CN117462696A - Nanometer immunity medicine for targeting neutrophil, preparation method and application - Google Patents
Nanometer immunity medicine for targeting neutrophil, preparation method and application Download PDFInfo
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Abstract
The invention relates to a nano immunity medicine for targeting neutrophils, a preparation method and application thereof, wherein a singlet oxygen response type shell formed by DSPE-TK-PEG is used as a carrier to coat the medicine, and a nano delivery system constructed by sialic acid of external targeting neutrophils formed by DSPE-PEG-SA is further provided with: photosensitizers PFODBT, NLG919 and modified ferrocene. The invention has mild synthesis conditions and simple and convenient operation; the X-ray response type multifunctional nano immune medicine targeting neutrophils has good anti-tumor treatment effect, has potential application value in the aspect of cancer treatment, and can be applied to mediated combination treatment.
Description
Technical Field
The invention belongs to the technical field of preparation of accurate anti-tumor therapeutic drugs, and particularly relates to an X-ray response type multifunctional nano immune drug targeting neutrophils, a preparation method and application thereof.
Background
Glioblastoma (GBM) is the most common primary tumor of the brain, accounting for 81% of Central Nervous System (CNS) malignancies. They are usually produced by glial cells or precursor cells and develop into astrocytomas, oligodendrogliomas, epitheliomas or oligodendrogliomas. Gliomas are classified into four grades according to the World Health Organization (WHO), with grade 1 and grade 2 gliomas being low grade gliomas and grade 3 and grade 4 gliomas being High Grade Gliomas (HGGs). In general, the higher the level, the worse the prognosis. GBM is the most common grade 4 glioma. The traditional radiotherapy and chemotherapy has poor treatment effect and large side effect due to the reasons of drug resistance, natural blood brain barrier, large-dose radiotherapy damage and the like. It is therefore highly desirable to devise a cancer strategy that can be combined with a variety of therapeutic modalities.
Immunotherapy shows great promise in cancer treatment, not only is the most important scientific breakthrough in the year of journal of science in 2013, but also the Nobel physiology or medical prize in 2018 was issued for related research. Tumor immunotherapy mainly includes immune checkpoint inhibitors, cell therapy, tumor vaccines, and the like. However, in clinical applications, only a small fraction of patients with advanced cancer respond to immune checkpoint inhibitor monotherapy, and multiple treatments may be combined to increase the efficacy of the immunotherapy and the patient response rate. The conversion to a biologically active form of a prodrug following a reactive reaction can greatly reduce drug off-target toxicity, which is also of particular concern in cancer treatment. Cleavable linkers or specific peptides, such as pH, GSH, and redox potential, that are sensitive to internal stimuli in the tumor microenvironment have been widely used to construct prodrugs. Through reasonable design of the nano platform, development of a novel and efficient nano platform with responsiveness is possible.
The presence of the blood brain tumor barrier (BBB) constitutes the greatest barrier to noninvasive delivery of therapeutic agents to brain tumors. Here, we propose a new approach to specifically modulate the BBB and deliver to tumor sites with precision to treat GBM. In order to allow the Semiconducting Polymer (SP) to deliver drugs precisely across the BBB to brain tumors in organisms, we utilize a drug-containing polymer comprising 1 O 2 The amphiphilic polymer (DSPE-TK-PEG 2000) responsive to the linker (ketal, TK) constructs a drug delivery vehicle, in addition we attach Sialic Acid (SA) specific targeting neutrophils at the polymer housing, which can accurately deliver drugs to tumor sites. The properties of the prepared intelligent nano delivery system are evaluated one by taking C6 (a kind of mouse glioma cells) as a model cell and a brain glioma model thereof, and the X-ray response type multifunctional nano immunity medicament for targeting the neutrophils prepared by the research has good chemical dynamic characteristics of X-ray response, in particular 1 O 2 The generation capacity, the chemical kinetics capacity of X-ray response and the like are expected to realize the accurate treatment of various tumors, so that the X-ray response type multifunctional nano immune medicine targeting the neutrophils has the clinical transformation potential.
