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Metal-free contrast agents: novel approaches

The joint expertise of CIBER-BBN Nanomol Group – NANBIOSIS U6 from at ICMAB-CSIC (José Vidal and Vega Lloveras) and NANBIOSIS U25 at UAB (Ana Paula Candiota), led to a recently published article in the prestigious journal Biomacromolecules

Brain tumours such as Glioblastomas are a challenge in the clinics and proper diagnosis and follow-up are crucial for patient outcome. Contrast agents are usually administered to patients for assessing blood brain barrier integrity and quantitation of enhancing areas are part of the clinical criteria for estimating response/relapse. However, most contrast agents currently used in clinics are based in metal elements such as Gadolinium and are not exempt of risks. In addition, due to the renal excretion route, administering such agents to some patients is contraindicated. Our work explored the potential of organic radicals anchored to dendrimers to act as contrast agents for glioblastoma studies, proposing a metal-free alternative for contrast enhanced glioblastoma studies. The article describes details of synthesis and characterization of these agents, as well as in vivo, ex vivo and in vivo magnetic resonance studies. The orthotopic immunocompetent GL261 glioblastoma murine model was used for in vivo and ex vivo studies. The novel contrast agent proved to be non-harmful for wild type mice and produced sustained and long lasting contrast in tumour-bearing mice, even in much lower doses in comparison with gadolinium administration.

The diagnosis and follow-up of high-grade brain tumours such as glioblastomas relies mostly in MRI, and contrast agents currently used are based on Gadolinium, which is not exempt of risks. The resarchers’ approach explores organic radicals anchored to dendrimers as a metal-free alternative to produce contrast enhancement in MRI, safer than Gadolinium-based compounds, and with translational potential.

Article of reference:
Zhang S, Lloveras V, Lope-Piedrafita S, Calero-Pérez P, Wu S, Candiota AP, Vidal-Gancedo J. Metal-Free Radical Dendrimers as MRI Contrast Agents fof Glioblastoma Diagnosis: Ex Vivo and In Vivo Approaches. Biomacromolecules. 2022 Jun 24. doi: 10.1021/acs.biomac.2c00088.

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How engineered protein helps Nanomedicine againts Cancer

The use of protein nanoparticles as biomaterials have been rising in recent years due to their characteristics: high biocompatibility, structural versatility, biodegradability and plasticity of design. We can later incorporate peptide ligands for specific targeting as fusion proteins and use these nanoparticles for targeted nanomedicine.

However, not all proteins can be used as scaffolds for targeted drug delivery, as they need to meet certain criteria. First, it is crucial that the proteins used as a scaffold allow site-specific drug conjugation. The stability and proteolysis resistance of these proteins is also important to remain assembled during the bloodstream circulation. In addition, the scaffolds must be biologically neutral, meaning that they should not interact with other human proteins that interfere with their capacity to reach and specifically deliver their cargo. The lack of immunogenicity of these proteins is also desired to avoid immune system recognition. And, ideally, the proteins used as a scaffold should not have post-translational modifications to ensure that they fold equally in both prokaryotic and eukaryotic cell factories for production.

The scaffolds that have all these properties have a better chance to both achieve a proper biodistribution and to successfully deliver their cargo molecules into the target cells. The Green Fluorescent Protein (GFP) satisfy most of the desired characteristics for a scaffold. Moreover, its intrinsic fluorescence allows the tracking of the protein distribution and intracellular localization both in vitro and in vivo.

The use of GFP as a protein scaffold for targeted drug delivery has been extensively studied in our group. We have been able to deliver cytotoxic drugs through our patented platform for targeted delivery. This platform consists of a cationic peptide ligand (T22) and a hexa-histidine peptide that act as self-assembling tags. T22 is a CXCR4 ligand that enables a targeted delivery to CXCR4+ cells, a receptor that is overexpressed in metastatic cancer cells. We have demonstrated previously in an in vivo model that more than the 85% of the administered product was accumulated in the tumor and that we could efficiently conjugate Floxuridine (a genotoxic antimetabolite) to our T22-GFP-H6 nanoparticles, resulting in a strong anti-metastatic activity.

