Targeting plant polypeptide drug-loaded particle and preparation method thereof

Abstract

The invention relates to the technical field of biomedical polymers, in particular to targeted plant polypeptide drug-loaded particles and a preparation method thereof, and the targeted plant polypeptide drug-loaded particles are wrapped by using targeted molecules and plant polypeptides through electrostatic adsorption and then crosslinking. Through the synergistic effect of electrostatic adsorption and cross-linking reaction, stable loading of targeting molecules (such as hydrophilic macromolecules and hydrophobic micromolecules) with different physicochemical properties is adapted. According to the method, the targeting efficiency is ensured, and meanwhile, the particle size uniformity and the drug controlled release performance of the drug-loaded particles are maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical macromolecules, and in particular to targeted plant polypeptide drug-loaded particles and a preparation method thereof. Background Art

[0002] Cancer, a serious threat to human health, has seen its morbidity and mortality rates rise annually. While traditional chemotherapy can inhibit or kill tumor cells to a certain extent, its lack of selectivity often leads to severe damage to normal tissues and a range of side effects, such as hair loss, nausea, and bone marrow suppression, significantly impacting patients' quality of life. Furthermore, traditional chemotherapy drugs are widely distributed throughout the body, have a short circulation time, and are easily metabolized and cleared by the liver and kidneys, making it difficult to maintain effective drug concentrations at the tumor site, thereby reducing therapeutic efficacy.

[0003] To overcome these challenges, targeted drug delivery systems have emerged. These systems utilize carriers through physical, chemical, or biological means to precisely deliver drugs to tumor tissue or cancer cells, thereby increasing local drug concentrations, enhancing therapeutic efficacy, and significantly reducing toxic side effects on normal tissues. Currently, commonly used targeting mechanisms include passive targeting (such as the EPR effect), active targeting (such as ligand-receptor recognition), and stimuli-responsive targeting (such as tumor microenvironment-specific stimuli such as pH, temperature, enzymes, and reductive properties).

[0004] Among the many delivery vehicles, natural polymers are ideal candidates for developing targeted drug delivery systems due to their excellent biocompatibility, biodegradability, and flexibility in chemical modification. By attaching ligands (such as folic acid, antibodies, and peptides) to the surface of polymer carriers, active targeting systems can be constructed to achieve specific recognition and binding to receptors highly expressed on the surface of tumor cells, thereby improving the precision and efficiency of drug delivery.

[0005] In existing technologies, the introduction of targeting molecules typically requires designing specific modification processes tailored to their physicochemical properties (such as hydrophilicity and molecular weight), resulting in complex approaches and a lack of universal applicability. For example, hydrophilic macromolecules (such as hyaluronic acid) require covalent coupling, while hydrophobic small molecules (such as folic acid) require hydrophobic interactions, making them difficult to integrate in the same process system.

[0006] Therefore, developing a universal method for preparing drug-loaded particles that can adapt to targeting molecules with different properties is a technical challenge that needs to be urgently solved in the current targeted drug delivery system. Summary of the Invention

[0007] The purpose of the present invention is to provide a targeted plant polypeptide drug-loaded particle, which achieves the stable loading of targeting molecules with different physical and chemical properties (such as hydrophilic macromolecules and hydrophobic small molecules) through the synergistic effect of electrostatic adsorption and cross-linking reaction, while ensuring the targeting efficiency while maintaining the particle size uniformity, drug loading capacity and release performance of the drug-loaded particles.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect of the present invention, a targeted plant polypeptide drug-loaded particle is provided, wherein the targeted plant polypeptide drug-loaded particle is prepared by encapsulating a targeting molecule on the outer surface of a redox-sensitive plant polypeptide drug-loaded particle; in the encapsulation, the mass ratio of the targeting molecule, the plant polypeptide, and the redox-sensitive plant polypeptide drug-loaded particle is 2-30:28-0:150; The redox-sensitive plant polypeptide drug-loaded particles are prepared using plant polypeptides and hydrophobic drugs as raw materials, introducing redox-sensitive monomers, and using a mixed solution of alcohol and water as solvent under the action of a cross-linking agent.

[0009] Preferably, in the targeting molecule encapsulation, the mass ratio of the targeting molecule and the plant polypeptide to the mass ratio of the redox-sensitive plant polypeptide drug-loaded particles is 30:150. If this ratio is too low, effective encapsulation cannot be achieved, while if this ratio is too high, the drug-loaded nanoparticles will be too large in size and the drug loading capacity will be reduced, which is not conducive to drug delivery. The total mass of the targeting molecule and the plant polypeptide is 30 parts, and the ratio between the targeting molecule and the plant polypeptide is determined by the particle size stability and targeting ability of the obtained targeted plant polypeptide drug-loaded particles.

