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Experimental Therapeutics |
Department of Otorhinolaryngology, Head and Neck Surgery [C. A., W. A., P. H.], Department of Experimental Oncology and Therapeutic Research [R. J. K., W. E.], and Institute for Medical Statistics and Epidemiology [S. W.], Klinikum rechts der Isar, Technical University of Munich, 81675 Munich; Physics-Department E 17, Technical University of Munich, 81675 Munich [F. G. P.]; Chemicell, 10777 Berlin [C. B.]; and Cecilien-Klinik, 33175 Bad Lippspringe [A. S. L.], Germany
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ABSTRACT |
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3500 mm3, FF-MTX was
injected intraarterially (i.a.; femoral artery) or i.v. (ear vein),
whereas an external magnetic field was focused on the tumor. FF-MTX
i.a. application with the external magnetic field resulted in a
significant (P < 0.05), complete, and
permanent remission of the squamous cell carcinoma compared with the
control group (no treatment) and the i.v. FF-MTX group, with no signs
of toxicity. The intratumoral accumulation of FFs was visualized both
histologically and by magnetic resonance imaging. Thus, our data show
that i.a. application of FF-MTX is successful in treating experimental
squamous cell carcinoma. This "magnetic drug targeting" offers a
unique opportunity to treat malignant tumors locoregionally without
systemic toxicity. Furthermore, it may be possible to use these
magnetic particles as a "carrier system" for a variety of
anticancer agents, e.g., radionuclides, cancer-specific
antibodies, and genes. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Targeting and prolonged retention of the FF complex at the target site reduces its reticuloendothelial system (RES) clearance and facilitates extravascular uptake. To optimize intratumoral magnetic particle concentration, several features need to be considered: (a) the particles should be of a size that allows sufficient attraction by the magnetic field and their introduction into the tumor or into the vascular system surrounding the tumor; (b) the magnetic fields should be of sufficient strength to be able to attract the magnetic nanoparticles into the desired area; (c) the FF complex should deliver and release a sufficient amount of anticancer agent; and (d) the method of injection should have good access to the tumor vasculature and should avoid clearance by the reticuloendothelial system ("first pass effect").
The purpose of the present study was to compare different application methods (i.v., i.a.) of magnetic drug targeting for the treatment of experimental VX-2 squamous cell carcinoma. Because FFs are visible histologically and by imaging techniques such as MRI, we also wished to demonstrate the morphological intratumoral distribution of these magnetic nanoparticles in conjunction with an external magnetic field focused on the tumor region.
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MATERIALS AND METHODS |
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Magnetic Nanoparticles (FFs).
The FFs used in the experiments were obtained from Chemicell (Berlin,
Germany; German patent application no. 19624426.9) and consisted of a
colloidal dispersion formed by wet chemical methods from iron oxides
and hydroxides to produce special multidomain particles (Table 1)
. The particles were surrounded by starch polymers for stabilization
under various physiological conditions and to allow chemoabsorptive
binding. MTX has cationic characteristics and combines (amine
groups of MTX-HCl with phosphate groups of the starch derivates) at a
pH of 7.4 (Fig. 1)
. The FF-MTX contained 6.5 mg of MTX per 10 ml. Because the drug bond
is reversible (ionic binding), desorption of the bound drug was
dependent on the physiological environment (pH, osmolality,
temperature) and could be varied by changing the blood electrolyte
concentration according to the specific need. In experiments,
desorption of MTX took place within 60 min (Fig. 2)
, which ensured that the drug could act freely once localized to the
tumor by the magnetic field. Pyrogenicity and sterility tests were
performed by the Pharmacy Department of the Virchow Medical School
(Humboldt-Universität, Berlin, Germany) according to good
manufacturing practice guidelines. The characteristics of the FF-MTX
are depicted in Table 1
(see also Figs. 1
and 2
).
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Animals.
The experimental animals were female New Zealand White rabbits
(2000–2500 g body weight, 12–15 weeks old; Charles River, Sulzfeld,
Germany) that were housed individually in a room with an artificial
12/12 h light/dark cycle (exposed to light from 0700 to 1900 h).
