Characterization of plasma-stabilized liposomes composed of dioleoylphosphatidylethanolamine and oleic acid

Vol.

162,

No.

July

14, 1989

1, 1989

BIOCHEMICAL

BIOPHYSICAL

AND

RESEARCH

COMMUNICATIONS Pages

326-333

CHARACTERIZATION OF PLASMA-STABILIZED LIPOSOMES COMPOSEDOF DIOLEOYLPHOSPHATIDyLETHANOLAMINE AND OLEIC ACID Dexi Liul,

Fan Zhou2 and Leaf Huang1r2*

1Department of Biochemistry, Program, University Received

and 2Cell, Molecular and Developmental Biology of Tennessee, Knoxville, TN 37996-0840

June 2, 1989

Summary: We have previously reported that small unilamellar liposomes (as200 composed of dioleoylphosphatidylethanolamine and oleic acid can be stabilized by incubating with normal human plasma (Liu and Huang, Biochemistry 1989, in press). The stabilized liposomes were very stable even under relatively harsh conditions such as extreme pH, high salt and trypsin treatment. of diphenylhexatriene showed that the stabilized Fluorescence depolarization liposome had a high microviscosity in the lipid core, which did not decrease even after the majority of proteins were removed by trypsin. These data suggest that plasma proteins inserted into the lipid bilayer are probably responsible for the stabilization activity. After i-v. injection into mouse, stabilized liposomes showed a relatively low affinity to liver and spleen as compared to a conventional liposome composition. a 1989 Academic Press, Inc. ml

Liposomes

composed of unsaturated

in

the serum or plasma,

in

the

membrane (for

lipoprotein (1).

interact

We have

unilamellar acid

review with

plasma proteins

such as the

membrane causing small

buffered or

characteristics solution, serum.

(2).

a rapid

(as200

included

high

density

liposome

and lipids

These liposomes

This behavior is just PC liposomes.

stable

are

but

m),

but become stabilized

commonly found for the cholesterol-free, that

of cholesterol

Blood proteins

that

(PC) are not

leakage

not

composed of dioleoylphosphatidylethanolamine

stability

human plasma

see 1).

reported

liposomes

show unusual

high concentrations

the liposome

recently

at 37'C in a simple normal

unless

phosphatidylcholine

large,

and

oleic

not stable

are

when incubated opposite

We have

are found in the stabilized

to also

with

what

reported

liposomes and that

*To whomcorrespondence should be addressed. Abbreviations: DOPE, dioleoylphosphatidylethanolamine; PC, phosphatidylcholine; DOC, deoxycholate; DPH, 1,6-diphenyl-1.3.5-hexatriene; ps, phosphatidylserine; OA, oleic acid; PBS, phosphate buffered saline. ooO6-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

326

is

Vol. 162, No. 1, 1989

BIOCHEMICAL

oleic

from liposomes by

some plasma component (probably

a few additional

observations

acid

is

extracted

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

albumin).

We now report

stabilized

liposomes which include the stability

under extreme conditions, biodistribution

the microviscosity

of the stabilized

of the

of the lipid

about stabilized

the

plasmaliposomes

core, and the &J m

liposomes.

MATERIALSANDMETHODS Dioleoyl PE, egg PC, PS were purchased from Avanti Polar Lipids, Inc. Oleic acid, cholesterol, inulin, calcein and trypsin were obtained from Sigma Chemical Co. Freshly collected humanplasma was obtained from Fort Sanders Regional Medical Center, Knoxville, TN. Tyraminyl inulin was synthesized according to Sommerman et al. (3) followed by standard radioiodination with 1251 performed by the chloramine T methods. Small unilamellar vesicles containing either calcein (50 mMin PBS which was isotonic to plasma) or [ 1251]tyraminyl inulin were prepared by sonication according to Liu and Huang (2). A trace amount of hexadecyl [3H]cholestanyl ether was included in the lipid to monitor the lipid. Calcein containing liposomes (20 mM) were incubated with an equal volume of humanplasma for 1 hour at 3l'C followed by gel chromatography using a Bio-Gel A 1.5m column to separate free plasma components from liposomes. To test the stability of liposomes, 200 ul of liposomes containing 0.4 pmole of lipids were mixed with either PBS, KC1 (1M in PBS) or trypsin containing buffer in a final volume of 400 ul. The ones mixed with PBS were adjusted to a desired pH by NaOH or HCl and left at room temperature for 1 hour. The fluorescence intensity was measured with a LS-5 fluorescence spectrophotometer. Release of the entrapped calcein from the liposomes was calculated according to Liu and Huang (2). Liposomes for protein association measurement were prepared in a simple PBS buffer and mixed with a Ficoll solution to a final Ficoll concentration of 20% incubation with plasma. The mixture was placed in a 15 ml COREX after centrifuge tube. 6 ml of 15% Ficoll were overlayed, followed by 0.5 ml PBS. The mixture was centrifuged in a Sorvall HB4 swing bucket rotor for 1 hour at The liposomes floated to the PBS/15% Ficoll 5000 rpm at room temperature. interface were collected and treated as described above. The liposomes were Protein content was determined by Lowry floated one more time after treatments. assay.