The domestic and foreign documents are searched, and no X-ray response type multifunctional nano immune medicine for targeting neutrophils is found to be used for accurate anti-tumor treatment.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a neutrophil-targeted X-ray response type multifunctional nano-immune medicament, which is mild in preparation condition and easy to operate, and the prepared neutrophil-targeted X-ray response type multifunctional nano-immune medicament has good dispersibility and colloid stability. The raw materials are environment-friendly, the preparation process is simple, and the method has industrial feasibility.
A nano immune medicine for targeting neutrophil is prepared from singlet oxygen response shell as carrier, medicine, and Sialic Acid (SA) as external delivering system for targeting neutrophil.
Preferably, the singlet oxygen responsive housing of the present invention is DSPE-TK-PEG; the targeted neutrophil is DSPE-PEG-SA.
Preferably, the neutrophil-targeted nanoimmune of the invention further comprises: photosensitizers PFODBT, NLG919 and modified ferrocene (Fe-modified).
A preparation method of a nano immune medicine for targeting neutrophils comprises the following steps:
(1) Synthesizing DSPE-PEG-SA, dissolving SA, EDC.HCL and NHS with methanol, and stirring at room temperature for 2h for activating carboxyl at one end; then adding DSPE-PEG-NH slowly into the above solution 2 Continuously stirring for 3d at room temperature; the resulting solution was evaporated in a rotary evaporator to methylene chloride; dialyzing the residual solution for 3d, and then freeze-drying to remove water to obtain white solid powder;
(2) Dissolving DSPE-PEG-SA, DSPE-TK-PEG, photosensitizer PFODBT, NLG919 and modified ferrocene in tetrahydrofuran;
(3) Adding the solution obtained in the step (2) into an aqueous solution of tetrahydrofuran under an ultrasonic condition, and carrying out ultrasonic treatment for 30min;
(4) Using a rotary evaporator to fully volatilize tetrahydrofuran;
(5) Filtering with PES membrane filter while stirring; and (5) loading the filtered solution into a dialysis bag for ultrafiltration dialysis to obtain the nano immune medicine.
The application of the nano immunity medicine targeting neutrophil is applied to the combined treatment of radiotherapy and chemotherapy and the tumor of immunotherapy.
The application of the nano-immune medicine targeting the neutrophils in the aspect of resisting tumors comprises the step of accurately delivering the nano-immune medicine to a tumor area by the targeting the neutrophils and the step of treating in-situ brain glioma.
Compared with the prior art, the nano immunity medicament for targeting neutrophils and the preparation method thereof are adopted:
(1) The preparation method is simple, the main material is convenient to obtain, and the biological safety is good;
(2) The nano immunity medicament prepared by the invention can precisely target a tumor area and effectively treat in-situ brain glioma;
(3) The invention uses novel materials, enhances the combined treatment effect through cascade reaction, and realizes the maximization of the curative effect of the medicine;
(4) The medicine wrapped by the invention has a controlled release effect, and can improve the safety, the effectiveness and the utilization rate of the medicine.
Drawings
FIG. 1 is a flow chart of a neutrophil-targeted X-ray responsive multifunctional nanoimmune prepared in accordance with the present invention;
FIG. 2 is a PN prepared according to the present invention FeN 、HPN Fe And HPN FeN Wherein a is PN prepared according to the present invention FeN ,HPN Fe And HPN FeN B is PN prepared by the invention FeN 、HPN Fe And HPN FeN Is a fluorescent emission spectrum;
FIG. 3 is PN prepared according to the present invention FeN 、HPN Fe And HPN FeN Wherein a is PN prepared according to the present invention FeN 、HPN Fe And HPN FeN B is PN prepared by the invention FeN 、HPN Fe And HPN FeN Is a hydrated particle size plot of (2);
FIG. 4 is a PN prepared according to the present invention FeN 、HPN Fe And HPN FeN Is a surface potential diagram of (2);
