Professor Department of Biomedical Engineering, Rochester Institute of Technology, Rochester, NY, United States
Introduction:: Microporous membranes are widely used in tissue barrier and co-culture models in the tissue-on-a-chip field. These membranes are commonly selected based on their pore size and porosity to limit transport across the barrier to small molecules or some cell types while excluding others and helping support the barrier monolayer. Membranes are sometimes engineered to be ultrathin to improve permeability and chemical and physical cross-talk between cells on opposite sides to recapitulate more complex tissue barriers microenvironments such as the blood-brain barrier. Research conducted over the past few decades increasingly shows physical and mechanical properties of the microenvironment surrounding cells, such as stiffness, influence cellular adhesion, morphology, migration, and differentiation. [2]. In order to establish a more biologically relevant cellular microenvironment, it is necessary to construct substrates using materials with mechanical properties in the physiological range. However, fabricating ultrathin porous membranes with soft materials limits membrane area and utility. One approach is to fabricate the membrane with commonly used microfabrication materials such as silicon nitride and subsequently modify the surface with a soft polymer brush. In the present study, we deposited poly(L-lysine)-g-poly(ethylene glycol)-biotin (PLL-g-PEG-biotin) as a surface coating to effectively soften ultrathin microporous silicon nitride membranes. We hypothesize that the non-crosslinked brush configuration of PLL-g-PEG-biotin molecules can attach to the non-porous regions of the surface without obstructing the pores. Additionally, the surface's desired physical and mechanical properties, such as stiffness, can be tuned by modifying the length, density, and microstructure of the PEG molecules.
Materials and Methods:: First, a 2x2 mm microporous silicon nitride membrane with a 0.5 μm pore size underwent oxygen plasma treatment to enable the electrostatic interaction of PLL-g-PEG-biotin on the surface. To optimize the attachment process, the membrane was subjected to oxygen plasma treatment for varying periods of time. Immediately afterward (< 2 h), 20 μL of 0.5 mg/mL PEG solution was introduced dropwise onto the surface and incubated at room temperature for 75 minutes. The PLL-g-PEG-biotin concentration and the incubation time were selected based on a previous investigation [1]. A qualitative analysis was conducted using scanning electron microscopy (SEM) to examine the porosities of a coated vs. non-coated membrane. Pore diameters were measured using ImageJ [3]. The surface was first modified with biotin, then 0.005 mg/mL of streptavidin-conjugated fluorophore was allowed to bind, excess dye was removed by rinsing, and fluorescence was quantified with a fluorescent microscope. The optimum green fluorophore concentration was determined. Fluorescent comparisons with an unmodified surface validated the coating process.
Results, Conclusions, and Discussions:: Plasma treatment for 1 minute was shown to provide an effective surface for PLL-g-PEG-biotin coating due to the generation of an adequate number of negative hydroxyl groups on the surface. Longer plasma treatments did not show improvements in the surface coating condition. SEM images of the coated membrane demonstrated that the application of the coating did not lead to clogging of the membrane pores, and the average pore diameter of the coated membrane was reduced by only 13% compared to the uncoated membrane, indicating minimal impact on overall porosity (Figure 1.A). This can be attributed to the brush-like conformation of PLL-g-PEG-biotin molecules without crosslinking with neighboring grafted polymers. Compared to non-crosslinked structure, crosslinked polymers would be expected to more easily clog pores by forming thick and dense films. To verify that the reduction in pore diameter was attributed to the formation of the PLL-g-PEG-biotin coating on the surface, a streptavidin-conjugated fluorophore was utilized to label the coated surface. Statistical analysis of the labeled coated surface demonstrated a significant difference in fluorescence intensity compared to the non-coated surface (Figure 1.B). These results confirm that the decrease in pore diameter was due to the formation of the PLL-g-PEG-biotin coating on the surface. Further studies will be conducted to characterize the physical and mechanical properties of the membrane surface using AFM and to explore potential effects on cell behavior on the soft substrate. Surface modification of the microporous membrane was achieved using brush-like polymer chains. The SEM images and the quantitative fluorescence data supported our hypothesis that the PLL-g-PEG-biotin molecules are effectively attached to the surface while preserving porosity. Stiffness measurement with AFM and cell behavior studies will evaluate the softening process.
[2] Cai, Lei, et al. "Injectable and biodegradable nanohybrid polymers with simultaneously enhanced stiffness and toughness for bone repair." Advanced Functional Materials 22.15 (2012): 3181-3190.
[3] Schneider, Caroline A., Wayne S. Rasband, and Kevin W. Eliceiri. "NIH Image to ImageJ: 25 years of image analysis." Nature methods 9.7 (2012): 671-675.
