POSITIVELY CHARGED NANOPARTICLES, USE THEREOF, AND PREPARATION METHOD THEREOF

Abstract

Provided are positively charged nanoparticles and use thereof. According to the nanoparticles of an aspect, cucurbituril may limit electrostatic attraction between a guest for cucurbituril and a metal salt, thereby generating nanoparticles with a uniform size. Accordingly, the nanoparticles may have an effect of being capable of efficient intracellular gene delivery.

Claims

1. A nanoparticle comprising a metal salt, cucurbituril, and a guest for cucurbituril.

2. The nanoparticle of claim 1, wherein the guest for cucurbituril is a compound represented by the following Formula 1: ##STR00002## wherein, in Formula 1, R.sub.1, R.sub.2, and R.sub.3 are each independently any one selected from the group consisting of hydrogen, C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 alkenyl, C.sub.1-C.sub.10 alkynyl, and benzyl; A is any one selected from the group consisting of substituted or unsubstituted C.sub.1-C.sub.40 alkyl, substituted or unsubstituted alkenyl, and substituted or unsubstituted alkynyl; and B is any one selected from the group consisting of a hydroxyl group, thiol, thioether, sulfone, sulfoxide, amine, and a silane group.

3. The nanoparticle of claim 2, wherein R.sub.1, R.sub.2, and R.sub.3 are each independently any one selected from the group consisting of hydrogen, C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 alkenyl, C.sub.1-C.sub.5 alkynyl, and benzyl; A is any one selected from the group consisting of substituted or unsubstituted C.sub.1-C.sub.15 alkyl, substituted or unsubstituted C.sub.1-C.sub.15 alkenyl, and substituted or unsubstituted C.sub.1-C.sub.15 alkynyl; and B is any one selected from the group consisting of a hydroxyl group and thiol.

4. The nanoparticle of claim 1, wherein the cucurbituril is cucurbit[5]uril, cucurbit[6]uril, cucurbit[7]uril, cucurbit[8]uril, or cucurbit[10]uril.

5. The nanoparticle of claim 1, wherein the metal salt is NaAuCl.sub.4.2H.sub.2O, HAuCl.sub.4.3H.sub.2O, NaAuBr.sub.4.xH.sub.2O, KAuCl.sub.4, NaAuCl.sub.4, HAuCl.sub.4, NaAuBr.sub.4, KAuBr.sub.4, HAuBr.sub.4, AuCl.sub.3, AuBr.sub.3, or AuCl.sub.3, and x is 1 to 5.

6. The nanoparticle of claim 1, wherein the nanoparticle is synthesized by reduction in an aqueous medium.

7. The nanoparticle of claim 1, wherein at least part of the guest for cucurbituril exists as a complex with cucurbituril.

8. The nanoparticle of claim 1, wherein the nanoparticle is positively charged.

9. The nanoparticle of claim 1, wherein the cucurbituril reduces electrostatic attraction between the guest for cucurbituril and the metal salt.

10. The nanoparticle of claim 1, wherein the nanoparticle has a uniform size.

11. The nanoparticle of claim 1, wherein the nanoparticle is for gene delivery.

12. A method of preparing a nanoparticle, the method comprising: mixing a metal salt, cucurbituril, and a guest for cucurbituril, and reducing the mixture obtained from the mixing.

13. The method of preparing a nanoparticle of claim 12, wherein the method is carried out in one pot.

14. The method of preparing a nanoparticle of claim 12, wherein the method is carried out in an aqueous medium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the USPTO upon request and payment of the necessary fee. The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0036] FIG. 1 is an illustration of a synthetic process of cationic gold nanoparticles (AuNPs) in the presence or absence of cucurbituril, and the size of AuNPs in each case;

[0037] FIG. 2 is an illustration of a synthetic process of 11-N,N,N-trimethyl amino undecylthiol (HS-C11-TMA) which is a guest compound for cucurbituril used in one embodiment of the present disclosure;

[0038] FIG. 3 is a graph showing .sup.1H NMR signals of HS-C11-TMA in the presence or absence of cucurbituril;

