Hydrophilic core-shell nanoparticles entrapped with TPP-DOX as well as preparation method and application of hydrophilic core-shell nanoparticles entrapped with TPP-DOX

Abstract

The invention belongs to the technical field of biological hospitals, and relates to a TPP-DOX entrapped hydrophilic core-shell nano-particle as well as a preparation method and application thereof. The nano-particle is formed by covalently binding (3-carboxypropyl) triphenylphosphonium bromide with amino of DOX through carboxyl to form a TPP-DOX compound and entrapping the TPP-DOX compound in an amphipathic multifunctional heparin compound nano-particle inner core through an ultrasonic emulsification method. According to the nanoparticles provided by the invention, the ES2 is used for inhibiting angiogenesis, the TOS is used for enhancing the stability and the synergistic anti-tumor effect, the GA is used for opening mitochondrial permeable pores, and the TPP-mediated mitochondrial accumulation and pH response drug release characteristics are combined, so that the anti-tumor effect of the adriamycin is remarkably enhanced, the toxicity is reduced, and the nanoparticles have important application value in solid tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a hydrophilic core-shell nanoparticle loaded with TPP-DOX, a preparation method thereof, and an application thereof. Background Art

[0002] Doxorubicin (DOX), as a broad-spectrum anti-tumor drug, its clinical application is limited by cardiotoxicity, systemic side effects, and tumor multi-drug resistance (MDR). Although traditional nanocarriers (such as liposomes, polymer micelles) can partially improve the drug delivery efficiency, there are still problems such as insufficient targeting, poor drug-loading stability, and difficulty in reversing drug resistance.

[0003] Heparin is a natural sulfated polysaccharide with excellent biocompatibility and anti-tumor metastasis activity. However, when directly used as a drug, it has a risk of bleeding, and when used alone as a nano-drug carrier, it is easily cleared quickly. At the same time, it lacks tumor targeting function. Studies have shown that chemically modifying heparin can endow it with anti-angiogenic, long-circulating, and targeting functions. For example, non-anticoagulant heparin (GSHP) prepared by destroying the anticoagulant structure combined with phenylboronic acid (PBA) can enhance tumor targeting, while linking D-α-tocopherol succinate (TOS) can improve liposolubility and the stability of nanoparticles. It has been found that glycyrrhetinic acid (GA), as a natural triterpenoid compound, has unique dual values in the fields of anti-tumor therapy and nano-drug carriers. The nano-carrier co-modified with GA and triphenylphosphine (TPP) can deliver drugs to mitochondria directionally, accelerate mitochondria-dependent apoptosis, and in addition, it can also synergistically act with anti-tumor drugs (such as doxorubicin, paclitaxel) to achieve a dual anti-tumor effect. In addition, the anti-angiogenic peptide ES2 (IVRRADRAAVP) can synergistically play an anti-tumor metastasis role with chemotherapy drugs by inhibiting the formation of new blood vessels.

[0004] Triphenylphosphine (TPP), as a mitochondrial targeting molecule, can increase the accumulation of drugs in mitochondria and reverse drug resistance. However, there is no report in the prior art on integrating non-anticoagulant heparin GSHP, ES2 peptide, TOS, GA, and doxorubicin modified with TPP loading into a multifunctional nano-delivery system. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a multifunctional heparin-based nanoparticle, which realizes the efficient delivery of doxorubicin, reverses drug resistance, and reduces side effects through ES2 inhibiting the formation of new tumor blood vessels, TOS improving liposolubility and enhancing the stability of nanoparticles, GA opening the mitochondrial permeability pore and playing a synergistic anti-tumor role, and mitochondrial targeting mediated by TPP.

