Preparation method and application of albumin nanoparticles

Abstract

The embodiment of the invention provides a preparation method and application of albumin nanoparticles. The preparation method comprises the following steps: dissolving albumin in water to obtain an albumin solution; adding ethanol into the albumin solution to obtain a mixed solution; and heating the mixed solution, stirring, taking a supernatant, and dehydrating the supernatant to obtain the albumin nanoparticles. According to the preparation method of the albumin nanoparticles provided by the embodiment of the invention, the ultrapure water is adopted to dissolve the albumin, the ethanol is added into the albumin solution to destroy the hydration layer on the surface of the albumin, and the albumin is heated to form a gel structure; and meanwhile, the albumin is broken and polymerized to form nanoparticles under the action of stirring shearing force and cavitation of gas and ethanol steam in water. Therefore, according to the preparation method provided by the embodiment of the invention, the nanoparticle forming time is shortened, the use amount of the organic solvent can be reduced, the process is simplified, and the albumin nanoparticles with low particle size and uniform particle size distribution can be obtained.

Description

Technical Field

[0001] The embodiments of the present application relate to the technical field of pharmaceutical preparations, and specifically to a method for preparing albumin nanoparticles and uses thereof. Background Art

[0002] Albumin has good biocompatibility and biodegradability, which makes it widely used in the medical field, especially in nanoformulations. Albumin nanoparticles (ANPs) are nanoparticles with albumin as the basic material, which have the following advantages: 1. Drug delivery: Albumin nanoparticles can be used as drug carriers to encapsulate hydrophobic drugs inside the nanoparticles, or to bind hydrophilic drugs to the surface of the nanoparticles to achieve targeted delivery and controlled release of drugs. 2. Biocompatibility: Albumin has good biocompatibility, is not easy to cause immune response, and can circulate in the body for a long time. 3. Targeting: Albumin nanoparticles can be modified with functional ligands (such as antibodies, peptides) on the surface to achieve targeted delivery to specific organs or cells. 4. Long circulation: Albumin nanoparticles have a long blood half-life, which can prolong the action time of drugs in the body.

[0003] At present, the traditional preparation methods of albumin nanoparticles mainly include thermal gelation and desolvation. Among them, the thermal gelation method is to dissolve the drug and albumin in an appropriate buffer, heat to form albumin thermal gel, and then cool to form nanoparticles. This method needs to rely on the buffer system, and the heating time is longer. Heat treatment may cause partial drug degradation. The desolvation method is to dissolve the drug and albumin in an organic solvent, add a desolvation solvent (such as water or buffer), and form nanoparticles through solvent replacement. However, residual organic solvent may affect the stability of the nanoparticles.

[0004] Therefore, finding new methods for preparing albumin nanoparticles is an urgent problem to be solved in this field. Summary of the invention

[0005] The purpose of the present application is to provide a preparation method and use of albumin nanoparticles.

[0006] To achieve the above objectives, the present application proposes the following technical solutions:

[0007] In a first aspect, the present application provides a method for preparing albumin nanoparticles, the preparation method comprising:

[0008] dissolving albumin in water to obtain an albumin solution;

[0009] adding ethanol to the albumin solution to obtain a mixed solution;

[0010] The mixed solution is heated and stirred, and the supernatant is taken out, and the supernatant is dehydrated to obtain albumin nanoparticles;

[0011] Wherein, the volume ratio of the ethanol to water is 2:14-7:14.

[0012] As an embodiment, the mixed solution is heated and stirred, and then the supernatant is taken out, and the supernatant is dehydrated to obtain albumin nanoparticles, comprising:

[0013] stirring the mixed solution at 60-80° C. to obtain an albumin nanoparticle solution;

[0014] The albumin nanoparticle solution was centrifuged and the supernatant was taken to obtain a nanoparticle dispersion;

[0015] The nanoparticle dispersion is dehydrated to obtain albumin nanoparticles.

[0016] As an embodiment, the stirring time of the mixed solution is 2-10 min.

[0017] As an embodiment, the stirring time of the mixed solution is 3-10 min.

[0018] As an embodiment, the volume ratio of ethanol to water is 2:14-6:14.

[0019] As an embodiment, the albumin is at least one of human serum albumin, bovine serum albumin, and recombinant human serum albumin.

[0020] As an embodiment, the concentration of the albumin solution is 2.5-20 mg / mL.

[0021] As an embodiment, the concentration of the albumin solution is 2.5-10 mg / mL.

[0022] As an embodiment, the ethanol is anhydrous ethanol.

[0023] In a second aspect, an embodiment of the present application provides use of albumin nanoparticles prepared by the preparation method described in the first aspect in the synthesis of targeted drugs, wherein the albumin nanoparticles serve as drug carriers.

