Volume-Phase Transitions in Surface-Tethered Networks and

Implications for Swelling Instabilities

Ryan Toomey

Department of Chemical & Biomedical Engineering

University of South Florida

Tampa, FL 33620

1-D Fibers 2-D Coatings 3-D Structures

5 μm 5 μm

Stimulus

Expanded Collapsed

Volume-Phase Transition

Built-In Actuation, External Cue Generates Macroscopic Response

Mechanical Micro-Switches and Actuators

Sensing Polymer Coatings Adjustable Foundations

“Buckling” Surface Coatings

PRL, 2009

PRL, 2010 10µm 10µm

Tunable Wrinkling

Surface Confined Hydrogels

Langmuir, 2010

Langmuir, 2007

Soft Matter, 2010

Soft Matter, 2013

Soft Matter, 2011

Surface-Attached Gel

Unconstrained Gel

L

L

L

L0

L0

L0

L

L0

L0

5/321 c

o

s NV

V

3/121 c

o

s NV

V

Implications of Constraints

Surface-attachment prevents attainment of

zero stress state

1E-3 0.01 0.1 11

10

100

De

gre

e o

f V

olu

me

tric

Sw

elli

ng

1/Nc

1E-4 1E-3 0.011

2

3

4

5

6

7

8

9

Lin

ear

Sw

ell R

atio

(L

/L0)

1/Nc

Unconstrained

Surface-Attached

Unconstrained

Surface-Attached

Unconstrained and Surface-Attached

Poly(dimethylacrylamide) networks

Volumetric Swelling

Linear Swelling

The surface-attached networks

experience a higher degree of

linear deformation than the

unconstrained networks

5/3

c

o

s NV

V

Vs

Vo

µ Nc

1/3

Toomey et al., Macromolecules (2004)

Can we use the phase diagram of linear polymers to predict the volume-phase transition

behavior in confined geometries?

Transition

Point

Th

ickn

ess

Stimulus

Analog

Digital

Goal: To rationally build intelligence into surface response

0.0 0.2 0.4 0.6 0.8 1.00.0

0.5

1.0

1.5

2.0

Spinodal

Coexistence Curve

2-Phase

1-Phase

T/

Polymer Volume Fraction

Critical Point

Phase Behavior of “Classical” Polymer

“Classical” Brushes

Polystyrene Brush in Cyclohexanone

Karim et al. (1994) Phys. Rev. Lett. 73:3407–10

0.0 0.2 0.4 0.6 0.8 1.0285

290

295

300

305

310

315

320

1-Phase

Polymer Volume Fraction

Coexistence Curve

Spinodal

Tem

pe

ratu

re (

K)

Critical Point

2-Phase

Phase Behavior of “Non-Classical” Polymer

Linear Poly(NIPAAm) Solution

Afroze et al. (2000) J. Mol. Struct. 554:55–68

“Non-Classical” Brushes

Poly(NIPAAm) Brush in D2O

Yim et al. (2005) Phys. Rev. E 72:051801

Phase Behavior of “Non-Classical” Polymer

0.0 0.2 0.4 0.6 0.8 1.0285

290

295

300

305

310

315

320

1-Phase

Polymer Volume Fraction

Coexistence Curve

Spinodal

Tem

pe

ratu

re (

K)

Critical Point

2-Phase

UV Cross-Linkable Copolymer Films

Benzophenone based monomers form

cross-links with free aliphatic groups

365 nm

Statistical copolymers comprising UV-sensitive benzophenone moieties are

deposited and photo-cross-linked: Strategy permits multilayer build-up of

several polymer types

• Activated at 365 nm (non-damaging)

• Provides both cross-linking and surface-attachment

• Cross-links in the presence of oxygen and water

O

R

HC

H

R

C R H

C O

R

C O

H

R

C RC O

H

R

365 nm

h

Photo-Crosslinking Benzophenone

BENZOPHENONE

Reflected

Beam

Neutron Reflection

Heated Jacket

Quartz

D2O

Poly(NIPAAm)

Incident

Beam

Heated Jacket

q q

Neutrons are incident at low angles

Reflection arises due to mismatch between averaged scattering length

densities of atomic nuclei in the direction normal to the interface

Measurement of Reflectivity versus provides “fingerprint” of

density profile with Angstrom scale resolution

Material SLD (x 106)Å2

Quartz

Poly(NIPAAm)

D2O

4.17 0.96 6.33

q sin4zq

• Plot Intensity vs.

