CN114099468A - Dry cell membrane camouflage nano-medicament based on immune regulation and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a stem cell membrane camouflage nano-drug based on immune regulation, a preparation method and application thereof, wherein the nano-drug is formed by loading a magnetic nano-material and a bone disease treatment drug on a stem cell membrane, and the stem cell membrane camouflage nano-drug can break through various physiological barriers to reach a bone disease focus area through immune evasion and a membrane lubrication interface and realize long-term residence through mutual fusion with local cells and tissues. The long-term immunoregulation treatment on the bone injury area is realized by combining the slow release of the medicine and generating the magnetic effect through the stimulation of an external magnetic field device, and the clinical development potential is very high.
Description
Technical Field
The invention relates to the field of bone-related disease diagnosis and treatment medicines, in particular to an immunomodulatory stem cell membrane camouflage nano-medicine, and further relates to a preparation method of the nano-medicine and application of the nano-medicine in treating bone diseases.
Background
Refractory bone diseases represented by osteoarthritis, osteoporosis and cartilage damage involve processes such as cartilage degradation, subchondral bone sclerosis and osteophyte formation, and are chronic degenerative diseases affecting joints characterized by inflammation, cartilage loss and bone changes. It is estimated that 15% of people over the age of 60 in the world today suffer from clinical symptoms of cartilage damage. Due to the scarcity of blood vessels, damaged cartilage is difficult to self-repair, presents as a degenerative and inflammatory process, and promotes the progression of diseases, however, the mechanisms of the causes and pathological processes of such diseases are still unknown. There is currently no effective treatment, and current treatments focus on symptomatic relief, by means including physical strength enhancement, weight management, and anti-inflammatory agents (non-steroidal anti-inflammatory drugs or glucocorticoids) primarily relieve inflammatory symptoms such as pain, and in severe cases joint transplants. There is still a lack of effective therapeutic techniques to inhibit cartilage degradation.
Some emerging new therapeutic technologies, such as small molecules and proteins, nucleic acid drug therapy, nanomaterials and regenerative medicine therapy, have development potential. But due to the high viscous microenvironment of the surface property of the material and the bone focus area, accurate delivery is difficult to realize; and because of the immune reaction, the material or medicine is easy to be removed, and the long-term residence has problems; and the dose-effect relationship of the materials is not clear, and the safe application of the dose is also risky. Among the regenerative medicine cell therapy methods, Mesenchymal Stem Cells (MSCs) are the most commonly used among them as cells having a regenerative effect. MSCs are known to be involved in wound repair, immunomodulation, and homing of various inflammatory areas. In related applications, it is also in the clinical trial testing phase. Stem cell therapies provide healthy and suitable newly differentiated tissue chondrocytes for cartilage regeneration, and some stem cell therapies have also entered preclinical or even clinical studies. Cell therapy, however, has some drawbacks. About 107The stem cells may cause scar formation and additional bone tissue formation, and the nature of stem cells from different sources may vary greatly, so that the number and type of clinically required stem cells remain to be determined.
It was reported that fragments of mesenchymal stem cells were phagocytosed by monocytes and macrophages in the lung, spleen, liver, kidney and lymph nodes after injection into mice. After phagocytosis of cytoplasmic components of living umbilical cord mesenchymal stem cells by monocytes and macrophages, gene programs of antigen presentation and co-stimulation can be down-regulated, and activation of helper T cells is functionally inhibited, so that a long-term inflammation regulation effect is exerted. Therefore, the mesenchymal stem cells can still play the role of immunoregulation after being eliminated, and the mechanism of long-term anti-inflammation through stem cell fragments such as stem cell membranes and the like provides a wider development idea for the long-term treatment of the stem cells in vivo. Based on the above problems, the cell membrane camouflaging technology has become an effective means for replacing cell therapy. It has been found that membrane-based nanovesicles of MSCs are obtained by extracting and separating the cell membranes of MSCs. The immune regulation performance of the MSCs, particularly the ability of homing inflammatory tissues and immune evasion are kept, and meanwhile, the host cells are not influenced, and the use is safe.
