Nano-composite hydrogel for radiosensitization after breast-conserving surgery for breast cancer and preparation method therefor

Abstract

A nano-composite hydrogel for radiosensitization after breast-conserving surgery for breast cancer and a preparation method therefor. The nano-composite hydrogel comprises a hydrogel loaded with mesoporus gold nanoparticles M@AuNPs. The preparation method is as follows: The starting materials include a modified chitosan solution, a modified hyaluronic acid solution, and M@AuNPs. M@AuNPs are dispersed in the modified hyaluronic acid solution and the modified chitosan solution is isovolumetrically mixed with the modified hyaluronic acid solution to give the hydrogel. An injectable hydrogel encapsulating nanoparticles. The injectable hydrogel has a structure uniformly encapsulating outer-layer modified gold nanoparticles, and is capable of implementing immunotherapy by means of the targeted sustained-release of therapeutic drugs and meanwhile achieving ray energy accumulation due to the higher atomic number of the gold nanoparticles, thereby effectively killing tumor cells at the tumor bed position. The strategy effectively reduces the radiation dose in the whole breast radiotherapy, and is beneficial to protecting normal tissues of the breast outside the tumor bed.

Description

A nanocomposite hydrogel for radiotherapy sensitization after breast-conserving surgery for breast cancer and its preparation method Technical Field

[0001] The present invention belongs to the field of biomedical polymer materials, and in particular relates to a nanocomposite hydrogel suitable for injectable radiotherapy localization sensitization after breast-conserving surgery for breast cancer and a preparation method thereof. Background Art

[0002] Breast cancer refers to a malignant tumor originating in the breast. According to data released by the World Health Organization's International Agency for Research on Cancer in 2023, breast cancer has replaced lung cancer as the world's leading cancer. In my country, there are approximately 420,000 new cases of breast cancer each year, with an incidence rate of approximately 7.7%, making it the most common malignant tumor. Breast-conserving surgery is relatively effective for early-stage breast cancer, often achieving a cure rate of over 90% while preserving the appearance of the breast. The "Guidelines for the Diagnosis and Treatment of Breast Cancer" (2018 edition) issued by the National Health Commission of China states that, in principle, all patients undergoing breast-conserving surgery require radiotherapy. Preventive radiotherapy and, if necessary, adjuvant chemotherapy are required after breast-conserving surgery to control the progression of the disease and prevent local recurrence and distant metastasis.

[0003] Currently, post-breast-conserving surgery radiotherapy consists of whole-breast irradiation plus boosted irradiation of the tumor bed or accelerated partial breast irradiation targeting the tumor bed alone. Currently, the most common method for determining the irradiation range of the tumor bed is to place a lead wire in the post-operative skin scar. This approach uses the size of the scar to determine the tumor bed's extent. However, the actual location of the tumor differs significantly from the post-operative scar, making it difficult to clearly define the accelerated irradiation zone in the tumor bed. This makes it difficult for normal tissue to avoid radiotherapy radiation. Because radiotherapy radiation is harmful to the human body, reducing the overall radiation dose can minimize damage to normal tissue.

[0004] Summary of the Invention

[0005] In response to the existing problem in the art that radiotherapy radiation after breast-conserving surgery for breast cancer poses a potential risk to normal tissue, the present invention aims to provide a radiotherapy sensitizing hydrogel with excellent biocompatibility and a preparation method, which is specifically achieved through the following technical solutions:

[0006] A nanocomposite hydrogel for radiotherapy sensitization after breast-conserving surgery for breast cancer, comprising a hydrogel loaded with mesoporous gold nanoparticles M@AuNPs.

[0007] A method for preparing a nanocomposite hydrogel for radiosensitization after breast-conserving surgery for breast cancer, wherein the raw materials include a modified chitosan solution, a modified hyaluronic acid solution and M@AuNPs;

[0008] The method comprises dispersing M@AuNPs in a modified hyaluronic acid solution at a material-liquid ratio of 1 mg:5 mL, and then uniformly mixing the modified chitosan solution and the modified hyaluronic acid solution in equal amounts to prepare a hydrogel.

[0009] The present invention uses natural biological polysaccharides as raw materials, which have the advantages of excellent biocompatibility, low price and easy availability. At the same time, the rich hydroxyl and amino groups in its structure can meet the Schiff base reaction conditions through simple modification. The resulting hydrogel can quickly form gel in situ and has a certain self-repair ability, which better meets the actual situation that the target position is easily squeezed.

[0010] Furthermore, the above method specifically includes the following steps:

[0011] 1) Modified chitosan powder was prepared into a 5 wt% A solution using sterile PBS solution;

[0012] 2) preparing the modified hyaluronic acid powder into a 10 wt% B solution using sterile PBS solution;

[0013] 3) Disperse M@AuNPs in solution B at a material-liquid ratio of 1 mg:5 mL;

[0014] 4) Mix equal amounts of Solution A and Solution B in a vial or use a syringe to mix them evenly into a gel.

