Rolf Schubert

University of Freiburg, Freiburg, Baden-Württemberg, Germany

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Publications (74)364.9 Total impact

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    ABSTRACT: Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein.
    The Journal of Cell Biology 09/2015; DOI:10.1083/jcb.201506085 · 9.83 Impact Factor
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    ABSTRACT: The spontaneous formation of lipid vesicles, in particular fatty acid vesicles, is considered an important physical process at the roots of cellular life. It has been demonstrated previously that the addition of fatty acid micelles to preformed vesicles induces vesicle self-reproduction by a growth-division mechanism. Despite multiple experimental efforts, it remains unresolved how vesicles rearrange upon the addition of fresh membrane-forming compounds, and whether solutes that are initially encapsulated inside the mother vesicles are evenly redistributed among the daughter ones. Here we investigate the growth-division of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles, which, following the addition of oleate micelles, form mixed oleate/POPC vesicles. Our approach is based on free-flow electrophoresis (FFE) and cryogenic transmission electronmicroscopy (cryo-TEM). Two new features emerge from this study. FFE analysis unexpectedly reveals that the uptake of oleate micelles by POPC vesicles follows two different pathways depending on the micelles/vesicles ratio. At low oleate molar fractions (<0.35), plain incorporation of oleate into pre-existing POPC vesicles is our dominant observation. In contrast, oleate-rich and oleate-poor daughter vesicles are generated from parent POPC vesicles when the oleate molar fraction exceeds 0.35. Cryo-TEM reveals that when ferritin-filled vesicles grow and divide, some vesicles contain ferritin at increased concentrations, others are empty. Intriguingly, in some cases, ferritin appears to be highly concentrated inside the vesicles. These observations imply a specific redistribution (partitioning) of encapsulated solutes among nascent vesicles during the growth-division steps. We have interpreted our observations by assuming that freshly added oleate molecules are taken-up preferentially (cooperatively) by oleate-rich membrane regions that form spontaneously in POPC/oleate vesicles when a certain threshold (oleate molar fraction ca. 0.35) is surpassed. The proposed cooperative mechanism could be based on differential microscopic constants for oleate/oleic acid dynamics in oleate-rich and oleate-poor membrane regions, which eventually generate populations of oleate-rich and oleate-poor vesicles.
    The Journal of Physical Chemistry B 09/2015; DOI:10.1021/acs.jpcb.5b05057 · 3.30 Impact Factor
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    ABSTRACT: To detect unstable atherosclerotic plaques early and noninvasively would be of great clinical interest. Activated platelets are an interesting molecular target for detecting early lesions or unstable plaques. We therefore developed an MRI contrast agent consisting of magnetoliposomes (ML) linked to an antibody (anti-LIBS) specifically targeting the ligand-induced binding site of the activated GPIIb/IIIa receptor of platelets. ML were prepared by dual centrifugation (DC). ML pegylation up to a total PEG content of 7.5 mol% positively influenced the stability and amount of entrapped SPIOs, and also reduced SPIO-membrane interactions, while higher PEG contents destabilized PEG-ML. Stable anti-LIBS-ML with high amounts of entrapped SPIOs (∼86%, ∼0.22 mol Fe/mol liposomal lipid) and high MRI sensitivity (relaxivity r2 = 422 s(-1) mM(-1) and r2(∗) = 452 s(-1) mM(-1)) were obtained by coupling anti-LIBS to ML in a two-step post-insertion technique. We confirmed specific binding to the GPIIb/IIIa receptor's activated conformation on activated human platelets and cell lines expressing activated GPIIb/IIIa receptor ex vivo. The immuno-ML obtained in this study constitute an important step towards developing a potentially human-compatible MRI contrast agent for the timely detection of plaque rupture by targeting activated platelets. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Biomaterials 06/2015; 53. DOI:10.1016/j.biomaterials.2015.02.088 · 8.56 Impact Factor
