Oral paclitaxel microcapsule capable of resisting multidrug resistance as well as preparation method and application of oral paclitaxel microcapsule

Abstract

The invention relates to the field of porous starch drug loading, in particular to an oral paclitaxel microcapsule capable of resisting multidrug resistance as well as a preparation method and application of the oral paclitaxel microcapsule. The preparation method comprises the following steps: (1) adsorbing paclitaxel by using porous starch; and (2) curcumin-paclitaxel co-amorphous preparation: pouring an acetic acid solution of curcumin into a plate of the product obtained in the step (1), uniformly dispersing, then carrying out solvent volatilization again, after complete volatilization, washing with an ethanol solution to remove unloaded paclitaxel, and centrifuging to obtain a precipitate, namely the oral paclitaxel microcapsule. The preparation method has the beneficial effects that the paclitaxel and the curcumin are loaded by the porous starch, so that the drug (paclitaxel) loading amount (35.35 + / -0.12%) of the porous starch is increased from the aspect of drug crystallization regulation and control, and the encapsulation efficiency reaches 100%. The constructed co-loading system obviously enhances the anti-tumor effect of paclitaxel in the aspect of overcoming drug resistance.

Description

Technical Field

[0001] This invention relates to the field of porous starch-loaded drugs, and particularly to an oral paclitaxel microcapsule for resisting multidrug resistance, its preparation method, and its application. Background Technology

[0002] Cancer, a major health threat in modern society, significantly impairs patients' quality of life. Chemotherapy is an important anti-cancer treatment; however, the clinical application of commonly used chemotherapeutic drugs such as paclitaxel faces severe challenges. Their inherent hydrophobicity leads to poor gastrointestinal stability, significant first-pass effect, and systemic toxicity, resulting in low oral bioavailability. More critically, cancer cells often develop multidrug resistance, becoming a major cause of treatment failure and relapse. The mechanisms of multidrug resistance (MDR) are complex, with P-glycoprotein (P-gp)-mediated drug efflux being particularly prominent. These limiting factors collectively weaken the in vivo efficacy of chemotherapeutic drugs, necessitating the development of new strategies to overcome delivery barriers and reverse tumor drug resistance, thereby improving treatment efficacy and patient prognosis.

[0003] Porous starch, as a biodegradable material, has become a promising carrier due to its high specific surface area, tunable pore structure, and excellent biocompatibility. Its three-dimensional network structure not only efficiently co-loads hydrophobic drugs (such as paclitaxel and curcumin) through hydrophobic interactions, but its nanoscale pores (10-100 nm) also restrict drug diffusion, inhibit crystal nucleation and growth, and promote highly dispersed drugs in an amorphous form, thereby significantly improving solubility and bioavailability. Notably, curcumin (CU) not only possesses anti-inflammatory and antioxidant activities, but its core value lies in its ability to effectively reverse tumor malignant transformation (MDR), for example, by inhibiting P-gp-mediated drug efflux and downregulating the NF-κB signaling pathway, thus sensitizing chemotherapeutic drugs such as paclitaxel.

[0004] However, (1) porous starch alone cannot effectively resist the corrosion and degradation of the gastrointestinal tract, resulting in a large amount of drug leakage in the upper digestive tract that cannot reach the colon area; (2) curcumin alone cannot improve the oral bioavailability of paclitaxel; and (3) the drug loading and encapsulation efficiency of porous starch for co-loading two hydrophobic drugs are still not ideal.

[0005] Chinese invention application CN 114948908 B discloses a method for preparing high-load oral paclitaxel capsules for colonic sustained release, belonging to the field of porous starch drug loading. The preparation method includes the following steps: (1) adding an ethanol solution of paclitaxel dropwise into an aqueous phase, drying to obtain amorphous paclitaxel microspheres; (2) redissolving the paclitaxel microspheres obtained in step (1) in an ethanol solution, dispersing the porous starch therein for adsorption, then evaporating the solvent in an oven, washing with an ethanol solution to remove unadsorbed paclitaxel, and centrifuging to obtain the precipitate as the paclitaxel-loaded porous starch; (3) dispersing the paclitaxel-loaded porous starch obtained in step (2) in a chitosan solution, and adding the solution dropwise to a phytic acid solution, stirring for 4 hours to obtain coated capsules. The above technical solution is green and environmentally friendly, can greatly reduce drug loss, efficiently load paclitaxel and achieve the effect of slow release in the colon.

