CN111281858B - Application of carbonyl iron sulfur cluster compound nano particles in preparation of medicine - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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- A61K9/513—Organic macromolecular compounds; Dendrimers
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61P35/04—Antineoplastic agents specific for metastasis
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Abstract
本发明属于纳米医学技术领域，公开了羰基铁硫簇合物纳米粒子在药物制备中的应用，具体是羰基铁硫簇合物纳米粒子(C8H4Fe2O6S2‑PSMA‑PEG NPs)在制备和筛选治疗/预防黑色素瘤肺转移药物中的应用，该纳米粒子具有以羰基铁硫簇合物(C8H4Fe2O6S2)为核、以双亲性嵌段聚合物(PSMA‑PEG)为包覆层的球型结构，利用其在肿瘤内特殊微环境下的化学动力学和CO气体治疗协同作用实现精准治疗黑色素瘤肺转移，且治疗效果显著。
The invention belongs to the technical field of nano-medicine, and discloses the application of carbonyl iron sulfide cluster nanoparticles in the preparation of medicines, in particular to carbonyl iron sulfide cluster nanoparticles (C 8 H 4 Fe 2 O 6 S 2 -PSMA-PEG) Application of NPs ) in the preparation and screening of drugs for the treatment/ prevention of melanoma lung metastasis The compound (PSMA-PEG) is a spherical structure with a coating layer, and the synergistic effect of its chemical kinetics and CO gas therapy in the special microenvironment of the tumor can be used to achieve precise treatment of melanoma lung metastases, and the therapeutic effect is remarkable.
Description
Technical Field
The invention belongs to the technical field of nano medicine, and particularly relates to a carbon dioxide (CO) release tabletRadiochemical kinetic carbonyl iron sulphur cluster compound nano particle (C)8H4Fe2O6S2PSMA-PEG NPs), a preparation method thereof and application thereof in preparing and screening medicaments for treating/preventing melanoma lung metastasis.
Background
Currently, 100 different types of human cancers are known, malignant melanoma, the most aggressive of the malignant tumors, generally does not present local recurrence, but may present local and distant metastases, especially lung metastases, while current treatment methods chemotherapy and radiotherapy are not ideal for such metastases and have significant side effects, and many patients with malignant melanoma die of tumor metastases. Importantly, the treatment and prevention of melanoma metastasis remain major challenges, and with the development of nanotechnology, there is an urgent need to research nanomaterials for cancer treatment and develop effective nanomedicines for the treatment and prevention of its metastasis.
Iron is a metal element which is abundant in the human body, plays an indispensable role in various biological processes such as oxygen supply and storage of hemoglobin, and ferrose for cell breathing, and inorganic nanomaterials have good therapeutic and imaging functions and have been widely studied, many of which are in clinical trial, and so far only iron-based nanoparticles have been approved for inorganic nanopharmaceuticals by the U.S. Food and Drug Administration (FDA). It was found that Fe2+Can utilize hydrogen peroxide (H) over-expressed in tumor cells2O2) Triggering Fenton reaction to generate hydroxyl free radical (. OH) with cytotoxicity, and achieving the purpose of tumor treatment by using chemokinetic therapy. However, the therapeutic effect of pure chemokinetic therapy on malignant tumors is still to be improved.
CO produced at low concentrations of heme-oxygenase in living organisms has cytoprotective activity, on the contrary, at very high concentrations, it is extremely toxic due to its accumulation in tissues. For example, due to the enrichment of CO molecules released in tumor tissue, a cytotoxic effect can be achieved at locally high concentrations of CO, and it can kill cancer cells, but not surrounding cells. Carbon monoxide gas is useful in therapeutic applications such as systemic pulmonary hypertension, heart, kidney and small intestine transplant rejection, as well as intestinal disease, hemorrhagic shock and lung injury. The non-specific release of CO in normal tissues reduces CO concentration in tumor cells, leading to metabolic and redox adaptations, eventually leading to drug resistance. How to trigger the release of CO to the target tissue, how to safely and effectively release CO gas in a controlled and targeted manner for therapeutic purposes remains a great challenge. Several years ago, Motterlini demonstrated that transition metal-carbonyl complexes can function as CO-releasing molecules (CORMs). This idea has been expanded since 2003 and the development of metal carbonyl complexes that can release CO with a high degree of control under carbonylation conditions has become an important discipline. The basic goal of carbon monoxide delivery systems is to quantitatively deliver carbon monoxide. Therefore, recent research in this area has focused on two main goals: (1) how to trigger the release of CO to the target tissue, (2) how to control the amount of release.
