Multi-walled carbon nanotube-physicochemical properties predict the systemic acute phase response following pulmonary exposure in mice

Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) has been linked to an increased risk of developing cardiovascular disease in addition to the well-documented physicochemical-dependent adverse lung effects. A proposed mechanism is through a strong and sustained pulmonary secretion of acute phase proteins to the blood. We identified physicochemical determinants of MWCNT-induced systemic acute phase response by analyzing effects of pulmonary exposure to 14 commercial, well-characterized MWCNTs in female C57BL/6J mice pulmonary exposed to 0, 6, 18 or 54 μg MWCNT/mouse. Plasma levels of acute phase response proteins serum amyloid A1/2 (SAA1/2) and SAA3 were determined on day 1, 28 or 92. Expression levels of hepatic Saa1 and pulmonary Saa3 mRNA levels were assessed to determine the origin of the acute phase response proteins. Pulmonary Saa3 mRNA expression levels were greater and lasted longer than hepatic Saa1 mRNA expression. Plasma SAA1/2 and SAA3 protein levels were related to time and physicochemical properties using adjusted, multiple regression analyses. SAA3 and SAA1/2 plasma protein levels were increased after exposure to almost all of the MWCNTs on day 1, whereas limited changes were observed on day 28 and 92. SAA1/2 and SAA3 protein levels did not correlate and only SAA3 protein levels correlated with neutrophil influx. The multiple regression analyses revealed a protective effect of MWCNT length on SAA1/2 protein level on day 1, such that a longer length resulted in lowered SAA1/2 plasma levels. Increased SAA3 protein levels were positively related to dose and content of Mn, Mg and Co on day 1, whereas oxidation and diameter of the MWCNTs were protective on day 28 and 92, respectively. The results of this study reveal very differently controlled pulmonary and hepatic acute phase responses after MWCNT exposure. As the responses were influenced by the physicochemical properties of the MWCNTs, this study provides the first step towards designing MWCNT that induce less SAA

Peptide functionalization of silicon for detection and classification of prostatic cells

The development of simple, rapid, and low costmethods for early detection, identification, andmeasurement ofmultiple biomarkers remains a challenge to improve diagnosis, treatment monitoring, and prognosis of cancer. Biosensing technology, combining the properties of biological systems with functional advanced materials, guarantees rapid, reproducible, and highly sensitive cell detection. In this study, we developed silicon-based biochips for prostate cancer PC3 cells detection by using cytokeratin 8/18 and Urotensin Receptor (UTR) as markers in order to obtain a biochip-based diagnostic system. Spectroscopic ellipsometry and fluorescence microscopy were used to characterize surface homogeneity and chemical properties. Cell detection was investigated by optical microscopy.Moreover, synthetic fluorescently labeled peptides were prepared and used for developing faster and lowercost identification assay compared with classic ELISA immunoassay. Results showed an effective immobilization of PC3 cells on silicon surface and the specific recognition of these cells by fluorescent Urotensin II (4-11). In conclusion, this strategy could be really useful as diagnostic system for prostate cancer

Patterning Large-Scale Nanostructured Microarrays on Coverslip for Sensitive Plasmonic Detection of Aqueous Gliadin Traces

(c) The Author/sUser-friendly devices for detecting low gliadin content in commercial foods are of extreme importance for people with gluten diseases. With this concern, the present work proposes a rapid and sensitive optical nanostructured microarrays platform for the detection of gliadin using specific anti-gliadin IgG antibodies immobilized on annealed gold nanostructures (AuNPs) obtained after the high annealing process (550◦C) of gold thin films evaporated on commercial glass coverslips. Localized Surface Plasmon Resonance (LSPR) immunosensing of gliadin in the range of 0.1 ppm to 1000 ppm is successfully achieved. In addition, the biofunctionalization protocol was used for gluten screening in five food complex products.Publishe

Magnetic Janus micromotors for fluorescence biosensing of tacrolimus in oral fluids

