Targeted therapeutics and nanodevices


 

Silvia Muro | Group Leader / ICREA Research Professor
Belén García Lareu | Postdoctoral Researcher
Marcelle Silva de Abreu | Postdoctoral Researcher
Josep Fumadó Navarro | PhD Student
Maximilian Loeck | PhD Student
Marco Vigo | PhD Student

About

Currently, a myriad of drug carriers or delivery systems have been developped to improve the overall performance and safety of therapautic agents. However, our ability to treat neurological maladies, genetic syndromes, cancer, etc., still remains a major challenge. One of the prime obstacles is our limited knowledge on the biological parameters that regulate the interaction of these systems with our tissues and, hence, our inability to gain non-invasive, efficient, and specific access within the body, its cells, and subcellular organelles.

To generate knowledge and tools to overcome this obstacle, we study the biological mechanisms ruling how our cells and tissues transport cargoes to precise destinations within our bodies, and apply this knowledge to the design of nanodevices for improved delivery of therapeutic agents to specific disease sites.

Biologically-Controlled Transport of Drug Carriers

Most current strategies aim to bind drug nanocarrier to specific cell-surface receptor; but after said binding takes place, receptor-associated signaling and transport processes take control. Instead, our laboratory can impart a drug carrier control over the biological events that occur beyond bindingWe have shown how using the same receptor, the kinetics, mechanism, and destination of a drug carrier can be modulated by: (a) varying its size, shape, and targeting valency; (b) varying the receptor epitope to which the carrier binds; (c) coupling carriers to signaling molecules; (d) combining targeting to several receptors; or (e) coupling targeting moieties with anti-phagocytic moieties on the surface of drug carriers.  

Transport of Drug Carriers Across Physiological Barriers

Crossing the linings that separate body and cellular compartments is paramount for efficient drug deliveryWe were first to identify a natural pathway regulated by ICAM-1, a cell adhesion molecule overexpressed by most cells under pathological states, which enables transcytosis across the epithelial barrier that separates the gastrointestinal tract from the bloodstream for oral delivery, and the blood-brain barrier that separates the bloodstream from the brain tissue for delivery of therapeutics against neurological diseases. We also work with DNA-built nanocarriers which enable uptake within cells and endosomal escape for delivery to the cytosol and other subcellular compartments.  

ICAM-1-targeted nanocarriers (anti-ICAM NCs) crossing the blood-brain barrier in an animal model. Fluorescence microscopy of cortical brain samples isolated from mice 30 min or 3 h after intravenous injection with green-fluorescent anti-ICAM NCs or control IgG NCs. Scale bar = 10 μm. Boxes = regions magnified 3-fold in the right panels. Arrows = NCs on the blood-facing surface of the blood-brain barrier. Open arrowheads = NCs on the brain-facing surface of the blood-brain barrier. Closed arrowheads = NCs inside of brain endothelial cells. Extracted from Manthe et al., Intertwined mechanisms define transport of anti-ICAM nanocarriers across the endothelium and brain delivery of a therapeutic enzyme. J Control Rel. 324 (2020):181-193).

 

 

Improving Treatment of Lysosomal Disorders

Pathologies due to monogenic deficiencies, such as the case of lysosomal disorders, are valuable models to study disease progression and therapeutic intervention because of their well-known etiologyunequivocal diagnosis, and availability regarding patient samples, diverse cell types, and animal models. Since these diseases associate to either acute or long-term effects depending on genetic severity, and they associate with neurodegeneration, cardiovascular, metabolic, and cancer-like syndromes, they represent excellent disease models. Consequently, we are applying targeted nanotechnology concepts to the treatment of genetic lysosomal disorders. Current therapies by i.v. enzyme infusion are only helpful for diseases where clearance cells and organs (liver, spleen, macrophages, etc.) are the main targets. Yet, delivery to other organs such as the brain, lungs, etc. hinders treatment for most of these diseases. Using types A and B Niemann-Pick, Fabry, and Gaucher diseases as examples, we are developing new therapeutic strategies to deliver therapeutic enzymes to all affected organs in animal models, which holds considerable translational potential. 

