Nano drug delivery system as well as preparation method and application thereof

Abstract

The invention relates to a nano drug delivery system as well as a preparation method and application thereof, and belongs to the technical field of nano biomedicine. The nano drug delivery system disclosed by the invention comprises gold nanoparticles coated with polydopamine and Bcl-2siRNA, wherein the Bcl-2siRNA is coated with polydopamine; the Bcl-2siRNA is loaded on the surfaces of the gold nanoparticles coated with the polydopamine. According to the invention, PDA-AuNPs is used as a carrier, Bcl-2 siRNA is loaded in the PDA-AuNPs, and a Bcl-2-PDA-AuNPs composite structure is constructed. Meanwhile, the composite structure is also loaded with a chemotherapeutic drug daunorubicin (DAU) and / or a photosensitizer thionine (THN), so that chemotherapy-photodynamic-photothermal-gene combined therapy is realized, and the problem of non-pump resistance drug resistance caused by Bcl-2 protein overexpression is solved.

Description

Technical Field

[0001] The present invention relates to the field of nano-biomedicine technology, and in particular to a nano-drug delivery system and a preparation method and application thereof. Background Art

[0002] Although chemotherapy, the primary treatment for cancer, has achieved remarkable success in clinical applications, it can easily lead to multidrug resistance (MDR) in tumor cells, thereby reducing the effectiveness of treatment. The resistance mechanisms of resistant cells mainly fall into two categories: pump resistance and non-pump resistance resistance. Pump resistance is mediated by the ABC transporter superfamily (such as MRP1 and BCRP). These proteins actively excrete chemotherapeutic drugs, reducing intracellular drug concentrations and thus reducing therapeutic efficacy. On the other hand, the main mechanism of non-pump resistance resistance involves overactivation of the B-cell lymphoma-2 (Bcl-2) protein. The anti-apoptotic mechanism mediated by this protein enables cells to resist apoptosis caused by chemotherapeutic drugs, thereby inducing drug resistance.

[0003] Although, Chen et al. (Chen B., Mei L., Fan RR, et al. Polydopamine-coated i-motif DNA / gold nanoplatforms for synergistic photothermal-chemotherapy[J]. Asian Journal of Pharmaceutical Sciences, 2023, 18(2): 100781. https: / / doi.org / 10.1016 / j.ajps.2023.100781) used the PDA-AuNPs structure to achieve the effective loading of the anthracycline chemotherapy drug DOX, thereby exerting an anti-cancer effect. Liu et al. (Liu W.J., Yu Y.Y., Cheng W., et al. DA structured high-performance photothermal / photodynamic thionin-synthetic melanin nanoparticles for rapid bactericidal and wound healing effects [J]. Advanced Healthcare Materials, 2023, 12 (21): 2203303. https: / / doi.org / 10.1002 / adhm.202203303) synthesized melanin nanoparticles through the polymerization reaction of THN and levodopa, showing excellent PDT and PTT effects, and are expected to be used in the treatment of wound healing caused by bacterial infection under low-power dual laser irradiation. However, none of them targets the above-mentioned drug resistance mechanism, and the more targeted and effective drugs developed for the treatment of cancer are developed. Moreover, the interaction and loading sequence between PDA-AuNPs and chemotherapy-phototherapy combination drugs are closely related to their efficacy. Therefore, the development of strategies such as targeted intervention and combined therapy is expected to become an effective way to overcome MDR and achieve multimodal synergistic treatment to improve efficacy. Summary of the Invention

[0004] The purpose of the present invention is to provide a nano drug delivery system and its preparation method and application. The present invention uses PDA-AuNPs as a carrier to load Bcl-2 siRNA into it to construct a Bcl-2-PDA-AuNPs composite structure ( Figure 2 At the same time, the composite structure will also be loaded with chemotherapy drug DAU and / or photosensitizer THN ( Figure 1 ), realize chemotherapy-photodynamic-photothermal-gene combined therapy, and overcome the non-pump resistance problem caused by overexpression of Bcl-2 protein.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a nano drug delivery system, which comprises gold nanoparticles with a polydopamine coating and Bcl-2siRNA; the Bcl-2siRNA is loaded on the surface of the gold nanoparticles with a polydopamine coating.

[0007] Preferably, the nano drug delivery system further comprises a chemotherapeutic drug and / or a photosensitizer;

[0008] The chemotherapy drug is daunorubicin, and the photosensitizer is thionine.

[0009] The present invention provides a method for preparing the nano drug delivery system, comprising mixing polydopamine-coated gold nanoparticles with activated thiol-modified Bcl-2 siRNA, stirring and reacting for 1 to 3 hours, and obtaining polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA;

[0010] The molar ratio of the polydopamine-coated gold nanoparticles to the activated thiol-modified Bcl-2 siRNA is 1:450-550.

[0011] Preferably, after obtaining the Bcl-2 siRNA loaded polydopamine coated gold nanoparticles, the method further comprises the following steps:

[0012] Polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA are mixed with chemotherapeutic drugs and / or photosensitizers to form a nanodrug delivery system.

[0013] Preferably, the preparation method of the activated thiolated Bcl-2 siRNA is as follows: mixing the thiolated Bcl-2 siRNA with tris(2-carboxyethyl)phosphine hydrochloride, stirring and reacting for 0.5 to 1.5 hours to obtain the activated thiolated Bcl-2 siRNA;

[0014] The molar ratio of the thiolated Bcl-2 siRNA to tris(2-carboxyethyl)phosphine hydrochloride is 1:50-150.

[0015] The present invention also provides the use of the nano drug delivery system or the nano drug delivery system obtained according to the preparation method in the preparation of a preparation for inhibiting the survival of cancer cells.

[0016] Preferably, the cancer cells include human breast cancer cells and human breast cancer adriamycin-resistant cells.

[0017] Preferably, the method for inhibiting the survival of cancer cells is: directly applying the drug delivery system to cancer cells or introducing laser irradiation into the drug delivery system to act on cancer cells together to achieve the inhibitory effect on cancer cells.

[0018] Preferably, the laser is a NIR laser.

[0019] The present invention also provides the use of the nano drug delivery system or the nano drug delivery system obtained according to the preparation method in the preparation of anti-tumor drugs.

[0020] The present invention has the following technical effects and advantages:

[0021] (1) The present invention utilizes PDA-AuNPs as a carrier to load Bcl-2 siRNA, successfully constructing a Bcl-2-PDA-AuNPs composite structure. Furthermore, this composite structure is also used to load the chemotherapy drug DAU and / or the photosensitizer THN, enabling chemotherapy-photodynamic therapy-photothermal therapy-gene multimodal combination therapy, overcoming the non-pump resistance drug resistance problem caused by Bcl-2 protein activation.

