Contrast Peter Reimer, Jack Wittenberg,

MD

Receptor of Liver

#{149} Ralph

MD

Weissleder,

J.

#{149} Thomas

Brady,

Imaging: Cancer’

A new

contrast agent for magnetic (MR) imaging, directed to asialoglycoprotein (ASG) receptors on hepatocytes, was used for detection of liver cancer in rats. Ultrasmall superparamagnetic (mean size, i2 nm) particles of iron oxide (USPIOs) were targeted to ASG receptors by coating particles with arabinogalactan (AG). Liver T2 relaxation times decreased more effectively after a single intravenous administration of AG-USPIO than after an equal dose of a conventional superparamagnetic liver MR contrast agent (AMI-25; mean size, 72 nm). Receptor affinity studies demonstrated that receptor-mediated attachment and subsequent cellular endocytosis do not occur in primary malignant (hepatocellular carcinoma) or metastatic (adenocarcinoma) tumors, because the surface ASG receptors are lost during malignant dedifferentiation. In vitro relaxation and in vivo MR imaging cxperiments of liver tumors show that targeting USPIO to hepatocytes rather than to the mononuclear phagocytic system allows a considerable dose reduction, increases tumom-liver contrast, and potentially allows distinction of ASG-positive (benign hepatocellular) and ASGnegative (malignant hepatocellular) tumors. resonance

Index terms: Contrast media, experimental studies #{149} Iron #{149} Liver neoplasms, diagnosis, 761.321, 761.332 #{149} Liver neoplasms, MR studies, 761.1214 #{149} Magnetic resonance (MR), contrast enhancement Radiology

1

From

Harvard

1990;

the

177:729-734

Department

Medical

School,

MD, PhD MD

of Radiology, 13th

sion requested June 26; revision tional Institutes of Health grant P.R. supported by the Deutsche R.W. C RSNA, 1990

St.

Bldg

MGH-NMR 149,

#{149} Albert

Application

C

S. Lee,

BA

.

to MR Imaging

tumors. We hypothesized that AGUSPIO would accumulate in normal hepatocytes but not in tumor cells, thus improving tumor-liver contrast. To prove this hypothesis, dose response studies, magnetic resonance (MR) imaging experiments with primary and secondary liver tumors, and cell-membrane receptor studies were performed. These results were compared with results obtained with a conventional superparamagnetic iron oxide (AMI-25; Advanced Magnetics, Cambridge, Mass), which has previously been used in clinical trials as a contrast agent for MR imaging of the liver.

superparamagnetic iron oxides with albumin (1), starch (2), and dextran (3,4) coating are rapidly phagocytosed by the mononuclear phagocytic system of liver and spleen. We have recently described a novel, hepatocyte-directed, ultrasmall supemparamagnetic iron oxide (AG-USPIO) preparation as a liver contrast agent (5). Iron oxide particles were targeted to asialoglycoprotein (ASG) receptors by coating particles with arabinogalactan (AG), a galactose-containing polysaccharide (6). ASG receptors have a high affinity for the terminal galactose groups (7), a concept previously exploited in the design of both diagnostic (8-9) and therapeutic drugs (10). The strategy of targeting superparamagnetic iron oxide to hepatocytes rather than to Kupffer cells allows a more homogenous tissue distribution, which potentially results in a greater tissue relaxivity. Previous biodistribution studies demonstrated selective accumulation of AC-USPIO in liver but not in othem organs of the mononuclear phagocytic system such as the spleen. Findings at electron microscopy confirmed preferential distribution of iron oxide to hepatocytes and showed selective binding of ACUSPIO to hepatocyte cell surface membranes. After internalization from surface receptors, AC-USPIO accumulates within intracellular hepatocyte lysosomes, where particles are degraded in a time-dependent fashion (5). Our study was designed to evaluate the potential of ASG receptom-directed AC-USPIO in detecting liver ONVENTIONAL

Center,

Charlestown,

received July 18; accepted no. CA 48279, PPG, and Forschungsgemeinschaft

Media

Massachusetts General Hospital and MA 02129. Received May 7, 1990; mcviJuly 23. Supported in part by the Naby the MGH-NMR development fund; (Re 758/1-1). Address reprint requests

