The properties of Caco-2 monolayers were compared on aluminium oxide and nitrocellulose permeable-supports. On nitrocellulose, Caco-2 cells displayed a higher rate of taurocholic acid transport than those cultured on aluminium oxide inserts. In addition, Caco-2 cells grown on these two inserts were not comparable with respect to cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of the aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands, offers a distinct advantage over nitrocellulose inserts for drug transport studies. The carrier-mediated uptake and transport of the imino acid (L-proline) and the acidic amino acids (L-aspartate and L-glutamate) have been studied. At pH7.4, L-proline uptake is mediated via an A-system carrier. Elevated uptake and transport under acidic conditions occurs by activation of a distinct carrier population. Acidic amino acid transport is mediated via a X-AG system. The flux of baclofen, CGP40116 andCGP40117 across Caco-2 monolayers was described by passive transport. The transport of three peptides, thyrotrophin-releasing hormone, SQ29852 and cyclosporin were investigated. Thyrotrophin-releasing hormone transport acrossCaco-2 monolayers was characterised by a minor saturable (carrier-mediated,approximately 25%) pathway, superimposed onto a major non-saturable (diffusional)pathway. SQ29852 uptake into Caco-2 monolayers is described by a major saturable mechanism (Km = 0.91 mM) superimposed onto a minor passive component. However, the initial-rate of SQ29852 transport is consistent with a passive transepithelial transport mechanism. These data highlight the possibility that itsbasolateral efflux is severely retarded such that the passive paracellular transportdictates the overall transepithelial transport characteristics. In addition, modelsuitable for investigating the transepithelial transport of cyclosporin A has been developed. A modification of the conventional Caco-2 model has been developed which has a calcium-free Ap donor-solution and a Bl receiver-solution containing the minimumcalcium concentration required to maintain monolayer integrity (100 μM). The influence of calcium and magnesium on the absorption of [14C]pamidronate was evaluated by comparing its transport across the conventional and minimum calciumCaco-2 models. Ap calcium and magnesium ions retard the Ap-to-Bl flux of pamidronate across Caco-2 monolayers. The effect of self-emulsifying oleic acid-Tween 80 formulations on Caco-2monolayer integrity has been investigated. Oleic acid-Tween 80 (1 0:1) formulations produced a dose-dependent disruption of Caco-2 monolayer integrity. This disruption was related to the oleic acid content of the formulation.

No description available.

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Pharmaceutics. Bibliography: leaves 163-178. Also available online.

A variety of dendrimers based on melamine are designed, synthesized, and evaluated for drug delivery systems. The synthesis of a dendrimer, including multiple copies of four orthogonally reactive groups, is described. The three groups on the surface are nucleophilic and include four free hydroxyl groups, four tert-butyldiphenylsilyl (TBDPS) ether groups, and sixteen amines masked as tert-butoxycarbonyl (BOC) groups. The core of the dendrimer displays two electrophilic monochlorotriazines. The dendrimer above is further manipulated for in vivo biodistribution: incorporation of the reporting groups Bolton-Hunter and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid); PEGylation for biocompatibility and size tuning. In preliminary biodistribution studies, dendrimers circulate in the blood for a longer time as the molecular weight increases, which is important to passively target tumor tissues via the EPR effect. Also, high uptake by the tumor tissues was observed in mice bearing prostate cancer xenografts. A drug delivery vehicle for the anticancer agent paclitaxel is described. This drug delivery vehicle contains sixteen molecules of paclitaxel via acid-labile ester linkage, two Bolton-Hunter groups, and sixteen monochlorotriazine groups for PEGylation. The in vitro drug release studies shows faster release of paclitaxel at lower pH in PBS.

