CN114522240A - Copper sulfide/manganese dioxide composite material and preparation method and application thereof - Google Patents
Copper sulfide/manganese dioxide composite material and preparation method and application thereof Download PDFInfo
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- CN114522240A CN114522240A CN202210100855.6A CN202210100855A CN114522240A CN 114522240 A CN114522240 A CN 114522240A CN 202210100855 A CN202210100855 A CN 202210100855A CN 114522240 A CN114522240 A CN 114522240A
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- copper sulfide
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- stem cells
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- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 title claims abstract description 241
- OMZSGWSJDCOLKM-UHFFFAOYSA-N copper(II) sulfide Chemical compound [S-2].[Cu+2] OMZSGWSJDCOLKM-UHFFFAOYSA-N 0.000 title claims abstract description 183
- 239000002131 composite material Substances 0.000 title claims abstract description 101
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 95
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- 238000005406 washing Methods 0.000 claims description 23
- 238000001035 drying Methods 0.000 claims description 20
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- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 description 8
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- BWFPGXWASODCHM-UHFFFAOYSA-N copper monosulfide Chemical compound [Cu]=S BWFPGXWASODCHM-UHFFFAOYSA-N 0.000 description 2
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- AMWRITDGCCNYAT-UHFFFAOYSA-L hydroxy(oxo)manganese;manganese Chemical compound [Mn].O[Mn]=O.O[Mn]=O AMWRITDGCCNYAT-UHFFFAOYSA-L 0.000 description 2
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- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K47/02—Inorganic compounds
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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Abstract
The invention discloses a copper sulfide/manganese dioxide composite material and a preparation method and application thereof. The copper sulfide/manganese dioxide composite material comprises copper sulfide, manganese dioxide coated on the surface of the copper sulfide, and stem cell targeting peptide modified on the surface of the manganese dioxide. The copper sulfide/manganese dioxide composite material has the capabilities of promoting the migration of MSCs, resisting inflammation and differentiating chondrogenic, also has the activities of superoxide dismutase and catalase, and can improve the cell activity of the MSCs in an RA oxidative stress microenvironment. Moreover, the stem cell targeting peptide on the surface can enhance the capability of the material in targeting mesenchymal stem cells and enhance the efficiency of the nano material engineered stem cells. The mesenchymal stem cells engineered by the copper sulfide/manganese dioxide composite material can obviously enhance the cell migration, anti-inflammatory and chondrogenic capacities of the stem cells, particularly the capacities of resisting an inflammatory environment and ROS on the cells.
Description
Technical Field
The invention belongs to the technical field of nano biomedical materials, and particularly relates to a copper sulfide/manganese dioxide composite material as well as a preparation method and application thereof.
Background
Rheumatoid arthritis, abbreviated as RA, is a chronic systemic autoimmune disease with a complex etiology, with a global prevalence of about 0.5% to 1%. According to estimation, RA patients in China reach at least over 500 thousands of people, and the traditional Chinese medicine is one of the most common joint bone diseases in clinic. The main pathological features of RA are systemic polyarticular synovium pannus formation and persistent synovitis, which further invades cartilage and bone, resulting in loss of joint function and extremely high disability rate. RA has no specific treatment means until now, and is mainly treated by drugs to relieve pain and delay the disease process. Currently, RA treatment drugs mainly comprise four types, namely drugs for relieving disease and rheumatism, glucocorticoid drugs, non-steroidal anti-inflammatory drugs and biological agents, but all have certain treatment limitations and disadvantages.
