A berberine protein and its use as a nanocarrier for delivering drugs

Abstract

The present application relates to a berberine protein and its use as a nanocarrier for drug delivery. Specifically, the present application provides a berberine protein, which is prepared by the following method: (1) extracting berberine with water to obtain a water extract; (2) sequentially centrifuging and filtering the water extract, and then dialyzing the filtrate to obtain the berberine protein. The berberine protein of the present application used as a nanocarrier for drug delivery can significantly improve the solubility, dissolution rate and oral bioavailability of docetaxel, thereby improving the oral application value of docetaxel.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically, to a berberine and its use as a nanocarrier for drug delivery. Background Technology

[0002] Docetaxel is a semi-synthetic taxane drug that primarily inhibits tumor cell proliferation by inducing tubulin polymerization into microtubules and inhibiting microtubule depolymerization. Docetaxel is one of the world's most valuable anti-cancer drugs, mainly used clinically to treat locally advanced or metastatic breast cancer. Due to its poor solubility and significant first-pass metabolism, docetaxel has extremely low oral bioavailability, therefore it is primarily administered by injection. However, injection administration leads to excessively high instantaneous blood concentrations, often causing severe adverse hematological reactions, primarily neutropenia. Furthermore, the cosolvent Tween 80 is added to docetaxel injections, which can cause hemolytic reactions and severe allergic reactions, manifesting as shock, dyspnea, hypotension, angioedema, and even death. Therefore, oral formulations of docetaxel with good bioavailability are currently a hot research topic.

[0003] Therefore, there is a need in this field to improve the oral bioavailability of docetaxel, thereby enhancing the value of oral docetaxel applications. Summary of the Invention

[0004] The purpose of this invention is to provide a berberine that, when used as a nanocarrier for drug delivery, can significantly improve the oral bioavailability of docetaxel, thereby enhancing the oral application value of docetaxel.

[0005] The first aspect of this invention provides berberine, which is prepared by the following method:

[0006] (1) Extracting Coptis chinensis with water to obtain an aqueous extract;

[0007] (2) The water extract was centrifuged and filtered in sequence, and the filtrate was dialyzed to obtain berberine.

[0008] Preferably, in step (1), the mass ratio of Coptis chinensis to water is 1:5-20, more preferably 1:5-15, even more preferably 1:8-12, and most preferably 1:10.

[0009] Preferably, in step (1), the extraction temperature is 80-100℃, more preferably 90-100℃, and even more preferably 100℃.

[0010] Preferably, in step (1), the extraction is carried out in boiling water.

[0011] Preferably, in step (2), the centrifugation temperature is 10-50℃, more preferably 10-40℃, even more preferably 15-30℃, even more preferably 20-25℃, and most preferably 22℃.

[0012] Preferably, in step (2), the centrifugal force is 500-1500g, more preferably 800-1200g, even more preferably 900-1100g, and most preferably 1000g.

[0013] Preferably, in step (2), the centrifugation time is 2-8 min, more preferably 4-6 min, and even more preferably 5 min.

[0014] Preferably, in step (2), the supernatant obtained by centrifugation is filtered.

[0015] Preferably, in step (2), the filtration is microporous membrane filtration.

[0016] Preferably, the pore size of the microporous filter membrane is 0.1-0.8 μm, more preferably 0.1-0.5 μm, even more preferably 0.1-0.3 μm, even more preferably 0.18-0.25 μm, and most preferably 0.22 μm.

[0017] Preferably, in step (2), the molecular weight cutoff for dialysis is 2500-4500 Da, more preferably 3000-4000 Da, even more preferably 3200-3800 Da, even more preferably 3400-3600 Da, and most preferably 3500 Da.

[0018] Preferably, in step (2), the berberine is obtained by freeze-drying after dialysis separation.

[0019] Preferably, in step (2), the molecular weight of the berberine is 20-40 Da, more preferably 25-35 Da, even more preferably 22-30 Da, even more preferably 26-27 Da, and most preferably 28 Da.

[0020] A second aspect of the present invention provides a use of berberine as described in the first aspect of the present invention for preparing nanocarriers for drug delivery.

[0021] Preferably, the nanocarrier is loaded with a drug.

[0022] Preferably, the nanocarrier is a nanoparticle.

[0023] Preferably, the nanoparticles are nanoparticles.

[0024] Preferably, the drug includes docetaxel.

[0025] A third aspect of the present invention provides a drug-loaded nanoparticle, wherein the drug-loaded nanoparticle comprises berberine and a drug as described in the first aspect of the present invention.

[0026] Preferably, the berberine is used as a carrier for loading the drug onto the nanoparticles.

[0027] Preferably, the nanoparticles are nanoparticles.

[0028] Preferably, the drug includes docetaxel.

