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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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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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Antitumoral nanoparticles with multiple activities, a close reality.

Conventional chemotherapeutics used to fight cancer promote off-target damage in cells and organs that are not affected by the disease. This major drawback may be overcome with the development of tumor-targeted therapies, in which the antitumoral drugs are selectively delivered to tumoral cells using the efficient recognition between a receptor overexpressed in these cells and its ligand, without promoting off-side effects in the rest of the body.

The group of Nanobiotechnology (NBT) from the Institut de Biotecnologia i Biomedicina (IBB-UAB), led by Prof. Antonio Villaverde, develops a new concept of pharmaceuticals based on protein nanoparticles, in close collaboration with the group of Oncogenesis and Antitumor Drugs (GOA) from the Institut d’Investigació Biomèdica Sant Pau (IIB-Sant Pau) and the group of Nucleic Acids Chemistry from the Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), respectively lead by Prof. Ramon Mangues and Prof. Ramon Eritja. This research is conducted in the frame of the Director plan of CIBER-BBN, an excellence center from the Instituto de Salud Carlos III, to which all the groups belong, being assisted by three Nanbiosis ICTS units (U1, Protein Production Platform; U18, Nanotoxicology Unit; U29, Oligonucleotide Synthesis Platform).

The generated pharmaceuticals are selective for metastatic stem cells, responsible of cancer propagation, recurrence and bad prognosis, that overexpress in their surface the CXCR4 receptor, present in 23 distinct human neoplasias. Using a precise protein engineering, we generate multi-functional protein nanoparticles that remain in the bloodstream for long times and selectively enter and destroy metastatic stem cells, thus contributing to stop cancer progression. In the last years, we have employed two main strategies in the development of antitumoral protein nanoparticles. On one side, current chemotherapeutics already used in clinics in non-targeted approaches, such as Floxuridine or Monomethyl Auristatin E, are chemically linked to targeted protein nanoparticles that serve as drug delivery systems and comprise an inert scaffold, a polyhistidine tag and a targeting peptide that directs their effect to the CXCR4-tumor. On the other side, the inert scaffold of our protein nanoparticles is replaced by toxins, venoms or other death-inducer proteins that confer the protein nanoparticle an intrinsic antitumoral activity, without the need of delivering chemical drugs. Both strategies are protected by intellectual property rights.

Recently, we have explored the possibility of combining both strategies to generate intrinsically toxic nanoparticles loaded with conventional chemotherapeutics in a single pharmacological entity. This way, we seek to potentiate their antitumoral effect and face the appearance of resistances in the tumor. In this initial step, the concept proposed has been demonstrated as fully feasible, as stable nanoparticles that contain both the toxin and the loaded chemotherapeutics were generated. Although these novel nanomaterials do not improve toxic antitumoral activities in CXCR4+ tumor cell lines, this research has been crucial to identify the main bottleneck of the technology, that is achieving a precise control of the drug-binding site in order to maintain the antitumoral activity of targeted toxins, which must act at the same time as active principle and as anchoring site for chemical drugs.

This novel platform recruits in a single pharmacological entity different therapeutic actions and may open a broad investigation field in the design of antitumoral drugs. The current results of this project have been published in the scientific journal Acta Biomaterialia and presented in the international conference NALS2022 by Eric Voltà-Durán.

By Eric Voltà-Durán

Reference article – Design and engineering of tumor-targeted, dual-acting cytotoxic nanoparticles  https://doi.org/10.1016/j.actbio.2020.11.018

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Colloidal phenomena in COVID-19

Transmission electron microscope image of SARS-CoV-2 (National Institute of Allergy and Infectious Diseases https://www.niaid.nih.gov)

The special volume (No. 55) of the journal Current Opinion in Colloid and Interface Science reviews the implications of colloidal science in the phenomenology of COVID-19, for which the techniques available in NANBIOSIS U12, “Nanostructured liquid characterization unit” , are relevant.

