A kind of nanocomposite sodium hyaluronate hydrogel and its preparation method and application

Abstract

The present application relates to a kind of nanocomposite sodium hyaluronate hydrogel and its preparation method and application.The present application specifically relates to a kind of preparation method of nanocomposite sodium hyaluronate hydrogel, comprising: preparation of oxidized sodium hyaluronate;Preparation of sodium hyaluronate-adipic acid dihydrazide;3.6% to 4.4% of oxidized sodium hyaluronate solution is mixed with 1.8% to 2.2% of sodium hyaluronate-adipic acid dihydrazide solution with 1:1, obtain Schiff base crosslinking hydrogel;Preparation of BMS-202 loaded liposome, the particle size of BMS-202 loaded liposome is 90nm to 300nm;BMS-202 loaded liposome and R837 are jointly loaded into Schiff base crosslinking hydrogel, obtain nanocomposite sodium hyaluronate hydrogel.The present application also relates to the application of nanocomposite sodium hyaluronate hydrogel in preparation for preventing breast cancer recurrence and treating postoperative breast defect breast filling repair material.

Description

Technical Field

[0001] This invention relates to the field of materials chemistry, specifically to a nanocomposite sodium hyaluronate hydrogel, its preparation method, and its applications. Background Technology

[0002] Breast cancer is the most common malignant tumor in women, and surgical resection (including breast-conserving surgery and total mastectomy) remains the core strategy for the clinical treatment of primary breast cancer. However, the risk of local recurrence after surgery is as high as 10%-41%, mainly due to residual microtumors or in situ regeneration, making postoperative recurrence prevention treatments (such as radiotherapy, chemotherapy, and immunotherapy) necessary. However, existing treatments have limitations in terms of efficacy and safety: radiotherapy is prone to causing tissue fibrosis, chemotherapy has significant systemic toxicity, and while immune checkpoint inhibitors (ICIs) can block the PD-1 / PD-L1 pathway to activate anti-tumor immunity, their efficacy is limited by the tumor immunosuppressive microenvironment (TME) and low drug delivery efficiency.

[0003] On the other hand, the tissue loss caused by mastectomy can easily trigger psychological trauma in patients, leading to a surge in demand for breast reconstruction. Currently, implant filling or autologous fat transplantation are commonly used in clinical practice, but these methods carry risks such as capsular contracture, displacement, and infection. Furthermore, postoperative radiotherapy can further increase postoperative complications and limit the options for preventing recurrence.

[0004] In recent years, injectable hydrogels have become ideal candidate materials for breast repair due to their minimally invasive implantation, adjustable mechanical properties, and biomimetic extracellular matrix (ECM) characteristics. Sodium hyaluronate (HA) hydrogels, with their excellent biocompatibility and shear-thinning properties, have been widely used as injectable fillers in the medical aesthetics field and have shown potential in tissue repair and controlled drug release. However, existing HA hydrogel products have limited functionality and lack a synergistic design that combines tissue repair and anti-tumor recurrence.

[0005] In the field of tumor immunotherapy, the clinical efficacy of immune checkpoint inhibitors (such as the representative small molecule inhibitor BMS-202) is often limited by the tumor immunosuppressive microenvironment, and one important influencing factor is the immunosuppressive effect mediated by M2 tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). M2 TAMs mediate tumor immune escape through multiple mechanisms, including promoting tumor angiogenesis, suppressing effector T cell activity, and promoting the secretion of immunosuppressive factors. Existing research indicates that repolarizing M2 TAMs to a pro-inflammatory M1 phenotype can effectively reshape the immunosuppressive microenvironment and enhance the response to ICI therapy. For example, Zhang et al. (Mol Cancer. 2023; 22:58) suggested that first using TAM repolarizing drugs to induce TAM polarization towards the M1 phenotype, inhibiting tumor immune escape, and creating a microenvironment favorable to immune response, can significantly enhance the anti-tumor immune response when subsequently using ICIs.

[0006] Studies have confirmed that repolarizing agents such as TLR agonists (e.g., R837) can effectively induce the M2 to M1 phenotype conversion. However, when used in combination with ICIs, traditional dosing regimens fail to achieve the desired spatiotemporal synergistic effect due to the differences in the pharmacokinetic characteristics of the two drugs. For example, Ashish Kulkarni et al. (ACS Nano, 2016 Oct 25) demonstrated that an "on-demand" dosing regimen (i.e., alternating injections of TLR agonists and ICIs every other day) is less effective than administering ICIs after completing a TLR agonist treatment cycle. Furthermore, off-target toxicity caused by systemic administration further limits its clinical application value.

