纳米生物学及其应用

·亓立峰浙江加州国际纳米技术研究院·前言：纳米微粒概述·Part

I:纳米药物与靶向设计·Part

II:肿瘤纳米分子影像与实时治疗·Part

III:纳米探针·前景展望What

is

Nano?Quantum

dots,

magnetic

NPFig1.

A

schemetic

of

nanoscale

materialPolymer

NPPhycoerythrin前言某种物质或元素纳米级别粒子:nano

Zinic;SiO2，nano

gold,CdSe/ZnS,Fe2O3,Polymer

NPDegradablePolymericmatrixnanospherenanocapsuleOily

or

aqueous

corePolymericmembrane前言化学法：(1)沉淀法/透析法把沉淀剂加入到盐溶液中反应后，（将沉淀热处理）得到纳米材料。其特点简单易行，但纯度低，颗粒半径大，聚合体纳米粒子、氧化物。(2)微乳液法/乳液扩散法两种互不相溶的溶剂在表面活性剂的作用下形成乳液，在微泡中经成核、聚结、团聚、（热处理）后得纳米粒子。其特点粒子的单分散和界面性好，聚合体纳米粒子、Ⅱ～Ⅵ族半导体纳米粒子多用此法制备纳米微粒制备方法前言纳米粒子表征方法测

量

方

法测量功能适用的尺寸范围使用的主要仪器离心沉降法等效直径>25nm高速离心机，分光光度计或暗场法光学系统气体吸附法（容量法或重量法）比表面积尺寸：约1～10nm比表面积：0.1～1000m2/gBET吸附装置或重量法装置光散射法平均直径>约3nm喇曼光谱仪X射线衍射峰宽法晶粒平均尺寸约<500nm常用于<50nmX射线衍射仪小角度X射线散射线晶粒平均尺寸约<100nmX射线衍射仪电子成像法（TEM）直接观察粒子形貌并测量粒径尺寸约>2nm透射电子显微镜扫描隧道显微镜法（STM）形貌与尺寸宽范围扫描隧道显微镜粒径分析粒径分布粒径分析仪荧光光谱法荧光图谱荧光光谱仪These

newly

formed

tumor

vessels

areusually

abnormal

in

form

and

architecture.They

are

poorly-aligned

defectiveendothelial

cells

with

wide

fenestrations,lacking

a

smooth

muscle

layer,

orinnervation

with

a

wider

lumen,

andimpaired

functional

receptors

forangiotensin

II.

Furthermore,

tumor

tissues

usually

lack

effective

lymphatic

drainage.All

these

factors

will

lead

to

abnormalmolecular

and

fluid

transport

dynamicsespecially

for

macromolecular

drugs.“enhanced

permeability

and

retention(EPR)-effect”

of

macromolecules

andlipids

in

solid

tumors./wiki/Enhanced_Per

meability_and_Retention_effectEnhanced

permeability

and

retention(EPR)

effect.

Long-circulating

drugcarriers

(1),

penetrate

through

the

leakyNamely,

this

phenomenon

was

coinedpathological

vasculature

(2),

into

thetumor

interstitium

(3),

and

degradethere,

releasing

a

free

drug

(4)

andcreating

its

high

local

concentration.AAPS

Journal.

2007;9(2):Article

15.DOI:10.1208/aapsj0902015Nanoparticles

:

clinicaladministration

of

anticancerdrugs.Advantages:√

Controlled

and

targeteddelivery

of

the

drug√

Reduced

systemic

side

effects√

Facilitated

extravasation

intothe

tumor

cells

(EPR

effect:enhanced

permeability

andretention)√

High

capability

to

crossvarious

physiological

barriersFig.3

Size

and

zeta

potential

of

CNP

and

CNP-CuL.

