KR20220036544A - Multi-functional mesoporous bioactive glass nanoparticle and manufacturing method thereof - Google Patents

Abstract

The present invention relates to multi-functional mesoporous bioactive glass nanoparticles and a method for producing the same, and to: a method for producing mesoporous bioglass nanoparticles; nanoparticles produced by the same; and a use thereof. Specifically, the present invention relates to: a method for producing mesoporous bioglass nanoparticles; nanoparticles produced by the same; and a composition using the nanoparticles. The method comprises the steps of: a) preparing a mixture by dissolving cetyltrimethylammonium bromide, 2-methoxyethanol, ethanol and an ammonia solution in water; b) adding calcium nitrate, tetraethyl orthosilicate and 3-aminopropyl triethoxysilane to the mixture and stirring the mixture thereof; c) obtaining particles produced by the addition and stirring; and d) thermally treating the particles at a temperature of 350-450℃.

Description

Multi-functional mesoporous bioactive glass nanoparticles and manufacturing method thereof

The present invention relates to multifunctional mesoporous bioactive glass nanoparticles and a method for manufacturing the same, and to a method for manufacturing mesoporous bioglass nanoparticles, nanoparticles prepared thereby, and use thereof.

Phototherapy using multimodal imaging has a significant effect on cancer cells. Due to the advantages of real-time observation of cancer sites and photoinduced destruction of cancer cells, it is evaluated as a promising strategy in the medical field because of its wide application in tumor treatment. Both photothermal (PTT) and photodynamic (PDT) techniques have better therapeutic efficacy compared to other techniques of the same target. This PTT and PDT therapy is based on the optical absorption and conversion of light energy into thermal energy or the production of highly toxic active ingredients such as reactive oxygen species (ROS) to destroy cancer cells. Active research has been conducted focusing on the development of light-responsive nanotheranostic biomaterials accompanied by these PTT and PDT effects. Recently, it has been reported for certain nanoterranostick biomaterials such as carbon and metal-based nanomaterials.

On the other hand, fluorescence-based nanotheranostic biomaterials may be useful for real-time imaging and observation in the diagnosis of cancer sites. For such real-time imaging and phototherapy, a number of nanomaterials such as semi-quantum dots, Au-based, CD-based, magnetic-based, etc. have been developed. However, disadvantages such as toxicity and high synthesis cost limit the use of these nanomaterials. However, since CD has properties such as low toxicity, specific tunable surface structure, ease of functionalization, good stability to light, tunable absorption-emission spectrum, etc., fluorescence imaging (FI), two-photon imaging (TP), and It can be a good option for multimodal imaging materials for Raman imaging (RI). In this regard, techniques for developing CD-based nanomaterials are known, such as a chemical method, a microwave method, a hydrothermal method, an ultrasonic method, an oxygen plasma method, and an electrochemical method. However, the present inventors used 3-aminopropyl triethoxysilane (APTES) as a surface modifier and a simple sol-gel method of heat treatment. Recently, imaging techniques such as TP spectroscopy using NIR wavelengths have been used for biomedical applications including cell imaging and cancer treatment. NIR laser light has a higher transmittance compared to visible light due to its high intensity and low absorption promoting deep tissue penetration. Research on other applications of carbon nanotheranostic biomaterials for TP imaging and theranostics is still ongoing. Besides these spectroscopic techniques based on absorption and emission of photons, another technique can be used Raman spectroscopy, which is based on inelastic scattering of photons. Raman spectra are more suitable for long-term analysis than FI due to their stability. Carbon-based nanomaterials such as CNT, GO, and CD show characteristic peaks in the Raman spectrum and can be used as non-destructive imaging materials for long-term cell imaging.

Chemotherapy is the main method of destroying cancer cells, but many anticancer drugs are hydrophobic and therefore have low water solubility, which may limit their intravenous administration. CDs are ineffective as carriers for loading anticancer drugs or other biomolecules for therapeutic purposes due to their small size and non-porous nature. Several studies have shown that when using mesoporous nanocarriers, hydrophobic drugs for intravenous administration can be easily loaded due to their excellent drug-holding ability. This approach can improve the efficacy of chemotherapy and reduce side effects. Among various silica-based nanomaterials, mesoporous bioglass can be a good option for targeted delivery due to its high surface area, tunable pore size, and good biocompatibility. Bioglass is based on calcium (Ca) and silica (Si) and is also used for bone and tissue regeneration. Mesoporous bioglass has a high surface area and thus a high drug-loading capacity, and can cause release to occur through changes in light, pH, temperature, etc.

The present inventors tried to develop a material for real-time monitoring, target delivery, and phototherapy, and succeeded in developing a CD-based bioglass nanotheranostic carrier through the sol-gel method using APTES and heat treatment at a specific temperature. .

Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869-10939.

The main object of the present invention is to provide a method that can be usefully used for real-time monitoring, targeted delivery, and phototherapy, and can easily produce carbon point-based mesoporous bioglass with excellent stability at low cost.

Another object of the present invention is to provide a carbon point-based mesoporous bioglass having excellent stability, which is manufactured by the method as described above, is applicable to real-time monitoring, target delivery, and phototherapy.

Another object of the present invention is to provide a composition using the carbon point-based mesoporous bioglass.

According to one aspect of the present invention, the present invention comprises the steps of: a) dissolving cetyltrimethylammonium bromide, 2-methoxyethanol, ethanol and aqueous ammonia in water to prepare a mixture; b) adding calcium nitrate, tetraethyl orthosilicate and 3-aminopropyl triethoxysilane to the mixture and stirring; c) obtaining particles resulting from said addition and stirring; and d) heat-treating the particles at 350 to 450° C.; provides a method for producing mesoporous bioglass nanoparticles comprising.

In the manufacturing method of the present invention, the molar ratio of Ca:Si is preferably 5:95 to 25:75.

In the production method of the present invention, the volume ratio of the tetraethyl orthosilicate to 3-aminopropyl triethoxysilane is preferably 1: 2 to 2: 1.

In the manufacturing method of the present invention, the stirring of step b) is preferably performed for 3 to 5 hours.

According to another aspect of the present invention, the present invention provides mesoporous bioglass nanoparticles prepared by the above manufacturing method.

