Composite porous scaffold, composite porous drug delivery system and application

Abstract

The application belongs to the field of drug delivery and particularly relates to a composite porous stent, a composite porous drug delivery system and application. The composite porous stent comprises crosslinked oxidized hyaluronic acid, methacrylated xanthan gum, gelatin and sodium alginate; the composite porous drug delivery system is constructed by loading hyaluronic acid-coated quercetin and ginsenoside Rh2 nanoparticles on the composite porous stent, and can precisely and targetedly deliver the loaded traditional Chinese medicine components to a lesion site, so as to achieve the purpose of high-efficiency, safe and targeted treatment of breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery, specifically relating to a composite porous scaffold, a composite porous drug delivery system, and its applications. Background Technology

[0002] Breast cancer is one of the most common malignant tumors among women worldwide. According to the latest statistics from the International Agency for Research on Cancer (IARC), as of 2022, breast cancer has become the leading cause of cancer-related death (23.8%) and mortality (15.4%) among women. Therefore, the prevention and treatment of breast cancer has become a major challenge in global public health. Currently, numerous studies have shown that traditional Chinese medicine (TCM) can effectively treat cancer, and that TCM has minimal irritation to the gastrointestinal tract, liver, and kidneys, and low toxicity to normal organs, allowing for long-term conditioning of the body. Quercetin (Que) is a typical antioxidant dietary flavonoid with antioxidant, antiviral, antitumor, and anti-inflammatory biological activities. It can participate in the prevention and treatment of breast cancer by regulating multiple signal transduction pathways. Ginsenosides (Gin) are tetracyclic triterpenoid monomers extracted from ginseng, a traditional Chinese medicine. They possess synergistic effects in detoxification, enhancing immunity, and inhibiting tumor cell proliferation. Numerous studies have shown that ginsenoside Rh2 (GRh2) can effectively treat tumors. Compared to the various side effects of radiotherapy and chemotherapy for tumors, Que and GRh2 are safer and more effective. Combining them may have a synergistic effect in reducing postoperative complications of breast cancer, promoting wound healing, and preventing tumor recurrence. However, Que and GRh2 have poor water solubility and low bioavailability, making them unsuitable for injection administration.

[0003] In recent years, drug delivery systems based on nanocarriers have attracted widespread attention in cancer treatment. Compared with carrier-free anticancer drugs, nanocarrier drug delivery systems can improve the solubility and biocompatibility of chemotherapeutic drugs and achieve cell internalization and tumor accumulation by enhancing retention effects. Therefore, it is crucial to develop a novel nano-drug delivery system based on a tissue-engineered composite porous scaffold model. This system should possess good mechanical properties and biocompatibility, and be able to simultaneously load HA@QueNPs and HA@GRh2 NPs. This nano-drug delivery system can maintain the drug at the tumor site, prolong drug release, and precisely and safely deliver the drug to the tumor lesion, thereby achieving an effective anti-tumor effect. As one of the three essential elements of tissue engineering, the selection of bioporous scaffold materials is also crucial. Summary of the Invention

[0004] To address the aforementioned technical challenges, this invention utilizes modified materials such as XaGMA-OHA to construct a composite porous scaffold drug delivery system loaded with HA@Que nanoparticles and HA@GRh2 nanoparticles. This system is designed for the efficient, targeted, and low-toxicity treatment of breast cancer, providing a novel approach for the preparation of nano-sized traditional Chinese medicine particles for breast cancer treatment.

[0005] A first aspect of the present invention provides a composite porous scaffold comprising xanthan gum methacrylated (XaGMA), oxidized hyaluronic acid (OHA), gelatin, and sodium alginate (SA), which are mixed and crosslinked to form the composite porous scaffold. The mass ratio of xanthan gum methacrylated (XaGMA), oxidized hyaluronic acid (OHA), gelatin, and sodium alginate (SA) is 0.125~0.5:0.25~0.75:1~2:1~2. In some embodiments, the mass ratio is 0.25:0.25:1.5:1.5, 0.25:0.25:1:1.5, or 0.25:0.25:1:2.

[0006] In some implementations, the crosslinking is performed using a three-stage crosslinking method, sequentially using CaCl2, an EDC / NHS / MES system, and photoinitiator I2959.

[0007] In some embodiments, the composite porous scaffold is prepared using the following steps: (A1) Dissolve and mix 0.125–0.5 w / v% of methacrylamide xanthan gum, 0.25–0.75 w / v% of oxidized hyaluronic acid, 1–2 w / v% of gelatin, and 1–2 w / v% of sodium alginate in water, and dry (e.g., freeze-dry) to obtain a composite scaffold. (A2) The composite scaffold was first cross-linked with 1-2 wt% CaCl2 solution for 1-6 h, then cross-linked a second time with a composite cross-linking agent solution of 20-200 mmol / L EDC, 20-200 mmol / L NHS and 20-200 mmol / L MES for 1-24 h, and finally cross-linked a third time with 0.1-1% photoinitiator I2959 for 10-120 s under UV light. After washing, it was dried (e.g., freeze-dried) to obtain the composite porous scaffold.

[0008] In some embodiments, the methacrylamide xanthan gum is synthesized by xanthan gum and methacrylic anhydride.

[0009] In some embodiments, the oxidized hyaluronic acid is synthesized by oxidizing hyaluronic acid with an oxidizing agent (such as sodium periodate, hydrogen peroxide, etc.).

[0010] A second aspect of the present invention also provides a composite porous drug delivery system, comprising: (a) the composite porous scaffold described in the first aspect, and (b) the composite porous scaffold loaded with hyaluronic acid-coated quercetin nanoparticles (hereinafter referred to as "HA@Que NPs"), and / or loaded with hyaluronic acid-coated ginsenoside Rh2 nanoparticles (hereinafter referred to as "HA@GRh2 NPs").

[0011] In some embodiments, the composite porous drug delivery system is prepared using the following steps: (B1) Hyaluronic acid-coated quercetin nanoparticles are prepared by adding a 0.5-2 w / v% aqueous solution of hyaluronic acid containing 0.1-2 w / v% EDC·HCl dropwise to a 0.1-1 w / v% organic solution of quercetin; and / or, hyaluronic acid-coated ginsenoside Rh2 nanoparticles are prepared by adding a 0.1-1 w / v% aqueous solution of hyaluronic acid containing 0.1-2 w / v% EDC·HCl dropwise to a 1-3 w / v% organic solution of ginsenoside Rh2. (B2) Dissolve and mix 0.125~0.5 w / v% of methacrylamide xanthan gum, 0.25~0.75 w / v% of oxidized hyaluronic acid, 1~2 w / v% of gelatin, and 1~2 w / v% of sodium alginate in water. Then add hyaluronic acid-coated quercetin nanoparticles and / or hyaluronic acid-coated ginsenoside Rh2 nanoparticles, mix well, and dry (e.g., freeze-dry) to obtain a drug-loaded composite scaffold. (B3) The drug-loaded composite scaffold was first cross-linked with 1-2 wt% CaCl2 solution for 1-6 h, then cross-linked a second time with a composite cross-linking agent solution of 20-200 mmol / L EDC, 20-200 mmol / L NHS and 20-200 mmol / L MES for 1-24 h, and finally cross-linked a third time with 0.1-1% photoinitiator I2959 for 10-120 s under UV light. After washing, it was dried (e.g., freeze-dried) to obtain the composite porous drug delivery system.

[0012] In some embodiments, during the preparation of hyaluronic acid-coated quercetin nanoparticles, the mass ratio of hyaluronic acid to quercetin is 1:5 to 5:1.

