Magnetic nanoparticles coupled to annexine, and utilization ther

Details Magnetic nanoparticles coupled to annexine, and utilization ther Magnetic nanoparticles coupled to annexine, and utilization ther

1998-06-11

2000-11-21

Chin, Christopher L.

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

Carrier is inorganic

436518, 436535, 436538, 436823, 422 50, 422 55, 422 57, 422 63, 422 681, 42218601, 422255, 422261, 435 4, 435 71, 428402, 428403, 427 211, 427 213, 427 214, 427216, 427331, 427337, 427338, G01N 33553, G01N 1506

Patent

active

061501816

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates to a new means making it possible to differentiate and/or separate components of a complex mixture, especially cells, still more particularly red corpuscles, and exhibiting at their surface a receptor specific for an effector coupled to magnetic particles. The surface complexing of the particles by dimercaptosuccinic acid (DMSA) of formula HOOC--CHSH--CHSH--COOH makes it possible to obtain a ferrofluid which is stable in aqueous medium. The invention also relates to an improvement to the process for preparation of these DMSA-treated ferrofluids, making the latter capable of being employed in a process of differentiation or of separation of cells or of molecules.
In what follows, the term "effector" or "ligand" means any molecule or macromolecule that is free or bonded to a structure which contains it or which carries it, and capable of directly or indirectly forming an affinity complex with another molecule or macromolecule.
Also in what follows the numbers within brackets refer to the literature references at the end of the description.


BACKGROUND OF THE INVENTION

Biomedical applications of colloidal solutions of magnetic particles, or ferrofluids, have developed essentially in three directions:
imaging (MRI) as contrast agents (1, 2, 3),
magnetic separation of various cells, organites or biological molecules (4, 5, 6, 7, 8, 9)
destruction of target cells by creation of a local hyperthermia under pulsating magnetic field (10).
The most extensively employed magnetic particles are the ferrites MFe.sub.2 O.sub.4 (including magnetite Fe.sub.3 O.sub.4) and maghemite .gamma. Fe.sub.2 O.sub.3. The surface of the particles must be conditioned in order to obtain colloidal solutions that are stable in physiological medium. In most cases the particles are coated with macromolecules such as carbohydrates like dextran (10, 12, 1), proteins like albumin (5, 8) or synthetic polymers like methacrylates and organosilanes (7, 9, 13). However, as Groman indicates, covering the particles with macromolecules of high molecular mass does not make it possible to obtain sols that are stable in the long term. The macromolecules separate from the particles, which then gradually aggregate. Other methods of conditioning the surface of the particles have been proposed, such as the use of molecules of low molecular mass, containing complexing groups such as phosphates, phosponates and carboxylates (2). Very particularly, hydroxylated polycarboxylic acids such as citric and tartaric acids and polycarboxylic acids containing thiol groups, like dimercaptosuccinic acid (DMSA) are complexed with the surface atoms of iron(III) (14). Each of these complexing molecules is bonded to one or more surface sites of the particles. The aqueous sols thus obtained are very stable in physiological conditions.
In many biomedical applications of the magnetic particles the latter must be coupled to a specific protein. Thus, in the case of cell sorting under magnetic field the particles must be capable of bonding specifically to the target cells. This recognition is often ensured by the formation of an antigen-antibody complex between a surface antigen of the target cell and an antibody bonded to the particles. The protein may be either adsorbed directly at the surface of the particles (4) or bonded covalently (5, 7, 13). The use of difunctional intermediate compounds such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (8, 14) makes it possible to bond an antibody strongly to a particle via a peptide bond and a disulfide bridge, without damage to the protein or to its antigenic properties (15).
French patent No. 2 662 539 (14) described a process for obtaining finely divided magnetic supports by controlled modification of particles filled with precursor ferrofluid, including a stage of treatment with an agent capable of modifying the nature of the surface of the said particles, and so as to make them stable in polar or nonpolar solvents in wide pH ranges and to endow them with a chemical or bi

REFERENCES:
patent: 4920061 (1990-04-01), Poynton et al.
patent: 5264157 (1993-11-01), Bidan et al.
patent: 5384073 (1995-01-01), Shigekawa et al.
patent: 5514340 (1996-05-01), Landsdorp et al.
patent: 5610274 (1997-03-01), Wong
patent: 5627036 (1997-05-01), Reutelingsperger
patent: 5817292 (1998-10-01), Snow et al.
Claude Sestier, et al., "Use of annexin V-ferrofluid to enumerate ertythrocytes damaged in various pathologies or during storage in vitro", Nov. 1, 1995, C.R. Acad. Sci. Paris, Sciences de la vie/Life sciences, 1995; vol. 318, No. 11, pp. 1141-1146.

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