Two articles to highlight in this special volume:

  • Airborne transmission of the virus through droplets, and the effect of evaporation and sedimentation. Airborne transmission is determined by the settling time, that is, the time it takes for droplets to be in the air before settling. Evaporation increases the settling time by reducing the mass of the droplets. In fact, the small droplets can, depending on their solute content, evaporate almost completely and remain in the air for a long time. Considering that viruses possibly remain infectious for hours in the form of aerosols, the formation of droplet nuclei can substantially increase the infectious viral airborne load. The article reviews the physical-chemical factors that control the evaporation and sedimentation times of droplets and play an important role in determining the risk of airborne infection. (https://www.sciencedirect.com/science/article/pii/S1359029421000558)

  • The interactions between surfactants and viruses, which act on different components such as the lipid envelope, the membrane proteins (envelope) and the nucleocapsid proteins. Surfactants play very important roles, either directly, as in disinfection, or as carrier components of drug delivery systems for prophylaxis or treatment. By designing tailor-made surfactants and consequently advanced formulations, an increasingly effective use of surfactants can be expected, either directly as antiviral compounds or as part of more complex formulations. (https://www.sciencedirect.com/science/article/pii/S1359029421000637)

In summary, colloid science can contribute in a multidisciplinary strategy to fight pandemics.

By Carlos Rodriguez Abreu, Scientific Director of NANBIOSIS U12

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Fabry Desease in the Rare Disease Day: A New Hope

WHY DO CELEBRATE TODAY THE INTERNATIONAL #RareDiseaseDay?

29 of February is a ‘rare’ date and February, a month with a ‘rare’ number of days, has become a month to raise awareness about rare diseases and their impact on patients’ lives.  Since 2008 thousands of events happen every year all around the world and around the last day of February with the aim of improving equity and reducing stigmatization for people who live with more than 6,000 rare diseases.

WHAT ARE RARE DISEASES

Rare diseases are pathologies or disorders that affect a small part of the population (less than 5 per 10,000 inhabitants) and generally have a genetic component. They are also known as orphan diseases.

Diseases present a series of particular symptoms, and it is very difficult to diagnose what their true cause is. These disorders or alterations that patients present must be evaluated by a specialist, depending on each case.

Today 5% of the world population suffer from them. This translated into numbers, corresponds to approximately 300 million affected.

A patient with a rare disease waits an average of 4 years to obtain a diagnosis, in 20% of cases it takes 10 or more years to achieve the proper diagnosis.

ORPHAN DRUGS

To combat this disease, patients need to be treated with so-called orphan drugs. They serve to prevent and treat pathology. Its composition is based on biotechnological compounds whose manufacture is very expensive and not profitable for companies. For this reason, cooperation of governments is needed as well as financial incentives to encourage pharmaceutical companies to develop and market medicines to make these treatments accessible to a greater number of people.

FABRY DISEASE

Fabry is one of the rare diseases that currently lack a definitive cure. Symptoms may include episodes of pain, especially in the hands and feet (acroparesthesias); small dark red spots on the skin called angiokeratomas; decreased secretion of sweat (hypohidrosis); opacity of the cornea (cataracts) and hearing loss. Internal organs such as the kidney, heart, or brain may be involved, resulting in progressive kidney damage, heart attacks, and strokes.

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients.

SMART 4 FABRY” EUROPEAN PROJECT

CIBER-BBN, through the researcher Nora Ventosa has coordinated the european project “Smart-4-Fabry” developed during 2017-2021, the proyect was undertaken by a consortium formed by ten partners, including private companies and public institutions in Europe and Israel, with a Horizon 2020 financial programme by the European Commission (H2020-NMBP-2016-2017; call for nanotechnologies, advanced materials, biotechnology and production; Proposal number: 720942-2).

In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability.

Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease.

Through a risk analysis and a Design of Experiments (DoE), researechers obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.

The new nanoformulation developed by Smart4Fabry for the treatment of Fabry disease achieved the ODD (Orphan Drug Designation) by the European Commission. The new nanomedicine is more effective and has a better biodistribution than the current treatments, based on enzyme replacement. The new nanomedicine is based on a nanovesicle that protects the enzyme and achieves a better cell internalisation, thus reducing the doses needed, the total cost and improving the quality of patients.

Four units of NANBIOSIS participated in the project:

– U1 Protein Production Platform (PPP) led by Neus Ferrer and Antony Villaverde at IBB-UAB for the production and purification in different expression systems for R&D purposes.

– U3 Synthesis of Peptides Unit led by Miriam Royo at IQAC-CSIC performed all the chemical process of the Smart-4-Fabry project, i.e. design and synthesis of peptides used as targeting ligands in the nanoliposome formulation.

