PEGylated hydroxyapatite zirconium oxide nanoparticles and method for producing the same

Abstract

The present invention relates to hydroxyapatite zirconium oxide nanoparticles synthesized and pegylated to enable biological applications, and to pegylated hydroxyapatite zirconium oxide nanoparticles and a method for manufacturing the same.

Description

Technology Field

[0001] The present invention relates to pegylated hydroxyapatite zirconium oxide nanoparticles and a method for producing the same. More specifically, the invention relates to pegylated hydroxyapatite zirconium oxide nanoparticles and a method for producing the same, wherein hydroxyapatite zirconium oxide nanoparticles are synthesized and pegylated to enable biological applications. Background Technology

[0002] Multifunctional nanoparticles (MF NPs) hold notable relevance in the biomedical field and are applied across a wide range of areas. These include applications in targeted drug delivery systems, detection, diagnostic imaging, and tissue engineering. Nanomaterials (NMs) can be customized with various materials and compositions to suit specific purposes. The adoption of MF NPs has significantly enhanced efficiency, ranging from disease prevention to diagnosis and treatment. One of the most serious diagnoses is age-related bone disease. Bone disease encompasses conditions that affect the skeletal system, leading to impaired mobility and death. Currently, the treatment of common skeletal diseases such as arthritis, osteoarthritis, osteosarcoma, and metastatic bone cancer lacks effective therapeutic options capable of balancing the alleviation of drug side effects with the achievement of desired treatment outcomes, particularly when compared to treatments for other diseases. Therefore, leveraging the unique characteristics of nanotechnology represents a promising approach, adding the need to integrate MF-NPs for further advancement. Hydroxyapatite (HAP) is a naturally occurring inorganic material with a chemical and crystalline composition very similar to the bones and teeth of humans and vertebrates. Due to these remarkable similarities, hydroxyapatite has been shown to be widely used as an implantable material in both orthopedic and dental applications. Furthermore, HAP crystals have been used as carriers in drug delivery systems due to their non-toxicity and excellent biocompatibility. HAP is a naturally occurring porous biomaterial with high adsorption capacity and strong binding affinity. Due to its unique properties, it is not only suitable for drug loading but is also ideal for interactions with various biological substances, including collagen, enzymes, proteins, and cells.

[0003] HAP holds significant potential as a versatile nanodrug delivery system carrier applicable to bone regeneration and the treatment of bone-related diseases. Previous studies have demonstrated the potential to extend drug half-lives and precisely control drug release kinetics by utilizing HAP NPs. This consequently enhances therapeutic efficacy while mitigating undesirable side effects. Of particular note is the intrinsic calcium-to-phosphate (Ca-P) ratio of HAP, which imparts osteoinductive properties characterized by osteoconduction and osteofusion. Furthermore, HAP exhibits the ability to upregulate genes responsible for bone formation. Nevertheless, the application of HAP has been limited primarily by its relatively lower strength and toughness compared to metals, making it unsuitable for applications requiring high drug loadings. Consequently, HAP is frequently used in combination with composite materials or polymers to broaden its spectrum of utility.

[0004] Conversely, zirconia oxide nanoparticles (ZrO2NPs) represent another category of bioceramic soft metals. ZrO2 is renowned for its excellent properties, including high strength, biocompatibility, chemical stability, and mechanical elasticity. Consequently, it has been widely applied in the development of implants and bone joint replacements. Furthermore, ZrO2NPs possess remarkable capabilities in oxygen ion transport, sustained stability, and resistance to temperature fluctuations. As a result, ZrO2 is finding increasingly valuable applications as a drug carrier, drug delivery system, and medium for drug targeting, expanding its utility beyond its role as a substitute material for hard tissues in the human body. The production of zirconia nanoparticles involves various techniques, including sol / gel methods, hydrothermal synthesis, hydrolysis, and microwave plasma processes. Hydroxyapatite (HAP) has emerged as a major focus of research as a means to deliver nanomedicines targeting bone regeneration and bone-related diseases. However, HAP has certain limitations, particularly its relatively weak mechanical strength. ZrO2 is biocompatible and strong enough to replace the body's hard tissues.

[0005] Recently, research has been conducted on HAP-based nanocomposites with improved mechanical and biological properties. Nevertheless, there has been a research gap in addressing the issue of improving nanoparticle aggregation and stability while mitigating the low hardness and fracture toughness associated with HAP when used as carriers for drug delivery systems, and there is a growing need to enhance the biocompatibility of nanoparticles. Prior art literature

[0006] Korean Registered Patent Publication No. 10-1345256 (Published Dec. 27, 2013), "Method for manufacturing zinc oxide nanostructures" The problem to be solved

[0007] To solve the above-mentioned problems, the present invention aims to provide hydroxyapatite zirconium oxide nanoparticles synthesized and pegylated to enable biological applications, as well as a method for manufacturing the same. means of solving the problem

[0008] To achieve the above objectives, the present invention comprises: a step (S10) for manufacturing hydroxyapatite nanoparticles (HAP) by heating bovine bone with purified water, treating it with acid and base, and heating it; a grinding synthesis method in which the 'hydroxyapatite nanoparticles (HAP)' from the hydroxyapatite nanoparticle manufacturing step (S10) and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating them' are mixed together, ground, and calcined; or a sol-gel synthesis method in which a 'zirconium chloride solution' is added to a solution in which the 'hydroxyapatite nanoparticles (HAP)' from the hydroxyapatite nanoparticle manufacturing step (S10) are dissolved, sodium hydroxide is added, and heating is performed. The present invention provides a method for manufacturing pegylated hydroxyapatite zirconium oxide nanoparticles, characterized by comprising: a step (S20) of manufacturing zirconium oxide nanoparticles (ZrO2NPs) through one or more of the above methods; and a step (S30) of manufacturing pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NPs) by adding PEG to the hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by the grinding synthesis method or the sol-gel synthesis method and ultrasonically treating them to produce pegylated hydroxyapatite zirconium oxide nanoparticles.

[0009] In addition, the hydroxyapatite nanoparticles (HAP) in the above hydroxyapatite nanoparticle manufacturing step (S10) are characterized by being prepared by heating a cow bone (BB) with purified water at a temperature of 80 to 90°C, immersing it in 1N HCl for 20 to 28 hours, neutralizing it by immersing it in purified water, immersing it in 1M NaOH for 20 to 28 hours, drying it at room temperature, and heating it at a calcination temperature of 5 to 700°C for 4 to 6 hours in a furnace set at a heating rate of 5°C / min.

[0010] In addition, the zirconium oxide nanoparticles (ZrO2NPs) in the hydroxyapatite zirconium oxide nanoparticle manufacturing step (S20) are characterized as being pulverized synthetic zirconium oxide nanoparticles (HZG) manufactured by mixing the 'hydroxyapatite nanoparticles (HAP)' from the hydroxyapatite nanoparticle manufacturing step (S10) and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating' in equal weight ratios, grinding them, and calcining them at a temperature of 500 to 700°C for 2 to 4 hours.

