Temperature-sensitive estradiol hydrogel composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a temperature-sensitive estradiol hydrogel composition, a preparation method and application. According to the composition, a temperature-sensitive responsive network is constructed by introducing a PCL-PEG-PCL triblock copolymer, modified chitosan, a penetration enhancer and a buffer regulation system, so that rapid gelling, controlled-release drug delivery and good retention under a body temperature condition are realized. Through systematic contrast experiments with a plurality of contrast samples, evaluation is carried out from four aspects of gelling performance, drug release behavior, degradation residual rate and adhesiveness, and results show that the composition in a preferable ratio has the comprehensive advantages of good gel stability, long drug release period, good biodegradability, lasting uterine cavity adhesion and the like; good post-operation anti-adhesion and local drug delivery application prospects are realized.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedical materials, specifically relating to a thermosensitive estradiol hydrogel composition, its preparation method, and its application. Background Technology

[0002] Estradiol (17β-estradiol), as an endogenous estrogen, is traditionally administered via oral, transdermal patch, injection, and suppository methods. However, each of these methods has certain limitations in clinical use. For example, oral formulations are susceptible to the first-pass effect, leading to reduced bioavailability; patches pose a risk of local skin irritation; injections have poor patient compliance; and traditional vaginal preparations such as suppositories or creams suffer from problems such as high fluidity, poor retention, and uneven drug release.

[0003] Although existing hydrogel technology has improved the mucosal delivery performance of estradiol to some extent, it still has the following shortcomings: (1) Some copolymer systems have unstable thermal response and inaccurate gelation temperature, resulting in incomplete gel formation during use; (2) Poor drug loading uniformity, estradiol is prone to agglomeration or precipitation; (3) Insufficient adhesion or biodegradation performance, which limits its long-term application; (4) Insufficient research on the permeability regulation mechanism, the drug's penetration ability in local mucosal tissues still needs to be improved.

[0004] Therefore, there is an urgent need to design a thermosensitive estradiol hydrogel composition with reasonable composition, suitable response temperature, stable drug loading, and controllable structure, and to establish a preparation method with high reproducibility and process optimization, so as to meet the growing technical demand for local hormone delivery systems in the field of reproductive health. Summary of the Invention

[0005] To address the above problems, the present invention aims to provide a thermosensitive estradiol hydrogel composition, wherein the composition comprises the following components by weight percentage:

[0006] (1) 10% to 30% of polycaprolactone-polyethylene glycol-polycaprolactone triblock copolymer PCL-PEG-PCL, wherein the number average molecular weight of polyethylene glycol PEG is 1000 to 4000 Daltons, and the mass ratio of PCL to PEG is (2 to 5): 1.

[0007] (2) 0.05% to 0.5% of 17β-estradiol, pre-dissolved in ethanol, is distributed in the composition by homogeneous dispersion;

[0008] (3) 1% to 4% of modified chitosan, wherein the modified chitosan is selected from carboxymethyl chitosan, hydroxypropyl chitosan or quaternized chitosan, and its molecular weight is 50 to 200 kDa, and the degree of substitution is controlled at 0.6 to 0.9.

[0009] (4) 0.1% to 1.0% of a penetration enhancer, wherein the penetration enhancer is one or a combination of menthol, caprylic / capric monoglyceride, and laurocapram;

[0010] (5) 0.2% to 1.5% of a thickener, wherein the thickener is hydroxypropyl methylcellulose (HPMC), carbomer or xanthan gum, used to adjust the room temperature viscosity and gel structure;

[0011] (6) 0.05% to 0.3% of a buffer regulator, selected from a mixture of sodium lactate and sodium dihydrogen phosphate, to control the pH of the resulting gel between 4.5 and 5.5;

[0012] (7) The remainder is deionized water.

[0013] The composition is a low-viscosity solution at ambient temperatures below or equal to 25°C, and automatically forms a spatial physical cross-linked structure when the temperature rises to 25°C or above, exhibiting thermotropic gelation behavior.

[0014] As a preferred technical solution, the synthesis method of the triblock copolymer PCL-PEG-PCL uses ε-caprolactone and PEG as reaction units, and carries out a ring-opening polymerization reaction at 120-150°C under the catalysis of stannous octoate for 12-24 hours. The polymerization product is dissolved in tetrahydrofuran, precipitated in anhydrous ethanol, filtered and dried to obtain a solid copolymer with a number average molecular weight controlled at 3000-8000 Daltons and a molecular weight distribution coefficient of less than 1.5.

[0015] As a preferred technical solution, the estradiol is introduced as follows: first, estradiol is dissolved in ethanol at a concentration of 10-20 mg / mL, and the temperature is kept not higher than 30°C. Then, it is slowly added dropwise to a mixed solution of PCL-PEG-PCL and chitosan. The mixture is then homogenized for 20-30 minutes by ultrasonic treatment at a frequency of 40 kHz and a power of 150 W. The particle size of the resulting estradiol microemulsion is controlled between 200 and 500 nm.

[0016] As a preferred technical solution, the chitosan derivative is dissolved in a weakly acidic aqueous solution at 4℃ to 10℃, and then mixed with PCL-PEG-PCL copolymer at a weight ratio of 1:5 to 1:10. The mixing process is carried out by using a magnetic stirrer to maintain a speed of 500 rpm for 4 to 6 hours. The resulting composite solution is uniform and transparent, and can undergo hydrogen bond entanglement and crosslinking with PEG segments, thereby enhancing the structural stability and retention performance of the subsequent gel formation.

