Anti-tumor dual-targeted nano drug delivery system, preparation method and application thereof

Abstract

The present invention belongs to the technical field of nano-delivery system and targeted therapy of ovarian cancer, and specifically relates to an anti-tumor dual-targeted nano-drug delivery system, a preparation method and its application. The anti-tumor dual-targeted nano-drug delivery system of the present invention is prepared by using polyamidoamine as a nano-carrier and simultaneously encapsulating therapeutic siRNA and paclitaxel using a nano-carrier; the surface of the nano-carrier is modified with hyaluronic acid and heparin; the therapeutic siRNA is siCXCR2 and siPD-L1. The anti-tumor dual-targeted nano-drug delivery system provided by the present invention can effectively target ovarian cancer cells, significantly inhibit their proliferation and migration, and co-load siCXCR2, siPD-L1 and paclitaxel to have a synergistic anti-cancer effect in vitro, showing a good synergistic anti-cancer effect. In addition, the nano-drug delivery system of the present invention has low cytotoxicity, good biosafety, and has good clinical application prospects in the preparation of anti-ovarian cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-delivery system and ovarian cancer targeted therapy, and specifically relates to an anti-tumor dual-targeted nano-drug delivery system, a preparation method and an application thereof. Background Art

[0002] Ovarian cancer (OC) is the most lethal gynecological malignancy. It is insidious and has no obvious symptoms in the early stage. Most ovarian cancer patients are not diagnosed until the late stage. Therefore, the treatment effect and prognosis are poor. It is highly sensitive to chemotherapy and is often treated with combined chemotherapy after surgery. Paclitaxel (PTX) is a commonly used chemotherapy drug for ovarian cancer, but it has no tumor targeting, high toxicity, and many adverse reactions, resulting in limited therapeutic effects. Most patients will eventually relapse, resulting in a reduced survival rate. Therefore, in order to improve the clinical outcome of ovarian cancer, it is urgent to explore new safe and effective treatment strategies.

[0003] The binding of programmed cell death-ligand 1 (PD-L1) and programmed cell death protein-1 (PD-1) can induce T lymphocyte apoptosis, inhibit anti-tumor immune response, and promote tumor cell growth and migration. Ovarian cancer immunotherapy represented by anti-PD-1 / PD-L1 monoclonal antibodies has gradually been applied in clinical practice, but due to the different expression rates of PD-L1 on different types of ovarian cancer, its clinical treatment effect also varies to a certain extent. CXC chemokine receptor 2 (CXCR2) is persistently overexpressed in ovarian cancer cells, which promotes tumor invasion and development and is a poor prognostic factor for ovarian cancer. Tumor cells can bind to CXCR2 in a paracrine and autocrine manner, recruit bone marrow-derived suppressor cells to aggregate into the tumor microenvironment (TME), and promote tumor angiogenesis, tumor cell growth and metastasis. Small interfering RNA (siRNA) can specifically inhibit the expression of pathogenic genes in cancer cells, effectively inhibit tumor growth, and generally does not interfere with normal gene expression, and can play the same role as targeted drugs.

[0004] Cluster of differentiation 44 (CD44) is an acidic glycoprotein and a natural receptor for hyaluronic acid (HA). It is often detected in ovarian cancer cells. Epidermal growth factor receptor (EGFR) is overexpressed in most ovarian cancers and has a high affinity for heparin (HEP). The addition of exogenous heparin can inhibit EGFR activation and hinder the progression of malignant tumors. Polyamidoamine (PAMAM) is a type of dendritic macromolecule with a positive zeta potential on the surface, making it a good candidate for delivering negatively charged molecules (including siRNA).

[0005] Combining immunotherapy and chemotherapy can improve the treatment effect of ovarian cancer and improve the prognosis and survival rate of patients. However, clinical chemotherapy drugs and targeted drugs are often limited by relatively low tumor targeting, strong toxic side effects and slow release of targeted drugs. In order to promote drug targeting and drug release in cancer cells, researchers have developed various types of nanocarriers. However, there is still a lack of a nano-delivery system that can effectively target ovarian cancer and can use nanocarriers as a medium to combine chemotherapy and immunotherapy to synergistically enhance the treatment effect of ovarian cancer. Summary of the invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide an anti-tumor dual-targeted nanodrug delivery system, which has good biosafety, manifested as active targeting guided by hyaluronic acid and heparin, and can simultaneously and accurately deliver chemotherapy drugs PTX and siCXCR2, siPD-L1 to ovarian cancer cells, and has good efficacy and high safety.

[0007] The present invention also aims to provide a method for preparing an anti-tumor dual-targeted nano drug delivery system, which has mild conditions and simple process and can effectively prepare an anti-tumor dual-targeted nano drug delivery system.

[0008] The present invention also aims to provide an application of an anti-tumor dual-targeting nano drug delivery system.

[0009] In order to achieve the above-mentioned purpose, the anti-tumor dual-targeted nano drug delivery system of the present invention is implemented by the following technical scheme:

[0010] An anti-tumor dual-targeted nano drug delivery system is prepared by using polyamidoamine as a nanocarrier and simultaneously encapsulating therapeutic siRNA and paclitaxel in the nanocarrier; the surface of the nanocarrier is modified with hyaluronic acid and heparin; the therapeutic siRNA is siCXCR2 and siPD-L1.

[0011] The anti-tumor dual-targeted nano drug delivery system provided by the present invention is characterized by active targeting guided by hyaluronic acid and heparin, and can accurately deliver chemotherapy drugs PTX and siCXCR2 and siPD-L1 to ovarian cancer cells. In in vitro cell experiments, the anti-tumor dual-targeted nano drug delivery system effectively interferes with the expression of PD-L1 and CXCR2 on ovarian cancer cells and simultaneously delivers PTX, showing excellent synergistic anti-cancer effects. In addition, the hyaluronic acid / heparin@polyamidoamine carrier has low cytotoxicity and good biosafety, which can effectively lay a theoretical and experimental foundation for the subsequent clinical transformation of ovarian cancer treatment.

