A biomimetic nanocarrier and its preparation method and application

Abstract

The present invention belongs to the technical field of anti-tumor drug preparation, specifically relating to a biomimetic nanocarrier, its preparation method, and application. The biomimetic nanocarrier comprises UCNPs, inclusions, manganese dioxide, and gold nanoparticles. The inclusions include a photosensitizer and / or DHA. The manganese dioxide has a hollow structure with pores on its surface. The manganese dioxide acts as a shell around the UCNPs, with a gap between the manganese dioxide and the UCNPs. The inclusions are dispersed within the gaps. The gold nanoparticles are dispersed within the pores and / or coated on the manganese dioxide shell. The biomimetic nanocarrier can catalyze the decomposition of H2O2 and MnO2 produced by glucose oxidation under specific conditions through gold nanoparticles (AuNPs), introducing a cascade synergistic reaction, in situ amplifying the production of ROS, and enhancing the effects of PDT and CDT.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug preparation, and in particular relates to a bionic nanocarrier and a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is a highly lethal liver malignancy, accounting for approximately 90% of primary liver cancers. The etiology of HCC is complex, including viral infection, liver fibrosis, and alcohol abuse. Due to its subtle early symptoms, insidious onset, and rapid progression, most patients are diagnosed in the advanced stages, posing significant challenges to its treatment.

[0003] Currently, chemotherapy drugs can inhibit a variety of tumor entities and show broad-spectrum and highly effective anti-tumor activity against a variety of tumors. However, the strong hydrophobicity, nonspecific distribution and rapid elimination of the drugs from the body have hindered their widespread application in cancer treatment. In addition, chemotherapy can also damage healthy cells.

[0004] Photodynamic tumor therapy is a hot research topic in cancer treatment because it primarily destroys the lesion while minimizing damage to surrounding normal tissue. Upconversion nanoparticles (UCNPs), a new generation of bioluminescent markers, offer significant advantages, including low excitation light energy, low background, high and stable luminescence intensity, and tunable emission wavelength. These properties make UCNPs promising for application in photodynamic tumor therapy. Summary of the Invention

[0005] Based on this, the present invention coats upconversion nanoparticles (UCNPs) with a manganese dioxide layer and gold nanoparticles, and incorporates a photosensitizer into the gaps between the manganese dioxide layer and the upconversion nanoparticles. The gold nanoparticles (AuNPs) can catalyze the H2O2 produced by glucose oxidation and the decomposition of MnO2 under specific conditions, introduce a cascade enhancement reaction, amplify the generation of ROS in situ, and enhance the effects of PDT and CDT.

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

[0007] On one hand, the present invention provides a biomimetic nanocarrier, which includes UCNPs, inclusions, manganese dioxide and gold nanoparticles, wherein the inclusions include a photosensitizer; the manganese dioxide has a hollow structure with pores on the surface; the manganese dioxide is wrapped around the UCNPs as a shell, and there is a gap between the manganese dioxide and the UCNPs; the inclusions are dispersed in the gap; and the gold nanoparticles are dispersed in the pores and / or coated on the outside of the manganese dioxide shell.

[0008] Preferably, the biomimetic nanocarrier satisfies one or more of the following conditions: (i) the photosensitizer is selected from one or more combinations of hematoporphyrin derivatives, mTHPC, chlorophyll-a, BPMppa, pheophorbide-a, dihydrochlorin e6, pyropheophorbide-a, or pyropheophorbide-a hexyl ether; (ii) the pore size is 3 nm to 10 nm; and (iii) the contents further include anti-tumor active substances.

[0009] More preferably, the above-mentioned anti-tumor active substance is DHA.

[0010] More preferably, the mass ratio of the inclusions to other components of the biomimetic nanocarrier is (0.1-0.5):1.

[0011] More preferably, the biomimetic nanocarrier is further connected with cRGD.

[0012] Another aspect of the present invention provides a method for preparing the biomimetic nanocarrier of the present invention, the preparation method comprising:

[0013] First, the preparation method of the biomimetic nanocarrier without cRGD includes: (1) modifying UCNPs@SiO2 with manganese dioxide to obtain USM; (2) etching SiO2 in USM to obtain UCNPs@H-MnO2; (3) incorporating the inclusions into UCNPs@H-MnO2 to obtain UMDC-1; (4) modifying UMDC-1 with gold nanoparticles to obtain UMDCA, i.e., the biomimetic nanocarrier.

[0014] Secondly, the preparation method of the biomimetic nanocarrier connected to cRGD includes: (1) modifying UCNPs@SiO2 with manganese dioxide to obtain USM; (2) etching SiO2 in USM to obtain UCNPs@H-MnO2; (3) incorporating the inclusions into UCNPs@H-MnO2 to obtain UMDC; (4) modifying UMDC with gold nanoparticles to obtain UMDCA; (5) mixing UMDCA and phospholipid polyethylene glycol to obtain a reactant, and then mixing the reactant with cRGD to obtain UMDCAc, that is, the biomimetic nanocarrier.

