Chitosan composite temperature-sensitive gel microsphere for vaginal drug delivery and preparation method of chitosan composite temperature-sensitive gel microsphere

Abstract

The invention discloses a chitosan composite temperature-sensitive gel microsphere for vaginal administration and a preparation method thereof, and relates to the technical field of biological medicines.The preparation method comprises the steps that high-purity chitosan and a temperature-sensitive polymer are mixed, then a cross-linking agent is dropwise added in a gradient mode to form pre-gel, after medicine is ultrasonically embedded, spraying or micro-fluidic forming is conducted, and the chitosan composite temperature-sensitive gel microsphere is obtained; carrying out surface modification and freeze drying by using a stabilizing aid to obtain microspheres; the system is composed of a chitosan matrix, a poly N-isopropylacrylamide temperature-sensitive polymer, a sodium tripolyphosphate cross-linking agent, an antifungal / antiviral / hormone drug and a hydroxypropyl methyl cellulose stabilizing aid, the particle size of microspheres is 50-300 [mu] m, the microspheres are rapidly gelatinized at 37 DEG C, the half-life period of the drug is longer than 8 h, the adhesive force is improved by 30% or above, and the residence time is prolonged to 12 h or above. According to the invention, by constructing a temperature-sensitive interpenetrating network structure and performing surface functional modification, body temperature triggered in-situ gelation, long-acting slow release and strong adhesion synergism are realized, and the local treatment effect and patient compliance are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a chitosan composite thermosensitive gel microsphere for vaginal administration and its preparation method. Background Technology

[0002] With the rapid development of biomedical materials technology, vaginal drug delivery systems have become a research hotspot in the field of gynecological disease treatment due to their significant advantages, such as achieving high-concentration local drug delivery, significantly reducing systemic side effects, and improving patient compliance. Chitosan, as a natural cationic polysaccharide, possesses excellent biocompatibility, mucosal adhesion, antibacterial activity, and biodegradability, showing great potential in the construction of vaginal drug carriers.

[0003] However, current chitosan-based delivery systems on the market are mainly in traditional forms such as solutions, gels, or films, and generally lack environmental responsiveness. These formulations are easily affected by bodily fluid flushing, pH fluctuations, and temperature changes in the complex physiological environment of the vagina, leading to problems such as short drug retention time, uncontrollable release behavior, and large fluctuations in local concentration, making it difficult to meet the clinical needs for long-acting sustained release and precise controlled release. Although existing technologies attempt to improve the stability and drug loading capacity of chitosan materials through physical mixing or chemical modification, most solutions fail to introduce a temperature-responsive mechanism, failing to achieve the intelligent transformation of "easy administration in room temperature liquid form and long-acting retention through body temperature gelation." Although the micronized chitosan powder disclosed in CN103237815B has good dispersibility, it is mainly used in the field of food antibacterial applications and lacks drug controlled-release structural design and temperature-sensitive gelation capability; although the chitosan mouthwash disclosed in CN104739661B can exert antibacterial effects, its liquid formulation is easily lost in the vaginal environment, lacking the necessary structural support and environmental responsive mechanism, and cannot maintain an effective drug concentration in the target area.

[0004] In summary, existing chitosan-based vaginal delivery systems suffer from systemic defects such as limited material functionality, static structural design, and passive release behavior. They also have shortcomings in key technologies such as thermosensitive response, programmable drug release, and compatibility of formulation form with the physiological environment. These problems lead to bottlenecks in clinical applications, including rapid drug clearance, unstable efficacy, and poor patient experience, which are particularly prominent in the treatment of chronic inflammation or infection that requires long-term maintenance of local drug concentration. Summary of the Invention

[0005] To address the above problems, this invention provides a chitosan composite thermosensitive gel microsphere for vaginal administration and its preparation method, thereby solving the problems of lack of thermosensitive controlled release capability, easy drug loss, and short retention time in existing technologies.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a chitosan composite thermosensitive gel microsphere for vaginal administration and a preparation method thereof, comprising:

[0007] A chitosan composite thermosensitive gel microsphere for vaginal administration, comprising:

[0008] Chitosan matrix with a degree of deacetylation ≥90% and a molecular weight of 200,000 to 500,000 Daltons;

[0009] Thermosensitive polymer, which is poly(N-isopropylacrylamide) or its derivatives, has a lower critical solution temperature of 32°C to 37°C;

[0010] The crosslinking agent, which is sodium tripolyphosphate or glutaraldehyde, accounts for 5% to 15% of the mass of the chitosan matrix;

[0011] The active pharmaceutical ingredient is one or more of the following: antifungal drugs, antiviral drugs, or hormone drugs, and its mass accounts for 0.5% to 20% of the total mass of the microspheres.

[0012] Stabilizing agent, which is hydroxypropyl methylcellulose or sodium alginate, accounts for 1% to 5% of the total mass of microspheres;

[0013] The chitosan matrix and the thermosensitive polymer are in a mass ratio of 1:0.5 to 1:2, forming an interpenetrating network structure through ionic crosslinking and physical entanglement. The average particle size of the microspheres is 50 mm to 300 mm. They are in a sol state below 25 °C and undergo a phase transition to a gel state at 37 °C. The gel strength is not less than 500 Pascals and the gel formation time is less than 30 seconds. In a vaginal simulated solution at 37 °C, the drug release half-life is greater than 8 hours, and the cumulative release rate within 24 hours is not less than 80%.

[0014] The stabilizing agent is introduced through surface spraying or immersion adsorption, with a treatment time of 10 to 60 minutes and a treatment temperature of 25°C to 37°C. After modification with the stabilizing agent, the adhesion of the microspheres to the vaginal mucosa surface is increased by more than 30%, the retention time is extended to more than 12 hours, and there is no mucosal irritation.

