KR20220092291A - Exosome-based fluorescent gold nanoclusters and method for application of cell imaging - Google Patents

Abstract

The present invention relates to exosome-based fluorescent gold nanoclusters, a method for manufacturing the same, and a cell imaging use of the fluorescent gold nanoclusters. The present invention provides the advantage that it can be produced by simply culturing a mixture of exosomes and Au ions at room temperature without harsh experimental conditions or toxic chemicals.

Description

Exosome-based fluorescent gold nanoclusters and cell imaging method using same {Exosome-based fluorescent gold nanoclusters and method for application of cell imaging}

The present invention relates to exosome-based fluorescent gold nanoclusters, a method for preparing the same, and use of the fluorescent gold nanoclusters for cellular imaging.

In recent years, fluorescent metal nanoclusters have been applied to develop bio-imaging or sensing technologies. In particular, fluorescent gold nanoclusters (AuNCs) using protein as a template are receiving great attention as bioimaging, sensing and drug delivery systems due to their excellent fluorescence properties and biocompatibility. Representative examples of this type of nanomaterial include gold, silver, copper and platinum nanoclusters synthesized through interaction with proteins or nucleic acids. AuNCs are usually formed from protein templates, whereas AgNCs and CuNCs are formed from nucleic acid templates. Bovine serum albumin was the first protein used to generate AuNCs and has been widely used for cell imaging and biomolecular detection. In addition, other protein templates such as insulin, lysozyme and transferrin were used for the synthesis of fluorescent AuNCs.

Exosome (exosome) refers to a small endoplasmic reticulum (50 ~ 150nm size) secreted from most cells through the endosomal pathway. Exosomes are receiving great attention in the medical field in that they encapsulate biomolecules such as proteins, nucleic acids, and lipids derived from parental cells. Since exosomes have been reported to contribute to cancer metastasis, much research effort has been focused on deciphering the information provided by exosomes for non-invasive cancer diagnosis in recent years. Exosomes containing various proteins have been reported, including origin-specific markers (EpCAM and MUC-1) and generic markers (HSP 70, TSG101, CD63 and CD81). In addition, exosomes with a stable phospholipid bilayer structure were used as a drug delivery method.

The present inventors were studying a method of using an exosome template to synthesize gold nanoclusters in an environmentally friendly way that does not require harsh conditions or toxic chemicals. Different exosome-based AuNCs (exo-AuNCs) were synthesized, and it was confirmed that the exosome-based AuNCs stained the nucleus, confirming that the exosome-based AuNCs of the present invention can be used for cell imaging.

Accordingly, the present inventors were studying a method of using an exosome template to synthesize gold nanoclusters in an environmentally friendly way that does not require harsh conditions or toxic chemicals, blue and red in neutral and alkaline pH ranges, respectively. Two different exosome-based AuNCs (exo-AuNCs) emitting light were synthesized, and it was confirmed that the exosome-based AuNCs based on the exosomes stained the nucleus, indicating that the exosome-based AuNCs of the present invention can be used for cell imaging. Confirmed.

Accordingly, it is an object of the present invention to provide a method for preparing an exosome-gold nanoparticle cluster comprising the following steps.

(i) preparing exosomes; and

(ii) mixing and culturing the exosome and gold ion (Au ion) salt;

Including, exosome-method for producing a gold nanoparticle cluster.

Another object of the present invention is to provide an exosome-gold nanoparticle cluster produced by the above method.

Another object of the present invention is to provide a cell imaging composition comprising the exosome-gold nanoparticle cluster.

In order to achieve the above object, the present invention is to provide a method for preparing an exosome-gold nanoparticle cluster comprising the following steps.

(i) preparing exosomes; and

(ii) mixing and culturing the exosome and gold ion (Au ion) salt;

Including, exosome-can provide a method for producing a gold nanoparticle cluster.

In order to achieve another object of the present invention, it is possible to provide an exosome-gold nanoparticle cluster produced by the present method.

In order to achieve another object of the present invention, the present invention can provide a cell imaging composition comprising the exosome-gold nanoparticle cluster.

Hereinafter, the present invention will be described in detail.

The present invention is to provide a method for preparing an exosome-gold nanoparticle cluster comprising the following steps.

