Bionic nanoparticle for photodynamic combined calcium overload and preparation method thereof

Abstract

The invention relates to the technical field of biological targeted delivery systems, and discloses a bionic nanoparticle for photodynamic combined calcium overload, which comprises a nanoparticle which is an indocyanine green-calcium phosphate nanoparticle; the fusion membrane is a composite membrane, and the composite membrane comprises a mitochondrial membrane and a neutrophile granulocyte membrane. The particle size of the nanoparticles prepared by a coprecipitation method is nanoscale, and the particle size of the nanoparticles coated with the two films is slightly increased. Under the wrapping of the two membranes, the ICG / CaP nanoparticles are endowed with the biological characteristics of long circulation time, targeting, good biocompatibility and the like, and the anti-tumor effect of a photodynamic therapy and the synergistic treatment effect of mitochondrial Ca < 2 + > overload are enhanced; the synergistic treatment effect not only can accurately target tumor cell mitochondria, but also can promote calcium ion release based on photodynamic force to form a cascade synergistic effect of photodynamic force and calcium overload, and provides a potential efficient strategy for synergistic treatment of photodynamic therapy and other anti-tumor strategies.

Description

Technical Field

[0001] The invention relates to the technical field of biological targeted delivery systems, and in particular to a bionic nanoparticle for photodynamic combined calcium overloading and a preparation method thereof. Background Art

[0002] Photodynamic therapy (PDT) is an emerging, non-invasive tumor treatment technology that uses specific light waves to excite photosensitizers to produce reactive oxygen species (ROS), thereby destroying the structure of tumor cells and inducing apoptosis. Compared with traditional cancer treatments (such as surgery, chemotherapy, and radiotherapy), PDT has the advantages of minimally invasive, strong local controllability, and fewer side effects during the treatment process. In addition, PDT can also be used in combination with other treatments (such as chemotherapy and targeted therapy) to further enhance the efficacy.

[0003] However, single PDT also has certain limitations. First, traditional PDT lacks efficient tumor targeting, which may cause damage to normal tissues during treatment. Secondly, tumor cell mitochondria have a higher tolerance to oxidative stress than normal cells, which makes the killing effect of PDT on tumor cells limited. Therefore, in order to improve the therapeutic effect of PDT, it is urgently needed to combine it with other treatment strategies to enhance its targeting to tumors and its destructive effect on tumor cell mitochondria.

[0004] At present, some studies have tried to use targeted delivery strategies to improve the specificity of photodynamic therapy, such as delivering photosensitizers to tumor sites through nanocarriers to enhance the selective killing of cancer cells. However, how to combine PDT with mitochondrial destruction strategies, such as calcium overload, to accurately cascade and destroy tumor cell mitochondria is a key challenge to further improve the efficacy of PDT. Summary of the invention

[0005] Technical Problems Solved by the Invention

[0006] To solve the problem of how photodynamic therapy can be combined with calcium ion overload strategy to accurately cascade and synergistically destroy tumor cell mitochondria and kill tumor cells

[0007] Technical solution adopted by the present invention

[0008] The present invention combines photodynamic therapy with calcium ion overloading strategy to achieve precise cascade synergistic destruction of tumor cell mitochondria, thereby improving the killing efficiency of tumor cells and providing a new and effective solution for the treatment of breast cancer.

[0009] This is because: mitochondrial calcium overload is a strategy that increases the calcium ion concentration in mitochondria, leading to loss of mitochondrial membrane potential, mitochondrial structural damage and cell apoptosis. Calcium ion overload can not only directly destroy mitochondrial function, but also enhance the sensitivity of mitochondria to ROS, thereby exacerbating cell apoptosis. Therefore, combining the ROS generated by PDT with mitochondrial calcium ion overload can form a double-hit effect, effectively improving the destructive effect on tumor cells.

[0010] To expand on this, it means:

[0011] First, the present invention provides a biomimetic nanoparticle for photodynamic combined calcium overloading, including nanoparticles, which are indocyanine green-calcium phosphate nanoparticles;

[0012] Fusion membrane, which is a composite membrane, the composite membrane includes mitochondrial membrane and neutrophil membrane.

