Browsing by Author "Yuan, Fan"

Three-dimensional (3D) tumor models aim to reduce the need for animal models for drug and gene delivery studies. However, many models are not conducive to environmental manipulation and may not be easily adapted for in situ microscopic analysis of transport phenomena. One goal of this study was to develop a 3D tumor model that can mimic 3D cell-cell interactions to mimic native tumor tissues.

To this end, a novel 3D microfluidics-based tumor model was created which allowed the overnight culture of a high density of tumor cells and could be used for small molecule penetration studies. This microfluidic device facilitated the loading of B16.F10 tumor cells in a densely-packed three-dimensional arrangement in a micro-channel which was accessible for nutrient supply via channels on either side through which culture media was continuously infused. Cell volume fraction in the micro-channel was determined via nuclear staining and counting of cells immediately after loading and after a 12-hr culture period. The average volume fraction of cells in this model was 0.32 immediately after loading and 0.26 after 12-hr culture. The values are comparable to cell volume fractions of the in vivo B16.F10 tumor previously measured in our lab. The reduction in cell volume fraction after overnight culture was due to the change in cell morphology to become more elongated after time in culture. Cell-cell adhesions appeared to have formed during culture, resulting in more uniform packing.

Sodium fluorescein dye was used as a drug analog and the extent of penetration of this fluorescent molecule through the cell compartment was assessed through microscopy. The dye was introduced on one side of the cell micro-channel and fluorescence images were captured for generation of concentration profiles in the cell compartment. Results showed that dye penetration through the cell chamber was greatly limited by the presence of the 3D cell culture and a linear concentration profile was achieved across the cell compartment. Also, the concentration of sodium fluorescein in the cell compartment of the 12-hr microfluidic cell culture was appreciably lower than the concentration in the cell compartment when the dye was introduced immediately after loading cells. These results suggest that the proposed tumor model shows significant resistance to dye penetration and could prove to be extremely useful for mimicking tumor tissue resistance to drug penetration via diffusion.

There are many barriers to gene delivery to tumors which highlight the importance of selecting an effective gene carrier system. Some pathogenic bacteria have been investigated as gene delivery vectors because of their innate ability to selectively proliferate in tumor environments. However, pathogenicity concerns arise when trying to achieve therapeutic levels of gene expression. It has been shown that non-pathogenic bacteria such as E. coli can be engineered to invade mammalian cells and participate as gene delivery vehicles. Hence, the second part of this research project involved the use of the newly developed microfluidic 3D tumor model previously described to visualize the transport behavior of invasive (inv+) and non-invasive (inv-) E. coli. The inv+ bacteria harbored a plasmid containing the inv gene encoding the protein invasin that binds to &beta₁ integrin receptors on the surface of mammalian cells resulting in the phagocytosis of invasin-expressing bacteria by normally non-phaogcytotic cells. Two tumor cells lines were used: B16.F10 and EMT6, which have been shown to differ in expression of &beta₁ integrins. The bacteria were also engineered to express mCherry for fluorescent detection.

A suspension of tumor cells and bacteria was loaded into the microfluidic device and cultured for 12 hrs before imaging bacteria distribution throughout the cell culture. Proliferation of inv+ bacteria was generally uniform throughout the cell compartment in the B16.F10 model and bacterial cells were primarily concentrated outside of cells. Bacteria that were internalized did not appear to migrate far from the plasma membrane of the tumor cell. The non-invasive bacteria proliferated to a much greater extent than the invasive form and this proliferation was also generally uniform throughout the cell compartment. Proliferation of both invasive and non-invasive bacteria in the EMT6 model was less uniform than in the B16.F10 model. Overall bacterial concentration appeared to be lower in the EMT6 model. Viability staining after bacterial infection showed that tumor cells in the 3D model were able to maintain viability despite bacterial cell proliferation.

An additional assay was conducted in culture plate wells to determine the effect of chemical factors secreted by tumor cells on bacterial cell proliferation. The results of this assay revealed that tumor cells may be secreting anti-microbial factors that inhibit the proliferation of bacteria and that the binding of invasin-expressing E. coli to tumor cells may further promote the release of these factors.

The results of this study suggest that tumor cell type plays a major role in the distribution and proliferation of bacteria in a 3D environment. The ability to visualize bacterial spread throughout a 3D tumor model will prove to be useful for observing the effect of various genetic modifications on the transport and gene delivery efficiency of E. coli.

