Preparation and application of nano delivery system for realizing broad-spectrum overcoming of tumor drug resistance by reversing tumor hypoxic slightly acidic environment

Abstract

The invention discloses preparation and application of a nano delivery system for loading chemotherapeutic drugs to relieve tumor drug resistance. According to the prepared nano delivery system with the red cell membrane wrapped with the copper peroxide, the tumor microenvironment is reversed, absorption of tumors to drugs is increased, excretion of the drugs is reduced, meanwhile, the nano delivery system efficiently acts on tumor cells in a targeting mode, the cell apoptosis effect of the tumors is further enhanced, and the excellent anti-tumor effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the preparation and application of a nano-delivery system loaded with chemotherapeutic drugs to alleviate tumor resistance. This type of nano-drug delivery system can be used for the treatment of tumors, and greatly improves the anti-tumor effect of chemotherapeutic drugs by reversing the hypoxic and slightly acidic environment of the tumor. Technical Background

[0002] The tumor microenvironment is composed of tumor cells, recruited host cells and other resident cells, as well as the corresponding cell secretions and components in the extracellular matrix. It enhances tumor survival by providing cancer stem cell niches for tumor cells, promoting tumor cell metabolism and programming, while reducing drug intake and promoting an immunosuppressive environment. These drug resistance mechanisms not only produce resistance to traditional tumor treatments, but also to chemotherapy and radiotherapy that are paired with them, greatly limiting the effectiveness of drugs in tumor treatment.

[0003] The typical characteristic of the tumor microenvironment is low oxygen concentration. Due to the high metabolic rate and low apoptosis rate of tumor cells, the rapid metabolism and volume growth of tumor cells make tumors consume more oxygen and the corresponding various material energy bases than normal cells, which further leads to the characteristics of low oxygen in tumor cells. At the same time, because the tumor is far away from the blood vessel end, a tumor hypoxic microenvironment is formed.

[0004] Another characteristic of the tumor microenvironment is that it is slightly acidic. In the hypoxic microenvironment of the tumor, due to its distance from the vascular end, glycolysis increases, and the tumor excretes a large amount of protons and lactic acid, resulting in a decrease in the pH value of the tumor microenvironment.

[0005] With people's understanding of tumor drug resistance and the development of drug research, more and more treatment options have been applied in the face of low therapeutic efficiency and poor prognosis of chemotherapy drugs in tumor treatment. Among them, the huge potential of nano-delivery systems has been developed. Nano-delivery systems increase the solubility and controlled release properties of drugs in the tumor microenvironment, while improving the stability of drugs and enhancing tumor targeting. They play an important role in enhancing the therapeutic effect of anti-tumor drugs and improving bioavailability.

[0006] Copper peroxide is the first choice for nanocarriers. Multifunctional nanocarriers can load drugs while increasing drug stability and drug enrichment in tumors. However, due to the different properties and functions of various nanomaterials, we found through research that copper peroxide nanoparticles have good stability and can produce oxygen and alkali, reversing the slightly acidic and hypoxic environment inside and outside the body, and have a strong ability to load drugs. At the same time, a layer of red blood cell membrane is wrapped on its surface, which can improve the biocompatibility of nanomaterials and increase the bioavailability of materials. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a nano-delivery system for alleviating tumor drug resistance, which is a tumor-targeted delivery system capable of high-load broad-spectrum chemotherapy drugs. This type of delivery system utilizes the oxygen- and alkali-producing properties of copper peroxide to reverse the tumor microenvironment, thereby alleviating drug resistance and providing new ideas for the research of anti-tumor drugs.

[0008] The preparation method of the nano-delivery system for alleviating tumor drug resistance of the present invention specifically comprises the following steps:

[0009] (1) Weigh 0.1705g CuCl2 and dissolve it in 100mL water to prepare a 0.01mol / L CuCl2 solution. Weigh 0.12g NaOH and dissolve it in 100mL water to prepare a 0.03mol / L NaOH solution. Take 20ml CuCl2 solution and 20ml NaOH solution in a flask and mix them thoroughly.