Tacrolimus (FK506) is a macrolide lactone immunosuppressive drug that is commonly used in transplanted patients to avoid organ rejection. FK506 exhibits high inter-and intra-patient pharmacokinetic variability, making monitoring necessary for organ graft survival. This work describes the development of a novel bioassay for monitoring FK506. The bioassay is based on using polycaprolactone-based (PCL) magnetic Janus micro motors and a recombinant chimera receptor that incorporates the immunophilin tacrolimus binding protein 1A (FKBP1A) tagged with Emerald Green Fluorescent Protein (EmGFP). The approach relies on a fluorescence competitive bioassay between the drug and the micromotors decorated with a carboxylated FK506 toward the specific site of the fluorescent immunophilin. The proposed homogeneous assay could be performed in a single step without washing steps to separate the unbound receptor. The proposed approach fits the therapeutic requirements, showing a limit of detection of 0.8 ng/mL and a wide dynamic range of up to 90 ng/mL. Assay selectivity was evaluated by measuring the competitive inhibition curves with other immunosuppressive drugs usually co-administered with FK506. The magnetic propulsion mechanism allows for efficient operation in raw samples without damaging the biological binding receptor (FKBP1A-EmGFP). The enhanced target recognition and micromixing strategies hold considerable potential for FK506 monitoring in practical clinical use.Ministerio de Ciencia e InnovaciónComunidad de Madri

Isolation of exosomes from whole blood by a new microfluidic device: proof of concept application in the diagnosis and monitoring of pancreatic cancer

Background: Exosomes are endocytic-extracellular vesicles with a diameter around 100 nm that play an essential role on the communication between cells. In fact, they have been proposed as candidates for the diagnosis and the monitoring of different pathologies (such as Parkinson, Alzheimer, diabetes, cardiac damage, infection diseases or cancer). Results: In this study, magnetic nanoparticles (Fe3O4NPs) were successfully functionalized with an exosome-binding antibody (anti-CD9) to mediate the magnetic capture in a microdevice. This was carried out under flow in a 1.6 mm (outer diameter) microchannel whose wall was in contact with a set of NdFeB permanent magnets, giving a high magnetic field across the channel diameter that allowed exosome separation with a high yield. To show the usefulness of the method, the direct capture of exosomes from whole blood of patients with pancreatic cancer (PC) was performed, as a proof of concept. The captured exosomes were then subjected to analysis of CA19-9, a protein often used to monitor PC patients. Conclusions: Here, we describe a new microfluidic device and the procedure for the isolation of exosomes from whole blood, without any need of previous isolation steps, thereby facilitating translation to the clinic. The results show that, for the cases analyzed, the evaluation of CA19-9 in exosomes was highly sensitive, compared to serum samples

How Nanophotonic Label-Free Biosensors Can Contribute to Rapid and Massive Diagnostics of Respiratory Virus Infections : COVID-19 Case

Altres ajuts: Generalitat de Catalunya /CERCAThis ACS article is provided to You under the terms of this Standard ACS AuthorChoice License. License: https://pubs.acs.org/page/policy/authorchoice_termsofuse.htmlThe global sanitary crisis caused by the emergence of the respiratory virus SARS-CoV-2 and the COVID-19 outbreak has revealed the urgent need for rapid, accurate, and affordable diagnostic tests to broadly and massively monitor the population in order to properly manage and control the spread of the pandemic. Current diagnostic techniques essentially rely on polymerase chain reaction (PCR) tests, which provide the required sensitivity and specificity. However, its relatively long time-to-result, including sample transport to a specialized laboratory, delays massive detection. Rapid lateral flow tests (both antigen and serological tests) are a remarkable alternative for rapid point-of-care diagnostics, but they exhibit critical limitations as they do not always achieve the required sensitivity for reliable diagnostics and surveillance. Next-generation diagnostic tools capable of overcoming all the above limitations are in demand, and optical biosensors are an excellent option to surpass such critical issues. Label-free nanophotonic biosensors offer high sensitivity and operational robustness with an enormous potential for integration in compact autonomous devices to be delivered out-of-the-lab at the point-of-care (POC). Taking the current COVID-19 pandemic as a critical case scenario, we provide an overview of the diagnostic techniques for respiratory viruses and analyze how nanophotonic biosensors can contribute to improving such diagnostics. We review the ongoing published work using this biosensor technology for intact virus detection, nucleic acid detection or serological tests, and the key factors for bringing nanophotonic POC biosensors to accurate and effective COVID-19 diagnosis on the short term

