Method for the preparation of albumin nanoparticles and uses thereof
By adding ethanol to the albumin solution and heating and stirring to form nanoparticles, the problems of long heating time and residual organic solvent in the existing technology are solved, and albumin nanoparticles with small and uniform particle size are prepared, which improves the stability of the drug and the targeted treatment effect.
Patent Information
- Application Number
- CN202411870214.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-10-17
- Estimated Expiration
- 2044-12-18
AI Technical Summary
Existing methods for preparing albumin nanoparticles have problems such as long heating time, possible drug degradation, and residual organic solvents affecting the stability of the nanoparticles.
Ethanol is added to the albumin solution, and a gel-like structure is formed through heating and stirring. Ethanol is used to destroy the hydration layer on the albumin surface, and nanoparticles are formed through stirring shear force and ethanol vapor cavitation, reducing the use of organic solvents and simplifying the process.
The nanoparticle formation time is shortened, the amount of organic solvent used is reduced, and albumin nanoparticles with small particle size and uniform particle size distribution are prepared, thereby improving the bioavailability and targeted therapeutic effect of the drug.
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Figure CN119656131B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The embodiment of the present application relates to the technical field of pharmaceutical preparation, in particular to a preparation method of albumin nanoparticles and application thereof. BACKGROUND
[0002] Albumin has good biocompatibility and biodegradability, so that it has wide application in the medical field, especially in nano-preparation. Albumin nanoparticles (ANPs) are nanoparticles taking albumin as basic material, which has 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 on the surface of the nanoparticles, so as to realize targeted delivery and controlled release of drugs. 2, biocompatibility: albumin has good biocompatibility and is not easy to cause immune response, and can circulate in the body for a long time. 3, targeting: albumin nanoparticles can realize targeted delivery to specific organs or cells by surface modification of functional ligands (such as antibodies, polypeptides). 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 method and desolvation method. Among them, the thermal gelation method is to dissolve the drug and albumin in a suitable buffer, heat to form albumin thermal gel, and form nanoparticles after cooling. This method needs to rely on the buffer system, and the heating time is longer, and the heat treatment may cause partial drug degradation. The desolvation method is to dissolve the drug and albumin in an organic solvent, add a desolvating agent (such as water or buffer), and form nanoparticles by solvent replacement. However, the residual organic solvent may affect the stability of the nanoparticles.
[0004] Therefore, it is an urgent problem to be solved in the art to find a new preparation method of albumin nanoparticles. SUMMARY
[0005] The purpose of the present application is to provide a preparation method of albumin nanoparticles and application thereof.
[0006] In order to achieve the above purpose, the embodiment of the present application proposes the following technical scheme:
[0007] In the first aspect, the embodiment of the present application provides a preparation method of albumin nanoparticles, which comprises:
[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 then the supernatant is taken, and the supernatant is dehydrated to obtain the albumin nanoparticles.
[0011] The volume ratio of the ethanol to the water is 2:14-7:14.
[0012] As an embodiment, the method of obtaining the albumin nanoparticles comprises the following steps:
[0013] The mixed solution is stirred at 60-80℃ to obtain an albumin nanoparticle solution.
[0014] The albumin nanoparticle solution is centrifuged to obtain a supernatant, and a nanoparticle dispersion is obtained.
[0015] The nanoparticle dispersion is dehydrated to obtain the 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 the ethanol to the 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, the embodiments of the present application provide a use of the albumin nanoparticles prepared by the preparation method of the first aspect as a drug carrier for synthesizing a targeted drug.
[0024] The embodiments of the present application have at least the following beneficial effects:
[0025] The method for preparing albumin nanoparticles provided in the examples of this application uses ultrapure water to dissolve albumin, then adds ethanol to the albumin solution to disrupt the hydration layer on the albumin surface. Heating causes the albumin to form a gel-like structure. Simultaneously, the shear force of stirring and the cavitation effects of the gas and ethanol vapor in the water cause the albumin to break and aggregate into nanoparticles. As a result, the preparation method of the examples of this application not only shortens the nanoparticle formation time, but also reduces the amount of organic solvent used and simplifies the process, resulting in albumin nanoparticles with a small particle size and uniform particle size distribution.
