Method for synthesizing a new ferrihydrite nano-photosensitizer and its antibacterial and anticancer use

Abstract

The present invention discloses a method for synthesizing a new ferrihydrite nano-photosensitizer, comprising steps of: weighing 303 mg of Fe(NO.sub.3).sub.3⋅9H.sub.2O solid dissolved fully in 30 ml of distilled water to prepare a 0.75 mM of Fe(NO.sub.3).sub.3 solution in water; adding PEG solid to the solution in water by stirring to fully dissolve at a molar ratio of PEG to Fe.sup.3+ of 1:1-1:50; stirring the obtained solution under heating at 75° C. in a water bath for 10-50 minutes, and then immediately cooling in an ice bath after removing; centrifuging and washing the cooled mixed solution at high speed under low temperature with the supernatant discarded, to obtain pellets as PEG-modified ferrihydrite nanoparticles (PEG-Fns). The PEG-Fns synthesized in the present invention can be controllably induced and reduced by blue light to release Fe.sup.2+, and then produce ⋅OH through Fenton reaction of Fe.sup.2+ and H.sub.2O.sub.2 in the cell, which induces cell oxidative damage, thereby achieving controllable anticancer and antibacterial purposes.

Claims

1. A method for synthesizing a new ferrihydrite nano-photosensitizer, comprising steps of: (1) weighing 303 mg of Fe(NO.sub.3).sub.3.Math.9H.sub.2 O solid dissolved fully in 30 ml of distilled water to prepare a 0.75 mM of Fe(NO.sub.3).sub.3 solution in water; (2) adding PEG solid as a modifier to the solution in water obtained in step (1) by stirring to fully dissolve at a molar ratio of PEG to Fe.sup.3+of 1:1-1:50; (3) stirring the solution obtained in step (2) under heating at 75° C. in a water bath for 10-50 minutes, and then immediately cooling in an ice bath after removing; (4) centrifuging the cooled mixed solution obtained in step (3) at high speed under low temperature with the supernatant discarded to obtain pellets; (5) washing the pellets obtained in step (4) by centrifuging with distilled water for three times to obtain another pellets as PEG-modified ferrihydrite nanoparticles.

2. The method for synthesizing a new ferrihydrite nano-photosensitizer according to claim 1, wherein the molar ratio of PEG to Fe.sup.3+is 1:5-1:30.

3. The method for synthesizing a new ferrihydrite nano-photosensitizer according to claim 2, wherein the molar ratio of PEG to Fe.sup.3+is 1:20.

4. The method for synthesizing a new ferrihydrite nano-photosensitizer according to claim 1, wherein stirring for 20 minutes under heating in a water bath in step (3).

5. A ferrihydrite nano-photosensitizer synthesized by the method of claim 1, wherein the ferrihydrite nano-photosensitizer has a particle size of 20-30 nm.

6. A ferrihydrite nano-photosensitizer synthesized by the method of claim 1, wherein the ferrihydrite nano-photosensitizer is in a highly dispersed state.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the particle size of PEG-Fns obtained with different PEG modification ratios;

(2) FIG. 2 shows the particle size of PEG-Fns obtained by heating for different time when the PEG modification ratio is PEG:Fe.sup.3+=1:20;

(3) FIG. 3 shows electron micrograph and energy spectrum results of the synthesized PEG-Fns when the optimal conditions of PEG:Fe.sup.3+=1:20 and heating for 20 minutes are selected;

(4) FIG. 4 shows that the synthesized PEG-Fns is specifically induced to release Fe.sup.2+ by blue light when the optimal conditions of PEG:Fe.sup.3+=1:20 and heating for 20 minutes are selected;

(5) FIG. 5 shows that when PEG-Fns synthesized under optimal conditions are incubated with cells at different concentrations, they can be effectively absorbed by cells in the mouse tumor cell proliferation inhibition experiment in vitro (scale bar: 50 μm);

(6) FIG. 6 shows changes of intracellular ROS level in the mouse tumor cell proliferation inhibition experiment in vitro;

(7) FIG. 7 shows changes of cell viability during the continued incubation in the mouse tumor cell proliferation inhibition experiment in vitro;

(8) FIG. 8 shows the damage of cells around light source during the continued incubation in the mouse tumor cell proliferation inhibition experiment in vitro;

(9) FIG. 9 shows the tumor recovery of mice in each group after treatment in the antitumor experiment in vivo;

(10) FIG. 10 shows weight changes of mice in each group during treatment in the antitumor experiment in vivo;

(11) FIG. 11 shows the number of clones of fungi and bacteria with different treatments in the antibacterial experiment;

(12) FIG. 12 shows the confocal microscope 3D images of biofilms composed of bacteria and fungi with different treatments in the antibacterial experiment;

(13) FIG. 13 shows the average thickness of biofilms composed of bacteria and fungi with different treatments in the antibacterial experiment;

(14) FIG. 14 shows images of wound areas of animals on days 0, 3, 7, and 11 in the wound healing experiment in vivo;

(15) FIG. 15 shows the average number of bacterial and microbial clones in different groups of wounds in the first three days after treatment in the wound healing experiment in animals;

(16) FIG. 16 shows the average wound area of different groups over time after treatment in the wound healing experiment in vivo.

