Hyperbranched cyclodextrin inclusion compound for adjusting tabular pseudopod and inflammatory microenvironment

Abstract

The invention discloses a hyperbranched cyclodextrin inclusion compound for adjusting tabular pseudopod and inflammatory microenvironment, and belongs to the technical field of medicines. The preparation method comprises the following steps: firstly, covalently coupling 2-(6-methoxynaphthalene-2-yl) acetic acid and a platinum tumor drug to obtain an anti-inflammatory tetravalent platinum prodrug, and then enabling the prodrug and cyclodextrin to form a cyclodextrin inclusion compound by virtue of host-guest interaction, thereby obtaining the nano delivery material capable of simultaneously inhibiting COX-2 and platy pseudopod. The cyclodextrin inclusion compound disclosed by the invention not only can coordinate and inhibit metastasis promoted by softening of ECM, but also can avoid cancer metastasis aggravated by hardening of ECM, and also can effectively kill cancer cells at the primary focus of cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a hyperbranched cyclodextrin inclusion compound for regulating lamellipodia and inflammatory microenvironment. BACKGROUND

[0002] Cervical cancer is one of the most common malignant tumors in women, and its incidence ranks second, posing a serious threat to women's health. Although cervical cancer screening and HPV vaccination have been widely used, due to the easy metastasis of cancer at the treatment stage, the overall survival rate of patients is still poor. About 46.4% of cervical cancer patients have a five-year survival rate of 91.5% after standard treatment. However, in the case of lymph node (LN) metastasis and distant metastasis, the five-year survival rate decreases to 57.4% and 16.5%, respectively. Traditional surgery and radiotherapy have achieved certain results, but still have side effects such as impaired fertility of patients. Therefore, it is urgent to develop new treatment methods to improve the cure rate of cancer and the quality of life of patients.

[0003] Chemotherapy has a core advantage in the field of cervical cancer treatment, which is to reduce tumor volume, control tumor metastasis and improve cure rate. Among them, platinum drugs are the first choice for chemotherapy drugs and have significant effects. OXaliplatin (OXa) as the third generation of platinum antitumor drugs can form adducts with DNA, interfere with the replication and transcription process of DNA, and then cause functional damage to DNA, leading to apoptosis of cancer cells, and showing outstanding antitumor effect. In the treatment of cervical cancer, the efficacy of single drug is often limited, especially in the process of cancer cell metastasis. Matrix metalloproteinases (MMPs) play a crucial role in the tumor inflammatory microenvironment. MMPs can degrade the extracellular matrix (ECM) of cancer cells, making the extracellular matrix of cancer cells "soft", and helping tumor cells to break through the tissue barrier. In the previous study, the inventors prepared a four-valent platinum prodrug DN-Pt(IV) composed of OXaliplatin and COX-2 inhibitor 2-(6-methoxynaphthalen-2-yl) acetic acid (DN) and a polyrotaxane carrier composed of α-cyclodextrin and methoxypolyethylene glycol polylactic acid-glycolic acid copolymer (mPEG-PLGA). The polyrotaxane carrier constructs DNPt@PPRI NPs for the treatment of colorectal cancer. After tumor cells uptake DNPt@PPRI NPs, in the high glutathione environment of the tumor, the axial covalent bond of the DN-Pt(IV) prodrug breaks down, releasing free DN. The released DN inhibits the expression of COX-2 and down-regulates the level of metastasis-related protein MMP-9, enhances OXa-induced apoptosis, and inhibits tumor metastasis. However, although the inhibition of MMPs can limit cancer cell metastasis to some extent, the persistent reduction of MMPs and the hardening of the extracellular matrix will enhance the invasiveness and migration ability of cancer cells by promoting epithelial-mesenchymal transition (EMT) and pseudopod formation. Therefore, how to effectively solve the "contradiction" of softening or hardening of the extracellular matrix of cancer cells to promote cancer metastasis has become a key research direction for inhibiting cervical cancer metastasis.

[0004] In order to solve the above-mentioned problems, a new drug delivery system needs to be designed, which can coordinate the inhibition of metastasis caused by the softening of ECM, while avoiding the aggravation of cancer metastasis caused by the hardening of ECM, and can effectively kill cancer cells at the primary lesion of cancer, so as to achieve the goal of effectively reducing cancer metastasis and improving treatment effect. SUMMARY

[0005] One of the purposes of the present application is to provide a cyclodextrin inclusion compound comprising a platinum drug, 2-(6-methoxynaphthalen-2-yl) acetic acid and cyclodextrin.

[0006] The platinum drug and 2-(6-methoxynaphthalen-2-yl)acetic acid are covalently coupled to form a prodrug, and then form an inclusion compound with cyclodextrin; the molar ratio of the platinum drug and 2-(6-methoxynaphthalen-2-yl)acetic acid is 1:1-1:2, and the molar ratio of the prodrug and cyclodextrin is 1:1-1:10.

[0007] Further, the platinum drug is cisplatin, carboplatin, oxaliplatin or lobaplatin.

[0008] Further, the cyclodextrin is β-cyclodextrin, dimethyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin or hyperbranched cyclodextrin.

