In vitro characterization of transport, release, and PC12 cell biocompatibility with microfabricated nanoporous silicon membranes

Current therapies for neuronal pathologies rely mainly on pharmaceutical treatment with drugs. With the benefits come a multitude of pitfalls; dramatic inter- and intra-patient variability, loss of efficacy with disease progression, and prohibitive side-effects from systemic administration are a few examples. With the use of micro and nanotechnology, it has been possible to create devices which may provide an effective, permanent treatment method for progressive diseases resulting from the loss of specific hormonal and biochemical transmission. These technologies have provided us the means to build structures which can influence and control cell actions and fates such as attachment, proliferation, differentiation, and senescence. Also, the material and physical properties of these devices can be manipulated to allow effective immunoisolation and the incorporation of activating components for the controlled release of desired cellular compounds. Silicon-based devices are easily manufactured in bulk, can be readily modified, and have shown high to moderate biocompatibility in certain applications. For this project, in vitro research was conducted on the integration of neurosecretory cell lines and microfabricated silicon nanoporous membranes to quantify design parameters for novel therapeutic immunoisolating macrocapsules. The PC12 pheochromocytoma clonal line was implemented for its differentiating capacity, fidelity to neuronal attributes, and cytochemical (catecholaminergic) properties. Cell attachment, proliferation, differentiation, and functionality were all investigated on these silicon membranes through various biocompatibility studies. Additionally, mass transport studies determining effective diffusion coefficients (Deff) for two different membrane designs were performed. The results demonstrate that these membranes, fabricated in similar ways but having different bulk size and structure, exhibit a comparable Deff for dopamine (DA). These studies elucidated other important considerations for optimizing the transport of small molecules. Exploring the level of transport resistance established by these membranes for small, water-soluble substances relative to their natural diffusion (in 'binary' aqueous solutions) has provided insight into what applications they may be useful for and what inherent limitations must be considered. Lastly, the release rates of the neurotransmitter DA and two of its major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, HVA), from biocapsules loaded with collagen-embedded PC12 cells and the concentrations obtainable outside the nanoporous membrane are also important parameters that were studied for these unique macroencapsulation devices. Characterization of small molecule release from the biocapsules was performed with respect to the physiological mechanisms of secretion and reuptake control by the encapsulated PC12 cells. Specifically, the release of these substances was monitored under conditions of basal secretion, 'activated' release (high [K+ ] balanced salt solution), and dopamine reuptake-inhibited release (blocking the dopamine transporter with nomifensine) to determine what mechanisms of cell physiology play an important role in biocapsule functionality.