Tetraether lipid derivatives and liposomes and lipid...

Details Tetraether lipid derivatives and liposomes and lipid... Tetraether lipid derivatives and liposomes and lipid...

1999-04-19

2001-11-13

Nguyen, Dave Trong (Department: 1633)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C424S450000, C435S320100, C435S069100

Reexamination Certificate

active

06316260

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to tetraether lipid derivatives, to liposomes and lipid agglomerates containing the inventive tetraether lipid derivatives, and to the use thereof.
Scientific research work requires a great number of methods for the transfection of cells in cell culture and multicellular organisms with nucleic acids. Conventional methods, such as electroporation, DEAE and “calcium phosphate”-supported transfection, microinjection or ballistic methods, have the drawback that the transfection efficiencies achieved by them are often poor, that the cell survival rates are very small and/or that they cannot be performed on multicellular organisms. Although viral and retroviral transfection systems are more efficient, they have risks of their own, such as an increased immune response or an uncontrolled integration into the target genome. Therefore, transfection of non-viral nucleic acids with the aid of liposomes, also called lipofection, is a successful and frequently employed alternative to the above-described methods.
Liposomes are artificially produced unilamellar or multilamellar lipid vesicles which enclose an aqueous interior. They are in general similar to biological membranes and, therefore, they are often easily integrated into the membrane structure after attachment to the membranes. During this membrane fusion the contents of the liposome interior is discharged into the lumen which is enclosed by the biological membrane. Alternatively, the liposomes are moved by endocytotic processes into the cytosol of the cell to be transfected; subsequently they are either destroyed in the cytosol or they interact as such with the nuclear membrane. In the last-mentioned case the compounds contained in the aqueous interior of the liposome are substantially protected against proteolytic or nucleolytic attacks.
Therefore, liposomes can be used as transport vehicles for substances, such as nucleic acids or pharmaceuticals. For instance, the cosmetic industry produces liposome-containing creams for skin care which transport active agents in a target-directed manner into the epidermis or lower cell layers. Natural lecithins of soybean or egg yolk and defined natural or artificial phospholipids, such as cardiolipin, sphingomyelin, lysolecithin, and others, are mainly used for the preparation of liposomes. Size, stability and absorbency as well as the release of the associated molecules are influenced by varying the polar head groups (choline, ethanolamine, serine, glycerine, inosite), the length as well as the saturation degrees of the hycrocarbon atom chains.
One of the essential drawbacks of the liposomes thus far known is their low stability. Even in a cooled state liposomes which are formed from normal bilayer-forming phospholipids are only durable for a short period of time. Although their storage stability can be increased e.g. by the inclusion of phosphatidic acid, the stability improved thereby is still inadequate for many purposes. Moreover, conventional liposomes are not acid-stable and are thus neither suited for the transport of pharmaceutical active agents which pass through the stomach after oral administration, nor for the liposome-supported DNA transfection under slightly acid pH conditions.
Liposome-forming lipid mixtures, such as Lipofectamin®, Lipofectin® or DOTAP®, are often used in mammalian cells for scientific or medicinal lipofections. Apart from the already mentioned drawbacks, their use requires an exact determination of a multitude of parameters (such as cell density, amount of nucleic acid, amount of the lipids, volume of the liposome batch, etc.) because there is only a very limited range of optimum parameters within which an adequate transfection efficiency can be achieved. As a result, transfections using commercial lipofection reagents become very troublesome and expensive. Furthermore, great variations between the individual charges can be observed in the above-mentioned products, which makes them hardly reliable in practice.
SUMMARY OF THE INVENTION
Therefore, it is the object of the present invention to prepare lipofection agents which exhibit an enhanced mechanical and chemical stability and thus a prolonged storage stability and which permit a simple and reliable use.
According to the invention this object is achieved by providing tetraether lipid derivatives (also abbreviated hereinafter as “TEL derivatives”) represented by the general formula (I):
wherein S
1
and S
2
may be the same or different and respectively have the following meaning:
and
Y may be —NR
2
R
3
or —N
⊕
R
4
R
5
R
6
;
X
1
and X
2
may be the same or different and are respectively selected independently of each other from the group comprising a branched or unbranched alkylene or alkenylene having 2 to 20 carbon atoms;
R
1
to R
6
may be the same or different and are respectively selected independently of each other from the group comprising hydrogen, branched or unbranched alkyl, alkenyl, aralkyl or aryl groups having 1 to 12 carbon atoms, wherein a respective one of the moieties R
2
to R
6
may further comprise an antibody against cell surface molecules or a ligand for cell surface receptors; and
n may be an integer between 0 and 10,
and modifications thereof formed by the formation of pentacycles in the basic tetraether skeleton.
The lipid skeleton of the tetraether lipid derivatives according to the invention consists of a 72-membered macrotetraether cycle, the basic skeleton of which is a dibiphytanyl-diethyl-tetraether heterocycle. In this skeleton the &ohgr;-carbon atoms of the phytanyl chains of two respective diether molecules are covalently linked to each other. Tetraether lipids are already known and have so far exclusively been detected in archaebacteria. In response, for instance, to the cultivation temperature, pentacycles can be formed within the dibiphytanyl chains, the pentacycles giving the lipid a specific physicochemical character. With each pentacyclization the basic skeleton loses two hydrogen atoms. A summary of all of the basic structures of archaebacterial lipids so far known can be found in Langworthy and Pond (System Appl. Microbiol. 7, 253-257, 1986).
According to the invention the naturally occurring lipid skeleton has now been derivatized to be suited for incorporation into liposomes or lipid agglomerates provided for transfection. To this end side chains are introduced that are positively charged either per se through the formation of quatemary ammonium salts or under physiological conditions, i.e. at a pH of 7.35 to 7.45. Lipids derivatized in this manner are particularly well suited for contacting negatively charged molecules, e.g. nucleic acid molecules, and for enclosing the same, for instance in liposomes. Since a possible intended use of the lipids according to the invention may be the formation of liposomes or lipid agglomerates for genetic therapeutic applications, the lipids according to the invention can additionally be coupled with molecules that enable the lipids to specifically dock onto special cells. Examples thereof are antibodies against cell surface antigens, in particular those that are selectively expressed on the target cells. Suited are also ligands for receptors which are selectively found on the surface of specific cells, as well as biologically active peptides which permit an organ- or cell-specific targeting in vivo (Ruoslati, Science, 1997). The latter have also been designated as ligands for the purposes of the present application.
The special advantage of the tetraether lipid derivatives according to the invention is that their lipid skeleton is devoid of any double bonds and therefore insensitive to oxidation. Furthermore, instead of the lipid ester bonds contained in lipids consisting of eubacteria and eukaryotes, it only contains lipid ether bonds which are not attacked—even at high proton concentrations as are e.g. found in the stomach.
In preferred embodiments the substituents S
1
and S
2
are the same at both ends of the basic tetraether lipid skeleton. Starting from n

Affiliated with

Also associated with

Rating

Tetraether lipid derivatives and liposomes and lipid... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Tetraether lipid derivatives and liposomes and lipid..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Tetraether lipid derivatives and liposomes and lipid... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2582400