ROS-responsive self-carrying oxygen nanoparticles and their applications

Abstract

The present invention belongs to the technical field of tumor treatment drugs, and specifically relates to a ROS-responsive self-carrying oxygen nanoformulation and its application. The preparation method of the nanoformulation includes: (1) using chitosan as a skeleton, grafting glycidol, PFO, Ce6 and TK-SN38 respectively to obtain chitosan derivatives; (2) using chitosan derivatives to encapsulate PFH to obtain SN38-Ce6-NPs; (3) saturating SN38-Ce6-NPs with oxygen to obtain a ROS-responsive self-carrying oxygen nanoformulation. After the SN38-Ce6-NPs@O2 was assisted by sonodynamic therapy, tumor growth was significantly inhibited, and a large amount of cell necrosis was found in tumor tissue sections, indicating that the treatment method has a good tumor growth inhibition effect; in addition, SN38-Ce6-NPs@O2 has good in vivo safety and will not affect the normal liver and kidney function of mice.

Description

Technical Field

[0001] The invention belongs to the technical field of tumor therapeutic drugs, and specifically relates to a ROS-responsive self-carrying oxygen nano preparation and application thereof. Background Art

[0002] Colon cancer is the third most common malignant disease in the world. It presents a serious state with strong insidiousness and high mortality. In the treatment of early colon cancer, surgery is the main treatment. However, due to the strong insidiousness of colon cancer, it is already in the late stage when it is discovered, and chemotherapy is needed to reduce the pain of patients. Therefore, the development of a safe and efficient treatment strategy has become an urgent need for modern medicine. Reactive oxygen species (ROS) are a class of active molecules in the human body, and its existence is a double-edged sword. Low levels of ROS are second messengers that regulate physiological functions, but excessive accumulation of ROS will exceed the antioxidant capacity of cells and cause cell death. Cancer cells' dependence on antioxidants makes them more vulnerable to attack by exogenous ROS or compounds that destroy the antioxidant system. Therefore, it has received considerable attention as a potential target for cancer treatment. Photodynamic therapy (PDT) has been widely studied in exogenous ROS-mediated tumor treatment due to its advantages such as minimal invasiveness, targeting, and induction of anti-tumor immune response. However, the effectiveness of PDT depends on the cytotoxicity produced by photosensitizers (PS) under light excitation. However, since light is easily absorbed and reflected during the penetration process, it is difficult to effectively reach the deep tumor site to activate the photosensitizer production. 1 O 2 . Sonodynamic therapy (SDT) is a treatment method developed from photodynamic therapy. It uses low-frequency ultrasound (US) as exogenous energy, which has stronger penetration and can reach deep tissues. It has become an alternative therapy to PDT. The anti-tumor mechanism of SDT is similar to that of PDT. After US activates the sonosensitizer in the tumor site, it transfers energy to the surrounding oxygen, generating a variety of reactive oxygen species (ROS), such as 1 O 2 , hydroxyl radical (·OH), superoxide radical (·O 2 These ROS can induce oxidative damage to cancer cells.

[0003] Solid tumors can cause O 2Diffusion is restricted and blood perfusion is insufficient, resulting in a hypoxic microenvironment inside the tumor tissue. Hypoxia not only promotes tumor proliferation, angiogenesis and metastasis, but also limits the efficiency of radiotherapy, chemotherapy, photodynamic therapy and sonodynamic therapy. In addition, the oxygen consumption behavior of SDT during treatment further aggravates the hypoxic environment in the tumor. Regarding how to overcome the problem of tumor hypoxia microenvironment, many strategies have been reported to alleviate tumor hypoxia. There are three important strategies to alleviate hypoxia: (1) Delivering exogenous oxygen to the tumor site through oxygen-carrying materials to alleviate hypoxia. (2) Alleviating hypoxia by decomposing endogenous hydrogen peroxide through catalase or nanozymes with catalase characteristics. (3) Alleviating hypoxia by reducing oxygen consumption. Perfluorocarbon (PFH) has become an artificial blood substitute as a high-quality oxygen-carrying material. It is reported that US can trigger PFH to burst and release oxygen to alleviate the tumor hypoxia environment, increase ROS generation, and enhance SDT.

[0004] Due to the complexity of tumor treatment, a single treatment method alone cannot completely inhibit tumor growth in clinical treatment. Therefore, a combination of multiple treatment methods is usually used to improve the effect of tumor treatment. In recent years, the combination of chemotherapy and SDT has shown excellent therapeutic effects and less toxic side effects in preclinical and clinical studies of tumors. Ultrasound-induced SDT can increase the permeability of blood vessels and tumor cell membranes, improve the concentration and distribution of drugs in tumors, and enhance the effect of chemotherapy. At the same time, chemotherapy drugs enhance the sensitivity of tumor cells to SDT, thereby achieving the synergistic anti-tumor effect of chemotherapy and SDT. However, the disordered release of chemotherapy drugs in the body and low targeting reduce the synergy between the two. In recent years, various strategies have been used to achieve controlled release of drugs. Among them, endogenous and exogenous stimulus-responsive release have become a hot topic and have made great progress. Endogenous response release includes pH, GSH, enzymes, DNA-specific sequence response, etc.; exogenous stimulus-responsive release includes light control, ultrasound stimulation, magnetic control release, etc. The mechanism of SDT is to produce excessive exogenous ROS through sonosensitizers under US stimulation. Therefore, ROS-responsive release becomes a new strategy to achieve the “toxicity reduction and efficacy enhancement” of drugs, which is expected to achieve the synergistic anti-tumor effect of deep chemotherapy and SDT. Summary of the invention

[0005] In view of the above problems, the present invention aims to provide a ROS-responsive self-carrying oxygen nanoparticle preparation. The nanoparticle preparation uses chitosan as a skeleton material, and is successively grafted with glycidol (Gly), perfluorooctanoyl chloride (Pentadecafluorooctanoyl chloride, PFO), sonosensitizer dihydrochlorin e6 (chlorin e6, Ce6) and TK-SN38 to obtain a Gly-CS-PFO-Ce6-SN38 chitosan derivative; then Gly-CS-PFO-Ce6-SN38 is used to encapsulate perfluorohexane (PFH) to prepare nanoparticles SN38-Ce6-NPs, and after oxygen saturation, self-carrying oxygen nanoparticles SN38-Ce6-NPs@O 2 The nanoformulation combined with ultrasound stimulation can alleviate tumor hypoxia and achieve precise and controllable release of drugs.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides a ROS-responsive self-carrying oxygen nanoformulation, and the preparation method thereof comprises: (1) using chitosan as a skeleton, grafting glycidol, PFO, Ce6 and TK-SN38 respectively to obtain a chitosan derivative Gly-CS-PFO-Ce6-SN38; (2) using the chitosan derivative Gly-CS-PFO-Ce6-SN38 to encapsulate PFH to obtain SN38-Ce6-NPs; (3) saturating the SN38-Ce6-NPs with oxygen to obtain a ROS-responsive self-carrying oxygen nanoformulation.

[0008] Another aspect of the present invention provides an anti-tumor drug preparation, which includes the ROS-responsive self-carrying oxygen nanoparticle preparation of the present invention.

[0009] In another aspect, the present invention provides a use of the ROS-responsive self-carrying oxygen nanoformulation of the present invention in the preparation of an anti-tumor drug preparation.

[0010] The beneficial effects of the present invention include at least:

[0011] (1) The results of dissolved oxygen experiment and oxygen release experiment show that the SN38-Ce6-NPs@O 2 Good oxygen dissolving capacity, able to quickly release oxygen and alleviate the oxygen-deficient environment; 1 O 2 The experimental results show that compared with Ce6-NPs and SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 More ROS can be produced under US stimulation.

[0012] (2) In vitro drug release experiments show that: the SN38-Ce6-NPs@O 2 The drug release was affected by H 2 O 2 The effect of concentration. Under US stimulation, the SN38-Ce6-NPs group and SN38-Ce6-NPs@O 2 The cumulative release amounts of the two groups within 48 hours were about 27% and 43%, and the cumulative release amounts of the two groups increased in a leap-like manner after each US stimulation, indicating that the nanoparticles can achieve precise and controllable release of drugs in vitro.

[0013] (3) The results of cell uptake experiments showed that: SN38-Ce6-NPs@O 2 The SN38-Ce6-NPs@O was successfully taken up by CT26 cells within 6 hours. The results of the experiment on alleviating cell hypoxia showed that: 2 It can relieve cell hypoxia. The results of intracellular ROS generation and mitochondrial membrane potential damage experiments showed that under US stimulation, SN38-Ce6-NPs@O 2 It can significantly increase the level of intracellular ROS, causing damage to the cell membrane potential, leading to mitochondrial damage, and further inducing cell apoptosis. At the same time, excessive ROS responsively releases SN38, thereby effectively killing tumor cells.

[0014] (4) The results of in vitro antitumor effect experiments showed that CT26 cells were treated with SN38-Ce6-NPs@O 2 After US stimulation, about 80% of cells died after 48 h of incubation, indicating that SN38-Ce6-NPs@O 2 It has a stronger inhibitory effect on CT26 cells.

[0015] (5) A colon cancer mouse model was successfully established, and the results of in vivo imaging of small animals showed that compared with Free Ce6, the SN38-Ce6-NPs@O 2 The nanoparticles can be passively targeted to the tumor site through the tumor EPR effect and prolong the retention time of the nanoparticles in the tumor.

[0016] (5) In vivo pharmacodynamic results showed that tumor-bearing mice received SN38-Ce6-NPs@O 2 When combined with US treatment, tumor growth can be inhibited to the greatest extent, with an inhibition rate of up to 89%.

