A nano-aluminum formulation of doxorubicin / zoledronic acid, its preparation method and application
By synthesizing nano-aluminum loaded with doxorubicin and zoledronic acid in an aqueous phase, the problem of short retention time of existing drugs at the lesion site was solved, achieving effective drug delivery and inhibition in the treatment of osteosarcoma, and simplifying the preparation process.
Patent Information
- Application Number
- CN202311144374.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-06
- Publication Date
- 2025-10-31
- Estimated Expiration
- 2043-09-06
AI Technical Summary
Existing small molecule drugs such as doxorubicin and zoledronic acid have short retention time at the lesion site and poor water solubility, which limits their application efficacy and causes toxic side effects. Nanotechnology provides a platform to improve solubility and release properties and for combination therapy, but the existing aluminum hydroxide nanomaterials have complex synthesis processes.
Nanoparticles of doxorubicin and zoledronic acid loaded with aluminum nanoparticles were prepared by an aqueous phase synthesis method. The pH of the aluminum salt aqueous solution was adjusted and the reaction was carried out. After centrifugation and washing, zoledronic acid and doxorubicin were loaded onto the aluminum nanoparticles to prepare nano-aluminum hydroxide sheets with a dynamic light scattering particle size of 200-250 nm.
This study achieved efficient phagocytosis and rapid entry into the nucleus of doxorubicin/zoledronic acid nano-aluminum formulation in osteosarcoma cells, inhibiting tumor cell growth. It also simplified the preparation process and optimized the drug loading, showing broad application prospects.
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Figure CN117045666B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of preparation and application of nanopharmaceuticals, and specifically relates to an aqueous phase synthesis of nano-aluminum and the doxorubicin / zoledronic acid nanopharmaceutical formulation and application based on the nano-aluminum. Background Technology
[0002] Osteosarcoma is the bone cancer with the highest incidence and mortality rate. Postoperative treatment of osteosarcoma often involves the use of one or more drugs for tumor control. Zoledronic acid, a compound with high calcium affinity and specific action on bone, is clinically used to reduce disease metastasis and pathological fractures in cancer patients. Although it does not directly improve survival, zoledronic acid can treat bone metastases caused by inhibiting osteoclast-induced bone resorption. Currently, zoledronic acid has become an important clinical drug in the treatment of osteosarcoma. Doxorubicin, a commonly used broad-spectrum anticancer drug, can effectively inhibit tumor cell growth and metastasis when used in combination with zoledronic acid and other drugs. However, small-molecule doxorubicin and zoledronic acid have problems such as poor water solubility and short retention time at the lesion site, which limit the efficacy of zoledronic acid and can lead to additional toxic side effects. Nanotechnology provides technical support for the development of these clinical drugs. On the one hand, nanomaterials can utilize their unique size to achieve enrichment at the tumor site through enhanced penetration and retention. On the other hand, nanomaterials improve the solubility and release properties of drugs and provide an ideal platform for the combined therapy of multiple drugs. Among them, aluminum nanomaterials, especially aluminum hydroxide nanomaterials, have been proven to be suitable for loading various drugs. Current synthesis of aluminum hydroxide nanomaterials mainly relies on high-temperature calcination, co-precipitation hydrothermal methods, etc., which are relatively complex processes. Therefore, this invention provides an aqueous-phase synthesis of aluminum nanomaterials and an doxorubicin / zoledronic acid nanomedicine formulation based on this aluminum nanomaterial, as well as its application. Summary of the Invention
[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a nano-aluminum formulation of doxorubicin / zoledronic acid, which addresses the shortcomings of the prior art.
[0004] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned nano-aluminum formulation of doxorubicin / zoledronic acid.
[0005] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned nano-aluminum formulation of doxorubicin / zoledronic acid.
[0006] To address the first technical problem mentioned above, this invention discloses a nano-aluminum formulation of doxorubicin / zoledronic acid, wherein doxorubicin and zoledronic acid are loaded onto nano-aluminum; the nano-aluminum is a nano-aluminum hydroxide sheet synthesized in an aqueous phase.
[0007] The dynamic light scattering particle size of the formulation is 200-250 nm, preferably 238-248 nm, more preferably 240-246 nm, and even more preferably 242-244 nm.
