Porphyrin-based coordinated molecular cage and its preparation method and application

Abstract

The present invention discloses a porphyrin-based coordinated molecular cage and a preparation method and application thereof. The pyridine functional group in an organic ligand containing a porphyrin skeleton is coordinated with a metal M having biological activity to obtain a molecular cage compound M6L3; the porphyrin ring of the molecular cage compound M6L3 further chelates or partially chelates the metal M' to obtain a homonuclear or heteronuclear porphyrin-based coordinated molecular cage M6[L(M' x )]3. The porphyrin-based coordination molecular cage prepared by the present invention can effectively inhibit the fluorescence quenching caused by the aggregation of porphyrin molecules, thereby greatly improving its photodynamic therapy efficacy, and by introducing a biologically active metal M, such as Pd 2+ , which can catalyze Fenton-like reactions, induce programmed ferroptosis of cells, and achieve dual anti-tumor effects. In addition, the unique cavity of the above molecular cage can synergistically load small molecule drugs to achieve a synergistic anti-cancer effect. At the same time, the phototoxicity of the porphyrin coordination molecular cage can be regulated by controlling the metal chelation rate of the porphyrin skeleton center, further improving the therapeutic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a porphyrin-based coordinated molecular cage, a nano-preparation, and a preparation method and application thereof. Background Art

[0002] Cancer treatment methods mainly include surgical resection, radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy. For example, early cancer can be treated by surgical resection or surgery combined with radiotherapy and chemotherapy. Advanced metastatic cancer can be treated with chemotherapy combined with targeted drugs to achieve the purpose of improving the quality of life and relieving symptoms. In addition, immune drugs such as antibody immunotherapy for PD-1, PD-L1 and CTLA-4 have greatly improved the overall benefit of patients, but the response rate of these immune checkpoint inhibitors (ICI) alone is only 19-47%. During immunotherapy, some patients can obtain a lasting immune response, while others will develop primary or secondary drug resistance. Therefore, research and exploration of new materials and new methods for the treatment of cancer still has important practical significance.

[0003] As an important supplement to classical cancer treatment methods, photodynamic therapy (PDT) is also a common therapy, which has been widely used in the treatment of malignant tumors such as melanoma, non-small cell lung cancer, and pancreatic cancer. PDT has the advantages of being non-invasive, having few side effects, and having high spatiotemporal selectivity. Some compounds based on porphyrin skeletons, such as Photofrin, Temoporfin, Photochlor, and Redaporfin, have been approved by the FDA and used in the clinical treatment of cancer. Current studies have shown that in addition to being an ablative cancer treatment, PDT can also mediate the body's anti-tumor immune response through the immunogenic death (ICD) or ferroptosis of tumor cells, ultimately improving the anti-tumor effect.

[0004] Porphyrin monomers can be coordinated with metals through self-assembly technology to prepare nanomaterials with high dispersion and stability, showing a stronger high permeability and long retention (EPR) effect, which can be used for PDT treatment of tumors in vivo. For example, Chen et al. prepared a nanoscale porphyrin-modified Fe-based metal-organic framework MIL-101(Fe)@TCPP. After this nanoagent enters tumor cells, under the stimulation of the acidic microenvironment of the tumor, MIL-101(Fe)@TCPP decomposes and releases free Fe 3+ ions and TCPP (Chen et al, ACS Appl. Mater. Interfaces 2021, 13, 45201-45213). 3+The ion-catalyzed Fenton reaction can effectively convert endogenous H2O2 into highly cytotoxic ·OH, while TCPP can also generate reactive oxygen species (ROS) in tumor tissues after being activated by light of a specific wavelength. This dual dynamic effect can significantly increase the level of ROS in tumor cells and synergistically induce oxidative damage to cells, thereby producing better tumor treatment effects. However, these porphyrin compounds do not contain expandable functional groups, which greatly limits the systematic research of such materials. At the same time, the carrier materials usually have large particles, which are not easy to disperse evenly, and the drug delivery efficiency is low. The constructed carrier system contains a large excess of Fe 3+ The source of TCPP, as well as the toxic side effects of another component, 2-aminoterephthalic acid, after the decomposition of the carrier material, are also worthy of attention. Although TCPP has a certain absorption in the infrared band, the absorption intensity is not large, which greatly limits the effectiveness of PDT. TCPP, as a small guest molecule with low solubility, is easily released explosively with the decomposition of the carrier skeleton and then quickly cleared, greatly reducing the potential applicability of the system.

[0005] The hypoxic microenvironment in solid tumors also has a certain negative impact on the efficacy of PDT. Based on this, Dai et al. proposed a simple strategy to ultrasonically disperse perfluorooctyl bromide (PFOB) liquid into porphyrin-grafted lipids (PGL) to form O2@PFOB@PGL nanoparticles (Dai et al, ACS Nano 2020, 14, 13569-13583), and then load O2 to prepare oxygen self-supplied PDT nanocarriers. This carrier has a high porphyrin / O2 loading rate and can deliver oxygen to hypoxic tumors without external stimulation, which is beneficial to increase singlet oxygen ( 1 O2) production, relieving tumor hypoxia and subsequently downregulating COX-2 expression. In animal experiments, such materials can significantly inhibit tumor growth and liver metastasis. In addition, O2@PFOB@PGL also exhibits excellent fluorescence (FL) and computed tomography (CT) imaging capabilities. Therefore, this oxygen self-supplementing PDT nanocarrier can not only ablate primary tumors, but also relieve tumor hypoxia under the guidance of FL / CT imaging to inhibit tumor metastasis. However, the rigid structure and hydrophobic plane of porphyrin often cause porphyrin to aggregate in solution and cause ACQ effect, which will reduce 1 O2 generation efficiency and PDT efficacy. In addition, due to the poor tissue penetration of the light source used, PDT treatment is limited to the superficial layer. Therefore, a single PDT treatment is prone to cause tumor recurrence and metastasis, and it is usually difficult to completely eliminate cancer cells.

[0006] In the above two self-assembly processes, porphyrin compounds are respectively connected to coordination polymers and organic nanocarriers by means of coordination bonds and covalent bonds to obtain nanoparticles with PDT therapeutic efficacy. In addition, porphyrin compounds can also directly participate in the self-assembly of small molecule coordination molecular cages as building blocks. The resulting nanoparticles have a high porphyrin loading rate and good PDT effect. For example, Chen et al. used TPP, DSTP and cPt as building units to synthesize a discrete organic platinum metal cage through coordination-driven self-assembly to integrate chemotherapy and PDT treatment (Chen et al., Nat. Commun. 2018, 9, 4335). The formation of the porphyrin coordination molecular cage effectively inhibits the aggregation fluorescence quenching (ACQ) effect of porphyrin, resulting in enhanced fluorescence intensity and 1 The improvement of O2 quantum yield is beneficial to NIR / FL imaging and PDT treatment. At the same time, different imaging functions can be achieved by chelating various metal ions through the porphyrin ring center. The obtained metal cage is encapsulated with a polyethylene glycol chain with RGD targeting to prepare nanoparticles, which can improve its biocompatibility, prolong blood circulation time and give nanoparticles active targeting ability to cancer cells, all of which lead to the excellent tumor inhibition effect of nanoparticles, effectively preventing tumor recurrence and tumor metastasis after a single treatment. However, the coordination molecular cage components constructed by Chen et al. are extremely complex, and also include the use of highly toxic PEt3 as a ligand. Not only is its synthetic feasibility low, but the toxic side effects during drug degradation should not be underestimated. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a porphyrin-based coordinated molecular cage and a preparation method and application thereof. The present invention uses a coordination method to synergistically assemble a porphyrin-based photosensitizer with a specific configuration and an anticancer active metal to form a triangular prism-shaped coordinated molecular cage compound. The assembled molecular cage has the ability to further load other drugs. At the same time, the molecular cage can be further coordinated with the metal through the functionalization of the ligand, and other targeting or water-soluble ligands can be introduced to achieve the water solubility and biocompatibility of the drug, as well as the tracing of the drug entering the body, and on this basis, the toxic and side effects of the drug are minimized.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a porphyrin-based coordination molecular cage, wherein the porphyrin-based coordination molecular cage M6[L(M' x )]3 has the following general structural formula:

[0010]

[0011] Among them, M is selected from one of Mn, Fe, Co, Ni, Cu, Zn, Pd, Pt, Ru, Rh, Ir, Os; M' is the same as or different from M and is selected from one of Mn, Fe, Co, Ni, Cu, Zn, Pd, Pt, Ru, Rh, Ir, Os;

[0012] R is -CH3 or -CH2OH;

[0013] L(M' x ) has a value of 0 < x ≤ 1. When x < 1, the porphyrin ring in the porphyrin-based coordination molecular cage is partially chelated with the metal M'.

