Self-folding polymer films were only recently proposed as an alternative method for the design of three-dimensional constructs. Due to the relative novelty of the approach, insufficient amount of data on the behavior of such systems is available in the literature. This study is bound to fill the gaps and give a deeper insight into the understanding of how and why different types of folding occur. In this study, four different types of folding of polymer bilayers are presented. Rectangles are one of the simplest geometrical forms and were therefore adopted as a convenient initial system for the investigation of the folding behavior of polymer bilayers. We chose PNIPAM for the active polymer, as it is a well-studied polymer with sharp Lower Critical Transition Temperature at around 33 C. For the passive layer, poly(methyl methacrylate) and poly(caprolactone) were chosen. The influence of different parameters of the system, such as polymer thickness and temperature was thoroughly investigated in order to be accounted for in later experiments. It was demonstrated that bilayers placed on a substrate start to roll from the corners due to quicker diffusion of water. Rolling from the long-side starts later but dominates at high aspect ratio. We showed that the main reasons causing a variety of rolling scenarios are (i) non-homogenous swelling due to slow diffusion of water in hydrogels and (ii) adhesion of polymer to a substrate until a certain threshold. Moreover, non-homogenous swelling determines folding in the first moments, while adhesion plays a decisive role at later stages of folding. After having understood the abovementioned basics, we decided to explore how those applied to more complex shapes. For the purpose, four- and six-arm stars were chosen, the main idea behind this being the creation of self-folding polymer capsules capable of encapsulation of microparticles and cells. Adjusting the polymer thickness and thus the radius of folding allowed creating structures, capable of reversible self-folding and unfolding. The possibility to reversibly encapsulate and release objects in the micro-range was demonstrated on the example of yeast cells. Noteworthy, the capsules were produced by means of the same process we used for the design of tubes – when compared to the folding of rectangles, it was the shape of the initial pattern and the folding radius that were changed; the mechanism was the same – simple one-step folding towards the center of the bilayer. Clearly the number of structures that can be generated by this method is fairly limited. The search for means to overcome this constraint led to the idea of hierarchical multi-step folding. Due to the edge-activation of the bilayers, the observed deformed shapes differ from the classical ones obtained by homogeneous activation. It was found that films could demonstrate several kinds of actuation behavior such as wrinkling, bending and folding that result in a variety of shapes. It was demonstrated that one can introduce hinges into the folded structure by proper design of the bilayer's external shape through diffusion without having to use site selective deposition of active polymers. Experimental observations led us to derive four empirical rules: 1) “Bilayer polymer films placed on a substrate start to fold from their periphery and the number of formed wrinkles/tubes decreases until the angle between adjacent wrinkles/tubes approaches 130°”; 2) “After the wrinkles along the perimeter of the film form tubes, further folding proceeds along the lines connecting the vertexes of the folded film”; 3) “The folding goes along the lines which are closer to the periphery of the films”; 4) „Folding of the rays may result in blocking of the neighboring rays if the angle between the base of the folded ray and the shoulders of the neighboring rays is close to 180°”. These rules were then applied to direct the folding of edge-activated polymer bilayers through a concrete example - the design of a 3D pyramid. One consequence of the second and third rules is that generally triangles are formed during the multi-step folding process. In order to create a cube, or any other 3D structure with non-triangular sides, an effective way to stop the folding along the lines, connecting neighboring vertexes had to be thought of. A possible solution would be the insertion of a rigid element inside the bilayer, perpendicular to the direction of folding. The solution of this problem was to design structures with pores. A pore would normally decrease the rigidity of a structure but in our case, a pore basically comprised an edge inside the structure and could thus form tubes which, as was already shown, exhibit much higher rigidity than a film. On the other hand, a pore, or many pores, would expose different parts of the active layer to the solvent and would strongly influence its swelling and, as a consequence, folding behavior. Hence, the influence of a pore on the swelling and the folding behavior of polymer bilayers had to be investigated. It was shown that pores of the right form and dimensions did indeed hinder the folding as intended. Instead, the polymer films took other ways to fold. As a result, despite the correctness of our reasoning, we failed to produce a cube by hierarchical folding of polymer bilayers. However, other sophisticated 3D objects were obtained, further increasing the arsenal of available structures, as well as giving an in-depth insight on the folding process.

