Method for preparing artificial target substance

Abstract

The present invention relates to a method for preparing an artificial target substance, said method comprising: producing an emulsion or suspension of the target substance, the emulsion or suspension comprising particles and / or microparticles of the target substance having an average particle size or microparticle size in the nanometer range, and concentrating the emulsion or suspension by a) adding a superabsorbent (2) to an initial first liquid volume (1) of the emulsion or suspension or adding a first liquid volume (1) to the superabsorbent (2), b) incubating a mixture formed by the superabsorbent (2) and the first liquid volume (1) for a first time period (t1), and removing the second liquid volume (4) from the liquid portion (3) of the mixture present after the incubation.

Description

Technical Field

[0001] The present invention relates to a method for preparing an artificial target substance. Background Art

[0002] Nanoparticles and nanomicroparticles are playing an increasingly important role in our lives. This concerns, for example, the use of these particles and microparticles for various applications in the pharmaceutical industry (e.g. in connection with encapsulated mRNA active ingredients), in food technology, or in the electrical engineering or electronics industry (e.g. in connection with quantum dots), and in this context also the production of these particles and microparticles.

[0003] Due to their small size and the associated changes in physical properties, nanoparticles or nanomicroparticles can be used to transport drugs to the desired organs, as they can penetrate the blood-brain barrier (e.g., suitable for use in pharmacology) and the skin (e.g., suitable for use in the cosmetics industry). Nanoparticles or nanomicroparticles are also used for diagnostic purposes - for example for color detection in rapid tests based on colloidal gold. They also stabilize foods and thereby ensure a longer shelf life.

[0004] Nanoparticles or nanoparticles are almost entirely anthropogenic substances. This means that the nanoparticles or nanoparticles have been produced by humans through industrial, commercial and public processes, rather than, for example, by biological organisms. Possible components include natural polymers such as albumin, but also biocompatible synthetic polymers such as polymethyl methacrylate, polyalkyl cyanoacrylates and copolymers, and various inorganic or organic particles.

[0005] Most of the manufacturing methods of nanoparticles are based on sol-gel methods, emulsion polymerization, interfacial polymerization and similar methods. At the end of the process, an emulsion of nanoparticles is present in a suitable solvent.

[0006] The concentration of the desired nanoparticles in the solvent naturally fluctuates during the production process and is often too low. The produced nanoparticles or nanoparticles must therefore be concentrated. However, due to the small size, this presents a problem. Concentration is usually performed by ultracentrifugation. This process is complex and, for large-scale industry, can usually only be performed in relatively small volumes. Some nanoparticles or nanoparticles may also be destroyed during centrifugation. Due to the tiny size of the nanoparticles or nanoparticles, filtration is not an option. Summary of the invention

[0007] The object of the present invention is to propose a method for the production of artificial target substances comprising nanoparticles or nanomicroparticles, which comprises a simple, rapid and generally applicable concentration method for the nanoparticles or nanomicroparticles.

[0008] This object is achieved in a surprisingly simple and generally applicable manner by means of the method defined in claim 1. Advantageous embodiments are listed in the dependent claims.

[0009] The technical solution is based on the use of so-called superabsorbents, with which any aqueous liquid, such as an emulsion or suspension, can be treated in order to concentrate the nanoparticles or nanoparticles contained in the liquid.

[0010] Superabsorbents (superabsorbent polymers, SAP) are plastics that are able to absorb several times their own weight in polar liquids. These are primarily water or aqueous solutions. When absorbing liquids, superabsorbents swell and form hydrogels. Hydrogels can be formed by crosslinked polymers of all polarities (e.g. polyacrylamide, polyvinylpyrrolidone, pullulan, gelatin, cellulose). However, copolymers of acrylic acid (acrylic acid, H2C=CH-COOH) or sodium acrylate (sodium salt of acrylic acid, H2C=CH-COONa) on the one hand and acrylamide on the other hand are usually used, where the ratio of the two monomers to one another can vary. In addition, so-called core crosslinkers (CXLs) are added to the monomer solution and connect the long-chain polymer molecules formed to one another in place via chemical bridges (also called crosslinks). Due to these bridges, the polymer is insoluble in water. This so-called base polymer may be subjected to so-called surface crosslinking (SXLs). Here, another chemical is applied to the surface of the particles, which, by heating, connects a second network only to the outer layer of the granules. This outer shell supports the swollen gel so that it also remains together under external loads (movement, pressure).