However, the therapeutic effect on bone diseases is still limited by using the independent stem cell membrane vesicles and the stem cell membrane to deliver drugs, and the reconstruction of the damaged bone region needs to be regulated by combining other means. In recent years, the application of magnetic micro-nano materials to the treatment of intractable diseases such as bones and the like has made an important progress: comprises a submillimeter-scale magnetic device to deliver stem cells for knee joint cartilage regeneration; the magnetic mechanical force acts to trigger cell reaction; the iron-based nanoparticles can promote the conversion of macrophages to M1 type with immune activity, and the like. The above advances indicate that magnetic effects are highly likely to have an immunomodulatory effect on the progression of bone disease. However, there are few reports of inducing immunological processes to repair bone injury diseases by magnetic effects including magnetic polarization, magnetocaloric effect, magnetic force, etc. The targeted nano-drug with multiple magnetic effects is utilized to carry out immune regulation on bone injury diseases, has great development prospect and brings eosin for the treatment of bone diseases.
Disclosure of Invention
In view of the above, one of the objectives of the present invention is to provide an immunomodulatory-based stem cell membrane camouflage nano-drug, which has significant magnetic effect, disease area targeting performance and immune evasion ability; the second purpose of the invention is to provide a preparation method of the stem cell membrane camouflage nano-drug based on immune regulation; the invention also aims to provide application of the stem cell membrane camouflage nano-drug based on immune regulation in preparation of a drug for treating bone diseases, wherein the nano-drug can be delivered in a targeted manner and stays in a diseased area for a long time, and the bone inflammation area is treated by utilizing double immune regulation of magnetic effect and drug.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the nano-drug is formed by loading a magnetic nano-material and a bone disease treatment drug on a stem cell membrane.
Preferably, the stem cell membrane is derived from one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord blood mesenchymal stem cells and neural stem cells.
Preferably, the bone disease is one or more of osteoporosis, osteoarthritis and cartilage damage.
Preferably, the bone disease treatment drug is a small molecule drug, a protein drug, a nucleic acid drug or an inorganic nano material.
Preferably, the small molecule drug is selected from but not limited to 2- ([1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid or strontium ranelate; the protein drug is selected from but not limited to fibroblast growth factor FGF-18 or dissuzumab; the nucleic acid micro ribonucleic acid 21-5p and 124-3p and inorganic nano material hydroxyapatite.
Preferably, the magnetic nano material is prepared from any one or more of ferroferric oxide, gamma-ferric oxide, cobaltosic oxide, manganese ferrite, zinc ferrite, cobalt ferrite, manganous oxide and manganese dioxide.
2. The preparation method of the stem cell membrane camouflage nano-drug based on immune regulation comprises the steps of preparing a stem cell membrane by using stem cells, loading a magnetic nano-material and a bone disease treatment drug into the stem cell membrane, and obtaining the stem cell membrane camouflage nano-drug with uniform size by ultrasonic or extrusion.
Preferably, the method for preparing the stem cell membrane comprises the steps of hypotonic treatment, homogenization and ultrasonic treatment of the stem cell, and extraction to obtain the stem cell membrane.
Preferably, the magnetic nanomaterial and the bone disease therapeutic drug loading method are selected from, but not limited to, an acoustic perforation method, an electroporation method, or a magnetic field induction method.
Preferably, the magnetic nanomaterial is prepared by a gas phase method, a liquid phase method or a solid phase method.
Preferably, the magnetic nanomaterial is prepared by a chemical coprecipitation method or a hydrothermal method.
3. The application of the stem cell membrane camouflage nano-drug based on immune regulation in preparing a drug for treating bone diseases.
