Supramolecular porphyrin phthalocyanine nano assembly as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine preparations, and particularly discloses a supramolecular porphyrin phthalocyanine nano assembly which is formed by assembling 5, 10, 15, 20-tetra (trimethylammonium phenyl) porphyrin and octacarboxylic phthalocyanine together. According to the supramolecular porphyrin-phthalocyanine nano assembly, water-soluble 5, 10, 15, 20-tetra (trimethylaminophenyl) porphyrin (TMAP) and octacarboxylic phthalocyanine are selected to be self-assembled into the porphyrin-phthalocyanine nano assembly, compared with single TAPc and TMAP, the porphyrin-phthalocyanine nano assembly has the advantages that the highest peak of an absorption wave band in a near-infrared region is shifted to the near-infrared region in a red-shift manner, an absorption spectrum is effectively widened, and the absorption efficiency is improved. The material has near-infrared response and excellent light stability, so that the material is more suitable for phototherapy; meanwhile, effective electron transfer is formed, I-type ROS is generated under illumination, I-type phototherapy antibacterial performance is achieved under near-infrared light, a remarkable sterilization effect is achieved, a biological membrane can be effectively damaged, wound tissue can be repaired, and wound healing can be effectively promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical preparations, and in particular to a supramolecular porphyrin phthalocyanine nanoassembly and a preparation method and application thereof. Background Art

[0002] Bacterial infections pose a major threat to human health. Since the birth of penicillin, the use of antibiotics has developed rapidly and saved countless lives; however, the long-term use or abuse of antibiotics has led to the development of bacterial resistance. Therefore, the development of new methods to treat bacterial infections has become an urgent need. Phototherapy, as a promising non-resistance alternative, is widely used in the treatment of bacterial infections. Phototherapy includes photodynamic therapy (PDT) and photothermal therapy (PTT). Photodynamic therapy refers to the interaction of photosensitizers with surrounding molecules under the excitation of specific light to produce cytotoxic ROS, thereby inducing cell death. Currently, most photodynamic therapies are based on the type II mechanism, which mainly involves the interaction of photosensitizers with oxygen molecules to produce singlet oxygen ( 1 O2) to kill cells. It can be seen that the type II PDT mechanism is highly dependent on oxygen. However, the microenvironment of bacterial infection is generally hypoxic, which limits the therapeutic effect of type II PDT. In contrast, type I PDT is less dependent on oxygen. This mechanism mainly produces highly cytotoxic type I ROS such as superoxide anions (O2 ·- ) and hydroxyl radicals (·OH). Therefore, compared with type II PDT, type I PDT has greater antibacterial therapeutic potential in hypoxic microenvironment.

[0003] Organic aggregate nanomaterials have shown broad application prospects in the field of antibacterial therapy due to their unique physical, chemical and biological properties. In recent years, most studies have focused on the development of effective type I photosensitizers. For example, in 2018, the Li group (Li, X; Lee, D; Huang, J.-D; Yoon, J. Phthalocyanine-Assembled Nanodots as Photosensitizers for Highly Efficient Type I Photoreactions in Photodynamic Therapy. Angew. Chem. Int. Ed, 2018, 57 (31), 9885-9890.) introduced (N, N-dimethylaminomethyl) phenoxy groups to the periphery of zinc phthalocyanine (ZnPc) to prepare ZnPc photosensitizers, which self-assembled into nanoparticles (ZnPc NPs) in water. ZnPc NPs produce a large amount of O2 under irradiation. ·-, effective bacterial inactivation was achieved through type I PDT; in 2022, Liu and their team (Shanshan Liu, Bingnan Wang, Yuewen Yu, et al. Cationization-Enhanced Type I and Type II ROS Generation for Photodynamic Treatment of Drug-Resistant Bacteria. ACS Nano, 2022, 16 (6), 9130-9141.) converted neutral molecules into cationic molecules through a cationization strategy, named CTBZPyI. CTBZPyI showed significantly enhanced electron separation and transfer capabilities, and could produce a large amount of type I ROS, especially ·OH with high cytotoxicity; in 2023, Liu's team designed a type I photosensitizer (LMBP), which can be self-assembled in situ to form J-aggregated nanofibers (LMBPJ). Compared with LMBP, the absorption band of LMBPJ is red-shifted to 740nm, and at the same time, significant O2 is produced under 730nm light. ·- , is a highly efficient type I photosensitizer. It can be seen that most type I photosensitizers require a carefully designed route, which is time-consuming and costly. Even most photosensitizers after synthesis are difficult to dissolve in water and need to be modified before they can be used in biological applications. Summary of the invention

