CN113398092A

CN113398092A - Bladder targeted drug-loaded exosome and application and drug for treating bladder diseases

Info

Publication number: CN113398092A
Application number: CN202110692320.8A
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: 2021-09-17
Also published as: CN114948899A

Abstract

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

Description

Bladder targeted drug-loaded exosome and application and drug for treating bladder diseases

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a bladder targeted drug-loaded exosome and a drug for applying and treating bladder diseases.

Background

The urinary bladder is an organ for storing and discharging urine of a human body, and the urinary bladder loses the normal functions for any reason, namely, the urinary bladder diseases occur due to the abnormality of the times of urination, the urine volume and the urine color. Common bladder disorders include bladder inflammation, bladder polyps, bladder muscle weakness, and bladder cancer. Bladder cancer is the most common malignant tumor of the urinary system and is one of ten common tumors of the whole body. The medicine takes the first place of the disease rate of genitourinary tumors in China, and the disease rate of the medicine is second to that of prostatic cancer in the West and is 2 nd.

Current cancer treatments, whether chemical or biological, involve three problems, the first: the medicine is easy to be degraded in the serum environment in vivo, has small molecular volume and is easy to be excreted; secondly, the method comprises the following steps: the drug is non-specifically distributed in the body to reduce the concentration of the target tissue; thirdly, the method comprises the following steps: the drug needs to overcome the physical barriers of the vessel wall and various tissues to reach the target cells. Therefore, there is a need for a drug delivery system, which has the characteristics of good biocompatibility, high stability, tissue targeting property and strong transmembrane delivery capability.

The targeted drug delivery system is a drug delivery system which selectively enriches drugs in focus or specific parts in vivo by using a carrier. The medicine can play the maximum role in the focus of infection, and the toxic and side effects of the whole body are reduced to the minimum degree. Comprises a passive targeting preparation: insoluble drug-loaded particles (such as liposome, nanocapsule and the like) enable the drug to enter a target region by means of phagocytosis of body cells; active targeting formulation: drug carriers are modified with antibodies and the like as "missiles" to target drug delivery. The targeting preparation can improve the drug effect, reduce the toxicity, improve the safety, effectiveness and reliability of the medicine and the compliance of the patient in the medicine, so the targeting preparation is increasingly paid extensive attention by the medical field at home and abroad. The development of nano-drug delivery systems has solved the above problems to some extent. However, the liposome or albumin-based nano-carrier cannot exist stably in blood, is easy to be eliminated by a reticuloendothelial system or a mononuclear phagocyte system, and lacks tissue targeting; the antibody modified carrier increases the cost of the medicine and is difficult to achieve the ideal targeting effect. In recent years, it has been discovered that exosomes can act as carriers, carrying drugs and delivering them to adjacent or distant cells. Compared with the liposome which is widely researched, the exosome shows greater advantages, and has good biocompatibility, biodegradability, low toxicity, stability and low immunogenicity. The preparation of an exosome drug-loading system with a bladder targeting effect is an ideal solution for treating bladder diseases.

Therefore, the preparation of a high-efficiency bladder targeting exosome drug-carrying system for treating bladder-related diseases is urgently needed.

Disclosure of Invention

The invention aims to solve the defects in the prior art, and provides a bladder targeted drug-loading exosome and a drug for application and treatment of bladder diseases.

In order to achieve the purpose, the invention is implemented according to the following technical scheme:

the first purpose of the invention is to provide a bladder targeted drug-loaded exosome, which is obtained by introducing a drug for treating bladder diseases into an exosome with bladder tissue targeting property, wherein the exosome with bladder tissue targeting property is derived from renal cells.

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

As a preferred embodiment of the present invention, the kidney cells include, but are not limited to, human kidney cells, animal kidney cells, human kidney cancer cells, animal kidney cancer cells, induced pluripotent stem cell-induced kidney cells.

As a preferred embodiment of the present invention, the human kidney cells include, but are not limited to, the normal human embryonic kidney cells HEK-293 and derivatives thereof: AD293, CRL-11268, HEK293-L, HEK293-T, GP2-293, GP2-293Luc, KiMA, HEK-293-6E, 293A, 293F, 293FT, 293H, 293-L.P, 293-mTLR5, 293T/17, 293XL-hTLR7, 293XL-hTLR9, 2V6.11, AAV-293, GP-293, Phix-293T, 293E, 293ET, 293Ad 5; human embryonic kidney cell CRL-11268; normal human kidney cells HKb-20, FC33, HK-2C, ProPakA.6, TCMK-1; the animal kidney cells include but are not limited to African green monkey kidney vero and derivatives thereof vero-E6, VREO/IgRCD4, VREO/IgR; african green monkey kidney fibroblast COS-1, COS-7, COS-3; african green monkey embryonic kidney cell MARC-145; vero cells CV-1; immortalized mouse mKF cells; mouse kidney cells MC53, MRGEC, TCMK-1, mRTEC; hamster kidney cells BHK, BHK-N, BHK-21, BHK-M; rat renal cells RK1, NRK-49F, HBZY-1, NRK-52E.