[0009] In a specific embodiment of the present application, the platinum drug is oxaliplatin. The synthesis route of oxaliplatin and 2-(6-methoxynaphthalen-2-yl)acetic acid covalently coupled to form a prodrug is as follows:

[0010]

[0011] The preparation process is as follows:

[0012] Step 1, oxaliplatin is added to 30% hydrogen peroxide, and the reaction is carried out at 60°C in the dark. The reaction solution is concentrated by rotary evaporation, and then dropped into cold ethanol to obtain a white precipitate. The volume ratio of the concentrated solution to cold ethanol is 1:10. The product is collected by vacuum filtration, washed with cold ethanol and cold ether, and then obtained as oxidized oxaliplatin OXA(OH)2.

[0013] Step 2, 2-(6-methoxynaphthalen-2-yl)acetic acid, N-hydroxysuccinimide, N,N'-dicyclohexyl carbodiimide, triethylamine are dissolved in acetonitrile, stirred and reacted, acetonitrile is removed by rotary evaporation, the product is dissolved in dichloromethane, extracted with pure water, the volume ratio of dichloromethane to pure water is 5:1, the organic layer is collected, anhydrous sodium sulfate is added, concentrated by rotary evaporation and dried to obtain N-hydroxysuccinimide-2-(6-methoxynaphthalen-2-yl) acetic acid ester DN-NHS ester.

[0014] Step 3, OXA(OH)2 and DN-NHS ester are added to anhydrous DMSO, and the reaction is carried out at room temperature in the dark. Wash with ether until a sticky beige precipitate appears. The precipitate is suspended in methanol and then added to ether. The volume ratio of methanol to ether is 1:10. The precipitate is collected by centrifugation and dried to obtain the prodrug.

[0015] The second object of the present application is to provide a preparation method of the above cyclodextrin inclusion compound. Specifically, the prodrug is first dissolved in N,N-dimethylformamide, then the cyclodextrin is dissolved in water, and then the prodrug solution is dropped into the cyclodextrin solution. After stirring, the solvent is removed by rotary evaporation, and the cyclodextrin inclusion compound is obtained by freeze-drying.

[0016] The third object of the present application is to provide the use of the above-mentioned cyclodextrin inclusion complex in the preparation of a tumor treatment drug.

[0017] Further, the tumor is cervical cancer, breast cancer, colorectal cancer, etc.

[0018] In order to achieve better tumor treatment effect and take into account the inhibition of metastasis, the present application constructs a combined system based on the clear anti-tumor activity of oxaliplatin (OXa) and COX-2 inhibitor (DN) which can reduce the degradation of cancer cells to extracellular matrix (ECM) by inhibiting matrix metalloproteinase (MMPs), and then block the metastasis process; in view of the key contradiction existing in the combination of MMPs inhibition and the continuous existence of matrix hardening which easily induces cancer cell epithelial-mesenchymal transition (EMT) and pseudopod formation, and reversely enhances its invasion and migration ability, the present application innovatively uses β-cyclodextrin to selectively include cholesterol in the hydrophobic cavity to destroy the structure of lipid raft (lipid raft is the necessary prerequisite for the formation of lamellar pseudopod, and lamellar pseudopod is the core power source of cancer cell movement) as a drug carrier; combined with the inclusion property of β-cyclodextrin which is only suitable for guest molecules with molecular weight less than 800 Da, a single DN is determined as the ligand of OXa to ensure the stability of inclusion. Since hydroxypropyl β-cyclodextrin (HP-β-CD) is generally safe, in the field of drugs, HP-β-CD is recognized by multiple authoritative agencies such as the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the China National Medical Products Administration (NMPA) as a pharmaceutical excipient, therefore HP-β-CD is selected as a carrier to improve the water solubility of the drug. At the same time, based on the same price effect of cyclodextrin polymer which can enhance the cholesterol removal capacity, and the advantage of hyperbranched structure which needs to meet the good water solubility of the carrier, hyperbranched cyclodextrin (10-poly-β-cyclodextrin, molecular weight 12000 Da, referred to as 12K-β-CD) is selected as the optimal carrier to construct the synergistic delivery system of OXa and DN.

[0019] The application provides a multifunctional cyclodextrin inclusion complex for double inhibition of cervical cancer metastasis. First, 2-(6-methoxynaphthalen-2-yl) acetic acid (DN) is covalently coupled with oxaliplatin (OXa) to obtain an anti-inflammatory tetravalent platinum prodrug DNPt(IV); then, DNPt(IV) is combined with hyperbranched cyclodextrin to form a cyclodextrin inclusion complex by host-guest interaction, so that a nano delivery material capable of simultaneously inhibiting COX-2 and lamellipodia is obtained. The prepared drug-loaded inclusion complex can on one hand cause DNA damage by OXa to achieve the effect of killing tumor cells, and on the other hand, DN can inhibit COX-2 and then down-regulate the expression of MMPs. At the same time, based on the homovalent effect, the hyperbranched cyclodextrin can effectively remove cholesterol in cancer cells and destroy the lipid raft structure to inhibit the formation of lamellipodia and reduce the movement ability of cancer cells. In this way, the metastasis promoted by the softening of ECM can be inhibited, the metastasis of cancer caused by the hardening of ECM can be avoided, and cancer cells can be effectively killed at the primary lesion of cancer.

