WO2014187878A1 - Étalons internes d'adn pour des analyses par micro-électrophorèse - Google Patents

Étalons internes d'adn pour des analyses par micro-électrophorèse Download PDF

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Publication number
WO2014187878A1
WO2014187878A1 PCT/EP2014/060483 EP2014060483W WO2014187878A1 WO 2014187878 A1 WO2014187878 A1 WO 2014187878A1 EP 2014060483 W EP2014060483 W EP 2014060483W WO 2014187878 A1 WO2014187878 A1 WO 2014187878A1
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lipid
capsules
dna
liposome
nucleic acid
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PCT/EP2014/060483
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English (en)
Inventor
Annie Madeleine Jeanne VIALLAT
Yannick Aman Baptiste Combet-Blanc
Katia COSENTINO
Michel DE MEO
Original Assignee
Institut National De La Sante Et De La Recherche Medicale (Inserm)
Centre National De La Recherche Scientifique (C.N.R.S)
Institut De Recherche Pour Le Developpement
Universite D'aix-Marseille
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Publication of WO2014187878A1 publication Critical patent/WO2014187878A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/586—Liposomes, microcapsules or cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard

Definitions

  • the present invention relates to capsules enclosing nucleic acid fragments of variable controlled sizes suitable to be used as an internal standard in micro-electrophoresis assays, such as single cell micro-electrophoresis assays.
  • DNA is an essential macromolecule in eukaryotic and prokaryotic cells, and its integrity is crucial for the survival of organisms. Any factor affecting the integrity of genomic DNA is genotoxic.
  • the development of analytical tools to assess DNA damage in living cells represents a fundamental issue in all research fields of biology.
  • the investigative techniques available so far, such as micronucleus test, comet assay and Ames test among the most known are increasingly used in many analytical fields such as biomonitoring of environmental quality and health, environmental toxicology, genotoxicity of chemical products on humans, cancer and degenerative diseases.
  • the single cell micro-electrophoresis assay also known as "comet assay” is one of the most sensitive analysis techniques of genotoxicity and is therefore increasingly recognized in the analytical field.
  • the results of the assay show variability issues related to the method of cell sample preparation and due to the analysis of comet images. As a major consequence, the results are difficult to compare over different laboratories, and quantitative measurements with this method are not yet possible.
  • the inventors developed an internal standard consisting of capsules encapsulating nucleic acid fragments of variable controlled sizes in an aqueous gel. The inventors discovered that during preparation of the capsules the formation of the shell is essential.
  • the shell which can be constituted of a monolayer, a lipid bilayer or a series of lipid bilayers, gives rise to capsules that are perfectly circularly shaped and that are homogenous in size.
  • the shell protects the core-enclosing an aqueous gel and the at least one nucleic acid- from aggregation, nucleic acid leakage and prevents the capsules from attaching to each other and to the wall of the tube in which they are prepared and/or stored.
  • the shell of the capsules of the invention is not necessary to the capsules to be an efficient standard during the electrophoresis assay and can thus be removed prior to use, for example by a centrifugation step in water.
  • the shell may be removed during the single cell micro-electrophoresis assay, in particular during the lysis step of the single cell micro-electrophoresis assay.
  • the capsules of the invention in particular capsules wherein the shell comprises or is constituted of a lipid bilayer or a surfactant monolayer, preferably a lipid bilayer, can be easily distinguished, due to their perfect circular head, the form of the comet and the intensity of the fluorescence emitted by the labeled nucleic acids in comparison to the studied cells and can thus be directly integrated into the samples to be analyzed.
  • Liposomes which have a lipid biomimetic membrane, were considered to be the best candidates for being used in the single cell micro-electrophoresis assay.
  • the smallest ones size 50 - 150 nm
  • Cell-size liposomes named giant lipid vesicles, which were available until now, are very fragile and thus not usable in comet assays.
  • the methods of preparation of these liposomes have several disadvantages. The simplest preparation technique is to hydrate a lipid film. Using this technique, the yields of vesicles and the encapsulation efficiency are low. Furthermore, the vesicle size cannot be controlled.
  • More recent inverted-emulsion techniques are based on the preparation of a water/oil emulsion. Microdroplets are produced and then forced, by centrifugation, to go through an oil/water interface and form vesicles. This method has high encapsulation efficiency and a controlled vesicle size when combined with a microfluidic method that gives monodisperse emulsions (Pautot, S. et al., 2003, Langmuir, 19: 2870). However, the efficiency of this technique is still very low because membranes are fragile, and many vesicles break during the centrifugation stage.
  • liposomes filled with nucleic acids suffer from a low stability, in particular during gel electrophoresis, which hampers their use as internal standards in micro-electrophoresis assays.
  • the inventors have unexpectedly shown that it was possible to produce stable capsules comprising a shell enclosing an elastic resistant gel filled with size-controlled nucleic acid fragments. To the inventors' knowledge such capsules have never been reported so far.
  • the inventors have unexpectedly shown that it was possible to produce stable giant lipid vesicles constituted by a lipid membrane enclosing an elastic resistant gel filled with size-controlled nucleic acid fragments.
  • liposomes To the inventors' knowledge such vesicles, herein called liposomes, have never been reported so far.
  • capsules comprising a surfactant monolayer enclosing an elastic resistant gel filled with size-controlled nucleic acid fragments are very stable and homogenous in size.
  • the inventors previously prepared gel-filled vesicles by encapsulating agarose (Viallat A. et al., 2004, Biophys. J., 86: 2179) or PolyNipam gels (Campillo, C. et al., 2007, Soft Matter, 3: 1421 ) using a simple hydration technique. Nevertheless, this preparation is not adapted to encapsulate nucleic acids. Moreover, agarose-filled vesicles were produced only in small quantities with low reproducibility and the method of preparation of PolyNipam vesicles was complicated and used toxic products. In the present invention the inventors achieved to obtain capsules encapsulating high concentrations of long calibrated nucleic acid fragments in an aqueous gel.
  • the inventors achieved to obtain liposomes encapsulating, inside a lipid bilayer, high concentrations of long calibrated nucleic acid fragments in an aqueous gel using a method comprising serial specific stages and involving double emulsion method and/or microfluidics.
  • the inventors achieved to obtain capsules encapsulating, inside a surfactant or lipid monolayer, high concentrations of long calibrated nucleic acid fragments in an aqueous gel.
  • the lipid bilayer may be formed in different steps of the protocol of the preparation of the liposomes. Firstly, a lipid monolayer may be formed during the formation of the droplets in the oil phase and a second monolayer may be formed when the obtained capsules with a lipid monolayer are transferred in their oil-lipid solution to an aqueous phase and centrifuged. The second lipid monolayer is then typically formed when the capsule having one lipid monolayer crosses the oil-aqueous solution interface during centrifugation. Therefore, in a further embodiment, the inventors obtained liposomes by transferring the capsules having a lipid monolayer to an aqueous phase, followed by centrifugation.
  • the inventors further showed that the capsules, comprising a lipid bilayer, a lipid monolayer or a surfactant monolayer, were reproducible with respect to their manufacture, stable, easy to handle and producible at low cost.
  • the inventors showed in particular that the capsules, were reproducible with respect to their manufacture, stable during at least 1 month, easy to handle and producible at low cost.
  • capsules having a lipid monolayer were reproducible with respect to their manufacture, stable during at least 6 month, easy to handle and producible at low cost.
  • the capsules having a lipid monolayer of the invention do not suffer from nucleic acid leakage, they do not aggregate and are stable during electrophoresis. Furthermore, the capsules comprising a lipid monolayer still form liposomes comprising a lipid bi-layer after centrifugation in water or in buffer or in saccharide solution, that are stable during electrophoresis.
  • capsules comprising a surfactant monolayer were reproducible with respect to their manufacture, stable during at least 6 weeks, preferably at least 10 weeks, easy to handle and producible at low cost.
  • the capsules of the invention comprising a surfactant monolayer do not suffer from nucleic acid leakage, they do not aggregate and are stable during electrophoresis.
  • the capsules of the invention in particular the liposomes of the invention, enclosing calibrated nucleic acids in an aqueous gel are particularly well suited to be used as internal standards in single cell electrophoresis assays by adding them directly to the eukaryotic or prokaryotic cell sample to be analyzed.
  • the inventors provided a quantitative comparison between the characteristic parameters of comets obtained from said capsules, in particular said liposomes, in various experimental conditions and those of comets of the studied cells obtained in the same experimental conditions.
  • the inventors designed a method of calibration providing a reference set of calculated parameters enabling a normalization of the calculated parameters from cell comets, thus allowing a quantitative comparison with data obtained from other single cell electrophoresis assays.
  • the capsule of the invention in particular the liposome of the invention, used as internal standard paves the way for broader genotoxicity tests on both prokaryotic and eukaryotic cells. Its use in single cell electrophoresis assays reduces the variability in intra- and inter-assay experiments and represents therefore an interesting approach for research in cancer, ecotoxicity and aging.
  • the present invention thus relates to a capsule comprising or consisting of a core enclosing an aqueous gel comprising at least one hydrophilic polymer dispersed in water or buffer and at least one nucleic acid.
  • the capsule further comprises a shell.
  • the shell may comprise or be constituted of at least one monolayer or at least one lipid bilayer.
  • the invention refers to a liposome comprising or consisting of:
  • lipid bilayer comprising at least one liposome-forming lipid
  • core enclosing an aqueous gel comprising at least one hydrophilic polymer dispersed in water or buffer and at least one nucleic acid.
  • the invention further relates to a method of preparation of said capsule comprising the following steps:
  • step c) preparation of an emulsion of the aqueous gel comprising at least one nucleic acid obtained in step b) in the oil solution obtained in step a), to obtain nucleic acid-aqueous gel droplets,
  • the method may further comprise an optional step f), wherein the capsules are transferred to an aqueous solution and the shell of the capsules obtained in step d) or e) might be removed.
  • the method may further comprise an optional step g) of adding at least one additional lipid monolayer to the lipid monolayer of the capsule obtained in step d) or e).
  • the present invention also concerns a capsule obtainable by said method of preparation.
  • the invention further relates to the use of said capsules as an internal standard in electrophoresis assays, wherein the electrophoresis assay may be a micro- electrophoresis assay and/or capillary electrophoresis assay.
  • the invention further provides a method of calibration of a single cell microelectrophoresis assay comprising:
  • capsule refers to an artificially-prepared capsule.
  • the "capsule” of the invention comprises a core enclosing an aqueous gel comprising at least one hydrophilic polymer dispersed in water or in buffer and at least one nucleic acid.
  • the capsule further comprises a shell.
  • the term "shell” refers to a selective barrier enclosing an aqueous gel comprising at least one hydrophilic polymer dispersed in water or in buffer and at least one nucleic acid.
  • the shell of the capsule of the invention may comprise or be constituted of at least one lipid bilayer or at least one monolayer.
  • the shell comprises or is constituted of at least one lipid bilayer or a series of lipid bilayers.
  • Liposomes may comprise one or more lipid bilayer(s).
  • capsules of the invention comprising a shell which comprises or is constituted of at least one lipid bilayer are called liposomes.
  • liposomes are often called vesicles. Therefore, as used herein, the terms "capsule with at least one lipid bilayer", “liposome” and “vesicle” can be used interchangeably.
  • the "liposome” of the invention is an artificially-prepared liposome.
  • the shell comprises or is constituted of at least one monolayer, wherein said monolayer preferably comprises at least one compound selected from the group constituted of a liposome-forming lipid, a surfactant and a lipid, preferably a liposome-forming lipid or a surfactant.
  • the capsule comprising a shell which comprises or is constituted of at least one monolayer is referred to as "capsule with a monolayer”.
  • the shell in particular the monolayer, more particularly the surfactant monolayer, may be removed after the preparation of the capsules, preferably prior to use, leaving the core of the capsule enclosing an aqueous gel comprising at least one hydrophilic polymer dispersed in water or in buffer and at least one nucleic acid.
  • the capsule core As used herein, the capsule without a shell is referred to as the "capsule core".
  • the size of the capsule of the invention is similar to that of a living cell.
  • the size may range from 1 to 200 ⁇ , in particular from 1 to 100 ⁇ .
  • the size of the capsule may be 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 m.
  • the size of the liposome of the invention may range from 5 to 200 ⁇ , in particular from 5 to 100 ⁇ .
  • the size of the liposome may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 m.
  • the size of the capsule with a monolayer in context of the invention may range from 1 to 100 ⁇ , in particular from 1 to 80 ⁇ , preferably 1 to 60 ⁇ .
  • the size of the capsule with a monolayer may be 1 , 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 m.
  • the capsules of the invention comprise a core enclosing an aqueous gel comprising at least one nucleic acid.
  • the capsule according to the invention comprises a core enclosing an aqueous gel comprising at least one hydrophilic polymer dispersed in water or in buffer and at least one nucleic acid.
  • the “core” refers to the inner part of the capsule, which may be enclosed by the shell.
  • the “core” refers to the inner part of the liposome enclosed by the lipid bilayer.
  • aqueous gel building the core of the capsule, is crucial for the stability of the capsules of the invention, in particular of the liposomes of the invention.
  • Capsules, in particular liposomes, comprising nucleic acids without an aqueous gel break or collapse once embedded in the gel for electrophoresis ( Figures 1 -2).
  • aqueous gel and/or “hydroqel” refers to a colloidal gel in which water or buffer is the dispersion medium and maintains a three dimensional structure.
  • hydrogels are formed by polymerization and cross- linking of a hydrophilic polymer in an aqueous solution to cause the solution to gel.
  • the aqueous gel of the invention thus comprises at least one hydrophilic polymer dispersed in water or buffer.
  • a "polymer” is a chemical compound or mixture of compounds consisting of repeating structural units created through a process of polymerization. Polymers according to the invention react from a finite branched network to an infinite branched network, forming an aqueous gel.
  • the "aqueous gel” refers to a polymer undergoing the transition from a finite branched network to an infinite branched network, but also to the infinite branched network itself. Elsewhere the finite branched network might be described as a “sol”. The transition from a finite branched network to an infinite branched network is also called “cross-linking” and is specified furthermore in the paragraph “cross- linking”.
  • aqueous gel can be further described as a polymeric, cross-linked network structure.
  • the aqueous gel is a substantially dilute cross-linked system, and is categorized principally as weak or strong depending on its flow behavior in steady-state (Ferry, J. D.,1980, Viscoelstic properties of polymers, John Wiley & sons, New York, page : 486-544).
  • the aqueous gel may be obtained from synthetic and/or natural polymer which can absorb and retain significant amount of water.
  • the aqueous gel according to the invention is a hydrophilic polymer dispersed in water or buffer.
  • the aqueous gel prevents, in addition to the shell, in particular in addition to the lipid-bilayer, defined in the section "shell" below, the at least one nucleic acid from diffusing outside the capsule, in particular outside the liposome. Furthermore, the aqueous gel typically enhances the stability of the capsule, in particular of the liposome.
  • the capsule of the invention in particular the liposome of the invention
  • a gel for use in an electrophoresis assay such as agarose or polyacrylamide
  • the aqueous gel of the capsule, in particular of the liposome once exposed to an electric field, allows the at least one nucleic acid to migrate within its polymeric network into the surrounding gel in which the capsule of the invention, in particular the liposome of the invention is embedded.
  • the aqueous gel preferably excludes a degradation of the nucleic acid enclosed in said aqueous gel.
  • the aqueous gel of the invention is not toxic.
