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Description  |
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FIELD OF THE INVENTION
This invention relates to the field of diagnosis and treatment of disease
and more specifically to the use of ferromagnetic, diamagnetic or
paramegnetic particles for such diagnoses and treatment.
BACKGROUND OF THE INVENTION
The efficacy of minute particles possessing ferromagnetic, paramagnetic or
diamagnetic properties for the treatment of disease, particularly cancer,
has been described by R. T. Gordon in U.S. Pat. Nos. 4,106,488 and
4,303,636. As exemplified therein, ferric hydroxide and gallium citrate
are used to form particles of a size of 1 micron or less and are
introduced into cells in the area to be treated. All cells in the sample
area are then subjected to a high frequency alternating electromagnetic
field inductively heating the intracellular particles thus resulting in an
increase in the intracellular temperature of the cells. Because the cancer
cells accumulate the particles to a greater degree than the normal cells
and further because of the higher ambient temperature of a cancer cell as
compared to the normal cells; the temperature increase results in the
death of the cancer cells but with little or no damage to normal cells in
the treatment area. The particles are optionally used with specific cancer
cell targeting materials (antibodies, radioisotopes and the like).
Ferromagnetic, paramagnetic and diamagnetic particles have also been shown
to be of value for diagnostic purposes. The ability of said particles to
act as sensitive temperature indicators has been described in U.S. Pat.
No. 4,136,683. The particles may also be used to enhance noninvasive
medical scanning procedures (NMR imaging).
As disclosed herein the particles of the subject invention are particularly
useful in light of the references cited above.
BRIEF DESCRIPTION OF THE INVENTION
This invention relates to the use of pharmacologically acceptable
ferromagnetic, paramagnetic and diamagnetic particles in the diagnosis and
treatment of disease. The particles possess magnetic properties uniquely
suited for treatment and diagnostic regimens as disclosed in U.S. Pat.
Nos. 4,106,488, 4,136,683 and 4,303,636. Enhanced magnetic properties
displayed by the particles disclosed herein include favorable magnetic
susceptibility and characteristic magnetic susceptibility vs. temperature
profiles. The enhanced magnetic properties displayed by the particles
result in increased sensitivity of response to an electromagnetic field
thereby permitting a more sensitive application of diagnostic and
treatment modalities based thereon. A further benefit is derived from the
chemical composition of said particles whereby intracellular accumulation
and compartmentalization of the particles is enhanced which also
contributes to the more sensitive application of diagnostic and treatment
modalities. Particles useful in light of the subject invention comprise
inorganic elements and compounds as well as organic compounds such as
metal-dextran complexes, metal-containing prosthetic groups, transport or
storage proteins, and the like. The organic structures may be isolated
from bacteria, fungi, plants or animals or may be synthesized in vitro
from precursors isolated from the sources cited above.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to the use of pharmacologically acceptable
ferromagnetic, diamagnetic and parmagnetic particles in the treatment and
diagnosis of disease. This invention relate to the synthesis and use of
said particles which mediate and measure alterations of intracellular
biophysical environment.
According to one form of the invention, selective treatment of cancer cells
is achieved without damaging normal cells. The process comprises
introducing minute particles into the interior of the cells of living
tissue, these particles being injected intravenously while suspended in an
appropriate solution are of a size generally having a diameter of
approximately 1 micron or less and being of a material with properties,
such as ferromagnetic, paramagnetic, or diamagnetic, so as to be
inductively heated when subjected to a high frequency alternating
electromagnetic field. Introducing the particles as described, the patient
is thereafter subjected to an alternating electromagnetic field to
inductively heat the particles sufficiently to raise the temperature of
the cells by an increment of 8.0.degree. to 9.5.degree. centigrade thus
killing the cancer cells without harming the normal cells. Further
selectivity and increased affinity of the cancer cells for these particles
may be achieved by incorporating specific radioisotopes or tumor specific
antibodies bound to these particles.
