CN117122692B - Targeting nano-carrier and preparation method and application thereof - Google Patents

Targeting nano-carrier and preparation method and application thereof Download PDF

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CN117122692B
CN117122692B CN202311109923.6A CN202311109923A CN117122692B CN 117122692 B CN117122692 B CN 117122692B CN 202311109923 A CN202311109923 A CN 202311109923A CN 117122692 B CN117122692 B CN 117122692B
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CN117122692A (en
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郭瑞
文荣
黄尚辉
冯龙宝
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Guangzhou Bioscience Co ltd
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Abstract

The invention relates to a targeting nano-carrier and a preparation method and application thereof, belonging to the technical field of biomedicine. The targeting nano-carrier comprises a black phosphorus nano-sheet and a modified PAMAM coated on the surface of the black phosphorus nano-sheet, wherein the modified PAMAM contains a diselenide bond and is modified with FA molecules. The targeting nano-carrier takes black phosphorus as a core framework and has a photo-thermal effect; secondly, the modified dendritic macromolecule PAMAM modifies the surface of the nano-material, so that the stability of the nano-material can be improved, and functional genes can be loaded; in addition, FA facilitates enrichment of nanomaterial in tumor cells. The nanometer material can be applied to medicines for treating liver cancer, or gene therapy and photothermal therapy, and provides a new idea for high-efficiency treatment of liver cancer.

Description

Targeting nano-carrier and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to a targeting nano-carrier and a preparation method and application thereof.
Background
Hepatocellular carcinoma (HCC) is one of the most common high mortality cancers worldwide. In recent years, the incidence of HCC worldwide has continuously increased, and is attracting more and more attention. The effective treatment of early liver cancer is operation, and the survival rate of the early liver cancer is 89-93% after 5 years of operation treatment. However, only a few HCC patients (about 20%) can be diagnosed early, most HCC patients (> 70%) are advanced, cannot be surgically resected, and can only be treated by other methods including liver transplantation, transcatheter Arterial Chemoembolization (TACE) and systemic chemotherapy. However, other treatment methods have limitations, so that the treatment effect of liver cancer is not ideal. Therefore, there is an urgent need to explore innovative therapeutic methods for liver cancer, and to remedy the deficiency of clinical treatment.
With the intensive research into long-chain non-coding rna (long-chain noncoding rna, lncRNAs), lncRNAs were found to play an important role in tumorigenesis, development, metastasis and apoptosis. Research on the role of lncRNAs in the occurrence and development of liver cancer is also possible to provide a new breakthrough for diagnosis and treatment of future liver cancer. At present, lncRNAs become important research hotspots in the field of tumor biology, but the research on the relationship between lncRNAs and tumors is still in the starting stage. CRNDE expression is significantly upregulated in most malignant tumors. CRNDE can participate in the biological process of related tumors by influencing epigenetic regulation and control of chromatin, expression of some important proteins, tumor specific signal paths and cell metabolism, so as to promote proliferation, migration and invasion of tumor cells, inhibit apoptosis and finally promote occurrence and development of tumors.
Currently, lncRNAs molecular technology has entered the experimental stage of human body, and is one of the most promising methods for treating various genetically mutated tumors. However, clinical use of lncRNAs remains a major challenge, such as instability in serum, susceptibility to nuclease degradation in the extracellular environment, electrostatic repulsion of negatively charged lncRNAs from negatively charged cell membranes, etc., which can severely affect transport and absorption of lncRNAs. Because of these characteristics, free lncRNAs are difficult to target directly to specific cells, and chemical modification of lncRNAs or loading of lncRNAs into protective carrier materials to improve stability of lncRNAs is one of the current research hotspots. Folic Acid (FA), a targeting motif that is widely studied, is a natural component that does not cause allergic reactions like many proteins. In addition, FA receptors are overexpressed in kidney, lung and breast cancer tissues, while expression levels are lower in normal tissues. Thus, FA functional nanoparticles can specifically enhance uptake of the composite pharmaceutical formulation by tumor cells via FA receptor-mediated endocytosis. Meanwhile, the FA-modified composite polymer nano-particles become potential carriers for tumor targeted drug delivery.
