Lycium barbarum polysaccharide-based SN38 active targeting oral preparation as well as preparation method and application thereof

Abstract

The invention relates to a lycium barbarum polysaccharide-based SN38 active targeting oral preparation for treating colon cancer, and belongs to the technical field of medicines. The preparation is prepared by taking SN38 as an anti-tumor active component, taking nano-micelles formed by self-assembly of an amphiphilic triblock copolymer synthesized by lycium barbarum polysaccharide, lipoic acid and chondroitin sulfate as an active targeting carrier, taking calcium alginate microspheres as an oral delivery carrier, and encapsulating the nano-micelles in the calcium alginate microspheres by utilizing a microfluidic technology. On one hand, active targeting is realized through ligand-receptor affinity interaction between chondroitin sulfate and a CD44 receptor highly expressed on the surface of a colon cancer cell; on the other hand, stimulation responsive release of SN38 is triggered by redox reaction of high-concentration glutathione (GSH) in a tumor microenvironment and disulfide bonds in lipoic acid molecules, and meanwhile, nano-micelles are protected by calcium alginate microspheres to avoid gastric acid damage and smoothly reach colon, so that precise oral targeted drug delivery is realized. The technical problems of poor water solubility, low oral bioavailability, serious gastrointestinal toxicity caused by non-specific distribution and the like of SN38 are effectively solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a Lycium barbarum polysaccharide-based SN38 active targeted oral preparation and a preparation method and application thereof. Background Art

[0002] Colon cancer (CC) is the third leading cause of cancer death. Although multimodal treatment strategies such as surgery, chemoradiotherapy, targeted therapy and immunotherapy have made certain progress in recent years, CC treatment still faces the key challenges of poor treatment effect and prominent adverse reactions. At present, the FOLFIRI regimen represented by intravenous injection of irinotecan (CPT-11) is one of the standard chemotherapy regimens in the clinical treatment of nearly 50% of patients with advanced CC. However, this method has significant defects: first, CPT-11 needs to be converted into its active form SN38 under the action of liver carboxylesterase, but the conversion rate is low (only about 10%), resulting in most drugs being unable to exert anti-tumor effects; secondly, intravenous administration causes SN38 to be nonspecifically distributed throughout the body, causing severe systemic toxicity. Among them, CPT-11-related intestinal toxicity is the most challenging adverse reaction in the implementation of the FOLFIRI regimen. About 30-40% of patients experience severe diarrhea during treatment, which becomes the main factor limiting the dose and seriously affects the implementation of clinical treatment plans. Therefore, effectively reducing the intestinal toxicity of CPT-11 while improving its efficacy is a key clinical problem that needs to be urgently addressed in the current chemotherapy of advanced CC.

[0003] As an active metabolite of CPT-11, SN38 has great research and development value in solving the current clinical application defects of CPT-11 due to its potent anti-tumor effect (its tumor cell killing efficacy is 100-1000 times higher than that of CPT-11). However, as a hydrophobic molecule, SN38 has key drawbacks such as low water solubility and high toxicity, which limit its further development and application. In recent years, with the rapid development of nanotechnology and oral targeted delivery systems, a technical foundation has been laid for further exploration of the clinical application potential of SN38. The oral targeted delivery system based on nanotechnology can effectively avoid the non-target toxicity after systemic administration while improving the water solubility of SN38. At the same time, oral administration can give full play to the "synergistic and toxic reduction" effect of LBP after the interaction with the flora in the colon.

[0004] The particularity of the colon cancer microenvironment has proposed new ideas for drug delivery design: abnormal pH value of tumor tissue, expression of special enzymes, and significantly increased glutathione (GSH) concentration can be used as important targets for designing stimulus-responsive drug release systems. Therefore, developing a new formulation that can overcome the physical and chemical defects of SN38 and achieve precise oral targeted delivery to colon cancer sites to improve its oral bioavailability is of great clinical significance for effectively reducing the intestinal toxicity of irinotecan while improving its efficacy. Summary of the Invention

[0005] The object of the present invention is to provide a Lycium barbarum polysaccharide-based SN38 active-targeting oral preparation, its preparation method and application to solve the above problems.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A Lycium barbarum polysaccharide-based SN38 active-targeting oral preparation, which preparation comprises:

[0008] (1) Using SN38 (7-ethyl-10-hydroxycamptothecin) as the anti-tumor active ingredient;

[0009] (2) Using a nano-micelle self-assembled from an amphiphilic triblock copolymer (CLL) synthesized from Lycium barbarum polysaccharide (LBP), lipoic acid (LA), and chondroitin sulfate (CS) as the active-targeting carrier, with SN38 encapsulated inside;

[0010] (3) Using a calcium alginate microsphere as the oral delivery carrier, and encapsulating the nano-micelle in the calcium alginate microsphere through microfluidic technology.

[0011] This preparation adopts a "nano-micelle - microsphere" double-layer delivery system design, and the active-targeting mechanism has dual specificities: on the one hand, it achieves active targeting through the ligand-receptor affinity between chondroitin sulfate and the highly expressed CD44 receptor on the surface of colon cancer cells; on the other hand, it utilizes the redox reaction between the high concentration of glutathione (GSH) in the tumor microenvironment and the disulfide bond in the lipoic acid molecule to trigger the stimulus-responsive release of SN38, achieving precise targeted drug delivery, effectively solving technical problems such as poor water solubility of SN38, low oral bioavailability, and severe gastrointestinal toxicity caused by non-specific distribution. This preparation realizes the effective enrichment of SN38 at the colon cancer site after oral administration, significantly improves the treatment effect, and reduces the systemic toxicity at the same time.

[0012] As a preferred technical solution of the present invention, the amphiphilic triblock copolymer (CLL) is synthesized by first synthesizing an amphiphilic diblock copolymer intermediate (LL) from Lycium barbarum polysaccharide and lipoic acid through an esterification reaction, and then further synthesizing it with chondroitin sulfate through an esterification reaction.

[0013] As a preferred technical solution of the present invention, in the amphiphilic diblock copolymer intermediate (LL), the mass ratio of Lycium barbarum polysaccharide to lipoic acid is 1:1 - 1:5, preferably 1:1 - 1:3; in the amphiphilic triblock copolymer, the mass ratio of chondroitin sulfate to the amphiphilic diblock copolymer intermediate (LL) is 1:2 - 2:1, preferably 1:1.