[0024] The embodiments of the present application have at least the following beneficial effects:

[0025] The preparation method of albumin nanoparticles provided in the embodiment of the present application adopts ultrapure water to dissolve albumin, and adds ethanol to the albumin solution to destroy the hydration layer on the albumin surface, and heats the albumin to form a gel-like structure, and simultaneously stirs the shear force and the cavitation effect of the gas and ethanol vapor in the water to cause the albumin to break and polymerize to form nanoparticles. Therefore, the preparation method of the embodiment of the present application not only shortens the time for nanoparticle formation, but also reduces the amount of organic solvent used and simplifies the process, that is, albumin nanoparticles with a low particle size and uniform particle size distribution can be obtained.

[0026] Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the particle size of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 1:14;

[0028] Figure 2 Schematic diagram of the particle size of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 2:14;

[0029] Figure 3 Schematic diagram of the particle size of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 3:14;

[0030] Figure 4 Schematic diagram of the particle size of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 4:14;

[0031] Figure 5 Schematic diagram of the particle size of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 5:14;

[0032] Figure 6 Schematic diagram of the particle size of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 6:14;

[0033] Figure 7 Schematic diagram of the particle size of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 7:14;

[0034] Figure 8 Schematic diagram of the particle size of nanoparticles obtained at a stirring temperature of 60° C. in Example 2;

[0035] Fig. 9 Schematic diagram of the particle size of nanoparticles obtained at a stirring temperature of 70° C. in Example 2;

[0036] Fig.10 Schematic diagram of the particle size of nanoparticles obtained at a stirring temperature of 80° C. in Example 2;

[0037] Fig.11Schematic diagram of the particle size of nanoparticles prepared when the albumin concentration is 2.5 mg / mL in Example 3;

[0038] Fig.12 Schematic diagram of the particle size of nanoparticles prepared when the albumin concentration is 5 mg / mL in Example 3;

[0039] Fig.13 Schematic diagram of the particle size of nanoparticles prepared when the albumin concentration is 10 mg / mL in Example 3;

[0040] Fig.14 Schematic diagram of the particle size of nanoparticles prepared when the albumin concentration is 20 mg / mL in Example 3;

[0041] Fig.15 Schematic diagram of the particle size of nanoparticles prepared when the albumin concentration is 30 mg / mL in Example 3;

[0042] Fig.16 Schematic diagram of the particle size of nanoparticles obtained with a heating and stirring time of 1 min in Example 4;

[0043] Fig.17 Schematic diagram of the particle size of nanoparticles obtained with a heating and stirring time of 2 min in Example 4;

[0044] Fig.18 Schematic diagram of the particle size of nanoparticles obtained when the heating and stirring time is 3 min in Example 4;

[0045] Fig.19 Schematic diagram of the particle size of nanoparticles obtained when the heating and stirring time is 5 min in Example 4;

[0046] Fig. 20 Schematic diagram of the particle size of nanoparticles obtained when the heating and stirring time is 10 min in Example 4;

[0047] Fig.21 This is a schematic diagram of the particle size results of nanoparticles obtained by ice water cooling in Example 5;

[0048] Fig. 22 This is a schematic diagram of the particle size results of the nanoparticles obtained at room temperature in Example 5;

[0049] Fig.23 This is a schematic diagram of the particle size results of the nanoparticles prepared in Comparative Example 1;

[0050] Fig.24 This is a schematic diagram of the particle size results of the nanoparticles prepared in Comparative Example 2;

[0051] Fig.25 Schematic diagram of the nanoparticle dispersion prepared in Example 1 with the ratio of ethanol to water being 3:14. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments will be described clearly and completely below in combination with the embodiments and drawings of the present application. Obviously, the embodiments described below are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0053] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0054] It should also be understood that the terms used in this specification of the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present application. As used in the specification of the embodiments of the present application and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0055] Some of the terms and materials involved in this embodiment are explained below to facilitate understanding by those skilled in the art.

[0056] Polydispersity index: The polydispersity index (PDI) is used to quantify the distribution of particle sizes in particulate materials. The PDI value usually varies between 0 and 1, which directly reflects the uniformity of the particle size. When the PDI value is close to 0, it means that the particle size distribution is very uniform and the size difference between the particles is very small; conversely, when the PDI value is close to 1, it indicates that the particle size distribution is extremely uneven and there are significant size differences between the particles. Generally, albumin nanoparticles with a small polydispersity index have better consistency and uniformity, which contributes to the stability of the physical and chemical properties of albumin nanoparticles.

[0057] The preparation method and application of the albumin nanoparticles of this embodiment will be described in detail below.

[0058] First, the preparation method of the albumin nanoparticles according to the first aspect of this embodiment is described.

