Specific magnetosome, method for the production and use thereof

Details Specific magnetosome, method for the production and use thereof Specific magnetosome, method for the production and use thereof

1999-09-01

2001-06-26

Clark, Deborah J. R. (Department: 1633)

Drug, bio-affecting and body treating compositions

In vivo diagnosis or in vivo testing

Magnetic imaging agent

C424S009100, C424S009200, C424S009321, C424S009340, C424S130100, C424S450000

Reexamination Certificate

active

06251365

ABSTRACT:

The invention relates to specific magnetosomes with magnetic particles of maximally 43-45 nm, method for the production and use thereof. Furthermore, it relates to magnetoliposomes which can be obtained from the said magnetosomes by liposomal encapsulation.
Fields of application of the invention are medicine and pharmaceutical industry.
It is known that superparamagnetic iron particles are applied in medical diagnostics as NMR contrast agents or in the form of immunoconjugates or as synthetic drug carriers. Matsunaga et al. described in 1989 magnetosomes obtained from the magnetic bacterium Magnetospirillum spec. ABMI (JP7-241192-A) and their use. However, these magnetosomes have the disadvantage that they are comparatively large, thus bringing about the danger that they may cause embolisms in medical use.
That is why it was the task of the invention to provide specific magnetosomes which are smaller than those known, thus
improving their medical use as regards reaching the envisaged targets in the body of the patient and
at the same time diminishing the danger of embolisms.
It was detected that magnetosomes with magnetic particles <50 nm are contained in the bacterium
Magnetospirillum gryphiswaldense.
To our surprise, it was possible to produce these specific magnetosomes of the magnetic bacterium
Magnetospirillum gryphiswaldense
on a semi-industrial scale.
Accordingly, the object of the invention are the magnetosomes themselves, the method of production/preparation and use thereof, preferably in medicine and pharmacy.
The invention is implemented according to the claims.
According to the invention magnetosomes are characterized by a magnetic oxide magnetite Fe
3
O
4
monocrystal with a maximum diameter of 43-45 nm surrounded by a phospholipid membrane. As a rule, they have a cubooctahedral shape.
The membrane consists preferably of phosphatidyl ethanolamine, phosphatidyl glycerol and phosphatidyl choline containing mainly the fatty acids palmitic acid, palmitoleic acid and oleic acid. The membrane consists preferably of 53±6% phosphatidyl ethanolamine, 38±6% phosphatidyl glycerol and 8.9±5% phosphatidyl choline where mainly the fatty acids palmitic acid (approx. 18.4%), palmitoleic acid (approx. 25.6%) and oleic acid (approx. 45.9%) are to be found.
A preferred variant of execution contains the magnetosomes as chains up to 100, preferably 10-60 magnetosomes and with a cationic surface charge. This chain form of magnetosomes increases the probability that antibodies and therapeutic agents may be correctly bound to them and become effective.
In addition, magnetosomes are also magnetosomes with additionally covalently bound antibodies or therapeutic agents bound to the magnetosome membrane through respective reactive groups.
Apart from that, the invention relates to a method for the production of these new magnetosomes. They are isolated from the magnetic bacterium
Magnetospirillum gryphiswaldense
according to a new fermentation method. For this purpose a new simple culture medium consisting of 0.3 g of KH
2
PO
4
, 1 g of Na acetate, 1 g of soybean peptone (Merck), 0.1 g of NH
4
Cl, 0.1 g of yeast extract, pH 6.9 which does not contain a complexing agent for iron is preferably used. The concentration of oxygen in the medium is maintained below 2%, later Na acetate and FeSO
4
are added. After approx. 30 hours the magnetic cells may be gathered. After subjecting the cells to a lysis the magnetosomes are obtained in a high output according to a new method by separating them from cell fragments and cell sap in a magnetic separation column by means of a strong, powerful permanent magnet (Sm-Neodyn) and purifying them by washing.