[0039] FIG. 4A is an image showing a mixture of AuCl.sub.4 with HS-C11-TMA@CB in which cucurbituril bound to HS-C11-TMA, FIG. 4B is a graph showing DLS result of analyzing a size distribution of AuNPs formed by mixing HS-C11-TMA@CB with AuCl.sub.4, and an image showing precipitation, FIG. 4C is a TEM image of AuNPs formed by mixing HS-C11-TMA@CB with AuCl.sub.4, FIG. 4D is an image of a mixture of AuCl.sub.4 with HS-C11-TMA without cucurbituril, FIG. 4E is a graph showing DLS result of analyzing a size distribution of AuNPs formed by mixing AuCl.sub.4 with HS-C11-TMA without cucurbituril, and an image showing precipitation, and FIG. 4F is a TEM image of AuNPs formed by mixing AuCl.sub.4 with HS-C11-TMA without cucurbituril;

[0040] FIG. 5 is a graph showing result of measuring a zeta potential of AuNP in the presence or absence of cucurbituril;

[0041] FIG. 6 is a graph showing DLS result of analyzing AuNPs prepared from AuCl.sub.4 and HS-C10-TMA@CB at a molar ratio of 2.5:1, 1.5:1, and 1:1;

[0042] FIG. 7 is a graph showing UV-vis absorption spectroscopy result of analyzing sizes of AuNPs prepared from AuCl.sub.4 and HS-C10-TMA@CB at a molar ratio of 2.5:1, 1.5:1, and 1:1;

[0043] FIG. 8 is a graph showing cytotoxicity of AuNP in the presence or absence of cucurbituril;

[0044] FIG. 9 shows PAGE results of siRNA/AuNP complexes obtained by mixing siRNA and AuNP in the presence or absence of cucurbituril;

[0045] FIG. 10 shows PAGE results of analyzing amounts of siRNA released from siRNA/AuNP complexes according to heparin concentrations, the siRNA/AuNP complexes obtained by mixing siRNA and AuNP in the presence or absence of cucurbituril;

[0046] FIG. 11 shows PAGE results of analyzing amounts of siRNA upon treating siRNA/AuNP complexes with RNase and heparin, the siRNA/AuNP complexes obtained by mixing siRNA and AuNP in the presence or absence of cucurbituril;

[0047] FIG. 12 shows fluorescence microscopy images of, clockwise from top left, GFP-HeLa cells cultured with siRNA/AuNP complexes in the presence of cucurbituril, GFP-HeLa cells cultured with siRNA/AuNP complexes in the absence of cucurbituril, GFP-HeLa cells cultured with only siRNA, and only GFP-HeLa cells cultured; and

[0048] FIG. 13 is a graph showing flow cytometry results of analyzing gene knockdown efficacy of siRNA/AuNP complexes in the presence or absence of cucurbituril.

DETAILED DESCRIPTION

[0049] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term and/or includes any and all combinations of one or more of the associated listed items. Expressions such as at least one of, when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

Preparation Example 1. Experimental Materials and Experimental Preparation

[0050] 1-1. Experimental Materials

[0051] Celltiter 96 aqueous one solution cell proliferation assay (MTS) kit was purchased from Promega Korea, Ltd. 10 Phosphate buffered saline (PBS), dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), and penicillin and streptomycin (PIS, 100) were purchased from WELGENE, Korea.

[0052] Tetrachloroauric acid (III) trihydrate (HAuCl.sub.4.3H.sub.2O), azobisisobutyronitrile (AIBN), thioacetic acid, trimethyl amine (NMe.sub.3), sodium methoxide (NaOMe), and cucurbituril (CB) hydrate were purchased from Sigma Aldrich. Sodium borohydride (NaBH.sub.4) was purchased from Acros Organics. 10-Undecenyl bromide was purchased from TCI, Japan. All chemicals were used without additional purification.

[0053] 1-2. DLS Measurement

[0054] Aqueous solutions of AuNP (w/CB) and AuNP (w/o CB) were separately prepared, and a zeta size and a zeta potential were measured using Malvern Zetasizer ZS series (UK). Mean data were obtained from three individual measurements.