[0014] As a preferred technical solution of the present invention, the mass ratio of the amphiphilic triblock copolymer to SN38 is 10:1 - 10:5, preferably 10:1 - 10:3.

[0015] As a preferred technical solution of the present invention, the particle size of the nanomicelles is 150 - 400 nm, preferably 200 - 250 nm, and the PDI is 0.01 - 0.5, preferably the PDI is 0.01 - 0.3.

[0016] As a preferred technical solution of the present invention, the concentration of sodium alginate in the calcium alginate microspheres is 1 - 3% (w / v), and the concentration of calcium chloride is 50 - 200 mM.

[0017] The preparation method of the oral preparation described above includes the following steps:

[0018] (1) Esterify lycium barbarum polysaccharide and lipoic acid in the presence of a catalyst to synthesize an amphiphilic diblock copolymer intermediate (LL);

[0019] (2) Synthesize an amphiphilic triblock copolymer (CLL) by esterifying the amphiphilic diblock copolymer intermediate (LL) obtained in step (1) with chondroitin sulfate;

[0020] (3) Use the nanoprecipitation method to combine the amphiphilic triblock copolymer obtained in step (2) with SN38, and use ultrasonic waves and stirring to self-assemble the amphiphilic triblock copolymer into nanomicelles encapsulating SN38;

[0021] (4) Mix the nanomicelle suspension obtained in step (3) with a sodium alginate solution, and drop the mixture into a calcium chloride solution through a microfluidic system to form calcium alginate microspheres encapsulating the nanomicelles.

[0022] As a preferred technical solution of the present invention, the specific method of step (1) is: dissolve lipoic acid and a coupling reagent in a solvent and stir well; add a dimethyl sulfoxide solution containing lycium barbarum polysaccharide and a catalyst; stir and react the mixed solution at room temperature, and after the reaction is completed, dialyze and freeze-dry with a dialysis bag to obtain the product;

[0023] Furthermore, as a specific implementation method, in step (1), the coupling reagent includes N,N'-dicyclohexylcarbodiimide (DCC); the solvent includes dichloromethane; the catalyst includes 4-dimethylaminopyridine (DMAP); lipoic acid and the coupling reagent are dissolved in the solvent and stirred at room temperature for 20 - 28 h, and after stirring is completed, the solution is filtered through a filter membrane to remove impurities formed during the reaction; the molar ratio of lipoic acid to the coupling reagent and the catalyst is 2:2:1; the mixed solution is stirred and reacted at room temperature for 36 - 72 h, then the solution is dialyzed with a dialysis bag for 18 - 36 h, and after freeze-drying, it is stored at -4°C;

[0024] The specific method of step (2) is as follows: Dissolve chondroitin sulfate, a coupling agent, and a catalyst in water, and stir at room temperature to form a chondroitin sulfate solution; dissolve the amphiphilic diblock copolymer intermediate in DMSO and stir under the same conditions; drop the chondroitin sulfate solution into the amphiphilic diblock copolymer intermediate solution under ice bath conditions, and further stir the mixture. After the stirring is completed, the mixture solution is dialyzed with a dialysis bag, and then washed with water to remove the unreacted chondroitin sulfate completely, and freeze-dried to obtain CLL.

[0025] Furthermore, as a specific implementation manner, in step (2), chondroitin sulfate, the coupling agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCL), and the catalyst 4-dimethylaminopyridine (DMAP) are dissolved in water according to a molar ratio of 1:1:0.8, and stirred at a speed of 300-500 rpm at room temperature for 1-5 h to form a chondroitin sulfate solution; dissolve the amphiphilic diblock copolymer LL of wolfberry polysaccharide-lipoic acid in DMSO and stir under the same conditions for 2 h; drop the chondroitin sulfate solution into the LL polymer solution under ice bath conditions, and further stir and react the mixture for 36-72 h. After the stirring is completed, the mixture solution is dialyzed with a dialysis bag with a cut-off molecular weight of 8000-14000 Da for 36-72 h, and then washed with water to remove the unreacted chondroitin sulfate completely, and freeze-dried to obtain CLL, which is stored at -4°C after freeze-drying.

[0026] As a preferred technical solution of the present invention, the specific method of step (3) is as follows: Dissolve the amphiphilic triblock copolymer (CLL) and SN38 in DMSO, then continuously stir at 200-500 rpm at room temperature for 0.5-2 h, then dropwise add deionized water, and continue to stir for 2-6 h. After completion, place the sample in a dialysis bag and dialyze for 18-32 h to obtain a suspension of SN38-loaded nanomicelles. After further ultrasonic and centrifugal treatment, nanomicelles loaded with SN38 are obtained.

[0027] As a preferred technical solution of the present invention, the specific method of step (4) is as follows: Add sodium alginate to the suspension of SN38-loaded nanomicelles to dissolve it, and drop it into a salt solution containing Ca 2+ , Ba 2+ or Fe 3+ to obtain the product.

[0028] The application of the above-mentioned oral preparation in the preparation of anti-tumor drugs, especially for the treatment of colon cancer. Through innovative material design and preparation process, this preparation realizes the effective enrichment of SN38 at the colon cancer site after oral administration, significantly improves the treatment effect, and reduces the systemic toxicity at the same time.

[0029] Compared with the prior art, the present invention has the following innovative points:

[0030] 1. Design of Multifunctional Triblock Copolymers

[0031] The present invention innovatively designs a triblock copolymer self-assembly system composed of Lycium barbarum polysaccharide, lipoic acid, and chondroitin sulfate, achieving multifunctional integration and overcoming the limitations of traditional SN38 delivery systems with simple components and single functions. Lycium barbarum polysaccharide provides good biocompatibility and potential immunomodulatory effects; the disulfide bond structure introduced by lipoic acid has specific responsiveness to the high-concentration GSH environment in colon cancer; chondroitin sulfate endows the system with CD44 targeting ability. This structural design not only solves the problem of poor water solubility of SN38 but also improves the selective enrichment of drugs at the tumor site through the synergistic action of multiple mechanisms, reduces system exposure, and decreases the toxic and side effects caused by nonspecific distribution, proposing a new strategy for the molecular design of anticancer drug delivery systems.

