Fabrication and properties of ultra-thin films (nanosheets) as innovative biomaterials

Shinji Takeoka

Dept. Life Science and Medical Bioscience,

Waseda University

polymer chemistry, biomaterials, DDS,

artificial red blood cells, artificial platelets,

molecular assembling science and technology

Electric devices, Biosensors,

Scaffolds for tissue engineering

(Platforms for surface

modification)

Researches on nanosheets fabricated in a bottom-up way

from a solid substrate

Researches on free-standing nanosheets

Development of novel

nanomaterials

(Well-designed synthetic

polymers and polymer network)

Nanosheet

Solid Substrate

Receptor part

Kunitake et. al., Nature Mater. 2006, 5, 494.

Research background

Visible Light Black Light

Skin

Adv. Mater. 2007, 19, 3549.

Huge aspect ratio (>106) ・ Small amount ・High flexibility ・ Large

contact area ・Non-covalent adhesion ・Heteromodification ・

Transparency

Meso-scale Size(mm-m order)

Nanosheet

subsrateFree-standing

nanosheet

Free-standing nanosheets for biomedical

applications

Medical Polymers (Biocompatible, Biodegradable)

Development of various kinds of nanosheets

Micron-sized nanosheets with microtechnology and nanobeads technology

Albumin nanosheet with heterosurfaces, huge nanosheets with microbeads

Colloid Surf. A-Physicochem. Eng. Asp., 318, 184-190 (2008).

Coll. Surf. A-Phys. Eng. Aspects, 334, 28-33 (2009).J. Biomed. Mater. Res. Part A, 89A, 233-241 (2009)..

Cytophobic, cytophilic

Preparation of the free-standing PLLA and

polysaccharide nanosheets

PVA

SiO2 Wafer

PLLA

Releasing

PLLA Nanosheet

PVA

SiO2 Wafer

Chitosan

Spin-coating assisted layer-by-layer (4500 rpm, 15 s, 10.5 pairs)

Casting

Releasing

Sodium Alginate

Polysaccharide Nanosheet

Control of the thickness of the polysaccharide

nanosheet by LbL and scooped states

High transparency and smoothness

AFM pictures

0

200

400

600

800

1000

1200

1400

1600

0 50 100 150 200 250 300

Layer pairs (-)

Polysaccharide nanosheets showing structural colors

Surface morphology scanned by AFM

30.2 + 4.3 nm (10.5 pairs)

Thin film interference theory

Single : orange Double : blue

Triple : green

Structural color change on SiO2 substrate

Colloids and Surface A: Physicochemical and Engineering Aspects, 334, 28-33 (2009).

Free-standing PLLA nanosheets (4 x 4 cm)

Evaluation of adhesive property of nanosheets

Scratch tester for thin films(Rhesca Co. Ltd.)

Thin film on a substrate is scanned with a diamond tip by

applying the increasing pressure. The signal of frictional

vibration just after breaking of the nanosheet was detected

as “critical loading”.

Coil

Magnet

Damper

Cantilever

ScratchingDiamond

tip

Microscratch detection part

Adhesion strength of the nanosheet dramatically increases when the

thickness becomes less than 200 nm with change of the trail.

This would be due to the lowering of the glass transition temperature.

0.00

0.05

0.10

0.15

0.20

0 50 100 150 200Thickness (nm)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0 250 500 750 1000125015001750Thickness (nm)

172 nm 77 nm

Drawn trail

383 nm

Cut-off trail1482 nm

100 mm

10/20

Adhesion strength of the polysaccharide nanosheet

Surgery, 148, 48-58 (2010).

Bulge test apparatus

Strain: s = (P x a2)/(4 x h x d)

Stress: e = (2 x d2)/(3 x a2)

Elastic modulus: E = s / e

Syringe pump

Pressure gauge

h

Evaluation of the mechanical strength of nanosheet

Tsukruk et. al., Adv. Funct. Mater. 2005.

AirP

ad

Steel chamber

Nanosheet

0.0 1.0 2.0 3.0 4.0 5.0

0

20

40

60

80

100

120

PN-35

PN-73

PN-114

Pressure (kPa)

0.000 0.002 0.004 0.006 0.008 0.010

0

10

20

30

40

50

60

70

80

90

PN-35

PN-73

PN-114

Strain (-)

Pressure-Deflection and Stress-Strain curves (f: 1 mm)

of polysaccharide nanosheets

Adv. Func. Mater., 19, 2560-2568 (2009).

