Plant-derived exosome-encapsulated drug-loaded calcium-based nanogenerator, preparation method thereof and anti-tumor application thereof

Abstract

The present application relates to a kind of plant source exosome-encapsulated drug-loaded calcium-based nanogenerator and preparation method and its application, the nanogenerator includes drug-loaded core, the drug-loaded core outside is coated with plant source exosome shell layer;Wherein, the plant source exosome shell layer is selected from one or more of curcuma source exosome, broccoli source exosome, artemisia annua source exosome, camellia source exosome.This nanogenerator of the present application has the ability of enhancing calcium overload and amplifying cell oxidative stress, the generator can improve the stability and biocompatibility of CaO2 While, overcome tumor cell antioxidant defense mechanism.By curcuma source exosome as protective layer wrapped outside CaO2, solve the problem of CaO2 In vivo blood circulation stability and dispersibility, increase the efficiency of the nanogenerator into tumor cell.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a drug-loaded calcium-based nanogenerator encapsulated by plant-derived exosomes, a preparation method thereof, and anti-tumor applications. Background Art

[0002] Cancer is a serious public health concern. According to the "2020 World Cancer Report" released by the World Health Organization and the International Agency for Research on Cancer, 19.29 million new cancer cases and 9.96 million cancer deaths were reported worldwide in 2020. In 2020, my country accounted for 4.57 million new cancer cases, accounting for 23.7% of the global total, and 3 million cancer deaths, approximately 30% of the global total. my country leads the world in both new cancer cases and deaths. The burden of malignant tumors in my country is increasing, necessitating breakthroughs in cancer treatment. Currently, clinical treatments for cancer primarily include surgery, radiotherapy, and chemotherapy. However, surgery carries a high risk of cancer recurrence; radiotherapy lacks specificity and selectivity for tumor cells and normal cells, leading to various adverse reactions in patients; and chemotherapeutic drugs suffer from poor solubility, insufficient tumor accumulation, and significant side effects. Therefore, the development of new and effective cancer treatment technologies is crucial.

[0003] Calcium peroxide nanoparticles (CaO2) are a commonly used calcium ion nanogenerator that can degrade in response to the weak acid environment of tumors, thereby releasing Ca 2+ CaO2 releases hydrogen peroxide (H2O2), which can increase the sensitivity of tumor cells to calcium overload and deplete intracellular glutathione, leading to more effective tumor cell killing. Under neutral pH conditions, the H2O2 produced by the slow decomposition of CaO2 is rapidly decomposed into oxygen and water by catalase, thus preventing it from inducing oxidative stress in normal cells. Furthermore, studies have reported that CaO2 has a high surface area and mesoporous structure, making it a promising candidate for drug loading. Consequently, CaO2 has been widely used as a calcium-based nanoparticle in anti-tumor research. However, prolonged exposure to aqueous solutions slowly degrades CaO2, and administration via the tail vein into the bloodstream can lead to increased blood calcium concentrations. Furthermore, CaO2 has poor particle size dispersion, making it difficult to ensure sufficient accumulation in tumor sites. Furthermore, the positively charged surface of CaO2 can induce hemolysis, inevitably causing damage to other organs. Therefore, improving the in vivo circulation stability of CaO2 is a key issue that must be addressed for its effective tumor treatment.

[0004] While existing calcium peroxide-based nanoreactors can be used to induce calcium overload in tumor cells, thereby exerting anti-tumor effects, they suffer from poor in vivo stability and slow degradation upon long-term exposure to water, potentially leading to hemolysis and other issues that hinder their further application. While CaO2's stability can be improved by further modifying its surface with materials to isolate it from the solution, these modified nanoreactors all combine Fenton reaction, photodynamics, or chemokinetics with calcium overload to produce higher concentrations of ROS for therapeutic purposes. However, excessive ROS poses the risk of damaging surrounding normal tissues. Furthermore, as ROS levels increase, tumor cells develop antioxidant defense mechanisms to adapt to high levels of ROS, rendering them resistant to ROS and unable to undergo apoptosis. This inherent tumor cell DNA damage repair mechanism can significantly reduce the efficacy of developed calcium-based nanoparticles in tumor treatment, necessitating the development of a method that can effectively treat tumors without harming tissues. Summary of the Invention

[0005] To solve the above problems, the present invention prepares the nanogenerator by loading the target drug in the drug-loaded core and then using plant-derived exosomes to encapsulate the drug-loaded core and the target drug. It is found that the nanogenerator can respond to degradation in a weak acid environment and produce a large amount of Ca in tumor cells. 2+ The release of the target drug, combined with H₂O₂, synergizes with CUR to induce mitochondrial calcium overload and ROS oxidative stress. Furthermore, the released target drug can overcome ROS damage tolerance within tumor cells, enhancing the lethality of ROS against tumor cells and achieving a synergistic calcium overload-induced tumor cell killing effect. Animal experiments demonstrated that encapsulation with plant-derived exosomes significantly reduced the hemolytic response of the drug-loaded calcium-based nanogenerator. The nanogenerator also demonstrated its ability to effectively inhibit tumor growth and improve mouse survival. This plant-derived exosome-encapsulated drug-loaded calcium-based nanogenerator is expected to serve as an effective anti-tumor nanoformulation that enhances calcium overload to amplify oxidative stress.

[0006] One object of the present invention is to provide a drug-loaded calcium-based nanogenerator wrapped with plant-derived exosomes, wherein the nanogenerator comprises a drug-loaded core, the mesopores of the drug-loaded core are loaded with target drugs, and the outer side of the drug-loaded core is coated with a plant-derived exosome shell;

[0007] The plant-derived exosome shell is selected from one or more of turmeric-derived exosomes (TDNPs), broccoli-derived exosomes, Artemisia annua-derived exosomes, and camellia-derived exosomes.

[0008] Preferably, the plant-derived exosome shell is turmeric-derived exosomes.

[0009] Specifically, curcumin (CUR) is an anti-tumor active substance. CUR can promote Ca 2+ Release from the endoplasmic reticulum to the cytoplasm and inhibition of Ca 2+ Increased mitochondrial Ca2+ released from the cytoplasm to the extracellular space 2+ Concentrations of CUR can synergistically induce mitochondrial calcium overload in calcium-based nanomaterials, leading to mitochondrial dysfunction, caspase-3 upregulation, and subsequent apoptosis. Calcium-based nanoparticles loaded with CUR achieve a synergistic calcium overload effect, but CUR's poor water solubility limits its bioavailability. The plant-derived exosomes extracted from edible turmeric in this invention inherently contain CUR. Similar in structure to animal-derived exosomes, their negatively charged surface structure, containing a phospholipid bilayer, can serve as a delivery vehicle for encapsulating drugs or nanoparticles. Compared to liposomes, these exosomes offer improved stability, biocompatibility, and low immunogenicity, enabling high cellular uptake through plasma membrane fusion. Compared to animal-derived exosomes, they are readily available, can be prepared in large quantities, and have excellent water solubility. More importantly, these CUR-encapsulated turmeric exosomes can also improve CUR's water solubility. Based on these properties, turmeric-derived exosomes are promising new delivery vehicles for combined use with calcium-based nanogenerators in tumor treatment.

