Encapsulated superparamagnetic particles

Stable, encapsulated superparamagnetic magnetite particles having a narrow particle size distribution with average particle diameters in the range of from about 50 .ANG. to about 350 .ANG. are prepared by forming an aqueous dispersion of magnetite particles having the above particle size characteristics in the presence of a surfactant, coacervating a mixture of gelatin and a carboxyl containing hydrophilic polymer such as gum arabic to form a thin coating of coacervate on the magnetite particles and crosslinking the coacervate coating with a gelatin hardener such as glutaraldehyde.

FIELD OF THE INVENTION 
This invention relates to magnetically responsive particles and to their 
use in systems in which the separation of certain molecules, 
macromolecules and cells from the surrounding medium is necessary or 
desirable. More particularly, the invention relates to methods for the 
preparation of magnetically responsive particles comprising a magnetite 
core surrounded by a very thin, stable, modified gelatin coating. If 
desired, a wide variety of organic and/or biological molecules may be 
coupled to the coating. The particles (coupled or uncoupled) can be 
dispersed in aqueous media without rapid gravitational settling and 
conveniently reclaimed from the media with a magnetic field. The process 
provided herein yields particles that are superparamagnetic; that is, they 
do not become permanently magnetized after application of a magnetic 
field. This property permits the particles to be redispersed without 
magnetic aggregate formation. Hence the particles may be reused or 
recycled. Stability of the coating of the invention as well as of the 
covalent attachment of molecules thereto also facilitates the use and 
reuse of the encapsulated superparamagnetic particles of the invention. 
The magnetically responsive particles of this invention may be coupled to 
biological or organic molecules with affinity for or the ability to adsorb 
or which interact with certain other biological or organic molecules or 
with cells. Particles so coupled may be used in a variety of in vitro or 
in vivo systems involving separation steps or the directed movement of 
coupled molecules to particular sites, including, but not limited to, 
immunological assays, other biological assays, biochemical or enzymatic 
reactions, affinity chromatographic purifications, nucleic acid 
hybridization, cell sorting, cell separation and diagnostic and 
therapeutic uses, including site specific drug delivery and magnetic 
resonance imaging. 
DESCRIPTION RELATIVE TO THE PRIOR ART 
Very small particles (50-350 .ANG. region) of normally ferromagnetic 
materials are unable to support magnetic domains and are called 
superparamagnetic. This means that they are weakly magnetic in the absence 
of an external magnetic field, but upon the application of an external 
magnetic field, become magnetic and agglomerate readily. The ease with 
which such particles become magnetized upon application of a magnetic 
field is directly proportional to their degree of magnetization, measured 
in emu/gm (electromagnetic units per gram). Their property of becoming 
demagnetized upon removal of the magnetic field is inversely proportional 
to their coercive force, measured in Oersteds (Oe). As a practical matter, 
materials (particles) that have a degree of magnetization of at least 
about 30 emu/gm and a coercive force of less than about 30 Oe can be 
considered superparamagnetic. Generally, the greater the magnetization and 
the lower the coercive force, the more usefully or "strongly" 
superparamagnetic the particles become. That is, less magnetic force is 
required to magnetize them and they lose their magnetic properties more 
rapidly upon removal of the outside magnetic force. Such particles have 
found many uses, ranging from mechanical seals and couplings to biological 
separations. 
A detailed review of pertinent prior art may be found in U.S. Pat. No. 
4,672,040, issued June 6, 1987. As noted therein, the use of magnetic 
separations in biological systems as an alternative to gravitational or 
centrifugal separations has been reviewed by B. L. Hirschbein et al., 
Chemtech, March 1982: 172-179 (1982); M. Pourfarzaneh, The Ligand 
Quarterly, 5(1): 41-47 (1982); and P.J. Halling and P. Dunnhill, Enzyme 
Microb. Technol., 2: 2-10 (1980). Several advantages of using magnetically 
separable particles as supports for biological molecules such as enzymes, 
antibodies and other bioaffinity adsorbents are generally recognized. For 
instance, when magnetic particles are used as solid phase supports in 
immobilized enzyme systems [see, e.g., P. J. Robinson et al., Biotech. 