[0029] In a fourth aspect, the present invention provides a method for preparing drug-loaded nanoparticles as described in the third aspect of the present invention, the method comprising the steps of:

[0030] (a) Dissolve berberine in water to obtain an aqueous solution of berberine, and adjust the pH of the aqueous solution of berberine to obtain a berberine solution;

[0031] (b) The drug is added to the berberine solution from step (a) and stirred to obtain drug-loaded nanoparticles.

[0032] Preferably, in step (a), the concentration of berberine in the aqueous solution is 0.8-1.2 mg / mL, more preferably 1.0 mg / mL.

[0033] Preferably, in step (a), the pH of the berberine aqueous solution is adjusted to 9-11, more preferably 9.5-10.5, and even more preferably 10.

[0034] Preferably, in step (a), the pH is adjusted using an aqueous sodium hydroxide solution.

[0035] Preferably, the concentration of the sodium hydroxide aqueous solution is 0.1-0.3 mM, more preferably 0.2 mM.

[0036] Preferably, in step (b), the drug comprises docetaxel in ethanol solution form.

[0037] Preferably, the concentration of docetaxel in the docetaxel ethanol solution is 20-40 mg / mL, more preferably 25-35 mg / mL, even more preferably 28-32 mg / mL, and most preferably 30 mg / mL.

[0038] Preferably, the volume ratio of the docetaxel ethanol solution to the berberine solution is 1:80-120, more preferably 1:90-110, even more preferably 1:95-105, and most preferably 1:97-102.

[0039] Preferably, in step (b), the stirring temperature is 15-30°C, more preferably 18-24°C, and even more preferably 22°C.

[0040] Preferably, in step (b), the stirring time is 10-14 hours, more preferably 11-13 hours, and even more preferably 12 hours.

[0041] Preferably, in step (b), the stirring is carried out under light-protected conditions.

[0042] Preferably, in step (b), the mixture is centrifuged after stirring.

[0043] Preferably, the centrifugation removes clumps.

[0044] Preferably, in step (b), the centrifugal force is 5000-15000g, more preferably 8000-12000g, even more preferably 9000-11000g, and most preferably 10000g.

[0045] Preferably, in step (b), the centrifugation time is 5-15 min, more preferably 8-12 min, and even more preferably 10 min.

[0046] In a fifth aspect, the present invention provides a composition comprising drug-loaded nanoparticles as described in the third aspect of the present invention.

[0047] Preferably, the composition is a pharmaceutical composition.

[0048] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0049] Preferably, the composition is a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

[0050] Preferably, the composition is an injectable formulation, an oral formulation, or a topical formulation.

[0051] Preferably, the dosage form of the composition is an oral solution.

[0052] Preferably, the injectable formulation is an intravenous injection formulation, an arterial injection formulation, an intratumoral injection formulation, a tumor intravascular injection formulation, or a tumor microenvironment injection formulation.

[0053] In a sixth aspect, the present invention provides the use of drug-loaded nanoparticles as described in the third aspect of the present invention for preparing compositions for the prevention and / or treatment of diseases.

[0054] Preferably, the disease is an indication for the drug.

[0055] Preferably, the drug includes an anticancer drug.

[0056] Preferably, the drug includes docetaxel.

[0057] Preferably, the disease includes a tumor.

[0058] Preferably, the composition is a pharmaceutical composition.

[0059] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0060] Preferably, the composition is a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

[0061] Preferably, the composition is an injectable formulation, an oral formulation, or a topical formulation.

[0062] Preferably, the dosage form of the composition is an oral solution.

[0063] Preferably, the injectable formulation is an intravenous injection formulation, an arterial injection formulation, an intratumoral injection formulation, a tumor intravascular injection formulation, or a tumor microenvironment injection formulation.

[0064] A seventh aspect of the present invention provides a method for preventing and / or treating a disease, characterized in that the method comprises the steps of:

[0065] By applying drug-loaded nanoparticles as described in the third aspect of the invention or compositions as described in the fifth aspect of the invention to a desired object, thereby preventing and / or treating diseases.

[0066] Preferably, the disease is an indication for the drug.

[0067] Preferably, the drug includes an anticancer drug.

[0068] Preferably, the drug includes docetaxel.

[0069] Preferably, the disease includes a tumor.

[0070] Preferably, the object includes a human or a non-human mammal.

[0071] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0072] Preferably, the application is by injection, oral administration, or topical application.

[0073] Preferably, the injection administration is intravenous injection, arterial injection, intratumoral injection, intravascular injection into the tumor, or injection into the tumor microenvironment.

[0074] Preferably, the intravenous injection is administered via an upper limb vein or a lower limb vein.