(i) preparing exosomes; and

(ii) mixing and culturing the exosome and gold ion (Au ion) salt;

Including, exosome-can provide a method for producing a gold nanoparticle cluster.

The present inventors found that tyrosine residues exhibit strong reducing ability at pH values higher than their pKa (about 10), and because the thiol group of cysteine residues binds tightly to Au ions, exosomes containing various proteins with tyrosine and cysteine residues are It was expected to mediate the formation of fluorescent gold nanoclusters. Accordingly, exosomes isolated from the present inventor's fetal bovine serum (6.25mg/mL; 1mL of 1X PBS) and HAuCl ₄ (1.25Mm; 1ml of DW) were mixed at room temperature for 5 minutes, and then NaOH was added was used to adjust the pH and incubated at 37° C., 200 rpm in a shaking incubator for 3 days. The exosome-gold nanoparticle clusters exhibited different fluorescence emission depending on the cultured pH. Blue fluorescent exo-AuNCs with maximum fluorescence intensity at 460 nm were synthesized at various pH values, and among them, the fluorescence intensity was the highest when cultured at pH7. Red fluorescent exo-AuNCs with maximum emission intensity at 670 nm were synthesized only at pH 11.5 (Fig. 1). Blue and red fluorescent exo-AuNCs with high fluorescence signals were generated only in the presence of both exosomes and Au ions (Fig. 2).

The exosome means a small vesicle of a membrane structure secreted from various cells. The diameter of the exosomes is approximately 30-100 nm, and the fusion of the polycystic body and the plasma membrane occurs and refers to vesicles that are released into the extracellular environment. The exo-bit of the present invention may be derived from fetal bovine serum (FBS).

The metal ion salt is chloro (trimethylphosphine) gold (AuClP(CH ₃ ) ₃ ), potassium tetrachloroaurate (III) (KAuCl ₄ ), sodium chloride (NaAuCl ₄ ), chloroauric acid (HAuCl ₄ ), bromide It may be sodium aurate (NaAuBr ₄ ), gold chloride (AuCl), gold (III) chloride (AuCl ₃ ), or gold bromide (AuBr ₃ ), preferably chloroauric acid (HAuCl ₄ ).

In step (ii), when culturing at pH 5.5 to pH 11.5 using hydrochloric acid and sodium hydroxide, when fluorescence is emitted at 460 nm, blue fluorescent exosome-gold nanoparticle clusters can be synthesized, preferably at pH 7.0 to pH 11.5, more preferably pH 7.0.

In step (ii), when culturing at a pH of 11 to 12 using sodium hydroxide, a red fluorescent exosome-gold nanoparticle cluster emitting fluorescence at 670 nm may be synthesized, preferably at a pH of 11.5. .

The present inventors tested the optimization conditions for the blue fluorescent exo-AuNCs and the red fluorescent exo-AuNCs, respectively. Both blue and red fluorescent exo-AuNCs showed the highest fluorescence intensity at the Au ion concentration of 1.25 mM (Fig. 3A). The fluorescence of blue fluorescent exo-AuNCs gradually increased until the 3rd day, and then the fluorescence did not increase any more, whereas the red fluorescent exo-AuNCs reached the highest fluorescence intensity after 1 day, after which the fluorescence intensity decreased. (Fig. 3B). The reason why the red fluorescent exo-AuNCs decreased after 1 day is that the high pH used to promote the formation of red fluorescent exo-AuNCs damages the exosomes. Based on the above optimization experiment, it was confirmed that day 3 and day 1 were optimal conditions for 1.25 mM Au ions and blue and red fluorescent exo-AuNCs, respectively.

The blue fluorescent exosome-gold nanoparticle cluster may be cultured for 1 to 5 days in step (ii) described above, preferably for 3 days.

The red fluorescent exosome-gold nanoparticle cluster may be cultured for 1 hour to 48 hours in step (ii) described above, preferably for 24 hours.

The gold ion salt may be included in a concentration of 0.312 mM to 5 mM, preferably 0.625 mM to 2.5 mM, and more preferably 1.25 mM.

