Thesis (Ph.D.) - University of Queensland, 2006. / Includes bibliography.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 70-80). Also available in electronic version.

The purpose of this project was to study the effects of anodic electrically stimulated silver-coated stainless steel implants and bacterial contamination in a canine radius fracture gap model. Twelve skeletally mature canines weighing 19.2-23.2 kg were used. Dogs were randomly assigned to into control and contaminated groups. A 5 mm ostectomy gap was made in both radii of each dog. One radius of each dog was stabilized with a silver-coated stainless steel bone plate and the other with an uncoated stainless steel bone plate. The ostectomy sites were inoculated with sterile PBS in 6 dogs and S. intermedius in the other 6 dogs. Each implant set was electrically stimulated with direct current for 20 minutes daily, for 10 days. Animals were treated with Cephalexin orally for 10 days. Radiographs were obtained at two week intervals. Animals were euthanized at 12 weeks and each plate was cultured. Radiographic, histologic and bacteriologic evaluations of each radius were performed. The difference in bone healing between silver-coated and stainless steel treated radii was determined for each dog by subjective radiographic evaluation and quantitative analysis of radiographic density using commercial software. At week 12, a significant decrease in bone healing was found on radiographic evaluations and quantitative bone area analysis of contaminated radii that were treated with electrically stimulated silver-coated implants (p-value 0.025 and 0.018 respectively). Inoculum or implant treatment type showed no significant difference on radiographic evaluations or quantitative bone area analysis. No significant differences were detected in radiographic evaluations of osteomyelitis or histologic evaluations of bone healing, inflammation and peri-implant resorption. Culture results were not indicative of S. intermedius inoculated osteomyelitis. The use of a bilateral radial ostectomy model allowed comparison of electrically stimulated silver-coated and uncoated implants within each dog. Bone healing in the stable 5 mm fracture gap radial ostectomy model, was progressive in both contaminated and non-contaminated situations. Our study found a negative effect on bone healing when electrically-stimulated silver stearate coated implant were used in S. intermedius contaminated radius ostectomies. This trend became apparent at 6 weeks post-operatively, but was not statistically evident until 12 weeks. Further studies are warranted to evaluate the appropriate protocol, the effects of, and indicated use of this treatment modality in contaminated fractures and clinical osteomyelitis situations. / Master of Science

It has been shown that prolonged systemic presence of a drug can cause a build up of that drug in the skin. This drug ‘reservoir’, if properly understood, could provide useful and important information about the recent drug-taking history of a patient. In this thesis we create three mathematical models which combine to explore the potential for a drug reservoir to form in the skin and be collected as a method of monitoring compliance. The first model is used to characterise timedependent drug concentrations in plasma and tissue following a customisable drug regimen. Outputs from this model provide boundary conditions for the second, spatio-temporal model of drug build-up and concentration profile in the skin. This then provides initial conditions for the final model which predicts the extraction. These models are then used to identify the scenarios which have the greatest potential for successfully monitoring patient compliance via the skin. We focus in particular on drugs that are highly bound as this will restrict their potential to move freely into the skin but which are lipophilic so that, in the unbound form, they would demonstrate an affinity to the outer layers of the skin (which are built around a lipid matrix). We highlight how this study might be used to inform future experimental design and data collection in order to provide relevant parameter estimates for reservoir formation and its potential to contribute to enhanced drug monitoring techniques.

Thesis (M. Sc.)--Memorial University of Newfoundland, 1997. / Bibliography: leaves 95-108.

Background: Understanding lactate levels can provide important information aboutour body’s condition. For athletes, this can improve their training and prevent earlyfatigue. In healthcare, monitoring lactate can provide valuable information and potentially prevent life-threatening episodes. Lactate can be measured non-invasively byanalyzing sweat. This is advantageous over the typical blood sampling since it is saferand pain-free. Sweat can be stimulated by using a method called iontophoresis. Itapplies a small current between two electrodes placed on the skin’s surface, deliveringsubstances to the inner layer of the skin. Objectives: The aim of this study was to design a device that implements iontophoresis to activate sweat production and uses a microfluidic system to collect thesweat and deliver it to a lactate sensor and provide a signal. Methodology: A device was designed in AutoCAD and 3D printed. It was improvedby trial and error. A sweat collecting test was performed to validate the iontophoresissystem. The efficiency of the microfluidic system was tested by recording the time ittakes to collect enough sweat to get a lactate signal. Finally, calibration tests wereperformed to validate the lactate signal in the form of batch-mode and flow-mode. Results: The sweat collection test produced 27 µL of sweat in 15 minutes and 49 µLin 30 minutes. The microfluidic system delivered sweat to the sensor and activated itin less than 3 minutes. The linearity of the batch-mode calibration, R2-value, was0.9994, and for the flow-mode it was 0.8908.Conclusions: The iontophoresis system stimulated sweat production, which themicrofluidic system delivered to the lactate sensor successfully. The lactate sensorwas implemented into the device, and a signal was detected. However, it could not becalibrated efficiently enough to display the electric signal as a lactate concentration.

