Porphyrin-based prismatic coordination molecular cage as well as preparation method and application thereof

Abstract

The invention discloses a porphyrinyl prismatic coordination molecular cage and a preparation method and application thereof.The preparation method comprises the steps that H2TMPP, Au (III) metal salt and Pd (OAc) 2 are dissolved in a solvent, an acidity regulator is added, after hydrothermal treatment, mother liquor is removed, obtained crystals are washed, and the porphyrinyl prismatic coordination molecular cage Pd-TMPP (Au) is obtained. 6 PdCl2 units and ligand combinations (H2TMPP) x [TMPP (Pd)] y [TMPP (Au)] zClz form 10 complexes in total, and the Pd-TMPP (Au) comprises at least 5 of the complexes. According to the invention, a Pd-TMPP (Au) hexagonal prism high-entropy coordination molecular cage based on Au (III) is synthesized by using a hydrothermal method, and the unique arrangement mode of the (metal) ligands in the cage inhibits pi-pi stacking of a porphyrin photosensitizer, so that the effect of PDT is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a porphyrin-based prismatic coordination molecular cage, a preparation method thereof and an application thereof. Background Art

[0002] Photodynamic therapy (PDT) is a new tumor treatment mode developed in recent years and has now become one of the emerging cancer treatment methods. PDT generates reactive oxygen species (ROS), such as singlet oxygen ( 1 1O2), hydroxyl radical (·OH) and superoxide radical (·O2 - ) by the reaction of excited photosensitizer and oxygen source to kill tumor cells. Selective uptake of photosensitizer by local lesions and appropriate wavelength of light irradiation, through photosensitizer-mediated and oxygen molecule participation, leads to oxidative damage, causing apoptosis and necrosis of target cells. Because it has almost no side effects of damaging normal tissue cells, it has unique advantages in clinic and has been widely used in the treatment of tumors such as esophageal cancer, non-melanoma skin cancer, cervical cancer, etc., and is expected to become a conventional adjuvant treatment means for various malignant tumors. At the same time, PDT can also induce the occurrence of tumor immunity after treatment, providing a new strategy for the effective treatment of some refractory malignant tumors.

[0003] In 1975, scientists first reported the work of using hematoporphyrin derivative (HpD) to inhibit mammary fat tumors in mice. After that, porphyrins and their derivatives have been successively used in the PDT process to achieve effective treatment of tumors, especially superficial tumors. Although some of the existing small molecule photosensitizers have been approved for clinical use, however, these porphyrin-based photosensitizers have poor selectivity for tumors and will accumulate highly in the skin, resulting in skin photosensitivity.

[0004] The porphyrin-based chelating ligand TPP in the Au(III)-porphyrin complexes reported by Che, Chung, and others can effectively stabilize Au(III) (C.-M. Che et al., Chem. Commun. 2003, 1718 - 1719; F.-R. Yang et al., Chem. Lett. 25, 2015, 3592 - 3596), and has excellent toxicity to multiple cell lines. However, its toxicity is limited to the drug itself, and the PDT potential of the porphyrin ligand itself remains to be explored. In addition, such planar single-molecule compounds are very likely to cause self-quenching of the excited state due to intermolecular π-π interactions and Au…Au interactions, thereby weakening the PDT effect. By combining the building units of Au(III)-porphyrin complexes with nanotechnology, composite functional materials can be obtained for the synergistic treatment of tumors. However, these composite materials often have high drug costs, low delivery efficiency, and potential side effects such as systemic toxicity and significant liver and kidney toxicity caused by premature drug release due to reasons such as overly complex components, weak interactions between multiple components (hydrogen bonds, π–π interactions, van der Waals forces, hydrophilic-hydrophobic interactions, etc.), and large particle sizes.

[0005] To solve the above problems existing in the prior art, the present invention is thus proposed. Summary of the Invention

[0006] In view of the above deficiencies in the prior art, the present invention provides a porphyrin-based prismatic coordination molecular cage and its preparation method and application. The present invention uses H2TMPP ligand, Au(III) metal salt, and Pd(OAc)2 as building units, and synthesizes a Pd-TMPP(Au) hexagonal prism high-entropy coordination molecular cage based on Au(III) by a hydrothermal method. The unique arrangement of these (metal) ligands in the cage inhibits the π-π stacking of the porphyrin photosensitizer, thereby improving the PDT effect.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides a porphyrin-based prismatic coordination molecular cage, and the porphyrin-based prismatic coordination molecular cage is denoted as Pd-TMPP(Au). 6 PdCl2 units and ligand combinations (H2TMPP) x [TMPP(Pd)] y [TMPP(Au)] z Cl z form a total of 10 kinds of complexes, and 0 ≤ x ≤ 3, 0 ≤ y ≤ 3, 0 ≤ z ≤ 3, x + y + z = 3, as shown in the following figure:

[0009]

[0010] The Pd-TMPP(Au) includes at least five of the complexes formed by six PdCl2 units and ligand combinations (H2TMPP). x [TMPP(Pd)] y [TMPP(Au)] z Cl z Theoretically, ten compounds can be produced by the random combination of three ligands. We identified five through spectroscopic data analysis, so this material is also called a high-entropy material.