[0013] In some embodiments, during the preparation of hyaluronic acid-coated ginsenoside Rh2 nanoparticles, the mass ratio of hyaluronic acid to ginsenoside Rh2 is 1:5 to 5:1.

[0014] In some embodiments, the composite porous drug delivery system contains quercetin at a content of 20-70 μg / mL and ginsenoside Rh2 at a content of 5-40 μg / mL.

[0015] A third aspect of the invention also provides the application of the composite porous scaffold in the preparation of composite porous drug-loaded systems.

[0016] A fourth aspect of the invention also provides the application of the composite porous drug-loaded system in the preparation of preventive or antitumor drugs.

[0017] In some implementations, the tumor is selected from breast cancer, liver cancer, stomach cancer, hematologic malignancy, lymphoma, etc., with breast cancer being preferred.

[0018] Compared with the prior art, the beneficial effects of the present invention include at least the following: 1. This invention uses methacrylated xanthan gum, oxidized hyaluronic acid, gelatin, and sodium alginate to prepare a composite porous scaffold through three cross-linking processes. It has excellent biocompatibility and can realistically simulate the in vitro breast cancer microenvironment. At the same time, it has excellent porosity, water absorption rate, and hydrophilicity, providing good material exchange and growth space for subsequent cell growth and proliferation on the composite porous scaffold.

[0019] 2. This invention uses a composite porous scaffold loaded with HA@Que NPs and HA@GRh2 NPs to prepare a composite porous drug delivery system, which can significantly inhibit the proliferation of MDA-MB-231 and MCF-7 cells, destroy the internal structure of breast cancer cell clusters, effectively kill tumor cells, and induce apoptosis of MDA-MB-231 and MCF-7 cells, thereby achieving the purpose of treating breast cancer. Attached Figure Description

[0020] Figure 1 The microstructure, pore size, and mechanical properties of the XaGMA / OHA / Gel / SA composite porous scaffolds are shown. (A) Microstructure of the nine composite porous scaffolds; (B) Pore size of the nine composite porous scaffolds; (C) Mechanical properties of the nine composite porous scaffolds. NS: p> 0.05, *** p <0.001.

[0021] Figure 2 The physicochemical properties of the XaGMA / OHA / Gel / SA composite porous scaffolds are shown. (A) FTIR of the three composite porous scaffolds and their components; (B) Contact angle of the three composite porous scaffolds; (C) Water absorption rate of the three composite porous scaffolds; (D) Porosity of the three composite porous scaffolds; (EF) Swelling rate of the three composite porous scaffolds; (G) Hemolysis rate of the three composite porous scaffolds; (H) Radar plot of the physical properties of the three composite porous scaffolds. NS: p >0.05, * p <0.05, *** p <0.001.

[0022] Figure 3The survival of MDA-MB-231 and MCF-7 cells after 3 days of culture on a composite porous scaffold is shown.

[0023] Figure 4 The proliferation of MDA-MB-231 and MCF-7 cells on the composite porous scaffold was shown. (AB) The proliferation of MDA-MB-231 and MCF-7 cells on the composite porous scaffold was examined using the CCK-8 assay, respectively; (CD) The growth and proliferation of MDA-MB-231 and MCF-7 cells on the composite porous scaffold were examined using laser confocal microscopy, respectively.

[0024] Figure 5 The comparison between HA@Que NPs and HA@GRh2 NPs is shown. 1 H NMR spectrum.

[0025] Figure 6 The morphology and particle size characterization of HA@Que NPs and HA@GRh2 NPs are shown. (AB) TEM morphology characteristics of HA@Que NPs and HA@GRh2 NPs; (CD) Particle size of HA@Que NPs and HA@GRh2 NPs; (EF) Potential of HA@Que NPs and HA@GRh2 NPs.

[0026] Figure 7 The SEM microstructure of each nano-drug delivery system is shown.

[0027] Figure 8 The effects of HA@Que NPs and HA@GRh2 NPs on the morphology of MDA-MB-231 (A) and MCF-7 (B) cells were investigated using SEM; the effects of HA@Que NPs and HA@GRh2 NPs on the survival of MDA-MB-231 (C) and MCF-7 (D) cells were investigated using the CCK-8 assay.

[0028] Figure 9 The study showed the killing effects of different drug delivery systems on MDA-MB-231(A) and MCF-7(B) cells for 5 days. Detailed Implementation

[0029] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0030] The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, which should be understood to include those close to them.

[0031] Example 1: Preparation and characterization of XaGMA and OHA (1) Preparation of XaGMA 5.5 g of EDC and 2.1 g of NHS were weighed and completely dissolved in 20 mL of DMSO at 40 °C. 2 mL of MAA was then added to this solution, and the mixture was stirred in the dark for 12 h. Simultaneously, 1 g of XaG was weighed and completely dissolved in 100 mL of 0.25 M PBS (pH 9.0) at 60 °C. Then, at 4 °C, the prepared MAA solution was added to the XaG solution in four 5 mL portions every 30 min, maintaining the pH of the XaG solution at 9.0 after each addition of MAA solution using 10× NaOH. The reaction was then allowed to proceed overnight at 4 °C, followed by the addition of 100 mL of 0.25 M PBS (pH 9.0) to terminate the reaction. Finally, the resulting product was placed in a dialysis bag (with a molecular weight cutoff of 8000-10000 Da) and dialyzed in ddH2O at a constant temperature of 37 °C for 5 days. After dialysis, dried XaGMA was obtained by freeze-drying for subsequent experiments. Modified XaGMA was synthesized from xanthan gum and methacrylic anhydride. XaG is a yellow powder, and a 1% XaG solution is a viscous, pale yellow substance. After methacrylation modification, the viscosity of the XaGMA solution was significantly reduced, turning milky white. After freeze-drying, XaGMA is a milky white, cotton-like solid material that is loose and soft.

[0032] (2) Preparation of OHA 1 g of hyaluronic acid (HA) was completely dissolved in 100 mL of ddH2O at 37 °C. After the HA solution cooled to room temperature, 0.563 g of NaIO4 (sodium periodate (NaIO4) to HA) was dissolved in the HA solution at a molar ratio of 1:1, and stirred at room temperature in the dark for 3 h. Then, 1 mL of ethylene glycol was added to the above solution to quench any unreacted NaIO4, and stirring was continued at room temperature in the dark for 1 h. Subsequently, the obtained product was placed in a dialysis bag (with a molecular weight cutoff of 8000-14000 Da) and dialyzed at 30 °C with ddH2O for 3 days. After dialysis, a small amount of product was taken, and an appropriate amount of silver nitrate solution was added. If no white precipitate formed, the dialysis was complete. The dialyzed product was centrifuged at 10000 rpm for 15 min, and the supernatant was collected by ultrafiltration and stored. Finally, dried OHA was obtained by freeze-drying for subsequent experiments. HA and sodium periodate are oxidized to synthesize OHA. HA is a white powder, and its solution is a colorless, viscous liquid. The OHA solution prepared by oxidation is a colorless, odorless, clear liquid. After freeze-drying, OHA appears as a white, cotton-like, fluffy, and porous substance.