[0010] When the number of plant peptides is 0 and the number of targeting molecules is 30, the targeting is better, but the stability is poor.

[0011] As a preferred embodiment, the mass ratio of the targeting molecule, the plant polypeptide, and the redox-sensitive plant polypeptide drug-loaded particles is 5:25:150.

[0012] Preferably, the plant polypeptide comprises at least one of millet polypeptide, soybean polypeptide, peanut polypeptide and corn polypeptide. The millet polypeptide, soybean polypeptide, peanut polypeptide and corn polypeptide can be directly purchased as commercial products.

[0013] Preferably, the hydrophobic drug includes at least one of doxorubicin or its hydrochloride, erlotinib or its hydrochloride, curcumin or its hydrochloride, paclitaxel or its hydrochloride, methotrexate or its hydrochloride, and ibuprofen or its hydrochloride.

[0014] Preferably, the targeting molecule includes at least one of hyaluronic acid, folic acid, RGD peptide, and chitosan.

[0015] In a second aspect of the present invention, a method for preparing the targeted plant polypeptide drug-loaded particles is provided, the method comprising the following steps: S1. Mixing the plant polypeptide and the aqueous solution containing the redox-sensitive structural monomer, adjusting the pH to 7.4-9.0, and continuing the reaction until the reaction is complete to obtain a mixed system; S2, adding the alcohol solution of the hydrophobic drug to the mixed system and mixing, and then adding the cross-linking agent until the reaction is complete to obtain a product; S3. After removing the alcohol solution from the product, the product is filtered, the remaining system is centrifuged, the supernatant is collected, and the supernatant is freeze-dried to obtain redox-sensitive plant polypeptide drug-loaded particles.

[0016] S4, dissolving the redox-sensitive plant polypeptide drug-loaded nanoparticles in an alcohol-water mixed solution, adding the targeting molecule, plant polypeptide and cross-linking agent, stirring evenly, and continuing the reaction; S5. After the reaction is completed, the alcohol solution is removed to obtain insoluble matter precipitated. The remaining system after filtration is centrifuged and the supernatant is freeze-dried to obtain targeted plant polypeptide drug-loaded particles.

[0017] Furthermore, when the hydrophobic drug is in the form of a hydrochloride salt, step S2 specifically includes: The alcohol solution of the hydrophobic drug is added to the mixed system and mixed evenly, and then a cross-linking agent is added. The pH of the solution is adjusted to 7.4-9.0 and the reaction is continued until the reaction is complete to obtain a product.

[0018] Sodium hydroxide or potassium hydroxide can be used to adjust the pH of the solution to 7.4-9.0.

[0019] Preferably, the mass ratio of the hydrophobic drug to the plant polypeptide is 1-8:20. If the mass ratio is too small, the drug content in the drug-loaded nanoparticles cannot meet the actual use requirements; if the mass ratio is too large, the drug cannot be completely encapsulated, resulting in large losses.

[0020] Preferably, in step S1, the ratio of the monomer containing the redox-sensitive structure to the plant polypeptide is in the range of 2-8:20. A ratio that is too small will not significantly affect the redox sensitivity; a ratio that is too large will be detrimental to the encapsulation of hydrophobic drugs and the formation of drug-loaded nanoparticles.

[0021] Preferably, in step S1, the mass ratio of the redox-sensitive monomer to the plant polypeptide is 2-8:20. If this mass ratio is too small, the redox performance of the drug-loaded nanoparticles is not significant; if this mass ratio is too large, the drug-loading performance of the drug-loaded nanoparticles is reduced and the structural stability is reduced.

[0022] The concentration of the aqueous solution containing the redox-sensitive structural monomer is in the range of 1 mg / mL to 4 mg / mL (preferably 2 mg / mL).

[0023] In the above technical solution, in step S1, the plant polypeptide and the aqueous solution containing the redox-sensitive structural monomer are mixed, and the pH of the solution is adjusted to 7.4-9.0 using sodium hydroxide or potassium hydroxide, and then the reaction is continued until the reaction is complete to obtain a mixed system; This is because the hydrochloric acid on the redox-sensitive structural monomer needs to be removed to expose the reactive groups, so the pH of the solution needs to be adjusted to 7.4-9.0.

[0024] Preferably, in step S2, the cross-linking agent is glutaraldehyde, and the ratio of the cross-linking agent to the plant polypeptide is between 10 μL / 20 mg and 80 μL / 20 mg. If this ratio is too small, the drug-loaded nanoparticle structure may not be well formed and may be relatively loose; if this ratio is too large, the carrier has a high degree of cross-linking and is dense, which may prevent the drug from being properly encapsulated and released.

