Pd-Zn dissimilar metal coordination molecular cage based on TMPP ligand and anti-tumor application

Abstract

The invention belongs to the field of anti-cancer drugs, and particularly relates to an anti-cancer porphyrin molecular cage structure and cytotoxic application of the anti-cancer porphyrin molecular cage structure to liver cancer. The invention discloses two coordination molecular cages Pd-TMPP (Cu) and Pd-TMPP (Zn), and detailed and accurate verification is carried out on the structural characteristics and element composition of the two coordination molecular cages through a series of tests. And then the two compounds are modified by hyaluronic acid so as to obtain targeting and water solubility. Experimental results show that the Pd-TMPP (Zn) has concentration-dependent cytotoxicity to HuH-7, Hep-G2 and PLC / PRF / 5, and the cytotoxicity is remarkably enhanced after the Pd-TMPP (Zn) is irradiated by infrared light.

Description

Technical Field

[0001] This invention belongs to the field of anticancer drugs, specifically relating to the molecular cage structure of anticancer porphyrins and their cytotoxic effects on liver cancer. Background Technology

[0002] Photodynamic therapy (PDT), as a controllable, non-invasive local treatment with few side effects, has attracted much attention and has become one of the emerging cancer treatment methods. PDT generates reactive oxygen species (ROS), such as singlet oxygen, through the reaction of excited-state photosensitizers and oxygen sources. 1 O2), hydroxyl radicals (•OH) and superoxide radicals (O2) •- This process uses photosensitizers to selectively take up photosensitizers at the local lesion site and is then exposed to light of an appropriate wavelength. Through photosensitizer-mediated oxidative damage involving oxygen molecules, it induces apoptosis and / or necrosis of target cells. Because it has almost no side effects of damaging normal tissue cells, it has unique advantages in clinical practice and has been widely used in the treatment of esophageal cancer, non-melanoma skin cancer, cervical cancer, and other tumors. It is expected to become a routine adjuvant therapy for various malignant tumors. Simultaneously, PDT treatment can also induce tumor immunity, providing a new strategy for the effective treatment of some refractory malignant tumors.

[0003] Porphyrin compounds are widely used as photosensitizers in the process of phototherapy (PDT). For example, Photofrin, Temoporfin, Photochlor, Redaporfin, and Talaporfin have been approved by the FDA and used in the clinical treatment of cancer. The introduction of transition metal ions, such as iron, manganese, copper, zinc, and gold, can enhance PDT or achieve combined therapeutic effects. However, for common porphyrin compounds, their PDT effect is always affected by many factors, including: (1) the excited-state aggregation-induced quenching (ACQ) effect caused by the π-π stacking of porphyrin planar and rigid structures; (2) the limitation of the porphyrin aromatic ring structure being difficult to dissolve in water; (3) the limitation of the penetration depth of the excitation light during PDT; (4) the dependence of PDT on oxygen and the limitation of the hypoxic microenvironment of the tumor; and (5) the selectivity of the photosensitizer to the tumor.

[0004] Although some small molecule photosensitizers have been approved for clinical use, these porphyrin-based photosensitizers have poor selectivity for tumors and are highly accumulated in the skin, causing the skin to be photosensitive. In addition, such planar monomolecular compounds are likely to cause self-quenching of the excited state due to intermolecular π-π interactions, thereby weakening the PDT effect. By combining porphyrin derivatives as functional modules with nanotechnology, complex functional materials can be obtained for the synergistic treatment of tumors. However, these complex materials often have unclear chemical components, such as molecular configuration, chiral configuration of the metal center, and accurate host-guest interaction sites, due to the complexity of the components. In addition, the interactions (hydrogen bonds, π-π interactions, van der Waals forces, and hydrophilic and hydrophobic interactions) between the multiple components of the multi-level assembled nanodrugs are weak, and the particle size is large, which leads to potential risks such as early release of the drug, systemic toxicity, significant liver and kidney toxicity, and low delivery efficiency. Finally, the presence of metal nanoparticles in the nanodrug delivery system is a concern for long-term immunotoxicity risks in clinical translation.

[0005] The construction of metal-organic hybrid systems, such as metal-organic frameworks and metal-organic coordination molecular cages, using porphyrin compounds as ligands and the surface functionalization of the resulting materials can effectively solve the above problems. SUMMARY

[0006] In the present disclosure, metal-porphyrin coordination molecular cages and their antitumor applications are disclosed. In order to further realize the synergistic effect of different metals in the antitumor process, heteronuclear metal-porphyrin coordination molecular cages and their corresponding antitumor activities are further studied.

[0007] In one aspect of the present application, the present application provides a TMPP ligand-based Pd-M heterometallic coordination molecular cage, wherein M is a divalent metal ion; preferably, M is Cu or Zn, i.e., a TMPP ligand-based Pd-Cu or Pd-Zn heterometallic coordination molecular cage Pd-TMPP(Cu) or Pd-TMPP(Zn).

