A method for preparing biocompatible dual-targeted drug delivery dimer nanoparticles

Abstract

The present invention discloses a method for preparing biocompatible dual-targeted drug delivery dimer nanoparticles, which belongs to the field of granular materials. The present invention uses a flash nanoprecipitation method based on the phase separation principle to prepare lac-polylactic acid dimer nanoparticles. In this process, a targeting molecule 1 targeting the cell membrane of cancer cells is modified on the polylactic acid hemisphere by molecular self-assembly; then a targeting molecule 2 targeting the mitochondria, an organ inside the cancer cell, is modified on the lac hemisphere by electrostatic interaction to prepare dual-targeted lac-polylactic acid dimer nanoparticles. The present invention realizes step-by-step targeted delivery by respectively modifying different targeting molecules on the surfaces of the two independent hemispheres of the dimer nanoparticles, wherein cancer cell membrane targeting can enhance the initial recognition and binding of nanoparticles to cancer cells, ensuring that the drug can smoothly enter the interior of the cell; mitochondrial targeting further guides the drug to the key subcellular organelles, exerting a stronger therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the field of granular 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 invasiveness of surgery, the toxic side effects of radiotherapy, and the multidrug resistance (MDR) of tumors to chemotherapy.

[0003] Multidrug resistance (MDR) in tumors refers to a phenomenon in which tumor cells, after continuous exposure to a specific drug, become simultaneously insensitive to multiple structurally / mechanistically unrelated chemotherapeutic agents. MDR significantly impairs the efficacy of chemotherapy. Mitochondria are bioenergetics and signaling organelles that play a crucial role in apoptosis, metabolism, and cancer progression. Recent studies have shown that directly targeting mitochondria can block the energy supply of MDR cells and activate apoptotic pathways by promoting the production of reactive oxygen species (ROS), thereby restoring the sensitivity of resistant cells to anticancer drugs. Therefore, mitochondria have been identified as a potential important target for the treatment of MDR tumors, and directing chemotherapeutic drugs to mitochondria has become an effective strategy to enhance chemotherapy efficacy. However, achieving drug targeting to mitochondria requires entry into the cell. In cancer therapy, the cell membrane is a critical barrier for drug entry. Therefore, ligand modification targeting cancer cell membranes is of great significance. By selectively modifying the surface of nanoparticles with ligands, the nanoparticle's recognition of cancer cells can be significantly enhanced, enhancing its binding to the cancer cell membrane and improving drug internalization efficiency, 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 still have shortcomings 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 are unable to achieve active and precise targeting. In addition, some nanoparticles achieve active targeting through single ligand modification, but their targeting effect is often less than ideal, especially when faced with the complex tumor microenvironment.

[0005] Traditional dual-targeting strategies typically involve simultaneously modifying two different targeting ligands on the surface of a single nanoparticle 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, thereby reducing the dual-targeting effect.

[0007] Steric hindrance effect: Simultaneously modifying 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 to allow 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 address the aforementioned issues, the present invention proposes a method for preparing biocompatible dual-targeted drug delivery dimeric nanoparticles. Based on the principle of phase separation, the present invention utilizes a flash nanoprecipitation method to prepare shellac-polylactic acid (PLA) dimer nanoparticles. During this process, targeting molecules (1) targeting cancer cell membranes are modified onto the PLA hemispheres through molecular self-assembly. Subsequently, targeting molecules (2) targeting mitochondria, an organ within cancer cells, are modified onto the shellac hemispheres through electrostatic interactions, resulting in dual-targeted shellac-PLA dimer nanoparticles. This method achieves sequential targeted delivery to both cancer cell membranes and mitochondria within cancer cells by modifying the two independent hemispheres with different targeting molecules. Targeted modification of the cancer cell membrane allows the modified cell membrane targeting molecules to specifically bind to overexpressed receptors on the cancer cell membrane, enhancing the nanoparticle's targeting efficacy to cancer cells through receptor-mediated endocytosis. This significantly improves the nanoparticle's initial recognition and binding to cancer cells, ensuring smooth drug entry into the cell. Subsequently, mitochondrial targeting further directs the drug to key subcellular organelles, resulting in enhanced accumulation within mitochondria and a stronger therapeutic effect.