[0014] Furthermore, the porphyrin-based coordination molecular cage is Pd6[TMPP(Pd x )]3 (0 < x ≤ 1), Pd6[TMPP(Fe)]3, Pd6[THPP(Pd)]3 or Pd6[THPP(Fe)]3.

[0015] Furthermore, the Pd6[TMPP(Pd x )]3 (0 < x ≤ 1) includes Pd6[TMPP(Pd 0.4 )]3, Pd6[TMPP(Pd 0.6 )]3, Pd6[TMPP(Pd)]3.

[0016] Furthermore, the molecular formula of the Pd6[TMPP(Pd 0.4 )]3 is C 132 H 96 Cl 12 N 24 Pd7, belonging to the orthorhombic system, with the space group Cmc21 and the unit cell parameters: α = 90°, β = 90°, γ = 90°,

[0017] Furthermore, the molecular formula of the Pd6[TMPP(Pd 0.6 )]3 is C 132 H 96 Cl 12 N 24 Pd 8.40 , belonging to the monoclinic system, with the space group P21 and the unit cell parameters: α = 90°, β = 106.113(3)°, γ = 90°,

[0018] Furthermore, the molecular formula of the Pd6[THPP(Pd)]3 is C 132 H 96 Cl 12 N 24Pd9 belongs to the monoclinic system, the space group is P21 / c, and the unit cell parameters are: α=90°, β=98.378(2)°, γ=90°,

[0019] Furthermore, the molecular formula of Pd6[TMPP(Fe)]3 is C 264 H 204 Cl 30 Fe6N 48 oeLh 12 , belongs to the triclinic system, the space group is P-1, and the unit cell parameters are: α=63.504(3)°, β=88.850(3)°, γ=85.741(4)°,

[0020] Furthermore, the molecular formula of Pd6[THPP(Pd)]3 is C 132 H 96 Cl 12 N 24 O 12 Pd9 belongs to the orthorhombic system, the space group is Pbca, and the unit cell parameters are: α=90°, β=90°, γ=90°,

[0021] Furthermore, the molecular formula of Pd6[THPP(Fe)]3 is C 132 H 108 Cl 12 Fe3N 24 O 18 Pd6 belongs to the trigonal crystal system, the space group is R-3c:H, and the unit cell parameters are: α=90°, β=90°, γ=120°,

[0022] The second aspect of the present invention provides a method for preparing the porphyrin-based coordination molecular cage described in the first aspect, comprising dissolving a ligand H2L, a metal M salt and a metal M' salt in a solvent, adding an acid regulator to the obtained mixed solution, and then subjecting the mixture to a temperature rise and fall treatment to precipitate a solid, and washing the solid to obtain the porphyrin-based coordination molecular cage; wherein the ligand H2L is selected from one of the compounds shown in the following structures:

[0023]

[0024] Furthermore, the metal M salt is preferably palladium acetate or palladium chloride; if K2[PdCl4] is used as the palladium salt, the target product cannot be obtained.

[0025] Furthermore, the metal M' salt is preferably ferric chloride hexahydrate, palladium acetate or palladium chloride.

[0026] Furthermore, the solvent is a mixed solvent obtained by mixing dichloromethane and methanol in a volume ratio of 1:3, or a mixed solvent obtained by mixing dichloromethane, chlorobenzene and methanol in a volume ratio of 1:1:6.

[0027] Furthermore, the acid regulator is acetic acid or benzoic acid, more preferably acetic acid.

[0028] In some preferred embodiments, the ligand H2L is H2TMPP, the metal M salt and the metal M' salt are both Pd(OAc)2, the molar ratio of H2TMPP to Pd(OAc)2 is 1:3, the solvent is a mixed solvent obtained by mixing dichloromethane and methanol in a volume ratio of 1:3, and the prepared porphyrin-based coordination molecular cage is Pd6[TMPP(Pd)]3.

[0029] In some preferred embodiments, the ligand H2L is H2TMPP, the metal M salt and the metal M' salt are both PdCl2, the molar ratio of H2TMPP to PdCl2 is 1:3, the solvent is a mixed solvent obtained by mixing dichloromethane and methanol in a volume ratio of 1:3, and the prepared porphyrin-based coordination molecular cage is Pd6[TMPP(Pd 0.6 )]3.

[0030] In some preferred embodiments, the ligand H2L is H2TMPP, the metal M salt and the metal M' salt are both PdCl2, the molar ratio of H2TMPP to PdCl2 is 1:3-4, for example, 1:3.73, the solvent is a mixed solvent obtained by mixing dichloromethane, chlorobenzene and methanol in a volume ratio of 1:1:6, and the prepared porphyrin-based coordination molecular cage is Pd6[TMPP(Pd 0.4 )]3.

[0031] In some preferred embodiments, the ligand H2L is H2TMPP, the metal M salt is Pd(OAc)2, the metal M' salt is FeCl3·6H2O, the molar ratio of H2TMPP to Pd(OAc)2 and FeCl3·6H2O is 1:2:1, the solvent is a mixed solvent obtained by mixing dichloromethane and methanol in a volume ratio of 1:3, and the prepared porphyrin-based coordination molecular cage is Pd6[TMPP(Fe)]3.

[0032] In some preferred embodiments, the ligand H2L is H2THPP, the metal M salt and the metal M' salt are both Pd(OAc)2, the molar ratio of H2THPP to Pd(OAc)2 is 1:5-6, the solvent is a mixed solvent obtained by mixing dichloromethane and methanol in a volume ratio of 1:3, and the prepared porphyrin-based coordination molecular cage is Pd6[THPP(Pd)]3.

[0033] In some preferred embodiments, the ligand H2L is H2THPP, the metal M salt is Pd(OAc)2, the metal M' salt is FeCl3·6H2O, the molar ratio of H2THPP to Pd(OAc)2 and FeCl3·6H2O is 1:2:1, the solvent is a mixed solvent obtained by mixing dichloromethane, chlorobenzene and methanol in a volume ratio of 1:1:6, and the prepared porphyrin-based coordination molecular cage is Pd6[THPP(Fe)]3.

[0034] Furthermore, the temperature rise and fall treatment is specifically as follows: firstly raise the temperature to 110-130°C, keep the temperature for 24-60h, and then reduce the temperature to 20-30°C at a rate of 4-5°C / h.

[0035] The third aspect of the present invention provides a use of the porphyrin-based coordination molecular cage described in the first aspect in the preparation of anti-tumor drugs.

[0036] Furthermore, the cavity of the porphyrin-based coordinated molecular cage can be cooperatively loaded with small molecule drugs, such as doxorubicin, cisplatin, etc.

[0037] The fourth aspect of the present invention provides a porphyrin-based coordination molecular cage nano-preparation, wherein the porphyrin-based coordination molecular cage and the amphiphilic copolymer described in the first aspect are dispersed in a solvent, added dropwise to water after ultrasound to obtain a micellar solution, and the porphyrin-based coordination molecular cage nano-preparation is obtained by dialysis.

[0038] Furthermore, the amphiphilic copolymer is preferably Pluronic F127.