FIG. 5 is a PN prepared in accordance with the present invention from TA solution FeN 、HPN Fe And HPN FeN Time-dependent fluorescence spectrum after hydroxyl radical reaction of (a) is TA at PN FeN Time-dependent fluorescence spectrum after reaction of medium hydroxyl radical, b is TA in HPN Fe Time-dependent fluorescence spectrum after reaction of medium hydroxyl radical, c is TA in HPN FeN Time-dependent fluorescence spectrum after the reaction of the medium hydroxyl radical;
FIG. 6 is a schematic diagram of the PBS treatment of C6 cells tested by CCK-8Buffer (control) and PN prepared according to the invention FeN 、HPN Fe And HPN FeN Cell viability after 24 hours of treatment;
FIG. 7 is a schematic diagram showing the CCK-8 assay of C6 cells in PBS buffer (control) and PN prepared according to the present invention FeN 、HPN Fe And HPN FeN Cell viability after 24 hours of treatment by X-ray irradiation;
FIG. 8 is a graph of NLG919 release percentage over different irradiation times;
FIG. 9 shows Western blot of C6 cells in PBS buffer (control) and PN prepared according to the invention FeN 、HPN Fe And HPN FeN The expression levels of intracellular GPX4 and ACSL4 after 12 hours of co-culture and X-ray irradiation treatment;
FIG. 10 is an inverted fluorescence microscope captured C6 cells with PBS and PN prepared according to the present invention FeN 、HPN Fe And HPN FeN Fluorescence images of intracellular ROS levels after 12 hours of co-incubation were X-rayed;
FIG. 11 shows Western blot of tumor-bearing mice injected intravenously with PBS buffer (control) and PN prepared according to the invention FeN 、HPN Fe And HPN FeN Then the expression quantity of GPX4 and ACSL4 in the tumor after X-ray irradiation;
fig. 12 is a graph showing changes in body weight of tumor-bearing mice within 14 days after treatment.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
As shown in figure 1, the preparation method of the X-ray response type multifunctional nano-immune medicine for targeting neutrophils comprises the following steps:
DSPE-PEG-SA was first synthesized, SA (22.5 mg,0.1 mmol), EDC. HCL (19.2 mg,0.1 mmol) and NHS (11.6 mg,0.1 mmol) were first dissolved in methanol, chamberThe mixture was stirred for 2 hours to activate the carboxyl group at one end. Then adding DSPE-PEG-NH slowly into the above solution 2 (30 mg,0.02 mmoL) in methylene chloride was stirred at room temperature for 3 days. The resulting solution was evaporated in a rotary evaporator to dichloromethane. Dialyzing the residual solution (molecular weight cut-off 1000D) for 3D, and then lyophilizing to remove water to obtain white solid powder;
wherein: SA: EDC HCL: the molar ratio of NHS is 1:3:3, a step of; DSPE-PEG-SA: DSPE-TK-PEG: photosensitizer PFODBT: fe-modified: the mass ratio of NLG919 is 10:10:0.5:0.5:0.5; tetrahydrofuran was 1mL in volume; the ratio of tetrahydrofuran to ultrapure water in the aqueous solution of tetrahydrofuran was 1:9, the total volume is 10mL; the time of the ultrasound is: 30min; the setting parameters of the rotary evaporator are as follows: 40 ℃,70rpm,1h; the molecular cut-off of the PES membrane filter device used was 0.22 μm; the molecular retention of the dialysis bag is 10000D; dialysis process parameters: room temperature, 36h.
Dissolving the DSPE-PEG-SA, DSPE-TK-PEG, photosensitizer PFODBT, fe-modified and NLG919 obtained in the steps in tetrahydrofuran, adding the solution into aqueous solution of tetrahydrofuran under ultrasonic condition after fully dissolving, and performing ultrasonic treatment; placing the solution into a rotary evaporator, and filtering by using a PES (polyether sulfone) membrane filter after tetrahydrofuran is fully volatilized; and (3) putting the filtered solution into a dialysis bag for dialysis to obtain a final material DSPE-PEG-SA/DSPE-TK-PEG@PFODBT-Fe-modified-NLG919.
Wherein: technological parameters of stirring reaction: room temperature, 24h; the molecular weight cut-off of the ultrafiltration tube is 50000D; technological parameters of ultrafiltration: 4 ℃,2700rpm,1h; repeating for a plurality of times: 4-6 times.
