GLUT-5 receptor targeted nanoparticles, and preparation method and application thereof

Abstract

The invention provides GLUT-5 receptor targeted nanoparticles and a preparation method and application thereof.The nanoparticles are fructose-doped manganese phosphate nanoparticles and are of an amorphous structure, the preparation method of the GLUT-5 receptor targeted nanoparticles comprises the steps that an organic phosphorus source and manganese salt are mixed, alkaline phosphatase is added under the condition that the pH ranges from 8.0 to 8.5, an enzymatic reaction is conducted, and the GLUT-5 receptor targeted nanoparticles are obtained. And the GLUT-5 receptor targeting type nanoparticles are obtained. The nano-particles Fr-MnP prepared by the method are spherical in appearance and uniform in size; a framework structure is MnP, so that the acid response sensitive drug release property is realized, and released Mn < 2 + > has a magnetic resonance enhanced developing effect and an effect of stimulating a Fenton reaction to promote tumor killing; fru is modified on the surface of MnP, so that the MnP can be specifically combined with an Fru receptor (GLUT-5) on the surface of osteosarcoma cells, the drug uptake of the osteosarcoma cells is increased, the bioavailability of Mn < 2 + > is improved, and the tumor diagnosis and treatment effects are enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to GLUT-5 receptor-targeted nanoparticles and a preparation method and use thereof. Background Art

[0002] Osteosarcoma is a common and fatal bone cancer in children, adolescents and young adults, with high metastatic rate and drug resistance. Existing treatments have limited effects, so the development of a therapeutic platform that integrates targeting, treatment and imaging functions is of great significance to improve the treatment of osteosarcoma. GLUT-5 transmembrane transporter, as a fructose transporter, is highly expressed in osteosarcoma cells, providing new possibilities for its targeted therapy.

[0003] Manganese (Mn) is an essential trace element involved in the synthesis of various metalloenzymes, normal bone tissue formation, and the maintenance of skeletal muscle activity. Mn-based nanomaterials with excellent biocompatibility provide multifunctional nanosystems for cancer diagnosis and treatment. They can be used as effective signal amplification contrast agents in magnetic resonance imaging (MRI). In addition, Mn 2+ The ions can catalyze endogenous H2O2 into hydroxyl radicals (·OH) through the Fenton reaction, which can be used to destroy the biological system of tumors. Therefore, manganese-based nanomaterials provide an instructive catalytic therapeutic strategy for ROS-mediated tumor apoptosis.

[0004] Improving the preparation methods of manganese-based nanomaterials is crucial to accelerate their clinical application. Various synthesis methods, including coprecipitation and hot water synthesis, have been used in the prior art to prepare manganese-based nanomaterials. However, the manganese-based nanomaterials prepared by these methods often require subsequent modification and drug loading before they can be used for tumor treatment. Therefore, it is of great significance to develop a simple one-step method to prepare multifunctional manganese-based nanomaterials for the treatment of osteosarcoma. Summary of the invention

[0005] The purpose of the present invention is to provide GLUT-5 receptor targeted nanoparticles and a preparation method and use thereof in view of the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The invention provides a GLUT-5 receptor targeted nanoparticle. The nanoparticle is fructose-doped manganese phosphate nanoparticle and has an amorphous structure.

[0008] The present invention also provides a method for preparing the above-mentioned GLUT-5 receptor targeted nanoparticles, which comprises mixing an organic phosphorus source with a manganese salt, adding alkaline phosphatase at a pH of 8.0-8.5, and performing an enzymatic reaction to obtain the GLUT-5 receptor targeted nanoparticles.

[0009] Furthermore, the enzymatic reaction is carried out at 37° C. and the reaction time is 4-12 hours.

[0010] Furthermore, the organic phosphorus source is disodium fructose diphosphate; and the manganese salt is manganese chloride.

[0011] Furthermore, the molar ratio of the organic phosphorus source to the manganese salt is (1.7-3):1.

