Composite Silica:Polypeptide Nanoparticles

Sibel Turksen, Brian Fong & Paul S. RussoMacromolecular Studies Group

Louisiana State University

NSF, ACS, LSU Coates Fund

Kasetsart UniversityBangkok, Thailand

Thursday, November 18, 2004

Fuzzballs

a silica interior and synthetic homopolypeptide exterior.

Homopolypeptide Shelltypically 100 nm thick

Silica (SiO2) core

typically 200 nm diameter

Optional superparamagneticinclusion

Why?The usual reasons for polymer-coated particles Stability studies, probe diffusion, standards, etc.The better reasons for polypeptide-coated

particles Should allow excellent shell thickness control. Shell is rigid spacer for assembling silica spheres. Astounding chemical versatility and functionality,

including chirality. Responsiveness and perfection of structures through

reproducible helix-coil transitions. Easily attach antibodies for recognition of cancer cells,

easily attach cancer-killing lytic peptides, too. When magnetic, good way to self-assemble all this

functionality

Our Little Corner of the World: Silica-Homopolypeptide Composite

Particles

Mostly…unstructured, random coil polymers

Co-Si-homopolypeptide composite systems Hierarchical structures Homopolypeptide shell – PBLG, PCBL (can be helix as shown, or coil?) Superparamagnetic – Fe3O4 or Co core

Silica-Stöber SynthesisHydrolysis of tetraethyl orthosilicate (TEOS)

