DE10237517B4 - Process for the enrichment or depletion of biomolecules from liquid or fluid media using layered double hydroxides and use of biomolecules attached or embedded in a layered double hydroxide as an inorganic vector - Google Patents

Abstract

Process for removing or obtaining or purifying at least one biomolecule, which contains nucleotides, nucleosides, amino acids, monosaccharides and / or fatty acids as building blocks, from a liquid or fluid medium with the aid of a layered double hydroxide, selected from the group of natural and synthetic hydrotalcites and the hydrotalcite-like compounds, wherein more than 50% of the total pore volume of the pores of the layered double hydroxide between 1.7 and 300 nm are formed by pores with a diameter of less than 20 nm, comprising the following steps: a. bringing the liquid or fluid medium containing the at least one biomolecule into contact with the layered double hydroxide, the layered double hydroxide being used in calcined and dry form, b. Enabling the attachment or incorporation of the at least one biomolecule onto or into the layered double hydroxide, c. Separating the attached or embedded biomolecule with the layered double hydroxide.

Description

The present invention relates to a process for the removal or recovery or purification of at least one biomolecule containing as building blocks nucleotides, nucleosides, amino acids, monosaccharides and / or fatty acids from a liquid or fluid medium with the aid of a layered double hydroxide, and the use of a the biomolecule incorporated or incorporated into the layered double hydroxide as an inorganic vector for introducing the biomolecule into cells, or as a pharmaceutical composition.

The use of certain layered double hydroxides as anion exchangers or adsorbents is known from the prior art.

For example, describes the EP 0 206 799 B1 the use of mixed metal hydroxides as anion exchangers, in particular for controlling dye migration in a liquid.

The EP 0 566 260 B1 describes a process for the treatment of liquids with hydrotalcites as sorbents, wherein it is essential to the invention that the sorbent is at least partially undried or freshly prepared or generated in situ in the treatment of a liquid medium.

The WO 01/49869 A1 describes compositions with nucleic acids incorporated into bilaminar mineral particles. Hydrotalcites are preferably used for the preparation of a medicament which is used to transfect cells with the embedded nucleic acids.

Similarly, the EP 0 987 328 A2 bio-inorganic hybrid compositions that can stably absorb biomaterials. The hybrid composition in this case has the formula [M ²⁺ _1-x N ³⁺ _x (OH) _2] [A _Bio ^n-] _{x / n} · yH ₂ O on. The use of layered double hydroxides as non-viral vectors for introducing DNA into cells is also disclosed in the reference Choy et al., Angew. Chem. 2000, 112 (22), pages 4208-4211. The reference Lotsch et al., Solid State Sciences 3 (2001), pages 883-886, deals with the separation of nucleoside monophosphates using preferential anion exchange intercalation in layered double hydroxides.

The intercalation of amino acids and sugars in layered double hydroxides is described for example in Aisawa and Narita, Zeoraito (2000) 17, pages 101-107.

In the DE 101 19 233 A1 describes a process for the preparation of hydrotalcite precursors or of hydrotalcites from compounds of divalent and trivalent metals, which comprises a) separate suspensions of the insoluble carbonates, oxides and / or hydroxides of the divalent metals and the oxides or hydroxides of the trivalent metals or b) subjecting a mixed suspension of these components to an intensive grinding until an average particle size (D ₅₀ ) of about 1 to 5 μm is reached, whereby a carbonate source is used when using the oxides or hydroxides of the divalent and the trivalent metals before, during or after the intensive grinding adding; in the case a) the separate suspensions are mixed together, wherein in both cases the intensive grinding is carried out until an amorphous hydrotalcite phase or characterized by a sharp X-ray diffraction hydrotalcite phase occurs, and the product obtained is separated, dried and optionally calcined.

In the WO 02/12255 A1 describes a process for the separation of a nucleoside phosphate of a solution, wherein the property of the nucleoside phosphate is used, to be able to intercalate in Schichtdappelhydroxide, is used. A first nucleoside phosphate can be separated from a second nucleoside phosphate by treating a solution containing both nucleoside phosphates with a layered double hydroxide, preferably intercalating the first nucleoside phosphate into the layered double hydroxide. The layered double hydroxide containing the first nucleoside phosphate can then be treated in such a way that the first nucleoside phosphate is recovered.

In the JP 10279307 A a composite of a layer of multiple hydroxide and a saccharide is described.

Ulibarri et al., Applied Clay Science 18 (2001), pages 17-27, Parker et al., Ind. Eng., Vol. Chem. Res. 34 (1995), pages 1196-1202, Pavan et al., Journal of Colloid and Interface Science 229 (2000), pp. 346-352, as well as the JP-A-63-132898 ,

However, the layer double hydroxides used according to the prior art as Anionenadsorptionsmittel are especially for the stable and reversible on or storage of biomolecules, eg. B. for attachment or depletion of biomolecules from liquid or fluid media, not sufficiently suitable. Thus, there is a constant need for improved materials in this field.