According to another aspect of the present invention, the present invention provides a composition for drug or biomolecule delivery comprising the nanoparticles.

According to another aspect of the present invention, the present invention provides a composition for bioimaging comprising the nanoparticles.

According to another aspect of the present invention, the present invention provides a photosensitizer composition for photodynamic therapy comprising the nanoparticles as an active ingredient.

According to another aspect of the present invention, the present invention provides a photothermal agent composition for photothermal treatment comprising the nanoparticles as an active ingredient.

According to another aspect of the present invention, the present invention provides a composition for theranostics comprising the nanoparticles as an active ingredient.

According to the manufacturing method of the present invention, strong multicolor fluorescence properties can be exhibited to effectively image cancer cells in vivo, and it can also be used for two-photon spectroscopy imaging and Raman spectroscopy imaging. , photothermal (PTT) and photodynamic (PDT) properties, efficient loading of biomolecules, transport to target and controlled release, and excellent biocompatibility carbon point-based mesoporous bioglass Nanoparticles can be prepared. In addition, the nanoparticles of the present invention can be used for drug or biomolecule delivery, bioimaging, photodynamic therapy, or photothermal therapy due to the above characteristics. It can be used for theranostics.

1 is (a) nanoparticles of the present invention (fBGn); (b) high doxorubicin loading and controlled release mechanism; and (c) schematic diagrams showing drug delivery, optical triple-mode imaging, and light-guided cancer therapy.
2 shows (a) the manufacturing process of nanoparticles (fBGn) of the present invention, and (b) low- and high-magnification TEM images of BGn (nanoparticles before heat treatment) and fBGn samples.
Figure 3 shows the properties of the nanoparticles (fBGn) of the present invention. (a) UV-vis spectra, (b) Raman spectra, (c) FT-IR spectra, (d) wide-scan XPS spectra, (e) narrow (C 1s) scans, and (f) narrow (N) 1s) scan.
4 shows (a) a sample of the present invention nanoparticles (fBGn) treated with a hydrofluoric acid (HF) solution and TEM images, (b) high-resolution TEM images, (c) PL emission spectra, (d) EPR spectra, (e) Shows the fluorescence mechanism.
5 shows the label-free fluorescence properties of nanoparticles (fBGn) of the present invention. (a) a schematic diagram showing that the nanoparticles (fBGn) of the present invention are produced by heat treatment of BGn, (b) emission spectra of fBGn prepared by treatment at different temperatures at an excitation wavelength of 360 nm, (c) at different temperatures The emission wavelength of the three peaks (P ₁ , P ₂ , and P ₃ ) measured, (d) the emission intensity of the three peaks (P ₁ , P ₂ , and P ₃ ) measured at different temperatures, (e) 2-D excitation-emission contour map of fBGn at different excitation wavelengths, (f) lifetime decay profile measured by monitoring emission at 500 nm, (g) two different concentrations of fBGn in living HeLa cells under bright field of the confocal fluorescence image.
6 shows the TP spectra of nanoparticles (fBGn) of the present invention excited with 800 nm laser light. Images of (a) emission spectra according to different temperatures at 800 nm excitation wavelength, (b) emission spectra according to different excitation wavelengths, and (c) fBGn under laser light excitation at 800 nm wavelength.
7 shows Raman images (RI) of HeLa cells and nanoparticles (fBGn) of the present invention on a silica wafer under laser light of a wavelength of 514 nm. (a) laser spot, (b) PL image with background, (c) PL image without background, (d) optical image, (e) Raman image of relative intensity of G with low intensity, (f) high intensity Raman images of the relative intensities of G with .
8 shows the PDT/PTT effect of nanoparticles (fBGn) of the present invention. (a) to (d) PTT light (808 nm) treatment of various concentrations of fBGn, (a) PTT thermal curve, (b) temperature change, (c) temperature NIR image, and (d) HeLa cells Relative cytotoxicity, (e) to (h) as a result of treating fBGn with PDT light (80 mW, 660 nm), (e) ¹ O ₂ -induced signal of fBGn irradiated with ESR spectra under dark and LED light conditions, (f) photolysis of DPBF, (g) ROS levels measured in HeLa cells treated with PBS, fBGn, fBGn+light irradiation, (h) cytotoxicity to HeLa cells, (i) and (j) PDT/PTT binding effect , (i) Confocal images and calcein AM/PI-stained fluorescence images of HeLa cells cultured by treatment with fBGn under various conditions, (j) Cytotoxicity to HeLa cells at various concentrations.
FIG. 9 shows the results of testing the loading and release of doxorubicin (DOX), an anticancer agent, using the nanoparticles (fBGn) and MSN (control) of the present invention. (a) anticancer drug loading, (b) loading and release mechanism, (c) DOX release as a function of pH (5.0 and 7.4) at 37°C, (d) cell viability of HeLa cells.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Terms such as first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another.

The method for preparing mesoporous bioglass nanoparticles of the present invention comprises: a) preparing a mixture by dissolving cetyltrimethylammonium bromide, 2-methoxyethanol, ethanol and aqueous ammonia in water; b) adding calcium nitrate, tetraethyl orthosilicate and 3-aminopropyl triethoxysilane to the mixture and stirring; c) obtaining particles resulting from said addition and stirring; and d) heat-treating the particles at 350 to 450°C.

In the production method of the present invention, preferably, the molar ratio of Ca:Si is 5:95 to 25:75, and more preferably 10:90 to 20:80.

In the production method of the present invention, preferably, the volume ratio of the tetraethyl orthosilicate: 3-aminopropyl triethoxysilane is 1: 2 to 2: 1, more preferably 1: 1.5 to 1.5: 1, More preferably, it is 1:1.2 to 1.2:1.

In the manufacturing method of the present invention, preferably, the stirring of step b) is performed for 1 to 10 hours, more preferably 2 to 8 hours, more preferably 3 to 5 hours.

In step a), 0.1 to 3 g of cetyltrimethylammonium bromide, 5 to 50 ml of 2-methoxyethanol, 2.5 to 25 ml of ethanol, and 0.5 to 5 ml of aqueous ammonia are preferably used based on 150 ml of water, more preferably Usually, 0.5 to 2 g of cetyltrimethylammonium bromide, 10 to 30 ml of 2-methoxyethanol, 5 to 15 ml of ethanol, and 1 to 3 ml of aqueous ammonia are used based on 150 ml of water.