[0033] Example 2: Preparation and concentration selection of XaGMA / OHA / Gel / SA composite porous scaffold (1) Nine sets of XaGMA / OHA / Gel / SA composite porous scaffolds (hereinafter referred to as "composite porous scaffolds") were prepared according to the concentration ratios of the four scaffold materials in Table 1. Nine 200 mL beakers were taken and labeled No. 1 to No. 9 on the side wall of each beaker. 100 mL of ddH2O was added to each beaker and they were heated in a 37℃ constant-temperature magnetic stirrer beforehand. The four scaffold materials, XaGMA, OHA, Gel and SA, were weighed out and added to the corresponding nine beakers. The beakers were stirred slowly for 2 h until the scaffolds were completely dissolved. The nine beakers were placed in an ultrasonic cleaner and sonicated for 2 h to completely remove air bubbles from the solution. Then, they were placed in a 37℃ constant-temperature magnetic stirrer and stirred slowly overnight. The scaffold solutions were labeled No. 1 to No. 9 on a 24-well plate. The solutions of the nine composite scaffolds were added to two-thirds of the corresponding wells of the 24-well plate. After ensuring that there were no air bubbles in the wells, the plates were pre-frozen at -20℃ for 24 h. The composite support was then placed in a freeze dryer for the first freeze drying, which lasted for 28 hours.

[0034] After the first freeze-drying, the composite scaffold was gently removed from the well plate, and its upper and lower surfaces were carefully cut off with a blade. The composite porous scaffold was then cross-linked using a triple cross-linking method. First, prepare 250 mL of 1%, 1.5%, and 2% CaCl2 solutions with a concentration ratio of SA:CaCl2 = 1:1. Add the 1.5% CaCl2 solution to wells 1, 5, and 9; add the 2.25% CaCl2 solution to wells 2, 6, and 7; and add the 3% CaCl2 solution to wells 3, 4, and 8. Let stand at room temperature for 6 hours. Second, weigh 100 mmol / L of crosslinking agents EDC, NHS, and MES, and dissolve them in 800 mL of 70% ethanol solution at 37°C. Remove the CaCl2 solution and add the EDC / NHS / MES crosslinking agent solution to a 24-well plate. Let stand at room temperature for 12 hours. Third, add the pre-prepared 0.25% photoinitiator I2959 to the 24-well plate and irradiate with 365 nm ultraviolet light for 1 minute in an ultraviolet curing chamber. After three crosslinking processes, the scaffolds were grouped and placed in 500 mL beakers, with an appropriate amount of ddH2O added. The beakers were then washed with water for 40 min on a speed-controlled shaker, with the ddH2O replaced every 20 min. Subsequently, the scaffolds were returned to their original 24-well plates and pre-frozen at -20°C for 24 h. Following this, the composite scaffolds underwent a second freeze-drying process for 24 h. After freeze-drying, nine sets of composite porous scaffolds were finally obtained.

[0035]

[0036] (2) The composite porous scaffold prepared by freeze-drying and triple cross-linking methods is a pale yellow and uniform cylinder. The composite porous scaffold is carefully cut into a cubic structure. The internal pore size of the composite porous scaffold is dense and uniform. The basis for the nano-drug delivery function of the XaGMA / OHA / Gel / SA composite porous scaffold lies in its ability to simulate the real physiological structure of breast cancer tissue, providing a real and reliable material basis for subsequent in vitro drug delivery research. Moreover, when implanted into the lesion site, it can better reduce the immunogenicity of the body and improve the safety of precise targeted treatment of breast cancer by drug release. Among them, the mechanical properties and pore size of breast cancer tissue are two of the most important physiological characteristics: First, due to the excessive proliferation of breast cancer cells, dense breast cancer tissue and microcalcifications or coarse calcifications are formed, making breast cancer tissue exhibit stronger mechanical strength. According to relevant studies, at a strain of 15%, the compressive strength of different types of breast cancer tissue is between 45-325 kPa. Secondly, moderate and uniform porosity is an important structure for ensuring the exchange of substances and metabolism between breast cancer cells and the external environment. This facilitates the uptake of large amounts of nutrients and oxygen by breast cancer cells, reduces the obstruction of metabolic waste by the matrix, and avoids excessive accumulation of substances such as lactic acid leading to microenvironment acidification. This provides a material basis for the rapid proliferation and growth of breast cancer cells. Furthermore, moderate porosity provides sufficient migration "channels" for the invasion of breast cancer cells. Existing research indicates that the pore size of breast cancer tissue is approximately between 50-200 μm. Therefore, the mechanical strength and pore size of nine composite porous scaffolds were tested to serve as an important basis for selecting the optimal concentration ratio of the three groups.

[0037] (3) Mechanical property testing: Samples with uniform texture were randomly selected from the 9 groups of composite porous scaffolds and gently cut into rectangular blocks of 8×7×6 mm, ensuring that each cut surface was regular and flat. The mechanical properties of the composite porous scaffolds were tested using a universal testing machine. The loading speed of the composite porous scaffold compression was 1 mm / min, and the maximum pressure was 50 N. When the stress reached 80%, the universal testing machine automatically stopped compressing the composite porous scaffold. During the test, the mechanical property test data of each sample were collected in real time, exported to Excel, and the compressive strength of the composite porous scaffold under 15% stress was summarized. In-depth analysis was performed using Origin plotting software. The results showed that when the strain was 15%, the mechanical strength of the first group was the lowest, at 105.50 ± 4.65 kPa; while the mechanical strength of the third group was the highest, reaching 1295.00 ± 134.08 kPa, followed by the seventh, fifth, and fourth groups. Figure 1 (A)

[0038] Pore ​​size detection: Nine sets of composite porous scaffolds were cut into 6×6×1 mm square slices. Multiple different regions of the composite porous scaffolds were randomly sampled at 4× field of view using a fluorescence inverted microscope. Sixty pores were randomly selected from each set of composite porous scaffolds and measured using ImageJ software. Further analysis was performed using Origin plotting software. It was found that the pore size of the nine sets of composite porous scaffolds ranged from 80 to 200 μm. Figure 1 (Middle B), which is basically within the pore size range of breast cancer tissue.

[0039] The pore structure of nine composite porous scaffolds was acquired using an inverted fluorescence microscope. ImageJ software was used to randomly select and calculate 60 pore sizes for each composite porous scaffold. Finally, Origin 2024 software was used for further analysis of the pore size data. The results are as follows: Figure 1 As shown in C.

[0040] Based on the mechanical properties and pore size of breast cancer tissue, and to evaluate the water solubility of the composite porous scaffold, the present invention ultimately selected the second, sixth, and eighth groups of composite porous scaffolds (hereinafter referred to as 0.25X0.25O1.5G1.5S, 0.5X0.5O1G1.5S, and 0.25X0.75O1G2S to represent the second, sixth, and eighth groups of composite porous scaffolds, respectively) for further evaluation of their physicochemical properties and cell biological behavior.

[0041] Example 3: Physicochemical properties and cell biological characterization of composite porous scaffolds 3.1 The following methods were used to characterize the physicochemical properties: Fourier transform infrared absorption spectroscopy (FTIR) detection: Randomly select homogeneous samples from each group of composite porous scaffolds and cut them into 6×6×6 mm cubes. Simultaneously, weigh appropriate amounts of dried scaffold materials such as XaGMA, OHA, Gel, and SA for later use. Mix three groups of composite porous scaffolds and scaffold materials with KBr, and grind them into a uniform powder using a mortar and pestle. Load the ground powder into a tableting mold and press it into a transparent sheet using a tablet press at 10-15 MPa. Place the prepared sample sheet on the sample holder of the Fourier transform infrared spectrometer for scanning, ensuring the scanning range is set to 4000-500 cm⁻¹. -1 Fourier transform infrared absorption spectrometer will simultaneously acquire infrared spectral data of the sample. Origin software will be used to process the acquired infrared spectral data in depth, analyze the characteristic absorption peaks in the spectra, determine the chemical bonds and functional groups of XaGMA, OHA, Gel, and SA in the composite porous scaffold, and compare them with standard spectra of XaGMA, OHA, Gel, and SA to analyze the interactions of each component in the composite porous scaffold and their influence on the spectral characteristics.