The rabbits were fed hard rabbit chow pellets (Altromin, Lage,
Germany), carrots, dry bread, and tap water.
Surgical Intervention.
Fragments of viable VX-2 tissue, 1 mm in size, were taken from the
tumor periphery in donor animals. These fragments were placed in a
special medium [RPMI 1640, 2.0 g/liter
NaHCO3, and L-glutamin
(Seromed); Biochrom, Berlin, Germany] and were immediately implanted
under sterile conditions into the hind limb of anesthetized recipient
rabbits (n = 26) in the supply area of the
femoral artery. The experiments were performed when the tumors had
reached a volume of approximately 3500 mm3
.
For application of the chemotherapy, the animals were anesthetized with
an i.m. injection of ketamine [35 mg/kg body weight (Narketan 10;
Chassot, Bern, Switzerland)] and xylazine [5 mg/kg body weight
(Xylapan; Chassot, Bern, Switzerland)], the femoral artery was
cannulized and an indwelling catheter [Venflon (0.8 mm); Ohmeda Co.,
Helsingburg, Sweden] was placed after separation of the femoral vein
and the saphenous nerve 2 cm distal to the inguinal furrow. The
FF-MTX and the MTX alone were administered by perfusor over a period of
10 min. To prevent thrombosis, prophylaxis consisting of heparin sodium
(heparin/natrium/25,000 IU Ratiopharm; Ratiopharm,
Ulm, Germany) was given preoperatively, once immediately
postoperatively, and twice daily for 5 days postoperatively (200 IU per
kg of body weight, s.c.).
Magnetic Field.
An electromagnet with a magnetic flux density of a maximum of 1.7 Tesla
was used to produce an inhomogeneous magnetic field. The magnetic flux
density was focused onto the region of the tumor with a specially
adapted pole shoe that was placed in contact with the surface of the
tumor. On the tip of the pole shoe, the gradient (Fig. 3
, yellow
arrows) has its maximum. Fig. 3
demonstrates the dependence of the magnetic flux density on the
distance to the pole shoe. A magnetic flux density of 1.7 Tesla was
estimated in the region of the tumor surface and at 10 mm below the tip
of the pole shoe, 1.0 Tesla (Fig. 3)
. The magnetic field was focused on
the tumor during FF infusion and for 60 min in total (Fig. 3)
.
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. Group 1 received an i.a. infusion of FF-MTX with the magnetic field at
a dose equivalent to 20 and 50% of the systemic dose MTX (group 1a and
group 1b, respectively). Group 2 received an i.a. infusion of MTX alone
without the magnetic field at doses equivalent to 20, 50, 75, and 100%
of the systemic dose. Group 3 received an i.a. infusion of FF alone
with the magnetic field at equivalent doses compared with groups 1a and
1b. Group 4 received an i.v. infusion of FF-MTX with the magnetic field
at doses equivalent to 20 and 50% of the systemic dose (group 4a and
group 4b, respectively). Group 5 received an i.a. infusion of FF-MTX
without the magnetic field at doses equivalent to 20 and 50% of the
systemic dose, and group 6 was the control group without treatment
(Table 2)
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Blood Samples.
Blood samples were drawn by venipuncture every week and centrifuged at
2000 x g within 2 h. Measurements of
clinical chemistry parameters (iron, alanine aminotransferase,
aspartate aminotransferase, 
-glutamyl transferase, alkaline
phosphatase, and lactate dehydrogenase; Hitachi 747 analyzer; Roche
Diagnostics, Mannheim, Germany) as well as the blood count parameters
(total and differential blood counts; Sysmex SE-9000 analyzer; Sysmex
GmbH, Norderstedt, Germany) were performed immediately after sampling.
Histological Evaluation, MRI.
Immediately after i.a. infusion of 50% FF-MTX into the femoral artery,
and after application of the magnetic field for a duration of 60 min,
one animal was killed and the tumor was removed and fixed in 3.7%
formalin. Five-µm thick paraffin sections of the tumor were cut and
stained with H&E. After the 3-month observation period, the remaining
animals were killed; and the tumor, liver, kidneys, spleen, lungs,
brain, and inguinal lymph nodes were removed and examined
histologically.