Despite these very promising results, GFP is an exogenous protein from Aequorea victoria and, consequently, triggers an immune response, which limits its clinical use. Thus, we needed to find a human protein that matches the exceptional properties of GFP as a protein scaffold. Fortunately, a non-fluorescent GFP-like protein has been described in humans and it corresponds to one of the three globular domains of Nidogen, a structural protein that binds to collagen IV, laminin and perlecan with high affinity. The globular domain G2 has a beta-barrel structure with a central alpha-helix that folds very similarly to the GFP, despite that these proteins share very low sequence identity. Notably, this domain does not have post-translational modifications that could interfere with its production and folding in prokaryotic cells.

However, perlecan and collagen IV binding sites have been reported within this G2 domain. Therefore, we needed to selectively mutate these binding sites in order to assure the biological neutrality of the nanoparticles. After a thorough structural analysis, we incorporated four different mutations to engineer a biologically neutral product that was named HSNBT. There were no differences detected between the wild-type G2 domain and the engineered HSNBT protein regarding the predicted structural epitopes, which suggested that the introduced mutations would not generate immunogenicity.

In order to validate the new scaffold, we used the above-mentioned patented platform with T22 and the hexa-histidine tag, replacing GFP for the new HSNBT scaffold. First, we characterized the resulting nanoparticles and we determined, both by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM), that they had a size of around 10 nanometers. Then, we observed that the T22-HSNBT-H6 nanoparticles were internalized effectively by CXCR4+ cells. This specificity was corroborated when we used a CXCR4 antagonist (AMD) and we saw a notable decrease of their internalization. Then, we successfully conjugated floxuridine to the nanoparticles (T22-HSNBT-H6-FdU) through the free lysine-amino groups of the protein and we demonstrated that the nanoconjugates had a potent cytotoxic effect in CXCR4+ cells.

Once we have validated these nanoconjugates in vitro, we tested them in a colorectal cancer mouse model. Notably, we saw an important tumor growth inhibition after several doses of these nanoconjugates. The inhibitory effect was slightly higher when using the new scaffold than with GFP. We also saw a significant increase in cell death bodies and caspase-3 activation in the tumor after the treatment with the nanoconjugates. Again, the effect was more potent with HSNBT as a scaffold than with GFP. Remarkably, the treatment did not result in any histological toxicity and there were no differences between the weight of the treated mice when compared to the untreated mice.

This technology is protected by 3 patents: The ligand to enter CXCR4+ cells (WO2012/095527), the nanoconjugates (EP17382461.6) and the human scaffold protein HSNBT, (EP19383201), all three licensed to Nanoligent SL.

All in all, these results confirm that the G2 domain of nidogen can be used as a protein scaffold for targeted drug delivery. Its performance both in vitro and in vivo not only matches the observed with GFP, but it is even more efficient than GFP when conjugated with floxuridine. Therefore, the engineered HSNBT protein shows a very exciting potential to be used in the development of protein-based nanomedicines.  

By Carlos Martínez Torró (NANBIOSIS U1 PPP)

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1st Forum of CIBER-BBN/NANBIOSIS and CSIC Nanomed Conection researchers

The Nanomed Conection of the Spanish Research Council (CSIC) and the Networking Biomedical Research Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), together with its singular infrastructure NANBIOSIS, have organised a Forum on Nanomedicine Research during the days 30 of June and 1st of July to be held at the Institute of Advanced Chemistry of Catalonia (IQAC-CSIC) in Barcelona. The event will be also transmitted on-line previous registration.

This is the first meeting gathering together scientists from CIBER-BBN and its ICTS NANBIOSIS and from the CSIC’ Nanomed Conection with a shared interest in Nanomedicine.

This two days meeting will allow researchers to present their works in progress, share their scientific concerns and needs and discuss the impact of nanomedicine in the emerging fields of drug delivery, diagnosis and therapy.

The programe, available in the web of the forum includes these sessions:

  • Nanobiotechnological solutions for diagnosis and therapy
  • Drug delivery nanosystems
  • Applications for oncology (I and II)
  • Nanomedicine & other frontier applications

Attendance to the Forum (in person / or online) is free prior registration in the web of the forum (following this link):

Registration will remain open until June 26.
We hope to see you there!

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NANBIOSIS in the new poster of ICTS map published by Spanish Goverment.

ICTS underpins the Spanish reputation for research excellence.

