A cascaded responsive time-controlled release three-layer microsphere and its preparation method
By designing cascaded responsive time-controlled release three-layer microspheres, and utilizing probiotics, pH response, and MMP-9 enzymatic hydrolysis to trigger multi-stage drug release, the challenge of tumor microenvironment regulation in the treatment of endometrial cancer was solved, achieving tumor clearance and microenvironment homeostasis repair, and providing a new treatment strategy.
Patent Information
- Application Number
- CN202610137286.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-30
- Publication Date
- 2026-05-26
AI Technical Summary
Existing treatments for endometrial cancer are unable to effectively intervene in and regulate the tumor microenvironment simultaneously, leading to treatment resistance and recurrence. Traditional treatment regimens cause significant damage to vaginal mucosal tissue, immunotherapy has limited efficacy, and chemotherapy drugs disrupt the balance of symbiotic flora.
A cascaded responsive time-controlled release three-layer microsphere was designed, comprising a PLGA drug-carrying layer, a sodium alginate-chitosan interlayer, and a probiotic shell. It was prepared by a W/O/W double emulsion method to achieve multi-stage drug release triggered by probiotic colonization, pH response, and MMP-9 enzymatic hydrolysis, thereby synergistically regulating the tumor microenvironment.
It achieves rapid probiotic colonization, acidic environment regulation, and long-acting chemotherapy drug release, effectively eliminating tumor cells, restoring microenvironment homeostasis, and reducing treatment resistance and recurrence.
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Figure CN122075417A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a cascaded responsive time-controlled release three-layer microsphere and its preparation method. Background Technology
[0002] Endometrial cancer, the fourth most common gynecological malignancy among women worldwide, is showing a continuous upward trend in incidence globally, particularly among young women. The pathogenesis of this disease is highly complex, involving the pathophysiological processes of multiple interacting factors, including genetic susceptibility, hormonal imbalances, metabolic disorders, and chronic inflammation. The tumor microenvironment (TME), as a key ecological niche for tumor cell survival and evolution, plays a decisive role in the malignant biological behaviors of endometrial cancer, including its development, invasion, metastasis, immune escape, and treatment resistance. Studies have shown that the progression and recurrence of endometrial cancer are closely related to a vicious cycle in the "microbiota-immunity-metabolism" triple regulatory network within the tumor microenvironment (TME): abnormal tumor proliferation first leads to a significant reduction in protective symbiotic flora such as lactobacilli in the vaginal microenvironment, thereby disrupting the integrity of the mucosal immune barrier; persistent dysbiosis further recruits immunosuppressive cells (such as M2 tumor-associated macrophages) and activates a pro-inflammatory storm (such as IL-6 / TNF-α), ultimately forming an immunosuppressive microenvironment; while metabolic reprogramming and extracellular matrix sclerosis jointly hinder drug delivery and induce treatment resistance. This pathological process indicates that the malignant progression of endometrial cancer is not simply a matter of tumor cell autonomy, but rather a dynamic malignant ecosystem integrating "microbiota dysbiosis, immunosuppression, matrix remodeling, and metabolic abnormalities." Therefore, purely tumor-killing therapies are unlikely to achieve lasting efficacy; simultaneous intervention and regulation of the tumor microenvironment are essential.
[0003] Currently, mainstream clinical treatment options include surgical resection, radiotherapy, chemotherapy, hormone therapy, and immune checkpoint inhibitors (such as PD-1 monoclonal antibodies). However, these strategies all have significant limitations: traditional surgery and radiotherapy, while directly reducing tumor size, severely damage vaginal mucosal tissue. Clinical data shows that this can lead to a sharp decline in lactobacillus abundance exceeding 90%, thereby inducing dysbiosis-related inflammation. The non-selective cytotoxic effects of chemotherapy drugs further disrupt the balance of symbiotic flora, weakening mucosal immune defense function. 30%-40% of patients develop treatment resistance and disease relapse within 3 months of treatment. Immunotherapy, represented by PD-1 inhibitors, is only effective for patients with microsatellite instability-high (MSI-H) type, and its efficacy is limited by both immunosuppressive cell infiltration and extracellular matrix fibrosis barriers in the TME, resulting in an overall response rate of less than 30%, with most responders developing secondary resistance within 6 months. Summary of the Invention
[0004] The purpose of this invention is to address the aforementioned technical problems by providing a cascade-responsive, time-controlled release three-layer microsphere for endometrial cancer and its preparation method. The microspheres of this invention utilize a synergistic triple mechanism of microbial regeneration, immune reversal, and tumor clearance, integrating tumor clearance and TME homeostasis repair strategies to achieve time-controlled release and sensitive response, effectively clearing tumors and regulating the TME to prevent recurrence. This strategy provides an important clinical alternative treatment option, particularly for young patients who wish to preserve fertility and elderly patients who cannot tolerate surgery.