ICAM-1 targeted DNA-made nanocarriers (anti-ICAM/3DNA) for drug delivery to the lungs. Cy3-labeled 3DNA targeted to ICAM-1 (anti-ICAM/Cy3-3DNA) or non-targeted controls (IgG/Cy3-3DNA) were intravenously injected in mice. Lungs were isolated and processed at sacrifice at 60 min. (A) Confocal microscopy shows Cy3 3DNA (red), while the targeting coat (antibody) is visualized using a FITC-secondary antibody (green). Arrowheads show colocalization of the antibody on the coat of 3DNA. (B) Confocal microscopy showing anti-ICAM/Cy3-3DNA (red) and lung endothelial cells visualized using polyclonal anti-PECAM-1+FITC-secondary antibody (green). Arrows indicate colocalization of Cy3-3DNA with endothelial PECAM-1. (Extracted from Roki et al, Unprecedently high targeting specific to lung ICAM-1 using 3DNA nanocarriers. J Control Release. 305 (2019):41-49)

Projects

National projects
CROSSTARGET · Desarrollo de nuevas herramientas traslacionales multi-especie para el direccionamiento de terapias con precisión de órgano y subcelular (2019 – 2021) Ministerio de Ciencia, Innovación y Universidades Silvia Muro
BBB2GATE · Control diferencial del transporte de vehiculos terapeuticos dentro versus a traves de la barrera hematoencefalica (2018 – 2020) MINECO Silvia Muro
NANO-GBA · Assessing the effects of glucocerebrosidase (GBA) alterations on receptor membrane nanoarchitecture to design improved nanomedicines(2020 – 2021) BIST Ignite Program Silvia Muro
Fundraising Projects
Campaña FasterFuture “A por el Parkinson”  (2019 – 2020) Fundraising Silvia Muro

Publications

Manthe, Rachel L., Loeck, Maximilian, Bhowmick, Tridib, Solomon, Melani, Muro, Silvia, (2020). Intertwined mechanisms define transport of anti-ICAM nanocarriers across the endothelium and brain delivery of a therapeutic enzyme Journal of Controlled Release 324, 181-193

The interaction of drug delivery systems with tissues is key for their application. An example is drug carriers targeted to endothelial barriers, which can be transported to intra-endothelial compartments (lysosomes) or transcellularly released at the tissue side (transcytosis). Although carrier targeting valency influences this process, the mechanism is unknown. We studied this using polymer nanocarriers (NCs) targeted to intercellular adhesion molecule-1 (ICAM-1), an endothelial-surface glycoprotein whose expression is increased in pathologies characterized by inflammation. A bell-shaped relationship was found between NC targeting valency and the rate of transcytosis, where high and low NC valencies rendered less efficient transcytosis rates than an intermediate valency formulation. In contrast, an inverted bell-shape relationship was found for NC valency and lysosomal trafficking rates. Data suggested a model where NC valency plays an opposing role in the two sub-processes involved in transcytosis: NC binding-uptake depended directly on valency and exocytosis-detachment was inversely related to this parameter. This is because the greater the avidity of the NC-receptor interaction the more efficient uptake becomes, but NC-receptor detachment post-transport is more compromised. Cleavage of the receptor at the basolateral side of endothelial cells facilitated NC transcytosis, likely by helping NC detachment post-transport. Since transcytosis encompasses both sets of events, the full process finds an optimum at the intersection of these inverted relationships, explaining the bell-shaped behavior. NCs also trafficked to lysosomes from the apical side and, additionally, from the basolateral side in the case of high valency NCs which are slower at detaching from the receptor. This explains the opposite behavior of NC valency for transcytosis vs. lysosomal transport. Anti-ICAM NCs were verified to traffic into the brain after intravenous injection in mice, and both cellular and in vivo data showed that intermediate valency NCs resulted in higher delivery of a therapeutic enzyme, acid sphingomyelinase, required for types A and B Niemann-Pick disease.

Keywords: Blood-brain barrier, ICAM-1-targeted nanocarriers, Targeting valency, Receptor-mediated transport, Lysosomal transcytosis destinations


Roki, N., Tsinas, Z., Solomon, M., Bowers, J., Getts, R. C., Muro, S., (2019). Unprecedently high targeting specificity toward lung ICAM-1 using 3DNA nanocarriers Journal of Controlled Release 305, 41-49

