WO2001049869A1 - Compositions comprising nucleic acids incorporated in bilaminar mineral particles - Google Patents
Compositions comprising nucleic acids incorporated in bilaminar mineral particles Download PDFInfo
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- WO2001049869A1 WO2001049869A1 PCT/FR2000/003702 FR0003702W WO0149869A1 WO 2001049869 A1 WO2001049869 A1 WO 2001049869A1 FR 0003702 W FR0003702 W FR 0003702W WO 0149869 A1 WO0149869 A1 WO 0149869A1
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- composition according
- nucleic acid
- formula
- dna
- composition
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- 229940069575 rompun Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
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- 238000001179 sorption measurement Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
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- 230000009466 transformation Effects 0.000 description 1
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- 238000005199 ultracentrifugation Methods 0.000 description 1
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- 229960001600 xylazine Drugs 0.000 description 1
- QYEFBJRXKKSABU-UHFFFAOYSA-N xylazine hydrochloride Chemical compound Cl.CC1=CC=CC(C)=C1NC1=NCCCS1 QYEFBJRXKKSABU-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1611—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
Definitions
- the present invention relates to compositions comprising at least one nucleic acid and a mineral particle having a structure with exchangeable sheets, their preparation process and their applications for the in vivo, in vitro and / or ex vivo transfection of nucleic acids .
- nucleic acids into cells that is to say of introducing them into cells so that the gene of interest which they carry is expressed there
- It may be the transfer of nucleic acids into cells in vitro, for example for the production of recombinant proteins, or in the laboratory for the study of the regulation of gene expression, the cloning of genes or any other manipulation involving DNA. It may also involve the transfer of nucleic acids into cells in vivo, for example for the production of vaccines, labeling studies or also therapeutic approaches. It may also involve the transfer of genes ex vivo into cells taken from an organism, with a view to their subsequent re-administration, for example for the creation of transgenic animals.
- compositions comprising a nucleic acid and a mineral particle having a structure with exchangeable sheets allow the transfer in vitro, ex vivo or in vivo of said nucleic acid into a cell and / or an organ with an efficiency greater than those obtained to date with conventional non-viral transfer techniques.
- the compositions of the invention further allow avoid the drawbacks associated with the use of viral vectors or physical transfection techniques.
- a first object of the present invention relates to the compositions comprising at least one nucleic acid and one mineral particle having a structure with exchangeable sheets.
- the term “mineral particles having a structure with exchangeable sheets” means mineral particles whose structure is based on a stack of sheets of composition M (OH) 2 similar to those well known in brucite (Mg (OH) 2 ), M representing a divalent metal, but in which part of the divalent metals is replaced by trivalent metals so as to make the sheets generally cationic.
- M brucite
- M representing a divalent metal
- the inter-sheet spaces include anionic entities solvated by water molecules.
- Such a structure is shown in Figure 1 and is also called "double lamellar hydroxide”.
- M u represents a divalent metal cation
- M 111 represents a trivalent metal cation
- X m ⁇ represents an interfoliar anion and m is an integer greater than or equal to 1
- x is between 0 and 1 strictly
- n is strictly greater at 0.
- Such mineral particles have already been described for applications in many fields such as catalysis or the environment. Indeed, they form nanostructured materials where molecular or colloidal species are interposed in a lamellar host structure.
- the properties of these structures can thus be used in heterogeneous or homogeneous catalysis on a support, in exchange and separation techniques, in particular optical isomers, for the design of membranes which may be selective for filtration and permeation, for trapping and controlled restitution of molecules, or for the design of electro-active materials and deposits, electrodes and electronic devices.
- 121 (6), 1399-1400 describes the preparation of hybrid nanoparticles composed of sheets of a Double Lamellar Hydroxide (HDL), pristine ( Mg 2 Al (NO 3 ) -LDH), between which are inserted monophoshated nucleosides or DNA fragments of herring testes.
- the nucleic molecules intercalated in the particles described in this article have a size less than 1000 base pairs. No use of these molecules is described.
- the mineral particles having a structure with exchangeable sheets usable within the framework of the present invention are often named after the nature of the major metal cations of the sheets: for example hydrotalcite (Mg-Al), pyroaurite (Mg-Fe) , stichtite (Mg-Cr) or takovite (Ni-Al).