[0017] The present invention also provides a method for preparing the thermosensitive estradiol hydrogel composition, comprising the following steps:

[0018] S1, Synthesis of triblock copolymers:

[0019] After drying PEG with a molecular weight of 1000-4000 under vacuum at 110°C for 8 hours, it was added to a reactor with ε-caprolactone at a molar ratio of 1:(20-40). Stannous octoate was added at a molar ratio of 0.1%-0.3% as a catalyst. The reaction was carried out under nitrogen protection at a constant temperature of 120-150°C for 12-24 hours. After the reaction was completed, the mixture was cooled to room temperature, dissolved in tetrahydrofuran, and precipitated three times in anhydrous ethanol. After filtration, the mixture was dried under vacuum to obtain a white PCL-PEG-PCL solid copolymer.

[0020] S2. Preparation of precursor system solution:

[0021] The copolymer was added to deionized water at 0-4°C at a mass ratio of 10% to 30%, and magnetic stirring was started at a speed of 400-600 rpm. Then, 1% to 4% of chitosan derivative and 0.2% to 1.5% of thickener were added, and stirring was continued at low temperature until completely dissolved. The resulting mixture was a homogeneous and transparent solution with good flowability.

[0022] S3, estradiol loading:

[0023] Estradiol was dissolved in ethanol at a concentration of 10–20 mg / mL and then slowly added dropwise to the above solution. The mixture was then treated with ultrasonic oscillation at 40 kHz for 20–30 minutes and kept homogeneously dispersed at 0–10 °C to allow estradiol to form a stable particulate structure in the system. Subsequently, a penetration enhancer (0.1%–1.0%) and a buffer regulator (0.05%–0.3%) were added, and the pH was adjusted to 4.5–5.5.

[0024] S4. Pretreatment and Static Aging of Gel Formation:

[0025] The uniformly mixed system is sealed and left to stand for 24 hours at 4°C to form a storage state hydrogel precursor. When used, the precursor is placed in a water bath at 37°C, which can quickly form a stable three-dimensional gel structure.

[0026] As a preferred technical solution, in the synthesis process of the polycaprolactone-polyethylene glycol-polycaprolactone triblock copolymer in step S1, unmodified end-group polyethylene glycol is used as an initiator, the end group of the polyethylene glycol is hydroxyl structure, and no end-capping agent, monofunctional initiator or copolymerization regulator with a closed structure is added in the polymerization reaction.

[0027] As a preferred technical solution, in step S2, before dissolving the chitosan derivative, a 0.1 mol / L acetic acid solution is used as a pretreatment medium. The solution is stirred in a 40°C water bath for 2 hours until completely dissolved, and then slowly added to a cooling aqueous solution while maintaining the pH of the solution between 4.8 and 5.2 to avoid molecular chain aggregation or precipitation.

[0028] As a preferred technical solution, the penetration enhancer in step S3 is a composite structure of caprylic / capric monoglyceride and menthol, wherein caprylic / capric monoglyceride accounts for 0.3% to 0.5% and menthol accounts for 0.2% to 0.4%. The two are pre-dissolved into a colorless and transparent liquid in a water bath at 50°C before being added to the main system, and stirred thoroughly for 30 minutes to obtain a penetration environment regulator with uniform molecular distribution.

[0029] The present invention also provides the use of the thermosensitive estradiol hydrogel composition in the preparation of a medicament for preventing postoperative re-adhesion of the intrauterine cavity.

[0030] Beneficial effects

[0031] This invention introduces a PCL-PEG-PCL triblock copolymer and modified chitosan to synergistically construct a thermosensitive network structure, enabling the composition to rapidly transform from a solution into a stable gel under body temperature conditions. This provides excellent thermoresponsiveness and gel integrity, ensuring precise positioning within the uterine cavity and the formation of a physical barrier. Secondly, by optimizing the proportion of penetration enhancers and the thickening system, the penetration efficiency and local bioavailability of estradiol in mucosal tissues are improved, achieving slow and sustained drug release and effectively prolonging the duration of efficacy. A buffering system maintains the stability of the gel microenvironment, preventing drug degradation and irritation, and enhancing biocompatibility. It exhibits stronger anti-adhesion properties and postoperative adjuvant repair capabilities, making it suitable for clinical application in postoperative intrauterine drug delivery in gynecological surgeries. Attached Figure Description

[0032] Figure 1 This is a schematic diagram showing the comparative experimental results (gelation properties) of the present invention;

[0033] Figure 2 This is a schematic diagram showing the comparative experimental results (cumulative percentage of drug release) of the present invention;

[0034] Figure 3 This is a schematic diagram showing the comparative experimental results (gel degradation residue rate) of the present invention;

[0035] Figure 4 This is a schematic diagram showing the comparative experimental results (retention capacity and adhesion) of the present invention. Detailed Implementation

[0036] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0037] Example 1 (T1)

[0038] This embodiment provides a thermosensitive estradiol hydrogel composition, which is mainly formulated using the lower limits of the parameters of the above-mentioned components. The purpose is to verify that the formulation still possesses good gelling properties and drug carrying capacity at the lowest dosage, making it suitable for adjuvant therapy in the postoperative anti-adhesion treatment of uterine cavity. The specific preparation process is as follows:

[0039] S1. Synthesis of the triblock copolymer PCL-PEG-PCL:

[0040] Polyethylene glycol (PEG) with a number average molecular weight of 1000 Daltons was selected and vacuum dried at 110℃ for 8 hours to remove adsorbed moisture. PEG and ε-caprolactone were weighed and added to a four-necked reaction flask equipped with a stirrer at a molar ratio of 1:20. 0.1 mol% stannous octoate (Sn(Oct)2) was added as a catalyst, and the reaction was carried out at 120℃ for 12 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, and tetrahydrofuran was added to fully dissolve the product. Subsequently, the product was precipitated three times in excess anhydrous ethanol to remove unreacted monomers and oligomers. After filtration and vacuum drying, a white powdery PCL-PEG-PCL copolymer was obtained with a number average molecular weight of approximately 3000 Da, a molecular weight distribution coefficient of less than 1.5, and hydroxyl structures retained at both ends.