The convergent strategy towards dendrimer synthesis is well-suited to generate macromolecules with a diverse periphery, at the expense of time and effort, while the divergent strategy has historically been effective at yielding higher generation dendrimers, although they are often plagued by impurities. Both the convergent and divergent routes were applied to the synthesis of melamine-based dendrimers, offering a comparison of the routes within a system. Generation-1 dendrons heterogeneously functionalized with Boc-protected amines and hydrazones were synthesized convergently and coupled to a generation-1 tris(piperazine) core to yield a generation-2 dendrimer bearing 18 Boc-amines and three hydrazones. Although the yield for the final coupling step was rather low (56%), the yields for all intermediate steps were quite high. Attempts toward obtaining a generation-3 dendrimer through this route were unsuccessful due presumably to steric hindrance. The materials obtained showed no impurities in their 1H and 13C NMR and mass spectra, although several chromatographic purifications were necessary throughout the synthesis. A divergent strategy based on addition of a dichlorotriazine monomer to polyamine cores was used to synthesize dendrimers of generations 1-5. All intermediates and dendrimers were either purified by precipitation, or did not need purification. 1H NMR spectroscopy indicated that reactions were complete up to G4-NH2 by integration, and mass spectroscopy confirmed that assignment. HPLC and GPC of Gn-Cl dendrimers showed sharp peaks for G1-G3, but G4-Cl appeared to have a small amount of impurities that are similar in size and polarity to the fully-substituted dendrimer. The G1-G3 dendrimers were confidently assigned as pure by conventional organic chemistry standards, but the assignment of purity to higher generations remained tentative. A G1-Cl dendrimer was functionalized with imidazole, and then deprotected and PEGylated with PEG5000 to yield a water soluble dendrimer. The imidazole-capped, Boc-protected dendrimer and the deprotected dendrimer were characterized by 1H and 13C NMR spectroscopy and mass spectrometry. The degree of PEGylation on the PEGylated material could not be definitively ascertained; however, the material is capable of solubilizing very hydrophobic Zn-phthalocyanines in water.

The convergent strategy towards dendrimer synthesis is well-suited to generate macromolecules with a diverse periphery, at the expense of time and effort, while the divergent strategy has historically been effective at yielding higher generation dendrimers, although they are often plagued by impurities. Both the convergent and divergent routes were applied to the synthesis of melamine-based dendrimers, offering a comparison of the routes within a system. Generation-1 dendrons heterogeneously functionalized with Boc-protected amines and hydrazones were synthesized convergently and coupled to a generation-1 tris(piperazine) core to yield a generation-2 dendrimer bearing 18 Boc-amines and three hydrazones. Although the yield for the final coupling step was rather low (56%), the yields for all intermediate steps were quite high. Attempts toward obtaining a generation-3 dendrimer through this route were unsuccessful due presumably to steric hindrance. The materials obtained showed no impurities in their 1H and 13C NMR and mass spectra, although several chromatographic purifications were necessary throughout the synthesis. A divergent strategy based on addition of a dichlorotriazine monomer to polyamine cores was used to synthesize dendrimers of generations 1-5. All intermediates and dendrimers were either purified by precipitation, or did not need purification. 1H NMR spectroscopy indicated that reactions were complete up to G4-NH2 by integration, and mass spectroscopy confirmed that assignment. HPLC and GPC of Gn-Cl dendrimers showed sharp peaks for G1-G3, but G4-Cl appeared to have a small amount of impurities that are similar in size and polarity to the fully-substituted dendrimer. The G1-G3 dendrimers were confidently assigned as pure by conventional organic chemistry standards, but the assignment of purity to higher generations remained tentative. A G1-Cl dendrimer was functionalized with imidazole, and then deprotected and PEGylated with PEG5000 to yield a water soluble dendrimer. The imidazole-capped, Boc-protected dendrimer and the deprotected dendrimer were characterized by 1H and 13C NMR spectroscopy and mass spectrometry. The degree of PEGylation on the PEGylated material could not be definitively ascertained; however, the material is capable of solubilizing very hydrophobic Zn-phthalocyanines in water.

A variety of dendrimers based on melamine are designed, synthesized, and evaluated for drug delivery systems. The synthesis of a dendrimer, including multiple copies of four orthogonally reactive groups, is described. The three groups on the surface are nucleophilic and include four free hydroxyl groups, four tert-butyldiphenylsilyl (TBDPS) ether groups, and sixteen amines masked as tert-butoxycarbonyl (BOC) groups. The core of the dendrimer displays two electrophilic monochlorotriazines. The dendrimer above is further manipulated for in vivo biodistribution: incorporation of the reporting groups Bolton-Hunter and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid); PEGylation for biocompatibility and size tuning. In preliminary biodistribution studies, dendrimers circulate in the blood for a longer time as the molecular weight increases, which is important to passively target tumor tissues via the EPR effect. Also, high uptake by the tumor tissues was observed in mice bearing prostate cancer xenografts. A drug delivery vehicle for the anticancer agent paclitaxel is described. This drug delivery vehicle contains sixteen molecules of paclitaxel via acid-labile ester linkage, two Bolton-Hunter groups, and sixteen monochlorotriazine groups for PEGylation. The in vitro drug release studies shows faster release of paclitaxel at lower pH in PBS.