– U6 Biomaterial Processing and Nanostructuring Unit led by Nora Ventosa at ICMAB-CSIC developed tasks related to the manufacture of the nanoliposome formulation of GLA enzyme and the physico-chemical characterization (this unit counts with plants at different scales, from mL to L, which allow process development by QbD and process scale-up, as well as instrumental techniques for assessment of particle size distribution, particle concentration, particle morphology and stability, and Z-potential) .

– U20 In Vivo Experimental Platform led by Ibane Abásolo at VHIR carried out the non-GLP preclinical assays of the project (in vivo efficacy, biodistribution and tolerance/toxicity assays).

PHOENIX: OPEN INNOVATION TEST BED

Researchers of CIBER-BBN and NANBIOSIS, led by Nora Ventosa, are currently participating in another european project, PHOENIX “Enabling Nano-pharmaceutical Innovative Products” in the framework of which this novel nanomedicine developed under the Smar4Fabry project and designed as Orphan Drug by the EMA, will be scaled-up and manufactured under GMP to enable its clinical testing.

Articles of reference:

Josep Merlo-Mas, Judit Tomsen-Melero, José-Luis Corchero, Elisabet González-Mira, Albert Font, Jannik N. Pedersen, Natalia García-Aranda, Edgar Cristóbal-Lecina, Marta Alcaina-Hernando, Rosa Mendoza, Elena Garcia-Fruitós, Teresa Lizarraga, Susanne Resch, Christa Schimpel, Andreas Falk, Daniel Pulido, Miriam Royo, Simó Schwartz, Ibane Abasolo, Jan Skov Pedersen, Dganit Danino, Andreu Soldevila, Jaume Veciana, Santi Sala, Nora Ventosa, Alba Córdoba, “Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method”, The Journal of Supercritical Fluids, Volume 173, 2021, 105204, https://doi.org/10.1016/j.supflu.2021.105204.

Judit Tomsen-Melero, Solène Passemard, Natalia García-Aranda, Zamira Vanessa Díaz-Riascos, Ramon González-Rioja, Jannik Nedergaard Pedersen, Jeppe Lyngsø, Josep Merlo-Mas, Edgar Cristóbal-Lecina, José Luis Corchero, Daniel Pulido, Patricia Cámara-Sánchez, Irina Portnaya, Inbal Ionita, Simó Schwartz, Jaume Veciana, Santi Sala, Miriam Royo, Alba Córdoba, Dganit Danino, Jan Skov Pedersen, Elisabet González-Mira, Ibane Abasolo, and Nora Ventosa. Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment, ACS Appl. Mater. Interfaces 2021, 13, 7, 7825–7838 ( https://doi.org/10.1021/acsami.0c16871).

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DNA nanotechnologists are in mourning

At the end of the XXth century Nanotechnology appeared as one of the more powerful technologies for the future. At that time material sciences were able to produce nanomaterials with exquisite size control and atomic force, microscopy was able to visualize objects in the nanoscale and photolithography arrived to their physical limits in the preparation of computer chips threatening Moore’s law. This empirical law saying that as transistor components shrank, the number per chip doubled about every 18 months, was acting from 1971  (Intel’s first chip) to billions in the present times. 

At these times, one crucial development was the discovery of the first self-assembling DNA structures, leaded by Ned Seeman, who died recently at age 75. Being a crystallographer interested in DNA-protein structures, Ned though that a good way to obtain crystals of DNA-protein complexes was to prepare crystal networks of DNA where proteins bind. (In the classical approach of obtaining protein crystals small oligonucleotides bind). In this way in 1982 he described the idea of making lattices from DNA junctions. In 1991 he obtained a DNA cube, the first tridimensional DNA nanostructure receiving the 1995 Feynman Prize in Nanotechnology. But the most impressive development was the so-called “DNA tile systems” published in 1998.

The figure shows a bidimensional array made by his former Ph.D. student Alejandra Garibotti in our laboratory in Barcelona. In the tile system two or more tiles (each one made out of 5 oligonucleotides) are designed to self-assemble one next to the other by their sticky ends making a large lattice or bidimensional crystal having a tunable shape and size defined by the tiles.

Later on in 2009, Ned was able to demonstrate the achievement of three-dimensional DNA crystals. These developments settle the foundations for the development of DNA origami, DNA computation, DNA nanoelectronics and DNA nanorobotics earning the Kavli Prize in Nanoscience in 2010. The immense creativity of Ned is not only an active value for mankind but also an example for old and new scientists.