[0007] Postoperative cavity filling after breast cancer resection requires hydrogels with excellent mechanical properties to maintain the cavity morphology, while the time-sequential release of drugs also needs to be regulated by cross-linked networks to control the diffusion rate. Therefore, there is an urgent need to develop a multifunctional implantable material that can dynamically adapt mechanical strength, self-healing properties, and drug diffusion rate through cross-linked network design, so as to simultaneously meet the mechanical adaptation requirements for postoperative cavity filling and the drug release requirements, thereby breaking through the bottleneck of postoperative breast cancer treatment. Summary of the Invention

[0008] The technical problem to be solved by this invention is how to prevent recurrence of breast cancer after surgical resection and to resolve breast defects.

[0009] Accordingly, the technical solution adopted by the present invention to solve its technical problem is, in the first aspect, a method for preparing nanocomposite sodium hyaluronate hydrogel, comprising the following steps:

[0010] Prepare oxidized sodium hyaluronate; prepare sodium hyaluronate-adipate dihydrazide; mix 3.6%-4.4% oxidized sodium hyaluronate solution with 1.8%-2.2% sodium hyaluronate-adipate dihydrazide solution at a 1:1 ratio to obtain Schiff base crosslinked hydrogel; prepare liposomes loaded with BMS-202 with a particle size of 90nm to 300nm; co-encapsulate the BMS-202-loaded liposomes and R837 into the Schiff base crosslinked hydrogel to obtain nanocomposite sodium hyaluronate hydrogel.

[0011] In some implementations, nanocomposite sodium hyaluronate hydrogel is used for breast tissue filling.

[0012] In some embodiments, a 3.8%-4.2% oxidized sodium hyaluronate solution is mixed with a 1.9%-2.1% sodium hyaluronate-adipic acid dihydrazide solution at a 1:1 ratio.

[0013] In some embodiments, a 4% oxidized sodium hyaluronate solution is mixed with a 2% sodium hyaluronate-adipic acid dihydrazide solution at a 1:1 ratio.

[0014] In some embodiments, sodium hyaluronate-adipic acid dihydrazide is prepared by the following method: sodium hyaluronate is activated with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and hydroxybenzotriazole in morpholine ethane sulfonate buffer, and then reacted with adipic acid dihydrazide, purified and dried.

[0015] In some implementations, liposomes loaded with BMS-202 are prepared via an ethanol injection method, specifically including the following steps: injecting DOPC, cholesterol, and PEG... 2000 The oil phase of BMS-202 was injected into ultrapure water at a constant rate of 0.1 mL / min to 0.5 mL / min using a syringe, and stirred continuously at 300 rpm to 700 rpm for 1 to 4 hours at 40℃-60℃.

[0016] The second aspect of the present invention also provides a nanocomposite sodium hyaluronate hydrogel, which is prepared by the preparation method of the first aspect of the present invention.

[0017] The third aspect of the present invention provides the application of nanocomposite sodium hyaluronate hydrogel in the preparation of breast filling and repair materials for preventing breast cancer recurrence, wherein the nanocomposite sodium hyaluronate hydrogel is prepared by the preparation method of the first aspect of the present invention.

[0018] In some implementations, the time difference between the cumulative release of R837 and the cumulative release of BMS-202 in the nanocomposite sodium hyaluronate hydrogel reaching 80% is greater than or equal to 8 days.

[0019] The fourth aspect of the present invention provides the application of nanocomposite sodium hyaluronate hydrogel in the preparation of breast filling and repair materials for preventing breast cancer recurrence and treating postoperative breast defects, wherein the nanocomposite sodium hyaluronate hydrogel is prepared by the preparation method of the first aspect of the present invention.

[0020] The beneficial effects of this invention are that the Schiff base crosslinked hydrogel prepared by crosslinking 3.6%-4.4% oxidized sodium hyaluronate and 1.8%-2.2% sodium hyaluronate-adipic dihydrazide via Schiff base crosslinking, after strain scanning, dynamic time, and dynamic frequency scanning tests, shows that the Schiff base crosslinked hydrogel has suitable injectability and mechanical strength for breast tissue filling. Simultaneously, the prepared hydrogel, after co-encapsulating R837 and BMS-202 liposomes, enables sequential delivery of the two drugs, thereby overcoming TME immunosuppression and achieving synergistic effects of the two drugs.

[0021] Therefore, the nanocomposite sodium hyaluronate hydrogel prepared by the method of the present invention can simultaneously take into account tissue filling and local anti-recurrence properties, and is suitable as a breast filling and repair material for preventing breast cancer recurrence and treating postoperative breast defects. Attached Figure Description

[0022] Figure 1 This is the 1H NMR spectrum of the oxidized sodium hyaluronate prepared according to the present invention.

[0023] Figure 2 This is the infrared spectrum of the oxidized sodium hyaluronate obtained by this invention.

[0024] Figure 3 This is the 1H NMR spectrum of sodium hyaluronate-adipic dihydrazide prepared in this invention.

