The delivery of nucleic acids at the tissue and cellular levels remains one of the major hurdles in this scientific area. Since nucleic acids are bulky macromolecules and unstable in the presence of nucleases, vehicles are required to compact them into nanosized particles, offer protection from degradation in vivo, and release the therapeutic cargo at the desired location. Polycationic vehicles are good candidates for these purposes since they can be chemically modified to tune the desired properties in nanoparticle formulations. We designed a family of trehalose-oligoethyleneamine copolymers that showed promising plasmid DNA (pDNA) transfection results in the presence of serum proteins. A diazidotrehalose monomer was copolymerized with linear oligoethyleneamines of varying length and containing alkyne end-groups via step-growth Cu(I)-catalyzed azide-alkyne cycloaddition polymerization resulting in a series of trehalose copolymers with a range of secondary amines (from 4 to 6) within the polymer backbone. Upon electrostatic complexation of the polycations and pDNA in aqueous media, nanosized particles were formed, and their sizes and zeta-potentials were characterized via dynamic light scattering (DLS). The glycopolymers were tested for pDNA binding, toxicity, cellular uptake, and transfection efficiency in vitro. Characterization of these polymers revealed a significant influence of minor structural modifications on bioactivity. In general, all of the polymers efficiently bind pDNA at low nitrogen to phosphate (N/P) ratios forming nanoparticles below 100 nm in size and demonstrated cellular uptake and transfection. Polymers comprised of trehalose moieties and four secondary amines in the repeat unit showed the greatest promise in pDNA delivery in vitro. Because of its large hydration volume, we hypothesize that trehalose contributes to particle stabilization in serum. The trehalose-based polymers with four secondary amines (Tr4) were subsequently modified with PEG (5kDa). This modification lead to the development of well-defined polymeric structures with PEG moieties selectively incorporated at the ends of linear trehalose-oligoethyleneamine polycations. The study of the effect of this modification on bioactivity revealed that there were no significant difference in the toxicity profiles within this series of PEGylated and non-PEGylated materials; however, overall results suggest that both modified and unmodified trehalose-oligoethyleneamine copolymers have a great promise for stem cell-based and regenerative therapies. / Ph. D.

Nucleic acid therapy holds real promise to offer less severe (lower side effects) as a treatment for life threatening and difficult to treat diseases such as cancer, heart disease or Alzheimer's disease. Theranostic nanomaterials that combine diagnostic imaging and therapeutic delivery, have potential to minimize the amount of time and dosage required for the treatment. This is achieved via delivery of nanoparticles that carry therapeutic payload as well as imaging agents; these agents need to circulate in the body longer due to its (larger) size and selectively accumulate in the tumor regions through the enhanced permeability and retention (EPR) effect. We have designed novel lanthanide (Gd, Tb or La) containing polymers with oligoethyleneamine and lanthanide chelating units to incorporate DNA binding and imaging agent functionality. Protonable amines along the polymer backbone electrostatically interact with DNA and compact it into a nanoparticle. These nanoparticles can be imaged both in vivo (Gd analogues, magnetic resonance imaging) and intracellularly (Tb chelation, fluorescence spectroscopy). Polymers were synthesized via step-growth polymerization to achieve a degree of polymerization of 18-24 for different analogues with varying amine number (three to six, N3-N6) along the backbone. Dynamic light scattering performed on the polyplexes (polymer-DNA complexes) indicate that they are in nanometer size range (50-80nm). All the polymers used to form polyplexes exhibited low toxicity to cultured human Glioblastoma cells (U-87) and showed variable transfection efficiency dependent on structure, comparable to G4 (sold as Glycofect"), a commercial transfection agent previously developed in our lab. This dissertation describes the first studies by the Reineke lab to monitor polyplex formation and destabilization using lanthanide resonance energy transfer (LRET). Polyplexes were formulated with Tb chelated N5 polymer and tetramethyl rhodamine (TMR) labeled pDNA, which are "LRET pairs". We observed decrease in luminescence intensity of Tb polymer (donor) in close proximity of TMR DNA (acceptor) in an intact polyplex at different N/P ratios. However, upon destabilization of polyplexes by addition of salt or heparin solution, the increase in distance between donor and acceptor resulted in increase in the luminescence intensity of Tb polymer. With the LRET technique, we are able to monitor formation and destabilization of polyplexes by monitoring change in luminescence of the donor chromophore (Tb). Polyplexes formulated with non-paramagnetic analogues (La chelated) of N4, polyethyleneimine (PEI) and G4 were studied using NMR to quantify free vs. bound polymer in a formulation. The amount of free polymer was measured by integrating the broad resonances from nanometer-sized particles (polyplexes) with narrow peaks from free polymer chains. This was supported by using an internal reference method to quantify free polymer amount from known internal reference concentration. We observed an increase in the amount of free polymer with N/P ratio for all three systems and both the methods showed comparable results. / Ph. D.

