Preparation method and application of asymmetric adhesion hydrogel powder for clinical treatment of pleural adhesion
By preparing asymmetric adhesive hydrogel powder, using formaldehyde-modified hyaluronic acid and 5-hydroxydopamine silk protein to cross-link to form an adhesive layer hydrogel, and loading simvastatin and miRNA-124 liposomes, the limitations of existing pleural adhesion treatment methods are overcome, efficient and stable drug release is achieved, surgical operations are simplified, and the treatment effect is improved.
Patent Information
- Application Number
- CN202511160712.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-19
- Publication Date
- 2025-10-14
AI Technical Summary
Existing treatments for pleural adhesions have problems such as limited mechanical isolation effect, poor targeting of drug intervention, high surgical complexity, and high risk of complications. It is difficult to effectively prevent and treat pleural adhesions. In addition, existing hydrogel materials are complex to operate in clinical applications and have unstable drug release.
An asymmetric adhesive hydrogel powder was prepared. The adhesive layer hydrogel was formed by cross-linking aldehyde-modified hyaluronic acid and 5-hydroxydopamine silk fibroin. Simvastatin and miRNA-124 liposomes were loaded to form a double-layer hydrogel, achieving local drug sustained release and rapid gelation, simplifying surgical operations.
It achieves efficient and stable drug release, reduces systemic side effects, simplifies surgical procedures, improves treatment effects, and reduces the risk of recurrence, making it suitable for the clinical treatment of pleural adhesions.
Smart Images
Figure CN120771105A_ABST
Abstract
Description
Background Art
[0001] Pleural adhesions are a common complication following thoracic surgery (e.g., cardiac bypass surgery, lobectomy), infection (tuberculosis, empyema), or trauma. They result from abnormal fibrin deposition and an inflammatory cascade triggered by damage to the pleural mesothelium. Pleural adhesions present a range of risks, including increased difficulty with secondary surgery: limited intraoperative visual field, increased operative time by more than 50%, and a significantly increased risk of bleeding and organ damage. These complications are particularly life-threatening for elderly patients or those with a history of thoracotomy (e.g., those with failed bioprosthetic valves requiring secondary valve replacement). Furthermore, lobectomy is the primary surgical procedure for malignant lung tumors. In the event of pleural adhesions, they must be dissected, which can be cumbersome depending on the severity of the adhesion. Severe pleural adhesions are occasionally encountered, resulting in prolonged operative time, increased blood loss, and a higher risk of tissue damage, sometimes necessitating conversion to thoracotomy during thoracoscopic surgery. Currently, there are several issues concerning pleural adhesions that need to be addressed. In clinical practice, 80% of patients experience pleural adhesions after surgery, making this a pressing issue. Furthermore, some pleural adhesions are caused by pleural inflammation, and long-term inflammatory pleural adhesions significantly impact lung function, a problem that cannot be ignored. Furthermore, in basic medicine, the molecular mechanisms underlying pleural adhesions remain largely unresolved, significantly hindering their prevention and treatment.
[0002] Due to the serious harm caused by pleural adhesions, medical professionals have been exploring effective means to prevent and treat them. However, current treatments have limitations and their effectiveness is limited. Initially, physical barriers (such as PLGA membranes and hyaluronic acid membranes) were used to prevent adhesions. However, this approach only provides mechanical isolation and cannot modulate the inflammatory microenvironment. Furthermore, the degradation cycle is misaligned with tissue repair. Therefore, physical barriers are generally used in conjunction with other methods as an adjunct. Pharmacological intervention is a commonly used treatment, but it also has drawbacks. Systemic drug delivery (such as anticoagulants and anti-inflammatory drugs) has poor targeting, while localized sustained-release materials (such as talc) can easily induce granulomas or new adhesions. Therefore, pharmacological interventions still need improvement. Furthermore, preoperative adhesion assessment relies on CT / ultrasound, but the sensitivity is only 38%-75%, making it difficult to accurately guide the surgical approach. Taking several common treatments as specific examples: 1. Chemical adhesiolysis has inconsistent efficacy and a long duration, carries high risks, and is suitable for a limited population. It is contraindicated in patients with impaired pulmonary function or those who cannot tolerate multiple invasive procedures. 