A kind of albumin nanoparticle and preparation method thereof

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to an albumin nanoparticle and a preparation method thereof. Currently, there is a lack of targeted preparations for the combined delivery of liquiritigenin and temozolomide in the treatment of brain glioma. The present invention carries liquiritigenin and temozolomide on human serum albumin nanoparticles, and connects a targeting polypeptide to the surface through a coupling agent to form active targeted albumin nanoparticles that can carry liquiritigenin and temozolomide at the same time, with uniform and stable particle size, simple preparation process, and high drug loading.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to albumin nanoparticles and a preparation method thereof. Background Art

[0002] Glioma is the most common brain tumor. It occurs in the neuroectoderm, so it can also be considered a neuroectodermal tumor or a neuroepithelial tumor. In terms of histological origin, gliomas originate from neural mesenchymal cells and neuronal cells including glia, ependyma, and choroid plexus epithelium. Among them, glial cells are the origin of most gliomas. Therefore, based on the histological origin and biological characteristics, various tumors that occur in the neuroectoderm are also habitually called gliomas.

[0003] Temozolomide (TMZ) is a new type of imidazole tetrazine alkylating agent and a standard first-line chemotherapy drug commonly used for malignant glioma. It can be rapidly converted into active compounds through non-enzymatic pathways in physiological environments and promote tumor cell apoptosis by inducing DNA methylation. TMZ is rapidly absorbed, easily penetrates the blood-brain barrier, and has good anti-glioma activity. Therefore, it has been widely used in the chemotherapy of central nervous system tumors, especially malignant gliomas. However, tumor cell resistance to TMZ often occurs, which is the main reason for the failure of chemotherapy in clinical practice.

[0004] Liquiritigenin (Liq) is a compound extracted from the rhizomes of licorice and licorice of the legume family. In recent years, Liq can also be obtained by artificial synthesis. Its alias is also called 7,4-dihydroxydihydroflavone, with a molecular formula of C15H12O4 and a molecular weight of 256.25. It is generally a yellow powder-like substance. Studies have found that Liq has certain effects on the cardiovascular system and the nervous system. In addition, it also has multiple pharmacological effects such as antiviral, antioxidant, anti-tumor and estrogenic effects. Because Liq can selectively supplement steroid hormone receptor co-activators to target genes, Liq can inhibit the proliferation of glioma cells (U138) and significantly promote their apoptosis, significantly enhancing the sensitivity of tumor cells to TMZ (PhD thesis of Jilin University, Study on the Sensitization Effect and Mechanism of Liquorice on Temozolomide-Induced Glioma Cell Apoptosis by Liquorice, Liu Xiaoyang).

[0005] In view of the synergistic anti-tumor effect of temozolomide and glycyrrhizin on glioma, it is urgent to develop a combined delivery system of the two drugs. However, it is difficult to ensure the directional enrichment of fat-soluble glycyrrhizin and water-soluble temozolomide at the tumor site. Moreover, if the drug treatment effect is maximized and the toxic side effects are reduced, it is necessary to target the drug to the tumor tissue and ensure that the two have similar in vivo behaviors. In the prior art, there is a lack of preparations for the combined directional delivery of the two. Summary of the invention

[0006] In view of the shortcomings of the prior art, the inventors provide an albumin nanoparticle and a preparation method thereof. By utilizing the advantages of the structures of glycyrrhizin, temozolomide and human serum albumin, water-soluble drugs and fat-soluble drugs can be simultaneously formed into nanoparticles with albumin, and then the Cys-Arg-Glu-Lys-Ala polypeptide that can target glioma cells is coupled with albumin to form a nanoformulation that can target the tumor site. The nanoparticles have uniform particle size, simple preparation process and high drug loading capacity.

[0007] The present invention specifically provides an albumin nanoparticle, which comprises a polypeptide Cys-Arg-Glu-Lys-Ala, human serum albumin, glycyrrhizin and temozolomide. The albumin nanoparticle is formed by carrying glycyrrhizin and temozolomide on human serum albumin, and the polypeptide Cys-Arg-Glu-Lys-Ala is connected to the human serum albumin by using a bifunctional coupling agent sulfo-SMCC containing an active ester of N-hydroxysuccinimide (NHS) and maleimide.

