Functional Multilayer Films with Controlled Nanostructures for in vitro Cell Studies

Abstract

Functional polymer thin films are of great interest for diverse potential applications due to their excellent productivity and cost-efficiency. Thorough understandings on the relationship between structures and physicochemical properties of polymer thin films play pivotal roles in developing the films for the fine purpose. Among many different types of functional polymer thin films, the multilayer films with controlled nanostructures has been recently highlighted in many biomedical applications such as controlled release platforms, disease diagnosis platforms, and tissue engineering. Particularly, in order to develop such novel biomedical platforms based on the functional multilayer films, in vitro cell study has to be performed, which provides perspectives on regulating the cell behaviors. In this thesis, we focus on the layer-by-layer (LbL) strategy for fine tuning of structures and properties of polymer thin films as well as for constructing polymer platforms for in vitro studies on metastatic cancer cell behavior. In particular, the external stimuli-triggered release mechanism of polymer multilayer films has been systematically investigated, in terms of changes in internal structures and physicochemical properties, in order to establish the ground for designing the controlled release of target active materials from thin film coatings. Furthermore, the functionalization of multilayer films with controlled nanostructures has been carefully examined in the viewpoint of the control of cell-matrix or cell-cell interactions. In Chapter 1, the control strategies for engineering internal structures and swelling properties of polymer multilayer films are introduced on the basis of the LbL deposition, because it has been the one of the most efficient methods for preparing functional multilayer platforms, taking advantage of various intermolecular interactions among paired species. The growth rates of bilayer thickness and the internal structures in multilayer films were greatly controlled by tuning the range of the intermolecular interactions between polymer chains (i.e., long-range electrostatic interactions or short-range hydrogen bonding) and by the film fabrication methods (i.e., dip- or spin-assisted LbL deposition). The internal structures of multilayer thin films in nanometer scale were systematically investigated by the neutron reflectivity (NR) measurements, as a function of LbL deposition techniques and the types of intermolecular interactions. Furthermore, it was demonstrated that the loop and tail conformations of partially charged weak PE chains preferentially capture water molecules within multilayer films as compared to fully charged and tightly bound PE chains with stretched conformations by NR along with quartz crystal microbalance with dissipation monitoring (QCM-D) measurements. In Chapter 2, we have designed the controlled release platforms based on polyelectrolyte (PE) blend multilayer films to investigate the release mode and kinetics at the nanoscale level. The model blend multilayer films are composed of positively charged layers with weak PE (linear poly(ethylenimine), LPEI) and negatively charged blend layers with mixtures of strong (poly(sodium 4-styrenesulfonic acid), PSS) and weak (poly-(methacrylic acid), PMAA) PEs. The blend multilayer films ([LPEI/PSS:PMAA]n) with well-defined internal structure were prepared by the spin-assisted LbL deposition method. The changes in nanostructures and physicochemical properties of the blend multilayer films were systematically studied as a function of blend ratio by NR, ellipsometer, AFM, FT-IR spectroscopy, and QCM-D. Since PSS strong PEs serve as robust skeletons within the multilayer films independent of external pH variation, the burst disruption of pure weak PE multilayer films was dramatically suppressed, and the release kinetics could be accurately controlled by simply changing the PSS content within the blend films. These release properties of blend multilayer films form the basis for designing the controlled release of target active materials from surfaces. In Chapter 3, we present the effect of molecular weight (MW) of PEs on the disintegration behavior of weak PE multilayer films consisting of LPEI and PMAA. The multilayer films prepared by the spin-assisted LbL deposition have well-ordered internal structures and also show the linear thickness growth behavior regardless of MWs of PMAA. The well-defined weak PE multilayer films were subject to disintegration into bulk solution when the electrostatic interactions between LPEI and PMAA layers were reduced by treatment at pH 2. However, we demonstrated the change in the disintegration mode and kinetics (i.e., from burst erosion to controlled surface erosion) as a function of MW of PMAA based on neutron reflectivity (NR) and quartz crystal microbalance with dissipation (QCM-D), revealing the correlation between the structural changes and the viscoelastic responses of the weak PE films upon pH treatment. Also, the unique swelling behavior as well as the significant increase in dissipation energy was monitored before the complete disintegration of the multilayer films containing high MW PMAA, which is believed to originate from their slow rearrangement kinetics within the film. We believe that the results shown in this study provide chain-level understanding as to the MW-dependence on pH-triggered disintegration mechanism of weak PE multilayer films. In Chapter 4, we have developed in vitro platforms for studying metastatic cancer cell behavior, based on the surface modification of LbL-assembled multilayer films with functional nanoparticles and biomolecules. The polymer multilayer platforms prepared by spin-assisted LbL deposition provide controlled surface charge and stable mechanical property in cell culture environments, which could offer easy surface modification with charged functional molecules while creating intimate cell-surface contacts. Gold nanoparticles (AuNPs) were employed to modify flat LbL surfaces and investigated the effect of nano-topographical cues on the metastatic cancer cell focal adhesion, shape and motility. Moreover, cellular signaling process with proteins in extracellular matrices (ECMs) was mimicked and analyzed by incorporating biomolecule-conjugated AuNPs onto LbL films. As a result, it is confirmed that the existence of nanotopographical features with a cell adhesion protein (fibroectin, Fn) is critical in inducing dramatic changes in metastatic cancer cell adhesion, protrusion, polarity and motility than the presence of the Fn on the flat multilayer surfaces. Also, the detachement signaling mediated by ephrinB3 was found to be more effective when the ephrinB3 were modified to the nanofeatured surfaces than flat surfaces. The results in this study would give insights on the basic understanding of tumor metastasis regulated by extracellular environmental signals. In Chapter 5, we have developed novel multilayered co-culture platforms with nanoporous cellulose acetate (CA) membranes for efficient in vitro analysis of cell-cell communications. The CA films designed in this chapter have high number density of well-defined nanopores and unique natures of transparency and transferability in cell culture environments. The transparent, transferable and nanoporous (TTN) CA membrane platforms allow for imaging and analyzing cells on each layer as well as mediating the paracrine communications between co-cultivated cells. The communications between human breast metastatic cancer cell (MDA-MB-231) and three different types of stromal cells [fibroblast (NIH-3T3), myoblast (C2C12), and human mesenchymal stem cell (hMSC)] via the TTN membrane were systematically investigated by cytokine and cell migration assays based on the high flexibility in stacking and destacking of the TTN platforms. The TTN membranes would address the issues from conventional membrane-separated cell co-culture platforms that lack the routes for cell-cell communications and direct cell-cell contact assay that does not offer the flexibility in studying cell-cell communications. We strongly believe that the research work in this thesis as to the engineering of the nanostructures and physicochemical properties in polymer thin films could eventually contribute to explore new perspectives on functional multilayer films and could also open up new possibilities to design flexible and multifunctional structures for numerous biological applications such as controlled release platforms and in vitro disease cell assay platforms.