Core cross-linked polymeric micelles (CCPMs) are designed to improve the therapeutic profile of hydrophobic drugs, reduce or completely avoid protein corona formation, and offer prolonged... Show moreCore cross-linked polymeric micelles (CCPMs) are designed to improve the therapeutic profile of hydrophobic drugs, reduce or completely avoid protein corona formation, and offer prolonged circulation times, a prerequisite for passive or active targeting. In this study, we tuned the CCPM stability by using bifunctional or trifunctional cross-linkers and varying the cross-linkable polymer block length. For CCPMs, amphiphilic thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) [pSar-b-pCys(SO2Et)] were employed. While the pCys(SO2Et) chain lengths varied from Xn = 17 to 30, bivalent (derivatives of dihydrolipoic acid) and trivalent (sarcosine/cysteine pentapeptide) cross-linkers have been applied. Asymmetrical flow field-flow fraction (AF4) displayed the absence of aggregates in human plasma, yet for non-cross-linked PM and CCPMs cross-linked with dihydrolipoic acid at [pCys(SO2Et)]17, increasing the cross-linking density or the pCys(SO2Et) chain lengths led to stable CCPMs. Interestingly, circulation time and biodistribution in mice of non-cross-linked and bivalently cross-linked CCPMs are comparable, while the trivalent peptide cross-linkers enhance the circulation half-life from 11 to 19 h. Show less
This thesis aimed to investigate core cross-linked polymeric micelles (CCPMs) and expand their potential for the delivery of hydrophobic drugs and co-factors. Applying polypept(o)ides as the... Show moreThis thesis aimed to investigate core cross-linked polymeric micelles (CCPMs) and expand their potential for the delivery of hydrophobic drugs and co-factors. Applying polypept(o)ides as the polymeric platform technology, the fundamental implications of secondary structure formation on ring-opening N-carboxyanhydride (NCA) polymerization and self-assembly were examined and optimized. CCPMs with functional core architectures serving external or disease-related stimuli were developed. To establish robust CCPM production, overcome drug resistance mechanisms, and explore therapeutic agents for immunomodulation, polymer science was combined with organic and inorganic chemistry. Show less
Core cross-linking of polymeric micelles has been demonstrated to contribute to enhanced stability that can improve the therapeutic efficacy. Photochemistry has the potential to provide spatial... Show moreCore cross-linking of polymeric micelles has been demonstrated to contribute to enhanced stability that can improve the therapeutic efficacy. Photochemistry has the potential to provide spatial resolution and on-demand drug release. In this study, light-sensitive polypyridyl-ruthenium(II) complexes were combined with polypept(o)ides for photocleavable core cross-linked polymeric micelles. Block copolymers of polysarcosine-block-poly(glutamic acid) were synthesized by ring-opening N-carboxyanhydride polymerization and modified with aromatic nitrile-groups on the glutamic acid side chain. The modified copolymers self-assembled into micelles and were cross-linked by cis-diaquabis(2,2'-bipyridine)-ruthenium(II) ([Ru(bpy)2(H2O)2]2+) or cis-diaquabis(2,2'-biquinoline)-ruthenium(II) ([Ru(biq)2(H2O)2]2+). Depending on the flexibility and hydrophobicity of the nitrile linker, either small spherical structures (Dh 45 nm, PDI 0.11) or worm-like micelles were obtained. The cross-linking reaction did not affect the overall size distribution but induced a change in the metal-to-ligand charge transfer peak from 482 to 420 nm and 592 to 548 nm. The cross-linked micelles displayed colloidal stability after incubation with human blood plasma and during gel permeation chromatography in hexafluoroisopropanol. Light-induced cleavage of [Ru(bpy)2(H2O)2]2+ was accomplished within 300 s, while [Ru(biq)2(H2O)2]2+ could not be completely released. Analysis in HuH-7 cells revealed increased cytotoxicity via micellar delivery of [Ru(bpy)2(H2O)2]2+ but mostly irradiation damage for [Ru(biq)2(H2O)2]2+. Further evaluation in ovo confirmed stable circulation pointing towards the future development of quick-release complexes. Show less
The secondary structure formation of polypeptides not only governs folding and solution self-assembly but also affects the nucleophilic ring-opening polymerization of alpha-amino acid-N... Show moreThe secondary structure formation of polypeptides not only governs folding and solution self-assembly but also affects the nucleophilic ring-opening polymerization of alpha-amino acid-N-carboxyanhydrides (NCAs). Whereby helical structures are known to enhance polymerization rates, beta-sheet-like assemblies reduce the propagation rate or may even terminate chain growth by precipitation or gelation. To overcome these unfavorable properties, racemic mixtures of NCAs can be applied. In this work, racemicS-(ethylsulfonyl)-dl-cysteine NCA is investigated for the synthesis of polypeptides, diblock and triblock copolypept(o)ides. In contrast to the polymerization of stereoregularS-(ethylsulfonyl)-l-cysteine NCA, the reaction ofS-(ethylsulfonyl)-dl-cysteine NCA proceeds with a rate constant of up tok(p) = 1.70 x 10(-3)L mol(-1)s(-1)and is slightly faster than the enatiopure polymerization. While the polymerization ofS-(ethylsulfonyl)-l-cysteine NCA suffers from incomplete monomer conversion and degrees of polymerization (DPs) limited to 30-40, racemic mixtures yield polypeptides with DPs of up to 102 with high conversion rates and well-defined dispersities (1.2-1.3). The controlled living nature of the ring-opening polymerization ofS-(ethylsulfonyl)-dl-cysteine NCA thus enables the synthesis of triblock copolymers by sequential monomer addition. This methodology allows for precise control over DPs of individual blocks and yields uniform triblock copolymers with symmetric molecular weight distributions at a reduced synthetic effort. Show less
The inverse electron demand Diets-Alder (IEDDA) reaction-initiated ligation between 1,2,4,5-tetra-zines (Tz) and trans-cyclooctenes (TCO) is one of the fastest bioorthogonal reactions known today... Show moreThe inverse electron demand Diets-Alder (IEDDA) reaction-initiated ligation between 1,2,4,5-tetra-zines (Tz) and trans-cyclooctenes (TCO) is one of the fastest bioorthogonal reactions known today and is therefore increasingly used for in vivo click chemistry. Described herein is the synthesis of Tz- and TCO-functionalised polypeptides, polypeptoids and polypeptide-block-polypeptoids (polypept(o) ides) by ring-opening polymerisation of the corresponding N-carboxyanhydrides using Tz- or TCO-functional amine initiators. Despite the reactivity of tetrazines, polymers with low dispersity and high end group integrity can be obtained as observed by gel permeation chromatography (GPC), nuclear magnetic resonance (NMR) spectroscopy and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Amphiphilic Tz-functionalised block copolypept(o)ides were used to prepare polymeric micelles and organic colloids by miniemulsion techniques, which may find an application as clearing agents in pretargeted nuclear imaging and therapy using efficient in vivo click chemistry. The reaction kinetics of the tetrazine ligation using the synthesised polymers and the accessibility of the Tz groups on the polymeric nanoparticles were evaluated using UV Vis and fluorescence correlation spectroscopy (FCS), and second-order rate constants were determined by stopped-flow spectrophotometry ensuring quantitative conversions in seconds at sub-millimolar concentrations (10-30 s). Show less
Dal, N.J.K.; Kocere, A.; Wohlmann, J.; Herck, S. van; Bauer, T.A.; Resseguier, J.; ... ; Fenaroli, F. 2020
The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real-time... Show moreThe zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real-time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse. Show less
