Preparation method and application of efficiently encapsulated pH-responsive drug-loaded liposome

Abstract

The invention provides a preparation method and application of a high-efficiency encapsulated pH-responsive drug-loaded liposome. According to the method, two-phase fluid of a medicine aqueous solution and a lipid component organic solution in a micro-channel is accurately controlled, and the efficiently encapsulated liposome is prepared from bottom to top by utilizing a micro-mixing technology. Compared with a conventional passive drug loading method which is relatively low in encapsulation efficiency in preparation of hydrophilic drug nano-liposomes, the method provided by the invention can improve the encapsulation efficiency to be close to 95% under optimized prescription and process conditions. According to the invention, by adjusting the proportion of each component in the prescription and the preparation process, the release behavior of liposome drugs such as adriamycin and the like can be adjusted, and the drug release shows pH responsiveness. Through efficient encapsulation of adriamycin and the like and pH-responsive release of the liposome dosage form, the effects of efficacy enhancement and toxicity reduction are achieved in the anti-tumor treatment aspect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and relates to a method for preparing a pH-responsive drug-loaded liposome with efficient encapsulation and its application. The present invention is a method for preparing a pH-responsive drug-loaded liposome with efficient encapsulation by micromixing and its application. The technology can also be used for preparing drug-loaded liposome preparations of other drugs that meet relevant conditions. Background Art

[0002] The anthracycline drug doxorubicin is one of the most important and widely used anti-tumor drugs. It is active against a variety of human tumors, including breast, ovarian, gastric, bladder and bronchial cancers, leukemias and lymphomas, and soft tissue sarcomas. The exact mechanism of action of doxorubicin is unclear, but it is known that it can insert into DNA base pairs, causing template disorder and steric occlusion, thereby inhibiting DNA synthesis and DNA-dependent RNA synthesis. Insertion can cause single-strand and double-strand breaks and sister chromatid exchanges. Doxorubicin disrupts the normal function of mitochondria and increases the production of free radicals and oxidative damage, which are highly destructive to cells (Int J Biol Macromol. 2014; 66: 144-150). Doxorubicin induces the formation of covalent topoisomerase II-DNA complexes, thereby inhibiting the religation part of the ligation-religation reaction in replicating DNA (ChemBiol. 2010; 17(5): 421-433). Doxorubicin also induces cancer cell senescence, autophagy, pyroptosis, ferroptosis, or necrosis, depending on the drug dose and cellular environment. Doxorubicin is active throughout the cell cycle, with maximum toxicity occurring in the S phase of DNA synthesis. At low drug concentrations, cells will continue to undergo the S phase and die in the G2 phase (Drug Saf. 2001; 24(12):903-20).

[0003] Similar to other cytotoxic anti-tumor drugs, the clinical application of doxorubicin is limited by toxicity, because toxicity may lead to inadequate medication or inability to re-administer medication in case of relapse. High cumulative doses of doxorubicin increase the probability of cardiotoxicity, which is the most prominent side effect of doxorubicin and one of the most dangerous dose-limiting toxicities of the drug; single-dose medication is usually limited by bone marrow suppression; hair loss, severe acute nausea and vomiting, and mucositis are other common adverse reactions of doxorubicin, which limit the use of doxorubicin in anti-tumor treatment. Therefore, anthracycline drug preparations with equivalent efficacy and higher safety will increase the therapeutic index of the drug and improve its overall clinical benefit (Ann Oncol. 2004 Mar; 15 (3): 440-9).

[0004] For the above reasons, researchers are committed to developing high-end doxorubicin preparations with the goal of retaining or improving drug efficacy while minimizing adverse reactions. Currently, two liposomal doxorubicin preparations have been approved for marketing: Myocet TM as well as Both formulations alleviated the acute cardiotoxicity of doxorubicin. The differences in doxorubicin release from liposomes after intravenous injection showed different drug clearance, biodistribution, antitumor activity, and toxicity. Compared with the same dose of free doxorubicin, both doxorubicin liposomes enhanced drug delivery to tumor sites and Shown to be superior to Myocet TM In animal models, The acute toxicity of Myocet TM Lower. Therefore It is a liposomal preparation of doxorubicin that is more popular in clinical practice.

[0005] It was developed by Sequus Pharmaceuticals in the United States and was the first nanoformulation approved by the FDA in 1995. It has obtained clinical indications for breast cancer, ovarian cancer, myeloma, and AIDS-related Kaposi's sarcoma. The drug is designed to balance the efficacy and toxicity of doxorubicin, reducing toxicity while ensuring efficacy. The formula composition is: hydrogenated soybean lecithin: cholesterol: distearoyl phosphatidylethanolamine-polyethylene glycol 2000 = 56:39:5 (molar ratio), each bottle The lipid concentration is about 16 mg / mL, the doxorubicin concentration is 2 mg / mL, and it is a translucent red suspension with a particle size of about 100 nm. The liposome carrier uses synthetic phospholipids, which are highly stable. The product is in the form of drug-loaded liposome injection, which is easy to use. It uses Stealth technology and has a long blood circulation time, so it has passive targeting to tumor tissue (J Control Release. 2012 Jun 10; 160(2):117-34).

[0006] The general preparation process of classic liposome preparation methods, such as thin film dispersion method, injection method and reverse evaporation method, can be summarized into two steps: the dispersion of phospholipid molecule monomers in organic solvents and subsequent assembly in an aqueous environment. The liposome formation mechanism determines that when encapsulating hydrophilic drugs, the encapsulation of drugs is limited by the volume of the aqueous phase in the liposome. This passive drug loading method results in the efficiency of liposome encapsulation of hydrophilic drugs being generally below 20% (Adv Drug Deliv Rev. 2020; 154-155: 102-122). The ammonium sulfate gradient method was creatively adopted to carry out active drug loading through the ammonium sulfate gradient formed inside and outside the liposome, and the encapsulation efficiency of doxorubicin was greatly improved to 98%.

