Glucosamine derivative nanoparticles and their production method and use

Abstract

To provide a glucosamine derivative nanoparticle and a method for preparation thereof, and use thereof.SOLUTION: By self-assembling glucosamine derivatives and ethanol into the form of nanoparticles, the skin penetration rate and cell absorption rate of glucosamine are improved, while the toxicity to cells and organisms is reduced. A conventional problem of poor absorption of glucosamine is improved by using glucosamine derivative nanoparticles. Furthermore, the glucosamine derivative nanopartides can be used as a delivery carrier to cover and bring specified ingredients into the cells or stratum corneum, thereby increasing the skin penetration rate and cell absorption rate of the specified ingredients.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to glucosamine derivative nanoparticles, and in particular to peptide-modified glucosamine derivative nanoparticles and their production method and use. [Background technology]

[0002] Glucosamine is a natural amino acid monosaccharide that exists in the connective tissues, cartilage, ligaments and other structures of the human body and serves as the raw material for synthesizing glycosaminoglycan and proteoglycan in the body. Glycosaminoglycan and proteoglycan are components of intercellular substances and are essential components for forming hyaluronic acid, which is deeply involved in maintaining the moisture content and elasticity of the skin and the health of articular cartilage, and are beneficial for maintaining the toughness, flexibility and elasticity of these parts, thus maintaining the health of the human connective tissues.

[0003] However, with conventional oral intake of glucosamine, the utilization efficiency is extremely low (approximately 19-26%) because it must go through liver metabolism and blood circulation before reaching connective tissue and being used by cells.

[0004] In addition, the method of applying glucosamine to the skin for absorption is poorly absorbed due to the influence of the stratum corneum, and although this can be improved by increasing the concentration of glucosamine, high concentrations are toxic to cells. For this reason, there are restrictions on the amount of glucosamine used and the related methods.

[0005] Therefore, how to increase the absorption rate of glucosamine without causing toxicity to cells is a problem that those skilled in the art would like to solve. Summary of the Invention [Problem to be solved by the invention]

[0006] The main objective of the present invention is to provide glucosamine derivative nanoparticles and their production method and use, which can enhance the skin penetration rate and cellular absorption rate of glucosamine by self-assembling glucosamine derivative and ethanol into the form of nanoparticles, while reducing the toxicity to cells. [Means for solving the problem]

[0007] In order to achieve the above object, the present invention discloses a method for producing glucosamine derivative nanoparticles, which comprises the steps of dissolving a glucosamine derivative in deionized water to form an aqueous solution of the glucosamine derivative, mixing the aqueous solution of the glucosamine derivative with ethanol, and vibrating the mixture for 5 to 10 minutes using an ultrasonic vibrator at a power of 40 to 50 W to form glucosamine derivative nanoparticles. The glucosamine derivative is formed by binding and reacting succinic anhydride with the amino terminal of glucosamine and the amino terminal of a peptide, and the chemical formula of the glucosamine derivative is: [ka] A is the peptide composed of 5 to 6 amino acids, and the peptide is one selected from the group consisting of KTTKS, CRGDS, and RRRRRR.

[0008] The present invention also discloses the use of glucosamine derivative nanoparticles to carry a specified component (e.g., glucosamine salt) to enter cells or the stratum corneum, and the glucosamine derivative nanoparticles enter the cells or the stratum corneum by peptides in the glucosamine derivative recognizing specific receptors on the surface of the cells (e.g., skin cells) (or by methods such as changing charge attraction, hydrophilicity / hydrophobicity), thereby increasing the skin penetration rate and cellular absorption rate of the glucosamine salt. [Brief description of the drawings]