Some CORMs release CO via simple hydrolysis by dissolution in aqueous buffer, while other methods of triggering CO from metal complexes have also been developed. For example, some enzyme-triggered CORM systems are based on and some redox CO producing molecules have been reported. Currently, one of the most promising methods for CO initiation from metal carbonyl complexes is by photoexcitation. The light-controlled release of CO gas is a commonly used method at present, and the effect of the light-controlled release of CO on the metastasis of the metastasis or the primary tumor in an organ is greatly restricted by the light penetration depth of the metastasis or the primary tumor in the organ. Therefore, the development of a specific and efficient CO-releasing nano-drug is still sought.
Disclosure of Invention
The main purpose of the invention is to provide carbonyl iron sulfur cluster compound nanoparticles (C) with CO release/chemical kinetics8H4Fe2O6S2-PSMA-PEG NPs) in the preparation and screening of drugs for treating/preventing melanoma lung metastasis, wherein the carbonyl iron-sulfur cluster compound nanoparticles have a carbonyl iron-sulfur cluster compound (C) represented by the following structural formula (I-a)8H4Fe2O6S2) A spherical structure which is used as a core and takes an amphiphilic block polymer (PSMA-PEG) shown as a structural formula (I-b) as a coating layer:
in a preferred technical scheme, C in the carbonyl iron sulfur cluster compound nano particles8H4Fe2O6S2And PSMA-PEG in a weight ratio of 1: 2.399, respectively; the carbonyl iron-sulfur cluster compound nanoparticles have the hydration kinetic diameter of 104nm, the surface potential of-37.9 mV, and the particle size of 58.28-61.62 nm in a dry state.
In the preferred technical solution, the carbonyl iron sulfide cluster nanoparticles can be obtained by a preparation method comprising the following steps:
(1) 160mg of polystyrene-co-polymaleic anhydride copolymer (PSMA, M1600 Da) and 110mg of aminopolyethylene glycol (H) were weighed out2N-PEG, M is 550Da) is added into a single-neck flask, 40mL of Tetrahydrofuran (THF) is added as a solvent, heating reflux reaction is carried out at 70 ℃ for 24h, light yellow PSMA-PEG solution is obtained, the solution with PSMA mass concentration of 4mg/mL is collected in a brown reagent bottle and stored at-20 ℃, and carbonyl iron sulfur cluster compound C is obtained8H4Fe2O6S2；
(2) Weighing 8mgC8H4Fe2O6S2Adding 40mL of LTHF as a solvent, stirring at normal temperature for 1h to completely dissolve the solution, adding 4mL of the preserved PSMA-PEG solution, stirring at room temperature to mix completely, rapidly injecting 80mL of pure water under stirring, stirring for 1h, dialyzing the stirred product for 48h to remove THF and C which is not successfully wrapped8H4Fe2O6S2And PSMA-PEG to obtain C8H4Fe2O6S2-PSMA-PEG NPs。
The invention also provides H for treating/preventing melanoma lung metastasis based on chemical kinetics and CO gas synergy2O2Responsive nanomaterial drug comprising any of the aboveThe carbonyl iron sulfur cluster compound nano particle takes the carbonyl iron sulfur cluster compound nano particle as an active ingredient. In a preferred embodiment, said H2O2Single use dose of said carbonyl iron sulfur cluster compound (C) in response to a nanomaterial drug8H4Fe2O6S2) The concentration of (A) is 0.01-10 mg/kg, more preferably 5.21mg/kg, the administration mode is injection, and the injection is performed once every three days.
Compared with the prior art, the invention realizes accurate treatment of melanoma lung metastasis by utilizing the chemical kinetics and the CO gas treatment synergistic effect of carbonyl iron sulfur cluster compound nanoparticles in a special microenvironment in a tumor, and has the main innovation that:
1) the carbonyl iron-sulfur cluster compound is wrapped in amphiphilic block high-molecular polystyrene-co-polymaleic acid-co-polyethylene glycol (PSMA-PEG), so that the carbonyl iron-sulfur cluster compound can be accumulated in the lung, and the accurate treatment effect is achieved.
2) By means of H2O2The Fenton reaction is promoted while CO release is triggered, so that the chemical kinetic treatment and the CO treatment are mutually cooperated, tumor cells but not normal cells are selectively killed, the CO can be effectively accumulated in the tumor due to the specific release of the CO at the tumor part, the effect of CO gas treatment is improved, CO poisoning is effectively avoided, and the toxic and side effects to organisms are small.
Drawings
FIG. 1 is C8H4Fe2O6S2The cluster compound (a) and the amphiphilic block polymer (b) have the structural formulas.