[0017] (6) The results of tumor pathological sections showed that: the SN38-Ce6-NPs@O 2A large amount of necrosis occurred in the tumor tissues of tumor-bearing mice in the +US group, indicating that this treatment strategy has a significant anti-tumor effect in vivo; in addition, the results of H&E staining of important organs of tumor-bearing mice, plasma biochemical indicators, and hemolysis experiments showed that SN38-Ce6-NPs@O 2 Good biological safety in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A synthetic route for chitosan derivatives;

[0019] Figure 2 For TK-SN38, add H 2 O 2 HPLC chromatogram after

[0020] Figure 3 Appearance of chitosan derivatives and nanoparticles in aqueous solution; a: CS, b: Gly-CS, c: Gly-CS-PFO, d: Gly-CS-PFO-Ce6, e: Gly-CS-PFO-Ce6-SN38;

[0021] Figure 4 Chitosan derivatives 1 HNMR spectra;

[0022] Figure 5 is the FITR spectrum of chitosan derivatives;

[0023] Figure 6 is the UV-vis spectrum of chitosan derivatives;

[0024] Figure 7 is the fluorescence emission spectrum of chitosan derivatives;

[0025] Figure 8 is the particle size of SN38-Ce6NPs with different ratios of perfluorohexane;

[0026] Fig. 9 The potential diagram of SN38-Ce6NPs with different ratios of perfluorohexane;

[0027] Fig.10 Transmission electron microscopy image of SN38-Ce6-NPs (scalebar=500nm);

[0028] Fig.11 SN38-Ce6-NPs@O 2 TEM image (scalebar=500nm);

[0029] Fig.12 is the oxygen dissolving capacity of different chitosan derivatives and SN38-Ce6-NPs;

[0030] Fig.13 For SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 Oxygen release graph;

[0031] Fig.14 Under the stimulation of US, H 2 Time-dependent UV-vis absorption spectra of O;

[0032] Fig.15 is the time-dependent UV-vis absorption spectrum of SN38-Ce6-NPs under US stimulation;

[0033] Fig.16 H 2 O, SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 Under US stimulation at different times 1 O 2 The generation of

[0034] Fig.17 H 2 O, SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 Under different time intensity US stimulation 1 O 2 The generation of

[0035] Fig.18 For different concentrations of H 2 O 2 SN38-Ce6-NPs@O after treatment 2 Cumulative drug release profile of

[0036] Fig.19 Figure 2 shows the structures of SN38-Ce6-NPs and SN38-Ce6-NPs@O under the stimulation of different concentrations of US. 2 Cumulative drug release profile of

[0037] Fig. 20 SN38-Ce6-NPs@O 2 Laser confocal image after 6h of co-incubation with CT26 cells;

[0038] Fig.21 is [Ru(dpp) 3 ]Cl 2 Fluorescence inverted image of staining to detect oxygen content in CT26 cells (bar: 50 μm);

[0039] Fig. 22 Fluorescence inverted image of DCFH-DA staining to detect ROS levels in CT26 cells (bar: 250 μm);

[0040] Fig.23 To semi-quantitatively analyze the intracellular ROS level;

[0041] Fig.24 Fluorescence inverted image of JC-1 staining to detect mitochondrial membrane potential damage in CT26 cells (bar: 50 μm);

[0042] Fig.25 The effect of different ultrasound time on CT26 cell viability;

[0043] Fig.26 The antitumor effect of self-carrying oxygen nanoparticles in vitro under normoxic conditions;

[0044] Fig. 27 The antitumor effect of self-carrying oxygen nanoparticles in vitro under hypoxic conditions;

[0045] Fig.28 This is a fluorescence inverted image of CT26 cells co-stained with Calcein-AM / PI (bar: 250 μm);

[0046] Fig.29 Schematic diagram for establishing the CT26 tumor-bearing mouse model;

[0047] Fig.30 SN38-Ce6-NPs@O were injected into the tail vein of CT26 tumor-bearing mice. 2 Later, the in vivo fluorescence imaging pictures at different time points, the yellow circle indicates the tumor area;

[0048] Fig.31 SN38-Ce6-NPs@O were injected into the tail vein of CT26 tumor-bearing mice. 2 Fluorescence semi-quantitative analysis of the tumor site;

[0049] Fig.32 SN38-Ce6-NPs@O 2 Fluorescence images of various organs and tumor tissues 48 hours after tail vein injection;

[0050] Fig.33 SN38-Ce6-NPs@O 2 Semi-quantitative analysis of fluorescence intensity of various organs and tumor tissues 48 hours after tail vein injection;

[0051] Fig.34 This is the tumor growth curve of CT26 tumor-bearing mice after different treatments;

[0052] Fig.35 is the tumor weight of CT26 tumor-bearing mice after different treatments;

[0053] Fig.36These are photos of tumors in CT26 tumor-bearing mice after different treatments;

[0054] Fig.37 This is the body weight change curve of CT26 tumor-bearing mice after different treatments;

[0055] Fig.38 H&E staining of tumor sections of CT26 tumor-bearing mice after different treatments (bar: 100 μm);

[0056] Fig.39 H&E staining of important organs of CT26 tumor-bearing mice after different treatments (bar: 200 μm);

[0057] Fig.40 is the level of serum alanine aminotransferase in CT26 tumor-bearing mice after different treatments (n=3);

[0058] Fig.41 is the level of serum aspartate aminotransferase in CT26 tumor-bearing mice after different treatments (n=3);

[0059] Fig.42 The levels of serum urea nitrogen in CT26 tumor-bearing mice after different treatments (n=3);

[0060] Fig.43 Serum lactate dehydrogenase levels in CT26 tumor-bearing mice after different treatments (n=3);

[0061] Fig.44 Serum creatinine levels in CT26 tumor-bearing mice after different treatments (n=3);

[0062] Fig.45 Photos of the hemolysis rate and red blood cell status of SN38-Ce6-NPs at different concentrations in CT26 tumor-bearing mice after different treatments. DETAILED DESCRIPTION

[0063] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0064] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0065] The embodiment of the present invention provides a ROS-responsive self-carrying oxygen nanoformulation, and the preparation method thereof comprises: (1) using chitosan as a skeleton, grafting glycidol, PFO, Ce6 and TK-SN38 respectively to obtain a chitosan derivative Gly-CS-PFO-Ce6-SN38; (2) using the chitosan derivative Gly-CS-PFO-Ce6-SN38 to encapsulate PFH to obtain SN38-Ce6-NPs; (3) saturating the SN38-Ce6-NPs with oxygen to obtain a ROS-responsive self-carrying oxygen nanoformulation.

[0066] It should be noted that the SN38-Ce6-NPs@O 2 , its particle size is 200-350nm, and its potential is 18mV. It should also be noted that SN38-Ce6-NPs@O 2 After reaching the tumor site through the tumor EPR effect in vivo, under US stimulation, PFH explodes and releases oxygen to relieve tissue hypoxia. At the same time, Ce6 accumulated deep in the tissue is activated to produce excessive ROS, causing cell apoptosis. Excessive ROS causes the TK bond to break, causing the nanoparticles to respond and release SN38; the released SN38 and ROS can both penetrate into the deep layer of tumor tissue, achieving a precisely controllable chemotherapy combined with SDT synergistic anti-tumor treatment effect.

[0067] In some specific embodiments, in the method for preparing the above ROS-responsive self-carrying oxygen nanoformulation, the chitosan is chitosan with a molecular weight of 50,000.

[0068] In some specific embodiments, in the preparation method of the above-mentioned ROS-responsive self-oxygen-carrying nanoformulation, in step (1), the preparation method of the chitosan derivative Gly-CS-PFO-Ce6-SN38 includes: (a) reacting chitosan with glycidol at 45°C-55°C to obtain Gly-CS; (b) reacting Gly-CS with PFO at room temperature to obtain Gly-CS-PFO; (c) reacting activated Ce6 with Gly-CS-PFO in the dark to obtain Gly-CS-PFO-Ce6; (d) reacting activated TK-SN38 with Gly-CS-PFO-Ce6 in the dark to obtain the chitosan derivative Gly-CS-PFO-Ce6-SN38.

[0069] In some specific embodiments, in the preparation method of the above-mentioned ROS-responsive self-carrying oxygen nanoformulation, in step (2), the preparation method of SN38-Ce6-NPs includes: mixing PFH and Gly-CS-PFO-Ce6-SN38 aqueous solution, and ultrasonic emulsification to obtain SN38-Ce6-NPs.

[0070] In some specific embodiments, in the preparation method of the above-mentioned ROS-responsive self-carrying oxygen nanoformulation, the mass ratio of PFH to Gly-CS-PFO-Ce6-SN38 is 4:1-32:1.

[0071] In some specific embodiments, in the method for preparing the above ROS-responsive self-carrying oxygen nanoformulation, the ultrasonic emulsification time is ≥ 3 min.

[0072] In some specific embodiments, in the preparation method of the above-mentioned ROS-responsive self-oxygen-carrying nanoformulation, in step (3), the preparation method of the ROS-responsive self-oxygen-carrying nanoformulation includes: passing oxygen through the SN38-Ce6-NPs solution for 10 minutes to obtain the ROS-responsive self-oxygen-carrying nanoformulation.

[0073] It should be noted that in the present invention, TK (Thioketal) and 7-ethyl-10-hydroxycamptothecin (SN38) are first used as raw materials to synthesize the ROS-responsive small molecule prodrug TK-SN38 through esterification reaction. Chitosan is used as the skeleton material, and glycido (Gly), perfluorooctanoyl chloride (Pentadecafluorooctanoyl chloride, PFO), sonosensitizer dihydrochlorin e6 (chlorin e6, Ce6) and TK-SN38 are successively grafted to obtain Gly-CS-PFO-Ce6-SN38 and other chitosan derivatives, and then through 1Secondly, Gly-CS-PFO-Ce6-SN38 was used to encapsulate perfluorohexane (PFH) to prepare nanoparticles SN38-Ce6-NPs, and then saturated with oxygen to obtain self-oxygen nanoparticles SN38-Ce6-NPs@O 2 To further investigate the particle size potential, morphology, dissolved oxygen and oxygen release behavior of self-carrying oxygen nanoparticles and the effects of US stimulation on the 1 O 2 Secondly, a mouse colon cancer CT26 cell model was established, and the in vitro performance of SN38-Ce6-NPs@O was investigated through uptake test, intracellular hypoxia relief test, intracellular ROS generation test, mitochondrial membrane potential damage test, cytotoxicity test, etc. 2 The killing effect and mechanism of CT26 cells under US stimulation were studied. Finally, a CT26 tumor-bearing mouse model was established to study the in vivo drug distribution, anti-tumor ability and biosafety of self-carrying oxygen nanoparticles.