- hydrotalcite Mg-Al
- Mg-Fe pyroaurite
- Mg-Cr stichtite
- Ni-Al takovite
- the general formula indicated above underpins the wide variety of mineral particles which it is possible to prepare by varying the nature of the two metal cations M 11 and M ⁇ , their respective proportions with possible extension to more than two types of cations (eg Mg, Zn and Al), the nature of the interfoliar anions, or the state of hydration.
- the richness of the general formula indicated above is further increased by a structural diversity resulting from the existence of polytypes differing by the sequence of stacking of the sheets as well as from the appearance of over-structures due for example to a classification cationic in the hydroxylated sheet.
- the divalent metal cations M ⁇ can be chosen for example from magnesium (Mg), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), cobalt (Co), copper (Cu), calcium (Ca) or even zinc (Zn).
- the divalent metal cation is magnesium.
- Trivalent metal cations M ⁇ may be chosen for example from aluminum (Al), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), cobalt (Co) or even gallium (Ga).
- the trivalent metal cation is aluminum.
- the two members of the couple M ⁇ and M can also correspond to the same element (for example Fe ⁇ / Fe ffl or Mn ⁇ / Mn ⁇ ).
- the interfoliar anions X ′′ may for example be chosen from halides, oxoanions, iso- and heteroanions, complex anions or even organic anions. Examples include fluorides, chlorides, bromides, carbonates , nitrates, sulfates or tetrahedral oxoanions such as CrO 2 " .
- the anion is chosen from carbonates
- x is strictly between 0 and 1, that is, 0 and 1 are excluded values.
- x is between 0.05 and 0.95, and preferably between 0.1 and 0.9. Even more preferably, x is between 0.2 and 0.85.
- x can be equal to 0.25.
- n this indicates the state of hydration of the particle in question and it is strictly greater than 0.
- n is greater than or equal to 0.1.
- n can be equal to 0.5.
- m is an integer greater than or equal to 1 which represents the charge of the interfoliar anion.
- m can be 1, 2, 3, 4, 5 or 6.
- Mineral particles having a structure with exchangeable sheets according to the present invention can be chosen for example from the hydrotalcite of formula Mg 6 Al 2 CO 3 (OH) ⁇ 6 .4H 2 O, the manasseite of formula Mg 6 Al 2 CO 3 (OH) ⁇ 6 .4H 2 O, the pyroaurite of formula Mg 6 Fe 2 CO 3 (OH) ⁇ 6 .4H 2 ⁇ , the stichtite of formula Mg 6 Cr 2 CO 3 (OH) ⁇ 6 .4H 2 O, the takovite of formula Ni 6 Al 2 CO 3 (OH) ⁇ 6 .4H 2 0, the reevesite of formula Ni 6 Fe 2 CO 3 (OH) ⁇ 6 .4H 2 ⁇ , or the comblainite of formula Ni 6 C ⁇ 2CO 3 (OH) 16 .4H 2 ⁇ .
- Other mineral particles having a structure with exchangeable sheets may also be suitable.
- the compositions according to the present invention comprise at least one nucleic acid and hydrotalcite
- the mineral particles having a structure with exchangeable sheets according to the present invention are either commercial, or they can be prepared according to methods which are similar to known methods known as “soft chemistry”.
- the preparation of the mineral particles according to the invention can be based on the controlled precipitation of aqueous solutions or suspensions containing both the metal cations intended to take place in the hydroxylated framework and the anions intended to occupy the interlamellar domains. Under these conditions, there is simultaneously construction of the interlamellar domains consisting of anions and water molecules, and of the sheet.
- the nature and texture of the phases obtained are highly dependent on the operating conditions (temperature, rate of addition and concentration of the reagents, pH control), but also on the geometry of the reactor and the state of prior association of the metal cations. in reagents (existence of oligomers).
- the preparation process ultimately always boils down to bringing together, at an appropriate concentration and reactivity, the species leading to the construction of the mineral particles under optimal conditions. These optimal conditions are determined on a case-by-case basis according to the conventional method of trial and error.
- nucleic acid is understood to mean both a deoxyribonucleic acid and a ribonucleic acid.
- They can be natural or artificial sequences, and in particular genomic DNA (gDNA), complementary DNA (cDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (RRNA), hybrid sequences or synthetic or semi-synthetic sequences, of modified or unmodified oligonucleotides.
