CN116178448A - Porphyrin-based coordination molecular cage and preparation method and application thereof - Google Patents

Porphyrin-based coordination molecular cage and preparation method and application thereof Download PDF

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CN116178448A
CN116178448A CN202310068656.6A CN202310068656A CN116178448A CN 116178448 A CN116178448 A CN 116178448A CN 202310068656 A CN202310068656 A CN 202310068656A CN 116178448 A CN116178448 A CN 116178448A
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porphyrin
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张文华
胡巧
马品品
王潇
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Abstract

The invention discloses a porphyrin-based coordination molecular cage, a preparation method and application thereof, wherein a pyridine functional group in an organic ligand containing a porphyrin skeleton is coordinated with a metal M with biological activity to obtain a molecular cage compound M 6 L 3 The method comprises the steps of carrying out a first treatment on the surface of the Molecular cage compound M 6 L 3 Further chelating or partially chelating the metal M' to obtain homonuclear or heteronuclearPorphyrin-based coordination molecule cage M 6 [L(M’ x )] 3 . The porphyrin-based coordination molecule cage prepared by the invention can effectively inhibit fluorescence quenching caused by aggregation among porphyrin molecules, thereby greatly improving photodynamic therapy efficiency, and the metal M with biological activity, such as Pd, is introduced 2+ Can catalyze Fenton-like reaction, induce programmed iron death of cells, and realize double tumor inhibiting effects. In addition, the unique cavity of the molecular cage can cooperatively load small molecular medicines to realize the synergistic anticancer effect, and the phototoxicity of the porphyrin coordination molecular cage can be regulated and controlled by controlling the metal chelating rate of the porphyrin skeleton center, so that the treatment effect is further improved.

Description

Porphyrin-based coordination molecular cage and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicine, in particular to a porphyrin-based coordination molecular cage, a nano preparation, a preparation method and application thereof.
Background
The treatment method of the cancer mainly comprises surgical excision, radiotherapy, chemotherapy, molecular targeted therapy, immunotherapy and the like. For example, early cancer can be treated by surgical excision or surgical combination with chemoradiotherapy. Advanced metastatic cancers can be treated by chemotherapy in combination with targeted drugs, thereby achieving the purposes of improving quality of life and relieving symptoms. In addition, antibody immunotherapy with immune drugs such as PD-1, PD-L1 and CTLA-4 greatly improves the overall benefit of the patient, whereas the response rate of these Immune Checkpoint Inhibitors (ICI) alone is only 19-47%. During the course of immunotherapy, some patients may acquire a sustained immune response, while others may develop primary or secondary resistance. Thus, research and exploration of new materials, new methods for the treatment of cancer remain of great practical importance.
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, pancreatic cancer, etc., as an important supplement to classical cancer treatment methods. PDT has the advantages of no wound, small side effect, high space-time selectivity and the like. Compounds based in part on porphyrin skeletons, such as Photofrin, temoporfin, photochlor, redaporfin, have been approved by the FDA and used in the clinical treatment of cancer. It has been demonstrated by current research that PDT, in addition to being a therapeutic treatment for ablative cancers, can mediate the anti-tumor immune response of the body through immunogenic death (ICD) or iron death (ferrovision) of tumor cells, ultimately enhancing the anti-tumor effect.
The porphyrin monomer can be coordinated with metal through a self-assembly technology to prepare a nano material with high dispersibility and stability so as to show stronger high-permeation long retention (EPR) effect, and can be used for PDT treatment of in vivo tumors. For example, chen et al prepared a nanoscale porphyrin-modified Fe-based metal-organic framework MIL-101 (Fe) @ TCPP, which upon entry into tumor cells, upon stimulation of the tumor acidic microenvironment, decomposed and released free Fe 3+ Ions and TCPP (Chen et al, ACS appl. Mater. Interfaces 2021,13,45201-45213). Fe (Fe) 3+ Ion-catalyzed Fenton reaction can effectively convert endogenous H 2 O 2 Conversion to OH, which is highly cytotoxic, and TCPP, upon activation by light of a specific wavelength, can also generate Reactive Oxygen Species (ROS) in tumor tissue. This dual dynamic effect can significantly increase ROS levels in tumor cells and synergistically induce oxidative damage to the cells, resulting in better tumor treatment. However, none of the porphyrin compounds contains expandable functional groups, which greatly limits the systematic research of the materials. Meanwhile, the carrier material is generally large in particle size, difficult to uniformly disperse and low in drug delivery efficiency. The carrier system is constructed to contain a large excess of Fe 3+ The toxic side effects of the source, as well as of the other component 2-aminoterephthalic acid after decomposition of the support material, are also alarming. TCPP has some absorption in the infrared band, but the absorption intensity is not large, which greatly limits the efficacy of PDT. The TCPP is taken as a guest small molecule with low solubility, is easy to rapidly remove after explosive release along with the decomposition of a carrier framework, and greatly reduces the potential applicability of the system.
The hypoxic microenvironment in solid tumors also has a negative impact on PDT efficacy. Based on this, dai et al propose a simple strategy to form O by ultrasonic dispersion of perfluorooctyl bromide (PFOB) liquid into porphyrin-grafted lipid (PGL) 2 PFOB@PGL nanoparticles (Dai et al ACS Nano 2020,14,13569-13583) followed by loading with O 2 To prepare PDT nano-carrier with oxygen self-supplying. The carrier has high porphyrin/O 2 The loading rate can deliver oxygen into anoxic tumors without external stimulation, which is beneficial to increasing single lineOxygen in state 1 O 2 ) Relieving tumor hypoxia and subsequently down regulating COX-2 expression. In animal experiments, the material can obviously inhibit the growth of tumors and liver metastasis. In addition, O 2 Pfob@pgl also exhibits excellent Fluorescence (FL) and Computed Tomography (CT) imaging capabilities. Therefore, the oxygen self-supplementing PDT nano-carrier not only can ablate primary tumors, but also can relieve tumor hypoxia under the guidance of FL/CT imaging to inhibit tumor metastasis. However, the rigid structure and hydrophobic plane of porphyrin tend to cause aggregation of porphyrin in solution to cause ACQ effect, which will decrease 1 O 2 Is effective in generating and PDT. In addition, PDT treatment is limited to shallow layers due to poor penetration of the light source tissue, so that single PDT treatment is prone to cause recurrence and metastasis of the tumor, and it is often difficult to completely remove cancer cells.
In the two self-assembly processes, porphyrin compounds are respectively connected into coordination polymers and organic nano carriers in a coordination bond and covalent bond mode, so that nano particles with PDT treatment efficacy are obtained. In addition, the porphyrin compound can be directly used as a construction unit to participate in the self-assembly of a small molecule coordination molecule cage, and the obtained nanoparticle porphyrin has high loading rate and good PDT effect. For example, chen et al synthesized a discrete organo-platinum metal cage for integrated chemotherapy and PDT treatment by coordination driven self-assembly with TPP, DSTP and cPt as building blocks (Chen et al, nat. Commun.2018,9,4335). The formation of porphyrin coordination molecule cage effectively inhibits the aggregation fluorescence quenching (ACQ) effect of porphyrin, resulting in the enhancement of fluorescence intensity and 1 O 2 the improved quantum yield facilitates NIR/FL imaging and PDT treatment. Meanwhile, different imaging functions can be realized by chelating various metal ions through the porphyrin ring center. The obtained metal cage is encapsulated by polyethylene glycol chains with RGD targeting to prepare the nano particles, so that the biocompatibility of the nano particles can be improved, the blood circulation time can be prolonged, the active targeting capability of the nano particles to cancer cells can be provided, the nano particles have excellent tumor inhibiting effect, and tumor recurrence and tumor metastasis after single treatment can be effectively prevented. However, the complex molecular cage component constructed by Chen et al is extremely complex and involves the following additional steps PEt with extremely toxic effect 3 As a ligand, the synthesis feasibility is low, and the toxic and side effects during drug degradation are not small.
Disclosure of Invention
The invention aims to solve the technical problem of providing a porphyrin-based coordination molecular cage, a preparation method and application thereof, wherein the porphyrin-based photosensitizer with a specific configuration and an anticancer active metal are cooperatively assembled by a coordination method to form a coordination molecular cage compound with a triangular prism shape, the assembled molecular cage has the capability of further loading other medicines, and meanwhile, the molecular cage can be further coordinated with the metal through the functionalization of the ligand, and other targeting or water-soluble ligands are introduced to realize the water solubility and biocompatibility of the medicines and the tracing of the medicines entering the body, so that the toxic and side effects of the medicines are minimized.