[0007] when The dose is 50 mg / m 2 When the drug is taken for 4 h, its AUC is about 300 times that of the free drug. The clearance rate and distribution volume are greatly reduced (at least 250 times and 60 times, respectively). Long-term circulation is conducive to more drug accumulation in tumor tissues. PEGylated liposome encapsulation can also reduce the concentration of free doxorubicin in the blood and may reduce the delivery of the drug to normal tissues, thereby reducing toxicity (Clin Pharmacokinet. 2003; 42(5): 419-36). A large-scale phase III clinical trial will Compared with doxorubicin as first-line treatment for patients with metastatic breast cancer, The efficacy is roughly equivalent to that of doxorubicin, but compared with free doxorubicin, The cardiac toxicity of The incidence of alopecia, nausea, vomiting and bone marrow suppression in treated patients was also lower than that in patients treated with doxorubicin (Ann Oncol. 2004 Mar; 15(3): 440-9).

[0008] Table 1. Cardiotoxicity of first-line treatments for patients with metastatic breast cancer

[0009] Stable and long-circulating liposomes While reducing the cardiac toxicity of doxorubicin, it has also brought new problems. According to the results of a Phase III clinical trial for the first-line treatment of patients with metastatic breast cancer, injection of The overall incidence of adverse reactions was comparable in the two treatment groups: The incidence of grade 3 or higher adverse reactions in the treatment group was even slightly higher than that in the doxorubicin group (Table 2). According to the Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0 published by the U.S. Department of Health and Human Services, grade 3 adverse reactions refer to adverse reactions that are severe or medically significant but not immediately life-threatening, which may lead to hospitalization or prolonged hospitalization, disability or limitation of self-care and daily living activities. When grade 3 or higher adverse reactions occur, adjuvant treatment, adjustment of drug dosage or discontinuation of medication are required according to actual conditions. During this clinical trial, Approximately 24% of patients in both treatment groups discontinued treatment due to drug toxicity.

[0010] Table 2. Medication-related adverse reactions

[0011] Unlike the common hair loss, nausea, vomiting and bone marrow suppression (anemia, leukopenia, neutropenia and thrombocytopenia) of doxorubicin, It is more likely to cause skin and mucosal adverse reactions (Table 3). Data from a Phase III clinical trial of first-line treatment for patients with metastatic breast cancer showed that hand-foot syndrome (HFS) is The most common adverse reactions were 48.4% in the doxorubicin group, compared with 1.9% in the adriamycin group. In a phase III study of recurrent epithelial ovarian cancer, 49% of patients receiving 50 mg / m every 4 weeks 2 of HFS occurred during treatment, with 22% of patients experiencing grade 3 or higher HFS. 4.2% of patients discontinued treatment due to HFS or other skin toxicity (J Clin Oncol. 2001; 19(14): 3312-3322). The exact mechanism of this adverse reaction is not fully understood, but it may be related to the accumulation of long-circulating liposomes in the extremities and the long-term slow release of encapsulated doxorubicin.

[0012] Table 3. Common adverse reactions of non-cardiac and blood system

[0013] In addition, in clinical trials for first-line treatment of patients with stage III metastatic breast cancer The efficacy of the group was comparable to that of the doxorubicin group, and did not show the expected superior therapeutic effect. The difficulty in drug release is related to the in vitro cell level studies have shown that It is difficult to release in cells, resulting in its cytotoxicity being lower than that of doxorubicin. The release of doxorubicin in liposomes can improve its anti-tumor efficacy in vivo (J Control Release. 2022 Oct: 350: 414-420 & 2013 May 28; 168(1): 61-9). Based on this phenomenon, Celsion Corporation in the United States has also developed the thermosensitive doxorubicin liposome ThermoDox, which aims to solve the problem of difficulty in releasing encapsulated drugs in liposomes under the conditions of active drug loading strategy. Summary of the invention

[0014] The purpose of the present invention is to provide a method for preparing pH-responsive drug-loaded liposomes with high efficiency encapsulation, which is a method for preparing pH-responsive drug-loaded liposomes with high efficiency encapsulation by micromixing. The specific operation is as follows: (1) dissolving the drug in a slightly acidic buffered saline solution, the buffered saline solution being used as the drug aqueous solution, and dissolving the lipid component in an organic solvent being used as the lipid component organic solution; (2) The two-phase solution is pumped into a microfluidic chip or micromixer via a syringe pump to complete mixing. During mixing, the lipid components are superior to the changes in the solution environment and initiate a "bottom-up" self-assembly process, during which the drug is encapsulated to form primary liposomes. (3) The primary liposomes are then dialyzed and ultrafiltered to remove unencapsulated free drugs, and the pH value is adjusted to a neutral pH of 7.0 using a neutral buffer (including but not limited to phosphate buffer, citric acid-sodium citrate buffer, Hanks' balanced salt solution, Earle balanced salt solution, etc.), and the drug is finally quantified to a target concentration to obtain the target liposomes.

[0015] The drug in step (1) is selected from drugs that remain protonated under acidic conditions (2.5 < pH < 6.5), such as doxorubicin hydrochloride, vinorelbine tartrate, and nintedanib. The present invention dissolves the drug in a slightly acidic buffered saline solution, at which time the drug molecule is protonated and carries a positive charge, and the drug concentration ranges from 0.1 to 10 mg / mL.

[0016] The buffered saline solution of step (1) includes, but is not limited to, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, acetic acid-sodium acetate buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, citric acid-sodium citrate buffer and the like, which are commonly used acidic buffered saline solutions. The pH value of the buffered saline solution ranges from 2.5 to 6.5, and the concentration ranges from 1 mM to 300 mM.

