Nano messenger for regulating mitochondrial calcium instability as well as preparation method and application of nano messenger

Abstract

The invention relates to the technical field of nano messengers, and discloses a nano messenger for regulating mitochondrial calcium instability and a preparation method and application thereof, the nano messenger takes polydopamine as a mineralization template, and the template is loaded with a calcium channel inhibitor, an autophagy inhibitor and metal calcium ions. According to the technical scheme, mitochondrial calcium overload in cells is aggravated by using calcium ions and Berbamine, an apoptosis pathway mediated by mitochondrial cytochrome C is activated, and breast cancer cells are induced to generate immunogenic cell death. Autophagy activation is accompanied in the ICD generation process, so that downstream signal channel transduction is started, and immune escape and metastasis of breast cancer cells are promoted. Therefore, by introducing the autophagy inhibitor, MHC-I molecules are prevented from being degraded by an autophagy lysosome way, more phagocytosis my signal-calreticulin CRT is promoted to be exposed on the surfaces of tumor cells, the anti-tumor immune response of the body is synergistically enhanced, and the purpose of inhibiting growth and metastasis of breast cancer is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of nano messengers, and in particular to a nano messenger for regulating mitochondrial calcium instability and a preparation method and application thereof. Background Art

[0002] Chemical messengers are considered to be important signaling molecules that regulate various metabolic activities of cells and play a key role in the diagnosis and treatment of various diseases. Studies in recent years have shown that chemical messengers, represented by gas molecules and metal ions, can have a significant impact on immune regulation and the metabolic activities and survival vitality of tumor cells. Therefore, the development of chemical messenger-based nanomedicines for combined cancer therapy is considered to be an anti-tumor treatment method with broad application prospects. However, the complexity of biological systems has brought many technical problems and obstacles to the practical application of chemical messengers.

[0003] 1. Limited regulatory therapeutic activity: The internal environment of biological systems is extremely complex, and multiple biological molecules interact with each other, resulting in chemical messengers generally showing limited regulatory therapeutic activity in the body. This complexity makes chemical messengers susceptible to interference from other biological molecules when transmitting signals, thereby affecting their regulatory effects. In addition, tumor cells express a variety of ion channels, and the presence of these ion channels partially weakens the regulatory effects of chemical messengers. In addition, the physiological metabolic processes of tumor cells themselves also consume chemical messengers, further reducing their anti-tumor efficacy.

[0004] 2. Stability issues: The stability of chemical messengers in the body is another urgent problem to be solved. Since chemical messengers are easily decomposed or inactivated in the biological environment, their regulatory activity is often difficult to maintain for a long time. This means that chemical messengers need to reach a sufficient concentration in a short period of time to play a therapeutic role, otherwise their effect will be greatly reduced.

[0005] 3. Targeting and toxicity: The distribution of chemical messengers in the body often lacks specificity, making it difficult to accurately reach the lesion site. This not only affects the therapeutic effect, but may also cause unnecessary damage to normal tissues. In addition, some chemical messengers may have toxic side effects at high doses, especially when used for a long time. How to balance the therapeutic effect and safety has become an important issue.

[0006] In order to solve the above technical problems and obstacles, it is of great significance to actively develop new chemical messengers and their delivery systems to achieve high stability, high targeting and low toxicity. Summary of the invention

[0007] The present invention aims to provide a nano messenger for regulating mitochondrial calcium instability and a preparation method and application thereof, so as to improve the stability and targeting of chemical messengers and reduce their toxic effects.

[0008] To achieve the above object, the present invention adopts the following technical solution: a nano messenger for regulating mitochondrial calcium instability, using polydopamine as a mineralization template, and the template is loaded with calcium channel inhibitors, autophagy inhibitors and metal calcium ions.

[0009] Preferably, as an improvement, the calcium channel inhibitor is berbamine.

[0010] Preferably, as an improvement, the autophagy inhibitor is U0126.

[0011] Preferably, as an improvement, the calcium ions are derived from CaCl2﹒2H2O.

[0012] Preferably, as an improvement, the particle size of the nano messenger is 149±4 nm.

