The primary objective of the proposed research is to synthesize new compounds that can be used to control lipid-mediated membrane fusion. An interdisciplinary project is described that will expand the range of materials available for accelerating this fundamentally important process. The proposed materials will be incorporated within guest membrane vesicles as masked, nonfusogenic compounds that will become fusogenic upon exposure to acidic or oxidative environments--a triggering process that is conceptually similar to pH-induced viral protein-based membrane fusion within acidic endosomes. Preliminary experiments have guided the design of cleavable vinyl ether-PEG lipids that promote membrane fusion after triggering vinyl ether bond degradation. Synthetic methodology developed in the PI's laboratory will be used to install vinyl ether linkages of tunable lability within a family of phase-segregating, cleavable PEG lipids. These compounds will contain hydrophobic rod segments that are masked on one end by a cleavable hydrophilic vinyl ether-PEG unit and anchored to the membrane on the other via a phase-segregating, hydrogen-bonded hydrophobic block. Mean- field single chain theory will be used to guide the design of PEG lipids that will remain dispersed prior to activation, but form a thermodynamically stable phase-separated state after triggering has occurred. Molecular dynamics simulations will be used to generate initial inputs for the proposed mean-field calculations. This fusogen library will then be tested for their ability to promote membrane fusion in model membrane systems upon PEG cleavage and insertion of the unmasked hydrophobic rod domains into apposed bilayers. HPLC analysis and fluorescence-based assays will be used to monitor the rates of PEG lipid cleavage, membrane lipid mixing, and vesicle contents mixing under acidic or oxidative triggering conditions. Physical characterization of the membrane structures, before and after triggering, will also be performed using 31P NMR, monolayer film balance, C-TEM/C-SEM, and DSC. The most efficient fusogens will be assayed, using flow cytometry and laser confocal microscopy techniques, for their ability to effect cytoplasmic release of plasmid DNA cargo in cells targeted to internalize these carriers via receptor mediated endocytosis.

Public Health Relevance

The primary objective of the proposed research is to synthesize new compounds that can be used to control lipid-mediated membrane fusion. An interdisciplinary project is described that will expand the range of materials available for accelerating this fundamentally important process. The proposed materials will be incorporated within guest membrane vesicles as masked, nonfusogenic compounds that will become fusogenic upon exposure to acidic or oxidative environments--a triggering process that is conceptually similar to pH-induced viral protein-based membrane fusion within acidic endosomes. The most efficient fusogens will be assayed, using flow cytometry and laser confocal microscopy techniques, for their ability to effect cytoplasmic release of plasmid DNA cargo in cells targeted to internalize these carriers via receptor mediated endocytosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087016-04
Application #
8214528
Study Section
Special Emphasis Panel (ZRG1-BST-G (02))
Program Officer
Okita, Richard T
Project Start
2009-04-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2012
Total Cost
$289,908
Indirect Cost
$51,362
Name
Purdue University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Kulkarni, Aditya; Badwaik, Vivek; DeFrees, Kyle et al. (2014) Effect of pendant group on pDNA delivery by cationic-*-cyclodextrin:alkyl-PVA-PEG pendant polymer complexes. Biomacromolecules 15:12-9
Uline, Mark J; Szleifer, Igal (2013) Mode specific elastic constants for the gel, liquid-ordered, and liquid-disordered phases of DPPC/DOPC/cholesterol model lipid bilayers. Faraday Discuss 161:177-91; discussion 273-303
Nap, R J; Szleifer, I (2013) How to Optimize Binding of Coated Nanoparticles: Coupling of Physical Interactions, Molecular Organization and Chemical State. Biomater Sci 1:814-823
Kulkarni, Aditya; DeFrees, Kyle; Schuldt, Ryan A et al. (2013) Multi-armed cationic cyclodextrin:poly(ethylene glycol) polyrotaxanes as efficient gene silencing vectors. Integr Biol (Camb) 5:115-21
Kim, Hee-Kwon; Thompson, David H; Jang, Ho Seong et al. (2013) pH-responsive biodegradable assemblies containing tunable phenyl-substituted vinyl ethers for use as efficient gene delivery vehicles. ACS Appl Mater Interfaces 5:5648-58
Kulkarni, Aditya; Verheul, Ross; Defrees, Kyle et al. (2013) Microfluidic Assembly of Cationic-?-Cyclodextrin:Hyaluronic Acid-Adamantane Host:Guest pDNA Nanoparticles. Biomater Sci 1:
Uline, Mark J; Schick, M; Szleifer, Igal (2012) Phase behavior of lipid bilayers under tension. Biophys J 102:517-22
Kulkarni, Aditya; Deng, Wei; Hyun, Seok-hee et al. (2012) Development of a low toxicity, effective pDNA vector based on noncovalent assembly of bioresponsive amino-*-cyclodextrin:adamantane-poly(vinyl alcohol)-poly(ethylene glycol) transfection complexes. Bioconjug Chem 23:933-40
Coon, Brian G; Crist, Scott; Gonzalez-Bonet, Andres M et al. (2012) Fibronectin attachment protein from bacillus Calmette-Guerin as targeting agent for bladder tumor cells. Int J Cancer 131:591-600
Park, Yoonjee; Luce, Adam C; Whitaker, Ragnhild D et al. (2012) Tunable diacetylene polymerized shell microbubbles as ultrasound contrast agents. Langmuir 28:3766-72

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