The object of the invention was therefore to provide a process for the removal or recovery or purification of at least one biomolecule from a liquid or fluid medium with the aid of a layered double hydroxide, which is a particularly efficient accumulation or incorporation of biomolecules, in particular for on or Depletion of biomolecules from liquid or fluid media allows. The disadvantages of the prior art should be avoided and a particularly effective interaction between the layered double hydroxide and the biomolecules should be ensured depending on the type of use.

This object is achieved by the method according to claim 1. Preferred embodiments can be found in the dependent claims.

According to the invention, a layered double hydroxide selected from the group of natural and synthetic hydrotalcites and hydrotalcite-like compounds is used, as described, for example, in the Catalysis today reference, Vol. 11 (2) of 2 December 1991, pages 173 to 301, are described ("hydrotacite-like compounds). Such compounds have a hydrotalcite-like structure.

For the preparation of the hydrotalcites it is possible in principle to use any process familiar to the person skilled in the art, as described, for example, in the above Catalysis today (loc. Cit.) On pages 173 to 301, in particular 201 to 212, in US Pat DE 20 61 114 , the US-A-5,399,329 and 5,578,286 , of the DE 101 19 233 or the WO 01/12570 is described.

Particularly preferred is a Schichtdoppelhydroxid (LDH) of the general empirical formula [M _1-x ²⁺ N _x ³⁺ (OH) ₂ ) ^{x +} [A ^n- ] _{x / n} · yH ₂ O, where M ^{2+ is} at least one divalent metal ion and N ^{3+ is} at least one trivalent metal ion, A ^{n is} at least one anion, x is a rational number between 0 and 1, n is a positive number and y is a positive number including 0.

In general, any divalent metal ion suitable for layered double hydroxides and familiar to those skilled in the art or a combination of two or more such metal ions may be used as M ²⁺ . In particular, M ^{2+ represents} one or more of Mg ^{2+ ,} Ca ²⁺ , Zn ²⁺ , Mn ²⁺ , Co ²⁺ , Ni ²⁺ , Fe ²⁺ , Sr ²⁺ , Ba ²⁺ and / or Cu ²⁺

In general, any trivalent cation suitable for layered double hydroxides and familiar to those skilled in the art or a combination of two or more such cations may be used as N ³⁺ . In particular, N ^{3+ represents} one or more of Al ³⁺ , Mn ³⁺ , Co ³⁺ , Ni ³⁺ , Cr ³⁺ , Fe ³⁺ , Ga ³⁺ Sc ³⁺ , B ³⁺ and / or trivalent Cations of rare earth metals.

According to a particularly advantageous embodiment of the invention, M ^{2+ is} magnesium and N ^{3+ is} aluminum. According to the invention, it is also possible to use those double-layer hydroxides (LDHs) which also contain monovalent cations, such as, for example, As Li ⁺ , which can replace the divalent cations partially or completely. Thus, monovalent cation layered double hydroxides are also encompassed by the present invention, especially those having the general formula below: [M _1-x ¹⁺ x ³⁺ (OH) ₂ ] ^{b +} [A ^n- ] _{x / n} · yH ₂ O, where M ^{1+ is} a suitable monovalent cation, in particular Li ⁺ , N ³⁺ , A ^n- , x and y are as defined above, and b = 2x-1.

The anion A ^n- is, in the uncalcined form of the layered double hydroxide, selected from carbonate, nitrate, halide, sulfate, phosphate and / or arsenate. Particularly preferred is the anion A ^n- in the uncalcined form carbonate.

According to a particularly advantageous embodiment of the invention, the layered double hydroxide is a hydrotalcite whose general empirical formula is between [Mg ₂ Al (OH) ₆ ] (CO ₃ ) _0.5 and [Mg _2.5 Al (OH) ₇ ] (CO ₃ ) is _0.5 and in particular the molecular formula [Mg _2.2 Al (OH) _6.4 ] (CO ₃ ) _0.5 is sufficient. With these hydrotalcites, particularly advantageous sorbents for biomolecules were obtained.

In the context of the present invention, it has surprisingly been found that a particularly advantageous attachment or incorporation of the biomolecule takes place when the layered double hydroxide is used in calcined form. In this case, the ability of the layered double hydroxides to regenerate the brucite-like structure (double layer structure) destroyed by the process of calcination is preferably used. If this happens in a solution, the anions contained in it are incorporated. This ability is commonly referred to as a "memory effect". It has surprisingly been found that the use of calcined layered double hydroxides leads to a particularly effective accumulation or incorporation of biomolecules.

The calcination conditions used for the calcination of layered double hydroxides are familiar to the person skilled in the art. Generally, the calcination can be done in an oven at 350 to 650 degrees Celsius over a period of 0.5 to 15 hours. Preferably, the calcination takes place at 450 to 600 degrees Celsius over a period of 0.5 to 5 hours.