[0015] Preferably, the above preparation method meets one or more of the following conditions: (a) The preparation method of UCNPs@H-MnO2 includes: mixing UCNPs@SiO2 and potassium permanganate, ultrasonicating, and centrifuging to obtain nanoparticles to obtain USM; then dissolving USM in a Na2CO3 aqueous solution, mixing and reacting to obtain UCNPs@H-MnO2; (b) The preparation method of UMDC includes: mixing UCNPs@H-MnO2 and the contents, ultrasonicating, and centrifuging to obtain UMDC; (c) The preparation method of UMDCA includes: dissolving UMDC and polyvinyl pyrrolidone in an organic solvent, ultrasonically stirring to obtain a suspension; mixing the suspension, NaBH4 and HAuCl4 to react, and centrifuging to obtain UMDCA.

[0016] Another aspect of the present invention provides an anti-tumor drug comprising the bionic nanocarrier of the present invention.

[0017] In another aspect, the present invention further provides a use of the bionic nanocarrier of the present invention in the preparation of anti-tumor drugs.

[0018] Preferably, the above-mentioned anti-tumor drug is an anti-hepatocellular carcinoma drug.

[0019] The beneficial effects of the present invention include:

[0020] (1) The biomimetic nanocarriers (UMDCA and UMDCAc) provided by the present invention can catalyze the decomposition of H2O2 and MnO2 produced by glucose oxidation under specific conditions through gold nanoparticles (AuNPs), introducing a cascade synergistic reaction. This not only amplifies the production of ROS in situ and enhances the effects of PDT and CDT, but also further strengthens the efficacy of PDT through the generated O2; that is, the cascade synergistic reaction mechanism can effectively overcome the limitations of the tumor microenvironment and provide new ideas for the treatment of complex tumors.

[0021] (2) The biomimetic nanocarrier provided by the present invention can target and deliver DHA and photosensitizer (such as Ce6) in combination, and can realize synergistic tumor treatment under the guidance of TEM response and NIR light control. This multimodal combined treatment strategy fully utilizes the advantages of each treatment method, significantly improves the treatment effect through synergistic effects, and solves the common problems in single treatment modes.

[0022] (3) The biomimetic nanocarrier provided by the present invention introduces DHA, which can alleviate the tumor hypoxia environment by targeting HIF-1α, which provides a new way to improve the efficacy of PDT. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Transmission electron microscopy (TEM) images and scanning electron microscopy (SEM) images of different nanomaterials; A, C, E, and G are transmission electron micrographs of UCNPs, UCNPs@SiO2, UCNPs@H-MnO2, and UMDCA, respectively; B, D, F, and H are scanning electron micrographs of UCNPs, UCNPs@SiO2, UCNPs@H-MnO2, and UMDCA, respectively;

[0024] Figure 2 Zeta potential diagram of different nanomaterials;

[0025] Figure 3 UV-visible absorption spectra of different nanomaterials;

[0026] Figure 4 Fourier transform infrared spectra of different nanomaterials;

[0027] Figure 5 This is the FTIR spectrum test diagram of nanomaterials UMDCA and UMDCAc;

[0028] Figure 6 This is the catalase (CAT) activity test result of UMDCA;

[0029] Figure 7 This is the glucose oxidase (GOD) activity test result of UMDCA;

[0030] Figure 8 This is the peroxidase (POD) activity test result of UMDCA;

[0031] Figure 9 Glutathione peroxidase (GPX) activity detection of UMDCA;

[0032] Figure 10 This is the test result of chemical kinetic effect of UMDCA;

[0033] Figure 11 This is the photodynamic effect test result of UMDCA;

[0034] Figure 12 CCK-8 experimental results of MHCC-97H cells cultured with different nanomaterials;

[0035] Figure 13 Trypan blue staining test results of MHCC-97H cells cultured with UMDCAc;

[0036] Figure 14 AO / EB live-dead cell staining test results of MHCC-97H cells cultured with different nanomaterials;

[0037] Figure 15Photos of tumors in mice injected with different nanomaterials;

[0038] Figure 16 Changes in tumor volume in mice injected with different nanomaterials;

[0039] Figure 17 HE staining analysis of mouse organs injected with different nanomaterials. DETAILED DESCRIPTION

[0040] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0041] 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", "comprise" and the like 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.

[0042] An embodiment of the present invention provides a biomimetic nanocarrier, which includes UCNPs, inclusions, manganese dioxide and gold nanoparticles, wherein the inclusions include a photosensitizer; the manganese dioxide has a hollow structure with pores on the surface; the manganese dioxide is wrapped around the UCNPs as a shell, and there is a gap between the manganese dioxide and the UCNPs; the inclusions are dispersed in the gap; and the gold nanoparticles are dispersed in the pores and / or coated on the outside of the manganese dioxide shell.

[0043] It should be noted that UCNPs in the present invention are all abbreviations of upconversion nanoparticles, referring to a type of upconversion nanoparticles and a plurality of upconversion nanoparticles.

[0044] It should also be noted that the manganese dioxide in the biomimetic nanocarrier can decompose under specific conditions to produce oxygen, further enhancing the effectiveness of PDT; gold nanoparticles have efficient and stable GOx-like catalytic activity, which can decompose glucose excessively taken up by TEM and tumor cells, regulate tumor glucose metabolism, and produce H2O2; that is, the biomimetic nanocarrier in the present invention introduces a cascade enhancement reaction, which not only amplifies the production of ROS in situ, but also enhances the effects of PDT and CDT; in addition, the catalytic activity of AuNPs can be rationally controlled by its surface hydrophobicity and subsequent interaction with glucose, specifically activating its enzyme-like catalytic ability in cancer cells.