[0042] Contact angle measurement of the composite porous scaffold: The contact angle of the composite porous scaffold was measured using the seated drop method. The composite porous scaffold was cut into 1×1×0.3 cm cuboids (n=3), ensuring that the upper and lower surfaces were flat. The composite porous scaffold was placed on a contact angle meter, and a 6 μL droplet was placed on the composite porous scaffold. The image of the droplet was captured by a high-resolution camera, and the contact angle was automatically measured by software.

[0043] Water absorption rate test of composite porous scaffolds: Each group of composite porous scaffolds was cut into 6×6×6 mm cubes (n=4). The weight of each composite porous scaffold when dry was recorded as W1. Then, each group of composite porous scaffolds was immersed in PBS for 6 h. The liquid on the surface of the composite porous scaffolds was gently wiped off with filter paper, and the weight of each composite porous scaffold was recorded as W2. The water absorption rate of each sample was calculated using the following formula: ; Porosity testing of composite porous scaffolds: The porosity of the composite porous scaffolds was tested using the ethanol substitution method. Each group of composite porous scaffolds was cut into 6×6×6 mm cubes (n=6), and the weight of each composite porous scaffold after drying was recorded as W. d The composite porous scaffold was placed in a centrifuge tube containing 5 mL of anhydrous ethanol. The scaffold was gently secured to the bottom of the tube with a glass rod, ensuring its integrity and preventing breakage. The centrifuge tube was then placed in a vacuum drying oven at 28°C for 24 hours to maintain a vacuum and ensure sufficient anhydrous ethanol to fill the pores of the scaffold. Next, 6 mL of anhydrous ethanol was added to a specific gravity bottle, its weight recorded as W1, and its volume as V1 (6 mL). The composite porous scaffold was then gently transferred from the centrifuge tube to the specific gravity bottle using tweezers. The weight of the specific gravity bottle at this point is recorded as W2, and its volume as V2. The porosity of the composite porous scaffold was calculated using the following formula: ; ρ: the density of anhydrous ethanol, ρ = 0.789 g / cm³ 3 .

[0044] (5) Detection of hemolysis rate of composite porous stent Each composite porous scaffold was cut into 40 mg cubes (n=3). 2 mL of mouse ocular blood was added to 98 mL of physiological saline to prepare a 2% (w / v) ocular blood diluent. The composite porous scaffolds were placed in 10 mL round-bottom centrifuge tubes with caps, and 5 mL of ocular blood diluent was added to each tube. 40 μL of physiological saline was added to 5 mL of ocular blood diluent as a negative control, and 40 mg of Triton X-100 was added to 5 mL of ocular blood diluent as a positive control. The samples were incubated at 37°C for 3 h and centrifuged at 1000 rpm for 10 min. The supernatant was added to a 96-well plate, and the OD value at 540 nm was measured using a microplate reader. The hemolysis of each scaffold was evaluated using the following formula: ; Among them, A s The OD value of the saline group, A t For Triton X-100 group OD values, A p This represents the OD value of the composite stent assembly.

[0045] 3.2 Physicochemical property characterization results Fourier transform infrared spectra of three sets of composite porous scaffolds and four types of scaffold materials show that ( Figure 2 (A), in the range of 3500-3000 cm -1 At this location, the hydroxyl groups (-OH) of XaGMA, OHA, Gel, and SA all exhibit stretching vibrations. Gel, however, shows a stretching vibration at 1750 cm⁻¹. -1 The stretching vibration of amide I (C=O) is present at this point, which is the strongest characteristic absorption band of the amide bond. The stretching vibration of the C=O group (-COOH) in OHA also shows a characteristic peak at this location. And at 1600 cm⁻¹... -1 and 1410 cm -1 The carboxylate ions (COO) of SA appeared at the location. - Asymmetric stretching vibration peaks and symmetric vibration peaks. Additionally, at 1100-1000 cm⁻¹... -1 The stretching vibrations of the ether bonds (COC) of the six-membered sugar rings in XaGMA, OHA, and SA were observed nearby. The composite porous scaffolds of 0.25X0.25O1.5G1.5S, 0.5X0.5O1G1.5S, and 0.25X0.75O1G2S not only retained the original groups of XaGMA, OHA, Gel, and SA, such as -OH and the six-membered sugar ring COC, but also showed stretching vibrations at 1543 cm⁻¹. -1Typical CN stretching vibrations were observed at all locations, indicating that the composite porous scaffolds of 0.25X0.25O1.5G1.5S, 0.5X0.5O1G1.5S, and 0.25X0.75O1G2S successfully generated amide bonds through EDC / NHS crosslinking. Because during CaCl2 crosslinking, Ca... 2+ It can react with sodium alginate (Na) + A displacement reaction occurs, at which point Ca 2+ It will cause carboxylate (COO) - 1410 cm -1 The symmetrical vibration peak shifted to a lower wavenumber, indicating that CaCl2 was successfully cross-linked to form a sodium alginate network structure, making the composite porous scaffold structure more stable and robust.

[0046] The contact angles of the composite porous scaffolds of 0.25X0.25O1.5G1.5S, 0.5X0.5O1G1.5S, and 0.25X0.75O1G2S at 10 s were 86.1 ± 0.4°, 75.3 ± 0.7°, and 88.7 ± 0.9°, respectively. Figure 2 (Figure B) indicates that all three scaffolds exhibit good hydrophilicity, which is closely related to the presence of a large number of hydrophilic groups -OH and -COOH in OHA, Gel and XaGMA, providing a material basis for good cell adhesion and permeation on the composite porous scaffold.

[0047] Secondly, the water absorption rates of the composite porous scaffolds of 0.25X0.25O1.5G1.5S, 0.5X0.5O1G1.5S, and 0.25X0.75O1G2S were 1067.47 ± 60.33%, 1425.74 ± 3.39%, and 925.83 ± 41.79%, respectively. The water absorption rates of all three composite porous scaffolds were above 900%. Figure 2 (C) This indicates that all three composite porous scaffolds possess excellent water absorption properties, enabling cells to adhere and grow better within the scaffolds and allowing a large amount of nutrients to penetrate deep into the scaffolds, ensuring normal cell growth and proliferation. The water absorption rate of all three composite porous scaffolds rapidly increased within 0-60 min, reaching over 600%, and then stabilized relatively within 360 min. The 0.5X0.5O1G1.5S and 0.25X0.75O1G2S composite porous scaffolds achieved a water absorption rate of 1200%, primarily due to their relatively high proportion of the hydrophilic material OHA. Figure 2(E, F). Additionally, the porosities of the composite porous scaffolds of 0.25X0.25O1.5G1.5S, 0.5X0.5O1G1.5S, and 0.25X0.75O1G2S were 93.03 ± 1.4%, 93.31 ± 1.17%, and 92.84 ± 0.72%, respectively, all above 90%. Figure 2 The data (D) indicates that the composite porous scaffold has excellent porous connectivity, which not only provides a good growth space for cells and avoids inhibiting division due to crowded growth space, but also facilitates the migration and diffusion of single cells or small cell clusters and the formation of tumor cell clusters. The excellent pore structure of the composite porous scaffold ensures the normal metabolism of tumor cells.

