Preparation method of biocompatible dual-targeting drug delivery dimer nanoparticles

Abstract

The invention discloses a preparation method of biocompatible dual-targeting drug delivery dimer nanoparticles, and belongs to the field of granular materials. The preparation method comprises the following steps: preparing shellac-polylactic acid dimer nano-particles by utilizing a flash nano-precipitation method based on a phase separation principle, and modifying a targeting molecule 1 of a targeting cancer cell membrane on a polylactic acid hemisphere through molecular self-assembly in the process; and then modifying targeting molecules 2 targeting organ mitochondria in cancer cells on the shellac hemispheres through electrostatic interaction to prepare the double-targeting shellac-polylactic acid dimer nanoparticles. Different targeting molecules are respectively modified on the surfaces of two independent hemispheres of the dimer nanoparticles to realize step-by-step targeting delivery, cancer cell membrane targeting can improve the preliminary recognition and combination of the nanoparticles to cancer cells, and the drug can smoothly enter the cells; and the drug is further guided to reach a key subcellular organelle part through mitochondrial targeting, so that a stronger treatment effect is achieved.

Description

Technical Field

[0001] The invention relates to the field of particle materials, and in particular to a method for preparing biocompatible dual-targeted drug delivery dimer nanoparticles. Background Art

[0002] Cancer is one of the most common fatal diseases worldwide. Traditional cancer treatments include surgical resection, radiotherapy, and chemotherapy. However, these methods have some limitations, such as the trauma of surgery, the toxic side effects of radiotherapy, and tumor multidrug resistance (MDR) to chemotherapy.

[0003] Tumor multidrug resistance (MDR) refers to a typical phenomenon in which tumor cells are insensitive to multiple structural / mechanism-independent chemotherapeutic drugs at the same time after continuous exposure to a specific drug. MDR greatly weakens the efficacy of chemotherapy. Mitochondria are bioenergetic and signaling organelles that play an important role in apoptosis, metabolism, and cancer progression. In recent years, studies have found that by directly targeting mitochondria, the energy supply of MDR cells can be blocked, and the apoptosis pathway can be activated by promoting the production of reactive oxygen species (ROS), thereby restoring the sensitivity of resistant cells to anticancer drugs. Therefore, mitochondria have been regarded as potential important targets for the treatment of MDR tumors, and directing chemotherapeutic drugs directly to mitochondria has become an effective strategy to improve the efficacy of chemotherapy. However, to achieve drug targeting to mitochondria, it is necessary to enter the cell first. In tumor treatment, the cell membrane is an important checkpoint for drugs to enter the cell, so ligand modification targeting cancer cell membranes is of great significance. By selectively modifying the ligand on the surface of nanoparticles, the nanoparticles can significantly improve their ability to recognize cancer cells and enhance their binding to cancer cell membranes, thereby improving the internalization efficiency of drugs and creating favorable conditions for subsequent drug delivery.

[0004] In recent years, the rapid development of nanotechnology has made nanoparticle-based drug delivery systems a new approach for anti-cancer treatment. However, existing nano drug carriers are still insufficient in achieving precise targeted delivery. Some nanomaterials only have passive targeting, relying on the enhanced permeability and retention effect (EPR) to accumulate at the tumor site, and cannot achieve active precise targeting. In addition, some nanoparticles achieve active targeting through single ligand modification, but their targeting effect is often not ideal, especially in the face of complex tumor microenvironments.

[0005] Traditional dual-targeting strategies usually modify two different targeting ligands on the surface of a single nanoparticle at the same time in order to achieve dual targeting of different targets. However, this strategy has the following disadvantages:

[0006] Mutual interference between ligands: Due to the differences in chain length, chemical structure or physical properties of the two ligands, when one ligand specifically binds to the target cell, the other ligand cannot fully exert its effect, thus reducing the dual-targeting effect.

[0007] Steric hindrance effect: Simultaneous modification of two ligands on the surface of a single nanoparticle may induce a steric hindrance effect, restricting the free arrangement and rotation of the ligands, thereby affecting their binding efficiency with target cell receptors and resulting in poor drug delivery.

[0008] Limited ligand selection: In order for two ligands to coexist on the same nanoparticle, the chemical compatibility and physical properties of the ligands need to be considered during the design, which limits the range of ligand selection and increases the complexity of design and preparation.