Bioeng., XV: 603-606 (1973)], the enzyme may be selectively recovered from 
media, including media containing suspended solids, allowing recycling in 
enzyme reactors. When used as solid supports in immunoassays or other 
competitive binding assays, magnetic particles permit homogeneous reaction 
conditions (which promote optimal binding kinetics and minimally alter 
analyte-adsorbent equilibrium) and facilitate separation of bound from 
unbound analyte, compared to centrifugation. Centrifugal separations are 
time consuming, require expensive and energy-consuming equipment and pose 
radiological, biological and physiological hazards. Magnetic separations, 
on the other hand, are relatively rapid and easy, requiring simple 
equipment. Finally, the use of non-porous adsorbent-coupled magnetic 
particles in affinity chromatography systems allows better mass transfer 
and results in less fouling than in conventional affinity chromatography 
systems. 
Although the general concept of magnetizing molecules by coupling them to 
magnetic particles has been discussed and the potential advantages of 
using such particles for biological purposes recognized, the practical 
development of magnetic separations has been hindered by several critical 
properties of magnetic particles developed thus far. 
Large magnetic particles [mean diameter in solution greater than 10 
microns(.mu.)]can respond to weak magnetic fields and magnetic field 
gradients; however, they tend to settle rapidly, limiting their usefulness 
for reactions requiring homogeneous conditions. Large particles also have 
a more limited surface area per weight than smaller particles, so that 
less material can be coupled to them. Examples of large particles are 
those of Robinson et al., [supra] which are 50-125.mu. in diameter, those 
of Mosbach and Anderson [Nature, 270: 259-261 (1977)]which are 60-140.mu. 
in diameter and those of Guesdon et al., [J. Allergy Clin. Immunol 61(1): 
23-27 (1978)] which are 50-160.mu. in diameter. 
Ferromagnetic materials in general become permanently magnetized in 
response to magnetic fields. Materials termed "superparamagnetic" 
experience a force in a magnetic field gradient, but do not become 
permanently magnetized. Crystals of magnetic iron oxides may be either 
ferromagnetic or superparamagnetic, depending on the size of the crystals. 
Superparamagnetic oxides of iron generally result when the crystal is less 
than about 350 .ANG.(0.035.mu.) in diameter; larger crystals generally 
have a ferromagnetic character. Following initial exposure to a magnetic 
field, ferromagnetic particles tend to aggregate because of magnetic 
attraction between the permanently magnetized particles, as has been noted 
by Robinson et al., [supra]. 
As described, for example, in U.S. Pat. No. 4,604,222, superparamagnetic 
particles are generally prepared by ball-milling magnetic powders for long 
periods of time, followed by tedious sieving and purification processes. 
As a result, they have been extremely costly and this has limited their 
applications. 
For use in biological separation, it is necessary to derivatize the 
particle so that functional groups, such as amine or carboxyl, are present 
at the surface for bonding to antibodies and the like. This has required 
the use of costly reagents, such as amine or carboxyl silanes, and the 
process of attachment to the magnetic particle is difficult. See U.S. Pat. 
Nos. 4,672,040 and 4,683,032. 
The preparation of magnetite by means of hydroxide addition to a solution 
of ferrous/ferric salts is well known. The concept of using a dispersing 
agent during or after the preparation to stabilize the magnetite particles 
has been reported, for example in U.S. Pat. No. 4,019,995 and is the basis 
for a commercial product ("Lignosite") manufactured by the Georgia Pacific 
Corporation. However, that product consists of magnetite particles that 
are appended to the lignin polymer chain and are not encapsulated. The 
ratio of lignin to magnetite is quite large, and the product does not 
appear to be suitable for biological work. 
Dispersible magnetic iron oxide particles reportedly having 300 .ANG. 
diameters and surface amine groups were prepared by base Precipitation of 
ferrous chloride and ferric chloride in the presence of polyethylene 
imine, according to Rembaum in U.S. Pat. No. 4,267,234. Reportedly, these 
particles were exposed to a magnetic field three times during preparation 
and were described as redispersible. The magnetic particles were mixed 
with a glutaraldehyde suspension polymerization system to form magnetic 
polyglutaraldehyde microspheres with reported diameters of 0.1.mu.. 