Mesenchymal Stem Cells (MSCs) have the capabilities of multi-lineage differentiation potential, self-renewal, tissue repair, anti-inflammation, low immunogenicity, targeting of inflammatory lesions and the like, and allogeneic MSCs can also keep activity in vivo due to the lack of human leukocyte class II antigens of the MSCs, so that the treatment of autologous and allogeneic MSCs can be used as a new direction for RA treatment. At present, a plurality of clinical researches on RA treatment by stem cells are carried out at home and abroad, and the completed clinical research results show that: after MSCs are intravenously injected to RA patients, no obvious side effect occurs, and symptoms such as arthralgia and the like are relieved to a certain extent. However, the results of the study also revealed that stem cells were ineffective in some patients or that symptoms rapidly recur, which may be associated with the inflammatory microenvironment specific to RA affected joints. In addition to pathological changes caused by synovial hyperplasia invasion, RA joints also have abnormal microenvironments such as oxidative stress and inflammatory cytokine infiltration, and the inflammatory microenvironments can directly cause the inactivation and apoptosis of MSCs. In addition, MSCs have problems of off-target, abnormal differentiation, and even tumor initiation after injection. If the activity of the MSCs in inflammatory environment and the capacity of cartilage differentiation and the like can be improved, the effect of stem cells on treating RA can be improved.
Disclosure of Invention
The invention aims to provide a copper sulfide/manganese dioxide composite material and a preparation method and application thereof aiming at the defects of the prior art. The copper sulfide/manganese dioxide composite material can be actively targeted into mesenchymal stem cells to form engineered mesenchymal stem cells, active oxygen can be effectively decomposed in an inflammatory environment, the cell migration, anti-inflammation, cartilage differentiation and other effects of MSCs are improved, good inflammation inhibition and cartilage repair effects are achieved, and the curative effect of stem cells in treating RA is effectively improved.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: the copper sulfide/manganese dioxide composite material comprises copper sulfide, manganese dioxide coated on the surface of the copper sulfide, and stem cell targeting peptide modified on the surface of the manganese dioxide.
The copper sulfide/manganese dioxide composite material has a core-shell structure, and copper sulfide nanoparticles with the core structure and manganese dioxide shells with the shell structure have the capabilities of promoting migration, anti-inflammation and chondrogenic differentiation of MSCs, and simultaneously have the activities of superoxide dismutase and catalase, so that the cell activity of the MSCs in an RA oxidative stress microenvironment can be improved. In addition, after the manganese dioxide wraps the copper sulfide particles, a manganese oxide shell with a mesoporous structure and the copper sulfide/manganese dioxide composite material are formed, so that the composite material has an efficient drug-carrying function, and can carry drugs to realize a slow-release effect. Moreover, the stem cell targeting peptide modified on the surface of the shell structure forms a chemical bond with the carboxyl on the surface of the manganese dioxide shell, and is combined with a specific site on the surface of the mesenchymal stem cell to enhance the capability of the material for targeting the mesenchymal stem cell and enhance the efficiency of the nano material engineered stem cell.
In the process of engineering the mesenchymal stem cells, after the copper sulfide/manganese dioxide composite material is added into a cell culture medium, the copper sulfide/manganese dioxide composite material firstly reaches the interior of the cells due to the peptide segment targeting effect of the surface stem cell targeting peptide; the stem cells internalized by the material can promote the biological activity of the stem cells and enhance the effect of resisting the external inflammatory environment of the mesenchymal stem cells due to the anti-inflammatory and antioxidant stress effects of the stem cells, and meanwhile, the chondrogenic effect of the mesenchymal stem cells is also obviously improved. The therapeutic effect of the engineered stem cells internalized by the copper sulfide/manganese dioxide composite material is well verified in-vivo and in-vitro models, and is remarkably stronger than that of pure mesenchymal stem cells.
The stem cell targeting peptide is prepared by adopting a standard solid-phase polypeptide synthesis method, the polypeptide sequence is VTAMEPGQ, and the C end of the polypeptide sequence is added with an amino-rich sequence GGGGKKKKKKK, so that the subsequent linking on the surface of the composite material is facilitated.
The invention claims a preparation method of the copper sulfide/manganese dioxide composite material, which comprises the following steps:
(1) dissolving copper salt, L-cysteine and sodium thiosulfate pentahydrate in water, heating, stirring, reacting, cooling, centrifuging, washing and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2And connecting the material with stem cell targeting peptide through EDC/NHS reaction to obtain the copper sulfide/manganese dioxide composite material.
In a preferred embodiment of the invention, in the step (2), the mass ratio of the copper sulfide to the potassium permanganate is 1: 0.5-1.5.