[0045] It should also be noted that the gold nanoparticles are dispersed in the pores and may also be coated outside the manganese dioxide shell. Dispersing the gold nanoparticles in the pores can prevent the premature leakage of the photosensitizer and / or DHA.

[0046] In some specific examples, the biomimetic nanocarrier satisfies one or more of the following conditions:

[0047] (i) The photosensitizer is selected from one or more combinations of hematoporphyrin derivatives, mTHPC, chlorophyll-a, BPMppa, pheophorbide-a, chlorin e6, pyropheophorbide-a, or pyropheophorbide-a hexyl ether; specifically, the photosensitizer in the present invention is well known in the art, such as the above-mentioned photosensitizers, preferably chlorin e6 (Ce6), which has a better effect than other photosensitizers;

[0048] (ii) the size of the pores is 3 nm to 10 nm, such as 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, or 9 nm;

[0049] (iii) The contents further include anti-tumor active substances; specifically, the anti-tumor active substances can be delivered to the tumor using the biomimetic nanocarrier of the present invention as a delivery carrier for administration.

[0050] It should be noted that the biomimetic nanocarrier may satisfy any one of the above conditions (i) to (iii), and preferably satisfies all of the above conditions simultaneously.

[0051] In some specific examples, the anti-tumor active substance is DHA (dihydroartemisinin).

[0052] It should be noted that DHA can alleviate the hypoxic environment of tumors by targeting HIF-1α. Dispersing DHA and photosensitizers in the gaps between UCNPs and manganese dioxide layers can target the delivery of DHA and photosensitizers, achieving synergistic tumor treatment under the guidance of TEM response and NIR light control. This multimodal combined treatment strategy fully utilizes the advantages of each treatment method, significantly improves the treatment effect through synergy, and solves common problems in single treatment modes, such as the limitation of therapeutic efficacy by adverse factors in the tumor microenvironment. In addition, DHA itself is an anti-tumor drug, but due to its hydrophobicity, it cannot effectively reach the tumor part to exert its efficient effect. The biomimetic nanocarrier in the present invention can deliver it to the tumor to improve its effectiveness.

[0053] In some specific examples, the mass ratio of the inclusions to other components of the biomimetic nanocarrier is (0.1-0.5):1, such as 0.1:1, 0.2:1, 0.3:1 or 0.4:1, etc.

[0054] It should be noted that if the contents contain both a photosensitizer and an anti-tumor active substance, the masses of the photosensitizer and the anti-tumor active substance may be the same or different, and the specific selection can be made according to needs.

[0055] In some specific examples, the biomimetic nanocarrier is further connected with cRGD.

[0056] It should be noted that cRGD, as a functional polypeptide, can specifically bind to integrin receptors on the surface of tumor cells. By connecting to biomimetic nanocarriers, targeted delivery to tumor cells can be achieved, thereby improving the accuracy and efficiency of treatment.

[0057] The present invention also provides a method for preparing the biomimetic nanocarrier of the present invention, the preparation method comprising:

[0058] First, the preparation method of the biomimetic nanocarrier without cRGD includes: (1) modifying UCNPs@SiO2 with manganese dioxide to obtain USM; (2) etching SiO2 in USM to obtain UCNPs@H-MnO2; (3) incorporating the inclusions into UCNPs@H-MnO2 to obtain UMDC-1; (4) modifying UMDC-1 with gold nanoparticles to obtain UMDCA, i.e., the biomimetic nanocarrier.

[0059] Secondly, the preparation method of the biomimetic nanocarrier connected to cRGD includes: (1) modifying UCNPs@SiO2 with manganese dioxide to obtain USM; (2) etching SiO2 in USM to obtain UCNPs@H-MnO2; (3) incorporating the inclusions into UCNPs@H-MnO2 to obtain UMDC; (4) modifying UMDC with gold nanoparticles to obtain UMDCA; (5) mixing UMDCA and phospholipid polyethylene glycol to obtain a reactant, and then mixing the reactant with cRGD to obtain UMDCAc, that is, a biomimetic nanocarrier.

[0060] In some specific examples, the above preparation method satisfies one or more of the following conditions:

[0061] (a) The preparation method of UCNPs@H-MnO2 includes: mixing UCNPs@SiO2 and potassium permanganate, ultrasonicating, and centrifuging to obtain nanoparticles to obtain USM; then dissolving the USM in a Na2CO3 aqueous solution, and mixing and reacting to obtain UCNPs@H-MnO2;

[0062] (b) The preparation method of UMDC includes: mixing UCNPs@H-MnO2 and the contents, sonicating, and centrifuging to obtain UMDC;

[0063] (c) The preparation method of UMDCA comprises: dissolving UMDC and polyvinyl pyrrolidone in an organic solvent, and ultrasonically stirring to obtain a suspension; mixing the suspension, NaBH4 and HAuCl4 for reaction, and centrifuging to obtain UMDCA.

