Protopanaxatriol nano-delivery system targeting twist1-ku70-sirt1 complex and its application in treatment of colon inflammation-related colorectal cancer

Abstract

The present application relates to the field of medicine, in particular to a nano delivery system of protopanaxatriol targeting Twist1-Ku70-Sirt1 complex and application of the nano delivery system in treatment of colon inflammation related colorectal cancer, the nano delivery system comprising: (a) protopanaxatriol nanocrystal; (b) lipoic acid modified chitosan; and (c) crosslinking agent; wherein the average particle size of the protopanaxatriol nanocrystal is 100-200 nm; the modification degree of the lipoic acid modified chitosan is 15-25%; the crosslinking agent is sodium tripolyphosphate; and the average particle size of the nano delivery system is 300-500 nm, the solubility and bioavailability of protopanaxatriol are significantly improved by preparing protopanaxatriol into nanocrystal.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to a protopanaxadiol nanodelivery system targeting the Twist1-Ku70-Sirt1 complex and its application in the treatment of colitis-associated colorectal cancer. Background Technology

[0002] Colitis-associated colorectal cancer (CAC) is one of the most serious complications of inflammatory bowel disease (IBD), with long-term chronic inflammation being a major risk factor for its development. Epidemiological data show that patients with IBD have a significantly higher risk of developing colorectal cancer than the general population, with the lifetime incidence of colorectal cancer in patients with ulcerative colitis reaching as high as 18%. Compared to sporadic colorectal cancer, CAC is characterized by earlier onset, higher incidence, and poorer prognosis, placing a significant burden on patients.

[0003] Current treatment options for cancer-associated abscess (CAC) mainly include anti-inflammatory therapy, chemotherapy, and surgical resection. However, these treatments have limited efficacy and are often accompanied by serious adverse reactions. Currently, there is a lack of targeted therapies against the specific pathological mechanisms of CAC, particularly drugs that can inhibit tumor stem cell characteristics and tumorigenesis. This is the main challenge facing CAC treatment.

[0004] Recent studies have revealed that various transcription factors and signaling pathways in the tumor microenvironment play important roles in the development and progression of colorectal cancer (CAC). Among them, Twist1, a key regulator of epithelial-mesenchymal transition (EMT), is highly expressed in various cancers and is associated with disease progression and poor prognosis. Studies have shown that in colorectal cancer, Twist1 can promote the maintenance of tumor cell stem cell characteristics, invasion and metastasis, and the development of drug resistance. Liu et al. (Oncotarget, 2017) reported that Twist1 promotes multidrug resistance in colorectal cancer by upregulating ATP-binding cassette transporters (such as ABCB1 and ABCC1). Chen et al. (ScientificReports, 2014) found that Sirt1 is highly expressed in colorectal cancer tissues and is significantly associated with poor patient prognosis, and that Sirt1 co-localizes with CD133, a marker of colorectal cancer stem cells.

[0005] At the molecular level, Roth et al. (Oncotarget, 2016) first reported the molecular mechanism by which SIRT1 and LSD1 competitively regulate Ku70 function, affecting DNA repair and the acquisition of drug resistance mutations in tumor cells. Their study showed that Ku70 is a key protein in the non-homologous end joining (NHEJ) DNA repair pathway, and SIRT1 enhances its DNA repair function by deacetyling Ku70. Furthermore, Cohen et al. (Science, 2004) confirmed that SIRT1 regulates cellular sensitivity to apoptotic stimuli by deacetyling Ku70. However, these studies have mainly focused on binary protein interactions, and in-depth research on the potential ternary protein complexes and their role in tumorigenesis is still lacking.

[0006] Traditional Chinese medicine (TCM) active ingredients have a long history and broad prospects in cancer treatment. Protopanaxatriol (20S-protopanaxatriol, PPT) is one of the main active metabolites of ginsenosides and has been proven to possess various pharmacological activities, including antitumor, anti-inflammatory, and antioxidant effects. Studies by Zhao et al. (Molecules, 2010) showed that PPT and its derivatives have significant inhibitory effects on the proliferation of various tumor cell lines. Wang et al. (Journal of Experimental & Clinical Cancer Research, 2019) reported that the combined use of 20(S)-protopanaxatriol (g-PPT) and EGFR-TKIs can overcome EGFR-TKI resistance by reducing SCD1-induced lipid accumulation. However, as a natural product, PPT suffers from poor water solubility, low bioavailability, and a short half-life in vivo, limiting its clinical application.

[0007] Nanoparticle delivery systems, as a novel drug delivery platform, have made significant progress in recent years in improving the bioavailability, targeting, and controlled release of poorly soluble drugs. Gao et al. (International Journal of Pharmaceutical Research, 2018) reviewed the application progress of nanoparticle delivery systems in the treatment of colorectal cancer, pointing out that nanoparticle delivery systems can significantly improve drug accumulation at the tumor site and reduce systemic toxicity. Chitosan, as a biocompatible and biodegradable natural polysaccharide, has been widely used in the construction of drug delivery systems. Liu et al. (International Journal of Nanomedicine, 2016) reported that chitosan nanoparticles can effectively improve drug release in the colon and enhance its therapeutic effect on colitis. However, there are currently no reports of combining PPT with targeted colonic delivery systems for targeted therapy of CAC.

[0008] Please refer to Figure 1In A-1D, the inventors discovered that Twist1, Ku70, and Sirt1 can form a ternary protein complex that promotes stem cell characteristics and CAC tumorigenesis. Through molecular docking technology, small molecule natural drugs targeting this complex were screened, revealing that PPT binds to tyrosine residue 320 of Ku70 and glutamate residue 32 of Sirt1, thereby inhibiting the formation of the ternary complex. However, the application of PPT is limited by its physicochemical properties and pharmacokinetic characteristics. Therefore, this invention aims to develop a nanodelivery system that improves the bioavailability and targeting of PPT to enhance its therapeutic effect on CAC. Summary of the Invention

[0009] The purpose of this invention is to provide a protopanaxadiol nanodelivery system targeting the Twist1-Ku70-Sirt1 complex, its preparation method, and its application in the treatment of colitis-associated colorectal cancer (CAC). The nanodelivery system of this invention can significantly improve the bioavailability and colon-targeting of protopanaxadiol, enhance its inhibitory effect on the formation of the Twist1-Ku70-Sirt1 complex, thereby more effectively reducing stem cell characteristics and CAC tumor development.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] In a first aspect, the present invention provides a protopanaxadiol nanodelivery system targeting the Twist1-Ku70-Sirt1 complex, comprising:

[0012] (a) Protopanaxadiol nanocrystals;

[0013] (b) Lipoic acid-modified chitosan; and

[0014] (c) Crosslinking agent;

[0015] The average particle size of the protopanaxadiol nanocrystals is 100-200 nm; the degree of modification of the lipoic acid-modified chitosan is 15-25%; the crosslinking agent is sodium tripolyphosphate; and the average particle size of the nanodelivery system is 300-500 nm.

[0016] In another preferred embodiment of the present invention, the protopanaxadiol nanocrystals are prepared by an ultrasound-assisted antisolvent precipitation method, wherein ethanol is used as a solvent, water as an antisolvent, and polyvinylpyrrolidone K30 or polysorbate 80 as a stabilizer.

[0017] In another preferred embodiment of the present invention, the chitosan has a molecular weight of 100-200 kDa and a degree of deacetylation ≥90%; the thioctic acid-modified chitosan is synthesized by EDC / NHS activation coupling method, wherein EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and NHS is N-hydroxysuccinimide.