[0015] Chitosan Chitosan is the product of removing some acetyl groups from the natural polysaccharide chitin. It has multiple physiological functions such as biodegradability, biocompatibility, non-toxicity, antibacterial properties, and immune enhancement. It is widely used in biomedical fields such as drug delivery.

[0016] Furthermore, the chitosan is selected from any one of carboxymethyl chitosan, hydroxyethyl chitosan, carboxyethyl chitosan and N-succinyl chitosan.

[0017] Furthermore, the modified chitosan is prepared according to the following method:

[0018] A certain amount of chitosan is weighed and dissolved in a 1-3% acetic acid solution, and then 300-600 mL of methanol is added and mechanically stirred; a certain amount of succinic anhydride is dissolved in acetone to prepare a succinic anhydride solution; the chitosan solution is dropped into the succinic anhydride mixture and allowed to stand overnight; the mixture is filtered, washed with ethanol, and filtered again to obtain a solid product; the solid product is dissolved in water, the solution pH is adjusted to 8-9, and then washed with excess acetone and ethanol, filtered, and freeze-dried to obtain a product.

[0019] Hyaluronic acid, a linear mucopolysaccharide, is widely distributed in the human body, plays an important physiological role, and is widely used in medical cosmetology and other aspects.

[0020] Furthermore, the modified hyaluronic acid is aldehyde-modified hyaluronic acid, which is specifically prepared by the following method: dissolving hyaluronic acid in water and adjusting the pH of the solution to 4-5; adding EDCI and stirring to dissolve it, then adding a propylene glycol hydrazide derivative and stirring and reacting at 20-60° C. for 1-28 hours; adjusting the pH of the reaction solution to 6-8 to terminate the reaction; dialyzing the reaction solution, adding sodium periodate solution, and stirring and reacting in the dark at 30-40° C. for 4-8 hours, adding ethylene glycol and stirring for 0.2-0.8 hours to terminate the reaction, and dialyzing and freeze-drying to obtain the product.

[0021] Gold nanoparticles have a higher atomic number and X-ray photon capture cross section, and can effectively aggregate into tumor tissue through passive targeting. At the same time, gold nanoparticles can be modified with an outer layer to simultaneously deliver drugs and deposit radiotherapy radiation energy.

[0022] Furthermore, M@AuNPs were prepared by the following method:

[0023] 1) Sodium citrate reduction: Pour 5-40 mg / mL chloroauric acid solution and 10-80 mg / mL sodium citrate aqueous solution into 50-150 mL of 90-100°C ultrapure water and stir for 10-80 min to obtain a gold nanoparticle AuNPs solution;

[0024] 2) Surface modification: The CTAB template method was used for preparation. The AuNPs solution was poured into 5-20 mL of a prepared 0.1-0.8 mol / L CTAB solution, and then 0.2-1.0 mL of tetraethyl silicate was added dropwise for 6-24 h. After washing, centrifugation, and drying, mesoporous gold nanoparticles M@AuNPs were obtained.

[0025] The injectable hydrogel obtained by the present invention uses a self-designed double syringe for in-situ gelation during gelation. The syringe has the advantage of adjustable component ratio and can fully mix the components, forming gel uniformly and effectively while uniformly encapsulating gold nanoparticles.

[0026] The hydrogel base of the present invention is selected from natural biopolysaccharides, which are cheap and easily available. After simple modification, they can meet the Schiff base reaction conditions and can quickly generate gel in situ at the injection site; the hydrogel relies on imine dynamic bond formation to have a certain self-repair ability, ensuring that the injection site can still recover certain mechanical properties after being squeezed; the hydrogel is evenly loaded with outer-layer modified gold nanoparticles, which can achieve radiotherapy sensitization while realizing targeted and controlled drug release, and can implement radiotherapy after breast-conserving surgery at a lower dose, effectively protecting normal breast tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG1 is a nanocomposite hydrogel obtained in Example 1 and its microscopic morphology (SEM);

[0028] FIG2 is a drug release curve of the hydrogel obtained in Example 2 in solutions with different pH values;

[0029] FIG3 is a fluorescence microscope photograph of tumor cells in the hydrogel-treated group obtained in Example 3 after staining with a reactive oxygen species (ROS) kit. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0031] The preparation scheme of N-succinyl chitosan is as follows: 2.0g of chitosan is accurately weighed into a three-necked flask, added to 80mL of acetic acid solution and mechanically stirred to dissolve, and after dissolution, 320mL of methanol is added and mechanical stirring is continued; 2.5g of succinic anhydride is dissolved in 25mL of acetone solution to prepare a succinic anhydride mixture, and the succinic anhydride mixture is added dropwise to the chitosan solution under intense mechanical stirring after 30 minutes of dropwise addition, and the reaction is continued at room temperature for 8 hours; the emulsion is filtered with a vacuum pump, and the reaction product is immersed in ethanol and filtered again, and washed with acetone and ethanol to obtain a crude product; the crude product is completely dissolved in water, and the solution is adjusted to pH = 9 with 5% NaOH solution, dialyzed, and freeze-dried to obtain the product.

