Preparation method and application of FD / USCZA nanoparticles with double-sided structure

Abstract

The invention discloses a preparation method and application of FD / USCZA nanoparticles with a double-sided structure, and belongs to the field of nano drug delivery systems. By means of up / down conversion nanoparticles D / UCNRs, the guiding effect of polyacrylic acid is utilized, a selective growth and modification method is combined to develop the simply prepared monodisperse double-sided structure FD / USCZA nanoparticles, and the obtained product is good in dispersity, uniform in particle size and good in biocompatibility and has very wide application prospects in the fields of drug delivery and the like. A zeolite imidazate framework material (ZIF-8) is used for wrapping ARV-771 organic molecules, DNA self-repairing related protein in tumor cells is degraded to enhance photodynamic therapy, and evaluation of the therapeutic effect is implemented. According to the invention, BRD4 inhibition and photodynamic therapy are combined to enhance oxidation therapy, and an effective strategy is provided to improve the curative effect of PDT.

Description

Technical Field

[0001] The invention relates to the field of nano drug delivery systems, and in particular to a preparation method and application of FD / USCZA nanoparticles with a Janus structure. Background Art

[0002] Photodynamic therapy (PDT) is a method that uses light of a specific wavelength to activate photosensitizers, causing them to transition from the ground state to the excited state and release reactive oxygen or other free radicals, inducing DNA double-strand breaks and initiating cell apoptosis. PDT has been widely used in the treatment of clinical malignant tumors due to its high selectivity, low invasiveness, rapid effect, few side effects, no obvious drug resistance, and easy combination with other treatment methods. However, the excitation wavelength of most existing photosensitizers is in the range of 630-690nm, which limits their application in deep tissues. In order to promote the application of photosensitizers in deep tissues, suitable transducers can be introduced to convert near-infrared light with strong penetration into visible light. Recent studies have found that DNA repair mechanisms may affect its efficiency. For example, bromodomain protein 4 (BRD4) participates in the occurrence and development of tumors by regulating non-transcriptional pathways such as DNA damage repair and telomere homeostasis to maintain genome stability. Therefore, combining BRD4 inhibition with photodynamic therapy (PDT) may be a promising strategy to enhance oxidative therapy. This combination may provide an effective strategy to improve the efficacy of PDT. Among them, protein degradation targeting chimeras (PROTACs) have attracted much attention because of their ability to regulate proteins that are difficult to target with traditional small molecules. Despite the above advantages of PROTACs, there are still some obstacles that hinder their clinical transformation. For example, PROTACs exhibit inefficient tumor accumulation due to their suboptimal pharmacokinetics and systemic side effects caused by target protein degradation. Therefore, it remains crucial to develop new prodrug delivery strategies to achieve efficient accumulation and in vivo activation of PROTACs at tumor sites to eliminate the potential nonspecific toxicity of PROTACs. Summary of the invention

[0003] The purpose of the present invention is to provide a preparation method and application of Janus-structured FD / USCZA nanoparticles to solve the problems existing in the above-mentioned prior art. The nanoparticles can be used to improve the efficiency of photodynamic therapy and the accuracy of tumor treatment.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a method for preparing Janus-structured FD / USCZA nanoparticles, comprising the following steps:

[0006] Adding chlorin E6 silane precursor to the modified D / UCNRs solution, adjusting the pH to alkaline, and then adding tetraethyl orthosilicate to obtain chlorin E6 modified D / UCNRs core-shell nanoparticles;

[0007] Dissolving zinc oxide in water, adding polyacrylic acid aqueous solution, and ultrasonicating to obtain polyacrylic acid-zinc solution; dropping the polyacrylic acid-zinc solution into the dihydrochlorin E6-modified D / USC core-shell nanoparticles, and ultrasonicating, and then slowly dropping isopropanol into the mixed solution for reaction to obtain D / USCPZ nanoparticles;

[0008] Adding ARV-771 and 2-methylimidazole to the D / USCPZ nanoparticles, stirring and reacting, to obtain D / USCZA nanomaterials;

[0009] After activating folic acid, the folic acid and D / USCZA nanomaterials are shaken to react to obtain Janus-structured FD / USCZA nanoparticles.

