Purpose: The purpose of this study was to investigate possible effects of different microemulsion structure-types and types of monomer used on the formation of poly(alkylcyanoacrylate) nanoparticles, the entrapment into and release of insulin from these formulations as well as the bioactivity of the insulin upon intragastric delivery of the insulin-loaded nanoparticles dispersed in the microemulsion template. Methods: For two different microemulsion systems consisting of water, isopropyl myristate and either sugar-based surfactants or a macrogol glyceride-based surfactant-mixture, pseudo-ternary phase diagrams were established. Microemulsion samples therein were identified and characterised with polarising light microscopy, viscosity and conductivity measurements, differential scanning calorimetry, cryo-field emission scanning electron microscopy and self-diffusion nuclear magnetic resonance to determine the microemulsion structure-type. Nanoparticles were prepared from various microemulsion templates by interfacial polymerisation using ethyl (2) cyanoacrylate and butyl (2) cyanoacrylate. Particle size distribution and surface charge were measured using photon correlation spectroscopy and electrophoretic mobility. The morphology of the particles was characterised by scanning and transmission electron microscopy. Insulin was used as a model protein and the amount entrapped into and released from the particles was determined using a reverse phase HPLC assay. A diabetic rat model was employed to examine the bioactivity of different nanoparticle-microemulsion formulations with blood glucose and serum insulin as parameters measured by a proprietary glucometer and enzyme-linked immunosorbent assays, respectively. Results: The microemulsion system based on sugar-surfactants only formed solution-type microemulsions which could not all satisfactorily be used as a polymerisation template in the presence of insulin. The system however also showed an environmentally responsive gelling behaviour which may be suitable for depot delivery. The macrogol glyceride-based microemulsion system resulted in microemulsions with a continuous transition from water-in-oil to oil-in-water droplet-types via the bicontinuous structure-type. Microemulsion samples of each structure-type could serve as nanoparticle templates and resulted in particles with similar properties. Entrapment efficiency of insulin into the nanoparticles was template and monomer dependent. However, insulin was found to interfere with the polymerisation leading to a high variability in entrapment and release kinetics of these drug delivery systems. The degree of interference depended on the type of monomer and the size of the aqueous pseudo-phase of the microemulsion template. The interpretation of the results was further complicated by a possible competitive polymerisation initiation of insulin with the surfactant-mixture. Upon intragastric administration of the insulin-loaded nanoparticles dispersed in the oil-in-water microemulsion template a significant reduction in blood glucose could be achieved for up to 30 hours. However, no significant serum insulin concentration was detectable. Conclusions: Structurally different microemulsion templates resulting in nanoparticles with similar properties may offer increased formulation flexibility, in that a microemulsion template can be chosen which best solubilises the drug. Thus the microemulsions investigated in this thesis may serve as nanoparticle templates for designing entrapment processes for peptides and proteins with a simple one-step preparation by interfacial polymerisation. However, only if one was able to optimise and control the factors leading to the high entrapment and release variability these nanoparticles on the basis of microemulsions might be promising carriers for the oral delivery of peptide and protein bioactives.

