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      • HARVEST
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      Development of Amino acid-Substituted Gemini Surfactant-Based Non-invasive Non-Viral Gene Delivery Systems

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      Permission to use published manuscripts or figures.docx (184.4Kb)
      Date
      2015-10-20
      Author
      Singh, Jagbir
      Type
      Thesis
      Degree Level
      Doctoral
      Metadata
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      Abstract
      Gemini surfactants are versatile gene delivery agents because of their ability to bind and compact DNA and their low cellular toxicity. The aim of my dissertation work was to develop non-invasive mucosal formulations of novel amino acid-substituted gemini surfactants with the general chemical formula C12H25(CH3)2N+-(CH2)3-N(AA)-(CH2)3-N+(CH3)2-C12H25 (AA= glycine, lysine, glycyl-lysine, lysyl-lysine). These compounds were formulated with a model plasmid DNA, encoding for interferon-γ and green fluorescent protein, in the presence of helper lipid, 1,2 dioleyl-sn-glycero-phosphatidyl-ethanolamine. Formulations were assessed in Sf 1 Ep epithelial cells. Among the novel compounds, plasmid/gemini/lipid (P/G/L) nanoparticles formulated using glycine- and glycyl-lysine substituted gemini surfactants achieved significantly higher gene expression than the parent unsubstituted compound. The key physicochemical properties, e.g. size, surface charge, DNA binding, and toxicity of P/G/L complexes were correlated with transfection efficiency. The presence of amino-acid substitution did not interfere with DNA compaction and contributed to an overall low toxicity of all P/G/L complexes, comparable to the parent gemini surfactant. A cellular uptake mechanistic study revealed that both clathrin- and caveolae-mediated uptake were major uptake routes for P/G/L nanoparticles. However, amino acid substitution in the gemini surfactant imparted high buffering capacity, pH-dependent increase in particle size, and balanced DNA binding properties. These properties may enhance endosomal escape of P/12-7NGK-12/L resulting in higher gene expression. Finally, the P/G/L complexes were incorporated into an in-situ gelling dispersion containing a thermosensitive polymer, poloxamer 407, and a permeation enhancer, diethylene glycol monoethyl ether (DEGEE). A 16% w/v poloxamer concentration produced a dispersion that gelled at body temperature and exhibited sufficient yield value to prevent formulation leakage from the vaginal cavity. The formulations were prepared with a model plasmid, encoding for red fluorescent protein, and administered topically to rabbit vagina. In agreement with our in vitro results, confocal microscopy revealed that glycyl-lysine substituted gemini surfactant exhibited higher gene expression compared to the parent unsubstituted gemini surfactant. This provided proof-of-concept for use of amino acid-substituted gemini surfactant in non-invasive mucosal (vaginal) gene delivery systems with potential therapeutic applications. These formulations will be developed with therapeutically relevant genes to assess their potential as genetic vaccines. In addition, new gemini surfactants will be developed by grafting other amino acids via glycine linkage to retain conformation flexibility and enhance endosomal escape of DNA complexes for higher transfection efficiency.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Pharmacy and Nutrition
      Program
      Pharmacy
      Supervisor
      Badea, Ildiko
      Committee
      Krol, Ed; Verrall, Ronald E.; El-Aneed, Anas; Alcorn, Jane
      Copyright Date
      August 2013
      URI
      http://hdl.handle.net/10388/ETD-2013-08-1189
      Subject
      Amino acid-substituted
      gemini surfactants
      plasmid
      non-viral gene delivery
      cellular uptake
      clathrin-mediated endocytosis
      caveolae-mediated endocytosis
      mucosal
      intravaginal administration
      gelling temperature
      rheology
      gene expression
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