1. 1. Engineering Di-Block Co-Polymers for Drug Delivery Ekta Sharma, Min Dai, Jin K. Montclare* Polytechnic Institute of NYUDepartment of Chemical and Biological Sciences 6 Metrotech Center, Brooklyn, NY 11201Sigma Xi Student Reserach Showcase 1
2. 2. Hypothesis: Generate Smart Biomaterials GNP hybridfor Drug Delivery Protein aggregation towards target cellsConversion :Light energy Thermal energyCurcumin boundProtein with GNPSigma Xi Student Research Showcase2
3. 3. Layout of Project :Sigma Xi Student Research Showcase 3
4. 4. Elastin-Like Polypeptide (E) - InverseTemperature Transition(VPGXG)n Elastin-Like Polypeptide (E) Repeats 5 Amino Acids X: Guest Residue; any Amino Acid but not Proline Possess Inverse Temperature TransitionPossible mechanisms for the phase transition of (VPGIG)40 proposed by Tirrell D.Chilkoti Group Website. http://www.chilkotilab.pratt.duke.edu/researchELP.htm (accessed January 6, 2012).Yamaoka, T.; Tamura, T.; Seto, Y.; Tada, S.; Hunugi, S.; Tirrell, D. A. Biomacromolecules, 2003, 4, 1680-16854Sigma Xi Student Research Showcase
5. 5. Coiled-Coil Domain of Cartilage Oligomeric Matrix Protein (C) - Binding COMP 4 epidermal type 3 globulargrowth factor repeat domain COMPcc or C CC(EGF) (T3) (TC) H2NCOOH2~6 ~30 73 VA, VD, Curcumin, Ca2+, Cl-AnaMar Website. http://www.compelisa.com/2/en/about-comp/what-is-comp.php (accessed December 19, 2012).Ozbek, S.; Engel, J.; Stetefeld, J. The EMBO Journal, 2002, 21, 5960-5968 Sigma Xi Student Research Showcase5
6. 6. Generate Smart Biomaterials Using E and C asBuilding BlocksSigma Xi Student Research Showcase 6
7. 7. DNA Transformation -PLASMID MAP PQE-30 LAC OPERON ANTIBIOTIC RESISTANCEE 1C HIS-TAG3806 bpGLYCEROL STOCK XL1-BlueELECTROPORATION AF-IQ Cells CE1 3816 bp Sigma Xi Student Research Showcase 7
8. 8. Cell Expression -Streak Colonies Small scale Expression Large scale ExpressionE1CCE1 glycerol stocksSuspend culture Colony pick-upstreaked on TSAAMP platein 1L LB and suspension in Luria Broth Incubation (370C) Induction with IPTG Overnight IncubationOvernight incubation (370C)37oC Overnight Incubation(370C)Sigma Xi Student Research Showcase 8
9. 9. SDS PAGE Gel- Expression GelOsmotic Shock Gel E 1CCE1Sigma Xi Student Research Showcase 9
10. 10. Extraction and Purification - Cell Lysis with Sonication 30% impulse 2 mins 15 seconds Centrifugation to separate cell lysates and ProteinsPurification using HPLC Ni Resin Column His residue of protein binds to Niions. Elution collected with differentpercentage of Buffer B(1%,5%,100%) Buffer A: 6 M urea, 50 mM Na2HPO4, 20 mM imidazole, pH 8.0 Buffer B: 6 M urea, 50 mM Na2HPO4, 200 mM imidazole, pH 8.0 Sigma Xi Student Research Showcase 10
11. 11. Purification Result -E1CCE115 Kb 15 KbMW : 14.1 KbMW: 13.9 Kb Sigma Xi Student Research Showcase11
12. 12. Quantification using MicroBCAPrinciple :Reduction of Cu to Cu1+: peptides containing three or more amino acid residues form a colored chelate 2+complex with cupric ions in an alkaline environment containing sodium potassium tartrate.Bicinchoninic acid (BCA) reaction : chelation of two molecules of BCA with one cuprous ion to givepurple color. Protocol Standards of BSA were made of concentration 40,20,10,5,2.5 ul On Microtiter plate - Std : 150 ul + 150 ul Reaction mixture Sample 15 ul + 135 ul of PB + 150 ul Working reagent (A:B:C 50:48:2) 2 hrs of incubation at 37 C.http://www.qcbio.com/pierce/BCA%20Protein%20Assay%20Reagent.htmSigma Xi Student Research Showcase 12
13. 13. Result : BSA Standard Absorbance Plot Concentration Calculation - CE1 E1CugmL223.3491705 141.420705 mgmL 0.223349171 0.14142071 MW 13941.72 13864.66molarity 1.60202E-05 1.02001E-05 Sigma Xi Student Research Showcase13
14. 14. Integration of InorganicMaterials with Organic Protein Polymers 14
15. 15. Controllable Metal Nanostructures AuAg alloyAu nanoparticles withnanosphere increasing Au concentration Au nanorods ofAu increasing aspect nanorodsratio AgAg nanoprisms with nanoprismsincreasing lateral sizeLiz-Marzn, L.M. Materials Today, 2004, 7, 26-31Sigma Xi Student Research Showcase 15
