Bionic nano-carrier as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of preparation of antitumor drugs, and particularly relates to a bionic nano-carrier as well as a preparation method and application thereof. The bionic nano-carrier comprises UCNPs, inclusions, manganese dioxide and gold nanoparticles, wherein the inclusions comprise a photosensitizer and / or DHA; the surface of the manganese dioxide is provided with holes; the manganese dioxide is used as a shell layer to wrap the UCNPs, and a gap is formed between the manganese dioxide and the UCNPs; the inclusions are dispersed in the gaps; and the gold nanoparticles are dispersed in the holes and / or coated outside the manganese dioxide shell layer. The bionic nano-carrier can catalyze decomposition of H2O2 and MnO2 generated by glucose oxidation under specific conditions through gold nanoparticles (AuNPs), a cascade synergistic reaction is introduced, ROS generation is amplified in situ, and PDT and CDT effects are enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-tumor drug preparation, and specifically 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 about 90% of primary liver cancer. The etiology of HCC is extremely complex, including viral infection, liver fibrosis or alcohol abuse. Since HCC has no obvious early symptoms, insidious onset and rapid progression, most patients are already in the late stage when diagnosed, which poses a great challenge to the treatment of HCC.

[0003] Currently, chemotherapy drugs can inhibit a variety of tumor entities and show broad-spectrum and high-efficiency anti-tumor activity against a variety of tumors. However, the strong hydrophobicity, non-specific 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 one of the hottest research directions in tumor therapy because it mainly destroys the lesion site while causing less damage to the surrounding normal tissues. Among them, upconversion nanoparticles (UCNPs), as a new generation of bioluminescent markers, have significant advantages such as low excitation light energy, low background, high and stable luminescence intensity, and adjustable emission wavelength; these characteristics make UCNPs show broad application prospects in the field of photodynamic tumor therapy. Summary of the invention

[0005] Based on this, the present invention, by coating upconversion nanoparticles (UCNPs) with a manganese dioxide layer and gold nanoparticles, and doping a photosensitizer between the manganese dioxide layer and the gaps between the upconversion nanoparticles, can introduce a cascade enhancement reaction by catalyzing the H2O2 produced by glucose oxidation and the decomposition of MnO2 under specific conditions through gold nanoparticles (AuNPs), thereby amplifying the generation of ROS in situ and enhancing 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 bionic nanocarrier, which comprises UCNPs, inclusions, manganese dioxide and gold nanoparticles, wherein the inclusions comprise a photosensitizer; the manganese dioxide is a hollow structure with holes 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 gaps; and the gold nanoparticles are dispersed in the holes and / or coated outside the manganese dioxide shell.

[0008] Preferably, the above-mentioned biomimetic nanocarrier meets 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 size of the pores is 3nm-10nm; (iii) the contents also include anti-tumor active substances.

[0009] More preferably, the above 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 above-mentioned bionic nanocarrier is further connected with cRGD.

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

[0013] First, the preparation method of the bionic nanocarrier without connecting 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 bionic nanocarrier.

[0014] Secondly, the preparation method of the bionic nanocarrier connected with 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, i.e., the bionic nanocarrier.

[0015] Preferably, the above preparation methods meet one or more of the following conditions: (a) The preparation method of UCNPs@H-MnO2 comprises: 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 comprises: mixing UCNPs@H-MnO2 and the inclusions, ultrasonicating, and centrifuging to obtain UMDC; (c) The preparation method of UMDCA comprises: 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 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 introduce a cascade enhancement reaction by catalyzing the H2O2 produced by glucose oxidation and the decomposition of MnO2 under specific conditions by gold nanoparticles (AuNPs). This not only amplifies the generation of ROS in situ and enhances the effects of PDT and CDT, but also further enhances the efficacy of PDT through the generated O2. That is, the cascade enhancement reaction mechanism can effectively overcome the limitations of the tumor microenvironment and provide a new idea for the treatment of complex tumors.