Fluorescence depolarization of DPH in liposome was measured by a method similar to that described by Shinitzky and Barenholz (4) by including 0.2% (mole/mole) of DPH in lipid composition. The anisotropy was calculated using the following equation: 111

- II

XG

Y=

where III is the fluorescence intensity parallel to and Ibis the intensity perpendicular to the plane of polarization of the excitation beam. G is the correction factor obtained using horizontally polarized light (G=II /III )Fluorescence was measured with excitation at 360 run and emission at 430 nm. 150 1.11[1251]tyraminyl inulin containing liposome (1 pmole lipid) was administered intravenously by tail vein injection into Balb/c mice (6-8 weeks old, male). After 5 hours, the mice were anaesthetized with diethyl ether and Blood collected was weighed for each mouse. The mice bled by eye puncture. The were then killed by cervical dislocation and all organs were collected. The total individual organ was counted for rz51-counts in a gamma counter. 327

Vol.

162,

No.

BIOCHEMICAL

1, 1989

AND

radioactivity in the blood was determined was 1.3% of the total body weight (5).

BIOPHYSICAL

RESEARCH

by assuming

the total

COMMUNICATIONS

blood

volume of

RESULTS AND DISCUSSION We have previously

shown that

small

(d6200 nm) sonicated

of DOPE and OA can be stabilized

by incubation

These

liposomes

amount of serum protein

lipid).

In

liposomes,

contain

order

loss of protein

1).

Incubation

test the

stability

of

fraction

Incubation protein, stabilized to

of

the

plasma-stabilized and examined

harsh conditions

(pH 10) or high salt (0.5M KCl)

calcein

solutions

in an acidic

(pH 3) solution

but less than 20% release of calcein. liposomes be compared

are

quite

even

with the original

release of entrapped calcein

Table

Treatmenta pn 7.4

stable

1.

These results in relatively

harsh

DOPE:OAliposomes which

of plasma-stabilized

Protein

Contentb

(a)

100 (n=9)

Calcein

Release=

conditions.

17.9 f 1.3

pH 10.0

99.9 f 6.7 (n=l2)

21.0 + 5.9

KC1

98.3 f 11.2 (n=9)

27.3.f

Trypsin

33.0 f

(%)

3.5

1.9 f 3.5

aPlasma-stabilized liposomes (2.5 umole in 0.5 ml PBS) were incubated with either equal volume of PBS with a final pH of 7.4, 3.0, 10.0 or with PBS containing 1 M KC1 at room temperature for 1 hour or with PBS containing 1.0 mg/ml trypsin for 1 hour at 37'C. bNormalized to the protein content of the plasma stabilized liposomes treated with the pH 7.4 buffer which was approximately 0.5 mg protein/mg lipid. =The pH was adjusted back to 7.4 and calcein release was measured fluorometrically inmediately after the treatment (n=4).

328

that

of the This

show a complete

0

73.8 + 2.8 (n=9)

a

incubation.

indicate

liposomes

pH 3.0

6.0 (n=12)

Only

caused about 30% loss

at pH below 5 (2).

Stability

(Table

for 1 hour

from liposomes.

(<30%) of the entrapped calcein was released during liposome

protein/mg

mg

(-0.5

from liposome and the release of entrapped

in alkaline

composed

normal plasma or serum (2).

room temperature did not cause any loss of protein

small

is

further

we have incubated them in relatively

the

at

to

a large

with

liposomes

Vol. 162, No. 1, 1989

BIOCHEMICAL

Table 2.

