Bio-inorganic compound capable of stable, solid-state storage of genes and preparation thereof

Disclosed is a bio-inorganic hybrid composite for retaining and carrying bio-materials with stability and reversible dissociativity, represented by the following chemical Formula I and a method for preparing the same. The bio-inorganic hybrid composite is prepared by (a) coprecipitating with an alkaline material an aqueous solution comprising a bivalent metal (M(II)) and a trivalent metal (N(III)) at a molar ratio satisfying the condition of 1/5.ltoreq.b/(a+b).ltoreq.1/2 wherein a and b represent a mole number of M and N, respectively, to form a stable layered double hydroxide in which anions are intercalated and (b) subjecting the intercalated anions to ion exchange reaction with a bio-material to compensate for layer charges of the layered double hydroxide. This composite is harmless to the body and artificially controls the appropriate expression of the bio-material retained therein. EQU [M.sup.2+.sub.1-x N.sup.3+.sub.x (Oh).sub.2 ][A.sub.BIO.sup.n- ].sub.x/n.yH.sub.2 O [I] wherein, PA1 M is a bivalent metal cation; PA1 N is a trivalent metal cation; PA1 A.sub.BIO is an anionic bio-material with n charges; PA1 x is a rational number between 0 to 1; and PA1 y is a positive number.

BACKGROUND OF THE INVENTION
 The present invention relates to a bio-inorganic hybrid composite which is
 able to retain and carry bio-materials with stability and reversible
 dissociativity. Also, the present invention is concerned with a method for
 preparing such a bio-inorganic hybrid composite.
 Generally, most inorganic compounds having lamellar structures, such as
 aluminosilicate, metal phosphate, etc, have a feature of being able to
 retain various materials in their interstices. In this regard, a variety
 of functional guest chemical species can be introduced into the
 interstices by subjecting the metal ions composing lattice layers to
 isomorphorous substitution to generate layer charges or modifying the
 lamellar in such a way that it is provided with physical and chemical
 absorptivity. Also, it is well known that the pore sizes of porous
 inorganic compounds, such as crosslink clay, MCM-41, etc, are controlled
 to physically adsorb selective sizes of molecules. Layered double
 hydroxides (hereinafter referred to as "LDH"), which are kinds of the
 inorganic compounds having lamellar structures, are called anionic clays,
 consisting of positively charged metal hydroxide layers between which
 anions counterbalancing the positive charges and water are intercalated.
 As a rule, these composites are represented by the following chemical
 Formula:
EQU [M.sup.2+.sub.1-x N.sup.3+.sub.x (OH).sub.2 ][A.sup.n- ].sub.x/n.yH.sub.2 O
 wherein,
 M is a bivalent metal cation, selected from alkaline earth metals or
 transition metals, such as Mg.sup.2+, Ni.sup.2+, Cu.sup.2+, Zn.sup.2+ and
 the like,
 N is a trivalent metal cation, selected from transition metals, such as
 Al.sup.3+, Cr.sup.3+, Fe.sup.3+, V.sup.3+, Ga.sup.3+ and the like,
 A is an anionic chemical species with n charges, such as NO.sub.3.sup.-,
 CO.sub.3.sup.2-, Cl.sup.-, SO.sub.4.sup.2-, metalate, anions of organic
 acids, etc,
 x is a rational number between 0 to 1; and
 y is a positive number.
 The layer charge densities of the composites can be controlled by changing
 the metal ratios according to x. Various anions, denoted by A in the LDH,
 can be easily introduced between the metal hydroxide layers through ion
 exchange reaction and coprecipitation.
