CN112138157B - Optically controlled granulocyte biological agent and preparation and application thereof - Google Patents

Optically controlled granulocyte biological agent and preparation and application thereof Download PDF

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CN112138157B
CN112138157B CN202011046199.3A CN202011046199A CN112138157B CN 112138157 B CN112138157 B CN 112138157B CN 202011046199 A CN202011046199 A CN 202011046199A CN 112138157 B CN112138157 B CN 112138157B
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李艳
董海青
李永勇
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Tongji University
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Abstract

The invention relates to an optically controlled granulocyte biological agent, and a preparation method and application thereof. The optically controlled granulocyte biological agent comprises granulocytes and a photosensitizer loaded in the granulocytes. Compared with the prior art, the invention constructs the light-controlled granulocyte (NE) capable of generating ROS under the excitation of light by loading photosensitizer in the granulocyte by an engineering meansP) The optical control granulocyte biological agent can be used for clinical tumor treatment, namely, the generation of a photosensitizer ROS in the optical control granulocyte is regulated and controlled through remote control of light, the local concentration of the ROS is improved, and the ROS killing and enhancing effect of the optical control granulocyte on tumors is realized.

Description

Optically controlled granulocyte biological agent and preparation and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an optically-controlled granulocyte biological agent as well as preparation and application thereof.
Background
Granulocyte-transfusion anti-cancer therapies have demonstrated potential in the clinic. Easy separation, large availability (accounting for 50-70% of white blood cells in blood), and the safety of allogenic blood transfusion, etc. lay the foundation for the wide clinical application. The granulocyte is used for the back transfusion of anticancer therapy, namely, high anticancer activity granulocytes in young and healthy people are collected and transfused into cancer patients, and the tumor is killed by using the mechanisms of Reactive Oxygen Species (ROS), degranulation and the like of the granulocytes. The therapy has shown anticancer efficacy in clinical studies, and a fraction of patients with one reinfusion can prolong survival from 6 months to 6 years. However, > 90% donor NE has insufficient tumor killing activity, greatly affecting its wide clinical application. Therefore, it is very urgent to develop a method for enhancing the killing activity of granulocytes to improve the immunotherapeutic effect thereof.
ROS, degranulation, etc. are the main killing means of granulocytes. The generation of a large amount of reactive oxygen species induces apoptosis or death of cells by damage to nucleic acids, proteins, biological membranes, and the like. The varying killing of granulocytes from different sources is associated with an insufficient efficiency of ROS production.
Therefore, how to improve the ROS killing capability of the granulocyte on the tumor is a technical problem to be solved at present.
Disclosure of Invention
The purpose of the invention is as follows: provides an optically controlled granulocyte biological agent and preparation and application thereof, and improves the ROS killing activity of the granulocyte on tumors through the construction of the engineered optically controlled granulocyte.
The purpose of the invention can be realized by the following technical scheme:
the present invention provides an optically controlled granulocyte biological agent, also known as light-controlled granulocyte (NE)P) The optically controlled granulocyte biological agent comprises granulocytes and a photosensitizer loaded in the granulocytes.
In one embodiment of the present invention, the photosensitizer is a nanometerized photosensitizer.
The photosensitizer is also called sensitizer, sensitizer and photocrosslinking agent. In photochemical reactions, it is a substance that transfers light energy to some reactant that is not sensitive to visible light to increase or enlarge its light sensitivity. The photosensitizer or its metabolite is a chemical substance selectively concentrated in the cells to be acted upon, and can produce photodynamic effect to destroy the target cells under the excitation of light with proper wavelength.
In one embodiment of the invention, the nanometerized photosensitizer is a serum protein modified photosensitizer.
In one embodiment of the present invention, the serum protein is selected from one or more of bovine serum albumin and human serum albumin.
In one embodiment of the invention, the photosensitizer is selected from one or more of chlorin e6, verteporfin, protoporphyrin ix, meso-tetra (4-carboxyphenyl) porphin, meso- (4-sulfophenyl) porphin tetrasodium salt, tetrasulfophenyl iron porphyrin, nickel phthalocyanine tetrasodium salt, or pyropheophorbide a.
