CN111135307B - Preparation method and application of drug delivery system based on CAR-T cells - Google Patents
Preparation method and application of drug delivery system based on CAR-T cells Download PDFInfo
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Abstract
The invention provides a drug delivery system based on CAR-T cells, which comprises the CAR-T cells, drug-loaded nano materials and anti-tumor drugs, wherein the anti-tumor drugs are encapsulated in the drug-loaded nano materials to form nano drugs, the CAR-T cells phagocytose the nano drugs, and chimeric antibodies on the CAR-T cells are utilized to target and locate specific antigens combined with the antibodies. The drug delivery system based on the CAR-T cells, which is prepared by the invention, can obviously improve the tumor targeting of the drug and increase the concentration of the drug at an action part, thereby enhancing the curative effect of the drug, reducing the distribution of the drug in peripheral organs and reducing the toxic and side effects of the drug. The invention develops the new application of the CAR-T cells, improves the economic value of the CAR-T cells, and provides theoretical and practical basis for the application of the CAR-T cells in a drug delivery system.
Description
Technical Field
The invention relates to the technical field of tumor treatment, in particular to a preparation method and application of a drug delivery system based on CAR-T cells.
Background
The primary condition of drug treatment of disease is that the drug is able to reach an effective drug concentration within the target tissue, but the target drug reaches the target tissue as a challenging process due to the various physiological barriers and potential toxic side effects that are limited in the body. Even some traditional medicines which are already used clinically at present have the problem that the treatment efficiency is limited and the toxic and side effects are serious due to poor pharmacokinetic behaviors. Therefore, in order to obtain a better therapeutic effect, a proper dose of the drug must be delivered to the site requiring treatment at a proper time and maintain a certain concentration to secure the therapeutic effect. In addition, in order to reduce the drug dosage and potential toxic side effects, it is desirable to minimize the tissue distribution of the drug outside the site of action and metabolic organs. Based on this, it is becoming particularly important to study and apply new drug delivery systems to improve drug delivery.
Cell-based drug delivery systems are a drug delivery platform that is effective in the treatment of diseases. The cells themselves are highly intelligent drug delivery devices that can sense various signals and migrate to desired locations, integrate various input signals in the microenvironment, and then perform corresponding complex actions. In addition, cell-based drug delivery systems, cells themselves have tumor-guiding capabilities, and are self-targeted, eliminating the need for reconnection of the targeted recognition molecule. In recent years, immune cells have attracted attention as drug delivery vehicles because they can migrate not only to sites of injury, inflammation or tumor by blood flow, but also have reduced immune clearance and can extend biological half-life.
In addition to the inherent targeting, drug delivery and lower immune clearance rate of immune cell drug delivery systems, CAR-T cells have the function of increasing the killing effect of CAR-T cells on tumor cells and the like, and are expected to become a more effective drug delivery system. Based on the above, we aim to develop a drug delivery system based on CAR-T cells and capable of actively targeting diseases, study the in vivo circulation time, the targeting effect of tumor tissues, the uptake of tumor cells and the in vivo anti-tumor activity, and lay a foundation for targeted treatment of tumors by drugs.
Disclosure of Invention
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a CAR-T cell based drug delivery system comprising CAR-T cells, drug-loaded nanomaterials, and an anti-tumor drug encapsulated in the drug-loaded nanomaterials to form a nanomaterials, which CAR-T cells engulf the nanomaterials and target specific antigens bound to the antibodies using chimeric antibodies on the CAR-T cells.
Preferably, the specific antigen comprises CD19, CD140a, EGFR, HER2.
Preferably, the chimeric antibody comprises a CD19 antibody, a CD140a antibody, an EGFR antibody, a Her2 antibody.
Preferably, the CAR-T cell is a CD19-CAR-T cell, a CD140a-CAR-T cell, an EGFR-CAR-T cell, a HER2-CAR-T cell.
Preferably, the drug-loaded nanomaterial is mesoporous silicon, liposome, polylactic-co-glycolic acid (PLGA), albumin, or exosome.
