L. Andrew Lyon

Peptide-functionalized nanogels for targeted siRNA delivery

A major bottleneck in the development of siRNA therapies is their delivery to the desired cell type or tissue, followed by effective passage across the cell membrane with subsequent silencing of the targeted mRNA. To address this problem, we describe the synthesis of core/shell hydrogel nanoparticles (nanogels) with surface-localized peptides that specifically target ovarian carcinoma cell lines possessing high expression levels of the Eph2A receptor. These nanogels are also demonstrated to be highly effective in the noncovalent encapsulation of siRNA and enable cell-specific delivery of the oligonucleotides in serum-containing medium. Cell toxicity and viability assays reveal that the nanogel construct is nontoxic under the conditions studied, as no toxicity or decrease in cell proliferation is observed following delivery. Importantly, a preliminary investigation of gene silencing illustrates that nanogel-mediated delivery of siRNA targeted to the EGF receptor results in knockdown of that receptor. Excellent protection of siRNA during endosomal uptake and endosomal escape of the nanogels is suggested by these results since siRNA activity in the cytosol is required for gene silencing.

Autonomic Self-Healing of Hydrogel Thin Films†

Self-healing materials have the ability repair themselves following damage. Over the past few decades, there has been a growing interest in materials that can self-heal, as this property can increase a material s lifetime, reduce replacement costs, and improve product safety. Self-healing systems can be made from a variety of materials, but polymers have been extensively explored because of their chemical and mechanical tunability, and the ability to create dynamic materials. Although the vast majority of these previous studies have explored healing processes in robust polymeric structures such as epoxy coatings and elastomers, more delicate architectures such as hydrogel thin films have not yet been studied as self-healing materials that can heal induced mechanical damage. Herein, we report autonomic self-healing polymeric thin films assembled from colloidal hydrogel building blocks. By employing a layer-by-layer polyelectrolyte approach in the fabrication, we have developed a material, which, upon exposure to water, undergoes rapid (on a timescale of seconds) healing of micrometer-sized defects that span the entire coated area (1 cm), with no apparent remnant damage even at submicron length scales. The self-healing properties displayed by these coatings enable the use of hydrated polymer films in applications where rough (e.g., surgical) handling and transient damage are inevitable, such as in biomedical implants. Most materials do not have the inherent ability to heal themselves, typically because their building blocks are organized into rigid architectures and therefore cannot migrate across defects that are longer than the molecular length scale, or because the molecular components are not chemically labile enough to reform bonds after rupture. In fact, most materials suffer from both these problems. However, several research groups have developed approaches to solve these issues. Materials that undergo reversible reactions between functional groups or weak interactions within the polymer matrix can successfully be mended following the introduction of a defect. 9] This approach is limited to particular chemical reactions and often the residual “dangling chains” will interact with other chains on a single side of the gap, as opposed to cross-gap interaction, thus preventing healing if the material is not mechanically reconnected soon after cutting. Another approach involves heating the polymer above its glass transition temperature (Tg), thereby increasing the mobility of the chains and causing rearrangement and molecular interdiffusion to promote “crack healing”. The obvious limitation to this approach is the need for the external application of heat, therefore truly autonomous healing is not possible. Other demonstrations involve filling a void by the release of healing agents or inhibitors into cracks, 13, 14] or by an induced phase separation of nanoparticles towards damaged areas. However, only limited numbers of the embedded reservoirs or nanoparticles are incorporated, and therefore it is unlikely that such materials could continue to heal after recurrent damage in the same area. Herein we describe a “self-healing” hydrogel film that can withstand repeated deformation and quickly recover its original structure when solvated with water. Hydrogels (cross-linked polymeric networks swollen with water) have been a topic of growing interest over the past twenty years because of their unique properties and the wide variety of applications in which they can be exploited. The films described below are fabricated by layer-by-layer (LbL) polyelectrolyte self-assembly, which has been demonstrated for a vast variety of materials, 21] thus offering universal utility to the method. In our approach, we employ spherical, sub-micrometer-sized hydrogel particles (microgels) as the main building block in the LbL assembly procedure to fabricate continuous, multilayered hydrogel films. We have previously demonstrated the use of microgels to fabricate 2D and 3D arrays on solid substrates by LbL assembly. In these earlier studies, it became apparent that a cationic linear polymer was able to penetrate the microgels and strongly cross-link the anionic acidic side chains within the microgels. Subsequent addition of another microgel layer resulted in a 3D coulombically cross-linked hydrogel network. These investigations led to an understanding of how linear polyelectrolytes can render individual microgels “sticky”, and also how the interplay of both strong and weak interactions impact the assembly and swelling properties of such materials. During the course of our previous studies, observations were made that suggested dynamic microgel reorganization within the films. Although these observations were not quantitatively explored at that time, they suggested the potential for defect healing properties. Thus, to more precisely investigate the response of microgel multilayers to controlled damage, the films were deposited on an elastomeric substrate, poly(dimethylsiloxane) (PDMS), which allowed for the controlled mechanical manipulation of the substrate and its associated microgel coating. Using a chemical treatment of PDMS that renders the surface of the [*] A. B. South, Prof. L. A. Lyon School of Chemistry and Biochemistry and Petit Institute for Bioengineering and Bioscience Georgia Institute of Technology Atlanta, GA 30332-0400 (USA) Fax: (+ 1)404-894-4090 E-mail: lyon@gatech.edu

Bioresponsive hydrogels for sensing applications

This Highlight presents some of the recent efforts in the design of bioresponsive hydrogels, and their application to biosensing. These efforts extend philosophically from early work on glucose responsive gels, with current studies being focused both on gel responsivity and transduction of that response such that true sensor applications can be realized. The future outlook for the field is also discussed.

