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

Abstract

The present invention discloses a preparation method and application of artificial peroxisomes LOX-Ce6-Mn; belonging to the field of nanomaterials and tumor treatment technology, the preparation steps are: firstly uniformly mixing prepared DCC, NHS, Ce6, and LOX in proportion, dialyzing with a dialysis bag after the reaction, adding an aqueous solution of MnCl2, adjusting the pH value, dialyzing, and drying to obtain LCM nanoparticles. In the present invention, the lactate oxidase activity, oxidase activity (OXD), peroxidase activity (POD), and catalase activity (CAT) of the LCM nanoparticles can constitute a cascade reaction, causing tumor cells to produce efficient reactive oxygen and consume intratumoral lactate and glutathione, causing tumor cells to undergo autophagy and apoptosis; in addition, the H2O2 catalyzed by LOX can be transported by Mn 2+ The mediated Fenton-like reaction converts hydroxyl radicals into highly toxic free radicals, further amplifying the oxidative damage of cancer cells. LCM nanoparticles are tumor-specific in killing tumor cells, effectively accumulating in tumor sites, with little killing effect on normal sites, and have good biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterials and tumor treatment technology, 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 therapeutic effects; namely, a multifunctional artificial peroxisomal LOX-Ce6-Mn (LCM) capable of inhibiting tumor growth through self-enhanced chemodynamic therapy (CDT) and photodynamic therapy (PDT) combined therapy, as well as its preparation method and application. Background Art

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

[0003] Peroxisomes are specialized organelles that regulate intracellular H2O2. They typically contain one or more enzymes, primarily oxidases, peroxidases, and catalases, which metabolize H2O2. Artificial peroxisomes hold great promise in disease treatment and hold promise as a replacement for natural peroxisomes.

[0004] Research has shown that lactate is not a metabolic waste product but rather a source of energy for cells. Depleting endogenous intratumoral lactate has become a promising approach for metabolic tumor therapy. LOX has attracted widespread interest in cancer diagnosis and treatment due to its specific catalytic activity towards lactate. The catalytic process generates large amounts of H2O2 and pyruvate. This H2O2 can serve as a substrate for nanozymes to exert their enzyme-like activity, further enhancing their catalytic activity.

[0005] Lactate oxidase, a natural enzyme, is highly selective but easily denatured and inactivated. Metal catalysts are broad-spectrum and highly efficient, but the reaction conditions are harsh, making it difficult to achieve high selectivity. Combining the respective advantages of enzymes and metal catalysts allows metal catalysis to be carried out under milder and greener conditions, allowing multiple steps of enzyme-catalyzed and metal-catalyzed reactions to be completed simultaneously in a single reactor under the same conditions. This new catalytic model enables targeted modification of enzymes, allowing enzymes to demonstrate 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 solution of the present invention is: a method for preparing artificial peroxisomal LOX-Ce6-Mn nanoparticles described in the present invention, the preparation steps of which are as follows: first, the prepared N,N'-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), photosensitizer (dihydrochlorin Ce6), and natural enzyme (lactate oxidase LOX) are evenly mixed in proportion, after reacting for 24 hours, dialyzed with a dialysis bag, and then dropwise added with an aqueous solution of a metal salt (manganese chloride MnCl2), and then the pH is adjusted with sodium hydroxide (NaOH). After dialysis and drying, LOX-Ce6-Mn nanoparticles are obtained; namely, LCM nanoparticles.

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

[0009] Furthermore, 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 allow the synthesized nanoparticles to have a PDT effect, wherein the photosensitizer is selected from one of porphyrin, indocyanine dye or dihydrochlorin E6 (Ce6), 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 the dialysis bag of 3000DA is preferably used for dialysis;

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

[0015] Furthermore, artificial peroxisomes with multi-enzyme activity are synthesized by doping metals, wherein the metal salts are 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, preferably freeze drying.