[0010] Preferably, the preparation method of the modified D / UCNRs comprises the following steps:

[0011] After mixing and stirring sodium hydroxide solution, oleic acid and ethanol, rare earth chloride mixed solution is added dropwise to the mixed solution, and finally ammonium fluoride solution is added, reacted at 210°C for 8 hours, cooled naturally and centrifuged, and washed alternately with anhydrous ethanol and water for 1-3 times, dispersed in chloroform, to obtain D / UCNRs nanoparticles;

[0012] The D / UCNRs nanoparticles and CTAB were stirred at 32° C. for 30 minutes, and then the temperature was raised to 65° C. to volatilize the chloroform in the solution, thereby obtaining CTAB-protected D / UCNRs nanoparticles, namely, modified D / UCNRs.

[0013] Preferably, the volume ratio of the sodium hydroxide solution, oleic acid, ethanol, rare earth chloride mixed solution and ammonium chloride solution is 1.5:5:5:2:1, the concentration of the sodium hydroxide solution is 0.2 g / mL, and the concentration of the ammonium chloride solution is 2 mol / L;

[0014] The rare earth chloride mixed solution is prepared by mixing yttrium chloride hexahydrate, ytterbium chloride hexahydrate, gadolinium chloride hexahydrate, erbium chloride hexahydrate and water in a mass volume ratio of 243 g:279 g:892 g:31 g:2 mL;

[0015] The volume ratio of the D / UCNRs nanoparticles to CTAB is 1:20, the concentration of the D / UCNRs nanoparticles is 10 mg / mL, and the concentration of the CTAB is 2M.

[0016] Preferably, the dihydrochlorin E6 silane precursor is obtained by dissolving dihydrochlorin E6 in DMSO, adding 3-aminopropyltriethoxysilane, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to react at room temperature in the dark for 12 hours; the ratio of the dihydrochlorin E6, DMSO, 3-aminopropyltriethoxysilane, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 2.2 mg: 0.5 mL: 12 μL: 6 mg: 4 mg.

[0017] Preferably, water is added to the modified D / UCNRs and ultrasonically dispersed to form a modified D / UCNRs solution; then, dihydrochlorin E6 silane precursor is added to the modified D / UCNRs solution, and after the pH is adjusted to 8, an ethanol solution of tetraethyl orthosilicate is added in 4-6 times, each time interval is 30 minutes; the ratio of the modified D / UCNRs solution, dihydrochlorin E6 silane precursor and the ethanol solution of tetraethyl orthosilicate is 20:0.03:0.25.

[0018] Preferably, the volume ratio of the aqueous solution of zinc oxide to the aqueous solution of polyacrylic acid is 100:1, the concentration of the aqueous solution of zinc oxide is 1 mg / mL, and the concentration of the aqueous solution of polyacrylic acid is 0.2 g / mL;

[0019] The volume ratio of the polyacrylic acid-zinc solution, the D / USC core-shell nanoparticles modified with dihydrochlorin E6 and isopropanol is 1:5:42; the ultrasonic time is 20-30 minutes, and the reaction condition is 32° C. for 12 hours.

[0020] Preferably, after dispersing the D / USC-PAA-Zn nanoparticles in methanol, adding a methanol solution of 2-methylimidazole and ARV-771, stirring and reacting at 70° C. for 30 minutes, the D / USCZA nanomaterial is obtained; the volume ratio of the methanol, the methanol solution of 2-methylimidazole and ARV-771 is 100:31:1, and the concentration of the methanol solution of 2-methylimidazole is 0.97 mg / mL.

[0021] Preferably, the method for activating folic acid comprises the following steps: placing a folic acid solution in DMSO, sonicating, then adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and activating for 12 hours; wherein the ratio of folic acid, DMSO, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 10 mg: 8 mL: 9 mg: 5 mg;

[0022] The shaking reaction time is 48 hours.

[0023] The invention also provides a Janus-structured FD / USCZA nanoparticle, which is prepared by the preparation method.

[0024] The present invention also provides the use of the Janus-structured FD / USCZA nanoparticles in any of the following items:

[0025] (1) Application in the preparation of drugs for treating breast cancer;

[0026] (2) Application in the preparation of drugs for increasing the sensitivity of breast cancer cells to photodynamic therapy;

[0027] (3) Application in the preparation of drugs for improving the efficacy of photodynamic therapy for breast cancer.

