カチオン性脂質とDNAからなる遺伝子導入剤の構造と機能の相関カチオン性脂質とDNAからなる遺伝子導入剤の構造と機能の相関

北九州市立大学 櫻井和朗

Background & AimsBackground & Aims

• Millions of biologically functional molecules have been synthesized or discovered from nature. Most of them turns out useless as drugs in practical sense, because of high toxicity.

• Necessary for a good vehicle to transport the molecules to their target, which is called Drug Delivery System (DDS) .

• In-situ structural characterization of DDS particles is a challenging p g gissue, owing to ultra-dilution and small amount.– Generally, 0.1-10 mg/mL (0.01-1 %)

Conventional SAXS set up:• Conventional SAXS set-up: – Need of high concentration Dens Packing effect– Low concentrationLow S/NLow concentration Low S/N

• We go to Synchrotron SAXS witha newly designed vacuum chamber.

e)2 0.8

1.0 Dens packing effect for thehard core potential.

(q)/N

(V s

pher

e

0.4

0.6 <n>=2 vol%5%

8%I(

qR1 2 3 4

0.2

00

8%

Vacuum chamber and cells Vacuum chamber and cells 

vacuum

air

Kapton Open able to wipe theKapton sample Open-able to wipe the inside taint of glass.

Conventional set-up Vacuum chamber

KaptonKapton

sample

Collaborated with Dr. Masunaga in SP8

Performance of the new setPerformance of the new set‐‐upup

Empty cell-S/N 10-100 times.-Low BG at high q.N k f th k t i d-No peaks from the kapton window

-Low noise around beam stopper

10Au particle10 nm

104

8

6

TRX+DLPC+pDNA

vacuum

10 nm

102

u.) .) 6

4

vacuum

100I(q) (

a.u

I(q) (

a.u

C×2

2air

Kaptonair

10-2

vacuum

airKapton

04.03.02.01.0

q -1 / nm5.00.01 0.1 1

q / nm -1

10 2

Materials: DDS vehiclesMaterials: DDS vehicles

Polymeric micelle DDSfor hydrophobic drug delivery

Lipoplex DDS for gene therapy

Amphipathic blockcopolymer Cationic lipids + amphisbaena lipidsAmphipathic blockcopolymer

+Hydrophobic Drug

Cationic lipids amphisbaena lipids

+pDNA

Hydrophobic Drug

-100-500 nm

Drug

Drug

DNA

DNA-templated highly ordered structure Core-shell10-100 nm

PEGPEG‐‐Poly(Asp,Bzl) MicellePoly(Asp,Bzl) MicelleCollaborating with Prof. YokoyamaKIST

CH3 O CH2CH2O CH2 NH COCHNH COCH2CHNH H117 3 0 25 0 75 24

Di‐Block Copolymer KIST

CH2COOR COOR117 3 0.25 0.75 24

PEG－P(Asp Bzl)

Drug

R = H (27.4 %) or CH2 (82.6 %)

LE540Drug

N

COOHLE540

NH3C

Neuroblastoma: Pre-incubation of SH-SY5Y human neuroblastoma cells with either RAR-pan-antagonist p gLE540 or MAP kinase kinase 1 (MEK-1) inhibitor PD98059.Breast Neoplasms (Breast Cancer) In ZR-75-1 human breast cancer cells, cotreatment of LE135 and LE540 with all-trans-RA inhibited all-trans-RA-induced apoptosis.

SAXS from LE540 loaded PEGSAXS from LE540 loaded PEG‐‐bb‐‐P(Asp,Bzl)P(Asp,Bzl)SPring-8, 40 B2

1000LE540 (wt% per Polymer)

30Low q region Middle&high q region

100

01.43.05.88 3

25 From P-Asp helix

10

8.3

20 Loading0 wt%

(a.u

.)

LE540LE540

1

15

10

1.4 wt%

3 0 wt%

I(q)

0.1

10

5

3.0 wt%

5.8 wt%

LE540Stacking(?)

