Artificial biomembranes stabilized over spin coated hydrogel scaffolds. Crosslinking agent nature induces wrinkled or flat surfaces on the hydrogel
Graphical abstract
Introduction
Tethered bilayer lipid membranes (tBLMs) have attracted large interest from current and modern research in the biotechnology field due to their wide application in technology development for tissue engineering, drug delivery, biosensors and protein channel transduction, among others (Drücker et al., 2014). Supported lipid bilayers (SLBs) is the most common and widely used configuration of tBLMs, corresponding to planar membranes deposited onto hydrophilic solid substrates separated with an ultrathin film of water (1–2 nm) (Rebaud et al., 2014). Thermal studies of these systems elucidate the behavior and properties of cell membranes, and – also – the efficiency of possible devices based on SLBs when are subjected to temperatures near ambient (González et al., 2012). In this way, the determination of bilayer phase/phase transition temperatures becomes in relevant information (Thiam et al., 2013). Some characterization techniques as magnetic resonance, Raman spectroscopy, Atomic Force Microscopy has been utilized for this purpose (Shlomovitz and Schick, 2013, Hain et al., 2013) allowing the detection of minimal structural changes in bilayer conformation such as thickness, smooth/roughness, molecule tilting and electric interaction between phospholipids (Jing et al., 2014). However, the complexity of SLBs systems lies in the low structural stability of the bilayer during formation processes and posterior characterization (Andrecka et al., 2013). In order to solve this problem, scaffolds for tBLMs are frequently used; the compound utilized as support must maintain an aqueous (moist) environment that increase their stability during long periods, particularly in unusual conditions (high temperatures, pressure variations, external mechanical stress and pH changes) (Luckey, 2014).
Polymer scaffolds enhance surface-bilayer interactions compared to several others materials commonly used such as aluminium, titanium, iron and silicon oxides (Nellis et al., 2011). Hydrogel is a kind of polymer, which has the capacity to absorb and retain large amounts of solvents into their structural network, being an excellent candidate for membrane support without direct linkage, significantly reducing the frictional coupling between membrane and solid substrate acting as “lubricant cushion” for the interface (Rebaud et al., 2014).
In this study, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was used for bilayer formation. This pulmonary surfactant present three characteristic phases; subgel (Lc), gel (Lβ) and liquid crystalline (Lα) with their respective transitions: laminar gel (Lβ́) and ripple gel (Pβ́), the superscript of prime means that lipid molecules are oriented tilted from the bilayer plane (Matsuki et al., 2013).
Different types of photo-polymerized hydrogel films, based on polyhydroxyethylmetacrylate (pHEMA), were used as scaffold for DPPC. The technique used to deposit the hydrogel was spin coating, producing a high surface homogeneity; posteriorly, the compounds were exposed to UV light (λ = 365 nm) in order to complete the polymerization. The systems formed by DPPC/Hydrogel films/Silicon wafer were characterized through different methods. Hydrogel film surface was analyzed using Field Emission gun Scanning Electron Microscopy (FE-SEM) in order to visualize the surface morphology. Atomic Force Microscopy (AFM) was used to detect surface structures dimensions and profiles. Simultaneously, heating cycles were applied during these measurements in order to obtain micrographies at different temperatures, this data was then used to calculate surface roughness for identification of DPPC phases with their respective transitions. Ellipsometric measurements were realized in every deposition step in order to obtain an appropriate thickness control, also, DPPC thickness variations were measured against temperature in order to corroborate phases and phase transitions temperatures detected via AFM.
Different surfaces morphologies were obtained according to the hydrogel type utilized as scaffold (undulated pattern in some cases or flat surface with some crystals structures in others). Patterns morphology and their dimensions are related to the ratio between monomer and crosslinking agent, to the polymerization technique utilized and with the deposition method, these processes generate a stress gradient between film surface and lower strata (Guvendiren et al., 2010). When hydrogel film show tightens wrinkles, DPPC bilayer is found highly packaged affecting their molecular mobility, disfavoring phase (transitions) occurrence. On the other hand, flat topography is ideal for detect thermal behavior of the surfactant and for conserve membrane stability during thermal cycles.
Section snippets
Materials
For hydrogel synthesis, the following precursors were utilized: 2-hydroxyethyl methacrylate (HEMA, 97%) containing monomethyl ether hydroquinone as inhibitor (≤250 ppm) as main monomer. Four different crosslinking agents were employed: di(ethylene glycol) dimethacrylate (DEGDMA, ≥95%) that includes monomethyl ether hydroquinone as inhibitor (300 ppm). Poly(ethylene glycol) diacrylate (PEGDA), with two different average molecular weights (Mn: 575 and 700 g/mol) and acrylamide (AAm, ≥99%) for
Results and discussion
After hydrogel film photo-polymerization it is necessary to place the sample into rough vacuum (10−3 torr) during 3 h in order to remove water (deswelling) that is trapped between polymer network spaces. According to the crosslinking agent used in the synthesis, different morphologies can be achieved, surface homogeneity (appropriate for ellipsometric measures) in some cases, and buckling in others. Wrinkled topography is generated due to deswelling process that induces a stress gradient between
Conclusion
Over a cleaned and hydrophilic silicon wafer surface, spin coating technique was used to deposit hydrogel films. Their thickness and surface homogeneity were affected by composite density/viscosity and monomers used in the reaction synthesis, among other factors. For HEMA–PEGDA700 films, a rough surface due to the high crosslinking degree, provided by PEGDA (wrinkle patterns), was detected. Similar situation is visualized for HEMA–PEGDA575 that present a disordered buckled surface
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
The authors gratefully acknowledge financial support from FONDECYT Grant No 11121281; Attraction and Insertion of Advanced Human Capital Program, PAI No 7912010031-CONICYT. Mr. Sarabia acknowledges the financial support given by CONICYT through the Doctoral Scholarship Grant. In addition, we wish to thank the following: LNLS-Brazilian Synchrotron National Light Laboratory together with LNNano-Brazilian Nanotechnology National Laboratory for the use of FE-SEM and AFM (Brazil-Campinas) and
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