MATERIALS Iron

AND

Oxide

METHODS

Preparations

Hepatocyte-directed

AC-USPIO

(Ad-

vanced Magnetics, Cambridge, Mass) was obtained by coating USPIO with AG (11), AG is known for its high specificity for the

asialoglycoprotein

tocytes.

receptor

on hepa-

The preparations-as

magnetophysical,

biologic,

cologic

properties-

USPIO

have

well and

and AGpreviously

of USPIO

been

as the

pharma-

described

(5,12). of 12.2 [SD]), sec, sec,

Briefly, AG-USPIO has a mean size nm ± 6.5 (standard deviation an Ri relaxivity of 23.3 mmol/L’an R2 relaxivity of 48.9 mmol/L’. and a blood half-life of 8 minutes T, 37#{176}C) (5). AG-USPIO was used at

(0.47 a concentration The

of 17 mg/mL.

efficacy

of AG-USPIO

in detection

of hepatic tumors was compared with that of a conventional, dextran-stabilized iron oxide (AMI-25) that has a mean particle size of 72 nm. Pharmacologic characteristics

have was mL.

to

(13) and

been used

in vivo

described

use (14) of this

previously.

at a concentration

agent

AMI-25

of 11.2 mg/

Abbreviations: AG arabinogalactan, ASF asialofetuin, ASG asialoglycoprotein, C/N contrast-to-noise ratio, CPMG Carr-PurcellMeiboorn-Gill, GRE gradient echo, SD standard deviation, SE spin echo, USPIO ultrasmall superparamagnetic iron oxide.

729

Animals A total relaxation

of 95 rats were used time measurements

MR imaging (n

(n

=

20),

4). Relaxation

or

time

Tumor

for either

(n

71),

receptor

Liver

studies

measurements

were used for dose response studies in male, retired breeder Sprague-Dawley rats (n = 41) (Charles River Breeding Laboratories, Wilmington, Mass, or Harlan Sprague Dawley, Indianapolis) and determination of relaxation times in tumor models in Fischer rats (n 15) and ACIAXC rats (n 15) (Harlan Sprague DawIcy). Different strains of rats were used because the models of liver metastasis (R3230AC tumor cells [Biomeasure, Hopkinton, Mass] in Fischer rats) and primary hepatocellular

ACI-AXC

carcinoma

rats) have

(H-4-II-E

been

well

Prf

Liver or Tumor

‘,

removed

from

was

rats, minced

performed

by

enzymatic

and

desegregation

in

Cells

+

cell fragments

Sonification,

in

Hanks buffered salt solution (Sigma Chemicals, St Louis), sieved through a 0.3 wire mesh, and resuspended in 20 mL of Hanks buffered salt solution. This solution contained tumor fragments and single cells. A total of 0.1-0.2 mL of this solution was then injected subcutaneously into host rats. In addition, intrahepatic inoculation

Mechanical

character-

ized. The properties of the H-4-II-E cell line, a poorly differentiated hepatoma (15-17), and the R3230AC cell line have been described in detail elsewhere (18). Tumor cell lines were propagated as follows: One-cubic-centimeter tumors

were

fragment

direct

centrifugation,

purification

Cell membranes

injec-

tion of the suspension into the liver with 25-gauge hypodermic needles (19). The rats were lightly anesthetized with ether. They were used for in vitro and in vivo experiments 7-10 days after tumor cell inoculation.

Receptor These

Affinity experiments

determine

whether

ceptors

would

bind

Experiments were tumor

performed

to

cell

re-

AG-USPIO

surface specifical-

ly or nonspecifically. Figure 1 summarizes the experimental procedures in a flow chart. Previous studies had shown that hepatic uptake of AG-USPIO is mcdiated specifically by ASG receptors on normal hepatocytes and can be blocked competitively with D(+)-galactose and asialoglycoproteins such as ASF (10,20-25). These initial observations were confirmed by results of a dose-dependent blockade

experiment

and

control

experi-

ments in which a “control blocker” (D(+)glucose and albumin) was used. Hepatocyte, H-4-II-E tumor cell, and R3230 tumor cell membrane suspensions were prepared with a modified technique that has been previously described (26,27). Normal livers from rats were perfused in vivo for 5 minutes by means of buffered salt solution (Hanks) with 0.05 mol/L of (N-[2-hydroxyethyl]piperazineN’-[2-ethanosulfonic acid]) (HEPES) and 0.5 mmol/L of ethylene glycol-bis($aminoethyl ether) N,N,N’,N’-tetraacetic acid (Sigma Chemicals, St Louis). After this initial perfusion to remove erythrocytes, the livers were perfused with colla-