[0012] The preparation method of the anti-tumor dual-targeting nano drug delivery system of the present invention comprises the following steps:

[0013] (1) mixing a methanol solution of paclitaxel with a polyamidoamine solution, and then evaporating to remove the methanol. The remaining solution is centrifuged. The supernatant obtained after centrifugation is a polyamidoamine / paclitaxel complex, which is recorded as PAM / PTX;

[0014] (2) Rotating and incubating the mixed solution of siCXCR2 and siPD-L1 with the polyamidoamine solution to obtain a polyamidoamine@siCXCR2 / siPD-L1 solution, denoted as PAM@siC / siP;

[0015] (3) PAM / PTX and PAM@siC / siP are mixed and stirred to obtain a PAM@siC / siP / PTX complex, and then a hyaluronic acid solution is added to the PAM@siC / siP / PTX complex and mixed and stirred, and then a heparin solution is added and mixed and stirred to obtain the anti-tumor dual-targeted nano drug delivery system, which is recorded as HA / HEP@PAM@siC / siP / PTX;

[0016] There is no distinction between the order of steps (1) and (2).

[0017] The preparation method of the anti-tumor dual-targeted nano drug delivery system of the present invention, on the one hand, siRNA is adsorbed by the electrostatic action of polyamidoamine (PAMAM) to obtain PAM@siC / siP, on the other hand, PAMAM is used to encapsulate the hydrophobic drug paclitaxel (PTX), and hyaluronic acid (HA) and heparin (HEP) with tumor targeting function are used to modify it successively, and the nano drug delivery system HA / HEP@PAM@siC / siP / PTX is successfully prepared. In the above preparation method, after PTX and PAMAM are stirred and reacted, PTX will be adsorbed in the dendritic voids of PAMAM, PAMAM and siRNA are electrostatically adsorbed and mixed with PAM / PTX, and hyaluronic acid is added to wrap PAM@siC / siP / PTX, which plays a protective role on the one hand, and hyaluronic acid has a targeting ability on the other hand. Heparin will replace the siRNA in PAM / siRNA to make it free to a certain extent. Therefore, the present invention adopts the method of first wrapping HA and then wrapping HEP to have the effect of targeted modification of nanoparticles.

[0018] Furthermore, in step (1), the concentration of the paclitaxel methanol solution is 4 mg / mL; the concentration of the polyamide amine solution is 4 mg / mL, and the solvent used for the polyamide amine solution is PBS; the volume ratio of the paclitaxel methanol solution to the polyamide amine solution is 1:1.

[0019] Furthermore, in step (2), the mixed solution of siCXCR2 and siPD-L1 is obtained by mixing siCXCR2 solution with siPD-L1 solution; the solvents used in the siCXCR2 solution and siPD-L1 solution are both DEPC water, and the concentration is 0.264ug / uL; the mixing volume ratio of siCXCR2 solution and siPD-L1 solution is 1:1.

[0020] Preferably, in step (2), the concentration of the polyamidoamine solution is 1 mg / mL, the solvent used for the polyamidoamine solution is PBS; and the mixed volume ratio of the mixed solution of siCXCR2 and siPD-L1 to the polyamidoamine solution is 7:20.

[0021] More preferably, the concentration of the solvent PBS is 0.01 M and the pH is 7.4.

[0022] As a more preferred scheme, in step (3), the mass ratio of paclitaxel in PAMAM / PTX to siCXCR2 and siPD-L1 in PAM@siC / siP (PTX:siCXCR2:siPD-L1) is 50:1:1; the hyaluronic acid solution is a PBS solution with a concentration of 4 mg / mL; and the heparin solution is a PBS solution with a mass concentration of 4 mg / mL.

[0023] Furthermore, in step (1), the mixing and stirring time is 10 to 15 hours; the centrifugation is performed at a speed of 3000 to 5000 rpm for 4 to 6 minutes; and in step (2), the rotary incubation is performed at a temperature of 37° C. and a speed of 300 to 500 rpm for 20 to 40 minutes.

[0024] Preferably, in the anti-tumor dual-targeting nano drug delivery system, the mass ratio of polyamidoamine, therapeutic siRNA, paclitaxel, hyaluronic acid and heparin is 1.2:0.09:1:1.03:0.17.

[0025] The present invention also provides the application of the anti-tumor dual-targeted nano drug delivery system, specifically the application in the preparation of therapeutic drugs targeting tumors.

[0026] Preferably, the tumor is ovarian cancer.

[0027] The beneficial effects of the present invention are:

[0028] The anti-tumor dual-targeted nano drug delivery system HA / HEP@PAM@siC / siP / PTX provided by the present invention adsorbs siRNA through the electrostatic effect of PAMAM, encapsulates the hydrophobic drug PTX, and simultaneously modifies HA and HEP with tumor targeting function, successfully preparing the nano drug delivery system HA / HEP@PAM@siC / siP / PTX, and characterizing it. TEM results show that the HA / HEP@PAM prepared by the present invention has uniform particle size, good dispersion, and a quasi-circular morphology, and can exist stably in the system. According to the standard curve of PTX, the drug loading of the PAM / PTX nanocarrier is 39.4%, and the encapsulation rate is 65%. The sustained release test of PTX shows that the cumulative release of PTX in HA / HEP@PAM@siC / siP / PTX under pH5 and pH7.4 is 56.08% and 47.18% respectively in 96h. After adding hyaluronidase, the cumulative release of PTX can reach 89.94% and 80.97%, proving that the drug release of HA / HEP@PAM@siC / siP / PTX has tumor microenvironment sensitivity and sustained release effect, and the continuous release of PTX by the nanosystem is beneficial to improve the long-term anti-tumor efficiency and improve the accumulation of drugs in the tumor site. In addition, the present invention finds through cytotoxicity test that under the action of nano empty carriers, the cell survival rate is higher than 90%, and the hemolysis test results show that the hemolysis rate of nano empty carriers and HA / HEP@PAM@siC / siP / PTX is less than 5%, proving that the nano material has biosafety and is a good drug delivery carrier.

