Twist1-Ku70-Sirt1 compound-targeted protopanaxatriol nano-delivery system and application thereof in treatment of colitis-related colorectal cancer

Abstract

The invention relates to the field of medicine, in particular to a protopanaxatriol nanometer delivery system targeting a Twist1-Ku70-Sirt1 compound and application of the nanometer delivery system in treatment of colorectal cancer related to colitis, and the nanometer delivery system comprises: (a) a protopanaxatriol nanometer crystal; (b) chitosan modified by lipoic acid; and (c) a cross-linking agent; wherein the average particle size of the protopanaxatriol nanocrystal ranges from 100 nm to 200 nm; the modification degree of the lipoic acid modified chitosan is 15 to 25 percent; the cross-linking agent is sodium tripolyphosphate; the average particle size of the nano delivery system is 300-500 nm, and the solubility and bioavailability of the nano delivery system are remarkably improved by preparing the protopanaxatriol into nano crystals.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a protopanaxatriol nano-delivery system targeting the Twist1-Ku70-Sirt1 complex and the application of the nano-delivery system in the treatment of colitis-associated colorectal cancer. Background Art

[0002] Colitis-associated colorectal cancer (CAC) is one of the most serious complications of patients with inflammatory bowel disease (IBD). Long-term chronic inflammation is the main risk factor for the occurrence of CAC. Epidemiological data show that the risk of colorectal cancer in patients with inflammatory bowel disease is significantly higher than that in the general population. Among them, the lifetime incidence of colorectal cancer in patients with ulcerative colitis is as high as 18%. Compared with sporadic colorectal cancer, CAC is characterized by early onset, multiple occurrences and poor prognosis, bringing a huge burden to patients.

[0003] The existing treatment regimens for CAC mainly include anti-inflammatory treatment, chemotherapy, surgical resection, etc. However, these treatment methods have limited efficacy and are often accompanied by serious adverse reactions. At present, there is a lack of targeted therapeutic drugs for the specific pathological mechanism of CAC in clinical practice, especially drugs that can inhibit the characteristics of cancer stem cells and tumorigenesis. This is the main challenge faced in the treatment of CAC.

[0004] In recent years, studies have found that various transcription factors and signaling pathways in the tumor microenvironment play important roles in the occurrence and development of CAC. Among them, Twist1, as 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 Twist1 can promote the maintenance of cancer cell stem cell characteristics, invasion and metastasis, and drug resistance in colorectal cancer. 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. (Scientific Reports, 2014) found that Sirt1 is highly expressed in colorectal cancer tissues and is significantly associated with poor prognosis of patients. Moreover, Sirt1 co-localizes with the colorectal cancer stem cell marker CD133.

[0005] At the molecular mechanism level, Roth et al. (Oncotarget, 2016) first reported the molecular mechanism by which SIRT1 and LSD1 competitively regulate the function of Ku70, affecting DNA repair and the acquisition of drug-resistant mutations in tumor cells. Studies have shown that Ku70 is a key protein in the non-homologous end joining (NHEJ) DNA repair pathway, and SIRT1 enhances its DNA repair function by deacetylating Ku70. In addition, Cohen et al. (Science, 2004) confirmed that SIRT1 regulates the sensitivity of cells to apoptotic stimuli by deacetylating Ku70. However, these studies mainly focused on the binary protein interaction level, and there is still a lack of in-depth research on potential ternary protein complexes and their roles in tumorigenesis.

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

[0007] As a novel drug delivery platform, the nano-delivery system has made remarkable progress in improving the bioavailability of poorly soluble drugs, enhancing targeting, and controlling release in recent years. Gao Jun et al. (International Journal of Pharmaceutical Research, 2018) reviewed the application progress of the nano-delivery system in the treatment of colorectal cancer and pointed out that the nano-delivery system can significantly increase the enrichment of drugs at the tumor site and reduce systemic toxicity. Chitosan, as a natural polysaccharide with good biocompatibility and biodegradability, 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 the release of drugs in the colon and improve its therapeutic effect on colitis. However, there is no report on combining PPT with a targeted colon delivery system for the targeted treatment of CAC.

[0008] Please refer to Figure 1A-1D. The present inventors found that Twist1, Ku70, and Sirt1 can form a ternary protein complex, which promotes stem cell characteristics and CAC tumorigenesis. By screening small molecule natural drugs that can target this complex using molecular docking technology, it was found that PPT can bind to tyrosine at position 320 of Ku70 and glutamate at position 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, the present invention aims to develop a nano-delivery system that can improve the bioavailability and targeting of PPT to enhance its therapeutic effect on CAC. Summary of the Invention

[0009] The object of the present invention is to provide a nano-delivery system of protopanaxatriol targeting the Twist1-Ku70-Sirt1 complex and its preparation method, as well as the application of this nano-delivery system in the treatment of colitis-associated colorectal cancer. The nano-delivery system of the present invention can significantly improve the bioavailability and colon targeting of protopanaxatriol, enhance its effect of inhibiting the formation of the Twist1-Ku70-Sirt1 complex, and thus more effectively reduce stem cell characteristics and CAC tumorigenesis.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] In the first aspect, the present invention provides a nano-delivery system of protopanaxatriol targeting the Twist1-Ku70-Sirt1 complex, including:

[0012] (a) Protopanaxatriol nanocrystals;

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

[0014] (c) A cross-linking agent;

[0015] Wherein, the average particle size of the protopanaxatriol nanocrystals is 100-200 nm; the modification degree of the thioctic acid-modified chitosan is 15-25%; the cross-linking agent is sodium tripolyphosphate; and the average particle size of the nano-delivery system is 300-500 nm.

[0016] In another preferred embodiment of the present invention, the protopanaxatriol nanocrystals are prepared by ultrasonic-assisted anti-solvent precipitation method, where ethanol is used as the solvent, water is used as the anti-solvent, and polyvinylpyrrolidone K30 or polysorbate 80 is used as the stabilizer.

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

[0018] In yet another preferred embodiment of the present invention, the mass ratio of the protopanaxatriol 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 yet another preferred embodiment of the present invention, the absolute value of the Zeta potential of the nano-delivery system is ≥30 mV, the polydispersity index is <0.3, the protopanaxatriol loading amount is ≥10%, and the encapsulation efficiency is ≥80%.

