Please read the abstract in the dissertation Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Physiology / unrestricted

In recent years, the application of microparticles in different fields including cosmetics, agriculture, pharmaceutics and biomedicine has been widely investigated.In this project, we aimed to improve the preparative methods for chitosan–triphosphate microparticles (Cs/TPP) for perspective application in the fields of cell encapsulation and controlled drug delivery. Prior to the preparation of chitosan-based microparticles, in order to confirm good biocompatibility and reproducibility, protocols have been established for the purification and characterisation of chitosan including the assessment of average molecular weight, protein content and degree of deacetylation.This study then primarily focused on the use of β-glycerophosphate (βGP) as an excipient, which is known to solubilize chitosan at neutral pH, thus allowing the preparation of chitosan microparticles (microspheres and toroidal) via ionotropic gelation under physiological conditions. The preparation of Cs-βGP/TPP microparticles was optimized varying several key process variables (concentration, flow rate, and frequency) and these microparticles were produced with a narrow size distribution (400 – 500 μm, spherical shape) and compared to Cs/TPP controls. The main result was the possibility to perform this process at neutral pH, although we have also demonstrated an improved toughness and cross-linking density of these microspheres as a result of the presence of β-glycerophosphate. Further, we have investigated the application of this method to a toroidal geometry, which provides advantages in terms of better transfer of oxygen and nutrients to any encapsulated materials. Cs/TPP and Cs-βGP/TPP ‘micro-doughnuts’ were also prepared and characterised. This research highlighted the evidence of a higher cross-linking density of these microparticles in comparsion with the spherical ones. In order to optimise the physicochemical characteristics of these microparticles for future applications as biomaterials, the surface of Cs/TPP and Cs-βGP/TPP microparticles was modified through an additional polyelectrolyte complexation with poly (sodium 4- styrene sulphonate) (PSS). The improved toughness and cross-linking density was confirmed by measuring the mechanical properties and solid content which indicated the successful complexation of PSS onto the surface of these microparticles.

The ability of a peptide vaccine derived from the human T-lymphotropic virus type 1 (HTLV-1) surface envelope glycoprotein protein (gp46) to mimic the native protein and elicit a protective immune response has been examined. This peptide construct, designated MVFMF2, comprises amino acids (aa) 175-218 of gp46 linked by a four residue turn (GPSL) to a promiscuous T-cell epitope from the measles virus fusion protein (MVF, aa 288-302). The peptide was structurally characterized by circular dichroism (CD) spectroscopy and was found to contain α-helical secondary structure. The immunogenicity of MVFMF2 in rabbits and mice was evaluated by direct ELISA and competitive ELISA using peptide constructs and the recombinant protein ACH-RE3 (aa 165-306). This peptide, when administered with adjuvant (N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine, nor-MDP) was immunogenic in an outbred population of both rabbits and mice. Furthermore, the peptide construct was encapsulated in biodegradable microspheres of poly(D,L-lactide-co-glycolide) to eliminate booster immunization and to examine adjuvant requirements. The data indicate that MVFMF2 shows enhanced immunogenicity when encapsulated in biodegradable microspheres. Inoculation of the encapsulated peptide produced a similar humoral response to that of the free peptide, but did not require the use of adjuvant. Elicited anti-rabbit and anti-mouse antibodies recognized whole viral preparations and the recombinant protein ACH-RE3 in ELISA assays. Additionally, inoculated rabbits exhibited enhanced reactivity to viral antigens by western blot compared to non-vaccinated controls. Although anti-rabbit and anti-mouse antibodies were capable of inhibiting syncytium formation at low dilutions, rabbits were not protected from cell-associated viral challenge. Future development of vaccines to HTLV-1 may need to incorporate the ability to elicit cell-mediated immune responses in order to protect against cell-associated viral infection.

No description available.

No description available.

To compare the optimum recoveries of an inoculation of <i>Salmonella enterica</i> serotype Typhimurium, fluorescent microspheres (1.0 μm diameter, carboxylate-modified, crimson FluoSpheres®, Molecular Probes, Eugene, OR), or a combination of both from stainless steel, three recovery methods, including a standard rinse, a one-ply composite tissue (Kimwipe®) or a sonicating brush were used. Findings were used to assess the effectiveness of fluorescent microspheres as surrogates for <i>S.</i> Typhimurium. For each method, ten coupons (304 grade, 2.5 x 8 cm) were inoculated with either 100 μl of a <i>S.</i> Typhimurium culture, or a solution of fluorescent microspheres, or both, at approximate concentrations of 10⁶. After drying for one hour, coupons were sampled using either a rinse of 100 ml of phosphate buffered saline solution (PBS) for one min, a Kimwipe® tissue method, or submerged in PBS and subjected to a sonicating brush for one min. After treatments, PBS solutions were analyzed using duplicate plate counting (<i>Salmonella</i>) or hemacytometry (microspheres). For microspheres and <i>Salmonella</i>, recovery by sonicating brush > rinse > Kimwipe® method. Additionally, the retention of microspheres on the steel ranged from 16 to 25% (mean from five coupons each recovery method). Microspheres yielded a significantly higher recovery rate (11 – 60%) than <i>Salmonella</i> (~1%) for each recovery method, therefore the microspheres used in this study, are not appropriate surrogates for <i>S.</i> Typhimurium for future recovery studies on stainless steel. However, due to their low standard deviations for their mean percent recovery, they hold the opportunity to provide better accuracy and reproducibility. / Master of Science