The concentration of the exosome may be included at a concentration of 1 mg/ml to 15 mg/ml, preferably at a concentration of 3 mg/ml to 9 mg/ml, more preferably at a concentration of 6.25 mg/mL may include

As a result of examining the characteristics of blue fluorescent exo-AuNCs and red fluorescent exo-AuNCs synthesized under the optimal conditions, both exo-AuNCs maintained the vesicle shape with a size of less than 200 nm, and the unique shape of the exosome endoplasmic reticulum was maintained. , The size and number of exo-somes were 136.2±3.1 nm and 2.01 Х 10 ¹⁰ particles/mL for blue fluorescence exo-AuNCs, 116 ± 5.9 nm and 1.63 Х 10 ¹⁰ particles/mL for red fluorescence exo-AuNCs, respectively, and blue fluorescence It was confirmed that exo-AuNCs and red fluorescent exo-AuNCs contained 8 (blue fluorescent exo-AuNCs) and 25 (red fluorescent exo-AuNCs) Au atoms, respectively. . The 4f _5/2 and 4f _7/2 binding energy peaks of Au ₈ NCs were 88.48 eV and 84.28 eV, respectively, and the Au ₂₅ NCs were 87.58 eV and 83.68 eV, respectively, indicating that Au ₈ NCs are relatively smaller than Au ₂₅ NCs. was confirmed (FIG. 4).

The present invention can provide an exosome-gold nanoparticle cluster produced by the above method.

The present invention may provide a composition for cell imaging, including the exosome-gold nanoparticle cluster.

The present inventors evaluated the cytotoxicity of exo-AuNCs using the MTT assay, and as a result, blue fluorescent exo-AuNCs up to 1 mg/mL MCF-7 (human breast cancer), HeLa (human cervical cancer), HT29 (human colorectal cancer) showed no cytotoxicity. At a concentration of 5 mg/mL, all three cell lines were highly toxic. Conversely, red fluorescent exo-AuNCs at concentrations up to 10 mg/mL did not show cytotoxicity. All three cell types grew better in the presence of red fluorescent exo-AuNCs at high concentrations, rather, it is interpreted that this compound had a beneficial effect on cell growth (Fig. 5). Overall, red fluorescent exo-AuNCs exhibited low toxicity to cells and good biocompatibility.

The present inventors performed cell imaging using red fluorescent exo-AuNCs. As a result, in all three cell lines, red fluorescent exo-AuNCs effectively stained nuclei in bright red as indicated ( FIG. 6 ). These results confirmed that fluorescent nanomaterials with positive charges can stain cell nuclei, and using this, fluorescent exo-AuNCs can be useful as novel bioimaging probes.

The method for preparing gold nanoclusters according to the present invention is a novel method for synthesizing fluorescent metal nanoclusters using an exosome template, and is produced by simply culturing a mixture of exosomes and Au ions at room temperature without harsh experimental conditions or toxic chemicals. It offers the advantage that this is possible. In addition, by optimizing the reaction conditions to produce blue (Au ₈ NC) and red light-emitting (Au ₂₅ NC) exo-AuNCs with emission wavelengths of 460 nm and 670 nm, respectively, these nanoclusters are less toxic to cells and beneficial to growth, so There is an effect that it can be used as a cell imaging composition having excellent compatibility.

