elastin-like polypeptides: biomedical applications...

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?