Preparation method and application of artificial peroxisome LOX-Ce6-Mn nanoparticles

Abstract

The invention discloses a preparation method of an artificial peroxisome LOX-Ce6-Mn and an application of the artificial peroxisome LOX-Ce6-Mn. Belongs to the technical field of nano materials and tumor treatment, and is prepared by the following steps: uniformly mixing prepared DCC, NHS, Ce6 and LOX in proportion, reacting, dialyzing by using a dialysis bag, adding an aqueous solution of MnCl2, adjusting the pH value, dialyzing, and drying to obtain the LCM nanoparticles. The lactate oxidase activity, the OXD activity, the POD activity and the CAT activity of the LCM nanoparticles can form a cascade reaction to cause tumor cells to generate efficient active oxygen and consume lactic acid and glutathione in the tumor, so that autophagy and apoptosis of the tumor cells occur; in addition, H2O2 generated through LOX catalysis can be converted into high-toxicity hydroxyl free radicals through Mn < 2 + > mediated Fenton-like reaction, and oxidative damage of cancer cells is further amplified. The LCM nanoparticles have tumor specificity for killing tumor cells, can be effectively accumulated at tumor parts, have a weak killing effect on normal parts, and have good biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials and tumor treatment, and relates to a preparation method and application of artificial peroxisomal LOX-Ce6-Mn (LCM) nanoparticles; specifically, it relates to a preparation method and application of artificial peroxisomal LOX-Ce6-Mn nanoparticles with multi-enzyme activity and tumor treatment effect; that is, a multifunctional artificial peroxisomal LOX-Ce6-Mn (LCM) that can inhibit tumor growth through self-enhanced chemical dynamics (CDT) and photodynamics (PDT) combined therapy, and its preparation method and application. . Background Art

[0002] Malignant tumors have high morbidity and mortality, posing a serious threat to human health. New treatments and therapeutic drugs are urgently needed. Innovative treatments based on nanomedicines have been widely studied due to their unique advantages over traditional treatments. These include chemodynamic therapy (CDT), photothermal therapy (PTT), and photodynamic therapy (PDT) to overcome the challenges faced by traditional medicine. The complexity, diversity, and heterogeneity of cancer make a single treatment method difficult, and the development of multimodal synergistic cancer treatment strategies has become a research hotspot.

[0003] Peroxisome is a special organelle that is a regulator of H2O2 in cells. It usually contains one or more enzymes, mainly oxidase, peroxidase, catalase and other enzymes that have H2O2 metabolism. Artificial peroxisomes are of great significance in disease treatment and are expected to replace natural peroxisomes.

[0004] Studies have shown that lactate is not a metabolic waste, but a source of energy for cells. Consuming endogenous intratumoral lactate has become a promising direction for tumor metabolic therapy. LOX has attracted widespread interest in cancer diagnosis and treatment due to its specific catalytic effect on lactate, and its catalytic process generates a large amount of H2O2 and pyruvate. These H2O2 can serve as substrates for nanozymes to exert enzyme-like activity, further improving enzyme-like catalytic activity.

[0005] Lactate oxidase, as a natural enzyme, has high selectivity, but is easily denatured and inactivated; metal catalysts are broad-spectrum and highly efficient, but the reaction conditions are harsh and it is difficult to achieve high selectivity. Combining the respective advantages of enzymes and metal catalysts, metal catalysis can be carried out under milder and greener conditions, allowing multiple steps of enzyme catalysis and metal catalysis to be completed simultaneously in one reactor and under one condition. This new catalytic mode achieves targeted transformation of enzymes, allowing enzymes to show considerable potential in various application scenarios. Summary of the invention

[0006] In view of the above problems, the present invention aims to provide an artificial peroxisome LOX-Ce6-Mn (LCM nanoparticle) with multi-enzyme activity and anti-tumor therapeutic effect, as well as a preparation method and application thereof.