  • Christoph Grapentin · Sabine Barnert · Rolf Schubert
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    ABSTRACT: Perfluorocarbon nanoemulsions (PFC-NE) are disperse systems consisting of nanoscale liquid perfluorocarbon droplets stabilized by an emulsifier, usually phospholipids. Perfluorocarbons are chemically inert and non-toxic substances that are exhaled after in vivo administration. The manufacture of PFC-NE can be done in large scales by means of high pressure homogenization or microfluidization. Originally investigated as oxygen carriers for cases of severe blood loss, their application nowadays is more focused on using them as marker agents in 19F Magnetic Resonance Imaging (19F MRI). 19F is scarce in organisms and thus PFC-NE are a promising tool for highly specific and non-invasive imaging of inflammation via 19F MRI. Neutrophils, monocytes and macrophages phagocytize PFC-NE and subsequently migrate to inflamed tissues. This technique has proven feasibility in numerous disease models in mice, rabbits and mini pigs. The translation to clinical trials in human needs the development of a stable nanoemulsion whose droplet size is well characterized over a long storage time. Usually dynamic light scattering (DLS) is applied as the standard method for determining particle sizes in the nanometer range. Our study uses a second method, analysis of transmission electron microscopy images of cryo-fixed samples (Cryo-TEM), to evaluate stability of PFC-NE in comparison to DLS. Four nanoemulsions of different composition are observed for one year. The results indicate that DLS alone cannot reveal the changes in particle size, but can even mislead to a positive estimation of stability. The combination with Cryo-TEM images gives more insight in the particulate evolution, both techniques supporting one another. The study is one further step in the development of analytical tools for the evaluation of a clinically applicable perfluorooctylbromide nanoemulsion.
    PLoS ONE 06/2015; 10(6):e0130674. DOI:10.1371/journal.pone.0130674 · 3.23 Impact Factor
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    ABSTRACT: Inflammation is a hallmark of cardiac healing after myocardial infarction and it determines subsequent cardiovascular morbidity and mortality. The aim of the present study was to explore whether inflammation imaging with two perfluorocarbon (PFC) nanoemulsions and fluorine magnetic resonance imaging ((19)F MRI) is feasible at 3.0 T with sufficient signal-to-noise ratio (SNR) using explanted hearts, an (19)F surface coil and dedicated MR sequences. Acute myocardial infarction (AMI) was induced by balloon angioplasty (50 min) of the distal left anterior descending artery in 12 pigs. One day thereafter, PFCs were injected intravenously to label circulating monocytes. Either emulsified perfluoro-15-crown-5 ether or already clinically applied perfluorooctyl bromide (PFOB) was applied. Four days after AMI and immediately after gadolinium administration, hearts were explanted and imaged with a 3.0 T Achieva MRI scanner. (19)F MRI could be acquired with an SNR of >15 using an in-plane resolution of 2 × 2 mm(2) within <20 min for both agents. Combined late gadolinium enhancement (LGE) and (19)F MRI revealed that (19)F signal was inhomogenously distributed across LGE myocardium reflecting patchy macrophage infiltration as confirmed by histology. In whole hearts, we found an apico-basal (19)F gradient within LGE-positive myocardium. The (19)F-positive volume was always smaller than LGE volume. Ex vivo experiments on isolated monocytes revealed that pig and human cells phagocytize PFCs even more avidly than mouse monocytes. This pilot study demonstrates that (19)F MRI at 3.0 T with clinically applicable PFOB is feasible, thus highlighting the potential of (19)F MRI to monitor the inflammatory response after AMI. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email:
    European Heart Journal – Cardiovascular Imaging 03/2015; 16(6). DOI:10.1093/ehjci/jev008 · 2.65 Impact Factor
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    ABSTRACT: -Non-invasive detection of deep venous thrombi and subsequent pulmonary thromboembolism are serious medical challenges and are difficult to detect by conventional ultrasound techniques. -Here we report a novel technique for the sensitive and specific identification of developing thrombi making use of background free (19)F MRI together with α2-antiplasmin peptide (α2(AP)) targeted perfluorocarbon nanoemulsions (PFCs) as contrast agent, which is cross-linked to fibrin by active factor XIII. Ligand functionality was ensured by mild coupling conditions using sterol-based post-insertion (SPIT). Developing thrombi with a diameter smaller than 0.8 mm could be unequivocally visualized in the murine vena cava inferior as hot spots in vivo by simultaneous acquisition of anatomical matching (1)H and (19)F MR images at 9.4 T with both excellent signal- and contrast-to-noise ratio (71±22 and 17±5, respectively). Furthermore, α2(AP)-PFC could be successfully applied for diagnosis of experimentally induced pulmonary thromboembolism. In line with the reported half-life of FXIIIa, application of α2(AP)-PFCs later than 60 min after thrombus induction did not any longer result in detectable (19)F MRI signals. Corresponding results were obtained in ex vivo generated human clots. Thus, α2(AP)-PFCs can visualize freshly developed thrombi which might still be susceptible to pharmacologic intervention. -Our results demonstrate that (1)H/(19)F MRI together with α2(AP)-PFCs is a sensitive non-invasive technique for the diagnosis of acute deep venous thrombi and pulmonary thromboemboli. Furthermore, ligand coupling by SPIT represents a unique platform for specific targeting of PFCs for in vivo (19)F MRI.
    Circulation 02/2015; 131(16). DOI:10.1161/CIRCULATIONAHA.114.010962 · 14.43 Impact Factor
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    ABSTRACT: Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.
    Nature Material 01/2015; 14(1):125-132. DOI:10.1038/nmat4118 · 36.50 Impact Factor
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    ABSTRACT: The aim of the present study was to develop film-coated tablets which release a minor amount of the active pharmaceutical ingredient (API) into the stomach and small intestine, yet show a sharp increase of drug release in the colon. Tablets containing the model drug Diclofenac-Na, microcrystalline cellulose as a filler (MT), as well as tablets consisting of Ludiflash(®) (LT), were both used as tablet cores, respectively. Either chitosan (CHI) alone or different ratios of chitosan and Kollicoat(®) Smartseal 30 D (KCSS) were applied onto these cores. The resulting film-coated tablets were analysed for swelling, drug dissolution and stability. In order to clarify whether the colon release is mainly enzyme-driven or pressure-controlled, the coated tablets were both tested in the colon microflora test (CMT), which simulates the enzyme environment within the colon, and using a bio-relevant dissolution apparatus mimicking the intraluminal pressures and stress conditions present in the gastrointestinal tract (GIT). CHI/KCSS (25:75) coated LTs showed a pressure-controlled site-specific drug release in the large intestine, while remaining intact in the upper GIT. CHI as well as CHI/KCSS (25:75) applied onto MTs, remained stable during the entire simulated bio-relevant dissolution transit of the GIT, but showed enzymatically controlled colon targeting in the CMT. These results could be confirmed for CHI/KCSS (25:75) film-coated MT tablets top-coated with an additional hydroxypropylmethycellulose (HPMC) layer and an Eudragit L 30 D-55 (EUL) layer to avoid the dissolution in the fasting stomach.