[0041] S2. Preparation of precursor system solution:

[0042] Under ice-water bath conditions, 10% of PCL-PEG-PCL was weighed and slowly added to deionized water at 0–4°C. The mixture was stirred continuously at 500 rpm for 2 hours with a magnetic stirrer to form a transparent milky white solution. Separately, 1% of carboxymethyl chitosan (molecular weight approximately 50 kDa, degree of substitution 0.6) was dissolved in 0.1 mol / L acetic acid solution and stirred in a 40°C water bath for 2 hours to form a pale yellow clear solution, which was then cooled. The chitosan solution was slowly mixed into the PCL-PEG-PCL solution at a mass ratio of 1:10, and stirring was continued for 4 hours to obtain a viscous, transparent composite solution. 0.2% hydroxypropyl methylcellulose (HPMC) was added as a thickener, and the mixture was stirred again for 1 hour until completely dissolved.

[0043] S3, Loading and Dispersion of Estradiol:

[0044] 0.05% (mass fraction) of 17β-estradiol was added to ethanol to a concentration of 10 mg / mL, and the solution was slowly stirred at room temperature until completely dissolved. This ethanol solution was then slowly added dropwise to the composite solution while continuously stirring, and ultrasonic dispersion was performed for 20 minutes using an ultrasonic homogenizer (40 kHz frequency, 150 W power). After treatment, the estradiol particle size was controlled to approximately 400 nm, forming a homogeneous microemulsion. Visual inspection revealed no stratification, no flocculation, and good transparency.

[0045] S4. Penetration enhancers and pH adjustment:

[0046] A composite penetration enhancer system was selected, comprising 0.3% caprylic / capric monoglyceride and 0.2% menthol. These were pre-dissolved in a 50°C water bath to form a colorless, transparent liquid, and then slowly added to the main system, stirring for 30 minutes to ensure uniform molecular distribution. Subsequently, 0.05% of a buffer conditioner was added. This buffer conditioner consisted of a 1:1 molar mixture of sodium lactate and sodium dihydrogen phosphate, ultimately adjusting the overall solution pH to 5.0.

[0047] S5. Aging and gelation treatment:

[0048] The prepared system was left to stand in a sealed environment at 4°C for 24 hours to stabilize the colloidal state and release internal stress.

[0049] Example 2 (T2)

[0050] This embodiment provides a thermosensitive estradiol hydrogel composition configured based on the upper limit of parameters, aiming to maximize the synergistic effect of various functional components and enhance the gel's response rate, drug stability, adhesion retention, and permeability under body temperature stimulation. The specific implementation steps are as follows:

[0051] S1. Synthesis of the triblock copolymer PCL-PEG-PCL:

[0052] Polyethylene glycol (PEG) with a number average molecular weight of 4000 Da was selected as the initiator and dried under vacuum at 110 °C for 8 hours. PEG and ε-caprolactone were added to the reactor at a molar ratio of 1:40, and stannous octoate catalyst was prepared with a molar ratio of 0.3%. The reaction system was carried out under nitrogen protection at 150 °C for 24 hours. After the reaction, the mixture was cooled to room temperature, and the product was dissolved in tetrahydrofuran and precipitated three times in anhydrous ethanol to remove impurities. Finally, the product was filtered and dried under vacuum to obtain a light yellow copolymer solid with a number average molecular weight of 8000 Da and a molecular weight distribution coefficient of less than 1.5. The PEG end groups retained the hydroxyl structure.

[0053] S2. Preparation of precursor system solution:

[0054] The obtained copolymer was added at a mass ratio of 30% to deionized water at 0°C and stirred on a magnetic stirrer at 600 rpm for 3 hours until fully dissolved, resulting in a system with a certain viscosity. 4% quaternized chitosan (molecular weight 200 kDa, degree of substitution 0.9) was weighed, dissolved in 0.1 mol / L acetic acid solution, and stirred in a 40°C water bath for 2 hours. This solution was then slowly added dropwise to the copolymer solution, ensuring thorough mixing at a mass ratio of 1:5, while maintaining the pH at 5.0 ± 0.2. Subsequently, 1.5% carbomer was added as a thickener, and the mixture was stirred for another 2 hours to obtain a viscous, clear, and transparent composite solution with good adhesion and film-forming properties.

[0055] S3, Loading and Dispersion of Estradiol:

[0056] 0.5% of 17β-estradiol was added to ethanol to prepare a 20 mg / mL solution, which was then fully dissolved under magnetic stirring at a constant temperature of 30 °C. This solution was then slowly added dropwise to the main solution system prepared in S2 while stirring. The system was then treated with an ultrasonic processor at 150 W power and 40 kHz frequency for 30 minutes at 0–10 °C to homogeneously disperse the estradiol into a microemulsion with a particle size of approximately 200 nm, resulting in a uniform and stable drug distribution.

[0057] S4. Penetration enhancers and pH adjustment:

[0058] Add 1.0% of a penetration enhancer mixture, comprising 0.5% caprylic / capric monoglyceride and 0.4% menthol. These two components are pre-dissolved in a 50°C water bath to form a homogeneous solution before being introduced into the main system. Stir continuously for 30 minutes to ensure complete integration. Finally, add 0.3% of a buffer regulator, composed of sodium lactate and sodium dihydrogen phosphate, to adjust the pH of the system to 5.5.

[0059] S5. Pretreatment and Aging of the Gelation Process:

[0060] The above mixture was placed in a sealed container at 4°C and left to stand for 24 hours to complete the initial aging of the colloidal network and the stabilization of its microstructure.

[0061] Example 3 (T3)

[0062] This embodiment provides a thermosensitive estradiol hydrogel composition configured based on optimal values ​​within a parameter range. Taking into account gelling properties, biocompatibility, drug loading capacity, and delivery effect, the composition is designed and prepared by selecting the optimal level of each component, striving to achieve the best balance between drug release, structural stability, and ease of operation. It is suitable for postoperative local drug delivery and tissue protection.