[0026] Additional aspects and advantages of the present application will be given in part in the following description, which will become apparent from the following description, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Schematic diagram of the particle size of nanoparticles prepared in Example 1 with an ethanol to water ratio of 1:14;
[0028] Figure 2 Schematic diagram of the particle size of nanoparticles prepared in Example 1 with an ethanol to water ratio of 2:14;
[0029] Figure 3 Schematic diagram of the particle size of nanoparticles prepared in Example 1 with an ethanol to water ratio of 3:14;
[0030] Figure 4 Schematic diagram of the particle size of nanoparticles prepared in Example 1 with an ethanol to water ratio of 4:14;
[0031] Figure 5 Schematic diagram of the particle size of nanoparticles prepared in Example 1 with an ethanol to water ratio of 5:14;
[0032] Figure 6 Schematic diagram of the particle size of nanoparticles prepared in Example 1 with an ethanol to water ratio of 6:14;
[0033] Figure 7 Schematic diagram of the particle size of nanoparticles prepared in Example 1 with an ethanol to water ratio of 7:14;
[0034] Figure 8 Schematic diagram of the particle size of nanoparticles prepared at a stirring temperature of 60° C. in Example 2;
[0035] Figure 9 Schematic diagram of the particle size of nanoparticles prepared at a stirring temperature of 70° C. in Example 2;
[0036] Figure 10 Schematic diagram of the particle size of nanoparticles prepared at a stirring temperature of 80° C. in Example 2;
[0037] Figure 11Particle size profile of nanoparticles prepared in Example 3 with albumin concentration of 2.5 mg / mL;
[0038] Figure 12 Particle size profile of nanoparticles prepared in Example 3 with albumin concentration of 5 mg / mL;
[0039] Figure 13 Particle size profile of nanoparticles prepared in Example 3 with albumin concentration of 10 mg / mL;
[0040] Figure 14 Particle size profile of nanoparticles prepared in Example 3 with albumin concentration of 20 mg / mL;
[0041] Figure 15 Particle size profile of nanoparticles prepared in Example 3 with albumin concentration of 30 mg / mL;
[0042] Figure 16 Particle size profile of nanoparticles prepared in Example 4 with heating and stirring time of 1 min;
[0043] Figure 17 Particle size profile of nanoparticles prepared in Example 4 with heating and stirring time of 2 min;
[0044] Figure 18 Particle size profile of nanoparticles prepared in Example 4 with heating and stirring time of 3 min;
[0045] Figure 19 Particle size profile of nanoparticles prepared in Example 4 with heating and stirring time of 5 min;
[0046] Figure 20 Particle size profile of nanoparticles prepared in Example 4 with heating and stirring time of 10 min;
[0047] Figure 21 Particle size profile of nanoparticles prepared in Example 5 with ice water cooling;
[0048] Figure 22 Particle size profile of nanoparticles prepared in Example 5 with room temperature standing;
[0049] Figure 23 Particle size profile of nanoparticles prepared in Comparative Example 1;
[0050] Figure 24 Particle size profile of nanoparticles prepared in Comparative Example 2;
[0051] Figure 25 Particle size profile of nanoparticles prepared in Example 1 with ethanol and water ratio of 3:14. DETAILED DESCRIPTION
[0052] The technical solutions in the embodiments will be described clearly and completely below in conjunction with the embodiments of the present application and the drawings. Obviously, the following described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application.
[0053] It should be understood that the terms "comprising" and "including" as used in the specification and the appended claims indicate the presence of the described features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.
[0054] It should also be understood that the terms used in the specification of the embodiments of the present application are only for the purpose of describing the 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, the singular forms "a", "an" and "the" are intended to include the plural forms unless the context clearly indicates otherwise.
[0055] The following first explains some terms and materials involved in the embodiments, so as to facilitate understanding by those skilled in the art.