Six h after 50% FF-MTX application with an external magnetic field, a MRI was performed on four tumor-bearing animals. Imaging was done with a 1.5-Tesla clinical MR scanner (ACS-NT; Philips, Best, the Netherlands). A fat-suppressed, T1-weighted turbo-spin echo sequence was used for imaging (TR 535; TE 20; echotrain length, 5).
Statistical Analysis.
The tumor volume was calculated using the formula for an elliptical
mass (1/6 
a2b, where
a = width on the horizontal axis and
b = length on the vertical axis). We
considered change of volumes as percentages of tumor volumes (100%)
found at day 0 (day of treatment). Statistical analysis for relative
tumor volumes was performed using the one sample Welch
t test (with a conservatively fixed value of 100% for the
control group) and a Welch t test for two independent
samples. For blood parameters (absolute values), we applied the
t test for two independent samples. The resulting two-sided
Ps were considered significant if 
0.05. The result was
considered significant at P = 0.01 or 0.05
and highly significant if <0.01. The Ps were calculated
using the Statistical Package for Social Sciences (SPSS) version 9.0
and Microsoft EXCEL version 97.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Figs. 4
5
6
7
8
9
10
) the tumor volume increased to 14.723 mm3
(median value) at 12 days, and palpable metastases appeared after 30
days. The animals of group 1a (Fig. 4
, •), treated i.a. with 20%
FF-MTX, had a 50% reduction in volume after 3–12 days (mean, 6 days)
and complete tumor remission between the 15th and 36th day (mean, 26
days) after treatment. This reduction in tumor volume was significant
by the 6th day (P = 0.047;
P < 
) and highly significant by the 15th
day (P < 0.001; P < ). The animals of group 1b (50% FF-MTX; Fig. 5
, 
) had a decrease in tumor volume similar to that of group 1a (Fig. 5
, ), with a 50% decrease in volume after 3–6 days (mean, 4.2
days) and complete tumor remission after 12–57 days (mean, 21.8 days).
The decrease in tumor volume was highly significant by the 6th day
(P = 0.001; P < ; Figs. 4
and 5
).
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, 
), and enlarged, palpable inguinal lymph nodes were found after 48
days. At higher doses (75 and 100%), complete remission of tumor
occurred at the 36th (75%) and 33rd day (100%; Fig. 6
, ).
The two group-3 animals (i.a. FF alone with the magnetic field, amount
of FFs alone equivalent to groups 1a and 1b) demonstrated a progressive
increase in tumor volume (Fig. 7
, 
) with palpable, enlarged inguinal lymph nodes (metastases) after
45 days.
The six animals of group 4 (i.v. injection via the ear vein of 20% and
50% FF-MTX with magnetic field) showed a slight tumor remission, but
the reduction of volume was not statistically significant in comparison
to the control group (Ps: group 4a 0.48- 0.70, group 4b
0.26- 0.96 (P > ; Fig. 8
, 
; Fig. 9
, 
).
The two animals of group 5 (i.a. FF-MTX 20 and 50%, without a magnetic
field) showed a discontinuation of tumor growth and no evidence of
metastases, but no remission of the tumor was seen (Fig. 10
, FF-MTX; 20%, ; FF-MTX 50%, ). At the time of treatment, <5%
of the animals showed a small necrotic fraction in the area of the
tumor area (Fig. 10)
.
Local and Systemic Effects.
Similar to the description in the literature (18)
, the
general condition of the control group animals (limited to two animals
for ethical reasons) worsened during the observation period, and the
animals developed pneumonia, which explains the increase of leukocytes
as seen in Fig. 11
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None of the animals of group 1 had any evident side effects such as
alopecia, ulcers, or muscular atrophy; and their general condition
(weight, food intake, excrement, urine, activity, fur condition)
remained normal during the whole 3-month observation period compared
with the physiological data of healthy animals (breeder’s statement by
Charles River, Sulzfeld, Germany). No significant changes in serum iron
or leukocyte values were seen in this group (Fig. 11a)
.