[0063] S1. Synthesis of the triblock copolymer PCL-PEG-PCL:

[0064] Polyethylene glycol (PEG) with a number average molecular weight of 3000 Da was selected and vacuum dried at 110 °C for 8 hours to remove moisture. PEG and ε-caprolactone were added to a three-necked flask at a molar ratio of 1:30, and 0.2 mol% stannous octoate was added as a ring-opening polymerization catalyst. Under a nitrogen atmosphere, the reaction was carried out at a constant temperature of 130 °C for 18 hours. After cooling to room temperature, the reaction product was dissolved in tetrahydrofuran and precipitated three times in anhydrous ethanol to remove oligomers and residual monomers. After filtration and vacuum drying, a light white powdery PCL-PEG-PCL copolymer was obtained, with a number average molecular weight controlled at around 6000 Da and a molecular weight distribution coefficient of approximately 1.3.

[0065] S2. Preparation of precursor system solution:

[0066] Weigh 20% of the triblock copolymer into deionized water at 0–4°C and stir at 500 rpm for 2 hours on a magnetic stirrer until completely dissolved. Dissolve 2.5% of hydroxypropyl chitosan (molecular weight approximately 120 kDa, degree of substitution 0.8) in 0.1 mol / L acetic acid aqueous solution beforehand. Stir in a 40°C water bath for 2 hours, then slowly add it dropwise to the copolymer solution, making its mass ratio with PCL-PEG-PCL 1:8. Continue stirring for 4 hours to form a transparent homogeneous solution. Then add 0.8% hydroxypropyl methylcellulose (HPMC) as a thickener and stir until fully dissolved to obtain a pretreated composite system with good flowability.

[0067] S3, Estradiol loading and homogeneous dispersion:

[0068] Weigh 0.25% of 17β-estradiol and dissolve it in ethanol at a concentration of 15 mg / mL, maintaining the temperature at room temperature not exceeding 30°C. Slowly add this solution dropwise to the precursor system prepared in S2, and homogenize it for 25 minutes at 8°C using a 40 kHz ultrasonic treatment device with a power of 150 W to form a stable microemulsion with a particle size controlled at around 300 nm.

[0069] S4. Penetration enhancers and pH adjustment:

[0070] Add 0.6% of a penetration enhancer complex, consisting of 0.4% caprylic / capric monoglyceride and 0.2% menthol. These two compounds are pre-dissolved in a 50°C water bath to form a clear liquid before being added to the system. Stir for 30 minutes to ensure uniform molecular layer distribution. Add 0.15% of a buffer regulator (sodium lactate to sodium dihydrogen phosphate molar ratio 1:1) to adjust the pH to 5.2 ± 0.1.

[0071] S4. Pre-gelling treatment and aging storage:

[0072] The prepared composite solution was sealed and stored in a refrigerator at 4°C for 24 hours to stabilize the structure and mature the system.

[0073] Comparative Example 1 (C1)

[0074] This comparative example aims to verify that the physical properties, gelling ability, and drug-carrying capacity of the system will be severely affected when key structural components are missing from the thermosensitive estradiol hydrogel composition. This comparative example is essentially the same as Example 3 (T3) in preparation method, with only one core difference: no modified chitosan is added (i.e., the modified chitosan content is 0%), while the remaining components are maintained at the preferred levels in Example 3 for control analysis.

[0075] Formula composition (by weight percentage):

[0076] Polycaprolactone-polyethylene glycol-polycaprolactone triblock copolymer (PCL-PEG-PCL): 20% (PEG number average molecular weight 3000 Da, PCL to PEG mass ratio 3.5:1);

[0077] 17β-estradiol: 0.25%, dissolved in 15 mg / mL ethanol solution;

[0078] Modified chitosan: 0% (missing);

[0079] Penetration enhancer: 0.6%, including caprylic / capric monoglyceride (0.4%) and menthol (0.2%);

[0080] Thickener (HPMC): 0.8%;

[0081] Buffer regulator (sodium lactate and sodium dihydrogen phosphate): 0.15%;

[0082] Deionized water: Top up to 100%.

[0083] Preparation process: Same as in Example 3 (T3), consisting of four steps:

[0084] S1: Synthetic triblock copolymer PCL-PEG-PCL;

[0085] S2: Dissolve the copolymer in low-temperature deionized water and add HPMC to form a composite precursor solution;

[0086] S3: Estradiol dissolved in ethanol is added, and the mixture is homogenized by ultrasound. A penetration enhancer and a buffer regulator are introduced to adjust the pH to 5.2.

[0087] S4: After 24 hours of cold aging, observe the gelation behavior at 37°C.

[0088] Comparative Example 2 (C2)

[0089] This comparative example aims to verify the effect of the dosage of key components exceeding the upper limit of the recommended parameter range on the physicochemical properties, gelling characteristics, and drug release behavior of the thermosensitive estradiol hydrogel composition. C2 is essentially the same as Example 3 (T3) in formulation and preparation method, except that the dosage of polycaprolactone-polyethylene glycol-polycaprolactone triblock copolymer (PCL-PEG-PCL) is increased to 40% (exceeding the specified upper limit of 30%), to analyze its effect on the system structure and performance.

[0090] Formula composition (by weight percentage):

[0091] Polycaprolactone-polyethylene glycol-polycaprolactone triblock copolymer (PCL-PEG-PCL): 40% (exceeding the limit);

[0092] Modified chitosan (hydroxypropyl chitosan, molecular weight 120kDa, degree of substitution 0.8): 2.5%;

[0093] 17β-estradiol: 0.25%, dissolved in 15 mg / mL ethanol;

[0094] Penetration enhancer: 0.6%, including caprylic / capric monoglyceride (0.4%) and menthol (0.2%);

[0095] Thickener (HPMC): 0.8%;

[0096] Buffer conditioner: 0.15%;

[0097] Deionized water: Top up to 100%.