[0013] Second, the present invention provides a method for preparing the aforementioned bionic nanoparticles, comprising the following steps:

[0014] S1 indocyanine green and sodium dihydrogen phosphate are mixed to obtain a system, calcium chloride is added dropwise to the system, stirring is maintained, sodium citrate is added, and the precipitate is centrifuged or dialyzed to obtain nanoparticles;

[0015] S2 extracts mitochondrial membranes and extracts neutrophil membranes;

[0016] The S3 mitochondrial membrane and neutrophil membrane were modified onto the surface of the nanoparticles by probe ultrasonic fusion to obtain bionic nanoparticles.

[0017] According to some preferred embodiments, in S2, mitochondrial membranes are extracted from mitochondria, and neutrophil membranes are extracted from bone marrow neutrophils, and the extraction method adopts density gradient centrifugation method.

[0018] According to some preferred embodiments, in S3, the mass ratio of mitochondrial membrane to neutrophil membrane is 1:1.

[0019] According to some preferred embodiments, in S1, the concentration of sodium citrate is 200-600 mM, the concentration of sodium dihydrogen phosphate is 80-150 mM, the concentration of indocyanine green is 0.5-1.5 mg / mL, and the concentration of calcium chloride is 80-150 mM.

[0020] According to some more preferred embodiments, in S1, the concentration of sodium citrate is 400 mM, the concentration of sodium dihydrogen phosphate is 120 mM, the concentration of indocyanine green is 1 mg / mL, and the concentration of calcium chloride is 100 mM.

[0021] According to some preferred embodiments, in S1, the volume ratio of sodium citrate, sodium dihydrogen phosphate, indocyanine green and calcium chloride is 3-8:1-5:0.5-2:3-8.

[0022] According to some more preferred embodiments, in S1, the volume ratio of sodium citrate, sodium dihydrogen phosphate, indocyanine green and calcium chloride is 5:2.5:1:5.

[0023] According to some preferred embodiments, in S3, the mass ratio of nanoparticles, mitochondrial membranes, and neutrophil membranes is 2:1 to 2:1 to 2. Specifically, it can be 2:2:1, 2:1:1, or 2:1:2.

[0024] Beneficial effects achieved by the present invention

[0025] (1) The fusion membrane used in the present invention includes a neutrophil membrane, which can transport drugs to the site of inflammation and treat inflammation through inflammatory factors and chemokine receptors on the membrane surface.

[0026] (2) The fusion membrane used in the present invention includes a mitochondrial membrane, and the mitochondrial membrane is used for coating. The nanomaterial coated with the mitochondrial membrane inherits the mitochondrial fusion protein of the mitochondrial membrane, and is fused with endogenous mitochondria through the mediation of the mitochondrial fusion protein, thereby targeting the mitochondria.

[0027] (3) The nanoparticles used in the present invention include indocyanine green and calcium chloride:

[0028] Indocyanine green (ICG) is a photodynamic therapy drug that is used as a fluorescent tracer in clinical practice;

[0029] Calcium phosphate (CaP) has good biocompatibility, controlled release and pH sensitivity. Calcium phosphate is stable at neutral pH and decomposes to produce Ca under acidic conditions. 2+ and PO4 3- , releasing excess Ca 2+ causing additional mitochondrial damage.

[0030] (4) The preparation method provided by the present invention is that the particle size of the nanoparticles prepared by the coprecipitation method is nanometer-level, and the particle size will be slightly increased after being coated with the two membranes. Under the wrapping of the two membranes, the ICG / CaP nanoparticles are not only endowed with biological characteristics such as long circulation time and targeting and good biocompatibility through the neutrophil membrane, but also achieve tumor mitochondrial targeting through the mitochondrial membrane, and can also promote the release of CaP calcium ions by downregulating mitochondrial pH under photodynamic therapy, thereby enhancing the anti-tumor effect of photodynamic therapy and mitochondrial Ca 2+Cascade synergistic therapeutic effect of overload; this synergistic therapeutic effect can not only cause calcium overload, but also accurately target the mitochondria of tumor cells. The PDT nanodrug delivery system provides a potential therapeutic strategy for the effective treatment of breast cancer in the clinic.