[0056] Polydispersity coefficient: The polydispersity coefficient (PDI) is used to quantify the distribution of particle size in a particle material. 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 indicates that the size distribution of the particles is very uniform, and the size difference between the particles is very small; on the contrary, when the PDI value is close to 1, it indicates that the size distribution of the particles is extremely uneven, and there is a significant size difference between the particles. Generally, albumin nanoparticles with a small polydispersity coefficient have better consistency and uniformity, which helps to stabilize the physical and chemical properties of the albumin nanoparticles.
[0057] The preparation method of the albumin nanoparticles of the embodiments and the use thereof will be described in detail below.
[0058] First, the preparation method of the albumin nanoparticles of the first aspect of the embodiments will be described.
[0059] Method of preparing albumin nanoparticles
[0060] In the prior art, the traditional preparation methods of albumin nanoparticles mainly include heat gelation method, desolvation method and emulsification method. Among them, the heat gelation method is to dissolve the drug and albumin in a suitable buffer, heat to form an albumin thermal gel, and form nanoparticles after cooling. This method needs to rely on the buffer system, and the heating time is longer, and the heat treatment may cause partial drug degradation. The desolvation method is to dissolve the drug and albumin in an organic solvent, add a desolvating agent (such as water or buffer), and form nanoparticles by solvent replacement. However, the residual organic solvent may affect the stability of the nanoparticles. The emulsification method is to disperse the albumin solution in an organic solvent that is not soluble with it, to form an emulsion system, and by adjusting the type and concentration of the emulsifier, the dispersed phase is formed into stable nanoparticles in the continuous phase; this method is simple and easy to operate, has high drug loading capacity, and is suitable for nanocrystallization of various drugs, but there are problems such as residual emulsifier and organic solvent.
[0061] In view of the deficiencies in the prior art, the present embodiment proposes a preparation method of 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) heating and stirring the mixed solution, taking the supernatant, and after dehydration treatment of the supernatant, obtaining 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 the present embodiment is ultrapure water.
[0066] In the present embodiment, by adding a specific volume ratio of ethanol to the albumin solution, the addition of ethanol can destroy the hydration layer on the surface of the albumin, and by heating, the albumin forms a gel-like structure, and at the same time, the shear force of stirring and the cavitation effect of gas in water and ethanol vapor make the albumin break and polymerize to form nanoparticles. Therefore, the preparation method of the present embodiment not only shortens the nanoparticle formation time, but also reduces the amount of organic solvent used and simplifies the process, that is, low-particle-size, uniformly distributed albumin nanoparticles can be obtained.
[0067] In the preparation method of the present embodiment, step (2) is a more critical step; as described above, the present embodiment is to destroy the hydration layer on the surface of the albumin by ethanol, and then to make the albumin break and polymerize to form nanoparticles by heating and stirring. 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 the embodiment is defined by the volume ratio of ethanol to water in step (1). The volume ratio of ethanol to water is 2:14-7:14. Within the range, the prepared nanoparticles have a small and uniform size. Outside the range, the nanoparticles may be non-uniform and / or have a large size.
[0069] For example, when the volume ratio of ethanol to water is 1:14, i.e., the amount of ethanol added is small, the polydispersity index of the prepared nanoparticles is 1, indicating that the size distribution of the nanoparticles is extremely non-uniform, and there is a significant size difference 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 prepared nanoparticles have a small and more uniform size.
[0071] For example, the volume ratio of ethanol to water in the embodiment 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 the embodiment is preferably anhydrous ethanol.
[0073] It should be noted that in step (2), after the ethanol is added to obtain the mixed solution, in order to avoid the volatilization of ethanol affecting the final result, the next step of heating and stirring operation 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 immediately after obtaining the mixed solution.
[0074] In the embodiment, the particle size and PDI of the nanoparticles can be detected by using a conventional known test method. For example, the dynamic light scattering (DLS) method can be used to detect the particle size and PDI of the nanoparticles. The method can be as follows: take the newly prepared nanoparticle dispersion liquid, draw 1.5 mL of the dispersion liquid into the sample cell of the nanoparticle size instrument using DLS, insert the sample cell into the sample holder, and select the particle size detection program for detection.
