A nucleic acid-based multichannel Ca 2+ Nano modulator and its preparation method and application

Abstract

The present invention discloses a nucleic acid-based multi-channel Ca 2+ Nanomodulators and their preparation methods and applications belong to the field of biomedical technology. The present invention uses a coprecipitation method to load the calcium ion efflux inhibitor curcumin into amorphous calcium phosphate, and then adsorbs DNA aptamers on its surface. The resulting ACP@C‑D can be used to promote the dual pathway of cancer treatment mediated by mitochondrial dysfunction, and has been shown to have the following advantages: (1) ACP@C‑D can disintegrate at the TME and release CUR and Ca 2+ , thereby improving the accuracy of treatment. (2)Ca 2+ The sudden increase in Ca 2+ (3) The released CUR can not only prevent Ca 2+ efflux and can also promote Ca 2+ From the endoplasmic reticulum to the mitochondria, further amplifying Ca 2+ In conclusion, this nanomodulator is a promising nanomedicine that can provide a valuable reference for mitochondria-based tumor therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and particularly relates to a nucleic acid-based multi-channel Ca 2+ Nano modulator and its preparation method and application. Background Art

[0002] Effective cancer treatment is an urgent challenge that can save millions of lives. In recent years, researchers have developed many methods to treat cancer, such as photothermal therapy, photodynamic therapy, immunotherapy, etc. Although certain results have been achieved, there are still some difficulties that restrict their application, such as limited oxygen concentration, insufficient near-infrared penetration, and cellular multidrug resistance. Mitochondria, as the "energy factory" of cells, provide energy for the normal life activities of cells. Mitochondria are also involved in processes such as apoptosis, differentiation, and cell information transmission, and have the ability to regulate cell growth and cell cycle. Studies have shown that abnormal mitochondrial metabolism and mitochondrial function are related to the proliferation and metastasis of tumor cells. Therapies targeting the disruption of mitochondrial function are becoming an attractive anti-cancer strategy.

[0003] Ca 2+ As the most important second messenger, it participates in regulating various physiological processes and is closely related to biological signal transduction. 2+ When the balance is disrupted, abnormal Ca 2+ Overload will trigger a series of mitochondrial disorders, including decreased mitochondrial membrane potential (MMP), decreased adenosine triphosphate (ATP) levels, and activation of mitochondrial-mediated apoptosis pathways. 2+ Overloading of intracellular Ca 2+ is gaining more and more attention in anticancer therapy. 2+ A certain concentration limits the Ca 2+ Therefore, it effectively increases intracellular Ca 2+ The concentration becomes Ca 2+ Overload is a key factor. Currently, increasing intracellular Ca 2+ There are two main methods: the first is to deliver exogenous Ca into the cell. 2+ , and the second is to block intracellular Ca 2+ However, single regulation has little effect on improving Ca efflux. 2+ Overloading has limited effect, while the combination of both regulation can significantly improve Ca 2+ overload.

[0004] Based on Ca 2+ Biomineralization is considered to be a highly biosafe cancer treatment strategy. This strategy usually uses chelators to collect free Ca 2+Through mineralization, a calcification layer is further formed on the surface of tumor cells or in organelles, blocking their normal functions and thus inhibiting cell growth and proliferation. It has been reported that nucleic acids are excellent Ca 2+ Chelating agent, the phosphate group in nucleic acid can be Ca 2+ Mineralization provides efficient and sufficient reaction sites, however, its application in living cells has not been reported. Summary of the invention

[0005] The present invention provides a nucleic acid-based multi-channel Ca 2+ Preparation method of nano-modulator, the multi-channel Ca 2 + The nano-modulator is prepared by loading ACP@C with Cy5.5-DNA having a nucleotide sequence as shown in SEQ ID NO.1.

[0006] Preferably, the multi-channel Ca 2+ The preparation method of the nano-modulator is as follows:

[0007] 45-55 μg ACP@C powder was dissolved in 0.5-1.5 mL 8-12 mM HEPES buffer, and then 1-3 μL 90-110 μM DNA was added. The mixture was incubated at room temperature for 0.8-1.2 h, and the precipitated ACP@CD was collected by centrifugation. The ACP@CD was a nucleic acid-based multi-channel Ca 2+ Nanomodulators.

[0008] More preferably, the usage amount of the ACP@C powder is 50 μg.

[0009] More preferably, the HEPES buffer is used in an amount of 1 mL and a concentration of 10 mM.

[0010] More preferably, the DNA is used in an amount of 2 μL and a concentration of 100 μM.

[0011] More preferably, the room temperature incubation time is 1 h.

[0012] More preferably, the preparation method of ACP@C is as follows: 0.8-1.2 mL of CUR with a concentration of 4-6 mg / mL is added to 27-31 mL of 0.8-1.2 mM Ca(NO3)2·4H2O solution, stirred at room temperature for 0.5-1.5 h, and then 27-31 mL of 0.5-0.7 mM (NH4)2HPO4 solution is slowly added to the above solution.