The invention uses ultraviolet visible absorption spectrum (UV-Vis), fluorescence spectrum, transmission Electron Microscope (TEM), ultraviolet visible absorption spectrum (UV-Vis), zeta potential, dynamic light scattering analysis (DLS) and other means to characterize the prepared nano immune medicine (PN) FeN 、HPN Fe And HPN FeN ) And determining the chemodynamic performance of the ultrasound response of a neutrophil-targeted X-ray responsive multifunctional nanoimmune drug by means of an OH detector (TA), and then evaluating a targeting by means of cell viability analysis (CCK-8 test)The cytotoxicity of the X-ray response type multifunctional nano-immune medicine of the neutrophil and the killing effect of the X-ray response type multifunctional nano-immune medicine of the targeted neutrophil on the cell. And finally, evaluating the therapeutic performance of the X-ray response type multifunctional nano immune medicine targeting the neutrophils through a tumor-bearing mouse.
Example 1
SA (22.5 mg,0.1 mmol), EDC. HCL (19.2 mg,0.1 mmol) and NHS (11.6 mg,0.1 mmol) were dissolved in methanol and stirred at room temperature for 2h to activate the carboxyl group at one end. Then adding DSPE-PEG-NH slowly into the above solution 2 (30 mg,0.02 mmoL) in methylene chloride was stirred at room temperature for 3 days. The resulting solution was evaporated in a rotary evaporator to dichloromethane. Dialyzing the residual solution (with a molecular weight cut-off of 1000D) for 3D, and then freeze-drying to remove water, thereby obtaining white solid powder DSPE-PEG-SA;
dissolving the DSPE-PEG-SA, DSPE-TK-PEG, photosensitizer PFODBT, fe-modified and NLG919 obtained in the steps in tetrahydrofuran, adding the solution into aqueous solution of tetrahydrofuran under ultrasonic condition after fully dissolving, and performing ultrasonic treatment; placing the solution into a rotary evaporator, and filtering by using a PES (polyether sulfone) membrane filter after tetrahydrofuran is fully volatilized; the filtered solution is put into a dialysis bag for dialysis to obtain the final material DSPE-PEG-SA/DSPE-TK-PEG@PFODBT-Fe-modified-NLG919 (HPN) FeN )(HPN Fe With HPN FeN The synthesis steps of (a) are similar to those described above. )
Test result 1
Referring to the test results of ultraviolet visible absorption spectrum (UV-Vis) and fluorescence absorption spectrum of FIG. 2 of the specification, PN prepared by the present invention was prepared by comparing FeN 、HPN Fe And HPN FeN Wherein a is PN prepared by the invention FeN 、HPN Fe And HPN FeN B is PN prepared by the invention FeN 、HPN Fe And HPN FeN Is a fluorescent emission spectrum; found PN prepared by the invention FeN 、HPN Fe And HPN FeN Similar absorption peaks at 550nm and 380nm, which are the photosensitizers PFODBTThus demonstrating that the delivery vehicle has no significant effect on the UV-visible light absorption of the photosensitizer PFODBT, PN prepared by the present invention FeN 、HPN Fe And HPN FeN The absorption peak at 275nm, caused by the immunological drug NLG919, illustrates the presence of NLG919 in the delivery system. PN prepared by comparison of the present invention FeN 、HPN Fe And HPN FeN The fluorescent absorption spectrum of (2) shows that PN prepared by the invention FeN 、HPN Fe And HPN FeN There is a similar absorption peak at 700nm, which is a characteristic peak of SP, thus indicating that the delivery vehicle has no significant effect on the fluorescence absorption of SP.
Test result 2
With reference to hydrodynamic particle size and Transmission Electron Microscope (TEM) test results of FIG. 3 of the specification, PN prepared by the present invention FeN 、HPN Fe And HPN FeN Is observed, wherein a is PN prepared according to the invention FeN 、HPN Fe And HPN FeN B is PN prepared by the invention FeN 、HPN Fe And HPN FeN Is a graph of the hydrated particle size of (3). Hydrodynamic particle size shows that PN prepared by the invention FeN 、HPN Fe And HPN FeN The hydrated particle sizes of (3) were 71.2nm, 68.9nm and 70.6nm, respectively, without significant differences. PN prepared by the invention FeN 、HPN Fe And HPN FeN TEM image of (C) shows PN prepared by the present invention FeN 、HPN Fe And HPN FeN Presenting uniformly dispersed spherical nanoparticles.
