CN115154618A - Mesoporous silica sustained-release preparation for cancerous ascites and preparation method thereof - Google Patents

Mesoporous silica sustained-release preparation for cancerous ascites and preparation method thereof Download PDF

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CN115154618A
CN115154618A CN202210848536.3A CN202210848536A CN115154618A CN 115154618 A CN115154618 A CN 115154618A CN 202210848536 A CN202210848536 A CN 202210848536A CN 115154618 A CN115154618 A CN 115154618A
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mesoporous silica
antibody
silica particles
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liposome
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王一飞
王运生
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Hangzhou Zhunxing Medical Technology Co ltd
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Hangzhou Zhunxing Medical Technology Co ltd
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Abstract

The invention provides a mesoporous silica sustained-release preparation aiming at cancerous ascites and a preparation method thereof, and aims to solve the problems of poor effect of drug in recognizing T cells, short activity maintenance time of the drug in a tumor microenvironment and poor anti-tumor effect in the prior art. The mesoporous silica sustained release preparation comprises mesoporous silica particles, wherein the mesoporous silica particles are loaded with cytokines and wrapped with biotinylated liposome; the biotinylated liposomes are loaded with a T cell function modulator. The preparation method comprises the steps of adsorbing cytokines by mesoporous silica particles to prepare the mesoporous silica particles loaded with the cytokines; wrapping biotinylated liposome on the surface of the silica particle loaded with the cytokine; on the surface of biotinylated liposome, a T cell function regulator is linked to biotin via streptavidin. The mesoporous silica sustained release preparation has good T cell recognition effect, long activity maintenance time of the drug in a tumor microenvironment and good anti-tumor effect.

Description

Mesoporous silica sustained-release preparation for cancerous ascites and preparation method thereof
Technical Field
The invention belongs to the technical field of treatment medicines for tumor immunocytes, and particularly relates to a mesoporous silica sustained-release preparation for cancerous ascites and a preparation method thereof.
Background
Normally, when tumor cells invade normal tissues of the body, the immune system eliminates them by recognizing antigens expressed on the surface of the tumor. However, tumor cells can evade attack by the body's immune system through a variety of mechanisms, which are critical to the development and metastasis of cancer. Tumor immune escape mechanisms mainly include: tumor cells down-regulate the expression of surface antigens to avoid the recognition of immune cells, the tumor cells highly express immune check points such as PD-1, CTLA-1 and the like to inhibit the tumoricidal activity of T cells, the tumor cells recruit or activate immunosuppressive cells such as MDSC and Treg into a tumor microenvironment through secreting immunosuppressive factors, and the cells secrete cytokines such as IL-10 and the like to inhibit tumor immune response. The tumor immunotherapy mainly achieves the treatment effect by improving the tumor microenvironment and reactivating immune response to kill tumor cells. Clinically, common tumor immunotherapy mainly comprises the step of delivering cell factors such as IL-2, IFN-gamma and the like and T cell function regulators such as anti-CD 3 antibodies, anti-PD-1 antibodies and the like to tumor parts to regulate immune response of organisms, so that tumor elimination is realized.
However, most of the existing immunopharmaceuticals mainly comprise: the cell factors and the like belong to macromolecular drugs, and have the problems of short half-life, quick release, easy initiation of toxic and side effects and the like. Therefore, how to accurately target these drugs to tumor sites and maintain the activity and anti-tumor effect of these drugs in the tumor microenvironment becomes a key issue to be solved currently.
Disclosure of Invention
The invention provides a mesoporous silica sustained-release preparation aiming at cancerous ascites and a preparation method thereof, and aims to solve the problems of poor effect of drug in recognizing T cells, short activity maintenance time of the drug in a tumor microenvironment and poor anti-tumor effect in the prior art.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a mesoporous silica sustained release preparation aiming at cancerous ascites, which comprises mesoporous silica particles, wherein the mesoporous silica particles are loaded with cytokines for regulating an organism to perform tumor immune response; the mesoporous silica particles are wrapped with biotinylated liposome for improving the biocompatibility of the mesoporous silica particles; the biotinylated liposomes are loaded with a T cell function modulator.
The further improved scheme is as follows: the cytokine comprises IL-2, and the cytokine further comprises at least one of IL-12, IL-21 and TGF-beta 1; the cytokines are mixed and loaded into the mesoporous silica particles by a physical adsorption method;
when IL-12 is added, the mass ratio of the IL-2 to the IL-12 is 1: 0.05-0.2;
when IL-21 is added, the mass ratio of the IL-2 to the IL-21 is 1: 0.05-0.3;
when TGF-beta 1 is added, the mass ratio of the IL-2 to the TGF-beta 1 is 1: 0.05-0.25.
