KR20220055550A - Preparation Method for Magnesium-doped Mesoporous Hydroxyapatite Nano particles - Google Patents

Abstract

The present invention relates to a method for preparing mesoporous hydroxyapatite nanoparticles based on biowaste and reinforced with biocompatible Mg ions. The method for preparing Mg ion-containing mesoporous hydroxyapatite nanoparticles includes the steps of: pulverizing Mytilus edulis to obtain calcium powder; dissolving the calcium powder in hydrochloric acid (HCl); mixing the resultant solution with magnesium nitrate; mixing the resultant mixture with a surfactant; adding/mixing a phosphate salt to/with the mixture; treating the mixture with a strong base; and aging the resultant mixture at room temperature.

Description

Method for preparing mesoporous hydroxyapatite nanoparticles containing magnesium ions {Preparation Method for Magnesium-doped Mesoporous Hydroxyapatite Nano particles}

The present invention relates to a method for producing mesoporous hydroxyapatite nanoparticles containing magnesium ions, and more particularly, biocompatible magnesium ions using biowaste mussel (scientific name: Mytilus edulis ) as a raw material. It relates to a method for preparing reinforced mesoporous hydroxyapatite nanoparticles.

Magnetic nanoparticles are raw materials for disease diagnosis biosensors, MRI contrast agents, hyperthermia, drug delivery systems, etc., and are safe medical technologies that can reduce side effects. At the same time, it is receiving attention in the bio and medical fields because it can simultaneously solve the requirements of medical technology, such as obtaining rapid diagnosis and treatment results, high accuracy, and degree of cure.

Recently, many magnetic nanoparticles have been developed as promising materials for medical applications. However, most are known to have potential in vivo toxicity issues.

On the other hand, hydroxyapatite (hydroxyapatite, hereinafter abbreviated as "HAp"), Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ is a calcium phosphate series and has excellent biocompatibility, so biomedical or industrial ceramic materials It is increasingly used as a raw material for

One of the biggest problems in synthesizing HAp nanoparticles at present is the problem of production cost caused by the use of expensive chemicals in manufacturing. In order to solve this problem, it is necessary to reduce production costs by using relatively inexpensive raw materials.

In addition, for example, HAp that can act as an antibiotic to improve the occurrence of various side effects such as peri-implant mucositis or peri-implantitis and sequelae after surgery due to, for example, microbial infection due to not strong adhesion after surgery, such as dental implants There is a need for materials.

As a method for synthesizing the HAp material as described above, a number of methods for using calcium carbonate as a precursor and converting it into a HAp material are being studied.

Calcium carbonate is a material constituting the exoskeleton (shell) of shellfish, eggshell (egg shell), fish bones, and coral. As a result, it is being treated as a large amount of bio-waste, and since it is very commonly available, it is used as a raw material for knife? More economical HAp nanoparticles can be manufactured by developing the provided process.

Accordingly, an object of the present invention is to provide an economical method for preparing HAp nanoparticles that can reduce manufacturing costs by using biowaste, particularly mussels, as an inexpensive calcium carbonate donor.

In addition, when HAp material is introduced into the living body by surgery, magnesium as a metallic element that can act as an antibiotic and antioxidant to overcome various surgical side effects and aftereffects caused by microbial infection. can be raised further.

Accordingly, another object of the present invention is to provide a method for producing HAp nanoparticles as a biomaterial capable of increasing biocompatibility and lowering side effects by introducing a magnesium component exhibiting an antibiotic action into HAp.

A method for producing mesoporous hydroxyapatite nanoparticles containing metal ions according to an embodiment of the present invention includes the steps of pulverizing mussels (scientific name: Mytilus edulis ) to obtain calcium powder; dissolving the calcium powder with hydrochloric acid (HCl); mixing magnesium nitrate with the solution; mixing a surfactant into the mixture; adding and mixing phosphate to the mixture; treating the mixture with a strong base; and aging the final mixture at room temperature.

Preferably, the mussels may be heat treated prior to grinding.

