A Bcl-2 siRNA@DOX core / shell fiber material for combination therapy of breast cancer and its preparation method

Abstract

This invention provides a Bcl-2siRNA@DOX core / shell fiber for combined breast cancer treatment and its preparation method, relating to the field of biomedical materials. The preparation method includes the following steps: S1: Polyvinylpyrrolidone and polylactic acid (PLA) are dissolved in a mixed solution of chloroform and methanol, stirred at room temperature to form a milky white homogeneous solution, and then ZIF-8@DOX is added to obtain a shell spinning solution. S2: Polyvinylpyrrolidone and polycaprolactone are dissolved in a mixed solution of chloroform and N,N-dimethylformamide, stirred at room temperature to form a milky white homogeneous solution, and MPB particles are added to obtain a core spinning solution. S3: The shell spinning solution and the core spinning solution are coaxially electrospun to obtain the Bcl-2siRNA@DOX core / shell fiber material. The preparation process is simple, the reaction conditions are mild, the energy consumption is low, the structure of the drug-loaded fiber is easily controlled, and the cost is low.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a Bcl-2siRNA@DOX core / shell fiber material for combined breast cancer treatment and its preparation method. Background Technology

[0002] Cancer is a malignant tumor that develops uncontrollably from cancer cells, seriously threatening human life and health. Breast cancer is the most common cancer among women, and common treatments include surgical resection, chemotherapy, and radiotherapy. Among these, adjuvant chemotherapy after surgical resection is the preferred standard strategy for treating breast tumors. However, because traditional chemotherapy is usually administered systemically via oral or injectable routes, the non-specific distribution of drugs during their journey to the tumor site often causes severe toxicity to normal tissues or cells, and systemic circulation results in low drug delivery efficiency. Furthermore, since cancer is a genetic disease caused by the accumulation of gene mutations, residual tumor cells not eliminated during surgery often lead to further gene mutations, resulting in an alarmingly high recurrence rate. Therefore, innovative therapies with higher tumor specificity and greater potency are needed to rapidly and effectively kill tumors. Gene therapy is a novel nucleic acid-based treatment method. Through vectors, designed exogenous nucleic acid sequences are introduced into target cells, which can regulate specific gene pathways, correct defective genes, and manipulate gene expression to achieve anti-tumor effects. Therefore, combining chemotherapy and gene therapy with surgical removal of breast tumors to treat breast cancer can inhibit or kill cancer cells through different cellular pathways, reduce tumor recurrence and metastasis, and improve postoperative treatment outcomes.

[0003] To address the side effects of traditional chemotherapy, using controlled drug delivery systems (DDS) to deliver therapeutic drugs is an effective strategy to improve the efficacy of postoperative adjuvant chemotherapy. Depending on the route of administration and mechanism of action, postoperative adjuvant chemotherapy can generally be divided into systemic and local therapies. Systemic therapy typically involves loading therapeutic molecules onto carrier materials through physical encapsulation, chemical covalent bonding, or electrostatic interactions. These nanocarriers can penetrate biological barriers such as tissues and cells, achieving passive targeting through the enhanced permeability and retention (EPR) effect at the tumor site. Then, specific surface-modified ligands enable active targeting of tumor cells, ultimately delivering the drug directly to the tumor site, thus reducing damage to normal tissues during systemic delivery. However, because the postoperative tumor site lacks a suitable environment for achieving the EPR effect, over-reliance on the EPR effect for systemic administration may reduce the cumulative concentration of the carrier in residual microtumors. DDS-based postoperative adjuvant local therapy generally refers to the direct delivery of drugs to the tumor site through implantable systems. This overcomes some of the shortcomings of systemic therapy because it can increase the cumulative drug concentration at the tumor site, reduce drug dosage and side effects on normal tissues or cells, and achieve continuous drug delivery to the tumor site. However, local combined delivery of chemotherapy drugs and nucleic acids remains challenging because it requires the construction of suitable implantable, multifunctional vectors.

[0004] Commonly used implantable local drug delivery systems for cancer treatment include prefabricated implants (cast implants, 3D-printed implants) and injectable implants. Cast implants are prepared using casting methods, which involve pouring a polymer solution or melt containing the drug into a casting, and then solidifying the precursor through solvent evaporation, physical cross-linking, or chemical methods to obtain the desired implant. Casting methods offer flexibility in material selection, allowing the use of thermoplastic biopolymers and natural / synthetic hydrogels; however, the controllability of structural aspects such as porosity, mechanical properties, and compositional distribution in cast implants still requires improvement. 3D printing technology for implants is a process of constructing 3D objects by depositing materials layer by layer on a computer-controlled build platform. It mainly includes various technologies such as inkjet printing, extrusion, cryogenic deposition modeling, and fused deposition modeling. The speed, flexibility (for complex geometries), and precise controllability of 3D printing technology have made it widely popular in personalized and precision medicine. Among various 3D printing technologies, inexpensive extrusion and fused deposition modeling (FDM) techniques are commonly used to fabricate 3D printed implants. However, extrusion and FDM printing often suffer from drawbacks such as nozzle clogging and interlayer debonding. Injectable implants typically involve mixing a polymer matrix and a therapeutic agent solution to create a low-viscosity fluid, which is then injected into the tumor site and cured in situ under various stimuli (such as light, temperature and pH changes, and in-situ crosslinking). Compared to prefabricated implants, injectable implants can fill any injection site, but these implants present challenges such as drug burst release, chemical crosslinking that can damage surrounding tissues, and relatively long curing times.