[0018] In another preferred embodiment of the present invention, the mass ratio of the protopanaxadiol nanocrystals to the lipoic acid-modified chitosan is 1:2 to 1:5, and the mass ratio of the lipoic acid-modified chitosan to sodium tripolyphosphate is 3:1 to 5:1.

[0019] In another preferred embodiment of the present invention, the absolute value of the Zeta potential of the nanodelivery system is ≥30mV, the polydispersity index is <0.3, the loading of protopanaxadiol is ≥10%, and the encapsulation efficiency is ≥80%.

[0020] In another preferred embodiment of the present invention, the nanodelivery system releases <20% protopanaxadiol within 4 hours in simulated gastric juice (pH 1.2) and small intestinal juice (pH 6.8), and >80% protopanaxadiol within 24 hours in simulated colonic juice (pH 7.4), exhibiting colon-targeted release characteristics.

[0021] Secondly, the present invention provides a method for preparing the above-mentioned nanodelivery system, comprising the following steps:

[0022] (a) Protopanaxadiol was dissolved in ethanol, a stabilizer was added, and the protopanaxadiol solution was added to ultrapure water under ultrasonic conditions to prepare protopanaxadiol nanocrystals.

[0023] (b) Lipoic acid was activated by EDC / NHS, and the activated lipoic acid was reacted with chitosan to prepare lipoic acid modified chitosan.

[0024] (c) The protopanaxadiol nanocrystals prepared in step (a) are dispersed in a solution containing lipoic acid-modified chitosan prepared in step (b), and then cross-linked with sodium tripolyphosphate solution to form a nanodelivery system; and

[0025] (d) Collect and purify the nanodelivery system.

[0026] Thirdly, the present invention provides the use of the nanodelivery system in the preparation of a medicament for treating colitis-associated colorectal cancer, wherein the nanodelivery system reduces the stem cell characteristics of cancer cells and tumorigenesis by targeting and inhibiting the formation of the Twist1-Ku70-Sirt1 complex.

[0027] In a preferred embodiment of the present invention, the drug is an oral formulation, and the dosage is equivalent to 10-20 mg / kg / day of protopanaxadiol, for the treatment of AOM-DSS-induced colitis-associated colorectal cancer, or in combination with an immune checkpoint inhibitor for the treatment of patients with colitis-associated colorectal cancer who have high expression of the Twist1-Ku70-Sirt1 complex.

[0028] The protopanaxadiol nanodelivery system provided by this invention has the following beneficial effects:

[0029] 1. By preparing protopanaxadiol into nanocrystals, its solubility and bioavailability are significantly improved. Protopanaxadiol is a poorly soluble compound with low bioavailability in conventional formulations. However, nanocrystal technology can reduce its particle size to 100-200 nm, greatly increasing the specific surface area and improving the dissolution rate, thereby improving its bioavailability.

[0030] 2. A nanodelivery system constructed by modifying chitosan with lipoic acid achieved colon-targeted delivery of protopanaxadiol. Lipoic acid modification enhanced the adhesion of chitosan to the intestinal mucosa, while chitosan could be degraded by colonic microbial enzymes, thereby specifically releasing the drug in the colon. In vitro release experiments showed that the system released <20% of the drug in simulated gastric and small intestinal fluids, while releasing >80% within 24 hours in simulated colonic fluid, demonstrating significant colon-targeting.

[0031] 3. Significantly improved the efficiency of protopanaxadiol in inhibiting the formation of the Twist1-Ku70-Sirt1 complex. Through the nanodelivery system, the concentration of protopanaxadiol in colonic tissue was significantly increased, with the colonic / blood drug concentration approximately four times higher than that of free protopanaxadiol. Immunoprecipitation experiments confirmed that the inhibitory effect of protopanaxadiol in the nanodelivery system on the formation of the Twist1-Ku70-Sirt1 complex was approximately 2.5 times higher than that of free protopanaxadiol.

[0032] 4. In an AOM-DSS-induced CAC animal model, the nanodelivery system of this invention significantly reduced the number and size of colon tumors. Compared with an equal dose of free ginsenoside triol, the nanodelivery system improved the reduction of tumor number by approximately 60% and the reduction of tumor volume by approximately 70%. Furthermore, histopathological analysis showed that the nanodelivery system more effectively reduced tumor atypia and invasiveness.

[0033] 5. The system demonstrated good safety, with no obvious toxic side effects observed. Long-term administration experiments showed that the nanodelivery system did not affect the weight gain of experimental animals, liver and kidney function indicators remained within the normal range, and no significant pathological changes were observed in major organs. In contrast, the group receiving an equal dose of free ginsenoside triol showed mild abnormalities in liver function indicators.

[0034] In summary, the protopanaxadiol nanodelivery system provided by this invention significantly enhances the inhibitory effect of protopanaxadiol on the formation of the Twist1-Ku70-Sirt1 complex by improving the solubility, bioavailability and colon targeting of protopanaxadiol, providing a new strategy and drug delivery system for targeted therapy of colitis-associated colorectal cancer. Attached Figure Description

[0035] Figure 1 A is a list of candidate compounds and their molecular docking scores;

[0036] Figure 1 B represents the molecular structure of PPT;

[0037] Figure 1 C shows the details of the interaction between PPT and key amino acid residues;

[0038] Figure 1 D represents the experimental results of PPT inhibiting the formation of the Twist1-Ku70-Sirt1 complex, showcasing the results of the co-immunoprecipitation (Co-IP) experiment;

[0039] Figure 1 E represents the in vitro experimental results of PPT inhibiting the characteristics of colorectal cancer cell stem cells, including in vitro spheroidization, colony formation, and soft agar colony formation experiments;

[0040] Figure 1 F is a schematic diagram of the AOM-DSS experimental scheme;

[0041] Figure 1 G represents a comparison of rectal morphology and tumor number in mice;

[0042] Figure 1 H represents the histopathological results of H&E staining. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that the embodiments described are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention.

[0044] Research and validation confirmed that Twist1 can form a complex with Ku70 and Sirt1, promoting stem cell properties and tumorigenesis of CAC. This finding suggests that the Twist1-Ku70-Sirt1 complex may serve as a potential therapeutic target for CAC. Small molecule natural drugs targeting the interaction interface of the Twist1-Ku70-Sirt1 complex were screened from a small molecule database using molecular docking technology. After comprehensive evaluation and analysis, 10 candidate compounds were selected (…). Figure 1 A), with PPT receiving the highest score ( Figure 1 A). The molecular structure of PPT is as follows: Figure 1 As shown in Figure B. Molecular docking results indicate that PPT binds to tyrosine (TRY) at amino acid position 320 of Ku70 and glutamic acid (GLU) at amino acid position 32 of Sirt1. Figure 1 C). Co-immunoprecipitation (Co-IP) experiments demonstrated that 20 μM PPT could inhibit the interaction of the Twist1-Ku70-Sirt1 complex in HCT116 cells overexpressing Twist1. Figure 1 D). Furthermore, in vitro spheroidization, colony formation, and soft agar colony formation experiments showed that, compared to the control group, PPT treatment significantly reduced the rates of in vitro spheroidization and colony formation (D). Figure 1 E). These data indicate that PPT can inhibit the stem cell characteristics of colorectal cancer cells. To further verify the role of PPT in CAC tumorigenesis, a CAC model was established by AOM-DSS induction. The control group and the PPT treatment group were given 0.5% CMC-Na and 20 mg / kg PPT every other day after intraperitoneal injection of AOM, respectively. Figure 1 F). Compared with the control group, PPT-treated mice showed reduced rectal prolapse and fewer macroscopically visible tumors. Figure 1 G). Furthermore, hematoxylin-eosin (HE) staining showed that, compared to control mice, PPT-treated mice exhibited reduced tumor infiltration and decreased atypia in their tissues. Figure 1 These results indicate that PPT inhibits stem cell characteristics and CAC tumorigenesis by regulating the interaction of the Twist1-Ku70-Sirt1 complex.

