CN115737591A - Platelet carrier, method for entrapping bortezomib in platelets and application - Google Patents
Platelet carrier, method for entrapping bortezomib in platelets and application Download PDFInfo
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- CN115737591A CN115737591A CN202211485052.3A CN202211485052A CN115737591A CN 115737591 A CN115737591 A CN 115737591A CN 202211485052 A CN202211485052 A CN 202211485052A CN 115737591 A CN115737591 A CN 115737591A
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Images
Abstract
The invention discloses a platelet carrier, a method for loading bortezomib in the platelet and application thereof; the method relates to the technical field of medicines, and the method comprises the steps of co-incubating platelets and a bortezomib solution to enable the platelets to wrap a large number of bortezomib drug molecules, reacting the platelets with tumor cells in vitro by utilizing the natural targeting effect of the platelets on the tumor cells, and enhancing the effect of killing the tumors.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a platelet carrier, a method for loading bortezomib in the platelet and application of the platelet carrier and the bortezomib.
Background
Multiple myeloma enables a group of malignant plasma cell clonal tumors which occur in B lymphocytes to account for the second place of the malignant tumors in the blood system, and has high malignancy and high invasiveness. In recent years, treatment of multiple myeloma is rapidly developed, and the addition of proteasome inhibitors, immunosuppressants, autologous stem cell transplantation and other means improves the complete remission rate of MM patients, but 80-90% of the MM patients still relapse and generate severe drug resistance, which affects the survival quality of the patients. By combining the recommendation of multiple myeloma diagnosis and treatment guidelines, the selectivity of domestic medicines and the economic bearing capacity of patients, the current treatment on MM patients is mostly the traditional chemotherapy means, while the affinity of chemotherapy medicines to tumor tissues is poor, the targeting is low, and the conventional treatment dosage generates obvious toxic and side effects on other normal tissues and organs. Therefore, the improvement of the targeting of the chemotherapeutic drug to MM, the increase of the accumulation of the chemotherapeutic drug in the targeted part and the achievement of the effects of high efficiency and low toxicity are the key for improving the treatment effect of MM and reducing the drug resistance.
Disclosure of Invention
In a first aspect, the object is: the invention aims to provide a platelet carrier, which is characterized in that a large amount of bortezomib drug molecules are wrapped by platelets through co-incubation of the platelets and a bortezomib solution, and the platelets react with tumor cells in vitro by utilizing the natural targeting effect of the platelets on the tumor cells to enhance the effect of killing the tumors.
A platelet carrier comprising: an inner core comprising a drug delivery matrix, wherein the drug delivery matrix comprises bortezomib; and a shell platelet membrane, the shell platelet membrane coating the inner core.
In some disclosures, the surface antigen of the outer shell platelet membrane comprises a self-recognition immunomodulatory protein selected from the group consisting of CD41 and CD 61.
In a second aspect, the object is: the invention aims to provide the application of the platelet carrier in the first aspect in preparing a medicament for treating multiple myeloma of a subject, wherein the platelets and a bortezomib solution are incubated together, so that a large number of bortezomib medicament molecules are wrapped by the platelets, and the natural targeting effect of the platelets on tumor cells is utilized to react with the tumor cells unexpectedly, so that the tumor killing effect is enhanced.
In a third aspect, the object is: the invention aims to provide a detection system of a chemotherapeutic drug, which is used for detecting a platelet carrier of the first aspect, wherein the platelet carrier is used for acting on multiple myeloma, and the platelet and a bortezomib solution are incubated together to enable the platelet to wrap a large amount of bortezomib drug molecules, so that the platelet can react with tumor cells unexpectedly by utilizing the natural targeting effect of the platelet on the tumor cells to enhance the effect of killing the tumor.
In a fourth aspect, the object is: the invention aims to provide a multiple myeloma detection device, which comprises: a processor and a memory; the memory is used for storing a computer program; the processor is connected with the memory and is used for executing the computer program stored in the memory to make the detection device execute the detection unit of the detection system of the chemotherapy medicament of the third aspect
In a fifth aspect, the object is: the invention aims to provide a tumor chemotherapy medicament, which comprises the platelet carrier of the first aspect and is used in a medicament for treating multiple myeloma of a subject.
In a sixth aspect, the object is: the invention aims to provide a method for entrapping bortezomib in platelets.
A method for entrapping bortezomib in platelets comprises dissolving bortezomib in dimethyl sulfoxide (DMSO) solution, and dissolving completely; incubating the platelet suspension and bortezomib to obtain a bortezomib mother solution; co-incubating the platelet suspension with bortezomib stock solution; the drug carrier was washed with PBS to remove unencapsulated free drug.
