Supramolecular protein-mediated assembly of brain extracellular matrix glycans

Introduction

A major paradigm that has dominated the drug delivery and tissue engineering communities is the development of bio-inspired hydrogels that mimic the intermolecular interactions and mechanical properties of physiological tissue. Glycosaminoglycans (GAGs) are polysaccharides that are critical structural components of the brain extracellular matrix (ECM). One particularly abundant brain GAG, hyaluronic acid (HA) (Figure 1A), has been made into many covalently modified derivatives that have been widely explored in drug delivery and tissue engineering¹. However, HA is the only supramolecular brain GAG², and other ECM components including the more abundant protein-linked GAG chondroitin sulfate (CS) have been comparatively under studied³. HA is often chemically functionalized because the high molecular weight polysaccharide does not gel on its own. Although functional materials based on covalently modified HA exhibit useful features for many applications, these gelation methods do not capture the physiological supramolecular interactions that are endogenously found in the brain extracellular matrix⁴.

Figure 1.

(A) Structures of chondroitin sulfate (CS) and hyaluronic acid (HA). (B) Relative surface charge densities of bovine serum albumin (BSA). Dataset 1: Dynamic light scattering and transmission electron microscopy data⁷.

In this work we examine the intermolecular interactions of two major biopolymer components of the brain, HA and CS, with the model protein bovine serum albumin (BSA) (Figure 1B). We exploit known electrostatic interactions between the BSA and GAG polymers^5,6 to generate structurally diverse protein-glycan complexes. We examine how these negatively charged biopolymers interact and self-assemble with BSA, and develop supramolecular systems formed from competing HA/BSA, HA/CS, and CS/BSA interactions.

Methods

Results and discussion

In this work we explored the electrostatically-driven self-assembly of charged proteins with negatively-charged polysaccharides endogenous to the human brain. To mimic the native interactions these polysaccharides have with surrounding proteins, we introduced BSA, which has electrostatic binding pockets complementary to anionic GAGs, analogous to the binding interfaces of ECM proteins. BSA and CS polymer solutions were mixed overnight and allowed to self-assemble into nanostructures (ESI). Two distinct populations of particles were observed to form (Figure 2, Dataset 1⁷), and were dissimilar to the flocculation of BSA aggregates alone. The smaller particles were characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM). DLS yielded an average particle diameter (D) of 51 ± 3 nm. These structures were stable for at least 2 days in the parent suspension (Figure S1A, Extended Data⁸). DLS autocorrelation data suggested that a large diameter species may also be present in the solution (Figure S1B, Extended Data⁸). TEM was used to characterize these self-assembled microparticles (Figure 2D). The analysis of these micrographs indicated the presence of two distinct populations of assembles, with the mean diameter of D = 60 ± 10 nm, that is consistent with DLS experiments, and an additional one of D = 1.5 ± 0.5 µm. In the brain, CS is covalently scaffolded onto peptide cores (e.g. aggrecan⁴) and binds with many ECM proteins through non-covalent, supramolecular interactions⁹. The supramolecular interactions between CS and BSA described here could provide insight into an electrostatic driving force that contributes to GAG aggregation and nanostructure formation in vivo.

Figure 2. Self-assembly of chondroitin sulfate (CS) and bovine serum albumin (BSA) particles.

(A) Schematic of the formation of dense CS-BSA particles. (B) DLS size plot of dynamic BSA aggregates (top) and CS-BSA particles (bottom). (C–D) TEM image of CS-BSA nanoparticles (C) and microparticles (D).

We then turned our attention to HA, a linear high molecular weight polysaccharide that is the only supramolecular GAG in human physiology. HA and BSA were mixed overnight and the electrostatic interactions between mixture components led the solution to self-assemble into a supramolecular gel (Figure 3, Dataset 1⁷). Supramolecular gels formed via electrostatic protein-polymer interactions with polysaccharide back-bones have been previously reported¹⁰. Solutions of HA alone were highly viscous but did not show gel-like properties. Oscillatory rheological measurements were used to probe the mechanics of this supramolecular gel (ESI). After introduction of BSA, electrostatic-driven network percolation resulted in a major stiffening effect and the formation of a gel with G' > 10 kPa. This material exhibited very clear shear-thinning and recovery behavior (Figure 3C).

Figure 3. Oscillatory rheological sweeps of 40 mg/mL hyaluronic acid (HA) and 50 mg/mL bovine serum albumin (BSA) gels.