[0008] In one embodiment of the present application, the structure of the TMPP ligand-based Pd-Cu heterometallic coordination molecular cage Pd-TMPP(Cu) is (PdCl2)6[TMPP(Cu)]3, and the chemical molecular formula is C 132 H 96 Cl 12 Cu3N 24 Pd 6。

[0009] In one embodiment of the present application, the TMPP ligand-based Pd-Zn heterometallic coordination molecular cage Pd-TMPP(Zn) has a structural formula of (PdCl2)6[TMPP(Zn)]3 and a chemical molecular formula of C 132 H 102 Cl 12 N 24 O3Pd6Zn3.

[0010] In one embodiment of the present application, two pairs of Pd 2+ (each pair of three) are combined with three TMPP(M) (M = Cu, Zn) to form a hexagonal prism structure, which is a monoclinic P21 / c space group and an orthorhombic Cmc21 space group.

[0011] In one aspect of the present application, the present application provides a hyaluronic acid modified TMPP ligand-based Pd-M heterometallic coordination molecular cage. In the present application, the applicant successfully constructed two palladium-based porphyrin coordination molecular cages Pd-TMPP(Cu) and Pd-TMPP(Zn) by assembling H2TMPP, Pd(OAc)2, Cu(OAc)2 / ZnCl2 using a hydrothermal method. The unique arrangement of these (metal) ligands in the cage inhibits the π-π stacking of porphyrin photosensitizers, thereby improving the effect of PDT. Subsequently, these coordination molecular cages were functionally modified with HA to impart excellent water solubility and targeting properties.

[0012] In one embodiment of the present application, the hyaluronic acid (HA) modified TMPP ligand-based Pd-M heterometallic coordination molecular cage is a hyaluronic acid modified Pd-TMPP(Zn), namely Pd-TMPP(Zn)-HA.

[0013] In one embodiment of the present application, the hyaluronic acid (HA) modified TMPP ligand-based Pd-M heterometallic coordination molecular cage is a hyaluronic acid modified Pd-TMPP(Cu), namely Pd-TMPP(Cu)-HA.

[0014] In the application, H2TMPP, Pd(OAc)2, Cu(OAc)2 / ZnCl2 are assembled by hydrothermal method, two palladium-based porphyrin coordination molecular cages Pd-TMPP(Cu) and Pd-TMPP(Zn) are successfully constructed. The hyaluronic acid (HA) functionalization modification is further carried out on the coordination molecular cages, which endows them with excellent water solubility and targeting property for HA receptor highly expressed liver cancer cell lines HuH-7, Hep-G2 and PLC / PRF / 5. The experimental results show that the HA modified Pd-TMPP(Zn), namely Pd-TMPP(Zn)-HA, has concentration-dependent cytotoxicity to HuH-7, Hep-G2 and PLC / PRF / 5, and after infrared light irradiation, the cytotoxicity is significantly enhanced; while the corresponding Pd-TMPP(Cu)-HA has poor cytotoxicity at the same concentration.

[0015] In one aspect of the application, the application provides a use of the TMPP ligand-based Pd-M heterometallic coordination molecular cage or the hyaluronic acid (HA) modified TMPP ligand-based Pd-M heterometallic coordination molecular cage for preparing an antitumor drug or an antitumor drug carrier.

[0016] In one embodiment of the application, the antitumor drug is a targeted drug.

[0017] In one embodiment of the application, the tumor is liver cancer.

[0018] In the application, H2TMPP ligand and Pd(OAc)2 are respectively coordinated and self-assembled with Cu(OAc)2 or ZnCl2 by hydrothermal method, two heteronuclear bimetallic coordination molecular cages Pd-TMPP(Cu) and Pd-TMPP(Zn) are obtained, and their successful synthesis is determined by X-ray single crystal diffraction (SCXRD), ultraviolet spectrum (UV-Vis), infrared spectrum (FT-IR), energy dispersive X-ray spectrum (EDS) and the like. Since Pd-TMPP(Cu) and Pd-TMPP(Zn) have poor solubility, they are further wrapped with sodium hyaluronate to obtain water-soluble nanoparticles. Transmission electron microscopy (TEM) results show that the wrapped coordination molecular cages form spherical nanoparticles, which effectively improve their solubility and stability in physiological environment.

[0019] (1) The π-π stacking caused by the excited state aggregation-induced quenching (ACQ) effect in the PDT process can be effectively inhibited by constructing a prismatic coordination molecular cage compound from a planar rigid structure porphyrin molecule.

[0020] (2) The Cu 2+ , Zn 2+Metal-chelated coordination molecular cages can significantly inhibit the deprivation of metal ions (such as Fe in heme) from biomolecules by porphyrin molecules during drug delivery. 2+ This is to reduce systemic toxicity during drug delivery.