[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. They are artificially synthesized peroxisomes with multiple enzyme activities and have ultra-high affinity for the substrate H2O2. They 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; it also 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 constitute 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; first, the LCM nanoparticles have high-intensity OXD and POD activities, show extremely high affinity for H2O2, and catalyze H2O2 into highly toxic ROS; at the same time, the LCM nanoparticles play a PDT role under 660nm excitation light, have a high efficiency of singlet oxygen generation rate, and the combined effect 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 exerting CAT activity, alleviate tumor hypoxia, and enhance the effect of PDT; and the LCM nanoparticles consume lactic acid through lactate oxidase, which is The generated H2O2 compensates for the lack of H2O2 within the tumor; 2. The LCM nanoparticles of the present invention have a powerful apoptotic effect on tumor cells at the cellular level through the combined effects of lactic acid consumption and CDT / PDT, effectively promoting ROS accumulation and inducing autophagy in tumor cells; 3. The LCM nanoparticles of the present invention were injected into mice via the tail vein. After a certain period of time, the tumor sites of the mice were irradiated with a 660nm laser, showing a more significant tumor growth inhibition effect than other control groups, demonstrating that the LCM nanoparticles can exert a powerful anti-tumor effect under the combined effects of lactic acid consumption and CDT / PDT; 4. The LCM nanoparticles of the present invention have good biocompatibility. In mouse experiments, HE staining results of major 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 This is a graph showing the relative cell viability of 4T1 cells after incubation with Ce6 and LCM nanoparticles under normal conditions and irradiation with 660 nm in an embodiment of the present invention;

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

[0034] Figure 8 1 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 an embodiment of the present invention;

[0035] Figure 9 Graph showing changes in body weight and tumor volume of mice in different groups treated with LCM nanoparticles for in vivo anti-tumor therapy according to an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The specific technical solutions of the present invention are 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), adding an aqueous solution containing a metal salt MnCl2 dropwise, and magnetically stirring for 1 hour after the addition is completed 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 loose product; i.e., 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 and particle size analysis by transmission electron microscopy and particle size analysis confirmed that the final product obtained was LCM nanoparticles, and its dispersibility was good and the particle size distribution was uniform, indicating that LCM nanoparticles can be prepared using the method provided by the present invention.

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

[0050] Figure 3 Schematic diagram of the 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 using H2O2 as substrate;

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

[0053] The POD-like activity of LCM nanoparticles was detected as follows: TMB was used as a substrate and the POD-like activity of LCM was measured 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. After the same time, the absorbance at 652 nm was measured in a 96-well plate.

[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 H2O2 concentration 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: PBS, LOX (1 mg / mL), and LCM (1 mg / mL) were reacted with lactic acid in a 15 mL ultrafiltration tube, respectively, using 2 mmol / L lactic acid as a substrate. The lactic acid concentration in the system after different reaction times was 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 production at the cellular level

[0061] Figure 6 Figure 2 shows the relative cell viability of 4T1 cells after incubation with Ce6 and LCM nanoparticles under normal conditions and irradiation with laser (660 nm, 3 min).

[0062] Figure 7 Figure 2 shows the relative cell viability of 4T1 cells after incubation with Ce6 and LCM nanoparticles under hypoxic conditions and irradiation with laser (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 using DCFH-DA and DHE as probes, respectively;

[0064] The cytotoxicity of LCM nanoparticles to tumor cells was detected at the cellular level as follows: CCK-8 assay was used to evaluate cytotoxicity; 4T1 cells were seeded in 96-well plates at a density of 1×10 4 Cells were cultured to 80% confluence. The medium was then replaced with various concentrations of LCM, Ce6, LCM+H2O2, and LCM+Lac for 24 h under normoxic and hypoxic conditions. The cells were then washed with PBS, incubated in fresh medium, and irradiated with a 660 nm laser for 3 min per well. The medium was then removed, and fresh medium with 10% CCK-8 was added, followed by an additional 2 h of incubation. Finally, absorbance was recorded at 450 nm in a 96-well plate.