[0009] Complex preparation process: The preparation process of traditional single-sphere dual-targeted nanoparticles is usually complicated, involving multi-step reactions and precise control. The process is cumbersome and time-consuming, further limiting its application potential. Summary of the invention

[0010] To solve the above problems, the present invention proposes a method for preparing biocompatible dual-targeted drug delivery dimer nanoparticles. The present invention uses a flash nanoprecipitation method based on the phase separation principle to prepare lac-polylactic acid dimer nanoparticles. In this process, the targeting molecule 1 targeting the cell membrane of cancer cells is modified on the polylactic acid hemisphere by molecular self-assembly; then the targeting molecule 2 targeting the mitochondria in the organ of the cancer cell is modified on the lac hemisphere by electrostatic interaction to obtain dual-targeted lac-polylactic acid dimer nanoparticles. The method realizes step-by-step targeted delivery to the cancer cell membrane and the mitochondria in the cancer cell by modifying different targeting molecules on the surfaces of the two independent hemispheres of the dimer nanoparticles. The targeted modification of the cancer cell membrane can utilize the modified cell membrane targeting molecule to specifically bind to the overexpressed receptor on the cancer cell membrane, enhance the effect of nanoparticles targeting cancer cells through receptor-mediated endocytosis, significantly improve the initial recognition and binding of nanoparticles to cancer cells, and ensure that the drug can smoothly enter the cell; then, mitochondrial targeting further guides the drug to the key subcellular organelles, showing enhanced accumulation in the mitochondria, and exerting a stronger therapeutic effect.

[0011] To achieve the above object, the present invention provides the following solutions:

[0012] A method for preparing biocompatible dual-targeted drug delivery dimer nanoparticles, wherein the drug delivery dimer nanoparticles are modified shellac-polylactic acid dimer nanoparticles, and the preparation method comprises the following steps:

[0013] (1) adding polylactic acid and polylactic acid modified with a targeting molecule 1 targeting a cell membrane of a cancer cell to an organic solvent containing shellac to obtain an organic phase solution;

[0014] (2) rapidly injecting the organic phase solution prepared in step (1) into heated deionized water, mixing evenly and then standing to obtain a dispersion of shellac-polylactic acid dimer nanoparticles modified with targeting molecule 1 targeting cancer cell membrane in deionized water;

[0015] (3) adding the targeting molecule 2 targeting the mitochondria of the cancer cell to the dispersion prepared in step (2), mixing evenly and then standing, centrifuging and washing to remove the targeting molecule 2 targeting the mitochondria of the cancer cell that has not been adsorbed, so that the lac hemisphere of the nanoparticle is modified with the targeting molecule 2 targeting the mitochondria of the cancer cell, and obtaining a dispersion of dual-targeted drug delivery dimer nanoparticles in deionized water.

[0016] As a preferred embodiment of the present invention, in step (1), the organic solvent is selected from tetrahydrofuran, and the mass range of the shellac added to 1 mL of tetrahydrofuran is 1-50 mg; further preferably, the mass range of the shellac added to 1 mL of tetrahydrofuran is 1-20 mg, and the shellac is a natural wrapping material, which is easily soluble in organic solvents such as ethanol and alkaline solutions such as sodium hydroxide aqueous solution, but is difficult to dissolve in neutral or acidic solutions.

[0017] As a preferred embodiment of the present invention, the mass range of the polylactic acid added in 1 mL of organic solvent is 1-50 mg; further preferred, the mass range of the polylactic acid added in 1 mL of organic solvent is 1-20 mg, and the polylactic acid is a synthetic polymer material that is easily soluble in tetrahydrofuran but poorly soluble in ethanol.

[0018] As a preference of the present invention, the molecular weight range of the polylactic acid is 1000-3000; further preferably, the molecular weight of the polylactic acid is 2000-3000.

[0019] As a preferred embodiment of the present invention, the polylactic acid modified targeting molecule 1 targeting cancer cell membrane includes RGD peptide-polyethylene glycol-polylactic acid, galactose-polyethylene glycol-polylactic acid, hyaluronic acid-polyethylene glycol-polylactic acid, etc., and the molecular weight of polyethylene glycol is 1000-8000; further preferably, the molecular weight of polyethylene glycol is 1000-4000.

[0020] As a preferred embodiment of the present invention, the mass fraction of the polylactic acid modified by the targeting molecule 1 targeting the cell membrane of cancer cells in the total polylactic acid is 1-10%; further preferred, the mass fraction of the polylactic acid modified by the targeting molecule 1 targeting the cell membrane of cancer cells in the total polylactic acid is 2.5-7.5%.

[0021] As a preferred embodiment of the present invention, in step (2), the preparation process needs to be carried out in a water bath (heated deionized water) at 70-90°C, and the generated particles can be stable at room temperature for a long time; further preferably, the water bath temperature is 80°C.