[0011] Preferably, the Pd-TMPP(Au) includes at least the following five: Pd6Cl 12 (H2TMPP)3, Pd6Cl 12 (H2TMPP)2[TMPP(Pd)], {Pd6Cl 12 (H2TMPP)2[TMPP(Au)]}Cl, {Pd6Cl 12 (H2TMPP)[TMPP(Pd)][TMPP(Au)]}Cl, and Pd6Cl 12 [TMPP(Pd)]2[TMPP(Au)]}Cl.

[0012] Preferably, the structure of the Pd-TMPP(Au) is a twin crystal structure with double arrows, and it crystallizes in the Cmc21 space group of the orthorhombic system.

[0013] The present invention also provides a preparation method for the above porphyrin-based prismatic coordination molecular cage. Dissolve H2TMPP, Au(III) metal salt, and Pd(OAc)2 in a solvent, add an acidic regulator, and then after hydrothermal treatment, remove the mother liquor and wash the obtained crystals to obtain the porphyrin-based prismatic coordination molecular cage Pd-TMPP(Au). H2TMPP is meso-tetrakis(6-methylpyridin-3-yl)porphyrin, and its structure is as follows:

[0014]

[0015] Preferably, the conditions for hydrothermal treatment are: first, heat from room temperature to 120 °C within 4–8 h, keep warm for 24–48 h, and cool to room temperature within 24–48 h.

[0016] Preferably, the Au(III) metal salt is at least one of HAuCl4·4H2O, HAuCl4·3H2O, and AuCl3;

[0017] The molar ratio of H2TMPP, Au(III) metal salt, and Pd(OAc)2 is 1:1:2.

[0018] Preferably, the solvent is a mixed solvent composed of methanol and dichloromethane with a volume ratio of 3:1.

[0019] Preferably, the acidic regulator is acetic acid (HAc).

[0020] The present invention also provides the application of the above porphyrin-based prismatic coordination molecular cage in the preparation of drugs for treating liver cancer. Specifically, Pd-TMPP(Au) can be used for the combined treatment of PDT and chemotherapy for liver cancer.

[0021] The present invention also provides a preparation method of a porphyrin-based prismatic coordination molecular cage nanoformulation. The above porphyrin-based prismatic coordination molecular cage is dissolved in dimethyl sulfoxide to obtain a mixed solution, and then the mixed solution is dropped into a sodium hyaluronate aqueous solution and stirred for 24–72 h, and then a porphyrin-based prismatic coordination molecular cage nanoformulation Pd-TMPP(Au)-HA is obtained through dialysis.

[0022] Preferably, the mass-volume ratio of the porphyrin-based prismatic coordination molecular cage to dimethyl sulfoxide is 2 mg / 1 mL.

[0023] Preferably, the preparation method of the sodium hyaluronate aqueous solution is to dissolve sodium hyaluronic acid in deionized water, and the mass-volume ratio of sodium hyaluronic acid to deionized water is 3 mg / 1 mL.

[0024] The present invention also provides a porphyrin-based prismatic coordination molecular cage nanoformulation prepared by the above preparation method.

[0025] The present invention also provides the application of a porphyrin-based prismatic coordination molecular cage nanoformulation in the preparation of drugs for treating liver cancer. In the present invention, Pd-TMPP(Au) is modified with sodium hyaluronate (HA), which can further form water-soluble nanoparticles and achieve targeting of HA receptor overexpressing liver cancer cell lines HuH-7, Hep-G2, and PLC / PRF / 5.