[0012] The present invention also provides the use of the GLUT-5 receptor-targeted nanoparticles in the preparation of drugs for treating osteosarcoma.

[0013] The present invention also provides a pharmaceutical composition, comprising the above-mentioned GLUT-5 receptor-targeted nanoparticles, and a pharmaceutically acceptable adjuvant or carrier.

[0014] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0015] The nanoparticles Fru-MnP prepared by the present invention are spherical in appearance and uniform in size; the skeleton structure is MnP, which makes it have acid response sensitive drug release performance, and the released Mn 2+ It has the effect of enhancing magnetic resonance imaging and stimulating Fenton reaction to promote tumor killing; Fru is modified on the surface of MnP so that it can specifically bind to the Fru receptor (GLUT-5) on the surface of osteosarcoma cells, increase the uptake of drugs by osteosarcoma cells, and improve the activity of Mn 2+ bioavailability, enhancing tumor diagnosis and treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The synthesis and characterization of Fru-MnP nanocomposites are shown; wherein, (A) schematic diagram of the biomineralization-inspired synthesis of Fru-MnP nanocomposites via an enzymatic reaction route, showing the cleavage of phosphate groups in FDP biomolecules catalyzed by ALP; (B) transmission electron microscopy (TEM) images and selected area electron diffraction (SEAD) patterns of Fru-MnP (a) and ordinary MnP control (b); (CE) X-ray diffraction (XRD) patterns, Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TG) of Fru-MnP and MnP; (F) concentrations of fructose and its derived molecules released by Fru-MnP; (G) cumulative amount of manganese released from Fru-MnP nanoparticles in PBS solutions with different pH values; (H) corresponding T1-weighted magnetic resonance (MR) imaging images of Fru-MnP, showing the changes in the molar concentration of manganese in the solution; (I) r1 relaxation rate of Fru-MnP as a function of the molar concentration of manganese in the solution.

[0017] Figure 2 The results of the in vitro interaction between Fru-MnP and osteosarcoma cells are shown; wherein, (A) the survival rate of 143b cells treated with different concentrations of Fru-MnP; (B) the survival rate of 143b cells treated with the same fructose content or the same manganese content in different high glucose or low glucose DMEM media, using FDP, MnCl2·4H2O, MnP and Fru-MnP, respectively; (C-D) the fluorescence intensity of DCFH in 143b cells co-cultured with FDP, MnCl2·4H2O, MnP and Fru-MnP in different high glucose or low glucose DMEM; (E) the fluorescence images of 143b cells treated with FDP, MnCl2·4H2O, MnP and Fru-MnP; (F) the expression of caspases detected by WB in 143b cells co-cultured with FDP, MnP and Fru-MnP.

[0018] Figure 3 The in vivo MRI imaging results of Fru-MnP are shown; wherein, (A) T1-weighted MRI images before and after injection; (B) changes in T / N contrast ratio of tumor, liver and kidney before and after injection of Fru-MnP (mean ± SD, n = 3).

[0019] Figure 4 The results of the in vivo antitumor activity of Fru-MnP in nude mice are shown; wherein, (A) relative tumor volume measured using a vernier caliper in different treatment groups (n=5); (B) tumor weights measured at the end of these treatments and their photographs; (C) average weight curves measured from mice; (D) results of HE staining, Ki-67 and caspase-3 staining of tumor tissues from these groups.

[0020] Figure 5 The results of in vivo toxicity evaluation of Fru-MnP are shown, including (A) HE images of major organs of mice treated with Fru-MnP (by intravenous injection) and untreated mice (control group); (B–E) routine blood analysis of untreated mice (blue columns) and mice treated with Fru-MnP (by intravenous injection) on days 7 and 14 (red and green columns); ALT and AST levels (F and G) and BUN and Cr levels (H and I) of untreated mice (control group) and Fru-MnP-treated mice on day 14.

