nanotechnology for intracellular nucleic acid delivery

Nanotechnology for Intracellular Nucleic Acid Delivery

Prof. Ke Zhang

1The screen versions of these slides have full details of copyright and acknowledgements

1


Prof. Ke ZhangDepartment of Chemistry and Chemical Biology

Northeastern University, Boston, MA

2

Diagnosis and detection

DNA microarraysPCR Bio-barcode assays

Verigene®

Nucleic acids – a biomedical revolution

RNA interference and antisense technologies

Therapeutics

Diagnosis and detection

DNA microarraysPCR Bio-barcode assays

Verigene®

Surprisingly, there are only a handful of therapeutics on the market…

CGGUAACGU

GCCAUUGCA

CGGUAACGU

GCCAGUGCA

CGGUCACGU

GCCAGUGCA

3

Nucleic acids as therapeutics

Negative charge

Physiochemical properties

Large!

Body clearance by kidney

Rapid nuclease degradation

Scavenger receptor capture

and intracellular degradation


Prof. Ke Zhang


4

Zamecnik et al., PNAS, 1978

Higashi et al.,J. Biochem., 1984

VA VV VAV1970 1980 1990 2000 2010

Synthetics

Nucleic acid delivery systems

Naked DNA Viral vectors

Peptides

Astriab-Fisher et al., Biochem.

Pharmacol., 1997

Liposomes

Wong et al.,Gene, 1980

Dendrimers

Haensler et al. Bioconjugate Chem., 1993

Laemmli et al., PNAS 1975

Rosi et al., Science,2006

Polymers Nano

Grillot-Courvalin et al.,Nat. Nanotechnol., 1998

Bacterial vectors

Biologics

5

Polymer chain Micelles

HydrophilicBlock

HydrophobicBlock

Self-assembly of amphiphilic block copolymers

Micelles

Block copolymer micelles

200 nm

Spheres

Block segment ratio → Interfacial curvature → Morphology

100 nm

Higher curvature

Lower curvature

0.2 m

Polymer chain

HydrophilicBlock

HydrophobicBlock

Self-assembly of amphiphilic block copolymers

Intra-NP forces

HelicesCylinders Discs Toroids

6

Nucleosome-mimicking polymer particles

Shell-crosslinkable• Positively charged

• Around 10 nm in diameter

Design principles:

O O O

Br

NH

NR1R2

O NH

NR1R2

O NH

NR1R2

O NH

NR1R2

O NH

NR1R2

O NH

NR1R2

O NH

NR1R2

O OO

Br

NH

NR1R2

O NH

NR1R2

O NH

NR1R2

O NH

NR1R2

O NH

NR1R2

O NH

NR1R2

O NH

NR1R2

OO

O

Br

NH

NR1R2

ONH

NR1R2

ONH

NR1R2

ONH

NR1R2

ONH

NR1R2

ONH

NR1R2

ONH

NR1R2

O O O

R

NH

NR1 R2

R1, R2 =H or alkyl

Self-assemble

poly(acrylamidoethylamine)-b-polystyrene


Prof. Ke Zhang


7

Cationic nanoparticles transfect cells

Cationic shell-xlinkedknedel-like nanoparticles

(cSCKs)

50 nm

Polyfect cSCK

EGFP transfection in HeLa cells

Lipo

fect

Polyf

ect

2:1

4:1

6:1

8:1

10:1

20:1

0

10

20

30

40

50

% c

ell t

ran

sfe

cte

d

N/P ratio

cSCK

Biomaterials, 2009

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The amine-rich shell of cSCKs

carries dual functions

Exposed amines –DNA complexation

Sterically hindered –Proton absorption

Why does it work, and how can one improve?

The amine-rich shell of cSCKs

carries dual functions

Exposed amines –DNA complexation

Sterically hindered –Proton absorption

vs.

N/P = 1N/P = 2

cSCK Homopolymer Cell0

150

300

450

600

750

900

Flu

ore

sc

en

ce (a.u

.)