[0074] Another embodiment of the present invention provides an anti-tumor drug preparation, which includes the ROS-responsive self-carrying oxygen nanoformulation of the present invention.

[0075] Another embodiment of the present invention provides an application of the ROS-responsive self-carrying oxygen nanoformulation of the present invention in the preparation of an anti-tumor drug preparation.

[0076] In some specific embodiments, the tumor is colon cancer.

[0077] It should be noted that colon cancer (CRC) is a highly prevalent malignant tumor. In the early stage of CRC, surgery is the main treatment. However, due to its strong occult nature, it is already in the late stage when it is discovered. Chemotherapy is needed to improve the survival rate of patients. However, the side effects of chemotherapy drugs in clinical practice are large, which seriously hinders the implementation of preoperative and postoperative chemotherapy policies. Therefore, it is urgent to develop a treatment method. Sonodynamic therapy (SDT) is a non-invasive treatment method that uses ultrasound (US) to stimulate sonosensitizers to produce cytotoxic reactive oxygen species (ROS) to kill tumor cells. However, as a solid tumor, CRC has metabolic disorders due to its malignant proliferation, which makes the tumor tissue in a hypoxic environment for a long time. As an oxygen-dependent treatment method, SDT is limited in its application due to the hypoxic environment of the tumor. Therefore, it is urgent to alleviate the problem of tumor hypoxia. In recent years, great research progress has been made in the combination of chemotherapy and sonodynamic therapy. However, the poor targeting and disordered release of chemotherapy drugs in the body and the hypoxic microenvironment of the tumor body reduce the synergistic effect of chemotherapy and SDT. Therefore, this study aims to construct a self-carrying oxygen nanoformulation with ROS-responsive drug release properties to achieve orderly drug release and alleviate hypoxia to enhance the synergistic effect of chemotherapy and SDT.

[0078] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0079] 1. Synthesis of chitosan derivative Gly-CS-PFO-Ce6-SN38

[0080] (I) Synthesis of Glycidyl Chitosan (Gly-CS)

[0081] like Figure 1 As shown in (A), 1g of chitosan (50,000 molecular weight, 95% deacetylation degree) was dissolved in 100mL of acetic acid solution (1%, v / v), stirred at room temperature for 48h, centrifuged (7000r / min, 15min), and filtered with a 0.22μm membrane to remove impurities. 5.38mL of glycidol was slowly added dropwise to 100mL of 1% chitosan solution, stirred at 50℃ and 1000rpm for 24h; deionized water was dialyzed for three days (water was changed every 8h) to remove the incompletely reacted glycidol. After freeze drying, yellow powder Gly-CS was obtained.

[0082] (II) Synthesis of chitosan derivative Gly-CS-PFO

[0083] Dissolve 100 mg of Gly-CS in 20 mL of deionized water, slowly drop 16 mg of perfluorooctanoyl chloride (PFO) into the Gly-CS solution and stir at room temperature for 24 hours. After the reaction is completed, dialyze with deionized water for three days (change the water every 8 hours) to remove the unreacted PFO. Freeze-dry to obtain a yellow powder. Freeze-dry to obtain a yellow powder of perfluorooctanoyl chloride-water-soluble chitosan (glycidochitosan pentadecafluorooctanoyl chloride, Gly-CS-PFO).

[0084] (III) Synthesis of chitosan derivative Gly-CS-PFO-Ce6

[0085] like Figure 1 As shown in (A), appropriate amounts of Ce6, EDC, and NHS were dissolved in DMSO in a molar ratio of 1:3:3, activated for 4 hours in the dark, and then 100 mg of Gly-CS-PFO was dissolved in 10 mL of deionized water. 15 mg of activated Ce6 was added to the Gly-CS-PFO solution and reacted for 48 hours in the dark. After the reaction, the unreacted Ce6 was removed by dialysis with deionized water for three days (changing the water every 8 hours). Freeze drying gave a yellow-green powder of dihydrochlorin e6-perfluorooctanoyl chloride-water-soluble chitosan (glycido chitosanpentadecafluorooctanoyl chloride chlorin e6, Gly-CS-PFO-Ce6).

[0086] (IV) Synthesis of chitosan derivative Gly-CS-PFO-Ce6-SN38

[0087] (1) Synthesis of ROS response bonds (TK)

[0088] After mixing an appropriate amount of anhydrous 3-mercaptopropionic acid and anhydrous acetone in a molar ratio of 2:1, a catalytic amount of trifluoroacetic acid was added, and then the mixture was transferred to a sealed round-bottom flask and stirred at room temperature for 6 hours. After the reaction was completed, it was cooled and crystallized in ice water, then washed three times with n-hexane and cold water, and filtered and dried to obtain a white powder, which is the ROS-responsive linker thioketal (TK).

[0089] (2) Synthesis of ROS-responsive prodrug (TK-SN38)

[0090] like Figure 1As shown in (B), appropriate amounts of SN38, TK, DMAP, and EDC were dissolved in anhydrous DCM in a molar ratio of 1:1:0.8:6, and the mixture was then transferred to a sealed round-bottom flask and stirred at room temperature for 6 hours. After the reaction was completed, it was extracted three times with saturated NaCl, and then the organic layer was dried over anhydrous sodium sulfate overnight and the solvent was removed by rotary evaporation. ROS-responsive TK-SN38 was obtained by preparative liquid phase separation and purification (purification chromatographic conditions were as follows: Basic C18 (5μm, 10×250mm) column; column temperature: room temperature; flow rate: 3mL / min; mobile phase: CH 3 OH:H 2 O(80:1) detection wavelength: 372nm; injection volume: 100μL).

[0091] (3) Grafting ROS-responsive prodrug TK-SN38 (Gly-CS-PFO-Ce6-SN38)

[0092] like Figure 1 As shown in (A), appropriate amounts of TK-SN38, EDC, and NHS were dissolved in DMSO at a molar ratio of 1:3:3, activated for 4 hours in the dark, and then 100 mg of Gly-CS-PFO-Ce6 was dissolved in 10 mL of deionized water. 30 mg of activated TK-SN38 was added to the Gly-CS-PFO-Ce6 solution and reacted for 48 hours in the dark. After the reaction, the unreacted TK-SN38 was removed by dialysis with deionized water for three days (changing the water every 8 hours). The green powder Gly-CS-PFO-Ce6-SN38 was freeze-dried.

[0093] 2. Characterization of chitosan derivative Gly-CS-PFO-Ce6-SN38

[0094] (I) ROS response characteristics of TK-SN38

[0095] The ROS response characteristics of TK-SN38 were characterized by high performance liquid chromatography, including: dissolving 1 mg of TK-SN38 in 1 mL of methanol, adding 1 mM hydrogen peroxide and reacting for 2 h, then removing the hydrogen peroxide by rotary evaporation, and detecting its ROS response ability after re-dissolving in methanol.

[0096] The retention times of SN38, TK-SN38 and degradation products are as follows Figure 2 As shown in the figure, the retention time of free SN38 was 4.1 min, the retention time of TK-SN38 was 5.4 min, and the addition of 1 mM H 2 O 2 After that, the retention time appeared at 4.4min. The results showed that TK-SN38 was 2 O 2Successful cleavage, with ROS response characteristics. The retention time of SN38 after TK-SN38 cleavage does not coincide with that of free SN38. The reason is that ketone thiolate (TK) is a subclass of thioether. When exposed to ROS, TK will be cleaved into groups containing thiol. The cleaved SN38 has a thiol group, which leads to a decrease in polarity and a later retention time.

[0097] (II) Appearance of chitosan derivatives

[0098] The appearance of chitosan derivatives is Figure 3 As shown: a is CS solution, b is Gly-CS solution, c is Gly-CS-PFO solution, d is Gly-CS-PFO-Ce6 solution, and e is Gly-CS-PFO-Ce6-SN38 solution. It was found that chitosan was obviously insoluble in water. After the introduction of glycidol, the solution became clear and uniform, indicating that the hydrophilic group was successfully introduced. The Gly-CS-PFO-Ce6 solution showed the green color of Ce6 itself, indicating that Ce6 was successfully introduced. The introduction of SN38 had no obvious change in the appearance of the solution.

[0099] (III) H NMR spectroscopy

[0100] CS, Gly-CS, Gly-CS-PFO, Gly-CS-PFO-Ce6, Gly-CS-PFO-Ce6-SN38 1 The HNMR results are as follows Figure 4 As shown, in the CS spectrum, 3.5-3.9ppm are the H signal peaks of the C-3, C-4, C-5, and C-6 methyl groups on the glucosamine ring. Compared with the CS spectrum, the signals at 3-3.5ppm in the spectra of Gly-CS, Gly-CS-PFO, Gly-CS-PFO-Ce6, and Gly-CS-PFO-Ce6-SN38 are mainly derived from the H signal peak on the amide bond formed after Gly, PFO, Ce6, and TK-SN38 combine with the free amino group of CS. The signals of Gly-CS at 2.0-2.3ppm and 4.4ppm are derived from the H signal peak of the hydroxyl group in glycidol. Since perfluorooctanoyl chloride has no hydrogen atoms, the spectrum of Gly-CS-PFO is similar to that of Gly-CS. Figure 1 The signal at 2-3ppm in the spectrum of Gly-CS-PFO-Ce6 is the H signal peak on Ce6. In the spectrum of Gly-CS-PFO-Ce6-SN38, the signals at 1.5-1.9ppm and 3.7ppm are from SN38 and C-20 -CH 2 CH 3 The signal at 2.5-3ppm comes from the H signal peak on the benzene ring of SN38. In summary, Gly, PFO, Ce6, and TK-SN38 were successfully grafted onto the CS skeleton.

[0101] (IV) Fourier transform infrared spectroscopy

[0102] Fourier transform infrared spectra (FTIR) of CS, Gly-CS, Gly-CS-PFO, Gly-CS-PFO-Ce6, and Gly-CS-PFO-Ce6-SN38 were measured by Fourier transform infrared spectrometer. The potassium bromide tablet method was adopted. After the potassium bromide was ground, it was dried at 120°C for 12h. The sample was mixed and ground with potassium bromide powder, and then tableted. The scanning wave number range was 4000~400cm-1, and the resolution was 4cm -1 .