(A) Schematic illustration dynamic network formation of HA and BSA. (B) Oscillatory rheological frequency sweep of HA solution alone and HA-BSA gels. (C) Oscillatory time sweep after 100% shear of HA-BSA gels and HA solution alone. (D) DLS size measurements of dynamic BSA particles alone and HA-BSA gel. Dataset 1: Rheology and dynamic light scattering data.

Upon introduction of dry CS into a HA/BSA matrix, a sharp decrease in stiffness was observed (Figure S3, Extended Data⁸). We hypothesize this effect is due to both interference from CS/BSA interactions and the reduction of entanglement of HA. DLS data showed that when combined with either HA or CS, BSA exhibited hierarchical assemblies that were not observed for BSA alone. Furthermore, when combined with HA, the BSA assemblies were more dynamic and polydispersed than with CS (Figure 3D, S2⁸).

We then explored whether these HA/CS/CS systems could be used to explore mass transfer of the model hydrophilic drug rhodamine B (rhodB). Many parenteral drug-delivery studies will monitor in vitro release kinetics into saline as a model for in vivo release. Here we explore the feasibility of monitoring release kinetics into a gel instead of saline (Figure 4, Extended Data⁸). A hydrophobic blend of poly(caprolactone) (PCL) chains at different molecular weights (ESI) loaded with rhodB was carefully added on top of the hydrogel phase (Figure 4, Extended Data⁸). We found that it was possible to monitor the interfacial concentration of rhodB in the hydrogel phase with UV-Vis spectroscopy. Interestingly, we observed a large bolus release of the hydrophilic drug from the hydrophobic phase to the hydrophilic phase at the interface until the same concentration was reached, subsequently an equilibrium or pseudo-steady-state concentration was reached after 14 hours. Such a system is potentially useful in modeling mass transfer of drugs between hydrophobic drug delivery materials and biological tissue such as the brain¹¹.

Figure 4. Measuring interfacial mass transfer kinetics between hyaluronic acid (HA)/bovine serum albumin (BSA)/chondroitin sulfate (CS) system and PCL blend.

(A) Picture of initial two phases measured via UV-Vis spectroscopy. (B) Concentration at interface after large bolus release over time. Hydrophobic phase drug concentration = 10 µg/mL. Average hydrophilic phase equilibrium concentration = 14.5 µg/mL. The periodic fluctuations in concentration were attributed to trapped air interfering with the interface. (Dataset 1: UV-Vis data.

Prior tissue engineering studies have largely neglected the question of how the exact composition of the brain ECM might inform efforts to create biologically-mimetic hydrogels. To estimate the physiological concentrations of CS and HA that occur in vivo (Figure 5, Dataset 1⁷), we summarize the literature for CS and HA measurements of mammalian brain tissue^12–19. These estimates clustered in the millimolar range for CS disaccharides, and 100 micromolar range for HA.

Figure 5. Summary of concentrations of hyaluronic acid (HA) and chondroitin sulfate (CS) in the brain of various species.

The concentration of HA in healthy tissue, glioblastoma, and astrocytoma is also plotted.

Conclusions

In this work we characterize supramolecular interactions between HA, CS, and BSA. We report nano- and microparticle self-assembly, gelation, and network interference be haviors. We also report on the mass transfer of a model drug from a hydrophobic phase into a glycan-based hydrogel. Finally, we summarize reported concentrations of HA and CS in different animal models. This report may inform the development of biomaterials for tissue engineering that capture or exploit supramolecular interactions between brain extracellular matrix glycans.

Data availability

Underlying data for this study is available from Open Science Framework

OSF: Dataset 1: Supramolecular Protein-Mediated Assembly of Brain Extracellular Matrix Glycans, https://doi.org/10.17605/OSF.IO/3BXQG⁷

Data is available under a CC0 1.0 License

Extended data

Electronic supplementary information (ESI) document is available from Open Science Framework.

OSF: Extended Data. Supramolecular Protein-Mediated Assembly of Brain Extracellular Matrix Glycans ESI https://doi.org/10.17605/OSF.IO/NG6H7⁸

The ESI contains the following Supplementary Figures -

Figure S1: Autocorrelation function of chondroitin sulfate (CS)/bovine serum albumin (BSA) NPs. (B) Time resolved DLS of CS/BSA NPs in Milli-Q water.

Figure S2: Time resolved dynamic light scattering of hyaluronic acid (HA)/bovine serum albumin (BSA) gels.

Figure S3: (A) Oscillatory frequency sweeps of hyaluronic acid (HA) systems loaded with bovine serum albumin (BSA) and with or without chondroitin sulfate (CS). (B) Time-resolved dynamic light scattering experiment of CS/BSA particles loaded with HA and sheared.

Extended data is available under a CC0 1.0 License