How wound environments trigger the release from Rifampicin-loaded liposomes.

BACKGROUND: Chronic wounds often contain high levels of proinflammatory cytokines that prolong the wound-healing process. Patients suffering from these conditions are likely to benefit from topical rifampicin therapy. Although recent research indicates considerable anti-inflammatory properties of the antibiotic, currently, there are no commercial topical wound healing products available. To address this medical need, a liposomal drug delivery system was developed. A mechanistic investigation outlined major influences of wound environments that affect the release kinetics and, as a consequence, local bioavailability. METHODS: Liposomes were prepared using the thin-film hydration method and subsequently freeze-dried at the pilot scale to improve their stability. We investigated the influence of oxidation, plasma proteins, and lipolysis on the in vitro release of rifampicin and its two main degradation products using the Dispersion Releaser technology. A novel simulated wound fluid provided a standardized environment to study critical influences on the release. It reflects the pathophysiological environment regarding pH, buffer capacity, and protein content. RESULTS: During storage, the liposomes efficiently protect rifampicin from degradation. After the dispersion of the vesicles in simulated wound fluid, despite the significant albumin binding (>70%), proteins have no considerable effect on the release. Also, the presence of lipase at pathophysiologically elevated concentrations did not trigger the liberation of rifampicin. Surprisingly, the oxidative environment of the wound bed represents the strongest accelerating influence and triggers the release. CONCLUSION: A stable topical delivery system of rifampicin has been developed. Once the formulation comes in contact with simulated wound fluid, drug oxidation accelerates the release. The influence of lipases that are assumed to trigger the liberation from liposomes depends on the drug-to-lipid ratio. Considering that inflamed tissues exhibit elevated levels of oxidative stress, the trigger mechanism identified for rifampicin contributes to targeted drug delivery.

Pharmaceutical Development of Nanostructured Vesicular Hydrogel Formulations of Rifampicin for Wound Healing.

Chronic wounds exhibit elevated levels of inflammatory cytokines, resulting in the release of proteolytic enzymes which delay wound-healing processes. In recent years, rifampicin has gained significant attention in the treatment of chronic wounds due to an interesting combination of antibacterial and anti-inflammatory effects. Unfortunately, rifampicin is sensitive to hydrolysis and oxidation. As a result, no topical drug product for wound-healing applications has been approved. To address this medical need two nanostructured hydrogel formulations of rifampicin were developed. The liposomal vesicles were embedded into hydroxypropyl methylcellulose (HPMC) gel or a combination of hyaluronic acid and marine collagen. To protect rifampicin from degradation in aqueous environments, a freeze-drying method was developed. Before freeze-drying, two well-defined hydrogel preparations were obtained. After freeze-drying, the visual appearance, chemical stability, residual moisture content, and redispersion time of both preparations were within acceptable limits. However, the morphological characterization revealed an increase in the vesicle size for collagen-hyaluronic acid hydrogel. This was confirmed by subsequent release studies. Interactions of marine collagen with phosphatidylcholine were held responsible for this effect. The HPMC hydrogel formulation remained stable over 6 months of storage. Moving forward, this product fulfills all criteria to be evaluated in preclinical and clinical studies.

Proton gradients from light-harvesting E. coli control DNA assemblies for synthetic cells.

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.

Heterologous expression of human norovirus GII.4 VP1 leads to assembly of T=4 virus-like particles.

Human noroviruses are a leading cause of acute gastroenteritis, yet there are still no vaccines or antivirals available. Expression of the norovirus capsid protein (VP1) in insect cells typically results in the formation of virus-like particles (VLPs) that are morphologically and antigenically comparable to native virions. Indeed, several different norovirus VLP candidates are currently used in clinical trials. So far, structural analysis of norovirus VLPs showed that the capsid has a T = 3 icosahedral symmetry and is composed of 180 copies of VP1 that are folded into three quasi-equivalent subunits (A, B, and C). In this study, the VLP structures of two norovirus GII.4 genetic variants that were identified in 1974 and 2012 were determined using cryo-EM. Surprisingly, we found that greater than 95% of these GII.4 VLPs were larger than virions and 3D reconstruction showed that these VLPs exhibited T = 4 icosahedral symmetry. We also discovered that the T = 4 VLPs presented several novel structural features. The T = 4 particles assembled from 240 copies of VP1 that adopted four quasi-equivalent conformations (A, B, C, and D) and formed two distinct dimers, A/B and C/D. The protruding domains were elevated ∼21 Šoff the capsid shell, which was ∼7 Šmore than in the previously studied GII.10 T = 3 VLPs. A small cavity and flap-like structure at the icosahedral two-fold axis disrupted the contiguous T = 4 shell. Overall, our findings indicated that GII.4 VP1 sequences assemble into T = 4 VLPs and these larger particles might have important consequences for VLP-based vaccine development.

Oral delivery of vancomycin by tetraether lipid liposomes.