[0098] Preparation process: Same as in Example 3 (T3):

[0099] S1: The triblock copolymer was vacuum dried after synthesis;

[0100] S2: The copolymer is added to cooled deionized water and stirred. Chitosan derivatives and HPMC are then added to form a precursor solution.

[0101] S3: Introduce ultrasonically dispersed estradiol, along with a penetration enhancer and a buffer regulator, to adjust the pH to 5.2;

[0102] S4: After refrigeration and aging, a gelation test was conducted in a 37°C water bath.

[0103] Comparative Example 3 (C3)

[0104] Comparative Example 3 (C3) aims to verify the effects of exceeding the recommended upper limit of the penetration enhancer dosage on the physical stability, biosafety, and drug release behavior of the thermosensitive estradiol hydrogel composition. C3 uses the same baseline formulation and preparation steps as Example 3 (T3), with modifications only to the dosage of the penetration enhancer, to assess the systemic consequences of overuse.

[0105] Formula composition (by weight percentage):

[0106] Polycaprolactone-polyethylene glycol-polycaprolactone triblock copolymer (PCL-PEG-PCL): 20% (PEG number average molecular weight 3000 Da, PCL:PEG mass ratio 3.5:1);

[0107] Modified chitosan (hydroxypropyl chitosan, molecular weight 120kDa, degree of substitution 0.8): 2.5%;

[0108] 17β-estradiol: 0.25%, dissolved in 15 mg / mL ethanol;

[0109] Penetration enhancer: 1.5% (exceeding limit), including caprylic / capric monoglyceride (1.0%) and menthol (0.5%);

[0110] Thickener (HPMC): 0.8%;

[0111] Buffer regulator (sodium lactate and sodium dihydrogen phosphate): 0.15%;

[0112] Deionized water: Top up to 100%.

[0113] Preparation process: Same as in Example 3 (T3):

[0114] S1: Synthetic triblock copolymer PCL-PEG-PCL;

[0115] S2: Add the copolymer, chitosan derivative and HPMC sequentially to low-temperature deionized water and stir to form a composite solution;

[0116] S3: Add estradiol dissolved in ethanol dropwise, and treat with ultrasound for 25 minutes to form microemulsion particles with a diameter of about 300 nm; then add a total of 1.5% of the penetration enhancer complex solution and stir for 30 minutes, and finally adjust the pH to 5.2;

[0117] S4: The system was left to stand at 4℃ for 24 hours, and the gelation behavior of the gelation precursor was observed in a water bath at 37℃.

[0118] Comparative Example 4 (C4)

[0119] Comparative Example 4 (C4) aimed to verify the effect of severely insufficient buffer regulator dosage (below the recommended lower limit) on the pH control capability, system stability, and drug release behavior of the thermosensitive estradiol hydrogel composition. C4 was consistent with Example 3 (T3) in all other components and preparation process, except that the buffer regulator dosage was reduced to 0.01%, significantly below the recommended lower limit of 0.05%, to assess the role of this factor in maintaining the acid-base balance of the microenvironment.

[0120] Formula composition (by weight percentage):

[0121] PCL-PEG-PCL triblock copolymer: 20% (PEG number average molecular weight 3000 Da, PCL:PEG mass ratio 3.5:1);

[0122] Modified chitosan (hydroxypropyl chitosan, molecular weight 120kDa, degree of substitution 0.8): 2.5%;

[0123] 17β-estradiol: 0.25%, dissolved in 15 mg / mL ethanol;

[0124] Penetration enhancer: 0.6%, including caprylic / capric monoglyceride (0.4%) and menthol (0.2%);

[0125] Thickener (HPMC): 0.8%;

[0126] Buffer regulator (sodium lactate and sodium dihydrogen phosphate, 1:1 molar ratio): 0.01% (significantly insufficient);

[0127] Deionized water: Top up to 100%.

[0128] Preparation process: Same as in Example 3 (T3):

[0129] S1: Synthesize the triblock copolymer to obtain a dry powder;

[0130] S2: Copolymer, chitosan, and HPMC are sequentially added to cooled deionized water and stirred to form a precursor solution;

[0131] S3: An ultrasonically dispersed estradiol microemulsion system was introduced, a penetration enhancer was added, and then only 0.01% of a buffer regulator was added. The pH of the system naturally drifted to approximately 6.0–6.2.

[0132] S4: Refrigerate and stand for 24 hours. Before use, observe the gelation behavior at 37°C.

[0133] Comparative Example 5 (C5)

[0134] Comparative Example 5 (C5) aims to compare the shortcomings of commonly used single thermosensitive material systems in the prior art in terms of drug delivery, gelation properties, and in vivo application, highlighting the innovation and comprehensive performance advantages of the multi-component synergistic thermosensitive estradiol hydrogel composition proposed in this invention. This comparative example references some existing technical literature and publicly reported basic formulations, constructing a control system using only a single thermosensitive material, Pluronic F-127 (polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer), to verify its limitations in intrauterine local drug delivery.

[0135] Formula composition (by weight percentage):

[0136] Pluronic F-127 (PEO-PPO-PEO, molecular weight approximately 12,600 Da): 25%;

[0137] 17β-estradiol: 0.25%, dissolved in ethanol (concentration 15 mg / mL);

[0138] Penetration enhancer: None added;

[0139] Modified chitosan: None added;

[0140] Thickener: None added;

[0141] Buffer: None added;

[0142] Deionized water: Top up to 100%.

