Bile acid-paclitaxel micromolecular prodrug, preparation method thereof, and construction method and application of self-assembled nanoparticles of bile acid-paclitaxel micromolecular prodrug

Abstract

According to the invention, a disulfide bond and carbon-carbon bond bridged bile acid-paclitaxel micromolecule prodrug is designed and synthesized, and a micromolecule prodrug self-assembled nano-drug delivery system is prepared on the basis of the disulfide bond and carbon-carbon bond bridged bile acid-paclitaxel micromolecule prodrug. The preparation method has the advantages that a nano precipitation method is adopted, the prodrug can be self-assembled in water to form the nanoparticles without using a carrier material, the preparation process is simple, and industrialization is easy; the amphiphilic micromolecular prodrug improves the water solubility of paclitaxel (PTX), has high drug loading capacity, and avoids adverse reactions possibly caused by carriers and auxiliary materials; when the nanoparticles are orally administered, bile acid can be used as a targeted switch of an ASBT receptor, and gastrointestinal permeability of the nanoparticles can be enhanced, so that the nanoparticles can be transferred and reabsorbed by ASBT mediation to enter intestinal cells and then enter a circulatory system, and oral bioavailability of PTX is enhanced; the tumor microenvironment reduction sensitivity of the connecting arm enables PTX to be specifically released at a tumor part, so that premature leakage of the medicine is avoided, the curative effect of the medicine is improved, and toxic and side effects are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of new excipients and new dosage forms of pharmaceutical preparations, and particularly relates to a bile acid-paclitaxel small molecule prodrug, a preparation method thereof, a construction method and application of self-assembled nanoparticles thereof. Background Art

[0002] Cancer treatment mainly relies on chemotherapy, and its administration methods are mainly injection and oral administration. Oral administration is more acceptable to the public because of its advantages such as stable blood drug concentration, fewer side effects, and repeatable administration. However, oral chemotherapy drugs still face many challenges in clinical applications. For example, taxane drugs, although they have been widely used in cancer treatment, are limited in oral administration due to poor water solubility and stability, low oral bioavailability, and large toxic and side effects. In addition, the complex biological barriers in the human body hinder the gastrointestinal absorption and penetration of chemotherapy drugs, making it impossible for the drugs to enter the circulatory system to exert their efficacy. Moreover, most drug delivery systems have low drug loading and poor stability, and it is difficult to be absorbed through the intestine and enter the body, so that the drugs cannot reach the tumor site to play an anti-cancer role. For poorly soluble drugs, a single administration strategy is not sufficient to overcome the multiple absorption barriers of oral drugs.

[0003] Bile acids are steroid substances synthesized by the liver from cholesterol. Its steroid nucleus consists of three six-membered rings and one five-membered ring. This structure endows bile acids with amphiphilicity, making them have high surface activity, which can reduce the surface tension of drug particles, increase their solubility in water, and thus improve the absorption of drugs. And bile acids and their intestinal transport proteins can be used as potential targets for oral administration due to their high capacity, safety and effectiveness, which can enhance the gastrointestinal permeability of drugs and improve the oral bioavailability of drugs, and are of great significance for the oral delivery of chemotherapy drugs.

[0004] Therefore, considering the characteristics that bile acids can improve the bioavailability and targeting of drugs, using tumor microenvironment-reducing sensitive disulfide bonds and non-reducing sensitive carbon-carbon bonds as linker arms, bile acid compounds are respectively linked to paclitaxel (PTX) to construct bile acid-paclitaxel prodrugs, and the drug release characteristics are investigated to verify the controlled release effect of different linker arms. This invention is of great necessity, feasibility and innovative significance for improving the treatment effect of tumors and reducing systemic toxicity.

[0005] Nano-delivery systems have been widely used in cancer treatment and offer great potential for oral administration. They have the advantages of improving drug solubility, prolonging the in vivo action time of drugs, and reducing the toxic and side effects of drugs. However, the preparation of most nano-delivery systems is relatively complex, and the addition of carrier materials and excipients increases the risk of drug administration, possibly resulting in unavoidable adverse reactions. In addition, most drug delivery systems have a low drug loading capacity and poor stability, making it difficult to be absorbed through the intestine and enter the body, so that the drug cannot reach the tumor site to play an anti-cancer role. Tumor microenvironment-responsive small molecule prodrug nano-delivery systems have improved the above-mentioned disadvantages to a certain extent, with a high drug loading capacity and safety, and can control the specific release of drugs in tumor tissues. However, for poorly soluble drugs, a single drug delivery strategy is not sufficient to overcome multiple oral drug absorption barriers. Bile acids and their intestinal transporters, due to their high capacity, safety, and effectiveness, can be used as potential targets for oral administration, can enhance the gastrointestinal permeability of drugs, improve the oral bioavailability of drugs, and are of great significance for the oral delivery of chemotherapeutic drugs.

[0006] Based on this, the present application was developed. Summary of the Invention

[0007] The object of the present invention and the technical problems to be solved are to provide and prepare a tumor microenvironment-sensitive bile acid-paclitaxel small molecule prodrug that combines an intestinal targeting strategy with prodrug nanotechnology, and self-assemble the prodrug into uniform prodrug nanoparticles, so as to achieve the oral administration of PTX, enhance its gastrointestinal permeability, have the effects of high drug loading capacity, good stability, and low toxic and side effects, and further improve the anti-tumor activity of the drug.

[0008] The object of the present invention is to design and synthesize a reduction-sensitive bile acid-paclitaxel small molecule prodrug, prepare a prodrug self-assembled nano-delivery system, and compare the effects of different linkers on the reduction responsiveness, drug release, cytotoxicity, pharmacokinetics, and anti-tumor activity of the prodrug.

[0009] The present invention achieves the above-mentioned invention object through the following technical solutions: A bile acid-paclitaxel small molecule prodrug, in which PTX and bile acid (taking UDCA as an example) are connected by a tumor microenvironment reduction-sensitive disulfide bond (a) or by a non-reduction-sensitive carbon-carbon bond (b), and it has any of the following structural formulas: R1 is independently selected from α-OH, b-OH, α-H, or b-H respectively; R2 is independently selected from α-OH, b-OH, α-H, or b-H respectively; X is independently selected from OH, NHCH2COOH or NHCH2CH2SO3H respectively.

[0010] As a preference, the above-mentioned bile acid-paclitaxel small molecule prodrug is preferably any of the compounds shown in the following structural formula: .

[0011] In the present invention, the abbreviations and chemical names of some compounds are as follows: DMAP: 4-dimethylaminopyridine; DCC: N,N'-dicyclohexylcarbodiimide; EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. DMF refers to N,N-dimethylformamide.

