Nabiha Saklayen

Intracellular Delivery Using Nanosecond-Laser Excitation of Large-Area Plasmonic Substrates

Efficiently delivering functional cargo to millions of cells on the time scale of minutes will revolutionize gene therapy, drug discovery, and high-throughput screening. Recent studies of intracellular delivery with thermoplasmonic structured surfaces show promising results but in most cases require time- or cost-intensive fabrication or lead to unreproducible surfaces. We designed and fabricated large-area (14 × 14 mm), photolithography-based, template-stripped plasmonic substrates that are nanosecond laser-activated to form transient pores in cells for cargo entry. We optimized fabrication to produce plasmonic structures that are ultrasmooth and precisely patterned over large areas. We used flow cytometry to characterize the delivery efficiency of cargos ranging in size from 0.6 to 2000 kDa to cells (up to 95% for the smallest molecule) and viability of cells (up to 98%). This technique offers a throughput of 50000 cells/min, which can be scaled up as necessary. This technique is also cost-effective as each large-area photolithography substrate can be used to deliver cargo to millions of cells, and switching to a nanosecond laser makes the setup cheaper and easier to use. The approach we present offers additional desirable features: spatial selectivity, reproducibility, minimal residual fragments, and cost-effective fabrication. This research supports the development of safer genetic and viral disease therapies as well as research tools for fundamental biological research that rely on effectively delivering molecules to millions of living cells.

Plasmonic Tipless Pyramid Arrays for Cell Poration

Improving the efficiency, cell survival, and throughput of methods to modify and control the genetic expression of cells is of great benefit to biology and medicine. We investigate, both computationally and experimentally, a nanostructured substrate made of tipless pyramids for plasmonic-induced transfection. By optimizing the geometrical parameters for an excitation wavelength of 800 nm, we demonstrate a 100-fold intensity enhancement of the electric near field at the cell-substrate contact area, while the low absorption typical for gold is maintained. We demonstrate that such a substrate can induce transient poration of cells by a purely optically induced process.

Dynamics of transient microbubbles generated by fs-laser irradiation of plasmonic micropyramids

We investigated the dynamics of microbubbles induced by fs-laser irradiation of plasmonic micropyramids in water. We simulated the localized plasmonic enhancement on the micropyramids using a finite-difference time-domain (FDTD) technique and experimentally confirmed the enhancement by observing the laser-induced damage pattern on the substrate. Finally, we experimentally observed the generation of micrometer-sized bubbles on our fabricated structures. We find that the maximum bubble diameter and bubble lifetime depend on power, exposure time, and repetition rate of the laser. The maximum bubble diameter increases with laser exposure time until a balance is reached between the surface tension and the pressure inside and outside the bubble.

Analysis of poration-induced changes in cells from laser-activated plasmonic substrates

Laser-exposed plasmonic substrates permeabilize the plasma membrane of cells when in close contact to deliver cell-impermeable cargo. While studies have determined the cargo delivery efficiency and viability of laser-exposed plasmonic substrates, morphological changes in a cell have not been quantified. We porated myoblast C2C12 cells on a plasmonic pyramid array using a 532-nm laser with 850-ps pulse length and time-lapse fluorescence imaging to quantify cellular changes. We obtain a poration efficiency of 80%, viability of 90%, and a pore radius of 20 nm. We quantified area changes in the plasma membrane attached to the substrate (10% decrease), nucleus (5 - 10% decrease), and cytoplasm (5 - 10% decrease) over 1 h after laser treatment. Cytoskeleton fibers show a change of 50% in the alignment, or coherency, of fibers, which stabilizes after 10 mins. We investigate structural and morphological changes due to the poration process to enable the safe development of this technique for therapeutic applications.

A comparison of inverted and upright laser-activated titanium nitride micropyramids for intracellular delivery

The delivery of biomolecules into cells relies on porating the plasma membrane to allow exterior molecules to enter the cell via diffusion. Various established delivery methods, including electroporation and viral techniques, come with drawbacks such as low viability or immunotoxicity, respectively. An optics-based delivery method that uses laser pulses to excite plasmonic titanium nitride (TiN) micropyramids presents an opportunity to overcome these shortcomings. This laser excitation generates localized nano-scale heating effects and bubbles, which produce transient pores in the cell membrane for payload entry. TiN is a promising plasmonic material due to its high hardness and thermal stability. In this study, two designs of TiN micropyramid arrays are constructed and tested. These designs include inverted and upright pyramid structures, each coated with a 50-nm layer of TiN. Simulation software shows that the inverted and upright designs reach temperatures of 875 °C and 307 °C, respectively, upon laser irradiation. Collectively, experimental results show that these reusable designs achieve maximum cell poration efficiency greater than 80% and viability greater than 90% when delivering calcein dye to target cells. Overall, we demonstrate that TiN microstructures are strong candidates for future use in biomedical devices for intracellular delivery and regenerative medicine.

