CN117100716A - Nano microsphere containing iNKT cells and preparation method thereof - Google Patents
Nano microsphere containing iNKT cells and preparation method thereof Download PDFInfo
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- CN117100716A CN117100716A CN202311092043.2A CN202311092043A CN117100716A CN 117100716 A CN117100716 A CN 117100716A CN 202311092043 A CN202311092043 A CN 202311092043A CN 117100716 A CN117100716 A CN 117100716A
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The invention provides a nano microsphere containing inKT cells and a preparation method thereof, belonging to the technical field of medicines, wherein the preparation method comprises the following steps: s1, preparing peripheral blood mononuclear cells; s2, preparing a culture medium; s3, cell culture; s4, embedding and modifying; s5, magnetic deposition. The prepared magnetic nano microsphere containing inKT cells can enable the medicine to directionally move to a lesion part under the action of an external magnetic field, and meanwhile, the medicine carrier for controlling the release of the targeted medicine has the characteristics of high efficiency and low toxicity, and the microsphere has the advantages of high content of inKT cells, good anti-tumor and anti-inflammatory effects, small toxic and side effects, good quality effect, capability of targeted medicine release and wide application prospect.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a nano microsphere containing inKT cells and a preparation method thereof.
Background
However, killer T cells (NKT cells) refer to lymphocytes expressing both NK cell surface. Shicd 56 (. NK 1.1) and T cell surface marker tcrαβ -CD3 complexes. NKT cells differentiate and develop in the thymus or embryo, mainly in the marrow, liver and thymus, and are present in small amounts in the spleen, lymph nodes and periphery.
NKT cells have the advantage of having the potential to activate various effector cell types simultaneously, including cd8+ T and NK cells, thereby eliminating MHC positive and MHC negative tumor cells. In addition, activated NKT cells can interact with CD1d receptor of immature DC cells via TCR (vα24jα18) to induce DC maturation, thereby allowing the DC cells to rapidly ingest WT1 tumor antigen polypeptides, and then present the fragments to cd4+ memory cells, establishing stable long-term anti-tumor immunity. Thus, NKT cell targeted therapies are considered ideal therapeutic approaches to combat cancer and prevent recurrent metastasis of tumors.
iNKT cells express the T Cell Receptor (TCR) vα24jα18, which interacts with CD1d on Antigen Presenting Cells (APC). In addition, iNKT cells produce ifnγ upon activation and interact with Dendritic Cells (DCs) through CD40L-CD40 interactions. In response, the DC produces IL-12, which then stimulates activation of the iNKT cells further, forming a positive feedback loop.
NKT cell biological function: mainly comprises immunoregulation and cytotoxin, after NKT cells are stimulated, a large amount of IL-4, IFN-gamma, GM-CSF, IL-13 and other cytokines and chemokines can be secreted, the immunoregulation is performed, and the NKT cells are one of bridges for connecting innate immunity and acquired immunity. After activation, NKT cells have NK cell-like cytotoxic activity, can dissolve NK cell-sensitive target cells, and the main effector molecules are perforin, fas ligand and IFN-gamma.
It is generally considered that lymphocytes constituting the immune system of the body are composed of three cell lines, one is T cells produced by thymus, one is B cells produced by differentiation of bone marrow, and one is natural killer cells (NK cells). Whereas the fourth lymphocyte-NKT cell was newly found to be present.
Discovery of NKT cells in 1986, the characteristic antigen receptor genes of NKT cells were cloned successfully. This gene is designated as Va14 gene and has unique structural characteristics unlike other T cell antigen receptor (TCR) genes. Two groups, fawlkes from the national institute of health and Budd in switzerland, 1987, reported that T cells in thymocytes were generally unable to express the receptor and only a portion of immature T cells were selected to express the V- β8.2 receptor. Subsequent studies demonstrated that such cells are not T cells, and considered to be receptors for NK cells, the number of such cell clusters was very small and physiologically unknown. In 1994, researchers of both groups found that the cells they reported were the same cells, and research from this NKT cell has attracted widespread attention.