FIG. 2 is C at various concentrations8H4Fe2O6S2The ultraviolet-visible absorption spectrum (left) of the cluster compound in THF was 0.00mM, 0.01mM, 0.02mM, 0.04mM, 0.08mM, 0.12mM, 0.16mM, 0.20mM in this order from bottom to top, and a linear relationship of concentration-absorbance based on this fit (right).
FIG. 3 is C8H4Fe2O6S2Transmission electron microscopy images of PSMA-PEG NPs (top) and their particle size statistics (bottom).
FIG. 4 is C8H4Fe2O6S2Hydration kinetic diameter (left) and Zeta potential characterization (right) of PSMA-PEG NPs.
FIG. 5 is C8H4Fe2O6S2Particle size stability of PSMA-PEG NPs in Water and physiological saline.
FIG. 6 is C8H4Fe2O6S2Chemical kinetics curves of reaction of PSMA-PEG NPs with hydrogen peroxide at different pH; wherein pH 5.2 (upper) and pH 7.4 (lower).
FIG. 7 is C8H4Fe2O6S2The UV-VIS absorption spectrum of the reaction of PSMA-PEG NPs with hydrogen peroxide as a function of time is the absorbance of the mixed solution at 0, 10, 20, 30, 40, 50, 60, 80, 90, 120, 150, 180, 210 and 840min from top to bottom.
FIG. 8 is a graph evaluating the ability of different materials to generate OH; wherein, the materials are as follows from bottom to top in sequence:
(1)MNPs：C8H4Fe2O6S2-PSMA-PEG NPs (50 μ M, calculated as Fe concentration);
(2)H2O2(0.1mM)；
(3)TMB(0.3mM)；
(4)MNPs(C8H4Fe2O6S2PSMA-PEG NPs, 50. mu.M, in Fe concentration) + H2O2(0.1mM)；
(5)MNPs(C8H4Fe2O6S2-PSMA-PEG NPs, 50 μ M, calculated as Fe concentration) + TMB (0.3 mM);
(6)H2O2(0.1mM)+TMB(0.3mM)；
(7)MNPs(C8H4Fe2O6S2PSMA-PEG NPs, 50. mu.M, in Fe concentration) + TMB (0.3mM) + H2O2(0.1mM)。
FIG. 9 is C8H4Fe2O6S2The influence of the concentration of PSMA-PEG NPs on the reaction rate of the reactionAnd (4) transforming.
FIG. 10 is a graph showing the effect of hydrogen peroxide concentration on the reaction rate of the reaction.
FIG. 11 is C8H4Fe2O6S2Electron spin resonance map of PSMA-PEG NPs in response to hydrogen peroxide. From bottom to top are: MNPs: c8H4Fe2O6S2PSMA-PEG NPs (50. mu.M, calculated as Fe concentration), H2O2(0.1mM)、MNPs+H2O2。
FIG. 12 is C8H4Fe2O6S2Profile of CO generation in response to PSMA-PEG NPs with hydrogen peroxide.
FIG. 13 is C8H4Fe2O6S2-cytotoxicity of PSMA-PEG NPs on different cells (calculated as cluster concentration); wherein the left picture is C8H4Fe2O6S2Cytotoxicity of PSMA-PEG NPs on mouse melanoma (B16-F10) cells, right panel C8H4Fe2O6S2-cytotoxicity of PSMA-PEG NPs on Human Umbilical Vein Endothelial Cells (HUVEC).
FIG. 14 is C8H4Fe2O6S2-detecting intracellular OH levels after incubation of cells with PSMA-PEG NPs using DCFH-DA as a fluorescent indicator; the left is the flow cytometry detection result, and the right is the laser confocal picture.
FIG. 15 is C8H4Fe2O6S2Laser confocal pictures of intracellular CO levels were detected after incubation of cells with PSMA-PEG NPs using COP-1 as a fluorescent indicator.
FIG. 16 is the body weight change of mice in vivo treatment experiment.
FIG. 17 is a photograph of the lungs of a mouse in a live treatment experiment; wherein, Saline represents a Control group, and MNPs represents a treatment group.