[0031] (5) The bionic nanoparticles provided by the present invention were found to have a targeting effect on cancer cells in a cell experiment on mouse breast cancer cells (4T1), and the accumulation amount in mitochondria was also higher. Furthermore, in a treatment study on an orthotopic tumor model of Babl / c Balb / c mice, it was found that the bionic nanoparticles had good tumor targeting and therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the particle size distribution diagram of nanoparticles;

[0033] Figure 2 This is the result of the Zeta potential measurement of the nanoparticles;

[0034] Figure 3 The graph is the change result of the particle size of nanoparticles and the polymer dispersibility index;

[0035] Figure 4 TEM image of ICG / CaP NPs;

[0036] Figure 5 TEM image of MN ICG / CaP;

[0037] Figure 6 The Coomassie Brilliant Blue staining results of Mm, NEM and MN ICG / CaP NPs;

[0038] Figure 7 This is the result of in vivo imaging observation of drug distribution (in vivo 2 hours after drug administration, the red dotted line is the tumor site);

[0039] Figure 8 This is the result of in vivo imaging observation of drug distribution (in vivo after 4 hours of drug administration, the red dotted line is the tumor site);

[0040] Fig. 9 This is the result of in vivo imaging observation of drug distribution (in vivo 8 hours after drug administration, the red dotted line is the tumor site);

[0041] Fig.10 This is a fluorescent imaging image of mouse organs;

[0042] Fig.11 This is the result diagram of quantitative analysis of drug fluorescence intensity in mouse tumor tissue;

[0043] Fig.12This is the result of CCK-8 detection of 4T1 cell survival rate (ICG / CaP NPs);

[0044] Fig.13 This is the result of CCK-8 detection of 4T1 cell survival rate (MN ICG / CaP NPs);

[0045] Fig.14 This is a graph showing the changes in tumor volume in mice treated with nanoparticles;

[0046] Fig.15 This is a photo of the actual tumor condition of breast cancer-bearing mice after MN ICG / CaP NPs combined with laser treatment;

[0047] Fig.16 This is the result diagram of ATP content in tumor tissue after MN ICG / CaP NPs combined with photodynamic therapy;

[0048] Fig.17 This is the result of mitochondrial membrane potential of tumor tissue after MN ICG / CaP NPs combined with photodynamic therapy;

[0049] Fig.18 This is the calcium concentration result of tumor tissue after MN ICG / CaP NPs combined with photodynamic therapy. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0051] Example 1

[0052] First, prepare 100mM calcium chloride solution, 400mM sodium citrate solution, 100mM sodium dihydrogen phosphate solution and 1mg / mL indocyanine green solution. Measure 5mL of calcium chloride solution and stir in a beaker, take 1mL of indocyanine green solution and slowly drop it into 2.5mL of disodium hydrogen phosphate, then slowly drop the disodium hydrogen phosphate solution containing ICG into the calcium chloride solution to form a precipitate, add 5mL of sodium citrate after 5min of reaction, stir for another 5min, and then centrifuge or dialyze the reaction solution at 20000g to obtain composite nanoparticles (centrifugation or dialysis can be performed by methods commonly used in the art). Then mix the obtained composite nanoparticles with mitochondrial membranes and neutrophil membranes (the mass ratio of nanoparticles: mitochondrial membranes: neutrophil is 2:2:1) and ultrasonicate to obtain MN ICG / CaP nanoparticles.

[0053] Example 2

[0054] The difference between this embodiment and embodiment 1 is that the mass ratio of nanoparticles: mitochondrial membranes: neutrophils is 2:1:1.

[0055] Example 3

[0056] The difference between this embodiment and embodiment 1 is that the mass ratio of nanoparticles: mitochondrial membrane: neutrophils is 2:1:2.

[0057] Test example

[0058] The nanoparticles prepared in Example 2 are the best experimental solution, and the experiments were carried out using the nanoparticles prepared therefrom.

[0059] 1. Preparation and characterization of MN ICG / CaP nanoparticles:

[0060] The ICG / CaP NPs and MN ICG / CaP NPs were characterized by particle size, potential, transmission electron microscopy and Coomassie or WB to prove the successful preparation of MN ICG / CaP nanoparticles.

[0061] in,

[0062] (1) Particle size distribution measurement: Use a laser particle size analyzer to measure the particle size distribution of nanoparticles. Take 200 μL of the prepared nanoparticles and add them to a four-way cuvette. Then add water to dilute it to 2 / 3 of the volume of the cuvette and carefully place it in the sample pool for measurement. Repeat the measurement 3 times for each sample. The results are as follows: Figure 1 shown.

