Bionic cancer cell membrane engineered cis-platinum nano-drug as well as preparation method and application thereof

Abstract

The invention discloses preparation and application of a bionic cancer cell membrane engineered cis-platinum nano anti-cancer drug, and belongs to the field of biological medicine. According to the nano-drug, a dual encapsulation strategy is developed by utilizing a cancer cell membrane. Firstly, cis-platinum is combined with a cancer cell membrane to form positively charged nano-particles, and then the separated nano-particles are compounded with the cancer cell membrane again. According to the strategy, the cis-platinum is tightly packaged in a cancer cell membrane by utilizing the natural characteristics of the cancer cell membrane, so that the cis-platinum is prevented from being attacked by reactive molecules during in-vivo circulation while targeting a homologous tumor, and the risks of drug inactivation and systemic toxicity are greatly reduced. The strategy abandons any artificial material intervention, and has the advantages of good biocompatibility, good targeted treatment effect, simple preparation process, low equipment requirement and good application prospect in the field of cancer treatment.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a bionic cancer cell membrane engineered cisplatin nano anticancer drug and a preparation method and application thereof. Background Art

[0002] Cisplatin is a very important first-line anti-cancer chemotherapy drug in clinical practice, playing a key role in more than 70% of clinical cancer treatment programs. However, there are a large number of macromolecular and small molecular nucleophilic substances in the human body, such as DNA bases, protein residues, anions, and sulfur-containing biological molecules, which are very easy to react with platinum ions. This is one of the main causes of cisplatin systemic toxicity and cisplatin inactivation, which seriously affects the clinical treatment of cisplatin.

[0003] In order to overcome the low efficacy and high toxicity caused by biodistribution, the tumor-targeted delivery of cisplatin highlights its important value. Its research is currently focused on the design and preparation of cisplatin nanoformulations. Due to the water solubility of cisplatin drugs, it is not suitable for conventional physical encapsulation in nanocarriers. The common countermeasure is to bond cisplatin to a targeted polymer carrier through coordination chemistry and then form a nanodrug. However, the complex synthesis of polymer carriers limits its clinical application. In addition, the targeted delivery of cisplatin faces other challenges, including the immune system identifying the drug carrier as a foreign body and clearing it in advance, the heterogeneity and complexity of tumors leading to inefficient targeting or even off-target, especially the inactivation of cisplatin caused by nucleophilic substances such as proteins and sulfur-containing biological molecules mentioned above.

[0004] In order to reduce the nonspecific binding of proteins, a protective hydrophilic corona composed of soluble polymer fragments (such as polyethylene glycol and polyanions) is usually designed on the surface of cisplatin nanoformulations to repel protein adsorption. However, this carrier molecule design cannot prevent the large number of small molecule nucleophiles present in the body from interacting with cisplatin. Taking cisplatin as an example, it is particularly easy to be rapidly neutralized by sulfur-containing compounds such as metallothioneins and glutathione. Therefore, the development of cisplatin nanomedicines that can achieve tumor targeting and resist interference from nucleophiles in the body is undoubtedly of great significance, although it is extremely challenging. In addition, the preparation process of this type of nanomedicine needs to be simplified for clinical application and transformation. Summary of the invention

[0005] The purpose of the present invention is to provide a bionic cancer cell membrane engineered cisplatin nano drug and its preparation method and application in the preparation of anticancer drugs. The nano drug has a bionic cell structure to isolate cisplatin, ensuring that it is not inactivated by nucleophilic substances during circulation in the body; at the same time, it can target homologous tumors, improve efficacy and reduce toxic side effects. The invention does not use any other artificial materials, has excellent biosafety, and has high feasibility for clinical transformation.

[0006] In order to solve the above problem, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a bionic cancer cell membrane-engineered cisplatin nano anticancer drug is provided, wherein the nano anticancer drug is formed by double encapsulation, wherein the first encapsulation in the double encapsulation is to combine cisplatin with a first cancer cell membrane to form positively charged nanoparticles, and the second encapsulation in the double encapsulation is to compound the positively charged nanoparticles with a second cancer cell membrane.

[0008] According to the above scheme, the cisplatin is an anticancer drug approved by the State Food and Drug Administration.

[0009] According to the above scheme, in the first encapsulation, the mass ratio of the first cancer cell membrane to cisplatin is 1:1-1:4.

[0010] According to the above scheme, in the second encapsulation, the mass ratio of the positively charged nanoparticles to the second cancer cell membrane is 1:1-1:4.

[0011] According to the above protocol, cancer cell membranes are extracted from cancer cells.

[0012] In the second aspect of the present invention, a method for preparing the above-mentioned bionic cancer cell membrane engineered cisplatin nano anticancer drug is provided, the method comprising:

[0013] Dispersing cancer cell membrane fragments and cisplatin in deionized water, respectively, to obtain cancer cell membrane dispersion and cisplatin solution;

[0014] The cancer cell membrane dispersion and the cisplatin solution are mixed, shaken and then centrifuged for the first encapsulation to obtain positively charged nanoparticles;

[0015] The positively charged nanoparticles are mixed with the cancer cell membrane dispersion, and then centrifuged after ultrasonic dispersion for secondary encapsulation to obtain a bionic cancer cell membrane engineered cisplatin nano anticancer drug.