[0004] In view of the above shortcomings, the present invention provides a supramolecular porphyrin phthalocyanine nanoassembly and a preparation method thereof. By adopting a supramolecular self-assembly strategy, 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin (TMAP) and octacarboxylphthalocyanine (TAPc) are assembled into a nanoassembly in water, which has excellent type I phototherapy antibacterial performance under near-infrared light. The technical scheme is as follows:

[0005] A supramolecular porphyrin phthalocyanine nanoassembly is co-assembled from 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin and octacarboxylphthalocyanine.

[0006] Preferably, in the supramolecular porphyrin phthalocyanine nanoassembly, the molar ratio of octacarboxylphthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in the nanoassembly is 1:0.5-1.5.

[0007] Preferably, in the supramolecular porphyrin phthalocyanine nanoassembly, the molar ratio of octacarboxyphthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in the nanoassembly is 1:1.

[0008] A method for preparing the supramolecular porphyrin phthalocyanine nanoassembly as described above comprises the following steps:

[0009] (1) preparing a dimethyl sulfoxide solution of octacarboxyphthalocyanine and a dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin respectively;

[0010] (2) Add the prepared octacarboxylphthalocyanine solution into water and mix well, then add 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin solution, and ultrasonicate for 2 to 5 minutes to obtain porphyrin phthalocyanine nanoassemblies.

[0011] Preferably, in the above-mentioned method for preparing supramolecular porphyrin phthalocyanine nanoassembly, in the step (1), the concentration of the dimethyl sulfoxide solution of octacarboxyphthalocyanine is 0.1-1 mM, and the concentration of the dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin is 0.1-1 mM.

[0012] Preferably, in the above-mentioned method for preparing supramolecular porphyrin phthalocyanine nanoassembly, in the step (2), the volume ratio of the octacarboxylphthalocyanine solution to water is 1:90-100.

[0013] On the other hand, the present invention also provides the use of the supramolecular porphyrin phthalocyanine nanoassembly in the preparation of type I photosensitizer.

[0014] On the other hand, the present invention also provides the use of the supramolecular porphyrin phthalocyanine nanoassembly in the preparation of antibacterial drugs.

[0015] On the other hand, the present invention also provides the use of the supramolecular porphyrin phthalocyanine nanoassembly in the preparation of drugs for inhibiting bacterial biofilm.

[0016] On the other hand, the present invention also provides the use of the supramolecular porphyrin phthalocyanine nanoassembly in the preparation of a drug for repairing wound tissue.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The supramolecular porphyrin phthalocyanine nanoassembly of the present invention selects water-soluble 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin (TMAP) and octacarboxylphthalocyanine to self-assemble into a porphyrin phthalocyanine nanoassembly. Compared with single TAPc and TMAP, the highest peak of the absorption band of the porphyrin phthalocyanine nanoassembly in the near-infrared region is red-shifted to the near-infrared region, effectively broadening the absorption spectrum, having a near-infrared response, and having excellent photostability, making it more suitable for phototherapy; at the same time, effective electron transfer is formed, type I ROS is generated under light, and it has type I phototherapy antibacterial properties under near-infrared light, has a significant bactericidal effect, can effectively destroy biofilms, can repair wound tissue, and effectively promote wound healing.

[0019] 2. The supramolecular porphyrin phthalocyanine nanoassembly of the present invention has a simple preparation process and a short preparation cycle, does not require expensive and complex equipment, and has readily available raw materials, which is conducive to the promotion and application of supramolecular porphyrin phthalocyanine nanoassembly. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 The hydrogen nuclear magnetic resonance spectrum of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin of the present invention;

[0022] Figure 2 The performance test results of the assembly in the present invention are as follows: a is a comparison diagram of the ultraviolet-visible absorption spectra of TAPc, TMAP and TAPc@TMAP in Example 1; b is a diagram of the ultraviolet-visible absorption spectra of assemblies with different ratios of TAPc and TMAP; c is a curve diagram of the relationship between the ratio of TAPc and TMAP and absorbance; d is a comparison diagram of the fluorescence spectra of TAPc, TMAP and TAPc@TMAP in Example 1; e is the fluorescence lifetime of TAPc, TMAP and TAPc@TMAP in Example 1; f is a transmission electron microscopy (TEM) image of TAPc@TMAP in Example 1; g is a dynamic light scattering (DLS) measurement result of TAPc@TMAP in Example 1; h and i are a diagram of the ultraviolet-visible absorption spectrum of TAPc@TMAP in Example 1 after continuous placement for 7 days and a curve diagram of placement time and absorbance, respectively.

