Development of Advanced Nanostar-Based Technologies for Diagnostic and Therapeutic Applications

Abstract

Plasmonic nanoparticles have been extensively studied for a wide range of biomedical applications, from analyte and biomarker detection to the treatment of solid tumors. The versatility of these nanoparticles arises from their strong, tunable interaction with light, known as localized surface plasmon resonance (LSPR). In this context, a plasmon refers to the collective oscillation of conduction electrons within a metallic nanoparticle when excited by light at its resonant frequency. When excited, localized surface plasmons generate intense local electromagnetic fields around the nanoparticle surface, significantly altering how light is absorbed and scattered. By modifying the nanoparticle morphology and composition, the LSPR frequency can be tuned, enabling the design of nanoparticle platforms tailored for specific biomedical applications.Among plasmonic nanoparticle morphologies, nanostar-based particles are considered the highest performing and most versatile. These particles consist of a core, from which branch-like protrusions extend outward, forming a star-shaped structure made entirely of gold. The high performance and versatility of this platform can be directly attributed to its star-like morphology. When the plasmon modes of the particle are excited, the free electron cloud is concentrated at the tips of the branches, generating exceptionally high electric fields. The dominant LSPR frequency of a nanostar can be tuned across much of the visible to near-infrared spectrum by adjusting parameters such as the core size, the number, length, thickness, and sharpness of the branches, and the metal composition of the structure. While plasmonic nanoparticles hold great potential in areas ranging from rapid, direct biomarker detection to photothermal transduction for tumor ablation, several fundamental limitations have thus far hindered their overall effectiveness. These limitations include batch-to-batch variability in optical characteristics, photo and thermal stability, and surface-enhanced Raman scattering (SERS) signal enhancement. This work details how these intrinsic challenges were addressed and demonstrates the potential of advanced nanostar-based technologies for diagnostic and therapeutic applications. First, an automated nanoparticle synthesis system was developed that enabled granular tuning and optimization of both gold nanostar (GNS) and bimetallic nanostar (BNS) particles. This system allowed our group to produce large volumes of nanoparticles with minimal batch-to-batch variability, supporting a range of other projects within the lab. Building upon this foundation, I developed the novel caged gold nanostar (C-GNS) morphology, which involved forming a hollow gold shell around the branch tips of a nanostar particle. This design successfully integrates the tunable optical properties of high-aspect-ratio nanoparticles with the potential for molecular encapsulation within hollow metallic structures. The C-GNS morphology was further refined by increasing the thickness of the gold shell, resulting in armored core-gold nanostars (AC-GNS). These particles addressed a key limitation of traditional high-aspect-ratio nanoparticles, as the geometric anchoring of the AC-GNS branch tips significantly enhanced photo and thermal stability. The C-GNS morphology provided the basis for creating silver-coated caged nanostars (Ag-CNS). These Ag-CNS particles generated surface-enhanced Raman scattering (SERS) signal enhancements several times greater than their gold-only counterparts, making them particularly well-suited for in vitro diagnostics. To demonstrate the utility of an advanced nanostar-based technology for diagnostic applications, a method to repeatably generate dense, homogeneous monolayers of BNS particles onto the surface of glass substrates was developed. A critically important first step in developing a biosensing platform. I then developed a method to functionalize the substrate-bound BNS with Inverse Molecular Sentinel (iMS) DNA probes and execute the miRNA biosensing assay. Several assay and probe design rules were established in the process to maximize the SERS signal generated by iMS probes and tune the dynamic range of the sensor, ultimately resulting in a limit of detection in the zeptomole range. Using miRNAs from plasma and enriched exosome samples derived from colorectal cancer patients, the substrate-based iMS platform demonstrated sensitivity values of 100% and 95.8% and specificity values of 100% and 100%, respectively. Further, all SERS signal increases values were strongly correlated with RT-PCR results, demonstrating the selectivity of the biosensing platform. The method was then adapted to detect miRNAs with diagnostic utility for Alzheimer’s disease, where again, the SERS signal increases were strongly correlated with next-generation sequencing results. The final portion of this work involved applying AC-GNS particles to detect solid tumors and treat them via nanoparticle-mediated photothermal therapy. First, a simulation pipeline that could be used to accurately predict the temperature increases within the tumor region during photothermal therapy was developed to aid in treatment outcome prediction. Then, the capability of molecular cargo encapsulation and superior photo stability of the AC-GNS morphology was leveraged to optically detect particles within the micro tumor environment, using several methods, such as fluorescence, hyperspectral SERS imaging, and photoacoustic computed tomography via diffractive acoustic tomography and full-view ring array. AC-GNS-mediated photothermal therapy of solid tumors was also rigorously investigated in several contexts using murine models. First, treatment feasibility was established using a bladder cancer single-flank tumor model, where temperatures easily reached therapeutic thermal dosages. Next, non-invasive photoacoustic thermometry was used to monitor the temperature throughout the tumor region during treatment and calculate the thermal dose received by each animal. This integrated study resulted in the long-term survival of all animals that received nanoparticle-mediated PTT. Further, at the predetermined study end point of 6 months, gross necropsies of the long-term survivors were unremarkable, and there was no statistical difference in hepatotoxicity-associated blood chemistry markers, indicating the biocompatibility of the AC-GNS platform. Finally, we investigated the efficacy of AC-GNS-mediated PTT and immunotherapy in a dual-flank tumor model. After performing PTT on one tumor, a majority of treated animals experienced a significant or complete reduction of the non-treated tumor, indicating a systemic immune response triggered by the combination photothermal immunotherapy.