KR20230156195A - Making Method For Si-Based Ni Nanoflower-Encapsulated Microsphere, Si-Based Ni Nanoflower-Encapsulated Microsphere Using The Same And Bactericide Method - Google Patents

Abstract

Antibiotic resistance is rapidly emerging in pathogenic microorganisms, threatening global public health. To alleviate this burgeoning problem, topical application of antibiotics may serve as an attractive solution. To achieve this, the material must be manufactured with a transport system. Hydrogels have diverse physical and chemical properties and mimic the extracellular matrix, making them promising materials for topical antibacterial applications. In the present invention, antibacterial silicon (Si)-based nickel (Ni) nanoflowers (Si@Ni) were synthesized and encapsulated in gelatin methacryloyl (GelMA) using microfluidic engineering and photo-crosslinking technology to produce uniform, micro-sized nanoflowers (Si@Ni). Hydrogel spheres (Si@Ni-GelMA) were constructed. X-ray diffraction, transmission electron microscopy, and scanning electron microscopy were used to characterize Si@Ni and Si@Ni-GelMA. Injectable Si@Ni-GelMA has excellent antibacterial properties against three different bacterial strains (Pseudomonas aeruginosa, Klebsiella pneumoniae, and methicillin-resistant Staphylococcus aureus (bacterial killing rate > 99.9%)) and against mouse embryonic fibroblasts. It was found to have negligible toxicity. Therefore, Si@Ni-GelMA has potential applications as a drug delivery carrier due to its injectability, cross-linking using visible light, degradability, biosafety, and excellent antibacterial properties.

Description

Manufacturing method for Si-based Ni nanoflower-encapsulated microspheres, Si-based Ni nanoflower-encapsulated microspheres produced thereby, and sterilization method {Making Method For Si-Based Ni Nanoflower-Encapsulated Microsphere, Si-Based Ni Nanoflower- Encapsulated Microsphere Using The Same And Bactericide Method}

The present invention relates to a method for producing Si-based Ni nanoflower encapsulated microspheres, the Si-based Ni nanoflower encapsulated microspheres produced thereby, and a sterilization method, and more specifically, to Si@Ni-GelMA microspheres. Preparation of Si-based Ni nanoflower encapsulated microspheres capable of sterilizing more than 99% of P. aeruginosa, K. pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA) at more than 2 mg·mL ^-1 Method, Si-based Ni nanoflower encapsulated microspheres prepared thereby, and sterilization method.

Infections caused by bacteria are a serious threat to human health, and antibiotics have been developed to treat infections. However, overuse and misuse of antibiotics has led to multidrug resistance, frustrating treatment outcomes and resulting in higher mortality rates. To reduce the risk of bacterial drug resistance, it is essential to design and control safe and efficient drug delivery systems and ensure long-term drug release. According to recent inventions, gold (Au), silver (Ag), nickel (Ni), copper (Cu), zinc oxide (ZnO), nickel oxide (NiO), copper oxide (CuO), titanium dioxide (TiO ₂ ) Many metal and metal oxide nanoparticles have been found to exhibit antibacterial properties. These metal and metal oxide nanoparticles are relatively small in size and have a large surface area to volume ratio, which allows them to interact more intensively with viruses, fungi, and bacterial membranes. More recently, Si-based nanocomposites have attracted increasing attention due to their inherent desirable properties, including high theoretical specific surface area and easy size and shape definition. These nano supports were encapsulated with metal nanoparticles, and their core-shell and yoke-shell structures were applied to the energy and medical fields, respectively. Nanoflower-like structures have a larger surface area than spherical or rod structures, making them more effective as antibacterial agents. However, these nanoparticles can also harm normal cells in the body, limiting their potential applications. Therefore, there is a need for effective delivery systems that increase drug effectiveness by reducing unwanted side effects and administering drugs closer to the target.

Hydrogels containing large amounts of water are cross-linked polymer networks that mimic native extracellular matrix (ECM) and are therefore promising materials as carriers for tissue engineering and drug and protein delivery. A variety of natural and synthetic polymers can be used to form promising hydrogel scaffolds. Collagen, chitosan, dextran, alginic acid, hyaluronic acid, and gelatin are among the natural polymers widely used to make hydrogel scaffolds. Gelatin derived from collagen is a promising material due to its abundance, low cost, good biocompatibility, good biodegradability, and low antigenicity. In the case of Gelatine Methacryloyl (GelMA), functionalization of gelatin with methacryloyl groups makes it possible to harden gelatin through optical or chemical crosslinking, which can adjust the physical and chemical properties of the hydrogel. If the hydrogel could be injected, it would be easier to introduce it into the target area because surgery would not be necessary. However, bulk hydrogels still have numerous drawbacks. For example, photocrosslinked hydrogels formed by exposure to UV or visible light are often heterogeneous in situ due to low skin penetration depth. Conversely, because heat-responsive hydrogels are formed through reversible physical cross-linking, they have poor mechanical properties and are highly likely to collapse in the body.

Hydrogel microspheres, also called microparticles or microgels, overcome the disadvantages of bulk hydrogels and have unique properties compared to bulk hydrogels, so they are widely applied in the biomedical field. Microsphere hydrogels retain most of their original properties as bulk hydrogels. As uniform microspheres are cross-linked in vitro, this means that the cross-linked microspheres can be injected into the body using a syringe. In addition, because the surface area of the microspheres is large, the adsorption capacity is improved, making it easy to load drugs. Another advantage is that microspheres can be manufactured using relatively inexpensive equipment, which can greatly reduce production costs.

In general, various techniques such as droplet microfluidics, flow lithography microfluidics, electrohydrodynamic co-spraying, photolithography, soft lithography-based imprinting, and micromolding have been used to fabricate hydrogel microspheres. In particular, droplet microfluidics has great advantages in producing monodisperse hydrogel microspheres with controllable size, narrow particle size distribution, and high reproducibility. Generally, the preparation of hydrogel microspheres consists of two steps. 1) Two or multiple non-flammable fluids (i.e., water-soluble polymer and oil) are injected into a microfluidic device and then meet at the junction to generate pre-crosslinked hydrogel droplets. 2) These water droplets are cured through chemical cross-linking or photo-crosslinking. Based on these steps, various hydrophilic drugs or cell suspensions are encapsulated in hydrogel microspheres.

