Dynamic assembly of ultrasoft colloidal networks enables cell invasion within restrictive fibrillar polymers.

In regenerative medicine, natural protein-based polymers offer enhanced endogenous bioactivity and potential for seamless integration with tissue, yet form weak hydrogels that lack the physical robustness required for surgical manipulation, making them difficult to apply in practice. The use of higher concentrations of protein, exogenous cross-linkers, and blending synthetic polymers has all been applied to form more mechanically robust networks. Each relies on generating a smaller network mesh size, which increases the elastic modulus and robustness, but critically inhibits cell spreading and migration, hampering tissue regeneration. Here we report two unique observations; first, that colloidal suspensions, at sufficiently high volume fraction (ϕ), dynamically assemble into a fully percolated 3D network within high-concentration protein polymers. Second, cells appear capable of leveraging these unique domains for highly efficient cell migration throughout the composite construct. In contrast to porogens, the particles in our system remain embedded within the bulk polymer, creating a network of particle-filled tunnels. Whereas this would normally physically restrict cell motility, when the particulate network is created using ultralow cross-linked microgels, the colloidal suspension displays viscous behavior on the same timescale as cell spreading and migration and thus enables efficient cell infiltration of the construct through the colloidal-filled tunnels.

Nanogels and Microgels: From Model Colloids to Applications, Recent Developments, and Future Trends.

Nanogels and microgels are soft, deformable, and penetrable objects with an internal gel-like structure that is swollen by the dispersing solvent. Their softness and the potential to respond to external stimuli like temperature, pressure, pH, ionic strength, and different analytes make them interesting as soft model systems in fundamental research as well as for a broad range of applications, in particular in the field of biological applications. Recent tremendous developments in their synthesis open access to systems with complex architectures and compositions allowing for tailoring microgels with specific properties. At the same time state-of-the-art theoretical and simulation approaches offer deeper understanding of the behavior and structure of nano- and microgels under external influences and confinement at interfaces or at high volume fractions. Developments in the experimental analysis of nano- and microgels have become particularly important for structural investigations covering a broad range of length scales relevant to the internal structure, the overall size and shape, and interparticle interactions in concentrated samples. Here we provide an overview of the state-of-the-art, recent developments as well as emerging trends in the field of nano- and microgels. The following aspects build the focus of our discussion: tailoring (multi)functionality through synthesis; the role in biological and biomedical applications; the structure and properties as a model system, e.g., for densely packed arrangements in bulk and at interfaces; as well as the theory and computer simulation.

The role of ions in the self-healing behavior of soft particle suspensions.

Impurities in crystals generally cause point defects and can even suppress crystallization. This general rule, however, does not apply to colloidal crystals formed by soft microgel particles [Iyer ASJ, Lyon LA (2009) Angew Chem Int Ed 48:4562-4566], as, in this case, the larger particles are able to shrink and join the crystal formed by a majority of smaller particles. Using small-angle X-ray scattering, we find the limit in large-particle concentration for this spontaneous deswelling to persist. We rationalize our data in the context of those counterions that are bound to the microgel particles as a result of the electrostatic attraction exerted by the fixed charges residing on the particle periphery. These bound counterions do not contribute to the suspension osmotic pressure in dilute conditions, as they can be seen as internal degrees of freedom associated with each microgel particle. In contrast, at sufficiently high particle concentrations, the counterion cloud of each particle overlaps with that of its neighbors, allowing these ions to freely explore the space outside the particles. We confirm this scenario by directly measuring the osmotic pressure of the suspension. Because these counterions are then no longer bound, they create an osmotic pressure difference between the inside and outside of the microgels, which, if larger than the microgel bulk modulus, can cause deswelling, explaining why large, soft microgel particles feel the squeeze when suspended with a majority of smaller particles. We perform small-angle neutron scattering measurements to further confirm this remarkable behavior.

Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach.