[0013] More preferably, the concentration of CUR is 5 mg / mL and the volume is 1 mL, the concentration of Ca(NO3)2·4H2O solution is 1 mM and the volume is 29 mL, and the concentration of (NH4)2HPO4 solution is 0.67 mM and the volume is 29 mL.

[0014] The present invention also provides a nucleic acid-based multi-channel Ca 2+ Nanomodulators.

[0015] The present invention also provides the above-mentioned nucleic acid-based multi-channel Ca 2+ Application of nanomodulators in the preparation of anti-tumor drugs.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention constructs a nucleic acid-based Ca 2+ Nanomodulators (ACP@CD) disrupt mitochondrial function in multiple ways for cancer treatment. Figure 1 As shown in the paper, the calcium ion efflux inhibitor curcumin (CUR) was loaded into amorphous calcium phosphate (ACP) by coprecipitation, and then Cy5.5-labeled DNA aptamer was adsorbed on its surface. The resulting ACP@CD can be used to promote the dual pathway of mitochondrial dysfunction-mediated cancer therapy and has been shown to have the following advantages: (1) ACP@CD is easy to synthesize and can be effectively loaded with drugs. (2) ACP@CD can disintegrate at the TME and release CUR and Ca 2+ , thereby improving the accuracy of treatment. (3)Ca 2+ The sudden increase in Ca 2+ (4) The released CUR can not only prevent Ca 2+ efflux and can also promote Ca 2+ From the endoplasmic reticulum to the mitochondria, further amplifying Ca 2+ (5) The mitochondrial targeting of Cy5.5 enriches DNA complexes in mitochondria and provides a source of Ca 2+ Provide sufficient and efficient chelation sites to achieve mitochondrial biomineralization. (6) All components of the constructed nanomodulator are biodegradable and bioabsorbable materials with good biosafety. In conclusion, this nanomodulator is a promising nanomedicine that can provide a valuable reference for mitochondrial-based tumor therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the preparation process and action mechanism of ACP@CD in Example 1.

[0019] Figure 2The detection results of ACP@C in Example 1, including (a) TEM image of ACP@C (scale = 200 nm); (b) EDS spectrum of ACP@C; (c) Fourier transform infrared spectra of ACP, CUR and ACP@C; (d) UV-visible absorption spectra of CUR, ACP@C and ACP@C+acid; (e) standard curve of CUR; (f) UV-Vis image of ACP@C supernatant before and after CUR loading.

[0020] Figure 3 The results of structural characterization of ACP@CD in Example 1, including (a) hydrodynamic sizes of ACP@C and ACP@CD; (b) fluorescence spectra of ACP, DNA, ACP-D, ACP@C and ACP@CD; (c) surface charges of ACP, ACP-D, ACP@C and ACP@CD; (d) condensation ability and mass ratio (nM:μg / mL) of Cy5.5-DNA and ACP@C determined by agarose gel electrophoresis; (e) loading efficiency of Cy5.5-DNA; (f) particle size analysis of ACP@C during incubation in PBS for 72 h.

[0021] Figure 4 The disintegration test results of ACP@C in Example 1, where (a) TEM images of ACP@C after culturing at pH = 5.4 for 1, 2, and 4 h (scale bar = 200 nm); (b) Ca 2+ Release curves of 1047 and 1047 CUR (c); data are presented as mean ± SD, n = 3 graphs.

[0022] Figure 5 The results of the 4T1 cell test of ACP@CD in Example 1, including (a) confocal microscopy image analysis of 4T1 cells after ACP@CD culture, scale bar = 20 μm; (b) flow cytometric analysis of ACP@CD absorption by 4T1 cells at different time periods.

[0023] Figure 6 The results of the 4T1 and L02 cell experiments of ACP@CD in Example 1, where (a) confocal microscopy images of 4T1 cells and L02 cells after 6 hours of ACP@CD treatment, blue fluorescence represents the nucleus, green fluorescence represents mitochondria, and red fluorescence represents Cy5.5 fluorescence, scale bar = 20 μm. Flow cytometry was used to measure the fluorescence intensity of 4T1 cells (b) and L02 cells (c).

[0024] Figure 7Results of the cytotoxicity test of ACP@CD in Example 1, including the effects of different treatment groups on L02 (a) and 4T1 (b) cell viability; (c) flow cytometry analysis of 4T1 cell apoptosis after treatment with different formulations; data are expressed as mean ± SD, n = 3.

[0025] Figure 8 This is the intracellular Ca level detection experiment in Example 1, where (a) the fluorescence images of the Ca level of 4T1 cells at different time points after ACP@CD treatment were detected using the Flou4-AM fluorescent probe. Scale bar = 20 μm. (b) The fluorescence images of the Ca level of 4T1 cells after treatment with different formulations were detected using the Flou4-AM fluorescent probe. Scale bar = 20 μm.

[0026] Fig. 9 The results of the mitochondrial damage test in Example 1, where (a) TMRM confocal microscopy image of mitochondrial membrane depolarization, scale bar = 20 μm; (b) infrared spectrum of 4T1 cells after ACP@CD treatment; (c) intracellular Ca2+ in 4T1 cells and L02 cells after ACP@CD treatment 2+ .