Preferably, the antitumor drug is one or more of doxorubicin, taxane, camptothecine, shallot and derivatives thereof.
The invention also provides application of the CAR-T cell-based drug delivery system in preparation of antitumor drugs.
Preferably, the tumor is any one of brain glioma, liver cancer, breast cancer, ovarian cancer, lung cancer, skin cancer or malignant melanoma.
The invention also provides a preparation method of the drug delivery system based on the CAR-T cells, which comprises the following steps:
(1) Preparing a drug-loaded nano material;
(2) Mixing the drug-loaded nano material prepared in the step (1) with an anti-tumor drug solution, and freeze-drying the mixture to obtain a nano drug;
(3) Transfecting T cells with the CAR plasmid introduced with the chimeric antibody encoding gene, culturing the transfected T cells in a culture medium containing interleukin 2, activating the T cells, and obtaining CAR-T cells targeting specific antigens;
(4) Co-culturing the CAR-T cells with the nanomedicine to form a CAR-T cell based drug delivery system.
Preferably, the specific antigen comprises CD19, CD140a, EGFR, HER2.
Preferably, the chimeric antibody is a CD19 antibody, a CD140a antibody, an EGFR antibody, a Her2 antibody.
Preferably, the CAR-T cell is a CD19-CAR-T cell, a CD140a-CAR-T cell, an EGFR-CAR-T cell, a Her2-CAR-T cell.
Preferably, the drug-loaded nanomaterial is mesoporous silicon, liposome, polylactic-co-glycolic acid (PLGA), albumin, or exosome.
Preferably, the antitumor drug is one or more of doxorubicin, taxane, camptothecine, shallot, etc. and derivatives thereof.
The invention has the beneficial effects that:
1. experiments prove that the CAR-T cell-based drug delivery system prepared by the invention can remarkably improve the tumor targeting of the drug and increase the concentration of the drug at an action part, thereby enhancing the curative effect of the drug, reducing the distribution of the drug in peripheral organs and reducing the toxic and side effects of the drug.
2. The invention develops the new application of the CAR-T cells, improves the economic value of the CAR-T cells, and provides theoretical and practical basis for the application of the CAR-T cells in a drug delivery system.
Drawings
FIG. 1 is an electron microscope image of the ferroferric oxide mesoporous silica core-shell structured material prepared in example 1 (wherein A is a scanning electron microscope image of the prepared material; and B is a transmission electron microscope element distribution diagram of the prepared material);
FIG. 2 is a flow cytometer detecting the intensity of DOX fluorescence in CAR-T cells (where A is CAR-T cells that do not phagocytose D-FMSNs, and B is CAR-T cells after co-incubation with D-FMSNs);
FIG. 3 determination of killing effect of CAR-T cell based drug delivery system on tumor cells;
FIG. 4 targeting assay of CAR-T cell based drug delivery system in animals;
figure 5 effect of CAR-T cell based drug delivery system on animal survival.
Detailed Description
In order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the present invention is described in further detail below with reference to the specific embodiments and the accompanying drawings.
EXAMPLE 1 preparation of CAR-T cell based drug delivery System of the invention
1. Preparation of medicine-carrying nano material-ferroferric oxide mesoporous silicon nano material
(1) 10mL of 0.25mol/L FeCl 3 ·6H 2 O and 10mL of 0.25mol/L FeSO 4 ·7H 2 Adding the O mixed solution into a flask, adding an appropriate amount of 1mol/L NH under the protection of nitrogen 3 ·H 2 Adding O solution into the reaction system to make p H of the system be more than or equal to 10, stirring vigorously for 30min, ending the reaction, repeatedly washing with distilled water until neutral, pouring out supernatant, and vacuum drying at 60deg.C to obtain Fe 3 O 4 And (3) nanoparticles.