[0008] The dynamic light scattering dispersibility index of the formulation is less than 1, preferably less than 0.7, and more preferably less than 0.4.
[0009] The mass fraction of zoledronic acid in the formulation is 10%-20%, preferably 13%-17%, and more preferably 15%.
[0010] The doxorubicin content in the preparation is 6.5%-10.5%, preferably 7.5%-9.5%, and more preferably 8.5%.
[0011] The method for preparing the aqueous-phase synthesized nano-aluminum hydroxide sheets includes the following steps:
[0012] (1) Adjust the pH of the aluminum salt aqueous solution to 5.0-7.0 using sodium hydroxide aqueous solution;
[0013] (2) The reaction of aluminum salt aqueous solution after pH adjustment;
[0014] (3) After the reaction is complete, centrifuge, wash, and disperse in water to obtain nano-aluminum aqueous solution.
[0015] In step (1), the aluminum salt is aluminum nitrate, preferably Al((NO3)3·9H2O); the concentration of the aluminum salt aqueous solution is 0.05-0.07M, preferably 0.06M; the concentration of the sodium hydroxide aqueous solution is 0.4-0.6M, preferably 0.5M; preferably, the pH is adjusted to 6.5; preferably, the sodium hydroxide aqueous solution is added dropwise to the aluminum salt aqueous solution under stirring conditions.
[0016] In step (2), the reaction is carried out under stirring, preferably with magnetic stirring at 700-800 rpm; preferably, the reaction temperature is room temperature; preferably, the reaction time is 20-40 minutes, preferably 30 minutes.
[0017] In step (3), the centrifugation is performed at 4800 rpm for 10 minutes at room temperature; the washing is performed by discarding the supernatant after centrifugation, adding 20 mL of pure water solution to the precipitate, and using a pipette to redisperse the precipitate evenly. After this, the precipitate is obtained again by centrifugation.
[0018] In step (3), the concentration of the nano-aluminum aqueous solution is 0.5-6.0 mg / mL, preferably 1-5 mg / mL; preferably, after the nano-aluminum aqueous solution has been left to stand at room temperature for 8 days, the dynamic light scattering particle size is 70-100 nm, preferably 75-91 nm, and more preferably 80-82 nm; preferably, after the nano-aluminum aqueous solution has been left to stand at room temperature for 8 days, the dynamic light scattering dispersibility index is below 0.15, preferably below 0.11, and more preferably below 0.06; preferably, after the nano-aluminum aqueous solution has been left to stand at room temperature for 8 days, the pH is 4-5.5, preferably 4.5-5.
[0019] To address the second technical problem mentioned above, this invention discloses a method for preparing the aforementioned doxorubicin / zoledronic acid nano-aluminum formulation, comprising the following steps:
[0020] S1: Add an aqueous solution of zoledronic acid with a pH of 4-6 to the aqueous solution of nano-aluminum, stir, and obtain zoledronic acid-nano-aluminum;
[0021] S2: Add the aqueous solution of doxorubicin to the aqueous solution of zoledronic acid-nano-aluminum, stir, centrifuge, and collect the doxorubicin / zoledronic acid nano-aluminum formulation.
[0022] In step S1, the pH of the zoledronic acid aqueous solution is 4.5-5.5, preferably 5; preferably, the concentration of the zoledronic acid aqueous solution is 0.5-1.9 mg / mL, preferably 0.7-1.5 mg / mL, preferably 0.9-1.1 mg / mL; preferably, the volume ratio of the zoledronic acid aqueous solution to the nano-aluminum aqueous solution is 1:0.8-1.2, preferably 1:1.
[0023] In step S1, the stirring time is 10-50 minutes, preferably 20-40 minutes, and more preferably 30 minutes; preferably, after stirring, centrifugation is performed to obtain nano-aluminum particles of doxorubicin / zoledronic acid; preferably, the centrifugation conditions are centrifugation at 8000-12000 rpm for 10-20 minutes at 4-25℃, preferably centrifugation at 9000 rpm for 20 minutes at 4℃.
[0024] In step S1, the dynamic light scattering particle size of the zoledronic acid-aluminum nanoparticles is 178-194 nm, preferably 182-190 nm, and more preferably 185-187 nm.