A Terapia Fotodinâmica (TFD) é uma modalidade terapêutica inovadora para o tratamento de tumores cutâneos. As ftalocianinas têm sido utilizadas como fotossensibilizantes sistêmicos devido à sua alta afinidade ao tecido tumoral e seu efeito acentuado quando irradiadas com luz. No entanto, a lipossolubilidade e o baixo coeficiente de partilha óleo/água dessas substâncias dificultam sua aplicação tópica, levando ao desenvolvimento de derivados carregados, como a zinco ftalocianina tetrassulfonada (ZnPcS4) na tentativa de aumentar sua solubilidade em água, bem como melhorar sua captação pelas células tumorais. Entretanto, moléculas carregadas têm dificuldades em atravessar o estrato córneo (EC), a principal barreira da pele. Como a iontoforese é uma técnica não-invasiva capaz de aumentar e controlar a penetração de moléculas carregadas na pele, ela parece ser uma alternativa para aumentar a penetração cutânea da ZnPcS4 nas camadas da pele onde os tumores estão presentes. Dessa forma, foram realizados experimentos in vitro de iontoforese de um gel hidrofílico da ZnPcS4 aplicado topicamente na presença e ausência de NaCl. Estudos de iontoforese in vivo também foram realizados, empregando ratos Wistar como modelo animal, bem como experimentos em cultura de células tumorais para avaliar a citotoxicidade do fármaco em estudo. O método analítico para quantificação do fármaco na pele foi validado quanto à linearidade, precisão, exatidão, sensibilidade e seletividade. A iontoforese catódica promoveu um aumento significativo da retenção da ZnPcS4 tanto no EC como na epiderme viável nos experimentos realizados na presença e ausência de NaCl em relação à aplicação passiva da formulação e de iontoforese anódica, sendo que ocorreu um aumento da quantidade do fármaco retido nas diferentes camadas da pele quando o sal foi retirado da formulação. Os estudos in vivo com a formulação também mostraram que a corrente elétrica aumentou a penetração do fármaco para as camadas mais profundas da pele em relação aos experimentos passivos de permeação, o que foi evidenciado pela intensidade de fluorescência do fármaco visualizada por microscopia confocal e pela quantidade de fármaco retido nas diferentes camadas da pele. Resultados em cultura de células tumorais A431 sugerem que a concentração de fármaco que chega à epiderme viável após experimentos de iontoforese catódica é capaz de matar mais de 90% dessas células tumorais quando a dose de irradiação de 5 J/cm2 é aplicada. Além disso, quando a corrente elétrica é aplicada em cultura celular, não foi observado nenhum aumento significativo da citotoxicidade da ZnPcS4, demonstrando que a aplicação de corrente elétrica não fez com que a entrada do fármaco para o interior das células tumorais aumentasse. Não restam dúvidas, no entanto, que a corrente elétrica aumentou a penetração do fármaco nas camadas profundas da pele, além de levar a uma distribuição homogênea da ZnPcS4 nessas camadas após 15 minutos de aplicação. / Photodynamic therapy (PDT) is an innovative therapeutic modality for the treatment of cutaneous tumors. The phthalocyanines have been used as systemic photosensitizing agents due to its high affinity to tumor tissues and its accentuated effect when irradiated with light. However, the lipophilicity and the low partition coefficient oil/water of these substances difficult its topical application, leading to the development of charged derivatives, such as the zinc phthalocyanine tetrasulfonated (ZnPcS4) in an attempt to increase the water solubility, as well as improve the drug uptake by tumor cells. However, charged molecules have difficulties to cross the stratum corneum (SC), the main barrier of the skin. As iontophoresis is a noninvasive technique able to improve and control the penetration of charged molecules through the skin, it seems to be an alternative for enhancing ZnPcS4 penetration into the deep layers of the skin, where cutaneous tumors reside. This way, in vitro iontophoresis experiments of a hydrophilic gel containing ZnPcS4 applied topically in the presence and absence of NaCl were performed. In vivo iontophoresis studies were also carried out employing Wistar rats as animal model, as well as experiments in culture of tumour cells to evaluate the cytotoxicity of the drug. The analytical method for the quantification of the drug in the skin was validated considering the parameters of linearity, precision, accuracy, sensitivity and selectivity. The cathodal iontophoresis promoted a significant increase in retention of ZnPcS4 in both SC and viable epidermis in the experiments conducted in the presence and absence of NaCl in relation to the formulation applied passively or by anodal iontophoresis. Therefore, there was an increase in the amount of the drug retained in different layers of the skin when salt was removed from the formulation in the cathodal iontophoresis. In vivo studies also demonstrated that the electrical current increased penetration of the drug to the deeper layers of the skin in relation to passive experiments, evidenced by the fluorescence intensity of the drug showed by confocal microscopy and by the amount of drug retained in the different layers of the skin. Results with A431 tumor cells suggest that the concentration of the drug that reaches the viable epidermis after cathodal iontophoresis is able to kill more than 90% of these tumor cells when the radiation dose of 5 J/cm² was applied. In addition, when the electric current was applied to the cells, it was not observed any significant increase of cytotoxicity, demonstrating that the electric current application did not increased the uptake of the ZnPcS4 by the tumor cells. There are no doubts, however, that the electric current increased the ZnPcS4 penetration to the deep layers of the skin and lead to a homogeneous distribution of ZnPcS4 in these layers after 15 minutes of application.