2. Talc pleurodesis carries the risk of respiratory complications. Non-homogeneous talc may trigger acute respiratory distress syndrome (ARDS), with a mortality rate of 1-4%. The risk of interstitial lung disease in patients treated with immune checkpoint inhibitors (ICIs) is as high as 32.7% (80% in those with baseline interstitial lung disease). Furthermore, talc pleurodesis has a high failure rate, exceeding 25% in patients with atelectasis or pleural thickening, and the risk increases threefold when the pleural fluid pH is less than 7.15. There is no significant difference in the failure rate between thoracoscopic spraying and chest tube instillation, both around 22%, but the former requires general anesthesia and increases the risk of the procedure. Furthermore, talc residue may persist and irritate the pleura, leading to chronic chest pain and pleural calcification. 3. Indwelling pleural catheters carry the risk of infection and complications. Long-term catheterization can result in infection rates of 5-10%, requiring frequent care. The incidence of catheter occlusion or migration is approximately 15%. The rate of pleural fixation is low, with spontaneous pleural fixation achieved in only 40-58% of cases with simple drainage. The recurrence rate of effusions is greater than 30%. Patients' quality of life is compromised, requiring drainage two to three times per week, leading to poor compliance, limited mobility, and increased psychological burden. Surgical procedures are invasive and carry postoperative risks. General anesthesia poses a higher risk for patients with cardiopulmonary insufficiency. The average postoperative hospital stay is seven days, and the recovery period is long. The recurrence rate is greater than 20% six months after adhesion lysis, with adhesions arising from tuberculosis or post-radiotherapy being more prone to regeneration. Furthermore, thoracoscopic laser lysis (eg, Nd:YAG) requires specialized equipment and is expensive. There is also the risk of intraoperative bleeding, particularly for dense vascular adhesions. Minimally invasive VATS requires high surgeon experience, and improper operation may damage the lungs or blood vessels. In addition, there are restrictions on special populations. For example, in CAPD (continuous peritoneal dialysis) patients with diaphragmatic leak, surgery may aggravate abdominal-thoracic communication, requiring combined adhesive closure (such as aldehyde-based glue), increasing the complexity of the operation.Therefore, the treatment of pleural adhesions still needs further exploration.
[0003] As a medical biomaterial, hydrogels have shown significant advantages in drug delivery, tissue engineering, wound repair and other fields due to their unique physicochemical properties and biological functions. Hydrogels have biomimetic structures and good biocompatibility, supporting cell migration, proliferation and differentiation. For example, chitosan-based hydrogels form porous scaffolds through electrostatic cross-linking, which promotes corneal epithelial cell survival rates of more than 95%. In addition, hydrogels have low immunogenicity, and hydrogels derived from natural polymers (such as hyaluronic acid and collagen) have excellent biocompatibility. Hydrogels can be designed to respond to stimuli such as pH, temperature, and ROS. For example, ROS-responsive polyvinyl alcohol hydrogels can target the release of copper peptides in diabetic wounds, with a controlled release rate of 90% within 72 hours. Hydrogels can also regulate drug release kinetics through cross-linking density. Thermosensitive chitosan hydrogels gel at body temperature and continuously release antimicrobial peptides to the site of infection. Hydrogels also offer excellent clinical operability. Light-cured hydrogels can be injected minimally invasively to adhere to wound surfaces, reducing surgical trauma. Tannic acid-containing hydrogels can self-repair fractured interfaces and strongly adhere to moist tissues (adhesion strength >30 kPa). These advantages give hydrogels a broad application prospect in clinical treatment. Summary of the Invention
[0004] The object of the present invention is to provide a method for preparing asymmetric adhesive hydrogel powder for clinical treatment of pleural adhesions, so as to solve the problems raised in the above background technology.