When the ferromagnetic, diamagnetic or paramagnetic particles described
above are employed as a cancer treating composition, the particle size of
the particles should be not greater than about 1 micron. Preferable
particle size would be less than the 1 micron size. When the minute
particles described are to be injected intravenously into the patient, the
use of any suitable compatible liquid vehicles is desirable. Aqueous
solution of any such body-acceptable materials as dextran, dextrose,
saline or blood, as well as water alone, can be used. The liquid vehicle
should sustain the particles in suspension for the subsequent injection.
Concentrations of such body-acceptable materials that may be useful are
those that are up to about 50% by weight in water. Usually a solution of
about 1% to 10% is adequate. The concentration of the particles in the
solution is not critical and is usually in a range between 50 to 75 mg/cc
of the solution.
The intravenous injection into the patient generally is in an amount such
that between 1 to 10 mg of the particles per kg of body weight of the
patient are injected at one time; however, up to approximately 20 to 45 mg
total dosage per kg of body weight is possible. The greater weight of the
patient, the higher the permissible dosage. The total amount of the dosage
is not critical though 2 to 3 injections, may be injected within a 24 to
72 hour period. The time span for the injections may vary greatly for
various patients and for various objectives. The minute particles
contained in the aqueous medium are transported through the bloodstream
and have been found to be phagocytized by the cancerous cells to a far
greater degree than, and in fact in some cases to the possible exclusion
of, their admittance into normal cells.
Electronmicrographs of the cancerous tissue have proven the selective
pickup of the magnetic particles by the cancer cells.
It has been found that the intracellular temperature of the cells may be
raised between 8.0.degree. centigrade and 9.5.degree. centigrade to cause
death in the cancer cell without damage being caused to the normal cells.
The inductive heating of the minute particles is achieved by using an
electronic oscillator operating in the high-frequency range which heats
the particles by subjecting them to an intense high-frequency field within
a large but otherwise conventional helical coil, field energy being
converted to heat through hysteresis losses and the resistive dissipation
of eddy currents. The helical inductive coil is of sufficient internal
diameter to permit the patient to pass within and of such length to
encompass the length of the patient. Generally, the interal diameter
should be at least 2 feet, but preferably would be greater than 3-6 feet
in diameter. No maximum diameter is known to exist except that required
from practical and economical considerations. Diameters of inductive coils
of greater than 6 feet have a preferential effect in the overall process
by providing more uniform flux gradient to the patient.
The frequency of the electromagnetic alternating high frequency field will
range from 1 hertz to 100 megahertz from 0.5 kilowatts per kilogram of
patient body weight 0.75 kilowatts of power per 1.0 kilograms of body
weight has been found to be particularly useful. In this power and
frequency range, the coil is selected to produce from 200-1000 oersteds,
preferably 550-650 oersteds.
The time necessary to inductively heat the minute particles held within the
cells to be treated depends substantially upon the frequency and the power
producing the alternating electromagnetic field and ultimately the
strength of the field produced. In general, it has been found that
subjecting the patient to 5 to 12 minutes or preferably 8 to 10 minutes of
the alternating electromagnetic field would be adequate to bring about the
necessary temperature rise of at least 8.0.degree. centigrade and that the
variables with respect to the type and concentration of the particles in
the vehicle and the electromagnetic treatment are not critical provided
that the necessary temperature is achieved.
In a further embodiment, the particles introduced intracellularly as
described may be used as a method of delivering a chemotherapeutic agent
primarily to the interior of the cancer cells by having the
chemotherapeutic agent encapsulated within said particles and released at
the proper time by application of the high frequency alternating
electromagnetic field thus solubilizing the said particles within the
cells.
These two embodiments are discussed in detail in U.S. Pat. Nos. 4,106,488
and 4,303,636 which are incorporated herein by reference.
It has recently been discovered that a wide range of ferromagnetic,
paramagnetic and diamagnetic particles which possess enhanced magnetic
characteristics and in combination with desirable structural properties
are particularly useful in light of the applications described above.