0.017 8 90.1

2 3 4 5 6 7 8 91

2 3 0654321

8.3 wt%

q / nm-140B2, 6.25 mg/mL (0.625 wt%)Exposure 10 min, camera length: 1.5m

Fitting with coreFitting with core‐‐shell sphereshell sphere

1000P(AspBzl)

calculated (fixed)

Loading rate = 0

100500

3]

10

(a. u.)450

rons/

nm

3

1

I(q)

(

400

ity

[ele

ct

0.1 Core‐shell sphere

5.2nm, 11.5 nm350

ron D

ensi

l t

PEG

0.017 8 9

0.12 3 4 5 6 7 8 9

12 3

q/nm-1

300

Ele

ctr

12840

solvent

calculated (fixed)

12840r/nm Adjustable parameters: shell RC, RS

LE540 loading 1.4 wt%LE540 loading 1.4 wt%core P(AspBzl) LE540(1 )

l f i f P(A B l)

1000

：volume fraction of P(Asp Bzl)［density P(Asp Bzl) =1.3 g/cm3]

Adjustable parameters: RC, RS

100500

10450

ons/

nm

3]

1

10

I(q)

(a. u.)

400y [e

lectr

o

1400

350n D

ensi

ty

0.1350

300

Ele

ctr

o RC=5.3nmRS=11.5 nm

0.017 8 9

0.12 3 4 5 6 7 8 9

12 3

q/nm-1

30012840

r/nmRC RS

core P(AspBzl) LE540(1 ) Fitting with a confined conditionFitting with a confined condition

：volume fraction of P(Asp Bzl)［density P(Asp Bzl) =1.3 g/cm3]

500LE540

0  wt%Adjustable parameters: RC, RS

450

nm

3] 8.3  wt%

LE540

400D

ensi

ty [

e/

0  wt% 8.3  wt%LE540

LE540

350Ele

ctr

on D

LE540

350

Core Shell Solvent300

121086420

r/nmCenter

Density change upon loadingDensity change upon loading

(1 ) Crystalline core

500

core P(AspBzl) LE540(1 )

480

m3]

d = 1 3

460nsi

ty [

e/nm dP(Asp Bzl) = 1.3 

g/cm3(crystalline)Crystalline core+ drug

460

ectr

on D

en

440Ele

dP(Asp Bzl) = = 1.1g/cm3 (amorphous) amorphous core

4201086420

[ ]

+ drug

LE540 [wt%]

Gene Carrier: pDNA/cationic lipid complexGene Carrier: pDNA/cationic lipid complex

O

OO

O

N

H+

DOTAP

OO

O

H

O

P

O

-O

ON

+

DOPC O

Safin a Science 2000 288 2035 2039Safinya, Science 2000 288, 2035-2039

micelleLipoplexPrevious workPrevious work

adding DNA Lamellar to lamellar transition

Biophysical Journal 77 1999 915–924Science 1997 275, 810-814 Phys. Rev. Lett. 1997 79, 2582-2585 Phys. Rev. E. 1998) 58, 889-904 Science 1998 281, 78-81Science 2000 288, 2035-2039SAXS profiles DOTAP/DOPC/pGL3 lipoplexes

Safinya (UCLA), Stanford Synchrotron Radiation LaboratoryTh i SAXS t ti (600 M) i h hith th l t f tiTheir SAXS concentration (600 mM) is much hither than normal transfection concentrations (ca., 0.08-0.004 mM).

Concentration and structureConcentration and structure

In order to measure SAXS from ultra-dilute solutions (less than 1 wt%, hopefully 0.1 wt%), we need a strong synchrotron source and a cell & set-up with low BG and high S/N.with low BG and high S/N.

Transfection SAXS

SAXS：15‐30 mMTransfection：1-10×10-3 mM

（ｍ

M)

α

t Con

c. （

β

Sal

t

Typical phase diagram for lipids. γ

δ

ε

Lipid Conc. （mM）

【micelle】q1 10 mM (0 5 wt%)

ReRe‐‐examination with the vacuum chamberexamination with the vacuum chamber

4

core-shell sphere【micelle】

0.9

1 8

q2q3 q4

10 mM (0.5 wt%)

102

103

104

2.95nm1.65nm

1.8

1.0N/P=2.7 3.6

100

101

10

/(a.u

)

core

shell2.21.2

10-2

10-1

10

I(q)/

【Lipoplex】

core-shell sphere

116.63.3

10-4

10-3

p p

lamellar structure with D = 7.0 nm micelle

core shell sphere model

5 6 7 8 91

2 3 4 5 6

q/nm-1

SAXS profiles of DOTAP/DOPC micelle and DOTAP/DOPC/pGL3 (5300 spherical micelleSAXS profiles of DOTAP/DOPC micelle and DOTAP/DOPC/pGL3 (5300 bp) lipoplex (10mM) as a function of N/P ratio. BL40B2, wavelength = 1Å, sample to detector = 0.7m, 3000 × 3000 IP, N/P ratio = nitrogen of DOTAP/ phosphate of DNA

spherical micelle↓

lamellar structure lipoplex

sandwiched between two bilayersTwo possible structuresTwo possible structures

<micelle> <lipoplex>lamella → lamella

+DNA< Safinya’s condition >

+cationic lipid

DNA DNA DNA

neuttral lipid

< more dilute ><micelle> <lipoplex>

< rearrangement of micelles >< deformation of micelles >

molecular

sphere → lamellaAttachment of DNA to the micelle surface induces structural transition