730

Radiology

#{149}

AG-USPIO

usplo

I

AG-USPIO

I

I Incubation,

I

Relaxation

USPIO

time

I

washing

measurement

Figure 1. The flow chart schematically summarizes the steps for the preparation of hepatocyte and tumor cell membranes. Membrane suspensions were incubated with AG-USPIO or Usplo in the presence or absence of blockers (D[+]-galactose [D(+)-gal] and asialofetuin [ASFJ) of the ASG receptor system. After repeated washing cycles, relaxation times of membrane suspensions were determined.

genase (100 U/mL) in Williams E medium (Sigma Chemicals) buffered with 0.05 mol/L of HEPES buffer. Livers and tumoms were then washed in complete medium and sieved through a 36-mesh stainless steel mesh. The resulting tissue fragments and cells were separately incubated in 40 mL of collagenase (at a concentration of 0.3 mg/mL) in complete medium before sonication was performed (10 W for three 20-second cycles on ice [Sonifier 450; Branson Ultrasonics, Dan-

bury, tant

Conn]). Subsequently, was washed twice by

tion and minutes.

centrifuged

Samples suspensions minutes tions

of the

block brane,

at 1,500 resulting

of D(+)-galactose

and complete

existing ASG or a “control

for

10

membrane

(0.1

ASF

supernaof dilu-

rpm

were then incubated at 37#{176}Cwith different

ma Chemicals) Chemicals)

the means

for 30 concentra-

mg/mL;

(2 mg/mL;

medium

receptors blocker”

(control)

Sig-

Sigma to

on the mem(D[+]-glu-

December

1990

Pr#{149}-Incubatlon

Post-Incubation Blockads

Post-IncubaUon Control

Hepatoma (H-uI-4-E)

Hepatocyte (SD)

Adenocarcinoma (R3230)

U

so

. 0 E

60

C,’

!

40

I-

! I

20

q .

“ !

q q

E

C 0

.$

#{149}

0

C

E 0

.

C.)

U

C.) D

-

D

b. 2. (a) T2 relaxation times (CPMG sequence) of concentrated hepatocyte cell membrane solutions (MS) at 37#{176}C and 0.47 T. AG-USPIO incubated with hepatocyte membrane receptor solution has a significantly lower T2 (third column) than solutions incubated after blockade of the receptor with D(+)-galactose (Gal) and ASF (second column). Competitive blocking with different concentrations demonstrates the dose-dependent relationship. Incubation with “control” blockers (D[+]-glucose [Gic]) and albumin [Mb]) does not affect the relaxation times of AG-USPIO (first and third columns). (b) T2 relaxation times (CPMG sequence) of aqueous hepatocyte or tumor cell membrane solutions at 37#{176}C and 0.47 T. AG-USPIO incubated with hepatocyte membrane receptor solution has a significantly lower T2 than solutions incubated after blockade of the receptor with D(+)-galactose and ASF (first column). No significant T2 changes are seen in the H-H-4-E tumor cell line (second column) after incubation with AG-USPIO. T2 of R3230AC (mammary adenocarcinoma) cell membranes are similar to those of H-I!4-E cells (second column). Note that there is no difference in T2 if tumor cell membrane solutions are incubated with or without blocker of the ASG receptor. Vertical bars in a and b represent the mean ± SD. Figure

cose and/or albumin, 0.1 mg/mL). Dccreasing concentrations of blockers (from 1 mg/mL to 0.0001 mg/mL of D[+J-galac-

weight.

tose and ASF) were

with

used

for the competi-

tive blocking experiment. After this incubation, either USPIO or AG-USPIO was added to the test tubes (10 Mmol iron per tube). After 30 minutes of incubation, samples were washed with buffer and ultracentrifuged twice to remove excessive unbound AG-USPIO and/or USPIO. Relaxation

were

times

then

of membrane

measured

solutions

as described

below.