The mechanism of action of the widely used in vivo ferric chloride (FeCl3) thrombosis model remains poorly understood; although endothelial cell denudation is historically cited, a recent study refutes this and implicates a role for erythrocytes. Given the complexity of the in vivo environment, an in vitro reductionist approach is required to systematically isolate and analyze the biochemical, mass transfer, and biological phenomena that govern the system. To this end, we designed an "endothelial-ized" microfluidic device to introduce controlled FeCl3 concentrations to the molecular and cellular components of blood and vasculature. FeCl3 induces aggregation of all plasma proteins and blood cells, independent of endothelial cells, by colloidal chemistry principles: initial aggregation is due to binding of negatively charged blood components to positively charged iron, independent of biological receptor/ligand interactions. Full occlusion of the microchannel proceeds by conventional pathways, and can be attenuated by antithrombotic agents and loss-of-function proteins (as in IL4-R/Iba mice). As elevated FeCl3 concentrations overcome protective effects, the overlap between charge-based aggregation and clotting is a function of mass transfer. Our physiologically relevant in vitro system allows us to discern the multifaceted mechanism of FeCl3-induced thrombosis, thereby reconciling literature findings and cautioning researchers in using the FeCl3 model.

An oil-in-water nanoemulsion enhances immunogenicity of H5N1 vaccine in mice.

To enhance the immunogenicity of the Influenza H5N1 vaccine, we developed an oil-in-water nanoemulsion (NE) adjuvant. NE displayed good temperature stability and maintained particle size. More importantly, it significantly enhanced IL-6 and MCP-1 production to recruit innate cells, including neutrophils, monocytes/macrophages and dendritic cells to the local environment. Furthermore, NE enhanced dendritic cell function to induce robust antigen-specific T and B cell immune responses. NE-adjuvanted H5N1 vaccine not only elicited significantly higher and long-lasting antibody responses, but also conferred enhanced protection against homologous clade 1 as well as heterologous clade 2 H5N1 virus challenge in young as well as in aged mice. The pre-existing immunity to seasonal influenza did not affect the immunogenicity of NE-adjuvanted H5N1 vaccine.

Ultrasoft microgels displaying emergent platelet-like behaviours.

Efforts to create platelet-like structures for the augmentation of haemostasis have focused solely on recapitulating aspects of platelet adhesion; more complex platelet behaviours such as clot contraction are assumed to be inaccessible to synthetic systems. Here, we report the creation of fully synthetic platelet-like particles (PLPs) that augment clotting in vitro under physiological flow conditions and achieve wound-triggered haemostasis and decreased bleeding times in vivo in a traumatic injury model. PLPs were synthesized by combining highly deformable microgel particles with molecular-recognition motifs identified through directed evolution. In vitro and in silico analyses demonstrate that PLPs actively collapse fibrin networks, an emergent behaviour that mimics in vivo clot contraction. Mechanistically, clot collapse is intimately linked to the unique deformability and affinity of PLPs for fibrin fibres, as evidenced by dissipative particle dynamics simulations. Our findings should inform the future design of a broader class of dynamic, biosynthetic composite materials.

Microgel mechanics in biomaterial design.

The field of polymeric biomaterials has received much attention in recent years due to its potential for enhancing the biocompatibility of systems and devices applied to drug delivery and tissue engineering. Such applications continually push the definition of biocompatibility from relatively straightforward issues such as cytotoxicity to significantly more complex processes such as reducing foreign body responses or even promoting/recapitulating natural body functions. Hydrogels and their colloidal analogues, microgels, have been and continue to be heavily investigated as viable materials for biological applications because they offer numerous, facile avenues in tailoring chemical and physical properties to approach biologically harmonious integration. Mechanical properties in particular are recently coming into focus as an important manner in which biological responses can be altered. In this Account, we trace how mechanical properties of microgels have moved into the spotlight of research efforts with the realization of their potential impact in biologically integrative systems. We discuss early experiments in our lab and in others focused on synthetic modulation of particle structure at a rudimentary level for fundamental drug delivery studies. These experiments elucidated that microgel mechanics are a consequence of polymer network distribution, which can be controlled by chemical composition or particle architecture. The degree of deformability designed into the microgel allows for a defined response to an imposed external force. We have studied deformation in packed colloidal phases and in translocation events through confined pores; in all circumstances, microgels exhibit impressive deformability in response to their environmental constraints. Microgels further translate their mechanical properties when assembled in films to the properties of the bulk material. In particular, microgel films have been a large focus in our lab as building blocks for self-healing materials. We have shown that their ability to heal after damage arises from polymer mobility during hydration. Furthermore, we have shown film mobility dictates cell adhesion and spreading in a manner that is fundamentally different from previous work on mechanotransduction. In total, we hope that this Account presents a broad introduction to microgel research that intersects polymer chemistry, physics, and regenerative medicine. We expect that research intersection will continue to expand as we fill the knowledge gaps associated with soft materials in biological milieu.

