Preparation method and application of MHI-148 chemically modified albumin-bound sorafenib nanoformulation

Abstract

The present invention relates to a preparation method and application of an albumin-bound sorafenib nanoformulation chemically modified by MHI-148, comprising the following steps: S1, synthesizing albumin chemically modified by MHI-148 (BSA-MHI148); S2, synthesizing BSA-MHI148@SRF nanoparticles; the albumin-bound sorafenib nanoformulation (BSA-MHI148@SRF) is used for anti-tumor treatment. The albumin-bound sorafenib nanoformulation (BSA-MHI148@SRF) of the present invention can simultaneously solve the problems of tumor hypoxia and high expression of PD-L1.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of an MHI-148 chemically modified albumin-bound sorafenib nanoformulation. Background Art

[0002] Photodynamic therapy (PDT) is a new treatment for diseases. It is non-invasive, highly controllable in time and space, and safe, and is considered to be a very promising cancer treatment. In the presence of molecular oxygen, PDT kills cancer cells by producing reactive oxygen species (ROS), but the hypoxic state of the tumor affects the production of ROS, resulting in limited efficacy of PDT on tumors. Although the "Warburg effect" is the main source of tumor energy, overexpression of mitochondrial-related oxidative phosphorylation (OXPHOS) in tumor cells still exists in many tumor cell lines, resulting in a large amount of physiological oxygen consumption, coupled with insufficient oxygen supply induced by immature and abnormal tumor blood vessels, which exacerbates tumor hypoxia.

[0003] In addition, in some cases, after photodynamic therapy, due to the production of interferon gamma, the expression of PD-L1 in the tumor increases significantly, weakening the killing effect of T lymphocytes, thereby inducing immune tolerance. Therefore, we urgently need to develop a drug that can simultaneously solve the problems of tumor hypoxia and high expression of PD-L1, which needs to be improved. Summary of the invention

[0004] In view of the above problems in the prior art, the purpose of the present invention is to overcome the deficiencies in the prior art and to provide a preparation method and application of an albumin-bound sorafenib nanoformulation chemically modified with MHI-148.

[0005] The present application provides a method for preparing an MHI-148 chemically modified albumin-bound sorafenib nanoformulation, comprising the following steps:

[0006] S1. Synthesis of MHI-148 chemically modified albumin (BSA-MHI148):

[0007] MHI-148 was dissolved in DMSO to form a solution A with a concentration of 10 mg / mL, and then the solution A was mixed with 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) under light-proof conditions for 1 h to activate MHI-148, and then the activated MHI-148 was mixed with albumin and dissolved in double distilled water to obtain a mixture A;

[0008] The obtained mixture A was stirred at room temperature for 6 h, and then dialyzed with pure H2O (relative molecular weight = 10 kDa) 5 times to remove free MHI-148, DMTMM and DMSO to obtain pure BSA-MHI148;

[0009] S2. Synthesis of BSA-MHI148@SRF nanoparticles:

[0010] Sorafenib was dissolved in DMF to form a solution B with a concentration of 12 mg / mL, and then solution B was added dropwise to the BSA-MHI148 solution. After stirring for 30 seconds, the free sorafenib was removed using an ultrafiltration cup (MW = 30 kDa) to obtain the MHI148 chemically modified albumin-bound sorafenib nanoformulation (BSA-MHI148@SRF).

[0011] Furthermore, the sorafenib includes one or more sorafenib derivatives.

[0012] Furthermore, the albumin is at least one of human serum albumin and bovine serum albumin.

[0013] Furthermore, the BSA-MHI148 is formed by condensing MHI-148 molecules with bovine serum albumin through DMTMM.

[0014] Furthermore, the concentration of the BSA-MHI148 solution in step S2 is 2 mg / mL, and the solvent is water.

[0015] Furthermore, the sorafenib in step S2 is added to the BSA-MHI148 solution by hanging drop, and the two are self-assembled to form a nanoformulation.

[0016] Furthermore, the mass ratio of MHI-148 to albumin in step S1 is 1:50.

[0017] Furthermore, the pure BSA-MHI148 prepared in step S1 is finally freeze-dried to prepare BSA-MHI148 powder. BSA-MHI148 is stored in the form of powder for reuse.

[0018] The invention discloses an application of a method for preparing an albumin-bound sorafenib nanoformulation chemically modified with MHI-148. The albumin-bound sorafenib nanoformulation (BSA-MHI148@SRF) is used for anti-tumor treatment.

[0019] In summary, this application includes the following beneficial technical effects:

[0020] 1. The BSA-MHI148@SRF nanoparticles used in the present invention can improve the hypoxic state of tumors and reverse the immunosuppression of the tumor microenvironment. The BSA-MHI148@SRF synthesized by a simple and economical chemical synthesis route can enhance the efficacy of photodynamic therapy while fighting tumors. Compared with the commercially available sorafenib tablets, it has better tumor targeting and less toxic side effects, and is expected to be used in the clinical treatment of cancer.