[0028] The present invention discloses the following technical effects:

[0029] (1) The present invention uses up / down conversion nanoparticles (D / UCNRs), utilizes the guiding effect of polyacrylic acid, and combines the selective growth and modification method to develop a simple preparation method for monodisperse Janus structure FD / USCZA nanoparticles. The obtained product has good dispersibility, uniform particle size, good biocompatibility, and has very broad application prospects in the fields of drug delivery. Zeolite imidazolate framework material (ZIF-8) is used to encapsulate ARV-771 organic molecules, enhance photodynamic therapy by degrading DNA self-repair-related proteins in tumor cells, and implement treatment effect evaluation; FD / USCZAs passively target mouse breast cancer cells after intravenous injection and release ARV-771 in tumor cells. ARV-771 can inhibit the expression of BRD4 protein. Downregulation of BRD4 protein expression inhibits the expression of RAD51 protein. Downregulation of these two proteins can hinder homologous recombination-mediated DNA repair, thereby causing DNA damage. The present invention combines BRD4 inhibition with photodynamic therapy (PDT) to enhance oxidative therapy, aiming to provide an effective strategy to improve the efficacy of PDT.

[0030] (2) The Janus-structured FD / USCZA nanoparticles prepared by the present invention have the following characteristics and advantages:

[0031] Blood circulation and targeting: The Janus structure reduces the adsorption of nanodrugs in the liver, increases the blood circulation time of nanodrugs and prolongs the circulation time of nanoparticles in the blood;

[0032] Drug delivery and release: Zeolite imidazolate skeleton-8 can protect drug molecules and prevent them from degradation in the environment, thereby maintaining the activity of the drug and improving the stability of the drug. Folic acid can target the folate receptors highly expressed on the cancer cell membrane, improving the targeting of nanomedicines;

[0033] Improving the sensitivity of PDT treatment: Introducing the PROTAC drug ARV-771 to degrade BRD4 protein and increase the sensitivity of tumor cells to PDT treatment;

[0034] Tissue penetration: Upconversion nanomaterials can respond to 980nm lasers with deeper penetration depth and less toxic side effects, and activate photosensitizers to achieve the effect of treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 The construction method and mechanism of action of Janus PROTAC nanodrug (FD / USCZA);

[0037] Figure 2 is the characterization of FD / USCZA; wherein, A is the near-infrared spectrum of D / UCNRs, B is the ultraviolet absorption spectrum of FD / USCZA, C is the Fourier transform infrared spectrum of FD / USCZA, D is the zeta potential of FD / USCZA, and E is the transmission electron microscope image of FD / USCZA;

[0038] Figure 3 This is the in vitro toxicity evaluation result of FD / USCZA; A is the light-controlled production of FD / USCZA 1 O2 schematic diagram, B is the fluorescence spectrum of FD / USCZA and the UV-visible absorption spectrum of Ce6, C is the DPBF measurement of singlet oxygen production of FD / USCZA nanoprobe, D is the electron paramagnetic resonance (EPR) spectroscopy evaluation of DMPO and 980nm laser after reaction with nanodrugs, and E is the light control of 4T1 cells treated with FD / USCZA 1 O2 production, F is the cytotoxic effect of different treatment groups on cells;

[0039] Figure 4 is the result diagram of the DNA damage mechanism mediated by FD / USCZA; wherein, A is the action mechanism diagram; B is the representative Western blot of BRD4, RAD51 and γH2AX expression under different conditions, CE is the representative immunoblot and quantitative analysis of BRD4, RAD51 and γH2AX in 4T1 cells (n=3), and F is the immunofluorescence image of γH2AX in 4T1 cells;

[0040] Figure 5The figure is the result of the anti-tumor animal experiment of FD / USCZA; A is the schematic diagram of chemotherapy / photodynamic therapy mediated by FD / USCZA nanomedicine, B is the real photo of tumor growth on the 0th and 16th day of mouse treatment, C is the representative photo of in vitro dissected tumor, D and E are the curves of the change of mouse body weight and tumor volume after different drug treatments, and F is the NIR-II FL image of 4T1 tumor-bearing mice treated with FD / USCZA via tail vein injection at different time points;