[0020] The beneficial effects of the application are as follows:

[0021] (1) The combination system of "hyperbranched cyclodextrin nanocarrier + anti-inflammatory oxaliplatin prodrug" is constructed, which takes into account the anti-tumor activity of the drug and the regulation function of the carrier, and breaks through the limitation of single drug.

[0022] (2) MMPs promote the invasion of tumor cells into surrounding tissues and into blood circulation by degrading extracellular matrix components and destroying tissue structure barriers, thereby significantly enhancing the metastasis ability, and are a key factor affecting tumor progression and prognosis. Precise blocking of the metastasis pathway can down-regulate MMP-9 by inhibiting COX-2, reduce extracellular matrix (ECM) degradation, and alleviate the problem of metastasis promotion caused by "softening".

[0023] (3) After MMPs are inhibited, the degradation of ECM is reduced, which leads to the increase of hardness, and in turn enhances the migration and metastasis ability of cancer cells. As a key power source, the inhibition of the formation and function of lamellipodia can effectively reduce the increase of cancer cell migration ability caused by ECM hardening, and provide a new idea for solving the paradox of MMPs inhibition. Based on the homovalent effect, hyperbranched cyclodextrin is selected as the carrier, the carrier destroys the lipid raft and inhibits the pseudopod to reduce the movement ability of cancer cells, offsets the risk of increased invasiveness caused by matrix hardening, and solves the problem of "soft and hard promoting metastasis".

[0024] (4) The preparation process is simple and efficient, and the required raw materials are easy to obtain, which provides favorable conditions for realizing large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 DSC spectra and FT-IR spectra of different drug-loaded systems.

[0026] Figure 2Figure 1 is a graph showing the inhibitory effect of different drugs on the cholesterol of U14 tumor cells.

[0027] Figure 3 Figure 2 is a graph showing the effect of different drugs on the lipid raft disruption of U14 tumor cells.

[0028] Figure 4 Figure 3 is a graph showing the effect of different drugs on the inhibition of lamellipodia of U14 tumor cells by laser confocal characterization.

[0029] Figure 5 Figure 4 is a graph showing the detection of COX-2 expression in U14 tumor cells treated with different drugs.

[0030] Figure 6 Figure 5 is a graph showing the detection of MMP-9 expression in U14 tumor cells treated with different drugs.

[0031] Figure 7 Figure 6 is a graph showing the results of a scratch test on U14 tumor cells treated with different drugs.

[0032] Figure 8 Figure 7 is a graph showing the results of an invasion test on U14 tumor cells treated with different drugs.

[0033] Figure 9 Figure 8 is a graph showing the results of tumor tissue proliferation and apoptosis in tumor-bearing mice.

[0034] Figure 10 Figure 9 is a graph showing the results of tumor lung metastasis in tumor-bearing mice. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present application will be described in detail below with reference to the following examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application.

[0036] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0037] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0038] The hyperbranched cyclodextrin (10-poly-β-cyclodextrin, molecular weight 12000 Da, referred to as 12K-β-CD) used in the present application was provided by Binzhou Zhiyuan Biotechnology Co., Ltd. of Shandong.

[0039] Example 1

[0040] A preparation method of a hyperbranched cyclodextrin inclusion delivery of a loaded oxaliplatin prodrug includes the following steps:

[0041] (1) First, accurately weigh oxaliplatin (300 mg, 0.759 mmol) into a 50 mL round bottom flask, add 30% hydrogen peroxide (20 mL, 177 mmol), heat and stir at 60 °C, avoid light reaction for 5 h, concentrate to 1 mL by rotary evaporation, drop the concentrated solution into 10 mL cold ethanol to obtain white precipitate, collect the product by vacuum filtration, wash with 2 mL cold ethanol and 2 mL cold ether for 3 times each, dry to obtain intermediate product oxaliplatin oxide 280 mg, yield 85.97%.

[0042] (2) Accurately weigh 2-(6-methoxynaphthalen-2-yl)acetic acid (216.3 mg, 1 mmol), NHS (173 mg, 1.5 mmol), DCC (227 mg, 1.1 mmol) into a 50 mL round bottom flask, add 15 mL acetonitrile, add TEA (110 μl, 0.791 mmol), stir at room temperature for 24 h, remove acetonitrile by rotary evaporation, dissolve the product in 200 mL dichloromethane, extract with 50 mL pure water for 3 times, collect the organic layer, add anhydrous sodium sulfate, concentrate by rotary evaporation and dry to obtain intermediate product DN-NHS ester 210 mg, yield 67.03%.