[0006] To achieve the above object, the technical solution provided by the present invention is: a hydrophilic core-shell type nanoparticle loaded with TPP-DOX, which is formed by covalently binding (3-carboxypropyl) triphenylphosphonium bromide to the amino group of DOX through a carboxyl group to form a TPP-DOX complex, and encapsulating it in the core of an amphiphilic multifunctional heparin complex nanoparticle by ultrasonic emulsification method; wherein, the amphiphilic multifunctional heparin complex nanoparticle is formed by sequentially connecting D-α-tocopherol succinate and glycyrrhetinic acid to a hydrophilic heparin derivative through an ester bond and self-assembling into a nanoparticle in an aqueous solution.

[0007] Preferably, the hydrophilic heparin derivative uses chemically modified anticoagulant-inactive heparin as the basic skeleton and sequentially connects an anti-angiogenic peptide through cystamine as a linker by an amide bond.

[0008] Preferably, the drug loading amount of DOX in the hydrophilic core-shell type nanoparticle loaded with TPP-DOX is 3-6%, and the encapsulation efficiency is 50-70%.

[0009] The present invention also provides a preparation method of the hydrophilic core-shell type nanoparticle loaded with TPP-DOX. The TPP-DOX complex and the amphiphilic multifunctional heparin complex nanoparticle are respectively dissolved in methanol and double-distilled water, and after the two are mixed evenly, they are stirred at room temperature and obtained after probe sonication.

[0010] Preferably, the preparation method of the amphiphilic multifunctional heparin complex nanoparticle includes: selecting an EDCI / NHS / DMAP reaction system, sequentially connecting a hydrophilic heparin derivative with D-α-tocopherol succinate and glycyrrhetinic acid through a pH-sensitive ester bond, and self-assembling the obtained amphiphilic conjugate into a nanoparticle in an aqueous solution.

[0011] Preferably, the preparation method of the hydrophilic heparin derivative includes the following steps: heparin is oxidized by sodium periodate and reduced by sodium borohydride in an alkaline environment to obtain low-molecular-weight non-anticoagulant heparin; the carboxyl group of the low-molecular-weight non-anticoagulant heparin is activated in an EDC / NHS system, and an anti-angiogenic peptide is connected through an amide reaction using cystamine as a linker.

[0012] The present invention further provides the use of the hydrophilic core-shell type nanoparticle loaded with TPP-DOX. The nanoparticle is used for preparing an anti-tumor drug; the anti-tumor drug is a tumor cell activity inhibitor or a tumor cell proliferation inhibitor.

[0013] The present invention also provides an anti-tumor drug, the drug contains an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is the hydrophilic core-shell type nanoparticle loaded with TPP-DOX.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The GGET(TD) nanoparticles provided by the present invention can achieve a multi-mechanism synergistic anti-tumor effect. On the one hand, the heparin-linked anti-angiogenic peptide ES2 specifically inhibits the VEGF signaling pathway, blocks the formation of new tumor blood vessels, and forms a dual anti-tumor effect of "starving the tumor + direct killing" with the cytotoxic effect of doxorubicin, significantly enhancing the anti-tumor effect; on the other hand, glycyrrhetinic acid (GA) and TPP synergistically target tumor mitochondria, bypass the cytoplasmic drug resistance mechanism, directly trigger the mitochondrial apoptosis pathway and then induce tumor cell apoptosis, which can not only improve the targeting effect but also reverse the multi-drug resistance of doxorubicin.

[0015] The GGET(TD) nanoparticles provided by the present invention can reduce toxicity through intelligent response release, and at the same time significantly improve stability and biocompatibility through carrier functionalization design. Under the action of the tumor microenvironment, the pH-sensitive ester bond and GSH-reducing-sensitive disulfide bond of the nanoparticles are broken, releasing doxorubicin, achieving precise controlled release and reducing systemic exposure. Compared with free doxorubicin, it has a more excellent anti-tumor effect, stronger hydrophilicity, smaller toxic and side effects, and shows better use effects and application values in the anti-tumor field. Brief Description of the Drawings