[0012] As a preference, the present invention provides a preparation method of the above-mentioned bile acid-paclitaxel small molecule prodrug (taking the ursodeoxycholic acid-paclitaxel prodrug as an example), the reduction-sensitive bile acid-paclitaxel small molecule prodrug (UDCA-SS-PTX): It includes the following steps: 1) Dissolve 3,3'-dithiobispropionic acid in anhydrous acetyl chloride, and reflux it under condensation at 55-70 °C for 2-5 h. The reaction solution is concentrated by rotary evaporation and precipitated with ice-cold diethyl ether to obtain the acid anhydride; 2) Dissolve the acid anhydride in an appropriate amount of anhydrous dichloromethane, add the catalyst DMAP and DCC or EDCI, and ice-bath for 1-2 h. Then add paclitaxel (PTX), and react at 25-35 °C for 24-48 hours. The intermediate product 1 is obtained by extraction; 3) Dissolve ursodeoxycholic acid (UDCA) in an appropriate amount of N,N-dimethylformamide DMF, add sodium bicarbonate to make the reaction condition weakly acidic, add p-methoxybenzyl chloride to protect the carboxyl group of UDCA, and react at 30-45 °C for 24-48 h to obtain the carboxyl-protected ursodeoxycholic acid (PMB-UDCA); 4) After dissolving the intermediate product 1 in anhydrous DMF, add the catalyst DMAP and DCC or EDCI, and ice-bath for 1-2 h. Then add PMB-UDCA, and react at 30-45 °C for 24-48 h. Dialyze and freeze-dry to obtain the carboxyl-protected prodrug; Dissolve the carboxyl-protected prodrug in an appropriate amount of dichloromethane, and deprotect the carboxyl group with trifluoroacetic acid or hydrochloric acid-methanol solution (preferably the volume ratio of hydrochloric acid to methanol is 1:2-5), and react at 30-45 °C for 1-2 h. The final product is obtained by column chromatography separation and purification; The above reactions are all carried out under the protection of an inert gas atmosphere.

[0013] Specifically, in step 1), the molar ratio of 3,3'-dithiobispropionic acid to anhydrous acetyl chloride can be 1:(2 - 5); in step 2), the molar ratio of PTX, acid anhydride, DMAP, and DCC or EDCI can be 1:(0.5 - 10):(0.5 - 3):(1 - 3); in step 3), the molar ratio of UDCA, sodium bicarbonate, and p-methoxybenzyl chloride can be 1:(1 - 1.5):1; in step 4), the molar ratio of intermediate product 1, DMAP, DCC, and PMB-UDCA can be 1:(1 - 3):(1 - 3):1.

[0014] As a preference, the present invention provides a preparation method of the above-mentioned bile acid-paclitaxel small molecule prodrug. For the non-reductive sensitivity bile acid-paclitaxel small molecule prodrug (taking UDCA-CC-PTX as an example), it comprises the following steps: a) Succinic anhydride is dissolved in anhydrous dichloromethane, catalyst DMAP and DCC or EDCI are added, and the mixture is ice-bathed for 1 - 2 h, then PTX is added, and the reaction is carried out at 25 - 35 °C for 24 - 48 h, and intermediate product 2 is obtained through extraction; b) UDCA is dissolved in an appropriate amount of DMF, sodium bicarbonate is added to make the reaction condition weakly acidic, p-methoxybenzyl chloride is added to carry out carboxyl protection on UDCA, and the reaction is carried out at 30 - 45 °C for 24 - 48 h to obtain PMB-UDCA; c) After intermediate product 2 is dissolved in anhydrous DMF, catalyst DMAP and DCC or EDCI are added, and the mixture is ice-bathed for 1 - 2 h, then PMB-UDCA is added, and the reaction is carried out at 30 - 45 °C for 24 - 48 h, followed by dialysis and lyophilization to obtain the carboxyl-protected prodrug; the carboxyl-protected prodrug is dissolved in an appropriate amount of dichloromethane, and the carboxyl protection is removed with trifluoroacetic acid or hydrochloric acid-methanol solution (preferably the volume ratio of hydrochloric acid to methanol is 1:2 - 5), and the reaction is carried out at 30 - 45 °C for 1 - 2 h, and the final product is obtained through column chromatography separation and purification. The above reactions are all carried out under the protection of an inert gas atmosphere.

[0015] Specifically, in step a), the molar ratio of PTX, succinic anhydride, DMAP, and DCC can be 1:(1 - 10):(0.5 - 3):(1 - 3); in step b), the molar ratio of UDCA, sodium bicarbonate, and p-methoxybenzyl chloride can be 1:(1 - 1.5):1; in step c), the molar ratio of intermediate product 2, DMAP, DCC or EDCI, and PMB-UDCA can be 1:(1 - 3):(1 - 3):1.

[0016] Further, as a preference, the PTX can be a taxane compound; the UDCA can also be replaced with bile acid compounds such as cholic acid, deoxycholic acid, chenodeoxycholic acid, Ursodeoxycholic acid p-methoxybenzyl ester, hyodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, taurohyodeoxycholic acid, or tauro Ursodeoxycholic acid.

[0017] The present invention also provides a self-assembled nanoparticle containing a bile acid-paclitaxel small molecule prodrug, and its preparation process is as follows: Dissolve a certain amount of the bile acid-paclitaxel small molecule prodrug or a mixture thereof with a hydrophobic fluorescent substance in an appropriate amount of organic solvent. Under stirring, slowly add this solution dropwise to water. The prodrug spontaneously forms uniform nanoparticles. Finally, use the reduced pressure distillation method or dialysis method to remove the organic solvent, and a self-assembled nanoparticle without organic solvent is obtained.

[0018] Further, the hydrophobic fluorescent substance can be doxorubicin, coumarin-6, DiR, DiL, Cy-5 or Cy-7, etc. The preferred hydrophobic fluorescent substances are doxorubicin and coumarin-6; the organic solvent can be ethanol, methanol, acetone, dimethylformamide or dimethyl sulfoxide, etc.

[0019] DiR: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide; DiL: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocyanine perchlorate; Cy-5: sulfonated cyanine 5 fluorescent labeling reagent; Cy-7: sulfonated cyanine 7 fluorescent labeling reagent.

[0020] Preparation method of small molecule prodrug self-assembled nanoparticles: Dissolve a certain amount of small molecule prodrug in an appropriate amount of organic solvent. Under stirring, slowly add this solution dropwise to water. The prodrug spontaneously forms uniform nanoparticles. Finally, use the reduced pressure distillation method or dialysis method to remove the organic solvent, and a nanoparticle solution without organic solvent is obtained.