[0006] However, the above-mentioned preparation methods for high-load oral paclitaxel capsules for colonic sustained release still have the following problems: (1) drug waste due to unsatisfactory encapsulation efficiency and drug loading; (2) capsule preparation involves many materials and complicated processes; and (3) cancer cells produce MDR, resulting in low bioavailability of paclitaxel. Therefore, it is necessary to propose a process for efficient loading-targeted delivery of paclitaxel that can overcome cancer MDR. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an oral paclitaxel microcapsule for resisting multidrug resistance, its preparation method and application. The obtained microcapsule has a high drug loading capacity and 100% encapsulation rate, and improves the oral bioavailability of paclitaxel in the colon, thus overcoming tumor MDR.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a method for preparing oral paclitaxel microcapsules for multidrug resistance, comprising the following steps: (1) Porous starch adsorbs paclitaxel: Porous starch was dispersed in an ethanol solution of paclitaxel, stirred until homogeneous for adsorption, and then poured into a petri dish for solvent evaporation. After complete evaporation, porous starch loaded with paclitaxel was obtained. (2) Preparation of co-amorphous curcumin-paclitaxel: Pour the curcumin acetic acid solution into a petri dish containing the product obtained in step (1), disperse it evenly, and then evaporate the solvent again. After complete evaporation, wash with ethanol solution to remove the unloaded paclitaxel. After centrifugation, the precipitate is the oral paclitaxel microcapsule.

[0009] Furthermore, in the above-mentioned method for preparing oral paclitaxel microcapsules for multidrug resistance, the porous starch in step (1) is enzymatically hydrolyzed porous starch, and the total amount of enzyme added is 1%-3% of the mass of the enzymatically hydrolyzed porous starch. The enzymes include α-amylase and glucosidase, and the volume ratio of α-amylase to glucosidase is 1:1-3. The hydrolysis time is 5-7 h, and the hydrolysis temperature is 45-60 °C.

[0010] Furthermore, in the above-mentioned method for preparing oral paclitaxel microcapsules for multidrug resistance, the concentration of paclitaxel in the ethanol solution of paclitaxel in step (1) is 10-40 mg / mL; The mass ratio of porous starch to paclitaxel in step (1) is 2-4:1; The adsorption in step (1) involves dispersing porous starch in paclitaxel solution and stirring at 37°C for 1.5-2.5 h. The temperature for solvent evaporation in step (1) is set to 45-55℃ and the evaporation time is 0.5-1h.

[0011] Furthermore, in the above-mentioned method for preparing oral paclitaxel microcapsules for multidrug resistance, the curcumin concentration in the acetic acid solution of curcumin in step (2) is 5-10 mg / mL.

[0012] Furthermore, in the above-mentioned method for preparing oral paclitaxel microcapsules for multidrug resistance, the mass ratio of curcumin to paclitaxel in step (2) is 1:4-8.

[0013] Furthermore, in the above-mentioned method for preparing oral paclitaxel microcapsules for multidrug resistance, the mass ratio of curcumin to paclitaxel in step (2) is 1:4-6.

[0014] Furthermore, in the above-mentioned method for preparing oral paclitaxel microcapsules for multidrug resistance, the mass ratio of curcumin to paclitaxel in step (2) is 1:5.

[0015] Furthermore, in the above-mentioned method for preparing oral paclitaxel microcapsules for multidrug resistance, the solvent evaporation temperature in step (2) is set to 45-55℃, and the evaporation time is 0.5-1h; In step (2), the volume ratio of the ethanol solution to the acetic acid solution is 10-15:1; In step (2), the centrifugation is performed at 4000-6000 r / min for 5-10 min.

[0016] Another technical solution provided by the present invention is: a method for preparing oral paclitaxel microcapsules with multidrug resistance to multidrug resistance.

[0017] Another technical solution provided by the present invention is: the application of the above-mentioned oral paclitaxel microcapsules with multidrug resistance in the preparation of colorectal cancer drugs.

[0018] The beneficial effects of this invention are as follows: (1) The present invention uses porous starch to support paclitaxel and curcumin. The method of the present invention is green and environmentally friendly, low in cost and biodegradable.

[0019] (2) The preparation method of the present invention improves the drug loading (paclitaxel) of porous starch by 35.35±0.12% from the perspective of drug crystallization regulation and achieves 100% encapsulation rate, which is important for expensive anticancer drugs.

[0020] After desolventization, the original paclitaxel molecules aggregate into elongated crystals via intermolecular hydrogen bonds and / or bridging networks. However, by co-loading curcumin to inhibit paclitaxel crystallization, it is transformed into an amorphous state during solvent evaporation (confirmed by XRD), significantly increasing the drug loading and achieving 100% encapsulation efficiency. Amorphous paclitaxel molecules are more easily accommodated by porous starch through pore adaptation and surface coating (confirmed by scanning electron microscopy), and tend to disperse in a molecular state during release, improving solubility.