[0011] In addition, the zirconium oxide nanoparticles (ZrO2NPs) in the hydroxyapatite zirconium oxide nanoparticle manufacturing step (S20) are characterized as sol-gel synthesized zirconium oxide nanoparticles (HZS) manufactured by a sol-gel synthesis method in which a 'zirconium chloride solution mixed with zirconium chloride in purified water in a volume ratio of 1000:100' is added to a 'solution in which hydroxyapatite nanoparticles (HAP) in purified water are dissolved in a volume ratio of 500:100' in the hydroxyapatite nanoparticle manufacturing step (S10), a 1M sodium hydroxide solution is added to adjust the pH to 7 to 9, and the mixture is heated at 70 to 90°C for 30 to 90 minutes.

[0012] In addition, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) in the pegylated hydroxyapatite zirconium oxide nanoparticle preparation step (S30) are characterized as pegylated pulverized hydroxyapatite zirconium oxide nanoparticles (PHZG) prepared by adding 'PEG-3350' to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by a pulverized synthesis method dispersed in purified water at a volume ratio of 100:50' in a volume ratio of 150:500, ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes.

[0013] In addition, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) in the pegylated hydroxyapatite zirconium oxide nanoparticle preparation step (S30) are characterized as pegylated sol-gel synthesized hydroxyapatite zirconium oxide nanoparticles (PHZS) prepared by adding 'PEG-3350' to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by sol-gel synthesis in purified water at a volume ratio of 100:50' in a volume ratio of 150:500, ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes.

[0014] In addition, the present invention provides pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) characterized by being obtained by adding PEG and ultrasonically treating zirconium oxide nanoparticles (ZrO2NPs) prepared through one or more of the following methods: a grinding synthesis method in which hydroxyapatite nanoparticles (HAP) prepared by heating bovine bone with purified water, treating with acid and base, and heating, and zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating; or a sol-gel synthesis method in which hydroxyapatite nanoparticles (HAP) prepared by heating bovine bone with purified water, treating with acid and base, and heating are mixed together, ground, and calcined; or a sol-gel synthesis method in which zirconium chloride solution and sodium hydroxide are added to a solution in which hydroxyapatite nanoparticles (HAP) prepared by heating bovine bone with purified water, treating with acid and base, and heating are dissolved and heated.

[0015] In addition, the above hydroxyapatite nanoparticles (HAP) are characterized by being prepared by heating bovine bone (BB) with purified water at a temperature of 80 to 90°C, immersing it in 1N HCl for 20 to 28 hours, neutralizing it by immersing it in purified water, immersing it in 1M NaOH for 20 to 28 hours, drying it at room temperature, and heating it in a furnace set at a heating rate of 5°C / min at a calcination temperature of 5 to 700°C for 4 to 6 hours.

[0016] In addition, the method for preparing pegylated hydroxyapatite zirconium oxide nanoparticles is characterized in that the zirconium oxide nanoparticles (ZrO2NPs) are prepared by a grinding synthesis method in which the 'hydroxyapatite nanoparticles (HAP)' and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating' are mixed in equal weight ratios, ground, and calcined at a temperature of 500 to 700°C for 2 to 4 hours.

[0017] In addition, the zirconium oxide nanoparticles (ZrO2NPs) are characterized as sol-gel synthesized zirconium oxide nanoparticles (HZS) prepared by a sol-gel synthesis method in which a 'zirconium chloride solution mixed with zirconium chloride in purified water at a volume ratio of 1000:100' is added to a 'solution in which hydroxyapatite nanoparticles (HAP) are dissolved in purified water at a volume ratio of 500:100', a 1M sodium hydroxide solution is added to adjust the pH to 7 to 9, and the mixture is heated at 70 to 90°C for 30 to 90 minutes.

[0018] In addition, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) are characterized as pegylated ground-synthesized hydroxyapatite zirconium oxide nanoparticles (PHZG) prepared by adding 'PEG-3350' to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by a ground-synthesized method dispersed in purified water at a volume ratio of 100:50' in a volume ratio of 150:500, ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes.

[0019] In addition, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) are characterized as pegylated sol-gel synthesized hydroxyapatite zirconium oxide nanoparticles (PHZS) prepared by adding 'PEG-3350' to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by sol-gel synthesis in purified water at a volume ratio of 100:50' in a volume ratio of 150:500, ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes. Effects of the invention

[0020] The pegylated hydroxyapatite zirconium oxide nanoparticles according to the manufacturing method of the present invention synthesize hydroxyapatite zirconium oxide nanoparticles and pegylate them to enable biological applications. Brief explanation of the drawing

[0021] FIG. 1 is an analysis image regarding the crystal structures of PHZG; PHZS;, HZG; HZS; ZrO2NP; and HAP according to an embodiment and a comparative example of the present invention. FIG. 2 is an FTIR analysis image of PHZG; PHZS;, HZG; HZS; ZrO2NP; HAP; according to an embodiment and a comparative example of the present invention. FIG. 3 is an image regarding particle size distribution (PDI) and zeta potential analysis of HZS NPs (a and b); HZG NPs (c and d); PHZS NPs (e and f) and PHZG NPs (g and h) according to embodiments and comparative examples of the present invention. FIG. 4 shows TEM images (a) of PHZS (A) and PHZG (B) according to an embodiment and a comparative example of the present invention, elemental mapping images for zirconium (b), calcium (c), oxygen (d), and phosphorus (e), and an elemental spectrum graph (f). FIG. 5 is an image relating to the thermogravimetric analysis (TGA) of PHZG according to an embodiment and a comparative example of the present invention. FIG. 6 is an image relating to a biocompatibility test (A); a fluorescence (a: AO / EB, b: RH123 c: PI) image analysis test (B); and a hemolysis analysis test (B) for PHZG; PHZS;, HZG; and HZS according to an embodiment and a comparative example of the present invention. Specific details for implementing the invention

[0022] The following detailed descriptions relating to the present invention refer to the accompanying drawings, which are embodiments in which the present invention may be practiced and are illustrated as examples of such embodiments. These embodiments are described in detail to sufficiently enable those skilled in the art to practice the present invention. It should be understood that various embodiments of the present invention are different but need not be mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be implemented in different embodiments without departing from the spirit and scope of the present invention in relation to one embodiment. Furthermore, it should be understood that the location or arrangement of individual components within each described embodiment may be changed without departing from the spirit and scope of the present invention.

[0023] Accordingly, the following detailed description is not intended to be taken in a limiting sense, and the scope of the invention is limited only by the appended claims, including all equivalents to those claimed therein, provided they are properly described. Similar reference numerals in the drawings refer to the same or similar functions across various aspects.

[0024] The terms used in this invention have been selected based on currently widely used general terms, taking into account their functions within the invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0025] In the present invention, when a part is described as “comprising” a certain component, this means that, unless specifically stated otherwise, it does not exclude different components but may include additional different components.

[0027] Hereinafter, pegylated hydroxyapatite zirconium oxide nanoparticles according to the present invention and a method for manufacturing the same will be described in detail.

[0029] A method for manufacturing pegylated hydroxyapatite zirconium oxide nanoparticles according to the present invention comprises: a hydroxyapatite nanoparticle manufacturing step (S10) of manufacturing hydroxyapatite nanoparticles (HAP) by heating bovine bone with purified water, treating it with acid and base, and heating it; a grinding synthesis method of mixing the 'hydroxyapatite nanoparticles (HAP)' from the hydroxyapatite nanoparticle manufacturing step (S10) with 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating them', grinding them together, and calcining them; or a sol-gel synthesis method of manufacturing by adding a 'zirconium chloride solution' and sodium hydroxide to a solution in which the 'hydroxyapatite nanoparticles (HAP)' from the hydroxyapatite nanoparticle manufacturing step (S10) are dissolved, and heating it. The method comprises: a step (S20) for manufacturing zirconium oxide nanoparticles (ZrO2NPs) through one or more of the following methods; and a step (S30) for manufacturing pegylated zirconium oxide nanoparticles (HAP-ZrO2NPs) by adding PEG to the hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by the grinding synthesis method or the sol-gel synthesis method and ultrasonically treating them.

