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Research Highlights

1. Non-viral Gene Delivery by Nanochannel Electroporation- 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. 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, molecular beacon, quantum dots and efficient delivery of large DNA directly into the nucleus using nanoparticle “bullets.” Dosage controlled delivery to multiple cells is not achievable with any existing techniques. NEP also leads to mass secretion of extracellular vesicles containing functional RNAs from transfected cells.

• 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).

• D. Gallego-Perez, L. Chiang, J. Shih, J. Ma, S. Kim, X. Zhao, X. Wang, P. Mao, K.J. Kwak, Y. Wu, L. Wu, G. Lafyatis, D.J. Hansford, I. Nakano, and L.J. Lee, “On-Chip Clonal Analysis of Glioma-Stem-Cell Motility and Therapy Resistance”, Nano Letters, 16(9), 5326-5332 (2016).

• D. Gallego-Perez, D. Pal, S. Ghatak, V. Malkoc, N. Higuita-Castro, S. Gnyawali, L. Chang, W-C Liao3, J. Shi, M. Sinha, K. Singh, E. Steen, A. Sunyecz, R. Stewart, J. Moore, T. Ziebro, R.G. Northcutt, M. Homsy, P. Bertani, W. Lu, S. Roy, S. Khanna, C. Rink, V.B. Sundaresan, J.J. Otero, L.J. Lee and C.K. Sen, "Topical Tissue Nano-transfection Mediates Non-viral Stroma Reprogramming and Rescue", Nature Nanotechnology, :10.1038/nnano.2017.134 (2017).

• Z. Yang, J. Shi, J. Xie, Y. Chen, J. Sun, T. Liu, Y. Zhao, X. Zhao, X. Wang, Y. Ma, V. Malkoc, C-L Chiang, Y. Fu, K.Joo Kwak, Y. Fan, C. Kang, C. Yin, J. Rhee, P. Bertani, J. Otero, W. Lu, A.S. Lee, W. Jiang, L. Teng, B.Y.S. Kim and L.J. Lee, “Large-Scale Generation of Functional mRNA Containing Exosomes via Cellular Nanoporation”, Nature Biomedical Engineering, 4, 69-83 (2020).

2. Targeted Delivery by Lipoplex Nanoparticles- The high mortality rates for many cancers can be attributed to late diagnosis, high recurrence rates, metastasis, and limited effectiveness of current therapeutic modalities. Although the aforementioned issues have been studied extensively, there is no current technology platform that allows for simultaneous detection, therapy and prognosis determination. MicroRNA (miRNA) dysregulation has been implicated in HCC development. These miRNAs regulate a network of tumor promoting and tumor suppressor genes that are critical for tumor cell survival, epithelial-to-mesenchymal and mesenchymal-to-epithelial transition (EMT/MET), escaping the host immune system, resistance to therapy, and angiogenesis. Modulating tumor cells and its microenvironment with miRNA replacement (MRT) and anti-miRNA therapy (AMT) can potentially inhibit tumor growth and sensitize tumor cells to existing therapy. A critical barrier to the clinical development of MRT/AMT is that oligonucleotides are sensitive to nucleases, subject to renal and reticuloendothelial system (RES) clearance with minimum membrane permeability. Delivery systems based on targeted lipid nanoparticles can potentially address these problems. My team has made significant contribution in this area.

• B. Yu, Y. Mao, L. Bai, S. May, A. Ramanunni, Y. Jin, X. Mo, C. Carolyn, K.K. Chan, D. Jarjoura, G. Marcucci, R.J. Lee, J.C. Byrd, L.J. Lee and N. Muthusamy, “Liposomal Targeted Delivery Overcomes Off-target Immunostimulatory Effects of RNA Oligonucleotide”, Blood, 121, 136-147 (2013).

• X. Huang, S. Schwind, B. Yu, R. Santhanam, H. Wang, P. Hoellerbauer, A. Mims, R. Klisovic, A.R. Walker, K.K. Chan, W. Blum, D. Perrotti, J.C. Byrd, C.D. Bloomfield, M.A. Caligiuri, R.J. Lee, R. Garzon, N. Muthusamy, L.J. Lee and G. Marcucci, “Targeted Delivery of microRNA-29b by Transferrin Conjugated Anionic Lipopolyplex Nanoparticles: A Novel Therapeutic Strategy in Acute Myeloid Leukemia”, Clinical Cancer Research, 19(9), 2275-2292 (2013).

