Dries Vercauteren - Academia.edu (original) (raw)

Papers by Dries Vercauteren

To restore inherited or acquired genetic defects or to silence malicious genes, nucleic acids can... more To restore inherited or acquired genetic defects or to silence malicious genes, nucleic acids can be introduced in somatic cells as an active compound, known as gene therapy. However, the nature of nucleic acids implicates packaging in advanced delivery systems that aid in the protection of their payload against enzymatic degradation and facilitate internalization and intracellular release into the target cells. Nanomaterials for nucleic acid delivery have been developed, however, these non-viral vectors still suffer from a relatively low efficiency in gene transfer. This lack of efficiency can be attributed to the combination of different extracellular and intracellular barriers, depending on the specific application. The future success of these nanomaterials, therefore, strongly depends on the careful scrutiny of these biological barriers, as this knowledge will support the rational design of stable, safe and effective multivalent delivery vectors that tackle all of the encountere...

Imaging & Microscopy, 2009

Obtaining information on the mobility of molecules in biomaterials is of interest to many fields ... more Obtaining information on the mobility of molecules in biomaterials is of interest to many fields in the life sciences, such as for cell biology and pharmacy. Advanced fluorescence microscopy techniques are available to this end and are constantly being improved. Here we will introduce the reader to three of the most important ones, FRAP, FCS and SPT, and briefly discuss how these techniques have been of use for our research in the drug delivery field.

Nanoscopy and Multidimensional Optical Fluorescence Microscopy, 2010

Ghent University Ghent University Academic Bibliography. ...

Virus Research, 2010

Statins, such as lovastatin, inhibit the cellular cholesterol biosynthesis. Addition of lovastati... more Statins, such as lovastatin, inhibit the cellular cholesterol biosynthesis. Addition of lovastatin to SK cells and subsequent infection with the alphaherpesvirus pseudorabies virus (PRV) did not affect the intracellular production of viral structural proteins but reduced virus titers. Addition of methyl-beta-cyclodextrin to deplete cholesterol from the viral envelope also resulted in reduced virus titers. Addition of exogenous cholesterol restored virus titers in both experimental assays. Further analysis showed that reducing cholesterol levels reduced both the infectivity of newly produced infectious virus and their stability, as assessed by determining virus titers immediately after treatment or upon storage of the virus at room temperature or frozen. This is the first report to demonstrate that cholesterol is involved in the stability of infectious alphaherpesvirus, and that treatment of host cells with statins reduces alphaherpesvirus titers. Hence, cholesterol is important for pseudorabies virus infectivity and stability.

Interactions between objects inside living cells are often investigated by looking for colocaliza... more Interactions between objects inside living cells are often investigated by looking for colocalization between fluorescence microscopy images that are recorded in separate colours corresponding to the fluorescent label of each object. The fundamental limitation of this approach in the case of dynamic objects is that coincidental colocalization cannot be distinguished from true interaction. Instead, correlation between motion trajectories obtained by dual colour single particle tracking provides a much stronger indication of interaction. However, frequently occurring phenomena in living cells, such as immobile phases or transient interactions, can limit the correlation to small parts of the trajectories. The method presented here, developed for the detection of interaction, is based on the correlation inside a window that is scanned along the trajectories, covering different subsets of the positions. This scanning window method was validated by simulations and, as an experimental proof of concept, it was applied to the investigation of the intracellular trafficking of polymeric gene complexes by endosomes in living retinal pigment epithelium cells, which is of interest to ocular gene therapy.

Virology, 2008

Alphaherpesviruses comprise closely related viruses of man and animal, including herpes simplex v... more Alphaherpesviruses comprise closely related viruses of man and animal, including herpes simplex virus, varicella-zoster virus and pseudorabies virus (PRV). Here, using methyl-beta-cyclodextrin and fluorescently tagged PRV, we directly show that depletion of cholesterol from the plasma membrane of host cells significantly reduces PRV entry. Cholesterol depletion did not reduce PRV attachment, but stalled virus particles at the plasma membrane before penetration of the cell. Cholesterol depletion results in destabilization of lipid raft microdomains in the plasma membrane, which have been shown before to be involved in efficient entry of different viruses. A significant fraction of PRV virions appears to localize juxtaposed to GM1, a lipid raft marker, during entry. Together, these data indicate that cholesterol and possibly cholesterol-rich lipid rafts may be important during PRV entry.