[0029] At the same time, the present invention verifies the uptake of HA / HEP@PAM@siC / siP / PTX by ovarian cancer cells at the cellular level. According to the high expression of CD44 and EGFR in the ovarian cancer cell membrane, the cell uptake test proves that compared with the simple chemotherapy drug PTX and therapeutic siRNA, the dual-targeted nano drug-carrying material modified with HA and HEP can more effectively target tumor cells and be better taken up by tumor cells. The Western Blot results show that under the action of the nanomaterials loaded with siCXCR2 and siPD-L1, the expression of ovarian cancer cell target genes CXCR2 and PD-L1 at the protein level is reduced, proving that the nanomaterials of HA / HEP@PAM@siC / siP / PTX have a gene silencing effect and inhibit the expression of target proteins. In the cell proliferation test and apoptosis staining test, compared with the blank control group and the PTX group alone, HA / HEP@PAM@siC / siP / PTX has a more obvious inhibitory effect on cell proliferation, which indicates that the combined application of chemotherapy and immunotherapy has a synergistic effect in anti-tumor activity. The scratch test results showed that HA / HEP@PAM co-loaded with siCXCR2, siPD-L1 and PTX could synergistically inhibit the migration of ovarian cancer ID8 cells, that is, silencing CXCR2 and PD-L1 inhibited the migration of cancer cells to a certain extent, indicating that the expression of CXCR2 and PD-L1 played a partial role in the metastasis of tumors. Western Blot was used to detect the expression level of apoptosis-related proteins in the treated ID8 cells. The results showed that HA / HEP@PAM@siC / siP / PTX could more effectively increase the expression of apoptotic proteins such as Caspase3 and Bax, while reducing the expression of anti-apoptotic protein Bcl-2, confirming that the nano-delivery system loaded with siCXCR2 and siPD-L1 enhanced the tumor cell killing effect of the chemotherapy drug PTX and promoted cell apoptosis. The effect of the nano-drug delivery system on the cytoskeleton after acting on cancer cells was studied by cytoskeleton experiments. The results showed that the nuclei of ovarian cancer cells acted on by HA / HEP@PAM@siC / siP / PTX were shrunken and fragmented, the cell microfilament skeleton was disordered, and the morphology was severely damaged. This shows that compared with simple PTX, the siCXCR2, siPD-L1 and PTX loaded in the nano-drug carrier material have a certain synergistic effect, have a better killing effect on ovarian cancer cells, and inhibit the growth and development of tumor cells.

[0030] Therefore, the anti-tumor dual-targeted nano drug delivery system provided by the present invention can effectively target ovarian cancer cells, significantly inhibit their proliferation and migration, and co-load siCXCR2, siPD-L1 and PTX have a synergistic anti-cancer effect in vitro, showing a good synergistic anti-cancer effect. In addition, HA / HEP@PAM has low cytotoxicity and good biosafety, and has good clinical application prospects in the preparation of anti-ovarian cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The results of gel electrophoresis and potential test of PAM@siRNA solutions with different nitrogen-phosphorus ratios in Experimental Example 1 are shown; (A) agarose gel electrophoresis retardation test: 1-7 represent N / P of 0:1, 1:1, 5:1, 10:1, 15:1, 20:1, and 30:1, respectively; (B) zeta potential;

[0032] Figure 2 The results of the particle size, potential and stability analysis of different nanomaterials in Experimental Example 2 are shown in Figure 2; (A) the particle size of the nano empty carrier HA / HEP@PAM and the nano composite HA / HEP@PAM@siC / siP / PTX; (B) the zeta potential of different nano composites; (C) the particle size stability measurement results;

[0033] Figure 3 Analysis of the drug loading performance of the PAM / PTX nanocarrier in Experimental Example 3: (A) full-band scanning of PTX; (B) standard curve of PTX;

[0034] Figure 4 is the in vitro release curve of PTX in Experimental Example 4;

[0035] Figure 5 This is a graph showing the hemolysis test results of the hollow carrier HA / HEP@PAM and the nanocomposite HA / HEP@PAM@siC / siP / PTX in Experimental Example 5;

[0036] Figure 6 This is a graph showing the effects of different concentrations of empty carrier HA / HEP@PAM and nanocomplex HA / HEP@PAM@siC / siP / PTX on the survival rate of ovarian cancer cells in Experimental Example 6;

[0037] Figure 7 Cell uptake diagrams of different nanocarriers in Experimental Example 7; (A) 2h uptake diagram; (B) 6h uptake diagram; a, b, c, d represent Control, Free DOX, FAM-siRNA, HA / HEP@PAM@FAM-siRNA / DOX, respectively;

[0038] Figure 8The expression results of CXCR2 and PD-L1 in ovarian cancer cells in Experimental Example 8 are shown in Figure 8; wherein: (A) protein level expression; (B) quantitative analysis; a, b, c, d, e, and f represent Control, HA / HEP@PAM, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, and HA / HEP@PAM@siC / siP / PTX, respectively;