DNADNA

DNA

molecularattraction

hydrophobic

structural transition.

DNADNA DNA DNA DNA DNA

region hydrophilicregion

The hydrophobic ion pairs go to the hydrophobic domain. This model can explain the N/P =1.

Direct Inv. Fourier TransformDirect Inv. Fourier Transform

N/P= 1 06 109

1st Density profile

N/P= 1.0

5 109 0

1

2( ) cos2

N

nn

A xx A nT

4 109

q2 3 4 5 6 7 8

4th 5th

6th

22

20 2( )NAI A

Fourier Transform

3 109

I(q)q

3 4 5 6 7 8 20

1

( )4 n

n

I q q A q nT

Scattering profile

22 109

2nd3rd

4th 5th 6th

Ai2: peak area

1 109 3rd

Svergun et al, Chem. Mater. 20000

0 1 2 3 4 5 6 7 8

q / nm-1

All possible combinationAll possible combinationClearAllT, r, i, An;T 6.866;An 13.237, 4.594, 0.764, 2.829, 1.386, 1.0233;

Combination N=26=64 3. 3 , .59 , 0. 6 , .8 9, .386, .0 33;

comb TableIntegerDigits2^6 i, 2, i, 1, 2^5;

dens : n1

61^combs, n 1AnnCos2

n

T r;

TablePlotdeni, r, 7, 7, PlotLabel combi, i, i, 1, 32

1520

1, 1, 1, 1, 1, 1, 1

1520

1, 1, 1, 1, 1, 0, 2

1520

1, 1, 1, 1, 0, 1, 3

1015

1, 1, 1, 1, 0, 0, 4

10

15

1, 1, 1, 0, 1, 1, 5

10

151, 1, 1, 0, 1, 0, 6

-6 -4 -2 2 4 6

-10-5

51015

,

-6 -4 -2 2 4 6

-10-5

510

,

-6 -4 -2 2 4 6

-10-5

510

,

-6 -4 -2 2 4 6

-10

-5

510

,

-6 -4 -2 2 4 6

-10

-5

5

10,

-6 -4 -2 2 4 6

-15

-10

-5

5,

151, 1, 1, 0, 0, 1, 7

15

1, 1, 1, 0, 0, 0, 820

1, 1, 0, 1, 1, 1, 9

1520

1, 1, 0, 1, 1, 0, 10

1520

1, 1, 0, 1, 0, 1, 1115

1, 1, 0, 1, 0, 0, 12

-6 -4 -2 2 4 6

-10

-5

5

10

,

-6 -4 -2 2 4 6

-10

-5

5

10

,

-6 -4 -2 2 4 6

-10-5

51015

,

-6 -4 -2 2 4 6

-10-5

51015

,

-6 -4 -2 2 4 6

-10-5

51015

,

-6 -4 -2 2 4 6

-10

-5

510

,

15

1, 1, 0, 0, 1, 1, 1315

1, 1, 0, 0, 1, 0, 1415

1, 1, 0, 0, 0, 1, 1515

1, 1, 0, 0, 0, 0, 1615

1, 0, 1, 1, 1, 1, 17

-6 -4 -2 2 4 6

-10

-5

5

10

15

,

-6 -4 -2 2 4 6

-10

-5

5

10

,

-6 -4 -2 2 4 6

-10

-5

5

10

,

-6 -4 -2 2 4 6

-10

-5

5

10

15

,-6 -4 -2 2 4 6

-15-10

-5

5

10

,

1, 0, 1, 1, 1, 0, 18 1, 0, 1, 1, 0, 1, 19 1, 0, 1, 1, 0, 0, 20 1, 0, 1, 0, 1, 1, 21 1, 0, 1, 0, 1, 0, 22