By Ramón Eritja, Scientific Director of NANBIOSIS U29, January 10th, 2022

U29.-Oligonucleotide-Synthesis-Platform
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The Medicine of the Future needs the Nanomedicine Revolution. This is why

The medicine of the future is an increasingly tackled topic. In the context of global concern for the sustainability of the health system (chronic diseases, new disorders, aging population and financing problems), nanomedicine could promote more affordable and personalized health care and improve the quality of life of the patients.

Between innovative techniques already implemented and concepts that evoke science fiction (nanobots, fluorescent particles working as spies, tiny Trojan horses introduced into our body …), nanomedicine generates great expectations.

Nanomedicine, what is it exactly?

Nanomedicine is the application of nanotechnology to medicine, that is, the use of nanotechnologic systems for the prevention, diagnosis or treatment of diseases, due to the particular properties that materials present on a nanometric scale. (Yes, although it seems strange, the same material has totally different attributes and behaviours when “nano” amounts of it are manipulated, what is very important in medicine, since many of the processes of the human body take place on a nanometric scale).

The current state, thanks to the previous effort.

When in 1959 Richard Feynmand, (Nobel Prize in Physics in 1965), gave his speech “There is a lot of space down there”, he opened the door to research at the nano scale: from 1nm to 100nm, this is one-millionth of a millimeter (10-9 meters); we are talking about the range of sizes resulting from dividing the diameter of a hair between 1,000 and 10,000, (or what a nail grows in a second).

Since the entry into the market of the first nanomedicine in 1995 (Doxil®, a drug encapsulated in liposomes for the treatment of cancer), nanoparticles or nanostructures have been developed for the controlled release of drugs in cancer and other pathologies, nanodevices have been created for disease diagnosis or nanomaterials have been designed for applications in regenerative medicine, and even messenger RNA vaccines for Covid-19, such as those from Pfizer and Moderna, are nanoformulated. Today there are on the market a hundred nanoformulated drugs all thanks to previous research and development of nanomaterials and nanoparticles over the last three decades.

The “Observatory of Trends in Medicine of the Future” promoted by the Roche Institute foundation has recently published a Report on Nanomedicine coordinated by Dr. Ramón Martínez Máñez, Professor of Inorganic Chemistry at the UPV and Scientific Director of the Centre for Networked Biomedical Research in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) in which Dr. José Becerra, Professor of Cell Biology at the University of Malaga and Principal Researcher at CIBER-BBN, BIONAND and IBIMA, Dr. Pilar Marco, Principal Investigator of the Nanobiotechnology group for the diagnosis (Nb4D) of the IQAC-CSIC and Coordinator of the Nanomedicine Research Program of the CIBER-BBN and Dr. María Jesús Vicent, Chief Researcher of the Therapeutic Polymers Laboratory and coordinator of the Advanced Therapies Area of ​​the Príncipe Felipe Research Center have participated as experts. The report was presented at the IV Conference “Anticipating the Medicine of the Future” on November 30, 2021 where a debate was held by the above mentioned in which various topics related to nanomedicine were discussed, such as its applications and barriers.

Nanomedicine applications of today and tomorrow

Nanomedicine is completely transversal, multidisciplinary and dependent on other disciplines, so its applications are multiple and complementary to other branches of knowledge such as artificial intelligence, but the following fields stand out fundamentally.

The design of nanomaterials that improve biocompatibility or biomechanical properties is investigated and can be used for the manufacture of implants that allow replacing portions of diseased tissue and that can even be designed in a personalized way attending to the individual response of each patient, minimizing the risk of rejection by the patient in regenerative medicine.

Nanoparticles are used to build highly sensitive nanodiagnostic platforms, which provide comprehensive biological information easily, quickly and economically at an increasingly early stage. Pilar Marco visualizes a future where “the diagnosis could be our molecular fingerprint, so that the detection of changes in said fingerprint could lead to the detection of a disease before the patient presents symptoms. In turn, this will contribute to prediction and prognosis since, if a large amount of information is available, it can be crossed with genetic information”.

Nanomedicine makes it possible to improve the pharmacokinetics and pharmacodynamics of current drugs, so that they specifically deploy their activity in diseased cells and tissues in a controlled way over time and crossing any biological barrier, which is called controlled drug release. According to Ramón Martínez “Any disease can be susceptible to use these systems to deliver a drug in the appropriate organ or tissue with the reduction of drug doses and side effects.”