[0021] (3) The preparation method of this invention simplifies the process and improves the drug loading stability and targeting of porous starch from the perspective of synergistic intermolecular forces. In particular, the process we proposed previously required a pretreatment step (i.e., amorphization of paclitaxel) and a subsequent coating preparation through the reaction of chitosan and phytic acid. The process of this invention achieves better encapsulation efficiency and drug loading while simplifying the process and the materials used. The formation of a multi-interaction network (FTIR / XPS confirmed) of porous starch (PS), paclitaxel (PTX) and curcumin (CU) is induced by solvent evaporation, including: π-π / R-π stacking between benzene rings and with starch pyran rings, OH-π hydrogen bonds and C=O / NH hydrogen bonds.

[0022] This synergistic effect not only enhances drug loading stability, but also utilizes the hydrophobic properties of curcumin to encapsulate the porous starch containing paclitaxel, resulting in almost no release in the stomach and very little release (<7%) in the small intestine, thus achieving colon-targeted delivery.

[0023] (4) The co-loading system constructed in this invention significantly enhances the antitumor effect of paclitaxel in terms of overcoming drug resistance. The synergistic effect of curcumin and porous starch-loaded paclitaxel (PTX-CU / PS) is enhanced through a dual mechanism: ① the sustained-release properties of porous starch, improved solubility of amorphous paclitaxel, and the synergistic cytotoxicity of curcumin increase the uptake rate of paclitaxel in Caco-2 cells to 80.30% and the apoptosis / necrosis rate; ② curcumin inhibits P-gp efflux activity (confirmed by Rhodamine 123 experiments), effectively reversing multidrug resistance to paclitaxel. This co-loading strategy based on natural porous starch provides a highly efficient synergistic delivery platform for colon-targeted chemoimmunotherapy. Attached Figure Description

[0024] Figure 1 Microscopic image (a) and particle diagram (b) of the process of preparing paclitaxel / curcumin co-amorphous microcapsules according to a specific embodiment of the present invention; Figure 2 XRD patterns of protopaclitaxel, protocurcumin, porous starch, and paclitaxel / curcumin co-amorphous microcapsules according to specific embodiments of the present invention; Figure 3 Electron micrograph of the co-amorphous microcapsules prepared in Example 1 of this invention; Figure 4 Electron microscopy image of the co-amorphous microcapsules prepared in Example 2 of this invention; Figure 5 Electron micrograph of the co-amorphous microcapsules prepared in Example 3 of this invention; Figure 6 Electron micrograph of the co-amorphous microcapsules prepared in Example 4 of this invention; Figure 7 Electron micrograph of the sample prepared according to Comparative Example 2 of the specific embodiments of the present invention; Figure 8 This is an electron microscope image of the sample prepared according to Comparative Example 3 of a specific embodiment of the present invention. Figure 9 XPS image of co-amorphous microcapsules according to a specific embodiment of the present invention; Figure 10 The image shows the FTIR spectrum of the co-amorphous microcapsules according to a specific embodiment of the present invention. Figure 11 The co-amorphous microcapsules prepared in Example 2 of this invention and the cell viability graphs of samples treated in Comparative Examples 1, 2 and 4 are shown. Figure 12 The diagram shows the paclitaxel uptake rate of the co-amorphous microcapsules prepared in Example 2 of this invention and the paclitaxel uptake rate of the cells treated with the sample of Comparative Example 4. Figure 13 The images show the co-amorphous microcapsules prepared in Example 2 of this invention and the CLSM diagram of multidrug resistance in cells treated with the sample of Comparative Example 4. Detailed Implementation

[0025] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0026] The following embodiments of the present invention utilize enzymatic hydrolysis of porous starch: A corn starch solution was dispersed in a sodium acetate buffer solution at pH 5.5 to obtain a 30% (w / v) corn starch milk. The ratio of glucosidase to α-amylase was set to 2:1 (v / v), with a total enzyme addition of 2% of the mass of the hydrolyzed porous starch. The hydrolysis time was 6 h, and the reaction was carried out at 50°C. After the hydrolysis reaction, the pH of the starch milk was adjusted to 3.0, and the system was neutralized after 15 min. Impurities were washed away with deionized water. Subsequently, the sample was dried in an oven at 40°C for 12 h. The final sample was pulverized and passed through a 100-mesh sieve for storage.

[0027] Test method: 1. Encapsulation efficiency (EE) and drug loading (DL) The content of paclitaxel in the prepared supernatant was determined at a wavelength of 230 nm using a UV-Vis spectrophotometer, and the EE and DL were calculated. The relationship between DL and EE is as follows: EE(%)=(M d -V s ×C d ) / M d ×100% DL(%)=(M d - V s ×C d ) / W ST ×100% Among them, M d V is the initial amount of paclitaxel. s C is the volume of the supernatant. d W represents the concentration of paclitaxel in the supernatant. st This is the dry weight of the precipitate. 2. Scanning electron microscope (SEM) The morphology of the particles was observed using electron microscopy and scanning electron microscopy. After drying, the samples were fixed to the column with double-sided tape, loose powder was blown off, and gold plating was performed under vacuum for 90 seconds. The entire operation was conducted under low vacuum conditions with an accelerating voltage of 15 kV.

