Liposomes, drug-loaded liposomes, and methods of making and using the same

Abstract

This invention discloses a liposome, a drug-loaded liposome, its preparation method, and its applications. The liposome comprises a liposome backbone and a targeting ligand; wherein the liposome backbone comprises the following components: phospholipids, cholesterol, cationic lipids, and PEGylated phospholipids; the cationic lipids are (2,3-dioleoyl-propyl)-trimethylammonium chloride and / or 1,2-dioleoyl-3-trimethylammonium propane; and the targeting ligands are sodium hyaluronate and / or hyaluronic acid. This liposome possesses excellent basic properties and excellent targeting ability; after encapsulating the active pharmaceutical ingredients (doxacin hydrochloride and berberine hydrochloride), its targeting ability can be further enhanced in practical applications, thereby increasing its efficacy.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to a liposome, a drug-loaded liposome, its preparation method and application. Background Technology

[0002] Liposomes are a class of nanoparticles with a bilayer membrane structure composed of natural or synthetic phospholipids, and they are widely used in drug delivery systems. Due to their excellent biocompatibility, controllability, and efficient drug loading capacity, liposomes have become one of the core research directions in modern drug delivery technology.

[0003] Doxorubicin hydrochloride (DOX) is a widely used chemotherapeutic drug for anti-tumor therapy, primarily exerting its anti-cancer effect by interfering with the DNA synthesis of tumor cells. However, the clinical application of doxorubicin is limited by its side effects, such as cardiotoxicity and non-targeting toxicity. Therefore, developing a carrier system that can effectively target tumor cells and improve the therapeutic effect of the drug is an important topic in doxorubicin research. Berberine hydrochloride (BBR) is a natural alkaloid with multiple biological activities, including antibacterial, anti-inflammatory, antioxidant, and anti-tumor effects. However, the clinical application of berberine hydrochloride is also limited by its low bioavailability, easy degradation, and difficulty in targeted delivery. Therefore, researching how to enhance the efficacy and biocompatibility of berberine hydrochloride through carrier systems has become a key direction in its drug development.

[0004] Although dual-drug-loaded liposomes containing both doxorubicin hydrochloride and berberine hydrochloride already exist, they also suffer from poor targeting.

[0005] Therefore, there is an urgent need for a highly targeted liposome to achieve the effective encapsulation and delivery of doxorubicin hydrochloride and berberine hydrochloride. Summary of the Invention

[0006] To address the aforementioned technical shortcomings of existing dual-drug-loaded liposomes simultaneously carrying doxorubicin hydrochloride and berberine hydrochloride, this invention provides a liposome, a drug-loaded liposome, its preparation method, and its applications. This liposome possesses excellent basic properties and significantly superior targeting; after encapsulating the active pharmaceutical ingredients (doxorubicin hydrochloride and berberine hydrochloride), its targeting ability and efficacy can be further enhanced in practical applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] The present invention provides a liposome comprising a liposome backbone and a targeting ligand; wherein the liposome backbone comprises the following components: phospholipids, cholesterol, cationic lipids and PEGylated phospholipids;

[0009] The cationic lipid is (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTMA).

[0010] The molar ratio of the phospholipids, cholesterol, cationic lipids and PEGylated phospholipids is (20-40):(15-35):(15-35):(1-10);

[0011] The targeting ligand is sodium hyaluronate and / or hyaluronic acid;

[0012] The mass ratio of the targeting ligand to the liposome backbone is 1:(5-20).

[0013] In this invention, the phospholipid refers to unPEGylated phospholipid.

[0014] In some embodiments, the molar ratio of the phospholipids, cholesterol, cationic lipids and PEGylated phospholipids is 40:35:20:5.

[0015] In some embodiments, the mass ratio of the targeting ligand to the liposome backbone is 1:(5-15), for example 1:10.

[0016] In some embodiments, the phospholipid is one or more of lecithin, soybean lecithin, egg yolk lecithin, sphingomyelin, and hydrogenated soybean lecithin, such as egg yolk lecithin.

[0017] In some embodiments, the PEGylated phospholipid is one or more of distearylphosphatidylethanolamine-polyethylene glycol 1000 (DSPE-MPEG1000), distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000), and distearylphosphatidylethanolamine-polyethylene glycol 5000 (DSPE-MPEG5000), preferably DSPE-MPEG2000.

[0018] In this invention, the molecular weight of the targeting ligand can be a conventional molecular weight in the art, such as 10 kDa.

[0019] In some embodiments, the liposomes have a particle size of 60-100 nm, for example 75 nm or 80 nm.

[0020] In some embodiments, the PDI of the liposomes is 0.1-0.3, for example 0.12 or 0.15.

[0021] In some embodiments, the zeta potential of the liposomes is +20 to -20 mV, preferably -10 to -20 mV, for example -12 mV or -13 mV.