[0007] The technical scheme of the present invention is: a method for preparing artificial peroxisome LOX-Ce6-Mn nanoparticles described in the present invention, and its preparation steps are as follows: comprising first mixing prepared N,N'-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), photosensitizer (dihydrochlorin Ce6), and natural enzyme (lactate oxidase LOX) in proportion, reacting for 24 hours, dialyzing with a dialysis bag, and then dropwise adding an aqueous solution of a metal salt (manganese chloride MnCl2), and then adjusting the pH with sodium hydroxide (NaOH), and obtaining LOX-Ce6-Mn nanoparticles after dialysis and drying; namely, LCM nanoparticles.

[0008] Furthermore, the synthesis method uses one of solvothermal method, self-assembly or biomineralization for synthesis, and preferably uses biomineralization to combine the compounds.

[0009] Further, the mass ratio of DCC to NHS is 2:1-3; preferably 2:1;

[0010] The mass ratio of the natural enzyme to the photosensitizer is 5:3-6, preferably 5:3.

[0011] Furthermore, a suitable photosensitizer is used to make the synthesized nanoparticles have the effect of PDT, and the photosensitizer is selected from one of porphyrin, indocyanine dye or dihydrochlorin E6 (Ce6), etc., preferably dihydrochlorin E6 (Ce6) as a near-infrared photosensitizer for synthesizing peroxisomes;

[0012] The nanoparticles are endowed with natural enzyme-like activity by using biological enzymes having natural enzyme activity. The natural enzyme is selected from one of glucose oxidase, lactate oxidase (LOX), catalase, cholesterol oxidase or urate oxidase, etc., preferably lactate oxidase.

[0013] Furthermore, the specifications of the dialysis bag include 500DA, 3000DA, 10000DA, etc., and a 3000DA dialysis bag is preferably used for dialysis;

[0014] The dialysis time is 8 hours to 36 hours, and the preferred dialysis time is 24 hours.

[0015] Furthermore, artificial peroxisomes with multi-enzyme activity are synthesized by doping metals, wherein the metal salt is selected from one or two metal salts such as ferric chloride, manganese chloride (MnCl2), and copper chloride, and manganese chloride is preferably used as the metal salt for doping peroxisomes.

[0016] Furthermore, the concentration of the aqueous solution containing the metal salt is 5-10 mg / mL, preferably 5 mg / mL.

[0017] Furthermore, the concentration of the NaOH solution used in the synthesis is 0.1-5 mol / L, preferably 1 mol / L NaOH solution;

[0018] During the synthesis process, the pH of the solution is adjusted to 11-13, preferably to 12.

[0019] Furthermore, the drying method is one of oven drying, vacuum drying and freeze drying, and freeze drying is preferred.

[0020] Furthermore, the present invention also provides LCM nanoparticles prepared by the preparation method.

[0021] Furthermore, the LCM nanoparticles prepared by the present invention have uniform particle size distribution and good stability.

[0022] Furthermore, the LCM nanoparticles prepared by the present invention have oxidase activity, peroxidase activity, catalase activity and lactate oxidase activity; can produce singlet oxygen under 660nm excitation light; and effectively generate ROS.

[0023] Furthermore, the LCM nanoparticles prepared by the present invention can produce effective toxicity and efficient ROS to tumor cells at the cellular level.

[0024] Furthermore, an artificial peroxisome LOX-Ce6-Mn nanoparticle was prepared and used in anti-tumor treatment.

[0025] Furthermore, the LCM nanoparticles prepared by the present invention have lactate oxidase-like activity, OXD-like activity, POD-like activity, and CAT-like activity, and are artificially synthesized peroxisomes with multi-enzyme activity, and have ultra-high affinity for the substrate H2O2, which can not only catalyze H2O2 in the tumor microenvironment to produce a large amount of ·OH to kill tumor cells, but also produce 1 O2, enhances the level of tumor oxidative stress and causes tumor cell apoptosis; at the same time, it has CAT-like activity to relieve intratumoral hypoxia and enhance the combined therapeutic effect of CDT and PDT; in addition, lactate oxidase can also consume intratumoral lactic acid; these effects can form a self-cascade platform to induce tumor cell autophagy and greatly enhance the killing effect on tumors.

