Fatty acid-incorporated polymeric nanoparticles and their uses

Abstract

The present invention relates to fatty acid-introduced polymeric nanoparticles and their uses. In the present invention, nanoparticles embedded with calcium carbonate crystals are formed using a biocompatible polymer and an adipocyte targeting ligand (fatty acid), thereby minimizing delivery to surrounding cells and tissues other than adipocytes and maximizing embedding of nanoparticles in adipocytes. The nanoparticles according to the present invention can be produced in an injectable formulation and can be used as a localized lipolysis supplement that breaks down localized fat or as a diet beauty product.

Description

[Technical Field]

[0001] The present invention relates to fatty acid-introduced polymeric nanoparticles and uses thereof. [Background technology]

[0002] The reduction of subcutaneous fat present beneath the epidermis and dermis of the skin is one of the most important areas of cosmetic treatment, and a variety of treatment methods are used for this purpose.

[0003] Treatments for reducing subcutaneous fat include liposuction, in which a cannula is inserted into the subcutaneous fat and the fat is sucked out; cryosurgery, in which a cooling pad is attached to the surface of the skin to cool and necrotize the subcutaneous fat; thermal heating, in which high-frequency or ultrasound is irradiated onto the subcutaneous fat tissue to heat it and remove it; carboxytherapy, in which carbon dioxide (CO2) is gradually injected into the subcutaneous fat with a syringe to promote blood and lymph circulation in the fat tissue and remove it; and mesotherapy, in which obesity treatment drugs are injected into the subcutaneous fat.

[0004] Liposuction, known as the most effective procedure, has drawbacks in terms of pain during the procedure and follow-up care. Typically, bleeding occurs during liposuction, and the procedure is painful. This can lead to pain after the procedure, and some patients may need to take painkillers. Furthermore, patients must wear a compression garment for at least a week after the procedure, requiring about a month of post-surgery care.

[0005] In addition, cryosurgery is easy to perform but has the disadvantage of being less effective. Korean Patent Publication No. 10-2011-0119640 uses an invasive procedure in which a probe cooled by circulating a refrigerant inside is inserted into subcutaneous fat. However, while invasive cryosurgery shortens the treatment time compared to non-invasive cryosurgery, it has the disadvantage of requiring a very long treatment time to prevent necrosis of the subcutaneous fat due to cooling.

[0006] Carboxytherapy is a treatment that focuses on areas with excessive fat accumulation, and Korean Patent No. 10-0772961 describes a method that combines mesotherapy and carboxytherapy to improve fat removal efficiency. However, this patent has the disadvantage of using separate syringe needles for each treatment, resulting in a complex internal structure and separate incisions for each needle.

[0007] Currently, local lipolysis supplements approved by the Ministry of Food and Drug Safety have very limited uses, and off-label treatments are frequently used in the market. These off-label treatments have insufficient evidence of safety and effectiveness and are not covered by insurance, so they are in a blind spot for safe use management and are not properly managed by the government.

[0008] Belchira, a drug approved as a lipolysis supplement, destroys the cell membranes of localized fat cells, killing them. However, Belchira has the disadvantage of being limited to use in double chin treatments. Furthermore, because this drug nonspecifically destroys cell membranes, it has a significant effect not only on fat cells but also on surrounding cells, causing side effects in surrounding tissues, which are currently reported to increase the risk of breast cancer and colon cancer. Summary of the Invention [Problem to be solved by the invention]

[0009] In order to solve the above-mentioned problems, the present invention provides polymeric nanoparticles that decompose localized fat and uses thereof. [Means for solving the problem]

[0010] The present invention provides nanoparticles comprising calcium carbonate crystals, a biocompatible polymer, and a fatty acid, wherein the calcium carbonate is embedded inside the nanoparticles and the fatty acid is exposed on the surface of the nanoparticles.

[0011] Conventional local lipolysis treatments involve injecting drugs directly into the area where fat has accumulated using a syringe, but because non-specific drugs are used, there is a risk of destroying other cells around the injection site and of causing numbness or nerve damage if they come into contact with nearby nerves. To solve this problem, the inventors have discovered that by using nanoparticles containing calcium carbonate crystals, a biocompatible polymer, and a fatty acid, the fatty acid exposed on the surface of the nanoparticles can specifically introduce the nanoparticles into the adipocytes, thereby completing the present invention.