Thesis (M. Pharm. (Pharmaceutics)) -- University of Limpopo, 2019 / Various approaches have been developed to retain dosage forms in the gastrointestinal tract. One of the commonly used approaches is the use of microspheres. Due to their intrinsic low density and small size, they are distributed throughout the gastrointestinal tract which improves drug absorption thus improving bioavailability. Ranitidine hydrochloride, an antiulcer drug is poorly absorbed from the lower gastrointestinal tract and has a short half-life of 2.5-3 hours. The aim of this study was to formulate and evaluate gastroretentive microspheres of ranitidine hydrochloride in order to extend gastric retention in the upper gastrointestinal tract, which may result in enhanced absorption and thus improved bioavailability. Pre-formulation studies were conducted to develop and validate the analytical method to identify and quantify ranitidine hydrochloride; to select the suitable polymers for further formulation development and; to determine the compatibility of the chosen polymers with ranitidine hydrochloride. The analytical method was validated and found to be sensitive, linear, precise and accurate. Preliminary formulations lead to the selection of ethyl cellulose and PEG 4000 as polymers and solvent evaporation as the method of manufacture. Compatibility studies were determined by DSC/TGA, FTIR and short-term accelerated studies and no incompatibilities were observed. Two prototype formulations of the preliminary formulations F24 and F26 were manufactured comprised of varying drug: polymer concentration. The microspheres were evaluated for morphology, particle size, flow properties, percentage yield, buoyancy and in vitro drug release. Both formulations resulted in spherical microspheres with good flow properties, high yield and buoyancy studies revealed that the microspheres would float immediately upon contact with the dissolution media and floating would continue for more than 8 hours. In vitro drug release studies revealed that polymer concentration greatly affected drug release. Dissolution kinetic studies revealed that formulation F24 and v F26 were best described by the Korsmeyer-Peppas and Higuchi kinetic models respectively. Formulation F26 was considered the best formulation, which comprised of a drug: PEG 4000 ratio of 1:2 w/w, as it yielded better in better drug encapsulation, better buoyancy results and had complete drug release.

The use of fluorescent imaging probes for the real time detection of cellular malfunctions, such as enzyme over expression has shown promise. Fluorescent dyes with absorption and emission values below 600 nm are limited in their in vivo applications due to high background auto-fluorescence and low resolution images. Employing near infrared (NIR) fluorophores such as cyanine dyes can overcome this disadvantage. Cyanine dyes can be synthesised using solution or solid-phase techniques with the use of solution phase chemistry allowing for larger scale and higher yielding reactions. Utilising a selection of functional groups and varying polymethine chain lengths a cyanine dye library with tuneable absorption and emission wavelengths was synthesised. This thesis gives the first detailed examples of how modifications on heptamethine cyanine dyes alter their cellular uptake and cellular toxicity. Furthermore, a NIR fluorescent microsphere is reported as well as NIR functionalised microspheres with the ability to be tracked within cells. Additional lines of work involved the synthesis of a fluorescent sensor for the visualisation of bacteria. Aminopeptidases are present within the peptidoglycan cell wall of Gram negative bacteria and therefore can be targeted for real time detection of bacteria to aid in the detection of infectious diseases. A coumarin based probe is reported which detects aminopeptidase in gram negative bacteria in vitro.

Photoreactive groups have been used in photoaffinity labelling of chemical macromolecules via the generation of highly reactive species upon short wave light irradiation. One of the most efficient photoreactive functional groups is trifluoromethyl aryldiazirine (TFMAD). This compound was synthesised as part of the work discussed in this thesis, making use of microwave irradiation to shorten reaction times (Chapter I). An investigation of properties allowed the development of three different applications for conjugation to biomolecules. The first application consisted of the development of an approach for generation of small-molecule microarrays, where a 2,000 compound library was immobilised onto the glass surface through carbene insertion. The microarray was then used to screen for potential binders to beta-transducin repeat containing protein (b-TrCP1) allowing the reduction of possible candidates to less than 25 compounds (Chapter II). The second application was the synthesis of two probes to allow the selective delivery of active compounds inside specific organelles or cells. The diazirine moiety was used as a rapid way to covalently capture a number of cargos. The approach allowed a peptoid and an anticancer drug to be conjugated to the two probes and their cell penetrability properties and therapeutic effect were studied, respectively (Chapter III). Finally, the insertion properties of TFMAD were used to develop approaches to attach DNA onto microspheres and the efficiency of this delivery system was evaluated (Chapter IV).

Narrowly dispersed amino-functionalised polystyrene microspheres, with a range of diameters, were successfully synthesised via emulsion and dispersion polymerisation. Fluorescent labelling allowed cellular translocation to be assessed in a variety of cell lines and was found to be very high, but controllable, whilst exhibiting no detrimental effect on cellular viability. In order to fully determine the mode of microsphere uptake, “beadfected” melanoma (B16F10) cells were studied using both chemical and microscopic methods. Uptake was found to be wholly unreliant upon energetic processes, with microspheres located cytoplasmically and not encapsulated within endosomes, an important characteristic for delivery devices. In order to demonstrate the effective delivery of exogenous cargo mediated by microspheres, short interfering (si)-RNAs were conjugated to beads and investigated for the gene silencing of enhanced green fluorescent protein (EGFP) in cervical cancer (HeLa) and embryonic (E14) stem cells. EGFP knockdown was found to be highly efficient after 48 – 72 hours. Dual-functionalised microspheres displaying a fluorophore (Cy5) and siRNA allowed only those cells beadfected with the delivery vehicle (and thus containing siRNA) to be assessed for EGFP expression, yielding an accurate assessment of microsphere-mediated gene silencing. In addition, by manipulation of the microsphere preparation conditions, micro-doughnuts and paramagnetic microspheres were produced and their cellular uptake assessed. Paramagnetic microspheres were found to enter cells efficiently and were subsequently used to bias the movement of beadfected cells in response to an externally applied magnet, while micro-doughnuts were found to exhibit cell selective properties and were noted to traffic specifically to the liver in vivo.