[0011] The product is traditionally used, for example, as a fine white granulate with a particle size of 100 to 1000 μm. It is mainly used in baby diapers, sanitary napkins, incontinence care products, bandages, and in small quantities in cable sheaths for deep-sea cables. Other areas of application are so-called gel beds, gel-forming extinguishing agents in firefighting, as mechanical stabilizers for flower arrangements in vases, or as additives to plant soils in order to store water continuously. However, potassium-neutralized acrylic acid is used here due to its better environmental compatibility. The use of superabsorbents as toys is known in the form of spherical particles, with names such as "Wasserperlen", "Aqua Beads" or "Water Beads". These are superabsorbents commercially available in the form of spheres of variable size (several sub-millimeters to several centimeters).

[0012] The present invention is based on the following unexpected observation: mixing a water sample with fluorescent nanoparticles having an average particle or microparticle size of 30 nm, after adding commercially available water pearl beads and after an incubation period during which the beads swell to several times their original volume, showed that the nanoparticles were not absorbed by the superabsorbent, but concentrated in the remaining residual liquid.

[0013] This observation shows that by using superabsorbents, and in particular superabsorbents commercially available in the form of so-called water pearl beads, various nanoparticles can be easily and quickly concentrated in liquid samples. In industrial production processes of nanoparticles or nanoparticles, these can be obtained in higher concentrations by this simple concentration possibility (for example, without the need for complex and inefficient ultracentrifugation or ultrafiltration).

[0014] Based on this observation, the object of the present invention can be achieved.

[0015] The method for producing an artificial target substance according to the present invention comprises:

[0016] - producing an emulsion or suspension of the target substance, wherein the emulsion or suspension contains particles and / or microparticles of the target substance having an average particle size or microparticle size in the nanometer range, and

[0017] - Concentrated emulsions or suspensions by:

[0018] a) adding a superabsorbent to an initial first liquid volume of an emulsion or suspension or adding a first liquid volume to a superabsorbent,

[0019] b) incubating for a first period of time a mixture obtained by mixing the superabsorbent and the volume of liquid, and

[0020] c) removing a second liquid volume from the liquid portion of the mixture present after the incubation.

[0021] "Anthropogenic substances" are substances that are not created by nature, but are created by humans, for example, through industrial, commercial or public processes. They include, for example, plastics, but also pesticides, pharmaceuticals, personal care products and industrial chemicals, as well as their degradation products and metabolites. Biomolecules (e.g., enzymes, DNA / RNA fragments, etc.) that are produced by microorganisms and have similar sizes in the nanometer range are not considered particles or granules of anthropogenic substances in this application.

[0022] In the above-described method according to the invention, the second liquid volume removed from the liquid portion of the mixture of liquid and superabsorbent present after incubation can be the entire remaining liquid portion. However, it is also possible to remove only a partial volume of a given liquid portion as the second liquid volume.

[0023] In the second liquid volume, the nanoparticles or nanomicroparticles are present in high concentration. The concentration or presence of the correct substance can be checked in a subsequent analysis, for example by means of a spectroscopic examination. However, the second liquid volume can also be further concentrated in one or more additional stages in the cascade method, for example by re-adding superabsorbent and / or re-adding to superabsorbent and re-incubating, or by means of conventional methods for concentrating the target substance - for example, by one of the methods specified at the beginning. If only a part of the liquid portion is removed as the second liquid volume, the target substance in the liquid portion of the mixture remaining after the removal can be further concentrated by re-incubating in a second time period. Both variants of the method can be carried out repeatedly so that a higher concentration of the target substance is obtained at each stage of the cascade concentration.

