CN113846047A - Kidney targeting drug-loaded exosome and application and drug for treating kidney diseases - Google Patents
Kidney targeting drug-loaded exosome and application and drug for treating kidney diseases Download PDFInfo
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- CN113846047A CN113846047A CN202110692827.3A CN202110692827A CN113846047A CN 113846047 A CN113846047 A CN 113846047A CN 202110692827 A CN202110692827 A CN 202110692827A CN 113846047 A CN113846047 A CN 113846047A
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Abstract
The invention belongs to the technical field of biological medicines, and discloses a kidney targeting drug-loaded exosome and a drug for applying and treating kidney diseases. The exosome secreted by the bladder cells can be enriched in the kidney without any modification, and the exosome secreted by the bladder cells is used, so that the problem of exosome yield is solved, and the exosome is good in application prospect; and the exosome secreted by the bladder cells can be loaded with different drugs or active molecules, and the drug delivery is carried out by targeting kidney tissues, so that the treatment effect of kidney diseases is improved, and the drug toxic and side effects are reduced.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a kidney targeted drug-loaded exosome and a drug for applying and treating kidney diseases.
Background
The kidney disease is a general term for common diseases seriously harming human health, and mainly comprises different types of nephritis, acute renal failure, kidney stones, renal cysts and the like. Renal tumors are one of the most common tumors in the human urogenital system, and the general therapeutic principle of renal cell carcinoma is that patients who do not have metastasis in the early stage or only locally progress are treated by surgery, patients who have no surgical chance of metastatic renal cancer in the late stage have surgery, and comprehensive treatment mainly based on internal medicine should be adopted. In the aspect of internal medicine treatment, a targeted treatment method of renal cell carcinoma is adopted, so that the overall life cycle and the like are greatly prolonged.
Currently, the FDA has approved 9 targeted therapeutic drugs for advanced renal cell carcinoma, which are: sunitinib, temsirolimus, pazopanib, axitinib, sorafenib, cabozantinib, everolimus, lenvatinib, and bevacizumab. The targeted therapy is a standard treatment scheme for the advanced renal clear cell carcinoma, and the median disease control time of a patient can reach about 30 months by alternately using a plurality of targeted drugs. The target medicine for treatment mainly acts through two signal pathways of VHL/HIF/VEGF and PI 3K/AKT/mTOR. The targeted drugs of kidney cancer are two classes, one is an anti-angiogenic tyrosine kinase inhibitor and the other is an mTOR inhibitor, and the sequential treatment is the rotation of the two classes of drugs. A large number of researches prove that the expression reduction of miR-1271 is closely related to kidney cancer, and miR-1271 can inhibit the proliferation and the metastasis of kidney cancer cells by inhibiting an mTOR signal pathway.
Although the kidney cancer targeting preparation has been developed to a certain extent in recent years, the kidney cancer targeting preparation has a therapeutic effect on a signal pathway and has strong toxic and side effects on normal cells and tissues. Poor targeting for the treatment of kidney cancer and other kidney-related diseases. Exosomes are used as drug-carrying systems, and can carry drugs and deliver the drugs to adjacent or distant cells due to good biocompatibility, biodegradability, low toxicity, stability and low immunogenicity.
Therefore, it is highly desirable to prepare a highly efficient drug-loaded system of kidney targeting exosomes for treating kidney tissue-related diseases.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a kidney targeted drug-loading exosome and a drug for applying and treating kidney diseases.
In order to achieve the purpose, the invention adopts the following technical scheme:
the kidney targeting drug-loaded exosome is obtained by introducing a drug for treating kidney diseases into a kidney tissue targeting exosome, and the kidney tissue targeting exosome is derived from bladder cells.
Of course, bladder cells can also be genetically modified or engineered to target kidney tissue, including but not limited to genetic modification, gene overexpression or deletion, molecular modification, and the like; exosomes of bladder cells may also be surface modified or engineered to target kidney tissue, including but not limited to surface protein modifications, surface protein alterations, surface small molecule modifications, and the like.
Preferably, the bladder cells include, but are not limited to, human bladder cells, animal bladder cells, human bladder cancer cells, animal bladder cancer cells, induced pluripotent stem cell-induced bladder cells.
