"Intracellular delivery of advanced biological and supramolecular agents holds great potential for fundamental and translational biomedical research, but it is staggeringly complex due to the natural biological barriers that have evolved to protect the cell. A plethora of viral, chemical, and physical approaches have been developed to facilitate delivery of biomolecules into different cell types. However, conventional methods are often unsuitable to deliver a broad range of bioactive agents into cells or require carriers such as viruses and peptides specific to the cargo molecules. For example, viruses are currently the most effective vector for DNA transfection, but they may raise severe biosafety issues such as immunogenicity and insertional mutagenesis; in addition, most lentiviral vectors have limited capacity to carry DNA (typically ~10kb). Chemical and physical delivery methods such as lipofection and bulk electroporation (EP) often suffer from high toxicity and low efficiency especially when applying on primary cells. All these limitations have hampered the progress towards safe, robust, and high-throughput intracellular delivery.
Hybrid interfaces between living cells and micro/nanomaterials have huge application potential in biotechnology, spanning from localized drug delivery and cellular interrogation to regenerative medicine and from intracellular probing and biosensing to neural computing. In particular, programmable vertically configured nanostructures are spurring scientific and technological advances in engineered cell–material interfaces, with potential for major improvements in complex cellular manipulation including intracellular delivery, biomolecular extraction/sampling, biosensing, and mechanotransduction.
To circumvent the limitations of conventional transfection methods, I have developed an innovative cellular disruptive nanotechnology, based on vertically aligned nanoneedle arrays. With their unprecedented nanoscale resolution, nanoneedles can negotiate localized biological barriers (e.g., cell membrane) with minimal invasiveness, permitting safe and efficient access to the cell interior for intracellular delivery. Using the versatile and powerful nanoneedle-enhanced devices, I have successfully delivered a wide range of biomolecules (including plasmids, ssDNAs, mRNAs, siRNAs, IgGs, proteins) into both adherent and non-adherent cells, including primary immune cells that are notoriously difficult to transfect. Specifically, I have delivered Cas9 ribonucleoprotein complex into mouse fibroblast cells to cleavage and knock out the target Hprt housekeeping gene. Moreover, by delivering an exonuclease-resistant PCR expression cassette encoding anti-CD19 CAR and GFP reporter (anti-CD19-CAR-GFP) into primary human T cells, I have realized CAR-T cell generation in vitro. When co-cultured with target lymphoma Raji cells, the nanoneedle-engineered CAR-T cells exhibited stronger anti-tumor efficacy suppressing Raji cell proliferation and function, compared with CAR-T cells produced by bulk electroporation. In addition, I have established the proof of concept to deliver transcription factors into fibroblasts to induce cellular reprogramming.
Such versatile and powerful nanoneedles has created a new paradigm exploiting the potential of such bio–nano interfaces to promote ex vivo cell engineering stem cell reprogramming. By delivering sophisticated biological effectors into target cells, it is highly promising to develop such nanoscale tool for advanced cellular technology and more significantly for engineering of living cell drugs."