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Cationic polymers are of broad interest in the biotechnology sector due to their potential use in drug conjugation, tissue engineering, gene delivery, water purification and chelation, and stimuli-responsive materials. Of particular interest in designing cationic polymers is control over their pKa—allowing for the presentation of a positive charge at specific pH, which is dependent on molecular weight, connectivity, and pendant group identity of the polymer. In this dissertation, we designed and synthesized a series of smoking-inspired monomers and polymers based on pyrrolidine, piperidine, and tocopherol—utilizing copolymerization and other polymer chemistry strategies to vary their molecular weight, dispersity, hydrophilicity/hydrophobicity, charge density, and pKa. These polymers are designed to mimic certain chemical structures present in tobacco alkaloids or other smoking-related compounds that have implications in human health. In this work, we explore the utility of amine-based polymers within and beyond the scope of innate immunity—playing important roles in solving bioengineering and tissue engineering problems. Exposure to certain chemical factors present in tobacco and smoking-related compounds leads to the elevated risk and greater symptom severity of several autoimmune conditions. While this link has remained mechanistically unclear, the presence of protein/DNA immune complexes in systemic lupus erythematosus (SLE) has been designated as an important marker of disease severity, seen more prevalently in smokers vs. never-smokers. The smoking-inspired polymer library developed in this study is used to mimic the features of these immune complexes. Investigation of these polymeric materials complexed with negatively-charged biological macromolecules shows notable differences in complex formation, biophysical structure, and subsequent innate immune activity. These results provide further evidence of the role of biophysical complex structure in innate immune activity, and the role that tobacco’s chemical structures may play in exacerbating disease. Additional work explores the interaction of DNA with cigarette smoke in vitro—demonstrating the formation of visible clew-like precipitates upon DNA exposure to cigarettes. These pellets, which resemble the precipitates formed by the condensation of oppositely-charged biomacromolecules, demonstrate a level of biophysical ordering and immune activity seen in immunomodulatory DNA complexes.

Despite tremendous progress within the field of oncology, highly metastatic forms of breast cancer remain particularly challenging to effectively treat. Systemically delivered chemotherapy with cytotoxic agents typically requires some convalescence time between treatments – allowing rapidly growing cancer types to develop resistance. Multidrug resistance, also known as pump-dependent, is particularly difficult to treat as it functions through overexpression of P-glycoprotein, an efflux pump which can produce resistance to a range of chemotherapeutics. We have previously developed the cationic micellar copolymer poly(lactide-co-glycolide)-g-poly(ethylenimine) (PgP) and demonstrated its capacity as a vector for gene therapy. Here, we examine the capacity of PgP in mediating co-delivery of siRNA targeting P-glycoprotein (siMDR1) and the anthracycline class drug doxorubicin to mitigate multidrug resistance in drug resistant triple-negative human cancer cells in vitro. The results of this project have shown that PgP can be used to successfully bind siRNA into a complex that protects from interaction with charged particles through heparin competition assay, and remains stable in serum conditions. Results of MTT assay assessing metabolic activity have shown PgP/siMDR1 complexes to exhibit minimal cytotoxicity in vitro in comparison to untreated human MDA-MB-435 cancer cells. Assessment of silencing following treatment with PgP/siMDR1 complexes formed at various nitrogen:phosphate (N/P) ratios has shown significant knockdown of MDR1 mRNA up 67%, in comparison to untreated groups of drug resistant MDA-MB-435 cells. PgP has also been shown to successfully load the hydrophobic chemotherapeutic doxorubicin, improving the toxicity of the drug in vitro. These results show the efficacy of PgP as a vehicle for delivery of both doxorubicin and siMDR1 to drug resistant cancer cells, and may have potential for use in co-delivery of siMDR1 and chemotherapeutics in metastatic cancer treatment. Future studies will include in vivo toxicity and antitumor studies in athymic mouse breast cancer models.

The transferrin (TfR) and epidermal growth factor receptors (EGFR) are known to be overexpressed on the surface of a wide variety of tumor cells. Therefore, the peptides B6 (TfR specific) and GE11 (targeted to the EGFR) were linked to the PAMAM (polyamidoamine) structure via a polyethylenglycol (PEG) 2 kDa chain with the aim of improving the silencing capacity of the PAMAM-based dendriplexes. The complexes showed an excellent binding capacity to the siRNA with a maximal condensation at nitrogen/phosphate (N/P) 2. The nanoparticles formed exhibited hydrodynamic diameters below 200 nm. The zeta potential was always positive, despite the complexes containing the PEG chain in the structure showing a drop of the values due to the shielding effect. The gene silencing capacity was assayed in HeLa and LS174T cells stably transfected with the eGFPLuc cassette. The dendriplexes containing a specific anti luciferase siRNA, assayed at different N/P ratios, were able to mediate a mean decrease of the luciferase expression values of 14% for HeLa and 20% in LS174T cells, compared to an unspecific siRNA-control. (p < 0.05). In all the conditions assayed, dendriplexes resulted to be non-toxic and viability was always above 75%.

