Loaded peptide and doxorubicin hydrochloride liposome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano-material drug delivery, in particular to a loaded peptide and doxorubicin hydrochloride liposome as well as a preparation method and application thereof. Comprising the following steps: (1) dissolving hydrogenated soybean lecithin and cholesterol in ethanol, and filtering with a filter membrane for later use; (2) dissolving peptide CH3 (CH2) 6CO-GIIKKIIKKI-NH2 and doxorubicin hydrochloride into pure water, and filtering with a filter membrane for later use; and (3) taking the phospholipid-cholesterol ethanol solution and the peptide-doxorubicin hydrochloride aqueous solution prepared in the step (2) as organic phases, and preparing the liposome loaded with peptide and doxorubicin hydrochloride by adopting a microfluidic method and controlling the total flow velocity and the flow velocity ratio of the aqueous phase to the organic phase. The prepared liposome is spherical, uniform in particle size, and good in biocompatibility and biodegradability. And no surfactant or dispersant is added in the preparation process, so that the toxicity to the body is further reduced.

Description

Technical Field

[0001] The present application relates to the technical field of nanomaterial drug delivery, and more specifically, to a liposome loaded with peptide and doxorubicin hydrochloride, and a preparation method and application thereof. Background Art

[0002] Doxorubicin hydrochloride (DOX-HCL), also known as doxorubicin hydrochloride, is an anthracycline antibiotic. It has a broad spectrum of antitumor activity and has inhibitory effects on many tumors. Therefore, it is widely used to treat sarcoma, non-Hodgkin's lymphoma, multiple myeloma, breast cancer, small cell lung cancer, gastric cancer, liver cancer, ovarian cancer, cervical cancer and other malignant tumors. It can also be used in combination with other anticancer drugs. However, its molecular structure is unstable and prone to hydrolysis, photolysis and other changes that reduce the efficacy. In addition, it has great side effects after intravenous injection. In addition to adverse reactions such as hair loss, bone marrow suppression and gastrointestinal toxicity, it can also cause severe cardiac toxicity, manifested as various arrhythmias. When the cumulative amount is large, irreversible myocardial damage or even congestive heart failure will occur. These toxic side effects have greatly limited the development and application of doxorubicin hydrochloride.

[0003] Liposomes are a type of cell membrane-like bilayer nanovesicles. In pharmacy, the liposome bilayer structure encapsulating nanodrugs is called a liposome vesicle.

[0004] Antitumor peptides (ACPs) are a class of polypeptides with anticancer activity. They are widely found in a variety of organisms, including mammals, plants, amphibians, insects and microorganisms. Antitumor peptides have many advantages in treating tumors, such as small molecular weight, simple structure, high anticancer activity, high selectivity, fewer side effects, multiple administration methods, and low risk of causing multidrug resistance. However, their disadvantages are also obvious. As proteins, they are prone to aggregation or enzyme degradation in normal physiological environments if used alone.

[0005] Microfluidics is a versatile technology used to process or manipulate microfluidics in channel structures with dimensions ranging from tens to hundreds of micrometers. Fluids controlled by microfluidics have unique properties, such as laminar flow and droplets. Utilizing these unique fluid phenomena, it is possible to finely control the mixing conditions of each solvent, precisely control the installation of nanostructures and command batch quality to achieve uniformity in the size distribution of synthesized particles. By adjusting the flow parameters of the fluid, such as the total flow rate, the flow rate ratio of different solutions, the lipid solution concentration, and the characteristic length in the microfluidic channel, nanoscale liposomes with smaller and narrower size distribution can be prepared. Summary of the invention

[0006] The present disclosure provides a peptide- and doxorubicin hydrochloride-loaded liposome and a preparation method and application thereof. The prepared liposome is spherical, has uniform particle size, and has good biocompatibility and biodegradability. In addition, the preparation process does not require the addition of surfactants and dispersants, further reducing toxicity to the body.

