A materials science framework for connecting protein stability with surface adsorption and coating applications

The formation of protein coatings at the solid-liquid interface via protein adsorption is broadly relevant for various materials science and biomedical applications such as implantable biomedical devices, biosensors and nanomedicine. The noncovalent adsorption behavior of protein molecules is governed by various interfacial forces between the protein molecule in solution and the solid surface, as well as by intramolecular forces which determine a protein’s folded structure and solution-phase conformational stability. Although a protein’s conformational stability has been closely linked to its extent of unfolding in the adsorbed state, it remains to be understood how this relationship may be exploited in order to control a protein’s adsorption behavior and adlayer properties to suit specific application needs. The objective of this thesis is to systematically interrogate the relationship between protein conformational stability and protein adsorption behavior such as adsorption uptake, adsorption rate, and the degree of adsorption-related denaturation and spreading. The overall hypothesis is that rationally tuning the conformational stability of protein molecules based on materials science concepts can influence protein adsorption and that the insights gained from this framework can be used to engineer improved protein coating applications. To test this hypothesis, serum albumins as model proteins with modulated conformational stabilities based on their structure and processing, in line with the materials science paradigm, were comparatively evaluated using an experimental approach that characterized the proteins’ solution-phase conformational stabilities by measuring their temperature-dependent aggregation profiles and degree of secondary structural unfolding, and also that which characterized the proteins’ adsorption behavior by measuring their adsorption uptake, adsorption rate and extent of surface-induced denaturation. It was revealed that primary structure variations between serum albumin proteins from different species affected protein conformational stability and adsorption behavior, and that bovine serum albumin (BSA) possessed the lowest conformational stability and greatest adsorption-related denaturation compared to that of human and rat origin. This provided mechanistic reasons for why BSA is so widely used in surface passivation (“blocking”) protocols for many biotechnological applications. A further investigation on several commercially available and differently processed BSA proteins revealed that they possessed significantly different conformational stabilities and adsorption behavior, depending on whether or not a purification step which removed fatty acids was performed. It was further determined that the doping of BSA proteins with fatty acids and other related amphipathic molecules generally increased protein conformational stability and could either increase or decrease adsorption uptake in a concentration-dependent manner, depending on the chemical structure of the amphipathic molecules and their binding strength with BSA protein. Finally, the experimentally observed differences in conformational properties and adsorption behavior of certain types of BSA proteins were correlated with their surface passivation performance. In summary, the findings in this thesis presented how materials science concepts can be used to rationalize the relationship between protein conformational stability and adsorption behavior and offer a broadly applicable approach to engineer protein coatings for various useful applications.Doctor of Philosoph

Nanoarchitectured air-stable supported lipid bilayer incorporating sucrose-bicelle complex system

Cell-membrane-mimicking supported lipid bilayers (SLBs) provide an ultrathin, self-assembled layer that forms on solid supports and can exhibit antifouling, signaling, and transport properties among various possible functions. While recent material innovations have increased the number of practically useful SLB fabrication methods, typical SLB platforms only work in aqueous environments and are prone to fluidity loss and lipid-bilayer collapse upon air exposure, which limits industrial applicability. To address this issue, herein, we developed sucrose-bicelle complex system to fabricate air-stable SLBs that were laterally mobile upon rehydration. SLBs were fabricated from bicelles in the presence of up to 40 wt% sucrose, which was verified by quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP) experiments. The sucrose fraction in the system was an important factor; while 40 wt% sucrose induced lipid aggregation and defects on SLBs after the dehydration-rehydration process, 20 wt% sucrose yielded SLBs that exhibited fully recovered lateral mobility after these processes. Taken together, these findings demonstrate that sucrose-bicelle complex system can facilitate one-step fabrication of air-stable SLBs that can be useful for a wide range of biointerfacial science applications.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Published versionThis work was supported by the Ministry of Education (MOE) in Singapore under Grant AcRF TIER1-2020-T1-002-032 (RG111/20). This work was also sup‑ ported by the China-Singapore International Joint Research Institute (CSIJRI). HT is supported by a SINGA graduate scholarship from the A*STAR Graduate Academy, Singapore

Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings

Bovine serum albumin proteins are used to fabricate antifouling coatings, but it is unclear which of these give the best coatings. Here, bovine serum albumin proteins from different purification processes are investigated, revealing that fatty acid-free proteins give superior antifouling properties

Elucidating how different amphipathic stabilizers affect BSA protein conformational properties and adsorption behavior

