Self-assembling auto-fluorescent amphiphiles : nano-sized platform technology for multi-purpose cellular targeting

Amphiphilic molecules emerged as versatile building blocks for the generation of nano-sized architectures in water, as they can be programmed to self-assemble into a wide range of different topologies. In this thesis the generation of auto-fluorescent heterovalent nano-sized structures was explored using two types of amphiphilic scaffolds: disc-shaped and linear amphiphiles self-assembling in water into columnar polymers or into amorphous spherical nanoparticles, respectively. Numerous applications for self-assembling nanostructures were reported in literature based on amphiphilic molecules, ranging from imaging to diagnostics, and from drug delivery to tissue engineering. Many of these applications require the capability of the supramolecular system to actively target specific cell surface receptors. This is typically achieved through decoration with bioactive epitopes such as small molecules, peptides, and proteins. As discussed in chapter 1, the bioactive epitopes can either already be part of the monomeric supramolecular building blocks (pre-functionalization) or introduced after self-assembly via covalent attachment to appending reactive groups (post-functionalization). Selective and multivalent binding of disc-shaped amphiphiles to bacterial receptors was previously shown through the introduction of three functional groups at the periphery of the ethylene oxide tails and subsequent functionalization with bioactive ligands. Here, to expand the library of scaffolds, amphiphiles containing either nine amine functionalities or a single amine, azide and propargyl group were synthesized. Their decoration with bioactive ligands such as peptides, carbohydrates, small molecules and fluorescent dyes using both amide coupling and copper-catalyzed azide-alkyne cycloaddition is described in chapter 2. The orthogonality of the copper-catalyzed azide-alkyne cycloaddition allowed the functionalization with unprotected ligands. Whereas the functionalization of discotics with a carbohydrate was quantitative, the coupling of peptides proceeded with at best 40% conversion. This was probably due to steric crowding of peripheral functionalities in the self-assembly inducing solvent, which is required for solubility of unprotected ligands. In contrast, discotics bearing a single amine emerged as a versatile non-sterically hindered scaffold for ligand attachment as they were rapidly and quantitatively functionalized with a range of peptidic- and non-peptidic ligands using both NHS ester and HBTU activation techniques under non-assembling solvent conditions. The ability to fine-tune the density and display of bioactive epitopes and thereby creating more complex dynamic and heterovalent structures without interfering with the self-assembling process is a key prerequisite for the development of a platform technology for targeting. A versatile and non-sterically hindered scaffold for ligand attachment, such as the presented discotic bearing a single amine, might constitute the basis for such a technology. The functionalization of this discotic leads to monovalent ligand functionalized discotics. The display of multiple ligands, which is important for enhanced binding affinities, will be accomplished upon self-assembly into columnar stacks. This so-called multivalency upon self-assembly has been probed with a number of monovalent ligand-functionalized discotics in chapter 3. Enzyme-linked lectin assay revealed a three-fold increase in binding activity compared with the non self-assembling counterpart. The self-assembly into a columnar stack and the accompanied display of multiple ligands was as well confirmed studying the binding of monovalent streptavidin to discotics functionalized with a single biotin using Förster resonance energy transfer and SDS-PAGE. The formation of heterovalent supramolecular polymers through dynamic intermixing of different functionalized building blocks was shown using mixtures of biotin and fluorescein functionalized discotics incubated with streptavidin coated magnetic beads. Thus the self-assembly into supramolecular polymers not only generates a multivalent, but as well a heterovalent system. The possibility to generate heterovalent supramolecular polymers via simple intermixing of discotics has a great potential in view of advanced biological applications, for example in the field of targeted imaging. To gain further inside into the dynamics of this intermixing process, discotics bearing a single O6 benzylguanine moiety were covalently post-functionalized with two