Polyglutaraldehyde microspheres have aldehyde groups on the surface which 
can form bonds to amino-containing molecules such as proteins. However, in 
general, only compounds which are capable of reacting with aldehyde groups 
can be directly linked to the surface of polyglutaraldehyde microspheres. 
Moreover, magnetic polyglutaraldehyde microspheres are not sufficiently 
stable for certain applications. 
Latex particles containing magnetite particles dispersed within the latex 
sphere are available in the micron (latex) range. See "Uniform Latex 
Particles" Seragen Diagnostics, Inc., April, 1986, supplement. These are 
derivatized to be used for antigen separation Large polyacrylamide-agarose 
particles containing finely divided magnetite are used for affinity 
chromatography. These particles are in the several-micron range. 
U.S. Pat. No. 4,582,622 describes the preparation of a "homogenous" 
magnetic particulate, having a particle size of 0.8-50 .mu.m by preparing 
an aqueous colloidal solution containing gelatin, a water soluble 
polysaccharide such as gum arabic, sodium polymetaphosphate and a 
ferromagnetic substance, adjusting the pH to 2.5 to 6 with an acid, and 
forming a water insoluble particulate by adding an aldehyde. The magnetic 
particulate is useful as a carrier to immobilize such biological proteins 
as antigens, antibodies, and enzymes for use in diagnostic assays. As 
shown in Example 8 (infra) however, the process of that patent, which does 
not use coacervation conditions as set forth herein, results in coarse 
aggregates having relatively large particle sizes, compared to the fine, 
discrete, paramagnetic coacervate coated magnetite particles of the 
present invention. As will be appreciated by those skilled in the art, 
smaller particles have larger surface areas per unit weight (or volume) 
than large particles, which, in turn, affords greater efficiency of use in 
that less small particle carrier than large particle carrier would be 
required to carry a given amount of immobilized biological protein. Also, 
the smaller size particles of the present invention are amenable to 
ingestion by animals for diagnostic or therapeutic use whereas the larger 
particles of U.S. Pat. No. 4,582,622 would be expected to be unsuitable 
for such use by virtue of their size. In addition, such large particles 
are not suitable for magnetic separation techniques such as that described 
in Example 1 (infra). 
SUMMARY OF THE INVENTION 
The present invention provides a method for preparing superparamagnetic 
particles of magnetite (Fe.sub.3 O.sub.4) that are encapsulated by a thin 
layer of mo gelatin The multifunctionality of the modified gelatin makes 
the attachment of biologically active compounds such as antibodies very 
facile. In addition, the encapsulation stabilizes these very small 
particles. 
The present invention also provides a method for preparing 
superparamagnetic magnetite particles that are encapsulated by a thin 
shell of a coacervate of gelatin and gum arabic (or another polymeric acid 
comprising recurring acid groups, preferably those selected from the group 
consisting of carboxylic acid groups and sulfonic acid groups). This 
procedure eliminates the need for extensive milling and grinding that have 
heretofore been widely used to prepare superparamagnetic particles. The 
encapsulation materials provide reactivity through the various functional 
groups present in the gelatin-gum arabic shell or coating.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, superparamagnetic magnetite 
particles with a narrow particle size distribution in the range of from 
about 50 .ANG. to about 350 .ANG. are prepared by adding a mixture of 
ferric and ferrous salts to water in amounts that provide a molar ratio of 
ferric to ferrous ions in the range of about 1.6 to 2.4 and in 
concentrations in the range of 0.01 to 1 molar, preferably about 0.05 to 
0.5 molar, more preferably about 0.1 molar; optionally, adding acid, e.g., 
sulfuric acid, to adjust the pH to less than about 1.5; preferably adding 
from about 0.1 to about 5% (wt/vol) of a surfactant solution; preferably 
purging the resulting solution of oxygen by bubbling therethrough an inert 
gas, preferably nitrogen, for a period of at least about 10 minutes, 
