Capsule endoscopy : diagnosis of intestinal localisation of systemic follicular B-cell non-Hodgkin lymphoma.

We report the case of a 58 year old man with occult obscure gastro-intestinal bleeding (OGIB) without other significant symptoms, in which systemic localisation of follicular B-cell non-Hodgkin lymphoma was discovered trough capsule endoscopy. This case reflects the clinical significance of performing capsule endoscopy in patients with OGIB.

Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient.

In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the "added value" we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms.

Screening poly(ethyleneglycol) micro- and nanogels for drug delivery purposes.

This study investigates poly(ethyleneglycol) (PEG)-based micro- and nanogels, with or without lipid coating, with the aim to slowly deliver encapsulated molecules. Hydroxyethylmethacrylated PEG (PEG-HEMA), PEG-HEMA with an oligo lactate spacer (PEG-lac-HEMA), and eight-armed PEG end capped with HEMA (* -PEG-HEMA) were used. PEG-lac-HEMA matrices degraded very fast (in terms of days), while it took about 1 month for linear PEG-HEMA and several months for * -PEG-HEMA hydrogels to become degraded. PEG-based microgels were made by use of an all aqueous technique and could be lipid-coated by mixing the microgels (made positively or negatively charged through copolymerization with respectively methacrylic acid and dimethyl aminoethyl methacrylate with a suspension of oppositely charged liposomes. The release of fluorescently labeled molecules incorporated in the PEG-based microgels could be clearly governed by the type of molecules used (lasting from hours to months). PEG-based nanogels could be made using liposomes as a nanoscopic mold, resulting in particles with a PEG gel core surrounded by a lipid coating. BSA could be easily encapsulated in the PEG nanogels which released the BSA over a period of about 1 week.

Pegylation of liposomes favours the endosomal degradation of the delivered phosphodiester oligonucleotides.

Liposomal vesicles have been widely investigated as carriers for the intracellular delivery of oligonucleotides (ONs). To avoid unspecific uptake by the reticulo endothelial system, 'pegylation' of the liposomes, by incorporating polyethyleneglycol (PEG) at the surface, has been an attractive strategy. While pegylation has a clear benefit on the systemic level, one could wonder if pegylation also benefits the delivery efficacy of liposomes at the intracellular level. We compared the intracellular distribution of non-pegylated and pegylated liposomes, with special attention to the integrity of the oligonucleotides they are carrying. After uptake in the cells, the non-pegylated liposomes efficiently escaped from the endosomes thereby releasing phosphodiester oligonucleotides (PO-ONs) in the cytoplasm of the cells. The PO-ONs were however rapidly degraded in the intracellular environment. In contrast to non-pegylated liposomes, pegylated liposomes failed in protecting the PO-ONs they were carrying, leading to rapid degradation of the PO-ONs in the endosomal compartment. Furthermore, the PEG chains inhibited the endosomal escape of the degraded ONs. These intracellular findings explain why pegylated liposomes failed in establishing an antisense effect.

Influence of free chains on the swelling pressure of PEG-HEMA and dex-HEMA hydrogels.

Insight in the osmotic behavior of degrading hydrogels is of great importance in the design of biodegradable hydrogels for biomedical applications. This study compares the degradation behavior of PEG-HEMA (hydroxyethylmethacrylated polyethylene glycol) and dex-HEMA (hydroxyethylmethacrylated dextran) hydrogels. The degradation of PEG-HEMA gels takes several months to over a year, while that of dex-HEMA gels takes only days or weeks. The faster degradation kinetics of dex-HEMA networks can be attributed to stabilization of the keto-enol form by hydroxyl groups. Upon degradation of PEG-HEMA and dex-HEMA hydrogels, respectively, free PEG and free dextran chains are produced. We investigated the effect of unattached PEG and dextran chains on the swelling pressure of the degrading gels. It is found that low molecular weight free chains significantly increase the swelling pressure. However, the contribution of higher molecular weight free chains (M(w)>10 kDa) is similar to that of the network chains.

Layer-by-layer coating of degradable microgels for pulsed drug delivery.

