Preparation of peptide-functionalized nanoparticles

Precision Delivery, Tailored Solutions: Peptide-Functionalized Nanoparticles for Next-Generation Therapies.

Scientific Leader / leaders of the project: Mariana Köber / Miriam Royo
Coordinator of the project: Guillem Vargas / Miriam Royo

Industrial gap/problem covered

One of the greatest challenges in nanomedicine is the delivery of therapeutically relevant amounts of actives to specific body tissues or organs, hampering the development of new therapies. To favor the delivery to specific target tissues or organs, the nanocarrier surface can be functionalized with moieties that specifically recognize targets that are predominantly present at the site of delivery. Peptides are one of the major classes of targeting molecules that are being investigated, and have gained increasing attention due to lower production costs compared to antibodies and easier processes to obtain functional chemical modifications. By now, many peptide sequences have been identified for different targets, and several modifications and chemical approaches can be applied to improve their selection, binding affinity, stability, and specificity.

Description

In this cutting-edge, we offer the preparation of tailored (lipid) nanoparticles that are functionalized with specific targeting peptides. The peptides are synthesized and characterized by HPLC-MS and HPLC and then are adequately modified to be conjugate on specific moieties/components (for example: lipids) that facilitate their integration into lipid nanoparticles and accessibility for binding, etc. The functionalized nanoparticles can be prepared using the scalable DELOS-SUSP method or microfluidics using the components that contain the targeting peptide, and purified using tangential flow filtration. A posterior physicochemical characterization can be performed using different techniques such as DLS, ELS, MADLS, NTA, ITC, etc.

These services are carried out by U3 (Synthesis of Peptides Unit) and U6 (Biomaterial Processing and Nanostructuring Unit), who have a strong background of collaboration for the preparation of different peptide functionalized nanoparticles:

Examples of scientific publications implementing this Cutting-Edge Biomedical Solution:

• Hierarchical Quatsome-RGD Nanoarchitectonic Surfaces for Enhanced Integrin-Mediated Cell Adhesion. M. Martinez-Miguel, M. Castellote-Borrell, M. Köber, A. Kyvik, J. Tomsen-Melero, G. Vargas-Nadal, J. Munoz, D. Pulido, E. Cristobal-Lecina, S. Passemard, M. Royo, M. Mas-Torrent, J. Veciana, M.I. Giannotti, J. Guasch, N. Ventosa, I. Ratera. ACS Applied Materials & Interfaces (2022), 14, 48179-48193.
• Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment. J. Tomsen-Melero, S. Passemard, N. García-Aranda, Z. Vanessa Díaz-Riascos, R. González-Rioja, J. Nedergaard Pedersen, J. Lyngsø, J. Merlo-Mas, E. Cristóbal-Lecina, J. L. Corchero, D. Pulido, P. Cámara-Sánchez, I. Portnaya, I. Ionita, S. Schwartz Jr., J. Veciana, S. Sala, M. Royo, A. Córdoba, D. Danino, J. S. Pedersen, E. González-Mira, I. Abasolo, and N. Ventosa. ACS Applied Materials & Interfaces (2021), 13(7), 7825-7838.
• Synthesis of Stable Cholesteryl-Polyethylene Glycol-Peptide Conjugates with Non-Disperse Polyethylene Glycol Length. E. Cristobal-Lecina, D. Pulido, P. Martin-Malpartida, M. J. Macias, F. Albericio, M. Royo. ACS Omega (2020), 5, 5508-5519.

Examples of projects implementing this Cutting-Edge Biomedical Solution:

• MyoQ: La Plataforma de Nanomedicina para terapias avanzadas contra las Distrofias Musculares Congénitas. Pla estratègic de recerca i innovació en Salut (PERIS), 2023. Budget: 100 k€. Coordinator: Dr. Cecilia Jiménez Mallebrera, Fundació Privada per a la Recerca i Docencia Sant Joan de Deu. Principal Investigators: Dr. Mariana Köber (ICMAB-CSIC) and Dr. Miriam Royo (IQAC-CSIC).
• Tag-Smartly: Targeted Quatsome nanocarriers for the delivery of microRNA for neuroblastoma therapy. CIBER-BBN Valorization project, 2020. Budget: 45 k€. Coordinator: Dr. Nora Ventosa / Dr. Mariana Köber (CB06/01/0033). Principal Investigator of CIBER-BBN group CB06/01/0074: Dr. Miriam Royo.
• Smart4Fabry: Smart Functional GLA-nanoformulation for Fabry Disease (ref 720942). European financed project. 01/01/2017-31/12/2020. Total budget: 5.844.508 €. Coordinator: Dr: Nora Ventosa. Principal Investigator of CIBER-BBN group CB06/01/0074: Dr. Miriam Royo.