[0027] Fig.10 The results of the effect of ACP@CD on ROS in Example 1, wherein (a) the DCFH-DA probe was used to analyze the ROS level produced by 4T1 cells after treatment with different samples, scale bar = 20 μm; (b) flow cytometry was used to analyze the ROS produced by 4T1 cells after treatment with different samples.

[0028] Fig.11 The results of the biological electron microscopy and WB experiments in Example 1, where (a) the microscope images of 4T1 cells before and after 12 hours of ACP@CD treatment. (b) Western blot analysis results of Caspase-3 (Casp-3) in 4T1 cells after the action of different preparations. (c) Gray analysis of protein blot.

[0029] Fig.12 This is the in vivo fluorescence imaging experiment in Example 1, where (a) the fluorescence images of 4T1 hormone mice were observed after intravenous injection of ACP@CD and free Cy5.5-DNA (2, 4, 8, 12, 24 and 48 h). (b) The corresponding average fluorescence intensity of the tumor area. (c) Fluorescence images of tumors and organs at the end of the study (48 h after injection) and (d) semi-quantitative analysis.

[0030] Fig.13This is the in vivo anti-tumor experiment in Example 1, where (a) is a schematic diagram of the mouse treatment cycle. (b) is the relative tumor change during the treatment. (c) is the average weight change of mice during the treatment. (d) is a digital photo of mice and ex vivo tumors at the end of treatment. (e) is the ex vivo tumor weight and (f) is the in vivo tumor inhibition efficiency.

[0031] Fig.14 H&E staining (scale bar = 50 μm), TUNEL and Caspase-3 immunofluorescence staining (scale bar = 20 μm) of tumor tissues resected from different treatment groups in Example 1, groups: 1) PBS; 2) ACP; 3) ACP-D; 4) ACP@C; 5) ACP@CD, data are expressed as mean ± SD, n = 5.

[0032] Fig.15 These are H&E staining photos of the heart, liver, spleen, lung and kidney tissues of mice in different treatment groups in Example 1 (scale bar = 50 μm). DETAILED DESCRIPTION

[0033] Example 1

[0034] 1.1 Reagents

[0035] DNA (SEQ ID NO.1: 5′-GCG GAC GAC CTG GGT GCT TGC TCACGG TGT CGG GAG GCGG-Cy5.5-3′) sequence was purchased from Bioengineering (Shanghai, China); calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) was purchased from Xilong Science (Guangdong, China); diammonium hydrogen phosphate ((NH4)2HPO4), HEPES buffer (pH 7.6), and sodium hydroxide (NaOH) were purchased from MacLean (Shanghai, China); curcumin (CUR) was purchased from J&K Chemical Co., Ltd. (Shanghai, China); mitochondrial dye MitoTrackerGreen, calcium detection kit (methyl molybdenum blue colorimetric method), and agarose were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; tetramethylrhodamine methyl ester (TMRM) was purchased from Aladdin (Shanghai, China); DAPI, Hoechst 33342 and DCFH-DA were purchased from Beyotime (Shanghai, China); MTT toxicity detection kit was purchased from Prilai (Beijing, China); V-FITC / PI apoptosis kit was purchased from Lianke Biotechnology (Hangzhou, China); 4T1 cell line was purchased from American Type Culture Collection (ATCC).

[0036] 1.2 Instruments

[0037] Transmission electron microscope (TEM-2100EX), fluorescence spectrophotometer (F-4600, Hitachi), UV spectrophotometer (Cary60), nanopotential particle size analyzer (NaNo-ZS90, Malvern), microplate reader (Epoch 2, BioTek, USA), laser confocal scanning microscope (Leica TCS SP8), flow cytometer (CutoFLEX, BackCoulter, USA), small animal three-dimensional multimodal imaging system (IVIS SpectrumCT, PerkinElmer, USA), etc.

[0038] 1.3 Preparation of ACP@CD nanoparticles

[0039] The ACP synthesis process is as follows: Ca(NO3)2·4H2O is used as the calcium source and (NH4)2HPO4 is used as the phosphorus source to prepare ACP nanoparticles by coprecipitation. First, Ca(NO3)2·4H2O is dissolved in 29mL of deionized water to form a 1mM solution, and then 29mL of (NH4)2HPO4 solution is added dropwise to the above solution, keeping the Ca / P molar ratio at 1.5, and adjusting the pH to 8 with 1M NaOH solution. After that, the resulting suspension is stirred at 30°C for 10min, aged at room temperature for 12h, the precipitate is collected and washed with deionized water, and repeated three times. The precipitate is dried in vacuum at room temperature to obtain white powder ACP.

[0040] Drug loading and release studies. 1 mL of CUR (5 mg / mL, CUR dissolved in ethanol) was added to 29 mL of 1 mM Ca(NO3)2·4H2O solution and stirred at room temperature for 1 h. Then, (NH4)2HPO4 solution was slowly added to the above solution, maintaining a Ca / P molar ratio of 1.5. After gentle stirring for 15 min in a water bath at 30 °C, the suspension was centrifuged at high speed (12000 rpm, 20 min) to collect ACP@C. The absorbance of the supernatant at 424 nm was measured by UV-visible spectrophotometry. The drug concentration in the supernatant was quantified according to the linear standard curve, and the drug loading (LC%) was calculated. To evaluate the in vitro release of ACP@C, ACP@C was placed in different pH (5.4, 7.4) (1 mL, 2 mg / mL), and 25 μL samples were aspirated at given time intervals for analysis by a calcium detection kit.