[0023] Figure 3 Graph showing the ROS detection results of H2O, TAPc, TMAP, methylene blue (MB) in Experimental Example 1 of the present invention and TAPc@TMAP in Example 1.

[0024] Figure 4 is the H2O, TAPc, TMAP in Experimental Example 1 of the present invention and TAPc@TMAP in Example 1 1 O2 detection result chart.

[0025] Figure 5 is the O2 of H2O, TAPc, TMAP in Experimental Example 1 of the present invention and TAPc@TMAP in Example 1 ·-Detection result diagrams: a is the H2O fluorescence spectrum; b is the TAPc fluorescence spectrum; c is the TMAP fluorescence spectrum; d is the TAPc@TMAP fluorescence spectrum; e is the fluorescence intensity comparison diagram; f is the ESR spectrum of TAPc@TMAP; g is the H2O fluorescence spectrum in hypoxic environment; h is the TAPc@TMAP fluorescence spectrum in hypoxic environment; i is the fluorescence comparison between hypoxic environment and normal environment.

[0026] Figure 6 The photothermal performance test results of Experimental Example 2 in the present invention: a is a comparison of the photothermal conversion capabilities of different concentrations of TAPc@TMAP under 808nm illumination; b is a comparison of the photothermal conversion capabilities of TAPc@TMAP (20μM) at different laser power densities; c is a comparison of the photothermal conversion capabilities of H2O, TAPc, TMAP and TAPc@TMAP; d is a comparison of the photothermal conversion capabilities of TAPc@TMAP after five irradiation cooling cycles; e is the photothermal conversion efficiency of TAPc@TMAP; f is a comparison of the photothermal conversion capabilities of H2O, TAPc, TMAP and TAPc@TMAP; g is the UV-visible absorption spectrum of ICG at different illumination times; h is the UV-visible absorption spectrum measured at different illumination times; i is a comparison of the absorbance of TAPc@TMAP and ICG at different illumination times.

[0027] Figure 7 These are the results of the in vitro antibacterial experiment of Test Example 3 in the present invention: a and b are comparisons of the bactericidal activity of different concentrations of TAPc@TMAP before and after laser irradiation; c and d are comparisons of the bactericidal activity of saline, TAPc, TMAP, and TAPc@TMAP (concentration of 30 μM) before and after laser irradiation; e and f are comparisons of the biofilm destruction effects of TAPc, TMAP, and TAPc@TMAP before and after laser irradiation; g is a scanning electron micrograph of MRSA treated differently.

[0028] Figure 8 The in vivo photodynamic antibacterial activity test results of Test Example 4 in the present invention: a and b are photographs of MRSA wound sites of mice in each group monitored over a 12-day period; c is the MRSA wound healing rate of mice in each group over a 12-day period; d and e are comparisons of the bactericidal activities of saline, TAPc, TMAP, and TAPc@TMAP; e and f are comparisons of the residual bacteria in the wound sites of mice in each group; f is the weight change of mice in each group; g is the wound tissue diagram of mice in each group after 12 days; h is the expression of inflammatory factors (TNF-α) in the wound tissue of mice in each group after 12 days; i is the expression of vascular growth factor (CD31) in the wound tissue of mice in each group after 2 days.

[0029] Fig. 9 It is the technical research roadmap of the present invention. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Unless otherwise defined, all professional terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention, etc. can be purchased from the market or can be prepared by existing methods.

[0031] Octacarboxylphthalocyanine (TAPc) was provided by University of Science and Technology Beijing. 2,7-Dichlorofluorescein diacetate (DCFH-DA) detection kit was purchased from Bio-Tech Biotech Co., Ltd., China; methicillin-resistant Staphylococcus aureus (MRSA) was purchased from Beijing Baozang Biotech Co., Ltd.