[0013] Preferably, as an improvement, a method for preparing a nano messenger for regulating mitochondrial calcium instability comprises the following steps:

[0014] Step 1, dissolve CaCl2﹒2H2O and dopamine in anhydrous ethanol, and stir magnetically to mix thoroughly to obtain system I;

[0015] Step 2: dissolving calcium channel inhibitor berbamine and autophagy inhibitor U0126 in dimethyl sulfoxide to obtain system II;

[0016] Step 3: Integrate System II into System I;

[0017] Step 4: Place the solution obtained in step 3 in a sealed box, and place an appropriate amount of NH4HCO3 powder around it, and react at room temperature overnight;

[0018] Step 5: Wash the solution obtained in step 4 and collect the precipitate to obtain the signal cascade calcium nano-messenger pCaBer+U0126.

[0019] Preferably, as an improvement, in step 1, the mass ratio of CaCl2﹒2H2O to dopamine is 5 to 10:1.

[0020] Preferably, as an improvement, in step 2, the mass ratio of the calcium channel inhibitor berbamine, the autophagy inhibitor U0126 and dopamine is 1:1:5-10.

[0021] Preferably, as an improvement, a nanosignal for regulating mitochondrial calcium instability is used in the preparation of anti-tumor drugs.

[0022] Preferably, as a refinement, the tumor is a breast cancer tumor.

[0023] The principle and advantages of this solution are: In the previous research, this technical solution found that when the intracellular Ca 2+ When the calcium increases, it can activate the mitochondrial calcium sensor (MICU1) to regulate the mitochondrial calcium ion uniporter (MCU) to transport mitochondrial Ca 2+ Uptake and activation of cell membrane Ca 2+ channels, promoting Ca 2+ excretion to maintain intracellular Ca 2+ Dynamic balance, weakening the regulatory effect of calcium chemical messengers. In addition, the inventors found that after immunogenic cell death occurs in breast cancer cells, autophagy is activated, which promotes the immune escape and metastasis of breast cancer cells, resulting in a weakened anti-tumor effect. Tumor cells can also promote immune escape or resist the regulatory effect of chemical messengers by activating autophagy, weakening the anti-tumor effect. Based on this, the inventors were inspired by the important influence of chemical messengers on immune regulation, tumor cell metabolic activity and survival vitality, and proposed a signal cascade calcium nanomessenger based on chemical messengers: in the preparation stage of nanomessenger, autophagy inhibitors are introduced to enhance anti-tumor immune response, and produce a better purpose of inhibiting breast cancer growth and metastasis, that is: using the abnormal calcium ion channel function of tumor cells, causing tumor cells to have calcium ion discharge dysfunction, and taking calcium channel inhibitor Berbamine to further prevent the discharge of calcium ions produced by nanomessenger degradation, aggravate intracellular mitochondrial calcium overload, activate mitochondrial cytochrome C-mediated apoptosis pathway, and induce immunogenic cell death (ICD) in breast cancer cells. Because the ICD process is accompanied by autophagy activation, the downstream signal pathway transduction is initiated, promoting breast cancer cell immune escape and metastasis. Therefore, by introducing autophagy inhibitors, MHC-I class molecules are prevented from being degraded by the autophagy lysosomal pathway, and more "eat me" signals, calreticulin CRT, are promoted on the surface of tumor cells to synergistically enhance the body's anti-tumor immune response, thereby achieving the purpose of inhibiting the growth and metastasis of breast cancer. In the technical research and development stage, the construction of the intracellular calcium overload system is the key to the success of the preparation of the nano messenger of this technical solution and the quality of the effect. In this process, the ratio of CaCl2﹒2H2O to dopamine needs to be strictly controlled. When the ratio of the two is improperly controlled, the obtained nano messenger particle size will be uneven, and the particle size will be larger at the micron level, which cannot achieve the expected effect of this technical solution. In addition, when constructing the calcium instability system, this technical solution uses calcium chloride dihydrate and intracellular physiological calcium. Since chloride ions are easy to remove, the dopamine-doped calcium carbonate formed in the final system can be decomposed to produce carbon dioxide to promote lysosomal escape; if it is replaced by other calcium ion sources such as calcium phosphate and calcium sulfate, it will cause the formation of dopamine-doped calcium phosphate or calcium sulfate instead of calcium carbonate in the system, which cannot be decomposed to produce carbon dioxide to promote lysosomal escape.

