CN113528427A

CN113528427A - Colorectal targeted drug-loaded exosome, application thereof and drug for treating colorectal diseases

Info

Publication number: CN113528427A
Application number: CN202110692366.XA
Authority: CN
Inventors: 姜海涛; 王天怡
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2021-10-22
Also published as: CN114958722B; CN114958722A

Abstract

The invention belongs to the technical field of biological medicines, and provides a colorectal targeted drug-loading exosome, application and a drug for treating colorectal diseases, wherein the colorectal targeted drug-loading exosome is obtained by introducing a drug for treating colorectal diseases into a colorectal tissue-targeted exosome, and the colorectal tissue-targeted exosome is derived from lung cells; the colorectal tumor treatment drug comprises the colorectal targeted drug-loading exosome. Compared with the prior art, the exosome secreted by the lung cells can be enriched in the colon and the colon without any modification, and the exosome derived from the lung cells is used, so that the problem of the yield of the exosome is solved, and the exosome has good application prospect; moreover, different drugs or active molecules can be loaded by exosomes derived from lung cells, and the drug delivery is carried out by targeting colorectal tissues, so that the treatment effect of colorectal diseases is improved, and the drug toxic and side effects are reduced.

Description

Colorectal targeted drug-loaded exosome, application thereof and drug for treating colorectal diseases

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a colorectal targeted drug-loading exosome, application thereof and a drug for treating colorectal diseases.

Background

The colon and rectum are part of the digestive system, and both form a long muscular tube, called the large intestine. Cancers that originate in the colon are referred to as colon cancer, while cancers that originate in the rectum are referred to as rectal cancer. Both may also be referred to as colorectal cancers. Colorectal cancer is a common malignancy in the gastrointestinal tract, the fourth largest tumor type in the world, resulting in the death of 50 million patients each year. The morbidity and mortality of colorectal cancer in China are respectively 27.3/10 ten thousand and 13.9/10 ten thousand, and the colorectal cancer is respectively located at the 5 th position of the morbidity and mortality of tumors, wherein patients over 45 years old account for 93.5 percent of the total sick population. The early symptoms are not obvious, and the symptoms such as defecation habit change, hematochezia, diarrhea and constipation alternation, local abdominal pain and the like are presented along with the increase of cancer, and the general symptoms such as anemia, weight loss and the like are presented at the late stage. The incidence and fatality rate of the cancer are only second to those of gastric cancer, esophageal cancer and primary liver cancer in digestive system malignant tumors.

In recent 50 years, despite rapid development of surgical techniques, the operative cure rate and 5-year survival rate of colorectal cancer are always about 50%, and the reason for failure of treatment is mainly high local recurrence rate, so comprehensive treatment must be considered for improving the treatment effect of colorectal cancer. In addition to surgical treatment, several drugs for colorectal cancer treatment have been marketed in recent years. Colorectal tumor targeted drug therapy is classified into three major categories according to the mechanism of action: the first acts on the tumor cell growth signal channel, and inhibits the proliferation of tumor cells by blocking the tumor cell growth signal. Represents the drug: cetuximab, panitumumab. The second one acts on the microenvironment for tumor growth (tumor angiogenesis), and the aim of 'starving the tumor' is achieved by blocking the blood supply of tumor cells and blocking the nutrition supply of the tumor cells. Represents the drug: bevacizumab, regorafenib. The third one acts on immune cells (T cells) of a patient, and enhances the recognition and killing effects of the immune cells on tumors by continuously activating the immune cells. Represents the drug: anti-PD-1 monoclonal antibody (Pembrolizumab). Although the medicines prolong the life of a patient to a certain degree, the medicines have obvious toxic and side effects because of weak targeting capability.

Good target medicine can carry medicine to enter the disease premonition part, and normal tissues and organs are not enriched with medicine. Therefore, the effective concentration of the medicine at the focus part can be improved, the curative effect is further improved, and the concentration of the medicine in normal tissues and organs can be reduced, so that the side effect of the medicine is reduced. Although targeted therapy has received much attention in the medical and pharmaceutical fields in recent years, there is a literature that targeting of drugs can be enhanced by using targeting groups (e.g., receptor-binding ligands specific to the surface of cancer cells) in combination with drug delivery systems, but clinical applications have shown that such targeting is of little effect. At present, no ideal drug targeting drug delivery system exists, which is a great scientific problem to be solved urgently in the aspect of cancer treatment.