Preferably, the animal bladder cell and animal bladder cancer cell in the animal including but not limited to monkey, mouse, rat, hamster; human bladder cells include, but are not limited to, normal human bladder cells, human bladder epithelial immortalized cells SV-HUC-1, human bladder epithelial cells HCV-29, human embryonic bladder tissue-derived cells CCC-HB-2; the human bladder cancer cells include, but are not limited to, human bladder squamous carcinoma cells SCaBER, human bladder transitional cell carcinoma cells T24, human bladder transitional cell papilloma cells RT4, human bladder transitional cell carcinoma SW780, human bladder cancer cells BIU-87 and human bladder cancer cells H/RB-CL 2.
Preferably, the drug for treating kidney disease includes, but is not limited to, siRNA, microRNA, protein, antibody, sunitinib, temsirolimus, pazopanib, axitinib, sorafenib, cabozantinib, everolimus, lenvatinib, and bevacizumab.
The step of introducing the drug for treating kidney diseases into the exosome with kidney tissue targeting is as follows:
putting a medicament for treating kidney diseases and exosome with kidney tissue targeting property into an electric rotor;
adopting exponential waves or square waves, and performing electrotransformation on a medicament for treating kidney diseases and an exosome with kidney tissue targeting property by using 50-300V voltage;
and heating the electrotransformation product in water bath, and centrifuging under centrifugal force to remove free medicine to obtain the purified kidney targeting medicine-carrying exosome.
The second purpose of the invention is to provide the application of the kidney targeting drug-loaded exosome in the preparation of the drug for treating kidney diseases.
The invention also provides a medicament for treating kidney diseases, which comprises any one of the kidney targeting medicament-loaded exosomes.
The invention has the beneficial effects that:
the embodiment of the invention provides a kidney targeting drug-loaded exosome and a drug for applying and treating kidney diseases, the kidney targeting drug-loaded exosome can be enriched in the kidney without any modification, and the exosome secreted by bladder cells is used, so that the problem of the output of exosome is solved, and the kidney targeting drug-loaded exosome has good application prospect; and the exosome secreted by the bladder cells can be loaded with different drugs or active molecules, and the drug delivery is carried out by targeting kidney tissues, so that the treatment effect of kidney 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 SV-HUC-1 human bladder epithelial immortalized cells.
FIG. 2 is a graph showing the particle size distribution of exosomes secreted by SV-HUC-1, an immortalized cell of human bladder epithelium.
FIG. 3 is a map of the secretion of exosomes secreted by SV-HUC-1, a PKH 67-labeled human bladder epithelial immortalized cell, in different tissues.
FIG. 4 is an in vitro assay of the antitumor activity of renal targeting exosomes loaded with microRNA-1271 mimic drug for the treatment of renal diseases.
FIG. 5 is an in vivo assay for the anti-tumor activity of renal targeting exosomes loaded with microRNA-1271 mimic drug for the treatment of renal disease.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. The specific embodiments described herein are merely illustrative of the embodiments of the invention and do not delimit the embodiments of the invention.
Example 1
The kidney targeting drug-loaded exosome is obtained by introducing a drug for treating kidney diseases into an exosome with kidney tissue targeting property, wherein the exosome with the kidney tissue targeting property is derived from bladder cells.
