Tripterygium wilfordii bionic drug delivery system based on fat uptake pathway as well as preparation method and application of tripterygium wilfordii bionic drug delivery system

Abstract

The invention relates to the technical field of medicines, in particular to a tripterygium wilfordii bionic drug delivery system based on a fat uptake pathway as well as a preparation method and application of the tripterygium wilfordii bionic drug delivery system. According to the bionic drug delivery system disclosed by the invention, the triptolide derivative is entrapped into lipid droplets of adipocytes through a co-incubation method, and the aim of resisting malignant melanoma is achieved by utilizing the characteristic that tumor cells naturally recruit and uptake fatty acid on the adipocytes. The bionic drug delivery system is mature in preparation process, efficient, good in anti-tumor effect and free of toxic and side effects on other tissues and organs, and is expected to be applied to treatment of malignant melanoma and other tumors.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medicines, in particular to a triptolide biomimetic drug delivery system based on a fat uptake pathway and a preparation method and application thereof. BACKGROUND

[0002] Malignant melanoma (MM) is a common skin and mucosal malignant tumor in clinical practice, which is characterized by easy invasion and metastasis, high mortality and poor prognosis. It is one of the malignant tumors with the fastest growth rate in the world, with an annual growth rate of 3%-5%. At present, the treatment of melanoma, early diagnosis can prolong the 5-year survival rate by surgical resection of primary tumor, and metastasis is easy to occur in melanoma at middle and late stages. Clinical studies have shown that the most common metastatic sites of melanoma are lung and brain (both accounting for 24%). Traditional treatment methods include chemotherapy and radiotherapy, but the effect is poor, and it is easy to have toxic side effects and drug resistance. In recent years, targeted therapy and immunotherapy have made certain progress in improving the quality of life and prolonging the survival of patients, but molecular targeted therapy has limited effect on other melanoma mutation types. The revolution of immunotherapy in the treatment of solid tumors began with the treatment of melanoma. Immunotherapy represented by PD-1 inhibitors, PD-L1 inhibitors and CTLA-4 inhibitors is one of the most important treatment methods at present. The effective rate of single-drug PD-1 inhibitor in melanoma patients in the United States is nearly 60%. The newly emerging immune checkpoint inhibitors, such as LAG-3 inhibitors, TIGIT inhibitors and TIM-3 inhibitors, are expected to improve the treatment efficiency of acral and mucosal melanoma in combination with PD-1 inhibitors, but the long-term safety of immunotherapy needs to be investigated, and the price is high. Cell and gene therapy (CGT) has developed rapidly in recent years and has shown outstanding therapeutic effect in the fields of genetic diseases, autoimmune diseases and malignant tumors. Among them, oncolytic virus therapy, TIL therapy, TCR-T therapy and CAR-T autologous cell therapy have achieved great results in the treatment of advanced melanoma, but the price of CGT treatment is high, and ordinary people are difficult to afford.

[0003] Triptolide (TP) is a epoxy diterpene lactone compound derived from the plant Tripterygium wilfordii Hook. f. of the family Celastraceae, which has the effects of anti-rheumatoid, anti-inflammatory, immunosuppression, and anti-fertility. In recent years, it has been found that triptolide has broad-spectrum antitumor activity, and the main mechanisms are: 1) TP can regulate the levels of Fas, caspase-3, caspase-8, caspase-9 and death receptor DR5, and induce tumor cell apoptosis; 2) TP can increase the production of ROS and Ca 2+1) TP can induce cell cycle arrest and apoptosis in tumor cells; 2) TP can induce endoplasmic reticulum stress and apoptosis; 3) TP can inhibit tumor cell proliferation by inducing cell cycle arrest; 4) TP can inhibit tumor cell invasion and migration by down-regulating the expression of metalloproteinase MMP-2 and MMP-9 through the PI3K / Akt pathway; 5) TP can reverse the multi-drug resistance of tumor cells by reducing the expression of multi-drug resistance protein (MRP) and multi-drug resistance gene (MDR); 6) TP can regulate tumor immunity by promoting the proliferation of T cells and B cells; 7) TP can inhibit tumor angiogenesis by inhibiting the expression of vascular endothelial growth factor A (VEGFA). However, the poor water solubility of TP limits its clinical application due to its poor drugability, fast elimination in vivo, narrow therapeutic window, and strong toxicity and side effects.