[0021] (3) First transition metal ions such as Cu 2+ Zn 2+ In chelates with porphyrins, the metal coordination configuration is often a five-coordinated tetrapyramidal or a six-coordinated octahedral. Furthermore, these ions are hard acids, making it easier for them to incorporate hard base ligands such as hyaluronic acid and folic acid with carboxylic acid functional groups during particle modification, thus forming a stable drug delivery system.

[0022] (4) Coordination molecular cage materials can greatly improve the biocompatibility and tumor targeting of the delivery system by coordinating with hyaluronic acid. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the synthesis of Pd-TMPP(Cu) and Pd-TMPP(Zn).

[0024] Figure 2(a) is a schematic diagram of the synthesis of Pd-TMPP(Cu)-HA.

[0025] Figure 2(b) is a schematic diagram of the synthesis of Pd-TMPP(Zn)-HA.

[0026] Figure 3 (a) and (c) are side views of the Pd-TMPP(Cu) and Pd-TMPP(Zn) crystal structures, respectively; (b) and (d) are top views of the Pd-TMPP(Cu) and Pd-TMPP(Zn) crystal structures, respectively.

[0027] Figure 4 The H2TMPP ligand in CDCl3 solvent in Example 1 1 H NMR spectrum.

[0028] Figure 5 Pd-TMPP(Zn) in CDCl3 solvent in Example 2 1 H NMR spectrum.

[0029] Figure 6(a) shows the MALDI-TOF MS image of Pd-TMPP(Cu) in CHCl3.

[0030] Figure 6(b) shows the MALDI-TOF MS plot of Pd-TMPP(Zn) in CHCl3.

[0031] Figure 7 This is the EDS mapping diagram for Pd-TMPP(Cu).

[0032] Figure 8 This is the EDS mapping diagram for Pd-TMPP(Zn).

[0033] Figure 9 Infrared spectra of H2TMPP, Pd-TMPP(Cu) and Pd-TMPP(Zn).

[0034] Figure 10 Ultraviolet spectra of H2TMPP, Pd-TMPP(Cu) and Pd-TMPP(Zn) (Inset: Local spectra with absorption values ​​magnified 4 times in the range of 480-680 nm).

[0035] Figure 11 In the diagram, (a) and (d) are the full spectra of Pd-TMPP(Cu) and Pd-TMPP(Zn), respectively; (b) is the Cu 2p spectrum of Pd-TMPP(Cu); (e) is the Zn 2p spectrum of Pd-TMPP(Zn); and (c) and (f) are the corresponding Pd 3d spectra of Pd-TMPP(Cu) and Pd-TMPP(Zn), respectively.

[0036] Figure 12 In the image, (a) and (b) are TEM images of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA, respectively.

[0037] Figure 13 In the image, (a) and (b) are the DLS spectra of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA, respectively.

[0038] Figure 14 In the diagrams (a) and (b), the zeta potentials of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA are respectively.

[0039] Figure 15 Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA solutions under laser irradiation (650 nm, 25 mW cm⁻¹) -2 The intensity variation of the lower Soret band.

[0040] Figure 16 (a) shows the infrared light intensity of DPBF in DMSO solution (650 nm, 25 mW cm⁻¹). -2 (a) The rate of change of absorbance at 417 nm under irradiation with time. (b) and (c) are the UV absorption intensities at 417 nm of DPBF dispersed in DMSO solutions of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA, respectively, under infrared light (650 nm, 25 mW cm⁻¹).-2 The curve of change under irradiation, (d) is Figure 16 Comparison of absorbance change rate curves in (a)-(c)

[0041] Figure 17 In the middle (a)-(f), the infrared light (650 nm, 25 mW cm⁻¹) is used for illumination with / without light. -2 Under the conditions of ), HuH-7 (…) was incubated with gradient concentrations of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA. Figure 17 (a and b), Hep-G2 ( Figure 17 (c and d) and PLC / PRF / 5 ( Figure 17 Bar chart of cell survival rates for e and f).

[0042] Figure 18 Rosup and PBS were used as positive and negative controls, respectively, to compare the effects of infrared light irradiation (650 nm, 25 mW cm⁻¹) with and without. -2 The diagram shows the ability of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA to generate ROS in HuH-7 cells under the condition of ). Detailed Implementation

[0043] Example 1: Synthesis of (PdCl2)6[TMPP(Cu)]3 (abbreviated as Pd-TMPP(Cu, the other examples are similar).

