A nanocarrier, drug-loaded nanoparticle, and a preparation method and application thereof

Abstract

The application discloses a kind of nano-carrier, drug-loaded nanoparticle and its preparation method and application, belong to biomedicine technical field.The present application is by using thioctic acid respectively with cholesterol and L-arginine methyl ester is reacted, obtains the nano-carrier of moderate particle size, the nano-carrier has better targeting, and drug loading is high, and has better bioavailability;Further, the above-mentioned nano-carrier is compounded, and the composite nano-carrier is obtained, and the composite nano-carrier has better target cell affinity;And the above-mentioned nano-carrier, composite nano-carrier can not only load small molecule drug, but also can load macromolecular polypeptide drug, thus, in preparation antitumor drug has better application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a nano carrier, drug-loaded nanoparticles and a preparation method and application thereof. BACKGROUND

[0002] Recently, the National Cancer Center released the 2024 National Cancer Report, and the data shows that the number of new cases and deaths of malignant tumors in China in 2022 has increased compared with previous years, and tumors are still a major public health problem in China. At present, the main means of treating cancer is still surgery, radiotherapy and chemotherapy, and doxorubicin (DOX) as a common chemotherapeutic drug has a great killing ability on normal cells while killing tumor cells, and has a very large toxic side effect, which greatly limits the clinical application of chemotherapeutic drugs.

[0003] Anti-tumor nano drugs can load small molecule chemotherapeutic drugs or biological macromolecules and other active ingredients to achieve better therapeutic and diagnostic effects. In recent years, nano carrier drug delivery systems based on polymers and liposomes and other forms have been widely studied. These systems have shown great application potential in tumor treatment due to their unique physical and chemical properties. Compared with traditional small molecule chemotherapeutic drugs or biological macromolecules, nano drugs can realize more ideal cell recognition and uptake by adjusting the surface properties and size, improve the bioavailability, and optimize the pharmacokinetics and distribution in the body, thereby effectively reducing the systemic toxicity and side effects.

[0004] However, the current nano carrier drug delivery system has problems such as poor targeting, low drug loading capacity and low bioavailability. SUMMARY

[0005] The purpose of the present application is to provide a nano carrier, drug-loaded nanoparticles and a preparation method and application thereof. In the present application, lipoic acid is reacted with cholesterol and L-arginine methyl ester respectively to obtain a nano carrier with a suitable particle size. The nano carrier has good targeting, high drug loading capacity and good bioavailability. Further, the nano carrier is compounded to obtain a composite nano carrier, which has good affinity to target cells. In addition, the nano carrier and the composite nano carrier can not only load small molecule drugs, but also load macromolecular polypeptide drugs, and therefore have good application prospects.

[0006] In a first aspect, the present application provides a nano carrier, which is obtained by reacting lipoic acid with cholesterol and L-arginine methyl ester respectively, and has the structure shown in the following formula (I) and / or formula (II):

[0007] 、 .

[0008] In the present application, the inventors have found that lipoic acid, as a natural small molecule, has good biocompatibility and antioxidant effect. Because it has abundant dynamic covalent disulfide bonds, the disulfide bonds are in a dynamic equilibrium state, so there is always some exchange between the bonding state and the cleavage state, and the disulfide bond can initiate chain exchange reaction under high temperature, ultraviolet light and redox reaction. Based on this, lipoic acid is used to modify cholesterol and L-arginine methyl ester respectively in the present application to obtain a nano-carrier with moderate particle size. The nano-carrier has good targeting, high drug loading capacity, and good bioavailability and biological responsiveness, so it can realize targeted delivery of drugs.

[0009] In a second aspect, the present application provides a composite nano-carrier, which is obtained by mixing the nano-carrier having the structure shown in formula (I) and the nano-carrier having the structure shown in formula (II) in a mass ratio of (1-5):(5-9).

[0010] In the present application, the inventors have further found that the lipoic acid-modified cholesterol nano-carrier is negatively charged, and the lipoic acid-modified L-arginine methyl ester nano-carrier is positively charged. After compounding the two nano-carriers, part of the charges can be neutralized, so that the composite nano-carrier obtained by compounding has stronger affinity with target cells. In addition, the lipoic acid-modified L-arginine methyl ester nano-carrier contains guanidino cations, which can enhance the cell penetration activity, so that the nano-carrier can enter the target cells more conveniently and quickly. The lipoic acid-modified cholesterol nano-carrier can provide hydrophobic interaction to enhance the stability of the nano-carrier and the interaction with the cell membrane. The synergistic effect of the above two nano-carriers improves the targeting and significantly improves the bioavailability of the loaded drug.

[0011] In a third aspect, the present application provides a preparation method of the above-mentioned nano-carrier, comprising the following steps: S1, using lipoic acid and cholesterol as raw materials, reacting under the action of a first catalyst and a first activator, and then separating and purifying to obtain lipoic acid-modified cholesterol; or using lipoic acid and L-arginine methyl ester as raw materials, reacting under the action of a second catalyst and a second activator, and then separating and purifying to obtain lipoic acid-modified L-arginine methyl ester; S2, dissolving the lipoic acid-modified cholesterol and the lipoic acid-modified L-arginine methyl ester in water respectively, and then self-assembling to obtain the nano-carrier.