BIOLIF: Artrodese lombar intersomática - abordagem não instrumentada

Tese de doutoramento em MedicinaSpinal fusion (SF) is a surgical procedure conducted to promote bone growth in-between spinal segments, supported by fixation hardware, and complemented by bone graft or bone substitute. There are recognized risks and complications associated with instrumentation, such as damage to surrounding tissues, neurological deficits, material failure or migration and non-union. In this thesis, a novel approach is proposed based on the development of an adhesive, biodegradable and injectable foam, with the purpose to avoid instrumentation in SF. Carbon dioxide foaming was explored as processing methodology to generate, within physiologically compatible conditions, polycaprolactone (PCL) foams with morphological characteristics equivalent to those found in trabecular bone. A three-dimensional, mechanically stable and bioactive composite of PCL+βTCP+Dexamethasone was foamed at 45ºC and 5 MPa. This optimized PCL processing opened the possibility for creating a porous foam, delivered directly into the intervertebral space through a surgical tool designed and built for this purpose. The adhesive properties of PCL were further improved through modification with polydopamine (pDA) and polymethacrylic acid (pMAA). After tensile testing, PCL pDA pMAA material–bone interface remained intact at both ends (adhesivity significantly superior to non-modified PCL, p<0.05). Further in vitro assays confirmed the formulation as non-cytotoxic and bioactive (calcium phosphate (CaP) layer formation). Lastly, the surgical feasibility of PCL pDA pMAA foaming and its biological performance for non instrumented spinal fusion were assessed in a 6-month survival study using an interbody fusion porcine model. Segmental instrumented arthrodesis was used as control group. Minimally invasive in situ foaming of PCL pDA pMAA (BIOLIF) was technically achieved, leading to reduced surgical time (p<0.05) as compared to instrumentation. Animals in BIOLIF approach demonstrated no surgical complications and a higher mobility (p<0.05) at immediate post-op. Spinal fusion was determined by a set of assessments including: i) bone volume/ tissue volume percentage (BV/TV), superior in BIOLIF group (p<0.05); ii) reduced range of motion and increased stiffness of the treated spinal segment, equivalent in both groups; and iii) a relatively well-organized newly formed osseous structure identified by histological analysis at BIOLIF samples. As conclusion, the results obtained in this work could open a new perspective for lumbar instrumentation-free spinal fusion using biologic solutions.A artrodese da coluna vertebral é um procedimento cirúrgico que visa a indução de crescimento ósseo entre segmentos vertebrais, utilizando sistemas de fixação e suplementação com enxerto ósseo ou substituto sintético. São reconhecidos riscos e complicações associados à instrumentação, incluindo, danos nos tecidos circundantes, compromisso neurológico, risco de mobilização ou migração do material e pseudartrose. Nesta tese, é proposta uma nova abordagem, baseada no desenvolvimento de uma espuma adesiva, biodegradável e injetável, de forma de realizar artrodese intersomática lombar sem recurso a instrumentação. A tecnologia supercrítica/subcrítica foi explorada para a produção de uma espuma de policaprolactona (PCL), em condições fisiologicamente compatíveis, com características morfológicas equivalentes às encontradas no osso trabecular. Foi possível obter a 45ºC e 5 MPa, uma estrutura tridimensional de PCL+βTCP+Dexametasona mecanicamente estável e com propriedades bioativas. Estas condições tornaram possível a extrusão da espuma diretamente no espaço intersomático, através de um instrumento cirúrgico desenvolvido para esse efeito. As propriedades adesivas do PCL foram otimizadas através da modificação do polímero com polidopamina (pDA) e ácido polimetacrílico (pMAA), que se demonstrou significativamente mais adesivo do que o PCL p<0,05 em ensaios mecânicos de tração. As propriedades citocompatíveis e bioativas da formulação foram confirmadas em ensaios in vitro. Por fim, a exequibilidade cirúrgica da extrusão da espuma de PCL pDA pMAA, e o seu desempenho biológico, foram avaliados num estudo de sobrevida de 6 meses usando o porco doméstico como modelo animal. Como grupo de controlo foi realizada artrodese intersomática instrumentada. Foi tecnicamente possível efetuar extrusão in situ de PCL pDA pMAA (BIOLIF) por via minimamente invasiva, sendo o tempo de procedimento cirúrgico significativamente inferior (p<0,05) ao grupo da instrumentação. Os animais do grupo BIOLIF não demonstraram complicações cirúrgicas e apresentaram uma maior mobilidade (p<0,05) no pós-operatório imediato. A qualidade da artrodese foi avaliada por um conjunto de parâmetros: i) a relação volume ósseo/ volume total (BV/TV), superior no grupo BIOLIF (p<0,05); ii) a redução da amplitude de movimento e o aumento da rigidez do segmento vertebral intervencionado, equivalente em ambos os grupos; e iii) uma estrutura óssea recém-formada relativamente bem-organizada no grupo BIOLIF, identificada por análise histológica. Em conclusão, os resultados obtidos neste trabalho podem abrir uma nova perspectiva para a utilização de soluções biológicas como forma de realizar artrodese intersomática lombar sem recurso a instrumentação.Portuguese Foundation for Science and Technology (FCT) - projects UIDB/50026/2020 and UIDP/50026/2020