[0048] International standards stipulate that the hemolysis rate of biomaterials should be less than 5%. The hemolysis rates of the three composite porous scaffolds were 0.61 ± 0.14%, 1.09 ± 0.32%, and 0.55 ± 0.22%, respectively. Figure 2 The three composite porous scaffolds (G) fully comply with international standards, indicating that they have good biocompatibility and are suitable as a platform for subsequent anticancer drug screening.

[0049] 3.3 The following methods were used for biological behavior characterization: (1) Cell culture on composite porous scaffold Human breast cancer cells MDA-MB-231 and MCF-7 (both purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were cultured in DMEM complete medium. Composite porous scaffolds were cut into 6×6×1 mm square slices, repeatedly immersed in 75% ethanol solution, and irradiated with UV for 30 min in a biosafety cabinet. 10 cells were seeded onto each of the three composite porous scaffolds. 5 Three days after seeding MDA-MB-231 and MCF-7 cells onto a composite porous scaffold, the survival of MDA-MB-231 and MCF-7 cells on the scaffold was assessed using live / dead cell staining. At 1, 3, 5, and 7 days of culture, the cell-composite porous scaffold constructs were subjected to CCK-8 analysis and biomarker staining to assess the proliferation of the two cell types on the composite porous scaffold.

[0050] (2) Cell survival on composite porous scaffold On day 3, MDA-MB-231 and MCF-7 cells exhibited good spreadability under Calcein-AM and Hoechst staining. Figure 3However, because MDA-MB-231 and MCF-7 cells can penetrate deep into the porous composite scaffold for growth, and because the exchange of nutrients and metabolic waste is difficult at this site, it is not easy to meet their rapid growth and proliferation needs, ultimately leading to the death of a small number of cells. This allows PI to enter the dead cells through the damaged cell membrane, bind to DNA, and emit sporadic fluorescence. In contrast, the good growth status of MDA-MB-231 and MCF-7 cells is mainly due to the excellent biocompatibility, porosity, and water absorption of the composite porous scaffold. The connections between the pores provide excellent channels for supplying nutrients needed for the rapid growth and proliferation of tumor cells. At the same time, the large amounts of metabolic waste such as lactic acid produced by tumor cell proliferation can be quickly discharged through this porous structure. Furthermore, the porous structure provides sufficient space for cell extension, growth, and proliferation, preventing contact inhibition from adversely affecting their growth. Gelatin, xanthan gum methacrylate, and oxidized hyaluronic acid have strong hydrophilicity, which endows the composite porous scaffold with excellent hydrophilicity, enabling it to adsorb a large amount of water and nutrient molecules, and provide the cells with the substances needed for growth in a timely manner.

[0051] (3) Cell proliferation on composite porous scaffold The proliferation of MDA-MB-231 and MCF-7 cells on three composite porous scaffolds at 1, 3, 5, and 7 days was quantitatively assessed using the CCK-8 assay. Figure 4 (AB). The results showed that the proliferation of both MDA-MB-231 and MCF-7 cells was relatively slow during culture for 1-3 days, but after culture for 5 days, the breast cancer cells exhibited excellent proliferation. This phenomenon is because the growth of MDA-MB-231 and MCF-7 cells on the composite porous scaffold requires a certain adaptation period. Therefore, their proliferation was relatively slow in the first 3 days of culture. After the MDA-MB-231 and MCF-7 cells adapted to the growth environment on the composite porous scaffold, their growth pattern shifted to proliferation as the main mode.

[0052] In addition, MDA-MB-231 was qualitatively examined using laser confocal microscopy. Figure 4 (C) and MCF-7 ( Figure 4The proliferation effect of MDA-MB-231 cells on a composite porous scaffold was observed after 1 and 5 days of culture. On day 1, MDA-MB-231 cells exhibited a long spindle shape, indicating good extensibility on the composite porous scaffold, and were sparsely distributed. With prolonged culture time, MDA-MB-231 and MCF-7 cells gradually aggregated and grew at the porous edges of the composite porous scaffold to facilitate nutrient absorption. On day 5, MDA-MB-231 and MCF-7 cells rapidly proliferated in certain areas of the composite porous scaffold, forming tumor spheroids. Therefore, MDA-MB-231 and MCF-7 cells maintained the rapid proliferation characteristic of malignant tumor cells on the composite porous scaffold.

[0053] Example 4: Preparation and Characterization of Drug-Loaded Nanoparticles 4.1 Preparation of drug-loaded nanoparticles 50 mg of HA was dissolved in 5 mL of ddH2O to form a homogeneous solution, and then 0.1 g of EDC•HCl was added. The solution was then stirred on a heated magnetic stirrer at 40 °C for 12 h. Separately, 50 mg of Quercetin was dissolved in 15 mL of DMSO at 37 °C, and then added dropwise to the HA solution in five separate additions, ensuring the reaction was carried out under sealed, light-protected, and low-temperature conditions for 16 h. The product was then dialyzed in ddH2O for 24 h using a dialysis bag (Mw = 8000-14000 Da), and centrifuged at 5000 rpm / min for 10 min to remove excess reagents and unreacted drugs. Finally, the supernatant was freeze-dried to obtain hyaluronic acid-coated quercetin nanoparticles (HA@Que NPs).

[0054] 50 mg of HA was dissolved in 10 mL of ddH2O, and 100 mg of EDC·HCl was added to the solution. The mixture was stirred at 40 °C for 12 h. Separately, 25 mg of GRh2 was dissolved in 1 mL of DMSO, and the activated HA solution was gradually added dropwise to the GRh2 solution. This coupling reaction was carried out under light-protected conditions at 0 °C with stirring for 16 h. Finally, the above products were dialyzed in ddH2O for 24 h using a dialysis bag (Mw = 8000-14000 Da), and centrifuged at 5000 rpm / min for 10 min to remove excess reagents and unreacted drugs. The supernatant was then freeze-dried to obtain hyaluronic acid-coated ginsenoside Rh2 nanoparticles (HA@GRh2NPs).

[0055] 4.2 Characterization of nanoparticles (1) Characterization methods Using D₂O as the solvent, the ¹H NMR spectra of Que, HA@Que NPs, GRh₂, and HA@GRh₂ NPs were detected using a 600 MHz liquid NMR spectrometer. The grafting ratio of Que to GRh₂ was then calculated using the ¹H NMR spectra.

[0056] HA@Que NPs and HA@GRh2 NPs were dissolved in ultrapure water and diluted to 0.5 mg / mL. 20 μL of each solution was dropped onto a copper grid and air-dried at room temperature. The internal structure of HA@Que NPs and HA@GRh2 NPs was then imaged at high resolution by exciting transmitted electrons using an electron beam from a transmission electron microscope.

[0057] Dissolve HA@Que NPs and HA@GRh2 NPs in ultrapure water and dilute to 0.1 mg / mL. Add 1 mL of the HA@Que NPs and HA@GRh2 NPs aqueous solution to a Malvern particle size analyzer and place it in the Malvern laser particle size analyzer to detect the nanoparticle size. Alternatively, add 1 mL of the HA@Que NPs and HA@GRh2 NPs aqueous solution to a Malvern potential analyzer to detect the potential value of the nanoparticles.