[0032] The 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin used in the following examples was prepared by the following method:

[0033] 78.4 mg of 5,10,15,20-tetrakis(dimethylaminophenyl)porphyrin and 1.4 g of iodomethane (CHI3) were dissolved in 20 mL of DMF, refluxed at 100 °C for 12 h, washed with DMF, acetonitrile and ether in turn, and dried in vacuo to obtain the product 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin (TMAP) (H NMR spectrum, see Figure 1 ), with a yield of 71%.

[0034] The NMR data are:

[0035] 1 H NMR (400MHz) in DMSO-d6, δ (ppm): 3.95 (s, 36H), 8.52-8.45 (m, 16H), 8.89 (s, 8H), -2.96 (s, 2H).

[0036] Example 1

[0037] The supramolecular porphyrin phthalocyanine nanoassembly of this embodiment is co-assembled by 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin and octacarboxylphthalocyanine, and the molar ratio of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin to octacarboxylphthalocyanine is 1:1.

[0038] This embodiment also provides a method for preparing a supramolecular porphyrin phthalocyanine nanoassembly, comprising the following steps:

[0039] (1) preparing a 1 mM solution of octacarboxyphthalocyanine (TAPc) in dimethyl sulfoxide (DMSO) and a 1 mM solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin (TMAP) in dimethyl sulfoxide (DMSO);

[0040] (2) Take 10 μL of the TAPc solution prepared in step (1) and dissolve it in 980 μL of water. After mixing, add 10 μL of the TMAP solution prepared in step (1) and sonicate for 2 min to obtain porphyrin phthalocyanine self-assembled nanoparticles (10 μM), i.e., supramolecular porphyrin phthalocyanine nanoassemblies, denoted as TAPc@TMAP.

[0041] In the following test example, the concentration of TAPc@TMAP is 20 μM (or 30 μM) and is prepared as follows: 20 μL (or 30 μL) of the TAPc solution prepared in step (1) is dissolved in 960 μL (or 940 μL) of water, mixed and then 20 μL (or 30 μL) of the TMAP solution prepared in the step is added, and ultrasonicated for 2 minutes.

[0042] Example 2

[0043] The supramolecular porphyrin phthalocyanine nanoassembly of this embodiment is co-assembled by 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin and octacarboxylphthalocyanine, and the molar ratio of octacarboxylphthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin is 1:0.6. The preparation method is the same as that of Example 1, except that the concentration of the dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in step (1) is 0.6 mM.

[0044] Example 3

[0045] The supramolecular porphyrin phthalocyanine nanoassembly of this embodiment is co-assembled by 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin and octacarboxylphthalocyanine, and the molar ratio of octacarboxylphthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin is 1:0.8. The difference is that the concentration of the dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in step (1) is 0.8 mM.

[0046] Example 4

[0047] The supramolecular porphyrin phthalocyanine nanoassembly of this embodiment is co-assembled by 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin and octacarboxylphthalocyanine, and the molar ratio of octacarboxylphthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin is 1:1.2. The difference is that the concentration of the dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in step (1) is 1.2 mM.

[0048] Example 5

[0049] The supramolecular porphyrin phthalocyanine nanoassembly of this embodiment is co-assembled by 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin and octacarboxylphthalocyanine, and the molar ratio of octacarboxylphthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin is 1:1.4. The difference is that the concentration of the dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in step (1) is 1.4 mM.

[0050] Comparative Example 1

[0051] The molar ratio of octacarboxylphthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in this comparative example is 1:0.2. The difference is that the concentration of the dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in step (1) is 0.2 mM.

[0052] Comparative Example 2

[0053] The molar ratio of octacarboxylphthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in this comparative example is 1:0.4. The difference is that the concentration of the dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in step (1) is 0.4 mM.

[0054] The performance of the assemblies prepared in Examples 1 to 5 and Comparative Examples 1 to 2 was tested, and the UV-visible absorption spectra were shown in Figure 2 a~c. As can be seen from the figure, 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin (TMAP) has a strong Soret band absorption at 380-450nm, with the maximum absorption at 410nm. Octacarboxylphthalocyanine (TAPc) has a wide absorption in the 550-750nm band, with the maximum absorption peak at 610nm. After TAPc and TMAP are mixed in aqueous solution, the strong absorption band of TMAP is retained, while the Q band absorption peak of TAPc is red-shifted from 610nm to 755nm, indicating that there is an interaction between TAPc and TMAP. With the addition of TMAP, the absorbance of the assembly at 755nm gradually increases. When an equal amount of TMAP is added, that is, when the molar ratio of TAPc to TMAP is 1:1, the absorbance tends to be flat.