In recent years, phototherapy (photothermal therapy, PTT) has become a rapidly developing new cancer treatment. Compared with the traditional tumor ablation method, the PTT has higher local effect and can be used for the areas with higher surgical difficulty. Black Phosphorus (BP) is a novel inorganic material, is an allotrope of white phosphorus and red phosphorus, and is mainly applied to the fields of lithium ion batteries, storage devices and the like. In recent years, it has been found that a two-dimensional lamellar structure of BP can be obtained by a graphene-like mechanical exfoliation method, and exhibits unique optical properties. BP was first used for photothermal therapy in the biomedical field. BP can generate high heat under the irradiation of near infrared laser (808 nm), and can be used for realizing high-efficiency and safe tumor photothermal treatment. Compared with the traditional inorganic nano material, BP can be oxidatively degraded into safe and nontoxic small molecules such as phosphate, phosphite and the like under physiological conditions.
Disclosure of Invention
Based on the above, it is necessary to provide a targeting nanocarrier to achieve targeting of liver cancer and improve the therapeutic effect of liver cancer.
The technical scheme of the invention is as follows: the targeting nano-carrier comprises a black phosphorus nano-sheet and a modified PAMAM coated on the surface of the black phosphorus nano-sheet, wherein the modified PAMAM contains a diselenide bond and is modified with FA molecules.
The nano-carrier takes black phosphorus as a core skeleton, and the carrier can have rich positive charges by modifying a modified polymer PMMA, so that the carrier can increase the load energy of a negatively charged functional gene such as a gene shCRNDE; the diselenide bond has GSH responsiveness, and black phosphorus is coated by the modified polymer PAMAM with GSH responsiveness, so that degradation can be prevented, and the stability of the nano carrier is improved; meanwhile, FA is modified into a guide molecule targeting the FA receptor on the liver cancer cell, so that the enrichment of the nano material in the tumor cell is promoted, and the high-efficiency combined treatment of the liver cancer tumor cell can be realized.
In one embodiment, the particle size of the targeting nano-carrier is 200nm, the zeta potential is 18mV, and the targeting nano-carrier has a good effect of killing liver cancer cells after gene loading.
The invention also provides a preparation method of the targeting nano-carrier, which comprises the following steps: (1) preparing black phosphorus nano-sheet BPNPs: dispersing black phosphorus in pure water, stirring, degassing the solution by flowing argon, then carrying out ultrasonic treatment on the mixed solution under ice bath condition to obtain brown dispersion, centrifuging the brown dispersion, collecting supernatant and further centrifuging the collected supernatant to obtain black phosphorus nanoplatelets BPNPs.
(2) Preparing PAMAM-se-FA comprising:
2.1 Synthesis of NH 2 -PEG-se-NH 2: dissolving selenocysteine hydrochloride in water, adding EDC and NHS into selenocysteine hydrochloride solution, and stirring for reaction; then drop wise add to NH 2 Stirring and reacting in PEG-COOH solution at room temperature, dialyzing, and freeze-drying to obtain NH 2 -PEG-SeSe-NH 2 ；
2.2 dissolving FA in DMSO, adding EDC and NHS into the FA solution, stirring at room temperature, adding the mixture into the PAMAM solution dropwise, stirring at room temperature for reaction, dialyzing, and freeze-drying to obtain PAMAM-FA;
2.3 adding EDC and NHS to the PAMAM-FA solution in water, stirring at room temperature, and adding dropwise to NH 2 -PEG-SeSe-NH 2 Stirring and reacting in water solution at room temperature, dialyzing, and freeze-drying to obtain PAMAM-SeSe-FA;
(3) Preparation of BP@PAMAM-SeSe-FA
Preparing a BPNPs aqueous solution, adding PAMAM-SeSe-FA, mixing, performing ultrasonic and stirring reaction to obtain the BP@PAMAM-SeSe-FA nano-carrier, namely the targeting nano-carrier.
The preparation method comprises the steps of firstly preparing black phosphorus nano-sheet BPNPs by adopting a liquid phase dissolution method; then taking the polymer as a core framework, and modifying the polymer PMMA-SeSe-FA to obtain the nano carrier BP@PAMAM-SeSe-FA, wherein the preparation method is simple and easy to operate and is beneficial to popularization.
In one embodiment, in the step (1), the mass ratio of black phosphorus to pure water is 10-30: 20-80 parts; the ultrasonic time is 6-24 hours; the centrifugal speed of the brown dispersion is 500 rpm-1000 rpm; the collected supernatant was centrifuged at 2000rpm to 4000rpm. Preferably, the mass ratio of black phosphorus to pure water is 25:50; the ultrasonic time is 12 hours; the brown dispersion was centrifuged at 1000rpm; the collected supernatant was centrifuged at 3750rpm.
