No description available.

No description available.

No description available.

<p>Hemophilia A and B are X-linked recessive bleeding disorders caused by the deficiency of coagulation factor VIII (FVIII) and Factor IX (FIX), respectively. Current treatment involves life-long protein replacement therapy which is invasive, expensive and inaccessible to the majority of hemophiliacs worldwide. Treatment is further compromised by the development of neutralizing antibodies. Thus, the development of an alternative treatment that is safer, cost effective and non-invasive that circumvents immune response induction is desirable.</p> <p>To this end, a chitosan-mediated gene therapy strategy delivered orally was developed to provide clinically relevant plasma expression of FVIII or FIX. Hemophilia A mice that ingested chitosan nanoparticles containing FVIII DNA transiently expressed canine FVIII reaching >100 mU one day post treatment, together with partial phenotypic correction. Residual FVIII activity was detected for several days. Repeated administration of nanoparticles restored FVIII expression for 4 weeks and reduced clotting time in treated mice. Interestingly, inhibitors and non-neutralizing antibodies were not detectable throughout the experiment.</p> <p>The immunomodulatory effects of chitosan-mediated oral gene delivery was investigated in naive hemophilia A mice and mice with pre-existing inhibitors. Administration of nanoparticles containing human FVIII DNA in naive mice suppressed systemic antibody responses and provided long-term tolerance to rhFVIII protein immunizations for at least 8 weeks. This tolerance was transferable to naive mice, suggesting development of regulatory T cells. In contrast, repeated oral nanoparticle administration was unable to suppress FVIII-specific antibody responses in hemophilia A mice with pre-existing inhibitors.</p> <p>Treatment of hemophilia B is challenged by a 25-50 fold higher therapeutic threshold. Nevertheless, hemophilia B mice fed chitosan nanoparticles containing CpG-FIXi plasmid transiently expressed therapeutically relevant human FIX >14mU/mL plasma.</p> <p>Chitosan nanoparticle formulation was optimized <em>in vitro</em> for improved transfection efficiency. Nanoparticles formulated at a chitosan:DNA charge ratio of >2:1 (N:P) provided DNA protection against proton and enzymatic degradation that mimic conditions of the stomach and intestine, respectively. The inclusion of 25 mM sodium acetate-acetic acid decreased transfection of HEK 293 cells 4-fold, while 50 mM sodium sulphate increased uptake by ~40%. Optimal transfection was achieved with chitosan chloride (CL 213) formulated at a charge ratio of 3:1 in 50 mM sodium sulphate.</p> <p>These findings suggest chitosan nanoparticles can provide clinically relevant FVIII and FIX transgene expression, which is amenable to a one-tablet-a-day dosing strategy. Taken together, chitosan-mediate gene therapy delivered orally is proposed as a potential non-invasive alternative strategy for hemophilia treatment and without inducing neutralizing and non-neutralizing antibody production.</p> / Doctor of Philosophy (PhD)