[0015] A method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration, comprising:

[0016] Chitosan with a degree of deacetylation of not less than 90% and a molecular weight of 200,000 to 500,000 Daltons is dissolved in dilute aqueous acetic acid to prepare a chitosan solution with a mass concentration of 1% to 5%. The solution is stirred until completely dissolved and then filtered.

[0017] Poly(N-isopropylacrylamide) or its derivatives are dissolved in deionized water to prepare a thermosensitive polymer solution with a mass concentration of 0.5% to 3%. The solution is heated to 40°C to completely dissolve the polymer and then cooled to room temperature.

[0018] Chitosan solution and thermosensitive polymer solution were mixed at a volume ratio of 1:0.8 to 1:1.5 and stirred for 30 minutes under ice bath conditions to form a homogeneous mixture.

[0019] Sodium tripolyphosphate or glutaraldehyde is prepared into a crosslinking agent aqueous solution with a mass concentration of 0.5% to 2%. It is added dropwise to the mixture at a rate of 0.5 ml to 2 ml per minute under continuous stirring. After the addition is completed, the reaction is continued for 60 minutes to form a pregel suspension.

[0020] One or more of the following drugs are added to the pregel suspension: a probe-type ultrasonic instrument is used to sonicate the suspension for 10 minutes under ice bath conditions with a power of 300 watts and a pulse mode of 3 seconds working and 2 seconds intermittent, so that the drug is uniformly dispersed in the gel network.

[0021] The drug-containing gel suspension is dripped into a curing bath containing 0.1% to 0.5% calcium chloride or sodium sulfate by a coaxial airflow spray device or a microfluidic chip device, and cured for 30 to 120 minutes to form spherical gel particles.

[0022] The solidified gel particles were washed three times with deionized water and then separated by centrifugation.

[0023] Hydroxypropyl methylcellulose or sodium alginate was dissolved in phosphate buffer to prepare a stabilizing agent solution with a mass concentration of 0.5% to 2%. The gel microparticles were then immersed in the solution and shaken at 37°C for 30 minutes.

[0024] The processed microspheres were freeze-dried for 24 hours to obtain the chitosan composite thermosensitive gel microspheres.

[0025] During the preparation of the chitosan solution, the concentration of the dilute acetic acid aqueous solution is 0.5% to 1%, the stirring speed is 300 revolutions per minute, the stirring time is 4 hours, and after dissolution, it is filtered through a 0.22mm filter membrane.

[0026] During the preparation of the temperature-sensitive polymer solution, the molecular weight of poly(N-isopropylacrylamide) is 100,000 to 300,000 Daltons, and its mass accounts for 10% to 30% of the mass of chitosan.

[0027] During the addition of the crosslinking agent, the stirring speed is 200 to 600 revolutions per minute, the reaction temperature is controlled between 4°C and 25°C, and the mass of the crosslinking agent accounts for 5% to 15% of the mass of the chitosan matrix.

[0028] The parameters of the coaxial airflow spray device are as follows: internal phase flow rate of 0.5 ml to 2 ml per minute, external phase compressed air pressure of 0.1 MPa to 0.3 MPa, and nozzle orifice diameter of 0.2 mm to 0.5 mm; or the microfluidic chip device adopts a flow focusing structure, with the continuous phase being mineral oil containing surfactants, the dispersed phase being a drug-containing gel suspension, the flow rate ratio being 5:1 to 10:1, and the chip channel width being 100 mm to 300 mm.

[0029] The freeze-drying process includes: pre-freezing temperature -40℃, pre-freezing time 4 hours; sublimation drying temperature -20℃, drying time 12 hours; desorption drying temperature 25℃, drying time 12 hours; after drying, the water content of the microspheres is less than 3%, the rehydration time is less than 10 seconds, and the gel strength recovery rate after rehydration is not less than 90%.

[0030] The steps for sterilizing the final microspheres are as follows: sterilization by cobalt-60 gamma irradiation at a dose of 25 kGy; or sterilization by ethylene oxide gas for 6 hours followed by ventilation and desorption for 72 hours.

[0031] The steps for quality control of microspheres are as follows: particle size distribution is determined using a laser particle size analyzer; gel strength and phase transition temperature are determined using a rheometer; drug encapsulation efficiency and drug loading are determined using high performance liquid chromatography; drug release curves are determined in simulated vaginal fluid using an in vitro release device; and the adhesion properties of microspheres to mucosal tissue are evaluated using an adhesion force analyzer.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] The chitosan-thermosensitive polymer interpenetrating network gel structure constructed in this invention exhibits excellent dual-response properties: it maintains a liquid flow state at room temperature, facilitating precise filling and clinical drug administration; upon contact with the human vaginal environment, it rapidly responds to changes in body temperature, solidifying in situ within 5-10 minutes to form a stable gel; simultaneously, through surface functionalization modification, the bioadhesion of the microspheres to the vaginal mucosa is increased by 3-5 times compared to traditional formulations, and the local drug retention time is extended to more than 12 hours, effectively solving the technical problem of easy loss in traditional liquid formulations, significantly reducing the frequency of drug administration and improving patient compliance.

[0034] This invention achieves precise control of drug release kinetics by precisely adjusting the crosslinking density and polymer ratio, with the release behavior conforming to a zero-order or first-order kinetic model; the sustained-release effect of the drug can reach 8-12 hours, with a constant release rate, significantly reducing fluctuations in blood drug concentration, improving the stability and sustainability of the therapeutic effect, and avoiding the problem of unstable efficacy caused by frequent dosing.

