Master Student Projects - Protein engineering of the CCMV coat protein
Viral protein nanocages, such as that of the cowpea chlorotic mottle virus (CCMV), are promising carriers for drug delivery applications or capsules for artificial organelle construction. The natural CCMV coat protein assembles spontaneously at pH 5.0 in the absence of its native RNA cargo, while it reversibly disassembles into dimers at pH 7.5. Via introduction of a stimulus-responsive elastin-like polypeptide (ELP) block at the N-terminus of the viral coat protein, we have established capsid assembly under physiological conditions (pH 7.5). As such we are very close to applying the CCMV capsid in vivo.
Summary of the projects and goals
In order to enhance the stability of the ELP-CCMV capsid in vivo, while maintaining its reversible disassembly behavior, the N-terminal ELP block can be modified via protein engineering. Based on a small library of ELP-CCMV variants that are currently available, the master student will design novel ELP-CCMV variants with intermediate hydrophobicity. Via the protein engineering toolbox, these variants can be produced and their assembly behavior and stability should then be evaluated and compared to the currently available variants. If successful, cellular uptake experiments of the assembled capsids can be performed.
Another option for protein engineering of the ELP-CCMV coat protein is to introduce a specific functional handle on the exterior surface of the capsid. This will allow for site-specific modification of the capsid exterior with functional groups that enhance capsid stability or in vivo targeting. In this project, the master student will identify positions on the capsid surface that are suitable for modification based on literature. Protein engineering will then be employed in order to introduce an amino acid with a specific handle, such as a cysteine. Lastly, capsid modification based on the reactive handle will be evaluated.
In these projects you will have the opportunity to gain experience with the following techniques (it is not a problem if you don’t have experience with these techniques yet):
- Protein engineering
- Protein expression
- Protein purification
- Analytical methods (i.e. Liquid chromatography-mass spectrometry (LCMS), size-exclusion chromatography (SEC), Dynamic light scattering (DLS), UV-vis spectroscopy, Mass spectrometry, Transmission electron microscopy (TEM))
- Capsid stability analysis
- Labelling via a reactive handle
- (Cellular uptake experiments)
Master Student Projects - Interior and exterior modification of the CCMV capsid
Viral protein nanocages, such as that of the cowpea chlorotic mottle virus (CCMV), are promising carriers for drug delivery applications or capsules for artificial organelle construction. For therapeutic applications a therapeutic agent should be encapsulated inside the capsids, which can be facilitated via modification of the capsid interior. In order to improve the in vivo stability and targeting, the exterior of the capsid can be modified with stabilizing or targeting agents.
Summary of the projects and goals
It has been shown that proteins can be successfully encapsulated via Sortase A-mediated N-terminal modification of CCMV. Hereto an LPXTG-tag (where X is any amino acid) must be present at proteins to encapsulate them via this Sortase reaction. In this project, the master student will develop a method to encapsulate enzymes in which for example, first a click-reaction between an LPXTG-peptide and a therapeutic enzyme is performed, followed by encapsulation of the enzyme via the Sortase reaction. If successful, in vitro studies will be performed to determine the activity of these encapsulated enzymes.
The effect of nucleic acid encapsulation into viral capsids represents a promising field of investigation for several applications such as gene delivery or catalytic activity. In this project the master student will experiment and compare encapsulation efficiency of oligonucleotides and nucleic acids of different lengths and the stabilizing effect of cargo loading. If successful, in vitro toxicity and efficacy studies can be performed.External functionalization of the particle surface could represent an interesting approach in order to increase capsid stability in versatile buffer conditions so expanding the system’s applicability for several purposes. In this project the master student will evaluate different covalent functionalization strategies reported in the literature in terms of applicability to the specific protein cage as well as efficiency and degree of functionalization. A comparison between modification strategies will be followed by the investigation of functionalized particle stability.
In order to control cellular uptake and the location in the body where a therapeutic agent will end up, specific targeting peptides can be used. In this project the master student will design cell-penetrating peptides (CPPs), endosomal escape peptides, and/or specific targeting peptides based on information that is available in literature. Next, these peptides will be synthesized and purified, after which they can be attached to the exterior of CCMV capsids. The degree of functionalization will then be determined as well as the cellular uptake behavior.
TechniquesIn these projects you will have the opportunity to gain experience with the following techniques (it is not a problem if you don’t have experience with these techniques yet):• Peptide synthesis• Protein expression• Protein purification• Chemical modification• Analytical methods (i.e. Liquid chromatography-mass spectrometry (LCMS), size-exclusion chromatography (SEC), Dynamic light scattering (DLS), UV-vis spectroscopy, Mass spectrometry, Transmission electron microscopy (TEM))• (Cellular uptake experiments)
Master Student Project – Molecular dynamics (MD) simulation on polymeric vesicle architecture
(Joint MSc project between Group Theory of Polymers and Soft Matter, Applied Physics and Bio-Organic Chemistry Group, Chemical Engineering and Chemistry)
Interest in fundamental research on the formation of polymeric vesicles
No MD simulation experience required
Polymersomes are an interesting group of polymeric (micro/nano) vesicles. These vesicles are formed due to the self-assembly of amphiphilic block copolymers into a well-defined nano/micro structure (100- 500 nm). Due to their ability to encapsulate cargo, e.g. antigen/peptides, it is possible to use polymersomes for drug delivery systems. By altering the composition of the block copolymer it is possible to control the polymersome size, membrane thickness and shape. For biological systems (uptake, cell response, etc.) the morphology (size, shape, membrane thickness) of these polymersomes is highly important and, consequently, we are interested in controlling these parameters. Traditionally, the morphology of assemblies of low molecular weight amphiphiles is predicted by using the packaging parameter p=v/(a0*lc) where v = volume of the hydrophobic chain, a0 = area of the hydrophilic head and lc = length of the hydrophobic chain. Generally vesicles are formed when 1/2 ≤ p ≤ 1.