  • the preparation of said aqueous gel may not include any toxic hydrophilic polymers. Hydrophilic polymers
  • hydrophilic polymers contain polar or charged functional groups, rendering them soluble in water.
  • hydrophilic polymers are well-known from the skilled person and include, without limiting them, polyurethane, poly(ethylene glycol), polypropylene glycol), poly(vinylpyrrolidone), xanthan, methyl cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hyaluronan, polyvinyl alcohol, acrylic acid, vinyl caprolactam, methacrylic acid, poly(acrylic acid), chitosan, ⁇ -glycerophosphate, 2-acrylamido-2- methylpropanesulfonic acid, carboxymethyl cellulose, collagenpolyesters, gelatin, polyphosphazenes, polypeptides, xanthan gum, chitosangum arabicxanthan, carrageenan, gellan, welan, guar gum, locust bean gum, arabic gum, pectin, alginate, heparin, carrageenans, ⁇ -carrageenan, guar gum, agar, aga
  • the at least one hydrophilic polymer is selected from the group consisting of hyaluronan, carboxymethyl cellulose, xanthan gum, chitosan gum, arabic xanthan, carrageenan, gellan, welan, guar gum, locust bean gum, arabic gum, pectin, alginate, heparin, ⁇ -carrageenan, agar, agarose, starch, modified starch, microcrystalline cellulose, and chitin.
  • the at least one hydrophilic polymer is agarose, in particular low melting agarose.
  • Hydrophilic polymers may be of synthetic and/or natural origin, wherein "natural” refers to a molecule found in nature.
  • Synthetic refers to a molecule not found in nature or not normally found in a human.
  • hydrophilic polymers that are of natural origin may be derivatized with functional groups.
  • hydrophilic polymers contain polar or charged functional groups, rendering them soluble in water. Therefore, in one embodiment, the hydrophilic polymers might be charged.
  • the at least one hydrophilic polymer might be in particular at least one polyanion and/or at least one polycation.
  • Polyanions are for example xanthan, alginate, carboxymethyl cellulose, and carrageenan.
  • a polycation is for example chitosan.
  • the at least one hydrophilic polymer is a mixture of at least two different hydrophilic polymers.
  • cross-linking refers to linking macromolecular chains together, initially leading to progressively larger branched yet soluble polymers depending on the structure and conformation of the starting material.
  • Cross-linking thus refers to the transition process of a polymer from a finite branched network to an infinite branched network. Said transition from a system with finite branched polymer to infinite molecules is called “cross-linking” and or "gelation”. In literature the expression 'sol-gel transition' might also be used.
  • cross-linking and “gelation” are thus used interchangeably.
  • Aqueous gels may be prepared by different cross-linking techniques described in literature and known to the skilled in the art, such as “physical cross-linking” (Hennink, W. E. and Nostrum, C. F. v., 2002, Advanced Drug Delivery Reviews 54, 13-36.), “chemical cross-linking” (Barbucci, R. et al., 2004, J. Biomater. Sci. Polymer Edn. 15, 607-619.), “grafting polymerization” (Said, H. M. et al. 2004, Reactive and Functional Polymers 61 , 397 ⁇ 104), and “radiation cross-linking” (Fei, B. et al., 2000, Journal of Applied Polymer Science 78, 278-283; Liu, P. et ai, 2002, Radiation Physics and Chemistry 63, 525-528).
  • physical cross-linking Haennink, W. E. and Nostrum, C. F. v., 2002, Advanced Drug Delivery Reviews 54, 13-
  • the preparation of an aqueous gel in step b) and the gelation in step d), in particular the gelation in step d) of the method of preparation of the invention comprise physical cross-linking, chemical cross-linking, grafting polymerization and/or radiation cross-linking, in particular physical cross-linking and/or chemical cross- linking, more particularly physical cross-linking.
  • the cross-linking preferably does not affect the at least one nucleic acid included in the aqueous gel.
  • nucleic acid is not modified, cross-linked or denatured.
  • "physical cross-linking” might comprise heating and cooling and/or adding di- or tri-valent counter ions, ionic interactions and/or changing of the pH.
  • the cross-linking preferably does not affect the cage that may surround the at least one nucleic acid.
  • physical cross-linking comprises heating and cooling.
  • heating might be applied during the preparation of an aqueous gel in step b) of the method of preparation of the invention and cooling might be applied preferably during the gelation in step d) of the method of preparation of the invention.
  • Heating refers to increasing the temperature of the hydrophilic polymer dispersed in water or buffer above room temperature, in particular increasing the temperature to a temperature of 25°C to 100°C, more particularly 30C to 70°C, particularly 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 °C, for 1 min to 2 h, in particular for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120 min.
  • Cooling refers to decreasing the temperature of the hydrophilic polymer dispersed in water or buffer below room temperature, in particular decreasing the temperature to a temperature of 25°C to 0 °C, more prticularly to 10°C to 0 °C, particularly to 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10°C, 10 min to 3h, in particular for 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120 140, 160, 180 min.
  • hydrophilic polymers form an aqueous gel by heating and cooling.
  • examples are gelatin, carrageenan and agarose.
  • the hydrophilic polymers might need the presence of ions for polymerization.
  • the physical cross-linking may comprise the addition of di- or tri-valent counter ions.
  • “Addition of di- or tri-valent counter ions” induces cross-linking due to the principle of gelling a polyelectrolyte solution with a multivalent ion of opposite charges (e.g.Ca 2+ + 2CI " ).
  • Some examples are, without being limited, Na + alginate, chitosan-polylysine (Bajpai, A. K. et at., 2008, Progress in Polymer Science 33: 1088-1 1 18) and chitosan-glycerol phosphate salt (Zhao, Q. et al., 2009, Carbohydrate Polymers 76 : 410-416) and chitosan- dextran hydrogels (Hennink, W. E. and Nostrum, C. F. v., 2002, Advanced Drug Delivery Reviews 54 13-36).
  • ions are added in step b) of the method of preparation of the invention.
  • ions might be added in the form of a buffer or in the form of a salt.
  • the capsule of the invention in particular the liposome of the invention, may additionally comprise ions and/or a buffer.
  • a “buffer” is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. Its pH changes very little when a small amount of strong acid or base is added to it and thus it is used to prevent changes in the pH of a solution. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications. Buffers used in the context of the invention might be PBS (Phosphate buffered saline) or TRIS (tris(hydroxymethyl)aminomethane).
  • the buffer consists of 100mM NaCI, 10mM Tris, 1 mM CaCI 2 , 1 mM MgCI 2 , 1 mM EDTA at pH8.0.
  • the buffer consists of 100mM NaCI, 20mM Tris, 1 mM CaCI 2 ,
  • An "ion” is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving the atom or molecule a net positive (cation) or negative (anion) electrical charge.
  • An ion consisting of a single atom is an atomic or monatomic ion; if it consists of two or more atoms, it is a molecular or polyatomic ion. Ions are further distinct according to the charge they carry. Therefore an ion can be a monovalent ion or bivalent (sometimes called divalent ion) or polyvalent ions. Ions may be without limitation F, CI " , ⁇ , NH 4 + S0 4 2+ Ca 2+ Mg 2+
  • the ion has a negative electrical charge.
  • physical cross-linking comprises ionic interactions.
  • ionic interactions refers to mixing of a polyanion with a polycation.
  • the underlying principle of this method is that polymers with opposite charges stick together and form soluble and insoluble complexes depending on the concentration and pH of the respective solutions.
  • polyanionic xanthan with polycationic chitosan WO 0004086.
  • the at least one hydrophilic polymer is a mixture of a polyanion and a polycation, in particular the mixture of polyanionic xanthan with polycationic chitosan.
  • Cross-linking by "changing the pH” can be obtained for example by lowering the pH of aqueous solution of polymers carrying carboxyl groups.
  • the at least one hydrophilic polymer is a hydrophilic polymer with a carboxyl group.
  • One example is a hydrogen-bound carboxymethyl cellulose network formed by dispersing carboxymethyl cellulose into 0.1 M HCI (Takigami, M. et al., 2007, Transactions of the Materials Research, Society of Japan, Vol 32, No 3 (32): 713-716).
  • the mechanism involves replacing the sodium in carboxymethyl cellulose with hydrogen in the acid solution to promote hydrogen bonding.
  • the hydrogen bonds induce a decrease of carboxymethyl cellulose solubility in water and result in the formation of an elastic hydrogel.
  • CM- chitosan carboxymethylated chitosan hydrogels prepared by cross-linking in the presence of acids or polyfunctional monomers, polyacrylic acid and polyethylene oxide (PEO-PAAc) based hydrogel prepared by lowering the pH to form H-bonded gel in their aqueous solution
  • PEO-PAAc polyacrylic acid and polyethylene oxide
  • xanthan-alginate mixed system molecular interaction of xanthan and alginate causes the change in matrix structure due to intermolecular hydrogen bonding between them resulting in formation of insoluble hydrogel network.
  • Cyhemical cross-linking refers to cross-linking of natural and synthetic polymers through the reaction of their functional groups (such as OH, COOH, and NH 2 ) with cross- linkers such as aldehyde.
  • Cross-linker refers to a chemical compound that links the polymers together due to its reaction with the functional groups.
  • the chemical cross-linking does not affect the at least one nucleic acid.
  • cross-linkers are to be excluded from the invention due to its undesired effect of cross-linking nucleic acids.
  • One such example is glutaraldehyde.
  • the aqueous gel included in the core of the capsules of the invention, in particular of the liposomes of the invention, preferably has pH ranging from 4 to 8, preferably 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 and most preferably, from 7 to 8, in particular 7, 7.2, 7.4, 7.6, 7.8, 8.
  • the pH of the hydrogel is preferably compatible with electrophoresis assays.
  • the concentration of the hydrophilic polymer within the hydrogel depends on the nature of hydrophilic polymer.
  • the concentration of the hydrophilic polymer is 0,001 % w/w to 80% w/w, in particular 0.01 % w/w to 30% w/w, particularly 0.1 % w/w to 30% w/w, more particularly 0.1 % w/w to 20% w/w.
  • the concentration is in particular more than 20%w/w, in particular 20%w/w to 40%w/w, particularly 20%w/w, 22%w/w, 24%w/w, 26%w/w, 28%w/w, 30%w/w, 32%w/w, 34%w/w, 36%w/w; 38%w/w, 40%w/w.
  • the concentration of pectin might be from 0.1 %w/w to 40%w/w, in particular 1 %w/w to 20%w/w, preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20%w/w.
  • the concentration of agarose might be from 0.01 %w/w to 3% w/w, in particular,
  • 0.3% to 2% preferably 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0%w/w.
  • nucleic acid refers to RNA or DNA, including cDNA, genomic DNA, recombinant DNA and synthetic DNA, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA, siRNA, micro-RNA, ribozymes comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A,” guanine “G,” thymine “T,” and cytosine “C”) or RNA (e.g. A, G, uracil "U,” and C). Nucleic acids can have any three-dimensional structure.
  • a nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense strand).
  • Non-limiting examples of nucleic acids include genomic DNA, genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant nucleic acids such as plasmid DNA, and branched nucleic acids.
  • a nucleic acid may contain unconventional or modified nucleotides.
  • the nucleic acid used in the context of the invention is DNA, wherein the DNA may be recombinant and/or genomic DNA.
  • Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources.
  • a recombinant DNA molecule is a hybrid molecule comprising at least two nucleotide sequences not normally found together in nature.
  • recombinant DNA examples include plasmids, viral vector DNA enclosed in capsids, cosmids, and artificial chromosomes.
  • Recombinant DNA may be for example Bacteriophage T5 DNA or lambda phage DNA or lambda DNA, wherein the Bacteriophage T5 DNA, lambda DNA or lambda phage DNA might be in particular commercially available.
  • Genomic DNA is the DNA found in the organisms genome and is passed on to an offspring as information necessary for survival. Genomic DNA applies specifically to the DNA stored on a complete set of nuclear DNA (i.e., the "nuclear genome”) but can also be applied to that stored within organelles that contain their own DNA, as with the "mitochondrial genome” or the “chloroplast genome”.
  • Genomic DNA may be, without limitation to it, from E.Coli or salmon sperm wherein the genomic DNA might be in particular commercially available.
  • the recombinant DNA and/or genomic DNA may be fragmented.
  • DNA fragments used in the context of the invention have a size of between 1 and 10,000kb, preferably between 1 and 1000 kb, more preferably between 10kb and 1000kb.
  • DNA fragments might be 10kb to 210kb, most preferably DNA fragments might be from 10kb to 50kb, 50 to 100 kbp, 100 to 150 kbp or 150 to 210 kbp.
  • DNA fragments might be from 10kb to 50kb such as 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50kb.
  • Techniques of fragmentation of nucleic acids include sonication, pipetting, vortexing and/or enzymatic digestion, in particular sonication, vortexing and/or enzymatic digestion.
  • fragmented DNA can be a mixture of DNA molecules of different lengths, thus a mixture of different sized fragments.
  • this mixture of different sized DNA fragments is a "DNA Ladder".
  • DNA Ladders are commercial available from, for example, New England Biolabs, Thermo Scientific, Promega, Bio Rad.
  • the "enzymatic digestion" of nucleic acids may be performed by the use of restriction enzymes. Techniques and protocols of enzymatic digestion of nucleic acid are well known to the skilled in the art.
  • the fragments of the nucleic acids might be purified before the preparation of a solution of at least one nucleic acid at step b1 ) of the method of preparation of the invention.
  • Vortexing for fragmentation of the nucleic acids might be performed during any step of the method of preparation of the invention, more particularly during any of steps b1 ) to b3) of the method of preparation of the invention.
  • vortexing may be performed at 4 Hz to 50 Hz for 30s to 10 min, preferably at 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 26, 28, 40, 42, 44, 46, 48, 50 Hz for preferably 1 s to 3 min, in particular from 10s to 3 min, more particularly for 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170,180 s.
  • vortexing might be performed by successive impulsions at 4Hz to 50Hz for 1 s to 30s.
  • genomic DNA is fragmented by vortexing at 10 Hz, 20Hz and 30Hz each time for 3min at 60 °C and/or at 40Hz for2min during step b2) and or b3) of the method of preparation of the invention.
  • genomic DNA may be fragmented in the vortex step of the preparation of the emulsion of step c) of the method of preparation of the invention.
  • the at least one nucleic acid present in the aqueous gel may have a concentration of 0.01 Mg/ ⁇ to 100 Mg/ ⁇ , preferably 0.05 Mg/ ⁇ to 10 Mg/ ⁇ .
  • the nucleic acid used in the context of the invention might be labeled for example with a fluorescence label. Fluorescent labeling techniques are known to the skilled in the art and include labeling with SYBR GREEN, SYTO 24, DAPI, Ethidium bromide, Propidium iodide. In a further embodiment the at least one nucleic acid may be surrounded by a cage.
  • a “cage” according to the invention is a structure that surrounds the nucleic acids and has three distinct surfaces such as the inside, outside and the interface.
  • the cage is different from the shell that the capsule of the invention can comprise.
  • the cage is preferably a protein cage.
  • a "protein cage” consists of protein subunits and is highly symmetric in its structure.
  • a protein cage may be purified and/or artificially assembled.
  • Protein cages may be for example virus capsids or other structurally related cage-like proteins such as ferritins, DNA-binding proteins from starved cells (dps), and heat shock proteins.
  • the cage is a viral capsid.