[0055] 1-3. UV Measurement

[0056] Aqueous solutions of AuNP (w/CB) and AuNP (w/o CB) were separately prepared, and UV spectra were recorded using a JASCO V-670 spectrometer.

[0057] 1-4. Transmission Electron Microscopy (TEM) Study

[0058] Each one drop of aqueous solutions of AuNP (w/CB) and AuNP (w/o CB) was put on a 300-mesh formvar/carbon-coated copper grid, and evaporated under ambient condition for 6 hours or longer. Samples were observed under TEM (JEM-1400) operated at 120 kV. Images were obtained using a BioTEM system. Data were analyzed using a Gatan Digital Micrograph program.

[0059] 1-5. Loading Capacity Test and Polyanionic Heparin Competitive Assay

[0060] For loading capacity test and polyanionic heparin competitive assay of siRNA/AuNP complex, polyacrylamide gel electrophoresis (PAGE) analysis was performed. 25 pmol of siRNA was incubated with various concentrations (0 g3 g) of AuNPs in 20 L of 1PBS. After 1 hr-incubation, a gel was stained with a SYBR gold staining reagent. For polyanionic heparin competitive assay, heparin (0 g50 g) was added to the siRNA/AuNP mixture to induce release of siRNA from AuNP, before gel electrophoresis.

[0061] 1-6. RNase Protective Assay

[0062] To evaluate siRNA protection from RNase-mediated degradation in the presence of AuNP, PAGE analysis was performed. RNase (25 g) was first incubated with siRNA or siRNA/AuNP complex in 20 L of PBS for 1 hr, followed by gel electrophoresis and SYBR gold staining.

[0063] 1-7. Cell Culture

[0064] Human cervical cancer cell line was cultured in DMEM containing 4.5 g/L of D-glucose, 10% FBS, 1% penicillin and streptomycin at 5% CO.sub.2 and 37 C.

[0065] 1-8. Cell Viability Assay

[0066] To assay cell viability of AuNP for biological application, GFP-HeLa cells (110.sup.4 cells/well) were prepared triplicate in a 96-well plate for 24 hr, and then incubated with various concentrations of AuNP in a complete medium. After 12-hr incubation, cells were washed with 1PBS, and then an MTS cell proliferation assay solution, together with a complete medium, was added thereto for 2 hr, and absorbance at 490 nm was measured using a microplate reader (TECAN, Infinite F200 Pro).

[0067] 1-9. Flow Cytometry

[0068] GFP-HeLa cells (310.sup.4 cells/well, 24-well plate) were treated with siRNA/AuNP complex in a serum-free medium for 12 hr (final volume; 500 L, [siRNA]=50 nM). Subsequently, the cell culture medium was replaced by a serum-containing fresh medium, followed by incubation for 12 hr. The cells were washed with 1PBS, and then treated with trypsin-EDTA for 3 min to collect cells. Then, 10% FBS was added to the collected cells, followed by centrifugation at 1,200 rpm for 3 min. Cells were finally washed with 1PBS, and cell fluorescence was measured using a flow cytometer, FACS Canto (Becton Dickinson, USA)

[0069] 1-10. Synthesis of HS-C11-TMA

[0070] Synthesis of 11-thioacetylundecyl bromide: 10-undecenyl bromide (1 g, 4.2 mmol) was dissolved in methanol, and then thioacetic acid (0.591 mL, 8.4 mmol) and AIBN (3.44 g, 21 mmol) were added thereto. The reaction mixture was refluxed at 70 C. for 15 hr. After completion of the reaction, 11-thioacetylundecyl bromide was purified by a silica gel column chromatography using an eluent with a ratio of hexane:ethyl acetate of 9:1.

[0071] Synthesis of N,N,N-trimethyl (11-mercaptoundecyl)-ammonium: 11-thioacetylundecyl bromide (1 g, 3.2 mmol) was added to NMe.sub.3 (0.57 g, 9.6 mmol) in methanol, followed by stirring at room temperature for 2 days. A desired compound was precipitated in hexane, and precipitates were washed with hexane several times, followed by purification.