Step c) may be performed in a manner to obtain a white precipitate produced by centrifugation after stopping the stirring of the mixture after step b).

In the manufacturing method of the present invention, preferably, a step of washing the particles is added between steps c) and d). In the process of obtaining the particles in step c), unnecessary substances such as cetyltrimethylammonium bromide may be obtained together with the particles. Wash using a mixed solution of ethanol.

Step d) is a step for generating a carbon point (CD) in the silica matrix of the mesoporous bioglass nanoparticles produced in step c), characterized in that it is heat-treated at 350 to 450°C. The carbon point is related to the fluorescence properties of the nanoparticles of the present invention, and these properties are related to the heat treatment temperature of step d). Accordingly, the heat treatment temperature is preferably set to 360 to 440 °C, more preferably 370 to 430 °C, more preferably 380 to 420 °C, and more preferably 390 to 410 °C.

According to the manufacturing method as described above, carbon dots are generated in the bioglass silica matrix, and nanoparticles having a high surface area and mesoporous properties can be prepared. At this time, the high surface area and mesoporous properties enable the efficient loading of drugs or biomolecules.

The nanoparticles of the present invention prepared by the above method are preferably spherical, and preferably have a size of 50 to 200 nm. Further, it preferably has a BET surface area of 600 to 700 m ² /g, preferably has a pore volume of 0.1 to 1 cm ³ /g, and preferably has a pore size of 2 to 5 nm. It also preferably has a carbon point in the size of 1 to 5 nm.

The nanoparticles of the present invention may exhibit strong fluorescence properties and multicolor fluorescence properties, and these fluorescence properties may be exhibited by the carbon point.

The nanoparticles of the present invention can emit different colors when the excitation wavelength is 405 nm (blue), 454 nm (green) and 546 nm (red), respectively. Accordingly, the nanoparticles of the present invention can be used as an imaging agent for multi-color bioimaging in bio-imaging applications, particularly in the field of biomedical science, and can be used as an active ingredient in a bio-imaging composition.

The nanoparticles of the present invention can emit light even at an excitation wavelength of near-infrared (NIR). For example, when the excitation wavelength is 800 nm, light having a wavelength of 450 to 550 nm may be emitted. Such near-infrared rays can easily penetrate deeper into tissues than visible light. Therefore, the nanoparticles of the present invention can enable more effective bio-imaging using near-infrared rays. Accordingly, the seventh invention also provides a biological imaging method using the nanoparticles of the present invention and using near-infrared light as an excitation wavelength. In this case, the near-infrared is preferably a near-infrared with a wavelength of 750 to 850 nm, more preferably a wavelength of 760 to 840 nm, more preferably a wavelength of 770 to 830 nm, more preferably a wavelength of 780 to 820 nm, more preferably 790 to 810 nm wavelength.

The nanoparticles of the present invention have two-photon fluorescence properties, so they can be used for two-photon spectroscopic imaging.

In addition, the nanoparticles of the present invention may exhibit a Raman effect, and thus may be used for Raman spectroscopic imaging.

Through the present invention, the photothermal (PTT) characteristics of the nanoparticles of the present invention were confirmed. For example, heat energy can be produced when irradiated with laser light of 808 nm, and sufficient heat energy can be produced to kill tumors or cancer cells. Therefore, the nanoparticles of the present invention can be used as a photothermal agent for photothermal therapy, and can be used as an active ingredient in a photothermal agent composition for photothermal therapy.

In addition, the photodynamic (PDT) characteristics of the nanoparticles of the present invention were also confirmed through the present invention. For example, when irradiated with LED light of 660 nm, ¹ O ₂ ROS (reactive oxygen species) can be produced, which can kill tumors or cancer cells. Therefore, the nanoparticles of the present invention can be used as a photosensitizer for photodynamic therapy, and can be used as an active ingredient in a photosensitizer composition for photodynamic therapy.

As described above, the nanoparticles of the present invention can exhibit both photothermal and photodynamic properties. Therefore, the nanoparticles of the present invention can be used as a photosensitizer-photothermal agent for photodynamic and photothermal combination-treatment, and can be used as an active ingredient in a photosensitizer-photothermal agent composition for photodynamic and photothermal combination-treatment. In particular, according to the present invention, compared to the case of separately used as a photothermal agent for photothermal treatment or a photosensitizer for photodynamic treatment, when used as a photosensitizer-photothermal agent of photodynamic and photothermal combination-treatment, tumors or cancer cells can be more effectively killed. can

In addition, it was confirmed that the loading and controlled delivery of drugs or biomolecules of the nanoparticles of the present invention were possible through the present invention. For example, the nanoparticles of the present invention can efficiently load doxorubicin (DOX), an anticancer agent, and a pH-dependent release is possible. The pH-dependent release properties show that controlled release of drugs or biomolecules is possible given that the environment of tumors or cancer cells is slightly acidic. Therefore, the nanoparticles of the present invention can be used as a drug or biomolecule delivery agent, and can be used as an active ingredient in a composition for drug or biomolecule delivery.

As described above, the nanoparticles of the present invention enable drug or biomolecule delivery, bioimaging, photodynamic therapy, and photothermal therapy. Therefore, the nanoparticles of the present invention can be used as a material for theranostics, which combines diagnosis and treatment, and can be used as an active ingredient in a composition for theranostics.

When administering the nanoparticles or composition of the present invention to the human body, the dosage may be adjusted according to the condition of the subject, administration route and dosage form, and those of ordinary skill in the art can use the present invention The dosage may be determined based on the presented efficacy test results and cytotoxicity test results, or any additional experiments.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

1. Materials and Methods

1.1. fBGn Nanoparticle Synthesis

Mesoporous bioglass nanoparticles (BGn) were prepared using the Stover method (45). 1 g of cetyltrimethylammonium bromide (CTAB), 20 ml of 2-methoxyethanol, 10 ml of ethanol, and 2 ml of aqueous ammonia were dissolved in 150 ml of water. After stirring for 30 minutes, Ca(NO ₃ ) ₂ .4H ₂ O, tetraethyl orthosilicate (TEOS), and APTES as a silane functionalizing agent were added to the mixture and stirred for an additional 4 hours. The molar ratio of Ca/Si was set to about 15:85, and the volume ratio of TEOS to APTES was set to 1:1. Water and ethanol were used to remove CTAB from the precipitated white powder, and finally, to obtain BGn nanoparticles, it was dried at 70° C. overnight. Afterwards, the generated BGn nanoparticles were heat treated at different temperatures of 250, 300, 350, 400, 450, 500, and 600° C. in the presence of air for 2 hours. The heat-treated nanoparticles are the final product of fBGn synthesis. .

