Nano-carrier, drug-loaded nanoparticles and preparation method and application of nano-carrier and drug-loaded nanoparticles

Abstract

The invention discloses a nano-carrier, a drug-loaded nanoparticle and a preparation method and application of the drug-loaded nanoparticle, and belongs to the technical field of biological medicine. According to the invention, lipoic acid is respectively reacted with cholesterol and L-arginine methyl ester to obtain the nano-carrier with moderate particle size, and the nano-carrier has better targeting property, high drug loading capacity and better bioavailability; furthermore, after the nano-carriers are compounded, a composite nano-carrier is obtained, and the composite nano-carrier has relatively good target cell affinity; moreover, the nano-carrier and the composite nano-carrier not only can load micromolecular drugs, but also can load macromolecular polypeptide drugs, so that the nano-carrier and the composite nano-carrier have a better application prospect in preparation of antitumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nanocarrier, drug-loaded nanoparticles, and a preparation method and application thereof. Background Art

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

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

[0004] However, current nanocarrier drug delivery systems have problems such as poor targeting, poor drug loading, and low bioavailability. Summary of the invention

[0005] The purpose of the present invention is to provide a nanocarrier, drug-loaded nanoparticles, and preparation methods and applications thereof. In the present invention, lipoic acid is used to react with cholesterol and L-arginine methyl ester respectively to obtain a nanocarrier with a moderate particle size, the nanocarrier has good targeting, high drug loading, and good bioavailability; further, after the above nanocarrier is compounded, a composite nanocarrier is obtained, the composite nanocarrier has good target cell affinity; and the above nanocarrier and composite nanocarrier can not only load small molecule drugs, but also load large molecule polypeptide drugs, therefore, it has good application prospects.

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

[0007] In the present invention, the inventors have found that lipoic acid, as a natural small molecule, has good biocompatibility and antioxidant effects. Because it has abundant dynamic covalent disulfide bonds, the disulfide bonds are in a dynamic equilibrium state, so there are always some exchanges between the bonding state and the cleavage state, and the disulfide bonds can trigger the exchange reaction of the chain under high temperature, ultraviolet light and redox reaction. Based on this, lipoic acid is used in the present invention to modify cholesterol and L-arginine methyl ester respectively to obtain a nanocarrier with a moderate particle size, and the nanocarrier has good targeting, high drug loading, and good bioavailability and biological responsiveness, therefore, it is possible to achieve targeted delivery of drugs.

[0008] In a second aspect, the present invention provides a composite nanocarrier, which is obtained by mixing the above-mentioned nanocarrier having a structure represented by formula (I) with the nanocarrier having a structure represented by formula (II) in a mass ratio of (1-5):(5-9).

[0009] In the present invention, the inventors further studied and found that the cholesterol nanocarrier modified with lipoic acid has a negative charge, and the L-arginine methyl ester nanocarrier modified with lipoic acid has a positive charge. After the two nanocarriers are compounded, part of the charge can be neutralized, so that the compounded composite nanocarrier has a stronger affinity with the target cells; in addition, the L-arginine methyl ester nanocarrier modified with lipoic acid contains guanidinium cations, which can enhance the cell penetration activity and facilitate the nanocarrier to enter the target cell more conveniently and quickly; and the cholesterol nanocarrier modified with lipoic acid can provide a hydrophobic effect, enhance the stability of the nanocarrier and the interaction with the cell membrane; the above two nanocarriers act synergistically, improve the targeting, and significantly improve the bioavailability of the loaded drug.

[0010] In a third aspect, the present invention provides a method for preparing the above-mentioned nanocarrier, 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 obtaining lipoic acid-modified cholesterol after separation and purification; 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 obtaining lipoic acid-modified L-arginine methyl ester after separation and purification; S2, dissolving lipoic acid-modified cholesterol and lipoic acid-modified L-arginine methyl ester in water respectively, and obtaining a nanocarrier after self-assembly.

[0011] In the present invention, the preparation process of lipoic acid-modified cholesterol is shown in the following formula (1):

[0012] The preparation process of lipoic acid-modified L-arginine methyl ester is shown in the following formula (2):

[0013] The preparation method provided by the invention is simple, the raw materials used are green, non-toxic, cheap and easily available, and have good biological safety, and therefore, are convenient for large-scale production and application.

[0014] 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 includes 4-dimethylaminopyridine, and the first activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0015] In some preferred embodiments, the molar ratio of lipoic acid, cholesterol, the first catalyst, and the first activator is 1:1:1:1.

[0016] In some embodiments, in step S1, reacting 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.

[0017] In some preferred embodiments, the reaction under the action of the first catalyst and the first activator specifically includes: reacting for 24 hours at room temperature and a rotation speed of 600 r / min.

