The fabrication of stimuli-responsive, multifunctional proteins comprised of self-assembling domains capable of forming defined nanometre scale structures has tremendous application in drug delivery. This coupled to inorganic material like Gold nanoparticles (GNP’s) can facilitate external triggers controlling binding and release of embedded agents within the self-assembling materials. This renders smart biomaterial suitable for drug delivery.

1. Engineering Di-Block Co-Polymers
for Drug Delivery
Ekta Sharma, Min Dai, Jin K. Montclare*
Polytechnic Institute of NYU
Department of Chemical and Biological Sciences
6 Metrotech Center, Brooklyn, NY 11201
Sigma Xi Student Reserach Showcase 1

2. Hypothesis: Generate Smart Biomaterials – GNP hybrid
for Drug Delivery
Protein aggregation
towards target cells
Conversion :
Light energy 
Thermal energy
Curcumin bound
Protein with GNP
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3. Layout of Project :
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4. Elastin-Like Polypeptide (E) - Inverse
Temperature Transition
(VPGXG)n
Elastin-Like Polypeptide (E) Repeats 5
Amino Acids
X: Guest Residue; any Amino Acid but
not Proline
Possess Inverse Temperature Transition
Possible 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, 1680-1685
4
Sigma Xi Student Research Showcase

5. Coiled-Coil Domain of Cartilage Oligomeric
Matrix Protein (C) - Binding
COMP 4 epidermal type 3 globular
growth factor repeat domain COMPcc or C
CC
(EGF) (T3) (TC)
H2N COOH
2~6 Å
~30 Å
73 Å
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
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6. Generate Smart Biomaterials Using E and C as
Building Blocks
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7. DNA Transformation -
PLASMID MAP
PQE-30
LAC OPERON
ANTIBIOTIC RESISTANCE E 1C
HIS-TAG 3806 bp
GLYCEROL STOCK
XL1-Blue
ELECTROPORATION
AF-IQ Cells
CE1
3816 bp
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8. Cell Expression -
Streak Colonies Small scale Expression Large scale Expression
E1CCE1 glycerol stocks Suspend culture
Colony pick-up
streaked on TSAAMP plate in 1L LB
and suspension
in Luria Broth
Incubation (370C)
Induction
with IPTG
Overnight
Incubation
Overnight incubation (370C)
37oC Overnight Incubation
(370C)
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9. SDS PAGE Gel-
Expression Gel Osmotic Shock Gel
E 1C CE1
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10. Extraction and Purification -
 Cell Lysis with Sonication –
• 30% impulse
• 2 mins 15 seconds
Centrifugation to separate
cell lysates and Proteins
Purification – using HPLC
• Ni Resin Column
• His residue of protein binds to Ni
ions.
• Elution collected with different
percentage 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
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11. Purification Result -
E1C CE1
15 Kb
15 Kb
MW : 14.1 Kb MW: 13.9 Kb
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12. Quantification using MicroBCA
Principle :
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 give
purple 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.htm
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13. Result :
BSA Standard Absorbance Plot
Concentration Calculation -
CE1 E1C
ugmL 223.3491705 141.420705
mgmL 0.223349171 0.14142071
MW 13941.72 13864.66
molarity 1.60202E-05 1.02001E-05
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14. Integration of Inorganic
Materials with Organic
Protein Polymers
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15. Controllable Metal Nanostructures
AuAg alloy
Au nanoparticles with
nanosphere increasing Au
concentration
Au nanorods of
Au increasing aspect
nanorods ratio
Ag Ag nanoprisms with
nanoprisms increasing lateral size
Liz-Marzán, L.M. Materials Today, 2004, 7, 26-31
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16. Potential of Gold Nanoparticles Based on