  • the viral capsid may be selected for example from the capsid of Cowpea Chlorotic Mottle Virus (CCMV), the capsid from Brome Mosaic Virus (BMV), the capsid from Tabacco Mosaic Virus(TMV), the capsid from Cowpea Mosaic Virus (CPMV) and the capsid of Bacteriophage T5.
  • the viral capsid is preferably the capsid of Bacteriophage T5.
  • the capsid may protect the nucleic acid from fragmentation during vortexing as for example during the vortex step of the preparation of the emulsion of step c) of the method of preparation of the invention.
  • the cage may provide an additional protection to prevent the at least one nucleic acid to diffuse outside the capsule.
  • the at least one nucleic acid is surrounded by a viral capsid, preferably by the capsid of Bacteriophage T5.
  • the capsule of the invention comprises a lipid- bilayer and a core enclosing an aqueous gel comprising at least one hydrophilic polymer dispersed in water or buffer and nucleic acids of 120 kbp surrounded by a capsid of bacteriophage T5.
  • the capsule of the invention comprises a surfactant monolayer and a core enclosing an aqueous gel comprising at least one hydrophilic polymer dispersed in water or buffer and nucleic acids of 120 kbp surrounded by a capsid of bacteriophage T5.
  • the capsule of the invention comprises a surfactant monolayer and a core enclosing an aqueous gel comprising at least one hydrophilic polymer dispersed in water or in a buffer and a mixture of genomic DNA and nucleic acids of 120 kbp surrounded by a capsid of bacteriophage T5.
  • Genomic DNA can be E. Coli DNA or Salmon sperm DNA from testes.
  • the cage can be destroyed.
  • the cage may be destroyed by detergents, in particular by cell lysis buffers used in the single cell electrophoresis assay.
  • the core of the capsule may further comprise a shell.
  • the "shell" of the capsule has an amphiphilic nature and due to its amphiphilic nature, is a selective barrier.
  • the shell prevents the at least one nucleic acid included in the core of the capsule, to diffuse outside the capsule.
  • nucleic acid included in the capsule should not be released before the capsule is voluntarily destroyed.
  • the shell preferably protects the capsules from aggregation.
  • the shell preferably prevents the capsules from attaching to each other and to the wall of the tube in which they are prepared and/or stored.
  • the shell enables the storage of the capsules in solution for more than 1 month, preferably more than 2 months, wherein, during said period, preferably no nucleic acid leaks and/or no aggregation of the capsules occurs.
  • the shell when the shell is a lipid monolayer it enables the storage of the capsules with a lipid monolayer in oil/lipid solution for at least 6 months, wherein, during said period, preferably no nucleic acid leaks and/or no aggregation of the capsules occurs.
  • the capsules comprising a lipid bilayer can be stored during a storage period in aqueous solution, wherein, during said period, preferably no nucleic acid leaks and/or no aggregation of the capsules occurs.
  • the capsules comprising a surfactant monolayer can be stored in oil/surfactant for at least 4 weeks, preferably at least 10 weeks, wherein, during said period, preferably no nucleic acid leaks and/or no aggregation of the capsules occurs.
  • the capsules of the invention are preferably prepared with a shell. During this preparation the shell gives rise to capsules that are perfectly circularly shaped and that are homogenous in size.
  • the shell may be removed.
  • the shell of the capsules can be destroyed.
  • the shell may be destroyed by detergents, in particular by cell lysis buffers used in the single cell electrophoresis assay.
  • the shell, in particular the monolayer can be destroyed by applying force, such as centrifugal force.
  • the shell in particular the monolayer, in particular the surfactant monolayer, can be destroyed by transferring the capsules from the oil phase, wherein they are stored, into an aqueous solution followed by mixing, for example by centrifugation.
  • the shell may comprise or be constituted of at least one monolayer or at least one lipid bilayer.
  • the at least one monolayer may comprise at least one compound selected from the group constituted of a liposome-forming lipid, a surfactant and a lipid.
  • the monolayer may comprise at least one compound selected from the group constituted of a liposome-forming lipid, a surfactant and a lipid, in particular a liposome-forming lipid and a surfactant.
  • the monolayer comprising at least one surfactant is herein referred to as a surfactant monolayer.
  • the monolayer comprising at least one surfactant may comprise more than one surfactant, preferably a mixture of two surfactants.
  • the surfactants that can be used for the preparation of the surfactant monolayer are defined in the section "Surfactants, lipids and proteins" herein below.
  • the monolayer comprising at least one liposome-forming lipid and/or at least one lipid is herein referred to as a lipid monolayer.
  • the monolayer comprising at least one liposome-forming lipid and/or at least one lipid may comprise more than one liposome- forming lipid or more than one lipid or a mixture of at least one liposome-forming lipid and at least one lipid.
  • the more than one liposome-forming lipid may be a mixture of liposome- forming lipids.
  • the more than one lipid may be a mixture of lipids.
  • liposome-forming lipids and lipids that can be used for the preparation of the lipid monolayer are defined in the section "liposome-forming lipid' and "Surfactants, lipids and proteins" herein below.
  • the monolayer prevents the at least one nucleic acid included in the core of the capsule to diffuse outside the capsule.
  • nucleic acid included in the capsule should not be released before the shell, or monolayer of the capsule is voluntarily destroyed, simultaneously with the cells to be analyzed, during the comet assay, using specific detergents and/or prior to use by centrifugation.
  • the monolayer preferably protects the capsule from aggregation.
  • the capsules comprising a monolayer can be stored in oil/surfactant or oil/lipid solution for preferably more than 1 month, wherein, during said period, preferably no nucleic acid leaks and/or no aggregation of the capsules occurs.
  • the capsules comprising a lipid monolayer can be stored in oil/lipid solution for preferably more than 6 month, wherein, during said period, preferably no nucleic acid leaks and/or no aggregation of the capsules occurs.
  • the capsules comprising a surfactant monolayer can be stored in oil/lipid solution for preferably more than 1 month, wherein, during said period, preferably no nucleic acid leaks and/or no aggregation of the capsules occurs.
  • the monolayer of the capsules can be destroyed by detergents, in particular by cell lysis buffers used in the single cell electrophoresis assay.
  • the monolayer, in particular the surfactant monolayer, of the capsules can be destroyed by centrifugal force, in particular by centrifugation in water.
  • the surfactant monolayer of the capsules can be destroyed by removing the capsules from the oil phase wherein they are stored and by transferring them in an aqueous solution followed by mixing, for instance by centrifugation in aqueous solution.
  • the capsule with a lipid monolayer can be used to prepare a capsule with a lipid bi-layer, according to step g) of the preparation of the capsules described herein below.
  • the at least one lipid bilayer may comprise at least one liposome-forming lipid.
  • the lipid-bilayer may further comprise at least one surfactant and/or at least on lipid.
  • the liposome-forming lipids, lipids and surfactants that can be used for the preparation of the lipid bilayer are defined in the sections "Liposome-forming lipid” and Surfactants, lipids and proteins" herein below.
  • the liposome or "capsule with at least one lipid bilayer" of the invention may comprise one or more lipid bilayer(s).
  • lipid bilayer can be used interchangeably with “membrane”.
  • the “membrane” or “lipid bilayer” of the liposome is, due to its hydrophobic nature, a selective barrier.
  • the liposome of the invention can be unilamellar or multilamellar.
  • Unilamellar liposomes are liposomes having a single lipid-bilayer, a so-called unilamellar membrane. Multilamellar liposomes have more than one membrane, each membrane being separated from the adjacent membrane by an aqueous layer.
  • the multilamellar liposome is a bilamellar liposome, wherein the membrane is bilamellar.
  • the lipid bilayer comprising at least one liposome- forming lipid prevents the at least one nucleic acid included in the core of the liposome, to diffuse outside the liposome.
  • the nucleic acid included in the liposome should not be released before the liposome is voluntarily destroyed, simultaneously with the cells to be analyzed, during the comet assay, using specific detergents.
  • the lipid bilayer preferably protects the liposome from aggregation.
  • the liposome is stable when it is suspended in an aqueous solution.
  • the lipid bilayer of the liposome can be destroyed by detergents, in particular by cell lysis buffers used in the single cell electrophoresis assay.
  • Liposome-forming lipid Liposome-forming lipid
  • the "liposome-forming lipid” is an amphiphilic molecule that is capable of forming liposomes, wherein an amphiphilic molecule comprises or consists of a hydrophobic tail and a hydrophilic head group.
  • Liposome-forming lipids might be natural or synthetic.
  • Liposome-forming lipids used in the context of the invention may form spontaneously, when in solution, in particular in aqueous solution, one lipid bilayer or a series of lipid bilayers, separated by water molecules.
  • the "liposome-forming lipids” may preferably form a monolayer of lipids.
  • the liposome forming-lipid preferably forms a lipid monolayer in step d) of the method of preparation and preferably forms a lipid bilayer when the capsule is centrifuged in aqueous solution.
  • At least one lipid and/or at least one surfactant can be added to at least one liposome-forming lipid to form a lipid monolayer or a lipid bilayer.
  • the liposome-forming lipid is chosen from the group constisting of phospholipids, glycolipids and sphingolipids, in particular from phospholipids.
  • Phospholipids are for example phosphatidylcholine (PC) such as L-a- phosphatidylcholine (Egg-PC), dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 -oleoyl-2-palmitoy-1 -sn-glycero-3-phosphocholine, 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1 ,2-dilauroyl-sn-glycero-3-phosphocholine (
  • the liposome-forming lipid used in the context of the invention is selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 -oleoyl-2-palmitoy-1 -sn-glycero-3-phosphocholine, and 1 ,2-dilauroyl-sn- glycero-3-phosphocholine (DLPC).
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • the liposome-forming lipid used in the context of the invention is L-a-phosphatidylcholine (egg-PC) and/or 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), more preferably 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
  • liposome-forming lipids derive from purified egg yolk, in particular phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidic acid (PA) and phosphatidylethanolamine (PE) may be derived from purified egg yolk.
  • synthetic phospholipids containing either altered aliphatic portions such as hydroxyl groups, branched carbon chains, cycloderivatives, aromatic derivatives, ethers, amides, polyunsaturated derivatives, halogenated derivatives or altered hydrophillic portions containing carbohydrate, glycol, phosphate, phosphonate, quaternary amine, sulfate, sulfonate, carboxy, amine, sulfhydryl, imidazole groups and combinations of such groups can be either substituted or intermixed with the above mentioned phospholipids and may be used as a liposome-forming lipid.
  • the liposome-forming lipid might contain a polyethylene glycol (PEG) group, in particular phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidic acid (PA) and phosphatidylethanolamine (PE) might contain a polyethylene glycol (PEG) group.
  • PEG polyethylene glycol
  • the liposome-forming lipid containing a polyethylene glycol (PEG) group might be in particular poly(ethyleneglycol)- phosphoethanolamine (PEG-PE).
  • the liposome-forming lipid might contain a biotin or avidin group to add adhesion properties to the lipid.
  • the at least one liposome-forming lipid may be a mixture or combination of more than one liposome-forming lipid, as defined above (at any ratio).
  • the at least one liposome-forming lipid is a mixture of at least two different liposome-forming lipids, as defined above, in particular a mixture of at least two liposome-forming lipids (at any ratio).
  • the mixture of liposome-forming lipids might be a mixture of phosphatidylcholines, a mixture of phosphatidylethanolamines or a mixture of phosphatidylcholines and phosphatidylethanolamines.
  • the mixture of liposome-forming lipids might be a mixture of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1 ,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC).
  • DPPC and DOPC might be mixed in a ratio (mol%) of 1 and 100 until 100 to 1 , for example in ratio of 30 and 70, 35 and 65, 40 and 60, 45 and 55, 50 and 50, 55 and 45, 60 and 40, 65 and 35, 70 and 30, in particular 50 and 50 mol%.
  • the at least one liposome-forming lipid is a mixture of egg- PC and PEG-PE. Said mixture might be prepared in a ratio of 98:2 mol% (egg-PC: PEG- PE).
  • the concentration of the liposome-forming lipid in the lipid oil solution can be between 0.05 mg/ml to 25 mg/ml, for example 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.5, 0.7, 0.8, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 25 mg/ml.
  • the at least one liposome-forming lipid is added during the preparation of the capsules, as further detailed in the section "method of preparation” below.
  • the at least one liposome-forming lipid may be added during the preparation of an oil solution in step a) of the method of preparation of the invention.
  • the at least one liposome-forming lipid is added first during the preparation of the oil solution in step a) and might be added in form of an additional oil-lipid solution during the step g) of the method of preparation of the invention.
  • the at least one liposome-forming lipid used in step a) and liposome-forming lipid used in step g) might be the same or might be different.
  • the shell may comprise or consist of at least one surfactant and/or at least one lipid.
  • the shell comprises or is constituted of at least one monolayer
  • the monolayer comprises at least one surfactant, at least one lipid or at least one liposome-forming lipid, preferably at least one liposome-forming lipid or at least one surfactant.
  • a “surfactant” is an amphiphilic molecule with a hydrophobic group and a hydrophilic group, wherein the amphiphilic molecule lowers the surface tension of a liquid, the interfacial tension between two liquids, and/or that between a liquid and a solid.
  • the surfactant might be used to prevent capsules from aggregation.
  • the at least one surfactant decreases fusogenicity of the capsules and therefore decreases aggregation.
  • the surfactant might further decrease coalescence and generates droplets of size similar to cells' sizes.
  • Surfactants are classified according to their polar head group, and include non- ionic surfactants and ionic surfactants encompassing anionic surfactants, cationic surfactants and zwitterionic surfactants.
  • the at least one surfactants may be a mixture of at least two different surfactants.
  • the at least one surfactant in the context of the invention is selected from the group consisting of non ionic surfactants such as polyglycerols, Tritons, polysorbates (Tweens) and different spans, such as triton X-100, tween 20, 40, 60, 80, span 20, 60, 80, polysorbat 20, 40, 60, 80, preferably polysorbats and spans, such as polysorbat (tween) 20, 40, 60, 80, span 20, 60, 80, in particular span80 and span 60, more preferably tween80 and span80.
  • non ionic surfactants such as polyglycerols, Tritons, polysorbates (Tweens) and different spans, such as triton X-100, tween 20, 40, 60, 80, span 20, 60, 80, polysorbat 20, 40, 60, 80, preferably polysorbats and spans, such as polysorbat
  • the inventors used a mixture of polysorbat 80 (tween80) and span80 to prepare a capsule having a surfactant monolayer.
  • the at least one surfactant is added during the preparation of the capsules, as further detailed in the section "method of preparation” below.
  • the at least one surfactant may be added during the preparation of an oil solution in step a) of the method of preparation of the invention.
  • the surfactant is added during the preparation of an oil solution in step a) of the method of preparation of the invention, in particular in step a3) of the method of preparation of the invention.
  • the at least one surfactant may be mixed with the oil in step a3).
  • the at least one surfactant might be added in a concentration of 0.01 % to 20% w/w, preferably, 0.1 to 10% w/w, more preferably 0.1 to 1 % w/w, in particular in concentrations of 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1 % w/w.
  • the lipid bilayer may additionally comprise at least one surfactant and/or at least one lipid.
  • the at least one surfactant or at least one lipid might be used to modulate the fluidity of the lipid-bilayer and to prevent liposomes from aggregation.
  • the at least one surfactant or the at least one lipid is preferably added during the preparation of the capsules, as further detailed in the section "method of preparation” below.
  • the at least one surfactant or the at least one lipid may be added during the preparation of an oil solution in step a) of the method of preparation of the capsules, more particularly during any of the steps a1 ) to a4) of the method of preparation of the invention.