Preferably, the photosensitizer is selected from one or more of chlorin e6, protoporphyrin IX or pyropheophorbide a.
More preferably, the photosensitizer is selected from chlorin e 6.
Chlorin e6(Chlorine6, Ce6) has a property of absorption peak at a wavelength of about 660nm, and has strong absorption in the near infrared region, so that it is suitable for developing photodynamic therapy for tumors. Ce6 is one of chlorophyll a degradation product derivatives, and its absorption spectrum is 600-800 nm, and maximum absorption wavelength is 660nm, so that the excitation light with longer wavelength can reach the tumor tissue with deeper position to activate the photosensitizer, and the therapeutic effect is generated by observing the therapeutic effect of cancer cells, which shows that the greater the maximum absorption wavelength of the photosensitizer, the deeper the therapeutic depth is, and the better the therapeutic effect is. Ce6 also has the characteristics of high singlet oxygen yield, high clearance speed in vivo, short skin retention time and the like.
The particle size of the nano photosensitizer can be regulated and controlled, and the particle size of the nano photosensitizer is distributed between 20nm and 500 nm.
In the present invention, the average particle diameter of the photosensitizers to be used for the nanocrystallization is 20 to 500nm, and may be, for example, 20nm, 30nm, 45nm, 50nm, 65nm, 70nm, 85nm, 90nm, 110nm, 120nm, 150nm, 200nm, 220nm, 250nm, 280nm, 310nm, 350nm, 380nm, 400nm, 420nm, 450nm, 480nm, or 500 nm;
preferably, the average particle size of the nano photosensitizer is 20-200 nm;
more preferably, the average particle size of the nanosized photosensitizer is 20 to 100 nm.
In one embodiment of the present invention, when the nanometerized photosensitizer is a serum protein modified photosensitizer, the serum protein modified photosensitizer is prepared by the following method:
1) adding a photosensitizer, condensing agents EDC and NHS into a reactor, reacting at room temperature, and pre-activating carboxyl of the photosensitizer;
2) then adding serum protein (BSA) into the reaction system, continuing the reaction at room temperature, and after the reaction is finished, dialyzing and purifying.
In one embodiment of the present invention, when the nanometerized photosensitizer is a serum protein modified photosensitizer, the serum protein modified photosensitizer is prepared by the following method:
1) adding a photosensitizer, condensing agents EDC and NHS into a reactor, reacting for 3 hours at room temperature, and pre-activating carboxyl of the photosensitizer;
2) then serum protein is added into the reaction system, the reaction is continued for 3 to 24 hours at room temperature, and after the reaction is finished, dialysis purification is carried out.
In one embodiment of the present invention, the molar ratio of the photosensitizer to EDC and NHS in the method for preparing the serum protein modified photosensitizer is 1: (1.1-3): (1-5);
preferably, the molar ratio of the photosensitizer to EDC and NHS in the preparation method of the serum protein modified photosensitizer is 1: (1.2-2): (1.5-3);
more preferably, the molar ratio of the photosensitizer to EDC and NHS in the preparation method of the serum protein modified photosensitizer is 1: 1.5: 2.
in one embodiment of the invention, the molar ratio of the serum protein to the photosensitizer in the serum protein modified photosensitizer is 1 (1-10); preferably, the molar ratio of the serum protein to the photosensitizer is 1: 7.
EDC is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, a water-soluble carbodiimide, and is used as a carboxyl-activating reagent in amide synthesis, as well as for activating phosphate groups, crosslinking proteins and nucleic acids, and for preparing immunoconjugates. The pH range of the coupling agent is 4.0-6.0 when the coupling agent is used, and the coupling agent is often used together with N-hydroxysuccinimide (NHS) or N-hydroxythiosuccinimide to improve the coupling efficiency.
NHS is N-hydroxy succinimide.
The invention also provides a preparation method of the optical control granulocyte biological agent, and the optical control granulocyte biological agent is obtained by constructing the granulocyte and the photosensitizer.
