Nanoparticles embedded with mitochondrial division inhibitor as well as preparation method and application of nanoparticles

Abstract

The invention relates to a nano particle for embedding a mitochondrial division inhibitor as well as a preparation method and application of the nano particle, and belongs to the technical field of nano particles. The technical problems that in the prior art, a mitochondrial division inhibitor is poor in fat solubility and not ideal in bioavailability and cycle half-life period are solved. The nano particle embedded with the mitochondrial division inhibitor is prepared from the mitochondrial division inhibitor, poly (D, L-lactic acid-co-glycollic acid) and sodium hyaluronate which are sequentially coated from inside to outside. The nano particles embedded with the mitochondrial division inhibitor can be applied to preparation of drugs for treating hepatocellular carcinoma. Through test detection, the release amount of the mitochondrial division inhibitor released from the nanoparticles embedded with the mitochondrial division inhibitor is higher than the release amount of the mitochondrial division inhibitor released from the nanoparticles embedded with the mitochondrial division inhibitor in a simulated in-vivo normal environment with the pH value of 7.4 under the condition of a simulated tumor microenvironment with the pH value of 5.0, and the mitochondrial division inhibitor has a stable release characteristic.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoparticles, and specifically relates to a nanoparticle encapsulating a mitochondrial fission inhibitor, a preparation method and an application thereof, and in particular relates to an application of the nanoparticle encapsulating a mitochondrial fission inhibitor in the preparation of a drug for treating hepatocellular carcinoma. Background Art

[0002] The mitochondrial fission inhibitor (Mdivi-1) has high selectivity and cell permeability. It can precisely inhibit the activity of dynamin-related protein 1 (Drp1), thereby affecting the fission process of mitochondria. Its mechanism of action is: Mdivi-1 crosses the cell membrane, inhibits yeast dynamin (Dnm1) and human Drp1 split guanosine triphosphate (GTP) enzyme activating proteins (DRPs), causing mitochondria to fuse into a nest-like structure.

[0003] The chemical structure and properties of Mdivi-1 make it a powerful tool for studying mitochondrial fission, especially in the fields of cell biology and drug development. By regulating the activity of Drp1, Mdivi-1 provides precise control of mitochondrial fission, providing new perspectives for understanding mitochondrial dynamics and disease models.

[0004] However, Mdivi-1 is currently used only as a tool drug, and due to its lipid solubility, its bioavailability and circulation half-life are not ideal. Summary of the invention

[0005] The present invention aims to solve the technical problems in the prior art that Mdivi-1 has poor lipid solubility, unsatisfactory bioavailability and circulation half-life, and provides a nanoparticle encapsulating a mitochondrial fission inhibitor, and a preparation method and application thereof.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows.

[0007] In a first aspect, the present invention provides a nanoparticle encapsulating a mitochondrial fission inhibitor, which is composed of a mitochondrial fission inhibitor (Mdivi-1), poly (D,L-lactic acid-co-glycolic acid) (PLGA) and sodium hyaluronate (HA-T) sequentially encapsulated from the inside to the outside.

[0008] Preferably, the number average molecular weight of the poly (D, L-lactic acid-co-glycolic acid) is 7000-17000 Da.

[0009] Preferably, in the poly(D,L-lactic acid-co-glycolic acid), the molar ratio of lactic acid (LA) to glycolic acid (GA) is 50:50.

[0010] Preferably, the number average molecular weight of the sodium hyaluronate is less than 10 kDa.

[0011] Preferably, the mass ratio of the mitochondrial fission inhibitor, poly (D, L-lactic acid-co-glycolic acid), and sodium hyaluronate is 1:2-5:10-20, and more preferably, 1:3:15.

[0012] In a second aspect, the present invention also provides a method for preparing the above-mentioned nanoparticles encapsulating a mitochondrial fission inhibitor, comprising the following steps: Step 1, stirring and mixing the polyvinyl alcohol (PVA) solution and sodium hyaluronate to obtain an aqueous phase; Step 2, dissolving the mitochondrial fission inhibitor and poly (D, L-lactic acid-co-glycolic acid) in an organic solvent to obtain an oil phase; Step 3, adding the oil phase dropwise to the water phase, and ultrasonically emulsifying under ice bath to obtain an emulsion; Step 4: Stir at room temperature in the dark, let stand to remove excess organic solvent, then centrifuge, wash the resulting precipitate to obtain nanoparticles encapsulating the mitochondrial fission inhibitor.

[0013] Preferably, in step 1, the concentration of the polyvinyl alcohol solution is 1-4 wt %.

[0014] Preferably, in step 1, the concentration of sodium hyaluronate in the aqueous phase is 1 wt %.

[0015] Preferably, in step 1, the stirring speed is 800-1000 rpm.