[0021] 2. The albumin-bound sorafenib nanoformulation of the present invention has a preparation process including the synthesis of bovine serum albumin chemically modified with MHI-148 (albumin is covalently modified with MHI148 molecules) and BSA-MHI148@SRF nanoparticles, and the particle size of the albumin-bound sorafenib nanoformulation is less than 200 nm. By combining sorafenib with albumin, the present invention has better safety, solubility and tumor targeting compared to commercially available sorafenib tosylate tablets, can be effective at extremely low concentrations, reduce the dosage of the drug, and thus greatly reduce toxic side effects.

[0022] 3. The present invention uses Sorafenib (Sorafenib, SRF) to mediate tumor vascular normalization to increase oxygen supply to reverse the hypoxic microenvironment of the tumor. SRF enhances oxygen perfusion by normalizing blood vessels, inhibits mitochondrial respiration to reduce oxygen consumption, and can also reverse the tumor immunosuppressive microenvironment by activating AMPK to downregulate the expression of PD-L1, thereby enhancing the efficacy of PDT treatment. Albumin, as a hydrophobic drug carrier, has the characteristics of tumor targeting, can increase the drug concentration in the tumor stroma, and improve the dosage of the drug. In addition, albumin, as an endogenous substance, can avoid the occurrence of allergic reactions. In this application, MHI-148 is connected to albumin through an amide bond to form BSA-MHI148. BSA-MHI148 can self-assemble with SRF through hydrophilic and hydrophobic self-assembly to form BSA-MHI148@SRF nanoparticles. This carefully designed nanoparticle significantly inhibits the growth of solid tumors. Compared with the sorafenib tosylate tablets used on the market, it has better solubility, tumor tissue targeting, biosafety and efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The preparation and characterization diagram of BSA-MHI148@SRF nanoparticles;

[0024] Figure 2 BSA-MHI148@SRF nanoparticles colocalize with mitochondria;

[0025] Figure 3Effects of SRF, MHI-148, BSA-MHI148, and BSA-MHI148@SRF on proliferation and apoptosis of MB49 cells;

[0026] Figure 4 The anti-tumor effect of BSA-MHI148@SRF combined with photodynamic therapy in vivo;

[0027] Figure 5 To evaluate the safety of BSA-MHI148@SRF nanoparticles in vivo. DETAILED DESCRIPTION

[0028] The following is combined with Figure 1-5 This application is described in further detail.

[0029] The present application discloses a method for preparing an albumin-bound sorafenib nanoformulation chemically modified with MHI-148.

[0030] Example 1: Preparation and characterization of BSA-MHI148@SRF The preparation method steps are as follows:

[0031] S1. Synthesis of MHI-148 chemically modified bovine serum albumin (BSA-MHI148):

[0032] 40 mg of MHI-148 was dissolved in 4 mL of DMSO (dimethyl sulfoxide) to form solution A, and then the solution A was mixed with 20 mg of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) under light-proof conditions for 1 h, and then the activated MHI-148 was mixed with 2 g of bovine serum albumin and dissolved in 200 mL of double distilled water to obtain mixture A. The obtained mixture A was stirred at room temperature for 6 h. Then it was dialyzed with pure H2O (relative molecular weight = 10 kDa) 5 times to remove free MHI-148, DMTMM and DMSO to obtain pure BSA-MHI148. Finally, BSA-MHI148 powder was prepared by vacuum freeze drying.

[0033] S2. Synthesis of BSA-MHI148@SRF nanoparticles

[0034] 3 mg of sorafenib was dissolved in 250 μL of DMF to form solution B, and then solution B was added dropwise to 50 mL of BSA-MHI148 solution (2 mg / mL, solvent is water). After stirring for 30 seconds, free sorafenib was removed with an ultrafiltration cup (MW = 30 kDa), and the MHI148 chemically modified albumin-bound sorafenib nanoformulation (BSA-MHI148@SRF) was obtained.

[0035] Figure 1Transmission electron microscopy images show the morphology of BSA-MHI148@SRF, which is spherical. The particle size and Zeta potential of BSA-MHI148@SRF nanoparticles are 145±10nm and -13.5±1.1mV, respectively. And within 7 days, there was no obvious change in particle size of BSA-MHI148@SRF nanoparticles in double distilled water, PBS and culture medium, indicating that BSA-MHI148@SRF nanoparticles may have an ideal blood circulation time in vivo.