FRET-pairing fluorescent proteins. Firstly, the covalent post-functionalization with proteins, ligands which are incompatible with the pre-functionalization strategy, was confirmed with several analytical techniques such as SDS-PAGE and LC-MS in chapter 4. The covalent protein conjugation at the same time leads to Förster resonance energy transfer from the auto-fluorescent discotic scaffold to the yellow fluorescent protein and allows on-line monitoring of the conjugation. At the same time the protein conjugation does not interfere with the self-assembling process, leading to a multivalent protein display on a supramolecular wire, as visualized via energy transfer from the cyan to the yellow fluorescent protein. Secondly, the system maintains its intermixing dynamics, which allows the formation of hetero-functionalized supramolecular protein-conjugated polymers through exchange of the protein-functionalized discotics over time. The supramolecular wires act as dynamic framework on which the two proteins can assemble and exchange in a dynamic manner, leading to effective protein interactions, as observed by energy transfer. The cellular uptake of amine-decorated discotics and the dependence of cellular uptake on the peripheral amine density were explored in chapter 5. Using the auto-fluorescence of the discotic scaffolds, their internalization was studied using live cell multiphoton fluorescence microscopy Discotics bearing three or nine amine groups at their periphery efficiently translocated through the plasma membrane via endocytosis. Additionally, the knowledge about the formation of intermixed supramolecular polymers obtained in chapter 3 and 4 was applied to generate multi-functional supramolecular polymers consisting of up to three different cell-permeable and non cell-permeable discotic monomers. Through intermixing with cell-permeable discotic monomers in the supramolecular polymer, the cellular uptake of non-cell permeable discotics was induced and each of the components could be individually visualized, demonstrating the potential of dynamic multi-component supramolecular polymers. The functionalization of self-assembling p-conjugated nanoparticles with bioactive epitopes, a prerequisite for applications in targeted multimodal imaging, was investigated in the last chapter. Upon microinjection into water, these linear and auto-fluorescent amphiphiles self-assemble into highly-fluorescent amorphous nanoparticles of 80-100 nm. Azide and mannose groups were introduced at the periphery of the ethylene glycol chains of the amphiphile and did not interfere with the self-assembly process. The binding of mannose functionalized nanoparticles to proteins and bacteria confirmed the accessibility of the introduced ligand. Co-assembly of different amphiphiles enabled the fine-tuning of ligand density, which was confirmed with Förster resonance energy transfer. Additionally, using copper catalyzed azide-alkyne cycloaddition reaction, azide bearing nanoparticles were post-functionalized with different ligands. Successful combination of both functionalization strategies via intermixing of mannose and azide bearing amphiphiles and subsequent copper catalyzed azide-alkyne cycloaddition led to heterovalent nanoparticles. Nano-sized columnar and spherical supramolecular assemblies were functionalized with a wide range of ligands such as carbohydrates, peptides, and proteins using both pre- and post-functionalization strategies. This allowed for expanding the ligand diversity at two independent stages in the fabrication process of these bioactive nano-structures. Supramolecular synthesis enabled the facile generation of complex heterovalent bioactive assemblies; in the case of nanoparticles via co-assembly of different amphiphiles and in the case of discotics via dynamic intermixing of building blocks between the supramolecular stacks. With this knowledge in hand advanced applications of complex multitargeting and multimodal supramolecular nano-sized structures in imaging can be envisioned; carrying for example several targeting ligands as well as an alternative imaging probe. The ability to tune the optical properties in the case of the nanoparticles should additionally enable multi-color imaging. At the same time, the self-assembling nature of these nanoparticles allows the incorporation of hydrophobic (drug) molecules and functionalized lipids, expanding the scope of functionalization strategies and with it of possible applications. The absence of unspecific adsorption of the bare scaffolds of both the disc-shaped and linear amphiphiles proves their broad potential as selective biological targeting tools

