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RESEARCH

Our research focuses on understanding and controlling the effects of additives on the physicochemical behavior of biological and synthetic macromolecules, micelles and nanoparticles in general (see figure below) in aqueous mixtures.

Our research is currently focusing on two specific phenomena here denoted as macromolecular condensation and particle diffusiophoresis. They are described below.

Macromolecular Condensation

Macromolecular condensation refers to the formation of macromolecule-rich spherical liquid microdroplets inside aqueous liquid media. It is a liquid-liquid phase separation (LLPS) transition, which has attracted great attention in the case of proteins. Protein condensation is typically observed by cooling protein aqueous solutions. Next figure illustrates the formation of a protein-rich droplet from cooling a protein-water solution. A picture with actual protein-rich micro-droplets is also included.

Protein condensation is a reversible process as expected for phase transitions. Next picture shows the opacification of a protein aqueous sample induced by cooling. Sample transparency is restored by heating. Click the link below the picture to watch the reversible opacification of a protein sample.

Click: Reversible opacification of a protein sample

Protein condensation is known to drive the formation of membraneless organelles inside living cells and it is believed to be an important intermediate for the formation of pathological protein aggregates. Protein-rich microdroplets are also known to be metastable with respect to protein crystallization and can become sites for the nucleation of protein crystals. Understanding and controlling the formation of protein droplets and their transformation into protein crystals or aggregates is important for

·         Protein crystallization, which is important for the characterization of protein three-dimensional structure by traditional X-ray crystallography or emerging crystallographic methods such as X-ray free-electron laser and micro-crystal electron diffraction techniques.

·         Preparation of protein-based materials with applications in catalysis, drug delivery and sensing.

·         Inhibition of protein aggregation in pharmaceutical formulations containing therapeutic proteins such as monoclonal antibodies.

·         Comprehending the role of protein condensation in biological processes and diseases associated with protein aggregation.

We have being interested in answering the following questions:

Can additives induce protein condensation?

Protein condensation is driven by protein-protein attractive interactions (e.g., electrostatic, van der Waals, hydrophobic) in water. It was thought to be a not-very-common phenomenon, occurring only for few proteins (e.g., eye-lens crystallins). It is believed that protein condensation in water is not observed for many protein cases because it would occur well below the freezing point of water. In other words, protein-protein attractive interactions are not sufficiently strong. Recently, protein condensation has been reported for many protein cases. One reason is related to the continuous production and characterization of many new recombinant proteins such as monoclonal antibodies. A second important reason can be linked to the comprehension of another phenomenon of macromolecular aqueous mixtures, known as macromolecular crowding. This is an alteration of the state of protein aqueous solutions created by the addition of “inert” synthetic or natural macromolecular additives. Macromolecular crowding introduces steric (excluded volume) interactions that increase protein-protein attractive interactions thereby favoring protein condensation. To appreciate this phenomenon, we consider an example of people crowding inside a room. The following figure shows a room with four tables (red rectangles) and eight persons (blue circles). The persons in the room will push tables together in order to maximize the space available to them. People and tables represent the crowding additive and the proteins, respectively.  Joining tables together represents protein condensation due to macromolecular crowding.

In one of our previous work (J. Phys. Chem. B 2007, p1222), we showed that polyethylene glycol (PEG) induces condensation of serum albumin by macromolecular crowding.

How can we use additives to control the fate of metastable protein-rich droplets?

In our research group, we are currently preparing aqueous mixtures in which protein condensation occurs and investigating how additives such as salts or organic molecules can control the fate of protein microdroplets (Int. J. Biol. Macromol. 2021, p519). Next picture shows the opacification of a protein aqueous sample induced by cooling due protein condensation. Upon heating, sample transparency is not recovered because protein-rich microdroplets act as nucleation sites for protein crystallization.

Click: Protein Condensation as a platform for protein crystallization

Can we observe condensation of other globular macromolecules?