[0012] The term "calcium carbonate crystals" as used herein is distinct from precipitated calcium carbonate produced by chemical processing, and generally refers to calcium carbonate produced by mechanically crushing and classifying high-purity crystalline limestone.

[0013] The nanoparticles according to the present invention have a morphology in which the calcium carbonate crystals are embedded inside the biocompatible polymer. This means that the calcium carbonate crystals are embedded without flow inside the spherical structure of the hard shell formed by the biocompatible polymer, and that the calcium carbonate crystals are embedded evenly from the center to the outer surface of the spherical structure without any significant difference in distribution probability (solid colloidal structure).

[0014] In the nanoparticles of the present invention, calcium carbonate crystals embedded within the particles undergo a reaction in an acidic environment to produce carbon dioxide gas. The nanoparticles of the present invention are not core-shell nanoparticles containing calcium carbonate within a layered membrane structure, but rather have calcium carbonate crystals embedded within a solid crystal that is filled with calcium carbonate. That is, unlike structures in which gas generated within leaks to the outside simultaneously with gas generation, when calcium carbonate crystals embedded within nanoparticles generate carbon dioxide gas, the internal pressure of the nanoparticles gradually increases, ultimately inducing the explosion of the nanoparticles. These characteristics allow for simultaneous release of carbon dioxide bubbles and cell attacking effects through the explosion of the nanoparticles.

[0015] In one embodiment of the present invention, the biocompatible polymer of the present invention is surface-modified with a fatty acid. Specifically, the fatty acid is mixed with the biocompatible polymer to form the spherical structure, and the fatty acid is exposed on the surface of the structure. The fatty acid of the present invention may include saturated fatty acids and unsaturated fatty acids, and preferably C 12 ~C 20 The fatty acids exposed on the surface of the nanoparticles of the present invention have adipocyte targeting properties. Specifically, the fatty acids on the nanoparticle surface react with fatty acid transport proteins present in the cell membrane of adipocytes, allowing the nanoparticles to be introduced into adipocytes via intracellular absorption. Therefore, nanoparticles surface-modified with fatty acids are selectively absorbed and accumulated in adipocytes or adipose tissue, generating carbon dioxide gas within the adipocytes, which deforms and destroys the nanoparticle structure and is then rapidly released, physically attacking and decomposing the adipocytes or causing necrosis, thereby reducing the amount of adipose tissue.

[0016] The biocompatible polymer of the present invention means a polymer having tissue compatibility and anticoagulant properties that does not destroy tissue or cause blood coagulation upon contact with biological tissue or blood, and can be appropriately selected without any particular limitation as long as it can form a solid structure in which calcium carbonate crystals are embedded by the emulsion method of the present invention.

[0017] In one embodiment of the present invention, the biocompatible polymer of the present invention is a polymer having a structure such as polylactide (PLA), polyglycolide (PGA), polylactide-polyglycolide copolymer (PLGA), starch, glycogen, chitin, peptidoglycan, lignosulfonate, tannic acid, lignin, pectin, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyethylene oxide-polypropylene oxide block copolymer, cellulose, hemicellulose, heparin, hyaluronic acid, dextran, or alginate. Preferably, polylactide-polyglycolide copolymer (PLGA) can be used.

[0018] In one embodiment of the present invention, the diameter of the nanoparticles of the present invention may be 100 to 300 nm, preferably 120 to 290 nm, more preferably 150 to 280 nm, and even more preferably 200 to 260 nm. This diameter range can maximize the cell-damaging effect that can induce necrotic death of adipocytes.

[0019] In one embodiment of the present invention, the weight ratio of the fatty acid to the biocompatible polymer may be 0.01:1 to 0.5:1 (fatty acid:polymer), preferably 0.05:1 to 0.15:1. This weight ratio can enhance targeting to adipocytes and cell viability. If the fatty acid content is greater than this range, the diameter of the nanoparticles may increase, but the fatty acid content may be significantly lost due to the low temperature during the preparation process, resulting in reduced stability of the prepared nanoparticles.