Finally, nanotechnology methods facilitate the fusion of diagnosis and therapy in the new medical field of theragnostic; diagnose and treat at the same time by understanding the biological response to treatments, that is, the administration of drugs whose molecules allow visualize how the drug is working.

Barriers faced by nanomedicine

In addition to the difficulties presented by nanomedicine in matters of regulation and industrial property, the aforementioned experts agree that one of the most important challenges is the standardization of manufacturing procedures and quality controls, investment is needed in infrastructures to fine-tune manufacturing and standardization systems (manufacturing of nanoparticles under GMP) and in collaboration with the private sector, which is crucial, to make nanomedicine reach the productive sector and society.

But there are also barriers in the research itself, and funding is needed to break them down. In nanomedicine research, cost / effectiveness analyses have to be focused on the long term. Professor José Becerra explains it very clearly: “Research topics become fashionable and it happens frequently that the years go by and administrations “get tired” of financing a certain field and this is a problem because if a tree is planted by a person who knows It takes ten years to bear fruit, this person has to take care of the tree, but if we give the tree care to someone who does not know about trees, probably this person will abandon the tree in five years … Scientific policies have to persevere in financing nano and accompany it with an improvement in the regulation of products and only then will companies invest in this area”.

At the end of the debate, Professor José Becerra celebrated that the Carlos III Health Institute opted, fifteen years ago, for the creation of a CIBER in Bioengineering, Biomaterials and Nanomedicine, as a tool for scientific policy, he also mentioned the NANBIOSIS platform created by CIBER-BBN, CCMIJU and BIONAND, recognized as ICTS by the Ministry and available for companies and researchers to produce and characterize bio and nanomaterials, and stated that “it is evident that it is not possible to advance in the transfer of knowledgy from nano to the clinic at the same rate as is done in other knowledge areas but to take care of this project is essential”.

Related news:

Nanomedicine in the Medicine of the future

The Nanomedicine Revolution

informe sobre nanomedicina

‘Point-of-care or PoC’ devices are able to directly detect the genetic material of the virus in just 30 minutes

A more effective nanomedicine has been developed for the treatment of Fabry rare disease

Nanomedicine: how to get drugs to the place where they have to act.

A new generation of devices for the rapid, cheap and easy diagnosis of candidemia

New Nanomedicines for the topical treatment of complex wounds

Sources of information:

Nanomedicine (European Nanotecnology Platform)

IV Jornada Anticipando la Medicina del Fututo

Nanomedicine Report

Nanomed Spain

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Microfluidic devices: The process of development

The development of microfluidic devices is very significant for in-vitro diagnostic devices, biomarkers and organ-on-a-chip applications.

The techniques, the materials and the equipment used for the fabrication of these devices, are as important as the strategy followed to obtain them. And at the Nanotechnology unit of NANBIOSIS U7– (MicrofabSpace and Microscopy Characterization Facilities of IBEC), we own the tools, the know-how and the expertise to accomplish such a goal. We regularly guide the process of design, in collaboration with the final user, we customize the technology needed to get the product and complete the fabrication of the microfluidic device, drawing up like this, a full plan addressed to the final application.

In this video, detailed explanations are provided of all the steps required for the fabrication of a microfluidic device. We go over the whole process, starting by the design of the device, the fundamentals of the Photo Lithography and Soft Lithography processes, to end up with a functional device ready for experiments.

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The Need to Determine the Therapeutic Window of Novel Targeted Anticancer Nanomedicines

The Nanotoxicology Unit of CIBER-BBN ICTS NANBIOSIS, leaded by Ramon Mangues at the Research Institute of the Hospital de Sant Pau is devoted to evaluate effectiveness and toxicity of novel nanoparticles.  This Unit advises clients on the need to study simultaneously anticancer activity and associated toxicity. Thus, preclinical evaluation of novel Nanomedicines is usually carried out performing studies that assess their therapeutic effect, separated from additional experiments devoted to evaluate the toxicity associated with treatment. The dosage used to assess the therapeutic effect, often, significantly differs from the one used to study toxicity, since one is aiming to know the biodistribution of the nanoparticle and whether it is able to control cancer growth, whereas the other tries to identify the maximal tolerated dosage that can be achieved without conferring severe toxicity or lethality.

However, to maximize the effectiveness of novel nanoparticles in the preclinical assessment and their subsequent clinical translation it is important to consider a crucial point of divergence between nanomedicines and classical low molecular weight drugs.

