Hyaluronic acid germanium, and preparation method and application thereof

Abstract

The application discloses hyaluronic acid germanium, belongs to the field of biological medicine and new materials. The application utilizes ethylenediamine bridging Ge-132 and hyaluronic acid to generate macromolecular hyaluronic acid germanium. The hyaluronic acid germanium can improve the activity and bioavailability of Ge-132, and has better antioxidant, anti-inflammatory and cell migration promoting effects. The application further provides a preparation method and application of the hyaluronic acid germanium.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and new materials, and specifically relates to a method for preparing germanium hyaluronic acid and its application. Background Technology

[0002] Hyaluronic acid (HA) is a natural macromolecular polysaccharide widely found in the connective tissue, skin, synovial fluid, and vitreous humor of the human and animal bodies. It possesses excellent biocompatibility, anti-inflammatory, and antioxidant properties, and is widely used in dermatology, ophthalmology, and arthritis treatment. Due to its ability to maintain skin moisture and elasticity, repair the skin barrier, and reduce wrinkles, HA is widely used in the cosmetic field. As a major component of synovial fluid, it effectively reduces cartilage friction and cushions impacts. Cross-linked hyaluronic acid (such as Synvisc) can be used to treat arthritis through intra-articular injection. Hyaluronic acid influences cell proliferation, differentiation, and migration by binding to receptors such as CD44, and its CD44 targeting ability makes it a common drug delivery carrier.

[0003] Organic germanium possesses unique medicinal and health-promoting functions. β-Carboxyethyl germanium sesquioxide (Ge-132) is a synthetically produced organic germanium widely used in medicine, food, health products, and cosmetics. Organic germanium often contains multiple Ge-O bonds, exhibiting strong oxidative dehydrogenation capabilities. It can efficiently capture hydrogen and some harmful ions from metabolic products, acting as an "oxygenator and waste remover." Ge-132 can enhance the activity of antioxidant enzymes in the body, thus scavenging excess free radicals. Ge-132 can stimulate the body to produce interferon, increasing the activity and number of immune cells such as macrophages, thereby enhancing the body's immune regulation. Simultaneously, Ge-132 can increase the number of hemoglobin and red blood cells in the blood and stimulate platelet production, reducing serum total lipid levels to some extent and affecting lipid metabolism. Furthermore, it has anti-inflammatory, anti-aging, and anti-tumor effects. Moreover, it is stable, highly water-soluble, and has few toxic side effects, and was initially widely used as a food additive and health product. Although Ge-132 has demonstrated anti-cancer, immunomodulatory, antioxidant, anti-inflammatory, and anti-aging functions in studies, its utilization rate and functional activity are low in practical applications, resulting in limited clinical efficacy. Furthermore, long-term or high-dose use can cause side effects such as nephrotoxicity. Therefore, further in-depth research is needed to modify and optimize the structure of Ge-132 to screen for drugs with superior activity, fewer toxic side effects, and higher bioavailability. Summary of the Invention

[0004] This invention provides a germanium hyaluronic acid material. The carboxyl group in the Ge-132 structure reacts with ethylenediamine to obtain an aminated Ge-132, which then reacts with the carboxylamide in hyaluronic acid to obtain hyaluronic acid-germanium. Utilizing the excellent biocompatibility, CD44 targeting, and anti-inflammatory activities of hyaluronic acid, combined with the immunomodulatory and other multifunctional activities of Ge-132, a multifunctional germanium hyaluronic acid material with good biocompatibility, targeting, anti-inflammatory, antioxidant, and immunomodulatory properties is obtained.

[0005] A germanium hyaluronic acid, characterized in that Ge-132 is bridged with hyaluronic acid by forming an amide bond with ethylenediamine.

[0006] A method for preparing germanium hyaluronic acid, characterized in that the method includes the following steps:

[0007] Step S1: Ge-132 is dispersed in chloroform and then reacted with 2-5 molar amounts of thionyl chloride at 40-60°C for 4-8 h. The solvent and excess thionyl chloride are removed by evaporation to obtain acyl chloride organic germanium.

[0008] Step S2: Under ice bath conditions, the acyl chloride organic germanium solution dissolved in dichloromethane is slowly added dropwise to 5-10 times the molar volume of ethylenediamine solution, and the reaction is carried out at room temperature for at least 12 hours to obtain the germanium-ethylenediamine compound after purification.