[0024] In summary, the beneficial effects of this technical solution are: this technical solution utilizes calcium ions (Ca2+ ) and Berbamine, aggravating mitochondrial calcium instability, activating the Cytochrome c-mediated apoptosis pathway, and inducing ICD in tumor cells. Since autophagy is activated when tumor cell-related antigens are exposed, causing residual breast cancer cells to escape immune system, the introduction of U0126 can inhibit autophagy, prevent MHC-I molecules from being degraded by the autophagy-lysosome pathway, and promote the exposure of more "eat me" signals—CRT—on the surface of tumor cells, producing a cascade-amplified breast cancer immunotherapy effect, thereby achieving the purpose of long-term and effective inhibition of breast cancer cell growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a scanning electron micrograph of the pCaBer+U0126-Her nanomessenger in an embodiment of the present invention (scale 100 nm).

[0026] Figure 2 This is a particle size distribution diagram of pCaBer+U0126-Her nano messenger in an embodiment of the present invention.

[0027] Figure 3 1 is the release curve of berbamine in different solutions of the pCaBer+U0126-Her nano messenger in the embodiment of the present invention.

[0028] Figure 4 This is the release curve of U0126 in the pCaBer+U0126-Her nano messenger in different solutions in the embodiment of the present invention.

[0029] Figure 5 This is the cell survival rate curve of 4T1-HER2 cells after being treated with various groups of nanomessengers in the example of the present invention.

[0030] Figure 6 This is a diagram of intracellular calcium ions in 4T1-HER2 cells after being treated with different groups of nanomessengers in an example of the present invention.

[0031] Figure 7 These are confocal images of intracellular autophagy / apoptosis in 4T1-HER2 cells after being treated with different groups of nanomessengers in an example of the present invention.

[0032] Figure 8 The expression of specific proteins in 4T1-HER2 cells after being treated with different groups of nanomessengers in the example of the present invention.

[0033] Fig. 9 The tumor weights of each group after being treated with different groups of nanomessengers in the examples of the present invention (*P<0.05, **P<0.01, ***P<0.001). DETAILED DESCRIPTION

[0034] The following is further described in detail through specific implementations, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0035] Example 1

[0036] A nanomessenger that regulates mitochondrial calcium instability is a signal cascade calcium nanomessenger pCaBer+U0126, which uses polydopamine (PDA) as a mineralization template. The mineralization template is loaded with calcium channel inhibitor berbamine, autophagy inhibitor U0126 and metal calcium ions. The particle size of the nanomessenger is 149±4nm.

[0037] A method for preparing a nano messenger for regulating mitochondrial calcium instability comprises the following steps:

[0038] Step 1: Dissolve 50 mg of CaCl2﹒2H2O and 10 mg of dopamine in anhydrous ethanol and mix thoroughly by magnetic stirring to obtain system I.

[0039] Step 2: Dissolve 2 mg of calcium channel inhibitor berbamine and 2 mg of autophagy inhibitor U0126 in dimethyl sulfoxide to obtain system II.

[0040] Step 3: slowly dropwise add the system II obtained in step 2 into the system I under magnetic stirring;

[0041] Step 4: Place the solution obtained in step 3 in a sealed box, and place an appropriate amount of NH4HCO3 powder around it, and react at room temperature overnight.

[0042] Step 5: Wash the solution obtained in step 4 by centrifugation with anhydrous ethanol and double distilled water, collect the precipitate, and obtain the signal cascade calcium nano-messenger pCaBer+U0126.

[0043] A nano messenger for regulating mitochondrial calcium instability is used in the preparation of anti-tumor drugs, specifically in the preparation of anti-breast cancer drugs.