[0039] The fifth aspect of the present invention provides a use of the porphyrin-based coordination molecular cage nano-preparation described in the fourth aspect in the preparation of anti-tumor drugs.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. The present invention uses a hydrothermal method to coordinate and drive self-assembly of a porphyrin-based photosensitizer with a specific configuration and an anticancer active metal to form a triangular prism-shaped coordination molecular cage compound, and synthesizes three porphyrin coordination molecular cage coordination compounds with different palladium chelation rates by adjusting the ratio of different solvents. The porphyrin-based coordination molecular cage can effectively inhibit the fluorescence quenching caused by the aggregation of porphyrin molecules, greatly improving the PDT efficacy. The assembled molecular cage has the ability to further load other drugs. At the same time, the molecular cage can further coordinate with the metal through the functionalization of the ligand, introduce other targeting or water-soluble ligands, realize the water solubility and biocompatibility of the drug, and the tracing of the drug entering the body, and on this basis, minimize the toxic and side effects of the drug in order to improve the solubility of the porphyrin coordination molecular cage. For example, the present invention uses an ultrasonic method to further encapsulate the obtained porphyrin coordination molecular cage in the hydrophobic core of the amphiphilic copolymer to obtain a spherical nanoformulation. The spherical nanoformulation has good photostability and exhibits good solubility and stability in physiological media. The hemolysis experiment shows that the hemolysis rate of this type of nanoformulation is less than 2%, and it has good biocompatibility and can be used in biological experiments.

[0042] 2. The present invention measures the related indicators of ferroptosis by treating cells with Pd6-TMPP (Pd)-F127, such as mitochondrial morphology and membrane potential, intracellular GSH and ROS levels, etc. The experimental results show that the mitochondrial membrane potential of cells treated with Pd6-TMPP (Pd)-F127 is significantly reduced, the mitochondrial morphology is damaged, the cristae are reduced, the GSH level is reduced, and the ROS production is increased. It can be seen that the palladium complex prepared by the present invention is similar to the iron compound, and can also mediate cell ferroptosis by catalyzing the occurrence of Fenton-like reactions. Therefore, the palladium porphyrin coordination molecular cage prepared by the present invention has the dual anticancer effects of PDT and mediating ferroptosis.

[0043] 3. The present invention makes a qualitative and quantitative comparison of the ability of three nanoparticles with different chelation rates to produce ROS in solution and in cells, and finds that the metal cage with a lower chelation rate has a stronger ability to produce ROS, and the metal cage with a low chelation rate exhibits lower dark toxicity and higher phototoxicity. The above conclusions have been proven in experiments such as cytotoxicity, cell apoptosis, and live-death staining. In the process of studying the dark toxicity mechanism, the present invention finds that by controlling the Pd6-TMPP (Pd x )-F127(x=1,0.6,0.4) 2+ The concentration can selectively kill cancer cells.

[0044] 4. The results of anti-tumor experiments in animals show that Pd6-TMPP (Pd 0.4)-F127 intake will not affect the normal physiological metabolic activities of mice, and has an inhibitory effect on tumors, effectively inhibiting tumor growth and improving the survival rate of mice. Combined with PDT treatment, it can completely inhibit tumor growth, and the survival rate of mice during treatment can reach 100%. Combined with the results of cell experiments, Pd6-TMPP (Pd 0.4 )-F127 further achieves in vivo tumor treatment by combining with PDT. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The structure of the porphyrin-coordinated molecular cage: (a) Pd6-TMPP (Pd 0.4 ), (b) side view of Pd6-TMPP(Pd 0.4 ), (c) Pd6-TMPP(Pd 0.6 ), (d) top view of Pd6-TMPP(Pd), (e) top view of Pd6-TMPP(Fe), (f) top view of Pd6-THPP(Pd), (g) top view of Pd6-THPP(Fe);

[0046] Figure 2 The UV absorption curves of H2TMPP and three palladium porphyrin coordination molecular cages with different chelation rates in DMSO;

[0047] Figure 3 The FT-IR spectra of three palladium porphyrin coordination molecular cages with different chelation rates;

[0048] Figure 4 TMPP-F127 and Pd6-TMPP (Pd x )-F127 (x = 1, 0.6, 0.4) nanoformulation synthesis schematic diagram;

[0049] Figure 5 (a) TMPP-F127, (b) Pd6-TMPP(Pd)-F127, (c) Pd6-TMPP(Pd 0.6 )-F127 and (d)Pd6-TMPP(Pd 0.4 )-TEM image of F127;

[0050] Figure 6 TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4 )-average particle size and zeta potential of F127;

[0051] Figure 7Within 0-10 minutes of laser irradiation, TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4 )-The curve of the absorption value of the aqueous solution of F127 at the maximum absorption wavelength (417nm) versus irradiation time;

[0052] Figure 8 (a) TMPP-F127, (b) Pd6-TMPP(Pd)-F127, (c) Pd6-TMPP(Pd 0.6 )-F127, (d)Pd6-TMPP(Pd 0.4 )-F127 average particle size change after dispersion in H2O, PBS, PBS + 10% FBS for 0h and 8h;

[0053] Fig. 9 (a) DPBF and (b) dispersed in TMPP-F127, (c) Pd6-TMPP(Pd)-F127, (d) Pd6-TMPP(Pd 0.6 )-F127, (e)Pd6-TMPP(Pd 0.4 ) The curve of the variation of the ultraviolet absorption intensity of -F127 in aqueous solution with the laser irradiation time; (f) The curve of the variation of the absorption value of DPBF at the maximum absorption wavelength with the irradiation time in the aqueous solution of the above four nanoparticles;

[0054] Fig.10 TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127, Pd6-TMPP(Pd 0.4 B16F10 cells treated with )-F127 for 24 h under (a) no light and (c) light (50 mW cm -2 Cell survival rate under (a) no-light and (b) light-irradiated conditions and their half-inhibitory concentration curves ( * P<0.05, ** P<0.01, *** P<0.001);

[0055] Fig.11 B16F10 cells were treated with TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127, Pd6-TMPP(Pd 0.4 )-Fluorescence microscopy images after incubation with F127;

[0056] Fig.12 B16F10 cells were treated with TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127, Pd6-TMPP(Pd 0.4 )-Flow cytometry after incubation with F127;

[0057] Fig.13 B16F10 cells were treated with four nanomaterials and DCFH-DA and irradiated with laser (650 nm, 50 mW cm -2 )Fluorescence microscope images after irradiation;

[0058] Fig.14 B16F10 cells were treated with four nanomaterials and DCFH-DA and irradiated with laser (650 nm, 50 mW cm -2 ) Flow cytometry images after irradiation;

[0059] Fig.15 B16F10 cells were treated with four nanomaterials and laser (650 nm, 25 mW cm -2 ) Fluorescence microscopy images after irradiation and incubation with Calcein-AM / PI probe;

[0060] Fig.16 B16F10 cells were treated with four nanomaterials in the presence or absence of laser (650nm, 25mW cm -2 ) Flow cytometry images after incubation with Annexinv-FITC / PI probe under irradiation conditions;

[0061] Fig.17 TMPP-F127 and Pd6-TMPP (Pd x )-F127 (x = 1, 0.6, 0.4) hemolysis test results;

[0062] Fig.18 The UV absorption curves of methylene blue (MB) after reaction with Pd6-TMPP(Pd)-F127(ab) and Pd(OAc)2(cd). The H2O2 content in a and c is 100μM, and the H2O2 content in b and d is 5μM.

[0063] Fig.19 Fluorescence microscopy images of B16F10 cells (a) and HEK293 cells (b) treated with materials and DCFH-DA without light.

