A benzalkonium chloride contraceptive gel for killing the AIDS pathogen and a preparation method thereof

Abstract

The application discloses a benzalkonium chloride contraceptive gel for killing AIDS pathogens and a preparation method thereof, and belongs to the technical field of biological medicine manufacturing. The benzalkonium chloride contraceptive gel is prepared from water, hydroxypropyl methyl cellulose, polyquaternium-10, modified cyclodextrin, disodium edetate, benzalkonium chloride and glycerol in a specific proportion. In the preparation, the hydroxypropyl methyl cellulose and the polyquaternium-10 are first dispersed by heating, and then the water solution of the modified cyclodextrin, the disodium edetate and the benzalkonium chloride is sucked into the mixture after cooling, and the glycerol is added, and the finished product is obtained through cooling homogenization and vacuum degassing. Compared with the prior art, the amino acid grafted modified cyclodextrin enhances the inclusion and mucosal adhesion performance, the obtained gel has a fast inactivation rate for the AIDS pathogens, a long vaginal retention time, and has the high-efficiency antiviral and long-acting barrier effects, and has low irritation and is safe and reliable.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical manufacturing technology, and in particular to a benzalkonium chloride contraceptive gel that kills HIV pathogens and its preparation method. Background Technology

[0002] With increasing public awareness of health protection, personal care products that combine contraception and prevention of sexually transmitted diseases are receiving growing attention. In existing technologies, benzalkonium chloride, as a cationic surfactant, is widely used in topical contraceptives and gynecological hygiene preparations due to its broad-spectrum bactericidal and sperm-inactivating properties. To improve drug bioavailability and local retention, cyclodextrin inclusion technology, bioadhesive polymers, and the combined use of various functional excipients have become research hotspots in this field. However, further improving the product's ability to instantly inactivate specific highly hazardous pathogens such as HIV, while simultaneously prolonging the effective retention time of the formulation on the vaginal mucosa, remains an important area of ​​ongoing research for those skilled in the art.

[0003] Patent CN118526443A discloses an acetic acid contraceptive gel, which uses acetic acid as the main active ingredient and is prepared with excipients such as poloxamer and hydroxypropyl methylcellulose. It forms a semi-solid polymer gel to immobilize and inactivate sperm, exerting a dual physical and chemical barrier effect. The main drawback of this approach is that acetic acid has limited selectivity for inactivating enveloped viruses, making it difficult to achieve efficient and complete inactivation of HIV pathogens within a short contact time. Patent CN118121621A discloses an anti-human papillomavirus (HPV) topical contraceptive gel, which combines octylphenyl polyol with benzalkonium chloride and β-glucan, achieving sperm immobilization and virus inactivation at relatively low concentrations. The main drawback of this approach is that it primarily targets HPV and has not specifically verified its efficacy against HIV pathogens. Furthermore, the durability of the multi-component compound system's mucosal adhesion in the complex vaginal microenvironment still has room for improvement.

[0004] In summary, existing technologies generally suffer from insufficient inactivation rates of HIV pathogens and inadequate gel retention time on the vaginal mucosa, resulting in difficulty in maintaining long-term efficacy. Therefore, there is an urgent need to develop a benzalkonium chloride contraceptive gel formulation that can rapidly and efficiently kill HIV pathogens during sexual intercourse while also possessing excellent vaginal mucosal adhesion properties to provide a long-lasting protective barrier. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a benzalkonium chloride contraceptive gel that kills HIV pathogens and its preparation method.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] A benzalkonium chloride contraceptive gel for killing HIV pathogens comprises the following ingredients in parts by weight:

[0008] 60-100 parts water, 1-5 parts hydroxypropyl methylcellulose, 0.1-0.5 parts polyquaternium-10, 0.5-2 parts modified cyclodextrin, 0.03-0.08 parts disodium edetate, 0.05-0.3 parts benzalkonium chloride, 3-8 parts glycerol;

[0009] The modified cyclodextrin is prepared by oxidizing γ-cyclodextrin, reacting it with amino acids, and then dialysis and drying.

[0010] The amino acid is at least one of glycine, serine, cysteine, lysine, arginine, and taurine.