In one embodiment of the invention, the granulocytes are associated withThe construction steps of the photosensitizer are as follows: incubating granulocyte with photosensitizer (preferably nano photosensitizer, more preferably serum protein modified photosensitizer, and more preferably bovine serum protein modified photosensitizer) to construct NEP；
The incubation concentration is 0.01-1mg/mL, wherein the incubation concentration refers to the concentration of a photosensitizer, and the volume ratio of the dosage of the granulocyte to the photosensitizer is 1:1 in the incubation process;
the incubation temperature is 20-40 ℃;
the incubation time is 10-60 min.
In one embodiment of the present invention, the step of constructing the granulocyte and the photosensitizer is: incubating granulocyte with photosensitizer (preferably nano photosensitizer, more preferably serum protein modified photosensitizer, and more preferably bovine serum protein modified photosensitizer) to construct NEP；
The incubation concentration is 0.1-0.8 mg/mL;
the incubation temperature is 22-39 ℃;
the incubation time is 10-30 min.
More preferably, the steps of constructing the granulocytes and photosensitizer are: incubating granulocyte with photosensitizer (preferably nano photosensitizer, more preferably serum protein modified photosensitizer, and more preferably bovine serum protein modified photosensitizer) to construct NEP；
The incubation concentration is 0.2-0.5 mg/mL;
the incubation temperature is 25-39 ℃;
the incubation time is 10-20 min.
The invention also provides the application of the optical control granulocyte biological agent in preparing a medicament for treating cancer. The optically controlled granulocyte biological agent has an effect of treating cancer.
In one embodiment of the invention, the cancer includes at least skin cancer, breast cancer.
The invention also provides a therapeutic use method of the optical control granulocyte biological agent, which specifically comprises the following steps: the optically controlled granulocyte biological agent is returned to the body of a cancer patient, and the enrichment of the optically controlled granulocyte biological agent at a tumor part is realized by utilizing the inherent chemotactic migration back-transfusion capability of the granulocyte to tumor tissues. When the concentration of the optically-controlled granulocyte biological agent at the tumor part is the maximum, the tumor part is irradiated by light, the generation of a photosensitizer ROS in the optically-controlled granulocyte biological agent is regulated and controlled through remote control of light (namely light-ROS light-controlled activation), the local concentration of ROS is improved, and the ROS killing and enhancing effect of the optically-controlled granulocyte on the tumor is realized.
In one embodiment of the present invention, the excitation light source used to illuminate the tumor site is a 650nm laser;
the illumination time is 4-30 min;
the illumination power density is 10-100mW/cm2；
Preferably, the excitation light source used is a 650nm laser;
the illumination time is 4-16 min;
the illumination power density is 10-100mW/cm2。
More preferably, the excitation light source used is a 650nm laser; the illumination time is 8-16 minutes; the illumination power density is 20-65mW/cm2。
Based on the principle that photosensitizers can efficiently generate ROS in vivo. The invention obtains the optical control granulocyte biological agent, which can be called as optical control granulocyte (NE), by loading photosensitizer in the granulocyte through engineering meansP) The optically controlled granulocyte biological preparation can generate ROS under the excitation of light, and can be used for preparing clinical tumor treatment biological preparations.
The invention loads photosensitizer in the granulocyte by means of engineering, namely light-controlled granulocyte (NE) capable of generating ROS under the action of light excitationP) The optical control granulocyte biological agent can be used for clinical tumor treatment, namely, the generation of a photosensitizer ROS in the optical control granulocyte is regulated and controlled through remote control of light, the local concentration of the ROS is improved, and the ROS killing and enhancing effect of the optical control granulocyte on tumors is realized.
Compared with the prior art, the protein BSA has the advantages that the protein BSA which has good biocompatibility and is easy to modify and is easy to phagocytize particulate matters is utilized to carry out nanocrystallization on the photosensitizer, so that the solubility of the photosensitizer in an aqueous solution and the uptake of the photosensitizer in cells are improved; thereby improving the ROS killing activity of the granulocyte on the tumor.