• Fringe spacing length

scale of sample

• Solution to the reflectivity

profile is non-unique

• Modeling profile determines

real space interpretation

zq

Neutron Reflection

Poly(NIPAAm-co-MaBP (3%))

Dry Layer Thickness = 320 Å

0.00 0.05 0.10 0.15 0.20 0.25 0.30-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 4000

1

2

3

4

SLD

(Å

-2)

x 1

06

z (Å)

Quartz Substrate

Lo

g (

R)

qz (Å

-1)

Against Dry Air

at 40 oC

0.00 0.05 0.10 0.15 0.20 0.25 0.30-9

-8

-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 4000

1

2

3

4

SLD

(Å

-2)

x 1

06

z (Å)

Quartz Substrate

Against D2O Vapor at 40

oC

Lo

g (

R)

qz (Å

-1)

Against Dry Air

at 40 oC

Neutron reflection of dry film

Neutron Reflection Profiles of Poly(NIPAAm-co-MaBP (3%))

Dry Layer Thickness = 320 Å

Scattering length density ~20% lower than expected. Suggests 2-3 water

molecules associated with each polymer segment

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

10-14

10-13

10-12

10-11

10-10

10-9

10-8

33oC

29oC

27oC

Re

fle

ctivity*q

4 z

Momentum Vector (Å-1)

15 oC

Best fit NR profiles for temperature range 15oC-33oC

Neutron reflection of wet films

Neutron Reflection Profiles of Poly(NIPAAm-co-MaBP (3%))

Dry Layer Thickness = 270 Å

• Approximately 2-3 D2 O molecules per polymer segment in the collapsed state

• Extended interface between swollen network and D2O

0 500 1000 1500 2000 25000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

15 oC

22 oC

25 oC

29 oC

31 oC

33 oC

42 oC

Volu

me

Fra

ctio

n

z (Angstroms)

Segment Density Profiles

Extended profile

due to surface buckling

15 20 25 30 35 40 450

200

400

600

800

1000

1200

<Z

> (

Å)

Temperature (oC)

Thickness versus Temperature

• Gradual transition between 15-29 oC followed by strong collapse

“Dry” layer thickness

Prediction of Discontinuity?

0.0 0.2 0.4 0.6 0.8 1.0285

290

295

300

305

310

315

320

1-Phase

Volume Fraction

Binodal (prediction)

Linear poly(NIPAAm) (experimental)

3% cross-linked network

Tem

pera

ture

(K

)

Critical Point

2-Phase

Avidyasagar et al. Macromolecules (2008)

20 25 30 35

1.0

1.5

2.0

2.5

3.0

3.5

4.0

<

Z>

/Zd

ry

Temperature (oC)

Effect of cross-link density

MaBP (10%)

MaBP (3%)

Poly(NIPAAm-co-MaBP (x%))

0.0 0.2 0.4 0.6 0.8 1.0285

290

295

300

305

310

315

320

2-Phase

3% cross-link denisty

10% cross-link density

1-Phase

Volume Fraction

Te

mp

era

ture

(K

)

Critical Point

Effect of cross-linking on phase behavior

Layers prepared to the left of the critical point can enter the 2-phase region, whereas

layers prepared to the right of the critical point remain in the 1-phase region

Effect of cross-linking on phase behavior

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8285

290

295

300

305

310

315

320

10% MaBP5% MaBP3% MaBP

1% MaBP

Volume Fraction

Tem

pera

ture

(K

)

Cloud Curve2-Phase

Repeat of previous experiments: photo-crosslinked samples above demixing

temperature to drive out water

Avidysagar et al. Soft Matter (2009)

Hydrophilic Hydration

Shells

Hydrophobic Hydration

Shells (Ordered Water) Release of ordered water cannot overcome

entropy loss with collapse of network

O NH

Low Temperatures Favor Swelling

Low temperatures favor swelling

O NH

Release of ordered water from hydrophobic shells overcomes

entropy loss with collapse of network

Hydrophilic hydration

shells stay intact?