[0016] Preferably, in step 2, the method of dissolving in the organic solvent is oscillating ultrasound.

[0017] Preferably, in step 2, the organic solvent is one or more of dichloromethane, chloroform and acetone.

[0018] Preferably, in step three, the ultrasonic conditions are: power 100-150W, ultrasonication 3s with intervals of 3s, and a total time of 5-10min.

[0019] Preferably, in step three, the volume ratio of the oil phase to the water phase is 1:4.

[0020] Preferably, in step 4, the stirring speed is 600-800 rpm, and the stirring time is 1-3 h.

[0021] Preferably, in step 4, the standing time is 12 hours.

[0022] Preferably, in step 4, the centrifugation temperature is 4°C, the centrifugation speed is 10000-13000xg, and the centrifugation time is 10-15min.

[0023] Preferably, in step 4, deionized water is used for washing more than three times.

[0024] In a third aspect, the present invention also provides the use of the above-mentioned nanoparticles encapsulating mitochondrial fission inhibitors in the preparation of drugs for treating hepatocellular carcinoma.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The nanoparticles encapsulating the mitochondrial fission inhibitor of the present invention present a core-shell spherical structure, Mdivi-1 is distributed inside the nanoparticles, PLGA serves as an oil phase to constitute the outer shell of the nanoparticles and as a carrier to load Mdivi-1, and HA-T is coated on the outermost layer to achieve targeting of tumor cells.

[0027] The preparation method of the embedded mitochondrial fission inhibitor of the present invention has a high embedding rate of Mdivi-1.

[0028] The nanoparticles encapsulating the mitochondrial fission inhibitor of the present invention can be used in the preparation of drugs for treating hepatocellular carcinoma. According to experimental detection, under the condition of pH=5.0 simulating the tumor microenvironment, the release amount of Mdivi-1 from the nanoparticles encapsulating the mitochondrial fission inhibitor is higher than the release amount under the condition of pH=7.4 simulating the normal environment in the body, and has a stable release characteristic. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 This is a graph showing the expression of mitochondrial quality control signal-related proteins regulated by the mitochondrial fission inhibitor (Mdivi-1) of the present invention. In the graph, mito represents mitochondria, DRP1 represents mitochondrial fission protein, TFAM represents mitochondrial biogenesis protein, BNIP3 represents mitochondrial autophagy receptor, BNIP3L represents mitochondrial autophagy receptor, COXIV represents cytochrome c oxidase V subtype 1, SNU-387 represents human liver cancer cells, Nor represents the normoxic group, Hy represents the hypoxic group, and Hy+Mdivi-1 represents the hypoxia plus mitochondrial fission inhibitor treatment group.

[0031] Figure 2 This is an electron microscope photograph of the nanoparticles encapsulating the mitochondrial fission inhibitor prepared in Example 1 of the present invention.

[0032] Figure 3This is a particle size distribution diagram of the nanoparticles encapsulating the mitochondrial fission inhibitor prepared in Example 1 of the present invention.

[0033] Figure 4 This is an in vitro drug release diagram of the nanoparticles encapsulating the mitochondrial fission inhibitor prepared in Example 1 of the present invention.

[0034] Figure 5 Flow cytometry was used to detect the uptake of nanoparticles containing mitochondrial fission inhibitors prepared in Example 1 of the present invention by Hep1-6 cells. A is a blank control, B is nanoparticles incubated for 1 hour, C is nanoparticles incubated for 2 hours, and D is nanoparticles incubated for 4 hours. 2 Represents the square of the correlation coefficient. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with embodiments.

[0036] Example 1

[0037] Step 1: Add 100 mg of PVA powder to 10,000 mg of deionized water and dissolve at 85-95°C to obtain a 1 wt% PVA solution;

[0038] Step 2: Add 100 mg of sodium hyaluronate (number average molecular weight 8000 Da) powder to 10000 mg of the PVA solution in step 1, and stir at 1000 rpm to dissolve to obtain an aqueous phase (W);

[0039] Step 3: Add 6 mg of Mdivi-1 and 18 mg of PLGA (number average molecular weight 10,000 Da) powder into 2 ml of dichloromethane (DCM), and oscillate and ultrasonicate to fully dissolve them to obtain an oil phase (O);

[0040] Step 4: slowly drop the oil phase (O) into the water phase (W) at a volume ratio of 1:4, and ultrasonically emulsify for 6 min under an ice bath at a power of 150 W and an interval of 3 s for 3 s to obtain an O / W emulsion;

[0041] Step 5. At room temperature, stir the O / W emulsion at 800 rpm for 3 h in the dark, let it stand for 12 h to remove the organic phase in the solution, centrifuge at 4°C and 12,000 x g for 10 min, collect the supernatant, and wash the precipitate three times with deionized water to remove PVA to obtain nanoparticles encapsulating mitochondrial fission inhibitors, recorded as HA@PLGA-Mdivi-1.

