The long-term goal is to use nanotechnology to regulate and control cellular signaling processes and behaviors. This thesis focuses on applications of nanotechnology to cellular biophysics research, i.e., developing new nanotechnology based methodologies and approaches to further our understanding of single cell biophysics. Specifically, the thesis tackles two specific problems: (a) developing a single cell mechanics based approach to regulate and control cellular motility; and (b) investigations of silver nanowire (AgNW) interactions with macrophages. Cell motility plays critical roles in many biophysical and physiological processes ranging from in vitro biomechanics, wound healing to cancer metastasis. This thesis introduces a new means to trigger and regulate motility individually using transient mechanical stimulus applied to designated cells. Using BV2 microglial cells, our investigations indicate that motility can be initiated, reproducibly and reliably, using mechanical compression of the cells. The impact of location and magnitude of the applied force on the movement of the cell is also discussed. A model to describe the process of mechano-induced cell motility was created, which involves high degrees of myosin activation to repair actin cortex breakages induced by the initial mechanical compression, leading to focal adhesion degradation, lamellipodia detachment, and finally cell polarization and movement. This approach is of generic importance to other cell types beyond BV2 cells, and has intrinsic advantages of being transient, non-toxic, and non-destructive, thus exhibits high translational potentials including mechano-based therapy. The interactions of silver nanowires (AgNWs) with cells are important with respect to their effects on the cells and the overall health of human and living entitles. With AgNW being used in a variety of commercial and research application, the potential for exposure is high, with inhalation exposure being a primary concern. Alveolar macrophages in lungs are among the first encounters with AgNWs, upon inhalation. These macrophage cells are responsible for clearing away foreign material. Thus, the interactions between AgNW and alveolar macrophages are of high importance. NR8383 cell line provides a good model for alveolar macrophages, and was used to study AgNW interactions in this thesis. Using laser scanning confocal microscopy (LSCM) combined with single cell compression, and AFM-based technique to probe the mechanical properties of cells, a number of AgNW-cell interactions were captured in live cells. Among them is frustrated phagocytosis. Previously known for microfibers and carbon nanotubes, first direct observations of frustrated phagocytosis of silver nanowires (AgNWs) among living cells in situ are reported by us and summarized in Chapter 4. Confocal imaging of AgNW frustrated phagocytosis revealed actin participation at the entry sites, whose behavior differ from microwire-induced frustrated phagocytosis. Combining confocal imaging with SCC, the impacts of frustrated phagocytosis on the cellular membrane and cytoskeleton were also measured. Using high resolution laser scanning confocal microscopy, the precise locations of AgNWs can be visualized upon their interactions with a macrophage cell line, NR8383 cells. Combined with membrane and actin tags, this investigation allows direct visualization of AgNWs-interactions. In addition to frustrated phagocytosis, direct piercing of AgNWs into cells were captured, where a portion of wires poked into a cell without significant structural perturbation of its local surroundings. Other situations include failed attempts by AgNWs to pierce due to membrane wrapping of the invading portion of the AgNWs to resist their entry. This study provides a comprehensive view of AgNW-cell interactions. The results benefit the understanding of potential adversary effects of AgNWs to health, and guide the development of preventative measures.

This thesis covers systemic nanomanufacturing efforts aimed at fabricating nano- and micro-structures containing lipids. In Chapter 3, a novel method for the controlled assembly of lipids is presented. The controlled assembly method utilizes an Atomic Force Microscopy (AFM)-based microfluidics system and relies on ultrafast evaporation rate and spatial confinement by the sub-femtoliter droplets. This enables the formation of lipid structures that deviate from the near-equilibrium state, such as ordered bilayers. In Chapter 4, AFM in conjunction with microfluidics delivery was utilized to produce three-dimensional (3D) lipid structures following a custom design. A variety of lipid 3D structures were successfully printed, including nanometer-thick disks, long linear spherical caps, three-layered stacking grids, and organizational chiral structures. Our high-resolution structural characterization reveals the spatial precision of the 3D nanoprinting in nanometers over the range of 0.5 mm. These results collectively demonstrate the promising potential of our designed technology and methodology for producing 3D structures ranging from the nanometer to continuum scale. In Chapter 5, the feasibility of one important application is demonstrated, i.e., the rehydration of the lipid constructs to form liposomes. While the thin film rehydration method has traditionally been used for liposome production, it typically requires post-rehydration handling for size and location selection. In contrast, this thesis work reports “in-situ” liposome production through the rehydration of lipid nanostructures, with demonstrated size control by varying the size of the lipid constructs. This investigation of the thesis work explores and demonstrates the possibility of programmable synthesis of biomimetic systems, including lipid bilayers, lipid nanoconstructs, and liposomes, all of which hold significant potential in material science, biomedical technology, and fundamental bio-research.