• Y. Wu, J. Ma, P Woods, N. Chesarino, L.J. Lee, S.P. Nana-Sinkam and I.C. Davis, DVM, “Selective Targeting of Alveolar Type II Respiratory Epithelial Cells by Anti-surfactant Protein-C Antibody-conjugated Lipoplexes”, J. Controlled Release, 203, 140-149 (2015).

• C-L Chiang, S. Goswami, F. Frissora, Z. Xie, P Yan, R. Bundschuh, L. Walker, X. Huang, R. Mani, X. Mo, S. Baskar, C. Rader, M. Phelps, G. Marcucci, J. Byrd, L.J. Lee, and N. Muthusamy, “ROR1-targeted Delivery of miR-29b Induces Cell Cycle Arrest and Therapeutic Benefit in vivo in CLL Mouse Model”, Blood, 134, 432-444 (2019).

3. Tethered Lipoplex Nanoparticle Biochips for Extracellular Vesicles Based Circulating RNA Biomarkers- Biomolecules such as miRNAs, lncRNAs, mRNAs and protein antigens can be useful as biomarkers for cancer and other diseases. Recent studies show that they are excreted by cells in the form of extracellular vesicles (EVs) including exosomes and can be detected in the blood. Our group has been actively engaged in establishing EVs as potential noninvasive biomarkers. EVs are known mobile elements that function as escape routes for proteins and RNAs from one cell (site of origin) to distant locations. Many studies have revealed that EVs cross talk and/or influence major disease-related pathways, such as hypoxia-driven EMT, angiogenesis, and metastasis, involving many cell types within the tumor microenvironment. However, existing methods based on next generation sequencing (NGS), hybridization microarrays, and qRT-PCR have limited sensitivity and require tedious and expensive sample preparation and detection procedures. They also need several hundred microliters of blood for EV analysis. This necessitates the sacrifice of animals in murine studies, and making it impractical for tumor monitoring in murine tumor model therapy trials. To address these issues, my lab has developed “tethered lipid nanoparticle (TLN)” technology that can be used for ultra-sensitive detection of miRNAs, lncRNA, mRNA and protein antigen targets in EVs. Analysis can be performed using only 20 µL of blood from a patient or mouse with minimal sample preparation requirement. This would enable “non-lethal” monitoring of tumor load in mice. We believe that serum/plasma EV miRNA/mRNA/membrane protein profiles can serve as detection, surveillance and prognostic biomarkers that can be used for early disease diagnosis and to monitor and predict disease response to therapy.

• Y. Wu, K.J. Kwak, K. Agarwal, A. Marras, C. Wang, Y. Mao, X. Huang, J. Ma, B. Yu, R.J. Lee, A. Vachani, G. Marcucci, J.C. Byrd, N. Muthusamy, K. Huang, C.E. Castro, M. Paulaitis, S.P. Nana-Sinkam and L.J. Lee, “Detection of Extracellular RNAs in Cancer and Viral Infection by Tethered Cationic Lipoplex Nanoparticles”, Analytical Chemistry, 85(23), 11265-11274 (2013).

L.J. Lee, Z. Yang, M. Rahman, J. Ma, K.J. Kwak, J. McElory, K. Shilo, C. Goparaju, L. Yu, W. Rom, T-K Kim, X. Wu, Y. He, K. Wang, H.I. Pass and S.P. Nana-Sinkam, “Extracellular mRNA Detected by Tethered Lipoplex Nanoparticle Biochip for Biomarker Development in Lung Cancer”, American Journal of Respiratory and Critical Care Medicine, 193(12), 1431-1433 (2016).

• Hu, Y. Sheng, K.J. Kwak and L.J. Lee, “A Signal-amplifiable Biochip Quantifies Extracellular RNAs for Early Cancer Detection", Nature Communication, 8(1),1683 (2017).

• J. Hu, K.J. Kwak, Y. Sheng and L.J. Lee,” Overhang Molecular Beacons Encapsulated in Tethered Cationic Lipoplex Nanoparticles for Detection of Single-point Mutation in Extracellular Vesicle-associated RNAs”, Biomaterials, 183:20-29 (2018).