[0045] Example 1: Preparation of protopanaxadiol nanocrystals

[0046] This invention first requires the preparation of protopanaxadiol (PPT) nanocrystals to improve their solubility and bioavailability. An ultrasound-assisted antisolvent precipitation method is used for preparation; this method is simple to operate, highly reproducible, and facilitates control over the particle size distribution of the nanocrystals.

[0047] Specifically, protopanaxadiol with a purity of not less than 98% (purchased from Sigma-Aldrich) was selected as the raw material, anhydrous ethanol (analytical grade, ≥99.5%) was used as the solvent, ultrapure water (resistivity ≥18.2 MΩ·cm, 25°C) was used as the antisolvent, and polyvinylpyrrolidone K30 (PVP K30, molecular weight approximately 40,000) was used as the stabilizer.

[0048] In the preparation process, 100 mg of protopanaxadiol was first dissolved in 10 mL of anhydrous ethanol, and 100 mg of PVP K30 was added simultaneously to achieve a PPT to PVP K30 mass ratio of 1:1. The mixture was then magnetically stirred (approximately 600 rpm) at room temperature until completely dissolved to form a clear solution. Subsequently, 100 mL of ultrapure water was added to a 250 mL beaker and the mixture was placed under ultrasonic conditions (power set to 400 W) while maintaining the temperature within the range of 10-15°C. It is noteworthy that temperature control is crucial for preventing the degradation of the heat-sensitive drug and controlling the crystallization process.

[0049] Next, the PPT ethanol solution was injected into stirred ultrapure water at a rate of 3 mL / min using a constant flow pump. During this process, due to the antisolvent effect, the solubility of PPT in the aqueous phase dropped sharply, leading to supersaturation and the formation of nanoscale crystals. The energy provided by ultrasound helped promote uniform nucleation and prevent the formation of large crystals. After injection, sonication was continued for 15 minutes to stabilize the formed nanocrystal suspension, at which point the solution was milky white.

[0050] To collect and purify the formed PPT nanocrystals, high-speed centrifugation (18,000 × g, 15 min, 4°C) was employed. Subsequently, the precipitate was washed three times with ultrapure water to thoroughly remove residual organic solvents and unbound stabilizers. Finally, the purified nanocrystals were freeze-dried (-50°C, 0.1 mbar, approximately 36 hours) to obtain a white to slightly yellow PPT nanocrystal powder, suitable for long-term storage and subsequent applications.

[0051] To characterize the prepared PPT nanocrystals, a series of analyses were performed. First, the particle size distribution was measured using dynamic light scattering (DLS), which showed that the average particle size of the PPT nanocrystals was 156 ± 12 nm, and the polydispersity index (PDI) was 0.22 ± 0.03, indicating a relatively uniform particle size distribution. Transmission electron microscopy (TEM) observations showed that the nanocrystals were nearly spherical, uniformly dispersed, and without obvious aggregation, which is beneficial for improving their stability and bioavailability.

[0052] Furthermore, X-ray powder diffraction (XRPD) analysis revealed that the prepared PPT nanocrystals exhibited a partially amorphous state with reduced crystallinity, which contributes to further improving their dissolution rate. The zeta potential measured in PBS buffer at pH 7.4 was -28.6 ± 2.4 mV, close to -30 mV, indicating good electrostatic stability of the nanocrystals. Storage stability studies showed that after 14 days of storage at 4°C, the particle size increased by no more than 5%, demonstrating the reliability of the preparation process and the good stability of the product.

[0053] Notably, the apparent solubility of the prepared PPT nanocrystals in PBS (pH 7.4) was increased by approximately 4.2 times compared to the original PPT crystals. This significant improvement is mainly attributed to the high specific surface area, low crystallinity, and surface stabilizing effect provided by PVP K30. These results demonstrate that nanocrystal technology is an effective strategy for improving the water solubility and bioavailability of PPT.

[0054] Example 2: Synthesis of lipoic acid modified chitosan

[0055] To construct a colon-targeted delivery system, suitable carrier materials are required. In this embodiment, lipoic acid-modified chitosan (LA-CS) was synthesized via EDC / NHS activation coupling. This modification enhances the interaction between chitosan and the intestinal mucosa, increases its retention time in the colon, while maintaining the good biocompatibility and degradability of chitosan.

[0056] For material selection, chitosan (purchased from Sigma-Aldrich) with a molecular weight of approximately 150 kDa and a degree of deacetylation as high as 95% was used to ensure sufficient amino groups for modification. High-purity lipoic acid (LA) (≥99%, CAS Registry No. 1077-28-7, R-isomer) was selected to improve synthesis efficiency and product quality. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were used as activating agents, both with a purity of not less than 98%.

[0057] The synthesis process begins with the preparation of a chitosan solution. 2 g of chitosan was added to 200 mL of a 1% (v / v) aqueous acetic acid solution to a final concentration of 1% (w / v). The mixture was stirred thoroughly at room temperature (approximately 5 hours) to ensure complete dissolution of the chitosan. Subsequently, a small amount of insoluble matter was removed by filtration, yielding a clear chitosan solution. It is noteworthy that the dissolution of chitosan is pH-dependent; under acidic conditions, its amino groups are protonated, resulting in a homogeneous solution.

[0058] Activation of lipoic acid is a crucial step in the synthesis process. 500 mg of lipoic acid was dissolved in 30 mL of acetonitrile (approximately 16.7 mg / mL), followed by the addition of 270 mg of EDC and 164 mg of NHS, resulting in a lipoic acid:EDC:NHS molar ratio of 1:1.2:1.2. Under nitrogen protection, the mixture was stirred at room temperature for 1.5 hours to fully activate the carboxyl groups of the lipoic acid. In this step, EDC reacts with the carboxyl groups to form an unstable O-acylurea intermediate, which in turn reacts with NHS to form a more stable reactive ester, facilitating subsequent coupling reactions with chitosan amino groups.

[0059] The coupling reaction is the core step in the entire synthesis. The activated lipoic acid solution was slowly added dropwise to 100 mL of chitosan solution at a rate of approximately 1 mL / min, maintaining a lipoic acid to chitosan mass ratio of 0.25:1. The reaction was carried out at room temperature with gentle stirring (approximately 200 rpm) to ensure thorough mixing without damaging the chitosan molecular structure. The reaction continued for 24 hours, allowing the lipoic acid to form stable amide bonds with the amino groups on the chitosan.

[0060] After the reaction was complete, the product was purified by dialysis. The reaction mixture was placed in a dialysis bag (molecular weight cutoff 12,000-14,000 Da) and dialyzed against 0.2 M NaOH solution for 48 hours, with the dialysate changed every 12 hours for a total of 4 times. Alkaline conditions helped neutralize the remaining acetic acid and remove unreacted small molecule components. The dialyzed solution was freeze-dried (-50°C, 0.1 mbar, 48 hours) to obtain a pale yellow, loose lipoic acid-modified chitosan (LA-CS) powder.

