WO2023002080A1 - Extraction et purification de protéines avec des nanoparticules magnétiques stabilisées avec des systèmes dendritiques carbosilanes - Google Patents

Extraction et purification de protéines avec des nanoparticules magnétiques stabilisées avec des systèmes dendritiques carbosilanes Download PDF

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WO2023002080A1
WO2023002080A1 PCT/ES2022/070471 ES2022070471W WO2023002080A1 WO 2023002080 A1 WO2023002080 A1 WO 2023002080A1 ES 2022070471 W ES2022070471 W ES 2022070471W WO 2023002080 A1 WO2023002080 A1 WO 2023002080A1
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group
npm
proteins
protein
integer ranging
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PCT/ES2022/070471
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Spanish (es)
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Mª Concepción GARCÍA LÓPEZ
Mª Luisa MARINA ALEGRE
Rafael GÓMEZ RAMÍREZ
F. Javier DE LA MATA DE LA MATA
Isabel PRADOS NIETO
Andrea BARRIOS GUMIEL
Javier SÁNCHEZ-NIEVES FERNÁNDEZ
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Universidad De Alcalá
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B63/00—Purification; Separation; Stabilisation; Use of additives
    • C07B63/04—Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/04—Esters of silicic acids

Definitions

  • the present invention refers to the extraction or purification of proteins using magnetic nanoparticles (NPM) of iron oxide (preferably magnetite; Fe 3 0 4 ), coated on their surface with dendritic molecules of carbosilane structure, which in turn are functionalized in its periphery with active groups that are preferably in anionic form.
  • NPM magnetic nanoparticles
  • This extraction is carried out in an aqueous medium without using organic solvents.
  • the present invention also relates to the method of reusing the NPM for said use.
  • Protein purification/extraction is a tedious step that usually requires the use of polluting and non-reusable reagents and solvents that are ultimately not sustainable (Wu, X., et al. Proteomics 2014, 14, 645). As a consequence of this fact, it is important to develop alternative strategies to the commonly used methods that use, for example, water as an extraction medium.
  • Magnetic nanoparticles have attracted much attention due to their large surface/volume ratio and because they can be separated or guided by an external magnetic field (Akbarzadeh, A. et al. Nanoscale Research Letters 2012, 7, 144).
  • those formed by iron oxides (magnetite, FesCL; maghemite, Fe2C>3) stand out for their biocompatibility.
  • its applicability in a certain field requires adequate functionalization.
  • the surface coating can favor its dispersion in the liquid state and, therefore, its use. Its applications include bioseparation, molecular detection or analyte concentration (Oksvold, MP et al. Methods Mol Biol 2015, 1218, 465).
  • NPMs coated with different ligands have been used in the purification of different enzymes and proteins (Farzi-Khajeh, H. et al. Colloids and Surfaces B: Biointerfaces 2019, 175, 644; Cimen, D. et al., Separation Science and Technology 2020, 55, 2259; Xu, J. et al. Molecules 2014, 19, 11465; Gu, H. et al. Chemical Communications, 2006, 9, 941; Nicolás, P. et al. Journal of Food Engineering, 2019 , 263, 380). In most of these studies, the validity of the application of the NPM with real samples but with standard proteins. In addition, none of these works has evaluated the possibility of reusing these materials.
  • dendritic molecules are hyperbranched molecules of arborescent construction, of a well-defined size and three-dimensional structure and that have uniform chemical properties due, in part, to their low polydispersity as a consequence of their controlled synthesis.
  • Dendrimers and dendrons of older generations present a spherical molecular topology. In both cases, their surface contains the active groups of these molecules.
  • focal point can be used to introduce a new active function or as an anchor to other systems, such as a NP.
  • MD or systems modified with them can interact with different biomacromolecules. This interaction will depend on the type of function that the MDs present in their periphery and is related to their multivalence, since they present a high number of functionalities on the same molecule.
  • dendritic systems with anionic or cationic groups interact with peptides or proteins depending on the charge of the dendritic system and the isoelectric point of the protein. Furthermore, this ability is maintained if these dendritic systems are incorporated into gold NPs (Vásquez-Villanueva, R. et al. Microchimica Acta 2019, 186, 508). These types of systems have been tested for the extraction of proteins and are capable of selectively interacting with some proteins based on their charge and size. Unfortunately, the separation of these MD-protein conjugates from the medium is not easy because they have similar behaviors to free proteins.
