Femtosecond Laser Pulse Interaction with Tissue for Attachment of Cells and Neurons, and Treatment of Retinoblastoma

• Author / Creator

  Katchinskiy, Nir

• This thesis explores novel applications of femtosecond laser pulses in the medical and biomedical research area. It focuses on three main themes: 1. Treatment of Retinoblastoma using cancer targeting gold nanorods. 2. Selective attachment of cells. 3. Selective attachment of neurons. An introduction to the current understanding of femtosecond laser pulse interactions with nanoparticles is provided, where, the reasoning for using cancer targeting gold nanorods for cancer treatment applications is detailed, as well as, the reasons for using femtosecond laser pulses. Additionally, an introduction to the current understanding of femtosecond laser pulse interaction with biological matter is provided. The non-linear multiphoton absorption and cascade ionization processes are discussed, as well as ionized electron densities, shockwave and cavitation bubble formation.
  The application of retinoblastoma targeting gold nanorods with femtosecond laser pulses towards treatment of retinoblastoma is presented. Retinoblastoma is a cancerous disease that affects the retina, and primarily affects young children. To date, the primary treatment goal of retinoblastoma is to save the child's life, while the preservation of the eye and its functionality are the secondary goals. Reoccurrence of tumors is mainly attributed to the persistence of cancer stem cells. EpCAM+ Y79 retinoblastoma cells behave like cancer stem cells and are recognized as cells that are resistant to treatment. Here, an effective technique to treat retinoblastoma cancer cells is presented, using femtosecond laser pulses and epithelial cell adhesion molecule (EpCAM) targeting gold nanorods (Au-NRs). Complete assessment of the optimal laser parameters required for the development of a translational retinoblastoma cancer treatment is provided. Both an MTS cellular metabolism assay and a fluorescence viability assay demonstrate cellular viability drop, to ≈10%. Right after laser irradiation the cellular membrane ruptures. Calculations and field emission scanning electron microscopy (FESEM) imaging show that Au-NRs reach melting temperature after laser pulse exposure. This treatment methodology could be developed treat chemotherapy resistant and radiation resistant cancers.
  The application of laser-induced cell-cell surgical attachment using femtosecond laser pulses is also reported. Attachment of single cells using sub-10 femtosecond laser pulses, with 800nm central wavelength delivered from a Ti:Sapphire laser, is demonstrated. To check that the cells did not go through a cell-fusion process, a fluorescent dye Calcein AM was used to verify that the fluorescent dye did not migrate from a dyed cell to a non-dyed cell. The mechanical integrity of the attached joint was assessed using an optical tweezer. The attachment of cells was performed without the induction of cell-cell fusion, with attachment efficiency of 95%. Cell-cell attachment was achieved by delivery of one to two trains of femtosecond laser pulses lasting 15 ms each. Then, an insight into the mechanism of femtosecond laser nanosurgical attachment of cells is provided. It is demonstrated that during the attachment of two retinoblastoma cells the phospholipid molecules of both cells hemifuse and form one shared phospholipid bilayer, at the attachment location. In order to verify the hypothesis that hemifusion takes place, transmission electron microscope images of the cell membranes of retinoblastoma cells were taken. It is shown that at the attachment interface, the two cell membranes coalesce and form one single membrane shared by both cells. Thus, further evidence is provided to support the hypothesis that laser-induced ionization process led to an ultrafast reversible destabilization of the phospholipid layer of the cellular membrane, which resulted in crosslinking of the phospholipid molecules in each membrane. This process of hemifusion occurs throughout the entire penetration depth of the femtosecond laser pulse train. Thus, the attachment between the cells takes place across a large surface area, which affirms our findings of strong physical attachment between the cells. The femtosecond laser pulse hemifusion technique can potentially provide a platform for precise molecular manipulation of cellular membranes.
  Finally, the application of attachment of neurons using femtosecond laser pulses is presented. Neuronal axons are connected to neuronal soma. Neuronal injury may cause an irreversible damage to cellular, organ and organism function. While preventing neural injury is ideal, it is not always possible. There are multiple etiologies for neuronal injury including trauma, infection, inflammation, immune mediated disorders, toxins and hereditary conditions.

• Subjects / Keywords

  • Nanosurgery
  • Neuron attachment
  • Cancer treatment
  • Nanorods
  • Femtosecond laser

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3Z02ZR17

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.