  • the at least one surfactant or the at least one lipid may be mixed with at least one liposome-forming lipid, prior to the mixing with the at least one organic solvent in step a1 ).
  • the at least one surfactant or at least one lipid might be added in a ratio (surfactant to liposome-forming lipid) of 1 to 100 mol% to 1 to 1000 mol%.
  • lipids are defined as hydrophobic or amphiphilic small molecules comprising the following eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits).
  • Lipids thus include liposome-forming lipids described above in the section
  • liposome-forming lipids Nevertheless not all lipids are capable of forming liposomes.
  • the at least one lipid and at least one liposome-forming lipid might thus be the same or different.
  • lipids include, without limitation, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), steroids, cholesterol, aliphatic amines such as long chain aliphatic amines and carboxylic acids, long chain sulfates and phosphates, dicetyl phosphate, butylated hydroxytoluene, tocophenol, retinol, sterols, such as cholesterol hemisuccinate, tocopherol hemisuccinate, ceramides, cationic lipids, monoacyl glycerol, diacyl glycerol, triacyl glycerol, fatty acids, fatty acid methyl esters, single-chain nonionic lipids, glycolipids, lipid-peptide conjugates and lipid-polymer conjugates and isoprenoid compounds.
  • aliphatic amines such as long chain aliphatic
  • the at least one lipid used in the context of the invention is selected from the group consisting of glycerophospholipids.
  • the lipid is not a charged lipid.
  • the at least one lipid may be a mixture of at least two different lipids.
  • Some molecules might be classified according to the definitions as a lipid as well as a surfactant, such as cholesterol.
  • the at least one lipid and the at least one surfactant might be as well the same or different.
  • the at least one lipid is added during the preparation of the capsules, as further specified under the paragraph "method of preparation” here below.
  • the at least one lipid may be added during the preparation of an oil solution in step a) of the method of preparation of the invention.
  • the at least one lipid may be used to form a lipid monolayer.
  • the at least one lipid may be added during the preparation of an oil solution in step a) of the method of preparation of the invention, more particularly during step a3) of the method of preparation of the invention.
  • the lipids might be used in a concentration of 0.05 mg/ml to 25 mg/ml in the lipid oil solution.
  • the lipid bilayer may additionally comprise at least one surfactant and/or at least one lipid.
  • the lipid may be mixed with the at least one liposome-forming lipid and/or optionally the surfactant, prior to the mixing with the at least one organic solvent in step a1 ).
  • the at least one lipid might decrease fusogenicity of the liposomes and therefore decrease aggregation.
  • lipids might be preferably added to the liposome-forming lipid in a ratio (mol%) of 1 and 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 3, 1 to 2 and 1 to 1 , or further 2 to 1 , 3 to 1 , 4 to 1 , 5 to 1 , 6 to 1 , 7 to 1 , 8 to 1 , 9 to 1 .
  • the at least one lipid is cholesterol.
  • cholesterol might be added at 1 -14 mol % to the at least one liposome-forming lipid.
  • Cholesterol might be added in particular at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 mol% to the at least one liposome-forming lipid, preferably at 10%mol.
  • the shell in particular the lipid bilayer, comprises additionally at least one protein.
  • a “protein” is a polypeptide which is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.
  • the at least one protein might be voluntarily added during the preparation of the capsule, in particular of the liposome, or might be present in the shell, in particular in the lipid bilayer, as a residue of the process of preparation of the liposome-forming lipid and/or the lipid, when said liposome-forming lipid and/or lipid used in the context of the invention are derived from natural sources.
  • the at least one protein may be added during the preparation of the capsules, as specified in the section "method of preparation" below.
  • the at least one protein may be added during the preparation of an oil solution in step a) of the method of preparation of the invention, more particularly during any of steps a1 ) to a4) of the method of preparation of the invention.
  • the at least one protein might be mixed with the at least one liposome-forming lipid, prior to the mixing with the at least one organic solvent in step a1 ).
  • the at least one protein may be added during preparation of an emulsion in step c) or in step g) of the method of preparation of the invention.
  • capsules of the invention in particular liposomes of the invention, were obtainable using a method of preparation following serial specific stages and involving double emulsion methods and/or microfluidics.
  • the invention therefore also relates to a method of preparation of the capsules, as defined herein above, comprising the following steps:
  • an aqueous gel comprising at least one hydrophilic polymer, as defined in the section “hydrophilic polymer” herein above, dispersed in water or buffer and at least one nucleic acid, as defined in the section “nucleic acids” herein above,
  • step c) preparation of an emulsion of the aqueous gel comprising at least one nucleic acid obtained in step b) in the oil solution obtained in step a), to obtain nucleic acid- aqueous gel droplets,
  • the method of preparation may further comprise an optional step f), wherein the capsules are transferred to an aqueous solution and the shell of the capsules obtained in step d) or e) might be removed.
  • step f) The method of preparation may be directly followed by the optional step f) or step f) may be applied after storage, just prior to use.
  • the optional step f) may remove the shell, in particular the lipid and surfactant monolayer, more particularly the surfactant monolayer.
  • the method may further comprise an optional step g) of adding at least one additional lipid monolayer to the lipid monolayer of the capsule obtained in step d) or e).
  • Step g) is further specified in the section "Production of the liposomes starting from capsules having a lipid monolayer" below.
  • the invention relates to a method of preparation of a capsule, in particular with a lipid monolayer, as defined herein above, comprising the following steps:
  • an aqueous gel comprising at least one hydrophilic polymer, as defined in the section “hydrophilic polymer” herein above, dispersed in water or buffer and at least one nucleic acid, as defined in the section “nucleic acids” herein above,
  • step c) preparation of an emulsion of the aqueous gel comprising at least one nucleic acid obtained in step b) in the oil solution obtained in step a), to obtain nucleic acid- aqueous gel droplets,
  • the invention also relates to a method of preparation of the liposomes, as defined herein above, comprising the following steps:
  • an aqueous gel comprising at least one hydrophilic polymer, as defined in the section “hydrophilic polymer” herein above, dispersed in water or buffer and at least one nucleic acid, as defined in the section “nucleic acids” herein above,
  • step c) preparation of an emulsion of the aqueous gel comprising at least one nucleic acid obtained in step b) in the oil solution obtained in step a), to obtain nucleic acid- aqueous gel droplets,
  • step d) or e) adding at least one additional lipid monolayer to the lipid monolayer of the capsule obtained in step d) or e), wherein the adding of at least one additional lipid monolayer is obtained by a centrifugation step further specified in the section "Production of the liposomes starting from capsules having a lipid monolayer" below, therby obtaining liposomes.
  • the preparation of an oil solution comprising at least one compound selected from the group constituted of a liposome-forming lipid, a surfactant and a lipid at step a) of the method of the invention, preferably comprises:
  • a1 optionally mixing of at least one compound selected from the group constituted of a liposome-forming lipid and a lipid as defined in the section "shell" herein above, with at least one organic solvent;
  • the preparation of an oil solution comprising at least one compound selected from the group constituted of a liposome-forming lipid and a lipid, at step a) of the method of the invention preferably comprises:
  • a1 mixing of at least one compound selected from the group constituted of a liposome- forming lipid and a lipid, as defined in the section "liposome-forming lipid” and the section “Surfactants, lipids and proteins” herein above, with at least one organic solvent, a2) evaporating of solvent to obtain a surface of the compound selected from the group constituted of a liposome-forming lipid and a lipid,
  • a "organic solvent” describes a substance that dissolves a solute resulting in a solution, wherein the solute according to the invention is at least one compound selected from the group constituted of a liposome-forming lipid and/or lipid, more preferably the at least one liposome-forming lipid.
  • organic solvents examples include acetaldehyde, acetone, acetonitrile, allyl alcohol, allylamine, 2-amino- 1 -butanol, 1 - aminoethanol, 2-aminoethanol, 2-amino-2-ethyl- 1 ,3-propanediol, 2-amino-2-methyl-1 - propanol, 3-aminopentane, N-(3-aminopropyl)morpholine, benzylamine, bis(2-ethoxy- ethyl)-ether, bis(2-hydroxyethyl)-ether, bis(2-hydropropyl)-ether, bis(2-methoxyethyl)- ether, 2-bromoethanol, meso-2,3-butanediol, 2-(2-butoxyethoxy)-ethanol, butylamine, sec- butylamine, tert-butylamine, 4-butyrolacetone, 2-
  • the at least one organic solvent is selected from the group consisting of chloroform, methanol, ethanol, acetonitrile and mixtures thereof.
  • the at least one organic solvent might be a mixture of at least two different organic solvents as defined above.
  • the at least one organic solvent is a mixture of two different organic solvents, wherein the ratio (vol to vol, before mixture) between the two different solvents might be a ratio of 1 and 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 3, 1 to 2 and 1 to 1 , or further 2 to 1 , 3 to 1 , 4 to 1 , 5 to 1 , 6 to 1 , 7 to 1 , 8 to 1 , 9 to 1 .
  • the at least one organic solvent is a mixture of chloroform and methanol, chloroform and ethanol, chloroform and acetonitrile.
  • Chloroform and methanol might be mixed in a ratio of 1 and 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 3, 1 to 2 and 1 to 1 , or further 2 to 1 , 3 to 1 , 4 to 1 , 5 to 1 , 6 to 1 , 7 to 1 , 8 to 1 , 9 to 1 , preferably 2 to 1 , 3 to 1 , 4 to 1 , 5 to 1 , 6 to 1 , 7 to 1 , 8 to 1 , 9 to 1 , 10 to 1 .
  • chloroform and methanol are used in a ratio of 9:1 .
  • mixing refers to an action leading to the state wherein one compound and/or composition is completely dissolved in another chemical composition and a homogeneous mixture composed of only one phase is obtained.
  • mixing comprises techniques such as pipetting, vortexing, stirring, mixing by repetitively inverting the upper and lower part of the tube containing the solution and/or shaking.
  • step a1 ) of the method of preparation of the invention refers to dissolving at least one compound selected from the group constituted of a liposome-forming lipid and a lipid, more preferably the at least one liposome-forming lipid completely with the at least one organic solvent.
  • solvent is suitable to dissolve a particular compound of the shell, such as a particular at least one liposome forming-lipid and/or lipid, preferably a particular liposome-forming lipid and in which ratio to use them.
  • step a1 Mixing of step a1 ) might be performed at a temperature between 15-40°C, depending on the boiling point of the at least one organic solvent, wherein the temperature should be below the boiling point of the at least one organic solvent, in particular mixing of step a) might be at 15°C to 25° C, preferably at room temperature.
  • a surfactant and/or lipid and and/or protein might be added either together and/or sequentially to the liposome-forming lipid prior to mixing with at least one organic solvent in step a1 ) of the method of preparation of the invention.
  • "Evaporating" of the solvent in step a2) of the method of preparation of the invention refers to vaporization or sublimation of the at least one organic solvent, leading to the removal of the at least one organic solvent from the at least one compound selected from the group constituted of a liposome-forming lipid and a lipid, preferably from the liposome-forming lipid.
  • Evaporating of solvent to obtain a surface of the at least one compound selected from the group constituted of a liposome-forming lipid and a lipid, preferably the at least one liposome-forming lipid in step a2) of the method of preparation of the invention, might be performed by techniques known to the skilled in the art.
  • the surface of the at least one liposome-forming lipid might further comprise a surfactant, lipid and/or protein.
  • some residual solvent might be left over, at a level of for example 0.01 -10%v/v, or 0.01 -5%v/v, or 0.01 -1 %v/v in comparison to the at least one organic solvent present in the mixture of step a1 ) of the method of preparation of the invention.
  • the evaporation of the at least one organic solvent might be obtained by open dish evaporation.
  • the evaporation can be obtained by reduced pressure evaporation.
  • a “reduced pressure evaporation” refers to a technique of evaporation wherein a vacuum is applied to the solution for a specific time, at a specific temperature, for example at lower temperatures ( ⁇ 4°C), at room temperature (RT) or at higher temperatures (>RT).
  • oil refers to a neutral, nonpolar chemical substance, that is a viscous liquid at ambient temperatures, and is immiscible with water but soluble in alcohols or ethers. Oils are known to have a high carbon and hydrogen content.
  • the oil is a mineral oil.
  • a mineral oil is any of various colorless, odorless, light mixtures of alkanes and might in the Ci 5 to C 40 range from a non-vegetable (mineral) source.
  • the mineral oil is commercial available, for example from Sigma (Ref.: M3516).
  • the mixing in step a4) of the method of preparation of the invention is preferably sonication.
  • the lipid in oil solution obtained in step a4) of the method of preparation of the invention has a concentration of at least one liposome-forming lipid of 0.1 mM to 2 mM, in particular 0.4 mM to 1 mM, preferably 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mM, most preferably 0.4, 0.42, 0.44, 0.46, 0.48, 0.5 mM.
  • oil solution may be prepared as follows.
  • a for example 100mg/ml stock solution of lipids (for example egg-PC: PEG-PE (both Sigma), for example 98:2 mol%) in preferably chloroform: methanol (typically, 9:1 ) is diluted for example 5 times in the same solvent in a flask.
  • the solvent is evaporated preferably under vacuum at room temperature for typically 1 hour obtaining a lipid film at the bottom of the flask and for example 20 ml of oil (for example mineral oil) is added.
  • the solution is then stored for example for 1 h preferably at room temperature. All these steps are preferably performed in a 10% humidity environment.
  • the solution is sonicated typically for 30 min in a bath at a temperature preferably lower than 40 °C, to obtain a stable lipid in oil solution of preferably 0.4 mM.
  • the solution can be used typically up to three weeks after preparation.
  • the at least one compound of the shell may be at least one surfactant.
  • the at least one surfactant does not need to be mixed with an organic solvent. Therefore, in one embodiment, the optional steps a1 and a2 may be omitted.
  • the preparation of an oil solution comprising at least one surfactant, at step a) of the method of the invention preferably comprises:
  • mixing in step a4) preferably refers to stirring.
  • the mixing may be performed at any temperature.
  • a liposome-forming lipid and/or lipid and/or protein as defined in the section "shell" herein above, might be added either together or sequentially to the surfactant.
  • the optional steps a1 and a2 might become necessary depending in particular on the nature and amount of the liposome-forming lipid and/or lipid.
  • oil-surfactant solution may be prepared by mixing for example 50% Tween80 and Span80 in a total concentration of 0.5% in mass in mineral oil and gently stirring at room temperature. Preparation of an aqueous gel
  • the preparation of an aqueous gel comprising at least one hydrophilic polymer dispersed in water or buffer and at least one nucleic acid, at step (b) of the method of preparation of the invention preferably comprises
  • nucleic acid as defined in the section "nucleic acid' herein above
  • b2 the preparation of an aqueous solution comprising at least one hydrophilic polymer, as defined in the section "hydrophilic polymer” herein above, mixed with the solution of at least one nucleic acid obtained at step b1 ) and
  • the nucleic acid used in step b1 might be fragmented, as defined in the section "nucleic acids" herein above, prior to step b1 ).
  • Techniques to prepare a solution of at least one nucleic acid are well known from the skilled person.
  • the solution of at least one nucleic acid, obtained at the end of step b1 ), has a nucleic acid concentration of 0.01 ⁇ g/ ⁇ L to 100 ⁇ g ⁇ L, preferably 0.01 ⁇ g/ ⁇ L to 50 ⁇ g ⁇ L, O.O ⁇ g ⁇ L to 20 ⁇ g/ ⁇ L or 0.01 ⁇ g/ ⁇ L to 10 ⁇ g ⁇ L, in particular 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1 ,1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6.5, 7.0, 7.5, 8, 8.5, 9, 9.5, 10 ⁇ g ⁇ L.
