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

archives@tulane.edu / Fibrotic diseases comprise up to 45% of deaths in the industrialized world. Few effective anti-fibrotic therapeutics exist, due in part to the lack of human-relevant preclinical models. The goal of this research was to improve the modeling of fibrotic diseases in microphysiological systems (MPS) by engineering quiescence in cultured human fibroblasts prior to MPS incorporation. To create an assay for testing this approach, a versatile organ chip was designed while optimizing workflow for production of the organ chip molds with an SLA 3D printer. After identifying 2D culture conditions that repress fibroblast activation, we tested the hypothesis that the 2D culture protocol would impact the fibrotic baseline in our MPS. 3D confocal microscopy and multi-metric image analysis of immunostaining for cellular and extracellular matrix (ECM) components via intensity and pattern quantification revealed the establishment of more physiological baseline for MPS fibrosis models. To test in a disease-relevant context, we created a model of the stromal reaction in lung cancer using our organ chip and demonstrated that our integrated MPS can be used to quantify the fibrosis-inducing effects of cancer cells that drive stromal reactions. / 1 / Max Wendell

abstract: This dissertation studies two outstanding microscale fluid mechanics problems: 1) mechanisms of gas production from the nanopores of shale; 2) enhanced mass flow rate in steady compressible gas flow through a micro-conduit. The dissertation starts with a study of a volumetric expansion driven drainage flow of a viscous compressible fluid from a small capillary and channel in the low Mach number limit. An analysis based on the linearized compressible Navier-Stokes equations with no-slip condition shows that fluid drainage is controlled by the slow decay of the acoustic wave inside the capillary and the no-slip flow exhibits a slip-like mass flow rate. Numerical simulations are also carried out for drainage from a small capillary to a reservoir or a contraction of finite size. By allowing the density wave to escape the capillary, two wave leakage mechanisms are identified, which are dependent on the capillary length to radius ratio, reservoir size and acoustic Reynolds number. Empirical functions are generated for an effective diffusive coefficient which allows simple calculations of the drainage rate using a diffusion model without the presence of the reservoir or contraction. In the second part of the dissertation, steady viscous compressible flow through a micro-conduit is studied using compressible Navier-Stokes equations with no-slip condition. The mathematical theory of Klainerman and Majda for low Mach number flow is employed to derive asymptotic equations in the limit of small Mach number. The overall flow, a combination of the Hagen-Poiseuille flow and a diffusive velocity shows a slip-like mass flow rate even through the overall velocity satisfies the no-slip condition. The result indicates that the classical formulation includes self-diffusion effect and it embeds the Extended Navier-Stokes equation theory (ENSE) without the need of introducing additional constitutive hypothesis or assuming slip on the boundary. Contrary to most ENSE publications, the predicted mass flow rate is still significantly below the measured data based on an extensive comparison with thirty-five experiments. / Dissertation/Thesis / Doctoral Dissertation Mechanical Engineering 2019

