Artificial biomembranes stabilized over spin coated hydrogel scaffolds. Crosslinking agent nature induces wrinkled or flat surfaces on the hydrogel

https://doi.org/10.1016/j.chemphyslip.2016.02.001Get rights and content

Highlights

  • Hydrogel films, deposited through spin coating technique, were used as scaffold for artificial biomembranes.

  • DPPC bilayer (artificial membrane model) was deposited over hydrogel films using Langmuir–Blodgett technique.

  • Wrinkled or flat surfaces were spontaneously formed in the top of hydrogel layers according to crosslinking agent nature.

  • DPPC phase and phase transitions were detected using ellipsometry and AFM techniques.

  • Hydrogel-cushioned biomembranes maintain their stability in time under uncomfortable conditions.

Abstract

Hydrogel films possess the ability of retain water and deliver it to a phospholipid bilayer mainly composed by DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine); moisture of the medium favors the stability of an artificial biomembrane when it is subjected to repetitive heating cycles. This hypothesis is valid when the hydrogel film, used as scaffold, present a flat surface morphology and a high ability for water releasing. On the other hand, when the sample presents a wrinkle topography (periodic undulations), free lateral molecular movement of the bilayer becomes lower, disfavoring the occurrence of clear phases/phase transitions according to applied temperature.

Hydrogel films were prepared using HEMA (hydroxyethylmetacrylate), different crosslinking agents and initiators. This reaction mixture was spread over hydrophilic silicon wafers using spin coating technique. Resultant films were then exposed to UV light favoring polymeric chain crosslinking and interactions between hydrogel and substrate; this process is also known to generate tensile stress mismatch between different hydrogel strata, producing out-of-plane net force that generate ordered undulations or collapsed crystals at surface level. DPPC bilayers were then placed over hydrogel using Langmuir–Blodgett technique. Surface morphology was detected in order to clarify the behavior of these films. Obtained data corroborate DPPC membrane stability making possible to detect phases/phase transitions by ellipsometric methods and Atomic Force Microscopy due to their high hydration level. This system is intended to be used as biosensor through the insertion of transmembrane proteins or peptides that detect minimal variations of some analyte in the environment; artificial biomembrane stability and behavior is fundamental for this purpose.

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

References (39)

  • J. Stamatoff et al.

    Amplitude of rippling in the pβ phase of dipalmitoylphosphatidylcholine bilayers

    Biophys. J.

    (1982)
  • F. Tokumasu et al.

    Atomic force microscopy of nanometric liposome adsorption and nanoscopic membrane domain formation

    Ultramicroscopy

    (2003)
  • F. Yarrow et al.

    AFM study of the thermotropic behaviour of supported DPPC bilayers with and without the model peptide WALP23

    Chem. Phys. Lipids

    (2011)
  • J. Andrecka et al.

    Direct observation and control of supported lipid bilayer formation with interferometric scattering microscopy

    ACS Nano

    (2013)
  • P.S. Cremer et al.

    Formation and spreading of lipid bilayers on planar glass supports

    J. Phys. Chem. B

    (1999)
  • S.F. Gilmore et al.

    Programmed bending reveals dynamic mechanochemical coupling in supported lipid bilayer

    PLoS One

    (2011)
  • S.F. Gilmore et al.

    Thermal annealing triggers collapse of biphasic supported lipid bilayers into multilayer islands

    Langmuir

    (2014)
  • C. González et al.

    Thermal behavior of 1,2-dipalmitoyl-sn-3-phosphoglycerocholine bi- and multi-layers, deposited with physical vapor deposition under ellipsometric growth control

    J. Chem. Phys.

    (2012)
  • C.M. González-Henríquez et al.

    In situ silver nanoparticle formation embedded into a photopolymerized hydrogel with biocide properties

    J. Nanostruct. Chem.

    (2014)
  • Cited by (0)

    View full text