In one embodiment, in step 2.1, the mass ratio of the raw materials is selenocysteine hydrochloride: deionized water: EDC: NHS: NH (NH) 2- PEG-cooh=5 to 15:2 to 8: 5-25: 5-20: 5 to 50, the reaction time of EDC and NHS with selenocysteine hydrochloride is 1 to 6 hours, and the selenocysteine hydrochloride solution and NH 2 The reaction time of the PEG-COOH is 6 to 24 hours; in the step 2.2, the mass ratio of FA to PAMAM is 1-10: 30-70 parts; in step 2.3, PAMAM-FA and NH 2 -PEG-SeSe-NH 2 The mass ratio of (2) is 10-30: 5.
preferentially, in the step 2.1, the mass ratio of the raw materials is that selenocysteine hydrochloride: deionized water: EDC: NHS: NH (NH) 2 PEG-COOH = 10:6:15:12:30, the reaction time of EDC and NHS with selenocysteine hydrochloride is 4h; selenocysteine hydrochloride solution and NH 2 The reaction time of PEG-COOH was 24h; in the step 2.2, the mass ratio of FA to PAMAM is 5:50; in the step 2.3, PAMAM-FA and NH 2 -PEG-SeSe-NH 2 The mass ratio of (2) is 20:5.
in one embodiment, in step (3), the mass ratio of BP to PAMAM-se-FA is 0.1 to 0.5:5 to 15; the ultrasonic time is 10-40 min; the reaction time is 2-8 h.
Preferably, in the step (3), the mass ratio of BP to PAMAM-se-FA is 0.375:10; the ultrasonic time is 30min; the reaction time was 6h.
In one embodiment, step (1) further comprises resuspending the obtained BPNPs in PBS solution to obtain a BPNPs-PBS solution, which is then stored at 4 ℃.
In one embodiment, in the step (3), the preparation of the aqueous solution of BPNPs is obtained by centrifuging the solution of BPNPs-PBS, removing the supernatant and re-suspending in water. Preferably, the centrifugal speed is 4000 rpm-8000 rpm; further preferably, the centrifugal speed is 6000rpm.
In still another aspect, the invention further provides application of the nano-carrier in preparing medicines for treating liver cancer, or gene therapy and photothermal therapy.
Compared with the prior art, the invention has the following beneficial effects:
the nano-carrier takes black phosphorus as a core skeleton, and the modified polymer PMMA is modified, so that the carrier has rich positive charges, and the loading capacity of the carrier on negatively charged functional genes such as shCRNDE is improved; the diselenide bond has GSH responsiveness, and is coated by the modified polymer PAMAM responded by GSH, so that black phosphorus degradation can be prevented, and the stability of the nano carrier is improved; meanwhile, FA is modified into a guide molecule targeting the FA receptor on the liver cancer cell, so that the enrichment of the nano material in the tumor cell is promoted, and the high-efficiency combined treatment of the liver cancer can be realized. The preparation method of the invention firstly adopts a liquid phase dissolution method to prepare the black phosphorus nano-sheet BPNPs; then taking the polymer as a core framework, and modifying the polymer PMMA-SeSe-FA to obtain the nano carrier BP@PAMAM-SeSe-FA, wherein the preparation method is simple and easy to operate and is beneficial to popularization. The nano-carrier can be applied to preparation of medicines for treating liver cancer or gene therapy and photothermal therapy.
Drawings
FIG. 1 is a transmission electron microscope image of black phosphorus nanoplatelets BPNPs and a nanocarrier BP@PAMAM-SeSe-FA according to an embodiment of the invention;
FIG. 2 shows Zeta potential test results;
FIG. 3 shows cytotoxicity test results;
FIG. 4 is a photo-thermal profile of BP@PAMAM-SeSe-FA at various concentrations;
FIG. 5 shows the results of cell viability test.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Definition:
BP: black phosphorus;
BPNPs: black phosphorus nanoplatelets;
EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride;
NHS: n-hydroxysuccinimide;
NH 2 -PEG-COOH: amino polyethylene glycol carboxyl group
PBS: phosphate buffer;
DMSO: dimethyl sulfoxide;
FA: folic acid;
PAMAM: polyamide-amine dendrimers.
The source is as follows:
the reagents used in the following examples, unless otherwise specified, are all commercially available; the methods used in the examples below, unless otherwise specified, are all conventional.