[0032] 2. Double-Layer Protective Delivery System Prepared by Microfluidic Technology

[0033] The present invention constructs a double-layer delivery structure of "nano micelles - calcium alginate microspheres" using microfluidic technology. This ingenious design effectively solves the multiple physiological barrier problems faced by oral SN38. The outer calcium alginate microspheres can remain stable in the gastric acid environment and selectively release the contents in the colon; the inner nano micelles further protect SN38 and achieve precise release within tumor cells. The application of microfluidic technology ensures uniform particle size distribution and high encapsulation efficiency during the preparation process, effectively overcoming the technical problems in traditional preparation methods. This double-layer design significantly improves the oral bioavailability of SN38, realizes full protection from oral administration to tumor-targeted delivery, and provides a technical breakthrough for the oral delivery of poorly soluble anticancer drugs.

[0034] 3. Precision Drug Release Strategy Triggered by the Colorectal Cancer Microenvironment

[0035] The present invention uniquely utilizes the characteristics of the colorectal cancer microenvironment to achieve the precise release of SN38. First, based on the pH environment characteristics of the colon, the calcium alginate microspheres selectively disintegrate in the colon; second, taking advantage of the high GSH concentration in tumors, the release of SN38 is triggered through the redox reaction of disulfide bonds; finally, the cell uptake is further enhanced through CD44 receptor-mediated targeted internalization. This multi-level and multi-mechanism synergistic release strategy significantly increases the drug concentration of SN38 at the tumor site while minimizing its exposure in normal tissues, effectively solving the serious gastrointestinal toxicity problem in traditional irinotecan treatment. This precision drug release strategy provides a new paradigm for the targeted delivery of oral chemotherapy drugs and has important clinical translation value. Description of the Drawings

[0036] Figure 1 It is the Fourier transform infrared spectroscopy (FT-IR) of CLL and its synthetic raw materials and intermediates;

[0037] Figure 2 1H-NMR spectra of CLL and its synthetic raw materials and intermediates;

[0038] Figure 3 XRD patterns of CLL and its synthetic raw materials and intermediates;

[0039] Figure 4 DSC curves of CLL and its synthetic raw materials and intermediates;

[0040] Figure 5 Critical micelle concentration (CMC) curve of CLL;

[0041] Figure 6 Particle size distribution diagram of CLLNPs;

[0042] Figure 7 TEM images of CLLNPs;

[0043] Figure 8 Cumulative release of SN38 from SN38 / CLLNPs in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), simulated colonic fluid (SCF) and SCF + GSH environments (n = 3) (GSH-mediated responsive release);

[0044] Figures 9 - 10 Cytotoxicity of LLNPs loaded with SN38 against colon cancer cell line CT-26 after 24 h and 48 h (detected by CCK8 method);

[0045] Figure 11 Cytotoxicity of CLL NPs loaded with SN38 against colon cancer cell line CT-26 after 24 h with / without GSH (enhanced cytotoxicity caused by GSH-mediated responsive release);

[0046] Figure 12 In vitro CT-26 cell uptake of CLLNPs and CS-mediated active targeting uptake;

[0047] Figure 13 Morphology and elemental analysis of SN38 / CLLNPsinMPs;

[0048] Figure 14 Stability of SN38 / CLL NPsinMPs in SGF, SIF and SCF environments under optical microscope (A) and apparent image (B);

[0049] Figure 15Drug release of SN38 / CLLNPsinMPs in SGF, SIF and SCF environments;

[0050] Figure 16 Ki-67 staining of tumor tissues after treating colon cancer mice with SN38 / CLLNPsinMPs;

[0051] Figure 17 TUNEL staining of tumor tissues after treating colon cancer mice with SN38 / CLLNPsinMPs. Detailed implementation mode

[0052] The present invention will be described in detail below with reference to specific embodiments.

[0053] The abbreviations in the embodiments are defined as follows:

[0054] LBP / LBPs: Lycium barbarum polysaccharide;

[0055] LA: Lipoic acid;

[0056] SN38: 7-Ethyl-10-hydroxycamptothecin;

[0057] CS: Chondroitin sulfate;

[0058] LL: Lycium barbarum polysaccharide-lipoic acid amphiphilic diblock copolymer;

[0059] LL NPs: Lycium barbarum polysaccharide-lipoic acid amphiphilic diblock copolymer self-assembled nanomicelles;

[0060] CLL: Amphiphilic triblock copolymer;

[0061] CLL NPs: Lycium barbarum polysaccharide-lipoic acid-chondroitin sulfate amphiphilic triblock copolymer self-assembled nanomicelles;

[0062] SN38 / LL NPs: Lycium barbarum polysaccharide-lipoic acid amphiphilic diblock copolymer self-assembled nanomicelles loaded with SN38;

[0063] SN38 / CLLNPs: Lycium barbarum polysaccharide-lipoic acid-chondroitin sulfate amphiphilic triblock copolymer self-assembled nanomicelles loaded with SN38;

[0064] SN38 / LL NPsinMPs: Calcium alginate microspheres loaded with SN38 / LLNPs;

[0065] SN / 38 / CLL NPsinMPs: Calcium alginate microspheres loaded with SN38 / CLLNPs.

[0066]

Preparation and characterization of SN38 / CLLNPs

[0067] (1) Preparation of CLL

[0068] Weigh 0.537 g of LA and a certain amount of N,N'-dicyclohexylcarbodiimide (DCC) and dissolve them in 5 ml of dichloromethane. Stir the solution at 300 rpm / min for 24 h at room temperature using a thermostatic magnetic stirring water bath. After stirring, filter the solution through a 0.45 μm filter membrane to remove the impurity of N-N dicyclohexylurea formed during the reaction. Then add LBP dissolved in 5 ml of dimethyl sulfoxide (DMSO) with a mass ratio of 1:1 - 1:5. Finally, add 4-dimethylaminopyridine (DMAP), where the molar ratio of LA:DCC:DMAP is 2:2:1, and further stir the resulting solution at the same rotation speed for 48 h at room temperature. Then dialyze the solution for 24 h using a pre-treated dialysis bag (cut-off molecular weight: 3500), and lyophilize to obtain LL. Dissolve CS, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and DMAP in 5 ml of deionized water and stir at 100 - 500 rpm at room temperature for 2 - 8 h. Dissolve the LL polymer in 5 ml of DMSO and stir for 1 - 4 h under the same conditions. Then add the CS solution dropwise to the LL polymer solution under ice bath conditions, and further stir the mixture for 36 - 72 h. After stirring, dialyze the mixture solution for 36 - 72 h using a dialysis bag with a cut-off molecular weight of 8000 - 14000 Da, and finally wash with water to remove the unreacted CS completely. Lyophilize to obtain CLL as the final product and store it at -4 °C for further use.