[0007] In a first aspect, the present disclosure provides a method for preparing liposomes loaded with peptide and doxorubicin hydrochloride, comprising the following steps:

[0008] (1) dissolving hydrogenated soybean lecithin and cholesterol in ethanol and filtering through a membrane for later use;

[0009] (2) dissolving the peptide CH3(CH2)6CO-GIIKKIIKKI-NH2 and doxorubicin hydrochloride in pure water, and filtering the membrane for later use;

[0010] (3) The phospholipid-cholesterol ethanol solution and the peptide-doxorubicin hydrochloride aqueous solution prepared in step (2) are used as the organic phase and pure water is used as the aqueous phase. The total flow rate and the flow rate ratio of the aqueous phase to the organic phase are controlled by a microfluidic method to prepare liposomes loaded with the peptide and doxorubicin hydrochloride.

[0011] Preferably, in step (1), the mass ratio of phospholipid to cholesterol is 1:1-4:1.

[0012] Preferably, in step (1), the mass ratio of phospholipid to cholesterol is 1:1-3:1.

[0013] Preferably, in step (1), the mass ratio of phospholipid to cholesterol is 2:1.

[0014] Preferably, in step (2), the peptide CH3(CH2)6CO-GIIKKIIKKI-NH2 is dissolved in pure water to prepare a peptide solution, the concentration of the peptide solution is 0.1-0.5 mg / mL, and doxorubicin hydrochloride is dissolved in pure water to prepare a doxorubicin hydrochloride solution, the concentration of the doxorubicin hydrochloride solution is 0.1-0.5 mg / mL.

[0015] Preferably, in step (2), the concentration of the peptide solution is 0.2-0.5 mg / mL, and the concentration of the doxorubicin hydrochloride solution is 0.2-0.5 mg / mL.

[0016] Preferably, in step (2), the concentration of the peptide solution is 0.2 mg / mL, and the concentration of the doxorubicin hydrochloride solution is 0.2 mg / mL.

[0017] Preferably, in step (3), the total flow rate of the microfluidic method is 4-64 mL / min.

[0018] Preferably, in step (3), the total flow rate of the microfluidic method is 20-64 mL / min.

[0019] Preferably, in step (3), the total flow rate of the microfluidic method is 20 mL / min.

[0020] Preferably, in step (3), in the microfluidic method, the flow rate ratio of the aqueous phase to the organic phase is 1:1-9:1.

[0021] Preferably, in step (3), in the microfluidic method, the flow rate ratio of the aqueous phase to the organic phase is 3:1-9:1.

[0022] Preferably, in step (3), in the microfluidic method, the flow rate ratio of the aqueous phase to the organic phase is 3:1.

[0023] Preferably, the particle size of the prepared liposome loaded with peptide and doxorubicin hydrochloride is 80-100 nm.

[0024] Preferably, the particle size of the prepared liposome loaded with peptide and doxorubicin hydrochloride is 100 nm.

[0025] In a second aspect, the present disclosure provides loaded peptide and doxorubicin hydrochloride liposomes prepared by the preparation method according to any one of claims 1 to 8.

[0026] In a third aspect, the present disclosure provides an application of a liposome loaded with a peptide and doxorubicin hydrochloride, and the use of the liposome loaded with a peptide and doxorubicin hydrochloride in the preparation of a drug for treating cancer.

[0027] In summary, this application has the following beneficial effects:

[0028] 1. The preparation method in this application is simple in process, easy to operate, flexible and controllable. With the help of microfluidic equipment, the preparation time is greatly shortened by adjusting different experimental parameters. The prepared liposomes have controllable particle size, uniform particle size, good biocompatibility and biodegradability, and good stability, which is conducive to its stable release in vivo. In addition, no surfactant and dispersant need to be added in the preparation process of the liposomes, which further reduces the toxicity.

[0029] 2. The liposomes prepared in the present application are spherical, with controllable particle size and uniform particle size of about 100 nm. The liposomes have good biocompatibility and biodegradability, good stability, which is conducive to their stable release in vivo, and no surfactant and dispersant need to be added during the preparation of the liposomes, which further reduces the toxicity.

[0030] 3. Compared with the commonly used thin film hydration method, high-pressure homogenization method, ethanol injection method, etc., the preparation method of the present application has a simpler process, more convenient operation, smaller batch differences, better repeatability, and the advantage of continuous production.