On the one hand, lipophilic small drug bidodistribute by passive diffusion, reaching similar concentration in tumor and non-tumor tissues. Besides, they display a steep dose/effect curve, so that higher doses reach higher anticancer effect (e.g. genotoxic drugs, such as 5-fluorouracil or cisplatin). Nevertheless, this increased effect, obtained intensifying the drug dosage, is achieved at the expense of higher toxicity, that is also dose dependent. In contrast, this situation differs in the case of nanomedicines that use targeted drug delivery, which are capable of selectively concentrating the payload drug delivered by the nanocarrier in target cancer cells, leading to an enhanced uptake in tumor tissue. This effect makes it unnecessary and inefficient to increase the nanomedicine dosage over the one that effectively kill target cells, while maintaining low the associated toxicity. This is because nanomedicines that exploit targeted drug delivery do not have a dose dependent increase in antitumor activity; whereas if administered at high dosage they lose selectivity in their delivery, triggering off-target toxicity, that is likely to be dose-dependent. Thus, increasing the dosage of targeted nanoparticles may increase off-target effects without increasing anticancer effectiveness. In this regard, administering a dosage higher than the one that reaches optimal therapeutic effect can only lead to unspecific internalization in non-target cells and subsequent toxicity.

Therefore, it is our opinion that the evaluation of the tumor and non-tumor tissues biodistribution and the assessment of the therapeutic effect is more informative if at the same time and in the same model is tested the associated toxicity. The testing of various dosage levels will determine which of the evaluated dosage achieves the highest therapeutic window, that is, the one that achieves effective cancer cell killing and optimal antitumor activity without associated toxicity, and the one for which an additional increase in dosage will not improve further the antitumor effect, while increasing instead its toxicity

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Printed sensors, a low cost alternative for clinical detection

In today’s society there is a great interest in developing new technologies that allow low-cost mass manufacturing, also called “rapid prototyping” or “additive manufacturing”. Rapid prototyping includes technologies such as sterolithography, 3D printing, laser sintering or printed electronics, among others. All of these share digital design and manufacturing from the consecutive addition of layers, that is, techniques that allow creating almost any shape or geometric feature in a very fast time.
Printed electronics consists of printing inks on different types of substrates such as textiles, plastics, papers or films to make them “smart”. This technique is presented as an alternative to traditional silicon that is being implemented in sectors as varied as health and wellness, automotive and transport, professional sports, safety and protection, packaging, architecture and construction, and technical textiles. Printed electronics is one of the new technologies that will have a long history throughout the functional electronic device manufacturing space, with a wide range of applications, electronic designs, processes and materials, compared to conventional electronic and microelectronics based manufacturing technology in silicon. It is expected that in the next decade it will be part of everyday life, with products such as electronic skin, electronic tissues and organs or architectural elements that respond to external stimuli.
Among the many areas of interest of these technologies, one of them is the development of low-cost sensors for the medical or environmental area. For example, in these disciplines, it is essential to get devices that can be very economical or even single-use to promote sustainable environmental control and personalized medicine. Specifically with inkjet technology, researchers from NANBIOSIS Unit 8 Micro-Nano Technology Unit belonging to the Institute of Microelectronics of Barcelona (IMB-CNM, CSIC /and CIBER-BBN), have developed over the last few years multiple materials, inks, substrates and sensors for the development of electrochemical sensors in micrometric dimensions.