[0044] Experimental Example 1 Scanning electron microscope

[0045] The nano-messenger prepared in Example 1 of the present invention was observed by scanning electron microscopy, and the results were as follows: Figure 1-2 As shown: the signal cascade calcium nano-messenger (pCaBer+U0126) is uniform, regular in shape, has an uneven surface and is spherical in shape, and the particle size is 149±4 nm.

[0046] Experimental Example 2

[0047] Experimental design and experimental method: The nanomessenger prepared in Example 1 of the present invention was resuspended and dispersed in 1 mL of PBS buffer with different pH values ​​(pH 6.5, pH 7.4 and PBS buffer containing 10% FBS), and then transferred into a pre-treated activated dialysis bag, and the bag mouth was sealed with a dialysis bag clamp, and placed in 10 mL of different buffer release media, and shaken in a constant temperature shaker at room temperature (180 rpm / min), and 0.5 mL of release medium solution was taken at different times (0 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h), and 0.5 mL of release medium was added to the release medium at the same time, keeping the volume constant. The release medium solution was centrifuged and the supernatant was taken, and the contents of berbamine and U0126 were detected by UV-visible spectrophotometer and high performance liquid chromatography, respectively, and the release amount was calculated, and the in vitro time-drug release curve under different pH conditions was drawn, and the experiment was repeated 3 times.

[0048] The experimental results are as follows Figure 3-4 As shown, within half an hour after the start of the drug release experiment, berbamine and U0126 in the nanomessenger were released rapidly at pH 6.5, with a release of about 35%, while only about 9% were released at pH 7.4 and 10% serum conditions. Until 4 hours, only about 12% were released, indicating that the nanomessenger was relatively stable at pH 7.4 and 10% serum conditions, and could be released in a pH-responsive manner under weakly acidic conditions (pH 6.5), and was pH acid sensitive.

[0049] Experimental Example 3

[0050] The experimental design and experimental methods are as follows:

[0051] (1) Experimental groups:

[0052] ①CaBer-Her calcium channel inhibitor group;

[0053] ②CaU0126-Her autophagy inhibitor group;

[0054] ③CaPDA-Her dopamine group;

[0055] ④pCaBer+U0126-Her synergistic treatment group;

[0056] (2) Breast cancer 4T1-HER2 cells were incubated with different concentrations (12.5, 25, 50, 100, 200 μg / mL) of the above-mentioned different groups of nanomessengers for 24 hours, and then the cell proliferation was detected by CCK-8 method.

[0057] The experimental results are as follows Figure 5As shown: After different groups of nanomessengers were co-incubated with 4T1-HER2 breast cancer cells for 24 hours, the cell survival rate of the pCaBer+U0126-Her group was the lowest, only 15.81±4.94%, while the cell survival rate of the CaBer-Her calcium channel inhibitor group was 31.10±2.38%, and the cell survival rates of the CaU0126-Her autophagy inhibitor group and the CaPDA-Her dopamine group were 49.47±2.35% and 64.41±3.71%, respectively, indicating that the efficacy of the synergistic treatment group was significantly better than that of other groups.

[0058] Experimental Example 4

[0059] The experimental design and experimental methods are as follows:

[0060] (1) Experimental groups:

[0061] ①PBS blank group;

[0062] ②CaBer-Her calcium channel inhibitor group;

[0063] ③CaU0126-Her autophagy inhibitor group;

[0064] ④CaPDA-Her dopamine group;

[0065] ⑤pCaBer+U0126-Her synergistic treatment group;

[0066] (2) Breast cancer 4T1-HER2 cells were digested with trypsin to form a single cell suspension, and 1×10 5 The cells were randomly divided into the above 5 groups and cultured overnight in a 37°C constant temperature incubator to allow them to fully adhere to the wall;

[0067] (3) The corresponding nanomessengers were added to the above groups of cells respectively. After incubation for 12 h, the cells were washed three times with PSB, and then Fluo-4 AM (1 μM) working solution was added to the confocal dish. The dish was placed in a cell culture incubator and incubated for 30 min. The Fluo-4 AM working solution was discarded, the cells were washed three times with sterile PBS, and DAPI staining solution was added. After staining for 10 min, the cells were washed three times with PBS, and then laser confocal microscopy was used to observe and collect the changes in intracellular calcium ions in each group of cells.