[0043] (3) Accurately weigh oxaliplatin oxide (644 mg, 1.5 mmol), DN-NHS ester (564 mg, 1.8 mmol) into anhydrous DMSO, react at 60 °C for 12 h in the dark, wash with ether until a sticky beige precipitate appears, suspend the precipitate in 1 mL methanol, add to ether, collect the precipitate by centrifugation and dry to obtain tetravalent platinum prodrug DNPt(IV). Purify the product by column chromatography (MeOH / EtOAc 2:3 v / v) to obtain white solid 412.35 mg, yield 43.89%. 1 HNMR (600 MHz, DMSO-d6) δ = 7.74 (d, 1H), 7.69 (d, 1H), 7.32 (dd, 1.4 Hz, 1H), 7.27 (d, 1H), 7.12 (dd, 1H), 4.07 (s, 1H), 3.86 (s, 3H), 3.65 (d, 2H), 3.31 (s, 3H), 2.46 (s, 2H), 2.05 (d, 1H), 1.43 (m, 3H), 1.24 (m, 1H).13C NMR (151 MHz, DMSO-d6) δ = 179.98, 163.81, 156.79, 132.77, 131.27, 128.82, 128.24, 128.11, 126.79, 126.35, 118.31, 105.59, 61.32, 55.01, 43.35, 30.62, 23.3. MS: m / z calculated for C 21H 26 N2O8Pt: 628.13371; found: 628.12606.

[0044] (4) Accurately weigh tetra-valent platinum prodrug DNPt(IV) (3.14 mg, 0.005 mmol), hyperbranched cyclodextrin 12K-β-CD (60 mg, 0.005 mmol), dissolve DNPt(IV) in 300 μL DMF, dissolve 12K-β-CD in 700 μL pure water, slowly drop DNPt(IV) solution into 12K-β-CD solution, stir at 60 °C for 12 h, remove solvent by rotary evaporation, re-dissolve in 1 mL pure water, filter the solution using 0.22 μm needle filter, freeze-dry the filtrate to obtain inclusion complex DNPt / 12K-β-CD.

[0045] Example 2

[0046] A preparation method of an oxaliplatin prodrug loaded cyclodextrin inclusion complex delivery, comprising the following steps:

[0047] (1) First, accurately weigh oxaliplatin (300 mg, 0.759 mmol) into a 50 mL round-bottom flask, add 30% hydrogen peroxide (20 mL, 177 mmol), heat and stir at 60 °C, avoid light reaction for 5 h, concentrate to 1 mL by rotary evaporation, drop the concentrated solution into 10 mL cold ethanol to obtain white precipitate, collect the product by vacuum filtration, wash with 2 mL cold ethanol and 2 mL cold ether for 3 times, dry to obtain intermediate product oxidized oxaliplatin 280 mg, yield 85.97%.

[0048] (2) Accurately weigh 2-(6-methoxynaphthalen-2-yl)acetic acid (216.3 mg, 1 mmol), NHS (173 mg, 1.5 mmol), DCC (227 mg, 1.1 mmol) into a 50 mL round-bottom flask, add 15 mL acetonitrile, add TEA (110 μl, 0.791 mmol), stir at room temperature for 24 h, remove acetonitrile by rotary evaporation, dissolve the product in 200 mL dichloromethane, extract with 50 mL pure water for 3 times, collect the organic layer, add anhydrous sodium sulfate, concentrate and dry to obtain intermediate product DN-NHS ester 210 mg, yield 67.03%.

[0049] (3) Accurately weigh the Oxaliplatin (644 mg, 1.5 mmol), DN-NHS ester (564 mg, 1.8 mmol) into anhydrous DMSO, react at 60 °C for 12 h in the dark, wash with ether until a sticky beige precipitate appears, suspend the precipitate in 1 mL of methanol, add to ether, centrifuge to collect the precipitate, dry to obtain the tetravalent platinum prodrug DNPt(IV); purify the product by column chromatography (MeOH / EtOAc 2:3 v / v) to obtain 412.35 mg of white solid, yield 43.89%. 1 HNMR (600 MHz, DMSO-d6) δ = 7.74 (d, 1H), 7.69 (d, 1H), 7.32 (dd, 1.4 Hz, 1H), 7.27 (d, 1H), 7.12 (dd, 1H), 4.07 (s, 1H), 3.86 (s, 3H), 3.65 (d, 2H), 3.31 (s, 3H), 2.46 (s, 2H), 2.05 (d, 1H), 1.43 (m, 3H), 1.24 (m, 1H).13C NMR (151 MHz, DMSO-d6) δ = 179.98, 163.81, 156.79, 132.77, 131.27, 128.82, 128.24, 128.11, 126.79, 126.35, 118.31, 105.59, 61.32, 55.01, 43.35, 30.62, 23.3. MS: m / z calculated for C 21 H 26 N2O8Pt: 628.13371; found: 628.12606.

[0050] (4) Accurately weigh the tetravalent platinum prodrug DNPt(IV) (3.14 mg, 0.005 mmol), hydroxypropyl-β-cyclodextrin HP-β-CD (30.8 mg, 0.020 mmol), dissolve DNPt(IV) in 300 μL of DMF, dissolve HP-β-CD in 1700 μL of pure water, slowly drop the DNPt(IV) solution into the HP-β-CD solution, stir at 60 °C for 12 h, remove the solvent by rotary evaporation, re-dissolve with 1 mL of pure water, filter the solution using a 0.22 μm needle filter, freeze-dry the filtered liquid to obtain the inclusion complex DNPt / HP-β-CD.

[0051] The inclusion complexes prepared in the above examples are detected as follows.