Whereas the particles described in U.S. Pat. Nos. 4,106,488 and 4,303,636
were selected primarily on the basis of their size and their ability to be
inductively heated; it is now appreciated that additional criteria must be
considered when selecting a particle for a particular application. In
selecting the particles of the instant invention the following magnetic
and physical characteristic were evaluated: magnetic permeability,
magnetic susceptibility, magnetic moment, Curie points, and thermal
conductivity. Magnetic permeability is a property of materials modifying
the action of magnetic poles placed therein and modifying the magnetic
induction resulting when the material is subjected to a magnetic field and
may be defined as the ratio of the magnetic induction in the substance to
the magnetizing field to which it is subjected. Magnetic susceptibility is
measured by the ratio of the intensity of magnetization produced in a
substance to the magnetizing force of intensity of the field to which it
is subjected. Magnetic moment is measured by the torque experienced when
it is at right angle to a uniform field of unit intensity. The value for
magnetic moment is given by the product of the magnetic pole strength by
the distance between the two poles. The Curie point represents the
temperature above which substances loose their ferromagnetic properties
Thermal conductivity relates to the ability of a substance to transfer
thermal energy and is known to be effective by temperature.
In addition to the physical and magnetic characteristics listed above,
other parameters must be evaluated. For ease of consideration these
additional parameters may be grouped in relation to the time course of
treatment. For example certain evaluations as to the efficacy of a
particular particle can be made prior to the introduction of said particle
to the subject, but such as selection must be modified by considerations
relating to the behavior of the particle during the treatment period, and
finally consideration must also be given to post-treatment parameters.
Pre-treatment parameters to be considered comprise, an evaluation of the
magnetic and physical properties of the particles, the composition and
solution properties displayed by the particles, and route of
administration of said particles. For example, if the particles are to be
delivered by intraveneous injection it would be important for the
particles to be in a stable colloidial suspension in aqueous media.
After introduction of the particles into the subject, the following
parameters become important; biocompatibility and toxicity considerations,
the rate and degree of cellular uptake of the particles, the specificity
of said uptake, the subcellular localization of particles once they have
been taken up by the cells, the modification of the magnetic properties as
a result of intracellular localization, and the effect upon magnetic
properties as a result of metabolic activity within cells. For example,
sulfactants may be profitably employed to reduce surface tension, and mask
groups contributing to zeta potentials thereby enhancing the uptake of
particles by the cells. In reference to sub-cellular localization it is
possible to specifically target particles to specific intracellular
locales by constructing the particle in the form of a molecular analog
(e.g., mimic) of an endogeneous compartmentalized cellular component. For
example by forming particles containing porphyrin moieties and providing
same to cells within the treatment area; the particles will accumulate
within porphyrin-rich area within the cells, i.e., mitochondria or
chloroplasts, and participate in the cellular reactions attendant thereto.
It is important to realize that as the structural complexity of the
particle increases not only is size a consideration but also the overall
shape and the conformation and configuration of various particle
components must be considered. With reference to the porphyrin-containing
particles mentioned above, it is known, for example, that the position of
the metal value relative to the plane of the porphyrin ring has important
consequences with respect to the metal's reactive properties. Further it
must be appreciated that the type and position of the side chains on the
porphyrin moiety can act to position the metal in a particular orientation
and as a corollary, the metal will have an affect of the conformation of
the side chains of the porphyrin as well.
The interactions at the particle surface-cellular environment interface are
also important. For example, induced magnetic moments can result from the
ordering which takes place at the particle surface.
Further particles which are subject to cellular metabolism will display a
change in magnetic characteristic as a result of said metabolism. Although
the intracellular effects upon metabolizable, organic metal-containing
particles as described above are important considerations it should be
remembered that the characteristics of less complex particles also
affected by intracellular localization. For example, the inductive heating
of the particles comprised of inorganic materials in suspension outside
the cell; generally transmit their effect through hysteresis owing to
their small size. However, after uptake of cells the individual particles
tend to cluster providing an overal "group" particles size whereby heating
due to eddy currents is also possible.
Thus, the subject invention not only provides an effective method for
monitoring the treatment phase so as to allow for treatment techniques
based upon limited increases in temperature (i.e., rises in temperature of
9.5.degree. C.), but also provides for an additional treatment technique.
