DILUTION-INDUCED COMPLEX COACERVATION AS A ROBUST, ENVIRONMENTALLY FRIENDLY SURFACE TREATMENT APPROACH FOR CELLULOSE MATERIALS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028643
• Grant No.: 2022-67022-38145
• Cumulative Award Amt.: $300,000.00
• Proposal No.: 2021-10440
• Project Start Date: Aug 31, 2022
• Project End Date: Aug 30, 2025
• Grant Year: 2022
• Program Code: [A1531]- Biorefining and Biomanufacturing

Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100

Performing Department
LSUAM | Col of SCI | Chemistry

Non Technical Summary
Cotton is one of the most valuable crops that contributes significantly to the global textile and apparel manufacturing market. Due to its versatility, sustainability, and performance, the global demand for cotton is expected to increase steadily. Cotton fabrics often need to be functionalized in order to impart desired properties, such as fire-retardancy, resistance to crease and microbial penetration. The treatments of cotton fabrics are challenging and could involve the use of hazardous chemicals and lead to increased waste production and energy consumption. With the growing demand for cotton fabrics and the increased emphasis on green and sustainable chemistry,more robust, reliable, and environmentally-friendly surface modification approaches for cotton fabrics are urgently needed.Complex coacervation is a form of liquid-liquid phase separation (LLPS) resulting from strongly associative interactions among oppositely charged macromolecules, including polymers, micelles, polysaccharides and proteins.Complex coacervates possess many unique properties when compared with that of conventional polymer complexes. For example, complex coacervates have extremely low surface tension, therefore,they spread easily onto both hydrophilic and hydrophobic surfaces under aqueous conditions. Therefore, based on the unique and peculiarproperties of complex coacervates,this proposal is focused on investigating and controlling the deposition of complex coacervates onto cellulose surfaces with the goal of developing a robust, stimuli-responsive and environmentally friendly surface treatment approach for cotton fabrics. The outcome of the proposed research will lead to the development of novel, robust and sustainable surface modification approach to improve the functionality and profitability of cotton fabrics.

Animal Health Component

40%

Research Effort Categories

Basic

50%

Applied

40%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
511	1719	2000	100%

Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
1719 - Cotton, other;

Field Of Science
2000 - Chemistry;

Keywords

cotton

stimuli-responsive

surface modification

Goals / Objectives
With the growing demand for cotton fabrics and the increased emphasis on sustainable chemistry, more robust and environmentally-friendly surface modification approaches are urgently needed for making functionalized cotton fabrics. Complex coacervation is a form of liquid-liquid phase separation (LLPS) resulting from strongly associative interactions among oppositely charged macromolecules, including micelles, polyelectrolytes, proteins, and polysaccharides. The dense liquid phase is known as the coacervate, which demonstrates elevated viscosity, yet extremely low surface tension. As a result, the coacervate can spread and adhere easily onto various surfaces underneath the water. Moreover, the network structure of complex coacervates makes them ideal carriers for a wide range of molecules including antimicrobial peptides/proteins, dyes, and fire retardant chemicals. These characteristics of coacervate permit their potential use as versatile, water-borne surface coatings for cellulose materials. Previous studies found that the complex coacervation can be induced by a decrease in the total macromolecular concentration (CT), this process is known as the Dilution-Induced Complex Coacervation (DICC). While complex coacervates represent a novel surface coating for cellulose materials, the DICC allows additional control of the deposition upon external stimuli, in this case, the simple dilution of the macromolecule solutions. Therefore, this proposal is focused on investigating and controlling the deposition of complex coacervates onto cellulose surfaces with the goal of developing a robust, stimuli-responsive, and environmentally friendly surface functionalization approach for cotton fabrics. To achieve this goal, the proposed research has two major objectives:1. To identify the effects of intrinsic (e.g. charge, size, and conformation of macromolecules) and extrinsic properties (e.g. temperature, ionic strength, and stoichiometry) on the phase boundary observed during DICC.2. To examine the deposition mechanism and, subsequently, the microstructure of complex coacervates as they deposit onto surfaces with varying hydrophobicity and roughness.

