HSP90 inhibitor albumin nano-drug and preparation method and application thereof

Abstract

The application discloses an albumin nano-drug based on HSP90 inhibitor and a preparation method and application thereof, and belongs to the technical field of medicines.The short peptide A6 is modified to human blood albumin through a connecting chain, and the human blood albumin modified by the A6 is self-assembled with geldanamycin derivative G2111 in the presence of an organic solvent, so that the G2111 is combined to the hydrophobic region of the human blood albumin through a non-covalent bond, effective loading of the G2111 is realized, the solubility of the geldanamycin derivative is improved, the drug is targetedly released into cancer cells with high expression of CD44, the targeted accumulation of the drug in tumor tissues is enhanced, damage to other normal tissues and cells caused by off-target effects of the drug is avoided, the albumin nano-drug can be simultaneously used for chemotherapy of leukemia and solid tumors, and has important practical significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to an albumin nano-drug based on an HSP90 inhibitor and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of generally presenting the context of the application. The information disclosed in this Background section is not to be necessarily taken as an acknowledgement or any form of suggestion that this information forms part of the prior art already known to a person skilled in the art.

[0003] Heat shock proteins (Hsps) are a group of highly conserved proteins synthesized by the body under stress conditions, and are important molecular chaperones in cells. According to the molecular weight, they can be divided into small molecular Hsp, Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100, etc. Among them, Hsp90 is one of the most active molecular chaperones in cells, and plays an important role in the occurrence of tumors, inflammation and immune-related diseases.

[0004] Studies have shown that Hsp90 is highly expressed in tumor cells and is in an activated state, while Hsp90 exists in an inactivated form in normal cells. Most of the client proteins regulated by Hsp90 are closely related to the occurrence and development of tumors, and by inhibiting Hsp90, the proliferation of cancer cells can be affected from multiple aspects and multiple pathways, thereby avoiding the problem of drug resistance that may be caused by single-target treatment. Therefore, Hsp90 has become a promising target for anti-tumor drugs, and in recent years, a large number of studies have reported that Hsp90 inhibitors are used for treating various cancers, such as esophageal cancer, chronic leukemia, acute leukemia and lymphoma, and have shown good anti-tumor activity. Geldanamycin is the first reported Hsp90 inhibitor, which is a natural product isolated from broth of radiating fungi in the 1970s. However, although geldanamycin has significant anti-tumor activity, it also has side effects such as strong hepatotoxicity. In order to reduce the adverse reactions such as hepatotoxicity, a series of geldanamycin derivatives have been synthesized. Although the hepatotoxicity of the geldanamycin derivatives has been reduced through structural modification, the problems of hepatotoxicity and poor water solubility of these drugs have greatly limited their application. SUMMARY

[0005] In order to solve the deficiencies of HSP90 inhibitors in cancer treatment in the prior art, the present application aims to provide an HSP90 inhibitor-based albumin nanodrug and a preparation method and application thereof, the nanodrug provided by the present application can increase the solubility of geldanamycin derivatives, target the release of the drug into cancer cells with high CD44 expression, enhance the characteristics of the drug in the target accumulation of tumor tissues, avoid the damage to other normal tissues and cells caused by the off-target effect of the drug, and can be used for the chemotherapy of leukemia and solid tumors at the same time.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] The first aspect of the present application provides an HSP90 inhibitor-based albumin nanodrug, which is composed of albumin, short peptide A6 and HSP90 inhibitor, the short peptide A6 is modified on the albumin, and the HSP90 inhibitor is loaded in the hydrophobic region of the short peptide A6 modified albumin.

[0008] Preferably, the albumin is human blood albumin, and the short peptide A6 is modified on the human blood albumin through a connecting chain. Further preferably, the connecting chain is 4-(N-maleimide methyl) cyclohexane carboxylic acid-N-succinimidyl ester.

[0009] Preferably, the short peptide A6 has a sequence of Ac-KPSSPPEEC-NH2.

[0010] Preferably, the HSP90 inhibitor is a geldanamycin derivative, which is G2111, and has a structural formula as shown in formula (I).

