Role of Polymer Concentration on the Release Rates of Proteins from Single- and Double-Network Hydrogels.

Controlled delivery of proteins has immense potential for the treatment of various human diseases, but effective strategies for their delivery are required before this potential can be fully realized. Recent research has identified hydrogels as a promising option for the controlled delivery of therapeutic proteins, owing to their ability to respond to diverse chemical and biological stimuli, as well as their customizable properties that allow for desired delivery rates. This study utilized alginate and chitosan as model polymers to investigate the effects of hydrogel properties on protein release rates. The results demonstrated that polymer properties, concentration, and crosslinking density, as well as their responses to pH, can be tailored to regulate protein release rates. The study also revealed that hydrogels may be combined to create double-network hydrogels to provide an additional metric to control protein release rates. Furthermore, the hydrogel scaffolds were also found to preserve the long-term function and structure of encapsulated proteins before their release from the hydrogels. In conclusion, this research demonstrates the significance of integrating porosity and response to stimuli as orthogonal control parameters when designing hydrogel-based scaffolds for therapeutic protein release.

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.

Modeling the Additive Effects of Nanoparticles and Polymers on Hydrogel Mechanical Properties Using Multifactor Analysis.

Interpenetrating networks (IPN)s have been conceived as a biomimetic tool to tune hydrogel mechanical properties to the desired target formulations. In this study, the rheological behavior of acrylamide (AAm) [2.5-10%] hydrogels crosslinked with N,N'-methylenebis(acrylamide) (Bis) [0.0625-0.25%] was characterized in terms of the saturation modulus affected by the interaction of silica nanoparticle (SiNP) nanofillers [0-5%] and dextran [0-2%] at a frequency of 1 Hz and strain rate of 1% after a gelation period of 90 min. For single-network hydrogels, a prominent transition was observed at 0.125% Bis for 2.5% AAm and 0.25% Bis for 5% AAm across the SiNP concentrations and was validated by retrospective 3-level factorial design models, as characterized by deviation from linearity in the saturation region (R2 = 0.86). IPN hydrogels resulting from the addition of dextran to the single network in the pre-saturation region, as outlined by the strong goodness of fit (R2= 0.99), exhibited a correlated increase in the elastic (G') and viscous moduli (G"). While increasing the dextran concentrations [0-2%] and MW [100 kDa and 500 kDa] regulated the increase in G', saturation in G" or the loss tangent (tan(δ)) was not recorded within the observed operating windows. Results of multifactor analysis conducted on Han plots in terms of the elastic gains indicate that amongst the factors modulating the viscoelasticity of the IPN hydrogels, dextran concentration is the most important (RDex = 35.3 dB), followed by nanoparticle concentration (RSiNP = 7.7 dB) and dextran molecular weight (RMW = 2.9 dB). The results demonstrate how the Han plot may be systematically used to quantify the main effects of intensive thermodynamic properties on rheological phase transition in interpenetrating networks where traditional multifactor analyses cannot resolve statistical significance.

Role of Polymer Concentration and Crosslinking Density on Release Rates of Small Molecule Drugs.

Over the past few years, researchers have demonstrated the use of hydrogels to design drug delivery platforms that offer a variety of benefits, including but not limited to longer circulation times, reduced drug degradation, and improved targeting. Furthermore, a variety of strategies have been explored to develop stimulus-responsive hydrogels to design smart drug delivery platforms that can release drugs to specific target areas and at predetermined rates. However, only a few studies have focused on exploring how innate hydrogel properties can be optimized and modulated to tailor drug dosage and release rates. Here, we investigated the individual and combined roles of polymer concentration and crosslinking density (controlled using both chemical and nanoparticle-mediated physical crosslinking) on drug delivery rates. These experiments indicated a strong correlation between the aforementioned hydrogel properties and drug release rates. Importantly, they also revealed the existence of a saturation point in the ability to control drug release rates through a combination of chemical and physical crosslinkers. Collectively, our analyses describe how different hydrogel properties affect drug release rates and lay the foundation to develop drug delivery platforms that can be programmed to release a variety of bioactive payloads at defined rates.

Multifunctional Hydrogel Nanocomposites for Biomedical Applications.

Hydrogels are used for various biomedical applications due to their biocompatibility, capacity to mimic the extracellular matrix, and ability to encapsulate and deliver cells and therapeutics. However, traditional hydrogels have a few shortcomings, especially regarding their physical properties, thereby limiting their broad applicability. Recently, researchers have investigated the incorporation of nanoparticles (NPs) into hydrogels to improve and add to the physical and biochemical properties of hydrogels. This brief review focuses on papers that describe the use of nanoparticles to improve more than one property of hydrogels. Such multifunctional hydrogel nanocomposites have enhanced potential for various applications including tissue engineering, drug delivery, wound healing, bioprinting, and biowearable devices.

Combined Effects of Confinement and Macromolecular Crowding on Protein Stability.