[0031] In the above hydroxyapatite nanoparticle manufacturing step (S10), a process is performed to manufacture hydroxyapatite nanoparticles (HAP) by heating cow bone with purified water, treating it with acid and base, and heating it.

[0032] The hydroxyapatite nanoparticles (HAP) in the above hydroxyapatite nanoparticle manufacturing step (S10) may be prepared by heating bovine bone (BB) with purified water at a temperature of 80 to 90°C, immersing it in 1N HCl for 20 to 28 hours, neutralizing it by immersing it in purified water, immersing it in 1M NaOH for 20 to 28 hours, drying it at room temperature, and heating it in a furnace set at a heating rate of 5°C / min to a calcination temperature of 5 to 700°C for 4 to 6 hours.

[0033] As a more specific example, the hydroxyapatite nanoparticles (HAP) in the above hydroxyapatite nanoparticle manufacturing step (S10) may be prepared by heating a cow bone (BB) with purified water at a temperature of 80 to 90°C, immersing it in 1N HCl for 20 to 28 hours, neutralizing it by immersing it in purified water, immersing it in 1M NaOH for 20 to 28 hours, drying it at room temperature, and heating it at a calcination temperature of 5 to 700°C for 4 to 6 hours in a furnace set at a heating rate of 5°C / min.

[0035] In the above hydroxyapatite zirconium oxide nanoparticle manufacturing step (S20), a process of manufacturing zirconium oxide nanoparticles (ZrO2NPs) is performed through one or more of the following methods: a grinding synthesis method in which the 'hydroxyapatite nanoparticles (HAP)' from the above hydroxyapatite nanoparticle manufacturing step (S10) and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating' are mixed together, ground, and calcined; or a sol-gel synthesis method in which a 'zirconium chloride solution' is added to a solution in which the 'hydroxyapatite nanoparticles (HAP)' from the above hydroxyapatite nanoparticle manufacturing step (S10) are dissolved, sodium hydroxide is added, and heated.

[0036] The zirconium oxide nanoparticles (ZrO2NPs) in the hydroxyapatite zirconium oxide nanoparticle manufacturing step (S20) above may be pulverized synthetic zirconium oxide nanoparticles (HZG) manufactured by mixing the 'hydroxyapatite nanoparticles (HAP)' of the hydroxyapatite nanoparticle manufacturing step (S10) and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating' in equal weight ratios, grinding them, and calcining them at a temperature of 500 to 700°C for 2 to 4 hours.

[0037] As a more specific example, the zirconium oxide nanoparticles (ZrO2NPs) in the hydroxyapatite zirconium oxide nanoparticle manufacturing step (S20) may be pulverized synthetic zirconium oxide nanoparticles (HZG) manufactured by mixing the 'hydroxyapatite nanoparticles (HAP)' of the hydroxyapatite nanoparticle manufacturing step (S10) and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating' in equal weight ratios, grinding them, and calcining them at a temperature of 600°C for 3 hours.

[0038] The zirconium oxide nanoparticles (ZrO2NPs) in the above hydroxyapatite zirconium oxide nanoparticle manufacturing step (S20) may be sol-gel synthesized zirconium oxide nanoparticles (HZS) prepared by a sol-gel synthesis method in which a 'zirconium chloride solution mixed with zirconium chloride in purified water in a volume ratio of 1000:100' is added to a 'solution in which hydroxyapatite nanoparticles (HAP) in purified water are dissolved in a volume ratio of 500:100' in the above hydroxyapatite nanoparticle manufacturing step (S10), a 1M sodium hydroxide solution is added to adjust the pH to 7 to 9, and the mixture is heated at 70 to 90°C for 30 to 90 minutes.

[0039] As a more specific example, the zirconium oxide nanoparticles (ZrO2NPs) in the hydroxyapatite zirconium oxide nanoparticle manufacturing step (S20) may be sol-gel synthesized zirconium oxide nanoparticles (HZS) manufactured by a sol-gel synthesis method in which 1000 mL of zirconium chloride mixed with 100 mL of purified water is added to the 'solution in which 500 mL of hydroxyapatite nanoparticles (HAP) are dissolved in 100 mL of purified water' of the hydroxyapatite nanoparticle manufacturing step (S10), 1 M sodium hydroxide solution is added to adjust the pH to 8, and the mixture is heated at 80°C for 60 minutes.

[0041] In the step (S30) for manufacturing pegylated hydroxyapatite zirconium oxide nanoparticles, a process is performed to produce pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) by adding PEG to hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by either the grinding synthesis method or the sol-gel synthesis method and ultrasonically treating them.

[0042] At this time, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) in the pegylated hydroxyapatite zirconium oxide nanoparticle manufacturing step (S30) may be pegylated ground-synthesized hydroxyapatite zirconium oxide nanoparticles (PHZG) prepared by adding 'PEG-3350' to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by a ground-synthesized method dispersed in purified water at a volume ratio of 100:50' in a volume ratio of 150:500, ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes.

[0043] As a more specific example, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) in the pegylated hydroxyapatite zirconium oxide nanoparticle preparation step (S30) may be pegylated ground-synthesized hydroxyapatite zirconium oxide nanoparticles (PHZG) prepared by adding 500mL of 'PEG-3350' to 150mL of '100mL of hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by a ground-synthesized method dispersed in 50mL of purified water', ultrasonically treating for 5 minutes, and centrifuging at a speed of 12,000rpm for 20 minutes.

[0044] Alternatively, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) in the pegylated hydroxyapatite zirconium oxide nanoparticle preparation step (S30) may be pegylated sol-gel synthesized hydroxyapatite zirconium oxide nanoparticles (PHZS) prepared by adding 'PEG-3350' in a volume ratio of 150:500 to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by sol-gel synthesis dispersed in purified water in a volume ratio of 100:50', ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes.

[0045] As a more specific example, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) in the pegylated hydroxyapatite zirconium oxide nanoparticle preparation step (S30) may be pegylated sol-gel synthesized hydroxyapatite zirconium oxide nanoparticles (PHZS) prepared by adding 500 mL of 'PEG-3350' to 150 mL of '100 mL of hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by sol-gel synthesis dispersed in 50 mL of purified water', ultrasonically treating for 5 minutes, and centrifuging at a speed of 12,000 rpm for 20 minutes.

[0047] In addition, the present invention provides pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) characterized by being obtained by adding PEG and ultrasonically treating zirconium oxide nanoparticles (ZrO2NPs) prepared through one or more of the following methods: a grinding synthesis method in which hydroxyapatite nanoparticles (HAP) prepared by heating bovine bone with purified water, treating with acid and base, and heating, and zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating; or a sol-gel synthesis method in which hydroxyapatite nanoparticles (HAP) prepared by heating bovine bone with purified water, treating with acid and base, and heating are mixed together, ground, and calcined; or a sol-gel synthesis method in which zirconium chloride solution and sodium hydroxide are added to a solution in which hydroxyapatite nanoparticles (HAP) prepared by heating bovine bone with purified water, treating with acid and base, and heating are dissolved and heated.