[0012] In the present application, the preparation process of lipoic acid-modified cholesterol is shown in the following formula (1):

[0013]

[0014] The preparation process of lipoic acid-modified L-arginine methyl ester is shown in the following formula (2):

[0015]

[0016] The preparation method is simple, the raw materials are green, non-toxic, cheap and easy to obtain, and has good biological safety, so that large-scale production and application are facilitated.

[0017] In some embodiments, in step S1, the molar ratio of lipoic acid, cholesterol, the first catalyst and the first activator is (0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5), and the first catalyst comprises 4-dimethylaminopyridine, and the first activator comprises 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride.

[0018] In some preferred embodiments, the molar ratio of lipoic acid, cholesterol, the first catalyst and the first activator is 1:1:1:1.

[0019] In some embodiments, in step S1, the reaction under the action of the first catalyst and the first activator specifically comprises: reacting at room temperature and a rotation speed of 500-700 r / min for 20-28 h.

[0020] In some preferred embodiments, the reaction under the action of the first catalyst and the first activator specifically comprises: reacting at room temperature and a rotation speed of 600 r / min for 24 h.

[0021] Understandably, the reaction is carried out in an organic solvent, and the organic solvent can be selected from conventional organic solvents. In the present application, the organic solvent is preferably dichloromethane.

[0022] In some embodiments, after separation and purification, the lipoic acid-modified cholesterol specifically comprises: adding a saturated sodium chloride solution to the obtained product and oscillating, washing the obtained lower liquid, and then performing vacuum concentration, continuing to add anhydrous methanol to the obtained solid powder to perform beating, and finally drying the obtained solid to obtain the lipoic acid-modified cholesterol.

[0023] In some embodiments, in step S1, the molar ratio of lipoic acid, L-arginine methyl ester, the second catalyst and the second activator is (1.5-2.5):(0.5-1.5):(0.5-1.5):(1.5-2.5), and the second catalyst comprises 4-dimethylaminopyridine, and the second activator comprises N,N-carbonyldiimidazole.

[0024] In some preferred embodiments, the molar ratio of lipoic acid, L-arginine methyl ester, the second catalyst and the second activator is 2:1:1:2.

[0025] In some embodiments, the reaction in the presence of the second catalyst and the second activator specifically comprises: reacting at room temperature for 6-12 hours at a rotation speed of 500-700 r / min.

[0026] In some preferred embodiments, the reaction in the presence of the second catalyst and the second activator specifically comprises: reacting at room temperature for 9 hours at a rotation speed of 600 r / min.

[0027] It can be understood that the reaction in the presence of the second catalyst and the second activator is carried out in an organic solvent, and the organic solvent can be selected from conventional organic solvents, and in the present application, the organic solvent is preferably N,N-dimethylformamide.

[0028] In some embodiments, the lipoic acid-modified L-arginine methyl ester obtained after separation and purification specifically comprises: adding ice ethyl ether to the obtained product to precipitate, washing the obtained precipitate after centrifugation, and drying to obtain the lipoic acid-modified L-arginine methyl ester.

[0029] In a fourth aspect, the present application provides a drug-loaded nanoparticle, which is obtained by loading a drug on the above-mentioned nanocarrier, the above-mentioned composite nanocarrier or the nanocarrier prepared by the above-mentioned any one preparation method.

[0030] In some embodiments, the drug comprises a small molecule drug and / or a large molecule polypeptide drug; wherein the small molecule drug comprises doxorubicin, and the large molecule polypeptide drug is selected from any one of the following: A1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; A2) a polypeptide having lipoic acid coupled to one end of an amino acid sequence as shown in SEQ ID NO: 1; A3) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2; and A4) a polypeptide having lipoic acid coupled to one end of an amino acid sequence as shown in SEQ ID NO: 2.

[0031] It can be understood that the small molecule drug and the large molecule polypeptide drug can be selected according to the type of target cells.

[0032] In a fifth aspect, the present application provides the use of the above-mentioned drug-loaded nanoparticle in the preparation of an anti-tumor drug.

[0033] The present application has the following beneficial effects: different from the prior art, the present application uses lipoic acid to react with cholesterol and L-arginine methyl ester respectively to obtain a nanocarrier with a moderate particle size, which has good targeting property, high drug loading capacity and good bioavailability; the above-mentioned nanocarrier can not only load small molecule drugs, but also load large molecule polypeptide drugs, and therefore has good application prospect in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Hydrodynamic size of DLA powder synthesized in Example 1 of the present application 1 H-NMR nuclear magnetic chart (CDCl3400Hz);

[0035] Figure 2 Hydrodynamic size of GLA powder synthesized in Example 1 of the present application 1 H-NMR nuclear magnetic chart (D2O 400Hz);

[0036] Figure 3 Hydrodynamic size of DLA nanocarriers in Example 1 of the present application under different pH conditions;

[0037] Figure 4 TEM of DLA nanocarriers in Example 1 of the present application under different pH conditions;

[0038] Figure 5 Hydrodynamic size of DLA nanocarriers in Example 1 of the present application under different concentration conditions;

[0039] Figure 6 TEM of DLA nanocarriers in Example 1 of the present application under different concentration conditions;

[0040] Figure 7 Hydrodynamic size of DLA nanocarriers in Example 1 of the present application under different time conditions;

[0041] Figure 8 TEM of DLA nanocarriers in Example 1 of the present application under different time conditions;