Republic of Korea Patent Publication No. 2020-0034696 Republic of Korea Patent Publication No. 2021-0062599 Republic of Korea Patent Publication No. 2013-0016933

Cho, K.; Wang, X.; Nie, S.; Chen, Z.G.; Shin, D.M. Therapeutic nanoparticles for drug delivery in cancer. Clin. Cancer Res. 2008, 14, 1310-1316. Maeda, H. The enhanced permeability and retention (EPR) effect in tumor vasculature: The key role of tumor-selective macromolecular drug targeting. Adv. Enzyme. Regul. 2001, 41, 189-207. Allen, T. M.; Cullis, P.R. Liposomal drug delivery systems: From concept to clinical applications. Adv. Drug Delivery. Rev. 2013, 65, 36-48.

In the present invention, antibacterial Si@Ni nanoflowers were synthesized and encapsulated in GelMA using microfluidics to produce uniform micro-sized hydrogel spheres (Si@Ni-GelMA). The properties of the synthesized Si@Ni nanoflowers are observed using scanning electron microscopy, transmission electron microscopy, and powder X-ray diffraction. A microfluidic system was used to generate GelMA-based droplets containing Si@Ni nanoflowers, followed by a photocrosslinking step to synthesize Si@Ni-GelMA microspheres. The size of the microspheres could be easily adjusted by changing the flow rate ratio of the water and oil phases. Additionally, Si@Ni-GelMA exhibits excellent antibacterial properties against three bacterial strains (Pseudomonas aeruginosa, Klebsiella pneumoniae, and methicillin-resistant Staphylococcus aureus (>99.9% killing rate)) and negligible cytotoxicity against mouse embryonic fibroblasts. . Herein, Si@Ni-GelMA microspheres can be used for tissue engineering and biomedical applications due to their biocompatibility, easy fabrication, injectability, degradability, biosafety, and excellent antibacterial properties.

A method for producing Si-based Ni nanoflower encapsulated microspheres as an active antibacterial carrier, which is an embodiment of the present invention, includes:

A first step of synthesizing gelatin methacryloyl (GelMA);

A second step of mixing Si@Ni nanoflowers with the gelatin methacryloyl to prepare a polymer precursor solution containing Si@Ni nanoflowers;

It includes a third step of producing Si@Ni-GelMA microspheres by crosslinking the polymer precursor solution mixed with Si@Ni nanoflowers.

Here, Si@Ni-GelMA microspheres refer to Si-based Ni nanoflower encapsulated microspheres.

In addition, the step of synthesizing gelatin methacryloyl (GelMA) in the first step,

Step 1-1 of dissolving gelatin by stirring in phosphate-buffered saline solution at 50°C to 70°C for 30 minutes to 3 hours;

Step 1-2 of stirring the mixture of step 1-1 at 40 ℃ to 60 ℃ for 2 to 4 hours and injecting methacrylic anhydride at 0.1 mL min ^-1 to 0.8 mL min ^-1 ;

Step 1-3 of stopping the reaction by mixing the second phosphate buffered saline solution at 35°C to 45°C with the mixture of step 1-2;

A 1-4 step of dialyzing the mixture of steps 1-3 through a dialysis membrane with distilled water at 35°C to 45°C;

Steps 1-5 of filtering the mixture of steps 1-4 with a filter;

Step 1-6 of freeze-drying the mixture of step 1-5 for 1 to 5 days at a temperature range of -90 ℃ to -70 ℃ and a pressure range of 50 mTorr to 300 mTorr;

It includes steps 1-7 of storing the freeze-dried mixture of steps 1-6 at a temperature range of -30 ℃ to -10 ℃ to obtain gelatin methacryloyl (GelMA).

In addition, the step of mixing Si@Ni nanoflowers with the gelatin methacryloyl of the second step to prepare a polymer precursor solution in which Si@Ni nanoflowers are mixed,

A 2-1 step of dissolving the gelatin methacryloyl (GelMA) produced in the first step in third phosphate buffered saline;

Step 2-2 of adding 3 to 10 mM of lithium phenyl-2,4,6-trimethylbenzoylphosphinate to the mixture of step 2-1;

Step 2-3 of adjusting the pH to 7.5 to 8.5 by mixing NaOH or HCl into the mixture of step 2-2;

Step 2-4 of mixing Si@Ni nanoflowers with the mixture of step 2-3 and sonicating the mixture at 50°C to 60°C for 10 to 50 minutes to obtain a polymer precursor solution containing Si@Ni nanoflowers; Includes.

In addition, the step of producing Si@Ni-GelMA microspheres by crosslinking the polymer precursor solution mixed with Si@Ni nanoflowers in the third step is,

Step 3-1 of mixing the polymer precursor solution mixed with the Si@Ni nanoflowers and a solvent containing paraffin oil;

An agent that forms emulsion droplets using the solution mixed in step 3-1 using a syringe, and irradiates the emulsion droplets with a wavelength of 400 nm to 450 nm, 100 mW/cm ² to 300 mW/cm ² to crosslink. Step 3-2;

and a 3-3 step of purifying the crosslinking mixture in step 3-2 with quaternary phosphate buffered saline to remove excess additives including paraffin oil.

The Si@Ni nanoflower, which is an embodiment of the present invention, is synthesized using chemical bath deposition (CBD) and reduction heating,

A first process of dispersing plasma-synthesized silicon nanoparticles in a first solvent containing water and ethanol and sonicating them to obtain a first solution;

A second step of dissolving Ni(CH ₃ COO) _2· 4H ₂ O and potassium peroxodisulfate in a second solvent containing water and ethanol to obtain a second solution;

A third process of obtaining a third solution by mixing the first service and the second solution at 100 rpm to 300 rpm;

Dilute NH _{3 ·} H ₂ O in deionized water and add 1 drop to the third solution ^. Fourth process of dropping at s- ¹ ;

A fifth process of washing the precipitate of the mixture of the fourth process with deionized water to remove SO ₄ ^2- , K ⁺ , Ni ²⁺ and CH ₃ COO ^- to obtain Si@NiOOH nanoflowers;

A sixth process of drying the Si@NiOOH nanoflowers obtained in the fifth process in an oven at 50 ° C to 70 ° C for 5 to 7 hours;

After the sixth process, a seventh process of reducing Si@Ni nanoflowers by heating for 10 to 40 minutes in a chemical vapor deposition (CVD) pipe with a 10 vol% H ₂ /90 vol% Ar gas mixture at 300 ° C to 500 ° C; Includes.