Project Methods
In order to better utilize DICC as a surface functionalization approach for cotton fabrics, we propose to perform preliminary studies first to understand the phase behavior and interfacial properties of various coacervation systems. Therefore, the proposed research has two major objectives:1: To identify the effects of intrinsic (e.g., charge, size and conformation of macromolecules), and extrinsic properties (e.g., temperature, ionic strength and stoichiometry) on the phase boundary observed during DICC.2: To examine the deposition mechanism and, subsequently, the microstructure of complex coacervates as they deposit onto surfaces with varying hydrophobicity and roughness.Objective 1: Impacts of intrinsic and extrinsic factors on the phase boundary observed during DICCPart I: Previously, we studied the effects of head groups on the phase behavior of micelle/polymer complexes. For this part of the research, we plan to examine if the coacervation region can be tuned by using polymers with varying charge patterns and molecular weight. For the proposed research, we will replace synthetic polymers withnatural biopolymers including chitosan and pectin because these materials are renewable, naturally antimicrobial and the charge/molecular weight of these molecules can be easily modified with a wide range of commercially available digestive enzymes. Since both chitosan and pectin are negatively charged under neutral pH conditions, cationic surfactant will be used to promote the electrostatic interactions between micelles and polymers. To identify the phase transition of various macromolecular complexes, we will monitor the turbidity change of sample solutions since the dispersion of complex coacervate droplets will lead to an increase in solution turbidity. Considering the important roles played by electrostatic interactions, our hypothesis is that the onset of DICC can be well-controlled by using polymers with varying charge pattern and molecular weight because both parameters contribute towards the electrostatic interactions between micelle and polymer.Part II: Previously, the phase boundary of the model micelle/polymer systems was determined by diluting the concentrated complexes with the same buffer that was used to prepare the individual micelle and polymer solutions. Therefore, this type of dilution is called iso-ionic dilution. During the dilution experiment, the critical CT was recorded for each micelle/polymer mixing ratio (see Figure 5). Although it is feasible to perform iso-ionic dilution to elucidate the effects of CT on the phase behavior of macromolecular complexes, when it comes to real-life applications, dilution will most likely be performed with tap water. Thus, not only will CT decrease, but the ionic strength and pH will change during the dilution process (depending on the regional water quality). Therefore, this part of the project will investigate the effects of ionic strength and pH on the phase behavior of the model micelle/polymer systems. Eventually, we will be able to identify the individual and collective effects of all changing variables on the phase behavior of macromolecular complexes upon dilution, and this fundamental knowledge will allow us to elucidate the origin and significance of various attractive and repulsive interactions that together lead to the formation of complex coacervates upon dilution.Objective 2: Characterize the deposition mechanism and hierarchical structure of complex coacervates as they adhere to cellulosePart III: In order to elucidate the deposition mechanism and the structure of deposited coacervates on the molecular level, we plan to perform a series of surface measurements, including quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), ellipsometry and NR measurements. Before any of these surface characterization measurements can be made, we will first develop a reliable method to produce model cellulose surfaces with reproducible thickness and roughness. There is a large body of research describing various protocols for preparing cellulose thin films on silica or gold substrates. We will initially follow the established protocol by Nylander et.al., where they prepared reproducible and uniform cellulose thin films using the spin-coating method. Once we have prepared the cellulose thin film, we will examine the composition and roughness of the cellulose surface with XPS and AFM, respectively. Based on the results, we will either adopt the established method, or make changes to the method to suit our needs. Our hypothesis is that we will be able to establish a method for preparing cellulose thin films with controlled thickness and roughness to mimic the cellulose surface of cotton fabrics. Although very unlikely, if the cellulose surface cannot be prepared, we will purchase fabricated silicon wafers with oxide layers of varying thickness and roughness, and use these to provide model hydrophilic surfaces to study the deposition and structure of complex coacervates at the solid-liquid interface.Part IV: Once we have prepared the cellulose thin film on a solid substrate, we will then characterize the deposition mechanism and the resulting microstructure of the surface-adhered coacervate layer when DICC takes place. For this part of the study, we will start with the model micelle/polymer system, and expand to other coacervation systems (including chitosan and pectin) with the progress made from Part I. We will perform QCM-D measurements first, and use this as a screening tool to determine the successful deposition of complex coacervates during DICC. QCM-D results will allow more convenient measurements of the phase boundaries of various macromolecular systems by looking at the abrupt changes in surface mass through the course of dilution. Moreover, changes in dissipation will allow the simultaneous characterization of the viscoelastic properties of the deposited coacervate layer. In addition to QCM-D experiments, we will perform both ellipsometry and NR measurements to probe the thickness, composition and microstructure of the coacervate layer. In particular, any preferred orientation or surface-interaction of the individual macromolecules can be revealed through the fitting of the ellipsometry/NR results. Our hypothesis is that with the combined use of QCM-D, Ellipsometry, NR and AFM techniques, we will be able to 1) identify factors that will benefit the spread and adhesion of coacervates, 2) understand how macromolecules are deposited and organized on the surface of cellulose materials upon DICC.