[0005] Besides cast implants, 3D-printed implants, and injectable implants, electrospun fibers are widely used in implantable local drug delivery systems due to their advantages such as high drug loading capacity, controllable drug release behavior, and simple processing. While there has been considerable research on electrospun implants for local cancer treatment, most studies focus on loading one or two chemical drugs. There are few electrospun implants that co-load chemical drugs and genes, and significant challenges remain in the co-delivery of chemical drugs and genes. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention utilizes coaxial electrospinning technology to construct a core / shell fibrous membrane in which a small interfering RNA (Bcl-2 siRNA) is located in the nuclear layer and the chemical drug doxorubicin hydrochloride (DOX) is located in the shell layer. The resulting fibrous material achieves co-loading of the drug and the gene, and exhibits a significant synergistic inhibitory effect on MCF-7 human breast cancer cells. This invention features a simple preparation process, low energy consumption, easily controllable drug loading system structure, and low cost.

[0007] This invention provides a method for preparing Bcl-2 siRNA@DOX core / shell fibrous material for combination therapy of breast cancer, the preparation method comprising the following steps:

[0008] S1: Polyvinylpyrrolidone and polylactic acid (PLA) are dissolved in a mixed solution of chloroform and methanol. The mixture is stirred at room temperature to form a milky white homogeneous solution. Then, ZIF-8@DOX is added under stirring and mixed evenly to obtain the shell spinning solution.

[0009] S2: Polyvinylpyrrolidone and polycaprolactone are dissolved in a mixed solution of chloroform and N,N-dimethylformamide and stirred at room temperature to form a milky white homogeneous solution. MPB particles are added under ultrasonic or vortex oscillation to obtain the core spinning solution.

[0010] S3: The shell spinning solution obtained in step S1 and the core spinning solution obtained in step S2 are coaxially electrospun to obtain Bcl-2 siRNA@DOX core / shell fiber material.

[0011] Another aspect of the present invention provides a Bcl-2 siRNA@DOX core / shell fiber material for combined treatment of breast cancer. The Bcl-2 siRNA@DOX core / shell fiber material is a fiber membrane composed of fibers loaded with Bcl-2 siRNA@DOX. The Bcl-2 siRNA@DOX core / shell fibers are continuous long fibers with a diameter of 0.35-5 μm.

[0012] Compared with the prior art, the present invention achieves at least one of the following beneficial effects:

[0013] (1) This invention utilizes coaxial electrospinning technology to construct a core / shell fibrous material with a small interfering RNA (Bcl-2 siRNA) located in the core layer and the chemical drug doxorubicin hydrochloride (DOX) located in the shell layer, co-loaded with both the gene and the drug. The preparation method is simple, the preparation conditions are mild, and the reaction conditions are simple and easy to control. The resulting fibrous material has a large drug loading capacity, and the preparation process does not affect the activity and efficacy of the drug.

[0014] (2) The preparation process of this invention is simple, the reaction conditions are mild, the energy consumption is low, the structure of the drug-loaded fiber is easy to control, and the cost is low.

[0015] (3) In the preparation method of the present invention, ZIF-8@DOX is pH sensitive, which enables the prepared core / shell fiber material to release DOX in response to the tumor micro-acidic environment, reducing damage to normal cells.

[0016] (4) The fiber material obtained by the present invention can be used as a local drug delivery preparation for cancer treatment; it can effectively overcome the excessive dependence on EPR effect of systemic drug delivery, increase the cumulative drug concentration at the tumor site, reduce the drug dosage and side effects on normal tissues or cells, and achieve continuous drug delivery at the tumor site; the local drug delivery preparation with co-loaded chemical drugs and genes has good application prospects in the field of combined tumor treatment. Attached Figure Description

[0017] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0018] Figure 1 The TEM image shows the Bcl-2 siRNA@DOX core / shell fiber material prepared in Example 1 of this invention;

[0019] Figure 2 The diagram shows the cell viability of human breast cancer cells MCF-7 after 24h and 48h of treatment with free DOX, free Bcl-2 siRNA, and three drug-loaded fiber membranes SF1, CF1, and CS.

[0020] Figure 3 The cumulative drug release of the Bcl-2 siRNA@DOX core / shell fibrous material prepared in Example 2 of this invention is shown in the figure.