[0021] Preparation method of small molecule prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances: Dissolve a certain amount of small molecule prodrug and hydrophobic fluorescent substance in an appropriate amount of organic solvent. Under stirring, slowly add this solution dropwise to water. The prodrug spontaneously forms uniform nanoparticles. Finally, use the reduced pressure distillation method or dialysis method to remove the organic solvent in the preparation, and a nanoparticle solution without any organic solvent is obtained.

[0022] The present invention also provides the application of the above-mentioned bile acid-paclitaxel small molecule prodrug or the self-assembled nanoparticle containing the bile acid-paclitaxel small molecule prodrug in a drug delivery system or the preparation of anti-tumor drugs (especially anti-hepatocellular carcinoma drugs, etc.).

[0023] The present invention also provides the use of the above-mentioned bile acid-paclitaxel small molecule prodrug or the self-assembled nanoparticles containing the bile acid-paclitaxel small molecule prodrug in systems such as injection administration, oral administration or topical administration.

[0024] The present invention discloses a bile acid-paclitaxel small molecule prodrug and a preparation method thereof. The prodrug is realized through two structural forms: one is that PTX is connected to bile acid through a tumor microenvironment-responsive disulfide bond, and the other is through a stable carbon-carbon bond. The structural feature is that the taxane compound can replace PTX, and the bile acid component can be selected from 12 bile acid derivatives including cholic acid, deoxycholic acid, etc. The preparation method adopts a stepwise condensation strategy, constructs a linker through an acid anhydride ring-opening reaction, controls the reaction selectivity through a carboxyl protection / deprotection process, and uses a DMAP, DCC or EDCI catalytic system to complete the coupling reaction under the protection of an inert gas atmosphere. This prodrug system has the dual advantages of tumor-targeted release characteristics and structural stability. It improves the water solubility and bioavailability of PTX through the lipophilicity of the bile acid carrier, and at the same time utilizes the reduction sensitivity of the disulfide bond to achieve tumor microenvironment-responsive release, improving the therapeutic effect while reducing systemic toxicity.

[0025] The bile acid-paclitaxel small molecule prodrug of the present invention is first discovered to be able to self-assemble into a uniform nano-system. The advantages of this nano-drug delivery system are as follows: (1) Adopting a one-step nano-precipitation method, the preparation process is simple and easy to industrialize; (2) The particle size is small and uniform, which is beneficial for the nanoparticles to accumulate in the tumor site through the EPR effect; (3) The amphiphilic small molecule prodrug improves the water solubility of PTX, has a high drug loading capacity, and avoids the adverse reactions that may be caused by carriers and excipients; (4) When the nanoparticles are orally administered, UDCA can act as a targeting "switch" for the ASBT receptor, enhancing the gastrointestinal permeability of the nanoparticles, enabling the nanoparticles to be transported and reabsorbed by ASBT-mediated transport into intestinal cells and then into the circulatory system, enhancing the oral bioavailability of PTX; (5) Through the sensitivity of the linker to the tumor microenvironment, PTX is specifically released at the tumor site, avoiding premature leakage of the drug, improving the drug efficacy and reducing the toxic and side effects.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1) A bile acid-paclitaxel small molecule prodrug is designed and synthesized, and the synthesis method is simple and feasible; 2) By designing and comparing different chemical linkers, the release of PTX at the tumor site is effectively promoted; 3) Uniform small molecule prodrug self-assembled nanoparticles are prepared, and the preparation method is simple and feasible, with a high drug loading capacity and good stability; 4) As a carrier, bile acids can enhance the accumulation of drugs at the target site through an active uptake mechanism mediated by bile acid receptors (such as receptors on the surface of intestinal or certain tumor cells). As a targeting switch for ASBT receptors, bile acids enhance the gastrointestinal stability and permeability of bile acid prodrugs, and improve the oral bioavailability of PTX; 5) The reduction sensitivity of the disulfide bond enables the prodrug to be cleaved under the action of GSH highly expressed in tumor cells, releasing PTX, significantly improving the anti-tumor effect of PTX and reducing its toxic side effects. Description of the Drawings

[0027] Figure 1 For the bile acid-paclitaxel small molecule prodrug UDCA-SS-PTX synthesized in Example 1 1 H NMR (DMSO- d 6, 400 MHz); Figure 2 For the bile acid-paclitaxel small molecule prodrug UDCA-CC-PTX synthesized in Example 2 1 H NMR (DMSO- d 6, 400 MHz); Figure 3 FT-IR diagrams of the bile acid-paclitaxel small molecule prodrug UDCA-SS-PTX (a) synthesized in Example 1 and the bile acid-paclitaxel small molecule prodrug UDCA-CC-PTX (b) synthesized in Example 2; Figure 4 Critical micelle concentration (n = 3) of the self-assembled nanoformulations of the bile acid-paclitaxel small molecule prodrug UDCA-SS-PTX (a) synthesized in Example 1 and the bile acid-paclitaxel small molecule prodrug UDCA-CC-PTX (b) synthesized in Example 2; Figure 5 Particle size (left) diagram and Zeta potential distribution (right) diagram of the prodrug nanoformulation USP-Nanos; Figure 6 Particle size (left) diagram and Zeta potential distribution (right) diagram of the prodrug nanoformulation UCP-Nanos; Figure 7 TEM diagrams of the nanoparticles of the two prodrug nanoformulations USP-Nanos (a) and UCP-Nanos (b), and the right diagram is the enlarged view of the box in the left diagram; Figure 8 Particle size changes of the two prodrug nanoformulations USP-Nanos and UCP-Nanos stored for 21 days (n = 3); Figure 9Particle size changes of two prodrug nanosystems USP-Nanos and UCP-Nanos after dilution at different multiples (n = 3); Figure 10 Reductive release performance diagrams of two prodrug nanosystems USP-Nanos and UCP-Nanos (n = 3); Figure 11 Cell survival rates of drugs in different groups after acting on Huh-7 cells for 24 h (a), 48 h (b), and on HepG2 cells for 24 h (c), 48 h (d); Figure 12 Apparent permeability coefficients of drugs in different groups during monolayer transport across Caco-2 cell monolayers at different time points (n = 3, ***P < 0.001); Figure 13 Tumor volume changes of mice in different drug groups (n = 6, ***P < 0.001). Detailed implementation manners

[0028] The technical solutions of the present invention will be further introduced in detail below in combination with embodiments, but the protection scope of the present invention is not limited thereto.

[0029] In the following embodiments, the raw materials used are all ordinary commercially available products that can be directly purchased or can be prepared by using conventional techniques in the art.

