CN108671237A - It is a kind of to enhance the carrier and its preparation method and application that drug targeting delivers - Google Patents

Abstract

The present invention relates to a kind of carrier of enhancing drug targeting delivering and its preparation method and applications, the carrier for enhancing drug targeting delivering is the leucocyte for being derived from the homologous allosome through pre- immunity inoculation, and the preparation method of the carrier of the enhancing drug targeting delivering includes the following steps：(1) pre- immune formulation is prepared, pre- immunity inoculation is carried out 1~3 time to homologous allosome；(2) leucocyte in the homologous allosome body of separation and Extraction；(3) target cell in leucocyte is detached, the carrier of drug targeting delivering is as enhanced.Compared with prior art, the present invention provides a kind of completely new thinking for enhancing cancer target, a kind of potential cell carrier platform not only can be provided to solve cancer target bottleneck, moreover it is possible to potential application of the further exploration immunocyte platform on this basis in antineoplastic target treatment, targeting mechanism etc..

Description

A carrier for enhancing targeted delivery of drugs, its obtaining method and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a carrier for enhancing targeted drug delivery and its obtaining method and application.

Background technique

The targeting feasibility of traditional nanomedicine in human body has been challenged. At present, the role of nano drug systems is limited to improving drug stability, reducing side effects, prolonging circulation time, etc., and the targeting effect is minimal. clinical research shows Compared with naked doxorubicin, nanomedicine has no obvious tumor enrichment advantage. In 2016, paclitaxel-based nano-drugs NK105, BIND-014, and CRLX-101[6] all failed in the third phase of clinical trials, and Bind Therapeutics, which was expected to become the world's first targeted nano-drug, also declared bankruptcy in the same year. The low targeting efficiency has been considered as an important reason for the failure of clinical transformation of many nanomedicines. More and more data show that even the targeting ligands that can actively recognize tumor tissues or cells on the surface of nanomedicine systems, such as antibodies, peptides, folic acid, etc., cannot change the abundance of drugs in tumor tissues. There are even reports that the introduction of targeting ligands may cause adverse effects, such as causing the size of nanoparticles to become larger, making them easier to be captured by the RES system, or changing the surface properties of nanoparticles, making it difficult for nanoparticles to enter tumor tissues. cell.

Regarding tumor targeting enhancement strategies, in addition to improving the tumor microenvironment, such as the normalization of tumor blood vessels, and cell membrane engineering, live cell carriers (Live cell carriers) are favored because they are not EPR-dependent. Leukocytes have special functions such as immune response, cell interaction, cell adhesion, and crossing physiological barriers to reach non-vascular areas, and are regarded as important new forces in the application of cellcarrier tumor targeting. In the past few years, researchers have used the inherent tumor chemotaxis of immune cells as a "Trojan horse" drug delivery system for targeted tumor delivery studies. ability (directed migration), but the targeting effect is not satisfactory in practical applications. How to enhance leukocyte tumor targeting is extremely important for tumor therapy.

Contents of the invention

The object of the present invention is to provide a carrier for enhancing targeted delivery of drugs and its obtaining method and application in order to solve the above-mentioned targeting bottleneck problem.

The purpose of the present invention can be achieved through the following technical solutions:

A carrier that enhances the targeted delivery of drugs is allogeneic leukocytes obtained from pre-immunization.

The white blood cells include one or more combinations of neutrophils, monocytes/macrophages, or NK cells.

The method for obtaining the carrier for enhancing targeted drug delivery comprises the following steps:

(1) Prepare a pre-immune preparation, and perform pre-immunization on the allogeneic body for 1 to 3 times;

(2) Separation and extraction of leukocytes in the allogeneic body;

(3) Separating the target cells in the white blood cells, which is the carrier to enhance the targeted delivery of drugs.

Since leukocytes include granulocytes, monocytes/macrophages or NK cells, which are mixed cells, it is necessary to separate the target cells inside, such as granulocytes or NK cells.

The pre-immunization preparation is a nano preparation containing an immune stimulant, or other dosage forms with the function of slow-release immune stimulant.

The immune stimulants include lipopolysaccharide receptor agonists or Toll-like receptor agonists, and the Toll-like receptor agonists are selected from unmethylated cytosine nucleotides-guanosine Acid (CpG) or resiquimod (Resiquimod, R848) etc.) or a combination of both.

The interval between inoculations was one week.

The method for separating and extracting the leukocytes in the allogeneic body in step (2) is as follows: it can be extracted from the bone marrow, or it can be collected by lavage after aseptic stimulation of the peritoneal cavity for several hours.

The carrier for enhancing targeted delivery of drugs is used as a carrier of therapeutic substances for targeted delivery of tumor sites, and the therapeutic carrier includes anti-tumor drugs or nano-medicines for tumor drugs.

