Method for preparing hypericin nanoparticles by using Span20 and hypericin nanoparticles prepared by method
By using Span20 to prepare hypericin nanoparticles, the problems of poor stability and low targeting of hypericin were solved, and the drug stability and targeting were improved and the effect of enhancing the inhibitory effect on liver cancer cells was achieved.
Patent Information
- Application Number
- CN202411862754.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-03-25
AI Technical Summary
The poor stability of hypericin in solution leads to its autopolymer being captured by the mononuclear macrophage system, reducing the target distribution ability and therapeutic effect of the drug.
Hypericin nanoparticles were prepared using the surfactant Span20, and nanoparticles with particle sizes between 30 and 80 nm were formed by mixing with Span20 aqueous solution, vortex, centrifugation, ultrasonic crushing and freeze-drying.
It effectively reduces the aggregation of hypericin, improves its stability and targeting, and enhances the inhibitory effect on liver cancer cells.
Smart Images
Figure CN119684104A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing hypericin nanoparticles by using a surfactant Span20 and the hypericin nanoparticles prepared by the method. Background Art
[0002] Hypericin (HY) is a naphthodianthrone compound extracted from Hypericumperforatum Linn., a Clusiaceae plant. It is considered to be the most biologically active substance in Hypericumperforatum Linn. extracts. It has photosensitizing activity and photodynamic effects, and can be used in photochemical diagnosis and treatment of tumors. Hypericin has an active electronic conjugated system structure, and it is easy to aggregate between molecules, resulting in poor stability in solution. Hypericin self-aggregates will be captured by the mononuclear macrophage system, and self-aggregates will increase the distribution of drugs in non-target organs, reduce the targeted distribution ability of drugs, and thus reduce their therapeutic effects. Summary of the invention
[0003] The invention provides a method for preparing hypericin nanoparticles by using a surfactant Span20. The method can reduce the aggregation of hypericin and improve its targeting.
[0004] The present invention provides a method for preparing hypericin nanoparticles using a surfactant Span20, comprising the following steps:
[0005] 1) Span20 and ultrapure water were mixed at a material-liquid ratio of 1 mg: 1-5 ml, and centrifuged to obtain a Span20 aqueous solution;
[0006] 2) Hypericin and Span 20 aqueous solution are mixed at a material-liquid ratio of 5-8 mg:1 ml, vortexed, centrifuged, ultrasonically crushed, and freeze-dried to obtain hypericin nanoparticles.
[0007] Preferably, in step 1), the centrifugation conditions are 1000-2000 r / min, 2-4° C., and centrifugation for 2-8 min.
[0008] Preferably, in step 2), the vortex rotation speed is 20 to 80 r / min, and the vortex time is 2 to 5 min.
[0009] Preferably, in step 2), the centrifugation conditions are 1000-2000 r / min, 2-4° C., and centrifugation for 2-8 min.
[0010] Preferably, in step 2), the ultrasonic power is 60-80 W, the ultrasonic temperature is 0-4° C., and the ultrasonic time is 10-15 h.
[0011] The invention also provides hypericin nanoparticles prepared by the method.
[0012] Compared with the prior art, the present invention has the following beneficial effects:
[0013] 1) Span20, as a surfactant, can form a charged ion layer on the surface of hypericin, which helps to control the size of hypericin nanoparticles to only 30-80 nm and avoid aggregation of hypericin molecules, greatly improving stability.
[0014] 2) The hypericin nanoparticles prepared by the present invention have high water solubility, high drug bioavailability, and good inhibitory effect on liver cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG1(a) is a transmission electron microscope image of hypericin nanoparticles; FIG1(b) is a transmission electron microscope image of hypericin nanoparticles; FIG1(c) is a scanning electron microscope image of hypericin nanoparticles; FIG1(d) is a high-resolution image of hypericin nanoparticles; FIG1(e) is a particle size distribution diagram of hypericin nanoparticles;
[0016] FIG. 2( a ) is a diagram showing the ultraviolet absorption spectra of hypericin nanoparticles and hypericin; FIG. 2( b ) is a diagram showing the particle size distribution of hypericin nanoparticles;
[0017] Figure 3(a) is the cell apoptosis rate of hypericin group 1; Figure 3(b) is the cell apoptosis rate of hypericin nanoparticle group 1; Figure 3(c) is the cell apoptosis rate of hypericin group 2; Figure 3(d) is the cell apoptosis rate of hypericin nanoparticle group 2. DETAILED DESCRIPTION
[0018] The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.