[0022] (2) The present invention synthesized and characterized Bcl-2-PDA-AuNPs nanocarriers. TEM, DLS, and UV-vis spectroscopy characterized the successful synthesis of Bcl-2-PDA-AuNPs. The interactions between Bcl-2-PDA-AuNPs and DAU and / or THN binary and ternary systems were studied using spectroscopic and calorimetric methods. The results showed that the binding strength of Bcl-2-PDA-AuNPs to DAU was greater than that to THN. In the ternary system, the preferential addition of THN to form a binary complex (Bcl-2-PDA-AuNPs + THN) with weaker binding was followed by the addition of DAU to achieve a higher drug loading.

[0023] (3) The therapeutic effect of the Bcl-2-PDA-AuNPs drug delivery system was evaluated through relevant cell experiments. The results showed that the ROS detection experiment confirmed that the system could effectively produce ROS in cells under NIR irradiation; the in vitro cytotoxicity experiment proved that the (Bcl-2-PDA-AuNPs+THN)+DAU+NIR system had a chemotherapy-photodynamic-photothermal-gene multimodal treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the structural formula of THN;

[0025] Figure 2 Schematic diagram of the self-assembly of Bcl-2-PDA-AuNPs and the loading and release of DAU and THN;

[0026] Figure 3TEM images of AuNPs, PDA-AuNPs and Bcl-2-PDA-AuNPs, as well as their particle sizes, Zeta potentials and UV-vis characterizations. A to C are TEM images of AuNPs, PDA-AuNPs and Bcl-2-PDA-AuNPs, respectively; D is the particle size image of the three; E is the Zeta potential image of the three; and F is the UV-vis characterization of the three.

[0027] Figure 4 is the standard curve of FAM-Bcl-2siRNA;

[0028] Figure 5 Figure 2 is the fluorescence spectrum of the supernatant after hybridization of FAM-Bcl-2 siRNA and PDA-AuNPs;

[0029] Figure 6 Fluorescence spectra of the interaction between DAU (1 μM) and Bcl-2-PDA-AuNPs (0-2.2 nM) or THN (1 μM) and Bcl-2-PDA-AuNPs (0-5.5 nM) at 298.2 K, 304.2 K, and 310.2 K, where A and D are at 298.2 K, B and E are at 304.2 K, and C and F are at 310.2 K;

[0030] Figure 7 Van't Hoff plot of the interaction between Bcl-2-PDA-AuNPs and DAU or THN, where A represents the interaction with DAU and B represents the interaction with THN;

[0031] Figure 8 Normalized fluorescence spectra of (Bcl-2-PDA-AuNPs+DAU)+THN and (Bcl-2-PDA-AuNPs+THN)+DAU at 298.2K, 304.2K and 310.2K, where A and D are at 298.2K, B and E are at 304.2K, and C and F are at 310.2K;

[0032] Figure 9 Van't Hoff plot of the interaction between (Bcl-2-PDA-AuNPs+DAU)+THN and (Bcl-2-PDA-AuNPs+THN)+DAU;

[0033] Figure 10 The particle size storage stability of Bcl-2-PDA-AuNPs in different media;

[0034] Figure 11ITC graphs of DAU or THN binding to Bcl-2-PDA-AuNPs, THN binding to Bcl-2-PDA-AuNPs+DAU, and DAU binding to Bcl-2-PDA-AuNPs+THN at 298.2 K, where A is the ITC graph of DAU binding to Bcl-2-PDA-AuNPs, B is the ITC graph of THN binding to Bcl-2-PDA-AuNPs, C is the ITC graph of THN binding to Bcl-2-PDA-AuNPs+DAU, and D is the ITC graph of DAU binding to Bcl-2-PDA-AuNPs+THN;

[0035] Figure 12 DSC graphs of Bcl-2-PDA-AuNPs, Bcl-2-PDA-AuNPs+DAU and / or THN complexes;

[0036] Figure 13 Inverted fluorescence microscopy images of ROS levels in MCF-7 / ADR cells detected by DCFH-DA probe, scale bar: 50 μm;

[0037] Figure 14 Figures 2A and 2B show the cytostatic effects of free THN and DAU loaded with PDA-AuNPs or Bcl-2-PDA-AuNPs, as well as their combined effects on MCF-10A, MCF-7, and MCF-7 / ADR cells (n=3), where A to C show the cytostatic effects on MCF-10A cells, D to F show the cytostatic effects on MCF-7 cells, and G to I show the cytostatic effects on MCF-7 / ADR cells.

[0038] Figure 15 Inverted fluorescence microscopy images of MCF-7 / ADR cells cultured with (Bcl-2-PDA-AuNPs+THN)+DAU for 6 h, with a scale bar of 50 μm. DETAILED DESCRIPTION

[0039] The invention provides a nano drug delivery system, which comprises gold nanoparticles with a polydopamine coating and Bcl-2siRNA; the Bcl-2siRNA is loaded on the surface of the gold nanoparticles with a polydopamine coating.

[0040] In the present invention, the nano drug delivery system further comprises a chemotherapeutic drug and / or a photosensitizer;

[0041] The chemotherapy drug is daunorubicin (DAU), and the photosensitizer is thionine (THN).

[0042] The present invention provides a method for preparing the nano drug delivery system, comprising mixing polydopamine-coated gold nanoparticles with activated thiol-modified Bcl-2 siRNA, stirring and reacting for 1 to 3 hours, and obtaining polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA;

[0043] The molar ratio of the polydopamine-coated gold nanoparticles to the activated thiol-modified Bcl-2 siRNA is 1:450-550.

[0044] In the present invention, the molar ratio of the polydopamine-coated gold nanoparticles to the activated thiolated Bcl-2 siRNA is preferably 1:480-520, more preferably 1:500, the stirring reaction time is preferably 1.5-2.5 h, more preferably 2 h, the stirring reaction speed is 400-600 rpm, preferably 450-550 rpm, more preferably 500 rpm, and the stirring reaction temperature is 20-30° C., preferably 22-28° C., more preferably 25° C.

[0045] In the present invention, the preparation method of the activated thiolated Bcl-2 siRNA is as follows: mixing the thiolated Bcl-2 siRNA with tris(2-carboxyethyl)phosphine hydrochloride, stirring and reacting for 0.5 to 1.5 hours to obtain the activated thiolated Bcl-2 siRNA;

[0046] The molar ratio of the thiolated Bcl-2 siRNA to tris(2-carboxyethyl)phosphine hydrochloride is 1:50-150.

[0047] In the present invention, the molar ratio of the thiolated Bcl-2 siRNA to tris(2-carboxyethyl)phosphine hydrochloride is preferably 1:80-120, more preferably 1:100. The concentration of tris(2-carboxyethyl)phosphine hydrochloride is 9-11 mM, preferably 9.5-10.5 mM, more preferably 10 mM. The stirring reaction time is preferably 0.75-1.25 h, more preferably 1 h, the stirring reaction temperature is 20-30°C, preferably 22-28°C, more preferably 25°C, and the stirring reaction speed is 100-200 rpm, preferably 120-180 rpm, more preferably 150 rpm.

[0048] In the present invention, after obtaining the polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA, the following steps are further included:

[0049] Polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA are mixed with chemotherapeutic drugs and / or photosensitizers to form a nanodrug delivery system.