[0041] In the above figures, I: PBS+980nm represents PBS+light; II: FD / USCZ represents treatment with FD / USCZ nanoparticles without drug loading; III: treatment with free ARV-771 injected into the tail vein; IV: FD / USCZA represents FD / USCZ nanoparticles loaded with drug ARV-771; V: FD / USCZ+980nm represents treatment with FD / USCZ nanoparticles without drug loading and light; VI: FD / USCZA+980nm: treatment with FD / USCZ nanoparticles loaded with drug ARV-771 and light. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The materials and instruments used in the following examples are all commercially available. Table 1 lists the main reagents and materials used in the following examples, and Table 2 lists the main instruments and equipment used in the following examples.

[0048] Table 1 Main reagents and materials required for the examples

[0049]

[0050]

[0051] Table 2 Main instruments and equipment required for the examples

[0052]

[0053] In view of the problems of insufficient light penetration depth in traditional PDT and limited therapeutic effect of DNA self-repair mechanism, the present invention proposes to increase the penetration depth by using upconversion light to activate photosensitizer, monitor the distribution of drugs in vivo in real time by NIR-II fluorescence, and use targeted protein degradation technology (PROTACs) to degrade BRD4 protein, thereby increasing the sensitivity of tumor cells to PDT treatment. Combined with JNPs nanoparticle delivery system, the efficiency of PDT in vivo is synergistically improved. The present invention specifically provides a Janus (Janus) PROTAC nanodrug for precise protein degradation enhanced photodynamic therapy, i.e., FD / USCZA nanoparticles, and its preparation method and application are further described below with specific examples.

[0054] Example 1 Preparation method of FA-D / USCZ nanoparticles (FD / USCZA)

[0055] 1. Preparation of D / UCNRs

[0056] Preparation of D / UCNRs includes the following steps: first, dissolving 0.3 g NaOH in 1.5 mL of deionized water, then adding 5 mL OA and 5 mL C2H5OH in sequence, and stirring for 30 minutes. Then, dissolving 0.0243 g YCl3·6H2O, 0.0279 g YbCl3·6H2O, 0.0892 g GdCl3·6H2O, and 0.0031 g ErCl3·6H2O in 2 mL of deionized water, first obtaining a rare earth chloride mixed solution, and then adding it dropwise to the solution in the previous step. Finally, adding 1 mL of 2 mol / L NH4F solution, transferring to a 20 mL reactor, tightening the reactor and placing it in an electric heated blast drying oven, reacting at 210°C for 8 hours. After heating, let it cool naturally to room temperature, then centrifuge the product at 8000 rpm for 10 minutes, wash it alternately with anhydrous ethanol and deionized water for 3 times, and disperse it in chloroform for later use.

[0057] 2. Preparation of D / USC core-shell nanoparticles

[0058] The preparation steps of D / USC-Ce6 nanodevices are as follows:

[0059] (1) Preparation of Ce6-silane precursor: Weigh 2.2 mg of dihydrochlorin E6 (Ce6) dye molecules and dissolve them in 0.5 mL of DMSO solution. Then add 12 μL of 3-aminopropyltriethoxysilane (APTMS), 6 mg of EDC-HCl and 4 mg of NHS and react for 12 hours in the dark. Then store at 4°C.

[0060] (2) Synthesis of hexadecyltrimethylammonium bromide (CTAB)-protected D / UCNRs: 400 μL of 10 mg / mL D / UCNRs nanoparticles protected by oleic acid were stirred with 8 mL of 0.2 M CTAB at 32 °C for 30 min, and then the temperature was raised to 65 °C to evaporate the chloroform in the solution to obtain CTAB-protected D / UCNRs nanoparticles.

[0061] (3) Preparation of D / USC: Add 20 mL of deionized water to the CTAB-protected D / UCNRs nanoparticles obtained in step (2), and then disperse them evenly by ultrasound. Add 30 μL of the Ce6-APTMSDMSO solution prepared in step (1). After that, adjust the pH value of the solution to 8 (the alkaline solution is 0.1 M NaOH), and then add 250 μL of 20% tetraethyl orthosilicate (TEOS) ethanol solution in 5 times (each time with an interval of 30 minutes), and stir at 32°C for 24 hours. Collect the product by centrifugation and wash it repeatedly with ultrapure water. The obtained Ce6-modified D / USC core-shell nanoparticles (D / USC) are dispersed in 1.6 mL of pure water for use (2.5 mg / mL).