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.

Chemosensitization of Cancer Cells by siRNA Using Targeted Nanogel Delivery

BackgroundChemoresistance 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.MethodsWe 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.ResultsTreatment 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).ConclusionThis 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.

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 dispersions 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 microgels 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.

Reduced Acute Inflammatory Responses to Microgel Conformal Coatings

Implantation of synthetic materials into the body elicits inflammatory host responses that limit medical device integration and biological performance. This inflammatory cascade involves protein adsorption, leukocyte recruitment and activation, cytokine release, and fibrous encapsulation of the implant. We present a coating strategy based on thin films of poly(N-isopropylacrylamide) hydrogel microparticles (i.e. microgels) cross-linked with poly(ethylene glycol) diacrylate. These particles were grafted onto a clinically relevant polymeric material to generate conformal coatings that significantly reduced in vitro fibrinogen adsorption and primary human monocyte/macrophage adhesion and spreading. These microgel coatings also reduced leukocyte adhesion and expression of pro-inflammatory cytokines (TNF-alpha, IL-1beta, MCP-1) in response to materials implanted acutely in the murine intraperitoneal space. These microgel coatings can be applied to biomedical implants as a protective coating to attenuate biofouling, leukocyte adhesion and activation, and adverse host responses for biomedical and biotechnological applications.

Self‐Healing Colloidal Crystals

Healing hands: A complex interplay between colloidal and polymeric energetics in microgel self-assembly behavior results in soft colloidal assemblies with self-healing properties. Repulsive soft spheres can adopt highly compressed conformations in colloidal crystalline lattices without directly contacting the nearest neighbors (see picture). This distant action is directly responsible for the self-healing of the assemblies.

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 Translocation through Pores under Confinement

In applications utilizing synthetic biomaterials, such as drug delivery, bioimaging, and tissue engineering, the material mechanical properties represent an important set of design parameters. Most studies of mechanical properties in biomaterials have focused on how cells interact with or move on surfaces of different rigidity in the context of mechanotransduction 10,11] and cell proliferation or differentiation. However, few studies have investigated the effects of the mechanical softness of nanoparticles in nanoor microbiological environments. It has been suggested, however, that the softness of nanoparticles may be relevant in processes such as phagocytosis or endocytosis. 14] This concept implies that cells are not only affected by the mechanics of large surfaces or interfaces but also by the rigidity of individual nanoparticles. The in vivo performance of nanoparticles is strongly dependent on a variety of biological processes, including lymphatic drainage, endocytosis, extravasation, and kidney filtration. It stands to reason that any process that has a rigid size dependence may also be dependent on the mechanical flexibility of the biomaterial. Therefore, it is necessary to consider mechanics when outlining the nanoparticle size restrictions relevant for certain processes. This aspect might especially be important when the process involves passage through small, well-defined pores, such as in renal filtration. Renal or glomerular filtration is one of two routes of clearance of biomaterials from the body for particles smaller than 500 nm. 16] The other clearance route is biliary clearance through the liver; however, in nanomedicine applications biliary clearance is generally bypassed owing to the small particle sizes typically used. Therefore, renal clearance is a desired mechanism of nanoparticle excretion. This mechanism requires passage through approximately 8 nm diameter pores (as defined by endothelial gaps) under a pressure differential of 40 to 80 Torr (0.7 to 1.5 psi). Obviously, for most carrier systems these figures of merit are not easily met and require the integration of degradability into the nanoparticle design or rigorous control over small particle sizes. In some cases, these modifications may negatively alter drug loading and release, circulation times, cell uptake, and cytotoxicity. Therefore, it may be desirable to develop a carrier system that has the ability to be excreted without additional design complexity. For a hard-sphere system, such as quantum dots, this stipulation implies a strict particle size limit, which may negatively impact payload or may result in clearance through lymphatic drainage. However, soft conformable nanoparticles that are able to deliver a large cargo yet are flexible enough to fit through small pores are a potentially attractive alternative. One example of such a construct is that of hydrogel colloids (i.e. nanogels or microgels), which are nanoparticles that can be dramatically compressed, owing to their significant network flexibility. Herein we describe the first demonstration of microgel translocation through cylindrical pores under pressure differentials relevant to renal filtration. We observe that microgel particles easily pass through such pores, even when the opening is more than tenfold smaller than the unperturbed microgel diameter. For this study, track-etch membranes were used as the model for pores in the renal system. As shown in Scheme 1, track-etch membranes were placed into gasket-