[0011] To achieve the above objectives, 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 that targets 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 well and then allowing to stand to obtain a dispersion of shellac-polylactic acid dimer nanoparticles modified with targeting molecule 1 targeting cancer cell membranes in deionized water;

[0015] (3) adding the targeting molecule 2 targeting the mitochondria of the cancer cell organ 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 organ 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 organ, 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. The shellac is a natural wrapping material that 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 preference of the present invention, the mass range of the polylactic acid added in 1 mL of organic solvent is 1-50 mg; further preferably, the mass range of the polylactic acid added in 1 mL of organic solvent is 1-20 mg. 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 that modifies the targeting molecule 1 that targets the cell membrane of cancer cells 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 preference 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 preferably, 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 preference 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 preference 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 preference of the present invention, in step (2), the volume percentage of the organic phase solution to deionized water is in the range of 1-7%; further preferably, the volume percentage of the organic phase solution to deionized water is in the range of 2-4%.

[0024] As a preference of the present invention, in step (2), the time range of standing still in the fume hood is 6-24 hours; further preferably, the time range of standing still 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 further comprises a hydrophobic drug, a functional nanomaterial, and / or a functional polymer. Further preferably, the hydrophobic drug comprises curcumin, paclitaxel, docetaxel, and camptothecin; the functional nanomaterial comprises magnetic nanoparticles and catalytic nanoparticles; and the functional polymer comprises a temperature-responsive polymer, a pH-responsive polymer, an enzyme-responsive polymer, and a light-responsive polymer.

[0028] As a preference of the present invention, in step (3), the time of standing still after mixing evenly is 4-12 hours, and further preferably, the time of standing still after mixing evenly is in the range of 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 to prepare dimer nanoparticles by coprecipitation and phase separation. At the same time, tumor cell membrane targeting molecules and tumor cell mitochondria targeting molecules are modified on the surface of the dimer nanoparticles through molecular self-assembly and electrostatic interaction to obtain 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 dimeric nanoparticle structure of the present invention is simpler and more efficient during preparation, with fewer steps and milder conditions, which not only reduces production costs but also improves the efficiency of achieving dual-targeting functions. Through this innovative design, the present invention has shown significant application prospects and potential advantages in the treatment of multidrug resistance in tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below with reference to the accompanying drawings and examples.

[0034] Figure 1 Schematic diagram of the method for preparing biocompatible dual-targeted drug delivery dimeric 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 for biocompatible dual-targeted drug delivery. DETAILED DESCRIPTION

[0037] The present invention will be further described and illustrated below in conjunction with specific embodiments. The embodiments are merely illustrative of the present disclosure and do not limit its scope. The technical features of the various embodiments of the present invention may be combined accordingly, provided that there is no conflict between them.

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

[0039] Refer to the 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 was dissolved in 1 mL of tetrahydrofuran organic solvent to obtain a 10 mg / mL shellac / tetrahydrofuran solution, and polylactic acid and hyaluronic acid-polyethylene glycol-polylactic acid were then added, where the molecular weight of the polyethylene glycol was 2000; the total concentration of polylactic acid was 10 mg / mL, and the mass fraction of the hyaluronic acid-polyethylene glycol-polylactic acid in the total polylactic acid was 2.5%.

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

[0043] Heat deionized water to 80°C. At this temperature, use a pipette and a 1-200 μL gel spotting tip (inner diameter 200 μm) to draw 100 μL of the organic phase solution obtained in step (1). 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] The dispersion of the hyaluronic acid-modified lac-polylactic acid dimer nanoparticles prepared in step (2) in deionized water is added with triphenylphosphine TPP 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. After mixing evenly, the mixture is 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] Refer to the 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 was 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 were added, where the molecular weight of the polyethylene glycol was 2000; the total concentration of polylactic acid was 10 mg / mL, and the mass fraction of hyaluronic acid-polyethylene glycol-polylactic acid in the total polylactic acid was 2.5%. Finally, 1 mg of the hydrophobic anticancer drug paclitaxel was added.