[0055] Depend on Figure 2 It can be seen from e that the fluorescence lifetime of TAPc@TMAP in Example 1 is shortened to 0.33ns, proving that an electron transfer process occurs between TAPc and TMAP. Figure 2 f Transmission electron microscopy (TEM) images and dynamic light scattering (DLS) measurements ( Figure 2 g) shows that TAPc@TMAP is uniformly distributed in spherical shape with an average particle size of 197.5 nm. Figure 2 h and Figure 2 As can be seen from Fig. 1, the absorbance of TAPc@TMAP aqueous solution at 755 nm did not change significantly within 7 days, indicating that TAPc@TMAP has good colloidal stability in aqueous solution.

[0056] In the following experimental examples, TAPc refers to octacarboxyphthalocyanine, TMAP refers to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin, and TAPc@TMAP refers to the porphyrinphthalocyanine self-assembled nanoparticles prepared in Example 1.

[0057] Test Example 1 Photodynamic Performance Test

[0058] 1.1 Reactive oxygen species (ROS) detection

[0059] Detection method: The ability of TAPc, TMAP and TAPc@TMAP to generate ROS in aqueous solution was detected by fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA entered cells through the cell membrane and was hydrolyzed into DCFH by esterase. When DCFH was exposed to ROS (O2 ·- , H2O2, ·OH) is oxidized to dichlorofluorescein (DCF), and then emits a significant fluorescence signal in the wavelength range of 500-650nm, thereby realizing the detection of ROS. Specific steps: Add photosensitizer (TAPc, TMAP, TAPc@TMAP, 30μM, 1mL) and DCFH (0.1mM, 10μL) into a quartz cuvette and mix evenly. Use an aqueous solution containing only 2,7-dichlorofluorescein as a control and a commercial photosensitizer methylene blue (MB) as a comparison, and then use 808nm (0.5W / cm 2 ) laser light was irradiated, and the fluorescence spectrum of DCFH (excitation at 480 nm) was recorded every 10 s.

[0060] Test results: The results are as follows Figure 3 As shown in the figure, under illumination, the fluorescence change of the aqueous solution containing DCFH is negligible. The fluorescence intensity of the DCFH solution containing TAPc@TMAP increases rapidly with the increase of illumination time, indicating that TAPc@TMAP can effectively generate ROS, while the fluorescence changes of TAPc and TMAP are negligible. It is worth noting that after 808nm laser irradiation for 60s, the DCFH fluorescence intensity of TAPc@TMAP is enhanced by about 11.8 times, which is twice that of the commercial photosensitizer methylene blue (MB), showing excellent ROS generation efficiency.

[0061] In order to further verify the type of ROS generated, SOSG was used as a probe to detect 1 The production of O2. Figure 4As shown in the figure, the fluorescence intensity of the SOSG solution containing TAPc@TMAP did not change significantly within 60 seconds of irradiation, with the aqueous solution containing only SOSG as the control, indicating that there was no 1 The results show that TAPc@TMAP is not a type II photosensitizer, but a type I photosensitizer, which is not possessed by single TAPc and single TMAP.

[0062] 1.2 O2 ·- Detection

[0063] Detection method: Detect O2 by using the interaction between dihydroethylenediamine (DHE) and DNA ·- , at O2 ·- In the presence of ethylenediamine, hydrogen is oxidized to form ethylenediamine, which is inserted into DNA and emits bright fluorescence at around 520-750nm. The specific steps are: mix the photosensitizer (TAPc, TMAP, TAPc@TMAP, 30μM, 1mL) and DHE (1mM, 10μL) in a quartz cuvette containing 250μg / mL ctDNA, and use a mixture containing only DHE and ctDNA as a control. Use 808nm (0.5W / cm 2 ) light source to irradiate the mixture, and record the fluorescence spectrum of DHE (excitation at 510 nm) every 10 s. Electron spin resonance (ESR) spectroscopy was used to detect O2 ·- DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was used as a spin trapping agent. 30 μL TAPc@TMAP (30 μM) was added with 30 μL DMPO (100 mM, methanol as solvent). After mixing well, the spin was detected by 808 nm (0.5 W / cm 2 ) laser irradiation, a certain amount of mixed liquid is sucked by capillary, placed in a quartz tube, and placed in the EPR sample chamber for O2 ·- Test.

