Intracellular Delivery with Laser-Irradiated Materials

Abstract

Diseases and disorders arise when cells begin to malfunction. To advance basic research in diseases and develop therapeutics, it is vital to understand and fix mechanisms at the cellular level. In the emerging field of nanomedicine, it is important to deliver biologically interesting cargo and molecules directly into relevant cells. Without cargo delivery, therapeutics cannot happen. Alongside medical applications, targeted delivery of cargo, such as probes, and nanodevices for imaging and labeling, is crucial for studying fundamental cell biology. Current established methods each come with respective strengths and weaknesses that fit different application needs. A challenge is the direct delivery into cellular cytoplasm with these key features: viability of the cells, throughput of the cells for therapeutic applications, and efficiency of the tool. In this thesis, different laser-activated materials for intracellular delivery of large biomolecules in the kDa-range are explored, such as thermoplasmonic gold nanopyramid substrates and particle-embedded PDMS microcuvette chambers. Macromolecules such as siRNA are successfully delivered, and concurrent delivery of multiple cargoes is achieved for human aortic valve interstitial cells in vitro. All platforms are excited by 11-ns laser pulses of 1064 nm wavelength to create transient pores in cells that allow materials in the environment to diffuse into the cytoplasm before the phospholipid bilayer membrane seals. Fluorescence imaging and flow cytometry are used to quantify the delivery efficiency and viability in a reproducible manner. Image analysis is used to characterize cell membrane sealing. Scanning electron microscopy, optical profilometry and hydrophone measurements are used to characterize effects generated by laser-irradiated materials in aqueous medium. The light-activated platforms discussed in this thesis can deliver important material directly into cells, furthering the field of nanomedicine in a cost-effective manner. There has been a lot of development in medicine and biomaterials, but we need to continue exploring methods and tools to get novel molecules into cells so they can reach their full potential for their targeted applications.