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
The following examplesThe carbonyl iron sulfur cluster compound nano particles adopted in the method are carbonyl iron sulfur cluster compound (C) with the structural formula shown as the following (I-a)8H4Fe2O6S2) A spherical structure which is used as a core and takes an amphiphilic block polymer (PSMA-PEG) shown as a structural formula (I-b) as a coating layer, and the weight ratio of the two is 1: 2.399, as shown in figure 1, the cluster compound has a weight average molecular weight (Mw) of 371.93 and contains two Fe and six carbonyl groups, and the structural formula shows that the carbonyl iron-sulfur cluster compound nano-particle has the potential of chemical kinetic therapy and gas therapy. The nano particles are obtained by the following preparation method, and the preparation method comprises the following specific steps:
first, preparation of PSMA-PEG: 160mg of polystyrene-co-polymaleic anhydride copolymer (PSMA, M1600 Da) and 110mg of aminopolyethylene glycol (H) were weighed out2N-PEG, M is 550Da) is added into a single-neck flask, 40mL of Tetrahydrofuran (THF) is added as a solvent, heating reflux reaction is carried out at 70 ℃ for 24h, light yellow PSMA-PEG solution is obtained, the solution with PSMA mass concentration of 4mg/mL is collected in a brown reagent bottle and is stored at-20 ℃, and C is obtained8H4Fe2O6S2；
Second step, preparation C8H4Fe2O6S2-PSMA-PEG NPs: weighing 8mgC8H4Fe2O6S2Adding 40mL of LTHF as a solvent, stirring at normal temperature for 1h to completely dissolve the solution, adding 4mL of the above preserved PSMA-PEG solution, stirring at room temperature to fully mix the two substances, rapidly injecting 80mL of pure water under stirring, continuously stirring for 1h, dialyzing the stirred product for 48h to remove THF and C which is not successfully wrapped8H4Fe2O6S2And PSMA-PEG to obtain C8H4Fe2O6S2-PSMA-PEG NPs。
The above-obtained C was subjected to the following test or characterization method8H4Fe2O6S2PSMA-PEG NPs were characterized in order to further define the physicochemical properties of the carbonyl iron-sulfur cluster compound nanoparticles.
(1)C8H4Fe2O6S2Standard curve in THF
THF was used as a solvent to prepare 0.2mM (Fe concentration) of C8H4Fe2O6S2The solution was a mother liquor, which was diluted to a concentration of 0.2mM, 0.16mM, 0.12mM, 0.08mM, 0.04mM, 0.02mM, and 0.01mM, respectively, and tested for UV-visible absorption curves.
(2)C8H4Fe2O6S2Compositional analysis of-PSMA-PEG NPs
Get C8H4Fe2O6S22mL of PSMA-PEG NPs solution is respectively placed in weighed uncovered boxes formed by folding aluminum foil paper, then the boxes are placed in a dryer filled with allochroic silica gel for drying for 3 days, and the weight of total solid solution in the nanoparticle solution is obtained after the solution is completely dried and weighed again. Then using a pipette to pipette 100 mu LC8H4Fe2O6S2-PSMA-PEG NPs solution, wherein moisture was lyophilized by freeze-drying, the obtained powder was dissolved in THF, quantified in a volumetric flask to 5mL, 3mL of the solution was taken and tested for its uv-visible absorption curve, and C was calculated from the concentration of the cluster compound in THF and the absorbance, and a linear equation of y 6.28+0.0121x was fitted with the absorbance at 324nm as the y-axis and the concentration as the x-axis8H4Fe2O6S2The concentration of Fe in PSMA-PEG NPs, as shown in FIG. 2.
Calculating cluster compound C by Fe element8H4Fe2O6S2(371.93Da) the weight of the amphiphilic polymer obtained by subtracting the weight of the polyvalent cluster compound from the weight of the total solid solution, C8H4Fe2O6S2The components of the-PSMA-PEG NPs are shown in Table 1. As can be seen from Table 1, C synthesized by the present invention8H4Fe2O6S2The content of iron element in the-PSMA-PEG NPs is 8.82%, the content of carbonyl iron-sulfur cluster compound is 29.30%, and the content of amphiphilic block polymer is 70.70%, which shows that after being wrapped by the amphiphilic block polymer, the nano materialHas moderate content of effective components.
TABLE 1
| Composition (I) | Content (wt.) |
| Iron element | 8.82％ |
| Carbonyl iron sulfur cluster compound | 29.30％ |
| Amphiphilic block polymers | 70.70％ |
(3)C8H4Fe2O6S2Morphological characterization of-PSMA-PEG NPs
As can be seen by transmission electron microscope, C8H4Fe2O6S2The PSMA-PEG NPs have good complete spherical morphology and good uniformity, and ensure the uniformity of the effect of the material during application, as shown in (upper) of FIG. 3; c8H4Fe2O6S2The particle size of the PSMA-PEG NPs in a dry state is 58.28-61.62 nm, as shown in figure 3 (lower).