The Si@Ni nanoflower of the core-shell structure, which is an embodiment of the present invention,

It has a Ni shell in the form of a nanoflower deposited on a Si nanoparticle core, and has a flower shape of 500 nm to 1 μm,

The Si nanoparticle core has a spherical diameter of 100 nm to 200 nm, and the Ni shell has a flower shape and a thickness of 10 nm to 30 nm,

The micropore volume (V _micro ) of Si nanoparticles according to the N ₂ adsorption-desorption isotherm obtained at -196°C is 0.005 to 0.007 cm ³ /g, and the mesopore volume (V _meso ) is 0.03 to 0.07 cm ³ /g. , the specific surface area (S _BET ) is 14 to 15 m ² /g, the pore size is 17 to 18 nm, and the micropore volume (V _micro ) of Si@Ni is 0.001 to 0.003 cm ³ /g, meso The pore volume (V _meso ) is 0.08 to 0.09 cm ³ /g, the specific surface area (S _BET ) is 23 to 24 m ² /g, and the pore size is 30 to 31 nm.

Si-based Ni nanoflower encapsulated microspheres of one embodiment of the present invention,

The oil phase flow rate (Q _o ) is fixed at 40 μL·min ^-1 and the aqueous phase flow rate (Q _a ) is manufactured under conditions of 2 to 4 μL·min ^-1 The diameter is 254.1±5.4 μm, improving injection performance through the syringe head.

In one embodiment of the present invention, a method for sterilizing more than 99% of Pseudomonas aeruginosa, K. pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA) uses Si-based Ni nanoflower encapsulated microspheres 2 It is possible at a concentration of mg·mL ^-1 to 10 mg·mL ^-1 .

According to the method for producing Si-based Ni nanoflower encapsulated microspheres of the present invention, the Si-based Ni nanoflower encapsulated microspheres produced thereby, and the sterilization method, Si@Ni-GelMA microspheres are used to kill Pseudomonas aeruginosa ( It can sterilize more than 99.9% of P. aeruginosa, K. pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA).

Figure 1 is a schematic diagram of Si@Ni carrying GelMA microspheres (Si@Ni-GelMA) for antibacterial applications.
Figure 2 shows Si@Ni manufactured using Si nanoparticles and Ni(II) acetate (Ni(CH ₃ COO) ₂ ).
Figure 3 is SEM and TEM images of Si and Si@Ni.
Figure 4 shows the nitrogen absorption of Si@Ni and Si nanoparticles versus relative pressure.
Figure 5 shows the pore size distribution of Si@Ni and Si nanoparticles.
Figure 6 shows gelatin methacryloyl (GelMA) reactivity and precursor solution preparation.
Figure 7 shows the microfluidic fabrication of Si@Ni-GelMA microspheres.
Figure 8 shows the synthesis of GelMA through the transesterification reaction of gelatin (Gel) and methacrylic anhydride (MA).
Figure 9 shows H-NMR spectra of gel and GelMA.
Figure 10 shows bright field images of GelMA microspheres with different aqueous phase flow rates.
Figure 11 shows the size of GelMA microspheres as a function of aqueous phase flow rate.
Figure 12 shows a bright field image of aqueous phase modified microspheres.
Figure 13 shows the size of GelMA microspheres as a function of oil phase flow rate.
Figure 14 shows a bright field image of Si@Ni-GelMA microspheres injected through the syringe after Si@Ni-GelMA was transferred to the syringe with a 24 gauge needle and injected into a Petri dish.
Figure 15 shows PXRD patterns of control GelMA, Si@Ni, and Si@Ni-GelMA.
Figure 16 shows FT-IR spectra of Si@Ni-GelMA and GelMA.
Figure 17 shows the TGA profiles of Si@Ni-GelMA and GelMA.
Figure 18 shows SEM images of control GelMA and Si@Ni-GelMA.
Figure 19 shows bright field images showing morphological changes in GelMA over 5 weeks of culture with collagenase.
Figure 20 shows enzymatic digestion of GelMA by 10 IU·mL ^-1 type II collagenase (n = 3).
Figure 21 shows the concentration of Ni ions released from GelMA, Si@Ni, and Si@Ni-GelMA in 1 mL DPBS.
Figure 22 shows representative images of bacteria grown under different conditions after 24 hours of incubation compared to the control, top (P. aeruginosa), middle (K. pneumoniae), bottom (MRSA), left to right: blank, GelMA; Si@Ni, Si@Ni-GelMA
Figure 23 shows the antibacterial rates of GelMA, Si@Ni, and Si@N-GelMA against P. aeruginosa, K. pneumoniae, and MRSA.
Figure 24 shows cytotoxicity tests of Si@Ni-GelMA (A) Live/Dead stain images after contact with control GelMA, Si@Ni, or Si@Ni-GelMA on days 1 and 3, respectively.
Figure 25 shows in vitro cytotoxicity of control GelMA, Si@Ni or Si@Ni-GelMA to MEF extracts. Scale bar: 200 ㎛

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. The preferred embodiments described below can be designed in various ways by those skilled in the art within the scope of the technical idea of the present invention.

1. Materials and Methods

1.1. ingredient

Gelatin (pork skin, type A, powder, gel strength ~300 g Bloom), methacrylic anhydride (MA), deuterium oxide (D ₂ O), ethyl alcohol (ABV 200, ≥99.5%), plasma-synthesized silicon nanoparticles (20 mg, particle size: 50~100 nm, purity: 99%), ammonium hydroxide solution (NH ₃ ^. H ₂ O), nickel(II) acetate (Ni(CH ₃ COO) ₂ ^. 4H ₂ O and ferrooxo Potassium disulfate (K ₂ S ₂ O ₈ ) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was purchased from Cellink Life Sciences (Boston, USA). Dialysis membrane was obtained from Spectrum Laboratory (Rancho Dominguez, CA, USA): DMEM (Dulbecco's Modified Eagle's Medium), Dulbecco's Phosphate Buffered Saline (DPBS), fetal bovine serum (FBS), and penicillin. /streptomycin, collagen I (rat tail) were purchased from Gibco (Grand Island, NY, USA), and the LIVE/DEAD™ viability/cytotoxicity kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). MTS assay kit (cell proliferation) was purchased from Abcam (Cambridge, MA, USA) Disposable plastic syringes, mouse embryonic fibroblast (MEF) and LIVE/DEAD assay kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). ) was purchased from.