[0103] FTIR of CS, Gly-CS-PFO, Gly-CS-PFO-Ce6, Gly-CS-PFO-Ce6-SN38 Figure 5 Shown: 3700-3000cm in CS spectrum -1 The stretching vibration peaks of OH on the hydroxyl group and NH on the amino group are shown in Figure 2. Comparing the peaks in this area in each spectrum, it can be seen that Gly-CS-PFO, Gly-CS-PFO-Ce6, and Gly-CS-PFO-Ce6-SN38 are significantly weaker than CS, indicating that -NH 2 Replaced. 1600cm -1 Nearby is the NH in-plane bending vibration peak on the free amino group of CS, which is significantly weakened in Gly-CS-PFO, Gly-CS-PFO-Ce6, and Gly-CS-PFO-Ce6-SN38, indicating that the reaction mainly occurs on the primary amino group on CS to form an amide bond. -1 The absorption peak at 40° is enhanced, which is the C=C stretching vibration peak of the benzene ring on SN38. In summary, it is proved that Gly, PFO, Ce6 and TK-SN38 are successfully grafted.

[0104] (V) Ultraviolet spectrometry

[0105] In order to prove whether Ce6 exists in chitosan derivatives, ultraviolet-visible spectra (UV-vis) of Gly-CS, Gly-CS-PFO, Gly-CS-PFO-Ce6, Gly-CS-PFO-Ce6-SN38, and Ce6 and SN38 solutions were measured by UV-visible spectrophotometer with a scanning range of 300-1000nm.

[0106] The UV-vis absorption spectra of Gly-CS, Gly-CS-PFO, Gly-CS-PFO-Ce6, Gly-CS-PFO-Ce6-SN38, and Ce6 solutions are shown in Figure 6 As shown, Gly-CS and Gly-CS-PFO have no obvious characteristic absorption peaks. Ce6 has S band absorption at 402nm and Q band absorption at 508, 540, and 674nm; SN38 has obvious ultraviolet absorption in the range of 350-450nm, which is consistent with the description in the literature. Compared with free Ce6, Gly-CS-PFO-Ce6 and Gly-CS-PFO-Ce6-SN38 both showed obvious Ce6 characteristic absorption peaks at 664nm, indicating that Ce6 was successfully grafted. Gly-CS-PFO-Ce6-SN38 has a higher absorption peak in the range of 350-400nm. It is believed that in addition to Ce6 absorption, SN38 also has absorption, indicating that Ce6 and SN38 are successfully grafted. In addition, Gly-CS-PFO-Ce6 and Gly-CS-PFO-Ce6-SN38 showed a slight blue shift at 664nm, which may be caused by the environmental effect brought about by the grafting of Ce6 onto CS, such as the influence of the amino and hydroxyl groups with lone pair electrons on CS on Ce6.

[0107] (VI) Fluorescence emission spectrum

[0108] In order to prove whether SN38 and Ce6 exist in chitosan derivatives, the fluorescence spectra (FS) of Gly-CS, Gly-CS-PFO, Gly-CS-PFO-Ce6, Gly-CS-PFO-Ce6-SN38, Ce6, and SN38 were measured by Multiskan FC microplate reader. The excitation wavelength was 358 nm and the scanning range was 400-850 nm.

[0109] The fluorescence emission spectra of Gly-CS, Gly-CS-PFO, Gly-CS-PFO-Ce6, Gly-CS-PFO-Ce6-SN38, Ce6 and SN38 solutions are shown in Figure 7As shown. Under 358nm wavelength light excitation, the fluorescence spectra of Gly-CS and Gly-CS-PFO show no clear fluorescence signal in the range of 400-850nm. Free Ce6 can produce obvious red fluorescence, so Ce6 has a characteristic maximum emission peak at 664nm. The fluorescence spectra of Gly-CS-PFO-Ce6 and Gly-CS-PFO-Ce6-SN38 show the characteristic emission peak of free Ce6 near 664nm, indicating that Ce6 has been successfully grafted. Free SN38 can emit obvious yellow-green fluorescence, so SN38 has a characteristic maximum emission peak at 558nm. The fluorescence spectrum of Gly-CS-PFO-Ce6-SN38 shows the characteristic emission peak of free SN38 near 558nm, indicating that SN38 has been successfully grafted.

[0110] In summary, the present invention successfully synthesized the ROS-responsive drug TK-SN38, and HPLC verified that TK-SN38 was ROS-sensitive. Secondly, Gly, PFO, Ce6, and TK-SN38 were successfully grafted to obtain Gly-CS-PFO, Gly-CS-PFO-Ce6, and Gly-CS-PFO-Ce6-SN38, and 1 The characterization by HNMR, FI-TR, UV-vis and FL spectra all indicated successful grafting.

[0111] 3. Self-carrying oxygen nanoparticles (SN38-Ce6-NPs@O 2 ) preparation and physical and chemical properties test

[0112] (I) Preparation of self-carrying oxygen nanoparticles (SN38-Ce6-NPs@O2)

[0113] 5 mg of Gly-CS-PFO-Ce6-SN38 was dissolved in 5 mL of deionized water to prepare a 1 mg / mL SN38-Gly-CS-PFO-Ce6 solution, and perfluorohexane (PFH) and Gly-CS-PFO-Ce6-SN38 were mixed in a mass ratio of 4:1, 8:1, 16:1, and 32:1, and ultrasonic emulsification was performed for 3 min (ultrasonic power 1000w, ultrasonic 20s, interval 20s, total ultrasonic emulsification 3 min) to obtain SN38-Ce6-NPs of different proportions. Then, oxygen was passed through the nanoparticle solution for 10 min to saturate the nanoparticle solution with oxygen, and self-carrying oxygen nanoparticles SN38-Ce6-NPs@O2 were obtained.

[0114] (II) Self-carrying oxygen nanoparticles (SN38-Ce6-NPs@O 2 ) Characterization

[0115] (1) Particle size and potential

[0116] NanoZS90 laser particle size analyzer was used to measure the SN38-Ce6-NPs@O with different mass ratios of Gly-CS-PFO-Ce6-SN38:PFH. 2 The particle size and potential of Figure 8 and Fig. 9 As shown in the figure, with the increase of the ratio of perfluorohexane, the particle size of SN38-Ce6-NPs gradually increased, and the particle size was between 250-620nm. The higher the concentration of perfluorohexane, the more conducive it is to carry oxygen. Since the particle size of 1:32 is too large and the EPR effect needs to be taken into account, the nanoparticles of 1:16 are selected as the optimal ratio. The Zeta potential is between 20m V and 26mV.

[0117] (2)TEM morphology

[0118] The SN38-Ce6-NPs and SN38-Ce6-NPs@O were observed by JEM-2000EX transmission electron microscope. 2 The TEM morphology is shown in Fig.10 and Fig.11 SN38-Ce6-NPs(A) and SN38-Ce6-NPs@O 2 (B) It is spherical under transmission electron microscopy, and the particle size is between 200 and 300 nm. The particle size in the transmission electron microscope is lower than that measured by the nanoparticle size analyzer because the nanoparticles have a hydration layer in water. However, when performing transmission electron microscopy, the nanoparticles need to be dried, which causes the hydration layer of the nanoparticles to disappear, and the nanoparticles shrink and become smaller in size. Both the nanoparticle size analyzer and the transmission electron microscope show that the particle size of the nanoparticles is uniform.

[0119] (III) Self-carrying oxygen nanoparticles (SN38-Ce6-NPs@O 2 ) Functional evaluation

[0120] (1) Oxygen dissolution capacity and oxygen release behavior of ROS-responsive self-carrying oxygen nanoparticles

[0121] ① Dissolved oxygen capacity

[0122] Another important sign of the tumor microenvironment is hypoxia caused by the irregular proliferation of tumor cells. Hypoxia is not only a factor that induces tumor metastasis, but also limits the therapeutic effect of oxygen-dependent treatments. Many reports have shown that perfluorohexane has a high oxygen carrying capacity. 2 It can reach the tumor site through blood circulation and alleviate the problem of tumor hypoxia. Therefore, the present invention has conducted a preliminary test on its oxygen load, as follows:

[0123] The dissolved oxygen experiment was divided into five groups: anaerobic H 2O group, Gly-CS group, Gly-CS-PFO group, Gly-CS-PFO-Ce6-SN38 group, SN38-Ce6-NPs group. 2 mL of solution from each group was oxygenated for 7 min, and the real-time oxygen concentration in the solution was measured using a dissolved oxygen meter, and the oxygen concentration was recorded every 10 s. The oxygen loading capacity of different chitosan derivatives and SN38-Ce6-NPs is shown in Fig.12 As shown, the dissolved oxygen content of SN38-Ce6-NPs reached about 26 mg / L, which was higher than that of water and chitosan derivatives, indicating that SN38-Ce6-NPs had good oxygen dissolving capacity.

[0124] ② Oxygen release behavior

[0125] The oxygen release experiment was divided into three groups: anaerobic H 2 O group, SN38-Ce6-NPs group, SN38-Ce6-NPs@O 2 Group. 4 mL of degassed water was placed in a glass bottle, and the gas in the bottle was discharged with a 50 mL syringe to simulate an oxygen-deficient environment. Then, 2 mL of oxygenated solution was quickly transferred to the bottle. A dissolved oxygen meter was used to measure the real-time oxygen concentration in the bottle. The oxygen concentration was recorded every 10 seconds.

[0126] SN38-Ce6-NPs@O 2 Oxygen release behavior Fig.13 As shown in the figure, after adding oxygen-saturated SN38-Ce6-NPs to deoxygenated water, the dissolved oxygen concentration quickly reached ~3.5 mg / L within 20 seconds, and then slowly rose to a maximum of ~4 mg / L after two minutes. The dissolved oxygen concentration of SN38-Ce6-NPs in the anoxic group hardly increased. The results show that SN38-Ce6-NPs@O 2 Shows good oxygen carrying and rapid oxygen release capabilities.