[0004] Designing TP as a prodrug can improve its drugability and reduce its toxicity to some extent. Structure-activity relationship studies have found that the hydroxyl group at C-14 can be used as a connection site to introduce water-soluble or targeted groups. Currently, TP water-soluble prodrugs include fatty acids, amino salts, and phosphates, and TP targeted prodrugs are conjugated with glucose or glucosamine. Except for phosphate TP prodrugs, which have entered clinical trials, other TP prodrugs have not been well enhanced and attenuated due to large steric hindrance and incomplete conversion. In 2012, the University of Minnesota proved the effectiveness of TP on pancreatic cancer through mouse models and prepared the phosphate TP water-soluble prodrug Minnelide, which has entered phase II clinical trials. Another TP analog, F60008, has completed phase I clinical trials for primary central nervous system tumors. In addition to traditional preparations such as liposomes and nanoparticles composed of artificially synthesized macromolecules, functional exosomes are also used as biomimetic carriers for drug delivery. Due to the difficulty of degrading artificially synthesized materials, there are often large liver and kidney toxicities, while biomimetic carriers have good biocompatibility and natural targeting, which have attracted more and more researchers' attention.

[0005] The tumor microenvironment is a dynamic and complex network comprising tumor cells, locally infiltrating immune cells, tumor-associated fibroblasts, myeloid-derived suppressor cells, and adipocytes (Apo). Adipose tissue within the tumor microenvironment is chronically exposed to hypoxia, acidity, and inflammatory infiltration, leading to phenotypic changes in its adipocytes: 1) enhanced lipolysis and cell atrophy; 2) gradual dedifferentiation and enhanced secretory function; and 3) increased signaling exchange with tumor cells, promoting tumor cell metabolism. These altered adipocytes are termed "tumor-associated adipocytes" (TAAs). They are typically found at the periphery of tumor invasion and are regulated by factors within the tumor microenvironment. Richard M. White et al., in their study of the relationship between adipocytes and melanoma, discovered that adipocyte-derived lipids can be transferred to melanoma cells via the FATP / SLC27A (fatty acid transfer protein) lipid transporter family expressed on the surface of tumor cells, promoting melanoma progression. Furthermore, the expression of FATP1 / SLC27A1 is significantly abundant in melanoma, mediating the uptake of long-chain fatty acids.

[0006] Therefore, I urgently need to find a drug that can effectively fight melanoma and deliver it to melanoma tissue in a targeted manner to fully exert its anti-tumor effect. Summary of the Invention

[0007] The purpose of this invention is to provide a biomimetic drug delivery system based on the fat uptake pathway of Tripterygium wilfordii, its preparation method, and its application.

[0008] The biomimetic drug delivery system of this invention takes the close connection between melanoma cells and adipocytes as the entry point for combating melanoma, and encapsulates triptolide derivatives into adipocytes to enhance the killing effect of the drug on melanoma cells, while having no obvious toxicity to normal tissues and organs, providing a reliable theoretical basis and experimental foundation for the clinical treatment of melanoma.

[0009] The aforementioned biomimetic drug delivery system utilizes the characteristics of tumor tissue in recruiting and degrading adipocytes, using lipid droplets within adipocytes as natural drug carriers for hydrophobic drugs, thus overcoming the difficulty in drug formation of triptolide (TP). Taking advantage of the characteristic that melanoma readily absorbs long-chain fatty acids, TP is designed as triptolide coupled with palmitic acid TP-SS-PA with disulfide bonds as the linking bond, overcoming the shortcomings of TP such as short half-life and high toxicity to normal tissues and organs.

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

[0011] In a first aspect, the present invention provides a Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway, wherein the Tripterygium wilfordii biomimetic drug delivery system uses mature adipocytes as drug carriers to encapsulate a triptolide derivative TP-SS-PA (triptolide prodrug, pTP); wherein the triptolide derivative TP-SS-PA is coupled to triptolide and palmitic acid using disulfide bonds as connecting arms.

[0012] Furthermore, the chemical structural formula of the triptolide derivative TP-SS-PA is shown in Formula I below:

[0013]

[0014] Furthermore, the aforementioned Tripterygium wilfordii biomimetic drug delivery system is prepared by co-incubating mature adipocytes with the Tripterygium wilfordii derivative TP-SS-PA.

[0015] Furthermore, the preparation method of the aforementioned Tripterygium wilfordii biomimetic drug delivery system involves co-incubating mature adipocytes with the Tripterygium wilfordii derivative TP-SS-PA, thereby encapsulating TP-SS-PA in lipid droplets of the adipocytes.

