A chitosan copper ion complex hydrogel, its preparation method and application

Abstract

The present invention discloses a chitosan copper ion complex hydrogel, a preparation method thereof and an application. The raw materials of the hydrogel include: a chitosan copper ion complex solution, sodium alginate, glycerol, sodium hyaluronate and an acidity regulator. The concentration of copper ions in the chitosan copper ion complex solution is 0.01% to 0.1%. The chitosan copper ion complex in the chitosan copper ion complex hydrogel of the present invention can induce the death of Candida albicans, so as to achieve the purpose of inhibiting or treating related infections or diseases caused by Candida albicans.

Description

Technical Field

[0001] This belongs to the field of pharmaceutical technology, and specifically belongs to a chitosan copper ion complex hydrogel, its preparation method and application. Background Art

[0002] Candida albicans, also known as Monilia albicans or Canidia albicans, is a common pathogenic fungus. It usually exists in the oral cavity, upper respiratory tract, intestine and vagina of normal people. Generally, the number is small in the normal body and does not cause diseases. When the body's immune function or general defense ability declines or the balance of mutual restraint among normal flora is disrupted, Candida albicans will multiply in large numbers and change its growth form (germ tube phase) to invade cells and cause diseases. Candida albicans can cause cutaneous candidiasis, mucosal candidiasis, visceral candidiasis, candidal exanthema, etc. Candidal infection of the nails can cause candidal paronychia. People who have been engaged in humid work for a long time may have Candida infection between fingers or toes, leading to candidal intertrigo. Some immunodeficiency or endocrine disorders, such as hypoparathyroidism and adrenal hypofunction, can induce chronic mucocutaneous candidiasis. Mucosal candidiasis is more common in thrush of infants and young children, genital candidiasis (including vulvovaginal candidiasis in women and balanoposthitis in men). The bacteria of visceral candidiasis can affect all organs of the body, causing pulmonary candidiasis, urinary tract infection, meningitis, endocarditis, candidal sepsis, etc. According to statistics, at least 75% of women worldwide will suffer from vulvovaginal candidiasis caused by Candida albicans, and there are recurrent cases. Most cases of candidiasis may be endogenous, and there are many factors that promote the occurrence of the disease. The main ones are as follows: conditions such as diabetes, tuberculosis, tumors, severe burns, organ transplantation, etc. can all reduce the body's resistance of patients and make them prone to Candida infection; long-term and large-scale use of antibiotics may result in bacterial substitution phenomenon or bacterial substitution syndrome, that is, after using antibiotics, the bacteria in the body that are sensitive to antibiotics may be inhibited or killed, while some Candida are not sensitive to antibiotics, which will cause the proliferation of drug-resistant strains, thus destroying the antagonistic balance among bacteria in the body; long-term use of corticosteroids can also lead to a decline in the body's immune function and is conducive to Candida infection. In addition to the above, exogenous infection cannot be ignored, that is, candidiasis can be infected by contacting external bacteria. Candidal infection of the nipple skin in lactating women mostly comes from infants with thrush, or may also be caused by the mother having vulvovaginal candidiasis. At present, the number of effective anti-Candida albicans drugs is limited, and coupled with the spread of Candida drug resistance, the number of cases caused by Candida albicans infection has been increasing year by year. Therefore, there is an urgent need to develop new antibacterial products.

[0003] Copper ions, as an essential element for the human body, are involved in many important physiological processes. It is a component of some enzymes that play a crucial role in maintaining normal metabolism in organisms. Copper ions can also promote redox reactions and electron transfer, and thus participate in processes such as energy metabolism and protein synthesis. "Copper-induced cell death" is a newly discovered form of cell death. Excessive copper promotes the abnormal oligomerization of copper-dependent lipoylated proteins in the tricarboxylic acid cycle and reduces the level of Fe-S cluster proteins to induce copper-induced cell death. Summary of the Invention

[0004] To solve the problems existing in the prior art, the present invention provides a chitosan copper ion complex hydrogel, a preparation method thereof and an application. By inducing the death of Candida albicans with the chitosan copper ion complex, the purpose of inhibiting or treating related infections or diseases caused by Candida albicans is achieved.