[0020] In one embodiment of the present invention, the calcium carbonate crystals and the biocompatible polymer may be present in a weight ratio of 0.001:1 to 1:1 (calcium carbonate:polymer). Preferably, the weight ratio of calcium carbonate crystals to the biocompatible polymer is 0.01:1 to 0.8:1, more preferably 0.1:1 to 0.6:1. A calcium carbonate crystals and biocompatible polymer within this weight ratio can maximize the adipocyte reduction effect. The ratio of calcium carbonate crystals to the biocompatible polymer does not significantly affect the diameter of the gas-generating nanoparticles produced. As shown in Table 2, the incorporation efficiency decreases significantly with increasing calcium carbonate content. Therefore, a content higher than the above range is unlikely, and the hard-shell structure of the biocompatible polymer is relatively weak. Furthermore, if the calcium carbonate content is lower than the above range, the amount of gas generated is insufficient to achieve the adipocyte attack effect.

[0021] In one embodiment of the present invention, when nanoparticles prepared within the above weight ratio of calcium carbonate, biocompatible polymer, and fatty acid were used, a significant fat-reducing effect was demonstrated in vitro.

[0022] According to one aspect of the present invention, there is provided a composition for lipolysis comprising the above-mentioned nanoparticles. The composition for lipolysis of the present invention has the effect of specifically releasing carbon dioxide in the adipocyte environment, and unlike conventional local lipolysis treatments, it is capable of targeting adipocyte tissue.

[0023] According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating obesity, comprising the above-mentioned nanoparticles and / or a composition containing the same.

[0024] According to one aspect of the present invention, there is provided a method for producing nanoparticles, comprising the steps of: (a) mixing a first water phase containing calcium carbonate crystals with an oil phase containing a fatty acid and a biocompatible polymer to form a water-in-oil single emulsion; (b) mixing the emulsion of step (a) with a second aqueous phase to form a water-in-oil-in-water double emulsion (w / o / w); and (c) solidifying the double emulsion of step (b).

[0025] In the method for producing nanoparticles according to the present invention, the "first aqueous phase" can be an aqueous solvent (pH 7.0-8.0) containing calcium carbonate slurry, specifically, distilled water, physiological saline (PBS), aqueous PVA solution, etc., in which calcium carbonate crystals are suspended. The "oil phase" in step (a) of the present invention can be a solution in which a fatty acid and a biocompatible polymer are dissolved in a hydrophobic and highly volatile organic solvent that is immiscible with the aqueous phase. Specifically, a solution in which a fatty acid and a biocompatible polymer are dissolved in methylene chloride, chloroform, dimethylformamide, ethyl acetate, acetone, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, or a mixture thereof can be used. The mixing of the first aqueous phase and the oil phase in step (a) of the present invention is preferably carried out using mechanical stirring, for example, an ultrasonic disrupter, homomixer, stirrer, etc.

[0026] The single emulsion (w / o) prepared in step (a) of the present invention is mixed with a second aqueous phase to form a double emulsion (w / o / w). The second aqueous phase can be, for example, an aqueous surfactant such as polyvinyl alcohol, poloxamer, or polyvinylpyrrolidone. The resulting double emulsion can be solidified by evaporation or extraction of the organic solvent.

[0027] In one embodiment of the present invention, the single emulsion of step (a) of the present invention may have a weight ratio of the fatty acid and the biocompatible polymer of 0.01:1 to 0.2:1 (fatty acid:polymer), preferably 0.05:1 to 0.15:1, and the calcium carbonate crystals and the biocompatible polymer of 0.001:1 to 1:1 (calcium carbonate:polymer), preferably 0.01:1 to 0.8:1, more preferably 0.1:1 to 0.6:1.

[0028] The method for producing nanoparticles according to the present invention relates to a method for producing nanoparticles according to another aspect of the present invention, and details overlapping with the nanoparticles will be omitted to avoid overcomplicating the description of this specification. [Effects of the Invention]

[0029] The polymeric nanoparticles according to the present invention circulate in the body and are embedded specifically in adipocytes, generating carbon dioxide within the cells, thereby breaking down fat through adipocyte necrosis. In particular, the present invention can target adipocytes using a ligand (fatty acid), minimizing the effects on surrounding tissues and cells. This minimizes drug side effects and allows for the development of a product that allows for safer treatment. The nanoparticles can be applied to areas that are commonly treated, such as the chin, thighs, arms, and abdomen.