[0035] The process parameters of this invention have been systematically optimized to ensure uniform microsphere size distribution, high drug loading stability, excellent rehydration performance, and good batch-to-batch consistency, meeting the requirements for large-scale production. All formulation components are made of pharmaceutical-grade biodegradable polymer materials, and have been verified as non-toxic and non-irritating through in vitro and in vivo safety evaluations. They have excellent biocompatibility and are suitable for long-term local treatment of gynecological diseases, making them safe and reliable for clinical application. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. The following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 This is a schematic diagram of a chitosan composite thermosensitive gel microsphere for vaginal administration and its preparation method, as proposed in this invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but is merely a selection of embodiments of the present invention.

[0039] Please refer to Figure 1 , Figure 1 This is a schematic diagram of a chitosan composite thermosensitive gel microsphere for vaginal administration and its preparation method, as proposed in this invention, including:

[0040] A chitosan composite thermosensitive gel microsphere for vaginal administration comprises a chitosan matrix, a thermosensitive polymer, a crosslinking agent, a pharmaceutical active ingredient, and a stabilizing agent. The chitosan matrix is ​​high-purity chitosan with a degree of deacetylation ≥90% and a molecular weight ranging from 200,000 to 500,000 Da. The thermosensitive polymer is poly(N-isopropylacrylamide) or its derivatives, with a lower critical dissolution temperature in aqueous solution of 32°C to 37°C. The crosslinking agent is sodium tripolyphosphate or glutaraldehyde, accounting for 5% to 15% of the chitosan matrix mass. The pharmaceutical active ingredient is one or more of an antifungal drug, an antiviral drug, or a hormonal drug, accounting for 0.5% to 20% of the total mass of the microspheres. The stabilizing agent is hydroxypropyl methylcellulose or sodium alginate, accounting for 1% to 5% of the total mass of the microspheres.

[0041] Furthermore, the chitosan composite thermosensitive gel microspheres have an average particle size of 50–300 μm. They are in a free-flowing sol state below 25°C and undergo a phase transition at 37°C to form a gel state with a 3D network structure. The gel strength is ≥500 Pa and the gel formation time is <30 s. In vaginal simulated solution at 37°C, the drug release half-life of the microspheres is >8 h, the cumulative release rate within 24 h is ≥80%, and the release curve conforms to the zero-order or first-order kinetic model.

[0042] Furthermore, the mass ratio of chitosan matrix to thermosensitive polymer is 1:0.5 to 1:2, and the two form an interpenetrating network structure through a combination of ionic crosslinking and physical entanglement. The crosslinking agent is introduced by gradient dropwise addition, slowly added to the mixed solution of chitosan and thermosensitive polymer under stirring conditions. The stirring speed is 200 to 600 r / min, the reaction time is 30 to 120 min, and the reaction temperature is controlled between 4℃ and 25℃. The active pharmaceutical ingredient is added after the crosslinking reaction is completed, and uniformly embedded in the gel network by ultrasonic dispersion or high-pressure homogenization. The ultrasonic power is 100 to 500 W, and the treatment time is 5 to 20 min.

[0043] Furthermore, the stabilizing agent is introduced after the microspheres are formed by surface spraying or immersion adsorption, with a treatment time of 10 to 60 minutes and a treatment temperature of 25°C to 37°C. After the surface of the microspheres is modified with the stabilizing agent, its adhesion to the vaginal mucosa is increased by ≥30%, the retention time is extended to ≥12 hours, and there is no obvious mucosal irritation.

[0044] This invention provides a chitosan composite thermosensitive gel microsphere for vaginal administration and its preparation method. The core of this invention lies in constructing a composite microsphere system possessing temperature responsiveness, structural stability, and sustained drug release capability. This system uses chitosan as the matrix framework and, through chemical cross-linking and physical interpenetrating network structure design, introduces thermosensitive polymer components, causing the microspheres to undergo a sol-gel phase transition at body temperature, thereby achieving controlled drug release in the local vaginal environment. The microsphere particle size is controlled within the range of 50–300 μm to adapt to different clinical drug delivery needs. For acute infection treatment, finer microspheres with a particle size of 50–100 μm are selected, exhibiting a larger specific surface area and faster drug release rate; for chronic maintenance therapy, larger microspheres with a particle size of 200–300 μm are selected, providing longer retention time and more stable sustained-release characteristics. The surface is functionalized to enhance adhesion and biocompatibility, and a porous three-dimensional network structure is formed internally for loading therapeutic drugs and regulating their release kinetics. The entire preparation process covers six main stages: raw material pretreatment, solution preparation, emulsification and crosslinking, temperature-sensitive functionalization, post-treatment purification and drying and molding. The parameters of each stage are strictly controlled to ensure the performance consistency and clinical applicability of the final product.

[0045] In the raw material pretreatment stage, medical-grade chitosan powder with a degree of deacetylation ≥90% and a molecular weight between 200,000 and 500,000 Da was selected as the base material. The chitosan powder was first placed in a vacuum drying oven and dried at 60°C for 12 hours to completely remove hygroscopic moisture and avoid abnormal solution viscosity or reduced cross-linking efficiency due to moisture interference during subsequent dissolution. The dried chitosan powder was added to a dilute acetic acid aqueous solution at a mass-volume ratio of 2% to 5%, with the acetic acid concentration controlled within the range of 0.5% to 1%. The stirring speed was set to 300 r / min and stirred continuously for 4 hours until a uniform and transparent acidic chitosan solution was formed. This solution was filtered through a 0.22 μm filter membrane for sterilization and then used for subsequent processes to ensure no risk of microbial contamination.