[0021] Figure 6 The schematic diagram shows the preparation process of GLUT-5 receptor-targeted nanoparticles Fru-MnP and their use in efficient tumor targeting and CDT treatment in mice. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0023] Example 1

[0024] This embodiment provides a method for preparing the nano drug Fru-MnP. Fru-MnP is synthesized by a one-step method (refer to Figure 1 A), the organic phosphorus source used was from disodium fructose diphosphate (C6H 12 Na2O 12 The hydrolysis of P2, Na2FDP) is as follows:

[0025] 6.6 g Na2FDP was dissolved in 24 ml deionized water, 1.9 g MnCl2·4H2O was dissolved in 16 ml deionized water, and then the two solutions were added dropwise to 960 ml deionized water; then 1.6 ml ALP (20 U mL^ -1 ) aqueous solution;

[0026] The mixed solution was magnetically stirred in a constant temperature water bath at 37°C. During the enzymatic reaction, the phosphate ions in the Na2FDP biomolecules were cleaved and the pH value gradually decreased. During the entire reaction process, the pH value should be maintained between 8.0 and 8.5 and adjusted by continuously adding a diluted 1M NaOH aqueous solution.

[0027] After the reaction, the prepared samples were collected by centrifugation, washed twice with anhydrous ethanol, washed twice with deionized water, and then freeze-dried for later use.

[0028] Example 2

[0029] The difference from Example 1 is that the molar ratio of the organic phosphorus source to the manganese salt is 3:1.

[0030] Example 3

[0031] The difference from Example 1 is that the molar ratio of the organic phosphorus source to the manganese salt is 1.7:1.

[0032] Comparative Example 1

[0033] Synthesis of MnP control sample: Na2HPO4 (0.0947 g) was dissolved in deionized water (100 ml), and then MnCl2·4H2O (0.190 g) was added. The resulting reaction solution was magnetically stirred, and the temperature and pH value were the same as in Example 1.

[0034] The Fru-MnP and the control sample MnP used in the following verification example are prepared by Example 1 and Comparative Example 1, respectively.

[0035] Verification Example 1

[0036] 1.1 X-ray powder diffraction (XRD) patterns were recorded using an X-ray diffractometer (Rigaku D / max 2550V, Cu Kα radiation, ), Fourier transform infrared (FTIR) spectra were recorded using a Fourier transform infrared spectrometer (FTIR-7600, Lambda Scientific, Australia). Scanning electron microscopy (SEM) (Hitachi S4800, Japan), transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS) (FEI Tecnai G2 F20, USA) and thermogravimetric analysis (TG) (STA-409 / PC, Netzsch, Germany) were also performed. In addition, the potential and particle size were measured using a potentiodynamic analyzer 7.3 (MalvernPanalytical, China).

[0037] The results are as follows Figure 1 BE shows:

[0038] TEM and SEAD images show ( Figure 1 B) The difference in structure between Fru-MnP and the control sample MnP. The MnP nanoparticles (b) have a crystalline structure and are larger than 200 nm in size, while the Fru-MnP nanoparticles (a) are amorphous microspheres with a diameter of about 20 nm. XRD spectrum ( Figure 1 C) confirmed the amorphous nature of Fru-MnP. FTIR analysis ( Figure 1 D) shows that PO4 3- The presence of fructose in Fru-MnP was confirmed by thermogravimetric analysis ( Figure 1 E) shows that the fructose content in Fru-MnP is close to 9.81%. Fru-MnP has good dispersibility, no obvious precipitation is observed in PBS, its zeta potential is about -33mV, and the particle size is about 135nm.

[0039] 1.2 Detection of manganese ion release from Fru-MnP:

[0040] The prepared Fru–MnP samples were investigated to confirm their pH-sensitive degradability. The experimental protocol was as follows: 30 mL of PBS solutions of different pH values ​​(pH 7.4, 6.5, and 5.4) were prepared in sealed containers. 10 mg of sample (dry powder) was dispersed in the above PBS solution. Subsequently, the temperature was controlled at 37 °C under constant shaking (140 rpm) (THZ-100, Shanghai Yiheng Instrument Co., Ltd., China). 2 mL of the culture medium was extracted at given time intervals for analysis, detected using an atomic absorption spectrometer (AAS, ZEEnit700P, Analytik JenaAG, ​​Germany), and replaced with PBS solution (2 mL).