[0042] Figure 9 Hydrodynamic size of DLA nanocarriers in Example 1 of the present application under different temperature conditions;

[0043] Figure 10 TEM of DLA nanocarriers in Example 1 of the present application under different temperature conditions;

[0044] Figure 11 Schematic diagram of synthesis of composite nanocarriers and drug loading thereof in Example 2 of the present application;

[0045] Figure 12 Hydrodynamic size of composite nanocarriers mixed in different proportions in Example 2 of the present application;

[0046] Figure 13 TEM of composite nanocarriers mixed in different proportions in Example 2 of the present application;

[0047] Figure 14Zeta potential diagram of the composite nanocarriers mixed in different proportions in Example 2 of the present application;

[0048] Figure 15 Drug loading rate and encapsulation efficiency diagram of D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles in Example 3 of the present application;

[0049] Figure 16 Drug release curve diagram of D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles in Example 3 of the present application;

[0050] Figure 17 Mass spectrum diagram of polypeptide drug T1 in Example 4 of the present application;

[0051] Figure 18 Mass spectrum diagram of lipoic acid modified polypeptide drug T2 in Example 4 of the present application;

[0052] Figure 19 Mass spectrum diagram of polypeptide drug T3 in Example 4 of the present application;

[0053] Figure 20 Mass spectrum diagram of lipoic acid modified polypeptide drug T4 in Example 4 of the present application;

[0054] Figure 21 Drug loading rate and encapsulation efficiency diagram of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles in Example 4 of the present application;

[0055] Figure 22 Drug release curve diagram of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles in Example 4 of the present application;

[0056] Figure 23 Survival rate diagram of DLA, GLA nanocarriers and D3G7 composite nanocarriers after incubation with cells in Example 5 of the present application;

[0057] Figure 24 Survival rate diagram of D-DOX, G-DOX, D3G7-DOX drug-loaded nanoparticles and free DOX after incubation with cells in Example 5 of the present application;

[0058] Figure 25 Survival rate diagram of polypeptide drugs T1, T2, T3 and T4 after incubation with cells in Example 5 of the present application;

[0059] Figure 26 Survival rate diagram of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles after incubation with cells in Example 5 of the present application;

[0060] Figure 27A Figure 6 is a laser confocal image of D3G7-DOX drug-loaded nanoparticles in Example 6 of the present application after incubation with cells;

[0061] Figure 27B Figure 6 is a laser confocal image of D3G7-DOX drug-loaded nanoparticles in Example 6 of the present application after incubation with cells; DETAILED DESCRIPTION

[0062] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0063] The experimental methods not specified in the embodiments are usually performed according to the conventional conditions and the conditions described in the manual, or according to the conditions suggested by the manufacturers. The general equipment, materials, reagents, etc. used are commercially available, unless otherwise specified.

[0064] Example 1 Synthesis of nanocarriers

[0065] 1.1 Synthesis of thioctic acid-modified cholesterol (DLA) nanocarriers

[0066] First, DLA was synthesized: 1 mmol of thioctic acid (TA), 1 mmol of cholesterol, 1 mmol of 4-dimethylaminopyridine (DMAP), and 1 mmol of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) were accurately weighed in a 100 mL round-bottom flask, 50 mL of dichloromethane (DCM) was added to the round-bottom flask, a magnetic stirrer was placed in it, and the reaction was stirred at room temperature at a speed of 600 r / min for 24 h. 25 mL of the reaction solution was poured into a separatory funnel, 150 mL of saturated sodium chloride solution was added, shaken well and degassed, and the lower liquid was collected in a pear-shaped bottle and washed by extraction three times. The remaining 25 mL of reaction solution was washed by extraction three times in the same way. The lower liquids collected twice were concentrated under reduced pressure to obtain a light yellow powder. 50 mL of anhydrous methanol was added to the light yellow powder to make a slurry, and then the solid-liquid mixture was collected in a 50 mL centrifuge tube and centrifuged at a speed of 8000 r / min for 10 min. The supernatant was poured out, and 50 mL of anhydrous methanol was added to make a slurry. This operation was repeated three times. Finally, the supernatant was poured out, and the solid was dried in a vacuum drying oven for more than 3 h to obtain DLA powder.

[0067] Then synthesize DLA nanocarriers: slowly drop 50 μL DLA solution (obtained by weighing 1 mg DLA powder into 500 μL tetrahydrofuran) into 950 μL Wahaha pure water under vortex, add 10 μL iodoacetamide solution with a concentration of 1 mg / mL (obtained by weighing 1 mg iodoacetamide into 1 mL dimethyl sulfoxide) to prevent depolymerization, add a magnet, stir at room temperature for 12 h, and obtain DLA nanocarriers.

[0068] 1.2 Synthesis of L-arginine methyl ester (GLA) nanocarriers modified by glutathione

[0069] First, synthesize GLA: accurately weigh 2 mmol glutathione, 2 mmol N,N-carbonyldiimidazole (CDI) into a 10 mL round-bottom flask, add 5 mL N,N-dimethylformamide (DMF), put into a magnet, stir at room temperature at a speed of 600 r / min for 0.5 h, then add 1 mmol L-arginine methyl ester, 1 mmol 4-dimethylaminopyridine, continue to stir at room temperature at a speed of 600 r / min for 9 h; slowly drop the reaction solution into diethyl ether, put into a refrigerator for 0.5 h to make it completely precipitate, then centrifuge at a speed of 8000 r / min for 10 min, pour the supernatant; wash with washing liquid (mix dichloromethane and ethylene glycol monopropyl ether according to a volume ratio of 1:2), precipitate yellow paste, then put into a centrifuge, centrifuge at a speed of 8000 r / min for 10 min, pour the supernatant, repeat the operation three times, and finally obtain yellow paste; put it into a vacuum drying box and dry for more than 3 h, and obtain GLA powder.