[0143] Preparation method:

[0144] Dissolve 25% of Pluronic F-127 slowly in deionized water at 4°C and stir for 12 hours until completely transparent;

[0145] Estradiol was dissolved in ethanol and then slowly added dropwise to the solution. The solution was then subjected to ultrasonic treatment for 20 minutes to form a drug microparticle dispersion system.

[0146] After refrigerating the system for 24 hours, the gelation process was observed and the performance was tested at 37°C.

[0147] Comparative experiment:

[0148] Experimental Objective: This experiment aims to verify the technical advantages of the thermosensitive estradiol hydrogel composition described in this invention across multiple key performance dimensions. Through systematic comparison with multiple comparative samples (C1-C5), the following scientific questions are clarified:

[0149] The effect of different formulation parameters (upper and lower limits and preferred values) on gelation behavior;

[0150] Does multi-component synergistic design help improve drug controlled release, adhesion, and system stability?

[0151] Does the proposed optimized ratio scheme (Example T3) have the best overall performance under actual simulation conditions?

[0152] Is there a significant improvement compared to existing single-material thermosensitive systems (such as F127)?

[0153] According to the plan requirements, 8 groups of experimental samples were set up:

[0154] Example 1 (T1): Lower limit group of parameters;

[0155] Example 2 (T2): Parameter upper limit group;

[0156] Example 3 (T3): Optimal formulation group;

[0157] Comparative Example 1 (C1): Unmodified chitosan group;

[0158] Comparative Example 2 (C2): Triblock copolymer excess group;

[0159] Comparative Example 3 (C3): Permeation enhancer exceeding the limit group;

[0160] Comparative Example 4 (C4): Buffer Insufficient Group;

[0161] Comparative Example 5 (C5): Conventional F127 material group (existing public technology).

[0162] The following key performance indicators are planned for this experiment to systematically evaluate sample performance:

[0163] 1. Gel-forming properties: Evaluate the system's thermosensitive response behavior, gel-forming time, gel structure integrity, and visible transparency at 37℃. This indicator reflects whether the system meets the prerequisites for application.

[0164] Experimental method: Each group of samples was stored at 4℃ for 24 hours; then placed in a 37℃ water bath, and the transition process from liquid to gel was timed and observed; the start time and completion time of gelation were recorded; and it was evaluated whether a stable, continuous, non-flowing gel structure was formed.

[0165] 2. Drug release behavior:

[0166] The hourly release rate and cumulative release curve of estradiol in simulated body fluids were determined to reflect the controlled release capability and delivery efficiency.

[0167] Experimental method: Take an equal mass of gel sample and place it in a dialysis bag. After sealing, suspend it in a buffer solution containing PBS (pH 5.5). Shake at a constant speed on a shaker at 37°C. Set sampling time points (1h, 4h, 8h, 12h, 24h, 48h). Take out the external solution each time and add an equal volume of fresh solution. Measure the estradiol concentration and calculate the cumulative release percentage.

[0168] 3. Gel degradation residue rate:

[0169] The degradation cycle and residual amount of the samples were observed in a simulated uterine cavity environment to evaluate their in vivo retention capacity and biodegradability.

[0170] Experimental methods: After gel formation, each group was placed in simulated uterine cavity fluid (pH 5.5) and allowed to stand at 37℃; samples were taken out at time points of day 3, day 7, day 10, and day 14; the samples were gently washed with deionized water and dried; the mass of residue was accurately weighed and the residue rate was calculated (residual mass / initial mass × 100%).

[0171] 4. Retention capacity and adhesion:

[0172] Assessing the duration of gel adhesion and anti-slip ability on simulated tissue surfaces is crucial for intrauterine application.

[0173] Experimental method: Prepare porcine uterine slices and place them on a slant device (initial tilt angle 30°); place the gel sample on its surface and observe for slippage; a retention time of 30 minutes is considered acceptable; gradually increase the tilt angle to 45° and observe the slippage distance and whether detachment occurs.

[0174] The experimental data are shown in Tables 1-4:

[0175] Table 1. Gelation performance / min

[0176] Grouping gelation initiation time (min) Gel formation completion time (min) Gel structural integrity Visual transparency T1 6 10 whole better T2 1 3 Rigid structure Slightly mixed T3 3 5 whole excellent C1 Ungelatinized Ungelatinized fail turbid C2 2 5 Brittle opaque C3 Uneven Unstable fracture micro flocculent C4 7 12 weak Uneven C5 5 8 loose Weak turbidity

[0177] Table 2 Cumulative percentage of drug release / %

[0178]

[0179]

[0180] Table 3. Gel degradation residue rate / %

[0181] Number of days 3 7 10 14 T1 85.2 72.4 18.7 3.6 T2 93.5 85.2 42.6 12.1 T3 88.6 70.2 28.7 6.3 C1 35.4 10.2 0.8 0 C2 95.3 89.1 70.6 45.3 C3 68.2 35.6 9.2 1.1 C4 70.4 51.2 22.8 7.9 C5 58 20.3 2.6 0.4

[0182] Table 4 Retention Capacity and Adhesion

[0183]

[0184]

[0185] Data Analysis:

[0186] 1. Analysis of gelling properties:

[0187] The gelation behavior of each group of samples was observed under a 37℃ water bath condition, and the results are as follows: Figure 1 As shown, Example 3 (T3) gels in approximately 3 minutes, forming a stable gel with a complete structure and high transparency within 5 minutes, exhibiting excellent temperature-sensitive response. Example 1 (T1) also gels, but the onset time is delayed to 6 minutes, and the completion time exceeds 10 minutes. The gel transparency and structural strength are slightly insufficient, indicating that gelation efficiency and film quality are limited under low-dose conditions. Example 2 (T2) gels rapidly within 1 minute, with a completion time of approximately 3 minutes, but the gel structure is relatively rigid, exhibiting some rigid shrinkage issues, which affects bioadhesion.