Based on the scheme as follows: under the condition of slow release of the proportion, the infiltration degree of TIL (human tumor infiltration lymphocytes) in a tumor microenvironment is stronger, and the ratio of CD8+ T cells which can effectively kill tumor cells is the highest.
The further improved scheme is as follows: the biotinylation liposome is prepared by mixing POPC and Cap PE; the molar ratio of the POPC to the Cap PE is 500-1100: 1.
The further improved scheme is as follows: the mass ratio of the mesoporous silica particles to the biotinylated liposome is 1: 0.81.2; the biotinylated liposomes are disposed in one or two layers.
Two layers of biotinylation liposome are arranged, so that the property of the mesoporous silica sustained release cytokine can be further enhanced, and the biocompatibility of the particles is improved.
The further improved scheme is as follows: the mesoporous silica particles are strip-shaped, the length of the mesoporous silica particles is 80 +/-40 mu m, the width of the mesoporous silica particles is 6 +/-2.5 mu m, and the size of the mesopores is 5 +/-2.5 nm.
Based on the scheme, the mesoporous silica particles adopt the strip shape and the size, so that the mesoporous silica particles can be prevented from being phagocytized by tumor cells (20 microns); the degradation period is increased and thus the sustained release time period is increased.
The further improved scheme is as follows: the modulators of T cell function comprise anti-CD 3 antibodies and the modulators of T cell function further comprise at least one of anti-PD-1 antibodies, CTLA-4 blockers, 4-1BB agonists, and aCCR8 antibodies; the T cell function regulator is connected to the surface of the biotinylated liposome in a mode of connecting streptavidin and biotin;
according to the mass ratio:
anti-CD 3 antibody to anti-PD-1 antibody =1: 0.2-2 when anti-PD-1 antibody is added;
when a CTLA-4 blocker is added, the anti-CD 3 antibody to CTLA-4 blocker =1: 0.2-2;
anti-CD 3 antibody: 4-1BB agonist =1: 0.1-1 when 4-1BB agonist is added;
anti-CD 3 antibody: aCCR8 antibody =1: 1-2 when an aCCR8 antibody is added.
Based on the scheme, the anti-CD 3 antibody is arranged on the outer side, so that T cells can be conveniently identified, and after the anti-CD 3 antibody identifies the T cells, the anti-PD-1 antibody, the CTLA-4 blocker, the 4-1BB agonist and the aCCR8 antibody are used for reactivating the immune response of a tumor microenvironment, activating the tumor killing activity of the T cells in the tumor immune microenvironment and improving the immune response capability of an organism; enhance the proliferation capacity of T cells, promote T cells to secrete effector molecules, and improve the tumor microenvironment in cooperation with cytokine drugs so as to achieve the anti-tumor curative effect.
In addition, the mesoporous silica lipid preparation is used for ascites in vitro T cell amplification from clinical patients in the proportion, and the effect is optimal. In this ratio, the preparation is used for amplifying T cells derived from mouse ascites tumor, and after the T cells are transfused back to the mouse, the anti-tumor effect is the best.
In a second aspect, the invention provides a preparation method of a mesoporous silica sustained-release preparation for cancerous ascites, which comprises the following steps:
s1, adsorbing a cytokine for regulating an organism to perform tumor immune response by using mesoporous silica particles, and preparing the mesoporous silica particles loaded with the cytokine;
s2, wrapping biotinylated liposome on the surface of the silica particle loaded with the cytokine;
and S3, connecting the T cell function regulator on the surface of the biotinylated liposome in a streptavidin and biotin connection mode.
The further improved scheme is as follows: the cell factor comprises IL-2, and the cell factor also comprises at least one of IL-12, IL-21 and TGF-beta 1; the cytokines are mixed and loaded into the mesoporous silica particles by a physical adsorption method;
when IL-12 is added, the mass ratio of the IL-2 to the IL-12 is 1: 0.05-0.2;
when IL-21 is added, the mass ratio of the IL-2 to the IL-21 is 1: 0.05-0.3;
when TGF-beta 1 is added, the mass ratio of the IL-2 to the TGF-beta 1 is 1: 0.05-0.25.
Based on the scheme as follows: under the condition of slow release of the proportion, the infiltration degree of TIL in a tumor microenvironment is stronger, and the ratio of CD8+ T cells which can effectively kill tumor cells is the highest.
The further improved scheme is as follows: the preparation method of the biotinylated liposome comprises the following steps:
POPC and Cap PE are mixed according to the molar ratio of 500-1100: 1, a liposome film is obtained through rotary evaporation, PBS is added for ultrasonic mixing, and biotinylation liposome with uniform size is obtained through physical extrusion.
