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 nearfield 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/cm 2 , 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. Precisely targeted femtosecond (fs) laser pulses have been used to disrupt cellular and sub-cellular structures with great accuracy for a variety of applications, including DNA and protein transfection across the cell membrane 1-4 , organelle manipulation 5,6 , axotomy 7 , and initiating controlled cell apoptosis 8,9. However, the need for tight focusing to achieve nano-scale precision limits the number of structures that can be manipulated during therapy, restricting the technique to small-scale research applications. In Plasmonic Laser Nanosurgery (PLN), the use of gold nanoparticles as local field enhancers holds promise as a scalable, low energy alternative to direct ultrashort (< 10 ps) laser irradiation for cellular manipulation 10,11. The increased near-field strengths and/or large absorption cross-sections due to surface plasmons substantially reduce thresholds for photomodification. Plasmonic effects localize peak laser intensity in the particles' vicinity, permitting more relaxed focusing conditions while maintaining nano-scale selectivity. We can also target molecularly-specific moieties with high selectivity 12 since the surface of gold nanoparticles can be functionalized with bio-specific agents, such as antibodies 13 , ligands 14 , and DNA sequences 15. These properties have the potential to reduce photo-treatment times and increase throughput, as large volumes of cells or tissue can be rapidly treated without needing for tight focusing on the desired target. More importantly, the substantial ablation threshold reduction would allow ablating deeper in a highly scattering tissue by reducing the probability of out-of-focus photodamage du...

Cell Membrane Perforation: Patterns, Mechanisms and Functions

“…Optoporation or photoporation employs microsecond, nanosecond, [39] picosecond, [40] and femtosecond (fs) [41][42][43] pulsed laser or a continuous laser [44,45] directed toward the cell membrane to form tiny pores. Traditionally, the cells are perforated by laser focusing on a tiny spot on the cell membrane, which is usually labor-intensive and energy-consuming, suitable mostly for single-cell operation, [46,47] but by combining microflow-cell setups with an fs laser, high throughput optoporation can be completed.…”

“…[45,51,52] By conjugating receptor-specific antibodies, [40] perforation on specific cells can be realized without out-of-focus photodamage due to strict self-focusing. [41] The mechanisms of nanomaterial-mediated optoporation vary with the laser fluence. If the laser fluence of nanosecond pulsed lasers or picosecond pulsed lasers is below the threshold of vapor nanobubbles (VBs) formation, the temperature of photothermal nanomaterials increases, heat distributes to the cell membrane causing phase-transition of the lipid-bilayer and even denaturation of the glycoproteins, and the cell membrane is perforated.…”

Nanosurgical Manipulation of Titin and Its M-Complex

“…Such nanoscale manipulation methods can be used to deform, displace, cut and mechanically modify biomolecular or cellular objects with nanometer precision, and they have contributed to the emergence of the concept of nanosurgery [27]. In nanosurgery, fast lasers [28][29][30], optical tweezers [31], or atomic force microscopy (AFM) [32] are employed for nanoscale manipulation and modification. AFM has been used for the nanomanipulation and nanodissection of a wide range of biological objects such as DNA [32][33][34][35], chromosomes [36], fibers [37,38], cells [39], and proteins [40][41][42][43].…”

Part II. Nanobubbles around plasmonic nanoparticles in terms of modern simulation modeling: what makes them kill the malignant cells?

“…It was also pointed out that the generation of VC near the membrane of a malignant cell could lead to its destruction followed by death (necrosis) [20,23]. The cell death can be triggered after a single laser pulse, while most hyperthermia methods use prolonged irradiation with a series of pulses [20,24]. Exposure to laser pulses can result in protein denaturation and mechanical stresses that may induce a mechanical damage in cell structures [25].…”