[0052] 1. FT-IR spectra, DSC spectra of different nano-drug delivery systems

[0053] Different cyclodextrin inclusion complexes were identified using Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). Figure 1 As shown in Figure A, the characteristic absorption peak of HP-β-CD is at 3400 cm⁻¹. 1 (-OH stretching vibration) and 1152 cm⁻¹ -1 (CO stretching vibration), the characteristic absorption peak of DNPt(Ⅳ) is at 1720 cm⁻¹. -1 (C=O tensile vibration) and 660cm -1 (Out-of-plane bending vibration of the aromatic ring CH); in the physical mixture, it includes both characteristic peaks of HP-β-CD and characteristic peaks of DNPt(Ⅳ); in the inclusion complex, the characteristic peaks of DNPt(Ⅳ) are significantly reduced, confirming the formation of the DNPt / HP-β-CD inclusion complex. Figure 1 As shown in Figure B, the characteristic absorption peak of 12K-β-CD is at 3400 cm⁻¹. -1 (-OH stretching vibration) and 1152cm -1 (CO stretching vibration), the characteristic absorption peak of DNPt(Ⅳ) is at 1720 cm⁻¹. -1 (C=O tensile vibration) and 1260cm -1 (CO stretching vibration of ester bond); In the physical mixture, it includes both the characteristic peaks of 12K-β-CD and the characteristic peaks of DNPt(Ⅳ); In the inclusion complex, the characteristic peaks of DNPt(Ⅳ) are significantly reduced, confirming the formation of the DNPt / 12K-β-CD inclusion complex.

[0054] like Figure 1 As shown in Figure C, DNPt(Ⅳ) has an exothermic peak at 223℃ and an endothermic peak at 253℃. The physical mixture of DNPt(Ⅳ) and HP-β-CD also contains the characteristic peak of DNPt(Ⅳ), but the DSC curve of the inclusion complex is similar to that of HP-β-CD. Compared with the physical mixture, no characteristic endothermic peak of DNPt(Ⅳ) appears in the inclusion complex, proving that DNPt(Ⅳ) has been included in HP-β-CD.

[0055] like Figure 1 As shown in Figure D, DNPt(Ⅳ) has an exothermic peak at 223℃ and an endothermic peak at 253℃. The physical mixture of DNPt(Ⅳ) and 12K-β-CD also contains the characteristic peak of DNPt(Ⅳ), but the DSC curve of the inclusion complex is similar to that of 12K-β-CD. Compared with the physical mixture, the inclusion complex does not show the characteristic endothermic peak of DNPt(Ⅳ), proving that DNPt(Ⅳ) has been included in 12K-β-CD.

[0056] 2. MTT assay to detect the inhibitory effect of different drugs on the activity of U14 and HeLa tumor cells.

[0057] To evaluate the killing ability of different formulations against cervical cancer cells, mouse cervical cancer cells U14 and human cervical cancer cells HeLa were used in the experiment to investigate the cytotoxicity of the inclusion complexes. Cells were stored at 8 x 10⁸ cells per well. 3 Cells were cultured at a density of 100 μL in 96-well plates for 24 h. Different concentrations of reagents were added to each well, with cells in 100 μL of FBS-free DMEM medium serving as a blank control. After co-incubation with the drug for 24 h, MTT solution (5 mg / mL) was added to each well. Cells were incubated in a cell culture incubator for 24 h, and the absorbance at 490 nm was measured using a multi-mode microplate reader, and cell viability was calculated. As shown in Table 1, DN showed weak anti-tumor activity. OXa had a certain killing effect on both types of cervical cancer cells, with the weakest effect against human cervical cancer cells (HeLa). Cell viability remained above 40% at different concentrations, reflecting the limitations of oxaliplatin treatment. When the two drugs were used in combination, the anti-proliferative effect was enhanced, indicating a synergistic effect. When cells were treated with DNPt / 12K-β-CD, the viability of both cell types decreased significantly, and the effect was stronger than that of DNPt / HP-β-CD. DNPt / 12K-β-CD showed an IC50 value for HeLa and U14 cells. 50 The values ​​were 13.97 times and 10.46 times that of OXa, respectively, indicating that it can achieve a therapeutic effect comparable to OXa at a lower dose, which can reduce the toxicity of the drug to normal tissues and organs and improve the killing effect on tumors.

[0058] Table 1. Inhibitory effect of different drugs on U14 and HeLa tumor cells as determined by MTT assay.

[0059]

[0060] FI(foldincrease)is defined as IC50(OXa) / IC50(DNPt / 12K-β-CD))

[0061] 3. Detect the cholesterol-inhibiting ability of different drugs on U14 tumor cells.

