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Elastin-like Polypeptides:Biomedical applications of
Sarah MacEwanWafa Hassouneh
February 19, 2010
Amino acids as building •20 natural amino acids
•Unique side chains provide functionality
•Cys disulfide bondschemical conjugationRedox sensitivity
•Lys amine reactive conjugationenzymatic crosslinking
•Arg bidendate H-bonding
•His" pH sensitivity pKa close to blood pH
chelation of metal cations
•Trp optical properties
Peptides for biological
• Biodegradation
• Biocompatability
• Biologically relevant functionalization
• Monodispersity
• Precise control of composition
Peptides as nanomaterialsPrimary Structure Higher Order
•Inherent stimulus-responsive properties of sequence
•Protease sites
•Encoded locations for modifications, intra- and inter-molecular interactions
•Stimulus responsive changes in intra- and inter-molecular interactions
•Conformational changes of single peptides
•Structural organization of multiple peptides
Stimulus responsive
Environmental stimuli can be extrinsic or intrinsic
• Temperature• Light• pH• Ionic strength• Reducing conditions• Enzymes• Biomolecular recognition
…respond to changes in their environment
Jiang et al. Tumor imaging by means of proteolytic activation of cell-penetrating peptides. Proc Natl Acad Sci USA (2004) vol. 101 (51) pp. 17867-72Kommareddy and Amiji. Poly(ethylene glycol)-modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery. Nanomedicine : nanotechnology, biology, and medicine (2007) vol. 3 (1) pp. 32-42
Bionanosyringe
Protease Targets
Reducible Encapsulants
Elastin-like polypeptides ELP =(V P G X G )n where X= guest residue (Any AA except P)
ELP exhibits an Inverse Phase Transition
T< Tt
Soluble Unimers
Tt< T
Insoluble Aggregate
Temp
Tt depends on X, MW, concentration, solvent, and solutes
ELP Unimers ELP Block Copolymers
ELP[ V1A8G7-n]
ELP[V5-n]
ELP[V5-n]
ELP[V5-n]
ELP[ V1A8G7-n]
ELP[ V1A8G7-n]
Meyer and Chilkoti. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. Biomacromolecules (2002) vol. 3 (2) pp. 357-67
Constructing ELPs: recursive directional ligation
Additional advantages of stimuli-responsive peptide polymers
SolubleContaminants
Hot Spin
Cold SpinInsoluble
Contaminants
Purified ELP
Simple means of purifying ELP unimers…
...or recombinant protein targets
Kim et al. Genetically engineered elastin-protein A fusion as a universal platform for homogeneous, phase-separation immunoassay. Anal Chem (2005) vol. 77 (8) pp. 2318-22
Triggered self-assembly of ELPSo
lubl
e U
nim
er
Changes in: Temperature Solution pH Ionic Strength
Agg
rega
te
Solu
ble
Uni
mer
Hyd
roge
lReaction withCrosslinker
•Local Drug depots•Targeted drug delivery•Molecular actuators
Solu
ble
Uni
mer
Mic
elle
Increase inTemperature
•Dynamic affinity modulation•Targeted drug delivery•Polyvalent protein display
•Tissue engineering hydrogels
Aggregation-assisted tumor
Olympusfluoview.com
Dreher et al. Thermal cycling enhances the accumulation of a temperature-sensitive biopolymer in solid tumors. Cancer Res (2007) vol. 67 (9) pp. 4418-24
Inc. Temp with local hyperthermia
CalmodulinCa+2
M13 peptideApo form
Calcium and peptide bound
Calcium bound
Two domains
Calcium binding loops
M13 peptide
Biomolecular Molecular binding events can trigger ELP transition Conformational changes upon binding influence local environment of exposed and buried residues
Kim, B., and Chilkoti, A. 2008. Allosteric Actuation of Inverse Phase Transition of a Stimulus-Responsive Fusion Polypeptide by Ligand Binding. Journal of the American Chemical Society 130: 17867-17873.
Biomolecular actuator
Kim, B., and Chilkoti, A. 2008. Allosteric Actuation of Inverse Phase Transition of a Stimulus-Responsive Fusion Polypeptide by Ligand Binding. Journal of the American Chemical Society 130: 17867-17873.
