Tailoring Therapeutic Responses via Engineering Microenvironments with a Novel Synthetic Fluid Gel

Abstract

This study reports the first fully synthetic fluid gel (SyMGels) using a simple poly(ethylene glycol) polymer. Fluid gels are an interesting class of materials: structured during gelation via shear-confinement to form microparticulate suspensions, through a bottom-up approach. Structuring in this way, when compared to first forming a gel and subsequently breaking it down, results in the formation of a particulate dispersion with particles "grown" in the shear flow. Resultantly, systems form a complex microstructure, where gelled particles concentrate remaining non-gelled polymer within the continuous phase, creating an amorphous-like interstitial phase. As such, these materials demonstrate mechanical characteristics typical of colloidal glasses, presenting solid-like behaviors at rest with defined yielding; likely through intrinsic particle-particle and particle-polymer interactions. To date, fluid gels have been fabricated using polysaccharides with relatively complex chemistries, making further modifications challenging. SyMGels are easily functionalised, using simple click-chemistry. This chemical flexibility, allows the creation of microenvironments with discrete biological decoration. Cellular control is demonstrated using MSC (mesenchymal stem cells)/chondrocytes and enables the regulation of key biomarkers such as aggrecan and SOX9. These potential therapeutic platforms demonstrate an important advancement in the biomaterial field, underpinning the mechanisms which drive their mechanical properties, and providing a versatile delivery system for advanced therapeutics.

Keywords: PEG; cell scaffolds; fluid gels; functionalization; soft-particle glasses.

Figure 1

Formation of synthetic microgel suspensions (SyMGels) via sheared gelation. a) Schematic representation for the fabrication of SyMGels, with proposed gelation mechanism: i) radical formation; ii) initiation and propagation, and; iii) restriction of gel growth through applied shear to form terminated particles. b) “Gelation” profiles for SyMGels prepared at mixing rate of either 300 or 700 rpm. Profiles were obtained by measuring the deviation from initial liquid height as a function of time as shown by the photographic representations at 0, 75, and 120 s (700 rpm). c) Determination of the gelled phase using centrifugation. Mass of continuous phase removed as a function of processing mixing rate from a 0.5 g aliquot after centrifugation at 17000 rcf for 10 min. All data presented is the average on n = 3, with error bars demonstrating the 95% confidence interval. Statistical analysis was conducted using one‐way ANOVA with significance denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2

Synthetic microgel particle shape and size. a) static light scattering data for SyMGels prepared at various mixing rates. i) Particle size distributions as a function of processing mixing rate and ii) volume weighted averages (D[4,3]) taken from the distributions showing a decreasing linear trend in size as a function of applied mixing. b) Optical micrographs obtained using phase contrast microscopy for diluted (1:4) SyMGel systems prepared at varying mixing rates. c) 3D stacked CSLM images rotated through 90° and 180° to show particle thickness (particles prepared at 300 rpm). Gelled particles were stained with Rhodamine 6G and images using a 543 nm laser. (Scale bars represent 100 µm). All data presented is the average on n = 3, with error bars demonstrating the 95% confidence interval.

Figure 3

Mechanical behavior of SyMGel systems. a) Amplitude sweep, stress controlled, for SyMGel suspensions prepared at either 300 or 700 rpm. b) Frequency sweeps obtained at 0.04 Pa stress for SyMGels prepared at 300 and 700 rpm. c) Storage moduli (G′) obtained via frequency sweeps (0.04 Pa stress), as a function of processing mixing rate. Lines of best fit added to each data set with equation of the line shown in the legend. d) Collapsed amplitude sweeps for SyMGels prepared at varying processing mixing rates. e) Change in Tanδ as a function of the processing mixing rate used during SyMGel curing. All data presented is the average on n = 3, with error bars demonstrating the 95% confidence interval.

Figure 4

Flow behavior of the SyMGel suspensions. a) Shear rate ramps for SyMGels prepared at mixing rates of 300 and 700 rpm obtained over a 1 min sweep. Fit to the Cross model applied and used to determine data presented in the table below it. b) Zero shear viscosity (η ₀) data obtained using the Cross fit plotted as a function of processing rate. c) Zero shear viscosity plotted as a function of the particle volume fraction (φ _gel) determined using the centrifugation data presented in Figure 1c. Fit to Mark‐Houwink equation for concentrated systems (M > M _c), η ₀ = K_T   M ^3.4, where K _T was used as a fitting factor and M has been replaced by the particle volume fraction, φ _gel. d) Table of data collated from fitting flow profiles to the Cross model where, η ₀ is the zero‐shear viscosity, η _∞ is the infinite shear viscosity, 1/C is the critical shear value to induce thinning, m is the thinning index, and R² is the statistical measure of fitting. All data presented is the average on n = 3, with error bars demonstrating the 95% confidence interval.

Figure 5

Controlling cellular microenvironments through functionalization. a) Schematic showing the proposed mechanism for the functionalization of SyMGel particles with bioactive molecules and ECM components. Mechanism is based on a typical Michael‐type reaction, reactions steps from reactants to product are highlighted in (i) through to (iii). b) Optical micrographs with fluorescent overlays of SyMGel particles (prepared at 300 rpm) functionalized with fibronectin (FN) and hyaluronic acid (HA). (Scale bar represents 100 µm). c) Micrograph of a FN‐SyMGel particle (prepared at 300 rpm) with chondrocytes adhered to the surface. (Scale bars represent 100 µm). d) Wnt‐SyMGel activation using a luciferase reporter line. Micrographs visually shows activation of the cells via fluorescence. (scale bar represents 150 µm). e) Control over key chondrocytic markers using, i) FN‐SyMGel and ii) HA‐SyMGel (300 rpm) microenvironments. All data presented is the average on n = 3, with error bars demonstrating the 95% confidence interval. Statistical analysis was conducted using two‐way ANOVA with significance denoted as * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 6

Complex fluid gel microstructure. Schematic diagrams of the complex fluid gel microstructure showing i) the system at the macroscale (inserts used to better depict the amorphous‐like interstitial phase and particle structures). ii) Diagram showing the various system interactions: * polymer‐polymer entanglements in the interstitial phase; ** particle‐particle interactions when in close proximity; *** particle‐polymer interactions formed at the particle interface and interstitial layers.