[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing an asymmetric adhesive hydrogel powder for the clinical treatment of pleural adhesions, comprising the following steps:
[0006] S1. Preparation of aldehyde-modified hyaluronic acid (HAA): Hyaluronic acid (HA) is completely dissolved in ultrapure water, NaIO4 is weighed according to a certain molar ratio of HA to sodium periodate (NaIO4) and dissolved in ultrapure water, and the dissolved NaIO4 solution is slowly added to the HA solution. The above system is stirred to react under light-proof conditions, and then ethylene glycol is added to the reaction solution, and stirring is continued at room temperature to terminate the reaction. Subsequently, the above reaction solution is placed in ultrapure water for light-proof dialyzation to remove excess reactants. After the dialysis is completed, the dialysate is collected and freeze-dried to obtain a white flocculent solid (HAA), which is stored in the dark at -20°C for subsequent experiments;
[0007] Preparation of S2.5-hydroxydopamine silk fibroin (5-HDP-SF): Silk fibroin (SF) was completely dissolved in ultrapure water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were added to the SF solution in a certain molar ratio. After the above system was stirred and reacted under light-proof conditions, 5-hydroxydopamine hydrochloric acid (5-HDP·HCl) was added in a certain molar ratio and stirred and reacted under light-proof conditions. Subsequently, the above reaction solution was dialyzed in ultrapure water in the dark to remove excess reactants. After the dialysis was completed, the dialyzate was collected and freeze-dried to obtain a white flocculent solid (5-HDP-SF). The freeze-dried powder was stored in a sealed container at -80°C in the dark for subsequent experiments;
[0008] S3. Preparation of simvastatin and miRNA-124 co-loaded liposomes: Simvastatin, cationic lipids (DOTAP), cholesterol, neutral phospholipids (DOPE) and PEG lipids were added to an eggplant-shaped flask, dissolved with a certain amount of chloroform, and then the eggplant-shaped flask was placed in a vacuum rotary evaporator to dry to form a thin film. After drying under vacuum conditions, ultrapure water was added to the eggplant-shaped flask for hydration. The hydrated liquid was then placed in an ice bath and ultrasonicated with a probe, and then extruded through a filter membrane to obtain simvastatin liposomes (S-Lipo). Subsequently, miRNA-124 was mixed with S-Lipo and incubated at room temperature to obtain simvastatin and miRNA-124 co-loaded liposomes (Sm-Lipo);
[0009] S4. The synthesized HAA, ε-polylysine·HCl, and 5-HDP-SF were completely dissolved in ultrapure water at a certain mass ratio, and then NaOH was slowly added to adjust the pH to about 7 to form an adhesion layer hydrogel. Sm-Lipo was dispersed into the adhesion layer hydrogel, and then freeze-dried to obtain an adhesion layer hydrogel powder (loaded liposomes). The synthesized HAA and 4-arm-PEG-SH were completely dissolved in ultrapure water at a certain mass ratio to form a non-adhesion layer hydrogel. The non-adhesion layer hydrogel was fully freeze-dried and powdered to obtain a non-adhesion layer hydrogel powder. The adhesion layer hydrogel powder was redissolved to obtain an adhesion layer hydrogel, and then the non-adhesion layer hydrogel powder was applied to the upper surface of the adhesion layer hydrogel at a certain mass ratio. An asymmetric adhesion layer hydrogel was obtained.
[0010] In the preferred technical solution, the HA molecular weight is 800 kDa, the concentration of the HA aqueous solution is 1%, the molar ratio of HA to NaIO4 is 1:1, the stirring reaction time under light-proof conditions is 20-30 hours, the ethylene glycol concentration is 0.1 M, the stirring reaction time under room temperature is 0.5-2 hours, and the light-proof dialysis time is 40-60 hours.
[0011] In the preferred technical solution, the concentration of the silk fibroin aqueous solution is 1%, the molar ratio of EDCI to NHS is 1:1.2:2.4, the stirring reaction time under light shielding condition is 30 min, the molar ratio of 5-HDP·HCl added in the system is 1:4, and the stirring reaction time under light shielding condition is 20-30 h.
[0012] In the preferred technical solution, the volume of chloroform used in the preparation of the liposome is 2-3 mL, the film forming condition is 37℃, the rotation speed is 100 rpm, the reaction time is 30-60 min, the vacuum drying time is 4-6 h, the volume of ultrapure water used in the hydration is 4-6 mL, the hydration condition is 37℃, the rotation speed is 15 rpm, the reaction time is 1 h, the ultrasonic time is 15 min, the pore size of the filter membrane used is 0.45 µm, the N / P ratio of miRNA-124 is 7, and the co-incubation time of S-Lipo and miRNA-124 is 30 min.
[0013] In the preferred technical solution, the mass ratio of HAA, ε-polylysine·HCl and 5-HDP-SF is 1:4:2, the concentration of NaOH added is 0.5-1 M, the concentration of simvastatin in Sm-Lipo is 20-30 µg / mL, the molecular weight of 4-arm-PEG-SH used is 10 kDa, the mass ratio of HAA and 4-arm-PEG-SH is 1:4, and the mass ratio of the non-adhesion layer hydrogel and the adhesion layer hydrogel is 1:2.