[0049] The above hydroxyapatite nanoparticles (HAP) are preferably prepared by heating bovine bone (BB) with purified water at a temperature of 80 to 90°C, immersing it in 1N HCl for 20 to 28 hours, neutralizing it by immersing it in purified water, immersing it in 1M NaOH for 20 to 28 hours, drying it at room temperature, and heating it at a calcination temperature of 5 to 700°C for 4 to 6 hours in a furnace set at a heating rate of 5°C / min.

[0050] The above zirconium oxide nanoparticles (ZrO2NPs) may be ground-synthesized zirconium oxide nanoparticles (HZG) prepared by a grinding synthesis method in which the above 'hydroxyapatite nanoparticles (HAP)' and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating' are mixed in equal weight ratios, ground, and calcined at a temperature of 500 to 700°C for 2 to 4 hours.

[0051] The above zirconium oxide nanoparticles (ZrO2NPs) may be sol-gel synthesized zirconium oxide nanoparticles (HZS) prepared by a sol-gel synthesis method in which a 'zirconium chloride solution mixed with zirconium chloride in purified water in a volume ratio of 1000:100' is added to a 'solution in which hydroxyapatite nanoparticles (HAP) are dissolved in purified water in a volume ratio of 500:100', a 1M sodium hydroxide solution is added to adjust the pH to 7 to 9, and the mixture is heated at 70 to 90°C for 30 to 90 minutes.

[0052] The pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) above may be pegylated ground-synthesized hydroxyapatite zirconium oxide nanoparticles (PHZG) prepared by adding ‘PEG-3350’ to ‘hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by a ground-synthesized method dispersed in purified water at a volume ratio of 100:50’ in a volume ratio of 150:500, ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes.

[0053] The pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) above may be pegylated sol-gel synthesized hydroxyapatite zirconium oxide nanoparticles (PHZS) prepared by adding 'PEG-3350' to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by sol-gel synthesis in purified water at a volume ratio of 100:50' in a volume ratio of 150:500, ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes.

[0055] Hereinafter, the effects of the pegylated hydroxyapatite zirconium oxide nanoparticles according to the present invention will be examined in detail through the following examples, comparative examples, and experimental examples.

[0057] 1. Materials and Methods

[0058] 1.1. Materials

[0059] Zirconium chloride, sodium hydroxide (NaOH), and polyethylene glycol (PEG-3350) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The water-soluble tetrazolium salt WST-8 Cellomax™ cell viability assay kit was purchased from MediFab, South Korea. Acridine orange (AO), ethidium bromide (EB), rhodamine 123 (Rh123), and propidium iodide (PI) were purchased from Sigma-Aldrich, South Korea. Hydrochloric acid (HCl) was purchased from Daejeong Chemical & Metal Co., Ltd., located in Siheung, South Korea. All cell culture media were purchased from Thermo Fisher Scientific, Waltham, Massachusetts, USA.

[0061] 1.2. pegylated Grinding synthesis hydroxyapatite Zirconium oxide nanoparticles ( PHZG Manufacturing of )

[0062] Pegylated pulverized synthetic hydroxyapatite zirconium oxide nanoparticles (PHZG) were prepared as an example according to the following process.

[0063] Step for preparing hydroxyapatite nanoparticles (S10): Bovine bone (BB) was heated with purified water at a temperature of 85°C, immersed in 1N HCl for 24 hours, neutralized by immersion in purified water, immersed in 1M NaOH for 24 hours, dried at room temperature, and heated at a calcination temperature of 600°C for 5 hours in a furnace set at a heating rate of 5°C / min to prepare hydroxyapatite nanoparticles (HAP).

[0064] Hydroxyapatite zirconium oxide nanoparticle preparation step (S20): Zirconium oxide nanoparticles (ZrO2NPs) were prepared by mixing the 'hydroxyapatite nanoparticles (HAP)' from the hydroxyapatite nanoparticle preparation step (S10) and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding 1M sodium hydroxide to a solution of 200mL of zirconium chloride mixed with 100mL of purified water and heating at 80℃ for 60 minutes' in equal weight ratios, grinding them, and calcining them at a temperature of 600℃ for 3 hours to produce ground-synthesized zirconium oxide nanoparticles (HZG).

[0065] Step for preparing pegylated hydroxyapatite zirconium oxide nanoparticles (S30): 150 mL of '100 mL of hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by grinding synthesis dispersed in 50 mL of purified water' was mixed with 500 mL of 'PEG-3350', sonicated for 5 minutes, and centrifuged at a speed of 12,000 rpm for 20 minutes to prepare pegylated ground-synthesized hydroxyapatite zirconium oxide nanoparticles (PHZG).

[0067] 1.3. pegylated Sol-gel synthesis hydroxyapatite Zirconium oxide nanoparticles ( PHZS Manufacturing of )

[0068] Pegylated sol-gel synthesized hydroxyapatite zirconium oxide nanoparticles (PHZS) were prepared as an example according to the following process.

[0069] Step for preparing hydroxyapatite nanoparticles (S10): Bovine bone (BB) was heated with purified water at a temperature of 85°C, immersed in 1N HCl for 24 hours, neutralized by immersion in purified water, immersed in 1M NaOH for 24 hours, dried at room temperature, and heated at a calcination temperature of 600°C for 5 hours in a furnace set at a heating rate of 5°C / min to prepare hydroxyapatite nanoparticles (HAP).

[0070] Step for preparing hydroxyapatite zirconium oxide nanoparticles (S20): To the 'solution in which 500 mL of hydroxyapatite nanoparticles (HAP) from the step for preparing hydroxyapatite nanoparticles (S10) is dissolved in 100 mL of purified water,' a 'zirconium chloride solution in which 1000 mL of zirconium chloride is mixed with 100 mL of purified water' is added, a 1 M sodium hydroxide solution is added to adjust the pH to 8, and the mixture is heated at 80°C for 60 minutes to prepare sol-gel synthesized zirconium oxide nanoparticles (HZS).

[0071] Step (S30) for preparing pegylated hydroxyapatite zirconium oxide nanoparticles: 150 mL of '100 mL of hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by sol-gel synthesis dispersed in 50 mL of purified water' was mixed with 500 mL of 'PEG-3350', sonicated for 5 minutes, and centrifuged at a speed of 12,000 rpm for 20 minutes to prepare pegylated sol-gel synthesized hydroxyapatite zirconium oxide nanoparticles (PHZS).

[0073] 1.4. Grinding-synthesized zirconium oxide nanoparticles ( HZG Manufacturing of )

[0074] Grinding-synthesized zirconium oxide nanoparticles (HZG) were prepared as a comparative example according to the following process.

[0075] Step for preparing hydroxyapatite nanoparticles (S10): Bovine bone (BB) was heated with purified water at a temperature of 85°C, immersed in 1N HCl for 24 hours, neutralized by immersion in purified water, immersed in 1M NaOH for 24 hours, dried at room temperature, and heated at a calcination temperature of 600°C for 5 hours in a furnace set at a heating rate of 5°C / min to prepare hydroxyapatite nanoparticles (HAP).