1.2. Synthesis of Si-based Ni composite nanoflowers (Si@Ni)

Si@Ni was synthesized using chemical bath deposition (CBD) and reduction heating. Plasma-synthesized silicon nanoparticles (20 mg) were dispersed in a beaker mixed with water (4 mL) and ethanol (2 mL) and sonicated for 2 hours (solution 1). Ni(CH ₃ COO) _2· 4H ₂ O (0.25 g) and potassium peroxodisulfate (0.05 g) were dissolved in a beaker containing water (4 mL) and ethanol (2 mL) (solution 2). Solution 1 and Solution 2 were mixed by magnetic stirring at 200 rpm (Solution 3). NH _{3 ·} H ₂ O (28-37%, 0.2 mL) was diluted in deionized water (10 mL), and the diluted solution was stirred and added dropwise (1 drop.s- ¹ ) to Solution 3. The yellowish solution gradually turned black. The reaction continued for 30 minutes after adding NH ₃ ·H ₂ O, and the precipitate was washed with a large amount of deionized water to remove other adsorbed ions (SO ₄ ^2- , K ⁺ , Ni ²⁺ , CH ₃ COO ^- , etc.). Brown Si@NiOOH nanoflowers were obtained, dried in an oven at 60°C for 6 hours, and then heated in a chemical vapor deposition (CVD) tube at 400°C with a 10vol% H ₂ /90vol% Ar gas mixture for 30 minutes to obtain a dark appearance. It was reduced to brown Si@Ni nanoflowers.

1.3. Preparation of Si@Ni encapsulated gelatin microspheres (Si@Ni-GelMA)

Step 1: Synthesis of gelatin methacryloyl (GelMA).

Gelatin was used as a substrate. To realize photopolymerization, modified gelatin was prepared by introducing a photoreactor. Specifically, gelatin (2g) was dissolved in DPBS (20 mL) by stirring at 60°C for 1 hour. Afterwards, the mixture was stirred at 50°C for 3 hours and MA (1.2 mL) was added at 0.5 mL min ^-1 . To stop the reaction, the solution was diluted 5-fold with heated DPBS (40 °C, 80 mL). The mixture was dialyzed in distilled water (40°C) for 7 hours using a dialysis membrane (Mw cutoff: 12-14 kDa). Next, it was passed through a filter (0.22 μm, Perkin Elmer, Waltham, MA, USA). The resulting solution was freeze-dried (Sentry 2.0, SP Scientific, Warminster, PA, USA) at -80 °C and 100 mTorr for 3 days and stored at -20 °C for subsequent use. The methacrylate groups on gelatin were characterized using nuclear magnetic resonance (NMR) spectroscopy in D ₂ O using a 400 MHz spectrometer (JEOL RESONANCE ECZ 400S JEOL, Tokyo, Japan).

Step 2: Preparation of Si@Ni mixed polymer precursor solution.

To prepare the prepolymer solution, GelMA was dissolved in DPBS to prepare a 5% (w/v) solution. Next, LAP dissolved in deionized water was added to this GelMA solution at a final concentration of 5 mM, and the pH of the polymer solution was adjusted to 8.0 using NaOH or HCl. Then, Si@Ni nanoflowers (0.2% (w/v)) were further mixed with the polymer solution and sonicated at 55°C for 30 minutes to form a homogeneous solution.

Step 3: Microfluidic generation of Si@Ni-GelMA microspheres.

A prepolymer solution was used to prepare Si@Ni-GelMA microspheres as the dispersed phase. The continuous phase contained 5 wt% Span80 paraffin oil. These two phases were injected into different microchannels, and the flow rate was finely adjusted by a syringe pump to obtain monodisperse droplets. The collected emulsion droplets were exposed to visible LED lighting (Jinsoft, Daegu, Korea) for 10 seconds (λ=400-450 nm, 200 mW/cm ² ) to cross-link the GelMA-based droplets. To remove excess additives, including paraffin oil and photoinitiator, the microspheres were extracted in washing water and purified in DPBS. All workpieces and reagents were kept sterile. For clarity, Si@Ni encapsulated GelMA microspheres and control GelMA microspheres without Si@Ni are denoted as Si@Ni-GelMA and GelMA, respectively. The resulting microspheres were photographed using an optical microscope, and their sizes were analyzed using ImageJ 1.53a software.

1.4. Characterization of Si@Ni

Si nanostructures were analyzed using scanning electron microscopy (SEM; Hitachi SU8230, Hitachi High-Tec, Tokyo, Japan) at 10 kV and transmission electron microscopy (TEM; Tecnai G2 F20 TWIN TMP, FEI, Hillsboro, OR, USA) at 120 kV. The morphologies of particles and Si@Ni nanoflowers were characterized. The Brunauer-Emmett-Teller (BET) method measured the specific surface area of the sample at 196 °C using a 3FLEX surface characterization analyzer and a physisorption analyzer (Micromeritics, Atlanta, Georgia). The Barrett-Joyner-Halenda (BJH) method was used to analyze the pore size distribution of the adsorption basin.

1.5. Characteristics of Si@Ni-GelMA

Powder The diffractometer was operated at a voltage of 40 kV and a current of 30 mA at a scan speed of 4°/min and an interval of 0.02°. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded using KBr pellets using an FTS 135 spectrometer from Bio-Rad (Hercules, CA, USA). Thermal properties were evaluated using thermal gravimetric analysis (TGA; TG 209 F3 Tarsus, NETZSCH, Selb, Germany). The morphological properties of lyophilized Si@Ni-GelMA and control GelMA were evaluated using SEM-EDS, and gold-palladium (Au-Pd) alloy was coated on the lyophilized sample for 30 seconds before SEM analysis. As part of the in vitro degradation invention, swelling was evaluated by culturing 5% (w/v) photo-crosslinked GelMA microspheres in DPBS for 24 hours. To mimic the physiological environment, GelMA microspheres were cultured with 0.5 ml of DPBS containing Type II Collagenase (10 UmL ^-1 ) in a 1.5 mL tube at 37 °C for 2 weeks. Activity was ensured by charging the enzyme solution at regular intervals, and the microspheres were observed under a microscope and their weight was measured at a certain point after removing the enzyme solution. Deterioration rate (DR) was calculated using Equation 1.

Equation 1

W _o is the initial weight, and W _t represents the weight at time t.

1.6. Metal ion release evaluation

The metal ion release potential of GelMA, Si@Ni or Si@Ni-GelMA was tested by adding 400 μg of GelMA, Si@Ni or Si@Ni-GelMA to 1 mL of PBS and stirring at 25°C for 6 to 48 hours. . This solution was centrifuged at 25°C and 5000 rpm for 5 minutes, and the supernatant was removed from the reaction tube. Inductively coupled plasma optical emission spectroscopy (ICP-OES; iCAP 7400 ICP-OES Duo, Thermo Fisher Scientific, Waltham, MA, USA) was used to measure metal ions emitted from the samples. The concentration of metal ions released into the medium is expressed in ppm.