[0026] The beneficial effects of the present invention are as follows:

[0027] (1) The porphyrin center has a similar coordination ability to Pd and Au. In this invention, H2TMPP ligand, Au(III) metal salt, and Pd(OAc)2 are used as building units, and a hexagonal prism coordination molecular cage Pd-TMPP(Au) based on Au(III) is synthesized by hydrothermal method. It is proved by various characterization means that the double-arrow-shaped polycrystal contains a coordination molecular cage with a high-entropy ligand combination. This molecular cage compound has both chemical toxicity and phototoxicity. Its phototoxicity stems from the special stacking mode of Au(III)-based porphyrin ligands in the molecular cage. The unique arrangement of (metal) ligands in Pd-TMPP(Au) in the cage inhibits the π-π stacking of porphyrin photosensitizers, thus improving the effect of PDT. Coupled with the cytotoxic Au(III) ions and the cancer cell specificity after being modified by HA, these characteristics together broaden the application prospects of Au(III)-based high-entropy coordination molecular cage materials in the biomedical field.

[0028] (2) In the current field of materials chemistry, "high-entropy" materials have attracted extensive attention due to their unique properties and have been widely applied in multiple fields such as energy materials and catalytic materials. This invention first introduces the "high-entropy" concept into the construction of coordination molecular cages. Specifically, by constructing a high-entropy system, the synergy of multiple functions within one material is realized, such as the mitochondrial targeting of ionic drugs, the unique anti-tumor mechanism of Au porphyrin compounds, the reactive oxygen process of Pd, and the photodynamic therapy process of uncoordinated ligands. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the drawings and embodiments:

[0030] Figure 1 : Schematic diagram of the synthesis of Pd-TMPP(Au) (taking {Pd6Cl 12 [TMPP(Au)]3} 3+ as an example);

[0031] Figure 2 : Crystal photograph of Pd-TMPP(Au);

[0032] Figure 3 : Molecular structure and crystal structure diagrams of H2TMPP(a), TMPP(Pd)(b), TMPP(Au)(c), and Pd-TMPP(Au)(d), where (d) and (e) take {Pd6Cl 12 [TMPP(Au)]3} 3+ as an example;

[0033] Figure 4 : EDS Mapping diagram of Pd-TMPP(Au) powder;

[0034] Figure 5: EDS Mapping diagrams of different parts of a single Pd-TMPP(Au) crystal;

[0035] Figure 6 : Six corresponding peaks in the full mass spectrum of Pd-TMPP(Au);

[0036] Figure 7 : 1H NMR of H2TMPP(a) and Pd-TMPP(Au)(b) in CDCl3; 1 1H NMR;

[0037] Figure 8 : Infrared spectra of H2TMPP and Pd-TMPP(Au);

[0038] Figure 9 : Ultraviolet spectra of H2TMPP and Pd-TMPP(Au) (Insert: Absorption curve magnified 1.5 times in the range of 475 - 700 nm);

[0039] Figure 10 : XPS full spectrum (a), Pd 3d spectrum (b), and Au 4f spectrum (c) of Pd-TMPP(Au);

[0040] Figure 11 : Schematic diagram for the synthesis of Pd-TMPP(Au)-HA (taking {Pd6Cl 12 [TMPP(Au)]3} 3+ as an example);

[0041] Figure 12 : TEM images of Pd-TMPP(Au)-HA;

[0042] Figure 13 : DLS diagrams of Pd-TMPP(Au)-HA;

[0043] Figure 14 : Zeta potential diagrams of Pd-TMPP(Au)-HA;

[0044] Figure 15 : UV-Vis intensity change diagram of the aqueous solution of Pd-TMPP(Au)-HA under laser irradiation (650 nm, 25 mW cm -2 ) at the Soret band;

[0045] Figure 16 : (a) UV absorption intensity change curves of DPBF and its dispersion in the DMSO solution of (b) Pd-TMPP(Au)-HA as a function of laser irradiation time at different times, and (c) Intuitive comparison of their generation of 1 O2 in aqueous solution through the rate of change of absorbance at the Soret band;

[0046] Figure 17 : Cell survival rate graphs of Pd-TMPP(Au)-HA in HuH-7, Hep-G2, and PLC / PRF / 5 under the condition of laser irradiation (650 nm, 25 mW cm -2 ) with or without light;

[0047] Figure 18 : Using Rosup and the culture medium as the positive control and negative control respectively, compare the ability of Pd-TMPP(Au)-HA to generate ROS in HuH-7 cells under the condition of laser irradiation (650 nm, 25 mW cm -2 ) with or without light. Detailed implementation manners

[0048] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with specific implementation manners and with reference to the accompanying drawings. It should be understood that these descriptions are exemplary and are not intended to limit the scope of the present invention. In addition, in the following descriptions, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention.