In order to solve the technical problems, the invention provides the following technical scheme:
in a first aspect, the present invention provides a porphyrin-based ligand cage M 6 [L(M’ x )] 3 Has the following structural general formula:
Figure BDA0004063048400000031
wherein 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;
R is-CH 3 or-CH 2 OH；
L(M’ x ) Wherein x has a value of 0<x is less than or equal to 1, and when x is less than 1, the porphyrin ring in the porphyrin-based coordination molecule cage is chelated with a metal M' part.
Further, the porphyrin-based coordination molecule cage is Pd 6 [TMPP(Pd x )] 3 (0<x≤1)、Pd 6 [TMPP(Fe)] 3 、Pd 6 [THPP(Pd)] 3 Or Pd (or) 6 [THPP(Fe)] 3 。
Further, the Pd 6 [TMPP(Pd x )] 3 (0<x.ltoreq.1) comprises Pd 6 [TMPP(Pd 0.4 )] 3 、Pd 6 [TMPP(Pd 0.6 )] 3 、Pd 6 [TMPP(Pd)] 3 。
Further, the Pd 6 [TMPP(Pd 0.4 )] 3 Has the molecular formula of C 132 H 96 Cl 12 N 24 Pd 7 Belongs to an orthorhombic crystal system, and the space group is Cmc2 1 The unit cell parameters are:
Figure BDA0004063048400000032
α＝90°，β＝90°，γ＝90°，/>
Figure BDA0004063048400000033
further, the Pd 6 [TMPP(Pd 0.6 )] 3 Has the molecular formula of C 132 H 96 Cl 12 N 24 Pd 8.40 Belonging to monoclinic system, the space group is P2 1 The unit cell parameters are:
Figure BDA0004063048400000034
α＝90°，β＝106.113(3)°，γ＝90°，/>
Figure BDA0004063048400000035
further, the Pd 6 [THPP(Pd)] 3 Has the molecular formula of C 132 H 96 Cl 12 N 24 Pd 9 Belonging to monoclinic system, the space group is P2 1 And/c, the unit cell parameters are as follows:
Figure BDA0004063048400000036
α＝90°，β＝98.378(2)°，γ＝90°，/>
Figure BDA0004063048400000037
further, the Pd 6 [TMPP(Fe)] 3 Has the molecular formula of C 264 H 204 Cl 30 Fe 6 N 48 O 6 Pd 12 Belongs to a triclinic system, the space group is P-1, and the unit cell parameters are as follows:
Figure BDA0004063048400000038
α＝63.504(3)°，β＝88.850(3)°，γ＝85.741(4)°，/>
Figure BDA0004063048400000039
further, the Pd 6 [THPP(Pd)] 3 Has the molecular formula of C 132 H 96 Cl 12 N 24 O 12 Pd 9 Belongs to an orthorhombic crystal system, the space group is Pbca, and the unit cell parameters are as follows:
Figure BDA00040630484000000310
α＝90°，β＝90°，γ＝90°，/>
Figure BDA00040630484000000311
further, the Pd 6 [THPP(Fe)] 3 Has the molecular formula of C 132 H 108 Cl 12 Fe 3 N 24 O 18 Pd 6 Belonging to a trigonal system, the space group is R-3c: H, and the unit cell parameters are:
Figure BDA00040630484000000312
α＝90°，β＝90°，γ＝120°，/>
Figure BDA0004063048400000041
in a second aspect, the invention provides a method for preparing a porphyrin-based ligand cage according to the first aspect, wherein the ligand H 2 Dissolving L, metal M salt and metal M' salt in a solvent, adding an acid regulator into the obtained mixed solution, then separating out solid after temperature rising and rising treatment, and washing the solid to obtain the porphyrin-based coordination molecular cage; wherein the ligand H 2 L is selected from the following structuresOne of the compounds is shown:
Figure BDA0004063048400000042
further, the metal M salt is preferably palladium acetate or palladium chloride; if K is adopted 2 [PdCl 4 ]As palladium salt, the target product was not obtained.
Further, the metal M' salt is preferably ferric chloride hexahydrate, palladium acetate or palladium chloride.
Further, 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.
Further, the acid regulator is acetic acid or benzoic acid, more preferably acetic acid.
In some preferred embodiments, the ligand H 2 L is H 2 TMPP, the metal M salt and the metal M' salt are Pd (OAc) 2 ，H 2 TMPP and Pd (OAc) 2 The molar ratio of the porphyrin-based ligand molecular cage is 1:3, the solvent is a mixed solvent obtained by mixing dichloromethane and methanol according to the volume ratio of 1:3, and the prepared porphyrin-based ligand molecular cage is Pd 6 [TMPP(Pd)] 3 。
In some preferred embodiments, the ligand H 2 L is H 2 TMPP, wherein the metal M salt and the metal M' salt are PdCl 2 ，H 2 TMPP and PdCl 2 The molar ratio of the porphyrin-based ligand molecular cage is 1:3, the solvent is a mixed solvent obtained by mixing dichloromethane and methanol according to the volume ratio of 1:3, and the prepared porphyrin-based ligand molecular cage is Pd 6 [TMPP(Pd 0.6 )] 3 。
In some preferred embodiments, the ligand H 2 L is H 2 TMPP, wherein the metal M salt and the metal M' salt are PdCl 2 ，H 2 TMPP and PdCl 2 The molar ratio of (2) is 1:3-4, for example, 1:3.73, the solvent is a mixed solvent obtained by mixing dichloromethane, chlorobenzene and methanol according to the volume ratio of 1:1:6, and the porphyrin-based coordination molecules are preparedThe sub-cage is Pd 6 [TMPP(Pd 0.4 )] 3 。
In some preferred embodiments, the ligand H 2 L is H 2 TMPP, the metal M salt is Pd (OAc) 2 The metal M' salt is FeCl 3 ·6H 2 O，H 2 TMPP and Pd (OAc) 2 、FeCl 3 ·6H 2 The molar ratio of O is 1:2:1, the solvent is a mixed solvent obtained by mixing dichloromethane and methanol according to the volume ratio of 1:3, and the prepared porphyrin-based coordination molecular cage is Pd 6 [TMPP(Fe)] 3 。
In some preferred embodiments, the ligand H 2 L is H 2 THPP the metal M and metal M' salts are Pd (OAc) 2 ，H 2 THPP and Pd (OAc) 2 The molar ratio of the porphyrin-based coordination molecule cage is 1:5-6, the solvent is a mixed solvent obtained by mixing dichloromethane and methanol according to the volume ratio of 1:3, and the prepared porphyrin-based coordination molecule cage is Pd 6 [THPP(Pd)] 3 。
In some preferred embodiments, the ligand H 2 L is H 2 THPP the metal M salt is Pd (OAc) 2 The metal M' salt is FeCl 3 ·6H 2 O，H 2 THPP and Pd (OAc) 2 、FeCl 3 ·6H 2 The molar ratio of O is 1:2:1, the solvent is a mixed solvent obtained by mixing dichloromethane, chlorobenzene and methanol according to the volume ratio of 1:1:6, and the prepared porphyrin-based coordination molecular cage is Pd 6 [THPP(Fe)] 3 。
Further, the temperature raising and lowering treatment specifically includes: heating to 110-130 ℃, preserving heat for 24-60 h, and then reducing the temperature to 20-30 ℃ at a cooling rate of 4-5 ℃/h.
The third aspect of the invention provides an application of the porphyrin-based ligand molecular cage in preparing an anti-tumor drug.
Further, the cavities of the porphyrin-based coordination molecule cages can cooperatively load small molecule drugs, such as doxorubicin, cisplatin and the like.
The invention provides a porphyrin-based coordination molecule cage nano preparation, which is prepared by dispersing the porphyrin-based coordination molecule cage and the amphiphilic copolymer in the first aspect in a solvent, performing ultrasonic treatment, dripping the solution into water to obtain a micelle solution, and performing dialysis.
Further, the amphiphilic copolymer is preferably pluronic F127.