[0009] Step S3: Add 2-5 molar amounts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 2-5 molar amounts of N-hydroxysuccinimide to the hyaluronic acid aqueous solution, and stir at room temperature for 30-120 minutes; then add 2-5 molar amounts of Ge-ethylenediamine and triethylamine, and stir at room temperature for at least 24 hours; the resulting solution is dialyzed and freeze-dried to obtain germanium hyaluronic acid material.

[0010] In step S1 of this invention, the addition of 2-5 times the molar amount of thionyl chloride is because excess thionyl chloride can accelerate the conversion of carboxyl groups to acyl chlorides and increase the solubility of Ge-132 in chloroform.

[0011] In step S2 of this invention, the addition of 5-10 times the molar amount of ethylenediamine solution is intended to maximize the 1:1 reaction between germanium chloride and ethylenediamine, preventing both amino groups at the ends of the ethylenediamine from undergoing amide reactions.

[0012] In step S3 of this invention, the purpose of adding 2-5 times the molar amount of EDC·HCl, N-hydroxysuccinimide, Ge-ethylenediamine and triethylamine is to enable Ge-ethylenediamine to bond to hyaluronic acid more efficiently and to achieve the control of the ratio of germanium compound and hyaluronic acid.

[0013] The use of germanium hyaluronic acid in the preparation of a drug for the prevention and treatment of skin diseases, the skin diseases including at least one of skin barrier damage, inflammatory skin diseases and immune skin diseases.

[0014] The use of germanium hyaluronic acid in the preparation of a drug for the prevention and treatment of skeletal system diseases, said skeletal system diseases including at least one of bone defects, osteoarthritis and rheumatoid arthritis.

[0015] A drug, wherein the active ingredient of the drug comprises germanium hyaluronic acid of the present invention, and the drug includes drugs for the prevention and treatment of skin diseases or drugs for the prevention and treatment of skeletal system diseases.

[0016] This invention utilizes the amide reaction between the carboxyl group on hyaluronic acid and the amino group on germanium-ethylenediamine to obtain germanium hyaluronic acid material. Germanium hyaluronic acid is Ge-132 bonded to a large molecule of hyaluronic acid, simultaneously possessing the bioactive functions of both Ge-132 and hyaluronic acid. Specifically, it combines the anti-inflammatory, antioxidant, and immunomodulatory functions of Ge-132 with the anti-inflammatory, antioxidant, lubricating, moisturizing, and targeted functions of hyaluronic acid. The combination of the two can synergistically exert superior functional activity. Compared to small-molecule Ge-132, large-molecule germanium hyaluronic acid has a longer in vivo residence time, which can to some extent solve the problems of low bioavailability, easy metabolism, and lack of targeted targeting associated with small-molecule Ge-132 drugs. Furthermore, it possesses anti-inflammatory, antioxidant, immunomodulatory, lubricating, and moisturizing effects, making it highly effective in treating diseases of the skin and skeletal system.

[0017] This invention provides germanium hyaluronic acid with anti-inflammatory, antioxidant, wound-healing, moisturizing, lubricating, and immunomodulatory effects, and it is effective in treating skin diseases and diseases of the skeletal system. The preparation method of this invention for germanium hyaluronic acid is simple, easy to implement, and low-cost, making it easy to industrialize and possessing potential economic, social value and application prospects. Attached Figure Description

[0018] Figure 1 The Fourier transform infrared spectroscopy results are shown for the germanium hyaluronic acid prepared in Example 1.

[0019] Figure 2 This is a diagram showing the effect of germanium hyaluronic acid prepared in Example 1 on scavenging reactive oxygen species in HUVEC cells.

[0020] Figure 3 This is a diagram showing the effect of germanium hyaluronic acid prepared in Example 1 on the migration of HUVEC cells.

[0021] Figure 4 The graph shows the results of germanium hyaluronic acid prepared in Example 1 in reducing the inflammatory factor IL-6 in macrophages. Detailed Implementation

[0022] Example 1: A method for preparing germanium hyaluronic acid, comprising the following steps:

[0023] Step S1: Weigh 1 mmol of Ge-132 and place it in a 25 ml flask. Add 5 ml of chloroform to disperse it, add 4 mmol of thionyl chloride, react at 60 °C for 4 h, and remove the solvent and excess thionyl chloride by rotary evaporation to obtain acyl chloride organic germanium.