[0010] Furthermore, the drug-loaded core is selected from one or more of calcium peroxide, calcium carbonate, and calcium fluoride.

[0011] Furthermore, the target drug is selected from one or more of TH588, TH287, (s)-Crizotinib, 3-lsomangostin, and MTH1-IN-2.

[0012] Furthermore, the particle size of the nanogenerator is 90-150 nm.

[0013] Furthermore, the mass ratio of the drug-loaded core, the target drug and the plant-derived exosome shell is 1:0.07-0.09:1.1-1.3.

[0014] In particular, this calcium-based nanogenerator delivers targeted drugs directly into tumor cells while ensuring stability and safety, resolving the ROS tolerance issue inherent in existing calcium-based nanomaterials and significantly enhancing the lethality of ROS against tumor cells. Finally, we experimentally demonstrated that this nanogenerator exhibits excellent anti-tumor therapeutic efficacy both in vitro and in vivo, with high in vivo biosafety. Therefore, this drug-loaded calcium-based nanogenerator encapsulated by turmeric-derived exosomes offers a promising approach for tumor treatment.

[0015] The present invention also provides a method for preparing the drug-loaded calcium-based nanogenerator wrapped with the plant-derived exosomes, comprising the following steps: encapsulating the drug-loaded core with the plant-derived exosome shell to obtain a nanogenerator.

[0016] Furthermore, the coating time is 25-30 minutes.

[0017] The present invention also provides the use of the plant-derived exosome-encapsulated drug-loaded calcium-based nanogenerator in the preparation of an anti-tumor drug delivery agent.

[0018] The present invention also provides the application of the plant-derived exosome-encapsulated drug-loaded calcium-based nanogenerator in synergistic anti-tumor effects.

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

[0020] 1. The nanogenerator of the present invention has the ability to enhance calcium overload and amplify cellular oxidative stress. The generator can overcome the antioxidant defense mechanism of tumor cells while improving the stability and biocompatibility of the drug-loaded core. By using plant-derived exosomes wrapped around the drug-loaded core as a protective layer, the problems of poor blood circulation stability and dispersibility of the nanogenerator in the body are solved, and the efficiency of the nanogenerator entering tumor cells is increased. At the same time, under the premise of ensuring stability and safety, the plant-derived exosomes deliver the drug-loaded core, target drugs and the plant-derived exosomes themselves to the tumor cells at the same time, overcoming the challenge of tumor cell DNA damage repair without generating excessive ROS, and ultimately achieving the therapeutic effect of efficiently killing tumor cells.

[0021] 2. The present invention designs a drug-loaded calcium-based nanogenerator encapsulated by plant-derived exosomes, which can be used for anti-tumor treatment without damaging body tissue. This technical method is simple to prepare, uses inexpensive raw materials, and is relatively low in cost. By using plant-derived exosomes as a shell, the solubility of the anti-tumor active substances contained therein can be improved, which not only helps to increase their bioavailability in the body, but also synergizes with the drug-loaded core to induce calcium overload in tumor cells, providing an ingenious approach for the application of plant-derived exosomes in tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 (a) is a transmission electron microscopy image of CaO2 nanoparticles;

[0023] Figure 1 (b) Transmission electron microscopy image of TDNPs nanoparticles;

[0024] Figure 1 (c) Transmission electron microscopy image of TDNPs@CaO2 nanoparticles, the black arrow represents the exosome membrane structure;

[0025] Figure 1 (d) SDS-PAGE gel electrophoresis of CaO2 nanoparticles, TDNPs nanoparticles and TDNPs@TH588@CaO2 nanogenerator;

[0026] Figure 1 (e) Element distribution diagram of TDNPs@TH588@CaO2 nanogenerator;

[0027] Figure 1 (f) Fluorescence co-localization imaging of CaO2 and TDNPs in the TDNPs@CaO2 nanoparticle system;

[0028] Figure 1 (g) is the nitrogen adsorption-desorption isotherm of CaO2 nanoparticles;

[0029] Figure 1 (h) Particle size distribution diagram of CaO2 nanoparticles and TDNPs@CaO2 nanogenerator;

[0030] Figure 1 (i) is the pore size distribution diagram of CaO2 nanoparticles;

[0031] Figure 1 (j) Zeta potential diagrams of TDNPs nanoparticles, CaO2 nanoparticles, CUR@CaO2 nanoparticles, TDNPs@CaO2 nanoparticles, and TDNPs@TH588@CaO2 nanogenerators;

[0032] Figure 1 (k) Hydrated particle size measurement results of TDNPs nanoparticles, CaO2 nanoparticles, CUR@CaO2 nanoparticles, TDNPs@CaO2 nanoparticles, and TDNPs@TH588@CaO2 nanogenerators (n=3, data are expressed as mean ± standard deviation);

[0033] Figure 1 (l) is the dispersion of CUR nanoparticles and TDNPs nanoparticles in aqueous solution;

[0034] Figure 2 (a) Transmission electron microscopy images of TDNPs@CaO2 nanoparticles incubated in pH 7.4 and pH 6.0 phosphate buffer for different times (0 h and 12 h).

[0035] Figure 2 (b) Calcium ion release curves of CaO2 nanoparticles and TDNPs@CaO2 nanoparticles after incubation in phosphate buffer at pH 7.4 and pH 6.0 for different times;

[0036] Figure 2(c) H2O2 generation curves of CaO2 nanoparticles and TDNPs@CaO2 nanoparticles after incubation in phosphate buffer at pH 7.4 and pH 6.0 for different times;

[0037] Figure 2 (d) Drug release curves of TDNPs@TH588@CaO2 nanogenerators incubated in phosphate buffer at pH 7.4 and pH 6.0 for different time periods;

[0038] Figure 3 (a) Quantitative analysis of the fluorescence intensity of cellular endocytosis of CaO2 nanoparticles and TDNPs@CaO2 nanoparticles after incubation with 4T1 tumor cells for different times;

[0039] Figure 3 (b) Cell activity of TH588, TDNPs nanoparticles, CaO2 nanoparticles, TDNPs@CaO2 nanoparticles, and TDNPs@TH588@CaO2 nanogenerators after incubation with 4T1 tumor cells for 24 h;

[0040] Figure 3 (c) The intracellular calcium ion content of TDNPs nanoparticles, CaO2 nanoparticles, and TDNPs@CaO2 nanoparticles after incubation with 4T1 tumor cells for 8 h;

[0041] Figure 3 (d) Confocal imaging of TH588, TDNPs nanoparticles, CaO2 nanoparticles, TDNPs@CaO2 nanoparticles, and TDNPs@TH588@CaO2 nanogenerators stained with ROS probes after incubation with 4T1 tumor cells for 8 h;