Different structures of non-viral cationic polymer delivery vehicles, including charge center type, molecular weight and degradability, could significantly affect toxicity, release of nucleic acid and transfection efficiency. Poly(glycoamidoamine)s (PGAAs) contained different carbohydrate and secondary amine moieties and showed high transfection efficiency to different cell lines in a nontoxic manner. The "proton sponge hypothesis" has attempted to relate the buffering capacity to endosomal release of polyethylenimine (PEI) based polyplexes, which could contribute to high transfection efficiency. Secondary amine structures rendered PGAAs buffering capacity around physiological pH. To test the feasibility of the mechanism for PGAAs, new no buffering capacity guanidine or methylguanidine containing poly(glycoamidoguanidine)s (PGAGs) were synthesized. PGAGs formed stable polyplexes with pDNA from N/P (# secondary amine or guanidine group on polymer backbone / # phosphate group on pDNA backbone) ratio 3. PGAG based polyplexes expressed low cytotoxicity and were internalized by 90% of cells at N/P 25. Furthermore, two PGAG based polyplexes showed higher transfection efficiency from N/P 5 to 30 than their PGAA based analogs. These data suggested the low transfection could be due to the difficulties to release pDNA from polyplexes; also, the "proton sponge theory" could not explain the higher transfection efficiency by some PGAGs. Degradation of delivery vehicles could potentially release pDNA in cells and increase transfection efficiency. PGAAs degraded rapidly at physiological conditions and the proposed mechanism was amide hydrolysis. Typically, amide groups are stable and hydrolyze slowly in absence of enzyme. Different models mimicking PGAAs were synthesized to study the fast hydrolysis. Amide groups showed asymmetric hydrolysis. Different hydrolysis behaviors suggested neighboring group participation of two terminal groups to induce rapid amide hydrolysis. These new models could potentially be used to design new polymer delivery vehicles with various degradation profiles. / Master of Science

Glioblastoma multiforme is the most aggressive form of malignant brain tumor with poor prognosis. The efficacy of brain cancer treatment by chemotherapeutics is limited by the blood-brain barrier (BBB) which allows less than 2% of the small molecules and blocks almost all the macromolecules to transport into the brain. Delivery of the large molecules such as proteins and nucleic acids across the BBB is a great challenge for brain-targeted drug delivery. To overcome this obstacle, cell-penetrating peptides (CPPs) were used as vectors for delivery of nucleic acids across the BBB targeting glioblastomas. The CPPs have shown such promising carriers to deliver various cargoes ranging from small molecules to large molecules into the cells. This thesis is focused on the development of glioblastoma-targeting vectors based on modifications of the CPPs and the targeting peptides. The peptide-based vectors were developed to improve the transport of the nucleic acids across the BBB and specifically target glioblastomas. In this thesis, a series of peptide-based vectors targeting glioblastomas were synthesized and modified with targeting peptides by either covalent conjugation or non-covalent complex formation. The delivery of plasmid DNA (pDNA) in the complex with the peptide-based vectors was studied in the in vitro model of the BBB. The role of receptors expressed on the BBB was investigated. Scavenger receptors class A and B were found to be expressed on the BBB, and they were involved in the delivery of the pDNA across the BBB model. Moreover, various targeting peptides were modified with hexaglutamate to form non-covalent complexes with the CPPs for small interfering RNA (siRNA) delivery to glioblastoma cells. The non-covalent complex of the CPP and the targeting peptide showed greater gene-silencing efficiency than the consecutively covalent conjugation of the CPP and the targeting peptide for siRNA delivery to glioblastoma cells. Lastly, a number of novel, amphipathic peptides were developed based on the model amphipathic peptide. The prediction of the biological effect of the designed peptides using quantitative structure-activity relationship model showed a correlation with the experimental data. Finally, the CPP-based nucleic acid delivery vectors with homing peptide strategy have a potential for the BBB shuttle and the future use as a glioblastoma-targeted drug carrier in the in vivo studies and the clinical applications. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

No description available.

