Sabrina S. Jedlicka

Sol-gel derived materials as substrates for neuronal differentiation: effects of surface features and protein conformation

This work demonstrates the ability of sol-gel derived materials to support the differentiation of neuronal cells, and investigates the physiochemical interactions between the surface and extracellular matrix proteins as a mediator of the effects of surface features on differentiation. We have applied fluorescence resonance energy transfer (FRET) spectroscopy to study the conformational changes of human serum fibronectin, a critical extracellular cell adhesion protein, after adsorption onto native and poly-L-lysine doped sol-gel derived silica thin films and bulk materials. The global conformation of fibronectin varied dramatically between native and organically modified materials and most interestingly between thin films and bulk materials of the same chemistry. A comparison of the surface topography of thin films and bulk materials by atomic force microscopy reveals that films of native silica have surface features less than the AFM tip size (<25 nm) while bulk materials of the same precursor chemistry have features ranging from 50–100 nm in size. Fibronectin assumed an inactive, globular, solution-like state on the larger feature size bulk gels and an active, fully extended fibrillar-like state on the smaller feature size films. Neither native nor PLL-doped bulk materials could support cell growth or neuronal differentiation of PC12 cells, in stark contrast to the thin films, which supported a robust neuronal phenotype. Morphological analysis and expression levels of the neuronal proteins β-tubulin and neurofilament, in addition to the FRET data, indicate that the effects of surface chemistry on fibronectin conformation, cellular adhesion, and differentiation are dependent upon the surface topography.

Liposome-Doped Nanocomposites as Artificial-Cell-Based Biosensors: Detection of Listeriolysin O

Listeriolysin O (LLO) is a pore‐forming hemolysin secreted by the foodborne pathogen Listeria monocytogenes and is required for bacterial virulence. Current detection methods for L. monocytogenes are time‐consuming, labor‐intensive, and expensive, which is impractical considering the limitations of food storage. To overcome these problems, we developed a liposome‐doped silica nanocomposite as a simple, inexpensive, and highly stable biosensor material that mimics existing whole‐cell assays for LLO. Small unilamellar liposomes containing fluorescent dyes were immobilized within porous silica using alcohol‐free sol‐gel synthesis methods. The immobilized liposomes served as cellular surrogates for membrane insertion and pore formation by LLO. The integrity of liposomes in the solid‐state sol‐gel glass was investigated by fluorescence quenching and leaching assays. The materials were stable for at least 5 months in ambient conditions. Both free and immobilized liposomes responded to LLO at pH 6.0 with concentration dependent kinetics. The pore formation of LLO in liposome‐doped silica composites displayed similar kinetic curves as free liposomes but with slower rates. LLO insertion into the immobilized liposomes was pH dependent. No increase in membrane permeability was observed at pH 7.4 for the liposome‐doped composites in the presence of LLO. Immobilized liposomes can detect LLO in ∼1.5 h using a steady state calibration and within 30 min using a kinetic calibration. These liposome silica composites potentially could be used for the detection of hemolysin producing L. monocytogenes as well as the many other bacteria that produce pore‐forming toxins.

Peptide ormosils as cellular substrates

Peptide-functionalized thin films exhibit significant potential for integration into implantable devices and cell-based technologies. A new type of neuroactive peptide-modified silica was developed using sol–gel reaction chemistry to produce thin films from four different peptide silane precursors. Peptide silanes containing binding sequences from laminin (YIGSR and KDI), fibronectin (RGD), and EGF repeats from laminin and tenascin (NID) were produced using standard solid-state FMOC peptide synthesis conditions and the covalent attachment of 3′-(aminopropyl)trimethoxysilane (APTMS), using carbonyldiimadazole (CDI) as a linking molecule. Precursor formation was confirmed with MALDI-MS. Thin films were produced by dip-coating using the peptide precursors combined with hydrolyzed tetramethoxysilane. Atomic force microscopy indicated that the surface topography was not affected by low concentrations of peptide precursor (0.0025 mol%), but higher concentrations of peptide precursor (0.01 mol%) resulted in features that were 50–75 nm in height. The height features observed were consistent in size with previously determined topographical morphology supportive of neuronal cell lines. The surfaces were biologically active and modulated the phenotype of the embryonic carcinoma stem cell line, P19. Combinations of the peptide silanes resulted in altered cell types after retinoic acid treatment. More neurons were observed on RGD/YIGSR and RGD/YIGSR/NID surfaces compared to tetramethoxysilane (TMOS) controls. More supporting cells were observed compared to collagen coated tissue culture plates. In addition, neurites were significantly longer on the peptide ormosils compared to controls. This work demonstrates a novel method for producing biologically active peptide ormosils using peptide-modified precursors.

Compartmentalized nanocomposite for dynamic nitric oxide release.

