Non-oxygen dependent photodynamic nano-material with M1 macrophage targeting function and application of non-oxygen dependent photodynamic nano-material

Abstract

The invention provides a non-oxygen dependent type photodynamic nano material with a function of targeting M1 macrophages and application of the non-oxygen dependent type photodynamic nano material. The non-oxygen dependent type photodynamic nano material comprises chitosan nano particles coated with porphyrin supramolecules and dextran sulfate sodium salt. The material regulates LSG excessive activation through a neural immune strategy of M1 macrophages in selective apoptosis left stellate ganglion (LSG), so as to prevent and treat malignant arrhythmia after myocardial infarction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology. Specifically, this invention relates to an oxygen-independent photodynamic nanomaterial with M1 macrophage targeting function and its application. Background Technology

[0002] Myocardial infarction is one of the most dangerous cardiovascular diseases, with its mortality primarily caused by malignant arrhythmias following the infarction. Post-infarction sympathetic overactivation is a major factor inducing malignant arrhythmias, and the left stellate ganglion is a key target point for sympathetic innervation of the heart. Currently, clinical treatment for LSG overactivation mainly involves LSG resection and nerve block, which primarily damage the nerves, easily leading to complications such as bleeding and Horner's syndrome. Furthermore, it eliminates the inherent physiological regulatory and protective functions of the nerves, limiting its widespread clinical application.

[0003] Studies have shown that sympathetic nerve activity can be regulated through neuroimmune interactions, and M1 macrophages are key participants in these interactions. Local depletion of M1 macrophages in the LSG can significantly reduce neuroinflammation and inhibit excessive LSG activation after myocardial infarction.

[0004] Preclinical studies typically employ macrophage depletion agents to consume macrophages. Besides commonly used clophosphate, antibodies and small molecules targeting specific receptors in macrophages are increasingly being used. However, these depletion agents still struggle to target only M1 macrophages in the LSG without consuming macrophages of the same phenotype in other tissues. Furthermore, macrophage depletion agents suffer from limitations such as the difficulty in real-time control of the degree of macrophage consumption and the regulation of the process through drug delivery systems. These shortcomings restrict the clinical application of macrophage consumption strategies in the field of sympathetic nervous system modulation.

[0005] Traditional photodynamic therapy generates reactive oxygen species (ROSs) under light irradiation, which can induce apoptosis and consumption of M1 macrophages, thereby inhibiting the overactivation of LSG and sympathetic nerves. The apoptosis process can be instantaneously controlled by switching on and off the light. However, the concentration of free oxygen in the tissue is usually not sufficient to sustain the needs of traditional photodynamic therapy. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems in related technologies. To this end, one objective of this invention is to provide a biodegradable nanomaterial with oxygen-independent and precisely targeting M1 macrophages, which regulates LSG overactivation through a neuroimmune strategy of selectively apoptotic M1 macrophages in the LSG, thereby preventing malignant arrhythmias after myocardial infarction. In this invention, after the nanoparticles are selectively taken up by M1 macrophages in the LSG, the process of generating photogenerated holes under near-infrared light does not require tissue oxygen participation, and the high oxidizing power of the photogenerated holes can induce M1 macrophage apoptosis. Furthermore, the degree of macrophage consumption can be precisely controlled in real time by a near-infrared light switch.

[0007] In one aspect, this invention proposes a nanomaterial that is oxygen-independent and precisely targets the function of M1 macrophages. According to an embodiment of the invention, the oxygen-independent and precisely targeted M1 macrophage nanomaterial comprises: chitosan nanoparticles coated with porphyrin supramolecular molecules and sodium dextran sulfate. This material regulates the overactivation of the left stellate ganglion (LSG) through a neuroimmune strategy of selectively apoptotic M1 macrophages in the LSG, thereby preventing malignant arrhythmias after myocardial infarction. In this invention, after the nanoparticles are selectively taken up by M1 macrophages in the LSG, the process of generating photogenerated holes under near-infrared light does not require tissue oxygen participation, and the high oxidizing power of the photogenerated holes can induce M1 macrophage apoptosis. Simultaneously, the degree of macrophage consumption can be precisely controlled in real time by a near-infrared light switch.