[0062] To evaluate the cholesterol-inhibiting ability of different drugs on cervical cancer tumor cells, U14 cervical cancer cells were used in the experiment. Cells were cultured at 3x10⁴ cells per cell line. 5Density seeded in 6-well plates, incubated in incubator for 24h, each well added OXa, DN, OXa+DN, DNPt / HP-β-CD, DNPt / 12K-β-CD solution prepared with DMEM medium without FBS, concentration of 2 μM, treated for 24h, GPO-Tinder enzymatic method was used to determine the content of cell cholesterol. Washed the cells with PBS twice, collected the cells by centrifugation at 2000 rpm. Added 0.1 mL lysis solution per million cells, mixed well by shaking and stood for 10 minutes for cell lysis. Mixed reagents R1 and R2 in a ratio of 4:1 and used immediately. The 5 mM cholesterol standard was diluted with anhydrous ethanol to different concentrations (such as 2500, 1250, 625, 312.5, 156, 78, 39 μmol / L). Took 190 μL working solution and added 10 μL blank control solution (anhydrous ethanol or distilled water), standard or sample per well. Incubated at 37°C for 20 minutes, then adjusted the blank control solution with distilled water or anhydrous ethanol, and measured the optical density value (OD value) of each well. Draw the standard curve, and calculate the cholesterol concentration in the sample by regression analysis. As shown in Figure 2 DNPt / 12K-β-CD can effectively reduce the cholesterol level of U14 cells, and the effect is better than DNPt / HP-β-CD.

[0063] 4. The ability of different drugs to destroy lipid rafts of U14 tumor cells

[0064] Since cholesterol is the main component of lipid rafts, depleting cholesterol can destroy the integrity of lipid rafts. In order to evaluate the ability of different drugs to destroy lipid rafts of cervical cancer cells, U14 cervical cancer cells were seeded in 35 mm glass bottom dishes at a density of 1 x 10 5 Density seeded in 6-well plates, incubated in incubator for 24h, each well added OXa, DN, OXa+DN, DNPt / HP-β-CD, DNPt / 12K-β-CD solution prepared with DMEM medium without FBS, concentration of 2 μM, treated for 24h, GPO-Tinder enzymatic method was used to determine the content of cell cholesterol. Washed the cells with PBS twice, collected the cells by centrifugation at 2000 rpm. Added 0.1 mL lysis solution per million cells, mixed well by shaking and stood for 10 minutes for cell lysis. Mixed reagents R1 and R2 in a ratio of 4:1 and used immediately. The 5 mM cholesterol standard was diluted with anhydrous ethanol to different concentrations (such as 2500, 1250, 625, 312.5, 156, 78, 39 μmol / L). Took 190 μL working solution and added 10 μL blank control solution (anhydrous ethanol or distilled water), standard or sample per well. Incubated at 37°C for 20 minutes, then adjusted the blank control solution with distilled water or anhydrous ethanol, and measured the optical density value (OD value) of each well. Draw the standard curve, and calculate the cholesterol concentration in the sample by regression analysis. As shown in Figure 3 DNPt / HP-β-CD has limited ability to destroy lipid rafts, which is due to its limited ability to remove cholesterol at low doses, while DNPt / 12K-β-CD can effectively destroy lipid rafts, which is due to the "homovalent effect" that can effectively deplete cholesterol.

[0065] 5. The ability of different drugs to inhibit the formation of lamellipodia of U14 tumor cells

[0066] Since lipid raft is a prerequisite for the formation of lamellipodia, disrupting lipid raft can effectively inhibit lamellipodia and reduce the migration and invasion ability of cancer cells. In order to evaluate the inhibitory ability of different drugs on lamellipodia of cervical cancer cells, U14 cervical cancer cells were seeded at a density of 1x10 5 After overnight incubation in 35mm glass bottom dishes, 2μM OXa, DN, OXa+DN, DNPt / HP-β-CD, DNPt / 12K-β-CD solutions prepared in FBS-free DMEM medium were added to each well for 24h. After washing with PBS, the cell nucleus was stained with DAPI and the cytoskeleton was stained with phalloidin, and analyzed by laser confocal microscopy. As shown in FIG. 2, compared with the control group and the DNPt / HP-β-CD group, DNPt / 12K-β-CD can effectively destroy lamellipodia, which is due to its effective lipid raft disruption ability. Figure 4

[0067] 6. COX-2 expression of U14 tumor cells under different drug treatments

[0068] In order to detect the COX-2 ability of U14 cells under different drug treatments, U14 was seeded at a density of 1x10 5 After overnight incubation in 35mm glass bottom dishes, 2μM OXa, DN, OXa+DN, DNPt / HP-β-CD, DNPt / 12K-β-CD solutions prepared in FBS-free DMEM medium were added to each well for 24h. After washing with PBS, the cell nucleus was stained with DAPI and the cytoskeleton was stained with phalloidin, and analyzed by laser confocal microscopy. As shown in FIG. 2, compared with the control group and the DNPt / HP-β-CD group, DNPt / 12K-β-CD can effectively destroy lamellipodia, which is due to its effective lipid raft disruption ability. Figure 5 As shown in FIG. 6, compared with the control group and the OXa group, the DN, double drug physical mixing group, DNPt / HP-β-CD and DNPt / 12K-β-CD four groups of drug treated cells, significantly down-regulated the COX-2 content in cells, and the effect of DNPt / 12K-β-CD was the most obvious. It is indicated that DNPt / 12K-β-CD inhibits COX-2 by DN released from the inclusion compound, and can obtain stronger inhibitory activity than free drug.