[0042] Figure 3 (e) Confocal imaging of TH588, TDNPs nanoparticles, CaO2 nanoparticles, TDNPs@CaO2 nanoparticles, and TDNPs@TH588@CaO2 nanogenerators stained with JC-1 probe after incubation with 4T1 tumor cells for 8 h;

[0043] Figure 3 (f) Cell apoptosis after TH588, TDNPs nanoparticles, CaO2 nanoparticles, TDNPs@CaO2 nanoparticles, and TDNPs@TH588@CaO2 nanogenerators were incubated with 4T1 tumor cells for 24 h;

[0044] Figure 4 (a) Schematic diagram of the experimental scheme for in vivo anti-tumor studies;

[0045] Figure 4 (b) Tumor growth curves of BALB / c mice after injection of nanoparticles in each group via tail vein;

[0046] Figure 4 (c) Images of tumors dissected from BALB / c mice 18 days after tail vein injection of nanoparticles in each group;

[0047] Figure 4 (d) is a statistical graph showing the average tumor weight of BALB / c mice;

[0048] Figure 4 (e) is a statistical graph showing the average spleen weight of BALB / c mice;

[0049] Figure 4 (f) H&E, TUNEL, Ki67, ROS, and MTH1 staining images of tumor sections of BALB / c mice injected with nanoparticles in each group via tail vein for 18 days;

[0050] Figure 4 (g) is a statistical graph showing the survival rates of BALB / c mice after tail vein injection of nanoparticles in each group;

[0051] Figure 5 (a) H&E staining images of the heart, liver, spleen, lung, and kidney of BALB / c mice after 18 days of tail vein injection of nanoparticles in each group.

[0052] Figure 5 (b)-(f) are the analysis of serum ALT, ALP, AST, BUN, and CREA concentrations;

[0053] Figure 5 (g) Monitoring of mouse body weight after administration;

[0054] Figure 5 (h) Hemolysis analysis of CaO2 nanoparticles, TDNPs@CaO2 nanoparticles and red blood cells after incubation for 1 h in vitro;

[0055] Figure 6 Schematic diagram of the preparation and treatment mechanism of the nanogenerator of the present invention;

[0056] Figure 7 is the standard curve of TH588 in methanol solution;

[0057] Figure 8 is the standard curve of TH588 in PBS solution containing 1.5 wt % Tween 80 and 5 wt % ethanol;

[0058] Figure 9 This is the standard curve of turmeric-derived exosomes in PBS. DETAILED DESCRIPTION

[0059] In order to more clearly illustrate the technical solutions of the present invention, the following examples are given. Unless otherwise stated, the raw materials, reactions and post-processing methods mentioned in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0060] The terms "preferred," "preferably," "more preferred," and the like, used herein, refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0061] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties to be obtained by the present invention.

[0062] TH588, purchased from Shanghai Taoshu Biological Co., Ltd. (TargetMol);

[0063] Curcumin (CUR) was purchased from Shanghai Macklin Company;

[0064] Anhydrous methanol, hydrogen peroxide (30%), and aqueous ammonia (25%-30%) were purchased from Guangzhou Chemical Reagent Factory;

[0065] Anhydrous calcium chloride was purchased from Aladdin Biochemical Co., Ltd.

[0066] Cell line: The 4T1 breast cancer cells used in this experiment are numbered ATCCCRL-2539 and are from the American Type Culture Collection (ATCC, USA);

[0067] Experimental Animals: This study used BALB / c mice (6-8 weeks old, female, weighing 16-18 g, SPF grade) purchased from Zhujiang Baishitong Biotechnology Co., Ltd., Animal Production License No.: SCXK(Yue)2020-0051, Animal Certification No.: 44822700019542. Mice had free access to water during the experiment and were housed in a clean area.

[0068] Example 1

[0069] Preparation of CaO2 nanoparticles

[0070] A CaCl2·2H2O aqueous solution (1 mL, 2 mol / L) was added to 60 mL of anhydrous methanol, and the mixture was vigorously stirred at room temperature for 5 min. Then, 300 μL of 30 wt% H2O2 was added dropwise and stirred for 5 min. NH3·H2O (400 μL, 25 wt%) was added to trigger the reaction. After stirring for 2 min, the resulting milky white solution was collected by centrifugation (20,000 g, 4°C, 10 min) and washed three times with anhydrous methanol to obtain CaO2 nanoparticles. The CaO2 nanoparticles were dispersed in 10 mL of anhydrous methanol and stored at 4°C for later use.

[0071] Figure 1 (a) is a transmission electron microscopy image of CaO2 nanoparticles, where it can be observed that the CaO2 has a relatively uniform spherical shape with an average diameter of about 80 nm. Figure 1 (g) is the nitrogen adsorption-desorption isotherm of CaO2 nanoparticles.

[0072] Example 2

[0073] Preparation of Turmeric-derived Exosome Nanoparticles (TDNPs)

[0074] Take 500g of freshly washed turmeric, spray it with alcohol and place it in a biosafety cabinet. After ultraviolet irradiation for 30min, turn it over and continue irradiation for 30min to sterilize it. After peeling in the biosafety cabinet, weigh it, add sterile PBS at a ratio of 1g / mL to squeeze the juice, filter it with gauze to remove the residue, collect the filtrate and put it into a 50mL centrifuge tube for centrifugation (3000g, 4℃, 20min), take the supernatant and continue centrifugation (10000g, 4℃, 40min), take the supernatant again and transfer it to an ultrahigh-speed centrifuge tube for ultrahigh-speed centrifugation. The supernatant was removed by centrifugation (150,000 g, 4°C, 1 h), and 5 mL of sterile PBS was added to the precipitate and resuspended by pipetting. The precipitate was evenly dispersed by ultrafiltration to obtain turmeric-derived exosomes (TDNPs) dispersed in sterile PBS. The prepared turmeric exosome solution was diluted 20 times with PBS, and the protein concentration contained in the turmeric-derived exosomes was detected using a BCA protein quantification kit. The concentration of the turmeric-derived exosomes was quantified based on the protein concentration. The turmeric-derived exosome solution was stored at -80°C.

[0075] Figure 1 (b) is the transmission electron microscopy image of TDNPs, where TDNPs with a clear membrane structure and a particle size of 60-180 nm can be observed.

[0076] Example 3

[0077] Preparation of TH588@CaO2 nanoparticles

[0078] A nanogenerator, comprising a drug-loaded core, the surface of which is loaded with a target drug;

[0079] Among them, the average diameter of the nanogenerator is about 90nm; the target drug is TH588, and the drug-loaded core is CaO2 nanoparticles.

[0080] The preparation method of the nanogenerator comprises the following steps:

[0081] A methanol solution containing 0.2 mL of 5 mg / mL CaO2 was diluted with methanol to 1 mg / mL at room temperature, and 0.2 mg of TH588 was added for drug loading. After 24 hours, the solution was centrifuged and washed twice with methanol to obtain TH588@CaO2. The drug loading capacity was measured by UV spectrophotometer and was 7.742%. Finally, 1 mL of anhydrous methanol was added to resuspend the solution and stored at 4°C for later use.