[0061] To confirm the structure and properties of the synthesized product, several characterization analyses were performed. Fourier transform infrared spectroscopy (FTIR) analysis revealed a new amide bond peak at 1655 cm⁻¹, consistent with the characteristic absorption peak of the amide bond formed by the successful coupling of lipoic acid and chitosan. Nuclear magnetic resonance (NMR) spectroscopy (1H NMR)... 1 H-NMR analysis further confirmed the presence of lipoic acid, showing characteristic peaks of lipoic acid in the range of 1.2-2.5 ppm, corresponding to its aliphatic methyl and methylene hydrogen.

[0062] Elemental analysis is an important method for determining the degree of modification. By measuring the sulfur content (3.84%) and combining it with the molecular structures of chitosan and lipoic acid, the degree of lipoic acid modification was calculated to be 21.2%, which is within the set target range (15-25%). This degree of modification can maintain the basic properties of chitosan while providing sufficient lipoic acid functional groups to enhance its interaction with biomembranes.

[0063] Regarding physicochemical properties, LA-CS retained the pH sensitivity of chitosan, remaining soluble in weakly acidic conditions (pH 4.0-6.0) at concentrations up to 10 mg / mL, while solubility decreased under neutral and alkaline conditions. This property is beneficial for protecting the stability of encapsulated drugs in the acidic environment of the stomach and enabling controlled release when the intestinal pH increases. A 2% (w / v) LA-CS solution had a viscosity of 452 ± 18 mPa·s at 25°C, suitable for nanoparticle preparation. The Zeta potential measured at pH 5.5 was +35.6 ± 2.1 mV, indicating that LA-CS possesses good electrostatic stability and mucosal adhesion.

[0064] Comprehensive characterization results show that lipoic acid-modified chitosan with a modification degree of 21.2% was successfully synthesized. This material retains the key properties of chitosan and gains new functions conferred by lipoic acid, making it an ideal carrier material for constructing colon-targeted nanodelivery systems.

[0065] Example 3: Construction of a protopanaxadiol nanodelivery system

[0066] Based on the protopanaxadiol (PPT) nanocrystals and lipoic acid-modified chitosan (LA-CS) prepared in the previous two examples, a protopanaxadiol nanodelivery system was constructed using the ionogel method. This method utilizes the electrostatic interaction between chitosan and polyvalent anions to form a nanoscale cross-linked network, and is a mild, efficient, and solvent-free preparation technique.

[0067] In terms of materials, PPT nanocrystals prepared in Example 1, LA-CS synthesized in Example 2, and high-purity sodium tripolyphosphate (STPP, purity ≥98%, purchased from Sigma-Aldrich) were used as crosslinking agents. Furthermore, phosphate buffered saline (PBS, pH 7.4) and ultrapure water were used as dispersion media and washing solvents.

[0068] First, a PPT nanocrystal dispersion needs to be prepared. 50 mg of the PPT nanocrystals obtained in Example 1 were added to 20 mL of PBS (pH 7.4) to achieve a concentration of 2.5 mg / mL. To obtain a homogeneous dispersion, the mixture was moderately sonicated at low temperature (approximately 10°C) (300 W power, 8 minutes). It is important to note that the control of sonication time and power is crucial; it is necessary to ensure sufficient dispersion of the nanocrystals while avoiding excessive sonication that could alter the crystal structure or cause aggregation.

[0069] Next, the LA-CS solution was prepared. 200 mg of the LA-CS obtained in Example 2 was slowly added to 50 mL of 1% (v / v) aqueous acetic acid solution to achieve a concentration of 4 mg / mL. The solution was gently stirred at room temperature until completely dissolved, and then the pH was adjusted to 5.8 using dilute sodium hydroxide solution. This pH value was chosen to consider both the solubility of LA-CS and to facilitate the subsequent ionic cross-linking reaction with STPP. After adjustment, the solution was filtered through a 0.45 μm filter membrane to remove any trace amounts of insoluble matter, yielding a clear LA-CS solution.

[0070] Nanoparticle formation is the core step in this embodiment. Under stirring conditions (approximately 700 rpm), 20 mL of PPT nanocrystal dispersion was slowly added to 50 mL of LA-CS solution at a constant rate of 2 mL / min. At this point, the mass ratio of PPT nanocrystals to LA-CS was 1:4. This ratio, optimized through multiple preliminary experiments, ensures high encapsulation efficiency and suitable particle size. After addition, stirring continued for 30 minutes to form a stable primary complex. This stage mainly relies on electrostatic attraction and hydrogen bonding to bind the negatively charged PPT nanocrystals to the positively charged LA-CS.

[0071] To further stabilize the nanodelivery system, STPP was used for ionic crosslinking. 50 mg of STPP was dissolved in 10 mL of ultrapure water to prepare a 5 mg / mL solution. Under continuous stirring (700 rpm), the STPP solution was added dropwise to the mixture at a constant rate of 1.5 mL / min. At this point, the mass ratio of LA-CS to STPP was 4:1. This ratio ensures an appropriate crosslinking density, preventing over-crosslinking from causing an overly dense system that could affect drug release, and under-crosslinking from reducing the stability of the nanoparticles. The crosslinking reaction was carried out at room temperature for 1.5 hours. During this time, the polyvalent phosphate ions in STPP ionically crosslinked with the amino groups on LA-CS, forming a three-dimensional network structure that encapsulated the PPT nanocrystals.

[0072] After the reaction was complete, the nanoparticle precipitate was collected by centrifugation (12,000 × g, 20 min, 4°C). To remove unencapsulated PPT and free STPP, the precipitate was washed three times with ultrapure water. Finally, the purified nanoparticles were redispersed in 10 mL PBS (pH 7.4) to form a homogeneous suspension, or prepared into a dry powder by freeze-drying (-50°C, 0.1 mbar, 36 h) for long-term storage. For the preparation of freeze-dried products, an appropriate amount of freeze-drying protectant (such as 5% mannitol) is usually added to prevent damage to the nanostructure during the freeze-drying process.

[0073] To characterize the prepared protopanaxadiol nanoparticle delivery system, a series of physicochemical property analyses were performed. First, the particle size distribution of the nanoparticles was measured by dynamic light scattering (DLS), showing an average particle size of 387 ± 24 nm and a polydispersity index (PDI) of 0.25 ± 0.04, indicating a uniformly sized nanoparticle delivery system. The zeta potential measured in PBS (pH 7.4) was +32.7 ± 1.9 mV, significantly higher than the ± 30 mV stability threshold, indicating good colloidal stability. Transmission electron microscopy (TEM) further confirmed that the nanoparticles exhibited a regular spherical shape and a smooth, uniform surface, which is beneficial for their circulation and distribution in vivo.

[0074] Drug loading parameters are important indicators for evaluating the performance of a drug delivery system. Using high-performance liquid chromatography (HPLC), the calculated loading amount (DL%) of PPT was 14.2 ± 0.8%, significantly higher than that of conventional formulations, which reduces the amount of carrier material used and improves dosing convenience. The encapsulation efficiency (EE%) reached 85.3 ± 2.2%, demonstrating the high efficiency and economy of the preparation process. These excellent loading parameters are mainly attributed to the use of PPT nanocrystals and optimized preparation process parameters.