  • NPM-MD modified NPMs
  • the present invention provides a method for the separation of proteins and peptides using iron oxide magnetic nanoparticles (NPM), preferably magnetite (FesCL), but without ruling out others such as maghemite (Fe2C>3).
  • NPMs are coated with dendritic molecules (MD) with a carbosilane structure that are functionalized on their periphery with anionic (for example, as carboxylate, sulfonate, or phosphate), cationic (ammonium groups), or neutral (carboxylic, sulfonic, and amino groups) groups ( Figures 1-4).
  • This type of NPM-MD corresponds to those already described in Barrios-Gumiel, A. et al. Colloids Surf. B 2019, 181, 360; or in the Spanish patent application ES2830873A1 ( Figure 5).
  • a first aspect of the present invention refers to an NPM-MD (hereinafter compound of the invention) comprising:
  • This nucleus composed of iron oxide, preferably magnetite (Fe 3 0 4 ) of nanoscopic size.
  • This nucleus can have a spherical, cylindrical, prism or other arrangement of its atoms, with at least one dimension between 1 and 1000 nm.
  • dendritic molecule is meant in the present invention a highly branched macromolecule where the growth units, branches or ramifications have a carbosilane skeleton and this dendritic molecule is functionalized in its external layer with anionic groups selected from carboxylate groups or sulfonates, with cationic groups selected from ammonium groups or with neutral groups selected from carboxylic, sulfonic and amino groups and comprising an anchoring chain of the formula -Si-(CH2)bR 1 -(CH2) a - where: the dendritic molecule is attached to the nucleus by the Si atom, a is an integer ranging from 0 to 10; b is an integer ranging from 1 to 10, preferably ranging from 1 to 5; Y
  • R 1 is selected from a urea, carbamate, thiocarbamate, thiourea group or a triazole group, preferably it is a urea group.
  • This dendritic compound is preferably a dendron ( Figures 1 and 3), the latter also called a dendritic wedge, which refers to a highly branched cone-shaped macromolecule and is defined by a focal point, the units, branches or ramifications of growth, which start from said focal point and the external layer, surface or periphery of said ramifications, which incorporates functional groups, and where:
  • the focal point of the dendron is the anchor chain of formula -Si-(CH 2 ) b -R 1 -(CH 2 ) a - where: the dendron is attached to the nucleus by the Si atom, a is an integer that it varies between 1 and 10, preferably between 1 and 5; yb and R 1 are defined in the above; Y
  • the outer layer of the dendron consists of the same or different units of the group of formula (I): where: R 2 is a (C 1 -C 4 )alkyl group, preferably R 2 is a methyl group; p is an integer and varies between 1 and 3, preferably p is 2;
  • R 3 is the following group -(CH2) c -S-(CH2)dR 4 ; c represents an integer ranging from 2 to 5; preferably c is 2 or 3; d represents an integer ranging from 1 to 10; preferably d varies between
  • R 4 is selected from -NR'R", -NR'R"R"', -COOR', -COO-, -S0 3 R' and -SOs, where R', R" and R'" represent independently an alkyl group (C 1 -C 4 ) or a hydrogen.
  • R 4 is a -CO 2 group, a -CO 2 H group or a -NMe 3 + group; and more preferably c is 2 or 3 and even more preferably d is 1 or 2.
  • this dendritic compound is preferably a carbosilane dendrimer
  • this dendrimer is heterofunctionalized (see for example WO 2014016460) and consists of an external layer, which has the same or different units from the group of formula (II): where: R 2 is a (C1-C4) alkyl group, preferably R 2 is a methyl group; p is an integer and varies between 1 and 3, preferably p is 2;
  • R 5 is the group -(CH 2 )cS-(CH 2 )dR 6 ; c represents an integer ranging from 2 to 5; preferably c is 2 or 3; d represents an integer ranging from 1 to 10; preferably d varies between 1 and 5;
  • R 6 is selected from -NR'R", -NR'R"R"', -COOR', -COO-, -S0 3 R' and -S0 3 , where R', R" and R'" represent independently a (C1-C4) alkyl group or a hydrogen, and with the proviso that at least one R 6 group of the external layer of the dendrimer consists of the anchoring chain of formula -Si-(CH 2 ) b -Ri- (CH 2 ) a - where the dendrimer is attached to the nucleus by the Si atom, ya, b and R1 are defined above.
  • R 6 is a -C0 2 group, a -C0 2 H group, or a -NMe3 + group, more preferably c is 2 or 3 and even more preferably d is 1 or 2. Therefore, this silicon atom present in said chain is the one that anchors the dendrimer to the surface of the NPM.