The urine of one animal in group 2 (50% MTX) showed blue-green
discoloration, and this animal developed mild alopecia in the region of
the digits after 48 days. Both animals with low-dose MTX (20 and 50%)
had a decrease in leukocyte values, but this was not statistically
significant (P = 0.29). Both of the group-2
animals with high-dose (75 and 100%) MTX had temporary blue-green
urine discoloration, as well as a unilateral alopecia (palmar region of
the digits to the knee joint) of the limb in which the tumor was
implanted developing after 33 days. This hairless area developed
cutaneous inflammation and ulceration, followed by mild alopecia of the
ipsilateral fore limbs and head. The musculature of the treated limb
became atrophic, and the circumference was noticeably smaller
(by 3 cm) at the end of the 3-month observation period. There
was no marked difference in the severity of the side effects between
the two animals, except for the fact that the animal with the higher
MTX dose (100%) developed the changes several days sooner. Group-2
animals with 50, 75, and 100% MTX steadily lost weight after an
initial lag-phase and were underweight at the end of the observation
period (mean value, 1800 mg below the lower reference values according
to the breeder’s statement; Charles River). These animals became
leucocytopenic (2.95 x 103
/µl) in the early phase (highly significant
drop; P = 0.004; Fig. 11b
), but
recovered slightly in the middle and late periods.
None of the animals of group 3 or 4 showed any significant changes in
serum iron (not shown in figures) or leukocyte counts (group 3, Fig. 11a
; groups 4a and 4b, Fig. 11a
) during
the observation period when compared with initial values.
Histological Findings.
Fig. 12
shows a whole-mount cross-section of the tumor that was excised just
after treatment. Brown-black granules, FF
particles distributed throughout the entire tumor. A higher
magnification of a blood vessel (Fig. 13)
shows that the intraluminal FF particles were concentrated and
deposited on the endothelium nearest to the magnetic field and were
separated from the erythrocyte pool, but, as can be seen from Fig. 14
, FF particles were also found in the tumor interstitium and in the
adjacent surrounding tissues as well (Fig. 15)
.
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In group 2, the VX-2 tumors of the two low-dose animals were 8.644 mm3 (50% MTX) and 2.497 mm3 (20% MTX) in size, with a large area of central necrosis and viable tumor at the periphery. The two animals with high-dose MTX (75 and 100%) had no viable tumor at the implantation site. None of the other investigated organs in the animals of group 2 (liver, kidneys, spleen, lungs, or brain) had any pathological changes.
The tumors of both animals of group 3 measured 13.324 mm3 and 17.649 mm3 , respectively, with a large area of central necrosis and viable tumor at the periphery. No FF particles were found within the tumor or in the surrounding musculature and skin. Some FFs were found in the spleen. Metastases were found in the inguinal lymph nodes and liver of both animals. None of the other investigated organs (kidneys, spleen, lungs, brain) had any pathological changes.
MRI.
Fig. 16, a and b
, show the left hind limb (implantation
site) of two rabbits that received 50% FF-MTX i.a. and i.v.,
respectively. The MRI was made 6 h after treatment. The tumor is
situated at the medial portion of the hind limb (dotted
circle), and the concentration of FF is seen by extinction of
signal. Fig. 16a
(i.a. FF-MTX) shows definite extinction of
signal and Fig. 16b
(i.v. FF-MTX) only a very discrete
signal extinction. The area marked f is at the head of the
femur and appears to be hypodense.