Successful transfection of genetically active materials is essential to gene delivery and genome editing. Electrotransfection, also known as electroporation, is a fast, safe, and convenient non-viral method for introducing materials such as proteins and nucleic acids into cells and tissues. It has been widely used in academic research, industrial manufacturing, and clinical therapeutics. Particularly, electrotransfection is one of the most commonly used method in gene delivery into mammalian cells. However, despite its many advantages comparing to other gene delivery methods, the application of electrotransfection is limited by inconsistent transfection efficiency, which is caused by the poor understanding of the mechanism of electrotransfection.

The goal of my research is to understand the fundamental biological mechanisms of electrotransfection and to develop novel strategies that can improve the transfection efficiency of gene delivery and genome editing. To this end, this study is divided into two phases. Phase 1 aims at understanding the key cellular components involved in the transport process. Phase 2 focuses on the development of strategies to enhance electrotransfection by controlling the biological pathways that are involved in electrotransfection.

In the first phase of my study, we investigated the dependence of electrotransfection efficiency on endocytosis. Data from this study demonstrated that macropinocytosis is involved in electrotransfection. The results revealed that electric pulses induced cell membrane ruffling and actin cytoskeleton remodeling. Using fluorescently labeled pDNA and a macropinocytosis marker (i.e., dextran), the study showed that electrotransfected pDNA co-localized with dextran in intracellular vesicles formed from macropinocytosis. Furthermore, electrotransfection efficiency was reduced significantly by lowering temperature or treatment of cells with a pharmacological inhibitor of Rac1 and could be altered by changing Rac1 activity. Taken together, the findings suggested that electrotransfection of pDNA involved Rac1-dependent macropinocytosis.

Second phase of this study focuses on the intracellular transport of plasmid DNA, especially the transport of DNA molecules towards degradative compartments. Our data elucidated that components in both endocytic and autophagic pathways are responsible for intracellular trafficking and processing of transfected materials such as pDNA. In addition, we also characterized a new type of vesicle named amphisome-like vesicle (ALB) and revealed its involvement in electrotransfection. Based on these findings, we propose a novel strategy to enhance electrotransfection by blocking degradative routes within the endocytic pathways, which led to the development of a new technique called transfection by redirection of endocytic and autophagic traffic (TREAT). Transfection of plasmid DNA (pDNA), messenger RNA (mRNA), sleeping beauty transposon system (SB), and different forms of CRISPR/Cas9 system by TREAT achieved superior efficiency in various cell lines including difficult-to-transfect human primary cells. In addition, we successfully applied TREAT method to improve clinically relevant applications including SB-based gene integration and CRISPR/Cas9-based editing of T cell receptor alpha constant (TRAC). In summary, we studied the biological mechanism of electrotransfection and developed a general, flexible, and reliable technique to enable highly efficient non-viral gene delivery and genome editing. Furthermore, the insights gained on the mechanism of electrotransfection provide better understanding of cellular response to exogenous materials. In the future, our study could potentially pave new paths for a wide range of research and therapeutic applications such as CRISPR/Cas9 mediated high-throughput loss-of-function gene screening analysis, correction of disease-related mutations, as well as genetic engineering of immune cells and stem cells for transplantation.

DNA vaccines have emerged as a promising platform for immunization due to their safety, scalability, and potential to induce both humoral and cellular immune responses. However, their efficacy in generating robust and long-lasting protective immunity has often fallen short of expectations. Various strategies, including viral and non-viral delivery methods, new adjuvants, and rational antigen design, are being explored to improve the immunogenicity of DNA vaccines. Despite all these efforts, to address the existing limitations and unlock the full potential of DNA vaccines as powerful tools in preventing infectious diseases and combating emerging pathogens, there is still a pressing need to develop efficient while safe approaches to boost the efficacy of DNA vaccines. In my thesis, I developed two methods to address this problem, with one of them being using sucrose-encapsulated lipid nanoparticles (LNPs) to interfere with the intracellular lysosomal degradation of DNA vaccines, and the other being using DNA cocktails composed of tunable amounts of vaccine DNA and cytokine DNA to controllably recruit and activate the antigen-presenting cells (APCs). Moreover, I also explored a previously undiscovered pathway by which antigens expressed at the vaccination site, such as skeletal muscle, are transported to immune cells residing in the draining lymph nodes. Together, my work demonstrated efficient methods to improve the efficacy of DNA vaccines while maintaining an excellent safety profile, and provided important clues of molecular mechanisms underlying antigen transport, which are critical in guiding the design of next-generation adjuvants for DNA vaccines targeting the antigen packaging and delivery.