Liposome

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

Fluorescence depolarization of DPHin liposcmes

Treatment

Fluorescence

Microviscosityf (poise)

AnisotropyC

PBS

0.121

* 0.002

1.205 f. 0.013

Plasm@

0.158

f 0.004

1.859 f 0.0.4

Plasma then trypsinb

0.160 f O.OOSe

1.901 f 0.034e

aLiposomes containing 0.2 mol 8 DPH were incubated with PBS or 50% plasma for 1 hr at 31°C, followed by chromatography through a Bio-Gel A1.5m column.

bPlasma-treated liposomeswere further incubated with trypsin (0.5 mg/ ml final) for 1 hr at 37OC. Trypsin inhibitor (2.5 mg/ml final) was added to stop the reaction. Liposomes were gel-filtered Bio-Gel A1.5m column to remove free proteins. CMeasured in 5 determinations. dp < 0.0002 when compared with values for PBS treatment. ep < 0.0001 when compared with values for PBS treatment, fCalculated according to Shinizky and Barenholz (9).

The stability

of

the stabilized

dependent on the associated proteins, 10% calcein

SDS-PAGEof trypsin-treated

major proteins

not

originally

stabilized

These results

into

was reDQTte!hby

the

lipid

a trypsin

liposomes has indicated

suggested that most proteins

liposomes are accessible to trypsin,

may be tightly

not

strongly than

treatment

that all

of

peptides which did not resolve well in the gel

does not depend on these proteins. peptides

apparently

the

associated with liposomes had been removed except some

low molecular-weight shown).

is

a

because the liposomes released less

even when 67% of the protein

(Table 1).

very

liposomes

through

Furthermore,

and the integrity

These peptides

with

of

could

the

liposome

the trypsin-resistant,

associated with the lipid

bilayer.

associated

(data

residual

membrane, perhaps

inserted

be important

liposome

for

stabilization. If

peptides

membrane of increase with lipid (61,

or a portion

lipcsomes,

significantly. bilayer

mellitin

(7),

the

of serum proteins microviscosity

are inserted

of the

into

membrane is

This phenomenonhas been observed in the

of several bilayer-inserting and apolipoproteins

(8). 329

proteins,

the

lipid

expected interactions

such as cytochrome

We have examined this

to

possibility

bg

Vol.

162,

No.

1, 1989

by measuring is widely

the fluorescence

used to measure

AND

DPH was observed

the lipid 1.9

core,

poise

DPH in

of a lipid

probably

membrane

due

Importantly,

increase

in the plasma-stabilized

calculated

according

to

insertion

the removal

with

the notion

which

could Two

that

important

(9),

This result

indicates

liposome

observations hydratable

head

monolayer

of a small

be easily

with

hydrated inserted

DOPE, bringing

apolipoproteins stabilizer, with

molecular

resistant

membrane. liposome these

to

bulky

they contain to

form

to the outer

acyl

trypsin

digestion

also

proteins

increase

membrane due to lipid-protein

by

trypsin

is consistent

peptides

or

proteins

chains,

(3)

the

outer

non-bulky

hydrophobic

portion

of the small

liposome

enriched

helices

which (8,121.

the microviscosity

of

the

would

liposome

of

the

promote

helices

"buried"

of the lipid

the

strongly

Insertion

These partly

that

interact

liposomes

bilayer.

they are

interactions.

poorly

amphipathic

such as lipids

these

Thus, we suspect

more stable

330

but

proteins

of the small

hypotheses.