[0011]

[0012] Formula (I)

[0013] Preferably, the HSP90 inhibitor is loaded in the hydrophobic region of the short peptide A6 modified albumin through a non-covalent bond.

[0014] The second aspect of the present application provides a preparation method of the above-mentioned HSP90 inhibitor-based albumin nanodrug, which comprises the following steps:

[0015] 1) Take human blood albumin, add a connecting chain, stir, add short peptide A6, continue to stir, wash the solution after reaction, and obtain short peptide A6 modified human blood albumin;

[0016] 2) Take the short peptide A6 modified human blood albumin, add a solution of geldanamycin derivative G2111, ultrasonically disperse, stir at room temperature to volatilize the organic solvent, centrifuge and filter, and obtain the HSP90 inhibitor-based albumin nanodrug.

[0017] The short peptide A6 modified human blood albumin is combined with the geldanamycin derivative G2111 by self-assembly in the presence of an organic solvent to non-covalently load the hydrophobic region of the human blood albumin, thereby preparing the HSP90 inhibitor albumin nanodrug.

[0018] Preferably, in step 1), the mass ratio of the human blood albumin, the connecting chain and the short peptide A6 is 400: 1-10: 10-100.

[0019] Preferably, the stirring temperature after adding the connecting chain is 25-30℃, and the stirring time is 25-35 min. To better maintain the activity of the short peptide A6, it is further preferred that the stirring temperature is 28℃, and the stirring time is 30 min.

[0020] Preferably, in step 1), the washing is performed using an ultrafiltration tube with a molecular weight cut-off of 30000 kDa.

[0021] Preferably, the solvent of the geldanamycin derivative G2111 solution is dichloromethane or trichloromethane; and the concentration of the geldanamycin derivative G2111 solution is 1-10 mg / ml. Further preferably, the mass ratio of the geldanamycin derivative G2111 to the human blood albumin is 1: 10-20.

[0022] The HSP90 inhibitor albumin nanodrug of the present application is used in the preparation of a leukemia or solid tumor related drug.

[0023] The present application has the following beneficial effects:

[0024] The short peptide A6 is modified to the human blood albumin through the connecting chain, and the A6 modified human blood albumin is self-assembled with the geldanamycin derivative G2111 in the presence of an organic solvent, so that the G2111 is non-covalently combined to the hydrophobic region of the human blood albumin, thereby realizing the effective loading of the G2111.

[0025] The present application provides a HSP90 inhibitor albumin nanodrug, a preparation method and application thereof. The prepared HSP90 inhibitor albumin nanodrug can improve the solubility of the geldanamycin derivative, release the drug into the cancer cells with high expression of CD44, enhance the targeted accumulation of the drug in the tumor tissue, avoid the damage to other normal tissues and cells caused by the off-target effect of the drug, and can be used for the chemotherapy of leukemia and solid tumors at the same time, which has important practical significance. BRIEF DESCRIPTION OF DRAWINGS

[0026] The drawings constituting a part of the specification of the present application are used to provide a further understanding of the present application, and the schematic embodiments of the present application and the description thereof are used to explain the present application, and do not constitute an improper limitation on the present application.

[0027] Figure 1 Transmission electron micrograph of A6-NP;

[0028] Figure 2 Particle size distribution of A6-NP;

[0029] Figure 3 Stability of A6-NP in vitro;

[0030] Figure 4 Inhibition rate of A6-NP on molm13 cells by CCK8 method;

[0031] Figure 5 Uptake experiment of A6-NP in molm-13. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0033] Example 1

[0034] A preparation method of a HSP90 inhibitor albumin nanoparticle drug, comprising the following steps:

[0035] Take 200 μL (10 g / 50 ml) of human blood albumin, add 100 μL of 4-(N-maleimide methyl) cyclohexane carboxylic acid-N-succinimidyl ester SMCC solution (1 mg / mL), place in a 28°C water bath and stir for 30 min, then add 500 μL of short peptide A6 solution (2 mg / mL) and continue to stir for 30 min. Collect the stirring liquid, wash three times with an ultrafiltration tube with a molecular weight cut-off of 30000 kDa, then add phosphate PBS buffer to 1 mL, then add 200 μL of G2111 dichloromethane solution (10 mg / mL), ultrasonic 4 times, immediately place in a 37°C water bath, stir at 1000 rpm / min for 20 min, then centrifuge at 1500 rpm for 10 min, take the supernatant, filter to remove unloaded drugs, and obtain the HSP90 inhibitor albumin nanoparticle drug A6-NP.