In recent years, the appearance of exosomes is expected to solve this problem. It is found that exosomes are carriers of information transfer between various tissues and cells of the body. It can transmit information in the body at a long distance. And the exosome can freely cross cell membranes, so that the medicine carrying system is ideal.

The chemotherapeutic drug has good antitumor activity, but has great toxic and side effects due to poor targeting property. For example: oxaliplatin and pentafluorouracil, and the like. Oxaliplatin (Oxaliplatin) is a 3 rd generation platinum anticancer drug and is a platinum compound of diaminocyclohexane, namely 1, 2-diaminocyclohexane group is used for replacing an amino group of cisplatin. The effect of the platinum-based drug is the same as that of other platinum-based drugs, namely DNA is taken as a target action site, and platinum atoms form cross connection with the DNA to antagonize the replication and transcription of the DNA.

Therefore, it is highly desirable to prepare a highly effective colorectal targeted exosome drug delivery system for the treatment of colorectal related diseases.

Disclosure of Invention

The embodiment of the invention aims to provide a colorectal targeted drug-loading exosome, application thereof and a drug for treating colorectal diseases, so as to solve the problems in the background technology.

In order to achieve the above object, the embodiments of the present invention provide a colorectal targeted drug-loading exosome, which is obtained by introducing a drug for treating colorectal diseases into a colorectal tissue-targeted exosome, and the colorectal tissue-targeted exosome is derived from lung cells.

Of course, the lung cells may also be genetically modified or engineered to target colorectal tissue, including but not limited to genetic modification, gene overexpression or deletion, molecular modification, and the like; exosomes of lung cells may also be surface modified or engineered to target colorectal tissues, including but not limited to surface protein modifications, surface protein alterations, surface small molecule modifications, and the like.

Preferably, the lung cells include, but are not limited to, human embryonic lung fibroblasts; human embryonic lung cells; human lung fibroblasts; human embryonic lung diploid cells; human lung cancer cells; an animal embryonic lung cell; animal embryonic lung fibroblasts; animal lung cells; an animal lung cancer cell; induced pluripotent stem cell-induced lung cells.

Preferably, the human embryonic lung fibroblasts include, but are not limited to, HFL-1, HELF, IMR-90; such human embryonic lung cells include, but are not limited to, MRC-5, WI-38, HLF; the human lung fibroblast includes but is not limited to HEL-2, HEL-1, CCL-153; the human embryo lung diploid cell is HLF-02; the animal embryonic lung cell; animal embryonic lung fibroblasts; animal lung cells; animals in animal lung cancer cells include, but are not limited to, monkeys, mice, hamsters, rats.

Preferably, the drug for treating colorectal diseases includes, but is not limited to siRNA, microRNA, protein, antibody, fluorouracil, oxaliplatin, irinotecan, capecitabine.

Preferably, the specific steps of introducing the drug for treating colorectal diseases into the exosomes with colorectal tissue targeting are as follows:

placing a medicament for treating colorectal diseases and an exosome with colorectal tissue targeting property into an electric rotor;

adopting exponential waves or square waves, and performing electric transformation on the medicaments for treating colorectal diseases and the exosomes with colorectal tissue targeting by using 50-300V voltage;

and (4) heating the electrotransformation product in water bath, and centrifuging to remove free drugs to obtain the purified colorectal targeted drug-loaded exosome.

The second purpose of the invention is to provide the application of the colorectal targeted drug-loading exosome in the preparation of drugs for treating colorectal diseases.

The invention also provides a medicine for treating colorectal diseases, which comprises the colorectal targeted medicine-carrying exosome.

In summary, due to the adoption of the technical scheme, the method has the following beneficial effects:

the embodiment of the invention provides a colorectal targeted drug-loading exosome and application thereof and a drug for treating colorectal diseases, the exosome secreted by lung cells can be enriched in the colorectal without any modification, and the exosome derived from the lung cells is used, so that the problem of the output of the exosome is solved, and the colorectal targeted drug-loading exosome has good application prospect; moreover, different drugs or active molecules can be loaded by exosomes derived from lung cells, and the drug delivery is carried out by targeting colorectal tissues, so that the treatment effect of colorectal diseases is improved, and the drug toxic and side effects are reduced.