[0030] Example 1 In this example, the synthesis route of the bile acid-paclitaxel small molecule prodrug (taking UDCA -SS-PTX as an example) is as follows: ; The synthesis method of the reduction-sensitive bile acid-paclitaxel small molecule prodrug (taking UDCA-SS-PTX as an example) provided in this example includes the following steps: 1) Dissolve 2 mmol of 3,3'-dithiobispropionic acid in 6 mmol of anhydrous acetyl chloride, reflux under condensation at 65 °C for 4 h, then concentrate the reaction solution by rotary evaporation and precipitate with ice-cold diethyl ether to obtain the acid anhydride. 2) Dissolve 0.1 mmol of the acid anhydride in 3 mL of anhydrous dichloromethane, add 0.1 mmol of the catalyst DMAP and 0.2 mmol of EDCI, cool in an ice bath for 1 hour, then add 0.2 mmol of PTX, and react at 25 °C for 48 hours. Extract with dichloromethane to obtain intermediate 1 (PTX-DTDPA); 3) Weigh 2 mmol of UDCA and dissolve it in 5 mL of DMF. Add 3 mmol of sodium bicarbonate to make the reaction condition weakly acidic. Add 2 mmol of p-methoxybenzyl chloride to protect the carboxyl group of UDCA, and react at 45 °C for 48 hours to obtain PMB-UDCA; 4) Dissolve 0.1 mmol of intermediate 1 in 5 mL of anhydrous DMF, add 0.1 mmol of DMAP and 0.2 mmol of EDCI as catalysts, and cool in an ice bath for 1 hour. Then add 0.1 mmol of PMB-UDCA and react at 45 °C for 48 hours. Dialyze (dialyze with deionized water for 24 h, and the molecular weight cut-off of the dialysis membrane is 1000 Da), and lyophilize (freeze at -60 °C for 24 h) to obtain the carboxyl-protected prodrug (PMB-UDCA-SS-PTX); dissolve the carboxyl-protected prodrug in 3 mL of dichloromethane, remove the carboxyl protection with 0.5 mL of trifluoroacetic acid, and react at 45 °C for 2 hours. Separate and purify by column chromatography (eluent: ethyl acetate: petroleum ether = 2:1, V:V) to obtain the final product (UDCA -SS-PTX, abbreviated as USP). The above reactions are all carried out under N2 protection.

[0031] Example 2 In this example, the synthesis route of the bile acid-paclitaxel small molecule prodrug (taking UDCA-CC-PTX as an example) is as follows: ; The synthesis method of the non-reduction-sensitive bile acid-paclitaxel small molecule prodrug (UDCA-CC-PTX) provided in this example includes the following steps: a) Dissolve 2 mmol of succinic anhydride in 15 ml of anhydrous dichloromethane, add 0.1 mmol of DMAP and 0.2 mmol of EDCI as catalysts, and cool in an ice bath for 1 hour. Then add 0.2 mmol of PTX and react at 25 °C for 48 hours. Extract to obtain intermediate 2 (PTX-SA); b) Dissolve 2 mmol of UDCA in 3 mL of DMF, add 3 mmol of sodium bicarbonate to make the reaction condition weakly acidic, add 2 mmol of p-methoxybenzyl chloride to protect the carboxyl group of UDCA, and react at 45 °C for 48 hours. Cool in an ice bath to precipitate a white solid, filter by suction to obtain the upper white solid, and dry it under vacuum to obtain PMB-UDCA; c) Dissolve 0.1 mmol of intermediate 2 in 5 mL of anhydrous DMF, add 0.1 mmol of catalyst DMAP and 0.2 mmol of EDCI, and ice-bath for 1 hour. Then add 0.1 mmol of PMB-UDCA and react at 45 °C for 48 hours. Dialyze (dialyze with deionized water for 24 h, the molecular weight cut-off of the dialysis membrane is 1000 Da), and freeze-dry (freeze at -60 °C for 24 h) to obtain the carboxyl-protected prodrug (PMB-UDCA-CC-PTX); dissolve the carboxyl-protected prodrug in 5 mL of dichloromethane, remove the carboxyl protection with 0.5 ml of trifluoroacetic acid, react at 45 °C for 2 hours, and separate and purify by column chromatography (eluent: ethyl acetate: petroleum ether = 2:1, V:V) to obtain the final product (UDCA-CC-PTX, abbreviation UCP). The above reactions are all carried out under N2 protection.

[0032] Example 3 The self-assembled nanoparticles containing bile acid-paclitaxel small molecule prodrugs provided by the present invention are prepared as follows: Dissolve the bile acid-paclitaxel small molecule prodrug or its mixture with a hydrophobic fluorescent substance in an organic solvent. Under stirring, slowly drop this solution into water, and the prodrug spontaneously forms uniform nanoparticles. Finally, remove the organic solvent in the preparation by vacuum distillation to obtain a self-assembled nanoparticle solution without organic solvents. The hydrophobic fluorescent substance is doxorubicin, coumarin-6, DiR, DiL, Cy-5 or Cy-7, and the preferred fluorescent substances are doxorubicin and coumarin-6. The organic solvent is ethanol, methanol, acetone, dimethylformamide or dimethyl sulfoxide. This example specifically gives the preparation processes of the following two preparations.

[0033] Preparation of UDCA-SS-PTX / UDCA-SS-PTX nanopreparation (i.e., USP-Nanos / UCP-Nanos): Precisely weigh 5 mg of the prodrug materials USP and UCP prepared in Examples 1 and 2 above and place them in EP tubes respectively. Add 0.2 mL of anhydrous ethanol to each and dissolve by ultrasonic treatment for later use. Another precisely measure 5 mL of ultrapure water and place it in a beaker. Control the rotation speed at 1000 r / min, and dropwise add the above prodrug material solutions to the ultrapure water one by one. The prodrug self-assembles into nanoparticles. Then remove the anhydrous ethanol by vacuum distillation at room temperature to obtain the prodrug nanopreparation.

[0034] Preparation of UDCA-SS-PTX / UDCA-SS-PTX solution (i.e., USP-Sol / UCP-Sol): Accurately weigh 5 mg of the prodrug materials USP and UCP prepared in Examples 1 and 2 above respectively, place them in EP tubes, add 0.5 mL of absolute ethanol to each, dissolve them by ultrasonic treatment, and set aside. Separately, accurately measure 375 mg of polyoxyethylated castor oil, add it to the above solutions, and dilute the above prodrug material solutions to 12 mL with physiological saline to obtain USP / UCP solution agents.