[0064] It should be noted that operations such as ultrasound and centrifugation in the above preparation method are well-known operating steps in the art and have no special meaning in the present invention.

[0065] An embodiment of the present invention further provides an anti-tumor drug, comprising the biomimetic nanocarrier of the present invention.

[0066] It should be noted that the biomimetic nanocarriers in the present invention can synergistically treat tumors through TEM response and NIR response. The biomimetic nanocarriers can be added with pharmaceutically acceptable carriers, such as buffer solutions, to prepare anti-tumor drugs, which can be administered through clinical methods such as tail vein injection.

[0067] The embodiment of the present invention further provides a use of the biomimetic nanocarrier of the present invention in the preparation of anti-tumor drugs.

[0068] In some specific examples, the anti-tumor drug is an anti-hepatocellular carcinoma drug.

[0069] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0070] 1. Preparation and Characterization of Bionic Nanocarriers

[0071] Example 1

[0072] (1)UCNPs(NaYF4:Yb 3+ (20%), Er 3+ Preparation of (2%) nanoparticles

[0073] In a 100 mL three-necked round-bottom flask, YCl3·6H2O, YbCl3·6H2O and ErCl3·6H2O were mixed with OA (99%) (oleic acid) and ODE (99%) (1-octadecene); the solution was heated to 150°C under vacuum and stirred for 90 minutes to form a homogeneous solution, and then cooled to room temperature; 10 mL of methanol solution containing NH4F (4 mmol, 148.16 mg) and NaOH (2.5 mmol, 100 mg) was slowly added to the flask and stirred for 45 minutes; then the solution was slowly heated to remove methanol and degassed at 100°C for 30 minutes; finally, the solution was heated to 300°C and kept under argon for 70 minutes; then cooled, and after the reaction system cooled to room temperature, the sample was washed with anhydrous ethanol and cyclohexane, centrifuged three times (8000 rpm, 15 minutes), and dried to obtain UCNPs; they were stored at room temperature for further use.

[0074] (2) Preparation of UCNPs@SiO2

[0075] A 50 mL cyclohexane solution containing 65 mg of nanoparticles (UCNPs) was mixed with Igepal Co520 (1.0 g, average molecular weight (Mn) of 441) and ultrasonicated (800 W) for 15 min; then ammonium hydroxide (NH4OH) solution (35%, 500 μL) was added, and the solution was vigorously shaken and ultrasonicated (800 W) for 20 min; finally, 375 μL of tetraethyl orthosilicate (TEOS) was injected into the solution under continuous stirring, stirred overnight, and the precipitate was collected by centrifugation (8000 rpm, 10 min) and washed three times with anhydrous ethanol; the obtained silica-coated nanoparticles (UCNPs@SiO2) were dried and stored at room temperature for further use.

[0076] (3) Synthesis of UHM

[0077] First, 10 μL of LTEOS was added dropwise to 2 mL of 20 mg of UCNPs@SiO2 dispersion (dispersant was water) and stirred continuously for 30 min. Then, 30 mL of 0.2 M KMnO4 solution was added dropwise to the suspension of the above solution (2 mL / min), mixed and ultrasonicated (800 W) for 1 h. After the ultrasonication, it was stirred overnight. The obtained nanoparticles were centrifuged and washed several times with deionized water to obtain USM. The prepared USM was then dissolved in 20 mL of Na2CO3 aqueous solution (0.5 M), gently stirred at 50 °C for 1 h, centrifuged at 6000 rpm for 10 min, and washed several times with water to obtain nanoparticles UHM (UCNPs@H-MnO2). The nanoparticles were vacuum dried and stored at room temperature for further use.

[0078] (4) Synthesis of UMDC

[0079] 10 mg of UHM was dispersed in 10 mL of anhydrous ethanol to obtain a UHM dispersion, 1 mg of DHA (dihydroartemisinin) was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain a DHA solution, and 1 mg of Ce6 was dissolved in 1 mL of DMSO to obtain a Ce6 solution; then the UHM dispersion, DHA solution, and Ce6 solution were mixed, ultrasonicated (800 W) for 5 min, vigorously stirred at 37°C for 24 h, and then the precipitate was collected by centrifugation, vacuum-dried, and stored at room temperature for future use.

[0080] (5) Synthesis of UMDCA

[0081] First, 13 mg of polyvinylpyrrolidone (PVP) and 20 mg of UMDC were uniformly dissolved in 10 mL of ethylene glycol (EG) and treated with ultrasound (800 W) for 15 min, followed by vigorous stirring at 90°C for 20 min to obtain a suspension; then, 1 mL of NaBH4 (120 mM) and 1 mL of HAuCl4 (24 mM) dissolved in ice water were slowly added to the above suspension in sequence; the mixed system was continuously mixed at 90°C for 60 min; and then the final product, UMDCA, was collected by centrifugation at 6000 rpm for 5 min and washed three times with deionized water / ethanol.