[0022] The present invention also provides a method for preparing liposomes, wherein the raw materials for the liposomes include raw materials for the liposome skeleton and a targeting ligand; wherein the raw materials for the liposome skeleton include phospholipids, cholesterol, cationic lipids and PEGylated phospholipids;

[0023] The cationic lipid is (2,3-dioleoyl-propyl)-trimethylammonium chloride and / or 1,2-dioleoyl-3-trimethylammonium propane;

[0024] The molar ratio of the phospholipids, cholesterol, cationic lipids and PEGylated phospholipids is (20-40):(15-35):(15-35):(1-10);

[0025] The targeting ligand is sodium hyaluronate and / or hyaluronic acid;

[0026] The mass ratio of the raw materials for the target ligand and the liposome matrix is ​​1:(5-20);

[0027] The liposome preparation method is selected from one or any combination of two of the following methods: microfluidic method, thin film dispersion method, injection method, reverse evaporation method, freeze-thaw method, and double emulsion method.

[0028] In some embodiments, the types and / or amounts of the phospholipids, cholesterol, cationic lipids, PEGylated phospholipids, targeting ligands, and liposome backbones are as defined above.

[0029] In some embodiments, the liposomes are prepared using a microfluidic method, which includes the following steps:

[0030] S1, organic phase and aqueous phase are mixed using microfluidic technology to prepare liposome framework solution, wherein:

[0031] The organic phase includes phospholipids, cholesterol, cationic lipids, PEGylated phospholipids, and organic solvents;

[0032] The aqueous phase includes ammonium sulfate and water;

[0033] S2. The liposome matrix solution, the targeting ligand, and water are mixed to prepare a liposome solution.

[0034] In this invention, microfluidic technology enables precise control of fluids at the micrometer scale, allowing for efficient and accurate mixing, distribution, and reaction of organic and aqueous phases at a tiny scale. Throughout the process, the particle size, morphology, and surface properties of the resulting liposome framework are controlled, thereby further improving drug delivery efficiency and targeting capability after drug loading. Specifically, the introduction of microfluidic technology in this invention further improves the preparation efficiency of liposomes and ensures consistent quality. The entire process is characterized by its simplicity, ease of operation, good flexibility, and excellent controllability. By applying the miniaturized, precise, and continuous characteristics of microfluidic technology, the preparation time is effectively shortened.

[0035] In some preferred embodiments, in step S1, the organic solvent is ethanol.

[0036] In some preferred embodiments, step S1, the mixing includes the following steps:

[0037] The organic phase and aqueous phase are pumped into the microfluidic device by syringe A and syringe B, respectively, and begin to mix upon contact to form the liposome skeleton solution.

[0038] Both syringe A and syringe B can be conventional syringes in the art.

[0039] The microfluidic device can be a conventional microfluidic device in the art; preferably, the microfluidic chip it contains is a split-merge structure liposome PDMS chip from SMIC, for example, model ZX-LS-300.

[0040] The flow rates of the organic phase and the aqueous phase are preferably 1:(1-9), more preferably 1:(1-5), for example 1:2, 1:3, or 1:4.

[0041] The total flow rate, calculated as the sum of the flow rates of the organic phase and the aqueous phase, is preferably 4-24 mL / min, more preferably 4-16 mL / min, for example 6, 8, 10, 12, 14, or 12 mL / min.

[0042] In some preferred embodiments, in step S1, the preparation of the organic phase includes dissolving the phospholipid, cholesterol, cationic lipid, or PEGylated phospholipid in the organic solvent.

[0043] The dissolution is preferably performed under ultrasound; the ultrasound duration is, for example, 0.5 h.

[0044] Preferably, the dissolution step is followed by a filtration step; the filtration is performed, for example, by an organic filter membrane; the pore size of the organic filter membrane is, for example, 0.22 μm; the organic filter membrane is, for example, a PVDF membrane.

[0045] In some preferred embodiments, step S1, the preparation of the aqueous phase includes dissolving the ammonium sulfate in the water.

[0046] Preferably, the dissolution step is followed by a filtration step; the filtration is performed, for example, by an aqueous filter membrane; the pore size of the aqueous filter membrane is, for example, 0.22 μm; the aqueous filter membrane is, for example, a PES membrane.

[0047] In some preferred embodiments, after step S1, a step of dialysis of the liposome skeleton solution is further included; the dialysis is performed, for example, in PBS buffer; the dialysis time is, for example, 8 hours.

[0048] In some preferred embodiments, step S2, the mixing step includes dissolving the targeting ligand in the water first, and then adding it dropwise into the liposome matrix solution.

[0049] Preferably, the dissolution step is followed by a filtration step; the filtration is performed, for example, by a filter membrane.

[0050] The mixing is preferably carried out at a rotational speed of 15 rpm.

[0051] The present invention also provides a liposome prepared by the method for preparing liposomes as described above.

[0052] In some implementations, the liposomes are as defined above.

[0053] The present invention also provides a drug-loaded liposome comprising the liposome as described above and a pharmaceutically active ingredient; the pharmaceutically active ingredient is doxorubicin hydrochloride and berberine hydrochloride.

[0054] In some embodiments, the encapsulation efficiency of doxorubicin hydrochloride in the drug-loaded liposome is not less than 85%, preferably 85%-95%, for example 94.4%, 93.4%, 92.7% or 87.8%.