[0044] H₂TMPP (2.0 mg, 0.003 mmol), Cu(OAc)₂ (0.5 mg, 0.003 mmol), and Pd(OAc)₂ (1.4 mg, 0.006 mmol) were dissolved in 2 mL of a 3:1 mixture of MeOH and CH₂Cl₂, with 60 μL of HOAc added as a modifier. The mixture was added to a glass tube and placed in a programmable oven. The temperature was increased from room temperature to 120 °C over 4 h, maintained at 120 °C for 48 h, and then cooled to room temperature over 24 h. The resulting crystals, after discarding the mother liquor, were washed 2-3 times with diethyl ether to obtain Pd-TMPP(Cu) (1.5 mg, yield calculated as 75% based on TMPP). FT-IR (ATR, cm⁻¹) -1):3455 (w), 2961 (w), 2920 (w), 2851 (w), 2161 (w), 2042 (vs), 1610 (w), 1541(s), 1498 (m), 1446 (w), 1374 (w), 1345 (w), 1299 (w), 1260 (m), 1209 (w),1139 (w), 1083 (m), 1029 (m), 1001 (s), 898 (w), 854 (w), 796 (vs), 718 (m),623 (w), 627 (w).

[0045] Example 2: Synthesis of (PdCl2)6[TMPP(Zn)]3 (Pd-TMPP(Zn)).

[0046] H₂TMPP (2.0 mg, 0.003 mmol), ZnCl₂ (0.4 mg, 0.003 mmol), and Pd(OAc)₂ (1.4 mg, 0.006 mmol) were dissolved in 2 mL of a 6:1:1 mixture of MeOH, CH₂Cl₂, and chlorobenzene, with 60 μL of HOAc added as a modifier. The mixture was added to a glass tube and placed in a programmable oven, heated from room temperature to 120 °C over 4 h, maintained at 120 °C for 48 h, and finally cooled to room temperature over 24 h. The resulting crystals, after discarding the mother liquor, were washed 2-3 times with diethyl ether to obtain Pd-TMPP(Zn) (1.8 mg, yield calculated as 90% based on TMPP). FT-IR (ATR, cm⁻¹) -1 ):3467 (w), 2921 (w), 2851 (w), 2184 (w), 2022 (w), 1609 (w), 1562 (w), 1527(w), , 1493(m), 1446 (m), , 1372 (w), 1338 (w), 1298 (m), 1259 (w), 1208 (w),1139 (m), 1076 (m), 1042 (m), 1014 (s), 995 (s), 914 (w), 989 (w), 850 (s),808 (vs), 793 (s), 718 (m), 625(w). The NMR data are: 1H NMR (400 MHz, CDCl3) δ 9.37(s, 12H), 8.83 (s, 12H), 8.62 (s, 12H), 8.36 (s, 12H), 7.57 (s, 12H), 3.81(s, 36H).

[0047] Schematic diagrams of the synthesis of Pd-TMPP(Cu) and Pd-TMPP(Zn) are shown below. Figure 1 As shown.

[0048] Example 3: Preparation of heteronuclear bimetallic coordination cage nanoparticles.

[0049] (1) Preparation of Pd-TMPP(Cu)-HA nanoparticles

[0050] 2.6 mg of Pd-TMPP(Cu) was dissolved in 1 mL of DMSO solution. This solution was then slowly added dropwise to an aqueous solution containing sodium hyaluronate (15 mg of sodium hyaluronate dissolved in 5 mL of deionized water), and stirred in a round-bottom flask for 72 h. Finally, this solution was dialyzed for 24 h in a dialysis bag with a molecular weight cutoff of 3500 Da to obtain Pd-TMPP(Cu)-HA. Distilled water was changed every 3 h during dialysis.

[0051] (2) Preparation of Pd-TMPP(Zn)-HA nanoparticles

[0052] Pd-TMPP(Zn)-HA was prepared using the same method described above.

[0053] The synthesis schematic diagrams of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA are shown in Figure 2(a) and Figure 2(b).

[0054] Example 4: Structural Confirmation.

[0055] (1) Crystal structure analysis and refinement

[0056] Single-crystal structures of Pd-TMPP(Cu) and Pd-TMPP(Zn) were collected using a Bruker APEX III CCD X-ray single-crystal diffractometer, with a Mo-Kα (λ = 0.71073 Å) monochromated with a graphite monochromator as the radiation source. The test data of the above compounds were then restored using the APEX III package, and all diffraction data were corrected for multi-scan absorption. The crystal structures of Pd-TMPP(Cu) and Pd-TMPP(Zn) were solved directly using the SHELXTL-2016 package. Subsequently, the coordinates of all non-hydrogen atoms were determined using a multi-round difference Fourier synthesis method, and anisotropy corrections for these non-hydrogen atoms were performed using the full matrix least squares method. Crystallographic data and refinement results are listed in Table 1.

[0057] Table 1. Important crystallographic data and refinement results of Pd-TMPP(Cu) and Pd-TMPP(Zn).

[0058]

[0059] Crystal structure characteristics of Pd-TMPP(Cu) and Pd-TMPP(Zn).