[0050] In the present invention, the polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA are mixed with chemotherapeutic drugs and / or photosensitizers as follows: the polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA are mixed with chemotherapeutic drugs to form a binary system, or the polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA are mixed with photosensitizers to form a binary system, or the polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA are mixed with chemotherapeutic drugs to form a binary system and then mixed with photosensitizers to form a ternary system, or the polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA are mixed with photosensitizers to form a binary system and then mixed with chemotherapeutic drugs to form a ternary system.

[0051] In the present invention, the preparation method of the gold nanoparticles with a polydopamine coating is as follows: (a) mixing activated double-terminated thiol polyethylene glycol with gold nanoparticles, fully fusing them by a freezing and thawing method, and after thawing, centrifuging to obtain a precipitate; (b) resuspending the precipitate in a Tris-HCl buffer to obtain polyethylene glycol-modified gold nanoparticles; (c) mixing a dopamine hydrochloride solution with the polyethylene glycol-modified gold nanoparticles, stirring and reacting for 1 to 3 hours, and obtaining a polyethylene glycol-modified gold nanoparticle solution with a PDA coating; and (d) centrifuging the polyethylene glycol-modified gold nanoparticle solution with a PDA coating to remove unreacted dopamine hydrochloride and other impurities, and obtaining polydopamine-coated gold nanoparticles.

[0052] In the present invention, activated double-terminal thiol polyethylene glycol is mixed with gold nanoparticles, and the mixture is fully fused by a freeze-thaw method. After thawing, the precipitate is centrifuged; the volume ratio of the activated double-terminal thiol polyethylene glycol to the gold nanoparticles is 1:0.5-1.5, preferably 1:0.8-1.2, and more preferably 1:1; the thawing is carried out in a natural thawing manner, the centrifugation temperature is 2-6°C, preferably 3-5°C, and more preferably 4°C; the centrifugation speed is 10000-14000 rpm, preferably 11000-13000 rpm, and more preferably 12000 rpm, and the centrifugation time is 10-20 min, preferably 12-18 min, and more preferably 15 min. The preparation method of the activated double-terminated thiol polyethylene glycol comprises: mixing tris(2-carboxyethyl)phosphine hydrochloride with double-terminated thiol polyethylene glycol and reacting for 0.5 to 1.5 hours to obtain the activated double-terminated thiol polyethylene glycol; the molar ratio of the tris(2-carboxyethyl)phosphine hydrochloride to the double-terminated thiol polyethylene glycol is 1:150 to 250, preferably 1:180 to 220, and more preferably 1:200; the concentration of the tris(2-carboxyethyl)phosphine hydrochloride is 9 to 11 mM, preferably 9.5 to 10.5 mM, and more preferably 10 mM. The synthesis method of the gold nanoparticles adopts the sodium citrate reduction method to synthesize the gold nanoparticles.

[0053] In the present invention, the precipitate is resuspended in Tris-HCl buffer to obtain polyethylene glycol-modified gold nanoparticles; the concentration of the Tris-HCl buffer is 9-11 mM, preferably 9.5-10.5 mM, more preferably 10 mM, and the pH of the Tris-HCl buffer is 7.5-9.2, preferably 8-9, more preferably 8.5.

[0054] In the present invention, a dopamine hydrochloride solution is mixed with polyethylene glycol-modified gold nanoparticles, and the mixture is stirred for reaction for 1 to 3 hours to obtain a polyethylene glycol-modified gold nanoparticle solution with a PDA coating; the volume ratio of the dopamine hydrochloride solution to the polyethylene glycol-modified gold nanoparticles is 1:1 to 3, preferably 1:1.5 to 2.5, and more preferably 1:2; the dopamine hydrochloride solution is prepared by dissolving dopamine hydrochloride in a Tris-HCl buffer to obtain a dopamine hydrochloride solution with a concentration of 0.5 to 1.5 mg / mL, the concentration of the dopamine hydrochloride solution is preferably 0.8 to 1.2 mg / mL, and more preferably 1.0 mg / mL, the concentration of the Tris-HCl buffer is 9 to 11 mM, preferably 9.5 to 10.5 mM, and more preferably 10 mM, and the pH of the Tris-HCl buffer is 7.5 to 9.2, preferably 8 to 9, and more preferably 8.5. The stirring reaction time is preferably 1.5 to 2.5 hours, more preferably 2 hours, the stirring reaction speed is 400 to 600 rpm, preferably 450 to 550 rpm, more preferably 500 rpm, and the stirring reaction temperature is 20 to 30°C, preferably 22 to 28°C, more preferably 25°C.

[0055] In the present invention, a solution of polyethylene glycol-modified gold nanoparticles with a PDA coating is centrifuged to remove unreacted dopamine hydrochloride and other impurities to obtain polydopamine-coated gold nanoparticles; the centrifugal speed is 10,000 to 14,000 rpm, preferably 11,000 to 13,000 rpm, and more preferably 12,000 rpm, and the centrifugal time is 10 to 20 minutes, preferably 12 to 18 minutes, and more preferably 15 minutes.

[0056] The present invention also provides the use of the nano drug delivery system or the nano drug delivery system obtained according to the preparation method in the preparation of a preparation for inhibiting the survival of cancer cells.

[0057] In the present invention, the cancer cells include human breast cancer cells and human breast cancer doxorubicin (DOX)-resistant cells.

[0058] In the present invention, the method of inhibiting the survival of cancer cells is: directly applying the drug delivery system to cancer cells or introducing laser irradiation into the drug delivery system to act on cancer cells together to achieve the inhibitory effect on cancer cells; the laser is NIR laser.

[0059] The present invention also provides the use of the nano drug delivery system or the nano drug delivery system obtained according to the preparation method in the preparation of anti-tumor drugs.

[0060] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0061] The oligonucleotide chains used in the present invention (as shown in Table 1, all purified by ULTRAPAGE), 10× PBS buffer (pH 7.4, cell culture grade), sodium citrate dihydrate (C6H5Na3O7·2H2O, purity ≥98%), and gold chloride tetrahydrate (HAuCl4·4H2O, purity ≥99%) were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). DAU (purity ≥98%) and THN (purity ≥90%) were purchased from J&K Biotechnology Co., Ltd. (Beijing, China). Dopamine hydrochloride (DA, purity 98.5%) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP, purity 98%) were provided by Yuanye Biotechnology Co., Ltd. (Shanghai, China). Double-terminated thiol-polyethylene glycol (SH-PEG 5000 ) was purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China). 2',7'-Dichlorofluorescein diacetate (DCFH-DA) was provided by Aladdin Reagent Co., Ltd. (Shanghai, China). Human breast cancer cells (MCF-7), human breast cancer DOX-resistant cells (MCF-7 / ADR), and human normal mammary epithelial cells (MCF-10A) were obtained from Pronocell Life Science Co., Ltd. (Wuhan, China). All experimental water was ultrapure water (>18.2 MΩ·cm) prepared using a Milli-Q water purification system (Millipore Elix5).