[0031] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of protection of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a process flow chart of the peptide- and doxorubicin hydrochloride-loaded liposomes prepared by the present invention;

[0033] Figure 2 It is the optimization diagram of the total flow rate of the microfluidic preparation parameters of liposomes loaded with peptides and doxorubicin hydrochloride;

[0034] Figure 3 is a diagram of the optimization of flow rate ratio of microfluidic preparation parameters of liposomes loaded with peptide and doxorubicin hydrochloride;

[0035] Figure 4 This is a graph showing the effect of the mass ratio of phospholipids and cholesterol on liposomes;

[0036] Figure 5 This is a graph showing the effect of doxorubicin hydrochloride concentration on drug-loaded liposomes;

[0037] Figure 6 is the effect of peptide concentration on drug-loaded liposomes;

[0038] Figure 7 is the stability graph of peptide- and doxorubicin hydrochloride-loaded liposomes at 4°C;

[0039] Figure 8 is the in vitro drug release profile of peptide- and doxorubicin hydrochloride-loaded liposomes;

[0040] Fig. 9 is the TEM image of liposomes loaded with peptide and doxorubicin hydrochloride;

[0041] Fig.10 is the biocompatibility graph of blank liposomes;

[0042] Fig.11 It is the cytotoxicity graph of drug-loaded liposomes. DETAILED DESCRIPTION

[0043] The present application is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0044] Example

[0045] Example 1

[0046] A preparation method of blank liposomes comprises the following steps:

[0047] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0048] (2) Pure water was passed through a water filter membrane (0.22 μm) and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the flow rate ratio of the aqueous phase to the organic phase was controlled to be 3:1.

[0049] (3) The total flow rate was set to 10 mL / min, and the liposomes were collected at the outlet and stored at 4 °C.

[0050] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 95 nm and the PDI was 0.16.

[0051] Example 2

[0052] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0053] (2) Pure water was passed through a water filter membrane (0.22 μm) and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the flow rate ratio of the aqueous phase to the organic phase was controlled to be 3:1.

[0054] (3) The total flow rate was set to 20 mL / min, and the liposomes were collected at the outlet and stored at 4 °C.

[0055] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 68 nm and the PDI was 0.07.

[0056] Depend on Figure 2 The particle size and PDI of liposomes prepared at different total flow rates are not much different when the flow rate is higher than 20 mL / min. Considering that a small flow rate is easier to control and saves more materials, the total flow rate was finally selected as 20 mL / min.

[0057] Example 3

[0058] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0059] (2) Pure water was passed through a water filter membrane (0.22 μm) and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the flow rate ratio of the aqueous phase to the organic phase was controlled to be 3:1.

[0060] (3) The total flow rate was set to 30 mL / min, and the liposomes were collected at the outlet and stored at 4 °C.

[0061] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 73 nm and the PDI was 0.08.

[0062] Example 4

[0063] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0064] (2) Pure water was passed through a water filter membrane (0.22 μm) and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the flow rate ratio of the aqueous phase to the organic phase was controlled to be 3:1.

[0065] (3) The total flow rate was set to 40 mL / min, and the liposomes were collected at the outlet and stored at 4 °C.

[0066] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 75 nm and the PDI was 0.074.

[0067] Example 5

[0068] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0069] (2) Pure water was passed through a water filter membrane (0.22 μm) and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the flow rate ratio of the aqueous phase to the organic phase was controlled to be 3:1.

[0070] (3) The total flow rate was set to 50 mL / min, and the liposomes were collected at the outlet and stored at 4 °C.

[0071] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 75 nm and the PDI was 0.08.

[0072] Example 6

[0073] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0074] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min.

[0075] (3) The flow rate ratio of the aqueous phase to the organic phase was controlled to be 1:1, and the liposomes were collected at the outlet and stored at 4°C.

[0076] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 1343 nm and the PDI was 0.70.