(4)C8H4Fe2O6S2Stability test of-PSMA-PEG NPs
Firstly, dynamic light scattering test C is adopted8H4Fe2O6S2Hydration kinetic diameter of PSMA-PEG NPs, and taking the concentrated C8H4Fe2O6S2-PSMA-PEG NPs fractionThe diluted nanoparticles were tested for their hydration kinetic diameter to assess their stability over time, using triplicate samples of water and saline. Dynamic light Scattering test C, as shown in FIG. 4 (left)8H4Fe2O6S2The hydration kinetic diameter of the PSMA-PEG NPs is 104nm, the Number value is 65nm, the particle size is close to that obtained by TEM statistics, and the smaller nanometer size is C8H4Fe2O6S2The application of the PSMA-PEG NPs in the organisms lays a good foundation; as shown in FIG. 4 (right), Zeta potential represents C8H4Fe2O6S2The surface potential of the PSMA-PEG NPs is-37.9 mV, because the PSMA surface has more carboxylate ions, and the surface charge repulsion among the nanoparticles is stronger, so that the nanoparticles have good stability. As shown in FIG. 5, C8H4Fe2O6S2As shown by the change of the hydration kinetic diameter of the PSMA-PEG NPs in secondary water and physiological saline along with time, the particle size of the nano particles is not changed basically in two weeks, which indicates that the nano particles have better stability in the secondary water and the physiological saline.
The invention C is further illustrated by the following specific examples8H4Fe2O6S2Application of PSMA-PEG NPs in preparation and screening of drugs for treating/preventing melanoma lung metastasis.
Example 1
8 4 2 6 2Chemical kinetics experiment of response of CHFeOS-PSMA-PEG NPs and hydrogen peroxide
(1) Effect of different pH values: acetic acid-sodium acetate (0.1M, pH 7.4, pH 5.2) and C were added to a buffer solution of an acetic acid-sodium acetate system, 3',5,5' -Tetramethylbenzidine (TMB) as an indicator for detecting hydroxyl radicals (· OH)8H4Fe2O6S2PSMA-PEG NPs (50. mu.M, calculated as Fe concentration), TMB solution (0.3mM), H2O2A solution (0.1mM) having a total volume of 3mL was mixedAfter mixing, the mixture was incubated in a water bath at 37 ℃ for 50min, and the absorbance of the mixed solution was measured as shown in FIG. 6.
The tumor microenvironment is slightly acidic (pH 5.2) and the normal tissue is neutral (pH 7.4). As can be seen from fig. 6, C is observed at pH 5.28H4Fe2O6S2the-PSMA-PEG NPs can react with hydrogen peroxide to form OH, but cannot form OH at pH 7.4, indicating that C of the present invention8H4Fe2O6S2The PSMA-PEG NPs can generate OH under the stimulation of a tumor microenvironment, so that the tumor cells are damaged, and the reaction can not occur in normal cells, so that the aim of reducing the toxic and side effects of the nano material on the normal tissues is fulfilled.
(2) Time dependence: acetic acid-sodium acetate (0.1M, pH 5.2), C were added in sequence8H4Fe2O6S2PSMA-PEG NPs (0.5mM, calculated as Fe concentration), H2O2A solution (1mM) was prepared in a total volume of 3mL, and after mixing the solutions, the mixture was incubated in a 37 ℃ water bath to measure the absorbance of the mixed solution at 0 th, 10 th, 20 th, 30 th, 40 th, 50 th, 60 th, 80 th, 90 th, 120 th, 150 th, 180 th, 210 th, and 840 th min, respectively, as shown in FIG. 7. C8H4Fe2O6S2PSMA-PEG NPs do react chemically, not catalytically, with hydrogen peroxide. The cluster compound is decomposed during the reaction process, the content of the cluster compound is gradually reduced along with the prolonging of the time, and C is obtained at 14h8H4Fe2O6S2The cluster compound in the PSMA-PEG NPs is decomposed by 78%, which shows that Fe (I) in the nano material in the invention can be oxidized by hydrogen peroxide in the tumor microenvironment to Fe (II) with higher valence and higher reactivity.