The aims of this study were to prepare poly (ethylcyanoacrylate) (PECA) nanoparticles on the basis of different types of microemulsions, to investigate the entrapment within and release of a bioactive from these particles and to establish a set of delivery systems with varying entrapment and release characteristics, thereby giving the formulator the opportunity of a more tailor-made approach in the development of a delivery system. Furthermore the scale up of particle preparation and the possible enhancement of the immunogenic properties of PECA particles by incorporation of the adjuvant Quil A was investigated. Methods: Four phase triangles were established and microemulsion samples, used as a template to prepare nanoparticles, were characterised by viscosity and conductivity measurements, polarising light microscopy, freeze fracture transmission electron microscopy (TEM), cryo field emission electron microscopy (cryo FESEM) and self-diffusion NMR to determine their microemulsion type (droplet, bicontinuous, solution type). PECA nanoparticles were prepared from different types of microemulsions by interfacial polymerisation. Particle size, polydispersity index (PI) and [zeta]-potential were measured by photon correlation spectroscopy and electrophoretic mobility respectively. Normal scanning electron microscopy (SEM) and cryo FESEM were used to visualise particles. Fluorescently labelled ovalbumin (FITC-OVA) was used as a model protein/antigen and entrapment within and release from nanoparticles was investigated. To scale up nanoparticle preparation an instrumental set-up with reactor, peristaltic pump and stirrer was used. A 2⁷ fractional factorial study was designed to observe possible factors or their interactions that could influence particle formation under scale up conditions. For an immunological study freeze dried formulations of PECA nanoparticles, having FITC-OVA and Quil A entrapped, were prepared, and activation and uptake of formulations by murine bone marrow derived dendritic cells (DCs) and T cells in vitro were monitored. Results: Results obtained from the measurements described above, for formulations from the four different phase triangles, indicated that microemulsions of w/o droplet, bicontinuous or solution type could be formed. It was possible to prepare PECA nanoparticles from all of the different types of microemulsions. Particles had an average size of 265 nm � 24, with an average PI of 0.18 � 0.05 and an average negative [zeta]-potential of -17 mV � -5. Particle size, PI and [zeta]-potential were not influenced by the type of microemulsion that was used as a polymerisation template. Entrapment and release were however influenced by the type of microemulsion and although entrapment of FITC-OVA was generally high for PECA particles, it was highest for particles prepared from a droplet type microemulsion. Entrapment could also be increased by increasing amounts of monomer. The rate of release was dependent on the amount of monomer used for polymerisation and the type of microemulsion used for particle preparation, with nanoparticles prepared from a w/o droplet type microemulsion showing the slowest release. Furthermore it was shown that particle preparation could be scaled-up with the instrumental set-up used in this study, but conditions need to be refined as the average particle size and polydispersity index were considerably larger (441 nm � 101, 0.68 � 0.14) when compared to particles prepared by the beaker-pipette method (see above). The adjuvant Quil A could efficiently be entrapped into PECA nanoparticles together with FITC-OVA. Incubation of DCs and T cells with the various formulations did, however, not result in increased uptake or activation. Conclusions: PECA nanoparticles with high entrapment efficiency of antigen and adjuvant can be prepared from different types of microemulsions. Particles show different rates of entrapment and release depending on the type of microemulsion used as a polymerisation template, possibly because two different types of nanoparticles form. Nanocapsules are believed to form on the basis of droplet type microemulsions and nanospheres form on the basis of bicontinuous and solution type microemulsions. Freeze dried formulations of PECA nanoparticles, containing Quil A and FITC-OVA, were not able to induce an immune response, which might be due to charge repulsion effects between the negatively charged PECA nanoparticles and the negatively charged surface of dendritic cells. Moreover, no adjuvant effect of Quil A was apparent, perhaps caused by total encapsulation of the compound into the particle matrix with no active groups extending out displaying adjuvanticity.

