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Femtosecond Plasmonic Laser Nanosurgery (fs-PLN) mediated by molecularly targeted gold nanospheres at ultra-low pulse fluences.

ABSTRACT: Plasmonic Laser Nanosurgery (PLN) is a novel photomodification technique that exploits the near-field enhancement of femtosecond (fs) laser pulses in the vicinity of gold nanoparticles. While prior studies have shown the advantages of fs-PLN to modify cells, further reduction in the pulse fluence needed to initiate photomodification is crucial to facilitate deep-tissue treatments. This work presents an in-depth study of fs-PLN at ultra-low pulse fluences using 47 nm gold nanoparticles, conjugated to antibodies that target the epithelial growth factor receptor and excited off-resonance using 760 nm, 270 fs laser pulses at 80 MHz repetition rate. We find that fs-PLN can optoporate cellular membranes with pulse fluences as low as 1.3 mJ/cm², up to two orders of magnitude lower than those used at lower repetition rates. Our results, corroborated by simulations of free-electron generation by particle photoemission and photoionization of the surrounding water, shed light on the off-resonance fs-PLN mechanism. We suggest that photo-chemical pathways likely drive cellular optoporation and cell damage at these off-resonance, low fluence, and high repetition rate fs-laser pulses, with clusters acting as local concentrators of ROS generation. We believe that the low fluence and highly localized ROS-mediated fs-PLN approach will enable targeted therapeutics and cancer treatment.

SUBMITTER: Eversole D

PROVIDER: S-EPMC7382507 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Femtosecond Plasmonic Laser Nanosurgery (fs-PLN) mediated by molecularly targeted gold nanospheres at ultra-low pulse fluences.

Eversole Daniel D Subramanian Kaushik K Harrison Rick K RK Bourgeois Frederic F Yuksel Anil A Ben-Yakar Adela A

Scientific reports 20200724 1

Plasmonic Laser Nanosurgery (PLN) is a novel photomodification technique that exploits the near-field enhancement of femtosecond (fs) laser pulses in the vicinity of gold nanoparticles. While prior studies have shown the advantages of fs-PLN to modify cells, further reduction in the pulse fluence needed to initiate photomodification is crucial to facilitate deep-tissue treatments. This work presents an in-depth study of fs-PLN at ultra-low pulse fluences using 47 nm gold nanoparticles, conjugate ...[more]

PMID: 32709944

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Project description:Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue’s native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model allows for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids’ response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24 h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first four hours upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves the same key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following acute airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level.

Project description:Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue's native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model might allow for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids' response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24 h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first 4 h upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following localized airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level.

Dataset Information

Femtosecond Plasmonic Laser Nanosurgery (fs-PLN) mediated by molecularly targeted gold nanospheres at ultra-low pulse fluences.

Publications

Femtosecond Plasmonic Laser Nanosurgery (fs-PLN) mediated by molecularly targeted gold nanospheres at ultra-low pulse fluences.

Similar Datasets

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