[0082] (6) Synthesis of UMDCAc

[0083] To improve the biocompatibility of UMDCA and connect targeting molecules, 1 mL of DSPE-PEG-2000-NHS solution (1 mg / mL) was added to 1 mL of UMDCA (1 mg / mL) aqueous solution, and the resulting mixture was stirred at room temperature overnight; the mixed solution was collected and washed with deionized water; then, 1 mL of cRGD aqueous solution (1 mg / mL) was added, and the mixture was reacted under vigorous stirring for 12 h; finally, free cRGD was removed by centrifugation at 10,000 rpm three times to obtain UMDCA-cRGD (UMDCAc).

[0084] The morphology and size distribution of the prepared UCNPs, UCNPs@SiO2, UCNPs@H-MnO2 and UMDCA were observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), respectively. Figure 1 As shown, the results show that UMDCA has an obvious hollow structure and a uniform overall size distribution, with a size of about 100 nm.

[0085] The prepared UCNPs, UCNPs@SiO2, UCNPs@H-MnO2, UMDC and UMDCA were tested for Zeta potential respectively. Figure 2 As shown, the results show that the polymer was successfully coated layer by layer on the nanoparticles.

[0086] DHA, Ce6, AuNPs and the prepared UCNPs, UCNPs@SiO2, UCNPs@H-MnO2, UMDC and UMDCA were tested by UV-visible absorption spectra. The results are as follows. Figure 3 As shown, the structure shows that the polymer is successfully coated layer by layer on the nanoparticles.

[0087] The prepared DHA, Ce6, AuNPs and the prepared UCNPs, UCNPs@SiO2, UCNPs@H-MnO2 and UMDCA were tested by Fourier transform infrared spectroscopy. The results are as follows. Figure 4 As shown in the results, the UCNPs nanoparticles coated with oleic acid (OA) have the highest peaks at 2924 and 2852 cm -1 A strong infrared band is shown at 3450cm, which belongs to the symmetrical and asymmetrical stretching vibration of methylene (-CH2) in oleic acid. -1 The broad bands on the left and right come from the stretching vibration of OH, at 1463 and 1557 cm -1 The band at 463cm comes from the vibration of carboxyl group (-COOH); after UCNPs are coated with SiO2, Si-O-Si bond (463cm -1 ) and asymmetrical telescopic (1078cm -1 ); for UHM, 1086cm-1 The broadband at 506 cm is sharply weakened, indicating that the hard template SiO2 shell is sacrificed and the Mn-O bond (506 cm -1 ), these results further proved that UMDCA was successfully prepared.

[0088] The cRGD, UNDCA and UNDCA-cRGD (UMDCAc) were tested by FTIR spectra. Figure 5 As shown in the results, the FTIR spectrum of UMDCAc is at 1630 cm -1 An additional peak compared to UMDCA was shown at , indicating successful ligation of the cRGD peptide.

[0089] Comparative Example 1

[0090] Preparation of UCNPs@H-MnO2@Ce6 (UMC): 10 mg of UCNPs@H-MnO2 prepared in Example 1 was dispersed in 10 mL of anhydrous ethanol to obtain a UHM dispersion, and 1 mg of Ce6 was dissolved in 1 mL of DMSO to obtain a Ce6 solution; then the UHM dispersion, DHA solution and Ce6 solution were mixed, ultrasonicated (800 W) for 5 min, vigorously stirred at 37°C for 24 h, and then centrifuged to collect the precipitate to obtain UCNPs@H-MnO2@Ce6, which was vacuum-dried and stored at room temperature for future use.

[0091] Comparative Example 2

[0092] Preparation of UCNPs@H-MnO2@DHA (UMD): 10 mg of UCNPs@H-MnO2 prepared in Example 1 was dispersed in 10 mL of anhydrous ethanol to obtain a UHM dispersion, and 1 mg of DHA was dissolved in 1 mL of DMSO to obtain a Ce6 solution; then the UHM dispersion, DHA solution, and Ce6 solution were mixed, ultrasonicated (800 W) for 5 min, vigorously stirred at 37°C for 24 h, and then centrifuged to collect the precipitate to obtain UCNPs@H-MnO2@DHA, which was vacuum-dried and stored at room temperature for future use.

[0093] Comparative Example 3

[0094] Preparation of UCNPs@H-MnO2@AuNPs (UMA): First, 13 mg of polyvinyl pyrrolidone (PVP) and 20 mg of UCNPs@H-MnO2 prepared in Example 1 were uniformly dissolved in 10 mL of ethylene glycol (EG) and treated with ultrasound (800 W) for 15 min, followed by vigorous stirring at 90 °C for 20 min to obtain a suspension; then 1 mL of NaBH4 (120 mM) and 1 mL of HAuCl4 (24 mM) dissolved in ice water were slowly added to the above suspension in sequence; the mixed system was continuously mixed at 90 °C for 60 min; then the final product UCNPs@H-MnO2@AuNPs was collected by centrifugation at 6000 rpm for 5 min and washed three times with deionized water / ethanol.