Realistic virtual lesion models are valuable in medical imaging applications including phantom design and observer studies. Radiologic diagnostic information rarely include lesion texture due to the fact that texture quantification is sensitive to changing imaging conditions. These effects are not well understood, in part, due to a lack of ground-truth phantoms with realistic textures. Internal tumor heterogeneity in nodules can be predictive of lesion malignancy but is not well understood and virtual lesion models will facilitate research in this area. The purpose of this study was to design and fabricate realistic virtual lung nodules with internal heterogeneity characteristics, and assess the variability as well as determine which imaging conditions provides the most accurate texture features compared to voxel-based 3D printed textured lesions for witch the true texture features are known.

We propose a texture synthesis method that accounts for the effects of the imaging system to mimic the appearance of texture in real nodules. Modulation Transfer Function blurring effects and noise contamination was included in the texture generation based on a 3D-Clustered Lumpy Background (3D-CLB). The governing parameters of the 3D-CLB were optimized using a Generic Algorithm with an objective function of Mahalanobis distance between synthesized textures and real lesion textures features. The resultant texture was objectively and visually similar to real nodules of the same heterogeneity category.

The heterogeneous lesion phantoms were designed with three shapes (spherical, lobulated, spiculated), two textures (homogenous, heterogeneous), and two sizes (diameter < 1.5cm, 1.5cm

Gene delivery has great potential to cure diseases. However, the field is currently limited by the ability to introduce genes into cells safely and efficiently. The two main categories for gene delivery are viral and non-viral methods. Viral methods generally have high efficiency for delivering genes to cells, but are limited by serious health risks and the size of genes that they can deliver. Non-viral vectors can overcome these problems. A non-viral method with great potential is electrotransfection, which is a physical method that involves the application of an electric field to introduce plasmid DNA (pDNA) into cells. The technique of electrotransfection has also been referred to as electroporation, electropermeabilization, electrogene transfer, and gene electroinjection in the literature [1]. Currently, electrotransfection is one of the most widely used physical approaches as it is simple to use, safe, and can result in successful delivery in some difficult-to-transfect cell lines, such as immune cells and stem cells. The goal of my research is to understand the largely unknown mechanisms by which the electric field delivers pDNA through the plasma membrane, cytosol, and nuclear membrane, which are largely unknown at present. With greater understanding on the underlying biological mechanisms of electrotransfection, we have been able to improve its efficiency.

Improvements in gene delivery methods have great potential for increasing the use and implementation of treatments such as gene therapy as well as techniques such as genome editing. Electrotransfection is a technique that has been widely used for gene delivery in both basic research and clinical applications. However, the efficiency of delivery remains low and unstable compared to viral methods. This is mainly because mechanisms of electrotransfection are still largely unknown. The main limitation that prevents wide-spread usage of electrotransfection in clinical practice is its relatively low efficiency; therefore, methods to enhance efficiency have potential for great impact. Prior enhancements in electrotransfection efficiency (eTE) were achieved by optimizing electric field parameters, including electric field strength, pulse number and duration, and pulse shape (square wave versus exponential decay). These methods do not consider the underlying mechanisms. The aims of this investigation are: (1) to understand mechanisms of pDNA endocytosis underlying electrotransfection, (2) to improve efficiency by overcoming physical barriers during intracellular trafficking and upon reaching the nucleus, and (3) to develop techniques that enhance efficiency by combining chemical delivery methods with electrotransfection.

The mechanistic studies focus on intracellular trafficking and transport through the nuclear envelope, as previous studies in our lab and others have examined the role of various endocytic pathways [2-5]. Specifically in relation to electrotransfection, the roles of intracellular trafficking involving endocytosis and the role of the nuclear envelope have been much less studied. To our knowledge, this work is the first examining the role of induction of endosomal escape in relation to electrotransfection. Endosomal escape is commonly believed to enhance chemical delivery methods, but we found it is detrimental when naked pDNA is being delivered. These mechanistic studies also involve studying the manipulation of microtubules to understand how recovery from depolymerization increases eTE by several fold. These are in fact some of the largest increases in eTE that we have observed.