[0075] Within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below can be combined with each other to form new or preferred technical solutions. Attached Figure Description

[0076] Figure 1 The difference in solubility between docetaxel and free docetaxel (DTX) in docetaxel nanoparticles (Nnps-DTX) (Mean±SD, n=3).**, p<0.01 vs DTX.

[0077] Figure 2 The difference in dissolution of docetaxel nanoparticles (Nnps-DTX) versus free docetaxel (DTX) in simulated gastric fluid (A) and simulated intestinal fluid (B) is represented by Mean±SD, n=3.**, p<0.01 vs DTX.

[0078] Figure 3 The drug-time curves (Mean ± SD, n = 5) of docetaxel in nanoparticles (Nnps-DTX) and free docetaxel (DTX) in rat systemic circulation after gavage administration. Detailed Implementation

[0079] This invention develops a berberine, which, when used as a nanocarrier for drug delivery, can significantly improve the solubility, dissolution rate, and oral bioavailability of docetaxel, thereby enhancing the oral application value of docetaxel.

[0080] the term

[0081] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only open-ended definitions but also semi-closed and closed definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0082] As used in this article, the English term for Dorcetaxel is Docetaxel.

[0083] As used in this article, the Latin name for Coptis chinensis is Coptis chinensis Franch.

[0084] In this invention, the term "prevention" refers to a method of preventing the onset of a disease and / or its accompanying symptoms or protecting a subject from acquiring a disease. As used herein, "prevention" also includes delaying the onset of a disease and / or its accompanying symptoms and reducing the subject's risk of contracting the disease.

[0085] In this invention, the term "treatment" includes delaying and stopping the progression of a disease, or eliminating the disease, without requiring 100% inhibition, eradication, and reversal.

[0086] Berberine

[0087] This invention provides a berberine, which is prepared by the following method:

[0088] (1) Extracting Coptis chinensis with water to obtain an aqueous extract;

[0089] (2) The water extract was centrifuged and filtered in sequence, and the filtrate was dialyzed to obtain berberine.

[0090] In a preferred embodiment of the present invention, in step (1), the mass ratio of Coptis chinensis to water is 1:5-20, more preferably 1:5-15, more preferably 1:8-12, and most preferably 1:10.

[0091] In a preferred embodiment of the present invention, in step (1), the extraction temperature is 80-100°C, more preferably 90-100°C, and even more preferably 100°C.

[0092] Preferably, in step (1), the extraction is carried out in boiling water.

[0093] In a preferred embodiment of the present invention, in step (2), the centrifugation temperature is 10-50°C, more preferably 10-40°C, more preferably 15-30°C, more preferably 20-25°C, and most preferably 22°C.

[0094] In a preferred embodiment of the present invention, in step (2), the centrifugal force is 500-1500g, more preferably 800-1200g, more preferably 900-1100g, and most preferably 1000g.

[0095] In a preferred embodiment of the present invention, in step (2), the centrifugation time is 2-8 min, more preferably 4-6 min, and even more preferably 5 min.

[0096] In a preferred embodiment of the present invention, in step (2), the supernatant obtained by centrifugation is filtered.

[0097] In a preferred embodiment of the present invention, in step (2), the filtration is microporous membrane filtration.

[0098] Preferably, the pore size of the microporous filter membrane is 0.1-0.8 μm, more preferably 0.1-0.5 μm, even more preferably 0.1-0.3 μm, even more preferably 0.18-0.25 μm, and most preferably 0.22 μm.

[0099] In a preferred embodiment of the present invention, in step (2), the molecular weight cutoff of the dialysis is 2500-4500 Da, more preferably 3000-4000 Da, more preferably 3200-3800 Da, more preferably 3400-3600 Da, and most preferably 3500 Da.

[0100] Preferably, in step (2), the berberine is obtained by freeze-drying after dialysis separation.

[0101] Preferably, in step (2), the molecular weight of the berberine is 20-40 Da, more preferably 25-35 Da, even more preferably 22-30 Da, even more preferably 26-27 Da, and most preferably 28 Da.

[0102] Drug-loaded nanoparticles and their preparation methods

[0103] This invention provides a drug-loaded nanoparticle, which includes berberine and a drug as described in this invention.

[0104] Preferably, the berberine is used as a carrier for loading the drug onto the nanoparticles.

[0105] Preferably, the nanoparticles are nanoparticles.

[0106] Preferably, the drug includes docetaxel.

[0107] The present invention also provides a method for preparing the drug-loaded nanoparticles described herein, the method comprising the steps of:

[0108] (a) Dissolve berberine in water to obtain an aqueous solution of berberine, and adjust the pH of the aqueous solution of berberine to obtain a berberine solution;

[0109] (b) The drug is added to the berberine solution from step (a) and stirred to obtain drug-loaded nanoparticles.