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

[0021] In a second aspect, the present invention provides a method for preparing the above nano-delivery system, comprising the following steps:

[0022] (a) Dissolve protopanaxatriol in ethanol, add a stabilizer, and under ultrasonic conditions, add the protopanaxatriol solution to ultrapure water to prepare protopanaxatriol nanocrystals;

[0023] (b) Activate lipoic acid with EDC / NHS, and react the activated lipoic acid with chitosan to prepare lipoic acid-modified chitosan;

[0024] (c) Disperse the protopanaxatriol nanocrystals prepared in step (a) in a solution containing the lipoic acid-modified chitosan prepared in step (b), and then add a sodium tripolyphosphate solution for crosslinking to form a nano-delivery system; and

[0025] (d) Collect and purify the nano-delivery system.

[0026] In a third aspect, the present invention provides the use of the nano-delivery system in the preparation of a drug for treating colitis-related colorectal cancer, wherein the nano-delivery system reduces the cancer stem cell characteristics and tumorigenesis of cancer cells 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 preparation, and the dosage is equivalent to 10 - 20 mg / kg / day of protopanaxatriol, which is used for the treatment of colitis-associated colorectal cancer induced by AOM-DSS, or is used in combination with an immune checkpoint inhibitor for the treatment of patients with colitis-associated colorectal cancer with high expression of the Twist1-Ku70-Sirt1 complex.

[0028] The protopanaxatriol nanodelivery system provided by the present invention has the following beneficial effects:

[0029] 1. By preparing protopanaxatriol into nanocrystals, its solubility and bioavailability are significantly improved. As a poorly soluble compound, protopanaxatriol has low bioavailability in conventional preparations. However, the 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. The nanodelivery system constructed by modifying chitosan with lipoic acid realizes the colon-targeted delivery of protopanaxatriol. The lipoic acid modification enhances the adhesion of chitosan to the intestinal mucosa, and at the same time, chitosan can be degraded by colonic microbial enzymes, thus specifically releasing the drug at the colon site. In vitro release experiments show that the drug release amount of this system in simulated gastric juice and small intestinal juice is <20%, while the release amount in simulated colonic juice is >80% within 24 hours, showing obvious colon targeting.

[0031] 3. The efficiency of protopanaxatriol in inhibiting the formation of the Twist1-Ku70-Sirt1 complex is significantly improved. Through the nanodelivery system, the concentration of protopanaxatriol in the colon tissue is significantly increased, and the colon / blood drug concentration ratio is increased by about 4 times compared with free protopanaxatriol. Co-immunoprecipitation experiments confirm that the inhibitory effect of protopanaxatriol in the nanodelivery system on the formation of the Twist1-Ku70-Sirt1 complex is increased by about 2.5 times compared with free protopanaxatriol.

[0032] 4. In the AOM-DSS-induced CAC animal model, the nanodelivery system of the present invention significantly reduces the number and size of colon tumors. Compared with an equal dose of free protopanaxatriol, the effect of this nanodelivery system in reducing the number of tumors is increased by about 60%, and the effect of reducing the tumor volume is increased by about 70%. In addition, histopathological analysis shows that this nanodelivery system more effectively reduces the atypia and infiltration degree of tumor tissues.

[0033] 5. It has good safety and no obvious toxic and side effects are observed. Long-term administration experiments show that this nanodelivery system does not affect the body weight gain of experimental animals, the liver and kidney function indexes are within the normal range, and no obvious pathological changes are found in the main organs. In contrast, there are mild abnormalities in liver function indexes in the group administered with an equal dose of free protopanaxatriol.

[0034] In summary, the protopanaxatriol nano-delivery system provided by the present invention significantly enhances the effect of inhibiting the formation of the Twist1-Ku70-Sirt1 complex by improving the solubility, bioavailability, and colon targeting of protopanaxatriol, providing a new strategy and drug delivery system for the targeted treatment of colitis-associated colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0036] Figure 1 B is 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 is the experimental result of PPT inhibiting the formation of the Twist1-Ku70-Sirt1 complex, showing the results of co-immunoprecipitation (Co-IP) experiments;

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

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

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

[0042] Figure 1 H is the histopathological result of H&E staining. DETAILED DESCRIPTION OF THE INVENTION

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

[0044] Through research and verification, it is confirmed that Twist1 can form a complex with Ku70 and Sirt1, promoting stem cell characteristics and tumorigenesis of CAC. This finding indicates that the Twist1-Ku70-Sirt1 complex may be a potential target for the treatment of CAC. Small molecule natural drugs that can target 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), in which PPT has the highest score ( Figure 1 A). The molecular structure of PPT is as Figure 1 shown in B. The molecular docking results show that PPT binds to tyrosine (TRY) at the 320th amino acid of Ku70 and glutamate (GLU) at the 32nd amino acid of Sirt1 ( Figure 1 C). Co-immunoprecipitation (Co-IP) experiments demonstrate that 20 μM of PPT can inhibit the interaction of the Twist1-Ku70-Sirt1 complex in HCT116 cells overexpressing Twist1 ( Figure 1 D). In addition, sphere formation assays, colony formation assays, and soft agar colony formation assays show that, compared with the control group, the formation rates of spheres and colonies in vitro are significantly reduced after PPT treatment ( Figure 1 E). These data indicate that PPT can inhibit the stem cell properties 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, the rectal prolapse of mice treated with PPT is reduced, and the number of visible tumors is also less ( Figure 1 G). In addition, hematoxylin-eosin (HE) staining shows that, compared with the control group of mice, the tumor infiltration in the tissues of mice treated with PPT is reduced and the atypia is decreased ( Figure 1 H). These results indicate that PPT inhibits stem cell properties and CAC tumorigenesis by regulating the interaction of the Twist1-Ku70-Sirt1 complex.