(3) Evaluation of the ability of different materials to generate OH
The experimental groups were as follows:
1)MNPs：C8H4Fe2O6S2-PSMA-PEG NPs (50 μ M, calculated as Fe concentration);
2)H2O2(0.1mM)；
3)TMB(0.3mM)；
4)MNPs(C8H4Fe2O6S2PSMA-PEG NPs, 50. mu.M, in Fe concentration) + H2O2(0.1mM)；
5)MNPs(C8H4Fe2O6S2-PSMA-PEG NPs, 50 μ M, calculated as Fe concentration) + TMB (0.3 mM);
6)H2O2(0.1mM)+TMB(0.3mM)；
7)MNPs(C8H4Fe2O6S2PSMA-PEG NPs, 50. mu.M, in Fe concentration) + TMB (0.3mM) + H2O2(0.1mM)。
The solutions of the above groups were added to acetic acid-sodium acetate (0.1M, pH 5.2) in sequence, keeping the total volume of the solutions at 3mL, after mixing them well, and after incubating in a water bath at 37 ℃ for 50min, the absorbance of the mixed solution was measured, and as shown in fig. 8, when hydrogen peroxide and TMB were present together, a small amount of OH was generated, while C was added8H4Fe2O6S2After the PSMA-PEG NPs, the reaction speed is greatly increased, because the addition of the nano material can react with more hydrogen peroxide, and the reaction speed is higher, thereby providing a good foundation for subsequent treatment experiments.
(4) Effect of different nanoparticle concentrations on the reaction Rate of the reaction
To acetic acid-sodium acetate (0.1M, pH 5.2) was added C in sequence separately8H4Fe2O6S2PSMA-PEG NPs (20, 40, 80. mu.M, calculated as Fe concentration) and H2O2A solution (0.1mM) having a total volume of 3mL was mixed, incubated in a 37 ℃ water bath, and the absorbance of the mixed solution was measured every 2min for 60min, as shown in FIG. 9. When the concentration of the fixed hydrogen peroxide is 0.1mM, the concentration of the nano material is changed, and the reaction rate is increased along with the increase of the concentration of the nano material, which shows that the generation of OH is dependent on the concentration of the nano material, and the concentration of the nano material is concentratedThe larger the degree, the larger the amount of. OH produced.
(5) Effect of different Hydrogen peroxide concentrations on the reaction Rate of the reaction
To acetic acid-sodium acetate (0.1M, pH 5.2) was added C in sequence separately8H4Fe2O6S2PSMA-PEG NPs (50. mu.M, calculated as Fe concentration) and H2O2Solutions (0.05, 0.1, 0.2mM) in a total volume of 3mL were mixed, incubated in a 37 ℃ water bath, and the absorbance of the mixed solution was measured every 2min for 60min, with the results shown in FIG. 10. When the concentration of Fe in the fixed nano material is 50 mu M and the concentration of hydrogen peroxide is changed, the reaction rate is sequentially increased along with the increase of the concentration of the hydrogen peroxide, which shows that the generation amount of OH is more and more along with the increase of the concentration of the hydrogen peroxide of a reaction substrate, and the nano material has good chemical kinetic treatment effect in a solution level.
(6) Electron spin resonance (EPR) detection of hydroxyl radicals
The capture agent was 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO, 100mM) and H was added during the test2O2(0.1mM) and C8H4Fe2O6S2PSMA-PEG NPs (50 μ M, calculated as Fe concentration), the test was performed in acetic acid-sodium acetate (0.1M, pH 5.2). The experiment was divided into three groups: 1) h2O2；2)C8H4Fe2O6S2-PSMA-PEG NPs(MNPs)；3)MNPs+H2O2The reaction time was 10min, as shown in FIG. 11. Nanomaterial + H in the figure2O2The occurrence of a quadruple peak with a peak area ratio of 1:2:2:1 is a characteristic peak generated after the DMPO captures the hydroxyl radical, and the simple hydrogen peroxide also has a quadruple peak because the hydrogen peroxide can generate a small amount of hydroxyl radical and the simple nanoparticles have no quadruple peak, which indicates that the nano material can generate the hydroxyl radical through the reaction with the hydrogen peroxide, and the nanoparticles can not generate the hydroxyl radical, thus indicating that the nano material can generate OH through chemical kinetics.
(7) Validation of CO production in response to Hydrogen peroxide
Phosphate buffer (0.1M, pH 7.4), myoglobin (Mb) (108 μ M), sodium dithionite (Na) were added in this order2S2O4)、C8H4Fe2O6S2PSMA-PEG NPs (50. mu.M, calculated as Fe concentration), H2O2(0.1mM), wherein VMb：VNa2S2O410: mb and Na were added before testing2S2O4Mixing uniformly, introducing nitrogen for 15min to remove oxygen in the reaction system, adding other solutions, and testing to ensure that the reaction system is in a closed state. The total volume of the solution is 3mL, the solution is uniformly mixed and then placed in a water bath kettle at 37 ℃ for incubation, the absorbance of the mixed solution at 500-600nm is tested every 5min, the release of CO in the nanoparticles excited by hydrogen peroxide is studied according to the test, and the monitoring time is 80min, as shown in FIG. 12. As can be seen from the figure, C8H4Fe2O6S2PSMA-PEG NPs can generate OH and release CO gas when reacting with hydrogen peroxide, thereby achieving the synergistic effect of the chemodynamic therapy and the gas therapy.