Drawings

FIG. 1 is a diagram of the detection of exosome marker proteins secreted by HFL-1 from human embryonic lung fibroblasts.

FIG. 2 is a graph showing the particle size distribution of exosomes secreted by human embryonic lung fibroblast HFL-1.

FIG. 3 is a map of PKH 67-labeled HFL-1 secreted exosomes in different tissues.

Figure 4 is an in vitro assay of colorectal targeting exosome antitumor activity loaded with oxaliplatin, a drug used for the treatment of colorectal disease.

Figure 5 is an in vivo assay of colorectal targeting exosome antitumor activity loaded with oxaliplatin, a drug used to treat colorectal disease.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the embodiments of the present invention are described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the embodiments of the invention and are not limiting of the embodiments of the invention.

Example 1

The embodiment of the invention provides a colorectal targeted drug-loading exosome, which is obtained by introducing a drug for treating colorectal diseases into a colorectal tissue-targeted exosome, wherein the colorectal targeted drug-loading exosome is derived from lung cells.

Human embryonic lung fibroblast cells HFL-1 (in this example, human embryonic lung fibroblast cells HFL-1 are used as an example) are collected.

The HFL-1 cell culture solution is cultured by exosome-free serum and a DMEM medium; centrifuging at 2000g centrifugal force for 10min, and collecting supernatant; centrifuging for 30min under the centrifugal force of 10000g, taking supernatant, and removing cell debris and precipitates; centrifuging the centrifuged cell culture solution under 100000g (g is gravity acceleration) for 2 h, re-suspending and collecting the precipitate with sterile PBS, and storing at 4 deg.C for a short period to obtain the target exosome for colon and rectum. The surface marker protein and particle size distribution of the extracted and separated colorectal targeted exosomes are shown in fig. 1 and fig. 2. Measuring the expression quantity of the HFL-1 cells and secreted exosomes thereof, and detecting the concentration of the secreted exosome protein of the HFL-1 cells by a BCA method to be 2.73 mu g/mu L.

Example 2

The exosomes secreted from the HFL-1 cells of example 1-2 above were taken, stained with PKH67, and the in vivo distribution of the exosomes secreted from the HFL-1 cells was traced, and male C57bl/6 mice (4-6 weeks) were purchased from Beijing Wakaukang Biotech GmbH, all of which were bred in SPF-grade facility. The method comprises the following specific steps:

100 mu g of exosome secreted by HFL-1 cells is taken, incubated with 1 mu of LPKH674 ℃ in the dark overnight, centrifuged for 2 h under 100000g (g is gravity acceleration) of centrifugal force, supernatant is discarded, and after being washed twice by PBS, the exosome secreted by the lung cells is resuspended by sterile PBS, and injected into a C57bl/6 mouse through tail vein. After 24h, the mice were anesthetized, and the heart, liver, spleen, lung, kidney, stomach and intestine of the mice were frozen and sectioned, and after Hoechst33342 stained the nuclei, the biodistribution of exosomes secreted from HFL-1 cells in each organ of the mice was observed. The results show that: the exosome secreted by the HFL-1 cell of this example is significantly enriched in colorectal tissue (fig. 3), and the targeting efficiency can reach as high as 65% -90%, which indicates that the colorectal targeting exosome prepared in this example has colorectal tissue targeting.

Example 3

The drugs for treating colorectal diseases include but are not limited to siRNA, microRNA, proteins, antibodies, fluorouracil, oxaliplatin, irinotecan, capecitabine. In this example, oxaliplatin, a drug used for treating colorectal cancer, is selected.

The drug for treating colorectal diseases is introduced into a colorectal targeted exosome to prepare the colorectal tumor targeted therapeutic drug, and the specific operation is as follows:

150 mu g of colorectal targeted exosome is mixed with oxaliplatin, and electrotransfer buffer solution can be PBS, DMEM, Cytomix and Tris-HCl, which is selected in the embodiment, PBS is used for complementing the colorectal targeted exosome to 150 mu L, and the colorectal targeted exosome is transferred to electrotransfers with different specifications (0.2 cm and 0.4 cm). Adopting different waveforms (exponential waves and square waves) and using different voltages (50V, 100V, 200V and 300V) to respectively carry out electrotransformation on the colorectal targeting exosomes and the oxaliplatin;

and (4) ultracentrifuging the electrotransformation product for 120 min under the centrifugal force of 100000g, and collecting the supernatant to measure the drug loading.