[0017] The lipid components of step (1) include natural phospholipids or synthetic phospholipids, cholesterol, anionic phospholipids and pegylated phospholipids, the mass ratio of the four components is 0-60:10-25:5-50:5-20, and the total concentration of the four lipid components in the organic solvent is 0.1-150 mg / mL, and the organic solvent includes but is not limited to ethanol, methanol, isopropanol, acetonitrile or ether, which are good solvents for lipid materials that can be completely mixed with aqueous solutions.

[0018] The natural phospholipids include but are not limited to egg yolk lecithin, soybean lecithin or PL-100M, and the synthetic phospholipids include but are not limited to hydrogenated soybean lecithin, dipalmitoyl lecithin, dierucoyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine and other phosphatidylcholine phospholipids, and oleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine or dimyristoyl phosphatidyl Phosphatidylethanolamine phospholipids such as acylethanolamine, anionic phospholipids include phosphatidylphosphatidic acid, phosphatidylinositol, phosphatidylglycerol, and phosphatidylserine phospholipids. In the specific solution environment after the drug aqueous solution and the phospholipid organic solution are mixed, the surface potential of the liposome containing this component is negative. Polyethylene glycol phospholipids include but are not limited to distearoyl phosphatidylethanolamine-polyethylene glycol 2000, dipalmitoyl phosphatidylethanolamine-methoxypolyethylene glycol 2000 and other commonly used pharmaceutical polyethylene glycol phospholipids.

[0019] The two-phase fluid described in step (2) is precisely controlled, the aqueous solution and the organic solution are transported by pumping, the flow rate of the two is 0.001L / min-1L / min, the flow rate ratio of the aqueous solution to the organic solution is 1-60:1, and the temperature during mixing is controlled at 0℃-65℃.

[0020] In the micromixing technology described in step (2), the aqueous solution and the organic solution are mixed in a micromixer. The types of micromixers include microfluidic chips, polymer material micromixers, and metal micromixers. The channel size range of the micromixer is 0.05-0.75 mm.

[0021] The micro mixer has a specific mixing structure inside, including a T-shaped, Y-shaped, cross-shaped preliminary mixing structure (in which the two-phase fluid completes preliminary mixing), and a Herringbone-shaped, Tesla-shaped, spiral-shaped auxiliary mixing structure (in which the two-phase fluid is completely mixed), and the two structures are connected by a pipeline. The two-phase fluid is quickly and completely mixed in the micro mixer, which helps to form nanoliposomes with uniform and controllable size.

[0022] The neutral buffer in step (3) includes, but is not limited to, phosphate buffer, citric acid-sodium citrate buffer, Hanks' balanced salt solution, Earle balanced salt solution, etc.

[0023] The drug-loaded liposomes are prepared in a "bottom-up" manner. When the drug aqueous solution and the lipid component organic solution are mixed in a micromixer, the lipid components immediately begin to self-assemble to form liposomes. The drug encapsulation mechanism includes: (1) the protonated positively charged drug is encapsulated by combining with the anionic phospholipids of one of the lipid components in an electrostatic interaction; (2) due to the change of the solvent environment, the good solvent environment of the drug is transformed into a relatively poor solvent environment, the solubility of the drug decreases, and the drug is encapsulated in the lipid bilayer structure of the liposome due to hydrophobic interaction; (3) part of the drug that is still dissolved in the solvent system is encapsulated in the inner aqueous phase by the liposome in a passive encapsulation manner.

[0024] The present invention uses micromixing technology to prepare doxorubicin liposomes in a "bottom-up" method by precisely controlling the flow of a two-phase fluid of a drug aqueous solution and a lipid component organic solution. The flow of the two-phase fluid is driven by a pump, and the flow rate and flow rate ratio of the two phases are adjusted according to actual needs. Micromixing occurs in a micromixer, and the structural characteristics and channel dimensions of the micromixer are adjusted according to actual needs and based on fluid simulation.

[0025] The present invention obtains doxorubicin liposomes with a suitable drug release rate by optimizing the ratio of each component in the prescription, the preparation process and the micromixer. The physicochemical properties of the doxorubicin liposomes can be affected by changing the ratio of four phospholipid components and the drug, the buffered salt solution, the flow rate and the flow rate ratio during micromixing, and the specifications of the micromixer. The final release behavior is determined by the above factors.

[0026] Another object of the present invention is to provide the use of the prepared liposomes in the preparation of pH-responsive doxorubicin liposome anti-tumor drugs. The doxorubicin liposomes prepared by the method of the present invention have a suitable drug release rate, while reducing the incidence of adverse reactions of doxorubicin drugs, and avoiding the active drug loading method. It can prevent HFS caused by chemotherapy and has advantages in anti-tumor efficacy.

[0027] The doxorubicin liposomes prepared by the present invention are pH responsive, relatively stable under neutral conditions, and exhibit slow drug release behavior, while exhibiting accelerated release behavior under weakly acidic conditions of the tumor microenvironment, which is beneficial to reducing the toxicity of the drug to normal tissues and enhancing the killing effect on tumor tissues.

[0028] The present invention designs and prepares pH-responsive doxorubicin liposomes Doxsome by micromixing, and develops a new generation of doxorubicin liposomes by improving and innovating the formulation and preparation process, taking efficient encapsulation and optimized drug release behavior as breakthrough points. Through the encapsulation of doxorubicin and the pH-responsive release of the liposome dosage form, the original doxorubicin and The incidence of adverse reactions is better than that of doxorubicin and anti-tumor efficacy.

[0029] The innovation of the present invention lies in that a method for preparing liposomes that efficiently encapsulate doxorubicin is adopted, which is different from the active drug loading method. The method gives the doxorubicin liposomes a certain degree of stability, and the drug release has pH responsiveness, which is relatively The invention can reduce the incidence of adverse reactions of doxorubicin while avoiding The invention has good clinical application prospects, involves preparation technology, has simple and mature process, controllable material cost, is stable and reliable, and is conducive to commercial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a microfluidic chip with Herringbone configuration.

