There is increased interest in porous silicon nanomaterials for biomedical applications due to their biodegradability, their biocompatibility, and their intrinsic photoluminescence. This thesis describes cargo loading chemistry, surface chemistry, molecularly targeted delivery and bioimaging applications using porous silicon nanomaterials.

After a brief introduction to porous silicon materials for biomedical applications, Chapter 2 describes a single-step procedure to simultaneously load and protect a model siRNA therapeutic in porous silicon nanoparticles (pSiNPs). Exogenous calcium ions precipitate with locally generated silicic acid to form calcium silicate, which serves to encapsulate the siRNA payload in pSiNPs. The target gene knockdown efficiency in vitro and target tissue accumulation of delivered siRNA in vivo are demonstrated.

Chapter 3 presents a facile chemical modification of the surface of the hydroxylated silicon nanostructure. The reaction, a ring-opening heterocyclic silane “click” reaction, is a rapid and efficient means to obtain high surface coverage while preserving the open pore structure and intrinsic photoluminescence of the original silicon nanostructure. This chemistry is sufficiently mild to maintain the activity of payload proteins.

Chapter 4 presents the example of pSiNPs as an imaging agent, which are targeted to tumor tissues in vivo using an iRGD peptide targeting probe, and the nanoparticles are imaged by two-photon microscopy. Superior photostability and low systemic toxicity are observed.

Chapter 5 discusses enhanced photoacoustic signals that can be obtained from indocyanine green (ICG) when it is encapsulated in pSiNPs. The photoacoustic response from ICG is enhanced 17-fold when it is sealed in pSiNPs. The substantially improved performance is attributed to the low thermal conductivity of pSiNPs and their ability to protect loaded ICG from photolytic degradation.

The efficient adsorption or catalytic degradation of toxic chemicals is desired to provide effective personal protection in both industrial and possible military situations. The possibility of both chemical warfare agents (CWAs, e.g., VX) and toxic industrial chemicals (TICs, e.g., NH3) being released underscores the need for new materials capable of sequestering and/or decontaminating these chemicals. For this purpose, we developed a new composite material by conjugating an inorganic molecule with nano-sized silica support. Specifically, zirconium (oxo) hydroxy methacrylate (Zr cluster) composed of a Zr6O4(OH)4 node and methacrylate backbones is polymerized on the surface of silica (SiO2) nanoparticles upon UV irradiation, and this composite material is tested for (1) catalytic hydrolysis of a CWA simulant, dimethyl p-nitrophenyl phosphate (DMNP) and (2) adsorption of a TIC, ammonia. The composite material displays superior catalytic performance towards DMNP hydrolysis compared to the "free" Zr cluster, and it also shows greater adsorption capacity for NH3 than the bare silica nanoparticles. The advantage of the low-cost and low-complexity of the synthesis is demonstrated in a notional clothing decontamination scenario, where the Zr-silica composite catalyst is embedded in fabric swatches and evaluated for its adsorption and decontamination capabilities.

ABSTRACT OF THE DISSERTATIONIntravitreal Therapeutic Nanoparticles for the Treatment of Posterior Segment Ocular Diseases Joel F. Grondek Doctor of Philosophy University of California, San Diego, 2021 Professor Michael J. Sailor, Chair Ocular therapeutic delivery affords challenges as unique as the diseases themselves. The human eye is anatomically compartmentalized, both within the organ itself and from the systemic vasculature typically used for drug delivery. Recent pharmaceutical developments have revolutionized the prognosis for ocular diseases, yet many therapies treating the posterior segment of the eye remain invasive and require frequent administration to maintain remission. Herein, we review the application of nanocarriers as intravitreally injected therapeutic delivery vehicles working toward enhanced retinal uptake to further improve current therapeutic outcomes and for the development future therapies and we present a novel approach to the treatment of diabetic retinopathy and diabetic macular edema. Chapter one provides introductory overview of the complex anatomy of the eye, routes of therapeutic administration, and current nanoparticle formulations designed to treat posterior segment diseases by intravitreal injection. Chapter two demonstrates a novel application of fusogenic porous silicon nanoparticles for intravitreal delivery of vascular endothelial growth factor siRNA using a rabbit model for diabetic retinopathy and diabetic macular edema. Chapter three summaries prior work focused on peptide targeted magnetic resonance imaging of myocardial infarction using a fibrin homing peptide. Here we demonstrate long circulating iron oxide nanoparticle chains call “nanoworms” can effectively target early fibrin associated with myocardial injury using a rat ischemia/reperfusion model.