  • the solution of at least one nucleic acid at step b1 ) has a nucleic acid concentration of 0.05 ⁇ g ⁇ L, 0.5 ⁇ g ⁇ L, 4 ⁇ g ⁇ L or 10 ⁇ g ⁇ L, preferably 0.05 ⁇ g ⁇ L, 0.5 ⁇ g ⁇ L, or 10 ⁇ g ⁇ L.
  • the nucleic acid might be dissolved in nuclease free water.
  • the pH or presence of ions such as K + , Na + , etc. or buffers may contribute to the stability of nucleic acids.
  • the solution might therefore preferably further comprise a buffer and/or ions as defined in section "nucleic acid' herein above.
  • the solution comprising the at least one nucleic acid is gently mixed, preferably per inversion, and stirred at 30°C to 70°C, preferably at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, in particular at 60 °C for 1 min to 120 min, preferably 10 min to 120 min, more preferably 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120 min in particular 15 or 60 min, preferably 60 min.
  • the solution of at least one nucleic acid might comprise in addition a nuclease inhibitor.
  • nuclease inhibitor inactivates nucleases and is commercially available.
  • the solution of at least one nucleic acid might comprise a cage as defined in the section "nucleic acids" herein above, that surrounds the nucleic acid.
  • the cage is a bacteriophage T5 capsid.
  • a hydrophilic polymer as defined in section "hydrophilic polymer” herein above is preferably mixed in the solution of at least one nucleic acid obtained at step b1 ).
  • the hydrophilic polymer is present in solution.
  • the aqueous solution according to step b2) comprising at least one hydrophilic polymer mixed with the solution of at least one nucleic acid obtained at step b1 ) preferably has a nucleic acid concentration of 0.0 ⁇ / ⁇ _ to 50 ⁇ g ⁇ L, 0.0 ⁇ / ⁇ _ to 20 ⁇ g ⁇ L or 0.0 ⁇ / ⁇ _ to 10 ⁇ g ⁇ L, in particular 0.01 , 0.05, 0.1 , 0.2, 0.25, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1 ,1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6.5, 7.0, 7.5, 8, 8.5, 9, 9.5, 10 ⁇ g ⁇ L, more particularly 0.25 ⁇ g/ ⁇ L or 2.5 ⁇ g ⁇ L.
  • the nucleic acid concentration in the aqueous gel, obtained in step b2) is for example 0.25 ⁇ g ⁇ L, 0.05 ⁇ g ⁇ L, 0.375 ⁇ g ⁇ L, 2.5 ⁇ g ⁇ L, 7.5 ⁇ g ⁇ L, more particularly 0.05 ⁇ g ⁇ L, 0.375 ⁇ g ⁇ L or 7.5 ⁇ g ⁇ L.
  • the concentration of the at least one hydrophilic polymer in the aqueous solution comprising at least one hydrophilic polymer mixed with the solution of at least one nucleic acid obtained in step b1 ) depends on the nature of said at least one hydrophilic polymer and has been specified in the paragraph "hydrophilic polymers".
  • low melting agarose might be added in a concentration of 0.3-2%
  • low melting agarose has a concentration of 0.8% w/vol.
  • the nucleic acid solution of step b1 ) is heated prior to addition of the at least one hydrophilic polymer in step b2). Heating might be required in particular in case of physical cross-linking when preferably comprising heating and/or cooling.
  • step b3) might depend on the mechanism of cross-linking used for the at least one hydrophilic polymer.
  • the solution of step b2) might be homogenized at a step b3) for a certain amount of time and at a certain temperature and followed by the addition of di- or tri-valent counter ions and/or pH adjustment followed by a short additional mixing period b3') and then proceeding with the emulsion step c).
  • the solution obtained at step b2) is heated to a specific temperature, as defined in the section "cross-linking" herein above, and the solution is then mixed at said specific temperature.
  • the hydrophilic polymer is low melting agarose.
  • the temperature of the solution of at least one nucleic acid obtained at step b1 ) is heated to 60 °C or 40 °C, preferably 60 °C, before bsig mixed with low melting agarose and then mixed at 60°C or 40°C, preferably 60 °C, fb 15 min or 30 min for homogenization.
  • an aqueous gel according to step b) of the method of the invention is prepared as follows.
  • DNA is dissolved in nuclease-free water or in buffer at for example 0.05 ⁇ g ⁇ L, 0.5 ⁇ g/ ⁇ L or 10 ⁇ g ⁇ L, gently mixed per preferably inversion and stirred at preferably 60 °C for example for 1 -2 h.
  • the solution is then mixed with low melting agarose (0.8% final concentration) at 60° C or 40° C to obtain a DNA-agaose solution at 0.05 ⁇ g ⁇ L, 0.375 ⁇ g/ ⁇ L or 7.5 ⁇ g ⁇ L respectively, and is left to homogenize at the same temperature for example for 30 min.
  • Preparation of an emulsion Preparation of an emulsion
  • the emulsion of step c) of the method of preparation of the invention is preferably obtained by mixing the aqueous gel comprising at least one nucleic acid obtained at step b) as defined in the section "preparation of an aqueous gel” herein above, in the oil solution obtained in step a) as defined in the section “preparation of an oil solution” herein above, to obtain nucleic acid -aqueous gel droplets.
  • step c) either at least one monolayer or at least one lipid bilayer will be formed, preferably at least one lipid bilayer will be formed, around the "nucleic acid - aqueous gel droplets", which might not already be fully polymerized, a process which will be completed during step d) of the method of preparation of the invention.
  • step c) either at least one surfactant monolayer or at least one lipid monolayer will be formed, around the "nucleic acid - aqueous gel droplets", which might not already be fully polymerized, a process which will be completed during step d) of the method of preparation of the invention.
  • nucleic acid - aqueous gel droplets describes a preliminary form of the capsules of the invention, in particular of the capsules comprising a shell of the invention, more particularly of the liposomes of the invention, in which the aqueous gel preferably undergoes the transition from an infinite to a finite network and the lipid bilayer is formed.
  • the "nucleic acid - aqueous gel droplets" describes a preliminary form of the capsules of the invention, in particular of the capsules comprising at least one surfactant monolayer or at least one lipid monolayer, in which the aqueous gel preferably undergoes the transition from an infinite to a finite network and the lipid bilayer is formed.
  • the mixing is achieved by vortexing, shaking or pipetting.
  • the vortexing might be done at 10 to 50 Hz for 30 s to 3 min, in one example for 1 min at 40 Hz.
  • the vortexing might be done for 30s at 40 Hz by applying 3 vortex pulses of 10s each.
  • glass-beads may be added during vortexing.
  • a cage protects the at least one nucleic acid from fragmentation at this step mixing step, in particular when the mixing is performed by vortexing.
  • the pipetting might comprise typically 10 to 60, preferably 30 successive slow aspirations and ejections.
  • nucleic acids-gel droplets may be prepared as follows.
  • the DNA-agarose solution from step b) are poured on the surface of, for example, 1 ml lipid-oil solution and the solution is vortexed for example for 1 min at for example 40 Hz to create a micro-emulsion of DNA-agarose droplets in the lipid-oil solution.
  • the micro-emulsion can also be obtained by preferably mixing the solution with a pipette by for example 30 successive slow aspirations and ejections.
  • nucleic acids-gel droplets preparation of an emulsion to obtain nucleic acids-gel droplets may be performed as follows.
  • DNA-agarose solution from step c) are poured on the surface of, for example, 1 ml surfactant-oil solution in for example a 2ml tube and the solution is vortexed for example for 30s or 1 min at for example 40 Hz to create a micro- emulsion of DNA-agarose droplets in the surfactant-oil solution.
  • 2 glass beads are added during vortexing. Gelation of the droplets to obtain capsules
  • said gelation or cross-linking occurs when solutions comprising a hydrophilic polymer are heated and cooled, in particular for galantine, carrageenan or agarose, in particular for low melting agarose. Heating and cooling refer to processes as defined in the section "cross-linking" herein above.
  • the gelation of the droplets to obtain capsules with a lipid monolayer in step d) is induced by decreasing the temperature to 4 ° C for at least 10 min.
  • the gelation of the droplets to obtain liposomes in step d) is induced by decreasing the temperature to 4 ° C for A least 1 0 min.
  • the gelation of the droplets to obtain capsules with a surfactant monolayer in step d) is induced by decreasing the temperature to 4 ° C for at least 2 h.
  • gelation of the nucleic-acid aqueous gel droplets is induced by decreasing the temperature to in particular 4° C for at least preferably 10 min.
  • the resulting capsules, in particular capsules comprising a shell, more particularly liposomes are left in the oil-lipid solution overnight for example at 4° C or at room temperature.
  • the diameter of the resulting droplets typically ranged from 5 ⁇ to 200 ⁇ , in particular from 5 ⁇ to 100 ⁇ , more particularly, the diameter of the resulting droplets is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 1 00 ⁇ .
  • capsules with a lipid monolayer are left in the oil-lipid solution overnight for example at 4° C or at room temperature
  • the diameter of the resulting droplets typically ranged from 5 ⁇ to 200 ⁇ , in particular from 5 ⁇ to 1 00 ⁇ , more particularly, the diameter of the resulting droplets is 5, 1 0, 20, 30, 40, 50, 60, 70, 80, 90, 100 ⁇ .
  • the resulting capsules comprising a surfactant monolayer are left in the surfactant-lipid solution overnight for example at 4° C or at room temperature.
  • the diameter of the resulting droplets typically ranged from 1 to 100 ⁇ , in particular from 1 to 80 ⁇ , preferably 1 to 60 ⁇ .
  • the size of the capsule with a surfactant monolayer may be 1 , 2, 4, 6, 8, 1 0, 1 2, 14, 16, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 ⁇ .
  • Capsules comprising a surfactant or lipid monolayer obtained by the above method of preparation are stable for over at least 1 months, preferably for at least 2 months to 2 years, in particular for 2, 3, 4, 5, 6, 8, 1 0, 1 2, 14, 16, 18, 20, 22, 24 months when kept suspended in the oil solution used for the emulsion.
  • “Stable” means that the capsules preferably do not aggregate and preferably do not leak any nucleic acid over at least 1 months, preferably for at least 2 months to 2 years, in particular for 1 , 3, 4, 5, 6, 8, 1 0, 12, 14, 16, 18, 20, 22, 24 months.
  • the capsules do not aggregate and the size and/or size distribution stays the same over at least 1 months, preferably for at least 2 months.
  • the capsules with a lipid monolayer obtained by the above method of preparation are stable for over at least 6 months, preferably for 6 months to 2 years, in particular for 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 months when kept suspended in the oil solution used for the emulsion in step d).
  • the capsules with a surfactant monolayer obtained by the above method of preparation are stable for over at least 1 months, preferably for 2 months to 2 years, in particular for 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 months when kept suspended in the oil solution used for the emulsion in step d).
  • Capsules with a lipid monolayer do not aggregate and the size and/or size distribution stays the same over at least 6 months.
  • Capsules with a lipid monolayer, after storage of 6 months may be used to prepare liposomes according to step g) of the method of preparation.
  • the liposomes obtained from capsules with a lipid monolayer of 6 months age typically have the same size and size distribution as liposomes obtained from capsules with a lipid monolayer just after their preparation.
  • the capsules according to the invention in particular the capsules comprising a shell according to the invention, more particularly the liposomes according to the invention, can be poly-disperse or mono-disperse.
  • Mono-disperse capsules are easily obtained after a sorting stage by using classical techniques.
  • the capsules preferably the capsules comprising a shell, more particularly the liposomes, might be selected according to their size in an optional step e) wherein the size selection of the capsules is achieved by centrifugation, extrusion, gelfiltration, microfluidics and/or cell sorting techniques, in particular centrifugation.
  • the capsules preferably the capsules comprising a shell, more particularly the liposomes, are mono-disperse.
  • the size selection of the capsules, in particular of liposomes, of step e) is achieved by centrifugation, wherein the centrifugation might be performed in water at 15 to 50x g for 1 -30 min, preferably at 32.5x g for 5-20 min, more preferably for 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 min.
  • the size sorting may be achieved in one example as follows:
  • Liposomes may be sorted for example by centrifugation at for example 32.5x g in typically water. After for example 5 min of centrifugation, liposomes at the bottom of the tube are collected and had a size of 50 to 200 ⁇ with a pick value at 100 ⁇ . After a further centrifugation of the supernatant solution for example 5 min (corresponding to a overall 10-minute centrifugation) the liposomes at the bottom of the tube ranged in size from 10-100 ⁇ with a pick value at 50 ⁇ . A third run centrifuging the supernatant for example for further 5-minutes (total of 15 min of centrifugation) gave raise to collected liposomes with a range in size of 4-50 ⁇ (with a pick value at 20 ⁇ ).
  • the lipid bilayer may be formed in different steps of the protocol of the preparation of the liposomes.
  • the first lipid monolayer may be formed during the formation of the droplets in the oil phase and the second monolayer may be formed when the droplets coated with one monolayer are centrifuged from the oil phase to an aqueous phase.
  • the additional lipid monolayer may be added when the capsule crosses the oil-aqueous solution interface during centrifugation.
  • the method of preparation may further comprise an optional step g) of adding an additional lipid monolayer to the lipid monolayer of the capsule obtained in step d) or e).
  • the additional lipid monolayer may be obtained by pouring a lipid-oil solution containing capsules on an aqueous solution, preferably water, buffer or saccharide solution, followed by centrifugation.
  • a lipid-oil solution containing capsules preferably water, buffer or saccharide solution
  • a lipid monolayer is typically built at the interface of the oil and the aqueous solution.
  • the capsules are typically centrifuged from the oil phase to an aqueous phase crossing this interface and an additional lipid monolayer typically surrounds the capsule, thus resulting in a lipid bilayer.
  • the lipid-oil solution refers to the oil solution comprising at least one compound selected from the group constituted of a liposome-forming lipid and a lipid obtained in step a). However, the lipid-oil solution containing capsules is obtained at step d) or e) of the preparation of capsules.
  • the lipid-oil solution containing capsules comprising a lipid monolayer is preferably placed at the top surface of an aqueous solution, typically water, buffer or a saccharide solution and centrifuged.
  • the capsules typically sediment in the lipid-oil solution, cross the lipid-oil-water interface, where they become coated by a second lipid monolayer and therefore turn into liposomes comprising a lipid bilayer.
  • the formed liposomes sediment in the water, buffer or saccharide solution.
  • 200 ⁇ _ of the oil- lipid solution containing capsules comprising a lipid monolayer are poured on the surface of 1 ml ultrapure water and centrifuged at 32.5xg for 20 min.
  • the lipid-oil solution containing capsules comprising a lipid monolayer may be mixed with another lipid-oil solution before centrifugation.
  • This other lipid solution can contain lipids or liposome-forming lipids that are of different nature than the ones used in step a) of the preparation of the capsules.
  • the resulting lipid bilayer of the liposomes may thus comprise monolayers that consist of different liposome-forming lipids and/or lipids.
  • the outer monolayer is typically constituted of the liposome-forming lipids and/or lipids added in step g).