 LDH and their derivatives, which are of technical importance in catalytic
 reactions, are put into spotlight as lamellar nano-composites useful in
 various fields, including separation technology, optics, medical science,
 and engineering, and active research has been directed to the composites
 of interest. For example, hydrotalcite, a mineral name for the compounds
 based on magnesium and aluminum with similar structures to those of LDH,
 represented by [Mg.sub.3 Al(OH).sub.8 ].sup.+
 [0.5CO.sub.3.multidot.mH.sub.2 O].sup.-, was investigated for the
 structure of intercalated carbonate anions and water by use of .sup.1 H
 and .sup.13 C-NMR ["Ordering of intercalated water and carbonate anions in
 hydrotalcite-An NMR study" Journal Physical Chemistry, 1994, 98,
 4050-4054]. A synthesis method for crosslinking precursors of the Mg.sub.3
 Al LDH intercalated with hydroxides and adipate through ion exchange
 reaction with the aid of polyoxometalate (P.sub.2 W.sub.18 O.sub.62.sup.6-
 or Co.sub.4 (H.sub.2 O).sub.2 (PW.sub.9 O.sub.34).sub.2.sup.10-) and assay
 results for the structural and thermal properties of the resulting
 composites are reported in ["Layered double hydroxides intercalated by
 polyoxometalate anions with keggin (.alpha.-H.sub.2 W.sub.12
 O.sub.40.sup.6-), dawson (.alpha.-P.sub.2 W.sub.18 O.sub.62.sup.6-), and
 finke (Co.sub.4 (H.sub.2 O).sub.2 (PW.sub.9 O.sub.34).sub.2.sup.10-)
 structures", Inorganic Chemistry, 1996, 35, 6853-6860]. Structural
 properties of some LDH are introduced, along with kinds and structures of
 possible metal cations and common intercalated anions in ["Crystal
 structures of some double hydroxide minerals", Mineralogical Magazine,
 1973, 39[304], 377-389]. A survey of LDH and their derivatives, including
 their historical backgrounds, possible-components (e.g., kinds of metal
 cations and intercalated anions), structural properties, and applicability
 is also found in ["Hydrotalcite-type anionic clays: Preparation,
 Properties and Applications", catalysis Today, 1991, 11, 173-301].
 Being useful as gene carriers, bacteria or cationic liposomes have a
 feasible possibility of causing side effects owing to their own toxicity
 and a problem of eliciting immune responses and showing poor expression
 rates.
 Therefore, there remains a need for a structure for retaining and carrying
 bio materials.
 SUMMARY OF THE INVENTION
 Based on the fact that genetic materials have a structural property of
 being negatively charged owing to phosphoric ions, the present invention
 has an object of preparing a bio-inorganic hybrid composite by
 intercalating genetic materials in interstices of LDH through anion
 exchange. LDH is dissolved in acidic conditions, but remains stable in
 neutral and alkaline conditions. This attribute allows genes or bio
 materials to be put into or taken from LDH, freely, thereby making it
 possible for LDH to function as a preserver and carrier for genes or other
 bio materials. In addition, the bio-inorganic hybrid composite according
 to the present invention has significant advantages over conventional
 bio-material carriers, e.g., bacteria or cation liposomes, in that the
 bio-inorganic hybrid composite is composed of metal hydroxides harmless to
 the body and artificially controls the appropriate expression of the
 genetic materials retained therein.
 The present invention has significance in that it is the first trial to
 make metal hydroxides used as a preserver and carrier for genes and
 bio-materials in the world.

DETAILED DESCRIPTION OF THE INVENTION
 Details are given of the present invention, below.
 The present invention provides a bio-inorganic hybrid composite for
 retaining and carrying genes and bio-materials with stability and
 reversible dissociativity, which is prepared by subjecting LDH to ion
 exchange with bio-materials. The bio-inorganic hybrid composite is
 represented by the following chemical Formula I:
EQU [M.sup.2+.sub.1-x N.sup.3+.sub.x (OH).sub.2 ][A.sub.BIO.sup.n-
 ].sub.x/n.yH.sub.2 O [I]
 wherein,
 M is a bivalent metal cation;
 N is a trivalent metal cation;
 A.sub.BIO is an anionic bio-material with n charges;
 x is a rational number between 0 to 1; and
 y is a positive number.
 Particularly, all alkaline earth metals and transition metals are available
 as M and N for which Mg and Al are preferably substituted, respectively.
 As for A.sub.BIO, its negative charges compensate for the layer charges of
 the hybrid composite and it may be nucleoside-5'-monophosphate,
 nucleoside-5'-triphosphate, or a gene material with a size of 500-1,000
 bp.
 A nucleoside-5'-monophosphate is a repeating unit composing a gene
 material. It has a nucleoside moiety consisting of a base and a sugar to
 which a phosphate group is attached in ester linkage. The phosphate group
 is negatively charged in aqueous solutions, thus providing the genetic
 materials, divided largely into DNA and RNA, with negative charges.
 The bio-inorganic hybrid composite of the chemical Formula I is prepared as
 follows.