[0049] Example 1 Synthesis of Pd-TMPP(Au)

[0050] Dissolve H2TMPP (2.0 mg, 0.003 mmol), HAuCl4·4H2O (1.2 mg, 0.003 mmol), and Pd(OAc)2 (1.4 mg, 0.006 mmol) in 2 mL of a mixed solvent (the volume ratio of methanol to dichloromethane is 3:1), and add the regulator HAc (60 μL). Add the above mixed solution into a Pyrex glass tube, place it in a programmable oven, heat it from room temperature to 120 °C within 4 h, then keep it at 120 °C for 48 h, and cool it to room temperature within 24 h. Remove the mother liquor, and wash the obtained crystals with ether 2 - 3 times to obtain Pd-TMPP(Au) (1.5 mg). FT-IR (ATR, cm -1 ): 2756(w), 2689(w), 2601(w), 2253(w), 1711(s), 1437(m), 1413(m), 1402(m), 1291(m), 1265(w), 1206(w), 995(m), 968(m), 951(w), 929(vs), 910(m), 791(vs), 747.(vs), 607(w). 11H NMR (400 MHz, CDCl3) δ 9.42 (d, J = 21.4 Hz, 12H), 8.85 (d, J = 44.0 Hz, 12H), 8.59 (d, J = 17.2 Hz, 12H), 8.35 (s, 12H), 7.62 (d, J = 8.4 Hz, 12H), 3.80 (s, 36H), -3.51 (s, 4H).

[0051] The diffraction data of Pd-TMPP(Au) prepared in Example 1 of the present invention were obtained on a Bruker D8 VENTURE X-ray single crystal diffractometer, using Mo-Kα monochromatized by a graphite monochromator as the radiation source. Subsequently, the Bruker SAINT program package was used to reduce the test data of the above compound, and all diffraction data were corrected for multi-scan absorption. To solve the twin data, 9464 diffractions with I / σ(I) > 10 were selected from all the collected data, and the diffraction points were split using the CELL_NOW program. The results showed that the rotation angle between the two twins was 165.6°. The data of the two components split by CELL_NOW were reduced, and then the crystal structure was solved and refined using one set of single crystal data.

[0052] The crystal structure of Pd-TMPP(Au) was solved by the direct method using the SHELXTL-2016 program package. Subsequently, the coordinates of all non-hydrogen atoms were determined by several rounds of difference Fourier synthesis, and the non-hydrogen atoms were anisotropically refined by full matrix least squares method. The crystal data of Pd-TMPP(Au) have been deposited in the Cambridge Crystallographic Data Centre (CCDC2422368).

[0053] The Pd-TMPP(Au) prepared in Example 1 was characterized, and the results are as follows:

[0054] (1) X-ray single crystal diffraction (SCXRD) data

[0055] X-ray single crystal diffraction (SCXRD) data of Pd-TMPP(Au) prepared in Example 1. Empirical formula C 264 H 192 Au3Cl 27 N 48 Pd 12 ; Formula weight 6861.49; Crystal system orthorhombic; Space group Cmc21; 30.46(2); 23.068(16); 28.45(2); α / ° 90; β / ° 90; γ / ° 90; 19985(25); Z 2; D c / (g cm –3 ) 1.140; μ(Mo–Kα) / mm –1 1.844; F(000) 6720; Unique reflections 10620; Observed data (I > 2σ(I)) 6223; R indexes [I > 2σ(I)] R1 = 0.0611, wR2 = 0.1295; GOF 1.047.

[0056] (2) Crystal characteristics of Pd-TMPP(Au)

[0057] Pd-TMPP(Au) is an extremely rare double-arrow crystal (as Figure 2 shown). We took the central part of the crystal and determined its structure by an X-single crystal diffractometer. Crystal structure analysis shows that the structure of Pd-TMPP(Au) is a twin structure, and it crystallizes in the Cmc21 space group of the orthorhombic system. In Pd-TMPP(Au), three TMPP(Au) metal ligands combine with six PdCl2 to form a hexagonal prism structure (as Figure 3 shown).