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DISCUSSION |
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and 5)
. The application was well tolerated by the animals, and
no signs of toxicity were detected. On the contrary, i.a. infusion of
the same doses, 20 and 50%, of MTX alone (group 2, Fig. 6
) resulted in
no reduction of tumor volume, and the animals developed metastases and
suffered from chemotherapeutic side effects. Only when the dose of MTX
alone was increased to 75% and 100%, was a tumor remission seen, but
this resulted in severe side effects (alopecia, ulcers, and
leukocytopenia as seen in Fig. 11b
). i.v. infusion of the
FF-MTX complex was also ineffective inasmuch as only a slight tumor
remission that was not statistically significant resulted (Figs. 8
and 9)
. The same was true of i.a. infused FF-MTX without an external
magnetic field, because the tumor remained at the same size, without
remission (Fig. 10)
. Thus, the combination of i.a. infusion with a
magnetic field was safe, effective, and well tolerated by the animals
and was very effective in treating the tumor even though the dose of
chemotherapeutic agent was markedly reduced. At present, i.a. delivery of chemotherapeutic agents is approved and well accepted for treatment of liver metastases (20) and has occasionally been used for other tumor types also (e.g., inoperable head and neck tumors); but it has often necessitated complicated, time-consuming operative procedures, including general anesthesia (21) . Experimentally, Swistel et al. (18) described encouraging results using i.a. chemotherapy for VX-2 squamous cell carcinoma. They achieved complete tumor remission after i.a. application of Adriamycin in four of six animals, whereas i.v. infusion of Adriamycin caused severe toxicity and resulted in complete remission in only two cases.
A potential complication that could arise with the use of FF compounds
is the fact that, with larger particles, embolization could occur,
preventing a sufficient concentration of the chemotherapeutic agent
from reaching the tumor. On the other hand, if the particles are too
small, the external magnetic field might not provide sufficient
attraction so that the particles are drawn into the tumor. The
particles used in the present study had a size of 100 nm. No
embolization was seen in the main vascular system of the tumor, and the
particles were attracted throughout the entire tumor including its
surface (Fig. 12)
. An additional helpful factor is that microvascular
permeability in neoplastic tissues is increased (8-fold compared with
normal tissue) as is diffusion (33-fold; Ref. 22
). Our
histological findings showing distribution of FF particles throughout
the tumor strongly support the concept that high-molecular-weight
substances such as chemotherapeutic agents or monoclonal antibodies can
be effectively targeted to tumor tissue. In addition, the fact that the
FF alone with a magnetic field failed to cause tumor remission (Fig. 7)
indicates that the therapeutic effect resulted from the action of the
chemotherapeutic agent itself, rather than intratumoral embolization by
the particles.
The electromagnet used for this study produced a magnetic flux density
of a maximum of 1.7 Tesla, which decreased depending on the distance to
the pole shoe (Fig. 3)
. The magnetic gradient can be seen as a
collection of vectors that point in the direction of increasing values
as shown in Fig. 3
(yellow arrows). The arrow sizes
correspond to the strength of the magnetic gradient. Both factors
(direction and magnitude) reflect the inhomogeneous character of
the magnetic field, which is of key importance for magnetic drug
targeting. In previous studies, it was suggested that a magnetic field
strength of 8000 Gauss (0.8 Tesla) is sufficient to exceed linear blood
flow in the intratumoral vasculature and allow 100% localization of
magnetic carrier containing 20% magnetite (23)
. In
contrast, Goodwin et al. (24)
applied MTCs i.a.
in a swine model, focusing a magnetic field of only 250-1000 Gauss
(0.025–0.1 Tesla; permanent neodymium magnet) to the desired
compartments in the liver and lungs. The depth of this MTC targeting
was 8–12 cm and the particle size was 0.5–5 µm. With this model,
MTCs with a predefined activity had a concentration of 67% in the
liver and 50% in the lung localized by the magnet.
The magnetic field strength with a maximum of 1.7 Tesla used in the
present investigation was the strongest ever applied for magnetic drug
targeting. We achieved a high concentration of FFs within the tumor
after i.a. infusion of FFs, which was seen by histological (Figs. 12
13
14
15)
and MRI (Fig. 16a)
methods. The VX-2 squamous cell
carcinoma in the present study was superficially exposed and had no
migratory motion, as was the case with the liver and lung targets
(breathing fluctuations) in the swine model of Goodwin et
al. (24)
. In addition these organs lie deeply in the
body cavity (8–12 cm from the body surface), greatly complicating
focusing of the magnetic flux density onto the tumor area. Two
approaches to overcome this problem are possible: (a) the
use of larger particles, as previously suggested by Lübbe and
Bergemann (25)
; or (b) the use of a stronger
magnetic field. The particles (FF-MTX) used in the present study were
100 nm in size (hydrodynamic diameter) and have shown good therapeutic
results in smaller animals (mouse, rat) as well (3
, 4)
.