[0041] Synthesis of ACP@CD. Dissolve 50 μg of ACP@C powder in 1 mL of 10 mM HEPES buffer (pH 7.6), then add 2 μL of 100 μM Cy5.5-DNA (sequence as shown in SEQ ID NO.1), incubate at room temperature for 1 h, centrifuge (5000 rpm, 10 min), and collect ACP@CD. Use a fluorescence spectrophotometer to measure the fluorescence intensity of the supernatant before and after the reaction, and calculate the amount of DNA adsorption based on the weakened fluorescence signal.

[0042] Synthesis of ACP-D: 50 μg of ACP powder was dissolved in 1 mL of 10 mM HEPES buffer (pH 7.6), and then 2 μL of 100 μM Cy5.5-DNA was added and incubated at room temperature for 1 h, followed by centrifugation (5000 rpm, 10 min) to collect ACP-D.

[0043] 1.4 Cell culture

[0044] 4T1 cells and L02 cells were cultured in a constant temperature and humidity incubator (37°C, 5% CO2) with DMEM medium containing 10% FBS and 5% penicillin-streptomycin. When the cell growth reached 85% of the culture dish, about 1 million cells were digested and centrifuged (800 rpm / min, 3 min), the upper culture medium was discarded, and clean cell culture medium was taken for washing, and then resuspended in 1 mL of cell culture medium for subsequent experiments.

[0045] 1.5 ACP@CD’s ability to generate ROS

[0046] 4T1 cells were plated at 1 × 10 5 4T1 cells were inoculated at a density of 100 μM on a confocal dish and cultured overnight. Then, 4T1 cells were treated with (1) control group (PBS); (2) ACP; (3) ACP-D; (4) ACP@C; (5) ACP@CD (the concentrations of ACP, ACP-D, ACP@C and ACP@CD were all 200 μg / mL), where ACP@CD was prepared according to 1.3. After the drugs were co-incubated with cells for 6 h, DCFH-DA (10 μM) was added and incubated for another 30 min. After washing three times with PBS, the ROS generation of each group of cells was observed under a laser confocal microscope (CLSM).

[0047] 1.6 TMRM mitochondrial damage study

[0048] 4T1 cells were plated at 1 × 10 5The cells were inoculated at a density of 1000 μg / mL on a confocal dish and cultured overnight. The cells were then treated with (1) control group (PBS); (2) ACP; (3) ACP-D; (4) ACP@C; (5) ACP@CD (the concentrations of ACP, ACP-D, ACP@C and ACP@CD were all 200 μg / mL) for 6 h. The cells were then labeled with 200 nM TMRM and analyzed under a confocal microscope (CLSM). ACP@CD was prepared at a DNA to ACP@C ratio of 250:50 (nM / μg).

[0049] 1.7 Determination of calcium content in cells

[0050] 4T1 cells and L02 cells were cultured at 2 × 10 5 The cells were inoculated at a density of 1000 μg / mL on six-well plates and cultured overnight. After incubation with ACP@CD (200 μg / mL) for 6 h, the cells were washed three times with PBS solution, digested and centrifuged, and then washed once with PBS. 2% (v / v) HNO3 was added, and the cells were dissolved by ultrasound, and the calcium detection kit was used for content analysis.

[0051] *Calcium content per cell = cell uptake (PG) / cell number. The number of 4T1 and L02 cells was determined by cell counter.

[0052] 1.8 Cellular uptake of ACP@CD

[0053] 4T1 cells and L02 cells were cultured at 1×10 5 The cells were inoculated with a density of 100 μg / mL on a confocal dish and cultured overnight. ACP@CD (200 μg / mL, ACP@CD was prepared according to 1.3) was added and incubated with the cells for 6 hours, then stained with Hochest33342 for 30 minutes, rinsed with PBS, and then stained with Mito Tracker Green for 30 minutes, washed with PBS 3 times, and observed by CLSM. In addition, the cellular uptake of ACP@CD was also measured by flow cytometry.

[0054] 1.9 Cell apoptosis

[0055] The cytotoxicity was determined by MTT assay. 4 4T1 cells and L02 cells were cultured overnight on 96-well plates, and different drugs with the same drug concentration and different concentration gradients of the same drug were analyzed in each well. Subsequently, 10 μL MTT Stock was added to each well per 100 μL of culture medium, and the culture medium was removed. 100 μL DMSO was added to each well, and the cells were incubated at room temperature for 10 minutes. After slight shaking, the absorbance at 570 nm was measured using a microplate reader.

[0056] In addition, flow cytometry was used to analyze cell apoptosis. Cells were treated with different drug concentrations for 24 hours, and then the cells were collected and analyzed by flow cytometry using the ANNEXIN V-FITC / PI apoptosis detection kit.