[0058] (2) Characterization results Que 1 The 1H NMR spectrum showed characteristic peaks induced by hydrogen atoms on the two aromatic rings between 5 and 8 ppm. HA is a linear polysaccharide formed by the alternating β-1,3 and β-1,4 glycosidic bonds of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc). Therefore, HA... 1 The 1H NMR spectrum will show a characteristic peak at 3-5 ppm caused by protons on the two six-membered sugar rings—GlcUA and GlcNAc. HA@Que NPs retain the characteristic peaks of both Que and HA. Figure 5 Similarly, the six-membered ring protons of GRh2 induce characteristic peaks at 5-9 ppm, and HA@GRh2 NPs retain the characteristic peaks of GRh2 at 8-12 ppm and the characteristic peaks of the two six-membered ring protons GlcUA and GlcNAc in HA at 3-5 ppm. Figure 5 (B) Calculations show that the grafting degree of HA@Que NPs is 5%, and the grafting degree of HA@GRh2 NPs is 6%.

[0059] TEM can be used to examine the morphological characteristics of HA@Que NPs and HA@GRh2 NPs. Figure 6Both HA@Que and HA@GRh2 NPs exhibit uniform particle size. Their hydrophilic outer shell is HA, which self-assembles to encapsulate hydrophobic Que and GRh2, forming relatively stable nanoparticles. Image J analysis shows that the particle sizes of HA@Que NPs and HA@GRh2 NPs are 41.24 ± 6.82 nm and 60.31 ± 5.43 nm, respectively. Figure 6 (CD). The electrical potentials of both nanoparticles, measured by Malvern particle size analyzer, were at -20 mV, indicating that the HA@Que NPs and HA@GRh2 NPs nanoparticle systems are relatively stable and not prone to aggregation. Figure 6 (EF). Therefore, both HA@Que NPs and HA@GRh2 NPs are uniformly sized and stable nanoparticles with stable drug release performance, and can be used for the preparation of subsequent drug delivery systems.

[0060] Example 5: Preparation and characterization of composite porous scaffold drug delivery system 5.1 Preparation of drug delivery system Based on cellular behavior assessment, breast cancer cells MDA-MB-231 and MCF-7 exhibited better cellular characteristics on a 0.5X0.5O1G1.5S composite porous scaffold. Therefore, the 0.5X0.5O1G1.5S composite scaffold was ultimately selected as the carrier for the drug evaluation system. According to the concentration ratio of 0.5X0.5O1G1.5S composite porous scaffold, four materials, XaGMA, OHA, Gel, and SA, were completely dissolved. Based on the results of nuclear magnetic resonance spectroscopy, the grafting degree of HA@Que NPs was calculated to be 5%, and that of HA@GRh2 NPs was 6%. Therefore, to ensure a scientific and reasonable evaluation of the antitumor effects of HA@Que NPs and HA@GRh2 NPs, 2.5 μg / mL Que, 50 μg / mL HA@Que NPs, 1.5 μg / mL GRh2, 25 μg / mL HA@GRh2 NPs, and HA@Que@GRh2 NPs (50 μg / mL HA@Que NPs + 25 μg / mL HA@GRh2) were added, respectively. The composite porous scaffold was used as the drug-loaded scaffold to prepare three cross-linked network systems: XaGMA / OHA / Gel / SA / Que (XOGS@Q), XaGMA / OHA / Gel / SA / GRh2 (XOGS@G), XaGMA / OHA / Gel / SA / HA@Que NPs (XOGS@HQ), XaGMA / OHA / Gel / SA / HA@GRh2 NPs (XOGS@HG), and XaGMA / OHA / Gel / SA / HA@Que@GRh2 NPs (XOGS@HQG).

[0061] 5.2 Physicochemical property characterization The microstructure of the drug-loaded systems was examined using scanning electron microscopy (SEM). Blank composite scaffolds, XOGS@HQ, XOGS@HG, and XOGS@HQG drug-loaded scaffolds were cut into 5×5×2 mm square slices. These samples were fixed to the sample stage using conductive adhesive, sputtered with gold for 60 s, and then the microstructure of each drug-loaded system was examined using SEM.

[0062] SEM characterization results are as follows: Figure 7As shown, the prepared 0.25X0.75O1G2S composite porous scaffold has a uniformly interconnected porous structure with a uniform pore size distribution and relatively smooth pore walls. Under 1000× high magnification, nanoparticles are visible in local areas of each drug-loaded system. The HA@Que NPs loaded on the XOGS@HQ drug-loaded system have a particle size between 37.7-49.4 nm (indicated by light-colored arrows and circles), while the HA@GRh2 NPs loaded on the XOGS@HQ drug-loaded system have a particle size between 52.9-68.1 nm (indicated by blue arrows and circles), consistent with the size distribution of the target loaded nanoparticles. The significant difference in particle size between the two drug-loaded systems is due to the difference in the size of the nanoparticles loaded on Que (Mr (C 15 H 10 O7)=302) and GRh2(Mr(C 36 H 62 The particle size of HA@Que NPs is smaller than that of HA@GRh2 NPs due to the molecular weight of O8(623). Additionally, two types of nanoparticles with different sizes were observed in the XOGS@HQ drug delivery system: HA@Que NPs and HA@GRh2 NPs. Due to their small size, these two types of nanoparticles did not exhibit significant aggregation, and the SEM results showed that the nanoparticles were successfully loaded onto the scaffold surface and pore walls, with the local loading state not disrupting the overall porous structure integrity of the scaffold. The successful loading of nanoparticles provides a structural basis for further investigation into the targeted release mechanism and local microenvironment regulation effects of the nanoparticles.

[0063] 5.3 Evaluation of the in vitro antitumor performance of the drug delivery system (1) Effect of drug delivery system on cell survival After MDA-MB-231 breast cancer cells, MCF-7 cells, and L929 fibroblasts were seeded into XOGS@Q, XOGS@G, XOGS@HQ, XOGS@HG, and XOGS@HQG drug delivery systems and cultured for 5 days, the cell-killing effects of HA@Que NPs and HA@GRh2 NPs were investigated using laser confocal microscopy. 10 cells were seeded onto each of the three composite porous scaffolds. 6MDA-MB-231, MCF-7, and L929 cells were cultured for 5 days. The cell-composite porous scaffold complexes of each group were stained with Calcein-AM, Hoechst, and PI. First, 7.5 μL of Calcein-AM, 150 μL of Hoechst, and 3 μL of PI were added to 3 mL of PBS and mixed thoroughly in a clean bench. Then, the cell-composite porous scaffold constructs were placed in the clean bench, the original culture medium in the 24-well plates was aspirated, and the mixed staining solution was added to each well. 300 μL of staining solution was added to each well according to the cell count. The plates were incubated at 37°C, 5% CO2 for 40 min, and then transferred to a laser confocal microscope for observation and photography to assess the survival of MDA-MB-231, MCF-7, and L929 cells in the drug-loaded system.

[0064] (2) Effects of drug delivery system on cell growth Breast cancer cells MDA-MB-231 and MCF-7 were seeded into XOGS@Q, XOGS@G, XOGS@HQ, XOGS@HG, and XOGS@HQG drug delivery systems, respectively, and cultured for 3 days. The inhibitory effects of HA@Que NPs and HA@GRh2 NPs on the growth of MDA-MB-231 and MCF-7 cells were investigated using laser confocal microscopy. First, 15 μL of Calcein-AM and 300 μL of Hoechst staining solution were added to 6 mL of PBS in a clean bench and mixed thoroughly. Then, the cell-composite porous scaffold construct was placed in the clean bench, the original culture medium in the 24-well plate was aspirated, and the mixed staining solution was added to each well (300-400 μL per well, depending on the cell number). The plates were incubated at 37°C, 5% CO2 for 40 min, and then observed and photographed under a laser confocal microscope to examine the inhibitory effects of the drug delivery systems on the two cell types.