  • alkyl refers in the present invention to aliphatic chains, straight or branched, having from 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert- butyl or sec-butyl preferably has 1 to 2 carbon atoms, more preferably the alkyl group is methyl or ethyl.
  • the compound of the present invention is preferably anionic, for example R 4 or R 6 formed by carboxylate groups (-C0 2 ). Therefore, the present invention not only includes the compounds themselves, but also any of their salts. Preferably the salts are sodium or potassium.
  • the present invention not only includes the compounds themselves, but any of their salts.
  • the salts are halide, which can be selected from chloride, bromide, iodide; or other types of anions such as triflate.
  • the salts are iodide and chloride.
  • a second aspect of the present invention includes the extraction of the proteins, which is carried out preferably using water, so that the use of organic solvents is avoided.
  • the present invention also describes the reuse of the NPM-MD.
  • the NPM-MD (preferably second generation, Figure 5) are dispersed in water and placed in contact for a determined time, preferably between 1 minute and 120 minutes, more preferably a few minutes, with the proteins to be extracted/ purify under certain pH conditions, preferably acidic pH, more preferably at a pH between 1.5 and 5, and even more preferably at pH 1.8, preferably using a NPM-MD:protein ratio between 1:1 and 1: 1000, which will depend on the size of the proteins to be extracted/purified (Figure 6).
  • this method has been used with different model proteins (eg lysozyme, myoglobin, concanavalin, bovine serum albumin), without ruling out others.
  • An external magnetic field eg a magnet
  • the magnet will remain in contact with the solution for a suitable time, preferably between 1 and 120 minutes.
  • the NPM-MD-protein conjugate is washed with water.
  • the present invention also refers to the release of the proteins or peptides from the conjugate formed with the NPM-MD, such that the biomacromolecule is obtained on the one hand and the NPM-MD system on the other.
  • different media were tested. These media and some conditions are indicated below, but others are not ruled out: 1 M urea, 0.1 M NaOH, 0.2% trifluoroacetic acid, 0.2% sodium dodecyl sulfate (SDS) at room temperature and at 100 °C and water at 100°C.
  • Figure 7 shows the profiles obtained by dodecylsulfate polyacrylamide gel electrophoresis (SDS, SDS-PAGE) corresponding to the proteins that were released from NPM-MD after treatment of the complex formed with the proteins with the aforementioned media and conditions.
  • SDS dodecylsulfate polyacrylamide gel electrophoresis
  • the release of the proteins or peptides from the conjugate formed with the NPM-MD is carried out in a medium selected from urea, NaOH, trifluoroacetic acid, sodium dodecyl sulfate at a temperature between 18°C and 100°C, water at about 100°C, C 4 -C 20 alkyl or aryl sulfonates, or sulfates.
  • a medium selected from urea, NaOH, trifluoroacetic acid, sodium dodecyl sulfate at a temperature between 18°C and 100°C, water at about 100°C, C 4 -C 20 alkyl or aryl sulfonates, or sulfates.
  • the present invention also shows that the use of the NPM-MD system obtained after releasing the protein can be used again in the extraction of new samples of biomacromolecules (Figure 8), an aspect that is not observed when NPM not coated with MD is used. .
  • biomacromolecules refers to proteins or peptides, preferably proteins, but without ruling out other systems formed by the union of amino acids.
  • NPM-MD consisting of a Fe 3 0 4 magnetite core and 2nd generation carbosilane dendron shells with carboxylic acid (Figure 1 top) or ammonium ( Figure 1 bottom) functions.
  • FIG. 7 Profiles obtained by SDS-PAGE corresponding to solutions (blanks) containing lysozyme (LYS), myoglobin (MYO), concanavalin (CONC) or bovine serum albumin (BSA) and solutions containing the proteins eluted from NPM-MD ( from 2nd generation with carboxylate groups) using different media and conditions: 1 M NaCI, 1 M urea, 0.2% NaOH, 0.2% SDS at room temperature and at 100 °C and water at 100 °C.
  • LYS lysozyme
  • MYO myoglobin
  • CONC concanavalin
  • BSA bovine serum albumin
  • Figure 8 Percentage of the proteins lysozyme (LYS), myoglobin (MYO), concanavalin (CONC) and bovine serum albumin (BSA) retained in NPM-MD (2nd generation with carboxylate groups) in three consecutive experiments.
  • LYS lysozyme
  • MYO myoglobin
  • CONC concanavalin
  • BSA bovine serum albumin