[0064] Fig. 20 Cell viability (a) and IC of B16F10 cells and HEK293 cells treated with Pd6-TMPP(Pd)-F127 for 24 h50 Graph (b);

[0065] Fig.21 is the content of GSH in B16F10 cells after incubation with different materials;

[0066] Fig. 22 CLSM images of B16F10 cells after incubation with different materials and staining with JC-1. JC-1 monomers emit green fluorescence, indicating that Δψ m Reduced. JC-1 aggregates in healthy mitochondria emit red fluorescence;

[0067] Fig.23 (a) TEM image of mitochondria in untreated B16F10 cells, (b) TEM image of mitochondria in B16F10 cells treated with Pd6-TMPP(Pd)-F127;

[0068] Fig.24 (a) TEM image of mitochondria in untreated HEK293 cells, (b) TEM image of mitochondria in HEK293 cells treated with Pd6-TMPP(Pd)-F127;

[0069] Fig.25 Pd6-TMPP(Pd 0.4 )-F127 in vivo anti-tumor effect. (a) Tumor growth inhibition curve; (b) Mouse weight change curve; (c) Survival rate curve of mice in each treatment group; (d) Tumor weight graph of each treatment group; (e) Tumor photos of each treatment group. DETAILED DESCRIPTION

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0071] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0072] Example 1

[0073] This embodiment relates to the preparation of a porphyrin-based coordinated molecular cage Pd6[TMPP(Pd)]3, and the specific operation is as follows:

[0074] H2TMPP (2 mg, 0.003 mmol) and Pd(OAc)2 (2 mg, 0.009 mmol) were dissolved in a mixed solvent of DCM / MeOH (v:v=0.5 mL:1.5 mL) and transferred to a Pyrex glass tube. HAc (20 μL) was then added to the above mixture as a regulator. The Pyrex glass tube was placed in a programmable oven, heated from 25°C to 120°C within 4 hours, then kept at 120°C for 48 hours, and cooled to 25°C within 24 hours. After filtration, the obtained solid mixture was dissolved in DCM, the filtrate was spin-dried, and the obtained solid was washed with MeOH 2-3 times to obtain a palladium porphyrin coordination molecular cage with a chelation rate of 100%, named Pd6-TMPP(Pd). IR (KBr disc; cm -1 ):3427(m),2921(w),1610(m),1546(w),1494(s),1447(m),1348(m),1296(m),12 32(w),1132(m),1086(m),1045(s),1010(vs),911(w),853(w),818(s),789(vs).

[0075] Example 2

[0076] This embodiment relates to a porphyrin-based coordination molecular cage Pd6[TMPP(Pd 0.6 )]3 preparation, the specific operation is as follows:

[0077] H2TMPP (2 mg, 0.003 mmol) and PdCl2 (1.6 mg, 0.009 mmol) were dissolved in a mixed solvent of DCM / MeOH (v:v=0.5 mL:1.5 mL) and transferred to a Pyrex glass tube. HAc (20 μL) was then added to the above mixture as a regulator. The Pyrex glass tube was placed in a programmable oven, heated from 25°C to 120°C within 4 hours, then maintained at 120°C for 48 hours, and cooled to 25°C within 24 hours. After filtration, the obtained solid mixture was dissolved in DCM, the filtrate was spin-dried, and the obtained solid was washed with MeOH 2-3 times to obtain a palladium porphyrin coordination molecular cage with a chelation rate of 100%, named Pd6-TMPP (Pd 0.6 ). IR (KBr disc; cm -1):3397(m),2920(w),2845(w),1610(s),1558(m),1494(s),1447(m),1372(m),1352(m ),1296(s),1226(m),1138(s),1062(m),1010(s),964(vs),894(m),847(m),795(vs).

[0078] Example 3

[0079] This embodiment relates to a porphyrin-based coordination molecular cage Pd6[TMPP(Pd 0.4 )]3 preparation, the specific operation is as follows:

[0080] H2TMPP (1 mg, 0.0015 mmol) and PdCl2 (1 mg, 0.0056 mmol) were dissolved in a mixed solvent of DCM / PhCl / MeOH (v:v:v=0.25 mL:0.25 mL:1.5 mL) and transferred to a Pyrex glass tube. HAc (20 μL) was then added to the above mixture as a regulator. The Pyrex glass tube was placed in a programmable oven, heated from 25°C to 120°C within 4 hours, then maintained at 120°C for 48 hours, and cooled to 25°C within 24 hours. After filtration, the obtained solid mixture was dissolved in DCM, the filtrate was spin-dried, and the obtained solid was washed with MeOH 2-3 times to obtain a palladium porphyrin coordination molecular cage with a chelation rate of 40%, named Pd6-TMPP (Pd 0.4 ). IR (KBr disc; cm -1 ):3415(m),2914(w),2845(w),2268(w),1610(s),1558(m),1500(s),1447(m),1372(m),13 48(m),1302(m),1261(m),1138(s),1086(s),1010(vs),964(s),894(w),853(w),795(vs).

[0081] Example 4

[0082] This embodiment relates to the preparation of a porphyrin-based coordinated molecular cage Pd6[TMPP(Fe)]3, and the specific operation is as follows:

[0083] H2TMPP (2 mg, 0.003 mmol), Pd(OAc)2 (1.4 mg, 0.006 mmol) and FeCl3·6H2O (1 mg, 0.003 mmol) were dissolved in a mixed solvent of DCM / MeOH (v:v=0.5 mL:1.5 mL) and transferred to a Pyrex glass tube. HAc (20 μL) was then added to the above mixture as a regulator. The Pyrex glass tube was placed in a programmable oven, heated from 25°C to 120°C within 4 hours, then kept at 120°C for 48 hours, and cooled to 25°C within 24 hours. After filtration, the obtained solid mixture was dissolved in DCM, the filtrate was spin-dried, and the obtained solid was washed with MeOH 2-3 times to obtain a palladium iron porphyrin coordination molecular cage, named Pd6-TMPP(Fe). IR (KBr disc; cm -1 ):3397(m),2568(w),2361(m),2164(w),2026(w),1979(w),1610(s),1562(w),1524(m),1489(s),1446(s),1375(m),1335( m),1301(s),1232(w),1206(s),1140(s),1080(m),1064(m),1024(s),1003(vs),901(m),854(m),796(vs),719(s),625(m).

[0084] Example 5

[0085] This embodiment relates to the preparation of a porphyrin-based coordinated molecular cage Pd6[THPP(Pd)]3, and the specific operation is as follows:

[0086] The phenylbutyrate of H2THPP (2 mg, 0.0015 mmol) and Pd(OAc)2 (2 mg, 0.0089 mmol) were dissolved in a mixed solvent of DCM / MeOH (v:v = 0.5 mL: 1.5 mL) and transferred to a Pyrex glass tube. HAc (20 μL) was then added to the above mixture as a regulator. The Pyrex glass tube was placed in a programmable oven, heated from 25°C to 120°C within 4 hours, then kept at 120°C for 48 hours, and cooled to 25°C within 24 hours. The resulting crystals were washed 2-3 times with anhydrous ether to obtain a palladium porphyrin coordination molecular cage, named Pd6-THPP (Pd).

[0087] Example 6

[0088] This embodiment relates to the preparation of a porphyrin-based coordination molecular cage Pd6[THPP(Fe)]3, and the specific operation is as follows:

[0089] H2THPP (2 mg, 0.003 mmol), Pd(OAc)2 (1.4 mg, 0.006 mmol) and FeCl3·6H2O (1 mg, 0.003 mmol) were dissolved in a mixed solvent of DCM / MeOH / PhCl (v:v:v = 0.25 mL: 1.5 mL: 0.25 mL) and transferred to a Pyrex glass tube. HAc (20 μL) was then added to the above mixture as a regulator. The Pyrex glass tube was placed in a programmable oven, heated from 25°C to 120°C within 4 hours, then maintained at 120°C for 48 hours, and cooled to 25°C within 24 hours. The resulting crystals were washed 2-3 times with anhydrous ether to obtain a palladium iron porphyrin coordination molecular cage, named Pd6-THPP(Fe). IR (KBr disc; cm -1 ):3366(m),2570(w),2162(w),2033(w),1980(w),1611(s),1524(w),1489(m),1447(m),1379(s),1352( m),1332(w),1266(w),1206(s),1135(w),1058(vs),1005(vs),903(w),858(m),800(s),718(m),671(w).