Immune function of NKT cells: the antigen recognized by T cells is a protein, and the antigen recognized by NKT cells is α -Gal-Cer, a so-called glycolipid, which is an important difference between the immune system and the general immune system. The differentiation of NKT cells differs from T cells in that they differentiate in extrathymus tissue 6 days prior to thymus formation in the initial fetal life. NKT cells are under-developed compared to T cells. NKT cells not only secrete Th1 and Th2 cytokines, but also have the same killing target cell effect as cd8+ killer T cells. NKT cells occupy an important place in the immune regulation system. NKT cells may have a number of relationships with diseases, and may be involved in the pathogenesis of autoimmune diseases, regulation of allergies, anti-tumor effects, and inhibition of parasitic infections.
Clinical trials for tumor treatment using iNKT cells have been conducted in recent years. Clinical data show that the amplified and cultured inKT cell adoptive immunotherapy tumor has no obvious side effect and has good clinical application prospect. However, due to the low iNKT cell content (about 0.01-1% of lymphocytes) in human peripheral blood, the current amplification method has the technical problems of large initial cell demand, long amplification period, low cell preparation purity, weak activity, large difficulty in targeted drug release and the like, and finally results in low clinical overall remission rate and unsatisfactory anti-tumor effect.
Disclosure of Invention
The invention aims to provide the nano microsphere containing the iNKT cells and the preparation method thereof, wherein the content of the iNKT cells in the microsphere is high, the microsphere has good anti-tumor and anti-inflammatory effects, and the like, has small toxic and side effects and good quality effect, can release medicine in a targeted way, and has wide application prospect.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of nano-microspheres containing iNKT cells, which comprises the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, and centrifuging to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding alpha-galactosylceramide and interferon-gamma into KBM581 culture medium, and uniformly stirring and mixing to obtain the culture medium;
s3, cell culture: adding peripheral blood human mononuclear cells in step S1 into the culture medium in step S2, and adjusting density to 2×10 6 Mixing uniformly/mL, incubating and culturing for 15-17 days, and collecting cell sap;
s4, embedding modification: adding sodium alginate and an emulsifying agent into the cell fluid in the step S3, uniformly stirring and mixing, quickly emulsifying by a membrane, dripping a calcium chloride solution, solidifying at normal temperature to obtain microspheres embedded with inKT cells, adding the microspheres into water, adding dopamine hydrochloride and a catalyst, heating and stirring for reaction to obtain modified embedded microspheres;
s5, magnetic deposition: adding the modified embedded microsphere prepared in the step S4 into water, adding ferric chloride and ferrous chloride, dropwise adding ammonia water, heating and stirring for reaction under the protection of inert gas, centrifuging, washing and drying to obtain the nano microsphere containing the iNKT cells.
As a further improvement of the invention, the rotational speed of the centrifugation is 3000-5000r/min and the time is 15-20min.
As a further improvement of the present invention, the addition amount of alpha-galactosylceramide in the KBM581 medium in the step S2 is 15-20. Mu.g/mL, and the addition amount of interferon-gamma is 500-1200U/mL.
As a further improvement of the present invention, the conditions of the incubation in step S3 are 36-38deg.C and 3-7v/v% CO 2 Is incubated in an incubator.
As a further improvement of the invention, in the step S4, the mass ratio of the cell sap to the sodium alginate to the emulsifying agent is 100:12-15:1-2, the emulsifying agent is lecithin, the concentration of the calcium chloride solution is 3-5wt%, the normal temperature curing time is 20-30min, the mass ratio of the microspheres embedded with the inKT cells to the dopamine hydrochloride to the catalyst is 10-15:17-20:1-2, the catalyst is Tris-HCl solution with pH=8.5-9, the heating and stirring reaction temperature is 45-50 ℃ and the time is 2-4h.