The fifth aspect of the invention provides an application of the porphyrin-based ligand molecular cage nano-preparation in preparing antitumor drugs.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, a porphyrin-based photosensitizer with a specific configuration and an anticancer active metal are subjected to coordination driving self-assembly by a hydrothermal method to form a coordination molecule cage compound with a triangular prism shape, and three porphyrin coordination molecule cage coordination compounds with different palladium chelation rates are synthesized by adjusting the proportion of different solvents. The assembled molecular cage has the capability of further loading other medicines, meanwhile, the molecular cage can be further coordinated with metal through functionalization of ligand, other targeting or water-soluble ligand is introduced, water solubility and biocompatibility of the medicines are realized, and tracing of the medicines entering the body is realized, and the toxic and side effects of the medicines are minimized on the basis so as to improve the solubility of the porphyrin coordination molecular cage. For example, the obtained porphyrin coordination molecule cage is further encapsulated in the hydrophobic core of the amphiphilic copolymer by adopting an ultrasonic method to prepare the spherical nano preparation, the spherical nano preparation has good light stability and good solubility and stability in a physiological medium, and the hemolysis experiment shows that the hemolysis rate of the nano preparation is less than 2 percent, and the nano preparation has good biocompatibility and can be used for biological experiments.
2. The invention is realized by the method of Pd 6 Measurement of iron death related indicators of TMPP (Pd) -F127 treated cells, such as mitochondrial morphology and membrane potential, intracellular GSH and ROS levels, and the like, shows Pd as a result of experiments 6 The mitochondrial membrane potential of cells after treatment with TMPP (Pd) -F127 was significantly reduced,mitochondrial morphology is impaired, cristae is reduced, GSH levels are reduced, and ROS production is increased. It is thus seen that the palladium complex and iron compound prepared by the present invention are similar and are also capable of mediating cell iron death by catalyzing the onset of Fenton-like reaction. Therefore, the palladium porphyrin coordination molecular cage prepared by the invention has double anticancer effects of PDT and mediated iron death.
3. The invention compares the ability of three nano particles with different chelation rates to generate ROS in solution and in cells qualitatively and quantitatively, and discovers that the metal cage with lower chelation rate has stronger ability to generate ROS, and the metal cage with lower chelation rate has lower dark toxicity and higher phototoxicity. The conclusion is proved in cytotoxicity experiments, apoptosis and dead and alive staining experiments and the like. And during the research of the mechanism of dark toxicity, the invention discovers that the Pd is controlled 6 -TMPP(Pd x ) Pd in F127 (x= 1,0.6,0.4) 2+ Can have selective killing effect on cancer cells.
4. As can be seen from the results of in vivo antitumor experiments 6 -TMPP(Pd 0.4 ) The ingestion of F127 does not affect the normal physiological metabolism of mice, plays a role in inhibiting tumors, can effectively inhibit the growth of the tumors and improve the survival rate of the mice, can completely inhibit the growth of the tumors by combining PDT treatment, and can reach 100 percent during the treatment period. Pd with the palladium content of 10 mug/mL can be selectively controlled by combining the cell experiment result 6 -TMPP(Pd 0.4 ) F127 further enables in vivo treatment of tumors by combination with PDT.
Drawings
FIG. 1 is a block diagram of a porphyrin-based coordination molecular cage: (a) Pd (Pd) 6 -TMPP(Pd 0.4 ) Side view of (b) Pd 6 -TMPP(Pd 0.4 ) (c) Pd in plan view 6 -TMPP(Pd 0.6 ) Top view of (d) Pd 6 -a top view of TMPP (Pd), (e) Pd 6 -top view of TMPP (Fe), (f) Pd 6 -a top view of THPP (Pd), (g) Pd 6 -a top view of THPP (Fe);
FIG. 2 is H 2 Ultraviolet absorption curves of TMPP and three palladium porphyrin coordination molecule cages with different chelation rates in DMSO；
FIG. 3 is a FT-IR spectrum of three palladium porphyrin coordination molecule cages with different chelation rates;
FIG. 4 shows Pd having different chelation rates for TMPP-F127 6 -TMPP(Pd x ) -a schematic synthesis of F127 (x= 1,0.6,0.4) nanoformulations;
FIG. 5 shows (a) TMPP-F127, (b) Pd 6 -TMPP(Pd)-F127、(c)Pd 6 -TMPP(Pd 0.6 ) F127 and (d) Pd 6 -TMPP(Pd 0.4 ) -a transmission electron micrograph of F127;
FIG. 6 is TMPP-F127 and Pd 6 -TMPP(Pd)-F127、Pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) -the average particle size and zeta potential of F127;
FIG. 7 shows TMPP-F127 and Pd in the range of 0-10min of laser irradiation 6 -TMPP(Pd)-F127、Pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) -the absorption value of an aqueous solution of F127 at the maximum absorption wavelength (417 nm) versus irradiation time;
FIG. 8 shows (a) TMPP-F127, (b) Pd 6 -TMPP(Pd)-F127，(c)Pd 6 -TMPP(Pd 0.6 )-F127，(d)Pd 6 -TMPP(Pd 0.4 ) -F127 at H 2 O, PBS, average particle size change graph after 0h and 8h of dispersion in PBS+10% FBS;
FIG. 9 shows (a) DPBF and (b) dispersed in TMPP-F127 and (c) Pd 6 -TMPP(Pd)-F127，(d)Pd 6 -TMPP(Pd 0.6 )-F127，(e)Pd 6 -TMPP(Pd 0.4 ) -a profile of uv absorption intensity in an aqueous F127 solution with laser irradiation time; (f) In the aqueous solution of the four nano particles, the DPBF has a change curve of absorption value at the maximum absorption wavelength along with irradiation time;
FIG. 10 is TMPP-F127, pd 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 )-F127，Pd 6 -TMPP(Pd 0.4 ) B16F10 cells 24h after F127 treatment were irradiated (a) without light and (c) with light (50 mW cm -2 Cell viability graph under 2 min) conditions (b) no illumination and (d) semi-inhibitory concentration profile under illumination * P<0.05， ** P<0.01， *** P<0.001)；
FIG. 11 shows B16F10 cells and TMPP-F127, pd 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 )-F127，Pd 6 -TMPP(Pd 0.4 ) Fluorescence microscopy pictures after incubation with F127;
FIG. 12 shows B16F10 cells and TMPP-F127, pd 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 )-F127，Pd 6 -TMPP(Pd 0.4 ) -flow cytometry after F127 incubation;
FIG. 13 shows that B16F10 cells were treated with four nanomaterials and DCFH-DA and were subjected to laser (650 nm,50mW cm -2 ) Fluorescent microscope pictures after irradiation;
FIG. 14 shows that B16F10 cells were treated with four nanomaterials and DCFH-DA and were subjected to laser (650 nm,50mW cm -2 ) A flow cytometer graph after irradiation;
FIG. 15 shows B16F10 cells treated with four nanomaterials and laser (650 nm,25mW cm) -2 ) Fluorescence microscope pictures after incubation with the Calcein-AM/PI probe after irradiation;
FIG. 16 shows the presence or absence of laser light (650 nm,25mW cm) after treatment of B16F10 cells with four nanomaterials -2 ) Flow cytometry after incubation with annexin v-FITC/PI probe under irradiation conditions;
FIG. 17 shows TMPP-F127 and Pd 6 -TMPP(Pd x ) -hemolysis experimental results of F127 (x= 1,0.6,0.4);
FIG. 18 shows Methylene Blue (MB) and Pd 6 -TMPP (Pd) -F127 (a-b) and Pd (OAc) 2 (c-d) ultraviolet absorption profile after the reaction. Wherein, a and c are shown as the drawing H 2 O 2 The content is 100 mu M, b and d are shown as the graph H 2 O 2 The content is 5 mu M;
FIG. 19 is a fluorescent microscope photograph of B16F10 cells (a) and HEK293 cells (B) treated with material and DCFH-DA in the absence of light;
FIG. 20 Pd-on B16F10 cells and HEK293 cells 6 Cell viability graph (a) and IC after 24h treatment with TMPP (Pd) -F127 50 Graph (b);
FIG. 21 shows the intracellular GSH content of B16F10 after incubation with different materials;