[0063] (2) Zeta potential measurement: 1.5 mL of the prepared nanoparticle solution was added to the sample cell, and the potential electrode was inserted. The surface Zeta potential of the nanoparticles was measured using a laser particle size analyzer. The results are shown in Figure 2. Figure 2 shown.

[0064] Depend on Figure 1 and Figure 2 It can be seen that:

[0065] Neutrophil membrane and mitochondrial membrane modified nanoparticles (MNICG / CaP NPs) with ICG / CaP nanoparticles as the core were prepared. Particle size measurement showed that the particle size of ICG / CaP NPs was 197.9±2.34nm and the zeta potential was -31.4±4.69mV; the particle size of MN ICG / CaP NPs (dialysis method) was 275.5±7.34nm and the zeta potential was -21.6±5.87mV; the particle size of MNICG / CaP NPs (centrifugation method) was 222.2±1.57nm and the zeta potential was -16.8±3.92mV. The encapsulation of cell membranes changed the particle size and zeta potential of the nanoparticles, resulting in an increase in the particle size of the nanoparticles and a decrease in the surface charge.

[0066] (3) In vitro stability

[0067] The prepared ICG / CaP NPs (centrifugation method) and MM ICG / CaP NPs (dialysis method) were placed in a 4°C environment and the changes in particle size and polymer dispersity index (PDI) were monitored every day for one week. Figure 3 shown.

[0068] Depend on Figure 3 It can be seen that:

[0069] The results showed that the particle size of MN ICG / CaP NPs (centrifugation method) varied within a small range, with the particle size remaining at around 220-250 nm, the PDI at around 0.2, the particle size being uniform, and the dispersion being good. Because the particle size of MN ICG / CaP NPs prepared by the dialysis method was relatively large and the stability was poor, the centrifugation method was used to obtain MN ICG / CaP NPs in the subsequent preparation.

[0070] (4) Transmission electron microscopy observation of nanoparticle morphology

[0071] The morphology of nanoparticles was studied by TEM. After the nanoparticles were purified and diluted 10 times, 20 μL was added to the copper mesh of the transmission electron microscope. After 1 hour, the sample was carefully dried from one side of the copper mesh with filter paper. After 3 seconds of staining with phosphotungstic acid, the sample was scanned by transmission electron microscope. The sample was magnified to a certain magnification, the voltage was 80 kV, and the photos were taken and recorded. The results are shown in the figure below. Figure 4 and Figure 5 shown.

[0072] Depend on Figure 4 and Figure 5 It can be seen that:

[0073] ICG / CaP nanoparticles are spherical particles with relatively uniform morphology under transmission electron microscopy and are consistent with the particle size measured by the particle size analyzer. Transmission electron microscopy (TEM) images show that after being wrapped by mitochondrial membranes and neutrophil membranes, ICG / CaP nanoparticles are surrounded by light gray circles. After being wrapped by the two membranes, the particle size will also increase appropriately, which is consistent with the results measured by the particle size analyzer above.

[0074] (5) Coomassie Brilliant Blue Staining

[0075] The specific preparation is as follows:

[0076] 1) Prepare the sample: Take the prepared cell membrane to measure the protein concentration, take a portion and add 5X loading buffer at a ratio of 4:1, mix thoroughly and centrifuge, boil at 100℃ for 10min, cool and centrifuge, and store at -80℃ for later use;

[0077] 2) Prepare SDS-PAGE gel: prepare 10% gel according to the gel preparation instructions;

[0078] 3) Prepare electrophoresis buffer: Prepare 1000 ml of electrophoresis buffer according to the instructions;

[0079] 4) Sample loading: The amount of protein loaded is 30 μg, and the sample is loaded using a sample injection needle;

[0080] 5) Electrophoresis: Set the initial voltage of electrophoresis to 80V. After the red protein band of Protein Maker appears, increase the voltage to 120V.

[0081] 6) Staining: Cut off the concentrated gel part of the gel, put the separation gel part into a staining box with an appropriate amount of Coomassie staining solution, and then place it on a horizontal shaker and shake it slowly at the minimum speed. Generally, staining at room temperature for 1 hour is sufficient;

[0082] 7) Decolorization. Usually, the staining is stopped when the color of the gel is close to that of the staining solution. The gel is decolorized and washed with a decolorizing solution at room temperature for 4-24 hours, during which the decolorizing solution is replaced 2-4 times.

[0083] 8) After destaining, soak in ultrapure water and finally perform gel imaging.