[0064] Test results: The results are as follows Figure 5 As shown by Figure 5 From a to e, it can be seen that the DHE fluorescence intensity of TAPc@TMAP at 605 nm increased significantly when the aqueous solution containing only DHE was used as the control, indicating that O2· - After 808nm light irradiation for 60s, the DHE fluorescence intensity of TAPc@TMAP increased by 19.3 times compared with that of DHE alone, indicating that TAPc@TMAP has excellent O2· - In addition, electron spin resonance (ESR) spectroscopy analysis was performed using DMPO (5,5-dimethyl-1-pyrroline-N-oxide) as a spin trapping agent. Figure 5f, Compared with the dark group, TAPc@TMAP showed superoxide anion (O2 ·- ) Typical ESR signal. Finally, the generation of O2 by TAPc@TMAP in hypoxic environment was detected. ·- The ability of Figure 5 As shown in g~i, in the hypoxic environment, the O2· - The generation efficiency is 0.55 times that in normoxic environment. The above results confirm that TAPc@TMAP has the ability to generate type I ROS and has great potential for photodynamic application in hypoxic environment.

[0065] Test Example 2 Photothermal Performance Test

[0066] Test method: To evaluate the photothermal performance of TAPc@TMAP, a 808nm (0.5W / cm 2 ) laser light was used to irradiate the aqueous solution of photosensitizer (TAPc, TMAP, TAPc@TMAP, 30 μM), and then the temperature change of the photosensitizer was recorded using a near-infrared thermal imager. The temperature change was recorded every 30 seconds. The photothermal conversion efficiency was calculated according to the following equation:

[0067] Q diss =10 3 m C ΔT / t

[0068] t=-lnθ·τ s ,

[0069] Where: T is the instantaneous temperature during cooling, Tmax is the maximum temperature, Tsurr is the ambient temperature, Qdiss is the thermal change of the solvent, ΔT is the temperature rise of water, t is the time required to cool from T to the ambient temperature, I is the laser power, A 808 is the absorbance of TAPc@TMAP at 808 nm, m is the mass of water, and C is the specific heat capacity of water (J / g / ℃).

[0070] Test results: See the results Figure 6 ,like Figure 6 As shown in Figure c, after 10 minutes of illumination, the temperature of TAPc and TMAP increased by only 3.4°C and 1.1°C, respectively. In contrast, under the same environment, the temperature of TAPc@TMAP increased from 26.8°C to 41.8°C (ΔT = 15°C), which is a large increase in temperature. The increase in temperature indicates that TAPc@TMAP can generate heat. Figure 6As shown in a and b, the temperature increase of TAPc@TMAP is positively correlated with the laser power density and the concentration of TAPc@TMAP. In a 20μM aqueous solution, the temperature of TAPc@TMAP increased by 11.1℃ after 600s of illumination, while in a 30μM aqueous solution, the temperature increased by 15.3℃. The power used was 0.5W / cm 2 After irradiation with laser light for 600s, the temperature of TAPc@TMAP increased by 12.6℃, while the power was 1W / cm 2 The temperature increased by 16.9°C when the temperature was increased by 16.9°C. These results show that the photothermal behavior of TAPc@TMAP is controllable. To further prove the photothermal performance of TAPc@TMAP, the photothermal conversion efficiency of TAPc@TMAP was calculated to be 28.9% ( Figure 6 e). In addition, after five irradiation-cooling cycles, TAPc@TMAP showed little degradation in water (Figure d), indicating the excellent photostability of TAPc@TMAP. Figure 6 From g~i, it can be seen that compared with the commercial photothermal reagent indocyanine green (ICG), the absorbance of TAPc@TMAP hardly changes with the increase of illumination time, while the absorbance of indole (ICG) decreases significantly.

[0071] Test Example 3 In vitro antibacterial test

[0072] Experimental method: The antibacterial activity of TAPc@TMAP was evaluated against methicillin-resistant Staphylococcus aureus (MRSA) by agar plate colony counting method. The culture medium of MRSA was diluted to about 0.5OD with nutrient broth. 600 The diluted bacterial solution was incubated with the sample and then the sample was exposed to 808nm (0.5W / cm 2 ) laser light irradiation for 30 minutes, after irradiation, the bacterial solution was diluted 100 times and coated on the nutrient agar plate, and finally placed in a 37°C constant temperature incubator for 24 hours, with the bacterial solution not treated with light as the control. The bactericidal activity of TAPc@TMAP at different concentrations (10μM, 20μM, 30μM) under light and dark conditions was tested, and the bactericidal activity of TAPc, TMAP and TAPc@TMAP at a concentration of 30μM was compared.