[0076] Hydroxyapatite zirconium oxide nanoparticle preparation step (S20): Zirconium oxide nanoparticles (ZrO2NPs) were prepared by mixing the 'hydroxyapatite nanoparticles (HAP)' from the hydroxyapatite nanoparticle preparation step (S10) and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding 1M sodium hydroxide to a solution of 200mL of zirconium chloride mixed with 100mL of purified water and heating at 80℃ for 60 minutes' in equal weight ratios, grinding them, and calcining them at a temperature of 600℃ for 3 hours to produce ground-synthesized zirconium oxide nanoparticles (HZG).

[0078] 1.5. Sol-gel synthesis Zirconium oxide nanoparticles ( HZS Manufacturing of )

[0079] Zirconium oxide nanoparticles (HZS) were prepared as a comparative example through a sol-gel synthesis process according to the following process.

[0080] Step for preparing hydroxyapatite nanoparticles (S10): Bovine bone (BB) was heated with purified water at a temperature of 85°C, immersed in 1N HCl for 24 hours, neutralized by immersion in purified water, immersed in 1M NaOH for 24 hours, dried at room temperature, and heated at a calcination temperature of 600°C for 5 hours in a furnace set at a heating rate of 5°C / min to prepare hydroxyapatite nanoparticles (HAP).

[0081] Step for preparing hydroxyapatite zirconium oxide nanoparticles (S20): To the 'solution in which 500 mL of hydroxyapatite nanoparticles (HAP) from the step for preparing hydroxyapatite nanoparticles (S10) is dissolved in 100 mL of purified water,' a 'zirconium chloride solution in which 1000 mL of zirconium chloride is mixed with 100 mL of purified water' is added, a 1 M sodium hydroxide solution is added to adjust the pH to 8, and the mixture is heated at 80°C for 60 minutes to prepare sol-gel synthesized zirconium oxide nanoparticles (HZS).

[0083] 1.6. Zinc oxide nanoparticles ( ZrO 2 Manufacturing of )

[0084] Zinc oxide nanoparticles (ZrO2) were prepared as a comparative example according to the following process.

[0085] Zirconium oxide nanoparticle (ZrO2NPs) preparation step (S10): Zirconium oxide nanoparticles (ZrO2NPs) were prepared by adding 1M sodium hydroxide to a solution of 200mL of zirconium chloride mixed with 100mL of purified water and heating at 80℃ for 60 minutes.

[0087] 1.5. hydroxyapatite nanoparticles HAP Manufacturing of )

[0088] Hydroxyapatite nanoparticles (HAP) as a comparative example were prepared according to the following process.

[0089] Step for preparing hydroxyapatite nanoparticles (S10): Bovine bone (BB) was heated with purified water at a temperature of 85°C, immersed in 1N HCl for 24 hours, neutralized by immersion in purified water, immersed in 1M NaOH for 24 hours, dried at room temperature, and heated at a calcination temperature of 600°C for 5 hours in a furnace set at a heating rate of 5°C / min to prepare hydroxyapatite nanoparticles (HAP).

[0091] 2. Experimental Method

[0092] 2.1. ZrO 2 , HZS , HZG , PHZS and PHZG's Characteristic analysis

[0093] To determine the particle size, dispersion, and ζ potential of ZrO2, HZS, HZG, PHZS, and PHZG NPs, they were placed in distilled water (1 mg / mL) and sonicated for 3 minutes. Subsequently, a membrane filter (0.45 μm) was used to remove aggregates, and analysis was performed using Dynamic Light Scattering (DLS) and Electrolytic Light Scattering (ELS) analysis (Malvern Panalytical Netherlands). The crystallographic characteristics of NPs and NCs were measured using an X-ray diffractometer (XRD, X'Pert PRO MPD, Almelo, Netherlands) with a scan range of 5°–80°. Fourier Transform Infrared Spectroscopy (FTIR, PerkinElmer Paragon 500 USA) was performed with a scan range of 500–4000 cm⁻¹. -1 Functional groups were identified. Detailed shapes and sizes of NPs and NCs were determined using a Field Emission Transmission Electron Microscope (FE-TEM) (JEOL-JSM 1200EX Japan). Additionally, the elemental composition of the nanoparticles was detected via X-energy dispersion analysis (EDS). The thermal stability of PHZG NPs was determined using a thermogravimetric analyzer (TGA, TA Instruments SDT Q600). Measurements were taken under analysis conditions set from 0°C to a maximum temperature of 1000°C with a heating rate of 10°C.

[0095] 2.2. In Vitro Cell Culture Analysis

[0096] Human embryonic kidney 293 cells (HEK293) were obtained from the Korean Culture Collection of the Republic of Korea. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) – high-glucose T-25 flasks supplemented with 10% FBS (fetal bovine serum) and 1% antibiotics (streptomycin and penicillin). Additionally, cells were maintained in a humidified 5% CO2 incubator at 37±1℃.

[0098] 2.3. Cell Viability Analysis

[0099] The cytotoxicity of HZS NP, HZG NP, PHZS NP, and PHZG NP was analyzed using the HEK-293 cell line. Briefly, HEK-293 cells at a density of 1.9 x 10⁶ 5 Flat-bottom 96-well plates containing 100 μL of DMEM were seeded and maintained in a 37°C CO2 (5%) incubator. Subsequently, 10 μL of different concentrations of HZS NP and HZG NP (3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, and 1000 μg / mL) were added to each well, and the plates were incubated in a CO2 incubator for 6 hours. After treatment, 10 μL of cell viability assay solution (WST; water-soluble tetrazolium salt) was added to each well, and the plates were incubated for 1 hour. After incubation, the 96-well plates were measured at 450 nm using a UV-vis spectrophotometer (SpectraMax®Plus 384 Microplate Reader from Molecular Devices), and cell viability (%) was compared to the control group. In addition, apoptosis (acridine orange; AO / ethidium bromide; EB), nuclear damage (propidium iodide; PI), and loss of mitochondrial membrane potential (rhodamine 123; Rh123) were measured using a fluorescence microscope. Briefly, HEK-293 cells were cultured in 24-well plates and then cultured for 12 hours under the same conditions. Subsequently, 50 μL of HZS NP, HZG NP, PHZS NP, and PHZG NP at a stock concentration of 250 μg / mL were added, and after incubation at 37 ± 1 °C for 6 hours, the cells were stained and observed using a fluorescence microscope.

[0101] 2.4. Hemolysis Analysis

[0102] The hematotoxicity of HZS NP, HZG NP, PHZS NP, and PHZG NP was confirmed in an in vitro hemolysis assay. Briefly, 1 mL of defibrotic sheep blood (Carlina, Korea) was mixed with 10 mL of PBS. Subsequently, red blood cells (RBCs) were separated by centrifugation at 2000 rpm for 10 minutes at 4°C and washed three times with phosphate-buffered saline (PBS) solution. The obtained RBCs were suspended in PBS (10 mL). For analysis, 200 μL of 4% RBCs were incubated with 200 μL of HZS NP, HZG NP, PHZS NP, and PHZG NP at 37°C for 60 minutes. Triton X-100 (1%) and PBS were used as positive and negative controls for hemolysis, respectively. Afterward, the cultured RBCs were centrifuged at 2000 rpm for 10 minutes, and the supernatant was measured at 540 nm using a UV-vis spectrophotometer (SpectraMax®Plus 384 Microplate Reader, Molecular Devices).