At higher temperatures hydrophobic forces dominate and drive collapse

High Temperatures Favor Collapse

4000 3000 2000 10000

2

4

6N-D

Amide II

N-H

Amide II

D2O

102OC (Saturated Vapor)

50OC (Liquid)

A

bsorb

ance

Wavenumber(CM-1)

H2O

Is water left in the collapse?

Isotopic substitution of N-H with D2O readily observed in collapsed state

0 10 20 30 40 50 60 70 80

1.0

1.5

2.0

2.5

3.0

3.5

4.0

S

we

llin

g R

atio

(H

w/H

dry)

Temperature(oC)

nPAAm

DEAAm

PNIPAAm

cPAAm

NVIBAAm

Effect of monomer structure

NH O

NH

O

NH O

NH O

10 20 30 40 50 60 70

1.0

1.5

2.0

2.5

3.0

3.5

4.0

poly(NVIBm)

poly(NVIBm)

poly(CPAAm)

poly(CPAAm)

H/H

dry

Temperature (oC)

Cross-linked Films of Poly(cPAAm) and poly(NVIBm)

Poly(NVIBAm) has a discontinuous transition with hysteresis. Poly(CPAAm) has a

continuous transition with no hysteresis. Both cross-linked with 3 % mole MnBP.

Soft Matter, 2013

Cloud Point Curves

0 5 10 15 20 25 30 35 40 45 5010

15

20

25

30

35

40

45

50

Poly(CPAAm)

Poly(NVIBm)

Tem

pera

ture

(oC

)

Polymer Weight %

Poly(NVIBm) has an off-zero critical point, allowing a discontinuous transition.

Poly(cPAAm) has a critical point near zero concentration, preventing a discontinuous

transition.

1700 1650 1600 1550 1500

0.0

0.5

1.0

1.5

2.0

Inte

nsity

Wavenumber (cm-1)

25 oC

40 oC

70 oC

2nd Order Derivarive of 70 oC

1700 1650 1600 1550 1500

0

1

2

Inte

nsity

Wavenumber (cm-1)

FTIR of poly(PVIBm) Coating

NH

O O2H

H2O

(Amide II)

(Amide I)

(Amide I)

(Amide II)

Subbands

1650 1625 1540

Two distinct hydrogen bonding populations are observed at Amide I. The populations

change through the phase transition

20 30 40 50 60 700.30

0.32

0.34

0.36

0.38

0.40

0.42

0.44

0.46

0.48

A1

65

0 / A

tota

l

Temperature oC

Change in area of

1650 sub-band

to total Amide I

1700 1650 1600 1550 1500

0.0

0.5

1.0

1.5

2.0

Inte

nsity

Wavenumber (cm-1)

25 oC

40 oC

70 oC

2nd Order Derivative 70 oC

FTIR of poly(CPAAm) coating

NH O O2H

O2H (Amide II)

(Amide I)

(Amide I) (Amide II)

1630

1540

In contrast to poly(NVIBm), only a single distinct sub-band found for the Amide I.

Phase diagram of (some) linear polymers may

serve to predict (or approximate) volume-phase

transition behavior in confined systems

In the single-phase region, swelling is determined

by a balance between chain elasticity and mixing

If the system enters the two-phase region, the

swelling jumps discontinuously to the polymer rich

binodal of the phase diagram

Summary … Part I

Adjustable Foundations

• Model of biological membranes – Barrier between bulk phases

• Current status – Bilayer on solid substrate easy to deposit but poor biological

mimic

– Bilayer on thick hydrated support difficult to deposit

• Solution: Adjustable cushion that provides a viable

surface for both deposition and natural mimicry

-1

0

1

2

3

4

5

6

7

0 50 100 150 200 250

Neutron Reflectometry Measurements of Polymer / Membrane (37 °C)

10-10

10-9

10-8

0 0.03 0.06 0.09 0.12 0.15

NIPAAm D2O

NIPAAm

Qz (Å-1)

Reflectivity (

R×

Q4)

SLD

(×

10

-6 Å

-2)

Thickness (Å)

Reflectivity Profiles

Scattering Length Density vs

Thickness

Poly(NIPAAm) (37° C)

Poly(NIPAAm) + DPPC (37° C)