[0060] The crystallographic data of Pd-TMPP(Cu) and Pd-TMPP(Zn) are shown in Table 1. Pd-TMPP(Cu) and Pd-TMPP(Zn) crystallize in the monoclinic P21 / c space group and the orthorhombic Cmc21 space group, respectively, and their molecular compositions and configurations are similar. In Pd-TMPP(Cu) and Pd-TMPP(Zn), two pairs of Pd 2+ (Each pair of three) combines with three TMPP(M) (M = Cu, Zn) to form a hexagonal prism structure, such as Figure 3 As shown.

[0061] Figure 3 (a) and (c) are side views of the Pd-TMPP(Cu) and Pd-TMPP(Zn) crystal structures, respectively; (b) and (d) are top views of the Pd-TMPP(Cu) and Pd-TMPP(Zn) crystal structures, respectively.

[0062] (2) Analysis of proton nuclear magnetic resonance (NMR) data

[0063] Because Pd-TMPP(Cu) is magnetic, it cannot be characterized by NMR. However, for Pd-TMPP(Zn), from... 1 As can be seen from H NMR, it is similar to the original H2TMPP ( Figure 4 Compared to ), in the formation of Pd-TMPP(Zn) ( Figure 5After that, the pyrrole proton peak at -2.82 ppm disappeared in H2TMPP, indicating that Zn 2+ The hydrogen atom on the pyrrole in H2TMPP was successfully replaced to form TMPP(Zn). The four methyl peaks at the ortho position of the pyridine N in H2TMPP are located at 2.95 ppm, while in Pd-TMPP(Zn) they shift to 3.81 ppm. Other... 1 The HNMR peaks also shifted accordingly.

[0064] (3) Analysis of matrix-assisted laser desorption / ionization time-of-flight mass spectrometry data

[0065] MALDI-TOF MS further confirmed the structures of Pd-TMPP(Cu) and Pd-TMPP(Zn). As shown in Figure 6(a), 3271.21 m / z belongs to ({Pd6Cl... 12 [TMPP(Cu)]3+H + As shown in Figure 6(b), 3278.15 m / z belongs to ({Pd6Cl) 12 [TMPP(Zn)]3 + H +}).

[0066] (4) Energy-dispersive X-ray spectroscopy characterization

[0067] Figure 7 and Figure 8 The images show the EDS and mapping diagrams for Pd-TMPP(Cu) and Pd-TMPP(Zn), respectively. The atomic ratio in Pd-TMPP(Cu) is Pd : Cu : Cl = 1.9 : 0.8 : 3.6 (equivalent to 6.0 : 2.5 : 11.3); the atomic ratio in Pd-TMPP(Zn) is Pd : Zn : Cl = 2.3 : 1.1 : 5.1 (equivalent to 6.0 : 2.9 : 13.3). This matches the Pd : Cu / Zn : Cl ratio (6.0 : 3.0 : 12.0) in both molecular formulas, and the elements are evenly distributed in the mapping diagram.

[0068] (5) Fourier transform infrared spectroscopy characterization

[0069] In FT-IR spectroscopy ( Figure 9 In ), H2TMPP is at 1593 cm. -1 The corresponding vibration is the stretching vibration of the C=N bond in pyridine, while in the Pd-TMPP(Cu) spectrum, due to the N and Cu bonds on pyridine... 2+ Coordination occurs, and the stretching vibrations after coordination redshift to 1610 cm. -1 H2TMPP at 964 cm.-1 The in-plane vibration peak of NH on the pyrrole at the corresponding porphyrin center is observed, but this vibration disappears in the Pd-TMPP(Cu) spectrum, indicating that the porphyrin center and Cu... 2+ Coordination and formation of Cu-N bonds occurred. Similar to Pd-TMPP(Cu), the stretching vibration of the C=N bond in Pd-TMPP(Zn) shifted to 1610 cm⁻¹. -1 The in-plane vibrational peak of NH on pyrrole disappears.

[0070] (6) Characterization by ultraviolet-visible absorption spectroscopy

[0071] In the UV-Vis spectrum ( Figure 10 ), free H2TMPP and Cu 2+ / Zn 2+ After chelation, the Soret band of the UV absorption peak underwent a blue shift, indicating that TMPP successfully chelated Cu. 2+ / Zn 2+ In the 500-750 nm range, the number of the four Q bands (516 / 552 / 592 / 648 nm) of H2TMPP decreases in Pd-TMPP(Cu) and Pd-TMPP(Zn), further indicating that the symmetry of the porphyrin ligands increases.

