Superparamagnetic iron oxide nanoparticles as a liver MRI contrast agent: Contribution of...

Magnetic Resonance Imaging. Vol. 7. pp. 619-627, 1989 0730-725X/89 $3.00 + .oO

Printed in the USA. All rights reserved. Copyright 0 1989 Pergamon Press plc

l Original Contribution

SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES AS A LIVER MRI CONTRAST AGENT: CONTRIBUTION OF MICROENCAPSULATION

TO IMPROVED BIODISTRIBUTION

D. POULIQUEN, R. PERDRISOT, A. ERMIAS, S. AKOKA,* P. JALLET, AND J.J. LE JEUNE

Laboratoire de Biophysique, FacultC de MCdecine, Angers, *Service d’IRM, Hopital Nord, Nantes, France

We have developed a new method of synthetizing superparamagnetic iron oxide nanoparticles, consisting in the modifications of Molday’s method, which ensures high relaxivity (2.4 10’ s-’ *M-l . L), good chemical stability, singular biodistribution and a considerable safety margin. The ED (Efficace Dose) to LD50 ratio is 2& instead of & for Gd-DTPA. In order to develop a magnetite-delivery system to the liver we have incorporated the nanoparticles into biodegradable synthetic microcapsules. Encapsulated 59Fe oxide nanopar- titles are injected into rats; in these conditions the sequestration is 9-fold greater in liver and 6 and 5 times lower in blood and carcase, respectively. This modification of the biodistribution enables the use of magnetite containing microcapsules at only 0.3 mg/kg iron to obtain an improved contrast in liver.

Keywords: Contrast agent; Superparamagnetic iron oxide; Nanoparticles; Microcapsules; Liver.

INTRODUCTION

A number of authors recently suggested using superparamagnetic iron oxide particles as a new contrast agent for magnetic resonance imaging (MRI). There are 3 main reasons for which magnetite would appear to be of great interest:

It has a high magnetic moment and can therefore be used at very low concentrations: 5-10 times lower than for Gd-chelates. Moreover, the effective dose (ED) to LDSO ratio is about fifty times lower than for Gd-DTPA.’ It causes predominantly Tz reduction. This must be taken into account, since MRI mostly uses Tz- weighted spin-echo pulse sequences. It consists of nanoparticles which, when coated with hydrophilic polymers, can be coupled to targeting molecules like monoclonal antibodies,2 polypeptides, and hormones.

Superparamagnetic iron oxide particles can also be incorporated into new drug delivery systems such as

microspheres, microcapsules or ghosts,576 unlike paramagnetic chelates whose diffusion processes de- pend on their very low molecular weight; in fact, these latter molecules must be chemically bound to the carrier7s8 causing modifications in stability and physicochemical properties.

To date, superparamagnetic iron oxide particles have been used as an intravenous contrast agent for MRI investigation of the reticuloendothelial system,‘-” liver’” or spleen13 and orally for gastro- intestinal tract explorations.‘4 Particle preparations differ from one author to another. Within them, great differences may appear in physical and biological properties. These characteristics relate to the dimen- sion of the particles and to the charge, hydrophobic- ity and structural properties of the coating polymer.15 In the last ten years, proteins,16y’7 lipids,” natural carbohydrate polymers,‘9p20 or synthetic polymers21- 24 have been successively used; but most publications refer to ferromagnetic particle preparations used for cell separation or electron microscopy. On the con- trary, iron oxide particles retained as contrast agent

RECEIVED 12/21/88; ACCEPTED 5/12/89. Acknowledgments-The authors wish to thank Mrs. M.

Moreau, G. Tanguy, and P. Legras for their excellent tech- nical assistance, and Mr. Filmon for electron microscopy.

This work was supported by research grant 874008 from

the Institut National de la Santk et de la Recherche Medicale (INSERM).

Address correspondence to D. Pouliquen, Laboratoire de Biophysique, Faculte de MCdecine, Angers.