[0022] As a preferred embodiment of the present invention, in step (2), the injection rate of the organic phase solution into the deionized water is 10-40 mL / min; further preferably, the injection rate is 15-25 mL / min.

[0023] As a preferred embodiment of the present invention, in step (2), the volume percentage range of the organic phase solution to deionized water is 1-7%; further preferably, the volume percentage range of the organic phase solution to deionized water is 2-4%.

[0024] As a preferred embodiment of the present invention, in step (2), the time range of standing in the fume hood is 6-24 hours; further preferably, the time range of standing in the fume hood is 8-12 hours.

[0025] As a preferred embodiment of the present invention, the targeting molecules 2 targeting mitochondria in cancer cells include triphenylphosphine TPP, DQA and rhodamine 123.

[0026] As a preferred embodiment of the present invention, the targeting molecule 2 targeting mitochondria in cancer cells is positively charged, and the ratio of the positive charge on the surface of the targeting molecule 2 targeting mitochondria in cancer cells to the negative charge on the surface of the lac hemisphere in the lac-polylactic acid dimer nanoparticles is greater than 1:1.

[0027] As a preferred embodiment of the present invention, the organic phase solution also includes hydrophobic drugs, functional nanomaterials and / or functional polymers. Further preferably, the hydrophobic drugs include curcumin, paclitaxel, docetaxel and camptothecin; the functional nanomaterials include magnetic nanoparticles and catalytic nanoparticles; the functional polymers include temperature-responsive polymers, pH-responsive polymers, enzyme-responsive polymers and light-responsive polymers.

[0028] As a preferred embodiment of the present invention, in step (3), the time of standing still after mixing evenly is 4-12 hours, and more preferably, the time of standing still after mixing evenly is 8-12 hours.

[0029] The beneficial effects of the present invention are as follows:

[0030] (1) The present invention uses biodegradable polylactic acid and shellac as materials, prepares dimer nanoparticles by coprecipitation and phase separation, and modifies tumor cell membrane targeting molecules and tumor cell mitochondria targeting molecules on the surface of the dimer nanoparticles by molecular self-assembly and electrostatic interaction, thereby obtaining biocompatible dual-targeted drug delivery dimer nanoparticles.

[0031] (2) Different from the traditional single-sphere dual-targeting strategy, the dimer structure of the present invention effectively avoids the interference problem between ligands, and at the same time achieves highly coordinated step-by-step targeted delivery through structural separation, so that the drug can reach the key parts of the tumor more accurately and significantly enhance the efficacy of the drug.

[0032] (3) The dimer nanoparticle structure of the present invention is more convenient and efficient in the preparation process, reducing the number of steps and using mild conditions, which not only reduces the production cost but also improves the efficiency of achieving dual-targeting functions. Through this innovative design, the present invention shows significant application prospects and potential advantages in the treatment of multidrug resistance of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0034] Figure 1 Schematic diagram of the method for preparing biocompatible dual-targeted drug delivery dimer nanoparticles;

[0035] Figure 2 Validation of polylactic acid hemispherical targeting molecule 1 modification for biocompatible dual-targeted drug delivery dimer nanoparticles;

[0036] Figure 3 Validation of dimeric nanoparticles modified with lac hemispherical targeting molecules 2 for biocompatible dual-targeted drug delivery. DETAILED DESCRIPTION

[0037] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0038] Example 1: Preparation of biocompatible dual-targeted drug delivery dimer nanoparticles

[0039] See attached Figure 1 The method of the present invention is used to prepare biocompatible dual-targeted drug delivery dimer nanoparticles, and the specific steps are as follows:

[0040] (1) Preparation of organic phase solution

[0041] 10 mg of shellac is dissolved in 1 mL of tetrahydrofuran organic solvent to obtain a 10 mg / mL shellac / tetrahydrofuran solution, and then polylactic acid and hyaluronic acid-polyethylene glycol-polylactic acid are added, the molecular weight of the polyethylene glycol is 2000; the total concentration of polylactic acid is 10 mg / mL, and the mass fraction of hyaluronic acid-polyethylene glycol-polylactic acid in the total polylactic acid is 2.5%.

[0042] (2) Polylactic acid hemisphere modified targeting molecule 1

[0043] Heat deionized water to 80°C, and at this temperature, use a pipette and a 1-200 μL gel spotting tip (inner diameter 200 μm) to absorb 100 μL of the organic phase solution obtained in step (1), and then inject it into a glass bottle containing 3 mL of heated deionized water at a rate of 15 mL / min, shake to mix evenly, and place it in a fume hood to stand for 12 hours to obtain a dispersion of hyaluronic acid-modified shellac-polylactic acid dimer nanoparticles in deionized water.