Pressure-Deflection curve (f: 6mm) of the nanosheets

0.0 kPa

2.0 kPa

4.0 kPa

1 mmPressure: P (kPa)

Def

lect

ion

: d

(m

m)

0.0 2.0 3.0 4.0 5.00

200

400

600

800

1000

1200

1.0

1400

0.005Strain: e (-)

Stre

ss: s

(MPa

)

0.0100.0000

40

80

120

E(75 nm): 9.6 + 3.1 GPa

Red line indicates that the nanosheet is well tolerated at the air pressure of

3kPa (30 cmH2O), corresponding to the normal respiratory pressure.

0 2 4 6 8 1012141618202224262830

0

10

20

30

40

50

60

70

80

90

100

Time (day)

Rem

ain

ing p

erc

enta

ge (

wt%

)

75 nm

41nm

11 days

7 days

T1/2 (41 nm): 7 days, T1/2 (75 nm): 11 days

Degradation of the polysaccharide nanosheets

with different thicknesses

PBS, 37oC

10 mm

Nanosheet 2 (75nm thickness)

PP mesh (f: 30 mm)

Nanosheet 2 (LbL)

Blood compatibility test

Blood

PP mesh fibers

Adv. Func. Mater., 19, 2560-2568 (2009).

16

Sealing effect of the polysaccharide nanosheet in a

lung perforation model (canine)

6 mm pleura

Sealed with the

(nanosheet + PVA) filmTissue-defect by punch Nanosheet adhered after

dissolution of PVA

lung

18 mm

Operation for a lung-defected beagle after operation

Adv. Func. Mater., 19, 2560-2568 (2009).

Nanosheet (Growth of fibroblast)

Histological sections of perforated lesion

(after 1 week)

Fibrin sheet (post-surgical adhesion)(h)

Chest wall

Fibrin sheet

Tissue adhesion

Fibrin sheet is used as a

conventional treatment of

pleural injury/ defect

But causes pleural adhesion.

Evaluation of PLLA nanosheet patching

to the incision site of mouse stomach

①

②

④

③

⑤

incision (ca. 1 cm)

PLLA nanosheet

stomach

Patching of PLLA/PVA composite film

PLA nanosheet

Dissolution of PVAfilm with PBS

Positive control: 5-6 needle suturing

Negative control: no suturing

PVAPLA nanosheet

Albert-Lembert

suturing

Adv. Mater., 21, 4388-4392 (2009).

baking

Si wafer

PVA (10wt%)PLA, PLGA solution

Spin coating

Peeling off Dissolution in water

Suspension in water

incision (ca. 1 cm)

PLA nanosheet

stomach

Overlapping of PLLA nanosheeton an incision of stomach and repair (mouse)

Nanosheet overlapping Suturing (5-7 needles)

0

20

40

60

80

100

120

1 70

Surv

ival

rat

io (

%)

Day32 4 65

5-7 needle suturing(N=6)

PLA nanosheet(N=9)

No treatment(N=6)

Drying

Preparation of a TC layer

Spin-coating of a PVAc layer

PVAc-TC-nanosheet

Removal of substrate

LbL of chitosan and Na alginate(10 pairs, 39 nm)

Preparation of a PVA film by casting Peeling off and inverting the LbL+PVA bilayered film

SiO2 substrate

Chitosan

Na alginatePVA

PVAcTetracycline (TC)

Antibiotic-loaded nanosheets for the treatment

of gastrointestinal tissue defects

Biomaterials, 31, 6269-6278(2010).

21

250 nmSubstrate

SEM

Sheet

TC

Confocul microscopyOH O

HO

NH2

OOOH

OH

N

OH

Tetracycline (TC) (Ex: 380 nm, Em: 510 nm)

Entry

PVAc-TC-nanosheet

TC-nanosheet

PVAc-nanosheet

Thickness (nm)

177±9

69 ±6

142 ±4

TC (mg/cm2)

6.2±0.5

5.6±0.3

0

ZOI (mm)

7.0±1.7

7.0±1.6

0

PVAc:100nmTC:30 nmNS:40 nm

Structure and function of antibiotic-loaded

nanosheets

ZOI: zone of inhibition

Needle (18 G)

Cecum

Stress StressNormal saline Nanosheet

Blacklight

Puncture hole

Patch nanosheet

(b)

(a)

(c)

Murine cecal puncture model :

(a) schematic representation

(b) macroscopic images

(c) location of TC under black light

Biomaterials, 31, 6269-6278 (2010).