(2) 2.5mg of Fe 3 O 4 Nanoparticles were added to 0.2mL of chloroform, and 2mL of cetyltrimethylammonium bromide (CTAB) aqueous solution (40 mg CTAB,54 mmol/L) was added to Fe 3 O 4 In the colloid solution, sealing with a cover, ultrasonic treating the mixture for 10min, and evaporating chloroform to obtain transparent well-dispersed Fe 3 O 4 Colloidal aqueous solution and named Fe 3 O 4 @CTAB。
(3) In a 100mL flask, 40mg CTAB was dissolved in 18mL of water, and 120uL of NaOH solution (2M) was added; then, 2ml of Fe obtained in the step (2) was added 3 O 4 Adding a @ CTAB colloidal solution to the reaction solution, vigorously stirring and raising the temperature of the solution to 70 ℃; 200uL of tetraethyl orthosilicate (TEOS) and 1.2mL of ethyl acetate were added dropwise to the solution, and after stirring for 2 hours, 40 uL of 3-aminopropyl triethoxysilane (APTES) was added dropwise to the solution and stirred for 2 hours again, then the solution was cooled to room temperature, centrifuged and washed 3 times with ethanol to obtain mesoporous silica nanoparticle particles.
(4) Adding the nanoparticles obtained in the step (3) to a solution containing 120mg of NH 4 NO 3 After this step was repeated twice, the nanoparticles were washed with deionized water and ethanol several times, and stirred at 60 ℃ for 1 hour.
The morphology and composition of the nano particles prepared in the step (4) are characterized by a transmission electron microscope, the result is shown in fig. 1, and the result can be seen from fig. 1A: the particle size of the prepared material is about 50 nm; from the elemental distribution analysis of fig. 1B, it can be seen that: the ferroferric oxide mesoporous silicon core-shell structure material is successfully prepared and is marked as FMSNs.
2. Preparation of nano-medicine
(1) 20mg of the ferroferric oxide mesoporous silica core-shell structure is added into 20ml of Doxorubicin (DOX) PBS solution, the solution is protected from light, and the solution is stirred overnight to obtain the doxorubicin nano-drug, which is marked as D-FMSNs.
(2) D-FMSNs were collected by centrifugation and lyophilized under vacuum. The efficiency of FMSNs loading DOX was measured by UV-Vis spectroscopy at λ=480 nm.
3. Preparation of CAR-T cells
First a CD140a CAR lentiviral vector was constructed comprising ScFv sequences from the clinical drug Olaratumab (VH and VL regions of Olaratumab are connected by a 3X GGGGS linker), CD8 hinge and transmembrane domains, 4-1BB and CD3 zeta intracellular domains. Plasmid vector DNA for CD140a CAR was optimized and synthesized by IGE gene company (guangzhou) and then cloned into lentiviral transfer vector slenti-EF 1a between NheI and agoi restriction sites using the Gibsson Assembly method. Peripheral Blood Mononuclear Cells (PBMCs) were then obtained by gradient centrifugation, T cells were sorted with Dynabeads magnetic beads and cultured in 1640 medium containing interleukin 2 to stimulate T cell growth. T cells were exposed to the supernatant containing anti-CD 140a-CAR lentiviral vector and conduction was initiated. These transduced cells were expanded and successful CAR gene transfer was determined by flow cytometry fluorescence sorting (FACS) analysis for CAR proteins to obtain CD140a CAR-T cells.
4. Preparation of CAR-T cell based drug delivery systems
The CAR-T cells and D-FMSNs nano-drug were incubated in fresh serum-free medium for one hour at 37℃and after centrifugation and washing 2 times with ice-cold PBS, a CAR-T+D-FMSNs suspension was obtained. Part of the CAR-T+D-FMSNs suspension was taken and DOX fluorescence in the CAR-T cells was detected by flow cytometry PE-A channel.
As can be seen from fig. 2A: the fluorescence intensity of CAR-T cells alone was 0.73%; from FIG. 2B, it can be seen that the fluorescence intensity of the CAR-T cells after co-incubation with D-FMSNs was 99.7%. The results indicate that D-FMSNs can enter CAR-T cells, and the drug delivery system based on CAR-T of the invention is successfully prepared.
