Vol. 179, No. 1, 1991 August 30, 1991

BIOCHEMICAL

A NOVEL CATIONIC

LIPOSOME REAGENT FOR EFFICIENT OF MAMMALIAN CELLS Xiang

Department

Received July

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 280-285

Gao and

of Biochemistry, Knoxville,

Leaf

Huang'

University 37996-0840

TN

TRANSFECTION

of Tennessee,

17, 1991

Summary: A novel cationic derivative of cholesterol, 3fl[N-(N',N'dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), has been synthesized and used to prepare sonicated liposomes with dioleoylphosphatidylethanolamine. This novel cationic liposome reagent facilitates efficient DNA mediated transfection in A431 human epidermoid carcinoma cells, A549 human lung carcinoma cells, L929 mouse fibroblast cells, and YPT minipig primary endothelial cells. The activity was greater than that of a commercial reagent, Lipofectin, and was approximately 4-fold less toxic than Lipofectin when assayed with A431 cells. The reagent is easy to synthesize and stable for at least 6 weeks. 0 1991Academic PIXSS,Inc.

Cationic reagents such

liposomes

for

reagent,

and ether of DOPE, in

animal

vitro

multistep

cells

DOTMA,

bonds. is

are in vitro

contains

This

and

which

expensive.

Although

been

prepared

they

are

here

a

relatively

novel

with

review,

transfection The first

see 1). quaternary

with

ammonium

an equimolar

in transfecting

a variety

in

The

synthesis

(2).

The commercial

vivo

(4).

low yield

contains

DOTMA and

several

other

commercially toxic

to

cationic

1To whom correspondence

(for

when mixed

and of relatively

Lipofectin,

nonviral

a double-chain

lipid,

effective (2,3)

efficient

the

is

available

cationic cells derivative

of

DOTMA

is

reagent,

also

(5-7).

cells

relatively reagents

have

amphiphiles, We describe which

can

be

be addressed.

Abbreviations: CAT, chloramphenicol 3@[N-(N'N I-dimethylaminoethane)-carbarmoyl dioleoylphosphatidylethanolamine; dioleoyloxy)propyl]-N,N,N-trimethyl 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in my form reserved.

DOPE,

liposome

cholesterol should

of mammalian

cationic

treated

amount

acetyltransferase; DC-Chol, cholesterol; DOPE, DOTMA, N-[1-(2,3ammonium chloride.

280

Vol.

179, No. 1, 1991

synthesized more

in

efficient

when compared

BIOCHEMICAL

a single

AND BIOPHYSICAL

step.

Liposomes

in transfection with

the

containing

and less

Lipofectin

RESEARCH COMMUNICATIONS

toxic

to the

this

lipid

treated

are cells

reagent.

Materials

and Methods

Materials: DOPE was purchased from Avanti Polar Lipids. Cholesteryl chloroformate and N,N-dimethylethylenediamine were obtained from Aldrich. Acetyl CoA, chloramphenicol and CAT were from Sigma. [3H] acetyl CoA (3.18Ci/mmol) was obtained from Amersham. Lipofectin reagent was from GIBCO BRL. Plasmid (constructed by Dr. Mark Magnuson of Vanderbilt pUCSV2CAT University) was amplified in E. Coli and purified by CsCl gradient ultracentrifugation method.(8) Synthesis of DC-Chol: DC-Chol was synthesized by a simple reaction from cholesteryl chloroformate and N,N-dimethylethylenediamine (Fig.1). A solution of cholesteryl chloroformate (2.25g, 5 mmol in 5ml dry chloroform) was added dropwise to a solution of excess N,Ndimethylethylendiamine (2m1, 18.2 mmol in 3ml dry chloroform) at ooc. After removal of solvent by evaporation, the residue was purified by recrystallization twice in absolute ethanol at 4OC and dried in vacua, yielding 0.545g of white powder of DC-Chol (21.8 %1. TLC yielded a single spot with Rf=0.57 (chloroform:methanol = 65:35); mp108°C; 'H NMR (25OMHz,CDCl,): 6, 2.18 (singlet, 6H, 6,2.36 (triplet,2H,(CH,),NCH,CH,NH); 6,3.21 (CH,) ,NCWH,) ; (quartet,2H,(CH,),NCH,C&NH); 6,5.18 (triplet, broad, lH, (CH,),NCH,CH,NHCO) . Preoaration of cationic lioosomes: A mixture of 1.2 pmol of DCChol and 0.8 umol of DOPE in chloroform was dried, vacuum desiccated, and'resuspended in lml sterile 20mM HEPES buffer (pH 7.8) in a sterile test tube. After hydration for 24 hours at 4OC, the dispersion was sonicated for 5-10 minutes in a bath sonicator to form liposomes with 150-200nm in average diameter. Cell cells

culture (from

and transfection: American Type Culture

A431 cells, Collection)

A549 cells, and L929 were maintained in D-

~>k3$mrH* + Pd+\

dimethylenthylenediaminc

ClC

cholesteryl chlorofonnate

3B[N-(N’,N’-dimethylaminoethane)-carbamoyl] Fiq.