[0104] 2.5. Statistical Analysis

[0105] Statistical differences were determined by one-way analysis of variance (ANOVA). All experiments were performed three times, and results were presented as means with standard deviations (SD). A p-value < 0.05 is considered statistically significant.

[0107] 3. Results and Discussion

[0108] 3.1. X-ray Diffraction Analysis

[0109] The crystallographic characteristics of HAP NP, HZS NP, HZG NP, and ZrO2NP were determined by XRD spectra (Fig. 1). Comparative analysis of crystal spectra confirmed similarities and differences between the samples. Specifically, ZrO2NPs exhibited diffraction peaks (2θ) at 30.22°, 35.08°, 50.41°, and 60.04° corresponding to crystal planes (101), (110), (200), and (211), respectively. JCPDS card number 49-1642. HAP NPs exhibited peaks at 25.84°, 31.78°, 32.14°, 32.96°, 39.81°, and 47.01° corresponding to crystal planes (002), (211), (112), (300), (310), and (222), respectively (Fig. 1). The crystal plane of HAP is similar to JCPDS card number 09-0432. Additionally, HZS NPs exhibited diffraction peaks (2θ) at 19.25°, 20.01°, 23.42°, 30.04°, 31.13°, 34.86°, 50.32°, and 60.1°. Similarly, HZG NPs exhibited diffraction peaks (2θ) at 25.61°, 30.02°, 31.52°, 32.71°, 50.16°, and 59.84° (Fig. 1). XRD confirmed that HZS NPs and HZG NPs exhibit distinct peaks at 2θ angles (°) identical to the diffraction angles observed in ZrO2. Furthermore, according to JCPDS no. 09-0432, both HZS NPs and HZG NPs exhibited peaks corresponding to HAP around 31.52° and 34.86°. This result confirmed that both samples contain ZrO2 and HAP. However, HZG NPs exhibit a relatively sharper peak than HZS NPs. Additionally, the crystallinity of the nanocomposite particles is higher than that of ZrO2 and HAP due to the dense network structure of PEG polymer chains. Moreover, a small crystallization peak is present in the XRD spectrum due to the calcium phosphate auxiliary signal.Previous studies have confirmed methods to increase the crystallinity of nanoparticles using PEG for targeted drug delivery and various biomedical applications. Accordingly, PEG has played a role in enhancing the physical properties of nanoparticles by increasing the crystallinity of HZS NPs and HZG NPs.

[0111] 3.2. FTIR Spectroscopy

[0112] The functional characteristics of HAP, HZS NP, HZG NP, and ZrO2NP are 400–4000 cm⁻¹ -1 It was studied in FTIR analysis using the scan range (Fig. 2). The FTIR spectrum of HAP NPs is 1456 cm⁻¹. -1 , 1411cm -1 and 873cm -1 It shows a small peak. This corresponds to the asymmetric extension of CO3 and the presence of carbonate groups in HAP. 1018 cm⁻¹ -1 , 599 cm -1 The sharp peaks at 562 and 562 are the phosphate group PO4 of hydroxyapatite 3- It is assigned to the symmetry extension of. The FTIR spectrum of HZS NPs is at 549 cm⁻¹, corresponding to the phosphate group of HAP. -1 and 1018cm -1 It showed a sharp peak at (Fig. 2a). 549-800 cm -1 The peaks in between correspond to the vibrations of the Zr-O bond. The FTIR spectra of HZG NPs are at 562, 598, 628, 962, 1026, 1087, and 1455 cm⁻¹. -1 A peak was observed at 562–800 cm⁻¹. -1 The sharp peak in between is attributed to the vibration of the Zr-O bond, at 1026 cm⁻¹ -1 The sharp peak is the stretching PO4 corresponding to the phosphate group of HAP. 3- It is attributed to. Additionally, in the FTIR spectrum of ZrO2NPs, 1373 cm⁻¹, corresponding to CH bending and Zr-O bonds, respectively... -1500-750cm -1 It was confirmed that a sharp peak exists at [location]. The spectral peaks of PHZS are at 542, 843, 962, 1030, 1279, 1342, 1466, and 2885 cm⁻¹. -1 It appeared at . Similarly, the FTIR spectra of PHZG were 562, 598, 628, 962, 1030, 1088, and 1456 cm⁻¹. -1 It appeared in (Fig. 2b). Upon closer examination, both PHZS and PHZG showed the same peaks at 840 (CH2 rocking), 960 (CH2 rocking, CH2 twisting), 1097 (CO, CC stretching), and 1466 (CH2 scissoring), which also correspond to the characteristic peaks of PEG.

[0114] 3.3. Particle Size and Zeta Potential Analysis

[0115] Zeta potential, representing the surface charge of nanoparticles, serves as an important parameter for evaluating nanoparticle stability. Particle size plays a crucial role in the initial stages of nanoparticle interaction with cell membranes. Furthermore, since the zeta potential and size of nanoparticles can influence toxicity, they allow for the assessment of potential risks and utility ranges. The surface charge, average particle size, and polydispersity index (PDI) of HZS, HZG, PHZS, and PHZG NPs were determined through dynamic light scattering (DLS) and electrophoretic light scattering (ELS) analyses (Fig. 3). For HZS NPs, the average nanoparticle size was 149.8 nm, the PDI was 0.086, and the zeta potential was -29.7 mV. Similarly, for HZS NPs, the average particle size was 122.8 nm, the PDI was 0.167, and the zeta potential was -25.7 mV (Fig. 3). Compared to HZG NPs, the particle size was smaller than that of HZS NPs, and the absolute value of the zeta potential was also lower. This was an important variable indicating stability and range of application when utilizing nanoparticles in biological applications. Furthermore, the average nanoparticle size of PEG-HZS was 130.3 nm, the PDI value was 0.122, and the zeta potential was -39.1 mV (Fig. 3). Similarly, the average nanoparticle size of PEG-HZG was 99.42 nm, the PDI value was 0.147, and the zeta potential was -27.9 mV. PEGylation was observed to improve the absolute value of the zeta potential and reduce the average particle size in both NPs. This indicates that PEGylation not only enhanced the colloidal stability of the nanoparticles but also demonstrated low toxicity and potential for use as a nano-carrier.

[0117] 3.4. TEM analyze

[0118] The morphological characteristics of PHZS NPs and PHZG NPs were observed using TEM (Fig. 4). HZS NPs exhibit two types of particles: relatively large spherical particles and small, dark black particles. Similarly, PHZG NPs displayed small, dense spherical NPs coated on large spherical NPs with smooth surfaces and a size of approximately 200 nm (Fig. 4). Thus, previous studies demonstrated that ZrO2 has a smooth surface polygon with an average diameter of 40 nm. HAPs are smooth, angular spherical shapes that are common to both PHZS NPs and HZG NPs. In dynamic light scattering (DLS) measurements, the average particle size was smaller in HZG NPs (122.8 d. nm) than in HZS NPs (149.8 d. nm) (Fig. 4). As a factor, HZS NPs have lower cohesion between ZrO2 and HZG NPs, unlike HZG NPs where ZrO2 is evenly attached to the spherical surface of the HAP. Elemental mapping and spectra confirmed the presence of Zr, Ca, O, and P in each nanoparticle (Fig. 4). Elemental mapping results confirmed that in both samples where the large particles were identified as HAP, Zr was present in relatively small, dense NPs on the surface. Additionally, the presence of Ca, O, and P was observed in the large particle regions associated with HAP. Furthermore, the morphology and structure of nanoparticles are considered critically important for their utilization as nanocarriers, i.e., therapeutic delivery systems. Drug loading capacity is significantly influenced by the morphological characteristics of the nanocarrier and the morphological characteristics of non-spherical particles, such as PHZS NPs and PHZG NPs; compared to spherical particles, non-spherical particles have a larger surface area for the same volume, leading to increased drug loading per unit. The use of PEG facilitates more efficient and localized drug release. This confirms that Zr-coated HAP NCS can be applied as a better formulation / propagation for bone-related treatments.

