In glioblastoma, a malignant primary brain tumor, liposomes have shown promise in pre-clinical and early phase clinical trials as delivery vehicles for therapeutics. However, external factors... Show moreIn glioblastoma, a malignant primary brain tumor, liposomes have shown promise in pre-clinical and early phase clinical trials as delivery vehicles for therapeutics. However, external factors influencing cellular uptake of liposomes in glioma cells are poorly understood. Heparin and heparin analogues are commonly used in glioma patients to decrease the risk of thrombo-embolic events. Our results show that heparin inhibits pegylated liposome uptake by U87 glioma and GL261 cells in a dose dependent manner in vitro, and that heparin-mediated inhibition of uptake required presence of fetal bovine serum in the media. In a subcutaneous model of glioma, Cy5.5 labeled liposomes could be detected with in vivo imaging after direct intra-tumoral injection. Ex-vivo analysis with flow cytometry showed a decreased uptake of liposomes into tumor cells in mice treated systemically with heparin compared to those treated with vehicle only. Show less
The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is a promising approach for anti-cancer immunotherapy by bridging innate and adaptive immunity. Recent evidence... Show moreThe cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is a promising approach for anti-cancer immunotherapy by bridging innate and adaptive immunity. Recent evidence suggests that chemotherapy-induced DNA damage can directly induce dendritic cell (DC) maturation and recruitment, which synergizes with STING activation to enhance anti-tumor effects. As an immunogenic cell death (ICD) inducer, oxaliplatin generates massive double-stranded DNA (dsDNA) crosslinks, release of tumor-associated antigens and promoting the "eat me" signal. STING activation improves antigen immunogenicity, which can promote T cell activation and infiltration. In this study, we developed liposomes encapsulating oxaliplatin and combine this formulation with a STING agonist (ADU-S100) for treating colorectal cancer. The liposomes efficiently inhibited the proliferation of tumor cells while induced ICD in CT26 colorectal cancer cells, which enhanced dendritic cell maturation and phagocytosis in vitro. The liposome-based immunochemotherapy exhibited the strongest efficacy, resulting in complete remission upon tumor inoculation. Mechanistic studies showed this potent anti-cancer effect was related to the significant recruitment of infiltrating CD8 and CD4 T cells, reduction of suppressive Treg cells, and a shift in the phenotype of tumor-associated suppressive macrophages that promote cancer to immune stimulating macrophages. Thus, our study demonstrated the potential of combining oxaliplatin-loaded liposomes with a STING agonist to reduce tumor growth by regulating the immunosuppressive state in the tumor. Show less
Gu, Z.L.; Silva, C.G. da; Hao, Y.; Schomann, T.; Camps, M.G.M.; Maaden, K. van der; ... ; Cruz, L.J. 2022
Therapeutic cancer drug efficacy can be limited by insufficient tumor penetration, rapid clearance, systemic toxicity and (acquired) drug resistance. The poor therapeutic index due to inefficient... Show moreTherapeutic cancer drug efficacy can be limited by insufficient tumor penetration, rapid clearance, systemic toxicity and (acquired) drug resistance. The poor therapeutic index due to inefficient drug penetration and rapid drug clearance and toxicity can be improved by using a liposomal platform. Drug resistance for instance against pemetrexed, can be reduced by combination with docetaxel. Here, we developed a specific liposomal formulation to simultaneously deliver docetaxel and pemetrexed to enhance efficacy and safety. Hydrophobic docetaxel and hydrophilic pemetrexed were co-encapsulated into pH-sensitive liposomes using a thin-film hydration method with high efficiency. The physicochemical properties, toxicity, and immunological effects of liposomes were examined in vitro. Biodistribution, anti-tumor efficacy, and systemic immune response were evaluated in vivo in combination with PD-L1 immune checkpoint therapy using two murine colon cancer models. In cellular experiments, the liposomes exhibited strong cytotoxicity and induced immunogenic cell death. In vivo, the treatment with the liposome-based drug combination inhibited tumor development and stimulated immune responses. Liposomal encapsulation significantly reduced systemic toxicity compared to the delivery of the free drug. Tumor control was strongly enhanced when combined with anti-PDL1 immunotherapy in immunocompetent mice carrying syngeneic MC38 or CT26 colon tumors. We showed that treatment with liposome-mediated chemotherapy of docetaxel and pemetrexed combined with anti-PD-L1 immunotherapy is a promising strategy for the treatment of colon cancers. Show less
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