[0011] Preferably, the amino acid is composed of arginine and cysteine ​​in a mass ratio of 0.5-2:0.5-2.

[0012] The preparation method of the benzalkonium chloride contraceptive gel that kills HIV pathogens is as follows:

[0013] Weigh water and inject it into a vacuum emulsification tank. Heat and stir. Sprinkle hydroxypropyl methylcellulose and polyquaternium-10 evenly into the vortex on the liquid surface within 5-15 minutes. After the addition is complete, degas under vacuum, turn off the heating, and stir to cool naturally. In a preparation tank, take water and add modified cyclodextrin, disodium edetate, and benzalkonium chloride in sequence. Stir until clear and transparent to obtain the drug solution. When the temperature in the emulsification tank drops to 30-35℃, reduce the stirring speed to 10-25 rpm. Suction the drug solution into the emulsification tank through vacuum suction. Then add glycerin and stir for 10-30 minutes. Turn on the external circulating cooling water to lower the material temperature to 18-25℃, circulate and homogenize. Finally, add water to adjust the total weight, stir for 5-20 minutes, degas under vacuum, and discharge to obtain benzalkonium chloride contraceptive gel.

[0014] The heating and stirring process involves heating to 80-90℃ and stirring at 20-40 rpm.

[0015] Each vacuum degassing step independently involves evacuating to -0.04 to -0.08 MPa and maintaining the vacuum for 3 to 10 minutes to eliminate air bubbles.

[0016] The cyclic homogenization is performed by homogenizing at 2000-4000 rpm for 1-5 minutes.

[0017] The modified cyclodextrin is prepared as follows:

[0018] S1. Take γ-cyclodextrin, add it to water, heat to 70-90℃ and stir until completely dissolved, then cool naturally to room temperature, add sodium periodate to the solution, stir and react at 20-30℃ for 60-90 minutes under light-protected conditions, then add glycerol and continue stirring for 10-20 minutes. Dialyze the resulting reaction solution to obtain aldehyde-modified cyclodextrin solution.

[0019] S2. Mix the aldehyde-modified cyclodextrin solution obtained in step S1 with amino acids, adjust the pH of the system to 5-6 with citric acid, and stir the reaction at 20-30℃ for 10-30 hours. After the reaction is completed, cool the reaction solution to 0-5℃, add 0.1-0.3 parts of sodium borohydride, and stir at 0-5℃ for 1-3 hours. Dialyze the reaction solution and dry it to obtain modified cyclodextrin.

[0020] The modified cyclodextrin can also be prepared by the following method, in parts by weight:

[0021] S1. Take γ-cyclodextrin, add it to water, heat to 70-90℃ and stir until completely dissolved, then cool naturally to room temperature, add sodium periodate to the solution, stir and react at 20-30℃ for 60-90 minutes under light-protected conditions, then add glycerol and continue stirring for 10-20 minutes. Dialyze the resulting reaction solution to obtain aldehyde-modified cyclodextrin solution.

[0022] S2. Mix the aldehyde-modified cyclodextrin solution obtained in step S1 with arginine, adjust the pH of the system to 4.5-5.5 with citric acid, and stir the reaction at 20-30℃ for 5-10 hours; then add cysteine ​​and continue stirring the reaction at 20-30℃ for 5-10 hours; after the reaction is completed, cool the reaction solution to 0-5℃, add 0.1-0.3 parts of sodium borohydride, and stir at 0-5℃ for 1-3 hours; dialyze the reaction solution and dry it to obtain modified cyclodextrin.

[0023] The dialysis in steps S1 and S2 is independent of each other. It involves using a dialysis bag with a molecular weight cutoff of 400-800 Da and dialysis with water for 40-72 hours, during which the external dialysis fluid is changed every 4-8 hours.

[0024] Existing benzalkonium chloride contraceptive gels generally suffer from slow HIV inactivation, short mucosal retention time, and low drug utilization. The main reason is that benzalkonium chloride lacks specific interactions with the virus and mucosa, and ordinary cyclodextrin excipients only provide limited physical inclusion. To solve these problems, this invention first oxidizes γ-cyclodextrin with sodium periodate, causing the vicinal diol structure of its glucose unit to open and generate an aldehyde group. Subsequently, the aldehyde group reacts with the amino group of the amino group in a Schiff base reaction to form an imine bond. Then, sodium borohydride reduces the unstable imine bond to a stable secondary amine covalent bond, allowing the amino acid to be firmly grafted onto the cyclodextrin backbone. This modified cyclodextrin combines the inclusion effect of the cyclodextrin cavity for benzalkonium chloride with the additional functional groups provided by the amino acid side chains, thus synergistically exerting both antiviral and mucosal adhesion effects within the gel matrix.