Intracellular cargo delivery with polymer substrates and nanosecond pulsed laser (Conference Presentation)

One major barrier to advancing fundamental studies of biological cargoes for clinical use has been effective delivery into the cytoplasm. Available methods such as electroporation, viral techniques, and liposomal reagents come with respective strengths and weaknesses depending on the application needs. We present a laser-based cargo delivery platform that combines 11-ns laser pulses and structured flexible polymer substrates to create transient pores in the plasma membrane of cells. Cells are grown on the substrates, and pores are induced form on the cells in the regions excited with nanosecond laser pulses—thus, allowing treatment selectivity in a population. The medium surrounding the cell contains the delivery cargoes in solution, and cargoes diffuse into the cell before the transient pores are sealed. Polymer-based substrates are a promising material for laser-based delivery methods because they are low-cost, have flexible spatial movements, and have simple fabrication techniques. We deliver cargos of various sizes. We use fluorescence imaging and flow cytometry to quantify the delivery efficiency and viability in a reproducible manner. We obtain delivery efficiencies of up to 40% with viabilities of 60% for calcein green in adherent cells such as HeLa and Panc-1. We also deliver molecules of up to 40 kDas and siRNA. We use scanning electron microscopy to study cell adherence and substrate surface morphology. Our data shows that polymer-based substrates can deliver biological material directly into cells in a cost-effective manner.

Self-Assembled Laser-Activated Plasmonic Substrates for High-Throughput, High-Efficiency Intracellular Delivery

Delivering material into cells is important for a diverse range of biological applications, including gene therapy, cellular engineering and imaging. We presented a plasmonic substrate for delivering membrane-impermeable material into cells at high throughput and high efficiency while maintaining cell viability. The substrate fabrication is based on an affordable and fast colloidal self-assembly process. When illuminated with a femtosecond laser, the light interacts with the electrons at the surface of the metal substrate, creating localized surface plasmons that form bubbles via energy dissipation in the surrounding medium. These bubbles come into close contact with the cell membrane to form transient pores and enable entry of membrane-impermeable material via diffusion. We performed proof of principle experiments using both a femtosecond laser and a nanosecond laser system. We used fluorescence microscopy to verify delivery of membrane-impermeable dye into HeLa CCL-2 cells and to verify cell viability after laser treatment. Our findings indicate that self-assembled plasmonic substrates may be an affordable, flexible tool for high-throughput, high-efficiency delivery of material into mammalian cells.

Reusable titanium nitride plasmonic microstructures for intracellular delivery (Conference Presentation)

Efficient drug and biomolecular delivery into cells is an important area of biomedical research. Intracellular delivery relies on porating cell membranes to allow exterior molecules to enter the cell efficiently and viably. Various methods, including optoporation, electroporation, and viral techniques, can deliver molecules to cells, but come with significant drawbacks such as low efficiency, low throughput, and low viability. We present a new laser-based delivery method that uses laser pulses to excite plasmonic, Titanium Nitride (TiN) microstructures for cell poration and offers high efficiency, throughput, and viability. TiN is a promising plasmonic material for laser-based delivery methods due to its high levels of hardness and thermal stability. We fabricate these microstructures by sputtering thin films of TiN on patterned sapphire substrates. We then optimize plasmonic enhancement and stability by investigating different fabrication conditions. We deliver dye molecules, siRNA, and microspheres to cells to quantify poration efficiency and viability by using flow cytometry and by imaging the target cells at defined time intervals post laser irradiation. Additionally, we study temperature effects via simulations and experiments, as well as oxidation of the TiN films over time. We also use scanning electron microscopy (SEM) techniques to study microstructure damage and cell adhesion. Overall, TiN presents a promising opportunity for use as a reusable material in future biomedical devices for intracellular biomolecular delivery and regenerative medicine.

Pulsed Laser-Activated Plasmonic Pyramids for Intracellular Delivery

We use pulsed laser-activated plasmonic micropyramids to deliver molecules to living cells with high efficiency, viability, and throughput. Cellular therapy holds great promise for applications in gene therapy and fundamental biomedical research, and it is essential to develop a universal delivery platform that can safely deliver biomolecules to different cell types effectively. Such a platform would be an important stepping stone towards treatment of hematologic diseases such as leukemia and primary immunodeficiency disorder treatments. An idea molecular delivery platform would exhibit advantages such as high delivery efficiency, low toxicity, minimal immune reaction, and reusability. None of the currently available commercial methods, such as viral-based or electroporation, offer all desirable characteristics at once. We present a new optical method for molecular delivery that uses laser-activated microstructures. Our micropyramids produce a strong plasmonic effect under laser illumination by focusing energy in a small volume at the tip of each pyramid. This leads to the formation of microbubbles which temporarily porate the cell membrane and allow dye molecules and siRNA to diffuse into the cytoplasm. We fabricate large-area micropyramid arrays using photolithograpy, anisotropic etching of silicon, metal deposition, and template stripping. The silicon pyramid templates can be used repeatedly to fabricate gold pyramids. We optimize our laser parameters for high efficiency delivery of small dye molecules like calcein (>80 %) at high cell viability (>90 %). Alongside small dyes, we also deliver different-sized fluorescently labeled dextrans (70 kDa–2000 kDa) and fluorescent microspheres. Our method delivers molecules with high efficiency and high cell viability in different cell types, and our substrates can be reused for repeated high efficiency poration. Our scalable technique offers an innovative approach to delivering molecules to living cells for important applications in regenerative medicine.