Recently, we reported on "self-rupturing" microcapsules which consist of a biodegradable dextran-based microgel surrounded by a polyelectrolyte membrane. Degradation of the microgel increases the swelling pressure in the microcapsules which, when sufficiently high, ruptures the surrounding polyelectrolyte membrane. The membrane surrounding the microgels is deposited using the layer-by-layer (LbL) technique, which is based on the alternate adsorption of oppositely charged polyelectrolytes onto a charged substrate. In this paper, we characterize with confocal microscopy, electrophoretic mobility, scanning electron microscopy and atomic force microscopy in detail the deposition and the properties of the LbL coatings on the dextran microgels. We show that by fine-tuning the properties of both the microgel core and the LbL membrane the swelling pressure which is evoked by the degradation of the microgel is indeed able to rupture the surrounding LbL membrane. Further, we show that the application of an LbL coating on the surface of the microgels dramatically lowers the burst release from the microcapsules and results in massive release at the time the microcapsules rupture.

The choice of a suitable oligosaccharide to prevent aggregation of PEGylated nanoparticles during freeze thawing and freeze drying.

In a previous study we have shown that the oligosaccharide inulin can prevent aggregation of poly(ethylene glycol) (PEG) coated plasmid DNA/cationic liposome complexes ("PEGylated lipoplexes") during freeze thawing and freeze drying [Hinrichs et al., 2005. J. Control. Release 103, 465]. By contrast, dextran clearly failed as stabilizer. These results were ascribed to the fact that inulin and PEG are compatible while dextran and PEG are not. In this study the stabilizing capacities of inulin and dextran (of various molecular weights) during freeze thawing and freeze drying of four different types of nanoparticles, each type with different amounts of PEG at their surface, were investigated. Freeze drying and freeze thawing of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleoyl-phosphatidyl-ethanolamine (DOPE) liposomes and egg phosphatidyl choline (EPC)/cholesterol (CHOL) liposomes showed that inulins are excellent stabilizers even for highly PEGylated liposomes while (especially higher molecular weight) dextrans dramatically lost their stabilizing capacity when increasing the degree of PEGylation of the liposomes. The same results were obtained for plasmid DNA/DOTAP/DOPE complexes. Finally, both inulin and dextran could prevent full aggregation of plasmid DNA/polyethylenimine (PEI) complexes independent whether PEI was PEGylated or not. It is concluded that inulins are preferred as stabilizers over dextrans for various types of PEGylated nanoparticles due to their compatibility with PEG.

Studying biophysical barriers to DNA delivery by advanced light microscopy.

Advanced light microscopy (ALM) has been intensively employed by biophysicists to reveal cellular mechanisms. As described in this review, ALM clearly has potential to enhance our understanding of the mechanisms that affect macromolecular therapeutics or nanoscopic drug vectors in biological environments. However, while in recent years confocal microscopy and related techniques became rather routinely used in drug delivery it remains challenging to extract reliable information on the biophysical behaviour of drug delivery systems from ALM measurements. This review discusses studies in which confocal imaging, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and fluorescence energy transfer were employed to reveal biophysical properties of DNA and DNA containing nanoparticles in extra- and intracellular media.

FRET-FCS as a tool to evaluate the stability of oligonucleotide drugs after intracellular delivery.

The intracellular degradation of single-stranded, double-labeled oligonucleotides (ONs) was studied by following the disappearance of Fluorescence Resonance Energy Transfer (FRET) between the rhodamine green and Cy5 fluorophores attached to respectively the 3' and 5' end of the ONs. The green and red fluorescence intensities upon rhodamine green excitation were monitored using the ultra-sensitive detectors of a dual-color Fluorescence Correlation Spectroscopy (FCS) instrument. The ratio of the red to green fluorescence (R/G ratio) as obtained from such FRET-FCS measurements showed to give accurate information on the integrity of the ONs, without the need for additional auto- or cross-correlation analysis of the registered fluorescence intensity fluctuations. Intracellular measurements revealed that most of the 40mer phosphodiester ONs were degraded before they entered the nucleus. For the 20mer phosphodiester ONs, this degradation occurred more slowly, and both intact and degraded ONs entered the nucleus. For the 20mer phosphorothioate ONs, no intracellular degradation was observed during the measured time period. The sensitive detection of the intracellular fluorescence by the FCS setup will be particularly useful in situations where the expected fluorescence is too low to be detected by FRET-imaging as may occur after intracellular delivery of ONs by cationic carriers.

Towards a better understanding of the dissociation behavior of liposome-oligonucleotide complexes in the cytosol of cells.