1.7. Antibacterial activity evaluation

The minimum bactericidal concentration (MBC) method was used to measure the antibacterial activity of control GelMA, Si@Ni, and Si@Ni-GelMA against three strains of bacteria: Pseudomonas aeruginosa (ATCC 15442), Bacillus pneumoniae (ATCC 4352), and Methi. Silin-resistant Staphylococcus aureus (MRSA; ATCC 33591) was determined. A blank Si@Ni-GelMA coated sheet (2 mg·mL ^-1 , 5x5±0.2 cm ² ) and a stemaker film were prepared and tested. Using the same procedure, GelMA and Si@Ni were also prepared for comparison. The test sample was wiped on the surface two to three times with gauze soaked in ethanol and then dried. Routine procedures were used to aliquot platelets of pre-cultured test bacteria containing 1-4x10 ⁵ colony forming units (CFU)·mL ^-1 . The Petri dish was placed with each sample with the test side facing up. Afterwards, 0.2 mL of test solution was dispensed into each sample using a pipette. The dropped test bacteria were covered with a film and spread by lightly pressing. Petri dishes containing samples inoculated with test bacteria and control GelMA were cultured at 37°C for 24 hours. After dispensing, the uncoated test samples containing the test strains were separated using sterilized tweezers to prevent each solution from flowing. Afterwards, soybeam casein was added to digest lecithin polysorbate medium (10 mL) to wash away the test bacteria. The washing solution was used to measure the number of viable cells. 1 mL of washing solution was added to 9 mL of physiological saline and mixed thoroughly. This procedure involves diluting the wash and plating wash (0.1 mL) on three nutrient agar plates and incubating them at 37°C for 24 hours. All experiments were repeated three times. The sterilization rate was calculated using Equation 2.

Equation 2

Here, A represents the CFU of bacteria cultured in the presence of a stomach film, and B represents the CFU of bacteria cultured in the presence of control GelMA, Si@Ni, or Si@Ni-GelMA.

1.8. Biocompatibility of Si@Ni-GelMA

(I) Cell culture: MEFs were cultured in high-glucose DMEM containing 10% fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL of streptomycin at 37°C in a CO ₂ humidified atmosphere. Medium was changed every two days, and cells were digested with trypsin and resuspended in fresh medium before confluence.

(II) Live/Dead analysis: The cytotoxicity of GelMA, Si@Ni, and Si@Ni-GelMA was evaluated using the direct contact method, and cells were grown on glass slides coated with Collagen I at a density of 5x10 ⁴ cells·cm ^-1 . Monolayers were grown and incubated for 3 hours.

After washing with DMEM to remove non-adherent cells, the glass slide was transferred to a 12-well plate and cultured at 37°C in a humidified incubator containing 5% CO ₂ . The control GelMA and Si@Ni-GelMA prepared the next day were carefully placed on the cell monolayer, alternately carefully mixed with Si@Ni (0.2% (w/v)), and then cultured for 1 or 3 days. Cells were analyzed using the Live/Dead assay kit, showing live cells with green fluorescence and dead cells with red fluorescence. Stained cells were observed using inverted fluorescence microscopy (IX83, Olympus, Center Valley, PA, USA). Cell viability was calculated using equation 3.

Equation 3

(III) MTS assay: Cell viability was also assessed using the MTS assay, which measures cell metabolism. In this analysis, GelMA, Si@Ni, and Si@Ni-GelMA were cultured in cell culture medium for 24 hours to prepare an extraction solution, and 5x10 ⁴ cells were seeded per well in a 24-well plate. After culturing at 37°C for 24 hours, the medium in each well was replaced with 200 μL of DMEM containing the extract. After culturing for an additional 24 hours, the medium containing the extract was carefully removed. Afterwards, fresh medium (200 μL) and MTS cell proliferation assay kit solution (20 μL) were added to each well. After culturing for an additional 4 hours using a microplate reader (Synergy H1, BioTek, Winooski, VT, USA), the absorbance was measured at 490 nm. Cell proliferation was quantified using a standard curve of cells.

1.9. Statistical analysis

All data were expressed as mean ± standard deviation using at least three samples. Statistically significant differences were evaluated by t-test, and statistical significance was (*) p < 0.05, (**) p < 0.01, and (**) p < 0.001.

2. Results and discussion

The overall goal of the present invention is to develop antibacterial properties and in vitro biocompatibility for various biomedical applications, as well as (1) antibacterial Si@Ni nanoflowers and photocrosslinkable gelatin methacryloyl (GelMA), (2) microfluidics and photonics. The goal was to fabricate Si@Ni-GelMA carrying Si@Ni through a cross-linking approach, (3) and verify the chemical and mechanical properties of Si@Ni-Gel (Figure 1).

2.1. Characterization of Si@Ni nanoflowers

Si@Ni nanoflowers were prepared as shown in Figure 2. In the CBD process, Si nanoparticles are encapsulated in a porous flower-shaped NiOOH shell (i.e., Si@NiOOH), and then Si@NiOOH is wrapped with a porous Ni composite sheet to reduce it to a core-shell structure in an atmosphere with Ar flow. It was heated to 350°C and then sequentially heated to 400°C in an atmosphere with a 10vol% H ₂ /90vol% Ar gas mixture flow to produce Si@Ni. As shown in Figure 3, Si nanoparticles are spheres with a diameter of 100-200 nm, and Si@Ni is a flower-shaped core of 500 nm to 1 μm with a thin nanoflower-shaped Ni shell uniformly deposited on the silicon core. -The shell structure is shown. Even after the additional annealing process, the flower-like shape was maintained stably. The thickness of the porous Ni shell layer was confirmed to be uniform at 10 to 30 nm, except for shrinkage due to Ni particle agglomeration (Figure 4). The pores and surface areas of Si nanoparticles and Si@Ni nanoflowers were determined by analyzing the N ₂ adsorption-desorption isotherm obtained at -196°C. Micropore volume (V _micro ), mesopore volume (V _meso ), and specific surface area (S _BET ) were determined by the BET method, and the average pore size was also determined from the adsorption basin by the BJH method. Figures 4 and 5 show N ₂ adsorption-desorption isotherms and pore size distribution curves. Based on the N ₂ adsorption curve for Si@Ni, the hysteresis loop between 0.5 and 1 (P/Po) indicates that Si@Ni has high mesoporosity and macroporosity after forming a shell with a Ni petal layer. indicates. In contrast, Si nanoparticles have much lower N ₂ adsorption, indicating their low porosity. As shown in Table 1, after Ni coating of Si nanoparticles, V _meso , S _BET and average pore size increased, forming Si@Ni.