[0028] 3. Laser Confocal Microscopy (CLSM) The morphology of paclitaxel-loaded porous starch was observed using a laser confocal microscope. Paclitaxel was labeled with Nile Red (0.1 mg / mL ethanol solution). Starch was labeled with fluorescein isothiocyanate (FITC; 0.5 mg / mL ethanol solution) and stained in the dark for 12 hours.

[0029] 4. X-ray diffraction (XRD) measurement The samples were analyzed using an X-ray diffractometer. The operating power was 2.2 kW, the scanning range was 5° to 45°, and the scanning speed was 6° / min.

[0030] 5. FTIR spectroscopy determination The spectrum of the sample was obtained using an FTIR spectrometer. The sample and KBr were completely ground at a ratio of 1:60. The sample was scanned 32 times at a temperature of 4000–400 cm⁻¹.

[0031] 6. X-ray photoelectron spectroscopy (XPS) measurement Before the experiment, the samples were pretreated by vacuum drying (50°C, 24 h) to eliminate the interference of moisture on the test results. XPS test conditions were as follows: monochromatic Al Ka ​​radiation (hv = 1486.6 eV, 150 W) was used as the X-ray source, and the base pressure was 10... -9 torr; wide-spectrum scanning used a transmission energy of 160 eV and a step size of 1 eV; narrow-spectrum scanning used a transmission energy of 40 eV and a step size of 0.1 eV; hybrid lens mode was used; the analysis area was 300 × 700 μm. 2 .

[0032] 7. Determination of upper gastrointestinal stability and simulated colonic release Samples containing equal amounts of drug were placed in sealed dialysis bags with a capacity of 8,000–14,000 Da and released for 2 hours and 4 hours in simulated gastric and intestinal fluids at 37 ± 0.5°C, respectively. The simulated gastric fluid (pH 1.2) consisted of 2.95 g / L sodium chloride, 8 mL / L hydrochloric acid, 3.2 g / L pepsin, and 3% (v / v) Tween-20. 2 mL of this release medium was collected at 30 min, 60 min, 90 min, and 120 min to determine the concentration of paclitaxel. The pH of the simulated gastric fluid system was adjusted to 6.8 to serve as the simulated intestinal fluid. The cumulative drug release in the medium was measured at 30 min, 60 min, 90 min, 120 min, 180 min, and 240 min. An equal volume of fresh release medium was added to fill the remaining volume after each sampling.

[0033] The concentration of paclitaxel was determined at a wavelength of 230 nm using a UV spectrophotometer, and the results were reported as the cumulative drug release at each time point.

[0034] 8. Tumor cell inhibition rate Human colon adenocarcinoma cells (Caco-2) were seeded in DMEM high-glucose medium containing 10% (v / v) fetal bovine serum, with 1% (v / v) penicillin-streptomycin and 1% (v / v) non-essential amino acids added. Cells were cultured in a 5% CO2 incubator at 37°C. The cytotoxicity of the samples to Caco-2 cells was detected using the CCK-8 assay: Caco-2 cell suspension (5 × 10⁻⁶ cells / mL) was... 4 Samples (samples / well) were seeded into 96-well plates and treated with 0.01, 0.1, 1, 10, 100, and 1000 μg / mL of the enzyme, respectively. After 48 hours of incubation, 10 μL of CCK-8 solution was added to each well, and the plates were incubated for another hour. The absorbance was measured at 450 nm using a microplate reader. The inhibition rate was calculated using the following formula: Inhibition rate = (OD) control -OD experiment ) / OD control ×100 Among them OD control The OD value represents the control group; OD experiment The values ​​represent the OD values ​​of each experimental group.

[0035] 9. Flow cytometer Paclitaxel uptake was quantified using flow cytometry. Cells were cultured at 5 × 10⁶ cells / cells. 5 Caco-2 cells were seeded at a density of cells / well in 6-well plates and cultured for 24 h. Cells were then treated with samples containing fluorescently labeled paclitaxel (0.1 mg / mL Nile Red-labeled paclitaxel) for 15 h. The culture medium was then removed, and cells were collected after trypsin digestion. Cells were resuspended in 500 μL PBS, and flow cytometry was used to detect paclitaxel uptake by Caco-2 cells in each group.