[0025] Figure 4 These are the strain scanning test results of four different Schiff base crosslinked hydrogels prepared in this invention.

[0026] Figure 5 These are the test results of dynamic time scanning of four different Schiff base crosslinked hydrogels prepared in this invention.

[0027] Figure 6 These are the test results of dynamic frequency scanning of four different Schiff base crosslinked hydrogels prepared in this invention.

[0028] Figure 7 These are the test results of the self-healing ability of the Gel4 Schiff base crosslinked hydrogel prepared in this invention.

[0029] Figure 8 This is a scanning electron microscope image of the Gel4 Schiff base crosslinked hydrogel prepared according to the present invention.

[0030] Figure 9 This is a transmission electron microscope image of the liposomes loaded with BMS-202 prepared according to the present invention.

[0031] Figure 10 The results are dynamic light scattering measurements of the liposomes loaded with BMS-202 prepared according to the present invention; wherein, A is the measurement result of particle size and its distribution; and B is the measurement result of zeta potential.

[0032] Figure 11 These are the strain scanning test results of the nanocomposite sodium hyaluronate hydrogel prepared in this invention.

[0033] Figure 12 These are the test results of dynamic time-scan of the nanocomposite sodium hyaluronate hydrogel prepared by this invention.

[0034] Figure 13 These are the test results of dynamic frequency scanning of the nanocomposite sodium hyaluronate hydrogel prepared by this invention.

[0035] Figure 14 These are the test results of the self-healing ability of the nanocomposite sodium hyaluronate hydrogel prepared by this invention.

[0036] Figure 15 This is the in vitro drug release curve of the nanocomposite sodium hyaluronate hydrogel prepared by this invention.

[0037] Figure 16 This is the in vitro drug release curve of sodium hyaluronate hydrogel crosslinked with 1,4-butanediol diglycidyl ether (BDDE). Detailed Implementation

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the technical field to which this invention pertains. The following definitions are supplementary to those definitions in the art and relate to this invention, but are not extrapolated to any relevant or unrelated situation, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in practical testing of the invention, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0039] In this document, the terms "one or more" and "at least one" are used interchangeably.

[0040] In this document, the terms "one or more" and "at least one" are used interchangeably.

[0041] The terms “including,” “comprising,” or “having”, when used before a step or element, indicate the addition of a further step or element, which is optional and not excluded.

[0042] As mentioned above, the present invention aims to address the shortcomings of existing technologies by providing a nanocomposite sodium hyaluronate hydrogel. Based on this, the present invention provides a method for preparing the nanocomposite sodium hyaluronate hydrogel.

[0043] The preparation method of the present invention includes the step of preparing sodium oxidized hyaluronic acid.

[0044] In a specific embodiment, oxidized sodium hyaluronate is prepared by oxidizing sodium hyaluronate. For example, sodium hyaluronate is oxidized with sodium periodate.

[0045] For example, oxidized sodium hyaluronate can be prepared by the following method: Sodium hyaluronate is dissolved in deionized water, and sodium periodate is added to form a homogeneous reaction system. The above oxidation reaction is carried out under light-protected conditions, the reaction temperature is maintained at 25℃±2℃, and the reaction is magnetically stirred for 24 hours. The reaction is terminated by adding diethylene glycol in an equimolar amount of sodium periodate, and stirring is continued for 1 hour. After dialyzing for 72 hours, the product, oxidized sodium hyaluronate, is obtained by freeze-drying.

[0046] The preparation method of the present invention includes the step of preparing sodium hyaluronate-adipic acid diacyl.

[0047] In the preparation method of the present invention, sodium hyaluronate-adipic dihydrazide is formed by grafting adipic dihydrazide (ADH) onto the main chain of sodium hyaluronate (HA) via a carbodiimide chemistry (EDC / NHS) reaction.

[0048] Exemplarily, sodium hyaluronate-adipate dihydrazide can be prepared by the following method: Sodium hyaluronate is dissolved in morpholine ethane sulfonate buffer (pH=6.5), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and hydroxybenzotriazole are added sequentially, and the mixture is activated at room temperature for 1 hour. Adipate dihydrazide is then added, and the reaction is continued at 25±2℃ for 24 hours. After the reaction is completed, the mixture is purified by dialysis for 96 hours, and after freeze-drying, the product sodium hyaluronate-adipate dihydrazide is obtained and stored at -20℃ protected from light.

[0049] The preparation method of the present invention includes the step of mixing sodium oxidized hyaluronic acid solution and sodium hyaluronate-adipic acid dihydrazide solution at a volume ratio of 1:1 to obtain Schiff base crosslinked hydrogel.

[0050] Oxidized sodium hyaluronate contains aldehyde groups, while sodium hyaluronate-adipic dihydrazide contains abundant hydrazine groups (hydrazine groups). The active hydrazine groups can undergo condensation reactions with aldehyde groups to generate Schiff bases, achieving controllable covalent cross-linking.