[0090] Example 7

[0091] The porphyrin-based coordinated molecular cages prepared in the above Examples 1 to 6 were characterized by single crystal X-ray diffraction (SCXRD). The structures of different porphyrin-based coordinated molecular cages are shown in the following figure: Figure 1 The corresponding crystallographic data and parameters are shown in Tables 1 and 2 below:

[0092] Table 1 Crystallographic parameters of the porphyrin-based coordinated molecular cages prepared in Examples 1 to 3

[0093]

[0094]

[0095] Table 2 Crystallographic parameters of porphyrin-based coordinated molecular cages prepared in Examples 4 to 6

[0096]

[0097]

[0098] In the above embodiments 1 to 3, Pd was prepared by adjusting the type of metal salt or solvent. 2+ The UV absorption curves of porphyrin-based metal cages with different chelation rates, H2TMPP and three palladium porphyrin coordination molecular cages with different chelation rates in DMSO are shown in Figure 2. Figure 2 As shown in the figure, the ultraviolet absorption peaks of H2TMPP are 418nm, 516nm, 550nm, 591nm, and 646nm. 0.4 ) are 418nm, 522nm, 553nm, 591nm, and 646nm, respectively. 0.6 ) are 418nm, 518nm, 550nm, 591nm, 646nm. The UV absorption peaks of Pd6-TMPP(Pd) are 411nm, 523nm, 555nm.

[0099] It can be seen that compared with the free H2TMPP, after the formation of the metal cage, the Soret band in the UV absorption peak has a red shift, indicating that the porphyrin ring center has successfully chelated the metal ion. 0.4 ) and Pd6-TMPP(Pd 0.6 ) The number of UV absorption peaks in the wavelength range of 500-700nm did not decrease, but when the palladium chelation rate at the center of the porphyrin ring was 100%, the UV absorption peak between 600-700nm disappeared, which is presumably due to the complete chelation of Pd by TMPP. 2+ , resulting in the fluorescence quenching of porphyrin.

[0100] Example 8

[0101] This example involves the preparation of some palladium-based porphyrin coordinated molecular cage nanoparticles. The synthesis schematic diagram is shown in Figure 4 As shown, the details are as follows:

[0102] (1) Synthesis of TMPP-F127

[0103] Pluronic F127 (10.6 mg, 0.0053 mmol) was added to a DMSO (1 mL) solution containing H2TMPP (4.5 mg, 0.0067 mmol). After complete dissolution, the resulting mixture was added dropwise to 10 mL of deionized water under ultrasonic conditions using an ultrasonic cell crusher. Finally, the micellar solution of TMPP-F127 was obtained by dialysis in a dialysis bag with a molecular weight cutoff of 2000 for 24 hours.

[0104] (2)Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4 )-Synthesis of F127.

[0105] Pluronic F127 (9.6 mg, 0.0048 mmol) was added to a flask containing Pd6-TMPP(Pd) (3.5 mg, 0.001 mmol), Pd6-TMPP(Pd 0.6 ) (3.5 mg, 0.001 mmol) and Pd6-TMPP (Pd 0.4 ) (3.5 mg, 0.0011 mmol) in DMSO (1 mL). After complete dissolution, the above mixture was added dropwise into 10 mL of deionized water under ultrasonic conditions using an ultrasonic cell crusher. Finally, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4 The micellar solution of )-F127 was obtained by dialysis in a dialysis bag with a molecular weight cut-off of 3500 for 24 hours.

[0106] TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4 )-F127 transmission electron microscopy image Figure 5 As shown, TMPP-F127 and three Pd6-TMPPs with different chelation rates (Pd x )-F127 (x = 1, 0.6, 0.4) are all spherical nanoparticles with a size of about 100 nm, which matches the hydrodynamic diameter measured by dynamic light scattering (DLS) ( Figure 6 ).

[0107] Example 9 Stability Study of Palladium-Based Porphyrin Coordination Molecular Cage Nanoformulation

[0108] (1) Photostability

[0109] Preparation of porphyrin with the same concentration (7 μg mL -1 ) of TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4 )-F127 aqueous solution, the above prepared solution was irradiated with a 650nm laser (100mWcm -2 ) and monitored the UV absorption curve of each solution within 0-10 min of irradiation. Figure 7 As shown, within 10 minutes of laser irradiation, the UV absorption curves of the four nanoparticles showed almost no change, proving that they have good photostability and will not undergo structural degradation under laser irradiation.

[0110] (2) Stability in physiological media

[0111] Dynamic light scattering (DLS) was used to monitor the binding of TMPP-F127 and three Pd6-TMPPs (Pd x The stability of the nanoparticles was evaluated by measuring the changes in the hydrodynamic diameter of )-F127 (x = 1, 0.6, 0.4) in different physiological media (H2O, PBS, PBS + 10% FBS). Figure 8 As shown, after the nanoparticles were dispersed in these three media for 8 hours, their particle size did not change much, indicating that the encapsulation of Pluronic F127 made the four prepared nanomaterials have good stability and dispersibility in different physiological media, providing conditions for conducting biological experiments.

[0112] Example 10 Extracellular ROS production of palladium-based porphyrin coordinated molecular cage nanoformulations

[0113] Since diphenylisobenzofuran (DPBF) can be oxidized and decomposed by ROS, resulting in a decrease in its UV absorbance, DPBF was used as a probe to detect ROS. The results showed that TMPP-F127 and three Pd6-TMPPs (Pd x )-F127 (x = 1, 0.6, 0.4). The same concentration of DPBF was added to TMPP-F127 and three Pd6-TMPP (Pd x )-F127 (x=1,0.6,0.4) aqueous solution, mixed evenly. In the mixed solution, the concentration of DPBF is 33μg / mL, and the concentration of porphyrin is 7μg / mL. Then a 650nm laser (100mW cm -2 ) was irradiated every 30 seconds, and the ability of DPBF to generate ROS was evaluated by monitoring the rate of decrease of the UV absorption peak of these four materials. Fig. 9 As shown in the figure, the decreasing rates of DPBF absorption values ​​are as follows: Pd6-TMPP (Pd 0.4 )-F127>Pd6-TMPP(Pd 0.6 )-F127>Pd6-TMPP(Pd)-F127>TMPP-F127. Compared with free H2TMPP, the ROS production was greatly improved after the formation of metal cages, and the metal cages with lower chelation rate had higher ROS production.

[0114] Example 11 Cytological study of palladium-based porphyrin coordinated molecular cage nanoformulations

[0115] (1) Cytotoxicity

[0116] The cytotoxicity of the materials to B16F10 cells was evaluated by 3-(4,5-dimethylthiazol-2'-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Two 96-well plates were prepared for the phototoxicity and dark toxicity experiments of the materials. 4 B16F10 cells were inoculated at a density of 10 cells / well and cultured at 37°C, 5% CO2 for 16 h. For the phototoxicity experiment, different concentrations of TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4 After the cells were incubated with serum-free DMEM solution of )-F127 for 12 h, the cells were irradiated with a laser at a wavelength of 650 nm (25 mW cm -1 , 2 min), and then the cells were further cultured for 12 h. For dark toxicity experiments, cells were directly cultured with the above materials at different concentrations for 24 h. Then MTT solution (200 μL, 0.5 mg mL -1 ) was added to each well. The cells were cultured at 37°C. After 4 h, the plates were turned over and the MTT solution was discarded. DMSO (100 μL) was added to dissolve the metabolite formazan crystals, and the mixture was shaken on a shaker for 5 min, and then the absorbance at 570 nm was measured using an ELISA reader. Cells without any treatment were used as controls.

[0117] Fig.10 (a)-(b) are TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127, Pd6-TMPP(Pd 0.4 The dark toxicity of the four materials is proportional to their concentration, and the higher the palladium chelation rate, the greater the dark toxicity of the porphyrin coordination molecular cage. Pd6-TMPP(Pd)-F127 has the highest dark toxicity, and its IC 50 The value was approximately 106.4 μg / mL (Table 3). Fig.10 (c)-(d) show that after laser irradiation, the cytotoxicity of all materials increased significantly, indicating that they all have good PDT effects.

[0118] Table 3 TMPP-F127 and three Pd6-TMPP (Pd x )-F127(x=1,0.6,0.4)IC 50 value

[0119]

[0120] As shown in Table 3, the lower the palladium chelation rate of the porphyrin coordination molecular cage, the greater the phototoxicity. 0.4 )-F127 has the highest phototoxicity, and its IC 50 The value was approximately 4.75 μg / mL, which was only 1 / 12 of that of TMPP-F127.