[0008] Sulfo-SMCC is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt.

[0009] Cys-Arg-Glu-Lys-Ala, abbreviated as CREKA, is composed of five amino acids: cysteine-arginine-glutamic acid-lysine-alanine. It can specifically bind to coagulation plasma proteins in tumor blood vessels and is a pentapeptide that can target the fibrin-fibronectin complex.

[0010] The albumin nanoparticles are prepared by the following method: temozolomide and human serum albumin are dissolved in an aqueous phase, glycyrrhizin is dissolved in ethanol as an oil phase, the pH of the aqueous phase is adjusted to be alkaline, the oil phase is added to the aqueous phase under stirring, ethanol is continuously added until the solution becomes turbid, glutaraldehyde is added after continuous stirring for cross-linking, ethanol is removed to obtain albumin nanoparticles, a coupling agent sulfo-SMCC is added and mixed, and then a polypeptide Cys-Arg-Glu-Lys-Ala is added and stirred to mix.

[0011] Preferably, the albumin nanoparticles are prepared by the following method: temozolomide and human serum albumin are dissolved in an aqueous phase, the concentration of human serum albumin is 20 mg / mL-50 mg / mL, the mass ratio of temozolomide to human serum albumin is 1:10-50, the pH of the aqueous phase is adjusted to 7.5-9.0, glycyrrhizin is dissolved in ethanol as an oil phase, the mass ratio of glycyrrhizin to human serum albumin is 1:10-50, the oil phase is added to the aqueous phase under stirring, ethanol is continued to be added and stirred until the solution becomes turbid, 3%-8% glutaraldehyde is added after continued stirring for cross-linking, the organic solvent is removed, and the coupling agent sulfo-SMCC is added and mixed, and the polypeptide Cys-Arg-Glu-Lys-Ala is added and stirred to obtain nanoparticles.

[0012] Further preferably, the albumin nanoparticles are prepared by the following method: temozolomide and human serum albumin are dissolved in an aqueous phase, the concentration of human serum albumin is 20 mg / mL-50 mg / mL, the mass ratio of temozolomide to human serum albumin is 1:10-50, the pH of the aqueous phase is adjusted to 7.5-9.0, glycyrrhizin is dissolved in ethanol as an oil phase, the mass ratio of glycyrrhizin to human serum albumin is 1:10-50, the oil phase is added to the aqueous phase under stirring, ethanol is continued to be added and stirred until the solution becomes turbid, 3%-8% glutaraldehyde is added after continuing to stir for cross-linking, the organic solvent is removed, and the coupling agent sulfo-SMCC is added and mixed for 1-2 hours, and the polypeptide Cys-Arg-Glu-Lys-Ala is added and stirred for 12-24 hours to obtain nanoparticles.

[0013] The stirring conditions of the peptide Cys-Arg-Glu-Lys-Ala ensure the stability of the peptide, and it is usually carried out in an ice bath.

[0014] The albumin nanoparticles are used for intravenous injection.

[0015] The albumin nanoparticles further contain a pH adjuster.

[0016] The nanomedicine also contains an osmotic pressure regulator.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) A kind of albumin nanoparticles co-loaded with liquiritigenin and temozolomide were prepared to achieve the water-soluble drug and lipid

[0019] Simultaneous encapsulation of soluble drugs;

[0020] (2) A method for preparing drug-co-loaded albumin nanoparticles is provided, which has a high drug encapsulation efficiency;

[0021] (3) Provided are albumin nanoparticles targeting tumor cells and a method for preparing the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Particle size distribution diagram of the nanoparticles of Example 3

[0023] Figure 2 : Transmission electron microscopy image of the nanoparticles of Example 1

[0024] Figure 3 :HPLC chromatogram of licorice

[0025] Figure 4 : Particle size variation over time of Example 4

[0026] Figure 5 : The polydispersity index PDI of Example 4 changes over time

[0027] Figure 6 :HPLC chromatogram of peptide CREKA DETAILED DESCRIPTION

[0028] The present invention is further described below by way of examples. It should be correctly understood that the examples of the present invention are provided only for illustrating the present invention, rather than limiting the present invention, and therefore, simple modifications of the present invention under the premise of the method of the present invention are within the scope of protection claimed by the present invention.