Basic information about nail behaviour, under passive and especially iontophoretic condition, lacks in the literature. Thus, this thesis aims to fill gaps in the nail understanding by studying the potential and feasibility of the application of iontophoresis to human nail. The iontophoretic and passive delivery of Sodium Fluorescein (SF) and Nile Blue Chloride (NBC) were studied, in vitro, in order to determine their pathways as well as their depth and uniformity of penetration into the nail. The permselective properties of the nail were investigated by characterizing the contribution of electroosmosis, using mannitol as a marker, and by studying the flux of two inorganic cations, sodium and lithium, during in vitro experiments. Finally, the feasibility of transungual iontophoresis and the extraction of sodium and chloride ions from the body through the nail plate were performed on a group of human volunteers. Iontophoresis led the fluorescent markers slightly deeper into the nail plate than passive diffusion. The delivery of the bianion and of the cation was not different. Both compounds mainly penetrated the nail via the transcellular pathway. Electroosmosis resulted only in a slight enhancement of the mannitol fluxes compared to passive diffusion and the fluxes presented high variability, especially at pH 7.4 and when the current was applied in the anode-to-cathode direction. The delivery of the two inorganic cations was significantly higher at pH 7.0 than at pH 4.0 and supported that nails hold a negative charge at physiological pH. Ions were easily extractable through the nail plate during in vivo iontophoresis and all volunteers' feedbacks supported iontophoresis as an acceptable technique. This thesis demonstrated the feasibility and potential of in vivo transungual iontophoresis.

This thesis investigates the potential for monitoring current and historic blood serum concentrations of amino acids via transdermal extraction using phenylalanine as a case study. This work furthers the field of non-invasive monitoring of amino acid disorders which have several advantages over invasive methods such as blood tests. In this thesis we derive models to simulate blood serum concentrations, the formation of the skin reservoir and, finally, transdermal extraction of amino acids under an applied electric field. Chapter 1 concerns itself with the biological background and sets up motivation of the thesis by discussing amino acids, associated amino acid disorders, the overarching clinical problem, skin structure and transdermal extraction methods. Chapter 2 then considers mathematical techniques utilised throughout the thesis. Chapter 3 formulates a model for the distribution of phenylalanine in blood serum. One compartment and two compartment approaches are considered in both a fasting state and a non-fasting state. We consider if these have a noticeable effect on the blood serum concentration of phenylalanine. Having obtained a model for the distribution of phenylalanine in blood serum, chapter 4 models the formation of reservoirs of amino acids in the skin. Prior work has identified the existence of such a reservoir, but its formation has not been addressed. The models developed consider the effect of the removal of outer layers of skin, the stratum disjunctum, and production of amino acids in the skin. Unknown parameters are estimated by comparing the model to in vivo and in vitro data. Chapter 5 and 6 are concerned with transdermal extraction under an applied electric field. Chapter 5 formulates the velocity induced by applying an electric field across a charged interface. Chapter 6 utilises these results for modelling extraction of compounds through the skin under an applied electric field.

This thesis investigated the possible use of transdermal iontophoresis in paediatric care, as an alternative strategy to the oral and intravenous routes. More specifically, the potential for non-invasive delivery of ranitidine, midazolam, and phenobarbital; and the clinical sampling of iohexol through the skin were examined. The feasibility for monitoring kidney function was assessed in vitro and in vivo using the glomerular filtration rate (GFR) marker, iohexol. Sampling of iohexol in vitro was sensitive to the changes in its subdermal concentration, and pharmacokinetic parameters estimated from skin sampling agreed well with reference subdermal values. Similar observations were confirmed in vivo in a pilot study performed in four children undergoing routine iohexol GFR test. Iontophoresis was well tolerated in all subjects and the marker was successfully extracted through the skin. In 3 of 4 subjects, the elimination rate constant estimated from skin sampling data agreed well with blood sampling results. This study demonstrated the potential of transdermal iontophoresis as a non-invasive sampling approach which could significantly improve the quality-of-life of children. Drug delivery by transdermal iontophoresis was examined in vitro for three commonly used paediatric medicaments: ranitidine, midazolam, and phenobarbital. Experiments used both intact and compromised pig skin to model the less resistant skin of premature babies. Iontophoretic delivery across intact skin was superior than passive delivery and optimised conditions were achieved by use of maximal molar fraction of the drug, higher current intensity, and appropriate vehicle pH. Pluronic® F-127 gels were suitable drug matrices for the iontophoretic delivery of ranitidine. Midazolam and phenobarbital transdermal delivery through partially compromised skin barriers was controlled by iontophoresis. Across highly compromised skin, however, passive diffusion increased drastically and iontophoretic control was lost. Overall, it was possible to deliver therapeutically meaningful fluxes of all three drugs with acceptable patch application area.