[Table 1]

2.2. Synthesis and characterization of GelMA

In the present invention, gelatin was used as the backbone polymer because it is well established as a biocompatible and biodegradable polymer. Furthermore, gelatin can be easily modified using functional groups including amine groups in the backbone. The methacrylation process is shown in Figures 6 and 8. Briefly, MA reacted with a reactive amine to introduce unsaturated bonds into the gelatin molecular chain. Later, GelMA can be cross-linked by free radical photopolymerization in an aqueous solution containing a photoinitiator. NMR spectra of gelatin and GelMA were obtained (Figure 9). Peak H _a (5.3 ppm and 5.6 ppm) represents the vinyl group of MA, peak H _b (1.87 ppm) corresponds to the methyl group of MA, and peak H _c (2.9 ppm) represents the signal of lysine on gelatin, indicating that GelMa synthesis was successful. Able to know. As a result of comparing the areas of each peak, the degree of methacrylation was found to be ~70%.

2.3. Fabrication and characterization of Si@Ni GelMA

In Figure 7, hydrogel droplets are generated by shear force of continuous flow in a water-in-oil (w/o) system. In the w/o emulsion system, Si@Ni dispersed GelMA solution and mineral oil were injected into the aqueous phase and oil phase, respectively. 5 wt% of GelMA was used because concentrations below 4 wt% inhibit hydrogel formation. Meanwhile, LAP was used as a photoinitiator due to its water solubility, cytocompatibility, and visible light initiation properties. Water droplet microspheres were collected in a Petri dish and exposed to light irradiation. To reduce cytotoxicity, visible LED lighting (λ=400-450 nm, 200 mW ^. cm ^-2 ) with an exposure time of 10 s was selected as the exposure condition. Free radicals are generated by LAP when exposed to visible LED light. These free radicals reacted with methacryloyl groups in GelMA to form carbon radicals, which propagated through other methacryloyl groups in GelMA to form chain-grown photopolymerized Si@Ni-GelMA (Figure 7).

2.4. Si@Ni-GelMA manufacturing optimization and injectability testing

The flow rate of a microfluidic system is one of the key parameters affecting the size and stability of the generated droplets. To observe the effect of aqueous phase flow rate (Qa) on the size of microspheres, the aqueous phase flow rate was adjusted to 2-8 μL·min ^-1 . The oil phase flow rate (Qo) was fixed at 40 μL·min ^-1 . As shown in Figures 10 and 11, the size of the microspheres increased from 254.1 ± 5.4 to 318.7 ± 6.8 μm. To evaluate the effect of oil phase flow rate, the oil phase flow rate was adjusted from 20 μL·min ^-1 to 80 μL·min ^-1 . The aqueous phase flow rate was fixed at 4 μL·min ^-1 . As shown in Figures 12 and 13, the size of the microspheres decreased from 331.2 ± 5.3 to 241.7 ± 4.2 μm. Therefore, both flow rates affected the overall size of GelMA microspheres. Micro-sized hydrogels are noteworthy because they can be injected using a syringe or catheter, minimizing the invasive delivery of cells, drugs, and organisms. Preformed bulk hydrogels are difficult to deliver with minimally invasive techniques because they must be shaped and implanted using surgical incisions. In general, bulk hydrogels are commonly used as homogeneous precursor solutions that exhibit liquid-like properties until gelation is induced by cross-linking. In contrast, micro-sized hydrogels are easy to inject due to their particle nature and small particle size. Therefore, the injectability of Si@Ni-GelMA microspheres was investigated for several potential applications, including wound healing, bioprinting, and anticancer agents. Once formed, Si@Ni-GelMA with a diameter of less than 250 μm can be easily injected through a 24-gauge syringe head, making it suitable for in situ minimally invasive tissue repair and maintaining its original shape and size (Figure 14).

2.5. Characterization of Si@Ni-GelMA

The compositions were characterized by analyzing the PXRD patterns and FT-IR spectra of GelMA, Si@Ni, and Si@Ni-GelMA. As shown in Figure 15, in the case of Si@Ni, high-intensity diffraction peaks were observed at 2θ = 28.3°, 47.2°, 55.9°, 75.7°, and 87.9°, corresponding to Si, and 2θ = 36°, corresponding to coated Ni. Additional peaks were observed at , 44°, and 52°. GelMA was observed as a large amorphous hump between 2θ = 20-25°. Si@Ni-GelMA showed a similar pattern to the control GelMA, and several peaks corresponding to Si@Ni were additionally observed. FT-IR spectral analysis was performed on control GelMA and Si@Ni-GelMA. Figure 16 shows the absorption peaks around 3400 cm ^-1 (contribution of -OH and -NH ² stretching) and 1630 cm ^-1 (contribution of amide I) in the control GelMA. In the case of Si@Ni-GelMA, it can be observed that the positions of all these bands have slightly changed. These PXRD patterns and FT-IR spectra indicate that Si@Ni is well integrated into the microspheres.

TGA is a continuous process that involves measuring the weight of a sample as its temperature increases in the form of programmed heating. Because TGA improves understanding of thermal decomposition behavior, TGA was used to investigate thermal decomposition and thermal decomposition of control GelMA and Si@Ni-GelMA. As shown in Figure 17, the weight of control GelMA and Si@Ni-GelMA decreased by about 11 wt% and 6.1 wt%, respectively, up to 200°C due to loss of absorbed water. At 250 °C, the percentile weight loss of control GelMA was 16.8 wt%, while that of Si@Ni-GelMA was 8.5 wt%. For GelMA, a weight loss of 50 wt% was observed at 323 °C and 453 °C for Si@Ni-GelMA, respectively. Additionally, the weight loss at 800°C was 93.6 wt% and 62.8 wt%, respectively. The addition of Si@Ni significantly improved the pyrolysis temperature.

To investigate the surface topography of control GelMA and Si@Ni-GelMA, the freeze-dried samples were characterized using SEM-EDS, and nano-sized Si@Ni was well encapsulated in the GelMA network, as shown in Figure 18. In addition, analysis by SEM-EDS was conducted to obtain the morphology and chemical composition of control GelMA and Si@Ni-GelMA. In more detail, C and O were evenly distributed throughout the two networks, and Si@Ni-GelMA Ni and Si elements were additionally discovered in the EDS spectrum.