Liposomes have established roles in drug delivery and cell membrane studies. Amongst other applications; they can also be used as analytical reagents, particularly in immunoassays. Liposomal immunoassays have potential advantages over alternatives; including sensitivity, speed, simplicity and relative reagent stability. The aim of these studies was to develop and evaluate novel examples of these assays. When liposomes entrapped the dye, Sulphorhodamine B, a shift in its maximum absorption wavelength compared to free dye was observed. This was attributed to dimerization of the dye at high concentrations. If the liposomes were disrupted, the released dye was diluted into the external buffer, and the dye's absorption spectrum reverted to that of free dye. After optimization of dye entrapment, immunoassays were developed using these liposomes. Albumin-coated liposomes were used in a model assay to measure serum albumin. This assay employed complement-mediated immunolysis, commonly used in liposomal immunoassays. The liposomes were lysed by anti-albumin and complement, and this could be competitively inhibited by serum albumin. To improve sensitivity, Fab' anti-albumin liposomes were prepared. These enabled measurement of urinary albumin by a complement-mediated immunoassay, but using a sandwich technique. Anti-albumin (intact) liposomes were shown to precipitate on gentle centrifugation after reaction with albumin. They were applied as a solid phase reagent in an heterogeneous immunoassay, using radioimmunoassay for urinary microalbumin as a model assay. Liposomes containing Sulphorhodamine B were also used in a more novel assay; for serum anticardiolipin antibodies. Cardiolipin-containing liposomes were prepared. These were lysable using magnesium ions. Anticardiolipin antibodies (IgG) were found to augment this lysis, enabling their estimation. Similar imprecision and acceptable correlation with a commercial enzyme-linked immunosorbent assay (ELISA) were obtained. The findings demonstrate Sulphorhodamine B release can be used as a marker in homogeneous colorimetric liposomal immunoassays; both in model assays and in potentially more useful clinical biochemistry applications.

Submicron sized vesicles consisting of single and double chain non-ionic surfactant mixtures were prepared by simple dispersion of surfactant dissolved in aqueous medium, or alternatively, injecting the surfactant dissolved in organic solvent into the aqueous phase. Drug entrapment values were measured by using a fluorescent marker, 5,6- Carboxyfluorescein, and drug release characteristics were evaluated in biological media (serum and plasma) as a function of surfactant composition and in the presence or absence of cholesterol. Surface charge measurements, zeta-potential, as a function of pH, gel electrophoresis and immunoblotting (ELISA) were performed in order to measure the interaction of components of the biological fluid with the prepared vesicles. It was found that all vesicles carried a negative charge and rapidly bound plasma protein, which included albumin and immunoglobulin G, thus affecting the latency of the entrapped marker. Uptake and degradation of niosomes (non-ionic surfactant vesicles) in a living, unicellular, eukaryotic micro-organism was also investigated. It was found that the rate of release of contents depended on the composition of the vesicles and was a function of enzymatic degradation within these organisms rather than an intracellular PH effect of the digestive organelle. An identical protocol was carried out with the well- characterised liposome system and their inherent stabilities under a variety of conditions directly compared with niosomes.

To ensure the effective and safe use of nanomaterials for medical applications, the biocompatibility of the materials must be tested with particular relevance to the environment in which the material is placed. In nanoparticle-based drug delivery, it is crucial to evaluate a nanoparticle???s biocompatibility to ensure minimal cytotoxicity. Of several types of nanoparticles, peptide-based nanoparticles have emerged as promising systems for targeted cancer therapy. Yet, the biocompatibility of many of these peptides and their assembled particles has not been studied. This thesis, summarizes the original contribution on the effective and safe use of the particular self/co-assembling, amino acid pairing peptides and some of their DEGylated forms (modified versions) as carriers for anticancer drug delivery application. Therefore, the biocompatibility of the self-assembling, amino acid pairing (AAP) peptides AC8, its two DEGylated forms, as well as two related peptides, EAK16-II and EK8, is systematically investigated. The toxicity of these peptides and their complexes with pirarubicin was tested against the human adenocarcinoma lung cancer cell line, A549.The biocompatibility of the peptide-drug co-assembling complexes is assessed and the potential of these five peptides as carriers for the hydrophobic anticancer drug pirarubicin is demonstrated. For the first time experimental results on cytotoxicity, haemolytic activity, red blood cell (RBC) aggregation, complement activation and anaphylotoxin activation as an end result of complement activity for these five AAP peptides is reported. AC8, the amino end DEGylated AC8 (NP-I) and EK might be strong candidates for hydrophobic drug delivery considering their lack of toxicity and the fact that they are not recognized as a foreign molecule, inducing no considerable immune reactions. These results provide a basis for in vivo experiments and predict minimal in vitro toxicity of these peptides based delivery systems.