Manganese metal complex, manganese metal nano platform, preparation method and application

Abstract

The invention provides a metal manganese complex, a metal manganese nano platform, a preparation method and application, and belongs to the technical field of chemical synthesis and biomedicine. The structural formula of the manganese complex is shown as a formula I. The method comprises the following steps: dropwise adding a MnCl2 aqueous solution into a THF solution containing Ce6, quickly stirring, carrying out ultrasonic treatment, and sufficiently aging to obtain preliminary Mn-Ce6NPs; and the Mn-Ce6NPs obtained in the first step is subjected to dialysis and centrifugation, and the metal manganese complex Mn-Ce6NPs is obtained. The nanoparticles have excellent chemiluminescence performance, and can specifically recognize the position of a suppository block. The prepared nanoparticles have a photodynamic and photo-thermal combined thrombolysis effect, and have an anti-oxidation function after thrombolysis, so that the purpose of treating ischemic stroke is achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of chemical synthesis and biomedical technology, and particularly relates to a metal manganese complex, a metal manganese nano platform, a preparation method and application. BACKGROUND

[0002] The high mortality rate of cardiovascular and cerebrovascular diseases urgently needs to develop non-invasive and controllable treatment strategies. Photodynamic therapy (PDT) and photothermal therapy (PTT) have shown potential in the field of thrombolysis due to their precise spatiotemporal control ability, but both have inherent defects: PDT is limited by tissue oxygen concentration and light source penetration depth, while PTT faces the risk of thermal damage and low energy conversion efficiency. Studies have shown that the synergistic integration of PTT and PDT can enhance local oxygen supply and promote drug penetration through thermal effects, achieving complementary synergies, but existing phototherapy agents still face bottlenecks such as poor biocompatibility, low photosensitizer loading efficiency, and uncontrollable metabolism.

[0003] Manganese-based nanoszymes have become an ideal candidate material for breaking through the above limitations due to their multivalent redox properties (Mn 2+ / Mn 3+ / Mn 4+ ) and enzyme-like catalytic activity. Compared with noble metal materials, its advantages are: ultra-long triplet state lifetime (> 100 μs) and high photothermal conversion efficiency (> 40%) can simultaneously enhance ROS generation and thermal energy output; manganese has innate biological safety as an essential element for the human body; and it can form a multifunctional composite system by stable loading of organic photosensitizers (such as Ce6) through coordination. However, traditional manganese-based nanomaterials have problems such as uneven size, uncontrollable surface chemistry, and unclear in vivo metabolic pathways, which seriously restrict their clinical translation. SUMMARY

[0004] The purpose of the present application is to provide a metal manganese complex, a metal manganese nano platform, a preparation method and application, which is a stimulus-responsive composite nanoparticle (Mn-Ce6NPs) constructed by the coordination of the carboxyl group of chlorin e6 (Ce6) and divalent manganese ions. The nanoparticle undergoes a specific ligand displacement reaction with overexpressed glutathione in the thrombus microenvironment, achieving controlled release of Ce6, thereby simultaneously activating the fluorescence imaging function and photodynamic therapy (PDT) effect at the lesion site. The preparation can realize the integration of thrombus-targeted therapy and fluorescence imaging, significantly improving the accuracy of diagnosis and treatment.

[0005] The present application first provides a metal manganese complex, the structural formula of which is shown as formula 1:

[0006]

[0007] The present application further provides a preparation method of a metal manganese complex, comprising the following steps:

[0008] Step one: add Ce6 into THF solution to obtain THF solution containing Ce6;

[0009] Step two: drop MnCl2 into THF solution containing Ce6 drop by drop, stir rapidly, and ultrasonic, and then obtain preliminary Mn-Ce6 NPs after aging sufficiently;

[0010] Step three: dialysis and centrifugation are performed on the preliminary Mn-Ce6 NPs obtained in step two to obtain metal manganese complex Mn-Ce6 NPs.

[0011] Preferably, the stirring temperature in step two is 20-30℃, and the stirring time is 15-24h.

[0012] Preferably, the aging temperature in step two is 20-30℃, and the aging time is 15-24h.

[0013] Preferably, the molar ratio of MnCl2 to Ce6 in step two is 1:1.

[0014] Preferably, the dialysis temperature in step three is 20-30℃, and the dialysis time is 15-24h.

[0015] Preferably, the centrifugation speed in step three is 3500-5000r / min, and the centrifugation time is 5-10min.

[0016] The application further provides a metal manganese nano platform comprising the metal manganese complex.

