DE19740092A1 - New liposome comprises lipid envelope based on natural or synthetic skin - Google Patents

Abstract

Liposomes are enveloped with a lipid phase which have a chemical composition corresponding to a skin layer or a specific mixture of phospholipids, cholesterol sulfate, free fatty acids, ceramides, sterols, triacylglycerols and sterol esters. Liposomes comprise an intraliposomal phase containing at least one nucleic acid, protein, carbohydrate, lipid and/or lower molecular weight compound enveloped with a lipid phase which has a chemical composition corresponding to a skin layer or which is a mixture (wt.%) of phospholipids (less than 2), cholesterol sulfate (2), free fatty acids (20), ceramides (20), sterols (43), triacylglycerols (neutral fats) (4) and sterol esters (9). Independent claims are also included for: (1) a process for the preparation of the liposomes by producing stratum corneum lipid and preparing the liposomes from this by ultrasonic treatment and/or detergent dialysis; (2) a pharmaceutical composition containing the liposomes and formulated for topical administration, optionally comprising a permeability enhancer and swelling agent; (3) a method for the transfection of cells using the liposomes by contacting the cells with liposomes (I); and (4) an inoculant containing the liposomes with an intraliposomal phase containing nucleic acids having genetic information for an antigen which produces an immune response or an antibody or antibody fraction specific for this antigen.

Description

The present invention relates to liposomes comprehensively a lipid phase forming a liposome shell and a intraliposomal phase enclosed by the lipid phase Process for their production and those that can be produced therefrom Liposomes, methods of transfecting cells, agents for topical administration and uses of the Liposo men and means.

With the increasing understanding of biological relationships at the molecular level, the most recent Ver past the molecular medicine, which the development new therapy concepts and drugs flows. This also applies to the skin, whose function goes far goes beyond the obvious barrier function and their diseases to a very special degree in many cases len are literally obvious.

One way of treating skin disorders is used in the insertion and expression of genes in the Epi dermis seen, d. H. the gene therapy of the epidermis forming cells. The are particularly important here Keratinocytes.

Various techniques are known in the art, Kera transfect tinocytes in situ. A possibility represents the intradermal injection, with it for the local Transfection, d. H. Expression of the introduced geneti material in a very narrowly delimited dermal Area is coming. Typically the expression is transient for 2 to 7 days and there is no integration of the ap plicated genetic material into the genome of the kerati nocytes. A major disadvantage of this technique be says that a corresponding transfection on the immediate injection area is limited and it is it is an invasive procedure that only qua qualified personnel under suitable hygienic conditions can be used.

Also be on the local application of genetic material the use of microprojectiles is restricted microscopic gold particles coated with DNA hen and using a so-called gene cannon directly into the Cells are introduced. This procedure is also with connected to a number of disadvantages, one of which is the use of the comparatively expensive gold particles as well as the required coating step, on the other hand, however, also in the use of the gene gun exist that have a not inconsiderable amount of equipment wall represents to be also qualified personnel ner operation and, moreover, not the desire success delivers.

Finally, topical procedures are still in the technology known in which the existing DNA is complexed directly in the cells that make up the epidermis are introduced target. For example, cationic lipids that to attach to the anionic DNA, as well as used receptor-mediated endocytosis. The first stayed said approach unsuccessful in practical application. The second approach, for example in the form of Transferri nofection, is due to the immunogenic components (es dissociation proteins from adenovirus or the hä magglutinating virus used by Japan) immunogenic and can therefore only be applied successfully a few times.

Finally, there are still those from cosmetics in technology well-known liposomes described in the aqueous gen inner phase contain the plasmid DNA. This too The rate only resulted in a small percentage of cells for successful transfection.

The present invention is based on the object Provide liposomes that have a high transfection rate of skin cells, and in particular keratinocytes.

Another object of the invention is to provide of methods of making such liposomes.

Finally, the present invention has the object based on a method for transfecting cells, and in particular keratinocytes, wel ches allows a high transfection rate.

The invention is also based on the object of a phar to provide a pharmaceutical composition containing the ge compared to the prior art improved application of Active ingredients, especially topical application of the same ben, allowed.

A further object of the invention is to provide means for topical administration of chemical compounds be to mount.

Furthermore, agents for gene therapy are intended according to the invention of skin cells, for the prophylaxis and / or treatment of Skin disorders, including warts, from sy stemic diseases and alopecia as well as means for Immunization will be disclosed.

According to the invention, the object is achieved by liposomes comprising a lipid phase forming a liposome shell and one intraliposomal enclosed in the lipid phase Phase, being the chemical composition of the lipid phase which corresponds to a layer of skin and the intraliposomal Phase contains at least one compound that is selected is from the group that includes nucleic acids, proteins, carbohydrates drate, lipids and low molecular weight compounds as well this includes composite molecules or structures.