[0127] ③Ultrasound stimulation generation 1 O 2

[0128] Ce6 is a common natural porphyrin compound that has the ability to generate ROS under US stimulation. The specific form of ROS generation is 1 O 2 , so it can be used as an effective sonosensitizer for SDT of tumors. In the present invention, DPBF is used as a probe to detect the generated 1 O 2 , evaluate the SN38-Ce6-NPs@O 2 The acoustic dynamic effects are as follows:

[0129] 1 O 2 The generation experiment was divided into four groups: anaerobic H2 O group, dihydrochlorin e6 (Ce6-NPs group), SN38-Ce6-NPs group, SN38-Ce6-NPs@O 2 The Ce6-NPs, SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 The solution was added to the DPBF solution and the mixture was stirred in the dark using US (1.0 MHz, 1 W cm -2 ) were ultrasonically stimulated, and the absorbance and absorption spectrum of DPBF at 410 nm were measured by UV spectrophotometer every 1 min. 2 The Ce6 concentration in the solution was 10 μg / mL, and the DPBF concentration was 25 μg / mL.

[0130] The results are as follows: It was found that 2 Under US stimulation in O, the characteristic band of DPBF hardly changed with the increase of ultrasound time ( Fig.14 ), in SN38-Ce6-NPs@O 2 Under US stimulation, the absorption peak of the characteristic band of DPBF decreased significantly ( Fig.15 ), indicating that ultrasound can effectively trigger the SN38-Ce6-NPs@O 2 produce 1 O 2 In order to verify the oxygen carried by PFH in ultrasound-induced 1 O 2 The key role of H 2 O, Ce6-NPs, SN38-Ce6-NPs and SN38-Ce6-NPs@O 2 of 1 O 2 The generated results are as follows Fig.16 As shown, after US irradiation, H 2 The absorbance of DPBF in the O group decreased by only about 4%. The absorbance of DPBF in the SN38-Ce6-NPs group decreased by about 36%, and the absorbance of DPBF in the SN38-Ce6-NPs@O 2 The group decreased by as much as 78%, indicating that SN38-Ce6-NPs can produce 1 O 2 , and after oxygenation, it can significantly improve 1 O 2 In addition to studying whether ultrasound time has an effect on 1 O 2 The effects of different ultrasound intensities on the generation of 1 O 2 The impact of the generation, the results are as follows Fig.17 As shown, as the US intensity increases 1 O 2 In summary, SN38-Ce6-NPs can effectively generate 1 O 2 , and secondly, oxygenation can increase 1 O 2 In addition, extending the ultrasonic time and power can also increase 1 O 2 The production of.

[0131] (2) ROS-responsive and ultrasound-responsive drug release experiments

[0132] ① ROS-responsive drug release experiment

[0133] The ROS level in cancer cells is tens to hundreds of times higher than that in normal cells, so the ROS-responsive drug delivery system provides more tumor-specific drug release. 2 The present invention uses H 2 O 2 Simulate the ROS conditions in tumor cells. The details are as follows: Take 2mL SN38-Ce6-NPs@O 2 The solution was placed in a dialysis bag, and the dialysis bag was placed in a centrifuge tube containing 10 mL PBS (pH = 7.4, containing 20% ​​acetonitrile), and the dialysis bag was completely immersed in PBS. The centrifuge tube was fixed on a constant temperature shaking incubator, and the temperature was shaken at 37°C at a speed of 100 rpm. Samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hours, and 0.2 mL PBS was sampled each time, and 0.2 mL blank PBS was added. The fluorescence value (Ex = 358 nm, Em = 426 nm) was measured with an enzyme marker, and the SN38 concentration was calculated according to the standard curve, and the cumulative release percentage was calculated and the release curve was drawn.

[0134] The results are as follows Fig.18 As shown, different concentrations of H 2 O 2 Processing SN38-Ce6-NPs@O 2 The drug cumulative release curve of SN38-Ce6-NPs@O 2 In the absence of H 2 O 2 In this case, there was almost no release of SN38 after 48 h of incubation. 2 O 2 When the concentrations were 0.1mM, 1mM and 10mM, it was found that the release of SN38 gradually increased. 2 O 2The higher the concentration, the more SN38 was released. After 48 h, the cumulative release of SN38 was 17%, 28% and 57%, indicating that SN38-Ce6-NPs@O 2 The SN38 release has good ROS response ability, and the SN38 release is related to H 2 O 2 The concentration was positively correlated.

[0135] ②Ultrasound stimulation response drug release experiment

[0136] In the study, it was demonstrated that SN38-Ce6-NPs@O 2 Under the action of US, it can quickly produce 1 O 2 After the release of SN38 has ROS response, the present invention continues to explore whether the TK bond is broken under US stimulation to achieve controlled release of drugs. Ultrasonic stimulation is performed every 3 hours to study its controlled release behavior of drugs. The specific details are as follows: 2mL SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 The solution was placed in a dialysis bag, which was then placed in a centrifuge tube completely immersed in 10 mL PBS (pH = 7.4, containing 20% ​​acetonitrile). US (1.0 MHz, 1 W cm -2 ) stimulation, and then fix the centrifuge tube on a constant temperature shaking incubator, keep constant temperature and shake at 37°C, and the speed is 100rpm. Samples are taken at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 25, 36, and 48h, and 0.2mL PBS is sampled each time, and 0.2mL PBS is added, and the fluorescence value (Ex=358nm, Em=426nm) is measured by an enzyme marker. The SN38 concentration is calculated based on the standard curve, and the cumulative release percentage is calculated and the release curve is drawn.

[0137] The results are as follows Fig.19 As shown in the figure, it was found that without US stimulation, the cumulative release of SN38 from SN38-Ce6-NPs within 48 hours was about 10%. Since SN38-Ce6-NPs itself undergoes a certain degree of hydrolysis in aqueous solution, SN38 is hydrolyzed, but under this hydrolysis, the release of SN38 is very small; however, within 48 hours, the SN38-Ce6-NPs group and SN38-Ce6-NPs@O 2 The cumulative release of SN38 in the two groups reached 27% and 43%, indicating that ROS was generated under US stimulation, which further led to the release of SN38; at the same time, in the presence of oxygen, the ROS yield increased, which increased the release of SN38. It is particularly noteworthy that each time US stimulation was performed, the cumulative release of SN38 increased in a leap-like manner, indicating that US stimulation can achieve the controlled release of SN38.

[0138] In summary, the present invention successfully prepared self-carrying oxygen nanoparticles SN38-Ce6-NPs@O 2 The particle size is within 200-300nm, with good polydispersity, uniformity and stability. 2 It has good oxygen dissolving capacity and oxygen releasing capacity, which can alleviate the hypoxic environment. The results of in vitro ROS generation study showed that SN38-Ce6-NPs@O 2 Compared with other groups, it can produce excessive ROS and achieve sonodynamic therapy. The in vitro release results show that the drug release of nanoparticles is ROS-sensitive, and more drugs are released as the ROS concentration increases. Under US stimulation, SN38-Ce6-NPs@O 2 The drug release can be achieved, and the drug release shows a jump-like increase under each US stimulation, indicating that the nanoparticles can achieve precise and controllable drug release.

[0139] IV. In vitro cell experiments with self-carrying oxygen nanoparticles

[0140] (I) Cellular uptake of self-oxygen-carrying nanoparticles

[0141] Laser confocal microscopy was used to investigate the effect of SN38-Ce6-NPs@O on CT26 cells. 2 Uptake. CT26 cells in the logarithmic growth phase were taken and 1×10 5 The cells were inoculated at a density of 100 / well in a 6-well plate containing a cell slide, cultured overnight, and after fully adhering to the wall, the original culture medium was aspirated, and 2 mL of fresh culture medium containing different nanoformulations was added to each well. The concentration of the nanoformulation was 200 μg / mL, of which the concentration of Ce6 was 10 μg / mL. After the experimental cells were cultured in the incubator for 6 hours, the culture medium was discarded. 1 mL of LDAPI dye (10 μg / mL) was added to each well at room temperature. After staining in the dark for 15 minutes, the dye was aspirated and discarded, and the PBS solution was washed 3 times to remove the remaining dye and unabsorbed nanoparticles. The cells were fixed with 4% paraformaldehyde fixative for 15 minutes, and the fixative was discarded. The cells were washed 3 times with PBS solution to wash away the fixative. After the cell slide was taken out, the slide was sealed. Laser confocal microscopy was used to observe SN38-Ce6-NPs@O in CT26 cells 2 The excitation wavelength of DAPI is 364nm and that of Ce6 is 664nm.

[0142] In this study, laser confocal microscopy was used to investigate the effect of CT26 cells on SN38-Ce6-NPs@O 2 Intake conditions, such as Fig. 20 SN38-Ce6-NPs@O 2The red fluorescence of Ce6 appeared in the cells after 2 h of treatment. As the incubation time increased, the red fluorescence of Ce6 in the cells increased, indicating that SN38-Ce6-NPs@O 2 It can be efficiently taken up by CT26 cells in a time-dependent manner.

[0143] (II) Effect of self-carrying oxygen nanoparticles in alleviating tumor hypoxia

[0144] In order to evaluate the SN38-Ce6-NPs@O 2 Relieve hypoxia in CT26 cells. 3 ]Cl 2 Detecting the oxygen content in cells, the indicator can emit red fluorescence. The lower the oxygen level, the brighter the red fluorescence. Therefore, the intracellular hypoxia can be detected according to its brightness; specifically as follows: [Ru(dpp) 3 ]Cl 2 Detection of intracellular oxygen content under normoxic and hypoxic conditions. Normoxic conditions: 21% O 2 , 5% CO 2 , 37°C, hypoxic conditions: 1% O 2 , 5% CO 2 , 37°C. Experimental groups: Control group, Control+US group, Ce6-NPs group, Ce6-NPs+US group, SN38-Ce6-NPs group, SN38-Ce6-NPs+US group, SN38-Ce6-NPs@O 2 Group, SN38-Ce6-NPs@O 2 +US group. CT26 cells in the logarithmic growth phase were collected and 1×10 5 The cells were inoculated at a density of 1000 cells / well in a 6-well plate. After culturing for 24 h and fully adhering to the wall, the original culture medium was removed and 2 mL of Ce6-NPs, SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 Fresh culture medium with different nanoparticles at a concentration of 200 μg / mL was added with 10 μL of oxygen indicator [Ru(dpp) 3 ]Cl 2 (concentration 100 μg / mL), and after incubation for 6 h, the US group was treated with ultrasound (1.0 MHz, 1 W / cm 2 , 3min), placed in an incubator and incubated for 6h, then the culture medium was discarded. The cells were washed three times with PBS solution to remove the remaining nanoparticles and oxygen indicator. The intracellular oxygen content was observed using an inverted fluorescence microscope.