Today, drugs are an integral part of healthy human life, with new drug entities being introduced every year in clinic. The advancement of drug development brings complexity and variation, in terms of both physical and chemical properties. Some of these physicochemical characteristics are many times suboptimal, eventually requiring robust delivery systems that can precisely deliver the drugs to the desired tissues. Although many materials have been studied for the generation of drug delivery systems, there is always a need for biomaterials with better properties that can translate into superior delivery systems. In this context, new drug delivery systems that are interface-engineered at materials level for better stability and delivery efficiency in vitro and in vivo are introduced in this dissertation. In the first part of the dissertation, novel oil/water interface-engineered amphiphilic block copolymer micelles that were previously introduced by our lab were assessed for their stability in the presence of various esterase enzymes present in serum and on blood vessel walls, normally encountered by drug delivery systems on route to the targeted tissues. I also assessed the vulnerability of the polymeric micelles in presence of enzymes typically present either inside the tumor cells or secreted in the tumor microenvironment. I revealed the selective stability of empty- and docetaxel-loaded polymeric micelles to enzymatic degradation en route/in tumors and I have correlated this selective stability with polymer structure and interfacial engineering mentioned above. The unique delivery capabilities of interfacial-engineered polymeric micelles were tested in vivo using a mouse model of triple negative breast cancer. We proved that our novel engineered triblock copolymer-based drug delivery systems are superior to similar delivery systems made out of standard diblock copolymer micelles and also to the clinically used Taxotere® formulation towards cancer cell killing and tumor treatment, without displaying any significant toxicity in experimental animals. The second part of the dissertation focuses on the development and assessment of a pyridinium-based pseudo-gemini surfactant that combined the high nucleic acid packaging capacity of pyridinium lipids with the high transfection efficiency of gemini surfactants while displaying a reduced associated cytotoxic effect. I have analyzed the temperature treatment on compaction of nucleic acids into lipoplexes and I have established a high temperature annealing method for this purpose. This novel formulation technique allowed a substantial reduction of the amount of amphiphiles required to compact a specific amount of nucleic acids. This in turn also reduced the cytotoxic effect associated with the use of pyridinium amphiphiles. The effect of inclusion of colipids to lipoplex compaction, the robustness and the transfection efficiency of the lipid/nucleic acid lipoplex systems were assessed in detail, and correlations between formulation composition and biological activity were established. I was also able to show for the first time that pyridinium pseudo-gemini surfactants were able to compact different types of nucleic acids, including pDNA, mRNA and siRNA at lower charge ratios than standard, state-of-the art formulations used for this purposes. I also showed that irrespective to the nucleic acid compacted within the lipoplexes, the novel amphiphiles can efficiently deliver the cargo into the targeted cells even in the presence of very high concentration of serum, a premise for future use of these amphiphiles and formulations in vivo. / Pharmaceutical Sciences

The synthesis of pyridinium cationic lipids, their counter-ion exchange, and the transfection of lipoplexes consisting of these lipids with firefly luciferase plasmid DNA (6.7 KDa), into lung, prostate and breast cancer cell lines was investigated. The transfection ability of these newly synthesized compounds was found to be twice as high as DOTAP/cholesterol and LipofectamineTM (two commercially available successful transfection agents). The compaction of the DNA onto silica (SiO2) nanoparticles was also investigated. For this purpose, it was necessary to study the stability and fusion studies of colloidal systems composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), a zwitterionic lipid, and mixtures of DMPC with cationic DMTAP (1,2-dimyristoyl-3-trimethylammonium-propane). / Chemistry

Cancer is currently the second most common cause of death in the world. Despite tremendous progress in the treatment of different forms of cancer, the five year survival rates for lung, colorectal, breast, prostate, pancreatic and ovarian cancers remain quite low. New therapies are urgently needed for the better management of these diseases. In this context, both therapeutic gene and drug delivery constitute promising approaches for cancer treatment and are addressed in this thesis. Focusing on gene delivery, we are proposing the use new pyridinium amphiphiles for obtaining gene delivery systems with improved stability and efficiency and low toxicity (Chapters 2 and 3). The main focus was on pyridinium gemini surfactants (GSs), which possess a soft charge, a high charge/mass ratio and a high molecular flexibility - all key parameters that recommend their use in synthetic gene delivery systems with in vitro and in vivo efficiency. In Chapter 2, we optimized a novel DNA delivery systems through interfacial engineering of pyridinium GS at the level of linker, hydrophobic chains and counterions. In Chapter 3, we tested the effects of blending pyridinium cationic GS into pyridinium cationic lipid bilayers and we have evaluated these blends towards plasmid DNA compaction and delivery process. We have also correlated the cationic bilayer composition with the dynamics of the DNA compaction process, and with transfection efficiency, cytotoxicity and internalization mechanism of resulted nucleic acid complexes. Toward improved drug delivery systems, we introduced new amphiphilic block copolymers synthesized from biocompatible and biodegradable segments. Although their capabilites for loading, transport and release of lipophilic substances stored in their hydrophobic cores are widely known, their stability in vivo is limited due to rapid degradation by esterases present in the body. In Chapter 4, we examined the possibility to increase the enzymatic stability of PEG-PCL macromolecular amphiphiles through interfacial engineering, in a process which separates the hydrophilic/hydrophobic interface from the degradable/non-degradable block interface. We evaluated the stability, toxicity, drug loading and release properties of these new polymers using docetaxel as a model chemotherapeutic drug. The results revealed how hydrophilic/ hydrophobic interface tuning can be used to adjust key properties of polymeric drug delivery systems of this type. / Pharmaceutical Sciences