[0036] Example 2: Co-localization of BSA-MHI148@SRF nanoparticles with mitochondria

[0037] Figure 2 A and B in the table represent the following information respectively:

[0038] Figure A shows the colocalization of BSA-MHI148@SRF nanoparticles and green fluorescent stained mitochondria 4 hours after treatment;

[0039] Panel B is the evaluation of the colocalization of BSA-MHI148@SRF nanoparticles with green-stained mitochondria;

[0040] In summary: Figure 2 This indicates that BSA-MHI148@SRF nanoparticles have mitochondrial targeting.

[0041] Example 3: Effects of SRF, MHI-148, BSA-MHI148, and BSA-MHI148@SRF on the proliferation and apoptosis of MB49 cells. The cells were treated with SRF, MHI-148, BSA-MHI148, and BSA-MHI148@SRF, respectively. Figure 3 A, B, C, D, E, and F represent the following information respectively:

[0042] Figure A: After the cells were incubated under normoxic conditions overnight, MB49 cells were treated with SRF, free MHI148, BSA-MHI148, and BSAMHI148@SRF (MHI148 was 2μM, SRF was 6.58μM) for 6h under hypoxic conditions (1% O2). Subsequently, the cells were irradiated with an 808nm xenon lamp (100mW / cm2, 10min) and incubated under normal conditions for 18h. The cells were then collected and stained with Annexin V-FITC / PI reagent at room temperature for 20min. Finally, the cells were washed three times with cold PBS and analyzed by flow cytometry. The results in Figure A show that compared with other groups, the cell apoptosis rate induced by BSA-MHI148@SRF nanoparticles combined with laser irradiation was the highest.

[0043] Figure B: MB49 cells were inoculated in a confocal culture dish and kept overnight under normoxic conditions (21% O2). The cells were then treated with SRF, free MHI-148, BSA-MHI148 and BSA-MHI148@SRF (MHI148 was 2μM, SRF was 6.58μM) under hypoxic conditions for 6h, and then irradiated with an 808nm laser (100mW / cm2, 10min). Subsequently, the apoptosis detection reagent was added to the culture dish for 30min, and live and dead cells were stained in the dark. Finally, the cells were washed three times and photographed with a laser confocal microscope. The results in Figure B show that BSA-MHI148@SRF nanoparticles produce more PI red fluorescence and less calcein green fluorescence in MB49 cells under laser irradiation, indicating that BSA-MHI148@SRF nanoparticles have the strongest cytotoxic effect on tumor cells.

[0044] Figure CD: The effect of BSA-MHI148@SRF nanoparticles on the expression of Bcl-2 protein, which plays an important role in cell proliferation, to further confirm its anti-tumor activity. The results in Figure CD show that after co-treatment of BSA-MHI148@SRF nanoparticles with laser irradiation, the expression level of Bcl-2 protein was significantly reduced, and the proliferation of tumor cells was significantly inhibited.

[0045] Figure EF: Cells were seeded in a 96-well plate containing culture medium. Subsequently, tumor cells were treated with different concentrations of SRF, free MHI-148, BSA-MHI148 and BSA-MHI148@SRF for 24 h under hypoxia (1% O2). After washing twice with PBS, the cells were irradiated with an 808 nm xenon lamp (100 mW / cm2, 10 min) and incubated for another 24 h. Finally, they were incubated with CCK-8 reagent for 3 h, and the absorbance of CCK-8 was detected.

[0046] The results in Figure E show that the cytotoxic effect of low-dose BSA-MHI148 on MB49 tumor cells without laser irradiation is negligible. In addition, the cytotoxicity of BSA-MHI148@SRF nanoparticles increases in a concentration-dependent manner with laser irradiation, which is due to the cell-killing effect of SRF encapsulated therein.

[0047] Figure F shows that BSA-MHI148@SRF nanoparticles under laser irradiation exhibited more significant cell growth inhibition than the same dose of MHI-148 or BSA-MHI148, which was due to the reversal of tumor hypoxia caused by SRF, thereby promoting ROS production.

[0048] In conclusion: Figure 3The results showed that compared with simple SRF and PDT (simple photodynamic therapy), BSA-MHI148@SRF combined with photodynamic therapy had significantly stronger cell killing ability and reduced the dosage.

[0049] Example 4: Anti-tumor effect of BSA-MHI148@SRF combined with photodynamic therapy in vivo

[0050] 2×106MB49 cells were injected subcutaneously in the right axilla of C57BL / 6 female mice. When the tumor volume reached about 70mm3, the mice were randomly divided into 8 groups (n=5). At the same time, normal saline, BSA, BSA-MHI148, BSA@SRF nanoparticles and BSA-MHI148@SRF nanoparticles (5mg / kg SRF, 2.5mg / kg MHI148) were injected through the tail vein, respectively. After 24h, the tumor sites of some mice were irradiated with 808nm laser (0.5W / cm2, 2min). The weight and tumor volume of mice were measured every 2 days, and the tumor volume of mice was calculated as: volume (mm~3) = 0.5×width 2×length. Finally, the mice were euthanized, and the tumors were removed and weighed.