 First, the metal cations, M(II) and N(III) are mixed in an aqueous solution
 at a molar ratio satisfying the condition of 1/5.ltoreq.b/(a+b).ltoreq.1/2
 wherein a and b represent a mole number of M and N, respectively. Then,
 the aqueous salt solution is coprecipitated using an alkaline material, to
 cause the isomophorous substitution of N(III) for M(II) of a brucite
 (Mg(OH).sub.2) structure, thereby leading to an induction of excess
 positive charges in lattice layers. To compensate for the generated
 charges, anions are intercalated from the aqueous solution in the layers
 to form stable LDH with intercalation of anions. The intercalated anions
 can be replaced with some bio-materials with negative charges, e.g.,
 nucleoside-5'-monophosphate, nucleoside-5'-triphosphate, and DNA or RNA
 with a stretch of 500-1,000 bp, to give the bio-inorganic hybrid composite
 of the chemical Formula I.
 In the present invention, the intercalation of negatively charged
 bio-materials in the interstices of LDH is stably maintained by the
 electrostatic attraction between two oppositely charged materials. The
 genetic materials intercalated, if required, can be reclaimed as being
 intact through ion exchange reaction or selective dissolution.
 The LDH used in the present invention is composed of metals nontoxic to the
 body. Particularly when magnesium and aluminum are used, that is, when the
 M and N in the chemical Formula I are Mg and Al, respectively, the
 resulting LDH are easily dissolved in acidic conditions and the ionized
 metals are known to be eliminated from the body through appropriate
 metabolism. In fact, hydroxides of magnesium and aluminum are widely used
 in antacids for protecting stomach walls. In addition, because metals are
 different in the solubility in acid conditions from one to another, the
 bio-inorganic hybrid composite of the present invention can be controlled
 in its dissolution through appropriate combinations of bivalent metals and
 trivalent metals, so that the expression time of the gene intercalated
 therein can be controlled.
 A better understanding of the present invention may be obtained in light of
 the following examples which are set forth to illustrate, but are not to
 be construed to limit the present invention. In the following examples,
 bio-inorganic hybrid composites according to the present invention were
 assayed for properties as follows:
 Assay Condition
 1) Assay Using IR Spectrum
 Sample pretreatment: Mixed with KBr and compressed into a disc.
 Measuring instrument: Spectrophotometer from Perkin-Elmer
 Measuring frequency range: 400-4,000 cm.sup.-1
 2) Assay Using Electrophoresis
 i) Assay for pH
 Sample pretreatment: pH controlled with HCl and electrophoresed at room
 temperature for 1 hour.
 Measuring method: Dyed with ethidium bromide and run on 0.7% agarose gel
 under an electric field.
 Measurement Range: pH 7 to 1.
 ii) Assay for Dnase I
 Sample pretreatment: Treated with Dnase I at room temperature for a period
 of 0.5, 1 and 24 hours. Treated with Dnase I at room temperature for 1
 hour, quenched in a solution (0.2 M NaCl, 40 mM EDTA, 1% SDS), and treated
 at pH 2 for 1 hour.
 Measuring method: Electrophoresed on 0.7% agarose gel.
 EXAMPLE I
 Mg(NO.sub.3).sub.2 and Al(NO.sub.3).sub.3 were combined at such an amount
 that the mole ratio of Mg(II) to Al(III) was 3:1. To this combined
 solution, a solution of 0.2 M NaOH in water was dropwise added, causing
 coprecipitation. The precipitates thus obtained were washed and
 centrifuged to synthesize LHD intercalated by nitrate
 (MgAl(NO.sub.3)--LDH, hereinafter referred to as NO.sub.3 --LDH). The
 whole procedure was conducted in a nitrogen atmosphere to prevent the
 NO.sub.3 --LDH from being contaminated with CO.sub.2.
 The NO.sub.3 --LDH was analyzed through X-ray diffraction and infrared
 spectrometry whose results are shown in FIGS. 1 (spectrum a) and 2
 (spectrum a), respectively.
 To a suspension of the NO.sub.3 --LDH in water (1 mg/1 ml H.sub.2 O), a
 solution of 10 mg of cytidine-5'-monophosphate (CMP) in boiled water was
 added, and they were allowed to undergo ion exchange reaction with each
 other for 24 hours in an incubating bath maintained at 50.degree. C. After
 completion of the ion exchange reaction, the product was washed three
 times with boiled water to completely remove excess phosphate and dried in
 an oven at 70.degree. C.
 Spectrometric analysis was conducted for the product with X-ray and
 infrared whose results are shown in FIGS. 1 (spectrum b) and 2 (spectrum
 b), respectively.