[0021] (2) The biomimetic nanocarrier provided by the present invention can target and co-deliver DHA and photosensitizer (such as Ce6), and can achieve 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 bionic 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 microscope images and scanning electron microscope images of different nanomaterials; A, C, E and G are transmission electron microscope images of UCNPs, UCNPs@SiO2, UCNPs@H-MnO2 and UMDCA respectively; B, D, F and H are scanning electron microscope images of UCNPs, UCNPs@SiO2, UCNPs@H-MnO2 and UMDCA respectively;

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

[0025] Figure 3 The 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 The catalase (CAT) activity test results of UMDCA;

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

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

[0031] Fig. 9 It is the detection result of glutathione peroxidase (GPX) activity of UMDCA;

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

[0033] Fig.11 This is the photodynamic effect test of UMDCA;

[0034] Fig.12 CCK-8 experimental conditions of MHCC-97H cells cultured with different nanomaterials;

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

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

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

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

[0039] Fig.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 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.

[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", "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.

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

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

[0044] It should also be noted that the manganese dioxide in the bionic 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 bionic nanocarrier in the present invention introduces a cascade enhancement reaction, which not only amplifies the generation 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 photosensitizer, preferably chlorin e6 (Ce6), which has better effects than other effects;

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

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

[0050] It should be noted that the bionic 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 above-mentioned 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 the efficacy of 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 play an efficient role. It can be delivered to the tumor through the bionic nanocarrier in the present invention 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 inclusions 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 bionic 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 bionic nanocarrier of the present invention, the preparation method comprising:

[0058] First, the preparation method of the bionic nanocarrier without connecting 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 bionic nanocarrier.

[0059] Secondly, the preparation method of the bionic nanocarrier connected with 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 bionic 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 USM in a Na2CO3 aqueous solution, mixing and reacting to obtain UCNPs@H-MnO2;

[0062] (b) The preparation method of UMDC includes: mixing UCNPs@H-MnO2 and the inclusions, ultrasonicating, 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 the 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] The embodiment of the present invention further provides an anti-tumor drug, comprising the bionic nanocarrier of the present invention.

[0066] It should be noted that the bionic nanocarriers in the present invention can synergistically treat tumors through TEM response and NIR response, and the bionic 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 also provides a use of the bionic 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 in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0070] 1. Preparation and characterization of biomimetic 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 uniform 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 and 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 an 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 into 2 mL of 20 mg of UCNPs@SiO2 dispersion (dispersant was water) and stirred for 30 min. Then, 30 mL of 0.2 M KMnO4 solution was added dropwise into the suspension of the above solution (2 mL / min), mixed and ultrasonicated (800 W) for 1 h. After the ultrasonication, the mixture was stirred overnight. The obtained nanoparticles were centrifuged and washed several times with deionized water to obtain USM. Then, the prepared USM was dissolved in 20 mL of Na2CO3 aqueous solution (0.5 M), stirred gently 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 UHM was dispersed in 10 mL anhydrous ethanol to obtain UHM dispersion, 1 mg DHA (dihydroartemisinin) was dissolved in 1 mL dimethyl sulfoxide (DMSO) to obtain DHA solution, and 1 mg Ce6 was dissolved in 1 mL DMSO to obtain 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 use.

[0080] (5) Synthesis of UMDCA

[0081] First, 13 mg of polyvinyl pyrrolidone (PVP) and 20 mg of UMDC were uniformly dissolved in 10 mL of ethylene glycol (EG), and subjected to ultrasonic treatment (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 washing 3 times with deionized water / ethanol.

[0082] (6) Synthesis of UMDCAc

[0083] In order 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 reacted for 12 h under vigorous stirring; finally, free cRGD was removed by centrifugation at 10,000 rpm for 3 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 the overall size distribution is uniform, 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 nanoparticle.

