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Ferromagnetic particles as contrast agent in T2 NMR imaging
Copyright
073&725X/86 I? I986 Prrgamon
$3.00 + .OO Journals Ltd.
0 Original Contribution
FERROMAGNETIC MAGNUS *Department
B.E.
PARTICLES
OLSSON,* BERTIL R.B.
of Radiation
AS CONTRAST
AGENT
IN T2 NMR IMAGING
PERSSON, * LEIF G. SALFORD~ AND ULF SCHR~DER~
Physics and INeurosurgery, University Hospital, and $Department The Wallenberg Laboratory, Lund University, Sweden
INTRODUCTION
for Microsphere Research,
weight) bovine serum albumin, at a pH of 7.0 Au. Albumin solution was preferred to plasma and serum for three reasons: (i) It is comparable to serum, but contains proteins of only one size. (ii) Its property will be almost the same between different batches. (iii) We “know” what is in the solution. Three different spheres were studied, containing 2.7%, 1.0% and 0.4% magnetite by weight. Free magnetite particles were also studied. The measuring volume was always 1 ml, and the samples were carefully suspended before measurement. Due to their small sizes, the spheres and the particles were easily maintained in suspension during the entire measurement period. In vivo NMR measurements were done in our homebuild imaging system, based on an ironcore frame magnet with a field of 0.07 T. Anesthetized wistar rats with an average weight of 380 -t 16 (1 SD) g were used. To make the spheres sensitive to external magnetic field gradients in vivo, the spheres contained approximately 20% Fe304 by weight. Magnetic spheres were targeted to the right hemisphere of the brain of rat by injection through the external carotid artery (Fig. l), with the right hemisphere placed in a magnetic field gradient of about 200 T/m. After 6 min, the magnetic field gradient was turned off, and after another 6 min the rats were sacrificed. The image of the head was obtained within 1 h. Studies of liver and spleen do not include the process of external targeting, since the spheres are in a natural way accumulated in these organs. These spheres were injected through vena femoralis, and the collection of the images started lo-20 min after injection. The rats were sacrificed after the imaging.
The idea of targeting pharmaceuticals, after their attachment to magnetically responsive particles, by mean of external magnetic fields has been suggested. I,’ To follow the in vivo distribution, NMR has been tried, and in those experiments the T2 relaxation time was found to be sensitive to trace amounts of magnetite. As a spin-off effect, the magnetic spheres were found to act as a T2 NMR contrast agent especially for the reticula-endothelial system (RES), where they are accumulated a few minutes after intravasal infusion. 3
Purpose of the study The purpose was to determine the feasibility of ferromagnetic contrast agents in T2 NMR in vivo measurements.
METHODS The magnetic microsphere we used consists of a biodegradable starch matrix with a diameter of about 1 pm, containing small grains of magnetite. Magnetite is an iron oxide, Fe304, and it is known as being ferromagnetic. If these small grains are used (diameter about 10 nm), the magnetized vector becomes unstable, and the magnetic property is no longer ferromagnetic but superparamagnetic. It is called this because the magnetic vector, now being unstable, fluctuates in the same way as for paramagnetic ions, and the “super” comes from the size of the magnetic vector or the magnetic moment since it is much larger than the magnetic moments of ordinary paramagnetic ions. In vitro measurements were carried out in a magnetic field strength of 0.25 T at a temperature of 37”C, (Praxis II NMR analyzer). The effects of the microspheres were studied in a solution of 8% (by
RESULTS The relaxation effects in albumin solution by the spheres and free magnetite particle are shown in Figs. 437
Magnetic Resonance Imaging 0 Volume 4, Number 5, 1986
438
Fig. 1. The microspheres are injected through the external carotid artery, ECA, and then carried through the internal carotid artery, ICA, to the right hemisphere of the brain where the spheres are trapped by an external magnetic field gradient, applied for a few minutes so the spheres will be adhered to the vascular wall.