[0057] 1.10 In vivo tumor models

[0058] First, a tumor mouse model was constructed. 1×10 6 4T1 cells were subcutaneously injected into the right abdomen of 4-6 week-old female BALB / c mice to establish a 4T1 subcutaneous tumor model. 3 At 4 pm, the mice were randomly divided into five groups (n=4). During the imaging experiments, the mice were anesthetized with 2.5% isoflurane in air at a flow rate of 0.3 L / min.

[0059] 1.11 Antitumor efficacy in vivo

[0060] BALB / c mice bearing 4T1 tumors were divided into five groups, including: (1) control group; (2) ACP group; (3) ACP-D group; (4) ACP@C group; (5) ACP@CD group. Treatment was performed through the tail vein, and the injection dose and injection frequency (injection dose was 2 mg / kg, 1, 3, 5, 7, 9, 11, 13 days of injection treatment) were consistent in each group. Tumor size and body weight (tumor volume = W 2 × L / 2, W = width, L = length), the relative tumor volume of each mouse was calculated as V / V0 (V0 is the tumor volume at the beginning of treatment). After the experiment, the mice were killed with CO2, and the tumors and organs were dissected and fixed with tissue fixative.

[0061] 1.12 Statistical analysis

[0062] Each experiment was performed at least three times. All data were expressed as mean ± standard deviation (SD) using GraphPad Prism software. Statistical analysis was performed using Student's t-test. Significant differences between groups were considered to be at a minimum of p < 0.05.

[0063] 2.1 Results and Discussion

[0064] 2.1.1 Design ideas

[0065] The invention adds curcumin (CUR) into amorphous calcium phosphate (ACP) by a coprecipitation method, and then adsorbs Cy5.5-labeled DNA on the surface of the ACP to construct an acid-responsive nucleic acid nano-modulator (ACP@CD).

[0066] ACP@CD can increase the Ca2+ Concentrations enhance tumor cell Ca 2+ Overloading and mitochondrial biomineralization can achieve the purpose of tumor treatment. Under the action of EPR, the nano-adjusting agent can be effectively accumulated in the tumor site and internalized by tumor cells. In an acidic environment, ACP@CD will disintegrate and release Ca 2+ and CUR, in Ca 2+ The concentration increased sharply and CUR inhibited Ca 2+ The combined effect of Ca 2+ outflow and Ca 2+ outflow in tumor cells enhances 2+ In addition, the mitochondrial targeting ability of Cy5.5 can enrich DNA in mitochondria, and the phosphate groups on DNA can be used as Ca 2+ This is the first report of providing sufficient chelation reaction sites to achieve mitochondrial biomineralization, leading to mitochondrial dysfunction and further promoting tumor cell death. This simple and functional strategy proposed in this topic will provide new insights into mitochondrial-based cancer therapy.

[0067] 2.1.2 Material structure characterization

[0068] 2.1.2.1 Structural characterization of ACP@C

[0069] The present invention obtains ACP@C by coprecipitation method, such as Figure 2 (a)-(b), according to the transmission electron microscopy (TEM) results, the average size of ACP@C is about 70nm, and the EDS spectrum shows that the Ca, P, C and O elements in ACP@C have a strong correlation, which is consistent with the elemental composition of ACP and CUR. In addition, the Fourier transform infrared spectroscopy Figure 2 (C) Verification of the successful synthesis of ACP@C, 563 cm -1 and 1033cm -1 The absorption peak at belongs to PO4 3- , indicating that the sample is calcium phosphate, 563cm -1 The unsplit peak at 850-1250cm -1 The absorption peaks in the range can be attributed to the OOC (peroxide) of the OO and CO modes of pure CUR. In addition, we verified the loading of CUR by UV-visible spectroscopy analysis. Figure 2 As shown in (d)-(f), after adding CUR, the emission peak of CUR appeared in ACP@C at 424nm. The CUR loading efficiency (LE) was calculated to be 69.35% based on the CUR standard curve and the change of supernatant fluorescence before and after CUR loading.

[0070] 2.1.2.2 Structural characterization of ACP@CD

[0071] like Figure 3 (a)-(b) show that after adsorbing DNA, the size of ACP@CD particles increases (about 100 nm). The changes in the Zeta potential after each step of synthesis are monitored. After adding CUR, the potential of ACP changes from positive to negative, and the potential becomes more negative after loading DNA. Figure 3 (c), the fluorescence detection results show that the fluorescence of Cy5.5 of ACP@CD is significantly enhanced compared with ACP@C, which confirms the successful loading of DNA. Figure 3 (d) The optimal mass ratio of DNA to ACP@C (nM:μg / mL) was determined by agarose gel electrophoresis. When the mass ratio of DNA to ACP@C was 200:50 (nM:μg / mL), DNA was almost completely adsorbed by ACP@C. Figure 3 (e), the calculated adsorption rate of DNA is 84.6%. Figure 3 (f). For further application in biomedicine, the storage stability of ACP@C was tested, and the hydrated particle size of ACP@C within 72h was tested. The results showed that the material had good stability within 72h. The above results show that ACP@CD was successfully prepared and has good stability.

