Oliver Kreft

Polyelectrolyte microcapsules for biomedical applications

In this paper we review the recent contributions of polyelectrolyte microcapsules in the biomedical field, comprising in vitro and in vivodrug delivery as well as their applications as biosensors.

Polymer microcapsules as mobile local pH-sensors

Polyelectrolyte microcapsules have been loaded with a pH-sensitive, high molecular weight SNARF-1-dextran conjugate. SNARF-1 exhibits a significant pH-dependent emission shift from green to red fluorescence under acidic and basic conditions, respectively. The unique spectral properties of the dye were maintained after the encapsulation. By investigating both human breast cancer cells and fibroblasts, we were able to follow the pH change of the local environment of SNARF-1-filled capsules during the transition from the alkaline cell medium to the acidic endosomal/lysosomal compartments. The incorporation of magnetite nanoparticles and an additional pH-insensitive fluorophore within the capsule shell resulted in a novel type of sensor system based on multifunctional polymer capsules.

Multicompartmental Micro- and Nanocapsules: Hierarchy and Applications in Biosciences

Multicompartmentalized micro- and nanocapsules allow simultaneous delivery of several vectors or biomolecules; they are the next generation of carriers with increased complexity. Here we overview multicompartment micro- and nanocapsules and present a road-map for future developments in the field. Four basic building block structures are demonstrated, three isotropic: concentric, pericentric, and innercentric, and one anisotropic: acentric. As an elaborate implementation of multicompartmentalization, an enzyme-catalyzed reaction inside the same capsule carrying both an enzyme and a substrate is shown. Applications of multicompartmentalized microcapsules for simultaneous multiple drug delivery in bio-medicine are discussed.

Cytotoxicity of nanoparticle-loaded polymer capsules

Cytotoxic effects of micrometer-sized polymer capsules composed out of alternating layers of polystyrenesulfonate (PSS) and polyallylamine hydrochloride (PAH) on a fibroblast cell line have been investigated with an adhesion assay. For the purpose of visualization with fluorescence nanometer-sized CdTe nanoparticles have been embedded in the walls of the capsules. Similar to free CdTe nanoparticles, toxic Cd-ions are also released from CdTe nanoparticles that have been embedded in capsules. At high capsule concentrations, the capsules start to sediment on top of the cells and thus impair cell viability.

Controlled intracellular release of peptides from microcapsules enhances antigen presentation on MHC class I molecules.

To understand the time course of action of any small molecule inside a single cell, one would deposit a defined amount inside the cell and initiate its activity at a defined moment. An elegant way to achieve this is to encapsulate the molecule in a micrometer-sized reservoir, introduce it into a cell, remotely open its wall by a laser pulse, and then follow the biological response by microscopy. The validity of this approach is validated here using microcapsules with defined walls that are doped with metallic nanoparticles so as to enable them to be opened with an infrared laser. The capsules are loaded with a fluorescent antigenic peptide and introduced into mammalian cultured cells where, upon laser-induced release, the peptide binds to major histocompatibility complex (MHC) class I proteins and elicits their cell surface transport. The concept of releasing a drug inside a cell and following its action is applicable to many problems in cell biology and medicine.

Functional analysis of cystathionine gamma-synthase in genetically engineered potato plants.

In plants, metabolic pathways leading to methionine (Met) and threonine diverge at the level of their common substrate, O-phosphohomoserine (OPHS). To investigate the regulation of this branch point, we engineered transgenic potato (Solanum tuberosum) plants affected in cystathionine γ-synthase (CgS), the enzyme utilizing OPHS for the Met pathway. Plants overexpressing potato CgS exhibited either: (a) high transgene RNA levels and 2.7-fold elevated CgS activities but unchanged soluble Met levels, or (b) decreased transcript amounts and enzyme activities (down to 7% of wild-type levels). In leaf tissues, these cosuppression lines revealed a significant reduction of soluble Met and an accumulation of OPHS. Plants expressing CgS antisense constructs exhibited reductions in enzyme activity to as low as 19% of wild type. The metabolite contents of these lines were similar to those of the CgS cosuppression lines. Surprisingly, neither increased nor decreased CgS activity led to visible phenotypic alterations or significant changes in protein-bound Met levels in transgenic potato plants, indicating that metabolic flux to Met synthesis was not greatly affected. Furthermore, in vitro feeding experiments revealed that potato CgS is not subject to feedback regulation by Met, as reported for Arabidopsis. In conclusion, our results demonstrate that potato CgS catalyzes a near-equilibrium reaction and, more importantly, does not display features of a pathway-regulating enzyme. These results are inconsistent with the current hypothesis that CgS exerts major Met metabolic flux control in higher plants.