[0041] The results are as follows Figure 1 G: Mn released in the first 4 hours at pH 7.4 2+ In the acidic solution of pH 6.5 and pH 5.4, the release amount was about 38% and 75%, respectively. After 12 hours, the cumulative manganese release at pH 5.4 exceeded 80%.

[0042] 1.3 Fru-MnP fructose release detection:

[0043] Under the action of hydrochloric acid, fructose generates hydroxymethyl acetaldehyde, whose maximum absorption wavelength is 291nm. Fructose concentration is 0-30μg mL -1 Beer's law is followed within the range. -1 A D-fructose standard solution was prepared in deionized water, and then 0, 0.5, 1, 2, 3, and 5 mL of the standard solution were added to 6 test tubes, respectively. 3 mL of concentrated hydrochloric acid was added to each test tube, and then deionized water was added until the final volume was 10 mL. Each test tube containing the mixed solution was incubated in boiling water for 8 minutes and then cooled with water. The final culture medium was analyzed using a UV-visible absorption spectrometer (Varian 100UV-VIS, Australia) at a wavelength of 291 nm. Fru-MnP was dissolved in deionized water and prepared into 1 mg mL -1 Fru-MnP solution. Then 0, 0.5, 1 and 2 mL of Fru-MnP solution were added to 4 test tubes respectively. 3 mL of concentrated hydrochloric acid was added to each test tube, and then deionized water was added until the final volume was 10 mL. Each test tube containing the mixed solution was incubated in boiling water for 8 minutes and then cooled with water. The final culture medium was analyzed using a Varian 100 UV-VIS spectrometer at a wavelength of 291 nm.

[0044] The results are as follows Figure 1 As shown in F: The fructose concentration was measured by UV spectrophotometer in an acidic solution, and the result showed that the fructose content in Fru-MnP was about 9.8%.

[0045] 1.4 Fenton reaction detection of Fru-MnP

[0046] The catalytic activity of Fru-MnP was evaluated by visual observation and by the degradation of MB. To obtain a visual observation, we followed the protocol of Fenton reaction for wastewater treatment. A 5-mL centrifuge tube was filled with 3 mL of the prepared MB dye solution (25 mg L -1 ), catalyst (Fru-MnP, 1 mg or MnCl2·4H2O, 2.5 mg) and 2 ml of 30 wt% H2O2 solution were shaken continuously for 48 h at pH = 3.0. Each catalytic process was carried out three times. MB concentration was measured at 663 nm using a UV-visible spectrophotometer. The generation of hydroxyl radicals in these catalyst suspensions was also detected by DMPO-trapped electron paramagnetic resonance (EPR) experiments. The final Fru-MnP concentration was 1.2 mmol L -1 The concentration of DMPO (5′,5-dimethyl-1-pyrroline N-oxide) was 100 mmol L -1 , the concentration of H2O2 in the phosphate solution at pH = 5 was 3%. Then 30 μL of the reaction solution was extracted and analyzed using a JES-FA200 spectrometer at 2, 4, 6, and 8 minutes.

[0047] We studied the 2+ The activity of the Fenton-type reaction driven by Fru-MnP to generate ·OH was observed in the experiment. O2 bubbles were visible in the experiment and significant degradation of methylene blue (MB) was observed. Electron paramagnetic resonance (EPR) experiments captured by DMPO confirmed that the signal of DMPO–·OH adducts could be detected after adding Fru-MnP, indicating that ·OH was effectively generated in the reaction. The results show that the Fenton-type reaction catalyzed by Fru-MnP has the potential for catalytic therapy of tumor cells.