[0070] Then synthesize GLA nanocarriers: slowly drop 50 μL GLA solution (obtained by weighing 1 mg GLA powder into 500 μL dimethyl sulfoxide) into 950 μL Wahaha pure water under vortex, add 10 μL iodoacetamide solution with a concentration of 1 mg / mL (obtained by weighing 1 mg iodoacetamide into 1 mL dimethyl sulfoxide) to prevent depolymerization, add a magnet, stir at room temperature for 12 h, and obtain GLA nanocarriers.

[0071] Performance characterization

[0072] Perform nuclear magnetic resonance hydrogen spectrum test on the DLA powder synthesized in step 1.1 and the GLA powder synthesized in step 1.2, and the results are shown in Figure 1 and 2 .

[0073] As can be seen from Figure 1 and 2 , the DLA powder and the GLA powder are both successfully prepared.

[0074] Exemplarily, the hydrated particle size and TEM of the DLA nanocarriers synthesized under different conditions are tested. The hydrated particle size is detected by a Malvern particle size analyzer, and the TEM test includes the following steps: 10 μL of the sample to be tested is dropped onto a copper mesh, the copper mesh is washed with water for 2 times after 10 min, then uranyl acetate is dropped onto the copper mesh for staining for 10 min, washed for 2 times, and then dried naturally, and then detected by a 120 kv high-resolution transmission electron microscope.

[0075] Specifically, first, the pH value of pure water is optimized, buffer solutions with pH values of 3, 4, 5, 6, 7, 8, 9, 10 and 11 are prepared (the pH value is adjusted by HCl and NaOH), 50 μL of DLA solution (1 mg of DLA powder is dissolved in 500 mL of tetrahydrofuran to obtain) is slowly dropped into 950 μL of the above-mentioned buffer solution with different pH values under vortex, 10 μL of iodoacetamide solution with a concentration of 1 mg / mL (1 mg of iodoacetamide is dissolved in 1 mL of dimethyl sulfoxide to obtain) is added to prevent depolymerization, a magnet is added, and stirring is performed at room temperature for 12 h to obtain DLA nanocarriers. The hydrated particle size and TEM results of the DLA nanocarriers under different pH conditions are shown in Figure 3 and 4 .

[0076] It can be seen from Figure 3 and 4 that when the pH value of the buffer solution is 7, the DLA nanocarrier has the smallest particle size and is well dispersed.

[0077] Then, the addition concentration of the DLA solution is optimized, and DLA tetrahydrofuran solutions with different concentrations (0.03, 0.05, 0.1, 0.15, 0.18, 0.2, 0.5, 1.0, 1.2 and 1.5 mg / mL) are prepared, 50 μL of the above-mentioned DLA tetrahydrofuran solution with different concentrations is slowly dropped into 950 μL of the buffer solution with a pH value of 7, 10 μL of iodoacetamide solution with a concentration of 1 mg / mL (1 mg of iodoacetamide is dissolved in 1 mL of dimethyl sulfoxide to obtain) is added to prevent depolymerization, a magnet is added, and stirring is performed at room temperature for 12 h to obtain DLA nanocarriers. The hydrated particle size and TEM results of the DLA nanocarriers under different concentration conditions are shown in Figure 5 and 6 .

[0078] It can be seen from Figure 5 and 6 that when the concentration of the DLA tetrahydrofuran solution is 0.15 mg / mL, the DLA nanocarrier has the smallest particle size and is well dispersed.

[0079] Then, the stirring time was optimized. 50 μL of DLA tetrahydrofuran solution (0.15 mg / mL) was slowly dropped into 950 μL of pH 7 buffer which was vortexed, 10 μL of iodoacetamide solution (1 mg / mL) was added to prevent depolymerization, a magnet was added, and the mixture was stirred at room temperature for different time (0, 2, 4, 8, 12, 24, 48, 72 h), respectively, to obtain DLA nanocarriers. The hydrated particle size and TEM results of the DLA nanocarriers under different stirring time conditions are shown in Figure 7 and 8 , respectively.

[0080] As can be seen from Figure 7 and 8 , when the stirring time is 4 h, the DLA nanocarriers have the smallest particle size and are well dispersed.

[0081] Finally, the stirring temperature was optimized. 50 μL of DLA tetrahydrofuran solution (0.15 mg / mL) was slowly dropped into 950 μL of pH 7 buffer which was vortexed, 10 μL of iodoacetamide solution (1 mg / mL) was added to prevent depolymerization, a magnet was added, and the mixture was stirred at different temperatures (5, 10, 20, 30, 40, 60, 70 °C) for 4 h, to obtain DLA nanocarriers. The hydrated particle size and TEM results of the DLA nanocarriers under different stirring temperature conditions are shown in Figure 9 and 10 , respectively.