[0121] The mechanism of light / dark toxicity of the above materials is explored mainly from the following aspects:

[0122] a. Detection of OH in solution

[0123] Palladium complexes can catalyze the Fenton-like reaction to convert H2O2 into ·OH. Figure 2 The UV absorption spectrum of Pd6-TMPP(Pd)-F127 shows that Pd6-TMPP(Pd)-F127 has no UV absorption in the range of 600-700nm. In order to avoid the interference of the absorption peak of the material on the experiment, Pd6-TMPP(Pd)-F127 was selected to study the dark toxicity of the palladium porphyrin coordination molecular cage. Methylene blue (MB) is a common colorimetric agent that can be degraded by ROS and measured at 663nm. Therefore, the ability to generate ROS can be evaluated by measuring the UV absorption curve of the mixed solution after Pd6-TMPP(Pd)-F127 with different palladium contents reacted with MB with different H2O2 concentrations (100μM and 5μM, simulating the H2O2 concentration in the tumor microenvironment and normal cell environment, respectively), and Pd(OAc)2 was selected as a control to further illustrate the catalytic effect of palladium ions.

[0124] Depend on Fig.18 It can be seen that when the palladium content is 10 μg / mL, the ultraviolet absorption peak of MB in the tumor microenvironment decreases, while the ultraviolet absorption peak in the normal cell environment has almost no change. From this, we can infer that Pd6-TMPP(Pd)-F127 has no toxic side effects on normal cells when the palladium concentration is 10 μg / mL, but can produce ROS in tumor cells and thus have a selective killing effect on tumor cells.

[0125] b. Intracellular ROS detection without light

[0126] DCFH-DA was used as a ROS probe, and the production of ROS in B16F10 cells and HEK293 cells after incubation with the material was observed using an inverted fluorescence microscope. Fig.19It can be seen that the green fluorescence of DCF was not detected in B16F10 and HEK293 cells treated with TMPP-F127 (porphyrin concentration of 67 μg / mL), indicating that TMPP-F127 cannot produce ROS in cells under dark conditions. The green fluorescence of DCF was detected in B16F10 cells treated with Pd6-TMPP(Pd)-F127 (porphyrin concentration of 67 μg / mL, palladium content of 10 μg / mL) and Pd(OAc)2 (palladium content of 10 μg mL), indicating that palladium complexes can catalyze the production of ROS in cells. It is worth noting that no green fluorescence was detected in HEK293 cells treated with Pd6-TMPP(Pd)-F127 under the same conditions, indicating that when the palladium content was 10 μg / mL, Pd6-TMPP(Pd)-F127 could only produce ROS in cancer cells but not in normal cells, thus further having a selective concentration-killing effect on cancer cells, which is consistent with Fig.18 The results are consistent.

[0127] c. Cytotoxicity experiment of Pd6-TMPP(Pd)-F127 on B16F10 / HEK293 cells

[0128] B16F10 cells and HEK293 cells were treated with Pd6-TMPP(Pd)-F127, and the cell survival rate was calculated after 24 hours. Fig. 20 As shown, when the concentration of Pd6-TMPP(Pd)-F127 was 10 μg / mL, the survival rate of HEK293 cells was as high as over 95%, while the survival rate of B16F10 cells was about 70%, which further quantitatively proved that Pd6-TMPP(Pd)-F127 had a selective killing effect on cancer cells.

[0129] d. Intracellular GSH content after Pd6-TMPP(Pd)-F127 treatment

[0130] TMPP-F127 (porphyrin concentration 67 μg mL -1 ), Pd6-TMPP(Pd)-F127 (porphyrin concentration was 67 μg mL -1 , palladium content 10μg mL -1 ), Pd(OAc)2 (palladium content 10 μg mL -1 ) were co-incubated with B16F10 cells for 24 h. Then the cell samples were treated according to the instructions of the GSH and GSSG detection kits, and the absorbance of the samples in the range of 405-414 nm was measured using an ELISA instrument. Fig.21As shown, when B16F10 cells were incubated with Pd6-TMPP(Pd)-F127 (porphyrin concentration was 67 μg / mL) and Pd(OAc)2 with a palladium content of 10 μg / mL for 24 hours, the intracellular GSH content was downregulated to about 40%, while TMPP-F127 did not show a significant effect at the same porphyrin concentration (67 μg / mL).

[0131] e. Mitochondrial membrane potential of cells after Pd6-TMPP(Pd)-F127 treatment

[0132] JC-1 is a widely used m It is an ideal fluorescent probe for mitochondria, and exhibits potential-dependent accumulation in mitochondria. One of the early signs of apoptosis is the decrease in mitochondrial membrane potential. As the mitochondrial membrane potential (MMP) of cells decreases, the JC-1 probe transforms from aggregates to monomers, and the fluorescence also changes from red to green. Fig. 22 It can be seen that compared with the PBS and TMPP-F127 groups, B16F10 cells treated with Pd6-TMPP(Pd)-F127 and Pd(OAc)2 showed strong green fluorescence, indicating a significant decrease in mitochondrial membrane potential.

[0133] f. TEM of mitochondrial morphology of cells after Pd6-TMPP(Pd)-F127 treatment

[0134] B16F10 and HEK293 cells were seeded in culture plates at a density of 3 million cells and incubated at 37°C for 24 h. Then, palladium with a concentration of 10 μg mL -1 The serum-free DMEM solution of Pd6-TMPP(Pd)-F127 was incubated for 8 hours, and serum-free DMEM without material was added as a control. The culture medium was removed and the cells were washed three times with PBS. The cells were collected in a 1.5 mL centrifuge tube, 2.5% glutaraldehyde was added, and fixed at 4°C overnight. The fixative was discarded, about 1 mL of agar was added, and it was allowed to stand for 3-4 minutes. Then, the cells were taken out and cut into 1×3×1 mm pieces with a blade. 3The slices were rinsed with PBS (0.1M, pH 7.0) three times for 15 minutes each time. The samples were then fixed with 1% osmic acid solution for 1 hour, and then rinsed with PBS (0.1M, pH 7.0) three times for 15 minutes each time. Next, the samples were dehydrated with gradient concentrations of ethanol solution (30%, 50%, 70%, 80%), each concentration was treated for 15 minutes, and then transitioned to 90% and 95% acetone solutions, each for 15 minutes, and finally treated twice with pure acetone, each for 20 minutes. Next, the samples were treated with a mixture of Spurr embedding agent and acetone with a volume ratio of 1:1 and 3:1 for 1 hour and 3 hours, respectively, and then treated with pure embedding agent overnight at room temperature. The samples were placed in a 0.5mL dry centrifuge tube containing Spurr resin and heated at 70°C for more than 9 hours. The samples were sliced ​​in a LEICA EM UC7 ultrasonic microtome, and the sections were stained with uranyl acetate and lead citrate for 5-10 min, respectively, and observed in a Hitachi H-7650 TEM.

[0135] Fig.23 The TEM images of mitochondria in B16F10 cells without any treatment (a) and induced by Pd6-TMPP (Pd)-F127 (palladium content of 10 μg / mL) (b). As can be seen from the TEM images, the mitochondria in B16F10 cells without any treatment are intact and the cristae are densely distributed, while the B16F10 cells treated with Pd6-TMPP (Pd)-F127 show severe mitochondrial damage and a decrease in the number of cristae, as indicated by the red arrows in the figure. Fig.24 TEM images of mitochondria in HEK293 cells without any treatment (a) and induced by Pd6-TMPP(Pd)-F127 (b). We can observe that compared with untreated HEK293 cells, the mitochondria of cells treated with Pd6-TMPP(Pd)-F127 (palladium content of 10μg / mL) are more complete in morphology and the structure is almost not damaged. This further proves that when the palladium content is 10μg / mL, Pd6-TMPP(Pd)-F127 can selectively kill cancer cells while being non-toxic to normal cells.

[0136] (2) Cellular uptake

[0137] Preparation of porphyrin with the same concentration (60 μg mL -1 )TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4)-F127 serum-free DMEM solution. B16F10 cells were incubated with the above-prepared solution at 37°C for 6 hours, the culture medium was removed and the cells were washed three times with PBS, and fixed with fresh 4.0% formaldehyde for 10 minutes. After washing away the formaldehyde with PBS, the cells were incubated with Hoechst 33258 staining solution for 5 minutes, washed twice with PBS, and the cell uptake was qualitatively compared and analyzed under an inverted fluorescence microscope. The cells were collected and the cell uptake of each material was quantitatively detected on a flow cytometer.