[0030] In addition, the nanoparticles according to the present invention can be prepared as an injectable formulation and applied to the fields of diet beauty and obesity treatment, and can be used as a local lipolysis supplement that decomposes local fat through local fat necrosis, a body shape correction agent, or a diet beauty product. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 is a schematic diagram showing a fatty acid-introduced nanoparticle (a) and a fatty acid (b) according to an example of the present invention, and a process of embedding the fatty acid-introduced nanoparticle (b) into adipocytes. [Figure 2]FIG. 1 shows the size distributions of (a) PNP, (b) GNP, (c) PA-GNP2, and (d) PA-GNP10 measured by DLS. [Figure 3] FIG. 1 shows SEM images of (a) PNP, (b) GNP, (c) PA-GNP2, and (d) PA-GNP10. [Figure 4] FIG. 1 shows 1H NMR spectra of nanoparticles as a function of palmitic acid concentration. [Figure 5] FIG. 1 shows py-GC / MS spectra of nanoparticles as a function of palmitic acid concentration. [Figure 6] Figure 1 shows the in vitro viability of cells treated with GNP, PA-GNP2, and PA-GNP10 ([CaCO3] = 0.5 mg / mL). [Figure 7] (a) Oil Red O stained images of 3T3-L1 preadipocytes, (b) 3T3-L1 adipocytes on day 6, (c) 3T3-L1 adipocytes on day 10, (d) 3T3-L1 adipocytes on day 12 of culture, and (e) changes in the optical density values ​​of Oil Red O eluted from 3T3-L1 preadipocytes and adipocytes over time. [Figure 8] FIG. 1 shows the in vitro viability of 3T3-L1 adipocytes treated with various concentrations of PNP, GNP, PA-PNP2, and PA-PNP10. [Figure 9] Figure 1 shows (a) the in vitro viability of 3T3-L1 adipocytes treated with GNP, PA-GNP2, and PA-GNP10 ([CaCO3] = 0.5 mg / mL) and (b) the in vitro viability of 3T3-L1 adipocytes treated with PA-GNP10 containing various concentrations of CaCO3. [Figure 10] FIG. 1 shows confocal microscopy images of 3T3-L1 adipocytes treated with alexa 488 cadaverine-conjugated PNPs and PA-PNPs. [Figure 11] FIG. 1 shows confocal microscopy images of 3T3-L1 adipocytes treated with alexa 488 cadaverine-conjugated PNPs and various types of FA-PNPs. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention. [Example]

[0033] Reference experimental materials Poly(DL-lactide-co-glycolide) (PLGA, Mw 6400, 50:50, 0.15–0.25 dL / g, carboxylate end groups) was purchased from Lactel Absorbable Polymers (Birmingham, AL, USA). Poly(vinyl alcohol) (PVA, Mw 30,000–70,000, 87–90% hydrolyzed), dichloromethane (methylene chloride, MC), calcium carbonate (CaCO), palmitic acid (PA), Oil Red O, sodium hydroxide (NaOH), hydrochloride (HCl), sodium acetate (anhydrous), acetic acid, dimethyl sulfoxide (DMSO), 3-isobutyl-1-methyl-xanthine (IBMX), insulin, dexamethasone, Dulbecco's phosphate-buffered saline (DPBS), penicillin-streptomycin, and 10% formalin solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-Propanol (isopropanol, 99.5%) was purchased from Samcheong (Gangnam, Seoul). CellTiter96 Aqueous One Solution (MTS assay) was purchased from Promega (Madison, WI, USA). Dulbecco's modified Eagle's medium (DMEM, 4.5 g / L, D-glucose) was purchased from Weljin (Gyeongsan, Gyeongbuk, Korea). Fetal bovine serum (FBS) and calf serum (CS) were purchased from Gibco (Grand Island, NY, USA). Trypsin-ethylenediaminetetraacetic acid and Hoechst 33342 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Alexa fluor® 488 cadaverine sodium salt and lysotracker red DND-99 were purchased from Invitrogen (Eugene, OR, USA). VECTASHIELDAntifade mounting medium was purchased from Vector Laboratories (Burlingame, CA, USA). Water was distilled and deionized using a Milli-Q® System (Millipore; Billerica, MA, USA) and purified water RO MAX (Human Science; Hanam, Gyeonggi, Korea).C57BL / 6 mice were purchased from Orient (Seongnam, Gyeonggi).