[0025] Preferably, in step S2, the alcohol solution is an ethanol-water solution, wherein the volume ratio of water to ethanol in the ethanol-water solution is 1:2-4. If this volume ratio is too low, the hydrophobic drug will precipitate from the water, resulting in a decrease in drug loading; if this volume ratio is too high, the solubility of the polypeptide carrier decreases, resulting in a decrease in yield.

[0026] Preferably, when the hydrophobic drug is in the form of a hydrochloride salt, step S2 specifically comprises: The alcohol solution of the hydrophobic drug is added to the mixed system and mixed evenly, and then a cross-linking agent is added. The pH of the solution is adjusted to 7.4-9.0 with sodium hydroxide or potassium hydroxide, and the reaction is continued until the reaction is complete to obtain a product.

[0027] This is because the hydrochloric acid on the hydrochloride drug needs to be removed to expose the reactive groups, so the pH of the solution needs to be adjusted to 7.4-9.0.

[0028] Preferably, when both the redox-sensitive monomer and the hydrophobic drug are in the hydrochloride form, the pH should be adjusted to 7.4-9.0 after the redox-sensitive monomer is introduced, and the reaction should continue for a period of time before the hydrophobic drug is added to adjust the pH. This is because the carrier must be formed first before the drug is encapsulated; adding the drug in the opposite order or simultaneously is not conducive to drug encapsulation.

[0029] Preferably, in step S2, the alcohol solution of the hydrophobic drug is added dropwise to the mixed system of step S1 at a dropping speed of 1-2 mL / min.

[0030] Preferably, the dehydrated dry particle size of the plant polypeptide drug-loaded particles under a transmission electron microscope is in the range of 30 nm-60 nm, and the hydrated particle size of the plant polypeptide drug-loaded particles under a dynamic light scattering analyzer is in the range of 200 nm-360 nm.

[0031] Plant peptides are natural macromolecules with excellent compatibility with human tissues. After entering the human body, under the action of microorganisms or enzymes, the molecular chains break and degrade, and are eventually converted into monomers in the body or metabolized into water and carbon dioxide.

[0032] Preferably, in step S4, the ratio of the mass of the targeting molecule to the mass of the added plant polypeptide drug-loaded particles is in the range of 0.01-0.2.

[0033] Preferably, in step S4, the targeting molecule includes at least one of hyaluronic acid, folic acid, RGD peptide and chitosan.

[0034] Preferably, in step S4, the ratio of the mass of the cross-linking agent to the added plant polypeptide drug-loaded particles is in the range of 0.5-2 μL / mg. Experiments have shown that exceeding this range will result in: Too little cross-linking agent (<0.5): The encapsulation layer is loose and the targeting molecules fall off (e.g., the drug loading in the folic acid group decreases by 50%); Too many cross-linkers (>2): The coating layer is dense, which inhibits drug release (for example, the release of the folic acid group at 10 mM GSH is reduced from 80% to 60%).

[0035] Preferably, in step S4, the cross-linking agent includes at least one of glutaraldehyde, glyoxal and o-phthalaldehyde.

[0036] Preferably, in step S4, the mass ratio or mass-to-volume ratio of the redox-sensitive plant polypeptide drug-loaded particles to the alcohol solution is 2-5 mg / mL, the alcohol solution is ethanol, and the volume ratio of water to ethanol is 1:2-4.

[0037] The present invention has the following advantages and beneficial effects: 1. The targeted plant polypeptide drug-loaded particles of the present invention are prepared by coating the outer surface of the redox-sensitive plant polypeptide drug-loaded particles with targeting molecules. Through the synergistic effects of electrostatic adsorption and cross-linking, they are suitable for the stable loading of targeting molecules with different physicochemical properties (such as hydrophilic macromolecules and hydrophobic small molecules). Key parameters include a mass ratio of targeting molecule to drug-loaded particles of 2-30:150 and a cross-linker ratio of 0.5-2 μL / mg. This method maintains particle size uniformity and controlled drug release while ensuring targeting efficiency. The synergistic effects of electrostatic adsorption and cross-linking between the targeting molecules and the redox-sensitive plant polypeptide drug-loaded particles result in excellent stability of the prepared targeted drug-loaded particles, resulting in an unexpected technical effect: stable and long-lasting drug release in PBS containing 10 μM GSH, which simulates normal human tissue.

[0038] 2. The targeted plant polypeptide drug-loaded particles of the present invention can not only improve the water solubility of hydrophobic drugs, reduce the toxicity of drugs to the human body, and improve the therapeutic efficiency of drugs; but also can be directed to target tumor cells, be taken up by tumor cells more quickly, and release drugs.

