CN111265491B

CN111265491B - Cell microparticle drug delivery system for entrapping allicin and preparation method and application thereof

Info

Publication number: CN111265491B
Application number: CN202010219886.4A
Authority: CN
Inventors: 刘玉萍; 陈彦; 瞿鼎; 刘聪燕
Original assignee: Jiangsu Provincial Insititute of Traditional Chinese Medicine
Current assignee: Jiangsu Provincial Insititute of Traditional Chinese Medicine
Priority date: 2020-03-25
Filing date: 2020-03-25
Publication date: 2022-05-20
Anticipated expiration: 2040-03-25
Also published as: CN111265491A

Abstract

The invention relates to a cell microparticle drug-loading system for encapsulating garlicin and a preparation method and application thereof. The invention also relates to a preparation method of the carrier system and application of the carrier system in preparation of anti-tumor metastasis medicaments, and the organ-targeted allicin-containing cell microparticles obtained according to the technical scheme provided by the invention have a remarkable inhibiting effect on melanoma lung metastasis, Lewis lung cancer lung metastasis and other various tumor metastases, and are a biocompatible traditional Chinese medicine anti-tumor metastasis medicament delivery system accurately targeting metastasis organs.

Description

Cell microparticle drug delivery system for entrapping allicin and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a drug loading system containing drug cell microparticles and loaded with allicin, a preparation method thereof and application of the drug loading system in resisting tumor lung metastasis.

Background

Tumor metastasis is a significant cause of clinical mortality, with about 90% of malignancies dying from distant metastasis. Tumor cells infiltrate into the surrounding tissues, spread to other tissues and organs of the body through the blood system and lymphatic system to form metastases, eventually leading to the death of the patient. The lung is the most prominent metastatic target organ of all malignancies, with about 30% -54% of them developing lung metastases in their natural course. Almost 1/3 patients who died from cancer were reported to have lung metastases at autopsy. The existing anti-metastasis treatment strategies are mostly limited to in-situ excision plus broad-spectrum chemotherapy but the reasons for poor effects mainly lie in that: chemotherapy drugs have poor selectivity and fail to recognize tumor cells of metastases, resulting in systemic body damage and immunosuppression.

Garlic organic sulfide garlicin is an active ingredient with the strongest anti-tumor activity of the traditional Chinese medicine garlic (Allium sativum L.), and the study of the preliminary period of the subject group finds that the garlicin has good potential of resisting tumor metastasis in vitro and in vivo: can inhibit migration and invasion of breast cancer cells; the number of tumor nodules in the lung of a nude mouse is reduced in an MDA-MB-231 nude mouse breast cancer in-situ metastasis model and an experimental metastasis model, and the far-end metastasis of cancer cells is inhibited; PAF-mediated lung metastasis was inhibited in an experimental model of melanoma in mice. The mechanism of inhibition of lung metastasis may be related to its intervention in the tumor microenvironment: the allicin can inhibit the activation of tumor cell MAPK, regulate the reconstruction of extracellular matrix, reduce the level of factors such as inflammatory factors TNF-alpha, IL-6 and the like by reducing hypoxia-induced tumor angiogenesis, inhibiting the transcriptional regulation of nuclear transcription factor NF-kappa B and the expression of MMP2/9, and the like. In conclusion, the allicin can not only directly inhibit the metastatic malignant biological behavior of the tumor cells, but also inhibit the occurrence of tumor metastasis by improving the action of the tumor microenvironment. However, the general poor physicochemical properties of allicin, volatility and instability of allicin, although the existing drug delivery system partially solves the problems, the existing drug delivery system still cannot realize accurate targeted delivery to the metastasis focus and better play the role of resisting tumor metastasis.