The antitumor drug can be selected from doxorubicin, daunorubicin, valrubicin, epirubicin, idarubicin, paclitaxel, docetaxel, cisplatin, carboplatin, oxali Platinum, camptothecin, vincristine, vinblastine, 5-fluorouracil (5-FU), mitomycin, cyclophosphamide, methotrexate, mitogen, topotecan, card Betabine, deoxyfluorouracil, irinotecan, tegafur, chlorambucil, belotecan, anastrolide, tamoxifen, Gleevec, fluorouracil, leuprolide , Flutamide, Milesinic Acid, Streptozocin, Vinorelbine, Hydroxymethine, Retinoic Acid, Nitrogen Mustard, Busulfan, Prednisone, Trimitone, Aspirin, Salicylates, Ibuprofen , Chapusheng, fenoprofen, called Limexin, phenylbutazone, nitrogen mustard, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide, cortisone, corticosteroids Steroids, gemcitabine, cedrol, or any combination thereof, or at least one of their derivatives, or their new formulations of nanomedicines.

After the carrier that enhances the targeted delivery of drugs is injected into the body, it has a strong tumor enrichment effect.

Compared with the existing tumor targeting efficiency, the tumor/liver, tumor/spleen and tumor/kidney enrichment ratios are 2-3 times higher than those obtained from conventional subjects without pre-immunization. The present invention provides a brand-new idea for enhancing tumor targeting. It not only provides a potential cell carrier platform for solving the bottleneck of tumor targeting, but also further explores the role of immune cell platform in anti-tumor targeted therapy. , targeting mechanisms and other potential applications.

Description of drawings

Figure 1 is a transmission electron microscope picture of nano-CpG used in the implementation case and a physical picture of the dispersion.

Figure 2 is a comparative picture of in vivo imaging in the case: (A) Tumor enrichment of granulocytes obtained from non-immune normal mice (B) Enrichment of granulocytes obtained from immunized mice at the tumor site.

Detailed ways

A carrier that enhances the targeted delivery of drugs is allogeneic leukocytes obtained from pre-immunization.

The white blood cells include one or more combinations of neutrophils, monocytes/macrophages, or NK cells.

The method for obtaining the carrier for enhancing targeted drug delivery comprises the following steps:

(1) Prepare a pre-immune preparation, and perform pre-immunization on the allogeneic body for 1 to 3 times;

(2) Separation and extraction of leukocytes in the allogeneic body;

(3) Separating the target cells in the white blood cells, which is the carrier to enhance the targeted delivery of drugs.

The pre-immunization preparation is a nano preparation containing an immune stimulant, or other dosage forms with the function of slow-release immune stimulant.

The immune stimulants include lipopolysaccharide receptor agonists or Toll-like receptor agonists, and the Toll-like receptor agonists are selected from unmethylated cytosine nucleotides-guanosine Acid (CpG) or resiquimod (Resiquimod, R848) etc.) or a combination of both.

The interval between inoculations was one week.

The method for separating and extracting the leukocytes in the allogeneic body in step (2) is as follows: it can be extracted from the bone marrow, or it can be collected by lavage after aseptic stimulation of the peritoneal cavity for several hours.

The carrier for enhancing targeted delivery of drugs is used as a carrier of therapeutic substances for targeted delivery of tumor sites, and the therapeutic carrier includes anti-tumor drugs or nano-medicines for tumor drugs.

The antitumor drug can be selected from doxorubicin, daunorubicin, valrubicin, epirubicin, idarubicin, paclitaxel, docetaxel, cisplatin, carboplatin, oxali Platinum, camptothecin, vincristine, vinblastine, 5-fluorouracil (5-FU), mitomycin, cyclophosphamide, methotrexate, mitogen, topotecan, card Betabine, deoxyfluorouracil, irinotecan, tegafur, chlorambucil, belotecan, anastrolide, tamoxifen, Gleevec, fluorouracil, leuprolide , Flutamide, Milesinic Acid, Streptozocin, Vinorelbine, Hydroxymethine, Retinoic Acid, Nitrogen Mustard, Busulfan, Prednisone, Trimitone, Aspirin, Salicylates, Ibuprofen , Chapusheng, fenoprofen, called Limexin, phenylbutazone, nitrogen mustard, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide, cortisone, corticosteroids Steroids, gemcitabine, cedrol, or any combination thereof, or at least one of their derivatives, or their new formulations of nanomedicines.

After the carrier that enhances the targeted delivery of drugs is injected into the body, it has a strong tumor enrichment effect.

Taking mice as an example, a new method for enhancing the targeting of white blood cell tumors in experimental mice is provided. The method includes the following steps:

(1) Prepare a suitable pre-immune preparation, and carry out pre-immunization on 4-13-week-old mice for 1-3 times, and the interval between vaccinations is one week, wherein the pre-immune preparation is a nano-sized preparation containing an immune stimulant or has Other dosage forms of sustained-release agonist function, agonists, including lipopolysaccharides, Toll-like receptor agonists (such as unmethylated cytosine nucleotide-guanine nucleotide (CpG), requimol Germany (Resiquimod, R848, etc.) one or two combinations;

(2) The peritoneal cavity of the mouse in step (1) was aseptically stimulated for several hours, and then collected by lavage to obtain white blood cells. The sterile stimuli include various sterile preparations, such as common thioglycollate at a certain concentration.