The research described in this thesis shows that hypercholesterolemia, a well-established risk factor for atherosclerosis, can impact skin lipid pool and barrier function already at young age. In... Show moreThe research described in this thesis shows that hypercholesterolemia, a well-established risk factor for atherosclerosis, can impact skin lipid pool and barrier function already at young age. In the field of atherosclerosis, we showed that the small peptide Lyp-1 can be used as a targeting molecule in liposomal formulations to deliver liver X receptor agonist to plaque resident foam cells/lipid-rich macrophages. Elucidation of the mechanisms underlying the intercommunication between plasma lipids and skin lipids may also bring valuable opportunities to prevent and treat dermatological pathologies in dyslipidemic patients; perhaps in combination with anti-atherogenic therapies. Thus, by deepening our knowledge we may improve our advice to the patients and ultimately improve their quality of life. 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
Alzheimer's Disease (AD) is the main contributor to the syndrome dementia, one for which no cure has yet been developed. One of the key issues with developing a cure is the presence of the blood... Show moreAlzheimer's Disease (AD) is the main contributor to the syndrome dementia, one for which no cure has yet been developed. One of the key issues with developing a cure is the presence of the blood-brain barrier (BBB), a restrictive network involving the cerebral vasculature which prevents passage of therapeutics into the brain. Currently many if not all of the clinically most advanced therapeutic avenues involve the use of antibodies to target the production, oligomerization and aggregation of the peptide Amyloid beta. In this thesis, an antibody variant derived from camelids, such as llamas, is examined. The llama antibody is devoid of the light chain, and therefore significantly smaller and easier to manipulate than conventional antibodies. Various strategies have been explored, including the use ofdirect intrathecal injections, liposomes and adeno associated virusses to allow the llama antibody fragment to enter and remain in the brain of mouse models of AD. Various imaging modalities are used to determine the success of BBB passage, including micro-SPECT, intravital multiphoton microscopy, in vivo whole body fluorescence imaging and ex vivo IHC and IF. The thesis supplies a foundation for delivery of llama antibody fragments into the brain. Show less
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
Current seasonal influenza vaccines rely on the induction of antibodies to neutralize the virus. However, influenza viruses frequently undergo genetic mutations due to antigenic drift and shift,... Show moreCurrent seasonal influenza vaccines rely on the induction of antibodies to neutralize the virus. However, influenza viruses frequently undergo genetic mutations due to antigenic drift and shift, altering the surface proteins hemagglutinin and neuraminidase to which antibodies usually bind. This could render vaccine-induced antibody responses ineffective, resulting in an ineffective influenza vaccine. Influenza vaccines based on the induction of T cell responses might be cross-reactive, since they target conserved influenza epitopes that do not tend to mutate. However, the peptide antigens that are able to induce such T cell responses are often poorly immunogenic. In this thesis, several formulation strategies are described that could improve the immunogenicity of influenza T cell peptide antigens. Using combinations of delivery systems and immunostimulators, the peptide antigens were able to induce influenza-specific T cell responses in mice. Furthermore, a model was developed that could predict the in vitro adjuvanticity of liposomes according to the liposomal lipid composition. In addition, the recent advances in influenza vaccine development are discussed. Finally, an alternative delivery system, the Bioneedle, was evaluated for the delivery of several influenza vaccines. Show less
Kroon, J.; Metselaar, J.M.; Storm, G.; Pluijm, G. van der 2014
The best form of protection against influenza is vaccination, in terms of efficacy to protect individuals and reduction of the social impact of epidemics on our human societies. Chapter 1 of this... Show moreThe best form of protection against influenza is vaccination, in terms of efficacy to protect individuals and reduction of the social impact of epidemics on our human societies. Chapter 1 of this thesis details the current influenza vaccines available and their lack of efficacy, and the current need for new adjuvanted influenza formulations. Pathogens are often particles and formulating antigens into nanoparticles (NP) results in systems that resemble the pathogens in terms of size, and notably can promote antigen uptake by dendritic cells (DC). The principal aim of the research in this thesis was to investigate how NP systems can act as an adjuvant for subunit influenza vaccine 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
