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Natural bioactive compounds have been studied for a long time for their chemopreventive and therapeutic potential in several chronic inflammatory diseases, including cancer. However, their physicochemical properties generally result in poor chemical stability and lack of in vivo bioavailability. Very few human clinical trials have addressed absorption, distribution, metabolism, and excretion of these compounds in relation to efficacy. This limits the use of these valuable natural compounds in the clinic. In this study, we examined caffeic acid (derivatives), carvacrol (derivatives), thymol, pterostilbene (derivatives), and N-(3-oxo-dodecanoyl)-l-homoserine lactone. These are natural compounds with strong anti-inflammatory properties derived from plants and bacteria. However, these compounds have poor water solubility or are chemically unstable. To overcome these limitations we have prepared liposomal formulations. Our results show that lipophilic 3-oxo-C(12)-homoserine lactone and stilbene derivatives can be loaded into liposomal lipid bilayer with efficiencies of 50-70%. Thereby, the liposomes solubilize these compounds, allowing intravenous administration without use of solvents. When compounds could not be loaded into the lipid bilayer (carvacrol and thymol) or are rapidly extracted from the liposomes in the presence of serum albumin (3-oxo-C(12)-homoserine lactone and pterostilbene derivatives), derivatization of the compound into a water-soluble prodrug was shown to improve loading efficiency and encapsulation stability. The phosphate forms of carvacrol and pterostilbene were loaded into the aqueous interior of the liposomes and encapsulation was unaffected by the presence of serum albumin. Chemical instability of resveratrol was improved by liposome-encapsulation, preventing inactivating cis-trans isomerization. For caffeic acid, liposomal encapsulation did not prevent oxidation into a variety of products. Still, by derivatization into a phenyl ester, the compound could be stably encapsulated without chemical degradation. Despite the instability of liposome-association of 3-oxo-C(12)-homoserine lactone and resveratrol, intravenous administration of these compounds inhibited tumor growth for approximately 70% in a murine tumor model, showing that simple solubilization can have important therapeutic benefits.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been receiving great attention lately due to their various biomedical applications, such as in MR imaging and image guided drug delivery. However, their systemic administration still remains a challenge. In this study, the ability of biodegradable thermosensitive polymeric micelles to stably encapsulate hydrophobic oleic-acid-coated SPIONs (diameter 5-10 nm) was investigated, to result in a system fulfilling the requirements for systemic administration. The micelles were composed of amphiphilic, thermosensitive, and biodegradable block copolymers of poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide dilactate] (mPEGb-p(HPMAm-Lac(2))) The encapsulation was performed by addition of one volume of SPIONs in THF to nine volumes of a cold aqueous mPEG-b-p(HPMAm-Lac(2)) Solution (0 degrees C; below the cloud point of the polymer), followed by rapid heating of the resulting mixture to 50 degrees C, to induce micelle formation ("rapid heating" procedure). Dynamic light scattering (DLS) measurements revealed that similar to 200 nm particles (PDI = 0.2) were formed, while transmission electron microscopy (TEM) analysis demonstrated that clusters of SPIONs were present in the core of the micelles. A maximum loading of 40% was obtained, while magnetic resonance imaging (MRI) scanning of the samples demonstrated that the SPION-loaded micelles had high r(2) and r(2)* relaxivities. Furthermore, the r(2)* values were found to be at least 2-fold higher than the r(2) values, confirming the clustering of the SPIONs in the micellar core. The particles showed excellent stability under physiological conditions for 7 days, even in the presence of fetal bovine serum. This, together with their ease of preparation and their size of similar to 200 nm, makes these systems highly suitable for image-guided drug delivery.

In recent years, colloidal systems (e.g. liposomes, nanoparticles and micelles) are increasingly applied as vehicles for controlled drug delivery purposes. Ideally, the encapsulation of hydrophobic drugs in a micellar core prolongs the systemic circulation and drug-loaded micelles selectively accumulate in diseased tissues as a result of the enhanced permeation and retention (EPR) effect. Furthermore, it is aimed for that the micelles dissociate only at the target site to allow the drug to exert a local therapeutic effect. Block copolymers comprising a hydrophilic part (e.g. mPEG) and a thermosensitive block (e.g. side-chain derivatised (meth)acrylamide backbone) self-assembled in water above their critical micelle temperature (CMT) into spherical micelles (60 - 80 nm). The hydrophobic side chains were cleaved off under physiological conditions, thereby increasing the hydrophilicity of the polymer. Ultimately, the CMT exceeded 37 C, and the polymer dissolved in water which led to micelle dissociation. The time required for micellar disassembly depended on the type of methacrylamide backbone and was 5 or 210 hours for N-(2-hydroxyethyl)methacrylamide (HEMAm) or N-(2-hydroxypropyl)methacrylamide (HPMAm), respectively. HPMAm-based micelles encapsulated a very hydrophobic photosensitiser (PS, used in the photodynamic therapy of localised tumours) up to a final concentration of ~ 2 mg/mL. In B16F10 and 14C cells, the PS-loaded micelles displayed a high photocytotoxicity, i.e. an IC50 of 3.0 +/- 0.2 nM in the presence of 10 % serum. Highly loaded micelles displayed a very good stability, also in the presence of 50 % serum, and the encapsulated PS was only released upon hydrolysis-induced micellar disassembly. Cellular uptake of rhodamine-labelled micelles by B16F10 or 14C cells demonstrated that micelles were only taken up to a very low extent (0.1 %). The stability of polymeric micelles was drastically enhanced by covalently crosslinking the micellar cores while retaining their biodegradability. After intravenous administration in mice, these stabilised micelles demonstrated a 6-fold higher tumour accumulation than non crosslinked particles. Moreover, the liver and spleen uptake was low, indicating that the small stabilised micelles successfully avoided macrophage uptake and extravasated via the EPR effect. However, administration of paclitaxel-loaded CCL micelles revealed that the drug was rapidly released and/or extracted from the stabilised micelles. Finally, the surface of fluorescently labelled and crosslinked micelles was decorated with thiol-reactive groups. These multifunctional particles can be used for the attachment of various targeting ligands to promote for instance cellular internalisation. In conclusion, the type of biodegradable thermosensitive micelles presented in this thesis can be used not only as a biocompatible and easy-to-load nanosized drug carrier with a high drug-loading capacity, but also embeds a controlled degradation mechanism. Especially the core crosslinked micelles display favourable characteristics in terms of circulation kinetics and tumour accumulation. Together with the potential targetability, these unique characteristics make biodegradable thermosensitive micelles very attractive as targeted drug delivery devices.