1.

Reaction

dimethylaminoethane)-carbamoyl]

scheme

for

cholesterol (DC-chol)

the synthesis of cholesterol (DC-Chol).

281

Z$[N-N',N'-

Vol.

179, No. 1, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MEM (high glucose), F12 nutrient mixture, and McCoy's medium, respectively, supplemented with 10% fetal calf serum. YPT minipig endothelial cells obtained from Drs. Mark Steward and Gary Nable, Howard Hughes Medical Institute, University of Michigan Medical Center, were grown in medium 199 containing 10% fetal calf serum. Cells were plated into 35mm plates the day before transfection. They were about 80% confluent. One to ten minutes before transfection, cationic liposomes were added to the bottom of a followed by the addition of Iml 12 X 75mm polystyrene culture tube, plasmid DNA diluted in McCoy's medium without serum. The mixture was added to the cells which had been washed once with serum free After 5 hours at 37OC, the incubation medium was replaced medium. with serum-containing medium and cells were harvested 36 hours after the transfection. CAT assay: CAT activity was determined by the benzene extraction method (9), with two modifications: 0.05pCi [3H] acetyl CoA and lnmol unlabelled acetyl CoA were used for each assay and the cell extracts were heated at 60°C for 5 minutes to inactivate interfering enzyme activity (10). Protein concentration was determined by Bio-Rad Protein Microassay method, using BSA as standard. Results Small prepared

liposomes with

ratio)

by

liposomes plasmid (Fig. cells.

the

more

in

efficient four

cells

highest detailed

nm in

of DC-Chol a

diameter)

and DOPE (4:6,

low

ionic

in mediating different

No appreciable Of the

showed for

sonication

DNA into 2).

(150-200

mixtures

are

and Discussion

of

cytotoxicity tested,

cultured

activity.

epidermoid These

be or 6:4,

easily molar These

buffer.

transfection

was noted

A431 human

transfection

5:5,

strength

the

types

could

of pUCSV2CAT mammalian

in the

transfected

carcinoma cells

cells

were

cells chosen

studies.

A 431

L 929 Cell

YPT

A 549

Lines

Fig. 2. Transfection activity of cationic liposomes containing DCChol and DOPE (6:4). The amount of liposomes used was 24 pg/ml for The amount of DNA used was A431 cells and 21 pg/ml for all others. 4 pg/ml for all types of cells. Data is shown as mean -t s.d.(n=3).

Vol.

BIOCHEMICAL

179, No. 1, 1991

AND EIIOPHYSICAL RESEARCH COMMUNICATIONS

8 0.4

Lipid

Concentration

c

(pghnt)

3. Transfection activity and toxicity of Lipofectin and liposomes composed of DC-Chol and DOPE (6:4) in A431 cells. Four pg/ml DNA was used for all wells. CAT activity is shown in squares and the amount of extractable protein is shown in circles. Cells were treated with either Lipofectin (open symbols) or liposomes composed of DC-Chol and DOPE (closed symbols). Data is expressed as mean _+s.d.(n=3). Fiq.

An pg/ml)

experiment with

(6:4).

various

that

was greater shape

was also

that

dependence

on

protein

indication

of

Lipofectin,

the

lipid

from

of

the

Lipofectin/DNA

complex

was quite

a IC,, value

of approximately

and DOPE (6:4) 25 pg/ml.

CAT activity Chol

and

of the cells DOPE

concentration, cells only

(6:4) the

was about 12% of

Lipofectin

It

the for

occurred

total

control maximal

cells

both

the

with 20

note

pg/ml

control. content

transfection

283

that

This for

cells

activity

as The

showing

being

the

At

of

the

DCthis

treated

is compared treated (at

at

maximal

containing

lipid.

content

of

composed of

IC,, value

liposomes

protein

amount

A431 cells,

the

to

reagent

cells.

Liposomes

with

is

determined

treated

lipid.

important

lipid).

It

showed a bell-

was also

to

toxic,

untreated

and DOPE

3).

The

the

toxic

at

DNA (4

of the new liposome

of

cellular

protein

(Fig.

concentration.

transfected

80% of the

the

is

of

DC-Chol

comparison

although

6 pg/ml

were less

amount

containing

treated

cytotoxicity

approximately

a fixed

activity

the

DC-Chol

using

used for

the transfection than

extractable

by

amounts of liposome

Lipofectin

apparent

an

was done

to

with

15 pg/ml

Vol.

179,

No.

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

40

0.6

0

.,.,.,.(.,.I. 2

1

0.2 3

DNA

Fiq. of

4.

