The effect of particle size on nanodiamond fluorescence and cellular function
Creators: name: Wilson, Emma
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 401807
subject: Nanomaterials

description: Over the past decade, the study of artificially created nanodiamonds for biological applications has rapidly increased. Detonation nanodiamonds (DNDs) have been widely studied for biomedical applications such as drug delivery due to their small size and uniform shape. While fluorescence from defects can be seen in most diamond nanoparticles stable nitrogen-vacancy (NV) fluorescence in nanodiamonds is currently established by irradiation and annealing of high-pressure high-temperature (HPHT) synthetic diamonds. The fluorescence is produced via the optical excitation of atomic defects, such as the negatively charged nitrogen-vacancy (NV–) centre, within the diamond crystal lattice. The NV– centre is sensitive to electrical and magnetic fields, and temperature changes. HPHT diamond can be ball-milled to create nanoparticles of diamond. Fluorescent nanodiamonds (FNDs) are attractive nanoscale tools with unique properties, making them highly desirable for bioimaging and biosensing applications.

Size fractionation by centrifugation separates the ball-milled diamond particles into different size groupings, but these different FND size categories can have a broad size distribution of tens of nanometres. Smaller particles (&lt;100 nm) are more desirable for biological applications. Larger nanoparticles can interfere with protein binding and function, either binding sterically to the functional site or blocking other functional pathways. Nanoparticle size can directly affect cellular processes in the case of other nanomaterials, including silicon dioxide and zinc oxide, while similar comprehensive studies have yet to be conducted on FNDs.

This thesis presents the most comprehensive and systematic biologically relevant examination of FND size. Using FNDs synthesised from the same starting material and with average particle diameters ranging from 10 nm to 140 nm, the particle size effect on colloidal properties, fluorescence characteristics, and biocompatibility is presented. This work provides valuable insight into the potential opportunities and limitations of FND as a function of particle size for biological imaging and optical sensing applications.

Chapter 1 introduces HPHT nanodiamonds’ synthesis, material characteristics, and optical properties. The chapter also provides an overview of the current biological applications that utilise the unique optical properties of FNDs.

Chapter 2 outlines the methods and materials used throughout this thesis, providing details on the instruments and techniques used. Also, a comparison of the U-87MG glioblastoma multiform cancer cell line and the HaCaT keratinocyte skin cell line used for the biological study are detailed.

Chapter 3 examines the effect of biological media on the colloidal properties and fluorescence of FNDs as a function of particle size. The highly negatively charged FNDs are colloidally stable in water. However, most in vitro studies are performed in cell culture media. Cell culture media contains salts and proteins, which can cause nanoparticle aggregates.

The relative contributions of scattering and absorption by the different-sized FNDs will be discussed. Due to diamonds' high refractive index, nanodiamonds can be highly light scattering. Scattering is dependent on particle size and concentration of nanodiamonds, potentially affecting the flow cytometry analysis used to study biocompatibility.

Chapter 4 examines the light scattering properties of the FNDs internalised by cells when observed using flow cytometry. Light scattering and fluorescence detection are crucial to flow cytometry analysis. FNDs can accumulate inside cellular vesicles forming large (100s of nanometres) sized objects. Light scattering increases as particle size increases, therefore FNDs taken up by the cell can affect flowcytometry analysis. How the FNDs light scattering properties and fluorescence emission can impact the reliability of flow cytometry will be discussed.

Chapter 5 focuses on the biocompatibility of FNDs as a function of particle size. How FND size and concentration affect cell viability, cell cycle, reactive oxygen species generation and apoptosis in U-87MG astrocyte cells and the HaCaT keratinocytes is investigated.

Chapter 6 examines the limitations and opportunities of FNDs for fluorescent markers. Fluorescence scanning confocal microscopy was used to explore the limit of detection for the different-sized FNDs in the U-87MG and HaCaT cells. Cell culture media cells contain autofluorescent molecules and this background fluorescence can make the reliable detection of the less bright FNDs challenging.

Some potential opportunities for utilising the photostability of NV fluorescence of the material considered within this thesis will be explored. At the same time, the autofluorescence of cells is examined, including its effect on limiting the usefulness of FNDs.
Chapter 7 summarises the findings of the previous chapters. While the biological community desires small particles to limit interference with proteins and other biological pathways, the poor signal-to-background ratio (SBR) of the small FNDs limits their usefulness as optical markers and sensors. The 50 nm and larger FNDs particles can be easily imaged in the cells, the 20 nm particles were not reliably detectable. 

The choice of FND based on size is highly dependent on the goal of the biological research. This thesis builds on our understanding of nanodiamonds' fluorescence and biological compatibility to help biologists select an appropriate material for their desired application. For example, if optical sensing or imaging is the aim, 50 nm or larger nanodiamonds can be reliably detected against cellular autofluorescence. However, the light scattering of the 140 nm particles, especially in cells, can impact the reliability of other fluorescence-based assays. Therefore, FNDs between 50 nm and 90 nm could be the optimum size range for most researchers interested in bioimaging and biosensing.