619

620 Magnetic Resonance Imaging 0 Volume 7, Number 6, 1989

for MRI must be superparamagnetic, nontoxic and biodegradable.

We report herein a new method of synthesizing superparamagnetic iron oxide nanoparticles with high relaxivity, singular biodistribution, and considerable safety margin. The development of biodegradable and nontoxic synthetic microcapsules loaded with these new superparamagnetic iron oxide nanoparticles is described for the improvement of liver MRI.

MATERIALS AND METHODS

Synthesis of superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles were generated by modifications of the Molday’s meth- od19; namely: Fe3+/Fe2+ molar ratio, Dextran T40 (Pharmacia, Uppsala, Sweden) concentration, titra- tion and heating parameters. The colloid was centrifuged at 1000 x g three times for 10 minutes and the final supernatant kept. Unbound Dextran was then separated from the nanoparticles by gel-filtration (sephacryl S 300) chromatography.”

The nanoparticles collected were finally dialyzed overnight using distilled water at 4°C to remove free NaCl and Na acetate and then successively filtered on 0.2 p and 0.1 p nylon filters (Nalgene). The solution can be kept at 4°C for at least 6 months.

Synthesis of magnetite-containing microcapsules Microcapsule preparation involved a water in oil

emulsion. The organic phase is made up of cyclohexane/chloroform (4/l; v/v) with span 85 as emulsifier. The aqueous phase containing the superparamagnetic iron oxide nanoparticles to be encapsulated is dis- persed during three minutes. Then the monomer (isobutyle cyanoacrylate) in the organic phase solution is introduced. The anionic polymerization begins at the interface between the aqueous microdroplets and the organic phase. Two minutes later, cyclohexane is added, and the stirring is stopped in the following minute. The capsules are collected by sedimentation and washed several times with aqueous phases containing Tween 20 as emulsifier to eliminate all organic solvents. Then they are washed three times with 0,15 M saline and stored. Encapsulation efficiency was evaluated with 59Fe oxide nanoparticles and transmission electron microscopy was used to check for intra- capsular iron oxide nanoparticles.

Physicochemical and morphometric analysis of the nanoparticles and microcapsules

Nanoparticle and microcapsule range of size distribution was analyzed with an N4M Nanosizer and a

Multisizer (Coultronics, France), respectively. Dextran and iron concentrations were measured by spectro- photometric methods (phenol/sulphuric acid at 490 nm,25 and HCl30% at 340 nm.26

For microcapsule evaluation, the suspension was dissolved in acetone, centrifuged, and the pellet measured in the same way as iron concentration was deter- mined. Nanoparticles or microcapsules were diluted in doubly distilled water and then gold-coated by cat- ionic vaporization (JEOL JFC 1100) to obtain scan- ning electron micrographs.

To obtain transmission electron micrographs, a drop of nanoparticle solution was deposited on to a carbon-coated microscope grid, and examined with a TEM JEOL 100 B apparatus. The microcapsule suspension was incorporated into a resin (Araldite) and, once solid, was cut into thin slices (60 nm).

Magnetite and microcapsule biodistribution To prepare 59Fe oxide nanoparticles, 200 PCi of

59FeC13 solution (0.15 M) were added to the iron chloride-Dextran mixture. Superparamagnetic iron oxide nanoparticles were separated, filtered, and encapsulated as described earlier.

To obtain early data on nanoparticle or microcapsule biodistribution, the solution was injected in- travenously into normal male Wistar rats at 1-mg iron/kg animal weight (40,000 cpm/animal). Half of the rats were sacrificed at 20 minutes and the other half at 2 hours postinjection. All the organs, the skin, muscles and bones (carcase) were counted with a gamma counter, and the results expressed as a percentage of the injected dose per organ as a function of time.

Assessment of LD50 Four groups of 5 female Swiss mice (mean weight:

25 g) were given IV injections of superparamagnetic iron oxide nanoparticles into the tail vein to determine LDSO. Each group received one of the following doses in a single injection: 1.4, 2.0, 2.6, 3.3 g/kg (total dry weight determination). Mortality was recorded 14 days postinjection. LDSO for microcapsules was investi- gated after injection of 150, 200, 250, and 300 mg/kg (total dry weight determination).