[0018] It is understandable that the reaction is carried out in an organic solvent, and the organic solvent can be selected from conventional organic solvents. In the present invention, the organic solvent is preferably dichloromethane.

[0019] In some embodiments, obtaining lipoic acid-modified cholesterol after separation and purification specifically includes: adding saturated sodium chloride solution to the obtained product for shaking, washing the obtained lower layer liquid, and then concentrating under reduced pressure, continuing to add anhydrous methanol to the obtained solid powder for pulping, and finally drying the obtained solid to obtain lipoic acid-modified cholesterol.

[0020] 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 includes 4-dimethylaminopyridine, and the second activator includes N,N-carbonyldiimidazole.

[0021] 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.

[0022] In some embodiments, in step S1, reacting 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.

[0023] In some preferred embodiments, the reaction under the action of the second catalyst and the second activator specifically includes: reacting for 9 hours at room temperature and a rotation speed of 600 r / min.

[0024] It is understandable that the reaction carried out under the action of the second catalyst and the second activator is carried out in an organic solvent, and the organic solvent can be a conventional organic solvent. In the present invention, the organic solvent is preferably N,N-dimethylformamide.

[0025] In some embodiments, obtaining lipoic acid-modified L-arginine methyl ester after separation and purification specifically includes: adding glacial ether to the obtained product for precipitation, washing the obtained precipitate after centrifugation, and drying to obtain lipoic acid-modified L-arginine methyl ester.

[0026] In a fourth aspect, the present invention provides a drug-loaded nanoparticle, wherein the drug-loaded nanoparticle is obtained by loading the drug on the above-mentioned nanocarrier, the above-mentioned composite nanocarrier or a nanocarrier prepared by any of the above-mentioned preparation methods.

[0027] In some embodiments, the drug includes a small molecule drug and / or a large molecule polypeptide drug; wherein the small molecule drug includes 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 the 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; A4) a polypeptide having lipoic acid coupled to one end of the amino acid sequence as shown in SEQ ID NO: 2.

[0028] It is understandable that small molecule drugs and large molecule polypeptide drugs can be routinely selected according to the type of target cells.

[0029] In a fifth aspect, the present invention provides the use of the above-mentioned drug-loaded nanoparticles in the preparation of anti-tumor drugs.