Natural proteins such as bovine serum albumin (BSA) are readily extracted from biological fluids and widely used in various applications such as drug delivery and surface coatings. It is standard practice to dope BSA proteins with an amphipathic stabilizer, most commonly fatty acids, during purification steps to maintain BSA conformational properties. There have been extensive studies investigating how fatty acids and related amphiphiles affect solution-phase BSA conformational properties, while it is far less understood how amphipathic stabilizers might influence noncovalent BSA adsorption onto solid supports, which is practically relevant to form surface coatings. Herein, we systematically investigated the binding interactions between BSA proteins and different molar ratios of caprylic acid (CA), monocaprylin (MC), and methyl caprylate (ME) amphiphiles-all of which have 8-carbon-long, saturated hydrocarbon chains with distinct headgroups-and resulting effects on BSA adsorption behavior on silica surfaces. Our findings revealed that anionic CA had the greatest binding affinity to BSA, which translated into greater solution-phase conformational stability and reduced adsorption-related conformational changes along with relatively low packing densities in fabricated BSA adlayers. On the other hand, nonionic MC had moderate binding affinity to BSA and could stabilize BSA conformational properties in the solution and adsorbed states while also enabling BSA adlayers to form with higher packing densities. We discuss physicochemical factors that contribute to these performance differences, and our findings demonstrate how rational selection of amphiphile type and amount can enable control over BSA adlayer properties, which could lead to improved BSA protein-based surface coatings.National Research Foundation (NRF)This work was supported by the National Research Foundation of Singapore through a Competitive Research Programme grant (NRF-CRP10-2012-07) and a Proof-of-Concept grant (NRF2015NRF-P0C0001-19) and by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (no. 2020R1C1C1004385)

Conformational stability as a quality attribute for the cell therapy raw material human serum albumin

Although human serum albumin (HSA) has been used for many decades, there is still a lack of suitable quality control (QC) attributes. Its current use as a raw material in gene-, cell- and tissue-therapies requires more appropriate functionally-relevant quality attributes and methods. This study investigated the conformational stability of serum albumin using circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) for evaluating the thermal sensitivity, and quartz crystal microbalance-dissipation (QCM-D) and localized surface plasmon resonance (LSPR) for assessing the adsorption behavior. Different serum albumin samples were used, encompassing plasma-derived HSA (pHSA), recombinant octanoate-stabilized HSA (rHSA) and bovine serum albumin (BSA). The melting temperature (T-m) as well as the onset temperature (T-onset) were obtained from the derivative curves of the temperature gradient CD data at 222 nm. The results from DLS, as well as from real-time QCM-D and LSPR silica-adsorption kinetic profiles confirmed the relatively higher conformational stability of the octanoate (fatty acid) containing rHSA, while the additional negative charge resulted in a lower amount adsorbed to the silica surface compared to the non-stabilized HSA and BSA. Adsorption studies further revealed that BSA has a lower conformational stability and undergoes more extensive adsorption-induced spreading compared to the non-stabilized HSA. Collectively, the temperature-based (CD and DLS) as well as adsorption-based biosensor (QCM-D and LSPR) approaches gave congruent and discriminatory information about the conformational stability of different serum albumins, indicating that these techniques provide information on valuable QC attributes

Comparing protein adsorption onto alumina and silica nanomaterial surfaces: clues for vaccine adjuvant development

Protein adsorption onto nanomaterial surfaces is important for various nanobiotechnology applications such as biosensors and drug delivery. Within this scope, there is growing interest to develop alumina- and silica-based nanomaterial vaccine adjuvants and an outstanding need to compare protein adsorption onto alumina- and silica-based nanomaterial surfaces. Herein, using alumina- and silica-coated arrays of silver nanodisks with plasmonic properties, we conducted localized surface plasmon resonance (LSPR) experiments to evaluate real-time adsorption of bovine serum albumin (BSA) protein onto alumina and silica surfaces. BSA monomers and oligomers were prepared in different water-ethanol mixtures and both adsorbing species consistently showed quicker adsorption kinetics and more extensive adsorption-related spreading on alumina surfaces as compared to on silica surfaces. We rationalized these experimental observations in terms of the electrostatic forces governing protein-surface interactions on the two nanomaterial surfaces and the results support that more rigidly attached BSA protein-based coatings can be formed on alumina-based nanomaterial surfaces. Collectively, the findings in this study provide fundamental insight into protein-surface interactions at nanomaterial interfaces and can help to guide the development of protein-based coatings for medical and biotechnology applications such as vaccines.We acknowledge support from the Samsung Research Fund, Sungkyunkwan University, 2019

Mechanistic aspects of the evolution of 3D cholesterol crystallites in a supported lipid membrane via a quartz crystal microbalance with dissipation monitoring