The further improved scheme is as follows: in step S2, during the process of coating biotinylated liposome on the surface of the silica microparticle loaded with cytokine: and mixing the mesoporous silica particles and the biotinylation liposome at room temperature for 1h according to the mass ratio of 1: 0.8-1.2 to obtain the silica particles with the biotinylation liposome wrapped on the surface.
The further improved scheme is as follows: the biotinylated liposomes were disposed in two layers.
Two layers of biotinylation liposome are arranged, so that the property of the mesoporous silica sustained release cytokine can be further enhanced, and the biocompatibility of the particles is improved.
The further improved scheme is as follows: the mesoporous silica particles are strip-shaped, the length of the mesoporous silica particles is 80 +/-40 mu m, the width of the mesoporous silica particles is 6 +/-2.5 mu m, and the size of the mesopores is 5 +/-2.5 nm.
The further improved scheme is as follows: the modulators of T cell function comprise anti-CD 3 antibodies and the modulators of T cell function further comprise at least one of anti-PD-1 antibodies, CTLA-4 blockers, 4-1BB agonists, and aCCR8 antibodies; the T cell function regulator is connected to the surface of the biotinylated liposome in a mode of connecting streptavidin and biotin;
according to the mass ratio:
anti-CD 3 antibody to anti-PD-1 antibody =1: 0.2-2 when anti-PD-1 antibody is added;
when a CTLA-4 blocking agent is added, the anti-CD 3 antibody to CTLA-4 blocking agent =1: 0.2-2;
anti-CD 3 antibody: 4-1BB agonist =1: 0.1-1 when 4-1BB agonist is added;
anti-CD 3 antibody: aCCR8 antibody =1: 1-2 when an aCCR8 antibody is added.
Based on the scheme: in the proportion, the mesoporous silica lipid preparation is used for ascites in vitro T cell amplification from clinical patients, and the effect is optimal.
The invention has the beneficial effects that:
the invention discloses a mesoporous silica lipid preparation aiming at cancerous ascites, wherein IL-2, IL-12, IL-21, TGF-beta 1 and other cytokines used for regulating an organism to carry out tumor immune response are loaded in mesoporous silica, and due to the physical characteristics of the mesoporous silica and the coating of mesoporous silica particles, biotinylation liposome is coated; the cell factors can be slowly released from the mesoporous silica particles (equivalent to a scaffold), so that the biological activity of the cell factors is prolonged; and cytokines such as IL-2 and the like are gradually released along with the degradation of the stent.
The surface of the mesoporous silica particle is wrapped with the biotinylation liposome similar to a cell membrane, so that the rigidity and hardness of the surface of the mesoporous silica particle are adjusted, and the biocompatibility of the mesoporous silica particle is improved.
Finally, connecting T cell function regulators such as an anti-CD 3 antibody, an anti-PD-1 antibody, a CTLA-4 blocker, a 4-1BB agonist, an aCCR8 antibody and the like on the surface of the biotinylated liposome; the anti-CD 3 antibody is arranged on the outer side and is convenient for recognizing T cells, after the anti-CD 3 antibody recognizes the T cells, the anti-PD-1 antibody, the CTLA-4 blocker, the 4-1BB agonist and the aCCR8 antibody are used for reactivating the immune response of a tumor microenvironment, enhancing the proliferation capacity of the T cells, promoting the T cells to secrete effector molecules, and improving the tumor microenvironment in cooperation with cytokine drugs to achieve an anti-tumor curative effect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and it will be apparent to those skilled in the art that other relevant drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a diagram showing the state of cells cultured for 2 days in the present invention.
FIG. 2 is a diagram showing the state of cells cultured for 5 days and 10 days, respectively, in the present invention.
FIG. 3 is a diagram showing the state of cells cultured for 17 days and 21 days, respectively, in the present invention.
FIG. 4 is a graph showing the change in the number of cells in the present invention.
FIG. 5 is a graph of the human ascites tumor effector T cell phenotype by flow analysis in accordance with the present invention.
FIG. 6 is a graph of human ascites tumor effector memory T cell phenotype by flow analysis in accordance with the present invention.
FIG. 7 is a graph of flow analysis of the depletion phenotype of human ascites tumor effector memory T cells in accordance with the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the invention without inventive step, are within the scope of the invention.
The first embodiment is as follows:
the embodiment provides a mesoporous silica slow-release preparation aiming at cancerous ascites, which comprises mesoporous silica particles, wherein the mesoporous silica particles are loaded with cytokines used for regulating the tumor immune response of an organism; the mesoporous silica particles are wrapped with biotinylated liposome for improving the biocompatibility of the mesoporous silica particles; the biotinylated liposome is loaded with a T cell function modulator.