[0025] In step S2, the concentration of the doxorubicin aqueous solution is 40-60 μg / mL, preferably 45-55 μg / mL, and more preferably 50 μg / mL; preferably, the concentration of the zoledronic acid-nano aluminum aqueous solution is 0.5-1.9 mg / mL, preferably 0.7-1.5 mg / mL, and more preferably 0.9-1.1 mg / mL; preferably, the volume ratio of the doxorubicin aqueous solution to the zoledronic acid-nano aluminum aqueous solution is 1:0.8-1.2, and more preferably 1:1.
[0026] In step S2, the stirring is carried out under light-protected conditions; preferably, the stirring time is 10-60 minutes, more preferably 20-45 minutes, and even more preferably 30 minutes; preferably, the centrifugation conditions are centrifugation at 8000-12000 rpm for 10-20 minutes at 0-10℃, preferably 4℃.
[0027] To address the third technical problem mentioned above, this invention discloses the application of the aforementioned doxorubicin / zoledronic acid nano-aluminum formulation in the preparation of products for the prevention and treatment of bone cancer.
[0028] The bone cancer mentioned includes osteosarcoma.
[0029] The products mentioned include pharmaceuticals.
[0030] In summary, this invention provides an doxorubicin / zoledronic acid-nano-aluminum, its preparation, and its application. The preparation process involved in this invention is a simple and mild aqueous phase reaction. The doxorubicin / zoledronic acid-nano-aluminum of this invention can achieve a drug loading of 8.5% / 15%, and can be effectively phagocytosed by osteosarcoma cells in vitro, exerting an inhibitory effect on osteosarcoma cell growth. The doxorubicin / zoledronic acid-nano-aluminum of this invention has broad application prospects in the treatment of osteosarcoma and other bone cancers.
[0031] Beneficial effects:
[0032] (1) The DOX / ZA-Al-nano of the present invention can exert a good effect of inhibiting tumor cell growth by promoting more doxorubicin to enter the cell and rapidly enter the cell nucleus, and can be used for the combined treatment of osteosarcoma.
[0033] (2) The preparation method of the present invention is simple, the reaction conditions are mild and easy to operate, and the preparation process and application scheme of nano-aluminum are optimized, which has the prospect of industrialization. Attached Figure Description
[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.
[0035] Figure 1The UV-Vis spectra of Al-nano, ZA, DOX, ZA-Al-nano, and DOX / ZA-Al-nano are shown.
[0036] Figure 2 TEM images of Al-nano, ZA-Al-nano, and DOX / ZA-Al-nano.
[0037] Figure 3 Infrared spectra of ZA, DOX, Al-nano, ZA-Al-nano, and DOX / ZA-Al-nano.
[0038] Figure 4 The phagocytic effects of LM8 cells on DOX, DOX+ZA, and DOX / ZA-Al-nano at different time points are shown. DOX+ZA represents a physical mixture of the drug in its free state.
[0039] Figure 5 The distribution of DOX and DOX / ZA-Al-nano in LM8 cells at different time points.
[0040] Figure 6 The growth inhibitory effects of DOX+ZA and DOX / ZA-Al-nano on LM8 cells are shown. DOX+ZA represents the drug in its free state or a physical mixture of drugs in their free state. The horizontal axis represents the concentration of each component in the added drug. Detailed Implementation
[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.
[0042] In this invention, the nano-aluminum, Al-nano, and aluminum hydroxide sheet nanomaterials all refer to nano-aluminum hydroxide.
[0043] Example 1
[0044] Weigh 600 mg of Al(NO3)3·9H2O and dissolve it in 25 mL of ultrapure water under magnetic stirring to prepare an aluminum solution. Separately weigh 400 mg of sodium hydroxide and dissolve it in 20 mL of ultrapure water to prepare a sodium hydroxide solution. Under magnetic stirring, add 8.8 mL of sodium hydroxide solution dropwise to the aluminum solution to adjust the reaction pH to 6.5. Using a similar method, the reaction pH of the solution can be adjusted within a certain range (5.0-7.0). After stirring at room temperature for 30 minutes, transfer the solution to a centrifuge tube and centrifuge at 4800 rpm for 10 minutes. After discarding the supernatant, resuspend the precipitate in 20 mL of deionized water and repeat the above centrifugation twice. The finally collected precipitate is resuspended in 20 mL of deionized water to obtain the nano-aluminum (Al-nano) solution. Take 5 mL of the solution, freeze-dry it, and weigh it to determine the mass of the powder. The concentration of the Al-nano solution was calculated as 4.02 ± 0.16 mg / mL based on the average mass of the three measurements (Table 1). The obtained Al-nano solution was left at room temperature for 8 days and used for subsequent preparations. At this time, the stable pH value of the Al-nano solution was approximately 4.7 (Table 1). The concentrations of Al-nano solutions obtained under other reaction pH conditions using a similar method are shown in Table 1.
