A delivery system for nucleoside analogs and methods of making and using the same

Abstract

The application provides a nucleoside analogue-loaded delivery system and a preparation method and application thereof, and relates to the technical field of medicines.In the application, a guisitebine delivery system with high encapsulation rate and high drug loading is obtained by loading guisitebine on a wrapping material obtained by hybridizing a platelet membrane and MSC extracellular vesicles through membrane engineering, and the system has excellent stability; the platelet membrane drug delivery system loaded with guisitebine can effectively improve the treatment effect of guisitebine on pancreatic cancer, and provides a new functional drug form for the treatment of pancreatic cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a delivery system loaded with nucleoside analogs and a preparation method and application thereof. BACKGROUND

[0002] Platelets are small pieces of cytoplasm that fall off from the cytoplasm of megakaryocytes matured from mammalian bone marrow. Recent studies have shown that platelet membranes can be used as a biomimetic coating to endow nanoparticles with the ability to avoid macrophage recognition, capture circulating tumor cells and locate inflammatory sites, and is an ideal drug carrier. However, there are problems in the direct wrapping of hydrophilic nucleoside analogs with platelet membranes, and although physical methods such as electroporation or ultrasound can improve drug loading efficiency, this process can easily damage the structure of the platelet membrane, affecting efficiency and treatment effect. In addition, high concentrations of nucleoside analogs can also interfere with platelet metabolism, affecting their normal activation, aggregation and release functions. Therefore, there are few related studies on the use of platelet membranes to load nucleoside analogs in the prior art. SUMMARY

[0003] The purpose of the present application is to provide a delivery system loaded with nucleoside analogs and a preparation method and application thereof, which uses MSC exosomes and platelet membranes to load gemcitabine, thereby breaking through to improve the loading efficiency and wrapping of gemcitabine, and the drug delivery system prepared by the method also has excellent stability and pancreatic cancer killing power.

[0004] In one aspect, the present application provides a preparation method of a delivery system loaded with nucleoside analogs, which comprises the following steps:

[0005] Step one, mixing and extruding platelet membranes and exosomes to obtain a hybrid material;

[0006] Step two, mixing nucleoside analogs with the hybrid material, and repeatedly freezing and thawing to fuse the nucleoside analogs with the hybrid material to obtain a delivery system loaded with nucleoside analogs.

[0007] Further, the exosomes are selected from one or more of MSC exosomes, NK cell exosomes and 293T cell exosomes.

[0008] Preferably, the exosomes are MSC exosomes.

[0009] Further, the nucleoside analogs are selected from one or more of gemcitabine, capecitabine, cytarabine and fludarabine.

[0010] Preferably, the nucleoside analogs are gemcitabine.

[0011] Further, the volume ratio of the platelet membranes to the exosomes is 1: (1-3).

[0012] Preferably, the volume ratio of the platelet membrane and the cell exosome is 1:2.

[0013] Further, the extrusion condition is 50-100 nm filter membrane extrusion for 5-15 times.

[0014] Preferably, the extrusion condition is 100 nm filter membrane extrusion for 10 times.

[0015] Further, the concentration of the nucleoside analog is 0.1-0.5 mg / mL.

[0016] Preferably, the concentration of the nucleoside analog is 0.1 mg / mL.

[0017] Further, the volume ratio of the nucleoside analog and the hybrid material is 1:(0.5-2).

[0018] Preferably, the volume ratio of the nucleoside analog and the hybrid material is 1:1.

[0019] Further, the number of repeated freeze-thawing is 3-10 times.

[0020] Preferably, the number of repeated freeze-thawing is 3 times.

[0021] Further, the method further comprises a centrifugation step.

[0022] Preferably, the centrifugation condition is 0-10 °C, 10000-20000 g centrifugation for 30-90 min.

[0023] More preferably, the centrifugation condition is 4 °C, 12000 g centrifugation for 60 min.

[0024] The purpose of the centrifugation step is to remove excess unbound drugs and hybrid materials, therefore, other separation methods can be used instead of centrifugation as long as the purpose of removing excess materials can be achieved.

[0025] Further, the method further comprises a buffer solution washing step.

[0026] Preferably, the buffer solution can be PBS buffer solution.

[0027] In a preferred embodiment, the method comprises the following steps:

[0028] Step one, mixing platelet membrane and MSC cell exosome at a volume ratio of 1:(1-3), 50-100 nm filter membrane extrusion for 5-15 times to obtain hybrid material;

[0029] Step two, dissolve gemcitabine in water, 0.1-0.5 mg / mL of gemcitabine is mixed with the hybrid material at a volume ratio of 1: (0.5-2), repeated freeze-thaw 3-10 times, after the end of centrifugation, the supernatant is discarded, and the drug-loaded composite drug delivery system is obtained.