[0058] (3) Energy-dispersive X-ray spectroscopy (EDS) characterization

[0059] Figure 4 The energy-dispersive X-ray spectroscopy diagram and Mapping diagram are for a large number of Pd-TMPP(Au) crystals crushed into powder. Overall, in Pd-TMPP(Au), Pd:Au:Cl = 2.6:0.6:5.4 (equivalent to 6.0:1.4:12.5), but it is lower than the expected Pd:Au:Cl = 6.0:3.0:15.0. Since the crystal morphology of Pd-TMPP(Au) is not uniform, and from the crystal data, Pd-TMPP(Au) grows in the form of twins. Therefore, we took a single crystal and analyzed the elemental composition of its main body and both ends again, and found that the distribution content of gold element in the two parts is not the same (as Figure 5As shown, very little or no gold element is distributed in the end part of the crystal, while it is concentrated in the central part of the crystal. The element distribution ratio is Pd:Au:Cl = 0.9:0.3:1.8 (equivalent to 6.0:2.0:12.0), which is much higher than that of the end without Au. It should be noted that the element distribution ratio of the whole crystal is Pd:Au:Cl = 0.6:0.1:1.1 (equivalent to 6.0:1.0:11.0). Assuming that the core of the central part of the crystal is rich in Au while the content of Au on the surface is less (there is no Au at the end of the crystal and the gold content on the surface of the whole crystal is low), a high-entropy coordination molecular cage with multiple ligand combinations is thus obtained.

[0060] (4) Data analysis of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS)

[0061] MALDI-TOF MS further proves the high entropy of Pd-TMPP(Au). In principle, the random combination of three porphyrin ligands (i.e., H2TMPP, TMPP(Pd), and TMPP(Au)) can produce a total of ten combinations, either neutral (without TMPP(Au)) or charged (with TMPP(Au)). And we can identify at least five ligand combinations (as Figure 6 shown), including 3087.50 m / z ([Pd6Cl 12 (H2TMPP)3 + H] + ), 3191.38 m / z ({Pd6Cl 12 (H2TMPP)2[TMPP(Pd)] + H} + ), 3281.37 m / z ({Pd6Cl 12 (H2TMPP)2[TMPP(Au)]} + ), 3386.25 m / z ({Pd6Cl 12 (H2TMPP)[TMPP(Pd)][TMPP(Au)]} + ), and 3490.11 m / z ({Pd6Cl 12 [TMPP(Pd)]2[TMPP(Au)]} + ).

[0062] (5) Data analysis of nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR)

[0063] As Figure 7 shown, it can be seen from 1 1H NMR that compared with the original H2TMPP ( Figure 7 a)), after the formation of Pd-TMPP(Au) ( Figure 7b), the six pyrrole proton peaks of H2TMPP at -2.82 ppm shifted to -3.51 ppm and decreased to four (the other two were removed by metal chelation), indicating that two-thirds of the porphyrin ligands remained free and one-third was chelated by Pd(II) or Au(III). There are four methyl groups at the ortho positions of each pyridine in H2TMPP, and the 1 H NMR peak positions of the 12 hydrogen atoms of the methyl groups are at 2.95 ppm, and the methyl displacement peaks of TMPP(Pd) in Pd-TMPP(Au) shifted to 3.80 ppm, and the 1 H NMR peaks on the porphine also showed corresponding shifts.

[0064] (6) Fourier transform infrared (FT-IR) spectroscopy characterization

[0065] As Figure 8 shown, in the FT-IR spectrum, the peak corresponding to the stretching vibration of the C=N bond on the pyridine of H2TMPP is at 1593 cm -1 , while in the spectrum of Pd-TMPP(Au), it shifted to 1612 cm -1 after metal coordination. At the same time, the in-plane vibration peak of N-H on the central pyrrole of the porphyrin corresponding to H2TMPP at 964 cm -1 reached its peak, and shifted to 1013 cm -1 and significantly weakened due to partial chelation at the center of the porphyrin ring.

[0066] (7) Ultraviolet-visible absorption (UV-Vis) spectroscopy characterization

[0067] As Figure 9 shown, it can be seen from the UV-Vis spectrum that after the formation of Pd-TMPP(Au), the Soret band (420 nm) of H2TMPP blue-shifted to 408 nm, indicating that the porphyrin skeleton had successfully chelated metal ions. In addition, the presence of an additional shoulder peak at around 420 nm and four Q bands in the wavelength range of 500 - 700 nm further confirmed the overall partial chelation at the porphyrin center.

[0068] (8) X-ray photoelectron spectroscopy (XPS) spectrum characterization

[0069] To determine the valence state of the metal in Pd-TMPP(Au), XPS tests were performed on the compound. Figure 10 Figure b is the Pd 3d spectrum of Pd-TMPP(Au). By comparing with the XPS spectra of Pd complexes in the literature, we found that the binding energies at 337.8 eV and 343.1 eV corresponded to Pd 3d 5 / 2 and Pd 3d 3 / 2Spin-orbit splitting, so the valence state of Pd in Pd-TMPP(Au) is +2. Since photoreduction is prone to occur during the photoemission process, the XPS of Au in Pd-TMPP(Au) exhibits a rather complex pattern ( Figure 10 c), and its binding energy is Au(III) (4f 7 / 2 87.2 eV; 4f 5 / 2 91.9 eV), Au(I) (4f 7 / 2 84.2 eV; 4f 5 / 2 91.4 eV), and Au(0) (4f 7 / 2 83.3 eV; 4f 5 / 2 88.0 eV). Combining with the crystal structure, we speculate that the valence state of Au in Pd-TMPP(Au) is +3.