[0045] Example 1: Preparation of protopanaxatriol nanocrystals

[0046] First, protopanaxatriol (PPT) nanocrystals need to be prepared in the present invention to improve its solubility and bioavailability. The ultrasonic-assisted anti-solvent precipitation method is used for preparation. This method is simple to operate, has strong repeatability, and is conducive to controlling the particle size distribution of nanocrystals.

[0047] Specifically, protopanaxatriol with a purity of not less than 98% (purchased from Sigma-Aldrich) is used as the raw material, absolute ethanol (analytical pure, ≥99.5%) is used as the solvent, ultrapure water (resistivity ≥18.2 MΩ·cm, 25°C) is used as the anti-solvent, and polyvinylpyrrolidone K30 (PVP K30, molecular weight about 40,000) is used as the stabilizer.

[0048] During the preparation process, first dissolve 100 mg of protopanaxatriol in 10 mL of absolute ethanol, and at the same time add 100 mg of PVP K30 to make the mass ratio of PPT to PVP K30 1:1. Under room temperature conditions, magnetically stir the mixture (rotation speed about 600 rpm) until it is completely dissolved to form a clear solution. Subsequently, add 100 mL of ultrapure water to a 250 mL beaker and place it under ultrasonic conditions (power set to 400 W), while controlling the temperature within the range of 10 - 15 °C. It should be noted that temperature control is crucial for preventing the degradation of thermosensitive drugs and controlling the crystallization process.

[0049] Next, use a constant flow pump to inject the above PPT ethanol solution into the stirred ultrapure water at a rate of 3 mL / min. During this process, due to the anti-solvent effect, the solubility of PPT in the aqueous phase drops sharply, resulting in supersaturation and the formation of nanoscale crystals. The energy provided by ultrasonic waves helps to promote uniform nucleation and prevent the formation of large crystals. After the injection is completed, continue ultrasonic treatment for 15 minutes to stabilize the formed nanocrystal suspension, and at this time the solution is milky white.

[0050] To collect and purify the formed PPT nanocrystals, a high-speed centrifugation (18,000×g, 15 minutes, 4 °C) method is used. Subsequently, wash the precipitate three times with ultrapure water to thoroughly remove the residual organic solvents and unbound stabilizers. Finally, freeze-dry the purified nanocrystals (-50 °C, 0.1 mbar, about 36 hours) to obtain a white to slightly yellow PPT nanocrystal powder, which is convenient for long-term storage and subsequent applications.

[0051] To characterize the prepared PPT nanocrystals, a series of analyses were carried out. First, the particle size distribution was measured using dynamic light scattering (DLS) method, and the results showed that the average particle size of PPT nanocrystals was 156 ± 12 nm, and the polydispersity index (PDI) was 0.22 ± 0.03, indicating a relatively uniform particle size distribution. The observation results of transmission electron microscopy (TEM) showed that the nanocrystals were nearly spherical, evenly dispersed, and there was no obvious aggregation, which was beneficial to improving their stability and bioavailability.

[0052] In addition, through X-ray powder diffraction (XRPD) analysis, it was found that the prepared PPT nanocrystals presented a partially amorphous state with reduced crystallinity, which helped to further improve 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 that the nanocrystals had good electrostatic stability. The storage stability study showed that when stored at 4 °C for 14 days, the particle size increased by no more than 5%, proving the reliability of the preparation process and the good stability of the product.

[0053] It is worth mentioning that compared with the original PPT crystals, the prepared PPT nanocrystals showed an approximately 4.2-fold increase in apparent solubility in PBS (pH 7.4). This significant improvement is mainly attributed to the high specific surface area, lower crystallinity of the nanocrystals, and the surface stabilization provided by PVP K30. This result indicates that nanocrystal technology is an effective strategy to improve the water solubility and bioavailability of PPT.

[0054] Example 2: Synthesis of Lipoic Acid-Modified Chitosan

[0055] To construct a colon-targeted delivery system, appropriate carrier materials need to be prepared. In this example, lipoic acid-modified chitosan (LA-CS) was synthesized by the EDC / NHS activation coupling method. This modification can enhance the interaction between chitosan and intestinal mucosa, increase the residence time in the colon, while maintaining the good biocompatibility and degradability of chitosan.

[0056] In terms of material selection, chitosan with a molecular weight of about 150 kDa and a deacetylation degree as high as 95% (purchased from Sigma-Aldrich) was used to ensure sufficient amino groups for modification. High-purity lipoic acid (LA) (≥99%, CAS registration number 1077-28-7, R-isomer) was selected to improve the synthesis efficiency and product quality. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were used as activation reagents, with a purity of not less than 98%.

[0057] The synthesis process starts with the preparation of the chitosan solution. 2 g of chitosan was added to 200 mL of 1% (v / v) acetic acid aqueous solution to make the final concentration 1% (w / v). Under room temperature conditions, this mixture was stirred thoroughly (about 5 hours) to ensure complete dissolution of chitosan. Subsequently, a small amount of insoluble matter was removed by filtration to obtain a clear chitosan solution. It should be noted that the dissolution of chitosan depends on the pH value. Under acidic conditions, its amino groups are protonated, thus dissolving to form a homogeneous solution.

[0058] The activation of lipoic acid is a key step in the synthesis process. 500 mg of lipoic acid was dissolved in 30 mL of acetonitrile (concentration about 16.7 mg / mL), and then 270 mg of EDC and 164 mg of NHS were added to make the molar ratio of lipoic acid:EDC:NHS 1:1.2:1.2. Under nitrogen protection, this mixture was stirred at room temperature for 1.5 hours to fully activate the carboxyl group of lipoic acid. In this step, EDC reacts with the carboxyl group to form an unstable O-acylurea intermediate, and then NHS reacts with this intermediate to form a relatively stable active ester, which is beneficial to the subsequent coupling reaction with the amino group of chitosan.

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

[0060] After the reaction was completed, the product was purified by dialysis. The reaction mixture was loaded into a dialysis bag (cut-off molecular weight 12,000 - 14,000 Da) and dialyzed against 0.2 M NaOH solution for 48 hours, with the dialysis solution being changed every 12 hours for a total of 4 times. The alkaline condition helped to neutralize the remaining acetic acid and remove unreacted small molecule components. The dialyzed solution was treated by freeze-drying (-50°C, 0.1 mbar, 48 hours) to finally obtain a light yellow porous lipoic acid-modified chitosan (LA-CS) powder.