[0030] The beneficial effects of the present invention are as follows: different from the prior art, the present invention uses lipoic acid to react with cholesterol and L-arginine methyl ester respectively to obtain a nanocarrier with a moderate particle size, the nanocarrier has good targeting, a high drug loading capacity, and good bioavailability; the above-mentioned nanocarrier can be loaded with not only small molecule drugs but also large molecule polypeptide drugs, therefore, it has good application prospects in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The DLA powder synthesized in Example 1 of the present invention is 1 H-NMR nuclear magnetic spectrum (CDCl3400Hz); Figure 2 The GLA powder synthesized in Example 1 of the present invention is 1 H-NMR (D2O 400Hz); Figure 3 This is a diagram of the hydrated particle size of the DLA nanocarrier in Example 1 of the present invention under different pH conditions; Figure 4 TEM images of the DLA nanocarrier in Example 1 of the present invention under different pH conditions; Figure 5 This is a diagram of the hydrated particle size of the DLA nanocarrier in Example 1 of the present invention under different concentration conditions; Figure 6 TEM images of the DLA nanocarriers in Example 1 of the present invention at different concentrations; Figure 7 This is a graph showing the hydrated particle size of the DLA nanocarrier in Example 1 of the present invention under different time conditions; Figure 8 TEM images of the DLA nanocarrier in Example 1 of the present invention under different time conditions; Fig. 9 This is a diagram of the hydrated particle size of the DLA nanocarrier in Example 1 of the present invention under different temperature conditions; Fig.10 TEM images of the DLA nanocarrier in Example 1 of the present invention under different temperature conditions; Fig.11 Schematic diagram of the synthesis of the composite nanocarrier and its drug loading in Example 2 of the present invention; Fig.12 This is a diagram of the hydrated particle size of composite nanocarriers mixed in different proportions in Example 2 of the present invention; Fig.13 TEM images of composite nanocarriers mixed in different proportions in Example 2 of the present invention; Fig.14 The Zeta potential diagram of the composite nanocarriers mixed in different proportions in Example 2 of the present invention; Fig.15 The drug loading rate and encapsulation rate of D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles in Example 3 of the present invention are shown in FIG. Fig.16 The drug release curves of D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles in Example 3 of the present invention; Fig.17 is the mass spectrum of the polypeptide drug T1 in Example 4 of the present invention; Fig.18 is the mass spectrum of the lipoic acid-modified polypeptide drug T2 in Example 4 of the present invention; Fig.19 is the mass spectrum of the polypeptide drug T3 in Example 4 of the present invention; Fig. 20 This is the mass spectrum of the lipoic acid-modified polypeptide drug T4 in Example 4 of the present invention; Fig.21 The drug loading rate and encapsulation rate result diagram of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles in Example 4 of the present invention; Fig. 22 The drug release curves of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles in Example 4 of the present invention; Fig.23 This is a graph showing the survival rate of cells incubated with DLA, GLA nanocarriers and D3G7 composite nanocarriers in Example 5 of the present invention; Fig.24 This is a graph showing the survival rate of D-DOX, G-DOX, D3G7-DOX drug-loaded nanoparticles and free DOX after incubation with cells in Example 5 of the present invention; Fig.25 This is a graph showing the survival rate of cells incubated with polypeptide drugs T1, T2, T3, and T4 in Example 5 of the present invention; Fig.26 This is a graph showing the survival rate results of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles after incubation with cells in Example 5 of the present invention; Fig.27A This is a laser confocal image of D3G7-DOX drug-loaded nanoparticles after incubation with cells in Example 6 of the present invention; Fig.27B This is a graph of the relative fluorescence intensity of D3G7-DOX drug-loaded nanoparticles after incubation with cells in Example 6 of the present invention. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0034] Example 1 Synthesis of Nanocarriers 1.1 Synthesis of lipoic acid-modified cholesterol (DLA) nanocarriers First, synthesize DLA: accurately weigh 1mmol lipoic acid (TA), 1mmol cholesterol, 1mmol 4-dimethylaminopyridine (DMAP), and 1mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) in a 100mL round-bottom flask, add 50mL dichloromethane (DCM) to the round-bottom flask, put in a magnetic bar, stir and react for 24h at room temperature and a speed of 600r / min; take 25mL of the reaction solution and pour it into a separatory funnel, add it to 150mL of saturated sodium chloride solution, shake it and release the gas, collect the lower layer of liquid in a pear-shaped bottle, extract and wash it three times, and the remaining 25mL of the reaction solution is stirred and reacted. The liquid was extracted and washed three times in the same way; the lower layer liquid collected twice was concentrated under reduced pressure to obtain a light yellow powder, 50 mL of anhydrous methanol was added to the light yellow powder for slurrying, and then the solid-liquid mixture was collected in a 50 mL centrifuge tube, centrifuged at a speed of 8000 r / min for 10 min, the supernatant was poured out, and 50 mL of anhydrous methanol was added for slurrying, and this operation was repeated three times; finally, the supernatant was poured out, and the solid was placed in a vacuum drying oven and dried for more than 3 h to obtain DLA powder.

[0035] Then, DLA nanocarriers were synthesized: 50 μL of DLA solution (1 mg of DLA powder was weighed and dissolved in 500 μL of tetrahydrofuran) was slowly dripped into 950 μL of vortexed Wahaha pure water, and 10 μL of 1 mg / mL iodoacetamide solution (1 mg of iodoacetamide was weighed and dissolved in 1 mL of dimethyl sulfoxide) was added to prevent depolymerization, a magnetic particle was added, and the mixture was stirred at room temperature for 12 h to obtain DLA nanocarriers.

[0036] 1.2 Synthesis of lipoic acid-modified L-arginine methyl ester (GLA) nanocarriers First, synthesize GLA: accurately weigh 2mmol lipoic acid and 2mmol N,N-carbonyldiimidazole (CDI) in a 10mL round-bottom flask, add 5mL N,N-dimethylformamide (DMF), put in a magnetic bar, stir for 0.5h at room temperature and a speed of 600r / min, then add 1mmol L-arginine methyl ester and 1mmol 4-dimethylaminopyridine, continue to stir and react for 9 hours at room temperature and a speed of 600 r / min; slowly drip the reaction solution into ice ether, put it in a refrigerator for 0.5 hour to completely precipitate it, and then centrifuge it at a speed of 8000 r / min for 10 minutes, and pour out the supernatant; use a washing liquid (dichloromethane and ethylene glycol monopropyl ether are mixed in a volume ratio of 1:2) to wash, precipitate a yellow paste, and then put it into a centrifuge, centrifuge it at a speed of 8000 r / min for 10 minutes, pour out the supernatant, repeat the operation three times, and finally obtain a yellow paste; put it into a vacuum drying oven and dry it for more than 3 hours to obtain GLA powder.

[0037] Then, GLA nanocarriers were synthesized: 50 μL of GLA solution (1 mg of GLA powder was weighed and dissolved in 500 μL of dimethyl sulfoxide) was slowly dripped into 950 μL of vortexed Wahaha pure water, and 10 μL of 1 mg / mL iodoacetamide solution (1 mg of iodoacetamide was weighed and dissolved in 1 mL of dimethyl sulfoxide) was added to prevent depolymerization, a magnetic particle was added, and the mixture was stirred at room temperature for 12 h to obtain GLA nanocarriers.

