Synthetic Approaches to Flexible Fluorescent Conjugated Polymers

Conjugated polymers (CPs) are intrinsically fluorescent materials that have been used for various biological applications including imaging, sensing, and delivery of biologically active substances. The synthetic control over flexibility and biodegradability of these materials aids the understanding of the structure-function relationships among the photophysical properties, the self-assembly behaviors of the corresponding conjugated polymer nanoparticles (CPNs), and the cellular behaviors of CPNs, such as toxicity, cellular uptake mechanisms, and sub-cellular localization patterns. Synthetic approaches towards two classes of flexible CPs with well-preserved fluorescent properties are described. The synthesis of flexible poly(p-phenylenebutadiynylene)s (PPBs) uses competing Sonogashira and Glaser coupling reactions and the differences in monomer reactivity to incorporate a small amount (~10%) of flexible, non-conjugated linkers into the backbone. The reaction conditions provide limited control over the proportion of flexible monomer incorporation. Improved synthetic control was achieved in a series of flexible poly(p-phenyleneethynylene)s (PPEs) using modified Sonogashira conditions. In addition to controlling the degree of flexibility, the linker provides disruption of backbone conjugation that offers control of the length of conjugated segments within the polymer chain. Therefore, such control also results in the modulation of the photophysical properties of the materials. CPNs fabricated from flexible PPBs are non-toxic to cells, and exhibit subcellular localization patterns clearly different from those observed with non-flexible PPE CPNs. The subcellular localization patterns of the flexible PPEs have not yet been determined, due to the toxicity of the materials, most likely related to the side-chain structure used in this series. The study of the effect of CP flexibility on self-assembly reorganization upon polyanion complexation is presented. Owing to its high rigidity and hydrophobicity, the PPB backbone undergoes reorganization more readily than PPE. The effects are enhanced in the presence of the flexible linker, which enables more efficient π-π stacking of the aromatic backbone segments. Flexibility has minimal effects on the self-assembly of PPEs. Understanding the role of flexibility on the biophysical behaviors of CPNs is key to the successful development of novel efficient fluorescent therapeutic delivery vehicles.

Sea organisms as a source of collagen-derived polymers for skin tissue engineering applications

Advanced therapies combating acute and chronic skin wounds are likely to be brought about using our knowledge of regenerative medicine coupled with appropriately tissue engineered skin substitutes. At the present time, there are no models of an artificial skin that completely replicate normal uninjured skin and they are usually accompanied by fibrotic reactions that result in the production of a scar. Natural biopolymers such as collagen have been a lot investigated as potential source of biomaterial for skin replacement in Tissue Engineering. Collagens are the most abundant high molecular weight proteins in both invertebrate and vertebrate organisms, including mammals, and possess mainly a structural role in connective tissues. From this, they have been elected as one of the key biological materials in tissue regeneration approaches, as skin tissue engineering. In addition, industry is constantly searching for new natural sources of collagen and upgraded methodologies for their production. The most common sources are skin and bone from bovine and porcine origin. However, these last carry high risk of bovine spongiform encephalopathy or transmissible spongiform encephalopathy and immunogenic responses. On the other hand, the increase of jellyfish has led us to consider this marine organism as potential collagen source for tissue engineering applications. In the present study, novel form of acid and pepsin soluble collagen were extracted from dried Rhopilema hispidum jellyfish species in an effort to obtain an alternative and safer collagen. We studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using pepsin extraction method (34.16 mg collagen/g of tissue). The isolated collagen was characterized by SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy.

Four methods for measuring the solubilities of gases and vapors in liquids and polymers

Peer reviewed

Crystal structures of two one-dimensional coordination polymers constructed from Mn2+ ions, chelating anions and flexible bipyridyl bridging ligands

Peer reviewed

Crystal structures of a family of layered coordination polymers containing divalent metal ions (Mn2+, Co2+, Ni2+ and Zn2+) and the 3-amino 4-methyl benzoate ion

Peer reviewed

Four methods for measuring the solubilities of gases and vapors in liquids and polymers

Peer reviewed

Crystal structures of two one-dimensional coordination polymers constructed from Mn2+ ions, chelating anions and flexible bipyridyl bridging ligands

Peer reviewed

Crystal structures of a family of layered coordination polymers containing divalent metal ions (Mn2+, Co2+, Ni2+ and Zn2+) and the 3-amino 4-methyl benzoate ion

Peer reviewed

Epigenetic reprogramming of imprinted genes in embryonic stem cells, fertilized and cloned embryos

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Emulsion-Based RIR-MAPLE Deposition of Conjugated Polymers: Primary Solvent Effect and Its Implications on Organic Solar Cell Performance.

Emulsion-based, resonant infrared matrix-assisted pulsed laser evaporation (RIR-MAPLE) has been demonstrated as an alternative technique to deposit conjugated polymer films for photovoltaic applications; yet, a fundamental understanding of how the emulsion target characteristics translate into film properties and solar cell performance is unclear. Such understanding is crucial to enable the rational improvement of organic solar cell (OSC) efficiency and to realize the expected advantages of emulsion-based RIR-MAPLE for OSC fabrication. In this paper, the effect of the primary solvent used in the emulsion target is studied, both experimentally and theoretically, and it is found to determine the conjugated polymer cluster size in the emulsion as well as surface roughness and internal morphology of resulting polymer films. By using a primary solvent with low solubility-in-water and low vapor pressure, the surface roughness of deposited P3HT and PCPDTBT polymer films was reduced to 10 nm, and the efficiency of P3HT:PC61BM OSCs was increased to 3.2% (∼100 times higher compared to the first MAPLE OSC demonstration [ Caricato , A. P. ; Appl. Phys. Lett. 2012 , 100 , 073306 ]). This work unveils the mechanism of polymer film formation using emulsion-based RIR-MAPLE and provides insight and direction to determine the best ways to take advantage of the emulsion target approach to control film properties for different applications.

Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

Epigenetic reprogramming of imprinted genes in embryonic stem cells, fertilized and cloned embryos

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.