SiOC2H5

H5C2O

OC2H5

OC2H5

C2H5OH

NH4OH

SiOOC2H5

OC2H5

OC2H5

SiH5C2O

OC2H5

OC2H5

TEOS

C2H5OHNH4OH

Si

O

O

SiOSi

OSi

O

OH

OH

OH

OHOH

OH

OHOH

OH OH

OHOH

TEOS

hydrolysis

Stöber

condensation

SEM & TEM of Silica Particles

Synthesis of Magnetite – FeSynthesis of Magnetite – Fe33OO44

FeCl3 FeCl2 NH4OH Fe3O4 NH4Cl+2 8 + 8+

Fe3O4

OH-

OH-

OH-

OH-

-OH

-OH

Fe3O4

-OH

-OH

-OH

-OH

-OH

-OH

NCH3 CH3

CH3

OMe

N+

N+

N+

N+N

+

N+

+

TMAtetramethylammonium hydroxide

Magnetic silica particles

Dark:Magnetic inclusions(~ 10nm) Gray:Glassy SiO2 matrix

TEM- Silica Coated Fe3O4

Superparamagnetic cobaltSuperparamagnetic cobalt

Co

cit –

cit –

cit –

+ NH2(CH2)3Si(OH)3Co

NH2(CH2)3Si(OH)2O –

NH2(CH2)3Si(OH)2O –

NH2(CH2)3Si(OH)2O –

+ Cit–

Co

N O

N O

N O

Stöber reaction

TEOS, APS, EtOHCo

Si

O2

OH –

OH –

OH –

OH –

+ H2O

TEM- Silica Coated Cobalt

Superparamagnetic Particles

Surface Functionalization

SiOMeMeO

(CH2)3NH2

OMe

Si

O

O

SiOSi

OSi

O

OH

O

OH

OHH

MeOH

H2O, NH3

(CH2)3NH2Si (OH)3Si

O HHO

Si

OH

Si OHOH

(CH2)3NH2

H2O, NH3, C2H5OH

Si

O

O

SiOSi

OSi

O

OH

O

OH

O

Si SiOO

(CH2)3NH2(CH2)3NH2

SiNH2(CH2)3

OH

OH

association

condensation

oligomers

adsorption on a particle

APTMS 3-aminopropyltrimethoxysilane

R = CH2CH2CO2CH2C6H5 for PBLG

R = (CH2)4NHCO2CH2C6H5 for PCBL

R

CH

CHNn

O

PCBL helix-coil transition @ 27 C in m -cresol

Homopolypeptides

PBLG best understood

homopolypeptide semiflexible

structure helix-coil transition

Synthesis of homopolypeptides

R' NH2

N

OO O

R H

R'N

N OH

O

OR

H

HCO2

R'N

NH

O

R

H

H

R'N

NH

O

R

H

HN

OO O

R HCO2

R'N

NN

O

OH

HH

HR

n

R

+

1 2 3 4

+ n

4 2 5

Summary: Particle Preparation

H2O , NH3

SiOMeMeO

(CH2)3NH2

OMe

CBL-NCA, monomer

NH2

NH2

NH2

NH2

NH2

NH2

SiOC2H5

H5C2O

OC2H5

OC2H5

O

O NH

NH

OO

O

Superparamagneticdomain

cit –

+ NH2RSi(OH)3N

SiO-

+ SiO2- N

SiO

SiOH

Cobalt particles

Is the shell covalently attached?

4000 3500 3000 2500 2000 1500 1000 500-2

0

2

4

6

8

10

12

14

16 (a)

sour ce: stobersIR

Figure 2aFong and Russo

stober

802

946

1628

Tra

nsm

ittan

ce /

%

Wavenumber / cm-1

4000 3500 3000 2500 2000 1500 1000 5002

4

6

8

10

12

14(b)

source: bf2cp33IR

Figure 2bFong and Russo

PBLG-coated silica

1551

1653

1736

Tra

nsm

ittan

ce /

%

Wavenumber / cm-1

4000 3500 3000 2500 2000 1500 1000 500

0

2

4

6

8

10 (c)

source: bf5ttIR p148

Figure 2cFong and Russo

DMFWashed

1391

1654

Tra

nsm

ittan

ce /

%

Wavenumber / cm-1

Almost certainly

(By the way, the polypeptide conformation is mostly -helix with some -sheet)

TGA/DTA

0 200 400 600 800 1000 1200-100

-80

-60

-40

-20

0

Fong and Russo

Figure 3

Mixed with 16K and 91K

PBLG, then isolated (2 curves)

Silica Spheres Alone

Composite Particle

PBLG

TG

/ %

T / oC

--Particles with ~ 23% by mass PBLG--Again, no evidence for binding of loose PBLG

Dynamic Light Scattering

Bigger ones may diffuse slower (solvent viscosity effects)Flat plots indicate excellent, latex-like uniformity

1.0 1.5 2.0 2.5 3.0 3.5 4.01.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Composite Particles

C18

H37

Spheres

Rh = 1750

Rh = 973

Rh = 990

Silica Spheres

Dap

p / 1

0-8 c

m2 s

-1

q2/1010 cm-2

Particle Characteristics

Silica Core Properties Radius from DLS: 97 nm Molar Mass: 4.5 x 109

Surface area: 15.6 m2/g

PBLG Shell Properties 78 nm. ~90% solvent / 10% polymer. Polymer density limited by crowding around

initiator sites.

Unfortunately, the shell thickness was not controlled by [M]/[I]. Why not?

Not all initiators are active: crowding

Challenges:

Controlling initiator density Attachment of ready-made polymers

Helix-coil Transition of PCBL

Matsuoka, M., Norisuye, T., Teramoto, A., Fujita, H. Biopolymers, 1973, 12,1515-1532

Early attempts showed NO change in the size of the particles—as if the shells were not responding.

We reasoned this might be due to overcrowding on the surface.

Si NH2

OMe

MeO

OMe

APTMS

Si NH

NH2

OMe

OMe

MeO

AEAPTMS

Si

OMe

MeO

OMe

CH3

MTMS

NH2 NH2

25% amino groups

Avoiding crowding

3-(2-furoyl) quinoline-2-carboxaldehyde (ATTO-TAG™ FQ)