The focus of my thesis is the development of an advanced methodology to create 3D nanostructures by design, and to demonstrate the control over geometry and chemical functionalities of the nanostructures produced. The driving motive behind this are the pressing need for 3D nanostructures in biomaterials development, modern nanodevices and biomedical applications. My approach is scanning probe microscopy-based nanolithography in combination with advanced surface chemistry. This thesis clearly demonstrated the concept and feasibility of the approach. While the power of 3D printing has proven to be a powerful tool in additive manufacture, extending the spatial precision to nanometer scale would lay the foundation for the next science and technology revolutions. Specific applications impacted by this work include surface science, catalysis, modern sensors, nanophotonics, and nanoelectronics.

Three significant goalposts are reported in this thesis followed by future prospective research. First focuses on mechanically sensitive molecules known as mechanophores, which have recently attracted much interest due to the need for mechanoresponsive materials. Maleimide−anthracene mechanophores located at the interface between poly- (glycidyl methacrylate) (PGMA) polymer brushes and Si wafer surfaces were activated locally using atomic force microscopy (AFM) probes to deliver mechanical stimulation. Each individual maleimide−anthracene mechanophore exhibits binary behavior: undergoing a retro-[4 + 2] cycloaddition reaction under high load to form a surface-bound anthracene moiety and free PGMA or remaining unchanged if the load falls below the activation threshold. In the context of nanolithography, this behavior allows the high spatial selectivity required for the design and production of complex and hierarchical patterns with nanometer precision. The high spatial precision and control reported in this work brings us closer to molecular level programming of surface chemistry, with promising applications such as 3D nanoprinting, production of coatings, and composite materials that require nanopatterning or texture control as well as nanodevices and sensors for measuring mechanical stress and damage in situ.

Following our success with creating structures by design using nanolithography and mechanophore chemistry, we set out to fabricate organizational chirality on surfaces, which has been an interest in chemistry and materials science due to the need for enantioselective catalysis, separation, and reactions. Current methods for production of organizational chirality are primarily based upon self-assembly of molecules. While powerful, the chiral structures produced are restricted to those dictated by reaction thermodynamics. This work introduces a method to create organizational chirality by design. Using atomic force microscopy in conjunction with our chosen surface chemistry, various chiral structures were designed and produced with nanometer precision, from simple chiral spirals to arrays of chiral nanofeatures to hierarchical chiral structures. The size, geometry, and organizational chirality faithfully follow the designs with a high degree of spatial control. The concept and methodology reported here provides researchers a new means to carry out organizational chiral chemistry, with the intrinsic advantages of chiral structures by design. The results open new and promising applications including organizational chiral sensors, 3D nanoprinting of chiral structures, enantiomeric separation, and enantiomeric heterogeneous catalysis.

Building upon the theme of controlled fabrication of nanostructures, we utilized controlled assembly to create structures by design. Our prior work has demonstrated the concept of controlled assembly of macromolecules such as star polymers [molecular weight (Mw) ∼383 kDa,hydrodynamic radius R ∼ 13.8 nm] in droplets. This work extends this concept to smaller molecules, in this case, poly(ethylene glycol) bis-tetrazine (PEGbisTz, Mw 8.1 kDa, R ∼1.5 nm). The key to controlled molecular assembly is to first deliver ultrasmall volumes (sub-fL) of solution containing PEG-bisTz to a substrate. The solvent evaporates rapidly due to the minute volume, thus forcing the assembly of solute, whose overall size and dimension are dictated by the initial liquid geometry and size. Using prepatterned surfaces, this work revealed that the initial liquid shape can be further tuned, and we could control the final assembly of solute such as PEGbisTz molecules. The degree of control was demonstrated by varying the micropatterns and delivery conditions. This work demonstrated the validity of controlled assembly for PEG-bisTz and enables three-dimensional (3D) nanoprinting of functional materials. The technology has promising applications in nanophotonics, nanoelectronics, nanocomposite materials, and tissue engineering.