[0031] Figure 2 It is a T-shaped mixer.

[0032] Figure 3 It is a Tesla-configured microfluidic chip.

[0033] Figure 4 It is a microfluidic chip with flow focusing configuration.

[0034] Figure 5 It is a spiral auxiliary mixing structure.

[0035] Figure 6 This is the in vitro release curve of Doxsome and Doxil.

[0036] Figure 7 These are the in vitro release curves of Doxsome and Doxil under different pH conditions.

[0037] Figure 8 is the in vitro cytotoxicity of different doxorubicin preparations.

[0038] Fig. 9 The intracellular distribution of doxorubicin of different doxorubicin preparations.

[0039] Fig.10 There are some differences in the physical and chemical properties of doxorubicin liposomes produced by different processes and formulations.

[0040] Fig.11 The inhibitory effect of doxorubicin liposomes with different processes and formulations on the growth of 4T1 breast cancer tumors.

[0041] Fig.12 The effect of doxorubicin liposomes with different processes and formulations on the body weight of mice bearing 4T1 breast cancer tumors.

[0042] Fig.13 The distribution of Doxsome in mice bearing 4T1 breast cancer tumors.

[0043] Fig.14 It is the inhibitory effect of Doxsome on 4T1 breast cancer tumor growth.

[0044] Fig.15 The effect of Doxsome on the body weight of mice bearing 4T1 breast cancer tumors.

[0045] Fig.16 It is the inhibitory effect of Doxsome on B16-F10 melanoma growth.

[0046] Fig.17 The effect of Doxsome on the body weight of mice bearing B16-F10 melanoma.

[0047] Fig.18 The effect of Doxsome on the survival of mice bearing B16-F10 melanoma.

[0048] Fig.19 These are H&E sections of the hearts of B16-F10 melanoma-bearing mice treated with different doxorubicin formulations.

[0049] Fig. 20 The effect of different doxorubicin preparations on body weight in Sprague Dawley rats.

[0050] Fig.21 The skin conditions of the limbs of Sprague Dawley rats in groups treated with different doxorubicin preparations. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is described in detail below in conjunction with the accompanying drawings and embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0052] Example 1 A method for preparing doxorubicin hydrochloride liposomes Prescription composition:

[0053] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ), self-assembled in a "bottom-up" manner to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 48.29±0.12nm, a PDI of 0.242±0.006, a surface potential of -15.8±0.66mV, and an encapsulation rate of 94.76%.

[0054] Example 2 Preparation of doxorubicin hydrochloride liposomes using different natural phospholipids (I) Prescription composition:

[0055] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of soybean lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 71.55±0.29nm, a PDI of 0.274±0.009, a surface potential of -10.3±1.39mV, and an encapsulation rate of 93.56%.

[0056] Example 3 Preparation of doxorubicin hydrochloride liposomes using different natural phospholipids (II) Prescription composition:

[0057] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of PL-100M, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 96.50±0.94nm, a PDI of 0.296±0.009, a surface potential of -16.2±0.93mV, and an encapsulation rate of 93.27%.

[0058] Example 4 Preparation of doxorubicin hydrochloride liposomes using different synthetic phospholipids (I) Prescription composition:

[0059] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of dipalmitoyl phosphatidylcholine, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 164.24±1.33nm, a PDI of 0.033±0.023, a surface potential of -16.7±0.45mV, and an encapsulation rate of 94.50%.

[0060] Example 5 Preparation of doxorubicin hydrochloride liposomes using different synthetic phospholipids (II) Prescription composition:

[0061] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of hydrogenated soybean lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 210.70±3.04nm, a PDI of 0.176±0.022, a surface potential of -15.0±0.51mV, and an encapsulation rate of 92.29%.

[0062] Example 6 Preparation of doxorubicin hydrochloride liposomes using different synthetic phospholipids (III) Prescription composition:

[0063] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of dioleoylphosphatidylethanolamine, cholesterol, egg yolk phosphatidylglycerol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 66.88±0.10nm, a PDI of 0.178±0.011, a surface potential of -15.2±0.93mV, and an encapsulation rate of 94.89%.

[0064] Example 7 Preparation of doxorubicin hydrochloride liposomes using different synthetic phospholipids (IV) Prescription composition:

[0065] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of distearoyl phosphatidylethanolamine, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 194.0±4.17nm, a PDI of 0.200±0.012, a surface potential of -41.2±5.60mV, and an encapsulation rate of 94.77%.

[0066] Example 8 Preparation of doxorubicin hydrochloride liposomes using different anionic phospholipids (I) Prescription composition:

[0067] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, 1,2-palmitoylphosphatidylglycerol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution (dimethyl sulfoxide assisted dissolution). The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 49.18±0.41nm, a PDI of 0.252±0.004, a surface potential of -15.8±0.72mV, and an encapsulation rate of 92.57%.

[0068] Example 9 Preparation of doxorubicin hydrochloride liposomes using different anionic phospholipids (II) Prescription composition:

[0069] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, distearoylphosphatidylglycerol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution (dimethyl sulfoxide assisted dissolution). The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ), self-assembled to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product doxorubicin hydrochloride liposomes, with a particle size of 66.88±0.10nm, a PDI of 0.178±0.0111, a surface potential of -12.7±0.82mV, and an encapsulation rate of 91.78%.

[0070] Example 10 Preparation of doxorubicin hydrochloride liposomes using different anionic phospholipids (III) Prescription composition:

[0071] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, dioleoylphosphatidylglycerol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution (dimethyl sulfoxide assisted dissolution). The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ), self-assembled to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product doxorubicin hydrochloride liposomes, with a particle size of 58.58±0.45nm, a PDI of 0.163±0.012, a surface potential of -16.7±0.45mV, and an encapsulation rate of 91.45%.