[0051] In some embodiments, the mass fraction of the oxidized sodium hyaluronate solution is 3.6% to 4.4%. Exemplary examples include 3.6%, 3.65%, 3.7%, 3.75%, 3.8%, 3.85%, 3.9%, 3.95%, 4%, 4.05%, 4.1%, 4.15%, 4.2%, 4.25%, 4.3%, 4.35%, or 4.4%.

[0052] In a preferred embodiment, the mass fraction of the oxidized sodium hyaluronate solution is 3.8% to 4.2%.

[0053] In a more preferred embodiment, the mass fraction of the oxidized sodium hyaluronate solution is 4%.

[0054] In some embodiments, the mass fraction of the sodium hyaluronate-adipate dihydrazide solution is 1.8% to 2.2%. Exemplarily, the mass fraction of the sodium hyaluronate-adipate dihydrazide solution is 1.8%, 1.85%, 1.9%, 1.95%, 2%, 2.05%, 2.1%, 2.15%, or 2.2%.

[0055] In a preferred embodiment, the mass fraction of the sodium hyaluronate-adipic dihydrazide solution is 1.9% to 2.1%.

[0056] In a more preferred embodiment, the sodium hyaluronate-adipic acid dihydrazide solution has a mass fraction of 2%.

[0057] In some embodiments, the Schiff base crosslinked hydrogel prepared by the present invention has the good mechanical strength and injectability required for breast tissue filling.

[0058] In some embodiments, the Schiff base crosslinked hydrogel prepared by the present invention exhibits good dynamic stability required for breast tissue filling.

[0059] It should be noted that the Schiff base crosslinked hydrogel prepared by this invention exhibits good biocompatibility, stability, and specific mechanical properties to simulate the feel and appearance of a natural substance. The main material used in the preparation of this invention is hyaluronic acid, whose main metabolic components—hyaluronic acid oligosaccharides / monosaccharides—are metabolic substances found in the human body, are non-toxic, and are biocompatible. Unreacted ADH molecules or their oxidation byproducts in hyaluronic acid-adipic acid dihydrazide are completely removed by dialysis. This invention is safe and non-toxic, unlikely to cause local inflammation, and its degradation products are safe and can be normally metabolized and excreted, demonstrating good biocompatibility.

[0060] Furthermore, the Schiff base crosslinked hydrogel prepared by this invention has a higher G' (storage modulus or elastic modulus), exhibiting more "solid" characteristics. It can withstand certain external forces (such as compression and gravity) and rebound while maintaining its shape, thus better mimicking the feel of human tissue. Moreover, its self-healing properties mean that even after deformation and breakage, the hydrogel tends to maintain its overall shape and is not prone to flow, migration, or displacement. Therefore, the Schiff base crosslinked hydrogel prepared by this invention is suitable for applications such as breast tissue filling, while differing in properties from hydrogels used in other tissues.

[0061] The preparation method of the present invention includes the step of encapsulating BMS-202 into liposomes to prepare liposomes encapsulated with BMS-202.

[0062] In some implementations, liposomes loaded with BMS-202 can be prepared by ethanol injection.

[0063] For example, DOPC, cholesterol, and PEG can be used. 2000 BMS-202 was mixed in a certain proportion to form a homogeneous oil phase, with ethanol as the solvent; the oil phase was slowly injected into water at a constant rate to form an oil-water mixture; the oil-water mixture was heated and mechanically stirred to completely remove the ethanol, and finally a suspension of liposomes loaded with BMS-202 was obtained.

[0064] Specifically, DOPC, cholesterol, and PEG can be included. 2000 BMS-202 was mixed in a certain proportion to form a homogeneous oil phase, with ethanol as the solvent. The oil phase was transferred to a syringe and slowly injected into ultrapure water at a constant rate to form an oil-water mixture. The oil-water mixture was heated and mechanically stirred, with the temperature controlled at 40℃-60℃ and the stirring rate at 300rpm-700rpm for 1-4 hours to completely remove the ethanol, finally obtaining a suspension of liposomes loaded with BMS-202.

[0065] In the specific implementation plan, DOPC:cholesterol:PEG 2000 The molar ratio range of BMS-202 is 1:(0.5-1.2):(0.1-0.5):(0.05-0.2).

[0066] In alternative embodiments, liposomes loaded with BMS-202 can also be prepared by other methods commonly used in the art, such as emulsification sonication.

[0067] In a specific implementation plan, the particle size of the liposomes loaded with BMS-202 is 90 nm to 300 nm.

[0068] In some implementations, the particle size of the prepared BMS-202-loaded liposomes is 180 nm to 220 nm.