The object is also achieved by liposomes comprising a lipid phase forming a liposome shell and an intraliposomal phase enclosed by the lipid phase, the liposome comprising the following components with regard to the lipid classes:

Phospholipids <2% by weight Cholesterol sulfate 2% by weight Free fatty acids 20% by weight Ceramides 20% by weight Sterols 43% by weight Triacylglycerols (neutral fats) 4% by weight Sterol esters 9% by weight

and the intraliposomal phase at least one compound contains, which is selected from the group that includes nuclein acids, proteins, carbohydrates, lipids and low molecular weight clear compounds as well as moles composed of these includes kules or structures.

Furthermore, the object is achieved according to the invention by a method wherein it is provided that a) Stratum cor neum lipid is obtained and b) from the stratum corneum Lipid under ultrasound treatment and / or detergent dialy se liposomes are produced.

The object is also achieved by liposomes which can be produced by the process according to the invention.

Furthermore, the object is according to the invention by a Method for the transfection of cells solved, the to transfecting cells with the liposo according to the invention men are brought into contact.

According to the invention, the object is also achieved by a pharma Zeutische composition dissolved, which the invention ole liposomes and a pharmaceutically acceptable carrier includes.

Furthermore, the object is according to the invention by a Means for topical administration of chemical compounds dissolved, the agent having a content of the invention Has liposomes.

Finally, the object is achieved by using the liposomes according to the invention, the pharma according to the invention zeutischen composition or the inventive Mit for gene therapy of skin cells.

Furthermore, the object is achieved by using the liposomes according to the invention, the pharma according to the invention zeutischen composition or the inventive Mit means for the prophylaxis and / or treatment of diseases the skin, including the treatment of warts, syste matic diseases and alopecia as well as use of the liposomes according to the invention, the according to the invention pharmaceutical composition or of the invention ßen agent for immunizing a host organism.

The problem is also solved by a vaccine that has a content of the liposomes according to the invention, where the liposomes are nucleic acids that carry the genetic In comprises formation for an antigen of interest, or Nucleic acids that contain the genetic information for comprises an antibody or an antibody fragment, wherein the antibody or the antibody fragment specific for is the antigen of interest.

In which a lipid phase that forms a liposome shell and one intraliposomal enclosed in the lipid phase Phase comprehensive liposome in which the chemical compound setting of the lipid phase that corresponds to a layer of skin, it can be provided that the skin layer is human epidermis is.

In a further embodiment it can be provided that the skin layer is the stratum corneum.

In the method according to the invention for the production of Liposomes can be provided that Stratum corneum-Li pid is obtained by enzymatic and / or stratum corneum mechanically removed from the skin and attached is then extracted into methanol / chloroform.

In one embodiment of the liposome according to the invention can be provided that the intraliposomal phase a aqueous phase is.

Furthermore, it can be provided that the intraliposomal Phase contains at least one chemical compound that is selected from the group consisting of nucleic acids, proteins ne, carbohydrates, lipids and low molecular weight compounds genes and molecules or structures composed of these doors included.

In addition, it can be provided that the connection is selected from the group, the growth factors, An tibiotics, pain reliever agents, agents containing the Affect cell division and / or vascularization, coagulating and anti-coagulating agents, cytokines, chemical attractant, enzymes, and combinations thereof grasps.

In a very particularly preferred embodiment is the chemical compound is a nucleic acid sequence.

In one embodiment of the method according to the invention for the transfection of cells can be provided that the cells to be transfected are skin cells.

In a preferred embodiment, the skin cells are selected from the group comprising the skin cells of the stratum cor neum, stratum granulosum and stratum spinosum as well as kera includes tinocytes.

In a very particularly preferred embodiment is provided that the cells in the method according to the invention for transfection are keratinocytes of the hair follicles.

In another, very particularly preferred embodiment are the cells of the method according to the invention for Tr Ansfection of epidermal Langerhans cells.

In the agent according to the invention for topical administration Chung chemical compounds can be provided that the agent is selected from the group consisting of creams, sal ben, gels, spray plasters and tinctures.

In a further preferred embodiment of the inven the means according to the invention is provided that the means white thereafter a content of at least one compound has, which is selected from the group that permeabili includes strength enhancers and swelling agents.

In the use according to the invention of the herein apparently th liposomes, the pharmaceutical composition or of the agent for gene therapy of skin cells can be provided be that the skin cells are in situ.

In an alternative embodiment of the above inven Proper use can be provided that the Skin cells are available ex vivo.