[0010] (2) Add 2.0 g PVP (polyvinyl pyrrolidone) and mix thoroughly by ultrasonication for 3 min, then stir at 800 rpm for 30 min at room temperature. After stirring completely, centrifuge at 8000 rpm for 5 min, remove the supernatant, add water and shake thoroughly by ultrasonication, continue centrifugation, and repeat 2-3 times.

[0011] (3) After centrifugation, remove the supernatant, continue to add water and ultrasonically oscillate, centrifuge at 11000 rpm for 10 min, remove the supernatant, add water and ultrasonically oscillate thoroughly, continue centrifugation, and repeat 2-3 times.

[0012] (4) After centrifugation, remove the supernatant and add an appropriate amount of water to obtain a copper peroxide (CP) solution. Take an appropriate amount of CP and an appropriate amount of red blood cell membrane for ultrasonic mixing. Transfer the solution to a syringe and filter with 0.8μm and 0.45μm filters respectively. Centrifuge the solution at 10000rpm for 5min. Remove the supernatant and add an appropriate amount of water for ultrasonic mixing to obtain the nano delivery system CP@M. Then add an excess of the chemotherapy drug to be loaded, control the water temperature, ultrasonicate for 40min, and centrifuge at 16000rpm for 10min.

[0013] (5) Remove the colored supernatant, add water and mix by ultrasonication, and continue centrifugation at 16000 rpm for 10 min until the supernatant is colorless. Finally, remove the supernatant, add an appropriate amount of water, and mix by ultrasonication to obtain the drug-loaded erythrocyte membrane-encapsulated copper peroxide nano-delivery system.

[0014] In the specific preparation, the mass ratio of CP to red blood cell membrane in step (4) is 1:1

[0015] The present invention has the following outstanding beneficial effects:

[0016] The present invention uses a specific material synthesis ratio and a method of extrusion and ultrasonic oscillation to synthesize a tumor active targeting nano delivery system of copper peroxide wrapped in an erythrocyte membrane as a carrier, and loads chemotherapeutic drugs to reverse the tumor microenvironment and achieve a nano delivery system that alleviates drug resistance. The above-mentioned erythrocyte membrane wrapped copper peroxide tumor active targeting nano delivery system alleviates drug resistance evaluation experiments also show that the delivery system can reverse the tumor microenvironment, achieve tumor resistance alleviation, and enable chemotherapeutic drugs to better play an anti-tumor role, while also greatly reducing the toxic and side effects of drugs, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a performance test diagram of CP in reversing the tumor microenvironment;

[0018] Figure 2 This is the detection diagram of DOX uptake by 4T1 cells in the hypoxia model (A) and the hypoxia relief model (B);

[0019] Figure 3 The graphs show the uptake of DOX by 4T1 cells in the micro-acid model (A) and the micro-acid relief model (B). DETAILED DESCRIPTION

[0020] The specific implementation of the present invention is further described below by way of examples. It should be understood that the following description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0021] Example 1: Preparation of Nanodelivery System CP

[0022] Weigh 0.1705g CuCl2 and dissolve it in 100mL water to prepare 0.01mol / L CuCl2 solution, and weigh 0.12gNaOH and dissolve it in 100mL water to prepare 0.03mol / L NaOH solution. Take 20ml CuCl2 solution and 20mL NaOH solution in a flask and mix them thoroughly. ) Add 2.0g PVP (polyvinyl pyrrolidone) and mix thoroughly for 3min, then stir at room temperature at 800rpm for 30min. After stirring completely, centrifuge at 8000rpm for 5min, remove the supernatant, add water and ultrasonically oscillate, continue centrifugation, and repeat 1-2 times. After centrifugation, remove the supernatant, continue to add water and ultrasonically oscillate, centrifuge at 11000rpm for 10min, remove the supernatant, add water and ultrasonically oscillate, continue centrifugation, and repeat 1-2 times. After centrifugation, remove the supernatant, add an appropriate amount of water, and obtain a copper peroxide (CP) solution.