Grafenoxid (GO) har potential att användas i läkemedelsleveransapplikationer. Dess stora specifika yta gör det intressant som en effektiv bärare och skyddare av olika aktiva substanser för genterapi, såsom DNA och mRNA. Denna studie har fokuserat på att undersöka förhållandena för att ladda negativt laddat mRNA på GO. Kitosan (CS) och linjär polyetylenimin (PEI) har preadsorberats på GO för att underlätta mRNA-adsorption. Studien undersökte vid vilka förhållanden zeta-potentialen av GO/polyelektrolyt för det negativt laddade GO blir positivt. Dessa komplexerades sedan med mRNA vid olika N/P-förhållanden. Dessutom bedömde studien mRNA-frisättningskapaciteten genom att reducera pH.  GO/CS-komplexet vid förhållandet 1:2 visade positiv zeta-potential med N/P-förhållandena som sträcker sig från 1:1 till 10:1 visade att all mRNA och polyA har adsorberat till komplexet. N/P-förhållandet 10:1 var den enda som uppnådde en neutral zeta-potential, vilket tyder på tillräckligt mRNA för mättnad. Genom att öka koncentrationen av CS, kunde zeta-potentialen skifta till positivt vilket potentiellt förbättrar transfektionseffektiviteten. Visade en förbättring i signalen av det fria mRNA ökade när GO/CS/mRNA-komplexet utsattes för ett mer surt pH. Detta tyder på en potentiell frisättning när vektorn transfekteras in i cellen, eftersom den transporteras till lysosomerna som kännetecknas av sin sura miljö. GO/PEI-komplex visade endast negativ zeta-potential vid GO:PEI-förhållanden som når upp till 1:10, och därmed kommer det negativt laddade mRNA inte att adsorbera på dessa GO/PEI-komplex.  Resultaten tyder på en lovande utgångspunkt för pre-formuleringen av GO/CS-komplexet för vidare forskning. Detta arbete ger ett bidrag för framtida studier inom detta område. / Graphene oxide (GO) has a potential to be used in drug delivery applications. The large surface-to-mass ratio makes it interesting as efficient carrier and protector of various substances aimed for therapy, including DNA and mRNA. This study has focused on determining the ideal conditions for loading negatively charged mRNA onto GO using chitosan (CS) and linear polyethyleneimine (PEI) to facilitate mRNA adhesion. This was achieved by examining at what ratios of GO/polyelectrolyte the zeta potential of the negatively charged GO becomes positive, which were then subjected to mRNA complexation at different N/P (nitrogen/phosphate) ratios. Moreover, the study assessed the mRNA release capability by altering the pH.  The GO/CS complex at ratio 1:2 showed positive zeta potential with the N/P ratios ranging from 1:1 to 10:1 presented 100% loading efficiency of the added nucleic acids. With the N/P ratio 10:1 standing out as it achieved a neutral zeta potential, suggesting enough mRNA for saturation. By increasing the concentration of CS, the zeta potential could shift to positive potentially enhancing transfection efficiency. During the release assessment, the GO/CS/mRNA complex displayed increased amount of unbound mRNA when subjected to a more acidic pH. This suggests potential release when transfected into the cell, as the vector is transported to the lysosomes characterized by their acidic environment. GO/PEI complexes demonstrated only negative zeta potential at GO:PEI ratios reaching to 1:10, and thus the negatively mRNA will not adsorb on these GO/PEI complexes.  The findings suggest a promising starting point for the pre-formulation of the GO/CS complex for further research. This work provides a solid foundation for future studies in this area.

Non-viral gene delivery agents, such as cationic polyelectrolytes, are attractive replacements to viruses due to the absence of potential immunogenic risk and the ability to tune their macromolecular structure. Although non-viral vectors possess numerous design advantages, several investigators have shown that transfer efficiencies are considerably lower when compared to viral vectors. The work reported in this dissertation aims to fundamentally understand the underlying structure-transfection relationships involved in polycation-mediated gene delivery. Efforts focused on the influence of molecular weight, macromolecular topology, carbohydrate modifications, and charge density on the overall transfection activity in vitro. Several families of polycations were synthesized in order to correlate chemo-physical characterization with transfection results. Results revealed that seemingly small changes in the structure of cationic polyelectrolytes can have profound consequences on their transfection activity. / Ph. D.