According to the invention, the calcined layered double hydroxide in dry form is brought into contact with the liquid or fluid medium containing the biomolecule (s), so that preferably during the reconstitution of the bilayer structure, the biomolecules contained in the liquid or fluid medium are bound to the layered double hydroxide embedded therein and / or enveloped in it. Without being limited to this theoretical mechanism, it is believed that during the reconstitution of the bilayer structure in addition to an intercalation in the interlayer spaces also an adsorption of the negatively charged biomolecules on the positively charged surface of the regenerated layered double hydroxide and a (partial) enveloping the biomolecules takes place. The newly formed bilayer structure is characterized by the presence of polyanionic biomolecules in the desired manner, d. H. in favor of a gentle and effective storage or storage, influenced.

In summary, it has been found that the use of calcined double layer hydroxides in dry form as sorbents leads to a particularly gentle and at the same time efficient attachment or incorporation of biomolecules to or into the layered double hydroxide, whereby at least partial envelopment of the biomolecules by the (Reconstituted) bilayer hydroxide is assumed.

According to the invention, it is particularly preferable to use a layered double hydroxide which was present in the carbonate form before the calcination, since this favors the "memory effect".

According to the invention, it has furthermore been found that a particularly efficient and at the same time gentle accumulation or incorporation of biomolecules is achieved if more than 50% of the total pore volume of the pores is between 1.7 and 300 nm, through pores with a layer double hydroxide used in calcined form Diameter of less than 20 nm are formed. These values are determined as indicated below.

It is assumed, without the invention being restricted thereto, that the reconstitution of the double-layer structure is favored with respect to the parallel or subsequent accumulation and incorporation of the biomolecules in the liquid or fluid medium while maintaining this pore volume distribution. This assumption is confirmed by the fact that, with a significantly smaller proportion of the pores having a diameter of less than 20 nm or a correspondingly higher proportion of the larger meso- and macropores in which (larger) biomolecules could be incorporated, the advantageous on or Incorporation of the biomolecules at the same size of Doppelschichthydroxidteilchen seems to be at least partially lost.

It has proved particularly favorable if more than 75% of the total pore volume of the pores between 1.7 and 300 nm are formed by pores having a diameter of less than 20 nm, in particular less than 10 nm.

According to a further preferred embodiment of the invention, in the case of the calcined layered double hydroxide used, the area of the micropores (up to 200 nm in diameter) is at least 50 m ² / g, in particular at least 60 m ² / g, particularly preferably at least 70 m ² / g. It is believed, without the invention being limited to this theoretical mechanism, that the biomolecules particularly preferably reside in the area of the micropores. Structurally, LDH surfaces of the order of magnitude above are due to a highly variable surface topology in the micropores. This also increases the statistical probability that the biomolecule in the region of the micropores interacts particularly strongly with the layered double hydroxide.

According to a further preferred embodiment of the invention, the proportion of micropores up to 200 nm on the total surface of the layered double hydroxide is at least 50%, preferably more than 60%, in particular more than 70%. A possible explanation for why particularly good results were obtained with such layered double hydroxides is provided by the above model concept, since the probability of the interaction between biomolecules and the micropores is directly correlated with their share of the total surface area.

It has been found that, in particular, larger or long-chain biomolecules according to the invention can be deposited or encased or enveloped ("packaged") in a particularly gentle and efficient manner. It is assumed that in the parallel to the reconstitution of Doppelschichthydroxid extending or subsequent accumulation or storage or envelopment of the biomolecules in the liquid or fluid medium, a particularly good "packaging" of biomolecules takes place, so that they better against those at the further processing of the fluid or fluid inevitable occurring in the biomolecules inevitably occurring shear forces and possible exposure of partial moieties are better protected against enzymatic cleavages.

According to a particularly preferred embodiment of the invention, the average particle size (D50) of the LDHs is between 0.3 and 1.5 μm, in particular between 0.5 and 1.0 μm. The D10 values of the particle size are preferably between 0.1 and 1.0 μm, in particular between 0.15 and 0.5 μm. The D90 values of the particle size are preferably between 0.5 and 4.0 μm, in particular between 1.0 and 3.0 μm. Adhering to these particle sizes together with the above porosimetry surprisingly results in particularly advantageous binding properties for biomolecules, in particular larger biomolecules.

According to a further preferred embodiment of the invention, the calcined layered double hydroxide has a BET surface area (determined to DIN 66131) of at least 50 m ² / g, in particular at least 100 m ² / g, particularly preferably at least 150 m ² / g. The high surface apparently promotes reconstitution or the advantageous accumulation and storage of the biomolecules.