Double-degradable responsive self-assembled multivalent arrays-temporary nanoscale recognition between dendrons and DNA

This article reports self-assembling dendrons which bind DNA in a multivalent manner. The molecular design directly impacts on self-assembly which subsequently controls the way these multivalent nanostructures bind DNA-this can be simulated by multiscale modelling. Incorporation of an S-S linkage between the multivalent hydrophilic dendron and the hydrophobic units responsible for self-assembly allows these structures to undergo triggered reductive cleavage, with dithiothreitol (DTT) inducing controlled breakdown, enabling the release of bound DNA. As such, the high-affinity self-assembled multivalent binding is temporary. Furthermore, because the multivalent dendrons are constructed from esters, a second slow degradation step causes further breakdown of these structures. This two-step double-degradation mechanism converts a large self-assembling unit with high affinity for DNA into small units with no measurable binding affinity-demonstrating the advantage of self-assembled multivalency (SAMul) in achieving highly responsive nanoscale binding of biological targets

Enantiomeric and Diastereomeric Self-Assembled Multivalent Nanostructures : Understanding the Effects of Chirality on Binding to Polyanionic Heparin and DNA

A family of four self-assembling lipopeptides containing Ala-Lys peptides attached to a C16 aliphatic chain were synthesised. These compounds form two enantiomeric pairs that bear a diastereomeric relationship to one another (C16-l-Ala-l-Lys/C16-d-Ala-d-Lys) and (C16-d-Ala-l-Lys/C16-l-Ala-d-Lys). These diastereomeric pairs have very different critical micelle concentrations (CMCs). The self-assembled multivalent (SAMul) systems bind biological polyanions as a result of the cationic lysine groups on their surfaces. For heparin binding, there was no significant enantioselectivity, but there was a binding preference for the diastereomeric assemblies with lower CMCs. Conversely, for DNA binding, there was significant enantioselectivity for systems displaying d-lysine ligands, with a further slight preference for attachment to l-alanine, with the CMC being irrelevant

Self-assembling auto-fluorescent amphiphiles : nano-sized platform technology for multi-purpose cellular targeting