-6 -4 -2 2 4 6

-15

-10-5

5

10

, , , , , ,

,-6 -4 -2 2 4 6

-15

-10

-5

5

10

, , , , , ,

,-6 -4 -2 2 4 6

-15

-10

-5

5

10

, -6 -4 -2 2 4 6

-20-15-10-5

510

, , , , , ,

, -6 -4 -2 2 4 6

-20-15-10-5

510

, , , , , ,

,

1 0 0 1 1 1 25

-6 -4 -2 2 4 6

-15

-10-5

510

1, 0, 1, 0, 0, 1, 23

, -6 -4 -2 2 4 6

-20-15-10-5

510

1, 0, 1, 0, 0, 0, 24

,-6 -4 -2 2 4 6

-15

-10

-5

5

10

1, 0, 0, 1, 1, 1, 25

,-6 -4 -2 2 4 6

-15

-10

-5

5

101, 0, 0, 1, 1, 0, 26

, -6 -4 -2 2 4 6

-15

-10

-5

5

101, 0, 0, 1, 0, 1, 27

,

-6 -4 -2 2 4 6

-15

-10

-5

5

101, 0, 0, 1, 0, 0, 28

, -6 -4 -2 2 4 6

-20-15-10-5

510

1, 0, 0, 0, 1, 1, 29

, -6 -4 -2 2 4 6

-20-15-10-5

510

1, 0, 0, 0, 1, 0, 30

,-6 -4 -2 2 4 6

-15-10

-5

5

10

1, 0, 0, 0, 0, 1, 31

, -6 -4 -2 2 4 6

-20-15-10-5

510

1, 0, 0, 0, 0, 0, 32

The most possible structureThe most possible structure

DNA

2 nm

DNA

Cationic lipid Neutral lipid

+++

5

101.5 nm

r / nm-4 -2 2 4

5

-10

-5

DNA

++

+

+ + + + + +

-15DNA

DNA

DNA+ + + +++

+ +

6.86 nm

4.8 nm

+ ++

6 86

Circular dichroic (CD) spectra is related DNA conformation

Anther evidence with CD Anther evidence with CD 

( ) p

DNA changes the conformation by changing surrounding environment.

6

8

10

DNA only

Lipoplex(N/P=1.2)

【DNA solution】

→ typical B-form DNAater rich

0

2

4

D[m

deg]

water-rich【Lipoplex】

→disappearing the positive 275 nm

254nm

-6

-4

-2230 250 270 290 310 330CD →disappearing the positive 275 nm

band and appearing the negative 254nm band

275nm

-8

6

wavelength[nm] Structual transition from B- to C-form DNA

CD spectra DNA solution and DOTAP/DOPC/pGL3 lipoplex(Lipoplex = 0.3mM, N/P=12)

Böttcher et al.J. Am. Chem. Soc., 1998Vol. 120, No. 1 12-17

This result suggests that the atmosphere surrounding DNA was changed to water poor condition by complexation.

Bilayer FusionBilayer Fusion DNA/cationic lipid complex: lipoplex

Supramolecular drug

Bilayer Fusion DNA/cationic lipid complex: lipoplex

Supramolecular drugSupramolecular drug

1. Cellular Up-Take: electrostatic2. Endosomal Escape: →fusion ?Micelle including a

drug in the

Micelle including a drug/lipid complex in the hydrophobic domain

Supramolecular drug

1. Cellular Up-Take: electrostatic2. Endosomal Escape: →fusion ?Micelle including a drug

in the hydrophobic

Micelle including a drug/lipid complex in the hydrophobic domain

3. Nucleus Ingestion : diffusiondrug in the hydrophobic domain Or , an inverted micelle

covered with normal layer How the DNA is included in the

3. Nucleus Ingestion : diffusionin the hydrophobic domain Or , an inverted micelle

covered with normal layer How the DNA is included in the micelle micelle is essential for the transportation between cellular vesicles.

is essential for the transportation between cellular vesicles.

Escape from endosomal vesicle

Vesicle bilayer

Escape from endosomal vesicle

Vesicle bilayer

endosomal vesicle vesicle

Drug is just Drug is just transported to the inside of bilayer.transported to the inside of bilayer.