[0095] 2. Enzyme Activity Verification of Cascade Catalytic System

[0096] (1) Enzyme activity verification

[0097] (1) Catalase (CAT) activity

[0098] The CAT-like activity of UMDCA was evaluated by measuring oxygen production using a JPB-607A dissolved oxygen meter. Specifically, before the experiment, all solutions were purged with argon for 30 minutes to completely remove O2 from the solution. Subsequently, the dissolved oxygen meter was used to measure the oxygen production in real time in a mixed solution (total volume of 4 mL aqueous solution) containing H2O2 solution (10 mM) and UMDCA at different concentrations (0, 50 μg / mL, 100 μg / mL, and 300 μg / mL). The measurement results are shown in Figure 2. Figure 6 As shown, the results showed that the level of generated O2 gradually increased with the increase of UMDCA concentration for alleviating hypoxia.

[0099] (2) Glucose oxidase (GOD) activity

[0100] The pH of the mixed solution was measured to confirm the production of gluconic acid after the nanoparticles reacted with glucose. The specific method was as follows: UMDCA and glucose were dissolved in deionized water (the concentration of UMDCA and glucose was 1 mg / mL) and incubated. During the incubation process, the pH value of the mixed solution was recorded using a real-time pH meter (Racitech). Figure 7 As shown, the results show that the pH value continues to decrease with time, and the decrease in pH value also confirms the generation of gluconic acid after reaction with glucose (generating gluconic acid and releasing H2O2).

[0101] The catalase (CAT) activity verification and glucose oxidase (GOD) activity verification showed that UMDCA exhibited good GOx mimetic activity, which was mediated by the O2 generation cascade of CAT mimetic activity.

[0102] (3) Peroxidase (POD) activity

[0103] In order to verify the peroxidase activity of the nanoparticles, the catalytic performance of UMDCA was verified by TMB colorimetry; specifically, 100 μL UMDCA (1 mg / mL) was mixed with 850 μL 0.1M HAc-NaAc buffer solution (pH 5.6), and then 50 μL TMB solution (16 mM) was added to obtain a 1 mL system; due to the generation of ·OH under weak acidic conditions, the absorption of ox-TMB at 652 nm gradually increased with the increase in the concentration of nanoparticles produced, which is a POD-like catalytic activity; different concentrations (0, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL and 1000 μg / mL) of UMDCA were mixed with H2O2 solution (10 mM) and measured by UV-visible absorption spectroscopy. The results are shown in Figure 2. Figure 8 As shown, the results showed that the OD652 of oxTMB increased with the increase of H2O2 concentration, indicating that UMDCA nanoparticles have peroxidase activity.

[0104] (4) Glutathione peroxidase (GPX) activity detection

[0105] The GSH depletion characteristics of the nanoparticles were determined using 5,5-dithio-2-nitrobenzoic acid (DTNB) as an indicator; GSH reacts with DTNB to generate yellow 5-thio-2-nitrobenzoic acid (TNB); DTNB has a significant absorption peak at 323nm. After the addition of GSH, the peak absorption at 323nm is significantly weakened, and a new peak appears at 412nm, indicating the generation of TNB. The results are as follows Figure 9 As shown, the results showed that the absorption peak of DTNB around 410 nm decreased significantly with the increase of incubation time, indicating that the GSH consumption ability of UMDCA nanomaterials has significant time dependence.

[0106] (2) Dynamic effects

[0107] (1) Chemical kinetic effect

[0108] In the Mn 2+ In the system of nanomaterials and H2O2, hydroxyl radicals (·OH) are generated through a Fenton-like reaction, which ultimately degrades methylene blue (MB). Therefore, the generation of hydroxyl radicals by UMDCA was evaluated using methylene blue as an indicator. The results are shown in Figure 2. Figure 10 As shown in Figure 3, the results showed that as the GSH content increased from 0 to 2 mM, MB degradation increased, the blue color gradually became lighter, and the composite material promoted the generation of ·OH.

[0109] The above-mentioned glutathione peroxidase (GPX) activity test and chemical kinetic effect test results showed that UMDCA has good GSH depletion and synergistic promotion capabilities.

[0110] (2) Photodynamic effect

[0111] 1,3-Diphenylisobenzofuran (DPBF) is a 1 A highly specific fluorescent probe for O2 (singlet oxygen), indicating 1 O2 can form endoperoxides and decompose; that is, 10 μL of 10 mM DPBF / DMSO solution was added to the UMDCA dispersion (2 mL, 50 μg / mL) to obtain a mixture; then a 980 nm laser (1.0 W / cm 2 ) irradiate the mixture and record the DPBF absorbance (at 410 nm) every minute until 10 min. Figure 11 As shown, the results show that under 980nm laser irradiation 1 The amount of O2 produced is time-dependent.

[0112] In summary, UMDCA can trigger multiple cascade catalytic reactions, promote the in situ amplification of ROS production, and effectively play an anti-tumor role.

[0113] 3. In vitro anti-tumor effect of the cascade catalytic system (cell assay)

[0114] (1) CCK-8 experiment

[0115] MHCC-97H cells (Infection Laboratory, Chongqing Medical University) were prepared into a suspension and seeded at a density of 5000 cells / well in a 96-well plate. After incubation for 24 h, the culture medium was removed. DMEM culture medium (Gibco) containing different concentrations and different materials for each group (UMC, UMD, UMA, UMDCA, and UMDCAc) was added to a volume of 100 μL and re-added to the 96-well plate. After incubation for 24 h, the culture medium was discarded. Then, a 980 nm laser was used for illumination (1.0 W / cm 2 , 5 min); after illumination, wash three times with PBS, add 100 μL of CCK-8 reagent, and incubate at 37°C for 1 h; add the same volume of PBS as the control group, and add only 100 μL of culture medium as the blank group; then measure the absorbance of each well at 450 nm, and calculate cell viability according to the following formula: Cell viability = (A treatment group - A blank group) / (A control group - A blank group) × 100%; where A represents absorbance, A treatment group is the absorbance of the treatment group, and so on; in addition, the control group is the control group.