[0072] 2.1.3 Drug disintegration experiment

[0073] ACP is also a smart multi-cargo carrier that can achieve pH-responsive and sustained release of drugs. We first explored the pH-responsive degradation of ACP@C. Figure 2 As can be seen in (d), the apparent absorption peak of CUR can be detected after adding acid. Figure 4 (a), we used transmission electron microscopy (TEM) to investigate the effect of acid on ACP@CD. After 1 h in acidic buffer, ACP@CD showed slight degradation, and obvious fragmentation and decomposition were observed after 4 h. Figure 4 As shown in (c), under the condition of pH 7.4, only a small amount of CUR was detected after 12 hours of culture, and its cumulative release was about 18.7%; while under the condition of pH 5.4, burst release of CUR was detected, and the release of CUR could reach 89.4%. Figure 4 (b), Ca 2+ The release curve of CUR was similar to that of CUR. After immersion in pH 5.4 buffer for 12 h, the Ca 2+ The concentration can reach 400μg / mL.

[0074] 2.1.4 Cellular level

[0075] 2.1.4.1 Ingestion and killing

[0076] The effective internalization of nanoparticles by tumor cells is an important guarantee for therapeutic efficacy. We studied the cellular uptake of nanomodulators and the mitochondrial targeting of Cy5.5-labeled DNA materials in 4T1 cells by confocal laser scanning microscopy (CLSM) and flow cytometry. Figure 5 As shown in (a), with the extension of incubation time, the red fluorescence of Cy5.5 gradually increased, indicating that the cell absorption behavior is time-dependent and sufficient to ensure a satisfactory therapeutic effect. It is worth noting that with the extension of incubation time, the number of red / yellow dots also increased, which means that ACP@CD is gradually enriched in mitochondria. Figure 5 As shown in (b), flow cytometry results also demonstrated the time dependence of fluorescence enhancement.

[0077] Breast cancer cells 4T1 and normal cells L02 were co-cultured with the nano-modulator ACP@CD. Figure 6 (a)-(c), confocal microscopy images (CLSM) and flow cytometry results show that strong fluorescence signals can be observed in 4T1 cells, but almost no fluorescence signals can be observed in L02 cells. These results indicate that the nanomodulator can effectively target tumor cells and accumulate in mitochondria, but cannot do so in normal cells, further verifying the therapeutic safety of the nanomodulator.

[0078] To gain a deeper understanding of the cytotoxicity of ACP@CD, we tested the in vitro cell viability of 4T1 cells and L02 cells using the MTT assay. Figure 7 (a) Even at a concentration of up to 200 μg / mL, no significant cytotoxicity was detected in L02 cells, which means that ACP@CD has excellent biosafety and low toxicity. Figure 7 (b) After co-incubation with 4T1 cells, the relative cell survival rates of the ACP-D group and the ACP@C group were 69.3% and 51.9%, respectively, indicating that the tumor killing ability of a single pathway is limited. In contrast, ACP@C caused more severe damage to tumor cells, with a cell survival rate of 30.3%, which may be due to the fact that Ca 2+ Overloading and DNA-mediated mitochondrial mineralization synergistically enhance the effects on mitochondrial dysfunction. Figure 7 (c), quantitative flow cytometric analysis of apoptosis further confirmed the therapeutic effect, ACP@CD caused significant necrosis of 4T1 cells (≈66.5%), while only about 45.6% and 30.0% of cells in the ACP@C and ACP-D groups were necrotized, respectively. In conclusion, ACP@CD, with its most efficient tumor-killing ability, will bring exciting opportunities to promote mitochondria-mediated cancer therapy.

[0079] 2.1.4.2 Mitochondrial damage

[0080] As mentioned above, ACP@CD will disintegrate in an acidic environment, so we used Ca 2+ Fluorescent probe (Fluo-4AM) was used to study the release of Ca from ACP@CD in living cells. 2+ If Figure 8 (a) We first monitored the intracellular Ca2+ expression in 4T1 cells at different time points (0, 1, 2, 4, and 6 h) after incubation with ACP@CD. 2+ With the extension of incubation time, the green fluorescence in the cells gradually increased, indicating that the intracellular Ca 2+ Then, if Figure 8 (b), we investigated the intracellular Ca2+ in 4T1 cells after treatment with different formulations. 2+ The CLSM results showed that after ACP@CD treatment, the green fluorescence in the mitochondria of cancer cells was the strongest, indicating that Ca 2+ The highest level.

[0081] In addition, if Fig. 9 (a), The distribution of mitochondria in cells was investigated by TMRM (tetramethylrhodamine methyl perchlorate) staining. Compared with other groups, cells treated with ACP@CD showed the weakest red fluorescence, indicating the least number of mitochondria and the most severe mitochondrial dysfunction. Fig. 9 As shown in (b), the mineralized cells have a wavelength of 563 cm -1 and 1033cm -1 There is an obvious absorption peak at PO4 3- The characteristic peaks of , indicating that calcium phosphate is deposited in the cells and calcium phosphate minerals are generated. Fig. 9 (c) shows that the intracellular Ca 2+ Compared with L02 cells, the content of Ca in 4T1 cells was detected. 2+ The concentration was higher and the biomineralization degree was higher, which is consistent with the results of previous studies.

