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An ellipsometric approach towards the description of inhomogeneous polymer-based Langmuir layers
(2016)
The applicability of nulling-based ellipsometric mapping as a complementary method next to Brewster angle microscopy (BAM) and imaging ellipsometry (IE) is presented for the characterization of ultrathin films at the air-water interface. First, the methodology is demonstrated for a vertically nonmoving Langmuir layer of star-shaped, 4-arm poly(omega-pentadecalactone) (PPDL-D4). Using nulling-based ellipsometric mapping, PPDL-D4-based inhomogeneously structured morphologies with a vertical dimension in the lower nm range could be mapped. In addition to the identification of these structures, the differentiation between a monolayer and bare water was possible. Second, the potential and limitations of this method were verified by applying it to more versatile Langmuir layers of telechelic poly[(rac-lactide)-co-glycolide]-diol (PLGA). All ellipsometric maps were converted into thickness maps by introduction of the refractive index that was derived from independent ellipsometric experiments, and the result was additionally evaluated in terms of the root mean square roughness, R-q. Thereby, a three-dimensional view into the layers was enabled and morphological inhomogeneity could be quantified.
The influence of terminal functionalization of oligo(epsilon-caprolactone)s (OCL) with phenylboronic acid pinacol ester or phenylboronic acid on the enzymatic degradation behavior at the air-water interface is investigated by the Langmuir monolayer degradation technique. While the unsubstituted OCL immediately degrades after injection of the enzyme lipase from Pseudomonas cepacia, enzyme molecules are incorporated into the films based on end-capped OCL before degradation. This incorporation of enzymes does not inhibit or suppress the film degradation, but retards it significantly. A specific binding of lipase to the polymer monolayer allows studying the enzymatic activity of bound proteins and the influence on the degradation process. The functionalization of a macromolecule with phenyl boronic acid groups is an approach to investigate their interactions with diol-containing biomolecules like sugars and to monitor their specified impact on the enzymatic degradation behavior at the air-water interface.
A series of multiblock copolymers (PDLCL) synthesized from oligo(omega-pentadecalactone) diol (OPDL) and oligo(epsilon-caprolactone) diol (OCL), which are linked by 2,2(4), 4-trimethyl-hexamethylene diisocyanate (TMDI), is investigated by the Langmuir monolayer technique at the air-water interface. Brewster angle microscopy (BAM) and spectroscopic ellipsometry are employed to characterize the polymer film morphologies in situ. PDLCL containing >= 40 wt% OCL segments form homogeneous Langmuir monofilms after spreading. The film elasticity modulus decreases with increasing amounts of OPDL segments in the copolymer. In contrast, the OCL-free polyesterurethane OPDL-TMDI cannot be spread to monomolecular films on the water surface properly, and movable slabs are observed by BAM even at low surface pressures. The results of the in situ morphological characterization clearly show that essential information concerning the reliability of Langmuir monolayer degradation (LMD) experiments cannot be obtained from the evaluation of the pi-A isotherms only. Consequently, in situ morphological characterization turns out to be indispensable for characterization of Langmuir layers before LMD experiments.
Three oligo[(rac-lactide)-co-glycolide] based polyesterurethanes (OLGA-PUs) containing different diurethane linkers are investigated by the Langmuir monolayer technique and compared to poly[(rac-lactide)-co-glycolide] (PLGA) to elucidate the influence of the diurethane junction units on hydrophilicity and packing motifs of these polymers at the air-water interface. The presence of diurethane linkers does not manifest itself in the Langmuir layer behavior both in compression and expansion experiments when monomolecular films of OLGA-PUs are spread on the water surface. However, the linker retard the evolution of morphological structures at intermediate compression level under isobaric conditions (with a surface pressure greater than 11 mN m(-1)) compared to the PLGA, independent on the chemical structure of the diurethane moiety. The layer thicknesses of both OLGA-PU and PLGA films decrease in the high compression state with decreasing surface pressure, as deduced from ellipsometric data. All films must be described with the effective medium approximation as water swollen layers.
The interplay of an enzyme with a multiblock copolymer PDLCL containing two segments of different hydrophilicity and degradability is explored in thin films at the air-water interface. The enzymatic degradation was studied in homogenous Langmuir monolayers, which are formed when containing more than 40 wt% oligo(epsilon-caprolactone) (OCL). Enzymatic degradation rates were significantly reduced with increasing content of hydrophobic oligo(omega-pentadecalactone) (OPDL). The apparent deceleration of the enzymatic process is caused by smaller portion of water-soluble degradation fragments formed from degradable OCL fragments. Beside the film degradation, a second competing process occurs after adding lipase from Pseudomonas cepacia into the subphase, namely the enrichment of the lipase molecules in the polymeric monolayer. The incorporation of the lipase into the Langmuir film is experimentally revealed by concurrent surface area enlargement and by Brewster angle microscopy (BAM). Aside from the ability to provide information about the degradation behavior of polymers, the Langmuir monolayer degradation (LMD) approach enables to investigate polymer-enzyme interactions for non-degradable polymers. (C) 2016 Elsevier Ltd. All rights reserved.