[0035] The NMR spectra of the bile acid-paclitaxel small molecule prodrugs UDCA-SS-PTX and UDCA-CC-PTX synthesized in Examples 1 and 2 are as Figure 1 , Figure 2 shown. From top to bottom in the spectra are PMB-UDCA, PTX-DTDPA (or PTX-SA), PMB-UDCA-SS-PTX (or PMB-UDCA-CC-PTX), and UDCA-SS-PTX (or UDCA-CC-PTX). In the NMR spectra of the intermediates PMB-UDCA-SS-PTX and PMB-UDCA-CC-PTX, the characteristic absorption peaks of the benzene ring of PTX-DTDPA or PTX-SA are at positions a, b, and c, the absorption peaks of methyl groups are m, o, p, r, s, and t, and the unique methylene peaks on the -SS- or -CC- bond connecting bridges are v and u. In the NMR spectra of the intermediates, the carboxyl peaks of PTX-DTDPA and PTX-SA disappear, and at the same time, the absorption peaks n' and o' of the benzene ring on PMB-UDCA, as well as the methyl peaks c', d', e', and p', appear, indicating that the esterification reaction has proceeded successfully. After that, the carboxyl protection of the intermediate is removed to obtain the final product. It can be seen from the figure that: in the NMR spectra of the final products, the carboxyl peaks of the ursodeoxycholic acid UDCA raw material medicine appear near 12.0 ppm, and the absorption peaks of the benzene ring in the protecting group PMBCL disappear, and other characteristic peaks in the compound all exist, indicating that the protecting group has been successfully removed to obtain the final product.

[0036] The infrared spectra of the bile acid-paclitaxel small molecule prodrugs UDCA-SS-PTX and UDCA-CC-PTX synthesized in Examples 1 and 2 are as Figure 3 shown. It can be seen from the figure that: in the spectrum of UDCA-SS-PTX, the stretching vibration peak of the benzene ring skeleton of paclitaxel PTX is at 1647 cm -1 , the stretching vibration of the carbonyl group after the ring opening of PTX and the acid anhydride is at 1724 cm -1 , the absorption peaks of methyl and methylene groups in UDCA and PTX are at 2857 cm -1 and 2933 cm -1 , and the peak at 3401 cm -1 is the stretching vibration peak of hydroxyl and carboxyl groups. Its infrared spectrum has the characteristic peaks of UDCA and PTX, so it is speculated that the synthesis is successful. Similarly, it is speculated that UDCA-CC-PTX is also successfully synthesized.

[0037] Determination of Critical Micelle Concentration To investigate the self-assembly ability of the two synthesized bile acid-paclitaxel small molecule prodrugs UDCA-SS-PTX and UDCA-CC-PTX, we used the pyrene-acetone method to determine the critical micelle concentration of the two prodrug nanosystems (USP-Nanos, UCP-Nanos). The two prepared prodrug nanosystems were diluted into a series of solutions with a concentration range of "1.0×10 -5 ~1.0 mg / mL". Then, 1 mL of pyrene-acetone solution with a concentration of 1 mg / mL was taken and placed in a brown vial, and placed in the dark to avoid light. After the acetone had completely evaporated, 10 mL of the above-diluted nanosystems with different concentrations was added to the brown vial. After sonication for 10 min, it was placed in the dark to avoid light for 24 h. Subsequently, a fluorescence spectrophotometer was used to measure the photometric value. The instrument parameters were set as (fluorescence excitation wavelength was 335 nm, emission wavelength range was 300~550 nm), and the photometric values of the samples at 384 nm and 373 nm were measured. Then, the logarithm of the sample concentration was used as the abscissa, and the photometric values of the samples at 384 nm and 373 nm were used as the ordinate to plot a graph, and the critical micelle concentration values of the two prodrug nanosystems were obtained. The results are shown in Figure 4 .

[0038] Figure 4 The critical micelle concentration results of the two prodrug nanosystems are shown as follows. As Figure 4 can be seen, when the preparation concentration is low, the ratio of I 384 / I 373 is relatively stable. When the preparation concentration exceeds the critical point, the ratio changes abruptly and increases with the increase of the concentration. At this time, the concentration corresponding to this critical point is the critical micelle concentration. After calculation, the critical micelle concentration values of the two prodrug nanosystems USP-Nanos and UCP-Nanos are 5.71 μg / mL and 5.95 μg / mL, respectively. The lower CMC value indicates that the nanoparticles have good stability after entering the body.

[0039] Particle Size and Distribution of Nanosystems The sizes of the two prepared prodrug nanosystems USP-Nanos and UCP-Nanos were measured using a laser particle size Zeta potentiometer. Before using the instrument, it was preheated for 30 min. Then, the sample was added to about 1 / 3 of the cuvette and placed in the measurement cell. After setting the measurement parameters, the measurement was started, and the particle size and PDI data were recorded. The results are shown in Figure 5 and 6 .

[0040] The particle size and potential distribution of the two nanosystems USP-Nanos and UCP-Nanos are as follows Figure 5, 6 As shown in the figure, it can be seen that the particle sizes of both USP-Nanos and UCP-Nanos nanoformulations are around 170 nm, and the PDI values are all less than 0.2. The particle sizes are relatively small and the distribution is relatively uniform. The Zeta potentials of USP-Nanos and UCP-Nanos are -22.7 mV and -28.1 mV respectively. The potentials are both negative and the absolute values are relatively large, suggesting that the formulations have good stability.

[0041] Morphological observation of nanoformulations Take appropriate amounts of the prodrug nanoformulations USP-Nanos and UCP-Nanos prepared, dilute them to appropriate concentrations (dilute the original formulations 10 times), then use a 10 μL pipette to aspirate the samples and drop them onto the copper grid to allow them to aggregate on the copper grid. After that, place them in a vacuum drying oven to dry, and use a transmission electron microscope to measure the morphology of the samples and take pictures for recording. The results are shown in Figure 7 .

[0042] The transmission electron micrographs of the nanoformulations USP-Nanos and UCP-Nanos are as shown in Figure 7 As shown in the figure. It can be seen from the figure that some of the nanoparticles are adhered, which may be due to excessive concentration during sample preparation resulting in particle overlap or aggregation of the nanoparticles caused by surface tension during the evaporation of water. However, it can be clearly observed from the figure that the morphologies of both nanoformulations are spherical-like, and the particle sizes are relatively uniform, indicating that the prodrug can self-assemble into nanoparticles.

[0043] Investigation of the stability of nanoformulations (1) Storage stability Place the prepared nanoformulations USP-Nanos and UCP-Nanos in vials and store them at 4°C and room temperature (n = 3) for 21 days. Measure and record the particle size changes of the samples on the 0th, 1st, 3rd, 6th, 9th, 12th, 15th, and 21st days respectively. The results are shown in Figure 8 .