As a preferred embodiment of the present invention, the drugs for treating bladder diseases include, but are not limited to, gemcitabine hydrochloride, microRNA, siRNA, proteins, antibodies, cisplatin, gemcitabine, mitomycin, BCG, and doxorubicin.

Further, the specific steps of introducing the drug for treating bladder diseases into the exosome with the bladder tissue targeting property are as follows:

1) placing a medicament for treating bladder diseases and exosomes with bladder tissue targeting into an electric rotor;

2) performing electrotransformation on a medicament for treating bladder diseases and exosomes with bladder tissue targeting by using an exponential or square wave and using 50-300V voltage;

3) and (3) after the electrotransformation product is subjected to water bath at 37 ℃ for 30min, centrifuging for 120min under 100000g of centrifugal force to remove free drugs, and obtaining the purified bladder targeted drug-loaded exosome.

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

The third purpose of the invention is to provide a medicine for treating bladder diseases, which comprises the bladder targeted medicine-carrying exosome.

Compared with the prior art, the exosome secreted by the kidney cells can be enriched in the bladder without any modification, and the exosome derived from the kidney cells is used, so that the problem of the yield of the exosome is solved, and the exosome has good application prospect; and the exosome from the kidney cell can be loaded with different drugs or active molecules, and the drug delivery is carried out by targeting bladder tissues, so that the treatment effect of bladder diseases is improved, and the drug toxic and side effects are reduced.

Drawings

FIG. 1 is a graph showing the detection of exosome marker proteins secreted by HEK-293 cells of normal human embryonic kidney.

FIG. 2 is a graph showing the particle size distribution of exosomes secreted by HEK-293 cells of normal human embryonic kidney.

FIG. 3 is a map of the secretion of HEK-293 exosomes from PKH 67-labeled human embryonic kidney cells in different tissues.

FIG. 4 is an in vitro assay of the antitumor activity of bladder targeted exosomes loaded with the drug gemcitabine hydrochloride used to treat bladder disease.

FIG. 5 is an in vivo assay of bladder targeting exosome antitumor activity loaded with the drug gemcitabine hydrochloride used to treat bladder disease.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

Example 1

The normal human embryonic kidney cells HEK-293 (this example takes the normal human embryonic kidney cells HEK-293 as an example) were collected. Centrifuging culture solution of normal human embryonic kidney cell HEK-293 (cultured by kidney cell culture solution without exosome serum and DMEM medium) for 10min under 2000g centrifugal force, 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 2h, re-suspending and collecting the precipitate with sterile PBS, and storing at 4 deg.C for a short period to obtain the bladder targeting exosome. The surface marker protein and particle size distribution of the bladder targeting exosome obtained by extraction and separation are shown in fig. 1 and fig. 2. The amount of exosome expressed by the kidney cells was measured, and the BCA method measured the concentration of exosome protein secreted by the kidney cells (2.85 μ g/μ L).

Example 2

Taking exosome secreted by HEK-293 of normal human embryonic kidney cells, staining the exosome with PKH67, and tracing the distribution of the exosome secreted by the kidney cells in vivo, wherein the method comprises the following specific steps:

taking 100 mu g of exosome secreted by the kidney cells HEK-293, incubating the exosome with 1 mu L of PKH 674 ℃ in the dark overnight, centrifuging for 2h under 100000g (g is the gravity acceleration) of centrifugal force, discarding supernatant, washing twice by PBS, resuspending the exosome secreted by the kidney cells by sterile PBS, injecting the exosome into a C57bl/6 mouse through tail vein, in the embodiment, a male C57bl/6(4-6 weeks) mouse is purchased from Beijing Huafukang Biotech GmbH, and all mice are cultivated in SPF-level facilities; after 24h, the mice are anesthetized, the liver, lung, spleen, bladder, kidney, stomach and intestine of the mice are taken for frozen section, and after Hoechst33342 stains cell nucleus, the biological distribution of exosomes secreted by the kidney cells in each organ of the mice is observed. The results show that: exosomes secreted by the kidney cells of this example appeared significantly enriched in bladder tissue (fig. 3), targeting efficiencies as high as 65% -90%. The bladder targeting exosome prepared in the example is demonstrated to have bladder tissue targeting.

Example 3

In this example, gemcitabine hydrochloride, a drug used to treat bladder disorders, was selected.