Qi,

et

al,

Colloids

Surfaces

A:

Physicochem

Eng

Aspects

2004;

251:

183190

;

Carbohydrate

Research

2004;

2693-270050

mV96

mVCNP-Cu13

mVChitosanCNPCNP-CuCNPPanel

of

celllineCell

lineCytotoxicity

(IC50,μg/mL)Chitosan

CNP

CNP-CuLiverL-02na

na

naColon

cancerCalo3201200(±

28(±3)

14(±2)27)Gastric

cancerBGC8231200(±35)

21(±2)

8

(±1)Liver

cancerBEL74021200(±22)

15(±2)

6

(±1)Values

are

means

of

three

experiments,

standard

deviation

is

given

inparenthese=

not

active).L.

Qi,

et

al,

Bioorganic

&

Medicinal

Chemistry

Letters

2005;

15:

1357-1399Table

1.

Cytotoxic

activity

of

chitosan,

CNP

(mean

particle

size

=

40

nm),

andCNP-Cu

against

different

cell

linesFig.5

CNP-induced

changes

of

MMP

and

zeta

potential

of

membrane.

A:

Loss

of

MMPand

zeta

potential;

Histogram

of

untreated

cells

(B)

and

cells

treated

with

100

μg/mL

CNP(C)CNP

could

neutralize

the

negative

surface

charge

of

BEL7402

cells

so

as

todamage

the

cell

membrane.

The

strong

dissipation

in

MMP

suggestsapossible

disruption

of

mitochondrial

membrane

after

treated

with

CNP.L.

Qi,

et

al,

Eur.

J.

Cancer

2007;

43:184-93Fatty

acidControl

(μg/mg)CNP

group

(μg/mg)C14:029.93±2.0130.29±2.21C16:0216.5±13.73215.51±5.41C18:0255.61±14.86252.11±10.99C18:1142.62±8.52108.66±5.11aC18:2226.22±8.29195.78±4.54

aC20:412.97±1.469.87±0.45

aaP<0.01

vs

ControlTable

2

Effects

of

CNP

on

fatty

acid

composition

ofmembrane

phosphoric

lipid

from

BEL7402

cellsFig.

6

Lipid

peroxidation

measured

asthiobarbituric

acid

reactive

substances(TBARs)

production

of

BEL7402

cells

non-treated

or

treated

for

24

h

with

variousdoses

of

CNP.Increased

ROS

(Reactive

oxygen

species)production

and

LPO

degree,

decreasedunsaturated

fattyacid

indicatedthat

CNPexerted

membrane

penetration

activity

byinductionof

LPOGroupsBWincrease

(g)TW

(g)TIR

(%)Control1.54±0.562.95±0.46-Chitosan1.78±0.972.24±0.41b2440

nm

CNP2.40±0.441.13±0.22b6270

nm

CNP2.38±0.481.21±0.29b59100

nm

CNP1.96±0.851.92±0.46b35Cisplatin

-2.3±0.99

0.56±0.13bSide

effects

of

chemotherapy81Table

4

Effect

of

CNP

with

different

particle

sizeon

BEL7402

tumor

cell

growth

in

nude

mice

byoral

administration

(1

mg/kg/day)Fig.

7

Effects

ofadministration

routes

onantitumor

efficacy

of

CNPagainstSarcoma-180

subcutaneous

tumorformation

and

growth

in

ICR

mice

Imaging:

cancer

diagnostics,

staging,radiation

planning,

and

evaluation

oftherapy.

Standard

clinical

imaging

modalities:CT,

MRI,

ultrasound,

limitation

indetection

(

<

0.5

cm),

anddistinguishing

between

benign

andcancerous

tumors.

Molecular

imaging:

Multifunctionalnanoparticles:

MNP,

QDs,

gold

NP,drug

delivery

(siRNA,

DNA,

drug),nanothermotherapy,

photodynamictherapy.(b)

Fluorescencespectraof

mouse

skin

and

QDs

with

UV

excitation.

Due

to

the

largeStokes

shift

of

QDs,

their

fluorescence

signals

can

be

shifted

to

a

spectral

region

where

the

autofluorescence

is

reduced.