[0072] (7) Characterization by X-ray photoelectron spectroscopy (XPS)

[0073] To determine the valence states of the metals in Pd-TMPP(Cu) and Pd-TMPP(Zn), XPS analysis was performed on both compounds. Figure 11 The Pd-TMPP(Cu) spectrum was compared with that of existing Cu 2p, Zn 2p, and Pd 3d spectra. In Pd-TMPP(Cu), 935.2 eV and 955.3 eV correspond to the 2p of Cu(II), respectively. 2 / 3 and 2p 1 / 2 The formation of these two peaks is due to the splitting of Cu(II) spin orbitals. The peaks at 943.6 eV and 963.4 eV also belong to Cu(II) because of its d... 9 The electronic configuration is easily photoreduced to produce a more stable d-structure. 10 The configuration, while 338.0 eV and 343.2 eV correspond to the 3d configuration of Pd(Ⅱ), respectively. 5 / 2 and 3D 3 / 2 In Pd-TMPP(Zn), 1021.3 eV and 1044.6 eV correspond to the 2p of Zn(II), respectively. 2 / 3 and 2p 1 / 2 337.8 eV and 343.0 eV correspond to the 3d-terminal values ​​of Pd(Ⅱ), respectively. 5 / 2 and 3D 3 / 2 .

[0074] (8) Transmission electron microscopy characterization

[0075] Pd-TMPP(Cu) and Pd-TMPP(Zn) were modified with sodium hyaluronate to generate nanoparticles Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA. TEM images of the two nanoparticles are shown below. Figure 12 As shown in figures a and b, Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA form spherical nanoparticles (approximately 100 nm) in water and are uniformly dispersed.

[0076] (9) Characterization of dynamic light scattering (DLS) and Zeta potential

[0077] To further determine the particle size of the two nanoparticles, characterization analysis was performed using DLS. The particle size of Pd-TMPP(Cu)-HA was approximately 147.7 nm. Figure 13 In the middle (a), the particle size of Pd-TMPP(Zn)-HA is approximately 110.1 nm. Figure 13 (b) 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 micelle hydration. The Zeta potential of Pd-TMPP(Cu)-HA is -37.0 mV ( Figure 14 In (a), the Zeta potential of Pd-TMPP(Zn)-HA is -22.4 mV. Figure 14 (b) Nanoparticles with negative zeta potential have physicochemical advantages as anticancer drugs / carriers.

[0078] (10) Photostability evaluation of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA

[0079] The porphyrin concentration was 7 μg / mL -1 After preparing the DMSO solutions of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA, they were placed in a cuvette and examined under infrared light (650 nm, 25 mW cm⁻¹). -2 The control cuvette was irradiated for 0-10 minutes, and the UV absorption curve in the 300-600 nm range was measured immediately after each minute of irradiation. The stability of the nanomedicine was determined by monitoring the changes in UV absorption of the drug at the Soret band using UV-Vis. Figure 15 As shown, the Soret band intensity of the two compounds did not change significantly within 10 min of irradiation, indicating that both Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA have good photostability in aqueous solution.

[0080] (11) In aqueous solution 1 O2 production study

[0081] Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA were suspended in a solution containing 1,3-diphenylisobenzofuran (DPBF, 33 μg / mL). -1 In the aqueous solution of , the porphyrin concentration in both solutions was 7 μg / mL. -1 Then, the prepared mixture was placed in a cuvette and examined under infrared light (650 nm, 25 mW cm⁻¹). -2 The cuvette containing the mixture was irradiated for different durations, and the decrease in UV absorption at 417 nm of the DPBF was measured using UV-Vis to determine the effect. 1 The generation of O2. For example... Figure 16 As shown, under the same conditions, the generation of Pd-TMPP(Zn)-HA nanoparticles... 1 The yield of O2 was greater than that of Pd-TMPP(Cu)-HA nanoparticles because Cu 2+ The fluorescence quenching that occurs during photoexcitation indicates that Pd-TMPP(Zn)-HA nanoparticles are a better PDT photosensitizer than Pd-TMPP(Cu)-HA nanoparticles.

[0082] Example 5: Performance Testing.

[0083] 5.1 Measure cytotoxicity using the CCK-8 assay.

[0084] HuH-7 cells in the logarithmic growth phase were used for experiments. After removing the culture medium, the cells were washed twice with PBS, and then 1 mL of 0.25% trypsin solution was added. The cells were digested in a cell culture incubator at 37°C for approximately 2 minutes. After observing that the cells became rounded under a microscope, 2 mL of serum-containing culture medium was added to terminate the digestion. The cell suspension was collected and pipetted to obtain a single-cell suspension of HuH-7 cells. The cell suspension was counted using a hemocytometer, and the counts were recorded at a rate of 1 × 10⁻⁶. 4 Cells were seeded at a density of 90 μL per well in 96-well plates. After incubating the 96-well plates under the above culture conditions for 24 h, the culture medium was replaced with 90 μL / well serum-free DMEM. Then, drug stock solutions were prepared by serially diluting high concentrations of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA solutions with serum-free DMEM. 10 μL of each compound solution was added to the cell culture solution sequentially, with each concentration repeated in 6 wells (n = 6). For the control group, the cell-containing culture medium was directly replaced with 100 μL of serum-free DMEM. After co-incubating the cells and drugs for 24 h, the cells were compared using infrared light (650 nm, 25 mW cm⁻¹). -2Irradiate cells for 5 min; cells for the dark toxicity experiment do not require light exposure. After culturing cells for 48 h, remove the original culture medium and add 100 μL of serum-containing DMEM complete medium to each well. Add 10 μL of CCK-8 to each well and incubate for approximately 2 h. Measure the optical density (OD) at 450 nm using a microplate reader. Finally, calculate the cell viability using the following formula.