preferably at least about 30 minutes; adding to the (preferably purged) 
solution (preferably, as rapidly as possible), with stirring, concentrated 
NaOH (or an equivalent hydroxide, for example, ammonium, potassium, or 
lithium hydroxide) in an amount in excess of 8 moles of hydroxide ion per 
mole of Fe.sup.++ present in the solution, to form particles of Fe.sub.3 
O.sub.4 having a particle size distribution in the above described range; 
washing the resultant magnetite particles with water, preferably with the 
assistance of magnetic separation, until the pH of the magnetite 
dispersion is within the range of about 10-11; depositing a 
gelatin/polymeric acid coating on the particles by coacervate; and 
crosslinking. Preferably the coacervation is effected by removing excess 
water, preferably with the aid of magnetic separation, from the magnetite 
dispersion that had been washed to pH 10-11 to form a concentrated 
dispersion of magnetite particles, adding the resulting magnetite 
dispersion, with stirring, to an aqueous solution, having a temperature of 
at least about 40.degree. C., of gelatin having an isoelectric point 
greater than about 8 and a polymeric acid (preferably gum arabic) 
comprising at least one recurring acid group, preferably selected from the 
group consisting of carboxylic acid groups and sulfonic acid groups, each 
of said gelatin and polymeric acid being present in a concentration of 
from about 1% to about 10% (w/vol) and the ratio of magnetite to the 
gelatin/polymeric acid mixture on a dry basis being from about 4:1 to 
about 1:1; and adjusting to coacervation conditions comprising a pH in the 
range of between about 4 and 5.5 and a concentration of the 
gelatin/polymeric acid mixture of less than about 2% (wt/vol); and the 
crosslinking is effected with a known gelatin hardener. 
Preferably, each of the gelatin and gum arabic (or other polymeric acid) is 
present in the aqueous solution of step (5) in a concentration of about 4% 
(w/vol) and the ratio of magnetite to the gelatin/polymeric acid mixture 
on a dry basis is preferably about 3:1. Preferably, the coacervation is 
effected by adjusting the pH with H.sub.2 SO.sub.4 (or another suitable 
acid such as acetic acid, or a strong mineral acid, e.g., HCl) to a pH of 
about 4.0 to 5.5, more preferably a pH of about 4.5; and adding the 
resulting suspension slowly (over a period of from about 5 to about 30 
minutes) with stirring, to a large excess of cold water maintained at a 
temperature below about 5.degree. C., the resulting suspension being 
preferably stirred for at least about 30 minutes to stabilize the 
coacervate coated particles. The crosslinking is preferably effected by 
rapidly adding to the suspension of stabilized coacervate coated particles 
a concentrated solution of glutaraldehyde or another gelatin hardener in 
such amount as to provide the equivalent of at least about 2 gms. of 
glutaraldehyde per 100 gm. of gelatin on a dry basis, so as to crosslink 
the gelatin in the coacervate coating on the magnetic particles; stirring, 
preferably for about 30 minutes, to assure completion of the crosslinking 
reaction; raising the pH to above 7 with a base such as NaOH; increasing 
the temperature slowly to about 20-25.degree. C. (ambient); and washing 
with water to remove unreacted glutaraldehyde. 
Preferably the starting ferric and ferrous salts are sulfates. However, 
other water soluble salts, such as chlorides or other halides, acetates 
and nitrates can be used. 
While the ratio of ferric to ferrous ion in the process of the present 
invention may be varied within the range of about 1.6 to about 2.4, it is 
presently preferred that the ratio be approximately 2 so as to provide 
substantially stoichiometric amounts to satisfy the equation: 
EQU 2Fe.sup.+++ +Fe.sup.++ +8(OH).sup.- .fwdarw.Fe.sub.3 O.sub.4 +4H.sub.2 O. 
Although sodium dodecyl sulfate is presently preferred for use as the 
surfactant in the process of the invention, other anionic surfactants and 
cationic surfactants are also useful and non-ionic surfactants are 
expected to be useful. A variety of such surfactants can be selected from 
McCutcheon's Emulsifiers and Detergents, McCutcheon Division, MC 
Publishing Co., Glen Rock, New Jersey, USA. 