In the picture: the new poster of the ICTS map in which NANBIOSIS facilities have been highlighed

The term Unique Scientific and Technical Infrastructure (ICTS) refers to facilities, resources, or services for the development of top-quality cutting-edge research, as well as the communication, exchange, and preservation of knowledge, the transfer of technology, and promotion of innovation. They are unique or exceptional in their fields, with a high cost of investment, maintenance, and operation, and are of a strategic importance that justifies their availability to all actors in the field of R&D&I. The ICTS share three fundamental characteristics; they are infrastructures with public ownership, unique and open to competitive access.

ICTS offer an opening capacity percentage of their essential services under ‘Competitive Open Access’ for the use by national and international public and private sector researchers, with the support of technical and administrative personnel of the ICTS. Infrastructures access is ruled by a public “Access Protocol” that describes the procedure and criteria for access to the infrastructure. The main features of ‘Competitive Open Access’ are that R + D + i quality of activities developed at the infrastructure should be proven and that requests for access should be prioritized on the basis of objective criteria.

The dissemination of ICTS and their capabilities is essential to provide Spanish and international researchers with access to a large base of quality services and facilities, a basic requirement for the development of excellent science.

The new posster pushised by the General Subdirectorate of Large
Scientific-Technical Facilities of the of the Sapanish Ministry of Science and Research Innovation helps to disseminate and understand the map of ICTS wich are located throughout the country

NANBIOSIS, is one of the five ICTS in the field of Health Sciences and Biotecnology

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How to accomplish researchers’ goals with Confocal Microscopy: the tools, the know-how and the expertise you need

NANBIOSIS Unit 17 (Confocal Microscopy) is a CIBER-BBN unit located in the Cell Culture Unit, CAI Medicine and Biology, Faculty of Medicine at the University of Alcala. This unit of the ICTS NANBIOSIS supports researchers interested on their different studies visualizing diverse samples as tissues, cells, bacterial biofilms, etc. This unit owns the tools, the know-how and the expertise to accomplish researchers’ goals either by transmission or reflection fluorescent.

We are happy of sharing this video in which researchers of Unit 17 show all the steps required for the visualization of the PV-1 molecule, also known as PLVAP, on the gut-endothelium of cirrhotic rats. We look at the whole process, starting by the sample selection following their preparation until its visualization by the confocal fluorescent microscopy, ending up with the analyze process.

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“The almighty graphene”, a podcast by Elisabet Prats

Elisabet Prats Alfonso, a researcher in the team coordinating NANBIOSIS U8 Micro– Nano Technology Unit explains in a podcast her most recent research based on the functionalization of chemical and biochemical sensor platforms as well as the characterization of materials such as graphene for both neuronal recording and biomarker detection. Her work is part of the Graphene Flagship project in which she collaborates with relevant European groups.

Eli Prat as a researcher Ph.D. in Chemistry and also dedicated to dissemination is a great exemple for the NANBIOSIS aim to encourage STEAM scientific vocations especially among girls.

In addition, she is the author, together with Helena González and Oriol Marimón, of the book Elementum and the great robbery of Nurú” (La Esfera de los Libros, 2020), a scientific novel aimed at children .

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Nora Ventosa appointed Research Professor under the Materials Science

Nora Ventosa, the Scientific Director of NANBIOSIS U6 of Biomaterial Processing and Nanostructuring Unit (and IP of the NANOMOL Group of CIBERBBN at ICMAB-CSIC) has been appointed Research Professor in the last call of the Spanish National Reserach Council (CSIC)

This elective process for the selection and appointment of career civil servant, by internal promotion, in the Scale of Research Professors of Public Research Organisms had oppen 6 new positions for Professors under the Materials Science.

Nora Ventosa is a Chemical Engineer by the Institut Químic de Sarrià (IQS) and Doctor in Chemistry by the Universitat Ramon Llull. Her research is focused on green chemistry, soft materials and nanobiochemistry. She is devoted to develop and apply new methodologies, based on compressed fluids, in order to gain control of molecular self-assembly in solution.

Prof. Ventosa is also co-founder of the spin-off Nanomol Technologies, vicepresident of the Societat Catalana de Química (SCQ-IEC), member of the Administration Council of ACCIÓ, the Catalan Government agency for the promotion of the competitiveness within companies, and President of the TECNIO Research Association. She has participated, organized and directed more than 50 research projects, funded by different national and international agencies, public, private and industrial, including the European projects Smart4Fabry (Smart multifunctional GLA-nanoformulation for Fabry disease) or Phoenix (Open Innovation Test Bed for Enabling Pharmaceutical Innovative Products).