[0075] Next, each step of the preparation method will be further described.
[0076] In step (1), the preparation of the albumin solution can be prepared by conventional stirring and mixing.
[0077] The present application obtains, through relevant tests, that the concentration of the albumin solution will affect the particle size and polydispersity coefficient of the nanoparticles. Among them, when the concentration of the albumin solution is in the range of 2.5-20 mg / mL, the prepared nanoparticles have smaller particle size and better uniformity. When the concentration reaches 30 mg / mL, the solution becomes gelatinous, suggesting that the preparation method of the present embodiment can also become a new method for preparing albumin nanogel, but the particle size distribution is not uniform when 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 smaller particle size and polydispersity coefficient.
[0079] Illustratively, the concentration of the albumin solution in the present 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, 20 mg / mL.
[0080] As an embodiment, the albumin used in the present embodiment can be at least one of human serum albumin, bovine serum albumin, and recombinant human serum albumin. Preferably, it is 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 simple heating and stirring treatment.
[0082] Among them, the step of obtaining the albumin nanoparticles after heating and stirring the mixed solution, and then dehydrating the supernatant includes:
[0083] (3.1) stirring the mixed solution at 60-80°C to obtain an albumin nanoparticle solution;
[0084] (3.2) obtaining a nanoparticle dispersion by centrifuging the albumin nanoparticle solution;
[0085] (3.3) obtaining albumin nanoparticles by dehydrating the nanoparticle dispersion;
[0086] 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 aggregate to form nanoparticles, thereby obtaining an albumin nanoparticle solution.
[0087] As an embodiment, within the temperature range of 60-80°C, this embodiment can produce nanoparticles with relatively low particle size and good uniformity; preferably, the temperature is 70°C.
[0088] Typically, after heating and stirring to obtain an albumin nanoparticle solution, the solution is cooled to room temperature before subsequent centrifugation and dehydration. This embodiment does not specifically limit the cooling method; the solution can be cooled naturally to room temperature or with the aid of an external force, such as accelerated cooling with ice water. In this embodiment, even with ice water cooling, the resulting nanoparticle size and uniformity are not significantly affected.
[0089] The present embodiment does not impose any specific limitation on 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-size nanoparticles; the supernatant in this embodiment is the nanoparticle dispersion.
[0091] The centrifugation operation can be performed using a conventional centrifuge; for example, centrifugation 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 are obtained after dehydration. The dehydration can be achieved by conventional drying or filtration; for example, filtration using a sterile filter membrane to filter the nanoparticle dispersion 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, 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 has an effect on the particle size and polydispersity coefficient of the albumin nanoparticles. When the stirring time is 2-10 min, the nanoparticles prepared have a lower particle size and better uniformity; when the heating time is less than 2 min, for example, 1 min, it can lead to uneven particle size distribution (a larger polydispersity coefficient); preferably, the stirring time of the mixed solution is 3-10 min, more preferably 5 min.
[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 prepare albumin nanoparticles with low particle size and uniform particle size distribution; wherein the particle size of the albumin nanoparticles is 50-270 nm, preferably 57-180 nm. That is, the albumin nanoparticles have better stability, and the uniformly distributed albumin nanoparticles can ensure uniform distribution of the drug in various parts of the body, thereby improving the bioavailability and therapeutic effect of the drug. Through uniform distribution, the drug can more effectively reach the action site, reduce the waste of the drug in the body, and improve the therapeutic effect; at the same time, the uniformly distributed nanoparticles can more effectively bind to receptors, improve the accumulation of the drug in specific parts, and further enhance the targeted therapeutic effect.
[0097] Secondly, the use of the second aspect of this embodiment will be described.
[0098] Use
[0099] As described above, the preparation method of the albumin nanoparticles provided in this embodiment can prepare 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 a targeted drug.
[0100] For example, the albumin nanoparticles are used as a drug carrier to prepare alesong mannitol agarose albumin nanoparticle, micafungin albumin nanoparticle, irinotecan liposome albumin nanoparticle, or paclitaxel albumin nanoparticle.
