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

K. Petkau - Milroy

Research output: ThesisPhd Thesis 1 (Research TU/e / Graduation TU/e)

403 Downloads (Pure)

Abstract

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.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Department of Biomedical Engineering
Supervisors/Advisors
  • Brunsveld, Luc, Promotor
Award date25 Jun 2012
Place of PublicationEindhoven
Publisher
Print ISBNs978-90-386-3163-9
DOIs
Publication statusPublished - 2012

Fingerprint

Amphiphiles
Ligands
Bearings (structural)
Azides
Scaffolds
Polymers
Self assembly
Amines
Nanoparticles
Proteins
Alkynes
Cycloaddition
Energy transfer
Imaging techniques
Copper
Epitopes
Mannose
Molecules
Display devices
Peptides

Cite this

Petkau - Milroy, K.. / Self-assembling auto-fluorescent amphiphiles : nano-sized platform technology for multi-purpose cellular targeting. Eindhoven : Technische Universiteit Eindhoven, 2012. 132 p.
@phdthesis{982bf09d522e45d5b83a6c4b39f84a73,
title = "Self-assembling auto-fluorescent amphiphiles : nano-sized platform technology for multi-purpose cellular targeting",
abstract = "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 F{\"o}rster 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 F{\"o}rster 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 F{\"o}rster 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.",
author = "{Petkau - Milroy}, K.",
year = "2012",
doi = "10.6100/IR733401",
language = "English",
isbn = "978-90-386-3163-9",
publisher = "Technische Universiteit Eindhoven",
school = "Department of Biomedical Engineering",

}

Petkau - Milroy, K 2012, 'Self-assembling auto-fluorescent amphiphiles : nano-sized platform technology for multi-purpose cellular targeting', Doctor of Philosophy, Department of Biomedical Engineering, Eindhoven. https://doi.org/10.6100/IR733401

Self-assembling auto-fluorescent amphiphiles : nano-sized platform technology for multi-purpose cellular targeting. / Petkau - Milroy, K.

Eindhoven : Technische Universiteit Eindhoven, 2012. 132 p.

Research output: ThesisPhd Thesis 1 (Research TU/e / Graduation TU/e)

TY - THES

T1 - Self-assembling auto-fluorescent amphiphiles : nano-sized platform technology for multi-purpose cellular targeting

AU - Petkau - Milroy, K.

PY - 2012

Y1 - 2012

N2 - 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.

AB - 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.

U2 - 10.6100/IR733401

DO - 10.6100/IR733401

M3 - Phd Thesis 1 (Research TU/e / Graduation TU/e)

SN - 978-90-386-3163-9

PB - Technische Universiteit Eindhoven

CY - Eindhoven

ER -