[0082] As can be seen from Figure 9 and 10 , when the stirring temperature is 30 °C, the DLA nanocarriers have the smallest particle size and are well dispersed.

[0083] Example 2 Synthesis of composite nanocarriers

[0084] Please refer to Figure 11 , which is a schematic diagram of the synthesis of composite nanocarriers and the loading of drugs. Specifically, the synthesis of the composite nanocarriers includes the following steps:

[0085] First, prepare DLA tetrahydrofuran solution with a concentration of 0.15 mg / mL and GLA dimethyl sulfoxide solution with a concentration of 0.15 mg / mL respectively, then respectively take the above solutions, prepare mixed solutions according to the volume ratio of DLA tetrahydrofuran solution and GLA dimethyl sulfoxide solution as 0:10, 1:9, 3:7, 5:5, 7:3, 9:1, 10:0, slowly drop 50 μL of the above mixed solution into 950 μL of pH 7 buffer solution which is vortexed, and add 10 μL of iodine acetamide solution with a concentration of 1 mg / mL (weigh 1 mg of iodine acetamide and dissolve it in 1 mL of dimethyl sulfoxide to obtain) to prevent depolymerization, add a magneton, stir for 4 h at a temperature of 30 ℃, and obtain the composite nanocarrier. The hydrated particle size, Zeta potential and TEM results of the composite nanocarrier after mixing at different ratios are shown in Figures 12-14 .

[0086] As can be seen from Figures 12-14 , when the mass ratio of DLA nanocarrier and GLA nanocarrier is (1-5):(5-9), the particle size of the composite nanocarrier is small and well dispersed; and when the mass ratio of DLA nanocarrier and GLA nanocarrier is 3:7, the particle size of the composite nanocarrier is the smallest and well dispersed, and the composite nanocarrier is named as D3G7.

[0087] Example 3: Loading of small molecule drugs and drug release test of nanocarriers and composite nanocarriers

[0088] First, the drug loading test was performed. Specifically, 2 mg of doxorubicin (DOX), 18 mg of DLA were dissolved in 500 μL of tetrahydrofuran and dimethyl sulfoxide mixed solution to obtain a carrier D-DOX solution; 2 mg of DOX, 18 mg of GLA were dissolved in 500 μL of dimethyl sulfoxide to obtain a carrier G-DOX solution; 2 mg of DOX, 5.4 mg of DLA and 12.6 mg of GLA were dissolved in 500 μL of tetrahydrofuran and dimethyl sulfoxide mixed solution to obtain a carrier D3G7-DOX solution. The above three carrier solutions were stirred at room temperature for 2 h in the dark, slowly dropped into 9.5 mL of Wahaha pure water with a pipette gun, and then reacted at room temperature for 12 h in the dark. The reaction completed mixed solution was placed in a dialysis bag with a molecular weight cut-off of 1000, and dialyzed in ultrapure water in the dark for 24 h, and the ultrapure water was replaced every 4 h. After dialysis, the solution was collected in a centrifuge tube, wrapped in tin paper to avoid light, and placed in a freeze dryer to freeze dry for 3 d to obtain D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles respectively. The drug loading rate and encapsulation efficiency of the D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles are shown in Figure 15 .

[0089] As can be seen from Figure 15As can be seen, all three types of drug-loaded nanoparticles have good drug loading and encapsulation efficiency; and the composite D3G7-DOX drug-loaded nanoparticles have even better drug loading and encapsulation efficiency.

[0090] Then, drug release testing was performed. Specifically, 1 mL of D-DOX, G-DOX, and D3G7-DOX drug-loaded nanoparticles were placed into dialysis bags with a molecular weight cutoff of 1000, tied at both ends with cotton thread, and immersed in PBS buffer. Three parallel experiments were performed for each carrier. The rotation speed of the isothermal shaking incubator was adjusted to 100 rpm and the temperature to 37°C. The test samples were placed in the shaking incubator for drug release testing, and timing was started. At specific time points (0, 1, 2, 4, 6, 9, 12, 24, 36, 48, and 72 h), 1 mL of the external solution was collected and labeled. After collection, the same volume of PBS solution was added. The fluorescence intensity of the collected sample solution was measured at 485 nm using an ELISA reader at specific time points. The drug concentration released at different time points was calculated by comparing the fluorescence intensity with the DOX standard curve, thus obtaining the drug release curve. The results are shown below. Figure 16 As shown.

[0091] from Figure 16 It can be seen that within 12 hours, all three types of drug-loaded nanoparticles were released relatively quickly, and the release then slowed down, with the release being almost complete after 48 hours; and the composite D3G7-DOX drug-loaded nanoparticles had a higher drug release rate.

[0092] Example 4: Loading macromolecular peptide drugs onto nanocarriers and composite nanocarriers and drug release testing

[0093] First, the large molecule polypeptide drug is synthesized, specifically including the following steps:

[0094] The synthesis of the KLAKLAKKLAKLAK (amino acid sequence as shown in SEQ ID NO: 1, T1) polypeptide includes the following steps:

[0095] ①Swelling resin: Weigh 300mg of Fmoc-D-Lys(BOC)-Wang Resin and put it into a polypeptide synthesis tube. Add activated N,N-dimethylformamide (DMF) and place it in a shaker to swell for 2.5~3h.