[0075] In vitro release behavior is an important basis for predicting the in vivo release characteristics of drugs. The drug release characteristics of the prepared nanodelivery system in different simulated physiological environments were studied using the dialysis bag method. The results showed that only 7.2±1.1% of PPT was released within 2 hours in simulated gastric juice (pH 1.2), and the cumulative release within 4 hours was less than 18.5±2.3% in simulated small intestinal juice (pH 6.8). However, the cumulative release reached as high as 82.4±3.6% within 24 hours in simulated colonic juice supplemented with colonic bacterial enzymes (pH 7.4). This significant pH-dependent release behavior and responsiveness to colonic enzymes fully demonstrate the colon-targeting characteristics of the constructed system, which can effectively reduce premature drug release and absorption loss in the upper gastrointestinal tract and increase its local concentration in the colon.

[0076] In terms of stability, the nanodelivery system exhibited a particle size change of no more than 10% and maintained a drug content of over 93% of its initial value within three months of storage at 4°C, demonstrating excellent physical and chemical stability. Even at room temperature of 25°C, the particle size increase was controlled within 15% within one month, and the drug content remained above 90% of its initial value, which is of great significance for practical applications and commercialization.

[0077] In summary, a protopanaxadiol nanodelivery system was successfully constructed using the ionogel method. This system exhibits ideal particle size, good stability, high drug loading capacity, and significant colon-targeted release characteristics, laying the foundation for subsequent in vivo studies.

[0078] Example 4: Inhibitory effect of protopanaxadiol nanodelivery system on the formation of Twist1-Ku70-Sirt1 complex

[0079] This embodiment aims to investigate the inhibitory effect of the protopanaxadiol nanodelivery system on the formation of the Twist1-Ku70-Sirt1 complex, and to compare it with free protopanaxadiol to verify the synergistic effect of the nanodelivery system. Human colon cancer HCT116 cells were selected as the research subject. This cell line is widely used in colon cancer research and has been reported to express high levels of Twist1 and Sirt1 proteins.

[0080] First, the cell culture conditions were optimized. HCT116 cells (purchased from ATCC, USA, catalog number CCL-247) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, maintained at 37°C and 5% CO2. At passage, when the cells reached approximately 80% confluence, they were digested with 0.25% trypsin-EDTA and reseeded. To ensure the reliability of the experimental results, all experiments used cells in the exponential growth phase, and the number of passages was controlled between 5 and 15.

[0081] In the drug treatment experiment, the following treatment groups were designed: (1) Control group: only an equal volume of PBS was added; (2) Low-dose free PPT group: 10 μM; (3) High-dose free PPT group: 20 μM; (4) Blank nanoparticle group: equivalent to the carrier concentration of the delivery system group; (5) Low-dose PPT nanodelivery system group: equivalent to 10 μM PPT; (6) High-dose PPT nanodelivery system group: equivalent to 20 μM PPT. The treatment time was 24 hours, and three parallel samples were set in each group to ensure the statistical significance of the results.

[0082] Cellular uptake studies are crucial for understanding the mechanisms of action of drug delivery systems. We investigated the cellular uptake behavior of a PPT nanodelivery system using a combination of confocal laser scanning microscopy (CLSM) and flow cytometry. For visualization, coumarin-6 was used to label PPT, and LA-CS membranes were labeled with DiI. Cell nuclei were stained with DAPI, and lysosomes were stained with LysoTracker Green. CLSM observations showed that after 2 hours of treatment, the PPT nanodelivery system significantly enhanced cellular uptake of PPT. The fluorescence signal was mainly distributed in the cytoplasm, with some co-localization with lysosomes, indicating that endocytosis is the primary uptake mechanism. Quantitative flow cytometry analysis further confirmed that the intracellular fluorescence intensity of the nanodelivery system group was approximately 3.2-fold higher than that of free PPT, indicating that the nanodelivery system significantly enhanced the cellular uptake efficiency of PPT.

[0083] To determine the effect of the nanodelivery system on the formation of the Twist1-Ku70-Sirt1 complex, the interaction of the three proteins was first investigated using co-immunoprecipitation (Co-IP). Specifically, total cellular protein was extracted using RIPA lysis buffer (containing protease and phosphatase inhibitors), and co-precipitation was performed using the Pierce™ Co-IP kit (Thermo Fisher Scientific). Using a Twist1 antibody as a decoy protein, the co-precipitated Ku70 and Sirt1 were detected by Western blotting. The results showed that in the control group, Twist1 significantly interacted with both Ku70 and Sirt1, confirming the formation of a complex. Compared to free PPT, the PPT nanodelivery system more effectively reduced the interaction between Twist1 and both Ku70 and Sirt1, with an approximately 2.5-fold increase in inhibition at the equivalent concentration (20 μM).

[0084] To further validate the above results, a dual immunofluorescence staining technique was employed. The treated cells were fixed and permeabilized, and then labeled with specific antibodies against Twist1 (rabbit anti), Ku70 (mouse anti), and Sirt1 (goat anti), respectively. They were then stained with secondary antibodies labeled with Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647, respectively. Confocal microscopy revealed significant co-localization of the three proteins in the control group, while the co-localization signal was significantly weakened in the PPT nanodelivery system-treated group, further confirming its inhibitory effect on complex formation.

[0085] In addition to protein-protein interactions, the effects of the PPT nanodelivery system on the expression levels of Twist1, Ku70, and Sirt1 were investigated. Western blot analysis showed that after 24 hours of treatment with the PPT nanodelivery system, Twist1 protein levels were significantly reduced (by approximately 45%), while the expression of Ku70 and Sirt1 showed no significant changes (reduced by approximately 12% and 8%, respectively). This indicates that the PPT nanodelivery system not only inhibited the interactions of the three proteins but also selectively reduced Twist1 expression, thereby further attenuating complex formation.

[0086] To investigate the relationship between inhibition of protein complex formation and cellular function, the effect of the PPT nanodelivery system on the stem cell characteristics of HCT116 cells was also evaluated. Using a suspension sphere culture method, the PPT nanodelivery system significantly inhibited the cells' ability to form spheres, reducing the number of spheres by approximately 68% and the sphere diameter by approximately 52% compared to the control group. Furthermore, flow cytometry analysis of the proportion of CD44+ / CD133+ double-positive cells (representing a subset of colon cancer stem cells) revealed that the positivity rate in the PPT nanodelivery system-treated group decreased from 16.8% in the control group to 4.2%, a reduction of 75%. These results indicate that by inhibiting the formation of the Twist1-Ku70-Sirt1 complex, the PPT nanodelivery system effectively weakens the stem cell characteristics of colon cancer cells.

[0087] In summary, this embodiment demonstrates that the protopanaxadiol nanodelivery system can significantly enhance cellular uptake of PPT, effectively inhibit the formation of the Twist1-Ku70-Sirt1 complex, and reduce the stem cell characteristics of colon cancer cells, providing a molecular mechanism basis for its application in the treatment of colitis-related colorectal cancer. Compared with free PPT, the nanodelivery system shows a significant synergistic effect, which may be related to its improved cellular uptake and stability.

[0088] Example 5: Antitumor effect of protopanaxadiol nanodelivery system in AOM-DSS-induced CAC animal model

[0089] This embodiment aims to evaluate the in vivo antitumor effects of the protopanaxadiol nanodelivery system, particularly its preventive and therapeutic effects on colitis-associated colorectal cancer (CAC). A mouse model of CAC induced by ginseng-dextran sulfate sodium (AOM-DSS) was selected. This model effectively simulates the disease progression from colitis to colorectal cancer in humans and is widely used in the evaluation of related drugs.

