Characterization of an ester-based core-multishell (CMS) nanocarrier for the topical application at the oral mucosa.

OBJECTIVES: Topical drug administration is commonly applied to control oral inflammation. However, it requires sufficient drug adherence and a high degree of bioavailability. Here, we tested the hypothesis whether an ester-based core-multishell (CMS) nanocarrier is a suitable nontoxic drug-delivery system that penetrates efficiently to oral mucosal tissues, and thereby, increase the bioavailability of topically applied drugs. MATERIAL AND METHODS: To evaluate adhesion and penetration, the fluorescence-labeled CMS 10-E-15-350 nanocarrier was applied to ex vivo porcine masticatory and lining mucosa in a Franz cell diffusion assay and to an in vitro 3D model. In gingival epithelial cells, potential cytotoxicity and proliferative effects of the nanocarrier were determined by MTT and sulphorhodamine B assays, respectively. Transepithelial electrical resistance (TEER) was measured in presence and absence of CMS 10-E-15-350 using an Endohm-12 chamber and a volt-ohm-meter. Cellular nanocarrier uptake was analyzed by laser scanning microscopy. Inflammatory responses were determined by monitoring pro-inflammatory cytokines using real-time PCR and ELISA. RESULTS: CMS nanocarrier adhered to mucosal tissues within 5 min in an in vitro model and in ex vivo porcine tissues. The CMS nanocarrier exhibited no cytotoxic effects and induced no inflammatory responses. Furthermore, the physical barrier expressed by the TEER remained unaffected by the nanocarrier. CONCLUSIONS: CMS 10-E-15-350 adhered to the oral mucosa and adhesion increased over time which is a prerequisite for an efficient drug release. Since TEER is unaffected, CMS nanocarrier may enter the oral mucosa transcellularly. CLINICAL RELEVANCE: Nanocarrier technology is a novel and innovative approach for efficient topical drug delivery at the oral mucosa.

Characterization of hyperbranched core-multishell nanocarriers as an innovative drug delivery system for the application at the oral mucosa.

BACKGROUND AND OBJECTIVES: In the oral cavity, the mucosal tissues may develop a number of different pathological conditions, such as inflammatory diseases (gingivitis, periodontitis) and autoimmune disorders (eg, oral lichen planus) that require therapy. The application of topical drugs is one common therapeutic approach. However, their efficacy is limited. Dilution effects due to saliva hinder the adherence and the penetration of drug formulations. Therefore, the bioavailability of oral topical drugs is insufficient, and patients may suffer from disease over years, if not life-long. MATERIAL AND METHODS: In the present study, we characterized core-multishell (CMS) nanocarriers for their potential use as drug delivery systems at oral mucosal tissues. For this purpose, we prepared porcine masticatory as well as buccal mucosa and performed Franz cell diffusion experiments. Penetration of fluorescently labeled CMS nanocarriers into the mucosal tissue was analyzed using confocal laser scanning microscopy. Upon exposure to CMS nanocarriers, the metabolic and proliferative activity of gingival epithelial cells was determined by MTT and sulforhodamine B assays, respectively. RESULTS: Here, we could show that the carriers penetrate into both mucosal tissues, while particles penetrate deeper into the masticatory mucosa. Electron paramagnetic resonance spectroscopy revealed that the 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy-labeled glucocorticoid dexamethasone loaded on to the CMS nanocarriers was released from the carriers in both mucosal tissues but with a higher efficiency in the buccal mucosa. The release from the nanocarriers is in both cases superior compared to the release from a conventional cream, which is normally used for the treatment of inflammatory conditions in the oral cavity. The CMS nanocarriers exhibited neither cytotoxic nor proliferative effects in vitro. CONCLUSION: These findings suggested that CMS nanocarriers might be an innovative approach for topical drug delivery in the treatment of oral inflammatory diseases.

Lipid nanoparticles for skin penetration enhancement-correlation to drug localization within the particle matrix as determined by fluorescence and parelectric spectroscopy.