Mesoporous silica nanoparticles (MSNPs) possess large surface areas and ample pore space that can be readily modified with specific functional groups for targeted binding of bioactive materials to be transported through cellular barriers. Engineered silica nanoparticles (ESNP) have been used extensively to deliver bio-active materials to target intracellular sites, including as non-viral vectors for nucleic acid (DNA/RNA) delivery such as for siRNA induced interference. The reverse process guided by the same principles is called “nanoharvesting”, where valuable biomolecules are carried out and separated from living and functioning organisms using nano-carriers. This dissertation focuses on ESNP design principles for both applications. To investigate the bioactive materials loading, the adsorption of antioxidant flavonoids was investigated on titania (TiO2) functionalized MSNPs (mean particle diameter ~170 nm). The amount of flavonoid adsorbed onto particle surface was a strong function of active group (TiO2) grafting and a 100-fold increase in the adsorption capacity was observed relative to nonporous particles with similar TiO2 coverage. Active flavonoid was released from the particle surface using citric acid-mediated ligand displacement. Afterwards, nanoharvesting of flavonoids from plant hairy roots is demonstrated using ESNP in which TiO2 and amine functional groups are used as specific binding sites and positive surface charge source, respectively. Isolation of therapeutics was confirmed by increased pharmacological activity of the particles. After nanoharvesting, roots are found to be viable and capable of therapeutic re-synthesis. In order to identify the underlying nanoparticle uptake mechanism, TiO2 content of the plant roots was quantified with exposure to nanoparticles. Temperature (4 or 23 °C) dependent particle recovery, in which time dependent release of ESNP from plant cells showed a similar trend, indicated an energy independent process (passive transport). To achieve the selective separation and nanoharvesting of higher value therapeutics, amine functionalized MSNPs were conjugated with specific functional oligopeptides using a hetero-bifunctional linker. Fluorescence spectroscopy was used to confirm and determine binding efficiency using fluorescently attached peptides. Binding of targeted compounds was confirmed by solution depletion using liquid chromatography–mass spectrometry. The conjugation strategy is generalizable and applicable to harvest the pharmaceuticals produced in plants by selecting a specific oligopeptide that mimic the appropriate binding sites. For related gene delivery applications, the thermodynamic interaction of amine functionalized MSNPs with double-stranded (ds) RNA was investigated by isothermal titration calorimetry (ITC). The heat of interaction was significantly different for particles with larger pore size (3.2 and 7.6 nm) compared to that of small pore particles (1.6 nm) and nonporous particles. Interaction of dsRNA also depended on molecular length, as longer RNA (282 base pair) was unable to load into 1.6 nm particles, consistent with previous confocal microscopy observations. Calculated thermodynamic parameters (enthalpy, entropy and free energy of interaction) are essential to design pore size dependent dsRNA loading, protection and delivery using MSNP carriers. While seemingly diverse, the highly tunable nature of ESNP and their interactions with cells are broadly applicable, and enable facile nano-harvesting and delivery based on a continuous uptake-expulsion mechanism.