[0005] As one aspect of the present invention, the present invention provides a cascaded responsive time-controlled release three-layer microsphere, wherein the microsphere has a three-layer structure, and the microsphere comprises, from the inside to the outside:
[0006] Polylactic acid-glycolic acid copolymer (PLGA) drug-carrying layer, sodium alginate-chitosan interlayer, and probiotic outer shell layer;
[0007] The PLGA drug-carrying layer contains PLGA as a drug carrier, paclitaxel encapsulated in PLGA, turmeric oil, and matrix metalloproteinase-9 (MMP-9) responsive peptide.
[0008] The sodium alginate-chitosan interlayer is composed of negatively charged sodium alginate and positively charged chitosan with pH-responsive properties.
[0009] The probiotic outer shell layer anchors the freeze-dried curly lactobacillus to the surface of the sodium alginate-chitosan interlayer through electrostatic adsorption.
[0010] The microsphere synthesis mechanism of this invention is as follows: Microspheres are prepared via a water / oil / water (W / O / W) double emulsion method. The inner aqueous phase (W1) dissolves the MMP-9 responsive peptide, while the oil phase (O) consists of an organic solution of paclitaxel, turmeric oil, and polylactic-co-glycolic acid copolymer (PLGA). Emulsification forms a primary emulsion (W1 / O), simultaneously encapsulating paclitaxel, turmeric oil, and the MMP-9 responsive peptide within the PLGA core. Then, sodium alginate is introduced into the outer aqueous phase of the double emulsion to construct negatively charged surface groups. Subsequently, pH-responsive chitosan is used to modify the microsphere surface, and freeze-dried curly Lactobacillus is anchored via electrostatic adsorption, ultimately forming a functionalized probiotic shell.
[0011] As a preferred embodiment of the microspheres described in this invention, the size of the microspheres is 200-230 nm.
[0012] As a preferred embodiment of the microspheres described in this invention, the microspheres possess cascaded responsiveness and time-controlled release characteristics, and their release process is divided into three stages:
[0013] (1) Within 0-2 h, the freeze-dried curly Lactobacillus in the outer shell of the probiotic is preferentially released and colonized;
[0014] (2) Within 2-6 h, in response to the acidic microenvironment formed after the probiotic colonization, the chitosan in the sodium alginate-chitosan interlayer responds and releases the traditional Chinese medicine Curcuma zedoaria oil.
[0015] (3) Within 6-72 h, in response to the overexpression of MMP-9 in the tumor microenvironment, the MMP-9 responsive peptide in the PLGA drug-loaded layer is enzymatically hydrolyzed, thereby releasing the chemotherapeutic drug paclitaxel in a long-term manner.
[0016] As a preferred embodiment of the microspheres described in this invention, the acidic microenvironment in stage (2) that triggers the release of Curcuma zedoaria oil has a pH value of 6.0.
[0017] As a preferred embodiment of the microspheres described in this invention, the concentration of MMP-9 required to trigger paclitaxel release in stage (3) is not less than 500 μg / mL.
[0018] As a preferred embodiment of the microspheres described in this invention: in the raw materials for preparing the microspheres, the concentration of paclitaxel is 2 mg / mL, the volume percentage concentration of turmeric oil is 0.04%, the concentration of PLGA in the organic phase is 50 mg / mL, and the concentration of MMP-9 responsive peptide in the aqueous phase is 2 mg / mL.
[0019] In another aspect, the present invention also provides a method for preparing cascade-responsive time-controlled release three-layer microspheres, specifically comprising the following steps:
[0020] (1) Preparation of PLGA core and interlayer: A solution of 2 mg / mL paclitaxel, 0.04% turmeric oil and 50 mg / mL PLGA / DCM was prepared as the oil phase, and 2 mg / mL MMP-9 responsive peptide was dissolved in pH 7.4 PBS as the inner aqueous phase. The oil phase and aqueous phase were combined at a volume ratio of 4:1 and ultrasonicated in an ice bath (100 W, 30 s) to prepare the W / O promulgated emulsion. 10 mL of 10 mg / mL sodium alginate solution was injected into the promulgated emulsion, and homogenized emulsification was carried out at 4 °C (6000 rpm, 2 min) to form a W / O / W double emulsion. After the solvent was evaporated, the microspheres were cross-linked in 20 mg / mL CaCl2 solution for 10 min to solidify.