  • the other lipid solution added in step g) is placed at the top of the water or buffer solution at least 15 minutes, preferably, 30 minutes, preferably one hour before adding the lipid-oil solution containing the capsules comprising a lipid monolayer. During this waiting time a monolayer has time to be formed at the interface between the lipid-oil solution and water or buffer or saccharide solution.
  • the invention further relates to the use of a capsule, in particular a liposome, as defined above, as an internal standard in electrophoresis assays.
  • Electrodes is a process which enables the sorting of molecules based on size.
  • molecules such as nucleic acids
  • the molecules being sorted are dispensed into a well in the gel material.
  • the gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move slowly through the gel while the smaller molecules move faster.
  • the macromolecules such as nucleic acids are thus separated according to their fragments, based on their size and charge.
  • the gel material suppresses the thermal convection caused by application of the electric field, furthermore it acts as a sieving medium, retarding the passage of molecules.
  • the gel material further serves to simply maintain the finished separation, so that a post electrophoresis stain can be applied.
  • the gel material used for nucleic acids may be agarose.
  • Said electrophoresis assay might be a micro-electrophoresis or a capillary electrophoresis assay, in particular a Single Cell Gel Electrophoresis assay also known as as the comet assay.
  • micro electrophoresis assay refers to traditional gel electrophoresis principles that have been transferred to a chip format.
  • Capillary electrophoresis is electrophoresis performed in a capillary tube.
  • An "internal standard” is a compound or substance different from the analyte that is added in a known amount to a sample comprising the analyte. This compound or substance can then be used for calibration.
  • the internal standard is a quantitative internal standard.
  • the capsules of the invention When used as an internal standard, the capsules of the invention, in particular liposomes of the invention, are typically mixed with the cells to be analyzed, before being submitted to an electrophoresis assay, as defined above which might be for example a neutral comet assay or alkaline comet assay.
  • an electrophoresis assay as defined above which might be for example a neutral comet assay or alkaline comet assay.
  • capsule comets in particular liposome comets
  • capsule comets are distinguishable from normal cell comets.
  • they can be used as an internal standard for comparing results in the same experimental conditions between different slides (as a reference for intra-assay variability) as well as results in different experimental conditions and different laboratories (as a reference for inter-assay reproducibility).
  • the capsules are preferably stored in the emulsion of step c) of the method of preparation of the invention.
  • the capsules Prior to use, the capsules are preferably transferred to an aqueous solution in step f). According to the optional step f), the capsules are transferred to aqueous solution and the shell of the capsules obtained in step d) or e) might be removed.
  • the capsules are preferably transferred in an aqueous solution, typically water, buffer or a saccharide solution and then mixed, preferably centrifuged for example at 32.5xg for 20 min.
  • aqueous solution typically water, buffer or a saccharide solution
  • the surfactant-oil solution containing capsules with a surfactant monolayer obtained in step d) or e) of the method of preparation of the invention are poured on the surface of water or a buffer or a saccharide solution, preferably ultrapure water, and immediately centrifuged at 32.5xg for 20 min.
  • the capsules After centrifugation, the capsules are typically gently mixed to re-suspend and they are immediately used for experiments.
  • capsules with a monolayer in particular capsules with a surfactant monolayer, may lose the surfactant monolayer during the step f) and are therefore transformed into capsule cores. After their transfer to aqueous solution, the capsules may be directly used as described below concerning the method of calibration.
  • the invention further relates to a method of calibration of a single cell microelectrophoresis assay comprising:
  • Calibration refers to a comparison between measurements.
  • the calibration refers to compare the comets of the capsule, in particular of the liposome, with the comets of cells.
  • the “Single Cell Gel Electrophoresis Assay”, also called “comet assay”, enables detecting single or double strand lesions of genomic DNA induced by genotoxic agents in individualized prokaryotic and eukaryotic cells. It was first developed by Ostling and Johansson in 1984 (Ostling, O. and Johanson, K.J. ,1984, Biochem. Biophys. Res. Commun., 123: 291-298) and later modified by Singh et al. in 1988 (Singh, N.P. et al., 1988, Exp. Cell Res. 175: 184-191 ).
  • cells of the biological sample are mixed with the capsule, in particular the liposomes, defined above in a step 1 ) of the method of calibration of a comet assay.
  • the capsules of the invention in particular the liposomes of the invention, might be mixed with the biological sample comprising cells to be analyzed in a ratio of 1 :10 to 1 :150, in particular 1 :10 to 1 :100, more preferred 1 :10 to 1 :70 (liposome to cells).
  • ⁇ _ For the mixing of the cells in one example blood (for example 20 ⁇ _) is diluted in for example 1 ml_ of RPMI 1640 with 10% Fetal Calf serum (FCS).
  • FCS Fetal Calf serum
  • Ficoll 100 ⁇ _
  • FCS Fetal Calf serum
  • lymphocytes 100 ⁇ _
  • the pellet of cells of the biological sample is then suspended with for example 75 ⁇ _ of water solution containing for example nucleic acid-agarose liposomes.
  • the method for the preparation of the biological sample depends on the cells of interest and is known to the skilled in the art.
  • the capsules of the invention in particular the liposomes of the invention, can be mixed with the cells in any condition or media required for the cells.
  • the cells may be treated prior to the comet assay, for example with a genotoxic compound.
  • the cells are treated with hydrogen peroxide, for example 30 ⁇ _ of a solution of 1 mM of H 2 0 2 in PBS " I X were added to the cell tube and left in ice for example for 5 min, cells were then centrifuged for example at 1326x g for typically 4 min and the pellet was collected and mixed as described with the liposomes.
  • hydrogen peroxide for example 30 ⁇ _ of a solution of 1 mM of H 2 0 2 in PBS " I X were added to the cell tube and left in ice for example for 5 min, cells were then centrifuged for example at 1326x g for typically 4 min and the pellet was collected and mixed as described with the liposomes.
  • a step 2) of the method of calibration of a single cell micro-electrophoresis assay is performed.
  • the single cell micro-electrophoresis assay encompasses the embedding of cells and caspules, in particular liposomes, on a microscope slide, usually comprising three layers of agarose in different concentrations and cell lysis and the electrophoresis.
  • the cells and capsules are embedded in agarose on a microscope slide and lysed with a lysing buffer comprising typically a detergent and high salt to form nucleotides containing supercoiled loops of DNA linked to the nuclear matrix.
  • a lysing buffer comprising typically a detergent and high salt to form nucleotides containing supercoiled loops of DNA linked to the nuclear matrix.
  • comet assays There are several different types of comet assays, which are mostly vary based on the pH of the electrophoresis step. Known to the skilled person are the neutral comet assay (pH around 8.3) and the alkaline comet assay (pH>13).
  • the capsule according the invention in particular the liposome according to the invention, might be used in a alkaline comet assay and a neutral comet assay.
  • Variations of the comet assay might be made by the skilled in the art, with respect to, for example, the embedding of the cells and liposomes, the lysis buffer used, the exact electrophoresis condition, the labeling or staining of the nucleic acids.
  • the neutral comet assay may be performed as follows for the detection of DNA double strands, similar to the description of Fairbain DW (1995, Mutation Res., 339: 37-59).
  • the microscope slides (for example from Superfrost Plus, Menzel- Glaser) may be prepared as following: The slide is dipped in a solution of preferably 1 .8% normal melting agarose dissolved in preferably PBS 1 x (without Mg ++ and Ca ++ ) and for example air dried preferably overnight at room temperature.
  • a solution (85 ⁇ ) of for example 0.8% low melting point agarose in PBS 1 x (without Mg ++ and Ca ++ ) kept at for example 45 °C is placed on the slide, covered with typically a 24 x 36 mm coverslip (Menzel-Glaser), and left to solidify on ice for example for 5 min.
  • step 1 The cell/capsule mixture obtained in step 1 ) of the method of calibration of a comet assay is then embedded in a next layer.
  • 75 ⁇ _ of water solution containing preferably DNA-agarose capsules and for example lymphocytes obtained in step 1 ) are mixed with preferably 75 ⁇ _ of for example 1 .6% low melting agarose in typically PBS, giving a mixture comprising cells, capsules and 0.8% low melting agarose in PBS.
  • the solution is gently mixed and for example 75 ⁇ _ are placed on the slide, sealed with the coverslip and left to solidify on ice typically for 5 min.
  • a third layer of for example 0.8 % low melting agarose (75 ⁇ _) is added to the slide and cooled down on ice for typically 5 min.
  • the cells may be treated prior to the electrophoresis, once they are embedded in the agarose gel.
  • the cells and capsules may be treated with a genotoxic compound.
  • the cells are treated with hydrogen peroxide.
  • the cells are preferably lysed. Therefore the coverslip is removed and the slides may be immersed in a lytic buffer, typically 2.5 M NaCI, 100 mM EDTA, 10 mM Tris-HCI, 1 % N-lauryl sarcosine, 3% Triton X100 and 10% DMSO, pH 10 for typically 30 min at preferably 4°C.
  • a lytic buffer typically 2.5 M NaCI, 100 mM EDTA, 10 mM Tris-HCI, 1 % N-lauryl sarcosine, 3% Triton X100 and 10% DMSO, pH 10 for typically 30 min at preferably 4°C.
  • the slides are then incubated in an electrophoresis tank with typically TBE 1 x buffer (89 mM Tris base, 89 mM boric acid and 2 mM Na 2 EDTA, pH 8.3) for in particular 10 min.
  • TBE 1 x buffer 89 mM Tris base, 89 mM boric acid and 2 mM Na 2 EDTA, pH 8.3
  • 2 replicate slides are preferably prepared, a control not submitted to electrophoresis, and slide treated by electrophoresis at typically 0.8 V/cm, 14 mA for preferably 25 min. After electrophoresis the slides are preferably neutralized using neutralization buffer, and stained by a fluorescent agent.
  • slides are rinsed in ultrapure water, immersed in for example absolute methanol to dehydrate the slides, and dried preferably overnight.
  • volumes of 75 ⁇ _ of for example ethidium bromide with for example a concentration of 2 ⁇ g mL are used to stain the DNA.
  • the lysis buffer of the comet assay typically comprises a detergent for cell lysis.
  • the lysis buffer comprises 2.5 M NaCI, 100 mM EDTA, 10 mM Tris- HCI, 1 % N-lauryl sarcosine, 3% Triton X100 and 10% DMSO, pH 10.
  • the shell of the capsule in particular the monolayer or the bilayer composing the capsules, in particular the liposomes of the invention can be easily destroyed by the same reagents used to lyse cell membranes during the comet assay.
  • the cage that might surround the nucleic acid may also be destroyed by the lysis buffer.
  • Fluorescent stains for DNA are known to the skilled in the art and may be for example propidium iodide, ethidium bromide, SYBR Green I (SYBR), YO-PRO-1 (YOPRO), TOTO-3 (TOTO), TO-PRO-3 (TOPRO) and/or DAPI.
  • step 3 of the method of calibration of a single cell micro-electrophoresis assay the comets of the cells of the biological sample and the comets of the capsules, in particular of the liposomes, are then detected.
  • Detection in the context of the invention refers also to detection of the comets by fluorescence microscope and includes the analysis of the comets.
  • Tail DNA% refers to the conventional analysis of the comets for example by using Tail DNA% and the application to the comets of the capsules, in particular of the liposomes, leading to the comparability of the test samples.
  • the common principle of the computer software for comet analysis is to decompose the picture of the comet into pixels and then to distribute values according to the intensity of the signal.
  • the values might be for example between 0-255 for a 8 bit image.
  • a segmentation step takes place in which the object is distinguished from the background.
  • the head and tail that were described above are defined.
  • the intensities are background corrected by subtracting the signals of the background to obtain background corrected intensities.
  • the DNA damage can be quantified by measuring either the displacement between the genetic material of the nucleus ('comet head') and the resulting 'tail' or by the intensities corresponding to the DNA present in those regions.
  • Tail moment and Tail DNA% are the most commonly parameters used to analyze comet assay results.
  • the background corrected intensities are integrated in order to define the total intensities of the different regions.
  • Tail DNA% 100 * Tail DNA Intensity/Cell DNA Intensity
  • the intensity of head and tail is given in the percentage of DNA in the tail of the comet. This method is the most commonly used to study and analyze comets in clinical and research studies.
  • Tail moment length is measured from the center of the head to the center of the tail.
  • Tail moment is the measure of tail length multiply by tail intensity. This number is not really popular among investigators since it does not provide the shape of the comet.
  • tail length measured in microns and is only useful at a low damage level since the length of tail tends not to increase proportional to the damage to the DNA.
  • the tail DNA%, the olive tail moment or extend tail moment or any other value derived from the comet can be used for referencing and/or calibration.
  • the inventors provide a complementary analysis of sample comets, which integrates data from capsules, in particular from liposomes, and provides information about specific short DNA fragments in the comet.
  • randomly selected cells and liposomes are scored on each slide and image capture is carried out for example with an Olympus BX53 fluorescent microscope typically equipped with ANDOR Luca S Electron multiplying CCD camera (excitation filter 515-560 nm, emission filter 590 nm).
  • the analyses is performed with for example ANDOR Komet software (version 6.0).
  • the same slides are analyzed for example with a Leica SP5 resonant scanner on a DMI 6000 with 20X.
  • step 3) of the method of calibration of a comet allows to obtain values characterizing the comets of the cells of the biological sample and the comets of the capsules, in particular of the liposomes, and can therefore be used in step 4) of the method of calibration, wherein the comets of the capsules, in particular of the liposomes, are mixed as a reference.
  • Referencing and/or “calibration” and/or “normalization” refers to adjusting values measured on different scales to a notionally common scale and may be used herein interchangeably.
  • values derived from cell comets and obtained in step 3) of the method of calibration of a comet may be adjusted to the same values derived from capsule comets, in particular liposome comets.
  • tail DNA%, olive tail moment or extend tail moment or any other value obtained from comet cells may be adjusted to the corresponding value obtained from the capsule comet, in particular from the liposome comet, called normalization.
  • any mathematical formula comprising the tail DNA% and/or the olive tail moment and/or extend tail moment of the cells and of the capsules, in particular of the liposomes, and further comprising multiplication and/or division of said values and optionally and further substraction and/or addition might be used.
  • Tail DNA% (TD value) of the cells is divided through the value of Tail DNA% for capsules, in particular for liposomes.
  • the same procedure might be applied to the olive tail moment and/or extend tail moment.
  • kits comprising at least one population of capsules of the invention.
  • Kits comprising at least one population of capsules of the invention find use as internal standard in electrophoresis assays as described above. Therefore, kits comprising at least one population of capsules of the invention find preferably use in the method of calibration described herein above.
  • the invention further relates to a kit for use as internal standard in electrophoresis assays.
  • a population of capsules herein refers to a homogenous group of capsules having the same characteristics.
  • a population of capsules thus refers to at least two capsules, preferably 100 to 100000 capsules, more particularly 100 to 10000, preferably 100 to 1000 capsules, for example 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 capsules having the same characteristics.
  • the characteristics may refer to any of the characteristics of capsules mentioned in the context of the invention herein above.
  • a population of capsules might refer to a capsule having the same particular size, comprising the same aquous gel, comprising the same at least one nucleic acid in the same concentration and comprising the same shell.
  • nucleic acids Possible nucleic acid sizes and/or concentrations are defined herein above in the section “nucleic acids”.
  • Preferable fragment sizes of the at least one nucleic acid may be from 10 to 50 kbp, 50 to 100 kbp, 100 to 150 kbp or 150 to 210 kbp. Capsule sizes are defined herein above in the chapter “Detailed description of the invention”.