1.2. Sample characterization

The shape and size of the nanoparticles were confirmed by JEOL-7100 TEM (transmission electron microscopy). Brunauer-Emmett-Teller (BET) surface area, pore-volume, and mesopore size distribution were measured with a surface area analyzer (2SI-MP-9 Quantachome Instruments). Surface binding chemistry and composition ratio were analyzed using X-ray photoelectron spectroscopy (XPS). A JES-FA200 EPR (electron paramagnetic resonance) spectrophotometer was used to analyze the presence of crystal lattice defects in nanomaterials. Raman spectra were obtained using a Raman spectrometer (LabRAM HR UV/vis/NIR) operated with a 550 nm red laser. Thereafter, the chemical bonding structure was analyzed using a Fourier transform infrared spectroscopic technique (FT-IR) (Varian 640-IR). Spectra related to optical absorption were obtained using a Cary100 Ultraviolet-visible-NIR (Ultraviolet-visible-NIR) spectrometer. One-photon excitation fluorescence or fluorescence properties and 2D contour excitation/emission maps of fBGn in aqueous solution were detected using a UV-vis filter at various wavelengths using a Jasco FP-6500 instrument. The decay lifetime was measured using an iHR320 spectrophotometer. TP excitation fluorescence spectroscopy of fBGn in aqueous solution was observed using a fiber spectrometer. An amplified Ti:sapphire laser system (SpectraPhysics, 50 fs, 1 kHz) with a tuning range of 550 to 900 nm was used as the light excitation source.

1.3. Confocal FI

HeLa cancer cells were inoculated on a glass chamber slide (1x10 ⁴ cells) and maintained at 37°C for 12 hours. Thereafter, the cell medium was replaced with a fresh cell medium containing 50 or 100 μg/ml of fBGn nanoparticles, and incubated at 37° C. for about 4 hours. Cells were washed with phosphate-buffered saline (PBS) and imaged using a confocal microscope with three different excitation wavelengths.

1.4. Raman imaging

Cells were cultured in α-MEM medium for 24 hours under appropriate culture and adherence conditions using 24-well plates (1x10 ⁴ cells/well). The cells were then treated with fBGn for about 6 hours. After incubation for 6 hours, the adhered cells were washed with PBS and treated with trypsin-EDTA to remove the cells from the plate and resuspended in PBS. Drops of cell suspension were added to the silicon wafer to avoid absorption of fBGn into the cell membrane or silicon wafer during use for RI.

1.5. Evolution of the PTT effect of fBGn

To investigate the PTT efficacy induced by 808nm laser irradiation (1W/cm ² ), fBGn was used at different concentrations (0, 25, 50, 100, 200, and 400 μg/ml). A thermal NIR imaging camera was used to record the temperature change of the fBGn solution for 6 min. After 6 min the laser light was turned off and the temperature decrease was recorded to determine PTT efficacy.

In vitro cytotoxicity was investigated using HeLa cells and standard CCK-8 methods. Cells were inoculated (1x10 ⁴ cells/well) in a 96-well plate and cultured for 24 hours to attach cells using α-MEM medium. The cells were re-treated with fBGn at various concentrations (0 to 800 μg/ml) for about 4 hours. Laser irradiation was performed at 808 nm for 5 minutes and then incubated for an additional 24 hours. Finally, the relative cytotoxicity of the cells was determined using standard CCK-8 methods.

1.6. Evolution of the PDT effect of fBGn

In order to determine singlet oxygen species ( ¹ O ₂ ) in biological systems, a highly reactive capture agent DPBF (1,3-diphenylisobenzofuran) was used as a standard reagent. In this experiment, fBGn nanoparticles were added to about 1 ml of a DPBF (100 μg/ml) solution and sonicated for 10 minutes in the absence of light. Thereafter, the mixture was irradiated with 660 nm (80 mW) LED light. The optical absorption (408 nm) of DPBF was measured at different irradiation times using a UV spectrophotometer.

ESR spectrum irradiation method was used to detect ¹ O ₂ ROS generated under the influence of irradiated light. TEMP (2,2,6,6-Tetramethylpiperidine) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) were used as trapping agents for ¹ O ₂ and O2 ^-● (or OH ^● ), respectively. Here, the fBGn solution was added to the TEMP or DMPO solution, and sonication was performed for 10 minutes in the absence of light. The ESR spectrum of the fBGn sample was measured before and after irradiation with LED light at room temperature.

The ROS assay kit was used to confirm the presence of ROS in vitro. Cancer cells were inoculated into a cover glass chamber and cultured in α-MEM medium for about 24 hours. Thereafter, the cells were treated with fBGn for 4 hours and then treated with DCFH-DA (2,7-dichloro-dihydro-fluorescein diacetate) for an additional 20 minutes. Cells were washed with Hanks' buffered saline and finally irradiated with light of 660 nm for 20 minutes. Thereafter, fluorescence images were obtained using a confocal microscope.

The cytotoxicity of fBGn in vitro was investigated using standard CCK-8 methods using HeLa cells. Under sterile conditions, the cells were inoculated into a 96-well plate (1x10 ⁴ cells/well) and cultured for 24 hours in α-MEM medium to allow the cells to adhere. The cells were then treated with fBGn at various concentrations (0 to 800 μg/ml) for 4 hours. Thereafter, the cells were irradiated with a 660 nm LED for 20 minutes and incubated for an additional 24 hours. Finally, the relative cytotoxicity was determined using standard CCK-8 methods.