Wherein the cytokine comprises IL-2, and the cytokine further comprises at least one of IL-12, IL-21, and TGF-beta 1; the cytokines are mixed and loaded into the mesoporous silica particles by a physical adsorption method;
when IL-12 is added, the mass ratio of the IL-2 to the IL-12 is 1: 0.05-0.2;
when IL-21 is added, the mass ratio of the IL-2 to the IL-21 is 1: 0.05-0.3;
when TGF-beta 1 is added, the mass ratio of the IL-2 to the TGF-beta 1 is 1: 0.05-0.25.
Wherein the biotinylated liposome is prepared by mixing POPC and Cap PE; the molar ratio of the POPC to the Cap PE is 500-1100: 1, preferably 1010: 1. Wherein POPC is the main component of the biological membrane, and Cap PE is a biotinylated phospholipid.
Wherein the mass ratio of the mesoporous silica particles to the biotinylation liposome is 1: 0.8-1.2; the biotinylated liposomes are disposed in one or two layers. In a preferred scheme, the mass ratio of the mesoporous silica particles to the biotinylation liposome is 1: 1; the biotinylated liposomes were disposed in two layers.
The mesoporous silica particles are synthesized by a template method, and the preparation method specifically comprises the following steps: the mesoporous silica particles are strip-shaped, the length of the mesoporous silica particles is 80 +/-40 mu m, the width of the mesoporous silica particles is 6 +/-2.5 mu m, and the size of the mesopores is 5 +/-2.5 nm.
Wherein the T cell function modulator is a mixture of anti-CD 3 antibody, anti-PD-1 antibody, CTLA-4 blocker, 4-1BB agonist and aCCR8 antibody; the T cell function regulator is connected to the surface of the biotinylated liposome in a mode of connecting streptavidin and biotin;
according to the mass ratio:
anti-CD 3 antibody to anti-PD-1 antibody =1: 0.2-2;
anti-CD 3 antibody to CTLA-4 blocker =1: 0.2-2;
anti-CD 3 antibody: 4-1BB agonist =1: 0.1-1;
anti-CD 3 antibody: ccr8 antibody =1: 1-2.
When in use, the mesoporous silica sustained-release preparation aiming at the cancerous ascites is injected into the cancerous ascites through the abdominal cavity, so as to achieve the treatment effects of improving the proliferation capacity and the tumor cell killing activity of T cells in the cancerous ascites, killing tumor cells in the cancerous ascites and reactivating the immune response of the tumor microenvironment.
The cytokines such as IL-2, IL-12, IL-21, TGF-beta 1 and the like loaded in the mesoporous silica are used for regulating the tumor immune response of an organism, and due to the physical characteristics of the mesoporous silica, the cytokines can be slowly released from the mesoporous silica particles, so that the biological activity of the cytokines is prolonged. Finally, connecting T cell function regulators such as an anti-CD 3 antibody, an anti-PD-1 antibody, a CTLA-4 blocker, a 4-1BB agonist, an aCCR8 antibody and the like on the surface of the biotinylated liposome; the anti-CD 3 antibody is arranged on the outer side and is convenient for recognizing T cells, and after the anti-CD 3 antibody recognizes the T cells, the anti-PD-1 antibody, the CTLA-4 blocker, the 4-1BB agonist and the aCCR8 antibody are used for activating the tumor killing activity of the T cells in a tumor immune microenvironment and improving the immune response capability of an organism.
The second embodiment:
the embodiment provides a preparation method of a mesoporous silica sustained-release preparation for cancerous ascites, which comprises the following steps:
s1, adsorbing a cytokine for regulating a tumor immune response of an organism by using mesoporous silica particles, and preparing the mesoporous silica particles loaded with the cytokine;
the cytokine comprises IL-2, and the cytokine further comprises at least one of IL-12, IL-21 and TGF-beta 1; the cytokines are mixed and loaded into the mesoporous silica particles by a physical adsorption method;
when IL-12 is added, the mass ratio of the IL-2 to the IL-12 is 1: 0.05-0.2;
when IL-21 is added, the mass ratio of the IL-2 to the IL-21 is 1: 0.05-0.3;
when TGF-beta 1 is added, the mass ratio of the IL-2 to the TGF-beta 1 is 1: 0.05-0.25.
The IL-12, IL-21 and TGF-beta 1 can be added into 1, 2 or 3 types simultaneously.
S2, wrapping biotinylated liposome on the surface of the silica particle loaded with the cytokine;
the preparation method of the biotinylated liposome comprises the following steps: POPC and Cap PE are mixed according to the molar ratio of 500-1100: 1, a liposome film is obtained through rotary evaporation, PBS is added for ultrasonic mixing, and biotinylation liposome with uniform size is obtained through physical extrusion.