DETAILED DESCRIPTION OF EMBODIMENTS

(17) In order to understand the above objective, features and advantages of the present invention more clearly, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that the embodiments of the application and the features in the embodiments can be combined with each other if there is no conflict.

(18) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the present invention. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments, and are not intended to limit the present invention.

Embodiment 1

(19) A method for synthesizing a new ferrihydrite nano-photosensitizer, comprising steps of:

(20) weighing 303 mg of Fe(NO.sub.3).sub.3.9H.sub.2O solid dissolved fully in 30 ml of distilled water to prepare a 0.75 mM of Fe(NO.sub.3).sub.3 solution in water; adding 125 mg of PEG solid to the solution in water by stirring to fully dissolve at a ratio of PEG to Fe.sup.3+ of 1:1, 1:2.5, 1:5, 1:10, respectively; stirring the obtained solution under heating at 75° C. in a water bath for 10 minutes, and then immediately cooling in an ice bath after removing; centrifuging the cooled mixed solution at high speed under low temperature with the supernatant discarded, and then washing the obtained pellets with distilled water for three times. The obtained pellets were the ferrihydrite nanoparticles with a particle size less than 50 nm. The particle size of PEG-Fns was shown in FIG. 1.

Embodiment 2

(21) A method for synthesizing a new ferrihydrite nano-photosensitizer, comprising steps of:

(22) weighing 303 mg of Fe(NO.sub.3).sub.3.9H.sub.2O solid dissolved fully in 30 ml of distilled water to prepare a 0.75 mM of Fe(NO.sub.3).sub.3 solution in water; adding 125 mg of PEG solid to the solution in water by stirring to fully dissolve at a molar ratio of PEG to Fe.sup.3+ of 1:20; stirring the obtained solution under heating at 75° C. in a water bath for 10 minutes, and then immediately cooling in an ice bath after removing; centrifuging the cooled mixed solution at high speed under low temperature with the supernatant discarded, and then washing the obtained pellets with distilled water for three times. The obtained pellets was the ferrihydrite nanoparticles with a particle size d≈100 nm. The particle size of PEG-Fns was shown in FIG. 2.

Embodiment 3

(23) A method for synthesizing a new ferrihydrite nano-photosensitizer, comprising steps of:

(24) weighing 303 mg of Fe(NO.sub.3).sub.3. 9H.sub.2O solid dissolved fully in 30 ml of distilled water to prepare a 0.75 mM of Fe(NO.sub.3).sub.3 solution in water; adding 125 mg of PEG solid to the solution in water by stirring to fully dissolve at a molar ratio of PEG to Fe.sup.3+ of 1:20; stirring the obtained solution under heating at 75° C. in a water bath for 15 minutes, and then immediately cooling in an ice bath after removing; centrifuging the cooled mixed solution at high speed under low temperature with the supernatant discarded, and then washing the obtained pellets with distilled water for three times. The obtained pellets were the ferrihydrite nanoparticles with a particle size (d) about 30 nm, some of which have a larger particle size d≈100 nm. The particle size of PEG-Fns was shown in FIG. 2.

Embodiment 4

(25) A method for synthesizing a new ferrihydrite nano-photosensitizer, comprising steps of:

(26) weighing 303 mg of Fe(NO.sub.3).sub.3.9H.sub.2O solid dissolved fully in 30 ml of distilled water to prepare a 0.75 mM of Fe(NO.sub.3).sub.3 solution in water; adding 125 mg of PEG solid to the solution in water by stirring to fully dissolve at a molar ratio of PEG to Fe.sup.3+ of 1:20; stirring the obtained solution under heating at 75° C. in a water bath for 20 minutes, and then immediately cooling in an ice bath after removing; centrifuging the cooled mixed solution at high speed under low temperature with the supernatant discarded, and then washing the obtained pellets with distilled water for three times. The obtained pellets were the ferrihydrite nanoparticles with d≈20 nm; thus the condition of PEG:Fe.sup.3+=1:20 and heating for 20 minutes selected in this embodiment was optimal. The particle size of PEG-Fns was shown in FIG. 2.