[0082] Wherein, the drug loading amount = (M TH588投料 -M TH588洗涤上清 ) / (M TH588投料 -M TH588洗涤上清 +M CaO2 )×100%.

[0083] Example 4

[0084] Preparation of TDNPs@CaO2 nanogenerator

[0085] A plant-derived exosome-encapsulated drug-loaded calcium-based nanogenerator, the nanogenerator comprising a drug-loaded core coated with a plant-derived exosome shell;

[0086] Among them, Figure 1 As shown in (c), the particle size of the nanogenerator is 90 nm; the drug-loaded core is CaO2 nanoparticles; and the plant-derived exosome shell is turmeric-derived exosomes;

[0087] The preparation method of the plant-derived exosome-encapsulated drug-loaded calcium-based nanogenerator comprises the following steps:

[0088] Take 0.2 mL of 5 mg / mL CaO2 methanol solution and centrifuge to collect the precipitate, add 2 mg of TDNPs, and adjust the CaO2 concentration to 1 mg / mL with PBS for coating to obtain TDNPs@CaO2. Take the supernatant to measure the ultraviolet absorbance. According to the standard curve of turmeric exosomes in PBS ( Figure 9 ) Calculate the supernatant concentration and add TDNPs@CaO2 to sterile PBS and store at 4°C for no more than 24 hours. It should be used for testing in a timely manner; wherein, the coating step is as follows: ice bath ultrasonication at 10% power for 2 seconds, off for 3 seconds, repeat for 1 minute, then incubate at 4°C for 15 minutes, and centrifuge (10000g, 4°C, 10 minutes);

[0089] According to the formula: Encapsulation rate = (M 初始TDNPs -M 上清TDNPs ) / (M 初始TDNPs -M 上清TDNPs +M CaO2 )×100%,

[0090] The coating rate of TDNPs@CaO2 was found to be 54.55%.

[0091] Figure 1 (h) Particle size distribution diagram of CaO2 nanoparticles and TDNPs@CaO2 nanogenerators (n=3, data are expressed as mean ± standard deviation).

[0092] Example 5

[0093] Preparation of TDNPs@TH588@CaO2 nanogenerator

[0094] See also Figure 6 , a drug-loaded calcium-based nanogenerator wrapped in plant-derived exosomes, the nanogenerator comprising a drug-loaded core, the surface of which is loaded with a target drug;

[0095] The outer side of the drug-loaded core is coated with a plant-derived exosome shell;

[0096] The particle size of the nanogenerator is 90 nm; the target drug is TH588, the drug-loaded core is CaO2 nanoparticles; and the plant-derived exosome shell is TDNPs.

[0097] The preparation method of the plant-derived exosome-encapsulated drug-loaded calcium-based nanogenerator comprises the following steps:

[0098] 0.2 mL of the methanol solution of TH588@CaO2 in Example 3 was centrifuged to collect the precipitate, 2 mg of TDNPs was added, and the concentration of TH588@CaO2 was adjusted to 1 mg / mL with PBS for coating to obtain TDNPs@TH588@CaO2. The supernatant was measured for ultraviolet absorbance. According to the standard curve of turmeric-derived exosomes in PBS ( Figure 9 ) Calculate the supernatant concentration;

[0099] The membrane encapsulation step is as follows: ultrasonication in an ice bath at 10% power for 2 seconds, then off for 3 seconds, repeating for 1 minute, incubating at 4°C for 15 minutes, and centrifuging (10,000 g, 4°C, 10 minutes).

[0100] According to the formula: Encapsulation rate = (M 初始TDNPs -M 上清TDNPs ) / (M 初始TDNPs -M 上清TDNPs +M CaO2)×100%,

[0101] The coating rate of TDNPs@CaO2 was found to be 54.55%.

[0102] Figure 1 (e) is the element distribution diagram of TDNPs@TH588@CaO2 nanogenerator. It can be seen that TDNPs@TH588@CaO2 nanogenerator has a uniformly distributed Ca, O, Cl, C and N element structure. It can be seen that the prepared nanogenerator contains CaO2 and encapsulated drug TH588 (with characteristic Cl element).

[0103] Figure 1 (i) is the pore size distribution diagram of CaO2 nanoparticles; it can be seen that the prepared CaO2 has a pore size of 109.7589m 2 / g specific surface area and 21.6745nm pore size, endowing it with drug loading potential, and through the standard curve of TH588 (such as Figure 7 The drug loading of TH588 was calculated to be 7.742%. The above experimental results confirm the successful construction of TDNPs@TH588@CaO2 nanogenerator.

[0104] Comparative Example 1

[0105] Preparation of CUR@CaO2 nanoparticles

[0106] A nanoparticle comprises a drug-loaded core, wherein the mesopores of the drug-loaded core are loaded with curcumin.

[0107] Wherein, the particle size of the nanoparticles is 814 nm;

[0108] The method for preparing the nanoparticles comprises the following steps:

[0109] At room temperature, 1 mg of the methanol solution of CaO2 prepared in Example 1 was diluted with methanol to 1 mg / mL, and 0.2 mg of CUR was added for drug loading. After 24 h, the mixture was centrifuged and washed twice with methanol to obtain CUR@CaO2 nanoparticles.

[0110] Figure 1 (j) Zeta potential diagrams of TDNPs nanoparticles, CaO2 nanoparticles, CUR@CaO2 nanoparticles, TDNPs@CaO2 nanogenerators, and TDNPs@TH588@CaO2 nanogenerators; Figure 1(k) Hydrated particle size measurements of TDNPs nanoparticles, CaO2 nanoparticles, CUR@CaO2 nanogenerators, TDNPs@CaO2 nanogenerators, and TDNPs@TH588@CaO2 nanogenerators (n = 3, data expressed as mean ± standard deviation). The successful construction of the TDNPs@TH588@CaO2 nanogenerator was further verified by measuring the zeta potential and hydrated particle size. The zeta potential of CaO2 before encapsulation was +20.5 mV and the hydrated particle size was 556.6 nm. However, the zeta potential of the TDNPs@CaO2 nanogenerator after encapsulation with turmeric-derived exosomes shifted to -15.8 mV and the hydrated particle size was 171 nm. The shift in zeta potential from positive to negative and the improved dispersibility of the hydrated particle size indicate successful encapsulation. Moreover, the particle size of CUR@CaO2 obtained after CaO2 was loaded with CUR was 814 nm, indicating that the strong hydrophobicity of CUR greatly reduced the dispersibility of CUR@CaO2. However, the hydrated particle size of TDNPs@CaO2 nanogenerator was much smaller than that of CUR@CaO2, which indicated that TDNPs was more conducive to reducing CaO2 than directly loading CUR.