    European Journal of Pharmaceutics and Biopharmaceutics 10/2014; 88(3). DOI:10.1016/j.ejpb.2014.09.006 · 3.38 Impact Factor
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    ABSTRACT: The potential of boron-containing lipids with three different structures, which were intended for use in boron neutron capture therapy, was investigated. All three types of boron lipids contained the anionic dodecaborate cluster as the headgroup. Their effects on two different tumor models in mice following intravenous injection were tested; for this, liposomes with boron lipid, distearoyl phosphatidylcholine, and cholesterol as helper lipids, and containing a polyethylene glycol lipid for steric protection, were administered intravenously into tumor-bearing mice (C3H mice for SCCVII squamous cell carcinoma and BALB/c mice for CT26/WT colon carcinoma). With the exception of one lipid (B-THF-14), the lipids were well tolerated, and no other animal was lost due to systemic toxicity. The lipid which led to death was not found to be much more toxic in cell culture than the other boron lipids. All of the lipids that were well tolerated showed hemorrhage in both tumor models within a few hours after administration. The hemorrhage could be seen by in vivo magnetic resonance and histology, and was found to occur within a few hours. The degree of hemorrhage depended on the amount of boron administered and on the tumor model. The observed unwanted effect of the lipids precludes their use in boron neutron capture therapy.
    International Journal of Nanomedicine 07/2014; 9(1):3583-90. DOI:10.2147/IJN.S65166 · 4.38 Impact Factor
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    ABSTRACT: Ultrasound is a common tool for clinical diagnosis due to its safety and economic. Especially the addition of ultrasound contrast agents leads to a high diagnostic reliability. In recent years ultrasound has been used as a trigger for directed drug delivery or to enhance thrombolysis. We developed a nanoscaled ultrasound contrast agent (NUSCA) to improve these applications. In the future drugs can be incorporated into this contrast agent to achieve a combination of ultrasound diagnosis and therapy. The aim of the present study is to elucidate the structure of the nanoscaled lipid formulations and a potential dependence of the ultrasound contrast enhancement on this structure. Our NUSCA is based on the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and the single-chained polyethylene glycol (40) stearate (PEG40S). In this study the effect of increasing concentrations of the single chained PEG40S on the structure of the lipid formulations was characterised using Dynamic Light Scattering, cryo-Transmission Electron Microscopy, Nuclear Magnetic Resonance spectroscopy, lipid monolayer studies and epifluorescence measurements. In addition, the ultrasound contrast enhancement for the formulations was determined in vitro. Dependence between structure and ultrasound contrast was found. All PEG40S concentrations lead to a mixture of liposomes and discoid micelles. With increasing PEG40S content the amount of micelles increased. Certain PEG40S concentrations lead to an ultrasound contrast superior to the contrast of the commercially available ultrasound contrast agent SonoVue(®).
    Colloids and surfaces B: Biointerfaces 05/2014; 117:206–215. DOI:10.1016/j.colsurfb.2014.02.029 · 4.15 Impact Factor
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    ABSTRACT: Since pharmacokinetic and pharmacodynamic activities of drugs are often related to their interactions with biomembranes, it is of high interest to establish an approach for the characterization of these interactions at the molecular level. For the present study beta-blockers (oxprenolol, propranolol, and acebutolol) were selected due to their well described nonspecific membrane effects (NME). Their interactions with model lipid membranes composed of palmitoyloleoylphosphatidylcholine (POPC) were studied using Time-Dependent Fluorescence Shift (TDFS) and Generalized Polarization (GP) as well as molecular dynamics (MD) simulations. Liposomal vesicles were labeled with fluorescent membrane polarity probes (Laurdan, Prodan, and Dtmac). Increasing beta-blocker concentrations (0-10 mM for acebutolol and oxprenolol, and 0-1.5 mM for propranolol) significantly rigidifies the lipid bilayer at the glycerol and headgroup level, which was detected in the steady-state and in the time-resolved fluorescence data. The effects of propranolol where considerably stronger than those of the two other beta-blockers. The addition of fluorescent probes precisely located at different levels within the lipid bilayer revealed the insertion of the beta-blockers into the POPC bilayer at the glycerol backbone level, which was further confirmed by MD simulations in the case of propranolol.