[0055] In some embodiments, the drug loading rate of doxorubicin hydrochloride in the drug-loaded liposome is not less than 8%, preferably 8%-20%, for example 8.1%, 8.6%, 16.8% or 19.1%.

[0056] In some embodiments, the encapsulation efficiency of berberine hydrochloride in the drug-loaded liposome is not less than 50%, preferably 50%-65%, for example 52.6%, 56.4%, 60.2% or 61.5%.

[0057] In some embodiments, the loading rate of berberine hydrochloride in the drug-loaded liposome is not less than 10%, preferably 10%-20%, for example 10.6%, 10.8%, 11.9% or 13.3%.

[0058] In some embodiments, the particle size of the drug-loaded liposomes is 60-100 nm, for example 78.31 nm, 81.12 nm, 83.54 nm, 90.32 nm or 80 nm.

[0059] In some embodiments, the PDI of the drug-loaded liposome is 0.05-0.2, for example 0.1.

[0060] In some embodiments, the zeta potential of the drug-loaded liposome is +20 to -20 mV, preferably -10 to -20 mV, for example -12.94 mV, -15.43 mV or -13 mV.

[0061] The present invention also provides a method for preparing drug-loaded liposomes, comprising the step of loading a drug active ingredient into the liposomes as described above using an active drug loading method; wherein the drug active ingredient is doxorubicin hydrochloride and berberine hydrochloride.

[0062] In some implementations, the drug-loaded liposomes are as defined above.

[0063] In some embodiments, the mass ratio of the liposome backbone, doxorubicin hydrochloride, and berberine hydrochloride is 5:(0.5-1.5):(0.5-1.5), for example, 5:1:0.5, 5:1:0.8, 5:1:1, or 5:1:1.2.

[0064] In some embodiments, the method for preparing the drug-loaded liposomes includes the following steps:

[0065] A. The organic phase and aqueous phase are mixed using microfluidic technology to prepare a liposome framework solution, wherein:

[0066] B. The liposome skeleton solution, the active pharmaceutical ingredient, and the solvent are mixed to obtain a first mixture;

[0067] C. The first mixture, the targeting ligand, and water are mixed to prepare a drug-loaded liposome solution;

[0068] In step A, the organic phase, aqueous phase, liposome skeleton solution, and mixture are as defined above.

[0069] In some preferred embodiments, in step B, the solvent is PBS.

[0070] In some preferred embodiments, step B, the mixing step includes dissolving the active pharmaceutical ingredient in the solvent first, and then mixing it with the liposome matrix solution.

[0071] In some preferred embodiments, in step B, the mixing temperature is 60-70°C, for example 65°C.

[0072] In some preferred embodiments, in step B, the mixing time is 40-50 minutes, for example, 45 minutes.

[0073] In some preferred embodiments, step C, the mixing step includes dissolving the targeting ligand in the water first, and then adding it dropwise into the first mixture.

[0074] Preferably, the dissolution step is followed by a filtration step; the filtration is performed, for example, by a filter membrane.

[0075] The mixing is preferably carried out at a rotational speed of 15 rpm.

[0076] The present invention also provides a drug-loaded liposome, which is prepared by the method for preparing drug-loaded liposomes as described above.

[0077] In some implementations, the drug-loaded liposomes are as defined above.

[0078] The present invention also provides the application of drug-loaded liposomes as described above in the preparation of antitumor drugs.

[0079] In some implementations, the antitumor drug includes an anti-ovarian cancer drug.

[0080] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0081] The reagents and raw materials used in this invention are all commercially available.

[0082] The positive and progressive effects of this invention are as follows:

[0083] The liposomes of this invention, containing cationic lipids and PEGylated phospholipids, possess excellent basic properties such as uniform particle size, good biocompatibility, and good biodegradability, while exhibiting significantly superior targeting. Furthermore, after encapsulating doxorubicin hydrochloride and berberine hydrochloride, while ensuring high drug loading and high encapsulation efficiency, they can further enhance targeting in practical applications (such as tumor cell uptake of DOX, tumor cell killing ability, ability to induce tumor cell apoptosis, and ability to inhibit tumor cell migration), prolong in vivo circulation time, and thus effectively enhance drug efficacy. Attached Figure Description

[0084] Figure 1 This is a TEM image of the drug-loaded liposomes in Example 1.

[0085] Figure 2 The image shows the results of the DOX cell uptake experiment in Example 2.

[0086] Figure 3 The figure shows the cytotoxicity experiment results in Example 2.

[0087] Figure 4 The figure shows the results of the apoptosis experiment in Example 2.

[0088] Figure 5 The image shows the results of the cell migration experiment in Example 2. Detailed Implementation

[0089] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0090] In the following examples and comparative examples, the main reagents used, unless otherwise specified, are all commercially available; among them, sodium hyaluronate has a molecular weight of 10 kDa.

[0091] Unless otherwise specified, the organic filter membranes used in the embodiments and comparative examples of this invention are all 0.22μm PVDF membranes, and the aqueous filter membranes used are all 0.22μm PES membranes.