Abstract: This thesis work is aimed at addressing some of the fundamental questions surrounding the intracellular fate of plasmid DNA in clinically-relevant primary cells, when delivered with lipid-modi!ed cationic polymers as gene carriers. We developed an optimized procedure for the transfection of primary cells, which included modi!cations designed to maximize the in vitro stability of the DNA-carrier complexes and enhance their transfection utility. These polymeric gene carriers bind to DNA through electrostatic interaction, which drives a self-assembly process that condenses DNA into sub-micron particles suitable for cellular uptake. This interaction is not DNA sequence or structurally speci!c, and thus able to package DNA molecules with different topologies and molecular weights with equal efficiency for uptake. However, circularized DNA performed better than its linearized equivalent in transfection, suggesting intracellular processing may be dependent on the physicochemical properties of the assembled complexes. Thus, we concentrated our effort on understanding the intracellular events leading to transfection. Using a linoleic acid substituted cationic polymer, we found that transfection efficiency is correlated with the amount of pDNA associated with the nucleus and that lipid-moieties is able to facilitate nuclear association of DNA to enhance transfection. Further, lipid modi!cation altered the uptake pathways of the complexes from ones that are predominantly driven by macropinocytosis to ones that are mediated by clathrin. Endosome escape continues to be an inefficient process with these polymeric gene carriers, suggesting methods to promote cytosolic release may be the most effective approach to enhance their transfection efficiencies.

Abstract: This thesis explores the capabilities of cationic polymers modified with lipids of different carbon chain length to deliver DNA molecules to primary cells and transformed cell lines. Our studies focus on two different polymers: polyethylenimine (PEI) and poly(L-lysine) (PLL). Firstly, PEI and PLL were conjugated to palmitic acid (C16). The delivery of plasmid DNA to rat bone marrow stromal cells (rat-BMSC) was evaluated by using a Green Fluorescent Protein gene expressing plasmid (pEGFP-N2) as a reporter system. The rationale for lipid substitution is to give the polymer an amphiphilic character so as to improve the transfection efficiency of native polymers by improving the DNA/polymer translocation through the phospholipid-rich cell membranes. In the case of PLL-C16, transfection efficiency was significantly increased (5 fold) as compared to native PLL, and it was significantly higher than commercially available cationic lipids (LipofectamineTM 2000 and FugeneTM). We further explore the use of other lipids with variable chain lengths (carbon chain length ranging from 8 to 18 saturated and unsaturated) in order to identify other candidates to enhance the gene delivery properties of the PLL. Lipid-modified PLL of high molecular weight (25 vs. 4 kDa) was found to be more effective in delivering plasmid DNA in rat-BMSC. We noted that C14-, C16- and C18-substituted PLL gave the most effective DNA delivery. Moreover, a correlation between the extent of lipid substitution and the plasmid DNA delivery efficiency was found Additionally, transgene expression by BMSC significantly increased when amphiphilic PLLs were used as compared to native PLL. The modified polymers were able to transfect the cells up to 7 days, after which the expression decreased. Encouraged by the successful transgene expression agents obtained by modifying low molecular weight PEI with the same series of lipids described above, we explored the possibility of modifying low molecular weight PEI (2 kDa) with longer lipids; saturated fatty acid (C22), trans fat (C18:1T) and essential fatty acids (C22:1, C22:6 and C18:3). Transfection efficiency proved to be cell dependent. Only the transformed 293T cells were able to express GFP compared to human-derived BMSC. The highest transfection efficiency was found with highly unsaturated lipid-substituted PEI (C18:3 and C22:6) and were able to increase transgene expression overtime (6 days). Furthermore, internalization studies indicated that effective transfection of these carries do not follow any known endocytosis pathway instead the DNA/carrier penetrates the plasma membrane directly.

In this research, a number of new approaches to increase the removal of NOM during the lime-soda softening process were investigated. Specifically, softening sludge recycling in the presence and absence of cationic polyelectrolytes, MgCl2 addition in the presence and absence of softening sludge, and the role of high magnesium lime were all evaluated. Recycling of softening sludge during lime softening was found to effectively improve NOM removal. A 554 mg/L sludge dose added with the stoichiometric lime-soda ash dose removed 28% of NOM present in a raw water as compared to only 10% NOM removal in the absence of sludge. In addition, Hydrophobic/Hydrophilic fractionation analyses, together with SUVA measurements indicated that hydrophobic fractions of NOM were preferentially removed. Further experiments showed that sludge recycling did not significantly affect pH, turbidity, hardness and level of inorganic elements in treated waters. The presence of polyelectrolyte CPAM was found to increase the sorption capacity of calcite particles for Aldrich humic acid by three times. Based on this, softening sludge was coated with CPAM and recycled during jar tests. Results showed that sludge coated with low doses of CPAM can improve NOM removal to level significantly higher than regular softening. However, the NOM removal level was comparable to that achieved by recycling bare softening sludge. The use of high magnesium hydrated lime and the addition of magnesium were found to be promising techniques to increase Mg(OH)2 precipitation and consequently NOM removal during the softening process. DOC removal increased as available magnesium in hydrated lime increased. A dolomitic quick lime sample, slaked at ambient pressure, however, hindered NOM removal due to lack of available magnesium and subsequent coprecipitation. As high as 43% NOM removal was achieved in the presence of 0.312 mM MgCl2 and 457 mg/L softening sludge dose as compared to only 13% for regular softening. DBP precursors were removed as indicated by THM concentration measurements. All the techniques discussed here can be implemented without any modifications in existing facilities or addition of new treatment step and/or equipments.