[0042] The performance of HA@PLGA-Mdivi-1 prepared in Example 1 was tested.

[0043] 1. Mitochondrial fission inhibitor (Mdivi-1)

[0044] Human liver cancer cells (SNU-387) were cultured under hypoxic conditions, and a mitochondrial fission inhibitor (Mdivi-1) was added. Mdivi-1 was purchased from MCE and is an effective mitochondrial fission / mitochondrial autophagy inhibitor. Mitochondrial proteins of human liver cancer cells were extracted and subjected to protein immunoblotting. Mitochondrial quality control signal-related proteins (DRP1, TFAM, BNIP3, BNIP3L) were detected. Cytochrome c oxidase V subtype 1 (COXIV) was used as a mitochondrial protein internal reference. The detection groups were divided into normoxic group (Nor), hypoxic group (Hy), hypoxia plus mitochondrial fission inhibitor treatment group (Hy+Mdivi-1), and the protein expression levels were shown in Table 1. Figure 1 As shown. Figure 1 It can be seen that under hypoxic conditions, the expression of DRP1, TFAM, BNIP3 and BNIP3L all increased. These changes indicate that hypoxia enhances mitochondrial quality control signals; and mitochondrial fission inhibitors can inhibit this phenomenon under hypoxic conditions, indicating that Mdivi-1 can inhibit mitochondrial quality control signals under hypoxic conditions.

[0045] 2. Transmission electron microscopy

[0046] The HA@PLGA-Mdivi-1 prepared in Example 1 was redispersed in water, and the HA@PLGA-Mdivi-1 was observed using a transmission electron microscope.

[0047] Transmission electron microscopy results Figure 2 As shown, from Figure 2 It can be seen that the nanoparticles encapsulating the mitochondrial fission inhibitor have a spherical structure and are covered with an obvious membrane structure, presenting a complete core-shell structure, and the outer layer HA-T is successfully modified.

[0048] 3. Average particle size and Zeta (electrokinetic) potential

[0049] The prepared HA@PLGA-Mdivi-1 was added dropwise to a nanoparticle size analyzer to measure the average particle size and Zeta potential.

[0050] The test results are as follows Figure 3 and Figure 4 As shown, from Figure 3 It can be seen that the average particle size of HA@PLGA-Mdivi-1 is 159.7±1.3nm. Figure 4 It can be seen that the Zeta potential is -14.3±1.2 mV (pH=7.4) and -8.75±0.19 mV (pH=5.0).

[0051] 4. Embedding rate

[0052] The embedding rate of Mdivi-1 in HA@PLGA-Mdivi-1 was detected by ultraviolet spectrophotometry. The ultraviolet absorbance of the supernatant collected in step 5 of the preparation process was measured at 297 nm, and the content of free Mdivi-1 was calculated.

[0053] The calculation formula for the encapsulation rate of Mdivi-1 in HA@PLGA-Mdivi-1 is: (total amount of Mdivi-1 input during preparation - content of Mdivi-1 in supernatant) / total amount of Mdivi-1 input during preparation × 100%; It was determined that the encapsulation rate of nanoparticles encapsulating mitochondrial fission inhibitors was 95.75%±2.35%.

[0054] 5. In vitro drug release performance

[0055] The prepared HA@PLGA-Mdivi-1 was weighed, 2 ml of PBS buffer (pH 7.4 or pH 5.0) was added, and the solution was transferred to a dialysis bag. The dialysis bag was then placed in PBS buffer (pH 7.4 or pH 5.0) containing 0.5 wt % Tween-80 and oscillated at 120 rpm on a 37 ° C constant temperature shaker; the solution outside the dialysis bag was taken out at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, and 48 h, and an equal volume of PBS buffer (pH 7.4 or pH 5.0) was added at the same time to determine the content of Mdivi-1 in the release medium.

[0056] The drug release amount was calculated using the cumulative content of Mdivi-1 and the release time. The formula for calculating the drug release amount is: ; Wherein, Ve is the volume of sample taken out each time, V0 is the total volume of the release medium, Ci is the concentration of the sample taken out each time, n is the number of times the sample is taken out, Cn is the concentration of the release medium after taking n samples, m Mdivi-1 is the cumulative content of Mdivi-1 in the release medium.

[0057] It was determined that the cumulative release of HA@PLGA-Mdivi-1 in an acidic environment (pH 5.0) was significantly higher than that in a neutral environment (pH 7.4). Figure 4 shown.