Drawings
FIG. 1 biocompatible construction of light-controlled granulocytes;
FIG. 2 photo-ROS response of light-controlled granulocytes;
FIG. 3 Security assessment;
FIG. 4 enhanced killing efficacy of light-controlled granulocytes against tumor cells;
FIG. 5 light-controlled granulocyte NEPTargeted enrichment of tumors;
FIG. 6 enhanced killing efficacy of light-controlled granulocytes on tumor cells in animal levels.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
The term "prepared from …" as used herein is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a dosage form, procedure, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such dosage form, procedure, method, article, or apparatus.
The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4," "1 to 3," "1-2 and 4-5," "1-3 and 5," and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
1) Preparation of Nanomethanized photosensitizer (P)
Adding 0.006mmol of photosensitizer Ce6, 0.009mmol of EDC and 0.012mmol of NHS into a reactor, reacting for 3 hours at room temperature, and pre-activating carboxyl of the photosensitizer; then adding 8mg Bovine Serum Albumin (BSA) into the reaction system, continuing the reaction at room temperature overnight, and dialyzing and purifying to form nano particles;
2) granulocyte harvesting
Selecting a BALB/c female mouse with 6-8 weeks, injecting 3% thioglycollate salt into the abdominal cavity, 3 mL/mouse, performing peritoneal lavage by PBS 6h after injection, collecting abdominal white blood cells, placing the abdominal white blood cells in an incubator for culture and incubation, removing adherent mononuclear macrophages, collecting supernatant to obtain granulocytes, and performing purity identification flow analysis by using antibodies such as Ly-6G, CD11b and the like to show that the purity of the extracted granulocytes is higher than 90% after the abdominal cavity is stimulated for 6h, and the extraction quantity can reach 1000 ten thousand/mouse.
3) Light-controlled granulocytes (NE)P) Construction of
Co-incubating the purified granulocyte NE and P, wherein the incubation concentration is 0.2mg/mL (the incubation concentration refers to the concentration of a photosensitizer, and the volume ratio of the dose of the granulocyte to the dose of the photosensitizer in the incubation process is 1: 1); the incubation temperature is 37 ℃, the incubation time is 20min, and the incubation medium is PBS.
The distribution of P in granulocytes by laser confocal observation using the inherent red fluorescence property of Ce6 is shown in FIG. 1.
Biocompatible construction of light-controlled granulocytes: it was confirmed that granulocyte NE could achieve high efficiency loading of P as shown in fig. 1 a. The effect of P loading on granulocyte viability (1b characterized by the cell viability dye calcein-AM), surface chemotactic migration Marker (1c) and chemotactic migration ability (1d) was minimal.
4) Light-controlled granulocyte light-ROS activation
Irradiating light-controlled granulocytes, and measuring the content change of ROS in the granulocytes before and after the light irradiation by using an ROS measuring kit, wherein the light irradiation time is 16 minutes, and the light irradiation power density is 20mW/cm2。
light-ROS response of light-controlled granulocytes: light-controlled granulocyte NEPUnder near infrared light excitation, ROS are produced (see fig. 2), and light-controlled granulocyte ROS production increases with increasing illumination time in a positive correlation.
Example 2
Light-controlled granulocyte light-ROS activation
Irradiating light-controlled granulocyte, and measuring ROS content change in the granulocyte before and after irradiation by ROS measuring kit, wherein the irradiation time is 4 minutes, and the irradiation power density is 20mW/cm2。
light-ROS response of light-controlled granulocytes: light-controlled granulocyte NEPUnder near infrared light excitation, ROS are produced (see fig. 2), and light-controlled granulocyte ROS production increases with increasing illumination time in a positive correlation.
The preparation of the photosensitizing agent (P) for nanocrystallization, the obtainment of granulocytes, and the construction of the light-controlled granulocytes (NEP) were the same as those in example 1.
Example 3
Light-controlled granulocyte light-ROS activation
Irradiating light-controlled granulocytes, and measuring the content change of ROS in the granulocytes before and after the light irradiation by using an ROS measuring kit, wherein the light irradiation time is 8 minutes, and the light irradiation power density is 20mW/cm2。
light-ROS response of light-controlled granulocytes: light-controlled granulocyte NEPUnder near infrared light excitation, ROS are produced (see fig. 2), and light-controlled granulocyte ROS production increases with increasing illumination time in a positive correlation.