- These nucleic acids can be, for example, of human, animal, plant, bacterial or viral origin. They can be obtained by any technique known to a person skilled in the art, and in particular by screening of banks, by chemical synthesis, or even by mixed methods including chemical or enzymatic modification of sequences obtained by screening of libraries. They can be chemically modified.
- deoxyribonucleic acids can be single or double stranded or else short oligonucleotides or longer sequences.
- the nucleic acids advantageously consist for example of plasmids, vectors, episomes or expression cassettes.
- deoxyribonucleic acids may in particular carry an origin of functional or non-functional replication in the target cell, one or more marker genes, transcription or replication regulatory sequences, genes of therapeutic interest, antisense sequences which may or may not be modified or even regions of binding to other cellular components.
- said nucleic acid has a size greater than 1000 base pairs. Even more preferably, it has a size of between approximately 1000 base pairs and approximately 2,000,000 base pairs (2 Mb), approximately 1,000 base pairs and approximately 100,000 base pairs (100 kb), and approximately 1,000 base pairs and approximately 20,000 base pairs (20 kb).
- the nucleic acid comprises one or more genes of therapeutic interest under the control of regulatory sequences, for example one or more promoters and a transcriptional terminator active in the target cells.
- the term “gene of therapeutic interest” in particular means any gene coding for a protein product having a therapeutic effect.
- the protein product thus coded can in particular be a protein or a peptide.
- This protein product can be exogenous homologous or endogenous with respect to the target cell, that is to say a product which is normally expressed in the target cell when the latter presents no pathology.
- the expression of a protein makes it possible for example to compensate for an insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or else to overexpress said protein.
- the gene of therapeutic interest can also code for a mutant of a cellular protein having, for example, increased stability or modified activity.
- the protein product can also be heterologous towards the target cell.
- an expressed protein can, for example, supplement or bring about a deficient activity in the cell, allowing it to fight against a pathology, or stimulate an immune response.
- trophic factors for example BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF, NT3, NT5 or HARP / pleiotrophin
- apolipoproteins for example ApoAI, ApoAIV or ApoE: see FR 93/05125
- dystrophin or a minidystrophin FR 91/11947
- the CFTR protein associated with cystic fibrosis for example p53, Rb, RaplA, DCC or k-rev : see FR 93/04745), the genes coding for factors involved in coagulation (Factors VII, VIII, IX),
- the nucleic acid of therapeutic interest can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs.
- Such sequences can, for example, be transcribed in the target cell into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308.
- the therapeutic genes also include the sequences coding for ribozymes, which are capable of selectively destroying target RNAs (EP 321,201).
- the nucleic acid can also contain one or more genes coding for an antigenic peptide, capable of generating in humans or animals an immune response.
- the invention allows the production of either vaccines or immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancers.
- These may in particular be antigenic peptides specific for the Epstein Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the "syncitia forming virus", d other viruses or tumor-specific antigenic peptides (EP 259,212).
- the nucleic acid preferably also comprises sequences allowing the expression of the gene of therapeutic interest and / or of the gene coding for the antigenic peptide in the desired cell or organ.
- These may be sequences which are naturally responsible for the expression of the gene considered when these sequences are capable of functioning in the infected cell. It can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic).
- they may be promoter sequences of eukaryotic or viral genes.
- they may be promoter sequences originating from the genome of the cell which it is desired to infect.
- they may be promoter sequences originating from the genome of a virus.
- promoters of the El A, MLP, CMV, RSV or HSV genes can be modified, for example, by adding activation or regulation sequences. They can also be inducible or repressible promoters.
- the nucleic acid can also comprise, in particular upstream of the gene of therapeutic interest, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell.
- This signal sequence may be the natural signal sequence of the therapeutic product, but it may also be any other functional signal sequence, or an artificial signal sequence.
- the nucleic acid may also have a signal sequence directing the synthesized therapeutic product to a particular compartment of the cell.
- compositions according to the present invention can be prepared by mixing an aqueous solution containing a mineral particle having a structure with exchangeable sheets and a solution containing the nucleic acids. More specifically, the compositions according to the present invention can be prepared by bringing the mineral particles having a structure with exchangeable sheets in aqueous solution at a pH close to neutral (for example between 6 and 8, and more preferably between 6.5 and 7 , 5), then by adding the aqueous solution thus obtained to a solution containing the nucleic acids or else by adding said solution containing the nucleic acids to the aqueous solution of mineral particles having a structure with exchangeable sheets.