[0085] ;

[0086] Where V% represents the percentage of cell viability, [A] experimental [A] represents the absorbance of cells treated with different drugs. blank [A] is the absorbance of the blank group. control It is the absorbance of cells that have not been treated with any drugs.

[0087] The CCK8 experiment for PLC / PRF / 5 and Hep-G2 cells is similar to that for HuH-7 cells, and will not be repeated here.

[0088] Select the liver cancer cell line HuH-7 ( Figure 17 (a and b), Hep-G2 ( Figure 17 (c and d) and PLC / PRF / 5 ( Figure 17 Figures e and f) were used to evaluate the cytotoxicity of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA under infrared light irradiation conditions. As shown in the figure, the cytotoxicity of Pd-TMPP(Cu)-HA was negligible compared to Pd-TMPP(Zn)-HA. The dark toxicity of Pd-TMPP(Zn)-HA was positively correlated with drug concentration. Under light irradiation, cytotoxicity increased significantly. The half-maximal inhibitory concentration (IC50) of Pd-TMPP(Zn)-HA on HuH-7 cells was [not specified in the original text]. 50 The concentration under light conditions is 0.13 μg / mL. -1 Hep-G2 was 0.25 μg / mL. -1 PLC / PRF / 5 is 0.14 μg / mL -1 (Table 2).

[0089] Table 2. IC50 of Pd-TMPP(Zn)-HA on different cell lines with / without infrared irradiation 50 value.

[0090]

[0091] 5.4 Detection of intracellular ROS.

[0092] 2 × 10 7One HuH-7 cell was seeded into each well of a 12-well plate at 1 mL. After culturing for 24 h, the medium was replaced with the same volume of serum-free medium. The same concentrations of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA were added to the corresponding wells to achieve a final porphyrin concentration of 3 μg / mL. -1 For the phototoxicity experiment, a plate of cells was exposed to infrared light (650 nm, 25 mW cm⁻¹). -2 Cells were irradiated for 5 min under light; cells for the dark toxicity experiment did not require light exposure and were cultured in a cell culture incubator for 24 h. Before detection, 1 μL of the positive control Rosup solution (50 mg / mL) was added to each well. -1 ), to achieve a final concentration of 5 μg mL -1 After culturing for 20 min, 1 μL of DCFH-DA was added to all experimental wells. After culturing in an incubator for 20 min, the cells were washed with serum-free medium to remove excess DCFH-DA. The fluorescence was then observed and recorded under an inverted fluorescence microscope.

[0093] DCFH-DA was used as a fluorescent probe for detecting ROS. Using HuH-7 cells as a representative example, and Rosup and PBS as positive and negative controls, the ROS levels generated by Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA were measured. DCFH-DA itself is non-fluorescent and can freely cross the cell membrane, be phagocytosed by cells, and then hydrolyzed into DCFH by intracellular esterases. Since DCFH cannot cross the cell membrane, the probe is easily loaded into the cell. The ROS generated by the nanoparticles within the cell can oxidize the non-fluorescent DCFH to generate green fluorescent DCF. The fluorescence of DCF can be detected to understand the intracellular ROS production status. Figure 18 As shown, neither Pd-TMPP(Cu)-HA nor Pd-TMPP(Zn)-HA produces ROS in the absence of light irradiation. In stark contrast, when irradiated with infrared light (650 nm, 25 mW cm⁻¹), ROS is generated. -2 After 3 min, Pd-TMPP(Zn)-HA produced more ROS, but Pd-TMPP(Cu)-HA produced weaker ROS, which is consistent with the conclusions of extracellular ROS experiments.

[0094] 5.5 Cellular uptake

[0095] HuH-7 cells were cultured in DMEM complete medium at a rate of 2 × 10⁶ cells per tray. 7The cells were seeded into six 10 mL cell culture dishes. Once the cells adhered and proliferated to approximately 95% confluence, the medium was replaced with serum-free medium. The group pre-treated with sodium hyaluronate was designated Pd-TMPP(Cu)-HA' / Pd-TMPP(Zn)-HA' to distinguish it from the directly administered Pd-TMPP(Cu)-HA / Pd-TMPP(Zn)-HA group. The Pd-TMPP(Cu)-HA' / Pd-TMPP(Zn)-HA group was pre-treated with 30 mg of sodium hyaluronate for 24 h before Pd-TMPP(Cu)-HA / Pd-TMPP(Zn)-HA was added. The Pd-TMPP(Cu)-HA / Pd-TMPP(Zn)-HA group received Pd-TMPP(Cu)-HA / Pd-TMPP(Zn)-HA directly, with a porphyrin concentration of μg / mL in the culture medium. -1 After adding the drug, cells were cultured for 2 h, 4 h, and 6 h, respectively. Cells were washed three times with PBS buffer, and then collected in 15 mL centrifuge tubes using trypsin digestion. The cells were centrifuged at 900 rpm for 3 min. The precipitate was washed twice with PBS, centrifuged again, and the cell pellet was collected. Cells were lysed with 0.5 mL of nitric acid, diluted to 5 mL with deionized water, filtered, and the mixture was analyzed by ICP-MS to determine the intracellular metal ion content.