Example 2
8 4 2 6 2CHFeOS-PSMA-PEG NPs cell experiment
(1) Evaluation of cytotoxicity: mouse melanoma (B16-F10) cells and Human Umbilical Vein Endothelial Cells (HUVEC) were selected. Collecting logarithmic phase cells, digesting with pancreatin, centrifuging after termination, and collecting to obtain cells with concentration of 5 × 104cells/mL, adding 100 μ L of cell suspension into each well of a 96-well plate, placing the plate in an incubator at 37 ℃ for incubation for 12h to ensure that the cells are fully attached, and diluting the material into clusters by using a high-sugar medium (DMEM) with the following concentration: 0.20, 40, 100, 200, 400 μ M C8H4Fe2O6S2PSMA-PEG NPs solution. And adding 100 mu L of prepared nano materials with different concentrations into a 96-well plate (5 parallel wells are arranged at intervals of concentration), and putting the 96-well plate into an incubator to continue incubation for 12 or 24 hours. After incubation, pipetteThe stock culture was washed once with PBS and then the blank was removed with a microplate reader. Then 100. mu.L of the prepared 3- (4, 5-dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide (MTT) solution was added to each well, the MTT solution was aspirated after incubation in an incubator for 4h, 150. mu.L of DMSO-solubilized formazan was added to each well, and the absorbance of each well was measured at 490nm using a microplate reader, as shown in FIG. 13. The left picture is C8H4Fe2O6S2Cytotoxicity of PSMA-PEG NPs on mouse melanoma (B16-F10) cells, C at a cluster concentration of 50. mu.M8H4Fe2O6S2PSMA-PEG NPs have obvious toxicity to B16-F10 cells, and after B16-F10 cells are incubated for 24 hours by using the nano material with the cluster concentration of 100 mu M, the survival rate of the cells is only about 20%, which shows that the nano material has obvious killing effect on B16-F10 cells. The right picture is C8H4Fe2O6S2The PSMA-PEG NPs have cytotoxicity to Human Umbilical Vein Endothelial Cells (HUVEC), and the nano material has lower toxicity to the HUVEC, which indicates that the nano material has lower toxic and side effects to normal tissues.
(2) Flow cytometric and confocal laser evaluation of intracellular ROS levels
Flow cytometry evaluation: the logarithmic phase of B16-F10 cells was prepared to 5X 10 with the prepared complete medium5cells/mL, then 2mL of B16-F10 cell suspension was added to each well of a six-well plate, and 5% CO was added2Culturing for 12h in a cell culture box to allow cells to adhere to the wall, sucking away old culture solution, and then sequentially adding 2mL of 50 μ M cluster compound nanoparticles (prepared by DMEM medium) into each hole, wherein the incubation time is 0h, 0.5h, 1h, 2h and 4h respectively. After the incubation was completed, the old culture solution was removed, PBS was added and washed three times, 0.5mL of EDTA-free pancreatin was added in order for 2min, 0.5mL of DMEM medium was added to terminate the digestion, the supernatant was aspirated after centrifugation, 1mL of 10 μ M DCFH-DA was added and incubated in an incubator for 30min (the centrifuge tube was shaken from time to mix the cells and the probe), PBS was added and washed three times, 1mL of PBS was added to the tube, and the experiment was performedThe group was added with 1. mu.L of active oxygen positive control and blown up evenly for use. The test uses 488nm excitation light, 525nm emission wavelength, flow cytometry is used to detect each group of samples, FL1 channel fluorescence intensity is collected, and the obtained data is processed by FlowJo 7.6.1 software.
Laser confocal evaluation: ROS in B16-F10 cells were detected using the ROS indicator DCFH-DA. Cells grown in log phase were taken and diluted to a density of 2X 106cells/mL, wherein 1mL of B16-F10 cells in each hole are transferred to a special laser confocal culture dish; at 5% CO2Culturing for 12h in a cell culture box to allow cells to adhere to the wall, sucking away old culture solution, and then sequentially adding 1mL of 50 mu M cluster compound nanoparticles (prepared by DMEM medium) into each dish, wherein the incubation time is 0h, 2h and 4h respectively. Subsequently, the sample was washed 3 times with PBS to wash off the excess nanomaterial. And adding DCFH-DA with the final concentration of 10 mu M into the culture dish, incubating for 30min in a dark place, discarding the culture solution, washing for 3 times by PBS (phosphate buffer solution), washing away redundant probes, adding 0.5mL of DMEM into the culture dish, observing the fluorescence in the cells by a laser confocal scanning microscope, and testing the emission wavelength of 525nm by adopting 488nm exciting light.