The irreversible formation of cholesterol monohydrate crystals within biological membranes is the leading cause of various diseases, including atherosclerosis. Understanding the process of cholesterol crystallization is fundamentally important and could also lead to the development of improved therapeutic strategies. This has driven several studies investigating the effect of the environmental parameters on the induction of cholesterol crystallite growth and the structure of the cholesterol crystallites, while the kinetics and mechanistic aspects of the crystallite formation process within lipid membranes remain poorly understood. Herein, we fabricated cholesterol crystallites within a supported lipid bilayer (SLB) by adsorbing a cholesterol-rich bicellar mixture onto a glass and silica surface and investigated the real-time kinetics of cholesterol crystallite nucleation and growth using epifluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) monitoring. Microscopic imaging showed the evolution of the morphology of cholesterol crystallites from nanorod- and plate-shaped habits during the initial stage to mostly large, micron-sized three-dimensional (3D) plate-shaped crystallites in the end, which was likened to Ostwald ripening. QCM-D kinetics revealed unique signal responses during the later stage of the growth process, characterized by simultaneous positive frequency shifts, nonmonotonous energy dissipation shifts, and significant overtone dependence. Based on the optically observed changes in crystallite morphology, we discussed the physical background of these unique QCM-D signal responses and the mechanistic aspects of Ostwald ripening in this system. Together, our findings revealed mechanistic details of the cholesterol crystallite growth kinetics, which may be useful in biointerfacial sensing and bioanalytical applications.National Research Foundation (NRF)This work was supported by the National Research Foundation of Singapore through a Competitive Research Programme grant (NRF-CRP10-2012-07) and a Proof-ofConcept grant (NRF2015NRF-POC0001-19)

Probing the Interaction of Dielectric Nanoparticles with Supported Lipid Membrane Coatings on Nanoplasmonic Arrays

The integration of supported lipid membranes with surface-based nanoplasmonic arrays provides a powerful sensing approach to investigate biointerfacial phenomena at membrane interfaces. While a growing number of lipid vesicles, protein, and nucleic acid systems have been explored with nanoplasmonic sensors, there has been only very limited investigation of the interactions between solution-phase nanomaterials and supported lipid membranes. Herein, we established a surface-based localized surface plasmon resonance (LSPR) sensing platform for probing the interaction of dielectric nanoparticles with supported lipid bilayer (SLB)-coated, plasmonic nanodisk arrays. A key emphasis was placed on controlling membrane functionality by tuning the membrane surface charge vis-à-vis lipid composition. The optical sensing properties of the bare and SLB-coated sensor surfaces were quantitatively compared, and provided an experimental approach to evaluate nanoparticle–membrane interactions across different SLB platforms. While the interaction of negatively-charged silica nanoparticles (SiNPs) with a zwitterionic SLB resulted in monotonic adsorption, a stronger interaction with a positively-charged SLB resulted in adsorption and lipid transfer from the SLB to the SiNP surface, in turn influencing the LSPR measurement responses based on the changing spatial proximity of transferred lipids relative to the sensor surface. Precoating SiNPs with bovine serum albumin (BSA) suppressed lipid transfer, resulting in monotonic adsorption onto both zwitterionic and positively-charged SLBs. Collectively, our findings contribute a quantitative understanding of how supported lipid membrane coatings influence the sensing performance of nanoplasmonic arrays, and demonstrate how the high surface sensitivity of nanoplasmonic sensors is well-suited for detecting the complex interactions between nanoparticles and lipid membranes.NRF (Natl Research Foundation, S’pore)Published versio

Lipid coating technology: a potential solution to address the problem of sticky containers and vanishing drugs

Pharmaceutical drugs and vaccines require the use of material containers for protection, storage, and transportation. Glass and plastic materials are widely used for packaging, and a longstanding challenge in the field is the nonspecific adsorption of pharmaceutical drugs to container walls – the so-called “sticky containers, vanishing drugs” problem – that effectively reduces the active drug concentration and can cause drug denaturation. This challenge has been frequently discussed in the case of the anticancer drug, paclitaxel, and the ongoing coronavirus disease 2019 (COVID-19) pandemic has brought renewed attention to this material science challenge in light of the need to scale up COVID-19 vaccine production and to secure sufficient quantities of packaging containers. To reduce nonspecific adsorption on inner container walls, various strategies based on siliconization and thin polymer films have been explored, while it would be advantageous to develop mass-manufacturable, natural material solutions, especially ones involving pharmaceutical grade excipients. Inspired by how lipid nanoparticles have revolutionized the vaccine field, in this perspective, we discuss the prospects for developing lipid bilayer coatings to prevent nonspecific adsorption of pharmaceutical drugs and vaccines and how recent advances in lipid bilayer coating fabrication technologies are poised to accelerate progress in the field. We critically discuss recent examples of how lipid bilayer coatings can prevent nonspecific sticking of proteins and vaccines to relevant material surfaces and examine future translational prospects.National Research Foundation (NRF)Published versionThis work was supported by the National Research Foundation of Singapore through a Proof-of-Concept grant (NRF2015NRF-POC0001-19) and by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (2020R1C1C1004385 and2020R1C1C1005523). In addition, this work was supported by Brain Pool Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2019H1D3A1A01070318). This research was also supported by the International Research & Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT(2020K1A3A1A39112724)