[0039] Fig. 9 The effects of different nano-drug delivery systems on the proliferation of ovarian cancer cells in Experimental Example 9; a, b, c, d, e, f, and g represent Control, HA / HEP@PAM, Free PTX, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, and HA / HEP@PAM@siC / siP / PTX, respectively;

[0040] Fig.10 The effects of different nano-drug delivery systems on the migration of ovarian cancer cells in Experimental Example 10; (A) scratch results; (B) quantitative analysis; a, b, c, d, e, f, g represent Control, HA / HEP@PAM, Free PTX, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, HA / HEP@PAM@siC / siP / PTX, respectively;

[0041] Fig.11 The effects of different nano-drug delivery systems on the expression of apoptosis-related proteins in Experimental Example 11; wherein, (A) expression of apoptosis-related proteins; (B) quantitative analysis; a, b, c, d, e, and f represent Control, HA / HEP@PAM, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, and HA / HEP@PAM@siC / siP / PTX, respectively. DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but they do not constitute a limitation on the protection scope of the present invention.

[0043] In the following examples, the mouse epithelial ovarian cancer ID8 cells involved were purchased from Cebio Biotech Co., Ltd.; polyamidoamine (PAMAM) was purchased from Weihai Chenyuan Molecular New Materials Co., Ltd.; paclitaxel (PTX) was purchased from Shanghai Ruiyong Biotechnology Co., Ltd.; doxorubicin (DOX) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; hyaluronic acid (HA) was purchased from Shandong Avicon Biotechnology Co., Ltd.; heparin (HEP) was purchased from Dalian Meilun Biotechnology Co., Ltd.; hyaluronidase was purchased from Bio Basic, Canada; DMEM high glucose medium was purchased from HyClone, USA; fetal bovine serum was purchased from Invigentech, USA; CCK8 kit was purchased from Hangzhou Omer Pharmaceutical Co., Ltd.; RIPA tissue / cell lysis buffer, FITC-labeled phalloidin, and Hoechst 33258 staining solution were purchased from Beijing Solarbio; Calcein AM / PI cell activity and toxicity detection kits were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; goat anti-rabbit secondary antibody, PD-L1 / CD724 antibody, CXCR2 antibody, Bax antibody, Bcl-2 antibody, and Caspase3 antibody were purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd. Other equipment and reagents not specifically specified were available from commercial sources.

[0044] The siRNAs involved in the following examples (including siCXCR2, siPD-L1, and negative control siRNA) were provided by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0045] Sense primer for CXCR2: 5′-CCAUCAUCUAUGCCUUUAUTT-3′;

[0046] Antisense primer: 5′-AUAAAGGCAUAGAUGAUGGTT-3′.

[0047] Sense primer for PD-L1: 5′-CCCACAUAAAAAACAGUUGTT-3′;

[0048] Antisense primer: 5′-CAACUGUUUUUUAUGUGGGTT-3′.

[0049] Negative control sense primer: 5'-UUCUCCGAACGUGUCACGUTT-3',

[0050] Antisense primer: 5′-ACGUGACACGUUCGGAGAATT-3′.

[0051] Example 1

[0052] The anti-tumor dual-targeted nano drug delivery system of this embodiment uses polyamidoamine (PAMAM) as a nanocarrier, and is prepared by using the nanocarrier to simultaneously encapsulate therapeutic siRNA and the chemotherapy drug paclitaxel; the surface of the nanocarrier is modified with hyaluronic acid and heparin; the therapeutic siRNA is siCXCR2 and siPD-L1.

[0053] The preparation method of the above anti-tumor dual-targeting nano drug delivery system comprises the following steps:

[0054] (1) A methanol solution of paclitaxel (concentration of 4 mg / mL, amount of 2 mL) and a polyamidoamine solution (concentration of 4 mg / mL, solvent of 0.01 M, pH 7.4, 1× PBS, amount of 2 mL) were mixed and stirred for 12 h, and then the methanol was evaporated to remove the methanol. The remaining solution was centrifuged (4000 rpm, 5 min). The supernatant obtained after centrifugation was the polyamidoamine / paclitaxel complex, denoted as PAM / PTX;

[0055] (2) Rotating and incubating the mixed solution of siCXCR2 and siPD-L1 with the polyamidoamine solution to obtain a polyamidoamine@siCXCR2 / siPD-L1 solution, denoted as PAM@siC / siP;

[0056] Among them, the mixed solution of siCXCR2 and siPD-L1 is obtained by mixing siCXCR2 solution (concentration of 0.264ug / uL, solvent DEPC water, dosage 35uL) with siPD-L1 solution (concentration of 0.264ug / uL, solvent DEPC water, dosage 35uL); the concentration of the polyamide amine solution is 1mg / mL, the solvent used for the polyamide amine solution is 1×PBS, and the dosage is 100uL; the conditions of rotation incubation are: temperature 37°C, incubation at 300rpm for 30min.

[0057] (3) PAM / PTX and PAM@siC / siP were mixed and stirred to obtain a PAM@siC / siP / PTX complex, and then a hyaluronic acid solution (4 mg / mL PBS solution, added volume of 209.1 uL) was added to the PAM@siC / siP / PTX complex and mixed and stirred for 4 hours, and then a heparin solution (4 mg / mL PBS solution, added volume of 34.85 uL) was added and mixed and stirred for 10 minutes to obtain the anti-tumor dual-targeted nano drug delivery system, which was recorded as HA / HEP@PAM@siC / siP / PTX. The mass ratio of paclitaxel in PAMAM / PTX to siCXCR2 and siPD-L1 in PAM@siC / siP was 50:1:1. In the anti-tumor dual-targeted nanodrug delivery system, the mass ratio of PAMAM, siRNA, paclitaxel, hyaluronic acid, and heparin is 1.2:0.09:1:1.03:0.17.