Effects of nanosheets

until 7 days after operation, (a) murine survival,

(b) the number of bacteria in the intraperitoneal lavage

(a)

0

20

40

60

80

100

0 1 2 3 4 5 6 7Time (day)

****

0

5

10

15

20

25

30

TC PVAcPVAc-TC

*

(b)

PVAc-TC-nanosheet (n=14)

TC-Nanosheet （n=11)

PVAc-Nanosheet (n=11)

Without nanosheet (n=5)

nanosheet

NOTES (Natural Orifice Transluminal Endoscopic Surgery), access to the target organs through holes made in stomach/ vagina /lung wall, etc.

Iron oxide nanoparticles-loaded nanosheet for

endoluminal surgery (with SSSA@IIT)

Non invasive, flexible, efficient methods for hole

closing are requested because current techniques

show many limitations

24Silvia et al., Langmuir, 27, 5589–5595 (2011).

Thermo-responsive polymers(pNIPAM)

LCST ?

Hydrodynamic transformation of a free-standing

nanosheet Induced by a thermo-responsive surface

Br BrBr

Bromo initiation ATRP of NIPAM

NH

CO

PMPDEA, CuBr

MeOH/water

Br

C

Br

O

TEA

THF

OH NH2 OH

SiO2

Peel-off with tweezers Release in water

PVA cast

Chitosan

Alginate

pNIPAM

41 nm

10.5 pair

47 nm

Prof. Advincula

(U. Houston, TX)

40 oC-225 oC-2

25 oC-1 40 oC-1

1 cm

ACS Appl. Mater. Inter., 1, 1404-1413 (2009).

Blue

Softening

Locating in the water

Orange

Stiffening

Locating on the air-water

interface

Reversible conversion of the color, property, and

location of the nanosheet by temperature change

Convenient method for surface modification by

patching a free-standing anti-biofouling nanosheet

J. Mater. Chem. (2011) in press

PAH

PC

MPC

MPCMPC

Fluorescent microscopic images of NIH-3T3 cells at the

interface between the FITC-labeled pMPC-nanosheets and

the intact cell culture dish after 72-hrs culture

Few immobilized thrombins are sufficient for platelet spreading(with LIMES, U. Bonn)

(37oC, 15 min)

Blood(without anti-coagulant)

A few immobilizing Thr molecules on the

nanosheet readily activate platelets.

This number is more than 1000-fold

lower than expected from experiments

in solution !!

Biophys. J. 100,1855- 1863 (2011).

29

Nanosheet（LbL)

16 x 15 cm

Dipping

Spray-coater Inside

Spray-Coating

4 x 4 cm

Manufacturing of polysaccharide nanosheets

in an industrial scale

Manufacturing of PLLA nanosheets

in an industrial scale

10 cm x 200 m x 60 nm as a free-standing state

Reconstruction of nanosheets from fragments

5 mm

10 mm

Casting from a fragmented nanosheets

suspension

Dipping in a fragmented nanosheets

suspension and drawing

Various researches and applications

• Nanoparticles(Ag, Au, Fe2O3)-loaded nanosheet as a new functional

materials.

• Drug-loaded nanosheets for periodontal, dermatological,

ophthalmological, and otorhinolaryngological applications.

• Polymorphic structures such as tube-like, bag-like, chip-like, ribbon-

like, porous structures by various methods such as spray-coating,

printing, phase-separation and removing, molding methods.

• Other applications such as cosmetics, foods, optoelectronics,

catalysts, environmental materials.

• Basic polymer physics such as crystallinity, thermal properties,

permeability, surface properties between substrate-side and air-side.

(Waseda Univ.) Prof. Osaka, Prof. Homma, Prof. Ikeda, Prof. Goda, Prof.

Asahi, Dr. Y. Okamura, Dr. T. Fujie, Mr. Akihiro Saito, MC, BC colleageus

(National Defense Medical College) Assoc. Prof. M.Kinoshita, Dr. H. Miyazaki,

Mr. S. Ohtsubo, Prof. D. Saitoh

(IIT@SSSA, Italy) Prof. P. Dario, Prof. A Menciassi, Dr. V. Mattori, Dr. V.

Pensabene

(U. Bonn, LIMES, Germany) Prof. T. Lang, Prof. Hoch, Dr. D. Walrafen, Dr. L.

