Sandwich Culture Platforms to Investigate the Roles of Stiffness Gradients and Cell-Matrix Adhesions in Cancer Cell Migration.

Given the key role of cell migration in cancer metastasis, there is a critical need for in vitro models that better capture the complexities of in vivo cancer cell microenvironments. Using both two-dimensional (2D) and three-dimensional (3D) culture models, recent research has demonstrated the role of both matrix and ligand densities in cell migration. Here, we leveraged our previously developed 2.5D sandwich culture platform to foster a greater understanding of the adhesion-dependent migration of glioblastoma cells with a stiffness gradient. Using this model, we demonstrated the differential role of stiffness gradients in migration in the presence and absence of adhesion moieties. Furthermore, we observed a positive correlation between the density of cell adhesion moieties and migration, and a diminished role of stiffness gradients at higher densities of adhesion moieties. These results, i.e., the reduced impact of stiffness gradients on adhesion-dependent migration relative to adhesion-independent migration, were confirmed using inhibitors of both mechanotransduction and cell adhesion. Taken together, our work demonstrates the utility of sandwich culture platforms that present stiffness gradients to study both adhesion-dependent and -independent cell migration and to help expand the existing portfolio of in vitro models of cancer metastasis.

Silver nanoparticle interactions with glycated and non-glycated human serum albumin mediate toxicity.

Introduction: Biomolecules bind to and transform nanoparticles, mediating their fate in biological systems. Despite over a decade of research into the protein corona, the role of protein modifications in mediating their interaction with nanomaterials remains poorly understood. In this study, we evaluated how glycation of the most abundant blood protein, human serum albumin (HSA), influences the formation of the protein corona on 40 nm silver nanoparticles (AgNPs) and the toxicity of AgNPs to the HepG2 human liver cell line. Methods: The effects of glycation on AgNP-HSA interactions were quantified using circular dichroism spectroscopy to monitor protein structural changes, dynamic light scattering to assess AgNP colloidal stability, zeta potential measurements to measure AgNP surface charge, and UV-vis spectroscopy and capillary electrophoresis (CE) to evaluate protein binding affinity and kinetics. The effect of the protein corona and HSA glycation on the toxicity of AgNPs to HepG2 cells was measured using the WST cell viability assay and AgNP dissolution was measured using linear sweep stripping voltammetry. Results and Discussion: Results from UV-vis and CE analyses suggest that glycation of HSA had little impact on the formation of the AgNP protein corona with protein-AgNP association constants of ≈2x107 M-1 for both HSA and glycated HSA (gHSA). The formation of the protein corona itself (regardless of whether it was formed from HSA or glycated HSA) caused an approximate 2-fold decrease in cell viability compared to the no protein AgNP control. While the toxicity of AgNPs to cells is often attributed to dissolved Ag(I), dissolution studies showed that the protein coated AgNPs underwent less dissolution than the no protein control, suggesting that the protein corona facilitated a nanoparticle-specific mechanism of toxicity. Overall, this study highlights the importance of protein coronas in mediating AgNP interactions with HepG2 cells and the need for future work to discern how protein coronas and protein modifications (like glycation) may alter AgNP reactivity to cellular organisms.

Silver Nanoparticle Surface Chemistry Determines Interactions with Human Serum Albumin and Cytotoxic Responses in Human Liver Cells.

Engineered nanomaterials (ENMs) are synthesized with a diversity of surface chemistries that mediate biochemical interactions and physiological response to the particles. In this work, silver engineered nanomaterials (AgENMs) are used to evaluate the role of surface charge in protein interactions and cellular cytotoxicity. The most abundant protein in blood, human serum albumin (HSA), was interacted with 40 nm AgENMs with a range of surface-charged coatings: positively charged branched polyethyleneimine (bPEI), negatively charged citrate (CIT), and circumneutral poly(ethylene glycol) (PEG). HSA adsorption to AgENMs was monitored by UV-vis spectroscopy and dynamic light scattering, while changes to the protein structure were evaluated with circular dichroism spectroscopy. Binding affinity for citrate-coated AgENMs and HSA is largest among the three AgENM coatings; yet, HSA lost the most secondary structure upon interaction with bPEI-coated AgENMs compared to the other two coatings. HSA increased AgENM oxidative dissolution across all particle types, with the greatest dissolution for citrate-coated AgENMs. Results indicate that surface coating is an important consideration in transformation of both the particle and protein upon interaction. To connect results to cellular outcomes, we also performed cytotoxicity experiments with HepG2 cells across all three AgENM types with and without HSA. Results show that bPEI-coated AgENMs cause the greatest loss of cell viability, both with and without inclusion of HSA with the AgENMs. Thus, surface coatings on AgENMs alter both biophysical interactions with proteins and particle cytotoxicity. Within this study set, positively charged bPEI-coated AgENMs cause the greatest disruption to HSA structure and cell viability.

Low-Dose Silver Nanoparticle Surface Chemistry and Temporal Effects on Gene Expression in Human Liver Cells.

Silver nanoparticles (AgNPs) are widely incorporated into consumer and biomedical products for their antimicrobial and plasmonic properties with limited risk assessment of low-dose cumulative exposure in humans. To evaluate cellular responses to low-dose AgNP exposures across time, human liver cells (HepG2) are exposed to AgNPs with three different surface charges (1.2 µg mL-1 ) and complete gene expression is monitored across a 24 h period. Time and AgNP surface chemistry mediate gene expression. In addition, since cells are fed, time has marked effects on gene expression that should be considered. Surface chemistry of AgNPs alters gene transcription in a time-dependent manner, with the most dramatic effects in cationic AgNPs. Universal to all surface coatings, AgNP-treated cells responded by inactivating proliferation and enabling cell cycle checkpoints. Further analysis of these universal features of AgNP cellular response, as well as more detailed analysis of specific AgNP treatments, time points, or specific genes, is facilitated with an accompanying application. Taken together, these results provide a foundation for understanding hepatic response to low-dose AgNPs for future risk assessment.

Three-Dimensional (3D) Printed Microneedles for Microencapsulated Cell Extrusion.

Cell-hydrogel based therapies offer great promise for wound healing. The specific aim of this study was to assess the viability of human hepatocellular carcinoma (HepG2) cells immobilized in atomized alginate capsules (3.5% (w/v) alginate, d = 225 µm ± 24.5 µm) post-extrusion through a three-dimensional (3D) printed methacrylate-based custom hollow microneedle assembly (circular array of 13 conical frusta) fabricated using stereolithography. With a jetting reliability of 80%, the solvent-sterilized device with a root mean square roughness of 158 nm at the extrusion nozzle tip (d = 325 µm) was operated at a flowrate of 12 mL/min. There was no significant difference between the viability of the sheared and control samples for extrusion times of 2 h (p = 0.14, α = 0.05) and 24 h (p = 0.5, α = 0.05) post-atomization. Factoring the increase in extrusion yield from 21.2% to 56.4% attributed to hydrogel bioerosion quantifiable by a loss in resilience from 5470 (J/m³) to 3250 (J/m³), there was no significant difference in percentage relative payload (p = 0.2628, α = 0.05) when extrusion occurred 24 h (12.2 ± 4.9%) when compared to 2 h (9.9 ± 2.8%) post-atomization. Results from this paper highlight the feasibility of encapsulated cell extrusion, specifically protection from shear, through a hollow microneedle assembly reported for the first time in literature.