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

[0051] Deionized water was heated to 80°C. At this temperature, 100 μL of the organic phase solution obtained in step (1) was aspirated using a pipette and a 1-200 μL gel spotting tip (inner diameter 200 μm). The organic phase solution was then injected into a glass bottle containing 3 mL of heated deionized water at a rate of 15 mL / min. The mixture was shaken to mix evenly. The mixture was placed in a fume hood and allowed to stand for 12 h to obtain a dispersion of hyaluronic acid-modified, paclitaxel-loaded lac-polylactic acid dimer nanoparticles in deionized water.

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

[0053] To the dispersion of the hyaluronic acid-modified, paclitaxel-loaded lac-polylactic acid dimer nanoparticles prepared in step (2) in deionized water, triphenylphosphine TPP was added according to a ratio of 1:2 between the negative charge on the surface of the lac hemisphere of the nanoparticles and the positive charge on the surface of the positively charged triphenylphosphine TPP. After mixing evenly, the mixture was allowed to stand for 8 hours, and the unadsorbed triphenylphosphine TPP was 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] Example 3:

[0055] According to the steps of Example 1, under the premise that other conditions remain the same, the hyaluronic acid-polyethylene glycol-polylactic acid is replaced with 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 with rhodamine 123.

[0056] Example 4:

[0057] According to the steps of Example 1, under the premise that other conditions are 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] Example 5:

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

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

[0061] Following the steps of Example 1, under the premise of keeping other conditions the same, the concentration of shellac and polylactic acid was kept the same to prepare shellac-polylactic acid dimer nanoparticles with and without modification of hyaluronic acid. The changes in the element content of the polylactic acid hemispheres before and after modification were analyzed by EDS spectroscopy. 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 mitochondria as an intracellular organelle

[0063] Following the steps of Example 1, under the premise of keeping other conditions the same, the concentration of shellac and polylactic acid was kept the same, and shellac-polylactic acid dimer nanoparticles modified with and without triphenylphosphine TPP were prepared. The changes in 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 cancer cell membrane 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 cancer cell membrane in the total polylactic acid is 1-10%; the targeting molecule 1 is selected from hyaluronic acid-polyethylene glycol-polylactic acid; (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 the mixture evenly and then allowing the mixture to stand to obtain a dispersion of shellac-polylactic acid dimer nanoparticles modified with targeting molecule 1 targeting cancer cell membranes in deionized water; (3) adding the targeting molecule 2 targeting the mitochondria of the cancer cell organ to the dispersion prepared in step (2), mixing evenly and then standing, centrifuging and washing, and removing the supernatant, so that the lac hemisphere of the nanoparticle is modified with the targeting molecule 2 targeting the mitochondria of the cancer cell organ, to obtain a dispersion of dual-targeted drug delivery dimer nanoparticles in deionized water; the targeting molecule 2 is selected from triphenylphosphine TPP.

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 2, characterized in that: The molecular weight of the polyethylene glycol is 1000-8000.

4. 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.

5. 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.

6. The method for preparing a biocompatible dual-targeted drug delivery dimer nanoparticle according to claim 5, characterized in that: The hydrophobic drugs include curcumin, paclitaxel, docetaxel and camptothecin; the functional nanomaterials include magnetic nanoparticles and catalytic nanoparticles; and the functional polymers include temperature-responsive polymers, pH-responsive polymers, enzyme-responsive polymers and light-responsive polymers.

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

8. Use of the dual-targeted drug delivery dimer nanoparticles prepared according to claim 1 in the preparation of tumor therapeutic drugs.

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