[0014] Compared with the prior art, the present application has the following beneficial effects:
[0015] 1. The hydrogel prepared in the present application simultaneously loads simvastatin and miRNA-124, and can synergistically treat postoperative pleural adhesion, with a curative effect far exceeding that of a single drug.
[0016] 2. The asymmetric adhesive hydrogel powder prepared in the present application can be directly applied, simplifying the intraoperative operation process, without the need for complex injection equipment, and shortening the operation time.
[0017] 3. The asymmetric adhesive hydrogel prepared in the present application can quickly gel in the body, avoiding the problem of loss due to physical factors, and ensuring the retention of the drug.
[0018] 4. The asymmetric adhesive hydrogel powder prepared in the present application is beneficial to maintaining the stability of the drug, and is easy to store and transport.
[0019] 5. The asymmetric adhesive hydrogel powder prepared in the present application has good biodegradability, and can be absorbed and metabolized in the body after completing drug delivery, without the need for secondary removal.
[0020] 6. The asymmetric adhesive hydrogel prepared by the present application directly acts on the wound surface with a high local concentration, thereby reducing the risk of systemic side effects.
[0021] 7. The asymmetric adhesive hydrogel prepared by the present application can effectively prevent the formation and secondary occurrence of pleural adhesion through the dual mechanisms of physical barrier and drug-loaded sustained release. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 A schematic diagram for preparing the asymmetric adhesive hydrogel in the present application.
[0023] Figure 2 A transmission electron microscope image of the Sm-lipo prepared by the present application.
[0024] Figure 3 A, B and C are scanning electron microscope images of the adhesive layer hydrogel, the non-adhesive layer hydrogel and the asymmetric adhesive layer hydrogel prepared by the present application, respectively.
[0025] Figure 4 A and B are the frequency scanning results of the dynamic rheological test of each group of hydrogels.
[0026] Figure 5 A and B are the compression stress-strain curve and tensile strength test results of each group of hydrogels.
[0027] Figure 6 A two-layer hydrogel and the adhesion work test results between the two-layer hydrogels.
[0028] Figure 7 A test result of the in vitro degradation and drug release performance of the asymmetric adhesive hydrogel.
[0029] Figure 8 A test result of the in vivo degradation performance of the asymmetric adhesive hydrogel.
[0030] Figure 9 A test result of the in vivo drug efficacy of the asymmetric adhesive hydrogel.
[0031] Figure 10 A photograph of the asymmetric adhesive hydrogel powder. DETAILED DESCRIPTION
[0032] Hereinafter, the present application will be further described in conjunction with the drawings and specific embodiments. It should be noted that, under the premise of no conflict, each embodiment described below or each technical feature can be combined with any other embodiment or technical feature to form a new embodiment. It should be understood that the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor are within the scope of protection of the present application.
[0033] See also Figure 1 The present invention provides a preparation method: a preparation method of asymmetric adhesive hydrogel powder for clinical treatment of pleural adhesions, characterized in that the method comprises the following steps:
[0034] S1. Preparation of aldehyde-modified hyaluronic acid (HAA): Hyaluronic acid (HA) is completely dissolved in ultrapure water, NaIO4 is weighed according to a certain molar ratio of HA to sodium periodate (NaIO4) and dissolved in ultrapure water, and the dissolved NaIO4 solution is slowly added to the HA solution. The above system is stirred to react under light-proof conditions, and then ethylene glycol is added to the reaction solution, and stirring is continued at room temperature to terminate the reaction. Subsequently, the above reaction solution is placed in ultrapure water for light-proof dialyzation to remove excess reactants. After the dialysis is completed, the dialysate is collected and freeze-dried to obtain a white flocculent solid (HAA). The freeze-dried powder is stored in the dark at -20°C for subsequent experiments;