The aims of this study were to prepare poly (ethylcyanoacrylate) (PECA) nanoparticles on the basis of different types of microemulsions, to investigate the entrapment within and release of a bioactive from these particles and to establish a set of delivery systems with varying entrapment and release characteristics, thereby giving the formulator the opportunity of a more tailor-made approach in the development of a delivery system. Furthermore the scale up of particle preparation and the possible enhancement of the immunogenic properties of PECA particles by incorporation of the adjuvant Quil A was investigated. Methods: Four phase triangles were established and microemulsion samples, used as a template to prepare nanoparticles, were characterised by viscosity and conductivity measurements, polarising light microscopy, freeze fracture transmission electron microscopy (TEM), cryo field emission electron microscopy (cryo FESEM) and self-diffusion NMR to determine their microemulsion type (droplet, bicontinuous, solution type). PECA nanoparticles were prepared from different types of microemulsions by interfacial polymerisation. Particle size, polydispersity index (PI) and [zeta]-potential were measured by photon correlation spectroscopy and electrophoretic mobility respectively. Normal scanning electron microscopy (SEM) and cryo FESEM were used to visualise particles. Fluorescently labelled ovalbumin (FITC-OVA) was used as a model protein/antigen and entrapment within and release from nanoparticles was investigated. To scale up nanoparticle preparation an instrumental set-up with reactor, peristaltic pump and stirrer was used. A 2⁷ fractional factorial study was designed to observe possible factors or their interactions that could influence particle formation under scale up conditions. For an immunological study freeze dried formulations of PECA nanoparticles, having FITC-OVA and Quil A entrapped, were prepared, and activation and uptake of formulations by murine bone marrow derived dendritic cells (DCs) and T cells in vitro were monitored. Results: Results obtained from the measurements described above, for formulations from the four different phase triangles, indicated that microemulsions of w/o droplet, bicontinuous or solution type could be formed. It was possible to prepare PECA nanoparticles from all of the different types of microemulsions. Particles had an average size of 265 nm � 24, with an average PI of 0.18 � 0.05 and an average negative [zeta]-potential of -17 mV � -5. Particle size, PI and [zeta]-potential were not influenced by the type of microemulsion that was used as a polymerisation template. Entrapment and release were however influenced by the type of microemulsion and although entrapment of FITC-OVA was generally high for PECA particles, it was highest for particles prepared from a droplet type microemulsion. Entrapment could also be increased by increasing amounts of monomer. The rate of release was dependent on the amount of monomer used for polymerisation and the type of microemulsion used for particle preparation, with nanoparticles prepared from a w/o droplet type microemulsion showing the slowest release. Furthermore it was shown that particle preparation could be scaled-up with the instrumental set-up used in this study, but conditions need to be refined as the average particle size and polydispersity index were considerably larger (441 nm � 101, 0.68 � 0.14) when compared to particles prepared by the beaker-pipette method (see above). The adjuvant Quil A could efficiently be entrapped into PECA nanoparticles together with FITC-OVA. Incubation of DCs and T cells with the various formulations did, however, not result in increased uptake or activation. Conclusions: PECA nanoparticles with high entrapment efficiency of antigen and adjuvant can be prepared from different types of microemulsions. Particles show different rates of entrapment and release depending on the type of microemulsion used as a polymerisation template, possibly because two different types of nanoparticles form. Nanocapsules are believed to form on the basis of droplet type microemulsions and nanospheres form on the basis of bicontinuous and solution type microemulsions. Freeze dried formulations of PECA nanoparticles, containing Quil A and FITC-OVA, were not able to induce an immune response, which might be due to charge repulsion effects between the negatively charged PECA nanoparticles and the negatively charged surface of dendritic cells. Moreover, no adjuvant effect of Quil A was apparent, perhaps caused by total encapsulation of the compound into the particle matrix with no active groups extending out displaying adjuvanticity.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Thesis (M.S.)--Duquesne University, 2009. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 82-90) and index.