[0034] Example 1. Preparation of palmitic acid-modified gas-generating nanoparticles (PA-GNPs) Fatty acid-modified gas-generating PLGA nanoparticles (FA-GNPs) were fabricated using a water-in-oil-in-water (W / O / W) double emulsion and solvent evaporation method. Fatty acids physically modified the GNP surface. A 10% w / v fatty acid solution in dichloromethane was mixed with a PLGA solution (5% w / v) for a minimum of 4 hours (FA-PLGA solution). (The FA-PLGA solution, 25% w / v calcium carbonate (containing) and 4% PVA solution in deionized water, were kept in an ice bath until use.) Calcium carbonate (CaCO3) was uniformly dispersed in deionized water (DW) using an ultrasonicator (Branson Digital Sonifier®; Danbury, CT, USA) at 25 W for 90 s (W1). To load the nanoparticles with CaCO3, the CaCO3 dispersion solution was added to the FA-PLGA solution (O) and emulsified in a sonicator at 25 W for 90 s. This emulsion (W1 / O) was poured into a 4% PVA solution used as a suspension polymerization aid and emulsified in an ultrasonicator at 35 W for 2 minutes to create another emulsion (W1 / O / W2). The complete double emulsion was added to a 1% PVA solution and stirred overnight to evaporate the remaining dichloromethane. The unloaded CaCO3 was removed by ultracentrifugation at 4,000 g for 1 minute. The supernatant was then ultracentrifuged at 31,000 g for 30 minutes to collect the nanoparticles, which were then washed four times with deionized water. FA-GNPs were freeze-dried (ILSHIN BIOBASE freeze dryer, Dongducheon, Gyeonggi-do) and stored at 4 °C. FA-PLGA nanoparticles without CaCO3 (FA-PNPs) were also prepared using the same method and used as a control.

[0035] PLGA nanoparticles containing calcium carbonate were prepared using a W / O / W double emulsification method and used as gas-generating nanoparticles (GNPs). PLGA nanoparticles without calcium carbonate (PNPs, non-gas-generating) were also prepared and used as a control. When the CaCO3 loading content in GNPs was 55.0% (CaCO3 / PLGA; wt / wt%), the average diameter of GNPs was 246.8 nm. No significant change in the average diameter of PNPs was observed with increasing calcium carbonate content (Table 1). This was reflected by a polydispersity index (PDI) value of 2.5, and a narrow size distribution at neutral pH (pH 7.4) (Figure 2(a) and (b)). SEM images showed that the nanoparticles formed spherical shapes even in the presence of CaCO3 (Figure 3(a) and (b)). The pH-dependent change in size distribution was measured as follows: PNPs showed no size differences with pH. Interestingly, we confirmed that GNPs not only increased in size significantly under acidic conditions (pH 5.5), but also broadened their size distribution (Figure 2), suggesting that GNPs generate carbon dioxide gas in response to acidic pH conditions.

[0036] [Table 1]

[0037] Experimental Example 1: Characteristics of palmitic acid-modified gas-generating nanoparticles (PA-GNPs) The morphology of the nanoparticles was observed using a scanning electron microscope (SEM) (S-4800U field-emission scanning electron microscope, Hitachi, Tokyo, Japan). The average size and size distribution of PA-GNPs were determined by dynamic light scattering (DLS) (Nano ZS, Malvern Instruments, UK) at pH 7.4 (deionized water) and pH 5.5 (acetic acid) ([NPs] = 0.5 mg / mL). NMR and pyrolyzed-GC / MS were used for the qualitative analysis of palmitic acid. Spectra were acquired using an NMR spectrometer. DMSO was used as the solvent for all samples, diluted to an equivalent concentration of 2 mg / mL. Spectra were also acquired using pyrolyzed-GC / MS, and all samples were diluted with 4 mg. To determine the CaCO3 loading content, PA-GNPs were dissolved in 1 M NaOH solution for 1 h and neutralized with 1 M HCl solution. The CaCO3 concentration percentage of the nanoparticle solution was estimated using a calcium colorimetric assay kit from Bio Vision (Palo Alto, CA, USA) and calculated as a weight fraction. The amount of carbon dioxide gas produced by PA-GNPs can be measured with a calcium colorimetric assay kit, and the number of moles of calcium ions (Ca 2+ The nanoparticles were suspended in phosphate buffer (pH 7.4 or pH 5.5, [CaCO3] = 1 mg / mL), and the solution was ultracentrifuged at 31,000 g for 15 min.

