PhD Scholar (Student) Indian Institute of Technology Madras Chennai, Tamil Nadu, India
Abstract: Effective intracellular delivery of biomolecules is crucial in analysing and engineering cell functions, for applications in therapeutic development, diagnostics, and drug delivery towards personalised medicine. Traditional bulk cell culture models employed so far for intracellular delivery, overlook the heterogeneity of the cell population. Whereas, single-cell patterning provides the opportunity to conduct statistically robust analyses and find vital biological information without compromising cellular variability. Single-cell patterning has not yet been used for investigating biomolecular delivery; instead, the emphasis has been on cell behaviour studies. We have explored the application of single-cell patterning as a viable platform for intracellular biomolecule delivery. We present a high-throughput single-cell patterning approach using microcontact printing, that uses a stamp to transfer a pattern of a liquid precursor onto a substrate. We fabricated micro-pillar polydimethyl siloxane stamp with different diameters (40 μm ~ 100 μm with 1 cm × 1 cm patterning area) and then imprint distinct proteins and finally pattern single-cell to small clusters of cells depending on the micro-pillar diameters. This approach has a patterning efficiency of 97-99 %, and is universal for any protein-cell combination. The second part of our work is utilizing this single-cell patterned platform as a intracellular biomolecule delivery. For this purpose, we have created an easy-to-fabricate and simple-to-use two- dimensional array of titanium micro-dish (TMD) device is used to facilitate near infrared mediated optoporation, which disrupts the cell plasma membrane, allowing biomolecules to enter into cells. By aligning this device on top of the patterned cells and exposing to infrared light pulses, we were able to acheive massively parallel optoporation-mediated intracellular delivery of small to very large biomolecules such as PI dye (668 Da), Dextran (3000 MW), SiRNA (20-24 bp), and enzyme (464 kDa). The delivery efficiency for PI dye, Dextran 3000, siRNA and enzyme for patterned cells are approximately 95±3%, 97±1%, 96±1% and 94±3%, with cell viability of 98±1%. Our approach is compact, robust, simple to print, and it could potentially be useful for single-cell therapy and diagnostics. As a result, the proposed method has proven to be of inevitable potential for studying medicines and diagnostics research, as well as creating single-cell level biological processes with high specificity and subcellular accuracy.
