Targeted delivery of functional nanoparticles (NPs) holds tremendous potential in diagnostics and therapeutics of vascular diseases and cancer. One of the major attributes is their ability of forming multiple bonds with target cells, thereby providing the flexibility for engineering desired adhesion strength. However, the multivalent binding of nanoparticles is still poorly understood, particularly from a dynamic perspective.

In this work, we first developed Nano adhesive dynamics (NAD) simulation to investigate the binding dynamics between an antibody-conjugated, 210-nm-diameter sphere and an ICAM-1-coated surface. In the NAD simulation, the particle motions are determined using the Langevin equation and the ICAM-1-Antibody bond association and dissociation are computed using Bell's law. The NAD simulation replicated the time-decay in NP detachment rate as found in previous flow chamber assays. We found that the decreased detachment rate resulted from the heterogeneity of NPs in bonding ability, which is inherent in NP multivalence. NPs detached preferentially from those unable to quickly accumulate bonds. This could serve as a selection mechanism to eliminate NPs confined to a lower bonding ability. In addition, we found that NP detachment was driven by mechanical forces due to NP thermal motions.

In order to understand the influence of NP bonding heterogeneity on adhesion dynamics, we further examined NP detachment behaviors at higher resolutions: at sub-population level, in which we grouped NPs with similar bonding ability as in one bond potential (BP) category; and further at bond level, in which we grouped NPs with the same bond number at a time point as in one bond state. At sub-population level, we found that the whole population detachment curve from NAD simulation data could be recovered via summing up fitted detachment curves of each BP category, fitted using mixture exponential distribution with two components. At bond level, we modeled NP transition between different bond states as a birth and death process (BD process), and the transition rate matrix of BD process was parameterized from NAD simulation data. Thereby we extracted detachment rates from the transition rate matrix using first passage times to reach the detached state. Combining different levels of analysis, we were able to provide a comprehensive study on multivalent nanoparticle adhesion dynamics.

Development of a modular Nanocarrier that contains multiple Fluorescent protein and receptor modification options would allow for optimal imaging and binding conditions for therapeutic studies. In order to achieve this, protein production of these custom plasmids need to be optimized for protein yield and accuracy. Using plasmid cloning of pRS 314 and pGEM 3z plasmid vectors, a library of mCherry, mRFP, eGFP and moxGFP fluorescent tagged plasmids are produced through yeast BJ5464 and E.coli BL21 DE3 expression systems. Comparisons between both expression systems are drawn based on fluorescence detected by Nanodrop, and Western blotting to determine yield and accuracy.

Aside from fluorescent modifications, peptides that unwind under force are explored to reduce mechanical forces on bonds, thus stabilizing adhesion of nanocarriers. These peptides are referred to as springy linkers. The second part of this study includes additional cloning of ggs50, flagelliform 50, HP35 and HP35st variants of springy linkers into the pRS vector through plasmid cloning and transformation. Springy Linker plasmids are also expressed to compare protein yield across plasmids containing Springy Linkers and plasmids without linkers.

Subsequent attempts at protein production of these newly cloned vectors indicate that cloning of all fluorescent protein variants in both pGEM and pRS were successful and protein production of a sample of both plasmid types in E.coli and yeast yielded protein of correct molecular weight, with E.coli producing a more robust band under TMB colorimetric detection. Production of plasmids containing Springy linker variants produced a lower protein yield when compared to plasmids without springy linkers under identical expression conditions. Instances of non-specific protein production were also observed in Springy Linker modified plasmids.

Despite production of protein of desired size, protein fluorescence was not detected without mechanical excitation, thus presenting a need to investigate protein folding and aggregation to restore expected fluorescent attributes of our protein.

Circulating tumor cells (CTCs) are cells that shed into the vasculature from a primary tumor and circulate in the bloodstream. CTCs can be used to elucidate the molecular characterization of the tumor cells and to gauge the efficiency of therapeutic treatment in metastatic carcinoma patients. They can also be used to determine the primary site of the tumor in areas where the tumor is undetectable with traditional oncological imaging. The detection of CTCs has a substantial value for prognostic and therapeutic implications, but they are not easily detected because of their low cell count. Because microfluidic devices are useful for cell detection and diagnosis, can be easily obtained, and are less invasive than tissue biopsies, we have developed a microfluidic platform to capture CTCs using multiple capture targets to achieve a higher cell capture. We can selectively isolate the cancer cells using specific antibodies to the antigen capture target on the surface of malignant cells. The capture efficiency was evaluated by the flow rate, cell count, and antibody immobilization. Cancer cell lines that were known to have high expression for targeted ligands, specifically HER2, EGFR, EpCAM, and MUC-1, were tested with antibodies specific to these ligands. We obtained capture efficiency with these different capture targets on a single channel. This allowed us to develop a device with four parallel capture channels to run in series with the anticipation of achieving higher cell capture.