[0016] Furthermore, the mature adipocytes are obtained by inducing differentiation from 3T3-L1 preadipocytes. Even further, the mature adipocytes are obtained by inducing differentiation from 3T3-L1 preadipocytes twice.

[0017] Furthermore, the drug loading capacity of the Tripterygium wilfordii biomimetic drug delivery system is 13.26±0.89μg / 10 6 cell.

[0018] In a second aspect, the present invention provides a method for preparing the Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway as described above, comprising the following steps: synthesizing the Tripterygium wilfordii derivative TP-SS-PA, inducing 3T3-L1 preadipocytes to differentiate into mature adipocytes Apo, and then co-incubating TP-SS-PA with the mature adipocytes to obtain the Tripterygium wilfordii biomimetic drug delivery system.

[0019] Furthermore, the method for co-incubating the mature adipocytes with TP-SS-PA is as follows: 1×10 6 Mature adipocytes were added to 2 mL of a PBS solution containing 40 μg / mL TP-SS-PA and incubated at 37°C and 5% CO2 for 1 h to construct the Tripterygium wilfordii biomimetic drug delivery system. Furthermore, the TP-SS-PA PBS solution was prepared by first dissolving TP-SS-PA in DMSO, then adding 0.5% Tween 80 to enhance the solubility, and finally diluting with PBS to the desired concentration.

[0020] Furthermore, the preparation method of the triptolide derivative TP-SS-PA is as follows: using palmitic acid as raw material, a disulfide bond is introduced by reacting 3,3-dithiodipropionic acid with acetic anhydride, and a palmitic acid derivative is synthesized by using 4-dimethylaminopyridine (DMAP) as a catalyst. The derivative is then coupled with triptolide (TP) through a disulfide bond, and the triptolide derivative TP-SS-PA (pTP) is prepared by a one-step esterification method.

[0021] Furthermore, the preparation method of the triptolide derivative TP-SS-PA includes the following steps: (a) mixing and stirring 3,3-dithiodipropionic acid and acetic anhydride at 50°C for 5 hours, and concentrating to obtain crude product intermediate 2; (b) adding intermediate 2 to cetyl alcohol and catalyst DMAP, stirring at 20°C for 12 hours, washing, purifying, and concentrating to obtain crude product intermediate 3; (c) mixing triptolide with intermediate 3, and adding condensing agents DMAP and DCC, stirring the mixture at 0°C for 0.5 hours, then stirring at 15°C for 12 hours, washing with saturated brine, concentrating under reduced pressure, and purifying to obtain triptolide derivative TP-SS-PA.

[0022] Furthermore, the method for preparing the mature adipocytes is as follows: 3T3-L1 preadipocytes are seeded in 6-well plates. When the cells grow to the confluence state, 2.5 mL of induction medium I (complete medium containing IBMX, dexamethasone, and insulin) is added to the cell culture medium and cultured for 48 h. Then, induction medium II (complete medium containing insulin) is replaced and cultured for another 48 h. Finally, the cells are cultured continuously in complete medium for 11 days to obtain mature adipocytes.

[0023] Furthermore, in the induction solution I, the final concentration of IBMX is 0.5 mol / L, the final concentration of dexamethasone is 2.5 mmol / L, and the final concentration of insulin is 10 μg / mL; in the induction solution II, the final concentration of insulin is 10 μg / mL.

[0024] A third aspect of the present invention provides the application of the Tripterygium wilfordii biomimetic drug delivery system described above in the preparation of a therapeutic drug for malignant melanoma.

[0025] The advantages of this invention are:

[0026] 1. The triptolide derivative in this invention uses palmitic acid as a target for melanoma. Since melanoma cells require the uptake of large amounts of fatty acids during proliferation, invasion, and metastasis, and palmitic acid is an essential fatty acid with high safety, the triptolide derivative TP-SS-PA is more easily taken up by tumor cells. The biomimetic drug delivery system of this invention encapsulates the triptolide derivative TP-SS-PA into lipid droplets of adipocytes through co-incubation, utilizing the natural recruitment and fatty acid uptake characteristics of adipocytes by tumor cells to achieve the purpose of combating malignant melanoma.

[0027] 2. The triptolide derivative TP-SS-PA in this invention uses disulfide bonds as linkages, which can be rapidly broken in tumor cells containing at least 10 times higher concentrations of GSH than normal cells and tissues, releasing the parent drug triptolide to kill tumor cells while reducing toxic side effects on normal tissues and organs.