[0069] (2) Characterization of CLL

[0070] The structure of the prepared amphiphilic triblock copolymer CLL of wolfberry polysaccharide - lipoic acid - chondroitin sulfate was characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance hydrogen spectroscopy (1H NMR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC); the critical micelle concentration (CMC) of the CLL polymer was determined by pyrene fluorescence spectroscopy.

[0071] (3) Synthesis and characterization of CLLNPs

[0072] Preparation of CLL blank nanomicelles by nanoprecipitation method: Weigh 10 mg of CLL polymer and dissolve it in 2 ml of dimethyl sulfoxide (DMSO). Then, continuously stir at a speed of 300 rpm / min for 1 h at room temperature, and slowly add 3 ml of ultrapure water dropwise. After stirring for 4 h, dialyze the mixture for 1 day using a dialysis bag (molecular weight cut-off: 3500) to remove DMSO, with a final concentration of 1 mg / ml. Then, sonicate the solution for 5 min under the condition of 120 W, centrifuge at 2500 rpm for 15 min, and take the supernatant to measure the particle size and observe the morphology of the nanoparticles using a transmission electron microscope. Finally, lyophilize the obtained solution and store it at -4 °C for further use. Synthesize LL blank nanomicelles in the same method as a control.

[0073] (4) Synthesis of SN38 / CLLNPs

[0074] Preparation of SN38C / LLNPs by nanoprecipitation method: Weigh 10 mg of CLL polymer and 1 mg of SN38 and dissolve them in 2 ml of DMSO. Continuously stir at a speed of 300 rpm / min for 1 h at room temperature, and then slowly add 3 ml of ultrapure water dropwise. Stir for 4 h, and then place the sample in a dialysis bag (molecular weight cut-off: 3500). After dialysis for 24 h, drug-loaded nanoparticles are obtained. Sonicate the drug-loaded nanoparticle solution for 5 min under the condition of 120 W, centrifuge at 2500 rpm for 15 min, and take the supernatant to measure its drug loading, encapsulation efficiency, and particle size. Synthesize SN38 / CLLNPs nanomicelles in the same method as a control.

[0075] (5) Determination of drug encapsulation efficiency and drug loading

[0076] When preparing SN38 / CLLNPs, collect the supernatant obtained by centrifugation, measure the absorbance value of SN38 in the supernatant using a UV-visible spectrophotometer, substitute it into the standard curve established above, and calculate the drug content of SN38 in the supernatant.

[0077] The drug encapsulation efficiency (EE) and drug loading (DL) of SN38 / LLNPs are calculated according to the following formulas:

[0078] Encapsulation efficiency (EE) % = weight of SN38 in nanoparticles / total drug dosage × 100%

[0079] Drug loading (DL) % = weight of SN38 in nanoparticles / weight of drug-loaded nanoparticles × 100%

[0080] (6) Prescription screening of SN38 / CLLNPs

[0081] Taking particle size, PDI index, drug loading and encapsulation efficiency as comprehensive indicators, single-factor experiments were carried out to optimize the ratio of LL to CS when preparing CLL and the ratio of CLL to SN38 when preparing SN38 / CLLNPs, respectively.

[0082] (7) In vitro responsive release of SN38 from SN38 / CLLNPs mediated by GSH

[0083] The dialysis bag diffusion method was used to evaluate the drug release behavior of the prepared nanoparticles. First, 1 ml of free SN38 (containing 0.5 mg of SN38), SN38 / LL NPs and SN38 / LL NPs + GSH (10 mM) were placed in a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da. Subsequently, the dialysis bag was immersed in 35 ml of PBS buffer of simulated gastric fluid (pH 1.2, 0 - 2 h), small intestinal fluid (pH 6.8, 2 - 6 h) and colonic fluid (pH 7.4, 6 - 48 h). Then, 0.5% w / v Tween 80 was added to the release medium to promote the dissolution of SN38. The mixture was shaken on a rotary shaker at 37 °C and 100 rpm. At predetermined time intervals: 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 16 h, 24 h, 48 h, 72 h, 96 h, 3 mL of the supernatant was collected and an equal volume of fresh buffer was added. The absorbance of SN38 at 380 nm was obtained using a UV-visible spectrophotometer. According to the amount of SN38 released, the cumulative release curve of SN38 was plotted.

[0084] [Performance verification of SN38 / CLLNPs at the cellular level]

[0085] (1) Evaluation of in vitro cytotoxicity of SN38 / CLLNPs

[0086] The CCK-8 method was used to evaluate the cytotoxicity of free SN38 / CLLNPs, with SN38 and SN38 / LL NPs as controls. The specific method was as follows: CT-26 cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 1 × 10 4 cells per well in 100 μL of RPMI-1640 medium containing 10% fetal bovine serum. After culturing the cells overnight at 37 °C in a 5% carbon dioxide atmosphere to allow cell adhesion, the medium was discarded and 100 μL of fresh medium containing free SN38, SN38 / LL NPs and SN38 / CLLNPs at concentrations of 0.1, 1.25, 2.5, 5, 10 μM was added. The cells were incubated for 24 and 48 hours. After incubation, the medium was discarded and the cells were washed twice with PBS. Subsequently, 100 μl of basal medium and 10 μl of CCK-8 were added to each well and incubated for another 2 h. The absorbance of each well at a wavelength of 450 nm was measured using a microplate reader.