Example 1
(1) Preparation of black phosphorus nano-sheet BPNPs
BP 25mg was dissolved in 50ml of pure water, and vigorously stirred. The solution was degassed with flowing argon to eliminate dissolved oxygen molecules and reduce oxidation during stripping. Then, the mixed solution was sonicated in an ice bath at a power of 250W (45 s on, 15s off for a total time of 12 h). The brown dispersion obtained was centrifuged at 1000rpm for 10min, the bulk BP was removed, and the supernatant containing BPNPs was collected. The collected supernatant containing BPNPs was then centrifuged at 3750rpm at 4℃for 30min to remove water. The pure BPNPs obtained were resuspended in PBS to give a solution of BPNPs-BPS for further use and stored at 4 ℃.
(2) Preparation of PAMAM-SeSe-FA
2.1 Synthesis of NH 2- PEG-SeSe-NH 2 :10 mg of selenocysteine hydrochloride is dissolved in 6mL of water, 15mg of EDC and 12mg of NHS are added into the selenocysteine hydrochloride solution, and the mixture is stirred for reaction for 4h. And adding the selenocysteine hydrochloride solution dropwise to 15mL of NH 2 The reaction was stirred at room temperature overnight in the PEG-COOH solution. Dialyzing for 24 hours, and freeze-drying to obtain NH 2 -PEG-SeSe-NH 2 。
2.2 Synthesis of PAMAM-FA
5mg of FA is dissolved in DMSO, 10.2mg of EDC and 8.5mg of NHS are added into the FA solution, the mixture is stirred for 4 hours at room temperature, then the mixture is added into 10mL of 50mg of PAMAM solution dropwise, the mixture is stirred at room temperature for reaction overnight, dialysis is carried out for 24 hours, and the PAMAM-FA is obtained after freeze drying.
2.3 Synthesis of PAMAM-SeSe-FA
20mg of the PAMAM-FA solution synthesized in 2.2 was taken in water, 18mg of EDC and 16.5NHS were added to the PAMAM-FA solution, stirred at room temperature for 4 hours, and then added dropwise to NH 2 -PEG-SeSe-NH 2 In the aqueous solution, the reaction was stirred at room temperature overnight. Dialyzing for 24 hours, and freeze-drying to obtain PAMAM-SeSe-FA.
(3) Preparation of nano carrier BP@PAMAM-SeSe-FA
0.5mL of 750. Mu.g/mL BPPs-BPS solution was centrifuged at 6000rpm for 30min, and the supernatant was removed and resuspended in 2mL of water. 10mg of PAMAM-SeSe-FA was added thereto, followed by sonication for 30 minutes and stirring for reaction for 6 hours. After the reaction was completed, the mixture was centrifuged (6000 rpm,30 min) and resuspended in 2mL of water for further use.
Characterization and testing:
characterization of topography
Fig. 1 is a transmission electron microscope image of black phosphorus nanoplatelets BPNPs obtained in the step (1) and finally synthesized nano carriers bp@pamam-se-FA, and it can be seen from the image that each group of black phosphorus nanoplatelets have a lamellar structure, the particle size concentration is about 200nm, and the lamellar structure of black phosphorus is not affected by modification of polymers.
Zeta potential test
FIG. 2 shows the results of Zeta potential tests of synthesized BPNPs, PAMAM-SeSe-FA, BP@PAMAM-SeSe-FA, and BP@PAMAM-SeSe-FA/shCRNDE samples loaded with genes by uniformly mixing BP@PAMAM-SeSe-FA aqueous solution with the genes and then complexing for 0.5h at room temperature to obtain BP@PAMAM-SeSe-FA/shCRNDE complex; the result shows that the potential of the BPNPs is-15.3 mV, and the potential of the BP@PAMAM-SeSe-FA after the PAMAM-SeSe-FA is modified is +18mV; the positive charge of BP@PAMAM-SeSe-FA is slightly reduced compared with that of PAMAM-SeSe-FA, because black phosphorus is negatively charged, and PAMAM-SeSe-FA is positively charged, and electrostatic interaction is carried out to obtain BP@PAMAM-SeSe-FA, so that the positive charge of BP@PAMAM-SeSe-FA is slightly reduced, but the positive charge is still kept at about +18mV, and the potential of BP@PAMAM-SeSe-FA/shCRNDE is +13.4mV after the genes are loaded, so that the nano system can well load the genes.