[0009] [Figure 1] 1 is a flow chart of the steps for making glucosamine derivative nanoparticles of the present invention. [Figure 2A] 1H-NMR spectrum of the glucosamine derivative (KTTKS) of the present invention. [Figure 2B] TEM image of glucosamine derivative nanoparticles (KTTKS) of the present invention. [Figure 3A] 1H-NMR spectrum of the glucosamine derivative (CRGDS) of the present invention. [Figure 3B] TEM image of glucosamine derivative nanoparticles (CRGDS) of the present invention. [Figure 4A] 1H-NMR spectrum of the glucosamine derivative (RRRRRR) of the present invention. [Figure 4B] TEM image of the glucosamine derivative nanoparticles (RRRRRR) of the present invention. [Figure 5A] 1 is a graph showing an experimental transmittance of the glucosamine derivative nanoparticles (KTTKS) of the present invention. [Figure 5B] 1 is a graph showing an experimental transmittance of the glucosamine derivative nanoparticles (CRGDS) of the present invention. [Figure 5C] 1 is a graph showing an experimental transmittance of the glucosamine derivative nanoparticles (RRRRRR) of the present invention. [Figure 6] FIG. 1 is a graph showing the experimental results of the cytotoxicity test of the present invention. [Figure 7] FIG. 1 is a graph showing the experimental results of the biological toxicity test of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In order to allow the examiner to more clearly understand the features and effects achieved by the present invention, the following examples are given.

[0011] In order to enhance the skin penetration rate and cell absorption rate of glucosamine, the present invention provides a glucosamine derivative and its preparation method, nanoparticles containing glucosamine derivative and its preparation method and use, thereby solving the problems arising from the prior art.

[0012] The properties, combining structures and methods encompassed by the present invention are further described below.

[0013] First, Figure 1 is a flow chart of the steps for producing the glucosamine derivative nanoparticles of the present invention. As shown in Figure 1, the method for producing the glucosamine derivative nanoparticles of the present invention includes the following steps:

[0014] S1: A glucosamine derivative is dissolved in deionized water to form an aqueous solution of the glucosamine derivative.

[0015] S2: After mixing ethanol and an aqueous solution of the glucosamine derivative, the mixture is vibrated for 5 to 10 minutes using an ultrasonic vibrator at a power of 40 to 50 W to form nanoparticles of the glucosamine derivative.

[0016] The method further includes the following method for producing a glucosamine derivative before step S1.

[0017] S11: At room temperature, glucosamine and succinic anhydride are added to an aqueous solution of N,N-dimethylformamide and N-methylmorpholine to react with each other, and after the reaction, the mixture is suction filtered to form glucosamine-succinic anhydride.

[0018] S12: Glucosamine-succinic anhydride and peptide are added to N,N-dimethylformamide, respectively, and then mixed and reacted at room temperature to form a yellow precipitate.

[0019] S13: The yellow precipitate is subjected to suction, filtration and centrifugation to obtain the glucosamine derivative.

[0020] Here, the glucosamine derivative of the present invention is formed by reacting succinic anhydride with the amino terminal of glucosamine and the amino terminal of a peptide, and the chemical formula of the glucosamine derivative thus prepared is: [ka] A is the peptide composed of 5 to 6 amino acids, and is produced by solid phase synthesis. The peptide in the present invention is one selected from the group consisting of KTTKS, CRGDS, and RRRRRR, but is not limited to KTTKS, CRGDS, and RRRRRR, and the peptide generally means a peptide having permeability, and is composed of 5 to 6 amino acids.

[0021] In addition, step S1 further includes a step of adding glucosamine salt to deionized water, so that the glucosamine derivative nanoparticles (particle size between 400-1000 nm) of the present invention can carry more of the glucosamine salt.

[0022] The following are examples of the present invention (examples of KTTKS, CRGDS, and RRRRRR, respectively): Example 1: Preparation of the glucosamine derivative nanoparticles (the KTTKS) First, the glucosamine and succinic anhydride (1 mmol each) in a molar ratio of 1:1 are placed in a double-neck flask containing a magnetic stirrer, 10 ml of N,N-dimethylformamide aqueous solution (N,N-dimethylformamide:water (V / V) = 9:1) is added, and then 0.2 ml of N-methylmorpholine is added and reacted at room temperature for 12 hours. After removing the water and most of the N,N-dimethylformamide using a high vacuum system, the remaining part is precipitated by adding acetic acid and left in a refrigerator at 4°C overnight. The precipitate is filtered by suction, washed with ice ether and deliquified, and the resulting white solid is glucosamine-succinic anhydride (yield is about 85%).