[0032] The preparation method of aldehyde-modified hyaluronic acid is as follows: 2 g of hyaluronic acid (sodium) is accurately weighed and dissolved in 200 mL of water, the pH of the resulting solution is adjusted to 4.5, and then 4 g of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) is added and stirred for 20 minutes to dissolve it, followed by adding 8 g of propylene glycol hydrazide derivative and stirring and reacting at room temperature for 3 hours, adjusting the pH of the reaction solution to 7.0, dialyzing the reaction solution and freeze-drying to obtain propylene glycol-modified hyaluronic acid (sodium); 2 g of propylene glycol-modified hyaluronic acid (sodium) is added to 200 mL of water, and then 40 mL of 10 wt% sodium periodate aqueous solution is added, stirring and reacting for 12 hours at room temperature in the dark, and then 5 mL of ethylene glycol is added dropwise and stirred for 1 hour to terminate the reaction. The reaction solution is dialyzed using a dialysis bag (3500 Da) for 48 hours and freeze-dried to obtain aldehyde-modified hyaluronic acid (sodium).

[0033] The preparation scheme of mesoporous gold nanoparticles is as follows: prepare 20.0 mg / mL chloroauric acid solution and 50.0 mg / mL sodium citrate solution, add 1 mL of chloroauric acid solution and 2 mL of sodium citrate solution to boiling ultrapure water, react for 1 hour, and then cool to obtain gold nanoparticle (AuNPs) solution; then pour the AuNPs solution into 8 mL of prepared 0.4 mol / L CTAB solution, and then add 0.2 mL of tetraethyl silicate and react for 24 hours. After washing, centrifugation, and drying, mesoporous gold nanoparticles (M@AuNPs) are obtained.

[0034] Example 1: Preparation of hydrogel system

[0035] N-succinyl chitosan powder was prepared into 5 wt% solution A using sterile PBS solution, and aldehyde-modified hyaluronic acid powder was prepared into 10 wt% solution B using sterile PBS solution. 1 mg of M@AuNPs was dispersed in the aldehyde-modified hyaluronic acid solution. 5 mL of each of solution A and B was evenly mixed into a gel in a vial or using a syringe.

[0036] The morphology (SEM) of the hydrogel prepared in this example is shown in Figure 1. It can be seen that after freeze-drying, the gel formed by N-succinyl chitosan / aldehyded hyaluronic acid-aldehyded iodixanol presents a good continuous, tight, porous network structure inside the gel, and the gelation is relatively uniform. M@AuNPs are evenly distributed in the hydrogel, indicating that M@AuNPs have been successfully loaded into the hydrogel.

[0037] Example 2: In vitro drug release test of nanocomposite hydrogel

[0038] The drug absorbance in the release solutions at different time intervals was measured using a UV spectrophotometer. N-succinyl chitosan powder was prepared into a 5wt% solution A using sterile PBS, and aldehyde-modified hyaluronic acid powder was prepared into a 10wt% solution B using sterile PBS. 1mg of M@AuNPs was dispersed in the aldehyde-modified hyaluronic acid solution. Solutions A and B were mixed uniformly in a vial or syringe to form a gel. The nanocomposite hydrogels were then transferred to dialysis bags and immersed in pH 5.7 or 7.4 solutions, respectively. In vitro drug release experiments were performed on a shaker at 37°C.

[0039] The drug release curve obtained from the test in this example is shown in Figure 2. As can be seen, after 6 hours of rapid release in the early stage, the release behavior gradually stabilizes, indicating a slow release of the drug. In addition, the drug has a faster release rate in an acidic environment, confirming the targeted drug delivery and sustained release characteristics of the hydrogel of the present invention.

[0040] Example 3: Testing of Hydrogel Radiotherapy Sensitization Effect

[0041] The killing of tumors by radiotherapy rays is mediated by the decomposition of intracellular water into ROS after irradiation. A ROS detection kit was used to measure the ROS levels of tumor cells to verify the radiosensitization effect of the nanocomposite hydrogel of the present invention. Huh-7 cells were seeded into 6-well plates and cultured for 24 hours. The control group received the original culture medium, while the experimental group received the culture medium containing the hydrogel described in Example 2. After another 24 hours of culture, the cells were irradiated with 5 Gy of radiation. Fluorescence staining using a reactive oxygen species detection kit was then performed to compare the reactive oxygen species levels in the experimental and control groups.