    European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 03/2014; 87(3). DOI:10.1016/j.ejpb.2014.03.013 · 3.38 Impact Factor
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    ABSTRACT: Classical Hodgkin's lymphoma (cHL)-affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell-cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL-derived extracellular vesicles might substitute for a direct cell-cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40-800 nm, as determined by cryo- and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30-dependent release of interleukin-8 in CD30L(+) eosinophil-like EoL-1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)-embedded culture of HRS cells, a network of actin and tubulin-based protrusions guided CD30(+) vesicles into the micro-environment. This network targeted CD30(+) vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle-containing network also in cHL-affected lymphoid tissue of both mixed-cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30-CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin-based CD30 antibody constructs. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
    The Journal of Pathology 03/2014; 232(4):405-14. DOI:10.1002/path.4306 · 7.43 Impact Factor
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    ABSTRACT: Inflammatory processes can reliably be assessed by (19) F MRI using perfluorocarbons (PFCs), which is primarily based on the efficient uptake of emulsified PFCs by circulating cells of the monocyte-macrophage system and subsequent infiltration of the (19) F-labeled cells into affected tissue. An ideal candidate for the sensitive detection of fluorine-loaded cells is the biochemically inert perfluoro-15-crown-5 ether (PFCE), as it contains 20 magnetically equivalent (19) F atoms. However, the biological half-life of PFCE in the liver and spleen is extremely long, and so this substance is not suitable for future clinical applications. In the present study, we investigated alternative, nontoxic PFCs with predicted short biological half-lives and high fluorine content: perfluorooctyl bromide (PFOB), perfluorodecalin (PFD) and trans-bis-perfluorobutyl ethylene (F-44E). Despite the complex spectra of these compounds, we obtained artifact-free images using sine-squared acquisition-weighted three-dimensional chemical shift imaging and dedicated reconstruction accomplished with in-house-developed software. The signal-to-noise ratio of the images was maximized using a Nutall window with only moderate localization error. Using this approach, the retention times of the different PFCs in murine liver and spleen were determined at 9.4 T. The biological half-lives were estimated to be 9 days (PFD), 12 days (PFOB) and 28 days (F-44E), compared with more than 250 days for PFCE. In vivo sensitivity for inflammation imaging was assessed using an ear clip injury model. The alternative PFCs PFOB and F-44E provided 37% and 43%, respectively, of the PFCE intensities, whereas PFD did not show any signal in the ear model. Thus, for in vivo monitoring of inflammatory processes, PFOB emerges as the most promising candidate for possible future translation of (19) F MR inflammation imaging to human applications. Copyright © 2013 John Wiley & Sons, Ltd.
    NMR in Biomedicine 03/2014; 27(3). DOI:10.1002/nbm.3059 · 3.04 Impact Factor
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    ABSTRACT: The delivery of nucleic acids like DNA or siRNA still represents a major hurdle, especially with regard to possible therapeutic applications in vivo. Much attention has been focused on the development of non-viral gene delivery vectors including liposomes or cationic polymers. Among them, polyethylenimines (PEIs) have been widely explored for the delivery of nucleic acids and show promising results. The combination of cationic polymers and liposomes (lipopolyplexes) for gene delivery may further improve their efficacy and biocompatibility, by combining the favourable properties of lipid systems (high stability, efficient cellular uptake, low cytotoxicity) and PEI (nucleic acid condensation, facilitated endosomal release). In this study, we systematically analyse various conditions for the preparation of liposome-polyethylenimine-based lipopolyplexes with regard to biological activity (DNA transfection efficacy, siRNA knockdown efficacy) and physicochemical properties (size, zeta potential, stability). This includes the exploration of lipopolyplex compositions containing different liposomes and different relevant branched or linear low-molecular weight PEIs. We establish optimal parameters for lipopolyplex generation, based on various PEIs, N/P ratios, lipids, lipid/PEI ratios and preparation conditions. Importantly, we also demonstrate that certain lipopolyplexes retain their biological activity and physicochemical integrity upon prolonged storage even at 37 °C and/or in the presence of serum, thus providing formulations with considerably higher stability as compared to polyplexes. In conclusion, we establish optimal liposome-polyethylenimine lipopolyplexes that allow storage under ambient conditions. This is the basis and an essential prerequisite for novel, promising and easy-to-handle formulations for possible therapeutic applications.