[0145] The results are as follows Fig.21As shown in the figure, it was found that under normoxic conditions, the cells in the Control group, Control+US group, Ce6-NPs group, Ce6-NPs+US group, SN38-Ce6-NPs group, and SN38-Ce6-NPs+US group all showed different degrees of red fluorescence, indicating that the CT26 cells cultured under normoxic conditions were still in a hypoxic environment, while the SN38-Ce6-NPs@O 2 Group and SN38-Ce6-NPs@O 2 The red fluorescence of the cells in the +US group almost disappeared, indicating that the nanoparticles successfully alleviated cell hypoxia. The oxygen content in the cells under hypoxic conditions was further investigated, and it was found that the red fluorescence in the cells of the Control group, Control+US group, Ce6-NPs group, Ce6-NPs+US group, SN38-Ce6-NPs group, and SN38-Ce6-NPs+US group was brighter than that in the normoxic group, indicating that the internal environment of CT26 cells cultured under hypoxic conditions was more hypoxic, while SN38-Ce6-NPs@O 2 Group and SN38-Ce6-NPs@O 2 There is still no red fluorescence in the cells of the +US group, indicating that SN38-Ce6-NPs@O 2 Nanoparticles can still maintain intracellular oxygen concentration under hypoxic conditions.

[0146] (III) Generation of intracellular ROS

[0147] The ROS probe DCFH-DA itself has no fluorescence. It is highly lipid-soluble and can freely pass through the cell membrane. It is hydrolyzed by esterases in the cell into a fluorescent substance DCFH, which can be oxidized by ROS in the cell to generate DCF that can emit green fluorescence. Therefore, DCFH-DA can be used to indicate the level of ROS in cells. The present invention uses a DCFH-DA reactive oxygen probe and a fluorescent inverted microscope to observe the ROS generation ability of different experimental groups in CT26 cells, specifically including: using a reactive oxygen detection kit (DCFH-DA) to detect the generation of ROS in cells under normoxic and hypoxic conditions; normoxic conditions: 21% O 2 , 5% CO 2 , 37°C; hypoxic conditions: 1% O 2 , 5% CO 2 The experiment was divided into 8 groups: Control group, Control+US group, SN38 group, Ce6-NPs group, Ce6-NPs+US group, SN38-Ce6-NPs group, SN38-Ce6-NPs+US group, SN38-Ce6-NPs@O 2 +US group. CT26 cells in the logarithmic growth phase were taken at 8×10 3The cells were inoculated at a density of 100 μL / well in a 96-well plate. After culturing for 24 h and fully adhering to the wall, the original culture medium was discarded and 100 μL of Ce6-NPs, SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 Fresh culture medium with different nanoformulation concentrations of 200 μg / mL was added after 6 h of incubation. Then, 10 μL of DCFH-DA probe (concentration 10 μg / mL) was added. After further incubation for 1 h, the US group was treated with ultrasound (1.0 MHz, 1 W / cm 2 After incubation for 15 min, the culture medium was discarded. The cells were washed with PBS solution for 3 times to remove the remaining nanoparticles and probes. The generation of ROS in the cells was observed under an inverted fluorescence microscope. Fig. 22 As shown. Under normoxic conditions, it was found that the cells in the Control group, Control+US group, SN38 group, Ce6-NPs group and SN38-Ce6-NPs group did not show obvious green fluorescence, while the green fluorescence signals of the Ce6-NPs+US group and SN38-Ce6-NPs+US group were enhanced, indicating that under US stimulation, Ce6-NPs and SN38-Ce6-NPs can produce ROS in cells, triggering the sonodynamic therapy effect. 2 +US group cells showed stronger green fluorescence, indicating that SN38-Ce6-NPs@O 2 Carry O 2 , ROS production was significantly enhanced after US stimulation. Further study of the ROS generation of cells in each drug group under hypoxia found that the ROS production of each group under hypoxia was significantly reduced compared with that under normal conditions, but SN38-Ce6-NPs@O 2 The +US group still has strong green fluorescence, indicating that SN38-Ce6-NPs@O 2 In an oxygen-deficient environment, more ROS can still be produced, thus better playing the role of SDT.

[0148] Finally, Image J software was used to semi-quantitatively analyze the fluorescence intensity of ROS levels in CT26 cells of each drug-treated group. Fig.23 As shown, the results were consistent with the fluorescence inversion images.

[0149] (IV) Changes in mitochondrial membrane potential of self-carrying oxygen nanoparticles

[0150] ROS can block the respiratory chain by oxidizing the mitochondrial membrane, causing mitochondrial dysfunction and inducing apoptosis. A decrease in mitochondrial membrane potential is a landmark event in the early stages of apoptosis. JC-1 is a fluorescent probe for changes in mitochondrial membrane potential. Normal mitochondria have high membrane potentials, and JC-1 presents red aggregates. Mitochondria have low membrane potentials after damage. JC-1 presents green monomers. The present invention uses JC-1 to detect mitochondrial membrane potential damage, specifically including: using a mitochondrial membrane potential detection kit (JC-1) to detect changes in mitochondrial membrane potential. The experiment was divided into 8 groups: Control group, Control+US group, SN38 group, Ce6-NPs group, Ce6-NPs+US group, SN38-Ce6-NPs group, SN38-Ce6-NPs+US group, SN38-Ce6-NPs@O 2 +US group. CT26 cells in the logarithmic growth phase were taken at 8×10 3 The cells were inoculated at a density of 100 μg / well in a 96-well plate. After culturing for 24 h, the original culture medium was discarded and 100 μL of fresh culture medium containing Ce6-NPs and SN38-Ce6-NPs was added. The concentration of different nanoformulations was 200 μg / mL. After incubation for 6 h, the US group was treated with ultrasound (1.0 MHz, 1 W / cm 2 After 6 hours of incubation, 5 μL of JC-1 dye (concentration 5 mg / L) was added, and the cells were incubated for 20 minutes. The cells were rinsed twice with PBS to remove the remaining nanoparticles and oxygen probes. The damage of mitochondrial membrane potential in the cells was observed using an inverted fluorescence microscope.

[0151] The results are as follows Fig.24 As shown in the figure, most of the cells in the Control group and Control+US group showed red fluorescence but no green fluorescence, indicating that US did not cause damage to the mitochondrial membrane potential. The cells in the Ce6-NPs group and SN38-Ce6-NPs group showed weak green fluorescence, but still had strong red fluorescence, indicating that most of their mitochondrial membrane potentials were normal; while the green fluorescence in the Ce6-NPs+US group and SN38-Ce6-NPs+US group was significantly enhanced, and the red fluorescence was significantly weakened, indicating that Ce6-NPs and SN38-Ce6-NPs produced a large amount of ROS after US stimulation, causing damage to the mitochondrial membrane potential; SN38-Ce6-NPs@O 2 The green fluorescence of the +US group was the strongest, and the red fluorescence almost disappeared, indicating that SN38-Ce6-NPs@O 2 Oxygen carrying further increases the efficiency of ROS generation, causing serious damage to mitochondrial membrane potential.

[0152] (V) In vitro antitumor effect of self-carrying oxygen nanoparticles

[0153] CT26 cells in the logarithmic growth phase were collected, digested with 0.25% trypsin, counted, and the cell concentration was adjusted. The cell suspension was inoculated into a 96-well plate (100 μL / well, with a cell density of 5×10 per well). 3 -6×10 3 ). 2 After culturing in a 37°C incubator for 24 h, the culture medium in the wells was poured out, fresh culture medium was added, and the wells were treated with ultrasound (1.0 MHz, 1 W / cm 2 ) After 30s, 60s, 120s, 180s, and 300s, place it in an incubator and continue to culture for 48h, discard the old culture medium, wash it with PBS three times, add 100μL of MTT solution (working concentration is 5mg / mL, i.e. 0.5% MTT), continue to culture for 4h, discard the culture medium, and add 100μL of DMSO to each well, and wait for the formazan precipitate to completely dissolve in the dark. Use an enzyme marker to test the absorbance value of each well at 570nm. Perform 3 groups of parallel tests for each concentration, and set the culture medium as the zero well and the cell group without nanoparticles as the control well. Calculate the survival rate of CT26 cells according to the formula of cell survival rate. The formula for cell survival rate is as follows:

[0154]

[0155] The results are as follows Fig.25 As shown, when the US intensity is 1W / cm 2 When the US intensity was 2W / cm 2 When the US time was between 0 and 60 s, the cell survival rate was high and there was no obvious damage to the cells. However, when the US time was between 120 and 300 s, the cell survival rate decreased significantly as the ultrasound time increased. Therefore, the US parameter for subsequent experiments was selected as 1 W / cm 2 , time 180s.

[0156] (VI) Evaluation of cytotoxicity of ROS-responsive self-carrying oxygen nanoparticles

[0157] To evaluate the SN38-Ce6-NPs@O 2 The in vitro antitumor effects of Ce6-NPs, SN38-Ce6-NPs, and SN38-Ce6-NPs@O were investigated using the MTT method under normoxic and hypoxic conditions. 2 The cytotoxicity of ultrasound or not on CT26 cells. Normoxia: 21% O 2 , 5% CO 2 , 37°C, hypoxic conditions: 1% O 2 , 5% CO 2, 37°C. Experimental groups: control group (Control group), control group supplemented with US (Control+US group), SN38 group, Ce6-NPs group, Ce6-NPs supplemented with US treatment group (Ce6-NPs+US group), SN38-Ce6-NPs group, SN38-Ce6-NPs supplemented with US treatment group (SN38-Ce6-NPs+US group), SN38-Ce6-NPs@O 2 The US treatment group (SN38-Ce6-NPs@O 2 +US group). CT26 cells in the logarithmic growth phase were collected, digested with 0.25% trypsin, counted, and the cell concentration was adjusted. The cell suspension was inoculated into a 96-well plate (100 μL / well, with a cell density of 5×10 3 -6×10 3 ). 2 After culturing in an incubator at 37 °C for 24 h, the culture medium in the wells was poured out and SN38, Ce6-NPs, SN38-Ce6-NPs and SN38-Ce6-NPs@O were added to the wells. 2 The cells were then placed in an incubator for 6 h and the SDT group was treated with ultrasound (1.0 MHz, 1 W / cm 2 ), the cells were placed in an incubator and cultured for 42 hours, the old culture medium was discarded, 100 μL of MTT solution (working concentration was 5 mg / mL, i.e., 0.5% MTT) was added after washing 3 times with PBS, and cultured for 4 hours, the culture medium was discarded, and 100 μL of DMSO was added to each well, and the formazan precipitate was completely dissolved in the dark. The absorbance value of each well at 570 nm was tested with an ELISA instrument, and the survival rate of CT26 cells was calculated using the above formula.