[0036] Example 2

[0037] A preparation method of a HSP90 inhibitor albumin nanoparticle drug, comprising the following steps:

[0038] Take 200 μL (10 g / 50 ml) human blood albumin, add 100 μL 4-(N-maleimide methyl) cyclohexane carboxylic acid-N-succinimidyl ester SMCC solution (2 mg / mL), place in a 28°C water bath and stir for 30 min, then add 500 μL short peptide A6 solution (4 mg / mL) and continue stirring for 30 min. Collect the stirring liquid, wash three times with an ultrafiltration tube with a molecular weight cutoff of 30000 kDa, then add phosphate PBS buffer to 1 mL, then add 200 μL G2111 dichloromethane solution (10 mg / mL), ultrasonic 4 times, immediately place in a 37°C water bath, stir at 1000 rpm / min for 20 min, then centrifuge at 1500 rpm for 10 min, take the supernatant, filter to remove unloaded drugs, and obtain HSP90 inhibitor albumin nanoparticle drug A6-NP.

[0039] Comparative Example 1

[0040] A preparation method of a HSP90 inhibitor albumin nanoparticle drug, compared with Example 1, human blood albumin is not modified with short peptide A6, and the obtained HSP90 inhibitor albumin nanoparticle drug is named NP.

[0041] Application Example 1

[0042] The performance of the HSP90 inhibitor albumin nanoparticle drug obtained in Example 1 is detected, and the detection content and detection results are as follows:

[0043] 1. Transmission electron microscopy observation of A6-NP

[0044] As Figure 1 The morphology of A6-NP is characterized by transmission electron microscopy (TEM), and A6-NP is round and uniformly dispersed. The core of A6-NP is dark in color, and the periphery is lighter in color, indicating that short peptide A6 is mainly dispersed in the periphery of the nanoparticle drug, and hydrophobic geldanamycin derivative G2111 is mainly loaded into the core of the nanoparticle drug.

[0045] As Figure 2 The particle size of A6-NP is measured by a particle size distribution instrument, and the particle size of A6-NP is about 180 nm, and the surface charge is negative.

[0046] 2. Stability of A6-NP

[0047] The prepared A6-NP is stored at 4°C, and the particle size is monitored at 0 days, 3 days and 7 days, respectively, to observe the stability of A6-NP. The results are as follows: Figure 3As shown, the particle size of A6-NP remained around 180 nm on days 3 and 7, with no significant difference from that on day 0, indicating that the prepared A6-NP has good stability.

[0048] 3. Inhibition rate of A6-NP on molm13 cells

[0049] Molm13 logarithmic phase cell suspension was seeded into 96-well plates at a density of 2 × 10⁴ cells per well. A6-NP was diluted to the required concentration according to the drug content, mixed thoroughly by pipetting, and added to the 96-well plates. The plates were sealed and incubated at 37°C for 48 h. The 96-well plates were then removed, and 10 μL of CCK8 solution was added to each well. The plates were incubated for another 4 h, and the absorbance of each well at 450 nm was measured using a microplate reader to calculate the cell inhibition rate.

[0050] Depend on Figure 4 It was found that both G2111 and A6-NP exhibited dose-dependent effects, meaning that the higher the concentration, the greater the cell-killing effect. Furthermore, compared to A6-NP, A6-NP showed a significantly higher inhibitory effect on cells than G2111, indicating that A6-NP can significantly enhance the in vitro activity against leukemia cells.