[0058] Test example

[0059] Experimental Example 1 Determination of siRNA loading ratio

[0060] The siRNA solution (solute is negative control siRNA, solvent is DEPC water, concentration is 0.264ug / uL) was mixed with a proportionally diluted 1×PBS solution of PAMAM, and incubated at 37°C, 300rpm for 30min to obtain a PAM@siRNA solution.

[0061] 10 μL of PAM@siRNA solution with different nitrogen-phosphorus ratios (nitrogen-phosphorus ratio is the molar ratio of nitrogen in PAMAM to phosphorus in siRNA) was prepared, mixed with 2 μL of loading buffer, and loaded in order, 1% agarose gel electrophoresis, placed in 1×TAE electrophoresis buffer, constant voltage 110V electrophoresis for 20 min, and the optimal siRNA loading ratio was obtained through gel imaging results. The results are shown in Figure 1 shown.

[0062] Depend on Figure 1 It can be seen that the optimal ratio of PAMAM-encapsulated siRNA was optimized by agarose gel electrophoresis ( Figure 1A), the nitrogen-phosphorus ratio is 0:1, 1:1, 5:1, 10:1, 15:1, 20:1, and 30:1 from left to right. On the gel plate, naked siRNA (lane 1) has a bright band; lanes 2-4 also have bands, and lanes 5-7 can obviously block the migration of siRNA, and no obvious bands are seen, indicating that when N / P is 15:1, PAMAM can block the migration of siRNA, which proves that PAMAM can load siRNA at this time. Therefore, the present invention initially selects a ratio of N / P of 15:1 to prepare PAM@siRNA solution.

[0063] The results of zeta potential analysis showed that ( Figure 1 B), the zeta potential of PAM@siRNA is negative when N / P is 1:1 to 10:1, while the potential is positive when N / P is 15:1 and 30:1. Considering the further loading of HA and HEP (both of which are negatively charged), the present invention finally selects N / P of 15:1 as the optimal condition.

[0064] Experimental Example 2 Particle size and stability analysis of HA / HEP@PAM@siC / siP / PTX

[0065] The un-drug-encapsulated nanocarrier HA / HEP@PAM was used as the control group. When preparing the control group, PAMAM, HA and HEP samples were first dissolved in PBS to make solutions (all with a concentration of 4 mg / mL), and then 243.95uL of PAMAM solution was mixed with 209.1uL of HA solution and stirred for 4h, and then mixed with 34.85uL of HEP solution and stirred for 10min to obtain HA / HEP@PAM nanocomplex.

[0066] During the particle size test, a drop of HA / HEP@PAM nanocarrier and HA / HEP@PAM@siC / siP / PTX of Example 1 of the present invention were taken respectively, and a copper mesh was placed therein, and then stained with uranyl acetate and dried. The morphology and size of the nanocomposite were analyzed by transmission electron microscopy, and the particle size was determined by a particle size analyzer. The potential of different nanomaterials (PAMAM, PAM@siRNA, PAM / PTX, HA / HEP@PAM, HA / HEP@PAM@siC / siP / PTX) was tested, as well as the stability of HA / HEP@PAM and HA / HEP@PAM@siC / siP / PTX materials stored in water for 7 days. The results are as follows: Figure 2 shown.

[0067] Depend on Figure 2A shows that the hydrated particle size of the nanocarrier HA / HEP@PAM is about 250 nm. In Example 1 of the present invention, the particle size remains basically unchanged after HA / HEP@PAM is used to encapsulate PTX and siRNA. The potential analysis results are shown in Figure 2 As shown in B, the potential of PAMAM is +21mV, the potential of PAM@siRNA is +9mV, and the potential of the complex PAM / PTX obtained by PAMAM loading PTX is +7mV. After PAMAM loading HA and HEP, the charge of the nano empty carrier obtained is reversed, and the zeta potential is negative. The zeta potential of the nanocomposite HA / HEP@PAM@siC / siP / PTX of the present invention is -25mV. The change in the potential of the above materials indicates that the composite nanomaterial of the present invention is successfully prepared. Figure 2 C shows that the particle size of the HA / HEP@PAM@siC / siP / PTX nanocarrier of Example 1 did not change significantly in water within 7 days, and the stability was good.

[0068] Experimental Example 3 Determination of drug loading of PAM / PTX nanocarriers

[0069] Determination of the maximum wavelength and standard curve of PTX: Dissolve paclitaxel powder in methanol to prepare paclitaxel solution as the test group, and methanol as the control group. Use an ultraviolet spectrophotometer to scan the full band of PTX and determine the absorbance at the maximum wavelength. Then measure the absorbance values ​​of PTX solutions with different concentration gradients at the maximum wavelength, repeat the measurement three times, and draw a standard curve.

[0070] Preparation of PAM / PTX complex and determination of PTX: Mix equal volumes of PTX methanol solution (4 mg / mL) and PAMAM PBS solution (4 mg / mL) and centrifuge at 4000 rpm for 5 min. Mix the above liquid with methanol / water (1:1, v / v) and stir for 12 h, centrifuge at 4000 rpm for 5 min, remove methanol by rotary evaporation, the supernatant is the PAM / PTX solution, and the precipitate is the PTX that was not successfully encapsulated. Collect the supernatant and precipitate, dissolve the precipitate with methanol, and then measure the ultraviolet absorption at the maximum wavelength. Use the standard curve of PTX to determine the content, and calculate the drug loading and encapsulation efficiency. The results are as follows: Figure 3 shown.

