Anirudha Singh

The independent roles of mechanical, structural and adhesion characteristics of 3D hydrogels on the regulation of cancer invasion and dissemination

Metastasis begins with the escape, or dissemination, of cancer cells from the primary tumor. We recently demonstrated that tumors preferentially disseminate into collagen I and not into basement membrane protein gels (Matrigel). In this study, we used synthetic polymer systems to define material properties that could induce dissemination into Matrigel. We first specifically varied rigidity by varying the crosslinking density of poly(ethylene glycol) (PEG) networks within Matrigel scaffolds. Increased microenvironmental rigidity limited epithelial growth but did not promote dissemination. We next incorporated adhesive signals into the PEG network using peptide-conjugated cyclodextrin (α-CDYRGDS) rings. The α-CDYRGDS rings threaded along the PEG polymers, enabling independent control of matrix mechanics, adhesive peptide composition, and adhesive density. Adhesive PEG networks induced dissemination of normal and malignant mammary epithelial cells at intermediate values of adhesion and rigidity. Our data reveal that microenvironmental signals can induce dissemination of normal and malignant epithelial cells without requiring the fibrillar structure of collagen I or containing collagen I-specific adhesion sequences. Finally, the nanobiomaterials and assays developed in this study are generally useful both in 3D culture of primary mammalian tissues and in the systematic evaluation of the specific role of mechanical and adhesive inputs on 3D tumor growth, invasion, and dissemination.

Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid

Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and coat medical devices.

Characterization of linear and branched polyacrylates by tandem mass spectrometry.

AbstractThe unimolecular degradation of alkali-metal cationized polyacrylates with the repeat unit CH2CH(COOR) and a variety of ester pendants has been examined by tandem mass spectrometry. The fragmentation patterns resulting from collisionally activated dissociation depend sensitively on the size of the ester alkyl substituent (R). With small alkyl groups, as in poly(methyl acrylate), lithiated or sodiated oligomers (M) decompose via free-radical chemistry, initiated by random homolytic C-C bond cleavages along the polymer chain. The radical ions formed this way dissociate further by backbiting rearrangements and β scissions to yield a distribution of terminal fragments with one of the original end groups and internal fragments with 2–3 repeat units. If the ester alkyl group bears three or more carbon atoms, cleavages within the ester moieties become the predominant decomposition channel. This distinct reactivity is observed if R = t-butyl, n-butyl, or the mesogenic group (CH2)11-O-C6H4-C6H4-CN. The [M+alkali metal]+ ions of the latter polyacrylates dissociate largely by charge-remote 1,5-H rearrangements that convert COOR to COOH groups by expulsion of 1-alkenes. The acid groups may displace an alcohol unit from a neighboring ester pendant to form a cyclic anhydride, unless hindered by steric effects. Using atom transfer radical polymerization, hyperbranched polyacrylates were prepared carrying ester groups both within and between the branches. Unique alkenes and alcohols are cleaved from ester groups at the branching points, enabling determination of the branching architecture. FigureMALDI-CAD tandem mass spectrum of the lithiated 4-mer from a hyperbranched polyacrylate. The fragments marked by green stars diagnose the branched architecture shown on top of the spectrum. The fragments marked by violet stars diagnose a different isomer.

PEG hydrogel degradation and the role of the surrounding tissue environment.

Poly(ethylene glycol) (PEG)‐based hydrogels are extensively used in a variety of biomedical applications, due to ease of synthesis and tissue‐like properties. Recently there have been varied reports regarding PEG hydrogels degradation kinetics and in vivo host response. In particular, these studies suggest that the surrounding tissue environment could play a critical role in defining the inflammatory response and degradation kinetics of PEG implants. In the present study we demonstrated a potential mechanism of PEG hydrogel degradation, and in addition we show potential evidence of the role of the surrounding tissue environment on producing variable inflammatory responses. Copyright