Cytotoxicity of cells
The cytotoxicity of the nanocarrier BP@PAMAM-SeSe-FA on BEL-7402 cells was evaluated by using a method of detecting the cell activity with CCK-8. The specific operation steps are as follows: BEL-7402 cells were first seeded into 96-well plates at a density of 5000 cells/well, and then placed in a carbon dioxide incubator for culture adherence overnight. Subsequently, the original medium was aspirated and replaced with fresh complete medium containing BP@PAMAM-SeSe-FA at different concentrations, selected from the range of BP@PAMAM-SeSe-FA at 5-300. Mu.g/mL, 5 in parallel. The cells were then incubated in an incubator for 24h, washed once with PBS and 100. Mu.L of fresh medium (containing 10% CCK-8) was added to each well. Incubation in incubator for a period of time, and finally detection and recording of absorbance at 450nm wavelength using a microplate reader, cell viability was calculated by the following formula: cell viability (%) = (experimental group absorbance-blank group absorbance)/(negative control group absorbance-blank group absorbance) ×100%. As shown in FIG. 3, the cell viability of BEL-7402 cells was 102.5% at a BP@PAMAM-SeSe-FA nanoparticle concentration of 10. Mu.g/mL, and it was found that the low concentration of black phosphorus nanoparticles did not affect cell proliferation. In addition, with increasing concentration of black phosphorus nanoparticles, the survival rate of BEL-7402 cells was slightly decreased but still kept high, and when the concentration of BP@PAMAM-SeSe-FA nanoparticles was 100. Mu.g/mL, the survival rate of cells was 97.4% in turn. When the concentration of BP@PAMAM-SeSe-FA nanoparticles is as high as 300 mug/mL, the cell survival rate of cells is still more than 90%, and even though the black phosphorus nanoparticles are very high, the cytotoxicity is still very low, which indicates that the nano-carrier provided by the embodiment of the invention has lower cytotoxicity.
Photothermal properties
The resulting nanocarriers BP@PAMAM-SeSe-FA were dispersed in water to prepare solutions of different concentrations (40, 80, 160 and 320. Mu.g/mL). At room temperature, 1mL of the above solutions with different concentrations are respectively added into a cuvette, a thermocouple thermometer is inserted, and the power is 1.5W/cm 2 The liquid surface was irradiated with near infrared light of 808nm for 5min, and the temperature was recorded every 10 s. And (3) plotting by taking time as an abscissa and temperature as an ordinate, and comparing the in-vitro photo-thermal effects of materials with different concentrations. Meanwhile, deionized water is used as a control group and recorded by an infrared thermal imager. Fig. 4 shows time-temperature curves of materials at different concentrations. It can be seen from the graph that the higher the concentration, the more pronounced the temperature rise. At a material concentration of 50. Mu.g/mL, the temperature was raised to 48.3℃over 5 min. The nano-carrier of the embodiment of the invention has good photo-thermal performance.
Inhibition of cell proliferation in vitro
In order to explore cytotoxicity of the nano gene vector in the embodiment of the invention under the conditions of genes and light and heat, liver cancer cells BEL-7402 are selected, 5 groups of experimental controls are established, and the following nano particles are respectively added to each group of cells for co-culture: shCRNDE, BP@PAMAM-SeSe-FA, BP@PAMAM-SeSe-FA+NIR, BP@PAMAM-SeSe-FA/shCRNDE+NIR, BP@PAMAM-SeSe-FA/shCRNDE, wherein the preparation of the loaded gene sample is as described above, followed by PBS set as control, wherein NIR stands for treatment with 808nm NIR near infrared laser irradiation, laser power density is 1.5W/cm 2 The illumination time was 5min, and the gene amount per well was 0.5. Mu.g. After the cells of each group are co-cultured for 24 hours under the same condition, the cell activity is detected by using a CCK-8 method cell proliferation detection kit. The results are shown in FIG. 5, in which 1.PBS,2.shCRNDE,3.BP@PAMAM-SeSe-FA,4.BP@PAMAM-SeSe-FA+NIR,5.BP@PAMAM-SeSe-FA/shCRNDE,6.BP@PAMAM-SeSe-FA/shCRNDE+NIR。
The result shows that the cell viability of the single material BP@PAMAM-SeSe-FA is not obviously different from that of the PBS group, the cell viability of the BP@PAMAM-SeSe-FA+NIR group is 65.3%, the cell viability of the BP@PAMAM-SeSe-FA/shCRNDE group is 50.9%, and the cell viability of the BP@PAMAM-SeSe-FA/shCRNDE+NIR group is 20.9%, so that the nano-carrier can kill liver cancer cells more only by combining genes with light and heat.