FIG. 22 is a CLSM image of B16F10 cells after incubation with different materials and staining with JC-1. JC-1 monomer emitted green fluorescence, indicating Deltapsi m And (3) lowering. JC-1 aggregates in healthy mitochondria fluoresce red;
FIG. 23 is a TEM image of mitochondria in untreated B16F10 cells, (B) Pd-passed 6 -TEM images of mitochondria in B16F10 cells after TMPP (Pd) -F127 treatment;
FIG. 24 is a TEM image of mitochondria in (a) untreated HEK293 cells, (b) Pd-passed 6 -TEM images of mitochondria in HEK293 cells after TMPP (Pd) -F127 treatment;
FIG. 25 is Pd 6 -TMPP(Pd 0.4 ) -in vivo antitumor effect of F127. (a) a tumor growth inhibition curve; (b) a mouse weight change profile; (c) survival curves for mice in each treatment group; (d) tumor weight map for each treatment group; (e) photographs of tumors from each treatment group.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Example 1
This example relates to a porphyrin-based coordination molecule cage Pd 6 [TMPP(Pd)] 3 The preparation of (2) is specifically carried out as follows:
will H 2 TMPP (2 mg, 0.003mmol) and Pd (OAc) 2 (2 mg,0.009 mmol) was 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 as a modifier to the above mixture. Glass tube of PyrexPlaced in a programmable oven, heated from 25 ℃ to 120 ℃ over 4 hours, then held at 120 ℃ for 48 hours, and cooled to 25 ℃ over 24 hours. Filtering, dissolving the obtained solid mixture with DCM, spin-drying the filtrate, and washing the obtained solid with MeOH for 2-3 times to obtain palladium porphyrin coordination molecular cage with 100% chelation rate, named Pd 6 -TMPP(Pd)。IR(KBr disc；cm -1 ):3427(m),2921(w),1610(m),1546(w),1494(s),1447(m),1348(m),1296(m),1232(w),1132(m),1086(m),1045(s),1010(vs),911(w),853(w),818(s),789(vs)。
Example 2
This example relates to a porphyrin-based coordination molecule cage Pd 6 [TMPP(Pd 0.6 )] 3 The preparation of (2) is specifically carried out as follows:
will H 2 TMPP (2 mg, 0.003mmol) and PdCl 2 (1.6 mg,0.009 mmol) was 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 as a modifier to the above mixture. The Pyrex glass tube was placed in a programmable oven, heated from 25 ℃ to 120 ℃ over 4 hours, then held at 120 ℃ for 48 hours, and cooled to 25 ℃ over 24 hours. Filtering, dissolving the obtained solid mixture with DCM, spin-drying the filtrate, and washing the obtained solid with MeOH for 2-3 times to obtain palladium porphyrin coordination molecular cage with 100% chelation rate, named Pd 6 -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)。
Example 3
This example relates to a porphyrin-based coordination molecule cage Pd 6 [TMPP(Pd 0.4 )] 3 The preparation of (2) is specifically carried out as follows:
will H 2 TMPP (1 mg,0.0015 mmol) and PdCl 2 (1 mg,0.0056 mmol) was dissolved in a mixed solvent of DCM/PhCl/MeOH (v: v=0.25 mL:0.25mL:1.5 mL) and transferred to a Pyrex glass tube. HAc (20 μl) was then added as a modifier to the above mixture. Placing Pyrex glass tube in programmable bakingIn the box, heat was applied from 25 ℃ to 120 ℃ over 4 hours, then hold at 120 ℃ for 48 hours, and cool to 25 ℃ over 24 hours. Filtering, dissolving the obtained solid mixture with DCM, spin-drying the filtrate, and washing the obtained solid with MeOH for 2-3 times to obtain palladium porphyrin coordination molecular cage with chelating rate of 40%, named Pd 6 -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),1348(m),1302(m),1261(m),1138(s),1086(s),1010(vs),964(s),894(w),853(w),795(vs)。
Example 4
This example relates to a porphyrin-based coordination molecule cage Pd 6 [TMPP(Fe)] 3 The preparation of (2) is specifically carried out as follows:
will H 2 TMPP(2mg，0.003mmol)、Pd(OAc) 2 (1.4 mg, 0.006mmol) and FeCl 3 ·6H 2 O (1 mg, 0.003mmol) was 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 as a modifier to the above mixture. The Pyrex glass tube was placed in a programmable oven, heated from 25 ℃ to 120 ℃ over 4 hours, then held at 120 ℃ for 48 hours, and cooled to 25 ℃ over 24 hours. Filtering, dissolving the obtained solid mixture with DCM, spin-drying the filtrate, and washing the obtained solid with MeOH for 2-3 times to obtain palladium iron porphyrin coordination molecular cage named Pd 6 -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)。
Example 5
This example relates to a porphyrin-based coordination molecule cage Pd 6 [THPP(Pd)] 3 The preparation of (2) is specifically carried out as follows:
will H 2 Phenylbutyrate of THPP (2 mg,0.0015 mmol), pd (OAc) 2 (2 mg,0.0089 mmol) was dissolved in a mixed solvent of DCM/MeOH (v: v=0.5 mL:1.5 mL) and transferred to a Pyrex glass tube. Then willHAc (20. Mu.L) was added as a regulator to the above mixture. The Pyrex glass tube was placed in a programmable oven, heated from 25 ℃ to 120 ℃ over 4 hours, then held at 120 ℃ for 48 hours, and cooled to 25 ℃ over 24 hours. Washing the obtained crystal with anhydrous diethyl ether for 2-3 times to obtain palladium porphyrin coordination molecular cage named Pd 6 -THPP(Pd)。
Example 6
This example relates to a porphyrin-based coordination molecule cage Pd 6 [THPP(Fe)] 3 The preparation of (2) is specifically carried out as follows:
will H 2 THPP(2mg，0.003mmol)、Pd(OAc) 2 (1.4 mg, 0.006mmol) and FeCl 3 ·6H 2 O (1 mg, 0.003mmol) was dissolved in a mixed solvent of DCM/MeOH/PhCl (v: v=0.25 mL:1.5mL:0.25 mL) and transferred to a Pyrex glass tube. HAc (20 μl) was then added as a modifier to the above mixture. The Pyrex glass tube was placed in a programmable oven, heated from 25 ℃ to 120 ℃ over 4 hours, then held at 120 ℃ for 48 hours, and cooled to 25 ℃ over 24 hours. Washing the obtained crystal with anhydrous diethyl ether for 2-3 times to obtain palladium iron porphyrin coordination molecular cage named Pd 6 -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)。
Example 7
X-ray single crystal diffraction (SCXRD) characterization was performed on the porphyrin-based coordination molecule cages prepared in examples 1 to 6, the structure of different porphyrin-based coordination molecule cages is shown in FIG. 1, and the corresponding crystallographic data and parameters are shown in tables 1 and 2 below:
TABLE 1 crystallographic parameters of porphyrin-based coordination molecular cages prepared in examples 1-3
Figure BDA0004063048400000091
Figure BDA0004063048400000101
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TABLE 2 crystallographic parameters of porphyrin-based coordination molecular cages prepared in examples 4 to 6
Figure BDA0004063048400000102
Figure BDA0004063048400000111
Pd was prepared by adjusting the kinds of metal salts or solvents in examples 1 to 3 2+ Porphyrin-based metal cages with different chelation rates H 2 The ultraviolet absorption curves of TMPP and three palladium porphyrin coordination molecule cages with different chelation rates in DMSO are shown in FIG. 2, and it can be seen from the graph that H 2 The ultraviolet absorption peaks of TMPP are 418nm,516nm,550nm,591nm, 640 nm in this order. Pd (Pd) 6 -TMPP(Pd 0.4 ) The ultraviolet absorption peaks of (a) are 418nm,522nm,553nm,591nm and 640 nm in sequence. Pd (Pd) 6 -TMPP(Pd 0.6 ) The ultraviolet absorption peaks of (C) are 418nm,518nm,550nm,591nm and 646nm in sequence. Pd (Pd) 6 The UV absorption peaks of TMPP (Pd) are in turn 4111 nm,523nm and 555nm.
From this, it can be seen that free H 2 Compared with TMPP, after metal cage formation, the Soret band in the ultraviolet absorption peak has red shift, which indicates that the porphyrin ring center successfully chelates metal ions. In addition, pd 6 -TMPP(Pd 0.4 ) And Pd (Pd) 6 -TMPP(Pd 0.6 ) The number of ultraviolet absorption peaks in the wavelength range of 500-700nm is not reduced, but when the palladium chelation rate at the center of porphyrin ring is 100%, the ultraviolet absorption peaks between 600-700nm disappear, presumably due to complete chelation of Pd by TMPP 2+ Resulting in fluorescence quenching of the porphyrin.