Test result 3
Referring to the potential test results of FIG. 4 of the specification, PN was determined using Dynamic Light Scattering (DLS) FeN 、HPN Fe And HPN FeN The surface potentials of (a) were-35.1 mV, -29.9mV and-33.3 mV, respectively, with no significant difference (FIG. 4).
Test result 4
Referring to the chemical kinetics test results of FIG. 5 of the specification, the PN prepared by the present invention was detected using an OH detection probe (terephthalic acid, TA) FeN 、HPN Fe And HPN FeN OH-producing ability of (C). PN prepared by the invention is detected by an OH detection probe (terephthalic acid, TA) FeN 、HPN Fe And HPN FeN Chemical kinetics of (c) are described. FIG. 5a is a TA solution in PN FeN Ultraviolet absorption spectrum of OH generated by the reaction; FIG. 5b is a TA solution in HPN Fe Ultraviolet absorption spectrum of the reaction to OH and FIG. 5c is a graph of TA solution in HPN FeN The reaction produced OH.
Test result 5
Referring to the CCK-8 cell viability test result of the figure 6 of the specification, the PN prepared by the invention is evaluated by taking C6 cells as model cells FeN 、HPN Fe And HPN FeN Effects of three nanoparticles on cell proliferation. Configuration of PN with different concentrations with sterile PBS FeN 、HPN Fe And HPN FeN Nanoparticle solution and sterilized overnight with ultraviolet radiation. Configuring PN FeN 、HPN Fe And HPN FeN Nanoparticle (PFODBT concentration of 3, 6, 12, 25, 50 μg/mL) solution, 5 compound wells are arranged in each group, a blank group is arranged at the same time, PBS buffer is added to the edge-most well to reduce the influence of evaporation, and then cells are placed at 37 ℃ and 5% CO 2 Culturing in a cell culture box for 24 hours. After culturing, sucking out the nano-particles, slowly washing with PBS for 2 times, preparing culture medium containing 10% CCK-8 solution, adding into the pore plate, avoiding generating bubbles during the adding process, placing the culture plate with CCK-8 added into 37 deg.C, 5% CO 2 Incubation in the incubator is continued for 2-4 hours. The 96-well plate was removed, the OD value of each well at a wavelength of 450nm was measured using an enzyme-labeled instrument, the effect of different concentrations of material on cell proliferation was compared against buffer PBS, and the viability of the cells was calculated from this value (as shown in fig. 6). PN, compared to PBS control group FeN 、HPN Fe And HPN FeN The effect on the viability of C6 cells was not significantly different at experimental concentrations of PFODBT ranging from 3 to 50. Mu.g/mL, with cell viability above 80%, which is a full indication of synthetic PN FeN 、HPN Fe And HPN FeN Has good biological safety, and can be used for in vivo treatment
Test result 6
Referring to the results of the in vitro combination therapy test of FIG. 7 of the specification, PN was evaluated using the CCK-8 method FeN 、HPN Fe And HPN FeN Therapeutic effect on C6 cells (fig. 7). As shown in the figure, C6 cells are mixed with phosphate buffer (PBS buffer), PN prepared by the invention FeN 、HPN Fe And HPN FeN X-ray irradiation (2 Gy,100 kv) was applied/not applied after 12 hours of co-cultivation. After 12 hours of co-culture at 37℃the survival rate of C6 cells was evaluated by CCK-8 method. As can be seen from FIG. 7, the X-ray irradiated PN is compared with other treatment groups FeN 、HPN Fe And HPN FeN Group C6 cells were significantly lower in viability and sonicated HPN FeN Group C6 showed lower survival due to the presence of NLG919 and SA. The experimental results demonstrate that HPN prepared in accordance with the present invention FeN Can effectively cause death of C6 cells under the irradiation of X-rays.