[0030] Preferably,

[0031] Step one, mix platelet membranes and MSC cell exosomes at a volume ratio of 1:2, extrude 10 times with a 100 nm filter membrane, and obtain a hybrid material;

[0032] Step two, dissolve gemcitabine in water, 0.1 mg / mL of gemcitabine is mixed with the hybrid material at a volume ratio of 1:1, repeated freeze-thaw, repeat the above freeze-thaw operation 3 times, after the end of centrifugation, the supernatant is discarded, and the drug-loaded composite drug delivery system is obtained.

[0033] In another aspect, the application also provides a delivery system loaded with nucleoside analogs prepared by the method.

[0034] The delivery system loaded with nucleoside analogs prepared by the method has high encapsulation efficiency and drug loading, and has stability and therapeutic efficacy.

[0035] The encapsulation efficiency is greater than or equal to 70%, and the highest can reach 75.20%.

[0036] There is no obvious change within at least 96 h, which can maintain long-term stability.

[0037] In another aspect, the application also provides a composition comprising the delivery system loaded with nucleoside analogs.

[0038] The composition described in the application can also add adjuvants, which can be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavorings, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integration agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.

[0039] The composition described in the application can be prepared by a general method, wherein one or more diluents or carriers can be added.

[0040] In another aspect, the application also provides the use of the delivery system loaded with nucleoside analogs or the composition described in the preparation of a medicament for treating pancreatic cancer.

[0041] The exosomes and platelet membranes are used to wrap gemcitabine in the application, which has better treatment effect than the gemcitabine wrapped by liposomes in the prior art.

[0042] The application has the following beneficial effects:

[0043] 1. The platelet membrane and MSC cell exosomes are hybridized to obtain a wrapping material loaded with gemcitabine through membrane engineering, a gemcitabine delivery system with high encapsulation efficiency and high drug loading is obtained, and the system has excellent stability.

[0044] 2. The gemcitabine-loaded drug delivery system can effectively improve the treatment effect of gemcitabine on pancreatic cancer, and provides a new functional drug form for pancreatic cancer treatment. DETAILED DESCRIPTION

[0045] In order to more clearly illustrate the overall concept of the application, the following will be described in detail in the form of examples. In the following description, a large number of specific details are given in order to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without one or more of these details. In other examples, some technical features known in the art are not described in order to avoid obscuring the application.

[0046] Before further describing the specific embodiments of the application, it should be understood that the scope of protection of the application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the application are for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the application. The test methods in the following examples are not specified, and are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers.

[0047] It should be noted that the terms used herein are only for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection according to the exemplary embodiments of the application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprise" and / or "include" are used in the specification, there is a feature, step, operation, device, component and / or combination thereof.

[0048] When the embodiments give numerical ranges, it is to be understood that unless the application specifically states to the contrary, each numerical range is a continuum, and that every number within the range and each point within the continuum are included. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Except in the Examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, particular or preferred methods and materials are described herein.

[0049] Unless otherwise specified, in the following examples, the reagents or instruments used are not noted by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0050] Among them, gemcitabine (CAS No.: 122111-03-9), capecitabine (CAS No.: 154361-50-9), cytarabine (CAS No.: 147-94-4), fludarabine (CAS No.: 75607-67-9) are purchased from Merck; MSC Cell Exosomes (Human) MSC Exosomes (Human) (Cat No.: 41220ES60), NK Cell Exosomes (Human) NK Exosomes (Human) (Cat No.: 41221ES60), 293T Cell Exosome 293T Exosomes (Cat No.: 41219ES60), Liposome (Cat No.: 40338ES08) are purchased from Yixing Bio.

[0051] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present application all use conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields.

[0052] In addition, the "water" described in the present application includes deionized water, distilled water, ion exchange water, double distilled water, high-purity water, pure water, and any feasible water that can be used in the field.

[0053] In the following examples, if there is no other special indication, it means wt%, that is, weight percentage.

[0054] The platelet membrane can be obtained by a general method by those skilled in the art. In a preferred embodiment, the method for preparing the platelet membrane comprises the following steps: collecting blood from the abdominal aorta of an adult SD rat, centrifuging the collected whole blood at 200 g for 20 min, centrifuging the supernatant at 1800 g for 20 min at room temperature, washing the precipitate repeatedly for 3 times, and centrifuging the precipitate at 1800 g for 5 min to obtain the platelets. The platelets are placed in a constant temperature water bath at 43°C for 30 min, and then repeatedly frozen and thawed for 3 times. The platelets are centrifuged at 8000 g for 15 min at room temperature to remove the supernatant, resuspended after washing, and treated by a water bath ultrasonic instrument at 20-25 KHz for 3 min to obtain the platelet membrane.