[0188] In comparison, C1, lacking modified chitosan, could not effectively construct a three-dimensional network structure and failed to complete the gelation process during the observation period; C2, although gelling within 5 minutes, produced a thick, easily cracked gel with an overly rigid structure; C3, due to the interference of the penetration promoter on the polymerization system formation, resulted in discontinuous gel breakage; C4's system lacked sufficient acid-base buffering capacity, causing delayed gelation and a loose structure; C5 (F127 single-component control) could form a gel within 5–8 minutes, but the colloid was soft, had poor transparency, and an unstable structure. In summary, Example 3 achieved ideal results in terms of gelation rate, structural integrity, and system transparency, demonstrating the positive effect of its optimized component ratio on temperature-sensitive gelation behavior.

[0189] 2. Drug release behavior analysis:

[0190] By measuring the release behavior of estradiol in each group of samples, such as Figure 2 As shown, Example 3 (T3) maintained a continuous and stable release process within 48 hours, with a cumulative release rate of 88%. The release curve exhibited typical sustained-release characteristics, indicating that its structure is conducive to long-term drug delivery and stable diffusion. Example 1 (T1) had a faster release rate in the first 8 hours, with 62% released after 12 hours and 82% after 48 hours. Its controlled-release capability was slightly lower than T3, belonging to the type with rapid release and a slightly shorter duration. Example 2 (T2) had a slow release in the early stages due to its dense system, with a cumulative release rate of only 40% after 8 hours and only 78% released after 48 hours, reflecting the phenomenon that drug diffusion is hindered in a high-concentration matrix.

[0191] Comparative analysis showed that C1 experienced a burst release of the drug due to its inability to form a gel structure, releasing 60% within 1 hour and nearly completely within 48 hours. C3 contained excessive permeation enhancer, inducing a rapid release of 92% of the drug within the first 12 hours, indicating that excessive permeation enhancer disrupted the controlled-release balance. C2, due to its excessively high colloid concentration, experienced a severely delayed drug release, with a cumulative release rate of only 35% after 48 hours, significantly lower than the example. C4 exhibited irregular release behavior, with rapid initial release followed by slower release in the later stages, reflecting that the lack of buffers affected drug release stability. C5 released only 65% ​​within 48 hours, resulting in insufficient release and an early plateau phase, failing to meet the need for continuous postoperative drug administration. In summary, T3 demonstrated significant advantages in release stability and long-term delivery, making it the preferred option for achieving controlled drug release.

[0192] 3. Analysis of gel degradation residue rate:

[0193] The degradation behavior of each group of samples was observed in simulated uterine fluid, such as... Figure 3 As shown, T3 retained 88.6% of its mass on day 3, decreased to 70.2% on day 7, and reached 6.3% by day 14, indicating that it can stably release the drug within a week and degrade essentially within two weeks, ensuring the treatment cycle while avoiding the risk of residue. T1 degraded relatively quickly, with a residue rate of only 72.4% on day 7 and decreasing to 3.6% on day 14. Although safe, its retention period was slightly shorter. T2 degraded the slowest, with 12.1% residue remaining on day 14, suggesting a certain risk of accumulation in the body.

[0194] In the comparative studies, C1 showed a residual rate of only 35.4% on day 3, which decreased to 10.2% on day 7, and was completely degraded by day 14, reflecting its lack of structural support. C2 degraded the slowest, with a residual rate as high as 45.3% after 14 days, indicating that the high copolymer concentration severely inhibited degradation. C3 exhibited an unstable degradation rate, with a residual rate of 35.6% on day 7, but rapidly decreasing to 1.1% by day 14, indicating that the uneven structure of the copolymer structure led to drastic fluctuations in degradation stages. C4 and C5 showed moderate degradation performance, but still did not match the structural balance of T3. Overall, T3 achieved a good match in terms of degradation cycle control, effectively supporting its application positioning for postoperative delivery.

[0195] 4. Retention capacity and adhesion analysis:

[0196] The test results using the pig uterine sloping model are as follows: Figure 4 As shown, T3 remained stably on a 30° incline for over 30 minutes, and its sliding distance was controlled within 8 mm under a 45° incline, demonstrating its excellent adhesion and physical bonding properties. Although T2 had the longest retention time (35 minutes), its surface spreading ability was insufficient due to the excessive viscosity of the colloid. T1, due to its relatively thin structure, could only maintain a retention time of 28 minutes, with a slightly higher sliding distance of 12 mm.

[0197] C1 exhibits almost no retention capacity, sliding down within 2 minutes with a slippage distance of 65mm; C3, due to the penetration agent disrupting interfacial tension, slips down to 52mm, exhibiting extremely poor adhesion; C2, although relatively thick, has poor adhesion and slides after 20 minutes; C4 begins to slip after 10 minutes of retention, and C5 only maintains retention for 8 minutes, demonstrating poor structural support. In summary, T3 achieves optimal retention stability due to its moderate gel strength and good elasticity, meeting the clinical requirements for intrauterine targeted drug administration.

[0198] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A thermosensitive estradiol hydrogel composition, characterized in that, The composition comprises the following components by weight percentage: (1) 10% to 30% of polycaprolactone-polyethylene glycol-polycaprolactone triblock copolymer PCL-PEG-PCL, wherein the number average molecular weight of polyethylene glycol PEG is 1000 to 4000 Daltons, and the mass ratio of PCL to PEG is (2 to 5):

1. (2) 0.05% to 0.5% of 17β-estradiol, pre-dissolved in ethanol, is distributed in the composition by homogeneous dispersion; (3) 1% to 4% of modified chitosan, wherein the modified chitosan is selected from carboxymethyl chitosan, hydroxypropyl chitosan or quaternized chitosan, and its molecular weight is 50 to 200 kDa, and the degree of substitution is controlled at 0.6 to 0.