In the context of the present invention, a biomolecule is understood as meaning a molecule which has, as building blocks, nucleotides or nucleosides (nucleobases), amino acids, monosaccharides and / or fatty acids. This group therefore includes the mono-, oligo- and polymers of these building blocks, in particular the oligonucleotides and nucleic acids, oligopeptides and proteins, oligo- and polysaccharides and / or the lipids and their above building blocks and their respective derivatives. Derivatives are understood to be all chemical modifications of the above molecules, which can furthermore be bound as polyanions to layered double hydroxides. In general, these will be modifications or derivatizations of the functional (side) groups of one or more building blocks of the biomolecules. However, in the context of the invention, derivatives are also detected in which the "backbone" of the biomolecule is modified, as for example in the case of the peptidic nucleic acids (PNA).

According to an advantageous embodiment of the invention, the biomolecules are DNA or RNA molecules in double-stranded or single-stranded form with one or more nucleotide units; Amino acids or oligopeptides and proteins as well as mono-, oligo- or polymeric carbohydrates and lipids, including glycoproteins and glycolipids, as long as they can be deposited or incorporated as (poly) anions on layered double hydroxides.

As mentioned above, with the aid of the present invention, biomolecules can be deposited or stored particularly gently on or in the layered double hydroxides. Of course, this advantage according to the invention is particularly evident in larger or longer-chain biomolecules, such as longer-chain oligonucleotides and nucleic acids or oligopeptides and proteins or long-chain saccharides.

One aspect therefore relates to the use of the above-defined layered double hydroxides for the removal or recovery of biomolecules from liquid or fluid media containing at least one larger or long-chain biomolecule as defined below. Preferably, the larger or long-chain biomolecule, if it is an oligopeptide or protein, has at least 5 amino acids, preferably at least 50 amino acids and more preferably at least 100 amino acids; if it is an oligonucleotide or a nucleic acid, at least 10 nucleotide building blocks, preferably at least 100 nucleotide building blocks and more preferably at least 1000 nucleotide building blocks; if it is an oligo- or polysaccharide, at least 3 monosaccharide, preferably at least 30 monosaccharide and more preferably at least 100 Monosaccharidbausteine on.

In a particularly advantageous aspect, the above composition thus contains at least one biomolecule having a molecular weight of at least 200 D, in particular 10 kD, particularly preferably at least 100 kD. Within the scope of the invention, the size of the biomolecule has no upper limits, so that even very large biomolecules with more than 500 kD or more than 1 MD can be deposited or stored in a particularly advantageous manner.

With regard to amino acids, the method according to the invention or the use of the calcined layered double hydroxides is particularly suitable for liquid or fluid media which have peptides or proteins with at least 5 amino acids, preferably at least 50 amino acids and more preferably at least 100 amino acids.

With regard to nucleic acids, the method according to the invention is particularly advantageous in the case of liquid or fluid media containing oligonucleotides or nucleic acids having at least 10 bases (pairs), preferably at least 100 bases (pairs), in particular at least 1000 bases (pairs).

With regard to carbohydrates, the method according to the invention is particularly advantageous in the case of liquid or fluid media containing oligonucleotides or nucleic acids with at least 3 monosaccharide units, preferably with at least 30 monosaccharide units and in particular at least 100 monosaccharide units.

Liquid or fluid media are understood to mean all media which bring into contact and thus enable the biomolecules to accumulate or deposit in the layered double hydroxide. In particular, these are in practice aqueous or water-containing media in the form of a solution, suspension, dispersion, colloidal solution or emulsion.

Typical examples are industrial or non-industrial wastewaters, process wastewaters, fermentation residues or media, biomolecule-containing media from medical or biological research, biomolecule-contaminated, liquid or fluid contaminated sites and the like.

The method according to the invention comprises the following steps: a) contacting the liquid or fluid medium containing at least one biomolecule which contains as building blocks nucleotides, nucleosides, amino acids, monosaccharides and / or fatty acids with the sorbent; b) enabling the attachment or incorporation of the at least one biomolecule to or in the layered double hydroxide; c) separating the attached or stored biomolecule with the layered double hydroxide; d) if appropriate, separation or desorption of the at least one biomolecule from the layered double hydroxide.

The method according to the invention for the attachment or incorporation of biomolecules onto or in layered double hydroxides can be used both for enrichment (that is to say increase of the concentration of the desired biomolecule) and enrichment (ie reduction of the concentration of the desired biomolecule).

If the method according to the invention is aimed at removal or disposal of biomolecules, the layered double hydroxide containing the biomolecules can be disposed of in a further step. The disposal can take place, for example, by thermal treatment to remove the layered double hydroxide containing the biomolecules, wherein after the thermal disintegration of the biomolecules, the layered double hydroxide can be disposed of particularly advantageously.

Thus, according to a first aspect of the invention, it is possible to selectively remove biomolecules from media. This plays an important role, for example, in wastewater treatment, since in most countries there are strict legal regulations on the removal of biomolecules from wastewater.