Regulating synthetic gene networks in 3D materials

Combining synthetic biology and materials science will enable more advanced studies of cellular regulatory processes, in addition to facilitating therapeutic applications of engineered gene networks. One approach is to couple genetic inducers into biomaterials, thereby generating 3D microenvironments that are capable of controlling intrinsic and extrinsic cellular events. Here, we have engineered biomaterials to present the genetic inducer, IPTG, with different modes of activating genetic circuits in vitro and in vivo. Gene circuits were activated in materials with IPTG embedded within the scaffold walls or chemically linked to the matrix. In addition, systemic applications of IPTG were used to induce genetic circuits in cells encapsulated into materials and implanted in vivo. The flexibility of modifying biomaterials with genetic inducers allows for patterned placement of these inducers that can be used to generate distinct patterns of gene expression. Together, these genetically interactive materials can be used to characterize genetic circuits in environments that more closely mimic cells’ natural 3D settings, to better explore complex cell–matrix and cell–cell interactions, and to facilitate therapeutic applications of synthetic biology.

Multifunctional aliphatic polyester nanofibers for tissue engineering

Electrospun fibers based on aliphatic polyesters, such as poly(ε-caprolactone) (PCL), have been widely used in regenerative medicine and drug delivery applications due to their biocompatibility, low cost and ease of fabrication. However, these aliphatic polyester fibers are hydrophobic in nature, resulting in poor wettability, and they lack functional groups for decorating the scaffold with chemical and biological cues. Current strategies employed to overcome these challenges include coating and blending the fibers with bioactive components or chemically modifying the fibers with plasma treatment and reactants. In the present study, we report on designing multifunctional electrospun nanofibers based on the inclusion complex of PCL-α-cyclodextrin (PCL-α-CD), which provides both structural support and multiple functionalities for further conjugation of bioactive components. This strategy is independent of any chemical modification of the PCL main chain, and electrospinning of PCL-α-CD is as easy as electrospinning PCL. Here, we describe synthesis of the PCL-α-CD electrospun nanofibers, elucidate composition and structure, and demonstrate the utility of functional groups on the fibers by conjugating a fluorescent small molecule and a polymeric-nanobead to the nanofibers. Furthermore, we demonstrate the application of PCL-α-CD nanofibers for promoting osteogenic differentiation of human adipose-derived stem cells (hADSCs), which induced a higher level of expression of osteogenic markers and enhanced production of extracellular matrix (ECM) proteins or molecules compared with control PCL fibers.

A hyaluronic acid-binding contact lens with enhanced water retention

PURPOSE As a main component of an artificial tear or eyedrop, hyaluronic acid (HA) prolongs water retention, slows tear removal, improves tear film stability, reduces protein adsorption at the ocular surface and permits uninterrupted blinking. Here, we hypothesized that the contact lens modified with an HA-binding peptide (HABpep) could locally bind and concentrate exogenous HA present in eyedrops to the modified contact lens surface, which exhibited superior water retention. METHODS To bind HA, a contact lens surface was covalently modified by HABpep with and without a poly(ethylene glycol) (PEG) spacer. Bound HA and its retention over time on the modified surfaces were evaluated by fluorescence measurements. A comparative water evaporation study was performed to determine water retention in an HA-bound contact lens. RESULTS Fluorescence studies showed that the contact lens was successfully modified by HABpep with or without a PEG spacer, and HA bound to the contact lens surface. Furthermore, the bound HA via HABpep significantly reduced water loss from the modified contact lens. CONCLUSION HABpep strategies that locally bind and concentrate HA to create a thin coating of a therapeutic molecule on surfaces could provide physical and biological benefits to treat ocular surface dysfunction. The surface bound HA via HABpep enhanced water retention in the modified contact lens.

Physically cross-linked pH-responsive hydrogels with tunable formulations for controlled drug delivery