[0044] The results of the particle size changes of the nanoformulations measured on the 0th, 1st, 3rd, 6th, 9th, 12th, 15th, and 21st days are as shown in Figure 8 As shown in the figure. It can be seen from the figure that the particle size changes of both nanoformulations do not exceed 20 nm after being placed at 4°C and room temperature for 21 days, indicating that both nanoformulations have good storage stability.

[0045] (2) Dilution stability Take appropriate amounts of the prepared nanoformulations USP-Nanos and UCP-Nanos, and dilute them with ultrapure water. The dilution factors are 5, 10, 25, 50, and 100 times respectively. Each group has three replicates in parallel, and measure and record their particle sizes. The results are shown in Figure 9 .

[0046] The particle size results of two nanopreparations, USP-Nanos and UCP-Nanos, after being diluted with ultrapure water by different multiples are shown as Figure 9 follows. It can be seen from the figure that the particle size changes of both nanopreparations, USP-Nanos and UCP-Nanos, are less than 10 nm, indicating that the samples still have good stability after dilution.

[0047] Release study under reducing conditions Four release media with different glutathione (GSH) concentrations were prepared, with concentrations of 0 μM, 10 μM, 10 mM, and 20 mM (containing 0.5% Tween 80). Take 1 mL of each of the prodrug nanopreparations, USP-Nanos and UCP-Nanos, and place them in a dialysis bag (MWCO = 1000 Da, n = 3). Place them in 20 mL of PBS release medium with different GSH concentrations at pH = 7.4 and incubate with shaking at 100 rpm at 37°C. Take 1 mL of samples at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 h, and supplement the same amount of release medium at the same time. Then filter the taken samples and detect the content of PTX in the samples by HPLC, and calculate the cumulative release amount of the prodrug preparation. The results are shown in Figure 10 .

[0048] The nanopreparation USP-Nanos contains disulfide bonds, which will break under certain concentrations of GSH, resulting in the destruction of the nanoparticle structure and the release of the free drug PTX. Therefore, we set four release media with GSH concentrations of 0 μM, 10 μM, 10 mM, and 20 mM, and used the dialysis method to determine the release of the free drug. At the same time, UCP-Nanos was used as a control to study the release behavior of the two preparations under reducing conditions. The release results are shown as Figure 10 follows. When the nanopreparation USP-Nanos was incubated for 48 h under low-concentration GSH (0 μM, 10 μM) conditions, less than 20% of the free paclitaxel (PTX) was released. However, after incubation for 48 h under high-concentration (10 mM, 20 mM) GSH conditions, the cumulative release of free PTX was 53% and 75% respectively, and the release amount increased with the increase of GSH concentration. For UCP-Nanos, the cumulative release amount was less than 15% regardless of the GSH concentration. The experimental results show that due to the presence of disulfide bonds, the USP prodrug can specifically release PTX in the highly reducing environment of tumor tissues and has good reduction sensitivity. Since the UCP prodrug does not contain disulfide bonds, its drug release is not affected by reducing conditions.

[0049] Cytotoxicity The logarithmically growing human hepatoma cells HepG2 and Huh-7 were diluted into cell suspensions with DMEM medium. After pipetting evenly, 100 μL was added to each well of a 96-well plate, so that the cell density in each well was 8×10 3 cells. After culturing in a constant temperature incubator at 37 °C for 24 h, the medium in the wells was discarded. The free drugs (UDCA or PTX solution, Taxol injection) and the preparations (USP-Nanos, UCP-Nanos, USP-Sol, UCP-Sol) were diluted with serum-free blank medium to prepare a series of solutions with concentrations of 0.9375, 1.875, 3.75, 7.5, 15 μg / mL. 100 μL of each solution was added to each well of the 96-well plate, with 6 replicates for each concentration. At the same time, the cell wells with blank DMEM medium added were used as the negative group, and the cell-free wells with blank DMEM medium added were used as the blank control group. After culturing the 96-well plate in an incubator at 37 °C for 24 h or 48 h, 10 μL of MTT solution with a concentration of 0.5% was added to each well of the plate under light-proof conditions. After continuing to culture in the incubator for 4 h, it was taken out, the supernatant was discarded, 100 μL of DMSO was added to each well, and it was incubated with shaking for 10 min to fully dissolve the crystals in the wells. Then, the OD values of each well were measured at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader. After that, the cell survival rate of each group of drugs was calculated according to the following formula. The results are shown in Figure 11 .

[0050] The cytotoxicity results of free drugs UDCA, PTX, Taxol injection and USP-sol, UCP-sol and USP-Nanos, UCP-Nanos preparations on HepG2 and Huh-7 cells for 24 h and 48 h are as shown in Figure 11 . Figure 11The results showed that the toxicity of each group of drugs to HepG2 and Huh-7 cells was concentration-dependent. Among them, Taxol injection showed the strongest cytotoxicity. The cell viability of HepG2 and Huh-7 cells treated with Taxol injection at a concentration of 15 μg / mL for 48 h was 15.40% and 15.98% respectively. The cytotoxicity of USP-sol was slightly lower than that of Taxol injection, and the cell viability was 19.8% and 34.4% respectively. This may be attributed to the potential cytotoxicity of excipients in Taxol injection and the slow release of drugs in the nanosystem. The prodrug was responsive to release free PTX under the action of GSH highly expressed in tumor cells, thus exerting cytotoxicity. The cytotoxicity of USP-Nanos was significantly stronger than that of free UDCA and PTX. It was speculated that the combination of the two drugs might play a synergistic role and the nanoparticles were more easily taken up by cells, so it had stronger cytotoxicity. In contrast, the cytotoxicity of UCP-Nanos and UCP-sol was lower than that of USP-Nanos and free PTX. This may be attributed to the non-reductive sensitivity of UCP, resulting in less drug release at the tumor site. This result was consistent with the previous in vitro release results.

