carolea, Cytotoxicity, Genotoxicity, Antibacterial activity

PIKfyve phosphoinositide kinase produces PtdIns(3,5)P2 and PtdIns5P and governs a myriad of cellular processes including cytoskeleton rearrangements and cell proliferation. The latter entails rigorous investigation since the cytotoxicity of PIKfyve inhibition is a potential therapeutic modality for cancer. Here we report the effects of two PIKfyve-specific inhibitors on the attachment/spreading and viability of mouse embryonic fibroblasts (MEFs) and C2C12 myoblasts. Importantly, 18-h treatment of adherent cells with YM201636 (800 nM) and apilimod (20 nM) in serum-containing culture media did not affect cell viability despite the presence of multiple cytoplasmic vacuoles, a hallmark of PIKfyve inhibition. Strikingly, at the same dose and duration the inhibitors caused excessive cytoplasmic vacuolation, initial suppression of cell attachment/spreading and subsequent marked detachment/death in serum-deprived cells. The remaining adherent cells under serum-deprived conditions had smaller surface area, lacked vinculin/actin-positive focal adhesions and displayed vacuoles occupying the entire cytoplasm. Serum or growth factors protected against PIKfyve inhibitor cytotoxicity. This protection required Akt activation evidenced by the abrogated beneficial effect of serum upon treatment with the clinically-relevant Akt inhibitor MK-2206. Moreover, Akt inhibition triggered cell detachment/death even in serum-fed adherent MEFs treated with apilimod. Intriguingly, BafilomycinA1 (H+-vacuolar ATPase inhibitor), which prevents the cytoplasmic vacuolation under PIKfyve perturbations, rescued all defects in attaching/spreading as well as in adherent cells under serum-starved or serum-fed conditions, respectively. Together, the results indicate that the cytotoxicity of PIKfyve inhibitors in MEFs and C2C12 myoblasts requires Akt suppression and excessive cytoplasmic vacuolation.

JTD Keywords: AKT, Cytotoxicity, MK-2206, PIKfyve, Ppilimod, YM201636

Pulmonary delivery of drugs for both local and systemic action has gained new attention over the last decades. In this work, different amphiphilic polymers (Soluplus®, Pluronic® F68, Pluronic® F108 and Pluronic® F127) were used to produce lyophilized formulations for inhalation of insulin. Development of stimuli-responsive, namely glucose-sensitive, formulations was also attempted with the addition of phenylboronic acid (PBA). Despite influencing the in vitro release of insulin from micelles, PBA did not confer glucose-sensitive properties to formulations. Lyophilized powders with aerodynamic diameter (<. 6. μm) compatible with good deposition in the lungs did not present significant in vitro toxicity for respiratory cell lines. Additionally, some formulations, in particular Pluronic® F127-based formulations, enhanced the permeation of insulin through pulmonary epithelial models and underwent minimal internalization by macrophages in vitro. Overall, formulations based on polymeric micelles presenting promising characteristics were developed for the delivery of insulin by inhalation. From the Clinical Editor: The ability to deliver other systemic drugs via inhalation has received renewed interests in the clinical setting. This is especially true for drugs which usually require injections for delivery, like insulin. In this article, the authors investigated their previously developed amphiphilic polymers for inhalation of insulin in an in vitro model. The results should provide basis for future in vivo studies.

JTD Keywords: Cytotoxicity, Inhalation, Permeability, Phagocytosis, Polymeric micelles, Protein delivery