Fernando

(U. Huston, USA) Prof. R. C. Advincula

“High-Tech Research Center” Project for Waseda University: matching fund

subsidy; grant-in-aid for Scientific Research (B) 21300181 from MEXT

Global Center of Excellence （Global COE）, Waseda University, "Practical Chemical Wisdom“, from MEXT

Hi-Tech Research Center, Waseda University, “Medicine/Science/Engineering Interdisciplinary Life Science Research Center”, from MEXT

Adaptable and Seamless Technology Transfer Program through Target-driven

R&D (A-STEP) from JST, Japan

A grant-in-aid for the Special Research Program from the National Defense

Medical College

Acknowledgments

Biopolis, Singapore

Clean room for nanoparticle preparation

Room for Microscopies

Room for animal study

Open labo

Evaluation of nanosheets as wound healing materials

Overlapping treatment of nanosheets of the perforated cecum

0 1 2 3 4 5 6 7 8

Time after operation (days)

Mouse v

iabili

ty

(%)

20

40

60

80

100

0

****

CF

U (

x 1

04)

Nanosheet PVA

200

400

600

800

1000

1200

1400

1600

0

*

Suture

*Nanosheet

PVA

Suture

(Surgery 2010;148:48-58)

Plastering treatment of nanosheets for the mouse burn skin

Second degree burn70C, 4 sec

Nanosheetplastering

Dissemination ofPseudomonas Aeruginosa

Evaluation of the treatment

Ps (+) / nanosheet (-)Ps (+) / nanosheet (+)

Hemostatic treatment of incision wound of the inferior vena cava

membrane (f: 8 mm) attached with a nanosheet

E. coli (1 x 106)

FBSOuter

Inner

E.coli

E.coli

100 mm

6 hrs

24 hrs

0

100

200

300

400

500

600

700

PVAc-TC-nanosheetPVAc-nanosheet

without nanosheet

**

6 hrs24 hrs

Antimicrobial permeability test

using a transmembrane assay

PVAc-TC-nanosheet PVAc-nanosheet

E. Coli were penetrating through the

nanosheet without TC and such invasion

was shut off by the TC layer.

TC release from the PVAc-TC-nanosheet with

different PVAc thickness under physiological

condition (37oC, pH7.4)

0

20

40

60

80

100

0 10 20 30 40 50 60

Time (min)

TC

rele

ase (

%)

0

20

40

60

80

100

0 1 2 3 4 5 6Time (hrs)

TC

rele

ase (

%)

0 nm

50 nm

100 nm

Fabrication procedure for a polysaccharide nanosheet

supported by a PVA film and its application

for a cecal defect caused by needle puncture.

Surgery, 148, 48-58 (2010).

Application of the polysaccharide nanosheet

to a murine cecal puncture model

Time-course study of murine survival after sealing the puncture

holes with nanosheet samples compared with suturing.

suture(square).nanosheet(circle)

PVA film (triangle)

The number of intraperitoneal bacterium one day after the treatment using a nanosheetfor the 0.8 mm2 hole.

0.5 mm2 puncture hole 0.8 mm2 puncture hole

mucosa

serosa

muscle

mucosa

serosamuscle

puncturepuncture

PVAc-nanosheetPVAc-TC-nanosheet

Histological sections of cecal punctured lesion after

treatment with nanosheet samples (2 weeks)

Instead of the typical accumulation/growth of fibroblasts around the site

of injury, lipocytes or fibroblasts specifically bridged the tissue-defect site

without any associated inflammatory reactions and tissue adhesion,

resulting in almost complete regeneration of the mucosal defect.

Introduction of Bio-molecular Assembly Science Labo.

OO

O

NH

CO (CH2)n CH3

O (CH2)n CH3

H3N

NH

NHNH3

+

+

Gene Transfection by Cationic Liposomes

Nanosheets: Ultra-thin Dressing Material

OO

O

NH

CO (CH2)n CH3

O (CH2)n CH3

H3N

H3N+

+BIO-MOLECULAR

ASSEMBLY Takeoka Lab.

NH

HN

N

O

HN

N

HN

O HN

NH

O

N

HN

N

NH

HN

ONH

O

O

N

NH

NNH

-OOC N

-OOC-OOC

COO-N

COO-COO-

Fluorescent

molecules

Ni2+ Ni2+

Scaffold

His-Tag Targeting Probes

20 mm 5 mm

Nanosheet(LbL)Nanosheet(coating) Nanosheet(hybrid)

Nanosheet (LbL)

Hydrophobic Drug

Hydrophobic (drug) +

Hydrophilic (sheet)

Nanosheet(Coat)

Hydrophobic Drug

Hydrophobic (drug) +

Hydrophobic (sheet)

LbLナノシート

Hydrophilic Drug

Protective layer

Hydrophobic (barrier) +

Hydrophilic (drug) +

Hydrophilic (sheet) +

Drug-loading nanosheets

Methods to prepare free-standing nanosheets

Sacrificial-layer method

Supporting-layer method

Substrate

PVA

Nanosheet

Nanosheet

SubstrateSacrificial layer

Peeling off and immersed into water

Immersed into water