- the solution containing the nucleic acids is preferably an isotonic solution in sodium chloride or in glucose.
- the mineral particles having a structure with exchangeable sheets and the nucleic acids are introduced into the composition in quantities such that the mass ratio between the mineral particles having a structure with exchangeable sheets and the nucleic acids is between 0.01 and 1000 , preferably between 0.01 and 500, more preferably between 0.01 and 100, and even more preferably between 0.5 and 100.
- the respective amounts of each component can be easily adjusted and optimized by l he skilled in the art by the usual method of trial and error, as a function of the mineral particle having a structure with exchangeable sheets used, of the nucleic acid, and of the desired applications (in particular of the cell type to be transfected).
- a subject of the invention is also the compositions as defined above for use as a medicament.
- the invention also relates to the use of the compositions as defined above for the transfer of nucleic acids into cells in vitro, in vivo or ex vivo. More specifically, the subject of the present invention is the use of the compositions as defined above for the preparation of a medicament intended to treat diseases, in particular diseases which result from a deficiency in a protein or nucleic product.
- the nucleic acid contained in said medicament encodes said protein or nucleic product, or constitutes said nucleic product, capable of correcting said diseases in vivo or ex vivo.
- Said medicament can also be a vaccine and in this case, the transfected nucleic acid induces an immune response.
- compositions according to the invention can be formulated for administration in particular by topical or cutaneous route, oral, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal or intraperitoneal.
- the compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into the desired organ, or for topical administration (on the skin and / or on the mucous membrane).
- compositions according to the invention may contain one or more adjuvants conventionally used in pharmacy.
- the doses of nucleic acids used for the injection as well as the number of administrations can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or the duration of the treatment sought.
- the mode of administration it may be either a direct injection into the tissues, for example at the level of tumors, or into the circulatory tract, or a treatment of cultured cells followed of their reimplantation in vivo, by injection or graft.
- the tissues concerned in the context of the present invention are for example the muscles, the skin, the brain, the lungs, the liver, the spleen, the bone marrow, the thymus, the heart, the lymph, the blood, the bones, cartilage, pancreas, kidneys, bladder, stomach, intestines, testes, ovaries, rectum, nervous system, eyes, glands or connective tissue.
- Another object of the present invention relates to a method for transferring nucleic acids into cells, comprising the following steps:
- composition comprising at least one nucleic acid and one mineral particle having a structure with exchangeable sheets as defined above, and
- the present invention relates to a method of therapeutic treatment of the human or animal body comprising the following steps:
- composition comprising at least one nucleic acid and one mineral particle having a structure with exchangeable sheets as defined above, and
- the contacting of the cells with the composition according to the present invention can be carried out by incubation of the cells with said composition or by injection of the composition into the organism or part of the organism.
- the incubation is preferably carried out in the presence of, for example, from 0.01 to 1000 ⁇ g of nucleic acid per 10 cells.
- doses of nucleic acid ranging from 0.01 to 10 mg can for example be used.
- compositions of the invention may further contain one or more pharmaceutically acceptable adjuvants.
- the adjuvant (s) are mixed beforehand with the aqueous solution containing the mineral particle having a structure with exchangeable sheets according to the invention and / or with the solution of nucleic acid (s).
- the present invention thus provides a particularly advantageous method for the transfer of nucleic acids, in particular for the treatment of diseases, comprising the administration in vivo or ex vivo of a nucleic acid coding for a protein or capable of being transcribed into a nucleic acid. able to correct said disease, said nucleic acid being associated with a mineral particle having a structure with exchangeable sheets as defined above under the conditions defined above.
- compositions according to the present invention are particularly useful for the transfer of nucleic acids into primary cells or into established lines. They can be, for example, fibroblastic, muscular, nervous cells (neurons, astrocytes, glial cells), hepatic, hematopoietic (for example lymphocytes, CD34, or dendritics) or epithelial, in differentiated or pluripotent forms (precursors).