[0069] For example, the particle size of the liposomes loaded with BMS-202 prepared is 90nm, 120nm, 140nm, 160nm, 180nm, 185nm, 190nm, 195nm, 200nm, 205nm, 210nm, 215nm, 220nm, 240nm, 260nm, 285nm or 300nm.

[0070] When the particle size of the liposomes prepared by this invention is within the above-mentioned range, R837 and BMS-202 can be released sequentially, resulting in better therapeutic effects.

[0071] The preparation method of the present invention includes the step of encapsulating BMS-202-loaded liposomes and R837 together into a Schiff base crosslinked hydrogel to obtain a nanocomposite sodium hyaluronate hydrogel.

[0072] BMS-202 is an effective PD-1 / PD-L1 complex inhibitor with CAS number 1675203-84-5. BMS-202 binds directly to PD-L1 and blocks the interaction between human PD-1 and PD-L1, thereby exhibiting anti-tumor activity.

[0073] R837, also known as imiquimod, is an immunomodulator with CAS number 99011-02-6. It can be used as an agonist of TLR7 (Toll-like receptor 7).

[0074] The inventors discovered in their research that the nanocomposite sodium hyaluronate hydrogel prepared by the method of this invention not only possesses the good mechanical strength, injectability, and dynamic stability required for breast tissue filling, but more importantly, as described above, it can also achieve the sequential release of R837 and BMS-202, resulting in better therapeutic effects.

[0075] As demonstrated in the examples below, when using common cross-linked sodium hyaluronate hydrogels containing both R837 and BMS-202 liposomes, sequential release of R837 and BMS-202 was not achieved. However, during the research, the inventors unexpectedly discovered that Schiff base cross-linked hydrogels prepared by cross-linking with 3.6%-4.4% oxidized sodium hyaluronate and 1.8%-2.2% sodium hyaluronate-adipic acid dihydrazide could achieve sequential release of the two drugs and had a good sustained-release effect (as demonstrated in Example 5).

[0076] The present invention further provides a nanocomposite sodium hyaluronate hydrogel prepared by the preparation method of the present invention.

[0077] The present invention further provides the application of the nanocomposite sodium hyaluronate hydrogel of the present invention in the preparation of breast filling and repair materials for preventing breast cancer recurrence.

[0078] This invention also provides the application of nanocomposite sodium hyaluronate hydrogel in the preparation of breast filling and repair materials for preventing breast cancer recurrence and treating postoperative breast defects.

[0079] In some embodiments, the time difference between the cumulative release of R837 and the cumulative release of BMS-202 in the nanocomposite sodium hyaluronate hydrogel of the present invention reaching 80% is greater than or equal to 8 days.

[0080] By creating the aforementioned time difference in the release of the two drugs, the efficacy of BMS-202, which is limited by M2 tumor-associated macrophage-dominated immunosuppression, can be reduced, maximizing its therapeutic effect and preventing breast cancer recurrence.

[0081] The following describes preferred embodiments of the present invention, but the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0082] Example 1: Preparation and characterization of oxidized sodium hyaluronate (OHA) and sodium hyaluronate-adipic acid dihydrazide (HA-ADH)

[0083] (1) Preparation of oxidized sodium hyaluronate (OHA):

[0084] Sodium hyaluronate was dissolved in deionized water, and sodium periodate was added to form a homogeneous reaction system. The reaction was carried out under light-protected conditions at a temperature of 25±2℃ with magnetic stirring for 24 hours. Then, diethylene glycol with an equimolar amount of sodium periodate was added to terminate the reaction. After stirring for another hour, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 Da. Dialysis was performed using flowing deionized water for 72 hours, with the dialysis medium being changed every 8 hours. After freeze-drying, the product oxidized sodium hyaluronate (OHA) was obtained and stored at -20℃ in the dark.

[0085] (2) Preparation of sodium hyaluronate-adipic acid dihydrazide (HA-ADH):

[0086] Sodium hyaluronate was dissolved in morpholine ethane sulfonate buffer (pH=6.5), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and hydroxybenzotriazole were added sequentially. The mixture was activated at room temperature for 1 hour. Adipate dihydrazide was added, and the reaction was maintained at 25±2℃ for 24 hours. After the reaction, the mixture was dialyzed with flowing deionized water for 96 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, with the dialysis medium changed every 8 hours. After freeze-drying, the product sodium hyaluronate-adipate dihydrazide (HA-ADH) was obtained and stored at -20℃ in the dark.

[0087] (3) Characterization of the product:

[0088] The product prepared in (1) was analyzed by proton nuclear magnetic resonance spectroscopy. 1 Qualitative detection was performed using 1H NMR and Fourier transform infrared spectroscopy (FTIR). The product prepared in (2) was analyzed using 1H NMR spectroscopy. 1 Qualitative detection was performed using H NMR.