[0072] Example 11 Preparation of doxorubicin hydrochloride liposomes using different anionic phospholipids (IV) Prescription composition:

[0073] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, dipalmitoylphosphatidylserine, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution (dimethyl sulfoxide assisted dissolution). The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ), self-assembled to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product doxorubicin hydrochloride liposomes, with a particle size of 93.56±0.47nm, a PDI of 0.245±0.008, a surface potential of -18.4±0.72mV, and an encapsulation rate of 91.65%.

[0074] Example 12 Preparation of doxorubicin hydrochloride liposomes using different anionic phospholipids (V) Prescription composition:

[0075] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, dipalmitoyl phosphatidic acid, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution (dimethyl sulfoxide assisted dissolution). The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ), self-assembled to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product doxorubicin hydrochloride liposomes, with a particle size of 77.80±0.30nm, a PDI of 0.252±0.010, a surface potential of -16.6±1.66mV, and an encapsulation rate of 93.15%.

[0076] Example 13 Preparation of doxorubicin hydrochloride liposomes using different buffered saline solutions (I) Prescription composition:

[0077] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (140 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 80.14±0.82nm, a PDI of 0.228±0.017, a surface potential of -36.1±1.21mV, and an encapsulation rate of 52.73%.

[0078] Example 14 Preparation of doxorubicin hydrochloride liposomes using different buffered saline solutions (II) Prescription composition:

[0079] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (280 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 108.4±1.25nm, a PDI of 0.199±0.013, a surface potential of -31.68±2.95mV, and an encapsulation rate of 28.72%.

[0080] Example 15 Preparation of doxorubicin hydrochloride liposomes using different buffered saline solutions (III) Prescription composition:

[0081] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium acetate buffer solution (280 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 187.3±2.40nm, a PDI of 0.270±0.017, a surface potential of -41.30±5.60mV, and an encapsulation rate of 25.98%.

[0082] Example 16 Preparation of doxorubicin hydrochloride liposomes using different buffered saline solutions (IV) Prescription composition:

[0083] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of PBS buffer solution (10 mM, pH 5.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 74.57±0.82nm, a PDI of 0.222±0.009, a surface potential of -12.1±4.72mV, and an encapsulation rate of 62.77%.

[0084] Example 17 Preparation of doxorubicin hydrochloride liposomes using different lipid formulations (I) Prescription composition:

[0085] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (280 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 86.70.0±1.16nm, a PDI of 0.264±0.015, a surface potential of -36.2±1.51mV, and an encapsulation rate of 42.98%.

[0086] Example 18 Preparation of doxorubicin hydrochloride liposomes using different lipid formulations (II) Prescription composition:

[0087] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (280 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 86.93±0.46nm, a PDI of 0.234±0.027, a surface potential of -24.9±1.16mV, and an encapsulation rate of 71.58%.

[0088] Example 19 Preparation of doxorubicin hydrochloride liposomes using different lipid formulations (III) Prescription composition:

[0089] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (280 mM, pH 3.5) as an aqueous solution; the prescribed amount of cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 133.6±1.92nm, a PDI of 0.197±0.008, a surface potential of -35.0±0.51mV, and an encapsulation rate of 59.35%.

[0090] Example 20 Different lipid formulations for the preparation of doxorubicin hydrochloride liposomes (IV) Prescription composition:

[0091] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (280 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of hot anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 128.2±0.98nm, a PDI of 0.201±0.008, a surface potential of -41.3±5.60mV, and an encapsulation rate of 46.82%.

[0092] Example 21 Preparation of doxorubicin hydrochloride liposomes using different lipid formulations (V) Prescription composition:

[0093] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (280 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of hot anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 159.0±4.00nm, a PDI of 0.131±0.022, a surface potential of 35.4±0.76mV, and an encapsulation rate of 51.13%.

[0094] Example 22 Preparation of doxorubicin hydrochloride liposomes using different lipid formulations (VI) Prescription composition:

[0095] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (280 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of hot anhydrous ethanol as a lipid component organic solution. The two were injected into the Herringbone configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 1 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free doxorubicin, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 214.0±3.03nm, a PDI of 0.150±0.030, a surface potential of -39.9±0.47mV, and an encapsulation rate of 59.86%.

[0096] Example 23 A method for preparing vinorelbine tartrate liposomes Prescription composition:

[0097] The prescribed amount of vinorelbine tartrate was dissolved in 15 mL of sodium citrate buffer solution (80 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into a Herringbone-configured microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing and form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove the unencapsulated free vinorelbine tartrate, and the pH value was adjusted to obtain the final product vinorelbine tartrate liposomes, with a particle size of 71.66 ± 2.30 nm, a PDI of 0.221 ± 0.013, a surface potential of -7.15 ± 1.15 mV, and an encapsulation rate of 79.97%.

[0098] Example 24 A method for preparing nintedanib liposomes Prescription composition:

[0099] The prescribed amount of nintedanib was dissolved in 15 mL of sodium citrate buffer solution (80 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into a Herringbone-configured microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing to form liposomes encapsulating nintedanib. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free nintedanib, and the pH value was adjusted to obtain the final product, nintedanib liposomes, with a particle size of 67.80 ± 1.34 nm, a PDI of 0.266 ± 0.021, a surface potential of -6.78 ± 1.85 mV, and an encapsulation rate of 96.92%.

[0100] Example 25 Method for preparing doxorubicin liposomes in a T-type mixer Prescription composition:

[0101] The prescribed amount of doxorubicin hydrochloride was dissolved in 150 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 50 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into a T-type mixer at a constant speed (12 mL / min: 4 mL / min) to complete micromixing ( Figure 2) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free hydrochloric acid, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 64.81±0.19nm, a PDI of 0.168±0.015, a surface potential of -30.00±1.63mV, and an encapsulation rate of 95.17%.