[0062] 3. Preparation of D / USCPZ Janus-structured nanoparticles

[0063] (1) Preparation of PAA-Zn: 1 mg ZnO was added to 2 mL deionized water at room temperature and ultrasonicated for 30 minutes to form a white suspension. 20 μL 0.2 g / mL polyacrylic acid (PAA) solution was added to the suspension under ultrasonication and ultrasonically dispersed for 45 minutes until a transparent solution was obtained, which was the PAA-Zn solution.

[0064] (2) Then, 0.5 mL of PAA-Zn solution was added dropwise to 0.25 mL of 2.5 mg / mL D / USC-Ce6 solution under shaking, followed by ultrasonic reaction for 30 minutes. 21 mL of isopropyl alcohol (IPA) was slowly dripped into the mixed solution and reacted at 32°C for 12 hours to obtain D / USCPZ nanoparticles.

[0065] (3) Preparation of D / USCZA Janus-structured nanoparticles

[0066] First, 0.725 mg of 2-methylimidazole (2-MIM) was dissolved in 0.775 mL of CH3OH, and 25 μL of 1 mg / mL ARV-771 (an effective BET degrader of PROTAC, with a chemical formula of C 49 H 60 ClN9O7S2) to obtain a mixed solution. Subsequently, D / USCPZ was dispersed in 2.5 mL CH3OH, and the mixed solution was added under stirring conditions. After stirring at 70°C for 30 minutes, stirring was stopped and the reaction was carried out in the dark for 12 hours to obtain the final product, D / USCZA Janus-structured nanoparticles.

[0067] 4. Preparation of FA-modified Janus nanoparticles

[0068] 25 mg of FA was dissolved in 20 mL of DMSO, and the mixture was sonicated for 45 min. Subsequently, 22.5 mg of NHS and 12.5 mg of EDC-HCl were added to activate the carboxyl groups of FA for 12 h. The mixture was filtered and transferred to a brown flask and stored at 4 °C for further use. Then, 1 mL of D / USCZA was added to the above solution and shaken for 48 h to covalently bond the amino groups of SiO2 with the carboxyl groups of FA to obtain the final product FA-D / UCSZA nanoparticles, i.e., FD / USCZA Janus nanoparticles.

[0069] The precise synthesis process and mechanism of action of FD / USCZA Figure 1 First, D / UCNRs were synthesized according to the above method. Figure 2As shown in Figure A, under 980nm laser irradiation, D / UCNRs emit at 550nm and 660nm, which can verify that D / UCNRs act as a converter of near-infrared light to visible light. In addition, D / UCNRs have a clear near-infrared second region (NIR-II) fluorescence emission peak with an emission wavelength of about 1550nm. This shows that D / UCNRs have good NIR-II fluorescence imaging capabilities and can be used for in vivo imaging. Next, the present invention adds TEOS, APTES and Ce6 to the above solution, and Methods Ce6-modified D / USC core-shell nanoparticles were prepared. Then, by controlling the ratio of isopropanol to water, ZIF-8 was selectively generated on one side by changing the interfacial energy of the D / USC surface. In order to achieve the targeting ability of JNP to 4T1 cells, FA ( Figure 1 ). Folate-targeted cancer therapy can deliver therapeutic drugs specifically to tumor cells with high expression of folate receptors. Figure 2 As shown in Figure B, the spectrum of FD / USCZA was studied and obvious characteristic peaks were found at 404 nm and 660 nm from Ce6, indicating that Ce6 was successfully loaded into the nanoparticles. The UV absorption spectrum of FD / USCZA has characteristic absorption peaks of FA at 283 and 352 nm. The Fourier transform infrared spectrum (FTIR) of FD / USCZA shows that the amide bond is at 1650 cm -1 The carbonyl stretching (blue line) of FD / USCZA is formed at 1701 cm -1 disappeared, indicating that FA was successfully coupled with D / USCZA ( Figure 2 C). Figure 2 As shown in Figure 2, the zeta potential of the resulting FD / USCZA was measured to monitor the progress of each step of the multilayer functionalization. The successful synthesis of the asymmetric FD / USCZA structure was confirmed by transmission electron microscopy images ( Figure 2 Middle E).