[0070] Example 2 Preparation of Pd-TMPP(Au)-HA Nanopreparation

[0071] Dissolve 2 mg of Pd-TMPP(Au) prepared according to the method of Example 1 in 1 mL of DMSO solution, and then slowly drop the above mixed solution into the sodium hyaluronate aqueous solution (15 mg of sodium hyaluronate dissolved in 5 mL of deionized water), and stir in a round-bottom flask for 72 h. Finally, dialyze this solution in a dialysis bag with a molecular weight cut-off of 3500 for 24 h to obtain Pd-TMPP(Au)-HA, and change the water every 3 h during dialysis.

[0072] Characterize and test the Pd-TMPP(Au)-HA nanopreparation prepared in Example 2 as follows:

[0073] (1) Transmission electron microscopy (TEM) characterization

[0074] Modify Pd-TMPP(Au) with sodium hyaluronate to generate nanoparticles Pd-TMPP(Au)-HA. The TEM image of the nanoparticles is as Figure 12 shown. It can be seen from the figure that Pd-TMPP(Au)-HA forms spherical nanoparticles in water and is uniformly dispersed.

[0075] (2) Dynamic light scattering (DLS) and Zeta potential characterization

[0076] To further determine the particle size of the two kinds of nanoparticles, we characterized and analyzed them by DLS. The particle size of Pd-TMPP(Au)-HA is about 202.8 nm (as Figure 13 shown). It can be seen that the obtained value is larger than the theoretical value measured by TEM, which is due to the phenomenon formed by the hydration of the micelles. The Zeta potential of Pd-TMPP(Au)-HA is -29.9 mV (asFigure 14 As shown, nanoparticles with negative Zeta potential are beneficial to the circulation stability during the drug delivery stage.

[0077] (3) Photostability of Pd-TMPP(Au)-HA

[0078] Prepare an aqueous solution of Pd-TMPP(Au)-HA with a porphyrin concentration of 7 μg / mL, and then irradiate the above aqueous solution with a laser at 650 nm (25 mW cm -2 ), and compare the intensity changes of their respective Soret bands through UV-Vis spectroscopy. As Figure 15 shown, there is no obvious change in the ultraviolet absorption intensity within 10 min of irradiation, indicating that Pd-TMPP(Au)-HA has good photostability in aqueous solution.

[0079] (4) Determination of 1 O2 content in solution

[0080] Using 1,3-diphenylisobenzofuran (DPBF) as a probe, measure the generation of 1 O2 in the aqueous solution of Pd-TMPP(Au)-HA. Add the DPBF solution with the same concentration to the aqueous solution of Pd-TMPP(Au)-HA. Among them, the concentration of DPBF is 33 μg / mL, and the porphyrin concentration is 7 μg / mL. Irradiate with a laser (650 nm, 25 mW cm -2 ) once every 1 min for a total of 6 min. DPBF can be 1 oxidized and decomposed by Figure 16 O2, resulting in a decrease in the absorption peak centered at 417 nm. As 1 shown, compared with the control group, there is significant

[0081] (5) Measurement of cytotoxicity by CCK8 method

[0082] Take HuH-7 cells in the logarithmic growth phase for experiments. After removing the culture medium, wash the cells twice with PBS, then add 1 mL of 0.25% trypsin solution, digest in a cell incubator at 37°C for about 2 min, and add 2 mL of serum-containing medium to terminate digestion after observing that the cells become round under the microscope. Collect the cell suspension and pipette to obtain a single-cell suspension of HuH-7. Count the cell suspension with a hemocytometer and use 1×10 4The cells were seeded in a 96-well plate at a density of 90 μL per well. After culturing the 96-well plate under the above culture conditions for 24 h, the medium was replaced with serum-free DMEM at 90 μL / well. Then, a drug stock solution was prepared, and the high-concentration Pd-TMPP(Au)-HA aqueous solution was serially diluted with serum-free DMEM medium. 10 μL of the above compound solution was added to the cell solution in sequence, with 6 replicates per concentration (n = 6). In the control group, the medium with cells was directly replaced with 100 μL of serum-free DMEM. After co-incubating the cells with the drug for 24 h, for the phototoxicity experiment, the cells were irradiated with a laser for 5 min, and the cells in the dark toxicity experiment were not irradiated. After the cells were cultured for another 48 h, the original medium was removed, and 100 μL of complete medium containing serum was added to each well to be tested. 10 μL of CCK8 was added to each well and then placed in an incubator for about 2 h. The OD value at 450 nm was measured with a microplate reader, and finally, the cell viability was calculated according to the formula.