With topical treatment of skin diseases, the requirement of a high and reproducible drug uptake often still is not met. Moreover, drug targeting to specific skin strata may improve the use of agents which are prone to cause local unwanted effects. Recent investigations have indicated that improved uptake and skin targeting may become feasible by means of nanoparticular systems such as solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and nanoemulsions (NE). Here we describe techniques to characterize drug loading to carrier systems and skin penetration profiles by using the lipophilic dye nile red as a model agent. Since the mode of drug association with the particle matrix may strongly influence the efficiency of skin targeting, parelectric spectroscopy (PS) was used to differentiate between matrix incorporation and attachment to the particle surface and fluorescence spectroscopy (FS) to solve dye distribution within NLC particles. Nile red was incorporated into the lipid matrix or the covering tensed shell, respectively, of SLN and NLC with all the lipids studied (Compritol, Precirol, oleic acid, Miglyol). In NLC, the dye was enriched in the liquid phase. Next, nile red concentrations were followed by image analysis of vertical sections of pigskin treated with dye-loaded nanoparticular dispersions and an oil-in-water cream for 4 and 8 h in vitro. Following the SLN dispersions, dye penetration increased about fourfold over the uptake obtained following the cream. NLC turned out less potent (

The small Gtpase ras is involved in growth factor-regulated expression of the alpha1 integrin subunit in PC12 cells.

PC12 cells interact with several growth factors (e. g. EGF, FGF, and NGF) via specific tyrosine receptor kinases, resulting in cell proliferation or neuronal differentiation. The small GTPase Ras is known to be involved in downstream signaling of these growth factor receptors. Furthermore, cell-matrix interactions mediated by integrins, as well as integrin-induced signaling, are also involved in growth factor-stimulated signal transduction in PC12 cells. In this study we determined the expression of the alpha1 integrin subunit in response to EGF and NGF in PC12 wild-type (wt) cells, and in PC12 cells overexpressing an inactive H-Ras protein (RasN17). In PC12 wt cells, alpha1 integrin expression is upregulated by EGF and NGF. Cell surface expression of alpha1beta1integrin is also enhanced in growth factor-treated cells. This upregulation leads to increased alpha1beta1-specific adhesion to collagen. In cells expressing the dominant-negative RasN17 variant, alpha1 integrin expression and alpha1beta1-specific adhesion remain unchanged in response to both growth factors.

alpha1 Integrin cytoplasmic domain is involved in focal adhesion formation via association with intracellular proteins.

Integrins are heterodimeric adhesion receptors consisting of alpha- and beta-subunits capable of binding extracellular matrix molecules as well as other adhesion receptors on neighbouring cells. These interactions induce various signal transduction pathways in many cell types, leading to cytoskeletal reorganization, phosphorylation and induction of gene expression. Integrin ligation leads to cytoplasmic protein-protein interactions requiring both integrin cytoplasmic domains, and these domains are initiation points for focal adhesion formation and subsequent signal transduction cascades. In previous studies we have shown that the very short cytoplasmic alpha1 tail is required for post-ligand events, such as cell spreading as well as actin stress-fibre formation. In the present paper we report that cells lacking the cytoplasmic domain of the alpha1 integrin subunit are unable to form proper focal adhesions and that phosphorylation on tyrosine residues of focal adhesion components is reduced on alpha1beta1-specific substrates. The alpha1 cytoplasmic sequence is a specific recognition site for focal adhesion components like paxillin, talin, alpha-actinin and pp125FAK. It seems to account for alpha1-specific signalling, since when peptides that mimic the cytoplasmic domain of alpha1 are transferred into cells, they influence alpha1beta1-specific adhesion, presumably by competing for binding partners. For alpha1 integrin/protein binding, the conserved Lys-Ile-Gly-Phe-Phe-Lys-Arg motif and, in particular, the two lysine residues, are important.

Biochemical engineering of the side chain of sialic acids increases the biological stability of the highly sialylated cell adhesion molecule CEACAM1.