Utilizing the diverse functionality of amino acids, a new class of amphiphilic graft copolymers has been synthesized, characterized, and explored for applications in biomaterials and nucleic acid delivery. This thesis research focused on the syntheses of oligopeptide-functionalized polyesters and polyolefins. Polyester functionalization was geared towards applications in biomaterials, tissue engineering, and drug delivery by incorporating sequences that promote cell-adhesion. These polyester- graft -oligopeptide materials were prepared by a 1,3-Huisgen cycloaddition reaction, "click" chemistry, of an azide-terminated oligopeptide (prepared by Fmoc-based solid phase peptide synthesis (SPPS)) and alkyne-containing polyester (synthesized by ring-opening polymerization). Following the syntheses of these materials, they were analyzed by nuclear magnetic resonance (NMR) and organic gel permeation chromatography (GPC). The oligopeptide-functionalized polyolefins were designed for nucleic acid complexation, and therefore the oligopeptide sequences were intended to incorporate positively-charged moieties ( e.g. , oligolysine) for DNA and short interfering RNA (siRNA) complexation. These graft copolymers, prepared by SPPS followed by ring-opening metathesis polymerization, have highly tunable structures that enable control over charge density and polymer backbone rigidity. Moreover, non-ionic hydrophilic grafts such as polyethylene glycol were integrated into these polyelectrolytes such that the charges along the polymer backbone are spaced accordingly while maintaining the hydrophilicity of the polymer. While numerous applications for such charged, "bio-tailored" materials can be envisioned, this work is geared towards positively-charged polyelectrolytes for their potential application in nucleic acid therapy, specifically the delivery of plasmid DNA and siRNA. These graft copolymers were characterized ( 1 H, 13 C NMR, organic and aqueous GPC), studied for their solution properties (static and dynamic light scattering), and investigated as polyplexes with plasmid DNA.

Le cancer est la principale cause de mortalité au Canada. Les taxanes (e.g. le paclitaxel et le docétaxel (DCTX)) constituent des remèdes efficaces contre une série de tumeurs solides telles que les cancers du sein, du poumon et de l’ovaire. Par ailleurs, des acides nucléiques (e.g. les oligonucléotides antisens (AON) ou les petits ARN interférents (siRNAs)), capables de supprimer sélectivement certains oncogènes impliqués dans la carcinogénèse, sont actuellement étudiés pour traiter une large gamme de cancers. Bien que l’activité des taxanes et des acides nucléiques soit bien établie sur des modèles humains et/ou animaux, plusieurs aspects physico-chimiques et cliniques restent encore à améliorer. Leur solubilité limitée (pour les taxanes), leur dégradation rapide dans le sang (pour les acides nucléiques), leur élimination précoce, leur absence de sélectivité et leur toxicité envers les tissus sains sont les principaux facteurs limitant leur efficacité. C’est pourquoi de nombreux efforts ont porté sur l’élaboration de systèmes de vectorisation ciblés à base de polymères, dans le but de surmonter les problèmes associés aux thérapies actuelles. Dans cette thèse, deux types de micelles polymères ont été développés pour la vectorisation de DCTX et d’acides nucléiques. D’une part, des micelles de poly(oxyde d’éthylène)-bloc-poly(oxyde de butylène/styrène) ont été étudiées pour la première fois pour solubiliser le DCTX et le protéger de l’hydrolyse. Ces polymères se sont révélés moins toxiques que le surfactant utilisé commercialement pour solubiliser le DCTX (i.e. polysorbate 80) et ont permis une libération prolongée du principe actif. D’autre part, deux systèmes différents de micelles polyioniques (PICM) ont été mis au point pour la vectorisation d’acides nucléiques. De nouveaux conjugués de poly(éthylène glycol) (PEG)-oligonucléotide ont été proposés pour la protection et la libération contrôlée d’AON. Lorsque ces conjugués ont été formulés avec des dendrimères de poly(amidoamine) (PAMAM), des complexes de taille homogène ont été obtenus. Ces PICM ont permis de prolonger la libération de l’AON et de le protéger efficacement contre la dégradation enzymatique. De