These investigations into fabrication of a variety of nanostructures demonstrated the success in creating, complex, hierarchical, and chiral structures with nanometer precision. This success lays the foundation for utilization of scanning probe lithography to create functional nanostructures out of new and exciting materials. Future investigations of this technology will focus on incorporating materials such as mithrene, a unique 3D material that possesses 2D properties, and anthraquinone modified cellulose nanocrystals. Combined with the methodology presented here, the development of these structures will be useful in future applications such as modern sensors, nanodevices, nanophotonics, and nanoelectronics.

A table-top microdevice was used to produce flavopiridol-loaded ultrasmall particles for pulmonary delivery to reduce pulmonary fibrosis and inflammation. Microparticles were image with the help of scanning electron microscopy to visualize their structure. The Loading capacity and encapsulation efficiency of flavopiridol in the microparticles were determine using a UV-Visible spectrophotometer. Commercial microfluidic piezo devices with different orifice diameter were optimized to get the preferred inhalable particle morphology. Optimized microparticles were subject to aerodynamic performance testing and moisture content analysis. The stability of microparticles were perform for up to 24 weeks. A high-speed camera with a long working distance lens was mounted to visualize the droplets size formed by a microfluidic piezo device. Synthesis parameters were optimized to produce microparticles, whose morphology, size, physio-chemical properties, aerodynamic performance, moisture content and release profiles met the criteria for inhalation.

The focus of this dissertation research is on the advanced methodology for drug delivery, which includes the synthesis and characterization of drug loaded delivery vehicles. Drug delivery is a rapidly advancing field, ranging from local injections for vaccine delivery to treatment of asthma via pulmonary delivery. There are many illnesses that are left untreated, because of the lack of means to deliver drugs effectively and safely, such as local sustained release to treat post-traumatic osteoarthritis (PTOA) or pulmonary delivery of anti-inflammatory therapeutics for treatment of coronavirus disease 2019 (COVID-19). In this dissertation, I will fill this void, including formulation, characterization, toxicity, and all the way to efficacy. There are three main components in this dissertation investigation. The first, is the development of a greener approach to producing polymer microparticles for local sustained release of flavopiridol. PLGA microparticles represent an important class of materials used for drug delivery. Current synthesis frequently uses conventional emulsion, where dichloromethane(DCM) is used as the organic phase solvent. Due to the health and environmental toxicity of DCM and its slow degradation, this work replaces DCM with a greener solvent, dimethyl carbonate(DMC). To attain narrow distribution of PLGA particle size, microfluidic flow focusing was chosen over conventional emulsion. This new approach successfully produced PLGA microparticles encapsulated with flavopiridol, a kinase inhibitor. These particles exhibit sustained release profile more desirable than the conventional counterparts. The cytotoxicity and activity tests have demonstrated high biocompatibility and efficacy of these PLGA particles. The high sustainability is also evaluated using simple E-Factor(sEF) and complete E-Factor(cEF). The lower health and environmental toxicities of DMC than DCM are evidenced by approximately one order of magnitude higher in lethal dose, i.e., 50%(LD50) values in rat, 5-fold faster degradation rate, and 30% higher GlaxoSmithKline(GSK) combined greenness value. The approach reported in this work shall provide a new and green means for drug delivery in general. The products enable local sustained delivery of flavopiridol for prevention of post-traumatic osteoarthritis, and anti-cancer therapy. The second development is focused on a new approach to quantify drug release kinetics from PLGA microparticles. Current approaches for quantifying drug release profiles from PLGA vehicles involve non-linear fitting using burst, degradation, and diffusion terms. The non-linear fitting is typically performed across the entire time spectrum, assuming that the contribution from each term is constant across the entire release profile. It is known for instance that burst release only occurs during the initial time of release, therefore prior assumption gives poor representation of physical meaning. To improve upon the current approach, a new model was developed by piecewise splitting the fitting terms over two-time regions during release of flavopiridol from PLGA microparticles. The time regions were identified via time dependent SEM imaging, through the analysis of particle morphological evolution during release. The application of our approach to these particles greatly improved and matched the current understanding of release mechanisms. Our approach was validated in larger particles, where new insight was revealed in the evolution of the fitting parameters across time regions. Particles were fabricated with a porous intra-particulate structure, as verified by AFM imaging, to test the genericness of our approach. The intra-particulate structure of these particles was revealed to cause the geometrical changes during release. Our approach further revealed the impact of particle shape change during release in contrast to particle fragmentation, especially through degradation and diffusion mechanisms. This new approach reported in this work, can be applied to drug release from polymer vehicles in general, which allows tunability of release profile to a higher degree. The third development is focused on the production of flavopiridol loaded inhalable ultra-small particles for pulmonary delivery. Treatment of inflammation causing diseases, like COVID-19, generally used an intravenous delivery, however it has been shown that the best course of delivery to the lungs is via inhalation. Production of inhalable particles containing therapeutics are well suited for treatment of lung inflammation and has been demonstrated as the best delivery pathway for pulmonary based diseases. In this work, we showed the development of inhalable particles containing flavopiridol, a CDK-9 inhibitor with demonstrated anti-inflammatory properties, for the first time reported. Formulations of DPPC, L-isoleucine, and flavopiridol were generated into ultra-small particles that presented a desired geometry and size, well suited for pulmonary delivery. The drug loading of the particles was investigated by utilizing UV-Visible spectroscopy, showing 99% efficiency in loading. The presence of DPPC and L-isoleucine were verified by comparing against known standards in ATR-FTIR spectroscopy. The produced particles were demonstrated to be dispersible using a newly developed sampling method for high resolution SEM imaging. The in vitro release of the particles matched the required release kinetics for pulmonary delivery Flavopiridol was found to be biologically active in the produced particles. This work enables the flavopiridol to be delivered to the lungs for treatment of inflammation caused by diseases like pulmonary fibrosis or COVID-19.