[0071] Depend on Figure 3 The full-band scanning results of PTX by UV spectrophotometer show that PTX has a maximum absorbance at 227nm ( Figure 3A). PTX was diluted with methanol to different concentrations, and the absorbance at 227 nm was measured by UV spectrophotometer. The standard curve of PTX was drawn with y=0.0742x-0.031, R2=0.99. The results showed that the linearity of PTX solution was good in the range of 1-13 μg / mL, and the drug loading of PTX in the nano drug delivery system was calculated to be 39.4% and the encapsulation efficiency was 65% according to the standard curve. Figure 3 B). This indicates that PAMAM has good performance in encapsulating PTX.

[0072] Experimental Example 4 Drug responsive release analysis of HA / HEP@PAM@siC / siP / PTX

[0073] 1 mL of the HA / HEP@PAM@siC / siP / PTX complex of Example 1 (containing 1 mg PTX, and with or without hyaluronidase HAase) was placed in a dialysis bag with a molecular weight cutoff of 3500, and then the dialysis bag was placed in 12 mL PBS (pH 5 and pH 7.4), 37°C, 100 rpm, 1 mL of the supernatant release medium was taken out on time between 0-96 h, and an equal amount of PBS was added at the same time, and finally the absorbance of PTX was measured, and the cumulative release rate was calculated according to the standard curve. The test results are shown in FIG. Figure 4 shown.

[0074] Depend on Figure 4 The in vitro drug release results of HA / HEP@PAM@siC / siP / PTX show that under pH 7.4, the cumulative release of PTX in 96h is 47.18%, and after adding hyaluronidase, the cumulative release of PTX can reach 80.97%; under pH 5, after adding hyaluronidase, the cumulative release of PTX increases from 56.08% to 89.94%. The results show that the release of nanomaterials in pH 5 buffer solution increases significantly under the action of hyaluronidase, and the drug release increases with time, which proves that the PTX release of HA / HEP@PAM@siC / siP / PTX has tumor microenvironment sensitivity and sustained release effect, that is, PTX release is faster in a mildly acidic tumor microenvironment than in normal tissue or blood.

[0075] Test Example 5 Hemolytic Analysis

[0076] During the test, take an appropriate amount of fresh blood into a 3mL centrifuge tube, centrifuge at 3000rpm / min for 5 minutes, discard the supernatant, and add an appropriate amount of saline to prepare a 2% red blood cell suspension. Prepare different concentrations of HA / HEP@PAM and HA / HEP@PAM@siC / siP / PTX of Example 1, add an equal volume of 2% red blood cell suspension, and use saline and deionized water as negative and positive controls. Let stand at room temperature for 4 hours and centrifuge at 3000rpm for 5 minutes. Collect the supernatant, measure the absorbance at a wavelength of 545nm, and calculate the hemolysis rate. The results are as follows: Figure 5 shown.

[0077] Figure 5 It can be seen that after incubating HA / HEP@PAM solution and HA / HEP@PAM@siC / siP / PTX solution with 2% red blood cell suspension at different concentrations at 37°C, the hemolysis rates of both solutions were less than 5%, indicating that the nanocomplex of the present invention has good biocompatibility and can be used in subsequent experimental studies.

[0078] Experimental Example 6 Cytotoxicity Analysis of HA / HEP@PAM

[0079] ID8 ovarian cancer cells with good growth status were collected and inoculated into 96-well plates. HA / HEP@PAM solutions with different concentration gradients were added. After culturing for 24 hours, the culture medium was discarded, and CCK8 detection solution (90 μL serum-free culture medium and 10 μL CCK8 solution per well) was added and placed in an incubator for 1 hour. The absorbance at 450 nm was measured with an ELISA reader to calculate the survival rate. The results are shown in Figure 6 shown.

[0080] Depend on Figure 6 The CCK8 test results show that although the survival rate of ID8 cells gradually decreases with the increase of drug concentration, the overall cell survival rate is still greater than 90%, indicating that the HA / HEP@PAM nanocarriers synthesized in the range of 0-100 μg / mL have no obvious cytotoxicity and good biosafety.

[0081] Experimental Example 7 Cellular Uptake Analysis of Nanomaterials

[0082] Since PTX is non-fluorescent and DOX has red fluorescence, both DOX and PTX are chemotherapy drugs. Using a carrier to encapsulate DOX can verify whether the nanocarrier delivers the drug into the cell. During the experiment, the collected ID8 cells with good growth status were inoculated into a 24-well plate. After the cells adhered to the wall, the culture medium was discarded and washed twice with PBS. 1 mL of culture medium, Free DOX, FAM-siRNA, and HA / HEP@PAM@FAM-siRNA / DOX were added respectively, and the cells continued to be cultured in the incubator. The fluorescence intensity was observed under a fluorescence microscope at 2h and 6h, and photos were taken. The results are shown in the figure. Figure 7 shown.

[0083] Depend on Figure 7 The fluorescence microscopy results show that due to the active targeting effect of HA and HEP, DOX and siRNA encapsulated by nanocarriers are more easily taken up by ovarian cancer cells than Free DOX and FAM-siRNA, and the fluorescence intensity is significantly higher than that of the non-targeted carrier group. And as time goes by, the fluorescence intensity increases, indicating that the uptake degree gradually increases, indicating that the nanocarriers modified with HA and HEP have a certain degree of targeting, and the dual-targeted nanocarriers can significantly increase the uptake rate of ID8 cells for encapsulated PTX and therapeutic siRNA.

[0084] Experimental Example 8 Effect of Nanocarrier-loaded siRNA on the Expression of PD-L1 and CXCR2 in Ovarian Cancer Cells

[0085] ID8 cells were counted and inoculated into six-well plates, and treated with the same concentration of HA / HEP@PAM, Free PTX, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, and HA / HEP@PAM@siC / siP / PTX of Example 1 for 24 hours, and the cell group with only culture medium was used as the control group. Proteins of cells in each group were extracted and quantified. The expression of CXCR2, PD-L1 and apoptosis proteins was detected by Western Blot, ECL luminescent liquid was prepared, the bands were exposed and photographed, and analyzed using Image J software. The results are shown in Figure 8 shown.