[0070] Example 2 Evaluation of the efficacy of FD / USCZA

[0071] 1. Through fluorescence spectrophotometry experimental analysis, it can be concluded that after 980nm laser excitation, the emission spectrum of FD / USCZA is highly matched with the absorption spectrum of Ce6, which can be used to generate ROS for PDT ( Figure 3 (A). Figure 3 As shown in B, in order to further verify 1 Photo-controlled generation of O2 using TEMP as 1 The formation of stable spin adducts can be followed by EPR measurement. 1 The generation of O2. Figure 3As shown in C, the FD / USCZA nanoprobe mixture has the ability to generate 1 O2's capabilities.

[0072] In order to investigate whether the tumor cells treated with FD / USCZA nanoprobes can produce 1 O2 was measured using the reactive oxygen species detection fluorescent dye DCHF-DA. First, after incubating FD / USCZA with 4T1 cells, a 980nm laser was used to illuminate the cells for 2 minutes at intervals of 2 minutes in the illumination group, and the illumination time for each well was 20 minutes. Then the dye DCHF-DA was added, and finally confocal fluorescence imaging of the above-treated cells was performed. Figure 3 As shown in D, in the absence of light, the tumor cells show almost no green fluorescence; in the case of light, they clearly show bright green fluorescence. This experimental result shows that under the condition of laser irradiation, the FD / USCZA constructed by the present invention can produce more reactive oxygen after entering the cells and being activated at 980nm.

[0073] 2. Based on the singlet oxygen production analysis results of FD / USCZA, FD / USCZA was co-cultured with mouse breast cancer cells (4T1), and the cytotoxicity of FD / USCZA was verified by cell cloning experiments and MTT method. In the absence of light, there were almost no dead cells in the PBS group and the FD / USCZ group; under light conditions, there were almost no dead cells in the PBS+980nm group, indicating that the effect of light on cells was negligible; while the number of dead cells in the FD / USCZ+980nm experimental group increased significantly, further indicating that after the FD / USCZA nanoprobe was incubated with cells, the therapeutic effect of tumor cells could be enhanced under light conditions. The FD / USCZA+980nm group had the greatest toxicity to cells, with a cell survival rate of about 9.14%. This proves that the cell survival rates of the single nanodrug chemotherapy group and the photodynamic therapy group were significantly higher than those of the combined treatment group.

[0074] Example 3 Study on the anti-tumor mechanism of FD / USCZA

[0075] After adding 200 μL of 2.5 mg / mL FD / USCZA to the cells, the cells were incubated for 12 hours. The cells were irradiated with a 980 nm laser for 2 minutes at intervals of 2 minutes in the illumination group, and the illumination time for each well was 20 minutes. The cells were washed 3 times, cultured in fresh medium for 12 hours, and then collected for Western blot analysis. Using β-actin as an internal reference, the expression of key proteins BRD4, RAD51, and γH2AX in the DNA repair pathway were analyzed by FD / USCZA. The experimental results are shown in Figure 2. Figure 4As shown in the results of western blot, compared with the PBS group (negative control group), it can be seen that the cells loaded with ARV-771 and FD / USCZA combined with light treatment showed lower BRD4 and RAD51 levels. In addition, after light treatment, the DNA damage-related protein γH2AX in the FD / USCZA experimental group was significantly upregulated. After normalization of the data and correlation analysis, Figure 4 It can be seen from CE that the presence of ARV-771 can effectively reduce the expression levels of BRD4 and RAD51 in cells. This is because ARV-771-mediated BRD4 deletion significantly inhibits the expression of RAD51. Downregulation of RAD51 will hinder homologous recombination-mediated DNA repair. The amount of γH2AX increases and persists after illumination, confirming that the combined effect of PDT and DNA repair inhibition enhances DNA damage. Immunofluorescence staining was used to detect the expression of γH2AX protein in the cell nucleus ( Figure 4 Middle F) Results showed that the level of γH2AX in cells in the light-irradiated group increased significantly; among them, the light-treated ARV-771 loaded with FD / USCZA had the strongest red fluorescence, which once again confirmed the inhibitory effect on DNA damage repair. In summary, ARV-771-mediated BRD4 degradation hindered DNA damage repair, thereby destroying the cell self-repair system and enhancing PDT.