- nucleic acids for example, fibroblastic, muscular, nervous cells (neurons, astrocytes, glial cells), hepatic, hematopoietic (for example lymphocytes, CD34, or dendritics) or epithelial, in differentiated or pluripotent forms (precursors).
- the present invention also includes other characteristics and advantages which will emerge from the examples and figures which follow, and which should be considered as illustrating the invention without limiting its scope.
- FIGURES Figure 1 idealized structure of the mineral particles having a structure with exchangeable sheets (double lamellar hydroxides) according to the present invention.
- Figure 2 schematic representation of the hydrotalcite Mg 6 Al 2 CO 3 (OH) ⁇ 6 .4H 2 O.
- Figure 3 measurement of the zeta potential of hydrotalcite particles alone in solution.
- Figure 4 measurement of the zeta potential of compositions containing hydrotalcite particles and DNA in a mass ratio equal to 0.5.
- Figure 5 Measurement of the percentage of DNA remaining in the supernatant relative to the initial amount of DNA introduced, as a function of the hydrotalcite / DNA mass ratio. The measurements were made for three different concentrations of DNA introduced: 20 ⁇ g of DNA / ml, 50 ⁇ g of DNA / ml, and 100 ⁇ g of DNA / ml.
- Figure 6 Electrophoresis on agarose gel of different DNA formulations.
- the column “a” constitutes the control (DNA not formulated and not subjected to nucleases)
- the column “b” represents the unformulated DNA subjected to nucleases
- the columns “C” to “f” represent hydrotalcite / DNA compositions at mass ratios of 0.125 or 0.25 or 0.5 or 1 which are subjected to nucleases.
- FIG. 7 visualization in the form of a histogram of the efficiency of gene transfer in vivo (by injection into the cranial tibial muscle of mice) of DNA / hydrotalcite compositions in different mass ratios, compared with the injection of naked DNA.
- Figure 8 Schematic representation of the plasmid pXL3031 used in DNA transfer experiments in cells
- mice used in the in vivo gene transfer experiments are 8 week old female C57B16 mice, divided into 9 groups of 12 mice.
- Mineral particle according to the invention used the mineral particle having a structure with exchangeable sheets used in the various tests is hydrotalcite in aqueous solution at a concentration of 20 g / 1 and at pH 7.
- This hydrotalcite is sold by the company Sud- Chemie AG (Germany).
- the plasmid used is pXL3031 which contains the read gene coding for luciferase under the control of the P / E CMV promoter of the cytomegalovirus, represented in FIG. 8. Its size is 3671 bp.
- the plasmid solution used contains 320 ⁇ g of DNA / ml in a 150 mM solution of sodium chloride (NaCl).
- the complexes are prepared by equivolumetric mixing of two solutions: one containing hydrotalcite and the other plasmid DNA.
- the purpose of this example is to show that DNA is capable of associating with a mineral particle having a structure with exchangeable sheets such as hydrotalcite.
- the measurement of the zeta potential provides information on the nature of the surface charge of a particle placed in a liquid.
- the zeta potential is not a direct measure of the surface charge, but is the potential that exists around the particle when it moves in a solution when it is subjected to an electric field. Therefore if the particles have a positive surface charge, the zeta potential, which is the only measurable quantity, will also be positive. Zeta potential plays a role determining in the colloidal stability of the particles in solution.
- the particles do not aggregate because forces of electrostatic repulsions keep them isolated.
- the particles which have a zeta potential close to zero are not stable because the Van Der Waals forces attract the particles together, and cause them to precipitate.
- the zeta potential of hydrotalcite and of hydrotalcite / DNA complexes was measured by a Coulter brand zetameter (Delsa 440 SX).
- the hydrotalcite solution contained 0.15 mg hydrotalcite / ml in a 20 mM solution of sodium chloride (NaCl), giving a conductivity of 2.3 mS / cm.
- the hydrotalcite / DNA complexes were prepared at concentrations of 250 ⁇ g of DNA / ml in a 20 mM solution of sodium chloride (NaCl), at a mass ratio of 0.5. The measurement was made by applying an electric field of 1.5 mA for 60 seconds.
- FIG. 3 shows the zeta potential of hydrotalcite alone before being mixed with DNA.
- This zeta potential is + 32 mV and therefore indicates an overall cationic surface charge, which is consistent with the structure and composition of the hydrotalcite (see Figures 1 and 2).