[0059] Preparation method of albumin nanoparticles

[0060] In the prior art, the traditional preparation methods of albumin nanoparticles mainly include thermal gelation, desolvation and emulsification. Among them, the thermal gelation method is to dissolve the drug and albumin in an appropriate buffer, heat to form albumin thermal gel, and cool to form nanoparticles. This method needs to rely on the buffer system, and the heating time is longer. Heat treatment may cause partial drug degradation. The desolvation method is to dissolve the drug and albumin in an organic solvent, add a desolvation solvent (such as water or buffer), and form nanoparticles by solvent replacement. However, residual organic solvent may affect the stability of nanoparticles. The emulsification method is to disperse the albumin solution in an organic solvent that is immiscible with it to form an emulsified system, and by adjusting the type and concentration of the emulsifier, the dispersed phase forms stable nanoparticles in the continuous phase; this method is simple and easy to operate, has a high drug loading capacity, and is suitable for the nano-ization of a variety of drugs, but there are problems such as residual emulsifiers and organic solvents.

[0061] In view of the shortcomings of the prior art, this embodiment provides a method for preparing albumin nanoparticles, which comprises:

[0062] (1) dissolving albumin in water to obtain an albumin solution;

[0063] (2) adding ethanol to the albumin solution to obtain a mixed solution;

[0064] (3) The mixed solution is heated and stirred, and the supernatant is taken out, and the supernatant is dehydrated to obtain albumin nanoparticles; wherein the volume ratio of ethanol to water is 2:14-7:14.

[0065] It can be understood that the water used in this embodiment is ultrapure water.

[0066] In this embodiment, by adding ethanol in a specific volume ratio to the albumin solution, the addition of ethanol can destroy the hydration layer on the albumin surface, and the albumin is formed into a gel-like structure by heating, and the stirring shear force and the cavitation effect of the gas and ethanol vapor in the water cause the albumin to break and polymerize to form nanoparticles. Therefore, the preparation method of this embodiment not only shortens the nanoparticle formation time, but also reduces the amount of organic solvent used and simplifies the process, that is, albumin nanoparticles with a small particle size and uniform particle size distribution can be obtained.

[0067] In the preparation method of this embodiment, step (2) is a key step; as mentioned above, in this embodiment, ethanol is used to destroy the hydration layer on the surface of albumin, and then heating and stirring are used to break and aggregate the albumin to form nanoparticles. The amount of ethanol added will directly affect the final particle size and polydispersity coefficient of the albumin nanoparticles.

[0068] Specifically, the amount of ethanol added in this embodiment is limited by the volume ratio of ethanol to water in step (1). The volume ratio of ethanol to water is 2:14-7:14; within this volume ratio range, the prepared nanoparticles have a small and uniform particle size, and a ratio outside this range may result in non-uniform and / or larger nanoparticles.

[0069] For example, when the volume ratio of ethanol to water is 1:14, that is, when less ethanol is added, the polydispersity coefficient of the prepared nanoparticles is 1, indicating that the size distribution of the nanoparticles is extremely uneven, and there are significant size differences between the particles.

[0070] As a preferred embodiment, the volume ratio of ethanol to water is 2:14-6:14. When the ratio of ethanol to water is 2:14-6:14, the particle size of the prepared nanoparticles is smaller and more uniform.

[0071] Illustratively, in this embodiment, the volume ratio of ethanol to water can be 2:14, 3:14, 4:14, 5:14, 6:14, or 7:14.

[0072] It can be understood that the ethanol used in this embodiment is preferably anhydrous ethanol.

[0073] It should be noted that in step (2), after adding ethanol to obtain a mixed solution, in order to prevent the volatilization of ethanol from affecting the final result, the next step of heating and stirring operations needs to be performed within a short time. For example, within 3 minutes, preferably within 2 minutes, more preferably within 1 minute, and most preferably, the next step of heating and stirring operations is performed immediately after obtaining the mixed solution.

[0074] In this embodiment, the particle size and PDI of the nanoparticles can be detected by conventional testing methods known in the art; for example, the particle size and PDI of the nanoparticles can be detected by dynamic light scattering (DLS). The method can be: take a newly prepared nanoparticle dispersion, draw 1.5 mL of the dispersion into a sample cell of a nanoparticle size analyzer using DLS, insert the sample cell into a sample holder, and select a particle size detection program for detection.

[0075] Below, each step of the above preparation method will be further described.

[0076] In step (1), the albumin solution can be prepared by conventional stirring and mixing.

[0077] The present application has concluded through relevant experiments that the concentration of the albumin solution affects the particle size and polydispersity coefficient of the nanoparticles. Among them, when the concentration range of the albumin solution is 2.5-20 mg / mL, the prepared nanoparticles have a smaller particle size and better uniformity. When the concentration reaches 30 mg / mL, the solution becomes a gel, indicating that the preparation method of this embodiment may also become a new method for preparing albumin nanogels, but the particle size distribution is not uniform when detected in the gel state.

