Artificial photosynthesis has recognised potential to produce green and sustainable fuels from earth-abundant resources such as water, carbon dioxide (CO2), and sunlight. In an artificial... Show moreArtificial photosynthesis has recognised potential to produce green and sustainable fuels from earth-abundant resources such as water, carbon dioxide (CO2), and sunlight. In an artificial photosynthetic system, two half-reactions, such as water oxidation and proton reduction or CO2 reduction, have to be combined. To achieve such a system, it is crucial to have: a) efficient light-harvesting by the photosensitiser, b) stable catalysts for the oxidation and the reduction reaction, c) unidirectional proton and electron transport between the oxidation and the reduction site, ideally by a recyclable electron relay, d) efficient charge separation, and e) a strong, photostable membrane that does not leak molecular components. In natural photosynthesis, these requirements are achieved altogether using compartmentalisation, which consists in embedding the key components of the system, i.e. for green plants the oxygen evolving complex, photosystem I and II, and the natural electron relays, around the lipid bilayer of the thylakoid membrane. The use of spherical lipid membranes (such as liposomes) as biological mimics of the thylakoid membrane is a promising approach to confine half-reactions, facilitate charge separation, and avoid charge recombination and other undesired side-reactions. In the research described in this thesis, it was attempted to realise a full artificial photosynthetic system based on liposomes and several of the key intermediate steps were achieved: 1) unidirectional electron transfer across a liposomal membrane from an electron donor encapsulated in the interior of the liposome to an electron acceptor located outside (Chapter 2), and 2) photocatalytic reduction of CO2 (Chapter 3) and of protons (Chapter 4) at the surface of liposomes. Special attention was paid in Chapter 2 and Chapter 5 to the question of the (photo)stability of the membrane and light-induced leakage. Show less
A structural investigation of coiled coil peptides used as membrane fusogens, mimicing naturally occuring coiled-coil fusion proteins. Synthetic modifications have been made to alter lipid... Show moreA structural investigation of coiled coil peptides used as membrane fusogens, mimicing naturally occuring coiled-coil fusion proteins. Synthetic modifications have been made to alter lipid attachment, secondary structure and to insert photoactive azobenzene moieties for active control over coiled coil structure. Finally, the underlying photocontrol mechanism investigated in coiled coil peptides is extended to beta-structured peptides, and was shown to be universally applicable. Show less
Dit proefschrift beschrijft een collectie aan alternatieve strategieën voor het begrijpen, ontwerpen en toepassen van lipide nanosystemen, waarin de rol van de bio-nano interacties centraal staan.... Show moreDit proefschrift beschrijft een collectie aan alternatieve strategieën voor het begrijpen, ontwerpen en toepassen van lipide nanosystemen, waarin de rol van de bio-nano interacties centraal staan. In het bijzonder wordt gekeken naar de interactie van RNA-lipide nanosystemen, bekend van de toepassing als vaccins en andere medicatie. Het onderzoeksgebied van de nanomedicatie kan gebruik maken van de specifieke voorbeelden die worden beschreven, maar er kan ook inspiratie worden opgedaan om de aanpak van onderzoek doen te verschuiven van een kostbare empirische aanpak naar rationeel gedreven ontwerpstrategie. Dit alles zal helpen in de ontwikkelen van nieuwe RNA therapieen in de toekomst. Show less
The work described in this dissertation contributes to a better mechanistic understanding of nanoparticles in vivo. To achieve that goal, we used the zebrafish as a highly predictive pre-screening... Show moreThe work described in this dissertation contributes to a better mechanistic understanding of nanoparticles in vivo. To achieve that goal, we used the zebrafish as a highly predictive pre-screening model of nanoparticles. This approach enables the investigation of the fundamental behavior of nanoparticles, correlation of the physicochemical properties of the formulated nanoparticles with their biodistribution and identification of important nano-bio interactions. Zebrafish established transgenic lines were used to study specific interactions. In addition, genetically modified zebrafish applying CRISPR/Cas9 were generated. These strategies not only show key mechanistic features of nanoparticles in circulation, but also promote the rational design of more efficient nanoparticles systems.After understanding the fundamental behavior of nanoparticles, this thesis describes the identification of a key interaction between stabilins receptors (expressed in liver sinusoidal endothelial cells) and nanoparticles. Next, the scope is changed to design nano-systems that target specific cell types showing liposomes capable of switching the surface charge in situ and in vivo using light as an external trigger and a rationally designed lipid nanoparticle formulation containing mRNA able to preferentially target the hepatic reticuloendothelial system. In addition, a phase-separated liposomes hijacking a lipase mediated transport to selectively target endothelial lipase in vivo was studied. Show less
Benne, N.; Leboux, R.J.T.; Glandrup, M.; Duijn, J. van; Lozano Vigario, F.; Neustrup, M.A.; ... ; Slütter, B. 2019
Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded... Show moreRegulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomescomposed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigenspecificTreg responses. We hypothesized that altering the rigidity of these liposomes while maintaining theirsize and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes isaffected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part bythe presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL(1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa),DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG(494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-deriveddendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation betweenliposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responsesin vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measureliposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system. Show less
Membrane fusion is a vital process in living organisms and is mediated by zipper-like proteins. This thesis describes the use of coiled-coil peptides, derived from these zipper-like proteins, to... Show moreMembrane fusion is a vital process in living organisms and is mediated by zipper-like proteins. This thesis describes the use of coiled-coil peptides, derived from these zipper-like proteins, to mediate fusion of liposomes. Designed mutations in the peptides and in the peptide-liposome connection influences the rate and the efficiency of the fusion process. The efficiency of the fusion process is found to be very dependent on peptide structures and peptide-membrane interactions. Crucial for efficient fusion are subtle membrane interactions and very helical peptide structures. This improves our understanding of the fusion mechanism and enables the rational design of new and better fusogens. The used peptides are also able to mediate fusion between liposomes and cells and are currently used to develop drug delivery systems. Show less
Hoekstra, M.; Geerling, J.J.; Jiskoot, W.; Eck, M. van 2018
Ruthenium complexes are promising prodrugs in photoactivated chemotherapy (PACT): to prevent systemic therapeutic side-effects, a non-toxic version of the drug is introduced in the body and is... Show moreRuthenium complexes are promising prodrugs in photoactivated chemotherapy (PACT): to prevent systemic therapeutic side-effects, a non-toxic version of the drug is introduced in the body and is only activated at the place of the tumor by means of visible light irradiation. However, most of these PACT compounds are only sensitive for UV or blue light, while this light does not permeate the body very well, in contrast to red or near-infrared light. To circumvent this problem, the principle of light-upconversion can be used to "upgrade" red light to blue light in a drug carrier such as a nanovesicle: the tumor is irradiated with red light, after which blue light is generated locally and used to activate the prodrug. Among the various methods of light-upconversion, triplet-triplet annihilation upconversion (TTA-UC) was selected as the most promising. In this thesis it is described that green-to-blue and red-to-blue upconverting nanovesicles were prepared. The red-to-blue upconverted light was successfully used to activate a ruthenium polypyridyl complex that was anchored to the same vesicle. Finally, the inherent oxygen-sensitivity of TTA-UC was greatly mitigated by the addition of water-soluble and biocompatible anti-oxidants. We expect that the results of this thesis will lead to exciting applications in PACT. Show less
Current energy-sources in the form of fossil fuels are quickly being depleted, while the demand of energy by society is increasing. In order to sustain this growth in energy demand, alternatives... Show moreCurrent energy-sources in the form of fossil fuels are quickly being depleted, while the demand of energy by society is increasing. In order to sustain this growth in energy demand, alternatives for the production of energy in a usable form are needed. One of such alternatives is to employ photocatalysis in order to use sunlight for the production of chemical fuels such as for example H2 or methanol. For the production of fuels, electrons are required that can be obtained by oxidizing water, as done by nature in a process called photosynthesis. The work in this thesis was inspired by this natural process; photosensitizers and water-oxidation catalysts were bound to lipid bilayers and their ability to photocatalytically oxidize water was studied in different conditions. The anchoring of compounds to a lipid bilayer leads to large differences in reactivity compared to homogeneous systems. In some cases, even the mechanism of the photocatalytic reaction changed upon membrane-anchoring of the constituents. In general, detailed experiments are described that fully characterize photocatalytic systems, because the mechanism of a reaction involving two different catalytic species is not straight-forward, and cannot be described by a single set of turn-over numbers. Show less
There are many diseases of the central nervous system (CNS), like Parkinson's disease, Alzheimer's disease, depression, schizophrenia, epilepsy, migraine headache, and HIV infection in the brain.... Show moreThere are many diseases of the central nervous system (CNS), like Parkinson's disease, Alzheimer's disease, depression, schizophrenia, epilepsy, migraine headache, and HIV infection in the brain. However, treatment is difficult since many drugs cannot reach the brain in sufficient quantities due to the existence of the blood-brain barrier (BBB). This barrier is situated at the interface of blood and brain and its primary function is to maintain the homeostasis of the brain. The research described in this thesis was initiated to meet the challenge of drug delivery across the BBB. The focus was to explore the possibilities for drug delivery to the brain via the transferrin receptor (TfR). Although drug delivery to the brain via the TfR is not selective, it is effective. A mechanistic approach was chosen in which the binding and association of Tf, Tf-drug conjugates and Tf-tagged liposomes by BCEC in vitro was determined. The efficiency of delivery depends mainly on the size of the cargo: a 40 kDa protein does not affect the association of Tf by the TfR, while a liposomal drug carrier (100 nm in diameter) affects both the association and the intracellular routing. Show less