[0072] Synthesis of 11-N,N,N-trimethyl amino undecylthiol (HS-C11-TMA): N,N,N-trimethyl (11-mercaptoundecyl)-ammonium (0.2 g, 0.7 mmol) was dissolved in 3 mL of methanol. 1 mL of 0.1 M sodium methoxide (NaOMe) in methanol was added, followed by stirring at room temperature for 30 min. A desired product (HS-C11-TMA) was precipitated in ether. Precipitates were washed with ether, and dried to obtain a final product.

Example 1. Preparation of Cationic AuNPs with Tunable Size

[0073] Cationic AuNPs with tunable size were prepared in an aqueous medium by one-pot synthesis based on host-guest chemistry. N,N,N-trimethyl-11-sulfanyl-1-undecanaminium chloride (HS-C11-TMA) (FIG. 2) was used as a cationic ligand. Cucurbituril was selected as a complementary host molecule due to its moderate water solubility, high binding affinity with cationic moieties, and compatibility in physiological environments. A complex of HS-C11-TMAs and CB was immediately produced due to modest binding affinity (Ka: 310.sup.5 M.sup.1) by mixing a molar ratio of 1:1 in water. As a host molecule, cucurbituril (CB) minimizes the electrostatic attraction between AuCl.sub.4.sup. anions and the positively charged ligand by threading guest alkylammonium cations. This shielding effect of CB on the ligands allows the mixture to be well dispersed, inhibiting ionic Au-ligand aggregation in an aqueous solution (FIG. 1). The host-guest complexation was confirmed by .sup.1H NMR spectroscopy analysis, in which the resonance signals attributed to the methylene groups (red circles and red stars) of the complex HS-C11-TMA@CB were shifted upfield relative to those of HSC11-TMA (FIG. 3). Then, direct reduction by adding NaBH.sub.4 produces cationic AuNPs with no diverse size and with narrow size distributions (FIG. 1). The size of the AuNPs may be tuned by controlling a ratio between AuCl.sub.4.sup. and the CB-threaded ligands.

[0074] FIG. 1 is an illustration of a synthetic process of cationic gold nanoparticles (AuNPs) in the presence or absence of cucurbituril, and the size of AuNPs in each case.

[0075] FIG. 2 is an illustration of a synthetic process of 11-N,N,N-trimethyl amino undecylthiol (HS-C11-TMA) which is the guest compound for cucurbituril used in one embodiment of the present disclosure.

[0076] FIG. 3 is a graph showing .sup.1H NMR signals of HS-C11-TMA in the presence or absence of cucurbituril.

[0077] Experimental Example 1. Analysis of Shielding Effect of Cucurbituril (CB) on Electrostatic Attraction

[0078] The interaction between HS-C11-TMAs and AuCl.sub.4.sup. anions in the presence and absence of CB was analyzed. As a result, in the absence of CB, HS-C11-TMAs produced insoluble precipitates in an aqueous solution due to the electrostatic attraction between AuCl.sub.4.sup. anions and the cationic moiety of the ligands, upon mixing with AuCl.sub.4.sup. (FIG. 4D). Meanwhile, in the mixture of AuCl.sub.4.sup. and HS-C11-TMA@CB, precipitates were hardly formed (FIG. 4A). This result suggests that CB threaded the quaternary ammonium group of the ligand, thereby preventing the formation of precipitates, which demonstrates the shielding effect of CB toward the electrostatic attraction.

[0079] To further investigate the shielding effect of CB on the synthesis of AuNP, a solution of NaBH.sub.4 was directly added to the mixture of AuCl.sub.4 and HS-C11-TMA in the presence or absence of CB. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses confirmed that polydisperse AuNPs were obtained in the absence of CB. In this case, a gold core had a diverse size distribution ranging from 4 nm to 100 nm, and a hydrodynamic diameter of the AuNPs ranged from 40 nm to 800 nm (FIGS. 4E an 4F). Due to the formation of larger AuNPs, a precipitate of AuNPs was observed after centrifugation of the mixture at 10000 rpm for 20 min (FIG. 4E). In contrast, cationic AuNPs with narrow size distributions were obtained in the presence of CB. In this case, the average size of the gold core was 5.51.7 nm, and the hydrodynamic diameter of AuNPs was 13.753.36 nm (FIGS. 4B and 4C).