[0048] 1.5 In vitro magnetic resonance detection of Fru-MnP

[0049] T1-weighted MRI was performed on Fru-MnP solutions (dissolved in PBS solutions under different conditions, pH 5.4, pH 6.5 and pH 7.4) on a Philips 3T MRI scanner. The average signal intensity in each well was different, so a nonlinear fit of the repetition time was performed to the changes. An inversion recovery sequence was also used to obtain T1. Finally, the r1 relaxation rate was determined by T1 mapping.

[0050] The results are as follows Figure 1 As shown in HI, Mn 2+By rapidly exchanging with surrounding water molecules, the MRI images are effectively enhanced, and the relaxation time of protons can be significantly reduced. Studies have found that Fru-MnP, as an MRI contrast agent, exhibits good performance under different pH conditions. At pH = 5.4, its longitudinal relaxation rate (r1) reaches 7.83mM -1 S -1 , which is significantly higher than that of MnP, and increases with decreasing pH. This indicates that Fru-MnP is more hydrophilic than MnP, and an acidic environment can enhance its relaxation rate. Compared with the commonly used clinical contrast agent gadolinium-diethylaminetetraacetic acid (Gd-DTPA), Fru-MnP provides better contrast enhancement and has the potential for tumor-specific MRI detection.

[0051] Verification Example 2

[0052] 2.1 Cell viability assay

[0053] Osteosarcoma 143b cells or MC3T3-E1 cells were cultured at 5 × 10 3 The cells were seeded into 96-well plates at the same density and cultured for 24 h. Subsequently, the culture medium was carefully removed, and the culture medium containing MnP or Fru-MnP nanoparticles was added to each well and incubated according to the designed concentration. MnCl2·4H2O or FDP was used as a positive control, and the blank control was pure culture medium without adding any compound. Cell viability was determined using the CCK-8 kit (Beyotime, China) and measured according to the instructions.

[0054] 2.2WB Detection

[0055] Human osteosarcoma 143 cells were treated with FDP, MnP and Fru-MnP (100 μg mL -1 , equivalent manganese concentration) to analyze the expression of apoptotic proteins. To study the expression of GLUT5 in different cells, well-cultured 143 cells and MC3T3-E1 cells were collected. After the cells were incubated on ice for 30 minutes, the supernatant was collected by centrifugation. The protein concentration was determined using the BCA method. After the same amount of protein was mixed with the loading buffer and heated, it was loaded into the SDS polyacrylamide gel, separated by electrophoresis, and then transferred to a polyvinylidene difluoride membrane. After the membrane was blocked with TBST at 4°C overnight, it was incubated with primary antibodies for caspase 3, caspase 8, and caspase 9 (Cell Signalling Technology, Inc.), and then a secondary antibody was used. The immunoreactive streaks were observed using an ECL kit (Thermo Scientific Pierce, USA) and a chemiluminescence system (Bio-Rad).

[0056] 2.3 Fluorescence staining

[0057] To investigate the changes in cell morphology, double immunofluorescence experiments were performed to detect the cytoskeleton. 143b cells or MC3T3-E1 cells were seeded on glass coverslips and incubated with FDP, MnCl2·4H2O, MnP, and Fru-MnP for 24 h, respectively. Red rhodamine (Cytoskeleton, Inc.) was used to stain the cytoskeleton at a concentration of 200 nM for 30 min at 37 °C. Subsequently, the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.3% Triton X-100. After three cold PBS rinses, the cells were stained with DAPI (Cytoskeleton, Inc.) solution. Cell morphology and differential expression of membrane protein GLUT5 were observed using a fluorescence microscope (Leica CTR6, Germany). Similar to the sample preparation described above, cells were fixed, permeabilized, and washed, then incubated with a primary antibody against GLUT5 (Santa Cruz Biotechnology Inc.) overnight at 4°C, followed by incubation with a secondary antibody (Alexa FluorPlus 488, Thermo Fisher). Finally, after DAPI staining, the expression of GLUT5 was detected using a Leica CTR6.