[0110] Specifically, the method for preparing drug-loaded nanoparticles according to the present invention is as described in the fourth aspect of the present invention above.

[0111] use

[0112] This invention provides the use of berberine in the preparation of nanocarriers for drug delivery.

[0113] Preferably, the nanocarrier is loaded with a drug.

[0114] Preferably, the nanocarrier is a nanoparticle.

[0115] Preferably, the nanoparticles are nanoparticles.

[0116] Preferably, the drug includes docetaxel.

[0117] The present invention also provides the use of drug-loaded nanoparticles as described herein for preparing compositions for the prevention and / or treatment of diseases.

[0118] Preferably, the disease is an indication for the drug.

[0119] Preferably, the drug includes an anticancer drug.

[0120] Preferably, the drug includes docetaxel.

[0121] Preferably, the disease includes a tumor.

[0122] The present invention also provides a method for preventing and / or treating diseases, the method comprising the steps of:

[0123] By applying the drug-loaded nanoparticles or compositions of the present invention to desired subjects, diseases can be prevented and / or treated.

[0124] Preferably, the disease is an indication for the drug.

[0125] Preferably, the drug includes an anticancer drug.

[0126] Preferably, the drug includes docetaxel.

[0127] Preferably, the disease includes a tumor.

[0128] Preferably, the object includes a human or a non-human mammal.

[0129] Preferably, the non-human mammal is a mouse, rat, rabbit, monkey, cow, horse, sheep, dog, cat, orangutan, or baboon.

[0130] Composition

[0131] The compositions described in this invention are preferably pharmaceutical compositions, and may include pharmaceutically acceptable carriers.

[0132] As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid, or gel fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" refers to the ability of the components and active ingredients in a drug or vaccine composition, as well as their intermingling, to not significantly reduce efficacy.

[0133] It should be understood that, in this invention, there are no particular limitations on the pharmaceutically acceptable carriers described. Materials commonly used in the art can be selected, or they can be prepared using conventional methods or purchased from the market. Some examples of pharmaceutically or vaccine-grade acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), buffers, chelating agents, thickeners, pH adjusters, transdermal penetration enhancers, colorants, flavoring agents, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0134] In a preferred embodiment of the present invention, the dosage form of the composition or preparation is a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

[0135] In a preferred embodiment of the present invention, the dosage form of the composition or preparation is an oral preparation, a topical preparation, or an injectable preparation.

[0136] Typically, the injectable formulation is an intramuscular injection formulation or a subcutaneous injection formulation.

[0137] The drug or vaccine formulation should be matched with the route of administration. The drug or vaccine of this invention can also be used with other synergistic therapeutic agents (including before, during, or after administration). When using a drug or vaccine composition or formulation, a safe and effective amount of the drug or vaccine is administered to the desired subject (e.g., human or non-human mammal), said safe and effective amount generally being at least about 10 micrograms per kilogram of body weight, and in most cases not exceeding about 8 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are within the scope of a skilled physician's expertise.

[0138] The main superior technical effects of this invention include:

[0139] (1) The present invention develops a berberine, which, when used as a nanocarrier for drug delivery, can significantly improve the solubility, dissolution rate and oral bioavailability of docetaxel, thereby improving the oral application value of docetaxel.

[0140] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the following specific embodiments are based on the present technical solution and provide detailed implementation methods and specific operation processes, but the scope of protection of the present invention is not limited to these embodiments.

[0141] Example 1

[0142] 1. Berberine was extracted from Coptis chinensis.

[0143] ①The decoction of Coptis chinensis was prepared by conventional method (the mass ratio of Coptis chinensis Franch. to water was 1:10, and the decoction was boiled twice with boiling water for 1 hour each time). The filtrates after the two decoctions were combined and dried under vacuum at 60℃ to obtain water extract powder, which was then stored in a cool and dry place.

[0144] ② Dissolve the Coptis chinensis water extract powder in pure water, centrifuge at 1000g for 5min at 22℃ to remove the undissolved part, and filter the supernatant through a 0.22μm microporous membrane to remove clumps;

[0145] ③ The filtrate was separated by dialysis (3500 Da molecular weight cutoff) to obtain berberine, which was then freeze-dried at -20℃ for later use. Electrophoresis experiment showed that the molecular weight of berberine was approximately 28 kDa.