[0021] Figure 4 The SEM image of the Bcl-2 siRNA@DOX core / shell fiber material prepared in Example 3 of the present invention is shown. Detailed Implementation

[0022] To more clearly illustrate the overall concept of the present invention, a detailed description will be provided below with reference to the accompanying drawings and examples.

[0023] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0024] In an exemplary embodiment of the present invention, the preparation method of Bcl-2 siRNA@DOX core / shell fiber material for combined breast cancer therapy includes the following steps:

[0025] S1: Polyvinylpyrrolidone (PVP) and polylactic acid (PLA) are dissolved in a mixed solution of chloroform and methanol. The solution is stirred at room temperature to form a milky white homogeneous solution. Then, DOX-loaded ZIF-8 (ZIF-8@DOX) is added under stirring and mixed thoroughly to obtain the shell spinning solution. The pH-responsive zeolite imidazole ester backbone-8 (ZIF-8) material, after DOX loading, can release more drug in response to the acidic environment of the tumor site, while reducing damage to normal tissues or cells.

[0026] S2: Polyvinylpyrrolidone and polycaprolactone (PCL) are dissolved in a mixed solution of chloroform and N,N-dimethylformamide (DMF). The solution is stirred at room temperature to form a milky white homogeneous solution. MPB particles are added under ultrasonic or vortex oscillation conditions to obtain the core spinning solution.

[0027] S3: The shell spinning solution obtained in step S1 and the core spinning solution obtained in step S2 are coaxially electrospun to obtain Bcl-2 siRNA@DOX core / shell fiber material.

[0028] The ZIF-8@DOX particles of this invention have a particle size of 96±20nm; the MPB particles have a particle size of 114±17nm.

[0029] Specifically, in step S1, the mass ratio of polyvinylpyrrolidone to polylactic acid (PLA) is 1:2-4, the volume ratio of methanol to chloroform is 1:5-7, the total concentration of polyvinylpyrrolidone and PLA is 10-15% (w / v), and the mass ratio of ZIF-8@DOX to polyvinylpyrrolidone is 1:9-30. Preferably, the mass ratio of ZIF-8@DOX to polyvinylpyrrolidone is 1:15-20.

[0030] In step S2, the mass ratio of polyvinylpyrrolidone (PVP) to polycaprolactone (PCB) is 1:3-7, the volume ratio of N,N-dimethylformamide (NDM) to chloroform is 1:3-6, the total concentration of PPVP and PCB is 6-10% (w / v), and the mass ratio of MPB to PPVP is 1:45-60. Preferably, the mass ratio of PPVP to MPB is 1:50-55.

[0031] Specifically, the coaxial electrospinning includes the following steps:

[0032] The spinning voltage is 15.0-23.0 kV, the core spinning solution injection rate is 0.1-0.2 mm / min, the shell spinning solution injection rate is 0.2-0.4 mm / min, the needle distance from the receiver is 10-20 cm, a fixed receiver is used to receive the spun fibers, the humidity in the spinning box is maintained within the range of 25-35%, and spinning is carried out at room temperature. In this invention, the room temperature is 25±5℃.

[0033] In another exemplary embodiment of the present invention, based on the above embodiments, the method for preparing MPB particles in step S2 includes the following steps:

[0034] A DEPC solution of Bcl-2 siRNA at a concentration of 0.08-0.12 μg / μL was mixed with polyethyleneimine-modified mesoporous polydopamine MPDA-PEI1.8k (MP), followed by shaking, room temperature incubation, and lyophilization to obtain MPDA-PEI1.8k@Bcl-2 siRNA (MPB) particles. The mass ratio of Bcl-2 siRNA to MP was 1:10-50. Preferably, the mass ratio of Bcl-2 siRNA to MP was 1:20-40. The DEPC water was ultrapure water (Grade I water) treated with DEPC (diethyl pyrocarbonate) and sterilized by high temperature and high pressure, and was tested to be free of impurities such as RNA, DNA, and protein.

[0035] Specifically, the preparation method of the MP includes the following steps:

[0036] Mesoporous polydopamine (MPDA) and polyethyleneimine (PEI1.8k) were mixed at a mass ratio of 1:12-28 in tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (Tris-HCl) at pH 8-9, stirred at room temperature in the dark, centrifuged, and dried to obtain MP.

[0037] Preferably, the mass ratio of MPDA to PEI1.8k is 1:16-24.

[0038] Specifically, the preparation method of the MPDA includes the following steps:

[0039] Poloxamer (F127) and 1,3,5-trimethylbenzene (TMB) were dissolved in a mixed solvent of water and ethanol. Tris(hydroxymethyl)aminomethane (Tris) and dopamine hydrochloride were added sequentially. The mixture was stirred, centrifuged, and dried at room temperature to obtain MPDA.