[0062] Table 1 Base sequences used to construct Bcl-2-PDA-AuNPs

[0063]

[0064] Example 1 Construction and characterization of Bcl-2-PDA-AuNPs

[0065] Synthesis of gold nanoparticles (AuNPs): The present invention synthesized gold nanoparticles with a particle size of about 13 nm by sodium citrate reduction method. Before the experiment, all glassware was washed with freshly prepared aqua regia (V HNO3 :V HCl=1:3) for 24 hours, ultrasonically rinse three times with ultrapure water, and then dry for later use. Heat 5 mL of 10 mM HAuCl4·4H2O and 45 mL of ultrapure water to boiling while stirring at 500 rpm. Rapidly add 5 mL of freshly prepared trisodium citrate solution (38.8 mM). The solution color changes from light yellow to light gray to purple-black to wine red. Continue boiling the solution for 10 minutes, then stir and cool to 25°C. Filter through a 0.22 μm microporous membrane and store in a brown glass bottle at 4°C in the dark.

[0066] Synthesis of polydopamine-coated gold nanoparticles (PDA-AuNPs): TCEP and SH-PEG 5000 Mix and react at a molar ratio of 1:200 for 1 h to activate SH-PEG 5000 , then the activated SH-PEG 5000 The mixture was mixed with an equal volume of AuNPs synthesized as described above and fully bound by freeze-thaw. After natural thawing at room temperature, the mixture was centrifuged at 12,000 rpm at 4°C for 15 minutes. The supernatant was discarded and the remaining material was suspended in Tris-HCl buffer (10 mM, pH 8.5) to obtain polyethylene glycol-modified gold nanoparticles (AuNPs-PEG). A solution of 1 mg / mL of DA dissolved in Tris-HCl buffer was mixed with AuNPs-PEG at a volume ratio of 1:2. The mixture was stirred at 500 rpm for 2 hours at room temperature to allow DA to polymerize on the AuNPs-PEG surface, forming a PDA coating. After the reaction, the mixture was centrifuged at 12,000 rpm for 15 minutes to remove unreacted DA and other impurities, resulting in a PDA-AuNPs solution.

[0067] Preparation of Bcl-2 siRNA-loaded polydopamine-coated gold nanoparticles (Bcl-2-PDA-AuNPs): To prepare the Bcl-2-PDA-AuNPs complex, thiolated Bcl-2 siRNA and tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 10 mM) were first mixed at a molar ratio of 1:100 and stirred at 150 rpm for 1 hour at 25°C to activate the thiolated Bcl-2 siRNA. The activated Bcl-2 siRNA was then mixed with PDA-AuNPs at a molar ratio of 500:1 and stirred at room temperature for 2 hours to achieve surface modification of the Bcl-2 siRNA on the PDA-AuNPs. The mixture was then centrifuged at 10,000 rpm for 20 minutes and reconstituted in 100 μL of 1× PBS (pH 7.4) to yield Bcl-2-PDA-AuNPs. Finally, the synthesized Bcl-2-PDA-AuNPs were characterized by TEM, DLS and UV-vis. The results are shown in Table 2 and Figure 3 .

[0068] Table 2 Particle size and Zeta potential values measured by DLS

[0069]

[0070] The assembly process of Bcl-2-PDA-AuNPs is as follows Figure 2 As shown. Figure 3 As shown in Figure A, the TEM image shows that the diameter of AuNPs is about 13-14 nm, spherical and uniform in size distribution. After PDA coating to form PDA-AuNPs, a clear core-shell nanostructure can be observed ( Figure 3 B); after the thiolated Bcl-2 siRNA was modified on the surface of PDA-AuNPs ( Figure 3 C), the size of the obtained Bcl-2-PDA-AuNPs further increased. According to the DLS results ( Figure 3 The hydrodynamic diameter of Bcl-2-PDA-AuNPs was 77.93±1.57nm and the Zeta potential was -53.90±0.39mV. Figure 3 F), during the step-by-step assembly process, the characteristic LSPR peak shifted from 520 nm to 536 nm to 558 nm, verifying the effective modification of Bcl-2 siRNA on the surface of PDA-AuNPs.

[0071] Example 2 Fluorescence spectroscopy experiment

[0072] 1. Experimental Methods

[0073] 1.1 Investigation of the amount of Bcl-2 siRNA modified on the surface of PDA-AuNPs

[0074] A standard curve was drawn by measuring the fluorescence intensity of FAM-labeled Bcl-2 siRNA (FAM-Bcl-2 siRNA) of known concentration (10-100 nM) provided by Sangon Biotech Co., Ltd. (Shanghai, China). After centrifugation of FAM-Bcl-2-PDA-AuNPs, the fluorescence intensity of the supernatant was measured, which was the concentration of free FAM-Bcl-2 siRNA. The ratio of the concentration of FAM-Bcl-2 siRNA bound to the surface of PDA-AuNPs to the concentration of FAM-Bcl-2-PDA-AuNPs was the amount of FAM-Bcl-2 siRNA modified on the surface of PDA-AuNPs. The results are shown in Figure 4 and Figure 5 .

[0075] 1.2 Fluorescence spectroscopy experiments of the Bcl-2-PDA-AuNPs+DAU / THN binary system

[0076] In the binary system fluorescence spectroscopy experiment, the experimental temperatures selected were 298.2K, 304.2K and 310.2K. The concentrations of DAU and THN were fixed at 1μM, and the concentration ranges of Bcl-2-PDA-AuNPs in the Bcl-2-PDA-AuNPs+DAU or THN binary systems were 0-2.20nM and 0-5.50nM. The fluorescence emission spectra of DAU or THN were collected after incubation at the corresponding temperature for 30min. The excitation wavelength of DAU was 495nm, and the emission spectrum was collected in the range of 515-680nm; the excitation wavelength of THN was 595nm, and the emission spectrum was collected in the range of 610-700nm. The results are shown in Figure 3. Figure 6 .

[0077] 1.3 Fluorescence spectroscopy experiments of the (Bcl-2-PDA-AuNPs+DAU)+THN and (Bcl-2-PDA-AuNPs+THN)+DAU ternary systems

[0078] Fluorescence spectroscopy experiments of the ternary system were performed at selected temperatures of 298.2 K, 304.2 K, and 310.2 K. 60 μL of DAU (5 μM) and 30 μL of Bcl-2-PDA-AuNPs (10 nM) were mixed and incubated at the corresponding temperature for 30 minutes to form a Bcl-2-PDA-AuNPs + DAU binary complex. 0-70 μL of THN stock solution (60 μM) was then added to the mixture, followed by 140-210 μL of PBS to a final volume of 300 μL. The mixture was incubated for an additional 30 minutes to form the (Bcl-2-PDA-AuNPs + DAU) + THN ternary system, and its fluorescence emission spectra were collected. For the (Bcl-2-PDA-AuNPs+THN)+DAU ternary system, 60 μL DAU (5 μM) and 60 μL Bcl-2-PDA-AuNPs (10 nM) were first added to form a binary complex, and then 0-110 μL of DAU stock solution (60 μM) was added. Finally, 70-180 μL of PBS was added to make the final volume 300 μL, and the incubation was continued for 30 minutes. The fluorescence measurement parameters of the ternary system are: DAU excitation wavelength is 495 nm, and the emission spectrum collection range is 515-680 nm; THN excitation wavelength is 595 nm, and the emission spectrum collection range is 610-700 nm. The results are shown in Figure 2. Figure 8 .