[0019] Example 1
[0020] 1) Mix 1 mg of Span 20 and 1 ml of ultrapure water, pipette and mix evenly, then place in a centrifuge and centrifuge at 1000 r / min and 2°C for 2 min to obtain a Span 20 aqueous solution.
[0021] 2) Mix the hypericin and Span20 aqueous solution at a solid-liquid ratio of 5 mg:1 ml, place in a vortex oscillator, and vortex for 2 minutes at 20 r / min; take out and place in a centrifuge, centrifuge for 2 minutes at 1000 r / min and 2°C; take out and place in an ultrasonic crusher, with an ultrasonic power of 60 W, an ultrasonic temperature of 0°C, and an ultrasonic time of 10 h, and freeze-dry to obtain hypericin nanoparticles.
[0022] The transmission electron microscope scanning analysis showed that the particle size distribution was uniform and the particle size was between 30 and 80 nm.
[0023] Example 2
[0024] 1) Mix 1 mg of Span 20 and 5 ml of ultrapure water, pipette and mix evenly, then place in a centrifuge and centrifuge at 2000 r / min and 4°C for 8 min to obtain a Span 20 aqueous solution.
[0025] 2) Mix hypericin and Span20 aqueous solution at a solid-liquid ratio of 8 mg:1 ml, place in a vortex oscillator, and vortex at 80 r / min for 5 minutes; take out and place in a centrifuge, centrifuge at 2000 r / min and 4°C for 8 minutes; take out and place in an ultrasonic crusher, with an ultrasonic power of 80 W, an ultrasonic temperature of 4°C, and an ultrasonic time of 15 hours, and freeze-dry to obtain hypericin nanoparticles.
[0026] The transmission electron microscope scanning analysis showed that the particle size distribution was uniform and the particle size was between 30 and 80 nm.
[0027] Example 3
[0028] 1) Mix 1 mg of Span 20 and 2 ml of ultrapure water, pipette and mix evenly, then place in a centrifuge and centrifuge at 1500 r / min and 3°C for 6 min to obtain a Span 20 aqueous solution.
[0029] 2) Mix the hypericin and Span20 aqueous solution at a solid-liquid ratio of 6 mg:1 ml, place in a vortex oscillator, and vortex at 60 r / min for 4 minutes; take out and place in a centrifuge, centrifuge at 1500 r / min and 3°C for 5 minutes; take out and place in an ultrasonic crusher, the ultrasonic power is 70 W, the ultrasonic temperature is 2°C, the ultrasonic time is 12 h, and freeze-drying is performed to obtain hypericin nanoparticles.
[0030] The transmission electron microscope scanning analysis showed that the particle size distribution was uniform and the particle size was between 30 and 80 nm.
[0031] Example 4
[0032] 1) Mix 1 mg of Span20 and 1 ml of ultrapure water, pipette and mix evenly, then place in a centrifuge and centrifuge at 1200 r / min and 3°C for 3 min to obtain a Span20 aqueous solution.
[0033] 2) Mix the hypericin and Span 20 aqueous solution at a solid-liquid ratio of 6 mg: 1 ml, place in a vortex oscillator, and vortex at 60 r / min for 4 minutes; take out and place in a centrifuge, centrifuge at 1800 r / min and 4°C for 5 minutes; take out and place in an ultrasonic crusher, with an ultrasonic power of 65 W, an ultrasonic temperature of 4°C, and an ultrasonic time of 14 hours, and freeze-dry to obtain hypericin nanoparticles.
[0034] The transmission electron microscope scanning analysis shows that the particle size distribution is uniform and the particle size is between 30 and 80 nm, as shown in FIG1 .
[0035] Experimental Example 1: UV-Vis absorption spectra of HYP and HYP NPs
[0036] The hypericin nanoparticles prepared in Example 4 were added with ultrapure water to prepare samples with concentrations of 80, 100, 120, 140, and 160 μg / mL, respectively.
[0037] Hypericin was added to ultrapure water to prepare a 50 μg / mL sample.
[0038] The UV-visible absorption spectra of the above samples in the wavelength range of 300-700nm are detected, as shown in Figure 2. The UV absorption spectra of hypericin and hypericin nanoparticles have two maximum absorption peaks, with strong absorption at 550nm and 595nm, and the strongest absorption peak is at 595nm. As shown in Figure 2, the maximum absorption wavelength has not changed, and the drug chemical structure of the hypericin nanoparticles itself has not changed.