 As apparent from the spectra of FIGS. 1 and 2, the interlayer distance in
 the NO.sub.3 --LDH composite was found to correspond to the size of CMP
 and the absorption IR peaks read were coincident with characteristic
 functional groups of CMP. Taken together, these data demonstrate that CMP
 is stably intercalated in the interstices of the LDH. Based on this
 result, a possible array of CMPs in the interstices of the LDH is
 suggested in FIG. 6 at panel a.
 EXAMPLE II
 The same procedure as in Example I was repeated using
 adenosin-5'-monophosphate (AMP).
 The NO.sub.3 --LDH suspension (1 mg/1 ml H.sub.2 O) obtained in Example I
 was added to a solution of 10 mg of AMP in boiled water, and they were
 allowed to undergo ion exchange reaction with each other for 24 hours in
 an incubating bath maintained at 50.degree. C. After completion of the ion
 exchange reaction, the product was washed three times with boiled water to
 completely remove excess phosphate and dried in an oven at 70.degree. C.
 Spectrometric analysis was conducted for the product with X-ray and
 infrared whose results are shown in FIGS. 1 (spectrum c) and 2 (spectrum
 c), respectively.
 As apparent from the spectra of FIGS. 1 and 2, the interlayer distance in
 the NO.sub.3 --LDH composite was found to correspond to the size of AMP
 and the absorption IR peaks read were coincident with characteristic
 functional groups of AMP, demonstrating that AMP was stably intercalated
 in the interstices of the LDH. Based on this result, a possible array of
 AMPs in the interstices of the LDH is suggested in FIG. 6 at panel b.
 EXAMPLE III
 The same procedure as in Example I was repeated using
 guanidine-5'-monophosphate (GMP).
 The NO.sub.3 --LDH suspension (1 mg/1 ml H.sub.2 O) obtained in Example I
 was added to a solution of 10 mg of GMP in boiled water, after which they
 were allowed to undergo ion exchange reaction with each other for 24 hours
 in an incubating bath maintained at 50.degree. C. After completion of the
 ion exchange reaction, the product was washed three times with boiled
 water to completely remove excess phosphate and dried in an oven at
 70.degree. C.
 Spectrometric analysis was conducted for the product with X-ray and
 infrared whose results are shown in FIGS. 1 (spectrum d) and 2 (spectrum
 d), respectively.
 As apparent from the spectra of FIGS. 1 and 2, the interlayer distance in
 the NO.sub.3 --LDH composite corresponded to the size of AMP and the
 absorption IR peaks read were coincident with characteristic functional
 groups of AMP, demonstrating that GMP was stably intercalated in the
 interstices of the LDH. Based on this result, a possible array of GMPs in
 the interstices of the LDH is suggested in FIG. 6 at panel c.
 EXAMPLE IV
 To a suspension of the NO.sub.3 --LDH in water (1 mg/1 ml H.sub.2 O)
 obtained in Example I, a solution of 10 mg of Herring testes DNA
 (hereinafter referred to as DNA) in boiled water was added, and they were
 allowed to undergo ion exchange reaction with each other for 24 hours in
 an incubating bath maintained at 50.degree. C. After completion of the ion
 exchange reaction, the product was washed three times with boiled water to
 completely remove excess DNA and dried in an oven at 70.degree. C.
 Spectrometric analysis was conducted for the product with X-ray and
 infrared whose results are shown in FIGS. 1 (spectrum e) and 2 (spectrum
 e), respectively.
 As apparent from the spectra of FIGS. 1 and 2, the interlayer distance in
 the NO.sub.3 --LDH composite was found to correspond to the size of DNA
 and the absorption IR peaks read were coincident with characteristic
 functional groups of DNA. Taken together, these data demonstrate that DNA
 is stably intercalated in the interstices of the LDH.
 With reference to FIG. 3, there is an electrophoresis result which gives
 information regarding the dissociation degrees of the DNA-LDH composite in
 dependence on pH. On lane 1 was run a DNA marker (.lambda./HindIII) which
 contained DNA fragments with sizes of 23.1, 9.4, 6.5, 4.3, 2.3 and 2.0 kb.
 The DNA-LDH composites were electrophoresed on lanes 4 to 10 after being
 treated at pH 7, 6, 5, 4, 3, 2 and 1, respectively. Also, a marker 500 bp
 in length and the DNA used in preparing the composite were run on lanes 2
 and 3, respectively.
 As seen in FIG. 3, no trails are found on lanes 4 to 8, indicating that the
 negative charges of DNA are compensated in the interstices of the LDH. On
 the contrary, traces electrophoresed in an electric field are found on
 lanes 9 and 10, both. Based on this electrophoresis analysis, it is
 believed that the layers of LDH collapse at less than pH 3, particularly
 at pH 2 or less, so that the DNA confined between the layers is released.