[0025] Testing revealed that while grafting a single amino acid can improve gel performance, each has its own emphasis: cysteine ​​exhibits outstanding anti-HIV activity due to its reducing sulfhydryl group, while arginine demonstrates excellent mucosal adhesion due to its strongly positively charged guanidine group. However, neither can simultaneously address both needs. Therefore, this invention proposes a combined grafting scheme of arginine and cysteine. Arginine anchors the gel to the mucosal surface through electrostatic adsorption, increasing local drug concentration and blocking viral adsorption. Simultaneously, cysteine ​​disrupts the HIV envelope protein structure through thiol-disulfide bond exchange. These two mechanisms complement and synergistically result in a gel with significantly higher viral inactivation rate and mucosal retention rate compared to single-amino acid approaches. Taurine and lysine, due to charge neutralization, show no synergistic effect, thus indirectly confirming the scientific validity of the arginine-cysteine ​​combination.

[0026] Based on the compounding scheme, research revealed that when arginine and cysteine ​​are reacted simultaneously with aldehyde-modified cyclodextrin, they compete for a limited number of aldehyde grafting sites. Furthermore, arginine exhibits significant steric hindrance, leading to uneven grafting and low utilization of active groups. To address this, this invention designs a stepwise grafting process: first, arginine is preferentially grafted onto the dominant sites at pH 5.0, and then cysteine ​​is grafted onto the remaining sites at natural pH. This process not only avoids competition and improves the grafting rate of both amino acids but also reduces the oxidative loss of cysteine ​​sulfhydryl groups during the reaction. The resulting modified cyclodextrin exhibits enhanced mucosal adhesion and anti-HIV activity in benzalkonium chloride contraceptive gel.

[0027] Compared with the prior art, the present invention has the following beneficial technical effects:

[0028] 1) This invention significantly improves the instantaneous inactivation rate and mucosal retention performance of benzalkonium chloride gel against HIV pathogens by modifying cyclodextrin with amino acid grafting, achieving a dual protective effect of rapid and efficient virus clearance and long-lasting physical barrier.

[0029] 2) This invention uses a combination of arginine and cysteine ​​grafting to endow the gel with a dual function of strong positive charge mucosal anchoring ability and thiol reduction to destroy the viral envelope. Under the synergistic effect, it greatly enhances antiviral activity and local adhesion persistence.

[0030] 3) This invention optimizes the stepwise grafting preparation process, effectively avoiding disordered competition of functional amino acids for active sites, improving the grafting rate and the retention rate of active groups, so that the gel has both excellent virus inactivation efficiency and vaginal mucosal affinity and safety. Detailed Implementation

[0031] Some material sources or parameters:

[0032] Hydroxypropyl methylcellulose, purity: methoxy content 22.0-24.0%, hydroxypropoxy content 7.5-9.5%, food grade, conforming to the quality standards for hydroxypropyl methylcellulose in USP, EP, and JP and the purity standards for the food additive hydroxypropyl methylcellulose (E464).

[0033] Benzalkonium chloride, CAS number 139-07-1, purity: 95-105%, pharmaceutical grade, conforming to the provisions of Part II of the Pharmacopoeia of the People's Republic of China.

[0034] Polyquaternium-10 (CAS:68610-92-4): White to off-white powder, purity ≥99%, nitrogen content 1.5~2.2%, viscosity (2% aqueous solution, 25℃) 300~500mPa·s, commercially available product.

[0035] Disodium edetate (CAS: 6381-92-6): White crystalline powder with a purity ≥99.0%, conforming to the pharmaceutical excipient standards of the 2020 edition of the Chinese Pharmacopoeia.