1.7. Effect of combining PTT and PDT in vitro

To investigate the combination of PDT/PTT efficacy of fBGn, cells were seeded into culture plates and cultured for 24 hours to adhere to cells in α-MEM medium under suitable conditions. Cells were treated with fBGn for 4 hours, 660 nm light for 20 minutes, and 808 nm laser for 5 minutes, respectively. After treatment, cells were co-stained with Live/Dead kit for 20 minutes and fluorescence images were obtained by confocal microscopy.

To investigate the antitumor effect of PDT and PTT binding, the cytotoxicity of fBGn was investigated using HeLa cells in the presence and absence of light. The cells were treated with fBGn for 4 hours and then irradiated with a light of 660 nm for 20 minutes and a laser of 808 nm for 5 minutes, respectively. After incubation, relative cytotoxicity was determined using standard CCK-8 methods.

1.8. Loading and release testing of anticancer drugs

Doxorubicin (DOX) is a commonly used anticancer drug. To load this DOX into MSN or fBGn nanoparticles, 1 mg of nanoparticles were added to 1 ml of DOX at different concentrations (80, 100, and 120 μg/ml) in the absence of light for 24 hours. Then, the solution was centrifuged and the absorbance was measured at 484 nm using a UV spectrophotometer. A standard spectral curve was then used to quantify the efficacy of DOX loading. To investigate the release curves of DOX from DOX-loaded nanoparticles (MSN and fBGn), two different pH values (5.0 and 7.4) were selected at various time intervals at 37°C. The mixture was centrifuged and the absorbance of the supernatant was measured at 484 nm for each time interval, the particles were resuspended in fresh phosphate buffer at each time point, and finally the DOX release efficiency of the nanoparticles was quantified using a standard curve.

1.9. In vivo bioimaging and biocompatibility

All animal experiments were performed in accordance with the guidelines of Korea and Dankook University Animal Protection Research Committee. In the first experiment, topical administration was performed, and fBGn nanoparticles (100 μl, in 2 mg/ml saline) were injected into the right body region of female nude mice. FIs were then recorded using a Maestro multispectral imaging system. In the second experiment, healthy nude mice were used to investigate biocompatibility in vivo. Different doses (5, 10, and 20 mg/kg) of fBGn solutions were intravenously injected through the tail vein of mice (n=3). Additionally, saline was used as a control. After 2 weeks, sacrifices were made to examine the effect of biocompatibility, and tissue samples were collected. Collected internal organs such as heart, lung, liver, spleen, and kidney were fixed in 4% formaldehyde, embedded in paraffin, and sliced. Thereafter, it was stained with H&E and examined under an optical microscope.

2. Results

2.1. Synthesis of CD-associated fBGn and its characterization

2A is a schematic diagram showing the fabrication of multicolor fluorescent fBGn. After heat treatment, BGn was converted to fluorescent BGn (fBGn) due to the generation of CD in the matrix. fBGn was prepared by a sol-gel method in the presence of APTES to form spherical mesoporous silica bioglass (BGn). After heat treatment, APTES produced CDs in silica bioglass silica matrix exhibiting multicolor fluorescence fBGn. Low- and high-magnification TEM images of BGn and fBGn samples containing APTES are shown in Figure 2b. Here, both the spherical BGn and fBGn have a size of 100±10 nm. The fBGn sample has numerous black dots distributed throughout the structure representing the CD inside the silica matrix. The N2 adsorption/desorption hysteresis loop showed that fBGn had a BET surface area of 650 m ² /g, a pore volume of 0.637 cm ³ /g, and a pore size of 3.07 nm. This high surface area and mesoporous size is useful for loading biomolecules.

Next, the characteristics of CD in fBGn were investigated. First, the UV-vis spectrum of the fBGn sample was analyzed as shown in FIG. 3a. fBGn showed two peaks at about 230 and about 340 nm, indicating the sp ² domain of the π-π* electron transition (C=C bond) and the unbound oxygen state of the n-π* electron transition (CO bond). it will indicate In addition, carbon-based nanomaterials have characteristic peaks in the fingerprint region in the Raman spectrum. As shown in FIG. 3b , two representative Raman peaks of fBGn, the D band at about 1400 cm ^-1 and the G band at about 1600 cm ^-1 , are characteristics of the Raman vibration modes sp ³ and sp ² , respectively. The D and G bands of the carbon structure represent disordered or carbon-related defects with amorphous and crystalline properties, respectively. The nature of the carbon structure depends on the strength ratio I _D / I _G . Next, the chemical structure was analyzed using the FT-IR spectrum as shown in FIG. 3C. fBGn is the Si-O bond (465 cm ^-1 ), Si-O-Si symmetric stretching (1095 cm ^-1 ), C=C stretching vibration (1414 cm ^-1 ), NH vibration (1560 cm ^-1 ), C=O/C =N shows Si- and C-related bands representing stretching oscillations, and CO stretching (880 cm ⁻¹ ). Further chemical composition, structure, and binding energy were analyzed using XPS with wide scan applied. As shown in FIG. 3d, the binding energies of Si, Ca, C, N, and O were shown. Figure 3e shows the C 1s spectra, which were fitted using the Gaussian method of dividing the main peak into four sub-peaks of CC, CN, C=O, and OC, showing the graphite structure of the sp ² bond. A high-resolution scan, N 1s spectra separated into three peaks: NH, (C)3-N, and CN, where nitrogen is mostly bound to carbon and different emission centers produce different local energy states is shown (FIG. 3f).