[0008] According to embodiments of the present invention, the above-mentioned oxygen-independent and precisely targeted M1 macrophage nanomaterials may further include at least one of the following additional technical features:

[0009] According to an embodiment of the present invention, the porphyrin is tetracarboxyphenylporphyrin.

[0010] According to an embodiment of the present invention, the chitosan has a molecular weight of 50-150 kDa.

[0011] In another aspect, the present invention provides a method for preparing oxygen-independent nanomaterials that precisely target the function of M1 macrophages. According to an embodiment of the present invention, the method includes:

[0012] S1: Porphyrin is dissolved in a strong base to obtain a porphyrin solution. A strong acid is added to the porphyrin solution to adjust the pH to neutral, thereby obtaining a porphyrin supramolecular photodynamic reagent.

[0013] S2: The porphyrin supramolecular photodynamic reagent is first mixed with sodium tripolyphosphate solution to obtain a mixed solution. The solution is then added to chitosan solution to obtain chitosan nanoparticles coated with porphyrin supramolecular molecules.

[0014] S3: A second mixing treatment is performed between sodium dextran sulfate and the chitosan nanoparticles coated with porphyrin supramolecular structures to obtain oxygen-independent nanomaterials that precisely target the function of M1 macrophages. This method is simple to operate. The material prepared according to this method regulates the overactivation of the left stellate ganglion (LSG) through a neuroimmunological strategy of selectively apoptotic M1 macrophages in the LSG, thereby preventing malignant arrhythmias after myocardial infarction. In this invention, after the nanoparticles are selectively taken up by M1 macrophages in the LSG, the process of generating photogenerated holes under near-infrared light does not require tissue oxygen participation. Furthermore, the high oxidizing power of the photogenerated holes can induce M1 macrophage apoptosis, and the degree of macrophage consumption can be precisely controlled in real time by a near-infrared light switch.

[0015] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0016] According to an embodiment of the present invention, the porphyrin is tetracarboxyphenylporphyrin.

[0017] According to an embodiment of the present invention, the strong base is KOH.

[0018] According to an embodiment of the present invention, the strong acid is HCl.

[0019] According to an embodiment of the present invention, the concentration of sodium tripolyphosphate is 2.0-4.0 mg / mL.

[0020] According to an embodiment of the present invention, the dosage of the porphyrin supramolecular photodynamic reagent is 5.0-10.0 μg / mL.

[0021] According to an embodiment of the present invention, the chitosan has a molecular weight of 50-150 kDa.

[0022] According to an embodiment of the present invention, the concentration of the chitosan solution is 0.25-0.8%.

[0023] According to an embodiment of the present invention, the dosage of sodium dextran sulfate is 2.0-10.0 μg / mL.

[0024] According to an embodiment of the present invention, the first mixing process is performed in the following manner:

[0025] The porphyrin supramolecular photodynamic reagent was mixed with sodium tripolyphosphate solution and then added dropwise to the chitosan solution.

[0026] According to an embodiment of the present invention, the second mixing process is performed in the following manner:

[0027] Sodium dextran sulfate and chitosan nanoparticles coated with porphyrin supramolecular molecules were rapidly stirred at low temperature, followed by low-speed centrifugation and washing.

[0028] According to an embodiment of the present invention, the low temperature is 0-5°C.

[0029] According to an embodiment of the present invention, the rapid stirring time is 30-90 minutes.

[0030] In another aspect of the invention, the invention also proposes the application of the aforementioned oxygen-independent and precisely targeted M1 macrophage function nanomaterials, or the oxygen-independent and precisely targeted M1 macrophage function nanomaterials prepared according to the aforementioned method, in the prevention and / or treatment of malignant arrhythmias.

[0031] According to embodiments of the present invention, the above application may further include at least one of the following additional technical features:

[0032] According to an embodiment of the present invention, the method includes injecting the aforementioned oxygen-independent and precisely targeting M1 macrophage function nanomaterial, or the oxygen-independent and precisely targeting M1 macrophage function nanomaterial prepared according to the aforementioned method, into the left stellate ganglion.

[0033] According to an embodiment of the present invention, the injection is performed via ultrasound-guided injection.

[0034] According to an embodiment of the present invention, the prevention and / or treatment of malignant arrhythmias is carried out by reducing LSG activity.