PE-,

and

structures

because

are

and

(2)

prime candidates

amphipathic

serum

interpreted

a small

surface

stabilization.

monolayer

and hence

lipid

and (2) only

(ll),

monolayer

discoid

packing

They would

bilayer.

has

in a concave

the plasma or serum are

phospholipids

tighter be

in

helix

the outer

of

the

We have

head group and relatively into

to

the mobility

by plasma or

(2).

of high curvature

about liposome

because

amphipathic

liposome

from 1.2

This result

(1) DOPE molecule

packed

of

reduced,

are stabilized,

(10) and relatively

DOPE are poorly

a richly

2).

stabilization

are stabilized that

polarization

of liposome.

of liposome

facts

group

like

can

the stability

see

significantly

into

of plasma proteins

(d5200 nm) liposomes

the

molecules

having

for

liposomes

by

that

is

(Table

which

review

had increased

does not remove the inserted

features

small

based

PC-,

trypsin

DPH,

The microviscosity

of most of the liposome-associated

be essential

(1) only

not

that

probe,

of fluorescence

to Shinizky

did not reduce the membrane microviscosity

COMMUNICATIONS

membrane (for

liposomes.

of plasma-treated

the

RESEARCH

of a hydrophobic

the microviscosity

by the plasma treatment. the

BIOPHYSICAL

depolarization

As can be seen in Table 2, a large

9). of

BIOCHEMICAL

would in

the

core of

the

We are currently

testing

BIOCHEMICAL

Vol. 162, No. 1, 1989 Table

3.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Biodistribution

of liposomes

in mousea

DOPE:OA (2:l) Serum Treated (n=5)

PC:chol:PS jlO:S:l)

Serum Treated Trypsinized (n=5)

and

Tissue

Untreated (n=5)

Blood Spleen Liver Lung Chest Intestine Heart Trunk Head Upper Limb Lower Limb Kidney Skin Tail

8.02f0.90 2.90f0.36 31.44f0.68 0.35f0.06 1.81+0.30 2.05f0.50 0.19f0.04 1.49f0.45 2.80f0.67 1.23f0.31 1.24f0.26 1.67f0.26 2.26kO.25 1.82f0.61

8.31f1.06 3.93f0.42 43.85k1.18 0.33f0.10 2.19kO.34 2.19f0.20 0.15to.05 2.15kO.31 2.51f0.29 1.15f0.29 1.30*0.13 0.96tO.09 2.09f0.63 2.47f0.98

7.07f0.41 2.68f0.32 40.88f1.41 0.28kO.03 1.92f0.2 2.15f0.33 0.13*0.03 2.55t0.44 2.08f0.15 0.82f0.20 0.84+0.19 0.86tO.07 1.65kO.43 6.19f3.2

2.73f0.72 3.55f0.68 24.77f3.22 0.09f0.02 0.52f0.16 0.94f0.07 0.34tO.52 1.05f0.40 1.70f0.15 0.90_+0.47 0.76f0.44 0.48f0.03 6.62f2.54 0.94f0.06

% Recovered

60.24k2.9

74.75*2.58

70.23k2.75

45.38t4.19

5.80k0.68

6.18t0.49

Res/Bloodb

4.34f0.60

Untreated (n=3)

11.00f3.72C

aData expressed as % injected dose. Liposomes were labeled with inulin. [ '*51]tyraminyl bRKS/blood = % doses in liver and spleen/% dose in blood. CSignificantly different from the other three, p< 0.0001 (F test). While tested

the molecular

a biological

vivo.

Since

with

property

plasma.

Balb/c

mice

following inulin.

of the

had given

The stabilized and the

stabilizer

stabilized

liposomes of

This

radioactive

marker

As can be seen

in Table

of injected

rapidly

dose).

with

and

tissues.

into

mouse showed

Blood

(tt=few

minutes)

approximately

8%.

DOPE:OA liposomes a similar

second

The rest

stabilized

biodistribution

331

into

the

had

mouse serum

with

a higher

later

by

tyraminyl and

free

the mouse

(3).

in

and

level

liver for

in various

with

of

mammals

compartment

spread

veins

[lz51]

accumulated

highest

of

tail

from

-in

to that

5 hours

by

excreted

mainly

have

stabilized

identical

marker,

not metabolizable

was the

were

was measured

is

we

biodistribution

liposomes

aqueous

3, DOPE:OA liposomes

unknown,

i.e.

injected

liposome

of an entrapped

still

activity

were

distribution

is

the

a stabilization

biodistribution

marker

is

liposome, model,

the

unencapsulated

distribution

of the

we used mouse as an animal

mouse serum which

human

31%

identity

other then

of

liver

(about liposome organs injected uptake.