[0076] Example 4 In vivo tumor imaging and anti-tumor assay in FD / USCZA animals

[0077] Mice were randomly divided into 4 groups (n=4), including (1) intravenous injection of normal saline (100 μL) and light irradiation (light group); (2) intravenous injection of FD / USZ (6.25 mg / kg); (3) intravenous injection of ARV-771 (3 mg / kg); (4) intravenous injection of FD / USCZA (6.25 mg / kg); (5) intravenous injection of FD / USZ (6.25 mg / kg) and light irradiation; (6) intravenous injection of FD / USCZA loaded with ARV-771 (6.25 mg / kg) and light irradiation. The light irradiation of groups 5 and 6 was (2.5 W / cm 2 , 20 minutes) was performed after intravenous injection of nanoparticles within 24 hours. 2 The mice in group (1) treated with 4% paraformaldehyde (20 minutes) were used as negative controls. The tumor volume and mouse body weight were measured every other day for 15 days. During the 16-day treatment period, the changes in mouse body weight and tumor volume were recorded, and digital photos were taken to record the actual tumor growth of mice on day 0 and day 16 of treatment (see the flowchart). Figure 5 (shown in A).

[0078] like Figure 5As shown in Figure BE, during the drug administration period, there was no significant change in the body weight of all mice, indicating that the drug had no toxic side effects. However, during the 16-day treatment period, there were significant differences in the changes in tumor volume between the drug-treated groups and the control group. The control group (1) had a nearly 15-fold increase in tumor volume over 16 days. The combined treatment group (5) began to show anti-tumor growth effects on day 4, and the tumor volume on day 16 was significantly smaller than the initial tumor (day 0), while the tumor volume of PBS-treated mice increased significantly. In the absence of light, the tumor growth of Ce6-treated mice was similar to that of PBS-treated mice, which was due to the low dark toxicity of Ce6. Interestingly, under light, Ce6 was able to inhibit tumor growth to a certain extent, verifying the light controllability of PDT. In addition, JNPs showed better anti-tumor effects compared with Free ARV-771, which was due to the improved drug delivery efficiency of FD / USCZA. After light exposure, FD / USCZA was shown to be the most effective treatment, resulting in superior tumor growth inhibition than other groups by improving drug delivery efficiency and enhancing PDT efficacy. To study the accumulation and retention of tumors in vivo, tumor-bearing BALB / c nude mice (n=3) were injected with FD / USCZA aqueous solution via the tail vein after anesthesia. Fluorescence images were captured at different times after injection (0, 1, 6, 12, 24, and 48 hours, respectively). The excitation wavelength was 980 nm and the collection range was 1400-1600 nm. Figure 5 As shown in Figure F, FD / USCZA began to show near-infrared fluorescence 12 hours after injection. The strong fluorescence emission of FD / USCZA at the tumor site was retained for at least 24 hours after injection, indicating that FD / USCZA has a significant long-term tumor retention effect.

[0079] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing Janus-structured FD / USCZA nanoparticles, characterized in that: The following steps are involved: Chlorin E6 silane precursor is added to the modified D / UCNRs solution, and tetraethyl orthosilicate is added after the pH is adjusted to be alkaline, thereby obtaining D / UCNRs core-shell nanoparticles modified with chlorin E6; Dissolving zinc oxide in water, adding polyacrylic acid aqueous solution, and ultrasonicating to obtain polyacrylic acid-zinc solution; dropping the polyacrylic acid-zinc solution into the dihydrochlorin E6-modified D / USC core-shell nanoparticles, and ultrasonicating, and then slowly dropping isopropanol into the mixed solution for reaction to obtain D / USCPZ nanoparticles; Adding ARV-771 and 2-methylimidazole to the D / USCPZ nanoparticles, stirring and reacting, to obtain D / USCZA nanomaterials; After activating folic acid, the folic acid and D / USCZA nanomaterials are shaken to react to obtain Janus-structured FD / USCZA nanoparticles.