Furthermore, it can be provided in the aforementioned use be that the skin cells are selected from the group the skin cells of the stratum corneum, stratum granulosum and Includes stratum spinosum and keratinocytes.

In a further embodiment it is provided that the Skin cells are keratinocytes of the hair follicles.

Furthermore, in one embodiment of the above inven proper use be provided that the skin cells len are epidermal Langerhans cells.

In the use according to the invention of the herein apparently th liposomes, the pharmaceutical disclosed herein Composition or the agent disclosed herein for Prophylaxis and / or treatment of skin disorders is provided in a preferred embodiment that the disease is selected from the group that geneti cal skin diseases, psoriasis, skin tumors and wounds includes.

In a particularly preferred embodiment, it is provided see the genetic skin disease is selected from the group that epidermolysis bullosa simplex, Epider molysis hyperkeratosis, palmoplantar keratosis, junction nale epidermolysis bullosa, dystrophic epidermolysis bullo see also includes lamellar ichthyosis and xeroderma pigmentosum.

In the use according to the invention of the herein apparently th liposomes, the pharmaceutical disclosed herein Composition or the agent disclosed herein for Treatment of systemic diseases is special ders preferred embodiment provided that the sy stemic disease is selected from the group that Hemophilia A and hemophilia B and collagen degeneration includes.

In the use according to the invention of the herein apparently th liposomes, the pharmaceutical disclosed herein Composition or the agent disclosed herein for Immunization of a host organism is special in one preferred embodiment provided that the host organ nism is a human organism.

The present invention is surprising Er knowledge based that with the liposomes according to the invention a particularly high rate of skin cell transfection, and especially of keratinocytes. The This effect appears due to the high degree of hydrophobicity the liposome shell, d. H. their lipid composition, be thing to be. DNA contained in the liposomes of the invention is encapsulated, the epidermal barrier of the Overcome skin and in the deeper layers of the epider mis absorbed and expressed.

The previously described passage ability of the invent liposomes according to the invention also allows one Release of compounds contained in the liposomes or drugs, including nucleic acids, in depth ren layers of the epidermis or the skin, which is both un pharmaceutically effective from the aspect of administration Connections as well as under the aspect of cosmetic Treating the skin is beneficial.

It is particularly noteworthy that when using of the liposomes according to the invention or the one disclosed herein th means and methods the nucleic acid application is repeatable several times and there is no occurrence of one Immune response, especially one against nakedness Small acid-directed comes up against what other trans fection techniques is a significant advantage.

Due to the high transfection rate, it is possible for the first time Lich, easily and reliably many skin cells to trans fify and thus lay the foundation for the Gene therapy benefits inherent to the skin actually too realize.

These advantages are, among other things, that, besides a usually causal therapy that the treated Tissue containing cells in the event of undesired side effects This can be easily removed and continues as a result the fact that an ectopic expression of the means of the nucleic acid introduced into liposomes according to the invention can be excluded the therapy as such any time can be interrupted.

In addition, the keratinocytes in particular are ver equally easy to gain as well as to expand in culture date and in technology there are also processes and co tel known to modulate the expression of a in Kerati nucleic acid sequence present in nocytes.

The intraliposomal phase can be an aqueous phase, it can be provided that this is a special Mi lieu, shaped by the addition of certain compounds, having. The aqueous phase can for example be one distinct pH, a specific buffer, or ions have concentrations that the stability, conforma tion and biological effectiveness of the ent hold - chemical - compound (s) influenced and thus for the Case that in the intraliposomal phase a transfect tion suitable nucleic acid construct is present the transfection rate with the lipo according to the invention so treated cells act. The design of a appropriate milieus in order to achieve the mentioned effects chen are known to those skilled in the art.

In the chemi contained in the intraliposomal phase connections are those where a chemical bond between at least two atoms exists and accordingly also include biochemical or biological compounds.

In this context, low molecular weight compounds are intended to include those be understood having a molecular weight of less than 10,000 and especially those that contain coals fabric included. Such connections should also Car carbohydrate acids, amino acids, carbohydrate monomers, lipids Finally, steroids, lactams, lactones and others conclude. Proteins are also intended to include peptides and generally polymers of at least two peptidically included other connected amino acids are understood.