The strong magnetic field was very efficacious in combination with
these particles, but additional experiments (which we have already
begun) should be performed using marked FFs to clarify the optimal
magnetic field strength and particle size. For example, to more
effectively treat in deep body cavities (i.e., pancreatic
cancer and so forth) rotating magnetic fields could be used to focus
the particles to the region of interest. It is also important that the
tumor has a sufficient blood supply so that the particles have access
to the particular area.
A remarkable feature of using ionically bound pharmaceuticals is that
the anticancer agents are able to desorb from the carrier (FF) after a
defined time span and the low-molecular-weight substances
(e.g., the molecular weight of MTX 517) can then pass
through the vascular wall or interstitium into the tumor cells. This is
important because once the FF-MTX complex has been directed to the
tumor by the magnetic field, the drug must dissociate to act freely
within the tumor. As shown in Fig. 2
, MTX desorbs from the FF after 30
min (half-life), and, therefore, 50% of the drug is free to act on the
tumor after 30 min.
Dextran-coated iron oxides have been shown to produce signal loss by
MRI and have been used as a contrast medium for the detection of
metastatic lymph nodes (negative contrast; Ref. 26
). We
found total signal loss and. therefore. a very high concentration of FF
by MRI after focusing by means of the magnetic field (Fig. 16a)
. Recent studies have shown that i.a. application of
radioactively labeled magnetic carriers with an external magnetic field
resulted in retention of at least 50% in the target site
(27)
. In comparison, after i.v. injection, only very
slight signal loss was seen, which indicates a very low concentration
(Fig. 16b)
. This underscores the advantage of i.a.
versus i.v. infusion in magnetic drug targeting.
Previous studies by Bacon et al. concerning FF with a particle size of 0.5–1.0 µm found no acute or chronic toxicity after the i.v. infusion of 250 mg of iron/kg of body weight in rats (28) , and 1–3 mg of iron/kg of body weight in humans have been shown to be safe as well (29) . This agrees with our findings, inasmuch as FF infusion was not associated with any signs of toxicity.
Magnetic microspheres loaded with the -emitting radioisotope
90Y have also been successfully used as a form of
radionuclide therapy. In one study, this compound was maneuvered within
the body of a mouse to a s.c. lymphoma, resulting in eradication of the
tumor (30)
. Magnetic fluids have also been used for the
so-called "magnetic fluid hyperthermia" that has been used to
control the local growth of murine mammary carcinoma (31)
.
Additional modification of the magnetic particles so that they could
bind monoclonal antibodies, lectins, peptides, hormones or genes could
make delivery of these compounds more efficient and also highly
specific. Therefore, magnetic particles could make important
contributions to molecular and cell biology (e.g., in
vitro transfection with genes), which would result in advances in
both basic science and clinical practice (32)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Margarete Ammon Foundation,
Munich, and grants from the Technical University of Munich, Germany. 
2 To whom requests for reprints should be
addressed, at Department of Otorhinolaryngology, Head and Neck Surgery,
Klinikum rechts der Isar, Technical University of Munich,
Ismaningerstrasse 22, 81675 Munich, Germany. Phone: 49-89-4140-2370;
Fax: 49-89-4140-4853; E-mail: C.Alexiou{at}lrz.tu-muenchen.de
3 The abbreviations used are: FF,
ferrofluid; i.a., intraarterial/intraarterially; MR, magnetic
resonance; MRI, MR imaging/image; MTX, mitoxantrone; MTX-FF, FF bound
to MTX; MTX-HCl, MTX hydrochloride; MTC, magnetic-targeted carrier.
Received 4/21/00. Accepted 10/ 3/00.
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REFERENCES |
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, i.v. FF-MTX 20% with
magnetic field; 
, i.v. FF-MTX 50% with magnetic field.
b, 
, i.a. FF-MTX 20% with magnetic
field; 
, i.a. FF-MTX 50% with magnetic field; 
, i.a. MTX 20%
and 50%; 
, i.a. MTX 75% and 100%.
