Porphyrin derivative iron complex nanoparticles and preparation method and application thereof

Abstract

The present invention provides a porphyrin derivative iron complex nanoparticle and its preparation method and application, belonging to the field of chemical synthesis and biomedical technology. The structural formula of the porphyrin derivative iron complex nanoparticle is as shown in Formula 1. The method mixes an FeCl3 aqueous solution with an aqueous solution of 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid, performs ultrasonic oscillation, and obtains a mixed solution; the mixed solution is placed under a magnetic stirring environment, so that the porphyrin derivative iron complex nanoparticle is fully aged to obtain an aged solution; the aged solution is dialyzed to obtain porphyrin derivative iron complex nanoparticle. The nanoparticles of the present invention have good photothermal and photodynamic properties, realize non-invasive photothermal / photodynamic synergistic thrombolysis, and simultaneously acts as a chemiluminescent substrate and acts on reactive oxygen species in the thrombus microenvironment for imaging, realizing integrated thrombosis diagnosis and treatment.

Description

Technical Field

[0001] The invention belongs to the technical fields of chemical synthesis and biomedicine, and particularly relates to porphyrin derivative iron complex nanoparticles and a preparation method and application thereof. Background Art

[0002] Thrombotic cardiovascular and cerebrovascular diseases, such as stroke, myocardial infarction and venous thromboembolism, are one of the serious diseases with the highest morbidity and mortality worldwide. Due to the high recurrence rate and mortality of thrombosis, there is an urgent need to study anti-thrombotic strategies. Surgical removal of blood clots is the main treatment method in clinical practice, but it often causes recurrence of thrombosis. Currently, intravenous thrombolytic drugs are a basic strategy to effectively relieve clinical symptoms and surgical risks. However, the half-life of traditional drugs is very short (usually only a few minutes), and most anti-thrombotic drugs have to be repeatedly administered in high doses within a short period of time, leading to adverse bleeding complications and serious neurotoxic effects. Therefore, finding effective methods to achieve efficient, safe and long-term thrombosis treatment is the focus and frontier of thrombosis diagnosis and treatment research.

[0003] Phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), is a non-invasive thrombolytic therapy modality that has attracted increasing attention due to its good efficacy and low drug resistance, and is considered an effective thrombolytic treatment method. Through the Landau damping effect, PTT converts light energy into heat energy, physically decomposing fibrin clots. At the same time, reactive oxygen species (ROS)-mediated PDT can destroy the fibrin skeleton of the blood clot, thereby preventing the fragments generated by PTT from causing secondary embolism in microvessels. Therefore, PTT / PDT synergistic thrombolytic therapy has been recognized as an effective non-invasive synergistic thrombolytic therapy mode. However, the current photosensitizers capable of PTT / PDT synergistic thrombolytic therapy are still limited by the low tissue penetration depth caused by the short excitation wavelength, making it difficult to achieve clinical application. Therefore, how to design and obtain photosensitizers that can achieve PTT / PDT synergistic thrombosis diagnosis and treatment under long-wavelength light source excitation remains a huge challenge.

[0004] To achieve precise diagnosis and treatment, in vivo imaging has become an indispensable technique in thrombosis treatment. Traditional imaging modalities, such as fluorescence imaging and photoacoustic imaging, require laser irradiation and are subject to interference from tissue autofluorescence. Chemiluminescence is a process in which reactants, products, or intermediates in a chemical reaction absorb the energy generated by the reaction, transition from the ground state to an excited state, and then release photons from the excited state back to the ground state. Chemiluminescence is a novel, real-time, optically independent imaging modality. It avoids autoluminescence noise interference, significantly improving the imaging signal-to-noise ratio and detection sensitivity, making it a promising approach for disease diagnosis. However, existing chemiluminescent photosensitizers still face several challenges, including poor biosafety, instability, and short-wavelength excitation. In recent years, semiconductor polymers and ethylenedioxyethane-based chemiluminescent materials have attracted considerable attention. Although their biocompatibility and functionality have been greatly improved, they are still limited by their short emission wavelength and high hydrophobicity. Therefore, developing near-infrared chemiluminescent materials with high biosafety and amenable functionalization is crucial for achieving high-contrast deep tissue imaging and clinical applications.