The mass ratio of the copper sulfide to the potassium permanganate is 1: and when the content of the active component is 0.5-1.5, a manganese dioxide shell with good wrapping property can be formed on the surface of the copper sulfide nano-particles, and the obtained copper sulfide/manganese dioxide composite material can remarkably enhance the cell migration, anti-inflammatory and chondrogenic capacities of stem cells, particularly the capacities of resisting an inflammatory environment and ROS on cells.
More preferably, the mass ratio of the copper sulfide to the potassium permanganate is 1:1.
When the mass ratio of the copper sulfide to the potassium permanganate is 1:1, the coating rate of manganese dioxide on copper sulfide is the highest, and the residual manganese is the least in the modification process, so that the coating is more favorable for enhancing the cell migration, anti-inflammation and chondrogenesis capacity of the material on stem cells, especially the capacity of resisting an inflammation environment and ROS on the influence of the cells.
In a preferred embodiment of the present invention, in the step (2), the reaction time is 0.5 to 2.0 hours.
The inventor finds that the reaction time of copper sulfide and potassium permanganate is critical to the formation of the core-shell structure and has a large influence on the binding force between manganese dioxide and copper sulfide in the core-shell structure through a large number of experiments. When the reaction time of copper sulfide and potassium permanganate is 0.5-2.0 h, the binding force between manganese dioxide and copper sulfide is large, and the copper sulfide/manganese dioxide composite material has the most obvious effects of resisting cell migration, inflammation and cartilage formation of stem cells, particularly resisting an inflammatory environment and resisting the influence of ROS on the cells.
More preferably, in the step (2), the reaction time is 1.0 h.
When the reaction time of the copper sulfide and the potassium permanganate is 1 hour, the CuS/MnO is2The materials react most completely, and no longer time or shorter time can obtain greater benefit.
As a preferred embodiment of the invention, in the step (1), the molar ratio of the copper salt, the L-cysteine and the sodium thiosulfate pentahydrate is 1 (1-3) to (1-3).
As a preferred embodiment of the present invention, in the step (1), the temperature for heating and stirring the reaction is 90 ℃ for 1 to 2 hours.
As a preferred embodiment of the present invention, in the step (3), CuS/MnO2The mass ratio of the material to the stem cell targeting peptide is (5-10): 1.
The application also claims a preparation method of the copper sulfide/manganese dioxide composite material engineered mesenchymal stem cells, which comprises the following steps:
s1: when the mesenchymal stem cells are cultured to be 100 percent fused, adding the copper sulfide/manganese dioxide composite material into a mesenchymal stem cell culture medium for culture;
s2: and then washing with phosphate buffer salt solution to obtain the copper sulfide/manganese dioxide composite material engineered mesenchymal stem cells.
In a preferred embodiment of the present invention, in step S1, the concentration of the copper sulfide/manganese dioxide composite material is 12.5 μ g/ml to 100 μ g/ml; the culture time is 2-4 h.
More preferably, in the step S1, the concentration of the copper sulfide/manganese dioxide composite material is 50 μ g/ml; the culture time is 4 h.
In the process of internalizing the copper sulfide/manganese dioxide composite material into the mesenchymal stem cells, the maximum cell uptake rate can be reached and the biological function of the mesenchymal stem cells can be promoted when the concentration is 50 mug/ml; meanwhile, the quantity of the nanoparticles taken up by the mesenchymal stem cells after 4 hours of culture is close to saturation.
The application also claims the application of the copper sulfide/manganese dioxide composite material in the preparation of rheumatoid arthritis medicines.