In this further embodiment, since the subject invention provides a means
for specific particle distribution and a sensing of the responsiveness to
the various treatment fields, high temperature treatment modalities are
also possible. The 9.5.degree. C. limitation as discussed supra is, of
course, predicated on the case situation in which particle distribution,
magnetic state and orientation were equal in all cancer cells and normal
cells under the treatment conditions. However, employing the improved
methods of the subject invention thereby affecting specific particle
distribution, orientation, differential magnetic susceptbility, timing and
other parameters described herein, between the cancer cells and the normal
cells within the target area, increases in the intracellular temperature
up to 100.degree. C. are possible without substantially damaging the
surrounding normal cells.
Irreversible cell death and biological alterations are induced by the
energy input to the particle and thereupon to the interior of the cancer
cell. Thus, the same energy input may be accomplished by application over
a long period of time with a consistent small temperature rise (8.degree.
to 9.degree. C. for 10 to 20 minutes) or when the same total amount of
energy is applied over a short period of time, a higher temperature
results (100.degree. C. for a few seconds).
Obviously, timing and energy parameters may be adjusted to provide a
spectrum of intracellular temperature which may be utilized depending upon
the treatment appropriate in specific cases.
Finally, with respect to post-treatment practice, consideration must be
given to the removal of the particles from the subject. The removal is
accomplished by natural excretory processes which may be supplemented with
chelating agents or metal efflux stimulating compositions.
Although the effective electromagnetic fields referred to herein have been
characterized as alternating electromagnetic fields, there is no evidence
which would preclude the use of electromagnetic fields of the oscillating
or pulsed type and such fields are contemplated by the subject invention.
Particularly useful particles include both inorganic elements and compounds
as well as metal-containing organic compounds. Inorganic elements and
compounds particularly well suited, owing to their favorable magnetic
parameters, comprise elements, such as dysprosium, erbium, europium,
gaolinium, holmium, samarium, terbium, thulium, ytterbium or yttrium and
compounds thereof such as dysprosium sulfate, erbium sulfate, europium
oxide, europium sulfate, gadolinium oxide, gadolinium sulfate, holmium
oxide, samarium sulfate, terbium oxide, terbium sulfate, thulium oxide,
ytterbium sulfide, yttrium oxide, yttrium sulfate, yttrium ferrioxide
(Y.sub.3 Fe.sub.5 O.sub.12) yttrium aluminum oxide (Y.sub.3 Al.sub.5
O.sub.12), other dimetallic compounds such as dysprosium-nickel,
dysprosium-cobalt, gadolinium-iron, ytterbium-iron, cobalt-samarium,
gadolinium-yttrium, and dysprosium-gallium, and actinide series element
and compounds thereof.
Metal-containing organic molecules useful for the application discussed
above, comprise particles of iron-dextrans such as FeOOH-dextran or
Fe.sub.3 O.sub.4 -dextran complexes and other dextran metal complexes
wherein the metal is selected from the group comprising cobalt, zinc,
chromium, nickel, gallium, platinum, manganese and rare earth metals such
as dyprosium, erbium, europium, gadolinium holmium, samarium, terbium,
thulium, ytterbium and yttrium, other dimetall compounds such as
dysprosium-nickel, dysprosium-cobalt, gadolinium-iron, ytterbium-iron,
cobalt-samarium, gadolinium-yttrium, and dysporsium hysprosium-gallium,
actinide series elements and compounds, ferric ammonium citrate, and
various iron transporting and chelating compounds such as enterochelin,
hydroxamates, phenolates, ferrichromes, desferri-ferrichromes, ferritin,
ferric mycobactins, and iron-sulfur proteins such as ferredoxin and
rubredoxin.
Particularly appropriate metal-containing organic structures for use with
the present invention are the metalloporphyrins such as etioporphyrins,
measoporphyrins, uroporphyrins, coprophyrins, protoporphyrins, and
dicarboxylic acid containing porphyrins and substituted porphyrins such as
tetraphenylporphyrin sulfonate (TPPS). Especially advantageous
protophoryrins comprise hematoporphyrins, chlorophylls, and cytochromes.