For potential in vivo applications of gelatin-based scaffolds as tissue engineering scaffolds and drug delivery carriers, the implanted material (suture, gauze or hydrogel) must be biodegradable so that a second surgery is not required for removal of the implant. . Therefore, we evaluated the biodegradability of GelMA microspheres against type II collagen enzymes due to their promising therapeutic and in vivo applications. GelMA microspheres were placed in a buffer solution containing 10 U·mL ^-1 collagenase and the degradation rate was monitored. GelMA microspheres showed minor deterioration particles within the first week. On the 10th day, the surrounding structure of GelMA began to collapse, creating larger cracks. Within 2 weeks, the number of GelMA decreased significantly and disappeared completely within 3 weeks (Figure 19). Additionally, the size of the spheres tended to increase over time, which may indicate a decrease in volume. Overall, the degradation of GelMA was slow and gradual. In accordance with the morphological changes (Figure 20), the residual weight of GelMA decreased over time.

2.6. Ion emission test

The ion release properties of control GelMA, Si@Ni, and Si@Ni-GelMA were tested in 400 μgmL ^-1 of DPBS at 25°C for 6, 12, 24, and 48 hours. The amount of Ni ions released from the sample was measured using ICP-OES. As shown in Figure 21, the concentration of Ni ions released from Si@Ni increased from 2.14 ppm for 6 hours to 8.72 ppm after 48 hours, indicating that Si@Ni maintained its flower-like structure intact in PBS. . In particular, the amount of Ni(II) ions released from Si@Ni-GelMA decreased by 5 times (1.79 ppm after 48 hours) compared to that released by Si@Ni. Si@Ni encapsulated in a polymer network may have been somewhat inhibited from direct contact with aqueous solutions, resulting in a decrease in metal ion release. Meanwhile, the amount of Ni(II) ions released from the control GelMA was negligible.

2.7. antibacterial activity

To investigate the bioactivity of antibacterial microspheres, control GelMA, Si@Ni, and Si@Ni-GelMA were tested in a concentration range of 200 μg·mL ^-1 to 2 mg·mL ^-1 . CFU of P. aeruginosa, K. pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA) were counted after 24 hours of incubation with Si@Ni nanoflowers, as shown in Figure 22, Figure 23, and Table 2. As shown, the bactericidal effect of the control group GelMA increased as follows: MRSA (2.7%)<P. aeruginosa (4.5%)<K. pneumoniae (8.9%). This is due to the low antibacterial properties of GelMA. On the other hand, Si@Ni nanoflowers had P. aeruginosa (97.6%) < MRSA (98.2%) < K. pneumoniae (98.3%) showed the highest antibacterial activity.

[Table 2]

Metal-coated nanoparticle surfaces can provide more effective bactericidal activity than metalloid Si nanoparticles. At a low MBC of 200 μg·mL ^-1 , the sterilization effect was higher than that of Ag/Cu-graphene. Meanwhile, the sterilization rate of Si@Ni-GelMA was 99.9% for all three types. These results indicate that Si@Ni-GelMA exhibits a synergistic bactericidal effect derived from the combination of GelMA and Si@Ni nanoflowers in all three tested species. As Si@Ni was encapsulated in GelMA microspheres, Si@Ni-GelMA released fewer Ni(II) ions compared to Si@Ni, but the sterilization rate of Si@Ni-GelMA at a concentration of 2 mg·mL ^-1 was 99.9. It was %. Therefore, it can be assumed that the sterilization process in Si@Ni-GelMA occurs when the antibacterial metal ions released from the nanoflower directly contact the cell membrane, rather than penetrating into the cells and killing bacteria in the process.

2.8. cytotoxicity

Finally, the biocompatibility of Si@Ni-GelMA was invented based on the direct contact method. MEFs have been used as a model cell line in in vitro experiments due to their important role in wound healing. To confirm the toxicity of control GelMA, Si@Ni, and Si@Ni-GelMA, three test samples were prepared in the same manner. In addition, as an additional positive control (blank), a MEF monolayer without contact was prepared, and as a negative control, a MEF monolayer in contact with a 10% EtOH solution was used. MEF viability was monitored using live/dead staining and colorimetric MTS assays. The fluorescence microscopy image in Figure 24 shows that the cell viability of blank, control GelMA, Si@Ni, and Si@Ni-GelMA samples was over 97% after one day of culture. In contrast, most MEF monolayer cells die upon exposure to EtOH. After dispensing, the cells gradually adhered, spread, and flattened on the surface, forming a confluent layer that survived for 3 days. These results indicate that only Si@Ni-GelMA and Si@Ni and GelMA are not toxic to cells. For further quantification, MTS analysis was used (Figure 25), and Si@Ni, control GelMA, and Si@Ni-GelMA extracted from the cell culture medium solution were serially diluted. For example, 100% refers to the original extraction medium, and 50% refers to a two-fold dilution of the initial extraction medium. After forming a MEF monolayer, the medium was changed to a medium containing the extract at the desired concentration, and the MEF monolayer was cultured. As found by MTS, Si@Ni-GelMA had good cytocompatibility, considering that the viability of MEFs was >95% in each case, but most MEFs died when exposed to EtOH, resulting in The low cytotoxicity of the Si@Ni-GelMA extract is again confirmed.

3. Conclusion

This invention demonstrates a facile and efficient approach to prepare strong and uniform micro-sized spheres containing Si@Ni nanoflowers using microfluidics and photocrosslinking techniques. The size of GelMA microspheres with well-defined 3D morphology could be easily controlled by adjusting the aqueous and oil phase flow rates. The chemical properties of Si@Ni-GelMA could be characterized using PXRD, FT-IR, TGA, SEM, and ICP-OES. In addition, Si@Ni-GelMA showed excellent antibacterial activity (>99.9% sterilization rate) against three strains (P. aeruginosa, K. penominiae, MRSA) at a concentration of 2 mg·mL ^-1 and against MEF. It showed negligible toxicity. Using visible light avoids potential biosafety issues associated with initiator toxicity or UV light. Additionally, the injectability and biodegradability of Si@Ni-GelMA make it a promising platform for tissue engineering and biomedical applications. To promote the use of Si@Ni-GelMA as a powerful and efficient antibacterial hydrogel for broad applications, further inventions are needed, including in vitro cell migration and in vivo wound closure.

In the above, preferred embodiments of the present invention have been shown and described, but the present invention is not limited to the specific embodiments described above, and may be used in the technical field to which the invention pertains without departing from the gist of the invention as claimed in the claims. Of course, various modifications can be made by those skilled in the art, and these modifications should not be understood individually from the technical idea or perspective of the present invention.