4. Cell Based Drug Delivery- Cell-based therapeutic strategy has been proposed as a tissue engineering application for several decades. Primary or cell line with certain product secretion can be used as “seed” cell for therapeutic function. Alternative resource of cells is from gene recombination-mediated methods. As most tissue or cellular transplants, the cellular grafts are subject to immunorejection in the absence of chronic immunosuppression. For cell-based device applications in vivo, the device must provide proper cell immunoprotection with minimal inflammatory response. Furthermore, the device should possess controllable degradation characteristics such that the implant does not need to be removed after use via invasive second surgery. My lab has conducted considerable research in this area.

• X. Zhang, H. He and L.J. Lee, “A Biodegradable, Immunoprotective, Dual Nanoporous Capsule for Cell-Based Therapies”, Biomaterials, 29, 4253-4259 (2008).

• H. He, V. Grignol, V. Karpa, C. Yen, K. Laperle, X. Zhang, N.B. Jones, M.I. Liang, G.B. Lesinski, W. Ho, W.E. Carson, III and L.J. Lee, “Use of a Nanoporous Biodegradable Miniature Device to Regulate Cytokine Release for Cancer Treatment”, Journal of Controlled Release, 151, 239-245 (2011).

• F. Yang, X. Zhang, A. Maiseyeu, G. Michai, R. Yasmeen, D. DiSilvestro, K.S., Maurya, M. Periasamy, V. Bergdall, C. Sen, S. Roy, L. J. Lee, S. Rajagopalan, and O. Ziouzenkova, ”The Prolonged Survival of Fibroblasts with Forced Lipid Catabolism in Visceral Fat Following Encapsulation in Alginate-poly-L-lysine”, Biomaterials, 33(22), 5638-5649 (2012).

• H. He, E. Luedke, X. Zhang, B. Yu, A. Schmitt, B. McClarren, V. Grignol, W.E. Carson III and L.J. Lee, “A Nanoporous Cell-Therapy Device with Controllable Biodegradation for Long-Term Drug Release”, Journal of Controlled Release, 165(3), 226-233 (2013).

5. Polymer Based Micro/Nanofluidics Biochips- Micro/nanotechnology is initiated from the electronics industry. It has been extended to micro-electro-mechanic system (MEMS) and nano-electro-mechanic system (NEMS) for producing miniature devices based on silicon and semi-conductor materials. However, the use of these hard materials alone is inappropriate for many biomedical devices. Soft polymeric materials possess many attractive properties such as high toughness and recyclability. Some possess excellent biocompatibility, are biodegradable, and can provide various biofunctionalities. Proper combinations of micro/nanoelectronics, polymers, and biomolecules can lead to new and affordable medical devices. My team has established a series of

non-cleanroom and cleanroom manufacturing techniques using biocompatible polymers, biomolecules, and nanoparticles as building blocks as well as micro/nanofluidics as a mechanism to design, synthesize, and fabricate biomedical and therapeutic devices. In addition to drug/gene delivery devices and cell-based constructs described earlier, we have also developed various biosensors/chips using advanced micro/nanofluidics concepts for medical diagnostics.

• J. Guan and L.J. Lee, “Generating Highly Ordered and Stretched DNA Arrays”, Proceedings of National Academy of Science, 102(51), 18321-18325 (2005).

• S. Wang and L.J. Lee, “Dynamic Assembly by Electrokinetic Microfluidics”, Journal of American Chemical Society, 129(2), 254-255 (2007).

• Y. Yang, D. Liu, L.J. Lee, and D.L. Tomasko, “Low Temperature Fusion of Polymeric Nanostructures Using Carbon Dioxide”, Advanced Materials, 19(2), 251-254 (2007).

• P. Boukany, O. Hemminger, S-Q Wang and L.J. Lee, “Molecular Imaging of Slip in Entangled DNA Solution”, Physical Review Letters, 105(2), 027802 (2010)

Shared Facility Highlights

  • Dynamic light scattering goniometry
  • Total internal reflection fluorescence (TIRF) microscopy
  • Bio-AFM