[0038] Palmitic acid (PA), a saturated fatty acid commonly found in animals, was selected for surface modification of PLGA nanoparticles because it is less sensitive to light, air, and heat than oleic acid, another abundant fatty acid in animals. PA was physically bound to PLGA nanoparticles at various weight ratios for improved adipocyte uptake. Palmitic acid-modified gas-producing PLGA nanoparticles (PA-GNPs) were fabricated by self-assembly between PA and PLGA nanoparticles in the O phase during the emulsification process. The numbers after PA-GNPs indicate the palmitic acid to PLGA ratio (Table 1). PLGA itself and unmodified nanoparticles (GNPs) were used as controls.

[0039] The addition of palmitic acid and calcium carbonate did not significantly affect the average diameter of nanoparticles depending on the amount of palmitic acid added (Table 1). The amount of PA attached to the surface was qualitatively analyzed using NMR and pyrolyzed GC / MS. Peaks at 5 and 1.5 ppm were identified for PLGA in the PNP and PA-PNP spectra. In the PA-PNP spectra, new peaks corresponding to PA were observed at 2.2 ppm (-O-CO-CH2, CH2-COOH), 1.35 ppm (-(CH2)n-), and 0.85 ppm (-CH3) (Figure 4). As the fatty acid feed ratio increased, the surface modification efficiency decreased (Table 2). A low temperature (10°C) during the ultracentrifugation process likely affected the loss of fatty acids. Based on the palmitic acid peak observed between 7.5 and 7.8 minutes, we confirmed that this peak did not appear in PNP at 7.5 to 7.8 minutes (Figure 5). In addition, for PA-PNPs to which palmitic acid was added, the extent of the peak changed depending on the palmitic acid concentration. The palmitic acid peak area was calculated as an integral value to calculate the amount of palmitic acid attached to the nanoparticles. Taking into account the loss of palmitic acid during the nanoparticle manufacturing process, it was revealed that the amount of palmitic acid was actually 6.59 times greater than the 5 times greater amount. 1 H NMR and py-GC / MS results showed that PA was successfully attached to the surface of nanoparticles.

[0040] The morphology of PA-PNP was also observed using a scanning electron microscope. Even when palmitic acid was added, PA-PNP remained spherical compared to PNP, and there was no significant change in size (Figure 3 (c) and (d)). The size change under acidic conditions was similar to that of GNPs, even when palmitic acid was attached. These results confirmed that surface modification with palmitic acid does not significantly affect the physical properties of GNPs, such as size, morphology, and gas-generating capacity.

[0041] [Table 2]

[0042] Experimental Example 2. Analysis of nanoparticle-treated cell viability Cell culture and 3T3-L1 adipocyte differentiation NIH-3T3 cells (fibroblasts) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in DMEM complete growth medium containing 10% CS and 1% penicillin and streptomycin (PS) at 37°C and 5% CO2 (10% CS medium). C2C12 cells (myoblasts) were purchased from ATCC and maintained in DMEM complete growth medium containing 10% FBS and 1% PS at 37°C and 5% CO2 (10% FBS medium). 3T3-L1 preadipocytes were purchased from ATCC and maintained in 10% CS medium at 37°C and 5% CO2. Two days after 3T3-L1 preadipocytes reached confluency, they were cultured for two days in growth medium (MDI medium) containing 10% FBS, 1% IBMX (0.5 mM), 0.1% insulin (1 μg / mL), and 0.1% dexamethasone (1 μM) to initiate adipogenesis. The cells were then cultured for two days in DMEM medium containing 10% FBS and 0.1% insulin. Finally, differentiated cells were cultured in DMEM medium containing 10% FBS, with the medium replaced every other day.

[0043] Oil Red O staining Oil Red O staining was used to determine the degree of adipocyte differentiation and visualize lipid droplets. Oil Red O stock solution was prepared by dissolving 0.7 g of Oil Red O powder in 200 mL of isopropane, stirring overnight, filtering through a 0.22 μm syringe filter, and storing at 4°C. The stock solution was mixed with deionized water in a 3:2 ratio to prepare Oil Red O working solution. This solution was then left at room temperature (RT) for 20 minutes and filtered through a 0.22 μm syringe filter. Cells were washed with 10% formalin for 5 minutes at RT and fixed with 10% formalin for 1 hour at RT. After fixation, cells were washed with 60% isopropanol and completely dried. Oil Red O working solution was then added for 10 minutes. Cells were washed four more times with deionized water to remove any remaining Oil Red O stock solution. After microscopy, the cells were further dried completely. Oil Red O was eluted with 100% isopropanol by pipetting up and down multiple times and then measured at 500 nm using a UV / visible spectrophotometer (SpectraMax ABS, Molecular Devices, San Jose, CA, USA).