Review of Palaeobotany and Palynology, 2011

Resting cysts of freshwater dinoflagellates in southeastern Georgian Bay (Lake Huron) ... Francin... more Resting cysts of freshwater dinoflagellates in southeastern Georgian Bay (Lake Huron) ... Francine MG McCarthy a,⁎,1, Kenneth Neil Mertens b,1, Marianne Ellegaard c, Keith Sherman d, Vera Pospelova e, Sofia Ribeiro c, Stephan Blasco f, Dries Vercauteren g a Department of ...

REPRODUCTION, 2009

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm–egg interaction in hum... more Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm–egg interaction in human. Recently, it has been demonstrated that Fn – when present during bovine IVF – strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (α5β1) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin α5on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin α5at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm–oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these resu...

REPRODUCTION, 2012

The necessity for early interaction between the embryo and the oviductal and/or uterine environme... more The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not ...

Palynology, 2014

Biostratigraphical investigations of Miocene deposits from the southern North Sea Basin, the Olig... more Biostratigraphical investigations of Miocene deposits from the southern North Sea Basin, the Oligocene and Miocene of the Bahamas, and the lower Pliocene of northern Iceland revealed the presence of new acritarch species. Halodinium eirikssonii n. sp. is recovered from the lower Pliocene Serripes Zone of the Tjörnes beds in northern Iceland, where its range is well constrained through magnetostratigraphy and biostratigraphy using dinoflagellate cysts. Leiosphaeridia spongiosa n. sp. is recovered from lower to upper Miocene deposits of the southern North Sea Basin and from upper Oligocene and Miocene deposits of the Bahamas. Palaeostomocystis orbiculata n. sp. appears to be restricted to the middle Miocene of the North Sea Basin.

Palynology, 2008

Four new dinoflagellate cyst species from the Lower and Middle Miocene strata of the Porcupine Ba... more Four new dinoflagellate cyst species from the Lower and Middle Miocene strata of the Porcupine Basin, offshore southwest Ireland, are formally described. Batiacasphaera edwardsiae sp. nov. was previously described under open nomenclature from the Miocene of the adjacent Rockall Plateau. Lejeunecysta challengerensis sp. nov. is recorded sporadically from the Burdigalian and Serravallian. Selenopemphix porcupensis sp. nov. and Trinovantedinium henrietii sp.

Nanomedicine, 2013

Aim: To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles... more Aim: To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles in the intact vitreous body. Materials & methods: Excised bovine eyes were prepared to preserve the fragile structure of the vitreous humor, while permitting high-resolution fluorescence microscopy and single-particle tracking analysis of intravitreally injected nanoparticles. This assay was validated by analyzing polystyrene beads and further employed to evaluate gene nanomedicines composed of poly(amido amine)s and plasmid DNA. Results: The assay was able to distinguish immobilized cationic nanoparticles from mobile PEGylated nanoparticles. PEGylation of the polyplexes resulted in a drastic improvement of their mobility. Conclusion: An ex vivo eye model is presented for studying nanoparticle mobility in intact vitreous humor by single-particle tracking microscopy. These results give important guidelines for developing gene- and drug-delivery nanomedicines that are compatible with intr...

Molecular Therapy, 2010

The work is done in Ghent, Belgium.

Journal of Controlled Release, 2008

as model drugs, and phosphate buffer [3]. Crosslinking was completed 30 min after the ATPS has be... more as model drugs, and phosphate buffer [3]. Crosslinking was completed 30 min after the ATPS has been exposed to UV-light (366 nm, 3 mW/cm 2). To characterize the hydrogel network regarding the influence of DS and molecular weight on the release profile fluorescence labelled lysozyme and FITC-dextran with varying molecular size (20 kDa, 70 kDa und 500 kDa) were incorporated and analysed regarding their release behaviour. The release studies were carried out under sink conditions at pH 7 and at a temperature of 37°C in a shaking bath. Results and discussion The produced spherical microparticles show a monomodal particle size distribution with an average size of about 10 µm (Fig. 2a, b). As expected, increasing the network density reduced the release rate. In addition, smaller molecules are retained to a lower extent than substances with higher molecular weight. This can be explained by the different degree of unhindered diffusion (Figs. 3 and 4). The data of the initial release suggests that hydrogels with DS 0.05 and 0.22 differ mostly in the amount of pores in the range of 6.6 nm (~70 kDa) (Fig. 4). Molecules which are bigger than that size were only released by degradation of the hydrogel, which was very slow in PBS at pH 7. To accelerate the biodegradation of the hydrogel and thus the release of FITCdextran, the loaded microspheres were incubated in sodium carbonate buffer at pH 9.6 and human serum respectively. In case of sodium carbonate buffer, the hydrolytically labile carbonate group of HES-HEMA is cleaved rapidly. A dependency on molecular weight and DS can be seen (Fig. 4). Incubating in human serum leads to a degradation of the hydrogel network due to the presence of the enzyme α-amylase, an endoamylase, which is able to degrade the HES-backbone at positions 1-4 and thereby accelerate the release (Fig. 5). Conclusion These studies show that the described method is well suited for the preparation of drug loaded hydrogel microspheres. The release of FITCdextran was bi-phasic, with a rapid release of predominantly small molecules by diffusion and a slow release after hydrolytic degradation of the hydrogel. Both phases are influenced by the degree of substitution of the polymer, which allows to tailor the release profile from HES-HEMA hydrogels.