[0005] Porphyrins and porphyrin derivatives exhibit excellent photodynamic and photothermal properties due to their high absorption capacity in the red region, thus showing promising application in phototherapy. Furthermore, porphyrins and porphyrin derivatives can be used as chemiluminescent substrates to interact with oxidative stress factors in the disease microenvironment for imaging, enabling integrated diagnosis and treatment. Furthermore, transition metal complex materials are highly sought after due to their rich excited-state properties, such as high luminescence quantum efficiency, ultra-long luminescence lifetime, highly stable luminescence, and tunable luminescence color. Porphyrins and porphyrin derivatives can be complexed with transition metal ions to form nanoparticles. Transition metal ions can significantly enhance the absorption capacity of porphyrins and porphyrin derivatives in the red region through coordination interactions. However, the use of porphyrin derivative-metal ion nanoparticles as photosensitizers for chemiluminescence imaging-guided PTT / PDT synergistic thrombolytic therapy has not been reported. Summary of the Invention

[0006] The present invention provides porphyrin derivative iron complex nanoparticles, their preparation method, and applications. Due to their excellent absorption in the long-wavelength region, these porphyrin derivative iron complex nanoparticles are used as phototherapy agents, enabling synergistic photothermal and photodynamic therapy. Furthermore, the porphyrin derivative iron complex nanoparticles provided herein can also function as chemiluminescent substrates, interacting with reactive oxygen species for imaging, enabling integrated diagnosis and treatment.

[0007] The present invention first provides a porphyrin derivative iron complex nanoparticle, the structure of which is shown in Formula 1:

[0008]

[0009] The present invention also provides a method for preparing porphyrin derivative iron complex nanoparticles, comprising the following steps:

[0010] Step 1: Mixing an aqueous solution of FeCl3 with an aqueous solution of 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid, and performing ultrasonic oscillation to obtain a mixed solution;

[0011] Step 2: placing the mixed solution of step 1 under a magnetic stirring environment to fully age the porphyrin derivative iron complex nanoparticles to obtain an aged solution;

[0012] Step 3: dialyze the aged solution obtained in step 2 to obtain porphyrin derivative iron complex nanoparticles.

[0013] Preferably, the ultrasonic oscillation time in step 1 is 10-30 minutes.

[0014] Preferably, the molar ratio of 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid to FeCl3·6H2O is 1:(0.5-3).

[0015] Preferably, the aging time in step 2 is 8-16 hours.

[0016] Preferably, the dialysis time in step three is 6-10 hours.

[0017] The present invention also provides the use of the porphyrin derivative iron complex nanoparticles described in Formula 1 as a photosensitizer in the preparation of drugs for thrombotic diseases.

[0018] Beneficial effects of the present invention

[0019] The present invention provides porphyrin derivative iron complex nanoparticles (Hp-Fe NPs), as well as their preparation method and application. Without an external excitation light source, the nanoparticles can chemically react with excess ROS in the thrombus microenvironment, generating chemiluminescence with high penetrating power, thereby achieving precise diagnosis. The Hp-Fe NPs provided by the present invention have the advantages of simple preparation and excellent photothermal and photodynamic properties. They exhibit excellent PTT / PDT synergistic thrombolytic effects in vivo, enabling non-invasive PTT / PDT synergistic thrombosis treatment. Furthermore, the nanoparticles act as chemiluminescent substrates, interacting with reactive oxygen species for imaging, enabling integrated diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the particle size of Hp-Fe NPs prepared in Example 1 of the present invention;

[0021] Figure 2 This is a scanning electron microscope image of Hp-Fe NPs prepared in Example 1 of the present invention;

[0022] Figure 3 Transmission electron microscopy image and elemental analysis test of Hp-Fe NPs prepared in Example 1 of the present invention;

[0023] Figure 4 This is the infrared absorption spectrum of Hp-Fe NPs prepared in Example 1 of the present invention;

[0024] Figure 5 The UV absorption spectrum and fluorescence emission spectrum of Hp-Fe NPs prepared in Example 1 of the present invention in solution;

[0025] Figure 6 UV absorption spectra of Hp-Fe NPs and DPBF prepared in Example 1 of the present invention under different conditions;

[0026] Figure 7 This is a photothermal data diagram of Hp-Fe NPs prepared in Example 1 of the present invention in solution;

[0027] Figure 8 This is a graph showing the chemiluminescence properties of Hp-Fe NPs prepared in Example 1 of the present invention;

[0028] Figure 9 This is the in vitro thrombolysis diagram of Hp-Fe NPs prepared in Example 1 of the present invention;

[0029] Figure 10 This is a graph showing the thrombolysis of Hp-Fe NPs prepared in Example 1 of the present invention in mice;

[0030] Figure 11 This is the laser speckle pattern of Hp-Fe NPs prepared in Example 1 of the present invention in mice. DETAILED DESCRIPTION

[0031] The present invention first provides a porphyrin derivative iron complex nanoparticle, the structure of which is shown in Formula 1:

[0032]

[0033] The present invention also provides a method for preparing porphyrin derivative iron complex nanoparticles, comprising the following steps:

[0034] Step 1: Mixing an aqueous solution of FeCl3 with an aqueous solution of 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid, and performing ultrasonic oscillation to obtain a mixed solution;

[0035] Step 2: placing the mixed solution of step 1 under a magnetic stirring environment to fully age the porphyrin derivative iron complex nanoparticles to obtain an aged solution;

[0036] Step 3: dialyze the aged solution obtained in step 2 to obtain porphyrin derivative iron complex nanoparticles.