Example 2 in vitro killing effect of CAR-T cell based drug delivery systems on tumor cells
In the embodiment, the toxicity of the drug delivery system taking the CAR-T cells as the carrier to U87 cells is detected by adopting a Luciferase method and a confocal experiment, and the in-vitro anti-tumor activity of the CAR-T cell drug delivery system is detected by adopting the following specific operations:
u87 glioma cells (supplied from Shanghai cell bank) were cultured in DMEM medium containing 10% foetal calf serum and 1% diabody, and placed at 37℃with 5% CO 2 Is replaced every other day, and after 2-3 days is digested with 0.25% trypsin (0.02% EDTA) for one passage. Taking logarithmic growth phase cells, conventionally sterilizing to obtain single cell suspension, counting, and adjusting the density of the cells to be detected to 5×10 3 Each well (edge wells are filled with sterile PBS), a U-shaped 96-well plate is spread, 200. Mu.L of medium is added to each well, and then the wells are placed at 37℃with 5% CO 2 Is cultured in a cell incubator. After 24h of total cell attachment, the original medium was discarded.
Cells were divided into experimental, control and blank groups. CAR-T+D-FMSNs suspensions were added to the experimental groups at 1:8,1:4,1:2,1:1,2:1,4:1,8:1, respectively, to tumor cell volumes; adding suspension of D-FMSNs complex in the same proportion into control group, and setting 5 compound holes in each proportion; blank groups were added to the medium. Three groups of cells were placed in an incubator for further culture for 24 hours, and the culture was terminated. Taking out the U-shaped 96-well plate, centrifuging at 1000rpm/min by a flat angle centrifuge, gently removing supernatant by a discharge gun, adding 100uL of reporter gene cell lysate per well, blowing and mixing uniformly cells, transferring the cells into a black opaque 96-well plate, adding 100uL of luciferase detection reagent per well, and immediately detecting a fluorescence value in a fluorescent illuminometer; and (3) calculating a killing proportion: taking a target cell hole without adding T cells as a reference, wherein the proportion of reduced fluorescence value is the killing percentage, and the formula is as follows: percent kill= (blank Kong Yingguang value-target Kong Yingguang value)/blank wells x 100%.
As can be seen from fig. 3: after co-culturing D-FMSNs, CAR-T+D-FMSNs and U87 human glioma cells respectively, the killing effect of the experimental group (CAR-T+D-FMSNs) on glioma cells is obviously higher than that of the control group (D-FMSNs) without CAR-T cells. The results show that the drug delivery system based on the CAR-T cells has remarkable tumor cell killing effect.
Example 3 targeting effect of CAR-T cell based drug delivery systems in animals
FMSNs-conjugated CY7 fluorescent label, 30mg FMSNs,345mg NHS,576mg EDC and 1.5mg CY7 were first mixed, added to 30mL PBS and stirred at room temperature in the absence of light for 24 hours. After stirring, the mixture was washed several times with PBS by centrifugation to obtain FMSNs-CY7. D-FMSNs-CY7, normal t+d-FMSNs-CY7 (T cells used in this group are Normal T cells, not containing anti-CD 140a CAR lentiviral vector) and CAR-t+d-FMSNs-CY7 were constructed according to the preparation method of the CAR-T cell drug delivery system described above. Then establishing a glioma in-situ model, and injecting 5X 10 into the brain right hemisphere of the NSG mouse by using a stereotactic fixing device 5 And (3) individual U87-Luc cells. Brain tumor growth was monitored by intraperitoneal injection of D-heparin potassium salt and observation of the luminescence intensity of U87-Luc cells in the brain using an in vivo imaging system (Caliper IVIS Spectrum). After 8 days after glioma cells were transplanted into mouse brain, 3 groups were randomized and given amounts of D-FMSNs-CY7, normal T+D-FMSNs-CY7 and CAR-T+D-FMSNs-CY7, respectively, via the tail vein. Subsequently, mice were anesthetized with 1-2% isoflurane containing 20% oxygen and observed and photographed using an IVIS spectroscopic imaging system 1,2, 4, 8 and 24 hours after dosing.
