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Protocol for manufacture of RDG conjugated Magnetite cationic Liposomes

Protocol for manufacture of RDG conjugated Magnetite cationic Liposomes. Andrew Naber Dan Knipmeyer. Objective. Develop protocol for enveloping magnetite nano-particles with liposomes bound to an RGD peptide sequence for cell specific integrin targeting. Concepts.

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Protocol for manufacture of RDG conjugated Magnetite cationic Liposomes

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  1. Protocol for manufacture of RDG conjugated Magnetite cationic Liposomes Andrew Naber Dan Knipmeyer

  2. Objective • Develop protocol for enveloping magnetite nano-particles with liposomes bound to an RGD peptide sequence for cell specific integrin targeting.

  3. Concepts • RGD peptides are protein sequences that are targeted to specific integrins. By targeting endothelial cells we hope to create a cell-magnetite complex that can bind to magnetic surfaces.

  4. Suggested Materials • Chloroform • Bath Sonicator • Vortexor • Gas tight syringe • Lipid Mix • N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG) • dilauroylphosphatidyl-choline (DLPC) • dioleoylphosphatidylethanolamine-N-[3-(2-pyridyldithio)-propionate] (PDP-DOPE) • Lipid marker DiI • 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.0) • 10 nm magnetite nano-particles • Specific RDG peptides • Round bottom flask

  5. Procedure • Dissolve the three lipids (in a 1:2:2 molar ratio) into chloroform in the round bottom flask. Do this at 10-20 lipid/ml organic solvent. • Remove solvent either through dry nitrogen method (volume<1ml) or through rotary evaporation. • Allow the chloroform to evaporate in a vacuum (overnight). Lipid film should coat the bottom of the flask. • Suspend the magnetite nano-particles in water. • Hydrate dried lipid layer on the bottom of the flask with nano-particle suspended water.

  6. Procedure cont. • Vortex the lipid/water/nano-particle solution for a while. • Sonicate solution for 30 mins at 28 W in a bath sonicator to reduce to single laminar liposomes. • Add 2ml of purified MCLs to the RGD peptides in a .55 peptide to 1 PDP-DOPE molar ratio. • Gently agitate this solution for 3.5 hours in a solution of 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.0).

  7. Illustration

  8. Purify the MCLs • At this point there will be a mixture of liposomes that contain magnetite, liposomes without magnetite, and magnetite nano-particles. • Place mixture in a magnetic stand and vacuum out particles that do not stick to the sides. • The remaining nano-particles in suspension should precipitate after a while.

  9. Next Phase • Cell Adhesion Test • Adhesion Test in flow chamber

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