[0017] The application further provides a preparation method of the metal manganese nano platform, comprising:

[0018] The metal manganese complex Mn-Ce6 NPs is added into DMSO, RGD is added, and the mixture is stirred at room temperature, and then dialysis purification is performed to obtain the metal manganese nano platform Mn-Ce6@RNPs.

[0019] The application further provides application of the metal manganese nano platform in preparation of a drug for treating ischemic cerebral stroke.

[0020] Advantages of the application

[0021] 1. The application provides a metal manganese complex, a metal manganese nano platform, a preparation method and application, wherein the metal manganese complex is formed by coordination of divalent manganese ions (Mn 2+ ) and chlorin e6 (Ce6) through carboxyl sites to form stable nanoparticles (Mn-Ce6 NPs). 2+ The Ce6 molecules in the metal manganese complex of the application are coordinated to Mn 2+The coordination network realizes high-density loading (>80% encapsulation rate), induces red shift of the absorption spectrum (>15 nm), and improves the photo-thermal conversion efficiency (>40%);

[0022] 2. Response release: The nanoparticles of the application specifically respond to glutathione (GSH concentration ≥10 mM) in the thrombus microenvironment, trigger coordination bond rupture, and controllably release Ce6, synchronously activating fluorescence imaging (650 nm emission) and photodynamic effect (ROS quantum yield ≥0.35);

[0023] 3. Treatment mechanism: The released Mn 2+ By clearing superoxide anions through SOD-like activity, and further regulating H2O2 levels through hydrogen peroxide-like or Fenton-like reactions, a self-regulating redox balance is established, which cooperates with photo-thermal ablation (ΔT>25℃) and PDT to achieve thrombus-targeted dissolution (in vitro thrombolytic rate >90%, in vivo thrombus mass reduction >75%). Experimental data show that the material can significantly improve the neurological function score of ischemic stroke model mice (mNSS decreased by ≥4 points) and reduce the cerebral infarction volume (reduced by >60%), confirming its clinical application value as a multi-modal diagnosis and treatment platform in thrombotic diseases. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Infrared spectrum of Mn-Ce6 NPs prepared for Example 1 of the application;

[0025] Figure 2 UV absorption spectrum of Mn-Ce6 NPs prepared for Example 1 of the application in aqueous solution;

[0026] Figure 3 Fluorescence emission spectrum of Mn-Ce6 NPs prepared for Example 1 of the application in aqueous solution;

[0027] Figure 4 Photo-thermal data of Mn-Ce6 NPs prepared for Example 1 of the application in aqueous solution;

[0028] Figure 5 Thermal imaging data of Mn-Ce6 NPs prepared for Example 1 of the application in aqueous solution;

[0029] Figure 6 Chemiluminescence property chart of Mn-Ce6 NPs prepared for Example 1 of the application;

[0030] Figure 7 In vitro targeting test chart of Mn-Ce6 NPs prepared for Example 1 of the application;

[0031] Figure 8 Antioxidant capacity test chart of Mn-Ce6@R NPs prepared for the application;

[0032] Figure 9 The hemolysis test diagram of the Mn-Ce6@R NPs prepared by the application;

[0033] Figure 10 The thrombolysis data diagram of the Mn-Ce6@R NPs prepared by the application under different conditions;

[0034] Figure 11 The photo-thermal imaging diagram of the thrombus position of the Mn-Ce6@R NPs prepared by the application in mice;

[0035] Figure 12 The chemiluminescence imaging diagram of the thrombus position of the Mn-Ce6@R NPs prepared by the application in mice.

[0036] Figure 13 The thrombolysis diagram of the Mn-Ce6@R NPs prepared by the application in mice.

[0037] Figure 14 The laser speckle diagram of the Mn-Ce6@R NPs prepared by the application in mice. DETAILED DESCRIPTION

[0038] The application first provides a metal manganese complex, the molecular formula is C 98 H 97 MnN 12 O 18 , the relative molecular mass is 1784.64 g / mol, and the structural formula is shown as formula 1:

[0039]

[0040] The application further provides a preparation method of the metal manganese complex, including the following steps:

[0041] Step one: Ce6 is added into a THF solution to obtain a THF solution containing Ce6;

[0042] Step two: MnCl2 is added drop by drop into the THF solution containing Ce6, and then rapid stirring and ultrasonic treatment are performed, and after sufficient aging, the preliminary Mn-Ce6 NPs are obtained;

[0043] Step three: the preliminary Mn-Ce6 NPs obtained in step two are subjected to dialysis and centrifugation to obtain the metal manganese complex Mn-Ce6 NPs.