[0044] (3) Lac hemisphere modified targeting molecule 2

[0045] To the dispersion of hyaluronic acid-modified lac-polylactic acid dimer nanoparticles prepared in step (2) in deionized water, triphenylphosphine TPP is added according to the ratio of the negative charge on the surface of the lac hemisphere of the nanoparticle to the positive charge on the surface of the positively charged triphenylphosphine TPP of 1:2, the mixture is evenly mixed and allowed to stand for 8 hours, and the unadsorbed triphenylphosphine TPP is washed off by centrifugation to obtain a dispersion of triphenylphosphine TPP-modified dual-targeted lac-polylactic acid dimer nanoparticles in deionized water.

[0046] Example 2: Preparation of biocompatible dual-targeted drug delivery dimer nanoparticles loaded with paclitaxel

[0047] See attached Figure 1 The method of the present invention is used to prepare biocompatible dual-targeted drug delivery dimer nanoparticles, and the specific steps are as follows:

[0048] (1) Preparation of organic phase solution

[0049] 10 mg of shellac is dissolved in 1 mL of tetrahydrofuran organic solvent to obtain a 10 mg / mL shellac / tetrahydrofuran solution, and then polylactic acid and hyaluronic acid-polyethylene glycol-polylactic acid are added, the molecular weight of polyethylene glycol is 2000; the total concentration of polylactic acid is 10 mg / mL, the mass fraction of hyaluronic acid-polyethylene glycol-polylactic acid in the total polylactic acid is 2.5%, and finally 1 mg of hydrophobic anticancer drug paclitaxel is added.

[0050] (2) Polylactic acid hemisphere modified targeting molecule 1

[0051] Heat deionized water to 80°C, and at this temperature, use a pipette and a 1-200 μL gel spotting tip (inner diameter 200 μm) to absorb 100 μL of the organic phase solution obtained in step (1), and then inject it into a glass bottle containing 3 mL of heated deionized water at a rate of 15 mL / min, shake to mix evenly, and place it in a fume hood to stand for 12 hours to obtain a dispersion of hyaluronic acid-modified, paclitaxel-loaded shellac-polylactic acid dimer nanoparticles in deionized water.

[0052] (3) Lac hemisphere modified targeting molecule 2

[0053] To the dispersion of hyaluronic acid-modified lac-polylactic acid dimer nanoparticles loaded with paclitaxel prepared in step (2) in deionized water, triphenylphosphine TPP is added according to the ratio of the negative charge on the surface of the lac hemisphere of the nanoparticle to the positive charge on the surface of the positively charged triphenylphosphine TPP of 1:2, the mixture is evenly mixed and allowed to stand for 8 hours, and the unadsorbed triphenylphosphine TPP is washed off by centrifugation to obtain a dispersion of triphenylphosphine TPP-modified dual-targeted lac-polylactic acid dimer nanoparticles loaded with paclitaxel in deionized water.

[0054] Embodiment 3:

[0055] According to the steps of Example 1, under the premise that other conditions remain the same, hyaluronic acid-polyethylene glycol-polylactic acid is replaced by RGD peptide-polyethylene glycol-polylactic acid, the total concentration of polylactic acid is 20 mg / mL, and the mass fraction of RGD peptide-polyethylene glycol-polylactic acid in the total polylactic acid is 5%; and triphenylphosphine TPP is replaced by rhodamine 123.

[0056] Embodiment 4:

[0057] According to the steps of Example 1, under the premise that other conditions remain the same, hyaluronic acid-polyethylene glycol-polylactic acid is replaced by galactose-polyethylene glycol-polylactic acid, the total concentration of polylactic acid is 35 mg / mL, and the mass fraction of galactose-polyethylene glycol-polylactic acid in the total polylactic acid is 7.5%; and triphenylphosphine TPP is replaced by DQA.

[0058] Embodiment 5:

[0059] According to the steps of Example 2, under the premise that other conditions are the same, 1 mg of hydrophobic anticancer drug paclitaxel is replaced by curcumin.

[0060] Example 6: Biocompatible dual-targeted drug delivery dimer nanoparticles polylactic acid hemispheres modified with targeting molecule 1 targeting cell membrane

[0061] According to the steps of Example 1, under the premise of other conditions being the same, the concentration of shellac and the concentration of polylactic acid were kept the same, and shellac-polylactic acid dimer nanoparticles with and without modification of hyaluronic acid were prepared respectively. The changes in the content of elements in the polylactic acid hemisphere before and after modification were analyzed by EDS spectrum, as shown in FIG. Figure 2 As shown, it was found that the oxygen content of the polylactic acid hemisphere increased, verifying that the hyaluronic acid modification was successful.

