A Nanotechnology Platform for Biomedical Image Enhancement

Ning GuJiangsu Laboratory for Biomaterials and Devices

State Key Laboratory of BioelectronicsSoutheast University, Nanjing, China

Email: guning@seu.edu.cnhttp://www.lmbe.seu.edu.cn/nano/

Location

Southeast University, Nanjing

Southeast University, founded in 1902

Main ContentsIntroduction

Micro CT and Au nanoparticles

Superparamagnetic iron oxide nanoparticle as

MRI contrast agents

FeFe33OO44 NPNP-- inclusion inclusion MicrobubblesMicrobubbles as Bi-

modality contrast agents for MRI and US

The action of The action of microbubblesmicrobubbles to cells under to cells under

ultrasound exposureultrasound exposure

Conclusions

The development of medical imagingD

iagn

ostic

tech

nolo

gy

Tissue and cell level Molecular and gene level

1892－2002Single modaltiy

NowadaysStructure and function imaging

FutureDiagnostic combined with therapeutic

1895 X-ray MRI, PET/CT, US

Nanomedicine

Time

anatomical structure level

EarlyClearSee

Nanomaterials and Molecular assembly

Molecular imagingImaging

Micro-MRI, Micro-PET/CT, US

NanotechnologyNanomaterial

Nanodevice or nano-scale functional structure

Nano-characterization and measurement

Biomedicine: Nanobiotechnology and Nanomedicine

Interaction and Bioeffects

Copyright ©Radiological Society of North America, 2001

Weissleder, R. et al. Radiology 2001, 219: 316-333

Schematics show prerequisites to in vivo molecular imaging

分子影像学

Molecular imaging refers to the characterization and measurement of biological processes at the cellular and/ or molecular level.

Micro CT and Au nanoparticles

Home made Micro X-CTwith high-speed algorithms software

The head of a Small Mouse

Chinese 1 yuan

Mouse 3D structure by Micro CT

Drug Analysis and Quality ControlThe CT images of the example pill

Au Nanoparticles

J Phys Chem C,2007; J Phys Chem C,2008

Au Nanoparticles: with diamater as 10nm， 20nm， 50nm

Synthesis of Au Nanomaterials (with various size and shapes)

Au NanoRods with 10 nm in diameter and AR:

3， 5， 7， 18

(1) DI-Water

(2) 5mg/ml 20nm Au Nanoparticles

• 5mg/ml 50nm Au Nanoparticles

• 5mg/ml Injection Iopromidi.

1234

44 KV 55 KV 66KV

139152156149

61696966

20242422

1234

1234

55 KV1

2

3

83

85

84

(1) 10mg/ml 20nm Au Nanoparticles

(2) 10mg/ml Au Rods(AR=3)

(3) 10mg/ml Injection Iopromidi.

Low concentration level

Superparamagnetic Iron OxidNanoparticles (MNPs) as MRI

Contrast Agents

Magnetic nanomaterials Diagnosis and therapy

Magnetic nanomaterialsNano-Devices based on Magnetic nanomaterialsCharacterization and Mesurment for Magnetic nanostructure

Detection and early diagnosis

Effective therapy：

•Carriers for Thermal and Chemical Therapy

•Magnetic target probe

•Monitors after treatments

•Combination?Eg., To Cancer and so on

•Sensor•Contrast

Solvent-thermal preparation method

D=12.5±1.1 nmD=12.5±1.1 nm D=22.5±2.2nmD=22.5±2.2nm

Size and size distribution

10 11 12 13 14 150.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Per

cent

age

diameter (nm)14 15 16 17 18 19 20 21

0.0

0.1

0.2

0.3

0.4

0.5

0.6Pe

rcen

tage

diameter (nm)

19 20 21 22 23 24 25 260.0

0.1

0.2

0.3

0.4

0.5

0.6

Pece

ntag

e

Diameter (nm)

D=17.3±1.6 nmD=17.3±1.6 nm

The size of MNPs can influence the MRI signal

R2 relaxation rate increases with the increase of MNPs size

Surface modification of Fe3O4 nanoparticles with DMSA

+DMSA

Aleate-Fe3O4 nanoparticlesDMSA-Fe3O4 nanoparticles

Double exchange

DMSA：2,3-dimercaptosuccinnic acid

PEG coating of MNPs

TEM images of 12.5 and 22.5 nm MNPs after PEG coating

Poly (ethylene glycol): PEG

EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride)