[0035] Preparation of S2.5-hydroxydopamine silk fibroin (5-HDP-SF): Silk fibroin (SF) was completely dissolved in ultrapure water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were added to the SF solution in a certain molar ratio. After the above system was stirred and reacted under light-proof conditions, 5-hydroxydopamine hydrochloric acid (5-HDP·HCl) was added in a certain molar ratio and stirred and reacted under light-proof conditions. Subsequently, the above reaction solution was dialyzed in ultrapure water in the dark to remove excess reactants. After the dialysis was completed, the dialyzate was collected and freeze-dried to obtain a white flocculent solid (5-HDP-SF), which was stored in a sealed container at -80°C in the dark for subsequent experiments;
[0036] S3. Preparation of simvastatin and miRNA-124 co-loaded liposomes: Simvastatin, cationic lipids (DOTAP), cholesterol, neutral phospholipids (DOPE) and PEG lipids were added to an eggplant-shaped flask, dissolved with a certain amount of chloroform, and then the eggplant-shaped flask was placed in a vacuum rotary evaporator to dry to form a thin film. After drying under vacuum conditions, ultrapure water was added to the eggplant-shaped flask for hydration. The hydrated liquid was then placed in an ice bath and ultrasonicated with a probe, and then extruded through a filter membrane to obtain simvastatin liposomes (S-Lipo). Subsequently, miRNA-124 was mixed with S-Lipo and incubated at room temperature to obtain simvastatin and miRNA-124 co-loaded liposomes (Sm-Lipo);
[0037] S4. The synthesized HAA, ε-polylysine·HCl, and 5-HDP-SF were completely dissolved in ultrapure water at a certain mass ratio, and then NaOH was slowly added to adjust the pH to about 7 to form an adhesion layer hydrogel. Sm-Lipo was dispersed into the adhesion layer hydrogel, and then freeze-dried to obtain an adhesion layer hydrogel powder (loaded liposomes). The synthesized HAA and 4-arm-PEG-SH were completely dissolved in ultrapure water at a certain mass ratio to form a non-adhesion layer hydrogel. The non-adhesion layer hydrogel was fully freeze-dried and powdered to obtain a non-adhesion layer hydrogel powder. The adhesion layer hydrogel powder was redissolved to obtain an adhesion layer hydrogel, and then the non-adhesion layer hydrogel powder was applied to the upper surface of the adhesion layer hydrogel at a certain mass ratio. An asymmetric adhesion layer hydrogel was obtained.
[0038] A method for preparing hydrogel powder for clinical treatment of pleural adhesions;
[0039] The HA molecular weight is 800 kDa, the concentration of the HA aqueous solution is 1%, the molar ratio of HA to NaIO4 is 1:1, the stirring reaction time under light-proof conditions is 20-30 hours, the ethylene glycol concentration is 0.1 M, the stirring reaction time under room temperature conditions is 0.5-2 hours, and the light-proof dialysis time is 40-60 hours.
[0040] The concentration of the silk fibroin aqueous solution is 1%, the molar ratio of the SF solution, EDCI and NHS is 1:1.2:2.4, the stirring reaction time is 30 minutes under light-proof conditions, the molar ratio of 5-HDP·HCl added to the system is 1:4, and the stirring reaction time is 20-30 hours under light-proof conditions.
[0041] The volume of chloroform used in the preparation of liposomes is 2-3 mL, the conditions for forming a thin film are 37° C., a rotation speed of 100 rpm, a reaction time of 30-60 min, and a vacuum drying time of 4-6 h. The volume of ultrapure water used for hydration is 4-6 mL, the hydration conditions are 37° C., a rotation speed of 15 rpm, a reaction time of 1 h, and an ultrasonic time of 15 min. The pore size of the filter membrane used is 0.45 μm, the N / P ratio of miRNA-124 is 7, and the co-incubation time of S-Lipo and miRNA-124 is 30 min.
[0042] The mass ratio of HAA, ε-polylysine·HCl and 5-HDP-SF is 1:4:2, the concentration of added NaOH is 0.5-1M, the concentration of simvastatin in Sm-Lipo is 20-30 μg / mL, the molecular weight of the 4-arm-PEG-SH used is 10 kDa, the mass ratio of HAA to 4-arm-PEG-SH is 1:4, and the mass ratio of the non-adhesive layer hydrogel to the adhesive layer hydrogel is 1:2.