According to a further preferred embodiment of the invention, the depletion or removal of biomolecules from culture media can also be carried out. For example, in bioreactors an undesirable increase in viscosity may occur due to the high concentration of biomolecules, in particular high molecular weight nucleic acids, contained in the medium. Here, an efficient and biocompatible removal of the interfering biomolecules from the Culture medium done. By adding the sorbent to the culture medium, the viscosity can also be adjusted to a desired level.

In the same way, it is often desirable to increase the concentration of biomolecules in a medium or to obtain these biomolecules in pure form as far as possible. For example, the recovery or purification of desired proteins, carbohydrates or nucleic acids from solutions has become standard procedures in biological and medical research. In this case, according to the invention, in a further step, the biomolecule can be desorbed or recovered from the layered double hydroxide, as a result of which the layered double hydroxide can optionally be reused.

According to a further aspect of the invention, the layered double hydroxide particles can be bonded to larger agglomerates, granules or shaped bodies via a suitable binder or applied to a support. The shape and size of such parent structures containing the primary layered double hydroxide particles will depend on the particular application desired. It is thus possible to use all shapes and sizes which are familiar to the person skilled in the art and which are suitable in individual cases. For example, agglomerates with a diameter of more than 10 .mu.m, in particular more than 50 .mu.m, may be preferred in many cases. In the case of a bed for chromatography columns and the like, a spherical shape of the agglomerates may be advantageous. A possible carrier is, for example, calcium carbonate.

It is also possible to use any binder known to the person skilled in the art as long as the attachment or incorporation of the biomolecules into or onto the layered double hydroxide particles is not excessively impaired or the stability of the particle agglomerates or shaped bodies required for the particular application is ensured. For example, but not limited to, can be used as binders: agar-agar, alginates, chitosans, pectins, gelatins, lupine protein isolates or gluten.

A further aspect of the present invention relates to the use of the compositions as inorganic vectors for introducing biomolecules into cells or as a pharmaceutical composition, in particular as a reservoir for the storage and controlled release of biomolecules, preferably of nucleic acids. Thus, it has surprisingly been found that the gentle and effective accumulation or incorporation of the biomolecules in or on the layered double hydroxides, which can be obtained by contacting the liquid or fluid medium containing the biomolecules with the calcined layered double hydroxide, also to a Particularly efficient introduction of these biomolecules can serve in prokaryotic or eukaryotic cells. Apparently, according to the method of the invention, biomolecules, in particular nucleic acids, can be packaged in a particularly advantageous manner for introduction into cells. The principal mechanism of such introduction of DNA-LDH nanohybrids is described, for example, in the reference Choy et al., Angew. Chem. 2000, 112 (22), pages 4207-4211, as well as in the EP 0 987 328 A2 to which reference is directed and which are hereby incorporated by reference into the specification. The use as a pharmaceutical composition, in particular as a reservoir for the storage and controlled release of biomolecules, preferably of nucleic acids, is as such in the WO 01/49869 to which reference is directed and which is hereby incorporated by reference into the specification.

The BET surface areas indicated here were determined according to DIN 66131.

The indicated (mean) pore diameters, volumes and areas were determined using a fully automatic nitrogen adsorption meter (ASAP 2000, Micrometrics) according to the manufacturer's standard program (BET, BJH, t-plot and DFT). The percentage figures for the proportion of specific pore sizes are based on the total pore volume of pores between 1.7 and 300 nm in diameter (BJH Adsorption Pore Distribution Report).

The invention will now be explained in more detail with reference to the following non-limiting examples.

• 1. HT-Granulat, erhältlich unter dem Handelsnamen CERATOFIX^®NA (HT-Granulat) von der Firma Süd-Chemie AG, ist ein Mg/Al-Hydrotalcit. Die Calcinierung erfolgte 4 h im Muffelofen bei 600°C, wobei über konzentrierter Schwefelsäure im Exsikkator abgekühlt wurde. Etwa 81% (76%) des Gesamtporenvolumens der Poren zwischen 1,7 und 300 nm wurden beim calcinierten Material durch Poren mit einem Durchmesser von weniger als 20 nm (10 nm) gebildet. Die Mikroporenfläche lag bei etwa 79 m²/g.
• 2. HT-16, erhältlich unter dem Handelsnamen CERATOFIX^®NA (HT-16) von der Firma Süd-Chemie AG, ist ein nicht-calcinierter Mg/Al-Hydrotalcit in der Nitratform.

The following hydrotalcites were used as starting materials, wherein the preparation of corresponding layered double hydroxides is also easily possible by methods familiar to the person skilled in the art (see above). The adjustment of the porosimetry and the surfaces of the layered double hydroxides can be carried out in this way in particular by routine tests for hydrothermal aftertreatment and for calcination.