[0045] Table 1. Al-nano solution concentration and stable pH under different reaction pH conditions.
[0046]
[0047] Example 2
[0048] Weigh 200 mg of zoledronic acid powder, add 199.3 mL of ultrapure water and 0.72 mL of sodium hydroxide solution (1 M), and dissolve thoroughly to obtain a zoledronic acid solution with a concentration of 1 mg / mL (pH 5.0). Separately, take an Al-nano solution that has been stored for 8 days and adjust its concentration to 0.5 mg / mL. Using a disposable dropper, add 200 mL of the above zoledronic acid solution to 200 mL of the above Al-nano solution and maintain the mixture for 30 minutes, keeping the magnetic stirrer at 700 rpm throughout the process. After stirring, transfer the solution to a centrifuge tube and centrifuge at 4°C and 9000 rpm for 20 minutes. Resuspend the resulting precipitate in 60 mL of deionized water to obtain an aqueous solution of ZA-Al-nano. Take 30 mL of the obtained ZA-Al-nano aqueous solution, freeze-dry it, and weigh the powder. Calculate the concentration of the ZA-Al-nano solution as 1 ± 0.06 mg / mL based on the average of the three weighings.
[0049] Example 3
[0050] A certain amount of doxorubicin hydrochloride powder was dissolved in deionized water to prepare a 50 μg / mL doxorubicin solution, which was stored in the dark. 2 mL of the doxorubicin solution was added dropwise to 2 mL of the ZA-Al-nano solution prepared in Example 2, and the solution was magnetically stirred at 700-800 rpm for 30 minutes at room temperature in the dark. All solutions were transferred to centrifuge tubes and centrifuged at 9000 rpm for 20 minutes at 4°C. The precipitate was collected and washed twice with deionized water. The final precipitate was resuspended in 4 mL of deionized water to obtain the DOX / ZA-Al-nano solution. The concentration of the solution was obtained by weighing the freeze-dried powder, which was 180 ± 24 μg / mL, corresponding to DOX and ZA contents of 14.8 ± 0.3 μg / mL and 27.4 ± 1.2 μg / mL (approximately 101 ± 4 μM), respectively.
[0051] This invention uses dynamic light scattering (DLS), zeta potential measurement, X-ray photoelectron spectroscopy (XPS) analysis, transmission electron microscopy (TEM) observation, ultraviolet-visible spectroscopy, and infrared spectroscopy to characterize the nano-aluminum (Al-nano), zoledronic acid-nano-aluminum (ZA-Al-nano), and doxorubicin / zoledronic acid-nano-aluminum (DOX / ZA-Al-nano) of this invention. Specific test results are as follows:
[0052] (1) Measurement of hydrated particle size and surface potential
[0053] The particle size and surface potential of aqueous-phase synthesized nano-aluminum (Al-nano), zoledronic acid-nano-aluminum (ZA-Al-nano), and doxorubicin / zoledronic acid-nano-aluminum (DOX / ZA-Al-nano) were determined by dynamic light scattering, and the results are shown in Table 2. By adjusting the pH of the solution during the synthesis process by changing the sodium hydroxide, stable nano-aluminum with a small particle size (81.2 nm, PDI 0.05) was obtained at pH 6.5, with a surface potential of +47.6 mV. The pH at which the Al-nano solution stabilized at this point was 4.7. The surface potential of ZA-Al-nano changed from +47.6 mV to -20.6 mV. This is because the zoledronic acid solution at pH 5.0 is negatively charged, and when loaded onto Al-nano, it can shield the positive charge, leading to a change in the surface potential of the material. Further loading with doxorubicin resulted in DOX / ZA-Al-nano, whose surface potential increased to -13.5 ± 0.6 mV, which is related to the positive charge generated by the ionization of amino groups on the DOX surface. Therefore, the change in surface potential before and after loading demonstrates the successful loading of ZA and DOX onto Al-nano. On the other hand, the particle sizes of the loaded ZA-Al-nano and DOX / ZA-Al-nano were 186.1 ± 1.6 nm and 243.4 ± 7.9 nm, respectively, showing a significant increase compared to Al-nano (81.2 ± 0.9 nm). This change in particle size also confirms the successful loading of ZA and DOX onto Al-nano. Furthermore, DOX / ZA-Al-nano exhibited good dispersibility (PDI 0.37 ± 0.04), thus making it a promising nanomaterial.