[0005] To achieve the above object, the present invention provides the following technical solution: A chitosan copper ion complex hydrogel, the raw materials including: a chitosan copper ion complex solution, sodium alginate, glycerol, sodium hyaluronate and an acidity regulator, and the copper ion concentration in the chitosan copper ion complex solution is 0.01% - 0.1%.

[0006] Further, in terms of mass percentage, 1% - 3% of sodium alginate, 1% - 3% of glycerol and 0.1% - 0.2% of sodium hyaluronate are added to every 100 mL of the chitosan copper ion complex solution, and the acidity regulator adjusts the pH value of the chitosan copper ion complex hydrogel to 3.8 - 4.5.

[0007] Further, the chitosan copper ion complex solution is a chitosan copper sulfate complex solution or a chitosan copper gluconate complex solution.

[0008] Further, the copper ion concentration in the chitosan copper ion complex solution is 0.05%.

[0009] Further, the acidity regulator is lactic acid.

[0010] The present invention also provides a preparation method of a chitosan copper ion complex hydrogel, and the specific steps are as follows:

[0011] S1 Dissolve chitosan in water and stir evenly to obtain a chitosan solution;

[0012] S2 Add a copper ion compound to the chitosan solution to form a chitosan copper ion complex solution;

[0013] S3 Add chitosan copper ion complex, glycerol, sodium hyaluronate, and sodium alginate to sterile ultrapure water and stir evenly to obtain a semi-finished chitosan copper ion complex hydrogel;

[0014] S4 adds an acidity regulator to the chitosan copper ion complex hydrogel semi-finished product, adjusts the pH value to 3.8 - 4.5, and obtains the chitosan copper ion complex hydrogel.

[0015] Further, in S2, the copper ion compound is copper gluconate or copper sulfate

[0016] The present invention also provides an application of the chitosan copper ion complex hydrogel in the treatment of bacterial infections caused by Candida albicans.

[0017] The present invention also provides a product for treating bacterial infections caused by Candida albicans, and this product contains the above-mentioned chitosan copper ion complex hydrogel.

[0018] Further, the bacterial infections caused by Candida albicans specifically include skin infections or reproductive system infections caused by Candida albicans.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] The present invention provides a chitosan copper ion complex hydrogel. The chitosan copper ion complex used has excellent antibacterial properties and can effectively kill Candida albicans. Components such as sodium alginate, glycerol, and sodium hyaluronate are all natural or substances with good biocompatibility, which are non-irritating to the skin, safe and reliable. By adding an acidity regulator to control the pH value of the hydrogel, it is beneficial to maintain the acid-base balance of the skin and enhance the antibacterial effect of the hydrogel. Using the chitosan copper ion complex hydrogel to kill Candida albicans shows a strong bactericidal effect, up to more than 99.9%. Compared with traditional antibiotics, the chitosan copper ion complex hydrogel is not likely to cause drug resistance problems, providing a new strategy for the treatment of Candida albicans infections.

[0021] The chitosan copper ion complex hydrogel of the present invention has a significant bactericidal effect on Candida albicans, can significantly shorten the treatment time and relieve the pain of patients; the product formula is mild and non-irritating, suitable for the treatment of sensitive skin and damaged skin; most of the components in the hydrogel are natural or biodegradable materials, which are environmentally friendly and in line with the concept of green environmental protection. The product can be prepared into easy-to-use dosage forms such as external creams and gels, which are convenient for patients to use by themselves. Description of the Drawings

[0022] Figure 1 Shown are the drop plate diagram and the survival rate statistical chart of Candida albicans after being treated with the chitosan copper gluconate complex aqueous solution;

[0023] Figure 2 Shown are the drop plate diagram and the survival rate statistical chart of Candida albicans after being treated with the chitosan copper sulfate complex aqueous solution;

[0024] Figure 3The inverted fluorescence microscope images of Candida albicans treated with aqueous solutions of chitosan copper sulfate complex and chitosan copper gluconate complex are shown;

[0025] Figure 4 The scanning electron microscope images of Candida albicans before and after treatment in Example 5 are shown;

[0026] Figure 5 The physical images of a chitosan copper ion complex hydrogel prepared in Examples 1-8 are shown;

