Tumor microenvironment response type cascade active oxygen generation nano platform as well as preparation method and application thereof

Abstract

The invention provides a tumor microenvironment response type cascade active oxygen generation nano platform and a preparation method thereof, the nano platform comprises a FeOOH nano spindle body, and the outer layer of the FeOOH nano spindle body is coated with tetrasulfide bond doped dendritic mesoporous organosilicon nanoparticles (DMOS NPs) to form nanoparticles DMOS (at) FeOOH. The nano platform disclosed by the invention can efficiently generate reactive oxygen species (ROS) in tumor tissues through cascade reaction, realizes selective killing of tumor cells, has relatively high specificity, a good treatment effect and relatively low side effects on normal tissues, and is a tumor treatment strategy with a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of nanotechnology, and in particular to a nano-platform for generating reactive oxygen species in a tumor microenvironment-responsive cascade manner, and a preparation method and application thereof. Background Art

[0002] In recent years, reactive oxygen species (ROS) have attracted much attention as an emerging anti-tumor therapeutic strategy because of their ability to induce apoptosis of tumor cells through oxidative stress. The main ways of using ROS to treat tumors include directly destroying tumor cells using reactive substances such as superoxide anions, hydroxyl radicals, and hydrogen peroxide. However, despite the significant anti-cancer potential of ROS, existing treatments still face some challenges, such as low efficiency of ROS generation, poor selectivity, and toxic side effects on normal tissues. Therefore, how to efficiently and selectively generate ROS and enhance its anti-tumor effect has become an urgent problem to be solved in this field.

[0003] By designing a multifunctional tumor microenvironment-responsive nanoplatform, it is possible to catalyze the production of ROS in the characteristic tumor microenvironment and reduce damage to normal tissues. However, the existing tumor microenvironment-responsive nanoplatform generates ROS through a one-step catalytic reaction, making it difficult to achieve selective killing of tumor tissues. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide a tumor microenvironment responsive cascade-generated reactive oxygen nanoplatform and a preparation method thereof, which can efficiently generate reactive oxygen species (ROS) in tumor tissues through cascade reactions and achieve selective killing of tumor cells.

[0005] According to the first aspect of the present invention, a tumor microenvironment responsive cascade nanoplatform for generating reactive oxygen species is provided, the nanoplatform comprising FeOOH nanospindles, the outer layer of the FeOOH nanospindles being coated with tetrasulfide bond-incorporated dendritic mesoporous organosilicon nanoparticles (DMOS NPs) to form nanoparticles DMOS@FeOOH.

[0006] As an optional implementation, the major diameter of the nanoparticle DMOS@FeOOH is 230 nm to 240 nm.

[0007] As an optional embodiment, the Zeta potential of the nanoparticle DMOS@FeOOH is -25 mV to -30 mV.

[0008] As an optional embodiment, the thickness of the dendritic mesoporous organosilicon nanoparticles doped with tetrasulfide bonds on the surface of the nanoparticle DMOS@FeOOH is 40 nm to 50 nm.

[0009] According to a second aspect of the present invention, there is provided a method for preparing the aforementioned tumor microenvironment responsive cascade reactive oxygen nanoplatform, comprising the following steps:

[0010] FeOOH nanospindles were prepared by hydrothermal method using FeCl3·6H2O as raw material.

[0011] Under the first temperature condition, FeOOH nanospindles, CTAB and NaSal are added to the TEA aqueous solution, and after stirring and mixing evenly, a mixed solution of TEOS and BTES is added dropwise to react. After the reaction is completed, the solid is collected by centrifugation, and the solid is washed with ethanol to remove residual reactants to obtain a crude product. The crude product is then dispersed in an HCl methanol solution, refluxed and stirred to remove the template, and FeOOH nanospindles (DMOS@FeOOH) with dendritic mesoporous organosilicon nanoparticles (DMOS NPs) doped with tetrasulfide bonds are obtained.