[0038] Performance Characterization The DLA powder synthesized in step 1.1 and the GLA powder synthesized in step 1.2 were tested by nuclear magnetic resonance hydrogen spectrum, and the results were as follows: Figure 1 and 2 shown.

[0039] from Figure 1 and 2 As can be seen, both DLA powder and GLA powder were successfully prepared.

[0040] For example, the hydrated particle size and TEM of DLA nanocarriers synthesized under different conditions were tested. The hydrated particle size was detected by a Malvern particle size analyzer, and the TEM test included the following steps: 10 μL of the sample to be tested was dripped onto a copper mesh, and the copper mesh was washed twice with clean water after 10 minutes, and then uranyl acetate was dripped onto the copper mesh for staining for 10 minutes, washed twice, and then tested using a 120kv high-resolution transmission electron microscope after natural air drying.

[0041] Specifically, the pH value of pure water was first optimized, and buffer solutions with pH values ​​of 3, 4, 5, 6, 7, 8, 9, 10, and 11 were prepared (the pH value was adjusted with HCl and NaOH), and 50 μL of DLA solution (obtained by weighing 1 mg of DLA powder and dissolving it in 500 mL of tetrahydrofuran) was slowly dripped into 950 μL of the vortexed buffer solutions with different pH values, and 10 μL of 1 mg / mL iodoacetamide solution (obtained by weighing 1 mg of iodoacetamide and dissolving it in 1 mL of dimethyl sulfoxide) was added to prevent depolymerization, a magnetic particle was added, and the mixture was stirred at room temperature for 12 h to obtain DLA nanocarriers. The hydrated particle sizes and TEM results of the DLA nanocarriers under different pH conditions were shown as follows: Figure 3 and 4 shown.

[0042] from Figure 3 and 4 It can be seen that when the pH value of the buffer is 7, the particle size of the DLA nanocarrier is the smallest and the dispersion is good.

[0043] Then, the concentration of DLA solution was optimized, and DLA tetrahydrofuran solutions of different concentrations were prepared (0.03, 0.05, 0.1, 0.15, 0.18, 0.2, 0.5, 1.0, 1.2, 1.5 mg / mL). 50 μL of the above DLA tetrahydrofuran solutions of different concentrations were slowly dripped into 950 μL of pH 7 buffer, and 10 μL of 1 mg / mL iodoacetamide solution (1 mg iodoacetamide was weighed and dissolved in 1 mL dimethyl sulfoxide) was added to prevent depolymerization. A magnetic particle was added, and the mixture was stirred 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 were shown as follows: Figure 5 and 6 shown.

[0044] from Figure 5 and 6 It can be seen that when the concentration of DLA tetrahydrofuran solution is 0.15 mg / mL, the particle size of DLA nanocarrier is the smallest and the dispersion is good.

[0045] Next, the stirring time was optimized, 50 μL of DLA tetrahydrofuran solution (0.15 mg / mL) was slowly dripped into 950 μL of vortexed pH 7 buffer, and 10 μL of 1 mg / mL iodoacetamide solution (1 mg of iodoacetamide was weighed and dissolved in 1 mL of dimethyl sulfoxide) was added to prevent depolymerization, and a magnetic bar was added. The mixture was stirred at room temperature for different times (0, 2, 4, 8, 12, 24, 48, and 72 h) to obtain DLA nanocarriers. The hydrated particle size and TEM results of the DLA nanocarriers under different stirring time conditions are shown as follows: Figure 7 and 8 shown.

[0046] from Figure 7 and 8 It can be seen that when the stirring time is 4h, the particle size of DLA nanocarriers is the smallest and the dispersion is good.

[0047] Finally, the stirring temperature was optimized, 50 μL of DLA tetrahydrofuran solution (0.15 mg / mL) was slowly dripped into 950 μL of vortexed pH 7 buffer, and 10 μL of 1 mg / mL iodoacetamide solution (1 mg iodoacetamide was weighed and dissolved in 1 mL dimethyl sulfoxide) was added to prevent depolymerization, and a magnetic particle was added. 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 as follows: Fig. 9 and 10 shown.

[0048] from Fig. 9 and 10 It can be seen that when the stirring temperature is 30°C, the particle size of the DLA nanocarrier is the smallest and the dispersion is good.