Self-folding polymer films were only recently proposed as an alternative method for the design of three-dimensional constructs. Due to the relative novelty of the approach, insufficient amount of data on the behavior of such systems is available in the literature. This study is bound to fill the gaps and give a deeper insight into the understanding of how and why different types of folding occur. In this study, four different types of folding of polymer bilayers are presented. Rectangles are one of the simplest geometrical forms and were therefore adopted as a convenient initial system for the investigation of the folding behavior of polymer bilayers. We chose PNIPAM for the active polymer, as it is a well-studied polymer with sharp Lower Critical Transition Temperature at around 33 C. For the passive layer, poly(methyl methacrylate) and poly(caprolactone) were chosen. The influence of different parameters of the system, such as polymer thickness and temperature was thoroughly investigated in order to be accounted for in later experiments. It was demonstrated that bilayers placed on a substrate start to roll from the corners due to quicker diffusion of water. Rolling from the long-side starts later but dominates at high aspect ratio. We showed that the main reasons causing a variety of rolling scenarios are (i) non-homogenous swelling due to slow diffusion of water in hydrogels and (ii) adhesion of polymer to a substrate until a certain threshold. Moreover, non-homogenous swelling determines folding in the first moments, while adhesion plays a decisive role at later stages of folding. After having understood the abovementioned basics, we decided to explore how those applied to more complex shapes. For the purpose, four- and six-arm stars were chosen, the main idea behind this being the creation of self-folding polymer capsules capable of encapsulation of microparticles and cells. Adjusting the polymer thickness and thus the radius of folding allowed creating structures, capable of reversible self-folding and unfolding. The possibility to reversibly encapsulate and release objects in the micro-range was demonstrated on the example of yeast cells. Noteworthy, the capsules were produced by means of the same process we used for the design of tubes – when compared to the folding of rectangles, it was the shape of the initial pattern and the folding radius that were changed; the mechanism was the same – simple one-step folding towards the center of the bilayer. Clearly the number of structures that can be generated by this method is fairly limited. The search for means to overcome this constraint led to the idea of hierarchical multi-step folding. Due to the edge-activation of the bilayers, the observed deformed shapes differ from the classical ones obtained by homogeneous activation. It was found that films could demonstrate several kinds of actuation behavior such as wrinkling, bending and folding that result in a variety of shapes. It was demonstrated that one can introduce hinges into the folded structure by proper design of the bilayer's external shape through diffusion without having to use site selective deposition of active polymers. Experimental observations led us to derive four empirical rules: 1) “Bilayer polymer films placed on a substrate start to fold from their periphery and the number of formed wrinkles/tubes decreases until the angle between adjacent wrinkles/tubes approaches 130°”; 2) “After the wrinkles along the perimeter of the film form tubes, further folding proceeds along the lines connecting the vertexes of the folded film”; 3) “The folding goes along the lines which are closer to the periphery of the films”; 4) „Folding of the rays may result in blocking of the neighboring rays if the angle between the base of the folded ray and the shoulders of the neighboring rays is close to 180°”. These rules were then applied to direct the folding of edge-activated polymer bilayers through a concrete example - the design of a 3D pyramid. One consequence of the second and third rules is that generally triangles are formed during the multi-step folding process. In order to create a cube, or any other 3D structure with non-triangular sides, an effective way to stop the folding along the lines, connecting neighboring vertexes had to be thought of. A possible solution would be the insertion of a rigid element inside the bilayer, perpendicular to the direction of folding. The solution of this problem was to design structures with pores. A pore would normally decrease the rigidity of a structure but in our case, a pore basically comprised an edge inside the structure and could thus form tubes which, as was already shown, exhibit much higher rigidity than a film. On the other hand, a pore, or many pores, would expose different parts of the active layer to the solvent and would strongly influence its swelling and, as a consequence, folding behavior. Hence, the influence of a pore on the swelling and the folding behavior of polymer bilayers had to be investigated. It was shown that pores of the right form and dimensions did indeed hinder the folding as intended. Instead, the polymer films took other ways to fold. As a result, despite the correctness of our reasoning, we failed to produce a cube by hierarchical folding of polymer bilayers. However, other sophisticated 3D objects were obtained, further increasing the arsenal of available structures, as well as giving an in-depth insight on the folding process.