The invention has the beneficial effects that: the stem cell membrane camouflage nano medicine based on immune regulation provided by the invention has the advantages that the stem cell membrane is loaded with the nano material with the magnetic effect and the bone inflammation treatment medicine, the physiological barrier can be broken through the magnetic targeting and chemotaxis, the bone inflammation treatment medicine can be accurately delivered to the bone inflammation tissue, the immune system can be well escaped from being cleared due to low immunogenicity, and after entering a diseased region, the stem cell membrane camouflage nano medicine can be rapidly fused into local cells and tissues due to the lubricating property of a membrane interface, so that the long-term residence in the diseased region is realized. Further regulating and controlling the immune response of the bone inflammation area based on the magnetic effect, combining the slow release of the therapeutic drug, and synergistically improving the pathological microenvironment of the bone inflammation area to achieve the final effect of continuously and radically treating bone-related diseases such as osteoporosis, osteoarthritis, cartilage injury and the like for a long time.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a schematic diagram of the mechanism of treatment of a diseased area by a stem cell membrane disguised nano-drug penetrating a biological barrier;
FIG. 2 is a flow chart of the process of masking the effect of the nano-drug by the stem cell membrane;
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 adipose-derived mesenchymal stem cell membrane-camouflaged ferroferric oxide and disitumumab nano-drug
The fat-derived mesenchymal stem cell membrane camouflage nano-drug comprises three components of a fat-derived mesenchymal stem cell membrane, ferroferric oxide nano-particles (with the size of 10-15nm) and a disulking monoclonal antibody (an anti-osteoporosis drug), wherein the size of the nano-drug is 100-200nm, the drug loading is 6-18mg/mL of iron content, and the disulking monoclonal antibody is 5-20 mg/mL.
The preparation process comprises the steps of firstly, extracting and preparing uniform stem cell membranes from adipose-derived mesenchymal stem cells by means of hypotonic, homogeneous and ultrasonic treatment; meanwhile, ferroferric oxide nano particles with good water dispersibility are prepared by a chemical coprecipitation method; and secondly, loading ferroferric oxide nanoparticles and the disitumumab into the stem cell membrane by using an acoustic perforation method, and obtaining the stem cell membrane camouflage nano-medicament with uniform size by using an extrusion method.
In the application of an osteoporosis mouse model, T2 magnetic resonance signal change in an osteoporosis region is monitored through magnetic resonance imaging within 48 hours by tail vein injection, and the signal-to-noise ratio of the region is gradually improved by 30-80%, which shows that the targeted enrichment of the nano-drug in a focus region is realized. After targeted delivery is determined to be complete, the osteoporotic region is magnetically stimulated using a variety of external magnetic field devices while magnetic resonance imaging observations are made periodically. After 2 weeks of application, collecting index information such as inflammatory related cytokines and related gene expression levels, and evaluating osteoporosis treatment conditions, specifically comprising: compared with the level before treatment, the production of bone morphogenetic protein BMP2 is improved by 40-60%, the mRNA expression of the nuclear factor kappa B receptor activator ligand RANKL is reduced by 55-70%, and the osteoporosis is obviously improved.
The treatment mechanism of this embodiment is shown in fig. 1. After injection, the injection rapidly breaks through physiological barriers such as cartilage tissue barrier and bone tissue under the action of a magnetic field and chemotaxis to reach a disease area, actively targets the bone tissue, stays for a long time, then is slowly released, and realizes diagnosis and treatment on a bone inflammation area by utilizing double immune regulation of magnetic effect and medicament, wherein the action process is shown in figure 2.
Example 2 bone marrow-derived mesenchymal stem cell membrane camouflaged manganese dioxide and strontium ranelate nano-drug
The marrow-derived mesenchymal stem cell membrane camouflage nano-drug comprises three components of a marrow-derived mesenchymal stem cell membrane, manganese dioxide nano-sheets (with the radial dimension of 50-85nm) and strontium ranelate (anti-osteoporosis drug), wherein the size of the nano-drug is 200-400 nm. The drug loading rate is 10-25mg/mL of manganese content and 50-70mg/mL of strontium ranelate.