Services involved:

1. Synthesis of targeting peptides and characterization (U3-S01). We offer advice on the selection of peptide ligands, not only in terms of affinity and recognition, but also in terms of synthesis. We synthesize the peptide appropriately modified (with a Cys) for incorporation into nanovesicles in the quantities (mg to g) and purity required.
2. Post-synthesis peptide modification for integration into nanoparticles (U3-S02). We offer the synthesis of a component of the nanovesicle (e.g. cholesterol) with the targeting peptide already incorporated into its structure in the quantities (mg to g) and purity required. These peptide-loaded components are used when the targeting/homing moieties are introduced at the same time as the nanovesicles are formed. We can also offer the modification of the targeting peptide sequences with different motifs to improve the affinity to the surface protein, such as cyclization, glycosylation, acetylation, etc., or the introduction of fluorescent probes to perform cell uptake studies.
3. Production of colloidal dispersions of targeted nanoparticles. We offer a scalable nanoparticle production technology based on compressed CO2 (DELOS-SUSP), to produce batch sizes of 25 – 300 mL (U6-S01). Alternatively, colloidal nanoparticle dispersions can be prepared using a microfluidic system (U6-S06). Cytotoxic compounds can be processed if required.
4. Purification and concentration of colloidal dispersions. Subsequently to the production of the colloidal dispersions of targeted nanoparticles, the dispersant medium can be exchanged or purified, and the dispersion can be concentrated (e.g. to perform studies in animal models), using a tangential flow filtration system (U6-S08).
5. Analysis of particle size, concentration and zeta-potential of nanoparticles (U6-S04). After production, the colloidal dispersions can be characterized in terms of hydrodynamic particle diameter, zeta-potential and particle concentration, using Dynamic Light Scattering (Zetasizer Nano & Ultra, Malvern Panalytical) or Nanoparticle Tracking Analysis (Nanosight NS300, Malvern Panalytical). In addition to light scatter, fluorescence signals can be detected, e.g. for subpopulation identification.
6. Characterization of biomolecular interactions by calorimetric measurements (U6-S14). Quantitative studies of binding affinities and thermodynamic parameters (e.g. reaction stoichiometry (n), enthalpy (ΔH) and entropy (ΔS)) can be performed marker-free and in-solution using Isothermal Titration Calorimetry (ITC).

Both units participate in the design and characterization of the nanoparticles.

• U3
• U6

Unit 3 is coordinated by the Multivalent Systems for Nanomedicine goup at IQAC-CSIC and has the equipment necessary to provide services of synthesis of peptides at different scales (mg to g), purification, characterization, and post-synthesis modification, such as, conjugation to proteins and fluorescent. It counts with laboratories for synthesis and other to carry out modification of the peptides during and post synthesis labels. Also counts with a laboratory equipped with several preparative and analytical chromatography systems for purification and characterization. This facility benefits from the wide experience of Dr. Fernando Albericio an Dra. Miriam Royo in the design and synthesis of peptides with specific biological activity and the introduction of modifications necessary for these peptides to be bound to therapeutic nanoconjugates and other molecules, either to take advantage of the pharmacological activity of the peptide itself or to facilitate the introduction of nanoconjugates or other molecules into the cells in order to reach the therapeutic target. Its location allows the access to the rest of services of the IQAC-CSIC.

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Our Unit 6 is located at the Instituto de Ciencia de Materiales de Barcelona (ICMAB-CSIC), in Barcelona, and under the coordination of Prof. Nora Ventosa, current director of NANOMOL Group, which is a research group with wide expertise and recognized excellence in the synthesis, processing and study of molecular and polymeric materials with chemical, electronic, magnetic and biomedical properties. It 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 is a simple one-step methodology, DELOS-SUSP, 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. The NANBIOSIS Unit 6 is works under the ISO9001 certification for standard quality control system.​