[0035] According to embodiments of the present invention, the present invention also proposes nanomaterials for the prevention and treatment of malignant arrhythmias after myocardial infarction using an oxygen-independent photodynamic system. According to embodiments of the present invention, the nanomaterials for the prevention and treatment of malignant arrhythmias after myocardial infarction using an oxygen-independent photodynamic system include nanoparticles coated with chitosan (CS) nanoparticles and loaded with sodium dextran sulfate (DSS) as a photodynamic reagent, wherein the oxidative holes generated under light irradiation can induce apoptosis.

[0036] According to an embodiment of the present invention, the outer layer of sodium dextran sulfate (DSS) can selectively target M1 macrophages. Therefore, after injection into the left stellate ganglion (LSG), it can selectively induce apoptosis of M1 macrophages in the LSG and reduce LSG activity after myocardial infarction.

[0037] According to an embodiment of the present invention, the amount of highly biocompatible PPS added is 50-100 μg / mL.

[0038] According to an embodiment of the present invention, the preparation of the nanoparticles includes the synthesis of a porphyrin supramolecular photodynamic reagent.

[0039] According to embodiments of the present invention, a method for preparing nanoparticles for preventing malignant arrhythmias after myocardial infarction during oxygen-independent photodynamic therapy is also provided. According to embodiments of the present invention, the method includes the following steps:

[0040] Synthesis of porphyrin supramolecular (PPS) photodynamic reagent: Porphyrin was dissolved under alkaline conditions, gradually adjusted to neutral pH, and then processed to obtain amorphous porphyrin supramolecular photodynamic reagent.

[0041] Preparation of nanoparticles: Porphyrin supramolecular and sodium tripolyphosphate were mixed and then added to a chitosan solution to form chitosan-coated porphyrin supramolecular nanoparticles (PPSC). Subsequently, they were reacted with sodium dextran sulfate, treated at low temperature, and then centrifuged and washed to obtain PPSCD nanoparticles.

[0042] According to an embodiment of the present invention, the porphyrin is tetracarboxyphenylporphyrin, the strong base is KOH, and the strong acid is HCl.

[0043] According to an embodiment of the present invention, the concentration of sodium tripolyphosphate is 2.0-4.0 mg / mL.

[0044] According to an embodiment of the present invention, the dosage of porphyrin supramolecular PPS is 5.0-10.0 μg / mL.

[0045] According to an embodiment of the present invention, the chitosan molecular weight is 50-150 kDa, and the chitosan solution concentration is 0.25-0.8%.

[0046] According to an embodiment of the present invention, the dosage of sodium dextran sulfate is 2.0-10.0 μg / mL.

[0047] According to an embodiment of the present invention, the aforementioned PPSCD is injected into LSG under ultrasound guidance to selectively consume M1 macrophages in LSG, reduce LSG activity after myocardial infarction, and prevent malignant arrhythmias.

[0048] This invention also provides the application of the aforementioned oxygen-independent and precisely targeted M1 macrophage function biodegradable nanomaterial in the prevention and treatment of malignant arrhythmias after myocardial infarction.

[0049] This invention discloses a non-oxygen-dependent and precisely targeted M1 macrophage function biodegradable nanomaterial (PPSCD) that significantly reduces the neural activity of the latent glomerular phage (LSG) after myocardial infarction. The nanomaterial is injected into the LSG under ultrasound guidance. Under near-infrared light irradiation, the nanoparticles generate highly oxidizing photogenerated holes, leading to apoptosis and consumption of M1 macrophages in the LSG. By reducing the release of pro-inflammatory cytokines, this invention aims to reduce LSG neural activity and prevent malignant arrhythmias after myocardial infarction.

[0050] Compared with the prior art, the present invention has at least one of the following significant advantages:

[0051] (1) The biodegradable nanomaterial (PPSCD) of the present invention has the function of being non-oxygen dependent and precisely targeting M1 macrophages. The oxidative vacancies generated by it under near-infrared light can induce apoptosis of M1 macrophages in LSG. Moreover, this process does not require tissue oxygen, thus solving the problem of dependence on tissue oxygen concentration in traditional photodynamic therapy.