Progress 08/31/23 to 08/30/24

Outputs
Target Audience:The primary audience for this research over the past reporting period has been academic researchers and professionals within the scientific community, focusing on sustainable practices and advanced material sciences. This group comprises faculty, postdoctoral researchers, and graduate students from various institutions where I have had the opportunity to present our findings. These presentations have allowed us to share advancements and gather feedback on the novel coacervate-based textile treatments we are developing. During 2023 and 2024, I actively engaged with this audience through a series of invited talks and seminars at prestigious conferences and universities.Notable among these was the seminar I delivered at Rensselaer Polytechnic Institute, NY, on the phase behavior of protein and polymer complexes. These engagements provided valuable platforms for disseminating research findings and fostering academic collaborations, enhancing the visibility and impact of our work within the scientific community. Looking ahead, our targeted audiences will expand to include industrial stakeholders interested in the practical applications of our research. Changes/Problems:During the grant period, a significant adjustment was made to our project timeline due to my maternity leave from January to April 2024, which necessitated a one-year non-cost extension. This leave coincided with a critical phase of our research, impacting our ability to conduct essential experiments and oversee data collection during this period. Consequently, the extension was crucial to compensate for the delays, allowing us the necessary time to thoroughly investigate our innovative formulations of complex coacervates. These formulations have shown promising capabilities for spreading and depositing on both hydrophilic and superhydrophobic surfaces, offering potential for patent applications and substantial commercial and scientific impact. The extension is enabling us to complete the studies effectively, aiming to enhance the final outcomes of our research, facilitate patent filing, and produce scientific publications. We have developed a detailed plan to accelerate the remaining work to prevent any further delays, ensuring the efficient use of the extended time. What opportunities for training and professional development has the project provided?This project has provided comprehensive opportunities for training and professional development to all team members, significantly enhancing their skills and professional capabilities. These opportunities range from hands-on laboratory training and national lab experiences to participation in professional conferences and direct mentorship. Laboratory and National Lab Training: Our team members, including undergraduate and graduate students, have received extensive training in advanced experimental techniques crucial to our research on complex coacervation. Specifically, students have had the opportunity to conduct experiments not only in our university lab but also at national labs where they accessed advanced characterization methods like Small-Angle X-ray Scattering (SAXS) and Small-Angle Neutron Scattering (SANS). This exposure to high-caliber facilities has provided them with a unique learning experience in handling sophisticated equipment and techniques, enhancing their understanding of material science and experimental physics. Mentorship and Skill Development: The project emphasizes one-on-one mentorship, where experienced researchers guide less experienced team members. This mentorship covers a wide range of skills from experimental design and data analysis to scientific writing and presentation skills. Particularly, three undergraduate students who have been directly involved in the research under this mentorship framework are gaining invaluable experience that enhances their readiness for advanced studies; two of these students have already commenced Ph.D. programs in chemistry at prestigious universities. Professional Development through Conferences: In terms of professional development, our students had the opportunity to attend the American Chemical Society (ACS) conference held in New Orleans in Spring 2024. This conference allowed them to present their research findings, interact with leading scientists in the field, and stay abreast of the latest scientific developments. Participation in such high-profile conferences not only aids in their academic growth but also helps in building essential professional networks. How have the results been disseminated to communities of interest?We have shared our research findings with the broader community through various outreach efforts, including our participation in Super Science Saturday in October 2023, attended by over a thousand students and their parents. At this event, I set up three simple polymer experiments to help students learn about what polymers are, how they behave in liquids, and their practical uses. These activities were hands-on, allowing students to directly interact with the experiments and see the results for themselves. By linking these polymer experiments to everyday examples, especially in food science, we made the science understandable and relevant. This helped spark curiosity among students and encouraged them to consider science and technology in their future learning and careers. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we intend to advance our scientific research by focusing on several critical areas. We will continue to explore the phase behavior of complex coacervation, with a particular emphasis on understanding how variations in micelle properties affect coacervation. This will involve detailed experiments to optimize the ratios of non-ionic to anionic surfactants within micelles, aiming to control their size, shape, and rigidity. Additionally, we will extend our on-surface studies to more comprehensively examine the deposition mechanisms and adhesion properties of coacervates on diverse surface types.These efforts are aimed at refining our approach to creating effective, environmentally-friendly surface treatments for cellulose-based materials.