[0044] According to the application, the MnCl2 is added drop by drop into the THF solution containing Ce6, and rapid stirring is carried out, the stirring temperature is preferably 20-30 DEG C, the stirring time is preferably 15-24 h, and ultrasonic treatment is carried out, the ultrasonic treatment time is preferably 3-5 min, and aging is carried out, the aging temperature is preferably 20-30 DEG C, and the aging time is preferably 15-24 h, to obtain the preliminary Mn-Ce6 NPs; the molar ratio of MnCl2 to Ce6 is preferably 1:1. -1 .

[0045] According to the application, the preliminary Mn-Ce6 NPs obtained above are subjected to dialysis, the dialysis bag has a pore size of 1000 molecular weight, the dialysis temperature is preferably 20-30 DEG C, and the dialysis time is preferably 15-24 h, to obtain the metal manganese complex Mn-Ce6 NPs containing large aggregates.

[0046] According to the application, the metal manganese complex Mn-Ce6 NPs containing large aggregates obtained above are subjected to centrifugation, the centrifugation speed is preferably 3500-5000 r / min, and the centrifugation time is preferably 5-10 min, to obtain the metal manganese complex Mn-Ce6 NPs after removing the large aggregates.

[0047] The application further provides a metal manganese nano platform, which comprises the metal manganese complex.

[0048] The application further provides a preparation method of the metal manganese nano platform, which comprises:

[0049] The metal manganese complex Mn-Ce6 NPs are added into DMSO, RGD is added, and stirring is carried out at room temperature, and the stirring time is preferably 12 h, to obtain the Mn-Ce6@RNPs through dialysis purification.

[0050] The application further provides an application of the metal manganese nano platform in the preparation of a drug for treating ischemic stroke.

[0051] The application is further described by the following examples, which do not limit the application in any way, and any modification or change made by those skilled in the art without departing from the technical solutions of the application will fall within the scope of the claims of the application.

[0052] Example 1: Preparation of Mn-Ce6 NPs, the preparation process is shown in Figure 1 Figure A:

[0053] Step one: Ce6 is added into a THF solution to obtain a THF solution containing Ce6.

[0054] Step 2: Add MnCl2 aqueous solution (0.16 mg, 9.9 mL) dropwise to Ce6 (5 mg, mL) -1 The sample was placed in a 0.1 mL THF solution and stirred rapidly for 15 h, followed by sonication for 3 min. After aging at 25 °C for 15 h, preliminary Mn-Ce6NPs were obtained.

[0055] Step 3: Dialyze the preliminary Mn-Ce6NPs obtained in Step 2. The dialysis bag has a pore size of 1000 molecular weight and the dialysis time is 15 h to obtain the manganese metal complex Mn-Ce6NPs containing large aggregates.

[0056] Step 4: Centrifuge the manganese metal complex Mn-Ce6NPs containing large aggregates obtained in Step 3 (3500 r / min, 5 min) to remove the large aggregates, yielding the manganese metal complex Mn-Ce6NPs. The infrared spectrum is shown below. Figure 1 As shown in B.

[0057] Comparative Example 1

[0058] The preparation process and conditions were the same as in Example 1, except that in step two, the MnCl2 aqueous solution (0.24 mg, 9.9 mL) was added dropwise to the Ce6 (5 mg mL⁻¹, 0.1 mL) THF solution, that is, the molar ratio of MnCl2 to Ce6 was 1.5:1.

[0059] Comparative Example 2

[0060] The preparation process and conditions were the same as in Example 1, except that in step two, the MnCl2 aqueous solution (0.48 mg, 9.9 mL) was added dropwise to the Ce6 (5 mg mL⁻¹, 0.1 mL) THF solution, that is, the molar ratio of MnCl2 to Ce6 was 3:1.

[0061] Comparative Example 3

[0062] The preparation process and conditions were the same as in Example 1, except that in step two, the MnCl2 aqueous solution (0.8 mg, 9.9 mL) was added dropwise to the Ce6 (5 mg mL⁻¹, 0.1 mL) THF solution, that is, the molar ratio of MnCl2 to Ce6 was 5:1.