We are also interested in another type of macromolecules known as dendrimers. These are branched tree-like synthetic polymers with a globular shape in solution, similar to that of proteins. Dendrimers have being investigated for their ability to host small molecules with potential applications as drug carriers, solubilizing and extracting agents and catalyst supports. We inquired whether dendrimer condensation occurs as in the case of proteins. In a previous work, we have shown that aqueous solutions of polyamidoamine (PAMAM) dendrimers with hydroxyl and amino surface functionalities undergo dendrimer condensation in the presence of sodium sulfate, a strong salting-out agent. To our knowledge, this represents the first experimental report on dendrimer LLPS. Not only dendrimer condensation occurs but it also exhibits an unusual thermal behavior: it can switch from being induced by cooling to being induced by heating as salt concentration increases (PCCP 2015, p28818).

    How is proximity to condensation conditions influencing aggregation processes?

A protein solution that is physically stable may be still susceptible to irreversible chemical processes such as dimerization, oligomerization and aggregation in general. These processes are involved in the formation of pathological aggregates.  We are interested in understanding how proximity to LLPS conditions influences aggregation. In one of our previous work, we mimicked protein oligomerization by adding small amounts of chemical crosslinkers. In the proximity of LLPS conditions, it was demonstrated that oligomerization raised LLPS temperature resulting in the rapid formation of spherical aggregates. In the absence of LLPS, aggregation was slow and results in the formation of branched aggregates (Langmuir 2008, p2799). The same approach was also extend to dendrimers. As illustrated in the figure below, a stable dendrimer-salt aqueous  sample is prepared with a composition near the LLPS boundary. A small amount of chemical crosslinking produces dendrimer dimers. This induces a shift in the LLPS boundary, which crosses sample coordinate, leading to sample LLPS. Dendrimer-rich droplets then produce crosslinked dendrimer nanospheres (Langmuir, 2017, p5482). We believe that oligomerization-induced LLPS may be valuable for the preparation of nanomaterials with large host capacity from building blocks such as small dendrimers.

Particle Diffusiophoresis

Diffusiophoresis rhymes with electrophoresis. We know that electrophoresis is the migration of a charged particle induced by a gradient of electric potential (electric field). This particle can be a protein, nucleic acid, a synthetic polyelectrolyte or a nanoparticle. Diffusiophoresis is instead the migration of a particle induced by a gradient of chemical potential of an additive such as a salt. This “chemical field” is established by imposing a gradient of additive concentration.

Diffusiophoresis has attracted much attention as a means to control particle motion in liquids. Specifically additive gradients could be used to achieve particle focusing and self-assembly, separation of different macromolecules, diffusion-based controlled release, enhancement or retardation of particle adsorption on surfaces and particle insertion into porous materials. Thus, diffusiophoresis could be exploited in applications such as

·         Protein separation and detection in microfluidics.

·         Enhance protein adsorption onto sensing surfaces (e.g., surface plasmon resonance).

·         Use of micelles and other nanoparticles in enhanced oil recovery and soil remediation.

·         Prevention of membrane fouling in reverse osmosis, forward osmosis and protein ultrafiltration

Diffusiophoresis has been observed for model colloidal particles such as polystyrene or silica particles in the presence of salt gradients. In these cases, this transport phenomenon can be explained by considering an elecrokinetic mechanism that requires particles to be electrically charged.

We have being interested in answering the following questions:

Does salt-induced diffusiophoresis occur for neutral particles?

Salt-induced diffusiophoresis can be driven by particle preference for water (preferential hydration). In other words, a hydrophilic particle may migrate along a salt gradient in order to reach the region with lower salt concentration and correspondingly higher water concentration. An important example of hydrophilic particles is represented by PEG polymer chains. Understanding PEG diffusiophoresis is also important for controlling the motion of PEG-based particles, such as micelles, vesicles, PEGylated proteins and PEG-coated nanoparticles.  In one of our previous work (Langmuir 2014, p4916), we used precision Rayleigh interferometry (see figure below) to show that a KCl concentration gradient induces PEG diffusiophoresis from high to low salt concentration.