[0092] Unless otherwise specified, the microfluidic chip contained in the microfluidic device used in the embodiments and comparative examples of the present invention is a split-merge structure liposome PDMS chip, which was purchased from Suzhou Zhongxin Qiheng Scientific Instruments Co., Ltd., and the product model is ZX-LS-300.

[0093] Experiment 1 - Effect of Total Flow Velocity

[0094] Liposomes without sodium hyaluronate modification and without drug loading were prepared at different total flow rates according to the following steps:

[0095] (1) Weigh the raw materials for the liposome skeleton: 253 mg (0.32 mol) of egg yolk lecithin, 113 mg (0.28 mol) of cholesterol, 117 mg (0.16 mol) of (2,3-dioleoyl-propyl)-trimethylammonium chloride and 117 mg (0.04 mol) of distearate phosphatidylethanolamine-polyethylene glycol 2000, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve each raw material, remove impurities in the solution using a 0.22 μm organic filter membrane to obtain the organic phase, and put it into a 10 mL syringe 1.

[0096] (2) Pass the ammonium sulfate aqueous solution (250 mmol / L) through an aqueous filter membrane to obtain the aqueous phase, and put it into syringe 2.

[0097] (3) Install syringe 1 and syringe 2 into the microfluidic device and control the total flow rate to 4, 6, 8, 10, 12, 14 and 16 mL / min.

[0098] (4) Control the flow rate ratio of the aqueous phase and the organic phase to 3:1, and collect the mixed solution at the outlet.

[0099] (5) Dialyze the above mixed solution in PBS buffer for 8 h to obtain a liposome solution without sodium hyaluronate modification and without drug loading.

[0100] Experiment 2 - Effect of the flow rate ratio of organic phase to aqueous phase

[0101] Liposomes without sodium hyaluronate modification and without drug loading were prepared at different flow rates according to the following steps:

[0102] (1) Weigh the raw materials for the liposome skeleton: 253 mg egg yolk lecithin, 113 mg cholesterol, 117 mg (2,3-dioleoyl-propyl)-trimethylammonium chloride and 117 mg distearate phosphatidylethanolamine-polyethylene glycol 2000, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve each raw material, remove impurities in the solution using a 0.22 μm organic filter membrane, and load it into a 10 mL syringe 1.

[0103] (2) Pass the ammonium sulfate aqueous solution (250 mmol / L) through an aqueous filter membrane and put it into syringe 2.

[0104] (3) Install syringe 1 and syringe 2 into the microfluidic device and control the total flow rate to 12 mL / min.

[0105] (4) Control the flow rate ratio of the aqueous phase and the organic phase to 1:1, 2:1, 3:1, 4:1, or 5:1, and collect the mixed solution at the outlet.

[0106] (5) After dialyzing the above mixed solution in PBS buffer for 8 hours, a liposome solution without sodium hyaluronate modification and without drug loading was obtained.

[0107] Experiment 3 - Effect of Doxorubicin Hydrochloride Dosage

[0108] Drug-loaded liposomes without sodium hyaluronate modification were prepared according to the following steps at different dosages of doxorubicin hydrochloride:

[0109] (1) Weigh the raw materials for the liposome skeleton: 253 mg egg yolk lecithin, 113 mg cholesterol, 117 mg (2,3-dioleoyl-propyl)-trimethylammonium chloride and 117 mg distearate phosphatidylethanolamine-polyethylene glycol 2000, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve each raw material, remove impurities in the solution using a 0.22 μm organic filter membrane, and load it into a 10 mL syringe 1.

[0110] (2) Pass the ammonium sulfate aqueous solution (250 mmol / L) through an aqueous filter membrane and put it into syringe 2.

[0111] (3) Install syringe 1 and syringe 2 into the microfluidic device and control the total flow rate to 12 mL / min.

[0112] (4) Control the flow rate ratio of the aqueous phase and the organic phase to 3:1, and collect the mixed solution at the outlet.

[0113] (5) After dialyzing the above mixed solution in PBS buffer for 8 hours, a liposome solution without sodium hyaluronate modification and without drug loading was obtained.

[0114] (6) Weigh 6 mg of berberine hydrochloride into 3 mL of PBS solution, control the amount of doxorubicin hydrochloride added, and add 0.5 mg, 0.8 mg, 1 mg and 1.2 mg of the solution respectively. Incubate with 2 mL of the unmodified and drug-free liposome solution obtained in step (5) at 65°C for 45 min to obtain a drug-loaded liposome solution without sodium hyaluronate modification.

[0115] Based on the preliminary exploration results of relevant parameters, the present invention prepared liposomes and drug-loaded liposomes in the following examples and comparative examples.

[0116] Example 1

[0117] (1) Weigh the raw materials for the liposome skeleton: 253 mg egg yolk lecithin, 113 mg cholesterol, 117 mg (2,3-dioleoyl-propyl)-trimethylammonium chloride and 117 mg distearate phosphatidylethanolamine-polyethylene glycol 2000, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve each raw material, remove impurities in the solution using a 0.22 μm organic filter membrane, and load it into a 10 mL syringe 1.

[0118] (2) Pass the ammonium sulfate aqueous solution (250 mmol / L) through an aqueous filter membrane and put it into syringe 2.