[0055] Example 1: Preparation method of a drug delivery system in which a drug is wrapped by a platelet membrane

[0056] The platelet membrane can be used as a biomimetic coating to endow the nanoparticles with the ability to avoid macrophage recognition, capture tumor cells in circulation, and locate inflammatory sites. However, the platelet membrane directly wrapped around the hydrophilic nucleoside analogs has the problem of poor wrapping. Although the physical methods such as electroporation or ultrasound can improve the drug loading efficiency, the process is easy to damage the structure of the platelet membrane, affecting the efficiency and treatment effect.

[0057] Therefore, in this embodiment, a plurality of components are used to form a composite drug delivery system together with the platelet membrane to explore a new method for loading nucleoside analogs.

[0058] The specific steps are as follows:

[0059] Step 1: The platelet membrane and the extracellular vesicles (MSC extracellular vesicles, NK extracellular vesicles, 293T extracellular vesicles) or liposomes are mixed at a volume ratio of 1:1, and then extruded 10 times through a 100 nm filter membrane using a micro-extruder to obtain a hybrid material.

[0060] Step 2: The nucleoside analogs (gemcitabine, capecitabine, cytarabine, fludarabine) are dissolved in water, and the nucleoside analogs at 0.1 mg / mL are mixed with the hybrid material at a volume ratio of 1:1. The mixture is repeatedly frozen and thawed, quickly frozen in a-80°C refrigerator for 30 min, and then quickly transferred to a 37°C water bath until it is thawed. The above freezing and thawing operation is repeated for 5 times. After the end, PBS buffer solution is added, and centrifuged at 12000 g for 60 min at 4°C. The supernatant is discarded to obtain the drug-loaded composite drug delivery system.

[0061] The drug encapsulation efficiency is detected by high performance liquid chromatography (HPLC), and the specific parameters and results are shown in Table 1.

[0062] Encapsulation Efficiency (EE) (%) = (initial concentration of drug solution - supernatant concentration) / initial concentration of drug solution x 100%.

[0063] Table 1

[0064]

[0065] As shown in the results of Table 1, the best wrapping effect can be achieved when using MSC exosomes and platelet membranes to load gemcitabine, and the encapsulation efficiency can reach more than 60%.

[0066] Example 2 Optimization of the preparation method of the drug delivery system of the drug wrapped by platelet membranes

[0067] This example further optimizes the preparation method of the drug-loaded composite drug delivery system based on Example 1. The volume ratio of platelet membranes to cell exosomes, the volume ratio of gemcitabine to hybrid materials, and the freezing number are adjusted and optimized. The specific adjustment parameters are shown in Table 2. Other experimental steps and encapsulation efficiency calculation methods are the same as in Example 1, and the results are shown in Table 2.

[0068] Table 2

[0069]

[0070] As shown in Table 2, the encapsulation efficiency of the drug delivery system of gemcitabine wrapped by platelet membranes after optimization can reach more than 70%.

[0071] In summary, the preparation method of the drug delivery system of gemcitabine wrapped by platelet membranes after optimization includes the following steps:

[0072] Step one, mix platelet membranes and MSC exosomes at a volume ratio of 1:2, use a micro-extruder to extrude through a 100 nm filter membrane for 10 times, and obtain hybrid materials;

[0073] Step two, dissolve gemcitabine in water, mix 0.1 mg / mL of gemcitabine with hybrid materials at a volume ratio of 1:1, perform repeated freeze-thawing, quickly place the mixture in a -80°C freezer for rapid freezing for 30 min, after freezing, quickly transfer to a 37°C water bath until it melts, repeat the above freeze-thawing operation for 3 times, after completion, add PBS buffer solution, centrifuge at 4°C, 12000 g for 60 min, discard the supernatant, and obtain the drug delivery system of gemcitabine wrapped by platelet membranes.

[0074] Example 3 Stability evaluation

[0075] In this embodiment, the platelet membrane-encapsulated gemcitabine drug delivery system sample obtained in Example 2 was placed in physiological saline, and the dissolution of gemcitabine at different time points (0 h, 12 h, 24 h, 48 h, 96 h) was observed. The concentration of gemcitabine in the supernatant was detected by high performance liquid chromatography (HPLC), and the concentration of gemcitabine stably encapsulated in the platelet delivery system was calculated. The stability of in vitro encapsulation was then observed, and the rate of change of encapsulation efficiency was calculated. The method for calculating the encapsulation efficiency was the same as in Example 1, and the formula for calculating the rate of change is as follows. The results are shown in Table 3.