[0080] Further, as a result of zeta potential measurement, a zeta potential of AuNPs synthesized with HS-C11-TMA@CB was +38.62.3 mV, and a zeta potential of AuNPs synthesized with HS-C11-TMA was +48.51.4 mV (FIG. 5).

[0081] These results indicate that CB prevented the cationic moieties of the ligands from electrostatically interacting with the AuCl.sub.4.sup. anions, conferring a shielding effect on the ligands for the synthesis of narrow-dispersed cationic AuNPs.

[0082] FIG. 4A is an image showing a mixture of AuCl.sub.4 with HS-C11-TMA@CB in which cucurbituril bound to HS-C11-TMA; FIG. 4B is a graph showing DLS result of analyzing a size distribution of AuNPs formed by mixing HS-C11-TMA@CB with AuCl.sub.4, and an image showing precipitation; FIG. 4C is a TEM image of AuNPs formed by mixing HS-C11-TMA@CB with AuCl.sub.4; FIG. 4D is an image of a mixture of AuCl.sub.4 with HS-C11-TMA without cucurbituril; FIG. 4E is a graph showing DLS result of analyzing a size distribution of AuNPs formed by mixing AuCl.sub.4 with HS-C11-TMA without cucurbituril, and an image showing precipitation; and FIG. 4F is a TEM image of AuNPs formed by mixing AuCl.sub.4 with HS-C11-TMA without cucurbituril.

[0083] FIG. 5 is a graph showing result of measuring a zeta potential of AuNP in the presence or absence of cucurbituril.

Experimental Example 2. Synthesis of AuNPs with Diverse Sizes

[0084] The size of AuNPs obtained in Example 2 may be tuned by controlling a molar ratio of AuCl.sub.4.sup. and the CB-threaded ligand. Thus, the sizes of AuNPs prepared by controlling the molar ratio of AuCl.sub.4.sup. and the HS-C10-TMA@CB at 2.5:1, 1.5:1 and 1:1 were analyzed by using DLS and UV-vis absorption spectroscopy. As a result of DLS measurements, the size of the cationic AuNPs synthesized at a ratio of 2.5:1, 1.5:1 and 1:1 exhibited 6.71.9 nm, 9.5 2.8 nm, and 13.73.4 nm, respectively, with narrow size distributions (FIGS. 6 and 7). Meanwhile, the UV-vis spectra showed the resonance wavelength of the surface plasmon on the AuNPs (FIG. 7). These results indicate that various cationic AuNPs may be synthesized by controlling the molar ratio between

[0085] AuCl.sub.4.sup. and the ligand through the shielding effect of CB.

[0086] FIG. 6 is a graph showing DLS result of analyzing AuNPs prepared from AuCl.sub.4 and HS-C10-TMA@CB at a molar ratio of 2.5:1, 1.5:1, and 1:1.

[0087] FIG. 7 is a graph showing UV-vis absorption spectroscopy result of analyzing sizes of AuNPs prepared from AuCl.sub.4 and HS-C10-TMA@CB at a molar ratio of 2.5:1, 1.5:1, and 1:1.

Experimental Example 3. Cytotoxicity Analysis of AuNPs

[0088] To examine cytotoxicity of AuNPs, cell viability of HeLa cells was evaluated using an MTS cell proliferation assay kit, after treatment with various concentrations of AuNPs for 24 hours (FIG. 8). As a result, 80% or more of the cells survived at a concentration of 0 g/mL to 62.5 g/mL of AuNPs, irrespective of the presence of CB. This result indicates that cationic AuNPs are biocompatible.

[0089] FIG. 8 is a graph showing cytotoxicity of AuNP in the presence or absence of cucurbituril.