Preferably, the surfactant is polyvinylpyrrolidone (Polyvinylpyrrolidone), and the phosphate is potassium dibasic (K ₂ HPO ₄ ).

Preferably, potassium hydroxide (KOH) may be used as the strong base.

Optionally, a microwave treatment step may be further included between the strong base treatment step and the aging step.

According to the manufacturing method of the mesoporous hydroxyapatite nanoparticles containing magnesium ions of the present invention, the manufacturing cost is due to the use of biowaste, especially mussels, as a biocompatible HAp material having non-toxic and antibacterial properties. It is possible to provide an economical method for manufacturing HAp nanoparticles that can reduce

In addition, according to the method for producing the mesoporous hydroxyapatite nanoparticles containing magnesium ions of the present invention, by introducing a magnesium component exhibiting antibiotic action into HAp, biocompatibility is improved, antibiotic action is enhanced, and side effects It is possible to provide a method for producing HAp nanoparticles as a biomaterial that can lower the

1 is a process flowchart sequentially illustrating a method for preparing mesoporous hydroxyapatite nanoparticles containing magnesium ions according to an embodiment of the present invention;
2 is an XRD pattern diagram for pure HAp powder (a), Mg-doped HAp powders, MgHAp-1 (b), MgHAp-2 (c) and MgHAp-3 (d);
3 is a Raman spectrum of HAp nanoparticles obtained according to an embodiment according to the present invention;
4 is an elemental mapping of HAp nanoparticles (pure HAp, MgHAp-1, MgHAp-2, MgHAp-3) samples obtained according to Examples according to the present invention;
Figure 5 shows morphological changes in MG63 cells treated and untreated with pure and Mg-doped HAp nanoparticles under an inverted-image contrast microscope;
Fig. 6(a) shows the cell viability after treatment with pure and Mg-doped HAp nanoparticles, and Fig. 6(b) shows the DNA content;
7 is a Hoechst fluorescence staining image of MG63 cells treated and untreated with pure and Mg-doped HAp nanoparticles;
8 is a view showing the antibiotic activity of commercial HAp, HAp nanoparticles (pure HAp, MgHAp-1, MgHAp-2, MgHAp-3) according to an embodiment of the present invention;
Figure 9 (a) is an image showing zebrafish embryos for each time after fertilization, Figure 9 (a) is pure HAp after 24 hpf (250 μg / ml), Figure 9 (b) is after 24 hpf (250 μg / ml) MgHAp-1 treated, FIG. 9(c) is MgHAp-3 treated after 48 hpf (250 μg/ml), FIG. 9(d) is pure HAp after 48 hpf (250 μg/ml), FIG. 9(e) is 48 MgHAp-1 treatment after hpf (250 μg/ml), Fig. 9(f) is MgHAp-3 treatment after 48 hpf (250 μg/ml), Fig. 9(g) is pure HAp after 72 hpf (250 μg/ml), 9(h) is MgHAp-1 treatment after 724 hpf (250 μg/ml), FIG. 9(i) is MgHAp-3 treatment after 72 hpf (250 μg/ml), FIG. 9(j) is time and concentration and It is a bar graph showing the percent mortality of zebrafish treated with HAp.

Hereinafter, a method for preparing mesoporous hydroxyapatite nanoparticles containing magnesium ions according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

The examples of the present invention are only provided to more completely explain the present invention to those of ordinary skill in the art, and the following examples may be modified in various ways, and the scope of the present invention is as follows It is not limited to an Example. Rather, these examples are provided so that this disclosure will be more thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

1 is a process flowchart sequentially illustrating a method for preparing mesoporous hydroxyapatite nanoparticles containing magnesium ions according to an embodiment of the present invention.

First, prepare the shells of mussels (Blue mussel: Mytilus edulis ), which are bio-waste as a calcium donor.

Given the high consumption of mussels worldwide, their shells are very readily available and are a natural source of calcium carbonate with a relatively high calcium content. Due to their high consumption, most mussel shells are dumped into the sea as natural waste, or in some cases landfills, resulting in contamination of the area.