Forensic toxicology is a critical field in which scientific techniques are employed in order to establish the presence or absence of pharmacological substances and/or their metabolites within an individual. The results of such analyses can have legal implications, and toxicology has a number of important applications, including post-mortem investigations, workplace drug testing, therapeutic drug monitoring, and impaired driving studies. The focus of this specific body of work is on the use of toxicology in the detection and quantification of drugs of abuse –specifically opioids - in biological samples. In recent years, there has been a surge in opioid abuse and the need for forensic toxicology labs to process samples from such cases quickly and accurately continues to increase. As a result, it is imperative to research different techniques and technologies that can be applied in toxicology to improve efficiency of sample processing while still remaining sensitive and specific. Many toxicology laboratories today use immunoassay techniques for screening, and utilize a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification. While these methods are established and reliable, the need to analyze an increasing number of samples in a more efficient time frame is essential, and with that, the need to develop and validate new analytical methods. This study sought to validate the use of Microchip Capillary Electrophoresis-Tandem Mass Spectrometry (CE-MS/MS) as a method for detecting and quantifying a panel of fourteen opioids. The experiments were run using a ZipChip (908 Devices, Boston, MA) as the separation scheme, which contains a small capillary where analytes are separated out by electrophoretic mobility - dictated largely by size and charge. These analytes were then ionized by electron spray ionization (ESI) at the end of the chip, and then detected, fragmented, and analyzed in a SCIEX 4500 Triple Quadrupole Mass Spectrometer (Framingham, MA). The analytical run time of the method evaluated was two and half minutes per sample. Calibration curves were run and the method was assessed for a number of validation parameters, including bias, precision, limit of detection, common analyte interferences, matrix interferences, and carryover, as recommended by the American Academy of Forensic Sciences Standards Board. The fourteen drugs and metabolites looked at in this study were 6-monoacetylmorphine, buprenorphine, codeine, dihydrocodeine, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidine (EDDP), fentanyl, heroin, methadone, morphine, naloxone, norfentanyl, oxycodone, oxymorphone, and tramadol. All standards were ordered from Cerilliant (Round Rock, TX), as well as deuterated internal standards used for quantification purposes. This study showed that as the method currently stands, it can reliably detect this panel of opioids at limits of detection between 1 and 15 ng/mL, with the exception of buprenorphine and morphine, for which the method appeared less sensitive. While some applications desire higher sensitivity than this, this level of detection could be very useful as a screening technique that is quick and also far more specific than current immunoassay screening techniques, and provide the additional advantage of quantification for samples at slightly higher concentrations. Quality control samples at 100 ng/mL and 150 ng/mL generally showed consistent results and acceptable levels of bias and precision, indicating that the method can be used to reliably quantify this panel of opioids at those concentrations. In addition, interference signals detected during analysis of other common analytes often encountered with opioids were negligible, with the exception of heroin and norfentanyl. Analysis of ten lots of urine for blank matrix interferences also demonstrated low potential for interference, with the exception of heroin. Finally, there was no evidence of significant carryover between samples, or interference from the deuterated internal standards. While some potential instrumentation issues such as mass spectrometer calibration prompt further study, the method shows promise for future use as a high throughput analysis tool in forensic toxicology labs. CE-MS/MS has the added benefit of not only faster run times, but significantly less sample consumption per run, and additionally, less sample preparation. CE is a viable separation scheme for metabolites and forensic applications, and could make large impacts as an effective way to analyze toxicological samples.

No description available.

This work describes the use of microfluidic technology and biomaterials in cancer research by mimicking the extracellular matrix (ECM) and development of drug delivery system. Initially, biomaterials such as Gelatin methacryloyl (GelMA) and collagen type I were combined to create a hydrogel composite able to mimic both healthy and cancerous ECM. The impact of the tumor microenvironment was analyzed by using the hydrogel inside of a pressurized microfluidic device and by tracking the movement of gold nanoparticles (GNPs). The GNPs showed a decrease in diffusion coefficient of 77% when analyzed in cancerous conditions. This investigation was further explored by analyzing the diffusion of charged GNPs in the same system, while also tracking cellular uptake. An inverse correlation between diffusion and cellular uptake was obtained for charged GNPs in breast cancer cells. Due to the tunable properties and biocompatibility of GelMA, this hydrogel was also employed in the development of pH-responsive drug delivery systems. Since GelMA contains a gelatin backbone, two responsive polymers (Polymers A and B) were synthesized. Microspheres of ~40 μm were fabricated in flow focusing microfluidic devices. Polymer A microspheres displayed a swelling increase of 167% in pH 6.0, while polymer B spheres showed a 296% swelling in pH 10. Considering the unique properties of the tumor microenvironment such as leaky vasculature and acid pH environment, polymer A was selected to be used in the production of nanocarriers. The behavior of this polymer in acidic environment illustrated its potential applicability as drug delivery systems to the tumor area. Polymer A nanogels displayed a uniform size of 74 ± 7 nm. Lastly, GNPs were added to the solution of polymer A, leading to the fabrication of GNPs-loaded nanogels, presenting a homogenous distribution of gold particles inside nanogels. / Graduate / 2023-07-05

The goal of this study is to design a protocol for the adherent cell culture within a novel microfluidic device. Microscale cell culture protocols were developed for loading cells using poly-L-lysine to enhance adherent cell culture of murine derived NIH 3T3 fibroblasts. This work sought to develop a method for adherent microculture by examining various sterilization, surface treatment, and seeding techniques. Using a vacuum suction loading technique, air plasma treatment and a poly-L-lysine surface treatment adherent cell culture was observed within the device. The work presented here is part of a collaborative effort that aims to develop protocols for the electrical and optical characterization of cell culture within a novel microfluidic device.