[0119] (3) Install syringe 1 and syringe 2 into the microfluidic device and control the total flow rate to 12 mL / min.

[0120] (4) Control the flow rate ratio of the aqueous phase and the organic phase to 3:1, and collect the mixed solution at the outlet.

[0121] (5) After dialyzing the above mixed solution in PBS buffer for 8 hours, a liposome solution without sodium hyaluronate modification and without drug loading was obtained.

[0122] (6) Weigh 6 mg of berberine hydrochloride into 3 mL of PBS solution, control the amount of doxorubicin hydrochloride added, add 1 mg to the solution, and incubate with 2 mL of the unmodified and drug-free liposome solution obtained in step (5) at 65°C for 45 min to obtain a drug-loaded liposome solution unmodified by sodium hyaluronate.

[0123] (7) Control the mass ratio of liposome backbone to sodium hyaluronate to be 10:1, and add sodium hyaluronate solution (dissolved in water, 100 μg / mL) to the drug-loaded liposome solution without sodium hyaluronate modification obtained in step (6) at a rotation speed of 15 rpm to obtain the drug-loaded liposome solution, denoted as HA-LP-DOX / BBR.

[0124] Example 2

[0125] The only difference from Example 1 is that the mass ratio of the liposome skeleton to sodium hyaluronate in step (7) is 20:1.

[0126] Example 3

[0127] The only difference from Example 1 is that the mass ratio of the liposome skeleton to sodium hyaluronate in step (7) is 15:1.

[0128] Example 4

[0129] The only difference from Example 1 is that the mass ratio of liposome skeleton to sodium hyaluronate in step (7) is 5:1.

[0130] Example 5

[0131] The only difference from Example 1 is that step (6) is not performed, i.e., no drug is loaded. The resulting sodium hyaluronate-modified but drug-free liposomes are denoted as HA-LP.

[0132] Comparative Example 1

[0133] The only difference from Example 1 is that berberine hydrochloride is not added in step (6), and the resulting drug-loaded liposomes are denoted as HA-LP-DOX.

[0134] Comparative Example 2

[0135] The only difference from Example 1 is that doxorubicin hydrochloride is not added in step (6), and the resulting drug-loaded liposomes are denoted as HA-LP-BBR.

[0136] Comparative Example 3

[0137] The only difference from Example 1 is that step (7) is not performed, i.e., sodium hyaluronate is not modified. The resulting drug-loaded liposomes without sodium hyaluronate modification are denoted as LP-DOX / BBR.

[0138] Example 1:

[0139] 1. Characterization of hydrated particle size, PDI and Zeta potential

[0140] (1) For exploratory experiment 1, the hydrated particle size and PDI of the liposomes without sodium hyaluronate modification and drug loading were determined by dynamic light scattering method (specifically by laser particle size analyzer). The results are shown in Table 1.

[0141] Table 1

[0142]

[0143] As shown in Table 1, with the increase of total flow rate, the particle size of the obtained liposomes without sodium hyaluronate modification and without drug loading gradually decreased in the range of 70nm-110nm, but the PDI was lower than 0.25. The PDI was the smallest when the total flow rate was 12 mL / min.

[0144] (2) For exploratory experiment 2, the hydrated particle size and PDI of the liposomes that were not modified with sodium hyaluronate and were not loaded with drugs were determined by dynamic light scattering method (specifically by laser particle size analyzer). The results are shown in Table 2.

[0145] Table 2

[0146]

[0147] As shown in Table 2, under different flow rate ratios, the particle size of the obtained liposomes without sodium hyaluronate modification and without drug loading was controlled in the range of 50nm-150nm, and the PDI was lower than 0.25. The PDI was the lowest when the flow rate ratio of aqueous phase to organic phase was 3:1.

[0148] (3) For Examples 1-4, the hydrated particle size and Zeta potential of the drug-loaded liposomes were determined by dynamic light scattering method (specifically by laser particle size analyzer), and the results are shown in Table 3.

[0149] Table 3

[0150]

[0151] As shown in Table 3, the particle size of the drug-loaded liposomes was controlled within the range of 75nm-100nm under different sodium hyaluronate inputs. As the sodium hyaluronate input increased, the Zeta potential gradually changed from a positive potential (about 10mV) to a negative potential (about -15mV). When the mass ratio of phospholipids to sodium hyaluronate changed from 10:1 to 5:1, the potential change tended to stabilize.

[0152] In addition, the physicochemical parameters of the sodium hyaluronate-modified liposomes obtained in Example 5 without drug loading were as follows: particle size of 75 nm, PDI of 0.12, and potential of -12 mV.

[0153] 2. Determination of encapsulation efficiency and drug loading rate

[0154] The encapsulation efficiency and drug loading rate of the drug-loaded liposomes without sodium hyaluronate modification obtained in Experiment 3 were tested. Methanol was added to different solutions of drug-loaded liposomes without sodium hyaluronate modification, and the mixture was sonicated for 20 min. Then, the encapsulation efficiency (EE%) and drug loading rate (LD%) of doxorubicin hydrochloride and berberine hydrochloride were determined by high-performance liquid chromatography (HPLC). The specific procedures are as follows:

[0155] (1) Inject the drug-loaded liposome solution into the upper layer of an ultrafiltration centrifuge tube (molecular weight cutoff of 30 kDa), set the refrigerated centrifuge time to 30 min, temperature to 4℃, and speed to 8000 rpm, and separate the free drug, denoted as F. drug .