Journal of Controlled Release, 2010

A great deal of attention in biopharmacy and pharmaceutical technology is going to the developmen... more A great deal of attention in biopharmacy and pharmaceutical technology is going to the development of nanoscopic particles to efficiently deliver nucleic acids to target cells. Despite the great potential of nucleic acids for treatment of various diseases, progress in the field is fairly slow. One of the causes is that development of suitable nanoscopic delivery vehicles is hampered by insufficient knowledge of their physicochemical and biophysical properties during the various phases of the transfection process. To address this issue, in the past decade we have developed and applied advanced fluorescence microscopy techniques that can provide a better insight in the transport and stability of nanoparticles in various biological media. This mini-review discusses the basic principles of fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT), and gives an overview of studies in which we have employed these techniques to characterize the transport and stability of nucleic acid containing nanoparticles in extracellular media and in living cells.

Journal of Controlled Release, 2012

The low transfection efficacy of non-viral gene delivery systems limits the therapeutic applicati... more The low transfection efficacy of non-viral gene delivery systems limits the therapeutic application of these vectors. Besides the inefficient release of the complexes or pDNA from endolysosomes into the cytoplasm or poor nuclear uptake, the nuclear and post-nuclear processing might unfavorably affect the transgene expression. Positively charged amphiphilic 1,4-dihydropyridine (1,4-DHP) derivatives were earlier proposed as a promising tool for the delivery of DNA into target cells in vitro and in vivo. However, the structure/activity relationship of these carriers is poorly understood as yet. In this work we studied the intracellular processing of complexes, composed of three structurally related 1,4-DHP derivatives, in a retinal pigment epithelial (ARPE-19) cell line. The pre-and post-nuclear processing of the complexes was quantified on the nuclear, mRNA and transgene expression level. Here we show that the interaction of 1,4-DHP complexes with the cell membrane temporarily increases the permeability of the ARPE-19 cell membrane for small molecular compounds. However, the main mechanism for internalization of 1,4-DHP complexes is endocytosis. We found that all examined derivatives are able to destabilize endosomal membranes by lipid exchange upon acidification. In addition, the buffering capacity of some of the compounds may contribute to the endosomal escape of the complexes as well through the proton sponge effect. Previously we reported that cellular uptake of 1,4-DHP complexes does not correlate with transgene expression. In this study we surprisingly revealed that there is no correlation between the amount of plasmids taken up by the cell and the amount of plasmids found in the cell nucleus. Furthermore, it was found that a high amount of plasmid in the nucleus does not ensure high mRNA expression, likely due to remaining interactions of the carrier with the plasmids. Neither did the expression of mRNA always result in the production of a functional protein, possibly due to the interaction of free carrier with intracellular components which are involved in the post-translational modification of protein and folding process. Overall, our data suggest that succeeding of both the pre-and the post-nuclear intracellular processes is equally essential for successful transgene expression.