Despite the outstanding progress in modern medicine, the oral delivery of peptide drugs is limited until today due to their instability in the gastrointestinal tract and low mucosa penetration. To overcome these hurdles, liposomes containing the specific tetraether lipid GCTE (glycerylcaldityltetraether lipid) were examined. For this purpose, the glycopeptide antibiotic vancomycin was used as model substance and liposomes were prepared by DAC (dual assymetric centrifugation). These liposomes showed a size and polydispersity index comparable to standard liposomes. A high encapsulation efficiency of 58.53±1.76% of the peptide drug vancomycin could be obtained as detected by HPLC. FCS analysis showed that in average each liposome contains 30 molecules of vancomycin. TEM and Cryo-EM micrographs verified the size and lamellarity of the liposomal formulations. Cytotoxicity tests in Caco-2 cells showed no significant cytotoxicity for all liposomal concentrations tested, indicating the good tolerability of these formulations. Furthermore, the use of sucrose as lyoprotector enabled the long term storage of the liposomal formulation for at least three months. The potency of this drug delivery system could be proven in an animal model using Wistar rats. One hour after oral application, 4.82±0.56% of the administered dose of vancomycin could be found in the blood as detected by immunoassay measurements. This transport did also not affect the integrity of the peptide as verified by immunoassay measurements. In combination with long term storage stability, this formulation appears to be a promising delivery system for oral application of peptide drugs.

Delivery of Copper-chelating Trientine (TETA) to the central nervous system by surface modified liposomes.

The existence of the blood-brain barrier (BBB) complicates the treatment of many central nervous system (CNS) disorders, including the copper storage disease, Wilson's disease. Its CNS symptoms represent a serious problem, since therapeutics for Wilson's disease do not cross the BBB. One strategy to overcome this obstacle is the transfer of drugs across the BBB with colloidal carrier systems like liposomes. The aim of the present study was to encapsulate triethylenetetramine (TETA), a copper chelating agent, into surface modified liposomes and to investigate their permeation across the BBB. Liposomes were modified with cationized bovine serum albumin or penetratin, a cell penetrating peptide. Liposomes were characterized regarding size, PDI, zeta potential and encapsulation efficiency. Size was between 139.4±1.9nm to 171.1±3.5nm with PDI's below 0.2. Zeta potentials of vectorized liposomes were at least 6.9mV higher than those of standard liposomes. Cryo-TEM micrographs displayed liposomal structure, integrity and the similarity of structure and size between loaded, unloaded, vectorized and non- vectorized liposomes. In vivo experiments in rats showed an up to 16-fold higher brain uptake of TETA in vectorized liposomes compared to free TETA or TETA in non-vectorized liposomes, proving successful brain delivery using target seeking surface modifications. Tissue analysis indicated TETA concentrations in the brain being high enough to treat Wilson's disease.

The RanBP2/RanGAP1*SUMO1/Ubc9 SUMO E3 ligase is a disassembly machine for Crm1-dependent nuclear export complexes.

Continuous cycles of nucleocytoplasmic transport require disassembly of transport receptor/Ran-GTP complexes in the cytoplasm. A basic disassembly mechanism in all eukaryotes depends on soluble RanGAP and RanBP1. In vertebrates, a significant fraction of RanGAP1 stably interacts with the nucleoporin RanBP2 at a binding site that is flanked by FG-repeats and Ran-binding domains, and overlaps with RanBP2's SUMO E3 ligase region. Here, we show that the RanBP2/RanGAP1*SUMO1/Ubc9 complex functions as an autonomous disassembly machine with a preference for the export receptor Crm1. We describe three in vitro reconstituted disassembly intermediates, which show binding of a Crm1 export complex via two FG-repeat patches, cargo-release by RanBP2's Ran-binding domains and retention of free Crm1 at RanBP2 after Ran-GTP hydrolysis. Intriguingly, all intermediates are compatible with SUMO E3 ligase activity, suggesting that the RanBP2/RanGAP1*SUMO1/Ubc9 complex may link Crm1- and SUMO-dependent functions.

Improved oral bioavailability of human growth hormone by a combination of liposomes containing bio-enhancers and tetraether lipids and omeprazole.

Liposomes for the oral delivery of human growth hormone (hGH) containing bio-enhancers and tetraether lipids were prepared by dual asymmetric centrifugation. Cetylpyridinium chloride (CpCl), d-α-tocopheryl polyethylene glycol 400 succinate, phenylpiperazine, sodium caprate or octadecanethiol were used as permeation enhancers. In vitro data showed that oligolamellar vesicles with average size in the range of 200-250 nm were formed. Performance of the formulations was investigated both ex vivo by confocal microscopy scans of sections of rat small intestine and in vivo by comparing the area under the plasma curve of hGH after oral or subcutaneous (s.c.) application. The microscopic data reveal an interaction between the liposomal formulation and the intestinal mucus layer. Particularly one formulation, which was designed to be mucus penetrative by addition of a high quantity of TPGS 400 and a ζ-potential close to 0 mV, showed a very strong mucus association in the duodenum and jejunum. Vesicles with CpCl 33% (mol/mol) led to a relative hGH bioavailability of 3.4% compared with s.c. control, whereas free hGH administered orally showed a bioavailability of only 0.01%.

Liposomal sphingomyelin influences the cellular lipid profile of human lymphoblastic leukemia cells without effect on P-glycoprotein activity.