[0146] 2. Screening and optimization of docetaxel nanoparticles

[0147] 2.1 Single-factor investigation of temperature

[0148] Berberine lyophilized powder was dissolved in water, shaken for 2 hours, and allowed to stand at 4°C for 12 hours. The solution was heated (65°C, 70°C, or 75°C) for 30 minutes, and then cooled to room temperature (22°C). Undissolved protein was removed by centrifugation at 1000g for 5 minutes at room temperature. The protein concentration in the supernatant was determined using a BCA kit and adjusted to 1 mg / mL. The pH of the solution was adjusted to 8 with sodium hydroxide or hydrochloric acid solution. Docetaxel ethanol solution was slowly added dropwise to the solution (the final ethanol concentration did not exceed 1%), and the drug concentration was controlled at 0.3 mg / mL. The solution was stirred for 12 hours to allow self-assembly of nanoparticles. Agglomerates were removed by centrifugation at 10000g for 10 minutes at room temperature to obtain a nanoparticle solution. The nanoparticle lyophilized powder was obtained by freeze-drying.

[0149] An appropriate amount of the above lyophilized powder was accurately weighed and a 1 mg / mL nanoparticle solution was prepared with pure water. The nanoparticle size, polydispersity index (PDI), and zeta potential were detected using dynamic light scattering (DLS) technology. The results (Table 1) show that with increasing temperature, there was no significant difference in particle size after heating at 65℃ and 70℃, while the particle size was larger at 75℃. The polydispersity index gradually increased. Stable drug-loaded nanoparticles were generated after treatment at all three temperatures. Therefore, 65℃ was determined to be the optimal heating temperature.

[0150] Table 1. Effect of different temperatures on nanoparticles (mean ± SD, n = 3)

[0151]

[0152] 2.2 Single-factor investigation of the initial concentration of berberine

[0153] Berberine lyophilized powder was dissolved in water, shaken for 2 hours, and allowed to stand at 4°C for 12 hours. The solution was heated (65°C) for 30 minutes and then cooled to room temperature (22°C). Undissolved protein was removed by centrifugation at 1000g for 5 minutes at room temperature. The protein concentration in the supernatant was determined using a BCA kit and adjusted to appropriate concentrations (0.3, 0.6, 0.8, 1, 3 mg / mL). The pH of the solution was adjusted to 8 with sodium hydroxide or hydrochloric acid solution. Docetaxel ethanol solution was slowly added dropwise to the solution (final ethanol concentration not exceeding 1%), and the drug concentration was controlled at 0.3 mg / mL. The solution was stirred for 12 hours to allow self-assembly of nanoparticles. Agglomerates were removed by centrifugation at 10000g for 10 minutes at room temperature to obtain a nanoparticle solution. The nanoparticle lyophilized powder was obtained by freeze-drying.

[0154] An appropriate amount of the above lyophilized powder was accurately weighed and a 1 mg / mL nanoparticle solution was prepared with pure water. The nanoparticle size, polydispersity index (PDI), and zeta potential were detected using dynamic light scattering (DLS) technology. The results (Table 2) show that within the range of 0.3-1 mg / mL, the nanoparticle size gradually decreased with increasing protein concentration, while the particle size increased with excessively high concentrations. The concentration of berberine had a relatively small effect on the dispersibility of the nanoparticles. The absolute value of the zeta potential decreased with increasing concentration. Therefore, 1 mg / mL was determined to be the optimal initial concentration of berberine.

[0155] Table 2. Effect of different protein concentrations on nanoparticles (mean ± SD, n = 3)

[0156]

[0157] 2.3 Single-factor investigation of drug loading ratio

[0158] Berberine lyophilized powder was dissolved in water, shaken for 2 hours, and allowed to stand at 4°C for 12 hours. The solution was heated (65°C) for 30 minutes and then cooled to room temperature (22°C). Undissolved protein was removed by centrifugation at 1000g for 5 minutes at room temperature. The protein concentration in the supernatant was determined using a BCA kit and adjusted to an appropriate concentration of 1 mg / mL. The pH of the solution was adjusted to 8 with sodium hydroxide or hydrochloric acid solution. Docetaxel ethanol solution was slowly added dropwise to the solution (final ethanol concentration not exceeding 1%), and the drug concentration was controlled (0.05, 0.1, 0.15, 0.2, 0.3, or 0.4 mg / mL). The solution was stirred for 12 hours to allow self-assembly into nanoparticles. Agglomerates were removed by centrifugation at 10000g for 10 minutes at room temperature to obtain a nanoparticle solution. The nanoparticle lyophilized powder was obtained by freeze-drying.

[0159] An appropriate amount of the above lyophilized powder was accurately weighed and a 1 mg / mL nanoparticle solution was prepared with pure water. The nanoparticle size, polydispersity index (PDI), and zeta potential were detected using dynamic light scattering (DLS) technology. The results (Table 3) show that within the range of 0.05-0.4, the particle size and absolute value of the potential gradually decreased with increasing drug loading ratio. The smallest particle size was 143.8 nm when the drug loading ratio was 0.2. However, with further increases in the drug loading ratio, both the particle size and the absolute value of the potential gradually increased. The drug loading ratio showed no significant trend in its effect on the polydispersity index. Considering size, PDI, and zeta potential, the optimal drug loading ratio was determined to be 0.3.