1 is a result of confirming the characteristics of exo-AuNCs synthesized according to the present invention, (A) is a result showing the effect of pH on the formation of blue and red fluorescent exo-AuNCs, (B) is a result showing the light emission at 360 nm Results showing the RFI of blue and red exo-AuNCs, (C) is the UV-Vis absorption spectrum result of exo-AuNCs.
Figure 2 is the result of synthesis of exo-AuNCs synthesized according to the present invention, (A) is the result of measuring the emission intensity at 460 nm of blue-emitting exo-AuNCs, (B) is the emission intensity of red-emitting exo-AuNCs at 670 nm is the measurement result.
3 is a result showing the optimal conditions for the synthesis of Exo-AuNCs, (A) is a result showing the effect of Au ion concentration on the formation of blue and red fluorescent exo-AuNCs (exosome concentration is 6.25 mg/mL After synthesizing with various Au ion concentrations (0.3125, 0.625, 1.25, 2.5 and 5 mM), the fluorescence intensity was calculated by dividing the fluorescence intensity by the maximum fluorescence intensity at the emission wavelengths of 460 nm (blue) and 670 nm (red). Both fluorescent exo-AuNCs were excited at 360 nm), (B) is the result showing the effect of reaction time on the formation of blue and red fluorescent exo-AuNCs (fluorescence intensity at different reaction times (0 to 7 days)) was calculated by dividing by the maximum fluorescence intensity at emission wavelengths of 460 nm (blue) and 670 nm (red). Both blue and red fluorescent exo-AuNCs were excited at 360 nm).
4 is a result of confirming the characteristics of exosomes, (A and B) are scanning electron microscope (FE-SEM) images of blue fluorescent exo-AuNCs (left; A) and red fluorescent exo-AuNCs (right; B) and Energy dispersive X-ray microanalysis results, (C and D) are the results of nanoparticle tracking analysis of blue fluorescent exo-AuNCs (left, C) and red fluorescent exo-AuNCs (right, D), (E) blue X-ray photoelectron spectroscopy spectra of fluorescent exo-AuNCs and red fluorescent exo-AuNCs.
5 is a result of confirming the cytotoxicity of Exo-AuNCs, (A) is a result of blue fluorescent exo-AuNCs, (B) is a result of red fluorescent exo-AuNCs. MCF-7, HeLa and HT29 cells were treated with various concentrations of blue fluorescent exo-AuNCs or red fluorescent exo-AuNCs (0.05, 0.1, 0.5, 1, 5 and 10 mg/mL) and then cultured for 24 hours.
6 is a fluorescence imaging photograph using red Exo-AuNCs. The cell membrane was counterstained with 5-dodecanoylaminofluorescein (500 μM), and the concentration of red fluorescent exo-AuNCs was 3.125 mg/mL.

Hereinafter, examples will be given to describe the present specification in detail. However, the embodiments according to the present specification may be modified in various other forms, and the scope of the present specification is not to be construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those of ordinary skill in the art.

Example 1. Materials of Experiment

Fetal bovine serum (FBS; Sigma-Aldrich, USA), ExoQuick-TC reagent (SBI, USA), phosphate buffered saline (PBS; Biosesang, Korea), gold(III) chloride trihydrate (HAuCl ₄ ; Sigma-Aldrich, USA) ), deionized sterile water (DW; Bioneer, Korea), Macrosep Advance Centrifugal Device (30kDa; Pall Corporation, USA), Micro BCA ^™ Protein Assay Kit (Thermo Scientific, USA), DMEM/high glucose (High Clone, USA), RPMI-1640 (Hyclone, USA), MEM (Welgene Korea), penicillin-streptomycin (Gibco, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT; Invitrogen) , USA) and 5-dodecanoylaminofluorescein (DAF; Invitrogen, USA) were used.

Example 2. Synthesis of Exo-AuNCs

Exosomes were isolated in FBS using ExoQuick-TC reagent according to the manufacturer's protocol. FBS was first centrifuged at 3000 x g for 15 min in a Macrosep Advance Centrifugal Device. The residue containing the concentrated exosomes was transferred to a new sterile tube and mixed with ExoQuick-TC reagent in a ratio of 5:1. After overnight incubation at 4°C, the mixture was centrifuged at 1500×g for 30 min. The supernatant was removed without disturbing the pellet formed at the bottom of the tube. Finally, the pellet containing exosomes was washed twice with 1x PBS, resuspended in PBS, and stored at -20 °C until next use. Exosomes (6.25 mg/mL) in 1 mL of 1x PBS (pH 7.4) and HAuCl ₄ (1.25 mM) in 1 mL of DW were mixed at room temperature for 5 minutes. Exosome concentrations were quantified using the Micro BCA ^™ Protein Assay Kit. The pH was adjusted using NaOH and incubated at 37° C., 200 rpm in a shaking incubator for 3 days. Absorbance and fluorescence spectra of the synthesized exo-AuNCs were measured using a Spectramax iD5 Multi-Mode Microplate Reader (Molecular Device, USA). 1A and B, blue fluorescent exo-AuNCs having the maximum emission intensity at 460 nm at various pH values were synthesized, and among them, blue exo-AuNCs cultured at pH 7 had the highest fluorescence intensity. Red fluorescent exo-AuNCs with maximum emission intensity at 670 nm were synthesized only at pH 11.5. Blue and red fluorescent exo-AuNCs with high fluorescence signals were generated only in the presence of both exosomes and Au ions (Fig. 2). No Au nanoparticles larger than AuNCs were formed using absorbance analysis (Fig. 1C).

