The drug development process is notoriously long and expensive. During preclinical studies, inaccurate prediction of pharmacokinetic properties such as the ability of a drug candidate to passively permeate cell plasma membranes contributes to the high failure rate of drug candidates during clinical trials. Passive drug permeability is currently predicted using in vitro techniques such as parallel artificial membrane permeability assays, or PAMPA. In PAMPA, drug transport is predicted between aqueous compartments via a synthetic filter filled with a phospholipid solution in an organic solvent. The lack of translatability of preclinical predictions to humans can be attributed, in part, to lack of biological similarly between models used for permeability prediction and cell plasma membranes in vivo. Here, I demonstrate a new method for pharmacokinetic prediction, built by using droplet interface bilayers (DIBs) as human-mimetic artificial cell membranes. DIBs are bilayer sections created at the interface of two aqueous droplets. In the literature, DIBs have been used as artificial cell plasma membranes to study, for example, electrophysiological properties, protein insertion, water permeability, and molecular transport. DIBs can be formed between droplets of differing composition such that one droplet can be used as a donor compartment and the other as an acceptor compartment for the quantification of molecular transport across the artificial cell membrane. DIBs have previously been used to measure the passive permeability of numerous fluorophores as well as the drugs caffeine and doxorubicin. However, the extent to which DIBs have been tuned to mimic human cell plasma membranes and transport across them is limited. I present here the use of microfluidic platforms for bespoke DIB formation, where variables such as temperature, bilayer composition, and droplet contents are customized to create biomimetic cells-on-a-chip. These artificial cells are then used to measure molecular transport with the aim of predicting permeability. In Chapter 2, I investigate the effectiveness of literature methods for the modification of polydimethylsiloxane (PDMS) microfluidic device channels for aqueous droplet formation and storage. While numerous techniques have been presented as mitigation strategies for common challenges in droplet microfluidics, it is not clear from the literature if any of these methods would be effective or necessary for the formation and analysis of DIBs. With the aim of facilitating aqueous droplet formation, I tested the effect of PDMS silanization using trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOS) on surface hydrophobicity and oleophobicity. To assess their effect on reducing the rate of aqueous droplet evaporation, I tested surface treatment of PDMS with Teflon AF or Aquapel. I also tested modifications to the device fabrication process by bonding a glass coverslip to the surface of the device and soaking the device in water overnight. To quantify changes in PDMS surface chemistry, I performed contact angle measurements, aqueous droplet formation experiments, and measurements of droplet size during on-chip storage. I determined that baking PDMS microfluidic devices at 65 C overnight produced channel surfaces which allowed for aqueous droplet formation and storage. In Chapter 3 I present a systematic study on the role of temperature in DIB formation using naturally derived phospholipids. The use of increased temperature to form DIBs using total lipid extracts has previously been demonstrated, but has never before been investigated systematically using naturally derived phospholipids and bespoke formulations thereof. I hypothesized that, in order to form complete phospholipid monolayers and DIBs, the microfluidic device must be held at the phase transition temperature of the phospholipids. Using a custom-built heating platform, I tested DIB formation over a range of temperatures to determine conditions which allowed DIB formation rather than droplet coalescence. I show that temperature is a key parameter for DIB formation using naturally derived phospholipids in a microfluidic device. In Chapter 4, I demonstrate the use of DIBs as a new type of pharmacokinetic compartment model for intestinal absorption. Using three-droplet networks, the components of which were designed to mimic the intestinal space, the enterocyte cytosol, and the blood, I measured fluorescein permeability across intestine-mimetic DIBs. The model was able to predict the transport of fluorescein more accurately than the current state-of-the-art technique, PAMPA. Chapter 5 describes the development of complex DIB models for pharmacologically relevant membranes as well as an investigation into novel methods of drug transport detection on-chip. I created a new DIB model for the small intestine, incorporating more components of the enterocyte plasma membrane such as cholesterol. Measurement of calcein permeability served as a control experiment, as calcein does not cross cell plasma membranes. Measurement of fluorescein permeability yielded a significantly shorter permeation half-life than was determined in Chapter 4, indicating an increase in permeability with the more complex, biomimetic phospholipid formulation. I also developed sex-specific models for intestinal absorption