[0037] The specific reaction process is as follows:

[0038]

[0039] According to the present invention, FeCl3·6H2O solid is placed in a test tube, distilled water is added to dissolve it and prepare an FeCl3 aqueous solution, 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid solid is placed in a test tube, a solvent is added to dissolve it, and the solvent is preferably tetrahydrofuran (THF), and then an aqueous solution is prepared. Then, the FeCl3 aqueous solution and 8,13-bis(1-hydroxyethyl)- An aqueous solution of 3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid is mixed, and the molar ratio of 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid and FeCl3·6H2O is preferably 1:(0.5-3). The two are subjected to ultrasonic oscillation, and the ultrasonic oscillation time is preferably 10-30 minutes. The mixture is fully mixed to obtain a mixed solution.

[0040] According to the present invention, the mixed solution after ultrasonication is added with a magnet and placed on a magnetic stirrer for stirring to fully age the porphyrin derivative iron complex nanoparticles, the aging time being preferably 8-16 hours, to obtain an aged solution;

[0041] According to the present invention, the aged solution is placed in a dialysis bag, which is then placed in distilled water for dialysis. The dialysis time is preferably 6-10 hours to remove impurities and obtain porphyrin derivative iron complex nanoparticles.

[0042] The present invention also provides the use of the porphyrin derivative iron complex nanoparticles described in Formula 1 as a photosensitizer in the preparation of drugs for treating thrombotic diseases.

[0043] The present invention is further described by way of examples below, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that can be easily implemented by a person skilled in the art will fall within the scope of the claims of the present invention.

[0044] Example 1

[0045] Step 1: Add 100 μL of THF to a test tube, then add 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid (0.000835 mmol, 0.5 mg), dissolve, and add distilled water to 1 mL. In another test tube, add FeCl3·6H2O (0.001221 mmol, 0.33 mg) and 2 mL of distilled water. After dissolution, pour the solutions from both test tubes into a 20 mL beaker and mix. Add 7 mL of distilled water. Place the beaker in an ultrasonicator and mix by sonication for 10 minutes to obtain a mixed solution.

[0046] Step 2: Add magnets to the sonicated solution and place it on a magnetic stirrer for stirring to fully age the nanoparticles in the mixed solution. The aging time is 10 hours.

[0047] Step 3: Place the aged solution in a dialysis bag, and place the dialysis bag in distilled water for 8 hours to remove impurities and obtain porphyrin derivative iron complex nanoparticles Hp-Fe NPs. Figure 1 shown.

[0048] Example 2

[0049] The preparation process and conditions are the same as those in Example 1, except that the aging time in step 2 is 8 h.

[0050] Example 3

[0051] The preparation process and conditions were the same as those in Example 1, except that the amount of FeCl3·6H2O added was (0.000814 mmol, 0.22 mg).

[0052] Example 4

[0053] The preparation process and conditions were the same as those in Example 1, except that the amount of FeCl3·6H2O added was (0.002442 mmol, 0.66 mg).

[0054] The performance of the porphyrin derivative iron complex nanoparticles prepared in Example 1 was characterized as follows:

[0055] 1. Preparation and properties of porphyrin derivative iron complex nanoparticles:

[0056] The particle size of Hp-Fe NPs in the present invention is as follows Figure 1 , Figure 1 is the particle size of the nanoparticles of the present invention ( Figure 1 ), as can be seen from the figure, the nanoparticles have a particle size of 91.06nm in aqueous solution. The nanoparticles are less than 100nm in size and are uniform in size, indicating good biocompatibility.

[0057] The nanoparticles in the present invention are morphologically Figure 2 and Figure 3 , Figure 2 This is a scanning electron microscope image of Hp-Fe NPs. It can be seen from the image that the nanoparticles are spherical and the size is consistent with the nanoparticle size test results.