[0082] Mitochondrial dysfunction is usually accompanied by the generation of ROS. In this regard, we used the DCFH-DA fluorescent probe to detect the level of ROS in cells after treatment with different formulas. Fig.10 As shown in (a)-(b), compared with the weak fluorescence of PBS, ACP, ACP-D or ACP@C groups, cells treated with ACP@CD showed the brightest green fluorescence, which is consistent with the results shown by flow cytometry, indicating that after the cells were treated with ACP@CD drugs, mitochondrial dysfunction occurred and a large amount of ROS was generated.

[0083] In addition, biological transmission electron microscopy (Bio-TEM) was used to observe the morphological changes of mitochondria in tumor cells in different treatment groups. Fig.11 As shown in (a), mitochondrial damage was most severe after ACP@CD treatment, with obvious swelling and cavitation of mitochondria, while the mitochondrial morphology of 4T1 cells was normal before treatment. Depolarization of mitochondrial membrane is often associated with the overflow of Cyto-c into the cytoplasm, which upregulates the expression of caspase-3. To this end, we detected the expression levels of proteins in cells after different treatments by Western blotting. Fig.11 As shown in (b)-(c), after treatment with different formulations, Caspase-3 in cells of all experimental groups was activated, and the most obvious one was in the ACP@CD group. 2+ Mitochondrial dysfunction due to overload and mitochondrial biomineralization has been demonstrated to promote efficient apoptosis in cancer cells.

[0084] 2.1.5 In vivo level

[0085] 2.1.5.1 Distribution in vivo

[0086] Inspired by the in vitro cytotoxicity and apoptosis studies, we further explored the tumor inhibitory effect of ACP@CD in vivo. Before biomedical applications, we first explored the accumulation and retention of ACP@CD in tumors. Fig.12 As shown in (a), the fluorescence signal of free Cy5.5-DNA gradually weakened over time, and almost no fluorescence signal of Cy5.5 was observed at the tumor site at 24 hours, indicating that free Cy5.5-DNA was quickly cleared by metabolic organs. However, within 2-8 hours after injection of ACP@CD, the fluorescence of Cy5.5 could be observed to gradually accumulate at the tumor site due to its enhanced permeability and retention (EPR) effect, making it a promising fluorescent imaging agent for direct tracking of tumor treatment. In addition, Fig.12 (b) Semi-quantitative analysis of fluorescence images of the tumor site showed that a clear Cy5.5 fluorescence signal appeared 2 h after ACP@CD injection, reached a peak at 8 h, and fluorescence could still be detected at the tumor site 48 h later. Fig.12 As shown in (c)-(d), the in vitro fluorescence images and semi-quantitative analysis after 48 hours showed that the tumors of mice in the ACP@CD group could emit strong fluorescence, indicating the effective accumulation and high retention of ACP@CD. In addition, obvious fluorescence could also be found in the liver and kidney, which may be due to the clearance effect of the endothelial reticular system.

[0087] 2.1.5.2 Evaluation of anti-tumor effects

[0088] The above experiments confirmed that ACP@CD has stronger tumor accumulation ability and better fluorescence imaging ability, so we were able to use the 4T1 tumor nude mouse model for anticancer evaluation. 3 The nude mice bearing 4T1 tumors were randomly divided into five groups (n=4 in each group). Fig.13 (a), ACP, ACP-D, ACP@C, and ACP@CD were intravenously injected on days 1, 3, and 5, respectively, and PBS was used as a control. To evaluate the antitumor efficacy, the body weight and tumor volume of the mice were measured serially.

[0089] like Fig.13 As shown in (b), after two weeks of observation (starting from the first day of injection), the tumors rapidly increased, and the tumor volume in the PBS group was 4 times the original. Although ACP-D and ACP@C have antitumor effects, their inhibitory effects are limited, reflecting that a single mitochondrial destruction pathway cannot produce satisfactory therapeutic effects. Compared with the other groups, ACP@CD can significantly reduce tumor burden and inhibit tumors for a long time, which may be due to the existence of three different factors: (1) pH-responsive degradation of ACP can effectively increase Ca 2+ concentration, thereby enhancing Ca 2+ Overload; (2) CUR can inhibit Ca 2+ efflux, by increasing endogenous Ca 2+ The concentration further enhanced the Ca 2+ (3) Mitochondrial-targeted Cy5.5 enriches DNA complexes in mitochondria, which can provide Ca 2+ Provides chelation sites, thereby promoting mitochondrial mineralization. Fig.13 As shown in (c), during the treatment, there was no significant change in body weight in each group, which proved the safety of the treatment. Fig.13 (e)-(f), the excellent therapeutic effect can be further visualized by comparing the tumor weight and tumor inhibition rate of the dissected tumor tissues after 14 days of monitoring. It is worth noting that the maximum inhibition rate of ACP@CD is 78.1%, which is higher than that of ACP-D (40.5%) or ACP@C (58.7%). Fig.13 (d) Photos of mice at the end of the treatment cycle and digital photos of ex vivo tumors showed that the tumors in the ACP@CD group were the smallest, fully demonstrating the good tumor inhibitory effect.