[0028] 3. In this invention, TP-SS-PA is encapsulated in adipocytes. The lipid droplets inside the adipocytes serve as natural carriers for hydrophobic drugs, while also exhibiting high drug loading capacity, good biocompatibility, and easier recognition by the adipose pathway of melanoma cells.

[0029] 4. The Tripterygium wilfordii biomimetic drug delivery system developed in this invention has a mature, efficient, and reproducible preparation process. It has significant in vitro and in vivo antitumor effects and no toxic side effects on other tissues and organs. It has good biosafety and provides a new strategy for the clinical treatment of melanoma. It is expected to be applied in the treatment of malignant melanoma and other tumors. Attached Figure Description

[0030] Figure 1 This is a synthetic route diagram of TP-SS-PA in Example 1.

[0031] Figure 2 shows the 1H NMR spectrum, HPLC spectrum, and mass spectrum of TP-SS-PA in Example 2.

[0032] Figure 3 The images shown are of 3T3-L1 preadipocytes, differentiated adipocytes, and adipocytes stained with Oil Red microscopy in Example 3, with a scale bar of 50 μm.

[0033] Figure 4The following is an example of the construction and characterization of the Tripterygium wilfordii biomimetic drug delivery system in Example 4, wherein: (A) is a standard curve of TP-SS-PA; (B) is the drug loading of adipocytes after co-incubation of different concentrations of TP-SS-PA with adipocytes for 60 min; (C) is the drug loading of adipocytes after co-incubation of TP-SS-PA at a concentration of 40 μg / mL with adipocytes for different durations; (D) is the activity of adipocytes after treatment with pTP and TP respectively (n=6); (E) is a fluorescence microscope image of adipocytes before and after drug loading; (F) is an in vitro release curve of adipocytes.

[0034] Figure 5 The following diagram shows the results of the investigation into the interaction mechanism between the Apo biomimetic drug delivery system and melanoma cells in Example 5: (A) is a diagram of the process of A375 cells causing lipolysis of adipocytes; (B) is the ratio of the area of ​​lipid droplets to the total field of view at each stage; (C) is a diagram of A375 cell migration; (D) is a diagram of A375 cell transmembrane count after co-culturing with adipocytes alone and the A375-adipocyte system for 24 hours; (E) is a diagram of adipocyte migration; and (F) is a diagram of adipocyte transmembrane count after co-culturing with A375 cells alone and the A375-adipocyte system for 24 hours.

[0035] Figure 6 The following are the results of the Western blot experiments on the marker cytokines in Example 6; (A) shows the Western blot analysis of proteins related to fatty acid uptake on the A375 cell membrane after co-culturing A375 cells with 3T3 cells, sodium oleate, and Apo, respectively; (B) shows the relative quantitative expression of related proteins in each group; (C) shows the Western blot analysis of FABP4 on the cell membranes of 3T3 cells and Apo, respectively after co-culturing A375 cells with 3T3 cells and Apo, respectively, in Example 6; (D) shows the relative quantitative expression of FABP4 in each group.

[0036] Figure 7 This is a colocalization fluorescence imaging of A375 cells and their uptake of lipid droplets (LDs) in Example 7. Scale bar: 20 μm.

[0037] Figure 8 shows the in vitro anti-melanoma effect evaluation of the pTP-Apo Tripterygium wilfordii biomimetic drug delivery system in Example 8; where: (A) is the constructed A375 cell subcutaneous xenograft model in nude mice; (B) fluorescence imaging of DiR-labeled Apo in tumor-bearing nude mice at 0, 1, 3, 6, and 12 h after subcutaneous injection next to the tumor; (C) solid tumors in each group in Example 8; (D) tumor growth inhibition rate in each group; (E) TUNEL fluorescence staining in Example 8; (F) tumor sections stained with H&E and Ki67, scale bar 50 μm; (G) H&E staining of major organs of each group of nude mice in Example 8, scale bar 50 μm. Detailed Implementation

[0038] The specific embodiments of the present invention will be described in detail below with reference to examples. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0039] Example 1: Synthesis of triptolide derivative TP-SS-PA

[0040] ① Synthesis of intermediate 2: Accurately weigh 3,3-dithiodipropionic acid (compound 1, 1.00 eq) and add it to acetic anhydride (2.30 eq). Stir at 50°C for 5 h in a magnetic stirrer. After the raw material is completely consumed by thin-layer chromatography (TLC), add an appropriate amount of toluene to the mixture for concentration. The crude product obtained is stirred together with an appropriate amount of petroleum ether at 15°C for 1 h to obtain intermediate 2 (yield 91.1%) as a white solid.