[0036] Glycerin (CAS: 56-81-5): A colorless, transparent, viscous liquid with a purity of ≥99.5%, conforming to international pharmacopoeia standards such as USP / EP / CP, and is of pharmaceutical injection grade.

[0037] Citric acid (CAS: 77-92-9): White crystalline powder with a purity of ≥99.5%, conforming to the pharmaceutical excipient standards of the 2020 edition of the Chinese Pharmacopoeia.

[0038] γ-Cyclodextrin (CAS:17465-86-0): White crystalline powder, purity ≥98%, commercially available analytical grade.

[0039] Sodium periodate (CAS: 7790-28-5): Colorless tetragonal crystals or white crystalline powder, purity ≥99%, commercially available analytical grade.

[0040] Glycine (CAS: 56-40-6): White crystalline powder, purity ≥ 98.5%, commercially available pharmaceutical grade product.

[0041] Serine (CAS: 56-45-1): White crystalline powder, purity ≥99%, commercially available pharmaceutical grade product.

[0042] Cysteine ​​(CAS: 52-90-4): White crystalline powder, purity ≥99%, commercially available pharmaceutical grade product.

[0043] Lysine (CAS: 56-87-1): White crystalline powder, purity ≥98%, commercially available pharmaceutical grade product.

[0044] Arginine (CAS: 74-79-3): White columnar crystals, purity ≥ 98%, commercially available pharmaceutical grade product.

[0045] Taurine (CAS: 107-35-7): White crystalline powder, purity ≥99%, commercially available pharmaceutical grade product.

[0046] In the embodiments and comparative examples of this invention, all raw materials are commercially available products.

[0047] Example 1

[0048] A method for preparing a benzalkonium chloride contraceptive gel that kills HIV pathogens is as follows, in parts by weight:

[0049] Weigh 55 parts of deionized water and inject it into a 200L vacuum emulsifying tank. Heat to 85℃ and stir at 25 rpm. Evenly sprinkle 2.5 parts of hydroxypropyl methylcellulose and 0.3 parts of polyquaternium-10 into the vortex on the liquid surface within 10 minutes. After sprinkling, evacuate to -0.06 MPa and maintain for 5 minutes to degas. Turn off the heating and allow the mixture to cool naturally while stirring. In a mixing tank, take 28 parts of deionized water and add 0.8 parts of modified cyclodextrin, 0.05 parts of disodium edetate, and 0.12 parts of benzene in sequence. Add benzalkonium chloride and stir until clear and transparent to obtain a drug solution. When the temperature in the emulsification tank drops to 34°C, reduce the stirring speed to 20 rpm and suck the drug solution into the emulsification tank by vacuum suction. Then add 5.0 parts of glycerin and stir for 15 minutes. Turn on the external circulating cooling water to lower the material temperature to 22°C. Turn on the homogenizer to circulate and homogenize for 3 minutes at 3000 rpm. Finally, add water to a total weight of 100 parts and stir for 10 minutes. Degas under a vacuum of -0.06 MPa for 5 minutes and discharge to obtain benzalkonium chloride contraceptive gel.

[0050] The modified cyclodextrin is prepared as follows, in parts by weight:

[0051] S1. Take 1 part of γ-cyclodextrin and add it to 100 parts of deionized water. Heat to 80°C and stir until completely dissolved. Then cool naturally to room temperature. Add 0.4 parts of sodium periodate to the solution and stir at 25°C for 80 minutes in the dark. Then add 2 parts of glycerol and continue stirring for 15 minutes. Put the resulting reaction solution into a dialysis bag with a molecular weight cutoff of 500 Da and dialyze with deionized water for 48 hours. Change the dialysate every 6 hours during the process to obtain the aldehyde-modified cyclodextrin solution.

[0052] S2. Mix the aldehyde-modified cyclodextrin solution obtained in step S1 with 0.2 parts of amino acids, adjust the pH of the system to 5.5 with citric acid, and stir at 25°C for 16 hours. After the reaction is completed, cool the reaction solution to 3°C, add 0.15 parts of sodium borohydride, and stir at 3°C ​​for 2 hours. Put the reaction solution into a dialysis bag with a molecular weight cutoff of 500 Da, dialyze with deionized water for 48 hours, and change the dialysate every 6 hours during the process. Dry the solution to obtain modified cyclodextrin.