[0138] The cell nuclei were stained with Hoechst 33258 staining solution, and red fluorescence of porphyrin was observed in B16F10 cells treated with the four materials. The co-localization of blue and red fluorescence in the merged image showed that they were successfully taken up by the cells into the cytoplasm. Fig.11 As shown in Figure 2, the fluorescence of Pd6-TMPP(Pd)-F127 is almost completely quenched due to the complete chelation of heavy metal ions at the center of the porphyrin ring. Fig.12 The results detected by flow cytometry were consistent.

[0139] (3) Intracellular ROS detection

[0140] 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the generation of ROS in B16F10 cells under illumination. -1 )TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4 )-F127 serum-free DMEM solution was cultured for 6 h. After washing three times with PBS, DCFH-DA (10 μM) was added and cultured for 30 min at 37°C and 5% CO2. The cells were washed three times with PBS and irradiated with a 650 nm laser for 2 min (25 mW cm -2 ). Subsequently, the cells were fixed with 4.0% formaldehyde for 10 minutes and then incubated with Hoechst 33258 staining solution for 5 minutes. Finally, the production of intracellular ROS was qualitatively observed using an inverted fluorescence microscope. The cells were collected and the intracellular ROS production after incubation of each material with the cells was quantitatively compared and analyzed on a flow cytometer.

[0141] The results are as follows Fig.13 As shown in the figure, compared with the control group, the green fluorescence of DCF can be detected in B16F10 cells after incubation with the four materials, indicating that they can all produce ROS in cancer cells. In addition, the ROS production of the four nanomaterials in cells was further quantitatively compared by flow cytometry. The ROS production in cells was as follows: Pd6-TMPP (Pd0.4 )-F127>Pd6-TMPP(Pd 0.6 )-F127>Pd6-TMPP(Pd)-F127>TMPP-F127, that is, compared with the H2TMPP ligand, the ROS production after the formation of the metal cage is significantly increased, and the metal cage with a lower chelation rate has a greater ROS production ( Fig.14 ), which is consistent with Fig. 9 The results of ROS production detection of the four nanomaterials in solution were consistent.

[0142] (4) Live cell / dead cell double staining experiment

[0143] B16F10 cells were treated with the same porphyrin concentration (30 μg mL -1 ) were co-incubated for 6 h and then heated at 650 nm (25 mW cm -2 ) laser irradiation for 4 min, then washed three times with PBS and stained with freshly prepared 1× staining solution (Calcein-AM 2 μM, PI 4.5 μM) for 15 min. Images were taken using an inverted fluorescence microscope.

[0144] Fig.15 The following are pictures of live and dead cells stained after incubation with four nanomaterials with the same porphyrin concentration (60 μg / mL) and laser irradiation. Green fluorescence indicates live cells, and red fluorescence indicates dead cells. As can be seen from the figure, cells incubated with TMPP-F127 and irradiated with laser showed a very high live cell ratio, indicating that its phototoxicity was low, while the three Pd6-TMPP (Pd x Most of the cells incubated with )-F127 (x=1, 0.6, 0.4) died after laser irradiation, indicating that they had high phototoxicity, which was consistent with the results of the MTT experiment.

[0145] (5) Cell apoptosis research

[0146] According to the apoptosis analysis kit, Annexinv-FITC / PI double staining method was used to detect cell apoptosis. Two 6-well plates were prepared for dark toxicity and phototoxicity experiments. B16F10 cells were seeded into 6-well plates at a cell density of 500,000 / well. For the phototoxicity experiment, laser (650nm, 25mW cm) was used after incubation for 8h. -2 ) for 1 minute. For dark toxicity experiments, they were directly incubated for 8 hours without laser irradiation. After incubation with the materials, the cells were digested with trypsin solution without EDTA, washed with PBS buffer and centrifuged. The cells were then resuspended in 500 μL Binding Buffer and stained with 5 μL Annexin V-FITC and 5 μL PI for 15 minutes in the dark. The stained cells were analyzed by flow cytometry.

[0147] The cell apoptosis was evaluated by Annexin V FITC / PI double staining. Fig.16 The figure shows the distribution of cell apoptosis and the apoptosis ratio at each stage after incubation with the material without laser (first row) and with laser (second row) irradiation. As can be seen from the figure, without laser irradiation, the survival rate of cells incubated with TMPP-F127 is higher, and the apoptotic cells account for only 4.76%, while incubated with three Pd6-TMPP (Pd x )-F127 (x=1, 0.6, 0.4) incubated with the four nanomaterials, the cell survival rate decreased, and the proportion of apoptotic cells increased. The proportion of apoptotic cells induced by Pd6-TMPP(Pd)-F127 was the highest, at 11.5%. Under 650nm laser irradiation, the cell survival rate decreased after incubation with the four nanomaterials, and the proportion of apoptotic cells increased. The cell survival rate of the metal cage with lower chelation rate was lower, and the proportion of apoptotic cells was larger. Among them, Pd6-TMPP(Pd 0.4 The proportion of live cells after )-F127 treatment was 25.8%, and the proportion of apoptotic cells was 34.51%.

[0148] (6) Evaluation of palladium content after cellular internalization

[0149] Inductively coupled plasma-mass spectrometry (ICP-MS) was used to evaluate the palladium content after cell internalization. Mouse melanoma cells (B16F10, about 2 million) were suspended in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (double antibody, P / S), transferred to a culture dish, and placed in a cell incubator at 37°C and 5% CO2 for culture. Two days later, the supernatant was removed, rinsed once with pancreatic enzyme containing 0.25% EDTA, and then 1 mL of pancreatic enzyme was added, and placed in a cell incubator for digestion for 3 minutes, and then 3 mL of DMEM containing serum was added to neutralize the pancreatic enzyme and centrifuged for 3 minutes (1200rpm). The supernatant was discarded, the cells were resuspended in DMEM medium, and seeded in a culture dish at a density of 3 million. After 24 hours, the cells proliferated rapidly in the culture dish, and the number of cells reached 10 million / plate. The three prepared Pd6-TMPP (Pd x )-F127 (x=1, 0.6, 0.4) were incubated with cells of the same number for 6 h, and the content of palladium ions in the mother solution before addition and after incubation was measured by ICP-MS to evaluate the cell chelation rate of Pd6-TMPP (Pd x )-F127 (x = 1, 0.6, 0.4) uptake. The test results are summarized in Table 4:

[0150] Table 4 Pd6-TMPP (Pdx Intracellular palladium content and uptake rate after treatment with )-F127 (x=1, 0.6, 0.4)

[0151] Material Palladium content in material (ppm) Intracellular palladium content (ppm) Uptake rate (%) <![CDATA[Pd6-TMPP(Pd)-F127]]> 5.4 0.8 15 <![CDATA[Pd6-TMPP(Pd 0.6 )-F127]]> 2.0 0.44 22 <![CDATA[Pd6-TMPP(Pd 0.4 )-F127]]> 1.6 0.42 26

[0152] As shown in Table 4, although the cell uptake rate of Pd6-TMPP(Pd)-F127 material is the lowest, due to the highest palladium content in its initial material, a higher palladium concentration accumulates in the cell compared with other materials. This also explains why Pd6-TMPP(Pd)-F127 has the greatest dark toxicity compared with other nanomaterials in the MTT dark toxicity experiment.