[0024] In an advantageous embodiment of the method, it is provided that the emulsion or suspension is produced by a sol-gel process, an emulsion polymerization process or an interfacial polymerization process. Alternatively, the emulsion or suspension is produced by crystallization or complex formation. These are established methods for producing nanoparticles or nanoparticles, wherein the nanoparticles or nanoparticles are present in the emulsion or suspension after the process is completed.

[0025] In the first stage, it is advantageous to reduce the initial sample volume by means of a superabsorbent using the method according to the invention, and in the subsequent second stage, further concentration of the target substance is carried out by means of conventional concentration techniques, such as filtration, ultrafiltration, precipitation reaction, ultracentrifugation or coating by means of the method described in EP2283026 B1. These known techniques can be used significantly more efficiently in the reduced liquid volume than in a more strongly diluted solution. Thus, the method according to the invention is suitable for significantly simplifying the known methods for concentrating nanoparticles or nano-microparticles for large volume sample liquids and / or sample liquids containing low concentrations of target substances. As mentioned above, in an advantageous embodiment, after removing the first sample, the method can include further concentration of the target substance in the removed first sample.

[0026] Further concentration of the target substance in the removed second liquid volume can be performed by means of filtration, ultrafiltration or precipitation reaction technology.

[0027] Alternatively, however, further concentration of the target substance in the removed second liquid volume can also be carried out again and optionally one or more times in cascade by carrying out the following method steps:

[0028] - adding a superabsorbent to the first sample or adding the first sample to the superabsorbent,

[0029] - incubating for a second period of time the mixture obtained by mixing the superabsorbent and the second liquid volume, and

[0030] - Removing the concentrated third liquid volume in the liquid portion of the mixture present after the incubation.

[0031] As described above, in the first stage of the cascade, the concentrated third liquid volume removed from the liquid portion remaining after incubation may comprise the entire volume of the liquid portion. Alternatively, the concentrated third liquid volume may be a partial volume of the remaining liquid portion.

[0032] The target substance can be further concentrated in the concentrated third liquid volume by means of filtration, ultrafiltration or precipitation reaction or in a further concentrated third liquid volume obtained by further concentration, in particular by means of a superabsorbent. This is advantageous if the volume of the concentrated first sample corresponds to only a few milliliters, for example 1 to 10 mL.

[0033] In the method according to the invention, the first liquid volume used initially, i.e. the emulsion or suspension, can contain a polar liquid, in particular as a main component. In an advantageous embodiment of the method according to the invention, the liquid volume can contain a polar solvent, in particular as a main component. For example, the liquid volume can consist of a polar solvent with a mass fraction of at least 50%. For example, the polar liquid or polar solvent can be water.

[0034] The target substance is a nanoparticle or nanoparticle. Such a substance consists of an artificial substance, in particular of at least one natural polymer, at least one biocompatible synthetic polymer, an inorganic material or an organic material.

[0035] As mentioned above, in an advantageous embodiment, the superabsorbent can be or include a plastic, which absorbs a certain proportion of the first liquid volume, for example a polar solvent such as water contained in the first liquid volume, to form a gel or hydrogel. Advantageously, the plastic is selected so that it does not substantially absorb nanoparticles or nanoparticles. This is the case, for example, in the above-mentioned superabsorbents made of the mentioned polymer and / or copolymer materials, such as the commercially available Wasserperlen, Waterbeads, etc.

[0036] The superabsorbent can be used in the form of particles, for example as a powder, as granules, or in the form of geometric bodies, in particular beads (spherical particles). It can thus be added to the liquid volume and / or the first sample in the form of such particles, or the liquid volume and / or the first sample can be added to the superabsorbent in this form. The diameter of the particles or beads can be between 100 and 5000 μm.

[0037] Advantageously, the superabsorbent is a commercially available superabsorbent sphere - for example, superabsorbent beads obtainable under the names "Aquabeads", "WaterBeads", "Wasserperlen", "Aquaperlen", "Hydrokugeln", "Gelkugeln".