In addition to the naturally occurring protoporphyrins which possess
either iron or magnesium-containing moieties, mixed-metal or di-metal
hybrid porphyrins may also be prepared. For example, by substituting an
alternative metal for the iron in hematoporphyrin, the advantages of the
porphyrin moiety (e.g., in terms of specificity of localization is
retained while the unique magnetic properties of the new metal enhance the
sensitivity of the substituted molecule. Suitable metals for purposes of
substitution comprise cobalt, manganese, zinc, chromium, gallium, nickel,
platinum and rare earth series of metals dysprosium, erbium, europium,
gadolinium, holmium, samarium, terbium, thulium, ytterbium and ytterium,
dimetallic compounds such as dysprosium-nickel, dysprosium-cobalt
gadolinium-iron, ytterbium-iron, cobalt-samarium, gadolinium yttrium,
dysprosium-gallium and actinide series elements and compounds thereof. The
substituted porphyrins are then optionally reacted with dextran to form a
metal-containing porphyrin dextran complex in particle form. Suitable
porphyrin acceptors comprise any dicarboxylic acid containing porphyrin,
such as protoporphyrins (e.g., hematoporphyrins), and the like.
The substitution reaction is carried out in vitro by reacting the desired
metal with the desired porphyrin in the presence of the enzyme
ferrochelatase (E.C. 4.99.1.1). Reaction conditions as described by Jones
and Jones (Biochem. J 113:507-14 (1969) or Honeybourne, et al. (FEBS
Lett.: 98:207-10(1979)) are suitable.
Particularly, advantageous particle systems include transferrin-based
particle systems wherein the particle system comprises an Fe.sub.3 O.sub.4
-transferrin dextran as well as other metal-transferrin dextran complexes
wherein the metal is selected from the group comprising cobalt, zinc,
chromium, nickel, gallium, platinium, manganese and rare earth metals such
as dyprosium, erbium, europium, gadolinium, holmium, samarium, terbium,
thulium, ytterbium and yttrium, other dimetallic compounds such as
dysprosium-nickel, dysprosium-cobalt, gadolinium-iron, ytterbium-iron,
cobalt-samariu, gadolinium-yttrium, and dysprosium-gallium, actinide
series elements and compounds. Additionally, metalloporphyrin-transferrin
wherein the metalloporphyins are those mentioned above.
Further useful particle systems include antibody-ferritin-Fe.sub.3 O.sub.4
complexes and other antibody-ferritin based systems where the Fe.sub.3
O.sub.4 is optionally substituted with a transition metal, rare earth
metal, metalloporphyrin or other ferromagnetic, diamagnetic or
paramagnetic particle wherein the antibody is of monoclonal or polyclonal
origin and is specifically reactive to the specific target organ or
cell-type desired.
Metallothionein-based particle systems and lectin-based systems are also
useful. In these systems either the metallothionein or lectin is used in
combination with Fe.sub.3 O.sub.4 or the transition metal, rare earth
metal, metalloporphyrin and ferromagnetic, diamagnetic or paramagnetic
particles as described above.
Specific metal-organic compound complexes are given in Table I.
