Light-Addressable Lipid-Based Nanocapsules.

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Overview

In this thesis a new tool for light-addressable delivery of biological stimuli based on synthetic lipid nanocapsules is explored. First, successful photolysis of the nanocapsules using far red (645nm) and near IR (710nm) light is presented. Using two-photon photolysis (710nm) carbachol is delivered to CHO-M1 cells. Then, using one-photon photolysis, the release of encapsulated dye as well as the delivery of two biological stimuli, carbachol and bradykinin, to CHO-M1 and PC12 cells respectively is demonstrated. In addition, the advantages of using far red (645nm) vs UV (355nm) light is established through a cell stress study by monitoring calcium levels of cells subjected to each wavelength of light.;Next, experimental evidence that rules out a photochemical pathway in favor of a photothermal mechanism in the far-red photolysis of dye-sensitized, lipid-vesicle based nanocapsules is presented. Photolysis efficiency was unaffected by the presence of radical inhibitors, and mass spectrometry measurements confirmed that the photolytic process did not produce dye radicals. Measurements of dye quantum yield in the lipid membrane showed an inverse correlation between quantum yield of the dye and photolysis efficiency of the vesicle. The result is consistent with the notion that a decrease in quantum yield translates into more vibrational relaxation and thermal motion of the dye molecules in the membrane and thus more efficient photothermal disruption of the vesicle. Furthermore, we observed that the decrease in quantum yield and increase in photolysis efficiency was caused by the formation of raft-like domains that clustered the dye molecules into concentrated regions. Based on this information, we were able to design new nanocapsules using ternary mixtures of lipid and cholesterol that promoted the formation of raft domains and dye clustering. These nanocapsules showed improved photolysis efficiency over the best results we obtained previously.;Finally, a new method by which molecules that are impermeable to cells are encapsulated in dye-sensitized nanocapsules for delivery into cells via endocytosis is described. Once inside the cells, the molecules are released from the nanocapsules into the cytoplasm with a single nanosecond pulse from a laser in the far red (645nm). We demonstrate this method with the intracellular release of the second messenger, IP3, in CHO-M1 cells, and report that calcium responses from the cells changed from a sustained increase to a transient spike when the average copy number of IP3 released is decreased below 50 molecules. We also demonstrate the delivery of a 23 kDa protein into Ba/F3 cells to inhibit a key player in the apoptotic pathway, BCR-ABL. We show that an average of 8 molecules of the inhibitor is sufficient to induce apoptosis in the majority of Ba/F3 cells.
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Product Details

  • BN ID: 2940043600455
  • Publisher: ProQuest LLC
  • Sold by: Barnes & Noble
  • Format: eTextbook
  • Pages: 74
  • File size: 4 MB

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