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Organic molecules with π electron systems and asymmetrical charge distribution are interesting tor use in quadratic nonlinear optics. Calix[4]arenes are cyclophanes that consist of four phenol moieties connected by methylene bridges. Functionalization of these phenols allows the combination of four D-π–A units within a single molecule. The increase in the number of D-π-A systems in a calix[4]arene molecule is accompanied by an enhancement of the molecular hyperpolarizability and the dipole moment and by a blue shift of the charge-transfer absorption band. Different substituted calix[4Jarene compounds have been synthesized and examined.

Nanotechnology provides the tools to influence biological processes on the cellular and molecular level, which offers great opportunities for medical applications, such as targeted imaging and drug delivery. Owing to their tremendous variability, polymeric nanoparticles can act as a versatile delivery vehicle for targeted imaging and controlled drug delivery. Hydrogel nanoparticles, or nanogels, are a class of biocompatible nanostructures, which can be used for imaging and delivery of therapeutics. Nanogels can be rendered responsive to a range of biophysical cues for triggered release and environment-responsive imaging. Moreover, zwiterionic nanogels show minimal nonspecifc interactions with proteins and cells, rendering them highly stable in biological environments and very suitable for targeting specifc tissues and cell types. This thesis describes the formation and evaluation of peptidomimetic zwiterionic poly(amido amine) nanogels as a suitable carrier for the targeted delivery of imaging agents and therapeutics.

Layer-by-layer (LbL) assembly is a versatile and economical method to coat existing biomaterials with multilayered thin films for added functionality. The technique requires at least two complementary macromolecules to be deposited alternately onto a surface, forming thin multiple layers with properties that are customizable through the deposition conditions such as pH, temperature, and ionic strength. As a class of synthetic peptidomimetic polymers that have shown significant promise as therapeutic delivery agent through the ability to interact with DNA, proteins, carbohydrates, and small drug molecules, poly(amido amine)s (PAA) are promising multilayer components. This thesis describes a series of reports on the use of PAA as a component for LbL assembly. The reports describe the study on structure-function relationship of the various PAAs to the build-up profiles, and properties and functionality of the resulting multilayers. The report starts with the pairing of PAAs with DNA to yield multilayer-coated surfaces that can facilitate cell transfection. The optimization of such surface-mediated cell transfection and its successful demonstration on 2D and 3D tissue engineering scaffolds from several common biomaterials are described in the last Chapter. The PAA-multilayers were also studied as drug-eluting surfaces. For this purpose, boronic ester formation was utilized to provide multi-responsive interactions with small drug compounds. In one Chapter, boronic acid-functionalized PAAs were paired with chondroitin sulfate, a natural biopolymer, to entrap alizarin red S, a model for catechol drugs. The multilayer-coated surfaces successfully promote intracellular uptake of polymer-complexed alizarin red S in vitro. In another Chapter, PAA was paired with poly(vinyl alcohol), an FDA-approved biomaterial, to entrap bortezomib, a drug for multiple myeloma. Depending on how the multilayers were fabricated, the therapeutic efficacy could be tuned to differently affect target cells depending on their spatial location. The results in this thesis indicate great promise for poly(amido amine)-based multilayered thin films.

Since 40 years, angiogenesis has been recognized to be a prominent factor in the development of solid tumors and has been regarded as an attractive target for cancer therapy. A promising strategy to interfere with diseases where (over)expression of specific genes contributes to the pathological process, such as angiogenesis, is gene silencing by RNA interference (RNAi). So far, clinical success of RNAi-based strategies to inhibit tumor angiogenesis have been limited due to (1) scarcity of clinically promising pharmacological targets and/or (2) inefficiency or toxicity of the siRNA carrier system. In this thesis, we attempted to improve both siRNA target and carrier. Therefore, we studied a novel small Rho GTPase protein, Rac1, as a potential target for anti-angiogenic therapy. Our results indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis and that knockdown of Rac1 using siRNA may represent an attractive approach to inhibit tumor angiogenesis and growth. At the same time, we tried to adapt and improve a novel class of biodegradable poly(amido amine)s, with built-in characteristics for intracellular release of its siRNA payload, for drug delivery purposes. By titrating the positive charges, these polymers were optimized for siRNA delivery. PEGylation of the polymers resulted in increased stability of siRNA polyplexes in salt and serum and further decreased their toxicity. Additionally, we modified a previously described lipid-based carrier for siRNA to target angiogenic endothelial cells. We show that these systems ensure cellular uptake and gene silencing in both murine and human endothelial cells. Further research should evaluate the use of these carriers for gene silencing in vivo. This thesis provides starting points for future research on the optimization of polymeric and lipidic carrier systems for silencing of genes involved in angiogenesis. This may ultimately lead to the development of safe delivery systems that enable therapeutic RNA interference in cancer therapy.