Tuberculosis(TB) was declared to be a global emergency in 1993, with South Africa declaring it to be the country’s top health priority in 1996, but ineffective treatment strategies have led to fewer than half of all treated patients in South Africa being cured. At present,paediatric treatment remains a problem, as the antitubercular preparations of rifampicin, isoniazid and pyrazinamide, that are currently available, were not initially designed for the treatment of paediatric TB patients, providing a motivation for this project. The aim of this project is thus the development of a microemulsion dosage form for the oral delivery of RIF(Rifampicin), INH(Isoniazid) and PZA(Pyrazinamide) in combination. RIF, INH and PZA were adequately characterised with reference to the monograph standards referenced and were found to be sufficiently pure to be used in subsequent work. A chromatographic system and conditions were selected and validated as being optimal for HPLC analysis of RIF, INH and PZA in combination, with a drug partitioning method for miglyol 812 developed and validated. Ternary and pseudo-ternary phase diagrams were constructed and reported, all employing miglyol 812 as the lipid. It was undoubtedly the imwitor 308 and crillet 3 combination o/w microemulsion system that proved most successful, maintaining homogeneity on dilution. The microemulsion used in formulation comprised imwitor 308 (27.63%), crillet 3 (27.63%), miglyol 812 23.68%) and water (21.06%). The stability of RIF, INH and PZA was investigated in aqueous solution, miglyol 812, corn oil, 10%m/v cremophor RH, 5%m/v imwitor 308, 10%m/v crillet 3 and 70%m/v sorbitol solution. Trends in the stability assessments conducted on RIF, INH and PZA were noted, with slight variation depending on the formulation component being evaluated. RIF invariably demonstrated temperature and oxidation dependent degradation in all vehicles, with a definite distinction possible between samples stored at 25, 40 and 600C over a 7 day trial period. A definite advantage of storing RIF solutions under nitrogen was observed, with these solutions showing less degradation over the course of the trial, than those stored under air. INH produced a pronounced increase in the degree of degradation of RIF, whereas PZA had a negligible effect on it’s stability. INH proved to be most stable in the 70%m/v sorbitol solution with no significant oxidation or temperature dependent degradation indicated. Temperature dependent degradation was only noticable when INH was in combination with RIF, most significant in crillet 3 solution. PZA was the most stable of the three drugs, remaining relatively unaffected by temperature and the presence of air, independent of the vehicle employed, although the drug remaining did decrease slightly in the presence of RIF.Due to drug dose specifications and solubility limitations, the final formulation assessed, only contained RIF and INH, despite INH and PZA having no significant effect on the stability of each other. The solubility of PZA in the lipid and aqueous components of the microemulsion was not great enough to achieve the required 500 mg/10ml dose, while RIF and INH could achieve the respective 150mg/10ml and 100mg/10ml dose. RIF stability was improved, as anticipated, with the incorporation of RIF into the internal phase decreasing contact with INH which has been shown to affect it’s stability. RIF behaved as predicted, possessing greater stability than shown in the individual formulation components, however, INH did not, being less stable in formulation in the absence of antioxidant, than in it’s presence. A novel microemulsion formulation capable of delivering the incompatible RIF and INH in combination, with numerous microemulsion systems mapped,with the ability of being used for the delivery of other lipophilic drugs and drug combinations, was produced.The final formulation provided valuable information into possible future improvements of the microemulsion to improve drug stability.