to investigate the effect of sex-based membrane differences on permeability. This relationship has never before been explored in the literature. In comparison to the initial intestinal phospholipid formulation, the sex-specific formulations contained acyl chain tail groups which have been found in different ratios in male and female cells. A significantly longer half-life for fluorescein permeability was found in female intestine-mimetic DIBs, mirroring the slower drug absorption observed in female patients. I also used DIBs to model blood-brain barrier permeability. I demonstrate this application using two different brain lipid extracts, polar and total brain lipids. Polar brain lipids have previously been used in PAMPA to predict blood-brain barrier permeability, but have been found to overpredict the permeation of charged molecules in comparison to custom lipid formulations which mimic the composition of human brain endothelial cells. Permeability measurements in DIBs formed using polar brain lipids gave results which agree with PAMPA, as DIBs formed using polar brain lipids were permeable to fluorescein, but those formed using total brain lipids were not. Blood-brain barrier-mimetic DIBs formed using either lipid extract are impermeable to calcein and FITC-dextrans (both 40 and 500 kDa). I also show the formation of the first DIBs to be created using a total lipid extract from human cells as well as their impermeability to calcein. The extract tested was prepared from testicular Sertoli cells, which exhibit properties similar to the blood-brain barrier, but future work will focus on extracts prepared from human brain endothelial cells. Finally, I explore new options for the on-chip detection of the transport of nonfluorescent molecules. To move away from reliance on fluorescent molecules for permeability measurements, I selected three fluorogenic molecular recognition agents (fluorescamine, Chromeo P540, and DimerDye 4) whose fluorescence signal is activated by amine groups. None of the tested methods proved to be viable in DIBs, potentially due to slow permeation, low quantum yield, and side reactions with phospholipids. Overall, I demonstrate here the microfluidic formation and application of several novel types of biomimetic DIBs to permeability prediction. My work shows that DIBs can be used to predict permeability and mirror effects observed in vivo. Future work will focus on the development of new methods for the detection of drug transport and the application of the pharmacokinetic compartment models presented to predicting drug permeability. Further work using total lipid extracts prepared from human cells will also be vital to enhancing the use of biomimetic DIBs as pharmacokinetic permeability prediction tools. As their biological similarity and capacity to accurately predict transport increase, so will the potential of DIBs to improve the accuracy and translatablity of preclinical drug development. / Graduate / 2023-08-09

Detta projekt drevs på uppdrag av Optikbutiken som behövde en tjänst där mankunde betala sin order med hjälp av kreditkort på deras aktuella hemsida. Det härprojektet gick ut på att integrera DIBS vilket är ett betalsystem för kreditkort.Utvecklingen skedde i Visual Studio miljö. De krav som ställdes upp för det härprojektet blev uppfyllda.

Virginia Axline developed a new field of child psychotherapy by applying a nondirective approach to the burgeoning experimentation of utilizing play in therapeutic work with children. While much biographical information is available regarding other leaders in the fields of counseling and psychology, historical research into Axline and her development of child-centered play therapy represent a gap in the literature. The purpose of the current study was to: 1) examine the professional contributions of Virginia Axline; 2) gather personal information regarding Axline that contributes to deeper understanding of her theory; and 3) identify life circumstances or events that influenced Axline's professional contributions. Historical methodology was utilized to locate and examine artifacts and materials necessary to create an interpretive biography of Axline's life and work, with a focus on her professional influences, experiences, and contributions. Historical methods utilized include historiography, oral history, and interpretive biography, with an emphasis on established and accepted source criticism and data synthesis processes. The research yielded a number of historically significant and previously unknown documents valuable to the field of CCPT including personal correspondence, academic writings, and interviews, as well as academic and government records. The research also established new information about and understandings of several of Axline's professional relationships. The research also calls into question the original authorship of scholarly contributions in the field of counseling for which Axline may have deserved, but not received credit. Included in the biography is information related to Axline's early life, higher education, career timeline, professional development, mentoring relationships, research interests, student perceptions, collegial relationships, personal hardships, professional interests and advocacy, teaching and learning methods, and her decline in later life.