Relaxation time measurements To determine Rl and R2 relaxivity, the Ti and T2

of successive dilutions in NaCl 0.15 M of both superparamagnetic iron oxide nanoparticle and microcapsule loaded with nanoparticles preparations were measured with a PC20 (Bruker, France) spectrometer at 0.47 T, 20 MHz and 4°C.

Superparamagnetic iron oxide nanoparticles as a liver MRI contrast agent l D. POULIQUEN ET AL. 62

SOP WEIGHT RESULTS PARTICLE RI.; NOT SPECIFIED SAMPLE IO

MEAN 46.9 NM SO. 12 NM c.v 26%

SIZE S.O. (NM1 (NM)

1: 46.9 12

OUST: 1%

SOP DIFFERENTIAL WEIGHT PARTICLE R.I.: NOT SPECIFIED

01

AMOUNT

loo?&

SIZE AMDUNT SIZE AMOUNT

10.0 0% 147 0% 14.7 c% 215 0% 21.5 .lK 316 0% 31.6 11% 464 0% 46.4 65% 661 0% 66.1 23% loo0 0% 100 .5x

A

15%

=I

10x

5%

0% 10 100 loo0

PARTICLE DIAMETER (NM) SDP DIFFERENTIAL WEIGHT

RUN PARAMETERS

SAMPLEID - 01

TEMPERATURE - ZODEGC vlsCDsllY I 1 CP REFRACTIVE INDEX - 1.333 ANGLE - 60.0 DEGREES

2.5 MICROSEC A 1 A

200 SECONOS M

.30 1.21 E+05

19:5536

SAMPLETIME I PRE-SCALE - RUN TIME -

COUNTS/ST. I COUNTS/SEC I ENDOF RUN -

w 23 8 Ei

h&n: 3.814e+OOum Median: 3.70Se+DDum Mode: 3.489eiODum

Variance: 7.832601 urn”2 S.D.: S.&De-01 urn C of Var: 23.205 %

Skewness: 9.36%01 right-skewed Kurtosis: 1.37Se+DO leptokurtic

Fixed and Variable Percentiles:

5% > 5.41Se+ODum 10% > 4.963e+OOum 15% > 4.693e+DOum 16% > 4.646etOOum 25% > 4.324e+DDum 37% > 4.001e+DOum 50% > 3.7D6e+DDum 75% > 3.14Se+DDum 84% > 2.916e+DCum

90% > 2.749e+ODum 95% > 2.589e+ODum

B

Fig. 1. Size distribution of (A) iron oxide nanoparticles and (B) microcapsules loaded with nanoparticles.


Table 1. Physicochemical properties of iron oxide nanoparticles and microcapsules loaded with nanoparticles

Parameters

Preparations Nanoparticles

Microcapsules

% iron Mean (w/w) diameter

30 48.9 nm

6 3.8 pm

(s-?&I) (s-tR;-r) RZ/Rl

2.7 lo4 2.4 10’ 8.9

0.8 lo4 3.4 104 4.3

B

Fig. 2. Electron micrographs of (A) iron oxide nanoparticles (x 100,000, 2 mm = 12.5 nm) and (B) microcapsules loaded with nanoparticles (X 10,000, 2 mm = 125 nm).

Tr was measured by a non-linear least squares fit calculation to 8 data points generated with an inver- sion-recovery pulse sequence.

Tz was obtained from 10 data points generated with a Meiboom-Gill-Carr-Purcell sequence.

Tissue relaxation times were measured after IV injection into normal male Wistar rats of superparamagnetic iron oxide nanoparticles at 0.5, 1 .O, 1.5, 2.0 mg

80.

60.

40.

7

20.