[0061] To confirm the structure and properties of the synthesized product, a number of characterization analyses were carried out. Fourier transform infrared spectroscopy (FTIR) analysis showed that a new amide bond peak appeared at 1655 cm^-1, which was consistent with the characteristic absorption peak of the amide bond formed by the successful coupling of lipoic acid and chitosan. Nuclear magnetic resonance hydrogen spectrum ( 1 1H-NMR) analysis further confirmed the presence of lipoic acid, showing that characteristic peaks of lipoic acid appeared at 1.2 - 2.5 ppm, corresponding to its aliphatic methyl and methylene hydrogens.

[0062] Elemental analysis is an important method for determining the modification degree. By measuring the sulfur content (3.84%) and combining the molecular structures of chitosan and lipoic acid, the calculated lipoic acid modification degree was 21.2%, which was within the set target range (15 - 25%). This modification degree could not only maintain the basic properties of chitosan but also provide sufficient lipoic acid functional groups to enhance its interaction with biological membranes.

[0063] In terms of physicochemical properties, it was found that LA-CS retained the pH sensitivity of chitosan, was soluble under weakly acidic conditions (pH 4.0 - 6.0) with a concentration up to 10 mg / mL, while its solubility decreased under neutral and alkaline conditions. This property was beneficial for protecting the stability of the encapsulated drug in the acidic environment of the stomach and achieving controlled release when the intestinal pH increased. The viscosity of 2% (w / v) LA-CS solution at 25°C was 452 ± 18 mPa·s, which was suitable for nanoparticle preparation. The Zeta potential measured at pH 5.5 was +35.6 ± 2.1 mV, indicating that LA-CS had good electrostatic stability and mucoadhesive ability.

[0064] The comprehensive characterization results indicate that lipoic acid-modified chitosan with a modification degree of 21.2% was successfully synthesized. This material retains the key properties of chitosan while obtaining new functions conferred by lipoic acid, making it an ideal carrier material for constructing a colon-targeted nanodelivery system.

[0065] Example 3: Construction of the protopanaxatriol nanodelivery system

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

[0067] In terms of materials, the PPT nanocrystals prepared in Example 1, the LA-CS synthesized in Example 2, and high-purity sodium tripolyphosphate (STPP, purity ≥ 98%, purchased from Sigma-Aldrich) were used as the cross-linking agent. In addition, phosphate buffer solution (PBS, pH 7.4) and ultrapure water were used as the dispersion medium and washing solvent.

[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 make the concentration reach 2.5 mg / mL. To obtain a uniform dispersion system, the mixture was moderately sonicated (power 300 W, time 8 minutes) under low-temperature conditions (about 10°C). It should be noted that the control of sonication time and power is crucial, as it is necessary to ensure the full dispersion of the nanocrystals while avoiding structural changes or aggregation of the crystals caused by excessive sonication.

[0069] Next, prepare the LA-CS solution. 200 mg of the LA-CS obtained in Example 2 was slowly added to 50 mL of 1% (v / v) acetic acid aqueous solution to make the concentration reach 4 mg / mL. Gently stir at room temperature until completely dissolved, and then adjust the pH value to 5.8 using dilute sodium hydroxide solution. This pH value selection takes into account both the solubility of LA-CS and is conducive to the subsequent ion cross-linking reaction with STPP. After adjustment, filter through a 0.45 μm filter membrane to remove possible trace insoluble substances, obtaining a clear LA-CS solution.

[0070] The formation of nanoparticles is the core step of this example. Under stirring conditions (rotation speed about 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 time, the mass ratio of PPT nanocrystals to LA-CS is 1:4, and this ratio has been optimized through multiple preliminary experiments to ensure a high encapsulation efficiency and appropriate particle size. After the addition was completed, stirring was continued for 30 minutes to form a stable primary complex. In this stage, the negatively charged PPT nanocrystals are mainly combined with the positively charged LA-CS through electrostatic attraction and hydrogen bond interactions.

[0071] To further stabilize the nanodelivery system, ion crosslinking was carried out using STPP. 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 above mixed system at a constant rate of 1.5 mL / min. At this time, the mass ratio of LA-CS to STPP is 4:1, and this ratio can ensure an appropriate crosslinking density, which will neither cause the system to be too tight due to excessive crosslinking and affect drug release, nor reduce the stability of the nanoparticles due to insufficient crosslinking. The crosslinking reaction was carried out at room temperature for 1.5 hours. During this period, the polyvalent phosphate ions in STPP underwent ion crosslinking with the amino groups on LA-CS to form a three-dimensional network structure, encapsulating the PPT nanocrystals therein.

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

[0073] To characterize the prepared protopanaxatriol nanodelivery system, a series of physicochemical property analyses were carried out. First, the particle size distribution of the nanoparticles was measured by dynamic light scattering (DLS). The results showed that the average particle size was 387±24 nm, and the polydispersity index (PDI) was 0.25±0.04, indicating that a nanodelivery system with uniform particle size was obtained. The Zeta potential measured in PBS (pH 7.4) was +32.7±1.9 mV, far higher than the stability threshold of ±30 mV, predicting good colloidal stability. Transmission electron microscopy (TEM) observation further confirmed that the nanoparticles presented regular spherical shapes with smooth and uniform surfaces, which was beneficial for their circulation and distribution in vivo.

[0074] Drug loading parameters are important indicators for evaluating the performance of delivery systems. Determined by high-performance liquid chromatography (HPLC), the drug loading (DL%) of PPT was calculated to be 14.2 ± 0.8%, which is much higher than that of conventional formulations, reducing the amount of carrier material used and improving the convenience of drug administration. 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 nano-delivery system in different simulated physiological environments were studied by the dialysis bag method. The results showed that only 7.2 ± 1.1% of PPT was released within 2 hours in simulated gastric fluid (pH 1.2), the cumulative release did not exceed 18.5 ± 2.3% within 4 hours in simulated intestinal fluid (pH 6.8), while the cumulative release was as high as 82.4 ± 3.6% within 24 hours in simulated colon fluid (pH 7.4) supplemented with colon bacterial enzymes. This significant pH-dependent release behavior and responsiveness to colon enzymes fully demonstrated the colon-targeting characteristics of the constructed system, effectively reducing the premature release and absorption loss of drugs in the upper digestive tract and increasing their local concentration at the colon site.