In the process of coating the biotinylated liposome on the surface of the silica microparticle loaded with the cytokine: the mesoporous silica particles and the biotinylation liposome are mixed for 1 hour at room temperature (20-30 ℃) according to the mass ratio of 1: 0.8-1.2, and the silica particles with the biotinylation liposome wrapped on the surfaces are obtained.
S3, connecting a T cell function regulator on the surface of the biotinylated liposome in a streptavidin-biotin connection mode;
the modulators of T cell function comprise anti-CD 3 antibodies and the modulators of T cell function further comprise at least one of anti-PD-1 antibodies, CTLA-4 blockers, 4-1BB agonists, and aCCR8 antibodies; the T cell function regulator is connected to the surface of the biotinylated liposome in a mode of connecting streptavidin and biotin;
according to the mass ratio:
anti-CD 3 antibody to anti-PD-1 antibody =1: 0.2-2 when anti-PD-1 antibody is added;
when a CTLA-4 blocking agent is added, the anti-CD 3 antibody to CTLA-4 blocking agent =1: 0.2-2;
anti-CD 3 antibody: 4-1BB agonist =1: 0.1-1 when 4-1BB agonist is added;
anti-CD 3 antibody: aCCR8 antibody =1: 1-2 when an aCCR8 antibody is added.
The invention is further illustrated below with reference to experiments:
1. mouse experiment
1.1 preparation of mesoporous silica particles:
adding 65ml deionized water, 2g P123 and 10ml HCL (37%) into a 250ml reaction flask, stirring the reaction solution for 1-2h under the conditions of 40 ℃ and 600r.p.m., then adding 4.6ml tetraethyl orthosilicate (TEOS) into the reaction solution, and stirring under the conditions of 40 ℃ and 600r.p.m.; after 20 hours, adjusting the stirring condition to 100 ℃, 600r.p.m, and continuing stirring for 20 hours; then carrying out vacuum filtration on the reaction solution, and drying the obtained mesoporous silica solid for 1-2h at room temperature; adding 250ml of absolute ethyl alcohol and 2.5ml of HCL (37%) into a 1L reaction bottle, stirring the mesoporous silica solid at 70 ℃ under the condition of 600r.p.m., filtering the reaction solution in vacuum after 24h, drying the obtained solid at 100 ℃ for 24h to obtain a mesoporous silica product, namely strip-shaped mesoporous silica particles, and sterilizing at high temperature.
1.2 preparation of biotinylated liposomes:
extracting 2.5mgPOPC, 3.6 mu g CAPPE and 500 mu l chloroform in a rotary bottle, and rotationally evaporating the reaction liquid for 1h under the condition of 50 ℃ water bath to obtain a biotinylation liposome film; the resulting biotinylated liposome membrane was added to 1ml of PBS and the biotinylated liposome suspension was vigorously mixed for 1 hour using a maximum speed vortex mixer. The biotinylated liposome suspension was removed and taken up in a pre-wetted syringe, followed by extrusion of the suspension using a biotinylated liposome extruder until the suspension became clear.
1.3 preparation of mesoporous silica sustained release preparation:
gently mixing 4 μ g IL-2, 0.8 μ g IL-12, 1.2 μ g IL-21 and 1 μ g TGF-beta 1 to prepare a cell factor mixture; taking 10mg of mesoporous silica particles and using 200 mul of deionized water for resuspending; then, 50. Mu.l of the mesoporous silica microparticle suspension was placed in an assembly tube, and the assembly tube was supplemented with a cytokine mixture and gently mixed, so that the cytokine was adsorbed at room temperature for one hour or more. Then 1ml of biotinylated liposome was added to the assembly tube and the biotinylated liposome was allowed to coat the mesoporous silica particles for one hour at room temperature. Centrifuging the material suspension at 700g for 5min, removing the supernatant by syringe, washing with 1ml PBS for 2 times, and blocking the precipitate obtained by the last washing with 500 μ l mesoporous silica microparticle blocking solution (0.25% BSA) at room temperature for 10min; gently mixing the material suspension, adding 3 mu g of streptavidin into the assembly tube, and uniformly mixing; streptavidin was allowed to bind to biotinylated lipids for 10 minutes at room temperature, gently mixed every 2 minutes (5 mixes total); mixing 5 μ g of anti-CD 3 antibody, 10 μ g of anti-PD-1 antibody, 10 μ g of CTLA-4 blocker, 5 μ g of 4-1BB agonist and 10 μ g of CCR8 antibody to obtain a T cell ligand mixture; add the T cell ligand mixture to the assembly tube and mix well, allowing the surface ligands to bind to streptavidin for 40 minutes at room temperature, gently mix every 10 minutes (5 mixes total); adding 500 mul of mesoporous silica particle sealing liquid, mixing uniformly, centrifuging for 5 minutes at 700g, and removing supernatant by using a 2ml disposable needle tube; the pellet was washed 2 times with 2ml PBS as above; the pellet from the last wash was resuspended in 1ml sterile PBS.