• 1. HT granules, available under the trade name NA CERATOFIX ^® (HT-granules) by Sud-Chemie AG, is a Mg / Al hydrotalcite. The calcination was carried out for 4 h in a muffle furnace at 600 ° C, being cooled over concentrated sulfuric acid in a desiccator. About 81% (76%) of the total pore volume of the pores between 1.7 and 300 nm was formed in the calcined material by pores having a diameter of less than 20 nm (10 nm). The microporous area was about 79 m ² / g.
• 2. HT-16, available under the trade name NA CERATOFIX ^® (HT-16) by Sud-Chemie AG, is a non-calcined Mg / Al hydrotalcite in the nitrate form.

The calcined hydrotalcites were added directly to the liquid medium containing the biomolecules to produce the hydroxide form.

For comparative activation of HT granules with acid, 28.6 μl of 99.8% acetic acid were added to 0.1 g of the hydrotalcite in the form of a 10% suspension in distilled water, mixed thoroughly and then centrifuged at 2500 rpm for 10 min. The residue was suspended in 5 ml of Tris buffer (10 mM Tris / HCl, pH 8.0) to remove the acid residues and recentrifuged.

As a further comparison, hydrotalcite HT-16 was converted from the nitrate form to the chloride form by washing 0.1 g of the dry material in 5 ml of buffer solution (10 mM Tris / HCl, 1M NaCl, pH 8.0), centrifuging to remove washed NaCl in 5 ml Tris buffer (10 mM Tris / HCl, 1M NaCl, pH 8.0) and again centrifuged (see Tables I and II, HT-16, washed).

1. Determination of DNA binding capacity

Each 0.1 g of the above Hydrotalcitproben to 5 ml of DNA solution (herring sperm DNA sodium or acid, Sigma, Schnelldorf) known concentration (2.5 mg / ml) was added and mixed until a uniform Suspension arises. The sample is then shaken for 1 h or 16 h at 250 rpm at room temperature. It is centrifuged for 15 minutes at 2500 rpm (the residue is used below in the elution experiments) and the supernatant is sterile filtered. If the buffer used does not contain EDTA then 10 μl 0.5 M EDTA solution is added to protect the samples from DNase contamination. The DNA concentration of the supernatant is determined photometrically on the basis of UV absorption at 260 nm (Sambrook et al, "Molecular Cloning - A Laboratory Manual", Vol. III, page C1, Cold Spring Harbor Laboratory Press, 2 ^nd Edition (1989) From the concentration difference, the binding capacity is calculated.

For the hydrotalcite samples described above, the static binding capacities for both the DNA acid and the DNA salt (see above) were determined.

As a buffer for preparing the DNA solutions, both a Tris buffer (10 mM Tris / HCl, pH 8) and a TE buffer (10 mM Tris / HCl, 1 mM EDTA, pH 8) were used. The determined capacities were identical for both buffer systems.

The capacities are always based on the weight of the material used. When activated with acid, the weight of the unactivated, dry form is indicated.

All experiments were carried out three times, the result being the mean of the three measurements given in Tables 1 and 11 below. Table I: Binding capacities of various hydrotalcites and DNA acid comparison systems hydrotalcite Activation pretreatment Capacity 16 h [mg · g ^-1 ] Capacity 1 h [mg · g ^-1 ] HT-16 dry 99.8 nb HT-16 washed 61.5 25.2 HT-granulate acetic acid 22.1 13.5 HT-granulate calcined 209.1 57.6 HT-granulate not activated 14.4 nb nb = not determined Table II: Binding capacity of various hydrotalcites and DNA salt comparison systems hydrotalcite Activation pretreatment Capacity 16 h [mg · g ^-1 ] Capacity 1 h [mg · g ^-1 ] HT-16 dry 28.6 nb HT-16 washed 26.5 17.6 HT-granulate acetic acid 8.1 4.5 HT-granulate calcined 205.3 52.7 HT-granulate not activated 11.8 nb nb = not determined

It turns out that when using the calcined hydrotalcite according to the invention, the binding capacities for DNA in both the acid form and the salt form are considerably higher than with non-activated or acid-activated hydrotalcites and with hydrotalcite in the nitrate form or in the chloride form.

Also a comparatively used calcined hydrotalcite, in which only about 42% of the total pore volume of the pores between 1.7 and 300 nm, formed by pores having a diameter of less than 20 nm due to another hydrothermal treatment during the preparation of calcined HT granules were (for comparison: calcined HT granules: more than 50%), shows a significantly lower binding capacities for DNA.

2. Elution of the bound DNA:

Before elution, wash the respective residue (see 1. above) with 5 ml Tris buffer (10 mM Tris / HCl, pH 8), then add the indicated volume of the following elution buffer and shake at 250 rpm for 16 h. It is centrifuged at 2500 rpm for 15 min and the supernatant is sterile filtered. The DNA concentration of the elution fraction is determined photometrically and the recovery rate is calculated.

