Synthesis of mucoadhesive thiol-bearing microgels from 2-(acetylthio)ethylacrylate and 2-hydroxyethylmethacrylate: novel drug delivery systems for chemotherapeutic agents to the bladder.

Thiol-bearing microgels have been synthesised from copolymerisation of 2-(acetylthio)ethylacrylate and 2-hydroxyethylmethacrylate, and subsequent deprotection using sodium thiomethoxide. The concentration of thiol groups on these microgels could be tailored by use of different molar ratios of the two monomers. These thiol-bearing microgels were shown to adhere to ex vivo porcine urinary bladder, which was correlated with their level of thiolation. By simply mixing solutions of thiol-bearing microgels and doxorubicin, high levels of drug loading into the microgels could be achieved. Thiol-bearing microgels controlled the release of doxorubicin in a time-dependent manner over several hours. These doxorubicin-loaded thiol-bearing microgels could have application in the treatment of early-stage bladder cancers. The method used represents a new 'bottom-up' approach for the synthesis of novel mucoadhesive microgels.

Antibacterial activity of Garcinia mangostana extracts on oral pathogens.

AIM: This study was conducted to evaluate the antibacterial activity of Garcinia mangostana (GM) extracts on oral pathogens. METHODS: The 95% ethanol and 70% acetone extracts of the pericarp of GM was prepared and standardized by determining the amount of α-mangostin, total phenolic compounds and tannins. The antibacterial activity of GM extracts against oral pathogens was investigated by using minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and time kill assay. Bacterial morphology was analyzed using scanning electron microscopy (SEM). RESULTS: The results indicated that the content of α-mangostin, total phenolic compounds and tannins of the both extracts were different. The 95% ethanol extract contained higher α-mangostin and total phenolic compounds. Whereas, the tannins of 70% acetone extract were significantly higher than 95% ethanol extract. The 95% ethanol extract exhibited a potent antibacterial activity with low MIC and MBC values compared to the acetone extract. The morphology of bacteria was significantly changed after treatment with extracts for 24 h. Furthermore, time kill assay revealed that bacterial cells were decreased within 2 h. CONCLUSION: GM extracts was effective against oral bacteria pathogens. The antibacterial activity was varied by the different extraction solvents and the distinction in the contents of the compounds among extracts. These findings indicated that GM extracts showed promising antibacterial activity against oral pathogens in vitro.

Comparison of the percutaneous absorption of hydrophilic and lipophilic compounds in shed snake skin and human skin.

The in vitro transdermal permeation of eight hydrophilic drugs (antipyrine, L-dopa, dopamine hydrochloride, diclofenac sodium, 5-fluorouracil, isoprenaline hydrochloride, nicorandil and morphine hydrochloride) and eight lipophilic drugs (aminopyrine, cyclobarbital, ibuprofen, indomethacin, isosorbide dinitrate, flurbiprofen, ketoprofen and lignocaine) was determined using shed snake skin of Elaphae obsoleta and human skin. The permeation parameters and physiological characteristics of the skin, e.g. the water and lipid content, and the thickness of shed snake skin and human skin were evaluated and compared. In shed snake skin, the permeability coefficients (P) of lipophilic drugs were in the same range as those through the human skin (0.9 to 1.8-times); whereas those of hydrophilic drugs were remarkably lower (3.3 to 6.1-times). The thickness and lipid content of shed snake skin and human stratum corneum were not significantly different (P > 0.05), whereas the water content of shed snake skin was significantly lower than that of human stratum corneum (P < 0.05). The lower permeability of shed snake skin for hydrophilic compounds might be caused by the lower porosity of skin strata. The results suggested a potential use of shed snake skin as barrier membrane for lipophilic compounds percutaneous absorption studies in vitro.

Effect of chitosan salts and molecular weight on a nanoparticulate carrier for therapeutic protein.

The objective of this study was to investigate the potential of chitosan salts as a carrier in the preparation of protein-loaded nanoparticles. Glutamic and aspartic acids were used to prepare chitosan salts of 35, 100, and 800 KDa. Nanoparticles of chitosan base, chitosan glutamate, and chitosan aspartate were produced by ionotropic gelation with sodium tripolyphosphate (TPP). Bovine serum albumin (BSA) was applied as a model protein at loading concentrations ranging from 0.2 to 2 mg/mL. The size of the nanoparticles, as measured by photon correlation spectroscopy, was in the range of 195 to 3450 nm, depending on type and molecular weight of chitosan. Nanoparticles prepared with higher molecular weight chitosan showed larger sizes. The encapsulation was controlled by the competition of BSA in forming ionic cross-linking with chitosan and by the entrapment of BSA during the gelation process. Higher BSA encapsulation efficiency (EE) was obtained for nanoparticles prepared with chitosan salts compared to those prepared with the base. The higher EE was a result of a higher degree of ionization, causing more active sites to interact with BSA. In addition, a higher and faster release of BSA from the nanoparticles into pH 7.4 buffer medium was observed for nanoparticles of the chitosan salts than was observed for nanoparticles of the chitosan base. The higher and faster release was attributed to higher EE and lower entrapment of BSA within the matrix of the nanoparticle during the gelation process. The influence of molecular weight on the property of nanoparticles exhibited different effects. The difference was a result of different organic acids used to prepare nanoparticles leading to the difference in polymer conformation and viscosity of organic acid solution. Therefore, this study showed that the characteristics of chitosan nanoparticles loaded with a protein drug could be readily modulated by changing the salt form or the molecular weight of the chitosan carrier.