  • Figure 10 Protein separation obtained by reverse phase high performance liquid chromatography from untreated cheese whey (whey protein) and after treatment with NPM-MD (2nd generation with carboxylate groups) at different NPM ratios -MD: protein.
  • NPM-MD for the purification of proteins extracted from a by-product of the olive industry, the olive bone
  • NPM-MD for the purification of proteins extracted from a by-product of the olive industry, the olive bone
  • the protein extract from the olive pits is obtained using the method described in patent ES2487115B1. Briefly, the olive stone is left to dry at room temperature and the seed is extracted from it by fracture by mechanical procedures. Once the olive seed has been extracted, it is crushed in a mill. Seed proteins are extracted using an extraction medium consisting of 125 mM Tris-HCl buffer (pH 7.5) containing sodium dodecyl sulfate and dithiothreitol.
  • the proteins go into aqueous solution and the solid residue is removed by centrifugation.
  • the extracted proteins are purified by precipitation with acetone for 24 h at 4 o C followed by centrifugation.
  • the purification of the extracted proteins is carried out using the NPM-MD at acidic pH, preferably between 1.5 and 5; using different NPM-MD:protein ratios.
  • These NPM-MD are like those described in Barrios-Gumiel, A. et al. Colloids Surf.
  • Figure 9 shows the separation of the proteins that remain in the extracts after treatment with NPM-MD, preferentially modified with second-generation dendrons with carboxylic groups or preferentially modified with second-generation dendrons with ammonium groups, at different ratios.
  • NPM-MD protein or after its precipitation with acetone.
  • NPM-MD:protein ratio When a 25:1 NPM-MD:protein ratio is used, it is possible to retain all the proteins in the extract, leaving none in the extract. The same is also observed when the proteins are precipitated with acetone, although in this case a longer time is necessary for the purification of the proteins (24 h), as well as the use of an organic solvent such as acetone or ethanol. .
  • the solutions are placed in contact with a magnet for a suitable time (about 10 min) that allows the separation of the NPM-MD-protein complex from the rest.
  • a surfactant such as sodium docedyl sulphate (SDS) of a determined concentration, for example 0.4%, at a determined temperature (for example at 100°C), for a determined time, for example 10 min.
  • SDS sodium docedyl sulphate
  • Cheese whey is a by-product of the cheese industry that contains proteins such as a-lactalbumin (a-LA) and b-lactoglobulins (b-LG (A + B)), which have a high biological and economic value.
  • the recovery of the proteins contained in this by-product can be done by filtration through membranes or spray-drying (Nicolás, P.; Ferreira, M. L.; Lassalle, V. Journal of Food Engineering, 2019, 263, 380).
  • Figure 10 shows the chromatographic separation of the main serum proteins (a-LA and b-LG (A + B)) that are not retained in NPM-MD, preferentially modified with second generation dendrons with carboxylic groups or modified preferably with second generation dendrons with ammonium groups, when added in NPM-MD:protein ratios between 1:1 and 25:1.
  • a-LA is partially retained while the retention of b-LG is not observed (A + B).
  • Protein retention increases when the NPM-MD:protein ratio is increased, observing the highest retention from a ratio of 10:1.
  • the NPM-MDs are dispersed in water and placed in contact for a few minutes (less than two minutes), with the proteins to be extracted/purified under conditions of pH determined (preferably acidic, pH 1.8) using a NPM-MD:protein ratio between 1:1 and 1:1000, which will depend on the size of the proteins to be extracted/purified.
  • Table 1 shows values of isoelectric points and molecular weight of model proteins (eg lysozyme, myoglobin, concanavalin, bovine serum albumin) that serve as examples, without ruling out others. Table 1. Molecular weights and isoelectric points of the proteins used.
  • NaCI is a reagent that is often used to break electrostatic interactions although, in the examples of this invention, it did not allow the release of the proteins.
  • SDS dodecyl sulfate
  • this invention shows that the use of the NPM-MD system obtained after releasing the protein can be used again in the extraction of new biomacromolecules.
  • Figure 8 shows the percentages of protein retained in the NPM-MD, modified with a second generation dendron with carboxylic groups, in three consecutive extractions. As can be seen, the NPM-MD can be used in successive extractions without losing its magnetism or its retention capacity, an aspect that is not observed when NPM not coated with MD is used.