[0024] Step S2: 10 mmol of ethylenediamine was placed in a 50 ml flask and dissolved in 20 ml of dichloromethane to form an ethylenediamine solution. 1 mmol of organogermanium acyl chloride obtained in step S1 was dissolved in 5 ml of dichloromethane to form an organogermanium acyl chloride solution. Under ice bath stirring conditions, the organogermanium acyl chloride solution was slowly added dropwise to the ethylenediamine solution. After the addition was complete, the reaction was allowed to proceed at room temperature for 12 h. The mixture was washed three times with 1 M hydrochloric acid solution, and the dichloromethane was removed by rotary evaporation to obtain the germanium-ethylenediamine compound.

[0025] Step S3: Add 5 times the molar amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 2 times the molar amount of N-hydroxysuccinimide to 50 ml of 10 mg / mL hyaluronic acid aqueous solution, and stir at room temperature for 30 minutes; add 2 times the molar amount of Ge-ethylenediamine and triethylamine respectively, and stir at room temperature for 24 h; place the solution in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze for 48 h, changing the solution 6 times. After dialyzing, freeze-dry the solution to obtain the germanium hyaluronic acid material of the present invention.

[0026] The germanium hyaluronic acid prepared in this embodiment is bridged with hyaluronic acid by forming an amide bond with ethylenediamine.

[0027] The germanium hyaluronic acid prepared in this embodiment was characterized, and the results are as follows: Figure 1 As shown. Compared to Ge-132, germanium-ethylenediamine at 1545.8 cm⁻¹ -1 2898.2 cm -1 and 3298.1 cm -1 The presence of characteristic peaks for amide bonds (-CONH-), CH, and NH bonds indicates that ethylenediamine was successfully bonded to Ge-132. Compared to HA, HA-Ge shows a peak at 1545.8 cm⁻¹. -1 2898.2 cm -1 Characteristic peaks also appeared at the HA, confirming the grafting of germanium-ethylenediamine onto HA. Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect the germanium content in three batches of HA-Ge samples prepared under the same conditions. The results showed that the average germanium content in HA-Ge was 0.685%, further confirming the binding of germanium-ethylenediamine onto HA.

[0028] The germanium hyaluronic acid prepared in this embodiment was tested for its ability to scavenge intracellular reactive oxygen species, promote cell migration, and reduce inflammatory factors.

[0029] 1. Assay on the effect of the prepared germanium hyaluronic acid on scavenging intracellular reactive oxygen species in HUVEC cells:

[0030] (1) Preparation of Germanium hyaluronic acid (HA-Ge) stock solution: Weigh 100 mg of germanium hyaluronic acid and add 1 ml of PBS to dissolve it completely. Filter the solution with a 0.22 µm filter to remove bacteria and set aside. Take 100 µL of the stock solution and use ICP-MS to detect the germanium content in the sample. The result showed that the germanium content was 0.685%.

[0031] (2) Select HUVEC cells in good growth condition, take 24-well plates, and use 3×10^ 4 Cells were seeded into plates at a density of cells / well, and then incubated at 37°C for 24 h. After that, different concentrations of HA-Ge (i.e., 2.5 mg / mL, 5 mg / mL and 10 mg / mL) were added and incubated for 12 h.

[0032] (3) Add 300 µM H2O2 and incubate for 30 min. Remove the culture medium and incubate with serum-free culture medium and 10 µM DCFH-DA probe at 37 °C for 30 min. Wash the cells twice with PBS to remove probes that have not entered the cells, and detect the intracellular ROS level using a fluorescence microscope;

[0033] The results are as follows Figure 2 As shown, from Figure 2 It is known that germanium hyaluronic acid (HA-Ge) can effectively remove H2O2-induced ROS in a concentration-dependent manner, and 10 mg / mL germanium hyaluronic acid can basically remove excessive ROS in cells.