Nitric oxide (NO) is an important cell-signaling molecule whose role in a variety of cellular processes such as differentiation and apoptosis depends strongly on its concentration and flux levels. This work describes and characterizes a novel nitric oxide releasing nanocomposite, capable of photostimulated NO flux that can by dynamically modulated in within a range of biological levels. This material mimics the common compartmentalization strategies used by living cells to achieve its novel features. The material is constructed by encapsulating a photosensitive nitric oxide donor within lipid vesicles with an average diameter of 150 nm. The vesicles are then doped into the interstitial liquid phase of a solid porous silica matrix, which has previously demonstrated biological compatibility and capabilities as a growth surface for mammalian cells. Stimulation by a light source produces a step increase in NO concentration within seconds. The NO flux at the surface of the material is measured to be 14 pmol-cm(-2) sec(-1) using a NO selective self-referencing amperometric microsensor. The NO concentration profile decreases with distance perpendicular to the surface as expected for diffusion from a surface through an aqueous environment. A pattern of one minute light pulses produced uniform pulses of increased NO concentration of one minute duration. A linear relationship exists between NO surface concentration and photon flux, and this relationship can be used to tune the material response.

CALIBRATION OF NEUROTRANSMITTER RELEASE FROM NEURAL CELLS FOR THERAPEUTIC IMPLANTS

In this work we quantified the in vitro calibration relationships between high frequency electrical stimulation and GABA and glutamate release in both mature retinoic acid differentiated P19 neurons and immortalized embryonic cortical cells engineered to express glutamic acid decarboxylase, GAD65. Extracellular glutamate and GABA was quantified by 2D gas chromatography and time of flight mass spectrometry after stimulation at varying amplitudes and frequencies. Amplitude sweeps resulted in a linear calibration for P19 neurons; the level of neurotransmitter varied over one order of magnitude from ~ 200 pg/neuron to ~ 1.2 ng/neuron for glutamate and ~ 1 ng/neuron to ~ 2 ng/neuron for GABA, depending on the stimulation amplitude. Frequency sweeps resulted in a peak release at 250 Hz for glutamate and 400 Hz for GABA in P19 cells. The GABA transporter inhibitor, nipecotic acid, increased extracellular GABA levels and decrease glutamate. In contrast the embryonic cortical cells had a strongly nonlinear dependency of release on stimulation amplitude, and a weak dependence on frequency. These cells had roughly equal extracellular glutamate and GABA levels after stimulation despite the expression of GAD65. In addition glutamate and GABA levels were insensitive to nipecotic acid. These results demonstrate an ability to calibrate and tune neurotransmitter release from neural cells using high frequency stimulation parameters.

Interactions Between Chemical Functionality and Nanoscale Surface Topography Impact Fibronectin Conformation and Neuronal Differentiation on Model Sol-gel Silica Substrates

Functional relationships between the biomaterial interface and extracellular matrix (ECM) proteins are intimately involved in cellular adhesion and function. Structural changes of ECM proteins upon adsorption to a surface alter the proteins biological activity by varying the availability of molecular binding sites. Recent work using native and organically modified sol-gel silica as a neuronal biointerface revealed that changes in surface nanotopography of bulk versus thin film materials result in dramatic differences in fibronectin structure, cell survival, and neuronal differentiation. In order to further investigate interactions between chemical functionality and surface topography, we evaluated the global conformation of human fibronectin adsorbed to seven different organically modified silica gels and thin films. Chemical functional groups were introduced into the materials either by altering the starting precursor or by doping with poly-l-lysine or polyethylenimine. Surface topography measurements by atomic force microscopy show that films have surface features less than 25 nm while bulk materials of the same precursor chemistry have features ranging from 50 – 100 nm in size. Fluorescence resonance energy transfer spectroscopy (FRET) revealed a strong interaction between surface topography and chemical functionality. Fibronectin remain globular on all bulk materials regardless of chemical modification. The same changes in precursors or dopant chemistry, however, induced changes in the conformation of fibronectin on the thin films. The differentiation of PC12 cells on the surface indicated a strong impact of the surface features and suggest a possible optimal fibronectin folding state.

Constant-Current Adjustable-Waveform Microstimulator for an Implantable Hybrid Neural Prosthesis

Microstimulation of neural tissue has become a widely-used technique for controlling neuronal responses with local electric fields as well as a therapeutic intervention for nervous system disorders such as epilepsy and Parkinsons disease. Of those afflicted by neurological diseases, many are or become tolerant to existing pharmaceuticals and are left with little recourse. Little is known about the necessary design criteria or efficacy of a hybrid neural prosthesis. Assessment of the potential clinical value of a hybrid electro-chemical neural prosthesis was performed through in vitro verification using a prototype microstimulator and P19 cell cultures. We constructed a printed circuit board (PCB) microstimulator as a prototype of a CMOS microstimulator ASIC that was subsequently fabricated in the IBM 7RF 0.18 mum process. Measured results for the prototype are described in this work. An output impedance of 237 kOmega, voltage compliance of 11.3 V, and a linear constant-current output up to +/-600 muA make this microstimulator system a viable option for an implantable hybrid neural prosthesis. Hybrid prostheses could uniquely affect neural modulation with linear glutamate release at physiological amplitudes and frequencies.