Earlier cancer detection and diagnosis is essential to prevent cancer mortality in nanomedicine and nanotechnology. Fluorescence and magnetic signals provide a way for earlier detection through imaging systems. Magnetic iron oxide nanoparticles have a superparamagnetism feature that allows them to act as contrast agents that can be detected through a magnetic resonance imaging system. These iron oxide cores have a polymer coating around them to provide stability, prevent aggregation, and allow for biocompatibility within the body. In addition, these functional coatings can have ligands and peptides for detection and therapy purposes. One functional coating is a polydiacetylene coating due to its chromatic and optical properties. When polymerized, it has the ability to change color in the visible spectrum to blue (not a fluorescent signal) and when heated, it changes to a red color (fluorescent signal). This way a strong and stable layer is formed around the iron oxide cores. These coatings are placed on the iron cores using a modified dual solvent exchange method, in which DMSO is slowly replaced by water without the use of organic solvents previous used. In addition, these nanoparticles can then be PEGylated, which provides a more stable and water soluble compound in aqueous solutions. Measurements can be taken through dynamic light scattering for size distributions and zeta potential and the Nanodrop for absorbance. Ideal sizes are about 30 nm for MNPs. Moreover, for future directions, there can be more molecules attached to the coated layers to use for molecular detection and analysis.

The focus of this paper is optimizing multivalent bond potential between antibody-coated nanoparticles to their respective receptor sites to create a nanoparticle diagnostic/drug/gene delivery system with high targeting efficacy. In recent work with Haun lab, a computational simulation of nanoparticle binding—formally the Nano Adhesive Dynamics (NAD)—was developed to simulate multivalent binding behavior between a nanoparticle decorated with antibodies and a planar surface covered in receptor targets. One of the main findings was that nanoparticles induced large forces on bonds which caused rupture. Rupture was primarily attributed to Brownian motion. Nanospring linker flagelliform—derived from spider silk—and biosensors HP35 and HP35st were chosen for study. HP35 and HP35st were successfully cloned and inserted into a pRS expression vector developed in previous work.

The development of nanobody research holds the potential to revolutionize targeted therapies. This study focuses on nanoparticles functionalized with recombinant Anti-HER2 nanobodies to specifically bind to cancer cells that overexpress HER2 receptors. Utilizing the high affinity and specificity of nanobodies, these nanoparticles are optimized to serve as precise tools for recognizing and adhering to cancer cells. This method not only facilitates the direct delivery of drugs to tumor sites but also minimizes damage to healthy tissues. This method involves constructing recombinant proteins with the 2Rb17c HER2 nanobody in the pRS-4420 vector and expressing the proteins in Saccharomyces cerevisiae. The recombinant nanobody proteins were successfully conjugated to nanoparticles. This study demonstrates the potential for future applications in targeting HER2-positive cells, offering promising prospects for enhanced binding efficacy in targeted therapies.

ABSTRACT OF THE THESISTitle By Alyssia Fossorier Master of Science in Biomedical Engineering University of California, Irvine, 2024 Professor Jered B. Haun, Chair

Cancer, particularly breast cancer, poses a significant global health challenge, affecting millions of women annually and remaining a leading cause of death. Early detection is crucial for improving outcomes, yet many cases go undiagnosed until advanced stages highlighting the need for accurate diagnostics. This thesis explores optical imaging and fluorescence lifetime imaging microscopy (FLIM) to enhance early detection in breast cancer.Two key aspects of fluorescence imaging are focused on: enhancing the stability of fluorescent dyes, specifically quantum dots, through silica coating, and analyzing fluorescence lifetime using the phasor approach. Silica coating improves the stability and biocompatibility of quantum dots by acting as a protective barrier preserving their optical properties and preventing aggregation. The phasor approach in FLIM is used to analyze the behavior of fluorescent dyes, revealing distinct fluorophore positions on the phasor plot and providing insights into fluorescence decay and self-quenching. Overall, the benefits of silica coating for dye stability and the insights gained through phasor plot analysis in FLIM can advance fluorescence imaging techniques. Future work should focus on applying these techniques to improve diagnostic accuracy in breast cancer analysis.