[0058] Figure 3 Transmission electron microscopy image of Hp-Fe NPs ( Figure 3 A) and elemental analysis test results ( Figure 3 B), from Figure 3 A) shows that the nanoparticles are hollow sphere structures. Figure 3 In Figure B), the upper figure shows the mapping element analysis test range from the center of the nanoparticle to the edge, the middle figure shows the specific test range, and the element distribution in the lower figure proves the presence of three elements, N, O, and Fe, in the nanoparticle.

[0059] Figure 4 is the infrared absorption spectrum of the nanoparticles, 3419 cm -1 is the stretching vibration absorption peak of OH bond, 3307cm -1 is the stretching vibration absorption peak of NH bond, 2970cm -1 , 2925cm -1 , 2851cm -1 They are the stretching vibration absorption peaks of the C-H bond, 1591 cm -1 and 1384cm -1 COO - Symmetric and antisymmetric stretching vibration absorption peaks, 674 cm -1 It is the stretching vibration absorption peak of Fe-O bond.

[0060] 2. Photophysical properties of porphyrin derivative iron complex nanoparticles:

[0061] Figure 5 It is the ultraviolet absorption and fluorescence emission of the Hp-Fe NPs of the present invention in aqueous solution. Figure 5 A) is its ultraviolet absorption spectrum, which has the characteristic ultraviolet absorption peak of porphyrin derivatives. 3+ With COO in porphyrin derivatives - The coordination effect occurs, which enhances the absorption of the Q band (500nm-700nm) of the porphyrin derivative. Figure 5 B) is the fluorescence emission spectrum. The nanoparticles have good red light emission at 600nm-700nm.

[0062] Figure 6 The photodynamic performance test of Hp-Fe NPs prepared by the present invention is as follows: Figure 6A) is the UV absorption spectrum of the prepared nanoparticles under the conditions of light and DPBF. 1,3-Diphenylisobenzofuran (DPBF) was used as an indicator to evaluate the ROS-generating ability of the nanoparticles. In the presence of both nanoparticles and DPBF, light degradation of DPBF and a decrease in absorbance indicate the generation of ROS. Figure 6 B) is the UV absorption spectrum of DPBF alone under light conditions, Figure 6 C) is the UV absorption spectrum of Hp-FeNPs and DPBF coexisting in the dark, and Figure 6 A) Compared with the control group, the nanoparticles have a good ability to generate ROS and can be used in subsequent thrombolysis tests.

[0063] 3. Photothermal testing of porphyrin derivative iron complex nanoparticles:

[0064] Figure 7 The photothermal properties of Hp-Fe NPs were investigated. Distilled water was used as a blank control, and Hp NPs (8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid nanoparticles) were used as a control group. Figure 7 A) Under the same laser power and irradiation time, the temperature of the blank control group remained almost unchanged, while the temperature of the nanoparticle group increased rapidly and exhibited better photothermal generation capability than the control group. Figure 7 B) measured the temperature changes of nanoparticles with different concentrations under the same power of 635nm laser irradiation. As the concentration of nanoparticles increases, the temperature of the system also rises significantly. In order to reduce the damage caused by excessively high power laser to the organism, Figure 7 C) The effect of laser power on system temperature was investigated. The higher the laser power, the higher the system temperature. When the laser power was 0.8W / cm 2 The solution temperature can rise to 61°C after 5 minutes of irradiation. Good photothermal stability is crucial for subsequent in vivo treatment. Figure 7 D) shows that the maximum temperature of the nanoparticles remains basically unchanged during 5 laser on-off cycles, and even increases slightly, indicating that the nanoparticles have good photothermal stability and can be used for photothermal therapy.

[0065] 4. Chemiluminescence experiment of porphyrin derivative iron complex nanoparticles:

[0066] Figure 8 For the relevant test of Hp-Fe NPs chemiluminescence of the present invention, Figure 8 A) is to add a variety of oxidants into the nanoparticles to explore the selectivity of the nanoparticles for the chemiluminescence of several oxidants. Compared with other types of oxidants, ONOO - It is a more effective chemiluminescence initiator. -In the study of the effect of the concentration of nanoparticles on the chemiluminescent signal, Figure 8 B) shows the chemiluminescent signal of the nanoparticles and ONOO - The concentration showed a very good linear correlation. Then the chemiluminescence spectrum of the nanoparticles was verified. Figure 8 As shown in Figure 3, the chemiluminescence wavelength of nanoparticles is between 620nm and 760nm, with the highest wavelength at approximately 720nm. Chemiluminescence can eliminate tissue autofluorescence and avoid photon scattering of excitation light, which is beneficial for increasing imaging depth. Figure 8 D) Add ONOO as shown - The sustained luminescence signal of the nanoparticles showed a decay half-life of about 3 min, which is conducive to subsequent in vivo imaging. Figure 8 E) The addition of nanoparticles to ONOO - The penetration depth and sensitivity of the chemiluminescence were then measured and compared with the fluorescence signal. It can be seen that the chemiluminescence and fluorescence signals of the nanoparticles decreased with the increase of the thickness of the chicken breast tissue. When the thickness of the chicken breast was 12 mm, there was still a chemiluminescence signal.