[0051] Cell transport Human colon adenocarcinoma cells Caco-2 in logarithmic growth phase were seeded in 12-well Transwell chambers at a density of 2×10 4 cells / well and cultured at 37 °C for about 21 days. During this period, the transepithelial electrical resistance (TEER) of the culture plate was measured every other day. When it was greater than 500 Ω·cm 2When it indicates that the in vitro intestinal epithelial monolayer membrane model is successfully constructed and can be used for cell transport research. First, discard the old culture medium in the culture plate, wash it three times with pre-warmed Hank's buffer, then add 0.5 mL and 1.5 mL of Hank's buffer to the upper chamber and the lower chamber respectively, place it in the incubator for 30 min and then discard it. Add the ASBT competitive inhibitor UDCANa solution (100 μg / mL, 0.5 mL) diluted with Hank's buffer to the chamber, discard it after 1 h in the incubator and wash the chamber 3 times with PBS. Then add the USP-Nanos and UCP-Nanos preparations (50 μg / mL, 0.5 mL) diluted with Hank's buffer. Absorb 300 μL of the receiving solution from the lower chamber at 30 min, 60 min, and 120 min and supplement an equal amount of Hank's buffer. Set three replicates for each group. At the same time, set the Taxol injection, USP-Nanos, and UCP-Nanos nanoparticles without adding the ASBT competitive inhibitor UDCANa solution as a control with the inhibitor group to study the intestinal epithelial transport mechanism of the nanoparticles. The collected samples are analyzed by HPLC injection, and the apparent permeability coefficient (Papp) of each group of samples is calculated according to the following formula, where dQ / dt is the drug permeability per unit time, A is the chamber membrane area (1.12 cm 2 ) and C0 is the initial concentration of the drug in the chamber. The results are shown in Figure 12 .

[0052] When the drug is administered orally, it needs to first pass through the intestinal epithelial cell layer and then enter the blood circulation system. The Caco-2 cell monolayer is a recognized model for evaluating intestinal absorption of drugs in vitro. Therefore, an intestinal epithelial monolayer membrane is constructed with Caco-2 cells as the model to evaluate the intestinal epithelial transport permeability of the nanoparticles, and the results are expressed by the apparent permeability coefficient. As can be seen from Figure 12 , the apparent permeability coefficients of each group of preparations are time-dependent. When Taxol injection is co-incubated with the Caco-2 cell monolayer for 2 h, its Papp value is 4.55×10 -6 cm / s, while the Papp values of the USP-Nanos and UCP-Nanos preparations are 11.59×10 -6 and 11.71×10 - 6cm / s, which is about 2.5 times higher than that of the Taxol injection, indicating that the USP-Nanos and UCP-Nanos formulations can significantly improve the intestinal epithelial transport ability of the drug. In addition, to investigate the role of ASBT in the transport and absorption of nanogranulocytes, the group added with the ASBT competitive inhibitor was used as the control. The results showed that after treatment with UDCANa, the Papp values of the USP-Nanos and UCP-Nanos formulations decreased to 6.87×10 -6 cm / s and 7.03×10 -6 cm / s respectively, indicating that the two prodrug nanosystems enhance the intestinal epithelial cell transport ability of the drug through the UDCA-ASBT pathway, and this result is consistent with the cell uptake result.

[0053] Oral pharmacokinetic experiment The SD rats were randomly divided into 4 groups with 5 rats in each group. They were fasted overnight in advance to exclude the influence of food on absorption. They were respectively given 1 mL of paclitaxel injection by intravenous injection (Taxol, i.v.), oral gavage of paclitaxel injection (Taxol, p.o.), nanosystem USP-Nanos (p.o.) and UCP-Nanos (p.o.), and the dose of PTX was 10 mg / kg. After dosing, blood samples were collected into heparinized test tubes at 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48 h. The blood samples were centrifuged at 8000 rpm for 5 min to obtain plasma samples, which were stored at -80 °C until analysis. The results are shown in Table 1.

[0054] Table 1 Pharmacokinetic parameters (n = 5) The pharmacokinetic parameters of each group are shown in Table 1. The results in Table 1 show that the USP-Nanos and UCP-Nanos formulations have similar pharmacokinetic behaviors. Compared with the Taxol oral group, their half-lives t 1 / 2 were extended from 11.68 h to 20.46 h and 18.08 h respectively, the maximum plasma concentration C max values were increased by 5.67 times and 5.42 times respectively, and the relative bioavailability Frel was increased by 7.76 times and 5.69 times respectively. Compared with the Taxol injection group, the absolute bioavailability Fabs of oral Taxol, USP-Nanos and UCP-Nanos were 2.98%, 23.11% and 16.96% respectively. These results indicate that the USP-Nanos and UCP-Nanos nanosystems can improve the oral bioavailability of PTX and prolong the drug circulation time.

[0055] Pharmacodynamic evaluation of nanosystems Inject 200 μL of mouse hepatocarcinoma cell H22 cell suspension (1×10 7 cells / mL) into the right axilla of Kunming mice to construct an H22 tumor-bearing mouse model. Observe the tumor growth. When the tumor volume grows to about 100 mm 3 , randomly divide the mice into 8 groups with 6 mice in each group. Inject Taxol via the tail vein, and orally administer Saline, UDCA, Taxol, UCP-Nanos, USP-Nanos (the dosing dose of PTX is 10 mg / kg for all). Additionally, set up a low-dose group (5 mg / kg) and a high-dose group (20 mg / kg) of USP-Nanos for oral administration. Administer the drugs via the tail vein injection once every other day, and administer the drugs orally once a day. Fast the mice for 12 h before oral administration. Measure the tumor volume (long diameter × short diameter × short diameter / 2) every day during the drug administration process. The drug administration period is 12 days, and sacrifice the mice one day after the last drug administration. Dissect the tumors of the mice, take pictures and weigh them to compare the anti-tumor effects of each group. The results are shown in Figure 13 .

[0056] Figure 13 Shown is the graph of the change in tumor volume during the treatment of mice. As Figure 13 can be seen, the change in the Saline group is the most obvious, followed by the UDCA group, and there are significant differences in the tumor volumes of each group. The results show that regardless of the dosing dose, the anti-tumor effect of the USP-Nanos group is stronger than that of the UCP-Nanos group. It is speculated that the carbon-carbon bond cannot rapidly release the drug in the tumor, so its anti-tumor activity is poor, while the reduction-sensitive bond disulfide bond breaks in the tumor site and rapidly releases the drug, thereby exerting an anti-tumor effect. The anti-tumor activity of the oral Taxol group is significantly weaker than that of the USP-Nanos group at the same dose. And it is worth noting that the anti-tumor activity of the high-dose USP-Nanos group is comparable to that of the Taxol group injected via the tail vein. This may be attributed to the relatively high oral bioavailability of the USP-Nanos nanoplatform. The prodrug can continuously release the drug in vivo to exert its effect. Generally speaking, the USP-Nanos nanoplatform has good in vivo anti-tumor activity, and the experimental results are consistent with the in vitro cytotoxicity results.