The cell microparticle is a kind of microvesicle secreted by cells and having a bilayer lipid membrane structure, and the diameter is about 100-1000 nm. The research shows that the extracellular vesicle is surrounded by a biological membrane consisting of membrane lipid and membrane protein, and the structure and the components of the vesicle are similar to those of a cell membrane. Wherein the membrane lipid component is enriched in cholesterol, ceramide, sphingomyelin and lipid raft structure compared with the cell membrane. The microparticles can transfer various carried proteins, mRNA, miRNA and the like to corresponding cells or tissues, and participate and maintain a plurality of normal physiological functions, such as tissue repair, immune monitoring, blood coagulation and the like. Recent research shows that the microparticles can be used as carriers of small molecule drugs for treating certain diseases. Cell microparticles exhibit several advantages as drug delivery vehicles: first, it is derived from the patient's own cells, thus reducing immunogenicity during drug delivery; secondly, as a membrane structure carrier, the microparticles have a structure similar to that of liposome, and can be fused with a target cell membrane through membrane protein on the surface of the microparticles, so that the loaded drug is directly delivered to a receptor cell, and the problems of drug degradation, cytotoxicity and the like caused by a cell phagocytosis-lysosome pathway are avoided; in addition, microparticles from different sources exhibit different characteristics due to the difference of donor cells, i.e. have specific organ affinity, for example, microparticles derived from lung metastatic tumor cells specifically reach the lung due to the surface expression of integrins α 6 β 1 and α 6 β 4, and are taken up by cells in the lung tissue of the host, such as lung epithelial cells, lung tumor cells, etc. Therefore, if a drug can be loaded in such microparticles having a specific functionality, it is possible to achieve a therapeutic effect of organ-targeted treatment of tumor metastasis.

Disclosure of Invention

The invention provides a drug delivery system for accurately targeting tumor metastasis, which loads allicin serving as an active ingredient with an anti-tumor metastasis effect into microparticles derived from high lung metastasis tumor cells.

Aiming at the above purpose, the invention provides the following technical scheme:

an allicin-entrapped cellular microparticle drug delivery system comprising microparticles extracted from organ-compatible cells and a drug having anti-tumor metastasis activity, which is garlic organosulfide.

Further, the cell types with organ affinity are lung metastatic tumor cells; can be mouse melanoma lung metastatic cells B16BL6, B16F10, mouse Lewis lung cancer cell LLC, breast cancer MDA-MB-231 tumor cells or mouse breast cancer cells 4T1, etc.

The organic sulfide of garlic can be one of diallyl trisulfide DATS, diallyl disulfide DADS, diallyl monosulfide DAS, allyl cysteine SAC and S-allyl-L-cysteine sulfoxide ACSO.

Another object of the present invention is to provide a method for preparing the above drug delivery system, comprising: taking lung metastasis tumor cells in logarithmic growth phase, stimulating the tumor cells to release microparticles, adding garlic organic sulfide solution to the tumor cells in a stimulation state for co-incubation with the cells, and after the incubation is finished, centrifuging cell culture supernatant to obtain garlic organic sulfide microparticle sediment, namely the cell microparticles encapsulating the allicin. In the prior art, the outer vesicles are generally extracted and then incubated with the medicine, but for garlic organosulfur compounds, the medicine-loading rate is low, and the concentration of the medicine after cracking is lower than a detection value. The mode of the invention gives certain stimulation to the cells without apoptosis, and simultaneously adds the medicine when the cells are in a stimulation state to promote the cells to generate more medicine-containing microparticles.

Furthermore, the invention adopts an ultraviolet irradiation mode to stimulate tumor cells; preferably, the ultraviolet irradiation time is 1-2 h, the release amount of the microparticles reaches a proper value within the time range, and cell death can occur after 2 h.

Further, the incubation concentration of the garlic organic sulfide solution is 10-200 mu M; preferably 60 to 120. mu.M.

Further, the co-incubation time is 1-24 h.

Further, after the incubation is finished, centrifuging cell culture supernatant, removing the precipitate, centrifuging the supernatant again, and obtaining the precipitate containing garlic organic sulfide microparticles.

The invention also aims to provide application of the drug delivery system in preparation of anti-tumor metastasis drugs.

The invention utilizes the cell microparticles derived from the high lung metastasis tumor cells to construct an allicin-cell microparticle drug delivery system, which has organ affinity (metastasis targeting) and a drug-loading function, effectively enhances the function of targeting metastasis and optimizes tissue affinity. The system improves the distribution of the medicine in lung tissues, and further shows more excellent treatment effect on tumor lung metastasis. Specifically, the anti-tumor lung metastasis activity of the allicin-drug-containing cell microparticles is obviously superior to that of a control naked drug, and extremely high subsequent application value and potential are shown.