[0042] Figure 3 shows fluorescence microscopic images of tumor cells before and after hydrogel treatment using a ROS kit. The M@AuNPs exhibited higher fluorescence brightness than the control group, confirming that low-dose irradiation with M@AuNPs can induce tumor cells to produce more ROS, thereby enhancing their ability to kill tumor cells.

[0043] This invention innovatively proposes a new strategy for comprehensive treatment after breast-conserving surgery for breast cancer. The design and preparation of an injectable hydrogel encapsulated with nanoparticles uniformly encapsulates a modified outer layer of gold nanoparticles, enabling the slow, targeted release of therapeutic drugs for immunotherapy. Furthermore, due to the higher atomic number of the gold nanoparticles, radiation energy is deposited, effectively killing tumor cells in the tumor bed. This strategy effectively reduces the radiation dose of whole-breast radiotherapy, protecting normal breast tissue outside the tumor bed.

Claims

1. A nanocomposite hydrogel for radiosensitization after breast-conserving surgery for breast cancer, characterized in that, It includes a hydrogel loaded with mesoporous gold nanoparticles M@AuNPs.

2. A method for preparing the nano-composite hydrogel for radiosensitization after breast-conserving surgery for breast cancer as described in claim 1, characterized in that, The raw materials include a modified chitosan solution, a modified hyaluronic acid solution and M@AuNPs; The method includes dispersing M@AuNPs in the modified hyaluronic acid solution according to the ratio of 1 mg: 5 mL of the material-liquid ratio, and then evenly mixing the modified chitosan solution and the modified hyaluronic acid solution in equal amounts to obtain the hydrogel.

3. The method according to claim 2, wherein Specifically, it includes the following steps: 1) Dissolve the modified chitosan powder in a sterile PBS solution to prepare a 5 wt% A solution; 2) Dissolve the modified hyaluronic acid powder in a sterile PBS solution to prepare a 10 wt% B solution; 3) Disperse M@AuNPs in the B solution according to the ratio of 1 mg: 5 mL of the material-liquid ratio; 4) Evenly mix an equal amount of the A solution and the B solution in a vial to form a gel or use a syringe to mix them evenly to form a gel.

4. The method according to claim 2 or 3, characterized in that The chitosan is selected from any one of carboxymethyl chitosan, hydroxyethyl chitosan, carboxyethyl chitosan, N-succinyl chitosan.

5. The method according to claim 2 or 3, characterized in that The modified chitosan is prepared according to the following method: Weigh a certain amount of chitosan, dissolve it in a 1-3% acetic acid solution, then add 300-600 mL of methanol and carry out mechanical stirring; dissolve a certain amount of succinic anhydride in acetone to prepare a succinic anhydride solution; drop the chitosan solution into the succinic anhydride mixture and let it stand overnight; filter the mixture, wash it with ethanol and filter it again to obtain a solid product; dissolve the solid product in water, adjust the pH of the solution to 8-9, then wash it with excessive acetone and ethanol, and then filter it and freeze-dry it to obtain the product.

6. The method according to claim 2 or 3, characterized in that The modified hyaluronic acid is aldehyde-functionalized hyaluronic acid, and it is specifically prepared according to the following method: dissolve hyaluronic acid in water and adjust the pH of the solution to 4-5; add EDCI and stir to dissolve it, then add propylene glycol hydrazide derivative and stir at 20-60 °C for 1-28 h; adjust the pH of the reaction solution to 6-8 to terminate the reaction; dialyze the reaction solution, then add sodium periodate solution and stir at 30-40 °C in the dark for 4-8 h, add ethylene glycol and stir for 0.2-0.8 h to terminate the reaction, and then dialyze and freeze-dry it to obtain the product.

7. The method according to claim 2 or 3, characterized in that M@AuNPs are prepared by the following method: 1) Sodium citrate reduction: Pour a 5-40 mg / mL chloroauric acid solution and a 10-80 mg / mL sodium citrate aqueous solution into 50-150 mL of ultrapure water at 90-100 °C and stir and react for 10-80 min to obtain a gold nanoparticle AuNPs solution; 2) Surface modification: Prepare it by the CTAB template method. Pour the AuNPs solution into a prepared 5-20 mL 0.1-0.8 mol / L CTAB solution, then add 0.2-1.0 mL of tetraethyl orthosilicate and react for 6-24 h. After washing, centrifuging and drying, mesoporous gold nanoparticles M@AuNPs are obtained.

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