This NSF award by the Biosensing /CBET program supports work by Professor Yu at University of Illinois at Urbana-Champaign to develop a set of robust, functional and readily-applicable nanowire needles/electrodes and a novel and powerful method for direct delivery of a trace but controlled amount of probes, such as QDs, nanoparticles and molecular probes, into the specific compartments in the same cell or different cells with minimal intrusiveness, bypassing the endocytic pathways.
More specifically, a new nanofabrication strategy will be applied for the practical and economical fabrication of the nanowire needle/electrode so to make it compatible with the general practice in bioscience laboratory and readily adaptable for broad use. Thiol-based conjugation chemistry for attaching probes onto nanowire needle/electrode surfaces will be designed and later exploited for realizing near instantaneous and targeted release of the attached probes inside cells or sub-cellular structures. We will further demonstrate the site specific delivery of magnetic nanoparticles and the intracellular mechanical studies with magnetic twisting cytometry to determine heterogeneity of stress propagation, important for understanding mechanotransduction pathways of cells. The development represents thus a potentially transformational improvement over the existing methods, and can open up novel venues to conjure innovative strategies, which otherwise would be impractical or even impossible, for the advanced biological studies of living cells. New tools developed in this proposal will be made available for members in the research community to significantly benefit the expanded studies of diversified and fundamental problems in cell biology. The method and technology developed in this project are also appealing subjects for the education of students and the public.
Nature of the project: The nature of this project is to develop new nanoneedle probes for detection of intracellular and intranuclear biological signals. A human body is made of many trillions of living cells that are fundamental to the function of the tissues and organs of the human being. Each cell has a plasma membrane (made of lipids and inserted with proteins) that selectively allows only certain molecules to pass through at certain time. Inside each cell, there is a nucleus in most cell types. The nucleus is where the DNA replication and mRNA transcription occur and is also enclosed with another membrane called nuclear envelope that allows limited molecules to pass across it at certain times. Therefore, it has been rather challenging to actively deliver molecules into the cytoplasm of the cell and it is even more difficult to deliver molecules into the nucleus. People have used different strategies to overcome this limitation by placing nano-sized quantum dots (QDs) on the plasma membrane and allowing the cell to engulf the QDs. However, this process is not controllable and many times the QDs are aggregated in large quantities inside the cytoplasm, causing undesirable negative effects on the cell function and too many QDs in a cell can become toxic to the cell. Outcomes of the project: Supported by this NSF grant, we have developed a nanoscale mechanochemical method to deliver single fluorescent quantum dots (QDs) into a specific location in the cytoplasm or the nucleus of a single individual living cell, using a membrane-penetrating nanoneedle. Since the nanoneedle is only ~50 nm in diameter, it causes little damage to the plasma membrane, nuclear envelope, and to the intracellular and intranuclear structures, thus overcoming the limitations of the existing microneedle technology. We also showed that the ability to deliver and track QDs and other molecules has opened the door for unconventional strategies for studying biological processes and biophysical properties in living cells with spatial and temporal precision, potentially with molecular resolution. This method has been published in Nano Letters (Yum et al, 2009). We then build on the nanoneedle technology and developed a controlled delivery and release method that uses a cargo-carrying nanoneedle (doubly served as a nanoscale electrode) to penetrate into the nucleus of a living cell. To overcome the limitation of the first generation of nanoneedles that depend on passive release of the QDs into the cytoplasm or the nucleus, we exploited an electrochemical means to directly break the incorporated electrochemically-active bonds by applying a small external electrical potential through the nanoneedle to rapidly release the attached cargo inside the nucleus, we then exploited an electrochemical means to directly break the incorporated electrochemically-active bonds by applying a small external electrical potential through the nanoneedle. We demonstrate that the release of the cargo is thus almost instantaneous and is externally controlled by adjusting the magnitude and duration of the applied potential. We further show the use of this active delivery method to deliver singly dispersed QDs into the nucleus. This new exciting technology opens the door to actively control delivery of QDs and other small biological molecules into the specific location of the cytoplasm or the nucleus. This technology has been published in Small (Yum et al, 2010). Stimulated by our nanoneedle approach, we developed a novel solid nanoneedle-based intracellular delivery method, named nano-mechanoporation, which uses a nanoneedle to mechanically slice the cell membrane to form a transient, localized nanoscales slit for transferring exogenous molecules into a living cell. We have demonstrated the delivery of various small molecules quickly in large quantities of fluorescent dextrans, phalloidin, and quantum dot nanoparticles through the localized nanoscale slit into living HeLa cells. We further show that the simple procedure of the nano-mechanoporation can be adapted to standard biological techniques and other nanotechnology-based methods, introducing new strategies for studying intracellular processes and biophysical properties of individual living cells and for delivering molecules into a single cell, causing little pain or damage to the cell plasma membrane. This technology has been awarded a US patent (Yum, Yu, and Wang, 2013). In summary, 3 novel unique methods of delivering single or multiple molecules into a single live cell have been developed using a nanoneedle. These approaches are useful for biological studies and may be extended for in vivo clinical applications in the future. Peer-reviewed original articles Kyungsuk Yum, Ning Wang and Min-Feng Yu. Electrochemically-Controlled Deconjugation and Delivery of Single Quantum Dots into the Nucleus of Living Cells, Small 6, 2109-2113 (2010). Kyungsuk Yum, Sungsoo Na, Yang Xiang, Ning Wang and Min-Feng Yu. Mechanochemical Delivery and Dynamic Tracking of Single Fluorescent Quantum Dots in the Cytoplasm and Nucleus of Living Cells. Nano Letters 9, 2193-2198(2009). Patent Yu M, Yum K, Wang N. Methods for Nano-Mechanoporation. US Patent No. 20,130,137,129. May 30, 2013.