Compared with the prior art, the invention has the following advantages:

1. the allicin is used as an active ingredient of the traditional Chinese medicine garlic with homology of medicine and food, has rich content and low economic cost, and has obvious anticancer activity and anti-tumor metastasis effect. The allicin can act on tumor cells, inhibit the migration and invasion capacity of the tumor cells and play a role in resisting tumor metastasis; the allicin can also act on a tumor microenvironment, and can inhibit infiltration of inflammatory cells in the tumor microenvironment, reduce release of inflammatory factors and improve the inflammatory degree of local tissues by resisting oxidation and inflammation, thereby realizing effective inhibition of tumor metastasis on the basis. Allicin is derived from medicinal and edible garlic, and has the advantages of low toxicity and multiple targets compared with the conventional medicaments at present.

2. The allicin-cell microparticle drug delivery system is reasonable in composition, and the microparticles are from the supernatant of tumor cells, are derived from a biological system, and have good biocompatibility and safety. The drug-loading system optimizes the tissue distribution characteristics, improves the distribution of the drug in a metastasis, enables the drug to be absorbed by tumor cells and lung epithelial cells of host cells of lung tissues, jointly generates the effect of resisting tumor metastasis from two aspects of inhibiting malignant biological behaviors of the tumor cells and intervening local tissue inflammatory microenvironment, and achieves the goal of synergistic preparation.

Drawings

FIG. 1 is a diagram showing the particle size distribution and morphology characteristics of DATS-microparticles of the present invention after they are dispersed in water.

FIG. 2 is a diagram showing the uptake of DATS-microparticles of the present invention by mouse melanoma cells B16BL6 cells and mouse lung epithelial cells TC-1 cells.

FIG. 3 is a tissue distribution diagram of DATS-microparticles of the present invention in a mouse melanoma lung metastasis model.

FIG. 4 is a graph showing the effect of DATS-microparticles of the present invention on a mouse melanoma lung metastasis model.

FIG. 5 is a graph showing the effect of DATS-microparticles of the present invention on the expression of inflammatory proteins in mouse lung tissue.

Detailed Description

The invention will be further elucidated by means of several specific examples, which are intended to be illustrative only and not limiting.

Example 1 preparation, characterization and drug-loading detection of DATS-drug-containing microparticles

Taking B16BL6 cells in logarithmic growth phase at 4X 10⁷Per mL, ultraviolet irradiating for 1 h, adding the concentrateAnd (3) continuously incubating DATS with the concentration of 60 mu M in the incubator for 10 h, collecting cell culture supernatant, centrifuging for 2 min at the temperature of 4 ℃ under the condition of 14000 g, discarding the precipitate, taking the supernatant, centrifuging for 70 min at the temperature of 4 ℃ under the condition of 14000 g, and obtaining precipitate containing DATS microparticles, namely DATS-drug-containing microparticles. The prepared DATS-drug-containing microparticles are white precipitates at the bottom of a centrifuge tube.

10 μ L of DATS-containing microparticles was diluted to 1 mL with pure water, and slowly poured into a sample cell of a Malvern particle size analyzer to measure the average particle size, Polydispersity (PDI) and potential of each DATS-microparticle. It was found that the particle size of the fine particles was (298.36. + -. 3.76) nm, the polydispersity was (0.53. + -. 0.18), the particle size distribution of the fine particles was broad (FIG. 1 a), and the Zeta potential was (-20.21. + -. 3.52) mV.

Absorbing 20 mu L of DATS-microparticles, respectively dripping the DATS-microparticles on a copper mesh loaded with a Formvar support film, sucking the DATS-microparticles dry after 5 s, then dripping 20 mu L of 1% phosphotungstic acid, sucking the DATS-microparticles dry after 5 s, drying the DATS-microparticles under an infrared lamp, and finally observing the form of the microemulsion particles under a JEM-2100 type transmission electron microscope. As observed by electron microscopy, DATS-MEs are spherical, have round appearance and are uniformly dispersed (FIG. 1 b).

The protein content of the drug-containing microparticles and the concentration of the cracked drug are respectively detected by BCA and HPLC, the drug loading rate is calculated, and the concentration of the cracked drug is 4.77 mu g/mL and the drug loading rate is 0.17 percent.

The concentration of organic sulfides in garlic was adjusted in the same manner, and the drug loading was as shown in table 1:

TABLE 1

Garlicin concentration (mu)M)	10	30	60	120	200	240
							Drug loading (%)	0.09± 0.01	0.12± 0.02	0.17± 0.01	0.18± 0.02	0.12± 0.02	0.03± 0.01

Garlic organosulfur concentrations below 10 μ M are low drug loads, while concentrations above 200 μ M induce tumor cell death.