Sphingomyelin (SM)/cholesterol liposomes are currently investigated as drug carriers in cancer therapy. However, no data is available on the influence of SM itself on P-glycoprotein (P-gp) mediated multidrug resistance. P-gp is at least partly located in sphingolipid-enriched lipid raft domains of the plasma membrane, and its activity depends on the lipid profile of the membrane, which could be altered by therapeutical SM liposomes. Therefore, the aim of this study was to analyze the effect of liposomal SM on P-gp activity, P-gp distribution in microdomains, SM content of the membrane domains, and sensitivity of human lymphoblastic CEM cells toward cytotoxic drugs in vitro. Assays were conducted in CEM and multidrug resistant CEM/ADR5000 cells. SM-only liposomes were prepared by a newly developed ethanol injection protocol and thoroughly characterized. Inclusion of SM into the membrane was analyzed by fluorescence microscopy and flow cytometry. Influence of SM liposomes on P-gp activity was assessed by rhodamine efflux and calcein assay, and sensitivity toward cytotoxic drugs was analyzed by flow cytometric 7-AAD staining. Influence on P-gp distribution was analyzed by Western blot after density gradient centrifugation. SM 16:0, 18:0, and 24:1 were quantified by liquid chromatography coupled to tandem mass spectrometry. P-gp was mainly located in nonraft fractions, which did not change upon liposome treatment. Liposomes increased SM 16:0 and SM 24:1 content in nonraft domains, but not in raft domains of multidrug resistant cells. SM-only liposomes did not influence P-gp activity and chemosensitivity. In conclusion, SM-only liposomes in therapeutic amounts did not influence P-gp mediated multidrug resistance in CEM cells.

Dimer ribbons of ATP synthase shape the inner mitochondrial membrane.

ATP synthase converts the electrochemical potential at the inner mitochondrial membrane into chemical energy, producing the ATP that powers the cell. Using electron cryo-tomography we show that the ATP synthase of mammalian mitochondria is arranged in long approximately 1-microm rows of dimeric supercomplexes, located at the apex of cristae membranes. The dimer ribbons enforce a strong local curvature on the membrane with a 17-nm outer radius. Calculations of the electrostatic field strength indicate a significant increase in charge density, and thus in the local pH gradient of approximately 0.5 units in regions of high membrane curvature. We conclude that the mitochondrial cristae act as proton traps, and that the proton sink of the ATP synthase at the apex of the compartment favours effective ATP synthesis under proton-limited conditions. We propose that the mitochondrial ATP synthase organises itself into dimer ribbons to optimise its own performance.

Structural organization of mitochondrial human complex I: role of the ND4 and ND5 mitochondria-encoded subunits and interaction with prohibitin.

Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of these subunits have significant pathogenic consequences in humans. However, the way these events affect complex I assembly is poorly documented. To understand the effects of particular mutations in ND subunits on complex I assembly, we studied four human cell lines: ND4 non-expressing cells, ND5 non-expressing cells, and rho degrees cells that do not express any ND subunits, in comparison with normal complex I control cells. In control cells, all the seven analysed nuclear-encoded complex I subunits were found to be attached to the mitochondrial inner membrane, except for the 24 kDa subunit, which was nearly equally partitioned between the membranes and the matrix. Absence of a single ND subunit, or even all the seven ND subunits, caused no major changes in the nuclear-encoded complex I subunit content of mitochondria. However, in cells lacking ND4 or ND5, very low amounts of 24 kDa subunit were found associated with the membranes, whereas most of the other nuclear-encoded subunits remained attached. In contrast, membrane association of most of the nuclear subunits was significantly reduced in the absence of all seven ND proteins. Immunopurification detected several subcomplexes. One of these, containing the 23, 30 and 49 kDa subunits, also contained prohibitin. This is the first description of prohibitin interaction with complex I subunits and suggests that this protein might play a role in the assembly or degradation of mitochondrial complex I.

Ineffective hematopoiesis linked with a mitochondrial tRNA mutation (G3242A) in a patient with myelodysplastic syndrome.

In a patient with refractory anemia with excess blasts (RAEB), a somatic mutation of mitochondrial transfer RNA(Leu(UUR)) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34(+) cells than in the unfractionated sample. Surprisingly, the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However, PB CD34(+) cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays, CD34(+) cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34(+) cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis, thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient.

The N-terminal cysteine pair of yeast sulfhydryl oxidase Erv1p is essential for in vivo activity and interacts with the primary redox centre.

Yeast Erv1p is a ubiquitous FAD-dependent sulfhydryl oxidase, located in the intermembrane space of mitochondria. The dimeric enzyme is essential for survival of the cell. Besides the redox-active CXXC motif close to the FAD, Erv1p harbours two additional cysteine pairs. Site-directed mutagenesis has identified all three cysteine pairs as essential for normal function. The C-terminal cysteine pair is of structural importance as it contributes to the correct arrangement of the FAD-binding fold. Variations in dimer formation and unique colour changes of mutant proteins argue in favour of an interaction between the N-terminal cysteine pair with the redox centre of the partner monomer.