[0078] As a preferred embodiment, when the concentration of the albumin solution is in the range of 2.5-10 mg / mL, the prepared nanoparticles have a smaller particle size and polydispersity coefficient.

[0079] Illustratively, the concentration of the albumin solution in this embodiment can be 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 5 mg / mL, 5.4 mg / mL, 5.8 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.2 mg / mL, 9.8 mg / mL, 10 mg / mL, 10.5 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 13.6 mg / mL, 14 mg / mL, 14.3 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 17.5 mg / mL, 18 mg / mL, 18.6 mg / mL, 19 mg / mL, and 20 mg / mL.

[0080] As an embodiment, the albumin used in this embodiment can be at least one of human serum albumin, bovine serum albumin, and recombinant human serum albumin, preferably human serum albumin and / or bovine serum albumin, and more preferably bovine serum albumin.

[0081] In step (3), after obtaining the mixed solution containing ethanol in step (2), the mixed solution is subjected to a simple heating and stirring treatment.

[0082] The step of heating and stirring the mixed solution, taking the supernatant, and dehydrating the supernatant to obtain albumin nanoparticles specifically comprises:

[0083] (3.1) stirring the mixed solution at 60-80° C. to obtain an albumin nanoparticle solution;

[0084] (3.2) After centrifugation of the albumin nanoparticle solution, the supernatant is collected to obtain a nanoparticle dispersion;

[0085] (3.3) After the nanoparticle dispersion is dehydrated, albumin nanoparticles are obtained;

[0086] Wherein, in step (3.1), the mixed solution is stirred at a temperature of 60-80°C. During this process, ethanol can destroy the hydration layer on the surface of albumin and form a gel-like structure of albumin by heating. At the same time, the stirring shear force and the cavitation effect of the gas and ethanol vapor in the water cause the albumin to break and polymerize to form nanoparticles, thereby obtaining an albumin nanoparticle solution.

[0087] As an implementation mode, within the temperature range of 60-80°C, the present embodiment can produce nanoparticles with relatively low particle size and good uniformity; preferably, the temperature is 70°C.

[0088] Usually, after heating and stirring to obtain the albumin nanoparticle solution, it will be cooled to room temperature before subsequent centrifugation and dehydration treatment. This embodiment does not specifically limit the cooling method, and it can be cooled to room temperature naturally, or it can be cooled with the help of external force, such as using ice water to accelerate cooling. In this embodiment, even if ice water cooling is used, it will not have a significant impact on the particle size and uniformity of the final nanoparticles.

[0089] The present embodiment does not specifically limit the stirring speed. For example, the stirring speed may be 500-1500 rpm / min.

[0090] Next, in step (3.2), the albumin nanoparticle solution is centrifuged, and the supernatant is the low-diameter nanoparticles; the supernatant in this embodiment is the nanoparticle dispersion.

[0091] The centrifugal operation can be performed using a conventional centrifuge; for example, the centrifugal operation is performed at a centrifugal speed of 8000-12000 rpm / min and a centrifugal time of 10-20 min to obtain a nanoparticle dispersion (supernatant).

[0092] In step (3.3), after obtaining the nanoparticle dispersion, the albumin nanoparticles can be obtained after dehydration treatment. The dehydration treatment can be achieved by conventional drying or filtration; for example, filtering with a sterile filter membrane, the nanoparticle dispersion is filtered through a sterile filter membrane to obtain nanoparticles with uniform particle size distribution.

[0093] Usually, for easy storage, the dehydrated nanoparticles are freeze-dried to remove ethanol to obtain albumin nanoparticles in powder form. If drug loading or further modification is required, the albumin nanoparticles are cooled to room temperature or lower temperature, or the freeze-dried powder is redissolved in water before subsequent operations.

[0094] It should be noted that the stirring time of the mixed solution also affects the particle size and polydispersity coefficient of the albumin nanoparticles. When the stirring time is 2-10 minutes, the prepared nanoparticles have a relatively low particle size and good uniformity; when the heating time is less than 2 minutes, for example, 1 minute, it may lead to uneven particle size distribution (larger polydispersity coefficient); preferably, the stirring time of the mixed solution is 3-10 minutes, more preferably 5 minutes.

[0095] Illustratively, the stirring time of the mixed solution in this embodiment can be 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min.

[0096] In summary, the preparation method of this embodiment can produce albumin nanoparticles with low particle size and uniform particle size distribution; wherein, the particle size of the albumin nanoparticles is 50-270nm, preferably 57-180nm. That is, the albumin nanoparticles have better stability, and the uniformly distributed albumin nanoparticles can ensure that the drug is evenly distributed in various parts of the body, thereby improving the bioavailability and therapeutic effect of the drug. Through uniform distribution, the drug can reach the site of action more effectively, reduce the waste of the drug in the body, and improve the therapeutic effect; at the same time, the uniformly distributed nanoparticles can bind to the receptor more effectively, increase the accumulation of the drug in a specific part, and further enhance the targeted therapeutic effect.