In this context, nucleic acid or nucleic acid sequence is intended to mean both DNA and RNA can be understood. The nucleic acid re (sequence) can be both naked and complex / asso present gracefully. It is also possible that the Nu small acid (sequence) as a single strand, double strand or exists as a triplet. The nucleic acid sequence can also be a ribozyme or an antisense nucleic acid. Therefore Furthermore, it can be naturally occurring as well recombinant. The nucleic acid (sequence) can go further Elements include both their transcription and Affect translation and its stability. So she can for example promoters, cell-specific as well as in Ducible, terminators and more extensive regulatory Include elements such as enhancers. In the end the nucleic acid (sequence) can also comprise elements, which the insertion of the nucleic acid (sequence) into the Chro mosome allow a transfected cell, or else Elements to prevent insertion of the Nucleic acid (sequence) enters the cellular genome. The latter can, for example, be particularly desirable be if only a transient expression of the trans Fified nucleic acid (sequence) is desired.

In principle, the nucleic acid (sequence) can be designed in this way exist that both at the RNA level and the Translation and transfection level the desired we kung is achieved. The for this purpose in the nucleic acid (-Se quenz) measures to be taken or the design a milieu within which the nucleic acid (sequence) has the corresponding properties or effects, such as a special configuration like the Z configuration are known to those skilled in the art.

Nucleic acid is also used herein to refer to oligonucleotides ver including antisense oligonucleotides to control the transcription and / or translation of nucleic acid sequences present in cells.

The transfection of cells using liposomes is the Known to those skilled in the art.

In the method according to the invention for the transfection of Cells is the use of the liposomes of the invention both advantageous in the event that the cells, out selects from the group, the skin cells of the stratum corneum, Stratum granulosum and stratum spinosum as well as keratinocytes th includes, or keratinocytes of the hair follicles or epi dermal Langerhans cells, in vivo as well as for the case that the aforementioned cells are transfected ex vivo.

From the above it follows that the fiction According to liposomes inherent advantages also and especially can then be realized if this is in a pharma chemical composition are included.

It has surprisingly been found that using the Liposomes according to the invention are generally pharmaceutical Compositions and in particular means for topical Administration of chemical compounds, which is particularly effective in releasing the in the chemical compound contained in the intraliposomal phase dung into the cell or the deeper layers of the epi dermis and dermis. In another In addition, a special aspect is also possible Form of depot administration contained in the liposomes to realize chemical compounds.

Under pharmaceutically acceptable carriers are, under at derem, water, buffer solutions and the like. In particular, this also includes those skilled in the art herein known carriers used in the manufacture of topical Application forms such as creams, ointments, gels, spray plasters and tinctures can be used.

Chemical compounds are intended to include those defined hereinabove ned connections are understood and include ins special those there, although not conclusive, be inscribed nucleic acids.

The means disclosed herein ultimately implement Approximate forms of the pharmaceutical composition according to the invention composition.

Both the pharmaceutical compositions described herein as well as the means for topical administration can also ent at least one active ingredient keep that is not in the liposomes it contains lies.

The agent for topical administration itself can thereby be designed as a cream, ointment, gel, spray plaster or tincture. These funds allow the inclusion of the he liposomes according to the invention in a matrix, which then, typi shearly on the skin, and - if necessary after an exposure time - the contact between cell and liposome enables. By distributing the liposomes in said Matrix can thus - statistically - also be used to transfek regulate the frequency of a single cell.

The above-mentioned topical administration forms allow in addition, that a comparatively large area of the Skin is treated accordingly, d. H. if in the intra liposomal phase nucleic acid sequences are present, a large area of skin cells can be transfected. Transfection, especially in its clinical application dung, is then particularly easy to carry out without specially qualified or special staff for this purpose hygienic conditions would be required.

The agents according to the invention can with regard to their Effectiveness, manageability and stability through modifications tion of the compounds forming the matrix of the same in manner known to those skilled in the art be formed. This includes the addition of further, for the formation of creams, ointments, gels, spray plasters and tinctures.

Both the liposomes as such and those they contain Tending agents according to the invention are particularly advantageous in gene therapy of skin cells due to the through them achievable transfection rates to be used. It is there possible that the corresponding skin cells, and in particular whose human keratinocytes, including those the hair follicle, as well as the epidermal Langerhans cells, in situ, d. H. as part of the patient's skin surface, exist, but ex vivo gene therapy is also possible lich if the skin cells or skin tissue, and here too again specifically the human keratinocytes, including ch those of the hair follicles, as well as the epidermal lan gerhans cells, in culture. It’s obvious Lich that in the in situ situation the invention Means containing nucleic acid in the intralipo somal phase associated with special benefits is where on the other hand, in the case of ex vivo gene therapy, in particular the Liposomes of the invention have the advantages disclosed herein le have a special degree.

In particular in the case of transient gene expression by means of the liposomes according to the invention introduced into the skin cells th nucleic acid (sequences) is a simple repetition development of the therapy as well as easy control of the therapy catch with a corresponding broad topical Application of the agent according to the invention possible without any problems lich. Especially under the aspect of repeated Be action is remarkable that against such into the biological system, for example the human Skin, nucleic acid introduced does not judge against this killed immune reaction is determined.