[0038] In a favorable method embodiment, the volume of the liquid portion remaining after the incubation step and thus the concentration of the target substance in the remaining liquid portion are controlled by the length of the incubation period or periods, by the size and amount of superabsorbent particles or superabsorbent spheres added to the sample liquid and / or to the initially used first liquid volume of the first sample, and / or by the temperature prevailing during the incubation.

[0039] Subsequent physical detection methods for the qualitative and / or quantitative detection of nanoparticles or nanoparticles in the removed liquid volume are also possible - for example, to determine whether certain nanoparticles or nanoparticles are present in the corresponding concentrated liquid volume or how high their concentration is. In emulsions or suspensions, these cannot be detected using physical detection methods because their concentration is too low. A microscope is used for qualitative and / or quantitative determinations. In this case, for example, the nanoparticles or nanoparticles are counted in segments of defined size. Alternatively, fluorescence measurement methods or spectroscopic measurement methods are used. For this purpose, the nanoparticles or nanoparticles must contain a fluorescent dye, which is added in particular before the concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be described in more detail below with reference to the accompanying drawings and some exemplary embodiments. These examples do not represent any limitation to the reagents and methods according to the present invention. In the accompanying drawings:

[0041] Figure 1 is a schematic representation of the cascade concentration of a target substance in a liquid;

[0042] a) liquid before the addition of superabsorbent;

[0043] b) liquid after addition of superabsorbent and incubation of the mixture;

[0044] c) a second volume of liquid removed from the mixture after adding additional superabsorbent and incubating the mixture;

[0045] d) a mixture of liquid and superabsorbent remaining, optionally after removal of the second liquid volume and after reincubation;

[0046] Figure 2 is a representation of samples and blank samples, some of which were obtained by means of concentration according to the invention and exposed to UV light; and

[0047] Figure 3 shows the evaluation of the measurement data:

[0048] a) Graphical representation of the mean of the measured values ​​in a bar graph;

[0049] b) Correlation between the degree of sample concentration and the increase in fluorescence. DETAILED DESCRIPTION

[0050] The use of superabsorbents for concentrating target substances in polar liquids such as water as solvent, in particular nanoparticles or nanomicroparticles, is very simple and generally available. Figure 1 a and Figure 1 b Briefly describe the appropriate method:

[0051] 1. adding a superabsorbent 2 to a volume of a liquid 1, in particular an aqueous liquid, or alternatively: adding the liquid 1 to the superabsorbent 2;

[0052] 2. incubating the mixture of liquid 1 and superabsorbent 2 for a first period of time t1 to reduce (reduce in volume) the liquid portion 3 of the mixture; and then

[0053] 3. Transferring at least a second liquid volume 4 of the liquid part 3 of the mixture as a sample into a new container for further processing.

[0054] Further processing can be, for example, a quantitative and / or qualitative detection of a target substance in the liquid. For example, the detection is performed using spectroscopic or microscopic methods.

[0055] By means of the type of superabsorbent used, by means of the amount of superabsorbent used, or by means of the incubation time and / or the incubation temperature, the degree of concentration and the speed of the process can be controlled very accurately.

[0056] With this method, the problem of concentration during the production of nanoparticles or nanoparticles can be easily solved. Figure 1 a and Figure 1 The method shown in b works without the following: equipment such as ultracentrifuges, expensive ultrafiltration membranes, complex processes such as PEG precipitation or precipitation reactions that are usually used for concentrated nucleic acids, etc. In addition, the method can be generally used with respect to the type of nanoparticles or nano-microparticles. Another advantage is that superabsorbents are nontoxic and safe and are also biodegradable usually. The method according to the present invention makes it possible to greatly simplify the research of nanoparticles or nano-microparticles at low concentrations.