Porous silicon photonic crystal-poly(vinylidiene fluoride) polymer composites were synthesized to fabricate a sensor exhibiting tunable hydrophobicity and stability in basic conditions. Poly(vinylidiene fluoride) polymer was infiltrated into porous silicon photonic crystals via the melt-cast method. Partial removal of the hydrophilic silicon template enabled tuning of the hydrophobicity of the silicon-polymer composites over a water contact angle range of 60°-110°. The composites exhibited much greater stability at pH 12 than did the free-standing porous silicon templates. The mechanism of dissolution of the silicon template was examined. In a separate study, perforated etching of silicon wafers was assessed for high-yield preparation of porous Si microparticles, with a focus on optimizing ultrasonication conditions. The effects of increasing the amount of cavitation on particle size and size distribution were assessed. Concentration of films during ultrasonication and duration of ultrasonication on particle were systematically investigated for efficient synthesis of ~70μm particles. The effects of pre-treatment by mechanical crushing on nucleation uniformity, on yield, and yield reproducibility were also assessed.

Porous silicon has been investigated as a novel material for biomedical applications since the early 1990s. Owing to its low toxicity profile and unique properties, it has been utilized in a variety of manners, including biomedical imaging and sustained delivery applications discussed within. In the first portion of this dissertation, quantum confined domains of silicon were used as ratiometric fluorescent probes, in which the long-lived excited states were harnessed to generate wavelength-dependent quenching motifs. To further evaluate the performance of porous silicon as a biomedical imaging agent, it was then compared and contrasted against a series of luminescent silicon nanocrystals to probe which unique properties are intrinsic to all silicon nanocrystals and which are imbued during the fabrication process. Finally, porous silicon microparticles were used to sustain the delivery of hormonal progestins to develop injectable contraceptives with the goal of reducing maternal mortality rates in sub-Saharan Africa. Owing to the anisotropic dissolution of porous silicon, hydrophobic progestin molecules were able to be released from the porous particle host in a highly linear fashion and for longer periods of time than the unprotected controls. The progestins were incorporated into the porous particle via a technique known as melt casting, in which molten drug infiltrates and then recrystallizes within the porous structure. Strategies for melt casting thermally instable drugs were also explored. Particles containing segesterone acetate were found to be non-toxic and well tolerated in a cohort of adult female Sprague-Dawley rats over an extended period of time.

By leveraging its structural, chemical and photonic properties, porous silicon materials were employed to develop platform drug delivery, theranostic and sensing systems. For drug delivery applications, oxidative and magnesium/calcium trapping strategies were investigated to physically immobilize small molecule and protein payloads within the silicon skeleton. The trapping chemistry utilized the pore volume of porous silicon for hosting payload so that high loading efficiency and near 0-order release kinetics was achieved. This strategy also alters the mechanism of payload release from diffusion to particle degradation and substantially minimizes or eliminates burst release. The intrinsic luminescence of silicon was employed as a self-reporting tool for real-time monitoring of payload release from the skeleton and activated by either thermal oxidation or aqueous aging. Correlations between pharmacokinetics and luminescence decay were demonstrated in both in vivo and in vitro studies for intraocular delivery applications.

Polymer-silicon composite photonic crystals were investigated for sensing applications by utilizing the change in structural color as a signal output. The molecular weight dependence on polymer melt infiltration into porous silicon templates enabled fabrication of free-standing polymer photonic crystals with different porosities and optical signatures. Porous silicon films were embedded into polymer catheter devices as sterilization sensors using simple manufacturing methods. Bacteria testing revealed that by wiping the tip of the catheter hub with an alcohol swab and upon observing a complete color change from green to red (and back to green) the catheter device was sterile and confirmed the sensing ability of the porous silicon film.