Figure 4a shows the treatment of the fBGn sample with 37% hydrofluoric acid (HF) to dissolve the silica-based glass and to make the CD observable through TEM. The TEM image shows black CD nanoparticles with background degradation of bioglass (Fig. 4a). In the inset, the size of the CD was about 1.6±0.4 nm. High-magnification TEM images show that CD has both crystalline and amorphous phases, as indicated by red and yellow circles, respectively (Fig. 4b). In addition, the fluorescence properties of fBGn before and after treatment with HF solution were investigated (Fig. 4c). The fluorescence intensity before and after HF treatment was found to be almost the same. This indicates that the strong fluorescence properties are due to the CD and not the silica matrix. Another possibility for fluorescence emission is due to crystal defects present in the silica matrix. Therefore, to confirm the fluorescence mechanism, EPR spectroscopy was used to investigate the crystal defects present in the material. EPR spectroscopy is a very sensitive technique used to identify crystal lattice defects in materials. Figure 4d shows that both samples show no EPR signal, indicating that the material has no silica-silica, silica-oxygen, and silica-carbon defects. This also demonstrates that the origin of the fluorescence is due to CD. Based on these results, the origin of the fluorescence mechanism of Fig. 4e is proposed. CD shows the full range of visible polyfluorescence of excitation wavelength-dependent emission. Polychromatic fluorescence depends on the surface oxidation edge state, the amorphous region, the nitrogen passivation of the surface state, and the crystalline region. Surface oxidation states are associated with carbon and oxygen bands that change with π or n-domain bonded electron transition band gaps. The polyfluorescence properties depend mainly on the surface state energy, which depends on the C-O, C=O, and O=C-OH emission modes. The surface energy state of the nitrogen-containing CD acts as a passivating material. This effective group is used to achieve a passivated state and to enable multifluorescence.

2.2. In vitro bioimaging and multicolor fluorescence of CD-associated fBGn

5a shows BGn heat-treated at different temperatures ranging from 250 to 600° C. to form CDs in silica matrix for polychromatic fluorescent fBGn. The fluorescence properties of fBGn were investigated using PL (photo-luminescence) measurement at λ _ex 360 nm ( FIG. 5b ). The fabricated samples had no fluorescence properties, but all heat-treated samples showed PL curves indicating the fluorescence properties. The PL curve showed three emission peaks, P ₁ , P ₂ , and P ₃ , with emission wavelengths ranging from 428 to 453, 469 to 481, and 511 to 513 nm, respectively ( FIGS. 5b to c ). The change in emission wavelength depends on the temperature at which fBGn is annealed. Due to the maximum generation of CD in the silica network, the maximum peak intensity was observed at 400 °C (Fig. 5d). After the higher temperature heat treatment, the CD burned completely in the silica matrix. Samples at 400° C. were selected for further experiments. Multiple peaks of the curve were fit using a Gaussian peak distribution, and the area of the three peaks was calculated using a specific wavenumber. The P ₁ , P ₂ , and P ₃ emission peaks have areas of 10.04, 28.25, and 61.71%, respectively. The area of the P ₃ emission peak was quite high and showed maximum fluorescence at 512 nm emission wavelength. Next, fBGn was stimulated with various wavelengths and the intensity of the emission peak was shown in a 2D contour diagram as shown in FIG. 5E. The maximum intensity of the emission was observed in the range of 470 to 550 nm. The lifetime decay profile was measured by monitoring the emission at 500 nm using the bi-exponential data and the calculated decay time of 6.8 ns (Fig. 5f). First, fBGn nanoparticles were dispersed in a buffer and stimulated with different wavelengths using PL spectroscopy. In addition, the full-color range of fBGn was observed with the naked eye at the corresponding excitation wavelength by irradiating xenon light. This fBGn can be used for multicolor imaging in biomedical applications. The application of such fBGn as an effective multicolor imaging material has been successfully demonstrated using cancer cells. First, HeLa cancer cells were inoculated into a culture plate and fBGn was treated in two different concentrations (50 and 100 μg/ml) for about 24 hours. Then, living cancer cells were observed using CLSM under three different excitation wavelengths of blue (405 nm), green (454 nm), and red (546 nm), respectively. It was observed that fBGn was present in the cytoplasm while emitting different colors. As the concentration of fBGn increased, the fluorescence intensity also increased due to the presence of more fBGn particles. Therefore, these fBGn materials can be used for multicolor bioimaging in biomedical fields.

2.3. Application of CD-Related fBGn to TP Spectroscopy for Bioimaging

Other unique properties of fBGn under the NIR wavelength of 800 nm were investigated using TP fluorescence spectroscopy. TP spectroscopy using NIR wavelengths is widely used for biomedical applications such as cell imaging and cancer treatment. Since NIR laser light has low absorption properties in soft tissue, it can penetrate deeper into the tissue more easily than visible light. Two spectroscopic techniques, one-photon and TP spectroscopy, were used to stimulate fBGn at two wavelengths (450 and 800 nm). Similar emission trends were observed at both excitation wavelengths. The TP emission spectra of fBGn were investigated at different temperature ranges using an excitation wavelength of 800 nm. The TP emission intensity in Fig. 6a shows that a strong emission peak was observed at about 500 nm, but the fluorescence intensity is dependent on the temperature at which fBGn was prepared. High TP emission intensity of the fBGn sample was observed at 400°C similar to that in PL spectroscopy due to the maximum generation of CD in the silica network. After high temperature heat treatment, the CD was completely burned out of the silica matrix. When the excitation wavelength of the laser light was changed from 800 to 600 nm, the emission intensity of fBGn at 400° C. was shown to decrease along with the emission peak at the same position as the excitation wavelength of the laser light was decreased (Fig. 6b). In addition, the TP spectra image of fBGn shown in green on the glass slide was analyzed (FIG. 6c). Thus, this fBGn system shows future biological applications in cellular imaging by TP spectroscopy, deep tissue imaging, and cancer theranostics.

2.4. CD-Related fBGn for Raman Spectroscopic Imaging

Carbon-based nanomaterials are most widely used for cancer theranostics and cellular imaging to aid in the detection and diagnosis of diseases. Recent studies show that techniques such as fluorescence, TP, and RI, which exhibit excellent spatial resolution, can be effectively used for cell imaging. RI is more stable for long-term monitoring compared to FI. Carbon-based nanomaterials such as CNT, GO, and CD have characteristic fingerprint regions in Raman spectra. In this study, cells were exposed to fBGn for about 6 hours and collected. A drop of cell suspension was placed on a silicon wafer. Figure 7a clearly shows the spherical shape of a single droplet of cells with a laser beam. A 514 nm laser beam was used and the fBGn nanoparticles were then stimulated with this wavelength, which appears red in the background due to the characteristic emission. Figure 7b shows that these results are in close agreement with PL measurements and confocal image studies. After removing the background, fBGn appears in red as in Fig. 7c. An optical image of the cells was also obtained (Fig. 7d). BGn with two typical Raman peaks is shown in the inset of Fig. 7e, where the D(1330 cm ⁻¹ ) and G (1595 cm ⁻¹ ) bands are characteristic of the Raman vibrational mode in the carbon structure. The G oscillation band was used as the cell scan probe, and the final image is the Raman image of the cell in Fig. 7e. Due to the increase in the peak intensity of the G band as the laser irradiation power was increased, it was found that the Raman signal was remarkably improved (Fig. 7f). Therefore, it is thought that the Raman image signal is highly dependent on the intensity of the laser beam and the concentration of fBGn. This indicates that fBGn was used for RI instead of FI in cells.