  • Inks: Most commercial inks are composed of a solvent that contains some material with insulating, conductive or semiconductor properties. As a general rule, an ink must be stable, with a particle size of several orders of magnitude smaller than the injector orifice, have a viscosity of less than 20 mPa s and a surface tension of less than 80 mN m-1. Although these values ​​may depend on the system in question. The final devices are obtained by selectively depositing in previously drawn areas, layer by layer, thin or thick structures on the substrates. They have worked with multiple commercial metallic inks such as gold, silver and platinum.
    Normally in an electrochemical system a noble material that is electrochemically stable is needed to be used in the working electrode and in the counter electrode, and for this gold or platinum are a good alternative. To make measurements with any electrochemical sensor, a reference electrode is essential since it is one that has a stable and constant potential over time and that allows us to reference our voltage value. We use the impression of an Ag / AgCl bilayer since it is one of the interfaces most used as a reference electrode. One of the main problems faced by miniaturization, however, is the rapid loss of the small volume of internal reference solution that these electrodes must have, which has a direct impact on their useful life and stability. For this, a polymeric membrane that can be printed was formulated, which allows the reference to have high performance compared to other commercial miniaturized reference electrodes (Ref1).
  • Substrates: a wide range of rigid, flexible, porous, plastic, fabric, etc. substrates can be used. However, the interaction of the ink with the substrate is crucial in determining the good quality of the printed pattern. For this reason, the properties of the inks are adapted for the different substrates with their own properties. For this reason, it is common practice to pretreat the substrate surface to improve hydrophobicity and adhesion issues mainly. It is common to use plastic substrates with a thickness of the order of microns that provide them with great flexibility and that are already specially treated to obtain excellent printing qualities. The deposition of uniform gold and silver conductive inks on porous substrates can be achieved by using a primer layer to seal the porosity of the membrane in specific and defined areas, with the aim of building a sensor device over the sealed area and leaving the rest of the intact substrate (Ref.2). With a paper substrate, alternatively we can print a silane ink, as a strategy that allows a monolayer of hydrophobic material to grow on the substrate and thus be able to obtain uniform lines of ink on its surface. (Ref.3)
  • Sensors developed: Dissolved oxygen (DO) (Ref.4) and pH (Ref.5) sensors have been developed using gold and platinum inks respectively, commercially available on plastic substrates. The inks have a specially designed formulation that allows their sintering at temperatures as low as 150 and 180 ° C for Au and Pt respectively. This is a key point in the development of low-cost sensors made on polymeric substrates or paper that cannot withstand high temperatures. These sensors integrate in a single platform all the basic elements for the registration of pH and DO, allowing measurements without any external electrode. DO is measured directly with a gold working electrode and pH sensors are achieved after electroplating an iridium oxide film on the platinum working electrode. In addition, this water-based platinum ink has another unique feature, it provides the electrode surface with high roughness, which promotes adhesion of the deposited sensor material, in this case iridium oxide. Long-term stability tests for more than 1 year demonstrate excellent stability of the mechanical sensor layer, and that it correlates perfectly with the different roughness of the printed platinum layer. Along the same lines and in relation to the development of inks, it has been possible to obtain a fully printed pH sensor based on a conductive polymer specially formulated to be printed by IJP . The measurements obtained with this ink have a good response in a wide pH range (pH 3 to 10) and the response in the physiological zone (pH 7-7.5) is well resolved, one of the main drawbacks of conductive polymers. We also present an IJP-printed electrochemical sensor for enzyme-free glucose analysis on flexible PEN substrate (Ref.6). In this case, CuO microparticles were used to modify the electrodes, and the detection of glucose was validated in concentrations that coincide with those of the tear fluid, which allows us to foresee applications in ocular diagnosis, where a painless control can be achieved and not invasive of diabetes by analyzing the glucose contained in tears.

(Ref.1): Moya A, Pol R, Martínez-Cuadrado A, Villa R, Gabriel G, Baeza M. Stable Full Inkjet-Printed Solid-State Ag/AgCl Reference Electrode. Analytical Chemistry 91 (2019) 15539-15546

(Ref.2): M. Ortega-Ribera; X. Guimerà; E. Sowade; M. Zea; X. Illa; E. Ramon; R. Villa; J. Gracia-Sancho; G. Gabriel. Online oxygen monitoring using integrated inkjet-printed sensors in a liver-on-a-chip system. Lab on a Chip. 18 – 14, pp. 2023 – 2035. 2018

(Ref.3): All Inkjet Printing Sensor Device on Paper: for Immunosensors Applications M Zea, A Moya, I Abrao-Nemeir, J Gallardo-Gonzalez, N Zine, A Errachid, … 2019 20th International Conference on Solid-State Sensors, Actuators and 

(Ref.4): Moya A, Sowade E, del Campo FJ, Mitra KY, Ramon E, Villa R, Baumann RR, Gabriel G. All-inkjet-printed dissolved oxygen sensors on flexible plastic Organic Electronics 39 (2016) 168-176

(Ref.5): Zea M, Moya A, Fritsch M, Ramon E, Villa R, Gabriel G Enhanced performance stability of iridium oxide based pH sensors fabricated on rough inkjet-printed platinum ACS Applied Materials & Interfaces 11 (2019) 15160-15169

(Ref.6): Romeo A, Moya A, Leung TS, Gabriel G, Villa R, Sánchez S. Inkjet printed flexible non-enzymatic glucose sensor for tear fluid analysis Applied Materials Today 10 (2018) 133-141

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