[0039] 3. The targeted plant polypeptide drug-loaded particles of the present invention retain their excellent properties after encapsulation. Furthermore, the preparation method is simple to operate, using targeting molecules and the plant polypeptide itself to encapsulate the drug-loaded particles, resulting in stable quality. The prepared drug-loaded particles have good targeting, controllable particle size, concentrated dispersion, and excellent stability, making them suitable for mass production.

[0040] 4. The plant polypeptide drug-loaded particles of the present invention have redox-sensitive response capabilities and can be released at designated locations, reducing the toxicity of drugs to the human body and improving the therapeutic effects of drugs. The targeted plant polypeptide drug-loaded particles of the present invention have targeting capabilities and can be specifically recognized and taken up by tumor cells, thereby improving the ability of drugs to be released at specific locations. The preparation of drug-loaded particles using natural polymers as materials can be used for a variety of wide applications such as controlled release of drugs, tissue engineering, and gene therapy. The preparation method of the present invention is simple to operate and has stable quality. The prepared drug-loaded particles have good targeting, controllable particle size, concentrated dispersion, and good stability, making them suitable for mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the hydrated particle size distribution diagram of the hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1; Figure 2 This is a stability diagram of the hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1; Figure 3 This is a transmission electron micrograph of the hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1; Figure 4The drug release performance of the hyaluronic acid-targeted millet polypeptide-loaded particles prepared in Example 1. The groups in the figure are PBS, ABS, PBS containing 10 μM GSH, PBS containing 10 mM GSH, and ABS containing 10 mM GSH. Figure 5 The cytotoxicity of the millet polypeptide vector used in Example 1 to BESA-2B cells and A549 cells; Figure 6 This is the hydrated particle size distribution diagram of the folic acid-targeted millet polypeptide drug-loaded particles prepared in Example 2; Figure 7 This is a stability diagram of the folic acid-targeted millet polypeptide drug-loaded particles prepared in Example 2; Figure 8 This is a transmission electron micrograph of the folic acid-targeted millet polypeptide drug-loaded particles prepared in Example 2; Figure 9 This figure shows the drug release performance of the folic acid-targeted millet polypeptide-loaded particles prepared in Example 2. The groups in the figure are PBS, ABS, PBS containing 10 μM GSH, PBS containing 10 mM GSH, and ABS containing 10 mM GSH. Figure 10 The cytotoxicity of the hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1 and the non-targeted millet polypeptide drug-loaded particles of Comparative Example 1 on BESA-2B cells (Figure A) and A549 cells (Figure B); Figure 11 Flow cytometric graphs of the A549 cell uptake effects of the hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1 and the non-targeted millet polypeptide drug-loaded particles in Comparative Example 1; Figure 12 This is a graph showing the results of circulating tumor cell uptake of the hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1 and the non-targeted millet polypeptide drug-loaded particles in Comparative Example 1; Figure 13 : Bar graph comparing the cellular uptake efficiency of different targeting molecules (hyaluronic acid, folic acid); DETAILED DESCRIPTION For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

[0042] Example 1: Plant polypeptide drug-loaded particles with hyaluronic acid as the targeting molecule Step S1, preparing a 2 mg / mL aqueous solution of cystamine hydrochloride, adjusting its pH to 8 using a 0.1 M NaOH aqueous solution, weighing 20 mg of millet polypeptide, adding 2 mL of the prepared aqueous solution of cystamine, and stirring evenly with a magnetic stirrer to obtain a mixed system; Step S2, preparing a 1.5 mg / mL doxorubicin hydrochloride ethanol solution, slowly dropping 4 mL of the doxorubicin hydrochloride ethanol solution into the mixed system, stirring evenly, adding a certain amount of 2.5 wt% glutaraldehyde, and then continuing to stir for 6 hours, then dropping a 0.1 M NaOH aqueous solution to adjust the pH to 8, and stirring for 24 hours to obtain the product; Step S3: vacuum-removing ethanol from the product, centrifuging it, and lyophilizing the supernatant to obtain millet polypeptide drug-loaded particles (i.e., redox-sensitive plant polypeptide drug-loaded particles).

[0043] Step S4: Take 15 mg of the prepared millet polypeptide drug-loaded nanoparticles, add 2 mL of deionized water and 4 mL of ethanol, stir evenly using a magnetic stirrer, add 0.5 mg of hyaluronic acid and 2.5 mg of millet polypeptide, stir for 20 h, then add 9 μL of glutaraldehyde and react for another 4 h. Step S5: After the reaction is completed, the ethanol is removed in vacuo, and the supernatant is taken and freeze-dried after centrifugation to obtain hyaluronic acid-targeted millet polypeptide drug-loaded particles.