When using the liposomes according to the invention, he agent according to the invention or the pharma according to the invention chemical composition for gene therapy of skin cells, and specifically from keratinocytes, including those the hair follicle, as well as Langerhans cells, is included basically possible through the nucleus that have been smuggled in acid sequences indicate a defect in the transfected cell to achieve a complementary effect, or the un desired expression of a gene present in the cell table information by means of the introduced by the liposomes led to suppress nucleic acid sequence.

The latter possibility also applies in the event that the liposomes according to the invention or the liposomes according to the invention Agent in the treatment of systemic diseases ver is turned. It comes about as a result of the liposomes elt transfection of the skin cells for the production of the through encoded the nucleic sequence introduced into the cell Gene product, which in turn becomes its biological we can unfold. It is possible that the ent speaking gene product from the avascular epidermis to the Released into the bloodstream and thus available systemically stands. Compared to the administration of appropriate proteins or peptides allows gene therapy of skin cells that the corresponding information transiently, typically over a range of 2 to 7 days, continuously in high local tissue concentration is expressed and ge thus guarantees against the administration of the peptide or protein as such a longer lasting kon constant titer, since the protein as such is only low has biological half-life.

In addition to treating real systemic diseases like For example, Hemophilia A and Hemophilia B are also Collagen degenerations using the erfindungsge moderate liposomes or the agent according to the invention, optionally cosmetic, treatable.

Collagen degenerations are also intended herein to include light matoses are understood in which there is UV exposure tion to the degeneration of collagen (and elastin) and thus wrinkles occur. Here the open here allows Barte gene therapy of skin cells or the invention ßen liposomes or the agent according to the invention are advantageous te effects, since due to the transient nature of the Gene therapy of the keratinocytes a reversibility of the before measures taken is given. Which result from it the advantages, especially in cosmetics, are open visibly. For example, a new collagen and Elastin production by the gene-treated skin cells conceivable, with the result that this for the skin, in particular whose appearance, so important structural substances endogenous can be formed.

When using the liposomes according to the invention for Treatment of warts lies in turn in the principle gene therapeutic treatment of the wart forming Skin cells, which typically infect with viral DNA are adorned. Accordingly, the liposomes can aforementioned cells are transfected and on molekula On this level, the event leading to wart formation is the last finally be suppressed. Has particularly proven itself here the interferon-α plasmid expression in warts that due to high intralesional levels of interferon-α protein leads to the complete regression of the papilloma.

Treatment of alopecia using the inven liposomes according to the invention are also based on the principle de that ultimately through gene therapy change the genotype and phenotype of the hair follicle cells or the corresponding keratinocytes the clinical picture represents, but can at least be reduced. Correspond corresponding expression of antagonists of the androgen receptors as well as cytokines relevant in the hair growth cycle (e.g. : TGF, Transforming Growth Factor; EGF - Epidermal Growth Factor) represent essential starting points for a sol technology.

The use of the er is of particular importance inventive liposomes in the immunization of a Host organism, where in this under host organism ins special mammals, including farm animals and domestic animals as well Zoo animals, such as cattle, pigs, horses, dogs, cats, advice animals, mice, monkeys and poultry are to be understood. Ultimately, the liposomes according to the invention can be used in all those animal systems apply in which Langerhans cells are present, especially those present in the skin that are able to produce antigen present. So can, for example with topical Auf Carrying of the liposomes according to the invention containing topical application form, such as a Cre me, a transfection of Langerhans cells with in the nucleic acid contained in the intraliposomal phase, with the effect that the said nucleic acid encoded antigen expressed in the Langerhans cells as well of these to stimulate the immune response on the cell surface is presented.

Thus, there is an effective way of creating a host organ immunization against any antigen that encoded directly or indirectly by a nucleic acid and presented by Langerhans cells on the cell surface can be.

Such an antigen against which one in the host organism Immune response to be generated is referred to herein as inter eating antigen.

Such immunization is not limited to the Presentation of antigen by the Langerhans cells be restricts what amounts to active immunization, but also enables passive immunization of the Shape that corresponding antibodies or fragments there of which are specific for an antigen of interest, by using the liposomes according to the invention, which are a for the nucleus encoding the antibody or a fragment thereof acid, expressed and transfected skin cells can be given to the bloodstream, similar to in the treatment of systemic diseases described above kungen.

Antibodies are also intended to include antibody derivatives be understood, so z. B. also those only from a ket te existing, truncated or due to the specificity of the antigen of interest specifically with regard to their bin construction site constructed corresponding molecules. Such Antibodies as well as their derivatives and others are the subject man known in this field.