[0021] (2) Preparation of probiotic shell: freeze-dried Lactobacillus curly powder was mixed at 1×10 9CFU / mL was reconstituted in sterile physiological saline containing 0.1% L-cysteine at 4 °C and activated by standing for 30 min. Simultaneously, the immobilized microspheres were immersed in a 3 mg / mL chitosan solution and shaken at room temperature for 30 min, then centrifuged to obtain positively charged microspheres. The microspheres and bacterial solution were mixed at a mass / volume ratio of 1:5, and 5 mM CaCl2 was added to enhance adsorption. The mixture was incubated at 4 °C with low-speed shaking at 100 rpm for 2 h. Finally, the microspheres were washed three times with 4 °C PBS (pH 7.4) to remove free bacterial cells, and then stored in a 100 mg / mL trehalose protectant via freeze-drying for long-term preservation.
[0022] As a preferred embodiment of the preparation method of the microspheres of the present invention: the viable count of the freeze-dried Lactobacillus curly powder in step (2) is ≥10. 11 CFU / g.
[0023] As another aspect of the present invention, the present invention provides an application of cascade-responsive time-controlled release three-layer microspheres in the preparation of drugs for treating endometrial cancer.
[0024] The advantages of this invention are:
[0025] 1. This invention simultaneously encapsulates the traditional Chinese medicine Curcuma zedoaria oil and the chemotherapy drug paclitaxel in the core and interlayer layers of microspheres using a W / O / W emulsion method and electrostatic adsorption method, and prepares a probiotic shell on the surface of the microspheres, thus constructing a three-layer core-shell structured microsphere. The microspheres of this invention exhibit pH and MMP-9 cascade responsive time-controlled release performance.
[0026] 2. Microspheres can release drugs sequentially. The release rate of probiotics reaches 98% within 0-2 hours, the cumulative release rate of traditional Chinese medicine reaches 83% within 2-6 hours, and the cumulative release rate of chemotherapy drugs reaches 96% within 6-72 hours. They can quickly regulate the microenvironment to acidity, have long-lasting anti-inflammatory effects, and continuously and powerfully kill tumor cells, ultimately achieving synergistic treatment of endometrial cancer and the tumor microenvironment.
[0027] 3. This invention utilizes microspheres with a three-layered core-shell structure for cascade-responsive time-controlled drug release to achieve anti-tumor effects and regulate the tumor microenvironment. This invention provides a new treatment strategy for endometrial cancer patients through a triple mechanism of microbial reconstruction, immune reversal, and tumor clearance, which synergistically promotes tumor clearance and microenvironment homeostasis restoration. Attached Figure Description
[0028] Figure 1 This is a schematic diagram of the structure of the microspheres of the present invention.
[0029] Figure 2 This is a transmission electron microscope (TEM) image of the microspheres of this invention.
[0030] Figure 3 This refers to the Zeta potential of the microspheres of this invention.
[0031] Figure 4 This describes the drug release behavior of the microspheres of the present invention.
[0032] Figure 5 This is a comparative example of the drug release behavior of the present invention. Figure 5 (a) is the stage one probiotic live bacteria release behavior; Figure 5 (b) is the release behavior of Curcuma zedoaria oil in stage two; Figure 5 (c) is the release behavior of the stage 3 chemotherapy drug paclitaxel.
[0033] Figure 6 This invention provides an evaluation of the live probiotic function of microspheres. Figure 6 (a) Characterization of serum E2 levels; Figure 6 (b) is its antibacterial properties; Figure 6 (c) is a characterization of pH regulation.
[0034] Figure 7 This invention relates to the functional evaluation of Curcuma zedoaria oil.
[0035] Figure 8 This invention relates to the in vivo antitumor performance evaluation of the microspheres.
[0036] Figure 9 This is an assessment of the biosafety of the microspheres in this invention. Detailed Implementation
[0037] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.