Our lab houses a unique instrument known as the “Gosting Diffusiometer”. This interferometer has reported the most precise diffusion coefficients of liquid mixtures. The Gosting diffusiometer was built by Prof. Louis J. Gosting and then enhanced and adapted for Rayleigh Interferometry by Dr. Donald G. Miller and Prof. John G. Albright. We also measure diffusion coefficients by dynamic light scattering (DLS).

Can we invert the roles?

A salt concentration gradient can induce diffusiophoresis of particles. However, a concentration gradient of particles can also induce motion of salt ions (see figure below). We denoted this mechanism as salt osmotic diffusion. We have experimentally characterized this mechanism and showed that it is theoretically linked to particle diffusiophoresis. Knowledge of salt osmotic diffusion allows us to identify the thermodynamic and hydrodynamic components of particle diffusiophoresis (see Langmuir 2015, p12210 for example).

Does salt-induced PEG diffusiophoresis follow the Hofmeister Series?

In 1888, Franz Hofmeister first reported that salt ions can be ranked according to their effectiveness in precipitating proteins from aqueous solutions. This ranking is known as the Hofmeister series. The  overall orders of the anions and cations are shown in the figure below. Ions on the left side tend to reduce protein solubility in water (salting-out) while ions on the right side tend to increase protein solubility (salting-in).

Nowadays, the Hofmeister series is investigated in many other processes distinct from protein solubility. it has also been shown to be valid for synthetic macromolecules. In one of our previous work, we showed that PEG diffusiophoresis follows the Hofmeister series. Furthermore, in salting-in conditions, ion binding gives rise to an electrical charge on PEG chains (Langmuir 2015, p1353).

Do osmolyte gradients cause diffusiophoresis?

Osmolytes are neutral organic molecules with low molecular weight that are soluble in water. Some osmolytes are shown in the figure below. Their name originated from their ability to influence solution osmotic pressure.

We showed that PEG diffusiophoresis occurs in the presence of TMAO and DEG from high to low osmolyte concentration while it is negligible in the presence of urea. Osmolyte osmotic diffusion was also characterize. PEG diffusiophoresis in the presence of TMAO is comparable to that observed in the presence of a strong salting-out agent (Langmuir 2018, p9525).

Does salt-induced diffusiophoresis occur for PEG-based nanoparticles?

We have characterized salt-induced diffusiophoresis of PEG chains. However, PEG also governs the interfacial properties of PEG-based nanoparticles. Our recent work has focused on the experimental characterization of diffusiophoresis of PEG-based micelles. Preliminary experimental results show that diffusiophoresis of PEG-based micelles in the presence of Na₂SO₄ can be approximately linked to that of PEG chains.

Does salt-induced diffusiophoresis occur for proteins?

Previous diffusiophoresis studies have demonstrated salt-induced diffusiophoresis for charged colloidal particles. Can this phenomenon occur also for charged proteins? We have reported the first experimental study on protein diffusiophoresis (J. Phys. Chem. B 2012, p12694). Specifically, we have characterized salt-induced diffusiophoresis of the protein lysozyme in the presence of NaCl. Salt osmotic diffusion was also characterized and can be linked to a thermodynamic phenomenon known as Donnan equilibrium. Experimental results show that salt-induced protein diffusiophoresis is governed by the electrokinetic mechanism at low salt concentration and the preferential-hydration mechanism at high salt concentration. Amplification of salt-induced diffusiophresis of protein is achieved in the presence of MgCl₂, due to cation binding to protein and salt large osmolarity (Langmuir 2020, p2635).

We have also theoretically examined protein diffusiophoresis, salt osmotic diffusion and the DLS protein diffusion coefficient in the context of multicomponent diffusion and the fundamental laws od non-equilibrium thermodynamics (Int. J. Heat Mass Transfer 2020, p120436).