The aforementioned immunization, especially if this due to the use of a topical application form becomes, has very special advantages of the shape that it is extremely easy to carry out in the sense that that no qualified personnel is required and esp Injek which is particularly hygienic in certain situations avoided. In addition, there are advantages parts regarding the durability of the invention appropriate vaccine comprising liposomes, which by the liposomes according to the invention, the pharmaceutical zu composition or the agents according to the invention dargest is ellt. This increased durability is essential due to the fact that not any protein-containing Material, be it the antigen in active immuni ation or the antibody or the antibody fragment in passive immunization, especially in for the In jection in a suitable manner, must be present.

From the fact that the vaccine in the narrower sense is only formed by the Langerhans cells and ultimately pre is presented, also requires a considerable amount of costs part because of the production of proteinaceous material can be dispensed with with subsequent purification.

From the example given below regarding the Production of the liposomes according to the invention are further Advantages and features of the invention disclosed, wherein

Fig. 1 is an electron micrograph of a freeze-fracture (FFEM) of human stratum corneum-lipid liposomes in preparation indicated by detergent dialysis,

Fig. 2 shows an electron micrograph of a freeze fracture (FFEM) of human stratum corneum lipid liposomes produced by ultrasound treatment,

Fig. 3 is a Nicomp size distribution analysis of stratum corneum-lipid liposomes prepared by ultrasonic treatment, shows an inverted-La place transform (bimodal distribution),

Figure 4 shows a Nicomp size distribution analysis of stratum corneum lipid liposomes produced by detergent dialysis with inverse Laplace transform (bimodal distribution)

Fig. 5 illustrates the zeta potential distribution of the stratum corneum produced by ultra-sonication lipid liposomes, and

Fig. 6 shows the result of transfection of human skin by means of plasmid DNA showing fiction, modern containing liposomes.

example 1 Manufacture of the liposomes of the invention Obtaining the stratum corneum lipid

From small surgical interventions The horny layer was enzymatically using pieces of skin Trypsinization gained and the total lipid after a mo modified Bligh & Dyer method extracted [Lasch, J .; J. Liposome Res. 4 (1), 93-106 (1994)]. Alternatively, ab scraped plantar cornea used.

The samples were extracted in the dark with chloroform / methanol (1: 1) with vigorous shaking in a nitrogen atmosphere. One third of the volume was added as PBS buffer in order to "salt out" cholesterol sulfate into the organic phase with NaCl. The mixture was vortexed and the phases separated by centrifugation. The lower organic phase was collected and dried _{under N 2.}

The lipid composition of the plantar stratum corneum was determined by measuring the areas under the reflection absorption curves after separation by HPTLC [(Automated Multiple Development HPTLC from CAMAG; Muttenz, Switzerland); Zel lmer, S .; Journal of Chromatography B, 691 (1997), 321-329] and calibration with the respective lipid on more than 100 test subjects. The staining is done using CuSO ₄ / H ₃ PO ₄ in 5% methanol.

In the following table 1 the different Li pid classes according to their percentage (mean values) specified:

Table 1

Average values of the various lipid classes forming the stratum corneum lipid, given in percent by weight

Manufacture of human stratum corneum lipid liposo men (hSCLLs)

HSCLLs were produced on the one hand by ultrasound, by making a lipid dispersion in PBS, pH 7.4, in the Branson So nifier was sonicated until it was transparent, the Be Sonication at intervals of 0.5 min under ice water cooling treatment took place.

Alternatively, the so-called detergent thinners were used the hSCLLs method. Specifically, cholate / - Mixed lipid micelles in a special rotary dialysis machine tor (Diachem AG, Lagnau / Zurich) converted into lipid vesicles.

The results of both methods are shown in FIGS. 1 and 2, FIG. 1 showing an electron micrograph (FFEM) of a freeze fracture of hSCLLs produced by detergent dialysis, and in FIG. 2 an electron micrograph (FFEM) of a freeze fracture of hSCLLs produced by ultrasound.

In both cases, the bar shown represents 100 nm and the circled arrows indicate the direction of vapor deposition.

The hSCLLs were further characterized with regard to their size distribution by photon correlation spectroscopy (PCS) with a Nicomp Submicron Particle Sizer, Model 370. The corresponding results are shown in FIGS. 3 and 4, FIG. 3 showing the results of the liposomes produced by sonication and FIG. 4 showing the liposomes produced by detergent dialysis.

From Fig. 3 it can be seen that 74.62% of the liposomes have a diameter of 160.6 nm, with a second, significantly smaller fraction, which makes up 25.38% of the liposomes, has a diameter of 416.7 nm.