[0027] Figure 6 The scanning electron microscope images of the hydrogels in Examples 1, 2, 4, and 6 are shown;

[0028] Figure 7 The statistical chart of the survival rate of Candida albicans after treatment in Examples 1 to 8 is shown;

[0029] Figure 8 The growth images of the mouse vaginal lavage fluid on the culture plate are shown;

[0030] Figure 9 The statistical chart of the results of the mouse vaginal lavage fluid on the culture plate is shown. Detailed implementation mode

[0031] The present invention will be further described below with reference to the accompanying drawings. The following examples are only used to more clearly illustrate the technical solution of the present invention and cannot be used to limit the protection scope of the present invention.

[0032] The present invention provides a chitosan copper ion complex hydrogel, and the raw materials include: chitosan copper ion complex solution, sodium alginate, glycerol, sodium hyaluronate, and acidity regulator. Among them, the concentration of copper ions in the chitosan copper ion complex solution is 0.01% - 0.1% by mass percentage. For every 100 mL of the chitosan copper ion complex solution, 1% - 3% of sodium alginate, 1% - 3% of glycerol, and 0.1% - 0.2% of sodium hyaluronate are added; an acidity regulator is added to adjust the pH value of the chitosan copper ion complex hydrogel within 3.8 - 4.5.

[0033] Preferably, the chitosan copper ion complex solution is a chitosan copper sulfate complex solution or a chitosan copper gluconate complex solution.

[0034] Preferably, the addition amounts of chitosan and copper ions in the chitosan copper ion complex solution are the same, and the concentration of copper ions is 0.05%.

[0035] Preferably, the acidity regulator is lactic acid.

[0036] Secondly, the present invention also provides a preparation method of a chitosan copper ion complex hydrogel, and the specific steps are as follows:

[0037] S1 Dissolve chitosan in water and stir evenly to obtain a chitosan solution;

[0038] S2 Add a copper ion compound to the chitosan solution to form a chitosan copper ion complex solution;

[0039] S3 Add glycerol, sodium hyaluronate, and sodium alginate to the chitosan copper ion complex solution and stir evenly to obtain a semi-finished chitosan copper ion complex hydrogel;

[0040] S4 Add lactic acid to the semi-finished chitosan copper ion complex hydrogel, adjust the pH value to 3.8 - 4.5, remove bubbles by ultrasound, and then perform aseptic filling to obtain a chitosan copper ion complex hydrogel.

[0041] Preferably, the copper ion compound is copper gluconate or copper sulfate.

[0042] The above chitosan copper ion complex hydrogel can be used in inhibiting Candida albicans infection, has a strong bactericidal effect on Candida albicans, up to more than 99.9%, and copper ions at a concentration of 0.01% - 0.1% in the chitosan copper ion complex hydrogel cause no harm to the human body. At the same time, it avoids the generation of a large number of drug-resistant Candida albicans due to the use of antibiotics.

[0043] The present invention provides a product for treating bacterial infections caused by Candida albicans. This product contains the above chitosan copper ion complex hydrogel, and the chitosan copper ion complex hydrogel can make the survival rate of Candida albicans less than 0.01%, providing a new treatment idea for the treatment of diseases caused by Candida albicans.

[0044] The above product can be prepared into external dosage forms, suppositories, etc., but is not limited to the above.

[0045] The bacterial infections caused by the above Candida albicans specifically include skin infections or reproductive system infections caused by Candida albicans, but are not limited to the above.

[0046] Example 1

[0047] A finished hydrogel, including the following steps:

[0048] S1 Add sodium alginate, glycerol, sodium hyaluronate, and lactic acid to sterile ultrapure water and stir evenly to obtain a semi-finished hydrogel. The addition amount of sodium alginate is 2%, the addition amount of glycerol is 2%, the addition amount of sodium hyaluronate is 0.1%, and the addition amount of lactic acid is 1.5%;

[0049] S2 Perform aseptic filling on the semi-finished hydrogel to make a finished hydrogel.