Design and Synthesis of Antithrombotic Liposomal Protein Conjugate

Recent advances in the molecular bases of haemostasis have highlighted that endothelial thrombomodulin (TM) plays a critical role in local haemostasis by binding thrombin and subsequently converting protein C to its active form (APC). In addition, the binding of thrombin to TM drastically alters the thrombin\u27s procoagulant activities to anticoagulant activities. The lipid bilayer in which it resides serves as an essential \u27cofactor\u27, locally concentrating and coordinating the appropriate alignment of reacting cofactors and substrates for protein C activation. On the other hand, liposomes have been extensively studied as cell membrane model as well as carrier for delivering certain vaccines, enzymes, drugs, or genes. In this study, antithrombotic liposomal TM conjugate was design and developed by combination of antithrombotic membrane protein TM into liposome through recombinant and bioorthogonal conjugation techniques, which providing a rational strategy for generating novel and potential antithrombotic agent. Namely, the liposomal TM conjugate mimics the native endothelial antithrombotic mechanism of both TM and lipid components and thus is more forceful than current antithrombotic agent.First, an efficient and chemoselective liposome surface functionalization method was developed based on Staudinger ligation, in which a model compound carbohydrate derivative carrying a spacer with azide was conjugated onto the surface of preformed liposomes carrying a terminal triphenylosphine in PBS buffer (pH 7.4) and at room temperature.Second, recombinant TM containing the EGF-like domains 456 with an azidohomoalaine at the C-terminal has been expressed and incorporated into liposome to form antithrombotic liposomal TM conjugate via Staudinger ligation. In addition, another chemically selective liposomal surface functionalization method for antithrombotic liposomal TM conjugated has been developed based on copper-free click chemistry, which provides an alternative approach to improving reaction efficiency and stability an