[0084] The results are as follows Figure 6 shown.

[0085] Depend on Figure 6 It can be seen that:

[0086] MN ICG / CaP NPs inherited the major proteins of mitochondrial membrane and neutrophil membrane, demonstrating the successful encapsulation of both membranes.

[0087] 2. In vivo targeting study of MN ICG / CaP NPs

[0088] In vivo imaging was used to study the pharmacokinetic properties of drugs. Fluorescence calibration was first performed on mice in the blank control group to ensure that mice that were not injected with drugs had no fluorescence. Breast cancer-bearing mice were divided into two groups: ICG / CaP NPs and MN ICG / CaP NPs. Each group of tumor-bearing mice was injected with 100 μL ICG / CaP NPs and MN ICG / CaP NPs (both at a dose of 2 mg / kg) through the tail vein. In vivo distribution of the drug was observed using in vivo imaging at fixed points at 2, 4, and 8 hours. The results are shown in Figure 2. Figure 7 (Administering for 2 hours), Figure 8 (administered for 4 hours), Fig. 9 (8h of administration) as shown.

[0089] Depend on Figure 7-Figure 9 It can be seen that:

[0090] In vivo imaging was used to observe the drug distribution in vivo at specific time points, and the results showed that MN ICG / CaP NPs were concentrated in the tumor site.

[0091] 3. In vitro targeting study of mouse organs

[0092] The mice were killed and their organs were taken out, and the fluorescence intensity of each organ was photographed by live imaging to observe its fluorescence distribution. The fluorescence images were quantitatively analyzed using ZEN software.

[0093] The results are as follows Figure 10-11 shown.

[0094] Depend on Figure 10-11 It can be seen that:

[0095] MN ICG / CaP NPs targeted to the tumor site at a higher content than ICG / CaP NPs.

[0096] 4. In vitro functional study of MN ICG / CaP NPs

[0097] CCK-8 detects 4T1 cell survival rate (killing effect on 4t1 cells)

[0098] 1) Cell plate: Take cells in logarithmic phase and plate them at 5×10 3 The cells were inoculated in a 96-well plate and allowed to adhere overnight for 12 hours. An appropriate amount of PBS was added around the plated cells to prevent errors caused by evaporation.

[0099] 2) Adding drugs to cells: When the cells grow to an appropriate density, carefully wash them twice with PBS to avoid washing away the cells. Add complete culture medium containing ICG / CaP NPs and MN ICG / CaP NPs.

[0100] 3) Add CCK8 working solution: After 24 hours of drug treatment, remove the culture medium and carefully wash once with PBS. The laser group was irradiated with laser (808nm, 1W / cm 2 ) for 5 minutes. After irradiation, CCK8 detection reagent and culture medium were prepared into CCK8 detection working solution in a ratio of 1:10. 110 μL detection solution was added to each well and incubated for 1-3 hours.

[0101] 4) Measure the absorbance of each well at 450 nm using a multifunctional microplate reader.

[0102] The results are as follows Figure 12-13 shown.

[0103] Depend on Figure 12-13 It can be seen that:

[0104] MN ICG / CaP NPs were highly cytotoxic under laser irradiation.

[0105] 5. Evaluation of the efficacy of combined PDT and MN ICG / CaP NPs therapy

[0106] Establishment of breast cancer tumor-bearing mouse model

[0107] 6-8 week old BALB / c female mice were anesthetized and 1×106 4T1 cells were subcutaneously injected into the fourth pair of mammary glands on the right side to establish a breast cancer tumor-bearing mouse model. Before the injection, the hair near the nipple was removed with a depilatory cream.

[0108] Treatment options

[0109] Starting from the 11th day after the injection of tumor cells, the corresponding drugs were given through the tail vein for treatment. The treatment cycle was once every other day. 4 hours after the drug administration, the tumor was irradiated with laser for 5 minutes (808nm, 1W / cm 2 ) for 21 days. On the 22nd day, all mice were euthanized for sampling and related tests.

[0110] Animal experiment group

[0111] Model:

[0112] ①Normal saline + Laser

[0113] ②ICG / CaP NPs+Laser

[0114] ③MN ICG / CaP NPs

[0115] ④MN ICG / CaP NPs+Laser

[0116] Record the weight and tumor volume of mice: During this period, the volume and tumor volume of mice were recorded every 2 days until the end of the anti-tumor study. The tumor volume was calculated using the formula: V = (L×W×W) / 2 (L: the maximum diameter of the tumor, W: the shortest diameter perpendicular to the length).