Test result 7
Referring to the NLG919 release test results of nano immune drug of FIG. 8 of the specification, 50. Mu.g/mL HPNFeN solution prepared by ultrapure water was irradiated (2 Gy,100 kv) with an X-ray radiotherapy apparatus for 0min, 2min, 4min and 6min respectively, and the release of NLG919 was detected with a high performance liquid chromatograph. As shown in fig. 8, it can be seen that the drug release percentages at X-ray irradiation times of 0min, 2min, 4min and 6min are 0.9%, 48%, 61% and 79%, respectively, and the results indicate that the longer the irradiation time is, the more sufficient the release of NLG919 is, indicating that the nanomaterial has an excellent controllable release effect.
Test result 8
Westernblot was used to determine expression levels of long chain acyl-CoA synthetase 4 (ACSL 4) and glutathione peroxidase (GPX 4) in C6 cells of different treatment groups to evaluate PN FeN 、HPN Fe And HPN FeN The effect of iron death is induced. C6 and PBS, PN prepared by the invention FeN 、HPN Fe And HPN FeN After 12 hours of co-culture, irradiation (2 gy,100 kv) was performed, and after 12 hours of treatment, cells were collected and washed with PBS, and expression of ACSL4 and GPX4 in the cells was measured by Western blot. From FIG. 9It can be seen that the irradiation treated PN FeN 、HPN Fe And HPN FeN The GPX4 expression level of the group is obviously lower than that of other treatment groups, and the ACSL4 expression level is obviously higher than that of the other treatment groups; this illustrates the PN of the irradiation process FeN 、HPN Fe And HPN FeN The group has better effect of inducing iron apoptosis.
Test result 9
PN obtained in example 1 was determined by reference to the in vitro iron apoptosis assay of FIG. 9 of the specification FeN 、HPN Fe And HPN FeN A solution was prepared at a concentration of 50. Mu.g/mL using sterile PBS buffer. A volume of 500. Mu.L was taken, containing 8X 10 4 Individual C6 cell suspensions were seeded in 24-well plates and cultured for 24h. Respectively adding sterile PBS and PN FeN 、HPN Fe And HPN FeN Four solutions, PBS group, served as a blank control, and the material was incubated with cells for 12h. The material in the 24-well plate was slowly aspirated with a pipette, washed 3 times with sterile PBS, added with DCFH-DA fluorescent probe diluted with DMEM medium, incubated for 30min, irradiated in the dark (2 Gy,100 kv), washed 3 times with PBS, added with 300 μLPBS, and immediately photographed for fluorescence of the DCFH-DA fluorescent probe using an inverted fluorescence microscope. The end result is shown in FIG. 10, where PBS group was irradiated, but no singlet oxygen was generated and PN was generated FeN 、HPN Fe And HPN FeN There was a clear green fluorescence, indicating that singlet oxygen was generated.
Test result 10
Referring to the results of the singlet oxygen production measurements shown in FIG. 10 of the specification, westernblot was used to determine the expression levels of ACSL4 and GPX4 in tumors of different treatment groups (FIG. 11), and to evaluate PN FeN 、HPN Fe And HPN FeN Iron death effects induced in vivo. Tumor-bearing mice were randomly divided into 6 groups (PBS group and HPN) FeN Group and irradiation-treated PBS group, PN FeN 、HPN Fe And HPN FeN Group), intravenous PBS, PN FeN 、HPN Fe And HPN FeN Irradiation treatment was administered (6 gy,100 kv)/not. The mice were euthanized, tumor tissues were removed, and the expression of ACSL4 and GPX4 in the tumors was determined using Westernblot,as can be seen from the figure, the irradiation-treated PN FeN 、HPN Fe And HPN FeN The GPX4 expression level of the group is obviously lower than that of other treatment groups, and the ACSL4 expression level is obviously higher than that of the other treatment groups; this illustrates the PN of the irradiation process FeN 、HPN Fe And HPN FeN The group has better effect of inducing iron apoptosis.
Test results 11
PN obtained in example 1 was described with reference to the in vivo iron apoptosis assay shown in FIG. 11 of the specification FeN 、HPN Fe And HPN FeN The nanoparticle is prepared into a solution with the PFODBT concentration of 300 mug/mL by using sterile PBS, 200 mug is taken and injected into a C6 tumor-bearing mouse through tail vein, and an X-ray radiotherapy instrument is used for irradiating the brain part of the mouse (6 Gy,100 kv) and is divided into a PBS group and HPN group FeN Group and PBS+X-ray, PN FeN +X-ray、HPN Fe +X-ray、HPN FeN The body weights of mice in the +X-ray six groups were recorded for 14 days thereafter, and as can be seen from FIG. 12, the body weights showed a smooth trend with no abnormalities.