Zhang, Xinran. / Thesis submitted in: December 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 80-87). / Abstracts in English and Chinese. / TITLE PAGE --- p.i / ABSTRACT --- p.ii / 中文摘要 --- p.v / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiv / ABBREVIATIONS --- p.xv / Chapter CHAPTER 1. --- Introduction / Chapter 1.1. --- Rationale of the Study --- p.1 / Chapter 1.2. --- Current technologies for formulating long-acting parenteral protein deliver system --- p.3 / Chapter 1.2.1. --- Chemical Modification --- p.3 / Chapter 1.2.2. --- Sustained-release formulation --- p.4 / Chapter 1.2.2.1. --- Phase separation method --- p.4 / Chapter 1.2.2.2. --- Solvent evaporation/extraction method --- p.5 / Chapter 1.2.2.3. --- Spray drying method --- p.6 / Chapter 1.2.2.4. --- Causes for protein instability --- p.6 / Chapter 1.2.2.4.1. --- Water/organic solvent interface --- p.6 / Chapter 1.2.2.4.2. --- Lyophilization --- p.8 / Chapter 1.2.2.4.3. --- Polymer --- p.11 / Chapter 1.2.2.4.4. --- Stabilizing additive --- p.13 / Chapter 1.3. --- Aqueous-aqueous emulsion technology --- p.17 / Chapter 1.3.1. --- Background --- p.17 / Chapter 1.3.2. --- Basic Principle --- p.17 / Chapter 1.3.3. --- Phase diagram --- p.18 / Chapter 1.3.4. --- Formation of aqueous-aqueous emulsion --- p.19 / Chapter 1.3.4.1. --- Introduction of a water-soluble charged polymer as stabilizer --- p.19 / Chapter 1.3.4.2. --- Freezing-induced phase separation --- p.20 / Chapter 1.3.5. --- General Protocol --- p.21 / Chapter 1.3.5.1. --- Introduction of a water-soluble charged polymeric stabilizer --- p.22 / Chapter 1.3.5.2. --- Freezing-induced phase separation --- p.22 / Chapter 1.3.6. --- Merits and limitations of the aqueous-aqueous emulsion technology --- p.23 / Chapter 1.3.7. --- Protein selection for the sustained release formulation --- p.25 / Chapter 1.4. --- Aims and scope of study --- p.26 / Chapter "CHAPTER 2," --- Materials and Methods / Chapter 2.1. --- Materials --- p.28 / Chapter 2.1.1. --- Proteins --- p.28 / Chapter 2.1.2. --- Polymers --- p.28 / Chapter 2.1.3. --- Media for TF-1 Cell Culture --- p.28 / Chapter 2.1.4. --- Chemicals and Solvents for Cell Proliferation Assay --- p.29 / Chapter 2.1.5. --- Other Chemicals and Solvents --- p.29 / Chapter 2.1.6. --- Materials for Cell Culture --- p.29 / Chapter 2.1.7. --- Materials for Reagent Kits --- p.30 / Chapter 2.2. --- Methods --- p.30 / Chapter 2.2.1. --- Determination of the Partition Coefficients of Proteins Between PEG and Dextran --- p.30 / Chapter 2.2.2. --- Preparation of Glassy Particles --- p.31 / Chapter 2.2.2.1. --- Standard Stable Aqueous-aqueous Emulsion Method --- p.31 / Chapter 2.2.2.2. --- Freezing-induced Phase Separation --- p.32 / Chapter 2.2.3. --- Preparation of Protein-loaded and Blank Microspheres Using S-o-w Solvent Extraction Technique --- p.32 / Chapter 2.2.4. --- Optical Microscopy and Scanning Electron Microscopy --- p.33 / Chapter 2.2.5. --- Determination of Protein Loading --- p.34 / Chapter 2.2.5.1. --- Within Dextran Particles --- p.34 / Chapter 2.2.5.2. --- Within PLGA microspheres --- p.34 / Chapter 2.2.6. --- Evaluation of Protein Structural Integrity and Bioactivity in Dextran Particles and PGLA Microspheres --- p.35 / Chapter 2.2.7. --- In vitro Release Study --- p.36 / Chapter 2.2.8. --- RhIFN Stability Determination under Simulated In Vitro Release Conditions --- p.37 / Chapter 2.2.8.1. --- In the Absence of PLGA --- p.37 / Chapter 2.2.8.2. --- In the Presence of PLGA --- p.37 / Chapter 2.2.9. --- MicroBCÁёØ Protein Assay --- p.38 / Chapter 2.2.10. --- Size Exclusion Chromatography (SEC) - High Performance Liquid Chromatography (HPLC) --- p.38 / Chapter 2.2.11. --- ELISA --- p.39 / Chapter 2.2.12. --- Bioactivity Assay --- p.40 / Chapter 2.2.12.1. --- RhIFN --- p.40 / Chapter 2.2.12.2. --- RhGM-CSF --- p.41 / Chapter CHAPTER 3. --- Results and Discussions / Chapter 3.1. --- Sustained-release RhIFN Formulation --- p.45 / Chapter 3.1.1. --- Partition Coefficient of RhIFN --- p.45 / Chapter 3.1.2. --- Formulation Based on the Standard Aqueous-aqueous Emulsion (SA-AE) Method With Sodium Alginate as Stabilizer --- p.45 / Chapter 3.1.2.1. --- Surface Morphology --- p.45 / Chapter 3.1.2.2. --- Formulation Characterization --- p.46 / Chapter 3.1.2.3. --- In Vitro Release of RhIFN from PLGA Microsheres --- p.54 / Chapter 3.1.3. --- Formulation Based on the Freezing-induced Phase Separation (FIPS) Technique without Sodium Alginate --- p.56 / Chapter 3.1.3.1. --- Formulation Characterization --- p.56 / Chapter 3.1.3.2. --- In Vitro Release of RhIFN from PGLA Microsphees --- p.59 / Chapter 3.2. --- RhIFN Stability Assessment under Simulated In Vitro Release Conditions --- p.63 / Chapter 3.2.1. --- In the Absence of PLGA --- p.63 / Chapter 3.2.2. --- In the Presence of PLGA --- p.65 / Chapter 3.3. --- Sustained-release RhGM-CSF Formulation --- p.68 / Chapter 3.3.1. --- Partition Coefficient Determination of RhGM-CSF Between PEG and Dextran --- p.68 / Chapter 3.3.2. --- Formulation Based on Freezing-induced Phase Separation --- p.68 / Chapter 3.3.2.1. --- Validation of MTT Assay Conditions --- p.69 / Chapter 3.3.2.2. --- Formulation Characterization --- p.71 / Chapter 3.3.2.3. --- In Vitro Release of RhGM-CSF from PLGA Microspheres --- p.75 / Chapter CHAPTER 4. --- Conclusion and Future Studies / Chapter 4.1. --- Conclusion --- p.78 / Chapter 4.2. --- Future Studies --- p.79 / References --- p.80

Se han desarrollado y optimizado dos nanoemulsiones que contienen Nistatina (N1 y N2) para su aplicación en piel y mucosa oral, respectivamente. Estas formulaciones tuvieron un tamaño de gota nanométrico y resultaron ser estables a lo largo del tiempo. Adicionalmente se determinó el comportamiento reológico de ambos sistemas resultando ser fluidos newtonianos. Se evaluó la actividad antifúngica in vitro, observado que la potencia de la nistatina aumentó al ser introducida en las nanoemulsiones, cuando se comparaba con la nistatina libre. El mecanismo de liberación de la nistatina de las nanoemulsiones siguió una cinética de orden uno para ambas formulaciones. Trás el ensayo de permeación ex vivo en piel humana con la nanoemulsion N1 se observó que la cantidad de fármaco permeada fue muy baja, pudiendo descartarse la posible aparición de efectos adversos a nivel sistémico. De la misma manera se realizó el ensayo de permeación ex vivo en mucosa oral porcina para la formulación N2 llegando a la misma conclusión que en el caso de la permeación en piel. En ambos casos la cantidad de fármaco retenido en el tejido (tanto en piel o mucosa) es suficiente para observar un efecto fungistático, fungicida y un prolongado efecto post-antifungico (PAFE). Con el fin de determinar el efecto de la nanoemulsion N2 sobre la mucosa oral se visualizó la ultra-estructura del tejido y no observándose variaciones significativas cuando se comparaban con el control (mucosa no tratada con la nanoemulsion). Las nanoemulsiones N1 y N2 son formulaciones prometedoras para el potencial tratamiento clínico de candidiasis muco-cutáneas. / Muco-cutaneous candidosis is a common opportunistic infection must be treated to prevent other tissue invasion. Nystatin is one of the most prescribed drugs to treat this pathology, but due to its physicochemical properties its pharmaceutical-technological requirements make it a challenge. The purpose of this work was the development and characterization of an optimal nystatin delivery system for the potential treatment of oral candidosis avoiding undesirable side effects and toxicity of potential systemic absorption. Two nanoemulsion (N1 and N2) was developed, evaluated and characterized. It has been formulated successfully as a stable nanoemulsion with a droplet size of 75 and 138 nm, respectively. First order release parameters were estimated using different mathematical approaches and ex vivo permeation of nystatin through human skin and porcine buccal mucosa were found no systemic effects would happen. Microbiologic studies performed revealed an enhanced antifungal effect of the nystatin loaded nanoemulsion. Also the evaluation of buccal mucosa ultrastructure by transmission electron microscopy methodology showing a harmless effect in the mucosa microstructure. We can infer that the selected nystatin nanoemulsion could be potentially used on candidosis infection under mucositis conditions.