[0117] The results are as follows Figure 14-15 shown.

[0118] Depend on Fig.14 It can be seen that:

[0119] In mice after different treatments, MN ICG / CaP NPs combined with photodynamic therapy can significantly inhibit tumor growth.

[0120] Depend on Fig.15 It can be seen that:

[0121] It can be clearly observed that the growth of the originally protruding tumor was inhibited after MN ICG / CaP NPs combined with photodynamic therapy.

[0122] 6. ATP level detection

[0123] (1) Sample preparation: A portion of the tumor tissue was lysed on ice with an appropriate amount of ATP lysis buffer. After sufficient lysis, the tissue was centrifuged at 4°C, 12,000 g for 5 minutes.

[0124] (2) Prepare standard curve solution: dilute ATP standard solution with ATP lysis buffer to different concentrations (0.01, 0.03, 0.1, 0.3, 1, 3, 10 μM) and place on ice for later use.

[0125] (3) Prepare ATP detection working solution: dilute ATP detection reagent at a ratio of 1:4 using ATP detection diluent, mix thoroughly, and place on ice away from light until ready for use.

[0126] (4) Take an opaque 96-well plate, add 100 μL of ATP detection working solution to each well, and leave at room temperature for 5 minutes.

[0127] (5) Add 20 μL of standard or sample to each well, gently shake the plate to mix, and detect the PLU value using the chemiluminescence method of a multifunctional microplate reader.

[0128] (6) The protein concentration of each sample was measured using the BCA method.

[0129] The results are as follows Fig.16 shown.

[0130] Depend on Fig.16 The results show that:

[0131] After MN ICG / CaP NPs combined with photodynamic therapy, the ATP content in tumor tissue was significantly reduced. And under the same laser irradiation conditions, the ATP content of the MN ICG / CaP NPs group was less than that of ICG / CaP NPs.

[0132] 7. Detection of mitochondrial membrane potential (JC-1) in tumor tissue

[0133] (1) Take fresh tumor tissue and wash it three times in an EP tube filled with pre-cooled PBS.

[0134] (2) Mince the tissue in PBS and centrifuge at 4°C, 500 g for 3 min to precipitate the minced tissue.

[0135] (3) Add an appropriate amount of trypsin digestion solution and digest at 37°C for 30 min. Vortex every 5 min to ensure complete digestion.

[0136] (4) Terminate the digestion with pre-cooled PBS and gently press the tissue suspension through a universal 70 μm cell strainer.

[0137] (5) After sieving, centrifuge at 4°C, 500 g for 10 min to collect the cells.

[0138] (6) Wash the cells twice with PBS, centrifuge at 4°C, 500 g for 10 min, and collect the washed cells.

[0139] (1) Prepare JC-1 staining working solution: dilute the JC-1 stock solution with staining buffer at a ratio of 1:200 for later use.

[0140] (3) Add an appropriate amount of staining working solution to each group of samples, mix thoroughly, and stain at 37°C for 30 minutes.

[0141] (4) Add the stained samples to an opaque 96-well plate.

[0142] (5) Detect the fluorescence intensity using a multifunctional microplate reader.

[0143] The results are as follows Fig.17 and as shown.

[0144] 8. Detection of calcium content in tumor tissue

[0145] (1) Preparation of tissue samples: (a) Cut the tissue into small pieces. (b) Dissolve the sample lysis buffer and mix well. (c) Add 100-200 μl of sample lysis buffer to every 20 mg of tissue. (d) After sufficient lysis, centrifuge at 10,000-14,000 g for 3-5 minutes at 4°C, take the supernatant, and place on ice for testing.

[0146] (2) Preparation of the kit: a. The detection buffer and the color developing solution should be equilibrated to room temperature before use. b. Preparation of standard products Preparation of calcium standard solution: Take 10 μl of 500 mM calcium standard, add 990 μl of deionized water, and mix thoroughly to obtain a 5 mM calcium standard solution. Dilute the calcium standard to prepare 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mM concentration standards.