Silica-homopolypeptide Composite Particles

DLS of Si-PCBL particles in DMF

0 1 2 3 4 5 6 70

50

100

150

200

250

300

350

400

Si-PCBL core shell particles

Rap

p nm

q2 / 1010 cm-1

Rapp= 251.6±1.42 nm

Helix-coil transition of Co-PCBL

250.00

275.00

300.00

325.00

350.00

375.00

400.00

425.00

0 5 10 15 20 25 30 35 40 45 50 55

Temperature / °C

Ra

pp

/ n

m

1st heating

1st cooling

2nd heating

2nd cooling

3rd heating

4th heating

250.00

275.00

300.00

325.00

350.00

375.00

400.00

425.00

0 5 10 15 20 25 30 35 40 45 50 55

Temperature / °C

Ra

pp

/ n

m

1st heating

1st cooling

2nd heating

2nd cooling

3rd heating

4th heating

3rd cooling

250.00

275.00

300.00

325.00

350.00

375.00

400.00

425.00

0 5 10 15 20 25 30 35 40 45 50 55

Temperature / °C

Ra

pp

/ n

m

1st heating

1st cooling

2nd heating

2nd cooling

3rd heating

4th heating

3rd cooling

Latex

It’s Alive!

0.000 0.001 0.002 0.003 0.004 0.005 0.00660

70

80

90

100

110

120

y=7628x + 68.2R=0.99804

Rap

p / n

m

[M] / g.mL-1

Si-PCBL

0.002 0.003 0.004 0.005 0.0060.00

0.02

0.04

0.06

0.08

0.10

2/2

[M] / g.mL-1

Si-PCBL in 3 weeks

This plot showspolydispersity

Hysteresis curve

M

-M

Magnetization

Magnetizationin opposite direction

SQUID- hysteresis plot of cobalt particles

-25

-20

-15

-10

-5

0

5

10

15

20

25

-60000 -40000 -20000 0 20000 40000 60000

Applied Field (Oe)

Mag

net

izat

ion

(em

u/g

)

Silica coated cobaltLatex iron oxide

SQUID- hysteresis plot of Co-PCBL

-0.015

-0.01

-0.005

0

0.005

0.01

0.015

-4000 -3000 -2000 -1000 0 1000 2000 3000 4000

Applied Field (Oe)

Ma

gn

eti

zati

on

(e

mu

/g)

Initial

3000 to 0 field

0 field to -3000

-3000 field to 0

0 field to 3000

Formation of colloidal crystals

Sufficiently dense suspensions assemble into colloidal crystals. With a size that matches that of visible light, diffraction results. Domains with different orientations result in different and quite pure colors.

~ 0.5 m

Colloidal Crystals (PCBL Shell)

Sufficiently dense suspensions assemble into colloidal crystals. With a size that matches that of visible light, diffraction results. Domains with different orientations result in different and quite pure colors.

~ 0.5 m

SiO2

~ 2 mm

Helical homopolypeptide shell

Why Study?Beautiful!Fun supramolecular synthesize &

characterize from nm to mm. Applies to optical devices,

better lasers, pigment-free paint, “smart colloids”, artificial muscle, separations technology

/ nm400 500 600 700

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

593 nm

568 nm

615 nm

Inte

nsi

ty

Transmittance measured on monochromator-equipped microscope

FWHM of line is ~ 16 nm, comparable to typical interference filters

Spectroscopic analysis of the crystal

Achieving population inversion gets progressively harder for shorter wavelengths; green < red.

8

3

12

12 A

B

E1

E2

A12 B12

Conclusions

Facile synthesis & excellent uniformity

Responsive shell

Hierarchical structures, conformal transitions

Potential applications —optical devices,

stationary phases for chiral separation, model

particles, artificial muscles, medical

treatments

Infinite variation with polypeptide chemistry

Future workHelix-coil transition effect on magnetizationCrosslinking particlesAsymmetric particlesApplication of different grafting techniques

Vapor deposition Grafting onto

Controlling cobalt chains-rodsInvestigation of colloidal crystalsParticles as probe diffusers

*NH

*

O

O

O

n

8

Ru

PhPCy3

PCy3

Cl

Cl

L4M RO

O

8

O

O

8

L4M R M

O

O

8

RL4

O

O

8

-dec-1-enyl-L-glutamate

benzylidene-bis(tricyclohexeylphosphine) dichlororuthenium

Crosslinking

Silica coating

N

N

N

N

N

N

N

NCA-monomer

crosslinking

Surface

Functionalization

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

HELIX COIL

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N