[0054] Table 2. Hydration diameter and surface potential of Al-nano, ZA-Al-nano, and DOX / ZA-Al-nano
[0055]
[0056] (2) X-ray photoelectron spectroscopy (XPS) analysis
[0057] XPS was used to analyze the Al and P elements of the obtained ZA-Al-nano. The results are shown in Table 3. Based on the ratio of zoledronic acid to nano-aluminum hydroxide P (2p), the mass ratio of zoledronic acid / nano-aluminum was calculated to be 1:2, and the drug loading rate of zoledronic acid was 15%, which was calculated by the following formula (1).
[0058] Zoledronic acid loading rate (%) = w 唑来膦酸 / (w 唑来膦酸 +w 纳米氢氧化铝 (1)
[0059] Table 3. Percentage of atomic molar concentration in particles obtained by XPS
[0060]
[0061] (3) Ultraviolet-Visible Spectroscopy Test
[0062] The ultraviolet-visible spectra of a series of nano-aluminum of the present invention are attached. Figure 1 As shown. Nano-aluminum and zoledronic acid showed increased UV absorption in the 200-240 nm range. After the addition of doxorubicin, DOX / ZA-Al-nano showed a characteristic absorption peak at 480 nm. The content of doxorubicin was calculated from the UV absorbance at 480 nm, and the drug loading of doxorubicin was calculated using the following formula (2), which was 8.5%.
[0063] Doxorubicin loading rate (%) = w 阿霉素 / (w 阿霉素 +w 唑来膦酸 +w 纳米氢氧化铝 (2)
[0064] (4) Observation by transmission electron microscopy (TEM)
[0065] A series of TEM images of nano-aluminum of the present invention are attached. Figure 2 As shown, the prepared DOX-ZA / Al-nano exhibits a classic near-hexagonal shape and good dispersibility. This result demonstrates the successful preparation of nano-aluminum by this invention, and that the loading of doxorubicin and zoledronic acid does not affect the morphology of the nano-aluminum hydroxide.
[0066] (5) Infrared spectroscopy analysis
[0067] The infrared spectra of a series of nano-aluminum hydroxides of the present invention are attached. Figure 3 As shown. The prepared nano-aluminum at 1646 cm⁻¹. -1 Corresponding to HO bending vibration, 738cm -1 526cm -1 This corresponds to the bending vibration of Al-O. The characteristic absorption peak of zoledronic acid standard is at 1580 cm⁻¹. -1 Tensile vibration of C=C in the imidazole ring, 1550 cm -1 and 1450cm -1 The CH stretching vibration in the imidazole ring, 1092-966 cm⁻¹ -1 The vibration of PO. When zoledronic acid is loaded onto Al-nano, the spectrum of ZA-Al-nano shows an increase in the characteristic absorption peak of zoledronic acid at 1580 cm⁻¹. -1 1550cm -1 1450cm -1 and 1092cm -1This indicates that zoledronic acid was successfully loaded onto Al-nano. The characteristic absorption peak of doxorubicin standard is at 1287 cm⁻¹. -1 The CH vibration of a single-oxygen six-membered ring, 816 cm⁻¹ -1 Weak out-of-plane vibrations of the single-membered oxygen ring. After doxorubicin was loaded onto ZA / Al-nano, the DOX / ZA-Al-nano spectrum showed an increase in the characteristic absorption peak of doxorubicin at 1287 cm⁻¹. -1 and 816cm -1 This indicates that doxorubicin was successfully loaded onto ZA-Al-nano.