[0089] The 1H NMR and IR spectra of oxidized sodium hyaluronate (OHA) are as follows: Figure 1 and Figure 2 As shown, it can be seen 1 New peaks appear at 4.6 ppm and 5.1 ppm in the ¹H NMR spectrum, and at 1726 cm⁻¹ in the infrared spectrum. -1 The presence of a characteristic aldehyde absorption peak confirms that sodium hyaluronate has been successfully oxidized to sodium oxyhyaluronate (OHA).

[0090] The 1H NMR spectrum of sodium hyaluronate-adipic dihydrazide (HA-ADH) is as follows: Figure 3As shown, it can be seen 1 The HNMR spectrum showed a characteristic peak of hydrazide at δ=2.2 ppm, which confirmed the successful modification to obtain sodium hyaluronate-adipic acid dihydrazide (HA-ADH).

[0091] Example 2: Preparation, performance testing and characterization of Schiff base crosslinked hydrogels

[0092] (1) Preparation of Schiff base crosslinked hydrogels:

[0093] The sodium oxidized hyaluronic acid (OHA) and sodium hyaluronate-adipate dihydrazide (HA-ADH) prepared in Example 1 were respectively prepared into solutions with different mass fractions using ultrapure water as solvent;

[0094] Four different mass fractions of oxidized sodium hyaluronate solution and different mass fractions of sodium hyaluronate-adipic acid dihydrazide solution were uniformly mixed at a volume ratio of 1:1 to obtain four different Schiff base crosslinked hydrogels. The composition of each group is as follows:

[0095] ① Gel1: 2%OHA, 2%HA-ADH;

[0096] ② Gel2: 2%OHA, 4%HA-ADH;

[0097] ③ Gel3: 4%OHA, 4%HA-ADH;

[0098] ④ Gel4: 4% OHA, 2% HA-ADH.

[0099] (2) Comparison of the properties of cross-linked hydrogels with different Schiff bases:

[0100] The mechanical properties of four different Schiff base crosslinked hydrogels were determined using a Harker Mars 40 rotational rheometer. Specifically, strain scanning was used to measure the elastic modulus G′ (i.e., storage modulus, the larger the value, the better the elasticity and the ability to store energy) and viscous modulus G″ (i.e., loss modulus, the larger the value, the better the viscosity, the greater the internal friction, the greater the energy loss, and the less likely the hydrogel is to return to its original shape after deformation under stress) of the hydrogels when a specific yield strain was reached. Furthermore, dynamic time scanning and dynamic frequency scanning were used to measure the changes in G′ and G″ of the four different Schiff base crosslinked hydrogels.

[0101] The test results of strain scanning are as follows Figure 4As shown, the elastic modulus G′ of the hydrogel decreases sharply when a specific yield strain is reached, indicating that the hydrogel has good injectability. The modulus measured in this region (usually the storage modulus G′) represents the initial structural strength of the material. When G′ remains stable, a higher G′ value indicates higher mechanical strength and greater hardness of the material. Therefore, it can be seen that Gel3 has the best mechanical strength, followed by Gel4, but Gel4 has a smaller G″ than Gel3, meaning that Gel4 is more likely to recover its original state after deformation under stress.

[0102] The test results for dynamic time scanning and dynamic frequency scanning are as follows: Figure 5 and Figure 6 As shown, the G′ and G″ of the four different Schiff base crosslinked hydrogels remained essentially unchanged, confirming the stability of the hydrogels.

[0103] The injectability of four different Schiff base crosslinked hydrogels was evaluated according to three levels: "excellent", "good" and "poor". The results are shown in Table 1 below.

[0104] Table 1

[0105] 2% OHA excellent 4% OHA excellent 2%HA-ADH good 4%HA-ADH Difference Gel1 good Gel2 Difference Gel3 Difference Gel4 good

[0106] Based on the mechanical strength and injectability results of the four different Schiff base crosslinked hydrogels, the Schiff base crosslinked hydrogel with a ratio of 4% OHA and 2% HA-ADH (Gel4) was selected for subsequent experiments.

[0107] (3) Self-healing ability of Schiff base crosslinked hydrogels:

[0108] The self-healing ability of Gel4 Schiff base crosslinked hydrogels was determined using a Hacker Mars40 rotational rheometer.

[0109] The self-healing ability test results are as follows: Figure 7 As shown, the strain transitions between 1% and 100% with a time interval of 1 minute. When the hydrogel is at 100% strain, G”>G’, and the hydrogel solid is destroyed; when the strain is restored to 1%, G’>G”, and G’ and G” remain stable, that is, the hydrogel can immediately restore its structure and remain stable, which indicates that the hydrogel of the present invention has strong self-healing ability.

[0110] (4) Characterization of Schiff base crosslinked hydrogels:

[0111] Its microstructure was imaged using a scanning electron microscope.