See

textfor

further

discussion.Size-tunable

light

emissionPhotostableSimultaneous

excitation

of

QDsLarge

Stokes

shiftFigure

13.

Schematic

illustration

of

a

multifunctional

quantum

dotcoated

with

amphiphilic

polymer.Fig.

14.

Schematic

diagram

showing

the

steps

of

siRNA-QD

in

membrane

binding,

cellular

entry,endosomal

escape,

capturing

by

RNA

binding

proteins,

loading

to

RNA-induced

silencingcomplexes

(RISC),

and

target

degradation.Fig.

16.

Schematic

drawing

of

the

hybrid

structure

of

QD

andamphipol

for

siRNA

delivery

and

real-time

imaging

in

live

cells.Qi

and

Gao,

ACS

nano,

2008,

2(7),

1403–1410,

2008Fig.

17

QD

loading

capacity

and

protection

of

siRNAmoleculesagainst

nuclease

degradation

determined

by

gel

electrophoresis.Qi

and

Gao,

ACS

nano,

2008,

2(7),

1403–1410Fig.

18

Gene

silencing

efficiency

of

siRNA

targeting

Her-2

using

QDPMAL

compared

with

the

classic

transfection

agents,

Lipofectamine

and

PEI.Qi

and

Gao,

ACS

nano,

2008,

2(7),

1403–1410纳米分子造影剂产品1）3TP的癌症诊断技术，基于MRI的诊断乳腺癌、前列腺癌，区分恶性/良性，〉5mm瘤块。主要以钆/氧化铁为组分核磁纳米造影剂Palatin技术公司2007年获批准新型造影剂neutrospec，neutrospec含有一个以锝为标记的抗cd15单克隆抗体，可以选择性的结合于参与免疫应答的嗜中性粒细胞。neutrospec被注射入血后，可与感染部位存在的嗜中性粒细胞相结合，使这些细胞被放射性示踪剂所标记。Part

III:纳米探针Fig.22

Modulating

signaling

pathways

and

probing

biological

interactions

withnanotopographyOligo**†*

†*

*Fig.

25.

Cytotoxicity

of

CNCdetermined

by

detection

ofLDH

release

from

BJ-5acells

cocultured

withextracts

of

CNC

for

72

h.Fig.

26Actin

localization

in

spreading

cells

with

timeControlCNCChitosan3

h3

d11

d3.2纳米纤维转染DNAFig.

27.

Human

embryonic

kidney

cell

line

(HEK

293T)

cultured

onSiNWs

(c)

with

and

(d)

without

PEI

treatment

prior

to

GFP

plasmiddeposition.

Yang

et.

al.

J.

AM.

CHEM.

SOC.2007,129,

7228-72293.3靶分子监测纳米探针Fig.

28.

Well-dispersed

antibody-nanoshell

conjugates

in

the

absence

of

analytepossess

a

well-defined

extinction

peak

in

the

near-IR.

In

the

presence

of

thecomplementary

analyte,

multiple

nanoshells

bind

to

the

analyte,

causingagglutination

and

acorresponding

reduction

in

the

extinction

peak.Methods

in

Molecular

Biology™

•

303NANOBIOTECHNOLOGYPROTOCOLSFig.

29.

Signal

detection

of

oligonucleotide-derivatized

NBCshybridized

with

complementary

Cy5

oligonucleotide.Methods

in

Molecular

Biology™

•

303NANOBIOTECHNOLOGYPROTOCOLS3.3纳米金荧光检测核苷酸序列Fig.

30.

Flow

chamber

setup

for

hybridization

and

visualization

of

fluorescence

signaon

the

nanoarray.

Optical

microscope

image

of

an

optical

fiber-bundle

based

nanoarray

containing

individual

300

nmdiameter

fibers;

(ii)

magnified

SEM

image

of

aregion

of