[0068] This invention employs fluorescence microscopy, flow cytometry, and MTT assays to examine the phagocytic efficacy and toxicity of doxorubicin / zoledronic acid-aluminum nanoparticles against mouse osteosarcoma cells (LM8 cells). Specific experimental and test results are as follows:
[0069] Example 4 Cell phagocytosis experiment
[0070] Mouse osteosarcoma cells (LM8) were selected as a model. The intensity of intracellular doxorubicin-related red fluorescence was measured by flow cytometry to investigate the in vitro uptake of doxorubicin / zoledronic acid-nano aluminum hydroxide by osteosarcoma cells. Specifically, LM8 cells in logarithmic growth phase and free from mycoplasma infection were co-cultured with doxorubicin / zoledronic acid-nano aluminum hydroxide (DOX / ZA-Al-nano), doxorubicin (DOX), or sterile water (control group) for 0.5-6 hours at 37°C and 5% CO2. After discarding the culture medium, the cells were washed three times with sterile PBS. Cells were digested with trypsin and collected, and the intensity of intracellular DOX-related red fluorescence was analyzed by flow cytometry.
[0071] Collect LM8 cells in the logarithmic growth phase, according to 2×10 4Cells were seeded at a density of 1:1 in 24-well plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was discarded, and the cells were washed 1-2 times with sterile PBS. 450 μL of medium was replaced in each well, and 50 μL of DOX / ZA-Al-nano aqueous solution, free DOX aqueous solution, a physically mixed solution of free DOX and ZA, or sterile water (control group) was added. The final concentrations of DOX and ZA in a 500 μL culture system were 7.4 μg / mL and 50 μM, respectively. The cells were incubated at 37°C with 5% CO2 for 0.5 h, 1 h, 2 h, 4 h, and 6 h. The culture medium was discarded, and the cells were washed 3 times with PBS. 100 μL of trypsin was added for digestion for approximately 2 min. Immediately afterward, approximately 1 mL of culture medium was added, and the cells were collected by pipetting and centrifugation at 1000 rpm for 3 min in a 1.5 mL centrifuge tube. The supernatant was removed, and the cells were resuspended in 100 μL of PBS and placed on ice. Using fluorescence resolution as a parameter, cell fluid was added as a sample to a flow cytometer for detection to obtain the results of cell phagocytosis of drugs.
[0072] As attached Figure 4 As shown, intracellular red fluorescence gradually accumulated over time, demonstrating that the uptake of DOX / ZA-Al-nano by LM8 cells gradually increased with time, and the uptake was higher than that of free doxorubicin at all time points. This indicates that the DOX / ZA-Al-nano of the present invention can promote greater entry of doxorubicin into cells, thereby exerting an anti-tumor effect.
[0073] Example 5: Fluorescence Microscopy Observation
[0074] Doxorubicin exerts its antitumor effect by inhibiting DNA synthesis; therefore, whether or not doxorubicin enters the cell nucleus significantly impacts its function. To more directly observe the intracellular distribution of the drug, fluorescence microscopy was used to observe the intracellular fluorescence distribution of LM8 cells after co-culturing with the drug for a period of time. Specifically, LM8 cells were co-cultured with DOX / ZA-Al-nano or doxorubicin (DOX) for 0.5–6 hours, followed by staining of the cell membrane and nucleus with the green fluorescent dye Dio and the blue fluorescent dye DAPI, respectively. After cell fixation, the distribution of doxorubicin-related red fluorescence within the cells was observed using fluorescence microscopy.
[0075] Place one 24mm prepared circular glass slide per well in a 24-well plate, and add 0.5mL of culture medium to each well for 24 hours. After discarding the soaking medium, collect LM8 cells in the logarithmic growth phase and divide them into 1×10⁻⁶ cells per well. 4Cells were seeded at a density of 1:1 on circular glass slides and incubated at 37°C with 5% CO2 for 24 hours. The culture medium was discarded, and the cells were washed 1-2 times with sterile PBS. 450 μL of medium was replaced in each well, and 50 μL of DOX / ZA-Al-nano aqueous solution or free DOX aqueous solution was added, with the final concentration of DOX in a 500 μL culture system being 7.4 μg / mL. The cells were then incubated at 37°C with 5% CO2 for 0.5 h, 1 h, 2 h, 4 h, and 6 h. The culture medium was discarded, and the cells were washed 1-2 times with sterile PBS. 200 μL of 4% paraformaldehyde fixative was added to each well, and the cells were incubated at room temperature for 15 min. The paraformaldehyde solution was discarded, and the cells were washed 1-2 times with sterile PBS. 200 μL of Dio cell membrane green fluorescent staining working solution (1×) was added, and the cells were incubated at 37°C in the dark for 15 min. Aspirate the Dio cell membrane staining solution, wash three times with sterile PBS, add one drop of DAPI staining solution (10 μL) to a glass slide, hook the coverslip out of the 24-well plate, press the cell-containing side onto the glass slide, and observe the cell morphology and intracellular fluorescence distribution using a fluorescence microscope.