2 and 3. At this low concentrations
the spin-spin
re-
(R2) is changed in the same way regardless of the magnetite content in the spheres, but free magnetite show a slightly stronger effect. The spinlattice relaxation rate (Rl) was almost unaffected by the spheres at micromolar concentration, but the effect of free magnetite particles is many times stronger than the effect of the spheres. The change in the spin-lattice relaxation rate, produced by paramagnetic agents, is due to magnetic dipole interaction, and this is proportional to re6, where r is the average distance between the paramagnetic agent and the hydrogen nucleus. For free magnetite particles, the distance is smaller than in the case of the particles being imbedded in starch matrices. Since the dependence of the distance goes as the sixth power, there is a marked difference in changing the relaxation rate between free magnetite particles and the starch matrix containing magnetite. The change in the spin-spin relaxation rate is due to inhomogeneities in the magnetic field created by the magnetite. The dependence of this effect to the distance is proportional to F3, and hence there is a smaller difference in the change of spin-spin relaxation rate between the spheres and the free magnetite particles. The changes in the relaxation rates are linear functions of the magnetite concentration, i.e. the changes in the rates are directly proportional to the concentration of the agent, and no saturation occurs. The slope of the lines is called the “relaxivity enhancement” and is mathematically described by laxation
rate
R=C[+R,,
,
where R = relaxation rate (s-l),
C = concentration (mM), f = relaxivity enhancement [(s mM)- ‘I, R. = relaxation rate at C = 0 (s-l). Table 1 show the experimentally found values of the relaxivity enhancement for some agents in albumin solution. Free magnetite strongly affects both Tl and T2, but magnetite in the starch matrix is just about only affecting the T2. Compared to Cd-DTPA, today used clinically,4 the spheres are, on a molar basis, about 30 times more effective in changing the spin-spin relaxation time. The spin-lattice relaxation time is more affected by Cd-DTPA than by the microspheres. Pure Gd3+ ions undergo chemical reactions in the albumin solution and therefore have a smaller relaxivity enhancement than Gd-DTPA. The in vivo measurements showed a marked response of the spheres in spin-echo images. At short
Table 1. Proton
relaxivity enhancement in solution (37”C, 0.25 T) Relaxivity [(s spin-spin
Free Fe30, Fe30, + starch Cd-DTPA Gd3+
800 230 7 3
8% albumin
enhancement
mM)-‘I spin-lattice 150 3 7 0.6
MAGNUS
Ferromagnetic particles as contrast agent 0
+ R2=1/T2
B. E.
Rl=l/T,
(5-1)
/
+
free
0
27%
A
10%
0
04
mt3g”ellle
4
-M-
439
OLSEN ET AL.
(s-1)
t
free
0
2.7X
-I-
A
1.0 x
-n-
Cl
0.4 x
-no-
0.25
T.
magnetite
-II-
%
-@I+
+ 025T.
+
37°C /
/’
37-C
/
n
L
0
10
20 pmole
30 (Fe,O,)
I,!
albumin
sol
Fig. 2. The diagram shows the spin-spin relaxation rate, R2, in a solution of albumin as a function of magnetic concentration. Three different microspheres were studied, containing 2.7%, 1.0% and 0.4% magnetite by weight, and free magnetite particle was also studied. The effect on the R2 relaxation rate is independent of the amount of magnetite in the spheres, but free magnetite will lower T2 somewhat
more.
1 0
t 10
30
20 p mole (Fe,O,)
lf
albumin sol.
Fig. 3. The diagram shows the spin-lattice relaxation rate, Rl, as for the R2 in Fig. 2. At these low concentrations, the microsphere showed no effect on the Tl relaxation time. Due to the closer distance between the free magnetite and the hydrogen nucleus, a marked increase of the relaxation rate occurs for free magnetite particles.
echo-times, the liver could not be discriminated from the surroundings, but with magnetite microspheres as a contrast agent the liver was clearly seen (Figs. 4-6). Targeting the magnetic microspheres to the right hemisphere of the brain was only partially successful. Most of the microspheres were accumulated in the muscle and not in the brain. However, in vitro measurements show, as can be seen in Fig. 7, that the T2 relaxation time is lowered in the right hemisphere when magnetic microspheres are targeted to this area. CONCLUSIONS By introducing a starch matrix with entrapped magnetite, we have created a specific T2 contrast agent, and since only trace amounts of magnetite are needed to change the relaxation time, we conclude that biodegradable starch spheres with magnetite particles can be used as a T2 NMR imaging contrast agent for the reticula-endothelial system. The possibility of targeting the spheres makes them a possible contrast agent for other organs as well. The starch matrix is completely biodegradable and gives no toxic
Fig. 4. A spin-echo NMR image of the abdomen of a rat. No contrast between various organs can be seen in this image. There are two reasons for this: (i) The slice thickness is approximately 1 cm, which is large on a rat. (ii) The spin-echo time was chosen 20 ms in order to emphasize the effect of the contrast agent. The repetition time was 2000 ms.