Example 3. Optimization of exo-AuNCs synthesis conditions

We optimized the conditions for exo-AuNCs synthesis. In addition to the pH value (Fig. 1A), it was hypothesized that the concentration of Au ions and the reaction time would be important for the formation of blue fluorescent exo-AuNCs and red fluorescent exo-AuNCs. Accordingly, optimization conditions for each synthesis were confirmed in blue fluorescent exo-AuNCs and red fluorescent exo-AuNCs by varying Au concentrations. Exosome concentration was fixed at 6.25 mg/mL, and exo-AuNCs were synthesized as in Example 2 at various Au ion concentrations (0.3125, 0.625, 1.25, 2.5, and 5 mM), and their fluorescence intensity was measured. As shown in Fig. 3A, both blue and red fluorescent exo-AuNCs exhibited the highest fluorescence intensity at the Au ion concentration of 1.25 mM. Optimization conditions for synthesizing exo-AuNCs of blue fluorescent exo-AuNCs and red fluorescent exo-AuNCs according to exosome incubation time were confirmed. As shown in Fig. 3B, the fluorescence of blue fluorescent exo-AuNCs gradually increased until the 3rd day and then the fluorescence did not increase any more, whereas the red fluorescent exo-AuNCs reached the highest fluorescence intensity after 1 day, and after that, The fluorescence intensity decreased. The reason for the decrease in fluorescence after 1 day is that the high pH used to promote the formation of red fluorescent exo-AuNCs damages the exosomes. Based on the above optimization experiment, it was confirmed that day 3 and day 1 were optimal conditions for 1.25 mM Au ions and blue and red fluorescent exo-AuNCs, respectively.

Example 4. Characterization of Exo-AuNCs

After synthesizing exo-AuNCs under the conditions optimized as in Example 3, exo-AuNCs were imaged using an electron microscope (FE-SEM; SU8010, Hitachi, Japan) to confirm that the unique shape of the exosomes was maintained. . 4A and 4B, both blue fluorescence (left; A) and red fluorescence (right; B) exo-AuNCs maintained vesicle shape with a size of less than 200 nm. Under optimal synthesis conditions (blue: 6.25 mg/mL exosomes, 1.25 mM Au ions, incubated for 3 days at 37°C, pH 7, red: 6.25 mg/mL exosomes, 1.25 mM Au ions, pH 11.5 at 37°C It was confirmed that exosomes can maintain the unique shape of the endoplasmic reticulum) under the conditions of 1 day. Through EDX analysis, it was confirmed that Au ions were present in both blue and red fluorescent exo-AuNCs. (Fig. 4B). Exo--AuNCs nanoparticle tracking analysis (NTA; NS300, Malvern-Panalytical, UK) was used to measure the size and number of exosomes. 4C and 4D, the size and number of exo-somes were 136.2±3.1 nm and 2.01 Х 10 ¹⁰ particles/mL for blue fluorescent exo-AuNCs, respectively, and 116 ± 5.9 nm and 1.63 Х 10 ¹⁰ for red fluorescent exo-AuNCs, respectively. particles/mL. It was confirmed that exo--AuNCs synthesis conditions did not change the physical properties of exosomes. X-ray photoelectron spectroscopy (XPS; K-Alpha, Thermo Fisher Scientific, USA) was used to determine the number of Au particles in blue and red fluorescent exo-AuNCs. It was confirmed that blue fluorescent exo-AuNCs and red fluorescent exo-AuNCs contained 8 (blue fluorescent exo-AuNCs) and 25 (red fluorescent exo-AuNCs) Au atoms, respectively. As shown in Figure 4E, Au ₈ NC and Au ₂₅ NC both had 4f _5/2 and 4f _7/2 binding energy peaks in common. The 4f _5/2 and 4f _7/2 binding energy peaks of Au ₈ NCs were 88.48 eV and 84.28 eV, respectively, and the Au ₂₅ NCs were 87.58 eV and 83.68 eV, respectively, indicating that Au ₈ NCs are relatively smaller than Au ₂₅ NCs. show that These results are consistent with the fact that Au ₂₅ NCs with a relatively larger size than Au ₈ NCs consist of an Au(0) core surrounded by Au(I), and the binding energy peak is smaller than that of Au ₈ NCs. Scanning electron microscopy, energy dispersive X-ray microanalysis, nanoparticle tracking analysis, and X-ray photoelectron spectroscopy were used to confirm that AuNCs were successfully formed in exosomes.