U6: Biomaterials processing and Nanostructuring unit

 Unit 6 of NANBIOSIS, on Biomaterials processing and Nanostructuring unit from CIBER-BBN, hosted at ICMAB-CSIC, with Scientific Director Nora Ventosa, from the Nanomol-Bio research group. The U6 gathers several laboratories, perfectly equipped, to perform the mission of this facility: the development, characterization, and large-scale production of molecular biomaterials of therapeutic or biomedical interest, with controlled micro-, nano- and supramolecular structure. One example of Key-Enabling-Technology (KET) available in this unit in collaboration with the SME Nanomol Technologies (spin off of CSIC) is a simple and green one-step methodology, DELOS, based on the use of compressed fluids (CF), such as CO2, to prepare particulate materials with precise and reproducible structural characteristics at micro-, nano- and supramolecular levels (size, shape, internal structural gradients, supra­molecular organization and crystalline purity). This example shows one of the singularities of this unit is that counts with CF–based plants at different scales, from mL to L, which allow process development by QbD and process scale-up. Recently U6 NANBIOSIS, together with the Soft Scientific and Technical Service, was accredited with the ISO9001 certification for the standard quality control system. 

Congratulations Nora!

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Improving quality of MR spectra from mouse brain. MRSI-detected pattern in glioblastoma patients

Work performed at Unit 25 of Nanbiosis ICTS of “NMR: Biomedical ApplicationsI” is being shown at the Joint annual meeting ISMRM-ESMRMB (May 7-12th) London, with the participation of CIBER-BBN group members Ana Paula Candiota, Silvia Lope-Piedrafita, Miquel Cabañas (abstract 1), Carles Arús, Gulnur Ungan, Margarida Julià-Sapé, Alfredo Vellido and Carles Majós (abstract 2).

In the first abstract, entitled “High resolution Multi-voxel spectroscopy using CSI-semi-LASER for mouse brain preclinical studies” we focused into improving quality of MR spectra obtained from mouse brain, a key factor when trying to pursue metabolomic-based biomarkers.

The second abstract, entitled “MRSI-detected pattern in glioblastoma patients one month after concomitant chemoradiotherapy” presented a study with a retrospective MRSI set of 31 glioblastoma patients and investigation of spectral patterns predictive of true progression or pseudoprogression.

The International Society for Magnetic Resonance in Medicine (ISMRM) and The European Society for Magnetic Resonance in Medicine and Biology (ESMRMB) are prestigious scientific societies devoted to magnetic resonance-based studies at international and European levels with participation of the most renowned scientifics in the field. This year, the international and european events are joined into a single event (https://www.ismrm.org/22m/)

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Induced pluripotent stem cells in disease modelling and experimental therapies: cardiovascular perspective

On the 20th of May, we will be receiving an international visit at the  Unversity of Zaragoza from Pr. Józef Dulak from Jagiellonian University, Kraków, Poland. The title of the talk will be “Induced pluripotent stem cells in disease modelling and experimental therapies: cardiovascular perspective“.

The invited lecture is programed within the framework of the European CISTEM project, in wich the University of Zaragoza participates through CIBER-BBN group TME lab NANBIOSIS U13 Tissue & Scaffold Characterization Unit.

The event will take place at 12:00 in the I3A SEMINAR (2nd floor) of Campus Rio Ebro, of University of Zaragoza

Induced pluripotent stem cells (iPSC) are generated by genetic reprogramming of somatic cells and thanks to the ability to differentiate into almost all cells types of the organism they offer the enormous possibilities for investigating disease mechanisms, drug sensitivity and safety and for experimental regenerative approaches.  iPSC thus became the indispensable tools of current medial biotechnology and received additional input thanks to the development of the CRISPR/Cas9 gene editing.

In this lecture PR. Józef Dulak will review his research in which iPSC and CRISPR/Cas9 gene editing is applyed for investigating the iPSC-differentiation to cardiomyocytes, endothelial cells and other cell types linked with the disease affecting vascular system, heart and the skeletal muscles. The special attention will be on discussing the potential of iPSC for diabetes and Duchenne muscular dystrophy disease modelling.