[0043] In some embodiments, the preparation method specifically includes: first preparing aldehyde-modified hyaluronic acid (HAA) and 5-hydroxydopamine silk protein (5-HDP-SF), and using a thin film method to prepare liposomes loaded with simvastatin and miRNA-124; mixing the synthesized HAA, ε-polylysine·HCl and 5-HDP-SF in a certain mass ratio and adding sodium hydroxide (NaOH) to adjust the pH to form an adhesion layer hydrogel; dispersing the prepared liposomes loaded with simvastatin and miRNA-124 into the adhesion layer hydrogel and then freeze-drying to obtain an adhesion layer hydrogel powder (loaded liposomes); mixing the synthesized HAA and four-arm thiol polyethylene glycol (4-arm-PEG-SH) in a certain mass ratio and freeze-drying to form a non-adhesion layer hydrogel powder; re-dissolving the adhesion layer hydrogel powder to obtain an adhesion layer hydrogel, and applying the non-adhesion layer hydrogel powder on the upper surface of the adhesion layer hydrogel in a certain mass ratio to obtain an asymmetric adhesion hydrogel (loaded liposomes).
[0044] Furthermore, the HA molecular weight is 800 kDa, the HA aqueous solution concentration is 1%, the molar ratio of HA to NaIO4 is 1:1, the stirring reaction time under light-proof conditions is 20-30 hours, the ethylene glycol concentration is 0.1 M, the stirring reaction time under room temperature is 0.5-2 hours, and the light-proof dialysis time is 40-60 hours;
[0045] Furthermore, the concentration of the silk fibroin aqueous solution is 1%, the molar ratio of the SF solution, EDCI and NHS is 1:1.2:2.4, the stirring reaction time is 30 minutes under light-proof conditions, the molar ratio of 5-HDP·HCl added to the system is 1:4, and the stirring reaction time is 20-30 hours under light-proof conditions;
[0046] Furthermore, the volume of chloroform used in the preparation of liposomes is 2-3 mL, the conditions for forming a thin film are 37° C., a rotation speed of 100 rpm, a reaction time of 30-60 min, a vacuum drying time of 4-6 h, the volume of ultrapure water used for hydration is 4-6 mL, the hydration conditions are 37° C., a rotation speed of 15 rpm, a reaction time of 1 h, an ultrasonic time of 15 min, the pore size of the filter membrane used is 0.45 μm, the N / P ratio of miRNA-124 is 7, and the co-incubation time of S-Lipo and miRNA-124 is 30 min;
[0047] Furthermore, the mass ratio of HAA, ε-polylysine·HCl and 5-HDP-SF is 1:4:2, the concentration of added NaOH is 0.5-1M, the concentration of simvastatin in Sm-Lipo is 20-30 μg / mL, the molecular weight of the 4-arm-PEG-SH used is 10 kDa, the mass ratio of HAA to 4-arm-PEG-SH is 1:4, and the mass ratio of the non-adhesive layer hydrogel to the adhesive layer hydrogel is 1:2.
[0048] In summary, the preparation method of the present invention comprises the following steps: ① Preparation of HAA; ② Preparation of 5-HDP-SF; ③ Preparation of simvastatin and miRNA-124 co-loaded liposomes; and ④ Preparation of asymmetric adhesive hydrogels. The present invention first prepares HAA and 5-HDP-SF, co-loading the two drugs using liposomes. Subsequently, a bilayer hydrogel (loaded with liposomes) is formed through different cross-linking methods, demonstrating a promising therapeutic effect for postoperative pleural adhesions.
[0049] The following further illustrates the technical solution of the present invention with reference to the accompanying drawings and several preferred embodiments. However, the experimental conditions and setting parameters herein should not be considered as limiting the basic technical solution of the present invention. Furthermore, the scope of protection of the present invention is not limited to the following embodiments.