[0023] Example 2: Preparation of Nano-delivery System CP@M

[0024] Take 1 mL of Example 1CP and 1 mL of red blood cell membrane and mix them ultrasonically. Transfer the solution to a syringe and filter with 0.8 μm and 0.45 μm filters respectively. Centrifuge the solution at 10,000 rpm for 5 min. Remove the supernatant, add an appropriate amount of water and mix ultrasonically to obtain the nano delivery system CP@M.

[0025] Example 3: Preparation of erythrocyte membrane-encapsulated copper peroxide-loaded drug nanodelivery system (CP@M@DOX)

[0026] Taking DOX as an example of a broad-spectrum chemotherapy drug, 1 mL of CP@M in Example 2 was added with 1 mL of DOX, the water temperature was controlled, ultrasonicated for 40 min, and centrifuged at 16000 rpm for 10 min. The colored supernatant was removed, water was added and ultrasonically mixed, and centrifuged at 16000 rpm for 10 min until the supernatant was colorless. Finally, the supernatant was removed, an appropriate amount of water was added, and ultrasonically mixed to obtain the erythrocyte membrane-wrapped copper peroxide-loaded DOX nanodelivery system CP@M@DOX.

[0027] Example 4: Evaluation of the Nano-delivery System CP in Reversing Tumor Microenvironment

[0028] The CP nanodelivery system was evaluated by detecting the changes in oxygen and alkali in PBS at different pH values. CP solution was quickly added to PBS at pH 5.0, 6.5, and 7.4, and the changes in the solution pH were recorded in real time. The alkali production capacity at different pH values ​​was obtained. After liquid sealing with paraffin in PBS at pH 5.0, 6.5, and 7.4, CP solution was quickly added to obtain the oxygen production capacity at different pH values. The amount of copper peroxide added was such that its concentration was 200 μg / mL.

[0029] The performance test results of reversing the tumor microenvironment show that ( Figure 1 ), in an acidic environment, CP has excellent oxygen and alkali production performance, and in an alkaline environment, the content of oxygen and alkali changes little. It can be seen that the CP nano delivery system can reverse the slightly acidic and hypoxic microenvironment of the tumor.

[0030] Example 5: Evaluation of the performance of erythrocyte membrane-encapsulated copper peroxide-loaded drug delivery system (CP@M@DOX) in alleviating hypoxia and reversing drug resistance with mild acidity

[0031] Taking DOX as an example of a broad-spectrum chemotherapy drug, 4T1 cells were inoculated on a six-well plate. After successful attachment, a DOX concentration of 5 μg / mL was selected based on the hypoxic endocytosis model, and 4T1 cells were cultured under different conditions, first hypoxia for 8 hours, and then the materials were added for co-culture for 2 hours (normoxia, DOX, CP@M@DOX, CP@M / DOX) (CP@M / DOX group added CP@M in advance, and then added DOX for co-culture for 2 hours). The cells were collected for flow cytometry analysis to detect the endocytosis of DOX by 4T1 cells.

[0032] 4T1 cells were inoculated on a six-well plate. After successful attachment, a 2-hour micro-acid endocytosis model was selected. 4T1 cells were divided into blank, DOX, CP@M@DOX, and CP@M / DOX groups and co-cultured with different culture media (pH 6.5, pH 6.8, and pH 7.4) for 2 hours. Cells were collected for flow cytometry analysis to detect the endocytosis of DOX by 4T1 cells.