[0023] Next, 0.3976 mmol of the resin containing the KTTKS (produced by solid phase synthesis) is put into a flask, 2 ml of the N,N-dimethylformamide is added, and the flask is placed on a shaker for 30 minutes. At the same time, 1.394 mmol of the glucosamine-succinic anhydride is put into another sample vial, and 2 ml of the N,N-dimethylformamide is added to dissolve it, and the solution in the sample vial is further injected into the flask. After reacting for 2 hours, a ninhydrin test is performed, and if the resin is a yellow precipitate, the reaction is confirmed to be complete. In addition, the yellow precipitate containing the glucosamine derivative (the KTTKS) is removed from the flask, suction filtered using a glass filter, and washed with ethanol and dichloromethane, and the resin is collected and dehydrated. A cleavage reagent (Trifluroacetic acid: Triisopropylsilane: Water = 95: 2.5: 2.5) is added to the dehydrated resin, and the flask is placed on a shaker and reacted at 150 rpm for 20 minutes. The filtrate is collected by suction filtration using a glass filter, followed by crystal precipitation with ice ether, the solution is transferred to a centrifuge tube and the solid is collected by centrifugation, the upper layer of ether is removed, and then ether is added again to repeatedly wash and centrifuge the precipitate, and this step is repeated three times in total. The solid is dehydrated using a high vacuum system to obtain the glucosamine derivative (Glu-SA-(KTTKS), a white solid product, 94% yield).

[0024] 1 H-NMR analysis confirmed the successful synthesis of Glu-SA-(KTTKS): 1 H-NMR(300MHz,D2O):4.33-4.17(m,4H),4.16-4.01(m,3H),3.78-3.69(m,2H),3.69-3.26(m,6H),2 .84(t,4H),2.46(m,2H),1.81-1.46(m,8H),1.40-1.21(m,4H),1.05(d,6H),0.67(t,J=6.2Hz,3H).

[0025] It was also confirmed that the 2.5 ppm position is the ethylene group linkage of the glucosamine derivative, and that the glucosamine is covalently bonded to the peptide at a specific amino group. 14 C and 15 The H in C was originally bound to an amino group (-NH2), but by binding to the glucosamine derivative, a peptide bond (-CONH-) is formed, resulting in a chemical shift that is more favorable for the formation of a covalent bond (as shown in Figure 2A).

[0026] Finally, 1 mg of the glucosamine derivative was dissolved in 4 ml of the deionized water, and 1 ml of ethanol (95%) was injected using an 18G syringe. The glucosamine derivative nanoparticles (KTTKS, particle size about 700-950 nm) were then obtained by vibrating the solution for 10 minutes at a power of 42 W using an ultrasonic vibrator (probe type ultrasonic). After initial evaluation using an optical microscope, the formation was verified using a TEM, and the observation results are shown in Figure 2B.

[0027] Example 2: Preparation of glucosamine derivative nanoparticles (CRGDS) 0.1568mmol of the resin containing CRGDS (produced by solid phase synthesis) is placed in a flask, 3ml of the N,N-dimethylformamide is added, and the flask is placed on a shaker for 30 minutes. At the same time, 0.6633mmol of the glucosamine-succinic anhydride (Example 1) is placed in another sample vial, 4ml of the N,N-dimethylformamide is added to dissolve, and the solution in the sample vial is injected into the flask. After reacting for 2 hours, a ninhydrin test is performed, and if the resin is the yellow precipitate, the reaction is completed.

[0028] Then, the yellow precipitate containing the glucosamine derivative (the CRGDS) is removed from the flask, and subjected to suction filtration and centrifugation (same as in Example 1 except that the shaking is performed for 2 hours). The solid is dehydrated in a high vacuum system to obtain the glucosamine derivative (Glu-SA-(CRGDS), a white solid product, 81% yield).