    Acta biomaterialia 02/2014; 10(6). DOI:10.1016/j.actbio.2014.02.037 · 6.03 Impact Factor
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    ABSTRACT: Abstract Poly(n-butyl-cyanoacrylate)-nanocapsules filled by perfluorodecalin (PFD) are proposed as potential oxygen carriers for blood substitute. The capsule dispersion is prepared via interfacial polymerisation from a PFD emulsion in water which in turn is generated by spontaneous phase separation. The resulting dispersion is capable of carrying approximately 10% of its own volume of gaseous oxygen, which is approximately half of the capacity of human blood. The volumes of the organic solvents and water are varied within a wide range, connected to a change of the capsule radius between 200 and 400 nm. The principal suitability of the capsule dispersion for intravenous application is proven in first physiological experiments. A total amount of 10 ml/kg body weight has been infused into rats, with the dispersion supernatant and a normal saline solution as controls. After the infusion of nanocapsules, the blood pressure as well as the heart rate remains constant on a normal level.
    Journal of Microencapsulation 10/2013; 31(3). DOI:10.3109/02652048.2013.843600 · 1.59 Impact Factor
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    ABSTRACT: Ultrasound is investigated as a novel drug delivery tool within cancer therapy. Non-thermal ultrasound treatment of solid tumours post i.v.-injection of drug-carrying liposomes may induce local drug release from the carrier followed by enhanced intracellular drug uptake. Recently, ultrasound-mediated drug release of liposomes (sonosensitivity) was shown to strongly depend on liposome membrane composition. In the current study the ultrasound-mediated drug release mechanism of liposomes was investigated. The results showed that differences in ultrasound drug release kinetics obtained for different liposomal compositions were caused by distinctive release mechanisms of the carriers. Two types of liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and hydrogenated soy l-α-phosphatidylcholine (HSPC) as main lipids, respectively, were recently shown to vary in sonosensitivity. Here, these liposomes were analyzed prior to and after a given ultrasound-exposure for their mean size, size distribution and morphology. Cryo-transmission electron microscopy, dynamic light scattering and asymmetric flow field-flow fractionation in combination with multi-angle light scattering revealed a significant change in mean size, size distribution and morphology of DOPE-based liposomes after ultrasound, pointing to an irreversible disruption of the vesicles and concomitant drug release. In contrast, the HSPC-based liposomes remained unchanged in size and structure after ultrasound application, indicating pore-mediated release mechanisms. The results show that the release mechanisms and interactions between ultrasound and liposomes depend on the liposome membrane-composition, explaining their sonosensitive properties.
    Journal of pharmaceutical and biomedical analysis 02/2013; 78-79C:118-122. DOI:10.1016/j.jpba.2013.01.043 · 2.98 Impact Factor
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    ABSTRACT: Highly ordered two or three dimensional mesophases in aqueous solution could be usefully obtained by using monoolein (MO) or diolein (DO) monomers. Nanostructures (also indicated as nanoparticles, NPs) of MO or DO containing different amounts (1%, 5%, 10% and 20%) of the synthetic amphiphilic gadolinium complex (C18)2DTPA(Gd) have been prepared and characterized for their relaxometric and structural behaviors. The nanostructure is found in the 110–200 nm range for all investigated systems, while the presence of the gadolinium containing monomer produces a partial loss of the cubic symmetry, as shown by Cryo-TEM images of NPs doped with 10% w/w of (C18)2DTPA(Gd). Gadolinium containing nanostructures display high relaxivity values (in the 10–15 mM−1 s−1 range at 25° and 20 MHz, with a further increase at 37 °C for DO based NPs), and interesting relaxometric properties for their possible use as MRI contrast agents. NPs containing 10% w/w of (C18)2DTPA(Gd) (MO3-NPs and DO3-NPs) have been also derivatized by introducing 3% wt of (C18)2–Peg3000–FA to obtain targeted aggregates (MO3-NP–FA, DO3-NP–FA). A preferential uptake efficiency of DO3-NP–FA in IGROV-1 cells with respect to DO-NPs without folic acid is observed, especially when cells are incubated with low concentrations of nanostructures or at short incubation times, thus indicating its potential use as a target-selective delivery system for MRI contrast agents on tumor cells overexpressing the folate receptor.