[0102] Example 26 Method for preparing doxorubicin liposomes in a Tesla configuration microfluidic chip Prescription composition:

[0103] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the metal Tesla configuration microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 3 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free hydrochloric acid, and the pH value was adjusted to obtain the final product doxorubicin hydrochloride liposomes, with a particle size of 84.00±1.42nm, a PDI of 0.230±0.019, a surface potential of -29.10±0.61mV, and an encapsulation rate of 92.51%.

[0104] Example 27 Method for preparing doxorubicin liposomes in a flow focusing configuration microfluidic chip Prescription composition:

[0105] The prescribed amount of doxorubicin hydrochloride was dissolved in 15 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 5 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into the flow focusing microfluidic chip at a constant speed (900 μL / min: 300 μL / min) to complete micromixing ( Figure 4 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free hydrochloric acid, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 107.30±0.85nm, a PDI of 0.194±0.011, a surface potential of -36.9±3.11mV, and an encapsulation rate of 91.78%.

[0106] Example 28 Preparation of doxorubicin liposomes using different flow parameters in a T-type mixer (I) Prescription composition:

[0107] The prescribed amount of doxorubicin hydrochloride was dissolved in 150 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 50 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into a T-type mixer at a constant speed (12 mL / min: 4 mL / min) to complete micromixing ( Figure 2 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free hydrochloric acid, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 64.81±0.19nm, a PDI of 0.168±0.015, a surface potential of -30.00±1.63mV, and an encapsulation rate of 95.17%.

[0108] Example 29 Preparation of doxorubicin liposomes using different flow parameters in a T-type mixer (II) Prescription composition:

[0109] The prescribed amount of doxorubicin hydrochloride was dissolved in 150 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 50 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into a T-type mixer at a constant speed (18 mL / min: 6 mL / min) to complete micromixing ( Figure 2 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free hydrochloric acid, and the pH value was adjusted to obtain the final product, doxorubicin hydrochloride liposomes, with a particle size of 44.81±0.19nm, a PDI of 0.068±0.015, a surface potential of -31.00±1.55mV, and an encapsulation rate of 90.27%.

[0200] Example 30 Preparation of doxorubicin liposomes using different flow parameters in a T-type mixer (III) Prescription composition:

[0201] The prescribed amount of doxorubicin hydrochloride was dissolved in 150 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 50 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into a T-type mixer at a constant speed (16 mL / min: 8 mL / min) to complete micromixing ( Figure 2 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free hydrochloric acid, and the pH value was adjusted to obtain the final product doxorubicin hydrochloride liposomes, with a particle size of 74.81±2.91nm, a PDI of 0.268±0.014, a surface potential of -29.55±1.78mV, and an encapsulation rate of 88.27%.

[0202] Example 31 Preparation of doxorubicin liposomes using different flow parameters in a T-type mixer (IV) Prescription composition:

[0203] The prescribed amount of doxorubicin hydrochloride was dissolved in 150 mL of sodium citrate buffer solution (4 mM, pH 3.5) as an aqueous solution; the prescribed amount of egg yolk lecithin, cholesterol, egg yolk phosphatidylglycerol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were dissolved in 50 mL of anhydrous ethanol as a lipid component organic solution. The two were injected into a T-type mixer at a constant speed (20 mL / min: 4 mL / min) to complete micromixing ( Figure 2 ) to form liposomes encapsulating doxorubicin. The obtained liposomes were dialyzed and ultrafiltered to remove unencapsulated free hydrochloric acid, and the pH value was adjusted to obtain the final product doxorubicin hydrochloride liposomes, with a particle size of 40.11±0.91nm, a PDI of 0.044±0.010, a surface potential of -27.88±2.64mV, and an encapsulation rate of 68.27%.

[0204] Example 32 In vitro release of pH-responsive doxorubicin liposomes Doxsome In this example, based on Example 1, 1 mL of the final product doxorubicin liposome Doxsome was placed in a dialysis bag with a specification of 8000-14000 Da, the dialysis bag was placed in a PBS solution with a pH of 7.4, and the sample was taken from the dialysis bag at a set time point in a 37°C constant temperature oscillation box. The doxorubicin content therein was determined by UV-spectrophotometer, the in vitro release behavior of Doxsome was investigated, and the results were compared with those of the dialysis bag. The release curves showed that under constant temperature and oscillation at 37°C, Doxsomes showed different The release behavior of Doxsome was continuous within 96 hours. After about 15% of the drug is released initially, no more is released ( Figure 6 ).

[0205] Example 33 In vitro pH-responsive release of doxorubicin liposomes Doxsome prepared by the optimal formulation and process The in vitro pH-responsive release of Doxsome was investigated in PBS solutions of pH 5.5, pH 6.5 and pH 7.4, respectively. The specific operation was the same as in Example 2. The release curves showed that Doxsome exhibited accelerated release behavior under weakly acidic conditions, and as the pH value further decreased, the drug release was further accelerated. The release behavior in the release medium with different pH values ​​did not change significantly ( Figure 7 ).

[0206] Example 34 Cytotoxicity of pH-responsive doxorubicin liposomes Doxsome 4T1 breast cancer, MCF7 human breast cancer, B16-F10 melanoma, and ID8 ovarian epithelial cancer cells were inoculated into 96-well plates. After overnight attachment, each cell type was divided into 4 groups and treated as follows: ① adding normal saline (Control), ② adding gradient concentrations of doxorubicin hydrochloride solution (Doxorubicin), ③ adding gradient concentrations of pH-responsive doxorubicin liposomes (Doxsome), ④ adding gradient concentrations of (Doxil), and the cell activity was detected by CCK8 method after 48 hours of co-incubation. At lower concentrations, Doxsome showed similar cytotoxicity to Doxorubicin, while the cytotoxicity of Doxil was significantly lower than that of Doxsome and Doxorubicin ( Figure 8 ), which proves that pH-responsive doxorubicin liposomes still maintain the cytotoxicity of doxorubicin in vitro. The in vitro cytotoxicity of liposomes was much lower than that of doxorubicin, and the difference in in vitro cytotoxicity may be due to the difference in release behavior between the two liposomes (P>0.05, ns; P≤0.05, *; P≤0.01, **; P≤0.001, ***; P≤0.0001, ****;).