In the embodiment, the drug for treating bladder diseases is introduced into the bladder target exosome to prepare the bladder tumor targeted therapeutic drug, and the specific operation is as follows:

1) 150 mu g of bladder target exosome is mixed with gemcitabine hydrochloride, and the electrotransfer buffer solution can be (PBS, DMEM, Cytomix, Tris-HCl) to supplement the mixture to 150 mu L, and the mixture is transferred to electrotransfers with different specifications (0.2cm 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 bladder target exosome and the gemcitabine hydrochloride;

2) and (4) ultracentrifuging the electrotransformation product for 120min under the centrifugal force of 100000g, and collecting the supernatant to measure the drug loading. As a result: for gemcitabine hydrochloride, 150 μ g of bladder targeting exosomes were electrotransformed at a voltage of 250V with the highest efficiency, which could be 35.7%.

Example 4

This example further demonstrates in vitro and in vivo experiments to examine the therapeutic effect of bladder cancer in bladder targeting exosomes loaded with gemcitabine hydrochloride, a drug used to treat bladder diseases, in this example, Balb/c nude mice (4-6 weeks) were purchased from beijing waukee biotechnology limited, all of which were bred in SPF-level facilities. The method comprises the following specific steps:

in vitro experiments:

spreading bladder cancer cell BIU-87 in 96-well plate at 5 × 10 per well³And respectively adding the bladder targeting exosome and the bladder targeting exosome loaded with the bladder tumor treatment drug gemcitabine hydrochloride into each cell, and detecting the killing effect of the bladder targeting exosome loaded with the bladder tumor treatment drug gemcitabine hydrochloride on the bladder cancer cell BIU-87 by MTT (methyl thiazolyl tetrazolium). In vitro experiments, the bladder targeting exosome loaded with the bladder tumor treatment drug gemcitabine hydrochloride can effectively kill tumor cells compared with a pure bladder targeting exosome, and the results are shown in fig. 4.

In vivo experiments:

in vivo experiments, a model of bladder cancer was established, and 5X 10 cells were used⁶Injecting BIU-87 into Balb/c nude mouse subcutaneously to grow to about 100mm³When the bladder target exosome carrying the bladder tumor treatment drug gemcitabine hydrochloride is injected into the tail vein, the injection is performed once every 3 days for 4 times, and then the tumor volume is measured every other day, wherein the tumor volume is 1/2 × a × b². a represents a long diameter, and b represents a short diameter.

In vivo experiments, as shown in fig. 5, the bladder targeting exosome loaded with the bladder tumor treatment drug gemcitabine hydrochloride can significantly reduce the tumor size of mice in a free antitumor drug group 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 bladder targeted drug-loaded exosome is obtained by introducing drugs for treating bladder diseases into an exosome with bladder tissue targeting property, wherein the exosome with the bladder tissue targeting property is derived from kidney cells.

2. The bladder-targeted drug-loaded exosome according to claim 1, wherein said kidney cells include but are not limited to human kidney cells, animal kidney cells, human kidney cancer cells, animal kidney cancer cells, induced pluripotent stem cell-induced kidney cells.

3. The bladder-targeted drug-loaded exosome according to claim 2, wherein the human kidney cells include but are not limited to normal human embryonic kidney cells HEK-293 and derivatives thereof: AD293, CRL-11268, HEK293-L, HEK293-T, GP2-293, GP2-293Luc, KiMA, HEK-293-6E, 293A, 293F, 293FT, 293H, 293-L.P, 293-mTLR5, 293T/17, 293XL-hTLR7, 293XL-hTLR9, 2V6.11, AAV-293, GP-293, Phix-293T, 293E, 293ET, 293Ad 5; human embryonic kidney cell CRL-11268; normal human kidney cells HKb-20, FC33, HK-2C, ProPakA.6, TCMK-1; the animal kidney cells include but are not limited to African green monkey kidney vero and derivatives thereof vero-E6, VREO/IgRCD4, VREO/IgR; african green monkey kidney fibroblast COS-1, COS-7, COS-3; african green monkey embryonic kidney cell MARC-145; vero cells CV-1; immortalized mouse mKF cells; mouse kidney cells MC53, MRGEC, TCMK-1, mRTEC; hamster kidney cells BHK, BHK-N, BHK-21, BHK-M; rat renal cells RK1, NRK-49F, HBZY-1, NRK-52E.

4. The bladder targeted drug-loaded exosome according to claim 1, wherein the drug for treating bladder disease comprises but is not limited to gemcitabine hydrochloride, microRNA, siRNA, proteins, antibodies, cisplatin, gemcitabine, mitomycin, BCG, doxorubicin.

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

6. The use of the bladder-targeted drug-loaded exosome of any one of claims 1-5 in the preparation of a medicament for treating a bladder disease.

7. A medicament for the treatment of bladder disease comprising the bladder-targeted drug-loaded exosome of any one of claims 1 to 5.