The imidazole ring offers great potential for a variety of applications including gene delivery vectors, ionic liquids, electromechanical actuators, and novel monomers and polymers. The imidazole ring provides a unique building block for these applications due to its thermal stability, aromatic nature, ability to form ionic salts, and ease of functionalization. Free radical polymerization of 1-vinylimidazole (1-VIm) and free radical copolymerizations with methyl methacrylate (MMA) and n-butyl acrylate (nBA) afforded homopolymers and copolymers with tunable solution and thermal properties. Aqueous SEC provided reproducible and reliable molecular weights for poly(1-VIm) in the absence of polymer aggregates. Analysis of the thermal properties revealed ideal random copolymers with MMA and non-ideal copolymers with nBA. Small angle X-ray scattering determined that the spacing between ionic groups remained constant with increased nonionic comonomer incorporation while the spacing between adjacent polymer backbones increased. Functionalization of 1-VIm with varying length alkyl halides and polymerization prepared a series of imidazolium homopolymers. Anion exchange reactions controlled the thermal and solution properties, and the bromide counteranion quantitatively exchanged to tetrafluoroborate (BF4), trifluoromethanesulfonate (TfO), and bis(trifluoromethanesulfonyl)imide (Tf2N). Thermogravimetric analysis revealed that thermal stability increased with decreased alkyl substituent length and larger counteranion size, and differential scanning calorimetry determined that glass transition temperature (Tg) decreased with increased alkyl substituent length and larger counteranion size. Electrochemical impedance spectroscopy determined the ionic conductivities of the imidazolium homopolymers, and analysis using the Vogel-Fulcher-Tammann equation revealed that the activation energy of ion conduction increased as alkyl substituent length increased. Polymer morphology determined using X-ray scattering also influenced the ionic conductivity. As the alkyl substituent length increased, the spacing between adjacent polymer backbones increased, which decreased the ionic conductivity due to the ion-hopping mechanism of ion conduction. Unsuccessful attempts to control the radical polymerization of 1-VIm led to the investigation of 1-(4-vinylbenzyl)imidazole (VBIm), which is a styrenic-based monomer with excellent propagating radical stability. Triblock copolymers incorporating VBIm monomer into a soft random copolymer center block and reinforcing, hard segment outer blocks provided a template for tuning the properties of the ionomer membranes for electroactive devices. Analysis of the morphology and mechanical properties using small angle X-ray scattering and dynamic mechanical analysis determined microphase separation and optimal mechanical properties for electromechanical transducer fabrication. Testing electromechanical transducers revealed superior performance relative to the benchmark Nafion®. Optimization of triblock copolymer design criteria through varying the comonomer ratio of VBIm and nBA in the soft center block, quaternization reactions, and ionic liquid introduction influenced mechanical properties and ionic conductivity. Higher percentages of VBIm and quaternization of VBIm in the random central block increased Tg and ionic conductivity. IL selectively incorporated into the imidazole-containing phases with no leakage observed for ionic systems, reduced the center block Tg, and increased ionic conductivity. Controlling charge density along poly(1-VIm) through well-defined alkylation reactions with 1-bromobutane provided a potential vector for nonviral gene delivery and polyanion binding. Analysis of DNA and heparin binding using gel electrophoresis revealed a decrease in N/P ratio with increased alkylation percentage. Dynamic light scattering indicated an increase in zeta potential with increasing alkylation percentages, and relatively uniform polyplex sizes in aqueous media. The MTT assay developed cytotoxicity profiles with little toxicity prior to 83% alkylation. Finally, the luciferase expression assay revealed inefficient nucleic acid delivery to multiple cell types. Synthesis of poly(1-VIm) vectors with glutathione conjugates provided an avenue for simultaneous therapeutic gene and anti-oxidant delivery in vitro. Cytotoxicity assays of cells pretreated with glutathione-conjugated poly(1-VIm) prior to oxidative stress showed that higher glutathione conjugation levels improved cell viability. / Ph. D.