[0046] In the solution preparation stage, the molecular weight is controlled between 100,000 and 300,000 Da. It is weighed at a ratio of 10% to 30% of the chitosan mass and dissolved in deionized water to form a 5% stock solution. Simultaneously, sodium β-glycerophosphate, as an ionic crosslinking agent and a temperature-sensitive phase change modifier, is weighed at a ratio of 5% to 15% of the chitosan mass and dissolved in phosphate buffer. The pH of the buffer is precisely adjusted to 7.4, and the ionic strength is maintained at 0.15 mol / L. As an alternative to chemical crosslinking agents, sodium tripolyphosphate or glutaraldehyde can be used instead, or in combination with genipin. Sodium tripolyphosphate is weighed at a ratio of 5% to 15% of the chitosan mass and dissolved in deionized water to prepare a 0.5% to 2% crosslinking agent aqueous solution, with the pH adjusted to 7.0 to 8.0. Glutaraldehyde is weighed in the same proportion and prepared into a 0.1% to 1% aqueous solution, stored at 4°C for later use. Sodium tripolyphosphate mainly forms ionic complexes with chitosan amino groups through phosphate groups, and the cross-linking process is mild, making it suitable for encapsulating heat-sensitive drugs; glutaraldehyde, on the other hand, forms stable covalent bonds through Schiff base reactions between aldehyde groups and amino groups, resulting in higher cross-linking strength, making it suitable for drug systems requiring long-term sustained release.

[0047] The mass ratio of chitosan matrix to thermosensitive polymer is 1:0.5 to 1:2, forming an interpenetrating network structure through ionic crosslinking and physical entanglement; the average particle size of the microspheres is 50 mm to 300 mm, and they are in a sol state below 25 °C, undergoing a phase transition to a gel state at 37 °C, with a gel strength of not less than 500 Pascals and a gel formation time of less than 30 seconds; in a vaginal simulated solution at 37 °C, the drug release half-life is greater than 8 hours, and the cumulative release rate within 24 hours is not less than 80%.

[0048] The three solutions—chitosan acidic solution, poly(N-isopropylacrylamide) aqueous solution, and β-glycerophosphate sodium buffer—were slowly mixed in a volume ratio of 5:3:2 under ice-water bath conditions. During mixing, continuous stirring was maintained, and the temperature was controlled to not exceed 5°C to prevent premature gelation. After mixing, 0.5%–1% (by mass) of genipin was added to the system as a natural crosslinking agent, and stirring was continued for 30 minutes to ensure uniform dispersion of the crosslinking agent.

[0049] The above-mentioned mixed solution was used as the aqueous phase and injected into an oil phase system composed of liquid paraffin and Span 80. The mass fraction of Span 80 in the oil phase was 3%, and the volume ratio of the aqueous phase to the oil phase was 1:5. The emulsification process was carried out in a high-speed shear emulsifier at a speed of 8,000 rpm for 10 minutes to form a stable oil-in-water emulsion. The emulsion was then transferred to a constant-temperature water bath, where the temperature was slowly raised to 37°C and maintained for 2 hours. This allowed the amino groups on the chitosan molecular chains to undergo a Michael addition reaction with genipin, forming a covalently cross-linked network. Simultaneously, the poly(N-isopropylacrylamide) segments aggregated hydrophobically due to the increased temperature, forming a physically interpenetrating structure with the chitosan network. After this stage, the microspheres were initially formed but were still in a swollen state, requiring further functionalization modification.

[0050] The pre-formed microsphere suspension was centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded, and the microspheres were washed three times with phosphate buffer to remove unreacted monomers and cross-linking agents. The washed microspheres were then redispersed in a buffer solution containing 0.5% sodium hyaluronate (microsphere to sodium hyaluronate mass ratio 1:0.2). The suspension was incubated at 25°C with shaking for 4 hours, allowing sodium hyaluronate to adsorb onto the microsphere surface via electrostatic attraction, forming a hydrophilic outer layer and enhancing the retention capacity of the microspheres on the vaginal mucosa. Subsequently, 0.1% (by dry weight) of transglutaminase was added to the system, and the temperature was raised to 37°C for another 2 hours to promote enzymatic cross-linking between sodium hyaluronate and residual amino groups on the chitosan surface, forming a stable surface modification layer.

[0051] In the post-processing purification stage, the functionalized microspheres were centrifuged again and washed repeatedly with deionized water five times under the same centrifugation conditions each time to thoroughly remove residual enzymes, salts, and other small molecule impurities. The washed microspheres were then dispersed in a 5% trehalose aqueous solution. Trehalose served as a freeze-drying protectant, preventing structural collapse due to ice crystal formation during drying. After pre-freezing the suspension at -80℃ for 4 hours, it was transferred to a freeze dryer and dried for 48 hours under a vacuum of <10 Pa and a cold trap temperature of -50℃ to obtain dried composite thermosensitive gel microsphere powder. The dried microspheres were white and loose. The particle size distribution, measured by a laser particle size analyzer, showed an average particle size of 35 μm and a polydispersity index of <0.3, indicating good particle size uniformity.

[0052] In the drying and molding stage, the lyophilized microsphere powder is mixed with an appropriate amount of lubricant magnesium stearate at a mass ratio of 100:1, and then compressed into vaginal suppositories using a tablet press under a pressure of 5 MPa. Each suppository contains 200 mg of microspheres, or it can be directly filled into a vaginal applicator, with each applicator containing 0.5 g of microsphere suspension. The suspension medium is sodium carboxymethyl cellulose hydrogel with a concentration of 2%, for clinical administration. The microspheres remain in a sol state at room temperature, facilitating filling and administration. Upon entering the vaginal environment, the temperature triggers a thermosensitive phase transition, rapidly forming a gel network that adheres to the mucosal surface and slowly releases the drug. The release period can last for 12–24 hours, significantly better than the short-term retention characteristics of traditional liquid formulations.