[0079] 2. Experimental Results

[0080] 2.1 Investigation of the modification amount of Bcl-2 siRNA

[0081] According to the standard curve of FAM-Bcl-2 siRNA ( Figure 4 ) and the fluorescence intensity of the FAM-Bcl-2-PDA-AuNPs supernatant ( Figure 5 ), it can be calculated that approximately 48 FAM-Bcl-2 siRNAs were modified on the surface of PDA-AuNPs.

[0082] 2.2 Fluorescence analysis of the Bcl-2-PDA-AuNPs+DAU / THN binary system

[0083] To compare the interaction between the Bcl-2-PDA-AuNPs+DAU or THN binary systems, fluorescence spectroscopy was used to study the interaction. The fluorescence spectra of DAU or THN with different concentrations of Bcl-2-PDA-AuNPs at three temperatures are shown in Figure 2. Figure 6 As shown, as the concentration of Bcl-2-PDA-AuNPs increased, the fluorescence intensity of DAU or THN was quenched to varying degrees, indicating that both drugs interacted with Bcl-2-PDA-AuNPs. For example, at 298.2K, when the concentration of Bcl-2-PDA-AuNPs was 1 nM, the fluorescence intensity of DAU and THN was quenched by 44.8% and 32.4%, respectively. Therefore, it can be inferred that under the same conditions, the binding strength of Bcl-2-PDA-AuNPs to DAU is stronger than that to THN.

[0084] In order to further explore the mechanism of interaction between Bcl-2-PDA-AuNPs and DAU or THN binary system, the Scatchard equation (1) was used to calculate the interaction K a and n values, and the results are listed in Table 3.

[0085]

[0086] Where r is the concentration of bound drug (C b ) and the total concentration of Bcl-2-PDA-AuNPs (M t ) ratio, C f is the free molar concentration of DAU or THN, which can be calculated using the following formula:

[0087]

[0088] Among them, C t is the total drug concentration, F represents the fluorescence intensity of DAU at 557 nm, and the constant k b and k f Can be obtained through experiments.

[0089] By comparing the n values, it can be found that the K of DAU loaded on Bcl-2-PDA-AuNPs constructed in the present invention is a and n values are higher than those of the ASOs-MUC1-TD structure (K a The order of magnitude is 10 5 M -1 , n value is between 30 and 40, Li Xinyu. Thermodynamic study of multifunctional DNA tetrahedron loaded chemotherapy / phototherapy combined drugs [D]. Shandong: Liaocheng University, 2024. DOI: 10.27214 / d.cnki.glcsu.2024.000169), indicating that the surface of Bcl-2-PDA-AuNPs has a higher THN binding site. However, compared with THN, the surface of Bcl-2-PDA-AuNPs has more DAU binding sites, indicating that DAU is more likely to bind to Bcl-2-PDA-AuNPs. According to the K of the binary system a The values indicate that Bcl-2-PDA-AuNPs have a strong binding force with DAU or THN, and the reaction process is exothermic. a The value means that the Bcl-2-PDA-AuNPs+DAU binary system has higher binding strength and stability.

[0090] By formula 3 and RlnK a Plot 1 / T ( Figure 7 ) calculated the enthalpy change (ΔH o ), entropy change (ΔS o ) and Gibbs free energy change (ΔG o ) values, and the results are listed in Table 3.

[0091]

[0092] ΔG o and ΔH o All < 0 and -ΔH o >TΔS°, indicating that the binding between Bcl-2-PDA-AuNPs and DAU or THN is a spontaneous, exothermic, and enthalpy-driven process. o <0 indicates that electrostatic binding, hydrogen bonding and van der Waals forces play a major role in the binding process; ΔS o >0 because there is a stronger interaction between Bcl-2-PDA-AuNPs and DAU or THN, and desolvation requires more energy.

[0093] 2.3 Fluorescence analysis of the (Bcl-2-PDA-AuNPs+DAU)+THN and (Bcl-2-PDA-AuNPs+THN)+DAU ternary systems

[0094] The interaction between the (Bcl-2-PDA-AuNPs+DAU)+THN and (Bcl-2-PDA-AuNPs+THN)+DAU ternary systems was analyzed by fluorescence spectroscopy. Figure 8 As shown in Figures AC, with the increase of THN concentration, the fluorescence intensity of the Bcl-2-PDA-AuNPs+DAU binary system gradually weakened, indicating that there was an interaction between THN and Bcl-2-PDA-AuNPs+DAU, and it was consistent with the "ternary approximate binary model", that is, the addition of the third component did not change the interaction mode between the first two components. According to the measured fluorescence intensity, n and K were obtained by fitting with Matlab software. a The values are listed in detail in Table 3. n and K of the (Bcl-2-PDA-AuNPs+DAU)+THN ternary system a The value is smaller than that of Bcl-2-PDA-AuNPs+DAU, which indicates that DAU, which binds strongly to Bcl-2-PDA-AuNPs, joins the drug binding site first and hinders the further binding of THN. The K value of the (Bcl-2-PDA-AuNPs+DAU)+THN ternary system a The value decreases with increasing temperature (Table 3), indicating that its interaction is similar to that of the binary system and is still a spontaneous process driven by exothermicity.

[0095] In contrast to the ternary system described above, where DAU was added first, when Bcl-2-PDA-AuNPs first bound to THN to form a Bcl-2-PDA-AuNPs+THN binary complex and then DAU was added, the fluorescence intensity increased continuously with increasing DAU concentration. This indicates competitive binding between DAU and THN, with DAU with stronger binding affinity displacing some of the bound THN and occupying the binding sites, consistent with the "competitive adsorption model":

[0096]

[0097] C i0 =C i +M t n i Θ i (5)

[0098] where Θ i is the ratio of the number of occupied sites of component i (DAU or THN) to the total number of sites, C i0 、C i are the total and free concentrations of DAU or THN, respectively, M t represents the total concentration of Bcl-2-PDA-AuNPs. n and K of the ternary system were obtained by fitting the competition model. aThe values are listed in Table 3. Taking 298.2K as an example, compared with the Bcl-2-PDA-AuNPs+THN or DAU binary system, the K values of the (Bcl-2-PDA-AuNPs+DAU)+THN and (Bcl-2-PDA-AuNPs+THN)+DAU ternary systems are a The values were reduced by 98.8% and 28.9%, respectively. K a The decrease in the value indicates that the binding of the first added drug will weaken the binding ability of the subsequent drug. However, for the ternary system, the preferential addition of THN to form the Bcl-2-PDA-AuNPs+THN binary complex and then the addition of DAU can achieve a higher drug loading. Figure 9 RlnK a The linear relationship diagram with 1 / T is shown in Table 3. ΔG of the ternary system o <0 and as the temperature increases, K a The value gradually decreases, indicating that the combination of the ternary system is a spontaneous and exothermic process.