[0207] Example 35 Intracellular distribution of pH-responsive doxorubicin liposomes Doxsome 4T1 breast cancer cells were seeded in a 6-well plate with a cell slide. After overnight attachment, they were divided into three groups and treated as follows: ① adding doxorubicin for 12 and 24 hours, ② adding pH-responsive doxorubicin liposomes (Doxsome) for 12 and 24 hours, and ③ (Doxil) for 12 and 24 hours. After incubation, Hoechest33342 and LysoTracker were used to stain the cell nucleus and lysosome, respectively, and confocal microscopy was used to observe the distribution of doxorubicin in the cells ( Fig. 9 ), the experimental results showed that co-localization of doxorubicin and the cell nucleus could be observed in doxorubicin and Doxsome, which means that doxorubicin has or is exerting a cytotoxic effect, and No fluorescence signal of doxorubicin was observed in cells treated for 12 h. Only weak doxorubicin fluorescence signals were observed in cells treated for 24 hours, and more of them were co-localized with lysosomes, failing to exert cytotoxic effects. This result proves that pH-responsive doxorubicin liposomes Doxsomes still maintain the cytotoxicity of doxorubicin in vitro. The in vitro cytotoxicity of liposomes is much lower than that of doxorubicin, which is due to the difference in the release behavior of the two liposomes.

[0208] Example 36 Comparative study of pH-responsive doxorubicin liposomes Doxsomes prepared by different formulation processes Five doxorubicin liposomes Doxsome-1-5 with different physical and chemical properties were prepared using different formulations and preparation processes. The main differences between them are the different particle sizes and the differences in stability and drug release rate caused by different formulations ( Fig.10 ). 6 The 4T1 breast cancer cells were inoculated into the mammary fat pad of adult female Balb / C mice to establish a tumor model. The mice were randomly divided into 6 groups (n=3) for comparative study of the effects of these doxorubicin liposomes on each other and on The anti-tumor efficacy and safety of different doxorubicin liposomes were different during the treatment period (doxorubicin hydrochloride dose: 5 mg / kg, twice a week). Fig.11 ), there were statistical differences among the groups, and different doxorubicin liposomes all caused a certain degree of weight loss ( Fig.12 ), but there was no statistical difference among the groups (P>0.05, ns; P≤0.05, *; P≤0.01, **; P≤0.001, ***;).

[0209] Example 37 Distribution of pH-responsive doxorubicin liposomes in vivo 1×10 6The 4T1 breast cancer cells were inoculated into the mammary fat pad of adult female Balb / C mice to establish a tumor model. 20 days after inoculation, 100 μL of Doxsome labeled with DiR fluorescent dye was injected into the tail vein. The distribution of liposomes in various organs of tumor-bearing mice was observed by small animal in vivo imaging at 8h, 24h, and 48h. The experimental results showed that the liposomes injected through the tail vein were mainly distributed in the liver, spleen, tumors, and limbs ( Fig.13 ).

[0210] Example 38 Investigation of the inhibitory effect of pH-responsive doxorubicin liposomes Doxsome on 4T1 breast cancer tumor growth 1×10 6 The 4T1 breast cancer cells were inoculated into the mammary fat pad of adult female Balb / C mice to establish a tumor model. Ten days after inoculation, the mice were randomly divided into 4 groups (n=8) and received the following treatments: ① saline group (Saline), 100 μL saline was injected into the tail vein twice a week; ② doxorubicin hydrochloride solution group (Doxorubicin), 100 μL free doxorubicin hydrochloride solution was injected into the tail vein twice a week (doxorubicin hydrochloride dose: 5 mg / kg); ③ pH-responsive doxorubicin liposome group (Doxsome), 100 μL pH-responsive doxorubicin liposome was injected into the tail vein twice a week (doxorubicin hydrochloride dose: 5 mg / kg); ④ Group (Doxil), tail vein injection 100 μL (doxorubicin hydrochloride dose: 5 mg / kg), twice a week, for a total of 3 weeks. Changes in body weight and tumor volume were recorded during treatment. The tumor volume curve showed that under the designed dosing regimen, pH-responsive doxorubicin liposomes showed the best tumor inhibition effect ( Fig.14 ), and no significant weight loss was caused in mice during the treatment ( Fig.15 )(P>0.05, ns; P≤0.05, *; P≤0.01, **; P≤0.001, ***;).

[0211] Example 39 Investigation of the inhibitory effect of pH-responsive doxorubicin liposomes Doxsome on the growth of B16-F10 melanoma 1×10 6The B16-F10 cells were inoculated subcutaneously in adult female C57b / c mice to establish a tumor model. Nine days after inoculation, the mice were randomly divided into 4 groups (n=8) and received the following treatments: ① saline group (Saline), 100 μL saline was injected into the tail vein three times a week; ② doxorubicin hydrochloride solution group (Doxorubicin), 100 μL free doxorubicin hydrochloride solution was injected into the tail vein three times a week (doxorubicin hydrochloride dose: 3 mg / kg); ③ pH-responsive doxorubicin liposome group (Doxsome), 100 μL pH-responsive doxorubicin liposome was injected into the tail vein (doxorubicin hydrochloride dose: 3 mg / kg) three times a week; ④ Group (Doxil), tail vein injection 100 μL (doxorubicin hydrochloride dose: 3 mg / kg), three times a week. The changes in body weight and tumor volume were recorded during the treatment period ( Fig.16 , 17 ), when the weight loss rate exceeds 15% or the tumor volume is greater than 1500mm 3 The mice were judged dead when the test results showed that the survival time of the Doxsome group was significantly improved compared with the Doxorubicin group, with a significant difference ( Fig.18 ), and doxorubicin-induced cardiotoxicity was observed in the heart H&E sections of mice in the Doxorubicin group, while no obvious cardiac damage was observed in the Doxsome group ( Fig.19 ).