[0058] 6. Cellular uptake experiment of HA@PLGA-Mdivi-1

[0059] Replace step 3 in Example 1 with the following: add 6 mg Mdivi-1, 18 mg PLGA (number average molecular weight 10,000 Da) powder and 3 mg Nile Red into 2 ml dichloromethane (DCM), oscillate and ultrasonicate to fully dissolve them to obtain an oil phase (O), and finally obtain HA@PLGA-Mdivi-1 loaded with Nile Red fluorescent dye.

[0060] Hep1-6 cells with good growth status were seeded in 6-well plates at a density of 6×10 5 Each well was incubated at 37°C overnight to allow the cells to adhere to the wall. HA@PLGA-Mdivi-1 loaded with Nile red fluorescent dye was added to each well and incubated for 1h, 2h and 4h. The cells were rinsed with PBS, digested and the cell suspension was collected, centrifuged at 800rpm for 5min, the supernatant was discarded, PBS was added for washing and centrifuged again, and then an appropriate amount of PBS was added to resuspend the cells. The cells were detected by flow cytometry. The results of blank control, nanoparticle incubation for 1h, nanoparticle incubation for 2h, and nanoparticle incubation for 4h were shown as follows: Figure 5 As shown in A, B, C, and D, after Hep1-6 cells were treated with HA@PLGA-Mdivi-1 loaded with Nile red fluorescent dye, the fluorescence intensity in the cells was significantly higher than that in the untreated group, and the fluorescence intensity increased over time. This indicates that HA@PLGA-Mdivi-1 with HA-T coating can be taken up by hepatocellular carcinoma cells and play a role in the cells.

[0061] Obviously, the above embodiments are merely examples for clear explanation, and are not intended to limit the embodiments. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. Nanoparticles encapsulating a mitochondrial fission inhibitor, characterized in that: It is composed of mitochondrial fission inhibitor, poly (D,L-lactic acid-co-glycolic acid) and sodium hyaluronate coated from the inside to the outside.

2. The nanoparticles encapsulating a mitochondrial fission inhibitor according to claim 1, characterized in that: The number average molecular weight of the poly (D, L-lactic acid-co-glycolic acid) is 7000-17000 Da; In the poly (D, L-lactic acid-co-glycolic acid), the molar ratio of lactic acid to glycolic acid is 50:

50.

3. The nanoparticles encapsulating a mitochondrial fission inhibitor according to claim 1, characterized in that: The number average molecular weight of the sodium hyaluronate is less than 10 kDa.

4. The nanoparticles encapsulating a mitochondrial fission inhibitor according to claim 1, characterized in that: The mass ratio of the mitochondrial fission inhibitor, poly (D, L-lactic acid-co-glycolic acid), and sodium hyaluronate is 1:2-5:10-20.

5. The method for preparing nanoparticles encapsulating a mitochondrial fission inhibitor according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1, stirring and mixing the polyvinyl alcohol solution and sodium hyaluronate to obtain an aqueous phase; Step 2, dissolving the mitochondrial fission inhibitor and poly (D, L-lactic acid-co-glycolic acid) in an organic solvent to obtain an oil phase; Step 3, adding the oil phase dropwise to the water phase, and ultrasonically emulsifying under ice bath to obtain an emulsion; Step 4: Stir at room temperature in the dark, let stand to remove excess organic solvent, then centrifuge, wash the resulting precipitate to obtain nanoparticles encapsulating the mitochondrial fission inhibitor.

6. The method for preparing nanoparticles encapsulating a mitochondrial fission inhibitor according to claim 5, characterized in that: In step 1, the concentration of the polyvinyl alcohol solution is 1-4wt%; In the aqueous phase, the concentration of sodium hyaluronate is 1wt%; The stirring speed is 800-1000 rpm.

7. The method for preparing nanoparticles encapsulating a mitochondrial fission inhibitor according to claim 5, characterized in that: In step 2, the dissolving in the organic solvent is carried out by oscillating ultrasound; The organic solvent is one or more of dichloromethane, chloroform and acetone.

8. The method for preparing nanoparticles encapsulating a mitochondrial fission inhibitor according to claim 5, characterized in that: In step 3, the ultrasonic conditions are: power 100-150W, ultrasonic 3s interval 3s, total time 5-10min; The volume ratio of the oil phase to the water phase is 1:

4.

9. The method for preparing nanoparticles encapsulating a mitochondrial fission inhibitor according to claim 5, characterized in that: In step 4, the stirring speed is 600-800 rpm, and the stirring time is 1-3 h; The standing time is 12h; The centrifugal temperature is 4°C, the centrifugal speed is 10000-13000xg, and the centrifugal time is 10-15min; Rinse with deionized water for more than three times.

10. Use of the nanoparticles encapsulating a mitochondrial fission inhibitor according to any one of claims 1 to 4 in the preparation of a drug for treating hepatocellular carcinoma.

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