[0038] Example 1: Preparation of responsive time-controlled release trilayer microspheres
[0039] (1) Preparation of PLGA core layer and interlayer: A solution of 2 mg / mL paclitaxel, 0.04% Curcuma zedoaria oil and 50 mg / mL PLGA / DCM was prepared as the oil phase, and 2 mg / mL MMP-9 responsive peptide was dissolved in pH 7.4 PBS as the inner aqueous phase. The oil phase and aqueous phase were combined at a volume ratio of 4:1 and ultrasonicated in an ice bath (100 W, 30 s) to prepare the W / O promulgated emulsion. 10 mL of 10 mg / mL sodium alginate solution was injected into the promulgated emulsion, and homogenized emulsified at 4 °C (6000 rpm, 2 min) to form a W / O / W double emulsion. After evaporation of the solvent, the microspheres were cross-linked in 20 mg / mL CaCl2 solution for 10 min to solidify.
[0040] (2) Preparation of probiotic shell: freeze-dried Lactobacillus curly powder (live count ≥10) 11 CFU / g) at 1×10 9 CFU / mL was reconstituted in sterile physiological saline containing 0.1% L-cysteine at 4 °C and activated by standing for 30 min. Simultaneously, the immobilized microspheres were immersed in a 3 mg / mL chitosan solution and shaken at room temperature for 30 min, then centrifuged to obtain positively charged microspheres. The microspheres and bacterial solution were mixed at a mass / volume ratio of 1:5, and 5 mM CaCl2 was added to enhance adsorption. The mixture was incubated at 4 °C with low-speed shaking at 100 rpm for 2 h. Finally, the microspheres were washed three times with 4 °C PBS (pH 7.4) to remove free bacterial cells, and then stored in a 100 mg / mL trehalose protectant via freeze-drying for long-term preservation.
[0041] In Example 1, a cascade-responsive, time-controlled release three-layer microsphere for endometrial cancer was prepared, with the structure as follows: Figure 1 As shown, from the inside out, the structure consists of PLGA drug-loaded layer (enzyme-responsive release of chemotherapy drugs), sodium alginate-chitosan interlayer (pH-responsive release of traditional Chinese medicine), and probiotic shell.
[0042] Comparative Example 1: Preparation of responsive time-controlled release trilayer microspheres without chemotherapeutic drug loading
[0043] The difference between this comparative example and Example 1 is that the microspheres were not loaded with the chemotherapy drug paclitaxel.
[0044] Comparative Example 2: Preparation of controlled-release trilayer microspheres without MMP-9 responsive peptide
[0045] The difference between this comparative example and Example 1 is that the microspheres were not loaded with the MMP-9 responsive peptide.
[0046] Comparative Example 3: Preparation of responsive time-sequential controlled-release microspheres without loading of Curcuma zedoaria oil
[0047] The difference between this comparative example and Example 1 is that the microspheres were not loaded with the traditional Chinese medicine Curcuma zedoaria oil.
[0048] Comparative Example 4: Preparation of Controlled-Release Microspheres Without Probiotic Shells
[0049] The difference between this comparative example and Example 1 is that the microspheres were not loaded with probiotics (Lactobacillus curvature).
[0050] Comparative Example 5: Preparation of Controlled-Release Trilayer Microspheres Loaded with Fire-Extinguishing Probiotic Shells
[0051] The difference between this comparative example and Example 1 is that the probiotic shell in the microspheres is prepared by adsorbing probiotics onto the surface of the microspheres after high-temperature inactivation.
[0052] Comparative Example 6: Preparation of Co-encapsulated Sustained-Release Microspheres
[0053] The difference between this comparative example and Example 1 is that the microspheres do not have a three-layer structure and do not have responsive time-controlled release performance.
[0054] Preparation of co-encapsulated microspheres: First, freeze-dried Lactobacillus curly powder was prepared at a ratio of 1×10⁻⁶. 9 CFU / mL was co-dispersed with 2 mg / mL paclitaxel and 2 mg / mL MMP-9 responsive peptide in pH 7.4 PBS (containing 0.1% L-cysteine protectant) at 4 °C, and 0.04% Curcuma zedoaria oil was added to form a composite inner aqueous phase. The composite inner aqueous phase was mixed with 50 mg / mL PLGA / DCM oil phase at a volume ratio of 1:4, and sonicated in an ice bath (100 W, 30 s) to prepare a W / O promulgated emulsion. The promulgated emulsion was then rapidly injected into a pre-cooled 10 mg / mL sodium alginate outer aqueous phase, and homogenized at 6000 rpm for 2 min at 4 °C to form a W / O / W double emulsion. The double emulsion was then added dropwise to a 20 mg / mL CaCl2 solution at 4 °C for cross-linking and curing for 10 min. After centrifugation to collect the microspheres, they were washed with PBS at 4 °C to remove free components, and the composite microspheres were stored in 100 mg / mL trehalose protectant and freeze-dried for long-term storage.