Journal of Controlled Release, 2012

In the field of nanomedicine, ample attention has been paid to the development of nanocarriers fo... more In the field of nanomedicine, ample attention has been paid to the development of nanocarriers for the intracellular delivery of therapeutic cargo, such as nucleic acids for gene therapy. The efficiency with which these non-viral carriers deliver their payload at the required intracellular site of action remains low. Despite extensive research on cellular attachment, endocytosis and intracellular trafficking of nanocarriers, clear-cut rules for the design of effective nanocarriers to improve nucleic acid transfer are still lacking. This is mainly caused by the cell type-dependence of this highly dynamic cellular processing, and to the lack of reliable methods to study these events. For these reasons there is a strong demand for the development and standardization of such methods in order to better understand the intracellular dynamics of nanomedicine processing and validate cellular and intracellular targeting strategies. This review aims at providing an overview of the different processes that are currently known to be involved in the cellular processing of nanomedicines, with a focus on cellular internalization mechanisms, as this has received a great deal of attention in the last couple of years. Furthermore, the intracellular hurdles that need to be overcome to allow efficient NA transfer will be critically discussed. In addition, an overview will be given of various methodologies that have been applied to unravel these cellular processing mechanisms, with a discussion on their strengths and weaknesses.

Journal of Controlled Release, 2012

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery o... more Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors.

FEMS Microbiology Ecology, 2010

Campylobacteriosis is the most frequently reported foodborne disease in the industrialized world,... more Campylobacteriosis is the most frequently reported foodborne disease in the industrialized world, mainly through consumption of contaminated chicken meat. To date, no information is available on the primary infection sources of poultry. In this study, the ability of five Campylobacter jejuni strains with different invasion potential towards Caco-2 cells to survive and replicate in the protozoan Acanthamoeba castellanii was tested under simulated in situ conditions (i.e. chicken broiler houses). Results indicate that environmental conditions play a crucial role in C. jejuni-A. castellanii interactions. Co-culture in general did not result in an increase of either bacteria or amoebae. However, co-culture with Acanthamoeba did result in a delayed decline and an increased long-term survival of Campylobacter. Bacterial strain-specific effects were observed, with higher survival rates for low-invasive strains. The presence of C. jejuni in general did not affect A. castellanii viability, except at 37 1C under microaerobic conditions, where the presence of the reference and low-invasive Campylobacter strains resulted in a significant decline in amoebal viability. Confocal laser scanning microscopy revealed that intra-amoebal campylobacters were not always colocated with acidic organelles, suggesting potential bacterial interference with digestive processes. As Acanthamoeba enhances the persistence of C. jejuni, the presence of the amoeba in broiler house environments may have important implications for the ecology and epidemiology of this food pathogen.

To restore inherited or acquired genetic defects or to silence malicious genes, nucleic acids can... more To restore inherited or acquired genetic defects or to silence malicious genes, nucleic acids can be introduced in somatic cells as an active compound, known as gene therapy. However, the nature of nucleic acids implicates packaging in advanced delivery systems that aid in the protection of their payload against enzymatic degradation and facilitate internalization and intracellular release into the target cells. Nanomaterials for nucleic acid delivery have been developed, however, these non-viral vectors still suffer from a relatively low efficiency in gene transfer. This lack of efficiency can be attributed to the combination of different extracellular and intracellular barriers, depending on the specific application. The future success of these nanomaterials, therefore, strongly depends on the careful scrutiny of these biological barriers, as this knowledge will support the rational design of stable, safe and effective multivalent delivery vectors that tackle all of the encountere...

Imaging & Microscopy, 2009

Obtaining information on the mobility of molecules in biomaterials is of interest to many fields ... more Obtaining information on the mobility of molecules in biomaterials is of interest to many fields in the life sciences, such as for cell biology and pharmacy. Advanced fluorescence microscopy techniques are available to this end and are constantly being improved. Here we will introduce the reader to three of the most important ones, FRAP, FCS and SPT, and briefly discuss how these techniques have been of use for our research in the drug delivery field.

Nanoscopy and Multidimensional Optical Fluorescence Microscopy, 2010

Ghent University Ghent University Academic Bibliography. ...

Virus Research, 2010

Statins, such as lovastatin, inhibit the cellular cholesterol biosynthesis. Addition of lovastati... more Statins, such as lovastatin, inhibit the cellular cholesterol biosynthesis. Addition of lovastatin to SK cells and subsequent infection with the alphaherpesvirus pseudorabies virus (PRV) did not affect the intracellular production of viral structural proteins but reduced virus titers. Addition of methyl-beta-cyclodextrin to deplete cholesterol from the viral envelope also resulted in reduced virus titers. Addition of exogenous cholesterol restored virus titers in both experimental assays. Further analysis showed that reducing cholesterol levels reduced both the infectivity of newly produced infectious virus and their stability, as assessed by determining virus titers immediately after treatment or upon storage of the virus at room temperature or frozen. This is the first report to demonstrate that cholesterol is involved in the stability of infectious alphaherpesvirus, and that treatment of host cells with statins reduces alphaherpesvirus titers. Hence, cholesterol is important for pseudorabies virus infectivity and stability.

