Electrostatic self-assembly of agro-sourced biomolecules: structural, thermodynamic study and development of stimulable carriers.

Context and Originality

The development of new systems capable of vectorizing active molecules to a defined target is a major research focus of the Laboratory of Biomolecule Engineering (LIBio). The originality of LIBio lies in the formulation of innovative vectors whose constituents are derived from renewable agro-resources. In this thesis, an original approach based on self-assembly via electrostatic interactions between polyelectrolytes and small ionized biomolecules will be explored.

Interactions between two polyelectrolytes can lead to the formation of various and complex colloidal structures (aggregates, coacervates, soluble complexes, gels, etc.)^1,2 depending on numerous parameters such as the chemical nature, molecular weight of the species involved, their relative proportions, pH, ionic strength, etc. Many agro-sourced polyelectrolytes, such as polysaccharides and proteins, have proven to be good candidates for forming such structures through liquid-liquid phase separation mechanisms, among others.³ The interest in these supramolecular assemblies lies in their sensitivity to environmental conditions, allowing control over the formed structures based on the physicochemical properties of the system. It is thus possible to develop assemblies responsive to external parameters such as pH, ionic strength, concentration, temperature, or physical actions like shear stress, opening prospects for controlled release of encapsulated actives. While the scientific literature is rich in examples of systems composed of two polyelectrolytes (mainly polysaccharides/proteins), it is much less so for systems composed of a polyelectrolyte and smaller charged molecules.

This thesis focuses on the study of self-assemblies between polysaccharides and peptides via electrostatic interactions. This work should open a new research avenue in the development of agro-sourced colloidal systems reactive to external stimuli and potential vectors for active molecules. The polysaccharides considered are chitosan and gum arabic, well-known substrates at LIBio, acting as a polycation and polyanion, respectively. The peptides will either be commercial or derived from parallel projects within partner laboratories of the Biomolecules 4 Bioeconomy (B4B) project, of which LIBio is an integral part.

The scientific questions associated with this project are as follows:

• What colloidal structures spontaneously form through electrostatic interactions between macromolecules and peptides?
• Is it possible to modulate these structures based on the physicochemical parameters of the medium (pH, ionic strength, temperature)?
• Can the formed structures be used for the encapsulation and controlled release of active biomolecules regardless of their properties?

This thesis will begin, like any PhD, with a bibliographic review of systems formed by electrostatic interactions between charged molecules derived from agro-resources, particularly polysaccharides. The choice of polysaccharides is fixed (chitosan and gum arabic), but the peptides will be selected based on the literature and intra- or inter-laboratory collaborations. Initial experiments will involve the simple mixing of aqueous solutions of a polysaccharide and a peptide (or a mixture of peptides). If turbidity is observed, characterization of the formed objects will be carried out using optical and/or electron microscopy, depending on their size, and light scattering (dynamic or static). Isothermal titration calorimetry experiments will be conducted to define the associated thermodynamic parameters. The stability of the formed colloidal structures will also be monitored using static multiple light scattering. Finally, modifications of the physicochemical conditions will highlight the system's response to external stimuli. It will also be possible to test ternary systems using the same methods to address the question:

• What is the impact of adding a charged molecule (polymer or peptide) to preexisting electrostatic assemblies?

For example, adding a peptide to a chitosan/gum arabic assembly (coacervate or soluble complex, mastered at LIBio) may lead to structural changes at the colloidal scale. Depending on the progress of the thesis, numerous systems can be explored to rationalize the formed structures based on the associated chemical and physicochemical parameters. 

Experimental Approaches

The experimental methods will focus on: Titration techniques (turbidimetry, isothermal titration calorimetry); Colloidal stability studies (static multiple light scattering, turbidimetry); Particle size and charge measurements (dynamic light scattering, laser granulometry); Nanoscale structural characterization (small-angle X-ray scattering [SAXS], access to the SOLEIL synchrotron [SWING beamline]). Modification of polysaccharides via enzymatic or chemical routes (mastered at LIBio)^4-6 is also possible to alter their chemical groups, enabling modulation of interactions with other biomolecules.

Future Perspectives

Encapsulation or purification applications will be tested at the end of the thesis, depending on results.