To elute the DNA from the layered double hydroxide, the following buffer was used: 0.5 M Na ₃ PO ₄ , pH 7.3, adjusted with Na ₃ PO ₄ (elution buffer). With this buffer, with a relative elution volume of 500 ml / g, the majority of the DNA could be eluted from the LDH used according to the invention and detected photometrically in the supernatant.

Thus, a reversible binding of the DNA is guaranteed to the hydrotalcite and recovery of the desired biomolecule in pure form possible. The separated layered double hydroxide can be used again, if appropriate after renewed calcination.

3. Binding of proteins using the example of BSA

The model protein used was BSA (fraction V, Sigma, Schnelldorf).

In each case, 0.1 g of the hydrotalcite samples given at the beginning was added to 5 ml BSA solution in TE buffer (c = 2 mg.ml ^-1 ) and shaken for 16 h at 250 rpm. After centrifuging for 10 min at 2500 rpm, the protein concentration in the supernatant was determined photometrically by measuring the absorbance at 280 nm. This experiment was also carried out three times.

Again, it has been found that when using the calcined hydrotalcites used in the present invention, the binding capacities for protein are considerably higher than in non-activated or acid-activated hydrotalcites and hydrotalcite in the nitrate form or the chloride form is the case.

4. Transfection of NIH-3T3 cells with hydrotalcite as inorganic vector

a) Cell cultivation:

For the transfection experiments, the cell line NIH-3T3 is used. These are adherently growing mouse embryo fibroblasts. The doubling time is about 20 h. The cells are cultured in the standard medium DMEM (with 4.5 g. 1 ^-1 glucose) with 10% NCS. The cultivation is carried out in an incubator at 37 ° C under humidified 5% CO ₂ atmosphere.

To prepare the stock culture, the thawed cell suspension is placed in a monolayer bottle (25 cm ² ) with 10 ml of medium. A direct determination of the cell count is not possible with adherently growing cells; a control of the growth rate takes place via the degree of coverage of the culture vessel. At about 90% confluence - usually after 3-4 days - the culture is implemented in order to avoid the overgrowth of the cells (Fokibildung). For this purpose, the medium is decanted off, added trypsin solution and incubated for 10 min in an incubator. The detached cell suspension is mixed with about 15 ml of serum-containing medium to block the trypsin. The centrifuged cells are resuspended in fresh medium and re-seeded at a dilution of 1:10.

b) transfection:

As an inorganic vector for the transfection of NIH-3T3 cells, the hydrotalcite samples indicated above were "loaded" with the plasmid pQBI25-fC1 (Qbiogene, Heidelberg) prior to transfection. For this, 10 mg of the respective hydrotalcite sample were weighed and washed with ethanol to sterilize. Subsequently, 2 ml of sterile, distilled water were added, vortexed briefly and centrifuged for 3 min at 5000 rpm. To the residue was added 1.5 ml of sterile plasmid DNA of concentration 1.45 ml.ml ^-1 and shaken at room temperature for 16 hours at 250 rpm.

The DNA hydrotalcite hybrid was suspended in 10 ml of distilled, sterile water. The transfection with hydrotalcite as a vector was carried out in each case with two different concentrations of DNA-hydrotalcite hybrid. The NIH-3T3 cells were seeded 24 hours before transfection at a density of 1-2 × 10 ⁵ cells cm ^-2 in 6-well plates and incubated in an incubator at 37 ° C. under humidified 5% CO ₂ . Atmosphere incubated. The indicated amounts each refer to a hole of the 6-hole plate. The analysis was carried out 48 h after the beginning of the transfection with the fluorescence microscope. Transfected cells can be recognized by the GFP production, when excited at 474 nm, they glow green when viewed in fluorescence microscopy.

Version 1:

Immediately before the experiment, the medium was removed and 1.5 ml of new medium added. 25 or 100 μl of the DNA hydrotalcite suspension were added and incubated for 3 hours in an incubator. Subsequently, the medium was changed and incubated for a further 45 h.

Variant 2:

Immediately before the experiment, the medium was removed and 1.5 ml of new medium added. 25 or 100 μl of the suspension was added and incubated for 24 h. Subsequently, the medium was changed and incubated for a further 24 h.

When using the hybrid according to the invention with the (reconstituted) calcined hydrotalcite significantly more successfully transfected cells could be detected by fluorescence compared to the hybrid compositions with the non-activated or acid-activated hydrotalcites and the hydrotalcite (HT-16) in the nitrate form or choroid form so that the DNA hydrotalcite hybrids according to the invention are suitable as inorganic vectors.