[0069] 7. MMP-9 expression of U14 tumor cells under different drug treatments

[0070] ​MMP-9 is expressed in a variety of cancers, and the expression level is associated with the invasive ability of the tumor. COX-2 and MMP-9 expression are closely related, and COX-2 inhibitors can down-regulate MMP-9 expression, thereby inhibiting cell invasion and metastasis. U14 was seeded at 3x10 5 After overnight culture, 2 μM OXa, DN, OXa+DN, DNPt / HP-β-CD, DNPt / 12K-β-CD solutions prepared in FBS-free DMEM medium were added to each well, and the treatment was performed for 24 h. The supernatant was collected, centrifuged at 3000 rpm for 15 min at 4°C, and the supernatant was collected. The MMP-9 protein was detected by Elisa method, and the standard and reagent were prepared according to the instructions. The standard solution or sample was added to the microwell plate, and incubated for 1 hour. Then, detection reagent A was added, incubated for 1 hour, and detection reagent B was added, incubated for 30 minutes. After each addition, the wells were washed 3 to 5 times with washing solution. Then, TMB substrate was added, incubated for 10-20 minutes, and protected from light. Finally, stop solution was added, mixed, and the absorbance was read at 450 nm wavelength. The MMP-9 concentration in the sample was calculated according to the standard curve. As shown in Figure 6 Compared with other groups, DNPt / 12K-β-CD significantly down-regulated the intracellular MMP-9 content, which was consistent with the COX-2 inhibition trend, indicating that DNPt / 12K-β-CD could release free DN in cells to effectively inhibit tumor metastasis.

[0071] 8. Wound healing experiment of U14 tumor cells under different drug treatment

[0072] The wound healing experiment was used to detect the inhibition of drug on tumor cell metastasis: U14 cells were seeded in a 6-well plate (3x10 5 Each well was added with 2 μM drug (OXa, DN, OXa+DN mixture, DNPt / HP-β-CD, DNPt / 12K-β-CD) prepared in FBS-free DMEM medium. After 24 h culture, the same method was used to take pictures (24 h). Image J software was used to quantitatively determine the width of the wound, and the healing rate was calculated. The experimental results are shown in Figure 7As shown, the cells of the control group had strong scratch healing ability, which was consistent with the enhanced cell migration ability during the progression of cervical cancer. The quantitative results after different drug treatments showed that the cell migration ability of the OXa group was similar to that of the control group, indicating that OXa had no obvious effect on tumor metastasis inhibition; the cell migration ability of the DN group and the physical mixture of the two drugs was weak, and it was speculated that the reason might be that the drug dose was too low to reach the effective inhibition dose; compared with the DNPt / HP-β-CD group, DNPt / 12K-β-CD effectively inhibited the scratch healing of tumor cells, showing that DNPt / 12K-β-CD had good anti-tumor metastasis potential in vitro.

[0073] 9. Invasion experiment of U14 tumor cells under different drug treatments

[0074] The cell invasion experiment was used to detect the inhibition of tumor cell metastasis by drugs: U14 cells were seeded in a 12-well plate at a density of 1.5×10 5 cells per well and incubated in a cell incubator overnight, and 2 μM drug (OXa, DN, OXa+DN mixture, DNPt / HP-β-CD, DNPt / 12K-β-CD) was added to each well in DMEM medium without FBS; after 24 h, the matrigel was first diluted at a ratio of 1:3 in DMEM medium without FBS and stored at -20°C for later use. Then the matrigel was thawed and mixed with a pre-cooled pipette tip to form a homogenate. The transwell chamber was inserted into the 24-well plate, and 100 μL of diluted matrigel was added to each chamber on ice, and incubated at 37°C in a cell incubator for 1 h. Then gently wash with DMEM medium without FBS to remove unbound matrigel. Counting plate was used for counting, and the cell density was diluted to 5×10 5 cells / mL using DMEM medium without FBS. 200 μL of cell suspension was taken and added to the transwell upper chamber coated with matrigel, and 600 μL of DMEM medium containing 10% FBS was added to the lower chamber, and the plate was placed in a cell incubator overnight. After 24 h, the cells were collected, and the cells on the transwell upper chamber membrane were gently scraped off with a PBS-wetted cotton swab, and 4% paraformaldehyde solution was added to fix for 15 min at room temperature. Then wash with PBS for 3 times, add 200 μL of 0.1% crystal violet staining solution to each transwell sample well, and stain for 15 min at room temperature. After completion, wash with PBS for 3 times, dry the transwell at room temperature, and then observe the cell staining under an inverted microscope and take pictures. After the experiment, Image J was used for quantitative determination, and the cell invasion rate was calculated. The experimental results are shown in Figure 8As shown, the cells of the control group had strong invasion ability. The quantitative results after different drug treatment of the cells showed that the OXa, DN group and the double drug physical mixing group weakly inhibited the metastasis ability of the cells, and compared with the DNPt / HP-β-CD group, the DNPt / 12K-β-CD more effectively inhibited the tumor cells from crossing the transwell polycarbonate membrane, showing that the DNPt / 12K-β-CD had good anti-tumor metastasis potential in vitro.