[0041] ② Synthesis of Intermediate 3: Intermediate 2 (1.00 eq) was dissolved in CH2Cl2, and hexadecyl alcohol (compound 2-1, 1.20 eq) and catalyst DMAP (2.30 eq) were added at 15 °C. The mixture was placed in a magnetic stirrer and reacted at 15 °C with stirring for 12 h. When TLC indicated that the starting material was completely consumed, the mixture was washed twice with acetic acid (5 mL) and then twice with purified water (5 mL). The organic phase was concentrated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography to obtain Intermediate 3 (69.1% yield) as a white solid.

[0042] ③ Synthesis of Tripterygium wilfordii derivative TP-SS-PA: Intermediate 3 (1.00 eq) was dissolved in an appropriate amount of dichloromethane, compound 3-1 (0.65 eq) and catalyst DMAP (1.00 eq) were added, and condensing agent DCC (1.00 eq) was added at 0℃. The mixture was first stirred at 0℃ for 0.5 h, and then stirred at 15℃ for 11.5 h. When the starting material was completely consumed by TLC, the pH of the mixture was adjusted to 5 with hydrochloric acid solution (1M). Then, the organic layer was extracted with dichloromethane (5 mL × 2), washed with saturated brine (5 mL), and concentrated under reduced pressure to obtain the crude product. The crude product was further purified by high performance liquid chromatography (pre-HPLC) (column: Phenomenex lμna C18 75*30 mm*3 μm; mobile phase: [water (0.04% HCl)-ACN]; B%: 75%-95%, 8.0 min). After purification, the organic solvent was removed, and the remaining aqueous solution was freeze-dried to obtain the final product, triptolide derivative TP-SS-PA (yield 17.3%), which was a yellow oily liquid.

[0043] Figure 1 This is the synthetic route diagram for TP-SS-PA in Example 1. The final product, TP-SS-PA, is a relatively viscous, pale yellow, oily liquid with a yield of 17.3% and a purity of 99.9%.

[0044] Example 2: Structural Identification of TP-SS-PA

[0045] Weigh an appropriate amount of the final product and dissolve it in methanol. Perform a full wavelength scan in the wavelength range of 190-600 nm using a UV-Vis spectrophotometer. Weigh a small amount of the final product, dissolve and dilute it in methanol, and determine its molecular weight using liquid chromatography-mass spectrometry (LC-MS) to identify it. Dissolve a small amount of the product in deuterated chloroform and determine the chemical shift of hydrogen in the product using nuclear magnetic resonance (NMR) spectrometry (ppm). 1H NMR is based on tetramethylsilane TMS (chemical shift = 0.00). The order of NMR data writing is peak type (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant J (Hz), and the number of hydrogens.

[0046] Figure 2 shows the 1H NMR spectrum, HPLC spectrum, and mass spectrum of TP-SS-PA in Example 2. The spectral interpretation results of the mass spectrum and 1H NMR spectrum of TP-SS-PA are as follows:

[0047] ESI-MS m / z: 750.3 [M+H] + ;

[0048] 1 H NMR (400MHz, CDCl3) δ: 5.10 (s, 1H), 4.68 (s, 2H), 4.13-4.16 (m, 2H), 3.84 (s, 1H) ,3.62(s,2H),3.56(m,2H),3.55-3.57(m,1H),3.48(d,J=3.2Hz,1H),2.68-2.71 (m,1H),2.17-2.31(m,1H),1.96-1.98(m,2H),1.91-1.98(m,2H),1.55-1.66(m, 3H), 1.12-1.20 (m, 27H), 1.07 (s, 3H), 0.97 (d, J = 6.8Hz, 3H), 0.84-0.89 (m, 6H).

[0049] Example 3: Induction of differentiation of mature adipocytes (Apo)

[0050] 3T3-L1 preadipocytes were threshed at a dose of 1.2 × 10⁻⁶. 6Cells were seeded at a density of [number] cells / well in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics. When cells reached confluence, incubation was continued for 24-48 hours to induce contact inhibition. Induction solution I (complete medium containing 0.5 mol / L IBMX, 2.5 mmol / L dexamethasone, and 10 μg / mL insulin) was added for the first induction (defined as day 0 of differentiation induction). Cells were cultured continuously at 37°C and 5% CO2 for 48 hours. On day 2, the medium was replaced with induction solution II (complete medium containing 10 μg / mL insulin) and cultured for another 48 hours. On day 4, the medium was replaced with complete medium and cultured for 11 days to obtain mature adipocytes. Mature adipocytes were collected on day 14 and stained with Oil Red O to observe the formation and distribution of lipid droplets.