Furthermore, magnetosomes according to the invention which are available packed in liposomes, forming themselves liposomes with other lipids or bound to the surface of liposomes are object of the invention. Such liposomes are
so-called classical liposomes (MLV, SUV, LUV)
so-called “stealth” liposomes (PEG)
micellar systems (e.g. SDS, triton, sodium cholate)
immunoliposomes containing e.g. antibodies or fab fragments against antigenes associated with diseases or adhesion molecules bound to the surface of the liposomes
so-called cationic liposomes (DAC-Chol, DOCSPER)
so-called fusogenic liposomes (reconstituted fusion proteins in liposomes).
Magnetoliposomes are prepared according to liposome preparation methods known per se, e.g. described in DE 41 34 158, DE 44 30 593, DE 44 46 937 and DE 196 31 189 with the magnetosomes being preferably added to the initial lipids.
The preferred modifications of magnetoliposomes and magnetosomes belonging to the invention are represented in Table 1 hereinafter.
TABLE 1
Magnetoliposomes
cationic
fusiogenic
classical
“stealth”
immuno
e.g.
e.g.
Magnetosomes
MLV
PEG
anti-CEA
DAC Chol/DOPE
HN, F protein
Immuno
Gener, or antisense oligonucleotide
SUV
anti Thy1.1
SP Chol/DOPE
(Sendai virus)
anti CEA
or ribozyme modified
LUV
anti CD44
DAC-Quat.
−> [pH 7]
anti CD44
(REV)
anti CD54
Chol/DOPE
synthetic
anti CD 54, CD56
anti CD56
DECSPER
fusion
anti CD30
anti CD30
proteins
anti CD31
HA influenza
virus [pH 5,2]
“cochelates”
The magnetosomes and magnetoliposomes according to the invention may contain specific antibodies additionally chemically coupled to their surfaces, one or a few therapeutic agents and radionuclides enclosed, i.e. encapsulated.
In addition, they, together with genetic material such e.g. plasmids, therapy genes, antisense oligonucleotides, ribozymes or gene diagnostic agents, may form cationic complexes suited for the transfer of genes.
These magnetosomes and magnetoliposomes according to the invention have a comprehensive spectrum of application. Owing to their magnetic properties they are per se (also unmodified) used as contrast agents for NMR examinations and as markers for mapping magnetic susceptibilities by means of a SQUID biomagnet meter and also as diagnostic agents for the detection of various diseases and focuses of inflammation or therapeutic agents as e. g. for purging (“taking out diseased cells”), as diagnostic agents for tumoral diseases or in lymphography, for inflammatory processes, for multiple sclerosis, Alzheimer disease and for Parkinson's disease or as a therapeutic agent against tumoral diseases, inflammatory processes and metabolic diseases.
Diagnostic agents are used preferably in the form of immunomagnetosomes or immunomagnetoliposomes. For this, antibodies or fab fragments against antigenes associated with diseases or adhesion molecules or ligands are covalently coupled to the magnetosome and magnetoliposome membrane through respective groups, preferably to phosphatidyl ethanolamine contained in the membrane through spacers of a differing length.
In particular, they are used as diagnostic agents for the detection of tumoral diseases or in lymphography, with among others anti CEA, anti CD44 being coupled to the magnetosome membrane or magnetoliposome membrane as a reagent. These antibody coupling products are also suited for detecting inflammatory processes such as arthroses (preferably with anti CD54, anti CD56) or for detecting multiple sclerosis or Alzheimer disease (preferably anti-&bgr;-amyloid, anti APOE4), Hogkin lymphoma cells (preferably with anti CD30) and Parkinson's disease.
The magnetosomes according to the invention are excellently suited for the so-called diagnostic applications.
It is necessary to use magnetoliposomes to simultaneously bring a therapeutic substance in relevant quantities to the target place. They are not only suited for coupling but also for enclosing therapeutic agents. In the case of magnetosomes therapeutic agents may be coupled only with a spacer being interconnected.
According to the invention an essential possibility of use is that therapeutic agents are coupled (magnetosomes) or coupled or enclosed (magnetoliposomes). These therapeutic agents may be enclosed in the membrane or in the aqueous interior of the liposomes depending on lipophilicity or hydrophilicity.
Thus, the following

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