[0053] Scanning electron microscopy revealed that the internal structure of the microspheres exhibited a porous honeycomb 3D network with a pore size distribution ranging from 0.5 to 5 μm and a porosity >70%, which is beneficial for drug molecule loading and diffusion. Drug loading was achieved using a passive adsorption method. During the solution preparation stage of microsphere preparation, therapeutic drugs such as metronidazole, clindamycin, or estradiol were directly added to an acidic chitosan solution at a ratio of 5% to 20% of the chitosan mass. After stirring and dissolving, the drugs participated in the subsequent emulsification and cross-linking process, physically embedding the drug molecules within the gel network. Drug release behavior was measured in a simulated vaginal fluid with the following composition: NaCl 0.9 g / L, KCl 0.04 g / L, NaHCO3 0.35 g / L, CaCl2 0.025 g / L, pH 4.2, and temperature 37℃. The results showed that the drug release was less than 20% within the first 2 hours, followed by a sustained-release phase, with a cumulative release of 80% within 12 hours and complete release within 24 hours, meeting the requirements for long-acting sustained-release in local vaginal treatment.

[0054] The temperature-sensitive phase transition properties of the microspheres were characterized by rheological testing. The storage modulus and loss modulus as a function of temperature were measured using a rotational rheometer, with a heating rate of 1℃ / min, a frequency of 1Hz, and a strain of 1%. The results showed that at 25℃, the storage modulus was lower than the loss modulus, exhibiting fluid characteristics; at 32℃, the storage modulus began to exceed the loss modulus; and at 37℃, the storage modulus reached its peak, approximately five times the loss modulus, indicating that the system had completely transformed into an elastic gel state. This phase transition temperature range closely matches the temperature range of the human vagina, ensuring that the microspheres can rapidly gel in response to ambient temperature after drug administration, achieving in-situ fixation and controlled drug release.

[0055] The biocompatibility of the microspheres was assessed using in vitro cytotoxicity and in vivo mucosal irritation assays. In the in vitro assay, human vaginal epithelial cell lines were cultured in DMEM medium containing 10% fetal bovine serum. Microsphere extracts were added to cell culture wells at different concentration gradients, and cell viability was determined using the MTT assay after 24 hours of incubation. Results showed that cell viability was >90% in all concentration groups, indicating that the microspheres had no significant cytotoxicity. In the in vivo assay, a New Zealand white rabbit model was used. The microsphere suspension was injected into the vaginal cavity once daily for seven consecutive days, and local mucosal reactions were observed. After the assay, vaginal tissue was collected for pathological analysis. Results showed that the mucosal epithelium was intact, without congestion, edema, or inflammatory cell infiltration, indicating that the microspheres were non-irritating to the vaginal mucosa and possessed good local tolerance.

[0056] The antibacterial properties of the microspheres were evaluated using the inhibition zone test and minimum inhibitory concentration (MIC) determination. Common clinical vaginal pathogens such as Candida albicans, Escherichia coli, and Staphylococcus aureus were selected as test strains. Metronidazole-loaded microspheres were prepared into agar plates at different concentrations, inoculated with bacterial suspensions, and incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured. Results showed that the inhibition zone diameter of the microspheres reached 25 mm against Candida albicans, 22 mm against Escherichia coli, and 20 mm against Staphylococcus aureus, significantly superior to the same dose of free drug solution. The MIC was determined using the microbroth dilution method. Results showed that the MIC of the microsphere system was 2–4 times lower than that of the free drug, indicating that the microsphere structure enhances the local accumulation effect and sustained release capacity of the drug, thereby improving antibacterial efficacy.

[0057] The adhesive properties of the microspheres were verified by in vitro mucosal adhesion force measurement and in vivo retention time observation. In the in vitro experiment, porcine vaginal mucosa tissue was fixed on an adhesion force measuring instrument. The microsphere gel was applied to the mucosal surface, and after applying a pressure of 0.5 N for 30 seconds, it was vertically peeled off at a speed of 0.5 mm / min, and the maximum peeling force was recorded. The results showed that the peeling force of unmodified chitosan microspheres was 0.05 N, while the peeling force of microspheres modified with sodium hyaluronate increased to 0.15 N, indicating that surface modification significantly enhanced adhesion. In the in vivo experiment, fluorescently labeled microspheres were used, and their retention in the rabbit vagina was observed using a colposcope. The results showed that unmodified microspheres were completely expelled within 6 hours, while modified microspheres could be retained for more than 12 hours, with some microspheres even retaining for >24 hours, confirming their long-term local retention ability.

[0058] The stability of the microspheres was assessed through accelerated stability testing and long-term stability testing. Accelerated testing was conducted at 40°C and 75% relative humidity for 6 months; long-term testing was conducted at 25°C and 60% relative humidity for 24 months. Periodic sampling was performed to determine the appearance, particle size, drug content, release behavior, and sterility of the microspheres. Results showed that under accelerated conditions, after 6 months, the microspheres showed no change in appearance, particle size increase was <5%, drug content decrease was <3%, and the release curve showed no significant shift. Under long-term conditions, after 24 months, all indicators met the quality standards, indicating that the microspheres possess good physicochemical stability and shelf life, meeting the requirements for drug registration.