[0052] (2) The biodegradable nanomaterial (PPSCD) of the present invention, which is non-oxygen dependent and precisely targets M1 macrophages, can selectively induce apoptosis of M1 macrophages in LSG and can precisely control the degree of apoptosis through light switching, thus solving the problem that traditional macrophage scavengers are difficult to quickly control the degree of macrophage consumption and the lack of control over the regulatory process. Attached Figure Description

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0054] Figure 1 Transmission electron microscope image of PPSCD nanomaterials;

[0055] Figure 2 Electrochemical impedance spectroscopy of PPSCD nanomaterials;

[0056] Figure 3 Photocurrent curves of PPSCD nanomaterials;

[0057] Figure 4 This demonstrates the selective uptake of PPSCD nanomaterials by M1 macrophages.

[0058] Figure 5 Selective apoptosis of M1 macrophages induced by PPSCD nanomaterials;

[0059] Figure 6 To assess the effectiveness of oxygen-independent photodynamic therapy for ventricular arrhythmias following acute myocardial infarction. Detailed Implementation

[0060] The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.

[0061] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0062] The present invention will now be described in further detail with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention in any way. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0063] Example 1

[0064] The preparation of a non-oxygen-dependent and precisely M1 macrophage-targeting degradable nanomaterial (PPSCD) includes the following steps: 0.5 g of tetracarboxyphenyl porphyrin is dissolved in 18 mL of KOH (1 M) solution, and 0.1 M HCl is added dropwise until the solution is neutral. After centrifugation, washing, and drying, porphyrin supramolecular photodynamic reagent (PPS) is obtained; the obtained PPS (7.5 μg / mL) is mixed with 2.5 mg / mL sodium tripolyphosphate solution, and added dropwise to 0.5% chitosan solution to obtain PPS-coated chitosan nanoparticles (PPSC); the obtained PPSC is rapidly stirred with 8.0 μg / mL sodium dextran sulfate at low temperature, and then centrifuged at low speed and washed to obtain PPSCD nanoparticles with dual functions of non-oxygen-dependent photodynamic therapy and M1 macrophage targeting.

[0065] Example 2

[0066] The preparation of a non-oxygen-dependent and precisely targeted M1 macrophage degradable nanomaterial (PPSCD) includes the following steps: 0.5 g of tetracarboxyphenyl porphyrin is dissolved in 18 mL of KOH (1 M) solution, and 0.1 M HCl is added dropwise until the solution is neutral. After centrifugation, washing, and drying, porphyrin supramolecular photodynamic reagent (PPS) is obtained; the obtained PPS (5.5 μg / mL) is mixed with 4.0 mg / mL sodium tripolyphosphate solution, and added dropwise to 1.0% chitosan solution to obtain PPS-coated chitosan nanoparticles (PPSC); the obtained PPSC is rapidly stirred with 10.0 μg / mL sodium dextran sulfate at low temperature, and then centrifuged at low speed and washed to obtain PPSCD nanoparticles with dual functions of non-oxygen-dependent photodynamic therapy and targeting M1 macrophages.

[0067] Example 3

[0068] RAW264.7 macrophages were incubated with 100 ng / mL lipopolysaccharide for 24 h to prepare an M1 type RAW264.7 macrophage model. M1 macrophages were incubated with PBS, PPS, CS, PPSC, and PPSCD (equivalent to 6 μg / mL PPSM) for 2 h, respectively. After fixation with 4% paraformaldehyde, the cells were stained with Hoechst 33342 and observed under confocal microscopy at 650 nm excitation. The results showed that compared with the PBS, CS, PPS, and PPSC groups, the uptake efficiency of M1 macrophages in the PPSCD group was significantly increased, and the red fluorescence intensity was stronger. Therefore, PPSCD has the ability to target M1 macrophages.

[0069] Example 4

[0070] RAW264.7 macrophages were infiltrated with 100 ng / mL lipopolysaccharide for 24 h to prepare an M1 type RAW264.7 macrophage model. M1 macrophages were divided into a control group (PBS only), a NIR light-only group, a PPSCD-only group, and a photodynamic (NIR+PPSCD) group. Flow cytometry results showed that there was no statistically significant difference in the apoptosis rate of M1 macrophages among the control group, the NIR light-only group, and the PPSCD-only group, while the apoptosis rate of M1 macrophages in the photodynamic group was significantly increased.