[0049] Example 2 Synthesis of composite nanocarriers See also Fig.11 , which is a schematic diagram of the synthesis of a composite nanocarrier and its drug loading. Specifically, the synthesis of the composite nanocarrier includes the following steps: First, a 0.15 mg / mL DLA tetrahydrofuran solution and a 0.15 mg / mL GLA dimethyl sulfoxide solution were prepared respectively, and then the above solutions were respectively taken to prepare mixed solutions according to the volume ratios of DLA tetrahydrofuran solution and GLA dimethyl sulfoxide solution of 0:10, 1:9, 3:7, 5:5, 7:3, 9:1, and 10:0, respectively. 50 μL of the above mixed solution was slowly dripped into 950 μL of a vortexed pH 7 buffer, and 10 μL of a 1 mg / mL iodoacetamide solution (1 mg of iodoacetamide was weighed and dissolved in 1 mL of dimethyl sulfoxide) was added to prevent depolymerization, and a magnetic particle was added. The mixture was stirred for 4 hours at a temperature of 30°C to obtain a composite nanocarrier. The hydrated particle size, Zeta potential and TEM results of the composite nanocarrier after mixing in different proportions are shown as follows: Figure 12-14 shown.

[0050] from Figure 12-14 It can be seen that when the mass ratio of DLA nanocarrier to GLA nanocarrier is (1-5): (5-9), the composite nanocarrier has a smaller particle size and is well dispersed; and when the mass ratio of DLA nanocarrier to GLA nanocarrier is 3:7, the composite nanocarrier has the smallest particle size and is well dispersed. This composite nanocarrier is named D3G7.

[0051] Example 3 Nanocarriers and composite nanocarriers loaded with small molecule drugs and drug release test First, a drug loading test was carried out. Specifically, 2 mg doxorubicin (DOX) and 18 mg DLA were weighed and dissolved in 500 μL of a mixed solution of tetrahydrofuran and dimethyl sulfoxide as a carrier D-DOX solution; 2 mg DOX and 18 mg GLA were dissolved in 500 μL of dimethyl sulfoxide as a carrier G-DOX solution; 2 mg DOX, 5.4 mg DLA, and 12.6 mg GLA were dissolved in 500 μL of a mixed solution of tetrahydrofuran and dimethyl sulfoxide as a carrier D3G7-DOX solution. The three carrier solutions were stirred at room temperature in the dark for 2 h, slowly added dropwise to 9.5 mL of Wahaha pure water with a pipette, and then placed at room temperature in the dark for 12 h. The mixed solution after the reaction was placed in a dialysis bag with a molecular weight cutoff of 1000, placed in ultrapure water for 24 h in the dark, and the ultrapure water was replaced every 4 h. After the dialysis, the solution was collected and placed in a centrifuge tube with tin foil to avoid light, and then placed in a freeze dryer for lyophilization for 3 days to obtain D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles, respectively. The drug loading rate and encapsulation rate of the D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles are shown in Figure 2. Fig.15 shown.

[0052] from Fig.15 It can be seen that the three drug-loaded nanoparticles have good drug loading rate and encapsulation rate; and the composite D3G7-DOX drug-loaded nanoparticles have better drug loading rate and encapsulation rate.

[0053] Then, a drug release test was conducted. Specifically, 1 mL of D-DOX, G-DOX and D3G7-DOX drug-loaded nanoparticles were loaded into dialysis bags with a molecular weight cutoff of 1000, respectively. The two ends were tied tightly with cotton thread and immersed in PBS buffer medium. Three parallel experiments were performed for each carrier. The speed parameter of the constant temperature oscillating shaker was adjusted to 100 rpm, and the temperature was adjusted to 37°C. The above-mentioned samples to be tested were placed in the shaker for drug release experiments and the timing was started. At specific time points (0, 1, 2, 4, 6, 9, 12, 24, 36, 48, 72 h), 1 mL of external solution was taken and marked. After taking, an equal volume of PBS solution was added. The fluorescence intensity of the collected sample solution was measured at 485 nm at a specific time point using an enzyme marker, and the drug concentration released at different time points was calculated by comparing the fluorescence intensity with the DOX standard curve to obtain the drug release curve. The results are shown in FIG. Fig.16 shown.

[0054] from Fig.16It can be seen that within 12 hours, the three drug-loaded nanoparticles released rapidly, then the release slowed down, and the release was basically complete after 48 hours; and the composite D3G7-DOX drug-loaded nanoparticles had a higher drug release rate.