[0077] Depend on Figure 3 It can be seen that the particle size and PDI of liposomes prepared at different flow rate ratios are similar. The particle size and PDI of liposomes prepared at flow rates above 20 mL / min are not much different. Considering that a small flow rate is easier to control and saves more materials, the total flow rate was finally selected as 20 mL / min.

[0078] Example 7

[0079] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0080] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min.

[0081] (3) The flow rate ratio of the aqueous phase to the organic phase was controlled to be 2:1, and the liposomes were collected at the outlet and stored at 4°C.

[0082] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 209 nm and the PDI was 0.24.

[0083] Example 8

[0084] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0085] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min.

[0086] (3) The flow rate ratio of the aqueous phase to the organic phase was controlled to be 3:1, and the liposomes were collected at the outlet and stored at 4°C.

[0087] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 68 nm and the PDI was 0.07.

[0088] Depend on Figure 3 It can be seen from the particle size and PDI of liposomes prepared at different flow rate ratios that the liposomes prepared with a flow rate ratio of 3:1 between the aqueous phase and the organic phase have better effects.

[0089] Example 9

[0090] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0091] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min.

[0092] (3) The flow rate ratio of the aqueous phase to the organic phase was controlled to be 4:1, and the liposomes were collected at the outlet and stored at 4°C.

[0093] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 73 nm and the PDI was 0.09.

[0094] Example 10

[0095] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0096] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min.

[0097] (3) The flow rate ratio of the aqueous phase to the organic phase was controlled to be 5:1, and the liposomes were collected at the outlet and stored at 4°C.

[0098] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 79 nm and the PDI was 0.23.

[0099] Embodiment 11

[0100] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0101] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min.

[0102] (3) The flow rate ratio of the aqueous phase to the organic phase was controlled to be 9:1, and the liposomes were collected at the outlet and stored at 4°C.

[0103] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 977 nm and the PDI was 0.79.

[0104] Example 12

[0105] (1) Weigh 20 mg of phospholipids and 20 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0106] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase is controlled to be 3:1.

[0107] (3) Collect the liposomes at the outlet and store at 4°C.

[0108] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 56 nm and the PDI was 0.12.

[0109] Example 13

[0110] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0111] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase is controlled to be 3:1.

[0112] (3) Collect the liposomes at the outlet and store at 4°C.

[0113] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 58 nm and the PDI was 0.07.

[0114] Depend on Figure 4 As shown in the figure, the particle size and PDI of liposomes prepared by phospholipids and cholesterol in different mass ratios show that the particle size and PDI are smaller when the mass ratio of phospholipids and cholesterol is 2:1. Finally, the mass ratio of phospholipids and cholesterol is selected to be 2:1.

[0115] Embodiment 14

[0116] (1) Weigh 30 mg of phospholipids and 10 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0117] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase is controlled to be 3:1.

[0118] (3) Collect the liposomes at the outlet and store at 4°C.

[0119] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 94 nm and the PDI was 0.05.

[0120] Embodiment 15

[0121] (1) Weigh 32 mg of phospholipids and 8 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials, use a 0.22 μm organic filter to remove impurities in the solution, and load the solution into a 20 mL syringe 1.

[0122] (2) Pure water is passed through a water filter membrane and loaded into syringe 2. Syringe 1 and syringe 2 are installed in a microfluidic device, and the total flow rate is controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase is controlled to be 3:1.

[0123] (3) Collect the liposomes at the outlet and store at 4°C.

[0124] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 193 nm and the PDI was 0.28.

[0125] Example 16

[0126] A preparation method of doxorubicin hydrochloride-loaded liposomes comprises the following steps:

[0127] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0128] (2) 2 mg of doxorubicin hydrochloride was dissolved in 20 mL of pure water, ultrasonicated for 0.5 h, filtered through an aqueous filter membrane, and the filtrate was loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0129] (3) Start the equipment, collect the liposomes at the outlet, and store them at 4°C.

[0130] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 61 nm and the PDI was 0.13.

[0131] Embodiment 17

[0132] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0133] (2) 4 mg of doxorubicin hydrochloride was dissolved in 20 mL of pure water, ultrasonicated for 0.5 h, filtered through an aqueous filter membrane, and the filtrate was loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0134] (3) Start the equipment, collect the liposomes at the outlet, and store them at 4°C.