Example 8
The embodiment relates to the preparation of a part of palladium-based porphyrin coordination molecule cage nano preparation, the synthetic schematic diagram is shown in fig. 4, and the specific steps are as follows:
(1) Synthesis of TMPP-F127
Pluronic F127 (10.6 mg,0.0053 mmol) was added to a solution containing H 2 TMPP (4.5 mg,0.0067 mmol) in DMSO (1 mL). After complete dissolution, the resulting mixture was added drop-wise to 10mL of deionized water using an ultrasonic cell disruptor under ultrasonic conditions. Finally, a micellar solution of TMPP-F127 can be obtained by dialysis for 24 hours in a dialysis bag with a molecular weight cut-off of 2000.
(2)Pd 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) -synthesis of F127.
Pluronic F127 (9.6 mg,0.0048 mmol) was added sequentially to the Pd-containing mixture 6 -TMPP(Pd)(3.5mg，0.001mmol)，Pd 6 -TMPP(Pd 0.6 ) (3.5 mg,0.001 mmol) and Pd 6 -TMPP(Pd 0.4 ) (3.5 mg,0.0011 mmol) in DMSO (1 mL). After complete dissolution, the mixture obtained above was added dropwise in sequence to 10mL of deionized water under ultrasonic conditions using an ultrasonic cell disruptor. Finally, pd 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) The micellar solution of F127 can be obtained by dialysis for 24 hours in a dialysis bag with a molecular weight cut-off of 3500.
TMPP-F127 and Pd prepared in the present example 6 -TMPP(Pd)-F127、Pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) As shown in FIG. 5, a transmission electron microscope of-F127 shows TMPP-F127 and three Pd having different chelation ratios 6 -TMPP(Pd x ) F127 (x= 1,0.6,0.4) are spherical nanoparticles, around 100 nm in size, matching the hydrodynamic diameter measured by Dynamic Light Scattering (DLS) (fig. 6).
EXAMPLE 9 stability study of Palladium-based porphyrin coordination molecule cage nanoformulation
(1) Light stability
Preparation of the same porphyrin concentration (7. Mu.g mL) -1 ) TMPP-F127, pd6-TMPP (Pd) -F127, pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) An aqueous solution of-F127, the solution prepared above was irradiated with a 650nm laser (100 mW cm -2 ) Irradiation and monitoringUV absorption curve of each solution over 0-10min of irradiation. The results are shown in fig. 7, and the ultraviolet absorption curves of the four nanoparticles hardly changed within 10 minutes of laser irradiation, which proves that the nanoparticles have good light stability and are not subject to structural degradation under the irradiation of laser.
(2) Stability in physiological Medium
Monitoring TMPP-F127 and three Pd with different chelation rates by Dynamic Light Scattering (DLS) test 6 -TMPP(Pd x ) -F127 (x= 1,0.6,0.4) in different physiological media (H 2 O, PBS, pbs+10% FBS) to evaluate the stability of the nanoparticles. As shown in FIG. 8, after the nanoparticles are dispersed in the three media for 8 hours, the particle size change is small, which indicates that the packaging of pluronic F127 ensures that the four prepared nano materials have good stability and dispersibility in different physiological media, and provides conditions for biological experiments.
Example 10 extracellular ROS production of Palladium-based porphyrin coordination molecule cage nanoformulations
Since diphenyl isobenzofuran (DPBF) can be oxidatively decomposed by ROS, resulting in a decrease in its ultraviolet absorbance, TMPP-F127 and three Pd having different chelation rates are quantitatively compared extracellular by using DPBF as a probe for detecting ROS 6 -TMPP(Pd x ) ROS production of F127 (x= 1,0.6,0.4). DPBF of the same concentration was added sequentially to TMPP-F127 and three Pd having the same porphyrin concentration 6 -TMPP(Pd x ) In an aqueous solution of F127 (x= 1,0.6,0.4), mixing homogeneously. In the mixed solution, the DPBF concentration was 33. Mu.g/mL, and the porphyrin concentration was 7. Mu.g/mL. Then using 650nm laser (100 mW cm -2 ) Once every 30 seconds, DPBF was evaluated for its ability to generate ROS by monitoring its rate of decrease in uv absorbance peaks in these four materials. As a result, as shown in fig. 9, the decrease rate of the DPBF absorption value was, in order: pd (Pd) 6 -TMPP(Pd 0.4 )-F127>Pd 6 -TMPP(Pd 0.6 )-F127>Pd 6 -TMPP(Pd)-F127>TMPP-F127, with free H 2 Compared to TMPP, ROS production is greatly improved after metal cage formation, and the lower the chelation rate, the higher the metal cage ROS production.
EXAMPLE 11 cytological investigation of Palladium-based porphyrin coordination molecule cage nanoformulations
(1) Cytotoxicity of cells
Cytotoxicity of the material against B16F10 cells was assessed by 3- (4, 5-dimethylthiazol-2' -yl) -2, 5-diphenyltetrazolium ammonium bromide (MTT) analysis. Two 96-well plates were prepared for phototoxicity and dark toxicity experiments of the materials. At 1.0X10 4 Cell/well density seeding of B16F10 cells at 37℃with 5% CO 2 Culturing for 16h under the condition. For phototoxicity experiments, the compositions containing TMPP-F127, pd at various concentrations 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) After 12 hours of incubation of the cells with serum-free DMEM solution of-F127, the cells were irradiated with a laser at a wavelength of 650nm (25 mW cm -1 2 min), and then the cells were further cultured for 12h. For the dark toxicity experiments, cells were directly cultured with the above materials at different concentrations for 24h. MTT solution (200. Mu.L, 0.5mg mL) was then added -1 ) To each well. Cells were cultured at 37 ℃. After 4h, the flap was turned over to discard the MTT solution. The metabolite formazan crystals were dissolved in DMSO (100 μl), shaken on a shaker for 5 minutes, and then absorbance at 570nm was measured with an enzyme-labeled instrument. Cells without any treatment were used as controls.
FIGS. 10 (a) - (b) are TMPP-F127, pd 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 )-F127，Pd 6 -TMPP(Pd 0.4 ) The cytotoxicity of the F127 material to B16F10 cells in the absence of light shows that all materials have smaller toxicity to B16F10 cells in the range of 70 mug/mL, indicating better biocompatibility and safety. The dark toxicity of the four materials is in direct proportion to the concentration of the four materials, and the higher the palladium chelation rate is, the higher the dark toxicity of porphyrin coordination molecule cage is, and Pd is 6 -TMPP (Pd) -F127 has the greatest dark toxicity, IC 50 The value was about 106.4. Mu.g/mL (Table 3). As can be seen from fig. 10 (c) - (d), cytotoxicity of all materials was significantly increased after laser irradiation, indicating that all had better PDT effect.
TABLE 3TMPP-F127 and three Pd 6 -TMPP(Pd x ) -IC of f127 (x= 1,0.6,0.4) 50 Value of
Figure BDA0004063048400000131
As is clear from Table 3, the lower the palladium chelation rate, the greater the phototoxicity of the porphyrin coordination molecule cage, pd 6 -TMPP(Pd 0.4 ) -F127 has the greatest phototoxicity and IC 50 The value was about 4.75. Mu.g/mL, which is only 1/12 of TMPP-F127.
The mechanism of the light/dark toxicity of the materials is explored, mainly from the following aspects:
a. detection of OH in solution
Palladium complex can catalyze Fenton-like reaction to make H 2 O 2 Is converted into OH. As can be seen from the ultraviolet absorption spectrum of FIG. 2, pd 6 TMPP (Pd) -F127 has no ultraviolet absorption in the range of 600-700nm, and Pd is selected for avoiding interference of absorption peaks of the material to experiments 6 -TMPP (Pd) -F127 to investigate the dark toxicity of palladium porphyrin coordination molecule cages. Methylene Blue (MB) is a common colorimetric agent that can be degraded by ROS and measured at 663 nm. Thus, by measuring Pd with different Pd contents 6 -TMPP (Pd) -F127 and having different H 2 O 2 Concentrations (100. Mu.M and 5. Mu.M, respectively, mimic H in tumor microenvironment and normal cellular environment) 2 O 2 Concentration) of the MB-reacted mixture at 663nm wavelength, and Pd (OAc) was selected for its ability to generate ROS 2 The catalysis of palladium ions is further illustrated for comparison.