[0029] Example 1

[0030] 1 mg of temozolomide and 10 mg of human serum albumin were dissolved in 1 mL of deionized water as the aqueous phase, and the pH was adjusted to 7.5 with sodium hydroxide. 1 mg of glycyrrhizin was dissolved in 1 mL of ethanol as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and ethanol was continued to be added and stirred until precipitation occurred. Then, 5% glutaraldehyde was added for cross-linking for 2 h, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 1 mg of sulfo-SMCC was added and stirred for 2 h, and then an equal molar ratio of CREKA polypeptide was added and stirred for 24 h.

[0031] Example 2

[0032] 1 mg of temozolomide and 30 mg of human serum albumin were dissolved in 1 mL of deionized water as the aqueous phase, and the pH was adjusted to 9.0 with sodium hydroxide. 1 mg of glycyrrhizin was dissolved in 1 mL of ethanol as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and ethanol was continued to be added and stirred until precipitation occurred. Then, 3% glutaraldehyde was added for cross-linking for 2 h, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 1 mg of sulfo-SMCC was added and stirred for 2 h, and then an equal molar ratio of CREKA polypeptide was added and stirred for 12 h.

[0033] Example 3

[0034] 1 mg of temozolomide and 50 mg of human serum albumin were dissolved in 1 mL of deionized water as the aqueous phase, and the pH was adjusted to 8.0 with sodium hydroxide. 1 mg of glycyrrhizin was dissolved in 2 mL of ethanol as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and ethanol was continued to be added and stirred until precipitation occurred. Then, 8% glutaraldehyde was added for cross-linking for 1 hour, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 5 mg of sulfo-SMCC was added and stirred for 1 hour, and then an equal molar ratio of CREKA polypeptide was added and stirred for 24 hours.

[0035] Example 4

[0036] 1 mg of temozolomide and 40 mg of human serum albumin were dissolved in 2 mL of deionized water as the aqueous phase, and the pH was adjusted to 10.0 with sodium hydroxide. 2 mg of glycyrrhizin was dissolved in 1 mL of ethanol as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and ethanol was continued to be added and stirred until precipitation occurred. Then, 5% glutaraldehyde was added for cross-linking for 3 h, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 3 mg of sulfo-SMCC was added and stirred for 2 h, and then an equal molar ratio of CREKA polypeptide was added and stirred for 24 h.

[0037] Comparative Example 1

[0038] 5 mg of temozolomide and 10 mg of human serum albumin were dissolved in 1 mL of deionized water as the aqueous phase, and the pH was adjusted to 7.5 with sodium hydroxide. 1 mg of glycyrrhizin was dissolved in 1 mL of ethanol as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and ethanol was continued to be added and stirred until precipitation occurred. Then, 5% glutaraldehyde was added for cross-linking for 2 h, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 1 mg of sulfo-SMCC was added and stirred for 2 h, and then an equal molar ratio of CREKA polypeptide was added and stirred for 24 h.

[0039] Comparative Example 2

[0040] 1 mg of temozolomide and 10 mg of hemoglobin protein were dissolved in 1 mL of deionized water as the aqueous phase, and the pH was adjusted to 7.5 with sodium hydroxide. 1 mg of glycyrrhizin was dissolved in 1 mL of ethanol as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and ethanol was continued to be added and stirred until precipitation occurred. Then, 5% glutaraldehyde was added for cross-linking for 2 h, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 1 mg of sulfo-SMCC was added and stirred for 2 h, and then an equal molar ratio of CREKA polypeptide was added and stirred for 24 h.