With increasing discoveries in genetic and biological pathways with respect to disease treatment, the potential for gene therapy is growing exponentially. In parallel, gene modulating tools have also expanded; catalyzed by the discovery of the RNA interference and zinc finger proteins in the 1990s, the 21st century has seen a variety of mechanisms for editing genetic expression (e.g. double-stranded oligonucleotides, zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENS), clustered regularly interspaced short palindromic repeats (CRISPR/Cas), etc.). However, a primary roadblock in enabling these gene editing tools for clinical translation is the biological clearance and degradation mechanisms that prevent the tools from reaching the target cells. The fusogenic porous silicon nanoparticles (FNPs) present an effective solution to this delivery challenge.

Chapter one provides introductory overview of gene therapy, their limitations, as well as the current state-of-the-art technologies with the aim of delineating materials design criteria of RNAi therapeutics. Furthermore, a brief analysis and discussion on trends of publications and clinical translations of RNAi therapy formulations are provided.

Chapter two details the synthesis protocol of fusogenic porous silicon nanoparticles. As the FNPs comprise of multi-layer structure (ie. payload-loaded pSiNPs, calcium silicate sealing, lipid bilayer encapsulation, targeting peptide decoration), the protocol requires delicate and precise handling for formation of stable nanoparticles. Examples of successful versus unsuccessful syntheses are presented, and critical steps to positive outcome are highlighted. The potential for variations and alternative optimizations to the protocol are discussed.

Chapter three delves into the material properties that enable FNP function, and the biological pathways that FNPs depend on for its unique cellular uptake and processing. The intracellular fate and metabolism of individual components of the FNP system (e.g. oligonucleotide payload, pSiNP core, and lipid bilayer shell) are also presented. The fundamental information gained from studying the material interaction at a single-cell level is used to inform the following chapters that deal with application of FNPs in disease models.

Chapters 4-6 demonstrate the FNPs as a platform technology that can be developed into a wide range of therapeutic formulations. By simple exchange of targeting peptides and the siRNA payload, the FNPs demonstrate successful therapeutic outcomes in mouse models of cancer (Chapter 4) and bacterial infections (Chapters 5 and 6).

For over 30 years, porous silicon as a material has been leveraged for its usefulness in biomedical and sensing applications. Its tunable structural features, low toxicity profile and readily modifiable surface render this material extremely useful for a wide variety of applications. By chemically modifying surface species, such as silicon hydrides and silicon oxides with silanes, the properties of porous silicon can be enhanced for its use for chemical sensing, biomedical imaging, and drug delivery.

After a brief introduction to porous silicon materials, the first part of this dissertation details surface-modified porous silicon photonic crystals for the chemical sensing of toxic vapors and nerve agents. Chapter 2 utilizes a dual-peak porous silicon photonic crystal embedded with specific for the selective detection of hydrogen fluoride (HF), hydrogen cyanide (HCN), and the chemical nerve agent diisopropyl fluorophosphate (DFP). The pore walls are rendered hydrophobic with octadecylsilane to aid with the loading of the colorimetric molecules while being insensitive to humidity fluctuations. This provides a robust means to develop a remote detection system for chemical agents. Chapter 3 employs the same photonic crystal, modified, however, with a specialized protein-based gatekeeper that is rendered semi-permeable only in the presence of HCN. This is one of the first novel designs of a bio-inorganic sensor capable of detecting chemical agents with high specificity and precision.

The second portion of the dissertation describes how surface-modified porous silicon nanoparticles can be applied in biomedical applications. The first project details the use of Anti-KIT protein DNA-aptamers decorated onto a fluorescently labelled porous silicon nanoparticle for the in vitro and in vivo imaging of gastrointestinal stromal tumors. This work provides an effective platform in which aptamer-conjugated porous silicon nanoparticle constructs can be used for the targeted imaging of KIT-expressing cancers. The final project utilizes hydrophobic porous silicon nanoparticles for the delivery of erucamide, a highly hydrophobic fatty acid amide, within the retina. By harnessing the versatility of porous silicon, erucamide’s target cells and mechanism of neurotrophic action can be identified.