Example 2 uptake of DATS-drug-containing cell microparticles by cells and in vivo tissue distribution

1. Study on uptake of DATS-microparticle drug delivery system by B16BL6 and TC-1 cells

Extracting blank microparticles: taking B16BL6 cells in logarithmic growth phase at 4X 10⁷Irradiating the cells per mL by ultraviolet for 1 h, continuously incubating the cells in an incubator for 10 h, collecting cell culture supernatant, centrifuging the cell culture supernatant for 2 min at the temperature of 4 ℃ at 14000 g, discarding the precipitate, taking the supernatant, centrifuging the supernatant for 70 min at the temperature of 4 ℃ at 14000 g, and obtaining Blank MPs. Preparation of PKH 26-DATS-microparticles: taking DATS-microparticles, diluting the same into total protein concentration of 800 ug/mL, taking 0.1 mL of DATS-MPs, re-suspending the DATS-microparticles in 0.5 mL of Diluent C as A, taking 2 mu L of PKH26 to be added into 0.5 mL of Diluent C as B, then mixing A and B, incubating for 3 min at 25 ℃, then adding equal volume of serum, terminating the reaction, then centrifuging the mixed solution of DATS-MPs under 14000 g, removing supernatant, re-suspending, washing 3 times by PBS,removing the unbound dye to obtain PKH 26-DATS-microparticles. B16BL6 and TC-1 cells are divided into 2 groups, Blank MPs and DATS-MPs are added respectively, after 2h, the culture medium in each hole is sucked out, PBS is washed for three times, the result of the uptake in each group of cells is observed under a fluorescence microscope, and the relative absorbance value of each group is measured by a flow cytometer. The results show that both TC-1 and B16BL6 cells were able to efficiently take up drug-containing microparticles, with B16BL6 having a stronger uptake capacity (FIG. 2).

2. Distribution of DATS-microparticle drug delivery system in mice

Establishing a model: taking B16BL6 cells in logarithmic growth phase, digesting with 0.25% pancreatin digestive juice containing EDTA, incubating for 2 min in an incubator, adding DMEM complete culture medium immediately to stop digestion when the cells are rounded under a microscope, transferring the cells into a centrifuge tube after the cells are blown uniformly, centrifuging for 5 min at 1000 r/min, discarding supernatant, collecting cells at the bottom of the centrifuge tube, preparing the cells into cell suspension with sterilized PBS solution, and adjusting the cell density to 1 × 10⁶and/mL. The prepared cell suspension is quickly inoculated. The left hind paw injection area of the mice was swabbed with an alcohol cotton swab, the cell suspension was blown evenly, and 0.05 mL of freshly prepared B16BL6 cell suspension was injected subcutaneously into the left hind paw of each C57BL/6 mouse rapidly with a 1.0 mL disposable medical syringe. 23 days after the mice are inoculated with tumor cells, performing in-situ tumor excision operation on the mice, namely performing amputation operation treatment on the left hind feet of the mice, sterilizing an operation table, a cotton rope and operation scissors, binding the upper parts of the ankles of the left hind feet of the mice by using the cotton rope, quickly cutting off the left hind feet of the mice along the lower part of the hemp rope by using the operation scissors, then sterilizing the amputation part by using iodophor, and promoting the mice to perform lung metastasis by performing the operation excision of the primary tumor.

Preparation of administration preparation: extracting blank microparticles: taking B16BL6 cells in logarithmic growth phase at 4X 10⁷Irradiating the cells per mL by ultraviolet for 1 h, continuously incubating the cells in an incubator for 10 h, collecting cell culture supernatant, centrifuging the cell culture supernatant for 2 min at the temperature of 4 ℃ at 14000 g, discarding the precipitate, taking the supernatant, centrifuging the supernatant for 70 min at the temperature of 4 ℃ at 14000 g, and obtaining Blank MPs. Preparation of PKH 26-DATS-microparticles: taking the DATS-micro-particles,diluting the two solutions to obtain a total protein concentration of 800 ug/mL, taking 0.1 mL of DATS-MPs, re-suspending in 0.5 mL of DiluentC to obtain A, taking 2 mu L of PKH26 to add into 0.5 mL of DiluentC to obtain B, mixing A and B, incubating at 25 ℃ for 3 min, adding an equal volume of serum, terminating the reaction, centrifuging the mixed solution of DATS-MPs at 14000 g, removing supernatant, re-suspending, washing with PBS for 3 times, and removing unbound dye to obtain PKH 26-DATS-microparticles.