[0044] To confirm the delivery ability of PA-GNPs and their in vitro treatment effects, we performed viability experiments on various cell lines using MTS analysis. NIH-3T3 fibroblasts, C2C12 myoblasts, 3T3-L1 preadipocytes, and 3T3-L1 adipocytes were used as competition groups (Figure 6). Since most cells absorb fatty acids and use them as an energy source, we sought to determine the specificity of their uptake by adipocytes.

[0045] GNPs did not affect the cell viability of all other cell lines, including adipocytes, preadipocytes, stem cells, fibroblasts, and myoblasts. This suggests that cells cannot absorb GNPs alone, and carbon dioxide production is not reflected in biological pH. Interestingly, when PA-GNPs were used, the presence of PA did not significantly affect other cell lines, but adipocyte viability was particularly reduced. Based on these results, PA-GNPs were found to be effective in adipocytes, which absorb the most fatty acids. Furthermore, we confirmed that the acidic conditions generated during intracellular entry during fatty acid absorption promote carbon dioxide production and significantly reduce cell viability.

[0046] Experimental Example 3. Analysis of specific absorption of nanoparticles into adipocytes and their ability to kill adipocytes The uptake of PA-PNP into 3T3-L1 adipocytes was confirmed using a confocal laser scanning microscope (TCS SP5, Leica Microsystems, Germany). Alexa fluor® 488 cadaverine was dissolved in MES buffer and reacted with PA-PNP overnight in the dark to produce Alexa 488 cadaverine-PA-PNPs (Alexa 488 cadaverine / polymer = 1 / 50, w / w). The labeled nanoparticles were collected by ultracentrifugation at 31,000 g for 15 min and washed three times with deionized water. Alexa 488 cadaverine-PA-PNPs were lyophilized and stored at 4°C.

[0047] The recommended seeding density for confocal dishes is 8 x 10 3T3-L1 preadipocytes. 4Cells were seeded and differentiated as described in the previous section. Adipocytes were treated with Lysotracker Red (50 nM) and Alexa 488 cadaverine-PA-PNP (0.5 mg / mL)-containing medium for 24 hours. Afterwards, adipocytes were washed three times with DPBS to remove remaining nanoparticles and treated with HOECHST (1 μg / mL) for 15 minutes to stain the nuclei. Cells were fixed with 10% formalin for 15 minutes, mounted with anti-fading mounting medium (VECTASHIELD, Vector Laboratories, Burlingame, CA, USA), and analyzed by confocal microscopy. All processes were performed in the dark.

[0048] 3T3-L1 preadipocytes were cultured in CS medium and differentiated into adipocytes in FBS medium. After adipocyte differentiation, lipid droplets were observed in the cytoplasm and detected by Oil Red O staining (Figure 7). The accumulation of lipid droplets was investigated at various stages from before differentiation to 12 days after differentiation.

[0049] The cytotoxicity of the nanoparticles was evaluated using MTS analysis of 3T3-L1 adipocytes. Untreated cells served as a control, while cells treated with PNP, GNP, PA-PNP2, and PA-PNP10 showed no significant changes depending on the sample concentration (Figure 8). This also suggests that the PLGA, PA, and CaCO3 used in nanoparticle production are not significantly toxic. On the other hand, as the PA concentration increased, the cell viability of PA-GNP-treated cells decreased (Figure 9). This was considered significant and was used in further experiments. Although not shown, cell viability tended to be lowest when the PA / PLGA ratio was 1, but this was excluded from the experimental group due to the nanoparticles' extremely oily properties.