[0063] The performance of the manganese metal complex prepared in Example 1 was characterized as follows:

[0064] 1. Photophysical properties of manganese metal complexes

[0065] The photophysical properties of Mn-Ce6NPs in this invention were determined in aqueous solution. Figures 2-3The UV absorption spectrum and fluorescence emission spectrum of the Mn-Ce6 NPs prepared in Example 1 and Comparative Examples 1-3 of the present application (wherein the ratio represents the molar ratio of MnCl2 and Ce6, and it is found from the figure that the absorption of the 1:1 ratio is significantly higher than that of other ratios after the red shift of the UV absorption peak, so we choose the 1:1 ratio for further testing), it can be seen from the figure that the Mn-Ce6 NPs show bright red light emission in aqueous solution, and the emission peak position is 650 nm. Its UV absorption spectrum shows two typical metal iridium complex absorption bands, and the strong UV absorption band is mainly due to the spin-allowed π-π* transition of the ligand, and the relatively weak absorption band is due to the charge transfer from metal to ligand (3MLCT). Figure 2 It can be seen from the UV absorption spectrum that after the preparation of manganese salt, the absorption intensity at 689 nm is significantly improved compared with Ce6, Figure 3 It can be seen from the fluorescence emission spectrum that the emission peak position is basically unchanged.

[0066] 2. Photothermal properties of metal manganese complex:

[0067] Figure 4 For the photothermal properties of Mn-Ce6 NPs of the present application, (A figure) compared with the control group, the drug group has better photothermal production capacity, and the concentration dependence of Mn-Ce6 NPs (B figure) under different concentrations, (C figure) under different powers, the stronger the power, the higher the heat production and temperature rise, and (D figure) after heating and cooling for five cycles, it can be seen that the drug has good photothermal cycling capacity.

[0068] Figure 5 For the thermal imaging figure corresponding to the photothermal data of Mn-Ce6 NPs, the solution temperature increases with the extension of illumination time under light condition.

[0069] 3. Chemiluminescence experiment of Mn-Ce6 NPs:

[0070] Figure 6 For the related data characterization of the chemiluminescence of Mn-Ce6 NPs of the present application. First, 6A figure can be seen that in the case of responding to different ROS, ONOO - has the strongest luminescence intensity, ONOO - as a marker substance can produce chemiluminescence in response to the drug, and light up the inflammatory site. 6B figure is a dot plot of chemiluminescence intensity at different concentrations, showing that the drug has the highest intensity of bioluminescence emission at 689 nm. Then 6C figure gives the measurement results of ONOO - and drug coexistence at different ratios, which shows a linear correlation. 6D figure shows that ONOO -The chemiluminescence intensity decreases over time when coexisting with the drug. Figure 6E shows that when chicken breast of varying thicknesses is used to cover the drug, the chemiluminescence penetration depth can reach up to 10 mm. This high penetration depth is beneficial for imaging tests of the drug at deep inflammatory sites. This has significant advantages over photosensitizers that are dependent on short-wavelength excitation light.

[0071] Figure 7 This invention provides an in vitro targeting assay for Mn-Ce6@R NPs. The obtained Mn-Ce6NPs were added to DMSO, along with 1 mg of RGD. The mixture was stirred at room temperature for 12 hours, and then purified by dialysis to obtain Mn-Ce6@R NPs (wherein Mn-Ce6@R NPs are Mn-Ce6NPs modified with -RGD). The drug was co-incubated with a thrombus, and the chemiluminescence intensity was measured at 2 hours, 4 hours, and 6 hours. The longer the co-incubation time with the drug, the higher the chemiluminescence intensity, demonstrating that the drug has good targeting ability for thrombi.

[0072] 4. Antioxidant performance experiment of Mn-Ce6@R NPs:

[0073] Figure 8 To test the antioxidant capacity of Mn-Ce6@R NPs of this invention, a kit was used for detection. When the drug concentration reached 1000 μg / mL, the DPPH consumption rate reached 70%. Figure 8 A). Co-incubation with hydrogen peroxide showed a consumption rate of nearly 60% ( Figure 8 B). The hydrogen peroxide content at the thrombus site is much higher than that in normal tissue. The consumption of hydrogen peroxide will effectively improve the maintenance of the thrombus site. At the same time, it is demonstrated that Mn-Ce6@R NPs have good antioxidant capacity and can have a good therapeutic effect on inflammation in the body.

[0074] 5. Hemolysis test of Mn-Ce6@R NPs:

[0075] Figure 9 For the hemolysis test of Mn-Ce6@R NPs of this invention, different concentrations of Mn-Ce6@R NPs (Figure A, where the small images in Figure A correspond to hemolysis photos at different concentrations on the horizontal axis) and Ce6 (Figure B) were incubated with red blood cells in hot water at 37°C for 2 hours. The PBS group was used as the negative control group and the water group was used as the positive control group. The hemolysis rate of the drug at 100 μg / mL was still less than 5%, which shows good biocompatibility.