[0096] ② Deprotection: First, rinse repeatedly (3 times) with dichloromethane (DCM) and N,N-dimethylformamide. Then, add a deprotecting agent (hexahydropyridine:DMF=1:4) to the polypeptide synthesis tube and shake on a shaker for 10 minutes to remove the Fmoc group on the amino acid.

[0097] ③Detection: rinse repeatedly with DCM and DMF alternately (3 times), then add A (0.5 g of indanone is dissolved in 10 mL of anhydrous ethanol), B (20 g of phenol is dissolved in 5 mL of anhydrous ethanol), C (0.1 g of ascorbic acid is dissolved in 5 mL of anhydrous ethanol) each one drop and a small amount of resin into 1.5 mL centrifuge tube, heat the centrifuge tube in boiling water for 1 min; if the color of the resin turns purple, it proves that the Fmoc protecting group is removed;

[0098] ④Coupling: weigh 10 times the amount of amino acid and benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU) relative to the loading capacity of the resin in a 15 mL centrifuge tube, then add 10 mL of coupling agent (DMF: N-methyl morpholine (NMM) = 19:1) and pre-react for 10 min, then pour into the polypeptide synthesis tube and react on the shaker for 2 h;

[0099] ⑤Detection: rinse repeatedly with DCM and DMF alternately, each solvent for 3 times, then add Kaiser text detection reagent, if the color of the resin does not change, it proves that the amino acid is coupled;

[0100] ⑥Cyclic coupling: repeat steps ②, ③, ④, ⑤, so that all amino acids are completely coupled, after detecting the complete coupling of the last amino acid, remove the Fmoc protecting group with the deprotection agent;

[0101] ⑦Detection: repeat step ③;

[0102] ⑧Contraction: shrink the resin with methanol for 15 min, then transfer the shrunk resin to a 10 mL serum bottle, and add a magnet to the bottle;

[0103] ⑨Lysis: add 3 mL of lysis solution (2.5% ultrapure water + 2.5% triisopropylsilane + 92.5% trifluoroacetic acid + 2.5% ethanedithiol) and magnetically stir in an ice bath for 2.5-3 h, then suction filter, rinse the serum bottle with TFA, then blow dry the TFA with nitrogen, add ethyl ether to precipitate the polypeptide in the ether, then transfer the polypeptide to a centrifuge tube, centrifuge at 8000 r / min at 4°C, repeat three times, then transfer the polypeptide to a 1.5 mL centrifuge tube and seal with sealing film, overnight, so that the ether evaporates completely, and finally obtain the polypeptide drug T1.

[0104] Synthesis of lipoic acid-KLAKLAKKLAKLAK (amino acid sequence as shown in SEQ ID NO: 1, T2) polypeptide, including the following steps:

[0105] First, repeat the steps 1-6 in the polypeptide drug T1, then couple the small molecule lipoic acid: take 4 times the amount of lipoic acid and 10 times the amount of HBTU in a 15 mL centrifuge tube, then add 10 mL of coupling agent and react for 10 min, then pour into the polypeptide synthesis tube and react overnight on a shaker; then repeat the steps 7-8 in the T1 synthesis procedure to obtain the lipoic acid-modified polypeptide drug T2.

[0106] The synthesis of the polypeptide KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL (amino acid sequence as shown in SEQ ID NO: 2, T3) includes the following steps:

[0107] Synthesis according to the steps in the polypeptide drug T1 to obtain the polypeptide drug T3.

[0108] The synthesis of the lipoic acid-KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL (amino acid sequence as shown in SEQ ID NO: 2, T4) polypeptide includes the following steps:

[0109] Synthesis according to the steps in the polypeptide drug T2 to obtain the lipoic acid-modified polypeptide drug T4.

[0110] Mass spectrometry was performed on the above-synthesized polypeptide drugs T1, lipoic acid-modified polypeptide drug T2, polypeptide drug T3, and lipoic acid-modified polypeptide drug T4, and the results are shown in Figures 17-20 .

[0111] As can be seen from Figures 17-20 , the polypeptide drugs T1, lipoic acid-modified polypeptide drug T2, polypeptide drug T3, and lipoic acid-modified polypeptide drug T4 were successfully synthesized.

[0112] Then, drug loading tests were performed. Specifically, first, the four polypeptides were labeled: 10 mg of T1, T2, and T3 were dissolved in 5 mL of pure water, 1 μL of Cy5-NHS was added under vortex, and then 2 μL of triethylamine was added to adjust the pH to about 8-10; 10 mg of T4 was dissolved in 5 mL of pure water, 1 μL of Cy5-NHS was added under vortex, and then 2 μL of triethylamine was added to adjust the pH to about 8-10, and 800 μL of acetonitrile was added to aid dissolution. After stirring in the dark for 2 h, the reaction mixture was placed in a dialysis bag with a molecular weight cutoff of 1000, and was dialyzed in ultrapure water for 24 h, with the dialysis water being changed every 4 h. After dialysis, the solution was collected in a centrifuge tube, wrapped in tin foil to avoid light, and placed in a freeze dryer for 3 days to obtain the Cy5-NHS-labeled four polypeptides.