The enzymatic degradation of oligo(epsilon-caprolactone) (OCL) based films at the air-water interface is investigated by Langmuir monolayer degradation (LMD) experiments to elucidate the influence of the molecular architecture and of the chemical structure on the chain scission process. For that purpose, the interactions of 2D monolayers of two star-shaped poly(epsilon-caprolactone)s (PCLs) and three linear OCL based copolyesterurethanes (P(OCL-U)) with the lipase from Pseudomonas cepacia are evaluated in comparison to linear OCL. While the architecture of star-shaped PCL Langmuir layers slightly influences their degradability compared to OCL films, significantly retarded degradations are observed for P(OCL-U) films containing urethane junction units derived from 2, 2 (4), 4-trimethyl hexamethylene diisocyanate (TMDI), hexamethylene diisocyanate (HDI) or lysine ethyl ester diisocyanate (LDI). The enzymatic degradation of the OCL based 2D structures is related to the presence of hydrophilic groups within the macromolecules rather than to the packing density of the film or to the molecular weight. The results reveal that the LMD technique allows the parallel analysis of both the film/enzyme interactions and the degradation process on the molecular level. (C) 2016 Elsevier Ltd. All rights reserved.
Enhancement of human induced pluripotent stem cells adhesion through multilayer laminin coating
(2019)
Bioengineered cell substrates are a highly promising tool to govern the differentiation of stem cells in vitro and to modulate the cellular behavior in vivo. While this technology works fine for adult stem cells, the cultivation of human induced pluripotent stem cells (hiPSCs) is challenging as these cells typically show poor attachment on the bioengineered substrates, which among other effects causes substantial cell death. Thus, very limited types of surfaces have been demonstrated suitable for hiPSC cultures. The multilayer coating approach that renders the surface with diverse chemical compositions, architectures, and functions can be used to improve the adhesion of hiPSCs on the bioengineered substrates. We hypothesized that a multilayer formation based on the attraction of molecules with opposite charges could functionalize the polystyrene (PS) substrates to improve the adhesion of hiPSCs. Polymeric substrates were stepwise coated, first with dopamine to form a polydopamine (PDA) layer, second with polylysine and last with Laminin-521. The multilayer formation resulted in the variation of hydrophilicity and chemical functionality of the surfaces. Hydrophilicity was detected using captive bubble method and the amount of primary and secondary amines on the surface was quantified by fluorescent staining. The PDA layer effectively immobilized the upper layers and thereby improved the attachment of hiPSCs. Cell adhesion was enhanced on the surfaces coated with multilayers, as compared to those without PDA and/or polylysine. Moreover, hiPSCs spread well over this multilayer laminin substrate. These cells maintained their proliferation capacity and differentiation potential. The multilayer coating strategy is a promising attempt for engineering polymer-based substrates for the cultivation of hiPSCs and of interest for expanding the application scope of hiPSCs.
Glycoproteins adsorbing on an implant upon contact with body fluids can affect the biological response in vitro and in vivo, depending on the type and conformation of the adsorbed biomacromolecules. However, this process is poorly characterized and so far not controllable. Here, protein monolayers of high molecular cohesion with defined density are transferred onto polymeric substrates by the Langmuir-Schaefer (LS) technique and were compared with solution deposition (SO) method. It is hypothesized that on polydimethylsiloxane (PDMS), a substrate with poor cell adhesion capacity, the fibronectin (FN) layers generated by the LS and SO methods will differ in their organization, subsequently facilitating differential stem cell adhesion behavior. Indeed, atomic force microscopy visualization and immunofluorescence images indicated that organization of the FN layer immobilized on PDMS was uniform and homogeneous. In contrast, FN deposited by SO method was rather heterogeneous with appearance of structures resembling protein aggregates. Human mesenchymal stem cells showed reduced absolute numbers of adherent cells, and the vinculin expression seemed to be higher and more homogenously distributed after seeding on PDMS equipped with FN by LS in comparison with PDMS equipped with FN by SO. These divergent responses could be attributed to differences in the availability of adhesion molecule ligands such as the Arg-Gly-Asp (RGD) peptide sequence presented at the interface. The LS method allows to control the protein layer characteristics, including the thickness and the protein orientation or conformation, which can be harnessed to direct stem cell responses to defined outcomes, including migration and differentiation. Copyright (c) 2016 John Wiley & Sons, Ltd.
Hydroxyl terminated oligo(epsilon-caprolactone) (OCL) monolayers were reversibly cross-linked forming two dimensional networks (2D) at the air-water interface. The equilibrium reaction with glyoxal as the cross-linker is pH-sensitive. Pronounced contraction in the area of the prepared 2DOCL films in dependence of surface pressure and time revealed the process of the reaction. Cross-linking inhibited crystallization and retarded enzymatic degradation of the OCLfilm. Altering the subphase pH led to a cleavage of the covalent acetal cross-links. The reversibility of the covalent acetal cross-links was proved by observing an identical isotherm as non-cross-linked sample. Besides as model systems, these customizable reversible OCL2D networks are intended for use as pHresponsive drug delivery systems or functionalized cell culture substrates.