[0062] Example 7: Validation of biocompatible dual-targeted drug delivery dimer nanoparticles modified with shellac hemispheres targeting intracellular organelles such as mitochondria and targeting molecule 2

[0063] According to the steps of Example 1, under the premise of keeping other conditions the same, the concentration of shellac and the concentration of polylactic acid were kept the same, and the shellac-polylactic acid dimer nanoparticles modified with and without triphenylphosphine TPP were prepared respectively. The changes in the particle potential before and after modification were detected by DLS, as shown in FIG. Figure 3 As shown, it was found that the absolute value of the negative potential became smaller after modification, indicating that a part of the negative charge was used for electrostatic interaction to bind triphenylphosphine TPP, verifying that the modification of triphenylphosphine TPP was successful.

Claims

1. A method for preparing biocompatible dual-targeted drug delivery dimer nanoparticles, characterized in that: The drug delivery dimer nanoparticles are modified shellac-polylactic acid dimer nanoparticles, and the preparation method comprises the following steps: (1) adding polylactic acid and polylactic acid modified with a targeting molecule 1 targeting a cell membrane of cancer cells to an organic solvent containing shellac to obtain an organic phase solution; the total concentration of the polylactic acid is 1-50 mg / mL, and the mass fraction of the polylactic acid modified with a targeting molecule 1 targeting a cell membrane of cancer cells in the total polylactic acid is 1-10%; (2) rapidly injecting the organic phase solution prepared in step (1) into deionized water at 70-90° C. at a rate of 10-40 mL / min, wherein the volume percentage of the organic phase solution to the deionized water is 1-7%; mixing well and then standing to obtain a dispersion of shellac-polylactic acid dimer nanoparticles modified with targeting molecule 1 targeting cancer cell membrane in deionized water; (3) adding the targeting molecule 2 targeting the mitochondria of the cancer cell to the dispersion prepared in step (2), mixing evenly and then standing, centrifuging and washing, removing the supernatant, so that the lac hemisphere of the nanoparticles is modified with the targeting molecule 2 targeting the mitochondria of the cancer cell, and obtaining a dispersion of dual-targeted drug delivery dimer nanoparticles in deionized water.

2. The method for preparing a biocompatible dual-targeted drug delivery dimer nanoparticle according to claim 1, characterized in that: The molecular weight of the polylactic acid is 1000-3000.

3. The method for preparing a biocompatible dual-targeted drug delivery dimer nanoparticle according to claim 1, characterized in that: The polylactic acid that modifies the targeting molecule 1 that targets the cell membrane of cancer cells is selected from any one of RGD peptide-polyethylene glycol-polylactic acid, galactose-polyethylene glycol-polylactic acid, and hyaluronic acid-polyethylene glycol-polylactic acid.

4. The method for preparing a biocompatible dual-targeted drug delivery dimer nanoparticle according to claim 2, characterized in that: The molecular weight of the polyethylene glycol is 1000-8000.

5. The method for preparing a biocompatible dual-targeted drug delivery dimer nanoparticle according to claim 1, characterized in that: The targeting molecule 2 targeting mitochondria in cancer cells is selected from any one of triphenylphosphine TPP, DQA and rhodamine 123.

6. The method for preparing a biocompatible dual-targeted drug delivery dimer nanoparticle according to claim 1, characterized in that: The targeting molecule 2 targeting mitochondria in cancer cells is positively charged, and the ratio of the positive charge on the surface of the targeting molecule 2 targeting mitochondria in cancer cells to the negative charge on the surface of the lac hemisphere in the lac-polylactic acid dimer nanoparticles is greater than 1:

1.

7. The method for preparing a biocompatible dual-targeted drug delivery dimer nanoparticle according to claim 1, characterized in that: The organic phase solution also includes hydrophobic drugs, functional nanomaterials and / or functional polymers.

8. The method for preparing a biocompatible dual-targeted drug delivery dimer nanoparticle according to claim 7, characterized in that: The hydrophobic drugs include curcumin, paclitaxel, docetaxel and camptothecin; the functional nanomaterials include magnetic nanoparticles and catalytic nanoparticles; the functional polymers include temperature-responsive polymers, pH-responsive polymers, enzyme-responsive polymers and light-responsive polymers.

9. The method for preparing a biocompatible dual-targeted drug delivery dimer nanoparticle according to claim 1, characterized in that: The concentration of shellac in the shellac-containing organic solvent is 1-50 mg / mL.

10. Use of the dual-targeted drug delivery dimer nanoparticles prepared according to claim 1 in tumor treatment.

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