[0026] The beneficial effects of the present invention are as follows: 1. The LCM nanoparticles of the present invention simultaneously have lactate oxidase activity, OXD-like, POD-like, and CAT-like activities, and these four enzyme activities can be connected in series to form a self-enhanced enzyme cascade reaction; firstly, the LCM nanoparticles have high-intensity OXD and POD activities, show extremely high affinity for H2O2, and catalyze the conversion of H2O2 into highly toxic ROS; at the same time, the LCM nanoparticles play a role of PDT under 660nm excitation light, and have a high efficiency of singlet oxygen generation rate. The combined action of CDT and PDT amplifies the oxidative stress level of tumor cells and enhances the effect of promoting tumor cell apoptosis; in addition, the LCM nanoparticles can also generate oxygen by playing a CAT activity, relieve tumor hypoxia, and enhance the effect of PDT; and the LCM nanoparticles consume lactic acid through lactate oxidase, The H2O2 produced thereby makes up for the deficiency of H2O2 in the tumor; 2. The LCM nanoparticles of the present invention have a strong effect of promoting tumor cell apoptosis at the cellular level by consuming lactic acid and acting in combination with CDT and PDT, effectively promoting ROS accumulation and inducing tumor cell autophagy; 3. The LCM nanoparticles of the present invention were injected into mice through the tail vein, and the tumor sites of the mice were irradiated with 660nm laser after a certain period of time, showing a more significant inhibitory effect on tumor growth than other control groups, proving that the LCM nanoparticles can exert a strong anti-tumor effect under the combined action of consuming lactic acid and CDT and PDT; 4. The LCM nanoparticles of the present invention have good biocompatibility. In the mouse experiment, the HE staining results of the main organs showed that the material had no long-term toxicity to mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a TEM image of the LCM nanoparticles prepared in Example 1 of the present invention;

[0028] Figure 2 is a particle size distribution diagram of LCM nanoparticles in an embodiment of the present invention;

[0029] Figure 3 is a structural diagram of the OXD and POD-like activities of LCM nanoparticles using TMB as a chromogenic substrate in an embodiment of the present invention;

[0030] Figure 4 is a structural diagram of the CAT activity of LCM nanoparticles using H2O2 as a substrate in an embodiment of the present invention;

[0031] Figure 5 is a structural diagram of the LOX activity of LCM nanoparticles using lactic acid as a substrate in an embodiment of the present invention;

[0032] Figure 6 is a diagram showing the relative cell viability of 4T1 cells after incubating them with Ce6 and LCM nanoparticles under normal conditions and irradiating them with 660 nm in an embodiment of the present invention;

[0033] Figure 7 is a diagram of the cell viability structure of the same treatment under hypoxic conditions in an embodiment of the present invention;

[0034] Figure 8 It is a fluorescence image of ROS generated by LCM nanoparticles at the cellular level under normal conditions and hypoxic conditions, respectively, using DCFH-DA and DHE as probes in the embodiment of the present invention;

[0035] Fig. 9 This is a graph showing changes in body weight and tumor volume of mice in different groups treated with LCM nanoparticles in vivo for anti-tumor therapy in an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The specific technical scheme of the present invention is further described in detail below with reference to specific examples.

[0037] Example 1 is a method for preparing LCM nanoparticles:

[0038] Step (1), dissolving DCC, NHS and photosensitizer Ce6 in DMSO in sequence to obtain solution A;

[0039] Step (2), after magnetic stirring for 1 hour, adding an aqueous solution of natural enzyme LOX to obtain solution B;

[0040] Step (3), after magnetic stirring for 24 hours, dialyze with a 3000DA dialysis bag for 24 hours to obtain solution C;

[0041] Step (4), dropwise adding an aqueous solution containing a metal salt MnCl2, magnetically stirring for 1 hour after the addition, to obtain a solution D, and adjusting the pH of the solution D to 12 with a 1 mol / L NaOH solution; after magnetic stirring for 24 hours, dialyzing with a 3000DA dialysis bag for 24 hours, and freeze-drying to obtain a product with a loose texture; namely, LCM nanoparticles.

[0042] The mass ratio of DCC to NHS used was 2:1, the concentration of MnCl2 solution was 5 mg / mL, and the mass ratio of LOX to Ce6 was 5:3.