[0065] In addition, the cytotoxic effects of each drug loading system on MDA-MB-231 and MCF-7 were quantitatively investigated using CCK-8 assay. MDA-MB-231 and MCF-7 breast cancer cells were seeded onto XOGS@Q, XOGS@G, XOGS@HQ, XOGS@HG, and XOGS@HQG drug loading systems, respectively, and cultured for 1, 3, 5, and 7 days. 220 μL of freshly prepared CCK-8 solution (i.e., a 1:10 mixture of CCK-8 and high-glucose DMEM medium) was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 3 h in the dark. After incubation, 100 μL of working solution was transferred from each well to a 96-well plate, and the absorbance was measured at 450 nm using a microplate reader.

[0066] (3) Effects of drug delivery system on cell morphology The effects of HA@Que NPs and HA@GRh2 NPs on the morphology and growth of MDA-MB-231 and MCF-7 cells were investigated using SEM. Ten cells were seeded onto XOGS@Q, XOGS@G, XOGS@HQ, XOGS@HG, and XOGS@HQG drug delivery systems, respectively. 5 MDA-MB-231 and MCF-7 cells were cultured for 3 and 10 days, respectively. A new 24-well plate was then prepared, and the cell-polyporous scaffold complex was carefully placed at the bottom of each well using forceps. 2.5% glutaraldehyde (25% glutaraldehyde stock solution diluted 10 times with PBS) was slowly added along the well walls using a pipette. The plate was then fixed at room temperature in the dark for 3 hours. After fixation, the fixative was removed, and the plate was dehydrated using a gradient ethanol solution method. 50%, 70%, 90%, and 100% ethanol solutions were slowly added along the well walls, changing the ethanol solution every 30 minutes. Finally, the 100% ethanol solution was removed, and the plate was left to dry completely at room temperature. It was then fixed to a sample platform with conductive adhesive, sputtered with gold for 90 seconds, and observed and images were acquired under a scanning electron microscope.

[0067] 5.4 Results of in vitro antitumor performance of the drug delivery system (1) Inhibitory effect on breast cancer cell growth MDA-MB-231 and MCF-7 cells were seeded into XOGS@Q, XOGS@G, XOGS@HQ, XOGS@HG, and XOGS@HQG drug delivery systems, respectively. The killing effects of HA@Que NPs and HA@GRh2 NPs on breast cancer cells were determined using the CCK-8 assay. The results showed that ( Figure 8 (CD) With prolonged culture time, the inhibitory effects of the XOGS@Q, XOGS@G, XOGS@HQ, XOGS@HG, and XOGS@HQG drug delivery systems on the growth of MDA-MB-231 and MCF-7 cells became increasingly significant. Compared to days 1-3, the survival rates of both breast cancer cell lines decreased significantly by day 5. More importantly, at days 1, 3, 5, and 7, the survival rates of MDA-MB-231 and MCF-7 cells on XOGS@HQ and XOGS@HG drug delivery systems were significantly lower than those on XOGS@Q and XOGS@G drug delivery systems, with XOGS@HQG exhibiting the strongest inhibitory effect on MDA-MB-231 and MCF-7. These results indicate that compared to XOGS@Q and XOGS@G, the XOGS@HQ, XOGS@HG, and XOGS@HQG drug delivery systems have superior cell growth inhibitory effects.

[0068] In addition, laser confocal microscopy was used to investigate the effects of HA@Que NPs and HA@GRh2 NPs on breast cancer cell survival. Results from Calcein-AM / Hoechst / PI staining showed that ( Figure 9 In the control group, MDA-MB-231 and MCF-7 cells exhibited excellent proliferation, showed aggregation, and had fewer dead cells. However, in the XOGS@Q, XOGS@G, XOGS@HQ, XOGS@HG, and XOGS@HQG drug delivery systems, both types of breast cancer cells showed varying degrees of cell death due to different drug effects.

[0069] The superior tumor-killing effects of XOGS@HQ and XOGS@HG are due to two main reasons: First, when Que and GRh2 are used alone in clinical treatment, their poor water solubility, short half-life, low bioavailability, and poor tumor targeting often result in poor therapeutic effects. In contrast, the core-shell structure of HA@Que NPs and HA@GRh2 NPs encapsulates hydrophobic quercetin and ginsenosides within the core, while HA acts as a hydrophilic shell covering surface, significantly improving drug water solubility. Second, the acidic environment within tumor cells (pH 4.5-5.5) can disrupt the nanoparticle structure, prompting Que and GRh2 to be rapidly released into the cell. This "pH-responsive release" pattern allows the drug to be released slowly in normal tissues but efficiently within tumor cells, thus effectively killing tumor cells. In contrast, single drugs, lacking a controlled-release mechanism, are easily and rapidly cleared from the cell, failing to achieve effective drug concentrations. Third, the hydrophilic shell of HA@Que NPs and HA@GRh2 NPs, as a natural linear polysaccharide, is widely involved in cell adhesion, migration, and signal transduction under physiological conditions. HA can target and bind to tumor cells by relying on CD44 and RHAMM receptors on the surface of tumor cells. Moreover, tumor cells can generate CD44 variants (such as CD44v6 and CD44v8-10) through selective splicing, making the HA binding site more fully exposed and increasing the binding affinity of HA by 10-100 times. In contrast, the expression levels of CD44 and RHAMM on the surface of normal cells are extremely low, and CD44 is the standard form (CD44s), which has a low binding affinity for HA. Fourth, after HA binds to CD44, it can promote the entry of nanoparticles into tumor cells through endocytosis. At the same time, HA molecules can bind to P-gp, inhibiting its drug pump function and reducing drug resistance. In summary, HA@Que NPs and HA@GRh2 NPs have excellent water solubility and drug controlled release, precise and efficient targeting and low drug resistance, which enable XOGS@HQ and XOGS@HG to have a highly efficient tumor cell growth inhibitory effect.

[0070] The XOGS@HQG drug delivery system exhibits the strongest antitumor activity, primarily due to the combined use of HA@Que NPs and HA@GRh2 NPs. Their precise synergy in molecular target complementarity, signaling pathway coordination, and drug resistance reversal allows for a more precise and efficient antitumor effect. Quercetin mainly targets tumor cell apoptosis pathways (such as downregulating the anti-apoptotic protein Bcl-2 and activating the pro-apoptotic protein Bax) and angiogenesis-related factors (such as inhibiting VEGF and bFGF), but its ability to regulate the tumor cell cycle is relatively weak. GRh2, on the other hand, can precisely arrest the tumor cell cycle at the G0 / G1 phase by downregulating Cyclin D1 and upregulating p21, while simultaneously inhibiting tumor invasion-related matrix metalloproteinases MMP-2 and MMP-9. The combined use of these two drugs can simultaneously induce apoptosis, cell cycle arrest, and anti-invasion, preventing tumor cells from escaping killing through "pathway compensation." Secondly, quercetin can inhibit upstream activators of PI3K and NF-κB (such as inhibiting IKKα / β phosphorylation), but its inhibition of downstream mTOR of AKT is weak; ginsenosides can directly downregulate the phosphorylation levels of AKT and mTOR, enhancing the pathway inhibition effect; the combination of the two can significantly weaken the proliferation and drug resistance of tumor cells and reverse the multidrug resistance phenotype.