[0111] Figure 1 (l) shows the dispersion of CUR and TDNPs in aqueous solution. Due to its low solubility in water, CUR settles to the bottom of the centrifuge tube, while TDNPs are evenly dispersed, resulting in a clear solution. This indicates that TDNPs are highly water-soluble and can improve the solubility of the CUR they contain.

[0112] Test Example 1

[0113] Electrophoresis analysis was performed on the TDNPs@CaO2 nanogenerator of Example 4, the TDNPs nanoparticles of Example 2, and the CaO2 nanoparticles of Example 1.

[0114] The TDNPs@CaO2 nanogenerator, TDNPs nanoparticles, and CaO2 nanoparticles were added to the loading buffer to achieve a volume ratio of 4:1. The samples were denatured by heating at 97°C for 10 minutes, loaded onto a 12 wt% SDS-PAGE gel for electrophoresis, and then imaged using a multifunctional molecular imager.

[0115] Figure 1 (d) is the SDS-PAGE gel electrophoresis diagram of CaO2 nanoparticles, TDNPs nanoparticles and TDNPs@CaO2 nanogenerators. The results of SDS-PAGE gel electrophoresis analysis showed that the protein bands of TDNPs@CaO2 nanogenerators were similar to those of simple TDNPs.

[0116] Co-localization test of TDNPs@CaO2 nanogenerator

[0117] The co-localization test of TDNPs@CaO2 nanogenerator was carried out using laser confocal microscopy (CLSM, Zeiss LSM 800, Germany) and Beckman Cytoflex flow cytometer. Figure 1 (e) shows that turmeric-derived exosome membranes were labeled with DiO (green) and CaO2 was labeled with Nile Red (red).

[0118] Figure 1 (f) is the fluorescence co-localization imaging of CaO2 and TDNPs in the TDNPs@CaO2 nanogenerator system. It can be seen that Nile red was used to label CaO2 and DiO was used to label TDNPs. The fluorescence co-localization of CaO2 and TDNPs was observed using a laser confocal microscope under excitation of 528nm and 484nm, respectively. The results show that the constructed TDNPs@CaO2 nanogenerator can simultaneously observe obvious red and green fluorescence signals, and has good co-localization.

[0119] Test Example 2

[0120] Investigation of the drug release performance of TDNPs@TH588@CaO2 nanogenerator under neutral and acidic conditions

[0121] Experimental method: The standard curve of TH588 was determined in PBS solution containing 1.5wt% Tween 80 and 5wt% ethanol;

[0122] 2.5 mg of TDNPs@TH588@CaO2 nanogenerators were dispersed in PBS solutions of pH 6 and pH 7.4 containing 1.5 wt% Tween 80 and 5 wt% ethanol, respectively. After shaking and incubating at 37 ° C for 0 h, 1.5 h, 3 h, 6 h, 9 h, and 12 h, 0.6 mL of the solution was centrifuged, the supernatant was measured for UV absorbance, and 0.6 mL of PBS buffer solution was added to the solution to obtain a standard curve of TH588 in 1.5 wt% Tween 80 and 5 wt% ethanol. The standard curve is shown in FIG. Figure 8 shown.

[0123] Test Example 3

[0124] Investigation of acid-responsive degradation and drug release capabilities

[0125] After confirming the successful preparation of TDNPs@TH588@CaO2 nanogenerator, this study then explored its pH-responsive release performance.

[0126] Test method:

[0127] Transmission electron microscopy was used to observe the degradation of TDNPs@CaO2 nanogenerators after incubation in 1 mL of 0.5 mg / mL phosphate buffer at pH 7.4 and pH 6.0 for 0 h and 12 h, respectively. Figure 2 (a) It can be seen that the degradation of TDNPs@CaO2 nanogenerator is obvious after incubation in acidic solution (pH 6.0) for 12 h, while the degradation of TDNPs@CaO2 nanogenerator remains basically unchanged after incubation under neutral conditions (pH 7.4) for 12 h. Under acidic conditions, the degradation of TDNPs@CaO2 nanogenerator will release Ca 2+ ( Figure 2 (b));

[0128] like Figure 2 (c) Figure 2 As shown in (d), the TDNPs@CaO2 nanogenerators encapsulated by turmeric-derived exosomes have a higher Ca content than CaO2 nanoparticles under acidic and neutral conditions. 2+ The release amount, H2O2 generation amount and TH588 release amount were slightly reduced, which may be due to the fact that the exosomes encapsulated the contact between CaO2 and phosphate buffer to a certain extent, thus enhancing the stability of CaO2. After incubation with pH 6.0 phosphate buffer for 12 h, the CaO2 of TDNPs@CaO2 nanogenerator 2+ The release amount reached 57.23%, and the amount of H2O2 generated was 95.2 μM. In the subsequent drug release experiment of TDNPs@TH588@CaO2 nanogenerator, according to the standard curve of TH588 (such as Figure 8 As shown in Figure 3, the release amount of TH588 was 75.94%. This shows that the constructed nanogenerator has good weak acid environment response degradation characteristics, as shown in Figure 3. Figure 2 Compared with other calcium-based nanodevice preparation technologies, since the turmeric-derived exosomes used in the present invention contain curcumin, the nanogenerator does not require the step of drug loading CUR, which greatly saves time and cost.

[0129] Test Example 4

[0130] We have previously verified that the TDNPs@CaO2 nanogenerator has good weak acid responsive degradation properties. Next, we will study the uptake of the TDNPs@CaO2 nanogenerator by 4T1 tumor cells.

[0131] Test method:

[0132] First, 50ug / mL of CaO2 labeled with Nile red dye and TDNPs@CaO2 nanogenerator (containing 50μg / mL of CaO2) were incubated with 4T1 tumor cells for 2h, 4h, and 8h, respectively, and then the cells were collected. Flow cytometry was used to quantitatively analyze the fluorescence intensity of the cells to examine the cellular uptake of nanoparticles.

[0133] Figure 3 (a) Quantitative analysis of the fluorescence intensity of cellular endocytosis of CaO2 and TDNPs@CaO2 nanogenerators after incubation with 4T1 tumor cells for different times (n=3, data are expressed as mean ± standard deviation); Figure 3 As shown in (a), compared with CaO2, the uptake of TDNPs@CaO2 nanogenerators showed a significant increase, and also had the characteristic of time-dependent uptake increase. This may be because the encapsulation of turmeric-derived exosomes improved the stability of CaO2, and the exosomes could fuse with the cell membrane, thereby promoting the cellular uptake of nanoparticles.