[0050] Example 2

[0051] A chitosan copper ion complex hydrogel, comprising the following steps:

[0052] S1: Dissolve chitosan in water and stir evenly. The addition amount of chitosan is 0.01%, and the water is 100 mL;

[0053] S2: Then add 0.01% copper gluconate to the chitosan solution to obtain a chitosan copper gluconate complex solution with a concentration of 0.01%;

[0054] S3: Then add glycerol, hyaluronic acid, and sodium alginate thereto and stir evenly to obtain a semi-finished chitosan copper gluconate complex hydrogel. Among them, the addition amount of sodium alginate is 2%, the addition amount of glycerol is 2%, and the addition amount of sodium hyaluronate is 0.1%;

[0055] S4: Then add lactic acid to the semi-finished chitosan copper gluconate complex hydrogel to adjust the pH value to 3.8 - 4.5;

[0056] S5: Then remove the bubbles from the semi-finished chitosan copper gluconate complex hydrogel by ultrasonic treatment, and after aseptic filling, obtain the finished chitosan copper ion complex hydrogel.

[0057] Example 3

[0058] A chitosan copper ion complex hydrogel, comprising the following steps:

[0059] S1: Dissolve chitosan in water and stir evenly. The addition amount of chitosan is 0.01%, and the water is 100 mL;

[0060] S2: Then add 0.01% copper sulfate to the chitosan solution to obtain a chitosan copper sulfate complex with a concentration of 0.01%;

[0061] S3: Then add glycerol, hyaluronic acid, and sodium alginate thereto and stir evenly to obtain a semi-finished chitosan copper sulfate complex hydrogel. Among them, the addition amount of sodium alginate is 2%, the addition amount of glycerol is 2%, and the addition amount of sodium hyaluronate is 0.1%;

[0062] S4: Then add lactic acid to the semi-finished chitosan copper sulfate complex hydrogel to adjust the pH value to 3.8 - 4.5;

[0063] S5: Then remove the bubbles from the semi-finished chitosan copper sulfate complex hydrogel by ultrasonic treatment, and after aseptic filling, obtain the finished chitosan copper ion complex hydrogel.

[0064] Example 4

[0065] The difference between Example 4 and Example 2 is that the concentration of the chitosan copper gluconate complex is 0.05%.

[0066] Example 5

[0067] The difference between Example 5 and Example 3 is that the concentration of the chitosan copper sulfate complex is 0.05%.

[0068] Example 6

[0069] The difference between Example 6 and Example 2 is that the concentration of the chitosan copper gluconate complex is 0.1%.

[0070] Example 7

[0071] The difference between Example 7 and Example 3 is that the concentration of the chitosan copper sulfate complex is 0.1%.

[0072] Example 8

[0073] The difference between Example 8 and Example 6 is that the addition amount of sodium alginate is 1%, the addition amount of glycerol is 1%, and the addition amount of sodium hyaluronate is 0.2%.

[0074] Example 9

[0075] The difference between Example 9 and Example 6 is that the addition amount of sodium alginate is 3%, the addition amount of glycerol is 3%, and the addition amount of sodium hyaluronate is 0.15%.

[0076] Example 10

[0077] The difference between Example 10 and Example 7 is that the addition amount of sodium alginate is 1%, the addition amount of glycerol is 1%, and the addition amount of sodium hyaluronate is 0.2%.

[0078] Example 11

[0079] The difference between Example 11 and Example 7 is that the addition amount of sodium alginate is 3%, the addition amount of glycerol is 3%, and the addition amount of sodium hyaluronate is 0.15%.

[0080] Figure 5 Shown is a physical diagram of a chitosan copper ion complex hydrogel prepared for Examples 1 to 8. It can be seen that as the chitosan copper ion complex increases, the viscosity of the hydrogel slightly increases.

[0081] To verify the antibacterial activity of copper ions against Candida albicans, the following tests were conducted:

[0082] 1. Two kinds of chitosan copper ion complex solutions were respectively prepared, including: aqueous solutions of chitosan copper sulfate complex and chitosan copper gluconate complex, and 2×10 6Candida albicans seed solution at CFU / mL, and then the aqueous solutions of chitosan copper sulfate complex and chitosan copper gluconate complex were respectively added to the seed solution to make the final concentration of the chitosan copper ion complex solution 0.01%, 0.02%, 0.05%, 0.1%, and a blank control group was set. Under the conditions of 30 °C and 200 rpm, after treatment for 3 hours, the total number of colonies was measured by the gradient drop plate experiment to explore the antibacterial effect of copper ions on Candida albicans.