[0120] 3.5. PHZG NP's thermal weight analyze

[0121] Thermogravimetric analysis (TGA) can be used to evaluate the composition and stability of NP formulations by monitoring changes in sample weight with temperature. Among PHZG NP and PHZS NP, PHZG NP and ZrO2, which contain the major components of HAP, were selected, and their thermal stability was confirmed through thermogravimetric analysis (Fig. 5). The mass change of PHZG NP with temperature was confirmed via TGA. The analysis results showed a gradual curve up to 700°C, with a weight loss of approximately 1.48%. Subsequently, PHZG NP experienced a relatively small weight loss (3.22%) between 700 and 1000°C. Overall, PHZG NP exhibited a weight loss of approximately 4.7% at a maximum temperature increase of 1000°C. The combination of HAP and ZrO2 enhanced the thermal stability of PHZG NP, and its weight did not change significantly with temperature.

[0123] 3.5. Analysis of Cell Viability and Fluorescence Staining

[0124] Ensuring biocompatibility is particularly important when applying NPs in drug delivery systems. Therefore, the cytotoxicity of PHZS NPs and PHZG NPs was verified in HEK-293 cells. The results showed that all nanoparticles (1000 μg / mL) reduced cell viability (approximately 58–75%) compared to the control group (Fig. 6A). At a concentration of 250 μg / mL, both NPs ensured >80% cell viability. This demonstrated that NP concentrations below 250 μg / mL did not cause more than 20% cell death, and at lower concentrations (below 125 μg / mL), they exhibited negligible toxicity, thereby ensuring biocompatibility. Additionally, necrotic and apoptotic cells were evaluated using AO / EB double staining; living cells appeared as green fluorescence, while apoptotic and necrotic cells appeared as red and orange fluorescence, respectively (Fig. 6B). The results indicate that neither PHZS nor PHZG NPs exhibited changes in apoptosis compared to control cells. Additionally, Rh123 staining was performed to confirm the loss of mitochondrial membrane potential mediated by the NPs. Neither NP showed a loss of mitochondrial membrane potential compared to the control group (Fig. 6B). PI staining was performed to determine the effects of PHZS NP and PHZG NP on the nucleus; the results showed that both samples exhibited low toxicity to the nuclei of HEK-293 cells compared to the control group (Fig. 6B). Thorough cytotoxicity analysis is required to assess the in vivo applicability of nanoparticles as nanocarriers for various biomedical purposes, particularly drug delivery. In this study, the cytotoxicity of PHZS NP and PHZG NP was evaluated using two analytical methods, and low cell mortality was observed. In a study on a hydroxyapatite / sodium alginate / chitosan composite for drug delivery, a high cell viability was demonstrated, with a survival rate of over 90% in normal human bronchial epithelial (16 HBE) cells at 100 μg / mL.Consistent with previous studies, the low cytotoxicity results obtained for PHZS NP and PHZG NP in this study indicate high biocompatibility and high potential as drug delivery carriers.

[0126] 3.6. HZS NP and HZG Evaluation of NP's hemolytic properties

[0127] Since in vivo hemolysis (destruction of red blood cells) can cause anemia, jaundice, and other pathological conditions, it is important to identify the hemolytic properties of nanoparticles. Nanoparticles pose a risk of causing toxicity to blood vessels due to their large surface area per unit volume. Therefore, the potential for hemolysis must be evaluated for all pharmaceuticals administered intravenously. The assessment of the hemocompatibility of nanomaterials is considered a critical factor for in vivo application. Hematotoxicity can be evaluated by identifying hemoosmotic disturbances and physical changes resulting from red blood cell hemolysis. Accordingly, in this study, the toxicity of HZS NP, HZG NP, PHZS NP, and PHZG NP to red blood cells was confirmed through hemolysis (Fig. 6C). As a result, it was confirmed that hematotoxicity was very low compared to the positive control group, even at the highest concentration of the NPs (1000 μg / mL) (p<0.05). Triton-X-100, the positive control, exhibited the highest hemolytic activity (>95%), while PBS, HZS NPs, HZG NPs, PHZS NPs, and PHZG NPs showed slight hemolytic activity (<10%) (Fig. 6C (p<0.001)). Although nanoparticles with sharp edges are more likely to damage red blood cells (RBCs), TEM observations in this study revealed that the surface morphology of the samples (HAP, ZrO2) was slightly rounded. Furthermore, as is known in previous studies, PEGylating NPs improves their hemocompatibility, so it was expected that they would not have a harmful effect on red blood cells. However, it was confirmed that hemocompatibility could be affected by the synthesis method of the HAP-ZrO2NPs used in this study, and analysis results confirmed low hemolytic activity. The low hemotoxicity suggests potential for use in intravenous administration, such as injectables. These results led to the conclusion that PHZS NP and PHZG NP have excellent blood compatibility.

[0129] 4. Conclusion

[0130] This study confirmed the successful synthesis and characterization of HZS, HZG, PEG-HZS (PHZS), and PEG-HZG (PHZG) nanoparticles, each of which, and particularly PHZS and PHZG, demonstrate significant potential for biomedical applications. XRD and FTIR analyses were used to verify the presence of HAP and ZrO2NPs in the resulting products and the PEGylation of HAP-ZrO2NPs. ELS and DLS analyses confirmed the negatively charged surfaces of both HZS NPs and HZG NPs, with average particle sizes of less than 150 nm. Notably, PEGylation further reduced the particle size of PHZG NPs and PHZS NPs, while simultaneously enhancing their stability. Cytotoxicity evaluations performed on HEK-293 cells showed that PHZS NPs and PHZG NPs did not induce cytotoxic effects. Furthermore, fluorescent cell staining experiments confirmed that HZS NP and HZG NP did not affect the nucleus or mitochondrial membrane of HEK-293 cells. In addition, the non-toxic properties of HZS NP and HZG NP against red blood cells (RBCs) were confirmed through the evaluation of hemolysis, a key cytotoxic factor. Consequently, PEGylated HAP-ZrO2NP demonstrated enhanced physicochemical stability and in vivo safety, offering promising potential as a drug delivery carrier for long-term drug administration in relation to bone diseases.