[0090] 2.1.5.3 Mechanism exploration

[0091] After treatment, tumor sections were histologically observed to understand pathological changes at the histological level. Fig.14, H&E staining results showed that the cells in the saline group and the ACP group had no obvious damage; on the contrary, severe tissue damage occurred in the ACP-D and ACP@C groups. The tissue damage in the ACP@CD group was the most serious. Similarly, the TUNEL assay results showed that ACP@CD caused the highest level of apoptosis and the largest number of green fluorescent spots in the field of view. ACP@CD can enhance mitochondrial dysfunction and activate the mitochondrial apoptosis pathway, thereby upregulating the expression of Caspase-3. Subsequently, we studied the expression of Caspase-3 by immunofluorescence. Immunofluorescence studies showed that the expression of Caspase-3 was higher after ACP@CD treatment, which was consistent with the results of Western blotting studies. In summary, ACP@CD can effectively inhibit cell proliferation and promote cell apoptosis by destroying mitochondria at multiple levels, and is expected to become an innovative calcium-based nanomedicine for tumor treatment.

[0092] 2.1.5.4 Security

[0093] Finally, the biosafety of the nanomodulator was tested by H&E staining of the main organs of mice in each treatment group. Fig.15 As shown, H&E staining of the main organs of mice in each treatment group showed no pathological abnormalities, indicating that ACP@CD and its mediated mitochondrial calcium mineralization therapy have good biosafety, which will be helpful for its future clinical application.

[0094] In summary, we successfully prepared nucleic acid-based Ca 2+ Nano-modulator ACP@CD, through mitochondrial Ca 2+ Overloading and mitochondrial biomineralization, realizing a multi-therapy approach to cancer treatment that focuses on promoting mitochondrial dysfunction. After systemic administration, this intracancer acid-responsive calcium nanomodulator can release Ca through material disintegration. 2+ and CUR inhibits Ca 2+ The combination of mitochondrial Ca 2+ and Ca 2+ can effectively enhance the Ca 2+ 2+ In particular, Cy5.5-DNA can efficiently aggregate in cancer cell mitochondria, and the nucleic acid framework can induce mitochondrial mineralization as a template for calcium phosphate mineralization in the mitochondrial matrix (pH = 8), which has never been reported before. Our results demonstrate that ACP@CD is an effective therapeutic agent with enhanced anticancer efficacy, reduced systemic toxicity, and good fluorescence. We believe that this precisely designed multifunctional Ca 2+ Nanomodulators have great potential for application in clinical practice for mitochondrial-targeted cancer therapy.

[0095] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A nucleic acid-based multi-channel Ca 2+ A method for preparing a nano modulator, characterized in that: The multi-channel Ca 2+ The nanomodulator was prepared by loading Cy5.5-DNA onto ACP@C, and the Cy5.5-DNA was 5′-GCG GACGACCTGGGTGCTTGCTCACGGTGTCGGGAGGCGG-Cy5.5-3′; The preparation method of ACP@C is as follows: 0.8-1.2 mL of curcumin CUR with a concentration of 4-6 mg / mL is added to 27-31 mL of 0.8-1.2 mM Ca(NO3)2·4H2O solution, stirred at room temperature for 0.5-1.5 h, and then 27-31 mL of 0.5-0.7 mM (NH4)2HPO4 solution is slowly added to the above solution.

2. The preparation method according to claim 1, characterized in that: The multi-channel Ca 2+ The preparation method of the nano-modulator is as follows: 45-55 μg ACP@C powder was dissolved in 0.5-1.5 mL 8-12 mM HEPES buffer, and then 1-3 μL 90-110 μM Cy5.5-DNA was added. The mixture was incubated at room temperature for 0.8-1.2 h, and the precipitated ACP@CD was collected by centrifugation. The ACP@CD is a nucleic acid-based multi-channel Ca 2+ Nanomodulators.

3. The preparation method according to claim 2, characterized in that: The usage amount of the ACP@C powder is 50 μg.

4. The preparation method according to claim 3, characterized in that: The HEPES buffer was used in an amount of 1 mL and a concentration of 10 mM.

5. The preparation method according to claim 4, characterized in that: The usage amount of the Cy5.5-DNA is 2 μL, and the concentration is 100 μM.

6. The preparation method according to claim 5, characterized in that: The room temperature incubation time is 1 h.

7. The preparation method according to claim 1, characterized in that: The concentration of the curcumin CUR is 5 mg / mL, and the volume is 1 mL; the concentration of the Ca(NO3)2·4H2O solution is 1 mM, and the volume is 29 mL; the concentration of the (NH4)2HPO4 solution is 0.67 mM, and the volume is 29 mL.

8. A nucleic acid-based multi-channel Ca2+ prepared by the preparation method according to any one of claims 1 to 7 2+ Nanomodulators.

9. The nucleic acid-based multi-channel Ca2+ system of claim 8. 2+ The use of a nano-modulator in the preparation of an anti-tumor drug is characterized in that: The tumor is 4T1 breast cancer.

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