[0055] Example 4 Nanocarriers and Composite Nanocarriers Loading Macromolecular Peptide Drugs and Drug Release Test First, the macromolecular polypeptide drug is synthesized, specifically, including the following steps: The synthesis of KLAKLAKKLAKLAK (amino acid sequence as shown in SEQ ID NO: 1, T1) polypeptide comprises the following steps: ① Swelling resin: Weigh 300 mg of Fmoc-D-Lys(BOC)-Wang Resin resin into a peptide synthesis tube, add activated N,N-dimethylformamide (DMF), and place it in a shaker to swell for 2.5~3 hours; ② Deprotection: First, rinse with dichloromethane (DCM) and N,N-dimethylformamide alternately (3 times), then add a deprotecting agent (hexahydropyridine:DMF=1:4) to the peptide synthesis tube, shake on a shaker for 10 minutes to remove the Fmoc group on the amino acid; ③ Detection: Wash repeatedly with DCM and DMF alternately (3 times), then take one drop each of A (0.5g ninhydrin dissolved in 10mL anhydrous ethanol), B (20g phenol dissolved in 5mL anhydrous ethanol), C (0.1g ascorbic acid dissolved in 5mL anhydrous ethanol) and a small amount of resin and add them to a 1.5mL centrifuge tube, and heat the centrifuge tube in boiling water for 1min; if the color of the resin turns purple, it proves that the Fmoc protecting group is removed; ④ Coupling: Weigh 10 times the equivalent of amino acid and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) relative to the resin loading into a 15mL centrifuge tube, then add 10mL of coupling agent (DMF:N-methylmorpholine (NMM) = 19:1) for pre-reaction for 10min, then pour into the peptide synthesis tube and place on a shaker for reaction for 2h; ⑤ Detection: Rinse repeatedly with DCM and DMF alternately, rinse with each solvent 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; ⑥ Cyclic coupling: Repeat steps ②, ③, ④, and ⑤ to fully couple all amino acids. After the last amino acid is fully coupled, remove the Fmoc protecting group with a deprotecting agent; ⑦ Detection: Repeat step ③; ⑧Shrinkage: Shrink the resin with methanol for 15 minutes, then transfer the shrunken resin to a 10 mL serum bottle and add a magnetic bar to the bottle; ⑨ Lysis: Add 3mL of lysis solution (2.5% ultrapure water + 2.5% triisopropylsilane + 92.5% trifluoroacetic acid + 2.5% ethanedithiol), stir magnetically in an ice bath for 2.5~3h, then filter, rinse the serum bottle with TFA, blow the TFA dry with nitrogen, add ice ether to precipitate the polypeptide in the ether, then transfer the polypeptide to a centrifuge tube, centrifuge at 4℃, 8000r / min, repeat three times, then transfer the polypeptide to a 1.5mL centrifuge tube, seal it with a sealing film, and leave it overnight to evaporate the ether, and finally obtain the polypeptide drug T1.

[0056] The synthesis of lipoic acid-KLAKLAKKLAKLAK (amino acid sequence as shown in SEQ ID NO: 1, T2) polypeptide comprises the following steps: First, repeat steps ①-⑦ in peptide drug T1, and then couple small molecule lipoic acid: weigh 4 equivalents of lipoic acid and 10 equivalents of HBTU in a 15mL centrifuge tube, then add 10mL of coupling agent to react for 10 minutes, then pour into a peptide synthesis tube and place on a shaker to react overnight; then repeat steps ⑧-⑨ in the synthesis steps of T1 to obtain lipoic acid-modified peptide drug T2.

[0057] The synthesis of KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL (amino acid sequence as shown in SEQ ID NO: 2, T3) polypeptide comprises the following steps: The peptide drug T3 is synthesized according to the steps in peptide drug T1.

[0058] The synthesis of lipoic acid-KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL (amino acid sequence as shown in SEQ ID NO: 2, T4) polypeptide comprises the following steps: The peptide drug T4 modified with lipoic acid was synthesized according to the steps in peptide drug T2.

[0059] Mass spectrometry was performed on the above-synthesized peptide drug T1, lipoic acid-modified peptide drug T2, peptide drug T3 and lipoic acid-modified peptide drug T4, and the results were as follows: Figure 17-20 shown.

[0060] from Figure 17-20 It can be seen that peptide drug T1, lipoic acid-modified peptide drug T2, peptide drug T3 and lipoic acid-modified peptide drug T4 were all successfully synthesized.

[0061] Then the drug loading test was carried out. Specifically, four polypeptides were labeled first: 10 mg T1, T2, and T3 were weighed and dissolved in 5 mL pure water, 1 μL Cy5-NHS was added in a vortex state, and then 2 μL triethylamine was added to adjust the pH to about 8-10; 10 mg T4 was weighed and dissolved in 5 mL pure water, 1 μL Cy5-NHS was added in a vortex state, and then 2 μL triethylamine was added to adjust the pH to about 8-10, and then 800 μL acetonitrile was added to assist dissolution. After the four groups of samples were stirred in the dark for 2 hours, the mixed solution after the reaction was placed in a dialysis bag with a molecular weight cutoff of 1000, placed in ultrapure water and dialyzed in the dark for 24 hours, and the ultrapure water was replaced every 4 hours. After the dialysis was completed, the solution was collected and placed in a centrifuge tube with tin foil to avoid light, and placed in a freeze dryer for freeze drying for three days to obtain four polypeptides labeled with Cy5-NHS.