[0029] 1 The successful synthesis of Glu-SA-(CRGDS) was confirmed by H-NMR: 1 H-NMR (300 MHz, DO) δ 4.56 (s, 1H), 4.33 (t, 1H), 4.25 (t, 1H), 4.17 (t, 1H), 3.79 (s, 2H), 3.72 (t, 2H), 3.04 (t, 2H), 2.75 (t, 2H), 2.46 (dd, 2H), 1.72 (m, 2H), 1.51 (m, 2H) (as shown in Figure 3A).

[0030] Finally, 1 mg of the glucosamine derivative was dissolved in 4 ml of the deionized water, and 1 ml of the ethanol (95%) was injected using an 18G syringe tube. The ultrasonic vibrator (probe type ultrasonic) was used to vibrate the glucosamine derivative nanoparticles (CRGDS, particle size: about 740-820 nm) for 5 minutes at a power of 42 W. After initial evaluation using an optical microscope, the production was verified using a TEM, and the observation results are shown in Figure 3B.

[0031] Example 3: Preparing glucosamine derivative nanoparticles (RRRRRR) 0.2026mmol of the RRRRRR resin (produced by solid phase synthesis) is placed in a flask, 2ml of the N,N-dimethylformamide is added, and then the flask is placed on a shaker for 30 minutes. At the same time, 0.249mmol of the glucosamine-succinic anhydride (Example 1) is placed in another sample vial, and 2ml of the N,N-dimethylformamide is added to dissolve, and then the solution in the sample vial is poured into the flask. After reacting for 2 hours, a ninhydrin test is performed, and if the resin is the yellow precipitate, the reaction is confirmed to be complete.

[0032] Then, the yellow precipitate containing the glucosamine derivative (RRRRRR) is removed from the flask, suction filtered, and centrifuged (same as in Example 1 except that the vibration time is changed to 2 hours). The solid is dehydrated in a high vacuum system to obtain the glucosamine derivative (Glu-SA-(RRRRRR), a white solid product, 78% yield).

[0033] 1 H-NMR analysis confirms successful synthesis of Glu-SA-(RRRRRR): 1 H-NMR (300 MHz, DO) δ 4.15 (m, 5H), δ 3.05 (t, 12H), δ 2.45 (m, 4H), δ 1.68 (m, 12H), 1.5 (m, 12H) (as shown in Figure 4A).

[0034] Finally, 1 mg of the glucosamine derivative was dissolved in 4 ml of the deionized water, and 1 ml of the ethanol (95%) was injected using an 18G syringe tube, and then the ultrasonic vibrator (probe type ultrasonic) was used to vibrate at a power of 42 W for 10 minutes to complete the glucosamine derivative nanoparticles (RRRRRR, particle size about 480-610 nm). After initial evaluation using an optical microscope, the production was verified using TEM, and the observation results are shown in Figure 4B.

[0035] In addition, the glucosamine derivative nanoparticles of the present invention (the KTTKS, the CRGDS or the RRRRRR) may be used alone or may be used to carry a specified component and penetrate the stratum corneum of the skin or enter cells.

[0036] Taking the cells as an example, the glucosamine derivative nanoparticles act on the cells through the peptides in the glucosamine derivative, and then, for example, bind to specific receptors on the surface (or by methods such as charge attraction or changing hydrophilicity or hydrophobicity), allowing the glucosamine derivative nanoparticles themselves (or the glucosamine derivative nanoparticles having the specified component (e.g., the glucosamine salt)) to enter the cells (e.g., skin cells, but skin cells are one embodiment of the present invention and are not limited to this, and refer to all cells that can absorb nanoparticles, for example, intestinal wall cells), thereby increasing the cellular absorption rate of the glucosamine.

[0037] Here, the binding with the specific surface receptor is, for example, that the CRGDS can identify and bind to a specific receptor on the surface of a cancer cell. Meanwhile, the charge attraction is, for example, RRRRRR when the peptide is positively charged. Since cell membranes are often negatively charged, the positive and negative charges attract each other, so that the glucosamine derivative nanoparticles are adsorbed on the surface of the cell membrane, and the probability that the drug will enter the cell by pinocytosis is increased. Finally, regarding changing the hydrophilicity, since known hydrophilic drugs are difficult to permeate (because the cell membrane is made of phospholipids), by modifying with the peptide (lipophilic peptide) selected in the present invention, the glucosamine derivative nanoparticles of the present invention are relatively easily bound to the cell membrane.