Compared with the prior art, the invention has the beneficial effects that: the copper sulfide/manganese dioxide composite material has the capabilities of promoting the migration, anti-inflammation and chondrogenic differentiation of MSCs, and simultaneously has the activities of superoxide dismutase and catalase, and can improve the cell activity of the MSCs in an RA oxidative stress microenvironment. And the stem cell targeting peptide on the surface can enhance the capability of the material in targeting mesenchymal stem cells and enhance the efficiency of the nano material engineering stem cells. In addition, the mesoporous structure and the core-shell structure of the composite material can synchronously realize the delivery and the release of the drug, and further enhance the biological curative effect of the stem cells. Compared with a pure stem cell, the mesenchymal stem cell engineered by the copper sulfide/manganese dioxide composite material can obviously enhance the cell migration, anti-inflammatory and chondrogenic capacities of the stem cell, particularly the capacities of resisting an inflammatory environment and ROS on the influence of the cells.
Drawings
FIG. 1 is a flow chart of the preparation of the copper sulfide/manganese dioxide composite material and a schematic diagram of the activity of the nanoenzyme;
FIG. 2 is a graph of CuS/MnO prepared in example 1 of the present invention2A TEM image of the material;
FIG. 3 is a graph of CuS/MnO prepared in example 1 of the present invention2XRD pattern of the material;
FIG. 4 is a graph of CuS/MnO prepared in example 1 of the present invention2A map of superoxide dismutase activity of the material;
FIG. 5 shows the addition of CuS/MnO prepared in example 12Materials and no addition of CuS/MnO2A comparison of catalase activity of the material;
FIG. 6 is a graph comparing the targeting performance of copper sulfide/manganese dioxide composites prepared in example 1 of the present invention and comparative example 1 on MSCs;
fig. 7 is a graph comparing migration performance of copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared according to example 1 and comparative example 1 of the present invention;
fig. 8 is a graph comparing cartilage differentiation performance of the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 of the present invention and comparative example 1;
fig. 9 is a graph comparing the anti-inflammatory performance of the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 of the present invention and comparative example 1;
fig. 10 is a graph comparing the cell activity of the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 of the present invention and pure mesenchymal stem cells in the hydrogen peroxide addition environment by neutral red staining detection;
FIG. 11 is a comparison graph of the DCFH-DA method for detecting the consumption of active oxygen in the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 and pure mesenchymal stem cells;
fig. 12a is a diagram of rat RA model for collagen-induced arthritis constructed by in vivo study of copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 and pure mesenchymal stem cells; fig. 12b is a rat RA model of adjuvant-induced arthritis of copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 versus simple mesenchymal stem cells.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
The preparation method of the copper sulfide/manganese dioxide composite material comprises the following steps:
(1) dissolving 188mg of copper nitrate and 363mg of L-cysteine in 200mL of deionized water, uniformly mixing to obtain a mixed solution, dissolving 744mg of sodium thiosulfate pentahydrate in 10mL of deionized water, slowly dripping into the mixed solution, stirring and reacting for 2 hours at 90 ℃, cooling to room temperature, centrifuging for 15 minutes at 4000rpm, trading and washing for 5 times by using deionized water or absolute ethyl alcohol, and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate according to the mass ratio of 1:1 for 1 hour, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2The material is connected with stem cell targeting peptide through EDC/NHS reaction to obtain the CuS/MnO of the copper sulfide/manganese dioxide composite material2The mass ratio of the material to the stem cell targeting peptide is 10: 1.
Example 2
The preparation method of the copper sulfide/manganese dioxide composite material comprises the following steps:
(1) dissolving 188mg of copper nitrate and 363mg of L-cysteine in 200mL of deionized water, uniformly mixing to obtain a mixed solution, dissolving 744mg of sodium thiosulfate pentahydrate in 10mL of deionized water, slowly dripping into the mixed solution, stirring and reacting for 2 hours at 90 ℃, cooling to room temperature, centrifuging for 15 minutes at 4000rpm, trading and washing for 5 times by using deionized water or absolute ethyl alcohol, and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate according to the mass ratio of 1:0.5 for 1 hour, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2The material is connected with stem cell targeting peptide through EDC/NHS reaction to obtain the CuS/MnO of the copper sulfide/manganese dioxide composite material2The mass ratio of the material to the stem cell targeting peptide is 10: 1.