[0158] The results are as follows Fig.26 As shown in the figure, under normoxic conditions, the cell viability of the 300μg / mL Ce6-NPs group was 90%, and the cell viability of the Ce6-NPs+US group was 74%. Compared with Ce6-NPs, the cell viability decreased by 16%, indicating that Ce6-NPs can only produce cytotoxic ROS and induce sonodynamic therapy under US stimulation. At the same dosage concentration, the SN38-Ce6-NPs group caused 37% cell death, which was similar to the results of the SN38 group, indicating that SN38-Ce6-NPs can only achieve limited chemotherapy effects through the slow release of SN38; the SN38-Ce6-NPs+US group caused 68% cell inhibition, indicating that under US stimulation, SN38-Ce6-NPs first produced cytotoxic ROS, triggered SDT, and further induced the ROS response release of SN38, and finally achieved the therapeutic effect of sonodynamic combined with chemotherapy; SN38-Ce6-NPs@O 2+US group cells caused 79% cell death, indicating that SN38-Ce6-NPs@O 2 Carry O 2 Under US stimulation, ROS production was increased, further inducing more SN38 release, and enhancing the anti-tumor effect of chemotherapy combined with sonokinesis. In addition, the cell cytotoxicity of each drug group increased significantly with the increase of concentration, indicating that Ce6-NPs and SN38-Ce6-NPs have a concentration-dependent effect on cell killing. The cytotoxicity of each drug group under hypoxia was further studied, and the results were as follows Fig. 27 Compared with normoxic conditions, the cell killing effects of each treatment group under hypoxic conditions decreased to varying degrees, indicating that hypoxia reduced the therapeutic effects of chemotherapy and SDT; SN38-Ce6-NPs@O 2 The cell killing effect of the +US group at concentrations of 100μg / mL and 200μg / mL was lower than that of the normoxia group, but when the concentration increased to 300μg / mL, the killing effect was consistent with that of the normoxia group, indicating that the inhibitory effect of hypoxia on chemotherapy and SDT can be alleviated only when the oxygen supply reaches a certain concentration.

[0159] 7. Cell Death and Viability Staining Analysis

[0160] Calcein-AM is a cell membrane permeable live cell dye that is hydrolyzed into calcein by lactonase after entering the cell and further binds to intracellular Ca 2+ Combined with Calcein-AM, a strong green fluorescence is produced. Since dead cells lack lactonase and cannot produce green fluorescence, Calcein-AM can only label living cells. It is often used in combination with pyridinium iodide (PI). Since the cell membrane of dead cells is damaged, PI can pass through the damaged cell membrane to reach the cell nucleus and combine with the DNA double helix region to produce a strong red fluorescence. Therefore, only dead cells are stained. The present invention uses Calcein-AM and PI to co-stain cells to evaluate the in vitro cancer cell killing effect, including: co-staining CT26 cells with Calcein-AM and PI to analyze the SN38-Ce6-NPs@O 2 In vitro antitumor effect; the experimental group was divided into 8 groups: Control group, Control+US group, SN38 group, Ce6-NPs group, Ce6-NPs+US group, SN38-Ce6-NPs group, SN38-Ce6-NPs+US group, SN38-Ce6-NPs@O 2 +US group. CT26 cells in the logarithmic growth phase were taken at 8×10 3 The cells were inoculated at a density of 100 μL / well in a 96-well plate. After culturing for 24 h and fully adhering to the wall, the original culture medium was discarded and 100 μL of Free SN38, Ce6-NPs, SN38-Ce6-NPs, SN38-Ce6-NPs@O2 Fresh culture medium with different nanoformulation concentrations of 200 μg / mL was added. After incubation for 6 hours, the cells were treated with US and placed in an incubator for 42 hours before the culture medium was discarded. 10 μL of 50 μM Calcein-AM and 10 μL of 10 μM PI were added and incubated for 15 minutes. The cells were washed three times with PBS to remove the remaining Calcein-AM and PI, and the cell survival was observed using an inverted fluorescence microscope.

[0161] Test results such as Fig.28 As shown. Control+US showed strong green fluorescence, indicating that US did not cause obvious cytotoxicity. Compared with the Ce6-NPs group, the Ce6-NPs+US group showed more red fluorescence but also more green fluorescence, indicating that a large number of cancer cells were 1 O 2 Survived after the attack. Although the SN38-Ce6-NPs group showed red fluorescence, there was still a large amount of green fluorescence, similar to the SN38 group, indicating that chemotherapy alone did not show sufficient cancer cell killing effect. Compared with SN38-Ce6-NPs, the increase in red fluorescence in the SN38-Ce6-NPs+US group indicated that the release of SN38 was further induced after triggering sonodynamic therapy, resulting in aggravated cell death and achieving synergistic killing of tumor cells by chemotherapy and SDT. Especially SN38-Ce6-NPs@O 2 After the +US group, the green fluorescence almost disappeared, and only red fluorescence was present, indicating that the cells were almost completely dead. 2 Oxygen delivery enhances the synergistic effect of chemotherapy and SDT.

[0162] In summary, the present invention uses mouse colon cancer cell CT26 as a model cell to conduct in vitro cell experiments. The cytotoxicity results show that SN38-Ce6-NPs@O 2 All of them have strong cell killing ability. The uptake experiment shows that SN38-Ce6-NPs@O 2 The SN38-Ce6-NPs@O were successfully taken up by CT26 cells within 2 h, and the uptake increased with the increase of incubation time. 2 Successfully alleviated cellular hypoxia. Even under hypoxic conditions, SN38-Ce6-NPs@O 2 Under normoxic and hypoxic conditions, SN38-Ce6-NPs@O 2 It can significantly increase the production rate of ROS, further induce stronger mitochondrial membrane potential apoptosis, and realize the combination of sonodynamic therapy and chemotherapy to synergistically kill tumor cells.

[0163] 5. In vivo anti-tumor study of self-carrying oxygen nanoparticles

[0164] In the present invention, PBS and free SN38 solution were used as controls to investigate the tumor-suppressing effect of self-carrying oxygen nanoparticles in CT26 colon cancer mice.

[0165] (I) Establishment of CT26 colon cancer mouse model

[0166] Schematic diagram of establishing CT26 tumor-bearing mouse model Fig.29 As shown, the specific steps include: resuscitating frozen CT26 cells and performing routine culture. After the cells reach the required number, the cells are collected by centrifugation. The cells are resuspended with PBS, counted, and diluted with physiological saline to 5×10 7 Healthy female BALB / C mice were inoculated with 0.1 mL of cell suspension subcutaneously in the axilla of the left forelimb of each mouse; the weight and tumor volume of the mice were recorded every day. When the tumor grew to 80 mm 3 ~120mm 3 The modeling was considered successful.

[0167] (II) Distribution of self-carrying oxygen nanoparticles in vivo

[0168] Take 6 tumors with a volume of 80-120 mm 3 The CT26 tumor-bearing mice were randomly divided into two groups. Experimental group: FreeCe6 group and SN38-Ce6-NPs@O 2 Group. Ce6 and SN38-Ce6-NPs@O were injected via tail vein 2 CT26 tumor-bearing mice were treated with Ce6 at a dose of 2.5 mg / kg. In vivo fluorescence images were recorded at 2, 4, 6, 8, 12, 24, and 48 h using a small animal imaging device. To eliminate the interference of autofluorescence, the main organs and tumors of the sacrificed mice were taken, and then the separated organs and tumors were imaged. Finally, the appropriate time for ultrasound action was determined by quantitatively analyzing the fluorescence intensity of the dissected mouse tumors.

[0169] Distribution of self-carrying oxygen nanoparticles in vivo Fig.30 , Fig.31 , Fig.32 and Fig.33 The results show that SN38-Ce6-NPs@O 2 The fluorescence intensity of tumor tissue in the group was much higher than that in the free Ce6 group. At the same time, the results of semi-quantitative analysis showed that SN38-Ce6-NPs@O 2 The fluorescence intensity of tumor tissue in the group was 9 times that of the free Ce6 group, indicating that SN38-Ce6-NPs@O 2Nanoparticles can efficiently accumulate in the tumor site for a long time. After the drug is injected into the tail vein, sufficient drug concentration in the tumor site is ensured, and then US stimulation is given to make it have a greater anti-tumor effect. Therefore, the treatment plan of injecting the drug into the tail vein once every 48 hours and giving US stimulation 24 hours later was selected.

[0170] (III) Anti-tumor effect of self-carrying oxygen nanoparticles

[0171] 48 tumors were collected and the tumor size was 80-120 mm. 3 The CT26 tumor-bearing mice were randomly divided into 8 groups (n=6 in each group), including PBS group, PBS+US group, SN38 solution group, Ce6-NPs group, Ce6-NPs+US group, SN38-Ce6-NPs group, SN38-Ce6-NPs+US group and SN38-Ce6-NPs@O 2 +US group, the first day of administration was recorded as day 0. Tumor-bearing mice in each group were injected with Ce6-NPs, SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 200 μL solution, of which the dosage of SN38 was 5 mg / kg and the dosage of Ce6 was 2.5 mg / kg; Ce6-NPs+US, SN38-Ce6-NPs+US and SN38-Ce6-NPs@O 2 The +US group received ultrasound treatment on the 1st, 3rd, 5th, 7th, 9th, and 11th days, with ultrasound parameters of 1.0 MHz, 1 W cm -2 The long diameter (L, mm) and short diameter (W, mm) of the tumor in each experimental group were measured and recorded every day with a vernier caliper, and the tumor volume (V) was calculated according to the following formula: t , mm 3 ):V t =L×W 2 / 2, continuously record V for 15 days t Data were collected and a tumor growth curve of time-tumor volume was drawn. The status of the mice was observed and the weight of the mice was weighed and recorded daily. After 15 days of administration, the tumor-bearing mice were killed by bleeding from the eyeballs. The blood was collected in a 1.5 mL Eppendorf tube treated with sodium heparin. The tumor mass and the heart, liver, spleen, lung, and kidney were carefully separated and washed with physiological saline. After weighing on an analytical balance, each group of tumors was photographed with a camera.