440
Magnetic Resonance Imaging 0 Volume 4, Number 5, 1986 72
t
(ms)
100
80
? ---
0
--.--.\_
60
+-------++
40
20
Fig. 5. The same spin-echo image as in Fig. 4 but taken 10 min after an injection through vena femoralis of 15mg magnetite per kg body weight. The rat is lying on its back and the cross-section is viewed from the head, so that the right side of the animal is to the right on the image. The liver is clearly seen in this image. The dim contour of the liver is due to the l-cm thickness of the slice.
0
Left
+
Right
hemisphere hemisphere
(magn
field)
0
c 0
a
4
mg Fe304
/ kg body
weight
Fig. 7. The diagram shows the spin-spin relaxation time, T2, in the right and left hemisphere of the rat brain as a function of the amount of magnetite targeted to the right hemisphere. The relaxation time is lowered in the right hemisphere, showing that microspheres indeed are trapped in the intended way.
Another interesting effect is the possibility of adding pharmaceuticals into the biodegradable microspheres and then targeting them with external magnetic field gradients and following the drug distribution with NMR imaging. REFERENCES
Fig. 6. This is a “real” image of the rat from Fig. 5. Comparison shows clearly that the microsphere acts as an NMR contrast agent.
byproducts as is the case when most covalently crosslinked matrices are degrading. The toxic effect of the magnetite is not yet examined in detail. However, preliminary studies in rats show little if any toxic effect of magnetite.
Widder, K.J.; Senyei, A.E. Magnetic microspheres: A vehicle for selective targeting of drugs. Pharm. Ther. 20:337-395; 1983. Schroder, U.; Stahl, A.; Salford, L. Crystallized carbohydrate spheres for slow release, drug targeting and cell separation. S.S. Davis, L. Illum, J.G. McVie and E. Tomlinson, eds. Microspheres and Drug Therapy. New York: Elsevier; 1984:427-437. Olsson, M.; Persson, B.; Salford, L.C.; et al. M.-A. Hopf and C.M. Bydder, eds. European Society of Magnetic Resonance in Medicine and Biology, Proceedings of the First Congress, 1985: 234-243. Felix, R.; Schorner, W.; Laniado, M.; et al. Diagnostic Value of I.V.-Application of Gadolinium-DTPA in Brain Lesions. Presented at Society of Magnetic Resonance in Medicine, 4th Annual Meeting, London, 1985.
073&725X/86 I? I986 Prrgamon
$3.00 + .OO Journals Ltd.
0 Original Contribution
FERROMAGNETIC MAGNUS *Department
B.E.
PARTICLES
OLSSON,* BERTIL R.B.
of Radiation
AS CONTRAST
AGENT
IN T2 NMR IMAGING
PERSSON, * LEIF G. SALFORD~ AND ULF SCHR~DER~
Physics and INeurosurgery, University Hospital, and $Department The Wallenberg Laboratory, Lund University, Sweden
INTRODUCTION
for Microsphere Research,
weight) bovine serum albumin, at a pH of 7.0 Au. Albumin solution was preferred to plasma and serum for three reasons: (i) It is comparable to serum, but contains proteins of only one size. (ii) Its property will be almost the same between different batches. (iii) We “know” what is in the solution. Three different spheres were studied, containing 2.7%, 1.0% and 0.4% magnetite by weight. Free magnetite particles were also studied. The measuring volume was always 1 ml, and the samples were carefully suspended before measurement. Due to their small sizes, the spheres and the particles were easily maintained in suspension during the entire measurement period. In vivo NMR measurements were done in our homebuild imaging system, based on an ironcore frame magnet with a field of 0.07 T. Anesthetized wistar rats with an average weight of 380 -t 16 (1 SD) g were used. To make the spheres sensitive to external magnetic field gradients in vivo, the spheres contained approximately 20% Fe304 by weight. Magnetic spheres were targeted to the right hemisphere of the brain of rat by injection through the external carotid artery (Fig. l), with the right hemisphere placed in a magnetic field gradient of about 200 T/m. After 6 min, the magnetic field gradient was turned off, and after another 6 min the rats were sacrificed. The image of the head was obtained within 1 h. Studies of liver and spleen do not include the process of external targeting, since the spheres are in a natural way accumulated in these organs. These spheres were injected through vena femoralis, and the collection of the images started lo-20 min after injection. The rats were sacrificed after the imaging.