[0087] (2) Influence of GSH-mediated responsive release on in vitro cytotoxicity

[0088] Studies have shown that GSH-OEt and BSO can increase and decrease intracellular glutathione levels respectively. Therefore, GSH-OEt and BSO were used to study the GSH-responsive release of intracellular nanodrugs. The specific steps are as follows: CT-26 cells were seeded in 96-well plates containing 100 μl of complete medium at a density of 1×10⁴ cells per well. After the cells adhered overnight, the medium was discarded, and CT-26 cells were pretreated with 1 mM BSO or 20 mM GSH-OEt for 2 h, and then fresh medium containing SN38 / LLNPs and SN38 / CLLNPs was added. The drug-loaded formulations were incubated at concentrations of 0.1, 1.25, 2.5, 5, 10 μM for 24 h. After incubation, the medium was discarded, and the cells were rinsed twice with PBS (pH 7.4, 0.01 M). Subsequently, 100 μl of basal medium and 10 μL of CCK-8 were added to each well and incubated for another 2 h. Untreated cells were used as controls. The absorbance of each well at a wavelength of 450 nm was measured using a microplate reader.

[0089] (3) Evaluation of the active targeting effect of CS-mediated CLLNPs on CD44 receptor

[0090] Coumarin-6 (C6), a fluorescent dye, was used instead of SN38, and C6 nanoparticles were prepared by the same preparation method. The uptake of C6 nanoparticles by CT-26 cells and the uptake mechanism were investigated. The fluorescence intensity in CT-26 cells was qualitatively observed by laser confocal microscopy. The specific method was as follows: CT-26 cells were seeded in confocal dishes at a density of 5×10 4 cells per well and cultured in a 5% carbon dioxide incubator for 24 h. After the cells were completely adherent, the original medium was discarded, and serum-free medium containing free C6, C6 / CLLNPs or C6 / LLNPs was added respectively, with a C6 concentration of 0.1 μg / ml. After the cells were cultured for 1 h, 2 h and 4 h respectively, the cells were fixed with 300 μL of 4% paraformaldehyde for 10 min, and then stained with DAPI (1 μg / ml) for 10 min. After treatment with an anti-fluorescence quencher, the penetration of cells at different culture times was observed using a confocal laser scanning microscope (CLSM). To further verify the blocking experiment of the active targeting effect of CS-mediated CD44 receptor, the cells were pretreated with CS for 0.5 h and then incubated with C6 / CLLNPs for 4 h. The cells were fixed with 4% paraformaldehyde for 10 min, and then stained with DAPI (1 μg / ml) for 10 min. After treatment with an anti-fluorescence quencher, the penetration of cells was observed using a confocal laser scanning microscope (CLSM).

[0091]

Preparation and Evaluation of SN38 / CLLNPsinMPs

[0092] (1) Preparation of SN38 / CLLNPsinMPs

[0093] SN38 / CLLNPsinMPs were prepared using microfluidic technology, and SN38 / LLNPsinMPs were prepared as a control. The specific preparation method is as follows: SN38 / LLNPs and SN38 / CLLNPs were separately dissolved in sodium alginate solution and sonicated for 5 min to form a uniformly dispersed NPs / Alg compound with a final concentration of 1% w / v (the higher the concentration, the easier the needle is to clog). Then, the sodium alginate solution was injected with a syringe, and the liquid droplets were dropped into 100 mM calcium chloride solution by gravity. Sodium alginate cross-linked with Ca 2+ to form SN38 / LLNPsinMPs and SN38 / CLLNPsinMPs. The Alg hydrogel microspheres were collected, and the remaining ions on the surface were washed three times with deionized water and stored at 4°C. Among them, the syringe needle was a flat needle (22G), the receiving distance was 20 cm, the flow rate was 100 mm / min, and the microspheres had good morphology and uniform particle size.

[0094] (2) Characterization of SN38 / CLLNPsinMPs

[0095] Particle size: The size of SN38 / CLLNPsinMPs was measured using a Malvern laser particle size analyzer.

[0096] Morphology: An appropriate amount of freeze-dried blank MPs, SN38 / LL NPsinMPs, and SN38 / CLL NPsinMPs were observed for the morphology and composition analysis of the microparticles by scanning electron microscopy combined with X-ray energy chromatography (SEM&EDS); and their microstructures were observed using an optical microscope.

[0097] (3) Determination of drug loading of SN38 / CLLNPsinMPs

[0098] Precisely weigh 3 portions of 5 mg of SN38 / CLLNPsinMPs and disperse them in a 10 ml mixed solvent of DMSO:PBS (1:9), and stir at 500 rpm / min in a 37°C constant temperature magnetic water bath for 24 h to completely dissolve the microspheres and release the drug. Measure the ultraviolet absorption of SN38 at 380 nm and calculate the drug loading.

[0099] (4) In vitro stability evaluation in the gastrointestinal environment

[0100] To study the stability of SN38 / CLLNPsinMPs during gastrointestinal delivery, they were placed separately in 5 mL of simulated gastric fluid (SGF pH 1.2), simulated intestinal fluid (SIF pH 6.8), and simulated colonic fluid (SCF pH 7.4), and shaken at 100 rpm at 37 °C. The morphology of SN38 / CLLNPsinMPs was observed using an optical microscope at 0, 2, 6, 12, and 24 h.

[0101] (5) In vitro release of SN38 from SN38 / CLLNPsinMPs

[0102] The dialysis bag diffusion method was used to evaluate the drug release behavior of the prepared microspheres. Dialysis bags (molecular weight cut-off 8000 - 14000 Da) containing 1 mL of free SN38, SN38 / CLLNPs, and SN38 / CLLNPsinMPs (SN38 content 0.5 mg) were immersed in 35 mL of simulated gastric fluid (pH 1.2, 0 - 2 h), and the remaining detection steps were the same as those for the drug loading determination method of SN38 / CLLNPs.

[0103]

Evaluation of the in vivo anti - colon cancer effect of SN38 / CLLNPsinMPs

[0104] (1) Establishment of a mouse colon cancer model

[0105] CT - 26 cells (5×10 6 ) were injected subcutaneously into 8 - week - old C57BL / 6J mice. After the tumor volume reached approximately 100 mm3, the mice were randomly grouped for treatment. The tumor volume (V) was calculated as V = ab2 / 2, where a is the longest length of the tumor and b is the longest width of the tumor.