[0050] Example 1
[0051] The preparation method of the asymmetric adhesive hydrogel powder for the clinical treatment of pleural adhesions provided in this embodiment comprises the following steps:
[0052] 1. Preparation of aldehyde-modified hyaluronic acid (HAA): Hyaluronic acid (HA) is completely dissolved in ultrapure water. According to a certain molar ratio of HA to sodium periodate (NaIO4), NaIO4 is weighed and dissolved in ultrapure water. The dissolved NaIO4 solution is slowly added to the HA solution. The above system is stirred to react under light-proof conditions, and then ethylene glycol is added to the reaction solution, and stirring is continued at room temperature to terminate the reaction. Subsequently, the above reaction solution is placed in ultrapure water for light-proof dialyzation to remove excess reactants. After the dialysis is completed, the dialysate is collected and freeze-dried to obtain a white flocculent solid (HAA), which is stored in the dark at -20°C for subsequent experiments;
[0053] 2. Preparation of 5-hydroxydopamine silk fibroin (5-HDP-SF): Silk fibroin (SF) was completely dissolved in ultrapure water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) were added to the SF solution in a certain molar ratio. After the above system was stirred and reacted under light-proof conditions, 5-hydroxydopamine hydrochloric acid (5-HDP·HCl) was added in a certain molar ratio and stirred and reacted under light-proof conditions. Subsequently, the above reaction solution was dialyzed in ultrapure water in the dark to remove excess reactants. After the dialysis was completed, the dialyzate was collected and freeze-dried to obtain a white flocculent solid (5-HDP-SF), which was stored in a sealed container at -80°C in the dark for subsequent experiments;
[0054] 3. Preparation of simvastatin and miRNA-124 co-loaded liposomes: Simvastatin, cationic lipids (DOTAP), cholesterol, neutral phospholipids (DOPE) and PEG lipids were added to an eggplant-shaped flask, dissolved with a certain amount of chloroform, and then the eggplant-shaped flask was placed in a vacuum rotary evaporator to dry to form a thin film. After drying under vacuum conditions, ultrapure water was added to the eggplant-shaped flask for hydration, and then the hydrated liquid was placed in an ice bath and ultrasonicated with a probe, and then extruded through a filter membrane to obtain simvastatin liposomes (S-Lipo). Subsequently, miRNA-124 was mixed with S-Lipo and incubated at room temperature to obtain simvastatin and miRNA-124 co-loaded liposomes (Sm-Lipo);
[0055] 4. The synthesized HAA, ε-polylysine·HCl, and 5-HDP-SF were completely dissolved in ultrapure water at a certain mass ratio, and then NaOH was slowly added to adjust the pH to about 7 to form an adhesion layer hydrogel. Sm-Lipo was dispersed into the adhesion layer hydrogel, and then freeze-dried to obtain an adhesion layer hydrogel powder (loaded liposomes). The synthesized HAA and 4-arm-PEG-SH were completely dissolved in ultrapure water at a certain mass ratio to form a non-adhesion layer hydrogel. The non-adhesion layer hydrogel was fully freeze-dried and powdered to obtain a non-adhesion layer hydrogel powder. The adhesion layer hydrogel powder was redissolved to obtain an adhesion layer hydrogel, and then the non-adhesion layer hydrogel powder was applied to the upper surface of the adhesion layer hydrogel at a certain mass ratio. An asymmetric adhesion layer hydrogel was obtained.
[0056] In summary, through the above technical scheme, the present invention first prepares aldehyde-modified hyaluronic acid (HAA) and 5-hydroxydopamine silk protein (5-HDP-SF), and uses a thin film method to prepare liposomes loaded with simvastatin and miRNA-124; the synthesized HAA, ε-polylysine·HCl and 5-HDP-SF are mixed in a certain mass ratio and sodium hydroxide (NaOH) is added to adjust the pH to form an adhesion layer hydrogel; the prepared liposomes loaded with simvastatin and miRNA-124 are dispersed into the adhesion layer hydrogel and then freeze-dried to obtain an adhesion layer hydrogel powder (loaded liposomes); the synthesized HAA and four-arm thiol polyethylene glycol (4-arm-PEG-SH) are mixed in a certain mass ratio and freeze-dried to form a non-adhesion layer hydrogel powder; the adhesion layer hydrogel powder is redissolved to obtain an adhesion layer hydrogel, and the non-adhesion layer hydrogel powder is applied to the upper surface of the adhesion layer hydrogel in a certain mass ratio to obtain an asymmetric adhesion hydrogel (loaded liposomes).
[0057] Figure 1This is a schematic diagram of the asymmetric adhesive hydrogel preparation process. First, aldehyde-modified hyaluronic acid (HAA) and 5-hydroxydopamine silk fibroin (5-HDP-SF) were prepared, and liposomes loaded with simvastatin and miRNA-124 were prepared using a thin film method. Subsequently, adhesive layer hydrogel and non-adhesive layer hydrogel were formed through different cross-linking methods, and the drug-encapsulated liposomes were loaded into the adhesive layer hydrogel.
[0058] Figure 2 The transmission electron microscope image of Sm-lipo shows that Sm-lipo has a clear vesicle structure.