The idea of targeting pharmaceuticals, after their attachment to magnetically responsive particles, by mean of external magnetic fields has been suggested. I,’ To follow the in vivo distribution, NMR has been tried, and in those experiments the T2 relaxation time was found to be sensitive to trace amounts of magnetite. As a spin-off effect, the magnetic spheres were found to act as a T2 NMR contrast agent especially for the reticula-endothelial system (RES), where they are accumulated a few minutes after intravasal infusion. 3
Purpose of the study The purpose was to determine the feasibility of ferromagnetic contrast agents in T2 NMR in vivo measurements.
METHODS The magnetic microsphere we used consists of a biodegradable starch matrix with a diameter of about 1 pm, containing small grains of magnetite. Magnetite is an iron oxide, Fe304, and it is known as being ferromagnetic. If these small grains are used (diameter about 10 nm), the magnetized vector becomes unstable, and the magnetic property is no longer ferromagnetic but superparamagnetic. It is called this because the magnetic vector, now being unstable, fluctuates in the same way as for paramagnetic ions, and the “super” comes from the size of the magnetic vector or the magnetic moment since it is much larger than the magnetic moments of ordinary paramagnetic ions. In vitro measurements were carried out in a magnetic field strength of 0.25 T at a temperature of 37”C, (Praxis II NMR analyzer). The effects of the microspheres were studied in a solution of 8% (by
RESULTS The relaxation effects in albumin solution by the spheres and free magnetite particle are shown in Figs. 437
Magnetic Resonance Imaging 0 Volume 4, Number 5, 1986
438
Fig. 1. The microspheres are injected through the external carotid artery, ECA, and then carried through the internal carotid artery, ICA, to the right hemisphere of the brain where the spheres are trapped by an external magnetic field gradient, applied for a few minutes so the spheres will be adhered to the vascular wall.
2 and 3. At this low concentrations
the spin-spin
re-
(R2) is changed in the same way regardless of the magnetite content in the spheres, but free magnetite show a slightly stronger effect. The spinlattice relaxation rate (Rl) was almost unaffected by the spheres at micromolar concentration, but the effect of free magnetite particles is many times stronger than the effect of the spheres. The change in the spin-lattice relaxation rate, produced by paramagnetic agents, is due to magnetic dipole interaction, and this is proportional to re6, where r is the average distance between the paramagnetic agent and the hydrogen nucleus. For free magnetite particles, the distance is smaller than in the case of the particles being imbedded in starch matrices. Since the dependence of the distance goes as the sixth power, there is a marked difference in changing the relaxation rate between free magnetite particles and the starch matrix containing magnetite. The change in the spin-spin relaxation rate is due to inhomogeneities in the magnetic field created by the magnetite. The dependence of this effect to the distance is proportional to F3, and hence there is a smaller difference in the change of spin-spin relaxation rate between the spheres and the free magnetite particles. The changes in the relaxation rates are linear functions of the magnetite concentration, i.e. the changes in the rates are directly proportional to the concentration of the agent, and no saturation occurs. The slope of the lines is called the “relaxivity enhancement” and is mathematically described by laxation
rate
R=C[+R,,
,
where R = relaxation rate (s-l),
C = concentration (mM), f = relaxivity enhancement [(s mM)- ‘I, R. = relaxation rate at C = 0 (s-l). Table 1 show the experimentally found values of the relaxivity enhancement for some agents in albumin solution. Free magnetite strongly affects both Tl and T2, but magnetite in the starch matrix is just about only affecting the T2. Compared to Cd-DTPA, today used clinically,4 the spheres are, on a molar basis, about 30 times more effective in changing the spin-spin relaxation time. The spin-lattice relaxation time is more affected by Cd-DTPA than by the microspheres. Pure Gd3+ ions undergo chemical reactions in the albumin solution and therefore have a smaller relaxivity enhancement than Gd-DTPA. The in vivo measurements showed a marked response of the spheres in spin-echo images. At short
Table 1. Proton
relaxivity enhancement in solution (37”C, 0.25 T) Relaxivity [(s spin-spin
Free Fe30, Fe30, + starch Cd-DTPA Gd3+
800 230 7 3
8% albumin
enhancement
mM)-‘I spin-lattice 150 3 7 0.6
MAGNUS
Ferromagnetic particles as contrast agent 0
+ R2=1/T2
B. E.
Rl=l/T,
(5-1)
/
+
free
0
27%
A
10%
0
04
mt3g”ellle
4
-M-
439
OLSEN ET AL.
(s-1)
t
free
0
2.7X
-I-
A
1.0 x
-n-
Cl
0.4 x
-no-
0.25
T.
magnetite
-II-
%
-@I+
+ 025T.