However, in case of polymeric amphiphiles the packaging parameter is too limited. Besides interactions of the copolymer with itself, neighboring copolymers and its environment, i.e. solvent and non-solvent, the folding of the polymer is an extremely important factor and is difficult to predict without modeling.
The goal of this master project is to use computer simulations to calculate the folding of the used block copolymers and its interactions, to correlate its composition to the morphology. Also, some of the formed polymersomes have semi-permeable membranes. By simulating membrane density this behavior can be explained and predicted. Another feature closely related to this permeability is the ability of the polymersome to change its shape by changing the environment (e.g. dialysis against salt). By altering the environment of an already assembled polymersome the change in morphology can be simulated (thanks to the change of interactions of the copolymers with the environment, pressure inside and/or change in membrane density changing the permeability). As we have experimentally access to a wide range of block copolymers, we can effectively validate the model with actual polymer assemblies.
Objectives of the Master student project:
As a Master student you will be given the task to execute MD simulation answering several important research questions. These MD simulations are conducted at an atomistic level giving detailed insight in the interactions of the system. Using these simulations it is possible to explain the behavior of the polymeric vesicles our group fabricates. After simulating these interactions at the atomistic level larger scale coarse grained simulations (DPD) can be done in a more detailed fashion.
Master Student Project - From amphiphilic copolymers to worm-like micelles and beyond
Master student, Interest in polymer synthesis and nanomedicine
In the past decades, a large number of novel formulations of drug delivery systems have been published. However, only a small percentage of them actually follow their way through clinical trials. One of the parameters of formulation we might forget in order to reach success, is particle morphology. Mimicking design principles and characteristics of natural objects, we can think of engineering functional nanomaterials with different morphologies. Small changes in the chemical components used in any nanoparticle fabrication process (building blocks, solvents, conditions as concentration/temperature/pH/salt behaviour, etc.) can have substantial effects upon the resulting structure (size, shape, stiffness, surface chemistry, etc.), which can obviously change the in vitro/in vivo performance.
Micelles are self-assembled nanoparticles formed through the hydrophobic re-arrangement of amphiphilic molecules into a thermodynamically favorable spheroid (Fig.1). The hydrophobic portion of the block copolymer will self-associate into the micellar core whereas the hydrophilic portion will form the external corona. Importantly, the micelle core is capable of encapsulating hydrophobic drugs. By varying the right parameters, micelles can undergo a shape change into worm-like structures (Fig.2). This is the aim of the project.
Based on the knowledge of our group, the student will synthesize and characterize different biodegradable amphiphilic block copolymers. Polymers will self-assemble into nanoparticles, aiming to produce worm-like micelles. Different characterization techniques (DLS, CryoTEM, fluorescence microscopy…) will be used to evaluate the shape and size of the nanoparticles. This system will further be developed and optimized in order to build a drug delivery vehicle. In vitro cell assays might also be performed.
Master Student Project - Microfluidic production of giant liposomes
Liposomes are used in as nanoparticles in drug delivery, cell transfection, and membrane models. Due to their similarity to natural cells (both have a phospholipid membrane), they are also used as models of natural cells. These so-called ‘artificial cells” can be loaded with enzymes, substrates, membrane proteins, fluorophores, etc. This versatility makes them ‘engineerable’, which explains their wide-spread use.
There are several established methods to make giant (> 1 um) liposomes, each with their own advantages and drawbacks. In recent years, the fabrication of these giant liposomes using microfluidics has proven to be very interesting due to its high reproducibility and high loading efficiency. For instance, it is possible to load giant liposomes with cell extract and a synthetic plasmid, permitting the production of your protein of interest in a cell-like vesicle. In our group, we want to load liposomes with enzymatic cascades, to create communicating colonies of artificial cells, or to use them to deliver nanoparticles to cells. Microfluidic fabrication of liposomes would greatly help us, since it is reliable, with high efficiency and throughput.
Based on literature reports, the student should design and develop a microfluidic chip to make giant liposomes in a reproducible manner. Different membrane constituents will be used to study the effect on the membrane properties, as well as a variety of loadings to create artificial cells with interesting properties. This project is suitable for students with a wide range of backgrounds, e.g. organic/supramolecular chemistry, but also molecular biology and engineering