[0086] Figure 8 The results of Western Blot showed that the expression of CXCR2 in ID8 cells decreased significantly after the action of HA / HEP@PAM@siC / PTX and HA / HEP@PAM@siC / siP / PTX groups; the protein expression of PD-L1 in ID8 cells decreased significantly under the action of HA / HEP@PAM@siP / PTX and HA / HEP@PAM@siC / siP / PTX test groups. The above results show that the nanomaterials loaded with siCXCR2 and siPD-L1 have obvious gene silencing effects and inhibit the expression of target proteins.

[0087] Experimental Example 9 Analysis of the effects of different nanocarriers on the proliferation of ovarian cancer cells

[0088] The collected ID8 cells were counted and inoculated into a 96-well plate, and solutions with different PTX concentrations and different experimental groups (HA / HEP@PAM, Free PTX, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, and HA / HEP@PAM@siC / siP / PTX in Example 1) were added. The cell group with only culture medium was the control group, and the pure culture medium group was the blank group. After 24 hours of incubation in the incubator, CCK8 detection solution was added and culture was continued for 1 hour. The absorbance at 450nm was measured with an enzyme marker to calculate the survival rate. The results are shown in Fig. 9 shown.

[0089] Depend on Fig. 9 The results of the cell proliferation inhibition test show that HA / HEP@PAM@siC / siP / PTX has a significant effect in inhibiting the proliferation of ovarian cancer cells. With the increase of carrier concentration, the survival rates of ovarian cancer cells in the HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, and HA / HEP@PAM@siC / siP / PTX groups gradually decreased, and the cell survival rate of HA / HEP@PAM@siC / siP / PTX decreased most significantly, indicating that simultaneous loading of PTX, siCXCR2, and siPD-L1 can effectively inhibit the proliferation of cancer cells.

[0090] Experimental Example 10 Analysis of the effects of different nanocarriers on ovarian cancer cell migration

[0091] Before the experiment, the marker pen and ruler were disinfected with alcohol and ultraviolet light, and horizontal lines were drawn on the back of a blank 6-well plate. After counting the collected ID8 cell suspension, they were inoculated into a six-well plate and cultured until the cells grew to about 95%. Parallel vertical lines were drawn with a 200μL pipette tip. The crossed-out cells were washed with PBS, the waste liquid was discarded, and serum-free culture medium, HA / HEP@PAM, Free PTX, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, and HA / HEP@PAM@siC / siP / PTX were added to the six-well plate respectively. The plates were observed and photographed under a microscope, and the time was recorded as 0h. After completion, the plates were put back into the incubator, and the width of the scratches at the same position was measured after 24h, and the scratch healing rate was calculated. The results are as follows: Fig.10 shown.

[0092] Depend on Fig.10The scratch test results showed that compared with the Control group, blank vector group and Free PTX group, the other experimental groups all showed a certain inhibitory effect on migration, and the inhibitory effect of HA / HEP@PAM@siC / siP / PTX was more obvious, indicating that under the same PTX concentration, the combined use of PTX and siCXCR2 and siPD-L1 has a stronger ability to inhibit the migration of ovarian cancer cells.

[0093] Experimental Example 11 Analysis of expression of apoptosis-related proteins in ovarian cancer ID8 cells

[0094] Western Blot test was used to detect the effect of different groups of experimental nanomaterials on the expression of apoptosis-related proteins such as Bcl-2, Caspase3, and Bax in ID8 cells. Fig.11 shown.

[0095] Depend on Fig.11 It can be seen that after HA / HEP@PAM@siC / siP / PTX treatment, the expression of apoptotic proteins Caspase3 and Bax was significantly enhanced, and the expression of anti-apoptotic protein Bcl-2 was significantly reduced, thereby inducing apoptosis of cancer cells.

[0096] Experimental Example 12 Analysis of the effect of HA / HEP@PAM@siC / siP / PTX on the viability of ovarian cancer cells

[0097] Calcein-AM / PI double staining test was used to analyze the expression of apoptosis-related proteins in ovarian cancer ID8 cells. The process was as follows: the collected ID8 cells with good growth status were inoculated into 24-well plates, and culture medium, HA / HEP@PAM, Free PTX, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, HA / HEP@PAM@siC / siP / PTX were added respectively. The cell group with only culture medium was used as the control group, and culture was continued for 24 hours. Calcein-AM / PI reaction solution was prepared under light-proof conditions, mixed thoroughly, 300uL of reaction solution was added to each well, and after culturing in the incubator for 30 minutes in the dark, the reaction solution was discarded, PBS was washed once, and the staining effect was observed under a fluorescence microscope and photographed for analysis.

[0098] The results of the Calcein-AM / PI double staining test showed that compared with the control group, the blank vector group, and the Free PTX group, the number of apoptotic cells in the other experimental groups after treatment of ovarian cancer cells increased, and the cell viability decreased, and the effect of HA / HEP@PAM@siC / siP / PTX was more obvious. These data show that HA / HEP@PAM@siC / siP / PTX can reduce the viability of ovarian cancer ID8 cells and has a strong sustained killing effect on cancer cells.