[0075] 10, Apoptosis results of tumor tissues of tumor-bearing mice

[0076] In order to verify the in vivo inhibition of tumor metastasis of DNPt / 12K-β-CD, lung tissues were subjected to HE staining to observe the lung tumor metastasis. The lung was fixed in 4% paraformaldehyde, paraffin-embedded to make sections, and subjected to HE staining, and the tumor metastasis inhibition ability of different groups of drugs was investigated according to the pathological state. The HE results 6 3 In order to verify the in vivo inhibition of tumor metastasis of DNPt / 12K-β-CD, lung tissues were subjected to HE staining to observe the lung tumor metastasis. The lung was fixed in 4% paraformaldehyde, paraffin-embedded to make sections, and subjected to HE staining, and the tumor metastasis inhibition ability of different groups of drugs was investigated according to the pathological state. The HE results Figure 9 As shown in A and B of the figure, the tumor volume and mass of the DNPt / 12K-β-CD group of mice were the smallest, showing good therapeutic effect, which was significantly better than that of the same dose of oxaliplatin; the TUNEL staining results (C) showed that the green fluorescence intensity of DNPt / 12K-β-CD was the strongest compared with other groups, and the number of apoptotic cells was the most, which indicated that DNPt / 12K-β-CD had good ability to induce tumor cell apoptosis and inhibit cell proliferation. Figure 9 As shown in A and B of the figure, the tumor volume and mass of the DNPt / 12K-β-CD group of mice were the smallest, showing good therapeutic effect, which was significantly better than that of the same dose of oxaliplatin; the TUNEL staining results (C) showed that the green fluorescence intensity of DNPt / 12K-β-CD was the strongest compared with other groups, and the number of apoptotic cells was the most, which indicated that DNPt / 12K-β-CD had good ability to induce tumor cell apoptosis and inhibit cell proliferation.

[0077] 11, Tumor lung metastasis map of tumor-bearing mice

[0078] In order to verify the in vivo inhibition of tumor metastasis of DNPt / 12K-β-CD, lung tissues were subjected to HE staining to observe the lung tumor metastasis. The lung was fixed in 4% paraformaldehyde, paraffin-embedded to make sections, and subjected to HE staining, and the tumor metastasis inhibition ability of different groups of drugs was investigated according to the pathological state. The HE results​Figure 10 ) The number of metastatic nodules in the control group was the largest, and the number of nodules in the OXa group was less, which may be because OXa has a certain killing effect on the primary tumor, so the tumor has weak ability to metastasize to the distal end. The number of metastatic nodules in the lung of the mouse treated by DN and the double-drug mixed group was less, which showed that DN had a certain inhibitory effect on tumor metastasis. Compared with the DNPt / HP-β-CD treatment group, no obvious metastatic nodules were observed in the DNPt / 12K-β-CD group, which showed that the final preparation group well controlled the tumor metastasis to the lung.

Claims

1. A cyclodextrin inclusion complex, characterized in that, The raw materials include platinum drugs, 2-(6-methoxynaphthalen-2-yl)acetic acid and cyclodextrin; The platinum drugs and 2-(6-methoxynaphthalen-2-yl)acetic acid are covalently coupled to form a prodrug, and then the prodrug is combined with cyclodextrin to form an inclusion compound; The molar ratio of the platinum drugs and 2-(6-methoxynaphthalen-2-yl)acetic acid is 1:1-1:2, and the molar ratio of the prodrug and cyclodextrin is 1:1-1:

10.

2. The cyclodextrin inclusion complex of claim 1, wherein The platinum drugs are cisplatin, carboplatin, oxaliplatin or lobaplatin.

3. The cyclodextrin inclusion complex of claim 1, wherein The cyclodextrin is β-cyclodextrin, dimethyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin or hyperbranched cyclodextrin.

4. The cyclodextrin inclusion complex of claim 3, wherein The cyclodextrin is β-cyclodextrin or hyperbranched cyclodextrin.

5. The cyclodextrin inclusion complex of claim 1, wherein The preparation process of covalently coupling the platinum drugs and 2-(6-methoxynaphthalen-2-yl)acetic acid to form a prodrug is as follows: Step 1, first add platinum drugs into 30% hydrogen peroxide, react in the dark, and then concentrate the reaction solution and precipitate with ethanol to obtain oxidized platinum; Step 2, dissolve 2-(6-methoxynaphthalen-2-yl)acetic acid, N-hydroxysuccinimide, N,N'-dicyclohexyl carbodiimide and triethylamine in acetonitrile, stir and react to prepare N-hydroxysuccinimide-2-(6-methoxynaphthalen-2-yl)acetic acid ester DN-NHS ester; Step 3, add oxidized platinum and DN-NHS ester to anhydrous DMSO, react in the dark, wash with diethyl ether until a sticky precipitate appears, suspend the precipitate in methanol, add to diethyl ether, centrifuge to collect the precipitate, and dry to obtain the prodrug.

6. A process for the preparation of a cyclodextrin inclusion complex as defined in any one of claims 1 to 5, characterized in that, First dissolve the prodrug in N,N-dimethylformamide, then dissolve cyclodextrin in water, then drop the prodrug solution into the cyclodextrin solution, stir, remove the solvent by rotary evaporation, and freeze-dry to obtain the cyclodextrin inclusion compound.

7. Use of the cyclodextrin inclusion compound of any one of claims 1-5 in the preparation of a tumor treatment drug.

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