[0113] Then the four polypeptides were loaded with drugs: 5.4 mg of DLA and 12.6 mg of GLA were weighed and dissolved in 500 μL of a mixed solution of DMSO and THF to prepare a D3G7 carrier solution, and then 2 mg of T1 was weighed and added to the D3G7 carrier solution, and the obtained carrier D3G7-T1 solution was stirred in the dark for 2 h; the other three polypeptides were operated according to the same method, and then the four groups of samples were dialyzed according to the above method, and finally freeze-dried to obtain D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles. The drug loading rate and encapsulation efficiency of the D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles are shown in Table 1. Figure 21

[0114] As can be seen from Table 1, the four drug-loaded nanoparticles have good drug loading rate and encapsulation efficiency; and the D3G7-T2 drug-loaded nanoparticles have better drug loading rate and encapsulation efficiency. Figure 21

[0115] Finally, the drug release test was performed. Specifically, 1 mL of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 were respectively loaded into dialysis bags with a molecular weight cutoff of 1000, and the two ends were tightly tied with cotton thread and then immersed in a PBS buffer medium. Three parallel experiments were performed for each carrier. The rotation speed parameter of the constant temperature shaking table was adjusted to 100 rpm, and the temperature was adjusted to 37°C. The above-mentioned samples to be tested were placed in the shaking table for drug release experiment and timing started. At specific time points (0.5, 1, 2, 4, 6, 9, 12, 24, 36, 48 h), 1 mL of external liquid was taken and labeled. After taking, the same volume of PBS solution was added. The fluorescence intensity of the collected sample solution was measured at 630 nm by using a microplate reader at a specific time point, and the drug concentration released at different time points was calculated by the fluorescence intensity and the standard curve of the corresponding Cy5-NHS labeled polypeptide, so as to obtain the drug release curve, as shown in Table 2. Figure 22

[0116] As can be seen from Table 2, within 12 h, the four drug-loaded nanoparticles are released relatively quickly, and then the release slows down, and after 48 h, the release is basically complete; and the D3G7-T2 drug-loaded nanoparticles have a higher drug release rate. Figure 22

[0117] Example 5 Cell toxicity test

[0118] 5.1 Cell toxicity test of DLA, GLA nanocarrier and D3G7 complex nanocarrier

[0119] ​​​​MTT method was used for cell toxicity detection, specifically, 200 μL PBS was added to the outer circle of the 96-well plate, and well-grown HeLa cells were inoculated in the 96-well plate, about 7000 cells per 100 μL of culture medium per well. Incubate in the incubator for 12 h, and then add 8 concentration gradients of DLA, GLA, D3G7 solution, set the maximum concentration to 150 μg / mL, set 5 replicate wells for each concentration, and set zero holes and control holes at the same time. After 24 h of cell-drug interaction, the culture medium was discarded and washed with PBS for 3 times. Then 100 μL of 0.05 mg / mL MTT solution was added to each well, and 2-3 h later, the absorbance of each well was measured at 490 nm using a microplate reader, and the optical density of each well was obtained, and the survival rate of the cells was calculated, and the results are shown in Figure 23 .

[0120] As can be seen from Figure 23 , DLA, GLA nanocarriers and D3G7 composite nanocarriers have low toxicity to cells, and therefore have good biological safety.

[0121] 5.2 D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles and cell toxicity test of free DOX

[0122] In step 5.1, MTT method was used for cell toxicity detection of D-DOX, G-DOX, D3G7-DOX drug-loaded nanoparticles and free DOX, and the maximum concentration of DOX in the nanocarrier was set to 30 μg / mL, and the survival rate of the cells was as shown in Figure 24 .

[0123] As can be seen from Figure 24 , when the concentration of DOX contained in the nanocarrier is 0.938 μg / mL or more, D-DOX, G-DOX, D3G7-DOX drug-loaded nanoparticles and free DOX have greater toxicity to cells, and compared with free DOX, drug-loaded nanoparticles have greater toxicity to cells. The above results show that D-DOX, G-DOX, D3G7-DOX drug-loaded nanoparticles have stronger toxicity to cells, which is conducive to killing tumor cells; and the drug-loaded nanoparticles have better drug utilization rate.

[0124] 5.3 Cell toxicity test of polypeptide drugs T1, lipoic acid modified polypeptide drugs T2, polypeptide drugs T3 and lipoic acid modified polypeptide drugs T4

[0125] In step 5.1, MTT method was used for cell toxicity detection of T1, T2, T3, T4, wherein the maximum concentration of polypeptide was set to 150 μg / mL, and the survival rate of the cells was as shown in Figure 25 .

[0126] As can be seen from Figure 25 , with the increase of the concentration of free polypeptide drug T1, T2, T3, T4, its toxicity to cells is enhanced.

[0127] 5.4 Cell toxicity test of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles

[0128] In step 5.1, MTT method was used to detect the cell toxicity of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4, wherein the maximum concentration of polypeptide in the carrier was set to 150 μg / mL, and the survival rate of cells was as shown in Figure 26 .

[0129] As can be seen from Figure 26 , compared with free polypeptide drugs T1, T2, T3, T4, D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles have stronger toxicity to cells, and the results show that the utilization rate of the drug can be improved by loading free polypeptide drugs.