I r

% A

a0

60

40

20

Blood Liver Spleen Kidney

t= 20 min

t=2H

Blood Liver Spleen Kidney Lung

I_

Fig. 3. Biodistribution of 59Fe oxide nanoparticles (0) and microcapsules loaded with nanoparticles (w), (A) 20 min and (B) 2 h after intravenous injection into rats.

iron/kg animal weight, and of microcapsules loaded with nanoparticles at 0.3, 0.7, 1.7 mg/kg. The animals were sacrified 20 mn postinjection, the plasma, spleen, kidney, lungs, and muscle were analyzed to determine the dose-to-response effect.

Superparamagnetic iron oxide nanoparticles as a liver MRI contrast agent 0 D. POULIQUEN ET AL. 623

Induction of hepatocarcinoma in rats Eight male Wistar rats (250 g initial body weight)

were used. Chemical-induced hepatocarcinomas were produced by addition of DENA (Sigma Chemical Co., St. Louis, USA) (100 ml/L) into drinking water during three months.27

MRI MR images of anaesthetized normal male Wistar

rats were acquired with a 1.5 T whole body imager (Siemens, West Germany) using a surface coil 8 cm in diameter. Superparamagnetic iron oxide nanoparticles and microcapsules loaded with nanoparticles were then injected at 0.3 mg iron/kg animal. The liver or kidney signal intensities were analyzed and compared with controls (no contrast agent injection) using the spin-echo multislice pulse sequence with the following timing parameters: TR = 1500 ms; TE = 45 ms; with 2- mm thickness frontal sections and a 15 cm x 15 cm field of view, 256 x 256 matrix (pixel = 0.6 mm).

MlCRocAPStiLES ~ I

40.

NANOPARIICLES I

35.

30.

7 25 ”

g

:I:‘---;

LIVER

MUSCLE

IUbNEY

LUNG

I g

8

1

0.5 19 1.5 2.0 Iron (mg/Kg) 1 2 3 DOSE (G/KG)

Fig. 4. Effect of iron concentration upon T, measure- Fig. 5. Acute lethal toxicity (LD50) after intravenous ments. administration of iron oxide nanoparticles in mice (g/kg).

RESULTS

Physicochemical properties of magnetite nanoparticles and microcapsules

Superparamagnetic nanoparticles consist of an iron oxide core and a Dextran coating, and have a high colloidal stability in solution even when the pH range is high (2 to 10). Stability is related to the low range of nanoparticle size distribution: lo-70 nm (Table 1 and Fig. 1) and to the uniformity of Dextran coating.

Since the iron oxide core size is 5 to 20 nm (Fig. 2), nanoparticles are assumed to be superparamagnetic.28 The relatively important Dextran coating (70% in weight) causes steric repulsion which neutralizes Van der Waals attraction.29

Comparison of magnetite nanoparticle and microcapsule biodistribution data

Figure 3 gives the early results for superparamagnetic 59Fe oxide nanoparticles and loaded microcapsules at 20 mn and 2 hours postinjection.

There is a considerable increase (9 times greater) in liver sequestration of encapsulated 59Fe oxide nanoparticles as opposed to free 59Fe oxide nanoparticles.

On the other hand, for microcapsules loaded with 59Fe oxide nanoparticles, the percentage of the dose injected in the blood and the carcase is six and five times lower, respectively, than with free s9Fe oxide nanoparticles.

At 2 hours postinjection, no discernible redistribu- tion of both product was noted: liver, spleen, and blood radioactivity remained constant.

The drop in 59Fe oxide nanoparticle concentration in the blood correlates with increased uptake in the spleen and carcase.

llORTAL,T”

(Z)

100

80

4


Dose-to-response relationship for the two methods

For a given dose of iron per kg animal weight (Wis- tar rats), the injection of encapsulated nanoparticles led to a greater decrease in T2 in the liver (Fig. 4) which is due to the difference in biodistribution data.

These results were obtained although relaxivity is lower with loaded microcapsules than with free nanoparticles.

In both cases, a linear dose-response relationship was obtained followed by a saturation effect as the injected dose increased.

LD 50 assessment The LD 50 for superparamagnetic iron oxide nano-

particles is very high (2.4 g/kg nanoparticles (Fig. 5)).