Boronic acid functionalized polymers are a very interesting class of materials capable of forming reversible covalent boronic esters. With boronic acids reversible binding of diols and polyols, including carbohydrates, is possible. Although such polymers are promising biomaterials, they are only rarely explored. Thus the goal of this research was to investigate the potential of boronic acid functionalized polymers for biomedical applications. In the first part of this thesis, phenylboronic acids were introduced into the side chains of disulfide containing poly(amido amine)s (SS-PAAs), a promising class of polymers for plasmid DNA and siRNA delivery. Polyplex formation and stability, and transfection efficiency and cell viability of these novel boronated polyplexes were evaluated in vitro. Their capacity for complexation with nucleic acids into polyplexes, polyplex stability, transfection efficiency, and cell viability of cells treated with these polymers were evaluated. These novel boronated polymers offer new possibilities for combined drug and gene delivery. Alizarin red was selected as model drug compound as it is capable of forming a reversible covalent fluorescent ester with boronic acids. Similarly, FITC-labeled dextran was used to form boronic esters. Both strategies allowed for the visualization of the polymeric nanoparticles and polyplexes and made it possible to study the particle uptake by confocal microscopy and FACS. In the second part of this thesis, boronic acid functionalized polymers were found to form strong gels with poly(vinyl alcohol). The thermo-reversible, pH-responsive, and glucose-responsive properties of these gels could be tuned by varying the architecture of the boronic acid and by selecting different polymeric backbones, including poly(amido amine)s, Jeffamines, and multi-arm PEGs. Altogether, it can be concluded that incorporating boronic acid moieties in polymers results in new materials (nanoparticles and hydrogels) with unique properties that may ultimately result in new therapies.

This study describes the design and development of multifuctional poly(amidoamine)s (PAAs) capable to form self-assembled nanocomplexes with peptides and proteins, as functional bioresponsive vectors for protein delivery to targeting cells in vitro and in vivo. The representative examples of this novel family of water-soluble PAAs have very promising properties to function as vectors for highly potent and non-toxic intracellular protein delivery. The synthesis procedure of these polymers is compatible with the use of a variety of functionalized amine monomers, allowing large variation in the structure of the polymers, and tuning of the chemical and biophysical properties that are of significance for efficient protein delivery. We have designed PAAs able to form nanosized complexes at neutral pH by a self-assembly process with model proteins, such as β-galactosidase, lysozyme, human serum albumin, ovalbumin, p24 and human insulin, and a model peptide for brain therapy. These nanocomplexes are stable at physiological pH and efficiently protect protein integrity (i.e. no denaturation occurs). Upon acidification of the solution after endosomal uptake, the pH decrease and consequently the change of charge lead to decreased protein-polymer interaction and thus a tendency for the nanoparticles to destabilize. Moreover, for nanocomplexes made from PAAs containing disulfide linkages in their polymeric backbone (SS-PAAs), fast disintegration of the complexes and release of the protein payload is observed in reductive solution, mimicking the intracellular environment. Profection experiments showed that proteins encapsulated in PAA nanoparticles were successfully internalized into the cells with remaining activity and excellent cell-viability. In addition of providing intracellular bioreduction, the presence of the disulfide groups also increased the mucoadhesive properties of the nanoparticles. In the presence of mucus secreting cells, we showed that the SS-PAA-based nanocomplexes were capable to successfully internalize protein into the cell cytosol with a much higher extent than nanoparticles based on PAAs lacking disulfide linkages. The results described in this thesis indicate that PAAs should create opportunities for novel applications in protein delivery for oral and nasal administration, as well as delivery to the brain.

Successes in RNA interference based therapies are still limited due to the lack of efficient delivery of the mediator, small interfering RNA (siRNA), to the targeted site. The key to success can be the delivery of the siRNA molecules by polymer-based carrier systems, since they can be chemically tailored for efficient siRNA encapsulation, protection and intracellular delivery. Recently, bioreducible poly(amido amine)s proved to be a promising class of polymers in the delivery of DNA. In this thesis the development of bioreducible poly(amido amine)s for the intracellular delivery of siRNA is reported. Polymer structure/function relationships on siRNA complexation/decomplexation, cellular delivery, gene silencing efficiency and cytotoxicity were extensively studied in vitro. The results of this thesis show great promise for future research on therapeutic applications with siRNA.

This thesis describes the design and development of bioreducible poly(amido amine)s as non-viral vectors for gene delivery in vitro and in vivo. The structural influences of these polymers on their physico-chemical properties and gene delivery properties, transfection capability and cytotoxicity in vitro are discussed in detail. Preliminary results on in vivo gene delivery to ovarian cancer cells with the bioreducible poly(amido amine)s are also described.