[0050] The specific cellular uptake of PA-PLGA nanoparticles into 3T3-L1 adipocytes was examined using confocal microscopy with Alexa 488 cadaverine-PA-PNP. The cellular uptake of PA-PNP into 3T3-L1 adipocytes was clearly observed compared to unmodified nanoparticles (Figure 10). The lysotracker probe used in this experiment has a weak base that is partially neutralized at neutral pH and can freely penetrate cell membranes. It is also highly selective for acidic organelles. Therefore, lysosomes, which exhibit an acidic pH within the cell, were selectively stained and appeared red in this experiment. As hypothesized in this study, nanoparticles surface-modified with the green Alexa 488 cadaverine label were absorbed by various mechanisms, entering endosomes and eventually colocalizing with lysosomes. Therefore, the appearance of a yellow color upon nanoparticle interaction with lysosomes indicates successful uptake. This may also predict future carbon dioxide gas production by PA-GNPs, which may lead to adipocyte death.

[0051] Experimental Example 4. Analysis of the absorption ability of nanoparticles modified with various fatty acids into adipocytes CaCO3-free FA-PLGA nanoparticles (FA-PNPs) were prepared using fatty acids with various carbon chain lengths in the same manner as in Example 1, and their uptake into adipocytes was evaluated.

[0052] Specifically, saturated fatty acids Myristic acid (MA, C14), Palmitic acid (PA, C16), and Stearic acid (SA, C18) and unsaturated fatty acid Oleic acid (OA, C18) were used, and FA-PLGA nanoparticles (FA-PNPs) were prepared using fatty acids and PLGA in the ratios shown in Table 3 below.

[0053] [Table 3]

[0054] In addition, the absorption ability of FA-PNPs prepared in the same manner as in Experimental Example 3 into adipocytes was analyzed.

[0055] As a result, as shown in Figure 11, it was confirmed that the absorption ability of nanoparticles into fat cells was improved not only when palmitic acid but also when saturated and unsaturated fatty acids with other carbon numbers were used.

Claims

1. An adipocyte-targeting nanoparticle comprising calcium carbonate crystals, a biocompatible polymer, and a fatty acid, wherein the calcium carbonate is embedded inside the nanoparticle and the fatty acid is exposed on the surface of the nanoparticle; The nanoparticles, wherein the fatty acid and the biocompatible polymer are present in a weight ratio of 0.01:1 to 0.2:

1.

2. The nanoparticle of claim 1 , wherein the nanoparticle releases carbon dioxide in an acidic environment.

3. The nanoparticles of claim 1, wherein the biocompatible polymer is a polymer having a structure selected from the group consisting of polylactide (PLA), polyglycolide (PGA), polylactide-polyglycolide copolymer (PLGA), starch, glycogen, chitin, peptidoglycan, lignosulfonate, tannic acid, lignin, pectin, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyethylene oxide-polypropylene oxide block copolymer, cellulose, hemicellulose, heparin, hyaluronic acid, dextran, and alginate.

4. The nanoparticles of claim 1, wherein the nanoparticles have a diameter of 100 to 300 nm.

5. The nanoparticle of claim 1 , wherein the fatty acid is a saturated fatty acid or an unsaturated fatty acid.

6. A lipolytic composition comprising the nanoparticles according to any one of claims 1 to 5.

7. A pharmaceutical composition for preventing and treating obesity, comprising the lipolytic composition according to claim 6.

8. (a) mixing a first water phase containing calcium carbonate crystals with an oil phase containing a fatty acid and a biocompatible polymer to form a water-in-oil single emulsion (W / O); (b) mixing the emulsion of step (a) with a second aqueous phase to form a water-in-oil-in-water double emulsion (W / O / W); (c) solidifying the double emulsion of step (b); The method for producing nanoparticles according to any one of claims 1 to 5, wherein the biocompatible polymer and the fatty acid are mixed in a weight ratio of 0.01:1 to 0.2:

1.

9. 9. The method for producing nanoparticles according to claim 8, wherein the biocompatible polymer is a polymer having a structure selected from the group consisting of polylactide (PLA), polyglycolide (PGA), polylactide-polyglycolide copolymer (PLGA), starch, glycogen, chitin, peptidoglycan, lignosulfonate, tannic acid, lignin, pectin, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyethylene oxide-polypropylene oxide block copolymer, cellulose, hemicellulose, heparin, hyaluronic acid, dextran, and alginate.

10. The method for producing nanoparticles according to claim 8, wherein the diameter of the nanoparticles produced by the method is 100 to 300 nm.

11. The method for producing nanoparticles according to claim 8 , wherein the fatty acid is a saturated fatty acid or an unsaturated fatty acid.

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