[0057] In the case of large volumes and / or strongly diluted liquids containing nanoparticles or nanoparticles at very low concentrations, cascade concentration of the target substance is possible. For example, in a first stage, the described method can be used together with the aforementioned steps 1 to 3 in order to concentrate the target substance. In a second stage, the volume of the second liquid volume 4 can be reduced for re-concentration of the target substance. This can be done by means of conventional filtration or precipitation methods or other conventional methods. Alternatively, however, such as Figure 1 As shown in FIG. 3 , the target substance in the second liquid volume 4 can also be re-concentrated by adding fresh superabsorbent 5 to the second liquid volume 4, or by transferring the first sample 4 to a new superabsorbent 5 and incubating it again within a second time period t2. Additionally or alternatively, Figure 1 d, by incubating the mixture for a third period of time t3, the liquid fraction 6 remaining in the mixture with the superabsorbent 2 after removal of the second liquid volume 4 can be further reduced. This leads to further swelling with an increase in the volume of the spheres consisting of superabsorbent 2 and a further volume reduction in the liquid fraction 6, which is accompanied by an increase in the concentration of the target substance in the liquid fraction 6.

[0058] In both alternative process paths, additional cascade concentration stages may follow.

[0059] Exemplary embodiments of the present invention will be described in detail below.

[0060] Example: Rhodamine B-filled latex nanoparticles (25.8 nm in diameter) in a water sample

[0061] The latex nanoparticles were provided by Fraunhofer Institute for Applied Polymer Research. The concentration of the latex particles in the stock solution was 2.02 M%. The particles were added to 500 mL of water sample and a 1:10,000 dilution was produced.

[0062] Subsequently, a superabsorbent in the form of commercially available "Waterbeads" was added to the water sample. During the concentration by absorption of water into the water beads, samples P0, P1, P2, each corresponding to a different concentration, were taken at different times.

[0063] The samples were divided into blank samples L0, L1, L2 for control (without latex nanoparticles) and samples P0, P1, P2 containing latex nanoparticles. Specifically, the samples were as follows:

[0064] -Blank sample L0: Ultrapure water without latex particles (500mL)

[0065] -Blank sample L1: Concentration of 500mL ultrapure water to 40mL ultrapure water

[0066] -Blank sample L2: Further concentration of L1 to 1 mL of ultrapure water

[0067] - Sample P0: 1:10000 dilution of latex particle stock solution in 500 mL ultrapure water

[0068] -Sample P1: Concentration of 500mL ultrapure water to 40mL ultrapure water

[0069] -Sample P2: Further concentration of P1 to 1 mL of ultrapure water

[0070] The particles in the corresponding samples are detected by measuring the fluorescence of the dye contained in the latex nanoparticles.

[0071] Figure 1 The qualitative detection of latex nanoparticles is shown. For this purpose, the corresponding samples P0, P1, P2 and blank samples L0, L1, L2 are transferred to UV-transparent measuring plates in triplicate. The measuring plates with samples are then exposed to UV light. Exposure to UV light causes fluorescence excitation in those samples where latex particles are present. No fluorescence is seen in blank samples L0, L1, L2, while for samples P0, P1, P2 comprising latex nanoparticles, an increase in fluorescence is seen as concentration increases.

[0072] Table 1 shows the quantitative measurement of the corresponding samples using the fluorescence measurement device. The measurements were performed by exciting the samples P0, P1, P2 and the blank samples L0, L1, L2 (each in triplicate) at 559 nm emission. Although the values ​​of the blank samples L0, L1, L2 remained low with concentration, the values ​​of the samples P0, P1, P2 containing nanoparticles increased in proportion to the degree of concentration.

[0073] Table 1:

[0074]

[0075] exist Figure 2 In , the measured values ​​listed in Table 1 are displayed or evaluated graphically. Figure 2 In a), the mean values ​​of the corresponding measured values ​​of the samples are shown as a bar graph. Figure 2 In b), the correlation between the degree of enrichment and the increase in fluorescence for samples P0 ("1"), P1 ("2") and P2 ("3") containing nanoparticles is shown.