In conclusion, the targeting nano-carrier provided by the invention takes black phosphorus as a core skeleton, and has a photo-thermal effect due to the excellent optical property; the modified polymer PMMA is modified by coating, so that the carrier has rich positive charges, and the capability of carrying negatively charged functional genes such as shCRNDE genes is improved; the diselenide bond has GSH responsiveness, so that the modified polymer PAMAM with GSH responsiveness is coated, thereby preventing black phosphorus from degradation and improving the stability of the carrier; meanwhile, FA is modified into a guide molecule targeting the FA receptor on the liver cancer cell, so that enrichment of the nano material in tumor cells is promoted. When the nano-carrier is applied, the combination of gene and photothermal therapy can be realized, so that the high-efficiency combined treatment of liver cancer is realized, and a new idea is provided for the high-efficiency treatment of liver cancer.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (7)

1. The preparation method of the targeting nano-carrier comprises a black phosphorus nano-sheet and a modified PAMAM coated on the surface of the black phosphorus nano-sheet, wherein the modified PAMAM contains a diselenide bond and is modified with FA folic acid molecules; the particle size of the nano carrier is 200nm, and the Zeta potential is 18mV; the preparation method is characterized by comprising the following steps:
(1) Preparing black phosphorus nano-sheet BPNPs: dispersing black phosphorus in pure water, stirring, degassing by flowing argon, then carrying out ultrasonic treatment on the solution under ice bath conditions to obtain brown dispersion, centrifuging the brown dispersion, collecting supernatant and further centrifuging the collected supernatant to obtain black phosphorus nanoplatelets BPNPs;
(2) Preparing PAMAM-se-FA comprising:
2.1 Synthesis of NH 2 -PEG-SeSe-NH 2 : dissolving selenocysteine hydrochloride in water, adding EDC and NHS into selenocysteine hydrochloride solution, and stirring for reaction; then drop wise add to NH 2 Stirring and reacting in PEG-COOH solution at room temperature, dialyzing, and freeze-drying to obtain NH 2 -PEG-SeSe-NH 2 ；
2.2 Dissolving FA in DMSO, adding EDC and NHS into the FA solution, stirring at room temperature, adding the mixture dropwise into the PAMAM solution, stirring at room temperature for reaction, dialyzing, and lyophilizing to obtain PAMAM-FA;
2.3 adding EDC and NHS to the PAMAM-FA solution in water, stirring at room temperature, and adding dropwise to NH 2 -PEG-SeSe-NH 2 Stirring and reacting in water solution at room temperature, dialyzing, and freeze-drying to obtain PAMAM-SeSe-FA;
(3) Preparation BP@ PAMAM-SeSe-FA:
preparing a BPNPs aqueous solution, adding PAMAM-SeSe-FA for mixing, and performing ultrasonic and stirring reaction to obtain the BP@ PAMAM-SeSe-FA nano-carrier, namely the targeting nano-carrier.
2. The preparation method according to claim 1, wherein the mass-volume ratio of black phosphorus to pure water in the step (1) is 10-30 mg: 20-80 ml; the ultrasonic time is 6 h-24 h; the centrifugal speed of the brown dispersion is 500 rpm-1000 rpm; the collected supernatant has a centrifugal speed of 2000rpm to 4000rpm.
3. The method according to claim 1, wherein,
in the step 2.1, the raw material ratio is selenocysteine hydrochloride: deionized water: EDC: NHS: NH (NH) 2 -PEG-COOH = 5-15 mg: 2-8 ml: 5-25 mg: 5-20 mg: 5-50 ml; the reaction time of EDC and NHS with selenocysteine hydrochloride is 1-6 h; selenocysteine hydrochloride solution and NH 2 The reaction time of PEG-COOH is 6-24 h;
in the step 2.2, the mass ratio of FA to PAMAM is 1-10: 30-70 parts.
4. The method according to claim 1, wherein in the step (3), the mass ratio of BPNPs to PAMAM-se-FA is 0.1 to 0.5:5 to 15; the ultrasonic time is 10-40 min; the reaction time is 2-8 h.
5. The method of claim 1, wherein step (1) further comprises re-suspending the obtained BPNPs in a PBS solution to obtain a BPNPs-PBS solution, and then storing at 4 ℃.
6. The method according to claim 5, wherein in the step (3), the preparation of the aqueous solution of BPNPs is carried out by centrifuging the solution of BPNPs-PBS, removing the supernatant and then re-suspending in water.
7. The method according to claim 6, wherein the centrifugal speed is 4000rpm to 8000 rpm.
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