[0160] Table 3. Effect of different drug loading ratios on nanoparticles (mean ± SD, n = 3)

[0161]

[0162] 2.4 Single-factor investigation of pH

[0163] Berberine lyophilized powder was dissolved in water, shaken for 2 hours, and allowed to stand at 4°C for 12 hours. The solution was heated (65°C) for 30 minutes and then cooled to room temperature (22°C). Undissolved protein was removed by centrifugation at 1000g for 5 minutes at room temperature. The protein concentration in the supernatant was determined using a BCA kit and adjusted to an appropriate concentration (1 mg / mL). The pH of the solution was adjusted to an appropriate value (8, 9, or 10) with sodium hydroxide or hydrochloric acid solution. Docetaxel ethanol solution was slowly added dropwise to the solution (the final ethanol concentration did not exceed 1%), and the drug concentration was controlled at 0.3 mg / mL. The solution was stirred for 12 hours to allow self-assembly of nanoparticles. Agglomerates were removed by centrifugation at 10000g for 10 minutes at room temperature to obtain a nanoparticle solution. The nanoparticle lyophilized powder was obtained by freeze-drying.

[0164] An appropriate amount of the above lyophilized powder was accurately weighed and a 1 mg / mL nanoparticle solution was prepared with pure water. The nanoparticle size, polydispersity index (PDI), and zeta potential were detected using dynamic light scattering (DLS) technology. The results (Table 4) show that within the pH range of 8-10, the higher the pH, the smaller the particle size and PDI, and the smaller the zeta potential. This indicates that at pH = 10, the prepared nanoparticles have a smaller particle size and better dispersibility and stability. Therefore, the optimal pH value for nanoparticle preparation was determined to be 10.

[0165] Table 4. Effect of different pH values ​​on nanoparticles (mean ± SD, n = 3)

[0166]

[0167] 2.5 Orthogonal Design Experiment Investigation

[0168] Four factors that significantly affect nanoparticle preparation (temperature, pH, drug loading ratio, and initial berberine concentration) were selected for orthogonal experiments. Each factor was designed with three levels, as shown in Table 5. The experiments were conducted using an orthogonal array, repeated three times, and the average value was taken.

[0169] Table 5. Orthogonal Design Factors and Levels

[0170]

[0171] The preparation method is as follows:

[0172] Berberine lyophilized powder was dissolved in water, shaken for 2 hours, and allowed to stand at 4°C for 12 hours. The solution was heated (65°C, 70°C, 75°C) for 30 minutes, and then cooled to room temperature (22°C). Undissolved protein was removed by centrifugation at 1000g for 5 minutes at room temperature. The protein concentration in the supernatant was determined using a BCA kit and adjusted to appropriate concentrations (0.6, 0.8, 1 mg / mL). The pH of the solution was adjusted to appropriate values ​​(8, 9, 10) with sodium hydroxide or hydrochloric acid solution. Docetaxel ethanol solution was slowly added dropwise to the solution (final ethanol concentration not exceeding 1%), and the drug concentration was controlled (0.2, 0.3, 0.4 mg / mL). The solution was stirred for 12 hours to allow self-assembly into nanoparticles. Agglomerates were removed by centrifugation at 10000g for 10 minutes at room temperature to obtain a nanoparticle solution. The nanoparticle lyophilized powder was obtained by freeze-drying.

[0173] An appropriate amount of the above lyophilized powder was accurately weighed and a 1 mg / mL nanoparticle solution was prepared with pure water. The nanoparticle size, polydispersity index (PDI), and zeta potential were detected using dynamic light scattering (DLS) technology. The results (Table 6) showed that the optimal conditions for particle size were A1B3C1D3; the optimal conditions for PDI were A1B1C1D3; and the optimal conditions for zeta potential were A2B1C2D3. Considering that particle size is one of the most important factors for nanoparticles, and combining the single-factor analysis, A1B3C2D3 was ultimately determined as the optimal preparation process for drug-loaded nanoparticles, namely, a heating temperature of 65℃, a protein concentration of 1 mg / mL, a drug loading ratio of 0.3, and a pH of 10.

[0174] Table 6. Four-factor, three-level orthogonal experiment (n=3) of temperature (A), protein concentration (B), drug loading ratio (C), and pH (D).