Suitable anionic surfactants include Triton 770, an alkylaryl polyether 
sulfate, sodium salt, sold by Rohm and Haas Co.; Triton X-200, an 
alkylaryl polyether sulfonate, sodium salt, sold by Rohm and Haas Co.; 
Triton GR-5M, dioctyl sodium sulfosuccinate, sold by Rohm and Haas Co.; 
Sterling AM, an ammonium lauryl sulfonate sold by Canada Packers, Inc.; 
Gafac RM-710, the free acid of a complex organic phosphate ester sold by 
GAF Corp.; and Witcolate, an alcohol ether sulfate sold by Witco Chem. 
Corp. 
Suitable cationic surfactants include dodecyltrimethylammonium chloride, 
Ammonyx DMCD-40, a lauryldimethyl amine oxide sold by Onyx Chem. Co.; 
Ammonyx T, a cetyl dimethyl benzyl ammonium chloride also sold by Onyx 
Chem Co.; Emcol CC55, a polypropoxy quaternary ammonium acetate sold by 
Witco Chem Corp.; Triton RW Series, cationic polyalkylene glycols sold by 
Rohm and Haas Co.; and Emulsifier 3, a quaternary ammonium chloride sold 
by Tomah Products, Inc. 
Suitable non-ionic surfactants include Surfactant 10G, a 
nonylphenoxypolyglycidol sold by Olin Chem Co.; and various Triton 
alkylaryloxy polyethoxy ethanols sold by Rohm and Haas Co., such as Triton 
X-100. 
It is preferred to use a substantial excess of hydroxide over the 
stoichiometric amount in step (2) above, preferably of the order of 10 
moles per mole of Fe.sup.++. While a ratio of NaOH/Fe.sup.++ in excess of 
10:1 can be used, there does not appear to be an advantage in doing so. 
While gum arabic is preferably used as the coacervating agent for the 
gelatin, another polymeric acid comprising recurring acid groups selected 
from the group consisting of carboxylic acid groups and sulfonic acid 
groups can be substituted for the gum arabic such as alginic acid, maleic 
acid, fumaric acid, citraconic acid, itaconic acid, crotonic acid, 
3-acrylamidopropane-sulfonic acid, 2-acrylamido-2-methylpropanesulfonic 
acid, 3-acryloyloxypropanesulfonic acid, styrenesulfonic acid, etc., 
typical comonomers being alkyl acrylates and alkyl methacrylates such as 
methyl methacrylate, ethyl acrylate, and vinyl monomers such as ethylene, 
e.g., partially hydrolyzed poly(ethylene-co-maleic anhydride), methyl 
vinyl ether, styrene, vinyl acetate, e.g., partially hydrolyzed poly(vinyl 
acetate-co-maleic anhydride), amides such as acrylamide, methacrylamide 
and N-isopropylacrylamide. The molecular weights of the polymers can range 
from about 5,000 to 300,000. 
When gum arabic is used, the preferred weight ratio of gelatin to gum 
arabic is 1:1, although this ratio can conveniently be within the range of 
2:1 to 1:2. When other polymeric acids, preferably polycarboxylic or 
polysulfonic acids, are substituted for gum arabic, the ratios can be 
adjusted accordingly. 
Similarly, while the presently preferred ratio of magnetite to coacervate 
on a dry basis is about 3:1, this ratio can conveniently be selected 
within the range of about 4:1 to 1:1. 
Preferably, the ratio of suspension to cold water in the quenching step 
described above is about 1 liter of suspension to about 8-10 liters of 
cold water. 
While glutaraldehyde is the presently preferred crosslinking agent for use 
in the process of this invention, other gelatin hardeners known to those 
skilled in the photographic arts can be substituted, with suitable 
adjustments as may be required to maintain equivalent stoichiometry. 
Typical useful gelatin hardeners include formaldehyde and dialdehydes such 
as succinaldehyde and glutaraldehyde as described in U.S. Pat. No. 