Binding M13 peptide results in return to globular shape, reverse ELP transition
Biomolecular ActuatorsInclusion of chromophores as guest residue imparts UV sensitivity to ELP
•Isomer switching of chromophore changes local environment of ELP
•UV sensitivity can be enhanced with alpha-cyclodextrans (α-CDs)
•Protection of chromophore depends on isomer conformationJ. Carlos Rodrigues-Cabello. Amplified Photoresponse of a p-Phenylazobenzene Derivative of an Elastin-like Polymer by alpha-Cyclodextrin: The
Amplifided delta Tt Mechanism. Advanced Materials (2002) vol. 14 (16) pp. 1151-1154
Triggered self-assembly of So
lubl
e U
nim
er
Changes in: Temperature Solution pH Ionic Strength
Agg
rega
te
Solu
ble
Uni
mer
Hyd
roge
lReaction withCrosslinker
•Local Drug depots•Targeted drug delivery•Molecular actuators
Solu
ble
Uni
mer
Mic
elle
Increase inTemperature
•Dynamic affinity modulation•Targeted drug delivery•Polyvalent protein display
•Tissue engineering hydrogels
Physical Crosslinking[VPAVG-(IPAVG)4][(IPAVG) 5]33
[VPAVG-(IPAVG)4][(IPAVG) 5]33[(VPGAG)2VPGEG(VPGAG)2
]20non-polar non-polar
polar
Wu et al. Deformation Responses of a Physically Cross-Linked High Molecular Weight Elastin-Like Protein Polymer. Biomacromolecules (2008) vol. 9 (7) pp. 1787-1794
ELP tri-block
Enzyme-activated gel
ELP[KV6-112]
ELP[QV6-112]
Gel with chondrocytes encapsulated after 28 days
ELP[QV6-112]
McHale, M.K., Setton, L.A., and Chilkoti, A. 2005. Synthesis and in vitro evaluation of enzymatically cross-linked elastin-like polypeptide gels for cartilaginous tissue repair. Tissue Engineering 11: 1768-1779.
Triggered self-assembly of So
lubl
e U
nim
er
Changes in: Temperature Solution pH Ionic Strength
Agg
rega
te
Solu
ble
Uni
mer
Hyd
roge
lReaction withCrosslinker
•Local Drug depots•Targeted drug delivery•Molecular actuators
Solu
ble
Uni
mer
Mic
elle
Increase inTemperature
•Dynamic affinity modulation•Targeted drug delivery•Polyvalent protein display
•Tissue engineering hydrogels
Temperature Controlled Polyvalence
Soluble Unimer
Aggregation of hydrophobic domains
Tt1
ELP[ V1A8G7-n]ELP[ V5-n]
Fluorophore Hydrophobic Domain
Hydrophilic Domain
Functional Residues
Tt2
Increase Temperature
T > Tt1
Targeting Receptors – Dynamic Affinity Modulation
Dreher et al. Temperature triggered self-assembly of polypeptides into multivalent spherical micelles. J Am Chem Soc (2008) vol. 130 (2) pp. 687-94
•Append hydrophilic block with low affinity ligand for overexpressed target receptors
•Low affinity of unimer decreases off-target uptake
•Temperature-triggered micelle assembly increases avidity for target tissue
Temp-regulated UV spectroscopy & Dynamic Light Scattering
Guanidine head group
phosphate
Arginine residue
Arginine-rich cell penetrating peptides
Uptake is non-specific and controlled by polyvalency
0 102
103
104
105
Fluorescence, a.u.
0
20
40
60
80
100
Nu
mb
er
of
Ce
lls
Abcam.comFlow Cytometry
Polyvalence-controlled cellular uptake Inc. Temp
Decreasing Polyvalency
Controlling polyvalency
ELP [V1A8G7] ELP [V5]
ELP micelle for multivalent presentation
Raise T Raise T
Monomer Micelle Aggregate
Hydrophilic HydrophobicProteinTt hydrophilic>> Tt hydrophobic
Trx ELP (1:1)Thioredoxin (Trx):11.7 kDa
Cryo-TEM Trx-ELP (1:1)10 μMFormed at 45o C and 80% humidty
No tilt 35o tilt
Micelle density regulationBAB triblock
• At low temp, core and shell morphology with a relatively loose water-filled core• At high temp, micelles are dehydrated and compact• reversible• decrease in micelle size and higher scattering intensity
α-helical structure to more β-sheet structure (IR and CD)
Drug-conjugate
•Localized conjugation of hydrophobic drug drives micelle assembly with hydrophobic drug buried in the nanoparticle core
•Cysteine residues allow chemical conjugation by maleimide chemistry
•Hydrazone linkers impart pH sensitivity for endosomal drug release
Mackay et al. Self-assembling chimeric polypeptide--doxorubicin conjugate nanoparticles that abolish tumours after a single injection. Nature materials (2009) vol. 8 (12) pp. 993-999
•Nanoparticle formation significantly changes pharmacokinetics, allowing 14-fold increase in drug accumulation in tumor tissue
•Dramatic increase in survival compared to free drug
Questions?