Amphiphilic molecules emerged as versatile building blocks for the generation of nano-sized architectures in water, as they can be programmed to self-assemble into a wide range of different topologies. In this thesis the generation of auto-fluorescent heterovalent nano-sized structures was explored using two types of amphiphilic scaffolds: disc-shaped and linear amphiphiles self-assembling in water into columnar polymers or into amorphous spherical nanoparticles, respectively. Numerous applications for self-assembling nanostructures were reported in literature based on amphiphilic molecules, ranging from imaging to diagnostics, and from drug delivery to tissue engineering. Many of these applications require the capability of the supramolecular system to actively target specific cell surface receptors. This is typically achieved through decoration with bioactive epitopes such as small molecules, peptides, and proteins. As discussed in chapter 1, the bioactive epitopes can either already be part of the monomeric supramolecular building blocks (pre-functionalization) or introduced after self-assembly via covalent attachment to appending reactive groups (post-functionalization). Selective and multivalent binding of disc-shaped amphiphiles to bacterial receptors was previously shown through the introduction of three functional groups at the periphery of the ethylene oxide tails and subsequent functionalization with bioactive ligands. Here, to expand the library of scaffolds, amphiphiles containing either nine amine functionalities or a single amine, azide and propargyl group were synthesized. Their decoration with bioactive ligands such as peptides, carbohydrates, small molecules and fluorescent dyes using both amide coupling and copper-catalyzed azide-alkyne cycloaddition is described in chapter 2. The orthogonality of the copper-catalyzed azide-alkyne cycloaddition allowed the functionalization with unprotected ligands. Whereas the functionalization of discotics with a carbohydrate was quantitative, the coupling of peptides proceeded with at best 40% conversion. This was probably due to steric crowding of peripheral functionalities in the self-assembly inducing solvent, which is required for solubility of unprotected ligands. In contrast, discotics bearing a single amine emerged as a versatile non-sterically hindered scaffold for ligand attachment as they were rapidly and quantitatively functionalized with a range of peptidic- and non-peptidic ligands using both NHS ester and HBTU activation techniques under non-assembling solvent conditions. The ability to fine-tune the density and display of bioactive epitopes and thereby creating more complex dynamic and heterovalent structures without interfering with the self-assembling process is a key prerequisite for the development of a platform technology for targeting. A versatile and non-sterically hindered scaffold for ligand attachment, such as the presented discotic bearing a single amine, might constitute the basis for such a technology. The functionalization of this discotic leads to monovalent ligand functionalized discotics. The display of multiple ligands, which is important for enhanced binding affinities, will be accomplished upon self-assembly into columnar stacks. This so-called multivalency upon self-assembly has been probed with a number of monovalent ligand-functionalized discotics in chapter 3. Enzyme-linked lectin assay revealed a three-fold increase in binding activity compared with the non self-assembling counterpart. The self-assembly into a columnar stack and the accompanied display of multiple ligands was as well confirmed studying the binding of monovalent streptavidin to discotics functionalized with a single biotin using Förster resonance energy transfer and SDS-PAGE. The formation of heterovalent supramolecular polymers through dynamic intermixing of different functionalized building blocks was shown using mixtures of biotin and fluorescein functionalized discotics incubated with streptavidin coated magnetic beads. Thus the self-assembly into supramolecular polymers not only generates a multivalent, but as well a heterovalent system. The possibility to generate heterovalent supramolecular polymers via simple intermixing of discotics has a great potential in view of advanced biological applications, for example in the field of targeted imaging. To gain further inside into the dynamics of this intermixing process, discotics bearing a single O6 benzylguanine moiety were covalently post-functionalized with two FRET-pairing fluorescent proteins. Firstly, the covalent post-functionalization with proteins, ligands which are incompatible with the pre-functionalization strategy, was confirmed with several analytical techniques such as SDS-PAGE and LC-MS in chapter 4. The covalent protein conjugation at the same time leads to Förster resonance energy transfer from the auto-fluorescent discotic scaffold to the yellow fluorescent protein and allows on-line monitoring of the conjugation. At the same time the protein conjugation does not interfere with the self-assembling process, leading to a multivalent protein display on a supramolecular wire, as visualized via energy transfer from the cyan to the yellow fluorescent protein. Secondly, the system maintains its intermixing dynamics, which allows the formation of hetero-functionalized supramolecular protein-conjugated polymers through exchange of the protein-functionalized discotics over time. The supramolecular wires act as dynamic framework on which the two proteins can assemble and exchange in a dynamic manner, leading to effective protein interactions, as observed by energy transfer. The cellular uptake of amine-decorated discotics and the dependence of cellular uptake on the peripheral amine density were explored in chapter 5. Using the auto-fluorescence of the discotic scaffolds, their internalization was studied using live cell multiphoton fluorescence microscopy Discotics bearing three or nine amine groups at their periphery efficiently translocated through the plasma membrane via endocytosis. Additionally, the knowledge about the formation of intermixed supramolecular polymers obtained in chapter 3 and 4 was applied to generate multi-functional supramolecular polymers consisting of up to three different cell-permeable and non cell-permeable discotic monomers. Through intermixing with cell-permeable discotic monomers in the supramolecular polymer, the cellular uptake of non-cell permeable discotics was induced and each of the components could be individually visualized, demonstrating the potential of dynamic multi-component supramolecular polymers. The functionalization of self-assembling p-conjugated nanoparticles with bioactive epitopes, a prerequisite for applications in targeted multimodal imaging, was investigated in the last chapter. Upon microinjection into water, these linear and auto-fluorescent amphiphiles self-assemble into highly-fluorescent amorphous nanoparticles of 80-100 nm. Azide and mannose groups were introduced at the periphery of the ethylene glycol chains of the amphiphile and did not interfere with the self-assembly process. The binding of mannose functionalized nanoparticles to proteins and bacteria confirmed the accessibility of the introduced ligand. Co-assembly of different amphiphiles enabled the fine-tuning of ligand density, which was confirmed with Förster resonance energy transfer. Additionally, using copper catalyzed azide-alkyne cycloaddition reaction, azide bearing nanoparticles were post-functionalized with different ligands. Successful combination of both functionalization strategies via intermixing of mannose and azide bearing amphiphiles and subsequent copper catalyzed azide-alkyne cycloaddition led to heterovalent nanoparticles. Nano-sized columnar and spherical supramolecular assemblies were functionalized with a wide range of ligands such as carbohydrates, peptides, and proteins using both pre- and post-functionalization strategies. This allowed for expanding the ligand diversity at two independent stages in the fabrication process of these bioactive nano-structures. Supramolecular synthesis enabled the facile generation of complex heterovalent bioactive assemblies; in the case of nanoparticles via co-assembly of different amphiphiles and in the case of discotics via dynamic intermixing of building blocks between the supramolecular stacks. With this knowledge in hand advanced applications of complex multitargeting and multimodal supramolecular nano-sized structures in imaging can be envisioned; carrying for example several targeting ligands as well as an alternative imaging probe. The ability to tune the optical properties in the case of the nanoparticles should additionally enable multi-color imaging. At the same time, the self-assembling nature of these nanoparticles allows the incorporation of hydrophobic (drug) molecules and functionalized lipids, expanding the scope of functionalization strategies and with it of possible applications. The absence of unspecific adsorption of the bare scaffolds of both the disc-shaped and linear amphiphiles proves their broad potential as selective biological targeting tools