Ultrasoft, highly deformable microgels.

Microgels are colloidally stable, hydrogel microparticles that have previously been used in a range of (soft) material applications due to their tunable mechanical and chemical properties. Most commonly, thermo and pH-responsive poly(N-isopropylacrylamide) (pNIPAm) microgels can be fabricated by precipitation polymerization in the presence of the co-monomer acrylic acid (AAc). Traditionally pNIPAm microgels are synthesized in the presence of a crosslinking agent, such as N,N'-methylenebisacrylamide (BIS), however, microgels can also be synthesized under 'crosslinker free' conditions. The resulting particles have extremely low (<0.5%), core-localized crosslinking resulting from rare chain transfer reactions. AFM nanoindentation of these ultralow crosslinked (ULC) particles indicate that they are soft relative to crosslinked microgels, with a Young's modulus of ∼10 kPa. Furthermore, ULC microgels are highly deformable as indicated by a high degree of spreading on glass surfaces and the ability to translocate through nanopores significantly smaller than the hydrodynamic diameter of the particles. The size and charge of ULCs can be easily modulated by altering reaction conditions, such as temperature, monomer, surfactant and initiator concentrations, and through the addition of co-monomers. Microgels based on the widely utilized, biocompatible polymer polyethylene glycol (PEG) can also be synthesized under crosslinker free conditions. Due to their softness and deformability, ULC microgels are a unique base material for a wide variety of biomedical applications including biomaterials for drug delivery and regenerative medicine.

Tunable swelling and rolling of microgel membranes.

The tunable swelling and rolling of films assembled via layer-by-layer (LbL) methods from poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) microgels and poly(ethylenimine) (PEI) have been systematically studied. Microgel/PEI films assembled at pH 7.4 display a high degree of in-plane swelling at low pH that dramatically increases the film area and drives self-delamination from the substrate to form a free-standing film. The degree of film swelling can be controlled by the size of microgels used in film fabrication. Taking advantage of this feature, self-rolled scrolls can be easily obtained from microgel/PEI films prepared from microgels of two different sizes. The rolling direction can be controlled by the assembly of different size microgels in different film strata, and the final shape of the scrolls can be controlled by scratching the desired film edges. The present work contributes to a deeper understanding of microgel/PEI film swelling properties and introduces a facile and novel method to prepare free-standing films and self-rolled scrolls.

Dynamic materials from microgel multilayers.

Multilayer coatings made from hydrogel microparticles (microgels) are conceptually very simple materials: thin films composed of microgel building blocks held together by polyelectrolyte "glue". However, the apparent simplicity of their fabrication and structure belies extremely complex properties, including those of "dynamic" coatings that display rapid self-healing behavior in the presence of solvent. This contribution covers our work with these materials and highlights some of the key findings regarding damage mechanisms, healing processes, film structure/composition, and how the variation of fabrication parameters can impact self-healing behavior.

Microgel film dynamics modulate cell adhesion behavior.

A material's mechanical properties greatly control cell behavior at the cell­substrate interface. In this work, we demonstrate that microgel multilayers have unique elastic and viscoelastic-like properties that can be modulated to produce morphological changes in fibroblasts cultured on the film. Protein adsorption is also examined and the data are contrasted with the number of cells adhered. The dynamic interaction of cell and substrate is only partially explained by conventional understanding of surface­receptor interactions and substrate elasticity. Viscoelasticity, a mechanical property not often considered, plays a significant role at cellular length and time scales for microgel films.

Ultrasoft platelet-like particles stop bleeding in rodent and porcine models of trauma.

Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly (N-isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.

Multifunctional nanogels for siRNA delivery.