In the preparation of the liposomes by means of detergent dialysis, as can be seen from FIG. 4, two populations of liposomes also emerged, with 87.3% of the liposomes having a diameter of 183.5 nm, compared to a second, albeit 12, 7% significantly smaller population, which has a diameter of 979.7 nm.

In both cases, the aforementioned data were obtained from the primary data under the following conditions:
Smoothing factor 3, minimum diameter 80 nm, plot size 39, collected pulses 8000 K in 30 min., Counting rate 300 Hz.

In Fig. 5 the determination of the zeta potential of the hSC-LLs is shown, which was determined with the Zeta Master Version PCS: v1.2 (Malvern Instruments, England) in the cell type ZEMO10 Cross Beam Mode (Lasch, J .; J. Liposome Res. 5 (1): 99-108 (1995).

As can be seen from FIG. 5, the zeta potential of the hSCLLs is -64 to -66 mV. This relatively high negative value can be attributed to the content of cholesterol sulfate.

The individual, different zeta potentials correspond the intensities are summarized again in Table 2.

Table 2

Zeta potential distribution (mV) of hSCLLs produced by sonication

Other methods of characterizing the lipid layer forming the liposome shell, such as, for example, the measurement of the phase transition temperature (T _m ), gave no results, which is due to the high content of cholesterol in the stratum corneum lipid.

Example 2 Transfection of human skin using the inventive Plasmid DNA-containing liposomes

50 µg of plasmid DNA were used with an appropriate proportion added to lyophilized lipid mixture, the lipid mixture herge in the manner explained in Example 1 was presented. With vigorous shaking, plasmid DNA-containing human stratum corneum liposomes were formed.

After appropriate pretreatment (Menk) or hydration of the stratum corneum with urea MENK (50 mM MOPS, 50 mM ED TA, 50 mM NaCl, 50 mM K ₃ PO ₄ ), the above-mentioned liposome mixture containing plasmid-DNA was applied. After 24 hours of incubation at 37 ° C., the corresponding skin samples were separated into their epidermal and dermal parts. The epidermal part was examined in a quantitative test for β-galactosidase protein activity. After lysis of the epidermis in lysis buffer with mechanical disintegration, 2 ml of the cell extract were mixed with reaction buffer (0.035 mmol galacton) chemiluminescent subtract (Tropix Bedford, Massachusetts). After incubation for one hour, emerald luminescence amplifier (Tropix Bedford, Massachusetts) was added and the chemiluminescence was measured in a chemiluminometer (e.g. Monolight 1005, Analytical Luminescence, San Diego, California). The β-galactosidase-specific activity in the epidermal extracts was determined as the ratio of the light units divided by the protein content. The negative control was obtained by non-β-galactosidase expressing plasmids (e.g. vector control DNA) under identical conditions. Any measurements were made in duplicates. The number of independent trials is shown in Fig. 6 for each of the treatment groups. The absolute amounts of β-galactosidase enzyme can be determined by comparing the obtained light unit values with a calibration curve constructed by adding known amounts of purified β-galactosidase enzyme to β-galactosidase negative epidermal cell extracts. In summary, as shown in FIG. 6, depending on the pretreatment, there is a highly significant increase in the amount of β-galactosidase marker protein produced in the epidermis.

An increase in the transfection efficiency or the amount of gene products obtained is within the scope of the usual Fä skills of a person skilled in the art, for example through further optimization or pretreatment with other penetration enhancers or making them available of suitable nucleic acid constructs possible.

The in the above description as well as in the claims The disclosed features of the invention can be either tent as well as in any combination for the realization the invention in its various embodiments men be essential.

Claims (35)

1. Liposome comprising a lipid phase forming a liposome shell and an intra liposomal phase enclosed by the lipid phase, characterized in that the chemical composition of the lipid phase corresponds to that of a skin layer and the intraliposomal phase contains at least one compound selected from the group which Nucleic acids, proteins, carbohydrates, lipids and low molecular weight compounds as well as molecules or structures composed of these. 2. Liposome according to claim 1, characterized in that the skin layer is human epidermis. 3. Liposome according to claim 1 or 2, characterized in that that the skin layer is the stratum corneum. 4. Liposome comprising a lipid phase forming a liposome shell and an intra liposomal phase enclosed by the lipid phase, characterized in that the liposome includes the following components with regard to the lipid classes:
Phospholipids: <2% by weight Cholesterol sulfate: 2% by weight Free fatty acids: 20% by weight Ceramides: 20% by weight Sterols: 43% by weight Triacylglycerols (neutral fats): 4% by weight Sterol esters: 9% by weight and the intraliposomal phase contains at least one compound selected from the group comprising nucleic acids, proteins, carbohydrates, lipids and low molecular weight compounds and molecules or structures composed of these. 5. A method for the production of liposomes, characterized geken nzeich that

• a) Stratum corneum lipid is obtained and
• b) liposomes are produced from the stratum corneum lipid under ultrasound treatment and / or detergent dialysis.