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Abstract

La présente invention concerne l'extraction ou la purification de protéines à l'aide de nanoparticules magnétiques (NPM) d'oxyde de fer (magnétite ; FesO4, recouvertes sur leur surface de molécules dendritiques à structure carbosilane, fonctionnalisées sur leur périphérie avec des groupes actifs qui, de préférence, se présentent sous forme anionique. Cette extraction est mise en oeuvre dans un milieu aqueux sans utiliser de dissolvants organiques. La présente invention concerne galement le procédé de réutilisation des NPM à cet effet.
PCT/ES2022/070471 2021-07-20 2022-07-19 Extraction et purification de protéines avec des nanoparticules magnétiques stabilisées avec des systèmes dendritiques carbosilanes WO2023002080A1 (fr)

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Publication number Priority date Publication date Assignee Title
ES2830873A1 (es) * 2019-12-04 2021-06-04 Univ Alcala Henares Nanoparticulas magneticas estabilizadas con sistemas dendriticos carbosilano y sus usos

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2830873A1 (es) * 2019-12-04 2021-06-04 Univ Alcala Henares Nanoparticulas magneticas estabilizadas con sistemas dendriticos carbosilano y sus usos

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BARRIOS-GUMIEL ANDREA, SEPÚLVEDA-CRESPO DANIEL, JIMÉNEZ JOSÉ LUIS, GÓMEZ RAFAEL, MUÑOZ-FERNÁNDEZ MARÍA ÁNGELES, DE LA MATA F. JAVI: "Dendronized magnetic nanoparticles for HIV-1 capture and rapid diagnostic", COLLOIDS AND SURFACES B: BIOINTERFACES, ELSEVIER AMSTERDAM, NL, vol. 181, 1 September 2019 (2019-09-01), NL , pages 360 - 368, XP093027616, ISSN: 0927-7765, DOI: 10.1016/j.colsurfb.2019.05.050 *
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