Further, the ratio of bortezomib to platelet volume is < 1.
Further, the incubation conditions of the platelet suspension and bortezomib are as follows: the temperature is 26 ℃, and the duration is 1h.
Further, the preparation method of the platelet comprises the following steps:
transferring the platelet-rich plasma to a 2mL centrifuge tube, centrifuging at 2500rpm for 5min, and removing the upper layer platelet-poor plasma, blood coagulation factors and plasma proteins;
adding PBS into the precipitate to wash twice; adding PBS buffer solution to gently resuspend the platelet sediment to obtain washed platelet suspension;
control platelet count to 10 8 one/mL.
Has the advantages that: the invention prepares the platelet suspension without damaging the function of the surface protein of the platelet, and then incubates the platelet with the bortezomib solution, so that the platelet wraps a large amount of bortezomib drug molecules, and the natural targeting effect of the platelet on tumor cells is utilized to react with the tumor cells unexpectedly and enhance the effect of killing the tumor.
Drawings
FIG. 1 is a platelet morphology map directly centrifuged from platelet rich plasma;
FIG. 2 is a platelet morphology map after bortezomib loading;
fig. 3 is a flow cytogram of the surface antigens CD41 and CD61 before and after platelet loading, wherein the left side is a simple platelet as a control, and the right side is a flow chart of the surface antigen after platelet loading with bortezomib.
Detailed Description
The technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the drawings in the embodiments of the present disclosure, and it is obvious that the described embodiments are only a part of the embodiments of the present disclosure, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without making any creative effort, shall fall within the protection scope of the present disclosure.
Example 1: preparation of washed platelet suspensions
In order to maintain the structural shape of platelets during the in vitro treatment process, avoid activation deformation, and achieve high platelet yield and high drug loading rate in the drug loading process, platelet activation needs to be strictly controlled, and fig. 1 is a platelet morphology chart obtained by directly centrifuging platelet-rich plasma. The preparation process comprises the following steps:
1. the platelet-rich plasma was transferred to a 2mL centrifuge tube, centrifuged at 2500rpm for 5min, and the upper layer of platelet-poor plasma was discarded along with coagulation factors and plasma proteins.
2. The pellet was washed twice with Phosphate Buffered Saline (PBS). Adding PBS buffer solution to gently resuspend the platelet sediment to obtain a washed platelet suspension.
3. Platelet count was controlled to 10 using a platelet counter 8 Per mL;
a platelet suspension is obtained.
Example 2: preparation of PLT-BTZ, FIG. 2 is a platelet morphology diagram after carrying bortezomib, the platelet morphology is complete, and organelles exist and are consistent with platelets without carrying drugs;
the preparation process comprises the following steps:
1. preparing BTZ mother liquor: weighing a certain weight of BTZ and dissolving the BTZ in a dimethyl sulfoxide (DMSO) solution;
2. adding a certain amount of BTZ mother liquor into a proper amount of platelet suspension by using a pipette, and gently blowing, beating and uniformly mixing; bortezomib to platelet volume ratio <1
3. The mixed solution is subjected to shading reaction for 1 hour at room temperature;
4. centrifuging the reacted mixed solution at the room temperature of 2500rpm for 6min, removing unbound free drug molecules, adding phosphate buffer solution for resuspension, centrifuging and washing for 3 times to obtain PLT-BTZ suspension, and standing and storing in a shading mode at the room temperature.
Example 3: characterization of PLT-BTZ
The preparation process comprises the following steps:
1. surface protein characterization before and after flow cytometry detection of platelet drug loading;
2. and (5) performing electron microscope characterization before and after platelet drug loading.
The prepared related characteristics, the electron micrographs of the related structural characteristics are shown in fig. 1 and fig. 2, and the flow analysis chart is shown in fig. 3:
as can be seen from transmission electron micrographs (figures 1 and 2) of the blood platelets carrying bortezomib, the cell structure of the blood platelets is complete, and organelles exist and are highly consistent with the blood platelets which are not carried with drugs.
From their flow charts (fig. 3 is a flow chart of the surface antigens CD41 and CD61 before and after platelet loading, wherein the left side is a simple platelet as a control, and the right side is a flow chart of the surface antigen after the platelet loading with bortezomib), it can be seen that the platelet surface mainly associated antigens CD41 and CD61 after the loading with bortezomib maintain high levels.