3,232,764; active esters such as described in U.S. Pat. No. 3,542,558; 
active halogen compounds such as described in U.S. Pat. Nos. 3,106,468, 
3,305,376 and 3,957,882; s-triazines such as described in U.S. Pat. No. 
3,325.287; aziridines such as described in U.S. Pat. No. 3,575,705; active 
olefins such as described in U.S. Pat. Nos. 3,490,911 and 3,640,720; 
vinylsulfones such as bis(vinylsulfonylmethyl) ether and 
bis(vinylsulfonyl)-methane as described in U.S. Pat. No. 3,841,872 and 
U.S. Pat. No. 3,539,644; halogen-substituted aldehyde acids such as 
mucochloric and mucobromic acids; and polymeric hardeners such as 
dialdehyde starches poly(acrolein-co-methacrylic acid); 
poly(styrene-co-2-chloroethylsulfonylmethylstyrene) and 
poly(styrene-co-vinylsulfonylmethylstyrene). 
The coacervate coated superparamagnetic particles of the present invention 
have a mean diameter in the range of from about 70 .ANG. to about 450 
.ANG., preferably from about 100 .ANG. to about 400 .ANG., more preferably 
from about 150 .ANG. to about 350 .ANG. and comprise magnetite particles 
having a mean diameter in the range of from about 50 .ANG. to about 350 
.ANG., preferably about 100 .ANG. to about 300 .ANG., more preferably 
about 150 .ANG. to about 250 .ANG., that are coated with a coating that is 
from about 20 .ANG. to about 100 .ANG. thick, preferably about 30 .ANG. to 
about 50 .ANG. thick, which coating comprises a crosslinked coacervate of 
gelatin with gum arabic or another polymeric acid, preferably one 
containing repeating units of a carboxylic acid or a sulfonic acid; the 
magnetite particles, before being coated, having a magnetization of 
greater than about 30 emu/gm, preferably greater than about 40 emu/gm, 
more preferably greater than about 50 emu/gm and a coercive force of less 
than about 30 Oe, preferably less than about 25 Oe, more preferably than 
about 20 Oe. The coated particles have a magnetization greater than about 
30 emu/gm preferably greater than about 40 emu/gm and a coercive force 
less than about 30 Oe, preferably less than about 25 Oe, more preferably 
less than about 20 Oe. The magnetization and coercive force values of the 
paramagnetic particles as set forth herein are values obtained by using a 
VSM meter while applying a magnetic field of 2500 Oe to the dry particles. 
As previously indicated, the coated superparamagnetic particles of the 
invention can be used in known techniques for separation of biological 
materials, as described, for example, in U.S. Pat. No. 4,672,040 and 
discussed in the Description Relative to the Prior Art hereinabove, as 
well as in drug delivery systems, for diagnostic imaging and in other 
applications wherein it is advantageous to use fine superparamagnetic 
particles having a narrow particle size distribution, particularly where 
biocompatability is important. 
The following examples are presented to illustrate the practice of &he 
present invention: 
EXAMPLE 1 
The pH of 100 ml of an equimolar mixture of ferrous and ferric sulfate was 
adjusted to 1.0 with 25% sulfuric acid. To this was added 10 ml. of a 4% 
gelatin solution acid processed (pI-9) whose pH had been adjusted to 0.8 
with sulfuric acid solution. The temperature was raised to 50.degree. C. 
and 25% sodium hydroxide solution was added over a period of five minutes 
to give a final pH of 12.5. During this time, the solution was stirred in 
the presence of air. The black suspension was separated magnetically and 
washed with distilled water. The washed magnetite precipitate was then 
mixed with 100 ml. of a solution containing 4% w/vol of each of gelatin 
and gum arabic. The gelatin was the same type as used in the magnetite 
preparation step described above. The mixture of magnetite and gelatin-gum 
arabic was agitated at 40.degree. C. and the pH was lowered to 4.5 with 
25% HCl. This mixture was then poured slowly into 500 ml. of water that 
was agitated rapidly at 4.5.degree. C. After 30 minutes, 20 ml. of 50% 
glutaraldehyde was added, and the gelatin coating was considered to be 
fully crosslinked and the encapsulated magnetite was washed several times 
by distilled water, using magnetic separation. 