[0041] Comparative Example 3

[0042] 1 mg of doxorubicin and 10 mg of human serum albumin were dissolved in 1 mL of deionized water as the aqueous phase, and the pH was adjusted to 7.5 with sodium hydroxide. 1 mg of paclitaxel was dissolved in 1 mL of ethanol as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and ethanol was continued to be added and stirred until precipitation occurred. Then, 5% glutaraldehyde was added for cross-linking for 2 hours, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 1 mg of sulfo-SMCC was added and stirred for 2 hours, and then an equal molar ratio of CREKA polypeptide was added and stirred for 24 hours.

[0043] Comparative Example 4

[0044] 1 mg of temozolomide and 10 mg of human serum albumin were dissolved in 1 mL of deionized water as the aqueous phase, and the pH was adjusted to 7.5 with sodium hydroxide. 1 mg of glycyrrhizin was dissolved in 1 mL of a mixed solution of ethyl acetate and acetone (volume ratio of 1:1) as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and the mixed solution was continued to be added and stirred until precipitation occurred. Then, 5% glutaraldehyde was added for cross-linking for 2 h, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 1 mg of sulfo-SMCC was added and stirred for 2 h, and then an equal molar ratio of CREKA polypeptide was added and stirred for 24 h.

[0045] Comparative Example 5

[0046] 1 mg of temozolomide and 90 mg of human serum albumin were dissolved in 1 mL of deionized water as the aqueous phase, and the pH was adjusted to 7.5 with sodium hydroxide. 1 mg of glycyrrhizin was dissolved in 3 mL of ethanol as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and ethanol was continued to be added and stirred until precipitation occurred. Then, 5% glutaraldehyde was added for cross-linking for 2 h, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 1 mg of sulfo-SMCC was added and stirred for 2 h, and then an equal molar ratio of CREKA polypeptide was added and stirred for 24 h.

[0047] Comparative Example 6

[0048] 1 mg of temozolomide and 50 mg of human serum albumin were dissolved in 1 mL of deionized water as the aqueous phase, and the pH was adjusted to 6.0 with hydrochloric acid. 1 mg of glycyrrhizin was dissolved in 1 mL of ethanol as the oil phase. The oil phase was added dropwise to the aqueous phase under magnetic stirring, and ethanol was continued to be added and stirred until precipitation occurred. Then, 5% glutaraldehyde was added for cross-linking for 2 h, and the organic solvent was removed by rotary evaporation to obtain nanoparticles. 1 mg of sulfo-SMCC was added and stirred for 2 h, and then an equal molar ratio of iRGD polypeptide was added and stirred for 24 h.

[0049] Verification Example

[0050] 1. The solutions obtained in Examples 1-4 and Comparative Examples 1-6 were filtered through a 0.45 μm microporous membrane, and the particle size and polydispersity index were monitored by a laser particle size analyzer. The nanoparticles were separated by high-speed centrifugation from the solution obtained in Example 1, washed three times with ultrapure water, diluted with water, and negatively stained with phosphotungstic acid, and the morphology was observed by transmission electron microscopy.

[0051] Table 1. Particle size and polydispersity index of various examples

[0052] Example Particle size (nm) Polydispersity Index (PDI) Example 1 61.5 0.103 Example 2 103.4 0.096 Example 3 113.5 0.109 Example 4 128.9 0.115 Comparative Example 1 257.6 0.354 Comparative Example 2 305.1 0.384 Comparative Example 3 402.9 0.406 Comparative Example 4 299.2 0.344 Comparative Example 5 395.2 0.411 Comparative Example 6 502.3 0.337

[0053] The results are as follows Figure 1 As shown in Figure 2, the particle size of the nanoparticles prepared by the present invention conforms to the normal distribution, and the nanoparticles are approximately spherical. The results in Table 1 show that the particle size of the nanoparticles obtained by the present invention is less than 200nm, and the polydispersity index is less than 0.2, and the particle size is uniform.

[0054] 2. The solutions obtained in Examples 1-4 and Comparative Examples 1-2 and 4-6 were separated into nano-drugs by high-speed centrifugation, washed with ultrapure water for 3 times, freeze-dried, a certain amount of the drug was weighed, 1 mL of dimethyl sulfoxide was added to break the emulsion, and the concentration of glycyrrhizin was detected by high performance liquid chromatography. The chromatographic column was Diamonsil Plus C18, 250×4.6 mm, 5 μm, the mobile phase ratio was acetonitrile: water = 90:10, the detection wavelength was 275 nm, the flow rate was 1 ml / min, the injection volume was 20 μl, and the drug encapsulation efficiency was calculated.

