RESUMO
There is a great need in the biomedical field to efficiently, and cost-effectively, deliver membrane-impermeable molecules into the cellular cytoplasm. However, the cell membrane is a selectively permeable barrier, and large molecules often cannot pass through the phospholipid bilayer. We show that nanosecond laser-activated polymer surfaces of commercial polyvinyl tape and black polystyrene Petri dishes can transiently permeabilize cells for high-throughput, diverse cargo delivery of sizes of up to 150 kDa. The polymer surfaces are biocompatible and support normal cell growth of adherent cells. We determine the optimal irradiation conditions for poration, influx of fluorescent molecules into the cell, and post-treatment viability of the cells. The simple and low-cost substrates we use have no thin-metal structures, do not require cleanroom fabrication, and provide spatial selectivity and scalability for biomedical applications.
Assuntos
Lasers , Polímeros , Sobrevivência Celular , Luz , PoliestirenosRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Sistemas de Liberação de Medicamentos , Endocitose , Terapia com Luz de Baixa Intensidade , Titânio/metabolismo , Células HeLa , Humanos , TemperaturaRESUMO
Intracellular delivery is crucial for cellular engineering and the development of therapeutics. Laser-activated thermoplasmonic nanostructured surfaces are a promising platform for high-efficiency, high-viability, high-throughput intracellular delivery. Their fabrication, however, typically involves complicated nanofabrication techniques, limiting the approach's applicability. Here, colloidal self-assembly and templating are used to fabricate large arrays of thermoplasmonic nanocavities simply and cost-effectively. These laser-activated substrates are used to deliver membrane-impermeable dye into cells at an efficiency of 78% and throughput of 30â¯000 cells min-1 while maintaining 87% cell viability. Proof-of-concept data show delivery of large cargoes ranging from 0.6 to 2000 kDa to cells without compromising viability.
RESUMO
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.
RESUMO
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.