CN112402607B - Method for selectively activating platelets - Google Patents
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- CN112402607B CN112402607B CN202011336304.7A CN202011336304A CN112402607B CN 112402607 B CN112402607 B CN 112402607B CN 202011336304 A CN202011336304 A CN 202011336304A CN 112402607 B CN112402607 B CN 112402607B
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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Abstract
The invention discloses a method for mediating the space-time selective activation of platelets loaded with photosensitizers, which relates to the technical field of medicinal chemistry and comprises the following steps of: s1, loading a photosensitizer by using platelets in vitro; and S2, laser irradiation is given in a specified space-time range, and the photosensitizer is induced to generate photodynamic action in the interior of the blood platelet, so that the blood platelet is selectively activated. The invention has simple flow, can activate the platelets in time-space selectivity according to the requirement, and can provide a basis for targeting tumor treatment by loading the photosensitizers on the platelets.
Description
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to a method for selectively activating platelets.
Background
At present, in vivo and in vitro researches, platelets are activated by adopting a direct treatment mode of a polymerization agent medicament, for example, the platelets are treated by thromboxane A2 (TXA 2), collagen, thrombin, adenosine Diphosphate (ADP) and the like with a certain concentration, and are induced to be activated, so that the coagulation reaction is further promoted, and thrombus can be induced to form in vivo. However, the greatest disadvantage of these methods is that: platelet activation occurs immediately after in vitro treatment with a multimerizing agent, lacking in time selectivity; platelets at various parts of the whole body are activated after in vivo administration of the multimerizing agent, and lack spatial selectivity (tissue selectivity).
The effect of photodynamic action on thromboxane release from rat platelets (Zhou Chuannong, 1996, 4 th edition of journal of Chinese laser medicine, 1993) is studied in quantitative ultrastructural study of early photodynamic injury of mouse platelets (Zhou Chuannong, 1992, 4 nd edition of journal of Chinese laser medicine, 1993) to activate platelets by intravascular administration of photosensitizers, but the effect is insufficient to load platelets with enough drugs, and has side effects on other cells of the body, and the effect of space-time controllability cannot be achieved. These are all detrimental to in vitro studies and in vivo selective treatment of various diseases with platelets.
Disclosure of Invention
In view of the drawbacks of the prior art, an object of the present invention is to provide a method for selectively activating platelets by loading a photosensitizer on platelets in vitro, and then selectively activating platelets by laser irradiation with a specific wavelength at a specific time or specific site. The main idea of the invention is to separate out the platelet, then load the photosensitizer into the platelet, and activate the platelet by utilizing the photodynamic effect generated by illumination when the platelet needs to be activated no matter in vitro or when the drug-loaded platelet is returned to the body. The invention discovers that the photosensitizer can activate the platelets well under the mediated photodynamic action after being loaded on the platelets, thereby achieving the purpose of time-space selective activation of the platelets.
In order to achieve the above purpose, the invention adopts the following technical scheme: a method of selectively activating platelets comprising the steps of: s1, loading a photosensitizer by using platelets in vitro; and S2, laser irradiation is given in a specified space-time range, and the photosensitizer is induced to generate photodynamic action in the interior of the blood platelet, so that the blood platelet is selectively activated.
Based on the technical scheme, the photosensitizer is a first-generation photosensitizer, a second-generation photosensitizer or various photosensitizers loaded by nano particles.
Based on the technical scheme, the first generation photosensitizer comprises hematoporphyrin derivatives and hematoporphyrin ether; the second generation photosensitizer comprises chlorins, phthalocyanine, dekka porphyrin, methylene blue and phenylporphyrin derivatives.
On the basis of the technical scheme, the platelets are platelets extracted from peripheral blood of mammals.
On the basis of the technical proposal, the blood platelets in the blood are separated, and the blood platelets are used after shaking and stabilizing for 1 hour; the photosensitizer is used to incubate with the platelets for at least 4 hours under the condition of continuous shaking at 25 ℃ and the incubation concentration of the photosensitizer is in the range of 0.5-10 mug/ml.
On the basis of the technical proposal, the platelets loaded with the photosensitizer are irradiated by laser in vitro, and the time is controlled within the range of 30 seconds to 1 minute.