DNA nanostructures hold great potential for drug delivery. However, their specific targeting is often compromised by recognition by scavenger receptors involved in clearance. In our previous study in cell culture, we showed targeting specificity of a 180 nm, 4-layer DNA-built nanocarrier called 3DNA coupled with antibodies against intercellular adhesion molecule-1 (ICAM-1), a glycoprotein overexpressed in the lungs in many diseases. Here, we examined the biodistribution of various 3DNA formulations in mice. A formulation consisted of 3DNA whose outer-layer arms were hybridized to secondary antibody-oligonucleotide conjugates. Anchoring IgG on this formulation reduced circulation and kidney accumulation vs. non-anchored IgG, while increasing liver and spleen clearance, as expected for a nanocarrier. Anchoring anti-ICAM changed the biodistribution of this antibody similarly, yet this formulation specifically accumulated in the lungs, the main ICAM-1 target. Since lung targeting was modest (2-fold specificity index over IgG formulation), we pursued a second preparation involving direct hybridization of primary antibody-oligonucleotide conjugates to 3DNA. This formulation had prolonged stability in serum and showed a dramatic increase in lung distribution: the specificity index was 424-fold above a matching IgG formulation, 144-fold more specific than observed for PLGA nanoparticles of similar size, polydispersity, ζ-potential and antibody valency, and its lung accumulation increased with the number of anti-ICAM molecules per particle. Immunohistochemistry showed that anti-ICAM and 3DNA components colocalized in the lungs, specifically associating with endothelial markers, without apparent histological changes. The degree of in vivo targeting for anti-ICAM/3DNA-nanocarriers is unprecedented, for which this platform technology holds great potential to develop future therapeutic applications.

Keywords: 3DNA, DNA nanostructure, Drug nanocarrier, Endothelial and lung targeting, ICAM-1, In vivo biodistribution


Manthe, R. L., Rappaport, J. A., Long, Y., Solomon, M., Veluvolu, V., Hildreth, M., Gugutkov, D., Marugan, J., Zheng, W., Muro, S., (2019). δ-Tocopherol effect on endocytosis and its combination with enzyme replacement therapy for lysosomal disorders: A new type of drug interaction? Journal of Pharmacology and Experimental Therapeutics 370, (3), 823-833

Induction of lysosomal exocytosis alleviates lysosomal storage of undigested metabolites in cell models of lysosomal disorders (LDs). However, whether this strategy affects other vesicular compartments, e.g., those involved in endocytosis, is unknown. This is important both to predict side effects and to use this strategy in combination with therapies that require endocytosis for intracellular delivery, such as lysosomal enzyme replacement therapy (ERT). We investigated this using δ-tocopherol as a model previously shown to induce lysosomal exocytosis and cell models of type A Niemann-Pick disease, a LD characterized by acid sphingomyelinase (ASM) deficiency and sphingomyelin storage. δ-Tocopherol and derivative CF3-T reduced net accumulation of fluid phase, ligands, and polymer particles via phagocytic, caveolae-, clathrin-, and cell adhesion molecule (CAM)-mediated pathways, yet the latter route was less affected due to receptor overexpression. In agreement, δ-tocopherol lowered uptake of recombinant ASM by deficient cells (known to occur via the clathrin pathway) and via targeting intercellular adhesion molecule-1 (associated to the CAM pathway). However, the net enzyme activity delivered and lysosomal storage attenuation were greater via the latter route. Data suggest stimulation of exocytosis by tocopherols is not specific of lysosomes and affects endocytic cargo. However, this effect was transient and became unnoticeable several hours after tocopherol removal. Therefore, induction of exocytosis in combination with therapies requiring endocytic uptake, such as ERT, may represent a new type of drug interaction, yet this strategy could be valuable if properly timed for minimal interference.


Muro, Silvia, (2018). Alterations in cellular processes involving vesicular trafficking and implications in drug delivery Biomimetics 3, (3), 19

Endocytosis and vesicular trafficking are cellular processes that regulate numerous functions required to sustain life. From a translational perspective, they offer avenues to improve the access of therapeutic drugs across cellular barriers that separate body compartments and into diseased cells. However, the fact that many factors have the potential to alter these routes, impacting our ability to effectively exploit them, is often overlooked. Altered vesicular transport may arise from the molecular defects underlying the pathological syndrome which we aim to treat, the activity of the drugs being used, or side effects derived from the drug carriers employed. In addition, most cellular models currently available do not properly reflect key physiological parameters of the biological environment in the body, hindering translational progress. This article offers a critical overview of these topics, discussing current achievements, limitations and future perspectives on the use of vesicular transport for drug delivery applications.