[0135] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 62 nm and the PDI was 0.10.

[0136] Depend on Figure 5 As shown, the particle size and PDI of liposomes prepared by different concentrations of doxorubicin hydrochloride show that when the drug concentration is 0.2 mg / mL, the prepared liposomes have better effect. Finally, the doxorubicin hydrochloride concentration is selected as 0.2 mg / mL.

[0137] Embodiment 18

[0138] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0139] (2) 6 mg of doxorubicin hydrochloride was dissolved in 20 mL of pure water, ultrasonicated for 0.5 h, filtered through an aqueous filter membrane, and the filtrate was loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0140] (3) Start the equipment, collect the liposomes at the outlet, and store them at 4°C.

[0141] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 74 nm and the PDI was 0.14.

[0142] Embodiment 19

[0143] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0144] (2) 8 mg of doxorubicin hydrochloride was dissolved in 20 mL of pure water, ultrasonicated for 0.5 h, filtered through an aqueous filter membrane, and the filtrate was loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0145] (3) Start the equipment, collect the liposomes at the outlet, and store them at 4°C.

[0146] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 84 nm and the PDI was 0.17.

[0147] Embodiment 20

[0148] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0149] (2) 10 mg of doxorubicin hydrochloride was dissolved in 20 mL of pure water, ultrasonicated for 0.5 h, filtered through an aqueous filter membrane, and the filtrate was loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0150] (3) Start the equipment, collect the liposomes at the outlet, and store them at 4°C.

[0151] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 94 nm and the PDI was 0.20.

[0152] Embodiment 21

[0153] A preparation method of a peptide- and doxorubicin hydrochloride-loaded liposome comprises the following steps:

[0154] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0155] (2) 2 mg of peptide and 4 mg of doxorubicin hydrochloride were dissolved in 20 mL of pure water, filtered through a water filter membrane, and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0156] (3) Start the equipment, collect liposomes at the outlet, and store at 4°C.

[0157] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 73 nm and the PDI was 0.10.

[0158] Embodiment 22

[0159] A preparation method of a peptide- and doxorubicin hydrochloride-loaded liposome comprises the following steps:

[0160] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0161] (2) 4 mg of peptide and 4 mg of doxorubicin hydrochloride were dissolved in 20 mL of pure water, filtered through a water filter membrane, and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0162] (3) Start the equipment, collect liposomes at the outlet, and store at 4°C.

[0163] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 74 nm and the PDI was 0.07.

[0164] Depend on Figure 6 As shown in the figure, the particle size and PDI of liposomes prepared by peptides of different concentrations show that when the concentration is 0.2 mg / mL, the prepared liposomes have better effect. Finally, the peptide concentration is selected to be 0.2 mg / mL.

[0165] Embodiment 23

[0166] A preparation method of a peptide- and doxorubicin hydrochloride-loaded liposome comprises the following steps:

[0167] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0168] (2) 6 mg of peptide and 4 mg of doxorubicin hydrochloride were dissolved in 20 mL of pure water, filtered through a water filter membrane, and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0169] (3) Start the equipment, collect liposomes at the outlet, and store at 4°C.

[0170] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 80 nm and the PDI was 0.12.

[0171] Embodiment 24

[0172] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0173] (2) 8 mg of peptide and 4 mg of doxorubicin hydrochloride were dissolved in 20 mL of pure water, filtered through a water filter membrane, and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0174] (3) Start the equipment, collect liposomes at the outlet, and store at 4°C.

[0175] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 81 nm and the PDI was 0.09.

[0176] Embodiment 25

[0177] (1) Weigh 26.6 mg of phospholipids and 13.3 mg of cholesterol, dissolve them in 10 mL of ethanol, sonicate for 0.5 h to completely dissolve the materials and drugs, use a 0.22 μm organic filter to remove impurities in the solution, and load it into a 20 mL syringe 1.