Application of iPSC-derived cardiomyocytes offer the chance for effective cell therapy of heart failure and this will be addressed in regard to recently published studies.

  1. Sci Rep. 2015 Feb 26;5:8597. doi: 10.1038/srep08597.
  2. Stepniewski J, et al., Dulak J. Heme oxygenase-1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells. IUBMB Life. 2018 Feb;70(2):129-142. doi: 10.1002/iub.1711. 2018 Jan 9.
  3. Kachamakova-Trojanowska N, Stepniewski J, Dulak J.  Human iPSCs-derived endothelial cells with mutation in HNF1A as a model of maturity-onset diabetes of the young. Cells. 2019 Nov 14;8(11). pii: E1440. doi: 10.3390/cells8111440.
  4. Stępniewski J, et al. Dulak J.  Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, in Contrast to Adipose Tissue-Derived Stromal Cells, Efficiently Improve Heart Function in Murine Model of Myocardial Infarction. Biomedicines. 2020 Dec 7;8(12):578. doi: 10.3390/biomedicines8120578.
  5. Jeż M, et al. Dulak J.  Role of Heme-Oxygenase-1 in Biology of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells. Cells. 2021 Mar 1;10(3):522. doi: 10.3390/cells10030522.
  6. Andrysiak K, Stępniewski J, Dulak J. Human-induced pluripotent stem cell-derived cardiomyocytes, 3D cardiac structures, and heart-on-a-chip as tools for drug research. Pflugers Arch. 2021 Jul;473(7):1061-1085. doi: 10.1007/s00424-021-02536-z. Epub 2021 Feb 24.
  7. Martyniak A, et al, Dulak J. Generation of microRNA-378a-deficient hiPSC as a novel tool to study its role in human cardiomyocytes. J Mol Cell Cardiol. 2021 Jul 28;160:128-141. doi: 10.1016/j.yjmcc.2021.07.007.  
  8. Kachamkova-Trojanowska N, Skoczek D, Dulak J,  Maturity Onset Diabetes of the Young – new approaches for disease modelling. Int J Mol Sci. 2021 Jul 14;22(14):7553. doi: 10.3390/ijms22147553.
  9. Andrysiak K, et al., Dulak J. Generation of DMBi002-A human induced pluripotent stem cell line from patient with Spinal muscular atrophy type 3. Stem Cell Res. 2021 Oct 13;57:102563. doi: 10.1016/j.scr.2021.102563. 
  10. Jelinkova S, Martyniak A, Dulak J, Stępniewski J.   Derivation of human pluripotent stem cell line via CRISPR/Cas9 mediated deletion of exon 3 LAMA2 gene (DMBi001-A-1) Stem Cell Res. 2021 Sep 2;56:102529. doi: 10.1016/j.scr.2021.102529
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OPEN SUBMISSION FOR JOURNAL SPECIAL ISSUE ON OLIGONUCLEOTIDE BASED THERAPIES

Dr. Ramon Eritja, Scientific Director of NANBIOSIS unit 29 of Oligonucleotide Synthesis Platform (OSP) and Dr. Santiago Grijalvo researcher at NANBIOSIS unit 12 of Nanostructured liquid characterization, from CIBER-BBN and IQAC-CSIC, together with Dr. Andreia F. Jorge, from Coimbra Chemistry Centre (CQC), acting as guest editors of journal Pharmaceutics, of MDPI Publisher, welcome authors to submit their articles on special issues on Recent Trends in Oligonucleotide Based Therapies.

In the past few decades, significant efforts have been made towards the clinical application of oligonucleotides. However, the potential of the therapeutic applications of RNA-based strategies have recently been spotlighted after the first approval of mRNA vaccines in response to COVID-19 pandemic. These molecules have the power to tackle targets that are usually considered to be “undruggable” by blocking the translation or transcription of a specific gene by stimulating the degradation of a particular messenger RNA.

This Special Issue aims to collect reviews, original research articles, and short communications covering innovative strategies in the design, synthesis, and characterization of therapeutic oligonucleotides as well as advances in their delivery based on nanotechnologies. Research concerning the study of sequence-specific protein–DNA/RNA interactions will be also considered.

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form.

For all the informations and instructions about the Special Issue, please visit this MDPI page

Deadline for submissions: 31 December 2022

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