[0043] Example 2 is the morphology characterization of LCM nanoparticles

[0044] Figure 1 is the transmission electron microscopy image of LCM nanoparticles;

[0045] Figure 2 is the particle size distribution diagram of LCM nanoparticles;

[0046] Depend on Figure 1 It can be seen that the LCM nanoparticles prepared by the above method are granular, have good dispersibility and uniform particle size distribution.

[0047] Depend on Figure 2 The particle size of the obtained LCM nanoparticles is 40nm-50nm.

[0048] In summary, the morphology analysis and particle size analysis by transmission electron microscopy and particle size analysis confirmed that the final product prepared was LCM nanoparticles, and it had good dispersibility and uniform particle size distribution, indicating that LCM nanoparticles can be prepared using the method provided by the present invention.

[0049] Example 3 is the detection of various enzyme activities of LCM nanoparticles

[0050] Figure 3 Schematic diagram of OXD- and POD-like activities of LCM nanoparticles using TMB as a chromogenic substrate;

[0051] Figure 4 Schematic diagram of CAT activity of LCM nanoparticles with H2O2 as substrate;

[0052] Figure 5 Schematic diagram of LOX activity of LCM nanoparticles using lactate as substrate;

[0053] The detection method of the POD-like activity of LCM nanoparticles is as follows: TMB was used as a substrate to measure the POD-like activity of LCM in the presence of H2O2; briefly, NaAc (pH = 4.55), TMB, LCM and H2O2 were mixed at room temperature, and a control group was set up, and the absorbance at 652 nm was measured in a 96-well plate at the same time after the same time;

[0054] Depend on Figure 3 It was found that LCM nanoparticles could convert the colorless TMB substrate into a blue oxidized state, indicating that LCM nanoparticles have effective oxidase and peroxidase activities, providing a strong basis for CDT treatment;

[0055] The CAT-like activity of LCM nanoparticles was detected as follows: CAT activity of LCM was measured in HAc-NaAc buffer solution (pH 7.0) at 37°C in the presence of H2O2 (0.4 M); the oxygen content in the solution was measured using a portable dissolved oxygen meter;

[0056] Depend on Figure 4 It can be seen that when the concentration of H2O2 remains unchanged, the higher the concentration of LCM nanoparticles, the more oxygen is produced, indicating that LCM nanoparticles can react with H2O2 to produce oxygen, which is expected to alleviate tumor hypoxia;

[0057] The method for detecting the LOX activity of LCM nanoparticles using lactic acid as a substrate is as follows: using 2 mmol / L lactic acid as a substrate, PBS, LOX (1 mg / mL), and LCM (1 mg / mL) are used to react with lactic acid in a 15 mL ultrafiltration tube, and the concentration of lactic acid in the system after different reaction times is detected using a lactic acid detection kit;

[0058] Depend on Figure 5 It was found that both LCM nanoparticles and LOX could effectively consume lactate compared with the Control group.

[0059] In summary, LCM nanoparticles have oxidase activity, peroxidase activity, catalase activity and lactate oxidase activity.

[0060] Example 4 is the effect of LCM nanoparticles on tumor cell viability and proliferation ability and the verification of ROS generation at the cellular level

[0061] Figure 6 The relative cell viability of 4T1 cells after incubation with Ce6 and LCM nanoparticles under normal conditions and irradiation with laser irradiation (660 nm, 3 min);

[0062] Figure 7 is the relative cell viability of 4T1 cells after incubation with Ce6 and LCM nanoparticles under hypoxic conditions and irradiation with laser irradiation (660 nm, 3 min);

[0063] Figure 8 Fluorescence images of ROS generated by LCM nanoparticles at the cellular level under normal and hypoxic conditions, detected by DCFH-DA and DHE as probes, respectively;

[0064] The method for detecting the cytotoxicity of LCM nanoparticles to tumor cells at the cellular level is as follows: the cytotoxicity was evaluated by CCK-8 method; 4T1 cells were seeded in 96-well plates at a density of 1×10 4 Cells were cultured to 80% confluence; then the medium was replaced with different concentrations of LCM, Ce6, LCM+H2O2, and LCM+Lac for 24 h under normoxic and hypoxic conditions; after that, the cells were washed with PBS, incubated in fresh medium, and irradiated with 660 nm laser for 3 min per well; then the medium was separated, fresh medium with 10% CCK-8 was added, and cultured for another 2 h; finally, the absorbance at 450 nm was recorded in a 96-well plate;