Combining supramolecular chemistry with biology

Supramolecular chemistry has primarily found its inspiration in biological molecules, such as proteins and lipids, and their interactions. Currently the supramolecular assembly of designed compounds can be controlled to great extent. This provides the opportunity to combine these synthetic supramolecular elements with biomolecules for the study of biological phenomena. This tutorial review focuses on the possibilities of the marriage of synthetic supramolecular architectures and biological systems. It highlights that synthetic supramolecular elements are for example ideal platforms for the recognition and modulation of proteins and cells. The unique features of synthetic supramolecular systems with control over size, shape, valency, and interaction strength allow the generation of structures fitting the demands to approach the biological problems at hand. Supramolecular chemistry has come full circle, studying the biology and its molecules which initially inspired its conceptio

Protein assembly along a supramolecular wire

Discotic molecules self-assemble into supramolecular wires that act as platforms for directed protein assembly via appended biotin functionalitie

Self-assembling multivalency : supramolecular polymers assembled from monovalent mannose-labelled discotic molecules

Supramolecular synthesis, the bottom-up construction of higher-order structures from monomeric building blocks, represents a flexible approach for the generation of multivalent materials. Here, monovalent building blocks decorated with a single bioactive ligand were synthesized. In water, these supramolecular elements self-assemble into columnar polymers that display multiple ligands. The supramolecular effect on the binding affinity was evaluated by using an enzyme-linked lectin assay, and the self-assembled architecture exhibited a stronger inhibitory power than that of the monovalent bioactive ligand. This so-called self-assembling multivalency enables the rapid and flexible generation of multivalent polymers from monovalent building blocks

Supramolecular chemical biology : bioactive synthetic self-assemblies

The regulation of recognition events in nature via dynamic and reversible self-assembly of building blocks has inspired the emergence of supramolecular architectures with similar biological activity. Synthetic molecules of diverse geometries self-assemble in water to target biological systems for applications ranging from imaging and diagnostics, through to drug delivery and tissue engineering. Many of these applications require the ability of the supramolecular system to actively recognize specific cell surface receptors. This molecular recognition is typically achieved with ligands, such as small molecules, peptides, and proteins, which are introduced either prior to or post self-assembly. Advantages of the non-covalent organization of ligands include the responsive nature of the self-assembled structures, the ease of supramolecular synthesis and the possibility to incorporate a multiple array of different ligands through premixing of the building blocks. This review aims to highlight the diversity of self-assembled nanostructures constructed from mono-disperse synthetic building blocks; with a particular focus on their design, selfassembly, functionalization with bioactive ligands and effects thereof on the self-assembly, and possible applications

Structure-activity relationship studies of miniproteins targeting the androgen receptor-coactivator interaction

A classical approach to treating prostate cancer uses antagonist ligands - so-called anti-androgens, such as bicalutamide - which block gene transcription through binding to a lipophilic pocket at the ligand binding domain of the androgen receptor (AR). An alternative strategy has been developed using compounds which directly target the surface charge clamp by mimicking the coactivator's highly conserved alpha-helical motif. Thus, to gain additional knowledge about the AR-coactivator interaction, the use of natural miniproteins as a source of novel AR-coactivator inhibitors incorporating the FXXLF motif was explored. Their stable well-defined alpha-helical secondary structures make miniproteins ideal candidates for development into AR-coactivator inhibitors. Therefore, starting from two potent miniprotein scaffold structures, identified from previous work, systematic point mutations aimed at improving AR affinity were introduced using solid-phase peptide synthesis (SPPS). Structure-activity relationship studies were performed, from which a number of high affinity inhibitors, typically in the low micromolar-to-high nanomolar range, with a ten-fold gain in potency compared with the reference compounds, were identified, thus highlighting the high potential for these scaffold

Combining supramolecular chemistry with biology

Supramolecular chemistry has primarily found its inspiration in biological molecules, such as proteins and lipids, and their interactions. Currently the supramolecular assembly of designed compounds can be controlled to great extent. This provides the opportunity to combine these synthetic supramolecular elements with biomolecules for the study of biological phenomena. This tutorial review focuses on the possibilities of the marriage of synthetic supramolecular architectures and biological systems. It highlights that synthetic supramolecular elements are for example ideal platforms for the recognition and modulation of proteins and cells. The unique features of synthetic supramolecular systems with control over size, shape, valency, and interaction strength allow the generation of structures fitting the demands to approach the biological problems at hand. Supramolecular chemistry has come full circle, studying the biology and its molecules which initially inspired its conceptio