Confinement and crowding have been shown to affect protein fates, including folding, functional stability, and their interactions with self and other proteins. Using both theoretical and experimental studies, researchers have established the independent effects of confinement or crowding, but only a few studies have explored their effects in combination; therefore, their combined impact on protein fates is still relatively unknown. Here, we investigated the combined effects of confinement and crowding on protein stability using the pores of agarose hydrogels as a confining agent and the biopolymer, dextran, as a crowding agent. The addition of dextran further stabilized the enzymes encapsulated in agarose; moreover, the observed increases in enhancements (due to the addition of dextran) exceeded the sum of the individual enhancements due to confinement and crowding. These results suggest that even though confinement and crowding may behave differently in how they influence protein fates, these conditions may be combined to provide synergistic benefits for protein stabilization. In summary, our study demonstrated the successful use of polymer-based platforms to advance our understanding of how in vivo like environments impact protein function and structure.

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.

Role of Nanoparticle-Polymer Interactions on the Development of Double-Network Hydrogel Nanocomposites with High Mechanical Strength.

Extensive experimental and theoretical research over the past several decades has pursued strategies to develop hydrogels with high mechanical strength. Our study investigated the effect of combining two approaches, addition of nanoparticles and crosslinking two different polymers (to create double-network hydrogels), on the mechanical properties of hydrogels. Our studies revealed that these orthogonal approaches may be combined to synthesize hydrogel composites with enhanced mechanical properties. However, the enhancement in double network hydrogel elastic modulus due to incorporation of nanoparticles is limited by the ability of the nanoparticles to strongly interact with the polymers in the network. Moreover, double-network hydrogel nanocomposites prepared using lower monomer concentrations showed higher enhancements in elastic moduli compared to those prepared using high monomer concentrations, thus indicating that the concentration of hydrogel monomers used for the preparation of the nanocomposites had a significant effect on the extent of nanoparticle-mediated enhancements. Collectively, these results demonstrate that the hypotheses previously developed to understand the role of nanoparticles on the mechanical properties of hydrogel nanocomposites may be extended to double-network hydrogel systems and guide the development of next-generation hydrogels with extraordinary mechanical properties through a combination of different approaches.

Solution effects on the self-association of a water-soluble peptoid.

A desire to replicate the structural and functional complexity of proteins with structured, sequence-specific oligomers motivates study of the structural features of water-soluble peptoids (N-substituted glycine oligomers). Understanding the molecular-level details of peptoid self-assembly in water is essential to advance peptoids' application as novel materials. Peptoid 1, an amphiphilic, putatively helical peptoid previously studied in our laboratory, shows evidence of self-association in aqueous solution. In this work, we evaluate how changes to aqueous solution conditions influence the self-association of 1. We report that changes to pH influence the fluorescence and CD spectroscopic features as well as the peptoid's interaction with a solvatochromic fluorophore and its apparent size as estimated by size exclusion chromatography. Addition of guanidine hydrochloride and ammonium sulfate also modulate spectroscopic features of the peptoid, its interaction with a solvatochromic fluorophore, and its elution in size exclusion chromatography. These data suggest that the ordering of the self-assembly changes in response to pH and with solvent additives and is more ordered at higher pH and in the presence of guanidine hydrochloride. The deeper understanding of the self-association of 1 afforded by these studies informs the design of new stimuli-responsive peptoids with stable tertiary or quaternary structures.

Exploring the Role of Nanoparticles in Enhancing Mechanical Properties of Hydrogel Nanocomposites.

Over the past few decades, research studies have established that the mechanical properties of hydrogels can be largely impacted by the addition of nanoparticles. However, the exact mechanisms behind such enhancements are not yet fully understood. To further explore the role of nanoparticles on the enhanced mechanical properties of hydrogel nanocomposites, we used chemically crosslinked polyacrylamide hydrogels incorporating silica nanoparticles as the model system. Rheological measurements indicate that nanoparticle-mediated increases in hydrogel elastic modulus can exceed the maximum modulus that can be obtained through purely chemical crosslinking. Moreover, the data reveal that nanoparticle, monomer, and chemical crosslinker concentrations can all play an important role on the nanoparticle mediated-enhancements in mechanical properties. These results also demonstrate a strong role for pseudo crosslinking facilitated by polymer⁻particle interactions on the observed enhancements in elastic moduli. Taken together, our work delves into the role of nanoparticles on enhancing hydrogel properties, which is vital to the development of hydrogel nanocomposites with a wide range of specific mechanical properties.

Trehalose effectiveness as a cryoprotectant in 2D and 3D cell cultures of human embryonic kidney cells.

Post cryopreservation viability of human embryonic kidney (HEK) cells under two-dimensional (2D) and three-dimensional (3D) culture conditions was studied using trehalose as the sole cryoprotective agent. An L9 (34) Taguchi design was used to optimize the cryoprotection cocktail seeding process prior to slow-freezing with the specific aim of maximizing cell viability measured 7 days post thaw, using the combinatorial cell viability and in-vitro cytotoxicity WST assay. At low (200 mM) and medium (800 mM) levels of trehalose concentration, encapsulation in alginate offered a greater protection to cryopreservation. However, at the highest trehalose concentration (1200 mM) and in the absence of the pre-incubation step, there was no statistical difference at the 95% CI (p = 0.0212) between the viability of the HEK cells under 2D and 3D culture conditions estimated to be 17.9 ± 4.6% and 14.0 ± 3.6%, respectively. A parallel comparison between cryoprotective agents conducted at the optimal levels of the L9 study, using trehalose, dimethylsulfoxide and glycerol in alginate microcapsules yielded a viability of 36.0 ± 7.4% for trehalose, in average 75% higher than the results associated with the other two cell membrane-permeating compounds. In summary, the effectiveness of trehalose has been demonstrated by the fact that 3D cell cultures can readily be equilibrated with trehalose before cryopreservation, thus mitigating the cytotoxic effects of glycerol and dimethylsulfoxide.