As is clear from FIG. 18, when the palladium content was 10. Mu.g/mL, the ultraviolet absorption peak of MB in the tumor microenvironment was decreased, while the ultraviolet absorption peak in the normal cell environment was hardly changed, so that it was estimated that Pd was 6 TMPP (Pd) -F127 has no toxic or side effect on normal cells at a palladium concentration of 10. Mu.g/mL, but is capable of generating ROS in tumor cells and thus has a selective killing effect on tumor cells.
b. Intracellular ROS detection in the absence of light
DCFH-DA was used as ROS probe by inversion fluorescenceLight microscopy was used to observe ROS production in B16F10 cells and HEK293 cells after incubation with the material. As can be seen from FIG. 19, no green fluorescence of DCF was detected in both B16F10 and HEK293 cells treated with TMPP-F127 (porphyrin concentration 67. Mu.g/mL), indicating that TMPP-F127 was unable to produce ROS in the cells under dark conditions. Through Pd 6 TMPP (Pd) -F127 (porphyrin concentration 67. Mu.g/mL, palladium content 10. Mu.g/mL) and Pd (OAc) 2 Green fluorescence of DCF was detected in B16F10 cells after treatment (palladium content 10. Mu.g mL), indicating that the palladium complex was able to catalyze the production of ROS in the cells. Notably, under the same conditions, through Pd 6 No green fluorescence was detected in HEK293 cells after TMPP (Pd) -F127 treatment, indicating Pd at a palladium content of 10. Mu.g/mL 6 TMPP (Pd) -F127 is only able to 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 the results of fig. 18.
c、Pd 6 Cytotoxicity experiment of TMPP (Pd) -F127 on B16F10/HEK293
Pd is adopted 6 -TMPP (Pd) -F127 treatment of B16F10 cells and HEK293 cells, cell viability was calculated for 24 h. As a result, as shown in FIG. 20, when Pd 6 At a concentration of TMPP (Pd) -F127 of 10. Mu.g/mL, HEK293 cells survived up to 95% or more, whereas B16F10 cells survived up to about 70%, further quantitatively demonstrating Pd 6 -TMPP (Pd) -F127 has a selective killing effect on cancer cells.
d、Pd 6 Intracellular GSH content after TMPP (Pd) -F127 treatment
TMPP-F127 (porphyrin concentration 67. Mu.g mL) -1 )，Pd 6 -TMPP (Pd) -F127 (porphyrin concentration 67. Mu.g mL) -1 The palladium content was 10. Mu.g mL -1 )，Pd(OAc) 2 (Palladium content was 10. Mu.g mL) -1 ) Incubated with B16F10 cells for 24h. The cell samples were then processed according to GSH and GSSG detection kit instructions and absorbance of the samples was measured with a microplate reader in the 405-414nm range. As a result, as shown in FIG. 21, when B16F10 cells were combined with Pd having a palladium content of 10. Mu.g/mL 6 TMPP (Pd) -F127 (porphyrin concentration 67. Mu.g/mL) and Pd (OAc) 2 After 24 hours incubation, intracellular GSH content was down-regulated to around 40%, whereas TMPP-F127 did not show a significant effect at the same porphyrin concentration (67. Mu.g/mL).
e、Pd 6 -mitochondrial membrane potential of TMPP (Pd) -F127 treated cells
JC-1 is a widely used method for detecting mitochondrial membrane potential Δψ m Exhibits a potential-dependent accumulation in mitochondria. One marker of early apoptosis is a drop in mitochondrial membrane potential. With a decrease in the Mitochondrial Membrane Potential (MMP) of the cells, the JC-1 probe turns from aggregates to monomers and fluorescence also turns from red to green. As can be seen from FIG. 22, pd was passed through the group of PBS and TMPP-F127 6 -TMPP (Pd) -F127 and Pd (OAc) 2 The B16F10 cells showed strong green fluorescence after treatment, indicating a significant decrease in mitochondrial membrane potential.
f、Pd 6 TEM of mitochondrial morphology of cells after treatment with TMPP (Pd) -F127
Both B16F10 and HEK293 cells were seeded at a density of 300 ten thousand cells in culture dishes and incubated for 24h at 37 ℃. Then adding a solution with a palladium content of 10 mug mL -1 Pd of (2) 6 -TMPP (Pd) -F127 in serum-free DMEM solution was incubated for 8h while serum-free DMEM without material was added as a control. The culture broth was removed and the cells were washed three times with PBS. Cells were collected in 1.5mL centrifuge tubes, 2.5% glutaraldehyde was added and fixed overnight at 4 ℃. Pouring out the fixing solution, adding about 1mL of agar, standing for 3-4min, taking out, and cutting into pieces of 1X 3X 1mm 3 The samples were rinsed three times with PBS (0.1M, pH 7.0) for 15min each. The samples were then fixed with 1% osmium acid solution for 1h and rinsed three times with PBS (0.1M, pH 7.0) for 15min each. Next, the samples were dehydrated with ethanol solutions of gradient concentration (30%, 50%,70%,80% in sequence), each concentration was treated for 15min, then transferred to 90% and 95% acetone solutions, respectively, for 15min, and finally treated with pure acetone twice for 20min each time. Then, the samples were treated with the mixed solution of Spurr embedding medium and acetone in volume ratio of 1:1 and 3:1 for 1h and 3h, respectively, and then treated with pure embedding medium at room temperature overnight. The sample was placed in 0.5mL dry-off containing Spurr resin Heating at 70deg.C for 9 hr or more in the tube. The samples were sectioned in a LEICA EM UC7 ultrasonic microtome, stained with uranium acetate and lead citrate for 5-10min, respectively, and observed in a Hitachi H-7650 type TEM.
FIG. 23 is a graph without any treatment (a) and Pd 6 TEM image of the mitochondria in B16F10 cells induced by TMPP (Pd) -F127 (palladium content 10. Mu.g/mL). From TEM images, the mitochondria in B16F10 cells without any treatment are complete in morphology, compact in cristae distribution and Pd-treated 6 B16F10 cells after TMPP (Pd) -F127 treatment showed severe mitochondrial damage, decreased numbers of cristae, as indicated by the red arrow in the figure with altered mitochondrial ultrafine morphology. FIG. 24 is a graph without any treatment (a) and Pd 6 TEM image of mitochondria in HEK293 cells induced by TMPP (Pd) -F127 (b). We can observe that in Pd compared to untreated HEK293 cells 6 The cell mitochondria morphology after TMPP (Pd) -F127 treatment (palladium content 10. Mu.g/mL) is relatively complete, and the structure is hardly destroyed. Further demonstrated is Pd at a palladium content of 10. Mu.g/mL 6 TMPP (Pd) -F127 is capable of selective cell killing of cancer cells, while being non-toxic to normal cells.
(2) Cellular uptake
Preparation of the same porphyrin concentration (60. Mu.g mL) -1 ) TMPP-F127, pd 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) Serum-free DMEM solution of-F127. B16F10 cells were incubated with the solution prepared above for 6 hours at 37 ℃, the culture broth was aspirated and the cells were then washed three times with PBS and fixed with fresh 4.0% formaldehyde for 10 minutes. After washing off formaldehyde with PBS, the cells were incubated with Hoechst 33258 staining solution for 5 minutes, washed 2 times with PBS, and analyzed for uptake by qualitative comparison under an inverted fluorescence microscope. The cells were collected and uptake of each material by the cells was quantitatively detected on a flow cytometer.
The nuclei were stained with Hoechst 33258 staining solution, red fluorescence of porphyrin was observed in the B16F10 cells treated with four materials, and it was confirmed that both of them were successfully taken up into the cytoplasm by the cells by co-localization of blue and red fluorescence in the combined figures.As shown in FIG. 11, pd was caused by complete chelation of heavy metal ions in the center of the porphyrin ring 6 Fluorescence of TMPP (Pd) -F127 was almost completely quenched, consistent with the results detected by the flow cytometer of FIG. 12.