Protoplasts are a prominent cell type for conducting gene transfection in the agricultural space in order to produce modified crops that can mature quicker or survive droughts. Traditional methods for isolation of protoplasts involve manually preparing leaves that are digested in an enzymatic solution containing cellulase and macerozyme for as long as six to eight hours. Understanding current techniques for isolation of protoplasts demonstrated there is prevailing need for a platform that can streamline this process, with quicker isolation times and less user interaction. Our lab has previously performed mammalian tissue digestion to isolate single cells with a microfluidic platform that utilizes mechanical shear forces in combination with enzymatic digestion. Initial tests were performed with the previously developed digestion device on tomato leaves, before being redesigned to suit leaf digestion by optimizing channel design and chamber holding volume. Tomato plants were used as our model because of their quick growth cycles, abundance and previous literature research. Multiple flow rates were tested to determine the ideal conditions for obtaining the highest yield and viability of protoplasts, through Trypan Blue staining. Protoplast yield and viability showed an increase over the digestion period, but a decrease with higher flow rates through the digestion device. Under optimal conditions the device yielded double the number of protoplasts in 2/3 the time compared to the traditional method, while maintaining equivalent viability. In order to streamline the preparation steps, a second preparation device was developed to prepare leaf samples in a quicker manner than the traditional method. The preparation device was then tested in conjunction with the digestion device. Cell yield and viability from using the leaf preparation device were shown to be equivalent to traditional preparation method with Trypan Blue staining. Cells were then cultured and tagged with calcein blue and calcofluor white, to demonstrate continued viability and cell wall formation.

Many studies have been done on different approaches for cancer therapy and one of the most promising fields is using nanoparticles as a drug delivery cancer therapeutic agent. The unique optical properties and high surface volume ratio of nanoparticles can improve the drug delivery system for not only cancer but other diseases. Moreover, nanoparticles can be attached to small proteins which provide the ability to target specific areas or inflamed tissues to minimize the death of healthy cells and tissues. Fusing the protein with fluorescent proteins also enables us to know the localization while targeting cells. Engineering a platform design for nanoparticles with adherent small proteins together with adding specific fluorescent proteins of interest on nanoparticles remains challenging. To achieve the goals, two different expression systems: E. coli and yeast protein expression system are being tested in this thesis. The E. coli expression system is known to have higher protein yield but with protein folding problems while the yeast expression system has lower yields but no protein folding problems which is easier for downstream applications. Therefore, the first part of this thesis is aiming to fuse different fluorescent proteins to an existing recombinant sequence which is construct in our lab and optimize the protein expression via E. coli and yeast expression system. The second part of the thesis is performing the protein yield testing of plasmids contains enzymatic tags. To perform the enzymatic test of our plasmid, we need to be sure that the enzymatic tags fused to our plasmid does not interact with the protein of interest which causes the protein yield to drop. Once we make sure that the enzymatic tags are not interfering the protein yield, enzymatic tests can be performed to know more about the enzyme activity our protein of interest and enzymes. Three different enzyme/tag systems are planned to be tested, Sfp synthase/S6 tag, and Sortase A/LPETG tag, and Lipoic Acid Ligase (LplA)/LAP2 tag. Different colors of fluorescent protein in this construct will create a library which enable us to have more options to choose while doing imaging and targeting in the future. It also creates a possibility to image multiple fluorescence simultaneously.

Biomarkers are intended to provide critical information to characterize the type/severity of the cancer, with the ultimate goal of providing information about treatment. However, their use in diagnostic applications is limited because they require the expensive and time consuming use of molecular probes. By using a technique that is label free and requires a simple and inexpensive microfluidic chip, we can capitalize on acute phenotypic differences that result from overexpression of glycoproteins that change the surface rheology of cells. Previous studies have shown that metastatic tumors overexpress bulky glycoproteins such as MUC1 which contributes to cancerous cell growth, adhesion, and survival. In our study, we quantified MUC1 expression in various cancer cell lines using flow cytometry and correlated this expression with the ability of cells to deform under shear forces during deformability cytometry experiments. Comparing the deformability data of cells from the same cell lines but with different levels of MUC1 expression provided compelling evidence that surface rheology effects can be captured using this technique. It has also been shown that the nucleus plays an important role in determining cell deformability vis-a-vis internal elasticity. Therefore, we also measured the nucleus size of these cell lines with fluorescence microscopy and compared the area of the nucleus to the area of the cytoplasm. Comparing the ratio of these two measurements in each cell line with the deformability values confirmed that nucleus size is indeed the key factor determining cell elasticity and must be accounted for when considering deformability and MUC1 expression.