Considering this, recycling mussel shells as waste and using them as a material for the synthesis of HAp is considered to be a very inexpensive alternative to expensive chemical methods.

The obtained mussel shells are washed with ordinary water or sterilized water, or heat-treated at a high temperature, for example, 550° C. if necessary.

After the mussels were dried, for example, milled by machine or pulverized with a mortar and pestle to obtain a powder state, and fine particles of calcium powder were obtained using a sieve (S1). The calcium powder thus obtained is used as a precursor for synthesizing the mesoporous hydroxyapatite of the present invention.

In the next step, the obtained calcium powder is dissolved in, for example, concentrated hydrochloric acid (HCl) to make a calcium chloride solution (S2). This calcium chloride solution may be filtered several times to remove impurities. In addition, a stirrer may be used in the dissolution step.

Magnesium (Mg) has many advantages compared to other doping metal ions reported to date. About 85-90% of Mg in the human body is present in living cells and tissues, and about 0.72, 1.23, and 0.44 wt% of Mg is present in bones, dentin and enamel of teeth, respectively.

In addition, Mg has been shown to play important roles in membrane transport, cell proliferation, mineral metabolism, bone calcification processes and cell signaling, and when deficient leads to diabetes, cardiovascular disease and hypertension. Therefore, considering the importance of Mg, its integration into HAp for better biological applications is very important.

In the present application, there is provided a method for synthesizing mesoporous hydroxyapatite nanoparticles in an environmentally friendly, inexpensive, and easy manner.

In order to dope the mesoporous hydroxyapatite with Mg as a metal that imparts non-toxic and antibacterial properties to the body, magnesium nitrate is added while stirring the calcium chloride solution obtained through the above process (S3).

Next, polyvinylpyrrolidone (PVP) as a surfactant is added to the solution doped with magnesium (Mg) (S4). PVP is used as an organic modifier for HAp synthesis, which has the function of making nanoparticles smaller and more porous.

Potassium dibasic phosphate (K ₂ HPO ₄ ) as a phosphate salt is slowly added to this solution using a stirrer (S5). The reason for adding phosphate is to add phosphate to the calcium obtained from the above-mentioned mussel shells, since HAp is basically a calcium phosphate series.

After the addition of phosphate, the pH of the solution is adjusted to 13 using potassium hydroxide (KOH) (S6).

Optionally, processing can be carried out in a microreactor to obtain the final mixture.

After the microwave treatment, when the reaction is completed, a final powder is obtained, aging is performed on it (S7), and the precipitate is filtered and washed, and the mesoporous hydroxyapatite containing magnesium ions according to the present invention is obtained. The synthesis of nanoparticles is completed.

(Example)

The collected mussel shells were washed with normal water, washed with sterile water, and stored for drying. After drying was completed, the mussel shells were ground into a fine powder, and about 11.2 g of mussel shell fine powder was dissolved in 33.4 ml of concentrated hydrochloric acid (HCl).

After adding hydrochloric acid, a clear solution of calcium chloride solution was observed, which was filtered several times to remove impurities.

After the filtration process, up to 100 ml of calcium chloride solution was obtained and used for HAp synthesis.

1 g of 100 ml of polyvinylpyrrolidone (PVP) solution was separately prepared and mixed with 100 ml of calcium chloride prepared above.

To this solution, 200 ml of potassium phosphate dibasic (K ₂ HPO ₄ ) solution (0.6M) was slowly added under magnetic stirring. After completion of the addition of dibasic potassium phosphate (K ₂ HPO ₄ ) solution, the pH of the solution was adjusted to basic 13 using potassium hydroxide (KOH) solution.

The obtained sol-state mixture was divided into two beakers and microwave-treated in a microwave oven at 900 W power for about 20 minutes, and after treatment, the sol was cooled by maintaining it at room temperature.