Example 3
The preparation method of the copper sulfide/manganese dioxide composite material comprises the following steps:
(1) dissolving 188mg of copper nitrate and 363mg of L-cysteine in 200mL of deionized water, uniformly mixing to obtain a mixed solution, dissolving 744mg of sodium thiosulfate pentahydrate in 10mL of deionized water, slowly dripping into the mixed solution, stirring and reacting for 2 hours at 90 ℃, cooling to room temperature, centrifuging for 15 minutes at 4000rpm, trading and washing for 5 times by using deionized water or absolute ethyl alcohol, and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate according to the mass ratio of 1:1.5 for 1 hour, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2The material is connected with stem cell targeting peptide through EDC/NHS reaction to obtain the CuS/MnO of the copper sulfide/manganese dioxide composite material2The mass ratio of the material to the stem cell targeting peptide is 10: 1.
Example 4
The preparation method of the copper sulfide/manganese dioxide composite material comprises the following steps:
(1) dissolving 188mg of copper nitrate and 363mg of L-cysteine in 200mL of deionized water, uniformly mixing to obtain a mixed solution, dissolving 744mg of sodium thiosulfate pentahydrate in 10mL of deionized water, slowly dripping into the mixed solution, stirring and reacting for 2 hours at 90 ℃, cooling to room temperature, centrifuging for 15 minutes at 4000rpm, trading and washing for 5 times by using deionized water or absolute ethyl alcohol, and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate according to the mass ratio of 1:1 for 0.5 hour, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2The material is connected with stem cell targeting peptide through EDC/NHS reaction to obtain the CuS/MnO of the copper sulfide/manganese dioxide composite material2The mass ratio of the material to the stem cell targeting peptide is 10: 1.
Example 5
The preparation method of the copper sulfide/manganese dioxide composite material of the embodiment includes the following steps:
(1) dissolving 188mg of copper nitrate and 363mg of L-cysteine in 200mL of deionized water, uniformly mixing to obtain a mixed solution, dissolving 744mg of sodium thiosulfate pentahydrate in 10mL of deionized water, slowly dripping into the mixed solution, stirring and reacting for 2 hours at 90 ℃, cooling to room temperature, centrifuging for 15 minutes at 4000rpm, trading and washing for 5 times by using deionized water or absolute ethyl alcohol, and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate according to the mass ratio of 1:1 for 1.5 hours, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2The material is connected with stem cell targeting peptide through EDC/NHS reaction to obtain the CuS/MnO of the copper sulfide/manganese dioxide composite material2The mass ratio of the material to the stem cell targeting peptide is 10: 1.
Example 6
The preparation method of the copper sulfide/manganese dioxide composite material of the embodiment includes the following steps:
(1) dissolving 188mg of copper nitrate and 363mg of L-cysteine in 200mL of deionized water, uniformly mixing to obtain a mixed solution, dissolving 744mg of sodium thiosulfate pentahydrate in 10mL of deionized water, slowly dripping into the mixed solution, stirring and reacting for 2 hours at 90 ℃, cooling to room temperature, centrifuging for 15 minutes at 4000rpm, trading and washing for 5 times by using deionized water or absolute ethyl alcohol, and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate according to the mass ratio of 1:1 for 2 hours, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2The material is connected with stem cell targeting peptide through EDC/NHS reaction to obtain the CuS/MnO of the copper sulfide/manganese dioxide composite material2The mass ratio of the material to the stem cell targeting peptide is 10: 1.
Example 7
The preparation method of the copper sulfide/manganese dioxide composite material comprises the following steps:
(1) dissolving 188mg of copper nitrate and 121mg of L-cysteine in 200mL of deionized water, uniformly mixing to obtain a mixed solution, dissolving 248mg of sodium thiosulfate pentahydrate in 10mL of deionized water, slowly dripping into the mixed solution, stirring and reacting at 90 ℃ for 1 hour, cooling to room temperature, centrifuging at 4000rpm for 15min, trading and washing for 5 times by using deionized water or absolute ethyl alcohol, and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate according to the mass ratio of 1:1 for 1 hour, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2The material is connected with stem cell targeting peptide through EDC/NHS reaction to obtain the CuS/MnO of the copper sulfide/manganese dioxide composite material2The mass ratio of the material to the stem cell targeting peptide is 5: 1.