[0016] Figure 1 Particle size of the hydrophilic core-shell nanoparticles encapsulating TPP-DOX prepared in Example 3 of the present invention; Figure 2 Morphology of the hydrophilic core-shell nanoparticles encapsulating TPP-DOX prepared in Example 3 of the present invention; Figure 3 Drug release curve of the hydrophilic core-shell nanoparticles encapsulating TPP-DOX prepared in Example 3 of the present invention; Figure 4 Inhibitory rate of the drug of the hydrophilic core-shell nanoparticles encapsulating TPP-DOX prepared in Example 3 of the present invention on B16F10 cells; Figure 5 Tumor mitochondrial targeting verification of the hydrophilic core-shell nanoparticles encapsulating TPP-DOX prepared in Example 3 of the present invention; Figure 6 Effect of the hydrophilic core-shell nanoparticles encapsulating TPP-DOX prepared in Example 3 of the present invention on tumor cell mitochondria; Figure 7 Effect of the hydrophilic core-shell nanoparticles encapsulating TPP-DOX prepared in Example 3 of the present invention on the migration of B16F10 cells. Detailed Description of the Invention

[0017] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be elaborated in detail below in conjunction with specific embodiments. It should be understood that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0018] The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can all be obtained through commercial channels. [[ID=..]]

[0019] To reduce the toxic and side effects of doxorubicin, improve its multidrug resistance, and enhance its tumor targeting ability, the present invention provides a class of hydrophilic core-shell nanoparticles drugs with heparin as the basic carrier and loaded with triphenylphosphine-modified doxorubicin.

[0020] Example 1: Preparation of hydrophilic heparin derivative GSHP-Cys-ES2 (GE) The preparation method is as follows: (1) Weigh GSHP, EDCI, and NHS according to a mass ratio of 1:2:4 and dissolve them in double-distilled water. After stirring evenly, activate for 30 min. Adjust the pH of the reaction solution to 7.41, add Cys, stir and react for 8 h, and then dialyze for 48 h. Collect the reaction solution and purify it by dialysis to obtain the GSHP-Cys conjugate.

[0021] (2) Weigh ES2, EDCI, and NHS according to a mass ratio of 1:3:2 and dissolve them in double-distilled water, stir and mix, and activate for 45 min. Weigh the GSHP-Cys and dissolve it in double-distilled water, slowly add it to the ES2 activation solution, mix well, and react at room temperature for 24 h. After the reaction is completed, dialyze with double-distilled water for 48 h, collect the reaction solution, and purify it by dialysis to obtain the GSHP-Cys-ES2 (GE) conjugate.

[0022] The structure of the hydrophilic heparin derivative (GE) is as follows: , denoted as Compound 1.

[0023] Example 2: Preparation of amphiphilic multifunctional heparin complex GA-GSHP-ES2-TOS (GGET) The preparation scheme is as follows: (1) Weigh TOS, EDCI, NHS, and DMAP according to a mass ratio of 4:3:2:1 and dissolve them in DMF, stir and activate for 3 h. After the activation is completed, add the GE formamide solution while stirring and continue to stir for 12 h. Collect the reaction solution and purify it by dialysis to obtain the GSHP-ES2-TOS (GET) conjugate.

[0024] (2) Weigh GA, EDCI, NHS, and DMAP according to the mass ratio of 3:5:3:1, dissolve them using DMF, stir and activate for 3 h. After the activation is completed, add the GET formamide solution while stirring and continue stirring for 12 h. Collect the reaction solution, purify it by dialysis to obtain the GA-GSHP-ES2-TOS (GGET) conjugate.

[0025] The structure of the amphiphilic multifunctional heparin complex (GGET) is as follows: , denoted as Compound 2.