1.4 establishment of animal model with cancerous ascites in mice
Recovering H22 cells (mouse liver cancer cells) in 37 ℃ water bath, transferring to a T25 bottle containing 5ml of DMEM complete culture medium for culture, and changing the culture solution after 1 day; after 3-4 days of culture, the cells were passaged once, and H22 cells were transferred to T75 flasks for further culture. Discarding the culture medium from T75 flask, washing with PBS for 2 times, supplementing 2ml pancreatin for 5min, adding 10ml DMEM, mixing completely, centrifuging to obtain cell precipitate, re-suspending the precipitate with PBS-free solution, and adjusting cell concentration to 21 × 10 7 Individual cells/ml. 0.2mL (about 2X 10) 7 Tumor cells) are respectively inoculated in 40 BALB/C mice for treating ascites tumor mice by using the intraperitoneal preparation;
after the mice are inoculated with H22 cells for 6 days, the ascites conditions of each mouse are observed and recorded, the ascites mice are randomly divided into 3 groups (a model group, a mesoporous silica sustained-release preparation treatment group and a drug treatment group), 8 mice in each group are treated, and tail part marks serial numbers.
The mice are inoculated with H22 cells for 6 days and then treated, each mouse of the model group is injected with 0.2ml of PBS in ascites, each mouse of the model group is injected with 0.2ml of assembled mesoporous silica sustained-release preparation in ascites, each mouse of the drug treatment group is injected with 0.2ml of tumor immunity drug mixture (4 mug of IL-2, 0.8 mug of IL-12, 1.2 mug of IL-21, 1 mug of TGF- beta 1, 5 mug of anti-CD 3 antibody, 10 mug of anti-PD-1 antibody, 10 mug of CTLA-4 blocker, 5 mug of 4-1BB agonist and 10 mug of CCR8 antibody are supplemented to 1ml with PBS and then mixed to prepare the tumor immunity drug mixture), and the mice of the 3 groups are injected once every 6-7 days, injected for 3 times totally for 18 days and the experiment lasts for 24 days. Recording the survival rate and the abdominal circumference of the mouse every 2 to 3 days; the data recorded are shown in the following table:
group of Survival rate Abdominal circumference (cm)
Model set 0/15 12.2±5.8
Mesoporous silicon dioxide sustained-release preparation treatment group 12/15 6.3±3.1
Drug treatment group 8/15 9.3±4.8
By recording data, the mesoporous silica sustained-release preparation treatment group has higher survival rate; in addition, the abdominal circumference of the mesoporous silica sustained-release preparation treatment group is smaller, which indicates that the abdominal water volume is less; the mesoporous silicon dioxide sustained release preparation has better anti-tumor effect.
In addition, although the medicinal components of the medicament treatment group are the same as those of the mesoporous silica sustained-release preparation treatment group, in the structure of the invention, the mesoporous silica particles are loaded with cytokines, the mesoporous silica particles are wrapped with biotinylated liposomes, and the biotinylated liposomes are loaded with T cell function regulators; the cell factor can be slowly released from the mesoporous silica particles, the biological activity of the cell factor is prolonged, the immune response of a tumor microenvironment is reactivated, the proliferation capacity of T cells is enhanced, T cells are promoted to secrete effector molecules, the immune response capacity of an organism is improved, and the tumor microenvironment is improved by cooperating with a cell factor medicament to achieve an anti-tumor curative effect.
2. In vitro clinical experiments:
for ascites tumors:
patient information: a female age of 31 years; ascites: 1L in total;
and (3) experimental operation:
(1) 200ml of ascites (containing tumor cells and TIL cells) are taken in a centrifuge tube and centrifuged for 8min under the condition of 400g, the cell sediment is resuspended by 25ml of erythrocyte lysate, the erythrocyte lysate is placed under the condition of 4 ℃ and incubated for 4min in the dark, then 20ml of PBS is supplemented and mixed evenly, the centrifugation is carried out, and the cell sediment is resuspended by 5ml of human TIL CM (Complete Medium).