[0055] Experimental Example 1: Transmission electron microscopy and zeta potential characterization were performed on the microspheres prepared in Example 1, Comparative Example 4, Comparative Example 5 and Comparative Example 6.
[0056] (1) Microstructure: The microspheres were diluted and dropped onto a copper grid and allowed to dry. The microstructure of the microspheres was characterized using a transmission scanning electron microscope (TEM). The results are as follows: Figure 2 As shown, the microspheres obtained in Example 1 have the following dimensions. Figure 2 As shown in (a), the microspheres are approximately 200-230 nm in size, with a clear three-layered core-shell spherical structure and a large amount of material loaded on their surface; while the microspheres prepared in Comparative Example 4 are as shown in (a). Figure 2 As shown in (b), although there is a clear core-shell spherical structure, the surface is not loaded with other substances. This is because, compared to Comparative Example 4, Example 1 additionally prepared a probiotic shell on the surface of the microspheres, resulting in a three-layer structure. Furthermore, the microspheres prepared in Comparative Example 6, as shown in [the diagram], exhibit a similar structure. Figure 2 As shown in (c), the microspheres exhibit a homogeneous morphology because Comparative Example 6 uses a co-encapsulation method to mix and coat the substances inside the microspheres. Therefore, Example 1 has a distinct three-layer core-shell structure, providing a feasible basis for responsive time-series controlled release performance.
[0057] (2) Zeta potential: The microspheres were ultrasonically dispersed in a small amount of deionized water, and the zeta potential of the microspheres was evaluated using a zeta potential meter. The results are as follows: Figure 3As shown, the Zeta potentials of the microspheres prepared in Example 1, Comparative Example 4, Comparative Example 5, and Comparative Example 6 are 25.3, -17.1, 23.4, and -18.9 mV, respectively. This is because the microspheres in Example 1 and Comparative Example 5, after being loaded with probiotic shells, have a positively charged surface, and the activity of the probiotics does not affect their physical structure. In contrast, the microspheres in Comparative Examples 4 and 6, which are not loaded with probiotic shells, have sodium alginate completely exposed on the surface, making the microspheres negatively charged. Therefore, the above experiments sufficiently demonstrate that the probiotic shell of Example 1 was successfully loaded onto the surface of the microspheres.
[0058] Experimental Example 2: Characterization of drug release performance of microspheres prepared in Example 1
[0059] To evaluate the drug release behavior of the microspheres prepared in Example 1, the analysis was conducted in three phases. Phase 1 involved placing the microspheres in 50 mL of pH 7.4 PBS medium for drug release, and at 0.5, 1, and 2 h, 3 mL of the test solution was collected and replaced with an equal volume of fresh replenishment solution. Phase 2 involved adjusting the pH of the drug release medium to 6 after 2 h to provide an acidic drug release environment (to simulate the pH regulation effect of probiotics), and at 3, 4, and 6 h, 3 mL of the test solution was collected and replaced with an equal volume of fresh replenishment solution. Phase 3 involved adding 500 μg / mL of MMP-9 protease to the drug release medium after 6 h (to provide a responsive environment for the MMP-9 responsive peptide), and at 8, 12, 24, 48, and 72 h, 3 mL of the test solution was collected and replaced with an equal volume of fresh replenishment solution. The test solutions from Phases 1, 2, and 3 were used to calculate the cumulative release rate of live probiotics, the cumulative release rate of the traditional Chinese medicine (Curcuma zedoaria oil), and the cumulative release rate of the chemotherapy drug (paclitaxel), respectively. The calculation of the cumulative release rate of probiotics requires centrifugation of the test solution, plate culture, and subsequent plate counting; the cumulative release rates of traditional Chinese medicine (turmeric oil) and chemotherapy drug (paclitaxel) require the use of a UV spectrophotometer to measure the absorbance of the test solution at 210 and 220 nm, respectively.