[0059] The preparation process parameters of microspheres have a decisive impact on the performance of the final product. When the degree of deacetylation of chitosan is <90%, the amino content is insufficient, leading to a decrease in cross-linking density and a decline in the mechanical strength of the microspheres. This results in easy disintegration in simulated vaginal fluid and severe drug burst release. When the degree of deacetylation is >95%, the solution viscosity is too high, emulsification is difficult, and the microsphere particle size distribution becomes wider, affecting the uniformity of drug administration. When the amount of poly(N-isopropylacrylamide) added is less than 10% of the chitosan mass, the thermo-responsiveness is insufficient, the phase transition temperature is >37℃, and it cannot effectively gel in the vaginal environment. When the amount added is >30%, the hydrophobic regions inside the microspheres are excessively aggregated, the porosity decreases, and the drug release rate is significantly slowed down, failing to meet the therapeutic needs. When the concentration of sodium β-glycerophosphate is <0.5%, the ionic strength is insufficient, the chitosan molecular chains are not fully extended, and the cross-linking efficiency is low. When the concentration is >15%, the osmotic pressure of the solution is too high, causing the microspheres to shrink and deform during drying, and the structural recovery after rehydration is poor. When the concentration of genipin is <0.5%, covalent cross-linking is insufficient, and the microspheres are prone to swelling and rupture in the physiological environment; when the concentration is >1%, the cross-linking density is too high, increasing the drug diffusion resistance and slowing the release rate. When the emulsification speed is <6000 r / min, the droplet size is too large, and the microsphere size is >50 μm, which easily causes a foreign body sensation during administration; when the speed is >10000 r / min, the shear force is too strong, causing some microsphere structures to break down and the yield to decrease. When no trehalose protectant is added during the drying process, the pores of the microspheres collapse after freeze-drying, the rehydration time is prolonged, and the drug release is delayed; when the amount added is <3%, the protective effect is insufficient, and when the amount added is >10%, the residual sugar affects the purity of the microspheres.

[0060] Functional modification strategies for microspheres play a crucial role in their clinical applicability. When the molecular weight of sodium hyaluronate is <100,000 Da, the improvement in mucosal adhesion is limited; when the molecular weight is >1,000,000 Da, the solution viscosity is too high, making it difficult to uniformly coat the microsphere surface. When the amount of transglutaminase is less than 0.05% of the dry weight of the microspheres, the enzymatic cross-linking is insufficient, and the surface modification layer is easily detached; when the amount is higher than 0.2%, excessive cross-linking leads to hardening of the microsphere surface, affecting drug release. When the amount of magnesium stearate added as a lubricant is less than a mass ratio of 100:0.5, the flowability of the microsphere powder is poor during tableting, and the tablets are prone to cracking; when the amount is higher than 100:2, the lubricant residue affects the contact between the microspheres and the mucosa, reducing adhesion performance. When the concentration of sodium carboxymethyl cellulose in the suspension medium is <1%, the microspheres are prone to sedimentation in the delivery device, resulting in uneven drug delivery; when the concentration is >3%, the system viscosity is too high, the injection resistance is large, and the patient finds it inconvenient to use.

[0061] The drug loading and release mechanism of microspheres involves multiple physicochemical processes. The distribution of drug molecules in the chitosan network is influenced by the drug's polarity, molecular weight, and the density of chitosan crosslinking. Hydrophilic small-molecule drugs, such as metronidazole, are mainly distributed in the aqueous pores of the gel network, with diffusion as the dominant release process. Hydrophobic large-molecule drugs, such as estradiol, are partially embedded in the hydrophobic microdomains of chitosan, and their release process is controlled by both diffusion and polymer dissolution. During the thermosensitive phase transition, the hydrophobic aggregation of poly(N-isopropylacrylamide) segments leads to the shrinkage of the network pores, creating dynamic resistance to drug diffusion and achieving temperature regulation of the release rate. In the vaginal environment, the sodium hyaluronate layer on the surface of the microspheres first absorbs water and swells, forming a hydrophilic barrier and delaying the initial burst release of the drug. Subsequently, the gel network shrinks at body temperature, further regulating the release rate. Finally, the chitosan backbone slowly degrades in the vaginal fluid, achieving complete drug release. This multiple controlled-release mechanism ensures that the drug maintains an effective local concentration while avoiding side effects caused by systemic absorption.

[0062] The clinical applications of microspheres cover the treatment of various gynecological diseases, including bacterial vaginosis, fungal vaginitis, and atrophic vaginitis. For acute infections, high-drug-load microspheres can be used to achieve rapid onset and long-term maintenance of action; for chronic inflammation or hormone replacement therapy, low-drug-load microspheres can be used to achieve stable sustained release and reduce the frequency of administration. Microspheres can be used alone or in combination with oral medications to form a synergistic local-systemic treatment regimen. The dosing frequency is adjusted according to the condition: once daily during the acute phase and once every two to three days during the maintenance phase, which is significantly better than the inconvenience of multiple daily administrations of traditional formulations. Patient compliance surveys show that over 90% of users find microsphere formulations convenient to use, experience no significant discomfort, and are willing to use them long-term.

[0063] A complete quality control system has been established for the large-scale production of microspheres. For each batch of raw chitosan, the degree of deacetylation, molecular weight, heavy metal content, and microbial limits are tested; for intermediate solutions, pH value, viscosity, and solid content are tested; and for finished microspheres, particle size distribution, water content, drug content, release rate, sterility, and endotoxin levels are tested. Key process parameters such as emulsification speed, crosslinking temperature, and drying vacuum degree are all monitored and automatically controlled online to ensure batch-to-batch consistency. Stability study data shows that the microspheres have a shelf life of up to 36 months under market packaging conditions, meeting the requirements for commercial production. The production process has been verified through pilot-scale amplification, with a single batch yield of up to 10 kg, demonstrating industrial-scale production capability.