The biological half-life time of many glycoproteins is regulated via terminal sialic acids. In this study we determined the half-lives of two different cell adhesion molecules, CEACAM1 and the alpha1-integrin subunit, in PC12-cells before and after biochemical engineering the side chain of sialic acids by the use of N-propanoylmannosamine. Both are transmembrane glycoproteins. While the immunoglobulin superfamily member CEACAM1 mediates homophilic cell-cell adhesion the alpha1-integrin subunit is involved in cell-matrix interactions. We found that the half-life of the highly sialylated CEACAM1 is increased from 26 to 40 h by replacement of the N-acetylneuraminic acid by the novel, engineered N-propanoylneuraminic acids, whereas the half-life of the alpha1-integrin subunit remains unaffected under the same conditions. This demonstrates that biochemical engineering not only modulates the structure of cell surface sialic acids, but that biochemical engineering also influences biological stability of defined glycoproteins.

Protein kinase C phosphorylates and regulates UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase.

UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase) is the key enzyme in the de novo synthesis pathway of neuraminic acid, which is widely expressed as a terminal carbohydrate residue on glycoconjugates. UDP-GlcNAc 2-epimerase is a bifunctional enzyme and catalyzes the first two steps of neuraminic acid synthesis in the cytosol, the conversion of UDP-N-acetylglucosamine to ManAc and the phosphorylation to ManAc-6-phosphate. So far, regulation of this essential enzyme by posttranslational modification has not been shown. Since UDP-N-acetylglucosamine is a cytosolic protein containing eight conserved motifs for protein kinase C (PKC), we investigated whether its enzymatic activity might be regulated by phosphorylation by PKC. We showed that UDP-GlcNAc 2-epimerase interacts with several isoforms of PKC in mouse liver and is phosphorylated in vivo. Furthermore, PKC phosphorylates UDP-GlcNAc 2-epimerase and this phosphorylation results in an upregulation of the UDP-GlcNAc 2-epimerase enzyme activity.

The cytoplasmic domain of the alpha1 integrin subunit influences stress fiber formation via the conserved GFFKR motif.

Integrins are heterodimeric transmembrane proteins that mediate substrate adhesion and migration but also the bidirectional transfer of information across the plasma membrane via their cytoplasmic domains. We addressed the question of whether the very short cytoplasmic tail of the alpha1 integrin subunit of alpha1beta1 integrin is required for alpha1beta1-specific adhesion, spreading, and migration. For this purpose we transfected the alpha1 integrin subunit and two cytoplasmically truncated alpha1 subunits into Chinese hamster ovary (CHO) cells. Elimination of the entire cytoplasmic domain of the alpha1 subunit does not affect adhesion but leads to inhibition of spreading and stress fiber formation. The defect in spreading could not be rescued by lysophosphatidic acid, which has been reported to stimulate actin stress fiber formation via Rho. Additionally, deletion of the entire cytoplasmic domain of the alpha1 subunit abolishes migration toward alpha1beta1-specific substrates. Migration and stress fiber formation are similar in CHO-alpha1 cells and CHO cells carrying an alpha1 subunit still containing the conserved GFFKR motif. So, the GFFKR motif of the alpha1 subunit is essential and sufficient for these processes.

Primary structure and expression analysis of human UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, the bifunctional enzyme in neuraminic acid biosynthesis.

N-Acetylneuraminic acid is a main constituent of glycoproteins and gangliosides. In many membrane-bound receptors it is the target for external stimuli. The key enzyme for its biosynthesis is the bifunctional enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, catalysing the first two steps of the biosynthesis in the cytosol. The rat enzyme was previously isolated and characterised. In this report we present the corresponding human cDNA sequence, compare it with the primary structure of the rodent enzyme, and report the analysis of its expression in different human tissues and cell lines.

C-CAM-mediated adhesion leads to an outside-in dephosphorylation signal.