Building upon previous research on Digital Light Projection (DLP) 3D printing for microfluidics, in this thesis I performed the detailed design and fabrication of a novel DLP 3D printer to increase resolution and device footprint flexibility. This new printer has a pixel resolution twice that of our group’s previous printers (3.8 μm vs 7.6 μm). I demonstrated a new state of the art for minimum channel width, reducing the minimum width to 15 μm wide (and 30 μm tall). This is an improvement over the previous smallest width of 20 μm. This printer also has the capacity to perform multiple spatially distinct exposures per printed layer and stitch them into one interconnected device. Image stitching enables printing devices with identical build areas to previous printers, and with smaller pixel pitch. I pursued validation of this stitching capacity by fabricating channel devices with features crossing the stitched image boundary, with the goal of printing channels that would flow fluid consistently and without leaking. To accomplish this, I began by characterizing the print parameters for successfully printing single microfluidics channels across the stitched image boundary, and then I explored the sensitivity of my method to multiple crossings of the image boundary by printing a stacked serpentine channel that crossed the stitched image boundary 392 times. This demonstrated that an arbitrary number of stitched boundary crossings are feasible and thus a high degree of complex device component integration across these boundaries is also possible. These developments will be useful in future research and design of 3D printed microfluidic devices.

Recent technologies in micro-devices for investigation of functional biology in a controlled microenvironment are continually growing and evolving. In particular, electric-field mediated microfluidic platforms are evolving technologies that have significant applications in drug delivery and cell migration investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge; in recent years, the positive impact of electric fields is being explored. The primary objectives of the dissertation are to design, fabricate, and employ two novel microfluidic platforms for drug delivery and cell migration in the presence of electric fields. Description of iontophoretic carboplatin delivery into the MDA-MB-231 triple-negative breast cancer cells and investigation of neutrophil electro taxis are two main aims of the dissertation. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemotherapeutics for tumor treatment not only because of their non-invasiveness but also due to their lower systematic toxicity compared to other drug delivery systems. While iontophoresis animal models are commonly being used for the development of new cancer therapies, there are some obstacles for precise control of the tumor microenvironment's chemoresistance and scaffold in the animal models. We employed experimental and computational approaches, the iontophoresis-on-chip and the fraction of tumor killed mathematical model, for predicting the outcome of iontophoresis treatment in a controlled microenvironment. Also, precise control over the cell electromigration is a challenging investigation which we will address in the second aim of the dissertation. Here, we developed a microfluidic platform to study the consequences of DC electric fields on neutrophil electromigration (electrotaxis), which has an application of directing neutrophils away from healthy tissue by suppressing the migration of neutrophils toward pro-inflammatory chemoattractant. / Doctor of Philosophy / Recent technologies in the micro-scale medical devices for diagnosis and treatment purposes are continually growing and evolving. Microfluidic platforms are reproducible devices with the dimensions from tens to hundreds of micrometers for manipulating and controlling fluids. In particular, electric-field mediated microfluidic platforms, are developing technologies that have significant applications in drug delivery and biological cell directional movement investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge. In recent years, the positive impact of electric fields is a significant advancement in drug delivery techniques. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemo drugs for tumor treatment not only because of their sensitivity but also to their lower systematic toxicity compared to injection or oral drug delivery. While iontophoresis animal models are conventional for the development of new cancer therapies, there are some obstacles to precise control of the tumor scaffold in the animal models. We also developed a novel microfluidic platform to study the consequences of DC electric fields on white blood cells' (WBC) directional movement, which has an application of directing WBC away from healthy tissue by suppressing the damage of WBC accumulation in healthy organs.

This research presents a novel capacitive-sensing-based method to measure fluid velocity for microfluidics devices. To illustrate the importance of fluid velocity measurement, a case study was first conducted for a split and recombine micromixer. The study underscored the influence of fluid velocity on micromixer efficiency and mixing quality. The proposed fluid velocity measurement method employs two capacitance sensing electrodes placed along the fluid channel, capable of detecting small capacitance changes as fluid passing through the sensing area. The relation between capacitance changes and fluid velocity in the proposed sensing structures was developed and hence used to estimate fluid velocity. The proposed technique does not require extensive bench equipment and is suitable for point-of-care applications. To validate our approach, we implemented a two-step 3D printing process, creating a Polylactic acid (PLA) micro platform with embedded graphene–PLA composite electrodes. The accuracy of the developed method was investigated by cross-verifying the obtained velocities with an optical measurement method. Most absolute percentage discrepancies between the results from the proposed method and the optical method are under 12%, validating the precision of the proposed method. Future research will focus on integrating this velocity measurement method into microfluidic devices produced using advanced microfabrication technologies.