[0073] The morphology of methicillin-resistant Staphylococcus aureus before and after illumination was observed using a scanning electron microscope. After the sample was incubated with the resistant bacteria, it was placed at 808nm (0.5W / cm 2 ) laser light for 30 minutes, centrifuged and fixed with 2.5% glutaraldehyde for 4 hours, centrifuged and discarded the supernatant, then washed with different gradients of ethanol (ethanol concentrations were 25%, 50%, 75%, 90%, 100%). Finally, the bacterial solution was freeze-dried, sprayed with gold and the morphological images of the bacteria were collected using SEM. The bacterial solution that was not treated with light was used as the control.

[0074] Crystal violet staining was used to determine the amount of bacterial biofilm, and the bacterial solution was adjusted to about OD 600 =0.2, take 200 μL of bacterial solution and inoculate it into a 96-well plate, and form a biofilm after 24 hours, then remove the culture medium, incubate the obtained biofilm with the sample, and then irradiate with 808nm laser for 30 minutes, discard the supernatant after centrifugation, add 200 μL of methanol to each well to fix the bottom biofilm, discard the methanol after 30 minutes, add 0.1% crystal violet, remove the crystal violet after 20 minutes, and wash the excess dye in the well with PBS solution. Finally, add 95% ethanol to dissolve the crystal violet bound to the biofilm, and measure the absorbance of the sample at 590nm after 20 minutes.

[0075] The experimental results are as follows Figure 7 As shown. Figure 7 As shown in a, before illumination, the number of bacterial colonies did not change significantly with the increase of TAPc@TMAP concentration, indicating that TAPc@TMAP has no dark toxicity. 2 , 30min), TAPc@TMAP showed a concentration-dependent antibacterial efficiency. When the concentration of TAPc@TMAP reached 30μM, its bactericidal rate reached 99.73% (see Figure 7 b) From Figure 7 c-d show that before and after laser irradiation, the colony counts of TAPc and TMAP did not change significantly compared with the control group, indicating that TAPc and TMAP did not show an inhibitory effect on the growth of MRSA and did not have type I phototherapy antibacterial properties; TAPc@TMAP had almost no bacteria after irradiation, indicating that TAPc@TMAP had a significant bactericidal effect on MRSA. Figure 7 g As can be seen, in both the control group and the non-irradiated group, the bacteria have regular body shapes, smooth surfaces, and intact cell walls. In contrast, the bacterial cell membranes treated with TAPc@TMAP and light were significantly wrinkled and collapsed, indicating that the cell integrity of the bacteria was damaged or destroyed. Figure 7 e It can be seen that under non-illumination conditions, there was no significant difference in the color and biomass of the biofilm between the control group and the TAPc@TMAP group. After illumination, the color of the biofilm in the TAPc@TMAP group became lighter and the biomass decreased significantly, indicating that the type I ROS generated by TAPc@TMAP under 808nm laser irradiation can effectively destroy the biofilm.

[0076] Test Example 4 In vivo photodynamic antibacterial activity test results

[0077] Experimental methods: All experiments were conducted in accordance with the relevant regulations of the Guangxi University of Chinese Medicine Research Institute. Male KM mice (24-28 g) were used for the experiment. A wound with a diameter of 1 cm was constructed on the back of the mice. Methicillin-resistant Staphylococcus aureus (MRSA, 10 8 CFU / mL) was used to treat the wound. One day later, a MRSA infection mouse skin wound model was induced. The mice were randomly divided into 4 groups and given saline, TAPc, TMAP, and TAPc@TMAP, respectively. 100 μL of saline, TAPc (30 μM), TMAP (30 μM), and TAPc@TMAP (30 μM) were subcutaneously injected into the infected area, respectively. After injection, 808 nm (0.5 W / cm 2 ) Laser light was used to irradiate the infected tissue of mice for 10 minutes, and saline was used as the control group. The wounds of mice were photographed on the 1st, 3rd, 5th, 7th, and 12th days, and the wound healing conditions of each treatment group were compared. After 12 days, mice from different treatment groups were collected for immunological component analysis.