16. 16. Potential of Gold Nanoparticles Based on Protein-Gold Nanoparticles InteractionProtein adsorptionon GNPs surfaceProtein misfolding Protein aggregationZhang, D.; Neumann, O.; Wang, H.l Yuwono, V. M.; Barhoumi, A.; Perham, M.; Hartgerink, J. D.; Wittung-Stafshede, P.; Halas, N.J. Nano Letters, 2009, 9, 666-671 Sigma Xi Student Research Showcase16
17. 17. Process of Templation - incubate with HAuCl43H2O for 5 mins 40:1 molar ration between Au andprotein reduce with NaBH4 2.5:1 ration to Au4HAuCl4 + 3NaBH4 -> 4Au + 6H2 + 3NaCl + 3BCl3 + 4HCl Gold binds to Histidine Uniform coatingSlocik, J. M.; Wright, D. W. Biomacromolecules, 2003, 4, 1135-1141.Sigma Xi Student Research Showcase 17
18. 18. Overview on Circular Dichroism -a form of light absorptionspectroscopy that measures thedifference in absorbance of right-and left-circularly polarized light bya chiral moleculeSigma Xi Student Research Showcase 18
19. 19. Circular Dichroism for Proteins All amino acids except glycine are chiral Phenomena exhibited due to e- transition n -> * centerd around 215-230 nm ; involves non-bonding electrons of O of the carbonyl -> * centerd around 185- 200 nm involves the -electrons of the carbonyl The intensity and energy of these transitions depends on and (i.e., secondary structure)http://www.informatics.indiana.edu/predrag/classes/2008springi619/week4_m.pdf - Andrs Szilgyi Sigma Xi Student Research Showcase 19
20. 20. Study-1 : Impact on Secondary Structure of P and PGNP1500025 CE1-GNP CE1GNP10000mrw (degCM2dmol-1) CE1wt CE1 GNPs decrease the CD reading E1C-GNP E1CGNP 5000E1Cwt E1 C 0 GNPs slightly increase the unordered structure for both E1C and-5000 CE1 -10000 180200 220 240 260Wavelength (nm)Sigma Xi Student Research Showcase20
21. 21. Measured Secondary Structure using CDSSTR 22000 E1C-GNP heating 17000E1CGNP 12000GNPCE1CE -GNP CE1 E1C 251 40 mrw (degCM2dmol-1) 12000 55 25 70 7000 95C4055 95C95 70957000 4 C 2000 4 C2000 12000 12000 -300095 -300095 CE1-GNP CE1-GNP 25257000 40-8000 700040 -8000 5555 7070 95 25 9525 -13000 2000-13000 2000 185 200 215 230 245260 185 200 215 230 245260Wavelength (nm) -3000-3000 E1CGNPCE1GNPFraction (%) -8000-8000BetaGNPs do not disturb theUnordered response of proteins to -13000 -13000 185 200 215 230 245260 Helix185 200 heat230215 245 260 Temperature () Sigma Xi Student Research Showcase21
22. 22. Study 2 : Impact of GNPs on Tt 1Uniformed Turbidity at 800 nm0.9E1C0.8 27 0.70.6 E1C-GNP E1C0.530 E1C-GNP CE10.4CE1-GNPCE1 CE1-GNP0.3 59 67 0.20.1 0020 40 60 80 100Temperature () Comparison between Tts of E1C and CE1 are consistent with library dataTt of both E1C and CE1 are enhanced after templation Enhanced Tt of CE1 after templation is greater than that of E1CSigma Xi Student Research Showcase 22
23. 23. Study 3 : Effect on Binding Ability Why Curcumin ? Hydrophobic natureCOMP domain forms hydrophobic pore which aids bindingof curcuminAnti-oxidant and anti-inflammatory propertiesPotential for cancer treatmentAggarwal, B. B.; Shishodia, S. (2004). "Suppression of the Nuclear Factor-B Activation Pathway by Spice-Derived Phytochemicals: Reasoning forSeasoning".Annals of the New York Academy of Sciences1030: 434441 Sigma Xi Student Research Showcase 23
24. 24. Result CCM to protein bindingProtein Samplemolar ratio CE11.60.9 : 1 E1C1.80.8 : 1CE1GNP 2.10.7 : 1E1CGNP 2.50.6 : 1 GNPs maintain the binding and releasing ability of E1C and CE1 to CurcuminSigma Xi Student Research Showcase 24
25. 25. Conclusions.. GNPs have impact on proteins conformation. GNPs also affect on the thermoresponsive properties of the proteins. The effect is greater on CE1 than E1C. GNPs do not eliminate binding ability of the proteins.GNPs have influence on proteins conformation andthermoresponsivity while still maintain the binding ability of theproteins. Through templation it is possible to control thethermoresponsivity of the proteins.Sigma Xi Student Research Showcase 25
26. 26. Thank you! and many morelab members!Sigma Xi Student Research Showcase 26