[0112] The microstructure of Gel4 was obtained by scanning electron microscopy, as shown in the image. Figure 8 As shown, it can be seen that it has a 3D network structure, which is a 3D cross-linked network of hydrogel, indicating that the hydrogel has been successfully cross-linked.

[0113] Example 3: Preparation and characterization of liposomes loaded with BMS-202

[0114] (1) Preparation of liposomes loaded with BMS-202:

[0115] (a) Oil phase preparation:

[0116] The following components were mixed in the specified proportions to form a homogeneous oil phase: 1 mL of DOPC solution at a concentration of 31 mg / mL; 1 mL of cholesterol solution at a concentration of 24 mg / mL; and 0.5 mL of PEG. 2000 A solution with a concentration of 24 mg / mL; 0.3 mL of BMS-202 solution with a concentration of 10 mg / mL;

[0117] (b) Aqueous phase dispersion:

[0118] The oil phase was transferred to a 1 mL syringe and slowly injected into 10 mL of ultrapure water at a constant rate of 0.5 mL / min to form an oil-water mixture.

[0119] (c) Solvent removal:

[0120] The oil-water mixture was heated and mechanically stirred at 50°C and 500 rpm for 4 hours to completely remove ethanol, ultimately obtaining a liposome suspension loaded with BMS-202.

[0121] (2) Characterization of liposomes loaded with BMS-202:

[0122] The prepared liposomes loaded with BMS-202 were imaged using transmission electron microscopy, and the particle size, PDI, and Zeta potential of the prepared liposomes loaded with BMS-202 were determined by dynamic light scattering.

[0123] Transmission electron microscopy image of liposomes as shown Figure 9 As shown in the figure, the liposomes are round or near-round vesicle structures, indicating that liposomes encapsulating BMS-202 were successfully obtained. Dynamic light scattering measurements are shown below. Figure 10 As shown in Figures A and B, the prepared liposomes have a particle size of 193.2 nm, a PDI of 0.1398, and a Zeta potential of 24.07 mV.

[0124] After demulsifying the liposomes with ethanol, the drug loading rate (DL) and encapsulation efficiency (EE) of the prepared liposomes were determined by high performance liquid chromatography. The test parameters are as follows:

[0125] The analytical column was a C18 column, the temperature was maintained at 40℃, the mobile phase was acetonitrile and 0.025M disodium hydrogen phosphate (pH adjusted to 3.8 with phosphoric acid) in a ratio of 60:40 (V:V); the detection wavelength was 230nm, the injection volume was 10μL, and the flow rate was 1mL / min.

[0126] Calculate the DL and EE of nanoparticles using the following formulas:

[0127] Drug loading rate (DL) = Mass of BMS-202 encapsulated / Total mass of all liposome components × 100%;

[0128] Encapsulation efficiency (EE) = Actual load of BMS-202 / Theoretical load of BMS-202 × 100%.

[0129] Ultimately, the encapsulation efficiency of the prepared liposomes was measured to be 85.50%, and the drug loading rate was 3.69%.

[0130] Example 4: Preparation and characterization of nanocomposite sodium hyaluronate hydrogel

[0131] (1) Preparation of nanocomposite sodium hyaluronate hydrogel:

[0132] Liposomes containing BMS-202 and R837 were co-encapsulated into Gel4 Schiff base crosslinked hydrogel to obtain nanocomposite sodium hyaluronate hydrogel.

[0133] (2) Characterization of nanocomposite sodium hyaluronate hydrogel:

[0134] The rheological properties of the nanocomposite sodium hyaluronate hydrogel were determined by a Harker Mars40 rotational rheometer using the same methods as in (2) and (3) of Example 2.

[0135] The test results of strain scanning are as follows Figure 11 As shown, the test results for dynamic time scanning and dynamic frequency scanning are respectively as follows: Figure 12 and Figure 13 As shown, the self-healing ability test results are as follows: Figure 14 As shown, the results of the nanocomposite sodium hyaluronate hydrogel are similar to those of the uncoated Schiff base crosslinked hydrogel.

[0136] Example 5: In vitro drug release experiment of nanocomposite sodium hyaluronate hydrogel

[0137] 150 μL of the nanocomposite sodium hyaluronate hydrogel prepared in Example 4 was placed into a 200-mesh nylon mesh bag. The nylon mesh bag was then transferred to a 5 mL glass bottle, and 2 mL of PBS (pH 6.5, with or without hyaluronidase) was added as the release medium. The bottle was then transferred to a constant-temperature shaker at 37°C and 100 rpm for the release test. At regular intervals, 0.2 mL of the release medium was taken out and quantitatively detected by high-performance liquid chromatography (HPLC), while an equal amount of fresh release medium was added. The cumulative release (CR) of R837 and BMS-202 at different time points was calculated according to Formula I.