The preparation of the photosensitizing agent (P) for nanocrystallization, the obtainment of granulocytes, and the construction of the light-controlled granulocytes (NEP) were the same as those in example 1.
Example 4
Biological safety investigation
Selecting 4-6 weeks BALB/c female mice, subcutaneously planting 5 xl 0 on the back of the mice54T1 cell suspension to establish breast cancer model. When the mouse tumor grows to about 100mm3Timely, intraperitoneal injection of NEPWhen NEPThe tumor tissue was illuminated when it reached the maximum enrichment concentration. Observing and recording the body weight of the mice every other day; after 2 weeks of the last illumination, blood is taken from the eye sockets to obtain mouse serum, the content of citrulline histone H3(CITH3) in the serum of each group of mice is measured, and whether systemic NETosis occurs in the therapy is judged; detecting the indexes of liver and kidney functions; killing the animal, weighing the weight of the mouse, dissecting a tumor body, a brain, a heart, a liver, a spleen, a lung and a kidney, weighing respectively, and calculating an organ index; the organs were fixed with 4% formaldehyde solution, and histological examination was performed by preparing tissue sections.
And (3) safety evaluation: analysis of liver and kidney function indices in blood of mice after treatment (a-E), analysis of CitH3 (f) and H & E stained sections of each major tissue (see FIG. 3).
Compared with a control group, the treatment of the optical control granulocyte biological agent does not cause pathological changes of liver and kidney function indexes of mice, citrulline histone H3(CITH3) content in serum of the mice and tissues such as heart, liver, spleen, lung, kidney and the like, and the treatment method has good biological safety.
Example 5
Evaluation of enhanced granulocyte killing Performance by photoactivation
NE (N-substituted amino acid) is synthesizedPNE, P (only generating ROS under illumination) and tumor cells are incubated together, the illumination time is set to be 8 minutes, and the illumination power density is set to be 20mW/cm2And evaluating the improvement of the killing performance of the granulocyte caused by light-operated activation by using various modes such as CCK8, a live-dead-dye kit, an apoptosis and necrosis kit and the like.
Enhanced killing efficacy of light-controlled granulocytes against tumor cells: the enhanced killing efficacy of light-controlled granulocytes against breast cancer cells 4T1(a) and melanoma cells B16-F10(B) is shown in fig. 4.
Example 6
Evaluation of enhanced granulocyte killing Performance by photoactivation
NE (N-substituted amino acid) is synthesizedPNE, P (only ROS are generated under illumination) and tumor cells are subjected to co-incubation, the illumination time is set to be 16 minutes, and the illumination power density is set to be 20mW/cm2And evaluating the improvement of the killing performance of the granulocyte caused by light-operated activation by using various modes such as CCK8, a live-dead-dye kit, an apoptosis and necrosis kit and the like.
Enhanced killing efficacy of light-controlled granulocytes against tumor cells: the enhanced killing efficacy of light-controlled granulocytes against breast cancer cells 4T1(a) and melanoma cells B16-F10(B) is shown in fig. 4.
The result shows that the enhanced killing effect on the tumor cells can be realized by using the light-ROS response characteristic of the light-controlled granulocyte under the excitation of near infrared light.
Example 7
NEPTargeted enrichment at tumor sites
Investigation of NE on a Small animal Living body imager with P-free granulocytes and P as controls and 4T1 breast cancer as a modelPNamely, the distribution and enrichment conditions of the nano photosensitizer-loaded granulocytes in vivo at different injection modes (tail vein and abdominal cavity) and different time points (0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h and 96h), especially the distribution in tumor tissues.
Light-controlled granulocyte NEPFor targeted enrichment of tumors, the circles indicate the location of the tumor as shown in FIG. 5.
The results show that the use of fine particlesThe inherent chemotactic migration ability of cells to tumor tissues can realize the light-operated granulocyte NEPTargeted enrichment at the tumor site.