[0083]

[0084] where V% is the percentage of cell viability, experimental is the absorbance of the cells treated with different drugs, blank is the absorbance of the blank group, control is the absorbance of the cells not treated with any drug.

[0085] The CCK8 experiments for PLC / PRF / 5 cells and Hep-G2 cells were similar to those for HuH-7 cells and will not be elaborated here.

[0086] The HCC cell lines HuH-7 ( Figure 17 a), Hep-G2 ( Figure 17 b), and PLC / PRF / 5 ( Figure 17 c) were selected to evaluate the cytotoxicity of Pd-TMPP(Au)-HA under the conditions of with or without laser irradiation. It can be seen that its dark toxicity is positively correlated with the drug concentration. Under light irradiation conditions, the cytotoxicity is significantly increased, showing a good PDT effect. The half-inhibitory concentration (IC50) of HuH-7 for Pd-TMPP(Au)-HA was 6.09 μg / mL and 0.05 μg / mL, that of Hep-G2 was 8.44 μg / mL and 0.02 μg / mL, and that of PLC / PRF / 5 was 26.37 μg / mL and 0.61 μg / mL.

[0087] (6) Detection of intracellular ROS

[0088] Using DCFH-DA as a fluorescent probe for detecting ROS, taking HuH-7 cells as a representative, and using Rosup and PBS as positive and negative controls respectively, the content of ROS generated by Pd-TMPP(Au)-HA was measured. The DCFH-DA probe itself does not have fluorescence characteristics, but it can easily cross the cell membrane and be taken up by cells. Once inside the cell, DCFH-DA is rapidly hydrolyzed by intracellular esterases to form DCFH. It should be noted that since DCFH cannot cross the cell membrane, it can be stably retained inside the cell, ensuring the effective loading of the probe. The ROS generated by nanoparticles inside the cell can oxidize non-fluorescent DCFH to generate fluorescent DCF with green fluorescence. In the intracellular environment, the ROS triggered by nanoparticles can oxidize non-fluorescent DCFH, and then generate DCF that emits green fluorescence. By measuring the fluorescence intensity of DCF, the generation of intracellular ROS can be visually evaluated. As Figure 18 shown, in the absence of light irradiation, the amount of ROS generated by Pd-TMPP(Au)-HA is relatively limited. However, when light irradiation (650 nm, 25 mW cm -2 , 5 min) is applied, the amount of ROS generated by Pd-TMPP(Au)-HA increases significantly, which is consistent with the experimental conclusion of extracellular ROS.

[0089] (7) Cell uptake

[0090] HuH-7 cells were seeded into six 10-mL cell culture dishes at a density of 2×10 7 per dish using DMEM complete medium. When the cells adhered and proliferated to a density of 95%, the medium was replaced with serum-free medium. One group pretreated with sodium hyaluronate was denoted as Pd-TMPP(Au)-HA’ to distinguish it from the direct drug application group Pd-TMPP(Au)-HA. In the Pd-TMPP(Au)-HA’ group, 30 mg of sodium hyaluronate was added to each dish and incubated for 24 h, and then Pd-TMPP(Au)-HA was added. In the Pd-TMPP(Au)-HA group, Pd-TMPP(Au)-HA was directly added. The porphyrin concentration in the medium was 2 μg / mL. After adding the drugs, the cells were cultured for 2 h, 4 h, and 6 h respectively. The cells were washed three times with PBS buffer solution, and the cells were collected in a 15-mL centrifuge tube by trypsin digestion method and centrifuged at 900 rpm for 3 min. The precipitate was washed twice with PBS, and the cell precipitate was collected after centrifugation. The cells were lysed with 0.5 mL of nitric acid and then diluted to 5 mL with deionized water, filtered, and the mixed solution was analyzed by ICP-MS to determine the content of metallic gold in the cells.