Quartz

10-10

10-9

10-8

0 0.025 0.05 0.075

1

2

3

4

5

6

0 200 400 600 800

Neutron Reflectometry Measurements of Polymer / Membrane (25 °C)

NIPAAm D2O

NIPAAm

Qz (Å-1)

Reflectivity (

R×

Q4)

SLD

(×

10

-6 Å

-2)

Thickness (Å)

Reflectivity Profiles

Scattering Length Density vs

Thickness

Poly(NIPAAm) (25° C)

Poly(NIPAAm) + DPPC (25° C)

Quartz

10-9

10-8

0 0.02 0.04 0.06 0.08 0.1

1

2

3

4

5

6

7

0 200 400 600 800

10-10

10-9

10-8

0 0.03 0.06 0.09 0.12 0.15

NIPAAm + DPPC

(before temp. cycle)

NIPAAm + DPPC (after)

Qz (Å-1)

Reflectivity (

R×

Q4)

SL

D (

×1

0-6

Å-2

)

Thickness (Å)

Qz (Å-1)

Re

fle

ctivity (

R×

Q4)

25 °C

32 °C

37 °C

25 °C

32 °C

37 °C

Polymer / Membrane Temperature Dependence

Smith et al. PRL (2009)

0.05 0.10 0.15 0.20 0.25 0.30

0.05

0.10

0.15

0.20

0.25

0.30

1.000E-61.687E-62.847E-64.804E-68.106E-61.368E-52.308E-53.894E-56.571E-51.109E-41.871E-43.157E-45.326E-48.988E-40.0015170.0025590.0043180.0072850.012290.020740.03500

Off-Specular Neutron Scattering

• Probes in-plane structure

• Can indentify 2 length scales

0.005 0.010 0.015 0.020 0.025 0.030

1E-4

1E-3

0.01

0.1

1

0.005 0.010 0.015 0.020 0.025 0.030

1E-4

1E-3

0.01

0.1

1

8 and 18 μm

pf (Å

-1)

pi (Å-1)

pi (Å-1)

Inte

nsity 18 μm

8 μm

Mikhail Zhernenkov (Lujan, Los Alamos National Labs)

Boris Toperverg (Ruhr-University Bochum and Nuclear Physics Institute)

3-D Structure at 25 °C

1000~ScaleLengthplaneofOut

ScaleLengthplaneIn

What is happening?

1. How tightly bound is the lipid bilayer to the poly(NIPAAm) layer?

2. Are concentration fluctuations in the bilayer independent of the

underlying poly(NIPAAm) layer?

3. Or are concentration fluctuations in the poly(NIPAAm) layer

transmitted to the bilayer?

Poly(NIPAAM) coatings were imaged with AFM, but no

discernable out-of-plane structure could be measured,

… however poly(NIPAAm) coatings of 100x thickness

present significant creasing effects

10 m We will deuterate the outer edge of the

poly(NIPAAm) coating to better understand fluctuations

In ultrathin filmsu

1 m 1 m

30 nm thick 180 nm thick

Biaxial stress results in bicusps

Effects of Surface Confinement

3 General Forms of Non-Uniform Swelling

Bulk Buckling

Differential Lateral Swelling

Edge Buckling

High Aspect Ratio Low Aspect Ratio

Bulk Buckling in pNIPAAm Structures

• Buckling occurs when: – compressive stresses overcome the energy required to

bend the structure

DuPont et al. Soft Matter (2010)

Linear Swelling Model

• Developed by Mora et al. (2006) • Describes onset and characteristics of bulk buckling • Linearization of Föppl–von Kármán equations

T. Mora et al. Eur. Phys. J. E (2006)

Buckling Onset: Pc = 0.867 w2/h2

Wavelength: = 3.256h

Model Assumptions

• σ =f(ε) is linear (stress is a linear function of strain) • kc is small (low magnitude strain)

Bulk Buckling in pNIPAAm Structures

Wavelength is only a function of height?

◊ - Dry Height ● - Swollen Height

DuPont et al. Soft Matter (2010)

Differential Lateral Swelling

Large Expansion of Structure’s Surface (ε: 0.1 – 3.5)

Low aspect ratio structures likely best for rapid cell release

50μm

Engineering Goal

• Culture organized μ-tissues of various shapes/sizes • Rapid • Minimize low temperature exposure

Develop Platform for: Organization, Rapid Release, and Direct Stamping of μ-Tissues

Cell Release from pNIPAAm Surfaces

• Thin film pNIPAAm surfaces (<30nm) • Very little geometric deformation

• Mostly normal to the surface

• Mostly hydration/dehydration of pNIPAAm chains

• Release is slow (>10 min)

μm

T<32°C

T>32°C

DuPont et al. Soft Matter, 2010.