[0099] Table 3 Thermodynamic parameters of the interaction between DAU and / or THN and Bcl-2-PDA-AuNPs obtained from fluorescence spectroscopy and ITC analysis

[0100]

[0101]

[0102] Example 3 DLS study

[0103] The particle sizes of the binary systems of Bcl-2-PDA-AuNPs+DAU or THN, the ternary systems of (Bcl-2-PDA-AuNPs+DAU)+THN and (Bcl-2-PDA-AuNPs+THN)+DAU obtained in Example 2 were determined by DLS. The results are shown in Table 4.

[0104] In addition, the synthesized Bcl-2-PDA-AuNPs were dispersed in various media and the storage stability was evaluated at 4°C. The particle size of the samples was measured by DLS at 0, 1, 3, 5 and 7 days, and the particle size stability was systematically analyzed. The results are shown in Figure 10 .

[0105] The particle size of the drug-loaded nanoparticles showed a significant correlation with drug loading efficiency. As shown in Table 4, the particle size of Bcl-2-PDA-AuNPs loaded with DAU and THN, both individually and in combination, showed an increasing trend. The particle size of the binary system of Bcl-2-PDA-AuNPs + DAU was slightly larger than that of Bcl-2-PDA-AuNPs + THN, positively correlated with drug loading. For the ternary system, the system in which the weakly bound THN was added first and then DAU had a larger particle size, which is consistent with the higher drug loading capacity of the (Bcl-2-PDA-AuNPs + THN) + DAU system.

[0106] Table 4 Particle size values of DAU and / or THN loaded by Bcl-2-PDA-AuNPs

[0107]

[0108]

[0109] To investigate the particle size stability of Bcl-2-PDA-AuNPs, they were placed in pure water, PBS buffer at pH 7.4 and 5.0, and DMEM culture medium containing 10% fetal bovine serum and stored at 4°C for 7 days to investigate their particle size stability. Throughout the storage period, the particle size fluctuation of Bcl-2-PDA-AuNPs was minimal, and there was no significant difference in particle size between the experimental groups ( Figure 10 This property is expected to provide a key guarantee for its application in tumor-targeted delivery.

[0110] Example 4 Calorimetric study

[0111] 1. Experimental Methods

[0112] 1.1 ITC Analysis

[0113] Using MicroCal ITC 200 Titration calorimeter, at 298.2K, ITC experiments were performed on the Bcl-2-PDA-AuNPs + DAU or THN binary system and (Bcl-2-PDA-AuNPs + DAU) + THN and (Bcl-2-PDA-AuNPs + THN) + DAU ternary systems obtained in Example 2. Among them, the concentrations of DAU and THN were both 300μM, and the concentration of Bcl-2-PDA-AuNPs was 28nM. The dilution heat of Bcl-2-PDA-AuNPs and DAU or THN was deducted from the binary system, and the dilution heat of the drug and the corresponding binary system was deducted from the ternary system. The data were fitted using the Origin 7.0 software supporting the instrument. The results are shown in Figure 11 .

[0114] 1.2DSC analysis

[0115] DSC analysis was performed using a MicroCal VP-DSC calorimeter. After baseline correction using PBS buffer solution, PBS was injected into the reference cell, and the sample cell contained Bcl-2-PDA-AuNPs, Bcl-2-PDA-AuNPs+DAU or THN, (Bcl-2-PDA-AuNPs+DAU)+THN, and (Bcl-2-PDA-AuNPs+THN)+DAU obtained in Example 2. The concentrations of DAU, THN, and Bcl-2-PDA-AuNPs were 5, 5, and 17 nM, respectively. The samples were degassed for 5 minutes before loading. The samples were scanned at a scanning speed of 90°C / h in the temperature range of 15 to 100°C, and the data were analyzed using the Origin 7.0 software that came with the instrument. The results are shown in the table. Figure 12 .

[0116] 1.3 In vitro ROS detection experiment

[0117] MCF-7 / ADR cells were used to detect ROS generation at the cellular level. 5 MCF-7 / ADR cells were seeded into 6-well plates at a density of 100 μg / well and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h. The culture medium was poured out and replaced with the Bcl-2-PDA-AuNPs, THN+DAU mixture, and (Bcl-2-PDA-AuNPs+THN)+DAU ternary complex obtained in Example 2, and cultured for another 6 h. The concentrations of Bcl-2-PDA-AuNPs, THN, and DAU were 25 nM, 1.25 μM, and 5 μM, respectively. Subsequently, a power of 100 mW / cm 2 The cells were irradiated with NIR light of 660 nm and a wavelength of 100 nm, with the irradiation time set to 0-10 min. Afterwards, DCFH-DA probe diluted to 10 μM in serum-free medium was added, and the cells were incubated at 37°C for 30 min. After the incubation, the cells were washed three times with PBS and finally placed under an inverted fluorescence microscope for fluorescence imaging. The level of ROS generation was determined by observing the fluorescence intensity. The results are shown in Figure 2. Figure 13 .

[0118] 1.4 In vitro cytotoxicity studies

[0119] The present invention uses the MTT method to detect the cytotoxicity of THN and / or DAU to MCF-10A, MCF-7 and MCF-7 / ADR cells before and after loading with PDA-AuNPs or Bcl-2-PDA-AuNPs. 3The three cell types were seeded at a density of 100 cells / well in 96-well plates and incubated for 24 hours. Subsequently, the medium was replaced with THN or DAU, THN + DAU, PDA-AuNPs + THN or DAU, (PDA-AuNPs + THN) + DAU, Bcl-2-PDA-AuNPs, Bcl-2-PDA-AuNPs + THN or DAU, and (Bcl-2-PDA-AuNPs + THN) + DAU, and the culture continued. For MCF-10A, MCF-7, and MCF-7 / ADR cells, the DAU concentrations in the added samples ranged from 0.1 to 2.0, 0.1 to 3.0, and 2 to 12 μM, respectively; the THN concentrations ranged from 0.1 to 2.0, 0.1 to 3.0, and 0.1 to 3.0 μM, respectively. In the binary system, the molar ratio of PDA-AuNPs or Bcl-2-PDA-AuNPs:THN was 1:50; while the molar ratio of PDA-AuNPs or Bcl-2-PDA-AuNPs:DAU was 1:250. In the ternary system, the molar ratio of PDA-AuNPs or Bcl-2-PDA-AuNPs:THN:DAU was 1:50:200. After incubation for 6 h, the cells were irradiated with a 660 nm laser (power density 100 mW / cm 2 ) All samples containing THN were irradiated for 5 min to investigate the PDT effect; in addition, a PDT or PTT control group was set up: 880 nm laser (power density 1.5 W / cm 2 ) for 10 minutes to investigate the combined effects of PDT and PTT. After laser irradiation, the cells were incubated for another 42 hours. 20 μL of MTT was added to replace the original solution. After 4 hours of incubation, the solution was replaced with 150 μL of DMSO. After shaking for 5 minutes, the absorbance was measured, and the cell inhibition rate (%) was calculated using Equation 6. The combined therapeutic index (CI) was calculated using Equation 7 to investigate the synergistic cytotoxicity of co-loading THN and DAU against cancer cells under different conditions:

[0120] Cell inhibition (%)=(AB) / A×100% (6)

[0121] CI=(D)1 / (D x )1+(D)2 / (D x )2 (7)

[0122] Where A and B are the absorbance of untreated cells and treated cells, respectively; (D x )1 and (D x )2 are the concentrations at which THN and DAU produce x% inhibitory effects (in the present invention, x% is 50%), (D)1 and (D)2 are the concentrations at which the combination produces the same therapeutic effect. CI>1, CI=1 and CI<1 correspond to antagonistic, additive and synergistic effects, respectively. The results are shown in Figure 14 With Table 6.

[0123] 1.5 In vitro cellular uptake studies

[0124] In order to further investigate the intracellular drug distribution of the (Bcl-2-PDA-AuNPs+THN)+DAU ternary system, the present invention cultured MCF-7 / ADR cells at a rate of 1×10 5 The cells were seeded in culture dishes at a density of 10 cells / mL and cultured conventionally for 24 hours. Subsequently, 500 μL (Bcl-2-PDA-AuNPs+THN)+DAU was added to each culture dish and incubated for 6 hours. Among them, the concentrations of Bcl-2-PDA-AuNPs, THN and DAU were 25 nM, 1.25 and 5 μM, respectively. After the incubation, the cell nucleus was first stained with DAPI staining solution to achieve specific labeling of the cell nucleus. After staining, the cells were fixed with 4% paraformaldehyde solution for 15 minutes. Finally, the cells were placed under an inverted fluorescence microscope for imaging observation, and the results are shown in FIG. Figure 15 .

[0125] 1.6 Statistical analysis

[0126] All experiments were repeated three times, and the final results are presented as "mean ± standard deviation". One-way analysis of variance was performed using SPSS 19.0 software. When the p value was less than 0.05, a significant difference between the groups was considered.

[0127] 2. Experimental Results

[0128] 2.1 ITC Research

[0129] Figure 11 The ITC diagrams of the binary system of Bcl-2-PDA-AuNPs+DAU or THN, the ternary system of (Bcl-2-PDA-AuNPs+DAU)+THN and (Bcl-2-PDA-AuNPs+THN)+DAU at 298.2K are shown in Table 3. By comparing ΔH o , ΔS o and ΔG o The values indicate that both the binary and ternary systems are spontaneously exothermic and mainly driven by enthalpy. In the binary system, the n and K of Bcl-2-PDA-AuNPs combined with DAU are a The values are higher than those of THN, indicating that the binding effect between DAU and Bcl-2-PDA-AuNPs is stronger. In the ternary system, there is still a phenomenon that the first addition of one drug affects the binding of subsequent drugs. The n and K values of the (Bcl-2-PDA-AuNPs+THN)+DAU ternary system are aThe value is higher than that of (Bcl-2-PDA-AuNPs + DAU) + THN, which is consistent with the conclusion of the fluorescence spectrum experiment of the ternary system.

[0130] 2.2 DSC study

[0131] The thermal denaturation curves measured by DSC are as Figure 12 shown. Table 5 shows the T m and ΔH cal values of Bcl-2-PDA-AuNPs, Bcl-2-PDA-AuNPs + DAU and / or THN complexes. The T m and ΔH cal values show the following pattern: Bcl-2-PDA-AuNPs < Bcl-2-PDA-AuNPs + THN < Bcl-2-PDA-AuNPs + DAU, indicating that the binding of Bcl-2-PDA-AuNPs with DAU or THN enhances the thermal stability of Bcl-2-PDA-AuNPs. The addition of DAU and THN increases the T m values of Bcl-2-PDA-AuNPs by 6.73 °C and 5.65 °C respectively, indicating that DAU or THN binds to Bcl-2-PDA-AuNPs in an intercalative binding mode. Among them, the T m and ΔH cal values of the Bcl-2-PDA-AuNPs + DAU system are higher because the binding between DAU and Bcl-2-PDA-AuNPs is tighter.

[0132] In the ternary system, (Bcl-2-PDA-AuNPs + DAU) + THN and (Bcl-2-PDA-AuNPs + THN) + DAU increase the T m of Bcl-2-PDA-AuNPs by 8.71 °C and 9.89 °C respectively. The change in the T m value is more significant than that in the binary system, and at the same time, the increase in the ΔH cal value is also more prominent. This fully indicates that the ternary system has higher stability than the binary system, and among these two ternary systems, the (Bcl-2-PDA-AuNPs + THN) + DAU ternary system has the best stability. Combining the results of fluorescence spectroscopy and calorimetry experiments, to ensure a high loading capacity of the two drugs on Bcl-2-PDA-AuNPs, during the subsequent joint drug loading research, THN with relatively weak binding ability can be preferentially added to form a stable binary complex with Bcl-2-PDA-AuNPs, and then the loading operation of DAU can be carried out.

[0133] Table 5 Thermodynamic parameters obtained from DSC experiments

[0134]

[0135] 2.3 In vitro ROS detection experiment

[0136] DCFH-DA was selected as a fluorescent probe. After entering the cell, it was hydrolyzed by cell membrane esterase to generate 2',7'-dichlorodihydrofluorescein (DCFH), which can be oxidized by ROS in the cell to the green fluorescent compound 2',7'-dichlorofluorescein (DCF). Its fluorescence intensity is positively correlated with the intracellular ROS level. Figure 13 As shown, as the laser irradiation time gradually increases, the fluorescence intensity shows a trend of gradual increase. The fluorescence intensity of different treatment groups follows the following order: blank cell group < Bcl-2-PDA-AuNPs < THN+DAU < (Bcl-2-PDA-AuNPs+THN)+DAU group. This result shows that the Bcl-2-PDA-AuNPs constructed by the present invention, after simultaneously loading DAU and THN into cells, can effectively induce cells to produce ROS under laser irradiation conditions to promote tumor cell apoptosis or necrosis. This shows that the system exhibits good photodynamic efficiency in PDT and is expected to become an effective means of tumor treatment.

[0137] 2.4 In vitro cytotoxicity studies

[0138] The cytotoxicity of THN and / or DAU alone and in combination before and after loading with PDA-AuNPs or Bcl-2-PDA-AuNPs on MCF-10A, MCF-7, and MCF-7 / ADR cells was systematically evaluated by MTT assay. Figure 14 As shown, the calculated IC 50 The IC values of free THN in MCF-10A and MCF-7 cells are shown in Table 6. 50 The values of THN and DAU were larger than those of DAU, indicating lower cytotoxicity against these two cell types; however, its inhibitory effect on MCF-7 / ADR was significantly superior to that of DAU. This is due to the high tolerance of MCF-7 / ADR cells to DAU due to multidrug resistance mechanisms, resulting in limited therapeutic efficacy. When the photosensitizer THN was used in combination with DAU, the killing effect on all three cell types was enhanced, confirming the therapeutic effect of CDT: ROS generated by THN under 660nm laser irradiation can disrupt the integrity of tumor cell membranes and promote the transmembrane transport of DAU, thereby enhancing therapeutic efficacy and overcoming tumor cell resistance to DAU.