TABLE I
Particle Complexes
Fe(III) Tetraphenylporphyrin sulfonate (TPPS.sub.4) Acetate
Fe(III) TPPS.sub.4 Acetate 4Na Salt (H.sub.2 O)
Fe(III) Mesoporphyrin IX Chloride
Fe(III) TPPS.sub.4 Chloride
Co TPPS.sub.4
Co(III) MesoTPPS.sub.4 Tetra Na Salt (acetate)
Fe Phthalocyanine Tetrasulfonate Tetra sodium salt
Tetra Sodium-meso-Tetra (4-sulfonato-phenyl) Porphine (12 hydrate)
Fe(III) Tetra (N-Methyl 4-Puridyl) Porphyrin Pentachloride
Fe Phthalocyanine
Hemin
Fe-Hematoporphyrin D. (HPD)
Fe-Acetoxyethyl vinyl Deuteroporphyrin
Fe-Protoporphyrin IX
Fe-Deuteroporphyrin 2,4 bis acetal
Mn-TPPS.sub.4
Co-N.sup.+ MTPyP
Mn-N.sup.+ MTPyP
Co-Mesoporphyrin X
Protohemin
Deuterohemin
Meso-tetra (4-N methyl pyridyl) hemin tetraiodide
Meso-tetra (4-carboxy phenyl) hemin
Ni-TPPS
Ni-HPD
Mn-Mesoporphyrin IX
Co-Protoporphyrin IX
Mn-Protoporphyrin IX
Sn-Protoporphyrin IX
Co-HPD
Mn-HPD
Gd-TPPS
Gd-HPD
Hematoporphyrin Mono-acetate-Fe
Ferretin-Fe
Ferredoxin-Fe(4)
Transferrin-Fe
Hematoporphyrin Diacetate-Gd
GdFe.sub.2 -TPPS.sub.4
GdFe.sub.2 -HPD
FeTPPS.sub.4 (OH.sub.2).sub.2 ]ClO.sub.4 --
FeTPP(OH.sub.2).sub.2 ]ClO.sub.4 --
Bisimidozole (FeTPPS)ClO.sub.4 --
Fe-nitrolacetate
Fetetrasulfinated phalocyanine
Rubrium-ferricytochrome/c
According to another embodiment of the invention, the ferromagnetic,
paramagnetic or diamagnetic particles described above are used for
diagnostic purposes whereby the magnetic characteristics of said particles
are correlated with the intracellular temperature of the cells within the
sample area. Experimental details of this application may be found in U.S.
Pat. No. 4,136,683 which is incorporated herein by reference.
One magnetic characteristic known to be temperature dependent is magnetic
susceptibility. Magnetic susceptibility is measured by the ratio of the
intensity of magnetization produced in a substance to the magnetizing
force or intensity of the field to which it is subjected. This magnetic
characteristic is routinely measured by magnetometer devices, such as a
vibrating magnetometer or a flux gate magnetometer. Therefore, by
measuring the magnetic susceptibility of particles at various
temperatures, it is quite simple to calibrate the magnetometer equipment
so that when it measures the magnetic susceptibility of the particles a
simple calibration will indicate the exact corresponding temperature of
the particle.
By way of illustrating the increased magnetic susceptibility of some of the
elements or compounds described above, the following table is provided:
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Mag. Sus.
Element or Compound
Temp (.degree.K.)
(10.sup.6 cgs)
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Iron Oxide (ref.)
293 +7,200
Dysprosium Oxide
287.2 +89,600
Dysprosium Sulfate
291.2 +92,760
Octahydrate
Erbium Oxide 286 +73,920
Erbium Sulfate 293 +74,600
Octahydrate
Europium 293 +34,000
Europium Oxide 298 +10,100
Europium Sulfate
293 +25,730
Holmium Oxide 293 +88,100
Holmium Sulfate 293 +91,600
Octahydrate
Terbium 273 +146,000
Terbium Oxide 288.1 +78,340
Terbium Sulfate 293 +76,500
Octahydrate
Thulium 291 +25,500
Thulium 296.5 +51,444
Ytterbium Sulfide
292 +18,300
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Thus, the enhanced magnetic characteristics displayed by the particles of
the subject invention results in an increase in the sensitivity of
response of said particles in an electromagnetic field thereby increasing
the overall sensitivity and control of the various diagnostic and
treatment modalities based thereon.
A further benefit is derived from the fact that some particle compositions
comprise a ferromagnetic, paramagnetic or diamagnetic component integrated
into a cell or organelle specific molecular structure, thereby permitting
efficient targeting and delivery of said particles to specific
intracellular compartments such as mitochondria, chloroplasts, nuclei,
vacuoles and the like.
EXAMPLE
Preparation of Fe.sub.3 O.sub.4 -Dextran-Transferrin Particles
An Fe-transferrin particle colloidal solution was prepared by combining
0.00638 g Fe(NH.sub.4).sub.2 (SO.sub.4).sub.2 .multidot.6H.sub.2 O water
solution with 0.00806 g citric acid, 1.0 cc of a 1M sodium phosphate
solution in water and 99 cc of water, the pH adjusted to 7.4 with dilute
sodium hydroxide or hydrochloric acid and 1 cc of the combination added to
10 mg of human transferrin obtained from Cappel Laboratories to obtain an
Fe-transferrin composition. The Fe-transferrin was dialyzed against a
0.01M sodium phosphate water solution adjusted to pH 7.4 and a pure
Fe-transferrin particle composition obtained. The dialysis was performed
against a cellophane membrane.