[0034] 2. Assay on the effect of the prepared germanium hyaluronic acid on the migration of HUVEC cells:

[0035] (1) Preparation of Germanium hyaluronic acid (HA-Ge) stock solution: Weigh 100 mg of germanium hyaluronic acid and add 1 ml of PBS to dissolve it completely. Filter the solution with a 0.22 µm filter to remove bacteria and set aside. Take 100 µL of the stock solution and use ICP-MS to detect the germanium content in the sample. The result showed that the germanium content was 0.685%.

[0036] (2) Preparation of hyaluronic acid (HA) and Ge-132 (Ge) stock solutions: Weigh 100 mg of hyaluronic acid and add 1 ml of PBS to dissolve it completely. Filter the solution through a 0.22 µm filter to sterilize it. Weigh 1.6 mg of Ge-132, add 1 ml of PBS, sonicate to dissolve it completely, and filter the solution through a 0.22 µm filter to sterilize it. The resulting solution has a germanium content of 0.685%.

[0037] (3) Select HUVEC cells in good growth condition and adjust the cell density to 5×10^5 cells using serum-free culture medium. 5 Approximately 200 µL of cell suspension per mL was added to the upper chamber of the transwell.

[0038] (4) Add 600 µL of culture medium containing 10% FBS to the lower chamber, and add different drugs such as HA, Ge-132, HA+Ge-132 and HA-Ge (HA concentration is 10 mg / mL, Ge concentration is 0.0685 mg / mL), and incubate in an incubator for 24 h;

[0039] (5) Remove the upper chamber, carefully wash with PBS, fix with paraformaldehyde for 15-30 min, wash with PBS, add 1 mL of crystal violet and stain for 15 min;

[0040] (6) Carefully wipe away the upper cells of the upper chamber with a cotton swab and take a picture with an upright microscope.

[0041] The results are as follows Figure 3 As shown, from Figure 3 It can be seen that, compared with HA, Ge-132 and HA+Ge-132 (a mixture of HA and Ge-132), HA-Ge exhibits a superior effect in promoting HUVEC cell migration at the same HA or Ge concentration. The above results indicate that HA-Ge exhibits superior activity in skin repair.

[0042] 3. Polarization effect of the prepared germanium hyaluronic acid on mouse mononuclear macrophage cell line (Raw264.7 cells):

[0043] (1) Select Raw264.7 cells in good growth condition, and use 1×10^ 6 The cells were seeded into 12-well plates at a density of cells / well, and then incubated at 37°C for 24 h. Then, different drugs, HA, Ge-132, HA+Ge-132 and HA-Ge (HA concentration 10 mg / mL, Ge concentration 0.0685 mg / mL), were added and incubated for 12 h.

[0044] (2) Then add 1µg / mL LPS and continue incubation for 24 h. Remove the culture medium, wash twice with pre-cooled PBS, lyse the cells thoroughly with Trizol reagent, and extract RNA using chloroform, isopropanol and ethanol according to standard procedures.

[0045] (3) The concentration and purity of RNA were determined using a nucleic acid quantification instrument. cDNA was synthesized according to the formula and conditions in the instructions of the reverse transcription kit and amplified using an RT-PCR instrument. The IL-6 gene was detected in this study, and the data were analyzed.

[0046] The results are as follows Figure 4 As shown, from Figure 4 It was found that LPS-induced Raw264.7 cells showed a significant upregulation of the inflammatory factor IL-6. Treatment with HA, Ge-132 and HA+Ge drugs did not reduce IL-6 expression, while HA-Ge treatment significantly reduced IL-6 expression, indicating that HA-Ge exhibited superior anti-inflammatory activity.

[0047] Example 2: A method for preparing germanium hyaluronic acid, comprising the following steps:

[0048] Step S1: Weigh 1 mmol of Ge-132 and place it in a 25 ml flask. Add 5 ml of chloroform to disperse it, add 2 mmol of thionyl chloride, react at 40 °C for 8 h, and remove the solvent and excess thionyl chloride by rotary evaporation to obtain acyl chloride organic germanium.

[0049] Step S2: 10 mmol of ethylenediamine was placed in a 50 ml flask and dissolved in 20 ml of dichloromethane to form an ethylenediamine solution. 1 mmol of organogermanium acyl chloride obtained in step S1 was dissolved in 5 ml of dichloromethane to form an organogermanium acyl chloride solution. Under ice bath stirring conditions, the organogermanium acyl chloride solution was slowly added dropwise to the ethylenediamine solution. After the addition was complete, the reaction was allowed to proceed at room temperature for 12 h. The mixture was washed three times with 1 M hydrochloric acid solution, and the dichloromethane was removed by rotary evaporation to obtain the germanium-ethylenediamine compound.