[0016] In the first encapsulation, the concentration of the cancer cell membrane dispersion is 0.5 mg / mL-1.5 mg / mL; the concentration of the cisplatin solution is 0.5 mg / mL-1.5 mg / mL.

[0017] In the third aspect of the present invention, there is provided a use of the above-mentioned bionic cancer cell membrane engineered cisplatin nanomedicine in the preparation of anticancer drugs.

[0018] Furthermore, the cancer treated by the anticancer drug includes at least one of breast cancer, colon cancer, and liver cancer.

[0019] The present invention has the following advantages and beneficial effects:

[0020] 1. The nano anticancer drug provided by the present invention utilizes the groups on the surface of the cancer cell membrane to complex with cisplatin, assembles into a positively charged nano core, and then assembles into a negatively charged nano drug through electrostatic interaction with the negatively charged cancer cell membrane. Double encapsulation can ensure that the encapsulated cisplatin is effectively isolated from the contact with nucleophilic substances in the body during the circulation of the nano drug, and at the same time has the homologous targeting effect of the cancer cell membrane, greatly reducing the risk of drug inactivation and systemic toxicity, and can achieve precise treatment of cancer.

[0021] 2. The nano anticancer drug provided by the present invention has simple ingredients, good biocompatibility, small toxic and side effects, obvious therapeutic effect, simple preparation process, low equipment requirements, and has good application prospects in the field of cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scanning electron microscope image of the nano anticancer drug prepared in Example 1.

[0023] Figure 2 This is a diagram showing the in vitro anticancer effect of the nano anticancer drug prepared in Example 1.

[0024] Figure 3 The efficacy of the nano-cisplatin anticancer drug prepared in Example 1 is tolerant to nucleophilic small molecules and proteins, wherein: A is the efficacy in the presence of glutathione; B is the efficacy in the presence of serum albumin (*P<0.05; **P<0.01; ***P<0.001).

[0025] Figure 4 This is the homologous tumor targeting effect of the nano anticancer drug prepared in Example 1 in tumor-bearing mice.

[0026] Figure 5 The nano anticancer drug prepared in Example 1 was used to treat tumor-bearing mice for 14 days and had an inhibitory effect on tumor growth (***P<0.001). DETAILED DESCRIPTION

[0027] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0028] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0029] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0030] The present application will be described in detail below with reference to embodiments and experimental data.

[0031] Example 1: Biomimetic cancer cell membrane engineered cisplatin nano anticancer drug and preparation method thereof

[0032] The present invention provides a method for preparing a bionic cancer cell membrane engineered cisplatin nano anticancer drug, the specific steps of which are as follows:

[0033] (1) Cancer cell membranes extracted from mouse breast cancer cells were dispersed in deionized water to form a 1 mg / mL dispersion.

[0034] (2) Dissolve cisplatin in deionized water to form a 1 mg / mL solution.

[0035] (3) The cisplatin solution and the cancer cell membrane dispersion are mixed at a mass ratio of 1:2 between the cancer cell membrane and cisplatin, shaken, and centrifuged to obtain positively charged nanoparticles.

[0036] (4) The positively charged nanoparticles are dispersed in deionized water to form a 1 mg / mL dispersion, and the dispersion is mixed with a cancer cell membrane dispersion at a mass ratio of 1:2, and subjected to ultrasound and centrifugation to obtain a biomimetic cancer cell membrane engineered cisplatin nano anticancer drug.

[0037] Figure 1 The scanning electron microscope image of the nano anticancer drug prepared in this example is shown in Figure 2. The prepared nano anticancer drug dispersion was added dropwise to a silicon wafer and observed after natural drying. Figure 1 As shown, the nanoparticles are regular spherical in shape and have a size of about 100 nanometers.

[0038] Example 2: In vitro anticancer experiment

[0039] Test method: Mouse breast cancer cells were seeded in a 96-well plate at a density of 6000 cells / well and cultured with 100 μL of culture medium for 24 hours. Then, 100 μL of the gradient dispersion of nanodrugs prepared with culture medium was added to each well. All cells were cultured at 37°C for 48 hours. Subsequently, 20 μL of 5 mg / m MTT (MTT dissolved in PBS buffer) was added to each well. After 4 hours of co-culture, the culture medium was aspirated and 150 μL of dimethyl sulfoxide was added. The absorbance at 570 nm in each well was measured by an enzyme-labeled instrument, and the cell survival rate was calculated to obtain the toxicity of the nano-anticancer drug to mouse breast cancer cells.

[0040] The results of the in vitro anticancer effects of nano anticancer drugs are as follows Figure 2 As shown, the survival rate of mouse breast cancer cells gradually decreased with the increase of nanomedicine concentration, showing a good anti-cancer effect.

