Post on 13-Aug-2020
transcript
Introduction
Methods and Materials• ELPs aliquots are kept frozen at -80C and once thawed they need to stay in
ice to make sure that they do not undergo preliminary Tt, to ensure the mostaccurate results.
• Both ELP stock solution and the drug solution, in this case Sildenafil (a wellknown vasodilator), are individually combined with 1xPBS buffer todetermine the baseline assembly and disassembly behaviour of both the ELPand the sildenafil individually.
• ELP is then combined with the sildenafil and then diluted into the 1xPBS tocreate a solution to test the assembly and disassembly of the drug-loadednanoparticles.
• A solution is then placed into the dynamic light scattering machine (DLS),and the intensity of the light scattering is measured, which is then used toderive volume. Figure 2 shows the special DLS cuvette.
• The DLS machine (Figure 3) measures Brownian Motion, the constantmovement of particles due to collisions with other particles and liquids, todetermine the size and therefore intensity of the particles.
Results
• Thank you to Dr. Larry Unsworth, Abdullah Alshememry, Scott Elgersma, and all the members of Dr. Unsworth’s team for all your help within the lab throughout this program.
• A special thank you to NSERC Promo Science for the funding for my research this summer.
• Thank you to the WISEST Program for granting me this opportunity.
• My lab partners, Rebecca Probst and Gabrielle Smith for their help and support throughout this experience.
Evaluation of Assembly and Disassembly Behaviour of Drug-Loaded Peptide Nanoparticles
Mackenzie Fix, Abdullah Alshememry, Larry D. UnsworthDepartment of Chemical and Material Engineering, University of Alberta
National Institute for Nanotechnology, Edmonton, Alberta
Conclusions
Elastin-Like Polypeptides (ELPs) are repetitiveartificial polypeptides. The most common of ELP is in theform (VPGXG)n where X is a guest residue, and n is thenumber of pentapeptide repeats. ELPs in the form of(VPGXG)n exhibit an inverse temperature phase transition(Tt), meaning that below this temperature they aresoluble in aqueous solution, and above the Tt theyundergo an aqueous demixing phase transition resultingin aggregation of the ELP. The tunable properties and theTt of the ELP make them well-suited for drug deliverywithin the body. Figure 1 shows the reversible phasetransition behavior of ELPs.
Literature Cited • MacEwan, S. R. and Chilkoti, A. (2010), Elastin-like polypeptides: Biomedical applications of tunable biopolymers.
Biopolymers, 94: 60–77. doi:10.1002/bip.21327
• Bahniuk MS, Alshememry A, Unsworth LD. High yield expression of short chain elastin-like polypeptides. Biotechniques. 2016
• Malvern Instruments LTD. (2003, 2004). Zetasizer Nano Series User Manual. Retrieved from http://www.biophysics.bioc.cam.ac.uk/files/Zetasizer_Nano_user_manual_Man0317-1.1.pdf
Figure 3: An image of the
DLS instrument used in
the lab.
DLS Instrument Theory• Brownian motion is the movement of particles due to random collisions with
the liquid that surrounds the particle, therefore the particles are always moving.
• An important part of Brownian motion for DLS is that small particles move quickly while large particles move slowly.
Figure 4: An image of scattered light
exhibiting bright and dark area
fluctuations as found in the Zetasizer
Nano Series User Manual (Malvern
Instruments LTD., 2003, 2004.)
Figure 2: An image of
the special cuvette
carrying the mixture of
ELP and Sildenafil
Figure 1: An image of the
reversible phase transition behavior of ELPs
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• Due to the movement of the particles, constructive and destructive phase addition of scattered light will lead to dark and bright area fluctuation (Figure 4), therefore it is said that the intensity at any particular point fluctuates.
• These fluctuations and the idea that large particles will lead to slow fluctuations and small particles will have quick fluctuations is what the DLS machine uses to calculate a size distribution.
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Graphs 1-6: These graphs are the baseline results of the heating and cooling behaviours
of ELP L40 and Sildenafil.
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0.1mg/ml ELP L40 with Sildenafil CoolingGraph 7 Graph 8
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Graphs 7-10: These graphs are the result of ELP combined with the Sildenafil at two
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3ELP L40 0.5mg/ml Heating
• The assembly behaviours of the 0.1mg/ml ELP L40 combined with the sildenafil solution varied from that of the assembly behaviours of just the ELP. The disassembly behaviours of the same ELP concentrations with sildenafil correlated with the ELP only solution, though the size of the ELP with the drug was larger and that might imply that the drug was loaded inside ELP particles.
• Similarly, the assembly behaviours of the 0.5mg/ml ELP L40 combined with the sildenafil solution related to the results of just the ELP though the size of the particles containing the drugs were much larger. The disassembly of the ELP with the sildenafil also correlated with that of the ELP only solution, though the particles were almost twice as large.
• The next step in this research is to determine quantitatively how much drug is loaded into ELP nanoparticles, and then evaluate the drug release profile from ELP nanoparticles/
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Acknowledgements
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