[0040] Bcl-2 siRNA is a small interfering RNA. Naked RNA is easily degraded by nucleases during in vivo delivery and has low transfection efficiency. Therefore, suitable vectors need to be designed for its in vivo delivery. PEI1.8k is a cationic polymer with many amino groups, which can adsorb RNA through electrostatic interactions. MPDA is a porous nanoparticle with excellent surface modification properties. By binding polyethyleneimine to MPDA through Michael addition or Schiff base reaction, its surface can be positively charged, allowing for the loading of large amounts of RNA through electrostatic adsorption.

[0041] Specifically, the preparation method of ZIF-8@DOX in step S1 may include the following steps:

[0042] Zinc nitrate hexahydrate, DOX, and 2-methylimidazole are dissolved in water, stirred at room temperature for 15-30 minutes, centrifuged, and dried at 50-70°C to obtain DOX-loaded ZIF-8. Preferably, the stirring time at room temperature is 24 minutes, and the drying can be carried out under vacuum at 60-65°C.

[0043] The mass ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:7-10, and the mass ratio of DOX to 2-methylimidazole is 1:80-100; the mass ratio of DOX to water is 1.5-2 g / L. Preferably, the mass ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:8-9, and the mass ratio of DOX to 2-methylimidazole is 1:83-95.

[0044] The Bcl-2 siRNA@DOX core / shell fiber material for breast cancer combination therapy prepared by this invention is a fiber membrane. The fiber membrane is composed of fibers loaded with Bcl-2 siRNA@DOX. These Bcl-2 siRNA@DOX core / shell fibers are continuous long fibers with a diameter of 0.35-5 μm. Preferably, the diameter is 1.5-5 μm. More preferably, the diameter is 2-4 μm. The Bcl-2 siRNA@DOX core / shell fiber material of this invention can achieve responsive release of DOX in the slightly acidic tumor environment of pH 4.5-5.5, reducing damage to normal cells.

[0045] Specifically, the preparation method of Bcl-2 siRNA@DOX core / shell fiber material for combination therapy of breast cancer includes the following steps:

[0046] (1) Dissolve zinc nitrate, DOX, and 2-methylimidazole in water, stir at room temperature for 15-30 minutes, and then centrifuge and dry to obtain DOX-loaded ZIF-8.

[0047] (2)a. Dissolve 0.36 g poloxamer and 420 μL 1,3,5-trimethylbenzene in a mixed solvent of 75 mL water and 60 mL ethanol. After stirring for 30 min, add 90 mg tris(hydroxymethyl)aminomethane and then add 60 mg dopamine hydrochloride. Stir at room temperature for 24 h, centrifuge, and vacuum dry at 50 °C to obtain MPDA.

[0048] b. 2.5 mg of MPDA and a certain mass of PEI1.8k were placed in 8 mL of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer at pH 8.5 and stirred at room temperature in the dark for 24 h. After centrifugation and drying at 25 °C, MP was obtained.

[0049] c. Dissolve Bcl-2 siRNA and MP in DEPC water at a concentration of 0.1 μg / μL. Vortex for 10 min and incubate at room temperature for 50 min to obtain MPB solution. Freeze-dry the MPB solution to obtain MPB particles.

[0050] (3) Dissolve 0.858g PVP and 2.002g PLA in a mixture of 19mL chloroform and 3mL methanol, stir at room temperature to form a milky white homogeneous solution, add ZIF-8@DOX under stirring, mix and disperse evenly to obtain a shell spinning solution; dissolve 0.16g PVP and 0.64g PCL in a mixture of 8mL chloroform and 2mL N,N-dimethylformamide, stir at room temperature to form a milky white homogeneous solution, add MPB particles under ultrasonic or vortex oscillation to obtain a core spinning solution. Coaxial electrospinning yields Bcl-2siRNA@DOX core / shell fiber material. Preferably, the mass of ZIF-8@DOX is 0.03-0.08g. More preferably, the mass of ZIF-8@DOX is 0.03-0.06g. Preferably, the mass of MPB particles is 2.0-3.6mg. More preferably, the mass of MPB particles is 2.8-3.2mg.

[0051] Example 1

[0052] The preparation method of Bcl-2 siRNA@DOX core / shell fibrous material for combination therapy of breast cancer includes the following steps:

[0053] (1) Dissolve 120 mg zinc nitrate hexahydrate, 12 mg DOX and 1 g 2-methylimidazole in 6.4 mL of water, stir at room temperature for 24 min, centrifuge and vacuum dry at 62 °C to obtain DOX-loaded ZIF-8.

[0054] (2)a. Dissolve 0.36 g poloxamer and 420 μL 1,3,5-trimethylbenzene in a mixed solvent of 75 mL water and 60 mL ethanol. After stirring for 30 min, add 90 mg tris(hydroxymethyl)aminomethane and then add 60 mg dopamine hydrochloride. Stir at room temperature for 24 h, centrifuge, and vacuum dry at 50 °C to obtain MPDA.