[0178] (2) 10 mg of peptide and 4 mg of doxorubicin hydrochloride were dissolved in 20 mL of pure water, filtered through a water filter membrane, and loaded into syringe 2. Syringe 1 and syringe 2 were installed in a microfluidic device, and the total flow rate was controlled to be 20 mL / min, and the flow rate ratio of the aqueous phase to the organic phase was 3:1.

[0179] (3) Start the equipment, collect liposomes at the outlet, and store at 4°C

[0180] The prepared liposomes were measured using a nanoparticle size analyzer, and the liposome particle size was found to be 89 nm and the PDI was 0.12.

[0181] Finally, the final preparation conditions of peptide-loaded and doxorubicin hydrochloride liposomes were determined to be a total flow rate of 20 mL / min, a flow rate ratio of aqueous phase to organic phase of 3:1, a mass ratio of phospholipid to cholesterol of 2:1, a doxorubicin hydrochloride concentration of 0.2 mg / mL, and a peptide concentration of 0.2 mg / mL.

[0182] Stability test of peptide-loaded and doxorubicin hydrochloride liposomes:

[0183] A portion of the liposomes stored at 4°C was taken out every other week, and the particle size and PDI thereof were measured.

[0184] Depend on Figure 6 It can be seen that the peptide- and doxorubicin hydrochloride-loaded liposomes have good stability for at least 6 weeks.

[0185] In vitro drug release assay of peptide- and doxorubicin hydrochloride-loaded liposomes:

[0186] The in vitro release test of doxorubicin hydrochloride was conducted by dialysis. Weigh 0.1g of drug-loaded liposomes accurately, add 10mL PBS (pH=5.6), and dissolve in water bath ultrasound. Mix well, put it in a dialysis bag, tie the bag tightly, and put it into a dissolution cup containing 100mL PBS for dialysis. The oscillator is kept rotating at 37℃ and 100r / min. Samples are taken at 0.5h, 1h, 2h, 6h, 12h, 24h, 36h, and 48h. After each sampling, the same volume of PBS is added to the original buffer. The released solution is filtered through a 0.22μm microporous membrane, and the drug concentration is detected by UV spectrophotometer to calculate the drug release.

[0187] In vitro drug release results Figure 7 As shown, the drug release reached more than 90% after 48 h.

[0188] Transmission electron microscopy (TEM) detection of peptides and doxorubicin hydrochloride liposomes:

[0189] Before TEM detection, the sample needs to be dialyzed in ultrapure water for 24 hours through a dialysis bag (molecular weight cutoff 3.5 kDa) until there is no organic solvent such as ethanol in the dialysis bag. The treated liposome sample is negatively stained with phosphotungstic acid staining solution, placed on a clean copper grid, dried, and finally observed and recorded using a transmission electron microscope.

[0190] like Figure 8 As shown in the figure, it can be seen that the drug-loaded liposomes have uniform particle size and are evenly dispersed, with a particle size of about 100 nm.

[0191] The biocompatibility test of blank liposomes includes the following:

[0192] (1) HFF-1 cells were seeded in a 96-well plate at a density of 5000 per well, 100 μL was seeded per well, and cultured for 18-24 h. The supernatant was discarded, and the liposomes were diluted to the required concentration of 100 μg / mL with DMEM, 200 μL was added to each well, and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0193] (2) After the incubation, 10 μL MTT (5 mg / mL) was added to each well and incubated in a 37°C, 5% CO2 cell culture incubator for 4 h. Finally, the supernatant was discarded, 150 μL DMSO was added to each well, and the cells were incubated in a constant temperature shaker at 160 rpm and 37°C for 15 min. Finally, the absorbance at 570 nm was measured using an ELISA reader.

[0194] from Fig. 9 It can be seen that the cell survival rate after adding blank liposomes is higher than 92%, indicating that the liposomes have good biosafety.