[0097] Next, the purpose of the second aspect of this embodiment will be described.

[0098] use

[0099] As mentioned above, the preparation method of albumin nanoparticles provided in this embodiment can produce albumin nanoparticles with low particle size and uniform particle size distribution. Based on this, the albumin nanoparticles provided in this embodiment can be used as a drug carrier for synthesizing targeted drugs.

[0100] For example, albumin nanoparticles are used as drug carriers to prepare asilicon mannitol agarose albumin nanoparticles, micafungin albumin nanoparticles, irinotecan liposome albumin nanoparticles or paclitaxel albumin nanoparticles.

[0101] Based on the advantages of albumin nanoparticles' low particle size and uniform particle size distribution, the corresponding targeted drugs have better stability, can improve the bioavailability and therapeutic effect of the drugs, and enhance the targeted therapeutic effect.

[0102] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate / explain the present application and are not used to limit the scope of the present application.

[0103] In the following examples, the materials, reagents and instruments used can be purchased from commercial sources unless otherwise specified.

[0104] In the following examples, the dynamic light scattering (DLS) method was used to detect the particle size and PDI of the nanoparticles; the specific method was as follows: take the newly prepared nanoparticle dispersion, absorb 1.5 mL of the dispersion into the sample cell of the nanoparticle size analyzer (model ZetasizerNano ZS90), insert the sample cell into the sample holder, and select the particle size detection program for detection.

[0105] Example 1

[0106] This embodiment provides a method for preparing albumin nanoparticles, the preparation method comprising:

[0107] (1) dissolving bovine serum albumin in 14 mL of ultrapure water to a concentration of 10 mg / mL; adding 1, 2, 3, 4, 5, 6, and 7 mL of anhydrous ethanol, respectively, to obtain a mixed solution;

[0108] (2) the mixed solution was immediately stirred at 70°C at 1000 rpm / min for 3 min to obtain an albumin nanoparticle solution, and then naturally cooled to room temperature;

[0109] (3) centrifuging the albumin nanoparticle solution at 10,000 rpm / min for 15 min, and taking the supernatant to obtain a nanoparticle dispersion;

[0110] (4) The nanoparticle dispersion was filtered through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.

[0111] The particle size (nm) and polydispersity coefficient of albumin nanoparticles are shown in Tables 1 and Figure 1-Figure 7 shown.

[0112] Table 1: Effect of ethanol and water ratio on particle size

[0113] Anhydrous ethanol: water 1:14 2:14 3:14 4:14 5:14 6:14 7:14 Particle size (nm) 28.64 57.11 79.96 95.92 140.50 178.40 264.10 Polydispersity coefficient 1 0.515 0.282 0.366 0.316 0.381 0.456

[0114] Figure 1-Figure 7 The schematic diagrams showing the effect of the ratio of ethanol and water on particle size are shown respectively; Figure 1 This is a schematic diagram of the particle size results of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 1:14; Figure 2 This is a schematic diagram of the particle size results of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 2:14; Figure 3 This is a schematic diagram of the particle size results of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 3:14; Figure 4 This is a schematic diagram of the particle size results of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 4:14; Figure 5This is a schematic diagram of the particle size results of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 5:14; Figure 6 This is a schematic diagram of the particle size results of nanoparticles obtained in Example 1 with a ratio of ethanol to water of 6:14; Figure 7 This is a schematic diagram of the particle size results of nanoparticles prepared in Example 1 with an ethanol to water ratio of 7:14.

[0115] For example, Fig.25 A schematic diagram of the nanoparticle dispersion prepared in Example 1 with an ethanol to water ratio of 3:14 is shown.

[0116] Combination Figure 1-Figure 7 From the test results in Table 1, it can be seen that when the volume ratio of ethanol to water is 2:14-7:14, within this volume ratio range, the particle size of the prepared nanoparticles is small and uniform; when the volume ratio of ethanol to water is 1:14, that is, when less ethanol is added, the polydispersity coefficient of the prepared nanoparticles is 1, indicating that the size distribution of the nanoparticles is extremely uneven and there are significant size differences between the particles.

[0117] Specifically, when the volume ratio of ethanol to water is 2:14-6:14, the prepared nanoparticles have smaller and more uniform particle sizes. Considering reducing the amount of ethanol and obtaining a smaller particle size product, 3:14 is the best choice.

[0118] Example 2

[0119] This example further verifies the effect of stirring temperature on the particle size and polydispersity coefficient of albumin nanoparticles.