To obtain real breakthroughs in antisense therapy, it is necessary to understand the cellular behavior of antisense delivery systems. Fluorescence fluctuation spectroscopy (FFS), which measures in time fluorescence fluctuations in the excitation volume of a microscope and which can thus be applied on a cellular scale, shows potential for this purpose. In this study dual color FFS was explored to characterize the complexation (association and dissociation) between Cy5-labeled oligonucleotides (Cy5-ONs) and FITC-labeled cationic liposomes (FITC-liposomes) in respectively buffer, cell lysate and the cytosol of Vero cells. In Hepes buffer the association of the Cy5-ONs to the FITC-liposomes could be clearly observed from the high peaks of Cy5- and FITC-fluorescence, which appeared simultaneously in the excitation volume. This was explained by the fact that in the complexed state many Cy5-ONs and FITC-liposomes are bound to each other and thus move together through the excitation volume thereby resulting in high fluorescence 'FITC/Cy5-peaks'. FFS measurements on FITC-liposome/Cy5-ONs complexes in cell lysate revealed that a minor part of the Cy5-ONs was released from the complexes. The major part of the Cy5-ONs remained in the complexes, which also seemed to aggregate in cell lysate. In agreement with the measurements in cell lysate, after microinjection of FITC-liposome/Cy5-ONs complexes in the cytosol of Vero cells a part of the Cy5-ONs was released (as Cy-ONs were detected by FFS in the nuclei) while the other part remained bound (as Cy5-peaks were frequently observed in the cytosol). As will be explained, the Cy5-peaks could be due both to Cy5-ONs clustered with cytosol components and Cy5-ONs still complexed to FITC-liposomes with quenched FITC-fluorescence.

Inulin is a promising cryo- and lyoprotectant for PEGylated lipoplexes.

The aim of this study was to investigate whether the oligosaccharides dextran and inulin are able to prevent aggregation of lipoplexes based on 1,2-dioleoyl-3-trimethylammonium-propane and dioleoylphosphatidyl-ethanolamine with and without distearoylphosphatidylethanolamine-polyethyleneglycol (PEGylated and nonPEGylated lipoplexes, respectively) during storage. The lipoplexes, dispersed in the oligosaccharide solution were frozen and subsequently stored at subzero temperature or freeze dried and subsequently stored at 37 degrees C. When lipoplexes in frozen dispersions were stored below the glass transition temperature of the maximally freeze concentrated fraction (Tg') of the oligosaccharide solutions severe aggregation of the nonPEGylated lipoplexes was prevented for 3 months by both inulin and dextran. However, while dextran failed to stabilize the frozen PEGylated lipoplexes (as in most cases full aggregation occurred in short time) inulin successfully protected them against aggregation. Compared to dextran, inulin was also a superior lyoprotectant of PEGylated lipoplexes: during freeze drying and subsequent storage at 37 degrees C of the dried powders for 3 months the PEGylated lipoplexes maintained their original size when dispersed in inulin matrices while in dextran matrices they fully aggregated in most cases. It is hypothesized that the aggregation of the PEGylated lipoplexes in dextran solutions is caused by the well known incompatibility between dextrans and PEG. This is further supported by the observation that inulins and PEG are compatible. It is concluded that oligosaccharides can prevent severe aggregation of nonPEGylated lipoplexes. The same holds for PEGylated lipoplexes provided that the oligosaccharide is compatible with PEG. Finally, this work also shows that the higher Tg' of oligosaccharides makes them more versatile cryoprotectants than disaccharides like sucrose or trehalose as the frozen dispersions can be stored at higher temperatures for prolonged periods of time. Furthermore, it is proposed that oligosaccharides are also more versatile lyoprotectants than the disaccharides because they can be exposed to higher relative humidities without passing the glass transition temperature.

Protection of oligonucleotides against nucleases by pegylated and non-pegylated liposomes as studied by fluorescence correlation spectroscopy.

Antisense phosphodiester oligonucleotides (ONs), complexed to carriers such as cationic liposomes, inhibit the production of proteins. The biochemical and biophysical phenomena that govern the extent of this inhibition are still not fully understood. Major biological barriers limiting a pronounced antisense effect are the cellular entry and endosomal escape of the ONs containing liposomes, the release of the ONs from the liposomes and the extra- and intracellular degradation of the ONs. In this paper we focus on the latter barrier and evaluate, by fluorescence correlation spectroscopy (FCS), to what extent phosphodiester ONs complexed to DOTAP/DOPE liposomes, are protected against degradation by nucleases. Liposomes studied were either with or without a polyethyleneglycol (PEG) moiety at the surface. Using non-pegylated liposomes the phosphodiester ONs were initially adequately protected when exposed to DNase I. Indeed, in the mechanism for lipoplex formation as suggested by others, the ONs become trapped between lipid bilayers and are therefore shielded from the environment. However, after a few hours the phosphodiester ONs no longer stayed intact. This was explained by a gradual fusion of the lipoplexes in time thereby spontaneously releasing phosphodiester ONs. Using pegylated liposomes, a substantial fraction of the phosphodiester ONs degraded immediately after exposing the complexes to DNase I. Based on experimental evidence we suggest that the presence of the PEG-chains influences lipoplex formation so that the ONs are not trapped between lipid bilayers and therefore remain accessible by the DNase I enzyme.