[0065] Depend on Figure 6 and Figure 7It was found 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 under normoxic conditions than when LCM was present alone, and the toxicity was further enhanced under 660nm laser irradiation, proving that the toxicity of LCM on 4T1 cells was greatly enhanced under the combined action of lactate consumption 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 both 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 ROS generation ability of LCM under different treatments at the cellular level was 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 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] The cells were incubated with 2.5 μg / mL of dapoxetine (2.5 μg / mL) and 200 μg / mL of Lac under normoxia and hypoxia for 24 h. The culture medium was then discarded and the cells were washed with PBS. The cells were irradiated with 660 nm laser for 3 min and then stained with DCFH-DA (30 μM) and DHE (30 μM) in the dark for 20 min, respectively. The DCF fluorescence excitation wavelength was 488 nm, and the DHE fluorescence excitation wavelength was 535 nm.

[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 generated 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] Figure 9Figure 2 is the change of tumor volume in mice of different groups treated with LCM nanoparticles in vivo;

[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; tumor size and body weight were measured and recorded every 2 days; after 16 days, the tumor and major 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 and the slices were sliced ​​with a thickness of 8 μm for histological evaluation.

[0073] Depend on Figure 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. At the end of the treatment, the tumors were too large for the mice to bear. In addition, the tumor volume in the free LOX group and the LCM group showed a significant trend of shrinkage. Especially in the LCM + laser irradiation group, the tumor almost disappeared after the end of treatment. The high tumor inhibition rate was due to the combined therapeutic effects of lactic acid consumption and CDT and 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, the combined treatment of lactate consumption produced by LCM and CDT, PDT can better inhibit tumor cell proliferation and mediate cell apoptosis; the combined treatment of lactate consumption produced by LCM and CDT, PDT can better inhibit tumor cell proliferation and mediate cell apoptosis.

[0075] In summary, a strategy different from the current conventional treatment method of inducing tumor cell apoptosis is to deplete intratumoral lactate and achieve self-enhanced CDT and PDT for tumor treatment, thereby inducing tumor cell autophagy, increasing tumor oxidative stress, and promoting tumor cell apoptosis. LCM nanoparticles are constructed from LOX, Ce6, and Mn through a biomimetic mineralization method. LOX can deplete intratumoral lactate 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. These two together promote the accumulation of ROS in the tumor, enhance the level of oxidative stress in the tumor, and ultimately lead to tumor cell apoptosis. Experiments have confirmed that LCM can also produce oxygen to alleviate intratumoral hypoxia. Moreover, the combined action of LCM depletion of intratumoral lactate and CDT and PDT can also induce tumor cell autophagy. Finally, the increased oxidative stress and the occurrence of autophagy further amplify the inhibitory effect on tumors. In addition, in vitro and in vivo experiments confirmed that the synthesized LCM nanoparticles can effectively accumulate at the tumor site and have good biocompatibility, showing good application prospects in the field of CDT and PDT treatment.

Claims

1. A method for preparing artificial peroxisome LOX-Ce6-Mn nanoparticles, characterized in that: The preparation steps include: uniformly mixing the prepared DCC, NHS, photosensitizer, and natural enzyme in proportion, reacting for 24 hours, dialyzing with a dialysis bag, and then dropwise adding an aqueous solution containing a metal salt. The pH value is then adjusted with a NaOH solution. After dialysis and drying, LOX-Ce6-Mn nanoparticles, i.e., LCM nanoparticles, are obtained. Wherein, the DCC is N,N'-dicyclohexylcarbodiimide; NHS is N-hydroxysuccinimide; The photosensitizer is chlorin e6; The natural enzyme is lactate oxidase LOX; The metal salt is manganese chloride.

2. The method for preparing artificial peroxisome LOX-Ce6-Mn nanoparticles according to claim 1, characterized in that: During the preparation process, 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 specification of the dialysis bag is 500DA, 3000DA or 10000DA; The duration of dialysis is 8 hours to 36 hours.

5. 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.

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

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

8. Use of artificial peroxisome LOX-Ce6-Mn nanoparticles prepared by the method according to any one of claims 1 to 7 in the preparation of anti-tumor drugs.

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