Example 8
NEPEvaluation of killing efficacy with enhanced animal levels
Mice were subcutaneously implanted 5 xl 0 on the back54T1 cell suspension to establish breast cancer model. The set groups are as follows: NEP+ light, P + light (ROS group only), NE and PBS control group. Tail vein or abdominal cavity injection of NE, NEPP, when NEPAnd when the tumor tissue reaches the maximum enrichment concentration, illuminating the tumor tissue. The enhanced killing efficacy of the light-controlled granulocytes on tumors at the animal level was evaluated by the change in tumor volume in each group of mice.
Enhanced killing efficacy of animal-level light-controlled granulocytes against tumor cells: mouse tumor volume change (a) and mouse survival results (b) are shown in FIG. 6. Compared with the control group, the treatment of the optical control granulocyte biological agent can obviously enhance the killing activity of the granulocyte on the tumor and prolong the survival time of the mouse.
The invention constructs light-controlled granulocyte (NE) by loading photosensitizer in granulocyte, namely clinical photodynamic therapy medicine capable of generating ROS under light excitation through an engineering meansP) Therefore, the generation of the photosensitizer ROS in the light-controlled granulocyte is regulated and controlled through remote control of light, the local concentration of the ROS is improved, and the ROS killing and enhancing effect of the light-controlled granulocyte on the tumor is realized.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims (10)

1. An optically-controlled granulocyte biological agent, which is characterized in that the optically-controlled granulocyte biological agent is a granulocyte in which a photosensitizer is loaded;
the photosensitizer is a nano photosensitizer which is a serum protein modified photosensitizer;
the particle size of the nano photosensitizer is distributed between 20nm and 200 nm;
the preparation method of the serum protein modified photosensitizer comprises the following steps:
1) adding a photosensitizer, condensing agents EDC and NHS into a reactor, reacting at room temperature, and pre-activating carboxyl of the photosensitizer;
2) adding serum protein into the reaction system, continuing the reaction at room temperature, and after the reaction is finished, dialyzing and purifying;
in the preparation method of the serum protein modified photosensitizer, the molar ratio of the photosensitizer to EDC and NHS is 1: (1.1-3): (1-5);
the molar ratio of the serum protein to the photosensitizer in the serum protein modified photosensitizer is 1 (1-10).
2. The optically controlled granulocyte biological agent according to claim 1, wherein the photosensitizer is selected from one or more of chlorin e6, verteporfin, protoporphyrin ix, meso-tetrakis (4-carboxyphenyl) porphine, meso- (4-sulfophenyl) porphine tetrasodium salt, tetrasulfophenylporphyrin, nickel phthalocyanine tetrasodium salt or pyropheophorbide a.
3. The optically-controlled granulocyte biological agent of claim 1, wherein the molar ratio of the photosensitizer to EDC and NHS in the serum protein-modified photosensitizer preparation method is 1: (1.2-2): (1.5-3).
4. The optically-controlled granulocyte biological agent of claim 3, wherein the molar ratio of the photosensitizer to EDC and NHS in the serum protein-modified photosensitizer preparation method is 1: 1.5: 2.
5. the optically-controlled granulocyte biological agent of claim 1, wherein the serum protein-modified photosensitizer comprises a serum protein and a photosensitizer in a molar ratio of 1: 7.
6. The method of claim 1, wherein the optically controlled granulocyte biological agent is obtained by co-incubating granulocyte with a photosensitizer.
7. The method of claim 6, wherein the incubation concentration is 0.01-1mg/mL, wherein the incubation concentration is the concentration of the photosensitizer;
the incubation temperature is 20-40 ℃;
the incubation time is 10-60 min.
8. The method of claim 7, wherein the incubation concentration is 0.1-0.8 mg/mL;
the incubation temperature is 22-39 ℃;
the incubation time is 10-30 min.
9. The method of claim 8, wherein the incubation concentration is 0.2-0.5 mg/mL;
the incubation temperature is 22-39 ℃;
the incubation time is 10-20 min.
10. Use of the optically-controlled granulocyte biologics of claim 1 for the manufacture of a medicament for the treatment of cancer.
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