The application of RNA interference to treat disease is an important yet challenging concept in modern medicine. In particular, small interfering RNA (siRNA) have shown tremendous promise in the treatment of cancer. However, siRNA show poor pharmacological properties, which presents a major hurdle for effective disease treatment especially through intravenous delivery routes. In response to these shortcomings, a variety of nanoparticle carriers have emerged, which are designed to encapsulate, protect, and transport siRNA into diseased cells. To be effective as carrier vehicles, nanoparticles must overcome a series of biological hurdles throughout the course of delivery. As a result, one promising approach to siRNA carriers is dynamic, versatile nanoparticles that can perform several in vivo functions. Over the last several years, our research group has investigated hydrogel nanoparticles (nanogels) as candidate delivery vehicles for therapeutics, including siRNA. Throughout the course of our research, we have developed higher order architectures composed entirely of hydrogel components, where several different hydrogel chemistries may be isolated in unique compartments of a single construct. In this Account, we summarize a subset of our experiences in the design and application of nanogels in the context of drug delivery, summarizing the relevant characteristics for these materials as delivery vehicles for siRNA. Through the layering of multiple, orthogonal chemistries in a nanogel structure, we can impart multiple functions to the materials. We consider nanogels as a platform technology, where each functional element of the particle may be independently tuned to optimize the particle for the desired application. For instance, we can modify the shell compartment of a vehicle for cell-specific targeting or evasion of the innate immune system, whereas other compartments may incorporate fluorescent probes or regulate the encapsulation and release of macromolecular therapeutics. Proof-of-principle experiments have demonstrated the utility of multifunctional nanogels. For example, using a simple core/shell nanogel architecture, we have recently reported the delivery of siRNA to chemosensitize drug resistant ovarian cancer cells. Ongoing efforts have resulted in several advanced hydrogel structures, including biodegradable nanogels and multicompartment spheres. In parallel, our research group has studied other properties of the nanogels, including their behavior in confined environments and their ability to translocate through small pores.

The polymer/colloid duality of microgel suspensions.

Colloidal dispersions have been studied for decades as a result of their utility in numerous applications and as models for molecular and atomic condensed phases. More recently, a number of groups have exploited in such studies submicrometer-sized hydrogel particles (microgels) that have environmentally tunable sizes. The experimental convenience of tuning the dispersion's colloidal volume fraction while maintaining a constant number density of particles provides a clear advantage over more tedious studies that employ traditional hard-sphere particles. However, as studies delved deeper into the fundamental physics of colloidal dispersions comprising microgel particles, it became abundantly clear that a microgel's utility as a tunable hard sphere was limited and that the impact of softness was more profound than previously appreciated. Herein we review the brief history of microgel-based colloidal dispersions and discuss their transition from tunable hard spheres to a class of soft matter that has revealed a landscape of physics and chemistry notable for its extraordinary richness and diversity.

Modulation of the deswelling temperature of thermoresponsive microgel films.

We demonstrate fine-tuning of the deswelling temperatures of thermoresponsive microgels within a biologically relevant range (30-40 °C). This was achieved by copolymerizing N-isopropylacrylamide and N-isopropylmethacrylamide (NIPAm and NIPMAm, respectively) in varying ratios; the parent homopolymers are well-known thermoresponsive polymers. Polyelectrolyte layer-by-layer (LbL) assemblies of these microgels retain the temperature response properties as demonstrated by temperature-dependent light scattering. Furthermore, films composed of more than one type of microgel building block were shown to have multiple temperature responses similar to those observed for the individual building blocks, permitting further tailoring of the temperature responsive interface. Additional experiments with mixed composition films, investigating multiple assembly processes, show that the location of the microgels within the film does not interfere with the temperature response. This suggests that microgels within the polyelectrolyte assembly behave independently of neighboring microgels with respect to their thermally induced deswelling.

Control of poly(N-isopropylacrylamide) microgel network structure by precipitation polymerization near the lower critical solution temperature.

Poly(N-isopropylacrylamide) (pNIPAm) microgels were synthesized by precipitation polymerization at temperatures ranging from 37 to 45 °C using redox initiator system ammonium persulfate (APS)/N,N,N',N'-tetramethylethylenediamine (TEMED) or photoinitiator 2,2'-azobis(amidinopropane) dihydrochloride (V50). Photon correlation spectroscopy (PCS) and atomic force microscopy (AFM) studies revealed that spherical microgels with narrow size dispersities can be obtained with these methods and that the resultant microgels have volume phase transition behaviors expected from their compositions. Additionally, the low-temperature redox initiator strategy produces microgels devoid of self-cross-linking, thereby permitting the synthesis of completely degradable microgels when using N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEA) as a cleavable cross-linker. We also demonstrate the potential utility of the approach in bioconjugate syntheses; in this case, avidin immobilization is demonstrated by one-pot copolymerization at low temperature.

One-pot synthesis of microcapsules with nanoscale inclusions.