[0060] The results are as follows Figure 4As shown, in a PBS environment at pH 7.4, the cumulative release rate of probiotic live bacteria reached 97.6% after 2 hours. In a PBS environment at pH 6, the traditional Chinese medicine began to be released, and its cumulative release rate reached 82.8% after 6 hours. When the drug release environment contained MMP-9 protease, the chemotherapy drug began to be released in a long-term effect, and the cumulative release rate reached 95.7% after 72 hours. This indicates that the drug release of the microspheres is divided into three stages: in the first stage, the probiotics release rapidly; in the second stage, the microspheres release the traditional Chinese medicine Curcuma zedoaria oil in response to an acidic environment; and in the third stage, the microspheres trigger the long-term release of the chemotherapy drug paclitaxel under the enzyme response. This is because the probiotic shell can rapidly release live probiotics when it comes into contact with the medium; in an acidic environment, chitosan responds to release the oil phase of the traditional Chinese medicine in the interlayer, and the lipid solubility of Curcuma zedoaria oil accelerates its release; in the environment of MMP-9 protease, the MMP-9 responsive peptide rapidly cleaves, releasing the chemotherapy drug paclitaxel in the core layer. Therefore, the microspheres prepared in Example 1 have cascade-responsive time-controlled release properties and have the potential to regulate TME and treat endometrial cancer in stages.
[0061] Experimental Example 3: Characterization of drug release performance of microspheres prepared in Comparative Examples 1-6
[0062] To compare the drug release behavior of the microspheres prepared in Comparative Examples 1-6, the differences in the release of probiotic live bacteria from the microspheres prepared in Comparative Examples 4-6 were compared in Stage 1. The differences in the release of traditional Chinese medicine from Comparative Examples 3 and 6 were compared in Stage 2, and the differences in the release of chemotherapy drugs from Comparative Examples 1, 2, and 6 were compared in Stage 3. The test methods were as described in Experimental Example 2.
[0063] Probiotic live bacteria release such as Figure 5 As shown in (a), neither Comparative Example 4 nor Comparative Example 5 released live probiotics from their microspheres, while Comparative Example 6 achieved a cumulative release rate of 62.8% after 2 hours. This is because Comparative Example 4 lacked a probiotic shell, and the probiotic shell of Comparative Example 5 was inactive. However, Comparative Example 6, with probiotics uniformly mixed inside the microspheres, was able to release live probiotics starting at 0 hours. (The release of traditional Chinese medicine is shown in Figure 6.) Figure 5 As shown in (b), the microspheres in Comparative Example 3 did not release any traditional Chinese medicine, while Comparative Example 6 achieved a release rate of 68.4% at 6 h. This is because Comparative Example 3 was not loaded with traditional Chinese medicine, while Comparative Example 6, due to the uniform mixing of Curcuma zedoaria oil within the microspheres, was able to release the traditional Chinese medicine starting at 0 h. Chemotherapy drugs were then released. Figure 5As shown in (c), the microspheres of Comparative Example 1 did not release the chemotherapeutic drug, while the release rates of Comparative Examples 2 and 6 reached 41.3% and 82.6% at 72 h, respectively. This is because Comparative Example 1 was not loaded with the chemotherapeutic drug; Comparative Example 2 was not loaded with the MMP-9 responsive peptide and could not respond to the MMP-9 protease in the environment. Its sustained release effect came from PLGA, and the drug release behavior was relatively slow; Comparative Example 6 was able to start releasing the chemotherapeutic drug paclitaxel from 0 h because the chemotherapeutic drug was uniformly mixed inside the microspheres.
[0064] In summary, in Comparative Example 6, because the encapsulating material is uniformly mixed inside the microspheres, the release of all three components begins from 0h. In contrast, the microspheres in Example 1 can release each component in a cascade-responsive, time-controlled manner.
[0065] Experimental Example 4: Performance testing of the probiotic live bacteria component in the microspheres prepared in Example 1, Comparative Example 4, and Comparative Example 5.
[0066] To evaluate the role of probiotics in blocking the vicious cycle of "estrogen-microbiota-tumor" by metabolizing estrogen (lowering serum E2 levels) and secreting antimicrobial peptides, as well as regulating the TME environment to acidity, the serum E2-lowering, antibacterial, and pH-regulating properties of the test solution after 2 hours of release in Phase 1 were characterized.
[0067] (1) Characterization of decreased serum E2 levels: The test solution released 2 hours after stage one was co-incubated with estradiol (E2), and the residual E2 level in the culture medium was detected. The results are as follows: Figure 6 As shown in (a), the E2 degradation rate of Example 1 reached 73%, while the degradation rates of Comparative Examples 4 and 5 were 0% and 2%, respectively. This is because Comparative Example 4 had no probiotic shell, and the probiotic shell of Comparative Example 5 was inactive and could not exert the effect of probiotics metabolizing estrogen.