(3) Separating target cells (neutrophils, monocytes/macrophages or NK cells, etc.) from leukocytes and injecting them into allogeneic tumor-bearing mice has a strong tumor enrichment effect.

Taking mice as an example, the possible mechanism of targeted enhancement involved: laboratory mice used in cell carrier research usually also live in a sterile environment, and the immune system of experimental mice has not been trained by pathogenic stimulation. It is understandable that it is difficult to develop and mature, and the chemotaxis and targeting of immune cells isolated from the body are not ideal. Immunostimulation of mice can significantly improve the responsiveness of the immune system of mice to inflammatory signals, thus greatly improving the ability to target tumors.

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

Example

1. Subcutaneously inject nano-CpG into 7-8w bable/c mice. The transmission electron microscope picture and physical picture of the dispersion liquid of the nano-CpG used are shown in Figure 1. Inject once or once a week, and inject 3 times in total. after times. The white blood cells in the body were collected by peritoneal lavage, and the neutrophils were separated and extracted.

2. Fluorescent labeling of neutrophils (Dir labeling)

3. Mice bearing 4T1 tumors were injected with about 5×10 ⁶ -10 ⁷ fluorescently labeled neutrophils per 200 uL when the tumor size was about 5-8 mm.

4. Inject neutrophils into allogeneic tumor-bearing mice, perform in vivo imaging at different intervals, and compare the granulocytes obtained from mice in the non-immune group. The targeting effect is shown in Figure 2 in detail. Figure 2 (A) is the tumor enrichment of granulocytes from non-immune normal mice, and Figure 2 (B) is the enrichment of granulocytes from immunized mice at the tumor site.

The above descriptions of the embodiments are for those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described here to other embodiments without creative efforts. Therefore, the present invention is not limited to the above-mentioned embodiments. Improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims (9)

1. a kind of carrier of enhancing drug targeting delivering, which is characterized in that it is to be derived from the homologous allosome through pre- immunity inoculation Leucocyte.

2. a kind of carrier of enhancing drug targeting delivering according to claim 1, which is characterized in that the leucocyte includes One or more combination in neutrophil leucocyte, Monocytes/Macrophages or NK cells.

3. the preparation method of the carrier of enhancing drug targeting delivering as claimed in claim 1 or 2, which is characterized in that including following Step：

(1) pre- immune formulation is prepared, pre- immunity inoculation is carried out 1~3 time to homologous allosome；

(2) leucocyte in the homologous allosome body of separation and Extraction；

(3) target cell in leucocyte is detached, the carrier of drug targeting delivering is as enhanced.

4. the preparation method of the carrier of enhancing drug targeting delivering according to claim 3, which is characterized in that described pre- immune Preparation is the nanosizing preparation containing immune agonist, or other dosage forms of agonist function are immunized with sustained release.

5. the preparation method of the carrier of enhancing drug targeting delivering according to claim 4, which is characterized in that described immune sharp Dynamic agent, including liopopolysaccharides receptor stimulating agent or Toll-like receptoroid agonist, the Toll-like receptoroid agonist are selected from not One or two kinds of combination in the cytidylic acid-guanylic acid or resiquimod that methylate.

6. the preparation method of the carrier of enhancing drug targeting delivering according to claim 3, which is characterized in that between inoculation every other week Phase is one week.

7. the preparation method of the carrier of enhancing drug targeting delivering according to claim 3, which is characterized in that step (2) institute The method for stating the leucocyte in the homologous allosome body of separation and Extraction is：Extracted from marrow, or by the sterile stimulation in abdominal cavity after, lavation Method, which is collected, obtains leucocyte.

8. the application of the carrier of enhancing drug targeting delivering as described in claim 1, which is characterized in that the enhancing drug targeting Carrier of the carrier of delivering as therapeutic substance, the therapeutic substance include antitumor drug or tumour medicine nanometer medicine Agent.

9. the application of the carrier of enhancing drug targeting delivering according to claim 8, which is characterized in that the antineoplastic Object, selected from Doxorubicin, daunorubicin, valrubicin, epirubicin, idarubicin, taxol, docetaxel, cis-platinum, card Platinum, oxaliplatin, camptothecine, vincristine, vincaleukoblastinum, 5 one fluorine urine crash smack one's lips, mitomycin, ring phosphorus phthalein amine, the cry of certain animals of first ammonia butterfly, rice It asks green onion to wake up, topotecan, capecitabine, deoxygenate general fluorine urine, Irinotecan, Tegafur, Chlorambucil, Belotecan, Ah that Bent mile, tamoxifen, Gleevec, fluorine urine is general, Leuprorelin, Flutamide, mile come bony acid, streptozotocin, vinorelbine, light Ji Mai, Retinoic acid, busulfan, prednisone, emerald green ketone, aspirin, salicylate, brufen, the general life of tea, fenoprofen, is named and pays attention at mustargen Mei Xin, bute, mustargen, dexamethasone, prednisolone, celecoxib, valdecoxib, aulin, cortisone, cortex Steroids, gemcitabine, cedrol or at least one of their arbitrary combination or their derivative or they receive Rice pharmaceutical dosage form.