[0053] The amino acid in question is glycine.

[0054] Example 2

[0055] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 1, except that the amino acid used in the preparation method of the modified cyclodextrin is serine.

[0056] Example 3

[0057] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 1, except that the amino acid used in the preparation method of the modified cyclodextrin is cysteine.

[0058] Example 4

[0059] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 1, except that the amino acid used in the preparation method of the modified cyclodextrin is lysine.

[0060] Example 5

[0061] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 1, except that the amino acid used in the preparation method of the modified cyclodextrin is arginine.

[0062] Example 6

[0063] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 1, except that the amino acid used in the preparation method of the modified cyclodextrin is taurine.

[0064] Example 7

[0065] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 1, except that the amino acids used in the preparation method of the modified cyclodextrin are arginine and cysteine ​​in a mass ratio of 1:1.

[0066] Example 8

[0067] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 1, except that the amino acids used in the preparation method of the modified cyclodextrin are taurine and lysine in a mass ratio of 1:1.

[0068] Example 9

[0069] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 7, except that the preparation method of the modified cyclodextrin is different.

[0070] The modified cyclodextrin is prepared as follows, in parts by weight:

[0071] S1. Take 1 part of γ-cyclodextrin and add it to 100 parts of deionized water. Heat to 80°C and stir until completely dissolved. Then cool naturally to room temperature. Add 0.4 parts of sodium periodate to the solution and stir at 25°C for 80 minutes in the dark. Then add 2 parts of glycerol and continue stirring for 15 minutes. Put the resulting reaction solution into a dialysis bag with a molecular weight cutoff of 500 Da and dialyze with deionized water for 48 hours. Change the dialysate every 6 hours during the process to obtain the aldehyde-modified cyclodextrin solution.

[0072] S2. Mix the aldehyde-modified cyclodextrin solution obtained in step S1 with 0.1 parts of arginine, adjust the pH of the system to 5.0 with citric acid, and stir at 25°C for 8 hours; then add 0.1 parts of cysteine ​​and continue stirring at 25°C for 8 hours; after the reaction is completed, cool the reaction solution to 3°C, add 0.15 parts of sodium borohydride, and stir at 3°C ​​for 2 hours; put the reaction solution into a dialysis bag with a molecular weight cutoff of 500 Da, dialyze with deionized water for 48 hours, changing the dialysate every 6 hours during the process, and dry to obtain modified cyclodextrin.

[0073] Comparative Example 1

[0074] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 1, except that the amino acid is not added in the preparation method of the modified cyclodextrin.

[0075] Comparative Example 2

[0076] The preparation method of a benzalkonium chloride contraceptive gel that kills HIV pathogens is basically the same as that in Example 1, except that the modified cyclodextrin is replaced with an equal amount of γ-cyclodextrin.

[0077] Test Example 1

[0078] Anti-HIV-1 viral inactivation activity test:

[0079] The inactivation effect of benzalkonium chloride contraceptive gels obtained in each example and comparative example on HIV-1 virus was detected by suspension quantitative inactivation test. The specific procedure was as follows: 1.0 g of each of the benzalkonium chloride contraceptive gels prepared in Examples 1-9 and Comparative Examples 1-2 was taken, and an equal volume of HIV-1 IIIB strain virus suspension (titer 2×10⁻⁶) was added. 7 TCID 50 / mL, containing 10% fetal bovine serum as an organic interferon), vortexed and incubated in a 25°C water bath for 1 minute, 2 minutes, and 5 minutes respectively; immediately after incubation, PBS containing 0.5% lecithin and 2% Tween 80 was added to neutralize and dilute the reaction to terminate the reaction. The infectivity titer (TCID) of residual virus in each group was determined using the MT-4 cell culture method. 50 / mL), and set up a virus positive control group with an equal amount of PBS instead of gel samples and a cell negative control group without virus; calculate the virus inactivation log value at each time point according to the following formula: inactivation log value = lg (virus positive control group titer) - lg (experimental group residual titer), each group of experiments was repeated 3 times and the average value was taken. The virus inactivation log value ≥ 4.00 was judged as complete inactivation.

[0080] The test results are shown in Table 1.