[0051] Figure 3 The images shown in Example 3 are fluorescence microscopy images of 3T3-L1 preadipocytes, differentiated adipocytes, and adipocytes stained with Oil Red O, scale bar 50 μm. After a 14-day induction differentiation process, 75%-85% of the 3T3-L1 preadipocytes successfully differentiated into mature adipocytes, which were stained red with Oil Red O dye. Multiple lipid droplets of different sizes were visible in the cytoplasm; these droplets were mostly round and distributed around the periphery of the adipocyte cytoplasm, giving the entire adipocyte a typical "ring-like" structure.

[0052] Example 4: Construction and Characterization of Tripterygium wilfordii Bionic Drug Delivery System

[0053] 1. Establishment of HPLC quantitative method for triptolide derivative TP-SS-PA (pTP)

[0054] Chromatographic column: Eclipse Plus C18 column (4.6mm×250mm, 5μm) Agilent; mobile phase: methanol:water (95:5); detection wavelength: 218nm; flow rate: 1.0mL / min; column temperature: 30℃; injection volume: 20μL.

[0055] TP-SS-PA sample solutions with concentrations of 2.2, 4.4, 8.8, 44, 88, 176, and 352 μg / mL were prepared using methanol. The solutions were injected and analyzed under the chromatographic conditions described above. Peak areas were recorded, and a linear regression was performed between peak area (Y) and concentration (X) to obtain the standard curve equation. Specificity, precision, recovery, and stability tests were then conducted.

[0056] 2. Effect of incubation drug concentration on cell drug loading

[0057] The mature adipocytes (Apo) after induced differentiation were digested and counted using trypsin, with 1×10⁻⁶ cells being counted. 6Mature adipocytes were added to 2 mL of PBS solution containing TP-SS-PA at concentrations of 5, 10, 20, 30, 40, and 50 μg / mL, respectively (TP-SS-PA was first dissolved in DMSO, then solubilized with Tween-80, and finally diluted to the desired concentration with PBS). The solutions were incubated at 37°C and 5% CO2 for 1 h, centrifuged at 3000 rpm for 5 min, and the adipocytes were separated from the supernatant. The supernatant was then centrifuged at 12000 rpm for 10 min and analyzed by liquid chromatography. The peak area of ​​free TP-SS-PA not loaded into adipocytes was used to calculate the amount of free drug in a standard curve. Finally, the effective drug loading content within the adipocytes was calculated using the formula: Adipocyte drug loading (DL) = (W... total -W free ) / N cell ×10 6 W total The drug concentration in the solution at the start of co-incubation; W free The content of free drug in the supernatant after incubation; N cell ×10 6 This represents the number of cells during co-incubation.

[0058] 3. Effect of incubation time on drug loading capacity in cells

[0059] Mature adipocytes after induced differentiation were digested and counted using trypsin, with 1×10⁻⁶ cells being counted. 6 Each cell was added to 2 mL of PBS solution containing 40 μg / mL TP-SS-PA and incubated at 37°C and 5% CO2 for 15, 30, 45, 60, 75, and 90 min, respectively. The cells were then centrifuged at 3000 rpm for 5 min to collect the adipocytes. Cells were treated as described above, and the drug loading on the adipocytes was calculated.

[0060] 4. Observe the morphology of adipocytes before and after drug loading using fluorescence microscopy.

[0061] Mature adipocytes after induced differentiation were digested and counted using trypsin, with 1×10⁻⁶ cells being counted. 6 Each cell was added to 2 mL of PBS solution containing 40 μg / mL TP-SS-PA and incubated at 37°C and 5% CO2 for 1 h. After centrifugation at 1000 rpm for 5 min, the drug-loaded adipocytes were collected, resuspended in DMEM, and transferred to 6-well plates for 12 h of culture. The morphology of the drug-loaded adipocytes and the distribution of lipid droplets were observed under a fluorescence microscope.