[0101] Based on the advantages of low particle size and uniform particle size distribution of the albumin nanoparticles, the corresponding targeted drug has better stability, can improve the bioavailability and therapeutic effect of the drug, and enhance the targeted therapeutic effect.
[0102] The present application will be further described 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 are commercially available unless otherwise specified.
[0104] In the following examples, the particle size and PDI of the nanoparticles are detected by dynamic light scattering (DLS) method; the specific method is as follows: take the freshly prepared nanoparticle dispersion solution, take 1.5 mL of the dispersion solution into the sample cell of the nanoparticle size analyzer (model Zetasizer Nano ZS90), insert the sample cell into the sample holder, and select the particle size detection program for detection.
[0105] Example 1
[0106] The present embodiment provides a preparation method of albumin nanoparticles, which comprises:
[0107] (1) Dissolve bovine serum albumin in 14 mL of ultrapure water at a concentration of 10 mg / mL; add 1, 2, 3, 4, 5, 6, and 7 mL of anhydrous ethanol respectively to obtain a mixed solution;
[0108] (2) Stir the mixed solution immediately at 70°C at 1000 rpm / min for 3 min to obtain an albumin nanoparticle solution, and cool it to room temperature naturally;
[0109] (3) Centrifuge the albumin nanoparticle solution at 10000 rpm / min for 15 min, and take the supernatant to obtain a nanoparticle dispersion solution;
[0110] (4) Filter the nanoparticle dispersion solution through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.
[0111] The test results of the particle size (nm) and polydispersity coefficient of the albumin nanoparticles are shown in Table 1 and Figures 1-7 .
[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] Figures 1-7 The effect of the ratio of ethanol and water on the particle size is shown in the schematic diagrams, respectively; wherein, Figure 1 is a schematic diagram of the particle size results of the nanoparticles prepared in Example 1 with the ratio of ethanol and water being 1:14; Figure 2 is a schematic diagram of the particle size results of the nanoparticles prepared in Example 1 with the ratio of ethanol and water being 2:14; Figure 3 is a schematic diagram of the particle size results of the nanoparticles prepared in Example 1 with the ratio of ethanol and water being 3:14; Figure 4 is a schematic diagram of the particle size results of the nanoparticles prepared in Example 1 with the ratio of ethanol and water being 4:14; Figure 5A diagram showing the particle size results of the nanoparticles prepared in Example 1 with a ratio of ethanol to water of 5:14 is shown. Figure 6 A diagram showing the particle size results of the nanoparticles prepared in Example 1 with a ratio of ethanol to water of 6:14 is shown. Figure 7 A diagram showing the particle size results of the nanoparticles prepared in Example 1 with a ratio of ethanol to water of 7:14 is shown.
[0115] Exemplarily, Figure 25 A diagram showing the nanoparticle dispersion prepared in Example 1 with a ratio of ethanol to water of 3:14 is shown.
[0116] In combination Figures 1-7 As can be seen from the test results in Table 1, when the volume ratio of ethanol to water is 2:14-7:14, the nanoparticles prepared in this volume ratio range have a smaller and more uniform particle size; when the volume ratio of ethanol to water is 1:14, i.e., when less ethanol is added, the polydispersity index of the prepared nanoparticles is 1, indicating that the size distribution of the nanoparticles is extremely uneven and there is a significant size difference between the particles.
[0117] Specifically, when the volume ratio of ethanol to water is 2:14-6:14, the prepared nanoparticles have a smaller and more uniform particle size. Considering the reduction of ethanol dosage and the obtaining of 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 index of albumin nanoparticles.
[0120] Specifically, the preparation method comprises:
[0121] (1) Dissolve bovine serum albumin in 14 mL of ultrapure water at a concentration of 10 mg / mL; add 4 mL of anhydrous ethanol to obtain a mixed solution;
[0122] (2) Stir the mixed solution at 1000 rpm / min immediately at 60°C, 70°C and 80°C respectively for 5 min to obtain an albumin nanoparticle solution, and naturally cool to room temperature;
[0123] (3) Centrifuge the albumin nanoparticle solution at 10000 rpm / min for 15 min, and take the supernatant to obtain a nanoparticle dispersion;
[0124] (4) Filter the nanoparticle dispersion through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.
