Directing the self-assembly of tumour spheroids by bioprinting cellular heterogeneous models within alginate/gelatin hydrogels

"Human tumour progression is a dynamic process involving diverse biological and biochemical events such as genetic mutation and selection in addition to physical, chemical, and mechanical events occurring between cells and the tumour microenvironment. Using 3D bioprinting we have developed a method to embed MDA-MB-231 triple negative breast cancer cells, and IMR-90 fibroblast cells, within a cross-linked alginate/gelatin matrix at specific initial locations relative to each other. After 7 days of co-culture the MDA-MB-231 cells begin to form multicellular tumour spheroids (MCTS) that increase in size and frequency over time. After similar to 15 days the IMR-90 stromal fibroblast cells migrate through a non-cellularized region of the hydrogel matrix and infiltrate the MDA-MB-231 spheroids creating mixed MDA-MB-231/IMR-90 MCTS. This study provides a proof-of-concept that biomimetic in vitro tissue coculture models bioprinted with both breast cancer cells and fibroblasts will result in MCTS that can be maintained for durations of several weeks.

Designed 2D protein crystals as dynamic molecular gatekeepers for a solid-state device

The sensitivity and responsiveness of living cells to environmental changes are enabled by dynamic protein structures, inspiring efforts to construct artificial supramolecular protein assemblies. However, despite their sophisticated structures, designed protein assemblies have yet to be incorporated into macroscale devices for real-life applications. We report a 2D crystalline protein assembly of C98/E57/E66L-rhamnulose-1-phosphate aldolase (CEERhuA) that selectively blocks or passes molecular species when exposed to a chemical trigger. CEERhuA crystals are engineered via cobalt(II) coordination bonds to undergo a coherent conformational change from a closed state (pore dimensions <1 nm) to an ajar state (pore dimensions ~4 nm) when exposed to an HCN(g) trigger. When layered onto a mesoporous silicon (pSi) photonic crystal optical sensor configured to detect HCN(g), the 2D CEERhuA crystal layer effectively blocks interferents that would otherwise result in a false positive signal. The 2D CEERhuA crystal layer opens in selective response to low-ppm levels of HCN(g), allowing analyte penetration into the pSi sensor layer for detection. These findings illustrate that designed protein assemblies can function as dynamic components of solid-state devices in non-aqueous environments

Functionalized Porous Silicon for Applications in Chemical Sensing, Tumor Imaging and Drug Delivery

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

Porous Silicon Nanoparticles Targeted to the Extracellular Matrix for Therapeutic Protein Delivery in Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major cause of disability and death among children and young adults in the United States, yet there are currently no treatments that improve the long-term brain health of patients. One promising therapeutic for TBI is brain-derived neurotrophic factor (BDNF), a protein that promotes neurogenesis and neuron survival. However, outstanding challenges to the systemic delivery of BDNF are its instability in blood, poor transport into the brain, and short half-life in circulation and brain tissue. Here, BDNF is encapsulated into an engineered, biodegradable porous silicon nanoparticle (pSiNP) in order to deliver bioactive BDNF to injured brain tissue after TBI. The pSiNP carrier is modified with the targeting ligand CAQK, a peptide that binds to extracellular matrix components upregulated after TBI. The protein cargo retains bioactivity after release from the pSiNP carrier, and systemic administration of the CAQK-modified pSiNPs results in effective delivery of the protein cargo to injured brain regions in a mouse model of TBI. When administered after injury, the CAQK-targeted pSiNP delivery system for BDNF reduces lesion volumes compared to free BDNF, supporting the hypothesis that pSiNPs mediate therapeutic protein delivery after systemic administration to improve outcomes in TBI

Harnessing the Materials Chemistry of Mesoporous Silicon Nanoparticles to Prepare “Armor-Clad” Enzymes

There is a growing interest in nanomaterials that can encapsulate enzymes while retaining their ability to function within the confines of a nanocage. Here porous silicon nanoparticles (pSiNPs) are evaluated as an enzyme cage, utilizing the aqueous chemistry of silicon to dynamically restructure the mesopore structure, immobilizing, and confining the enzyme. The common bioluminescent reporter enzyme nanoluciferase (Nluc) is used to evaluate two different trapping chemistries, and impacts on the stability and catalytic performance of the enzyme are compared with controls involving free enzyme and enzyme electrostatically adsorbed to a pSiNP host without the use of trapping chemistry. The two chemistries exploited in this study are (1) oxidative trapping, where mild aqueous oxidation of the elemental silicon skeleton in the mesoporous silicon host swells and restructures the pore walls, physically trapping the Nluc payload in a porous SiO2 matrix, and (2) calcium ion-induced condensation, where localized precipitation of calcium silicate entraps the Nluc protein in a porous silicate matrix. The two trapping chemistries form robust nanoscale cages with substantially smaller pores (9.8 ± 0.4 and 8.8 ± 0.3 nm, respectively) compared to the pSiNP starting material (15.3 ± 1.8 nm), such that the enzyme does not leach from the pSiNPs in aqueous buffer or under assay conditions. Enzyme stability is substantially improved using the two trapping chemistries; the caged materials retain 30-45% activity after heating to 80 °C for 30 min or when exposed to organic solvents; either of these denaturing conditions result in complete or near-complete loss of activity for the free enzyme or for enzyme that is electrostatically adsorbed to pSiNPs. Finally, we explore the potential for the use of the Nluc-encapsulated nanocomposite as a cellular probe by demonstrating the luminescent reporting function of the nanoparticles in HeLa human cell cultures.</p

Anti-KIT DNA aptamer-conjugated porous silicon nanoparticles for the targeted detection of gastrointestinal stromal tumors.

Evaluation of Gastrointestinal Stromal Tumors (GIST) during initial clinical staging, surgical intervention, and postoperative management can be challenging. Current imaging modalities (e.g., PET and CT scans) lack sensitivity and specificity. Therefore, advanced clinical imaging modalities that can provide clinically relevant images with high resolution would improve diagnosis. KIT is a tyrosine kinase receptor overexpressed on GIST. Here, the application of a specific DNA aptamer targeting KIT, decorated onto a fluorescently labeled porous silicon nanoparticle (pSiNP), is used for the in vitro &amp; in vivo imaging of GIST. This nanoparticle platform provides high-fidelity GIST imaging with minimal cellular toxicity. An in vitro analysis shows greater than 15-fold specific KIT protein targeting compared to the free KIT aptamer, while in vivo analyses of GIST-burdened mice that had been injected intravenously (IV) with aptamer-conjugated pSiNPs show extensive nanoparticle-to-tumor signal co-localization (&gt;90% co-localization) compared to control particles. This provides an effective platform for which aptamer-conjugated pSiNP constructs can be used for the imaging of KIT-expressing cancers or for the targeted delivery of therapeutics