[0064] The technological advantages of the microspheres are reflected in multiple dimensions. Compared to traditional chitosan powder, the microspheres of this invention are thermosensitive and can gel in situ at body temperature, achieving local fixation and sustained release of the drug. Compared to chitosan mouthwash, the microspheres of this invention are solid formulations, avoiding liquid loss and prolonging the drug's action time. Compared to ordinary gels, the microspheres of this invention have controllable particle size, and surface modification enhances adhesion, resulting in more stable drug release. The technical effects have been confirmed by multiple in vitro and in vivo tests, including precise matching of the thermosensitive phase transition temperature to the vaginal environment, extended drug release cycle to 24 hours, 3-fold increase in mucosal adhesion, 2-4 times improvement in antibacterial efficacy, zero local irritation, and storage stability of up to 36 months.

[0065] A method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration, comprising the following steps:

[0066] Chitosan was dissolved in a dilute aqueous acetic acid solution to prepare a chitosan solution with a mass concentration of 1% to 5%. The solution was stirred until completely dissolved, and then filtered to remove insoluble matter.

[0067] The thermosensitive polymer is dissolved in deionized water to prepare a thermosensitive polymer solution with a mass concentration of 0.5% to 3%. The solution is heated to 40°C to completely dissolve the polymer and then cooled to room temperature for later use.

[0068] Chitosan solution and thermosensitive polymer solution were mixed at a volume ratio of 1:0.8 to 1:1.5 and stirred for 30 minutes under ice bath conditions to form a homogeneous mixture.

[0069] The crosslinking agent was prepared into an aqueous solution with a mass concentration of 0.5% to 2%, and added dropwise to the above mixture at a rate of 0.5 to 2 mL / min under continuous stirring. After the addition was completed, the reaction was continued for 60 min to form a pregel suspension.

[0070] The active pharmaceutical ingredient was added to the pregel suspension and ultrasonically treated for 10 minutes in an ice bath using a probe-type ultrasonic instrument with a power of 300W and a pulse mode of 3 seconds on and 2 seconds off, so that the drug was uniformly dispersed in the gel network.

[0071] The above-mentioned drug-containing gel suspension is dripped into a curing bath containing calcium chloride or sodium sulfate at a mass concentration of 0.1% to 0.5% through a coaxial airflow spray device or a microfluidic chip device, and the curing time is 30 to 120 minutes to form spherical gel particles.

[0072] The solidified gel microparticles were washed three times with deionized water for 10 minutes each time, and the microspheres were collected after centrifugation.

[0073] The stabilizing agent was dissolved in phosphate buffer to prepare a solution with a mass concentration of 0.5% to 2%. The microspheres were then immersed in the solution and shaken at 37°C for 30 minutes. After removal, they were freeze-dried for 24 hours to obtain the final chitosan composite thermosensitive gel microsphere product.

[0074] Furthermore, the internal phase flow rate of the coaxial airflow spray device is 0.5–2 mL / min, the external phase compressed air pressure is 0.1–0.3 MPa, and the nozzle orifice diameter is 0.2–0.5 mm; the microfluidic chip device adopts a flow focusing structure, the continuous phase is mineral oil containing surfactant, the dispersed phase is the above-mentioned drug-containing gel suspension, the flow rate ratio is 5:1–10:1, and the chip channel width is 100–300 μm.

[0075] Furthermore, the freeze-drying process includes a pre-freezing temperature of -40℃ and a pre-freezing time of 4 hours, a sublimation drying temperature of -20℃ and a drying time of 12 hours, and a desorption drying temperature of 25℃ and a drying time of 12 hours. After drying, the microspheres have a water content of <3%, a rehydration time of <10 seconds, and a gel strength recovery rate of ≥90% after rehydration.

[0076] Furthermore, the preparation method also includes a step of sterilizing the final microspheres by irradiation with cobalt-60 gamma rays at a dose of 25 kGy, or by sterilization with ethylene oxide gas for 6 hours and ventilation desorption time of 72 hours.

[0077] Furthermore, the preparation method also includes a step of quality control of the microspheres, which involves using a laser particle size analyzer to determine the particle size distribution, using a rheometer to determine the gel strength and phase transition temperature, using high performance liquid chromatography to determine the drug encapsulation rate and drug loading, using an in vitro release device to determine the drug release curve in simulated vaginal fluid, and using an adhesion force tester to evaluate the adhesion performance of the microspheres to mucosal tissue.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations will be apparent to those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A chitosan composite thermosensitive gel microsphere for vaginal administration, characterized in that, include: Chitosan matrix with a degree of deacetylation ≥90% and a molecular weight of 200,000 to 500,000 Daltons; Thermosensitive polymer, which is poly(N-isopropylacrylamide) or its derivatives, has a lower critical solution temperature of 32°C to 37°C; The crosslinking agent, which is sodium tripolyphosphate or glutaraldehyde, accounts for 5% to 15% of the mass of the chitosan matrix; The active pharmaceutical ingredient is one or more of the following: antifungal drugs, antiviral drugs, or hormone drugs, and its mass accounts for 0.5% to 20% of the total mass of the microspheres. Stabilizing agent, which is hydroxypropyl methylcellulose or sodium alginate, accounts for 1% to 5% of the total mass of microspheres; The chitosan matrix and the thermosensitive polymer are in a mass ratio of 1:0.5 to 1:2, forming an interpenetrating network structure through ionic crosslinking and physical entanglement. The microspheres have an average particle size of 50 mm to 300 mm, are in a sol state below 25 °C, and undergo a phase transition to a gel state at 37 °C. The gel strength is not less than 500 Pascals, and the gel formation time is less than 30 seconds. In a vaginal simulated solution at 37 °C, the drug release half-life is greater than 8 hours, and the cumulative release rate within 24 hours is not less than 80%.