[0012] As an optional embodiment, the process of preparing FeOOH nanospindles by hydrothermal method using FeCl3·6H2O as raw material includes:

[0013] A FeCl3·6H2O solution is dissolved in deionized water and vigorously stirred at a second temperature until the solution becomes turbid. The obtained turbid solution is centrifuged to collect the product, the product is washed to obtain FeOOH nanospindles, and the FeOOH nanospindles are redispersed in deionized water for later use.

[0014] As an optional implementation, the second temperature is 70° C. to 80° C., and the stirring time is 2 h to 4 h.

[0015] As an optional embodiment, the volume ratio of TEA to deionized water is (2.0-3.5):1, the mass ratio of FeOOH, CTAB and NaSal is (1:5:3)-(1:10:6), and the mass ratio of TEOS, BTES and CTAB is (1:1:0.2)-(1:1.5:0.4).

[0016] As an optional embodiment, the first temperature is 70° C. to 80° C., the stirring time after adding FeOOH nanospindles, CTAB and NaSal is 1 h to 2 h, and the reaction time after adding the mixed solution of TEOS and BTES is 10 h to 24 h.

[0017] According to a third aspect of the present invention, there is provided a use of the aforementioned tumor microenvironment responsive cascade reactive oxygen species nanoplatform in the preparation of anti-tumor drugs.

[0018] From the above technical solutions of the present invention, it can be seen that the tumor microenvironment responsive cascade generation of reactive oxygen nanoparticles proposed in the present invention contains tetrasulfide bonds in the DMOS on the surface of the nanoparticles DMOS@FeOOH. When these nanoparticles enter tumor cells or tumor microenvironment, the high concentration of glutathione environment can break the tetrasulfide bonds through its strong reducing property, resulting in the structural degradation of the nanoparticles and the release of H2S gas; the H2S gas will react with the Fe in the FeOOH nanocore in the nanoparticles. 3+ Reaction occurs, promoting Fe 3+ Reduction to Fe 2+ ;Fe 2+ Reactive oxygen (·OH, etc.) is generated by reacting with H2O2 through the Fenton reaction, thereby improving the efficiency of reactive oxygen generation through cascade and selectively killing tumor cells.

[0019] In addition, the nanoplatform of the present invention can be enriched in the tumor site through the enhanced permeability and retention effect (EPR effect), thereby increasing the concentration of local reactive oxygen species, thereby enhancing the anti-tumor effect while reducing toxicity to normal tissues, and has broader clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a scanning electron microscope image of DMOS@FeOOH in an example of the present invention.

[0021] Figure 2 It is the element mapping diagram of DMOS@FeOOH in the example of the present invention.

[0022] Figure 3 is the EDS graph of DMOS@FeOOH in the example of the present invention.

[0023] Figure 4 : is the Zeta potential diagram of DMOS@FeOOH in the example of the present invention.

[0024] Figure 5 is the glutathione (GSH) consumption capacity of DMOS@FeOOH in the example of the present invention at different concentrations.

[0025] Figure 6 is the glutathione (GSH) consumption capacity of DMOS@FeOOH in the example of the present invention at different times.

[0026] Figure 7 is the ability of DMOS@FeOOH in the example of the present invention to produce H2S.

[0027] Figure 8 The ability of DMOS@FeOOH in the present invention to produce H2S in mouse 4T1 breast cancer cells.

[0028] Fig. 9 Graph showing the time-dependent change in the generation of active oxygen by DMOS@FeOOH in the example of the present invention.

[0029] Fig.10 This is a diagram showing the therapeutic effect of DMOS@FeOOH in the present invention on mouse 4T1 breast cancer cells and normal mouse embryonic fibroblast 3T3 cells.

[0030] Fig.11 This is a diagram showing the in vivo therapeutic effect of DMOS@FeOOH in the present invention on 4T1 breast cancer in mice. DETAILED DESCRIPTION

[0031] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

[0032] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways.

[0033] Tumor cells often show higher GSH levels, which is due to their strong adaptability to oxidative stress during rapid proliferation.