For microcapsules LD50 is ten times lower (250 mg/kg) and is in good agreement with published results for polyisobutyl cyanoacrylate polymers.30-32

A4RI investigation When compared with the controls, who had not

received any magnetite, contrast was more enhanced with microcapsules than with nanoparticles at the dose of 0.3 mg/kg (Fig. 6).

DISCUSSION

The high R2 relaxivity of the superparamagnetic iron oxide nanoparticles (2,4 lo5 s-l .M-’ .L) and the R2/Rl ratios (8.8) (Fig. 7) can be explained by the translational diffusion of water molecules across the

Fig. 6. Spin echo (TR: 1500 ms, TE: 45 ms) of rat liver with hepatocarcinoma nodules (arrows); before (A and C) and after administration of iron oxide nanoparticles (B) or microcapsules loaded with nanoparticles (D) (0.3 mg/kg iron).


Ti-’ (s-1)

RI = 2.7 104

0

: PC RI = 0.8 104

.,,, Q. . . . .,.“’ ,P. .’ . . . ,,

w ‘9 1,s Iroll (mM.L-1)

Fig. 7. Relaxivities (s-’ .M-’ .L) of iron oxide nanoparticles (nP) and microcapsules loaded with nanoparticles (PC) at 20 MHz and 4°C.

high magnetic field gradient that magnetite nanocrys- tals produce. 33 The relaxivity of microcapsules loaded with superparamagnetic iron oxide nanoparticles de- creases sevenfold (Fig. 7), as compared to free nanoparticles and the R2/Rl ratio becomes half this value. The increased distance between the magnetite core and the water molecules in the environment could explain this fall (Fig. 2).

The singular biodistribution of the nanoparticles in rats may be explained by the very low mean diameter, which is less than 50 nm; while for the different preparations already known this value is from 80-100’3*34 to 500-1000 nm.35,36 We could also mention that the size range for the nanoparticles described here is rather limited (30 to 100 nm), which differs from other current preparations (5 to 500 nm).37

Although for encapsulated superparamagnetic iron oxide nanoparticles the relaxivity R2 is divided by 7 and the R2/Rl ratio by 2, the main hepatic biodistribution observed allows to selectively modify the signal intensity of the liver after injection of only 0,3 mg/kg iron. This dose is therefore lower than the dose re- quired with superparamagnetic iron oxide nanoparticles, to obtain a 50% decrease of the signal intensity in the liver.

The choice of microcapsules formulation, instead of microspheres takes into account the parameters involved in the relaxivity of the product:

l porosity of the microcapsule membrane and the relative importance of water content: a free diffusion process of water molecules occurs more easily into microcapsules than into microspheres

l distance between the iron oxide core of the nanoparticles and water molecules is smaller into microcapsules than into microspheres. In the case of microspheres the iron oxide nanoparticles are “embedded”38 into the matrix which increases the total volume of the coating polymer.

Thus, the higher relaxivity of microcapsules loaded with superparamagnetic iron oxide nanoparticles could explain the differences in the iron doses used in the two cases. In fact, for magnetite albumin microspheres, compared to microcapsules, a five-fold higher iron dose is injected to obtain a 40% decrease of T2 in liver.38

The approach mentioned in this study to modulate the biodistribution of our singular superparamagnetic iron oxide nanoparticles differs from the use of cur-


rent larger particles known to be predominantly taken up by the liver but currently used at higher iron doses/kg animal: 18 pmo139 to 50 pmo1,40,41 that is

three- to ten-fold more the iron dose used in this study

with loaded microcapsules. Some authors have fur- thermore mentioned great differences in ED50 values

for the same product.42 Further studies may be of interest concerning the

importance of the polymer structure and superparamagnetic iron oxide nanoparticle content, respectively, in the biodistribution and efficiency of the contrast

agent. The future development of this new kind of contrast agent may take into account the research of

new biodegradable polymeric systems with better biocompatibility.43’44

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