Designing Peptide Amphiphiles to Facilitate Drug Delivery through ...

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Designing Peptide Amphiphiles to Facilitate Drug Delivery through ...

Peptide Amphiphiles in Drug DeliveryLipid BilayerHydrophilic drugsHydrophobic drugsPeptide acts as:•Drug•Targeting agentDiameter~100 nmDiameter~10 nm


Endosomal Escape for Drug ReleaseParticles enter the cellform endosomesLysosomes fuse withendosomesenzymatic degradationdrop in pHWaste is eliminated from cellhttps://eapbiofield.wikispaces.com/file/view/2-60.gifOverall Project Goal: Design drug delivery system thatreleases drug before degradation


Pore-Forming PeptidespH = 7 pH = 6Modified from http://www.dermaviduals.de/cms/upload/bilder/Publikationen/StarkeWirkung2.jpg• Acidic pH Pores formed in membrane• Dye is released through pores before degradation• Can measure change in fluorescence


Peptide Amphiphile DesignPeptide (GALA): WEAALAEALAEALAEHLAEALAEALEALAA(K)• At pH 6.0, 10 (+/-2) peptides aggregate to form pores in the membraneGALA Peptide AmphiphileWEAALAEALAEALAEHLAEALAEALEALAA(K)


Questions1. Do peptide amphiphiles form pores?2. At what concentration?3. Are peptide amphiphiles pH sensitive?


Sample PreparationMake liposomes and encapsulatecalcein dyeDialysis: remove excess dye fromliposome solutionDynamic Light Scattering Liposomes diffract light Measure diffusion coefficient Calculate diameter = 130 nm


Fluorescence Measurements Test 7 concentrations each of peptide and amphiphileLiposomesolutionPeptide orAmphiphileHClSurfactantStep #1 Step #2Step #3Step #4


• Need 0.5x10 -8 mol/L to form pores at pH 6• See dye leakage above 1.0x10 -7 mol/L at pH 7• At pH 6, amount of dye released increases significantly


• At pH 7 need 1.0x10 -8 mol/L to form pores• At pH 6 need 0.5x10 -9 mol/L to form pores Little difference after pH drop to 6


Peptide: pH dependent and threshold concentration is higher•Amphiphile: pH independent and threshold concentration is lower


Conclusions1. Do peptide amphiphiles form pores? Dye is released-pore formation-disrupting the membrane2. At what concentration? 0.5x10 -9 mol/L at pH 6 1.0x10 -8 mol/L at pH 73. Are peptide amphiphiles pH sensitive? Little to no pH sensitivity


Future Projects Repeat experiment to gain more statistical confidence Encapsulate peptide or amphiphile in liposome Compare secondary structure of peptide and amphiphile Test effect of tail length and peptide-tail linkage


Reflections Unexpected Results Precision at nano-scale Patience &Perseverance


AcknowledgementsThank you to… My amazing mentor, Amanda Trent All those who helped me out in the lab and myresearch Brian Lin, Rachel Marullo Graduate Advisor: Matt Tirrell CNSI AR Advisors Lubi, Anthony, and Herb


Designing Peptide Amphiphiles toFacilitate Drug Delivery throughEndosomal EscapeRashi SinghAmanda TrentProfessor TirrellBiomolecular Science and Engineering ProgramApprentice Researchers 2009


Why Amphiphiles?• Mixed micelle therapeutic peptide + pore-formingpeptide in one micelle• Self-assembly may promote pH-independent poreformation


The Process: How to Make LiposomesDissolve lipids in a solventLipid filmAdd buffer with dye & agitateFormation of liposomes(Multi-layered)Extrude to make single-layered

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