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U.S. Patent Jun.5,1984 _ Sh_¢_et6of6 4,452,773
I I I I I I I I I I
1 MAGNETIC IRON-DEXTRAN MICROSPHERES i ‘The present invention relates to colloidal sized particles composed of a magnetic iron oxide core coated 5
with a water-soluble polysaacharide or a derivative thereof having pendant functional groups.
BACKGROUND OF THE INVENTION AND
_ DISCUSSION OF THE PRIOR ART 10
Development of procedures for generating monoclonal antibodies against specific cells is described for
.instance.in.K6hler,.G. and Milstein,._C. (1975) “Continu
ous cultures of fused cells secreting antibody of predefmed specificity”, Nature 256, 495-497. These proce- 15 dures have underlined the need to develop new and improved immunological labeling techniques to detect and separate specific cells. An approach initiated by the present inventor in collaboration with Dr. A. Rembaum and S.P.S. Yen and described in Molday, R.S., 'Yen, 20 S.P.S. and Rembaum, A. (1977) “Application of magnetic microspheres in labeling and separation of cells, “Nature 268, 437-438 involved the synthesis of magnetic microspheres by cobalt 'y-irradiation of iron oxide colloidal particles in the presence of hydrophilic and 25 hydrophobic methacrylate monomers. These microspheres were coupled to immunoglobulin and used to label and separate cells by magnetic means. These magnetic reagents, however, were limited in application due to difficulties in synthesis and purification of the micro- 30 spheres and, more important, susceptability to aggregation and nonspecific binding to certain types of cells. Kronick, P. L., Campbell, G., Joseph, K. (1978) “Magnetic microspheres prepared by redox polymerization used in a cell separation based on gangliosides”, Science 35 200, 1074-1076 prepared similar magnetic polymeric particles, but these also appeared under the electron microscope as "aggregated material on cell surfaces. Albumincoated microspheres have also been prepared for use as drug-carriers, Widder, K., Flouret, G. and 40 Senyei, A. (1979) Magnetic microspheres: “Synthesis of a novel parenteral drug carrier”, J. Pharm. Sci. 68, 79-82, but these reagents are relatively large in size, approximately 1 micron (104 A) in diameter and, therefore, are limited as general reagents for cell labeling. 45
U.S., Pat. No. 3,970,518 of Giaever relates to the magnetic separation of biological particles such as cells, bacteria or viruses and makes use of magnetic particles coated with a layer of antibodies to the particles to be separated. The antibody coated magnetic particles 50 contact a mixed population including the particles to be separated. The particles to be separated attach to the antibodies present on the magnetic particles, the magnetic particles are magnetically separated and the separated particles are subjected to a cleaving reaction to 55 separate the required biological particles from the antibody-coated magnetic particles. The magnetic particles used can be ferromagnetic, ferrimagnetic or superparamagnetic. Suitable magnetic materials include oxides such as, for example, ferrites, perovskites, chromites 60 and magnetoplumbites. The particles can range in size from colloidal to about 10 microns.
U.S. Pat. No. 4,018,886 of Giaever relates to a diagnostic method for determining the presence or absence of select proteins in low concentration in a liquid sam- 65 ple. A plurality of finely-divided magnetic particles, each of which is coated with a layer directly bonded thereto of first protein molecules specific to the select
protein, is dispersed in the liquid sample. The select protein, if present attaches to the protein bonded to the magnetic particles. The magnetic particles are magnetically retrieved, washed and then treated with a cleaving agent solution in direct contact with a metallized surface. The select protein, if present, detaches from the protein-coated magnetic particles and attaches to the metallized surface, which is examined for presence of the select protein. The magnetic particles which are said to be useful are those useful in U.S. Pat. No. 3,970,518 and the size range for the particles is again colloidal to about 10 microns. In the only example use is made of nickel particles about 1 micron (104 A) in diameter. Synthesis of these particles is difficult and the particles have been found to have a tendency to aggregation during protein coupling and cell labeling proce-’ ures.