[0175]

[0176] 3. Preparation and efficacy of docetaxel nanoparticles

[0177] 3.1 Preparation of Docetaxel Nanoparticles

[0178] 32.0 mg of berberine lyophilized powder was dissolved in 6.4 mL of water, shaken at 22 °C for 2 h, and then allowed to stand at 4 °C for 12 h. The solution was heated at 65 °C for 30 min, cooled to 22 °C, and then centrifuged at 10000 g for 10 min to remove undissolved protein. The protein concentration in the supernatant was determined to be 2.34 mg / mL using a BCA kit. 6 mL of this berberine solution was taken and water was added to a final volume of 14.04 mL to adjust the protein concentration to 1 mg / mL. The pH of the berberine solution was adjusted to 10 with 0.2 mM sodium hydroxide. 0.14 mL of a 30 mg / mL docetaxel ethanol solution was slowly added dropwise to 13.86 mL of the berberine solution to ensure a final ethanol concentration not exceeding 1% and a docetaxel concentration of 0.3 mg / mL. The solution was stirred at 22 °C in the dark for 12 h to allow self-assembly of nanoparticles. The agglomerates were removed by centrifugation at 10000g for 10 min at 22℃ to obtain a nanoparticle solution, which was then freeze-dried to obtain docetaxel nanoparticle freeze-dried powder.

[0179] The evaluation of the efficacy of docetaxel nanoparticles includes the following indicators.

[0180] ① Nanoparticle size and Zeta potential

[0181] An appropriate amount of the above-mentioned lyophilized docetaxel nanoparticle powder was accurately weighed, and a 1 mg / mL nanoparticle solution was prepared with pure water. The nanoparticle size, polydispersity index (PDI), and zeta potential were detected by dynamic light scattering technology. The results showed that the obtained docetaxel nanoparticles had a particle size of 137.3 nm, a PDI of 0.46, and a zeta potential of -19.4 mV.

[0182] ② Dorset sample size and encapsulation efficiency

[0183] Take 2 mL of docetaxel nanoparticle suspension and place it in a 10 mL volumetric flask. Add methanol to make up the volume, weigh the flask, sonicate for 30 min, and then make up the missing weight with methanol. Take 1 mL of the solution and pass it through a 0.45 μm organic membrane. Take the filtrate for quantitative analysis to determine the mass of docetaxel in the nanoparticles.

[0184] Take 2 mL of docetaxel nanoparticle suspension and place it in a 4.5 mL ultrafiltration centrifuge tube (molecular weight cutoff 3500 Da). Centrifuge at 1000 g for 20 min, collect the lower filtrate, dilute with methanol, and determine the mass of free docetaxel.

[0185] Each sample size is 3. Encapsulation efficiency (EE) and drug loading (DL) are calculated using the following formulas, where m 总 It is the total mass of the drug, m 游离 It is the free drug mass, m 载体 The mass of the carrier in the nanoparticles:

[0186] EE = (m 总 -m 游离) / m 总 *100%

[0187] DL = (m 总 -m 游离 ) / m 载体 *100%

[0188] The results showed that the docetaxel encapsulation efficiency in the docetaxel nanoparticles reached 77.6±8.5%, and the drug loading was 6.8±1.9%.

[0189] ③ Effect on the saturated solubility of docetaxel

[0190] Free docetaxel and docetaxel nanoparticle lyophilized powder were taken separately, and their supersaturated solutions were prepared. The solutions were shaken at 37℃ and 100 rpm for 24 h, allowed to stand for 2 h, and then centrifuged at 20000 g for 10 min. The supernatant was collected, and the concentration of docetaxel was determined by LC-MS / MS. The saturated solubility was calculated. The number of samples in each group was 3.

[0191] The quantitative analysis method for docetaxel by LC-MS / MS is as follows:

[0192] Add the sample, internal standard, and three times the sample volume of acetonitrile to a centrifuge tube. Vortex thoroughly, centrifuge, and aspirate the supernatant. Add an equal volume of pure water, mix well, and inject for analysis. The chromatographic column was an ACQUITYUPLCBEHC18 (1.7 μm, 2.1 × 100 mm); mobile phase A was 1 mM ammonium formate-water, and mobile phase B was methanol; gradient elution: 0 min, 60% B; 1 min, 60% B; 3.5 min, 90% B; 4.0 min, 60% B; 5.5 min, 60% B; column temperature: 40℃; flow rate: 0.3 mL / min; injection volume: 10 μL. Mass spectrometry was performed using an ESI ion source; multiple reaction detection mode in positive ion mode; the docetaxel detection ion pair was determined to be m / z 808.4→527.4, and the internal standard paclitaxel detection ion pair was determined to be m / z 854.2→569.2. This method has been validated and is suitable for the quantitative analysis of docetaxel.

[0193] result( Figure 1 The results showed that the solubility of docetaxel in docetaxel nanoparticles was approximately 13.9 times that of the free docetaxel control group (p<0.01), indicating that nanoparticles can significantly improve the solubility of docetaxel.