[0125] The test results of the particle size (nm) and polydispersity index of the albumin nanoparticles are shown in Table 2 and Figures 8-10 .
[0126] Table 2: Effect of temperature on particle size
[0127] Temperature (°C) 60 70 80 Particle size (nm) 113.2 93.86 107.9 Polydispersity coefficient 0.344 0.353 0.405
[0128] Figures 8-10 The effect of stirring temperature on particle size is shown in the schematic diagram, wherein, Figure 8 The particle size results of nanoparticles prepared at a stirring temperature of 60°C are shown in the schematic diagram; Figure 9 The particle size results of nanoparticles prepared at a stirring temperature of 70°C are shown in the schematic diagram; Figure 10 The particle size results of nanoparticles prepared at a stirring temperature of 80°C are shown in the schematic diagram.
[0129] In combination with Figures 8-10 and the test results in Table 2, it can be seen that in the temperature range of 60-80°C, the nanoparticles with lower particle size and better uniformity can be prepared in this embodiment; in general, 70°C is the best condition.
[0130] Example 3
[0131] This embodiment 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 ultrapure water at a concentration of 2.5, 5, 10, 20, and 30 mg / mL, respectively; add 4 mL of anhydrous ethanol to obtain a mixed solution;
[0134] (2) Stir the mixed solution at 1000 rpm / min at 70°C immediately for 5 min to obtain an albumin nanoparticle solution, and naturally cool to room temperature;
[0135] (3) High-speed centrifuge the albumin nanoparticle solution at 10000 rpm / min for 15 min, and take the supernatant to obtain a nanoparticle dispersion;
[0136] (4) Filter the nanoparticle dispersion through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.
[0137] The test results of the particle size (nm) and polydispersity coefficient of the albumin nanoparticles are shown in Table 3 and Figures 11-15 .
[0138] Table 3: Effect of bovine serum albumin concentration on particle size
[0139]
[0140] Figures 11-15 The effect of bovine serum albumin concentration on particle size is shown in the schematic diagram, wherein, Figure 11A diagram showing the particle size results of the nanoparticles prepared with an albumin concentration of 2.5 mg / mL; Figure 12 A diagram showing the particle size results of the nanoparticles prepared with an albumin concentration of 5 mg / mL; Figure 13 A diagram showing the particle size results of the nanoparticles prepared with an albumin concentration of 10 mg / mL; Figure 14 A diagram showing the particle size results of the nanoparticles prepared with an albumin concentration of 20 mg / mL; Figure 15 A diagram showing the particle size results of the nanoparticles prepared with an albumin concentration of 30 mg / mL.
[0141] In combination Figures 11-15 As shown in Table 3 and the test results, when the concentration of the albumin solution ranges from 2.5 mg / mL to 20 mg / mL, the prepared nanoparticles have a smaller particle size and better uniformity. When the concentration reaches 30 mg / mL, the solution becomes gel-like, indicating that this embodiment can also be a new method for preparing albumin nanogels, but the particle size distribution is not uniform in the gel state.
[0142] Further, when the concentration of the albumin solution ranges from 2.5 mg / mL to 10 mg / mL, the prepared nanoparticles have a smaller particle size and polydispersity coefficient.
[0143] Example 4
[0144] This embodiment further verifies the effect of heating and stirring time on the particle size and polydispersity coefficient of the albumin nanoparticles.
[0145] Specifically, the preparation method comprises:
[0146] (1) Dissolve bovine serum albumin in 14 mL of ultrapure water at a concentration of 10 mg / mL; add 4 mL of anhydrous ethanol to obtain a mixed solution;
[0147] (2) Immediately stir the mixed solution at 1000 rpm / min at 70°C for 1, 2, 3, 5, and 10 minutes, respectively, to obtain an albumin nanoparticle solution, and naturally cool to room temperature;
[0148] (3) Centrifuge the albumin nanoparticle solution at 10000 rpm / min for 15 minutes, and take the supernatant to obtain a nanoparticle dispersion;
[0149] (4) Filter the nanoparticle dispersion through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.