- FIG. 4 represents the zeta potential of hydrotalcite / DNA complexes with a mass ratio of 0.5. In this case, the zeta potential is - 41 mV.
- the surface charge has therefore been modified and is now generally anionic, which indicates that the anionic DNA molecules have associated on the surface of the hydrotalcite thus modifying its surface charge.
- the purpose of this example is to show that mineral particles having a structure with exchangeable sheets such as hydrotalcite associate with DNA. Adsorption isotherms are achieved by mixing increasing amounts of hydrotalcite with a constant amount of DNA. Hydrotalcite / DNA mixtures are prepared by equivolumetric mixing of a solution containing DNA and a solution containing hydrotalcite. All of these mixtures are then ultracentrifuged at 50,000 rpm for 10 minutes (Ultracentrifuge: Beckman TL100). The graph in FIG. 5 indicates that the quantity of DNA present in the supernatant gradually decreases with the increase in the hydrotalcite / DNA mass ratio. At a hydrotalcite / DNA ratio of 50 w / w, there is no longer any DNA present in the supernatant.
- the purpose of this example is to show that DNA is protected against enzymatic degradations when it is associated with a mineral particle having a structure with exchangeable sheets such as hydrotalcite.
- the injections were carried out bilaterally in the cranial tibial muscle of mice at the rate of 25 ⁇ l / muscle, ie 4 ⁇ g of DNA / muscle.
- the animals are anesthetized with 250 ⁇ l of Ketamine / Xylazine intraperitoneally (3.9 ml of imalgene 1000, 0.6 ml Rompun at 2%, and 40.5 ml of a solution of sodium chloride NaCl at 150 mM ). Then the animals are shaved and injected into the two cranial tibial muscles.
- Luciferase activity is measured using a luciferase test kit (Promega) and a Dynex MLX luminometer. The reading of this activity is measured in RLU ("Relative Light Unit"). in vivo transfection into the muscle
- results of the in vivo gene transfer into the muscle are represented in FIG. 7. These results indicate that at the hydrotalcite / DNA mass ratio of 0.5 an increase in the luciferase activity by a factor of 14 is obtained compared to naked DNA. More generally, the DNA / hydrotalcite compositions have an improved transfection efficiency compared to that obtained by injection of naked DNA.
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Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IL15047600A IL150476A0 (en) | 1999-12-30 | 2000-12-27 | Compositions comprising nucleic acids incorporated in bilaminar mineral particles |
AU28604/01A AU2860401A (en) | 1999-12-30 | 2000-12-27 | Compositions comprising nucleic acids incorporated in bilaminar mineral particles |
CA002395742A CA2395742A1 (en) | 1999-12-30 | 2000-12-27 | Compositions comprising nucleic acids incorporated in bilaminar mineral particles |
EP00993726A EP1246931A1 (en) | 1999-12-30 | 2000-12-27 | Compositions comprising nucleic acids incorporated in bilaminar mineral particles |
MXPA02006528A MXPA02006528A (en) | 1999-12-30 | 2000-12-27 | Compositions comprising nucleic acids incorporated in bilaminar mineral particles. |
JP2001550397A JP2003519241A (en) | 1999-12-30 | 2000-12-27 | Composition containing nucleic acid incorporated in double-layered mineral particles |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR99/16707 | 1999-12-30 | ||
FR9916707A FR2803206A1 (en) | 1999-12-30 | 1999-12-30 | New composition, used in in vitro or ex vivo cellular transfection, comprises a nucleic acid and a mineral particle with an exchangeable layer structure |
US18567900P | 2000-02-28 | 2000-02-28 | |
US60/185,679 | 2000-02-28 |
Publications (1)
Publication Number | Publication Date |
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WO2001049869A1 true WO2001049869A1 (en) | 2001-07-12 |
Family
ID=26235199