Electron microscope examination showed the particles to be 100-150 .ANG. in 
diameter, with a very minor 40-50 .ANG. fraction. Elemental analysis was 
used to determine the gelatin-gum arabic content, and estimates of shell 
thickness of 31 .ANG. were calculated (assuming the magnetite particle 
diameter to be 100 .ANG.). Magnetic evaluation is shown in FIG. 1. 
It can be readily seen from the magnetization curve that no hysteresis 
exists. 
Magnetic separation was demonstrated by inserting a plug of steel wool 
(fine grade) approximately 3 cm long and 1.5 cm ID into the stem of a 
small plastic powder funnel. The stem was placed between the poles of a 
small horseshoe magnet (800 gauss), and the encapsulated magnetite 
solution poured into the funnel. Clear liquid drained out. Removing the 
magnet and pouring the clear liquid into the funnel caused the particles 
to be removed from the steel wool. The recovery was excellent, which is 
further proof that the particles are superparamagnetic. 
EXAMPLE 2 
Example 1 was repeated, but 0.5% of dodecyltrimethylammonium chloride was 
used as a surfactant/dispersing agent instead of gelatin in the 
preparation of the magnetite. The encapsulation step involving gelatin and 
gum arabic was carried out exactly as described in Example 1, and the 
results were comparable. 
EXAMPLE 3 
This example illustrates the hydrophobizing of the gelatin-gum arabic shell 
so that the encapsulated Fe.sub.3 O.sub.4 particles have an affinity for 
non-water-miscible organic solvents such as toluene or ethylbenzene, thus 
making them useful in non-aqueous systems such as ferro fluids. (See, for 
example, U.S. Pat. No. 3,531,413.) 
One gram of the wet coagulum of Example 1 was mixed with 15 ml. of water 
and 1 ml. of Quilon M (DuPont), and the mixture was shaken for five days. 
Another sample, which consisted of the unencapsulated magnetite 
preparation was also treated in this manner. The samples were then 
decanted and rinsed several times with water, decanting magnetically 
between rinses. They were then rinsed three times with methanol, decanting 
magnetically between rinses, after which they were mixed with 
ethylbenzene. Comparison with untreated samples of encapsulated magnetite 
and unencapsulated magnetite particles that were washed in the same manner 
showed that only the Quilon treated encapsulated magnetite had much slower 
sedimentation rates. This indicates that the Quilon had reacted with the 
surface of these magnetic samples and had attached so that the surfaces 
were now hydrophobic. 
Quilon is a chrome complex sold by the DuPont Corporation in which myristic 
acid is coordinated with trivalent chromium. The commercial solution (in 
isopropanol) contains 5.7% (by weight) chromium and 11.7% fatty acid. The 
anion is chloride (7.8%). Quilon is generally used to impart water 
repellancy to paper and fabric. 
Other hydrophobizing agents such as alkyl titanites, silanes and borates 
could also be used. 
EXAMPLE 4 
A suspension of magnetite particles that had been encapsulated by gelatin 
gum arabic coacervate as described in Example 1 was prepared mixing 2.34 
grams of a concentrated dispersion (13% wt/vol, dry basis) of the 
particles with 25 ml. of distilled water. This was stirred for 3 minutes 
in a high speed (Virtis) mixer at 23,000 rpm for 3 minutes. Twenty-five 
ml. of a 2% solution of benzoquinone was added and the pH raised to 11.1 
with sodium hydroxide. This mixture was shaken for 18 hours, after which 
it was magnetically separated and washed three times with distilled water, 
followed by two washes with methanol. It was dried at 75.degree. C. 
Combustion analysis showed that the gelatin-gum arabic shell had reacted 
with the benzoquinone so that 14% of the modified shell was benzoquinone. 