Interactions between objects inside living cells are often investigated by looking for colocaliza... more Interactions between objects inside living cells are often investigated by looking for colocalization between fluorescence microscopy images that are recorded in separate colours corresponding to the fluorescent label of each object. The fundamental limitation of this approach in the case of dynamic objects is that coincidental colocalization cannot be distinguished from true interaction. Instead, correlation between motion trajectories obtained by dual colour single particle tracking provides a much stronger indication of interaction. However, frequently occurring phenomena in living cells, such as immobile phases or transient interactions, can limit the correlation to small parts of the trajectories. The method presented here, developed for the detection of interaction, is based on the correlation inside a window that is scanned along the trajectories, covering different subsets of the positions. This scanning window method was validated by simulations and, as an experimental proof of concept, it was applied to the investigation of the intracellular trafficking of polymeric gene complexes by endosomes in living retinal pigment epithelium cells, which is of interest to ocular gene therapy.

Virology, 2008

Alphaherpesviruses comprise closely related viruses of man and animal, including herpes simplex v... more Alphaherpesviruses comprise closely related viruses of man and animal, including herpes simplex virus, varicella-zoster virus and pseudorabies virus (PRV). Here, using methyl-beta-cyclodextrin and fluorescently tagged PRV, we directly show that depletion of cholesterol from the plasma membrane of host cells significantly reduces PRV entry. Cholesterol depletion did not reduce PRV attachment, but stalled virus particles at the plasma membrane before penetration of the cell. Cholesterol depletion results in destabilization of lipid raft microdomains in the plasma membrane, which have been shown before to be involved in efficient entry of different viruses. A significant fraction of PRV virions appears to localize juxtaposed to GM1, a lipid raft marker, during entry. Together, these data indicate that cholesterol and possibly cholesterol-rich lipid rafts may be important during PRV entry.

Review of Palaeobotany and Palynology, 2011

Resting cysts of freshwater dinoflagellates in southeastern Georgian Bay (Lake Huron) ... Francin... more Resting cysts of freshwater dinoflagellates in southeastern Georgian Bay (Lake Huron) ... Francine MG McCarthy a,⁎,1, Kenneth Neil Mertens b,1, Marianne Ellegaard c, Keith Sherman d, Vera Pospelova e, Sofia Ribeiro c, Stephan Blasco f, Dries Vercauteren g a Department of ...

REPRODUCTION, 2009

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm–egg interaction in hum... more Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm–egg interaction in human. Recently, it has been demonstrated that Fn – when present during bovine IVF – strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (α5β1) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin α5on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin α5at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm–oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these resu...

REPRODUCTION, 2012

The necessity for early interaction between the embryo and the oviductal and/or uterine environme... more The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not ...

Palynology, 2014

Biostratigraphical investigations of Miocene deposits from the southern North Sea Basin, the Olig... more Biostratigraphical investigations of Miocene deposits from the southern North Sea Basin, the Oligocene and Miocene of the Bahamas, and the lower Pliocene of northern Iceland revealed the presence of new acritarch species. Halodinium eirikssonii n. sp. is recovered from the lower Pliocene Serripes Zone of the Tjörnes beds in northern Iceland, where its range is well constrained through magnetostratigraphy and biostratigraphy using dinoflagellate cysts. Leiosphaeridia spongiosa n. sp. is recovered from lower to upper Miocene deposits of the southern North Sea Basin and from upper Oligocene and Miocene deposits of the Bahamas. Palaeostomocystis orbiculata n. sp. appears to be restricted to the middle Miocene of the North Sea Basin.

Palynology, 2008

Four new dinoflagellate cyst species from the Lower and Middle Miocene strata of the Porcupine Ba... more Four new dinoflagellate cyst species from the Lower and Middle Miocene strata of the Porcupine Basin, offshore southwest Ireland, are formally described. Batiacasphaera edwardsiae sp. nov. was previously described under open nomenclature from the Miocene of the adjacent Rockall Plateau. Lejeunecysta challengerensis sp. nov. is recorded sporadically from the Burdigalian and Serravallian. Selenopemphix porcupensis sp. nov. and Trinovantedinium henrietii sp.