[0106] (2) Evaluation of the in vivo anti - tumor effect of SN38 / CLLNPsinMPs

[0107] The mice with colon cancer models were randomly divided into 3 groups and intragastrically administered with 0.5% sodium carboxymethylcellulose saline as the solvent, intragastrically administered with 20 mg / kg of free SN38, 20 mg / kg of SN38 / LL NPsinMPs, and SN38 / CLLNPsinMPs. The mice were administered the drug once every other day, and the tumor volume was measured. After 14 days of treatment, the mice were sacrificed, the tumor tissues were dissected, weighed, and then the tumor tissues were collected to evaluate the proliferation of the tumor tissues using anti - ki - 67 antibody. The terminal - deoxynucleotidyl - transferase - mediated dUTP nick - end - labeling (TUNEL) apoptosis detection kit was used to identify apoptotic cells in the tumor, and the analysis was performed using a fluorescence microscope.

[0108]

Experimental characterization results of CLL

[0109] (1) Fourier transform infrared spectroscopy (FT-IR)

[0110] As Figure 1 shown in the infrared spectrum, the 1639 cm-1 in CS belongs to the asymmetric stretching vibration peak of C=O; compared with the ester group peak at 1736 cm-1 of the LL polymer, a new ester group peak appears at 1743 cm-1 in the CLL polymer, and the blue shift of the infrared peak proves the successful synthesis of the CLL polymer.

[0111] (2) 1H nuclear magnetic resonance spectroscopy (1H NMR)

[0112] As Figure 2 , in the 1H NMR spectrum of CS, the sugar chain proton peak of CS is at 3.21 - 4.6 ppm, and the acetyl proton peak is at 1.88 ppm. Compared with the 1H NMR spectrum of LL, there is no obvious peak shift in the 1H NMR spectrum of CLL, probably because the input amount of CS is small and the molecular weight is small, so the chemical shift value is not obvious. However, the peak intensity at 1.00 - 1.81 ppm in the 1H NMR spectrum of CLL becomes weaker, indirectly indicating that CS may be grafted onto the LL polymer structure.

[0113] (3) X-ray diffraction (XRD)

[0114] To further verify the successful synthesis of the CLL polymer, XRD was used for further structural verification. The results are as Figure 3 shown. The X-ray diffraction peak of CS is an amorphous peak. Compared with the XRD peak of the LL polymer, the diffraction peak of the CLL polymer has a weaker intensity and a broader peak shape. Thus, it can be obtained that CS is successfully grafted onto the LL polymer structure.

[0115] (4) Differential scanning calorimetry (DSC)

[0116] As Figure 4 shown, in the DSC spectrum, CS shows a broad peak at 100 - 150 °C. After grafting onto the LL polymer, this peak disappears, and a sharp endothermic peak appears at 99.67 °C, further proving the synthesis of the CLL polymer.

[0117] (5) Critical micelle concentration (CMC)

[0118] Using pyrene as a hydrophobic fluorescent probe, the CMC of the CLL polymer was detected by fluorescence method. As Figure 5 shown, the CMC of the CLL polymer is 0.029 mg / ml, and the CMC is lower than that of sodium dodecyl sulfate, proving that the CLL polymer is more likely to form nano micelles.

[0119]

Prescription screening and characterization results of SN38 / CLLNPs

[0120] (1) Screening the optimal reaction ratio of LL and CS by single - factor method

[0121] Taking the particle size and PDI of the self - assembled nanomicelles as indicators, the optimal reaction ratio of CS and LL was screened. The results are shown in Table 1. When the mass ratio of CS and LL was 1:1, the average particle size of the formed nanomicelles was 178.1 ± 0.47 nm, and the PDI was 0.225 ± 0.010, which met the requirements of the nanodrug delivery system, with a narrow distribution range and good stability. Therefore, the mass ratio of CS and LL of 1:1 was used to prepare CLL in subsequent studies.

[0122] Table 1 Screening the optimal ratio of CS and LL polymers by single - factor method (n = 3)

[0123]

[0124] (2) Screening the optimal dosing ratio by single - factor method

[0125] Taking the particle size, PDI, DL(%), and EE(%) of CLLNPs loaded with SN38 as comprehensive indicators, the optimal dosing ratio of CLL and SN38 was screened. The results are shown in Table 2. When the mass ratio of CLL and SN38 was 10:2, the drug - loading rate was 9.51 ± 0.08%, and the encapsulation efficiency was 57.03 ± 0.50%. Compared with other groups, it had a higher encapsulation efficiency and a smaller particle size. The dosing ratio of CLL and SN38 of 10:2 was preferably used for subsequent studies.

[0126] Table 2 Screening the optimal dosing ratio of SN38 and CLL by single - factor method (n = 3)

[0127]

[0128] (3) Particle size and morphology characterization of LL NPs

[0129] The particle size measurement results are shown in Table 2. The average particle size of CLL NPs was 176.8 ± 2.63 nm, and the PDI was 0.135 ± 0.02; the average particle size of SN38 / CLLNPs was 214.6 ± 9.53 nm, and the PDI was 0.267 ± 0.02. Particle size measurement Figure 6 . Under the transmission electron microscope, CLLNPs were spherical and evenly distributed (as Figure 7 )

[0130] (4) Drug - loading rate and encapsulation efficiency of SN38 in SN38 / CLL NPs

[0131] As shown in Table 2, the drug loading and encapsulation efficiency of SN38 / CLLNPs were detected by ultraviolet-visible spectrophotometry. The optimal drug loading of the selected SN38 / CLLNPs was 9.51±0.08%, and the encapsulation efficiency was 57.03±0.50%.

[0132] (5) In vitro responsive release of SN38 / CLL NPs mediated by GSH

[0133] As Figure 8 shown, the release amount of free SN38 in the whole release system was significantly higher than that of SN38 / CLL NPs, which was 1.8 times that of SN38 / CLL NPs. SN38 / CLLNPs were slowly released in the gastrointestinal environment. In addition, the release amount of SN38 from SN38 / CLL NPs in the environment with additional GSH for 24 h was 49.74%, which was 1.4 times that of SN38 / CLLNPs without GSH, indicating that SN38 / CLLNPs had GSH responsiveness.

[0134] [Performance evaluation results of SN38C / LL NPs at the cellular level]

[0135] (1) In vitro cytotoxicity of SN38 / CLL NPs

[0136] Taking free SN38 and SN38 / LLNPs as controls, the in vitro cytotoxicity of SN38 / CLL NPs against CT-26 cells at 24 h and 48 h was evaluated by the CCK-8 method. As Figures 9 - 10 shown, the inhibitory effect of SN38 / CLL NPs on CT-26 cells was significantly stronger than that of the free drug SN38 and SN38 / LL NPs, and showed concentration- and time-dependence.