[0132] Although the present invention has been described together with the accompanying drawings, this is merely one example among various embodiments containing the gist of the invention, and its purpose is to enable those skilled in the art to easily implement it; it is clear that the present invention is not limited to the embodiments described above. Accordingly, the scope of protection of the present invention should be interpreted by the claims below, and all technical ideas within an equivalent scope by modification, substitution, replacement, etc., without departing from the gist of the invention shall be included within the rights of the present invention. Furthermore, it is clarified that some components in the drawings are provided in an exaggerated or reduced size compared to the actual form to more clearly explain the configuration. Explanation of the symbols

[0133] (S10): Hydroxyapatite nanoparticle manufacturing step (S20): Manufacturing step of hydroxyapatite zirconium oxide nanoparticles (S30): Step for manufacturing pegylated hydroxyapatite zirconium oxide nanoparticles

Claims

Claim 1 A hydroxyapatite nanoparticle manufacturing step (S10) for manufacturing hydroxyapatite nanoparticles (HAP) by heating cow bones with purified water, treating them with acid and base, and heating; a grinding synthesis method for mixing, grinding, and calcining the 'hydroxyapatite nanoparticles (HAP)' from the hydroxyapatite nanoparticle manufacturing step (S10) with 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating'; or a sol-gel synthesis method for manufacturing zirconium oxide nanoparticles (ZrO2NPs) by adding a 'zirconium chloride solution' and sodium hydroxide to a solution in which the 'hydroxyapatite nanoparticles (HAP)' from the hydroxyapatite nanoparticle manufacturing step (S10) are dissolved, and heating; and a hydroxyapatite zirconium oxide nanoparticle manufacturing step (S20) for manufacturing zirconium oxide nanoparticles (ZrO2NPs) through one or more of the above methods. A method for preparing pegylated hydroxyapatite zirconium oxide nanoparticles, characterized by comprising: a step (S30) of preparing pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) by adding PEG to hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by either the grinding synthesis method or the sol-gel synthesis method and ultrasonically treating them. Claim 2 A method for preparing pegylated hydroxyapatite zirconium oxide nanoparticles according to claim 1, wherein the hydroxyapatite nanoparticles (HAP) in the hydroxyapatite nanoparticle preparation step (S10) are prepared by heating bovine bone (BB) with purified water at a temperature of 80 to 90°C, immersing it in 1N HCl for 20 to 28 hours, neutralizing it by immersing it in purified water, immersing it in 1M NaOH for 20 to 28 hours, drying it at room temperature, and heating it in a furnace set at a heating rate of 5°C / min at a calcination temperature of 5 to 700°C for 4 to 6 hours. Claim 3 A method for manufacturing pegylated hydroxyapatite zirconium oxide nanoparticles according to claim 1, wherein the zirconium oxide nanoparticles (ZrO2NPs) in the hydroxyapatite zirconium oxide nanoparticle manufacturing step (S20) are pulverized synthetic zirconium oxide nanoparticles (HZG) manufactured by mixing the 'hydroxyapatite nanoparticles (HAP)' of the hydroxyapatite nanoparticle manufacturing step (S10) and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating' in equal weight ratios, grinding, and calcining at a temperature of 500 to 700°C for 2 to 4 hours. Claim 4 A method for preparing pegylated hydroxyapatite zirconium oxide nanoparticles according to claim 1, wherein the zirconium oxide nanoparticles (ZrO2NPs) in the hydroxyapatite zirconium oxide nanoparticle preparation step (S20) are sol-gel synthesized zirconium oxide nanoparticles (HZS) prepared by adding a 'zirconium chloride solution mixed with purified water in a volume ratio of 1000:100' to a 'solution in which hydroxyapatite nanoparticles (HAP) are dissolved in purified water in a volume ratio of 500:100' in the hydroxyapatite nanoparticle preparation step (S10), adding a 1M sodium hydroxide solution to adjust the pH to 7 to 9, and heating at 70 to 90°C for 30 to 90 minutes. Claim 5 A method for manufacturing pegylated hydroxyapatite zirconium oxide nanoparticles according to claim 1, wherein the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) in the pegylated hydroxyapatite zirconium oxide nanoparticle manufacturing step (S30) are pegylated ground-synthesized hydroxyapatite zirconium oxide nanoparticles (PHZG) prepared by adding 'PEG-3350' to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by a ground-synthesizing method dispersed in purified water at a volume ratio of 100:50' in a volume ratio of 150:500, ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes. Claim 6 A method for preparing pegylated hydroxyapatite zirconium oxide nanoparticles according to claim 1, wherein the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) in the pegylated hydroxyapatite zirconium oxide nanoparticle preparation step (S30) is pegylated sol-gel synthesized hydroxyapatite zirconium oxide nanoparticles (PHZS) prepared by adding 'PEG-3350' in a volume ratio of 150:50 to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by sol-gel synthesis dispersed in purified water at a volume ratio of 100:50', ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes. Claim 7 Pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) characterized by being obtained by adding PEG and ultrasonically treating zirconium oxide nanoparticles (ZrO2NPs) prepared through one or more of the following methods: a grinding synthesis method in which hydroxyapatite nanoparticles (HAP) prepared by heating bovine bone with purified water, treating with acid and base, and heating, and zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating; or a sol-gel synthesis method in which hydroxyapatite nanoparticles (HAP) prepared by heating bovine bone with purified water, treating with acid and base, and heating are dissolved, zirconium chloride solution, sodium hydroxide, and heated. Claim 8 In claim 7, the hydroxyapatite nanoparticles (HAP) are characterized by being prepared by heating bovine bone (BB) in purified water at a temperature of 80 to 90°C, immersing it in 1N HCl for 20 to 28 hours, neutralizing it by immersion in purified water, immersing it in 1M NaOH for 20 to 28 hours, drying it at room temperature, and heating it in a furnace set at a heating rate of 5°C / min at a calcination temperature of 5 to 700°C for 4 to 6 hours. Claim 9 In claim 7, the zirconium oxide nanoparticles (ZrO2NPs) are pegylated hydroxyapatite zirconium oxide nanoparticles (HZG), characterized in that the zirconium oxide nanoparticles (ZrO2NPs) are prepared by a grinding synthesis method in which the 'hydroxyapatite nanoparticles (HAP)' and 'zirconium oxide nanoparticles (ZrO2NPs) prepared by adding sodium hydroxide to a zirconium chloride solution and heating' are mixed in equal weight ratios, ground, and calcined at a temperature of 500 to 700°C for 2 to 4 hours. Claim 10 In claim 7, the zirconium oxide nanoparticles (ZrO2NPs) are pegylated hydroxyapatite zirconium oxide nanoparticles (HZS) prepared by a sol-gel synthesis method in which the zirconium oxide nanoparticles (ZrO2NPs) are prepared by adding a 'zirconium chloride solution mixed with zirconium chloride in purified water at a volume ratio of 1000:100' to a 'solution in which hydroxyapatite nanoparticles (HAP) are dissolved in purified water at a volume ratio of 500:100', adding a 1M sodium hydroxide solution to adjust the pH to 7 to 9, and heating at 70 to 90°C for 30 to 90 minutes. Claim 11 In claim 7, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) are pegylated ground-synthesized hydroxyapatite zirconium oxide nanoparticles (PHZG) prepared by adding 'PEG-3350' in a volume ratio of 150:500 to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by a ground-synthesizing method dispersed in purified water at a volume ratio of 100:50', ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes. Claim 12 In claim 7, the pegylated hydroxyapatite zirconium oxide nanoparticles (HAP-ZrO2NP) are pegylated sol-gel synthesized hydroxyapatite zirconium oxide nanoparticles (PHZS) prepared by adding 'PEG-3350' in a volume ratio of 150:500 to 'hydroxyapatite zirconium oxide nanoparticles (ZrO2NPs) obtained by a sol-gel synthesis method dispersed in purified water at a volume ratio of 100:50', ultrasonically treating for 3 to 7 minutes, and centrifuging at a speed of 10,000 to 14,000 rpm for 10 to 30 minutes.

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