EXAMPLE 5 
A suspension of encapsulated magnetite was prepared as described in Example 
1 and mixed with a solution of glutaraldehyde (1%). This was shaken for 
21/2 days after which it was magnetically separated and washed with water 
(3 cycles). The washed particles were mixed with a 2% gelatin (isoelectric 
point 8.3) solution and stirred at 40.degree. C. for 18 hours. It was then 
magnetically separated and washed 3 times with 35.degree. distilled water. 
This was followed by two methanol washes. Analysis showed that the 
gelatin-gum arabic shell had doubled in weight due to the coupling of 
gelatin in the solution with the glutaraldehyde activated particle 
surface. 
EXAMPLE 6 
A suspension of encapsulated magnetite was prepared as described in Example 
1 and mixed with a solution containing 2% gum arabic and 5% butanediol 
bis(glycidyl) ether. This was shaken for 6 days after which it was 
separated magnetically and washed with water. This was followed by a 
methanol wash. Analysis showed that the particles had reacted with a 
substantial amount of gum arabic through the epoxide coupling to the 
encapsulated magnetite particle. The shell had undergone a 50% increase in 
weight. 
EXAMPLE 7 
Two hundred ml. of a solution that is 1 molar in both ferrous and ferric 
sulfate were prepared and the resultant mixture made 1% in sodium dodecyl 
sulfate. The solution was purged of dissolved oxygen by passing nitrogen 
through for 30 minutes at 25.degree. C. To the rapidly stirred mixture, 40 
gms. of sodium hydroxide in 80 ml. water was rapidly added and the 
stirring continued under nitrogen for one hour. After this period of time, 
the reaction mixture was poured into an excess of water. The magnetite was 
separated magnetically and washed with distilled water until the pH of the 
wash water was below 11. The reaction mixture was identified as Fe.sub.3 
O.sub.4 by means of X-Ray diffraction. The magnetization and coercive 
force values were: 56.9 emu/g and 15.4 Oe respectively. A coercive force 
below 30 Oe is indicative of a superparamagnetic material. 
The wet magnetite paste was dispersed in 200 ml. of a gelatin gum arabic 
solution in which each of the gelatin and gum arabic was present in a 
concentration of 4% wt/vol. The gelatin had an isoelectric point of 
approximately 8.5. The mixture was agitated at 40.degree. C. and the pH 
lowered to 4.5 with 25% sulfuric acid. This mixture was then poured slowly 
into 2 liters of water that was agitated rapidly at 4.degree. C. After 30 
minutes, 4 ml. of 50% glutaraldehyde was added and the suspension of 
coacervated magnetite was stirred for a additional 30 minutes at 4.degree. 
C. The pH was then raised to 10 with sodium hydroxide and the solution 
allowed to come to room temperature. At this point, the coacervate shells 
were fully crosslinked, and the encapsulated magnetite was washed several 
times with distilled water at room temperature using magnetic separation 
between the washes. Electron microscopic examination 
showed the particles to be 100-150 .ANG. in diameter with a very minor 
40-50 .ANG. fraction. Elemental analysis was used to determine the 
gelatin-gum arabic content and estimates of shell thickness of 31 .ANG. 
were calculated assuming the magnetite particle diameter to be 100 .ANG.. 
EXAMPLE 8 
U.S. Pat. No. 4,582,622 describes the encapsulation of magnetic particles 
by a process which involves the deposition of a mixture consisting of 
gelatin, gum arabic, and sodium polymetaphos-phate. The procedure set 
forth in Example 1 of the patent was followed with the substitution of 
sodium dodecyl sulfate for the surfactants there used (alkylsulfomaleate, 
sodium oleate, and Demol Ep). 
Three variations with regard to the quantity of magnetite were made: (1) 
the concentration used in Example 1 of the patent, which is very low, (2) 
a 5 fold and (3) a 10 fold increase therefrom. 
All particles prepared showed very coarse aggregation with dimensions 
ranging from 1 to 10 microns. Separation using the steel wool funnel as 
described in Example 1 (of this application) was unsuccessful in that the 
particles could not be washed off the steel wool after the magnetic field 
had been removed. 
The invention has been described in detail with particular reference to 
preferred embodiments thereof, but it will be understood that variations 
and modifications can be effected within the spirit and scope of the 
invention.