[0147] (3) Drawing of standard curve and sample determination: b. Add 50 μl of standard or sample to each well of a 96-well plate. The sample can also be any volume of 2-50 μl and be made up to 50 μl with deionized water. The sample volume is recorded as V. c. Add 150 μl of detection working solution to each well and mix well. d. Incubate at room temperature in the dark for 5 minutes. e. Measure the absorbance at 575 nm with an ELISA reader to make a standard curve.

[0148] (4) Calculation of calcium content in the sample: a. Calculate the average absorbance of each concentration group in the standard group, subtract the absorbance of the blank control group, and the absorbance of each concentration standard is obtained. b. Draw a standard curve with the calcium ion amount of the calcium standard as the horizontal axis and the absorbance as the vertical axis. c. Calculate the calcium content in the test well based on the standard curve. d. Calculate the calcium concentration in the sample according to the formula.

[0149] The results are as follows Fig.18 shown.

[0150] Depend on Fig.17 and Fig.18 It can be seen that:

[0151] The decrease of mitochondrial membrane potential is a landmark event in the early stage of apoptosis. After MN ICG / CaP NPs combined with photodynamic therapy, the mitochondrial membrane potential in tumor tissue decreased significantly. And under the same laser irradiation conditions, the membrane potential of the MN ICG / CaP NPs group decreased much more than that of ICG / CaP NPs. MN iCa NPs combined with photodynamic therapy significantly increased the calcium content in tumor tissue and was the best group among the four groups.

Claims

1. A biomimetic nanoparticle for photodynamic therapy combined with calcium overloading, characterized in that: include Nanoparticles, which are indocyanine green-calcium phosphate nanoparticles; Fusion membrane, which is a composite membrane, the composite membrane includes mitochondrial membrane and neutrophil membrane.

2. A method for preparing biomimetic nanoparticles for photodynamic combined calcium overloading as claimed in claim 1, characterized in that: The steps include: S1 indocyanine green and sodium dihydrogen phosphate are mixed to obtain a system, calcium chloride is added dropwise to the system, stirring is maintained, sodium citrate is added, and the precipitate is centrifuged or dialyzed to obtain nanoparticles; S2 extracts mitochondrial membranes and extracts neutrophil membranes; The S3 mitochondrial membrane and the neutrophil membrane were modified onto the surface of the nanoparticles by ultrasonic fusion of the probe to obtain bionic nanoparticles.

3. The method for preparing biomimetic nanoparticles for photodynamic combined calcium overloading according to claim 2, characterized in that: In S2, mitochondrial membranes were extracted from mitochondria, and neutrophil membranes were extracted from bone marrow neutrophils by density gradient centrifugation.

4. The method for preparing biomimetic nanoparticles for photodynamic combined calcium overloading according to claim 2, characterized in that: In S3, the mass ratio of mitochondrial membrane to neutrophil membrane was 1:

1.

5. The method for preparing biomimetic nanoparticles for photodynamic combined calcium overloading according to claim 2, characterized in that: In S1, the concentration of sodium citrate is 200 to 600 mM, the concentration of sodium dihydrogen phosphate is 80 to 150 mM, the concentration of indocyanine green is 0.5 to 1.5 mg / mL, and the concentration of calcium chloride is 80 to 150 mM.

6. The method for preparing biomimetic nanoparticles for photodynamic combined calcium overloading according to claim 5, characterized in that: In S1, the concentration of sodium citrate was 400 mM, the concentration of sodium dihydrogen phosphate was 120 mM, the concentration of indocyanine green was 1 mg / mL, and the concentration of calcium chloride was 100 mM.

7. The method for preparing biomimetic nanoparticles for photodynamic combined calcium overloading according to claim 2, characterized in that: In S1, the volume ratio of sodium citrate, sodium dihydrogen phosphate, indocyanine green and calcium chloride is 3-8:1-5:0.5-2:3-8.

8. The method for preparing biomimetic nanoparticles for photodynamic combined calcium overloading according to claim 7, characterized in that: In S1, the volume ratio of sodium citrate, sodium dihydrogen phosphate, indocyanine green, and calcium chloride is 5:2.5:1:

5.

9. The method for preparing biomimetic nanoparticles for photodynamic combined calcium overloading according to any one of claims 2 to 8, characterized in that: In S3, the mass ratio of nanoparticles, mitochondrial membranes, and neutrophil membranes is 2:1-2:1-2.

10. A biomimetic nanoparticle for photodynamic combined with calcium overloading obtained by the preparation method according to any one of claims 2 to 9.

Images