As can be seen from fig. 4: glioma mice were randomly grouped and given treatment injections of D-FMSNs-CY7, normal t+d-FMSNs-CY7 and CAR-t+d-FMSNs-CY7, respectively, wherein the CAR-t+d-FMSNs-CY7 group was able to significantly aggregate at the glioma site in the brain of mice. The result shows that the drug delivery system based on the CAR-T cells has obvious glioma targeting effect, and the concentration of the drug at the action part is improved.
Example 4 therapeutic Effect of CAR-T cell based drug delivery System in animals
Establishing glioma in-situ model, and injecting 5×10 into brain right hemisphere of NSG mouse by using stereotactic fixing device 5 And (3) individual U87-Luc cells. Brain tumor growth was monitored by intraperitoneal injection of D-heparin potassium salt and observation of the luminescence intensity of U87-Luc cells in the brain using an in vivo imaging system (Caliper IVIS Spectrum). 8 days after glioma cells were transplanted into the mouse brain, groups were randomized, and received intravenous injections, respectively, as follows: (1) D-FMSNs; (2) CAR-T+D-FMSNs (1X 10 per mouse) 7 Individual cells); survival time was recorded for each group.
As can be seen from fig. 5: the results show that the CAR-T cell-based drug delivery system can remarkably improve the survival time of an in-situ glioma mouse model, and has remarkable anti-tumor effect compared with a control group (D-FMSNs).
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (7)
1. A CAR-T cell based drug delivery system, comprising a CAR-T cell, a drug-loaded nanomaterial, and an anti-tumor drug encapsulated in the drug-loaded nanomaterial to form a nano-drug, the CAR-T cell phagocytosing the nano-drug, targeting a specific antigen bound to the antibody with a chimeric antibody on the CAR-T cell;
the CAR-T cells are CD140a-CAR-T cells;
the drug-loaded nano material is ferroferric oxide mesoporous silicon; the antitumor drug is doxorubicin;
the preparation method of the nano-drug comprises the following steps:
1) Adding the ferroferric oxide mesoporous silicon core-shell structure into doxorubicin PBS solution, and stirring overnight in dark to obtain nano-drug D-FMSNs;
2) Centrifuging to collect nano-drug D-FMSNs, and freeze-drying under vacuum;
the CAR-T cells and nano-drug D-FMSNs were incubated in fresh serum-free medium at 37 ℃ for one hour, centrifuged and washed with PBS to obtain a suspension of CAR-T+D-FMSNs, forming a CAR-T cell based drug delivery system.
2. The CAR-T cell-based drug delivery system of claim 1, wherein the specific antigen is CD140a.
3. The CAR-T cell-based drug delivery system of claim 1, wherein the chimeric antibody is a CD140a antibody.
4. Use of a CAR-T cell based drug delivery system according to claim 1 for the preparation of an anti-tumor drug.
5. The use of claim 4, wherein the tumor is any one of brain glioma, liver cancer, breast cancer, ovarian cancer, lung cancer, skin cancer, or malignant melanoma.
6. A method of preparing a CAR-T cell based drug delivery system according to claim 1, comprising the steps of:
(1) Preparing a drug-loaded nano material;
(2) Mixing the drug-loaded nano material prepared in the step (1) with an anti-tumor drug solution, and freeze-drying the mixture to obtain a nano drug;
(3) Transfecting T cells with the CAR plasmid introduced with the chimeric antibody encoding gene, culturing the transfected T cells in a culture medium containing interleukin 2, activating the T cells, and obtaining CAR-T cells targeting specific antigens;
(4) Co-culturing the CAR-T cells with the nanomedicine to form a CAR-T cell based drug delivery system.
7. The method of claim 6, wherein the specific antigen is CD140a; the chimeric antibody is a CD140a antibody; the CAR-T cells are CD140a-CAR-T cells; the drug-loaded nano material is ferroferric oxide mesoporous silicon; the antitumor drug is doxorubicin.
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