[0076] As attached Figure 5 As shown, the DOX / ZA-Al-nano reported in this invention exhibits significant intranuclear red fluorescence distribution after 0.5 hours of co-culturing, and this fluorescence persists until 6 hours of co-culturing. In contrast, free doxorubicin only shows significant intranuclear red fluorescence after 1 hour of co-culturing. This demonstrates that the DOX / ZA-Al-nano of this invention can promote the rapid entry of doxorubicin into the cell nucleus, thereby exerting its antitumor effect.
[0077] Example 6: Antitumor effect of DOX / ZA-Al-nano
[0078] The inhibitory effect of DOX / ZA-Al-nano on LM8 cells was detected by the thiazolyl blue reaction (MTT assay). LM8 cells in logarithmic growth phase were co-cultured with DOX / ZA-Al-nano or control material for 24 hours, then washed with sterile PBS, and cultured for another 4 hours with medium containing 0.5 mg / mL MTT solution. Then, 100 μL of dimethyl sulfoxide was added to each well, and after complete dissolution of the formed formazan, the UV absorbance of each sample at 570 nm was recorded using a microplate reader. The specific steps are as follows:
[0079] Collect LM8 cells in logarithmic growth phase that are free from mycoplasma contamination, at a rate of 4 × 10⁻⁶ cells / cells. 3Cells were seeded at a density of 180 μL per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. 20 μL of DOX / ZA-Al-nano aqueous solution or a physically mixed aqueous solution of free DOX and ZA was added to each well. The cell culture plates were then incubated at 37°C with 5% CO2 for another 24 hours. After removing the original culture medium, the cells were rinsed with sterile PBS, and then cultured for another 4 hours with medium containing 0.5 mg / mL MTT solution. 100 μL of dimethyl sulfoxide was added to each well, and the plates were placed in a cell culture incubator and incubated at 37°C for another 3 hours. Under an optical microscope, the deep purple formazan was observed to be completely dissolved. The absorbance at 570 nm was measured using a microplate reader; the fluorescence value reflects the number of viable cells.
[0080] As attached Figure 6 As shown, the DOX / ZA-Al-nano of the present invention can more effectively inhibit the growth of osteosarcoma cells compared with the physical mixture of DOX+ZA.
[0081] Based on the above experimental results, it can be concluded that the DOX / ZA-Al-nano of the present invention has a uniform and stable size, can be taken up by osteosarcoma cells, and promotes the rapid entry of the anti-tumor drug doxorubicin into the cell nucleus to exert its effect, ultimately achieving a good effect of inhibiting cancer cell growth, and has good application prospects.
[0082] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.