[0076] In terms of stability, within 3 months of storage at 4°C, the particle size of the nano-delivery system changed by no more than 10%, and the drug content remained above 93% of the initial value, indicating good physical and chemical stability. Even at room temperature (25°C), the particle size growth was controlled within 15% within 1 month, and the drug content remained above 90% of the initial value, which is of great significance for practical applications and commercialization.

[0077] In summary, a nano-delivery system of protopanaxatriol was successfully constructed by the ion gel method. This system has an ideal particle size, good stability, high drug loading, and obvious colon-targeted release characteristics, laying a foundation for subsequent in vivo studies.

[0078] Example 4: Inhibitory effect of protopanaxatriol nano-delivery system on the formation of Twist1-Ku70-Sirt1 complex

[0079] This example aims to study the inhibitory effect of the protopanaxatriol nano-delivery system on the formation of the Twist1-Ku70-Sirt1 complex and compare it with free protopanaxatriol to verify the synergistic effect of the nano-delivery system. Human colon cancer HCT116 cells were selected as the research object. 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, and maintained at 37°C and 5% CO2. At passage, when the cells grew to approximately 80% confluence, they were digested with 0.25% trypsin-EDTA and replanted. To ensure the reliability of the experimental results, all experiments were performed using cells in the exponential growth phase, and the passage number was controlled between 5 and 15 generations.

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

[0082] Cell uptake studies are crucial for understanding the mechanism of action of drug delivery systems. The cell uptake behavior of the PPT nanodelivery system was investigated using a combination of confocal laser scanning microscopy (CLSM) and flow cytometry. To achieve visualization, coumarin-6 was used to label PPT, and DiI was used to label the LA-CS membrane. The cell nuclei were stained with DAPI, and the lysosomes were stained with LysoTracker Green. The CLSM observation results showed that after 2 hours of treatment, the PPT nanodelivery system significantly enhanced the cell uptake of PPT, and the fluorescence signal was mainly distributed in the cytoplasmic region, with partial co-localization with lysosomes, indicating that the endocytic pathway was the main uptake mechanism. Quantitative analysis by flow cytometry further confirmed that compared with free PPT, the intracellular fluorescence intensity in the nanodelivery system group increased by approximately 3.2-fold, indicating that the nanodelivery system significantly enhanced the cell uptake efficiency of PPT.

[0083] To determine the effect of the nano-delivery system on the formation of the Twist1-Ku70-Sirt1 complex, the interaction of the three proteins was first studied using co-immunoprecipitation (Co-IP) technology. In the specific operation, total cell proteins were extracted using RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors), and co-immunoprecipitation was performed using the Pierce™ Co-IP kit (Thermo Fisher Scientific). Twist1 antibody was used as the bait protein, and co-precipitated Ku70 and Sirt1 were detected by Western Blot. The results showed that in the control group, there was an obvious interaction between Twist1 and Ku70 and Sirt1, confirming that the three could form a complex. Compared with the free PPT, the PPT nano-delivery system more effectively reduced the interaction between Twist1 and Ku70 and Sirt1. At an equivalent concentration (20 μM), the inhibitory effect was increased by about 2.5 times.

[0084] To further verify the above results, double immunofluorescence staining technology was also used. The treated cells were fixed, permeabilized, and labeled with specific antibodies against Twist1 (rabbit anti-), Ku70 (mouse anti-), and Sirt1 (goat anti-), and then stained with secondary antibodies labeled with Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647, respectively. The results of confocal microscopy showed that there was obvious co-localization of the three proteins in the control group, while the co-localization signal in the group treated with the PPT nano-delivery system was significantly weakened, further confirming its inhibitory effect on complex formation.

[0085] In addition to protein interaction, the effect of the PPT nano-delivery system on the expression levels of Twist1, Ku70, and Sirt1 was also concerned. Western Blot analysis showed that after 24 hours of treatment with the PPT nano-delivery system, the level of Twist1 protein was significantly reduced (reduced by about 45%), while the expression changes of Ku70 and Sirt1 were not obvious (reduced by about 12% and 8%, respectively). This indicates that the PPT nano-delivery system not only inhibits the interaction of the three proteins but also selectively reduces the expression of Twist1, thereby further weakening the formation of the complex.

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

[0087] In summary, this example demonstrated that the protopanaxatriol nanodelivery system could significantly enhance the 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-associated colorectal cancer. Compared with free PPT, the nanodelivery system showed a significant synergistic effect, which might be related to its improved cellular uptake and stability.

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

[0089] This example aimed to evaluate the antitumor effect of the protopanaxatriol nanodelivery system in vivo, especially its preventive and therapeutic effects on colitis-associated colorectal cancer (CAC). An azoxymethane-dextran sulfate sodium (AOM-DSS)-induced CAC mouse model was selected. This model can well simulate the disease process from human colitis to colorectal cancer and is widely used in the evaluation research of related drugs.

[0090] Animal experiments were conducted under conditions approved by the Experimental Animal Ethics Committee. Male C57BL / 6 mice aged 6 - 8 weeks (body weight 20 - 25 g) were purchased from a certain experimental animal center. All animals were housed in a SPF-level environment with a constant temperature of (22 ± 2°C), a constant humidity of (55 ± 5%), and a 12-hour light / dark cycle, and had free access to food and water. After one week of acclimation to the environment, the mice were randomly divided into 6 groups of 10 each: (1) normal control group: not given any treatment; (2) model group: induced by AOM-DSS but not given drugs; (3) free PPT low-dose group: 10 mg / kg / day; (4) free PPT high-dose group: 20 mg / kg / day; (5) PPT nanodelivery system low-dose group: equivalent to 10 mg / kg / day PPT; (6) PPT nanodelivery system high-dose group: equivalent to 20 mg / kg / day PPT.