[0068] The experimental results are as follows Figure 6As shown: the intracellular Ca2+ (green fluorescence) in the pCaBer+U0126-Her group increased significantly, followed by the CaBer-Her calcium channel inhibitor group, while the intracellular calcium ions in the CaU0126-Her autophagy inhibitor group and the CaPDA-Her dopamine group were significantly weakened, indicating that berberine can effectively inhibit the outflow of intracellular calcium ions, causing intracellular calcium overload. At the same time, by combining with U0126, the pCaBer+U0126-Her signal cascade calcium nanomessenger can further exert a synergistic cytotoxic effect.

[0069] Experimental Example 5

[0070] Monodansylcadaverine (MDC) is one of the most commonly used fluorescent probes for cell autophagy detection. It can specifically label autophagosomes by capturing ions and specifically binding to membrane lipids. Propidium iodide (PI) can quickly stain necrotic cells or cells that have lost cell membrane integrity in the late stage of apoptosis, showing red fluorescence.

[0071] The experimental design and experimental methods are as follows:

[0072] (1) Experimental groups:

[0073] ①PBS blank group;

[0074] ②CaBer-Her calcium channel inhibitor group;

[0075] ③CaU0126-Her autophagy inhibitor group;

[0076] ④CaPDA-Her dopamine group;

[0077] ⑤pCaBer+U0126-Her synergistic treatment group;

[0078] (2) Breast cancer 4T1-HER2 cells were digested with trypsin to form a single cell suspension, and 1×10 5 The cells were randomly divided into the above 5 groups and cultured overnight in a 37°C constant temperature incubator to allow them to fully adhere to the wall;

[0079] (3) The corresponding nanomessengers were added to the above groups of cells respectively. After incubation for 12 h, the cells were washed three times with PSB, and then the MDP / PI staining working solution was added to the confocal dish. The dish was placed in a cell culture incubator and incubated for 30 min. The MDP / PI staining working solution was discarded, the cells were washed three times with sterile PBS, and then DAPI staining solution was added. After staining for 10 min, the cells were washed three times with PBS, and then laser confocal laser was used to observe and collect the changes in autophagy and cytotoxicity of each group of cells.

[0080] The experimental results are as follows Figure 7 As shown: the red fluorescence PI in the pCaBer+U0126-Her group was the strongest, and its green fluorescence MDC was also less than that in the CaBer-Her group and the CaPDA-Her group. Although the CaBer-Her group caused more cell apoptosis (red fluorescence), its autophagy also increased significantly. The above results indicate that the pCaBer+U0126-Her signal cascade calcium nanomessenger can effectively inhibit autophagy and cause cell apoptosis.

[0081] Experimental Example 6

[0082] The experimental design and experimental methods are as follows:

[0083] (1) Experimental groups:

[0084] ①Blank control group;

[0085] ②CaBer-Her calcium channel inhibitor group;

[0086] ③CaU0126-Her autophagy inhibitor group;

[0087] ④CaPDA-Her dopamine group;

[0088] ⑤pCaBer+U0126-Her synergistic treatment group;

[0089] (2) Breast cancer 4T1-HER2 cells were digested with trypsin to form a single cell suspension and cultured at 5×10 4 The cells were inoculated at a density of 10 cells / well in a sterile six-well plate and randomly divided into the above 5 groups, and then incubated in a cell constant temperature incubator for 24 hours;

[0090] (3) The corresponding nanomessengers were added to the above groups of cells, incubated for 24 h, and then washed three times with PSB. Then, the cell proteins of each group were extracted and Western blot experiments were performed. The antibodies SQSTM1 / P62 protein, ATG5 protein, Cytochrome c, Pro-Caspase 3 / p17 / p19, and GAPDH used in the experiment were diluted according to the antibody instructions.