[0062] 5. Effects of drug loading on adipocyte activity

[0063] Mature adipocytes were seeded and cultured in 96-well plates. Control group adipocytes received no treatment; experimental groups were treated with different concentrations of TP-SS-PA and TP solution (prepared in DMEM), with TP-SS-PA concentrations set at 10, 20, 30, 40, and 50 μg / mL, and TP concentrations set at 0.01, 0.02, 0.05, 0.1, and 0.2 μg / mL, respectively, and cultured overnight. Cell viability after treatment with the two drugs was determined using the CCK-8 assay.

[0064] 6. Drug stability and in vitro release after loading onto adipocytes

[0065] Mature adipocytes were digested and counted, with 1×10⁻⁶ cells counted. 6 One cell line was added to PBS solution containing 40 μg / mL TP-SS-PA and incubated at 37°C and 5% CO2 for 60 min. The supernatant was collected, and the drug loading was calculated. The cells were divided into two groups, with eight drug-loaded adipocytes in each group. The A375 group of drug-loaded adipocytes were cultured in 6-well cell culture chambers (0.4 μm) with 1×10⁶ cells / mL PBS solution. 6 A375 cells were co-incubated in the upper chamber, while drug-loaded adipocytes were in the lower chamber. The control group was cultured in DMEM. Both groups of drug-loaded adipocytes were incubated at 37°C and 5% CO2. Supernatants were collected at 0 min, 2 min, 5 min, 10 min, 30 min, 60 min, 90 min, 120 min, and 180 min, respectively. After processing, the supernatants were analyzed by liquid chromatography, and the peak areas were used to obtain a standard curve to calculate the drug concentration and the cumulative drug release rate at each time point.

[0066] Figure 4 (A) is the standard curve of TP-SS-PA in Example 5. The linear range of the quantitative analysis method for TP-SS-PA is 2.2-352 μg / mL, and the standard curve is Y = 22.85X - 0.0092, R0. 2=1. The results show that TP-SS-PA exhibits good linearity within its linear range. The intra-day precision RSDs for low, medium, and high concentrations of TP-SS-PA were 2.13%, 0.46%, and 0.08%, respectively. The inter-day precision RSDs were 1.77%, 0.47%, and 0.09%, respectively, indicating that the method has good precision and can meet the determination requirements. The relative recoveries of TP-SS-PA at all concentrations were between 99.0% and 101.0%, with RSDs < 2%, demonstrating stable relative recoveries. The RSD of the TP-SS-PA solution within 12 hours was 0.56%, indicating that the TP-SS-PA solution is stable within 12 hours. (B) Drug loading in adipocytes after co-incubation with different concentrations of TP-SS-PA for 60 min; (C) Drug loading in adipocytes after co-incubation with TP-SS-PA at a concentration of 40 μg / mL for different durations. The results showed that the drug loading in adipocytes increased with increasing TP-SS-PA concentration during co-incubation, reaching a maximum at a concentration of 50 μg / mL. The maximum drug loading was also observed after 60-75 min of incubation. After incubation exceeding 90 min, the drug loading decreased instead of increasing. (D) Adipocyte viability (n=6) after treatment with pTP and TP respectively. The CCK8 experiment showed that when the pTP concentration was below 40 μg / mL, adipocyte viability remained at approximately 90%, with minimal impact on the cells. At a concentration of 50 μg / mL, viability decreased significantly. Therefore, the pTP concentration used for drug loading should be at least below 40 μg / mL. Therefore, the appropriate drug co-culture concentration was 40 μg / mL, the co-culture time was 60 min, and the drug loading was 13.26 ± 0.89 μg / 10. 6 cells.

[0067] Figure 4 (E) Fluorescence microscopy images of adipocytes before and after drug loading. It was observed that after drug loading, the cell morphology, state, and size of adipocytes did not change significantly, still exhibiting the classic "ring-like" structure. The morphology and distribution of lipid droplets were also unaffected by drug loading. (F) In vitro release curves of adipocytes. The results showed that adipocytes released pTP at a faster rate in the release medium, reaching a cumulative release rate of 48.72% within 30 minutes. In contrast, the cumulative release rate of adipocytes co-incubated with A375 was 56.39%. After 120 minutes, the drug release in both groups tended to plateau. The cumulative release rate of adipocytes co-incubated with A375 reached 73.19% at 180 minutes, while the cumulative release rate of the control group adipocytes was slightly lower at 69.19% (P<0.01). The possible reason is that A375 cells promote lipolysis of adipocytes, which to some extent facilitates drug release from drug-loaded adipocytes.