[0081] Table 1

[0082] Experimental protocol 1-minute viral inactivation log 2-minute virus inactivation log 5-minute virus inactivation log Example 1 2.52 3.28 4.15 Example 2 2.68 3.45 4.32 Example 3 4.25 5.12 5.85 Example 4 3.15 3.88 4.62 Example 5 3.42 4.15 4.88 Example 6 2.45 3.12 3.95 Example 7 5.18 5.95 6.62 Example 8 2.88 3.55 4.28 Example 9 5.45 6.22 6.88 Comparative Example 1 2.12 2.85 3.62 Comparative Example 2 1.65 2.28 3.05

[0083] Test Example 2

[0084] Gel in vitro vaginal mucosal adhesion test:

[0085] Isolated porcine vaginal mucosa tissue (2.0±0.2 mm thick, taken from 3-5 cm from the vaginal opening) was cut into 1 cm × 1 cm squares. After gently rinsing with physiological saline to remove surface secretions, the tissue was fixed to the bottom of a culture dish. 0.5 g of benzalkonium chloride contraceptive gel obtained in Examples 1-9 and Comparative Examples 1-2 was evenly spread on the mucosal surface. The culture dish was placed in a constant temperature and humidity chamber at 37°C and 90% relative humidity for 4 hours. After removal, the mucosal surface was rinsed with PBS buffer (pH 4.5) preheated to 37°C at a flow rate of 5 mL / min for 30 seconds. The residual gel on the mucosal surface after rinsing was collected, and the benzalkonium chloride content was determined by high performance liquid chromatography. The mucosal retention rate was calculated using the following formula: Mucosal retention rate (%) = (residual drug amount after rinsing / initial drug amount) × 100%. Six samples were measured in parallel for each group, and the average value was taken.

[0086] The relevant test data are summarized in Table 2.

[0087] Table 2

[0088] Experimental protocol Mucosal retention rate (%) Example 1 38.5 Example 2 42.3 Example 3 41.8 Example 4 52.6 Example 5 61.2 Example 6 36.4 Example 7 68.5 Example 8 45.2 Example 9 72.8 Comparative Example 1 32.4 Comparative Example 2 24.6

[0089] Test Example 3

[0090] Vaginal mucosal irritation safety test:

[0091] Thirty-six healthy female rabbits, weighing 2.0-2.5 kg, were randomly divided into 12 groups of 3 rabbits each, corresponding to the benzalkonium chloride contraceptive gels obtained in Examples 1-9 and Comparative Examples 1-2, respectively. A blank control group (physiological saline) was also included. Following the "Repeated Vaginal Mucosal Irritation Test" method in the "Disinfection Technical Specifications" (2002 edition), 0.5 mL of the test gel was injected into the vagina of each group of rabbits daily for 5 consecutive days. The animals were sacrificed 24 hours after the last administration, and vaginal tissue was dissected for macroscopic observation and histopathological examination. The irritation response was scored based on the degree of mucosal epithelial cell degeneration, necrosis, inflammatory cell infiltration, and vascular congestion, and the irritation index was calculated. The vaginal mucosal irritation index of the benzalkonium chloride contraceptive gels obtained in Examples 1-9 and Comparative Examples 1-2 was less than 1.0, falling into the non-irritating category; the irritation index of the blank control group was 0. The results indicate that the benzalkonium chloride contraceptive gels prepared in each example and comparative example of this invention meet the safety standards for vaginal mucosal administration.