[0134] Test Example 5

[0135] After demonstrating that TDNPs@CaO2 nanogenerators had good cellular uptake, we used the MTT assay to evaluate the cytotoxicity of CaO2 NPs, TDNPs NPs, TH588, TDNPs@CaO2 nanogenerators, and TDNPs@TH588@CaO2 nanogenerators;

[0136] Test method:

[0137] 4T1 tumor cells were plated in 96-well plates at 7 × 10 cells per well. 3 The cells were cultured at a density of 100 μL at 37 ° C for 18 h. Afterwards, 100 μL of 200 μg / mL CaO2 nanoparticles, 100 μL of 240 μg / mL TDNPs nanoparticles, 100 μL of 16.78 μg / mL TH588 nanoparticles, 100 μL of TDNPs @ CaO nanogenerator (containing 200 μg / mL of CaO2), and 100 μL of TDNPs @ TH588 @ CaO2 (containing 200 μg / mL of CaO2) nanogenerator DMEM culture medium were added, and incubated in a 37 ° C cell culture incubator for 24 h. The drug solution was carefully discarded, and 100 μL of blank DMEM containing 10 μL of MTT was added to each well and incubated in a 37 ° C cell culture incubator in the dark for 4 h. Subsequently, 100 μL of formazan solution was added and incubated in the dark for at least 4 h to dissolve the generated purple crystals, and the absorbance of each well was measured at 570 nm using a microplate reader;

[0138] Figure 3(b) Cell viability of CaO2 nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO2 nanogenerators, and TDNPs@TH588@CaO2 nanogenerators after incubation with 4T1 tumor cells for 24 h (n=3, data are expressed as mean ± standard deviation); Figure 3 As shown in (b), after incubation with cells for 24 h, the cell viability of the TDNPs@CaO2 nanogenerator and TDNPs@TH588@CaO2 nanogenerator treatment groups were 38.62% and 13.42%, respectively, which had significantly improved ability to inhibit the growth activity of 4T1 tumor cells compared with the CaO2 nanoparticles, TDNPs nanoparticles and TH588 treatment groups.

[0139] Test Example 6

[0140] We used Fluo 4-AM probe to study intracellular calcium ion content and investigate the anti-tumor mechanism of TDNPs@CaO2 and TDNPs@TH588@CaO2;

[0141] Test method:

[0142] 4T1 tumor cells were plated at 1 × 10 5 The cells were seeded at a density of 100 μg / mL in a confocal culture dish and cultured for 18 hours. 4T1 tumor cells were then incubated with 1 mL of 100 μg / mL CaO2 nanoparticles, 1 mL of 120 μg / mL TDNPs nanoparticles, and 1 mL of TDNPs@CaO2 nanogenerators (containing 100 μg / mL CaO2) in a 37°C cell culture incubator for 8 hours. Subsequently, the cells were washed three times with PBS and stained with 1 mL of Fluo 4-AM (4 μM) solution for 30 minutes in the dark. After trypsin digestion, the cells were collected for flow cytometry testing.

[0143] Figure 3 (c) Intracellular calcium ion content of CaO2 nanoparticles, TDNPs nanoparticles, and TDNPs@CaO2 nanogenerators after incubation with 4T1 tumor cells for 8 h (n=3, data are expressed as mean ± standard deviation); Figure 3 As shown in (c), the intracellular Ca2+ of TDNPs@CaO2 nanoparticles was studied after incubation with 4T1 tumor cells for 8 h. 2+ The experimental results show that the intracellular Ca 2+ The content was significantly higher than that of cells treated with TDNPs nanoparticles and CaO2 nanoparticles. This may be due to the good cell endocytosis ability of TDNPs@CaO2 nanogenerators and the ability of CUR carried in TDNPs nanoparticles to inhibit Ca 2+ out of the cell, which together leads to a significant increase in intracellular Ca2+ content.

[0144] Test Example 7

[0145] DCFH-DA fluorescent probe was used to detect intracellular ROS levels;

[0146] Test method:

[0147] 4T1 tumor cells were plated at 2 × 10 5 The cells were seeded at a density of 100 μg / mL in a confocal culture dish and cultured for 18 hours. Then, 1 mL of 100 μg / mL CaO2 nanoparticles, 1 mL of 120 μg / mL TDNPs nanoparticles, 1 mL of 8.39 μg / mL TH588 nanoparticles, 1 mL of TDNPs@CaO2 nanogenerators (containing 100 μg / mL CaO2), and 1 mL of TDNPs@TH588@CaO2 nanogenerators (containing 100 μg / mL CaO2) were incubated with 4T1 tumor cells in a 37°C cell culture incubator for 8 hours. The cells were washed three times with PBS, the cell nuclei were stained with Hoechst 33342, and the intracellular fluorescence was observed using CLSM.

[0148] Figure 3 (d) is the confocal imaging of CaO2 nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO2 nanogenerators, and TDNPs@TH588@CaO2 nanogenerators stained with ROS probes after incubation with 4T1 tumor cells for 8 h; Figure 3 As shown in (d), the strongest green fluorescence was observed in 4T1 tumor cells treated with TDNPs@CaO2 nanogenerators and TDNPs@TH588@CaO2 nanogenerators, indicating that these two nanoparticles can produce a large amount of ROS after treating cells, and the encapsulated drug TH588 does not affect the ability of nanoparticles to produce ROS.

[0149] Test Example 8

[0150] Since a sudden increase in intracellular calcium content and the generation of a large amount of ROS can lead to mitochondrial calcium overload, we then used the JC-1 probe to detect changes in mitochondrial membrane potential.

[0151] Test method: 4T1 tumor cells were cultured at 2×10 5The cells were seeded at a density of 100 μg / mL in a confocal culture dish and incubated for 18 h. Then, 4T1 tumor cells were incubated with 1 mL of 100 μg / mL CaO2 nanoparticles, 1 mL of 120 μg / mL TDNPs nanoparticles, 1 mL of 8.39 μg / mL TH588 nanoparticles, 1 mL of TDNPs@CaO2 nanogenerator (containing 100 μg / mL CaO2 content), and 1 mL of TDNPs@TH588@CaO2 nanogenerator (containing 100 μg / mL CaO2 content) in a 37°C cell culture incubator for 8 h. The cells were washed three times with PBS, stained with a JC-1 kit, and then the cell nuclei were stained with Hoechst33342, and the intracellular fluorescence was observed using CLSM.

[0152] Figure 3 (e) is the confocal imaging of CaO2 nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO2 nanogenerators, and TDNPs@TH588@CaO2 nanogenerators stained with JC-1 probe after incubation with 4T1 tumor cells for 8 h; Figure 3 As shown in (e), the strongest green fluorescence was observed in 4T1 tumor cells after treatment with TDNPs@TH588@CaO2 nanogenerators. This may be because the CaO2 encapsulated in turmeric-derived exosomes can enhance the intracellular calcium ion content and ROS level, while the encapsulated TH588 can inhibit the activity of MTH1 protein and block the oxidative repair of DNA, thereby enhancing the sensitivity of mitochondrial DNA to ROS, which further affects the function of mitochondria and causes a significant decrease in mitochondrial membrane potential.