Embodiment 5

(27) A method for synthesizing a new ferrihydrite nano-photosensitizer, comprising steps of:

(28) weighing 303 mg of Fe(NO.sub.3).sub.3.9H.sub.2O solid dissolved fully in 30 ml of distilled water to prepare a 0.75 mM of Fe(NO.sub.3).sub.3 solution in water; adding 125 mg of PEG solid to the solution in water by stirring to fully dissolve at a molar ratio of PEG to Fe.sup.3+ of 1:50; stirring the obtained solution under heating at 75° C. in a water bath for 20 minutes, and then immediately cooling in an ice bath after removing; centrifuging the cooled mixed solution at high speed under low temperature with the supernatant discarded, and then washing the obtained pellets with distilled water for three times. The obtained pellets were the ferrihydrite nanoparticles.

(29) The experimental effect evaluation of the ferrihydrite nano photosensitizer obtained in the above Embodiment 4

(30) 1. Mouse Tumor Cell Proliferation Inhibition Experiment In Vitro

(31) The cells used in this experiment were SCC-7 cells, which were mouse squamous epidermal carcinoma cells.

(32) The cells grown in the logarithmic phase were seeded in a 96-well plate at a density of 1×10.sup.4/well, and adhered overnight in a cell incubator at 37° C. and 5% CO.sub.2. The cells were incubated with PEG-Fns diluted with culture medium to concentrations of 0 μM, 80 μM, 160 μM, and 320 μM (using the amount of Fe.sup.3+ in PEG-Fns as the concentration unit) for 12 hours, with three replicates for each concentration. Thereafter, the medium was replaced with phenol red-free medium, stimulated with blue light for 30 minutes. The H2DCFDA probe was used to detect the level of ROS in the cells, while continuing to incubate with cells at different times, and the CCK-8 kit was used to detect changes in cells activity; the cells grown in the logarithmic phase were seeded in a confocol cell culture dish, and then the cells were treated with the above method. The cell survival around light source was observed by confocal microscopy (Leica TCS SP8) after double-stained with Calcein-AM/PI.

(33) As shown in FIGS. 6, 7 and 8, when the final concentration of PEG-Fns reached 320 μM, the intracellular ROS level caused by the blue light system increased by about 30% compared with the group without PEG-Fns. After 24 hours, the cell viability dropped to 30%, and continued to decline, and approached 100% cell killing rate. Such killing did not extend to areas outside the light source, indicating that PEG-Fns itself was non-toxic to cells.

(34) 2. Antitumor Experiment In Vivo

(35) Twelve healthy female Balb/c mice, SPF grade, inoculated with tumor cells at an amount of 5×10.sup.4after shaving their hair in the groin. Treatment was started until achieving the tumor diameter d=5 mm (10 days): 3 mice/group, animals were randomly divided into PEG-Fns treatment group (intratumoral injection of 5 μmol PEG-Fns/mouse), blue light treatment group (blue light stimulation at tumor site 30 minutes), PEG-Fns+blue light treatment group (intratumor injection of 5 μmol PEG-Fns/mouse, blue light stimulation at tumor site for 30 minutes after 48 hours), and control group (intratumoral injection of the same amount of saline).

(36) As shown in FIGS. 9 and 10, the tumors of the mice in the PEG-Fns+blue light treatment group were obviously scabs after 7 days, and they were substantially cured after 14 days, and the weight of the mice stabilized at about 24 g during the treatment, and there was no significant difference between groups. It was illustrated that the synthesized PEG-Fns of the present invention could effectively kill tumors in mice in the blue light system.

(37) 3. Antibacterial Experiment 1

(38) Candida albicans in the logarithmic growth phase were collected and diluted to 10.sup.6 CFU mL.sup.−1 with modified YPD liquid medium, with adding 25 mL of bacterial solution (final concentration: PEG-Fns 400 μM, H.sub.2O.sub.2 0.5 mM) to a 50 mL culture flask which was incubated under shanking in blue light at 30° C. for 5 hours. 1 mL of bacterial solution was centrifuged at 4000 rpm for 3 minutes with removal 900 μL of supernatant, and the remaining 100 μL of supernatant was resuspended and coated on the sandcastle solid medium. After 12 hours, the antibacterial effect was analyzed.

(39) As shown in FIG. 11, PEG-Fns+H.sub.2O.sub.2+Light could significantly kill bacteria and fungi, and the lethality to bacteria and fungi was greater than 90%, indicating that PEG-Fns had strong antifungal and bacteria capacity.