Protein-Gold Nanoparticles Interaction
Protein adsorption
on GNPs surface
Protein misfolding
Protein aggregation
Zhang, 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
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17. Process of Templation -
• incubate with HAuCl4•3H2O for 5 mins
• 40:1 molar ration between Au and
protein
• reduce with NaBH4 2.5:1 ration to Au
4HAuCl4 + 3NaBH4 -> 4Au + 6H2 + 3NaCl + 3BCl3 + 4HCl
• Gold binds to Histidine
 Uniform coating
Slocik, J. M.; Wright, D. W. Biomacromolecules, 2003, 4, 1135-1141.
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18. Overview on Circular Dichroism -
a form of light absorption
spectroscopy that measures the
difference in absorbance of right-
and left-circularly polarized light by
a chiral molecule
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19. Circular Dichroism for Proteins
• All amino acids except glycine
are chiral
• Phenomena exhibited due to e-
transition
n -> π* centerd around 215-230
nm ;
involves non-bonding
electrons of O of the carbonyl
• π -> π* centerd around 185-
200 nm
involves the π-electrons of
the carbonyl
The intensity and energy of
these transitions depends on φ
and ψ (i.e., secondary structure)
http://www.informatics.indiana.edu/predrag/classes/2008springi619/week4_m.pdf - András Szilágyi
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20. Study-1 : Impact on Secondary Structure of P
and P•GNP
15000
25 ℃
CE1-GNP
CE1•GNP
10000
�mrw (deg∙CM2∙dmol-1)
CE1wt
CE1
GNPs decrease the CD reading
E1C-GNP
E1C•GNP
5000 E1Cwt
E1 C
0
GNPs slightly increase the
unordered structure for both E1C and
-5000
CE1
-10000
180 200 220 240 260
Wavelength (nm)
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21. Measured Secondary Structure using CDSSTR
22000
E1C-GNP heating
17000
E1C•GNP 12000 •GNP
CE1CE -GNP CE1
E1C 25 1
40
�mrw (deg∙CM2∙dmol-1)
12000 55 25
70
7000 95○C 40
55
95○C 95 70
95
7000
4 ○C
2000
4 ○C
2000
12000 12000
-3000 95 ℃
-3000
95 ℃
CE1-GNP CE1-GNP
25 25
7000 40 -8000 7000 40
-8000 55 55
70 70
95
25 ℃ 95
25 ℃
-13000 2000 -13000 2000
185 200 215 230 245 260 185 200 215 230 245 260
Wavelength (nm)
-3000
-3000
E1C•GNP CE1•GNP
Fraction (%)
-8000
-8000
Beta GNPs do not disturb the
Unordered response of proteins to
-13000 -13000
185 200 215 230 245 260 Helix
185 200 heat230
215 245 260
Temperature (℃)
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22. Study – 2 : Impact of GNPs on Tt
1
Uniformed Turbidity at 800 nm
0.9
E1C
0.8
27 ℃
0.7
0.6 E1C-GNP
E1C
0.5
30 ℃ E1C-GNP
CE1
0.4 CE1-GNP
CE1 CE1-GNP
0.3
59 ℃ 67 ℃
0.2
0.1
0
0 20 40 60 80 100
Temperature (℃)
Comparison between Tts of E1C and CE1 are consistent with library data
Tt of both E1C and CE1 are enhanced after templation
Enhanced Tt of CE1 after templation is greater than that of E1C
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23. Study – 3 : Effect on Binding Ability
Why Curcumin ?
 Hydrophobic nature
COMP domain forms hydrophobic pore which aids binding
of curcumin
Anti-oxidant and anti-inflammatory properties
Potential for cancer treatment
Aggarwal, B. B.; Shishodia, S. (2004). "Suppression of the Nuclear Factor-κB Activation Pathway by Spice-Derived Phytochemicals: Reasoning for
Seasoning". Annals of the New York Academy of Sciences 1030: 434–441
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24. Result
CCM to protein binding
Protein Sample molar ratio
CE1 1.6±0.9 : 1
E1C 1.8±0.8 : 1
CE1•GNP 2.1±0.7 : 1
E1C•GNP 2.5±0.6 : 1
GNPs maintain the binding and releasing ability of E1C and CE1 to
Curcumin
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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 and
thermoresponsivity while still maintain the binding ability of the
proteins. Through templation it is possible to control the
thermoresponsivity of the proteins.
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26. Thank you!
and many more
lab members!
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