[0036] 10. Multidrug resistance Rhodamine 123, a fluorescent P-gp substrate, is readily expelled from cancer cells with high MDR expression and was used to assess P-gp activity. Cells were pretreated with simulated digested microcapsules for 1 hour, followed by incubation with 10 μM Rhodamine 123 for 90 min. Cells were then washed twice with 1× buffer and loaded into wash buffer. The fluorescence intensity of Rhodamine 123, paclitaxel, and curcumin was measured using CLSM.

[0037] Example 1 A method for preparing oral paclitaxel microcapsules for multidrug resistance includes the following steps: (1) Porous starch adsorbs paclitaxel: Porous starch was dispersed in an ethanol solution of paclitaxel (30 mg / mL) at a mass ratio of 2:1 to paclitaxel (dry weight). The mixture was stirred until homogeneous and adsorbed for 2 hours. Then, the mixture was poured into a petri dish and allowed to evaporate the solvent at 55°C for 0.5 hours (the solvent evaporation temperature was set to 45-55°C and the evaporation time was 0.5-1 hours). After complete evaporation, porous starch loaded with paclitaxel was obtained. (2) Preparation of co-amorphous curcumin-paclitaxel The curcumin acetic acid solution (8 mg / mL) was poured into a petri dish containing the product obtained in step (1). The mass ratio of curcumin to paclitaxel (dry weight) was 1:4. After uniform dispersion, the solvent was evaporated again (the solvent evaporation temperature was set to 45-55℃, and the evaporation time was 0.5-1h). After complete evaporation, the unloaded paclitaxel was removed by washing with ethanol (the volume of the washing agent ethanol was 10-15:1 of the original solvent acetic acid). After centrifugation (the centrifugation was 4000r / min for 10min), the precipitate was the paclitaxel / curcumin@porous starch microcapsules, i.e., oral paclitaxel microcapsules for multidrug resistance.

[0038] The obtained paclitaxel microcapsules were subjected to performance testing. The results showed that the DL of paclitaxel was as high as 32.17±0.44%; the EE of paclitaxel was as high as 100%. Figure 3 A large amount of curcumin can be seen coated on the surface of porous starch; there is almost no release in the stomach; the release in the small intestine is 3.22±1.14% after 4 hours, which can achieve the effect of targeting the colon. Example 2 The mass ratio of curcumin to paclitaxel (dry weight) in step (2) of Example 1 was adjusted to 1:5. All other steps remained the same as in Example 1, resulting in high-load oral paclitaxel capsule microcapsules.

[0039] The obtained paclitaxel microcapsules were subjected to performance testing. The results showed that the DL of paclitaxel was as high as 35.35±0.12%; the EE of paclitaxel was as high as 100%. Figure 4 A large amount of curcumin can be seen coated on the surface of porous starch; there is almost no release in the stomach; the release in the small intestine is 6.97±0.81% after 4 hours, which can achieve the effect of targeting the colon.

[0040] Example 3 The mass ratio of curcumin to paclitaxel (dry weight) in step (2) of Example 1 was adjusted to 1:6. All other steps remained the same as in Example 1, resulting in high-load oral paclitaxel capsule microcapsules.

[0041] The obtained paclitaxel microcapsules were subjected to performance testing. The results showed that the DL of paclitaxel was as high as 35.60±1.02%; the EE of paclitaxel was as high as 100%. Figure 5 It can be seen that a large number of paclitaxel microparticles occupy the pores of porous starch; there is almost no release in the stomach; the release in the small intestine after 4 hours is 12.49±2.86%, which can achieve the effect of targeting the colon.

[0042] Example 4 The mass ratio of curcumin to paclitaxel (dry weight) in step (2) of Example 1 was adjusted to 1:7. All other steps remained the same as in Example 1, resulting in high-load oral paclitaxel capsule microcapsules.

[0043] The obtained paclitaxel microcapsules were subjected to performance testing. The results showed that the DL of paclitaxel reached 16.22±2.17%; the EE was only 49.94±2.69%. Figure 6 The presence of paclitaxel microparticles occupying the pores of porous starch indicates that the amount of curcumin added was too small. Although it promoted the amorphization of paclitaxel, it could not form an outer coating. The ethanol washing step reduced the EE. The amount released in the stomach was 13.09±1.47%; the amount released in the small intestine after 4 hours was 37.91±1.04%, which could not achieve the effect of targeting the colon.

[0044] Comparative Example 1: Propaclitaxel The original paclitaxel was directly subjected to cytotoxicity experiments. Figure 11 As can be seen, the cancer cells it incubates exhibit multidrug resistance.