[0076] The data from the experiments clearly show that the fluorescent latex nanoparticles of the sample do not diffuse into the utilized superabsorbent ("Waterbeads"), but remain in the external solution, and the concentration of the latex nanoparticles thereby increases continuously and in correspondence with the degree of concentration. This allows the nanoparticles or nanomicroparticles produced in industrial processes to be optimally concentrated. Industrial processes include, for example, sol-gel processes, emulsion polymerization processes or interfacial polymerization processes. This produces an emulsion or suspension consisting of a liquid portion and nanoparticles or nanoparticles. The average size of the nanoparticles or nanoparticles is less than one micron. Alternatively, the suspension can also be produced by crystallization or complex formation. Adding the emulsion or suspension to the superabsorbent causes the superabsorbent to absorb the liquid of the emulsion or suspension, so that the nanoparticles or nanoparticles are present in high concentration in the remaining residue.

[0077] Reference numerals list

[0078]

Claims

1. A method for producing an artificial target substance, comprising: - producing an emulsion or suspension of the target substance, said emulsion or suspension comprising particles and / or microparticles of the target substance having an average particle size or microparticle size in the nanometer range, and - Concentrating the emulsion or suspension by: a) adding a superabsorbent (2) to an initial first liquid volume (1) of the emulsion or suspension or adding the first liquid volume (1) to the superabsorbent (2), b) incubating the mixture formed by the superabsorbent (2) and the first liquid volume (1) for a first period of time (t1), and c) Removing a second liquid volume (4) from the liquid portion (3) of the mixture present after the incubation.

2. The method according to claim 1, wherein the emulsion or suspension is produced by a sol-gel method, an emulsion polymerization method or an interfacial polymerization method.

3. The method of claim 1, wherein the emulsion or suspension is produced by crystallization or complex formation.

4. The method according to any one of the preceding claims, The method also includes additionally concentrating the artificial target substance in the removed second liquid volume (4).

5. The method according to claim 4, The further concentration of the target substance in the removed second liquid volume (4) is carried out by filtration, ultrafiltration or precipitation reaction technology.

6. The method according to claim 4, The further concentration of the target substance in the removed second liquid volume (4) is again performed in the following manner: - adding a superabsorbent (5) to the second liquid volume (4) or adding the second liquid volume (4) to the superabsorbent (5), - incubating for a second period of time (t2) the mixture obtained by mixing said superabsorbent (5) and said second liquid volume (4), and - Removing the concentrated third liquid volume from the liquid portion of said mixture present after incubation.

7. The method according to claim 6, In this case, the target substance is further concentrated in the third liquid volume, in particular by means of a superabsorbent.

8. The method according to any one of the preceding claims, The first liquid volume (1) contains a polar liquid, such as water.

9. The method according to claim 8, The particles or microparticles of the artificial target substance are composed of at least one natural polymer, at least one biocompatible synthetic polymer, an inorganic material or an organic material.

10. The method according to any one of claims 1 to 9, The superabsorbent (2, 5) comprises a plastic that absorbs a portion of the liquid volume - water - thereby forming a hydrogel.

11. The method according to claims 9 and 10, The plastic material does not substantially absorb any particles or microparticles contained in the anthropogenic target substance.

12. The method according to any one of claims 1 to 11, The superabsorbent (2, 5) is used in particulate form, for example as a powder, as granules, or in the form of geometric bodies, in particular spheres.

13. The method according to any one of claims 1 to 12, The superabsorbents (2, 5) are used in the form of, in particular, commercially available "waserperlen", "hydrokugeln", "aquaperlen", "aquabeads", "waterbeads" or "gelkugeln".

14. The method according to any one of claims 1 to 13, The volume of the liquid fraction remaining after incubation is controlled by the length of one or more time periods (t1, t2) of the incubation and / or by the type and / or amount of the superabsorbent (2, 5) and / or by the temperature prevailing in the mixture during the incubation.

15. The method according to claim 14, The superabsorbent (2, 5) is used in particulate form, for example as a powder, as granules, or in the form of geometric bodies, in particular spheres, and the volume of the remaining liquid fraction after incubation is controlled by the size and / or number of the particles.

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