2. The chitosan composite thermosensitive gel microspheres for vaginal administration according to claim 1, characterized in that, The stabilizing agent is introduced by surface spraying or immersion adsorption, with a treatment time of 10 to 60 minutes and a treatment temperature of 25°C to 37°C. After modification with the stabilizing agent, the adhesion of the microspheres to the vaginal mucosa surface is increased by more than 30%, the retention time is extended to more than 12 hours, and there is no mucosal irritation.

3. A method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration, applied to the chitosan composite thermosensitive gel microspheres for vaginal administration as described in any one of claims 1-2, characterized in that, include: Chitosan with a degree of deacetylation of not less than 90% and a molecular weight of 200,000 to 500,000 Daltons is dissolved in dilute aqueous acetic acid to prepare a chitosan solution with a mass concentration of 1% to 5%. The solution is stirred until completely dissolved and then filtered. Poly(N-isopropylacrylamide) or its derivatives are dissolved in deionized water to prepare a thermosensitive polymer solution with a mass concentration of 0.5% to 3%. The solution is heated to 40°C to completely dissolve the polymer and then cooled to room temperature. The chitosan solution and the thermosensitive polymer solution are mixed at a volume ratio of 1:0.8 to 1:1.5 and stirred for 30 minutes under ice bath conditions to form a homogeneous mixture; Sodium tripolyphosphate or glutaraldehyde is prepared into a crosslinking agent aqueous solution with a mass concentration of 0.5% to 2%, and added dropwise to the mixture at a rate of 0.5 ml to 2 ml per minute under continuous stirring. After the addition is completed, the reaction is continued for 60 minutes to form a pregel suspension. One or more of the following drugs—antifungal, antiviral, or hormonal—are added to the pregel suspension, and the mixture is ultrasonically treated for 10 minutes in an ice bath at 300 watts with a pulse mode of 3 seconds on and 2 seconds off, so that the drug is uniformly dispersed in the gel network. The drug-containing gel suspension is dripped into a curing bath containing 0.1% to 0.5% calcium chloride or sodium sulfate by a coaxial airflow spray device or a microfluidic chip device, and cured for 30 to 120 minutes to form spherical gel particles. The solidified gel particles were washed three times with deionized water and then separated by centrifugation. Hydroxypropyl methylcellulose or sodium alginate was dissolved in phosphate buffer to prepare a stabilizing agent solution with a mass concentration of 0.5% to 2%. The gel microparticles were then immersed in the solution and treated with shaking at 37°C for 30 minutes. The processed microspheres were freeze-dried for 24 hours to obtain the chitosan composite thermosensitive gel microspheres.

4. The method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration according to claim 3, characterized in that, During the preparation of the chitosan solution, the concentration of the dilute acetic acid aqueous solution is 0.5% to 1%, the stirring speed is 300 revolutions per minute, the stirring time is 4 hours, and after dissolution, it is filtered through a 0.22 mm filter membrane.

5. The method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration according to claim 4, characterized in that, During the preparation of the temperature-sensitive polymer solution, the molecular weight of poly(N-isopropylacrylamide) is 100,000 to 300,000 Daltons, and its mass accounts for 10% to 30% of the mass of chitosan.

6. The method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration according to claim 5, characterized in that, During the addition of the crosslinking agent, the stirring speed is 200 to 600 revolutions per minute, the reaction temperature is controlled between 4°C and 25°C, and the mass of the crosslinking agent accounts for 5% to 15% of the mass of the chitosan matrix.

7. The method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration according to claim 6, characterized in that, The parameters of the coaxial airflow spray device are: internal phase flow rate of 0.5 ml to 2 ml per minute, external phase compressed air pressure of 0.1 MPa to 0.3 MPa, and nozzle orifice diameter of 0.2 mm to 0.5 mm; or the microfluidic chip device adopts a flow focusing structure, with the continuous phase being mineral oil containing surfactant, the dispersed phase being a drug-containing gel suspension, the flow rate ratio being 5:1 to 10:1, and the chip channel width being 100 mm to 300 mm.

8. The method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration according to claim 7, characterized in that, The freeze-drying process includes: pre-freezing temperature -40℃, pre-freezing time 4 hours; sublimation drying temperature -20℃, drying time 12 hours; desorption drying temperature 25℃, drying time 12 hours; after drying, the water content of the microspheres is less than 3%, the rehydration time is less than 10 seconds, and the gel strength recovery rate after rehydration is not less than 90%.

9. The method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration according to claim 8, characterized in that, It also includes a step of sterilizing the final microspheres: sterilization by cobalt-60 gamma irradiation at a dose of 25 kGrey; or sterilization by ethylene oxide gas for 6 hours followed by ventilation and desorption for 72 hours.

10. The method for preparing chitosan composite thermosensitive gel microspheres for vaginal administration according to claim 9, characterized in that, It also includes steps for quality control of microspheres: using a laser particle size analyzer to determine particle size distribution, using a rheometer to determine gel strength and phase transition temperature, using high performance liquid chromatography to determine drug encapsulation efficiency and drug loading, using an in vitro release device to determine the drug release curve in simulated vaginal fluid, and using an adhesion tester to evaluate the adhesion performance of microspheres to mucosal tissue.

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