[0071] Example 5

[0072] Ten adult male beagle dogs were randomly divided into a control group (n=6) and a photodynamic therapy (PDT) group (n=6). An equal volume of PPSCD was injected into the LSG of the dogs in the PDT group via ultrasound-guided microneedle injection. Following NIR irradiation, LSG tissue temperature, neural activity, and function were measured at baseline and after myocardial infarction. Compared with the control group, the LSG activity in the PDT group was significantly reduced.

[0073] Example 6

[0074] Ten adult male beagle dogs were randomly divided into a control group (n=6) and a photodynamic therapy (PDT) group (n=6). An equal volume of PPSCD was injected into the left anterior descending coronary artery (LSG) of the dogs in the PDT group under ultrasound guidance to embolize the LSG and establish an acute myocardial infarction model. Post-infarction malignant arrhythmias were recorded. Compared with the control group, the PDT group showed a significant reduction in post-infarction malignant arrhythmias.

[0075] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0076] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A nanomaterial that is oxygen-independent and precisely targets the function of M1 macrophages, characterized in that, include: Chitosan nanoparticles coated with porphyrin supramolecular molecules and sodium dextran sulfate.

2. The oxygen-independent and precisely targeted M1 macrophage function nanomaterial according to claim 1, characterized in that, The porphyrin is tetracarboxyphenylporphyrin; Optionally, the chitosan has a molecular weight of 50-150 kDa.

3. A method for preparing oxygen-independent nanomaterials that precisely target the function of M1 macrophages, characterized in that, include: S1: Porphyrin is dissolved in a strong base to obtain a porphyrin solution. A strong acid is added to the porphyrin solution to adjust the pH to neutral, thereby obtaining a porphyrin supramolecular photodynamic reagent. S2: The porphyrin supramolecular photodynamic reagent is first mixed with sodium tripolyphosphate solution to obtain a mixed solution. The solution is then added to chitosan solution to obtain chitosan nanoparticles coated with porphyrin supramolecular molecules. S3: The sodium sulfated dextran salt is mixed with the chitosan nanoparticles coated with porphyrin supramolecular molecules in a second mixing process to obtain nanomaterials that are non-oxygen dependent and precisely target the function of M1 macrophages.

4. The method according to claim 3, characterized in that, The porphyrin is tetracarboxyphenylporphyrin; Optionally, the strong base is KOH; Optionally, the strong acid is HCl.

5. The method according to claim 3, characterized in that, The concentration of sodium tripolyphosphate is 2.0-4.0 mg / mL; Optionally, the dosage of the porphyrin supramolecular photodynamic reagent is 5.0-10.0 μg / mL; Optionally, the chitosan has a molecular weight of 50-150 kDa; Optionally, the concentration of the chitosan solution is 0.25-0.8%.

6. The method according to claim 3, characterized in that, The dosage of sodium dextran sulfate is 2.0-10.0 μg / mL.

7. The method according to claim 3, characterized in that, The first mixing process is performed in the following manner: The porphyrin supramolecular photodynamic reagent was mixed with sodium tripolyphosphate solution and then added dropwise to the chitosan solution.

8. The method according to claim 3, characterized in that, The second mixing process is carried out in the following manner: Sodium dextran sulfate and chitosan nanoparticles coated with porphyrin supramolecular molecules were rapidly stirred at low temperature, followed by low-speed centrifugation and washing. Optionally, the low temperature is 0-5°C; Optionally, the rapid stirring time is 30-90 minutes.

9. The use of the oxygen-independent and precisely targeting M1 macrophage function nanomaterial as described in claim 1 or 2, or the oxygen-independent and precisely targeting M1 macrophage function nanomaterial prepared by the method according to any one of claims 3-8, in the prevention and / or treatment of malignant arrhythmias.

10. The application according to claim 9, characterized in that, This includes injecting the oxygen-independent and precisely targeted M1 macrophage nanomaterials as described in claim 1 or 2, or the oxygen-independent and precisely targeted M1 macrophage nanomaterials prepared by the method according to any one of claims 3-8, into the left stellate ganglion; Optionally, the injection is performed via ultrasound guidance; Optionally, the prevention and / or treatment of malignant arrhythmias is carried out by reducing LSG activity.

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