[0044] The hydrated particle size distribution of the hyaluronic acid targeted millet polypeptide drug-loaded particles prepared in Example 1 is shown in FIG. Figure 1 shown; from Figure 1 It can be seen that the hydrated particle size of hyaluronic acid-targeted millet polypeptide drug-loaded particles is 250 nm.

[0045] The stability of the hyaluronic acid targeted millet polypeptide drug-loaded particles prepared in Example 1 is as follows Figure 2 shown; from Figure 2 It can be seen that within the measured 120 h, the particle size and distribution of the drug-loaded particles changed little, indicating good stability.

[0046] The transmission electron microscopy image of the hyaluronic acid targeted millet polypeptide drug-loaded particles prepared in Example 1 is as follows: Figure 3 As shown; the hyaluronic acid-targeted millet polypeptide drug-loaded particles have a regular spherical shape and a dry particle size of approximately 180 nm.

[0047] The in vitro drug release performance of the hyaluronic acid targeted millet polypeptide drug-loaded particles prepared in Example 1 is as follows: Figure 4 As shown in the figure, it can be seen that the drug release rate of hyaluronic acid-targeted millet polypeptide drug-loaded particles changes significantly when the pH environment and GSH concentration change, showing good dual sensitivity to pH and redox, and the drug release is fastest under acidic and 10 mM GSH conditions.

[0048] The toxicity of the millet polypeptide carrier prepared in Example 1 to BESA-2B cells and A549 cells is as follows: Figure 5 As shown. Figure 5It can be clearly observed that the millet polypeptide carrier exhibits extremely low cytotoxicity.

[0049] From the above, it can be seen that Example 1 is a combined effect of electrostatic adsorption and cross-linking, and the obtained hyaluronic acid-targeted millet polypeptide drug-loaded particles have good stability, show slow drug release in 10 μM GSH PBS simulating normal human tissue, and can respond to pH environment and redox environment.

[0050] Example 2: Plant polypeptide drug-loaded particles with folic acid as the targeting molecule Step S1, preparing a 2 mg / mL aqueous solution of cystamine hydrochloride, adjusting its pH to 8 using a 0.1 M NaOH aqueous solution, weighing 20 mg of millet polypeptide, adding 2 mL of the prepared aqueous solution of cystamine, and stirring evenly with a magnetic stirrer to obtain a mixed system; Step S2, preparing a 1.5 mg / mL doxorubicin hydrochloride ethanol solution, slowly dropping 4 mL of the doxorubicin hydrochloride ethanol solution into the mixed system, stirring evenly, adding a certain amount of 2.5 wt% glutaraldehyde, and then continuing to stir for 6 hours, then dropping a 0.1 M NaOH aqueous solution to adjust the pH to 8, and stirring for 24 hours to obtain the product; Step S3: vacuum-removing ethanol from the product, centrifuging it, and lyophilizing the supernatant to obtain millet polypeptide drug-loaded particles (i.e., redox-sensitive plant polypeptide drug-loaded particles).

[0051] Step S4: Take 15 mg of the prepared millet polypeptide drug-loaded nanoparticles, add 2 mL of deionized water and 4 mL of ethanol, stir evenly using a magnetic stirrer, add 0.5 mg of folic acid and 2.5 mg of millet polypeptide, stir for 20 h, then add 9 μL of glutaraldehyde and react for another 4 h. Step S5: After the reaction is completed, the ethanol is removed in vacuo, and the supernatant is lyophilized after centrifugation to obtain folic acid-targeted millet polypeptide drug-loaded particles.

[0052] The hydrated particle size distribution of folic acid-targeted millet polypeptide drug-loaded particles prepared in Example 2 is shown in FIG. Figure 6 shown; from Figure 6 It can be seen that the hydrated particle size of hyaluronic acid-targeted millet polypeptide drug-loaded particles is 255 nm.

[0053] The stability of the folic acid-targeted millet polypeptide drug-loaded particles prepared in Example 2 is as follows: Figure 7 shown; from Figure 7 It can be seen that within the measured 120 h, the particle size and distribution of the drug-loaded particles changed little, indicating good stability.

[0054] The transmission electron microscopy image of the folic acid-targeted millet polypeptide drug-loaded particles prepared in Example 2 is as follows: Figure 8As shown in the figure, the folic acid-targeted millet polypeptide drug-loaded particles have regular morphology and are spherical, with a dry particle size of approximately 80 nm.