Claims (20)

A process for the removal or recovery or purification of at least one biomolecule containing as building blocks nucleotides, nucleosides, amino acids, monosaccharides and / or fatty acids from a liquid or fluid medium with the aid of a layered double hydroxide selected from the group of natural and synthetic hydrotalcites and hydrotalcite-like compounds, wherein more than 50% of the total pore volume of the pore of the layered double hydroxide is formed between 1.7 and 300 nm by pores having a diameter of less than 20 nm, comprising the following steps: a. contacting the liquid or fluid medium containing the at least one biomolecule with the layered double hydroxide, wherein the layered double hydroxide is used in calcined and dry form, b. Allowing the accumulation or incorporation of the at least one biomolecule on or in the layered double hydroxide, c. Separation of the attached or embedded biomolecule with the Schichtdoppelhydroxid. A method according to claim 1, characterized in that the method further comprises the step of: d. Separation or desorption of the at least one biomolecule from the layered double hydroxide. A method according to claim 1, characterized in that more than 65%, in particular more than 75 of the total pore volume of the pores between 1.7 and 300 nm are formed by pores having a diameter of less than 20 nm, in particular less than 10 nm. Method according to one of the preceding claims, characterized in that the at least one biomolecule is selected from the group of nucleic acids, proteins, polysaccharides and / or lipids and derivatives thereof. Method according to one of the preceding claims, characterized in that the at least one biomolecule is selected from oligo- and polymers based on nucleotides, amino acids and carbohydrates. Method according to one of the preceding claims, characterized in that it is the liquid or fluid medium is a colloidal solution, suspension, dispersion, solution or emulsion. Method according to one of the preceding claims, characterized in that the average particle size (D ₅₀ ) of the layered double hydroxide is not more than 1 micron, and preferably between 0.3 .mu.m and 0.8 .mu.m. Method according to one of the preceding claims, characterized in that the layered double hydroxide was present in the carbonate form before calcination. Process according to one of the preceding claims, characterized in that the layered double hydroxide is a hydrotalcite whose general empirical formula lies between [Mg ₂ Al (OH) ₆ ] (CO ₃ ) _0.5 and [Mg _2.5 Al (OH) ₇ ] ( CO ₃ ) is _0.5 and in particular the molecular formula [Mg _2.2 Al (OH) _6.4 ] (CO ₃ ) _0.5 is sufficient. Method according to one of the preceding claims, characterized in that the calcined layered double hydroxide has a BET surface area of at least 50 m ² / g, in particular at least 100 m ² / g, particularly preferably of at least 150 m ² / g. Method according to one of the preceding claims, characterized in that it is the liquid or fluid medium to an aqueous medium, preferably a fermentation medium, a fermentation residue from the cell culture, process water, or a waste water with at least one biomolecule as in any one of the preceding claims defined acts. Method according to one of the preceding claims, characterized in that in a further step, the layered double hydroxide containing the biomolecules is disposed of. Method according to one of claims 1 to 11, characterized in that in a further step, the biomolecule is desorbed or recovered from the layered double hydroxide, whereby the layered double hydroxide can optionally be used again. Method according to one of claims 1 to 7 and 10 to 13, characterized in that the layered double hydroxide has the following general empirical formula: [M _1-x ²⁺ N _x ³⁺ (OH) ₂ ] ^{x +} [A ^n- ] _{x / n} · yH ₂ O, wherein M ^{2+ is} at least one divalent metal ion and N ^{3+ is} at least one trivalent metal ion, A ^{n is} at least one anion, x is a rational number between 0 and 1, and y is a positive number including 0. Process according to Claim 14, characterized in that in the layered double hydroxide the bivalent metal ions are wholly or partly replaced by monovalent cations, in particular lithium, and the layered silicate preferably has the following general empirical formula: [M _1-x ¹⁺ N _x ³⁺ (OH) ₂ ] ^{b +} [A ^n- ] _{x / n} · yH ₂ O wherein M ^{1+ is} a suitable monovalent cation, in particular lithium, and N ³⁺ , A ^n- , x and y have the meaning given in claim 14, and b = 2x-1. Method according to one of the preceding claims, characterized in that the Schichtdoppelhydroxid an average particle size (D ₅₀ ) between 0.3 and 1.5 .mu.m, in particular between 0.5 and 1.0 microns preferably has the D ₁₀ values of the particle size between 0.1 and 1.0 .mu.m, in particular between 0.15 and 0.5 .mu.m, and preferably the D ₉₀ values of the particle size between 0.5 and 4.0 .mu.m, in particular between 1.0 and 3.0 microns lie. Method according to one of the preceding claims, characterized in that the layered double hydroxide in the form of particle aggregates, granules or shaped bodies of layered double hydroxide is used with the binder. Use of the according to step b) or c) of claim 1 in the layered double hydroxide or embedded biomolecule as an inorganic vector for introducing the biomolecule into cells, or as a pharmaceutical composition. Use according to claim 18, wherein the biomolecule deposited or incorporated into the layered double hydroxide according to step b) or c) of claim 1 as a reservoir for the storage and controlled release of biomolecules. Use according to claim 18, wherein the biomolecule is a nucleic acid.