[0212] Example 40 Safety Study of pH Responsive Doxorubicin Liposomes Healthy Sprague Dawley rats were randomly divided into 4 groups: ① saline group (Saline), n = 3; ② doxorubicin hydrochloride solution group (Doxorubicin), n = 6; ③ pH-responsive doxorubicin liposome group (Doxsome), n = 6; ④ Group (Doxil), n=6. The corresponding preparations were injected into the tail vein for 2 weeks, and the total dose of doxorubicin injected into each Sprague Dawley rat in the administration group was 15 mg / kg. During the administration period and one week after the completion of the administration, the weight changes of the Sprague Dawley rats were recorded ( Fig. 20 ) and observed the skin condition of the limbs. According to the change curve, during the observation period, except for the saline group, the weight loss of Sprague Dawley rats in the Doxsome group was the smallest, and no obvious changes in the skin of the limbs were observed, while obvious HFS symptoms such as redness, swelling, scabs, and peeling of the limbs were observed in the Doxil group ( Fig.21 ).

Claims

1. A method for preparing pH-responsive drug-loaded liposomes with high efficiency encapsulation, characterized in that: This is accomplished by following these steps: (1) dissolving the drug in a slightly acidic buffered saline solution, the buffered saline solution being used as the drug aqueous solution, and dissolving the lipid component in an organic solvent being used as the lipid component organic solution; (2) The two-phase solution is pumped into a microfluidic chip or a micromixer via a syringe pump to complete mixing. During mixing, the lipid components are superior to the changes in the solution environment and initiate a "bottom-up" self-assembly process, and in this process, the encapsulation of doxorubicin hydrochloride is completed to form primary liposomes; (3) The primary liposomes were then dialyzed and ultrafiltered to remove unencapsulated free drugs, and the pH value was adjusted to a neutral pH of 7.0 using a neutral buffer. The drugs were finally quantified to a target concentration of 2 mg / mL to obtain the target drug-loaded liposomes.

2. The preparation method according to claim 1, characterized in that: The drugs in step (1) are doxorubicin hydrochloride, vinorelbine tartrate, and nintedanib, and the drug concentration range is 0.1-10 mg / mL.

3. The preparation method according to claim 1, characterized in that: The buffered salt solution of step (1) includes, but is not limited to, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, acetic acid-sodium acetate buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, citric acid-sodium citrate buffer, and other acidic commonly used buffered salt solutions. The buffered salt solution has a pH value in the range of 2.5-6.5 and a concentration of 1 mM-300 mM.

4. The preparation method according to claim 1, characterized in that: The lipid components of step (1) include natural phospholipids or synthetic phospholipids, cholesterol, anionic phospholipids and pegylated phospholipids, the mass ratio of the four components is 0-60:10-25:5-50:5-20, and the total concentration of the four lipid components in the organic solvent is 0.1-150 mg / mL, and the organic solvent includes but is not limited to ethanol, methanol, isopropanol, acetonitrile or ether that is completely mixed with the aqueous solution.

5. The preparation method according to claim 4, characterized in that: The natural phospholipids include, but are not limited to, egg yolk lecithin, soybean lecithin or PL-100M, and the synthetic phospholipids include, but are not limited to, hydrogenated soybean lecithin, dipalmitoyl lecithin, dierucoyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine and other phosphatidylcholine phospholipids, as well as oleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine or Phosphatidylethanolamine phospholipids such as dimyristoylphosphatidylethanolamine; anionic phospholipids include phosphatidylphosphatidic acid, phosphatidylinositol, phosphatidylglycerol, and phosphatidylserine phospholipids. In the solution environment after the drug aqueous solution and the phospholipid organic solution are mixed, the surface potential of the liposome containing this component is negative. The PEGylated phospholipids include but are not limited to distearoylphosphatidylethanolamine-polyethylene glycol 2000 or dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000.

6. The preparation method according to claim 1, characterized in that: The precise control of the two-phase fluid in step (2) is characterized in that the aqueous solution and the organic solution are transported by pumping, the flow rate of the two is 0.001L / min-1L / min, the ratio of the flow rate of the aqueous solution to the organic solution is 1-60:1, and the temperature during mixing is controlled at 0°C-65°C.

7. The preparation method according to claim 1, characterized in that: In the micromixing technology described in step (2), the aqueous solution and the organic solution are mixed in a micromixer. The types of micromixers include microfluidic chips, polymer material micromixers or metal micromixers. The channel size range of the micromixer is 0.05-0.75mm.

8. The preparation method according to claim 7, characterized in that: The mixing structure inside the micromixer includes a T-shaped, Y-shaped or cross-shaped, and a Herringbone-shaped, Tesla-shaped, and spiral-shaped auxiliary mixing structure after preliminary mixing. The two structures are connected by a pipeline, and the two-phase fluid is quickly and completely mixed in this micromixer, which helps to form nanoliposomes with uniform and controllable size.

9. The preparation method according to claim 1, characterized in that: The neutral buffer solution in step (3) includes, but is not limited to, phosphate buffer, citric acid-sodium citrate buffer, Hanks' balanced salt solution, and Earle's balanced salt solution.

10. Use of the drug-loaded liposomes prepared by the method of claim 1 in preparing pH-responsive anti-tumor drugs.

Images