tumor-bearing

Relaxation

time rats were

administration

of AG-USPIO

a concentration

mol/kg. guination. diately,

ments hour

Time

after

trometer (PC-20 Conn) operating

times were meawith an MR spec-

Minispec; IBM, Danbury, at 0.47 T at 37#{176}C. Prior to

each measurement the spectrometer was tuned and calibrated. Ti was measured from eight data points generated by an inversion-recovery pulse sequence. T2 was measured from 10 data points by means of a Carr-Purcell-Meiboom-Gill

(CPMG) pulse sents

pulse

sequence

with

an inter-

time of 1 msec. Each value the mean of three separate

repremeasure-

ments. In Vitro

Relaxation

MR relaxation

times

Studies of liver

and tumor

tissue were measured to determine the magnetic effects of AG-USPIO and AMI25 in these tissues. Liver relaxation times in normal Sprague-Dawley rats were measured

as part

of the

dose

response

study by use of either AG-USPIO 25 with a concentration of 0, 2.5, or 20.0 mol of iron per kilogram

Volume

177

Number

#{149}

3

or AM!5.0, 10.0, of body

dow”

5 or 10

venous

MR

were

hematoxylin-eosin

Imaging was performed

with

superconducting

with

a 0.6-

a section

Muimages

thickness

of

4 mm and a 128 X 256 matrix. A head coil with a field of view of 16 cm was used;

stain

study

or absence

dimensions were 0.6 X 1.2 X 4 mm. Spin-echo (SE) images were obtained with 250/20, 500/30, 1,500/40, and 1,500/ 80 (repetition time [TRJ msec/echo time [TEl

msec)

pulse

sequences;

averaged and two.

the

number

was, respectively, Gradient-echo

six, (GRE)

images were obtained with a 120/18 sequence, a flip angle of 60#{176}, and eight signals averaged. Homogeneous regions of interest (more than 100 pixels) within individual livers and tumors were used to calculate tumor-liver contrast-to-noise tios (C/Ns) (mean signal intensity mor minus mean signal intensity divided

by

the

noise). All in vivo

were

standard

MR imaging

performed

raof tuof liver

deviation

of

of hepatic

the pres-

tumors.

Analysis

Differences C/N among

in relaxation different study

in rats anesthetized injection

(35 mg/kg). All and 60 minutes half-lives) after

(more than three the administration

with

of pentobarbital

imaged

before blood of ci-

times and groups were

evaluated statistically with the nonparametric Wilcoxon signed rank test (28). RESULTS Receptor

Affinity

Studies

Membrane receptor studies performed to study whether cell membranes or hepatocyte bind

AG-USPIO

by

a receptor

were tumor would system

(Fig i). The T2 relaxation time of medium containing hepatocyte ASG membrane receptors was significantly (P < .05) shorter (13.4 msec ± 1.7) after incubation with AC-USPIO than before incubation (i22.7 msec ± i5.7)

experiments

intraperitoneal

rats were

intra-

correlative

for

to determine

Statistical

pixel

of signals four, two,

after

magnet

GE Medical Systems, Transverse multisection

obtained

is 1-6 hours

administration of either agent. After the MR imaging experiment, the liver of each animal was removed, fixed in buffered 10% formaldehyde for 24 hours, and individually processed with

ence

MR imaging

or AMI-25. Previous studthat the optimum “win-

for imaging

histologic

waukee). Ti and T2 relaxation sured in milliseconds

ther AG-USPIO ies have shown

or AMI-25

of either

death.

(Technicare,

Measurements

in after

All rats were killed with exsanOrgans were removed immeand relaxation time measurewere performed at 37#{176}C within 1

T (25.i-MHz)

Relaxation

measurements performed

(Fig

2b).

This

is best

explained

by the retention of AC-USPIO on membrane receptors despite repeated washing cycles. When ASC membrane receptors on hepatocytes were blocked with i mg/mL of D(+)-galactose and ASF prior to incubation Radiology

731

#{149}

Table 1 Relaxation AMI-25

Times

of Different

Tissues

before

and after

Intravenous After

Before Administration (n3)

Tissue

Liver

Doses

of Different

Administration, 5ornol

of AG-USPIO

After

and

Administration,

10imol

AG-USPIO (n3)

AMI-25 (n3)

AG-USPIO (n3)

AMI-25 (n=3)

(CDF)

Ti T2

307 39.7

Liver

±27.0 ± 1.2

252 23.4

± ±

4.9 0.6*

298 33.2

±13.2 ± 0.9

0.7

250±7.4 30.1 ±

230 ± 18.6 ±

6.1* 0.6*

266 28.2

±33.4 ± 0.9

223±4.9* 19.4 ±

1.7*

234±5.9 25.6 ±

(AC!)

