Application of single crystalline Au nanowire for injection of genetic information

Abstract

During several decades, nanomaterials have achieved outstanding results in various research fields due to their intrinsic properties such as electrical, optical, mechanical properties and their nano dimension. Among diverse nanomaterials, gold (Au) nanostructures including Au nanoparticles, Au nanorods, Au nanowires and so on have some advantages

high biocompatibility, facile surface modification through gold-sulfur interaction, chemical inertness and high-reproducibility in synthetic process. Therefore, the Au nanomaterials have been valuable tools for biological application such as imaging, sensing, gene and drug delivery, and analysis of cellular behavior. Especially, due to their high biocompatibility and membrane permeability, Au nanomaterials have been used to efficiently deliver a sort of cargos in living cells such as siRNA, nucleic acids, and drugs.

Especially, recent studies have reported that nanowires can significantly improve the efficiencies of biochemical processes. Because nanowires (NWs) can transport electrical and optical signals/stimuli and biomaterials into living organisms, they can form nano-sized functional biointerfaces. Particularly, the NWs can reach a specific target in a cell or tissue with nanoscale spatial resolution and minimize the invasiveness owing to ultrathin diameters. In this dissertation, synthesis of single-crystalline 1-dimensional Au nanowire by chemical vapor deposition (CVD) and application of a fabricated Au NW injector (Au NWI) as a biological nanomaterial are reported. By introducing the Au NWI for injection of genetic information into mouse embryos or porcine spermatogonial stem cells (pSSCs), which is a first step for producing transgenic animals, it achieves improved results compared with conventional methods for injecting genetic information. This dissertation consists of as follows. Chapter 1 demonstrates a synthesis method of single-crystalline 1-dimensional Au nanowires (Au NWs) by chemical vapor transport method and electrochemical, mechanical and structural characteristics of the Au NWs. In addition, it describes the fabrication process of Au NW injector (Au NWI) for biological application of 1-dimensional single-crystalline Au NWs as a nano-biomaterials and electrochemical properties of the fabricated Au NWI.

In chapter 2, the development of nanoinjection system by introducing a 1-dimensional Au NWI for delivering plasmid into the pronucleus of a mouse embryo is reported. Since a zygote, which is a fertilized 1-cell stage embryo, has two physical barriers (cytoplasmic membrane and zona pellucida), the direct delivery of plasmids into a zygote pronucleus (PN) is more difficult than other mammalian cells. To direct delivery of the exogenous gene into a PN of an embryo, a glass capillary which has μm diameter has been generally used. However, it could induce physical damages on embryos by a quite thick diameter. In addition, the chemical damages could be induced by a chemical buffer solution which is delivered with plasmids. On the other hand, a Au NWI can directly deliver exogenous genes into the PN of an embryo by applying an electric pulse without using the extracellular buffer. To penetrate the two physical barriers with minimal disruption of the embryo, I defined the optimal diameter and length of the Au NWI. By using the optimized Au NWI, a green fluorescent protein (GFP)-encoding plasmids were delivered and the expression of GFP was confirmed in blastocysts stage embryos. It was found that the mosaicism highly reduced in the Au NWI injected embryos compared with the micropipette injected embryos. Since the frequent occurrence of the mosaicism has been a very important issue in the efficient production of the transgenic mouse, this result suggests that our Au NWI can improve the delivery efficiency with suppressed mosaicism and it can become very useful nano-biomaterial.

Chapter 3 demonstrates a successful genetic modification of porcine spermatogonial stem cells (pSSCs) via an electrically responsive Au nanowire injector (E-R Au NWI). In this study, pSSCs were adopted as an exogenous gene carrier. The SSCs can self-renew infinitely and differentiate into sperm cells through spermatogenesis in the seminiferous tubule of the testis. As a result, the genetically modified pSSCs can be a highly useful vehicle for transmitting transformed genetic traits to the next generation and moreover, they can achieve efficient germline transmission, resulting in efficiently producing transgenic animals. The E-R Au NWI can noninvasively interface with the nucleus of the pSSC. It can directly deliver plasmid into the nucleus by applying an electrical stimulus. After 1 day from the delivery, successful expression of the GFP was confirmed and maintenance of SSC characteristic was confirmed by analyzing a GFRα1 which is a SSC-specific marker. Especially, when compared to the transfection efficiencies of conventional nonviral vector-based gene delivery methods such as jetPEI, Lipofectamine, and electroporation, the E-R Au NWI-based method improved the pSSC transfection efficiency by at least 6.7-fold and even up to 46.7-fold. Furthermore, transgenic pSSCs containing the human bone morphogenetic protein 2 gene were successfully obtained by using E-R Au NWIs. This result suggests that the E-R Au NWI enables the efficient genetic modification of pSSCs and can be employed to produce diverse kinds of transgenic pigs.