Candidate Profile

The candidate must hold a Master’s degree (BAC+5) in physical chemistry or biochemistry with experience in: Peptides (extraction, purification, characterization), Polyelectrolytes, Colloidal systems

They should have a strong affinity for laboratory work and be able to integrate well into a research team.

Keywords: Colloids, polysaccharides, peptides, interactions, coacervation

Required/Developed Skills During the PhD: Peptide characterization (chromatographic methods, LC-MS/MS); Biopolymer characterization (Fourier-transform infrared spectroscopy, size-exclusion chromatography, nuclear magnetic resonance); Characterization of nano- and micro-objects (size, structure, charge); Thermodynamic parameters of interactions; Small angle scattering (SAXS)

Website of the laboratory: http://libio.univ-lorraine.fr/

Website of the Université de Lorraine : https://www.univ-lorraine.fr/

Laboratory located in Nancy, Grand EST region (1h30 of Paris by train), France : https://www.nancy.fr/accueil

Bibliography:

(1)         Vuillemin, M. E.; Michaux, F.; Muniglia, L.; Linder, M.; Jasniewski, J. Gum Arabic and Chitosan Self-Assembly: Thermodynamic and Mechanism Aspects. Food Hydrocoll. 2019, 96, 463–474. https://doi.org/10.1016/j.foodhyd.2019.05.048.

(2)         Delmas, C.; Michaux, F.; Durand, A.; Jasniewski, J. Polysaccharide–Polysaccharide Complexation: Phase Behavior and Destabilization Mechanisms in Gum Arabic–Chitosan Systems. Food Hydrocoll. 2026, 171, 111809. https://doi.org/10.1016/j.foodhyd.2025.111809.

(3)         Aberkane, L.; Jasniewski, J.; Gaiani, C.; Scher, J.; Sanchez, C. Thermodynamic Characterization of Acacia Gum−β-Lactoglobulin Complex Coacervation. Langmuir 2010, 26 (15), 12523–12533. https://doi.org/10.1021/la100705d.

(4)         Vuillemin, M. E.; Michaux, F.; Seiler, A.; Linder, M.; Muniglia, L.; Jasniewski, J. Polysaccharides Enzymatic Modification to Control the Coacervation or the Aggregation Behavior: A Thermodynamic Study. Food Hydrocoll. 2022, 122, 107092. https://doi.org/10.1016/j.foodhyd.2021.107092.

(5)         Vuillemin, M. E.; Muniglia, L.; Linder, M.; Bouguet-Bonnet, S.; Poinsignon, S.; Dos Santos Morais, R.; Simard, B.; Paris, C.; Michaux, F.; Jasniewski, J. Polymer Functionalization through an Enzymatic Process: Intermediate Products Characterization and Their Grafting onto Gum Arabic. Int. J. Biol. Macromol. 2021, 169, 480–491. https://doi.org/10.1016/j.ijbiomac.2020.12.113.

(6)         Adam, A. A.; Jasniewski, J.; Vuillemin, M. E.; Simard, B.; Burgain, J.; Badin, R.; Muniglia, L.; Michaux, F. Enzymatic Mediated Modification of Gum Arabic by Curcumin Oxidation Products: Physicochemical and Self-Assembly Study. Food Hydrocoll. 2022, 126, 107451. https://doi.org/10.1016/j.foodhyd.2021.107451.

The Laboratoire d’Ingénierie Des Biomolécules (Laboratory of Biomolecule Engineering) from the University of Lorraine (EA 4367) is located in ENSAIA. LIBio has been ISO 9001 certified since 2008 and will celebrate 40 years of existence in 2025.

For the five-year period 2024-2028, the LIBio scientific project is focused on the structuring of complex biosourced systems for increased functionalities. The scientific objective is to manage the biotic and abiotic interactions of biosourced systems in complex environments in order to increase their functional properties. Our approaches tend towards the creation of architectures with biosourced constituents and simplified formulation.

The candidate must hold a Master’s degree (BAC+5) in physical chemistry or biochemistry with experience in: Peptides (extraction, purification, characterization), Polyelectrolytes, Colloidal systems

They should have a strong affinity for laboratory work and be able to integrate well into a research team.