[0087] The prepared DHA, Ce6, AuNPs, 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 a high affinity for oleic acid at 2924 and 2852 cm -1 There is a strong infrared band at 3450cm -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 asymmetric telescopic (1078cm -1 ); for UHM, 1086cm-1 The broadband at 506 cm-1 is sharply weakened, indicating that the hard template SiO2 shell is sacrificed and the Mn-O bond (506 cm-1) appears. -1 ), these results further proved that UMDCA was successfully prepared.

[0088] The cRGD, UNDCA and UNDCA-cRGD (UMDCAc) prepared were tested by FTIR spectra. The results are as follows Figure 5 As shown in the results, the FTIR spectrum of UMDCAc is 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 the precipitate was collected by centrifugation 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 the precipitate was collected by centrifugation 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 subjected to ultrasonic treatment (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 washing 3 times with deionized water / ethanol.

[0095] 2. Enzyme activity verification of cascade catalytic system

[0096] (I) Enzyme activity verification

[0097] (1) Catalase (CAT) activity

[0098] The CAT-like activity of UMDCA was evaluated by measuring the 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 of 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 used to alleviate 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 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 (Ray Magnetics). 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] Through the validation of catalase (CAT) activity and glucose oxidase (GOD) activity, it can be seen that UMDCA exhibits good GOx mimetic activity through the O2 generation cascade mediated by CAT mimetic activity.

[0102] (3) Peroxidase (POD) activity

[0103] In order to verify the peroxidase activity of the nanoparticles, the TMB colorimetric method was used to verify the catalytic performance of UMDCA; 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 weakly 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; after mixing different concentrations (0, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL and 1000 μg / mL) of UMDCA with H2O2 solution (10 mM), the results were measured by UV-visible absorption spectroscopy. 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 nanoparticles were determined using 5,5-dithio-2-nitrobenzoic acid (DTNB) as an indicator; GSH reacted with DTNB to generate yellow 5-thio-2-nitrobenzoic acid (TNB); DTNB had an obvious absorption peak at 323nm. After adding GSH, the peak absorption at 323nm was significantly weakened, and a new peak appeared at 412nm, indicating the generation of TNB. The results are as follows Fig. 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 capacity of UMDCA nanomaterials has a significant time dependence.

[0106] (II) Kinetic effect

[0107] (1) Chemical kinetic effect

[0108] In the presence of Mn 2+ In the system of nanomaterials and H2O2, hydroxyl radicals (·OH) are generated through a Fenton-like reaction, which eventually 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. Fig.10 As shown, 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. Fig.11 As shown, the results show that under 980nm laser irradiation 1 The amount of O2 generated 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 cascade catalytic system (cell test)

[0114] (I) CCK-8 experiment

[0115] MHCC-97H cells (Infection Laboratory, Chongqing Medical University) were made into suspension and seeded in a 96-well plate at a density of 5000 cells / well. After incubation for 24 hours, the culture medium was removed. DMEM culture medium (Gibco) containing different concentrations and different materials of each group (UMC, UMD, UMA, UMDCA and UMDCAc) was added to a volume of 100 μL; added back to the 96-well plate, incubated for 24 hours, and then the culture medium was discarded. Then, a 980 nm laser was used for illumination (1.0 W / cm 2 , 5min); after illumination, wash three times with PBS, add 100μL CCK-8 reagent, and incubate at 37℃ for 1h; add the same volume of PBS as the Control group, and only add 100μL culture medium as the blank group; then measure the absorbance of each well at 450nm, and calculate the cell viability according to the following formula: cell viability = (A treatment group-A blank group) / (A control group-A blank group)×100%; wherein 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 Fig.12As shown in the figure (in the figure, NIR means laser irradiation), the results showed that almost all the MHCC-97H cells in the control group (PBS+NIR, i.e., Control group) survived. Compared with other groups, UMDCAc showed more obvious cytotoxic effects 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] (ii) Trypan blue staining