Human bladder epithelial immortalized cell SV-HUC-1 (in this example, human bladder epithelial immortalized cell SV-HUC-1) was collected. The SV-HUC-1 cell culture solution is cultured by exosome-free serum and a DMEM culture 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 for 2 h under 100000g of centrifugal force, re-suspending and collecting the precipitate by using sterile PBS, and storing the precipitate in a temperature environment of 4 ℃ for a short time to obtain a kidney targeting exosome; the obtained kidney targeting exosome surface marker protein is extracted and separated, and the particle size distribution is shown in figure 1 and figure 2; measuring the expression quantity of the exosome secreted by the SV-HUC-1, and detecting the concentration of the exosome protein secreted by the bladder cells by a BCA method to be 3.02 mu g/mu L;
example 2
The method takes the exosome secreted by the SV-HUC-1 cell, stains the exosome with PKH67, and traces the distribution of the exosome secreted by the SV-HUC-1 cell in vivo, and comprises the following specific steps:
taking 100 mu g of exosome secreted by SV-HUC-1 cells, incubating the exosome with 1 mu L of PKH67 in the dark at the temperature of 4 ℃ overnight, centrifuging for 2 h under the centrifugal force of 100000g, discarding the supernatant, washing with PBS twice, then resuspending the exosome secreted by the SV-HUC-1 cells with sterile PBS, injecting the exosome into a C57bl/6 mouse through a tail vein (in the embodiment, the selected male C57bl/6 (4-6 weeks) mouse is purchased from Beijing Huafukang biotech GmbH, and all mice are cultured in SPF-level facilities); after 24h, anesthetizing the mouse, taking the heart, the liver, the spleen, the lung, the kidney, the stomach and the intestine of the mouse to make frozen sections, staining the nucleus by Hoechst33342, and observing the biological distribution of the exosome secreted by the SV-HUC-1 cell in each organ of the mouse; the results show that: the exosome secreted by the bladder cells in this example is obviously enriched in kidney tissues (fig. 3), and the targeting efficiency can reach 65% -90%, which indicates that the kidney targeting exosome prepared in this example has kidney tissue targeting;
example 3
In the embodiment, the drug microRNA 1271 mice for treating the kidney cancer disease is selected.
In the embodiment, a medicament for treating kidney diseases is introduced into a kidney target exosome to prepare a kidney tumor targeted therapeutic medicament, and the specific operation is as follows:
step 1: 150 mu g of kidney targeting exosome is mixed with the microRNA 1271 micic, the electrotransfer buffer solution can be (PBS, DMEM, Cytomix and Tris-HCl) to complement the mixture to 150 mu L, and the mixture 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 kidney target exosomes and the microRNA 1271 mimic;
step 2: 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 the microRNA 1271 micic, 150 mu g of kidney target exosome and the microRNA 1271 micic are subjected to electrotransformation under the voltage of 250V, the efficiency is highest and can reach 24.9%. The results show that: the microRNA 1271 micic is successfully loaded into an exosome secreted by an SV-HUC-1 cell;
example 4
This example further verifies that in vitro and in vivo experiments detect the therapeutic effect of the renal cancer by loading the microRNA 1271 micic drug for treating renal diseases into renal target exosomes.
The method comprises the following specific steps:
in vitro experiments:
renal carcinoma cells ACHN were plated in 96-well plates at 5X 10 per well3Adding kidney targeting exosome (control) and tumor carrying kidney into each cellThe kidney targeting exosome of the treatment drug microRNA 1271 micic, and the MTT detects the killing effect of the kidney targeting exosome carrying the renal tumor treatment drug microRNA 1271 micic on renal cancer cells; in vitro experiments, the kidney targeting exosome loaded with the renal tumor treatment drug can effectively kill tumor cells compared with a pure kidney targeting exosome, and the results are shown in fig. 4.
In vivo experiments:
in vivo experiments, a kidney cancer model was established, and 4X 10 cells were used6A single ACHN cell was injected subcutaneously into Balb/c nude mice (4-6 weeks) purchased from Beijing Huafukang Biotech, Inc., all grown at SPF-level facility, in this example) and allowed to grow to approximately 100mm3In the time, the renal target exosome loaded with the renal tumor treatment drug microRNA 1271 micic is injected into the tail vein, the renal target exosome is injected 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(ii) a a represents a long diameter, and b represents a short diameter.
In an in vivo experiment, as shown in fig. 5, the renal targeting exosome (microRNA 1271 mim-exosome) loaded with the renal tumor treatment drug microRNA 1271 mim can significantly reduce the tumor size of a mouse compared with a free antitumor drug group (microRNA 1271 mim) with 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 (7)
1. The kidney targeting drug-loaded exosome is obtained by introducing a drug for treating kidney diseases into a kidney tissue targeting exosome, wherein the kidney tissue targeting exosome is derived from bladder cells.
2. The kidney-targeted drug-loaded exosome according to claim 1, wherein said bladder cells include, but are not limited to, human bladder cells, animal bladder cells, human bladder cancer cells, animal bladder cancer cells, induced pluripotent stem cell-induced bladder cells.