[0055] The in vitro drug release performance of the folic acid-targeted millet polypeptide-loaded particles prepared in Example 2 is as follows: Figure 9 As shown in the figure, it can be seen that the drug release rate of folic acid-targeted millet polypeptide drug-loaded particles changes significantly when the pH environment and GSH concentration change, showing good dual sensitivity to pH and redox, and the drug release is fastest under acidic and 10 mM GSH conditions.

[0056] From the above, it can be seen that Example 1 is a combined effect of electrostatic adsorption and cross-linking, and the obtained folic acid-targeted millet polypeptide drug-loaded particles have good stability, show slow drug release in 10 μM GSH PBS simulating normal human tissue, and can respond to pH environment and redox environment.

[0057] Example 3: Plant polypeptide drug-loaded particles with hyaluronic acid as the targeting molecule The targeting molecule provided in this embodiment is a plant polypeptide drug-loaded particle of hyaluronic acid, wherein the targeting molecule, The mass ratio of the plant polypeptide to the redox-sensitive plant polypeptide drug-loaded particles is 2:28:150. The other steps are the same as those in Example 1.

[0058] Example 4: Plant polypeptide drug-loaded particles with hyaluronic acid as the targeting molecule The targeting molecule provided in this embodiment is a plant polypeptide drug-loaded particle of hyaluronic acid, wherein the targeting molecule, The mass ratio of the plant polypeptide to the redox-sensitive plant polypeptide drug-loaded particles is 30:0:150. The other steps are the same as those in Example 1.

[0059] Example 5: Plant polypeptide drug-loaded particles with folic acid as the targeting molecule The targeting molecule provided in this embodiment is a plant polypeptide drug-loaded particle of folic acid, wherein the targeting molecule, The mass ratio of the plant polypeptide to the redox-sensitive plant polypeptide drug-loaded particles is 2:28:150. The other steps are the same as those in Example 2.

[0060] Comparative Example 1: No targeting molecule added The difference between this comparative example and Example 1 and Example 2 is that no targeting molecule is added, and only millet polypeptide is used for packaging. The specific operation steps are as follows: Step S1, prepare a 2 mg / mL aqueous solution of cystamine hydrochloride, adjust its pH to 8 using a 0.1 M NaOH aqueous solution, weigh 20 mg of millet polypeptide MP, add 2 mL of the prepared aqueous solution of cystamine, and stir evenly with a magnetic stirrer to obtain a mixed system; Step S2: prepare a 1.5 mg / mL doxorubicin hydrochloride ethanol solution, slowly add 4 mL of doxorubicin hydrochloride ethanol solution at a constant rate, stir evenly, add a certain amount of 2.5 wt% glutaraldehyde, react for 6 hours, add 0.1 M NaOH aqueous solution to adjust the pH to 8, and continue stirring for 24 hours to obtain the product.

[0061] Step S3: vacuum-removing ethanol from the product, centrifuging it, and lyophilizing the supernatant to obtain millet polypeptide drug-loaded particles (i.e., redox-sensitive plant polypeptide drug-loaded particles).

[0062] Step S4: Take 15 mg of the prepared millet peptide drug-loaded nanoparticles, add 2 mL of deionized water and 4 mL of ethanol, stir evenly with a magnetic stirrer, add 3.0 mg of millet peptide, stir for 20 h, then add 9 μL of glutaraldehyde and react for another 4 h. Step S5: After the reaction is completed, the ethanol is removed in vacuo, and the supernatant is lyophilized after centrifugation to obtain non-targeted millet polypeptide drug-loaded particles.

[0063] Comparative Example 2 In this comparative example, except that in step S4, the mass ratio of the targeting molecule to the drug-loaded particles was 0.1:150 (specifically, 0.01 mg of hyaluronic acid and 2.99 mg of millet polypeptide were added), the other steps were the same as those in Example 1.

[0064] Comparative Example 3 In this comparative example, except that in step S4, the mass ratio of the targeting molecule to the drug-loaded particles was 50:150 (specifically, 5.0 mg of hyaluronic acid was added), the other steps were the same as those in Example 1.

[0065] The hyaluronic acid-targeted millet polypeptide drug-loaded particles obtained in Comparative Example 3 are partially insoluble in water (poor water solubility).

[0066] Comparative Example 4 In this comparative example, except that in step S4, the ratio of the cross-linking agent was 0.1 μL / mg (specifically, 1.8 μL of 2.5 wt % glutaraldehyde), the other steps were the same as those in Example 2.

[0067] The hydrated particle size of the folic acid-targeted millet polypeptide drug-loaded particles obtained in Comparative Example 4 became larger, reaching 356 nm, making it difficult to enter cells through passive targeting.

[0068] Comparative Example 5 In this comparative example, except that in step S4, the ratio of the cross-linking agent was 5 μL / mg (specifically, 90 μL of 2.5 wt% glutaraldehyde), the other steps were the same as those in Example 2.