[0118] Under normal circumstances, cells can exclude this active dye (Trypan blue) and not be stained; but when cell activity is lost or the cell membrane is incomplete, Trypan blue can pass through the denatured cell membrane, bind to DNA, and stain it blue; MHCC-97H cells were cultured in Dulbecco's modified chickpea medium (DMEM medium, (Gibco)) containing high glucose, and added with 10% fetal bovine serum, and cultured in a 37°C, 5% CO2 incubator; then a certain concentration of 200μg / mL UMDCAc was added and cultured for 24h to obtain a cell suspension, and the cell suspension was mixed with Trypan blue working solution (0.4%) at a volume ratio of 9:1 (Trypan blue final concentration 0.04%); the staining was observed under a microscope: blue cells were dead cells, and colorless and transparent cells were live cells; in addition, the same volume of PBS was added as the Control group.

[0119] Test results such as Fig.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 and 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 a 6-well plate and incubated in a cell culture incubator (the culture medium was Gibco's DMEM medium) for 24 h; then the original culture 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 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, 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 of cell suspension and dropped it on the slide, added 2μL of anti-fluorescence quencher (Biyuntian) without DAPI, 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 Fig.14 As shown, the results showed that in AO / EB live-dead cell staining, UMDCAc had the strongest red fluorescence and the largest number of dead cells, followed by UMDCA; indicating that UMDCA and UMDCAc can induce effective cell death through cascade catalytic reactions combined with enhanced GSH consumption.

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

[0124] In vivo studies, tumors were inoculated subcutaneously in female Balb / c mice, and UMC, UMD, UMA, UMDCA, and UMDCAc were injected into the tail vein. The subcutaneous tumors were irradiated with near-infrared light at a wavelength of 980 nm, and the tumor size was analyzed to evaluate the anti-tumor effect of UMDCAc nanomaterials in vivo. The specific process is as follows:

[0125] MHCC-97H cells (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 for in vivo experiments (n=5): 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, and the mouse body weight was recorded and the length and width of the mouse 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: Fig.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 min and rest for 2 min, with a total irradiation of 15 min per day); the tumor volumes after different drug administration days are shown in Table 1 (the numerical units are all mm 3 ), and make a graph such as Fig.16 shown.

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

[0129]

[0130] From the above table 1 and Fig.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: Fig.17 As shown, the results show that UMDCA and UMDCAc in the present invention have no obvious effect on organs and have high biological safety.

[0132] 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. A bionic nanocarrier, characterized in that: The bionic nanocarrier includes UCNPs, inclusions, manganese dioxide and gold nanoparticles, wherein the inclusions include a photosensitizer; the manganese dioxide is a hollow structure with holes on the surface; the manganese dioxide is wrapped around the outside of the UCNPs as a shell, and there is a gap between the manganese dioxide and the UCNPs; the inclusions are dispersed in the gaps; and the gold nanoparticles are dispersed in the holes and / or coated outside the manganese dioxide shell.

2. The bionic nanocarrier according to claim 1, characterized in that: The biomimetic nanocarrier meets 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, chlorin e6, pyropheophorbide-a or pyropheophorbide-a hexyl ether; (ii) The size of the pores is 3nm-10nm; (iii) gap is 10nm-15nm; (iv) The contents also include anti-tumor active substances.

3. The bionic nanocarrier according to claim 2, characterized in that: The mass ratio of the inclusions and other components of the bionic 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 nanocarriers were also linked to cRGD.

5. The method for preparing the bionic 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 the inclusions into UCNPs@H-MnO2 to obtain UMDC; (4) modifying UMDC with gold nanoparticles to obtain UMDCA, i.e., a bionic nanocarrier.

6. The method for preparing the bionic 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 and phospholipid polyethylene glycol to obtain a reactant, and then mixing the reactant with cRGD to obtain UMDCAc, i.e., a bionic 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, 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 inclusions, ultrasonicating, and centrifuging to obtain UMDC; (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.

8. An anti-tumor drug, characterized in that: The bionic nanocarrier comprises the bionic nanocarrier according to any one of claims 1 to 4.

9. Use of the bionic nanocarrier according to 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.

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