[0039] Experimental Example 2: CCK-8 kit to detect the effects of HYP and HYP NPs on liver cancer Bel-7402 cells and HepG2 cells
[0040] Take 1 ml of Bel-7402 cells and HepG2 cells in logarithmic growth phase and in good condition, add 0.5 ml of trypsin to digest, then add 2 ml of RPMI-1640 complete medium to Bel-7402 cells and 2 ml of DMEM complete medium to HepG2 cells to terminate digestion, centrifuge and resuspend, count, and adjust the Bel-7402 cells and HepG2 cells to 0.8×10 5 / ml. Bel-7402 cells and HepG2 cells were inoculated into 96-well plates, 100μl per well, and cultured in a 37℃, 5% CO2 cell culture incubator for 24h. Bel-7402 cells were divided into 2 groups, namely hypericin group 1 and hypericin nanoparticle group 1; HepG2 cells were divided into 2 groups, namely hypericin group 2 and hypericin nanoparticle group 2.
[0041] Hypericin and hypericin nanoparticles (prepared in Example 4) were added to the hyperforin group and the hyperforin nanoparticle group, respectively. The whole process was protected from light. After completion, the cells were placed in a carbon dioxide incubator and incubated for 24 h in the dark. After incubation, each group was irradiated with laser (595 nm, 0.25 mW·cm -2 After light treatment, the cells in hypericin group 1 and hypericin nanoparticle group 1 were replaced with RPMI-1640 complete medium, and the cells in hypericin group 2 and hypericin nanoparticle group 2 were replaced with DMEM complete medium, and the culture was continued for 24 h.
[0042] After the culture is completed, add 10 μl CCK-8 solution (5 mg / ml) to each well, continue incubation for 0.5 h, and use a microplate reader to detect the absorbance value (optimal density, OD) of the cells at 450 nm. Calculate the cell apoptosis rate based on the OD value. The calculation formula of the cell apoptosis rate is: cell apoptosis rate = 1-(absorbance value of each experimental group / absorbance value of the blank control group) × 100%. The test results are shown in Figure 3.
[0043] according to Figure 3(a) , 3(b) When the apoptosis rate of liver cancer Bel-7402 cells combined with hypericin monomer and photodynamic therapy reached 50%, the hypericin concentration needed to reach 109.6μg / ml; while when the apoptosis rate of liver cancer Bel-7402 cells combined with hypericin nanoparticles and photodynamic therapy reached 50%, the hypericin nano-ion concentration only needed to reach 58.84ng / ml. It can be seen that the killing effect of hypericin nanoparticles on Bel-7402 cells is far better than that of hypericin monomer.
[0044] according to Figure 3(c) , 3(d) , the apoptosis rate of HepG2 cells by hyperforin monomer combined with photodynamic therapy reached 50%, and the hyperforin concentration needed to be close to 127.1μg / ml; while when hyperforin nanoparticles were combined with photodynamic therapy, the concentration of hyperforin nanoparticles only needed to be 49.35ng / ml. It can be seen that the killing effect of hyperforin nanoparticles on HepG2 cells is far better than that of hyperforin monomer.
Claims
1. A method for preparing hypericin nanoparticles using surfactant Span20, characterized in that: The following steps are involved: 1) Span20 and ultrapure water were mixed at a material-liquid ratio of 1 mg: 1-5 ml, and centrifuged to obtain a Span20 aqueous solution; 2) Hypericin and Span 20 aqueous solution are mixed at a material-liquid ratio of 5-8 mg:1 ml, vortexed, centrifuged, ultrasonically crushed, and freeze-dried to obtain hypericin nanoparticles.
2. The method for preparing hypericin nanoparticles using surfactant Span20 according to claim 1, characterized in that: In step 1), the centrifugal conditions are 1000-2000 r / min, 2-4° C., and 2-8 min.
3. The method for preparing hypericin nanoparticles using surfactant Span20 according to claim 1, characterized in that: In step 2), the vortexing speed is 20 to 80 r / min, and the vortexing time is 2 to 5 min.
4. The method for preparing hypericin nanoparticles using surfactant Span20 according to claim 1, characterized in that: In step 2), the centrifugation conditions are 1000-2000 r / min, 2-4° C., and 2-8 min.
5. The method for preparing hypericin nanoparticles using surfactant Span 20 according to claim 1, characterized in that: In step 2), the ultrasonic power is 60-80 W, the ultrasonic temperature is 0-4° C., and the ultrasonic time is 10-15 h.
6. A hypericin nanoparticle prepared by the method of claims 1 to 5.