[0034] Based on this, the present invention designs a cascade reaction nanoplatform that efficiently generates ROS by utilizing the characteristics of high concentration of GSH in the tumor microenvironment. It has high specificity, good therapeutic effect and low side effects on normal tissues. It is a tumor treatment strategy with broad application prospects.

[0035] In an exemplary embodiment of the present invention, a tumor microenvironment-responsive cascade-generated reactive oxygen nanoplatform is provided, the nanoplatform comprising FeOOH nanospindles, the outer layer of which is coated with tetrasulfide-bond-incorporated dendritic mesoporous organosilicon nanoparticles (DMOS NPs) to form nanoparticles DMOS@FeOOH.

[0036] As an optional example, the major diameter of the nanoparticle DMOS@FeOOH is 230 nm to 240 nm.

[0037] As an optional example, the Zeta potential of the nanoparticle DMOS@FeOOH is -25 mV to -30 mV.

[0038] As an optional example, the thickness of the dendritic mesoporous organosilicon nanoparticles doped with tetrasulfide bonds on the surface of the nanoparticle DMOS@FeOOH is 40 nm to 50 nm.

[0039] In another exemplary embodiment of the present invention, a method for preparing the aforementioned tumor microenvironment responsive cascade reactive oxygen species nanoplatform is provided, comprising the following steps:

[0040] FeOOH nanospindles were prepared by a hydrothermal method: FeCl3·6H2O solution was dissolved in deionized water and vigorously stirred at a second temperature until the solution became turbid, the turbid solution was centrifuged to collect the product, the product was washed to obtain FeOOH nanospindles, and the FeOOH nanospindles were redispersed in deionized water for later use.

[0041] Surface-coated tetrasulfide-doped dendritic mesoporous organosilicon nanoparticles (DMOS NPs): Under a first temperature condition, FeOOH nanospindles, CTAB and NaSal are added to a TEA aqueous solution, and after stirring and mixing evenly, a mixture of TEOS and BTES is added dropwise to react. After the reaction is completed, the solid is collected by centrifugation, and the solid is washed with ethanol to remove residual reactants to obtain a crude product. The crude product is then dispersed in an HCl methanol solution, refluxed and stirred to remove the template, and FeOOH nanospindles (DMOS@FeOOH) with surface-coated tetrasulfide-doped dendritic mesoporous organosilicon nanoparticles (DMOS NPs) are obtained.

[0042] As an optional example, the concentration of the FeCl3·6H2O aqueous solution is 20 mg / mL, the second temperature is 70°C to 80°C, and the stirring time is 2h to 4h.

[0043] As an optional example, the volume ratio of TEA to deionized water is (2.0-3.5):1, the mass ratio of FeOOH, CTAB and NaSal is (1:5:3)-(1:10:6), and the mass ratio of TEOS, BTES and CTAB is (1:1:0.2)-(1:1.5:0.4).

[0044] As an optional example, the first temperature is 70° C. to 80° C., the stirring time after adding FeOOH nanospindles, CTAB and NaSal is 1 h to 2 h, and the reaction time after adding the mixed solution of TEOS and BTES is 10 h to 24 h.

[0045] In other exemplary embodiments of the present invention, there is also provided a use of the aforementioned tumor microenvironment responsive cascade reactive oxygen species nanoplatform in the preparation of anti-tumor drugs.

[0046] The GSH-responsive nanoplatform of the present invention can specifically trigger a cascade reaction in the tumor microenvironment to generate high concentrations of ROS. This GSH-responsive strategy has the following advantages:

[0047] Efficient generation of ROS: The metal ions and other reactants in the nanoplatform can accelerate the reaction and efficiently generate ROS under the high GSH concentration of tumor cells, thereby greatly improving the efficiency of ROS generation.

[0048] Selective killing of tumor cells: Since the GSH concentration in the tumor microenvironment is generally high, the strategy of the present invention can ensure that the generation of ROS is limited to the tumor site. Compared with traditional ROS generation methods, the nanoplatform of the present invention can effectively avoid damage to normal tissues, because the GSH level in normal cells is relatively low, it is difficult to trigger the cascade reaction of the platform, thereby achieving specific killing of tumor cells.