As a further improvement of the invention, in the step S5, the mass ratio of the modified embedded microspheres to the ferric chloride to the ferrous chloride to the ammonia water is 25-30:16-16.4:12.5-12.7:5-7, the concentration of the ammonia water is 22-25wt%, and the temperature of the heating and stirring reaction is 60-70 ℃ for 2-4 hours.
As a further improvement of the invention, the method specifically comprises the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, and centrifuging for 15-20min at 3000-5000r/min to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding 15-20 mug/mL of alpha-galactosylceramide and 500-1200U/mL of interferon-gamma into KBM581 medium, and stirring and mixing uniformly to obtain the medium;
s3, cell culture: adding peripheral blood human mononuclear cells in step S1 into the culture medium in step S2, and adjusting density to 2×10 6 Mixing with 3-7v/v% CO at 36-38deg.C 2 Is a constant temperature boxAfter 15-17 days of medium incubation and culture, collecting cell sap;
s4, embedding modification: adding 12-15 parts by weight of sodium alginate and 1-2 parts by weight of lecithin into 100 parts by weight of the cell fluid obtained in the step S3, stirring and mixing uniformly, emulsifying by a rapid membrane, dripping 10 parts by weight of 3-5wt% of calcium chloride solution, solidifying at normal temperature for 20-30min to obtain microspheres embedded with inKT cells, adding 10-15 parts by weight of microspheres embedded with inKT cells into 100 parts by weight of water, adding 17-20 parts by weight of dopamine hydrochloride and 1-2 parts by weight of catalyst, heating to 45-50 ℃, stirring and reacting for 2-4h to obtain modified embedded microspheres;
the catalyst is Tris-HCl solution with pH=8.5-9;
s5, magnetic deposition: adding 25-30 parts by weight of the modified embedded microsphere prepared in the step S4 into 100 parts by weight of water, adding 16-16.4 parts by weight of ferric chloride and 12.5-12.7 parts by weight of ferrous chloride, dropwise adding 5-7 parts by weight of 22-25wt% ammonia water, heating to 60-70 ℃ under the protection of nitrogen, stirring and reacting for 2-4h, centrifuging for 15-20min at 3000-5000r/min, washing, and drying to obtain the nano microsphere containing iNKT cells.
The invention further provides the nano microsphere containing the iNKT cells, which is prepared by the preparation method.
The invention further provides the use of a nanoparticle comprising iNKT cells as described above for the preparation of a medicament for the diagnosis and treatment of cancer, degenerative diseases, brain diseases, cardiovascular diseases, infectious diseases, transplantation-related diseases, autoimmune diseases, or for the treatment of tumors, brain diseases, cardiovascular diseases, degenerative diseases, infectious diseases, transplantation-related diseases, cirrhosis and other diseases characterized by fibrosis, diseases characterized by recurrent fetal loss, intrauterine fetal death, neonatal diseases, congenital and acquired coagulation dysfunction, genetic diseases, autoimmune diseases, or for pain relief.
The invention further provides application of the nano microsphere containing iNKT cells in preparing medicines for diagnosing and treating tumors and related diseases of complications thereof.