[0092] After introducing amino acids for grafting modification during the preparation of modified cyclodextrin, the virus inactivation activity and mucosal retention rate of Examples 1-6 were significantly improved compared with the aldehyde-modified cyclodextrin without added amino acids in Comparative Example 1. This may be because the present invention first oxidizes γ-cyclodextrin with sodium periodate to open the vicinal diol structure of its glucose unit to generate an aldehyde group. Subsequently, the aldehyde group reacts with the amino group of the amino acid in a Schiff base reaction to form an imine bond. Then, the unstable imine bond is converted into a stable secondary amine covalent bond by sodium borohydride reduction, so that the amino acid is firmly grafted onto the cyclodextrin backbone. The introduction of amino acids not only improves the water solubility and dispersibility of cyclodextrin, but more importantly, it provides additional functional groups to the cyclodextrin molecule. The amino, carboxyl, hydroxyl, or thiol groups on the amino acid side chains can specifically interact with HIV envelope proteins or vaginal mucosal epithelial cells, thereby enhancing the delivery efficiency and targeted retention ability of benzalkonium chloride. In Example 3, cysteine ​​grafting was used, with its side chain containing an active thiol group (-SH). This thiol group has strong reducing properties and can directly react with the disulfide bonds in the HIV envelope glycoprotein gp120 through a thiol-disulfide exchange reaction, disrupting the spatial conformation of the viral surface protein. By blocking the binding of the virus to the CD4 receptor of the host cell, and with the thiol group also enhancing the penetration efficiency of benzalkonium chloride through the viral lipid envelope, Example 3 showed a higher viral inactivation log in Test Example 1. In the mucosal adhesion test of Test Example 2, the gel modified with arginine in Example 5 showed a better mucosal retention rate. This may be because the arginine side chain contains a strongly basic guanidine group, which is fully protonated and carries a strong positive charge under the physiological pH environment of the vagina. It can generate a strong electrostatic adsorption with the negatively charged sialic acid residues and glycosaminoglycans on the surface of vaginal mucosal epithelial cells, which significantly prolongs the retention time of the gel on the mucosal surface.

[0093] Example 7 uses a 1:1 mass ratio of arginine and cysteine ​​for modification. Its virus inactivation effect and mucosal retention rate are significantly better than those of Example 3 (cysteine ​​alone) and Example 5 (arginine alone). This may be because the two amino acids exert a synergistic effect. The guanidino group of arginine, through electrostatic adsorption, enriches the gel on the mucosal surface where the virus is located, increasing the local drug concentration. Simultaneously, its positive charge can bind to the negatively charged region of the HIV envelope gp120, blocking virus adsorption. Meanwhile, the thiol group of cysteine ​​directly reduces the disulfide bonds in the viral envelope. Both taurine and lysine work together through two different mechanisms—physical blocking and chemical destruction—to disrupt the viral structure, resulting in a synergistic inactivation effect. In contrast, Example 8, which used a combination of taurine and lysine, did not show a synergistic effect. This is because taurine contains sulfonic acid groups and carries a negative charge. These groups form an internal salt or neutralize the charge with the positively charged ε-amino group of lysine in solution. This not only weakens the positive charge advantage of lysine but may also change the charge distribution on the gel surface, leading to a decrease in mucosal adhesion. Therefore, the test data of Example 8 is only close to the level of lysine alone and fails to demonstrate the advantages of the combination.

[0094] Compared to the simultaneous feeding in Example 7, the stepwise grafting process in Example 9 further improved the logarithmic virus inactivation rate and mucosal retention rate. This is because the stepwise reaction avoids competition between the two amino acids for the limited aldehyde sites on the aldehyde-modified cyclodextrin. Under slightly acidic conditions at pH 5.0, the α-amino group of arginine has strong nucleophilicity and preferentially forms a Schiff base with the aldehyde group, occupying the advantageous grafting sites with less steric hindrance. After treatment, the pH will increase, and then cysteine ​​will be added. At this point, arginine has been stably grafted, and cysteine ​​can graft at the remaining aldehyde sites. Moreover, this pH is closer to the isoelectric point of cysteine, which is beneficial to protecting the reducing activity of its side chain thiol groups and reducing oxidation loss. Therefore, the stepwise process improves the grafting rate of the two amino acids and the retention rate of active groups, so that the final gel has both stronger mucosal adhesion and virus inactivation activity.

Claims

1. A benzalkonium chloride contraceptive gel that kills HIV pathogens, characterized in that, Includes the following raw materials in parts by weight: 60-100 parts water, 1-5 parts hydroxypropyl methylcellulose, 0.1-0.5 parts polyquaternium-10, 0.5-2 parts modified cyclodextrin, 0.03-0.08 parts disodium edetate, 0.05-0.3 parts benzalkonium chloride, 3-8 parts glycerol; The modified cyclodextrin is prepared by oxidizing γ-cyclodextrin, reacting it with amino acids, and then dialysis and drying.