[0137] (2) Responsive release mediated by GSH enhances the cytotoxicity of SN38 / CLLNPs

[0138] Taking CT-26 cells as a model, the intracellular GSH response of SN38 / CLLNPs was studied. As Figure 11 shown, the survival rate of cells without any pretreatment was significantly lower than that of cells treated with BSO, while the survival rate of cells treated with GSH-oet was significantly lower than that of cells pretreated with BSO and untreated cells. After treatment with GSH-oet, the survival rate of cells treated with SN38 / CLLNPs decreased significantly, confirming that the responsive release mediated by GSH significantly enhanced the cytotoxicity of SN38 / CLLNPs.

[0139] (3) Active targeting effect of CS-mediated CLLNPs on CD44 receptor

[0140] Coumarin-6 (C6) was used instead of SN38, and the uptake of free C6, C6 / LLNPs, and C6 / CLLNPs by CT-26 tumor cells was qualitatively observed by CLSM (the blue and green fluorescence signals are the nuclear dye DAPI and C6, respectively). At the same time, pretreatment with free CS was used as a control for competitive binding to CD44 to further confirm the CS-mediated active targeting effect of CD44. As Figure 12 shown, after co-incubation for 4 h, the green fluorescence of C6 / CLLNPs was the brightest, followed by C6 / LLNPs, while that of free C6 was the weakest, indicating that in addition to the passive targeting effect of the nanocarrier similar to C6 / LLNPs, C6 / CLLNPs also have the CS-mediated active targeting effect of the CD44 receptor. After CS pretreatment, the green fluorescence intensity of C6 / CLLNPs decreased significantly, further confirming that CLLNPs have the CS-mediated active targeting effect of the CD44 receptor.

[0141]

In vitro verification results of SN38 / CLLNPsinMPs

[0142] (1) Results of particle size and morphology characterization

[0143] The particle size of SN38 / LLNPsinMPs was measured to be 1.44 ± 0.43 μm and the PDI was 0.760 ± 0.17 using a Malvern particle size analyzer; the particle size of SN38 / CLLNPsinMPs was 3.29 ± 0.79 μm and the PDI was 0.280 ± 0.01. Under an optical microscope, SN38 / CLLNPsinMPs had a core-shell structure, indicating that SN38 / CLLNPs were successfully encapsulated in sodium alginate hydrogel microspheres ( Figure 13 D). SEM&EDS analysis showed (as Figure 13 A-13C, 13E-13F), due to water loss during freeze-drying, SN38 / CLLNPsinMPs were in a shrunken ellipsoidal shape; only C, O, and Ca elements were present in the blank microspheres, while in the structures of SN38 / LLNPsinMPs and SN38 / CLL NPsinMPs, in addition to C, O, and Ca elements, there was also the S element present in LA and CS, thus confirming that SN38 / LLNPs and SN38 / CLLNPs were successfully encapsulated in sodium alginate microspheres. In addition, the S element content in SN38 / CLLNPsinMPs was higher than that in SN38 / LLNPsinMPs, confirming that its S element not only came from LA but also from CS, further indirectly proving the successful synthesis of CLL and the successful encapsulation of SN38 / CLLNPs in the microspheres.

[0144] (2) Drug loading of SN38 in SN38 / CLL NPsinMPs

[0145] The drug loading of SN38 in SN38 / CLL NPsinMPs is shown in Table 3.

[0146] Table 3 Particle size and drug loading of SN38 / LLNPsinMPs and SN38 / CLLNPsinMPs (n = 3)

[0147]

[0148] (3) In vitro stability evaluation in the gastrointestinal environment

[0149] To study the stability of calcium alginate microspheres (MPs) in the gastric and small intestinal environments, SN38 / CLL NPsinMPs were placed in different simulated digestive fluids for 24 h. Their morphological changes at different time intervals were observed through microscopic images. As Figure 14 shown in A, the morphology of SN38 / CLLNPsinMPs did not change significantly after 24 h in SGF, demonstrating its stability under gastric pH conditions. In SIF, SN38 / CLL NPsinMPs did not change significantly within 6 h, but began to swell at 12 h. According to the physiological characteristics of the gastrointestinal tract, the retention time of gastrointestinal contents in the stomach and small intestine does not exceed 12 h. It can be speculated that SN38 / CLLNPsinMPs can maintain structural stability during oral transport in the stomach and small intestine. In SCF, SN38 / CLLNPsinMPs swelled rapidly at 6 h, became blurred in outline at 12 h, and the integrity of SN38 / CLL NPsinMPs was damaged at 24 h, indicating that SN38 / CLLNPsinMPs can be rapidly decomposed and released in colon fluid. Since SN38 / CLLNPsinMPs are translucent spherical microparticles and it is not easy to observe their changes in the simulated gastrointestinal environment with the naked eye, SN38 was replaced with C6, encapsulated into nanoparticles, and then C6 / CLLNPsinMPs were prepared to observe their morphological changes during gastrointestinal transport. As Figure 14 shown in B, the results were consistent with those observed under an optical microscope.

[0150] (4) In vitro release of SN38 in SN38 / CLLNPsinMPs

[0151] The in vitro release of SN38 in SN38 / CLLNPsinMPs is as Figure 15As shown, compared with free SN38, the release of SN38 / CLL NPs in MPs was very low in SGF and SIF. Only 5.13% was released in SGF, and it was gradually released in SIF. The release of free SN38 in SIF was 2.5 times that of SN38 / CLL NPs in MPs. SN38 / CLL NPs in MPs maintained a basically intact structure in SGF and gradually lysed in SIF. The release amount in SCF reached 39.4% after 96 hours. SN38 / CLL NPs were slowly released in the whole system, and the release amount at 96 h was 50.37%, which was 1.28 times that of SN38 / CLL NPs in MPs. It was thus confirmed that calcium alginate microspheres (MPs) could protect SN38 / CLL NPs from being destroyed in the gastric acidic environment and reach the colon smoothly.