PEG modified MNPs influence the cellular uptake

Iron uptake by RAW264.7

12.5nm MNPs 22.5nm MNPs

Chitosan (CS) modified MNPs

a) DMSA@MNPs（17nm）

b) CS@MNPs（17nm）

c) CS-DMSA@MNPsaggregates（100nm）

a-COO-

OH

OH

SH

HS

O

O

b -NH4+

O

OH

O

OH

AcHN

O O

HONHAc

HO

O

OH

O

OH

AcHN

O O

HONHAc

HOc

OH

OH

SH

HS

O

O

-COO-

Co-precipitation method

Modified MNPs influence the cancer cellular uptake

0 20 40 60 80 100 120 1400

10

20

30

40

50

60

KB cell 8h

iron

per c

ell/p

g

concentration(ug/ml)

DMSA@MNPs CS@MNPs CS-DMSA@MNPs

0 20 40 60 80 1000

10

20

30

40

50

60SMMC-7721 8h

iron

per c

ell/p

g

concentration(ug/ml)

DMSA@MNPs CS@MNPs CS-DMSA@MNPs

Determination of the various MNPs for interaction with KB and SMMC-7721 cells. The cells were incubated with different

concentration of MNPs for 8h. Results are shown as mean±SD.

In vitro MRI experiments

T2* imaging of different cell numbers when labeled with

DMSA@MNPs, CS@MNPs, CS-DMSA@MNPs.

CS-DMSA@MNPs has the strongest MRI T2

* signal intensity.

T2* singal enhanced1. The amount by cell uptake2. The aggregation of MNPS

enhance the MRI signal

Higher positive charge

SI of T2*（2Χ105

cell)

31.8550.2

15.2263.7

++++14.55.188.585.7CS_DMSA@MNPs

+++10.24.981.016.5CS@MNPs

+-31.45.188.517.3DMSA@MNPs

Iron uptakeZeta potential/mV (pH 7.4)

Aggregate index

Hydrodynamic size/nm

Mean size/nm

Higher aggregation

More uptaken MNPsby cancer cells

Better MRI signal detection

Summary of Magnetic NP for MRI

Anti-Sperm Protein 17 Immunomagnetic Nanoparticles

J. Nanosci. Nanotechnol. 2008, 8, 2341–2346

The anti-Sp17 mAb was grafted on the surface of carboxylated PEG coated MNPs by

covalent bonds.

TEM image of the carboxylated PEG coated MNPs

Magnetization curve of (a) naked MNPs,

and (b) carboxylated PEG coated MNPs

Magnetic Microbubbles as MRI and US contrast agents

Ultrasound images ~ MRI、 X-ray CT

Low cost when compared with X-CT and MRInon-invasiveness Real-time imaging featuresmore and more widely used in clinical applications

.

Rationale of US imaging

Leen E, et al. Eur Radiol 2004；Miller D L, et al. Ultrasound Medicine, 2008

MicrobubblesMicrobubblesUltrasoundBiological

tissue

Biological effectsthermal

cavitation

radiation

US therapy

……

Imaging

brighter

Aggregation

The preparation of FeThe preparation of Fe33OO44 NPNP-- inclusion inclusion microbubblesmicrobubbles

Microscopy observation of mcirobubble

Microscopy images：(a)Non-Fe3O4 inclusion microbubbles, (b- d) Fe3O4-inclusion microbubbles

5μm

a

5μm

b

5μm

c

5μm

d

The ultrasound imaging in the different samples in vitro (A), Control;(B) the multiple emulsion bubbles without SPIO; (C ) the multiple emulsion bubbles with SPIO

Tab.1 The mean grey scale within ROI measured by

using an ultrasound imaging system.