Example 5. Confirmation of cytotoxicity of blue fluorescent exo-AuNCs and red fluorescent exo-AuNCs

We evaluated the cytotoxicity of exo-AuNCs using the MTT assay. MCF-7 (human breast cancer), HeLa (human cervical cancer), and HT29 (human colorectal cancer) cell lines were purchased from the Korea Cell Line Bank (KCLB; Korea) and cultured at 5% CO ₂ , 37 °C. MCF-7 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. HeLa and HT29 cells were cultured under the same conditions as MCF-7 cells, except that RPMI 1640 medium was replaced with MEM and DMEM, respectively. MCF-7, HeLa and HT29 cells were seeded in RPMI 1640, MEM and DMEM in 96-well culture plates (1×10 ⁴ per well), respectively. After incubation for 24 hours at 5% CO ₂ , 37° C., the cells were washed twice with DPBS and fresh medium containing various concentrations of exo-AuNCs (0.05, 0.1, 0.5, 1, 5 and 10 mg/ml). transferred and incubated for 24 hours. The concentration of Exo-AuNCs was based on the concentration of exosomes quantified using the Micro BCA ^™ Protein Assay Kit. Then, 20 μL of MTT solution (5 mg/mL) was added and the cells were incubated for 4 hours. Finally, 10% SDS was added and cells were incubated for an additional 24 h. The absorbance signal generated at 595 nm was measured to determine the cell viability (%). Different cell lines were treated with blue exo-AuNCs and red fluorescent exo-AuNCs at different concentrations (0.05, 0.1, 0.5, 1, 5 and 10 mg/mL). As shown in FIG. 5A, blue fluorescent exo-AuNCs at a concentration of up to 1 mg/mL did not show cytotoxicity. However, both cell lines treated with 5 mg/mL and 10 mg/mL were toxic. Conversely, red fluorescent exo-AuNCs at concentrations up to 10 mg/mL did not show cytotoxicity. All three cell types grew better in the presence of red fluorescent exo-AuNCs at high concentrations, suggesting that this compound had a beneficial effect on cell growth. Overall, red fluorescent exo-AuNCs exhibited low toxicity to cells and good biocompatibility.

Example 6. Cell imaging using blue fluorescent exo-AuNCs and red fluorescent exo-AuNCs

We performed cell imaging using red fluorescent exo-AuNCs. MCF-7, HeLa and HT29 cells were seeded in black 96-well culture plates (1×10 ⁴ cells per well) and cultured for 24 hours. After washing 3 times with DPBS and fixing with 4% paraformaldehyde for 15 minutes, the cells were incubated with exo-AuNCs (3.125 mg/mL) for 6 hours. After washing the cells 3 times with DW, the cells were counterstained with DAF (500 μM) for 15 min and washed 3 times with DW. Finally, images were obtained using a fluorescence microscope (KI-2000F; Korea Lab Tech) under the following conditions. Filter cube 1 (excitation: 330-385 nm; barrier: 420 nm) was used to image exo-AuNCs with excitation and emission wavelengths. 360 nm and 670 nm, respectively; Filter Cube 2 (excitation: 450–480 nm; barrier: 515 nm) was used to image the DAF with excitation and emission wavelengths of 460 nm and 520 nm, respectively. The reason for using red fluorescent exo-AuNCs is that their fluorescence properties and biocompatibility are superior to those of blue light emission. After staining the cells with red fluorescent exo-AuNCs, the cell membranes were counterstained with DAF. In all three cell lines, red fluorescent exo-AuNCs effectively stained nuclei as indicated by bright red (Fig. 6). These results show that positively charged fluorescent nanomaterials can stain cell nuclei. The above experimental results show that fluorescent exo-AuNCs can be useful as novel bioimaging probes.