, U.S. Pat. No. 4,230,685 of Senyei et al. is concemed with magnetic separation of cells and the like and with microspheres for use therein. It discusses the teaching of U.S. Pat. No. 3,970,518 and says that there is no literature verification that uncoated. magnetic particles can be made to bind effectively with antibodies. It refers to published procedures in which particles of magnetic material are contained in microspheresformed from polymers which can be coupled to antibodies. Mention is made of magnetically responsive microspheres formed from acrylate polymer, such as hydroxyethyl methacrylate, or polyacrylamide-agarose microspheres. Such microspheres can be chemically coupled to antibodies with glutaraldehyde or other di-aldehyde. One described procedure involves the chemical attachment of diaminoheptane spacer groups to the microspheres, which are then chemically linked to the antibodies by the glutaraldehyde reaction. Senyei et al state that although effective bonding of the antibodies can be obtained, such procedures are difficult since aggregation of microspheres can readily occur and the preparative procedure is time consuming. Further, random attachment of the antibodies to the magnetic particle means that that portion of the antibody which binds to the antigen, the Fab region, may not be available for binding. Senyei et al. propose to overcome these various disadvantages by using magnetically responsive microspheres having staphylococcal Protein A associated with the surfaces thereofclt is known that staphylococcal Protein A selectively binds to antibodies through the F c region of the antibodies which is remote from the Fab region. Consequently the antibodies are arranged in oriented attachment with the Fab arms of the antibodies extending outwards. To attach the staphylococcal Protein A to the magnetic microspheres use is made of a polymer matrix material which does not mask the antibody binding sites of Protein A. The preferred matrix material is albumin but other materials mentioned are other amino acid polymers and synthetic polymers such as acrylate polymers. Examples mentioned are methyl methacrylate, hydroxyethyl methacrylate, methacrylic acid, ethylene glycol dimethacrylate, agarose polymers, polyacrylamide polymers or mixtures of such polymers. Albumin is the only polymer matrix material whose use is demonstrated in a working example. According to column 4 lines 24 to 27, the microspheres of Senyei et al. range in size from 0.2 to 100 mi.crons (20()0 to 106 A) in diameter preferably from about 0.5 to 2.0 microns (5000 to 2 X 104 A).
SUMMARY OF THE INVENTION '
-The present invention relates to colloidal sized-particles of ferromagnetic iron oxide (Fe3O4) coated with a water-soluble polysaccharide or a» reactive derivative thereof ' having pendant functional groups, and to~.'a process for preparing such particles. The particles are prepared by mixing the water-soluble polysaccharide or a derivative thereof having pendant functional groups with an aqueous solution containing ferrous and ferric salts, adding alkali to the solution andseparating polysaccharide-_» or polysacchride-derivative coated ferro
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, The particles of the invention _.have various useful properties. They can readily mono-dispersed andare stable against aggregation and degradation under_physiological conditions. They can be frozen and thawed without suffering adverse effects. As stated,,_th_'_e'y.are colloidal in size and a diameter of abo'ut,°l00_i,to _70_0 K, more particularly about 300 to about 400 A is preferred, with an electron dense core of about 10 to 20 nm (1(_)0 to 200 .&)L'The particles have a ’magne__tic moment a_nd_are electron dense so they can be used as visual markers in scanning and transmission electron microscopy.‘ They _a'r'e'compatible with cells and other biological material and have functional reactive surface‘ groups, i.e.‘ the hydroxyl groups present in the saccharide moieties of the polysaccharide orfunctional groups derived from those hydroxyl groups. The particles are not themselves toxic and do not bond to cells nonspecifically. They can be bonded covalently to antibodies and to other biospecific molecules, for instance cells, enzymes, toxins, hormones, lectins, growth factors, nucleic acids, drugs and radioisotopes for use in a wide range of biomedical research studies and clinical procedures. In particular, they can be used to label specifically cells or. other biological material. The properties of the particles permit separation of labeled cells or antigens either by means of a magnetic field or by centrifugation. The particles can be bonded to cytotoxic agents or drugs to serve as site-specific carriers for the agents or drugs, targeted by means of a magnetic field. .. »
Thus, in one aspect the invention provides a method of labeling cells, enzymes, toxins, hormones, lectins, growth factors, nucleic acids or radioisotopes which comprises attaching to the cells, enzymes, toxins, hormones, lectins, growth factors, nucleic acids or radioisotopes colloidal sized particles of ferromagnetic iron oxide coated with a polysaccharide or a derivative thereof having pendant functional groups, the particles being attached to the cells, enzymes, toxins, hormones, lectins, growth factors, nucleic acids or radioisotopes via the functional group.