[0038] Taking the stratum corneum as an example, the glucosamine derivative nanoparticles have their hydrophilicity and hydrophobicity changed according to their own properties (the peptide), and since the glucosamine derivative nanoparticles themselves (or the glucosamine derivative nanoparticles having the specified component (e.g., the glucosamine salt)) are modified with the lipophilic peptide, they can permeate the stratum corneum, thereby increasing the skin penetration rate of the glucosamine.

[0039] Here, if the designated component is oil-soluble, it is dissolved in the ethanol in advance, and then the glucosamine derivative nanoparticles coated with the oil-soluble designated component are produced in step S2. If the designated component is water-soluble, the water-soluble designated component and the glucosamine derivative are dissolved together in the deionized water as in step S1, and then the glucosamine derivative nanoparticles coated with the water-soluble designated component are produced in step S2.

[0040] The relevant experimental data are as follows: 1. The coverage of the glucosamine salt: 1 mg of the glucosamine derivative (the KTTKS, the CRGDS or the RRRRRR) is dissolved in 4 ml of an aqueous solution containing 0.25 mg / ml of the glucosamine salt, and placed in a sample vial. 1 ml of the ethanol (95%) is quickly injected using an 18G syringe tube, and the vial is vibrated for 10 minutes (5 minutes in the case of the CRGDS) at a power of 42 W using an ultrasonic vibrator (probe type ultrasonic) to complete the preparation of the glucosamine derivative nanoparticles coating the glucosamine salt (the KTTKS, the CRGDS or the RRRRRR).

[0041] In addition, 5 ml of the glucosamine derivative nanoparticle solution was placed in a centrifuge tube and centrifuged at 6,000 rpm and 4°C for 30 minutes using an ultra-high speed centrifuge, after which the supernatant was collected and the glucosamine was quantified by adjusting the RI of the HPLC, and the coverage rate of the nanoparticles was calculated.

[0042] First, the glucosamine salt was prepared into 0.03125mg / ml, 0.0625mg / ml, 0.125mg / ml, 0.25mg / ml and 0.5mg / ml aqueous solutions, and ethanol was used as the mobile phase. The analysis conditions were: mobile phase 95% ethanol:H2O=1:4; flow rate: 1ml / min. 0.4ml of the standard solution was taken with a microsyringe tube and injected into HPLC. In addition, 0.2ml of sample (C2: concentration of glucosamine in the supernatant after centrifugation) was taken with a microsyringe tube and injected into HPLC, and the mobile phase was also 95% ethanol:H2O=1:4. The integrated area value obtained by analyzing this sample was substituted into the calibration curve, and the coverage rate (ER) of the glucosamine derivative nanoparticles was calculated using the formula ER(%)=(C1-C2) / C1*100%, and the results are shown in Table 1. Here, C1 is the original concentration of glucosamine in the sample solution before coating, and C2 is the concentration of glucosamine in the supernatant after centrifugation. [Table 1] As a result, all of the glucosamine derivative nanoparticles of the present invention were able to carry more of the glucosamine salt as a carrier, and among them, Glu-SA-RRRRRR had the best effect, reaching a high value of 42.5%.

[0043] In addition, since the glucosamine derivative nanoparticles of the present invention have a significant number of -OH groups at their glucosamine ends, a Malvern Zetasizer Nano-ZS90 particle size analyzer was further used to test whether these -OH groups can form hydrogen bonds with the unmodified glucosamine salt, thereby carrying more of the glucosamine salt, and the results are shown in Table 2. [Table 2] As a result, when the glucosamine derivative nanoparticles (CRGDS and RRRRRR) of the present invention were coated with excess glucosamine salt, the particle size increased from 782 and 545 nm to 881 and 784 nm, respectively, proving that the glucosamine derivative nanoparticles of the present invention can bind to the unmodified glucosamine salt without any change in particle size over time. The change in particle size supports the fact that the glucosamine derivative nanoparticles can bind to the specified component by utilizing the intermolecular attraction of hydrogen bonds or non-hydrogen bonds to form a delivery carrier with a new structure.