[0153] Test Example 9

[0154] To further verify the anti-tumor effect of TDNPs@TH588@CaO2, we used Annexin-V FITC / PI kit to measure the cell apoptosis of CaO2 nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO2 nanogenerators, and TDNPs@TH588@CaO2 nanogenerators after incubation with 4T1 tumor cells for 24 h;

[0155] Test method:

[0156] 4T1 tumor cells were plated at 2 × 10 54T1 tumor cells were seeded at a density of 100 cells / mL in a 6-well plate and incubated for 18 hours. Then, 2 mL of 200 μg / mL CaO2 nanoparticles, 2 mL of 240 μg / mL TDNPs nanoparticles, 2 mL of 16.78 μg / mL TH588 nanoparticles, 2 mL of TDNPs@CaO2 nanogenerators (containing 100 μg / mL CaO2), and 2 mL of TDNPs@TH588@CaO2 nanogenerators (containing 100 μg / mL CaO2) were incubated with 4T1 tumor cells for 24 hours. After incubation with the Annexin V-FITC / PI apoptosis detection kit for 15 minutes in the dark, cell apoptosis was analyzed by flow cytometry.

[0157] Figure 3 (f) Cell apoptosis of 4T1 tumor cells after incubation of CaO2 nanoparticles, TDNPs nanoparticles, TH588, TDNPs@CaO2 nanogenerators, and TDNPs@TH588@CaO2 nanogenerators for 24 hours. P values ​​calculated by t-test: *p < 0.05, **p < 0.01, ***p < 0.001;

[0158] like Figure 3 As shown in (f), the apoptosis rate of 4T1 tumor cells was significantly increased after treatment with TDNPs@TH588@CaO2 nanogenerators, indicating that the synergistic effect of calcium overload caused by TDNPs@TH588@CaO2 nanogenerators amplifying oxidative stress and inhibiting DNA damage repair exerted an excellent anti-tumor effect.

[0159] Test Example 10

[0160] To evaluate the antitumor efficacy of TDNPs@TH588@CaO2 in vivo, we established a subcutaneous tumor inoculation model of 4T1 breast cancer tumor cells using BALB / c mice;

[0161] The specific method is as follows: before the 6th day of administration, the right lower abdomen of each mouse was depilated, and 100 μL of 1×10 6 4T1 breast cancer tumor cells were placed in a PBS solution and then reared and observed as Figure 4 As shown in (a).

[0162] When the tumor volume reaches 80 mm 3Afterwards, BALB / c mice were randomly divided into PBS, TH588, CaO2 nanoparticles, TDNPs nanoparticles, TDNPs@CaO2 nanogenerators and TDNPs@TH588@CaO2 nanogenerators groups, with 6 mice in each group. The mice were injected through the tail vein on days 0, 2, 4, 6 and 8, and the tumor volume (L / 2*W*W) was recorded and calculated, and the body weight of the mice was detected.

[0163] The dosages calculated based on drug loading and coating efficiency are: TH588 dosage is 0.05 mg / animal, CaO2 dosage is 0.6 mg / animal, and TDNPs dosage is 0.72 mg / animal.

[0164] The dosage of TDNPs@CaO2 nanogenerator and TDNPs@TH588@CaO2 nanogenerator is calculated based on the CaO2 content: 0.6 mg / animal.

[0165] Figure 4 (b) Tumor growth curves of BALB / c mice after tail vein injection of nanoparticles in each group (n=6, data are expressed as mean ± SD); Figure 4 (c) Images of tumors dissected from BALB / c mice 18 days after tail vein injection of nanoparticles in each group; Figure 4 (d) is a statistical graph showing the average tumor weight of BALB / c mice;

[0166] The results are as follows Figure 4 As shown in (b)-(d), after 18 days of treatment, compared with the PBS blank control group, the therapeutic effects of the TH588, TDNPs nanoparticles, and CaO2 nanoparticles treatment groups were weaker, while the TDNPs@CaO2 nanogenerator treatment group had obvious anti-tumor efficacy, with a tumor inhibition rate of 67.55%. Importantly, the TDNPs@TH588@CaO2 nanogenerator treatment group showed the best therapeutic effect, with a tumor inhibition rate of 87.3%, indicating that TDNPs@TH588@CaO2 nanogenerator has significant synergistic anti-tumor effects in vivo.

[0167] Figure 4 (e) is a statistical graph of the average spleen weight of BALB / c mice (n=6, data are expressed as mean ± SD); when tumor growth in the tumor model induces spleen enlargement, such as Figure 4As shown in (e), compared with the PBS blank control group, the spleen weight of the TDNPs@CaO2 nanogenerator-treated group decreased to 59.07% of that of the PBS group, which had a certain degree of effect in alleviating splenomegaly; and the spleen weight of the TDNPs@TH588@CaO2 nanogenerator-treated group decreased to 37.07% of that of the PBS group, and the effect of alleviating splenomegaly was the most obvious, which also verified the tumor treatment effect of TDNPs@TH588@CaO2 nanogenerator.

[0168] Figure 4 (f) H&E, TUNEL, Ki67, ROS and MTH1 staining images of tumor sections of BALB / c mice injected with nanoparticles in each group via tail vein for 18 days; Figure 4 As shown in (f), the H&E and immunofluorescence staining results of tumor sections showed that the tumor cells in the TDNPs@TH588@CaO2 nanogenerator-treated group had the most obvious vacuolation, nuclear shrinkage, cytoplasm permeabilization and other phenomena. TUNEL immunofluorescence staining showed that the green fluorescence of the tumor cells in the TDNPs@TH588@CaO2 nanogenerator-treated group was the most obvious. Ki67 immunofluorescence staining showed that the red fluorescence of the tumor cells in the TDNPs@TH588@CaO2 nanogenerator-treated group was also the most significantly weakened. These results further indicate that the treatment with TDNPs@TH588@CaO2 nanogenerator causes the most obvious apoptosis and necrosis of tumor cells.

[0169] In addition, by observing the tumor ROS fluorescence sections, it was found that the TDNPs@CaO2 nanogenerator treatment group and the TDNPs@TH588@CaO2 nanogenerator treatment group showed the strongest green fluorescence signal, indicating that the oxygen stress level of tumor cells in these two groups was higher after treatment, which may be the cause of tumor cell apoptosis. Tumor tissue MTH1 immunofluorescence sections showed that the TDNPs@CaO2 nanogenerator treatment group showed the strongest red fluorescence, which is most likely because the TDNPs@CaO2 nanogenerator enhanced intracellular calcium overload and H2O2 production, thereby leading to extensive DNA damage in tumor cells, and thereby stimulating tumor cells to overexpress MTH1 protein to prevent DNA from oxidative damage. Therefore, the strongest MTH1 expression signal was observed in the TDNPs@CaO2 nanogenerator group in the MTH1 immunofluorescence sections (such as Figure 4(f) As shown in the fifth row). The weakest red fluorescence was observed in the TDNPs@TH588@CaO2 nanogenerator-treated group. This is because the TH588 released by the nanogenerator inhibits the expression of MTH1 protein, hindering the DNA damage repair of tumor cells, thereby making DNA intolerant to ROS and amplifying ROS-induced DNA oxidative damage, thereby enhancing the inhibitory effect of calcium overload and oxidative stress on tumors. We also analyzed the survival rate of mice after treatment. Mice treated with the TDNPs@TH588@CaO2 nanogenerator group achieved the highest survival rate (71.43%), which was much higher than that of other treatment groups (such as Figure 4 (g)).