6. The method according to claim 5, characterized in that The stratum corneum lipid is obtained by removing the stratum corneumatically and / or mechanically removed from the skin will carry and then extra in methanol-chloroform is here. 7. Liposome, producible according to a method according to a of the preceding claims. 8. liposome according to any one of claims 1 to 4 and 7, characterized characterized in that the intraliposomal phase is an aqueous The current phase is. 9. liposome according to any one of claims 1-8, characterized indicates that the compound is selected from the group pe, the growth factors, antibiotics, pain relievers Agents, agents that promote cell division and / or vas cularization affecting, coagulating and anti-coagu lating agents, cytokines, chemical attractants, enzymes me, and combinations thereof. 10. Liposome according to any one of claims 1-9, characterized records that the compound is a nucleic acid sequence. 11. A method for the transfection of cells, thereby marked draws that the cells to be transfected with a Liposome according to any one of the preceding claims in contact to be brought. 12. The method according to claim 11, characterized in that that the cells to be transfected are skin cells. 13. The method according to claim 12, characterized in that that the skin cells are selected from the group that Skin cells of the stratum corneum, stratum granulosum and Includes stratum spinosum and keratinocytes. 14. The method according to any one of claims 11-13, characterized ge indicates that the cells are keratinocytes of the hair follicles are. 15. The method according to any one of claims 11-13, characterized ge indicates that the cells are epidermal Langerhans cells are. 16. A pharmaceutical composition comprising liposomes according to one of the preceding claims and a pharma Zeutically acceptable carrier. 17. Means for topical administration, marked by a content of liposomes according to one of the preceding the claims. 18. Means according to claim 17, characterized in that the agent is selected from the group consisting of creams, sal ben, gels, spray dressings and tinctures. 19. Means according to claim 17 or 18, characterized net that there is still a salary of at least one Has compound which is selected from the group which comprises permeability enhancers and swelling agents. 20. Use of the liposomes according to one of the preceding Claims, the pharmaceutical composition or the Agent according to one of the preceding claims for genthe rapie of skin cells. 21. Use according to claim 20, characterized in that that the skin cells are in situ. 22. Use according to claim 20, characterized in that that the skin cells are ex vivo. 23. Use according to any one of claims 20-22, characterized characterized in that the skin cells are selected from Group, the skin cells of the stratum corneum, stratum granu losum and stratum spinosum as well as keratinocytes. 24. Use according to any one of claims 20-23, characterized characterized that the skin cells are keratinocytes of the hair follicles are. 25. Use according to any one of claims 20-24, characterized characterized that the skin cells are epidermal Langerhans Cells are. 26. Use of the liposomes according to one of the preceding Claims, the pharmaceutical composition or the Means according to one of the preceding claims for prophylaxis lax and / or treatment of skin disorders. 27. Use according to claim 26, characterized in that that the disease is selected from the group that ge Netic skin diseases, psoriasis, skin tumors and wounds that includes. 28. Use according to claim 27, characterized in that that the genetic skin disease is selected from Group, epidermolysis bullosa simplex, epidermolysis hyperkeratosis, palmoplantar keratosis, junctional epi dermolysis bullosa, dystrophic epidermolysis bullosa, la mellar ichthyosis and xeroderma pigmentosum. 29. Use of the liposomes according to one of the preceding Claims, the pharmaceutical composition or the Agent according to one of the preceding claims for treating lung of warts. 30. Use of the liposomes according to one of the preceding Claims, the pharmaceutical composition or the Agent according to one of the preceding claims for treating treatment of systemic diseases. 31. Use according to claim 30, characterized in that that the systemic disease is selected from Group, the hemophilia A and hemophilia B as well as collagen includes degenerations. 32. Use of the liposomes according to one of the preceding Claims, the pharmaceutical composition or the Agent according to one of the preceding claims for treating treatment of alopecia. 33. Use of the liposomes according to one of the preceding Claims, the pharmaceutical composition or the Means according to one of the preceding claims for immuni sation of a host organism. 34. Use according to claim 33, characterized in that that the host organism is a human organism. 35. Vaccine, characterized in that the vaccine a content of liposomes according to one of the preceding Claims, wherein the liposomes nucleic acid, the the genetic information for an anti of interest gene, or nucleic acid that encompasses the genetic Information for an antibody or an antibody question ment, wherein the antibody or the antibody question ment is specific for the antigen of interest.