[0071] (2) Effects on breast cancer cell morphology MDA-MB-231 and MCF-7 cells were seeded onto XOGS@Q, XOGS@G, XOGS@HQ, XOGS@HG, and XOGS@HQG drug delivery systems, respectively. The effects of HA@Que NPs and HA@GRh2 NPs on the morphology of MDA-MB-231 and MCF-7 cells were investigated by SEM on day 1 and day 3 of culture, respectively.

[0072] The results showed that, compared with the control group, the nanoparticles released by the XOGS@HQ, XOGS@HG, and XOGS@HQG drug delivery systems all exhibited significant inhibitory effects on the proliferation of both MDA-MB-231 and MCE-7 cell clusters, resulting in a decrease in cell number. However, the MDA-MB-231 cell clusters showed collapse and shrinkage, while the MCE-7 cell clusters did not show significant collapse and shrinkage. The main reasons are: 1. The HA receptor CD44 is expressed at a higher level in MDA-MB-231 cells, resulting in a higher HA-mediated targeted delivery rate and a greater likelihood of apoptosis; 2. The two cell types have different anti-apoptotic mechanisms. MCF7, as an ER-positive, less invasive cancer cell, tends to achieve proliferation through "stable survival," for example, by highly expressing Bcl-2 family proteins. These proteins can inhibit the release of apoptosis-related factors such as cytochrome C from mitochondria, thereby directly blocking the mitochondrial apoptosis pathway and failing to effectively trigger the apoptosis program.

[0073] (3) Effects on breast cancer cell survival Laser confocal microscopy was used to investigate the effects of HA@Que NPs and HA@GRh2 NPs on MDA-MB-231 ( Figure 9 (A) and MCF-7 ( Figure 9 (B) Effect on cell survival. Calcein-AM / Hoechst / PI staining results showed that in the control group, both MDA-MB-231 and MCF-7 cells exhibited significant proliferative activity, displaying a typical cell aggregation growth pattern with a low proportion of dead cells. In stark contrast, both breast cancer cell types showed varying degrees of cell death in different drug delivery systems (XOGS@Q, XOGS@G, XOGS@HQ, XOGS@HG, and XOGS@HQG). Notably, HA@Que NPs and HA@GRh2 NPs exhibited significant cytotoxic effects, leading to significant disruption of the membrane integrity of numerous MDA-MB-231 and MCF-7 cells. This cell membrane damage allowed PI to enter dead cells and bind tightly to DNA, emitting a strong red fluorescent signal. Further analysis indicated that the hyaluronic acid-coated drug system exhibited stronger cytotoxicity compared to the simple scaffold-based drug delivery system. More importantly, the synergistic effect of quercetin and ginsenoside Rg3 in the hyaluronic acid coating system achieved optimal killing effect on breast cancer cells. Comprehensive experimental results confirm that HA@Que NPs and HA@GRh2 NPs can specifically recognize and efficiently kill breast cancer cells through hyaluronic acid-mediated targeting, providing a new strategy for targeted therapy of breast cancer.

Claims

1. A composite porous support, characterized in that, The composite porous scaffold comprises xanthan gum methacrylated (XaGMA), oxidized hyaluronic acid (OHA), gelatin (Gel), and sodium alginate (SA), which are cross-linked after mixing to form the composite porous scaffold. The mass ratio of xanthan gum methacrylated (XaGMA), oxidized hyaluronic acid (OHA), gelatin (Gel), and sodium alginate (SA) is 0.125~0.5:0.25~0.75:1~2:1~2.

2. The composite porous support as described in claim 1, characterized in that, The crosslinking is performed using a three-stage crosslinking method, which sequentially uses CaCl2, an EDC / NHS / MES system, and photoinitiator I2959 for crosslinking.

3. The composite porous support as described in claim 1 or 2, characterized in that, Prepared using the following steps: (A1) Dissolve and mix 0.125–0.5 w / v% of methacrylamide xanthan gum, 0.25–0.75 w / v% of oxidized hyaluronic acid, 1–2 w / v% of gelatin, and 1–2 w / v% of sodium alginate in water, and dry (e.g., freeze-dry) to obtain a composite scaffold. (A2) The composite scaffold was first cross-linked with 1-2 wt% CaCl2 solution for 1-6 h, then cross-linked a second time with a composite cross-linking agent solution of 20-200 mmol / L EDC, 20-200 mmol / L NHS and 20-200 mmol / L MES for 1-24 h, and finally cross-linked a third time with 0.1-1% photoinitiator I2959 for 10-120 s under UV light. After washing, it was dried (e.g., freeze-dried) to obtain the composite porous scaffold.

4. The composite porous support as described in claim 3, characterized in that, The methacrylamide xanthan gum is synthesized by xanthan gum and methacrylic anhydride; the oxidized hyaluronic acid is synthesized by hyaluronic acid and an oxidizing agent.

5. A composite porous drug delivery system, characterized in that, include: (a) The composite porous scaffold according to any one of claims 1-4, and (b) Hyaluronic acid-coated quercetin nanoparticles loaded on the composite porous scaffold, and / or hyaluronic acid-coated ginsenoside Rh2 nanoparticles loaded on the scaffold.

6. The composite porous drug delivery system as described in claim 5, characterized in that, Prepared using the following steps: (B1) Hyaluronic acid-coated quercetin nanoparticles are prepared by adding a 0.5-2 w / v% aqueous solution of hyaluronic acid containing 0.1-2 w / v% EDC·HCl dropwise to a 0.1-1 w / v% organic solution of quercetin; and / or, hyaluronic acid-coated ginsenoside Rh2 nanoparticles are prepared by adding a 0.1-1 w / v% aqueous solution of hyaluronic acid containing 0.1-2 w / v% EDC·HCl dropwise to a 1-3 w / v% organic solution of ginsenoside Rh2. (B2) Dissolve and mix 0.125~0.5 w / v% of methacrylamide xanthan gum, 0.25~0.75 w / v% of oxidized hyaluronic acid, 1~2 w / v% of gelatin and 1~2 w / v% of sodium alginate in water, then add hyaluronic acid-coated quercetin nanoparticles and / or hyaluronic acid-coated ginsenoside Rh2 nanoparticles, mix evenly, and dry to obtain a drug-loaded composite scaffold; (B3) The drug-loaded composite scaffold was first cross-linked with 1-2 wt% CaCl2 solution for 1-6 h, then cross-linked a second time with a composite cross-linking agent solution of 20-200 mmol / L EDC, 20-200 mmol / L NHS and 20-200 mmol / L MES for 1-24 h, and finally cross-linked a third time with 0.1-1% photoinitiator I2959 for 10-120 s under UV light. After washing and drying, the composite porous drug delivery system was obtained.

7. The composite porous drug delivery system as described in claim 6, characterized in that, In the preparation of hyaluronic acid-coated quercetin nanoparticles, the mass ratio of hyaluronic acid to quercetin is 1:5 to 5:1; in the preparation of hyaluronic acid-coated ginsenoside Rh2 nanoparticles, the mass ratio of hyaluronic acid to ginsenoside Rh2 is 1:5 to 5:

1.

8. The composite porous drug delivery system as described in claim 6, characterized in that, In the composite porous drug delivery system, the quercetin content is 20~70 μg / mL, and the ginsenoside Rh2 content is 5~40 μg / mL.

9. The application of the composite porous scaffold according to any one of claims 1-4 in the preparation of composite porous drug-loaded systems.

10. The composite porous drug delivery system according to any one of claims 5-8, used in the preparation of preventive or antitumor drugs.

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