[0068] (2) Antibacterial properties: The antibacterial activity of the sterilized test solution against *Escherichia coli* was detected by observing the inhibition zone. Results are as follows: Figure 6 As shown in (b), the inhibition zone diameter of Example 1 reached 13 mm, indicating that its probiotics were active, while the inhibition zones of Comparative Examples 4 and 5 were less than 1 mm, indicating that they had no active probiotics. This is because Comparative Example 4 had no probiotic shell, and the probiotic shell of Comparative Example 5 was inactive, so it was impossible for the probiotics to exert their antibacterial effect by secreting antimicrobial peptides.
[0069] (3) pH adjustment performance: The pH value of the test solution was tested using an electronic pH meter, and the results are as follows: Figure 6 As shown in (c), the pH of PBS at 0 was 7.4. After 2 hours, the pH of Example 1 was 5.8, which was significantly reduced, while the pH of Comparative Examples 4 and 5 was 7. This is because the probiotics in Example 1 had good activity, Comparative Example 4 had no probiotic shell, and the probiotic shell in Comparative Example 5 had no activity, so the probiotics could not play a role in regulating pH.
[0070] In summary, the probiotics in Example 1 exhibit good activity and can block the vicious cycle of "estrogen-microbiota-tumor" and regulate the TME environment to acidity by metabolizing estrogen (reducing serum E2 levels) and secreting antimicrobial peptides.
[0071] Experimental Example 5: Characterization of the ferroptosis-inducing properties of microspheres prepared in Example 1 and Comparative Example 3 in cancer cells.
[0072] To evaluate the ferroptosis-inducing properties of Curcuma zedoaria oil in cancer cells, cancer cells were treated with a 6-hour phase II assay solution. Lipid ROS levels in the cancer cells were measured, and fluorescence images were captured, with average fluorescence intensity recorded. The results are as follows: Figure 7 As shown, the average fluorescence intensity of the control group was 50.8 au, while that of Example 1 was 276.3 au, which is 5.4 times that of the control group. The average fluorescence intensity of Example 3 was 66.2 au, which is 4.2 times that of Example 1. This is because the turmeric oil loaded in Example 1 can significantly increase lipid ROS, while it has no effect on Example 3. In summary, the microspheres prepared in Example 1 can release turmeric oil under acidic conditions to induce ferroptosis in cancer cells.
[0073] Experimental Example 6: Characterization of the in vivo antitumor properties of the microspheres prepared in Example 1
[0074] To evaluate the antitumor properties of the microspheres, female thymic Nu / Nu mice (6-8 weeks old) were subcutaneously injected with Hec50co cells (1 × 10⁶ cells per mouse). 6 (Number of cells). When palpable tumors appeared in mice (day 18 post-tumor challenge), mice were randomly assigned to three treatment groups: control group (PBS, n=6), comparative group (microspheres prepared in Example 1, n=6), and microspheres prepared in Example 1 (n=6). The drugs were administered three times via tail vein intravenously on days 10, 13, and 16 post-tumor challenge. Each dose consisted of 100 μL PBS and 100 μL microspheres (microspheres dissolved in PBS at 50 mg / mL) / PBS (control). Body weight and tumor volume were monitored twice weekly for four weeks following the last administration (i.e., day 50 post-tumor challenge). On the last day of the study (day 50 post-tumor challenge), mice were euthanized, tumors were collected for photography, and body weight was assessed.
[0075] The results are as follows Figure 8 As shown, the tumor weights of the control group, Comparative Example 1, and Example 1 were 1.3, 0.8, and 0.5 g, respectively. The tumor volume of Example 1 was significantly reduced because the chemotherapeutic drug paclitaxel in Example 1 exerted its antitumor effect. Although the tumor volume of Comparative Example 1 decreased to some extent, it was not significant because it did not contain the main chemotherapeutic drug. Therefore, the microspheres of Example 1 have significant antitumor properties.
[0076] Experimental Example 7: In vivo safety characterization of the microspheres prepared in Example 1
[0077] To assess the biosafety of the microspheres prepared in Example 1, mice were euthanized on the last day of the experiment (day 50 post-tumor attack), and vital organs (heart, kidney, liver, lung, and spleen) were harvested. After H&E staining, the vital organs were fixed, embedded, and subjected to histological examination. In the control group and the Example 1 group, as... Figure 9 As shown, we did not detect necrosis or significant tissue damage, including apoptosis, nuclear cleavage, pyknosis, or inflammatory cell infiltration, indicating that the dose used was safe and had no obvious toxicity.
[0078] In summary, the cascaded responsive time-controlled release microspheres of this invention have the ability to release in a time-controlled manner. Experiments have shown that they have good acid environment responsiveness, MMP-9 protease responsiveness, three-stage controlled release, estrogen metabolism, secretion of antimicrobial peptides, induction of cancer cell ferroptosis, antitumor properties, and biosafety. They are expected to be widely used in the treatment of endometrial cancer and in the regulation of the tumor microenvironment.