[0194] ④ Effect of docetaxel on dissolution in artificial gastric and intestinal fluids

[0195] The dissolution of docetaxel nanoparticles in simulated gastric and intestinal fluids was determined by dialysis. The molecular weight cutoff of the dialysis bag was 3500 Da. The dialysis bags were first soaked in pure water, boiled for 10 min, and then rinsed three times with pure water before use. 2.0 mL each of the docetaxel nanoparticle dispersion (containing 0.2 mg / mL docetaxel) and the free docetaxel solution (containing 0.2 mg / mL docetaxel) used as a control were accurately measured and placed in the dialysis bag. Place the dialysis bag in 900 mL of artificial gastric fluid (16.4 mL of dilute hydrochloric acid, approximately 800 mL of water, and 10 g of pepsin, shake well, and dilute with water to 1000 mL) or artificial intestinal fluid (6.8 g of potassium dihydrogen phosphate, dissolved in 500 mL of water, pH adjusted to 6.8 with 0.1 M sodium hydroxide solution; separately, 10 g of pancreatin, dissolved in an appropriate amount of water, mix the two solutions, and dilute with water to 1000 mL). Set the rotation speed to 50 rpm. At the set time points (referring to the pharmacopoeia: 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, and 2 h for artificial gastric fluid; 5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, and 6 h for artificial intestinal fluid), aspirate 1 mL of dissolution medium at a fixed position, and immediately replenish with an equal volume of blank dissolution medium. Filter the sample through a 0.22 μm filter membrane. The filter membrane was saturated with 5 mL of filtrate before first use. Samples were stored at -80°C, appropriately diluted, and the concentration of docetaxel in the samples was determined using the aforementioned LC-MS / MS method, and the cumulative release percentage was calculated. Three samples were used in each group.

[0196] like Figure 2 As shown, the dissolution rate of docetaxel in docetaxel nanoparticles in artificial gastric and intestinal fluids was significantly better than that of the free docetaxel control group.

[0197] ⑤ Effects of docetaxel on rat plasma pharmacokinetics

[0198] Ten normal rats were randomly divided into two groups of five each, based on their body weight (220-240g). Each group was administered either free docetaxel (20mg / kg) or docetaxel nanoparticles (equivalent to 20mg / kg of docetaxel) via gavage (1mL / 100g body weight). At designated time points following administration (0.125, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 12h), approximately 0.2mL of blood was collected from the retro-orbital venous plexus to prepare plasma. After protein precipitation, the docetaxel concentration in each plasma sample was determined by LC-MS / MS to assess its effect on the pharmacokinetics of docetaxel in rat plasma.

[0199] like Figure 3As shown in Table 7, compared with the control group, the pharmacokinetics of docetaxel in the gavage nanoparticle group were significantly improved, mainly manifested in the shorter time to peak concentration (T0). max ) earlier, and pharmacokinetic parameter C max and AUC 0-12h Significantly increased.

[0200] Table 7. Pharmacokinetic parameters of docetaxel (Nnps-DTX) and free docetaxel (DTX) in the systemic circulation of rats after gavage administration (Mean±SD, n=5).

[0201]

[0202] *, p < 0.05, **, p < 0.01 vs DTX;

[0203] T max The time required to reach maximum blood drug concentration;

[0204] T 1 / 2 Half-life;

[0205] C max Maximum blood drug concentration

[0206] AUC 0-12h The area under the blood drug concentration curve from 0 to 12 hours;

[0207] MRT 0-12h This represents the average dwell time.

[0208] The above description is an implementation scheme designed for one case of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A drug-loaded nanoparticle, characterized in that, The drug-loaded nanoparticles include berberine and docetaxel; The berberine mentioned above is prepared by the following method: (1) Extracting Coptis chinensis with water to obtain an aqueous extract; (2) The water extract was centrifuged and filtered in sequence, and the filtrate was dialyzed to obtain berberine; The drug-loaded nanoparticles are prepared by the following method, which includes the following steps: (a) Dissolve berberine in water to obtain an aqueous solution of berberine, and adjust the pH of the aqueous solution of berberine to obtain a berberine solution; (b) Add docetaxel to the berberine solution from step (a) and stir to obtain drug-loaded nanoparticles; In step (a), the pH is adjusted with an aqueous sodium hydroxide solution to adjust the pH of the berberine aqueous solution to 9-11; In step (b), the stirring temperature is 15-30℃; In step (b), the stirring time is 10-14 hours; In step (b), the mixture is centrifuged after stirring.

2. A composition, characterized in that, The composition comprises the drug-loaded nanoparticles as described in claim 1.

3. Use of the drug-loaded nanoparticles as described in claim 1 for preparing a composition for treating tumors; The tumor in question is locally advanced or metastatic breast cancer.

4. The use as described in claim 3, characterized in that, The composition is a pharmaceutical composition; The composition is an oral preparation.

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