(2) Counting: in total 1.5X10 8 Individual cells (tumor cells and TIL cells), 1.5X10 were taken from them 7 Putting the TIL cells into a T75 bottle, adding the mesoporous silica sustained-release preparation aiming at the cancerous ascites and the human TIL CM, uniformly mixing, and then putting the mixture into an incubator to culture for a plurality of days.
(3) Liquid changing: after 4 days of culture, half of the medium was replaced.
FIG. 1 is a photograph of various cell contents after two days of culture; in the figure, the circles are TIL cells and the spindle (similar to the bar) is a tumor cell.
Referring to fig. 2:
after 5 days of culture, TIL cells began to expand and tumor cells began to expand.
After 10 days of culture, TIL number increased, but the expansion rate was slow, TIL cells tended to concentrate around tumor cells, the number of tumor cells increased, and the condition was good.
Referring to fig. 3:
after 17 days of culture, the TIL cells began to expand greatly and had more aggregates, and the tumor cells were observed to decrease greatly under the microscope.
After 21 days of culture, TIL cells expanded largely and the clump volume became large, at which time no tumor cells were seen under microscope observation.
The amount of cellular changes is shown in FIG. 4: the initial seeding rate is 1.5X10 7 (ii) a The cell number after 14 days of culture was 8X10 7 (ii) a The cell number after 21 days of culture was 3.6X10 8 。
Human ascites tumor TIL cell phenotype was analyzed by flow cytometry:
referring to fig. 5:
after 7 days of culture, the content of CD4+ is 22.72%; after 21 days of culture, the content of CD4+ is 76.11%;
after 7 days of culture, the content of CD8+ is 42.12%; after 21 days of culture, the content of CD8+ was 18.54%.
Although the content of CD8+ was reduced, the total content of effector T cells (CD 4+ and CD8 +) was changed from 66.84% to 94.65%.
Referring to fig. 6:
after 7 days of culture, the content of effector memory T cells (CD 45RO + CCR 7-) was 47.7%; after 21 days of culture, the content of effector memory T cells (CD 45RO + CCR 7-) is 83.74%;
referring to fig. 7:
the content of PD-1 (a marker of T cell depletion) after 7 days of culture was 2.09%; after 21 days of culture, the content of PD-1 is PD-1:5.41 percent;
the content of TIM-3 (marker of T cell depletion) after 7 days of culture was 0.79%; after 21 days of culture, the content of TIM-3 was PD-1:5.33 percent.
It follows that T cell depletion is always maintained at a low level.
In conclusion, the mesoporous silica sustained release preparation prepared by the invention has a growth effect on effector T cells and effector memory T cells, and can improve the killing effect of cancer cells; in addition, the mesoporous silica sustained release preparation prepared by the invention can enable the exhaustion of T cells to be always maintained at a lower level, and is beneficial to maintaining the killing effect of cancer cells.
The invention is not limited to the above alternative embodiments, and any other various forms of products can be obtained by anyone in the light of the present invention, but any changes in shape or structure thereof, which fall within the scope of the present invention as defined in the claims, fall within the scope of the present invention.

Claims (10)

1. A mesoporous silica sustained release preparation aiming at cancerous ascites is characterized in that: the cell-mediated immunity-enhancing agent comprises mesoporous silica particles, wherein the mesoporous silica particles are loaded with cell factors for adjusting the tumor immune response of an organism; the mesoporous silica particles are wrapped with biotinylated liposome for improving the biocompatibility of the mesoporous silica particles; the biotinylated liposomes are loaded with a T cell function modulator.
2. The mesoporous silica sustained-release preparation for cancerous ascites according to claim 1, wherein: the cytokine comprises IL-2, and the cytokine further comprises at least one of IL-12, IL-21 and TGF-beta 1; the cytokines are mixed and loaded into the mesoporous silica particles by a physical adsorption method;
when IL-12 is added, the mass ratio of the IL-2 to the IL-12 is 1;
when IL-21 is added, the mass ratio of the IL-2 to the IL-21 is 1;
when TGF-beta 1 is added, the mass ratio of the IL-2 to the TGF-beta 1 is 1.
3. The mesoporous silica sustained-release preparation for cancerous ascites according to claim 1, wherein: the biotinylation liposome is prepared by mixing POPC and Cap PE; the molar ratio of the POPC to the Cap PE is 500-1100.
4. The mesoporous silica sustained-release preparation for cancerous ascites according to claim 1, wherein: the mass ratio of the mesoporous silica particles to the biotinylated liposome is 1.8-1.2; the biotinylation liposome is arranged into one layer or two layers;
the mesoporous silica particles are strip-shaped, the length of the mesoporous silica particles is 80 +/-40 mu m, the width of the mesoporous silica particles is 6 +/-2.5 mu m, and the size of the mesopores is 5 +/-2.5 nm.