[0170] In summary, the TDNPs@TH588@CaO2 nanogenerator constructed in this study has excellent synergistic anti-tumor therapeutic ability in vivo.

[0171] Test Example 11

[0172] To verify the biosafety of the nanogenerator in mice, this study analyzed mice after being treated with different treatment groups.

[0173] Test method:

[0174] Before treatment, a 4T1 tumor cell subcutaneous tumor inoculation model was established using BALB / c mice (see Test Example 10 for the inoculation model). Six days before administration, the right lower abdomen of the mice was depilated, and 100 μL of a PBS solution containing 1 million 4T1 breast cancer tumor cells was subcutaneously injected into the right lower abdomen of the depilated area using a 1 mL sterile syringe. When the tumor volume reached 80 mm, the mice were injected with 100 μL of PBS solution containing 1 million 4T1 breast cancer tumor cells. 3 The mice with successfully implanted tumors were randomly divided into PBS, TH588, CaO2 nanoparticles, TDNPs nanoparticles, TDNPs@CaO2 nanogenerators, and TDNPs@TH588@CaO2 nanogenerators groups, with 6 mice in each group. The mice were injected into the tail vein on days 0, 2, 4, 6, and 8, and the tumor volume was recorded and calculated (L / 2*W*W), and the weight of the mice was detected.

[0175] The dosages calculated based on drug loading and coating efficiency are: TH588 dosage is 0.05 mg / animal, CaO2 dosage is 0.6 mg / animal, and TDNPs dosage is 0.72 mg / animal.

[0176] The dosage of TDNPs@CaO2 nanogenerator and TDNPs@TH588@CaO2 nanogenerator is calculated based on the CaO2 content: 0.6 mg / animal.

[0177] After the end of the drug administration, the tumor volume was recorded and calculated every other day, and the weight of the mice was detected. After 18 days, blood was collected through the eyeball for blood biochemistry evaluation. The tumor and the heart, liver, spleen, lung, and kidney were dissected and removed. The tumor and spleen were photographed and weighed. Then, the tumor and various important organs were stained with H&E, TUNEL and Ki67 sections, and MTH1 was immunofluorescently labeled. In order to investigate the generation of ROS in the tumor site after drug administration, 24 hours after the end of the two tail vein injections, 80μM DCFH-DA probe was injected into the tumor. The animals were killed 1 hour later, and the tumors were dissected and placed in a -80℃ refrigerator for 15 minutes. Frozen sections were then prepared and fluorescently scanned.

[0178] Figure 5 (a) H&E staining images of major organs (heart, liver, spleen, lung and kidney) of BALB / c mice injected with nanoparticles in each group via tail vein for 18 days; Figure 5 As shown in (a), no obvious lesions and necrosis were observed in H&E sections of major organs (heart, liver, spleen, lung, and kidney) in all treatment groups compared with the PBS group.

[0179] Figure 5 (bf) Analysis of serum ALT, ALP, AST, BUN, and CREA concentrations (n=6, data are expressed as mean ± SD).

[0180] like Figure 5 As shown in (bf), in blood biochemistry tests, the levels of aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea nitrogen (BUN) and creatinine (CREA) in the serum of mice in the TDNPs@TH588@CaO2 nanogenerator-treated group were not significantly different from those in healthy mice, indicating that the treatment did not cause liver toxicity and kidney toxicity in mice.

[0181] Figure 5 (g) Monitoring of the body weight of mice after administration (n=6, data are expressed as mean ± standard deviation); Figure 5 As shown in (g), the body weight of mice in each treatment group showed a healthy growth trend during the treatment period, indicating that TDNPs@TH588@CaO2 has good in vivo biosafety.

[0182] Test Example 12

[0183] Hemolysis test

[0184] After incubating 0.5 mL of TDNPs@CaO2 nanogenerators with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, and 1000 μg / mL and 0.5 mL of 1000 μg / mL CaO2 nanoparticles with 0.5 mL of red blood cell suspension at 37°C for 1 h, the samples were centrifuged (1000 g, 10 min), and the absorbance of the supernatant at 570 nm was detected using a microplate reader.

[0185] 0.5 mL of PBS and 0.5 mL of 0.1 wt% Triton X-100 solution were incubated with 0.5 mL of red blood cell suspension as negative and positive controls, respectively.

[0186] The hemolysis rate was calculated according to the following formula: Hemolysis rate (%) = (A 样品 -A 阴性 ) / (A 阳性 -A 阴性 )×100%.

[0187] A 样品 、A 阴性 and A 阳性 Represent the absorbance of sample, negative control and positive control, respectively.

[0188] Figure 5 (h) Hemolysis analysis of different nanoparticles after 1 h of incubation with erythrocytes in vitro (n=3, data are expressed as mean ± SD). P values ​​were calculated by t-test: *p<0.05, **p<0.01, ***p<0.001.

[0189] like Figure 5 As shown in (h), hemolysis experiments demonstrate that the TDNPs@CaO2 nanogenerator significantly improves the hemolysis of CaO2. This is likely due to the encapsulation of turmeric-derived exosomes, which reduces the positive surface potential of CaO2 and improves biocompatibility, thereby reducing the adsorption and toxicity of the nanoparticles to blood cells. In summary, our constructed nanogenerator has good in vivo biosafety and does not cause significant hepatotoxicity or nephrotoxicity after treatment.

[0190] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0191] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A drug-loaded calcium-based nanogenerator encapsulated by plant-derived exosomes, characterized in that: The nanogenerator comprises a drug-loaded core, the mesopores of the drug-loaded core are loaded with target drugs, and the outer side of the drug-loaded core is coated with a plant-derived exosome shell; Wherein, the plant-derived exosome shell is turmeric-derived exosomes; The drug-loaded core is calcium peroxide; The target drug is TH588; The particle size of the nanogenerator is 90-150nm; The mass ratio of the drug-loaded core, the target drug, and the plant-derived exosome shell is 1:0.07-0.09:1.1-1.

3.

2. The method for preparing the drug-loaded calcium-based nanogenerator encapsulated by plant-derived exosomes according to claim 1, characterized in that: The method comprises the following steps: encapsulating the drug-loaded core with the plant-derived exosome shell to obtain a nanogenerator.

3. The method for preparing the drug-loaded calcium-based nanogenerator wrapped with plant-derived exosomes according to claim 2, characterized in that: The coating time is 25-30 minutes.

4. Use of the plant-derived exosome-encapsulated drug-loaded calcium-based nanogenerator according to claim 1 in the preparation of an anti-tumor drug delivery agent.

Images