[0058] The results are as follows Figure 2 As shown in Figure 2, Fru-MnP exhibited significant cytotoxicity in osteosarcoma (143b) cells, and its IC 50 Values ​​approach 100 μg mL after 24 hours -1 , and showed stronger cytotoxicity in a low glucose environment. When co-cultured with GLUT5-negative MC3T3-E1 cells, Fru-MnP did not cause obvious cell death, demonstrating its selective uptake ability for GLUT5-positive cells. Mechanistic studies showed that Fru-MnP could promote apoptosis of 143b cells through ROS generation and activation of caspases.

[0059] Verification Example 3

[0060] 3.1 In vivo MRI detection of Fru-MnP

[0061] Fru-MnP nanocomposites were dissolved in PBS and tested for in vivo MRI imaging at different concentrations using a Philips 3T scanner for T1-weighted imaging. The following spin echo parameters were used: TE = 8 ms, TR = 450 ms, number of acquisitions (NEX) = 4, slice thickness 1 mm, field of view (FOV) 120 × 120 mm2, matrix size 256 × 256. In a preliminary experiment, tumor-bearing mice were injected with 10% chloric acid hydrate (0.03 ml g-1 After anesthesia with 100 μg / mL Fru-MnP solution, the mice were scanned by injecting different concentrations of Fru-MnP solution through the tail vein. -1 , volume 100 μL), a follow-up scan of T1-weighted MRI images was performed.

[0062] The results are as follows Figure 3 As shown in the figure, before and after the injection of Fru-MnP into the tail vein of mice, the T1-weighted MRI images of GLUT5-positive osteosarcoma 143b were significantly higher than those before injection 5 minutes after injection, and gradually reached a peak 1 hour after injection. The effect can be maintained at a high level for several days and then slowly decline, and it drops to the baseline level after 168 hours. At the same time, the MRI signal ratio of the liver increased after injection and reached a peak at 12 hours, and then quickly fell back to the baseline level, which was significantly lower than the tumor. The MRI signal ratio of the kidney increased significantly after injection and remained at a high level until 5 days after injection.

[0063] 3.2 Monitoring of anti-tumor effects in Fru-MnP mouse model

[0064] The mice were weighed every 3 days, and the relative volume of the tumor was measured using a vernier caliper. The tumor volume was calculated according to the formula: Volume (mm 3 )=(length×width2) / 2. All mice were euthanized at 14 days, and the tumors were removed, weighed, fixed, and subjected to histological examination. The tumor growth status and organ tissue damage were observed by HE staining, and immunohistochemical analysis was performed to detect the expression of Ki-67 and caspase-3. In addition, blood biochemical analysis of tumor-bearing rats (including white blood cell count WBC, red blood cell count RBC, platelet PLT and hemoglobin Hb; creatinine Cr and urea nitrogen BUN to evaluate renal function; and atropine aminotransferase ALT and aspartate aminotransferase AST to evaluate liver function) was performed to determine the toxic effects of the nanomedicine.

[0065] After 14 days of treatment, the internal organs from mice (control group and Fru-MnP intravenous injection group) were stained with HE and images were obtained. Fresh blood was drawn from SD rats and centrifuged for 3 minutes (5000 rpm). The supernatant was discarded and the red blood cells were retained. The red blood cells were washed with saline and centrifuged three times, and finally suspended in 2% saline. Fru-MnP powder was also suspended in saline. The experiment was divided into the following groups: positive group (RBC), 25μg mL -1 (RBC+Fru-MnP), 50 μg mL -1 (RBC+Fru-MnP), 100 μg mL -1 (RBC+Fru-MnP), 200 μg mL -1(RBC+Fru-MnP). All groups were shaken at 37°C for 3 h and centrifuged again for 3 min (10,000 rpm), and the UV-Vis spectra (570 nm) of the supernatant were measured. The final concentration was 25 μg mL -1 , 50 μg mL -1 , 100 μg mL -1 and 200 μg mL -1 The hemolysis test was repeated on three SD rats.