Design and Synthesis of Antithrombotic Liposomal Protein Conjugate

Recent advances in the molecular bases of haemostasis have highlighted that endothelial thrombomodulin (TM) plays a critical role in local haemostasis by binding thrombin and subsequently converting protein C to its active form (APC). In addition, the binding of thrombin to TM drastically alters the thrombin\u27s procoagulant activities to anticoagulant activities. The lipid bilayer in which it resides serves as an essential \u27cofactor\u27, locally concentrating and coordinating the appropriate alignment of reacting cofactors and substrates for protein C activation. On the other hand, liposomes have been extensively studied as cell membrane model as well as carrier for delivering certain vaccines, enzymes, drugs, or genes. In this study, antithrombotic liposomal TM conjugate was design and developed by combination of antithrombotic membrane protein TM into liposome through recombinant and bioorthogonal conjugation techniques, which providing a rational strategy for generating novel and potential antithrombotic agent. Namely, the liposomal TM conjugate mimics the native endothelial antithrombotic mechanism of both TM and lipid components and thus is more forceful than current antithrombotic agent.First, an efficient and chemoselective liposome surface functionalization method was developed based on Staudinger ligation, in which a model compound carbohydrate derivative carrying a spacer with azide was conjugated onto the surface of preformed liposomes carrying a terminal triphenylosphine in PBS buffer (pH 7.4) and at room temperature.Second, recombinant TM containing the EGF-like domains 456 with an azidohomoalaine at the C-terminal has been expressed and incorporated into liposome to form antithrombotic liposomal TM conjugate via Staudinger ligation. In addition, another chemically selective liposomal surface functionalization method for antithrombotic liposomal TM conjugated has been developed based on copper-free click chemistry, which provides an alternative approach to improving reaction efficiency and stability an

[99mTc]Tc-PentixaTec: development, extensive pre-clinical evaluation, and first human experience

Purpose The clinical success non-invasive imaging of CXCR4 expression using [(68) Ga]Ga-PentixaFor-PET warrants an expansion of the targeting concept towards conventional scintigraphy/SPECT with their lower cost and general availability. To this aim, we developed and comparatively evaluated a series of Tc-99m-labeled cyclic pentapeptides based on the PentixaFor scaffold.Methods Six mas(3)-conjugated CPCR4 analogs with different 4-aminobenzoic acid (Abz)-D-Ala-D-Arg-aa(3) linkers (L1-L6) as well as the corresponding HYNIC- and N-4-analogs of L6-CPCR4 were synthesized via standard SPPS. Competitive binding studies (IC50 and IC(50)inv) were carried out using Jurkat T cell lymphoma cells and [I-125]FC-131 as radioligand. Internalization kinetics were investigated using hCXCR4-overexpressing Chem-1 cells. Biodistribution studies and small animal SPECT/CT imaging (1 h p.i.) were carried out using Jurkat xenograft bearing CB17/SCID mice. Based on the preclinical results, [Tc-99m]Tc-N-4-L6-CPCR4 ([Tc-99m]Tc-PentixaTec) was selected for an early translation to the human setting. Five patients with hematologic malignancies underwent [Tc-99m]Tc-N-4-L6-CPCR4 SPECT/planar imaging with individual dosimetry.Results Of the six mas(3)-conjugated peptides, mas(3)-L6-CPCR4 (mas(3)-dap-r-a-Abz-CPCR4) showed the highest CXCR4 affinity (IC50 = 5.0 & PLUSMN; 1.3 nM). Conjugation with N-4 (N-4-L6-CPCR4) further improved hCXCR4 affinity to 0.6 & PLUSMN; 0.1 nM. [Tc-99m]Tc-N-4-L6-CPCR4 also showed the most efficient internalization (97% of total cellular activity at 2 h) and the highest tumor accumulation (8.6 & PLUSMN; 1.3% iD/g, 1 h p.i.) of the compounds investigated. Therefore, [Tc-99m]Tc-N-4-L6-CPCR4 (termed [Tc-99m]Tc-PentixaTec) was selected for first-in-human application. [Tc-99m]Tc-PentixaTec was well tolerated, exhibits a favorable biodistribution and dosimetry profile (2.1-3.4 mSv per 500 MBq) and excellent tumor/background ratios in SPECT and planar imaging.Conclusion The successive optimization of the amino acid composition of the linker structure and the N-terminal Tc-99m-labeling strategies (mas(3) vs HYNIC vs N-4) has provided [Tc-99m]Tc-PentixaTec as a novel, highly promising CXCR4-targeted SPECT agent for clinical application. With its excellent CXCR4 affinity, efficient internalization, high uptake in CXCR4-expressing tissues, suitable clearance/biodistribution characteristics, and favorable human dosimetry, it holds great potential for further clinical use