[0076] 6. In vitro thrombolysis experiment of Mn-Ce6@R NPs:

[0077] Figure 10For the in vitro thrombolytic test of the Mn-Ce6@R NPs of the application, the Mn-Ce6 NPs group can simultaneously open the PDT / PTT dual-mode thrombolysis under laser irradiation compared with the control group, Figure 10 Figure A shows that the thrombolytic rate reaches about 70%, Figure 10 Figure B shows the absorbance of hemoglobin and fibrin after thrombolysis, wherein PBS+L represents the thrombolysis of a thrombus in PBS under light irradiation, UK represents the thrombolysis of a thrombus in UK, and Mn-Ce6@R NPs+L represents the thrombolysis of a thrombus in the Mn-Ce6@R NPs solution under light irradiation. The experimental results show that the Mn-Ce6@R NPs have good in vitro thrombolytic capacity.

[0078] 7. Mouse in vivo experiment of the Mn-Ce6@R NPs:

[0079] Figure 11 For the in vivo photothermal imaging test of the Mn-Ce6@R NPs of the application, when the drug is accumulated at the thrombus position, the temperature at the thrombus position gradually increases with the light irradiation time recorded by an infrared thermal imager.

[0080] Figure 12 For the in vivo chemiluminescence imaging of the Mn-Ce6@R NPs of the application, after tail vein administration, the drug circulates to the thrombus position with the extension of time, and the chemiluminescence phenomenon is obvious under light irradiation, and the accumulation degree reaches the maximum at 60 min, and the thrombolytic treatment can be started.

[0081] Figure 13 For the in vivo thrombolytic test of the Mn-Ce6@R NPs of the application, after the thrombus is formed in vivo, the drug circulates to the thrombus position after administration, and the thrombus position is irradiated with laser, and the in vivo thrombolytic rate reaches 72%, which has good thrombolytic effect.

[0082] Figure 14 For the record of blood flow when the Mn-Ce6@R NPs of the application are used for laser speckle monitoring of the thrombolysis process, after the thrombus is formed in vivo, the thrombolytic rate of the Mn-Ce6@R NPs group gradually increases with the extension of light treatment time, the thrombus gradually dissolves, and the blood flow gradually recovers. Unlike the common thrombolytic drug UK enzyme, which may cause secondary embolization after thrombolysis, leading to a decrease in blood flow, the administration group Mn-Ce6@R NPs does not appear to have re-embolization in thrombolysis, which proves that the drug has good PDT / PTT capacity to effectively prevent re-embolization and performs well in in vivo treatment.

Claims

1. A manganese metal complex, characterized in that, Its structural formula is shown in Equation 1:

2. The method for preparing a manganese metal complex according to claim 1, characterized in that, Includes the following steps: Step 1: Add Ce6 to the THF solution to obtain a THF solution containing Ce6; Step 2: Add MnCl2 dropwise to a THF solution containing Ce6, stir rapidly, sonicate, and then age thoroughly to obtain preliminary Mn-Ce6NPs; Step 3: Dialyze and centrifuge the preliminary Mn-Ce6NPs obtained in Step 2 to obtain the manganese metal complex Mn-Ce6NPs.

3. The method for preparing a manganese metal complex according to claim 2, characterized in that, The stirring temperature in step two is 20-30℃, and the stirring time is 15-24h.

4. The method for preparing a manganese metal complex according to claim 2, characterized in that, The aging temperature in step two is 20-30℃, and the aging time is 15-24 hours.

5. The method for preparing a manganese metal complex according to claim 2, characterized in that, The molar ratio of MnCl2 to Ce6 in step two is 1:

1.

6. The method for preparing a manganese metal complex according to claim 2, characterized in that, The dialysis temperature described in step three is 20-30℃, and the dialysis time is 15-24h.

7. The method for preparing a manganese metal complex according to claim 2, characterized in that, The centrifugation speed in step three is 3500-5000 r / min, and the time is 5-10 min.

8. A metallic manganese nanoplatform, comprising the metallic manganese complex of claim 1.

9. The method for preparing the manganese nanoplatform according to claim 8, characterized in that, include: The above-mentioned manganese complex Mn-Ce6NPs were added to DMSO, RGD was added, and the mixture was stirred at room temperature and purified by dialysis to obtain the manganese nanoplatform Mn-Ce6@RNPs.

10. The use of the manganese nanoplatform of claim 9 in the preparation of drugs for treating ischemic stroke.

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