[0045] Comparative Example 2: Porous starch directly loaded with paclitaxel Porous starch was dispersed in an ethanol solution of paclitaxel (30 mg / mL) with a mass ratio of porous starch to paclitaxel (dry weight) of 2:1. The mixture was stirred and adsorbed for 2 hours. Then, it was poured into a petri dish and the solvent was evaporated at 55°C for 0.5 hours. After complete evaporation, the unloaded paclitaxel was removed by washing with ethanol solution. The precipitate after centrifugation was the porous starch loaded with paclitaxel. The obtained paclitaxel-loaded porous starch was subjected to performance testing. The results showed that the DL of paclitaxel was only 4.30±0.11%; the EE of paclitaxel was only 12.50±0.44%. Figure 7 A small amount of paclitaxel crystals could be seen adhering to the surface of porous starch; the release rate in the gastrointestinal tract was as high as 55.71±3.40%. Figure 11 In this study, cancer cells incubated with paclitaxel directly loaded with porous starch developed multidrug resistance, and the survival rate of cancer cells remained above 80%.

[0046] Comparative Example 3 Porous starch was dispersed in an ethanol solution of paclitaxel (30 mg / mL) with a mass ratio of 2:1 of porous starch to paclitaxel (dry weight). The mixture was stirred and adsorbed for 2 h. Then, it was poured into a petri dish and the solvent was evaporated at 55 °C for 0.5 h. After complete evaporation, porous starch loaded with paclitaxel was obtained. An ethanol solution of menthone (8 mg / mL) was poured into a petri dish containing the product obtained in step (1). The mass ratio of menthone to paclitaxel (dry weight) was 1:4. After dispersion, the solvent was evaporated again. After complete evaporation, the unloaded paclitaxel was washed with an ethanol solution. After centrifugation, the precipitate was paclitaxel-menthone@porous starch microcapsules.

[0047] The obtained paclitaxel-loaded porous starch was subjected to performance testing. The results showed that the DL of paclitaxel was only 5.70±0.28%; the EE of paclitaxel was only 15.21±0.61%. Figure 8 Paclitaxel can be seen as large, strip-shaped crystals adhering to the porous starch; the release rate in the stomach and small intestine is as high as 52.42±1.06%.

[0048] Comparative Example 4: The technical solution of the inventor's original application (authorization announcement number CN 114948908 B). (1) Preparation of amorphous paclitaxel With constant stirring, an ethanol solution of paclitaxel (20 mg / mL) was added dropwise to three times the volume of aqueous phase, and then dried at 55 °C to obtain amorphous paclitaxel microspheres.

[0049] (2) Porous starch adsorbs paclitaxel The paclitaxel microspheres prepared in step (1) were redissolved in an ethanol solution to form a mixture with a concentration of 20 mg / mL. Porous starch and paclitaxel microspheres were dispersed in the mixture at a mass ratio of 3:1. The mixture was stirred at 37 °C for 4 h and then the solvent was evaporated at 40 °C. Unadsorbed paclitaxel was removed by washing with 10 times the volume of ethanol solution in the mixture. The precipitate was the porous starch loaded with paclitaxel.

[0050] (3) Preparation of chitosan-coated capsules The porous starch (600%, w / w) loaded with paclitaxel prepared in step (2) was dispersed in chitosan solution (5%, w / v, g / mL). After being dispersed evenly, phytic acid solution (2.5%) with a volume of 10 times that of chitosan was added dropwise. The mixture was stirred for 4 hours and then centrifuged at 3000 r / min. The precipitate was dried in an oven at 40℃ for 6 hours to obtain coated capsules.

[0051] The obtained paclitaxel microcapsules were subjected to performance testing. The results were as follows: the DL of paclitaxel was 30.17±0.15%; the EE of paclitaxel reached 90.50±0.31%. Figure 8Numerous paclitaxel microparticles can be observed occupying the pores of porous starch; almost no release occurs in the stomach; the release in the small intestine over 4 hours is 14.27 ± 1.81%, achieving a controlled slow release effect in the colon; however, in… Figure 11 In this study, cancer cells incubated with chitosan-coated capsules developed multidrug resistance, and the cancer cell survival rate remained above 82%. Figure 12 Paclitaxel uptake in mid-cells was only 45.8%. Figure 13 The uptake fluorescence of Rhodamine 123 is very weak. like Figure 1 As shown, porous starch is dispersed in a paclitaxel solution for adsorption. After solvent evaporation, the paclitaxel recrystallizes into strip-shaped crystals. The situation is drastically different when curcumin solution is added to the system; the strip-shaped crystals dissolve. As the solvent evaporates, paclitaxel enters the starch pores, while curcumin collapses to the surface of the porous starch, forming multilayered enrichment. The diffraction patterns of paclitaxel and curcumin change to an amorphous state. Figure 2 Paclitaxel has the following appearance: Figure 5 The spherical shape with a diameter of <1μm located in the porous starch pores, rather than... Figure 6 Long, strip-shaped crystals of paclitaxel.