Keywords: Cellular vesicles, Vesicle fusion, Fission and intracellular trafficking, Drug delivery systems and nanomedicines, Transcytosis and endocytosis of drugs carriers, Disease effects on vesicular trafficking, Drug effects on vesicular trafficking, Role of the biological environment


Equipment

  • Dynamic Light Scattering & ζ-Potential: Malvern’s Zetasizer Ultra.
    Determination of the size, concentration, mobility, ζ-potential of nanoparticles, including high resolution size and MW distribution, non-invasive retrodispersion optics for wide concentration ranges, a fluorescent filter wheel to minimize artifacts from fluorescent samples, multiangle scattering (back, side, and forward scatter) to determine particle concentration without calibration, and constant current mode to measure high conductivity simples.
  • Gel Permeation Chromatography: Malvern’s OMNISEC.
    Detector system (refraction index) of 640 nm y 50 mW, as well low (<10°) angle for highly sensitive determination of the absolute MW of polymers and proteins, upgradable to measure viscosity, branching, etc. Chromatography module, regulable pump, degasifier, sampler adaptable to many sample formats, temperature control for high MW polymers, columns/pre-columns for separation of polymers and proteins, and fraction collector.
  • Confocal-epifluorescence microscope: Zeiss LSM 700 microscope with 100 mW max output, 400-650 wavelength range, and IIIb class laser; 10x: EC Plan-NEOFLUAR, 20x: LD Plan-NEOFLUAR, 50x: EC Epiplan-NEOFLUAR, and 100x/1.3 oil: Plan-NEOFLUAR objectives; AxioCam Mrm camera; XLmulti S1 incubator system, and Zen 2 blue software.

Collaborations

  • Dr. Alexander Andrianov, University of Maryland, MD, USA.
  • Dr. Yu Chen, University of Maryland College Park, MD, USA.
  • Dr. Mandy Esch, National Institutes for Standards and Technology, Gaithersburg, MD, USA.
  • Dr. Robert Getts, Genisphere LLC, Hatfield, PA, USA.
  • Dr. Hamid Ghandehari, University of Utah, UT, USA.
  • Dr. Janet Hoenicka, Sant Joan de Deu Hospital, Barcelona, Spain.
  • Dr. Christopher Jewell, University of Maryland College Park, MD, USA.
  • Dr. Joe Kao, University of Maryland Baltimore, MD, USA.
  • Dr. Peter Kofinas, University of Maryland College Park, MD, USA.
  • Dr. Juan Marugan and Dr. Wei Zheng, National Institutes of Health, Rockville, MD, USA.
  • Dr. Vladimir Muzykantov, University of Pennsylvania, Philadelphia, PA, USA.
  • Dr. Gianfranco Pasut, University of Padova, Padova, Italy.
  • Dr. Edward Schuchman, Mount Sinai School of Medicine, New York, NY, USA.
  • Dr. Brigitte Stadler, Aarhus University, Denmark.
  • Dr. Maria Jesus Vicent, Principe Felipe Research Center, Valencia, Spain.
  • Dr. Alberto Fernández de las Nieves, ICREA & University of Barcelona, Spain.
  • Dr. Xavier Rodó, ICREA & ISGlobal, Barcelona, Spain.
  • Dr. Antonio Alcamí, CNB & CSIC, Madrid, Spain.
  • Dr. Dolores Ledesma, CBM & CSIC, Madrid, Spain.
  • Dr. Ricardo Feldman, University of Maryland, Baltimore, MD, USA.
  • Dr. Ramón Farré, Clinic Hospital & University of Barcelona, Spain
  • Dr. Jesús Villarrubia, Ramón y Cajal Hospital, Madrid, Spain

News/Jobs

 

Investigadores del IBEC hallan una nueva forma de transportar eficazmente fármacos al cerebro

Un grupo internacional de investigadores liderados por la profesora ICREA Silvia Muro del Instituto de Bioingeniería de Catalunya (IBEC) y la Universidad de Maryland (Estados Unidos) ha identificado una nueva forma de transportar fármacos al cerebro, uno de los grandes desafíos de la ciencia farmacéutica actual, lo que podría ayudar a diseñar nuevos tratamientos para enfermedades neurológicas tales como el Parkinson o el Alzheimer.

Para la elaboración del trabajo, publicado esta semana en la prestigiosa revista Journal of Controlled Release, los expertos unieron un anticuerpo capaz de reconocer la proteína ICAM-1 –una molécula expresada en la superficie de los vasos sanguíneos- a una serie de nanopartículas poliméricas que pueden transportar un fármaco e inyectarlo por vía intravenosa.

Read more…