• 3D Micro-scale Surface Structures • High geometric deformation

• In all spatial dimensions

• Induces stress in overlying cell sheet

• Lateral swelling of the gel

Types of Organized μ-Tissues μ-Tissue Building Blocks

100μm 1000μm

Closely spaced arrays (4 - 8μm) Wide, isolated, structures (20 - 100μm)

100μm

Aligned Sheets

100μm

50μm

“fiber-like” μ-tissues

“ribbon-like” μ-tissues

Release is Fast

Testing Detachment Mechanism

• Thin film release: requires metabolic activity • Release inhibited by NaN3 (Sodium Azide)

– Inhibits cytochrome c in mitochondria (ATP production)

• Hypothesis: Cell release is mechanical in nature – Surface strain (pNIPAAm structure)

– Strain can be controlled by crosslink density

Sodium Azide • Inhibits ATP production • Diminishes metabolic activity

Y-27632 • Inhibits Rho-associated protein kinases

(ROCKs) • Allows depolymerization of stress fibers • Reduces cell stiffness

Strain Induced Cell Detachment

Control

Sodium Azide (NaN3)

Y-27632 (ROCK inhibitor)

pre-swell

post-swell

pre-swell

post-swell

Cell Release from pNIPAAm Structures

pre-swell post-swell (3 min)

Control

NaN3

(+)

Y-27632 (+)

Surface Strain > 0.3

μ-contact Printing of Aligned μ-tissues

• Strain induced cell detachment allows for rapid direct stamping of μ-tissues

NIH 3T3 fibroblasts transferred to fn treated PDMS (~5 minutes)

NIH 3T3 fibroblasts cultured atop pNIPAAm structures

50μm

μ-contact Printing of Aligned μ-tissues Stamping of various shapes by controlling pNIPAAm structure geometry

100 μm

Cultured Stamp Post-printing Printed μ-microtissue

μ-tissue “Ribbons”

75μm width

Printed μ-microtissue

50 μm

Cultured Stamp

μ-tissue “Strands”

20μm width

μ-contact Printing of Aligned μ-tissues

• Global pattern transfer of cells

500 μm

Post Transfer Alignment

Alignment is Gradually Lost after 4 Hours

58

0 h

ou

rs

4 h

ou

rs

Cell Culture (Summary)

• Alignment of NIH 3T3 fibroblasts on micron-scale pNIPAAm structures

• Ranging from 1 - 30μm thick, 4 - 100μm wide

• Rapid release of aligned μ-tissues • Release occurs in the time scale of swelling (~ 3min) • Temperature reduction minimized (28°C)

• Release of μ -tissues is primarily mechanical in nature • Does not require metabolic activity • Cell contractility effects detachment

• Micron-scale pNIPAAm structures as a platform for direct stamping of μ-tissues

• μ-tissues maintain organization upon transfer

Final Remarks…

• Soft “messy” systems with externally cued responses can show rich range of behavior

• Can we control, understand, and predict “specificity” in response and behavior?

• How do we control differential swelling in confined geometries to be able to produce 3D patterns using standard 2D processing techniques?

Ajay Vidyasagar (Ph.D.) – phase behavior

Leena Patra (Ph.D.) – phase behavior

Samuel DuPont (Ph.D.) – confined structures and cells

Alejandro Castellanos (Ph.D.)

Martiza Muniz (Ph.D.)

Vinicio Carias (Ph.D.)

Ophir Ortiz (Ph.D.)

Gulnur Efe (Ph.D.)

Ryan Cates (M.S.)

Carlos Bello (M.S.)

Prof. Nathan Gallant

USF, Mechanical Engineering

Camille and Henry Dreyfus Foundation

Draper Laboratory

NSF CMMI 107671

NSF CAREER DMR 0645574

NSF EEC 0530444

USF-BITT Center of Excellence

SmartMaterialsResearchGroup