The invention has the following beneficial effects: the prepared magnetic nano-microsphere containing inKT cells can move the medicine to a lesion site in a directional way under the action of an external magnetic field, and meanwhile, the medicine carrier for controlling the release of the targeted medicine has the characteristics of high efficiency and low toxicity, and the prepared nano-microsphere containing inKT cells can co-express T cell receptors and NK cell receptors, contains abundant CD4+ and DN NKT cells, has a unique restricted expression TCR library, can accept lipid antigens presented by CD1d, plays a role in inherent immunity, has high content of inKT cells in the microsphere, has good anti-tumor, anti-inflammatory and other effects, has small toxic and side effects and good quality effect, can target medicine release, and has wide application prospect.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a preparation method of nano-microspheres containing iNKT cells, which specifically comprises the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, and centrifuging at 3000r/min for 15min to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding 15 mug/mL of alpha-galactosylceramide and 500U/mL of interferon-gamma into KBM581 medium, and stirring and mixing uniformly to obtain the medium;
s3, cell culture: adding peripheral blood human mononuclear cells in step S1 into the culture medium in step S2, and adjusting density to 2×10 6 Mixing with/mL, 36℃and 3v/v% CO 2 After 15 days of incubation and culture in an incubator, collecting cell sap;
s4, embedding modification: adding 12 parts by weight of sodium alginate and 1 part by weight of lecithin into 100 parts by weight of the cell liquid obtained in the step S3, stirring and mixing uniformly, emulsifying by a rapid membrane, wherein the pore diameter of the membrane is 500nm, dropwise adding 10 parts by weight of 3wt% calcium chloride solution, curing at normal temperature for 20min to obtain microspheres embedded with inKT cells, adding 10 parts by weight of microspheres embedded with inKT cells into 100 parts by weight of water, adding 17 parts by weight of dopamine hydrochloride and 1 part by weight of catalyst, heating to 45 ℃, stirring and reacting for 2h to obtain modified embedded microspheres;
the catalyst is Tris-HCl solution with pH=8.5;
s5, magnetic deposition: and (3) adding 25 parts by weight of the modified embedded microsphere prepared in the step (S4) into 100 parts by weight of water, adding 16 parts by weight of ferric chloride and 12.5 parts by weight of ferrous chloride, dropwise adding 5 parts by weight of 22wt% ammonia water, heating to 60 ℃ under the protection of nitrogen, stirring and reacting for 2 hours, centrifuging for 15 minutes at 3000r/min, washing, and drying to obtain the nano microsphere containing inKT cells.
Example 2
The embodiment provides a preparation method of nano-microspheres containing iNKT cells, which specifically comprises the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, and centrifuging at 5000r/min for 20min to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding 20 mug/mL of alpha-galactosylceramide and 1200U/mL of interferon-gamma into KBM581 medium, and stirring and mixing uniformly to obtain the medium;
s3, cell culture: adding peripheral blood human mononuclear cells in step S1 into the culture medium in step S2, and adjusting density to 2×10 6 Mixing well at 38deg.C and 7v/v% CO 2 After incubation and culture for 17 days in an incubator, collecting cell sap;
s4, embedding modification: adding 15 parts by weight of sodium alginate and 2 parts by weight of lecithin into 100 parts by weight of the cell liquid obtained in the step S3, stirring and mixing uniformly, emulsifying by a rapid membrane, wherein the pore diameter of the membrane is 500nm, dropwise adding 10 parts by weight of 5wt% calcium chloride solution, curing for 30min at normal temperature to obtain microspheres embedded with inKT cells, adding 15 parts by weight of microspheres embedded with inKT cells into 100 parts by weight of water, adding 20 parts by weight of dopamine hydrochloride and 2 parts by weight of catalyst, heating to 50 ℃, stirring and reacting for 4h to obtain modified embedded microspheres;
the catalyst is Tris-HCl solution with pH=9;
s5, magnetic deposition: adding 30 parts by weight of the modified embedded microsphere prepared in the step S4 into 100 parts by weight of water, adding 16.4 parts by weight of ferric chloride and 12.7 parts by weight of ferrous chloride, dropwise adding 7 parts by weight of 25wt% ammonia water, heating to 70 ℃ under the protection of nitrogen, stirring and reacting for 4 hours, centrifuging for 20 minutes at 5000r/min, washing, and drying to obtain the nano microsphere containing inKT cells.