[0138] (I)

[0139] Where C i denoted as i, where i is the mass concentration of R837 or BMS-202 in the release medium at the end of time interval i; V0 is the initial volume of the release medium; V is the volume of the release medium at which the concentration of R837 or BMS-202 is measured during each extraction; and m is the initial mass of R837 or BMS-202 in the dialysis bag.

[0140] Sampling was performed at intervals until the basic release of R837 and BMS-202 was completed, and the cumulative release over time was plotted.

[0141] Meanwhile, a sodium hyaluronate hydrogel cross-linked with 1,4-butanediol diglycidyl ether (BDDE) and liposomes co-encapsulated with R837 and BMS-202 was used as a control group. The hydrogel was prepared by reconstituted and lyophilized BDDE-cross-linked sodium hyaluronate hydrogel with R837 and BMS-202-encapsulated liposomes in aqueous solution. Cumulative release tests were performed using the same method, and curves showing cumulative release over time were plotted.

[0142] The release curve of the nanocomposite sodium hyaluronate hydrogel of the present invention is as follows: Figure 15 As shown, the release curve of the control group is as follows: Figure 16 As shown, it can be seen that the release trends of the two drugs in the BDDE cross-linked sodium hyaluronate hydrogel of the control group are almost the same, and sequential release cannot be achieved, and the release tends to slow down after about 10 days. In contrast, in the nanocomposite sodium hyaluronate hydrogel of the present invention, R837 is released before BMS-202. The time difference between the cumulative release of R837 reaching 80% and the cumulative release of BMS-202 reaching 80% is about 10 days. It can be seen that the nanocomposite sodium hyaluronate hydrogel of the present invention can achieve sequential release of the two drugs and has a good sustained-release effect.

[0143] This document uses specific embodiments to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and central idea of ​​the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall under the protection of the claims of the present invention.

Claims

1. A method for preparing a nanocomposite sodium hyaluronate hydrogel, characterized in that, Includes the following steps: Preparation of oxidized sodium hyaluronate; Preparation of sodium hyaluronate-adipic dihydrazide; A 3.6%-4.4% sodium oxidized hyaluronic acid solution and a 1.8%-2.2% sodium hyaluronic acid-adipic acid dihydrazide solution were mixed at a 1:1 ratio to obtain a Schiff base crosslinked hydrogel. Liposomes loaded with BMS-202 were prepared. The particle size of the BMS-202-loaded liposomes was 90 nm to 300 nm. The BMS-202-loaded liposomes were prepared by an ethanol injection method, specifically including: [the following steps are described in the original text, which is incomplete and requires further context to translate accurately.] 2000 The oil phase of BMS-202 was injected into ultrapure water at a constant rate of 0.1 mL / min to 0.5 mL / min using a syringe, and stirred continuously at 300 rpm to 700 rpm for 1 to 4 hours at 40℃-60℃. The liposomes containing BMS-202 and R837 were co-encapsulated into the Schiff base crosslinked hydrogel to obtain the nanocomposite sodium hyaluronate hydrogel.

2. The preparation method according to claim 1, characterized in that, Mix 3.8%-4.2% sodium oxidized hyaluronic acid solution with 1.9%-2.1% sodium hyaluronic acid-adipic acid dihydrazide solution at a 1:1 ratio.

3. The preparation method according to claim 1, characterized in that, Mix 4% sodium oxidized hyaluronic acid solution with 2% sodium hyaluronic acid-adipic acid dihydrazide solution at a 1:1 ratio.

4. The preparation method according to claim 1, characterized in that, The sodium hyaluronate-adipic acid dihydrazide is prepared by the following method: sodium hyaluronate is activated with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and hydroxybenzotriazole in morpholine ethane sulfonic acid buffer, and then reacted with adipic acid dihydrazide. After purification, the mixture is dried.

5. A nanocomposite sodium hyaluronate hydrogel, characterized in that, The nanocomposite sodium hyaluronate hydrogel is prepared by the preparation method according to any one of claims 1 to 4.

6. The application of nanocomposite sodium hyaluronate hydrogel in the preparation of breast filling and repair materials for preventing breast cancer recurrence, characterized in that, The nanocomposite sodium hyaluronate hydrogel is prepared by the preparation method according to any one of claims 1 to 4; The time difference between the cumulative release of R837 and the cumulative release of BMS-202 in the nanocomposite sodium hyaluronate hydrogel reaching 80% is greater than or equal to 8 days.

7. The application of nanocomposite sodium hyaluronate hydrogel in the preparation of breast filling and repair materials for preventing breast cancer recurrence and treating postoperative breast defects, characterized in that, The nanocomposite sodium hyaluronate hydrogel is prepared by the preparation method according to any one of claims 1 to 4.

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