An iron oxide dextran particle having a particle size of less than one
micron was prepared by mixing 10 cc of 50% Dextran T-40 with 10 cc of an
aqueous solution containing 1.51 g FeCl.sub.3 .multidot.6H.sub.2 O and
0.64 g FeCl.sub.2 .multidot.4H.sub.2 O. This mixture was stirred and
titrated to pH 10.5 by the addition of 7.5% NH.sub.4 OH and heated to
60.degree. C. in a water bath for 20 minutes. Particles of Fe.sub.3
O.sub.4 -dextran were obtained and removed by centrifugation at a force of
600 g for 5 minutes. The particles are contained in the supernatant and
the supernatant subjected to the same centrifugation two more times. The
particles were then separated by taking the supernatant from the
centrifuge and contacting it with a chromatographic column comprising
Sephacryl 300 (obtained from the Pharmacia Company). The column was eluted
with an aqueous buffer of 0.1M NaC.sub.2 H.sub.3 O.sub.2 mixed with 0.15M
NaCl adjusted to a pH of 6.5. The Fe.sub.3 O.sub.4 -dextran particles were
thus removed by the elution step.
The Fe.sub.3 O.sub.4 -dextran particles were then oxidized by mixing 5 cc
of the particles at a concentration of 10 mg/cc as a colloidal solution in
aqueous NaC.sub.2 H.sub.3 O.sub.2 /NaCl buffer with 5 mM NaIO.sub.4 and
were stirred for one hour at 25.degree. C. This mixture was dialyzed
against a 20 mM sodium borate water solution at a temperature of 4.degree.
C. and a pH of 8.5. The dialysis was conducted against a cellophane
membrane. After the dialysis, the Fe.sub.3 O.sub.4 -dextran particles were
recovered and 5 cc of these particles in suspension in the aforesaid
NaC.sub.2 H.sub.3 O.sub.2 /NaCl buffer (the particles being at a
concentration 10 mg/cc) 1 cc of the Fe-transferrin particles and 4.72 mg
of sodium borohydride as an aqueous 0.25M solution were mixed for 5
minutes to produce Fe-dextran-transferrin particles after which this
mixture was contacted with a Sephacryl 300 chromatographic column to
remove any Fe-dextran particles. The column was eluted with 20 mM of a
sodium phosphate buffer containing 0.15M NaCl at a pH of 7.4. The product
eluted from the column comprised Fe.sub.3 O.sub.4 -dextran-transferrin
particles. These particles were assayed for protein via the Biuret
reaction (Bovine Serum Albumin standard). Iron was analyzed by means of a
Carey 14 spectrophotometer and it was determined that the peak of the
protein concentration corresponded to the peak of the colored Fe
adsorption after the Sephacryl 300 separation indicating the transferrin
had coupled to the iron-dextran particle.
When the Fe.sub.3 O.sub.4 -dextran-transferrin particles are contacted with
a Phenyl-Sepharose column (in lieu of the Sepharyl 300 column) the
Fe.sub.3 O.sub.4 -dextran-transferring particles remain attached while the
Fe.sub.3 O.sub.4 -dextran particles pass through. Subsequently, the
Fe.sub.3 O.sub.4 -dextran-transferrin particle is eluted off the column by
lowering the ionic strength e.g. by contacting the column with water or
using an ion with a less salting-out effect or increased chaotropic effect
on altering the pH all of which is known in the art.
Electrophoresis may also be used to isolate the Fe.sub.3 O.sub.4
-dextran-transferrin particles from the Fe.sub.3 O.sub.4 -dextran
particles.
The Fe.sub.3 O.sub.4 -dextran-transferrin particles thus obtained are used
in the treatment and diagnosis of disease as described herein and have a
particles size of less than about 1 micron in diameter.
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