A new cationic lipidOur new cationic lipidOur new cationic lipid

O

H2NO

Benzyl amine (BA)

JPA2006 287855JPA2006-287855

Preparation of micelle

Di l iEvaporate H2N

O

O

BA

OPOO

ONH3+ O

O

-

Dissolve in chloroform

pchloroform

2 O

O OODOPE

O 3mMPBS and

O OPOO

ON+ O

ODLPC-

sonicationLiposome

DNA

Li l

mixing

Lipoplex

micelle Lipoplex (after complexed with DNA)

How to evaluate DNA transfectionHow to evaluate DNA transfection

Cell cultivating LipoplexDNA

Liposome or micelle

24h

DNAFITC coding

Add

48h Endosomalescape

Exchange medium Endosome

FITC24h

Measure the amount of ProteinMeasure the amount of protein by fluoresces mRNA

Protein

BA遺伝子導入効率 Primary amineNot dispersed well

Gene Transfection and composition  Gene Transfection and composition  

A各組成でのlipoplexを肝癌細胞株(HepG2 Cells)に投与し、発現された蛍光タンパク質を発光量により定量した

Clear

AB

2 52 5××101055B

oduc

ed

2.52.5××101055

Clear solution

質発現

量

2.52.5××1010DLPCDOPE

BAprot

ein

pro

DLPCDOPE

Li l

Commercially available conventional

タン

パク質

BA

Lipofectamine

BA

amou

nt o

f

Lipofectamine

Lipoplex

DNA

transfection regent

Lipofectamine

0.860.86××101055The

a 0.40.4××101055

HepG2

DOPE DLPCFig 1 Lusiferase assay

DOPE DLPC0.2 0.4 0.6 0.8

The efficiency strongly depends on theFig.1 Lusiferase assay

発現の最も大きかったB-lipoplexと発現がほとんど見られなかったA-lipoplexの構

造を検討

The efficiency strongly depends on the composition

BASAXS from A (poor transfection)

Lamella A

105N/P

1.1

Lamella + cylinder

DLPCDOPE

104

1.1

2.64

3 3

cylinder

DLPCDOPE

102

103 3.3

4 4(q) /

a.u

.

Sphere -> Cylinder -> Lamella

101

10 4.4

6.6

I(

Sphere+cylinder

1

100 8.8

26.4

sphere10-1

3 4 5 6 7 81

2 3 4 5 6

1

Fig.2 SAXS of A-lipoplex

1q / nm-1

Fitting the data

( ) ( )sI q P qLipid micelle:

( ) ( ) ( ) ( )I q P q S q conP q

from factor of core-shell sphere

Mixture of lipid ( ) ( ) ( ) ( )C h sI q P q S q conP q pmicelle and hexagonally packed

li d structural factor forcylinder structural factor for hexagonal packing with the second kind imperfection.

from factor of three layer cylinder

FittingFitting

104

105N/P

1.1

2.64 678

1000(a)

(b)

103

10

3.3

a.u. 3

4

56

rc=1.3

rc=1.4/ a.u

.

101

1024.4I(q

) / a

100

2

rc=1.5

I(q) /

100

10 6.6

8.8

26 45678

100

10-1

3 4 5 6 7 81

2 3 4 5 6

26.43

4

3 4 5 6 7 8 91

2 3

/ -1

Figure 3. SAXS profile of A-lipoplex

1q / nm-1 q / nm 1

Before After0.2BA 1 h l

Model for formation of the complexModel for formation of the complex

10.32

0.18BANeutral lipid 1 The complex SAXS can

be fitted by a three layer cylinder that

t i hi h l t DNA

1 contains a high electron density domain at the core. The core may be DNADNA.

0.6nm 1.6nm 1.4nm1.4nm0.5nm

DNADNA

Cl-

Core-shell sphere6.65nm 6.4nm

Core-shell sphere

Double layer inverted cylinder Lamella including DNA in the hydrophobic domain

BA

SAXS  at the best transfection: point BSAXS  at the best transfection: point B

1000

B100

DLPCDOPE

B

10

N/P1.65

I(q) /

a.u

.

103.3

I

The lipid already form a cylinder before adding DNA. Upon addition of

1 8.8

g pDNA, the cylinder peak becomes more sharp and the higher order peaks appear. The peak position essentially

3 4 5 6 7 81

2 3 4 5 6

is same. This indicates that the cylinder structure does not change. The order of the structure is enhanced

Fig.4 SAXS at B-lipoplex

1q / nm-1 by addition of DNA

10.545 10.42Before Afte

r(N/P=1 65)

Model for the formation of complexModel for the formation of complex

1. 1. (N/P=1.65)

0 6 1 6 0 7 1 60.6nm 1.6nm 0.7nm 1.6nm

DNACl- DNA

7 186.85nm

7.18nm