Block copolymer self-assembly and co-assembly : shape function and application

Amphiphilic block copolymers can, in selective solvents such as water, assemble into various shapes and architectures. Among those, polymer vesicles, polymer micelles and polymer fibers are very popular structures in current nanotechnology. These objects each have their own particular properties and can serve as containers or templates for different nanotechnological applications. Polymer vesicles, for example, can encapsulate both hydrophobic and hydrophilic molecules, and are therefore considered as attractive candidates for drug delivery, nanoreactors and also microtemplates. Polymeric micelles are usually significantly smaller. Their hydrophobic cores are typically used to solubilize hydrophobic drugs or carry biomolecules. Polymer fibers by construction are long continuous entities. Because of this, they easily form gels at relatively low concentrations and may find functions as templates/substrates in biological applications. In some cases the can carry or conduct charges which may trigger their use in electronic devices. In reality, many applications of these objects require stimuli-responsive properties, and very importantly for biomedical applications, the block copolymer building blocks need to have zero or very low toxicity and preferably biocompatibility. It is a big challenge to assemble functional molecules into stable nanostructures with desired size and shape. To achieve these goals, (bio)functional polymers chains have to be internalized in the objects. For this purpose two distinct routes are proposed. One of them is self-assembly of functional block copolymers into a defined nanoparticle, which is a direct approach. In this case some preliminary work is required because the (bio)functional groups have to be chemically incorporated in the block copolymer. Alternatively, more than one type of copolymer can be co-assembled into large objects with fixed stoichiometry, each with their own functionalities. In these coassembled objects, we combine functionalities of its constituents in a controlled way. Both approaches are applied in this dissertation and results from both routes are promising. Pluronic polymers are an interesting class of materials that in water self-assemble in association colloids of various sizes and shapes. These polymers are highly temperature sensitive. One member of the Pluronics family forms unilamellar vesicles, namely L121, but these vesicles are extremely hard to work with. This is because they are extremely fragile and termally unstable. Much work that is reported in this dissertation has resulted from attempts to improve the stability of L121 vesicles. It was found that these vesicles now can also act as a template for the assembly of stimuli-responsive polypeptide block copolymers. These co-assembled vesicles can serve as multifunctional containers for transport of several biomolecules in vivo. The assembly rules apply to several other systems as well. This dissertation is divided into three parts, and a short overview for each part is given below. Part 1: Self assembly In Chapter 2 it is described how Pluronic L121 vesicles can be stabilized by means of a polymerized network of pentaerythritol tetraacrylate (PETA). The permanent interpenetrating network allows the Pluronic L121 copolymers to reversibly associate with the vesicles. The size of the vesicles can easily be varied from at least 100 nm to 1 μm, depending on the pore size of the extrusion membrane. The stabilized vesicles can retain their sizes for more than one month at room temperature. In Chapter 3 it was shown that such Pluronic L121 vesicles, stabilized by means of a network of polymerized PETA can become negatively charged upon mixing with polyacrylic acid (PAA). This can be used to immobilized these vesicles onto glass as well as onto mica surfaces in combination with Mg2+ ions that are used as bridge between the two negatively charged components, polyelectrolyte and the surface. This permits relatively easy observations with CLSM. It was found that the vesicles can change their shape to tubular vesicles upon addition of another micelle-forming Pluronic block copolymer. The latter shape transition also occurs for corresponding conditions in the bulk. Part 2: Co-assembly In Chapter 4 is described how small unilamellar vesicles spontaneously form in simple mixtures of a lamellae-forming block copolymer PB10PE10 and a micelleforming block copolymer PB10PE18. The co-assembled vesicles are rather monodisperse with a core radius down to 30 nm. Furthermore, it was found that under these conditions the polymersomes coexist with mixed spherical micelles. The polydispersity of the vesicles as well as their sizes are well controlled both by the mixing ratio of the components as by the polymer concentrations used. In Chapter 5, a molecularly detailed self-consistent field theory is used to study the assembly characteristics of binary polymer mixtures in a selective solvent. We mimic the binary mixture of the lamellae-forming copolymer PB10PE10 mixed with the micelle-forming PB10PE18 species. It is possible to arrive at a mixing ratio where small unilamellar vesicles coexist with many spherical micelles. We argue that the vesicle size can be tuned by the copolymer mixing ratio. For still modest PE18/PE10 ratios, one enters a regime where the micelles are the dominant species and these micelles buffer the chemical potential of the copolymers in the system. In such a situation one can store more PE18 chains in smaller vesicles than in larger ones. Hence, upon an increase of the amount of PB10PE18 in the system, the vesicles evolves towards small sizes. In addition the SCF model explains many other salient properties of these systems. Hence, the SCF analysis gives strong support for the expectation that in binary polymer mixtures it is possible to have thermodynamically stable small unilamellar vesicles. In Chapter 6, a new type of co-assembled, highly-ordered conjugated nanotape, made from either positively or negatively charged triblock peptide copolymers, and a conjugated zwitterionic polythiophene derivative (PTT) is described. The interaction of these compounds results in a change of the conformation and the electronic structure of the polythiophene. Morphological studies show that the co-assembled nanotapes are longer at a higher concentration and a higher PTT/triblock peptide copolymer ratio. In Chapter 7, a similar species was prepared from this conjugated zwitterionic polythiophene (PTT) derivative and a low molecular weight gelator. The inter action of these compounds results in complex assemblies with very large aspect ratios, wherein the polythiophenes molecules are in an extended, planar conformation. Morphologic studies have shown that the co-assembled nanowires adopt a helical structure that forms well-defined nanowires with lengths up to several μm long. These nanowires can be rather simply and reversibly manipulated by external stimuli, such as pH. Part 3: Applications In an attempt to further improve the working window of L121 vesicles, in Chapter 8, Pluronic L121 vesicles were stabilized by both PETA and Pluronic P85 micelles. The double-stabilized Pluronic L121 vesicles are stable to at least 33 ºC. We can routinely control the size of these vesicles by extruding them through an appropriately sized filter before the interpenetrating polymer network is formed. Furthermore, we can covalently polymerize fluorescent molecules in the interpenetrating network, and these stabilized fluorescent vesicles can be easily taken up by cells. This system is potentially useful for molecular imaging, biological assays, as biomarker, for cell-labeling, etc. In Chapter 9, a study on the co-assembly of a genetically designed peptide block copolymer with Pluronic vesicles to form templated protein vesicles is described. Now, several oppositely charged biological polyelectrolytes can interact with the peptide in the vesicle and become incorporated into the protein polymer. The structural features of the protein polymersome are investigated in detail, and the functionalized protein vesicles can be simply internalized in human cells. This method provides an intelligent approach to incorporate multifunctional materials for drug delivery and gene delivery applications. In Chapter 10, Pluronic micelles are stabilized by an interpenetrating network, which keeps them stable for months. Furthermore, fluorescent molecules are covalently polymerized in the interpenetrating network, and these stabilized fluorescent micelles can be easily taken up by HeLa cells and remain integrated in the cytoplasm. FCS studies of these micelles in living cells indicate that the diffusion properties are significantly different from those of micelles dissolved in aqueous solution. These results may be used to probe the local viscosity in the cell, because there is sufficient evidence that the micelles do not change their size and shape after internalisation. In Chapter 11, it is shown that similar nanoparticles can act as nanometersized thermal sensors. Within a short temperature range, the emission of the nanoparticles changes dramatically and reversibly as a function of temperature. These stabilized fluorescent micelles can be easily taken up by HeLa cells, where they can be used as intracellular nanosized thermometers