[0116] The test results are as follows Figure 12As shown in the figure (NIR indicates laser irradiation), the results showed that almost all MHCC-97H cells in the control group (PBS+NIR, i.e., Control group) survived. Compared with other groups, UMDCAc showed more obvious cytotoxic effect on tumor cells under 980nm NIR irradiation. This enhanced effect may be attributed to the targeting effect of cRGD, which leads to the accumulation of more nanoparticles in cells, thereby enhancing the cytotoxic effect.

[0117] (2) Trypan blue staining

[0118] Under normal circumstances, cells reject this active dye (trypan blue) and remain unstained. However, when cell viability is lost or the cell membrane is incomplete, trypan blue can penetrate the denatured cell membrane and bind to DNA, staining it blue. MHCC-97H cells were cultured in Dulbecco's modified chickpea medium (DMEM, Gibco) containing high glucose and 10% fetal bovine serum in a 37°C, 5% CO2 incubator. UMDCAc was then added at a concentration of 200 μg / mL and cultured for 24 hours to obtain a cell suspension. The cell suspension was then mixed with trypan blue working solution (0.4%) at a volume ratio of 9:1 (final trypan blue concentration of 0.04%). Staining was observed under a microscope: blue cells indicated dead cells, while colorless and transparent cells indicated live cells. In addition, the same volume of PBS was added to serve as a control group.

[0119] The test results are as follows Figure 13 As shown, the results showed that the UMDCAc group had more blue dead cells than the Control group, indicating that UMDCAc could cause the loss of MHCC-97H cell activity and damage the MHCC-97H cell membrane.

[0120] (III) AO / EB live-dead cell staining

[0121] Acridine orange (AO) / ethidium bromide (EB) staining was used to observe the killing effect of nanoparticles; specifically, MHCC-97H cells were first inoculated into 6-well plates and incubated in a cell culture incubator (Gibco's DMEM medium) for 24 hours; then the original medium was removed, and DMEM medium (Gibco) containing different materials was added to each group (UMC, UMD, UMA, UMDCA, and UMDCAc) (NIR irradiation: 980 nm, 1.0 W / cm 2, 5min), incubated together for 12h, discarded the culture medium, washed three times with PBS; and set up the Control group; then used 500μL trypsin to digest the cells, collected the cells by centrifugation, diluted and blown evenly with 90μL 1X buffer (AO / EB kit of Shanghai Maokang Biotechnology Co., Ltd.), added 5μL AO staining solution and 5μL EB staining solution, mixed gently, stained at room temperature in the dark for 8min, took 10μL cell suspension and dropped it on the slide, and added 2μL DAPI-free anti-fluorescence quencher (Biyuntian), then pressed with a coverslip, sealed with nail polish, and then observed the distribution of live and dead cells in different treatment groups under an inverted fluorescence microscope.

[0122] The results are as follows Figure 14 As shown, the results showed that AO / EB live-dead cell staining had the strongest red fluorescence of UMDCAc and the largest number of dead cells, followed by UMDCA; this indicated that UMDCA and UMDCAc could induce effective cell death through a cascade catalytic reaction combined with enhanced GSH consumption.

[0123] IV. Anti-tumor effect of cascade catalytic system in vivo (animal experiments)

[0124] In vivo studies were conducted in female Balb / c mice subcutaneously inoculated with tumors and then injected via the tail vein with UMC, UMD, UMA, UMDCA, and UMDCAc. The subcutaneous tumors were irradiated with 980nm near-infrared light, and tumor size was analyzed to evaluate the in vivo anti-tumor effects of the UMDCAc nanomaterial. The specific procedures are as follows:

[0125] MHCC-97H cells in the logarithmic growth phase (5×10 6 The cells were suspended in 100 μL PBS (Biosharp, pH 7.4) and injected subcutaneously into the right shoulder of 5-6 week old female nude mice. The survival of the mice and the growth of the tumor were observed and monitored after inoculation. 3 -100mm 3 The nude mice were randomly divided into 6 groups (n=5) for in vivo experiments, and the groups were as follows: Control (injected with the same volume of PBS, 100 μL), UMC, UMD, UMA, UMDCA and UMDCAc; the above groups were intravenously injected with different groups of drugs (injection volume was 100 mL / mouse, 200 μg / mL), and 24 hours after administration, the mice were irradiated with 980 nm laser (1.0 W / cm 2 ) Mouse tumor site; The treatment course was 18 days, with drug injection every 3 days. The weight of the mice was recorded and the length and width of the tumor were measured. The tumor volume was calculated according to the following formula: Tumor volume (mm 3) = (length × width × width) / 2; the mice were killed on the 18th day after nanomaterial treatment, and the organs (heart, liver, spleen, lung, kidney) and tumors were collected respectively; the tumors were photographed, and the inhibitory ability of different groups of drugs on tumor growth was evaluated.