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2000/003702 WO2001049869A1 (en) | 1999-12-30 | 2000-12-27 | Compositions comprising nucleic acids incorporated in bilaminar mineral particles |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP1246931A1 (en) |
JP (1) | JP2003519241A (en) |
AU (1) | AU2860401A (en) |
CA (1) | CA2395742A1 (en) |
IL (1) | IL150476A0 (en) |
MX (1) | MXPA02006528A (en) |
WO (1) | WO2001049869A1 (en) |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2826279A1 (en) * | 2001-06-26 | 2002-12-27 | Aventis Pasteur | VACCINE COMPOSITION COMPRISING A HYDROTALCITE TYPE COMPOUND |
DE10237518A1 (en) * | 2002-08-16 | 2004-02-26 | Süd-Chemie AG | Removing biomolecules from liquid media, useful for purification, product recovery or preparation of vectors, comprises retention on hydrotalcite of controlled particle size |
WO2010060110A1 (en) * | 2008-11-24 | 2010-05-27 | Northwestern University | Polyvalent rna-nanoparticle compositions |
JP2010524964A (en) * | 2007-04-17 | 2010-07-22 | バクスター・インターナショナル・インコーポレイテッド | Nucleic acid microparticles for pulmonary delivery |
US7897051B2 (en) | 2005-12-16 | 2011-03-01 | Sud-Chemie Ag | Method for separating proteins from liquid media |
US8999947B2 (en) | 2005-06-14 | 2015-04-07 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
US9376690B2 (en) | 2009-10-30 | 2016-06-28 | Northwestern University | Templated nanoconjugates |
US9506056B2 (en) | 2006-06-08 | 2016-11-29 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
US9890427B2 (en) | 2007-02-09 | 2018-02-13 | Northwestern University | Particles for detecting intracellular targets |
US9889209B2 (en) | 2011-09-14 | 2018-02-13 | Northwestern University | Nanoconjugates able to cross the blood-brain barrier |
US10098958B2 (en) | 2009-01-08 | 2018-10-16 | Northwestern University | Delivery of oligonucleotide functionalized nanoparticles |
US10837018B2 (en) | 2013-07-25 | 2020-11-17 | Exicure, Inc. | Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use |
US11213593B2 (en) | 2014-11-21 | 2022-01-04 | Northwestern University | Sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates |
US11364304B2 (en) | 2016-08-25 | 2022-06-21 | Northwestern University | Crosslinked micellar spherical nucleic acids |
US11433131B2 (en) | 2017-05-11 | 2022-09-06 | Northwestern University | Adoptive cell therapy using spherical nucleic acids (SNAs) |
US11957788B2 (en) | 2014-06-04 | 2024-04-16 | Exicure Operating Company | Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications |
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EP0681833A2 (en) * | 1994-05-11 | 1995-11-15 | Dott Research Laboratory | Nasally administrable compositions |
WO1997023241A1 (en) * | 1995-12-21 | 1997-07-03 | Quest International B.V. | Particle compositions |
EP0987328A2 (en) * | 1998-09-11 | 2000-03-22 | Jin Ho Choy | Bio-inorganic hybrid composite capable of storing and carrying genes and preparation thereof |
-
2000
- 2000-12-27 IL IL15047600A patent/IL150476A0/en unknown
- 2000-12-27 WO PCT/FR2000/003702 patent/WO2001049869A1/en not_active Application Discontinuation
- 2000-12-27 CA CA002395742A patent/CA2395742A1/en not_active Abandoned
- 2000-12-27 AU AU28604/01A patent/AU2860401A/en not_active Abandoned
- 2000-12-27 JP JP2001550397A patent/JP2003519241A/en not_active Withdrawn
- 2000-12-27 EP EP00993726A patent/EP1246931A1/en not_active Withdrawn
- 2000-12-27 MX MXPA02006528A patent/MXPA02006528A/en unknown
Patent Citations (3)
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EP0681833A2 (en) * | 1994-05-11 | 1995-11-15 | Dott Research Laboratory | Nasally administrable compositions |
WO1997023241A1 (en) * | 1995-12-21 | 1997-07-03 | Quest International B.V. | Particle compositions |
EP0987328A2 (en) * | 1998-09-11 | 2000-03-22 | Jin Ho Choy | Bio-inorganic hybrid composite capable of storing and carrying genes and preparation thereof |
Non-Patent Citations (2)
Title |
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CHOY JH. ET AL.: "Inorganic Layered Double Hydroxides as Nonviral Vectors", ANGEW CHEM INT ED ENGL 2000 NOV 17;39(22):4041-4045., XP002167062 * |