2. The benzalkonium chloride contraceptive gel for killing HIV pathogens as described in claim 1, characterized in that, The amino acid is at least one of glycine, serine, cysteine, lysine, arginine, and taurine.

3. The benzalkonium chloride contraceptive gel for killing HIV pathogens as described in claim 1, characterized in that, The amino acid is composed of arginine and cysteine ​​in a mass ratio of 0.5-2:0.5-2.

4. A method for preparing a benzalkonium chloride contraceptive gel for killing HIV pathogens as described in any one of claims 1-3, characterized in that, The method is as follows: Weigh water and inject it into a vacuum emulsification tank. Heat and stir. Sprinkle hydroxypropyl methylcellulose and polyquaternium-10 evenly into the vortex on the liquid surface within 5-15 minutes. After the addition is complete, degas under vacuum, turn off the heating, and stir to cool naturally. In a preparation tank, take water and add modified cyclodextrin, disodium edetate, and benzalkonium chloride in sequence. Stir until clear and transparent to obtain the drug solution. When the temperature in the emulsification tank drops to 30-35℃, reduce the stirring speed to 10-25 rpm. Suction the drug solution into the emulsification tank through vacuum suction. Then add glycerin and stir for 10-30 minutes. Turn on the external circulating cooling water to lower the material temperature to 18-25℃, circulate and homogenize. Finally, add water to adjust the total weight, stir for 5-20 minutes, degas under vacuum, and discharge to obtain benzalkonium chloride contraceptive gel.

5. The method as described in claim 4, characterized in that, The heating and stirring process involves heating to 80-90℃ and stirring at 20-40 rpm.

6. The method as described in claim 4, characterized in that, Each vacuum degassing step independently involves evacuating to -0.04 to -0.08 MPa and maintaining the vacuum for 3 to 10 minutes to eliminate air bubbles.

7. The method as described in claim 4, characterized in that, The cyclic homogenization is performed by homogenizing at 2000-4000 rpm for 1-5 minutes.

8. The method as described in claim 4, characterized in that, The modified cyclodextrin is prepared as follows: S1. Take γ-cyclodextrin, add it to water, heat to 70-90℃ and stir until completely dissolved, then cool naturally to room temperature, add sodium periodate to the solution, stir and react at 20-30℃ for 60-90 minutes under light-protected conditions, then add glycerol and continue stirring for 10-20 minutes. Dialyze the resulting reaction solution to obtain aldehyde-modified cyclodextrin solution. S2. Mix the aldehyde-modified cyclodextrin solution obtained in step S1 with amino acids, adjust the pH of the system to 5-6 with citric acid, and stir the reaction at 20-30℃ for 10-30 hours. After the reaction is completed, cool the reaction solution to 0-5℃, add 0.1-0.3 parts of sodium borohydride, and stir at 0-5℃ for 1-3 hours. Dialyze the reaction solution and dry it to obtain modified cyclodextrin.

9. The method as described in claim 4, characterized in that, The modified cyclodextrin can also be prepared by the following method: S1. Take γ-cyclodextrin, add it to water, heat to 70-90℃ and stir until completely dissolved, then cool naturally to room temperature, add sodium periodate to the solution, stir and react at 20-30℃ for 60-90 minutes under light-protected conditions, then add glycerol and continue stirring for 10-20 minutes. Dialyze the resulting reaction solution to obtain aldehyde-modified cyclodextrin solution. S2. Mix the aldehyde-modified cyclodextrin solution obtained in step S1 with arginine, adjust the pH of the system to 4.5-5.5 with citric acid, and stir the reaction at 20-30℃ for 5-10 hours; then add cysteine ​​and continue stirring the reaction at 20-30℃ for 5-10 hours; after the reaction is completed, cool the reaction solution to 0-5℃, add 0.1-0.3 parts of sodium borohydride, and stir at 0-5℃ for 1-3 hours; dialyze the reaction solution and dry it to obtain modified cyclodextrin.

10. The method as described in claim 8 or 9, characterized in that, The dialysis in steps S1 and S2 is independent of each other. It involves using a dialysis bag with a molecular weight cutoff of 400-800 Da and dialysis with water for 40-72 hours, during which the external dialysis fluid is changed every 4-8 hours.