[0096] CD44 is a hyaluronic acid receptor that is overexpressed in various cell lines, including HuH-7, Hep-G2, and PLC / PRF / 5. Therefore, pretreatment of CD44 receptor-containing cells with hyaluronic acid before drug therapy may reduce drug uptake efficiency. Using HuH-7 as a representative cell line, ICP-MS was used to estimate cellular uptake efficiency with and without sodium hyaluronic acid pretreatment. As shown in Tables 3 and 4, regardless of sodium hyaluronic acid pretreatment (cells pretreated with sodium hyaluronic acid are represented as Pd-TMPP(Cu)-HA' and Pd-TMPP(Zn)-HA'), cellular drug uptake increased with increasing incubation time. However, the uptake of Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA by pretreated HuH-7 cells was significantly lower than that by untreated cells. These results clearly demonstrate that the hyaluronic acid-modified drugs Pd-TMPP(Cu)-HA and Pd-TMPP(Zn)-HA have targeting effects.

[0097] Table 3. Copper content (ppb) in HuH-7 cells under different treatments.

[0098]

[0099] Table 4. Zinc content (ppb) in HuH-7 cells under different treatments.

[0100]

[0101] In this application, two coordination molecular cages, Pd-TMPP(Cu) and Pd-TMPP(Zn), were successfully constructed, and their structural characteristics and elemental composition were thoroughly and accurately verified through a series of tests. Subsequently, both compounds were modified with hyaluronic acid to obtain targeting and water solubility. Experimental results showed that Pd-TMPP(Zn) exhibited concentration-dependent cytotoxicity against HuH-7, Hep-G2, and PLC / PRF / 5, and its cytotoxicity was significantly enhanced after infrared irradiation. In contrast, Pd-TMPP(Cu) showed poor cytotoxicity at the same concentration. This may be because porphyrin, as a strong-field ligand, can stabilize the high-valence state of Cu. 2+ This makes Cu 2+ Difficult to reduce to Cu + This makes it difficult to trigger a Fenton-like reaction within the cell. Furthermore, Cu 2+ The photoexcitation process may also cause fluorescence quenching, which further weakens its effect as a PDT agent.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Pd-M heterometallic coordination molecular cage based on TMPP ligands, wherein M is a divalent metal ion; M is Zn, namely Pd-Zn heterometallic coordination molecular cage Pd-TMPP(Zn) based on TMPP ligand; The structural formula of the Pd-Zn heterometallic coordination molecular cage Pd-TMPP(Zn) based on TMPP ligands is (PdCl2)6[TMPP(Zn)]3, and its chemical formula is C 132 H 102 Cl 12 N 24 O3Pd6Zn3.

2. The Pd-M heterometallic coordination molecular cage based on TMPP ligands according to claim 1, wherein the Pd-M heterometallic coordination molecular cage based on TMPP ligands contains two pairs of Pd... 2+ (each pair of Pd) 2+ Three ions combine with three TMPP (Zn) to form a hexagonal prism structure, which is a monoclinic crystal system. P twenty one / c Space group and orthorhombic crystal system Cmc 21 Space Group.

3. A hyaluronic acid-modified Pd-M heterometallic coordination molecular cage based on TMPP ligands as described in claim 1.

4. The hyaluronic acid-modified Pd-M heterometallic coordination molecular cage based on TMPP ligand according to claim 3, wherein the hyaluronic acid-modified Pd-M heterometallic coordination molecular cage based on TMPP ligand is hyaluronic acid-modified Pd-TMPP(Zn), i.e., Pd-TMPP(Zn)-HA.

5. The use of the Pd-M heterometallic coordination molecular cage based on TMPP ligand as described in claim 1 or the hyaluronic acid (HA) modified Pd-M heterometallic coordination molecular cage based on TMPP ligand as described in claim 3, for the preparation of therapeutic antitumor drugs or antitumor drug carriers.

6. The antitumor drug according to claim 5 is a targeted drug.

7. The use according to claim 5, wherein the tumor cell line is a liver tumor cell line.

8. The use according to claim 5, which exhibits concentration-dependent cytotoxicity against HuH-7, Hep-G2 and PLC / PRF / 5, with enhanced cytotoxicity upon irradiation with infrared light.

Images