Ti T2

263±7.1 41.6 ±

Tumor

241±3.9* 26.1 ±

1.0

1.9

0.8

(R3230AC)

Ti T2 Tumor Ti

777 75.1

±41.3 ± 8.7

803 75.7

±49.5 ± 8.1

796 75.4

±43.2 ± 5.8

777 76.2

±17.2 ± 6.0

782 77.6

± 17.4 ± 6.7

655 63.0

±i3.0 ± 1.4

657 ±li.2 64.2 ± 3.7

657 63.1

±12.9 ± 7.5

657 65.6

±14.1 ± 4.4

651 63.6

± 16.5 ± 3.9

(H-4-!!-E)

T2 Note-Relaxation .

Administration

are expressed

times

lower

(P

C/N

of Different

Significantly

Table 2 Tumor-Liver

in milliseconds (mean and SD). the relaxation time obtained after administration

< .05) than

Doses,

Contrast

Agents,

and Pulse After

of AMI-25.

Sequences

Administration, 5Mmol

After

Administration, 10imol

Before

Administration PulseSequences SE 250/20

120/18

7.9 19.2 35.0 39.7

18.6 ±

3.3

Note.-Six signals were averaged in the SE 250/20 sequence, sequences. Data are expressed as mean plus or minus SD. * Significantly (P < .05)lower than AMI-25. t Significantly (P < .005) lower than AMI-25.

AG-USPIO,

nificantly i22.7 msec

± ± ± ±

AG-USPIO (n5)

(n5)

1.2 4.6 5.Ot 2.5*

2.3 8.7 16.8 20.1

± ± ± ±

5.3 1.7 4.3 2.2

75.3 ± 4.2*

40.4

± 4.8

T2 values

were

higher (69.9 msec ± 15.7, P < .05).

sig-

± 3.4 vs This ef-

four

USPIO

signals

because

in the

SE 500/30

of incomplete

sequence,

incubation D(+)-glucose in control

Dose USPIO function

response.-The effect of AGand AMI-25 on T2 of liver as a of administered dose is

shown

in Figure

medium and solutions

was

(Fig 2b). When albumin were used no receptor

seen

(Fig

of primary tumor cell suspensions

to those

of hepatocyte

(Fig

The

2b).

membranes

overall

of the

centration.

T2 values

membrane

After

of the

differ from in Figure

lower

AG-USPIO, the mary hepatocellular msec ± 9.2) nor

2a). (H-4-II-E) and (R3230AC) were similar

membrane solutions those of the solutions

cause

2a be-

con-

incubation

with

T2 of neither

the

carcinoma

the

pri(479.0

metastatic

adeno-

Relaxation

3. The

of the

those

dicating

732

test

of the contamination

Radiology

#{149}

± 12.8*

58.7

± 5.5

samples

control

in relaxation compared

samples, with

free

inAG-

and

two

signals

SE 1,500/40

and

in the

demon-

1.1 1.4 2.7 1.6

1,500/80

20

of AGof liver

Dosi

Figure

fore and different 41).

for clinical The liver

times

imaging T2 values

(14,29) obtained

(Fig 3). in

USPIO Table crease

and

after

administration

sizes. tumors.times

be-

AG-

or AMI-25 are summarized 1. There is a significant dein liver Ti and T2 relaxation

3.