[0090] Animal experiments were conducted in accordance with the approval of the Laboratory Animal Ethics Committee. Male C57BL / 6 mice (weighing 20-25g) aged 6-8 weeks were selected and purchased from a laboratory animal center. All animals were housed in an SPF-grade environment with constant temperature (22±2°C), constant humidity (55±5%), and 12-hour light / dark alternation, with free access to food and water. After one week of acclimatization, the mice were randomly divided into 6 groups of 10 mice each: (1) Normal control group: no treatment was given; (2) Model group: AOM-DSS induced but no drug was administered; (3) Low-dose free PPT group: 10 mg / kg / day; (4) High-dose free PPT group: 20 mg / kg / day; (5) Low-dose PPT nanodelivery system group: equivalent to 10 mg / kg / day PPT; (6) High-dose PPT nanodelivery system group: equivalent to 20 mg / kg / day PPT.

[0091] The CAC model was established following the classic method with slight modifications. Specifically, mice (excluding the normal control group) were first given a single intraperitoneal injection of argentin (AOM, 12 mg / kg body weight). One week later, they were given sodium dextran sulfate (DSS, 2.5% w / v) dissolved in drinking water for three cycles. Each cycle consisted of 7 days of drinking DSS solution followed by a 14-day recovery period with ordinary drinking water. This alternating administration induced chronic colitis, which gradually developed into colorectal cancer. The entire experimental period lasted 10 weeks.

[0092] Drug treatment began on the same day as the first DSS administration, via gavage. The drug was dispersed in a 0.5% sodium carboxymethyl cellulose (CMC-Na) solution, with an administration volume of 10 mL / kg body weight, once daily until the end of the experiment. The normal control group and the model group received the same volume of 0.5% CMC-Na solution. Throughout the experiment, changes in mouse body weight, general condition, fecal characteristics, and rectal prolapse were recorded weekly.

[0093] At the end of the experiment, mice were euthanized after blood collection, and colon tissue was removed and colon length was measured (colonic shortening is an important indicator of colitis). The colon was then longitudinally dissected, and its contents were gently washed away in PBS. The number of tumors was carefully counted and tumor size (long axis × short axis) was measured under a dissecting microscope. A portion of the colon tissue was fixed in 4% paraformaldehyde for histopathological analysis, while the remaining tissue was rapidly frozen in liquid nitrogen for subsequent molecular biological assays.

[0094] The results showed that, compared with the normal control group, the model group mice exhibited significant weight loss, colon shortening, rectal prolapse, and tumor formation, confirming that AOM-DSS successfully induced the CAC model. Regarding therapeutic efficacy, the high-dose PPT nanodelivery system group showed the most significant anti-tumor effect. Compared with the model group, the number of colon tumors in this group mice decreased by approximately 73.5% (from an average of 12.4±2.1 to 3.3±0.9), and the total tumor volume decreased by approximately 85.2%. Notably, this effect was significantly superior to the free high-dose PPT group, which showed a reduction of approximately 41.9% in tumor number and 53.6% in total tumor volume. Even the low-dose PPT nanodelivery system group showed anti-tumor effects comparable to the free high-dose PPT group, demonstrating that the nanodelivery system significantly improved the bioavailability and therapeutic efficacy of PPT.

[0095] Histopathological analysis (HE staining) further supported these results. The model group mice exhibited significant inflammatory cell infiltration, crypt structure disorder, epithelial cell atypia, and tumor invasion in their colon tissue, while these pathological changes were significantly alleviated in the PPT nanodelivery system treatment groups, especially the high-dose group. Based on semi-quantitative scores of inflammation severity, ulcer area, crypt structure, and epithelial cell atypia, the total pathological score of the high-dose PPT nanodelivery system group was approximately 68.7% lower than the model group and approximately 42.3% lower than the free high-dose PPT group.

[0096] To investigate the molecular mechanism, immunohistochemical staining was performed on colon tissue. The results showed that, compared with the model group, the expression of Twist1 was significantly reduced in the PPT nanodelivery system-treated group, and the co-localization of Twist1 with Ku70 and Sirt1 was significantly decreased. Simultaneously, the expression of the proliferation marker Ki-67 and the stem cell markers CD44 and CD133 was also significantly reduced, indicating that the PPT nanodelivery system weakens tumor cell proliferation and stem cell characteristics by inhibiting the formation of the Twist1-Ku70-Sirt1 complex.

[0097] Pharmacokinetics and tissue distribution of protopanaxadiol (PPT) in vivo were also studied. LC-MS / MS was used to determine the concentrations of PPT in blood and tissues at different time points (0.5, 1, 2, 4, 8, 12, 24, and 48 hours after administration). The results showed that compared with free PPT, the PPT nanodelivery system significantly increased the colonic tissue exposure of PPT, with a colonic / blood drug concentration ratio increased by approximately 4.2 times. Particularly in the distal colon (within 5 cm of the anus), the PPT concentration reached the therapeutically effective level and was maintained for a longer period. This result strongly demonstrates the colonic targeting capability of the PPT nanodelivery system and explains its enhanced antitumor effect.

[0098] Safety evaluation showed no significant toxic side effects in any treatment group. During the 10-week administration period, there were no significant abnormalities in body weight, food intake, or general behavioral activity. Complete blood counts and liver and kidney function indicators were within normal ranges, and pathological sections of major organs (heart, liver, spleen, lungs, and kidneys) showed no significant abnormalities. Notably, two mice in the high-dose free PPT group exhibited mild liver function abnormalities (mildly elevated ALT and AST), while no such abnormalities were observed in the PPT nanodelivery system at the same dose group, indicating that the nanodelivery system can also reduce systemic drug toxicity.

[0099] In summary, this embodiment demonstrates that the protopanaxadiol nanodelivery system exhibits significant antitumor activity in an AOM-DSS-induced CAC animal model, with effects significantly superior to an equal dose of free protopanaxadiol. Its mechanism of action is related to the inhibition of Twist1-Ku70-Sirt1 complex formation, reducing tumor cell proliferation and stem cell characteristics. Furthermore, this nanodelivery system demonstrates good colon-targeting ability and safety, providing important preclinical evidence for clinical application.

[0100] Example 6: Study on the combined treatment of protopanaxadiol nanodelivery system and immune checkpoint inhibitors

[0101] Given the groundbreaking progress of immune checkpoint inhibitors in the treatment of various tumors, and the key role of the tumor microenvironment in the development of colitis-associated colorectal cancer (CAC), this embodiment aims to explore the synergistic antitumor effect and potential mechanism of the combined use of protopanaxadiol nanodelivery system and anti-PD-1 antibody.

[0102] A similar AOM-DSS-induced CAC mouse model to that in Example 5 was used, but the grouping was adjusted. Mice were randomly divided into 8 groups of 8 mice each: (1) normal control group; (2) model group; (3) anti-PD-1 antibody group: 5 mg / kg, intraperitoneal injection, once every 3 days; (4) free PPT group: 20 mg / kg / day, orally; (5) PPT nanodelivery system group: equivalent to 20 mg / kg / day PPT, orally; (6) free PPT + anti-PD-1 antibody combination group; (7) PPT nanodelivery system + anti-PD-1 antibody combination group; (8) isotype control antibody group: 5 mg / kg, intraperitoneal injection, once every 3 days. The administration regimen was similar to that in Example 5, with anti-PD-1 antibody (clone number RMP1-14, purchased from BioXCell) or isotype control antibody administered from the second DSS cycle.