[0055] Table 2. Encapsulation efficiency of licorice in various examples

[0056]

[0057]

[0058] The results are shown in Table 2. The nano drug encapsulation rate of glycyrrhizin in the nano preparation prepared by the present invention can reach more than 85%.

[0059] 1. The solutions obtained in Examples 1-5, Comparative Examples 1-2 and 4-6 were separated into the aqueous phase of the nanoparticles by ultrafiltration centrifugation. 1 ml of the mother liquor in the upper layer was added with 4 ml of DMSO for demulsification. The contents of free drug and demulsified sample drug temozolomide in the lower aqueous phase were detected by ultraviolet spectrophotometer, respectively. The content detection wavelength was 329 nm, and the encapsulation efficiency of temozolomide was calculated.

[0060] Table 3. Encapsulation efficiency of temozolomide in various examples

[0061] Example Encapsulation rate (%) Example 1 76.2 Example 2 88.1 Example 3 82.8 Example 4 83.3 Example 5 72.4 Comparative Example 1 20.2 Comparative Example 2 19.3 Comparative Example 4 32.1 Comparative Example 5 38.9 Comparative Example 6 25.6

[0062] The results are shown in Table 3. The nanoparticles prepared by the present invention have a high temozolomide encapsulation rate of more than 70%.

[0063] 3. After filtering the nanoparticle solution obtained in Example 4 through a microporous membrane, the particle size and polydispersity index (PDI) were tested every day. Figure 4-5 As described above, within 7 days of storage, the particle size of the nanoparticles changed stably and the PDI remained less than 0.2.

[0064] 4. When the polypeptide is added in Example 1, 0.1 mL is taken and centrifuged, and the polypeptide content in the supernatant is detected by high performance liquid chromatography. After the reaction is completed, 0.1 mL is taken and centrifuged again to detect the polypeptide content in the supernatant. The polypeptide detection method is: chromatographic column DiamonsilPlus C18, 250×4.6mm, 5μm, mobile phase A 0.1% trifluoroacetic acid: B acetonitrile, injection volume is 10 microliters, detection wavelength is 220nm, and gradient elution method is as follows:

[0065]

[0066] The chromatogram results of the peptide are as follows Figure 6 As shown, the retention time of the peptide is about 12.5 minutes. After the coupling is completed, the chromatographic peak of the peptide disappears.

Claims

1. An albumin nanoparticle, characterized in that: The albumin nanoparticles contain a polypeptide Cys-Arg-Glu-Lys-Ala, human serum albumin, glycyrrhizin, and temozolomide. The albumin nanoparticles are formed by carrying glycyrrhizin and temozolomide on human serum albumin. The polypeptide Cys-Arg-Glu-Lys-Ala is connected to human serum albumin using a bifunctional coupling agent sulfo-SMCC containing an active ester of N-hydroxysuccinimide (NHS) and maleimide; The albumin nanoparticles are prepared by the following method: temozolomide and human serum albumin are dissolved in an aqueous phase, the concentration of human serum albumin is 10 mg / mL-50 mg / mL, the mass ratio of temozolomide to human serum albumin is 1:10-50, the pH of the aqueous phase is adjusted to 7.5-9.0, glycyrrhizin is dissolved in ethanol as an oil phase, the mass ratio of glycyrrhizin to human serum albumin is 1:10-50, the oil phase is added to the aqueous phase under stirring, ethanol is continuously added and stirred until the solution becomes turbid, 3%-8% glutaraldehyde is added after continuous stirring for cross-linking, the organic solvent is removed, a coupling agent sulfo-SMCC is added and mixed, and then the polypeptide Cys-Arg-Glu-Lys-Ala is added and stirred to obtain the nanoparticles.

2. The albumin nanoparticles according to claim 1, characterized in that The albumin nanoparticles are used for intravenous injection.

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