[0055] b. 2.5 mg MPDA and 40 mg PEI1.8k were placed in 8 mL of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer at pH 8.5 and stirred at room temperature in the dark for 24 h. After centrifugation and drying at 25 °C, MP was obtained. The mass ratio of MPDA to PEI1.8k was 1:16.

[0056] c. Dissolve 1 mg of Bcl-2 siRNA and 40 mg of MP in DEPC water to obtain a Bcl-2 siRNA concentration of 0.1 μg / μL. Vortex for 10 min and incubate at room temperature for 50 min to obtain an MPDA-PEI1.8k@Bcl-2 siRNA (MPB) solution. Freeze-dry the MPB solution to obtain MPB particles. The mass ratio of Bcl-2 siRNA to MP is 1:40.

[0057] (3) Dissolve 0.858 g of polyvinylpyrrolidone and 2.002 g of polylactic acid in a mixture of 19 mL of chloroform and 3 mL of methanol. Stir at room temperature to form a milky white homogeneous solution. Add 0.05 g of ZIF-8@DOX under stirring, and mix and disperse evenly to obtain the shell spinning solution. The mass ratio of polyvinylpyrrolidone to polylactic acid is 1:2.3, the volume ratio of methanol to chloroform is 1:6.3, the total concentration of polyvinylpyrrolidone and polylactic acid is 13%, and the mass ratio of ZIF-8@DOX to polyvinylpyrrolidone is 1:17.2.

[0058] 0.16 g PVP and 0.64 g PCL were dissolved in a mixed solution of 8 mL chloroform and 2 mL N,N-dimethylformamide. The solution was stirred at room temperature to form a milky white homogeneous solution. 3.0 mg MPB particles were added under ultrasonic or vortex conditions to obtain the core spinning solution. The mass ratio of PVP to PCL was 1:4, the volume ratio of N,N-dimethylformamide to chloroform was 1:4, the total concentration of polyvinylpyrrolidone and polycaprolactone was 8% (w / v), and the mass ratio of MPB to PVP was 1:53.3.

[0059] (4) The electrospinning solution obtained in step (3) is coaxially electrospinned using an electrospinning machine. The spinning voltage is set to 19.0 kV, the core spinning solution injection rate is 0.1 mm / min, the shell spinning solution injection rate is 0.2 mm / min, the needle is 15 cm away from the receiver, the spun fibers are received using a fixed receiver, the humidity in the spinning box is maintained at 30%, and spinning is carried out at room temperature to obtain Bcl-2 siRNA@DOX core / shell fiber material.

[0060] refer to Figure 1 As shown, Figure 1 The image shows a TEM image of the Bcl-2 siRNA@DOX core / shell fiber material prepared in Example 1. As can be seen from the image, the Bcl-2 siRNA@DOX core / shell fiber material has a distinct core / shell structure.

[0061] As a control experiment, the core spinning solution and the shell spinning solution were subjected to uniaxial electrospinning to obtain SF1 fiber material with single DOX loading and CF1 material with single Bcl-2 siRNA loading.

[0062] The Bcl-2 siRNA@DOX core / shell fiber material prepared in Example 1 was subjected to a cytotoxicity test on human breast cancer MCF-7 cells. The specific steps are as follows:

[0063] The cytotoxicity of free DOX, free Bcl-2 siRNA, and three drug-loaded fibrillary membranes (SF1, CF1, and Bcl-2 siRNA@DOX nucleo / shell fibers, referred to as the CS group) to human breast cancer cells MCF-7 was determined using the CCK-8 assay. The specific procedure was as follows: MCF-7 cells were cultured in T25 flasks using DMEM complete medium (containing 12% serum and 1% penicillin-dextrin). MCF-7 cells in the logarithmic growth phase were collected, washed twice with cell-grade PBS (containing 3% penicillin-dextrin), digested with trypsin, centrifuged (100 rpm, 5 min), and the supernatant was discarded. The cells were then resuspended in complete medium and thoroughly mixed. Cells were counted using a hemocytometer, and seeded into 96-well plates at 9000 cells per well with 200 μL of complete medium. The plates were incubated overnight at 37°C in a 5% CO2 incubator. Once the cell density reached 60%-80%, free DOX, free Bcl-2 siRNA, and three drug-loaded fiber membranes (SF1, CF1, and CS groups) were added. The DOX concentration was 5 μg / mL, and the RNA concentration was 33 ng / mL. A blank control group and a control group were also included, with six replicates per group. After culturing for 24 and 48 hours, the fiber membranes were removed, and the culture medium in the wells was aspirated. 100 μL of freshly prepared complete culture medium containing 10% CCK-8 was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance was measured at 450 nm using a microplate reader, and the viability of MCF-7 cells was calculated using the following formula.

[0064]

[0065] Among them, A b Absorbance of the blank well; A c Absorbance of the control well; A s The absorbance of the experimental well is shown.