The mice were divided into a normal mouse group and a melanoma lung metastasis model mouse group, and were administered with microparticles of PKH26-free and PKH26, respectively. The method comprises the steps of carrying out abdomen unhairing treatment on a mouse one day in advance, after fasting and water prohibition are carried out for 24 hours, taking 0.5 mL of each of a PKH26-free group and a PKH 26-microparticle group, injecting the groups into the mouse through tail vein injection, placing the mouse into a cavity with 200 mmHg of isoflurane for anesthesia after administration, after 5-10 minutes, horizontally placing the prone position of the mouse into a recording dark box of a living body imaging system of the mouse, respectively carrying out living body imaging recording after administration for 1, 4 and 8 hours, dissecting each group of the mouse after 8 hours, taking lungs of the mouse, and observing the fluorescence distribution condition of the lungs of the mouse in the recording dark box.

The results are shown in FIG. 3, in vivo imaging of mice, no matter in normal mice or in mice of a melanoma lung metastasis model, significant differences are difficult to see between the PKH26-free group and the PKH 26-microparticle group at 1, 4 and 8 h, but after 8 h of mouse dissection, in an infrared image of the lung of the mice, it can be seen that in the normal mice and the mice of the melanoma lung metastasis model, the free PKH26 dye group does not accumulate in the lung of the mice, and the PKH26 microparticles are all observed in the lung to show that fluorescence shows that the mouse melanoma cell B16BL6 has good lung targeting effect in the normal mice or the mice of the melanoma lung metastasis.

Example 3 evaluation of the therapeutic Effect of DATS-microparticles on Lung metastasis of melanoma in mice and intervention of inflammatory microenvironment

The C57BL6 mice with the weight of about 18g are adaptively bred for 7 days, and then injected with 5X 10 mice from tail vein⁵B16BL6 melanoma cells/mL, mice were inoculated with tumor cells, and groups of mice were randomly divided into model groups, blankMicroparticle group, DATS-microparticle and DATS nude drug group, each group was 5. The weight of the black mouse was measured every day, the state and death of the animal were observed and recorded, administration was started 23 days later, intraperitoneal injection was performed at an administration concentration of 1.8 mg/kg, administration was performed every other day, eye ball was removed 14 days later to obtain blood, the black mouse was sacrificed by cervical spine removal, dissected, lung tissue of the mouse was taken, and visceral index (tissue/weight) was calculated. The result shows that the DAS-microparticles can effectively inhibit lung metastasis and reduce lung weight. Subjecting the stripped lung tissue to H&E staining, the results showed that the DATS-microparticles reduced the number of lung nodules more than the naked drug, reduced the tumor area of lung metastasis, and significantly reduced lung metastasis (fig. 4). Further examination of lung tissue for inflammatory proteins MPO, fibrinectin, MRP8, etc. revealed that the DATS-microparticles of the present invention could reduce the expression of inflammatory proteins (fig. 5).

Claims

1. A cell microparticle drug delivery system entrapping allicin, comprising microparticles extracted from cells having affinity to organs and a drug having anti-tumor metastasis activity, the drug being allicin; the cell type with organ affinity is lung metastatic tumor cells;

the preparation method of the medicine carrying system comprises the following steps:

taking lung metastasis tumor cells in logarithmic phase, carrying out ultraviolet irradiation for 1-2 h to stimulate the tumor cells to release microparticles, adding an allicin solution to the tumor cells in a stimulation state to carry out co-incubation with the cells, and after the incubation is finished, centrifuging cell culture supernatant to obtain allicin-containing microparticle precipitates, namely allicin-encapsulated cell microparticles.

2. The drug delivery system of claim 1, wherein the concentration of the allicin solution incubated is 10-200 μ M.

3. The drug delivery system of claim 1, wherein the concentration of the allicin solution incubated is 60-120 μ M.

4. The drug delivery system of claim 1, wherein the co-incubation time is 1-24 hours.

5. The drug delivery system of claim 1, wherein after incubation, the cell culture supernatant is centrifuged, the pellet is discarded, and the supernatant is centrifuged again to obtain the pellet containing allicin microparticles.

6. The use of the drug delivery system of claim 1 in the preparation of a medicament for the treatment of tumor metastasis to the lung.