This dissertation demonstrates the methodology for drug delivery, through the production and characterization of flavopiridol loaded delivery vehicles for treatment of inflammation in the knee joint capsule via local sustained release or in the lungs via pulmonary delivery, which drives momentum even further into filling the void.

Molecular Modeling for 3D Printing and Biological Applications Abstract This dissertation covers several molecular modeling efforts applied across length and time scales. These techniques are primarily applied to two major categories: 3D printing (chapters 2-4) and biological applications (chapters 5-7). These computational efforts were part of a collaborative effort to improve the experimental design process for these two applications and have corresponding experimental validation. We first present the application of multi-scale simulation techniques to the process of controlled assembly, which is a critical pre-cursor to enabling 3D printing at the nano-scale. This controlled assembly process depends on the complex interplay between surface, solute, and solvent interactions during the evaporation process. In order to actually model the assembly that takes place during this evaporation process a computational software and methodology had to be developed to replicate evaporation of mixed solvent systems and account for potential hydrogen bonding shells. This resulting methodology was tested for different evaporation rates and scales and is presented in Chapter 2. Chapters 3 and 4 are application papers using this methodology. Chapter 3 focuses on the assembly of small linear sugar molecules analyzed using atomistic molecular dynamics simulations. This atomistic resolution allows the probing of phase transitions these sugars undergo as they assemble into various morphological structures during evaporation. Chapter 4 focuses on the coarse-grained interactions between the phospholipid POPC, various surface types, and mixed solvents. This work investigated the specific contributions of surface type and solvent type on assembled features as a function of evaporation. The final chapters of the work 5-7, focus on the virus that causes COVID-19, SARS-CoV-2. While no longer a part of an additive manufacturing process, these chapters remain a part of a collaborative effort to apply computational tools towards the experimental design process of a specific application, the development of a diagnostic or therapeutic tool. These chapters focus on the evaluation of SARS-CoV-2 spike protein, and specifically the receptor binding domain (RBD) and its interactions with the human angiotensin-converting enzyme 2 (hACE2) receptor. These structures were simulated using atomistic molecular dynamics, evaluated for the structural stability under different glycosylations that would derive from different synthesis methods for treatment (chapter 5). The interactions between the two during binding were evaluated using molecular force pulling simulations, with specific focus on analyzing the hydrogen bonding, electrostatic, and van der waals interactions between the two (chapter 6). This work demonstrated that they glycosylation contributes directly and indirectly to strengthening the interaction with hACE2. Finally the structural stability of various spike truncations synthesized in Chinese hamster ovary cells for use in therapeutic / diagnostic applications were analyzed in chapter 7.