(tmoI

Flag)

T2 relaxation times of liver beafter intravenous administration of doses of AG-USPIO or AMI-25 (n

after

administration of 5 .&mol or 10 imol (P < .05) of AGUSPIO, and of liver T2 after administration of iO mol of AMI-25 (P < .05). However, the decrease in liver T2 after administration of AG-USPIO is significantly greater than that after administration of AMI-25 at both doses investigated (P < .05). Relaxation times of either primary hepatocellular carcinoma or metastatic adenocarcinoma did not significantly de-

(P < .05)

Spmague-Dawley rats (Fig 3) differ only minimally from those in Fischer or AC! rats (Table 1), probably be-

fore

with

97.0

T2 (from 35.4 msec ± 2.4 to 24.5 msec ± 0.7) similar to the decrease caused by a dose of 20 mol/kg of AMI-25 (from 35.4 msec ± 2.4 to 22.4 msec ± 0.4 msec), the dose originally used

was

decrease

data

strate that a dose of 5 mol/kg USPIO results in a decrease

cause of different sample Relaxation times of liver Liver and tumor relaxation

times

± ± ± ±

Studies

carcinoma (479.0 msec ± 11.5) decreased to the extent that the T2 of the hepatocyte suspension did. Theme

a smaller

9.7 13.1 25.9 26.7

wash-

In Vitro

T2 values secondary membrane

16.8 ± 4.4 25.3 ± 4.4t 50.7 ± 5.i 52.9 ± i.8

ing.

fect was clearly dependent on the total amount of blocking agent in the

blockade

AMI-25 (n=5)

with

60#{176} flip angle

with

AMI-25

(iz=5)

-14.2 ± 10.5 -1.5 ± 1.1 11.7 ± 10.7 15.3 ± 3.2

SE 500/30 SE 1,500/40 SE 1,500/80 GRE

AG-USPIO

(n20)

in

December

1990

Figure imaged

4. (a) Tumor (R3230AC)-liver with SE 250/20 sequence

signals

averaged

quence

with

(top

four

row),

signals

model six

with

SE 500/30

se-

averaged

(middle

row), and SE 1,500/40 sequence with two signals averaged (bottom row). These sequences were performed before administration (first column), after administration of 5 omol (middle column), and after administration of 10 mol (right column) of AG-

USPIO.

: -

‘l;_

Note

the excellent

tumor

(f)-liver

(1)

contrast at low doses (5 tmol) in mixed Tiand T2-weighted pulse sequences (SE 500/ 30 with four signals averaged). (b) Tumorliver model imaged with an identical protocol as in a but after administration of AM!25 instead of AG-USPIO. Note that the liver appears less hypointense for any given

pulse

sequence

than

liver contrast sequence than USPIO.

in a. As a result,

is lower for any after administration

tumor-

given

pulse of AG-

,

:‘

mor-liver

.

‘:

C/N

higher dose preparation.

was

ministration

a.

of 5 mol

USPIO, tumor-liver ally higher than of AMI-25.

4

higher

at the

of either iron Interestingly,

oxide after

Fe/kg

ad-

of AG-

C/N was genertwice the dose

with

.1#{149}

DISCUSSION 4

the feasibilto ASG receptors on hepatocytes (5). Hepatocyte ASG receptors (400,000-500,000 receptors per cell) are responsible for the plasma clearance of desialylated glycoprotein by the liver. The directability of iron oxide was achieved by coupling galactose terminals in the form of AG Previously

ity

A

to

of

we

USPIOs.

AG-USPIO

predominantly cells

of

reported

USPIO

targeting

accumulates

in hepatic

liver

but

not

in

parenchymal other

organs

or

the the

mononuclear phagocytic system at low doses used for MR imaging (5). Our present and previous studies (5) confirm that AG-USPIO is selectively taken up by hepatocytes via the ASG receptor

system.

take

of iron

reduction

the

in of

by

the

AG-USPIO

administration

of either

duction in liver (Fig 4b), resulting

conspicuity MR

Imaging

Experiments

In vivo MR imaging experiments were performed in rats with implanted tumors by use of both SE and GRE sequences (Fig 4). Visual observation demonstrates that, at equal dose, AGUSPIO

Volume

(Fig

177

4a)

provides

#{149} Number

greater 3

re-

signal than AMI-25 in improved tumor on both SE and GRE im-

ages. Quantitative tumor-liver C/N measurements are summarized in Table 2. Maximum tumor-liver contrast was achieved with the GRE pulse Sequence (5 mol/kg AG-USPIO: 75.3 msec ± 4.2; 10 mol/kg AG-USPIO: 97.0 msec ± 12.8). As expected, tu-

USPIO AMI-25

USPIO

oxide

T2 caused

in

larger

3) despite

(Fig

The

increased

is thought

even

spatial

at

the

tocytes

the are

more

because targeted.

cells so

that

mass) (