[0076] Change rate (%) = (initial encapsulation rate - final encapsulation rate) / initial encapsulation rate * 100%.

[0077] Table 3 Stability Results

[0078]

[0079] As shown in Table 3, the encapsulation efficiency of the samples did not change significantly, proving that the sample system was stable.

[0080] Example 4: Experiment on the inhibitory effect on pancreatic cancer cell proliferation

[0081] Log-growing AsPC-1 human metastatic pancreatic adenocarcinoma cells were selected and treated with 1×10⁻⁶ cells. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates. The culture medium was growth medium (RPMI-1640 [PM150110] + 10% FBS [164210] + 1% P / S [PB180120]). The seeded cells were incubated overnight at 37°C with 5% CO2. The old culture medium was discarded, and the experimental group was replaced with a platelet membrane-coated 10 µM gemcitabine drug delivery system sample (prepared according to the preferred scheme in Example 2). An equal volume of growth medium was added to the negative control group. Three parallel wells were prepared at each level. After culturing for another 24 h, the relative proliferation rate of cells was detected by the CCK-8 assay (CCK-8 kit). The relative proliferation rate (RGR) was the ratio of the absorbance of the experimental group to the absorbance of the negative control group (NC). The results are shown in Table 4.

[0082] Table 4. Relative cell proliferation rate (%)

[0083]

[0084] As shown in Table 4, the platelet membrane-encapsulated gemcitabine drug delivery system can effectively inhibit the proliferation of pancreatic cancer cells.

[0085] Example 5 Animal Experiment

[0086] Cultivate AsPC-1 human metastatic pancreatic adenocarcinoma cells to the logarithmic growth phase, subcutaneously inoculate the right flank of BALB / c nude mice, and the inoculation day is defined as day 0. After 7 days of inoculation, when the tumor volume grows to 100-150 mm, it is determined that the modeling is successful, and a pancreatic cancer mouse model is obtained.

[0087] The pancreatic cancer mice were randomly divided into three groups, including a negative control group (NC), a positive control group (PC), and a sample group, with the same number of mice in each group. On days 7, 10, 13, 16, 19, and 22, the sample group was intravenously injected with the drug delivery system of platelet membrane-coated gemcitabine prepared according to the preferred scheme of Example 2 containing 5 mg / kg of gemcitabine, the positive control group was intravenously injected with liposome-coated gemcitabine containing 5 mg / kg of gemcitabine, and the negative control group was injected with the same amount of 5% glucose. During the treatment, the tumor volume, body weight, and survival status of the nude mice were recorded before each administration. Seven days after the last administration, the tumor volume, body weight, and survival status of the nude mice were recorded until the end of treatment (day 30). After the end of treatment, the tumors were collected, and the tumor inhibition rate was calculated according to the following formula, and the results are shown in Table 5.

[0088] Tumor inhibition rate (%) = (1 - sample group tumor weight or positive control group tumor weight / negative control group tumor weight) x 100.

[0089] Table 5

[0090]

[0091] As can be seen from Table 5, the drug delivery system of platelet membrane-coated gemcitabine can effectively inhibit pancreatic cancer.

[0092] The above only describes the embodiments of the present application and is not intended to limit the present application. Various modifications and changes can be made to the present application by those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of the claims of the present application.

Claims

1. A method of preparing a delivery system loaded with nucleoside analogs for the treatment of pancreatic cancer, characterized in that, The method comprises the following steps: Step one, mixing platelet membrane and MSC exosomes at a volume ratio of 1: (1-3), extruding 5-15 times through a 50-100 nm filter membrane to obtain a hybrid material; Step two, dissolving gemcitabine in water, mixing 0.1-0.5 mg / mL gemcitabine with the hybrid material at a volume ratio of 1: (0.5-2), repeating freezing and thawing 3-10 times, centrifuging and discarding the supernatant after the end to obtain a nucleoside analogue-loaded delivery system.

2. The nucleoside analogue-loaded delivery system prepared by the method of claim 1.

3. A composition characterized in that, The composition comprises the nucleoside analogue-loaded delivery system of claim 2.

4. Use of the nucleoside analogue-loaded delivery system of claim 2 or the composition of claim 3 in the preparation of a medicament for treating pancreatic cancer.

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