[0083] Results: Through Figure 1 and Figure 2 The results showed that at the same concentration, both the chitosan copper gluconate complex and the chitosan copper sulfate complex had inhibitory effects on Candida albicans, and the antibacterial effect of the chitosan copper sulfate complex was better than that of the chitosan copper gluconate complex. And the inhibitory effect on Candida albicans could reach more than 99.9%.

[0084] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the chitosan copper gluconate complex and the chitosan copper sulfate complex against Candida albicans could also be obtained. The MBC value of the chitosan copper gluconate complex against Candida albicans was 0.1%, and the MBC value of the chitosan copper sulfate complex against Candida albicans was 0.05%.

[0085] 2. To further exclude the possibility of Candida albicans producing VBNC (viable but non-culturable), the treatment groups and control groups of the chitosan copper gluconate complex and the chitosan copper sulfate complex loaded with PI probes were observed by inverted fluorescence microscopy technology.

[0086] Results: As Figure 3 The results showed that after 3 hours of treatment with the chitosan copper gluconate complex and the chitosan copper sulfate complex, compared with the control group, most cells showed red fluorescence, indicating that the cells were in a dead state. Verification with an inverted fluorescence microscope showed that the results were consistent, that is, almost all the cells treated with the chitosan copper gluconate complex and the chitosan copper sulfate complex showed red fluorescence. The results were consistent with the above test results. In addition, it was verified in the experiment that no VBNC cells were produced during this process.

[0087] 3. Phenotypic analysis of Candida albicans and hydrogel treated with the chitosan copper ion complex solution was carried out by scanning electron microscopy (SEM).

[0088] Results: As Figure 4 showed, after 3 hours of treatment with the chitosan copper ion complex solution, the morphology of Candida albicans changed significantly, from the original smooth surface to wrinkled, and there would also be a phenomenon of rupture. This indicated that the introduction of the chitosan copper ion complex solution would destroy the morphology of Candida albicans.

[0089] Figure 6 The results showed that Example 1 was an alginate hydrogel, and it could be seen that the structure of the alginate hydrogel was irregular, while Examples 2, 4, and 6 were chitosan copper ion complex hydrogels, and its shape was relatively regular and neat, and it could be seen that the chitosan copper ion complex hydrogel was flaky.

[0090] 4. To verify the performance of the chitosan copper ion complex hydrogel of the present invention, antibacterial activity analysis was carried out on each example: Prepare a Candida albicans seed solution of 2×10 6 CFU / mL, and take the hydrogels of Examples 1 to 8 for treatment respectively. The total number of fungal colonies was measured by the gradient drop plate experiment to explore the antibacterial effects of Examples 1 to 8 on Candida albicans.

[0091] Results: From Figure 7 The results showed that it could be seen that Examples 6 and 7 had good bactericidal effects, and the bactericidal rates were both above 99.9%, having good antibacterial application prospects.

[0092] 5. To further verify the performance of the chitosan copper ion complex hydrogel of the present invention, the following zoological tests were specifically carried out on Examples 4, 5, 6, and 7 to further illustrate its effects:

[0093] (1) Establish a mouse vaginal infection model:

[0094] Select Kunming mice, female, 8 - 10 weeks old, 36 in number. Take 30 mice and start subcutaneous injection of estradiol benzoate for estrogenization in each mouse 6 days before inoculation. The injection specification is 0.1 mL, 1 mg / mL. Inject once every 2 days, and a total of 3 injections are carried out. Take out the mice on the 6th day, lavage their vaginas (0.9% NaCl), observe the lavage fluid under an optical microscope to see if there are mouse vaginal epithelial cells. If so, use a micropipette to take 40 μL, 1×10 7 CFU / mL of Candida albicans and inoculate it into the mouse vagina, with the insertion depth of about 1 - 1.5 cm. After injection, invert the mouse for 5 minutes to prevent the bacterial fluid from flowing out. Inject once a day for a total of 5 days. On the sixth day, lavage the mouse vagina with 100 μL, 0.9% NaCl, fully mix the lavage fluid, take 10 μL and smear it on a clean glass slide for microscopic examination. Observation of more exfoliated cells or hyphae growth is positive; and inoculate the lavage fluid on Sabouraud solid medium and culture it at 30 °C for about 30 hours, and perform colony forming unit counting. Counting above 10 5 CFU / mL is positive; meeting the above conditions means the model is successfully established.