After the sol was cooled, the sol was washed several times with deionized water to remove unreacted residues. Washing was performed using a centrifuge with a rotation speed of 4000 rpm.

After washing, the final product was stored in a hot air oven and completely dried at about 100° C. for 12 hours. After drying, the collected white powder was pulverized again using a mortar and pestle and made into a fine powder denoted "pure HAp".

Different molar ratios were calculated for doping Mg in HAp and the synthesis was performed similarly to the above procedure in the presence of PVP. The molar ratios of Ca:Mg were 0.99:0.01, 0.97:0.03 and 0.90:0.1 M, respectively.

The magnesium nitrate calculated as above was combined during the preparation of the calcium chloride solution using a magnetic stirrer, then PVP and dipotassium phosphate (K ₂ HPO ₄ ) were added, the pH was adjusted to 13, followed by microwave treatment according to a similar procedure. and washing until a final powder is obtained. The prepared Mg-doped samples were named “MgHAp-1”, “MgHAp-2” and MgHAp-3, respectively.

(characteristic evaluation)

Figure 2 shows the XRD patterns for pure HAp powder (a), Mg-doped HAp powder, MgHAp-1 (b), MgHAp-2 (c) and MgHAp-3 (d). 2θ values are 38.97°(002), 42.56°(102), 43.70°(210), 48.25°(211), 49.04°(112), 50.27°(300), 52.03°(202), 61.58°(310). , 64.55° (311), 72.26° (222), 73.93° (312), 77.17° (213), 79.45° (321), 81.64° (104) and 83.75° (401), the XRD pattern of the ICCD of HAp -XRD It was found to be in good agreement with the standard data (09-0432). In addition, it could be concluded that the obtained HAp was formed in a hexagonal shape.

Raman spectroscopy analysis is one of the progressive approaches, providing a broad spectrum of secondary phase development during nanoparticle formation. The Raman spectrum of the obtained nanoparticles is shown in FIG. 3 . As a result, the stable band and main band of each sample appeared at 964 cm ^-1 , indicating ν ₁ stretching oscillations for the PO ₄ ^3- group. Also, a weak band appeared at 1052 cm ^-1 , indicating the ν ₃ stretching mode of the PO ₄ ^3- peak.

In addition, it shows that the vibrational bands of ν ₁ - ν ₄ exactly match the reference bands obtained for the HAp samples synthesized with different approaches. In addition, Mg-doped (MgHAp-1, MgHAp-2, MgHAp-3) samples also showed a band similar to that of pure HAp, but the peak intensity was reduced by adding Mg. The results of Raman spectroscopy analysis show that Mg was effectively incorporated into the HAp crystal without disturbing the original structure.

Figure 4 shows the elemental mapping of the obtained pure HAp, MgHAp-1, MgHAp-2, MgHAp-3 samples. This result clearly shows the pure HAp elements of carbon (C), oxygen (O), calcium (Ca), and phosphorus (P) based on the composition ratio of each element. In addition, Table 1 shows the directional distribution of elements in the obtained sample. The obtained samples show that the element content varies according to the nanoparticles.

The Ca/P concentrations of each HAp particle were 1.65, 1.56, 1.51, and 1.33 for pure HAp, MgHAp-1, MgHAp-2 and MgHAp-3, respectively. It clearly demonstrates that the nanoparticles obtained are in a non-stoichiometric form of HAp nanoparticles. The non-stoichiometric HAp had a Ca/P concentration of less than 1.65 with the addition of the Mg component.