[0078] Experimental results:

[0079] Photos of MRSA wound sites in each group of mice monitored over a 12-day period are shown below. Figure 8 As shown in a and b, the infected wound area gradually decreased with the increase of treatment time. Among them, the TAPc@TMAP group showed the fastest reduction in wound area. By the 12th day, the wound healing rate reached 92.2% ( Figure 8 c) Samples were collected from the wound site during the treatment to evaluate the antibacterial properties of TAPc@TMAP. Figure 8 As shown in d and e, the residual bacteria in the TAPc@TMAP group were significantly lower than those in the control group on days 2, 5, and 8. The above results show that TAPc@TMAP can significantly kill bacteria and promote the healing of infected wounds.

[0080] The weight changes of mice in each group during the treatment are shown in Figure f. It can be seen from the figure that during the 12-day treatment period, the weight of mice gradually increased, confirming the safety of TAPc@TMAP in vivo. After 12 days of treatment, the mice were killed, and the wounds and important organs of the mice were stained with hematoxylin and eosin (H&E). The results are shown in Figure g. The H&E staining results showed that the control group showed obvious inflammatory cell infiltration. On the contrary, the inflammatory cell infiltration in the TAPc@TMAP tissue was reduced, fibrous tissue proliferated, and new capillaries were formed. Subsequently, the expression of inflammatory factors (TNF-α) and angiogenic growth factors (CD31) in the wound tissue was further analyzed by immunohistochemical evaluation. The results are shown in Figures h and i. Taking the control group as the control, TNF-α was greatly reduced and CD31 was greatly increased in the wound tissue of TAPc@TMAP. In contrast, the wound tissues of other groups still showed a large number of inflammatory factors. The above results show that TAPc@TMAP has the best degree of wound tissue repair and can effectively promote wound healing. In addition, histological examination of heart, liver, spleen, lung, and kidney sections of TAPc@TMAP revealed no obvious pathological abnormalities compared with the control group, once again confirming the biosafety of TAPc@TMAP in vivo.

[0081] All the above experiments were repeated at least three times, and all data are expressed as mean ± standard deviation (SD). The results were statistically analyzed using one-way analysis of variance (ANOVA). Statistical significance is indicated as follows: *p<0.05, **p<0.01, and ***p<0.001.

[0082] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A supramolecular porphyrin phthalocyanine nanoassembly, characterized in that: The nano assembly is co-assembled by 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin and octacarboxylphthalocyanine.

2. The supramolecular porphyrin phthalocyanine nanoassembly according to claim 1, characterized in that: The molar ratio of octacarboxyl phthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in the nano-assembly is 1:0.5-1.

5.

3. The supramolecular porphyrin phthalocyanine nanoassembly according to claim 2, characterized in that: The molar ratio of octacarboxyl phthalocyanine to 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin in the nanoassembly is 1:

1.

4. A method for preparing a supramolecular porphyrin phthalocyanine nanoassembly according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) preparing a dimethyl sulfoxide solution of octacarboxyphthalocyanine and a dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin respectively; (2) Add the prepared octacarboxylphthalocyanine solution into water and mix well, then add 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin solution, and ultrasonicate for 2 to 5 minutes to obtain porphyrin phthalocyanine nanoassemblies.

5. The method for preparing the supramolecular porphyrin phthalocyanine nanoassembly according to claim 4, characterized in that: In the step (1), the concentration of the dimethyl sulfoxide solution of octacarboxylphthalocyanine is 0.1 to 1 mM, and the concentration of the dimethyl sulfoxide solution of 5,10,15,20-tetrakis(trimethylammoniumphenyl)porphyrin is 0.1 to 1 mM.

6. The method for preparing the supramolecular porphyrin phthalocyanine nanoassembly according to claim 4, characterized in that: In the step (2), the volume ratio of the octacarboxyphthalocyanine solution to water is 1:90-100.

7. Use of the supramolecular porphyrin phthalocyanine nanoassembly according to any one of claims 1 to 3 in the preparation of type I photosensitizer.

8. Use of the supramolecular porphyrin phthalocyanine nanoassembly according to any one of claims 1 to 3 in the preparation of antibacterial drugs.

9. Use of the supramolecular porphyrin phthalocyanine nanoassembly according to any one of claims 1 to 3 in the preparation of drugs for inhibiting bacterial biofilm.

10. Use of the supramolecular porphyrin phthalocyanine nanoassembly according to any one of claims 1 to 3 in preparing a drug for repairing wound tissue.

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