[0044] 2. Cell penetration ability experiment: In the process of producing the glucosamine derivative nanoparticles, first, the glucosamine derivative (1 mg) is dissolved in a fluorescent dye (rhodamine) containing 1 mg of rhodamine per 4 ml, and then the glucosamine derivative nanoparticles are produced.

[0045] Cell culture: Human skin keratinocytes (HaCaT) are inoculated into Dulbecco's modified Eagle's medium high glucose (DMEM) containing 10% fetal bovine serum and 1% penicillin / streptomycin, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. After aspirating the culture medium from the Petri dish, the cells are washed three times with phosphate buffered saline (PBS), the liquid is removed, trypsin-EDTA is added, and the cells are cultured in an incubator at 37°C and 5% CO2 for 3 to 5 minutes to detach the cells. The cell suspension is transferred to a 15 ml centrifuge tube, centrifuged at 3,000 rpm for 3 minutes, the supernatant is removed, and 6 ml of DMEM is added. Separately, a 6-well cell culture plate is prepared, a cover glass is placed in each well, 1 ml of cell solution and 2 ml of DMEM are added to each well, and the plate is placed in an incubator at 37°C and 5% CO2 for culture.

[0046] After 24 hours, observe the cell proliferation under a microscope to confirm that the number of cells in the four corners and center of the cover glass reaches at least 70%. Then, remove the culture medium in each well, wash twice with PBS, and move the cover glass to another clean and sterile 6-well plate, add the rhodamine-coated glucosamine derivative nanoparticles, and place in an incubator to culture for 4 hours. Then, remove the culture medium in the well plate, wash the cells with PBS, and remove the liquid. Fix the cells with PBS containing 4% paraformaldehyde for 10 minutes, remove the liquid and wash with PBS. Add 0.2% tritonX-100 and react in the dark for 5 minutes. Remove the liquid and wash the cells with PBS. Add 4',6-diamidino-2-phenylindole (DAPI) and react in the dark for 5 minutes to stain the cell nuclei (blue-purple fluorescent substance P). Remove the liquid and wash the cells with PBS. Then, move the cover glass onto a slide glass and observe under a fluorescent microscope.

[0047] After 4 hours of co-culture, the nanoparticles were observed by optical and fluorescent microscopes. Figures 5A-5C (magnification 10x20x0.45, scale 10μm) are experimental diagrams of the transmittance of the glucosamine derivative nanoparticles (KTTKS) / (CRGDS) / (RRRRRR) of the present invention, respectively. Note that Figure 5A is Glu-SA-KTTKS, Figure 5B is Glu-SA-CRGDS, and Figure 5C is Glu-SA-RRRRRR. Furthermore, it was shown that after rhodamine was coated with the glucosamine derivative nanoparticles of the present invention, rhodamine could efficiently enter cells, and the red fluorescent substance R (which is rhodamine) was clearly observed in the cytoplasmic region of the cells, confirming that the glucosamine derivative nanoparticles of the present invention can not only enter cells, but also act as a carrier to carry the specified component (all drugs, functional components, etc. that can be carried by nanoparticles are carried as the specified component, and are not limited to the glucosamine salt exemplified in the present invention) to enter the cells (for example, the skin cells, but the skin cells are one example exemplified in the present invention and are not limited thereto, and any cell that can absorb the absorbable nanoparticles can be used, such as intestinal wall cells) and accumulate therein.

[0048] 3. Toxic effects on cells (Taking HaCaT as an example): 10 HaCaT cells 5The cells were seeded in a 96-well plate at a density of 1000 cells / well, allowed to attach overnight, and then the culture medium was removed. The lyophilized peptides (KTTKS, CRGDS, and RRRRRR) and the glucosamine derivative nanoparticles (Glu-SA-KTTKS, Glu-SA-CRGDS, and Glu-SA-RRRRRR) were redissolved in serum-free medium (final concentration 1,000 ppm), and serially diluted using serum-free medium to obtain 500, 250, and 125 ppm. Then, 100 μl of each concentration of sample was placed in the well plate (repeated four times, and the group to which the serum-free medium was added was used as the control group). After 24 hours of culture, the medium was removed, and 10 μl of CCK-8 and 100 μl of PBS were added to each well, and the reaction was allowed to continue for 2 to 3 hours at 37°C, and the absorbance value was then read at a wavelength of 450 nm. The cell viability was expressed as the percentage of the absorbance value of each experimental group relative to the absorbance value of the control group. Here, the cell viability of the control group was taken as 100%.