[0065] Depend on Figure 6 and Figure 7It can be seen that the toxicity of different treatments on 4T1 cells showed concentration dependence; and when H2O2 and Lac coexisted with LCM, the cytotoxicity was significantly higher than that of LCM alone under normoxic conditions, and the toxicity was further enhanced under 660nm laser irradiation, proving that the toxicity of LCM to 4T1 cells was greatly enhanced under the combined action of consuming lactic acid and CDT and PDT; it is worth noting that Ce6 showed toxicity to laser-irradiated cells under normoxic or hypoxic conditions, indicating that the efficiency of PDT is highly dependent on oxygen content; although the cytotoxicity was reduced under hypoxic conditions, laser-irradiated LCM showed the best cytotoxicity under normal and hypoxic conditions; this shows that oxygen content has little effect on the toxicity of LCM, suggesting that its own oxygen production capacity enhances cytotoxicity;

[0066] The method for detecting the ability of LCM to generate ROS under different treatments at the cellular level is as follows: 4T1 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin; for fluorescence imaging, 4T1 cells (5×10 per well) were placed in 4 Cells) were seeded in 12-well plates for 24 h and then incubated with LCM (12.5 μg / mL), Ce6 (3.125 μg / mL), LCM (12.5 μg / mL) + H2O2 (300 μM), and LCM (1

[0067] 2.5μg / mL)+Lac (200μg / mL) was incubated under normoxia and hypoxia for 24h; then the culture medium was discarded and washed with PBS; after the cells were irradiated with 660nm laser for 3min, they were stained with DCFH-DA (30μM) and DHE (30μM) in the dark for 20min respectively; the DCF fluorescence excitation wavelength was 488nm, and the DHE fluorescence excitation wavelength was 535nm.

[0068] Depend on Figure 8 It can be seen that when there is no excitation light irradiation and H2O2 is used as a substrate, LCM can produce a small amount of ROS, indicating that LCM can react with H2O2 to produce ROS; however, the ROS produced by LCM under excitation light irradiation is more significant, indicating that LCM nanoparticles can promote the accumulation of ROS in tumor cells under the combined action of consuming lactic acid and CDT and PDT.

[0069] In summary, LCM nanoparticles consume lactic acid and produce oxygen through the combined effects of CDT and PDT to relieve tumor hypoxia, and produce a large amount of ROS to amplify the oxidative stress of the tumor, ultimately promoting tumor apoptosis.

[0070] Example 5 is a study on the anti-tumor effect of LCM nanoparticles in vivo

[0071] Fig. 9The graphs of tumor volume changes in mice of different groups treated with LCM nanoparticles in vivo for anti-tumor effects;

[0072] The specific implementation steps are as follows: 4-week-old female tumor-bearing mice were randomly divided into 5 groups (n=5): (1) control group (2) Ce6+light group (3) LOX group (4) LCM group (5) LCM+light group, and LCM (15 mg / kg) was intravenously injected every other day, and light treatment was performed 6 hours and 24 hours after intravenous injection; the tumor size and body weight were measured and recorded every 2 days; after 16 days, the tumor and the main organs of the mice were removed and fixed with 4% paraformaldehyde solution; H&E staining, immunofluorescence staining and immunohistochemistry staining were used, and the paraffin blocks were embedded with a section thickness of 8 μm for histological evaluation.

[0073] Depend on Fig. 9 It was found that during the entire treatment process, the tumors in the PBS group and the free Ce6 + light irradiation group grew rapidly, and at the end of the treatment, the tumors were so large that the mice could not bear it; in addition, the tumor volume in the free LOX group and the LCM group showed a significant trend of shrinking; especially in the LCM + laser irradiation group, the tumor almost disappeared after the treatment; the high tumor inhibition rate was due to the combined therapeutic effects of lactic acid consumption and CDT, PDT; the free LOX and LCM groups had obvious inhibitory effects on tumors, which may be due to the effects of lactic acid consumption and CDT, respectively.