(40) 4. Antibacterial Experiment II

(41) Escherichia coli or Staphylococcus aureus in logarithmic growth phase were collected and diluted to 10.sup.6 CFU mL.sup.−1 with LB liquid medium (1% peptone, 0.5% yeast powder, 1% NaCl, pH 7.0). Sterile cover slips with a diameter of 10 mm were placed in a 24-well plate, which was added 1 mL of E. coli or Staphylococcus aureus diluent, incubating in an incubator at 37° C. for 48 hours, with exchanging the medium every 24 hours. After 48 hours, the medium was removed, and the coverslips were washed twice with 10 mM pH 7.0 PBS buffer. 1 mL of LB liquid medium containing PEG-Fns and H.sub.2O.sub.2 (final concentration: PEG-Fns 400 μM, H.sub.2O.sub.2 100 mM) was added and continued to incubate for 12 hours in an incubator at 37° C. under blue light. After blue light treatment for 12 hours, it was washed twice with PBS buffer to remove free bacteria, stained with Acridine Orange (AO) staining solution at 37° C. for 30 minutes in the dark, and washed twice with PBS buffer. The antibacterial effect was analyzed by a confocal microscope (Leica TCS SP8).

(42) Experimental results illustrated that the synthesized PEG-Fns of the present invention could significantly kill bacteria in the presence of blue light and H.sub.2O.sub.2, with a lethality rate close to 90%, indicating that PEG-Fns had strong antibacterial capacity.

(43) Candida albicans in the logarithmic growth phase were collected and diluted to 10.sup.6 CFU mL.sup.−1 with YPD liquid medium (1% yeast powder, 2% peptone, 2% glucose, pH 5.6), and sterile cover slips with a diameter of 10 mm were place in a 24-well plate. 1 mL of Candida albicans dilution was added into the 24-well plate, incubating in an incubator at 30° C. for 48 hours, with exchanging the medium every 24 hours. The medium was removed after 48 hours. The cover slips were washed twice with 10 mM pH 7.0 PBS buffer, adding 1 mL of modified YPD liquid medium (400 μM PEG-Fns, 100 mM H.sub.2O.sub.2) under blue light and continuing to incubate in an incubator at 30° C. for 12 hours. After blue light treatment for 12 hours, it was washed twice with PBS buffer to remove free bacteria, stained with AO staining solution at 30° C. for 30 minutes in the dark, and washed twice with PBS buffer. The antibacterial effect was analyzed by a confocal microscope (Leica TCS SP8).

(44) As shown in FIG. 12, it could be seen from the 3D photo of the confocal microscope that the antibacterial effects of PEG-Fns, blue light, and H.sub.2O.sub.2 alone were not obvious, and the combination of these three factors could significantly kill the fungus with a lethality rate close to 90%, indicating that PEG-Fns had strong antifungal capacity.

(45) As shown in FIG. 13, PEG-Fns treatment alone could promote the growth of bacteria and fungi from the average thickness of the biofilm composed of bacteria and fungi, furthermore, the treatment of blue light and H.sub.2O.sub.2 alone had no obvious effect on the growth of bacteria and fungi. However, compared with the control group, the PEG-Fns+H.sub.2O.sub.2+Light group could significantly kill bacteria and fungi. In summary, PEG-Fns could promote the killing of bacteria and fungi by H.sub.2O.sub.2 in the blue light system.

(46) 5. Wound Healing Experiment in Animals

(47) Twenty-four Kunming mice aged 6-8 weeks were selected and divided into four groups, each with 6 mice. Animals were shaved on the would establishment and acclimated for a day. Two circular wounds with a diameter of 4 mm were established on the back of mice. The control groups were as follows: the wound without treatment; the wound treated with 20 μL of 700 μM PEG-Fns; the wound irradiated with blue light for 1 hour. In the experimental group, the wound was treated with 20 μL of 700 μM PEG-Fns and irradiated under blue light for 1 hour. Each group was treated once every 24 hours and photographed and recorded on days 0, 3, 7, and 11, respectively.

(48) It could be seen from FIG. 15, the number of bacterial clones in the wound was significantly reduced in the experimental group compared with the control group. It could also be seen from the figure that PEG-Fns treatment alone could also reduce the number of bacterial clones in wounds, however, the colony of the clones was larger possibly due to adsorption effects of PEG-Fns, thus the true antibacterial effect of the PEG-Fns group alone could not be accurately measured.

(49) It could be seen from FIGS. 14 and 16 that the wound areas of mice in the experimental group were significantly reduced compared with the control group on Day 7. Except for the control group, the wound areas of the other three groups were substantially healed on Day 11. We could also see from the figure that the PEG-Fns alone and blue light could also promote wound healing possibly due to promote the immune system. In summary, the Fe.sup.2+ released by PEG-Fns irradiated with blue light could react with H.sub.2O.sub.2 produced from wounds in mice to inhibit the growth of wound bacteria and promote wound healing.

(50) Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention but not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, an ordinary person skilled in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.