[0051] Figure 4 The results showed that the BSA-MHI148@SRF nanoparticles combined with laser irradiation group had a good anti-tumor effect, indicating that SRF can enhance the PDT efficacy of MHI148.

[0052] Example 5: Safety evaluation and hemolysis test of BSA-MHI148@SRF nanoparticles in vivo

[0053] Fresh blood from C57BL / 6 mice was taken and mixed with the anticoagulant sodium citrate. The blood was then centrifuged at 2000rpm for 4min to obtain red blood cells. Subsequently, different concentrations of BSA-MHI148@SRF nanoparticles were added to the red blood cell suspension to determine its hemolytic activity. At the same time, physiological saline was added as a negative control and distilled water as a positive control. All samples were placed at 37°C for 2h and then centrifuged to obtain the supernatant. Finally, the ultraviolet absorption of the supernatant at 541nm was recorded, and the corresponding hemolysis rate was calculated.

[0054] After the mice were treated with different methods, they were killed two weeks later and their main organs were taken for hematoxylin-eosin staining. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (CR) were detected using a kit to evaluate the toxic and side effects of the BSA-MHI148@SRF nanoformulation.

[0055] Figure 5The results showed that the BSA-MHI148@SRF nanoformulation had excellent blood compatibility because it did not induce severe hemolysis of red blood cells. In addition, the BSA-MHI148@SRF nanoparticles had no significant effect on the tissue structure of major organs such as the heart, liver, spleen, lungs, and kidneys. Therefore, the BSA-MHI148@SRF nanoparticles have excellent biosafety and have great clinical application value in tumor treatment.

[0056] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing an albumin-bound sorafenib nanoformulation chemically modified with MHI-148, characterized in that: The steps include: S1. Synthesis of MHI-148 chemically modified albumin (BSA-MHI148): MHI-148 was dissolved in DMSO to form a solution A with a concentration of 10 mg / mL, and then the solution A was mixed with 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) under light-proof conditions for 1 h to activate MHI-148, and then the activated MHI-148 was mixed with albumin and dissolved in double distilled water to obtain a mixture A; The obtained mixture A was stirred at room temperature for 6 h, and then dialyzed against pure H2O for 5 times to remove free MHI-148, DMTMM and DMSO to obtain pure BSA-MHI148; S2. Synthesis of BSA-MHI148@SRF nanoparticles: Sorafenib was dissolved in DMF to form a solution B with a concentration of 12 mg / mL, and then solution B was added dropwise to the BSA-MHI148 solution. After stirring for 30 seconds, the free sorafenib was removed using an ultrafiltration cup with a MW of 30 kDa to obtain the MHI148 chemically modified albumin-bound sorafenib nanoformulation (BSA-MHI148@SRF).

2. The method for preparing a MHI-148 chemically modified albumin-bound sorafenib nanoformulation according to claim 1, characterized in that: The sorafenib includes one or more sorafenib derivatives.

3. The method for preparing a MHI-148 chemically modified albumin-bound sorafenib nanoformulation according to claim 2, characterized in that: The albumin is at least one of human serum albumin and bovine serum albumin.

4. The method for preparing a MHI-148 chemically modified albumin-bound sorafenib nanoformulation according to claim 3, characterized in that: The BSA-MHI148 is formed by condensing MHI-148 molecules with bovine serum albumin through DMTMM.

5. The method for preparing a MHI-148 chemically modified albumin-bound sorafenib nanoformulation according to claim 4, characterized in that: The concentration of the BSA-MHI148 solution in step S2 is 2 mg / mL.

6. The method for preparing a MHI-148 chemically modified albumin-bound sorafenib nanoformulation according to claim 5, characterized in that: The sorafenib in step S2 is added to the BSA-MHI148 solution by hanging drop, and the two are self-assembled to form a nanoformulation.

7. The method for preparing a MHI-148 chemically modified albumin-bound sorafenib nanoformulation according to claim 5, characterized in that: The mass ratio of MHI-148 to albumin in step S1 is 1:

50.

8. The method for preparing a MHI-148 chemically modified albumin-bound sorafenib nanoformulation according to claim 5, characterized in that: The pure BSA-MHI148 prepared in step S1 is finally subjected to vacuum freeze drying to prepare BSA-MHI148 powder.

9. The use of the MHI-148 chemically modified albumin-bound sorafenib nanoformulation prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The MHI-148 chemically modified albumin-bound sorafenib nanoformulation (BSA-MHI148@SRF) is used for preparing drugs for anti-tumor treatment.

Images