[0103] The results showed that, compared with the single-drug treatment group, the combination of the PPT nanodelivery system and the anti-PD-1 antibody exhibited a significant synergistic anti-tumor effect. The number and total volume of tumors in this group were reduced by approximately 86.4% and 92.3% respectively compared to the model group, far superior to the PPT nanodelivery system single-drug group (reductions of approximately 72.6% and 84.5%, respectively) and the anti-PD-1 antibody single-drug group (reductions of approximately 45.3% and 58.7%, respectively). Notably, the effect of the free PPT combined with the anti-PD-1 antibody group was also enhanced, but not as significantly as that of the PPT nanodelivery system combination group.

[0104] To explore the mechanism of synergistic effects, the tumor microenvironment was analyzed in depth. Flow cytometry analysis of infiltrating immune cells in colon tissue revealed that combined treatment with the PPT nanodelivery system and anti-PD-1 antibody significantly increased the proportion and activity of CD8+ T cells (manifested as increased expression of IFN-γ and granzyme B), while decreasing the proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Furthermore, the proportion of M1 macrophages increased while the proportion of M2 macrophages decreased in the tumor tissue of the combined treatment group, indicating a shift in the tumor microenvironment from immunosuppressive to immunoactivating.

[0105] Immunohistochemical and immunofluorescence analyses also supported these results. Compared with the monotherapy group, the combination therapy group showed increased CD8+ T cell infiltration, decreased PD-1 and PD-L1 expression, and reduced expression of the tumor cell proliferation marker Ki-67 in tumor tissue. Furthermore, a further reduction in the co-localization of Twist1 with Ku70 and Sirt1 was observed in the combination therapy group, indicating that the inhibitory effect of the PPT nanodelivery system on the Twist1-Ku70-Sirt1 complex was maintained and possibly enhanced in the combination therapy.

[0106] To further understand the molecular mechanisms, RNA sequencing analysis was performed on colon tissue. Compared with the monotherapy group, the combination therapy group showed upregulated expression of genes related to T cell activation, antigen presentation, cytotoxicity, and cytokine signaling, while downregulated expression of genes related to tumor promotion, immunosuppression, and cell stemness. Pathway enrichment analysis revealed significant activation of the NFκB signaling pathway, JAK-STAT signaling pathway, and T cell receptor signaling pathway in the combination therapy group, which may be the molecular basis for the synergistic anti-tumor effect.

[0107] The effects of different dosing sequences on the efficacy of combination therapy were also investigated. Results showed that sequential therapy, administering the PPT nanodelivery system for 3 days followed by anti-PD-1 antibody, was more effective than simultaneous administration or administration of the anti-PD-1 antibody first. This suggests that the PPT nanodelivery system may enhance the efficacy of subsequent immune checkpoint inhibitors by pre-modulating the tumor microenvironment.

[0108] Safety evaluation showed that the combination therapy was well tolerated. No increased incidence of immune-related adverse events (irAEs) was observed in the combination therapy group compared to the anti-PD-1 antibody monotherapy group. It is noteworthy that previous reports have suggested that certain combination therapy regimens may increase the risk of immune-related adverse events, such as the combination of immune checkpoint inhibitors with certain chemotherapy drugs. However, in this study, after a 10-week treatment cycle, mice in the PPT nanodelivery system combined with the anti-PD-1 antibody group did not exhibit common immune-related adverse events such as abnormal weight loss, skin lesions, or exacerbation of colitis. Blood biochemistry indicators showed that liver function (ALT, AST) and kidney function (BUN, Cr) were within the normal range and showed no significant differences compared to the monotherapy group and the normal control group.

[0109] In addition, cytokine profiling was performed, measuring serum levels of pro-inflammatory cytokines such as IL-6, TNF-α, IL-17A, and IL-2, and anti-inflammatory cytokines such as IL-10. The results showed that compared to the anti-PD-1 antibody monotherapy group, the levels of pro-inflammatory cytokines in the combination therapy group were not significantly increased; in fact, the levels of IL-6 and TNF-α were even reduced, which may be related to the anti-inflammatory effect of the PPT nanodelivery system. This result further supports the safety of the combination therapy, indicating that the PPT nanodelivery system not only does not exacerbate the excessive immune activation that may be caused by immune checkpoint inhibitors, but may instead balance the immune response through its anti-inflammatory effect.

[0110] The effects of the combined therapy on normal mucosal tissue were also evaluated. Histopathological examination showed no significant inflammatory response or tissue damage in non-tumor tissues adjacent to normal colonic mucosa, indicating that the combined therapy had good tissue selectivity. This may be due to the colon-targeting nature of the PPT nanodelivery system, which allows the drug to be released and act primarily at the lesion site, reducing the impact on normal tissue.

[0111] Synergy index (CI) analysis in preclinical pharmacology is an important method for evaluating the value of combination therapy. CI values ​​for different dose combinations were calculated using the Chou-Talalay method. The results showed that the CI value for the PPT nanodelivery system and the anti-PD-1 antibody was 0.72, indicating a significant synergistic effect (CI < 1 indicates synergy). This result provides an important reference for the design of combination therapy regimens in subsequent clinical studies.

[0112] In summary, this embodiment demonstrates that the combined use of the protopanaxadiol nanodelivery system and the anti-PD-1 antibody exhibits significant synergistic anti-tumor effects and good safety. Its mechanism of action involves the inhibition of the Twist1-Ku70-Sirt1 complex and the improvement of the tumor immune microenvironment. This combination therapy strategy provides a new approach and approach for the treatment of colitis-associated colorectal cancer, particularly suitable for patients who do not respond well to single immune checkpoint inhibitors. The superiority of the sequential dosing regimen also provides a basis for optimizing the order of clinical medication.

[0113] Example 7: Research on the Extended Application of the Protopanaxadiol Nanodelivery System

[0114] Based on the foregoing embodiments, this embodiment aims to explore the potential application of the protopanaxadiol nanodelivery system in other intestinal disease models, and to further study its molecular mechanisms related to the Twist1-Ku70-Sirt1 complex.

[0115] First, the preventive and therapeutic effects of the PPT nanodelivery system in a DSS-induced colitis model were investigated. Unlike the AOM-DSS model, the simple DSS-induced model mainly presents with colitis rather than tumors, which is closer to the early stage of inflammatory bowel disease. Acute colitis was induced by 3% DSS solution for 7 consecutive days, followed by a 14-day recovery period with normal drinking water. Mice were divided into a prevention group (administered 3 days before DSS administration) and a treatment group (administered after DSS cessation), with a dose equivalent to 20 mg / kg / day of PPT.

[0116] The results showed that in the prevention group, the PPT nanodelivery system significantly alleviated symptoms such as DSS-induced weight loss, colonic shortening, and fecal bleeding, with a Disease Activity Index (DAI) score reduced by approximately 65% ​​compared to the model group. Histopathological analysis revealed a significant reduction in colonic mucosal inflammation and ulcer area, and a decrease in inflammatory cell infiltration in the prevention group. In the treatment group, the PPT nanodelivery system accelerated the recovery from colitis, manifested as faster weight gain during the recovery period and enhanced colonic mucosal repair. These results indicate that the PPT nanodelivery system not only possesses anti-tumor effects but also has the potential to prevent and treat colitis, which is of significant importance for tumor prevention in high-risk patients with inflammatory bowel disease.