[0051] 4. A6-NP uptake experiment in molm13 cells

[0052] Logarithmic growth phase molm13 cells were seeded in 6-well culture plates and cultured in RPMI-1640 with 10% fetal bovine serum for 24 hours. Then, 1 mL of A6-NP loaded with fluorescein C6 was added to the plates. After culturing the cells in an incubator for another 0.5 h, the cells were washed three times with PBS buffer to remove extracellular adsorbed nanoparticles. The cell nuclei were stained with Hoechst 33342. Finally, the obtained samples were observed under a fluorescence microscope to observe the nanoparticle uptake.

[0053] The results are as follows Figure 5 As shown, the green fluorescence is emitted by Ce; the blue fluorescence is emitted by the cell nucleus stained with Hoechst 33342. The figure shows that the green fluorescence is almost distributed throughout the entire cell, indicating that the nanoparticles are not only distributed in the cytoplasm but can also enter the cell nucleus; Figure 5 The green fluorescence of the A6-NP group was significantly stronger than that of the NP without the short peptide A6 target modification, indicating that A6-NP has strong targeting ability for molm13 cells under the action of short peptide A6, and can enable a large number of drug-loaded nanoparticles to enter leukemia cells.

[0054] The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.

Claims

1. A preparation method of a geldanamycin derivative G2111 albumin nanodrug, characterized by, The geldanamycin derivative G2111 albumin-based nanomedicine is composed of human blood albumin modified by a short peptide A6 and the geldanamycin derivative G2111, and the G2111 is loaded in the hydrophobic region of the human blood albumin modified by the short peptide A6. The preparation method of the geldanamycin derivative G2111 albumin-based nanomedicine comprises the following steps: 1) taking human blood albumin, adding 4-(N-maleimide methyl) cyclohexane carboxylic acid-N-succinimide ester, stirring, adding a short peptide A6, continuing to stir, washing the solution after reaction, and obtaining human blood albumin modified by the short peptide A6; the mass ratio of human blood albumin, 4-(N-maleimide methyl) cyclohexane carboxylic acid-N-succinimide ester and the short peptide A6 is 400: 1-10: 10-100; 2) taking the human blood albumin modified by the short peptide A6, adding a geldanamycin derivative G2111 solution, ultrasonic dispersion, stirring at room temperature, centrifugation, and filtration, to obtain the geldanamycin derivative G2111 albumin-based nanomedicine; the solvent of the geldanamycin derivative G2111 solution is dichloromethane or trichloromethane; the concentration of the geldanamycin derivative G2111 solution is 1-10 mg / mL; the mass ratio of the geldanamycin derivative G2111 and human blood albumin is 1:10-20; The short peptide A6 has the sequence of Ac-KPSSPPEEC-NH2. The structure of the geldanamycin derivative G2111 is shown in formula (I): Formula (I).

2. The method for preparing the G2111 albumin nanodrug based on geldanamycin derivative according to claim 1, characterized in that, The geldanamycin derivative G2111 is loaded in the hydrophobic region of the human blood albumin modified by the short peptide A6 by a non-covalent bond.

3. The method for preparing the G2111 albumin nanodrug based on geldanamycin derivative according to claim 1, characterized in that, In step 1), the stirring temperature after adding 4-(N-maleimide methyl) cyclohexane carboxylic acid-N-succinimide ester is 25-30℃, and the stirring time is 25-35 min. Alternatively, in step 1), the washing is washing by using an ultrafiltration tube with a molecular weight cut-off of 30000 kDa.

4. The method for preparing the G2111 albumin nanodrug based on geldanamycin derivative according to claim 1, characterized in that, In step 1), the stirring temperature after adding 4-(N-maleimide methyl) cyclohexane carboxylic acid-N-succinimide ester is 28℃, and the stirring time is 30 min.

5. The application of the geldanamycin derivative G2111 albumin-based nanomedicine prepared by the preparation method of the geldanamycin derivative G2111 albumin-based nanomedicine in claim 1-4 in the preparation of leukemia drugs.

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