Claims (8)

A first step of synthesizing gelatin methacryloyl (GelMA);
A second step of mixing Si@Ni nanoflowers with the gelatin methacryloyl to prepare a polymer precursor solution containing Si@Ni nanoflowers;
A method for producing Si-based Ni nanoflower encapsulated microspheres as an active antibacterial carrier, comprising a third step of crosslinking a polymer precursor solution mixed with Si@Ni nanoflowers to prepare Si@Ni-GelMA microspheres. In claim 1,
The step of synthesizing gelatin methacryloyl (GelMA) in the first step is,
Step 1-1 of dissolving gelatin by stirring in phosphate-buffered saline solution at 50°C to 70°C for 30 minutes to 3 hours;
Step 1-2 of stirring the mixture of step 1-1 at 40 ℃ to 60 ℃ for 2 to 4 hours and injecting methacrylic anhydride at 0.1 mL min ^-1 to 0.8 mL min ^-1 ;
Step 1-3 of stopping the reaction by mixing the second phosphate buffered saline solution at 35°C to 45°C with the mixture of step 1-2;
A 1-4 step of dialyzing the mixture of steps 1-3 through a dialysis membrane with distilled water at 35°C to 45°C;
Steps 1-5 of filtering the mixture of steps 1-4 with a filter;
Step 1-6 of freeze-drying the mixture of step 1-5 for 1 to 5 days at a temperature range of -90 ℃ to -70 ℃ and a pressure range of 50 mTorr to 300 mTorr;
An active antibacterial agent comprising: a 1-7 step of obtaining gelatin methacryloyl (GelMA) by storing the freeze-dried mixture of steps 1-6 in a temperature range of -30°C to -10°C; Method for preparing Si-based Ni nanoflower encapsulated microspheres as carriers. In claim 2,
The step of mixing Si@Ni nanoflowers with the gelatin methacryloyl of the second step to prepare a polymer precursor solution in which Si@Ni nanoflowers are mixed,
A 2-1 step of dissolving the gelatin methacryloyl (GelMA) produced in the first step in third phosphate buffered saline solution;
Step 2-2 of adding 3mM to 10mM of lithium phenyl-2,4,6-trimethylbenzoylphosphinate to the mixture of step 2-1;
Step 2-3 of adjusting the pH to 7.5 to 8.5 by mixing NaOH or HCl into the mixture of step 2-2;
Step 2-4 of mixing Si@Ni nanoflowers with the mixture of step 2-3 and sonicating the mixture at 50°C to 60°C for 10 to 50 minutes to obtain a polymer precursor solution containing Si@Ni nanoflowers; A method for producing Si-based Ni nanoflower encapsulated microspheres as an active antibacterial carrier, comprising: In claim 3,
The step of producing Si@Ni-GelMA microspheres by crosslinking the polymer precursor solution mixed with Si@Ni nanoflowers in the third step is,
Step 3-1 of mixing the polymer precursor solution mixed with the Si@Ni nanoflowers and a solvent containing paraffin oil;
An agent that forms emulsion droplets using the solution mixed in step 3-1 using a syringe, and irradiates the emulsion droplets with a wavelength of 400 nm to 450 nm, 100 mW/cm ² to 300 mW/cm ² to crosslink. Step 3-2;
A 3-3 step of purifying the crosslinking mixture in step 3-2 with quaternary phosphate buffered saline to remove excess additives containing paraffin oil. Si-based Ni as an active antibacterial carrier comprising a. Method for manufacturing nanoflower encapsulated microspheres. In claim 1,
The Si@Ni nanoflower is synthesized using chemical bath deposition (CBD) and reduction heating,
A first process of dispersing plasma-synthesized silicon nanoparticles in a first solvent containing water and ethanol and sonicating them to obtain a first solution;
A second step of dissolving Ni(CH ₃ COO) _2· 4H ₂ O and potassium peroxodisulfate in a second solvent containing water and ethanol to obtain a second solution;
A third process of obtaining a third solution by mixing the first service and the second solution at 100 rpm to 300 rpm;
Dilute NH _{3 ·} H ₂ O in deionized water and add 1 drop to the third solution ^. Fourth process of dropping at s- ¹ ;
A fifth process of washing the precipitate of the mixture of the fourth process with deionized water to remove SO ₄ ^2- , K ⁺ , Ni ²⁺ and CH ₃ COO ^- to obtain Si@NiOOH nanoflowers;
A sixth process of drying the Si@NiOOH nanoflowers obtained in the fifth process in an oven at 50 ° C to 70 ° C for 5 to 7 hours;
After the sixth process, the seventh step is to reduce Si@Ni nanoflowers by heating in a chemical vapor deposition (CVD) pipe with a 10 vol% H ₂ /90 vol% Ar gas mixture at 300 ℃ to 500 ℃ for 10 to 40 minutes. A method for producing Si-based Ni nanoflower encapsulated microspheres as an active antibacterial carrier, comprising: a process. It has a nanoflower-shaped Ni shell deposited on a Si nanoparticle core, and is a Si@Ni nanoflower with a flower-shaped core-shell structure of 500 nm to 1 μm,
The Si nanoparticle core has a spherical diameter of 100 nm to 200 nm, and the Ni shell has a flower shape and a thickness of 10 nm to 30 nm,
The micropore volume (V _micro ) of Si nanoparticles according to the N ₂ adsorption-desorption isotherm obtained at -196 ℃ is 0.005 to 0.007 cm ³ /g, and the mesopore volume (V _meso ) is 0.03 to 0.07 cm ³ /g. , the specific surface area (S _BET ) is 14 to 15 m ² /g, the pore size is 17 to 18 nm, and the micropore volume (V _micro ) of Si@Ni is 0.001 to 0.003 cm ³ /g, meso Si with a core-shell structure, characterized in that the pore volume (V _meso ) is 0.08 to 0.09 cm ³ /g, the specific surface area (S _BET ) is 23 to 24 m ² /g, and the pore size is 30 to 31 nm. @Ni Nanoflower. Si-based Ni nanoflower encapsulated microspheres,
While the oil phase flow rate (Q _o ) is fixed at 40 μL·min ^-1 , the aqueous phase flow rate (Q _a ) is controlled to 2 to 4 μL·min ^-1 ,
Si-based Ni nanoflower encapsulated microspheres with improved injection performance through a syringe head characterized by a diameter of 254.1 ± 5.4 μm. Si-based Ni nanoflower encapsulated microspheres against P. aeruginosa, K. pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) at concentrations of 2 mg·mL ^-1 to 10 mg·mL ^-1 How to sterilize more than 99%.