[0052] Electron micrographs of the paclitaxel / curcumin co-amorphous microcapsules prepared in Examples 1, 2, and 3 are shown below. Figure 3-5 As shown, in Examples 1, 2, and 3, the paclitaxel microspheres were accumulated in the pores of porous starch, and a large amount of curcumin was coated on the surface of the porous starch, while in Comparative Example 2, paclitaxel was directly adsorbed (…). Figure 7 No curcumin was observed in the pores; only a small number of elongated crystals and irregular debris were found on the surface. This is consistent with their DL. Furthermore, after the curcumin addition ratio was reduced in Example 4 ( Figure 6 Although amorphous paclitaxel and a certain amount of drug loading were obtained, a portion of the paclitaxel that lost its curcumin coating was washed away, resulting in low DL and encapsulation efficiency, and failing to achieve colon-targeted delivery. In fact, this process is tailored to menthone, and when menthone was poured into a petri dish of the product obtained in step (1) in Comparative Example 3 for solvent evaporation, the curcumin effect was not obtained. Figure 8 Secondly, comparing with the existing patent of the reference team in Example 4, the DL of the obtained paclitaxel was 30.17±0.15%; the encapsulation efficiency of paclitaxel was only 90.50±0.31%, and the release amount in the small intestine after 4 hours reached 14.27±1.81%, especially in... Figure 11 Cancer cells incubated with this preparation showed significant multidrug resistance: cell viability remained above 82%, while paclitaxel uptake was as low as 45.8%. Figure 12 Meanwhile, the fluorescence intensity of cellular uptake of Rhodamine 123 was extremely weak (). Figure 13This further confirms the occurrence of multidrug resistance in tumors. The above results highlight the significant advantages of the process described in this invention.

[0053] like Figure 3-5 As shown, the paclitaxel / curcumin co-amorphous microcapsules in Examples 1, 2, and 3 are spherical particles with a tightly encapsulated shell, containing porous starch loaded with paclitaxel inside. Figure 9-10 Intermolecular interaction analysis revealed that solvent evaporation induces a multi-interaction network between porous starch (PS), paclitaxel (PTX), and curcumin (CU) (FTIR / XPS confirmed), including π-π / R-π stacking between benzene rings and with the pyran ring of starch, OH-π hydrogen bonds, and C=O / NH hydrogen bonds. This synergistic effect not only enhances drug loading stability but also utilizes the hydrophobicity of curcumin to encapsulate the porous starch carrying paclitaxel, resulting in minimal release in the stomach and very little release in the small intestine, achieving colon-targeted delivery. Figure 11 The cytotoxicity results showed that cell survival gradually decreased with increasing microcapsule concentration, dropping below 40% at a concentration of 5 mg / mL. In contrast, cells treated with protopaclitaxel (Comparative Example 1), paclitaxel directly adsorbed from porous starch (Comparative Example 2), and the sample obtained from the existing patent (Comparative Example 4) all maintained survival rates above 80%, indicating the development of cellular resistance. Figure 12 Flow cytometry analysis showed that the paclitaxel uptake rate of cells incubated with amorphous microcapsules was as high as 80.3%.

[0054] P-gp is an ATP-dependent efflux pump highly expressed in various tumor cells (such as drug-resistant cancer cells), which can pump drugs or dyes (such as Rh123) out of the cell, reducing their intracellular concentration. Figure 13 As shown, the uptake of Rhodamine 123 (green fluorescence) in the paclitaxel / curcumin co-amorphous microcapsule treatment group was significantly greater than that in control group 4, suggesting that the uptake of Rhodamine 123 increased in the presence of curcumin, while P-gp efflux was inhibited. It is evident that the paclitaxel uptake in the co-amorphous microcapsule treatment group was significantly greater than that in control group 4. From the perspective of cell nuclear state (blue-white fluorescence stained with DAPI), control group 4 mainly consisted of early apoptotic cells, exhibiting nuclear condensation, darker staining, or crescent-shaped aggregation of nuclear chromatin on one side of the nuclear membrane; the co-amorphous microcapsule treatment group mainly consisted of late apoptotic cells, characterized by nuclear fragmentation into round bodies of varying sizes, surrounded by the cell membrane, i.e., apoptotic bodies. Therefore, co-amorphous microcapsules can increase the intracellular accumulation of the substrate and improve the bioavailability of paclitaxel. In summary, the parameter limitations of each step in the preparation method of the oral paclitaxel microcapsules for multidrug resistance in this application also have the following effects: 1. Enzymatic hydrolysis conditions: α-amylase:glucosidase = 1:1-3, total enzyme amount 1%-3%, enzymatic hydrolysis at 45-60℃ for 5-7 hours; Maintaining optimal enzyme activity at 45-60℃ and balancing pore development and structural stability for 5-7 hours is crucial. Too short a time results in sparse pores (drug loading <15%), while too long a time leads to starch granule disintegration (decreased drug loading stability). Technical necessity: This parameter combination is core to obtaining a high-drug-loading porous carrier, significantly increasing drug loading compared to commercially available porous starch (reaching 35.35% in Example 2).