[0041] Example 3: Detection of the effect of nano anticancer drugs in isolating cisplatin from binding to nucleophilic molecules and proteins in vivo

[0042] Mouse breast cancer cells were seeded in a 96-well plate at a density of 6000 cells / well and cultured with 100 μL of culture medium for 24 hours. Cisplatin and the prepared nano-drugs were mixed with glutathione and serum protein for 6 hours. Then, the concentration gradient dispersion was prepared in the culture medium and added to each well. All cells were cultured at 37°C for 48 hours. Subsequently, 20 μL of 5 mg / mL MTT (MTT dissolved in PBS buffer) was added to each well. After 4 hours of co-culture, the culture medium was aspirated and 150 μL of dimethyl sulfoxide was added. The absorbance at 570 nm in each well was measured by an ELISA instrument, and the cell survival rate was calculated to obtain the effect of the nano anticancer drug in isolating cisplatin from the binding of nucleophilic molecules and proteins in the body.

[0043] The effect of testing the nano anticancer drug in isolating cisplatin from the binding of nucleophilic molecules and proteins (glutathione or serum albumin) in the body is shown in the figure below. Figure 3 As shown, at the same concentration of nucleophilic substances, the ability of nanomedicine to kill cancer cells is significantly greater than that of the small molecule drug cisplatin, reflecting its excellent ability to tolerate nucleophilic molecules and proteins.

[0044] Example 4: In vivo anticancer experiment

[0045] Mouse breast cancer cells (4T1) and mouse colon cancer cells (CT26) were inoculated subcutaneously on the right back of 6-week-old female BALB / c mice (5×10 5 When the tumor grows to 150 mm 3 The cy5-labeled nano anticancer drug (10 mg / kg) was intravenously injected into mice bearing 4T1 and CT26 tumors, and then the mice were monitored in real time using an in vivo imaging system at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 hours to detect the fluorescence intensity at the tumor site of the mice.

[0046] The fluorescence intensity of mouse tumors is shown in Figure 4 As shown in the figure, after 48 hours, the fluorescence was significantly enriched in the 4T1 tumor site. During the entire observation process, the fluorescence in the 4T1 tumor was significantly higher than that in the CT26 tumor, and the trend became more and more obvious. The prepared nano anticancer drug showed significant tumor targeting and homologous tumor selectivity.

[0047] Mouse breast cancer cells (4T1) were inoculated subcutaneously on the right back of 6-week-old female BALB / c mice (5×10 5 When the tumor grows to 65mm 3 At 10:00 pm, nano anticancer drugs (10 mg / kg) were intravenously injected into mice bearing 4T1 tumors, and the size of the tumors was recorded on time.

[0048] The effect of the nano anticancer drug on inhibiting tumor growth after 14 days of treatment in tumor-bearing mice is shown in the figure below. Figure 5 As shown, the nano anticancer drug significantly inhibited the growth of tumors.

[0049] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0050] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0051] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A bionic cancer cell membrane engineered cisplatin nanomedicine, characterized in that: The nano anticancer drug is formed by double encapsulation, wherein the first encapsulation in the double encapsulation is to combine cisplatin with a first cancer cell membrane to form positively charged nanoparticles, and the second encapsulation in the double encapsulation is to combine the positively charged nanoparticles with a second cancer cell membrane.

2. The nano drug according to claim 1, characterized in that In the first encapsulation, the mass ratio of the first cancer cell membrane to cisplatin is 1:1-1:

4.

3. The nano drug according to claim 1, characterized in that In the second encapsulation, the mass ratio of the positively charged nanoparticles to the second cancer cell membrane is 1:1-1:

4.

4. A method for preparing the bionic cancer cell membrane engineered cisplatin nanomedicine according to any one of claims 1 to 3, characterized in that: The method comprises: Dispersing cancer cell membrane fragments and cisplatin in deionized water, respectively, to obtain cancer cell membrane dispersion and cisplatin solution; The cancer cell membrane dispersion and the cisplatin solution are mixed, shaken and then centrifuged for the first encapsulation to obtain positively charged nanoparticles; The positively charged nanoparticles are mixed with the cancer cell membrane dispersion, and then centrifuged after ultrasonic dispersion for secondary encapsulation to obtain a bionic cancer cell membrane engineered cisplatin nano anticancer drug.

5. The preparation method according to claim 4, characterized in that: In the first encapsulation, the concentration of the cancer cell membrane dispersion is 0.5 mg / mL-1.5 mg / mL; the concentration of the cisplatin solution is 0.5 mg / mL-1.5 mg / mL.

6. The preparation method according to claim 4, characterized in that: The mass ratio of the cancer cell membrane in the cancer cell membrane dispersion to the cisplatin in the cisplatin solution is 1:1-1:

4.

7. The preparation method according to claim 4, characterized in that: In the second encapsulation, the mass ratio of the positively charged nanoparticles to the cancer cell membranes in the cancer cell membrane dispersion is 1:1-1:

4.

8. The preparation method according to claim 4, characterized in that: The cancer cell membrane fragments are extracted from cancer cells.

9. Use of the bionic cancer cell membrane engineered cisplatin nanomedicine according to any one of claims 1 to 3 in the preparation of anticancer drugs.

10. The use according to claim 9, characterized in that: The cancer treated by the anticancer drug includes at least one of breast cancer, colon cancer and liver cancer.

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