Example 4: (5) After the blood platelet wraps the bortezomib, the peripheral neurotoxicity and systemic toxic and side effects of the bortezomib are greatly reduced
The invention directly wraps the anti-Tumor drug by the platelet, has no intermediate action of other compound particles, the platelet is used as a carrier of the anti-Tumor drug, and the passive targeting of the Tumor cell is realized by utilizing the platelet aggregation (TCIPA) induced by the Tumor cell, and the surface protein of the platelet is used as the target of the Tumor cell.
The platelet serving as a carrier for loading chemotherapeutic drugs has a plurality of advantages in the aspect of tumor treatment, and the specific expression is that (1) the platelet has high drug loading, and generally 10 encapsulation per unit can be achieved 5 The magnitude order of magnitude of drug molecules, and the synthetic nano-materials with the same volume only contain 500; (2) Platelets have a blood circulation time of about 7 to 10 days and a long accumulation time in the body; (3) The blood platelet belongs to the self-component of the organism, can avoid being eliminated by immune cells, and has good biocompatibility; (4) Platelets which are not utilized or complete the metabolic cycle can be clear through normal metabolic pathways such as liver, kidney, skin, respiration and the like, so that the toxic and side effects on the body are small; (5) The platelet membrane phospholipid bilayer can realize simultaneous encapsulation of various medicines, so that the anti-tumor effect is greatly enhanced; and (6) the platelet has natural targeting effect on tumor cells.
Therefore, the bionic drug targeting carrier prepared by encapsulating bortezomib based on the platelet as the carrier has high drug loading capacity and high targeting property, the anti-tumor effect is enhanced by targeting and combining tumor cells, the preparation process is simple, and the technology can be developed into practical clinical application in the platelet drug loading technology.
In the description herein, references to the description of "one embodiment," "an example," "a specific example," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the disclosure. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing illustrates and describes the general principles, principal features, and advantages of the present disclosure. It will be understood by those skilled in the art that the present disclosure is not limited to the embodiments described above, which are presented solely for purposes of illustrating the principles of the disclosure, and that various changes and modifications may be made to the disclosure without departing from the spirit and scope of the disclosure, which is intended to be covered by the claims.
Claims (10)
1. A platelet carrier, comprising: an inner core comprising a drug delivery matrix, wherein the drug delivery matrix comprises bortezomib; and a shell platelet membrane, the shell platelet membrane coating the inner core.
2. The platelet carrier of claim 1, wherein the surface antigen of the outer shell platelet membrane comprises a self-recognizing immunomodulatory protein selected from the group consisting of CD41 and CD 61.
3. Use of a platelet carrier according to any one of claims 1 to 2 in the manufacture of a medicament for treating multiple myeloma in a subject.
4. A detection system for a chemotherapeutic drug, comprising a detection unit for detecting a platelet carrier according to any one of claims 1 to 2 for effecting multiple myeloma.
5. A multiple myeloma detection device characterized by comprising: a processor and a memory; the memory is used for storing a computer program; the processor is coupled to the memory and configured to execute the computer program stored in the memory to cause the detection device to execute the detection unit of the detection system for chemotherapeutic drugs according to claim 4.
6. A tumor chemotherapeutic agent comprising the platelet carrier according to any of claims 1-2.
7. A method for entrapping bortezomib in platelets is characterized in that bortezomib is dissolved in dimethyl sulfoxide (DMSO) solution and fully dissolved to obtain bortezomib mother liquor; co-incubating the platelet suspension with bortezomib stock solution; the drug carrier was washed with PBS to remove unencapsulated free drug.
8. The method of claim 7, wherein the ratio of bortezomib to platelet volume is < 1.
9. The method of claim 7, wherein the platelet suspension is incubated with the bortezomib stock solution under conditions selected from the group consisting of: the temperature is 26 ℃, and the duration is 1h.
10. The method of claim 7, wherein the platelets are prepared by a method comprising:
transferring the platelet-rich plasma to a 2mL centrifuge tube, centrifuging at 2500rpm for 5min, and discarding the upper layer of platelet-poor plasma, blood coagulation factors and plasma proteins;
adding PBS to the precipitate for washing twice; adding PBS buffer solution to gently resuspend the platelet sediment to obtain washed platelet suspension;
control platelet count to 10 8 one/mL.
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| CN115227667A (en) * | 2022-05-23 | 2022-10-25 | 苏州大学 | Preparation method of bortezomib-loaded human monocyte exosome and application of bortezomib-loaded human monocyte exosome in preparation of multiple myeloma treatment drugs |
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2022
- 2022-11-24 CN CN202211485052.3A patent/CN115737591A/en active Pending
Patent Citations (8)
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| CN104771762A (en) * | 2015-03-23 | 2015-07-15 | 南京大学医学院附属鼓楼医院 | Preparation method of antitumor drug carrying platelet drug loading system |
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