The preparation process of the bone marrow-derived mesenchymal stem cell membrane is similar to that of the adipose-derived mesenchymal stem cell membrane in example 1; simultaneously, preparing a manganese dioxide nanosheet with good water dispersibility by adopting a hydrothermal method; and then loading the manganese dioxide nanosheets and the strontium ranelate into the dry cell membrane by an electroporation method, and obtaining the dry cell membrane camouflage nano-drug with uniform size by an ultrasonic method.
The therapeutic application to the osteoporosis mouse model was similar to the procedure of example 1. The signal-to-noise ratio of T2 magnetic resonance in the affected area gradually increases to 20-50% within 48 hours. After treatment by magnetic field effect and the like, the content of the type I collagen in the serum and the content of the pyridinoline and the deoxypyridinoline are respectively reduced by 30-55 percent, 40-60 percent and 45-70 percent compared with the level before treatment, which shows that the osteoporosis is obviously improved.
Example 3 umbilical cord blood-derived mesenchymal stem cell membrane-disguised zinc ferrite and hydroxyapatite nano-drug
The umbilical cord blood-derived mesenchymal stem cell membrane camouflage nano-drug comprises three components of an umbilical cord blood-derived mesenchymal stem cell membrane, zinc ferrite nanoparticles (with the size of 100-800 nm) and hydroxyapatite (a bone injury repair drug with the size of 80-20nm), wherein the size of the nano-drug is 500-800 nm. The drug loading rate is 20-50mg/mL of iron content and 10-20mg/mL of phosphorus content.
The preparation process of the umbilical cord blood-derived mesenchymal stem cell membrane is similar to the preparation process of the adipose-derived mesenchymal stem cell membrane in example 1; simultaneously, preparing zinc ferrite nanoparticles and hydroxyapatite nanoparticles with good water dispersibility by a hydrothermal method respectively; and secondly, loading the zinc ferrite nanoparticles and the disitumumab into the dry cell membrane by using a magnetic field induction method, and obtaining the dry cell membrane camouflage nano-medicament with uniform size by using an ultrasonic crushing method.
In the application of a cartilage injury mouse model, T2 magnetic resonance signal change of a cartilage injury area is monitored by magnetic resonance imaging within 60 hours through tail vein injection, and the signal-to-noise ratio of the area is gradually improved by 30-60%. The bone injury area is then magnetically stimulated with various external magnetic field devices while periodically performing magnetic resonance imaging observations. Collecting index information such as inflammation-related cytokine and related gene expression level, and evaluating the condition of cartilage repair, specifically comprising: compared with the level before treatment, the generation amount of the bone specific alkaline phosphatase ALP is increased by 40-60%, and the expression amounts of genes such as cartilage/bone related markers RUNX2, AGG, OCN, SOX9 and the like are increased by 30-65%, which indicates that cartilage injury is well repaired.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (12)
1. The immune regulation-based stem cell membrane camouflage nano-drug is characterized in that: the nano-drug is formed by loading a magnetic nano-material and a bone disease treatment drug on a dry cell membrane.
2. The immunomodulatory-based stem cell membrane camouflaged nano-drug of claim 1, wherein: the stem cell membrane is derived from one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord blood mesenchymal stem cells and neural stem cells.
3. The immunomodulatory-based stem cell membrane camouflaged nano-drug of claim 1, wherein: the bone disease is one or more of osteoporosis, osteoarthritis and cartilage injury.
4. The immunomodulatory-based stem cell membrane camouflaged nano-drug of claim 1, wherein: the bone disease treatment drug is a small molecule drug, a protein drug, a nucleic acid drug or an inorganic nano material.
5. The immunomodulatory-based stem cell membrane camouflaged nano-drug of claim 1, wherein: the small molecule drug is selected from but not limited to 2- ([1, 1-biphenyl ] -4-yl carbamoyl) benzoic acid or strontium ranelate; the protein drug is selected from but not limited to fibroblast growth factor FGF-18 or dissuzumab; the nucleic acid micro ribonucleic acid 21-5p and 124-3p and inorganic nano material hydroxyapatite.