Impacts
What was accomplished under these goals? In pursuit of our research objectives, we have achieved significant progress across both primary aims. For Aim 1, we conducted extensive experiments to examine the effects of non-ionic surfactant molecules on the phase boundary of a coacervating system composed of polymers and micelles formed from mixed surfactant micelles. By adjusting the ratios between non-ionic and anionic surfactant molecules within the micelles, we were able to control their size, shape, and rigidity. These modifications in micellar properties led to distinct variations in the phase boundary observed in different micelle/polymer systems. Our findings suggest that micelles with higher charge density and reduced flexibility are preferable for expanding the coacervation region during Dilution-Induced Complex Coacervation (DICC). For Aim 2, we discovered that the micelle/polymer coacervates we developed not only deposit on hydrophilic surfaces but also exhibit enhanced spreading and adhesion on hydrophobic surfaces. We are currently conducting detailed on-surface studies, including Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and contact angle measurements, to better understand the underlying mechanisms of this behavior. The ability of our coacervates to adhere effectively to hydrophobic surfaces significantly broadens their potential applications, extending well beyond the functionalization of cotton fabrics.

Publications

Progress 08/31/22 to 08/30/23

Outputs
Target Audience:During the reporting period, I was invited to give presentations on several occasions. The major ones include: 1) Presentation at the Southern Soft Matter Annual Symposium hosted by the University of Southern Mississippi at Hattiesburg, MS. 2) Presentation at the American Crystallography Association Annual Conference in Portland, OR. 3) Presentation at the Oak Ridge National Laboratory User Group Meeting in Knoxville, TN. 4) Several presentations were delivered to student groups here at LSU, including NOBCChE (National Organization for the Professional Advancement of Black Chemists and Chemical Engineers) and SASE (Society of Asian Scientists and Engineers). Beyond these presentations, I also engaged with research scientists at the USDA Southern Regional Research Center in New Orleans to explore potential collaboration opportunities for our project. Changes/Problems:For the proposed research, my intention was to hire a postdoctoral researcher who could collaborate with me to expedite the progress of the project. However, the search for a qualified postdoctoral researcher was not successful. Fortunately, my graduate students, motivated by their research, collectively made substantial progress on the project. Consequently, I requested a rebudget to reallocate the salary initially designated for the postdoctoral researcher to fund the summer salary of my graduate students. What opportunities for training and professional development has the project provided?1. Graduate students in our group have gained extensive hands-on experience conducting small-angle scattering experiments at various national synchrotron and neutron facilities. 2. Graduate students have also been accepted into various workshops and schools to develop systematic knowledge in small-angle scattering techniques. 3. Graduate students in our group have had the opportunity to present their research at the ACS Southwest Regional Meeting. 4. Our group has recruited several undergraduate students with interests in chemistry and agricultural science to participate in our research. We have provided them with training on a broad range of characterization instruments relevant to our research. The success of our training is evident in publications where undergraduate students have made significant contributions; they are co-authors on several papers published in the last reporting period. How have the results been disseminated to communities of interest?I presented our research on complex coacervation and the utility of small-angle scattering techniques for studying biomaterials at various workshops, conferences, and through virtual and in-person discussions with research scientists from both academia and research institutes. I have also established connections with experts in engineering and chemical synthesis to broaden the scope of this proposed research. What do you plan to do during the next reporting period to accomplish the goals?During the first reporting period, our students received systematic training in techniques relevant to the proposed research, and their work has laid the foundation for future experiments, propelling the project forward. We will continue to explore the intrinsic properties of coacervate systems and investigate how they influence the viscoelastic properties of coacervates formed upon DICC. Additionally, we will delve deeper into the effects of crowding on the surface deposition mechanisms of coacervates. We are currently working on creating surfaces with varied roughness and hydrophilicity. Once we have established an optimized protocol for producing these surfaces, we will employ the established characterization methods (NR and QCMD) to investigate the assembly mechanism and hierarchical structure of various complex coacervates deposited onto these surfaces upon dilution. By the end of the project period, we anticipate developing a robust delivery system suitable for testing on cotton fabrics.