[0091] CD44, as a specific receptor for hyaluronic acid, is overexpressed in a variety of cell lines (including HuH-7, Hep-G2, and PLC / PRF / 5). Therefore, pretreatment of cells containing CD44 receptors with hyaluronate followed by drug treatment may reduce the drug uptake efficiency. We used inductively coupled plasma mass spectrometry (ICP-MS) to estimate the cellular uptake efficiency of drugs with or without pretreatment with sodium hyaluronate, using the representative cell line HuH-7. As shown in Table 1, regardless of whether pretreatment with sodium hyaluronate (cells pretreated with sodium hyaluronate are denoted as Pd-TMPP(Au)-HA’), the amount of drug taken up by the cells increased with the prolongation of the incubation time. However, the uptake of Pd-TMPP(Au)-HA by HuH-7 cells pretreated with sodium hyaluronate was significantly lower than that of untreated cells. These results clearly demonstrate that the drug Pd-TMPP(Au)-HA modified with hyaluronic acid has a targeting effect.

[0092] Table 1. Gold content (ppb) in HuH-7 cells under different treatment methods

[0093]

[0094] It should be understood that the above specific embodiments of the present invention are only used for illustrative explanation or interpretation of the principle of the present invention, and do not constitute a limitation on the present invention. Therefore, any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and scope of the present invention shall be included within the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. A porphyrin-based prismatic coordination molecular cage, characterized in that, The porphyrin-based prismatic coordination molecular cage is denoted as Pd-TMPP(Au), which is composed of 6 PdCl2 units and the ligand combination (H2TMPP) x [TMPP(Pd)] y [TMPP(Au)] z Cl z A total of 10 complexes are formed, where 0 ≤ x ≤ 3, 0 ≤ y ≤ 3, 0 ≤ z ≤ 3, and x + y + z = 3, as shown in the figure below: The Pd-TMPP(Au) includes at least five of the complexes formed by six PdCl2 units and ligand combinations (H2TMPP) x [TMPP(Pd)] y [TMPP(Au)] z Cl z ​ 2. The porphyrin-based prismatic coordination molecular cage according to claim 1, characterized in that, The Pd-TMPP(Au) includes at least the following five: Pd6Cl 12 (H2TMPP)3, Pd6Cl 12 (H2TMPP)2[TMPP(Pd)], {Pd6Cl 12 (H2TMPP)2[TMPP(Au)]}Cl, {Pd6Cl 12 (H2TMPP)[TMPP(Pd)][TMPP(Au)]}Cl and Pd6Cl 12 [TMPP(Pd)]2[TMPP(Au)]}Cl.

3. The porphyrin-based prismatic coordination molecular cage according to claim 1, wherein The structure of Pd-TMPP(Au) is a twin crystal structure with double arrows, and it crystallizes in the Cmc21 space group of the orthorhombic system.

4. A method for preparing the porphyrin-based prismatic coordination molecular cage according to any one of claims 1-3, characterized in that, H2TMPP, Au(III) metal salt and Pd(OAc)2 are dissolved in a solvent, and an acidic regulator is added. Then, after hydrothermal treatment, the mother liquor is removed, and the obtained crystals are washed to obtain the porphyrin-based prismatic coordination molecular cage Pd-TMPP(Au).

5. The preparation method according to claim 4, characterized in that, The conditions for hydrothermal treatment are as follows: first, the temperature is raised from room temperature to 120 °C within 4 - 8 h, kept at this temperature for 24 - 48 h, and then cooled to room temperature within 24 - 48 h.

6. The preparation method according to claim 4, wherein The Au(III) metal salt is at least one of HAuCl4·4H2O, HAuCl4·3H2O, and AuCl3; The molar ratio of H2TMPP, Au(III) metal salt and Pd(OAc)2 is 1:1:

2.

7. Use of the porphyrin-based prismatic coordination molecular cage according to any one of claims 1 - 3 in the preparation of a drug for treating liver cancer.

8. A preparation method of a porphyrin-based prismatic coordination molecular cage nanoformulation, characterized in that, The porphyrin-based prismatic coordination molecular cage according to any one of claims 1 - 3 is dissolved in dimethyl sulfoxide to obtain a mixed solution. Then, the mixed solution is dropped into an aqueous solution of sodium hyaluronate and stirred for 24 - 72 h, and then the porphyrin-based prismatic coordination molecular cage nano - preparation Pd-TMPP(Au)-HA is obtained through dialysis.

9. A porphyrin-based prismatic coordination molecular cage nanomedicine, characterized in that, Prepared by the preparation method described in claim 8.

10. Use of the porphyrin-based prismatic coordination molecular cage nano - preparation according to claim 9 in the preparation of a drug for treating liver cancer.

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