[0049] As can be seen from Figure 6, as the concentration of glucosamine increases, the cell viability tends to decrease slightly. However, the cell viability of the glucosamine derivative nanoparticles of the present invention (KTTKS, CRGDS or RRRRRR) increases with increasing added concentration (ppm), indicating that the properties of the glucosamine and its effect on cells are changed by modifying the glucosamine with the peptide.

[0050] 4. Toxic effects on living organisms (using zebrafish embryos as an example): Zebrafish were kept in a fish tank in a low-temperature incubator (28°C) with a photoperiod of 14 hours of light and 10 hours of darkness, and were fed once in the morning and once in the evening. When it became necessary to collect fertilized zebrafish eggs, the photoperiod was changed to on the previous day, and the zebrafish were alternately fed with larval food and powdered shellless brine shrimp eggs every 2 hours to ensure sufficient nutrition for the zebrafish.

[0051] For the experiment, 0 hpf (hours post-fertilization) zebrafish larvae were selected for the drug administration experiment and placed in a 6-well culture plate (30 larvae / well, replicated three times). The suspended water in the 6-well culture plate was removed, and the glucosamine salt (Glu), the peptide (the KTTKS, the CRGDS, and the RRRRRR), the glucosamine derivatives (Glu-SA-(KTTKS), Glu-SA-(CRGDS), Glu-SA-(RRRRRR)) and the glucosamine derivative nanoparticles (Glu-SA-KTTKS, Glu-SA-CRGDS, Glu-SA-RRRRRR) were freeze-dried into powder form and redissolved in deionized water, and 3 ml of the powder was added to the culture plate and cultured in a low-temperature incubator in the dark. After 24 hours, the liquid in the well plate was removed and clean suspended water was immediately poured in. The survival rate of the fry was calculated, and the test results are shown in FIG. 7.

[0052] After the nanoparticles are formed, the glucosamine can be coated, so that the partially exposed peptide is advantageous in permeating cells, and even at the same concentration, the survival rate of zebrafish embryos increases to 81% (100 ppm), 86% (50 ppm) and 88% (50 ppm), demonstrating that the glucosamine derivative nanoparticles of the present invention can reduce toxicity to living organisms.

[0053] As can be seen from the above experimental data, the present invention uses glucosamine derivatives (glucosamine modified with succinic anhydride and then bound to a peptide with permeability) and ethanol to form nanoparticles through self-assembly, and the nanoparticles and the permeable peptides allow the glucosamine derivative nanoparticles to be easily absorbed into the stratum corneum of the skin and to enter and be absorbed into cells, further solving the problem of poor absorption of glucosamine.

[0054] Furthermore, the glucosamine derivative nanoparticles of the present invention can also act as carriers to carry larger amounts of the required specified ingredient (e.g., glucosamine salt), and can deliver and absorb the specified ingredient into cells or the stratum corneum without causing toxicity to cells and organisms.

[0055] Thus, compared to commercially available glucosamine, the glucosamine derivative nanoparticles of the present invention have a better absorption effect without causing toxicity to organisms / cells, and can achieve good glucosamine absorption rates whether administered orally or by skin application.

[0056] Therefore, the present invention is truly novel, inventive and industrially applicable, and satisfies the requirements for a patent application set forth in the Patent Act of Japan. We hope to file a patent application in accordance with the law and to be granted the rights at an early date.