Example 3
The embodiment provides a preparation method of nano-microspheres containing iNKT cells, which specifically comprises the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, centrifuging at 4000r/min for 17min to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding 17 mug/mL of alpha-galactosylceramide and 900U/mL of interferon-gamma into KBM581 medium, and stirring and mixing uniformly to obtain the medium;
s3, cell culture: adding peripheral blood human mononuclear cells in step S1 into the culture medium in step S2, and adjusting density to 2×10 6 Mixing well at 37℃and 5v/v% CO 2 After incubation and culture for 16 days in an incubator, collecting cell sap;
s4, embedding modification: adding 13.5 parts by weight of sodium alginate and 1.5 parts by weight of lecithin into 100 parts by weight of the cell fluid obtained in the step S3, stirring and mixing uniformly, emulsifying by a rapid membrane, wherein the pore diameter of the membrane is 500nm, dropwise adding 10 parts by weight of 4wt% calcium chloride solution, curing at normal temperature for 25min to obtain microspheres embedded with inKT cells, adding 12 parts by weight of microspheres embedded with inKT cells into 100 parts by weight of water, adding 18.5 parts by weight of dopamine hydrochloride and 1.5 parts by weight of catalyst, heating to 47 ℃, and stirring and reacting for 3h to obtain modified embedded microspheres;
the catalyst is Tris-HCl solution with pH=8.7;
s5, magnetic deposition: adding 27 parts by weight of the modified embedded microsphere prepared in the step S4 into 100 parts by weight of water, adding 16.2 parts by weight of ferric chloride and 12.6 parts by weight of ferrous chloride, dropwise adding 6 parts by weight of 23.5wt% ammonia water, heating to 65 ℃ under the protection of nitrogen, stirring and reacting for 3h, centrifuging for 17min at 4000r/min, washing, and drying to obtain the nano microsphere containing inKT cells.
Comparative example 1
The difference from example 3 is that polydopamine modification is not performed in step S4.
The method comprises the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, centrifuging at 4000r/min for 17min to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding 17 mug/mL of alpha-galactosylceramide and 900U/mL of interferon-gamma into KBM581 medium, and stirring and mixing uniformly to obtain the medium;
s3, cell culture: adding peripheral blood human mononuclear cells in step S1 into the culture medium in step S2, and adjusting density to 2×10 6 Mixing well at 37℃and 5v/v% CO 2 After incubation and culture for 16 days in an incubator, collecting cell sap;
s4, embedding modification: adding 13.5 parts by weight of sodium alginate and 1.5 parts by weight of lecithin into 100 parts by weight of the cell fluid obtained in the step S3, stirring and mixing uniformly, emulsifying by a rapid membrane, wherein the pore diameter of the membrane is 500nm, dropwise adding 10 parts by weight of 4wt% calcium chloride solution, and curing for 25 minutes at normal temperature to obtain microspheres embedded with inKT cells;
s5, magnetic deposition: adding 27 parts by weight of the microspheres embedded with iNKT cells prepared in the step S4 into 100 parts by weight of water, adding 16.2 parts by weight of ferric chloride and 12.6 parts by weight of ferrous chloride, dropwise adding 6 parts by weight of 23.5wt% ammonia water, heating to 65 ℃ under the protection of nitrogen, stirring and reacting for 3h, centrifuging for 17min at 4000r/min, washing, and drying to obtain the nano microspheres containing iNKT cells.
Comparative example 2
In comparison with example 3, the difference is that step S5 is not performed.
The method comprises the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, centrifuging at 4000r/min for 17min to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding 17 mug/mL of alpha-galactosylceramide and 900U/mL of interferon-gamma into KBM581 medium, and stirring and mixing uniformly to obtain the medium;
s3, cell culture: adding peripheral blood human mononuclear cells in step S1 into the culture medium in step S2, and adjusting density to 2×10 6 Mixing well at 37℃and 5v/v% CO 2 After incubation and culture for 16 days in an incubator, collecting cell sap;
s4, embedding modification: adding 13.5 parts by weight of sodium alginate and 1.5 parts by weight of lecithin into 100 parts by weight of the cell fluid obtained in the step S3, stirring and mixing uniformly, emulsifying by a rapid membrane, wherein the pore diameter of the membrane is 500nm, dropwise adding 10 parts by weight of 4wt% calcium chloride solution, curing at normal temperature for 25min to obtain microspheres embedded with inKT cells, adding 12 parts by weight of microspheres embedded with inKT cells into 100 parts by weight of water, adding 18.5 parts by weight of dopamine hydrochloride and 1.5 parts by weight of catalyst, heating to 47 ℃, stirring and reacting for 3h to obtain modified embedded microspheres, namely nano microspheres containing inKT cells;
the catalyst was Tris-HCl solution at ph=8.7.