As a result: for oxaliplatin, 150 mu g of colorectal targeting exosome and oxaliplatin are subjected to electrotransformation under the voltage of 250V, and the efficiency is the highest and can reach 34.3%. The results show that: oxaliplatin was successfully loaded into exosomes secreted by HFL-1 cells.

Example 4

In this example, Balb/c nude mice (4-6 weeks) were purchased from Beijing Huafukang Biotechnology GmbH, all of which were cultured in SPF-level facilities. The method comprises the following specific steps:

in vitro experiments:

the colorectal cancer cells HCT-116 cells were plated in 96-well plates at 5X 10 per well³Adding colorectal target exosome (control group) and Oroxa carrying colorectal tumor treatment drug into each cellUtilizing the colorectal targeting exosome of platinum, and detecting the killing effect of the colorectal targeting exosome carrying oxaliplatin serving as a colorectal tumor treatment drug on colon cancer cells by MTT. In vitro experiments, the colorectal targeted exosome loaded with the colorectal tumor treatment drug can effectively kill tumor cells compared with a pure colorectal targeted exosome, and the results are shown in fig. 4.

In vivo experiments:

in vivo experiments, colorectal cancer model was established, and 5 × 10⁶A single HCT-116 cell was injected subcutaneously into Balb/cNuded mice until they grew to approximately 100mm³In the meantime, colorectal targeted exosomes loaded with oxaliplatin as a colorectal tumor treatment drug were injected into the tail vein once every 3 days for 4 times, and then tumor volumes were measured every other day, with tumor volumes =1/2 × a × b². a represents a long diameter, and b represents a short diameter.

In vivo experiments, as shown in fig. 5, the colorectal targeting exosome (oxaliplatin-exosome) loaded with oxaliplatin as a colorectal tumor treatment drug can significantly reduce the tumor size of a mouse compared with a free antitumor drug group (oxaliplatin) with an equal dose.

It will be evident to those skilled in the art that the embodiments of the present invention are not limited to the details of the foregoing illustrative embodiments, and that the embodiments of the present invention are capable of being embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the embodiments being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. The colorectal targeted drug-loaded exosome is obtained by introducing drugs for treating colorectal diseases into a colorectal tissue-targeted exosome, and the colorectal tissue-targeted exosome is derived from lung cells.

2. The colorectal targeted drug-loaded exosome of claim 1, wherein the lung cells include but are not limited to human embryonic lung fibroblasts; human embryonic lung cells; human lung fibroblasts; human lung cells; human embryonic lung diploid cells; human lung cancer cells; an animal embryonic lung cell; animal embryonic lung fibroblasts; animal lung cells; an animal lung cancer cell; induced pluripotent stem cell-induced lung cells.

3. The colorectal targeted drug-loaded exosome of claim 2, wherein the animal of the animal embryonic lung cell, the animal embryonic lung fibroblast, the animal lung cell and the animal lung cancer cell includes but is not limited to monkey, mouse, rat, hamster; the human embryonic lung fibroblasts include but are not limited to HFL-1, HELF, IMR-90; such human embryonic lung cells include, but are not limited to, MRC-5, WI-38, HLF; the human lung fibroblast includes but is not limited to HEL-2, HEL-1, CCL-153; the human embryo lung diploid cell is HLF-02.

4. The colorectal targeted drug-loaded exosome according to claim 1, wherein the drug for treating colorectal disease includes but is not limited to siRNA, microRNA, protein, antibody, fluorouracil, oxaliplatin, irinotecan, capecitabine.

5. The colorectal targeted drug-loaded exosome according to claim 1, wherein the specific steps of introducing the drug for treating colorectal diseases into the colorectal tissue-targeted exosome are as follows:

6. The use of a colorectal targeted drug-loaded exosome according to any one of claims 1-5 in the preparation of a medicament for the treatment of colorectal disease.

7. A medicament for the treatment of colorectal disorders comprising a colorectal targeted drug-loaded exosome according to any one of claims 1 to 5.