[0059] Figure 3 Scanning electron microscope images of each group of hydrogels were taken. From the images, it can be seen that both the adhesion layer and the non-adhesion layer hydrogels have obvious porous structures, but there are obvious differences in pore size and density. The image of the asymmetric adhesion hydrogel shows that there is a clear dividing line between the two layers of hydrogel (red dotted line).
[0060] Figure 4 The frequency scanning results of the dynamic rheological test of each group of hydrogels can be seen from the image. It can be seen that the storage modulus is significantly greater than the loss modulus, which proves the stable formation of the cross-linked network.
[0061] Figure 5 The compressive stress-strain curves and tensile strength test results of each group of hydrogels are shown in Figure A. From the compressive stress-strain curves in Figure A, it can be seen that the compressive fracture strength of each group of hydrogels can reach 10 3 Pa, which proves that the prepared hydrogel has a high hardness. From the tensile strength test results in Figure B, it can be seen that the tensile strength of each group of hydrogels can reach 2x10 3 Pa, proving that the prepared hydrogel has high toughness.
[0062] Figure 6 This is a test of the adhesion performance of asymmetric hydrogels. Figure A shows that the adhesion strength of the adhesive layer hydrogel is significantly higher than that of the non-adhesive layer hydrogel, proving the asymmetric adhesion of the hydrogel. The results in Figure B show that the two groups of hydrogels have a high adhesion strength, proving that the two layers of hydrogels can be stably bonded.
[0063] Figure 7 A and B show the in vitro degradation and drug release performance test results of the asymmetric adhesive hydrogel, respectively. The in vitro degradation test results in Figure A demonstrate that the asymmetric adhesive hydrogel can slowly degrade over 21 days, which is beneficial for long-term retention. The drug release test results in Figure B demonstrate that the asymmetric adhesive hydrogel can slowly release the drug over 14 days, demonstrating that the hydrogel can serve as a drug reservoir and exert a sustained anti-fibrotic effect.
[0064] Figure 8The results of the in vivo degradation performance test of the asymmetric adhesive hydrogel show that the asymmetric adhesive hydrogel can remain subcutaneously for at least 15 days and can be completely degraded, proving that the prepared asymmetric adhesive hydrogel has good biodegradability and biosafety.
[0065] Figure 9 The results of in vivo efficacy testing of the asymmetric adhesion hydrogel show that compared with the model group, the pleural adhesion symptoms in the experimental group treated with the hydrogel were significantly alleviated, indicating that the prepared hydrogel can be used in the clinical treatment of pleural adhesions and has a good therapeutic effect.
[0066] Figure 10 Figure 1 is an illustration of the abstract. A physical image of asymmetrically adherent hydrogel powder, which rapidly gels both in vivo and in vitro.
[0067] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Various changes made by ordinary technicians in this field based on the above concepts without creative work fall within the scope of protection of the present invention.
Claims
1. A method for preparing an asymmetric adhesive hydrogel powder for clinical treatment of pleural adhesions, characterized in that: The preparation was carried out according to the following steps: first, aldehyde-modified hyaluronic acid (HAA) and 5-hydroxydopamine silk fibroin (5-HDP-SF) were prepared, and liposomes loaded with simvastatin and miRNA-124 were prepared using a thin film method; the synthesized HAA, ε-polylysine·HCl and 5-HDP-SF were mixed in a certain mass ratio and sodium hydroxide (NaOH) was added to adjust the pH to form an adhesion layer hydrogel; the prepared liposomes loaded with simvastatin and miRNA-124 were dispersed into the adhesion layer hydrogel and then freeze-dried to obtain adhesion layer hydrogel powder (loaded liposomes); the synthesized HAA and four-arm thiol polyethylene glycol (4-arm-PEG-SH) were mixed in a certain mass ratio and freeze-dried to form non-adhesion layer hydrogel powder; the adhesion layer hydrogel powder was redissolved to obtain adhesion layer hydrogel, and the non-adhesion layer hydrogel powder was applied to the upper surface of the adhesion layer hydrogel in a certain mass ratio to obtain an asymmetric adhesion hydrogel (loaded liposomes).
2. The asymmetric adhesion hydrogel powder prepared by the preparation method according to claim 1.
3. Use of the asymmetric adhesive hydrogel powder according to claim 2 in the clinical treatment of pleural adhesions.