The rat cell-cell adhesion molecule C-CAM, a member of the carcinoembryonic antigen family, was shown to be expressed in various isoforms, differing in the length of the cytoplasmic domain. The long isoform C-CAML inhibits the growth of different malignant cells. Several studies suggest that it is involved in the mechanism of signal transduction. So far no direct correlation between C-CAM function and C-CAM phosphorylation has been reported. In the present study we addressed the question of whether C-CAM-mediated adhesion is accompanied by changes in phosphorylation of the cytoplasmic domain of C-CAM. It was demonstrated that C-CAML is constitutively phosphorylated in adherent growing cells as well as in cells growing in suspension. In contrast, C-CAML-mediated cell aggregation is accompanied by a 40% reduction in C-CAML phosphorylation compared with nonaggregated cells. The same dephosphorylation was achieved by antibody-induced clustering of C-CAML in the plasma membrane. Phosphorylation and dephosphorylation indicate a C-CAM-mediated outside-in signalling induced by cell-cell adhesion.

Tissue expression and amino acid sequence of murine UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase.

Neuraminic acids are widely expressed as terminal carbohydrates on glycoconjugates and are involved in a variety of biological functions. The key enzyme of N-acetylneuraminic acid synthesis is UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, which catalyses the first two steps of neuraminic acid biosynthesis in the cytosol. In this study we report the complete amino acid sequence of the mouse UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase. The ORF of 2166 bp encodes 722 amino acids and a protein with a predicted molecular mass of 79.2 kDa. Northern blot analysis and in situ hybridization revealed that UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase is expressed at early stages during development and in all tissues investigated with a maximal expression in the liver.

Expression of the polysialyltransferase ST8SiaIV: polysialylation interferes with adhesion of PC12 cells in vitro.

Addition of polysialic acid (PSA) to the neural cell adhesion molecule, NCAM, represents a unique posttranslational modification. Polysialylation of NCAM is developmentally regulated and associated with neural regeneration and plastic processes, as well as learning and memory. Two enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, are known to be involved in the polysialylation of NCAM. Both enzymes are individually capable of catalyzing polysialylation of NCAM, but their time of occurrence and their tissue expression are different. In this study the influence of polysialylation on the nerve growth factor-induced differentiation of PC12 cells was investigated. For this purpose, PC12 cells, which endogenously express NCAM, were transfected with ST8SiaIV to produce, for the first time, a stable polysialylated PC12 cell. We demonstrate that integrin-dependent adhesion to collagen I is reduced in PSA-expressing PC12 cells. Furthermore, polysialylated cell membranes as matrix are a poor substrate for the adhesion and differentiation of PC12 cells, compared with normal cell membranes.

Carcinoembryonic antigen-related cell-cell adhesion molecule C-CAM is greatly increased in serum and urine of rats with liver diseases.

C-CAM (rat cell CAM/human CD66a) is ubiquitous and multifunctional. It is involved in intercellular adhesion, signal transduction and cell growth inhibition. Structurally, it is related to the carcinoembryonic antigen. In the present study serum, bile and urine of rats with liver diseases were analyzed for the presence of cell CAM. After bile duct ligation and during galactosamine (GalN) hepatitis we found that large amounts of liver membrane-bound C-CAM are secreted or shed into blood. The serum level of another liver membrane-bound protein, LI-cadherin, is not increased. It was shown that C-CAM is also present in bile fluid, and for the first time that C-CAM is present in the urine of rats with liver diseases. A particularly high concentration was measured in the urine of rats suffering from GalN hepatitis.

Focal adhesion kinase pp125FAK and the beta 1 integrin subunit are constitutively complexed in HaCaT cells.

Binding of integrins to the extracellular matrix (ECM) activates various signal transduction pathways and regulates gene expression in many cell types. Integrin-dependent cytoplasmic protein/protein interactions are necessary for activation of those signal transduction cascades. In our studies we investigated a possible association of pp125FAK, an adhesion involved tyrosine kinase, with the integrin beta 1 subunit. Further we wanted to know to which extent protein tyrosine phosphorylation affects cell adhesion to the ECM and the possible beta 1 integrin/pp125FAK complex. We were able to show that in HaCaT cells (a human keratinocyte derived cell line) the integrin beta 1 subunit is associated with tyrosine kinase pp125FAK. This association was observed in ECM-adherent cells and nonadherent cells and is independent of tyrosine phosphorylation. However, cell adhesion of HaCaT cells to specific substrates requires tyrosine phosphorylation since genistein treatment that blocks phosphorylation of many cellular proteins as pp125FAK led to a reduced substrate adhesion.