CHOY, JIN-HO ET AL: "Intercalative Nanohybrids of Nucleoside Monophosphates and DNA in Layered Metal Hydroxide", J. AM. CHEM. SOC. 121(6), 1399-1400, 17 February 1999 (1999-02-17), XP002146837 * |
Cited By (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2826279A1 (en) * | 2001-06-26 | 2002-12-27 | Aventis Pasteur | VACCINE COMPOSITION COMPRISING A HYDROTALCITE TYPE COMPOUND |
WO2003000284A1 (en) * | 2001-06-26 | 2003-01-03 | Aventis Pasteur | Vaccine composition comprising a hydrotalcite-type compound |
DE10237518A1 (en) * | 2002-08-16 | 2004-02-26 | Süd-Chemie AG | Removing biomolecules from liquid media, useful for purification, product recovery or preparation of vectors, comprises retention on hydrotalcite of controlled particle size |
US8999947B2 (en) | 2005-06-14 | 2015-04-07 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
US10370661B2 (en) | 2005-06-14 | 2019-08-06 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
US9719089B2 (en) | 2005-06-14 | 2017-08-01 | Northwestern University | Nucleic acid functionalized nonoparticles for therapeutic applications |
US7897051B2 (en) | 2005-12-16 | 2011-03-01 | Sud-Chemie Ag | Method for separating proteins from liquid media |
US10370656B2 (en) | 2006-06-08 | 2019-08-06 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
US9506056B2 (en) | 2006-06-08 | 2016-11-29 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
US9890427B2 (en) | 2007-02-09 | 2018-02-13 | Northwestern University | Particles for detecting intracellular targets |
JP2010524964A (en) * | 2007-04-17 | 2010-07-22 | バクスター・インターナショナル・インコーポレイテッド | Nucleic acid microparticles for pulmonary delivery |
US9844562B2 (en) | 2008-11-24 | 2017-12-19 | Northwestern University | Polyvalent RNA-nanoparticle compositions |
US9139827B2 (en) | 2008-11-24 | 2015-09-22 | Northwestern University | Polyvalent RNA-nanoparticle compositions |
US10391116B2 (en) | 2008-11-24 | 2019-08-27 | Northwestern University | Polyvalent RNA-nanoparticle compositions |
WO2010060110A1 (en) * | 2008-11-24 | 2010-05-27 | Northwestern University | Polyvalent rna-nanoparticle compositions |
US10098958B2 (en) | 2009-01-08 | 2018-10-16 | Northwestern University | Delivery of oligonucleotide functionalized nanoparticles |
US11633503B2 (en) | 2009-01-08 | 2023-04-25 | Northwestern University | Delivery of oligonucleotide-functionalized nanoparticles |
US9757475B2 (en) | 2009-10-30 | 2017-09-12 | Northwestern University | Templated nanoconjugates |
US9376690B2 (en) | 2009-10-30 | 2016-06-28 | Northwestern University | Templated nanoconjugates |
US9889209B2 (en) | 2011-09-14 | 2018-02-13 | Northwestern University | Nanoconjugates able to cross the blood-brain barrier |
US10398784B2 (en) | 2011-09-14 | 2019-09-03 | Northwestern Univerity | Nanoconjugates able to cross the blood-brain barrier |
US10837018B2 (en) | 2013-07-25 | 2020-11-17 | Exicure, Inc. | Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use |
US10894963B2 (en) | 2013-07-25 | 2021-01-19 | Exicure, Inc. | Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use |
US11957788B2 (en) | 2014-06-04 | 2024-04-16 | Exicure Operating Company | Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications |
US11213593B2 (en) | 2014-11-21 | 2022-01-04 | Northwestern University | Sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates |
US11364304B2 (en) | 2016-08-25 | 2022-06-21 | Northwestern University | Crosslinked micellar spherical nucleic acids |
US11433131B2 (en) | 2017-05-11 | 2022-09-06 | Northwestern University | Adoptive cell therapy using spherical nucleic acids (SNAs) |
Also Published As
Publication number | Publication date |
---|---|
JP2003519241A (en) | 2003-06-17 |
CA2395742A1 (en) | 2001-07-12 |
EP1246931A1 (en) | 2002-10-09 |
AU2860401A (en) | 2001-07-16 |
IL150476A0 (en) | 2002-12-01 |
MXPA02006528A (en) | 2002-11-29 |
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