[0079] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.
Claims
1. A cascaded responsive time-controlled release three-layer microsphere, characterized in that, The microspheres have a three-layer structure, comprising, from the inside out: PLGA drug-carrying layer, sodium alginate-chitosan interlayer, and probiotic outer shell layer; The PLGA drug-carrying layer includes PLGA as a drug carrier, paclitaxel encapsulated in PLGA, turmeric oil, and MMP-9 responsive peptide. The sodium alginate-chitosan interlayer is composed of negatively charged sodium alginate and positively charged chitosan with pH-responsive properties. The probiotic outer shell layer anchors the freeze-dried curly lactobacillus to the surface of the sodium alginate-chitosan interlayer through electrostatic adsorption.
2. The microspheres according to claim 1, characterized in that: The microspheres have a size of 200-230 nm.
3. The microspheres according to claim 1, characterized in that, The microspheres possess cascade response and time-controlled release characteristics, and their release process consists of three stages: (1) Within 0-2 h, the freeze-dried curly Lactobacillus in the outer shell of the probiotic is preferentially released and colonized; (2) Within 2-6 h, in response to the acidic microenvironment formed after the probiotic colonization, the chitosan in the sodium alginate-chitosan interlayer responds and releases the traditional Chinese medicine Curcuma zedoaria oil. (3) Within 6-72 h, in response to the overexpression of MMP-9 in the tumor microenvironment, the MMP-9 responsive peptide in the PLGA drug-loaded layer is enzymatically hydrolyzed, thereby releasing the chemotherapeutic drug paclitaxel in a long-term manner.
4. The microspheres according to claim 3, characterized in that, The acidic microenvironment in stage (2) that triggers the release of Curcuma zedoaria oil has a pH of 6.
0.
5. The microspheres according to claim 3, characterized in that, The concentration of MMP-9 required to trigger paclitaxel release in stage (3) is no less than 500 μg / mL.
6. The microspheres according to claim 1, characterized in that, In the raw materials for preparing the microspheres, the concentration of paclitaxel is 2 mg / mL, the volume percentage concentration of turmeric oil is 0.04%, the concentration of PLGA in the organic phase is 50 mg / mL, and the concentration of MMP-9 responsive peptide in the aqueous phase is 2 mg / mL.
7. A method for preparing microspheres as described in any one of claims 1-6, characterized in that, The preparation method includes the following steps: (1) Preparation of PLGA core layer and interlayer: 2 mg / mL paclitaxel, 0.04% Curcuma zedoaria oil and 50 mg / mL PLGA / DCM solution were prepared as oil phase, and 2 mg / mL MMP-9 responsive peptide was dissolved in pH 7.4 PBS as inner aqueous phase. The oil phase and aqueous phase were combined at a volume ratio of 4:1 and ultrasonicated at 100 W for 30 s in an ice bath to prepare W / O primary emulsion. 10 mL of 10 mg / mL sodium alginate solution was injected into the primary emulsion and homogenized and emulsified at 4°C and 6000 rpm for 2 min to form W / O / W double emulsion. After the solvent was evaporated, the microspheres were cross-linked in 20 mg / mL CaCl2 solution for 10 min to solidify. (2) Preparation of probiotic shell: freeze-dried Lactobacillus curly powder was mixed at 1×10 9 CFU / mL was reconstituted in sterile physiological saline containing 0.1% L-cysteine at 4 °C and activated by standing for 30 min. Simultaneously, the immobilized microspheres were immersed in a 3 mg / mL chitosan solution and shaken at room temperature for 30 min. After centrifugation, positively charged microspheres were obtained. The microspheres and bacterial solution were mixed at a mass / volume ratio of 1:5, and 5 mM CaCl2 was added to enhance adsorption. The mixture was incubated at 4 °C with low shaking at 100 rpm for 2 h. Finally, the microspheres were washed three times with PBS at 4 °C and pH 7.4 to remove free bacterial cells. The composite microspheres were then stored in a 100 mg / mL trehalose protectant and freeze-dried for long-term preservation.
8. A method for preparing the same according to claim 7, characterized in that: The viable count of the freeze-dried Lactobacillus curly powder in step (2) is ≥10. 11 CFU / g.
9. The use of the microspheres according to any one of claims 1-6 in the preparation of a drug for treating endometrial cancer.