Nanomedicine, 2013

Aim: To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles... more Aim: To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles in the intact vitreous body. Materials & methods: Excised bovine eyes were prepared to preserve the fragile structure of the vitreous humor, while permitting high-resolution fluorescence microscopy and single-particle tracking analysis of intravitreally injected nanoparticles. This assay was validated by analyzing polystyrene beads and further employed to evaluate gene nanomedicines composed of poly(amido amine)s and plasmid DNA. Results: The assay was able to distinguish immobilized cationic nanoparticles from mobile PEGylated nanoparticles. PEGylation of the polyplexes resulted in a drastic improvement of their mobility. Conclusion: An ex vivo eye model is presented for studying nanoparticle mobility in intact vitreous humor by single-particle tracking microscopy. These results give important guidelines for developing gene- and drug-delivery nanomedicines that are compatible with intr...

Molecular Therapy, 2010

The work is done in Ghent, Belgium.

Journal of Controlled Release, 2008

as model drugs, and phosphate buffer [3]. Crosslinking was completed 30 min after the ATPS has be... more as model drugs, and phosphate buffer [3]. Crosslinking was completed 30 min after the ATPS has been exposed to UV-light (366 nm, 3 mW/cm 2). To characterize the hydrogel network regarding the influence of DS and molecular weight on the release profile fluorescence labelled lysozyme and FITC-dextran with varying molecular size (20 kDa, 70 kDa und 500 kDa) were incorporated and analysed regarding their release behaviour. The release studies were carried out under sink conditions at pH 7 and at a temperature of 37°C in a shaking bath. Results and discussion The produced spherical microparticles show a monomodal particle size distribution with an average size of about 10 µm (Fig. 2a, b). As expected, increasing the network density reduced the release rate. In addition, smaller molecules are retained to a lower extent than substances with higher molecular weight. This can be explained by the different degree of unhindered diffusion (Figs. 3 and 4). The data of the initial release suggests that hydrogels with DS 0.05 and 0.22 differ mostly in the amount of pores in the range of 6.6 nm (~70 kDa) (Fig. 4). Molecules which are bigger than that size were only released by degradation of the hydrogel, which was very slow in PBS at pH 7. To accelerate the biodegradation of the hydrogel and thus the release of FITCdextran, the loaded microspheres were incubated in sodium carbonate buffer at pH 9.6 and human serum respectively. In case of sodium carbonate buffer, the hydrolytically labile carbonate group of HES-HEMA is cleaved rapidly. A dependency on molecular weight and DS can be seen (Fig. 4). Incubating in human serum leads to a degradation of the hydrogel network due to the presence of the enzyme α-amylase, an endoamylase, which is able to degrade the HES-backbone at positions 1-4 and thereby accelerate the release (Fig. 5). Conclusion These studies show that the described method is well suited for the preparation of drug loaded hydrogel microspheres. The release of FITCdextran was bi-phasic, with a rapid release of predominantly small molecules by diffusion and a slow release after hydrolytic degradation of the hydrogel. Both phases are influenced by the degree of substitution of the polymer, which allows to tailor the release profile from HES-HEMA hydrogels.

Journal of Controlled Release, 2010

A great deal of attention in biopharmacy and pharmaceutical technology is going to the developmen... more A great deal of attention in biopharmacy and pharmaceutical technology is going to the development of nanoscopic particles to efficiently deliver nucleic acids to target cells. Despite the great potential of nucleic acids for treatment of various diseases, progress in the field is fairly slow. One of the causes is that development of suitable nanoscopic delivery vehicles is hampered by insufficient knowledge of their physicochemical and biophysical properties during the various phases of the transfection process. To address this issue, in the past decade we have developed and applied advanced fluorescence microscopy techniques that can provide a better insight in the transport and stability of nanoparticles in various biological media. This mini-review discusses the basic principles of fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT), and gives an overview of studies in which we have employed these techniques to characterize the transport and stability of nucleic acid containing nanoparticles in extracellular media and in living cells.