Additionally, some small molecules can be added as a pretreatment or even in the pulsing buffer to enhance eTE in a manner that is simple and efficient for the user. Importantly, these molecules mostly consist of sugars that are already FDA approved and some have been used as vaccine adjuvants. We have begun to study the mechanisms by which these molecules increase eTE, but a greater understanding of the mechanisms and what characteristic of these molecules allows them to enhance delivery will be essential to finding additional molecules that may be similar yet able to increase the efficiency further and/or improve viability. Finally, the in vivo studies will be essential to translating these improvements into clinical trials. Thus, the goal of my research has been two-fold: one is to understand the molecular mechanisms of DNA transport through the plasma membrane, cytosol, and nuclear envelope; and the other is to develop new approaches to improve delivery efficiency based on results from the mechanistic study.

Electric field mediated gene delivery (EFMGD) or electrotransfection is a popular, non-viral gene delivery method that has been used in a variety of studies and applications ranging from basic cell biology research to clinical gene therapy. Yet, the mechanism(s) by which electrotransfection facilitates DNA delivery across the cell membrane into the cell and its subsequent intracellular transport across the cytosolic space towards the nucleus have been insufficiently studied and still remain controversial. Understanding these mechanisms and characterizing the intracellular journey of pDNA is important for understanding the physiological barriers of EFMGD within the cell, which can be used to engineer better solutions to overcome these barriers with the ultimate goal of improving the transfection efficiency of this technology.

Conventional thought in the field assumes that such transport modes as diffusion, electrophoresis, and electro-osmosis, which govern the entry of small molecules into cells through electric field-generated transient membrane pores, also apply to electric field-mediated delivery of therapeutic DNA. We propose that electrically-induced gene transfer into cells is governed by an alternative, more active mode of transport that entails the involvement of cellular endocytic processes. It is our hypothesis that pulsed electric field generate these membrane pores which interact with nearby DNA molecules; but that actual DNA translocation across the membrane is driven by endocytosis, which consequently, then, also plays a role in the intracellular transport of the DNA. To this end, we first investigated the dependence of electrotransfection efficiency (eTE) on binding of plasmid DNA (pDNA) to plasma membrane. Binding concentrates DNA molecules in the vicinity of the cell membrane, which should theoretically result in a greater number of DNA-membrane interactions during pulsed electric field, more internalized DNA, and ultimately, higher eTE values. We demonstrated that supplementing the electrotransfection buffer with divalent cations (Ca2+ and Mg2+) is an effective method of promoting pDNA adsorption to the cell membrane. This cation-mediated increase in DNA adsorption to the cellular membrane resulted in a consequent increase in eTE, up to a certain threshold concentration for each cation. To determine the timeframe for completion of pDNA internalization following pulse treatment, trypsin treatment was applied to cells at different timepoints after electrotransfection to strip off any residual, membrane-bound pDNA that had not been internalized. Trypsin treatment at 10 min post electrotransfection still resulted in a significant reduction in eTE, indicating that the time period for complete cellular uptake far exceeded the lifetime (~ 10 msec) of electric field-induced transient pores. The role of endocytosis was further probed by noting the effect on eTE when cells were treated with three endocytic inhibitors (chlorpromazine, genistein, dynasore) targeting different internalization mechanisms or silenced of dynamin expression using specific, small interfering RNA (siRNA). siRNA silencing and all three pharmacological inhibitors yielded substantial and statistically significant reductions in the eTE. Taken together, these findings suggest that the mechanism of electric-field mediated DNA internalization entails: (i) binding of pDNA to cell membrane and (ii) endocytosis of membrane-bound pDNA.

The same strategies of pharmacological endocytic inhibition and siRNA silencing was used to further explore and compare electric field-induced pDNA internalization in additional cell lines that differ in terms of cell type, proliferation rates, proliferative capacity (i.e. primary versus immortalized/cancer line), etc. in order to determine whether endocytosis is a universally implicated mechanism across many cell lines. Results showed different endocytic pathways to be recruited for pDNA uptake in a cell-dependent manner and that one or multiple pathways may contribute to uptake within a cell line.

Taken together, the studies presented in this dissertation provide both indirect and direct evidence suggesting an endocytic role in the translocation of pDNA across the cell membrane and its intracellular routing towards the nucleus for EFMGD. These seminal findings could potentially lead to better understanding of the intracellular barriers encountered by EFMGD, more strategic optimization of electrotransfection parameters than the trial-and-error approach currently used, and enhanced transfection efficiencies.