[0057] The above description is merely a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. All equivalent changes and modifications of the shape, structure, features, and spirit described in the claims of the present invention are intended to fall within the scope of the claims of the present invention. [Explanation of symbols]

[0058] S1~S2 steps P Blue-violet fluorescent material R red fluorescent material

Claims

1. A method for producing glucosamine derivative nanoparticles, comprising the steps of: dissolving a glucosamine derivative in deionized water to form an aqueous solution of the glucosamine derivative; and mixing the glucosamine derivative aqueous solution with ethanol, and then vibrating the mixture with an ultrasonic vibrator at a power of 40 W to 50 W for 5 to 10 minutes to form glucosamine derivative nanoparticles. The glucosamine derivative is formed by reacting succinic anhydride with the amino end of glucosamine and the amino end of a peptide, and the chemical formula of the glucosamine derivative is 【Chemistry 1】 and A method for producing said peptide, wherein A is composed of 5 to 6 amino acids, and said peptide is one selected from the group consisting of KTTKS, CRGDS and RRRRRR.

2. Prior to the step of dissolving the glucosamine derivative in deionized water, Adding the glucosamine and the succinic anhydride to an aqueous solution of N,N-dimethylformamide and N-methylmorpholine at room temperature to react with each other, and then suction filtering to form glucosamine-succinic anhydride; adding the glucosamine-succinic anhydride and the peptide to N,N-dimethylformamide respectively, and then mixing and reacting at room temperature to form a yellow precipitate; The method of claim 1, further comprising the steps of subjecting the yellow precipitate to suction filtration and centrifugation, and then obtaining the glucosamine derivative.

3. The method of claim 1 , wherein the step of dissolving the glucosamine derivative in deionized water further comprises the step of adding a glucosamine salt to the deionized water in advance.

4. A glucosamine derivative nanoparticle formed by self-assembly of a glucosamine derivative and ethanol, The method for producing the glucosamine derivative nanoparticles comprises: dissolving the glucosamine derivative in deionized water to form an aqueous solution of the glucosamine derivative; and injecting the ethanol into the aqueous solution of the glucosamine derivative, and then vibrating the aqueous solution of the glucosamine derivative with an ultrasonic vibrator at a power of 40 to 50 W for 5 to 10 minutes to form the glucosamine derivative nanoparticles. The glucosamine derivative is formed by reacting succinic anhydride with the amino terminal of glucosamine and the amino terminal of a peptide, The chemical formula of the glucosamine derivative is 【Chemistry 2】 and Glucosamine derivative nanoparticles, wherein A is a peptide composed of 5 to 6 amino acids, and the peptide is one selected from the group consisting of KTTKS, CRGDS and RRRRRR.

5. 5. The glucosamine derivative nanoparticle of claim 4, further coated with a specified component which is a glucosamine salt.

6. The glucosamine derivative nanoparticles according to claim 4, wherein the particle size of the glucosamine derivative nanoparticles is between 400 and 1,000 nm.

7. A method for forming a carrier for delivering a specified ingredient to a cell or the stratum corneum using glucosamine derivative nanoparticles, The designated component is a lipophilic or hydrophilic functional component, The method for producing the glucosamine derivative nanoparticles comprises: Dissolving the glucosamine derivative in deionized water to form an aqueous solution of the glucosamine derivative; After mixing ethanol with the aqueous solution of the glucosamine derivative, the mixture is vibrated for 5 to 10 minutes at a power of 40 to 50 W using an ultrasonic vibrator to form the glucosamine derivative nanoparticles; The glucosamine derivative is formed by reacting succinic anhydride with the amino terminal of glucosamine and the amino terminal of a peptide, The chemical formula of the glucosamine derivative is 【Chemistry 3】 and A method, wherein A is the peptide composed of 5 to 6 amino acids, and the peptide is one selected from the group consisting of KTTKS, CRGDS and RRRRRR.

8. The method of claim 7, wherein the glucosamine derivative nanoparticles enter the cells by a method in which the peptides in the glucosamine derivative recognize specific receptors on the surface of the cells and change charge attraction or hydrophilicity / hydrophobicity.

9. The method of claim 7, wherein the glucosamine derivative nanoparticles enter the stratum corneum by changing their hydrophilicity and hydrophobicity.

10. The method of claim 7, wherein the specified ingredient is a glucosamine salt.

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