Based on the technical proposal, platelets loaded with photosensitizer are injected into blood vessels, and laser irradiation is given to specific tissue organ parts after 2-24 hours.
On the basis of the technical scheme, the specific tissue organ part is a tumor metastasis focus or a tumor wound after surgical excision.
On the basis of the technical scheme, the wavelength range of the laser is the optimal wavelength of the corresponding photosensitizer.
The invention has the beneficial effects that:
the invention can load a large amount of photosensitizer on the platelets, selectively activate the platelets in specific tissues and organs (space) when needed (time), provides a new strategy for timing activation of the platelets in vitro and targeted activation of the platelets of specific tissues in vivo, and also provides a foundation for selectively activating the platelets in tumor tissues to form thrombus for treating tumors.
Drawings
Fig. 1 is a schematic flow chart of the technical scheme of the invention.
FIG. 2 shows the result of detecting that platelets loaded with a photosensitizer are enriched in a large amount of drug by flow cytometry and activated under specific laser irradiation in the present invention.
Detailed Description
The technical scheme and the beneficial effects of the invention are more clear and definite by further describing the specific embodiments of the invention with reference to the drawings in the specification. The embodiments described below are exemplary by referring to the drawings for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Referring to FIG. 1, the present invention provides a method for selectively activating platelets, comprising the steps of:
s1, loading a photosensitizer by using platelets in vitro; specifically, the platelets are platelets extracted from peripheral blood of mammals, including, but not limited to, mice, rats, rabbits, and human platelets. The photosensitizer is a first generation photosensitizer, a second generation photosensitizer or various photosensitizers loaded by nano particles. Further, the first generation photosensitizers include hematoporphyrin derivatives and hematoporphyrin ethers; the second generation photosensitizer comprises chlorins, phthalocyanines, texaphyrins, methylene blue and phenylporphyrin derivatives.
And S2, laser irradiation is given in a specified space-time range, and the photosensitizer is induced to generate photodynamic action in the interior of the blood platelet, so that the blood platelet is selectively activated. Specifically, the wavelength range of the laser is the optimum wavelength corresponding to the photosensitizer.
In the step S1, blood platelets in blood are separated, and the blood platelets are used after shaking and stabilization for 1 hour; the photosensitizer is used to incubate with the platelets for at least 4 hours under the condition of continuous shaking at 25 ℃ and the incubation concentration of the photosensitizer is in the range of 0.5-10 mug/ml.
In step S2, the platelets loaded with the photosensitizer may be irradiated with laser light in vitro for a time period within the interval of 30 seconds to 1 minute.
In step S2, platelets loaded with a photosensitizer may be injected into a blood vessel, and laser irradiation may be applied to a specific tissue organ site after 2 to 24 hours. In particular, the specific tissue organ site is a tumor metastasis or a tumor wound after surgical excision.
The invention is further illustrated by the following two examples.
Example 1
A method of selectively activating platelets comprising the steps of:
A. reacting 0.1g of ferric triacetylacetone with 10ml of glycidol at 140 ℃ for 24 hours, and purifying the product to obtain nano ferric oxide-polyglycerol; measuring and steaming 0.1ml of purified nano ferric oxide-polyglycerol to obtain powder, adding 5mg of chlorin e6 to dissolve in aqueous solution with a pH value of 7.4, reacting for 24 hours at 37 ℃, and purifying the product to obtain the nano ferric oxide-polyglycerol-chlorin e6 compound.
B. 2ml of peripheral blood of the mice is extracted, 180g is centrifuged for 15 minutes to obtain platelet-rich plasma, then 100g is centrifuged for 10 minutes, the supernatant is taken, 800g of the supernatant is centrifuged for 15 minutes to obtain platelet sediment, the platelets are resuspended by using a culture medium, and the platelet sediment is used after shaking and stabilizing at room temperature for 1 hour.
C. The nano ferric oxide-polyglycerol-chlorin e6 and the extracted mouse blood platelets are incubated for 6 hours under the condition of continuous shaking at 25 ℃, and the concentration of doxorubicin in the drug during incubation is 2 mug/ml.
D. After 6 hours, platelets were successfully loaded with nano iron oxide-polyglycerol-chlorin e6, and irradiated with a 690nm laser at 0.5w power for 30 seconds at a spot diameter of 2cm and a distance of 1cm from the platelets, if necessary.