[0050] Step S3: Add 5 times the molar amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 2 times the molar amount of N-hydroxysuccinimide to 50 ml of 10 mg / mL hyaluronic acid aqueous solution, and stir at room temperature for 120 minutes; add 2 times the molar amount of Ge-ethylenediamine and triethylamine respectively, and stir at room temperature for 24 h; place the solution in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze for 48 h, changing the solution 6 times. After dialyzing, freeze-dry the solution to obtain the germanium hyaluronic acid material of the present invention.

[0051] Example 3: A method for preparing germanium hyaluronic acid, comprising the following steps:

[0052] Step S1: Weigh 1 mmol of Ge-132 and place it in a 25 ml flask. Add 5 ml of chloroform to disperse it, add 4 mmol of thionyl chloride, react at 50 °C for 6 h, and remove the solvent and excess thionyl chloride by rotary evaporation to obtain acyl chloride organic germanium.

[0053] Step S2: 10 mmol of ethylenediamine was placed in a 50 ml flask and dissolved in 20 ml of dichloromethane to form an ethylenediamine solution. 1 mmol of organogermanium acyl chloride obtained in step S1 was dissolved in 5 ml of dichloromethane to form an organogermanium acyl chloride solution. Under ice bath stirring conditions, the organogermanium acyl chloride solution was slowly added dropwise to the ethylenediamine solution. After the addition was complete, the reaction was allowed to proceed at room temperature for 12 h. The mixture was washed three times with 1 M hydrochloric acid solution, and the dichloromethane was removed by rotary evaporation to obtain the germanium-ethylenediamine compound.

[0054] Step S3: Add 5 times the molar amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 2 times the molar amount of N-hydroxysuccinimide to 50 ml of 10 mg / mL hyaluronic acid aqueous solution, and stir at room temperature for 30 minutes; add 5 times the molar amount of Ge-ethylenediamine and triethylamine respectively, and stir at room temperature for 24 h; place the solution in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze for 48 h, changing the solution 6 times. After dialyzing, freeze-dry the solution to obtain the germanium hyaluronic acid material of the present invention.

Claims

1. A germanium hyaluronic acid, characterized in that, Ge-132 is bridged with hyaluronic acid by forming an amide bond with ethylenediamine.

2. A method for preparing germanium hyaluronic acid, characterized in that, The method includes the following steps: Step S1: Ge-132 is dispersed in chloroform and then reacted with 2-5 molar amounts of thionyl chloride at 40-60°C for 4-8 hours. The solvent and excess thionyl chloride are removed by evaporation to obtain acyl chloride organic germanium. Step S2: Under ice bath conditions, the acyl chloride organic germanium solution dissolved in dichloromethane is slowly added dropwise to 5-10 times the molar volume of ethylenediamine solution, and the reaction is carried out at room temperature for at least 12 hours to obtain the germanium-ethylenediamine compound after purification. Step S3: Add 2-5 molar amounts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 2-5 molar amounts of N-hydroxysuccinimide to the hyaluronic acid aqueous solution, and stir at room temperature for 30-120 minutes; then add 2-5 molar amounts of Ge-ethylenediamine and triethylamine, and stir at room temperature for at least 24 hours; the resulting solution is dialyzed and freeze-dried to obtain germanium hyaluronic acid material.

3. The application of germanium hyaluronic acid as described in claim 1 in the preparation of a drug for preventing and treating skin diseases, wherein the skin diseases include at least one of skin barrier damage, inflammatory skin diseases, and immune skin diseases.

4. The application of germanium hyaluronic acid as described in claim 1 in the preparation of a medicament for preventing and treating skeletal system diseases, wherein the skeletal system diseases include at least one of bone defects, osteoarthritis, and rheumatoid arthritis.

5. A medicament, wherein the active ingredient of the medicament comprises germanium hyaluronic acid as described in claim 1, wherein the medicament includes a medicament for preventing or treating skin diseases or a medicament for preventing or treating skeletal system diseases.

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