N/P=5

N/P=8

N/P=10

N/P=12

N/P=15

N/P=20

N/P=25

cell

9

Variation of cSCK shell composition

cSCK-pa100 cSCK-pa50-ta50 cSCK-ta100

cSCK-pa50-ca50 cSCK-ta50-ca50

Increasing tertiary amine

Incre

asin

g c

arb

oxylic a

cid

These changes allow for independent variation

in buffering capacity and binding affinity


Prof. Ke Zhang


10

Modulating binding affinity/buffering capacity

cSCK-pa100 cSCK-pa50-ta50 cSCK-ta100

cSCK-pa50-ca50 cSCK-ta50-ca50

N/P = 2 N/P = 8

N/P > 32 N/P > 32

N/P = 4

N/P = 8 N/P = 32

Decreasingbufferingcapacity

11

Polyf

ect

Lipo

fect

amin

eCel

l

cSCK-p

a 100

cSCK

-pa 75

-ta25

cSCK

-pa 50

-ta50

cSCK

-pa 25

-ta75

cSCK

-ta10

0

0

200

400

600

800

1000

1200

Mean

flu

ore

scen

ce (

a.u

.)

Controls

N/P = 4

N/P = 6

N/P = 10

N/P = 20

N/P = 30

N/P = 40

Improved transfection efficiency

Reduction of binding with DNA

improves transfection efficiency

Transfection in HeLa cells

Biomaterials, 2010; J. Nuc. Acids., 2012; Nanomedicine NBM, 2013;

Nuc. Acid Ther., 2013; Org. Biomol. Chem., 2013

Polyfect Lipofectamine2k

cSCK-pa100 cSCK-pa25ta75

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Oligonucleotide delivery with cSCKs

Luciferase pre-mRNA splicing correction

assay using HeLa pLuc 705 cells

Antisense (ON705)

Luc iferase Luciferase

Defective luciferase

Active luciferase

Kang, S. H.; Cho, M. J.; Kole R.

Biochemistry 1998, 37, 6235

Luc iferase3’ 5’

Pre-mRNA

mRNA

Olig

ofect

amin

e

Polyf

ect

Unt

reat

ed

cSCK-p

a 100

cSCK-p

a 75-ta

25

cSCK-p

a 50-ta

50

cSCK-p

a 25-ta

75

cSCK-ta

100

cSCK-p

a 70-c

a 30

cSCK-ta

80-c

a 20

0

200

400

600

800

Lu

min

esce

nce (

a.u

.)

+/- Controls N/P = 4 N/P = 6 N/P = 10 N/P = 20 N/P = 30 N/P = 40


Prof. Ke Zhang


13

Delivery peptide nucleic acids (PNAs)

Luciferase splice correction assay

HN

O

PNA

SHN

O

PNAS

R=

Cleavable

Non-cleavable

Mol. Pharm. 2009

Non-cleavable Cleavable

RT-PCR

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An overlooked aspect…

Arrangement of the Nucleic Acids

Physiochemical Properties

of the Carrier

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NaCl

12 h

Thiol-modified oligonucleotide

12 h

High density spherical nucleic acids

Thiol-modified oligonucleotide

12 h

NaCl

12 h

Densely functionalized

(~100 DNA strands/13 nm particle)

Property Conjugate Free nucleic acid

Melting transition

Cooperative andnarrow (~ 2-8 °C)

Broad (~ 20 °C)

StabilityResistance

to nucleasesRapid degradation

Binding Keq = 1.8 x 1014 Keq = 1.8x1012

Lytton-Jean et al. Adv. Mater. 2009, 21, 706-709

Elghanian et al. Science 1997, 1078-1080

Seferos et al. Nano Lett. 2009, 308-311

Lytton-Jean et al. J. Am. Chem. Soc. 2005, 12754-12755


Prof. Ke Zhang


16

Conjugates are taken up by cells

Giljohann et al. Nano Lett. 2007, 7, 3818-3821

Oligonucleotides per AuNP

Au

NP

s (

x1

05)

pe

r c

ell

BSA-coated AuNPs

Oligo-modifiedAuNP

C166 Endothelial Mouse Cells

Despite being negatively charged, oligo-modified AuNPs

are internalized by C116 cells in high quantities

Cell uptake is dependent on surface DNA density

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Enter all cell lines and primary cells studied

• Breast (SKBR-3, MCF-7, MDA-

MB-231, AU-565, MCF-10A)

• Brain (U87, LN229, U118)

• Bladder (HT-1376, 5637, T24)

• Colon (LS513)

• Cervix (HeLa, SiHa)

• Skin (C166, KB)

• Kidney (MDCK)

• Blood (Sup T1, Jurkat)

• Leukemia (K562)

• Liver (HepG2)

• Kidney (293T)

• Ovary (SKOV-3, CHO)

• Fibroblast (NIH3T3)

• Macrophage (RAW264.7)