6. The immunomodulatory-based stem cell membrane camouflaged nano-drug of claim 1, wherein: the magnetic nano material is prepared from any one or more of but not limited to ferroferric oxide, gamma-ferric oxide, cobaltosic oxide, manganese ferrite, zinc ferrite, cobalt ferrite, manganous oxide and manganese dioxide.
7. The method for preparing the immunomodulatory-based stem cell membrane camouflage nano medicine, according to any one of claims 1 to 6, is characterized in that: the stem cell membrane is prepared by using the stem cell, then the magnetic nano material and the bone disease treatment drug are loaded into the stem cell membrane, and the stem cell membrane camouflage nano drug with uniform size is obtained by ultrasonic or extrusion.
8. The method for preparing the immunomodulatory-based stem cell membrane camouflaged nano-drug according to claim 7, wherein the method comprises the following steps: the method for preparing the stem cell membrane comprises the steps of hypotonic treatment, homogenization and ultrasonic treatment of the stem cell, and the stem cell membrane is obtained through extraction.
9. The method for preparing the immunomodulatory-based stem cell membrane camouflaged nano-drug according to claim 7, wherein the method comprises the following steps: the magnetic nano material and the bone disease treatment drug loading method are selected from but not limited to an acoustic perforation method, an electroporation method or a magnetic field induction method.
10. The method for preparing the immunomodulatory-based stem cell membrane camouflaged nano-drug according to claim 7, wherein the method comprises the following steps: the magnetic nano material is prepared by a gas phase method, a liquid phase method or a solid phase method.
11. The method for preparing the immunomodulatory-based stem cell membrane camouflaged nano-drug according to claim 7, wherein the method comprises the following steps: the magnetic nano material is prepared by a chemical coprecipitation method or a hydrothermal method.
12. Use of the immunomodulatory-based stem cell membrane camouflage nano-drug of any one of claims 1 to 6 in the preparation of a medicament for treating bone diseases.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114681396A (en) * | 2022-03-07 | 2022-07-01 | 苏州科技大学 | Magnetic traditional Chinese medicine nanogel and preparation method and application thereof |
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| US20150313995A1 (en) * | 2012-12-14 | 2015-11-05 | Medivation Technologies, Inc. | Magnetic Retention of Regenerative Cells for Wound Repair |
| CN111265549A (en) * | 2020-03-02 | 2020-06-12 | 苏州大学 | Surface PD-L1 molecule over-expressed mesenchymal stem cell membrane coated bionic nanoparticle and preparation and application thereof |
| CN111588705A (en) * | 2020-05-29 | 2020-08-28 | 复旦大学附属华山医院 | Mesenchymal stem cell membrane camouflage magnetic drug-loaded nanoparticle and preparation method and application thereof |
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| US20150313995A1 (en) * | 2012-12-14 | 2015-11-05 | Medivation Technologies, Inc. | Magnetic Retention of Regenerative Cells for Wound Repair |
| CN111265549A (en) * | 2020-03-02 | 2020-06-12 | 苏州大学 | Surface PD-L1 molecule over-expressed mesenchymal stem cell membrane coated bionic nanoparticle and preparation and application thereof |
| CN111588705A (en) * | 2020-05-29 | 2020-08-28 | 复旦大学附属华山医院 | Mesenchymal stem cell membrane camouflage magnetic drug-loaded nanoparticle and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114681396A (en) * | 2022-03-07 | 2022-07-01 | 苏州科技大学 | Magnetic traditional Chinese medicine nanogel and preparation method and application thereof |
| CN114681396B (en) * | 2022-03-07 | 2024-04-26 | 苏州科技大学 | Magnetic traditional Chinese medicine nanogel and preparation method and application thereof |
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