[0066] like Figure 4 According to the results, mice treated with MnP and Fru-MnP showed a significant decrease in tumor weight and volume at the end of treatment (day 14), with the average tumor weight of mice receiving intravenous injection of Fru-MnP being 0.52 g, much lower than the 1.482 g in the control group. At the same time, pathological analysis results showed that 143b tumor cells in the control and FDP groups grew well, while MnP and Fru-MnP-treated groups showed significant nuclear chromatin condensation and enlargement of tumor necrotic tissue. Immunohistochemical analysis showed that Ki-67 expression was significantly reduced in the MnP and Fru-MnP groups, while caspase-3 expression was upregulated, indicating that Fru-MnP has a significant anti-cancer effect, inhibits cell proliferation by reducing Ki-67 expression, and promotes apoptosis by activating the caspase-3 signaling pathway.

[0067] 3.3 Biosafety

[0068] like Figure 5 As shown, Fru-MnP did not cause significant hemolysis in vivo, and tumor-bearing mice did not die of disease or toxicity at the end of the experiment, and there was no weight loss compared with the control group. HE staining images of liver, heart, lung, kidney, spleen and intestinal tissues of mice treated with Fru-MnP did not show pathological changes compared with the control group. The results of routine blood analysis showed that the various indices of white blood cells, red blood cells, hemoglobin and platelets were within the normal range, indicating that Fru-MnP was safe at a 14-day therapeutic dose. Liver function markers (such as AST, ALT) and renal function (such as serum creatinine and urea nitrogen) did not show significant differences, indicating that Fru-MnP has good liver and kidney compatibility. In summary, Fru-MnP exhibits excellent biosafety and negligible systemic toxicity, and has the prospect of becoming a potential effective nanomedicine for the treatment of osteosarcoma in the future.

[0069] Figure 6 The schematic diagram shows the preparation process of GLUT-5 receptor-targeted nanoparticles Fru-MnP and their use in mice for efficient tumor targeting and CDT treatment:

[0070] By studying clinical human osteosarcoma samples, it was confirmed that GLUT5 is highly expressed on the cell membrane of osteosarcoma cells; thanks to the strong interaction between fructose and overexpressed GLUT5 on the cell membrane, Fru-MnP is selectively taken up by osteosarcoma cells through receptor-mediated endocytosis; subsequently, Mn released from Fru-MnP 2+ ions through Fenton-type reactions leading to chemokinetic therapy; meanwhile, Mn 2+ The T1 signal enhancement of ions provided tumor-specific MRI capabilities to monitor the CDT process; in in vivo experiments, Fru-MnP showed a strong anti-cancer effect, leading to the inhibition of cell proliferation by reducing the expression of Ki-67 and enhancing cell apoptosis by activating the caspase-3 signaling pathway.

[0071] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A GLUT-5 receptor-targeted nanoparticle, characterized in that: The nanoparticles are fructose-doped manganese phosphate nanoparticles and have an amorphous structure.

2. A method for preparing GLUT-5 receptor-targeted nanoparticles as claimed in claim 1, characterized in that: The method comprises mixing an organic phosphorus source with a manganese salt, adding alkaline phosphatase under a pH condition of 8.0-8.5, and carrying out an enzymatic reaction to obtain GLUT-5 receptor-targeted nanoparticles.

3. The preparation method according to claim 2, characterized in that: The enzymatic reaction is carried out at 37° C. for 4-12 hours.

4. The preparation method according to claim 2, characterized in that: The organic phosphorus source is disodium fructose diphosphate; and the manganese salt is manganese chloride.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the organic phosphorus source to the manganese salt is (1.7-3):

1.

6. Use of the GLUT-5 receptor-targeted nanoparticles according to claim 1 in the preparation of a drug for treating osteosarcoma.

7. A pharmaceutical composition, characterized in that The invention comprises the GLUT-5 receptor-targeted nanoparticles according to claim 1, and a pharmaceutically acceptable excipient or carrier.

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