Development of Hybrid Materials and Nanostructures Based on Biopolymers for Nucleic Acid/Drug Delivery

Abstract

A large number of polymers have been utilized in various fields due to the flexibility in which their compositions, molecular weight, and structures. Especially biocompatible polymers which have no toxic or injurious effect on biological systems are called ‘biopolymers’. Over the past decade, biopolymer-based systems have attracted much attention in gene and drug delivery. This is mainly because polymers can be easily custom designed to overcome extracellular/intracellular obstacles of delivery process and to attain the desired effective properties of therapeutic agents. Nevertheless there are needs for more intelligent delivery systems to treat various diseases. In this thesis, novel strategies for the advanced, polymer-based delivery systems are presented. In Part 1, it gives a brief overview of nucleic acid/drug delivery systems based on biopolymers.In Part 2, polymer-inorganic nanoparticle hybrid materials were developed as multifunctional nucleic acid delivery carriers. I presented that those hybrid systems exhibited not only intrinsic properties of each component but also synergistic effects on gene transfer efficacy. In Chapter 1 on Part 2, cationic polymer-superparamagnetic iron oxide nanoparticles (SPION) hybrid materials were prepared to achieve an external magnetic field guided, local gene delivery and a magnetic resonance (MR) imaging. Low molecular weight (LMW) branched polyethylenimine (BPEI) were chemically conjugated on the surface of SPION, resulted in BPEI-SPION. BPEI-SPION efficiently loaded plasmid DNA (pDNA) by electrostatic binding interaction and formed cloud-like particle (magnetoplex) composed of several discrete BPEI-SPIONs. This magnetoplex showed much higher transfection efficacy in prostate cancer cell line in the presence of external magnetic fields compared to in the absence of magnetic fields. By time-dependent transfection study, I demonstrated that the rapid sedimentation of magnetoplexes on the cell surface by external magnetic field drove the increased cellular uptake of it and thereby it exhibited significant increase in transfection results. In addition, this rapid and targeted magnetofection render the successful delivery of a therapeutic gene (interleukin-10 coded pDNA) into primary human vascular endothelial cells with high cell viability. Meanwhile BPEI-SPIONs internalized in cancer cells were traced by T2-weighted gradient echo MR imaging in dose-dependent manner.In Chapter 2 on Part 2, a multifunctional polymer-silica nanotube (SNT) hybrid was presented for nucleic acid delivery and dual imaging. Due to their distinct structure property, SNTs were differently functionalized: their inner void was filled with quantum dots (Q-dot) and iron oxide nanoparticles for fluorescence and MR imaging and their outer surface were decorated with LMW BPEI for loading gene respectively. As BPEI-SNT had high cationic charge density on the surface by chemically conjugated ~107 molecules of BPEI, it could form compact complex with pDNA. BPEI-SNT exhibited much higher degree the gene transport efficacy and the gene expression efficiency in cancer cell line compare to unconjugated LMW BPEI and bare SNT. Moreover the behavior of BPEI-SNT/pDNA complex at cellular level was studied by confocal florescence microscopy using green Q-Dot in the BPEI-SNT. Finally the enhanced MR imaging of BPEI-SNT treated cancer cells revealed the potential of BPEI-SNT to act multiple modality nanoparticle. In Part 3, self-assembled nanostructures based on polymer-drug conjugates were developed for intracellular drug delivery. I proposed a new perspective on two molecules, small interfering RNA (siRNA) and paclitaxel (PTX), that those have dual potency as the therapeutic drug and the building block to fabricate nanoconstructs when they were grafted to polymer chains. In Chapter 1 on Part 3, reductively responsive and siRNA-incorporated nanoconstructs (NCs) were presented. This NC was composed with biocompatible natural polymer (dextran) and siRNA conjugates. By the sequence-specific hybridization of two complementary single strand siRNAs which were conjugated on the dextran via disulfide linkage, the spherical NCs were successfully formed and it had an average diameter of ~300 nm. The siRNAs formulated in NC were physically protected from serum enzymes and efficiently transported into cancer cells compared to free siRNA. In reductive cytoplasmic condition, the NCs were disintegrated and simultaneously released double stranded siRNA due to cleavage of disulfide linkages. As a result, the NCs lead efficient gene silencing without cytotoxicity. I demonstrated that only reducible NCs could induce sequence-specific siRNA-mediated gene knockdown. Furthermore a peptide aptamer (peptamer) was introduced on the NC to achieve specific cancer cell line targeted siRNA delivery. In Chapter 2 on Part 3, inclusion complex mediated and water-dispersible nanoparticles (NPs) were constructed to deliver PTX into cancer cells. β-cyclodextrin (CD) and PTX containing hydroxyl groups were grafted to two kinds of poly(maleic anhydride) via forming ester linkages. Thus Poly-CD and Poly-PTX conjugates were prepared. By mixing those two conjugates (CD:PTX=1:1) in water-ethanol solution, the pCD::pPTX self-assembled NPs were successfully formulated. It was due to the formation of inclusion complexes between CD and PTX and the entanglement of polymer chains. The diameter of this NP was around 55 nm determined by TEM image. I validated that pCD::pPTX was highly robust owing to the multiplicity of the CD::PTX host-guest complexes by measuring the association constant. Moreover the molecular dynamic simulation provided structural details that support the stability of pCD::pPTX NPs compare to that of CD::PTX complexes. The pCD::pPTX NP released PTX in physiological condition by the hydrolysis of ester linkages and/or the dissociation of PTX from the inclusion complexes. Finally, I demonstrated that pCD::pPTX NPs induced a higher cytotoxicity than free PTX in three cancer cell lines because of its stable structure, high aqueous solubility, and the enhanced release rate of PTX.