[0069] Experimental Example 1 The cumulative drug release rate and particle size of Examples 1-2 and Comparative Examples 1-5 in PBS containing 10 mM GSH were measured over 24 hours. The results are shown in Table 1.

[0070] The drug loading amount was determined by dissolving an appropriate amount of freeze-dried sample in deionized water and measuring the absorbance of the drug-loaded particles at a wavelength of 485 nm using an ultraviolet spectrophotometer to obtain the drug loading amount.

[0071] The cumulative drug release rate was determined as follows: 3 mg of freeze-dried drug-loaded particles were dissolved in each of the five media listed above, placed in a dialysis bag with a molecular weight cutoff of 3500, and then placed in 25 mL of the corresponding medium. The mixture was shaken on a thermostat set at 37°C. Subsequently, 3 mL of sample solution was removed from the outer medium of the dialysis bag at regular intervals and replaced with an equal amount (3 mL) of fresh medium to maintain a constant volume. The fluorescence intensity of the sample solution was measured using a fluorescence spectrophotometer at an excitation wavelength of 480 nm and an emission wavelength of 555 nm. The DOX concentration was calculated using a standard curve, and the cumulative release rate of DOX in the different media was calculated using the formula.

[0072] Hydrated Particle Size Determination Method: Dynamic light scattering was used to analyze the potential, particle size, and stability of the drug-loaded particles. A 1.0 mg / mL solution of the drug-loaded particles was prepared, filtered through a 0.45 μm filter membrane, and the average value was obtained by three measurements.

[0073] Table 1

[0074] From Table 1 we can see that: In Comparative Example 2, the mass ratio of the targeting molecule to the drug-loaded particles was 1:150, which was lower than the range of 2-30:150 in the present invention. The results showed that the encapsulation rate of the targeting molecule was too low, and the cellular uptake efficiency was greatly reduced ( Figure 13 ); In Comparative Example 3, the mass ratio of the targeting molecule to the drug-loaded particles was 50:150, which was greater than the range of 30:150 in Example 2 of the present invention. The obtained hyaluronic acid-targeted millet polypeptide drug-loaded particles were partially insoluble in water (poor water solubility); In Comparative Example 4, the ratio of the cross-linking agent added was 0.1 μL / mg, which was lower than the range of 0.5-2 μL / mg in the embodiment of the present invention. The hydrated particle size of the obtained folic acid-targeted millet polypeptide drug-loaded particles became larger, 356 nm, and it was difficult to enter the cells through passive targeting; In Comparative Example 5, the ratio of cross-linking agent added was 5 μL / mg, which was higher than the range of 0.5-2 μL / mg in the embodiment of the present invention. The response effect to the redox environment was reduced, and the cumulative drug release rate in PBS containing 10 mM GSH for 24 h was 60.9%; As can be seen from the above, the mass ratio of the targeting molecule to the drug-loaded particles in the methods provided by the present invention ranges from 2-30:150. If this ratio is too low, the targeting effect is not significant; if it is too high, an insoluble precipitate forms. The ratio of the crosslinker added in the methods provided by the present invention ranges from 0.5-2 μL / mg. If this ratio is too low, the drug-loaded particles become larger in size; if it is too high, the coating becomes dense, inhibiting drug release.

[0075] The cytotoxicity and targeting effect of the hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1 and Comparative Example 1 and the non-targeted millet polypeptide drug-loaded particles are as follows: Figure 10 The cytotoxicity of the hyaluronic acid-targeted millet polypeptide-loaded particles prepared in Example 1 and the non-targeted millet polypeptide-loaded particles in Comparative Example 1 to BESA-2B cells and A549 cells (HA / MMP1 is millet polypeptide-loaded particles, MMP is non-targeted millet polypeptide-loaded particles, Figure A is BESA-2B cells, and Figure B is A549 cells); Figure 10 It can be seen that hyaluronic acid-targeted millet peptide has lower toxicity to BESA-2B normal cells and better inhibitory effect on A549 tumor cells.

[0076] The cytotoxicity of the hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1 and Comparative Example 1 was compared with the targeting effect of the non-targeted millet polypeptide drug-loaded particles.

[0077] Figure 11 Flow cytometric graph of the A549 cell uptake effect of the hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1 and the non-targeted millet polypeptide drug-loaded particles in Comparative Example 1 (HA / MMP1 is the millet polypeptide drug-loaded particles, MMP is the non-targeted millet polypeptide drug-loaded particles, A and B are two different expressions of the flow cytometry results); Figure 11 It can be seen that under the same culture conditions, A549 tumor cells more efficiently absorb hyaluronic acid-targeted millet polypeptide drug-loaded particles and effectively accumulate anti-tumor drugs.