[0195] The cytotoxicity (MTT) assay of peptide- and doxorubicin hydrochloride-loaded liposomes includes the following:

[0196] (1) The cancer cells Mcf-7 were inoculated in a 96-well plate at a density of 5000 per well, 100 μL per well, and cultured for 18-24 hours. The supernatant was discarded, and the peptide-loaded and doxorubicin hydrochloride liposomes were diluted with DMEM to a concentration of 0-10 μg / mL of doxorubicin hydrochloride, 200 μL per well were added, and the cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0197] (2) After the incubation, 10 μL MTT (5 mg / mL) was added to each well and incubated in a 37°C, 5% CO2 cell culture incubator for 4 h. Finally, the supernatant was discarded, 150 μL DMSO was added to each well, and the cells were incubated in a constant temperature shaker at 160 rpm and 37°C for 15 min. The absorbance at 570 nm was measured by an enzyme reader.

[0198] Depend on Fig.11 It can be seen that when the drug concentration of peptide and doxorubicin hydrochloride liposomes is 10 μg / mL, the killing rate of cancer cells is as high as 72%, and it has an anti-cancer effect, which is better than that of naked doxorubicin hydrochloride at the same concentration.

[0199] The above is only an exemplary embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present disclosure should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A method for preparing liposomes loaded with peptide and doxorubicin hydrochloride, characterized in that: The following steps are involved: (1) dissolving phospholipids and cholesterol in ethanol, filtering through a membrane for later use, and preparing solution 1; (2) dissolving the peptide CH3(CH2)6CO-GIIKKIIKKI-NH2 and / or doxorubicin hydrochloride in pure water, filtering the membrane for later use, and preparing solution 2; (3) The solution 1 prepared in step (1) and the solution 2 prepared in step (2) are mixed to prepare a phospholipid-cholesterol ethanol solution and a peptide-doxorubicin hydrochloride aqueous solution, wherein the phospholipid-cholesterol ethanol solution and the peptide-doxorubicin hydrochloride aqueous solution are used as organic phases and pure water is used as aqueous phase. A microfluidic method is used to control the total flow rate and the flow rate ratio of the aqueous phase to the organic phase to prepare liposomes loaded with the peptide and doxorubicin hydrochloride.

2. The method for preparing the liposome loaded with peptide and doxorubicin hydrochloride according to claim 1, characterized in that: In step (1), the mass ratio of phospholipid to cholesterol is 1:1-4:

1.

3. The method for preparing the liposome loaded with peptide and doxorubicin hydrochloride according to claim 1, characterized in that: In step (2), the peptide CH3(CH2)6CO-GIIKKIIKKI-NH2 is dissolved in pure water to prepare a peptide solution, the concentration of the peptide solution is 0.1-0.5 mg / mL, and doxorubicin hydrochloride is dissolved in pure water to prepare a doxorubicin hydrochloride solution, the concentration of the doxorubicin hydrochloride solution is 0.1-0.5 mg / mL.

4. The method for preparing liposomes loaded with peptide and doxorubicin hydrochloride according to claim 1, characterized in that: In step (3), the total flow rate of the microfluidic method is 4-64 mL / min.

5. The method for preparing liposomes loaded with peptide and doxorubicin hydrochloride according to claim 1, characterized in that: In step (3), the total flow rate of the microfluidic method is 20-64 mL / min.

6. The method for preparing liposomes loaded with peptide and doxorubicin hydrochloride according to claim 1, characterized in that: In step (3), in the microfluidic method, the flow rate ratio of the aqueous phase to the organic phase is 1:1-9:

1.

7. The method for preparing liposomes loaded with peptide and doxorubicin hydrochloride according to claim 1, characterized in that: In step (3), in the microfluidic method, the flow rate ratio of the aqueous phase to the organic phase is 3:1-9:

1.

8. The method for preparing liposomes loaded with peptide and doxorubicin hydrochloride according to claim 1, characterized in that: The particle size of the prepared liposome loaded with peptide and doxorubicin hydrochloride is 80-100 nm.

9. The peptide- and doxorubicin hydrochloride-loaded liposomes prepared by the method according to any one of claims 1 to 8.

10. Use of a loaded peptide and doxorubicin hydrochloride liposomes, the use of the loaded peptide and doxorubicin hydrochloride liposomes according to claim 9 in the preparation of a drug for treating cancer.

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