E. After 1 hour of irradiation, platelet activation was detected using a flow cytometer.
Example 2
A method of selectively activating platelets comprising the steps of:
A. chlorin e6 was dissolved in PBS solution at PH 7.4.
B. 4ml of peripheral blood of the rat is extracted, platelet sediment is obtained by a gradient centrifugation method, the platelets are resuspended by using a culture medium, and the platelets are used after shaking and stabilizing for 1 hour at room temperature.
C. Chlorin e6 was added to the extracted rat platelets and incubated for 4 hours with constant shaking at 25 ℃ and the concentration of doxorubicin in the incubated drug was 1 μg/ml.
D. After 4 hours, platelets were successfully loaded with chlorin e6.
E. Platelets loaded with chlorin e6 are injected into tumor-bearing mice through tail veins, and tumors are surgically excised.
F. After 2 hours, the tumor excision wound part is irradiated with a 690nm laser with the spot diameter of 2cm and the distance from 1cm to the tumor tissue for 60 seconds under the power of 0.5w, so that the medicine carrying platelets at the incision part can be activated.
As shown in FIG. 2, since chlorin e6 carries near infrared fluorescence, it can be detected in the APC channel of the flow cytometer, and thus the uptake of the photosensitizer chlorin e6 by platelets (increased APC fluorescence value) is detected by the flow cytometer. CD62p expression was significantly increased on the surface of platelet membranes given chlorin e6 but not laser 690nm for 30 seconds compared to platelets of the control group (without any treatment) and the group given chlorin e6 but not laser 690 nm. CD62p, also known as p-selectin, is a characteristic marker of platelet activation, and the CD62p antibody used carries FITC fluorescence and is therefore detectable in the FITC channel when detected using a flow cytometer. In the left panel, the ordinate is the average value of the in-platelet APC fluorescence, reflecting the loading of chlorin e6 in the platelets; in the right panel, the ordinate is the average value of FITC fluorescence on the platelet membrane surface, reflecting the expression of CD62p on the platelet membrane surface.
In the description of the present invention, a description of the terms "one embodiment," "preferred," "example," "specific example," or "some examples," etc., means 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 invention, and a schematic representation of the terms described above in the present specification does 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 invention is not limited to the embodiments described above, but a number of modifications and adaptations can be made by a person skilled in the art without departing from the principle of the invention, which modifications and adaptations are also considered to be within the scope of the invention. What is not described in detail in this specification is prior art known to those skilled in the art.
Claims (5)
1. An application of platelets loaded with a photosensitizer in preparing a medicine for photodynamic therapy of tumors, which is characterized by comprising the following steps:
s1, loading a photosensitizer on platelets extracted from peripheral blood of a mammal in vitro; separating platelets in blood, and using after shaking and stabilizing for 1 hour; incubating the mixture with the platelet for at least 4 hours under the condition of continuous oscillation at 25 ℃ by using a photosensitizer, wherein the incubation concentration of the photosensitizer is in the range of 0.5-10 mug/mL;
the photosensitizer is chlorin e6;
and S2, irradiating laser to induce the photosensitizer to generate photodynamic action in the blood platelet, so as to selectively activate the blood platelet.
2. Use of a photosensitizer-loaded platelet according to claim 1 for the preparation of a photodynamic therapy tumor drug, characterized in that: in step S2, platelets loaded with the photosensitizer are irradiated with laser light outside the body for a time within the interval of 30 seconds to 1 minute.
3. Use of a photosensitizer-loaded platelet according to claim 1 for the preparation of a photodynamic therapy tumor drug, characterized in that: in step S2, platelets loaded with the photosensitizer are driven into blood vessels, and laser irradiation is performed on specific tissue organ sites after 2-24 hours.
4. Use of a photosensitizer-loaded platelet according to claim 3 for the preparation of a photodynamic therapy tumor drug, characterized in that: the specific tissue organ part is a tumor metastasis focus or a tumor wound after surgical excision.
5. Use of a photosensitizer-loaded platelet according to claim 1 for the preparation of a photodynamic therapy tumor drug, characterized in that: the wavelength range of the laser is the optimal wavelength corresponding to the photosensitizer.
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| CN202011336304.7A CN112402607B (en) | 2020-11-25 | 2020-11-25 | Method for selectively activating platelets |
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