(3) Intracellular ROS detection
The production of ROS in B16F10 cells under light was measured using 2, 7-dichlorofluorescein diacetate (DCFH-DA). The cells were treated to the same porphyrin concentration (60. Mu.g mL) -1 ) TMPP-F127, pd 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) Serum-free DMEM solution of-F127 for 6h. After three washes with PBS, DCFH-DA (10. Mu.M) was added at 37℃and 5% CO 2 Incubate for 30 minutes. The cells were washed three times with PBS and irradiated with a laser at 650nm for 2 minutes (25 mW cm -2 ). Subsequently, the cells were fixed with 4.0% formaldehyde for 10 minutes and incubated with Hoechst 33258 staining solution for 5 minutes. Finally, intracellular ROS production was qualitatively observed using an inverted fluorescence microscope. The cells were collected and analyzed by quantitative comparison on a flow cytometer for intracellular ROS production after incubation of the individual materials with the cells.
As a result, as shown in fig. 13, green fluorescence of DCF was detected in B16F10 cells after incubation with four materials, compared to the control group, indicating that they were all capable of producing ROS in cancer cells. In addition, the ROS production of four nanomaterials in cells was further quantitatively compared by flow cytometry, and the intracellular ROS production was in turn: pd (Pd) 6 -TMPP(Pd 0.4 )-F127>Pd 6 -TMPP(Pd 0.6 )-F127>Pd 6 -TMPP(Pd)-F127>TMPP-F127, i.e. with H 2 The significantly improved ROS production after metal cage formation compared to TMPP ligand, and the lower the sequestration rate, the greater the metal cage ROS production (fig. 14), which is consistent with the results of the ROS production detection of the four nanomaterials in solution of fig. 9.
(4) Double-staining experiment of live cells/dead cells
B16F10 cells and cells with the same porphyrin concentration (30. Mu.g mL) -1 ) Is incubated for 6h at 650nm (25 mW cm -2 ) Irradiating for 4min under laser, then washing the cells three times with PBS, and staining with freshly prepared 1XWorking solution (Calcein-AM 2. Mu.M, PI 4.5. Mu.M) was stained for 15min. The images were taken using an inverted fluorescence microscope.
FIG. 15 is a photograph of stained dead living cells after incubation and laser irradiation of four nanomaterials with the same porphyrin concentration (60 μg/mL), wherein green fluorescence indicates living cells and red fluorescence indicates dead cells. As can be seen, the cells incubated with TMPP-F127 and irradiated with laser showed a very high proportion of living cells, indicating a low phototoxicity compared to three Pd 6 -TMPP(Pd x ) Cells incubated with F127 (x= 1,0.6,0.4) had mostly died after laser irradiation, indicating that they had higher phototoxicity, consistent with the results of the MTT experiment.
(5) Apoptosis study
According to the apoptosis analysis kit, an annexin v-FITC/PI double staining method is adopted to detect apoptosis. Two 6-well plates were prepared for use as dark toxicity and phototoxicity experimental studies, respectively. B16F10 cells were seeded into 6-well plates at a cell density of 50 ten thousand per well and incubated for 8h for phototoxicity experiments with a laser (650 nm,25mW cm -2 ) For 1 minute of irradiation, for dark toxicity experiments, the direct incubation for 8 hours was not irradiated with laser light. Cells incubated with the material were digested with pancreatin solution without EDTA, washed with PBS buffer and centrifuged. Cells were then resuspended in 500. Mu.L Binding Buffer and stained with 5. Mu.L of 5-Lannexin V-FITC and 5. Mu.L of PI in the dark for 15 minutes. Stained cells were analyzed by flow cytometry.
Apoptosis of cells was assessed by using an Annexin V FITC/PI double staining method. FIG. 16 is a graph showing apoptosis profile and ratio of apoptosis at various stages after incubation with material with flow cytometry in the absence of laser (first row) and with laser (second row) irradiation. As can be seen from the graph, the cell viability after incubation with TMPP-F127 was higher in the absence of laser irradiation, and apoptotic cells accounted for only 4.76%, but three Pd 6 -TMPP(Pd x ) -cell viability was reduced after incubation with F127 (x= 1,0.6,0.4), apoptotic cell proportion was increased, pd 6 The highest proportion of apoptosis induced by TMPP (Pd) -F127 was 11.5%. Under 650nm laser irradiation, the cell survival rate is reduced and apoptosis is realized after the cell is incubated with four nano materialsThe proportion of cells is increased, and the lower the chelation rate is, the lower the cell survival rate is after metal cage treatment, the larger the proportion of apoptotic cells is, wherein Pd 6 -TMPP(Pd 0.4 ) The proportion of living cells after F127 treatment was 25.8% and the proportion of apoptotic cells was 34.51%.
(6) Assessment of Palladium content after intracellular internalization
The palladium content after internalization of the cells was assessed using inductively coupled plasma-mass spectrometry (ICP-MS). Mouse melanoma cells (B16F 10, about 200 ten thousand) were suspended in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin (diabody, P/S) and transferred to petri dishes, placed at 37℃with 5% CO 2 Is cultured in a cell incubator. After two days, the supernatant was removed, rinsed once with 0.25% EDTA-containing pancreatin, then added with 1mL pancreatin, and placed in a cell incubator for digestion for 3min, then added with 3mL serum-containing DMEM to neutralize pancreatin, and centrifuged for 3min (1200 rpm). The supernatant was discarded, the cells resuspended in DMEM medium and plated at a density of 300 ten thousand. After 24h, the cells proliferated rapidly in the dishes, reaching 1000 tens of thousands/dish. Three Pd with the same porphyrin concentration (30. Mu.g/mL) were prepared 6 -TMPP(Pd x ) After 6h incubation of F127 (x= 1,0.6,0.4) with cells of the same number, the differential chelation rate Pd of the cells was assessed by ICP-MS to measure the content of intracellular palladium ions before addition and after incubation 6 -TMPP(Pd x ) -ingestion of F127 (x= 1,0.6,0.4). The test results are summarized in table 4:
TABLE 4Pd 6 -TMPP(Pd x ) Intracellular palladium content and uptake after treatment with F127 (x= 1,0.6,0.4)
Material Palladium content (ppm) in the material Intracellular palladium content (ppm) Uptake (%)
Pd 6 -TMPP(Pd)-F127 5.4 0.8 15
Pd 6 -TMPP(Pd 0.6 )-F127 2.0 0.44 22
Pd 6 -TMPP(Pd 0.4 )-F127 1.6 0.42 26
As can be seen from Table 4, although the cells are specific to Pd 6 The uptake of the TMPP (Pd) -F127 material was the lowest, but due to the highest palladium content in its initial material, a higher accumulation of palladium concentration in the cell was caused compared to other materials, which also explains the Pd compared to other nanomaterials in the dark toxicity test of MTT 6 -TMPP (Pd) -F127 was the most darkly toxic cause.
(6) Hemolysis experiment
Hemolysis assays were performed on the material at a concentration ranging from 0.05 to 0.5mg/mL to evaluate four nanomaterials (TMPP-F127, pd 6 -TMPP(Pd)-F127，Pd 6 -TMPP(Pd 0.6 ) F127 and Pd 6 -TMPP(Pd 0.4 ) -F127). The specific operation is as follows: the mice were subjected to an eyeball removal experiment to obtain blood, the blood was collected in a 1.5mL centrifuge tube pre-placed with heparin sodium, transferred into a 10mL centrifuge tube and added with PBS liquid to 8mLAbout, 3200rpm is centrifuged for 6min. After washing to the supernatant to be colorless, red blood cells are diluted by PBS according to the required amount, and the mixture is uniformly mixed to prepare the cell suspension. 500. Mu.L of the solution was sequentially added to 500. Mu.L of four nanomaterial PBS solutions at concentrations of 0.05, 0.1, 0.2, 0.4, and 0.5mg/mL, respectively, while PBS and water were set as negative and positive controls, respectively. Incubation was carried out at 37℃for 4h, removed, centrifuged at 3200rpm for 6min, and absorbance at 540nm was measured for porphyrin and supernatant at the same concentration using a microplate reader to subtract the effect of porphyrin absorbance. The calculation formula of the hemolysis rate of each group of experimental three complex holes is as follows:
Hemolysis ratio (%) = [ (a) tes -A neg )/(A pos -A neg )]×100％
Fig. 17 shows the results of the hemolysis experiments of the four nanomaterials, and shows that after the four nanomaterials are incubated with erythrocytes for 4 hours at 37 ℃, the hemolysis rate of the four nanomaterials is less than 2% in the concentration of 0.5mg/mL, which also shows that the nanomaterials have better biocompatibility and are suitable for further biological application.