9. (4) 0.1% to 1.0% of a penetration enhancer, wherein the penetration enhancer is one or a combination of menthol, caprylic / capric monoglyceride, and laurocapram; (5) 0.2% to 1.5% of a thickener, wherein the thickener is hydroxypropyl methylcellulose (HPMC), carbomer or xanthan gum, used to adjust the room temperature viscosity and gel structure; (6) 0.05% to 0.3% of a buffer regulator, selected from a mixture of sodium lactate and sodium dihydrogen phosphate, to control the pH of the resulting gel between 4.5 and 5.5; (7) The remainder is deionized water. The composition is a low-viscosity solution at ambient temperatures below or equal to 25°C, and automatically forms a spatial physical cross-linked structure when the temperature rises to 25°C or above, exhibiting thermotropic gelation behavior.

2. The thermosensitive estradiol hydrogel composition according to claim 1, characterized in that, The synthesis method of the triblock copolymer PCL-PEG-PCL uses ε-caprolactone and PEG as reaction units, and carries out ring-opening polymerization at 120-150°C under the catalysis of stannous octoate for 12-24 hours. The polymerization product is dissolved in tetrahydrofuran, precipitated in anhydrous ethanol, filtered and dried to obtain a solid copolymer with a number average molecular weight controlled at 3000-8000 Daltons and a molecular weight distribution coefficient of less than 1.

5.

3. The thermosensitive estradiol hydrogel composition according to claim 1, characterized in that, The estradiol is introduced as follows: first, estradiol is dissolved in ethanol at a concentration of 10-20 mg / mL, and the temperature is kept not higher than 30°C. Then, it is slowly added dropwise to a mixed solution of PCL-PEG-PCL and chitosan. The mixture is then homogenized for 20-30 minutes by ultrasonic treatment at a frequency of 40 kHz and a power of 150 W. The particle size of the resulting estradiol microemulsion is controlled between 200 and 500 nm.

4. The thermosensitive estradiol hydrogel composition according to claim 1, characterized in that, The chitosan derivative is dissolved in a weakly acidic aqueous solution at 4℃ to 10℃ and then mixed with PCL-PEG-PCL copolymer at a weight ratio of 1:5 to 1:

10. The mixing process is carried out by stirring with a magnetic stirrer at a speed of 500 rpm for 4 to 6 hours. The resulting composite solution is uniform and transparent and can undergo hydrogen bonding and cross-linking with the PEG segment, thereby enhancing the structural stability and retention performance of the subsequent gel formation.

5. A method for preparing the thermosensitive estradiol hydrogel composition according to any one of claims 1-4, characterized in that, The following steps are involved: S1, Synthesis of triblock copolymers: After drying PEG with a molecular weight of 1000-4000 under vacuum at 110°C for 8 hours, it was added to a reactor with ε-caprolactone at a molar ratio of 1:(20-40). Stannous octoate was added at a molar ratio of 0.1%-0.3% as a catalyst. The reaction was carried out under nitrogen protection at a constant temperature of 120-150°C for 12-24 hours. After the reaction was completed, the mixture was cooled to room temperature, dissolved in tetrahydrofuran, and precipitated three times in anhydrous ethanol. After filtration, the mixture was dried under vacuum to obtain a white PCL-PEG-PCL solid copolymer. S2. Preparation of precursor system solution: The copolymer was added to deionized water at 0-4°C at a mass ratio of 10% to 30%, and magnetic stirring was started at a speed of 400-600 rpm. Then, 1% to 4% of chitosan derivative and 0.2% to 1.5% of thickener were added, and stirring was continued at low temperature until completely dissolved. The resulting mixture was a homogeneous and transparent solution with good flowability. S3, estradiol loading: Estradiol was dissolved in ethanol at a concentration of 10–20 mg / mL and then slowly added dropwise to the above solution. The mixture was then treated with ultrasonic oscillation at 40 kHz for 20–30 minutes and kept homogeneously dispersed at 0–10 °C to allow estradiol to form a stable particulate structure in the system. Subsequently, a penetration enhancer (0.1%–1.0%) and a buffer regulator (0.05%–0.3%) were added, and the pH was adjusted to 4.5–5.

5. S4. Pretreatment and Static Aging of Gel Formation: The uniformly mixed system is sealed and left to stand for 24 hours at 4°C to form a storage state hydrogel precursor. When used, the precursor is placed in a water bath at 37°C, which can quickly form a stable three-dimensional gel structure.

6. The preparation method according to claim 5, characterized in that, In the synthesis of the polycaprolactone-polyethylene glycol-polycaprolactone triblock copolymer in step S1, unmodified end-group polyethylene glycol is used as an initiator. The end groups of the polyethylene glycol are hydroxyl structures. No end-capping agent, monofunctional initiator or copolymerization regulator with a closed structure is added during the polymerization reaction.

7. The preparation method according to claim 5, characterized in that, Before dissolving the chitosan derivative in step S2, a 0.1 mol / L acetic acid solution was used as a pretreatment medium. The solution was stirred in a 40°C water bath for 2 hours until completely dissolved. Then, it was slowly added to a cooling aqueous solution, and the pH of the solution was maintained between 4.8 and 5.2 to avoid molecular chain aggregation or precipitation.

8. The preparation method according to claim 5, characterized in that, The penetration enhancer mentioned in step S3 is a composite structure of caprylic / capric monoglyceride and menthol, wherein caprylic / capric monoglyceride accounts for 0.3% to 0.5% and menthol accounts for 0.2% to 0.4%. The two are pre-dissolved in a 50°C water bath to form a colorless and transparent liquid before being added to the main system, and stirred thoroughly for 30 minutes to obtain a penetration environment regulator with uniform molecular distribution.

9. The use of the thermosensitive estradiol hydrogel composition according to any one of claims 1-4 in the preparation of a medicament for preventing postoperative re-adhesion of the intrauterine cavity.

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