[0033] After a certain period of hypoxia and mild acid treatment, the uptake of DOX by 4T1 cells was observed in the hypoxia group (H) and the normoxia group (N). Figure 2 As shown in A, it was found that the uptake of DOX by 4T1 cells was limited in the hypoxia group. Figure 2 As shown in B, normoxia group, DOX group, CP@M@DOX group, and CP@M / DOX group were added for hypoxic culture, and it was found that the uptake of DOX by 4T1 cells increased, which can prove that CP@M can increase the sensitivity of 4T1 cells to DOX under hypoxic conditions; Figure 3 As shown in A, it was found that the lower the pH value, the less DOX uptake by 4T1 cells. In the slightly acidic pH 6.5 mitigation model, after CP@M treatment, as shown in Figure 3 As shown in B, it was found that the uptake of DOX by 4T1 cells increased, which proved that CP@M could increase the sensitivity of 4T1 cells to DOX under slightly acidic conditions.

Claims

1. A method for preparing a nano-delivery system that can achieve broad-spectrum tumor drug resistance by reversing the hypoxic and slightly acidic environment of tumors, characterized in that: Copper peroxide nanoparticles are used as carrier materials and are wrapped by red blood cell membranes to obtain a red blood cell membrane-wrapped copper peroxide-loaded drug nano-delivery system that has the effect of alleviating tumor drug resistance.

2. The method for preparing the nano-delivery system of red blood cell membrane-wrapped copper peroxide loaded drug according to claim 1, characterized in that: The specific steps include: 1) Weigh 0.1705g CuCl2 and dissolve it in 100mL water to prepare 0.01mol / L CuCl2 solution, weigh 0.12g NaOH and dissolve it in 100mL water to prepare 0.03mol / L NaOH solution; take 20ml CuCl2 solution and 20mL NaOH solution in a flask and mix them thoroughly; add 2.0g polyvinyl pyrrolidone, mix thoroughly by ultrasonication for 3min, and stir at room temperature at 800rpm for 30min; after stirring completely, centrifuge at 8000rpm for 5min, remove the supernatant, add water for ultrasonication and oscillate thoroughly, continue centrifugation, and repeat 1-2 times; after centrifugation, remove the supernatant, continue to add water for ultrasonication and oscillate, centrifuge at 11000rpm for 10min, remove the supernatant, add water for ultrasonication and oscillate thoroughly, continue centrifugation, and repeat 1-2 times; after centrifugation, remove the supernatant, add appropriate amount of water to obtain a copper peroxide solution; 2) Take 1 mL of copper peroxide solution and 1 mL of red blood cell membrane and mix them by ultrasonication; The solution was transferred into a syringe and filtered using 0.8 μm and 0.45 μm filters respectively; the solution was centrifuged at 10,000 rpm for 5 min; the supernatant was removed, and an appropriate amount of water was added for ultrasonic mixing to obtain a nano delivery system; 3) Take 1 mL of the nano-delivery system and add an excess of the chemotherapy drug to be loaded, control the water temperature, sonicate for 40 minutes, and centrifuge at 16000 rpm for 10 minutes; remove the colored supernatant, add water and sonicate to mix, continue to centrifuge at 16000 rpm for 10 minutes until the supernatant is colorless; finally, remove the supernatant, add an appropriate amount of water, and sonicate to obtain a nano-delivery system of red blood cell membrane-wrapped copper peroxide loaded with drugs.

3. The method for preparing the nano-delivery system of erythrocyte membrane-encapsulated copper peroxide loaded drug according to claim 2, characterized in that: In step 1), the mass ratio of CuCl2 to NaOH is 1.42:

1.

4. The method for preparing the nano-delivery system of erythrocyte membrane-encapsulated copper peroxide loaded drug according to claim 2, characterized in that: In step 1), the mass ratio of the copper peroxide solution to the red blood cell membrane is 1:

1.

5. Use of a nano-delivery system containing copper peroxide loaded with a drug and wrapped with red blood cell membranes prepared according to claims 1 to 4 in the preparation of drugs for treating tumor diseases.

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