plus, des polymères de poly(oxyde d’éthylène)-bloc-poly(méthacrylate de propyle-co-acide méthacrylique) ont été incorporés afin de conférer des propriétés acido-sensibles aux PICM. Dans ces micelles, formées de ce dernier polymère formulé avec le dendrimère PAMAM, des oligonucléotides (AON et siRNA) ciblant l’oncogène Bcl-2 ont été encapsulés. L’internalisation cellulaire fut assurée par un fragment d’anticorps monoclonal (Fab’) situé à l’extrémité de la couronne de PEG. Après l’internalisation cellulaire et la protonation des unités d’acide méthacrylique sous l’effet de l’acidification des endosomes, les micelles se sont affranchies de leur couronne. Elles ont ainsi exposé leur cœur composé d’acide nucléique et de dendrimère PAMAM, qui possède une charge positive et des propriétés endosomolytiques. En effet, ces PICM acido-sensibles ciblées ont permis d’augmenter la biodisponibilité des acides nucléiques vectorisés et se sont avérées plus efficaces pour silencer l’oncoprotéine Bcl-2 que les micelles non ciblées ou que le dendrimère de PAMAM commercial seul. Finalement, les nanovecteurs polymères présentés dans cette thèse se révèlent être des systèmes prometteurs pour la vectorisation des anticancéreux et des acides nucléiques. / Cancer is considered as the leading cause of premature death in Canada. Taxanes (e.g. paclitaxel and docetaxel (DCTX)) are effective against a range of solid tumors including breast, lung, and ovarian malignancies. In addition, nucleic acids (e.g. antisense oligonucleotides (AON) and short interfering RNA (siRNA)) which are capable of selectively suppressing oncogenes involved in carcinogenesis are currently being investigated for the treatment of a wide variety of cancers. Although the activity of taxanes and nucleic acid drugs is well-established in human and/or animal models, several physicochemical and clinical issues still need to be addressed. Low aqueous solubility (i.e. taxanes), rapid degradation in the blood (i.e. nucleic acids), fast clearance, non-selectivity and toxicity to normal tissues are limiting factors to their effectiveness. Hence, many efforts have been focused on developing targeted polymeric delivery systems to overcome the problems associated with the current therapies. In this thesis, two types of polymeric micelles have been developed for the delivery of DCTX and nucleic acids. On the one hand, poly(ethylene oxide)-block-poly(butylene oxide/styrene oxide) micelles were tested for the first time to solubilize and protect DCTX from hydrolytic degradation. The polymers showed less toxicity than the surfactant used commercially to dissolve DCTX (i.e. polysorbate 80) and released the drug in a sustained fashion. On the other hand, two different systems of polyion complex micelles (PICM) were developed for the sustained release and intracellular delivery of nucleic acids. Novel poly(ethylene glycol) (PEG)-oligonucleotide conjugates were assessed to protect AON against degradation and release them in a sustained manner. When these conjugates were mixed with poly(amidoamine) (PAMAM) dendrimers, monodisperse PICM were formed. These PICM further slowed down AON release and significantly protected it against enzymatic degradation. In addition, the incorporation of poly(ethylene oxide)-block-poly(propyl methacrylate-co-methacrylic acid) was exploited to impart pH-sensitivity to PAMAM-based PICM. This system was composed of the previous copolymer mixed with PAMAM dendrimer. Such PICM were loaded with AON or siRNA targeting the Bcl-2 oncogene. Micelles uptake by the cancer cells was mediated by a monoclonal antibody fragment (i.e. Fab') positioned at the extremity of the PEG corona. Upon cellular uptake and protonation of the methacrylic acid units in the acidic endosomal environment, the micelles lost their corona, thereby exposing their positively-charged endosomolytic PAMAM/nucleic acid core. The targeted, pH-sensitive PICM were found to increase the intracellular bioavailability of the entrapped nucleic acids and knock down the Bcl-2 oncoprotein more than either non-targeted micelles or commercial PAMAM dendrimers. The polymeric nanocarriers reported in this thesis appear to be promising vehicles for the delivery of anticancer drugs and nucleic acids.

No description available.