V(+)-fibronectin expression and localization prior to gastrulation in Xenopus laevis embryos.

The V-region represents one of three alternatively spliced segments in Xenopus fibronectin. Here, we identify this V-region as binding epitope of the monoclonal antibody (MAb 6D9) that we generated against Xenopus plasma fibronectin. By the use of this antibody we obtained new results that change the present view of the fibronectin expression pattern before gastrulation: (1) the V(+)-fibronectin is the major isoform expressed during early development since only a single fibronectin band is found in Western blots up to tadpole stages. (2) In contrast to previously published data we demonstrate that fibronectin expression is induced by progesterone during oocyte maturation. (3) During cleavage stages the protein is stored in the cytoplasm where it is predominantly associated with plasma membranes. Immunoelectronmicroscopy reveals that V(+)-fibronectin is present at the surface of animal pole blastomeres and secreted into intercellular spaces. This extracellular localization of fibronectin is predominantly observed in the marginal zone, surrounding single cells of the outer cell layer baso-laterally. In the vegetal hemisphere V(+)-fibronectin is restricted to the cytoplasm and accumulated at plasma membranes. With the onset of gastrulation the intracellular and membrane associated fibronectin disappears and fibronectin becomes detectable at the blastocoel roof. Since reaggregation of dissociated blastula cells was not blocked by addition of GRGDS peptide or antibodies against fibronectin, we assume that the early expression and secretion of fibronectin serves as store to allow a rapid matrix assembly with onset of mesodermal cell migration.

Effects of heat shock on the pattern of fibronectin and laminin during somitogenesis in Xenopus laevis.

Heat shock causes partial disruption of the segmentation pattern during somitogenesis in Xenopus as well as in other vertebrates. However, Xenopus undergoes a different type of somite formation than that of most vertebrates: Somites are formed by rotation of cell blocks out of the paraxial mesoderm. We attempted to determine whether or not the segmentation disorder following heat shock is caused by an altered pattern of fibronectin and/or laminin, that could then effect the rotation of cell blocks. Therefore, we carried out heat shock experiments and analyzed the distribution of both ECM proteins in correlation to the position of somitic cells. Our results reveal that heat shock causes an incorrect deposition of fibronectin as well as laminin during somite formation. This leads to an intermingling of cells from different segments and to an anchorage of cells at the lateral matrix. Immunoblots show that the defects in the pattern of these ECM proteins do not correlate with a decrease of both proteins. However, immunohistological staining patterns demonstrate that oversized blocks of 20-cell width, instead of the normal ones of about 9-cell width, are separated out of the paraxial mesoderm following heat shock treatment. This indicates that the altered pattern of fibronectin and laminin might be a secondary effect caused by incorrect segregation and detachment of cell blocks during somitogenesis. Since anchorage of somitic cells is mostly affected by the altered distribution of fibronectin and laminin, it is more likely that both ECM proteins function in anchorage of migrating presomitic cells and in maintaining of segment borders rather than in stimulating cell rotation movements.

Two different mRNAs coding for identical elongation factor 1 alpha (EF-1 alpha) polypeptides in Xenopus laevis embryos.

Two related but clearly different cDNA clones corresponding to elongation factor 1 alpha (EF-1 alpha) mRNAs were isolated from a Xenopus laevis gastrula-stage library. Whereas the nucleotide sequences of these two cDNAs differ within the coding region at 49 out of 1386 positions (3.5%), the derived amino acid sequences are completely identical, thereby indicating a substantial evolutionary constraint on this translation factor. Southern-blot analysis of genomic DNA suggests that, besides the two closely related EF-1 alpha genes investigated in this study, other more-distantly related genes may exist in the X. laevis genome. Transcription of EF-1 alpha genes during oogenesis and embryonic development was studied by Northern-blot analysis and by in situ hybridizations. A high amount of EF-1 alpha mRNA was detected in previtellogenic oocytes. At later stages of embryonic development, EF-1 alpha mRNA was found to be accumulated in translationally active tissues.