[0026] Example 3: Preparation of TPP-DOX compound and encapsulation in nanoparticles The preparation scheme is as follows: (1) Weigh TPP, NHS, and EDCI according to the mass ratio of 9:5:8, dissolve them in DCM, and stir at room temperature for 40 min. Dissolve DOX in a small amount of DCM, add triethylamine, and stir for a period of time. Slowly add the solution containing DOX to the activated TPP solution, stir at room temperature for 24 h, and finally separate and purify the target product by thin-layer chromatography. (2) Dissolve GGET and TD in a double-distilled water / methanol mixed solution according to the mass ratio of 5:1, mix them using a magnetic stirrer for 2 h. Then, subject the mixed solution to probe sonication (100 W, 2 s, 4 s) and dialysis purification, filter through a membrane, and lyophilize to obtain GGET(TD) nanoparticles.

[0027] Example 4: Particle size determination and morphological observation of GGET(TD) nanoparticles The experimental method is as follows: (1) Dissolve the nanoparticles in double-distilled water to prepare a solution with a concentration of 3 - 5 mg / mL, and measure the particle size using a dynamic light scattering instrument.

[0028] (2) Dissolve the nanoparticles in an aqueous solution, take an appropriate amount of the solution, drop it on a copper grid and stain it, and observe using a transmission electron microscope.

[0029] GGET(TD) exists in the form of a nano-spherical structure in an aqueous solution, with a particle size (diameter) of 160 - 170 nm and good dispersibility, as shown in Figure 1 and Figure 2 shown.

[0030] Example 5: Release of chemotherapeutic drugs from GGET(TD) in an acidic environment The experimental method is as follows: Dissolve GGET(TD) in double-distilled water at a concentration of 1 mg / mL and place it in phosphate buffer solutions with different pH values. Collect the release solutions at specific time points and detect the content of DOX in them, and calculate the cumulative drug release amount.

[0031] GGET(TD) has good acid-sensitive properties and can respondently release DOX in an acidic environment, as Figure 3 shown.

[0032] Example 6: Cytotoxicity of GGET(TD) against B16F10 The experimental method is as follows: Collect B16F10 cells in the logarithmic growth phase, then adjust to an appropriate concentration, and inoculate them on a 96-well plate at a number of 1×10 4 per well, and culture them overnight in a cell incubator. Add different drug groups to the cell culture plate and incubate for 48 h, and add CCK-8 to detect the inhibition rate of GGET(TD) against B16F10 cells.

[0033] As Figure 4 shown, compared with the control group, GGET(TD) can effectively inhibit the proliferation of B16F10 cells.

[0034] Example 7: Mitochondrial targeting of GGET(TD) The experimental method is as follows: Collect B16F10 cells in the logarithmic growth phase, then adjust to an appropriate concentration, and inoculate them in a confocal dish at a number of 5×10 4 per well, and culture them overnight in a cell incubator. Add different drug groups to treat the cells for 8 h. Then, stain the cells with a mitochondrial fluorescent probe and Hoechst staining solution, and observe the fluorescence inside the cells using a laser confocal microscope.

[0035] As Figure 5 shown, compared with the fluorescence signals after incubating with DOX and TPP-DOX alone, the fluorescence signal of doxorubicin in the GGET(TD) group highly overlaps with the mitochondrial labeling signal, indicating that DOX significantly enters the mitochondria. The experimental results show that GGET(TD) has good tumor mitochondrial targeting. Example 8: Changes in mitochondrial membrane potential after incubation with GGET(TD) The experimental method is as follows: Collect B16F10 cells in the logarithmic growth phase, then adjust to an appropriate concentration, and inoculate them on a 12-well plate at a number of 5×10 4 per well, and culture them overnight in a cell incubator. Add different drug groups to treat the cells for 8 h. Then, stain the cells with JC-1 staining working solution, and observe the fluorescence inside the cells using an inverted fluorescence microscope.

[0036] As Figure 6 shown, compared with the control group, the red fluorescence in the GGET(TD) group was significantly reduced, while the green fluorescence was significantly enhanced, indicating that GGET(TD) could effectively reduce the mitochondrial membrane potential and thus induce apoptosis of tumor cells.