2.5. PTT and PDT effects of CD-related fBGn in synergistic cancer therapy

The PTT effect is one of the most common properties exhibited by carbon-based nanomaterials. The aqueous solution of fBGn showed different temperatures at different fBGn concentrations when laser light (808 nm) was irradiated for up to 600 seconds (FIG. 8a). The temperature of the aqueous solution increased from 7.6 to 38.6 °C at fBGn concentrations ranging from 25 to 400 µg/ml, which is considered significant compared to the case of irradiation with nanoparticles-free water exhibiting a ΔT of about 2.4 °C (Fig. 8b). Next, the time-dependent temperature curve was recorded until the fBGn aqueous solution reached the temperature of the stable stage and cooled to room temperature. Based on the heating-cooling data, the PTT conversion equation was used to calculate the PTT efficacy. The obtained PTT efficacy was about 32.4% based on the heat-cool data, which was similar to the previous report. In addition to this, NIR images of the fBGn aqueous solution were recorded at different concentrations of fBGn after irradiating for 600 seconds using laser light with a wavelength of 808 nm (FIG. 8c). In addition, the PTT efficacy of fBGn was quantitatively investigated and toxicity studies were conducted. As shown in FIG. 8D , the cytotoxicity of HeLa cells decreased as the concentration increased under laser light irradiation, whereas the quantitative PTT efficacy of fBGn was the ability to destroy cancer cells in the presence of CD in the bioglass. CD produced sufficient thermal energy to effectively kill cancer cells under laser light irradiation.

ESR spectroscopy was used to investigate the PDT ability of fBGn in vitro according to ¹ O ₂ ROS emission on LED light (660 nm) irradiation. The produced ¹ O ₂ or OH ^● was detected using TEMPO and BMPO, which are appropriate spin trapping materials, respectively (FIG. 8e). As shown in the ESR spectrum, TEMPO induced a ¹ O ₂ signal in fBGn under LED light irradiation and no ¹ O ₂ signal was generated in the dark. Therefore, BMPO did not detect any signal because it generated other ROS signals with fBGn. These results show the ability of fBGn to produce ¹ O ₂ in the presence of laser light and no other ROS were detected. In addition, the generation of ¹ O ₂ signal by fBGn was confirmed by a chemical trapping method using DPBF, a fluorescent molecule, as a trapping material for absorption of ¹ O ₂ signal. As shown in Fig. 8f, the DPBF absorption intensity of fBGn at 408 nm due to the decomposition of DPBF reacted with ¹ O ₂ under light irradiation gradually decreased as the LED light exposure period increased. These results indicate the ability of fBGn to generate ¹ O ₂ in the presence of LED light. ^{The 1} O ₂ production rate or quantum yield was calculated by linear fitting of DBPF absorption and irradiation times. ^{The 1} O ₂ quantum yield was 0.86, similar to the previous report. In addition, detection of the ¹ O ₂ ROS level of fBGn in HeLa cancer cells was analyzed using the ROS assay kit. As shown in Fig. 8g, HeLa cells treated with fBGn showed enhanced green fluorescence under LED light. Conversely, no green signal was detected in the absence of fBGn. Since fBGn nanoparticles exhibit fluorescence, green fluorescence was observed in the presence of fBGn, but this fluorescence was not observed equally in the cytoplasm compared to fluorescence according to LED light irradiation. ¹ O ₂ ROS is produced in HeLa cancer cells under light irradiation. Therefore, all in vitro PDT results indicate that ¹ O ₂ ROS is released by fBGn under light irradiation. In addition, the PDT efficacy of fBGn was quantitatively analyzed and a cytotoxicity test was performed. As shown in FIG. 8h , on laser light irradiation, it was shown that the viability of HeLa cells decreased as the concentration of fBGn increased, whereas the significant PDT efficacy of fBGn could effectively kill cancer cells due to the presence of CD in the bioglass. CD produced sufficient ¹ O ₂ ROS to effectively kill cancer cells under 808 nm light irradiation.

The effect of PDT/PTT binding treatment on HeLa cancer cells according to fBGn in vitro was investigated using the LIVE/DEAD assay ( FIG. 8i ). The method of co-staining these HeLa cells with calcein-AM (green) and PI (red) can be used to differentiate live and dead cells. In the three groups (cells, fBGn, and light irradiation), no cell death was observed (no red fluorescence) as all cells survived and exhibited green fluorescence. However, cells treated with fBGn showed intracellular autofluorescence of fBGn in the absence of irradiation. These results indicate that cell medium or water does not have the ability to generate ¹ O ₂ ROS to induce apoptosis under light irradiation. Because 660 nm LED light (PDT) or 808 nm laser (PTT) irradiation results in the generation of sufficient ¹ O ₂ ROS or thermal energy to effectively kill cancer cells, HeLa cells are irradiated with either 660 nm LED light (PDT) or 808 nm laser (PTT). ) was destroyed in the presence of irradiation and fBGn. When compared with the case where PDT and PTT were separately applied, the cell death rate was rapidly increased by the combination of PDT/PTT therapy. The intensity of red fluorescence was significantly increased, indicating complete cell death. The binding of fBGn to PDT/PTT can effectively kill cancer cells under light irradiation. Additionally, the PDT/PTT efficacy of fBGn was quantitatively investigated from the LIVE/DEAD assay. As shown in FIG. 8j , in vitro PDT/PTT in the presence of HeLa cells and without fBGn did not show a decrease in cell viability under light irradiation. However, a decrease in the viability of HeLa cells was observed when exposed to wavelength 660 nm light (PDT) or 808 nm laser (PTT). The combination of PDT/PTT showed a significant decrease in cell viability compared to other treatment groups. The efficacy enhanced by PDT/PTT binding of fBGn effectively destroyed cancer cells under light irradiation. The observed maximal cell death indicates the high therapeutic potential of fBGn in combination therapy.