[0120] Specifically, the preparation method comprises:

[0121] (1) dissolving bovine serum albumin in 14 mL of ultrapure water to a concentration of 10 mg / mL; adding 4 mL of anhydrous ethanol to obtain a mixed solution;

[0122] (2) the mixed solution was immediately stirred at 60°C, 70°C, and 80°C at 1000 rpm / min for 5 min to obtain an albumin nanoparticle solution, and then naturally cooled to room temperature;

[0123] (3) centrifuging the albumin nanoparticle solution at 10,000 rpm / min for 15 min, and taking the supernatant to obtain a nanoparticle dispersion;

[0124] (4) The nanoparticle dispersion was filtered through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.

[0125] The particle size (nm) and polydispersity coefficient of albumin nanoparticles are shown in Table 2 and Figure 8-Figure 10 shown.

[0126] Table 2: Effect of temperature on particle size

[0127] Temperature(℃) 60 70 80 Particle size (nm) 113.2 93.86 107.9 Polydispersity coefficient 0.344 0.353 0.405

[0128] Figure 8-Figure 10 A schematic diagram showing the effect of stirring temperature on particle size is shown, where: Figure 8 Schematic diagram of the particle size results of nanoparticles prepared at a stirring temperature of 60°C; Fig. 9 Schematic diagram of the particle size results of nanoparticles prepared at a stirring temperature of 70°C; Fig.10 Schematic diagram of the particle size results of nanoparticles prepared at a stirring temperature of 80°C.

[0129] Combination Figure 8-Figure 10 From the test results in Table 2, it can be seen that within the temperature range of 60-80°C, the present embodiment can produce nanoparticles with relatively low particle size and good uniformity; in general, 70°C is the best condition.

[0130] Example 3

[0131] This example further verifies the effect of albumin concentration on the particle size and polydispersity coefficient of albumin nanoparticles.

[0132] Specifically, the preparation method comprises:

[0133] (1) Dissolve bovine serum albumin in 14 mL of ultrapure water to concentrations of 2.5, 5, 10, 20, and 30 mg / mL, respectively; add 4 mL of anhydrous ethanol to obtain a mixed solution;

[0134] (2) the mixed solutions were immediately stirred at 70°C for 5 min at 1000 rpm / min to obtain albumin nanoparticle solutions, and then naturally cooled to room temperature;

[0135] (3) centrifuging the albumin nanoparticle solution at 10,000 rpm / min for 15 min, and taking the supernatant to obtain a nanoparticle dispersion;

[0136] (4) The nanoparticle dispersion was filtered through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.

[0137] The particle size (nm) and polydispersity coefficient of albumin nanoparticles are shown in Table 3 and Figure 11-Figure 15 shown.

[0138] Table 3: Effect of BSA concentration on particle size

[0139]

[0140] Figure 11-Figure 15 Schematic diagram showing the effect of bovine serum albumin concentration on particle size, where: Fig.11This is a schematic diagram of the particle size results of nanoparticles prepared at an albumin concentration of 2.5 mg / mL; Fig.12 This is a schematic diagram of the particle size results of nanoparticles prepared at an albumin concentration of 5 mg / mL; Fig.13 This is a schematic diagram of the particle size results of nanoparticles prepared at an albumin concentration of 10 mg / mL; Fig.14 This is a schematic diagram of the particle size results of nanoparticles prepared at an albumin concentration of 20 mg / mL; Fig.15 Schematic diagram of the particle size results of nanoparticles prepared with an albumin concentration of 30 mg / mL.

[0141] Combination Figure 11-Figure 15 As shown in the test results of Table 3, when the concentration of the albumin solution is in the range of 2.5-20 mg / mL, the prepared nanoparticles have a smaller particle size and better uniformity. When the concentration reaches 30 mg / mL, the solution becomes a gel, indicating that this embodiment may also become a new method for preparing albumin nanogels, but the particle size distribution is not uniform when detected in the gel state.

[0142] Furthermore, when the concentration of the albumin solution is in the range of 2.5-10 mg / mL, the prepared nanoparticles have a smaller particle size and polydispersity coefficient.

[0143] Example 4

[0144] This example further verifies the effect of heating and stirring time on the particle size and polydispersity coefficient of albumin nanoparticles.

[0145] Specifically, the preparation method comprises:

[0146] (1) dissolving bovine serum albumin in 14 mL of ultrapure water to a concentration of 10 mg / mL; adding 4 mL of anhydrous ethanol to obtain a mixed solution;

[0147] (2) the mixed solution was immediately stirred at 70°C and 1000 rpm / min for 1, 2, 3, 5, and 10 min to obtain an albumin nanoparticle solution, and then naturally cooled to room temperature;

[0148] (3) centrifuging the albumin nanoparticle solution at 10,000 rpm / min for 15 min, and taking the supernatant to obtain a nanoparticle dispersion;

[0149] (4) The nanoparticle dispersion was filtered through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.