[0150] The test results of the particle size (nm) and polydispersity coefficient of the albumin nanoparticles are shown in Table 4 and Figures 16-20 .
[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] Figures 16-20 The heating stirring time effect on particle size is shown in the schematic diagram, wherein, Figure 16 The heating stirring time is 1 min, and the particle size of the nanoparticles prepared is shown in the schematic diagram; Figure 17 The heating stirring time is 2 min, and the particle size of the nanoparticles prepared is shown in the schematic diagram; Figure 18 The heating stirring time is 3 min, and the particle size of the nanoparticles prepared is shown in the schematic diagram; Figure 19 The heating stirring time is 5 min, and the particle size of the nanoparticles prepared is shown in the schematic diagram; Figure 20 The heating stirring time is 10 min, and the particle size of the nanoparticles prepared is shown in the schematic diagram.
[0154] In combination with Figures 16-20 and the test results of Table 4, when the heating time is 2-10 min, the nanoparticles prepared have lower particle size and better uniformity; when the heating time is less than 2 min, the particle size distribution may be uneven; and the optimal time is 5 min.
[0155] Example 5
[0156] This embodiment synchronously verifies the cooling method effect on the particle size and polydispersity coefficient of the albumin nanoparticles.
[0157] Specifically, the preparation method comprises:
[0158] (1) Dissolve bovine serum albumin in 14 mL ultrapure water, and the concentration is 10 mg / mL; add 2 mL anhydrous ethanol to obtain a mixed solution (two parts);
[0159] (2) Stir the mixed solution at 1000 rpm / min at 70℃ for 3 min, respectively, to obtain an albumin nanoparticle solution; one part is cooled to room temperature in ice water, and one part is naturally placed to cool to room temperature;
[0160] (3) High-speed centrifuge the albumin nanoparticle solution at 10000 rpm / min for 15 min, and take the supernatant to obtain a nanoparticle dispersion;
[0161] (4) Filter the nanoparticle dispersion through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.
[0162] The test results of the particle size (nm) and polydispersity coefficient of the albumin nanoparticles are shown in Table 5 and Figures 21-22 .
[0163] Table 5: Cooling method effect on particle size
[0164] Cooling method Ice water cooling Room temperature standing Particle size (nm) 98.96 93.86 Polydispersity coefficient 0.352 0.353
[0165] Figures 21-22 The figure shows the effect of cooling method on particle size; wherein, Figure 21 The figure shows the particle size results of the nanoparticles prepared by ice water cooling; Figure 22 The figure shows the particle size results of the nanoparticles prepared by room temperature standing.
[0166] In combination with Figures 21-22 The test results of Table 5 and Table 6 show that whether the nanoparticles are prepared by the method of the present embodiment or by the hot gel method, the particle size is similar, and the particle size is small but more uniform. In the hot gel method, the nanoparticles are not uniformly distributed, and the particle size is large.
[0167] Comparative Example 1
[0168] Comparative Example 1 provides a method for preparing albumin nanoparticles by a hot gel method, which comprises:
[0169] (1) Dissolve bovine serum albumin in 14 mL of ultrapure water to obtain a mixed solution with a concentration of 10 mg / mL;
[0170] (2) Heat the mixed solution at 70°C for 20 min, and cool it on ice to room temperature to obtain an albumin nanoparticle solution;
[0171] (3) Centrifuge the albumin nanoparticle solution at 10000 rpm / min for 15 min, and take the supernatant to obtain a nanoparticle dispersion;
[0172] (4) Filter the nanoparticle dispersion through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.
[0173] The test results of the particle size (nm) and the polydispersity coefficient of the albumin nanoparticles are shown in Table 6, Figure 3 and Figure 23 .
