PDMAEMA Modified Nanoparticles With Different Morphologies As Effective Gene Vectors

Gene therapy provides new treatments for some inherited and acquired diseases by introducing genetic materials (DNA or RAN), which will modulate cellular responses and functions by expressing exogenous proteins or inhibiting induction of a particular gene, to target cells. However, in the process of delivering genes into target cells, nucleic acid is vulnerable to damage or enzymatic digestion by nucleases. So the key of gene therapy is to design an delivery system that can transfer nucleic acid drugs to target cells efficiently. Viral vectors and nonviral vectors are two categories of gene vectors. Traditional viral vectors show high transfection efficiency but also significant toxicity and immunogenicity. Nonviral vectors, such as lipids, polycations, have increasingly attracted much attention due to their biocompatibility and potential for large-scale production. However, unlike viral analogues, nonviral gene vectors have to overcome numerous extra-and intracellular obstacles, such as cellular uptake and endosomal escape, before they deliver nucleic acid drugs to destination. So the effectiveness and safety are the two major problems of delivery system. In addition, medical imaging plays an indispensable role in the clinical diagnosis and treatment process. So many studies have focused on designing gene vectors with the function of imaging. Well-defined cationic poly((2-dimethylamino)ethyl methacrylate) (or PDMAEMA) can spontaneously condense negatively charged DNA into compact nanocomplexes, protect plasmid DNA from enzymatic degradation by nucleases, and facilitate cellular transfection. PDMAEMA with high molecular weight as gene vector demonstrates good transfection performance but at the same time show high cytotoxicity. Nanoparticles with unique morphology are easier internalized by cells, and the higher efficiency of cellular internalization will improve transfection efficiency. Therefore, on account of the key issues and trends of the gene therapy, we fabricated a series of PDMAEMA modified efficient gene vectors that based on nanoparticles with unique morphology.Layered double hydroxides (LDHs), known as anion host-guest-layered nanoparticles, is a potential inorganic matrix for gene delivery due to its characters of layer and ion-exchangeable. However, the unsatisfactory transfection performance mediated by LDH needs to be further developed. It was reported that redox-responsive gene vectors containing disulfide bonds reveal excellent transfection performance. Disulfide bonds can be cleaved by reducing reagents in tumor tissues, such as glutathione (GSH). Redox-responsive vectors remained stable in extracellular fluids, but rapidly collapsed in tumor cells with the cleavage of disulfide bonds and release the cargo. In this dissertation, surface-initiated atom transfer radical polymerization (ATRP) of DMAEMA was applied to tailor the functionality of LDH surfaces and produce a series of well-defined layered LDH-based gene vectors (termed as LDH-PDs). ATRP initiation sites that containing disulfide bonds were introduced onto LDH surfaces in advance. The prepared LDH-PDs showed stronger capacity to condense plasmid DNA (pDNA) and much higher transfection efficiency in COS7 and HepG2 cell lines than the pristine LDH nanoparticles. Furthermore, the layered LDH-PDs can enhance cellular uptake obviously. This study reveals that PDMAEMA modified LDH is a meaningful gene vector for gene delivery systems.Carbon nanotubes (CNT) with admirable penetrability and encapsulation efficiency showed bright prospect in the field of drug delivery systems. Biocompatible rodlike cellulose nanocrystals (CNCs) were reported as an alternative of CNT in medical field due to their excellent physicochemical properties. In addition, rodlike nanoparticles are internalized faster than spherical nanoparticles, and our recent work also proved that rodlike nanoparticles have a significant effect on gene transfection. So based on the research of PDMAEMA modified LDH-based gene vectors, CNCs were modified with disulfide bond-linked PDMAEMA brushes for effective gene delivery applications. A variety of CNC-graft-PDMAEMA gene vectors (termed as CNC-SS-PDs) with different molecular weights of PDMAEMA side chains were produced. The gene condensation ability, redox-response, cytotoxicity, transfection efficiency, and in vitro/in vivo antitumor activities of CNC-based gene vectors were studied detailedly. The experimental results showed that, under reducible conditions, PDMAEMA chains can be cleaved from CNCs easily; and the needlelike shape of CNC matrix have important impact on cellular uptake. The CNC-SS-PDs exhibited outstanding transfection efficiency and low cytotoxicity. The antitumor effect of CNC-based gene vectors was assessed by a suicide gene/prodrug system (cytosine deaminase/5-fluorocytosine, CD/5-FC) in vitro and in vivo.It has been great interest to combine imaging and gene delivery for cancer therapy. Gold nanoparticles (Au NPs) can act as contrast agent due to the high atomic number, electron density, and X-ray absorption coefficient. In order to impart computed tomography (CT) imaging function to CNCs, based on previous research, Au NPs as CT contrast agents were in-situ formed on the CNC-based carriers by using the amine groups of PDMAEMA chains as reducing and protective agents. The gold (Au) nanoparticle-conjugated heterogeneous polymer brush-coated CNCs were proposed via different controllable polymerization techniques for effective biomedical applications. One bi-functional CNC-based initiator was first prepared to initiate both ATRP and reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(poly(ethylene glycol)ethyl ether methacrylate) (PPEGEEMA) and PDMAEMA brushes were individually grafted from CNCs. The cationic PDMAEMA chains complex genes effectively, while the uncharged PPEGEEMA brushes spread outwards and reduce the cytotoxicity significantly. The in vitro CT imaging, gene condensation ability, cytotoxicity, gene transfection, and cellular uptake of CNC-based vectors were investigated in detail. Such effective CNC-based gene vectors with gold nanoparticle-conjugated heterogeneous polymer brushes would be one promising multifunctional therapy system.In conclusion, we fabricated a series of efficient gene vectors that based on PDMAEMA modified nanoparticles with different morphologies. Satisfactory experimental results of redox-responsive gene vectors and multifunctional gene vector were obtained. The research about gene vectors in this dissertation may provide new strategies to develop promising gene vectors and useful information for the application of nanoparticles in biomedical areas.