Cell lines

Primary cells Epithelial tumor model

15m

• Brain (Rat hippocampus

neurons, astrocytes, glial cells)

• Bladder

• Blood (Mouse erythrocytes,

PBMC, T-cells)

• Pancreas (Mouse beta Islets)

• Skin (Mouse)

Rat hippocampal neurons

SKBR-3 cells

15m

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Targeting cancer cells by antibodies

Anti-her2 antibody

Rapid cell uptake (<4h) >90% knockdown at pM concentrations

JACS 2012


Prof. Ke Zhang


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Nucleic acids as transfection agent

• Sharp melting transition

• Nuclease stability

• Increased binding

• High cell uptake

• Gene regulation

?

20

Novel chemistry for hollow particle synthesis

KCN

Gold nanoparticles serve as both

the template and the catalyst

JACS 2010

Nanopods

Nanopods from 40 nm templates

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TEM of nanopods from 40 nm templates


Prof. Ke Zhang


22

Hollow spherical nucleic acids

3’ HS-(T-alkyne)10-Binding region- 5’

T-alkyne =

Self-crosslinking, “diblock” oligonucleotide:

JACS 2011

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hSNA MW increases with template size

10 nm template 30 nm template

TEM of hSNAs

Hollow SNAs

10 nm template 30 nm template

TEM of hSNAs

DNA densities are identical

Size 1 10 100 1000 (nm)

Num

ber

%

DLS number-average size distribution

Dh(n) = 30.5 ± 2.5 nm

Dh(n) = 51.5 ± 4.7 nm

hSNA MW increases with template size

Size 1 10 100 1000 (nm)

Num

ber

%

DLS number-average size distribution

Dh(n) = 30.5 ± 2.5 nm

Dh(n) = 51.5 ± 4.7 nm

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hSNAs have sharp melting transitions

Complementary hSNAs hybridize

and form aggregates upon mixing

500 525 550 575 600 625 650

0

200

400

600

800

1000

Norm

aliz

ed

em

issio

n

Emission Wavelength (nm)

Cy3-hSNA

FITC-hSNA Mixture @ RT

Mixture @ 80 oC

FRET signals observed at RT

0 10 20 30 40 50 60 700.0

0.2

0.4

0.6

0.8

1.0

No

rma

lize

d A

bs (

a.u

.)

Temperature (oC)

hSNA (260 nm) Free DNA (260 nm) AuNP-DNA (520 nm)

hSNA produce sharp melting transitions


Prof. Ke Zhang


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hSNA slow down nuclease degradation

Schematic of nuclease degradation assay

DNA degradation following addition of DNAse I

t½(hSNA) = 4.8 x t½ (free DNA)

0 20 40 60 800

2

4

6

8

10

Free DNA

AuNP-DNA conjugate

hSNA

DN

A d

up

lex

de

gra

de

d (n

M)

Time (min)

26

hSNAs enter cells in high quantities

SCC12 cell uptake of Cy5-labeled hSNAs

Oligo-modifiedAuNP

Cy5-labeled hSNA

SCC12 cells incubated with 1 nM Cy5-labeled hSNAs for 24 h

(Right) Cell uptake of DNA-AuNP conjugates and hSNAsquantified by using radiolabeled alkyne-DNA

0

2x106

4x106

6x106

5 nM 10 nM

Nu

mb

er

of

NP

s/c

ell

Incubation concentration

AuNP-DNA

hSNA

1 nM

27

Gene regulation control using hSNAs

EGFR mRNA levels EGFR protein levels

Very low cytotoxicity

RNA

DNA

AuNP

KCN

RNA

Chimeric “diblock” oligo

siRNA hSNA

0

20

40

60

80

100

Re

lati

ve

mR

NA

le

ve

l

100 1000 10000

0

25

50

75

100

Lipofectamine 2000

hSNA

Su

rviv

al%

(No

rma

lize

d t

o n

on

-tre

ate

d)

Total DNA (ng)


Prof. Ke Zhang


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Acknowledgement

Prof. Karen L. Wooley

Prof. John-Stephen

A. Taylor

Zhou Li

Huafeng Fang

Gang Shen

Yuefei Shen

Prof. Chad A. Mirkin

Joshua I. Cutler

Dan Zheng

Evelyn Auyeung

Liangliang Hao

Jian Zhang

Fei Jia

Alex Lu

Travis Xu

Thank You!

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nanotechnology for intracellular nucleic acid delivery

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