Comparative example 3
In comparison with example 3, the difference is that step S4 and step S5 are not performed.
The method comprises the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, centrifuging at 4000r/min for 17min to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding 17 mug/mL of alpha-galactosylceramide and 900U/mL of interferon-gamma into KBM581 medium, and stirring and mixing uniformly to obtain the medium;
s3, cell culture: adding peripheral blood human mononuclear cells in step S1 into the culture medium in step S2, and adjusting density to 2×10 6 Mixing well at 37℃and 5v/v% CO 2 After incubation in an incubator for 16 days, the cell fluid was collected.
Test example 1
Planting SMMC-7721 cells in armpit of experimental nude mice, 2×10 7 And randomly dividing the tumors into 7 groups when the tumors grow to be 3mm in diameter, and taking 14 tumors into the groups through tail veins. Examples 1 to 3 and comparative examples 1 to 3, the obtained products were prepared into 50mg/L aqueous solutions with physiological saline, 2 mL/dose was administered, 2 times a week, and the model group was administered with an equivalent amount of physiological saline. Meanwhile, the armpit strong bandage of the nude mice is used for fixing the magnet, the experimental period is 2 weeks, and the tumor volume inhibition rate and the tumor weight inhibition rate are observed. The results are shown in Table 1.
TABLE 1
As shown in the table above, the nano-microspheres containing iNKT cells prepared in examples 1-3 of the present invention have excellent tumor inhibition effect.
Compared with example 3, in comparative example 1, the magnetic deposition of the magnetic ferroferric oxide is unstable without polydopamine modification in step S4, and the prepared nano-microsphere containing iNKT cells has low magnetism, so that the effect of targeted therapy is reduced.
In comparative example 2, step S5 was not performed compared with example 3, and thus the prepared iNKT cell-containing nanoparticle had no magnetism and no targeting quality effect.
Comparative example 3 compared with example 3, the iNKT cell-containing fluid alone was easily dispersed without being embedded without performing steps S4 and S5, and the tumor-suppressing effect was significantly reduced.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The preparation method of the nano microsphere containing the inKT cells is characterized by comprising the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, and centrifuging to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding alpha-galactosylceramide and interferon-gamma into KBM581 culture medium, and uniformly stirring and mixing to obtain the culture medium;
s3, cell culture: adding peripheral blood human mononuclear cells in step S1 into the culture medium in step S2, and adjusting density to 2×10 6 Mixing uniformly/mL, incubating and culturing for 15-17 days, and collecting cell sap;
s4, embedding modification: adding sodium alginate and an emulsifying agent into the cell fluid in the step S3, uniformly stirring and mixing, quickly emulsifying by a membrane, dripping a calcium chloride solution, solidifying at normal temperature to obtain microspheres embedded with inKT cells, adding the microspheres into water, adding dopamine hydrochloride and a catalyst, heating and stirring for reaction to obtain modified embedded microspheres;
s5, magnetic deposition: adding the modified embedded microsphere prepared in the step S4 into water, adding ferric chloride and ferrous chloride, dropwise adding ammonia water, heating and stirring for reaction under the protection of inert gas, centrifuging, washing and drying to obtain the nano microsphere containing the iNKT cells.
2. The method according to claim 1, wherein the centrifugal speed is 3000-5000r/min for 15-20min.
3. The method according to claim 1, wherein the amount of α -galactosylceramide added to the KBM581 medium in step S2 is 15-20. Mu.g/mL, and the amount of interferon- γ added is 500-1200U/mL.
4. The method according to claim 1, wherein the incubation conditions in step S3 are 36-38deg.C and 3-7v/v% CO 2 Is incubated in an incubator.