[0095] (2) Animal grouping and drug administration:

[0096] After the successful establishment of the murine vaginitis model, the mice were grouped and administered drugs, divided into 6 groups with 6 mice in each group, namely:

[0097] Normal group mice (6 mice): For normal mice without modeling, 100 μL of 0.9% NaCl was injected into the vagina daily and observed.

[0098] Untreated group mice (6 mice): After successful modeling, the mice were not treated and observed daily.

[0099] Example 4 group (6 mice): After successful modeling, 100 μL of the hydrogel of Example 4 was injected into the vagina of the mice daily and observed.

[0100] Example 5 group (6 mice): After successful modeling, 100 μL of the hydrogel of Example 5 was injected into the vagina of the mice daily and observed.

[0101] Example 6 group (6 mice): After successful modeling, 100 μL of the hydrogel of Example 6 was injected into the vagina of the mice daily and observed.

[0102] Example 7 group (6 mice): After successful modeling, 100 μL of the hydrogel of Example 7 was injected into the vagina of the mice daily and observed.

[0103] Results: Figure 8 、 Figure 9 The results showed that Candida albicans would not grow in the vaginal lavage fluid of normal mice, while a large amount of Candida albicans would be present in the vaginal lavage fluid of the mice after successful modeling. After 7 days of treatment with the chitosan copper ion complex hydrogel, compared with the untreated group mice, the amount of Candida albicans decreased significantly. Among them, the effects of Example 6 and Example 7 were the best, and the bactericidal rates were both above 99.9%, showing good therapeutic effects.

Claims

1. Use of a chitosan copper ion complex hydrogel in the preparation of a drug for treating vaginal fungal infections caused by Candida albicans, characterized in that, The raw materials of the chitosan copper ion complex hydrogel include: chitosan copper ion complex solution, sodium alginate, glycerol, sodium hyaluronate and acidity regulator. The copper ion concentration in the chitosan copper ion complex solution is 0.1%; By mass percentage, 1% - 3% of sodium alginate, 1% - 3% of glycerol and 0.1% - 0.2% of sodium hyaluronate are added to every 100 mL of the chitosan copper ion complex solution. The acidity regulator adjusts the pH value of the chitosan copper ion complex hydrogel to 3.8 - 4.5; The chitosan copper ion complex solution is a chitosan copper sulfate complex solution or a chitosan copper gluconate complex solution; Among them, the chitosan copper gluconate complex is in flake shape; The MBC value of the chitosan copper gluconate complex against Candida albicans is 0.1%; The MBC value of the chitosan copper sulfate complex against Candida albicans is 0.05%; The inhibitory effects of both the chitosan copper gluconate complex and the chitosan copper sulfate complex on Candida albicans are above 99.9%; The acidity regulator is lactic acid.

2. Use of a chitosan copper ion complex hydrogel according to claim 1 in the preparation of a drug for treating vaginal fungal infections caused by Candida albicans, characterized in that, The preparation steps of the chitosan copper ion complex hydrogel are as follows: S1 Dissolve chitosan in water and stir evenly to obtain a chitosan solution; S2 Add a copper ion compound to the chitosan solution to form a chitosan copper ion complex solution; S3 Add the chitosan copper ion complex, glycerol, sodium hyaluronate and sodium alginate to sterile ultrapure water and stir evenly to obtain a semi-finished chitosan copper ion complex hydrogel; S4 Add an acidity regulator to the semi-finished chitosan copper ion complex hydrogel and adjust the pH value to 3.8 - 4.5 to obtain the chitosan copper ion complex hydrogel; Among them, the copper ion compound is copper gluconate or copper sulfate.

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