5. The mesoporous silica slow-release preparation aiming at cancerous ascites according to claim 1, which is characterized in that: the modulators of T cell function comprise anti-CD 3 antibodies and the modulators of T cell function further comprise at least one of anti-PD-1 antibodies, CTLA-4 blockers, 4-1BB agonists, and aCCR8 antibodies; the T cell function regulator is connected to the surface of the biotinylated liposome in a mode of connecting streptavidin and biotin;
according to the mass ratio:
when anti-PD-1 antibody was added, anti-CD 3 antibody: anti-PD-1 antibody =1:0.2 to 2;
anti-CD 3 antibodies, when CTLA-4 blocking agent is added: CTLA-4 blocker =1:0.2 to 2;
anti-CD 3 antibodies, when added to a 4-1BB agonist: 4-1BB agonist =1:0.1 to 1;
anti-CD 3 antibody when ccr8 antibody is added: ccr8 antibody =1:1 to 2.
6. A preparation method of a mesoporous silica sustained-release preparation aiming at cancerous ascites is characterized by comprising the following steps:
s1, adsorbing a cytokine for regulating an organism to perform tumor immune response by using mesoporous silica particles, and preparing the mesoporous silica particles loaded with the cytokine;
s2, wrapping biotinylated liposome on the surface of the silica particle loaded with the cytokine;
and S3, connecting the T cell function regulator on the surface of the biotinylated liposome in a streptavidin and biotin connection mode.
7. The method for preparing the mesoporous silica sustained-release preparation for cancerous ascites according to claim 6, wherein the cytokine comprises IL-2, and the cytokine further comprises at least one of IL-12, IL-21 and TGF- β 1; the cytokines are mixed and loaded into the mesoporous silica particles by a physical adsorption method;
when IL-12 is added, the mass ratio of the IL-2 to the IL-12 is 1;
when IL-21 is added, the mass ratio of the IL-2 to the IL-21 is 1;
when TGF-beta 1 is added, the mass ratio of the IL-2 to the TGF-beta 1 is 1.
8. The preparation method of the mesoporous silica sustained release preparation for cancerous ascites according to claim 6, wherein the preparation method of the biotinylated liposome comprises the following steps:
POPC and Cap PE are mixed according to the molar ratio of 500-1100.
9. The method for preparing the mesoporous silica sustained release preparation for cancerous ascites according to claim 6, wherein in the step S2, in the process of coating the biotinylated liposome on the surface of the silica microparticle loaded with the cytokine: and mixing the mesoporous silica particles and the biotinylation liposome at room temperature for 1h according to the mass ratio of 1.8-1.2 to obtain the silica particles with the biotinylation liposome wrapped on the surface.
10. The method for preparing a mesoporous silica sustained release preparation for cancerous ascites according to claim 6, wherein the T cell function regulator comprises an anti-CD 3 antibody, and the T cell function regulator further comprises at least one of an anti-PD-1 antibody, a CTLA-4 blocker, a 4-1BB agonist and an aCCR8 antibody; the T cell function regulator is connected to the surface of the biotinylated liposome in a mode of connecting streptavidin and biotin;
according to the mass ratio:
when anti-PD-1 antibody was added, anti-CD 3 antibody: anti-PD-1 antibody =1:0.2 to 2;
anti-CD 3 antibodies, when CTLA-4 blocking agent is added: CTLA-4 blocker =1:0.2 to 2;
anti-CD 3 antibodies, when added to a 4-1BB agonist: 4-1BB agonist =1:0.1 to 1;
anti-CD 3 antibody when ccr8 antibody is added: ccr8 antibody =1:1 to 2.
CN202210848536.3A 2022-07-19 2022-07-19 Mesoporous silica sustained-release preparation for cancerous ascites and preparation method thereof Pending CN115154618A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109789092A (en) * 2016-07-13 2019-05-21 哈佛学院院长等 Antigen presenting cell simulates bracket and its preparation and application
CN110898215A (en) * 2019-12-06 2020-03-24 郑州大学 Preparation method and application of anti-tumor vaccine based on cell microvesicles
CN111012761A (en) * 2019-12-24 2020-04-17 中南大学湘雅医院 Drug-loaded microsphere, anti-tumor drug and preparation method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109789092A (en) * 2016-07-13 2019-05-21 哈佛学院院长等 Antigen presenting cell simulates bracket and its preparation and application
CN110898215A (en) * 2019-12-06 2020-03-24 郑州大学 Preparation method and application of anti-tumor vaccine based on cell microvesicles
CN111012761A (en) * 2019-12-24 2020-04-17 中南大学湘雅医院 Drug-loaded microsphere, anti-tumor drug and preparation method

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