[0172] Tumor growth curves of CT26 tumor-bearing mice after different treatments Fig.34As shown. In the CT26 tumor-bearing mouse model, the PBS group and the PBS+US group had similar growth curves, and there was no difference between the two. The tumor inhibition rate of the Ce6-NPs group was about 31%, and when US stimulation was given, the tumor inhibition rate was about 53%, indicating that Ce6-NPs inhibited tumor growth under US stimulation, indicating that sonodynamics has a therapeutic effect. The tumor inhibition rate of the SN38-Ce6-NPs group was about 39%, which was similar to that of the SN38 group, indicating that SN38-Ce6-NPs only exerted a chemotherapeutic effect by slowly releasing SN38; the tumor inhibition rate of the SN38-Ce6-NPs+US group was about 72.5%, indicating that SN38-Ce6-NPs produced ROS after US stimulation, inducing the response release of SN38 under stimulation, and achieving the anti-tumor effect of chemotherapy combined with sonodynamic therapy; SN38-Ce6-NPs@O 2 The tumor inhibition rate of the +US group increased to about 89%, and its anti-tumor effect was higher than that of SN38-Ce6-NPs+US. This is because SN38-Ce6-NPs@O 2 Carrying oxygen successfully alleviates the problem of tumor hypoxia, increases ROS production, further promotes the release of more SN38, and enhances the synergistic effect of chemotherapy and sonodynamic therapy.

[0173] In addition, to further study the anti-tumor effect of each drug administration group, after the observation period, the tumor tissues of mice in each group were collected, photographed and the tumor weight was counted. The results are as follows: Fig.35 and Fig.36 SN38-Ce6-NPs@O 2 Although the +US treatment group could not completely eliminate the tumor, it showed the best therapeutic effect among all treatment groups. At the same time, the statistical results of tumor weight were similar to the conclusions of tumor growth curve. 2 It carries oxygen, relieves tumor hypoxia, and enhances sonodynamic therapy, while combining with chemotherapy to achieve anti-tumor effects. Fig.37 The body weight changes of mice in each drug-treated group showed no significant difference among the groups.

[0174] (IV) Tumor tissue section morphology

[0175] The tumor was fixed with 4% paraformaldehyde fixative for 48 hours, then embedded in paraffin to prepare 5 μm thick sections, and stained with H&E. Finally, the pathological changes were observed under a fluorescent upright microscope. H&E staining of tumor sections of CT26 tumor-bearing mice after different treatments (bar: 100 μm) Fig.38 The results showed that the SN38 group, Ce6-NPs group, Ce6-NPs+US group, SN38-Ce6-NPs group, SN38-Ce6-NPs+US group and SN38-Ce6-NPs@O 2+US groups showed different degrees of cell necrosis. Especially SN38-Ce6-NPs@O 2 The number of purple cell nuclei in the tumor tissue of the +US group decreased sharply, the rapidly proliferating tumor cells were effectively eliminated, and the damaged and necrotic areas were more, indicating that in O 2 In the presence of US, chemotherapy combined with sonodynamic therapy can induce tumor cell necrosis to the greatest extent, which is consistent with the anti-tumor conclusion in vivo.

[0176] (V) Safety Experiment

[0177] (1) Pathology of various organs of mice

[0178] The mouse heart, liver, spleen, lung, and kidney were fixed with 4% paraformaldehyde fixative for 48 h, then embedded in paraffin to prepare 5 μm thick sections, and stained with H&E. Finally, the pathological changes were observed under a fluorescent upright microscope. Fig.39 The results showed that no obvious pathological abnormalities were found in the heart, liver, spleen, lung and kidney of mice in each treatment group. 2 No effect on important tissues of mice.

[0179] (2) Mouse plasma parameters and hemolysis

[0180] Plasma samples were taken from each mouse to detect the levels of AST, ALT, LDH, BUN, and Cr in the mouse plasma; the detection was performed according to the instructions of the corresponding kits (the kits were all from Nanjing Jiancheng Bioengineering Institute).

[0181] In addition, the blood was centrifuged at 5000 r / min and 4°C for 15 min. 1 mL of H 2 O, PBS, SN38-Ce6-NPs@O 2 (100μg / mL, 200μg / mL, 300μg / mL), incubate the mixture at 37℃ for 8h, and then centrifuge to obtain the supernatant. Measure the supernatant by UV-visible spectroscopy, and record the absorbance at 540nm. The hemolysis rate is calculated according to the formula:

[0182]

[0183] Where A is different concentrations of SN38-Ce6-NPs@O 2 Absorbance of hemoglobin, A PBS is the absorbance of PBS on hemoglobin.

[0184] The levels of alanine aminotransferase (A), aspartate aminotransferase (B), urea nitrogen (C), lactate dehydrogenase (D), and creatinine (E) in the serum of CT26 tumor-bearing mice after different treatments (ALT and AST are important enzymes for amino acid metabolism in the liver. When the values ​​of ALT and AST are high, it indicates that the liver is damaged to a certain extent. LDH is an important indicator reflecting heart function. When the heart is damaged, the LDH level is high. BUN and Cr are important indicators reflecting kidney function, indicating that their levels will be abnormally high when the kidneys are damaged) (n=3) as well as the hemolysis rate and red blood cell status of different concentrations of SN38-Ce6-NPs are shown in the figure. Fig.40 , Fig.41 , Fig.42 , Fig.43 and Fig.44 The results showed that there was no significant difference in the levels of AST, ALT, LDH, BUN, and Cr in the serum of each nanoparticle administration group, indicating that Ce6-NPs, SN38-Ce6-NPs, and SN38-Ce6-NPs@O 2 It did not cause damage to the heart, liver, and kidney function of mice. In addition, the hemolysis rate and red blood cell morphology under physiological conditions were further studied. Fig.45 The results show that SN38-Ce6-NPs@O 2 The hemolysis rate of (100μg / mL-300μg / mL) was less than 9%. No significant changes were observed in the morphology of red blood cells, which further indicated that SN38-Ce6-NPs@O 2 It has good biocompatibility and biosafety.

[0185] In summary, in the present invention, tumor modeling was successfully completed by injecting CT26 cells into the axilla of BALB / c female mice; SN38-Ce6-NPs@O 2 After 6 hours, they successfully accumulated in the tumor site. In CT26 tumor-bearing mice, SN38-Ce6-NPs@O 2 After sonodynamic therapy, tumor growth was significantly inhibited, and a large number of cell necrosis phenomena were found in tumor tissue sections, indicating that this treatment method has a good tumor growth inhibition effect; in addition, Ce6-NPs, SN38-Ce6-NPs, SN38-Ce6-NPs@O 2 It has good in vivo safety and will not affect the normal liver and kidney functions of mice.

[0186] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. ROS-responsive self-carrying oxygen nanoparticles, characterized in that: The preparation method comprises: (1) using chitosan as a skeleton, grafting glycidol, perfluorooctanoyl chloride, Ce6 and TK-SN38 respectively to obtain a chitosan derivative Gly-CS-PFO-Ce6-SN38; (2) using the chitosan derivative Gly-CS-PFO-Ce6-SN38 to encapsulate perfluorohexane to obtain SN38-Ce6-NPs; (3) saturating the SN38-Ce6-NPs with oxygen to obtain a ROS-responsive self-carrying oxygen nanoparticle preparation; In step (1), the preparation method of the chitosan derivative Gly-CS-PFO-Ce6-SN38 includes: (a) reacting chitosan with glycidol at 45°C-55°C to obtain Gly-CS; (b) reacting Gly-CS with perfluorooctanoyl chloride at room temperature to obtain Gly-CS-PFO; (c) reacting activated Ce6 with Gly-CS-PFO in the dark to obtain Gly-CS-PFO-Ce6; (d) reacting activated TK-SN38 with Gly-CS-PFO-Ce6 in the dark to obtain the chitosan derivative Gly-CS-PFO-Ce6-SN38; In step (2), the preparation method of SN38-Ce6-NPs includes: mixing perfluorohexane with a Gly-CS-PFO-Ce6-SN38 aqueous solution, and ultrasonic emulsifying to obtain SN38-Ce6-NPs; The structural formula of TK-SN38 is as follows:

2. The ROS-responsive self-carrying oxygen nanoparticle preparation according to claim 1, characterized in that: The chitosan is chitosan with a molecular weight of 50,000.

3. The ROS-responsive self-carrying oxygen nanoparticle preparation according to claim 1 or 2, characterized in that: The mass ratio of perfluorohexane to Gly-CS-PFO-Ce6-SN38 is 4:1-32:

1.

4. The ROS-responsive self-carrying oxygen nanoparticle preparation according to claim 1 or 2, characterized in that: The duration of phacoemulsification was ≥3 min.

5. The ROS-responsive self-carrying oxygen nanoformulation according to claim 1 or 2, characterized in that: In step (3), the method for preparing the ROS-responsive self-oxygen-carrying nanoformulation includes: passing oxygen through the SN38-Ce6-NPs solution for 10 minutes to obtain the ROS-responsive self-oxygen-carrying nanoformulation.

6. An anti-tumor drug preparation, characterized in that: The invention comprises the ROS-responsive self-carrying oxygen nanoformulation as claimed in any one of claims 1 to 5.

7. Use of the ROS-responsive self-carrying oxygen nanoformulation according to any one of claims 1 to 5 in the preparation of an anti-tumor drug preparation, wherein the tumor is colon cancer.

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