[0055] 2. Paclitaxel ethanol concentration: 10-40 mg / mL, porous starch: paclitaxel = 2-4:1; Concentration threshold effect: adsorption saturation is slow at <10 mg / mL (drug loading <20%), and paclitaxel crystallizes prematurely during solvent evaporation at >40 mg / mL (drug loading fluctuates ±5%); mass ratio optimization: drug loading is 32.17% at 2:1 (Example 1), and drops to below 25% at 4:1. 2-4:1 balances carrier capacity and drug utilization.

[0056] 3. Curcumin:Paclitaxel = 1:4-6; Curcumin molecules insert into the spaces between the benzene rings of paclitaxel via π-π stacking, disrupting its crystalline structure (XRD amorphous peaks). Figure 2 Meanwhile, hydrophobic chains coat the starch surface ( Figure 3-5 Electron microscopy). Critical ratio verification: 1:5 (Example 2): Drug loading 35.35% + 100% encapsulation rate + colon targeting (small intestine release <7%). 1:7 (Example 4): Insufficient curcumin, partial crystallization of paclitaxel, encapsulation rate plummeted to 49.94% ( Figure 6 Irreplaceability: Compared to menthone (Comparative Example 3), which lacks a benzene ring structure, it cannot inhibit crystallization (drug loading is only 5.7%).

[0057] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing oral paclitaxel microcapsules for multidrug resistance, characterized in that, Includes the following steps: (1) Porous starch adsorbs paclitaxel: Porous starch was dispersed in an ethanol solution of paclitaxel, stirred until homogeneous for adsorption, and then poured into a petri dish for solvent evaporation. After complete evaporation, porous starch loaded with paclitaxel was obtained. (2) Preparation of co-amorphous curcumin-paclitaxel: Pour the curcumin acetic acid solution into a petri dish containing the product obtained in step (1), disperse it evenly, and then evaporate the solvent again. After complete evaporation, wash with ethanol solution to remove the unloaded paclitaxel. After centrifugation, the precipitate is the oral paclitaxel microcapsule.

2. The method for preparing oral paclitaxel microcapsules for multidrug resistance according to claim 1, characterized in that, The porous starch in step (1) is enzymatically hydrolyzed porous starch. The total amount of enzyme added is 1%-3% of the mass of the hydrolyzed porous starch. The enzymes include α-amylase and glucosidase. The volume ratio of α-amylase to glucosidase is 1:1-3. The hydrolysis time is 5-7 hours. The hydrolysis temperature is 45-60℃.

3. The method for preparing oral paclitaxel microcapsules for multidrug resistance according to claim 1, characterized in that, The concentration of paclitaxel in the ethanol solution of paclitaxel described in step (1) is 10-40 mg / mL; The mass ratio of porous starch to paclitaxel in step (1) is 2-4:1; The adsorption in step (1) involves dispersing porous starch in paclitaxel solution and stirring at 37°C for 1.5-2.5 h. The temperature for solvent evaporation in step (1) is set to 45-55℃ and the evaporation time is 0.5-1h.

4. The method for preparing oral paclitaxel microcapsules for multidrug resistance according to claim 1, characterized in that, In step (2), the curcumin concentration in the acetic acid solution of curcumin is 5-10 mg / mL.

5. The method for preparing oral paclitaxel microcapsules for multidrug resistance according to claim 1, characterized in that, The mass ratio of curcumin to paclitaxel in step (2) is 1:4-8.

6. The method for preparing oral paclitaxel microcapsules for multidrug resistance according to claim 1, characterized in that, The mass ratio of curcumin to paclitaxel in step (2) is 1:4-6.

7. The method for preparing oral paclitaxel microcapsules for multidrug resistance according to claim 1, characterized in that, The mass ratio of curcumin to paclitaxel in step (2) is 1:

5.

8. The method for preparing oral paclitaxel microcapsules for multidrug resistance according to claim 1, characterized in that, In step (2), the solvent evaporation temperature is set to 45-55℃, and the evaporation time is 0.5-1h; In step (2), the volume ratio of the ethanol solution to the acetic acid solution is 10-15:1; In step (2), the centrifugation is performed at 4000-6000 r / min for 5-10 min.

9. Oral paclitaxel microcapsules for multidrug resistance prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the oral paclitaxel microcapsules for multidrug resistance as described in claim 9 in the preparation of a colorectal cancer drug.

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