    01/2013; 1(5):617-628. DOI:10.1039/C2TB00329E
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    ABSTRACT: The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells, and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRβ chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin (SM) were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol- and SM-binding to the TCRβ chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen, and thus might be involved in enhanced sensitivity of memory compared to naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.
    Journal of Biological Chemistry 10/2012; 287(51). DOI:10.1074/jbc.M112.386045 · 4.57 Impact Factor
  • S Simon · R Schubert
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    ABSTRACT: Phospholipids are widely used excipients for pharmaceutical formulations, such as for preparing biphasic systems or to solubilize or encapsulate poorly soluble drugs. The present study investigates a new property of this class of substance: its ability to inhibit the efflux transporter Pglycoprotein (P-gp). P-gp is expressed in the intestinal epithelium, thereby significantly impairing the systemic absorption of various pharmaceutically active substances. The phospholipid screening performed in this study involved derivatives with different headgroups and fatty acid residues and a number of experimental parameters. For in vitro studies we carried out transport experiments and calcein accumulation assays in Caco-2- and MDCKII mdr1 and wildtype cell lines. The three compounds which displayed significant P-gp inhibition in both assays and in Caco-2 as well as in MDCKII mdr1, consisted of phosphatidylcholine (PC) and either two saturated fatty acid residues of eight (8:0 PC) or ten carbon atoms (10:0 PC), or of two unsaturated docosahexaeonic acid residues (cis-22:6 PC).Supported by P-gp ATPase activity measurements, 8:0 and 10:0 PC were assumed to function as direct P-gp inhibitors interacting with the transporter probably in their monomeric state, whereas a different, as yet unknown mechanism of action applied for cis-22:6 PC.Because of their proven ability to significantly inhibit P-gp in vitro, these phospholipids shall further be elucidated in vivo, whether they may truly serve to increase the bioavailability of orally applied drugs with a P-gp substrate character.
    Biochimica et Biophysica Acta 06/2012; 1821(9):1211-23. DOI:10.1016/j.bbalip.2012.06.001 · 4.66 Impact Factor
  • Zeljka Vanić · Sabine Barnert · Regine Süss · Rolf Schubert
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    ABSTRACT: The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These pH-sensitive liposomes were prepared by incorporating two different PEG lipids: distearoylphosphatidylethanolamine (DSPE)-PEG₂₀₀₀ was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG₁₁₀₀ was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG₂₀₀₀ or sterol-PEG₁₁₀₀ into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG₁₁₀₀ in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.
    Journal of Liposome Research 12/2011; 22(2):148-57. DOI:10.3109/08982104.2011.633267 · 1.82 Impact Factor

Publication Stats

2k Citations
364.90 Total Impact Points


  • 1997–2015
    • University of Freiburg
      • • Institute of Pharmaceutical Sciences
      • • Institute of Macromolecular Chemistry
      Freiburg, Baden-Württemberg, Germany
  • 2008
    • Heinrich-Heine-Universität Düsseldorf
      Düsseldorf, North Rhine-Westphalia, Germany
  • 2002
    • Clinic for Tumor Biology Freiburg
      Freiburg, Baden-Württemberg, Germany
  • 1998
    • Humboldt-Universität zu Berlin
      Berlín, Berlin, Germany