[0078] Figure 12 Figure 1 shows the results of circulating tumor cell uptake of hyaluronic acid-targeted millet polypeptide drug-loaded particles prepared in Example 1 and non-targeted millet polypeptide drug-loaded particles in Comparative Example 1 (HA / MMP1 is millet polypeptide drug-loaded particles, MMP is non-targeted millet polypeptide drug-loaded particles, from left to right are DAPI nuclear staining, DOX fluorescence, bright field, DOX fluorescence and DAPI nuclear staining cell superposition); Figure 12 It can be seen that circulating tumor cells can more effectively absorb hyaluronic acid-targeted millet polypeptide drug-loaded particles, which is conducive to faster drug entry into cells for drug release.

[0079] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0080] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0081] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A targeted plant polypeptide drug-loaded particle, characterized in that: The targeted plant polypeptide drug-loaded particles are prepared by encapsulating the outer surface of the redox-sensitive plant polypeptide drug-loaded particles with the targeting molecule and the plant polypeptide through electrostatic adsorption and cross-linking reaction; in the encapsulation, the mass ratio of the targeting molecule, the plant polypeptide, and the redox-sensitive plant polypeptide drug-loaded particles is 2-30:28-0:150; The redox-sensitive plant polypeptide drug-loaded particles are prepared using plant polypeptides and hydrophobic drugs as raw materials, introducing redox-sensitive monomers, and using a mixed solution of alcohol and water as solvent under the action of a cross-linking agent.

2. The redox-sensitive plant polypeptide drug-loaded particles according to claim 1, characterized in that In the package, the ratio of the mass of the targeting molecule and the plant polypeptide to the mass of the redox-sensitive plant polypeptide drug-loaded particles is 30:

150.

3. The targeted plant polypeptide drug-loaded particle according to claim 1, characterized in that: The targeting molecule includes at least one of hyaluronic acid, folic acid, RGD peptide, and chitosan; the plant polypeptide includes at least one of millet polypeptide, soybean polypeptide, peanut polypeptide, and corn polypeptide; and the hydrophobic drug includes at least one of doxorubicin or its hydrochloride, erlotinib or its hydrochloride, curcumin or its hydrochloride, paclitaxel or its hydrochloride, methotrexate or its hydrochloride, and ibuprofen or its hydrochloride.

4. A method for preparing the targeted plant polypeptide drug-loaded particles according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: S1. Mixing the plant polypeptide and the aqueous solution containing the redox-sensitive structural monomer, adjusting the pH to 7.4-9.0, and continuing the reaction until the reaction is complete to obtain a mixed system; S2. Adding an alcohol solution of a hydrophobic drug to the mixed system and mixing uniformly, then adding a cross-linking agent until the reaction is complete to obtain a product, which is purified and freeze-dried to obtain redox-sensitive plant polypeptide drug-loaded particles; S3. Dissolving the redox-sensitive plant polypeptide drug-loaded particles in an alcohol-water mixture, adding targeting molecules, plant polypeptides and cross-linking agents, forming a coating layer through electrostatic adsorption and cross-linking reaction, and purifying and freeze-drying to obtain targeted plant polypeptide drug-loaded particles.

5. The preparation method according to claim 4, characterized in that In step S3, the ratio of the volume of the targeting molecule to the mass of the added redox-sensitive plant polypeptide drug-loaded particles is in the range of 0.01-0.

5.

6. The preparation method according to claim 4, characterized in that The targeting molecule includes at least one of hyaluronic acid, folic acid, RGD peptide and chitosan.

7. The preparation method according to claim 4, characterized in that In step S3, the ratio of the volume of the cross-linking agent to the mass of the plant polypeptide is in the range of 0.5-2 μL / mg.

8. The preparation method according to claim 4, characterized in that In step S3, the cross-linking agent includes at least one of glutaraldehyde, glyoxal or o-phthalaldehyde.

9. The preparation method according to claim 4, characterized in that In step S3, the mass volume ratio of the redox-sensitive plant polypeptide drug-loaded particles to the alcohol-water mixture is 2-5 mg / mL, the alcohol solution is ethanol, and the volume ratio of water to ethanol is 1:2-4.

10. The preparation method according to claim 4, characterized in that When the hydrophobic drug is in the form of hydrochloride, step S2 specifically includes: The alcohol solution of the hydrophobic drug is added to the mixed system and mixed evenly, and then a cross-linking agent is added. After adjusting the pH to 7.4-9.0, the reaction is continued until the reaction is complete to obtain a product.

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