[0037] Example 9: Effect of GGET(TD) on the migration of B16F10 cells The experimental method was as follows: B16F10 cells in the logarithmic growth phase were collected, then adjusted to an appropriate concentration, and inoculated on a 12-well plate at a density of 1×10 5 cells per well and cultured overnight in a cell incubator. A 200 μL pipette tip was used to scratch each well of the 6-well plate. The culture medium was discarded, and the cells were washed with PBS buffer. The scratch area was photographed with an inverted microscope. Different drug groups were added to the cell culture plate and incubated for 48 h. The change in the scratch width was observed again with an inverted microscope and photographed, and the cell migration rate was calculated.

[0038] As Figure 7 shown, compared with the control group, after incubation with different drug groups for 48 h, the GGET(TD) group could significantly inhibit the migration of B16F10 cells.

[0039] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A hydrophilic core-shell nanoparticle loaded with TPP-DOX, characterized in that: The nanoparticle is formed by covalently binding (3-carboxypropyl) triphenylphosphonium bromide to the amino group of DOX through a carboxyl group to form a TPP-DOX complex, and encapsulating it in the core of an amphiphilic multifunctional heparin complex nanoparticle by ultrasonic emulsification; wherein, the amphiphilic multifunctional heparin complex nanoparticle is formed by sequentially connecting D-α-tocopherol succinate and glycyrrhetinic acid to a hydrophilic heparin derivative through ester bonds, and self-assembling into nanoparticles in an aqueous solution.

2. The hydrophilic core-shell nanoparticles encapsulating TPP-DOX according to claim 1, wherein: The hydrophilic heparin derivative uses chemically modified anticoagulant-inactive heparin as the basic backbone, and sequentially connects the anti-angiogenic peptide through cystamine as a linker by an amide bond.

3. The hydrophilic core-shell nanoparticles encapsulating TPP-DOX according to claim 1, characterized in that: The drug loading of DOX in the hydrophilic core-shell nanoparticle encapsulating TPP-DOX is 3-6%, and the encapsulation efficiency is 50-70%.

4. The preparation method of the hydrophilic core-shell nanoparticles encapsulating TPP-DOX according to any one of claims 1-3, characterized in that: The TPP-DOX complex and the amphiphilic multifunctional heparin complex nanoparticle are respectively dissolved in methanol and double-distilled water, and after mixing them evenly, stirring is carried out at room temperature, and then it is obtained after probe sonication.

5. The preparation method according to claim 4, characterized in that: The preparation method of the amphiphilic multifunctional heparin complex nanoparticle includes: selecting an EDCI / NHS / DMAP reaction system, and sequentially connecting the hydrophilic heparin derivative to D-α-tocopherol succinate and glycyrrhetinic acid through pH-sensitive ester bonds, and the obtained amphiphilic conjugate self-assembles into nanoparticles in an aqueous solution.

6. The preparation method according to claim 5, wherein: The preparation method of the hydrophilic heparin derivative includes the following steps: heparin is oxidized by sodium periodate and reduced by sodium borohydride in an alkaline environment to obtain low-molecular-weight non-anticoagulant heparin; the carboxyl group of the low-molecular-weight non-anticoagulant heparin is activated in an EDC / NHS system, and cystamine is used as a linker to connect the anti-angiogenic peptide through an amide reaction.

7. Use of the hydrophilic core-shell nanoparticles encapsulating TPP-DOX according to any one of claims 1-3, characterized in that, The nanoparticle is used for preparing an anti-tumor drug; the anti-tumor drug is a tumor cell activity inhibitor or a tumor cell proliferation inhibitor.

8. An anti-tumor drug, characterized in that, The drug contains an active ingredient and a pharmaceutically acceptable carrier, and is characterized in that: the active ingredient is the hydrophilic core-shell nanoparticle encapsulating TPP-DOX according to any one of claims 1-3.

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