5. The preparation method according to claim 1, wherein in step S4, the mass ratio of the cell sap, sodium alginate and emulsifier is 100:12-15:1-2, the emulsifier is lecithin, the concentration of the calcium chloride solution is 3-5wt%, the curing time at normal temperature is 20-30min, the mass ratio of the microspheres embedding iNKT cells, dopamine hydrochloride and catalyst is 10-15:17-20:1-2, the catalyst is Tris-HCl solution with ph=8.5-9, the temperature of the heating and stirring reaction is 45-50 ℃ for 2-4h.
6. The preparation method according to claim 1, wherein in the step S5, the mass ratio of the modified embedded microspheres to the ferric chloride to the ferrous chloride to the ammonia water is 25-30:16-16.4:12.5-12.7:5-7, the concentration of the ammonia water is 22-25wt%, and the temperature of the heating and stirring reaction is 60-70 ℃ for 2-4h.
7. The preparation method according to claim 1, characterized by comprising the following steps:
s1, preparing peripheral blood mononuclear cells: taking peripheral blood, anticoagulating heparin, separating by using lymphocyte separation medium to obtain peripheral blood mononuclear cells, washing by using phosphate buffer solution, and centrifuging for 15-20min at 3000-5000r/min to obtain peripheral blood human mononuclear cells;
s2, preparing a culture medium: adding 15-20 mug/mL of alpha-galactosylceramide and 500-1200U/mL of interferon-gamma into KBM581 medium, and stirring and mixing uniformly to obtain the medium;
s3, cell culture: will be in step S1Adding peripheral blood human mononuclear cells into the culture medium in step S2, and adjusting the density to 2×10 6 Mixing with 3-7v/v% CO at 36-38deg.C 2 Incubating in an incubator for 15-17 days, and collecting cell sap;
s4, embedding modification: adding 12-15 parts by weight of sodium alginate and 1-2 parts by weight of lecithin into 100 parts by weight of the cell fluid obtained in the step S3, stirring and mixing uniformly, emulsifying by a rapid membrane, dripping 10 parts by weight of 3-5wt% of calcium chloride solution, solidifying at normal temperature for 20-30min to obtain microspheres embedded with inKT cells, adding 10-15 parts by weight of microspheres embedded with inKT cells into 100 parts by weight of water, adding 17-20 parts by weight of dopamine hydrochloride and 1-2 parts by weight of catalyst, heating to 45-50 ℃, stirring and reacting for 2-4h to obtain modified embedded microspheres;
the catalyst is Tris-HCl solution with pH=8.5-9;
s5, magnetic deposition: adding 25-30 parts by weight of the modified embedded microsphere prepared in the step S4 into 100 parts by weight of water, adding 16-16.4 parts by weight of ferric chloride and 12.5-12.7 parts by weight of ferrous chloride, dropwise adding 5-7 parts by weight of 22-25wt% ammonia water, heating to 60-70 ℃ under the protection of nitrogen, stirring and reacting for 2-4h, centrifuging for 15-20min at 3000-5000r/min, washing, and drying to obtain the nano microsphere containing iNKT cells.
8. An iNKT cell-containing nanoparticle prepared by the method of any one of claims 1-7.
9. Use of the iNKT cell-containing nanoball of claim 8 in the manufacture of a medicament for the diagnosis and treatment of cancer, degenerative diseases, brain diseases, cardiovascular diseases, infectious diseases, transplantation-related diseases, autoimmune diseases, or for the treatment of tumors, brain diseases, cardiovascular diseases, degenerative diseases, infectious diseases, transplantation-related diseases, cirrhosis and other diseases characterized by fibrosis, diseases characterized by recurrent fetal loss, intrauterine fetal death, neonatal diseases, congenital and acquired coagulation dysfunction, genetic diseases, autoimmune diseases, or for pain relief.
10. Use of the iNKT cell-containing nanoball of claim 8 in the manufacture of a medicament for diagnosis and treatment of tumor and its complications related diseases.
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