  • the at least one population of capsules may at least 2, 3, 4, 5, 6, 7, 8, 9, 10 populations of capsules.
  • At least two populations may be mixed.
  • kit may comprise:
  • the capsules with a monolayer are stored in the emulsion of step c) of the method of preparation of the invention.
  • the liposomes may be stored in an aqueous solution.
  • the kit may further comprise instructions for the use of said kit.
  • Said instructions may relate to i) Material supplied, and/or
  • the kit can also comprise examples of the comet analysis for the different capsules supplied.
  • Figure 1 is a set of photography showing giant lipid vesicles encapsulation DNA without an aqueous gel in solution: a) phase contrast observation; b) Fluorescence microscopy (DNA labeled with DAPI) c) DNA labeled with syto 24.
  • the liposomes encapsulating DNA have a round shape and comprise the DNA.
  • Figure 2 is a set of photography showing the different stability of giant lipid vesicles encapsulating DNA without an aqueous gel inside the vesicle and liposomes encapsulating DNA with an aqueous gel inside the liposome when embedded in an agarose matrix. Most of the giant lipid vesicles without an aqueous gel break or collapse as presented in a).
  • Figure 4 is a set of photography showing liposomes encapsulating salmon testes DNA after 140 days after their preparation and storage in lipid/oil solution. Phase contrast images of the liposomes (a), DNA labeling with DAPI (b) and the lipid layer with nile red (c) confirming the stability of liposomes over time.
  • Figure 5 is a set of photography showing liposomes of different sizes sorted after 5, 10, 15 min (from left to right) of centrifugation at 32.5x g in water.
  • Figure 6 is a set of photography showing liposomes (L) and lymphocytes in agarose matrix 0.8% w/w, DNA has been labeled with ethidium bromide.
  • Figure 7 is a set of photography showing comet assay of liposomes containing DNA fragments from salmon testes (a) and E. Coli (b) after membrane lysis and electrophoresis.
  • Figure 8 is a photography of a liposome together with an intensity profile showing the process of DNA fragment identification of an average known size on a comet of a liposome (salmon testes genomic). Top: the comet, middle: intensity profile, bottom: comparison and normalization with PFG intensity bands.
  • Figure 9 is a schematic representation of the protocol to prepare capsules with a monolayer of surfactants, including the individual steps of preparation and further including the transfer step in aqueous solution prior to use.
  • Figure 10 is a photography showing a phase contrast microscopy image of capsules with a surfactant monolayer.
  • the surfactant monolayer consists of a mixture of the surfactants Tween 80/ Span 80.
  • the capsules further comprise a mixture of genomic E. coli DNA (2.5 mg/mL) and lambda phage DNA (48kbp) (0,25mg/ml) together with an aqueous gel.
  • the capsules with a monolayer of surfactants encapsulating DNA have a round shape.
  • Figure 11 is a histogramm displaying the size distribution of the capsules with a surfactant monolayer.
  • the surfactant monolayer consists of a mixture of the surfactants Tween 80/ Span 80.
  • the capsules further comprise a mixture of genomic E. coli DNA (2.5 mg/mL) and lambda phage DNA (48kbp) (0,25mg/ml) together with an aqueous gel.
  • the size distribution represents the sizes obtained without any sorting step.
  • Figure 12 is a set of photographs (a,b) showing fluorescence images of capsules and Trevigen cells after lysis before electrophoresis.
  • the capsules comprise an aqueous gel with a mixture of genomic E. coli DNA (2.5 mg/ml) and Lambda phage DNA (48kbp) (0,25mg/ml) and a surfactant monolayer (Tween 80/ Span 80). It is possible to distinguish the capsules from Trevigen cells before and after lysis by adjusting the microscope set-up, in contrast phase conditions. Capsules can be observed with the 40x objective but not T- cells. The DNA appears to diffuse more in Trevigen cells. Fluorescence intensities of DNA and diameters of beads are in the same range as that for cells.
  • Figure 13 is a set of photographs (a,c) a capsule prepared with a monolayer of surfactants together with an intensity profile.
  • the capsule were prepared with a surfactant monolayer and comprise genomic E. Coli DNA (2 ⁇ 9/ ⁇ _) together with capsids of bacteriophage T5 (120Kpb, 2 ⁇ g/ ⁇ L) (a and b) and genomic E. Coli DNA ⁇ g ⁇ L) and lambda phage DNA (48kbp, 1 ⁇ g ⁇ L) (c and d).
  • Figure 14 is a set of photographs showing fluorescence images of comets in alkaline conditions on capsules encapsulating a mixture E. Coli (2mg/ml) and capsids of phage T5 (2 mg/ml) comprising a 50% tween/span monolayer and standards sold by Trevigen. Conditions: lysis time:1 h30 , electrophoresis 20min
  • This example describes the preparation of capsules according to the invention, in particular, the preparation of liposomes according to the invention and the preparation of capsules comprising a surfactant monolayer according to the invention.
  • the continuous phase of the emulsion was prepared with mineral oil (Sigma) and lipids.
  • mineral oil Sigma
  • lipids A mixture of egg-PC (Sigma) and PEG-PE (Sigma) (98:2 mol %) was used as liposome-forming lipids.
  • Various lipids or mixtures of lipids can be used, either charged or not charged, and cholesterol or proteins can be added as well.
  • the continuous phase of the emulsion was prepared with mineral oil (Sigma) and surfactants.
  • a mixture of 50% Tween 80 and 50% Span 80 in a total concentration of 0.5% in mass was diluted in mineral oil.
  • the Oil-Tween80/Span80 solution was made by direct mixing of the surfactants with the oil under gentle stirring at room temperature. The mixture could be used immediately after preparation.
  • DNAs of different sizes were used such as: lambda DNA markers (8- 50 kb, Fermentas), lambda PFG DNA (50-1 ,000 kb, Biolabs, New England), lambda DNA markers (8-50 kb, Fermentas), lambda phage DNA (48 kbp), capsids of phage T5 DNA (wildtype) (120kbp), genomic salmon testes DNA (Sigma) and genomic E.coli DNA (Affymetrix). All those DNAs and mixtures of these DNAs were successfully encapsulated in liposomes.
  • the preparation of the liposomes of the invention involves 4 steps.
  • Step 1 Preparation of lipid-oil solution
  • DNA was dissolved in nuclease-free water (Promega) at 0.05 ⁇ g ⁇ L, 0.5 ⁇ g ⁇ L or 10 ⁇ g ⁇ L, gently mixed per inversion and stirred at 60°C for 1 -2 h. This solution was then mixed with a solution of low melting agarose (0.8% final concentration) at 60 °C to obtain a DNA-agarose solution at 0.05 ⁇ g ⁇ L, 0.375 ⁇ g ⁇ L or 7.5 ⁇ g ⁇ L respectively, and was left to homogenize at the same temperature for 30 min.
  • nuclease-free water Promega
  • DNA prepared according point 2 was successively vortexed at 10 Hz, 20 Hz and 30 Hz each time for 3 min at 60 °C and finally at 40 Hz for 2 min at 60 °C. The solution was then left to homogenize for 30 min at the same temperature.
  • Step 3 Preparation of the emulsion of nucleic acid-agarose beads
  • 5 ⁇ _ of the DNA-agarose solution were poured on the surface of 1 ml lipid-oil solution in a 2 ml tube and the solution was vortexed for 1 min at 40 Hz to create a micro- emulsion of DNA-agarose droplets in the lipid-oil solution.
  • the micro-emulsion could also be obtained by mixing the solutions with a pipette by 30 successive slow aspirations and ejections.
  • Gelation of the nucleic-acid aqueous gel beads was induced by decreasing the temperature at 4°C for at least 10 min.
  • the resulthg capsules were capsules comprising a lipid monolayer. They were left in the oil-lipid solution overnight (either at 4°C or room temperature).
  • the diameter of the resulting W/O droplets ranged from 10 ⁇ to 100 ⁇ . They were stored in the lipid-oil solution.
  • DNA fragments of different sizes from lambda DNA markers (8-50 kb), lambda PFG DNA in agarose solution (50-1000 kb), and eukaryotic and eukaryotic genomic DNAs (salmon sperm and E. coli respectively) were successfully encapsulated in the liposomes of the invention.
  • encapsulation of DNA fragments of calibrated size may be an additional advantage for calibrating comet tails, it is not a mandatory step.
  • Pulse field gel electrophoresis (PFGE) of the liposomes containing genomic DNA shows that DNA undergoes some mechanical breakings following the vortex step during the preparation of the DNA-aqueous gel beads. Fragments of different sizes are generated in a very reproducible way, the most intense of which is at 21 kb (Data not shown). These fragments can serve as a size reference. Time and frequency of vortexing as well as pipetting stages can be appropriately adjusted to modulate the size range and the number of produced DNA fragments. Fragmentation of salmon DNA depended on frequency and time of vortex.
  • the number of nucleotides inside a liposome of 10 ⁇ radius is equal to
  • the number of nucleotides in a eukaryotic cell is about 6 * 10 9 and taking account both volumes of eukaryote cell and liposome. It can be concluded that DNA concentration in liposomes is comparable to DNA concentration inside eukaryotic cells.
  • DNA retention efficiency In order to estimate the DNA retention efficiency in the liposomes, the inventors measured DNA concentration in the supernatant aqueous solution after centrifugation of liposomes. They found that this concentration measured by optical density method was 0.005 ⁇ 0.003 ⁇ g ⁇ L over an initial concentration of 7.5 ⁇ g ⁇ L (data from sperm salmon DNA). DNA retention efficiency is therefore higher than 99%.
  • the inventors have studied the stability of capsules comprising a lipid monolayer (precursor to liposomes) over a long period in terms of aggregation phenomena and leakage of encapsulated DNA in solution. They further studied their ability to form liposomes after storage.
  • Capsules with lipid monolayer were stored in oil/lipid solution were transformed into liposomes according to step g) of the method of fabrication as described in Materials and Methods and the liposomes were analyzed over time either at 4°C or room temperature (Figure 4).
  • Figure 4 illustrate liposomes encapsulating PFG DNA after 140 days of storage in oil/lipid solution, respectively. Epifluorescence observation of DNA (Figure 4b, center) shows that no apparent leakage of DNA occurred after 140 days.
  • lipids in the oil solution which act as surfactant, stabilizes the emulsion by creating a lipid layer around the nucleic acids- aqueous gel beads ( Figure 4c, below) and avoids aggregation phenomena, as verified also by phase contrast microscopy ( Figure 4a, above).
  • the very simple emulsion preparation method used here produces liposomes poly-disperse in sizes, with liposome size ranging from less than 20 ⁇ up to 200 ⁇ , and a mean diameter of 60 ⁇ . Nonetheless, a monodisperse distribution can be obtained by sorting the size of the liposomes.
  • the inventors simply sort the size of polydisperse liposomes by adjusting the duration of centrifugation applied to the liposome solution ( Figure 5).
  • Other methods can also be applied, like the extrusion of liposomes through a polycarbonate filter of desired pore size.
  • Figure 5 illustrates liposomes of different sizes sorted after 5, 10, 15 min (from left to right) of centrifugation at 32.5x g in water. After 5 min of centrifugation, liposomes at the bottom of the tube were collected for microscope analysis. A very large distribution of liposome size (between 50 and 200 ⁇ in diameter) was observed, with a pick value at 100 ⁇ . The supernatant solution was centrifuged again for 5 min (overall 10 min centrifugation) and liposomes at the bottom of the tube were collected for analysis. In this case, the resulting liposome size range was 10-100 ⁇ with a pick value at 50 ⁇ . A third run of a 5-minute centrifugation was applied to the remaining solution (for a total of 15 min of centrifugation) and liposomes were collected resulting in a size range of 4-50 ⁇ (pick value at 20 ⁇ ).
  • Sorting efficiency will be increased by the use of recent microfluidics approaches, which have turned out to be very efficient in sorting vesicles and beads of the same size range as liposomes (Srivastav, A. et al., Microfluids and Nanofluids, 13 (5), 697-701 ).
  • the emulsion method turns out to be very convenient in terms of yield of liposome per preparation, as all the DNA-agarose solution inside the aqueous solution, results in liposomes. Considering a mean liposome radius of 30 ⁇ , we can conclude by basic calculation that the total number of liposomes in the oil/water solution is in the order of 10 4 .
  • This example shows the use of liposomes of the invention as internal standards in comet assays.
  • Microscope slides (Superfrost Plus, Menzel-Glaser) were used for the assay and prepared as following: the slide was dipped in a solution of 1 .8% normal melting agarose dissolved in PBS 1 x (without Mg 2+ and Ca 2+ ) and air dried overnight at room temperature. A solution (85 ⁇ ) of 0.8% low melting point agarose in PBS 1 x (without Mg 2+ and Ca 2+ ) kept at 45 °C was placed on the slide, covered with 24 x 36 mm coverslip (Menzel-Glaser), and left to solidify on ice for 5 min.
  • Blood (20 ⁇ _) was diluted in 1 ml_ of RPMI 1640 with 10% Fetal Calf serum (FCS).
  • Ficoll 100 ⁇ _ was added at the bottom of the tube and cells were centrifuged at 1326x g for 4 min.
  • Lymphocytes 100 ⁇ _ were collected at the edge of the Ficoll layer, suspended in RPMI 1640 with 10% FCS and centrifuged again at 1326x g for 4 min.
  • the pellet was then suspended with 75 ⁇ _ of water solution containing DNA-Agarose liposomes and 75 ⁇ _ of 1 .6% low melting agarose in PBS.
  • the solution was gently mixed and 75 ⁇ _ were placed on the slide, sealed with the coverslip and left to solidify on ice for 5 min.
  • a third layer of 0.8% low melting agarose (75 ⁇ _) was added to the slide and cooled down on ice for 5 min.
  • 30 ⁇ _ of a solution 1 mM of H 2 0 2 in PBS 1 X were added to the cell tube and left in ice for 5 min, cells were centrifuged at 1326x g for 4 min and the pellet was collected.
  • 75 ⁇ _ of a solution 10 or 100 ⁇ of H 2 0 2 in PBS 1 X was placed on the slide and sealed with a coverslip for 5 min. Control slides were also covered with 75 ⁇ _ of a solution of PBS 1 X for 5 min in order to have the same experimental conditions for all the slides.
  • the slides were immersed in a lytic buffer (2.5 M NaCI, 100 mM EDTA, 10 mM Tris-HCI, 1 % N-lauryl sarcosine, 3% Triton X100 and 10% DMSO, pH 10) for 30 min at 4°C. Slides were then incubated in an electrophoresis tank with TBE 1 x buffer (89 mM Tris base, 89 mM boric acid and 2 mM Na2EDTA, pH 8.3) for 10 min. For each sample, 2 replicate slides were prepared, a control not submitted to electrophoresis, and slide treated by electrophoresis at 0.8 V/cm, 14 mA for 25 min.
  • a lytic buffer 2.5 M NaCI, 100 mM EDTA, 10 mM Tris-HCI, 1 % N-lauryl sarcosine, 3% Triton X100 and 10% DMSO, pH 10.
  • DNA-agarose solutions 5 ⁇ _ of the DNA-Agarose solution at 7.5 mg/ml was diluted to 100 ⁇ g ml in a solution of low melting agarose 0.8% (final concentration) and left to homogenize at 60 °C.
  • 1 ml of mineral oil was added to the solution and vortexed at 40 Hz for 1 min.