A three-dimensional tissue culture device previously described by Elliott et al. 1 is tested for its capacity to mimic cancer interstitial fluid pressure and interstitial pH. The device described by Elliott et al. is a three-channel system; a central channel that contains cells, and two side channels that act as model capillaries. The potential for variation of the interstitial fluid pressure was determined by measuring the resistances with respect to the various channels. It was determined that the unmodified device is incapable of mimicking physiological tumor pressures. The ability to vary pH was performed by using a pH sensitive florescent and by infusing a pH 5.9 solution into the side channels, before checking if the device could maintain this pressure for a long time period during constant perfusion of media into the device. It was observed that the florescence of the pH sensitive molecule decreased upon infusion of an acidic solution, as it should upon exposure to acid. However, no conclusions can be drawn from these tests, as the florescent molecule was no longer viable after the 12 hour perfusion of media. Further experiments are needed before any conclusions can be reached about the device's potential.

Promising applications in the design of various biological systems hold critical implications as heralded in the rising field of synthetic biology. But, to achieve these goals, the ability to synthesize and screen in situ DNA constructs of any size or sequence rapidly, accurately and economically is crucial. Today, the process of DNA oligonucleotide synthesis has been automated but the overall development of gene and genome synthesis and error correction technology has far lagged behind that of gene and genome sequencing. What even lagged behind is the capability of screening a large population of information on a single cell, protein or gene level. Compartmentalization of single cells in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with quantitative readouts. However these single-emulsion droplets are incompatible with aqueous phase analysis and are not controllable through molecule transports.

This thesis presents the development of a multi-tool ensemble platform targeted at high-throughput gene synthesis, error correction and screening. An inkjet oligonucleotide synthesizer is constructed to synthesize oligonucleotides as sub-arrays onto patterned and functionalized thermoplastic microchips. The arrays are married to microfluidic wells that provide a chamber to for enzymatic amplification and assembly of the DNA from the microarrays into a larger construct. Harvested product is then amplified off-chip and error corrected using a mismatch endonuclease-based reaction. Bacterial cells baring individual synthetic gene variants are encapsulated as single cells into double-emulsion droplets where cell populations are enriched by up to 1000 times within several hours of proliferation. Permeation of Isopropyl-D-1-thiogalactopyranoside (IPTG) molecules from the external solution allows induction of target gene expression. The induced expression of the synthetic fluorescent proteins from at least ~100 bacteria per droplet generates clearly distinguishable fluorescent signals that enable droplets sorting through fluorescence-activated cell sorting (FACS) technique. The integration of oligo synthesis and gene assembly on the same microchip facilitates automation and miniaturization, which leads to cost reduction and increases in throughput. The capacity of double emulsion system (millions discrete compartments in 1ml solution) combined with high-throughput sorting by FACS provide the basis for screening complex gene libraries for different functionality and activity, significantly reducing the cost and turn-around time.

The trabecular meshwork (TM) is comparable to a bioactive filter that plays a major role in regulating outflow of aqueous humor of the eye and setting intraocular pressure (IOP). TM dysfunction may lead to ocular hypertension which is the major risk factor in glaucoma. Although the outflow properties of the TM have been assessed over the last sixty years, very little work has been done assessing its mechanical properties. Therefore, the major goals of these studies were two-fold: (1) to determine the relationship between mechanical properties of TM, specifically the bulk Young's modulus, and outflow function in normal and glaucomatous eyes, and (2) to establish a method and possible animal model for future testing of this relationship.

Outflow function was assessed by constant pressure perfusion in enucleated eyes at four pressure levels (10, 20, 30, and 40 mmHg) to determine outflow facilities and variability in outflow resistance with pressure elevation. A micro-strain analyzer (MSA) was used to determine the circumferential bulk Young's modulus of the TM post-perfusion. Based on their relative ease of availability, pigs and rats were explored as possible animal models. Due to the small size of rat eyes, atomic force microscopy (AFM) was used to assess the Young's modulus of TM rather than with a MSA.

We found that there was a relationship with better outflow function and a stiffer TM in normal eyes. Additionally, glaucomatous TM was found to be much softer and more variable than normal TM. Unfortunately, porcine TM did not serve as a good model for the bulk Young's modulus of human TM, presumably due to anatomical difference in its outflow pathway. Lastly, we were able to establish a new method for measuring the Young's modulus of rat TM for future work to determine potential mechanism for evaluating stiffness changes that may be associated with glaucoma.