[0062] Then, the drug loading of the four peptides was performed: 5.4 mg DLA and 12.6 mg GLA were first weighed and dissolved in 500 μL DMSO and THF mixed solution to prepare D3G7 carrier solution, and then 2 mg T1 was weighed and added to the D3G7 carrier solution, and the obtained carrier D3G7-T1 solution was stirred for 2 h in the dark; the other three peptides were operated in the same way, and 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 rate of the D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles are shown in Figure 2. Fig.21 shown.

[0063] from Fig.21 It can be seen that the four drug-loaded nanoparticles have good drug loading rate and encapsulation rate; and D3G7-T2 drug-loaded nanoparticles have better drug loading rate and encapsulation rate.

[0064] Finally, a drug release test was conducted. 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. The two ends were tied tightly with cotton thread and immersed in PBS buffer medium. Three parallel experiments were performed for each carrier. The speed parameter of the constant temperature oscillating shaker was adjusted to 100 rpm, and the temperature was adjusted to 37°C. The above-mentioned samples to be tested were placed in the shaker for drug release experiments and the timing was started. At specific time points (0.5, 1, 2, 4, 6, 9, 12, 24, 36, 48 h), 1 mL of external solution was taken and marked. After taking, an equal volume of PBS solution was added. The fluorescence intensity of the collected sample solution was measured at 630 nm at a specific time point using an enzyme marker, and the drug concentration released at different time points was calculated by comparing the fluorescence intensity with the standard curve of the corresponding Cy5-NHS-labeled peptide, thereby obtaining a drug release curve. The results are shown in FIG. Fig. 22 shown.

[0065] from Fig. 22 It can be seen that within 12 hours, the four drug-loaded nanoparticles all released rapidly, then the release slowed down, and the release was basically complete after 48 hours; and D3G7-T2 drug-loaded nanoparticles had a higher drug release rate.

[0066] Example 5 Cytotoxicity Test 5.1 Cytotoxicity test of DLA, GLA nanocarriers and D3G7 composite nanocarriers The MTT method was used for cytotoxicity detection. Specifically, after adding 200 μL PBS to the outermost circle of the 96-well plate, the well-grown HeLa cells were inoculated in the 96-well plate, and about 7,000 cells were inoculated in 100 μL of culture medium in each well. Incubate in an incubator for 12 hours. After it adheres to the wall, add 8 concentration gradients of DLA, GLA, and D3G7 solutions. The maximum concentration was set to 150 μg / mL, and 5 replicate wells were set for each concentration. Zero wells and control wells were also set. After the cells acted with the drug for 24 hours, the culture medium was discarded and carefully rinsed with PBS three times. Then 100 μL of 0.05 mg / mL MTT solution was added to each well. After 2-3 hours, the absorbance intensity of each well was measured at 490 nm using an enzyme reader to obtain the optical density of each well, thereby calculating the cell survival rate. The results are shown in Fig.23 shown.

[0067] from Fig.23 It can be seen that DLA, GLA nanocarriers and D3G7 composite nanocarriers have low toxicity to cells and therefore have good biosafety.

[0068] 5.2 Cytotoxicity test of D-DOX, G-DOX, D3G7-DOX drug-loaded nanoparticles and free DOX Similar to step 5.1, the cytotoxicity of D-DOX, G-DOX, D3G7-DOX loaded nanoparticles and free DOX was tested by MTT method, where the maximum concentration of DOX in the nanocarrier was set to 30 μg / mL. The cell survival rate results are shown in Fig.24 shown.

[0069] from Fig.24It can be seen that when the concentration of DOX contained in the nanocarrier is above 0.938 μg / mL, D-DOX, G-DOX, D3G7-DOX drug-loaded nanoparticles and free DOX are more toxic to cells, and compared with free DOX, drug-loaded nanoparticles are more toxic to cells. The above results show that D-DOX, G-DOX, and D3G7-DOX drug-loaded nanoparticles are more toxic to cells and are easier to kill tumor cells; and drug-loaded nanoparticles have better drug utilization.

[0070] 5.3 Cytotoxicity test of peptide drug T1, lipoic acid-modified peptide drug T2, peptide drug T3 and lipoic acid-modified peptide drug T4 Similar to step 5.1, the cytotoxicity of T1, T2, T3, and T4 was tested by MTT method, where the maximum concentration of the peptide was set to 150 μg / mL. The cell survival rate results were as follows: Fig.25 shown.

[0071] from Fig.25 It can be seen that as the concentration of free peptide drugs T1, T2, T3, and T4 increases, their toxicity to cells increases.