[0152]

In vivo anti - colon cancer effect of SN38 / CLL NPs in MPs

[0153] (1) Significantly enhanced anti - colon cancer proliferation effect

[0154] Tumor - bearing mice were respectively given free SN38, SN38 / LL NPs in MPs and SN38 / CLL NPs in MPs by gavage. After 14 days of treatment, tumor tissues were taken for Ki67 immunohistochemical staining (IHC). As Figure 16 shown, the Ki - 67 expression in the SN38 / CLL NPs in MPs group was significantly higher than that in the SN38 / LL NPs in MPs group and the free SN38 group, indicating that NPs in MPs had a significantly enhanced anti - tumor cell proliferation effect.

[0155] (2) Significantly enhanced effect of inducing apoptosis of colon cancer cells

[0156] Tumor - bearing mice were respectively given free SN38, SN38 / LL NPs in MPs and SN38 / CLL NPs in MPs by gavage. After 14 days of treatment, tumor tissues were taken to further evaluate their effect of inducing tumor cell apoptosis by terminal - deoxynucleotidyl - transferase dUTP nick end - labeling (TUNEL) staining. The results showed that ( Figure 17 ), compared with the free SN38 group and SN38 / LL NPs in MPs, the SN38 / CLL NPs in MPs group had significantly enhanced apoptotic colon cancer cells (enhanced red fluorescence), proving that SN38 / CLL NPs in MPs had a significantly enhanced effect of inducing apoptosis of colon cancer cells.

[0157] Based on the above results, it can be seen that the lycium barbarum polysaccharide-based SN38 active targeting preparation prepared by the present invention can achieve effective enrichment of SN38 at the colon cancer site after oral administration, significantly improve the therapeutic effect, and reduce systemic toxicity, which helps to solve the technical problems such as poor water solubility of SN38, low oral bioavailability, and severe gastrointestinal toxicity caused by non-specific distribution, and has significant technological progress.

[0158] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A Lycium barbarum polysaccharide-based SN38 active targeted oral preparation, characterized in that: The preparation includes: (1) SN38 is the anti-tumor active ingredient; (2) The nanomicelles formed by self-assembly of an amphiphilic triblock copolymer (CLL) synthesized from Lycium barbarum polysaccharide (LBP), lipoic acid (LA), and chondroitin sulfate (CS) were used as active targeting carriers, and SN38 was encapsulated inside them; (3) Calcium alginate microspheres are used as oral delivery carriers, and the nano-micelles are encapsulated in the calcium alginate microspheres by microfluidic technology.

2. The oral preparation according to claim 1, characterized in that The amphiphilic triblock copolymer (CLL) is synthesized by esterifying Lycium barbarum polysaccharide and lipoic acid to form an amphiphilic diblock copolymer intermediate (LL), which is then further synthesized by esterifying with chondroitin sulfate.

3. The oral preparation according to claim 2, characterized in that In the amphiphilic diblock copolymer intermediate (LL), the mass ratio of the wolfberry polysaccharide to lipoic acid is 1:1-1:5, preferably 1:1-1:3; In the amphiphilic triblock copolymer, the mass ratio of the chondroitin sulfate to the amphiphilic diblock copolymer intermediate (LL) is 1:2-2:1, preferably 1:

1.

4. The oral preparation according to claim 1, characterized in that The mass ratio of the amphiphilic triblock copolymer to the SN38 is 10:1-10:5, preferably 10:1-10:

3.

5. The preparation according to claim 1, characterized in that The particle size of the nano micelle is 150-400 nm, preferably 200-250 nm, and the PDI is 0.01-0.5, preferably 0.01-0.

3.

6. The oral preparation according to claim 1, characterized in that The concentration of sodium alginate in the calcium alginate microspheres is 1-3% (w / v), and the concentration of calcium chloride is 50-200 mM.

7. A method for preparing the oral preparation according to claim 1, characterized in that: The following steps are involved: (1) esterifying Lycium barbarum polysaccharide with lipoic acid in the presence of a catalyst to synthesize an amphiphilic diblock copolymer intermediate (LL); (2) synthesizing an amphiphilic triblock copolymer (CLL) by esterifying the amphiphilic diblock copolymer intermediate (LL) obtained in step (1) with chondroitin sulfate; (3) using a nanoparticle coprecipitation method with the amphiphilic triblock copolymer obtained in step (2) and SN38, and using ultrasound and stirring to allow the amphiphilic triblock copolymer to self-assemble into nanomicelles encapsulating SN38; (4) mixing the nanomicelle suspension obtained in step (3) with a sodium alginate solution, and dropping the mixture into a calcium chloride solution through a microfluidic system to form calcium alginate microspheres encapsulating the nanomicelles.

8. The preparation method according to claim 7, wherein the specific method of step (1) is: dissolving lipoic acid and a coupling reagent in a solvent and stirring and mixing; adding a dimethyl sulfoxide solution containing wolfberry polysaccharides and a catalyst; stirring the mixed solution at room temperature for reaction, dialyzing with a dialysis bag after the reaction is completed, and freeze-drying to obtain; The specific method of step (2) is as follows: dissolving chondroitin sulfate, a coupling agent and a catalyst in water, stirring at room temperature to form a chondroitin sulfate solution; dissolving the amphiphilic diblock copolymer intermediate in DMSO, stirring under the same conditions; dripping the chondroitin sulfate solution into the amphiphilic diblock copolymer intermediate solution under ice bath conditions, further stirring the mixture, dialyzing the mixture solution with a dialysis bag after stirring, then washing with water to remove the unreacted chondroitin sulfate, and freeze-drying to obtain an amphiphilic triblock copolymer (CLL).

9. The preparation method according to claim 7, wherein the specific method of step (3) is as follows: dissolving the amphiphilic triblock copolymer (CLL) and SN38 in DMSO, and then continuously stirring at room temperature at 200-500 rpm for 0.5-2 h, then dropping deionized water, and continuing stirring for 2-6 hours. After the end, placing the sample in a dialysis bag, dialyzing for 18-32 hours to obtain a SN38-loaded nanomicelle suspension, and further ultrasonicating and centrifuging to obtain the SN38-loaded nanomicelles; The specific method of step (4) is as follows: sodium alginate is added to the nanomicelle suspension containing SN38 to dissolve, and Ca-containing 2+ , Ba 2+ or Fe 3+ of salt solution.

10. Use of the oral preparation according to claim 1 in the preparation of anti-tumor drugs.

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