Molecular Nanosyringe Nanosyringe for for Molecular pH- - - PowerPoint PPT Presentation

Molecular Molecular Nanosyringe Nanosyringe for for pH pH-

• Triggered Injection of

Triggered Injection of Molecules into Cells Molecules into Cells

Oleg Andreev1,2, Donald Engelman2, Yana Reshetnyak1,2

1Physics Department, University of Rhode Island

• 2Dept. of Molecular Biophysics and Biochemistry, Yale University

Presented at the International Congress of Nanotechnology-October 31-November 3, 2005 San Francisco

BRC peptide BRC peptide

Properties of BRC peptide: Properties of BRC peptide:

• water-solubility;
• spontaneous insertion;
• fast insertion (seconds);
• reversible insertion;
• pH-dependent insertion into

bilayer in a form of transbilayer alpha-helix

• Trp fluorescence is sensitive to

binding and insertion

n l m pH>6.0 pH<6.0

• H+

+ H+

G G E Q N P G G E Q N P I Y I Y W W A R Y A D A R Y A D W W L F T T P L L L L D L A L L V L F T T P L L L L D L A L L V D A D E G T D A D E G T

TM

cell membrane

The titration of the BRC with The titration of the BRC with liposomes at pH 7.4 and 5.0 liposomes at pH 7.4 and 5.0

The titration was monitored by changes in intensity of tryptophan fluorescence of the BR-C peptide excited at 295 nm

• 200

200 400 600 800 1000 1200 1400 1600 0.0 0.2 0.4 0.6 0.8 1.0

• pH 7.4
• pH 5.0

Fluorescence intensity, a.u. Concentration of liposomes (POPC), M

Acidic environment is created in many diseased tissues: cancer heart infarction or stroke atherosclerosis inflammation

What is the energy What is the energy of binding

• f binding

and insertion of the BRC and insertion of the BRC peptide into lipid peptide into lipid bilayer bilayer? ?  G Gbinding

binding 

 -

• 4.5 kcal/mol

4.5 kcal/mol  G Ginsertion

insertion 

 -

• 3

3 kcal/mol kcal/mol

What is the topology of the What is the topology of the BRC peptide insertion? BRC peptide insertion?

The topology of insertion was evaluated by using the fact that membrane-impermeable dithionite ion (S2O4

• 2 ) can chemically modify

the NBD fluorophore and quench its fluorescence

50 100 150 200 250

Fluorescence, a.u.

Time, sec

S2O4

• 2

triton

50 100 150 200 250

Fluorescence, a.u.

Time, sec

triton pH3 S2O4

• 2

POPC pH8

Topology of insertion Topology of insertion

NO2

N-terminus

• utside

inside

• NBD

N N-

• terminus of the BRC

terminus of the BRC peptide inserted into peptide inserted into liposomes is liposomes is outside

• utside

What is the oligomeric state of the What is the oligomeric state of the BRC peptide in solution and BRC peptide in solution and liposomes at neutral and low pHs? liposomes at neutral and low pHs? The BRC peptide in 95% is a The BRC peptide in 95% is a MONOMER in solution on the MONOMER in solution on the surface and inserted into surface and inserted into membrane at concentrations membrane at concentrations <40 <40µ µg/ml g/ml

healthy healthy diseased diseased tissue tissue tissue tissue blood blood

pH7.4 pH < 7.0 C pH7.4 pH < 7.0

• S-S

HS- HS- S-S- S-S-

N

General concept of Molecular General concept of Molecular Nanosyringe Nanosyringe

The pH dependent insertion of the BRC The pH dependent insertion of the BRC peptide and delivery of the peptide and delivery of the dansyl dansyl dye dye into into HeLa HeLa cells cells

The pictures were taken on the two-photon confocal microscope BioRad MRC-1024 with excitation at 740 nm.

pH7.0

Synthesis of the Synthesis of the BRC BRC-

• S

S-

• S

S-

• phalloidin

phalloidin-

• TRITC construct

TRITC construct

Phalloidin



TRITC

pH pH-

• Selective translocation of the

Selective translocation of the Phalloidin Phalloidin-

• TRITC by the BRC peptide into

TRITC by the BRC peptide into human ( human (HeLa HeLa) and mouse prostate ) and mouse prostate (TRAMP (TRAMP-

• C1) and breast (JC) cancer cells

C1) and breast (JC) cancer cells

untreated cells pH7.4, 37ºC pH6.5, 37ºC pH6.5, 4ºC

FACS analysis FACS analysis

The ability to wash out non The ability to wash out non-

• cleavable

cleavable BRC BRC-

• phalloidin

phalloidin-

• TRITC construct with

TRITC construct with buffer at pH 7.4 ruled out the buffer at pH 7.4 ruled out the endocytotic endocytotic pathway of molecules translocation pathway of molecules translocation

Cell toxicity Cell toxicity Membrane leakage Membrane leakage assay assay assay assay

Cell toxicity and membrane leakage Cell toxicity and membrane leakage tests ruled out the possibility of tests ruled out the possibility of formations of pores in membrane formations of pores in membrane

The translocation

• f

phalloidin inside the cells led to inhibition of cytoskeleton dynamics and loss of ability of cells to contract and change shape in response to treatment by EDTA/trypsin dissociation solution.

Biological effects of the Biological effects of the translocated translocated into cells phalloidin into cells phalloidin

Multinucleation was

• bserved

after 48 hours after treatement of cells with BRC-S-S-Phalloidin at pH6.5 for 1 hour followed by a change of the medium to DMEM

Whole Whole-

• body Fluorescence Imaging

body Fluorescence Imaging

To study of the distribution of the BRC peptide in mice and it accumulation in tumor by whole-body fluorescence imaging

computer camera controller lamp lamp dark box

excitation filter emission filter

CCD-Camera

Conclusion Conclusion

BRC peptide is a first example of novel class of transmembrane peptides for pH-selective delivery and translocation of molecules into cells

Acknowledgement Acknowledgement

Don Engelman,

Department of Molecular Biophysics and Biochemistry, Yale University

Srikanth Sandugu

Graduate student, Physics Department, URI

Oleg Andreev,

Physics Department., URI