+
37°C /
/’
37-C
/
n
L
0
10
20 pmole
30 (Fe,O,)
I,!
albumin
sol
Fig. 2. The diagram shows the spin-spin relaxation rate, R2, in a solution of albumin as a function of magnetic concentration. Three different microspheres were studied, containing 2.7%, 1.0% and 0.4% magnetite by weight, and free magnetite particle was also studied. The effect on the R2 relaxation rate is independent of the amount of magnetite in the spheres, but free magnetite will lower T2 somewhat
more.
1 0
t 10
30
20 p mole (Fe,O,)
lf
albumin sol.
Fig. 3. The diagram shows the spin-lattice relaxation rate, Rl, as for the R2 in Fig. 2. At these low concentrations, the microsphere showed no effect on the Tl relaxation time. Due to the closer distance between the free magnetite and the hydrogen nucleus, a marked increase of the relaxation rate occurs for free magnetite particles.
echo-times, the liver could not be discriminated from the surroundings, but with magnetite microspheres as a contrast agent the liver was clearly seen (Figs. 4-6). Targeting the magnetic microspheres to the right hemisphere of the brain was only partially successful. Most of the microspheres were accumulated in the muscle and not in the brain. However, in vitro measurements show, as can be seen in Fig. 7, that the T2 relaxation time is lowered in the right hemisphere when magnetic microspheres are targeted to this area. CONCLUSIONS By introducing a starch matrix with entrapped magnetite, we have created a specific T2 contrast agent, and since only trace amounts of magnetite are needed to change the relaxation time, we conclude that biodegradable starch spheres with magnetite particles can be used as a T2 NMR imaging contrast agent for the reticula-endothelial system. The possibility of targeting the spheres makes them a possible contrast agent for other organs as well. The starch matrix is completely biodegradable and gives no toxic
Fig. 4. A spin-echo NMR image of the abdomen of a rat. No contrast between various organs can be seen in this image. There are two reasons for this: (i) The slice thickness is approximately 1 cm, which is large on a rat. (ii) The spin-echo time was chosen 20 ms in order to emphasize the effect of the contrast agent. The repetition time was 2000 ms.
440
Magnetic Resonance Imaging 0 Volume 4, Number 5, 1986 72
t
(ms)
100
80
? ---
0
--.--.\_
60
+-------++
40
20
Fig. 5. The same spin-echo image as in Fig. 4 but taken 10 min after an injection through vena femoralis of 15mg magnetite per kg body weight. The rat is lying on its back and the cross-section is viewed from the head, so that the right side of the animal is to the right on the image. The liver is clearly seen in this image. The dim contour of the liver is due to the l-cm thickness of the slice.
0
Left
+
Right
hemisphere hemisphere
(magn
field)
0
c 0
a
4
mg Fe304
/ kg body
weight
Fig. 7. The diagram shows the spin-spin relaxation time, T2, in the right and left hemisphere of the rat brain as a function of the amount of magnetite targeted to the right hemisphere. The relaxation time is lowered in the right hemisphere, showing that microspheres indeed are trapped in the intended way.
Another interesting effect is the possibility of adding pharmaceuticals into the biodegradable microspheres and then targeting them with external magnetic field gradients and following the drug distribution with NMR imaging. REFERENCES
Fig. 6. This is a “real” image of the rat from Fig. 5. Comparison shows clearly that the microsphere acts as an NMR contrast agent.
byproducts as is the case when most covalently crosslinked matrices are degrading. The toxic effect of the magnetite is not yet examined in detail. However, preliminary studies in rats show little if any toxic effect of magnetite.
Widder, K.J.; Senyei, A.E. Magnetic microspheres: A vehicle for selective targeting of drugs. Pharm. Ther. 20:337-395; 1983. Schroder, U.; Stahl, A.; Salford, L. Crystallized carbohydrate spheres for slow release, drug targeting and cell separation. S.S. Davis, L. Illum, J.G. McVie and E. Tomlinson, eds. Microspheres and Drug Therapy. New York: Elsevier; 1984:427-437. Olsson, M.; Persson, B.; Salford, L.C.; et al. M.-A. Hopf and C.M. Bydder, eds. European Society of Magnetic Resonance in Medicine and Biology, Proceedings of the First Congress, 1985: 234-243. Felix, R.; Schorner, W.; Laniado, M.; et al. Diagnostic Value of I.V.-Application of Gadolinium-DTPA in Brain Lesions. Presented at Society of Magnetic Resonance in Medicine, 4th Annual Meeting, London, 1985.