[0156] (2) Add 5 times the volume of methanol to the drug-loaded liposome solution, sonicate for 20 min using an ultrasonic probe, dilute appropriately, and record as T. drug .

[0157] (3) T was determined by HPLC. drug and F drug The concentration of the drug is then calculated using the following formula:

[0158] EE (%) = (T) drug - F drug ) / T drug ×100%;

[0159] LD (%) = T drug / T 脂质 ×100%;

[0160] Among them, T 脂质 The total mass of the liposome skeleton in a drug-loaded liposome.

[0161] The results are shown in Table 4. Under different mass ratios (different amounts of doxorubicin hydrochloride), the encapsulation efficiency of berberine hydrochloride (BBR) can be guaranteed to be above 52%, and the drug loading rate can be guaranteed to be above 10%. The encapsulation efficiency of doxorubicin hydrochloride (DOX) can be guaranteed to be above 87%, and the drug loading rate can be guaranteed to be above 8%.

[0162] Table 4

[0163]

[0164] Based on the above calculation formula, even if sodium hyaluronate is further modified, the drug encapsulation rate and drug loading rate in the obtained sodium hyaluronate-modified drug-loaded liposomes will not change. That is, the drug encapsulation rate and drug loading rate in the sodium hyaluronate-modified drug-loaded liposomes (liposome skeleton: berberine hydrochloride: doxorubicin hydrochloride = 5:1:1) obtained in Examples 1-4 are the same as the specific data in the table.

[0165] 3. Observation using transmission electron microscopy

[0166] Take an appropriate amount of the drug-loaded liposome solution from Example 1, drop it onto a copper sheet and dry it. Then, add an equal amount of phosphotungstic acid for staining for 3 minutes, dry it, and take a picture using a transmission electron microscope. The results are shown in the figure. Figure 1 .

[0167] The results showed that the drug-loaded liposomes were generally spherical, and the hydrated particle size was consistent with the results measured by the laser particle size analyzer.

[0168] Example 2:

[0169] 1. Cell uptake experiment

[0170] To study the cellular uptake kinetics of DOX, A2780 cells were fed at a dose of 8 × 10⁻⁶. 4 Cells were seeded at a density of cells / well on coverslips of 12-well plates and cultured overnight in RPMI-1640 basal medium (Gibco). The medium was then replaced with serum-free medium containing 2 mL of DOX, DOX / BBR, HA-LP-DOX / BBR (Example 1), HA-LP (Example 5), HA-LP-DOX (Comparative Example 1), or LP-DOX / BBR (Comparative Example 3). DOX / BBR was a mixed solution of the two drugs; and in this experiment, the concentrations of DOX and BBR in the DOX / BBR group were controlled to be consistent with the DOX and BBR loadings in Example 1. Cells were washed with PBS at predetermined time intervals (4, 8, and 12 hours), and the nuclei were stained with Hoechst. Finally, cells were fixed with 4% paraformaldehyde and imaged using a fluorescence microscope.

[0171] The results are as follows Figure 2As shown, after 12 hours, the fluorescence intensity of HA-LP-DOX / BBR was significantly higher than that of the other groups, indicating that HA-LP-DOX / BBR can effectively achieve slow release of DOX and increase DOX uptake.

[0172] 2. Cytotoxicity assay

[0173] The cytotoxic effects of DOX / BBR, HA-LP-DOX / BBR (Example 1), HA-LP (Example 5), HA-LP-DOX (Comparative Example 1), HA-LP-BBR (Comparative Example 2), and LP-DOX / BBR (Comparative Example 3) on A2780 cells were investigated using the Cell Counting Kit-8 (CCK-8) method (Beyotime). DOX / BBR was a mixed solution of the two drugs; and in this experiment, the concentrations of DOX and BBR in the DOX / BBR group were controlled to be consistent with the drug loadings of DOX and BBR in Example 1.

[0174] The experimental results are shown in Figure 3 Table 5 shows that the HA-LP-DOX / BBR prepared in Example 1 has the strongest tumor-killing effect on A2780 tumor cells, and the cascade enzyme plays the main killing role.

[0175] Table 5

[0176]

[0177] 3. Apoptosis experiment

[0178] The in vitro apoptosis-inducing ability of 0.2 mL DOX / BBR, HA-LP-DOX / BBR (Example 1), HA-LP (Example 5), HA-LP-DOX (Comparative Example 1), HA-LP-BBR (Comparative Example 2), and LP-DOX / BBR (Comparative Example 3) to A2780 cells was determined using the Annexin V-FITC / PI apoptosis detection kit (Elabscience). DOX / BBR was a mixed solution of the two drugs; and in this experiment, the concentrations of DOX and BBR in the DOX / BBR group were controlled to be consistent with the drug loading in Example 1.