Comparative example 1
The preparation method of the copper sulfide/manganese dioxide composite material comprises the following steps:
(1) dissolving 188mg of copper nitrate and 363mg of L-cysteine in 200mL of deionized water, uniformly mixing to obtain a mixed solution, dissolving 744mg of sodium thiosulfate pentahydrate in 10mL of deionized water, slowly dripping into the mixed solution, stirring and reacting for 2 hours at 90 ℃, cooling to room temperature, centrifuging for 15 minutes at 4000rpm, trading and washing for 5 times by using deionized water or absolute ethyl alcohol, and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate according to the mass ratio of 1:1 for 1 hour, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2The material is connected with a non-mesenchymal stem cell targeting peptide segment through EDC/NHS reaction to obtain the copper sulfide/manganese dioxide composite material; measured as GP-CuS @ MnO2(ii) a The CuS/MnO2The mass ratio of the material to the non-mesenchymal stem cell targeting peptide fragment is 10: 1.
Test example 1
FIG. 1a is a flow chart showing the preparation of the copper sulfide/manganese dioxide composite material, wherein CuS @ MnO is shown2Is CuS/MnO2A material; VQ is a stem cell targeting peptide; VQ-CuS @ MnO2Copper sulfide/manganese dioxide composite material, abbreviated as VCM. As can be seen from the figure a, the copper sulfide/manganese dioxide composite material is CuS @ MnO2From C having a core-shell structureuS/MnO2Materials and modifications in CuS/MnO2Stem cell targeting peptides on the surface of the material.
FIG. 1b is a schematic diagram showing the nanoenzyme activity of the copper sulfide/manganese dioxide composite; as can be seen from the figure, CuS/MnO2The material has superoxide dismutase activity and catalase activity.
FIG. 2 is a graph of CuS/MnO prepared in example 1 of the present invention2A TEM image of the material; as can be seen from the figure, the CuS/MnO prepared in example 12The material has a core-shell structure and the particle size is 150 nm.
FIG. 3 is a graph of CuS/MnO prepared in example 1 of the present invention2XRD pattern of the material; by comparison with the CuS standard card, the copper sulfide characteristic diffraction data prepared in example 1 conforms to the characteristics of CuS, and the manganese dioxide shell has an amorphous structure.
TABLE 1CuS/MnO2SOD enzyme activity at a concentration of 50. mu.g/ml and H at 300 seconds2O2Result of reduction
From the results of FIG. 4, FIG. 5 and Table 1, it can be seen that CuS/MnO described in the present application2The material has superoxide dismutase and catalase activity. Furthermore, as can be seen from FIG. 4, the increase in SOD enzyme activity was not significant and there was no significant statistical difference with the increase in material concentration. FIG. 5(a) is a schematic diagram showing the addition of CuS/MnO prepared in example 12Materials and no addition of CuS/MnO2A graph comparing the reduction of hydrogen peroxide in catalase activity of the material; FIG. 5(b) is a graph showing addition of CuS/MnO prepared in example 12Materials and no addition of CuS/MnO2A graph comparing the amount of oxygen produced in the catalase activity of the material; in the figure H2O2+CuS@MnO2To add said CuS/MnO2Material, H2O2+ PBS is not added with CuS/MnO2A material. As can be seen in FIG. 5, the hydrogen peroxide is present in the CuS/MnO2After the material is added, the hydrogen peroxide removal rate is higher along with the time extension;at the same time, the oxygen production also increased with time and hydrogen peroxide removal, indicating that CuS/MnO2The material has better catalase activity.
Test example 2
In this test example, the targeting effect of the copper sulfide/manganese dioxide composite on MSCs and the migration, anti-inflammation, chondrogenic differentiation, and other properties of mesenchymal stem cells engineered with the copper sulfide/manganese dioxide composite were tested.