[0057] In summary, the present invention uses the method of nanoprecipitation. The bile acid-based paclitaxel small molecule prodrug can self-assemble into nanoparticles in water without using a carrier material, and the preparation process is simple and easy to industrialize. The amphiphilic small molecule prodrug improves the water solubility of paclitaxel, has a high drug loading capacity, and avoids the adverse reactions that may be caused by carriers and excipients. When the nanoparticles are orally administered, UDCA can serve as a targeting "switch" for the ASBT receptor, enhance the gastrointestinal permeability of the nanoparticles, enable the nanoparticles to be transported and reabsorbed mediated by ASBT into intestinal cells and then enter the circulatory system, and enhance the oral bioavailability of paclitaxel. The tumor microenvironment reduction sensitivity of the linker enables paclitaxel to be specifically released at the tumor site, avoids premature leakage of the drug, significantly improves the drug efficacy and reduces the toxicity and side effects.

Claims

1. A bile acid-paclitaxel small molecule prodrug, characterized in that, It has any of the following structural formulas: R1 is independently selected from α-OH, b-OH, α-H or b-H respectively; R2 is independently selected from α-OH, b-OH, α-H or b-H respectively; X is independently selected from OH, NHCH2COOH or NHCH2CH2SO3H respectively.

2. The preparation method of the bile acid-taxol small molecule prodrug according to claim 1, characterized in that, It includes the following steps: 1) Dissolve 3,3'-dithiobispropionic acid in anhydrous acetyl chloride, reflux and condense at 55-70 °C for 2-5 h, concentrate the reaction solution by rotary evaporation and precipitate with ice ether to obtain the acid anhydride; 2) Dissolve the acid anhydride in an appropriate amount of anhydrous dichloromethane, add DMAP and DCC or EDCI and ice-bath for 1-2 h, then add paclitaxel (PTX), react at 25-35 °C for 24-48 hours, and obtain intermediate 1 by extraction; 3) Dissolve ursodeoxycholic acid (UDCA) in an appropriate amount of DMF, add sodium bicarbonate to make the reaction condition weakly acidic, add p-methoxybenzyl chloride to protect the carboxyl group of UDCA, and react at 30-45 °C for 24-48 h to obtain carboxyl-protected ursodeoxycholic acid (PMB-UDCA); 4) After dissolving intermediate 1 in anhydrous DMF, add DMAP and DCC or EDCI and ice-bath for 1-2 h, then add PMB-UDCA, react at 30-45 °C for 24-48 h, dialyze and freeze-dry to obtain the carboxyl-protected prodrug; dissolve the carboxyl-protected prodrug in an appropriate amount of dichloromethane, remove the carboxyl protection with trifluoroacetic acid or hydrochloric acid-methanol solution, react at 30-45 °C for 1-2 h, and obtain the final product by column chromatography separation and purification; the above reactions are all carried out under the protection of an inert gas atmosphere.

3. The preparation method of the bile acid-paclitaxel small molecule prodrug according to claim 2, wherein, In step 1), the molar ratio of 3,3'-dithiobispropionic acid to anhydrous acetyl chloride is 1:(2-5); in step 2), the molar ratio of PTX, acid anhydride, DMAP and DCC or EDCI is 1:(0.5-10):(0.5-3):(1-3); in step 3), the molar ratio of UDCA, sodium bicarbonate and p-methoxybenzyl chloride is 1:(1-1.5):1; in step 4), the molar ratio of intermediate 1, DMAP, DCC and PMB-UDCA is 1:(1-3):(1-3):

1.

4. The preparation method of the bile acid-paclitaxel small molecule prodrug according to claim 1, characterized in that, It includes the following steps: a) Dissolve succinic anhydride in anhydrous dichloromethane, add DMAP and DCC or EDCI and ice-bath for 1-2 h, then add PTX, react at 25-35 °C for 24-48 h, and obtain intermediate 2 by extraction; b) Dissolve UDCA in an appropriate amount of DMF, add sodium bicarbonate to make the reaction condition weakly acidic, add p-methoxybenzyl chloride to protect the carboxyl group of UDCA, and react at 30-45 °C for 24-48 h to obtain PMB-UDCA; c) Dissolve the intermediate 2 in anhydrous DMF, add DMAP and DCC or EDCI, and react at ice bath for 1 - 2 h. Then add PMB - UDCA and react at 30 - 45 °C for 24 - 48 h. After dialysis and lyophilization, the carboxyl - protected prodrug is obtained. Dissolve the carboxyl - protected prodrug in an appropriate amount of dichloromethane, remove the carboxyl protection with trifluoroacetic acid or hydrochloric acid - methanol solution, and react at 30 - 45 °C for 1 - 2 h. The final product is obtained by column chromatography separation and purification. All the above reactions are carried out under the protection of an inert gas atmosphere.

5. The preparation method of the bile acid-taxol small molecule prodrug according to claim 4, wherein In step a), the molar ratio of PTX, succinic anhydride, DMAP and DCC is 1:(1 - 10):(0.5 - 3):(1 - 3); in step b), the molar ratio of UDCA, sodium bicarbonate and p - methoxybenzyl chloride is 1:(1 - 1.5):1; in step c), the molar ratio of intermediate 2, DMAP, DCC or EDCI and PMB - UDCA is 1:(1 - 3):(1 - 3):

1.

6. The preparation method of the bile acid-paclitaxel small molecule prodrug according to any one of claims 2 to 5, characterized in that, The UDCA can be replaced by cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid p - methoxybenzyl ester, hyodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, taurohyodeoxycholic acid, or tauro - ursodeoxycholic acid.

7. A self-assembled nanoparticle containing a bile acid-paclitaxel small molecule prodrug, characterized in that, The preparation process is as follows: Dissolve a certain amount of the bile acid - paclitaxel small - molecule prodrug described in claim 1 or a mixture thereof with a hydrophobic fluorescent substance in an appropriate amount of organic solvent. Under stirring, drop this solution into water, and the prodrug spontaneously forms uniform nanoparticles. Finally, the organic solvent is removed by vacuum distillation or dialysis to obtain self - assembled nanoparticles.

8. The self-assembled nanoparticles containing bile acid-paclitaxel small molecule prodrug according to claim 7, wherein, The hydrophobic fluorescent substance is doxorubicin, coumarin - 6, DiR, DiL, Cy - 5 or Cy - 7; the organic solvent is ethanol, methanol, acetone, dimethylformamide or dimethyl sulfoxide.

9. Use of the bile acid - paclitaxel small - molecule prodrug described in claim 1 or the self - assembled nanoparticles containing the bile acid - paclitaxel small - molecule prodrug described in any one of claims 7 - 8 in a drug delivery system or in the preparation of an anti - tumor drug.

10. Use of the bile acid - paclitaxel small - molecule prodrug described in claim 1 or the self - assembled nanoparticles containing the bile acid - paclitaxel small - molecule prodrug described in any one of claims 7 - 8 in an injection - based, oral - based or topical drug delivery system.

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