The in vitro evaluation of 'azo containing polysaccharide gels' for colon delivery.

This study reports on 'azo-polysaccharide gels', more specifically azo-inulin and azo-dextran gels, for colon drug delivery. Compared with azo-hydrogels which can be only degraded by reduction of the azo-groups, this study evaluates whether, in vitro, azo-polysaccharide gels can be degraded through both reduction of the azo-groups in the crosslinks as well as enzymatic break down of the polysaccharide backbone. The azo-polysaccharide gels were synthesized by radical crosslinking of a mixture of methacrylated inulin or methacrylated dextran and N,N'-bis(methacryloylamino)azobenzene (B(MA)AB) and were characterized by dynamic mechanical analysis and swelling measurements. Azo-dextran gels could be obtained from methacrylated dextran having low degree of substitution but not from lowly substituted methacrylated inulin. Increasing the amount of B(MA)AB resulted in denser azo-inulin and azo-dextran networks. Compared with their swelling in dimethylformamide, all azo-dextran gels became more swollen in water while azo-inulin gels shrank upon exposure to water, indicating a more hydrophobic character of the azo-inulin gels. Break down of the inulin and dextran chains in the azo-polysaccharide gels by inulinase and dextranase, respectively, was observed. However, the degradation of azo-dextran gels by dextranase seemed to be more pronounced than the degradation of the azo-inulin gels by inulinase. In rat caecal content medium, reduction of the azo function in azo-inulin gels was not observed. This may be attributed to a low partitioning of nicotinamide-adenine dinucleotide phosphate (NADP(+)) in the gels.

Optimisation of an in vitro procedure for the determination of the enzymatic inhibition potency of multifunctional polymers.

An in vitro procedure for the determination of the inhibition potency of multifunctional polymers towards the proteolytic enzyme trypsin was optimised. Carbopol((R)) 934P was used as the reference polymer. The enzymatic reaction was optimised and the HPLC method was validated. The optimal substrate concentration and enzymatic activity were determined aiming at extracting the linear or steady-state part of the metabolite concentration versus time curve of the enzymatic degradation reaction. A substrate concentration of 20 mmol/l N-alpha-benzoyl-L-arginine-ethylester and an enzymatic activity of 30 enzymatic units trypsin/ml were used. The degree of trypsin inhibition was expressed by the inhibition factor (IF), defined as the ratio of the enzymatic reaction rate without a polymer (control) to the reaction rate in the presence of a polymer. During the optimisation of the trypsin inhibition assay, formation of an ion complex between the substrate and the poly(acrylic acid) was observed. The complex formation was concentration dependent, but the influence on the enzymatic reaction was negligible as long as an excessive substrate concentration was present in the reaction medium. The optimised method allows to characterize, evaluate and compare the in vitro trypsin inhibition strength for most multifunctional polymers.

Biodegradable hydrogels based on stereocomplex formation between lactic acid oligomers grafted to dextran.

A novel hydrogel system in which crosslinking is established by stereocomplex formation between lactic acid oligomers of opposite chirality is proposed. To investigate the feasibility of this novel system, we first investigate whether there is an operation window where lactic acid oligomers in either the D- or L-form do not give a crystalline phase, whereas in a blend of the D- and L-form stereocomplex formation occurs. Therefore, D- and L-lactic acid oligomers with different degrees of polymerization (DP) were prepared and analyzed using DSC. It was shown that crystallinity was present in D- or L-oligomers with DP > or = 11. On the other hand, in blends of D- and L-oligomers of lactic acid crystallinity (stereocomplexation) was already observed at a DP > or = 7. In the next step, L- and D-lactic acid oligomers were coupled via their terminal hydroxyl group to dextran, yielding dex-(L)lactate and dex-(D)lactate, respectively. Upon dissolving each product in water separately and mixing the solutions, a hydrogel is formed at room temperature as demonstrated by rheological measurements. The storage modulus of the obtained hydrogel strongly decreased upon heating to 80 degrees C, while it was restored upon cooling to 20 degrees C demonstrating the thermo-reversibility and the physical nature of the cross-links. The storage modulus of the gels depends on the degree of polymerization of the lactate acid grafts and their degree of substitution on dextran. Interestingly, gel formation was favored when one lactic oligomer was coupled via its hydroxyl group whereas the oligomer of opposite chirality was coupled via its carboxylic acid group. This is ascribed to the parallel packing of the oligomers in stereocomplexes.