In addition, the obtained MgHAp-1, MgHAp-2, and MgHAp-3 results clearly show the appearance of the magnesium component in the sample. The components of Mg/(Ca+Mg)% for the obtained sample were 1.32, 5.80 and 8.99 for MgHAp-1, MgHAp-2 and MgHAp-3. It is judged that the proposed concentration and the resulting concentration are similar. In addition, it can be seen that doping of Mg occurred in the HAp nanoparticles.

sample code
Element (atomic %)

Ca/P ratio
Mg/(Ca+Mg)
% Ca P O C Mg Pure HAp 31.2 18.9 41.0 8.9 - 1.65 0 MgHAp-1 29.8 19.0 42.5 8.3 0.4 1.56 1.32 MgHAp-2 27.6 18.2 43.3 9.2 1.7 1.51 5.80 MgHAp-3 25.3 18.9 42.2 11.1 2.5 1.33 8.99

Elemental composition of pure and Mg-doped HAP samples

On the other hand, the porosity and crystal size of HAp particles play an important role in nutrient flow, cell adhesion, and development, and a higher surface area is required for osteocytes bonding and tissue remodeling.

MG63 cells (human osteoblast lines) were used to characterize the differentiation of the obtained HAp nanoparticles. MG63 cells were exposed to six different concentrations of HAp nanoparticles (25-250 μg/ml) to evaluate cell viability.

Figure 5 is a diagram of morphological changes in MG63 cells treated with or without pure and Mg-doped HAp nanoparticles under an inverted-image contrast microscope. It clearly shows that there is no morphological change due to the addition of pure HAp and Mg-doped HAp nanoparticles when compared to control cells (untreated).

In order to analyze the viability of the cells, MTT analysis and DNA estimation (Hoechst staining) were performed (Figs. 6(a) and 6(b)). Pure HAp and Mg-doped HAp nanoparticles showed a viability of 80% or more even at a maximum exposure rate of 250 μg/ml [Fig. 6(a)]. Similarly, the DNA estimation results were consistent with the MTT analysis confirming the cellular suitability of nanoparticles. Fluorescence staining images of MG63 cells also showed intact cell nuclei, confirming that pure HAp and Mg-doped HAp nanoparticles were non-toxic to MG63 cells (see FIG. 7 ).

The antimicrobial activity of the obtained HAp nanoparticles was tested against human infection causing bacterial strains and the results are shown in FIG. 8 and Table 2. The pure HAp samples did not show any antibacterial properties. In addition, the obtained Mg-doped HAp nanoparticles were Staphylococcus aureus (MTCC 7443), Bacillus subtilis (MTCC 1133), Pseudomonas aeruginosa (MTCC 2581) and Escherichia coli (MTCC 1692, such as each). It has better antibacterial properties against human bacterial infections, and all human infections by bacteria were inhibited by Mg-doped samples.

microbe
Inhibition area [mean ± standard deviation (mm)
pure HAp
(100μg/ml) Commercial HAp
(100μg/ml) MgHAp-1
(100μg/ml) MgHAp-2
(100μg/ml) MgHAp-3
(100μg/ml) Staphylococcus aureus (MTCC 7443)
-
-
6.66±0.57
10.66±1.15
12.33±0.57 Bacillus subtilis
(MTCC 1133)
-
-
10.33±1.15
10.66±0.57
13.33±1.52 Pseudomonas aeruginosa
(MTCC 2581)
-
-
8.66±0.57
10.33±0.57
12.66±1.15 coli
(MTCC 1692)
-
-
9.33±0.57
12.33±0.57
14.33±0.57

Antibacterial activity of HAp nanoparticles

For the samples, the MgHAp-1 sample shows a low region of inhibition and the MgHAp-3 sample shows a higher region. It was also found that the antibacterial properties were also greater here. Therefore, the repression region is different for each bacterium.

The difference in properties is due to the cell wall components of the pathogen and the external charge of the HAp nanoparticles. This is because the antibacterial effect occurs where there are various mechanisms, such as the destruction of cell membranes due to the interaction of nanoparticles with external domains through electrochemical reactions.

Another reason may be the uptake of nanoparticles, which occurs due to a lack of cellular enzymes that occur in cell proliferation.

Therefore, Mg-doped HAp nanoparticles increase the preventive mechanism for disease development after implant surgery. It can be said that the antibiotic potential due to the Mg contained in the HAp nanoparticles is very large.