 In recognition of the moving to the same distance as in the control DNA
 running on lane 3 upon electrophoresis, DNAs on lanes 9 and 10 are
 believed to be preserved intact within the LDH.
 With reference to FIG. 4, there is an electrophoresis result which gives
 information regarding the preservation of DNA in the composite through
 DNase I treatment. The DNA markers run on lanes 1, 2 and 3 were the same
 as in Example I. Along with intact DNA-LDH, which was not treated with
 DNases, on lane 4, only DNAs were electrophoresed on lane lanes 5, 7 and 9
 after being treated with DNase I for 0.5, 1 and 24 hours, respectively,
 while the DNA-LDH composites treated with DNase I for 0.5, 1 and 24 hours
 were allowed to run in an electric field on lanes 5, 7 and 9,
 respectively. The DNA electrophoresed on lane 11 came from the DNA-LDH
 composite after it was treated with DNase I for 1 hour and then, with a
 solution to quench the enzyme and finally, allowed to stand for 1 hour at
 pH 2.
 The electrophoresis patterns on lanes 6, 8 and 10, as seen in FIG. 4, are
 similar to that on lane 4, indicating that the DNA intercalated in the
 interstices of the composite are maintained intact. This result is quite
 contrary to the electrophoresis results on lanes 5, 7 and 9, which were
 obtained after DNAs alone were treated in the same conditions as that for
 lane 4. Confirming the result of lanes 6, 8 and 10, the electrophoresis
 pattern on lane 11, which was obtained after the same sample as on lane 8
 was treated to quench the DNase I, allowed stand at pH 2 for 1 hour to
 release the DNA from the interstices of the LDH and electrophoresed,
 showed that the DNA within LDH was preserved intact. The procedure in
 which DNA is intercalated in the interstices of LDH, treated with DNase I,
 and preserved intact is illustrated in FIG. 7. Therefore, the
 electrophoresis data of FIG. 4 demonstrate that the LDH according to the
 present invention is a very stable carrier for DNA and other bio-materials
 and has a characteristic of being easily dissociated.
 EXAMPLE V
 The NO.sub.3 --LDH suspension (1 mg/1 ml H.sub.2 O) obtained in Example I
 was added to a solution of 10 mg of ATP in boiled water, and they were
 allowed to undergo ion exchange reaction with each other for 24 hours in
 an incubating bath maintained at 50.degree. C. After completion of the ion
 exchange reaction, the product was washed three times with boiled water to
 completely remove excess phosphate and dried in an oven at 70.degree. C.
 X-ray diffraction analysis data for the product are given in FIG. 5
 (spectrum b). As apparent from the diffraction data, the 001 peak, which
 is indicative of interlayer distances in a solid, was moved toward a lower
 angle compared with the starting material, indicating that an expansion
 occurs between layers. Because the decreased angle extent was found to
 correspond to the thickness of ATP, it was demonstrated to be stabilized
 in LDH.
 EXAMPLE VI
 The NO.sub.3 --LDH suspension (1 mg/1 ml H.sub.2 O) obtained in Example I
 was added to a solution of 10 mg of an antisense single strand of RNA in
 boiled water, and they were allowed to undergo ion exchange reaction with
 each other for 24 hours in an incubating bath maintained at 50.degree. C.
 After completion of the ion exchange reaction, the product was washed
 three times with boiled water to completely remove excess phosphate and
 dried in an oven at 70.degree. C.
 X-ray diffraction analysis data for the product are given in FIG. 5
 (spectrum c). As apparent from the diffraction data, the 001 peak, which
 is indicative of interlayer distances in a solid, was moved toward a lower
 angle compared with the starting material, indicating that an expansion
 occurs between layers. Because the decreased angle extent was found to
 correspond to the thickness of ATP, it was demonstrated to be stabilized
 in LDH.
 As described hereinbefore, the present invention provides a bio-inorganic
 hybrid composited which can preserve and carry genes and is nontoxic to
 the body and easily dissociated.
 The present invention has been described in an illustrative manner, and it
 is to be understood that the terminology used is intended to be in the
 nature of description rather than of limitation. Many modifications and
 variations of the present invention are possible in light of the above
 teachings. Therefore, it is to be understood that within the scope of the
 appended claims, the invention may be practiced otherwise than as
 specifically described.