The particles of the invention have many applications in the field of medicine, as will be appreciated from the above. Their uses are not confined to the field of medicine, however. They can, for instance, be used in environmentalvresearch and operations. The particles can be coupled to proteolytic enzymes to be used to digest
iundesirable chemical agents. For example, cholera
toxin can be digested with the enzyme pronase attached t'o"par“ticles of the invention. The particles are small enough to remain in suspension due to Brownian movement, but can readily be recaptured and concentrated using magnetic fields. In another aspect, therefore, the invention provides a method of cleaning water contaminated with an undesirable chemical agent which comprises adding to the water colloidal sized particles of
The water-soluble polysaccharide has pendant functional groups in the form of hydroxyl groups. These hydroxyl groups can be oxidized to aldehyde groups, and a' preferred derivative of the polysaccharide is one containing aldehyde groups. It is not necessary that all the hydroxyl groups are oxidized to aldehyde groups. The aldehyde groups can be reacted with primary aini_ne,‘groups present on a molecule to form a Schiff base bonding the particle, to the molecule to which the ainine group is attached. To increase the stability of the bond it is preferred to subject the Schiff base to reduction. The preferred reducing agent is sodium borohydride but other reducing agents can of course be used.
‘The molecule bearing the primary amine group which reacts with the aldehyde group to form the Schiff base can be, for example, an antibody which is specific to a particular antigen. Particles of the invention _to which the antibody is covalently bonded can be introduced into a population of antigens including the particular antigen to which the antibody is specific. There..will be formed an antigen-antibody conjugate. ,Th_is conjugate is attached to the ferromagnetic particles and so can be separated from other antigens by magnetic means. Subsequently the antigen-antibody conjugate can be dissociated to release the required antigen. Methods of dissociating the conjugate include reaction with sodium thiocyanate or urea, acidification, for example with formic acid, and digestion with a .proteolytic enzyme such as trypsin. ,
The molecule bearing the primary amine group which reacts with the aldehyde to form the Schiff base can be an a,a)-alkylene diamine, for instance diamineethane or diaminoheptane. Reaction to form a Schiff base, _followed by reduction if required, results in a polysaccharide derivative having pendant famine groups. A wide variety of molecules including drugs, proteins, toxins, radio-isotope labeled compounds, fluorescent dyes, etc. can be bonded directly to amine groups or coupled to the amine groups using mild chemical agents and reactions, for example coupling with a dialdehyde such as glutaraldehyde. Further reaction with an a,w-dialdehyde, for instance glutaraldehyde, forms Schiff base between the amine group attached to the polysaccharide and an aldehyde group of the dialdehyde. This produces a polysaccharide derivative which again has pendant aldehyde functional groups and which can react, for instance, with an amine group present on an antibody as described above.
_ The amino-containing m0lecule- can be fluorescein isothiocyanate, which is a fiuorescentdye. .
The polysaccharide derivative can be one which contains cleavable bonds. Cleavage bonds can be introduced by reacting the polysaccharide with a bifunctional crosslinking agent, for example dimethyl 3,3’dithiobispropionimidate, prior to reacting the polysaccharide with the molecule to which it is to be attached. When it is desired to spearate the polysaccharide from the molecule the cleavable bond is cleaved with a suitable reagent. ln the case of dimethyl 3,3'-dithiobispro