[0091] The CAC model was established with reference to the classical method, with slight modifications. Specifically, mice except for the normal control group were first given a single intraperitoneal injection of azoxymethane (AOM, 12 mg / kg body weight). One week later, dextran sulfate sodium (DSS, 2.5% w / v) was administered in drinking water for a total of 3 cycles. Each cycle included 7 days of drinking DSS aqueous solution, followed by a 14-day recovery period with normal drinking water. This alternating administration could induce chronic colitis and gradually develop into colorectal cancer, and the entire experimental period was 10 weeks.

[0092] Drug treatment started on the same day as the first DSS administration and was given by gavage. The drug was dispersed in 0.5% sodium carboxymethylcellulose (CMC-Na) solution, and the administration volume was 10 mL / kg body weight, once a day until the end of the experiment. The normal control group and the model group were given an equal volume of 0.5% CMC-Na solution. During the entire experiment, the body weight changes of the mice were recorded weekly, and the general status, fecal characteristics, and rectal prolapse were observed.

[0093] At the end of the experiment, the mice were euthanized after blood collection, and the colon tissues were removed and the colon length was measured (colon shortening is an important indicator of colitis). Subsequently, the colon was longitudinally dissected, the contents were gently washed in PBS, and the number of tumors was carefully counted and the tumor size (long axis × short axis) was measured under a dissecting microscope. Part of the colon tissue was fixed in 4% paraformaldehyde for histopathological analysis, and the remaining tissue was quickly frozen in liquid nitrogen for subsequent molecular biology detection.

[0094] The results showed that compared with the normal control group, the mice in the model group showed obvious weight loss, colon shortening, rectal prolapse, and tumor formation, confirming that the AOM-DSS successfully induced the CAC model. In terms of the treatment effect, the high-dose group of the PPT nanodelivery system showed the most significant anti-tumor effect. Compared with the model group, the number of colon tumors in this group of mice decreased by about 73.5% (from an average of 12.4 ± 2.1 to 3.3 ± 0.9), and the total tumor volume decreased by about 85.2%. It is worth noting that this effect was significantly better than that of the high-dose free PPT group, in which the number and total volume of tumors decreased by about 41.9% and 53.6% respectively. Even the low-dose group of the PPT nanodelivery system showed an anti-tumor effect comparable to that of the high-dose free PPT group, demonstrating that the nanodelivery system significantly improved the bioavailability and treatment effect of PPT.

[0095] Histopathological analysis (HE staining) further supported the above results. In the colon tissues of the model group mice, obvious inflammatory cell infiltration, crypt structure disorder, epithelial cell atypia, and tumor invasion were observed. However, in the PPT nanodelivery system treatment group, especially the high-dose group, these pathological changes were significantly alleviated. According to the semi-quantitative scores of the degree of inflammation, ulcer area, crypt structure, and epithelial cell atypia, the total pathological score of the high-dose group of the PPT nanodelivery system was decreased by approximately 68.7% compared with the model group and by approximately 42.3% compared with the high-dose group of free PPT.

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

[0097] The pharmacokinetics and tissue distribution of protopanaxatriol in vivo were also studied. The LC-MS / MS method was used to determine the concentration of PPT in blood and tissues at different time points (0.5, 1, 2, 4, 8, 12, 24, 48 hours after administration). The results showed that compared with free PPT, the PPT nanodelivery system significantly increased the exposure of PPT in colon tissues, and the colon / blood drug concentration ratio was increased by approximately 4.2 times. Especially in the distal colon (within 5 cm from the anus), the concentration of PPT reached the effective level required for treatment and could be maintained for a long time. This result strongly demonstrated the colon targeting of the PPT nanodelivery system and explained its enhanced anti-tumor effect.

[0098] Safety evaluation showed that no obvious toxic and side effects occurred in all treatment groups. During the 10-week drug administration period, there were no obvious abnormalities in indicators such as body weight, food intake, and general behavioral activities. The blood routine and liver and kidney function indicators were within the normal range, and no obvious abnormalities were seen in the pathological sections of the main organs (heart, liver, spleen, lung, kidney). It is worth noting that 2 mice in the high-dose group of free PPT showed mild abnormalities in liver function indicators (slight elevation of ALT and AST), while no such abnormalities were seen in the same-dose group of the PPT nanodelivery system, indicating that the nanodelivery system can also reduce the systemic toxicity of the drug.

[0099] In summary, this example demonstrates that the original panaxatriol nano-delivery system exhibits significant anti-tumor effects in the AOM-DSS-induced CAC animal model, and the effect is significantly better than that of the same dose of free panaxatriol. Its mechanism of action is related to inhibiting the formation of the Twist1-Ku70-Sirt1 complex and weakening the proliferation and stem cell characteristics of tumor cells. At the same time, this nano-delivery system has good colon targeting and safety, providing an important pre-clinical basis for clinical application.

[0100] Example 6: Joint Therapy Study of Panaxatriol Nano-Delivery System and Immune Checkpoint Inhibitor

[0101] In view of the breakthrough progress of immune checkpoint inhibitors in the treatment of various tumors and the key role of the tumor microenvironment in the occurrence and development of colitis-associated colorectal cancer (CAC), this example aims to explore the synergistic anti-tumor effect and its potential mechanism of the combined use of the panaxatriol nano-delivery system and anti-PD-1 antibody.

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

[0103] The results showed that, compared with the single-agent treatment groups, the combination group of PPT nano-delivery system and anti-PD-1 antibody exhibited significant synergistic anti-tumor effects. The number and total volume of tumors in the mice of this group were reduced by approximately 86.4% and 92.3% respectively compared with the model group, far better than the single-agent group of PPT nano-delivery system (reduced by approximately 72.6% and 84.5% respectively) and the single-agent group of anti-PD-1 antibody (reduced by approximately 45.3% and 58.7% respectively). It is worth noting that the effect of the combination group of free PPT and anti-PD-1 antibody was also enhanced, but not as significant as that of the combination group of PPT nano-delivery system.