Impacts
What was accomplished under these goals? To accomplish the primary objective of advancing the application of DICC as a stimuli-responsive and environmentally friendly delivery platform for cotton fabrics, it is critical to gain insights into 1) the intrinsic and extrinsic properties that delineate the phase boundary of the coacervating system, and 2) the interfacial behavior of complex coacervates upon interaction with diverse surfaces. For Aim 1, we have discovered that the introduction of macromolecular crowding agents, such as polyethylene glycol, can markedly broaden the phase boundary of a model protein and polymer coacervation system. This finding is significant as it mitigates the extensive empirical exploration required for identifying systems that exhibit an expanded coacervation region. The inclusion of crowding agents has been shown to broaden the phase boundary of various coacervation systems, including protein-polymer, micelle-polymer, and polymer-polymer systems. Consequently, the versatility of crowding agents is universal, applicable to an extensive array of systems. This discovery has led to the publication of a paper, recently accepted by Biomacromolecules (DOI is currently unavailable). Progressing on this path, we will continue to investigate the influence of the intrinsic properties such as the size, shape, and surface charge of macromolecules on the phase behavior of their complexes. For Aim 2, understanding the interfacial behavior of complex coacervates necessitates a reliable and robust characterization technique capable of probing of coacervate deposition onto diverse surfaces in situ. Given that the structures of coacervates can be modified by pH, ionic strength, temperature, and concentration, traditional characterization methods involving prior sample preparation (including drying, heating, or chemical modifications) are not suitable for analyzing the structures of coacervate materials. Accordingly, we have developed a robust characterization methodology combining neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCMD) to scrutinize the assembly of protein and polysaccharide complexes at the solid-liquid interface. While the QCMD results confirm the successful adsorption of protein and polysaccharide on the surface, model fitting to the NR results enables the determination of thickness, composition, hydration level of the deposited coacervate layer, and the arrangement of protein and polysaccharide molecules within the surface-deposited matrix structure. This breakthrough lays the foundation for upcoming experiments designed to methodically examine the coacervate structures formulated by various systems and deposited on diverse surfaces. This research has been featured in Langmuir (Langmuir 2022, 38, 41, 12551-12561).

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Biswas, S., Melton, L. D., Nelson, A. R., Le Brun, A. P., Heinrich, F., McGillivray, D. J., & Xu, A. Y. (2022). The assembly mechanism and mesoscale architecture of proteinpolysaccharide complexes formed at the solidliquid interface. Langmuir, 38(41), 12551-12561.
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Xu, A. Y., Blanco, M. A., Castellanos, M. M., Meuse, C. W., Mattison, K., Karageorgos, I., ... & Curtis, J. E. (2023). Role of DomainDomain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt. The Journal of Physical Chemistry B.
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Biswas, S., Hecht, A., Noble, S., Huang, Q., Gillilan, R., & Xu, A. Understanding the Impacts of Molecular and Macromolecular Crowding Agents on Protein-Polymer Complex Coacervates. Biomacromolecules, just accepted, 2023