[0066] refer to Figure 2 As shown, Figure 2 This study investigated the cytotoxicity of free DOX, free Bcl-2 siRNA, and three drug-loaded fiber membranes (SF1, CF1, and CS) against human breast cancer cells MCF-7. (Reference) Figure 2 As can be seen, in this embodiment, the survival rates of MCF-7 cells after 48 hours of treatment with drug-loaded fiber membranes SF1, CF1, and CS were 49.42%, 60.31%, and 27.99%, respectively. This indicates that compared with single drugs, the nuclear / shell fiber membrane co-loaded with Bcl-2 siRNA and DOX can synergistically inhibit human breast cancer MCF-7 cells and improve the therapeutic effect on cancer cells.

[0067] Example 2

[0068] A method for preparing Bcl-2 siRNA@DOX core / shell fibrous material for combination therapy of breast cancer includes the following steps:

[0069] (1) Dissolve 110 mg zinc nitrate hexahydrate, 10 mg DOX and 1 g 2-methylimidazole in 6 mL of water, stir at room temperature for 24 min, centrifuge and dry under vacuum at 63 °C to obtain ZIF-8@DOX.

[0070] (2)a. Dissolve 0.36 g poloxamer and 420 μL 1,3,5-trimethylbenzene in a mixed solvent of 75 mL water and 60 mL ethanol. After stirring for 30 min, add 90 mg tris(hydroxymethyl)aminomethane and then add 60 mg dopamine hydrochloride. Stir at room temperature for 24 h, centrifuge, and vacuum dry at 50 °C to obtain MPDA.

[0071] b. 2.5 mg MPDA and 50 mg polyethyleneimine were placed in 8 mL of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 8.5), stirred at room temperature in the dark for 24 h, centrifuged, and dried at 25 °C to obtain MP. The mass ratio of MPDA to PEI1.8k was 1:20.

[0072] c. Dissolve 1 mg Bcl-2 siRNA and 35 mg MP in DEPC water to obtain a Bcl-2 siRNA concentration of 0.1 μg / μL. Vortex for 10 min and incubate at room temperature for 50 min to obtain an MPB solution. Freeze-dry the MPB solution to obtain MPB particles. The mass ratio of Bcl-2 siRNA to MP is 1:35.

[0073] (3) Dissolve 0.858g PVP and 2.002g PLA in a mixture of 19mL chloroform and 3mL methanol, and stir at room temperature to form a milky white homogeneous solution. Add 0.06g ZIF-8@DOX under stirring, and mix and disperse evenly to obtain the shell spinning solution. The mass ratio of ZIF-8@DOX to PVP is 1:14.3.

[0074] 0.16 g PVP and 0.64 g PCL were dissolved in a mixed solution of 8 mL CHCl3 and 2 mL LDMF. The solution was stirred at room temperature to form a milky white homogeneous solution. 2.9 mg MPB particles were added under ultrasonic or vortex conditions to obtain the core spinning solution. The mass ratio of MPB to PVP was 1:55.2.

[0075] (4) The electrospinning solution obtained in (3) was coaxially electrospinned using an electrospinning machine. The spinning voltage was set to 21.0 kV, the core spinning solution injection rate was 0.2 mm / min, the shell spinning solution injection rate was 0.4 mm / min, the needle distance from the receiver was 17 cm, the spun fibers were received using a fixed receiver, the humidity in the spinning box was maintained at 32%, and spinning was carried out at room temperature to obtain Bcl-2 siRNA@DOX core / shell fiber material.

[0076] refer to Figure 3 As shown, Figure 3 This is a graph showing the cumulative drug release of Bcl-2 siRNA@DOX core / shell fiber material, determined using the isothermal oscillation method. From... Figure 3 As can be seen, in this embodiment, the cumulative DOX release of the Bcl-2 siRNA@DOX core / shell fiber material within 72 hours was 60.66% and 81.3% under pH conditions of 7.4 (simulating normal physiological environment) and 5.0 (simulating slightly acidic environment of tumor area), respectively. This indicates that the ZIF-8 carrier can release more DOX under acidic conditions, which is beneficial to achieve the response release of DOX in the slightly acidic environment of tumor and reduce damage to normal cells.

[0077] Example 3

[0078] The preparation method of Bcl-2 siRNA@DOX core / shell fibrous material for combination therapy of breast cancer includes the following steps:

[0079] (1) Dissolve 100 mg zinc nitrate hexahydrate, 10 mg DOX and 1 g 2-methylimidazole in 5.5 mL of water, stir at room temperature for 24 min, centrifuge and vacuum dry at 63 °C to obtain DOX-loaded ZIF-8.

[0080] (2)a. Dissolve 0.36 g poloxamer and 420 u LTMB in a mixed solvent of 75 mL water and 60 mL ethanol. After stirring for 30 min, add 90 mg tris(hydroxymethyl)aminomethane and then add 60 mg dopamine hydrochloride. Stir at room temperature for 24 h, centrifuge, and vacuum dry at 50 °C to obtain MPDA.