Differential spectrophotometric determination of tyrosine and tryptophan in pharmaceutical amino acid solutions.

A method is proposed for the simultaneous determination of tyrosine and tryptophan in solutions by differential spectrophotometry. The concentrations are calculated from the measurement of the absorbance of the amino acid mixture in an alkaline medium and from the differential absorbance of the alkaline solution against the acid solution at 294.4 nm. A comparison with three other well-known methods is discussed.

The physical properties of biogels and their permeability for macromolecular drugs and colloidal drug carriers.

Macromolecular drugs, either free or complexed with colloidal drug carriers, have created a great deal of interest during the last decade. If one wants to administer these new therapeutics via the oral, nasal, and cervical routes or through the conductive airways, one of the first barriers to overcome is the mucus layer that adheres to the related epithelia. In this review, the physicochemical properties of biogels, macromolecular drugs, and colloidal drug carriers that play a major role in transport through biogels are reviewed. Also, methods of studying the mobility of macromolecular drugs and colloidal drug carriers in and through biogels are addressed.

An oral controlled release matrix pellet formulation containing nanocrystalline ketoprofen.

A controlled release pellet formulation using a NanoCrystal colloidal dispersion of ketoprofen was developed. In order to be able to process the aqueous NanoCrystal colloidal dispersion into a hydrophobic solid dosage form a spray drying procedure was used. The in vitro dissolution profiles of wax based pellets loaded with nanocrystalline ketoprofen are compared with the profiles of wax based pellets loaded with microcrystalline ketoprofen and of a commercial sustained release ketoprofen formulation. Pellets were produced using a melt pelletisation technique. All pellet formulations were composed of a mixture of microcrystalline wax and starch derivatives. The starch derivatives used were waxy maltodextrin and drum dried corn starch. Varying the concentration of drum dried corn starch increased the release rate of ketoprofen but the ketoprofen recovery remained problematic. To increase the dissolution yield surfactants were utilised. The surfactants were either added during the production process of the NanoCrystal colloidal dispersion (sodium laurylsulphate) or during the pellet manufacturing process (Cremophor RH 40). Both methods resulted in a sustained but complete release of nanocrystalline ketoprofen from the matrix pellet formulations.

In vivo evaluation of matrix pellets containing nanocrystalline ketoprofen.

The aim of this study was to evaluate the in-vivo behaviour of matrix pellets formulated with nanocrystalline ketoprofen after oral administration to dogs. No significant differences in AUC-values were seen between pellet formulations containing nanocrystalline or microcrystalline ketoprofen and a commercial ketoprofen formulation (reference: Rofenid 200 Long Acting). C(max) of the formulations containing nano- or microcrystalline ketoprofen was significantly higher compared to reference, whereas t(max) was significantly lower. The in-vivo burst release observed for the spray dried nanocrystalline ketoprofen matrix pellets was reduced following compression of the pellets in combination with placebo wax/starch pellets. These matrix tablets sustained the ketoprofen plasma concentrations during 5.6 and 5.4 h for formulations containing nano- and microcrystalline ketoprofen, respectively.

Abetalipoproteinemia or Bassen-Kornzweig syndrome. Clinical, biochemical and electrophysiological features of two cases.

The clinical, biochemical and electrophysiologic features of two patients with abetalipoproteinemia, a 17 year old boy and his sister of 14, are reported. They are the second and third reports of this disease in Belgium. Diagnosis was made by the Apo-B deficiency in their serum and the normal levels in their parents'. According to other investigators we revealed in both cases deficiency of other apoproteins, indicating, that the metabolic defect affects all classes of plasma lipoproteins. EMG findings showed axonal neuropathy. Somatosensory evoked potentials demonstrated dorsal column dysfunction. Findings were consistent with the known neuropathology of abetalipoproteinemia and of vitamin E deficiency syndromes. The therapeutic role of vitamin E is discussed.