Manipulation of thiol contents in plants

Summary. As sulfur constitutes one of the macronutrients necessary for the plant life cycle, sulfur uptake and assimilation in higher plants is one of the crucial factors determining plant growth and vigour, crop yield and even resistance to pests and stresses. Inorganic sulfate is mostly taken up as sulfate from the soil through the root system or to a lesser extent as volatile sulfur compounds from the air. In a cascade of enzymatic steps inorganic sulfur is converted to the nutritionally important sulfur-containing amino acids cysteine and methionine (Hell, 1997; Hell and Rennenberg, 1998; Saito, 1999). Sulfate uptake and allocation between plant organs or within the cell is mediated by specific transporters localised in plant membranes. Several functionally different sulfate transporters have to be postulated and have been already cloned from a number of plant species (Clarkson et al., 1993; Hawkesford and Smith, 1997; Takahashi et al., 1997; Yamaguchi, 1997). Following import into the plant and transport to the final site of reduction, the plastid, the chemically relatively inert sulfate molecule is activated through binding to ATP forming adenosine-5′-phosphosulfate (APS). This enzymatic step is controlled through the enzyme ATP-sulfurylase (ATP-S). APS can be further phosphorylated to form 3′-phosphoadenosine-5′-phosphosulfate (PAPS) which serves as sulfate donor for the formation of sulfate esters such as the biosynthesis of sulfolipids (Schmidt and Jäger, 1992). However, most of the APS is reduced to sulfide through the enzymes APS-reductase (APR) and sulfite reductase (SIR). The carbon backbone of cysteine is provided through serine, thus directly coupling photosynthetic processes and nitrogen metabolism to sulfur assimilation. L-serine is activated by serine acetyltransferase (SAT) through the transfer to an acetyl-group from acetyl coenzyme A to form O-acetyl-L-serine (OAS) which is then sulhydrylated using sulfide through the enzyme O-acetyl-L-serine thiol lyase (OAS-TL) forming cysteine. Cysteine is the central precursor of all organic molecules containing reduced sulfur ranging from the amino acid methionine to peptides as glutathione or phytochelatines, proteines, vitamines, cofactors as SAM and hormones. Cysteine and derived metabolites display essential roles within plant metabolism such as protein stabilisation through disulfide bridges, stress tolerance to active oxygen species and metals, cofactors for enzymatic reactions as e.g. SAM as major methylgroup donor and plant development and signalling through the volatile hormone ethylene. Cysteine and other metabolites carrying free sulfhydryl groups are com-monly termed thioles (confer Fig. 1). The physiological control of the sulfate reduction pathway in higher plants is still not completely understood in all details. The objective of this paper is to summarise the available data on the molecular analysis and control of cysteine biosynthesis in plants, and to discuss potentials for manipulating the pathway using transgenic approaches.

Engineering of cysteine and methionine biosynthesis in potato

Summary. Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato.

A Novel Flow-Cytometry-Based Assay for Cellular Uptake Studies of Polyelectrolyte Microcapsules

A flow-cytometry-based assay is presented with which the uptake of polyelectrolyte capsules can be quantified. The cavity of the capsules is loaded with the pH-sensitive dye SNARF, which emits in the red and green in alkaline and acidic environments, respectively. By recording the fluorescence intensities in the red and green channels, the localization of capsules associated with cells can be determined. Capsules adherent to the outer cell membrane fluoresce in the red due to the alkaline pH of the cell medium, whereas capsules internalized by cells fluoresce in the green due to the acidic pH in the endosomal/lysosomal/phagosomal compartments in which incorporated capsules are located. Adding the SNARF readout to the scattering signal typically derived with flow cytometry analysis allows for a more detailed quantitative analysis of particle uptake, which can also distinguish between adherent and ingested particles.

Approaches towards understanding methionine biosynthesis in higher plants

Summary. Plants are able to synthesise all amino acids essential for human and animal nutrition. Because the concentrations of some of these dietary constituents, especially methionine, lysine, and threonine, are often low in edible plant sources, research is being performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their transport, synthesis and accumulation in plants. This knowledge can be used to develop strategies allowing a manipulation of crop plants, eventually improving their nutritional quality.This article is intended to serve two purposes. The first is to provide a brief review on the physiology of methionine synthesis in higher plants. The second is to highlight some recent findings linked to the metabolism of methionine in plants due to its regulatory influence on the aspartate pathway and its implication in plant growth. This information can be used to develop strategies to improve methionine content of plants and to provide crops with a higher nutritional value.