Referring to the results of the survival rate and the weight monitoring of the mice shown in the figure 12 of the specification, the weight shows a stable increasing trend, and no abnormal situation exists.
Claims (10)
1. A nano immune medicine for targeting neutrophil is characterized in that a singlet oxygen response type shell is used as a carrier to coat the medicine, and a nano delivery system constructed by sialic acid of the targeting neutrophil is externally connected.
2. The neutrophil-targeted nanoimmune of claim 1, wherein the singlet oxygen-responsive shell is DSPE-TK-PEG; the targeted neutrophil is DSPE-PEG-SA.
3. The neutrophil-targeted nanoimmune drug of claim 1, comprising: photosensitizers PFODBT, NLG919 and modified ferrocene.
4. A method for preparing a neutrophil-targeted nanoimmune based on the method according to claims 1-3, characterized by comprising the steps of:
(1) Synthesizing DSPE-PEG-SA, dissolving SA, EDC.HCL and NHS with methanol, and stirring at room temperature for 2h for activating carboxyl at one end; then adding DSPE-PEG-NH slowly into the above solution 2 Continuously stirring for 3d at room temperature; the resulting solution was evaporated in a rotary evaporator to methylene chloride; dialyzing the residual solution for 3d, and then freeze-drying to remove water to obtain white solid powder;
(2) Dissolving DSPE-PEG-SA, DSPE-TK-PEG, photosensitizer PFODBT, NLG919 and modified ferrocene in tetrahydrofuran;
(3) Adding the solution obtained in the step (2) into an aqueous solution of tetrahydrofuran under an ultrasonic condition, and carrying out ultrasonic treatment for 30min;
(4) Using a rotary evaporator to fully volatilize tetrahydrofuran;
(5) Filtering with PES membrane filter while stirring; and (5) loading the filtered solution into a dialysis bag for ultrafiltration dialysis to obtain the nano immune medicine.
5. The method of claim 4, wherein SA in the step (1) is 22.5mg,0.1mmol; EDC & HCL is 19.2mg,0.1mmol; NHS 11.6mg,0.1mmol; SA dissolved in methanol: EDC HCL: the molar ratio of NHS is 1:3:3, a step of; DSPE-PEG-NH 2 30mg,0.02mmoL; the dialysis molecular weight cut-off was 1000D.
6. The method of claim 4, wherein the step (2) comprises dissolving DSPE-PEG-SA, DSPE-TK-PEG, photosensitizers PFODBT, fe-modified and NLG919 in tetrahydrofuran, DSPE-PEG-SA: DSPE-TK-PEG: photosensitizer PFODBT: fe-modified: the mass ratio of NLG919 is 10:10:0.5:0.5:0.5; an aqueous solution of tetrahydrofuran, the ratio of tetrahydrofuran to ultrapure water being 1:9, total volume 10mL.
7. The method for preparing a neutrophil-targeted nano-immune medicament according to claim 4, wherein the time of rotary evaporation in the step (4) is 2-3 hours; the molecular cut-off of the PES membrane filter unit used was 0.22. Mu.m.
8. The method for preparing the neutrophil-targeted nano-immune medicament according to claim 4, wherein the conditions of the membrane dialysis in the step (5) are as follows: the molecular weight cut-off of the dialysis bag is 10000D; the dialysis time was 36h; the stirring conditions are as follows: stirring for 24h at room temperature.
9. The method for preparing a neutrophil-targeted nano-immune drug according to claim 4, wherein the retention amount of the ultrafiltration tube used in the ultrafiltration in the step (5) is 50000D; the centrifuge parameters were: 2700rpm,4℃for 1h.
10. The application of the nano-immune medicine targeting neutrophils is characterized in that the nano-immune medicine is applied to the combined treatment of radiotherapy and chemotherapy and the tumor of immunotherapy.
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