[0104] To explore the mechanism of synergy, an in-depth analysis of the tumor microenvironment was conducted. By flow cytometry analysis of infiltrating immune cells in colon tissues, it was found that the combined treatment of 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 reducing the proportions of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). In addition, in the tumor tissues of the combination treatment group, the proportion of M1 macrophages increased, while the proportion of M2 macrophages decreased, indicating a shift of the tumor microenvironment from an immunosuppressive type to an immune-activated type.

[0105] Immunohistochemical and immunofluorescence analyses also supported the above results. Compared with the single-agent treatment group, the infiltration of CD8+ T cells in the tumor tissues of the combination treatment group increased, the expressions of PD-1 and PD-L1 decreased, and at the same time, the expression of the tumor cell proliferation marker Ki-67 decreased. In addition, it was also observed that the co-localization of Twist1 with Ku70 and Sirt1 in the combination treatment group was further reduced, indicating that the inhibitory effect of the PPT nanodelivery system on the Twist1-Ku70-Sirt1 complex was maintained and possibly enhanced in the combination treatment.

[0106] To further understand the molecular mechanism, RNA sequencing analysis was performed on colon tissues. Compared with the single-agent treatment group, the gene expressions related to T cell activation, antigen presentation, cytotoxicity, and cytokine signaling were up-regulated in the combination treatment group, while the gene expressions related to tumor promotion, immunosuppression, and cell stemness were down-regulated. Pathway enrichment analysis showed that the NFκB signaling pathway, JAK-STAT signaling pathway, and T cell receptor signaling pathway were significantly activated in the combination treatment group, which might be the molecular basis for the synergistic anti-tumor effect.

[0107] The effect of different administration sequences on the combination treatment was also investigated. The results showed that the sequential treatment regimen of administering the PPT nanodelivery system for 3 days first and then combining with the anti-PD-1 antibody had a better effect than the simultaneous administration or the regimen of administering the anti-PD-1 antibody first. This indicated that the PPT nanodelivery system might enhance the effect of subsequent immune checkpoint inhibitors by pre-regulating the tumor microenvironment.

[0108] Safety evaluation showed that the combination treatment regimen was well tolerated. Compared with the group treated with anti-PD-1 antibody alone, no increase in the incidence of immune-related adverse events (irAEs) was observed in the combination treatment group. Notably, it has been reported in the past that certain combination treatment regimens may increase the risk of immune-related adverse events, such as the combination of immune checkpoint inhibitors with certain chemotherapeutic drugs. However, in this study, after a 10-week treatment cycle, the mice in the group treated with the combination of PPT nanodelivery system and anti-PD-1 antibody did not show common immune-related adverse events such as abnormal weight loss, skin damage, and exacerbation of colitis. Blood biochemical indexes showed that liver function (ALT, AST) and kidney function (BUN, Cr) were within the normal range, and there was no significant difference compared with the single-drug treatment group and the normal control group.

[0109] In addition, cytokine profiling was performed to measure the levels of pro-inflammatory cytokines such as IL-6, TNF-α, IL-17A, IL-2 and anti-inflammatory cytokines such as IL-10 in serum. The results showed that compared with the group treated with anti-PD-1 antibody alone, the levels of pro-inflammatory cytokines in the combination treatment group did not increase significantly, and even the levels of IL-6 and TNF-α decreased, which may be related to the anti-inflammatory effect of the PPT nanodelivery system. This result further supported the safety of the combination treatment regimen, 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 balance the immune response through its anti-inflammatory effect.

[0110] The effect of combination treatment on normal mucosal tissues was also evaluated. Histopathological examination showed that no obvious inflammatory reaction or tissue damage was observed in the non-tumor tissues adjacent to the normal colonic mucosa, indicating good tissue selectivity of the combination treatment. This may be due to the colon-targeting property of the PPT nanodelivery system, which enables the drug to be mainly released and act at the lesion site, reducing the impact on normal tissues.

[0111] The combination index (CI) analysis in preclinical pharmacology is an important method to evaluate the value of combined drugs. The CI values of different dose combinations were calculated according to the Chou-Talalay method, and the results showed that the CI value of the PPT nanodelivery system and anti-PD-1 antibody was 0.72, indicating an obvious synergistic effect (CI < 1 indicates synergy). This result provided an important reference for the design of the combination treatment regimen in subsequent clinical studies.

[0112] Based on the above results, this example demonstrated that the combined use of the protopanaxatriol nano-delivery system and anti-PD-1 antibody has 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 combined treatment strategy provides new ideas and solutions for the treatment of colitis-associated colorectal cancer, especially for patients who respond poorly to single immune checkpoint inhibitors. The superiority of the sequential administration regimen also provides a basis for optimizing the clinical drug administration sequence.

[0113] Example 7: Extended Application Research of the Protopanaxatriol Nano-Delivery System

[0114] Based on the foregoing examples, this example aims to explore the potential application of the protopanaxatriol nano-delivery system in other intestinal disease models and further study its related molecular mechanisms of the Twist1-Ku70-Sirt1 complex.

[0115] First, the preventive and therapeutic effects of the PPT nano-delivery system in a DSS-induced colitis model were studied. Different from the AOM-DSS model, the simple DSS-induced model mainly shows colonic inflammation rather than tumors, being closer to the early stage of inflammatory bowel disease. Acute colitis was induced with a 3% DSS solution for 7 consecutive days, followed by 14 days of recovery with normal drinking water. The mice were divided into a preventive group (administered drugs 3 days before DSS administration) and a treatment group (administered drugs after DSS cessation), with a dose equivalent to 20 mg / kg / day of PPT.

[0116] The results showed that in the preventive group, the PPT nano-delivery system significantly alleviated symptoms such as DSS-induced weight loss, colon shortening, and fecal bleeding, and the disease activity index (DAI) score was reduced by approximately 65% compared to the model group. Histopathological analysis showed that the area of colonic mucosal inflammation and ulcers in the preventive group was significantly reduced, and the infiltration of inflammatory cells was alleviated. In the treatment group, the PPT nano-delivery system accelerated the recovery of colitis, manifested as faster weight gain during the recovery period and enhanced repair of the colonic mucosa. These results indicate that the PPT nano-delivery system not only has anti-tumor effects but also has the potential to prevent and treat colitis, which is of great significance for tumor prevention in high-risk inflammatory bowel disease patients.