2. The preparation method according to claim 1, characterized in that The preparation method of the modified D / UCNRs comprises the following steps: After mixing and stirring sodium hydroxide solution, oleic acid and ethanol, rare earth chloride mixed solution is added dropwise to the mixed solution, and finally ammonium fluoride solution is added, reacted at 210°C for 8 hours, cooled naturally and centrifuged, and washed alternately with anhydrous ethanol and water for 1-3 times, dispersed in chloroform, to obtain D / UCNRs nanoparticles; The D / UCNRs nanoparticles and CTAB were stirred at 32° C. for 30 minutes, and then the temperature was raised to 65° C. to volatilize the chloroform in the solution, thereby obtaining CTAB-protected D / UCNRs nanoparticles, namely, modified D / UCNRs.

3. The preparation method according to claim 2, characterized in that: The volume ratio of the sodium hydroxide solution, oleic acid, ethanol, rare earth chloride mixed solution and ammonium chloride solution is 1.5:5:5:2:1, the concentration of the sodium hydroxide solution is 0.2 g / mL, and the concentration of the ammonium chloride solution is 2 mol / L; The rare earth chloride mixed solution is prepared by mixing yttrium chloride hexahydrate, ytterbium chloride hexahydrate, gadolinium chloride hexahydrate, erbium chloride hexahydrate and water in a mass volume ratio of 243 g:279 g:892 g:31 g:2 mL; The volume ratio of the D / UCNRs nanoparticles to CTAB is 1:20, the concentration of the D / UCNRs nanoparticles is 10 mg / mL, and the concentration of the CTAB is 2M.

4. The preparation method according to claim 1, characterized in that: The dihydrochlorin E6 silane precursor is obtained by dissolving dihydrochlorin E6 in DMSO, adding 3-aminopropyltriethoxysilane, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to react at room temperature in the dark for 12 hours; the ratio of the dihydrochlorin E6, DMSO, 3-aminopropyltriethoxysilane, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 2.2 mg: 0.5 mL: 12 μL: 6 mg: 4 mg.

5. The preparation method according to claim 1, characterized in that: Add water to the modified D / UCNRs and disperse them by ultrasonic to form a modified D / UCNRs solution; then add dihydrochlorin E6 silane precursor to the modified D / UCNRs solution, adjust the pH to 8, and then add an ethanol solution of tetraethyl orthosilicate in 4-6 times, with an interval of 30 minutes each time; the ratio of the modified D / UCNRs solution, dihydrochlorin E6 silane precursor and the ethanol solution of tetraethyl orthosilicate is 20:0.03:0.

25.

6. The preparation method according to claim 1, characterized in that: The volume ratio of the aqueous solution of zinc oxide to the aqueous solution of polyacrylic acid is 100:1, the concentration of the aqueous solution of zinc oxide is 1 mg / mL, and the concentration of the aqueous solution of polyacrylic acid is 0.2 g / mL; The volume ratio of the polyacrylic acid-zinc solution, the D / USC core-shell nanoparticles modified with dihydrochlorin E6 and isopropanol is 1:5:42; the ultrasonic time is 20-30 minutes, and the reaction condition is 32° C. for 12 hours.

7. The preparation method according to claim 1, characterized in that: After dispersing the D / USC-PAA-Zn nanoparticles in methanol, adding a methanol solution of 2-methylimidazole and ARV-771, stirring and reacting at 70° C. for 30 minutes, the D / USCZA nanomaterial is obtained; the volume ratio of the methanol, the methanol solution of 2-methylimidazole and ARV-771 is 100:31:1, and the concentration of the methanol solution of 2-methylimidazole is 0.97 mg / mL.

8. The preparation method according to claim 1, characterized in that: The method for activating folic acid comprises the following steps: placing a folic acid solution in DMSO, ultrasonicating, then adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and activating for 12 hours; wherein the ratio of folic acid, DMSO, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 10 mg: 8 mL: 9 mg: 5 mg; The shaking reaction time is 48 hours.

9. A Janus-structured FD / USCZA nanoparticle, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the Janus-structured FD / USCZA nanoparticles as claimed in claim 9 in any of the following: (1) Application in the preparation of drugs for treating breast cancer; (2) Application in the preparation of drugs for increasing the sensitivity of breast cancer cells to photodynamic therapy; (3) Application in the preparation of drugs for improving the efficacy of photodynamic therapy for breast cancer.

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