Test samples: materials prepared in examples 1-7 and comparative example 1;
the copper sulfide/manganese dioxide composite material engineered mesenchymal stem cells comprise the following steps:
s1: when the mesenchymal stem cells are cultured to 100 percent fusion, 50 mu g/ml of test sample is added into the mesenchymal stem cell culture medium to be cultured for 4 hours;
s2: and then washing the cells for three times by using phosphate buffer solution to obtain the copper sulfide/manganese dioxide composite material engineered mesenchymal stem cells.
FIG. 6 is a graph comparing the targeting performance of composites prepared in example 1 and comparative example 1 to MSCs; in the figure, VQ-CuS @ MnO2Copper sulfide/manganese dioxide composite, GP-CuS @ MnO, prepared for example 12For the composite material prepared in comparative example 1, PBS was phosphate buffered saline and FITC was fluorescein isothiocyanate. FIG. 6a is a confocal microscope image comparing the targeting performance of composites prepared in example 1 and comparative example 1 to MSCs; as can be seen from FIG. 6a, VQ-CuS @ MnO2Relative to GP-CuS @ MnO2And stem cells can be targeted more effectively. FIG. 6b is a graph of FITC fluorescence intensity versus cell number for composites prepared in example 1 and comparative example 1; FIG. 6c is a graph comparing FITC fluorescence intensity of composites prepared in example 1 and comparative example 1, and it can be seen from FIGS. 6b and 6c that more VQ-CuS @ MnO was present2Reaching the inside of the mesenchymal stem cell; FIG. 6d is an inductively coupled plasma mass spectrum, VQ-CuS @ MnO, of the composite materials prepared in example 1 and comparative example 12The copper content in the cells is also obviously higher than GP-CuS @ MnO2And (4) grouping. Thus, the knot according to FIG. 6Fruit description of VQ-CuS @ MnO2Can effectively target stem cells, thereby improving the nano-engineering efficiency.
Fig. 7 is a graph comparing migration performance of the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 with that of pure mesenchymal stem cells; fig. 7a is a graph showing scratch experiment results and healing rates of the copper sulfide/manganese dioxide composite material engineered mesenchymal stem cells prepared in example 1 and simple mesenchymal stem cells, and fig. 7b is a graph showing transwell experiment results and cell numbers of the copper sulfide/manganese dioxide composite material engineered mesenchymal stem cells prepared in example 1 and simple mesenchymal stem cells. According to fig. 7, the migration of the cells of the mesenchymal stem cells engineered by the copper sulfide/manganese dioxide composite material is obviously enhanced compared with that of the pure MSCs.
Fig. 8 is a graph comparing cartilage differentiation performance of the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 of the present invention with that of pure mesenchymal stem cells; as can be seen from fig. 8, the marker gene of cartilage differentiation was significantly up-regulated in the mesenchymal stem cells engineered with the copper sulfide/manganese dioxide composite prepared in example 1, compared to pure MSCs.
Fig. 9 is a graph comparing the anti-inflammatory performance of the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 of the present invention with that of pure mesenchymal stem cells; as can be seen from fig. 9, the marker gene of the anti-inflammatory effect of the mesenchymal stem cells engineered with the copper sulfide/manganese dioxide composite material prepared in example 1 is significantly up-regulated compared to pure MSCs.
Table 2 results of cell activities of copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in examples and comparative examples and pure mesenchymal stem cells in an environment of hydrogen peroxide addition
| Cell Activity (%) | |
| Example 1 | 80.8 |
| Example 2 | 75.4 |
| Example 3 | 82.5 |
| Example 4 | 74.9 |
| Example 5 | 84.4 |
| Example 6 | 86.3 |
| Example 7 | 76.3 |
| Comparative example 1 | 72.1 |
| Simple mesenchymal stem cells | 57.9 |
Fig. 10 is a graph comparing the cell activity of the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 with that of pure mesenchymal stem cells in the environment of hydrogen peroxide addition. The results according to table 2 and fig. 10 show that: the mesenchymal stem cell engineered by the copper sulfide/manganese dioxide composite material prepared by the embodiment has the advantages that the material in the cell has enzyme activity, the antioxidant stress function of the mesenchymal stem cell is obviously enhanced, and the cell activity is obviously improved, so that the strategy of the mesenchymal stem cell engineered by the copper sulfide/manganese dioxide composite material can effectively help the stem cell to resist the influence of RA inflammation microenvironment.