[0081] b. 2.5 mg MPDA and 45 mg polyethyleneimine were placed in 8 mL of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer at pH 8.5 and stirred at room temperature in the dark for 24 h. After centrifugation and drying at 25 °C, MP was obtained. The mass ratio of MPDA to PEI1.8k was 1:18.

[0082] c. Dissolve 1 mg of Bcl-2 siRNA and 30 mg of MP in DEPC water to obtain a Bcl-2 siRNA concentration of 0.1 μg / μL. Vortex for 10 min and incubate at room temperature for 50 min to obtain an MPB solution. Freeze-dry the MPB solution to obtain MPB particles. The mass ratio of Bcl-2 siRNA to MP is 1:30.

[0083] (3) Dissolve 0.858g PVP and 2.002g PLA in a mixture of 19mL chloroform and 3mL methanol, and stir at room temperature to form a milky white homogeneous solution. Add 0.04g ZIF-8@DOX under stirring, and mix and disperse evenly to obtain the shell spinning solution. The mass ratio of ZIF-8@DOX to PVP is 1:21.5.

[0084] 0.16 g PVP and 0.64 g PCL were dissolved in a mixed solution of 8 mL chloroform and 2 mL LDM. The solution was stirred at room temperature to form a milky white homogeneous solution. 3.1 mg MPB particles were added under ultrasonic or vortex oscillation to obtain the core spinning solution. The mass ratio of MPB to PVP was 1:51.6.

[0085] (4) The electrospinning solution obtained in (3) is coaxially electrospinned using an electrospinning machine. The spinning voltage is set to 20.0 kV, the core spinning solution injection rate is 0.2 mm / min, the shell spinning solution injection rate is 0.4 mm / min, the needle is 18 cm away from the receiver, the spun fibers are received using a fixed receiver, the humidity in the spinning box is maintained at 34%, and spinning is carried out at room temperature to obtain Bcl-2 siRNA@DOX core / shell fiber material.

[0086] refer to Figure 4 As shown, Figure 4 This is a SEM image of the Bcl-2 siRNA@DOX core / shell fibrous material prepared in this embodiment. (Reference) Figure 4 As can be seen, the Bcl-2 siRNA@DOX core / shell fiber material prepared by this invention is composed of continuous long fibers with an average fiber diameter of 3.25±0.73um.

[0087] Example 4

[0088] A method for preparing Bcl-2 siRNA@DOX core / shell fibrous material for combination therapy of breast cancer includes the following steps:

[0089] (1) Dissolve 125 mg zinc nitrate hexahydrate, 10.5 mg DOX and 1 g 2-methylimidazole in 5.8 mL of water, stir at room temperature for 24 min, centrifuge and dry under vacuum at 63 °C to obtain ZIF-8@DOX.

[0090] (2)a. Dissolve 0.36 g poloxamer and 420 μL 1,3,5-trimethylbenzene in a mixed solvent of 75 mL water and 60 mL ethanol. After stirring for 30 min, add 90 mg tris(hydroxymethyl)aminomethane and then add 60 mg dopamine hydrochloride. Stir at room temperature for 24 h, centrifuge, and vacuum dry at 50 °C to obtain MPDA.

[0091] b. 2.5 mg MPDA and 52 mg polyethyleneimine were placed in 8 mL of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer at pH 8.5 and stirred at room temperature in the dark for 24 h. After centrifugation and drying at 25 °C, polyethyleneimine-modified mesoporous polydopamine MP was obtained. The mass ratio of MPDA to PEI1.8k was 1:20.8.

[0092] c. Dissolve 1 mg Bcl-2 siRNA and 25 mg MP in DEPC water to obtain a Bcl-2 siRNA concentration of 0.1 μg / μL. Vortex for 10 min and incubate at room temperature for 50 min to obtain an MPB solution. Freeze-dry the MPB solution to obtain MPB particles. The mass ratio of Bcl-2 siRNA to MP is 1:25.

[0093] (3) Dissolve 0.858g PVP and 2.002g PLA in a mixture of 19mL chloroform and 3mL methanol, and stir at room temperature to form a milky white homogeneous solution. Add 0.05g ZIF-8@DOX under stirring, and mix and disperse evenly to obtain the shell spinning solution. The mass ratio of ZIF-8@DOX to PVP is 1:17.2.

[0094] 0.16 g PVP and 0.64 g PCL were dissolved in a mixed solution of 8 mL chloroform and 2 mL N,N-dimethylformamide (DMF). The solution was stirred at room temperature to form a milky white homogeneous solution. 3.2 mg MPB particles were added under ultrasonic or vortex oscillation to obtain the core spinning solution. The mass ratio of MPB to PVP was 1:50.