Serum mannan binding protein inhibits mannosylated liposome-mediated transfection to macrophages.

The effects of serum mannan binding proteins (MBP) in the transfection of plasmid DNA/Man-liposome complex via mannose receptor-mediated endocytosis was studied in vitro using cultured mouse peritoneal macrophages. Plasmid DNA encoding luciferase gene was complexed with cationic mannosylated liposomes (Man-liposomes), composed of cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and dioleoyl phosphatidylethanolamine (DOPE). The transfection efficiency, as well as the binding and uptake of the plasmid DNA/Man-liposome complex, was investigated with or without serum MBP. The in vitro transfection efficiency of the complex was significantly reduced on increasing the amount of serum MBP. In addition, the cellular association of the complex was also reduced. These results indicate that serum MBP specifically binds to the mannose moieties on the complex and suppresses its cellular uptake, resulting in inhibition of the gene transfection in macrophages. Such an interaction is an obstacle to mannose receptor-mediated in vivo gene transfer to mannose receptor-positive cells using mannosylated gene carriers.

In vivo recognition of mannosylated proteins by hepatic mannose receptors and mannan-binding protein.

In vivo recognition of mannosylated proteins by hepatic mannose receptors and serum mannan-binding protein (MBP) was investigated in mice. After intravenous administration, all three different (111)In-mannosylated proteins were taken up mainly by liver, and uptake was saturated with increasing doses. (111)In-Man-superoxide dismutases and (111)In-Man(12)- and (111)In-Man(16)-BSA had simple dose-dependent pharmacokinetic profiles, whereas other derivatives ((111)In-Man(25)-, -Man(35)-, and -Man(46)-BSA and (111)In-Man-IgGs) showed slow hepatic uptake at <1 mg/kg. Purified MBP experiments in vitro indicated that these derivatives bind to MBP in serum after injection, which interferes with their hepatic uptake. To quantitatively evaluate these recognition properties in vivo, a pharmacokinetic model-based analysis was performed for (111)In-Man-BSAs, estimating some parameters, including the Michaelis-Menten constant of the hepatic uptake and the dissociation constant of MBP, which correlate to the affinity of Man-BSAs for mannose receptors and MBP, respectively. The dissociation constant of Man-BSA and MBP decreased dramatically with increasing density of mannose, but the Michaelis-Menten constant of hepatic uptake of Man-BSA was not so sensitive to the change in density. This suggests that the in vivo recognition of MBP has a stronger cluster effect than that of mannose receptors. Differences obtained here are due to the unique arrangement of carbohydrate recognition domains on each mannose-specific lectin available for mannosylated ligand recognition.

Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages.

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.

Pharmacokinetic analysis of lectin-dependent biodistribution of fucosylated bovine serum albumin: a possible carrier for Kupffer cells.

To examine the potential utility of fucosylation of drug carriers for targeted drug delivery to Kupffer cells, the pharmacokinetics of (111)In-labeled fucosylated bovine serum albumin (Fuc-BSA) with different numbers of fucose residues (11, 16, 25, 31 or 41) was studied. After intravenous injection in mice, all (111)In-Fuc-BSAs were mainly delivered to the liver and their hepatic uptake became saturated when the dose was increased. Of these derivatives, only (111)In-Fuc41-BSA showed a slow plasma elimination at low doses, suggesting an interaction with blood components. Examination of binding conditions as well as electrophoretic analysis of the binding components indicated that the serum-type mannan binding protein (MBP) is responsible. Kupffer cells, which possess fucose receptors, showed the highest uptake of (111)In-Fuc41-BSA, followed by endothelial cells and hepatocytes. The hepatic uptake of (111)In-Fuc41-BSA was inhibited by co-injection of Gal42-BSA, but not by Man46-BSA. On the other hand, excess Fuc41-BSA inhibited the hepatic uptake of (111)In-Man46-BSA, while (111)In-Gal42-BSA did not: These findings suggest that not only the fucose receptors on Kupffer cells but also other lectins are involved in the biodistribution of Fuc-BSAs. To understand how the degree of fucose modification affects the binding affinity of Fuc-BSA with hepatic lectins and serum MBP, a pharmacokinetic analysis was performed based on a physiological model. The Michaelis constant of the hepatic uptake of (111)In-Fuc-BSA decreased with an increasing number of fucose units, and the intrinsic hepatic clearance of (111)In-Fuc25-, (111)In-Fuc31- and (111)In-Fuc41-BSAs was close to, or much greater than, the hepatic plasma flow rate, indicating efficient hepatic uptake of these derivatives. These results suggest that fucosylation is a potentially useful method making drug carriers selective for Kupffer cells, although extensive modification might result in retarded delivery due to binding to other lectins like MBP.