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This thesis is focused on the design and development of an approach, allowing the fabrication of biocompatible/biodegradable self-rolled polymer tubes, which are sensitive to stimuli at physiological conditions, can be homogenously filled with cells and are able to self-assemble into a complex 3D construct with uniaxially aligned pores. These constructs are aimed to recreate the microstructure of tissues with structural anisotropy, such as of muscles and bones. The approach consists of two steps of self-assembly. As a first step, cells are adsorbed on the top of an unfolded bilayer; triggered rolling results in a parallel encapsulation of cells inside the tubes. As a second step, the formed self-rolled tubes with encapsulated cells can be assembled in a uniaxial tubular scaffold. Three polymer systems were designed and investigated in the present work in order to allow triggered folding of the bilayer. These systems allow either reversible or irreversible tube formation. The possibility to encapsulate microobjects inside self-rolled polymer tubes was demonstrated on the example of silica particles, yeast cells and mammalian cells. At conditions when bilayer film is unfolded, particles or cells were deposited from their aqueous dispersion on the top of bilayer. An appropriate change of conditions triggers folding of the bilayer and results in encapsulation of particles or cells inside the tubes. One way swelling of an active polymer allows irreversible encapsulation of cells in a way that tubes do not unroll and cells cannot escape. It was demonstrated that encapsulated cells can proliferate and divide inside the tubes for a long period of time. Since used polymers are optically transparent, encapsulated cells can be easily observed using optical and fluorescent microscopy. Reversible swelling of an active polymer provides the possibility to release encapsulated objects. It was demonstrated that in aqueous media microtubes possessing small amount of negatively charged groups on external walls self-assemble in the presence of oppositely charged microparticles that results in a formation of 3D constructs. In obtained aggregates tubes and therefore pores were well-aligned and the orientation degree was extremely high. Moreover, the approach allows the design of porous materials with complex architectures formed by tubes of different sorts. The assembly of cell-laden microtubes results in a formation of uniaxial tubular scaffold homogeneously filled with cells. The results presented in this work demonstrate that the proposed approach is of practical interest for biotechnological applications. Self-rolled tubes can be filled with cells during their folding providing the desired homogeneity of filling. Individual tubes of different diameters could be used to investigate cell behaviour in confinement in conditions of structural anisotropy as well as to mimic blood vessels. Due to their directionality tubes could be used to guide the growth of cells that is of interest for regeneration of neuronal tissue. Reversibly foldable films allow triggered capture and release of the cells that could be implemented for controlled cell delivery. In perspective, self-assembled 3D constructs with aligned pores could be used for bottom-up engineering of the scaffolds, mimicking such tissues as cortical bone and skeletal muscle, which are characterized by repeating longitudinal units. Such constructs can be also considered as a good alternative of traditional 2D flat cell culture.

This thesis is focused on the design and development of an approach, allowing the fabrication of biocompatible/biodegradable self-rolled polymer tubes, which are sensitive to stimuli at physiological conditions, can be homogenously filled with cells and are able to self-assemble into a complex 3D construct with uniaxially aligned pores. These constructs are aimed to recreate the microstructure of tissues with structural anisotropy, such as of muscles and bones. The approach consists of two steps of self-assembly. As a first step, cells are adsorbed on the top of an unfolded bilayer; triggered rolling results in a parallel encapsulation of cells inside the tubes. As a second step, the formed self-rolled tubes with encapsulated cells can be assembled in a uniaxial tubular scaffold. Three polymer systems were designed and investigated in the present work in order to allow triggered folding of the bilayer. These systems allow either reversible or irreversible tube formation. The possibility to encapsulate microobjects inside self-rolled polymer tubes was demonstrated on the example of silica particles, yeast cells and mammalian cells. At conditions when bilayer film is unfolded, particles or cells were deposited from their aqueous dispersion on the top of bilayer. An appropriate change of conditions triggers folding of the bilayer and results in encapsulation of particles or cells inside the tubes. One way swelling of an active polymer allows irreversible encapsulation of cells in a way that tubes do not unroll and cells cannot escape. It was demonstrated that encapsulated cells can proliferate and divide inside the tubes for a long period of time. Since used polymers are optically transparent, encapsulated cells can be easily observed using optical and fluorescent microscopy. Reversible swelling of an active polymer provides the possibility to release encapsulated objects. It was demonstrated that in aqueous media microtubes possessing small amount of negatively charged groups on external walls self-assemble in the presence of oppositely charged microparticles that results in a formation of 3D constructs. In obtained aggregates tubes and therefore pores were well-aligned and the orientation degree was extremely high. Moreover, the approach allows the design of porous materials with complex architectures formed by tubes of different sorts. The assembly of cell-laden microtubes results in a formation of uniaxial tubular scaffold homogeneously filled with cells. The results presented in this work demonstrate that the proposed approach is of practical interest for biotechnological applications. Self-rolled tubes can be filled with cells during their folding providing the desired homogeneity of filling. Individual tubes of different diameters could be used to investigate cell behaviour in confinement in conditions of structural anisotropy as well as to mimic blood vessels. Due to their directionality tubes could be used to guide the growth of cells that is of interest for regeneration of neuronal tissue. Reversibly foldable films allow triggered capture and release of the cells that could be implemented for controlled cell delivery. In perspective, self-assembled 3D constructs with aligned pores could be used for bottom-up engineering of the scaffolds, mimicking such tissues as cortical bone and skeletal muscle, which are characterized by repeating longitudinal units. Such constructs can be also considered as a good alternative of traditional 2D flat cell culture.