[0130] Example 6 In vitro cell uptake test of D3G7-DOX drug-loaded nanoparticles

[0131] The uptake of D3G7-DOX drug-loaded nanoparticles in Hela cells was analyzed by laser confocal microscope (CLSM). Specifically, first, Hela cell suspension was inoculated in 3 confocal dishes (1 x 10 5 cells / well), and after incubation for 24 hours for adherent growth, the culture medium was aspirated, washed with PBS for 3 times, and then D3G7-DOX solution with a prepared adriamycin concentration of 2 μg / mL was added to the culture dish, 2 mL of drug-containing medium was added to each dish. The drug action time gradient of the 3 confocal dishes was set to 2 h, 6 h and 12 h, respectively. After the drug action was completed, PBS was washed for 3 times, and then PBS was gently aspirated, and 1 mL of 4% paraformaldehyde tissue fixing solution was added. After 30 minutes of fixation, the fixing solution was discarded, and then PBS was washed for 3 times, and finally CLSM was used to obtain the fluorescence signal (red fluorescence) of DOX under the excitation of 488 nm wavelength, so as to observe the drug uptake in cells, and the results are shown in Figure 27A and 27B .

[0132] As can be seen from Figure 27A and 27B , with the increase of incubation time, the relative fluorescence intensity in cells is gradually enhanced, and the results show that the D3G7-DOX drug-loaded nanoparticles are easy to be taken up by cells.

[0133] To sum up, by using lipoic acid to react with cholesterol and L-arginine methyl ester respectively, the present application obtains a nano-carrier with moderate particle size, which has good targeting property, high drug loading capacity and good bioavailability; further, by compounding the nano-carrier, a composite nano-carrier is obtained, which has good affinity to target cells; and the nano-carrier and the composite nano-carrier can not only load small molecule drugs, but also load macromolecular polypeptide drugs, thus having good application prospect.

[0134] It should be noted that each of the above embodiments belongs to the same inventive concept, and the description of each embodiment has its own emphasis. If the description in an individual embodiment is not exhaustive, the description in other embodiments can be referred to.

[0135] The above-described embodiments only express the implementation of the present application, and the description is more specific and detailed, but it should not be construed as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the scope of protection of the present application patent should be subject to the appended claims.

Claims

1. A composite nanocarrier, characterized in that, The composite nanocarrier is obtained by mixing a nanocarrier having the structure shown in Formula (Ⅰ) with a nanocarrier having the structure shown in Formula (Ⅱ) at a mass ratio of (1-5):(5-9); The structures shown in equation (Ⅰ) and equation (Ⅱ) are as follows: 、 。 2. The composite nanocarrier according to claim 1, characterized in that, The method for preparing the nanocarrier having the structure shown in Formula (Ⅰ) includes the following steps: S1. Using lipoic acid and cholesterol as raw materials, the reaction is carried out under the action of the first catalyst and the first activator, and after separation and purification, lipoic acid modified cholesterol is obtained. S2. The cholesterol modified with lipoic acid is dissolved in water and self-assembled to obtain a nanocarrier with the structure shown in formula (I).

3. The composite nanocarrier according to claim 2, characterized in that, In step S1, the molar ratio of the lipoic acid, the cholesterol, the first catalyst, and the first activator is (0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5), and the first catalyst comprises 4-dimethylaminopyridine, and the first activator comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

4. The composite nanocarrier according to claim 2, characterized in that, In step S1, the reaction under the action of the first catalyst and the first activator specifically includes: reacting for 20-28 hours at room temperature and a rotation speed of 500-700 r / min.

5. The composite nanocarrier according to claim 1, characterized in that, The method for preparing the nanocarrier having the structure shown in Formula (II) includes the following steps: S1. Using lipoic acid and L-arginine methyl ester as raw materials, the reaction is carried out under the action of a second catalyst and a second activator, and after separation and purification, lipoic acid-modified L-arginine methyl ester is obtained. S2. The thioctic acid-modified L-arginine methyl ester is dissolved in water and self-assembled to obtain a nanocarrier with the structure shown in formula (II).

6. The composite nanocarrier according to claim 5, characterized in that, In step S1, the molar ratio of the lipoic acid, the L-arginine methyl ester, the second catalyst, and the second activator is (1.5-2.5):(0.5-1.5):(0.5-1.5):(1.5-2.5), and the second catalyst comprises 4-dimethylaminopyridine, and the second activator comprises N,N-carbonyldiimidazole.

7. The composite nanocarrier according to claim 5, characterized in that, In step S1, the reaction under the action of the second catalyst and the second activator specifically includes: reacting for 6-12 hours at room temperature and a rotation speed of 500-700 r / min.

8. A drug-loaded nanoparticle, characterized in that, The drug-loaded nanoparticles are obtained by loading drugs onto composite nanocarriers according to any one of claims 1-7.

9. The drug-loaded nanoparticles according to claim 8, characterized in that, The drugs include small molecule drugs and / or large molecule peptide drugs; The small molecule drug includes doxorubicin, and the large molecule polypeptide drug is selected from any of the following: A1) A polypeptide with the amino acid sequence shown in SEQ ID NO: 1; A2) A polypeptide with lipoic acid coupled to one end of the amino acid sequence shown in SEQ ID NO: 1; A3) A polypeptide with the amino acid sequence shown in SEQ ID NO: 2; A4) A polypeptide with thioctic acid coupled to one end of the amino acid sequence shown in SEQ ID NO:

2.

10. The use of the drug-loaded nanoparticles as described in claim 8 or 9 in the preparation of antitumor drugs.

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