[0126] On day 18, the tumor images of the mice were as follows: Figure 15 As shown, the results showed that UMDCA and UMDCAc had a significant inhibitory effect on tumor growth, and the inhibitory effect was significantly better than that of the Control group, UMC group (UCNPs@H-MnO2@Ce6), UMD group (UCNPs@H-MnO2@DHA) and UMA group (UCNPs@H-MnO2@AuNPs); especially in the UMDCAc group, the tumor size was significantly reduced compared with the Control group.

[0127] In addition, the laser irradiation (laser irradiation power is 1.0w / cm 2 The time was irradiation for 1 minute and rest for 2 minutes, with a total irradiation of 15 minutes per day); the tumor volumes after different administration days are shown in Table 1 (the values ​​are all in mm 3 ), draw a graph as Figure 16 shown.

[0128] Table 1 Changes in tumor volume of mice in different groups

[0129]

[0130] From the above table 1 and Figure 16 It can be seen that UMC, UMD, UMA, UMDCA and UMDCAc all have certain inhibitory effects on tumors compared with the Control group, especially the UMDCA and UMDCAc groups, among which the UMDCAc group has the best inhibitory effect.

[0131] In addition, HE staining analysis was performed on organs (heart, liver, spleen, lung, and kidney), and the results were as follows: Figure 17 As shown, the results show that UMDCA and UMDCAc in the present invention have no obvious effect on organs and have high biosafety.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. 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 solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A biomimetic nanocarrier, characterized in that: The biomimetic nanocarrier includes UCNPs, inclusions, manganese dioxide and gold nanoparticles, the inclusions include a photosensitizer; the manganese dioxide has a hollow structure with pores on the surface; the manganese dioxide is wrapped around the UCNPs as a shell, and there is a gap between the manganese dioxide and the UCNPs, the gap is 10nm-15nm; the inclusions are dispersed in the gap; the gold nanoparticles are dispersed in the pores and / or coated outside the manganese dioxide shell.

2. The biomimetic nanocarrier according to claim 1, characterized in that The biomimetic nanocarrier meets one or more of the following conditions: (i) a photosensitizer selected from one or more of hematoporphyrin, mTHPC, chlorophyll-a, BPMppa, pheophorbide-a, chlorin e6, pyropheophorbide-a, or pyropheophorbide-a hexyl ether; (ii) The size of the pores is 3 nm-10 nm; (iv) The contents also include anti-tumor active substances.

3. The biomimetic nanocarrier according to claim 2, characterized in that The mass ratio of the inclusions to other components of the biomimetic nanocarrier is (0.1-0.5):1; and / or the anti-tumor active substance is DHA.

4. The biomimetic nanocarrier according to any one of claims 1 to 3, characterized in that The biomimetic nanocarrier is also linked to cRGD.

5. The method for preparing the biomimetic nanocarrier according to any one of claims 1 to 3, characterized in that: The preparation method includes: (1) modifying UCNPs@SiO2 with manganese dioxide to obtain USM; (2) etching SiO2 in USM to obtain UCNPs@H-MnO2; (3) incorporating inclusions into UCNPs@H-MnO2 to obtain UMDC; (4) modifying UMDC with gold nanoparticles to obtain UMDCA, a biomimetic nanocarrier.

6. The method for preparing the biomimetic nanocarrier according to claim 4, characterized in that: The preparation method includes: (1) modifying UCNPs@SiO2 with manganese dioxide to obtain USM; (2) etching SiO2 in USM to obtain UCNPs@H-MnO2; (3) incorporating the inclusions into UCNPs@H-MnO2 to obtain UMDC; (4) modifying UMDC with gold nanoparticles to obtain UMDCA; (5) mixing UMDCA with phospholipid polyethylene glycol to obtain a reactant, and then mixing the reactant with cRGD to obtain UMDCAc, i.e., a biomimetic nanocarrier.

7. The preparation method according to claim 5, characterized in that The preparation method meets one or more of the following conditions: (a) The preparation method of UCNPs@H-MnO2 includes: mixing UCNPs@SiO2 and potassium permanganate, sonicating, and centrifuging to obtain nanoparticles to obtain USM; then dissolving the USM in a Na2CO3 aqueous solution, and mixing and reacting to obtain UCNPs@H-MnO2; (b) The preparation method of UMDC includes: mixing UCNPs@H-MnO2 and the contents, sonicating, and centrifuging to obtain UMDC; (c) A method for preparing UMDCA includes: dissolving UMDC and polyvinyl pyrrolidone in an organic solvent, and ultrasonically stirring to obtain a suspension; mixing the suspension, NaBH4, and HAuCl4 for reaction, and centrifuging to obtain UMDCA.

8. An antitumor drug, characterized in that: The biomimetic nanocarrier comprises the biomimetic nanocarrier according to any one of claims 1 to 4.

9. Use of the biomimetic nanocarrier according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that The anti-tumor drug is an anti-hepatocellular carcinoma drug.

Images