Vol. 162, No. 1, 1989

BIOCHEMICAL

which were trypsin-treated

Serum-stabilized

liposomes

showed a similar

biodistribution.

is

generally

The RES/blood ratio

accompanied with a higher level

This

coated the

result

ones stabilized treated

identical

protein

proteins

serve

preparations

are expected.

a commonly used lipid

similar

liposomes

Allen

independently relatively

On the contrary,

affinity

i.e.

et

al

developed long period of

prolonged

(15)

from

each

animal,

serum again,

showed nearly

If someof the

egg PC:cholesterol:PS

These results

associated

liposomes which time.

liposome

showed a

significantly that the

serum-

long period

and Papahadjopoulos

would

stay

in

low

of

time.

(13)

circulation

have for

of

a

liposome

such as ganglioside GM1. The present liposome system

circulation

looks

other

of liposome of

One of the important ingredients

Nevertheless, time.

tumor accumulation (13).

DOPE:OA liposomes

three

(lO:S:l),

indicate

as

serum-stabilized

the biodistribution

and Gabizon

liposomes with prolonged circulation of

for the

composedof DOPE:OA show a relatively

does not contain any glycolipids. of

the

for RESwith a RFS,fblood ratio

originally

composition is glycolipids

level

SDS-PAGE of

to RES and hence stay in blood for a relatively

Recently,

different

degrees of RES uptake of these

than those of the other three.

affinity

usually

The RES/blood ratios

into the blood of the

and exposed to

composition,

higher

stabilized

is

as opsonins for RES uptake, as has been suggested by (13,14),

greater

index for the relative

of RESuptake

bands on the gel (data not shown).

investigators

significantly

once injected

trypsin

(PES) (for

because the DOPE:OAliposomes would be

with serum -in vitro. with

also

and spleen

system

are not significantly

is not surprising

with the sameproteins,

liposomes

signs

is a sensitive

in the blood (13).

DOPE:OAliposome preparations

other.

injection

The uptake of liposomes in liver

of liposome to the RES, because lower level

three

before

regarded as uptake by the reticuloendothelial

recent review see 1). affinity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

favorable

the liposomes already show the

This is

halflife

an important

generally

be further

because

show a relatively

Thus, the drug delivery and will

result

potential evaluated

high of

in

future

experiments. ACKNOWLEDGMENT: This work is supported by NIH grants CA 24553 and AI 25834. 332

the

162,

No.

1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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Senior, J. H. (1988) in "Critical ReviewTM in Therapeutic Drug Carrier Systems," Stephen D. Bruck ed. CRCPress, Inc., Boca Raton, FL. Liu, D. and Huang, L. (1989) Biochem., in press. Sommerman,E. F., Pritchard, P. H. and Cullis, P. R. (1984) Biochem. Biophys. Res. Commun.122, 335-343. Shinitzky, M. and Barenholz, Y. (1974) J. Biol. Chem. 249, 2652-2657. wu, H. s., Robbins, J. C., Bugianesi, R. L., Ponpipom, M. M. and Shen, T. Y. (1981) Biochim. Biophys. Acta 674, 19-26. Enoch, H. G., Fleming, P. J. and Strittmatter, P. (1979) J. Biol. Chem. 254,

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Dasseux, J. L., Faucon, J. F., Lafleur, M., Pezolet, M. and Dufourcq, J. (1984) Biochim. Biophys. Acta 775, 37-50. GUO, L. S. S., Hamilton, R. L., Goerke, J., Weinstein, J. N. and Havel, R. J. (1980) J. Lipid Res. 21, 993-1003. Shintzky, M. and Barenholz, Y. (1978) Biochim. Biophys. Acta 515, 367-394. Cullis, P. R. and de Kruijff, B. (1979) Biochim. Biophys. Acta 559, 399-420. Tate, M. W. and Gruner, S. M. (1987) Biochem. 26, 231-236. Tall, A. R. and Small, D. M. (1977) Nature (London) 265, 163-164. Gabizon, A. and Papahadjopoulos, D. (1988) Proc. Natl. Acad. Sci. USA 6949-6953.

Moghimi, S. M. and Patel, H. M. (1988) FEBS lett. 233, 143-147. Allen, T. M. (1988) in "Liposomes in the Therapy of Infectious Diseases and Cancer," Lopez-Berenstein, G. and Fidler, I. J., eds. Allen R. Liss, Inc., New York.

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