  • the absence of surfactants (lipids) made the emulsion very unstable and after 30 min at 60 °C a two phases (oil/DNA-agarose) solution was again visible. From this DNA-agarose solution, gel plugs 1 .5x5 mm were produced and stored in EDTA 0.5 M until use.
  • the plugs were equilibrated in 0.5 x TBE buffer (50 mM Tris, 50 mM boric acid, 1 mM EDTA) for 15 min at room temperature.
  • 0.5 x TBE buffer 50 mM Tris, 50 mM boric acid, 1 mM EDTA
  • Each agarose block as well as molecular weight markers were placed in the well of a 1 % PFGE agarose gel (Sigma) in 0.5 x TBE.
  • the pulsed field gel separation was made on a CHEF -DR II apparatus (Bio-Rad Laboratories, Inc) with pulses ranging from 5 to 25 sec at a voltage of 5 V/cm and switch angle of 120 ° for 18 h at 14°C. Gels were stained vith ethidium bromide and analyzed using a Gel-Doc 2000 system (Bio-Rad Laboratories).
  • Liposomes 50 ⁇ - of liposomes in water were mixed with 50 ⁇ - of 1 % PFGE agarose gel (Sigma) in K36 at 37 °C and gel plugs 1 5x5 mm were produced.
  • the pellet was suspended in 50 ⁇ PBS 1 X at 37 °C and 50 ⁇ of 1 % PFGE agarose gel (Sigma) in K36 at 37°C. From this solution, gel plugs 1 .5x5 mm wee produced.
  • the plugs were immersed in a lytic buffer (2.5 M NaCI, 100 mM EDTA, 10 mM Tris- HCI, 1 % N-lauryl sarcosine, 3% Triton X100 and 10% DMSO, pH 10) for 3 h in ice.
  • the plugs were then incubated in TBE 1 x buffer (89 mM Tris base, 89 mM boric acid and 2 mM Na 2 EDTA, pH 8.3) for 30 min and stored in 0.5 M EDTA until use.
  • liposomes are initially transferred in water and immediately used.
  • Liposomes and lymphocytes cells were mixed in the same agarose matrix used for cell electrophoresis, without damaging them (no breaking of liposomes and no leakage of the encapsulated material; Figure 6).
  • This last aspect is intrinsically related to the amount of DNA (number of fragments at a specific size), which is higher in large liposomes. This affects the intensity not only over the z direction, but also over the xy plane, as observed by confocal microscopy measurements.
  • Liposome comets have been characterized with the classical tools used for the comet assay, % Tail DNA (%TD) and Olive moment (OTM). For three different slides, each one containing at least six comets from liposomes in the size range 50-80 ⁇ , the averaged value for comets in the same slide is 28,00 ⁇ 4,78 (%TD) and 32,75 ⁇ 3,22 (OTM) (values of comets from salmon DNA liposomes).
  • Liposomes comets can be used as an internal standard for comparing results in the same experimental conditions between different slides (as a reference for intra-assay variability) as well as results in different experimental conditions and different laboratories (as a reference for inter-assay reproducibility).
  • Variability in the same experimental condition can arise mainly from non- homogeneity of the gel layers, of the fluorescent dye in different slides, and/or non- homogeneity of the electric field in the electrophoresis tank.
  • results from different slides can be different even for cells having the same level of damage.
  • the inventors report the results of the comet assay performed on lymphocytes cells treated with 10 mM of hydroxide peroxide, for two different slides (table 1 ). Even being in the same experimental conditions, % TD values between the two slides are different (table 1 ).
  • Preparation of liposomes encapsulating DNA fragments of defined sizes thus enables to obtain more information on sizes and quantity of DNA fragments present in the comet of the studied cells.
  • the size of DNA fragments, which are resolved in the studied comet is determined by comparing their position along the comet to that of DNA fragments of defined size along the liposome comet.
  • the liposome comets present a maximum of the intensity profile corresponding to a DNA fragment size of 21 kb, as found by PFG analysis.
  • the corresponding distance of migration from the comet head enables to identify the presence of 21 kb-DNA fragments.
  • Liposomes comprising a nucleic acid mixture of genomic E. coli DNA (0.25 mg/mL) and Lambda phage DNA (0,25mg/ml), or genomic E. coli DNA (2.5 mg/mL) and Lambda phage DNA (0,25mg/ml) were prepared according to the protocol described in the paragraph "2. 1 Method for the preparation of liposomes".
  • Step 3 of the "Preparation of the emulsion of nucleic acid-agarose beads” was modified and the solution was vortexed as described in 2.1 for 1 min at 40 Hz or, as modified, for 1 min at 40Hz with two glass beads to create a micro-emulsion of DNA-agarose droplets in the lipid-oil solution.
  • the liposomes were then used in the neutral comet assay as described and the comets were analyzed. The results show that adding two glass beads does not affect the shape of the beads and their comets (Data not shown).
  • the inventors thus conceived, prepared and tested new calibrated liposomes that encapsulate high concentrations of long calibrated DNA fragments entrapped into micro hydrogel liposomes surrounded by a double lipid layer. These liposomes are reproducible with respect to their manufacture, stable during at least 6 months, easy to handle and should be produced at low cost. These liposomes are relevant and particularly well suited to be used as internal standards for the comet assay.
  • the liposomes using for comet assays provides a reference set of calculated parameters (% TD or OTM for instance) and enables a normalization of the calculated parameters from cell comets. Thus, for the first time, a quantitative comparison with all comet assay results becomes possible.
  • Example 3 Use of capsules with a surfactant monolayer in comet assays
  • the preparation of the capsules with a surfactant monolayer thus involves the step as described herein below:
  • Step 1 Preparation of surfactant-oil solution
  • the continuous phase of the emulsion was prepared with mineral oil (Sigma) and surfactants.
  • a mixture of 50%Tween 80 and 50%Span 80 in a total concentration of 0.5% in mass was diluted in mineral oil.
  • the Oil-Tween80/Span80 solution was made by direct mixing of the surfactants with the oil under gentle stirring at room temperature. The mixture could be used immediately after preparation.
  • Step 2 Preparation of DNA-agarose solution
  • DNA such as genomic E. Coli DNA (4600kbp), was dissolved in nuclease-free water (Promega). Lambda phage DNA (48 kbp) was dissolved in 10mM Tris, 1 mM EDTA at pH 8.0. T5 phage DNA with capsids (121 kbp) was mixed in a buffer 100mM NaCI, 10mM Tris Ph 7 environ, 1 mM CaCI 2 , 1 mM MgCI 2 .
  • Solutions were prepared at concentrations 0.25 ⁇ g ⁇ L, 2 ⁇ g ⁇ L and 2.5 ⁇ g ⁇ L and were gently mixed per inversion and stirred at 60 °C for 1 -2 h (at 40 °C for T5 phageDNA to preserve the integrity of the capsids).
  • DNA solutions or a mixture of two DNA solutions were then mixed with a solution of low melting agarose (0.8% final concentration) at 60 °C (at 40 °C for T5 phage DNA) to obtain a DNA-agarose solution and was left to homogenize at the same temperature for 30 min. .
  • Step 3 Preparation of the emulsion of nucleic acid-agarose beads
  • 5 ⁇ _ of the DNA-agarose solution were poured on the surface of 1 ml surfactant-oil solution in a 2 ml tube and the solution was vortexed for 30s at 40 Hz by applying 3 vortex pulses of 10s each (instead of 1 min at 40 Hz) to create a micro-emulsion of DNA- agarose droplets in the surfactant-oil solution.
  • the resulting capsules comprising a monolayer of surfactant were left in the surfactant-oil solution at 4°C until use.
  • the diameter of the resulting droplets ranged from 2 ⁇ to 60 ⁇ .
  • Step 4 Transfer of the capsules to an aqueous solution
  • Alkaline comet assay was performed for the detection of DNA single strands.
  • Microscope slides (Superfrost Plus, Menzel-Glaser) were used for the assay and prepared as following: the slide was dipped in a solution of 2.0% normal melting agarose dissolved in PBS 1 x (without Mg 2+ and Ca 2+ ) and air dried overnight at room temperature. Then, a sandwich of three agarose gels was prepared by successive depositions and gelling of each layer on the slide.
  • the bottom layer consisted of a solution (85 ⁇ ) of 0.8% normal melting point agarose in PBS 1 x (without Mg 2+ and Ca 2+ ), kept at 45 °C, which was placed on the slide, covered with 24 x 36 mm coverslip (Menzel-Glaser), and left to solidify on ice for 5 min.
  • the second layer contained the capsules or the capsules /cells mixture. It was prepared as following. A volume of 75 ⁇ _ of the water suspension containing capsules was gently mixed with cells to be studied suspended in 75 ⁇ _ of 1 .0% low melting agarose in PBS. Then 75 ⁇ _ of the resulting suspension was placed above the first layer after removal of the coverslip.
  • This layer was sealed with a new coverslip and left to gel on ice for 5 minutes.
  • the third layer of 0.5 % low melting agarose (75 ⁇ _) was added to the slide at the top of the two other layers (after removal of the coverslip), covered with a coverslip and cooled down on ice for 5 min. After removal of the coverslip, the slides were immersed in a lysis buffer (2.5 M NaCI, 100 mM Na 2 EDTA, 10 mM Tris-HCI pH 10, 1 % Triton X-100 and 10% DMSO) for 90 min at 4°C.
  • a lysis buffer 2.5 M NaCI, 100 mM Na 2 EDTA, 10 mM Tris-HCI pH 10, 1 % Triton X-100 and 10% DMSO
  • the slide was the immersed in an alkaline solution (1 mM Na 2 EDTA, 300 mM NaOH) for 20 min at room temperature in a horizontal electrophoresis unit.
  • an alkaline solution (1 mM Na 2 EDTA, 300 mM NaOH) for 20 min at room temperature in a horizontal electrophoresis unit.
  • two replicate slides were prepared, a control slide, not submitted to electrophoresis, and a slide treated by electrophoresis at 0.8V.cm 25 V, 300 mA.
  • the slides were neutralized with 0.4 M Tris-HCI at pH 7.5, rinsed with ultrapure water and dipped into 100% methanol (HPLC grade purity solvent). Thereafter, the slides were dried at room temperature overnight and kept in a dry atmosphere until analysis. DNA staining was performed with 50 ml_ of ethidium bromide solution (2 mg/mL).
  • Capsules comprising a surfactant monolayer were prepared with different DNA such as genomic DNA of E. Coli, genomic DNA of Salmon testes, lambda phage DNA (48 kbp) and capsids of Phages T5 with phage T5 DNA (wild type)(120 kbp).
  • the DNA in capsids of Phages T5 (wild type)(120 kbp) allow using supercoiled DNA in a capsid, wherein the DNA is partly protected during the vortex stage.
  • the phase contrast microscopy image of the capsules comprising a surfactant monolayer in solution show that the capsules are spherical (Fig. 10).
  • the capsules may be stored in oil-surfactant and comprised a surfactant monolayer at the surface of an agarose (0.8%w/w) gel.
  • the inventors explored the impact of different DNA concentrations on the fluorescence of the capsules comprising a surfactant monolayer. In this context they studied the comparability of the obtained capsules with comets of the standards sold by Trevigen (lymphocytes). DNA concentration
  • capsules comprising a surfactant monolayer having a nucleic acid concentration of 2 to 4 mg/ml yield a fluorescent intensity (before
  • Capsules comprising a surfactant monolayer and encapsulating E.Coli DNA, E.
  • Coli DNA and lambda phage DNA 48kbp
  • E. Coli DNA and Capsids of Phage T5 120Kpb were prepared according to the protocol described above. Comets were observed for all of them in the Neutral comet assay as described above in Example 2.
  • Capsules comprising a surfactant monolayer comprising genomic E.Coli DNA (2 mg/ml) and capsules comprising a surfactant monolayer comprising a mixture of genomic E. Coli DNA () and Capsids of Phage T5 with phage T5 DNA (120Kpb) were prepared and used together with standards sold by Trevigen in the Neutral Comet Assay (Fig. 13). The different characteristics of the comets, such as %Tail DNA and OTM, were measured for the capsules and are listed in table 2.
  • the inventors further prepared capsules comprising a surfactant monolayer encapsulating genomic E.Coli DNA (2mg/ml) and Lambda Phage T5 Capsids with Phage T5 DNA(120kbp) (2 mg/ml) and performed, together with the standards sold by Trevigen, a comet assay in standard alkaline conditions. 4 different conditions were tested, in condition 1 and 2 the lysis time was 30min and electrophoresis time either 10 min (Cond. 1 ) or 20 min (Cond. 2), in condition 3 and 4 the lysis time was 1 h30min and the electrophoresis time either 10 min (cond. 3) or 20 min (cond. 4). Comets were observed in the four cases see Figure 14. All the obtained comets had suitable sizes and intensities The different characteristics of the comets, such as %Tail DNA and OTM, were measured for 6 capsules of the same slide and one Trevigen cell and are listed in table 3.
  • Table 3 Alkaline comet assay analysis of 5 capsules comprising a surfactant monolayer an comprising genomic E.Coli DNA (2mg/ml) and Lambda Phage T5 Capsids with Phage T5 DNA(120kbp) (2 mg/ml) measured in different experimental conditions.
  • the inventors thus conceived, prepared and tested new calibrated capsules comprising either a monolayer of surfactant or lipid bilayer that encapsulate high concentrations of long calibrated DNA fragments or chromosomal DNA entrapped into a core of hydrogel.
  • These capsules comprising a monolayer of surfactant or lipid bilayer are reproducible with respect to their manufacture, are stable during at least 1 month, easy to handle and should be produced at low cost.
  • capsules comprising a monolayer of surfactant or lipid bilayer are relevant and particularly well suited to be used as internal standards for the comet assay.
  • the capsules used for comet assays provides a reference set of calculated parameters (% TD or OTM for instance) and enables a normalization of the calculated parameters from cell comets. Thus, for the first time, a quantitative comparison with all comet assay results becomes possible.

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Abstract

La présente invention concerne des capsules renfermant des fragments d'acides nucléiques de tailles contrôlées variables à utiliser en tant qu'étalon interne dans des analyses par micro-électrophorèse, par exemple des analyses par micro-électrophorèse sur cellule unique.
PCT/EP2014/060483 2013-05-21 2014-05-21 Étalons internes d'adn pour des analyses par micro-électrophorèse WO2014187878A1 (fr)

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IT202100002537A1 (it) * 2021-02-05 2022-08-05 Kyme Nanoimaging Srl Processo microfluidico per la preparazione di nanostrutture liposomiche caricate con idrogel

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JPWO2018181420A1 (ja) * 2017-03-29 2020-02-06 大日本住友製薬株式会社 ワクチンアジュバント製剤
JP7169968B2 (ja) 2017-03-29 2022-11-11 住友ファーマ株式会社 ワクチンアジュバント製剤
US11833247B2 (en) 2017-03-29 2023-12-05 Sumitomo Pharma Co., Ltd. Vaccine adjuvant formulation
IT202100002537A1 (it) * 2021-02-05 2022-08-05 Kyme Nanoimaging Srl Processo microfluidico per la preparazione di nanostrutture liposomiche caricate con idrogel
WO2022167536A2 (fr) 2021-02-05 2022-08-11 Kyme Nanoimaging Srl Procédé microfluidique pour la préparation de nanostructures de liposomales chargées d'hydrogel
WO2022167536A3 (fr) * 2021-02-05 2022-09-15 Kyme Nanoimaging Srl Procédé microfluidique pour la préparation de nanostructures de liposomales chargées d'hydrogel

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