[0150] The particle size (nm) and polydispersity coefficient of albumin nanoparticles are shown in Table 4 and Figure 16-Figure 20 shown.

[0151] Table 4: Effect of heating and stirring time on particle size

[0152] Stirring time (min) 1 2 3 5 10 Particle size (nm) 88.15 113.2 95.92 93.86 101.9 Polydispersity coefficient 1 0.356 0.366 0.353 0.367

[0153] Figure 16-Figure 20 The figure shows the effect of heating and stirring time on particle size, where: Fig.16 This is a schematic diagram of the particle size results of nanoparticles obtained with a heating and stirring time of 1 min; Fig.17 This is a schematic diagram of the particle size results of nanoparticles obtained with a heating and stirring time of 2 minutes; Fig.18 This is a schematic diagram of the particle size results of nanoparticles obtained with a heating and stirring time of 3 minutes; Fig.19 This is a schematic diagram of the particle size results of nanoparticles obtained with a heating and stirring time of 5 minutes; Fig. 20 Schematic diagram of the particle size results of nanoparticles prepared with a heating and stirring time of 10 minutes.

[0154] Combination Figure 16-Figure 20 From the test results in Table 4, it can be seen that when the heating time is 2-10 min, the prepared nanoparticles have a lower particle size and better uniformity; heating time less than 2 min may lead to uneven particle size distribution; the optimal time is 5 min.

[0155] Example 5

[0156] This example simultaneously verifies the effect of cooling method on the particle size and polydispersity coefficient of albumin nanoparticles.

[0157] Specifically, the preparation method comprises:

[0158] (1) Dissolve bovine serum albumin in 14 mL of ultrapure water to a concentration of 10 mg / mL; add 2 mL of anhydrous ethanol to obtain a mixed solution (two portions);

[0159] (2) The mixed solutions were immediately stirred at 70° C. and 1000 rpm / min for 3 min to obtain albumin nanoparticle solutions; one portion was cooled to room temperature in ice water, and the other portion was left to cool to room temperature naturally;

[0160] (3) centrifuging the albumin nanoparticle solution at 10,000 rpm / min for 15 min, and taking the supernatant to obtain a nanoparticle dispersion;

[0161] (4) The nanoparticle dispersion was filtered through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.

[0162] The particle size (nm) and polydispersity coefficient of albumin nanoparticles are shown in Table 5 and Figure 21-22 shown.

[0163] Table 5: Effect of cooling method on particle size

[0164] Cooling method Ice water cooling Leave at room temperature Particle size (nm) 98.96 93.86 Polydispersity coefficient 0.352 0.353

[0165] Figure 21-22 A schematic diagram showing the effect of cooling method on particle size is shown; wherein, Fig.21 Schematic diagram of the particle size results of nanoparticles obtained by ice water cooling; Fig. 22 Schematic diagram of the particle size results of the nanoparticles prepared at room temperature.

[0166] Combination Figure 21-22 From the test results in Table 5, it can be seen that whether or not the rapid cooling is performed with the aid of external force has no obvious effect on the particle size in this embodiment. Therefore, the preparation method of this embodiment does not rely on external force cooling, which can reduce the energy consumption of the preparation process; for example, in mass production, the energy consumption caused by cooling can be greatly reduced.

[0167] Comparative Example 1

[0168] Comparative Example 1 provides an albumin nanoparticle prepared by a thermal gelation method, wherein the preparation method comprises:

[0169] (1) dissolving bovine serum albumin in 14 mL of ultrapure water at a concentration of 10 mg / mL to obtain a mixed solution;

[0170] (2) heating the mixed solution at 70° C. for 20 min and cooling it to room temperature on ice to obtain an albumin nanoparticle solution;

[0171] (3) centrifuging the albumin nanoparticle solution at 10,000 rpm / min for 15 min, and taking the supernatant to obtain a nanoparticle dispersion;

[0172] (4) The nanoparticle dispersion was filtered through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.

[0173] The test results of the particle size (nm) and polydispersity coefficient of albumin nanoparticles are shown in Table 6. Figure 3 and Fig.23 shown.

[0174] Table 6: Particle size test results of Example 1 and Comparative Example 1

[0175]

[0176] Figure 3 and Fig.23 The effect of the two methods on particle size is shown in Table 6; Fig.23 This is a schematic diagram of the particle size results of the nanoparticles prepared in Comparative Example 1.