Experimental Example 4. Analysis of Gene Delivery Efficacy of AuNPs

[0090] To demonstrate biomedical function of the synthesized cationic AuNPs, intracellular-delivery experiment was performed. To allow facile monitoring of the gene delivery efficacy, siRNA that knockdowns green fluorescence proteins (GFP) was used as a biomacromolecule for delivery. First, the formation of the siRNA/AuNP complex and the siRNA loading capacity of the AuNPs synthesized in the absence or presence of CB were evaluated by polyacrylamide gel electrophoresis (PAGE) analysis. Upon mixing siRNA and AuNPs in a phosphate buffered solution, siRNA/AuNPs complexes were formed via electrostatic interaction between the siRNA and the cationic AuNPs. The siRNA/AuNPs complexes produced by mixing 25 pmol of siRNA and different amounts of AuNPs prepared with and without CB (0 g, 1.5 g, and 3 g) were loaded into the gel. As a result of electrophoresis, the band intensity of the siRNA was found to decrease with increasing the amount of cationic AuNPs, confirming the formation of the siRNA/AuNP complex. The PAGE results indicated that 25 pmol of siRNA is loaded with 2 g of cationic AuNPs (FIG. 9).

[0091] Further, a polyanionic heparin competitive assay and a ribonuclease (RNase) protective assay were carried out to confirm the conditional release of siRNA from AuNPs and chemical stability of the complexed siRNA. As a result, as shown in FIG. 10, the band intensity of siRNA increased with increasing the amount of heparin, facilitating the release of siRNA from AuNPs. The PAGE analysis performed after stepwise incubation of RNase and heparin with the siRNA/AuNPs complexes showed their protective capability toward RNase-mediated degradation, due to condensation of siRNA with the cationic AuNPs (FIG. 11). These results indicate that the quaternary ammonium-functionalized AuNPs may be used as gene delivery carriers.

[0092] Next, down-regulation of GFP expression induced by the siRNA/AuNPs complexes in GFP-expressing HeLa (GFP-HeLa) cells was quantitatively evaluated. The cells were incubated with siRNA/AuNPs complex composed of 50 nM siRNA and 4 g/mL AuNPs for 12 hr, followed by removing the medium and further incubating with a fresh serum-containing medium for 12 hr. The relative gene expression level of GFP was evaluated by monitoring the fluorescent intensity of the cells via microscope imaging and flow cytometry. As a result, as shown in FIG. 12, the green fluorescent intensities decreased in the siRNA/AuNPs-treated cells, as compared with those treated with AuNPs only. This result indicates a knockdown of the green fluorescent proteins via siRNA transfection.

[0093] Further, the flow cytometric data representing cell population versus fluorescent intensity further confirmed that the gene silencing efficiency of the siRNA/AuNPs complexes in which AuNPs were prepared with CB exhibited higher knockdown efficacy of 20.4% than 13.3% of those containing AuNPs prepared in the absence of CB (FIG. 13). These results indicate that the cationic AuNPs with a narrow size distribution which were prepared with CB may perform better gene transfection efficacy than cationic AuNPs with a wide size distribution.

[0094] FIG. 9 shows PAGE results of siRNA/AuNP complexes obtained by mixing siRNA and AuNP in the presence or absence of cucurbituril.

[0095] FIG. 10 shows PAGE results of analyzing amounts of siRNA released from siRNA/AuNP complexes according to heparin concentrations, the siRNA/AuNP complexes obtained by mixing siRNA and AuNP in the presence or absence of cucurbituril.

[0096] FIG. 11 shows PAGE results of analyzing amounts of siRNA upon treating siRNA/AuNP complexes with RNase and heparin, the siRNA/AuNP complexes obtained by mixing siRNA and AuNP in the presence or absence of cucurbituril.

[0097] FIG. 12 shows fluorescence microscopy images of, clockwise from top left, GFP-HeLa cells cultured with siRNA/AuNP complexes in the presence of cucurbituril, GFP-HeLa cells cultured with siRNA/AuNP complexes in the absence of cucurbituril, GFP-HeLa cells cultured with only siRNA, and only GFP-HeLa cells cultured.

[0098] FIG. 13 is a graph showing flow cytometry results of analyzing gene knockdown efficacy of siRNA/AuNP complexes in the presence or absence of cucurbituril.

[0099] According to nanoparticles and a method of preparing the nanoparticles according to an aspect, cucurbituril may limit electrostatic attraction between a guest for cucurbituril and a metal salt, and thus nanoparticles with a uniform size may be formed by a single reaction. Accordingly, the nanoparticles may have an effect of being capable of efficient intracellular gene delivery

[0100] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.