[0095] (4) The electrospinning solution obtained in (3) was coaxially electrospinned using an electrospinning machine. The spinning voltage was set to 18.0 kV, the core spinning solution injection rate was 0.15 mm / min, the shell spinning solution injection rate was 0.3 mm / min, the needle distance from the receiver was 19 cm, the spun fibers were received using a fixed receiver, the humidity in the spinning box was maintained at 28%, and spinning was carried out at room temperature to obtain Bcl-2 siRNA@DOX core / shell fiber material.

[0096] In this embodiment, the cytotoxicity assay method for human breast cancer MCF-7 cells prepared in this embodiment was used to conduct a cytotoxicity assay on the Bcl-2 siRNA@DOX core / shell fiber material. In this embodiment, the survival rate of MCF-7 cells treated with the Bcl-2 siRNA@DOX core / shell fiber material for 48 hours was 26.5%, which shows a high killing effect on cancer cells.

[0097] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for preparing Bcl-2 siRNA@DOX core / shell fiber materials for combined treatment of breast cancer, characterized in that, The preparation method comprises the following steps: S1: polyvinylpyrrolidone, left-handed polylactic acid is dissolved in a mixed solution of chloroform and methanol, stirred at room temperature to form a milky white homogeneous solution, then ZIF-8@DOX is added under stirring condition, and the shell spinning solution is obtained after mixing uniformly; S2: polyvinylpyrrolidone, polycaprolactone is dissolved in a mixed solution of chloroform and N,N-dimethylformamide, stirred at room temperature to form a milky white homogeneous solution, and MPB particles are added under ultrasonic or vortex oscillation to obtain a core layer spinning solution; S3: the shell spinning solution obtained in step S1 and the core layer spinning solution obtained in step S2 are coaxially electrospun to obtain a Bcl-2siRNA@DOX core / shell fiber material; The preparation method of ZIF-8@DOX in the step S1 comprises the following steps: Zinc nitrate hexahydrate, DOX and 2-methyl imidazole are dissolved in water, stirred at room temperature for 15-30 min, centrifuged, dried at 50-70 DEG C to obtain ZIF-8 loaded with DOX; The mass ratio of the zinc nitrate hexahydrate to the 2-methyl imidazole is 1:7-10, the mass ratio of the DOX to the 2-methyl imidazole is 1:80-100, and the mass of the DOX and the volume of water are 1.5-2 g / L; The preparation method of MPB particles in the step S2 comprises the following steps: The DEPC solution of Bcl-2siRNA with a concentration of 0.08-0.12 ug / uL is mixed with MP, then oscillation, room temperature incubation and freeze-drying are sequentially performed to obtain MPB particles; The preparation method of MP comprises the following steps: MPDA and PEI1.8k are stirred at room temperature in the dark in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a pH of 8-9 at a mass ratio of 1:12-28, centrifuged and dried to obtain MP.

2. The production method according to claim 1, characterized by, The mass ratio of polyvinylpyrrolidone to left-handed polylactic acid in the step S1 is 1:2-4, the volume ratio of methanol to chloroform is 1:5-7, the total concentration of polyvinylpyrrolidone and left-handed polylactic acid is 10-15%, and the mass ratio of ZIF-8@DOX to polyvinylpyrrolidone is 1:9-30; The mass ratio of polyvinylpyrrolidone to polycaprolactone in the step S2 is 1:3-7, the volume ratio of N,N-dimethylformamide to chloroform is 1:3-6, the total concentration of polyvinylpyrrolidone and polycaprolactone is 6-10%, and the mass ratio of MPB to polyvinylpyrrolidone is 1:45-60.

3. The preparation method according to claim 1, characterized in that, The mass ratio of Bcl-2siRNA to MP is 1:10-50.

4. The method of claim 1, wherein, The mass ratio of the zinc nitrate hexahydrate to the 2-methyl imidazole is 1:8-9, and the mass ratio of the DOX to the 2-methyl imidazole is 1:83-95.

5. The preparation method according to claim 3, characterized in that, The mass ratio of the Bcl-2siRNA to the MP is 1:20-40.

6. The method of claim 1, wherein, The mass ratio of the MPDA to the PEI1.8k is 1:16-24.

7. The preparation method according to claim 1, characterized in that, The coaxial electrospinning comprises the following steps: The spinning voltage is 15.0-23.0kv, the core layer spinning solution injection rate is 0.1-0.2mm / min, the shell layer spinning solution injection rate is 0.2-0.4mm / min, the needle distance from the receiver is 10-20cm, the fixed receiver is used to receive the spinning fiber, and the humidity in the spinning box is maintained in the range of 25-35%, and the spinning is carried out at room temperature.

8. The breast cancer combination therapy Bcl-2 siRNA@DOX core / shell fibrous material prepared by the method according to any one of claims 1-7, characterized in that, The Bcl-2siRNA@DOX core / shell fiber material is a fiber membrane composed of Bcl-2siRNA@DOX-loaded fibers, and the Bcl-2siRNA@DOX-loaded core / shell fibers are continuous long fibers with a diameter of 0.35-5um.

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