[0091] The experimental results are as follows Figure 8 As shown: After treatment with the signal cascade calcium nano-messenger pCaBer+U0126-Her, the expression of intracellular SQSTM1 / P62 protein and ATG5 protein was significantly downregulated, while the expression of cytochrome c protein and Caspase 3 / p17 / p19 protein was significantly upregulated, activating cytochrome c-mediated mitochondrial apoptosis. The other groups of nano-messengers also activated cytochrome c-mediated mitochondrial apoptosis to varying degrees.

[0092] Experimental Example 7 In vivo animal experiment

[0093] Experimental design: An orthotopic 4T1-HER2 female mouse model of breast cancer was established, and the mice were then randomly divided into five groups, with five mice in each group. The experimental grouping of the five groups is as follows:

[0094] Normal saline group, CaBer-Her calcium channel inhibitor group; CaU0126-Her autophagy inhibitor group; CaPDA-Her dopamine group; pCaBer+U0126-Her synergistic treatment group.

[0095] When the tumor volume grows to 80 mm 3 At 1:100 μL of the above preparations (5 mg per mouse) were injected into the female mice through the tail vein. The drugs were administered once every 3 days for a total of 6 times. After the 21-day treatment, the tumors of the mice in each group were weighed.

[0096] The experimental results are as follows Fig. 9 As shown: the tumor volume and tumor weight of the pCaBer+U0126-Her group were the smallest, while the tumor weight of the CaBer-Her group was 0.822±0.08g, and the tumor weight of the CaU0126-Her group was 1.336±0.05g. Although there was a significant difference in tumor weight compared with the normal saline group (1.638±0.07g), the tumor volume growth and weight growth were larger than those in the synergistic treatment group, which showed that the effectiveness of single calcium overload therapy or single autophagy inhibition in treating breast cancer is limited.

[0097] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A nano-messenger for regulating mitochondrial calcium instability, characterized in that: Polydopamine is used as the mineralization template, and the template is loaded with calcium channel inhibitors, autophagy inhibitors and metal calcium ions.

2. A nano messenger for regulating mitochondrial calcium instability according to claim 1, characterized in that: The calcium channel inhibitor is berbamine.

3. A nano messenger for regulating mitochondrial calcium instability according to claim 2, characterized in that: The autophagy inhibitor is U0126.

4. A nano messenger for regulating mitochondrial calcium instability according to claim 3, characterized in that: The calcium ions are derived from CaCl2﹒2H2O.

5. A nano messenger for regulating mitochondrial calcium instability according to claim 4, characterized in that: The particle size of the nanomessenger is 149±4nm.

6. The method for preparing a nano messenger for regulating mitochondrial calcium instability according to any one of claims 1 to 5, characterized in that: The steps include: Step 1, dissolve CaCl2﹒2H2O and dopamine in anhydrous ethanol, and stir magnetically to mix thoroughly to obtain system I; Step 2: dissolving calcium channel inhibitor berbamine and autophagy inhibitor U0126 in dimethyl sulfoxide to obtain system II; Step 3: Integrate System II into System I; Step 4: Place the solution obtained in step 3 in a sealed box, and place an appropriate amount of NH4HCO3 powder around it, and react at room temperature overnight; Step 5: Wash the solution obtained in step 4 and collect the precipitate to obtain the signal cascade calcium nano-messenger pCaBer+U0126.

7. The method for preparing a nano messenger for regulating mitochondrial calcium instability according to claim 6, characterized in that: In step 1, the mass ratio of CaCl2﹒2H2O to dopamine is 5 to 10:

1.

8. The method for preparing a nano messenger for regulating mitochondrial calcium instability according to claim 7, characterized in that: In step 2, the mass ratio of calcium channel inhibitor berbamine, autophagy inhibitor U0126 and dopamine is 1:1:5-10.

9. Use of a nanosignal for regulating mitochondrial calcium instability according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

10. The use of a nanosignal for regulating mitochondrial calcium instability according to claim 9 in the preparation of an anti-tumor drug, characterized in that: The tumor is a breast cancer tumor.

Images