[0049] Avoiding drug resistance mechanisms: Tumor cells have a strong ability to scavenge ROS due to their unique redox state. Therefore, by using the GSH level in the tumor microenvironment as a trigger mechanism, it is not only possible to enhance the local concentration of ROS, but also to effectively overcome the tolerance of tumor cells to ROS.

[0050] Enhanced therapeutic effect: The production of a variety of ROS through cascade reactions, such as superoxide anions, hydroxyl radicals, and hydrogen peroxide, not only enhances the anti-tumor effect, but may also produce synergistic effects with other treatment modes (such as ferroptosis, phototherapy, etc.), thereby improving the overall effect of the treatment.

[0051] For better understanding, the present invention is further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0052] Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0053] Example 1

[0054] [Synthesis and characterization of spindle-shaped FeOOH coated with tetrasulfide-incorporated dendritic mesoporous organosilica nanoparticles (DMOS@FeOOH)]

[0055] FeOOH nanospindles were prepared by a hydrothermal method: 0.68 g of FeCl3·6H2O was dissolved in 50 mL of deionized water to obtain a uniform and stable solution, which was then vigorously stirred at 80 °C for 2 h until the solution became turbid. The product was collected by centrifugation at 8000 rpm for 30 min, washed three times, and then redispersed in deionized water for subsequent use.

[0056] Preparation of surface-coated tetrasulfide-incorporated dendritic mesoporous organosilicon nanoparticles (DMOS NPs): 61 μL of TEA was dissolved in 25 mL of water and gently stirred at 80 °C for 0.5 h. Subsequently, 20 mg of FeOOH nanospindles, 380 mg of CTAB and 168 mg of NaSal were added to the solution and continued to stir at 80 °C for 2 h. Next, a mixture of TEOS (1 mL) and BTES (0.8 mL) was added dropwise and kept at 80 °C for 12 h. The product was collected by centrifugation and washed three times with ethanol to remove residual reactants. Then, the product was dispersed in 2 M HCl methanol solution and stirred at 75 °C for 6 h to remove the template. This process was repeated three times.

[0057] The obtained solution was photographed by scanning electron microscope, elemental analysis, EDS measurement, Zeta potential and other characterizations. The results are as follows Figure 1-4 shown.

[0058] Figure 1 The synthesized DMOS@FeOOH showed a spindle structure with a long diameter of about 230nm to 240nm and a coating layer on the surface with a thickness of about 40nm to 50nm; Figure 2 The element distribution map shows that C, Si and S elements are mainly concentrated in the outer layer, while Fe element is distributed in the nanocore, and O element is evenly distributed throughout the nanoplatform; accordingly, Figure 3 The EDS spectra further confirmed that these elements were detected; Figure 4 The surface charge of DMOS@FeOOH is shown to be -25.64 mV.

[0059] It can be seen that the present invention successfully synthesized DMOS@FeOOH.

[0060] Example 2

[0061] [Detection of reaction with glutathione in vitro]

[0062] Quantitative determination by UV-visible absorption spectroscopy: DMOS@FeOOH nanoparticles (2 mg / mL) were incubated with GSH aqueous solution (10 mM) at 37 °C and stirred at different time points (0, 0.5, 1, 3, 6, 9 and 12 h). Furthermore, different concentrations of DMOS@FeOOH nanoparticles (0, 100, 200, 400, 600, 800, 1000, 2000 μg / mL) were incubated with GSH solution at 37 °C for 6 h. After centrifugation at 15,000 rpm for 20 min, DTNB solution was added and incubated at 25 °C for 5 min, and the absorbance change at 412 nm was recorded. The results are as follows Figure 4-5 shown.

[0063] Figure 5 and Figure 6 It is the research result of concentration and time dependence. It can be seen from the figure that with the increase of nanoparticle concentration and reaction time, the absorbance of DTNB at 412nm is on a downward trend, indicating that its ability to remove GSH is enhanced, which also shows that the DMOS@FeOOH of the present invention can react with GSH.