93.4±4.757.9±3.829.2±4.1mean grey scale

bubbles withSPIO

bubbles withoutSPIO

degassed and deionized

waterSamples

Ultrasound Imaging in vitro: Statistic

ROI

ROI

Materials Letters, 2008, 62: 121-124

ROI

In vivo US imaging: Rabbit Liver

Ultrasound image of rabbit liver showing the enhanced contrast by using SPIO microbubbles (a) Conventional ultrasound image pre- injection of the microbubble contrast agent and (b) Post-injection( after 2, 6, 10min)of microbubble contrast agent.

d

ROI

b

ROI

a

ROI

c

ROI

The substantial contrast enhancement was observed with the injected contrast agent (microbubbles with SPIO) compared to the image of the control case obtained pre-injection of the contrast agent. After intravenous injection of the multiple emulsion microbubbles in the rabbit, the brightness in the liver increased over the time.

It was found that within the first 1min after injection, the grey scale was increased rapidly. Then the brightness of images increased gradually, then decreased. The SPIO- inclusion bubbles have better image enhancement. Therefore the SPIO indeed had the contributions to the ultrasound imaging enhancement.

Mean grey scale in rabbit liver vs time after injecting microbubbles

Phys. Med. Biol, 2008, 53: 6129-6141

With SPIO

Without SPIO

The influence of Fe3O4nanoparticles in the shell

Different NPs- embedded shell viscoelastic characterization Different US imaging effects

The visoelastic parameter values

With the increasing concentration of Fe3O4 nanoparticles in the shell, the scattering cross-sections increase at first and then decrease. Therefore, by depositing appropriate solid nanoparticles on the surface of microbubbles, the echo character of the microbubble response can be changed significantly.

189.54532.02E-011.85E+01180.23

257.24731.81E-011.73E+01145.24

423.60881.63E-011.61E+01122.85

594.84771.58E-011.56E+01105.69

608.86281.51E-011.31E+0186.47

594.84771.50E-011.25E+0154.23

456.62671.47E-011.27E+0133.14

409.45321.32E-011.32E+0112.06

356.64641.28E-011.55E+015.73

265.59761.87E-011.70E+010

Scattering cross section（μm2）

Viscosity parameter μs（Pa）

Elastic parameterGs（MPa）Fe3O4 concentration（μg/ml）

increasedecrease

Microbubbles concentration influences the MRI

SPIOBubbleTotal RRR 222 +≈

R2 Total contributed by SPIO Fe3O4 nanoparticles embedded in EMBs is greater than that contributed by the free SPIO Fe3O4 nanoparticles in the solution of the same concentration when volume fraction is greater.

In vivo MRI imaging

Although the T2 signal negative enhancement of MRI immediately starts to appear when the two types of microbubbles are injected, the SNR time-course of SPIO-inclusion microbubbles has longer negative enhancement than mcirobubbles non-SPIO-inclusion.

0min 10min 30min 60min

120min100min80min70min

ROI1

ROI4 ROI3

ROI2

Biomaterials, 2009, 30: 3882-3890

The action of The action of microbubblesmicrobubbles to cells to cells under ultrasound exposureunder ultrasound exposure

Microbubbles as ultrasound imaging and drug delivery systems

Microbubbles structure:

N2 as ultrasound imaging mediam

NBD- cholesterol as model drug

Setups of the in vitro ultrasound exposure:

different acoustic pressure to interact microbubbleswith cells

The schematic diagram of ultrasound exposure apparatus

A focusing transducer(radius=9.2 mm and focal length=8 mm) of 1 MHz was used.

Different ultrasound energy exposure leads to different uptake efficiency

The mean fluorescence intensity initially increases as psp increases and

reaches the maximum when psp = 0.25 MP and then decreases.

Cell observation by SEM

A B

D

FE

C

2μm（A）control, without micobubble, US；（B）control, without microbubble, with 0.25MPa/40S US；（C）-（F）

with only microbubble，,Psp=0.19，0.25，0.38 and 0.48MPa/40S/US

A B

C D

FE

500nm

In drug delivery applications of ultrasound, the

choice of adequate acoustic pressure amplitude is

important. If psp is too small, sonoporation will not be

induced. If psp is too great, nonreparable sonoporation

may be induced; the disruption of cell membranes can

be too great to be repaired by self-sealing.

The membrane repair

ConclusionsNew approaches to realize the designed micro- or nano-structure to be the nanoprobe for imaging enhancement;

The Action of functional nanoparticles to Bio-object, such as DNA, protein, cells;

The microbubbles structure embedded nanoparticles in the shell can be used as multimodality or multifunction carriers.