[0177] Combination Figure 3 , Fig.23 From the test results in Table 6, it can be seen that the method of Example 1 has similar particle sizes as the simplified thermal gel method, both of which are smaller, but more uniform; among them, the nanoparticles obtained by the thermal gel method are unevenly distributed. Figure 6 It can be seen that the majority of particles are larger than 100 nm. Therefore, the method of this embodiment has obvious advantages, and the heating time is shorter, which reduces energy consumption.

[0178] Comparative Example 2

[0179] Comparative Example 2 provides an albumin nanoparticle prepared by a desolvation method, wherein the preparation method comprises:

[0180] (1) dissolving bovine serum albumin in 14 mL of ultrapure water at a concentration of 10 mg / mL to obtain a mixed solution;

[0181] (2) stirring the mixed solution at 1000 rpm / min, and adding anhydrous ethanol dropwise while stirring until the solution produces blue opalescence, thereby obtaining an albumin nanoparticle solution;

[0182] (3) centrifuging the albumin nanoparticle solution at 10,000 rpm / min for 15 min, and taking the supernatant to obtain a nanoparticle dispersion;

[0183] (4) The nanoparticle dispersion was filtered through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.

[0184] The test results of the particle size (nm) and polydispersity coefficient of albumin nanoparticles are shown in Table 7. Figure 3 and Fig.24 shown.

[0185] Table 7: Particle size test results of Example 1 and Comparative Example 2

[0186]

[0187] Figure 3 and Fig.24 The effect of the two methods on particle size is shown in Table 7; wherein, Fig.24 This is a schematic diagram of the particle size results of the nanoparticles prepared in Comparative Example 2.

[0188] Among them, the desolvation method requires the addition of ethanol in a volume approximately equal to that of water to produce blue opalescence, and the end time is difficult to distinguish with the naked eye, making it difficult to ensure the repeatability of the experiment, and the particle size obtained is often too large.

[0189] Combination Figure 3 , Fig.24 From the test results in Table 6, it can be seen that compared with the simplified desolvation method, the preparation method of Example 1 has smaller and more uniform particle size of the prepared albumin nanoparticles, is easier to control, has high repeatability, and uses less ethanol.

[0190] In summary, this embodiment adds ethanol in a specific volume ratio to the albumin solution, and the addition of ethanol can destroy the hydration layer on the albumin surface, and the albumin forms a gel-like structure by heating, and the stirring shear force and the cavitation effect of the gas and ethanol vapor in the water cause the albumin to break and polymerize to form nanoparticles. Therefore, the preparation method of this embodiment not only shortens the nanoparticle formation time, but also reduces the amount of organic solvent used and simplifies the process, that is, albumin nanoparticles with a small particle size and uniform particle size distribution can be obtained.

[0191] The technical solutions provided in the embodiments of the present application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present application. At the same time, for those skilled in the art, according to the embodiments of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present application.

Claims

1. A method for preparing albumin nanoparticles, characterized in that: The preparation method comprises: dissolving albumin in water to obtain an albumin solution; adding ethanol to the albumin solution to obtain a mixed solution; The mixed solution is heated and stirred, and the supernatant is taken out, and the supernatant is dehydrated to obtain albumin nanoparticles; Wherein, the volume ratio of the ethanol to water is 2:14-7:

14.

2. The method for preparing albumin nanoparticles according to claim 1, characterized in that: The mixed solution is heated and stirred, and then the supernatant is taken out. After the supernatant is dehydrated, the albumin nanoparticles are obtained, comprising: stirring the mixed solution at 60-80° C. to obtain an albumin nanoparticle solution; The albumin nanoparticle solution was centrifuged and the supernatant was taken to obtain a nanoparticle dispersion; The nanoparticle dispersion is dehydrated to obtain albumin nanoparticles.

3. The method for preparing albumin nanoparticles according to claim 2, characterized in that: The stirring time of the mixed solution is 2-10 min.

4. The method for preparing albumin nanoparticles according to claim 2 or 3, characterized in that: The stirring time of the mixed solution is 3-10 min.

5. The method for preparing albumin nanoparticles according to claim 1, characterized in that: The volume ratio of the ethanol to water is 2:14-6:

14.

6. The method for preparing albumin nanoparticles according to claim 1, characterized in that: The albumin is at least one of human serum albumin, bovine serum albumin, and recombinant human serum albumin.

7. The method for preparing albumin nanoparticles according to claim 1, characterized in that: The concentration of the albumin solution is 2.5-20 mg / mL.

8. The method for preparing albumin nanoparticles according to claim 7, characterized in that: The concentration of the albumin solution is 2.5-10 mg / mL.

9. The method for preparing albumin nanoparticles according to claim 1, characterized in that: The ethanol is anhydrous ethanol.

10. Use of the albumin nanoparticles prepared by the preparation method according to any one of claims 1 to 9 in synthesizing targeted drugs, characterized in that: The albumin nanoparticles are used as drug carriers.

Images