[0064] Example 3

[0065] [In vitro H2S generation capacity test]

[0066] In order to quantitatively determine the generation of H2S, different concentrations of DMOS@FeOOH nanoparticles (1 mg / mL and 2 mg / mL) were dispersed in a GSH aqueous solution (10 mM) and stirred at different time points (0, 0.5, 1, 3, 6, 9, 12, 24, and 48 h). Then, the supernatant was collected by centrifugation and mixed with a zinc acetate / sodium acetate mixed solution (mass ratio of 4:1). Then, DMPD·2HCl and FeCl3 were added to form methylene blue, and incubated at 37°C for 15 min. The absorbance change at 664 nm was detected, and the concentration of H2S was calculated using the Na2S standard curve. The results are shown in Figure 2. Figure 6 shown.

[0067] Figure 7 It is based on the quantitative analysis results of the H2S ELISA kit. After 48 h, the H2S released by DMOS@FeOOH at concentrations of 1 mg / mL and 2 mg / mL were approximately 139.55 μM and 201.4 μM, respectively, which indicates that the DMOS@FeOOH of the present invention can react with GSH to generate H2S.

[0068] Example 4

[0069] [In vivo H2S generation capacity test]

[0070] Detection of H2S production in tumor cells. 4T1 cells were cultured at 1×10^ 4 / well were inoculated into 6-well plates and cultured overnight in an incubator; treated with DMOS@FeOOH for 24 hours, washed with PBS three times to remove residual nanomaterials, and then added with WSP-5 solution (50 μM) and incubated for 30 minutes; washed with PBS three times, and recorded images using a fluorescence microscope. The results are shown in Figure 8 shown.

[0071] Figure 8 This is the fluorescence change diagram of the probe WSP-5. Only after DMOS@FeOOH treatment can the obvious green fluorescence of WSP-5 be observed, indicating that H2S is produced in the cells.

[0072] Example 5

[0073] [Detection of reactive oxygen species (·OH) induced by DMOS@FeOOH]

[0074] Different concentrations of DMOS@FeOOH nanoparticles (0, 25, 50, 100, 200, 400, 600, 800, 1000 μg / mL) were dissolved in 10 mM GSH solution and incubated at 37°C for 24 h. Then, the supernatant was collected by centrifugation and reacted with H2O2. TMB solution in dimethyl sulfoxide was added, and the generation of ·OH was determined by detecting the absorbance change at 652 nm. In addition, the generation of ·OH was detected at different pH values ​​(pH = 5.0, 6.5 and 7.4). At the same time, DMOS@FeOOH nanoparticles (2 mg / mL) were incubated with GSH aqueous solution (10 mM) at pH = 5.0 and stirred at different time points (0, 1, 3, 6, 9, 12 and 24 h), and the absorbance change at 652 nm was also detected. The results are shown in the figure. Fig. 9 shown.

[0075] Fig. 9 The absorbance of TMB at 652 nm changes with time. As time goes by, the absorbance value increases, indicating that the generation of ·OH increases with time, indicating that the DMOS@FeOOH of the present invention can induce the generation of active oxygen (·OH).

[0076] Example 6

[0077] [The therapeutic effect of DMOS@FeOOH on breast cancer 4T1 at the cellular and in vivo levels]

[0078] 4T1 cells were plated at 1 × 10 ^4 The cells were seeded at a density of 100 μg / mL in a 96-well plate. After incubation for 12 h, fresh culture medium containing different concentrations (0-500 μg / mL) of DMOS@FeOOH was incubated with the cells for 24 h. Subsequently, 100 μL of MTT solution was added to each well and cultured for another 4 h at 37 °C in the dark. After that, 100 μL of dimethyl sulfoxide was added to each well to dissolve the formed purple crystals, and the absorbance at 490 nm was recorded using a microplate reader.