2.6. Anti-cancer drug loading and controlled delivery for chemotherapy

Another important method for destroying cancer cells is the efficient and targeted delivery of anticancer agents in chemotherapy. The ability to deliver drugs to the target site without a small loss of drug and the structure of nanoparticles are important for developing effective mesoporous nanoparticles for specific site mass delivery. To achieve this, fBGn with a mesoporous structure was used for anticancer drug loading and controlled delivery. 9A shows the drug loading efficacy of MSN and fBGn. The loading efficiency of fBGn was compared using MSN without Ca ²⁺ ions in the silica lattice as a control. The mesoporous properties of MSN and fBGn were almost the same because their preparation methods were similar. This was used for loading DOX, an anticancer agent that has a positive charge and can electrostatically interact with the negative charge of fBGn. DOX was encapsulated within the mesopores of fBGn. Three different concentrations of 80, 100, and 120 μg/ml were chosen. fBGn (1 mg) was added at each concentration for about 24 hours. It was found that the loading increased with increasing concentration. The loading potency of fBGn and MSN was about 92 and 67%, respectively, indicating that fBGn has a higher potency compared to MSN. The loading efficiency was significantly improved due to Ca ²⁺ ions ( FIG. 9b ). The figure clearly shows higher drug loading and controlled release due to Ca ²⁺ ions. DOX molecules exhibit two types of electrostatic interactions with Ca ²⁺ ions and Si-OH groups. This is the main reason why fBGn has a higher drug loading capacity compared to MSN without Ca ²⁺ ions. Additionally, drug release into fBGn at pH (7.4 and 5.0) conditions was investigated. The environment of tumor cells is rather acidic with a pH range of 4.0 to 5.8, indicating that pH-dependent release is a major factor for therapeutic delivery. Nanoparticles were incorporated into endosomes and lysosomes in cancer cells that were slightly acidic at pH 4.0 to 5.8. Figure 9c shows drug release from fBGn and MSN for 48 h under two different pHs. Because of the strong bonding between Ca ²⁺ ions and drug molecules, MSN showed more drug release than fBGn under acidic conditions. Drug molecules were released faster under acidic conditions compared to normal conditions. This indicates that fBGn has higher loading efficacy and controlled drug release properties that can be used in cancer chemotherapy due to the effect of Ca ²⁺ ions. Additionally, drug molecule delivery into HeLa cells was investigated in a cytotoxicity assay using the CCK-8 assay. HeLa cells were treated with different concentrations of fBGn, DOX, and fBGn-DOX for 24 hours. As shown in FIG. 9D , there was no significant cytotoxic effect on HeLa cells up to a concentration of 320 μg/ml of fBGn. In the concentration range of 5-40 μg/ml, free DOX showed slightly lower cytotoxicity than fBGn-DOX. Within the first 24 hours, drug release of fBGn-DOX was not sufficient to destroy cells at any particular concentration. Conversely, concentrations of fBGn-DOX (80 to 640 μg/ml) showed higher cell viability compared to free DOX, indicating that fBGn-DOX drug release was sufficient to destroy cancer cells. Such fBGn-based controlled release could be used for cancer chemotherapy in the near future.

2.7. In vivo imaging and biocompatibility

FI is one of the most important requirements for observation and real-time imaging in the process of diagnosing the location of cancer. fBGn was suspended in saline and used for in vivo imaging in a nude mouse model. First, we observed emission images of fBGn under the corresponding excitation wavelengths. This result indicates that fBGn has polychromatic fluorescence. Fluorescence images were detected in nude mice by local injection of fBGn. These results indicate that fBGn has excellent properties for real-time imaging and phototherapy and can be used for effective cancer treatment.

In vivo compatibility was used to determine the clinical viability of fBGn nanoparticles. The in vivo biocompatibility of fBGn using various doses (0, 5, 10, and 20 mg/kg) was evaluated by IV injection into nude mice. Two weeks later, mice were sacrificed and tissues of major organs were collected. Thereafter, biocompatibility was analyzed using H&E staining. The various doses of fBGn did not show a significant difference compared to the control saline. fBGn nanoparticles showed high histocompatibility in almost all organs.

Figure 1c shows that fBGn can be used in various biomedical applications such as real-time theranostic applications in FI, RI, TP FI, controlled drug delivery, combined PTT and PDT synergistic cancer therapy. Phototherapy using multimodal imaging is a promising strategy in the medical field, which can significantly improve the quality of tumor therapy with advantages such as real-time observation of cancer location and light-induced destruction of cancer cells.

Although the present invention has been shown and described in relation to specific preferred embodiments, the present invention may be modified and changed in various ways without departing from the technical features or fields of the present invention provided by the following claims. It will be apparent to one of ordinary skill in the art.

Claims (10)

a) preparing a mixture by dissolving cetyltrimethylammonium bromide, 2-methoxyethanol, ethanol and aqueous ammonia in water;
b) adding calcium nitrate, tetraethyl orthosilicate and 3-aminopropyl triethoxysilane to the mixture and stirring;
c) obtaining particles resulting from said addition and stirring; and
d) heat-treating the particles at 350 to 450° C.; Mesoporous bioglass nanoparticles manufacturing method comprising a. According to claim 1,
A manufacturing method, wherein the molar ratio of Ca:Si is 5:95 to 25:75. According to claim 1,
The tetraethyl orthosilicate: the volume ratio of 3-aminopropyl triethoxysilane is 1: 2 to 2: 1, the manufacturing method. According to claim 1,
A method of carrying out the stirring of step b) for 3 to 5 hours. A mesoporous bioglass nanoparticles prepared by the method of any one of claims 1 to 4. A composition for drug or biomolecule delivery comprising the nanoparticles of claim 5 . A composition for bioimaging comprising the nanoparticles of claim 5 . A photosensitizer composition for photodynamic therapy comprising the nanoparticles of claim 5 as an active ingredient. A photothermal agent composition for photothermal treatment comprising the nanoparticles of claim 5 as an active ingredient. A composition for theranostics comprising the nanoparticles of claim 5 as an active ingredient.

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