A variety of pH responsive hydrogels possessing macroporous interiors resembling a honey-comb framework with a continuous skin on the surface have been developed by free radical aqueous copolymerization of acrylic acid (AAc) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) (poly(AAc-co-DMAEMA) (PAD) hydrogels). This one step polymerization process makes scaling-up easier for mass production. Our formulations, being devoid of any chemical cross-linkers, remained dimensionally stable in buffer solutions of pH 1.2–7.4 with interlocked nanogels being identified as the building blocks of the network structures. Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), uniaxial compression testing and scanning electron microscopy (SEM) were used to characterize the hydrogels (PADs). Compressive elastic modulus and compressive strength of the swollen hydrogels at pH 7 were found to vary with composition from ∼3 to ∼11 kPa and ∼178 to ∼206 kPa, respectively. The swollen gels showed fairly strong viscoelastic behaviour and underwent deformation from ∼70% to 85% before failure, indicating the formation of robust 3D structures of PADs. Preliminary investigation into the biocompatibility of our hydrogels done by cytotoxicity assays using HeLa and McCoy mouse fibroblast cell lines have revealed that they are non-cytotoxic, paving the way for further biomedical applications. Swelling behaviour and release kinetics of bovine serum albumin (BSA) were investigated in various buffer solutions that mimic the pH-metric hierarchy in the gastrointestinal (GI) tract. Equilibrium swelling ratio was found to vary from 171% (mass) to 2027% (mass) depending on the pH and composition of hydrogels. Different compositions of PAD systems were investigated to verify the possibility of tailor-making the drug release behaviour of PAD formations.

Biodynamic performance of hyaluronic acid versus synovial fluid of the knee in osteoarthritis

Hyaluronic acid (HA), a natural biomaterial present in healthy joints but depleted in osteoarthritis (OA), has been employed clinically to provide symptomatic relief of joint pain. Joint movement combined with a reduced joint lubrication in osteoarthritic knees can result in increased wear and tear, chondrocyte apoptosis, and inflammation, leading to cascading cartilage deterioration. Therefore, development of an appropriate cartilage model that can be evaluated for its friction properties with potential lubricants in different conditions is necessary, which can closely resemble a mechanically induced OA cartilage. Additionally, a comparison of different models with and without endogenous lubricating surface zone proteins, such as PRG4 promotes a well-rounded understanding of cartilage lubrication. In this study, we present our findings on the lubricating effects of HA on different articular cartilage model surfaces in comparison to synovial fluid, a physiological lubricating biomaterial. The mechanical testings data demonstrated that HA reduced average static and kinetic friction coefficient values of the cartilage samples by 75% and 70%, respectively. Furthermore, HA mimicked the friction characteristics of freshly harvested natural synovial fluid throughout all tested and modeled OA conditions with no statistically significant difference. These characteristics led us to exclusively identify HA as an effective boundary layer lubricant in the technology that we develop to treat OA (Singh et al., 2014).

Hydrothermal synthesis of nitrogen doped graphene nanosheets from carbon nanosheets with enhanced electrocatalytic properties

Synthesis of nitrogen doped graphene nanosheets (NGS) with controlled structure is a burgeoning issue in the field of materials chemistry. Herein, we for the first time report a novel, green, low-temperature, hydrothermal synthesis of two dimensional (2D), porous, NGS from carbon nanosheets (CNS) as precursor materials. Hydrothermal reduction of CNS not only imparts crystallinity to the material but also results in a substantial removal of oxygen functionalities, forming NGS. The as-synthesized NGS displayed excellent electrochemical sensing properties with high selectivity and sensitivity for the detection of dopamine (DA) in human urine samples. NGS can easily distinguish the electrochemical oxidation peak for DA from that of uric acid (UA), which is a common interferent for DA detection. The electrochemical oxidation peak current of DA linearly varies within the concentration range of 0.1 to 100 μM, having remarkable sensitivity (2.661 μA μM−1) and a lower detection limit upto 100 nM, with a correlation coefficient (R2) of 0.9880. The separation of electrooxidation peak potential for DA–UA is extraordinarily high (0.37 ± 0.05 V). In contrast to carbonized carbon material (CCM), reduced graphene oxide (rGO) and other carbon materials, the current response for NGS is three folds higher at the same DA concentration with higher electrocatalytic response. Furthermore, superior electrochemical sensitivity and selectivity along with enhanced electrocatalytic properties of NGS for DA detection is metal/metal oxide free, which are usually employed for electrode modification to achieve higher current response. Henceforth, we believe that NGS synthesis offers great promises for creating a revolutionary new class of nanostructured electrodes for biosensing applications.