We developed a novel method for the synthesis of fluorescent AuNCs using exosome templates. It differs from the methods used in most exosome-related studies that focus on biomarker analysis or drug delivery. By optimizing the reaction conditions, we succeeded in synthesizing blue (Au8NC) and red luminescent (Au25NC) exo-AuNCs with emission wavelengths of 460 nm and 670 nm, which were successfully characterized using various analytical tools, respectively. In particular, fluorescent exo-AuNCs were produced by simply culturing a mixture of exosomes and Au ions at room temperature without harsh experimental conditions or toxic chemicals. In addition, red fluorescent exo-AuNCs showed large Stokes migration, were not cytotoxic, and were successfully used for nuclear staining in various cell lines. Given the numerous unique functions of exosomes, exo-AuNCs can serve as a versatile platform for bioimaging. We also demonstrated that AuNCs were successfully formed in exosomes using scanning electron microscopy, energy dispersive X-ray microanalysis, nanoparticle tracking analysis, and X-ray photoelectron spectroscopy. Importantly, it was found that red fluorescent exo-AuNCs had greater Stokes shift and stronger fluorescence intensity than blue fluorescent exo-AuNCs. Moreover, blue fluorescent exo-AuNCs were cytotoxic at high concentrations (≥5 mg/mL), whereas blue and red fluorescent exo-AuNCs were compatible with MCF-7, HeLa and HT29 cells. Red fluorescent exo-AuNCs successfully stained nuclei and were compatible with membrane staining dyes. This is the first study to synthesize fluorescent nanomaterials using exosomes for cellular imaging applications. Because exosomes are secreted from almost all types of cells and are naturally generated, the proposed method is a strategy for low-cost production of versatile nanomaterials. can be

Claims (10)

(i) preparing exosomes; and
(ii) mixing and culturing the exosome and gold ion (Au ion) salt;
Including, exosome-method for producing a gold nanoparticle cluster.
According to claim 1, wherein the gold ion salt is chloro(trimethylphosphine)gold (AuClP(CH ₃ ) ₃ ), potassium tetrachloroaurate (III) (KAuCl ₄ ), sodium aurate (NaAuCl ₄ ), auric acid chloride (HAuCl ₄ ), sodium bromide (NaAuBr ₄ ), gold chloride (AuCl), gold(III) chloride (AuCl ₃ ) or gold bromide (AuBr ₃ ), a method for preparing exosome-gold nanoparticle clusters.
According to claim 1, wherein in the step (ii) using hydrochloric acid and sodium hydroxide, when culturing at a pH of 5.5 to 11.5 conditions, when fluorescence is emitted at 460 nm, a blue fluorescent exosome-gold nanoparticle cluster is synthesized, exo A method for preparing mottle-gold nanoparticle clusters.
The method of claim 3, wherein the blue fluorescent exosome-gold nanoparticle cluster is cultured for 1 to 5 days in step (ii) of claim 1, exosome-a method for producing a gold nanoparticle cluster.
According to claim 1, wherein the red fluorescent exosome-gold nanoparticle clusters emitting fluorescence at 670 nm are synthesized when cultured at pH 11 to pH 12 using sodium hydroxide in step (ii). A method of making gold nanoparticle clusters.
The method of claim 5, wherein the red fluorescent exosome-gold nanoparticle cluster is cultured for 1 hour to 48 hours in step (ii) of claim 1, exosome-a method for producing a gold nanoparticle cluster.
According to claim 1, wherein the gold ion salt comprises a concentration of 0.312 mM to 5 mM, exosome-method for producing a gold nanoparticle cluster.
The method of claim 1, wherein the concentration of the exosomes comprises a concentration of 1 mg/ml to 15 mg/ml, exosomes-a method for producing a gold nanoparticle cluster.
The exosome-gold nanoparticle cluster prepared by the method of claim 1 .
The composition for cell imaging, comprising the exosome-gold nanoparticle cluster of claim 9.

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