[0174] Table 6: Particle size test results of Example 1 and Comparative Example 1
[0175]
[0176] Figure 3 and Figure 23 The figure shows the effect of the two methods in Table 6 on particle size; wherein, Figure 23 The figure shows the particle size results of the nanoparticles prepared by Comparative Example 1.
[0177] In combination with Figure 3 , Figure 23 and the test results of Table 6, the method of Example 1 has a similar particle size compared with the simplified hot gel method, and the particle size is small but more uniform; wherein, the nanoparticles prepared by the hot gel method are not uniformly distributed, and the particle size is large.Figure 6 It can be seen that the particles with actual size greater than 100 nm account for the majority. Therefore, the method of the present embodiment has obvious advantages, and the heating time is shorter, and the energy consumption is reduced.
[0178] Comparative Example 2
[0179] Comparative Example 2 provides an albumin nanoparticle prepared by a desolvation method, and the preparation method comprises:
[0180] (1) Dissolve bovine serum albumin in 14 mL of ultrapure water to obtain a mixed solution with a concentration of 10 mg / mL;
[0181] (2) Stir the mixed solution at 1000 rpm / min, and add anhydrous ethanol dropwise while stirring until the solution produces a blue opalescence to obtain an albumin nanoparticle solution;
[0182] (3) High-speed centrifuge the albumin nanoparticle solution at 10000 rpm / min for 15 min, and take the supernatant to obtain a nanoparticle dispersion;
[0183] (4) Filter the nanoparticle dispersion through a 0.22 μm sterile filter membrane to obtain albumin nanoparticles.
[0184] The test results of the particle size (nm) and the polydispersity coefficient of the albumin nanoparticles are shown in Table 7, Figure 3 and Figure 24 .
[0185] Table 7: Particle size test results of Example 1 and Comparative Example 2
[0186]
[0187] Figure 3 and Figure 24 show a schematic diagram showing the effect of the two methods in Table 7 on the particle size; wherein, Figure 24 is a schematic diagram of the particle size results of the nanoparticles prepared in Comparative Example 2.
[0188] Wherein, the desolvation method requires adding about the same volume of ethanol as water to produce a blue opalescence, and the end timing is difficult to distinguish by the naked eye, making it difficult to ensure the repeatability of the experiment, and the particle size of the obtained particles is often large.
[0189] In combination with Figure 3 , Figure 24 and the test results in Table 6, it can be seen that the preparation method of Example 1 has smaller and more uniform particle size of the prepared albumin nanoparticles compared with the simplified desolvation method, is easy to control, has high repeatability, and uses less ethanol.
[0190] In summary, this embodiment adds ethanol at a specific volume ratio to an albumin solution. The addition of ethanol disrupts the hydration layer on the albumin surface, and heating causes the albumin to form a gel-like structure. Simultaneously, the shear force from stirring and the cavitation effects of the gas and ethanol vapor in the water cause the albumin to break down and aggregate into nanoparticles. Consequently, 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, resulting in albumin nanoparticles with a small particle size and uniform particle size distribution.
[0191] The above is a detailed introduction to the technical solutions provided in the embodiments of the present application. 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 may be changes in the specific implementation methods and application scope. 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; the concentration of the albumin solution is 2.5-10 mg / mL; 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 ethanol to water is 2:14-7:14; The mixed solution is heated and stirred, and the supernatant is taken out. The supernatant is dehydrated to obtain albumin nanoparticles, 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 collected to obtain a nanoparticle dispersion; After the nanoparticle dispersion is dehydrated, albumin nanoparticles are obtained; Wherein, the stirring time of the mixed solution is 2-10 min.
2. The method for preparing albumin nanoparticles according to claim 1, wherein The stirring time of the mixed solution is 3-10 min.
3. The method for preparing albumin nanoparticles according to claim 1, wherein The volume ratio of the ethanol to water is 2:14-6:
14.
4. The method for preparing albumin nanoparticles according to claim 1, wherein The albumin is at least one of human serum albumin, bovine serum albumin, and recombinant human serum albumin.
5. The method for preparing albumin nanoparticles according to claim 1, wherein The ethanol is anhydrous ethanol.