Zebra fish embryos have been used because of their high transparency and maximum embryo production that occurs in stage 1 and are very similar to human genes. In vitro evaluations were performed on pure HAp and Mg-doped HAp (MgHAp-1, MgHAp-2 and MgHAp-3).

Pure HAp and Mg-doped HAp were treated at various concentrations (25-250 μg/ml) for toxicity evaluation. In zebrafish, the increased HAp ratio shows that the toxicological effect increases as the embryo death rate increases. It was concluded that mortality was lowered by 96 hours.

Fish embryo poisoning was performed in vitro by diluting pure HAp and various ratios of Mg-doped HAp in Hanks' solution. Zebrafish embryos were analyzed microscopically during their early development. Embryos hatched from HAp exposed at 24, 48, 72 and 96 h were clearly visible at 40 fold under the microscope shown in FIG. 9 . Embryos were found to have delayed hatching during particle exposure. The hatching and mortality rates were evaluated based on microscopic observations.

The mortality rate of embryos 48 hours after fertilization is shown in FIG. 9 . The obtained HAps were inherently less toxic compared to other nanoparticles. Figure 9 shows that increasing the proportion of particles up to 25-250 μg/ml decreases the mortality rate at 24 h and 48 h hpf. The embryonic death rate is low because the particle size of the obtained HAps is smaller, the proportion of surface area is increased and it is non-toxic. The previous formation stages of the embryo, including the increased proportion of HAp, are observed under the microscope and can be clearly seen in FIG. 9 .

In Fig. 9(j), while pure HAp showed a death rate of 6.1%, MgHAP-1 showed a low mortality rate of 2.9%, MgHAp-2 had the highest mortality rate of 7.3%, and MgHAp-3 had a mortality rate of 72 It shows a mortality rate of 4.1% within hours.

Many embryo hatchings indicated at early developmental stages are enhanced through the obtained HAps. After 72 h, the hatched embryos were observed under a microscope to clearly identify the development and anomalies of the tail, head, and eyes when treated with HAps samples. During previous exposures to the embryo, the phenotype was not seen. It can be seen that the nontoxic effect of the pure HAp and Mg-doped HAp according to the present application was clearly evaluated in zebrafish at an earlier stage of embryonic formation.

The nanoparticles or magnetic nanoparticles comprising magnesium of the present invention can be used in biomedical applications such as drug targeting, hyperthermia cancer treatment, and magnetic resonance imaging (MRI). In addition, it has various application fields as dental bone supplements and drug delivery agents, cancer cell targeting agents, cancer cell killing agents, and the like.

In the above, the present invention has been described based on preferred embodiments of the present invention, but the present invention is not limited to the embodiments of the above-described features, and the technical field to which the present invention belongs without departing from the gist of the present invention as claimed in the claims Anyone with ordinary skill in the art can make various modifications, of course, such changes and modifications are construed as being within the scope of the claims.

Claims (5)

mussels (scientific name: Mytilus edulis ) to obtain calcium powder by grinding (S1);
dissolving the calcium powder with hydrochloric acid (HCl) (S2);
mixing the solution with magnesium nitrate (S3);
mixing a surfactant in the mixture (S4);
adding and mixing phosphate to the mixture (S5);
treating the mixture with a strong base (S6);
A method for producing mesoporous hydroxyapatite nanoparticles containing metal ions, comprising the step (S7) of aging the final mixture at room temperature. The method of claim 1,
Method for producing mesoporous hydroxyapatite nanoparticles containing magnesium ions, further comprising the step of heat-treating the mussels before the step S1. The method of claim 1,
The surfactant is polyvinylpyrrolidone and the phosphate is potassium dibasic (K ₂ HPO ₄ ), a method for producing mesoporous hydroxyapatite nanoparticles containing magnesium ions. The method of claim 1,
Method for producing mesoporous hydroxyapatite nanoparticles containing magnesium ions, using potassium hydroxide (KOH) as the strong base. The method of claim 1,
A method for producing mesoporous hydroxyapatite nanoparticles containing magnesium ions, further comprising a microwave treatment step between steps S6 and S7.

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