3. The kidney-targeted drug-loaded exosome according to claim 2, wherein the animal bladder cell and the animal bladder cancer cell include but are not limited to monkey, mouse, rat, hamster; human bladder cells include, but are not limited to, normal human bladder cells, human bladder epithelial immortalized cells SV-HUC-1, human bladder epithelial cells HCV-29, human embryonic bladder tissue-derived cells CCC-HB-2; the human bladder cancer cells include, but are not limited to, human bladder squamous carcinoma cells SCaBER, human bladder transitional cell carcinoma cells T24, human bladder transitional cell papilloma cells RT4, human bladder transitional cell carcinoma SW780, human bladder cancer cells BIU-87 and human bladder cancer cells H/RB-CL 2.
4. The kidney-targeted drug-loaded exosome according to claim 1, wherein the drug for treating kidney disease comprises but is not limited to siRNA, microRNA, protein, antibody, sunitinib, temsirolimus, pazopanib, axitinib, sorafenib, cabozantinib, everolimus, lenvatinib and bevacizumab.
5. The kidney-targeted drug-loaded exosome according to claim 1, wherein the step of introducing the drug for treating kidney diseases into the kidney tissue-targeted exosome comprises:
putting a medicament for treating kidney diseases and exosome with kidney tissue targeting property into an electric rotor;
using exponential wave or square wave, and applying 50-300V voltage to convert medicine for treating kidney diseases and exosome with kidney tissue targeting effect
And heating the electrotransformation product in water bath, and centrifuging under centrifugal force to remove free medicine to obtain the purified kidney targeting medicine-carrying exosome.
6. The use of a kidney-targeted drug-loaded exosome according to any one of claims 1-5 in the preparation of a medicament for treating kidney disease.
7. A medicament for the treatment of kidney disease comprising a kidney-targeted drug-loaded exosome according to any one of claims 1 to 5.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110692827.3A CN113846047A (en) | 2021-06-22 | 2021-06-22 | Kidney targeting drug-loaded exosome and application and drug for treating kidney diseases |
| CN202210709469.7A CN115029297A (en) | 2021-06-22 | 2022-06-21 | Kidney targeting drug-loading exosome, application thereof and drug for treating kidney diseases |
| PCT/CN2022/100034 WO2022268056A1 (en) | 2021-06-22 | 2022-06-21 | Tissue-targeting exosomes and application thereof |
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| CN202210709469.7A Pending CN115029297A (en) | 2021-06-22 | 2022-06-21 | Kidney targeting drug-loading exosome, application thereof and drug for treating kidney diseases |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114699398A (en) * | 2022-04-26 | 2022-07-05 | 中国人民解放军陆军特色医学中心 | Inhibitor for treating renal clear cell carcinoma |
| WO2022268056A1 (en) * | 2021-06-22 | 2022-12-29 | 姜海涛 | Tissue-targeting exosomes and application thereof |
| CN116832171A (en) * | 2023-06-05 | 2023-10-03 | 中国人民解放军总医院第八医学中心 | Exosome drug-carrying system for targeted damage of kidney tissue, construction method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015120150A1 (en) * | 2014-02-05 | 2015-08-13 | Stc.Unm | Exosomes as a therapeutic for cancer |
| US20170121685A1 (en) * | 2015-11-02 | 2017-05-04 | Tigenix S.A.U. | Mesenchymal stem cell-derived exosomes and their uses |
| CN105769916B (en) * | 2016-04-29 | 2019-07-23 | 南京大学 | Application of the source for mesenchymal stem cells excretion body in preparation treatment preeclampsia drug or preparation |
| KR102053065B1 (en) * | 2018-06-27 | 2019-12-06 | 가톨릭대학교 산학협력단 | pH sensitive anti-cancer exosome composition using hyaluronic acid and doxorubicin |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022268056A1 (en) * | 2021-06-22 | 2022-12-29 | 姜海涛 | Tissue-targeting exosomes and application thereof |
| CN114699398A (en) * | 2022-04-26 | 2022-07-05 | 中国人民解放军陆军特色医学中心 | Inhibitor for treating renal clear cell carcinoma |
| CN116832171A (en) * | 2023-06-05 | 2023-10-03 | 中国人民解放军总医院第八医学中心 | Exosome drug-carrying system for targeted damage of kidney tissue, construction method and application thereof |
| CN116832171B (en) * | 2023-06-05 | 2024-03-05 | 中国人民解放军总医院第八医学中心 | Exosome drug-carrying system for targeted damage of kidney tissue, construction method and application thereof |
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