As shown in FIG. 14C8H4Fe2O6S2After the PSMA-PEG NPs incubate cells, DCFH-DA is used as a fluorescence indicator to detect the intracellular OH level, the detection result of the flow cytometry in the left picture shows that the fluorescence is strongest after the nanoparticles incubate the B16-F10 cells for 2h, which indicates that the intracellular ROS level is higher after 2h of incubation, and the laser confocal data in the right picture is matched with the experimental result of the flow cytometry. The cell level proves that the nano material of the invention can generate OH by reaction with hydrogen peroxide in cancer cells, and has the effect of chemodynamic treatment.
(3) Determination of CO levels in cells
CO in B16-F10 cells was detected using CO indicator COP-1. Cells grown in log phase were taken and diluted to a density of 2X 106cells/mL, wherein 1mL of B16-F10 cells in each hole are transferred to a special laser confocal culture dish; at 5% CO2Culturing for 12h in a cell culture box to allow cells to adhere to the wall, sucking off old culture solution, and sequentially adding 1mL of 50 μ M cluster compound to each dishThe incubation time of the nanoparticles (prepared by DMEM medium) is 0h, 1h and 2h respectively. Subsequently, the sample was washed 3 times with PBS to wash off the excess nanomaterial. Then adding COP-1 and PdCl with final concentration of 10 mu M into the culture dish respectively2After incubating the mixed solution for 30min in the dark, the culture solution is discarded, PBS is used for washing for 3 times, redundant probes are washed away, and finally 0.5mL of DMEM is added into a culture dish, and the fluorescence in the cells is observed by a laser confocal scanning microscope. The test uses 488nm excitation light, 525nm emission wavelength, C8H4Fe2O6S2Laser confocal pictures of CO levels in cells detected with COP-1 as a fluorescent indicator after incubation of cells with-PSMA-PEG NPs are shown in fig. 15. As can be seen from the figure, the fluorescence of the nanoparticles is strongest after the nanoparticles are incubated for 2h in B16-F10 cells, which indicates that the intracellular CO level is higher after the nanoparticles are incubated for 2 h. The cell level proves that the nano material can react with hydrogen peroxide in cancer cells to generate CO gas, and has the effect of gas treatment.
Example 3
In vivo therapeutic experiment
Treatment was divided into two groups: (1) control group (Saline) and (2) treatment group (MNPs), the concentration of the material injected into the mice was 1.4mM (calculated as the cluster concentration), i.e., 5.21 mg/kg. The mice were treated by intravenous injection of nanomaterials starting on day four, followed by a second treatment on day 7, a third treatment on day 10, a fourth treatment on day 13 and a treatment ending on day 16, starting at the time of intravenous injection of the mice B16-F10 cells, designated D0. One mouse was dissected before each injection of nanomaterial (day 4, day 7, day 10, day 13, day 16), its lungs were removed, lung weights were weighed, photographs were taken, and body weight monitoring was performed on mice each day, with the results shown in fig. 16 and 17.
As can be seen from the change in body weight of the mice in the in vivo treatment experiment in fig. 16, the body weight of the mice did not change significantly during the treatment period. FIG. 17 is a photograph of the lungs of a mouse in an in vivo treatment experiment; wherein, Saline represents a Control group, and MNPs represents a treatment group (note: the lungs of the mice in the picture are all bleached by 3 percent hydrogen peroxide solution). Dissecting mice of a control group and an experimental group respectively on the fourth day of injecting tumor cells into tail veins of the mice, and finding that no obvious melanoma is generated in the lungs of the mice on the fourth day (namely, the first treatment) and no melanoma metastasis is generated in other parts of the mice; at the seventh day (i.e., the second treatment), melanoma was visible in the lungs of the mice, with no apparent difference between the experimental and control groups; at day ten (i.e., the third treatment), the control group showed an increase in melanoma, and the experimental group showed a significant decrease; on the thirteenth day, all mice were dissected, and it was found that the number of melanoma nodules was significantly increased in the control mice and significantly decreased in the experimental mice, thus indicating that the nanoparticles had significant therapeutic effects on lung metastasis of melanoma.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
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