EXAMPLE 12Pd 6 -TMPP(Pd 0.4 ) In vivo anti-tumor experiment of F127
(1) Establishment of animal tumor model
Under aseptic conditions, a cell suspension of B16F10 (100. Mu.L, 2X 10) was subcutaneously injected in the right region of BALB/c nude mice 6 And (b) until the average tumor volume reaches 90mm 3 At that time, a grouping experiment was performed, and this day was set as day 0.
(2) Tumor growth inhibition experiments
Nude mice with similar tumor sizes were selected and randomly divided into three groups: PBS, pd 6 -TMPP(Pd 0.4 )-F127、Pd 6 -TMPP(Pd 0.4 ) F127+pdt, 5 per group. Mice received PBS and Pd by tail vein injection 6 -TMPP(Pd 0.4 )-F127(0.5.00mg Pd/kg)、Pd 6 -TMPP(Pd 0.4 ) F127+PDT (0.5.00 mg Pd/kg), 3 times per week. For Pd 6 -TMPP(Pd 0.4 ) F127+PDT group, laser treatment for 10 min (650 nm,100mW cm) -2 5 minutes of irradiation every 10 seconds), co-administration and laserTreatment is carried out for 17 days. During the treatment period, the mice were weighed daily and the tumor size was measured. The calculation formula of the tumor volume is: (Long) X (Wide) 2 /2。
As shown in fig. 25 (b), the body weight of each group of mice did not significantly change before and after treatment, indicating that the intervention treatment did not affect the normal physiological metabolic activity of the mice. From the tumor growth inhibition curve of FIG. 25 (a) and the tumor weight map of FIG. 25 (d), it can be seen that a single Pd was received as compared to the PBS-injected control group 6 -TMPP(Pd 0.4 ) The tumor growth of the mice subjected to the-F127 chemotherapy is inhibited to a certain extent, and the uptake of the medicaments by cancer cells is increased due to the EPR effect of the nano particles, so that Pd is caused 6 -TMPP(Pd 0.4 ) The inhibition effect of F127 nanoparticles on tumors was better than PBS, and the survival rate of mice was higher (FIG. 25 (c)). Further, as can be seen from FIG. 25, pd 6 -TMPP(Pd 0.4 ) F127 after treatment in combination with PDT can already completely inhibit tumor growth and the survival rate of mice during treatment is 100%. However, due to the limited penetration of the laser, the penetrating cancer cells are far from the epidermis and complete removal of the primary tumor is hardly achieved with only a single photodynamic therapy.
Pd in FIG. 25 6 -TMPP(Pd 0.4 ) F127+L represents Pd as described above 6 -TMPP(Pd 0.4 )-F127。
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (10)

1. A porphyrin-based coordination molecule cage, which is characterized in that the porphyrin-based coordination molecule cage M 6 [L(M’ x )] 3 Has the following structural general formula:
Figure FDA0004063048390000011
wherein 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;
r is-CH 3 or-CH 2 OH；
L(M’ x ) Wherein x has a value of 0<x is less than or equal to 1, and when x is less than 1, the porphyrin ring in the porphyrin-based coordination molecule cage is chelated with a metal M' part.
2. The porphyrin-based coordination molecule cage of claim 1, wherein the porphyrin-based coordination molecule cage is Pd 6 [TMPP(Pd x )] 3 (0<x≤1)、Pd 6 [TMPP(Fe)] 3 、Pd 6 [THPP(Pd)] 3 Or Pd (or) 6 [THPP(Fe)] 3 。
3. The porphyrin-based coordination molecule cage of claim 2, wherein the Pd 6 [TMPP(Pd x )] 3 (0<x.ltoreq.1) comprises Pd 6 [TMPP(Pd 0.4 )] 3 、Pd 6 [TMPP(Pd 0.6 )] 3 、Pd 6 [TMPP(Pd)] 3 ；
The Pd is 6 [TMPP(Pd 0.4 )] 3 Has the molecular formula of C 132 H 96 Cl 12 N 24 Pd 7 Belongs to an orthorhombic crystal system, and the space group is Cmc2 1 The unit cell parameters are:
Figure FDA0004063048390000012
α＝90°，β＝90°，γ＝90°，
Figure FDA0004063048390000013
Figure FDA0004063048390000014
the Pd is 6 [TMPP(Pd 0.6 )] 3 Has the molecular formula of C 132 H 96 Cl 12 N 24 Pd 8.40 Belonging to monoclinic system, the space group is P2 1 The unit cell parameters are:
Figure FDA0004063048390000015
α＝90°，β＝106.113(3)°，γ＝90°，/>
Figure FDA0004063048390000016
the Pd is 6 [THPP(Pd)] 3 Has the molecular formula of C 132 H 96 Cl 12 N 24 Pd 9 Belonging to monoclinic system, the space group is P2 1 And/c, the unit cell parameters are as follows:
Figure FDA0004063048390000017
α＝90°，β＝98.378(2)°，γ＝90°，/>
Figure FDA0004063048390000018
4. the porphyrin-based coordination molecule cage of claim 2, wherein the Pd 6 [TMPP(Fe)] 3 Has the molecular formula of C 264 H 204 Cl 30 Fe 6 N 48 O 6 Pd 12 Belongs to a triclinic system, the space group is P-1, and the unit cell parameters are as follows:
Figure FDA0004063048390000019
Figure FDA00040630483900000110
α＝63.504(3)°，β＝88.850(3)°，γ＝85.741(4)°，
Figure FDA00040630483900000111
The Pd is 6 [THPP(Pd)] 3 Has the molecular formula of C 132 H 96 Cl 12 N 24 O 12 Pd 9 Belongs to an orthorhombic crystal system, the space group is Pbca, and the unit cell parameters are as follows:
Figure FDA0004063048390000021
α＝90°，β＝90°，γ＝90°，/>
Figure FDA0004063048390000022
Figure FDA0004063048390000023
the Pd is 6 [THPP(Fe)] 3 Has the molecular formula of C 132 H 108 Cl 12 Fe 3 N 24 O 18 Pd 6 Belonging to a trigonal system, the space group is R-3c: H, and the unit cell parameters are:
Figure FDA0004063048390000024
α＝90°，β＝90°，γ＝120°，
Figure FDA0004063048390000025
5. a process for preparing a porphyrin-based ligand cage according to any one of claims 1 to 4, wherein ligand H is selected from the group consisting of 2 Dissolving L, metal M salt and metal M' salt in a solvent, adding an acid regulator into the obtained mixed solution, then separating out solid after temperature rising and rising treatment, and washing the solid to obtain the porphyrin-based coordination molecular cage; the ligand H 2 L is selected from one of the compounds shown in the following structures:
Figure FDA0004063048390000026
6. the method of claim 5, wherein 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, wherein the temperature raising and lowering treatment specifically comprises: heating to 110-130 ℃, preserving heat for 24-60 h, and then reducing the temperature to 20-30 ℃ at a cooling rate of 4-5 ℃/h.
8. Use of a porphyrin-based ligand molecular cage according to any one of claims 1-4 in the preparation of an antitumor drug.
9. A porphyrin-based coordination molecule cage nano preparation, which is characterized in that the porphyrin-based coordination molecule cage and amphiphilic copolymer of claims 1-4 are dispersed in a solvent, are added into water dropwise after ultrasonic treatment to obtain micelle solution, and are dialyzed to obtain the porphyrin-based coordination molecule cage nano preparation.
10. Use of the porphyrin-based ligand molecular cage nano-preparation of claim 9 in preparing antitumor drugs.
CN202310068656.6A 2023-02-06 2023-02-06 Porphyrin-based coordination molecular cage and preparation method and application thereof Pending CN116178448A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117659432A (en) * 2024-02-01 2024-03-08 四川大学 Porous nickel porphyrin-based hydrogen bond organic framework material and preparation method and application thereof
CN117659432B (en) * 2024-02-01 2024-04-02 四川大学 Porous nickel porphyrin-based hydrogen bond organic framework material and preparation method and application thereof

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