[0074] In summary, compared with PDT treatment alone and CDT treatment alone, lactate consumption produced by LCM and combined treatment with CDT and PDT can better inhibit tumor cell proliferation and mediate cell apoptosis; lactate consumption produced by LCM and combined treatment with CDT and PDT can better inhibit tumor cell proliferation and mediate cell apoptosis.

[0075] In summary, a strategy different from the current conventional treatment method for inducing tumor cell apoptosis is to consume intratumoral lactic acid and realize self-enhanced CDT and PDT therapy to treat tumors, induce tumor cell autophagy, increase tumor oxidative stress, and promote tumor cell apoptosis; LCM nanoparticles are constructed by LOX, Ce6, and Mn through a biomimetic mineralization method; LOX can consume intratumoral lactic acid and reduce the energy supply of tumor cells; Mn participates in the Fenton reaction to produce ROS, and Ce6 produces singlet oxygen under the action of PDT. The two together promote the accumulation of ROS in the tumor, enhance the level of oxidative stress in the tumor, and finally lead to tumor cell apoptosis; experiments have confirmed that LCM can also produce oxygen to relieve intratumoral hypoxia, and LCM can also induce tumor cell autophagy under the combined action of consuming lactic acid and CDT and PDT; finally, the increase of oxidative stress and the occurrence of autophagy further amplify the inhibitory effect on tumors; in addition, in vitro and in vivo experiments have confirmed that the synthesized LCM nanoparticles can achieve effective accumulation in the tumor site, have good biocompatibility, and have good application prospects in the field of CDT and PDT treatment.

Claims

1. A method for preparing artificial peroxisomal LOX-Ce6-Mn nanoparticles, characterized in that: The preparation steps are as follows: the prepared DCC, NHS, photosensitizer and natural enzyme are mixed evenly in proportion, after reacting for 24 hours, dialyzed with a dialysis bag, and then an aqueous solution containing a metal salt is added dropwise, and then the pH value is adjusted with a NaOH solution. After dialysis and drying, LOX-Ce6-Mn nanoparticles, namely LCM nanoparticles, are obtained.

2. The method for preparing artificial peroxisome LOX-Ce6-Mn nanoparticles according to claim 1, characterized in that: During the preparation process, one of solvothermal method, self-assembly or biomineralization is selected for synthesis.

3. The method for preparing artificial peroxisome LOX-Ce6-Mn nanoparticles according to claim 1, characterized in that: The mass ratio of DCC to NHS is 2:1-3; The mass ratio of the natural enzyme to the photosensitizer is 5:3-6.

4. The method for preparing artificial peroxisome LOX-Ce6-Mn nanoparticles according to claim 1, characterized in that: The photosensitizer is selected from one of porphyrin, indocyanine dye or dihydrochlorin E6; The natural enzyme is selected from one of glucose oxidase, lactate oxidase, catalase, cholesterol oxidase or urate oxidase.

5. The method for preparing artificial peroxisome LOX-Ce6-Mn nanoparticles according to claim 1, characterized in that: The specifications of the dialysis bag include 500DA, 3000DA, and 10000DA; The duration of dialysis is 8 hours to 36 hours.

6. The method for preparing artificial peroxisomal LOX-Ce6-Mn nanoparticles according to claim 1, characterized in that: The metal salt is selected from one or two of ferric chloride, manganese chloride or copper chloride.

7. The method for preparing artificial peroxisome LOX-Ce6-Mn nanoparticles according to claim 1, characterized in that: The concentration of the aqueous solution containing the metal salt is 5-10 mg / mL.

8. The method for preparing artificial peroxisome LOX-Ce6-Mn nanoparticles according to claim 1, characterized in that: The concentration of the NaOH solution used is 0.1-5 mol / L; The pH value of the solution is adjusted to 11-13 during the synthesis process.

9. The method for preparing artificial peroxisomal LOX-Ce6-Mn nanoparticles according to claim 1, characterized in that: The drying method is one of oven drying, vacuum drying and freeze drying.

10. Use of artificial peroxisome LOX-Ce6-Mn nanoparticles prepared by any one of the preparation methods described in claims 1-9 in anti-tumor treatment.

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