[0067] 5. In vitro thrombolytic experiment of porphyrin derivative iron complexes:

[0068] Figure 9 This is an in vitro thrombolytic test of the Hp-Fe NPs of the present invention. When the nanoparticle group is irradiated by a 635nm laser, the solution temperature rises rapidly, and under the action of the PDT / PTT dual mode, a significant thrombolytic effect is produced. Figure 9 A) is a comparison of blood clot size before and after light irradiation. The upper picture shows the blood clots of each group before light irradiation, and the lower picture shows the blood clots of each group after light irradiation. 2 After 40 minutes of 635 nm laser irradiation, the thrombus mass in the PBS group decreased the least, while the thrombus mass in the Hp-Fe NPs group decreased the most. Figure 9 B) is the blood clot lysis rate (%). Compared with the Hp NPs group, the thrombolysis rate of the Hp-Fe NPs group was higher, reaching 70%. Figure 9 C) Fibrin and hemoglobin levels were measured in the supernatant after thrombolysis. The amounts of hemoglobin and fibrin released into the supernatant during clot dissolution showed a similar trend to the dissolution efficiency. The supernatant of the Hp-Fe NPs group contained the highest levels of red blood cells and fibrin, demonstrating the excellent in vitro thrombolytic activity of Hp-Fe NPs.

[0069] 6. In vivo experiments on mice using porphyrin derivative iron complex nanoparticles:

[0070] Figure 10This is a graph showing the in vivo thrombolysis test of Hp-Fe NPs of the present invention. The present invention used a FeCl3-induced rat carotid artery thrombosis model. After the arterial thrombosis model was established, laser irradiation was performed and PDT / PTT dual-mode thrombosis treatment was initiated. After treatment, the thrombus in the experimental group was significantly dissolved.

[0071] Figure 11 This is a record of blood flow during the laser speckle monitoring thrombolysis process of the nanoparticles of the present invention. After thrombosis is induced, the blood flow changes are monitored after administration through the tail vein. Figure 11 A)-C) are the laser speckle color images and their corresponding black and white images of PBS, Hp NPs and Hp-Fe NPs groups, respectively. Figure 11 A) In the PBS treatment group, there was no significant change in blood flow after treatment with 635 nm laser. Figure 11 B) Hp NPs treatment group. Figure 11 C) is the Hp-Fe NPs experimental group. Figure 11 D) shows the blood perfusion of mice under different conditions. The figure shows that the experimental group has effective thrombus removal. Blood perfusion gradually recovered to about 70% within 2 hours, proving that re-embolism can be effectively prevented.

Claims

1. A porphyrin derivative iron complex nanoparticle, characterized in that: Its structure is shown in Formula 1:

2. The method for preparing porphyrin derivative iron complex nanoparticles according to claim 1, wherein: The following steps are involved: Step 1: Mixing an aqueous solution of FeCl3 with an aqueous solution of 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid, and performing ultrasonic oscillation to obtain a mixed solution; Step 2: placing the mixed solution of step 1 under a magnetic stirring environment to fully age the porphyrin derivative iron complex nanoparticles to obtain an aged solution; Step 3: dialyze the aged solution obtained in step 2 to obtain porphyrin derivative iron complex nanoparticles.

3. The method for preparing porphyrin derivative iron complex nanoparticles according to claim 2, wherein: The ultrasonic oscillation time in step 1 is 10-30 minutes.

4. The method for preparing porphyrin derivative iron complex nanoparticles according to claim 2, wherein: Solid FeCl3·6H2O was placed in a test tube, and distilled water was added to dissolve it to prepare an FeCl3 aqueous solution, wherein the molar ratio of the 8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2,18-dipropionic acid to FeCl3·6H2O was 1:(0.5-3).

5. The method for preparing porphyrin derivative iron complex nanoparticles according to claim 2, wherein: The aging time in step 2 is 8-16 hours.

6. The method for preparing porphyrin derivative iron complex nanoparticles according to claim 2, wherein: The dialysis time in step three is 6-10 hours.

7. Use of the porphyrin derivative iron complex nanoparticles according to Formula 1 of claim 1 as a photosensitizer in the preparation of drugs for thrombotic diseases.

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