Journal of Controlled Release, 2012

The low transfection efficacy of non-viral gene delivery systems limits the therapeutic applicati... more The low transfection efficacy of non-viral gene delivery systems limits the therapeutic application of these vectors. Besides the inefficient release of the complexes or pDNA from endolysosomes into the cytoplasm or poor nuclear uptake, the nuclear and post-nuclear processing might unfavorably affect the transgene expression. Positively charged amphiphilic 1,4-dihydropyridine (1,4-DHP) derivatives were earlier proposed as a promising tool for the delivery of DNA into target cells in vitro and in vivo. However, the structure/activity relationship of these carriers is poorly understood as yet. In this work we studied the intracellular processing of complexes, composed of three structurally related 1,4-DHP derivatives, in a retinal pigment epithelial (ARPE-19) cell line. The pre-and post-nuclear processing of the complexes was quantified on the nuclear, mRNA and transgene expression level. Here we show that the interaction of 1,4-DHP complexes with the cell membrane temporarily increases the permeability of the ARPE-19 cell membrane for small molecular compounds. However, the main mechanism for internalization of 1,4-DHP complexes is endocytosis. We found that all examined derivatives are able to destabilize endosomal membranes by lipid exchange upon acidification. In addition, the buffering capacity of some of the compounds may contribute to the endosomal escape of the complexes as well through the proton sponge effect. Previously we reported that cellular uptake of 1,4-DHP complexes does not correlate with transgene expression. In this study we surprisingly revealed that there is no correlation between the amount of plasmids taken up by the cell and the amount of plasmids found in the cell nucleus. Furthermore, it was found that a high amount of plasmid in the nucleus does not ensure high mRNA expression, likely due to remaining interactions of the carrier with the plasmids. Neither did the expression of mRNA always result in the production of a functional protein, possibly due to the interaction of free carrier with intracellular components which are involved in the post-translational modification of protein and folding process. Overall, our data suggest that succeeding of both the pre-and the post-nuclear intracellular processes is equally essential for successful transgene expression.

Journal of Controlled Release, 2012

In the field of nanomedicine, ample attention has been paid to the development of nanocarriers fo... more In the field of nanomedicine, ample attention has been paid to the development of nanocarriers for the intracellular delivery of therapeutic cargo, such as nucleic acids for gene therapy. The efficiency with which these non-viral carriers deliver their payload at the required intracellular site of action remains low. Despite extensive research on cellular attachment, endocytosis and intracellular trafficking of nanocarriers, clear-cut rules for the design of effective nanocarriers to improve nucleic acid transfer are still lacking. This is mainly caused by the cell type-dependence of this highly dynamic cellular processing, and to the lack of reliable methods to study these events. For these reasons there is a strong demand for the development and standardization of such methods in order to better understand the intracellular dynamics of nanomedicine processing and validate cellular and intracellular targeting strategies. This review aims at providing an overview of the different processes that are currently known to be involved in the cellular processing of nanomedicines, with a focus on cellular internalization mechanisms, as this has received a great deal of attention in the last couple of years. Furthermore, the intracellular hurdles that need to be overcome to allow efficient NA transfer will be critically discussed. In addition, an overview will be given of various methodologies that have been applied to unravel these cellular processing mechanisms, with a discussion on their strengths and weaknesses.

Journal of Controlled Release, 2012

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery o... more Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors.

FEMS Microbiology Ecology, 2010

Campylobacteriosis is the most frequently reported foodborne disease in the industrialized world,... more Campylobacteriosis is the most frequently reported foodborne disease in the industrialized world, mainly through consumption of contaminated chicken meat. To date, no information is available on the primary infection sources of poultry. In this study, the ability of five Campylobacter jejuni strains with different invasion potential towards Caco-2 cells to survive and replicate in the protozoan Acanthamoeba castellanii was tested under simulated in situ conditions (i.e. chicken broiler houses). Results indicate that environmental conditions play a crucial role in C. jejuni-A. castellanii interactions. Co-culture in general did not result in an increase of either bacteria or amoebae. However, co-culture with Acanthamoeba did result in a delayed decline and an increased long-term survival of Campylobacter. Bacterial strain-specific effects were observed, with higher survival rates for low-invasive strains. The presence of C. jejuni in general did not affect A. castellanii viability, except at 37 1C under microaerobic conditions, where the presence of the reference and low-invasive Campylobacter strains resulted in a significant decline in amoebal viability. Confocal laser scanning microscopy revealed that intra-amoebal campylobacters were not always colocated with acidic organelles, suggesting potential bacterial interference with digestive processes. As Acanthamoeba enhances the persistence of C. jejuni, the presence of the amoeba in broiler house environments may have important implications for the ecology and epidemiology of this food pathogen.