[0179] like Figure 4 As shown, the HA-LP-DOX / BBR group had the strongest apoptosis-inducing effect on A2780 cells compared to other groups (this can be seen by calculating the sum of Q2 and Q3 regions, with the lowest sum of Q2+Q3 being 60.93).

[0180] 4. Cell migration experiment

[0181] A2780 cells were seeded in six-well plates. The effects of 0.2 mL DOX / BBR, HA-LP-DOX / BBR (Example 1), HA-LP (Example 5), HA-LP-DOX (Comparative Example 1), HA-LP-BBR (Comparative Example 2), and LP-DOX / BBR (Comparative Example 3) on A2780 cell migration were measured. The scratch area after 24 h was analyzed using ImageJ software. DOX / BBR was a mixed solution of the two drugs; and in this experiment, the concentrations of DOX and BBR in the DOX / BBR were controlled to be consistent with the drug loading in Example 1.

[0182] Experimental results are as follows Figure 5 As shown, the results indicate that the migration rate of the HA-LP-DOX / BBR group was low at 7.35%, suggesting that HA-LP-DOX / BBR can significantly inhibit the migration of A2780 cells.

[0183] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A liposome, characterized in that, The liposomes include a liposome skeleton and a targeting ligand; wherein the liposome skeleton is composed of the following components: phospholipids, cholesterol, cationic lipids and PEGylated phospholipids; The cationic lipid is (2,3-dioleoyl-propyl)-trimethylammonium chloride and / or 1,2-dioleoyl-3-trimethylammonium propane; The molar ratio of the phospholipids, cholesterol, cationic lipids and PEGylated phospholipids is (20-40):(15-35):(15-35):(1-10); The targeting ligand is sodium hyaluronate and / or hyaluronic acid; The mass ratio of the targeting ligand to the liposome backbone is 1:(5-20).

2. The liposomes as described in claim 1, characterized in that, The liposomes satisfy one or more of the following conditions: (1) The molar ratio of the phospholipids, cholesterol, cationic lipids and PEGylated phospholipids is 40:35:20:5; (2) The mass ratio of the targeting ligand to the liposome backbone is 1:(5-15), for example 1:10; (3) The phospholipid is one or more of lecithin, soybean lecithin, egg yolk lecithin, sphingomyelin and hydrogenated soybean lecithin, such as egg yolk lecithin; (4) The PEGylated phospholipid is one or more of distearylphosphatidylethanolamine-polyethylene glycol 1000, distearylphosphatidylethanolamine-polyethylene glycol 2000 and distearylphosphatidylethanolamine-polyethylene glycol 5000, preferably distearylphosphatidylethanolamine-polyethylene glycol 2000; (5) The liposomes have a particle size of 60-100 nm, for example 75 nm or 80 nm; (6) The PDI of the liposomes is 0.1-0.3, for example 0.12 or 0.15; (7) The zeta potential of the liposome is +20 to -20mV, preferably -10 to -20mV, for example -12mV or -13mV.

3. A method for preparing liposomes, characterized in that, The raw materials for the liposomes include raw materials for the liposome skeleton and targeting ligands; wherein, the raw materials for the liposome skeleton include phospholipids, cholesterol, cationic lipids and PEGylated phospholipids; The cationic lipid is (2,3-dioleoyl-propyl)-trimethylammonium chloride and / or 1,2-dioleoyl-3-trimethylammonium propane; The molar ratio of the phospholipids, cholesterol, cationic lipids and PEGylated phospholipids is (20-40):(15-35):(15-35):(1-10); The targeting ligand is sodium hyaluronate and / or hyaluronic acid; The mass ratio of the raw materials for the target ligand and the liposome matrix is ​​1:(5-20); The liposome preparation method is selected from one or any combination of two of the following methods: microfluidic method, thin film dispersion method, injection method, reverse evaporation method, freeze-thaw method, and double emulsion method.

4. The method for preparing liposomes as described in claim 3, characterized in that, The method for preparing the liposomes satisfies one or more of the following conditions: (1) The types and / or amounts of the phospholipids, cholesterol, cationic lipids, PEGylated phospholipids, targeting ligands and liposome backbones are as defined in claim 2; (2) The liposomes are prepared by a microfluidic method, which includes the following steps: S1, organic phase and aqueous phase are mixed using microfluidic technology to prepare liposome framework solution, wherein: The organic phase includes phospholipids, cholesterol, cationic lipids, PEGylated phospholipids, and organic solvents; The aqueous phase includes ammonium sulfate and water; S2. The liposome matrix solution, the targeting ligand, and water are mixed to prepare a liposome solution.