Claims
1. The application of an doxorubicin / zoledronic acid nano-aluminum formulation in the preparation of products for the prevention and treatment of bone cancer, characterized in that, The doxorubicin / zoledronic acid nano-aluminum formulation uses nano-aluminum to support doxorubicin and zoledronic acid; The nano-aluminum is a nano-aluminum hydroxide sheet synthesized in aqueous phase. The preparation method includes the following steps: (1) adjusting the pH of the aluminum salt aqueous solution to 5.0-7.0 with sodium hydroxide aqueous solution; (2) reacting the aluminum salt aqueous solution after pH adjustment; (3) centrifuging, washing, and dispersing in water after the reaction to obtain a nano-aluminum aqueous solution. The preparation method of the doxorubicin / zoledronic acid nano-aluminum formulation includes: S1: adding an aqueous solution of zoledronic acid with a pH of 4-6 and a concentration of 0.5-1.9 mg / mL to the aqueous solution of nano-aluminum, wherein the volume ratio of the aqueous solution of zoledronic acid to the aqueous solution of nano-aluminum is 1:0.8-1.2, stirring for 10-50 minutes, and centrifuging at 8000-12000 rpm for 10-20 minutes at 4-25℃ to obtain zoledronic acid-nano-aluminum; S2: adding an aqueous solution of doxorubicin with a concentration of 40-60 μg / mL to the aqueous solution of zoledronic acid-nano-aluminum, wherein the volume ratio of the aqueous solution of doxorubicin to the aqueous solution of zoledronic acid-nano-aluminum is 1:0.8-1.2, stirring for 10-60 minutes, and centrifuging at 8000-12000 rpm for 10-20 minutes at 0-10℃ to obtain the doxorubicin / zoledronic acid nano-aluminum formulation; The nano-aluminum formulation of doxorubicin / zoledronic acid has a dynamic light scattering particle size of 200-250 nm and a dynamic light scattering dispersibility index of less than 1; the mass fraction of zoledronic acid in the nano-aluminum formulation of doxorubicin / zoledronic acid is 10%-20%, and the mass fraction of doxorubicin is 6.5%-10.5%.
2. The application according to claim 1, characterized in that, The nano-aluminum formulation of doxorubicin / zoledronic acid has a dynamic light scattering particle size of 238-248 nm and a dynamic light scattering dispersibility index of less than 0.7; the mass fraction of zoledronic acid in the formulation is 13%-17%, and the mass fraction of doxorubicin is 7.5%-9.5%.
3. The application according to claim 1, characterized in that, The nano-aluminum formulation of doxorubicin / zoledronic acid has a dynamic light scattering particle size of 240-246 nm and a dynamic light scattering dispersibility index of less than 0.4; the mass fraction of zoledronic acid in the formulation is 15%, and the mass fraction of doxorubicin is 8.5%.
4. The application according to claim 1, characterized in that, In step (1), the aluminum salt is Al((NO3)3·9H2O); the concentration of the aluminum salt aqueous solution is 0.05-0.07M; the concentration of the sodium hydroxide aqueous solution is 0.4-0.6M; and the pH is adjusted to 6.
5.
5. The application according to claim 1, characterized in that, In step (2), the reaction is carried out under stirring; the reaction temperature is room temperature; and the reaction time is 20-40 minutes.
6. The application according to claim 1, characterized in that, In step (3), the concentration of the nano-aluminum aqueous solution is 0.5-6.0 mg / mL; after the nano-aluminum aqueous solution is left to stand at room temperature for 8 days, the dynamic light scattering particle size is 70-100 nm, the dynamic light scattering dispersibility index is below 0.15, and the pH is 4-5.
5.
7. The application according to claim 1, characterized in that, In step (3), the concentration of the nano-aluminum aqueous solution is 1-5 mg / mL; after the nano-aluminum aqueous solution is left to stand at room temperature for 8 days, the dynamic light scattering particle size is 70-100 nm, the dynamic light scattering dispersibility index is below 0.15, and the pH is 4-5.
5.
8. The application according to claim 1, characterized in that, In step S1, the pH of the zoledronic acid aqueous solution is 4.5-5.5; the concentration of the zoledronic acid aqueous solution is 0.7-1.5 mg / mL; and the volume ratio of the zoledronic acid aqueous solution to the nano-aluminum aqueous solution is 1:
1.
9. The application according to claim 1, characterized in that, In step S1, the stirring time is 20-40 minutes; the dynamic light scattering particle size of the zoledronic acid-nano aluminum is 178-194 nm.
10. The application according to claim 1, characterized in that, In step S2, the concentration of the doxorubicin aqueous solution is 45-55 μg / mL; the concentration of the zoledronic acid-nano aluminum aqueous solution is 0.5-1.9 mg / mL; and the volume ratio of the doxorubicin aqueous solution to the zoledronic acid-nano aluminum aqueous solution is 1:
1.
11. The application according to claim 1, characterized in that, In step S2, the stirring is carried out under light-protected conditions; the stirring time is 20-45 minutes.
12. The application according to claim 1, characterized in that, The bone cancer includes osteosarcoma; the product includes pharmaceuticals.