Temperature responsive poly(N-isopropylmethacrylamide) (pNIPMAm) microgel capsules around 1 µm containing multiple poly(N-isopropylacrylamide) (pNIPAm) nanoinclusions were prepared. This structure was achieved through the addition of a cross-linked pNIPMAm shell to stable, monodispersed aggregates of pNIPAm chains. This one-pot synthetic approach resulted in core/shell microgels at high temperature wherein only the shell (pNIPMAm) component contained stable, covalent cross-links between chains. Thus, upon decreasing the temperature following synthesis, the majority of the encapsulated pNIPAm chains escaped from the shell, resulting in nearly hollow microcapsules. Remnant pNIPAm segments in the microcapsule then form nanoparticulate inclusions upon raising the temperature.

Multicompartment core/shell microgels.

Multiresponsive poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-AAc) microgels containing mechanically and thermodynamically decoupled poly(N-isopropylmethacrylamide) (pNIPMAm) cores have been prepared. To achieve this structure, pNIPMAm microgels were used as templates in the synthesis of an N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEA) cross-linked pNIPMAm inner shell. A pNIPAm-AAc outer shell was then added, resulting in "core/double-shell" microgels. Erosion of the inner shell via periodate-mediated cleavage of the 1,2-diol bond in DHEA produced multiresponsive core/shell microgels with independent cores. The striking structural changes and unique multiresponsivity achieved in microgels prepared via this approach illustrate the potential of multifunctional, multicomponent delivery vehicles that do not suffer from antagonistic interferences arising when different functional components are introduced within a single particle.

Monitoring the erosion of hydrolytically-degradable nanogels via multiangle light scattering coupled to asymmetrical flow field-flow fractionation.

We describe the synthesis and characterization of degradable nanogels that display bulk erosion under physiologic conditions (pH = 7.4, 37 degrees C). Erodible poly(N-isopropylmethacrylamide) nanogels were synthesized by copolymerization with N,O-(dimethacryloyl) hydroxylamine, a cross-linker previously used in the preparation of nontoxic and biodegradable bulk hydrogels. To monitor particle degradation, we employed multiangle light scattering and differential refractometry detection following asymmetrical flow field-flow fractionation. This approach allowed the detection of changes in nanogel molar mass and topology as a function of both temperature and pH. Particle erosion was evident from both an increase in nanogel swelling and a decrease in scattering intensity as a function of time. Following these analyses, the samples were recovered for subsequent characterization by direct particle tracking, which yields hydrodynamic size measurements and enables number density determination. Additionally, we confirmed the conservation of nanogel stimuli-responsivity through turbidity measurements. Thus, we have demonstrated the synthesis of degradable nanogels that erode under conditions and on time scales that are relevant for many drug delivery applications. The combined separation and light scattering detection method is demonstrated to be a versatile means to monitor erosion and should also find applicability in the characterization of other degradable particle constructs.

Chemosensitization of cancer cells by siRNA using targeted nanogel delivery.

BACKGROUND: Chemoresistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity to chemotherapeutic agents have the potential to increase drug efficacy while reducing toxic effects on untargeted cells. Targeted cancer therapy by RNA interference (RNAi) is a relatively new approach that can be used to reversibly silence genes in vivo by selectively targeting genes such as the epidermal growth factor receptor (EGFR), which has been shown to increase the sensitivity of cancer cells to taxane chemotherapy. However, delivery represents the main hurdle for the broad development of RNAi therapeutics. METHODS: We report here the use of core/shell hydrogel nanoparticles (nanogels) functionalized with peptides that specially target the EphA2 receptor to deliver small interfering RNAs (siRNAs) targeting EGFR. Expression of EGFR was determined by immunoblotting, and the effect of decreased EGFR expression on chemosensitization of ovarian cancer cells after siRNA delivery was investigated. RESULTS: Treatment of EphA2 positive Hey cells with siRNA-loaded, peptide-targeted nanogels decreased EGFR expression levels and significantly increased the sensitivity of this cell line to docetaxel (P < 0.05). Nanogel treatment of SK-OV-3 cells, which are negative for EphA2 expression, failed to reduce EGFR levels and did not increase docetaxel sensitivity (P > 0.05). CONCLUSION: This study suggests that targeted delivery of siRNAs by nanogels may be a promising strategy to increase the efficacy of chemotherapy drugs for the treatment of ovarian cancer. In addition, EphA2 is a viable target for therapeutic delivery, and the siRNAs are effectively protected by the nanogel carrier, overcoming the poor stability and uptake that has hindered clinical advancement of therapeutic siRNAs.