Transfection

DC-Chol liposomes

and were

various transfection activity activity

per

were

for

with at

at

higher

previously

been

(2,6),

and is

related

by the

excess

DNA.

extractable concentrations,

the of

medium. cell

excess

the

total

(Fig.

Also

probably

3 and DC-Chol,

mixed

with

to

A431

cells

for

the

due

to

observed the of

in

4 is

treated

cells.

was

observed

the

toxicity

excess

cell

treated

pg/ml)

was

dispersions

only

charges

total At in

amount

of

low

DNA

the

treated

liposomes

to

cells

(21-24

curve

cationic

the

close

cationic

protein

mU CAT

activities

cationic

of the

liposomes

10

lipid

of the Fig.

the

about

cationic

the

transfection

The bell-shaped

other

in

the

transfection

DNA concentrations,

of

were

to

oversaturation

shown

and as

(6:4)

lipid,

Lower

cytotoxicity

were

amount

for

pg/ml

activity (m) Data is expressed

added

total

pg/ml

four

CAT

DOPE

and

DNA.

from

amounts

and

composed

Twenty

concentration

pg/ml

to the

DNA can mask optimal

24

of liposomes

cells. Both

are shown.

DNA

observed

At higher

protein

the

similar

is

8

DNA concentrations.

some

This

A431 wells.

(0)

DNA

4

protein

cells.

all

For

7

Cre/ml,

DC-Chol

4).

well

observed

has

in

plasmid

increased

6

and toxicity

(6:4)

Protein

of

(Fig.

5

Concentration

containing

amounts

4

activity

DOPE used

amount of extractable mean f s.d.(n=3). Liposomes

COMMUNICATIONS

normal

amounts

indicating

that Using

1ipOSOItIeS.

and

in

DNA (4 pg/ml),

reduced

by

about

20%

4). containing

amphiphiles

with

a carbamoyl an ester

bond, bond

284

is much more

(Farhood

et al.,

stable

than

unpublished

Vol.

179, No. 1, 1991

Indeed,

data).

liposomes

transfection

activity Other

shown). labile

ester

ammonium

biodegradable

which

be

amphiphiles we have

can be synthesized is

more

Lipofectin. gene

It therapy

at

not

to

hydrolysis

reaction.

hydrolysed

by the

ether

prepared

a stable

by a simple, in

for

bonds,

due

transfection

a

to

other

and would such

hand,

not

be the

reagent The new toxic

experiments

References Felgner,

P.L.

and Ringold,

G.M.

(1989)

Nature.

337,

387-

388. 2.

Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M. and Danielsen, M. (1987) Proc. Natl. Acad. Sci. USA 84, 7413-

3.

Muller, S.R., Sullivan, P.D., S.C. (1990) DNA and Cell Biol. Bringham, K-L., Meyrick, B., and King, G. (1989) Am. Jr., 1, 95-100. Pinnaduwage, P., Schmitt, Biochim. Biophys. Acta. 985, Rose, J.K., Buonocore, L. BioTechniques. 10, 520-525. Ito, A., Miyazoe, R., Mitoma, Kunitake, T. (1990) Biochem. Maniatis, T., Fritsch, E.F.

7417. 4. 5. 6. 7. 8.

Molecular 1.33-1.45,

9. 10 . 11.

12.

than and

(12).

Acknowledcnnents: This work was supported by NIH grant AI 29893. thank Joan Filyaw for expert word processing and J. Gregory for help with NMR measurements.

1.

be

as DOTMA.

method. less

the

should

transfection

and

not

containing

On the

This

full

(data

be stable

inexpensive

transfection

be valuable

6 weeks

esterases

cells.

containing

and DOPE retained

amphiphiles

expected

entered

RESEARCH COMMUNICATIONS

4°C for

cationic

effective should

DC-Chol

storage

are

once having

summary,

reagent

in

can

those

In

(11)

catalyzed

bond

for

after

bond

AND BIOPHYSICAL

containing

metabolizable

group

carbamoyl

case

BIOCHEMICAL

Clegg, D.O. and Feinstein, 9, 221-229. Christman, B., Berry, L.C., J. Respir. Cell. Mol. Biol. L. and Huang, L. 33-37. and Whitt, M.A.

(1989) (1991)

J., Akao, T., Osaki, T. and Intern. 22, 235-241. and Sambrook, J. (1989) in Cloning; A Laboratory Manual, (2nd Ed.), pp. Cold Spring Harbor, New York. S.K., Green III, P.P. and Fowlkes, D.M. (1987)

Nordeen, DNA 6, 173-178. Sleigh, M.J. (1986) Anal. Biochem. 156, 251-256. Leventis, R. and Silvius, J.R. (1990) Biochim. Biophys. Acta. 1023, 124-132. Plautz, G-E., Stewart, M-J., Gao, X., Huang, L. and Nabel, G.J. (1991) submitted for publication.

We his