Droplet Microfluidic Platform for the Determination of Single-Cell Lactate Release.

Cancer cells release high levels of lactate that has been correlated to increased metastasis and tumor recurrence. Single-cell measurements of lactate release can identify malignant cells and help decipher metabolic cancer pathways. We present here a novel droplet microfluidic method that allows the fast and quantitative determination of lactate release in many single cells. Using passive forces, droplets encapsulated cells are positioned in an array. The single-cell lactate release rate is determined from the increase in droplet fluorescence as the lactate is enzymatically converted to a fluorescent product. The method is used to measure the cell-to-cell variance of lactate release in K562 leukemia and U87 glioblastoma cancer cell lines and under the chemical inhibition of lactate efflux. The technique can be used in the study of cancer biology, but more broadly in cell biology, to capture the full range of stochastic variations in glycolysis activity in heterogeneous cell populations in a repeatable and high-throughput manner.

Three-dimensional matrix stiffness and adhesive ligands affect cancer cell response to toxins.

There is an immediate need to develop highly predictive in vitro cell-based assays that provide reliable information on cancer drug efficacy and toxicity. Development of biomaterial-based three-dimensional (3D) cell culture models as drug screening platforms has recently gained much scientific interest as 3D cultures of cancer cells have been shown to more adequately mimic the in vivo tumor conditions. Moreover, it has been recognized that the biophysical and biochemical properties of the 3D microenvironment can play key roles in regulating various cancer cell fates, including their response to chemicals. In this study, we employed alginate-based scaffolds of varying mechanical stiffness and adhesive ligand presentation to further explore the role of 3D microenvironmental cues on glioblastoma cell response to cytotoxic compounds. Our experiments suggested the ability of both matrix stiffness and cell-matrix adhesions to strongly influence cell responses to toxins. Cells were found to be more susceptible to the toxins when cultured in softer matrices that emulated the stiffness of brain tissue. Furthermore, the effect of matrix stiffness on differential cell responses to toxins was negated by the presence of the adhesive ligand RGD, but regained when integrin-based cell-matrix interactions were inhibited. This study therefore indicates that both 3D matrix stiffness and cell-matrix adhesions are important parameters in the design of more predictive in vitro platforms for drug development and toxicity screening.

Experimental Investigation of Mechanical and Thermal Properties of Silica Nanoparticle-Reinforced Poly(acrylamide) Nanocomposite Hydrogels.

Current studies investigating properties of nanoparticle-reinforced polymers have shown that nanocomposites often exhibit improved properties compared to neat polymers. However, over two decades of research, using both experimental studies and modeling analyses, has not fully elucidated the mechanistic underpinnings behind these enhancements. Moreover, few studies have focused on developing an understanding among two or more polymer properties affected by incorporation of nanomaterials. In our study, we investigated the elastic and thermal properties of poly(acrylamide) hydrogels containing silica nanoparticles. Both nanoparticle concentration and size affected hydrogel properties, with similar trends in enhancements observed for elastic modulus and thermal diffusivity. We also observed significantly lower swellability for hydrogel nanocomposites relative to neat hydrogels, consistent with previous work suggesting that nanoparticles can mediate pseudo crosslinking within polymer networks. Collectively, these results indicate the ability to develop next-generation composite materials with enhanced mechanical and thermal properties by increasing the average crosslinking density using nanoparticles.

A novel 2.5D culture platform to investigate the role of stiffness gradients on adhesion-independent cell migration.

Current studies investigating the role of biophysical cues on cell migration focus on the use of culture platforms with static material parameters. However, migrating cells in vivo often encounter spatial variations in extracellular matrix stiffness. To better understand the effects of stiffness gradients on cell migration, we developed a 2.5D cell culture platform where cells are sandwiched between stiff tissue culture plastic and soft alginate hydrogel. Under these conditions, we observed migration of cells from the underlying stiff substrate into the alginate matrix. Observation of migration into alginate in the presence of integrin inhibition as well as qualitative microscopic analyses suggested an adhesion-independent cell migration mode. Observed migration was dependent on alginate matrix stiffness and the RhoA-ROCK-myosin-II pathway; inhibitors specifically targeting ROCK and myosin-II arrested cell migration. Collectively, these results demonstrate the utility of the 2.5D culture platform to advance our understanding of the effects of stiffness gradients and mechanotransductive signaling on adhesion-independent cell migration.

Function, structure, and stability of enzymes confined in agarose gels.

Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.