Fig. 11 is a comparison graph of the DCFH-DA method for detecting the consumption of active oxygen in the mesenchymal stem cells engineered with the copper sulfide/manganese dioxide composite material prepared in example 1 and the pure mesenchymal stem cells. The results show that: the composite material engineered mesenchymal stem cells have remarkably improved cell activity in the cells due to the remarkable antioxidation function and the capability of promoting the cell activity, so that the strategy of the composite material engineered mesenchymal stem cells is proved to be capable of effectively helping the stem cells to resist the influence of RA inflammation microenvironment.
Fig. 12a is a rat RA model diagram of collagen-induced arthritis constructed by in vivo studies of the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 and pure mesenchymal stem cells, and fig. 12b is a rat RA model of adjuvant-induced arthritis of the copper sulfide/manganese dioxide composite engineered mesenchymal stem cells prepared in example 1 and pure mesenchymal stem cells. White arrows indicate synovial invasion, black arrows indicate articular cartilage damage. The H & E staining results according to fig. 12 show that: the composite material engineered mesenchymal stem cells can effectively treat RA through tail vein injection, and the curative effect is obviously superior to that of simple MSCs.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. The copper sulfide/manganese dioxide composite material is characterized by comprising copper sulfide, manganese dioxide coated on the surface of the copper sulfide and stem cell targeting peptide modified on the surface of the manganese dioxide.
2. The method of preparing the copper sulfide/manganese dioxide composite material according to claim 1, comprising the steps of:
(1) dissolving copper salt, L-cysteine and sodium thiosulfate pentahydrate in water, heating, stirring, reacting, cooling, centrifuging, washing and drying to obtain copper sulfide;
(2) mixing and reacting the copper sulfide obtained in the step (1) with potassium permanganate, centrifuging, washing and drying to obtain CuS/MnO2A material;
(3) the CuS/MnO obtained in the step (2)2And connecting the material with stem cell targeting peptide through EDC/NHS reaction to obtain the copper sulfide/manganese dioxide composite material.
3. The method for preparing the copper sulfide/manganese dioxide composite material according to claim 2, wherein in the step (2), the mass ratio of copper sulfide to potassium permanganate is 1: 0.5-1.5.
4. The method for preparing the copper sulfide/manganese dioxide composite material according to claim 3, wherein in the step (2), the reaction time is 0.5-2.0 h.
5. The method of claim 3, wherein in the step (1), the molar ratio of the copper salt, L-cysteine and sodium thiosulfate pentahydrate is 1 (1-3) to (1-3).
6. The method for preparing a copper sulfide/manganese dioxide composite material according to claim 3, wherein in the step (1), the temperature for heating and stirring reaction is 90 ℃ and the time is 1-2 h.
7. The method of claim 3, wherein in step (3), the copper sulfide/manganese dioxide composite is formed of CuS/MnO2The mass ratio of the material to the stem cell targeting peptide is (5-1)0):1。
8. The copper sulfide/manganese dioxide composite engineered mesenchymal stem cell of claim 1, comprising the steps of:
s1: when the mesenchymal stem cells are cultured to be 100 percent fused, adding the copper sulfide/manganese dioxide composite material into a mesenchymal stem cell culture medium for culture;
s2: and then washing with phosphate buffer salt solution to obtain the copper sulfide/manganese dioxide composite material engineered mesenchymal stem cells.
9. The copper sulfide/manganese dioxide composite engineered mesenchymal stem cell of claim 8, wherein in the step S1, the concentration of the copper sulfide/manganese dioxide composite is 12.5 μ g/ml to 100 μ g/ml; the culture time is 2-4 h.
10. Use of the copper sulfide/manganese dioxide composite material according to claim 1 for the preparation of a medicament for rheumatoid arthritis.
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