[0099] Experimental Example 13 Analysis of the effect of HA / HEP@PAM@siC / siP / PTX on the skeleton of ovarian cancer cells

[0100] The collected ID8 cells with good growth status were inoculated into 24-well plates, and culture medium, HA / HEP@PAM, Free PTX, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, and HA / HEP@PAM@siC / siP / PTX were added respectively. The cell group with only culture medium was used as the control group and cultured for 24 hours. 4% paraformaldehyde was added according to the reagent instructions and fixed at room temperature. 0.5% Triton X-100 was used to permeabilize the cells at room temperature. FITC-Phalloidin working solution and Hoechst 33258 were added successively, and the cells were incubated in the incubator in the dark for 30 minutes, the working solution was discarded, and the cells were washed with PBS. The staining was observed under a fluorescence inverted microscope and photographed for analysis.

[0101] The results of the cytoskeleton test show that the microfilament skeleton of the cells in the Control group and the blank vector group is evenly arranged, and the structure and morphology are normal. The number of ovarian cancer cells treated with Free PTX, HA / HEP@PAM / PTX, HA / HEP@PAM@siC / PTX, HA / HEP@PAM@siP / PTX, and HA / HEP@PAM@siC / siP / PTX decreased, the cells shrank, the cell nuclei were fragmented, and the morphology was slender. In addition, the skeleton of ovarian cancer cells in the HA / HEP@PAM@siC / siP / PTX group was more disordered, indicating that the proliferation inhibition and apoptosis promotion effects on cancer cells were stronger.

[0102] In summary, the anti-tumor dual-targeted nano drug delivery system provided by the present invention can effectively target ovarian cancer cells, effectively inhibit their proliferation and migration, and co-load siCXCR2, siPD-L1 and PTX have a synergistic anti-cancer effect in vitro, showing a good synergistic anti-cancer effect. In addition, HA / HEP@PAM has low cytotoxicity and good biosafety, and has good clinical application prospects in the preparation of anti-ovarian cancer drugs.

Claims

1. An anti-tumor dual-targeting nano drug delivery system, characterized in that: The anti-tumor dual-targeted nano drug delivery system is prepared by using polyamidoamine as a nanocarrier and using the nanocarrier to simultaneously encapsulate therapeutic siRNA and paclitaxel; the surface of the nanocarrier is modified with hyaluronic acid and heparin; the therapeutic siRNA is siCXCR2 and siPD-L1; The preparation method of the anti-tumor dual-targeting nano drug delivery system is as follows: (1) mixing a methanol solution of paclitaxel with a polyamidoamine solution, and then evaporating to remove the methanol. The remaining solution is centrifuged. The supernatant obtained after centrifugation is a polyamidoamine / paclitaxel complex, which is recorded as PAM / PTX; (2) Rotating and incubating the mixed solution of siCXCR2 and siPD-L1 with the polyamidoamine solution to obtain a polyamidoamine@siCXCR2 / siPD-L1 solution, denoted as PAM@siC / siP; (3) PAM / PTX and PAM@siC / siP are mixed and stirred to obtain a PAM@siC / siP / PTX complex, and then a hyaluronic acid solution is added to the PAM@siC / siP / PTX complex and mixed and stirred, and then a heparin solution is added and mixed and stirred to obtain the anti-tumor dual-targeted nano drug delivery system, which is recorded as HA / HEP@PAM@siC / siP / PTX; There is no distinction between the order of steps (1) and (2).

2. The anti-tumor dual-targeting nano drug delivery system according to claim 1, characterized in that: In step (1), the concentration of the paclitaxel methanol solution is 4 mg / mL; the concentration of the polyamide amine solution is 4 mg / mL, and the solvent used for the polyamide amine solution is PBS; the volume ratio of the paclitaxel methanol solution to the polyamide amine solution is 1:

1.

3. The anti-tumor dual-targeting nano drug delivery system according to claim 1, characterized in that: In step (2), the mixed solution of siCXCR2 and siPD-L1 is obtained by mixing siCXCR2 solution with siPD-L1 solution; the solvents used in the siCXCR2 solution and siPD-L1 solution are both DEPC water, and the concentration is 0.264ug / uL; the mixing volume ratio of siCXCR2 solution and siPD-L1 solution is 1:

1.

4. The anti-tumor dual-targeting nano drug delivery system according to claim 1, characterized in that: In step (2), the concentration of the polyamidoamine solution is 1 mg / mL, and the solvent used for the polyamidoamine solution is PBS; the mixed volume ratio of the mixed solution of siCXCR2 and siPD-L1 to the polyamidoamine solution is 7:

20.

5. The anti-tumor dual-targeting nano drug delivery system according to claim 1, characterized in that: In step (3), the mass ratio of paclitaxel in PAMAM / PTX to siCXCR2 and siPD-L1 in PAM@siC / siP (PTX:siCXCR2:siPD-L1) is 50:1:1; the hyaluronic acid solution is a PBS solution with a concentration of 4 mg / mL; The heparin solution is a PBS solution with a mass concentration of 4 mg / mL.

6. The anti-tumor dual-targeting nano drug delivery system according to claim 1, characterized in that: In step (1), the mixing and stirring time is 10 to 15 hours; the centrifugation is performed at a speed of 3000 to 5000 rpm for 4 to 6 minutes; in step (2), the rotary incubation is performed at a temperature of 37° C. and a speed of 300 to 500 rpm for 20 to 40 minutes.

7. The anti-tumor dual-targeting nano drug delivery system according to claim 1, characterized in that: In the anti-tumor dual-targeted nano drug delivery system, the mass ratio of polyamidoamine, therapeutic siRNA, paclitaxel, hyaluronic acid, and heparin is 1.2:0.09:1:1.03:0.

17.

8. An application of the anti-tumor dual-targeting nano drug delivery system as claimed in claim 1, characterized in that: Application in the preparation of therapeutic drugs targeting tumors.

9. The use of the anti-tumor dual-targeting nano drug delivery system according to claim 8, characterized in that: The tumor is ovarian cancer.

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