[0117] Secondly, the role of the PPT nanodelivery system in other intestinal tumor models expressing the Twist1-Ku70-Sirt1 complex was investigated. Screening various intestinal tumor cell lines revealed that, in addition to HCT116, the human gastric cancer cell line MGC-803 and the human small intestinal adenocarcinoma cell line SIA also expressed high levels of Twist1, Ku70, and Sirt1, and these three compounds showed co-localization. In vitro experiments showed that the PPT nanodelivery system also exhibited significant inhibitory effects on proliferation and reduced stem cell characteristics in these two cell lines, with IC50 values ​​equivalent to 12.4 μM and 15.8 μM PPT, respectively. This result expands the potential indications for the PPT nanodelivery system, suggesting its potential effectiveness against various gastrointestinal tumors.

[0118] Third, through gene knockdown and overexpression experiments, the key role of the Twist1-Ku70-Sirt1 complex in the mechanism of action of the PPT nanodelivery system was investigated in depth. In HCT116 cells, the expression of Twist1, Ku70, and Sirt1 was knocked down, respectively. The results showed that in Twist1 knockdown cells, the anti-proliferative and anti-stem cell properties of the PPT nanodelivery system were significantly weakened, while this weakening was less pronounced in Ku70 or Sirt1 knockdown cells. Conversely, in Twist1 overexpression cells, the effect of the PPT nanodelivery system was enhanced. This indicates that Twist1 is the primary target of the PPT nanodelivery system, while Ku70 and Sirt1 may play auxiliary roles.

[0119] Further mechanistic studies revealed that the PPT nanodelivery system not only inhibits the formation of the Twist1-Ku70-Sirt1 complex through direct binding but also affects downstream signaling pathways of the complex. Phosphorylated proteomics analysis showed that treatment with the PPT nanodelivery system regulated multiple signaling pathways related to EMT and stem cell characteristics, including the Wnt / β-catenin, Notch, and Hedgehog pathways. In particular, the PPT nanodelivery system significantly reduced β-catenin nuclear translocation and TCF / LEF-mediated transcriptional activity, which may be one of the important mechanisms by which it inhibits tumor stem cell characteristics.

[0120] Finally, the PPT nanodelivery system was explored as a biomarker for predicting and screening patients for treatment response. By analyzing the expression levels and co-localization of Twist1, Ku70, and Sirt1 in tumor tissues from mice in each experimental group, it was found that tumors with high Twist1 expression and high co-localization of these three markers responded better to the PPT nanodelivery system. This finding provides a potential predictive biomarker for patient selection in future clinical trials.

[0121] In summary, this embodiment further expands the application scope and mechanism research of the original ginsenoside nanodelivery system, providing more comprehensive experimental evidence for its clinical translation in the treatment of inflammatory diseases of the digestive tract and tumors.

[0122] This invention provides a protopanaxadiol nanodelivery system targeting the Twist1-Ku70-Sirt1 complex and its application in the treatment of colitis-associated colorectal cancer. By preparing protopanaxadiol into nanocrystals and constructing a colon-targeted delivery system using lipoic acid-modified chitosan, the bioavailability and targeting of protopanaxadiol are significantly improved. This nanodelivery system effectively inhibits the formation of the Twist1-Ku70-Sirt1 complex, reduces the stem cell characteristics of colon cancer cells, and exhibits significant anti-tumor effects in animal models. Furthermore, this system, when used in combination with immune checkpoint inhibitors, demonstrates synergistic anti-tumor effects and good safety, providing a new strategy for the comprehensive treatment of colitis-associated colorectal cancer.

[0123] Although specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the described specific embodiments. Those skilled in the art should understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the basic ideas and principles of the present invention, and such changes, modifications, substitutions, and variations are still within the protection scope of the present invention.

Claims

1. A protopanaxadiol nanodelivery system targeting the Twist1-Ku70-Sirt1 complex, characterized in that, The nanodelivery system includes: (a) Protopanaxadiol nanocrystals, wherein the protopanaxadiol nanocrystals are prepared by an ultrasonic-assisted antisolvent precipitation method, wherein ethanol is used as a solvent, ultrapure water is used as an antisolvent, and polyvinylpyrrolidone K30 or polysorbate 80 is used as a stabilizer. (b) Lipoic acid-modified chitosan, wherein the chitosan has a molecular weight of 100-200 kDa and a degree of deacetylation ≥90%, and wherein the lipoic acid-modified chitosan is synthesized via an EDC / NHS activated coupling method, wherein EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and NHS is N-hydroxysuccinimide; and (c) A crosslinking agent, wherein the crosslinking agent is sodium tripolyphosphate; The nanocrystals of protopanaxadiol have an average particle size of 100-200 nm; the lipoic acid-modified chitosan has a modification degree of 15-25%; the nanodelivery system has an average particle size of 300-500 nm; the mass ratio of protopanaxadiol nanocrystals to lipoic acid-modified chitosan is 1:2 to 1:5, and the mass ratio of lipoic acid-modified chitosan to sodium tripolyphosphate is 3:1 to 5:1; the nanodelivery system has an absolute zeta potential ≥30 mV, a polydispersity index <0.3, a protopanaxadiol loading ≥10%, and an encapsulation efficiency ≥80%. In the nanodelivery system, protopanaxadiol binds to tyrosine at position 320 of Ku70 and glutamate at position 32 of Sirt1, thereby inhibiting the formation of the Twist1-Ku70-Sirt1 ternary protein complex.

2. The nanodelivery system according to claim 1, characterized in that, The nanodelivery system releases <20% protopanaxadiol within 4 hours in simulated gastric juice (pH 1.2) and small intestinal juice (pH 6.8), and >80% protopanaxadiol within 24 hours in simulated colonic juice (pH 7.4) with added colonic bacterial enzymes, demonstrating colon-targeted release characteristics.

3. A method for preparing the nanodelivery system according to claim 1 or 2, characterized in that, Includes the following steps: (a) Protopanaxadiol was dissolved in ethanol, a stabilizer was added, and the protopanaxadiol solution was added to ultrapure water under ultrasonic conditions to prepare protopanaxadiol nanocrystals. (b) Lipoic acid was activated by EDC / NHS, and the activated lipoic acid was reacted with chitosan to prepare lipoic acid modified chitosan. (c) The protopanaxadiol nanocrystals prepared in step (a) are dispersed in a solution containing lipoic acid-modified chitosan prepared in step (b), and then cross-linked with sodium tripolyphosphate solution to form a nanodelivery system; and (d) Collect and purify the nanodelivery system.

4. The use of the nanodelivery system of claim 1 or 2 in the preparation of a medicament for treating colitis-associated colorectal cancer, wherein the nanodelivery system functions through the following mechanism: protopanaxadiol binds to tyrosine residue 320 of Ku70 and glutamate residue 32 of Sirt1, targeting and inhibiting the formation of the Twist1-Ku70-Sirt1 ternary protein complex, reducing the stem cell characteristics of cancer cells and tumorigenesis; wherein the medicament is an oral formulation, administered at a dose equivalent to 10-20 mg / kg / day of protopanaxadiol, for the treatment of AOM-DSS-induced colitis-associated colorectal cancer.

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