[0153] (6) Hemolysis test

[0154] Hemolysis assays were performed to evaluate the performance of four nanomaterials (TMPP-F127, Pd6-TMPP(Pd)-F127, Pd6-TMPP(Pd 0.6 )-F127 and Pd6-TMPP(Pd 0.4 )-F127) blood compatibility. The specific operation is as follows: perform eye removal experiment on mice to collect blood, collect the blood into a 1.5mL centrifuge tube pre-placed with sodium heparin, transfer to a 10mL centrifuge tube and add PBS liquid to about 8mL, and centrifuge at 3200rpm for 6min. After washing until the supernatant is almost colorless, dilute the red blood cells with PBS as needed, and mix well to obtain a cell suspension. Take 500μL and add it to 500μL of PBS solutions of four nanomaterials with concentrations of 0.05, 0.1, 0.2, 0.4, and 0.5mg / mL in turn, and set PBS and water as negative and positive controls respectively. Incubate at 37°C for 4h, take out, centrifuge at 3200rpm for 6min, and use an enzyme marker to measure the absorbance of porphyrin and supernatant at 540nm of the same concentration to deduct the influence of porphyrin absorbance. Each group of experiments has three replicates, and the hemolysis rate is calculated as follows:

[0155] Hemolysis rate (%) = [(A tes -A neg ) / (A pos -A neg )]×100%

[0156] Fig.17 The figure shows the hemolysis test results of the above four nanomaterials. As can be seen from the figure, at 37°C, after incubation with red blood cells for 4 hours, the hemolysis rates of the four nanomaterials at a concentration of 0.5 mg / mL were all less than 2%, which also shows that this type of nanomaterials has good biocompatibility and is suitable for further biological applications.

[0157] Example 12 Pd6-TMPP (Pd 0.4 )-F127 in vivo anti-tumor experiment

[0158] (1) Establishment of animal tumor model

[0159] Under sterile conditions, B16F10 cell suspension (100 μL, 2×10 6 When the average tumor volume reaches 90 mm 3 When the experiment was performed, the group experiment was carried out and this day was set as day 0.

[0160] (2) Tumor growth inhibition experiment

[0161] Nude mice with tumors of similar size were randomly divided into three groups: PBS, Pd6-TMPP (Pd 0.4 )-F127、Pd6-TMPP(Pd 0.4 )-F127+PDT, 5 mice in each group. Mice received PBS, Pd6-TMPP (Pd 0.4 )-F127(0.5.00mg Pd / kg), Pd6-TMPP(Pd 0.4 )-F127+PDT (0.5.00 mg Pd / kg), administered 3 times a week. 0.4 )-F127+PDT group, laser treatment was performed for 10 minutes (650nm, 100mW cm) 4 hours after each administration. -2 , irradiation every 10 seconds for 5 minutes), and the drug administration and laser treatment lasted for 17 days. During the treatment, the mice were weighed and the tumor size was measured every day. The formula for calculating the tumor volume is: (length) × (width) 2 / 2.

[0162] like Fig.25 As shown in (b), there was no significant change in the body weight of mice in each group before and after treatment, indicating that the intervention treatment did not affect the normal physiological metabolic activities of mice. Fig.25 (a) Tumor growth inhibition curve and Fig.25 (d) shows that compared with the control group injected with PBS, the tumor weight of the mice receiving a single Pd6-TMPP (Pd 0.4 )-F127 chemotherapy in mice showed a certain degree of inhibition of tumor growth, and due to the EPR effect of nanoparticles, the drug uptake by cancer cells increased, resulting in Pd6-TMPP (Pd 0.4 )-F127 nanoparticles have a better tumor inhibition effect than PBS, and the survival rate of mice is higher ( Fig.25 (c)). In addition, Fig.25 It can be seen that Pd6-TMPP (Pd0.4 )-F127 can completely inhibit tumor growth after combined with PDT treatment, and the survival rate of mice during treatment is 100%. However, due to the limited penetration of lasers and the infiltrated cancer cells being far away from the epidermis, it is almost impossible to completely eliminate the primary tumor by single photodynamic therapy.

[0163] Fig.25 Pd6-TMPP(Pd 0.4 )-F127+L refers to the above Pd6-TMPP(Pd 0.4 )-F127.

[0164] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A porphyrin-based coordinated molecular cage, characterized in that: The porphyrin-based coordinated molecular cage M6[L(M' x )]3 has the following general structural formula: wherein M is Pd; M' is the same as or different from M and is selected from Fe or Pd; R is -CH3 or -CH2OH; L(M' x ) wherein the value of x is 0.4≤x≤1. When x<1, the porphyrin ring in the porphyrin-based coordination molecular cage is partially chelated with the metal M'.

2. The porphyrin-based coordination molecular cage according to claim 1, characterized in that: The porphyrin-based coordination molecular cage is Pd6[TMPP(Pd x )]3(0.4≤x≤1), Pd6[TMPP(Fe)]3, Pd6[THPP(Pd)]3 or Pd6[THPP(Fe)]3.

3. The porphyrin-based coordination molecular cage according to claim 2, characterized in that: The Pd6[TMPP(Pd x )]3(0.4≤x≤1) including Pd6[TMPP(Pd 0.4 )]3、Pd6[TMPP(Pd 0.6 )]3, Pd6[TMPP(Pd)]3; The Pd6[TMPP(Pd 0.4 )]3 has a molecular formula of C 132 H 96 Cl 12 N 24 Pd7 belongs to the orthorhombic system, the space group is Cmc21, and the unit cell parameters are: α=90°, β=90°, γ=90°, The Pd6[TMPP(Pd 0.6 )]3 has a molecular formula of C 132 H 96 Cl 12 N 24 Pd 8.40 , belongs to the monoclinic system, the space group is P21, and the unit cell parameters are: α=90°, β=106.113(3)°, γ=90°, The molecular formula of Pd6[THPP(Pd)]3 is C 132 H 96 Cl 12 N 24 Pd9 belongs to the monoclinic system, the space group is P21 / c, and the unit cell parameters are: α=90°, β=98.378(2)°, γ=90°, 4. The porphyrin-based coordination molecular cage according to claim 2, characterized in that: The molecular formula of Pd6[TMPP(Fe)]3 is C 264 H 204 Cl 30 Fe6N 48 oeLh 12 , belongs to the triclinic system, the space group is P-1, and the unit cell parameters are: α=63.504(3)°, β=88.850(3)°, γ=85.741(4)°, The molecular formula of Pd6[THPP(Pd)]3 is C 132 H 96 Cl 12 N 24 O 12 Pd9 belongs to the orthorhombic system, the space group is Pbca, and the unit cell parameters are: α=90°, β=90°, γ=90°, The molecular formula of Pd6[THPP(Fe)]3 is C 132 H 108 Cl 12 Fe3N 24 O 18 Pd6 belongs to the trigonal crystal system, the space group is R-3c:H, and the unit cell parameters are: α=90°, β=90°, γ=120°, 5. A method for preparing the porphyrin-based coordinated molecular cage according to any one of claims 1 to 4, characterized in that: The ligand H2L, the metal M salt and the metal M' salt are dissolved in a solvent, an acid regulator is added to the obtained mixed solution, and then a solid is precipitated after the temperature is raised and lowered, and the solid is washed to obtain the porphyrin-based coordination molecular cage; the ligand H2L is selected from one of the compounds shown in the following structures:

6. The preparation method according to claim 5, characterized in that: The metal M salt is palladium acetate or palladium chloride; the metal M' salt is ferric chloride hexahydrate, palladium acetate or palladium chloride; the solvent is a mixed solvent obtained by mixing dichloromethane and methanol in a volume ratio of 1:3, or a mixed solvent obtained by mixing dichloromethane, chlorobenzene and methanol in a volume ratio of 1:1:6; the acid regulator is acetic acid or benzoic acid.

7. The preparation method according to claim 5, characterized in that: The temperature rise and fall treatment is specifically as follows: firstly raise the temperature to 110-130° C., keep the temperature for 24-60 hours, and then reduce the temperature to 20-30° C. at a rate of 4-5° C. / h.

8. Use of the porphyrin-based coordination molecular cage according to any one of claims 1 to 4 in the preparation of anti-melanoma drugs.

9. A porphyrin-based coordinated molecular cage nanoparticle preparation, characterized in that: The porphyrin-based coordinated molecular cage and the amphiphilic copolymer described in claims 1 to 4 are dispersed in a solvent, added dropwise to water after ultrasound to obtain a micellar solution, and the porphyrin-based coordinated molecular cage nano preparation is obtained by dialysis.

10. Use of the porphyrin-based coordinated molecular cage nanoformulation according to claim 9 in the preparation of anti-melanoma drugs.

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