[0079] 4T1 cells (1×10 ^6 The tumor model was established by subcutaneous injection of 100 μg / ml into the right back of Balb / c mice. 3The tumor-bearing mice were randomly divided into 2 groups (n = 8 in each group) and given the following treatments by intravenous injection: (1) control group, (2) DMOS@FeOOH group (20 mg / kg). The tumor volume and body weight of all groups were measured every two days. The tumor volume was calculated as [(length × width ^2 ) / 2], the result is as follows Figure 10-11 shown.

[0080] Fig.10 It is a therapeutic effect at the cellular level. DMOS@FeOOH has a significant killing effect on tumor cells, but not on normal cells. After adding GSH to normal cells, DMOS@FeOOH has a killing effect, indicating that its killing effect is GSH-responsive. Fig.11 This is a diagram of the in vivo treatment effect. The results show that compared with the control group, DMOS@FeOOH can significantly inhibit tumor growth.

[0081] Therefore, it can be demonstrated that the DMOS@FeOOH of the present invention can selectively kill tumor cells.

[0082] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. A tumor microenvironment-responsive cascade-generated reactive oxygen nanoparticle platform, characterized in that: The nano-platform comprises a FeOOH nano-spindle, the outer layer of which is coated with dendritic mesoporous organosilicon nanoparticles (DMOS NPs) doped with tetrasulfide bonds to form nanoparticles DMOS@FeOOH.

2. The tumor microenvironment responsive cascade reactive oxygen species nanoplatform according to claim 1, characterized in that: The major diameter of the nanoparticle DMOS@FeOOH is 230nm~240nm.

3. The tumor microenvironment responsive cascade reactive oxygen species nanoplatform according to claim 1, characterized in that: The Zeta potential of the nanoparticle DMOS@FeOOH is -25mV~-30mV.

4. The tumor microenvironment responsive cascade reactive oxygen species nanoplatform according to claim 1, characterized in that: The thickness of the dendritic mesoporous organosilicon nanoparticles doped with tetrasulfide bonds on the surface of the nanoparticles DMOS@FeOOH is 40nm to 50nm.

5. A method for preparing a tumor microenvironment responsive cascade reactive oxygen species nanoplatform according to any one of claims 1 to 4, characterized in that: The following steps are involved: FeOOH nanospindles were prepared by hydrothermal method using FeCl3·6H2O as raw material. Under the first temperature condition, FeOOH nanospindles, CTAB and NaSal are added to the TEA aqueous solution, and after stirring and mixing evenly, a mixed solution of TEOS and BTES is added dropwise to react. After the reaction is completed, the solid is collected by centrifugation, and the solid is washed with ethanol to remove residual reactants to obtain a crude product. The crude product is then dispersed in an HCl methanol solution, refluxed and stirred to remove the template, and FeOOH nanospindles (DMOS@FeOOH) with dendritic mesoporous organosilicon nanoparticles (DMOS NPs) doped with tetrasulfide bonds are obtained.

6. The preparation method according to claim 5, characterized in that: The process of preparing FeOOH nanospindles by hydrothermal method using FeCl3·6H2O as raw material includes: A FeCl3·6H2O solution is dissolved in deionized water and vigorously stirred at a second temperature until the solution becomes turbid. The obtained turbid solution is centrifuged to collect the product, the product is washed to obtain FeOOH nanospindles, and the FeOOH nanospindles are redispersed in deionized water for later use.

7. The preparation method according to claim 6, characterized in that: The second temperature is 70°C to 80°C, and the stirring time is 2h to 4h.

8. The preparation method according to claim 5, characterized in that: The volume ratio of TEA to deionized water is (2.0-3.5):1, the mass ratio of FeOOH, CTAB and NaSal is (1:5:3)-(1:10:6), and the mass ratio of TEOS, BTES and CTAB is (1:1:0.2)-(1:1.5:0.4).

9. The preparation method according to claim 5, characterized in that: The first temperature is 70° C. to 80° C., the stirring time after adding FeOOH nanospindles, CTAB and NaSal is 1 h to 2 h, and the reaction time after adding the mixed solution of TEOS and BTES is 10 h to 24 h.

10. Use of the tumor microenvironment responsive cascade reactive oxygen species nanoplatform according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.

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