[0072] 5.4 Cytotoxicity test of D3G7-T1, D3G7-T2, D3G7-T3 and D3G7-T4 drug-loaded nanoparticles Similar to step 5.1, the cytotoxicity of D3G7-T1, D3G7-T2, D3G7-T3, and D3G7-T4 was tested by MTT method, where the maximum concentration of the peptide contained in the vector was set to 150 μg / mL. The cell survival rate results were as follows: Fig.26 shown.

[0073] from Fig.26 It can be seen that compared with free polypeptide drugs T1, T2, T3, and T4, D3G7-T1, D3G7-T2, D3G7-T3, and D3G7-T4 drug-loaded nanoparticles are more toxic to cells. The results show that the utilization rate of the drug can be improved by loading the free polypeptide drug.

[0074] Example 6 In vitro cellular uptake test of D3G7-DOX drug-loaded nanoparticles The uptake of D3G7-DOX-loaded nanoparticles in Hela cells was analyzed by confocal laser scanning microscopy (CLSM). Specifically, Hela cell suspensions were first inoculated in 3 confocal dishes (1×10 5 / well), incubate for 24 hours to allow it to adhere to the wall and grow, then aspirate the culture medium, wash three times with PBS, and then add the pre-prepared D3G7-DOX solution with a doxorubicin concentration of 2μg / mL to the culture dish, and add 2mL of drug-containing culture medium to each dish. The drug action time gradients of these three confocal dishes were set to 2h, 6h, and 12h, respectively. After the drug action is over, wash three times with PBS, then gently aspirate the PBS, and add 1mL of 4% paraformaldehyde tissue fixative. After fixation for 30 minutes, discard the fixative, wash three times with PBS, and finally use CLSM to excite at a wavelength of 488nm to obtain the fluorescence signal (red fluorescence) of DOX, so as to observe the intracellular drug uptake. The results are as follows. Fig.27A and 27B shown.

[0075] from Fig.27A and 27B It can be seen that with the increase of incubation time, the relative fluorescence intensity inside the cell gradually increased. The results showed that the D3G7-DOX drug-loaded nanoparticles were easily taken up by cells.

[0076] In summary, the present invention uses lipoic acid to react with cholesterol and L-arginine methyl ester respectively to obtain a nanocarrier with a moderate particle size, and the nanocarrier has good targeting, a high drug loading capacity, and good bioavailability; further, after the above nanocarriers are compounded, a composite nanocarrier is obtained, and the composite nanocarrier has good target cell affinity; and the above nanocarriers and composite nanocarriers can not only load small molecule drugs, but also load large molecule polypeptide drugs, and therefore have good application prospects.

[0077] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0078] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A nanocarrier, characterized in that: The nanocarrier is obtained by reacting lipoic acid with cholesterol and L-arginine methyl ester, and has a structure shown in the following formula (I) and / or formula (II): 、 。 2. A composite nanocarrier, characterized in that: The composite nanocarrier is obtained by mixing the nanocarrier having a structure represented by formula (I) in claim 1 with the nanocarrier having a structure represented by formula (II) in a mass ratio of (1-5):(5-9).

3. A method for preparing a nanocarrier as claimed in claim 1, characterized in that: The steps include: S1, using lipoic acid and cholesterol as raw materials, reacting under the action of a first catalyst and a first activator, and obtaining lipoic acid-modified cholesterol after separation and purification; 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 obtaining lipoic acid-modified L-arginine methyl ester after separation and purification; S2, dissolving the lipoic acid-modified cholesterol and the lipoic acid-modified L-arginine methyl ester in water respectively, and obtaining a nanocarrier after self-assembly.

4. The method for preparing a nanocarrier according to claim 3, 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 includes 4-dimethylaminopyridine, and the first activator includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

5. The method for preparing a nanocarrier according to claim 3, characterized in that: In step S1, reacting 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.

6. The method for preparing a nanocarrier according to claim 3, 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 includes 4-dimethylaminopyridine, and the second activator includes N,N-carbonyldiimidazole.

7. The method for preparing a nanocarrier according to claim 3, characterized in that: In step S1, reacting 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 on the nanocarriers according to claim 1, the composite nanocarriers according to claim 2, or the nanocarriers prepared by the preparation method of any one of claims 3-7.

9. The drug-loaded nanoparticles according to claim 8, characterized in that: The drugs include small molecule drugs and / or large molecule polypeptide drugs; Wherein, the small molecule drug includes doxorubicin, and the macromolecular 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 the amino acid sequence shown in SEQ ID NO: 1; A3) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2; A4) A polypeptide having lipoic acid coupled to one end of the amino acid sequence shown in SEQ ID NO:

2.

10. Use of the drug-loaded nanoparticles according to claim 8 or 9 in the preparation of anti-tumor drugs.

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