[0139] After THN and / or DAU were loaded with PDA-AuNPs or Bcl-2-PDA-AuNPs, their cytotoxicity to normal cells MCF-10A was weakened, while their toxicity to the other two tumor cells was enhanced. On the one hand, this is because the drugs loaded by nanocarriers can be enriched in the tumor site through the enhanced permeability and retention effect (EPR) and passively targeted to the tumor site, so that the accumulation of drugs in tumor cells is significantly higher than that in normal tissues; on the other hand, PDA-AuNPs are pH-sensitive and can accelerate drug release in the acidic microenvironment of the tumor, while the release in normal tissues is slow. Therefore, the drug is released at high concentrations in tumor cells, enhancing the killing effect; while in normal cells, the toxicity is weakened due to the reduced drug exposure. In addition, compared with single or combined drug systems loaded with PDA-AuNPs, the Bcl-2-PDA-AuNPs drug-loaded system has stronger cytotoxicity: compared with the Bcl-2-PDA-AuNPs-loaded THN, DAU, THN+DAU and PDA-AuNPs drug-loaded systems, IC 50 The values decreased by 20.33%, 35.54%, and 36.21% in MCF-7 cells, respectively; and by 18.09%, 36.43%, and 48.30% in MCF-7 / ADR cells, respectively. Furthermore, the efficacy of the Bcl-2-PDA-AuNPs drug delivery system was far superior to that of the free drug. This synergistic effect is primarily attributed to the synergistic effect of Bcl-2 gene silencing and chemotherapeutic drugs. Studies have shown that the anti-apoptotic protein Bcl-2, highly expressed in tumor cells, can contribute to tumor cell resistance to chemotherapeutic drugs by inhibiting apoptotic signaling through the mitochondrial pathway. Notably, the system showed no significant increase in toxicity to normal cells. This is due to the low expression level of Bcl-2 protein in normal cells, limiting the gene silencing effect and thus avoiding excessive damage to normal tissues.

[0140] To explore the potential of synergistic therapy, NIR laser (808 nm) irradiation was introduced to evaluate the PTT efficacy of the Bcl-2-PDA-AuNPs drug delivery system. The experimental results showed that after NIR laser (808 nm) irradiation, the IC of the (Bcl-2-PDA-AuNPs+THN)+DAU ternary system on MCF-7 and MCF-7 / ADR cells was 50The values were further reduced by 29.31% and 31.58% compared with the non-irradiated group, confirming the synergistic effect of photothermal effect with chemotherapy and PDT. CI value analysis showed that the CI values of the (Bcl-2-PDA-AuNPs+THN)+DAU+NIR system for MCF-7 and MCF-7 / ADR cells were 0.46 and 0.17, respectively, both less than 0.5, indicating that the system exhibited significant synergistic effects and significant reversal capabilities for MDR in tumor cells, especially drug-resistant cancer cells. In summary, the (Bcl-2-PDA-AuNPs+THN)+DAU+NIR system has a multimodal therapeutic effect of chemotherapy-photodynamic-photothermal-gene.

[0141] Table 6 IC values of free THN and DAU loaded with PDA-AuNPs or Bcl-2-PDA-AuNPs in MCF-10A, MCF-7 and MCF-7 / ADR cells as single and combined drugs determined by MTT assay 50 and CI values

[0142]

[0143]

[0144] 2.5 In vitro cellular uptake studies

[0145] like Figure 15 As shown, after DAPI staining, the nuclei of MCF-7 / ADR cells exhibit blue fluorescence, while the green fluorescence of DAU and the red fluorescence of THN are mostly concentrated in the nucleus. Upon cell incorporation, (Bcl-2-PDA-AuNPs+THN)+DAU is effectively internalized into the nucleus, releasing a large amount of the drug, effectively killing cancer cells and demonstrating excellent therapeutic efficacy. Therefore, the (Bcl-2-PDA-AuNPs+THN)+DAU nanoparticles constructed in this invention are a multifunctional nanocarrier that integrates chemotherapy, gene therapy, photothermal therapy, and PDT.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A nano drug delivery system, characterized in that: The nano drug delivery system comprises gold nanoparticles with a polydopamine coating and Bcl-2 siRNA; the Bcl-2 siRNA is loaded on the surface of the gold nanoparticles with a polydopamine coating.

2. The nano drug delivery system according to claim 1, characterized in that: The nano drug delivery system further includes a chemotherapeutic drug and / or a photosensitizer; The chemotherapy drug is daunorubicin, and the photosensitizer is thionine.

3. The method for preparing the nano drug delivery system according to claim 1, characterized in that: The polydopamine-coated gold nanoparticles were mixed with the activated thiol-modified Bcl-2 siRNA and stirred for 1 to 3 hours to obtain polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA; The molar ratio of the polydopamine-coated gold nanoparticles to the activated thiol-modified Bcl-2 siRNA is 1:450-550.

4. The preparation method according to claim 3, characterized in that After obtaining the polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA, the following steps are further included: Polydopamine-coated gold nanoparticles loaded with Bcl-2 siRNA are mixed with chemotherapeutic drugs and / or photosensitizers to form a nanodrug delivery system.

5. The preparation method according to claim 3, characterized in that The preparation method of the activated thiolated Bcl-2 siRNA is as follows: mixing the thiolated Bcl-2 siRNA with tris(2-carboxyethyl)phosphine hydrochloride, stirring and reacting for 0.5 to 1.5 hours to obtain the activated thiolated Bcl-2 siRNA; The molar ratio of the thiolated Bcl-2 siRNA to tris(2-carboxyethyl)phosphine hydrochloride is 1:50-150.

6. Use of the nano drug delivery system according to claim 1 or 2, or the nano drug delivery system obtained according to the preparation method according to any one of claims 3 to 5, in the preparation of a preparation for inhibiting the survival of cancer cells.

7. The application according to claim 6, characterized in that: The cancer cells include human breast cancer cells and human breast cancer adriamycin-resistant cells.

8. The use according to claim 6, characterized in that The method for inhibiting the survival of cancer cells is: directly applying the drug-carrying system to cancer cells or introducing laser irradiation into the drug-carrying system to act on cancer cells together, thereby achieving an inhibitory effect on cancer cells.

9. The application according to claim 8, characterized in that: The laser is an NIR laser.

10. Use of the nano drug delivery system according to claim 1 or 2 or the nano drug delivery system obtained according to the preparation method according to any one of claims 3 to 5 in the preparation of anti-tumor drugs.

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