5. The method for preparing liposomes as described in claim 4, characterized in that, The method for preparing the liposomes satisfies one or more of the following conditions: (1) In step S1, the organic solvent is ethanol; (2) In step S1, the mixing includes the following steps: the organic phase and the aqueous phase are pumped into the microfluidic device by syringe A and syringe B respectively, and they begin to mix upon contact to form the liposome skeleton solution; The flow rates of the organic phase and the aqueous phase are preferably 1:(1-9), more preferably 1:(1-5), for example 1:2, 1:3, 1:4; The total flow rate, calculated as the sum of the flow rates of the organic phase and the aqueous phase, is preferably 4-24 mL / min, more preferably 4-16 mL / min, for example 6, 8, 10, 12, 14, 12 mL / min. (3) In step S1, the preparation of the organic phase includes dissolving the phospholipid, cholesterol, cationic lipid, and PEGylated phospholipid in the organic solvent; The dissolution is preferably performed under ultrasound; the ultrasound duration is, for example, 0.5 hours. Preferably, the dissolution step is followed by a filtration step; the filtration is performed, for example, using an organic filter membrane; the pore size of the organic filter membrane is, for example, 0.22 μm; the organic filter membrane is, for example, a PVDF membrane; (4) In step S1, the preparation of the aqueous phase includes dissolving the ammonium sulfate in the water; Preferably, the dissolution step is followed by a filtration step; the filtration is performed, for example, using an aqueous filter membrane; the pore size of the aqueous filter membrane is, for example, 0.22 μm; the aqueous filter membrane is, for example, a PES membrane; (5) After step S1, the step of dialyzing the liposome matrix solution is further included; the dialyzing is performed, for example, in PBS buffer; the dialyzing time is, for example, 8 hours. (6) In step S2, the mixing step includes dissolving the targeting ligand in the water first, and then adding it dropwise into the liposome skeleton solution.

6. A liposome, characterized in that, The liposomes are prepared by the method for preparing liposomes as described in any one of claims 3-5.

7. A drug-loaded liposome, characterized in that, The drug-loaded liposome comprises a liposome as described in any one of claims 1, 2 and 6 and a pharmaceutically active ingredient; the pharmaceutically active ingredient is doxorubicin hydrochloride and berberine hydrochloride.

8. The drug-loaded liposome according to claim 7, characterized in that, The drug-loaded liposomes meet one or more of the following conditions: (1) In the drug-loaded liposomes, the encapsulation efficiency of doxorubicin hydrochloride is not less than 85%, preferably 85%-95%, for example 94.4%, 93.4%, 92.7% or 87.8%; (2) In the drug-loaded liposomes, the drug loading rate of doxorubicin hydrochloride is not less than 8%, preferably 8%-20%, for example 8.1%, 8.6%, 16.8% or 19.1%; (3) In the drug-loaded liposomes, the encapsulation efficiency of berberine hydrochloride is not less than 50%, preferably 50%-65%, for example 52.6%, 56.4%, 60.2% or 61.5%; (4) In the drug-loaded liposomes, the loading rate of berberine hydrochloride is not less than 10%, preferably 10%-20%, for example 10.6%, 10.8%, 11.9% or 13.3%; (5) The particle size of the drug-loaded liposomes is 60-100 nm, for example 78.31 nm, 81.12 nm, 83.54 nm, 90.32 nm or 80 nm; (6) The PDI of the drug-loaded liposome is 0.05-0.2, for example 0.1; (7) The zeta potential of the drug-loaded liposome is +20 to -20 mV, preferably -10 to -20 mV, for example -12.94 mV, -15.43 mV or -13 mV.

9. A method for preparing drug-loaded liposomes, characterized in that, The method for preparing the drug-loaded liposomes includes the step of loading a drug active ingredient into the liposomes as described in any one of claims 1, 2 and 6 using an active drug loading method; the drug active ingredient is doxorubicin hydrochloride and berberine hydrochloride.

10. The method for preparing drug-loaded liposomes as described in claim 9, characterized in that, The method for preparing the drug-loaded liposomes satisfies one or more of the following conditions: (1) The mass ratio of the liposome skeleton, doxorubicin hydrochloride and berberine hydrochloride is 5:(0.5-1.5):(0.5-1.5), for example 5:1:0.5, 5:1:0.8, 5:1:1 or 5:1:1.2; (2) The method for preparing the drug-loaded liposomes includes the following steps: A. The organic phase and the aqueous phase are mixed using microfluidic technology to prepare a liposome framework solution, wherein: B. The liposome skeleton solution, the active pharmaceutical ingredient, and the solvent are mixed to obtain a first mixture; C. The first mixture, the targeting ligand, and water are mixed to prepare a drug-loaded liposome solution.

11. The method for preparing drug-loaded liposomes as described in claim 10, characterized in that, The method for preparing the drug-loaded liposomes satisfies one or more of the following conditions: (1) In step A, the organic phase, aqueous phase, liposome skeleton solution, and mixture are as defined in claim 4 or 5; (2) In step B, the solvent is PBS; (3) In step B, the mixing step includes dissolving the active pharmaceutical ingredient in the solvent first, and then mixing it with the liposome matrix solution; (4) In step B, the mixing temperature is 60-70°C, for example 65°C; (5) In step B, the mixing time is 40-50 minutes, for example, 45 minutes; (6) In step C, the mixing step includes dissolving the targeting ligand in the water first, and then adding it dropwise into the first mixture.

12. A drug-loaded liposome, characterized in that, It is prepared by the method for preparing drug-loaded liposomes as described in any one of claims 9-11.

13. The use of a drug-loaded liposome as described in any one of claims 7, 8 and 12 in the preparation of an antitumor drug.

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