[0117] Secondly, the effect of the PPT nanodelivery system was investigated in other intestinal tumor models expressing the Twist1-Ku70-Sirt1 complex. By screening a variety of intestinal tumor cell lines, it was found that in addition to HCT116, the human gastric cancer cell line MGC-803 and the human small intestinal adenocarcinoma cell line SIA also expressed relatively high levels of Twist1, Ku70, and Sirt1, and there was co-localization among the three. In vitro experiments showed that the PPT nanodelivery system also exhibited significant inhibitory effects on the proliferation and stem cell characteristics of these two cell lines, with IC50 values equivalent to 12.4 μM and 15.8 μM PPT, respectively. This result expanded the potential indication range of the PPT nanodelivery system, indicating that it may be effective against a variety of digestive tract tumors.

[0118] Thirdly, 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 expressions of Twist1, Ku70, and Sirt1 were knocked down respectively. The results showed that in Twist1-knockdown cells, the anti-proliferative and anti-stem cell characteristics of the PPT nanodelivery system were significantly weakened, while in Ku70- or Sirt1-knockdown cells, the degree of weakening was smaller. On the contrary, in Twist1-overexpressing cells, the effect of the PPT nanodelivery system was enhanced. This indicates that Twist1 is the main target of the PPT nanodelivery system, while Ku70 and Sirt1 may play auxiliary roles.

[0119] Further mechanism studies showed that the PPT nanodelivery system not only inhibited the formation of the Twist1-Ku70-Sirt1 complex by direct binding, but also affected the downstream signaling pathways of the complex. Through phosphoproteomic analysis, it was found that after treatment with the PPT nanodelivery system, multiple signaling pathways related to EMT and stem cell characteristics were regulated, including the Wnt / β-catenin, Notch, and Hedgehog pathways. In particular, the PPT nanodelivery system could significantly reduce the nuclear translocation of β-catenin and the transcriptional activity mediated by TCF / LEF, which may be one of the important mechanisms for its inhibition of tumor stem cell characteristics.

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

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

[0122] The present invention provides a panaxatriol nanodelivery system targeting the Twist1-Ku70-Sirt1 complex and its application in the treatment of colitis-associated colorectal cancer. By preparing panaxatriol into nanocrystals and using lipoic acid-modified chitosan to construct a colon-targeted delivery system, the bioavailability and targeting of panaxatriol are significantly improved. This nanodelivery system can effectively inhibit the formation of the Twist1-Ku70-Sirt1 complex, reduce the stem cell characteristics of colon cancer cells, and exhibit significant anti-tumor effects in animal models. In addition, the combination of this system with immune checkpoint inhibitors has a synergistic anti-tumor effect and good safety, providing a new strategy for the comprehensive treatment of colitis-associated colorectal cancer.

[0123] Although the 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 idea and principle of the present invention, and these changes, modifications, substitutions, and variations are still within the protection scope of the present invention.

Claims

1. A protopanaxatriol nanodelivery system targeting the Twist1-Ku70-Sirt1 complex, characterized in that, The nano-delivery system includes: (a) Protopanaxatriol nanocrystals; (b) Lipoic acid-modified chitosan; and (c) A crosslinking agent; wherein, the average particle size of the protopanaxatriol nanocrystals 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.

2. The nano-delivery system according to claim 1, wherein The protopanaxatriol nanocrystals are prepared by an ultrasonic-assisted anti-solvent precipitation method, wherein ethanol is used as the solvent, water is used as the anti-solvent, and polyvinylpyrrolidone K30 or polysorbate 80 is used as the stabilizer.

3. The nano-delivery system according to claim 1, wherein The molecular weight of the chitosan is 100 - 200 kDa, and the deacetylation degree is ≥90%; the lipoic acid-modified chitosan is synthesized by the EDC / NHS activation coupling method, wherein EDC is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and NHS is N-hydroxysuccinimide.

4. The nano-delivery system according to claim 1, wherein The mass ratio of the protopanaxatriol 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.

5. The nano-delivery system according to claim 1, characterized in that, The absolute value of the Zeta potential of the nano-delivery system is ≥30 mV, the polydispersity index is <0.3, the loading amount of protopanaxatriol is ≥10%, and the encapsulation efficiency is ≥80%.

6. The nano-delivery system according to claim 1, wherein The nano-delivery system releases <20% of protopanaxatriol within 4 hours in simulated gastric juice (pH 1.2) and small intestinal juice (pH 6.8), and releases >80% of protopanaxatriol within 24 hours in simulated colon juice (pH 7.4), showing colon-targeted release characteristics.

7. A method for preparing the nano-delivery system according to any one of claims 1-6, characterized in that, It includes the following steps: (a) Dissolve protopanaxatriol in ethanol, add the stabilizer, and under ultrasonic conditions, add the protopanaxatriol solution to ultrapure water to prepare protopanaxatriol nanocrystals; (b) Activate lipoic acid through EDC / NHS, and react the activated lipoic acid with chitosan to prepare lipoic acid-modified chitosan; (c) Disperse the protopanaxatriol nanocrystals prepared in step (a) in a solution containing the lipoic acid-modified chitosan prepared in step (b), and then add a sodium tripolyphosphate solution for crosslinking to form a nano-delivery system; and (d) Collect and purify the nano-delivery system.

8. Use of the nano-delivery system according to any one of claims 1 - 6 in the preparation of a drug for the treatment of colitis-associated colorectal cancer, wherein the nano-delivery system reduces the cancer stem cell characteristics and tumorigenesis of cancer cells by targeting and inhibiting the formation of the Twist1-Ku70-Sirt1 complex.

9. The application according to claim 8, wherein The drug is an oral preparation, and the dosage is equivalent to 10 - 20 mg / kg / day of protopanaxatriol, and is used for the treatment of colitis-associated colorectal cancer induced by AOM-DSS.

10. The application according to claim 8, wherein The drug can be used in combination with an immune checkpoint inhibitor for the treatment of patients with colitis-associated colorectal cancer with high expression of the Twist1-Ku70-Sirt1 complex.

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