(382d) Non-Viral Gene and Biomolecule Delivery By Nanochannel Electroporation

Non-viral Gene and Biomolecule Delivery by Nanochannel Electroporation

L. James Lee^1,2,3,4,6, Daniel Gallego–Perez², Xi Zhao^1,2, Lingqian Chang^2,3, Keliang Gao², Veysi Malkoc², Nick Chiang^2,4, Xiaomeng Huang^2,4, John C. Byrd⁴, Raj Muthusamy⁴, Wu Lu^2,5 and Chandan Sen⁶

¹Department of Chemical and Biomolecular Engineering
²Center for Affordable Nanoengineering of Polymeric Medical Devices
³Department of Biomedical Engineering
⁴Comprehensive Cancer Center
⁵Department of Electrical and Computer Engineering
⁶Center for Regenerative Medicine and Cell Based Therapy
The Ohio State University

The ability to deliver precise amounts of biomolecules and nanofabricated probes into living cells offers tremendous opportunities for biological studies and therapeutic applications.  It may also play a key role in the non-viral generation of engineered stem cells and induce pluripotent stem cells with high efficiency and non-carcinogenic properties. Currently-available transfection approaches are heavily dependent upon diffusion- and endocytosis-based mechanisms, which results in highly stochastic transfection. Dosage controlled delivery to multiple cells is not achievable with any existing techniques. We have overcome this problem by developing a new technology, nanochannel electroporation* (NEP) allowing transfection of many small sized and delicate cells with precise control over dose and timing. Cell mortality from NEP is virtually zero. We show dose control effects on a variety of transfection agents such as oligonucleic acids for cancer therapy, molecular probes for intra-cellular biomarker detection and large plasmid for cell reprogramming.

We first describe the principle and design of a series of new NEP systems based on electrokinetic flows and electroporation forces. They include a 2D NEP-optical tweezers platform for single cell transfection*, a 3D DEP-NEP platform for large scale cell transfection, and a 3D NEP patch for in vivo gene transfection. For 3D NEP chips, a properly-engineered and massively parallel ordered array of Si nanochannels, capable of handling/transfecting ~60,000 cells/cm², was fabricated using cleanroom technologies. Positive dielectrophoresis (pDEP) was used to selectively place cells on the nanochannel outputs, thus allowing for controlled and highly efficient transfection. Single-cell dosage control capabilities were demonstrated using a wide variety of small and large molecules. We then present our on-going work on several important medical applications using those NEP systems.

Short interfering RNA (siRNA) has been widely used to silence its targets through complementary-binding to the target mRNA. The conventional delivery methods such as bulk electroporation and nanocarriers cannot provide uniform delivery and are stochastic in nature. The variation in delivery to each cell may cause different downstream consequences, making it difficult to understand the fundamentals of cell response to different drug dosages. An anti-apoptotic gene, MCL-1 was found elevated in some subset of Acute Myeloid Leukemia (AML) patients who harbor with FMS-like tyrosine kinase-3 (FLT3) mutation and have poor survival. We used our 2D NEP method to deliver MCL-1 siRNA to individual AML cells with controlled dosage, which revealed different thresholds of MCL-1 siRNA induced apoptosis to AML cells with or without FLT3 mutation. We were able to measure delivered MCL-1 siRNA and endogenous mRNA level and their dynamic changes by molecular beacon and single cell qRT-PCR and revealed the behavior of MCL-1 siRNA as a therapeutic agent at single cell level.

For the 3D DEP-NEP platform, the first example is the isolation and interrogation of living leukemic CLL stem cells in vitro from CLL patients. We used the 3D NEP technology to deliver molecular probes to identify rare cancer initiating cells with unique intracellular markers, and then interrogate their stemness and drug resistance in vitro, The second example is in vitro cell reprogramming. Although cell reprogramming holds great promise for regenerative medicine, safety concerns and/or the stochastic nature of viral or non-viral transduction approaches have hampered clinical translation. Here we report on the development of a novel non-viral nanotechnology-based platform that allows for deterministic large-scale cell transfection with single-cell resolution. The superior capabilities of such technology were demonstrated using a well-established induced neuron reprogramming model via overexpression of Brn2, Ascl1, and Myt1l (BAM). Our approach resulted in reprogramming performance similar to viral methodologies and much better than existing non-viral methods. Furthermore, increased neuronal complexity could be tightly tailored by varying the BAM ratio and substantially enhanced by adding patterning genes to the BAM cocktail because of the easy control of cocktail receipt and dosage. Although the mechanism remains obscure, we observed significant stroke recovery in mice when NEP induced neurons were implanted in the mouse brain. In addition, the high-throughput NEP system allows living cell interrogation of the reprogramming process.  We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, is regulated by the S-phase cyclin CCNA2, and that at least some neurons induced through BAM-mediated neuronal reprogramming pass through a nestin-positive progenitor cell stage.

The NEP patch is designed for in vivo gene transfection. In vivo cell reprogramming has major clinical implications. Ideally, a patient’s own tissue could be used as a prolific bioreactor to generate specific cells or tissue with minimal immunorepulsion and contamination. Today, non-viral reprogramming has rarely been achieved in vivo and little is known about the reprogramming mechanism within tissues. Herein we report a facile and yet powerful NEP patch technology to non-virally transfect and remodel/reprogram naturally-exposed or surgically-accessible tissue surfaces. Experiments with mouse skin and muscle show that by transfecting combinations of reprogramming genes into the outermost cell layers, secondary mechanisms could be triggered to propagate such transfection throughout the tissue via paracrine-like signaling, presumably mediated by extracellular vesicles (EVs) pre-loaded with a plethora of bioactive factors in addition to the transfected gene mRNAs. Overexpression of these genes led to tailored tissue remodeling and localized cellular reprogramming into different fates, e.g. induced neurons, brown adipocytes, and endothelium, depending on the specific gene combination used. Finally we demonstrate that skin transfection-activated reprogramming (STAR) could effectively revascularize ischemic tissues and limbs in mice.

*P. E. Boukany, A. Morss, W-C Liao, B. Henslee, X. Zhang, B. Yu, X. Wang, Y. Wu, H.C. Jung, L. Li, K. Gao, X. Hu, X. Zhao, O. Hemminger, W. Lu, G. Lafyatis and L.J. Lee, “Nanochannel Electroporation Delivers Precise Amounts of Biomolecules into Living Cells”, Nature Nanotechnology, 6, 747-754 (2011), research highlight in Nature Methods, 8, 996-997 (2011).