[0068] Example 5: Investigation into the interaction mechanism between the Apo biomimetic drug delivery system and melanoma cells

[0069] 1. Lipolytic activity of A375 cells on adipocytes

[0070] 3T3-L1 preadipocytes were divided into 1×10 6 / mL was seeded into 6-well plates, and after reaching confluence, differentiation was induced. Mature adipocytes were obtained after 12 days. Cell culture chambers (0.4μm) were placed in 6-well plates, and 1×10⁶ cells were added to the upper chamber. 6 A375 cells were co-cultured at a concentration of 1000 mL / min, with three replicates per group. At days 0, 4, 8, and 12, one well was selected for Oil Red O staining of adipocytes, and cell morphology was recorded using a fluorescence microscope.

[0071] 2. Recruiting of adipocytes by A375 cells

[0072] The cell density of A375 was adjusted to 2.5 × 10⁻⁶. 5 The culture medium was inoculated at a rate of 100 μL / mL into the upper chamber of a 24-well Transwell plate, 200 μL per well. The lower chamber was used for the control group (600 μL of complete culture medium) and the Apo group (1.8 × 10⁻⁶ μL of complete culture medium). 5 One fat cell, A375-Apo group plus 0.9×10 5 One fat cell and 0.9 × 10 5 After co-culturing A375 cells for 24 hours, the chambers were removed, and cells that had not penetrated the membrane were wiped off with cotton swabs. The cells were fixed with 4% paraformaldehyde for 15 minutes, washed twice with PBS, stained with 0.1% crystal violet for 15 minutes, washed with PBS, dried, and photographed using a fluorescence microscope. The number of cells that had penetrated the membrane was counted in 5 random fields. The number of cells that had penetrated the membrane was compared among the three groups.

[0073] 3. Adipocyte tropism towards A375 cells

[0074] The density of adipocytes was adjusted to 2.5 × 10⁻⁶. 5 The culture medium was inoculated at a rate of 1.8 × 10⁶ cells / mL into the upper chamber of a 24-well Transwell plate, 200 μL per well. The lower chamber was used for the control group, with 600 μL of complete culture medium added, and for the A375 group, 1.8 × 10⁶ cells / mL. 5 One fat cell, A375-Apo group plus 0.9×10 5 One fat cell and 0.9 × 10 5 After co-culturing A375 cells for 24 hours, the chambers were removed, and cells that had not penetrated the membrane were wiped off with cotton swabs. The cells were fixed with 4% paraformaldehyde for 15 minutes, washed twice with PBS, stained with 0.1% crystal violet for 15 minutes, washed with PBS, dried, and photographed using a fluorescence microscope. The number of cells that had penetrated the membrane was counted in 5 random fields. The number of cells that had penetrated the membrane was compared among the three groups.

[0075] Figure 5 (A) is a diagram illustrating the lipolysis process of adipocytes by A375 cells in Example 5. (B) shows the ratio of lipid droplet area to total field of view at each stage. After co-incubation of mature adipocytes with A375 cells, the number of lipid droplets gradually decreased over time, with larger droplets remaining. The possible reason is that smaller droplets were lipolyzed by A375 cells first, while larger droplets were gradually lipolyzed into smaller droplets, leaving only a few droplets by day 12. On days 0, 4, 8, and 12 of co-incubation, the ratios of adipocyte lipid droplet area to total field of view were 60.56±1.75%, 51.10±0.74%, 31.8±4.17%, and 14.4±2.77%, respectively. These results indicate that A375 cells can gradually lipolyze adipocytes. Therefore, it is speculated that if adipocytes are used to load drugs, A375 cells can help release the drug from the adipocytes, allowing it to be taken up by tumor cells and exert its therapeutic effect.

[0076] Figure 5 (C) is a diagram of A375 cell migration in Example 5. (D) is the cell count of A375 cells after co-culturing with adipocytes alone and the A375-adipocyte system for 24 hours. (E) is a diagram of adipocyte migration. (F) is the cell count of adipocytes after co-culturing with A375 cells alone and the A375-adipocyte system for 24 hours. The results showed that the number of A375 cells migrating through the membrane was relatively small under complete culture medium or co-culture conditions with adipocytes alone and A375 cells alone. However, when A375 cells were co-cultured with the A375-adipocyte system, the number of A375 cells migrating through the membrane increased significantly, showing a significant difference from the control group. The possible reason is that when A375 cells and adipocytes are present simultaneously, the secretion of a certain factor promotes the migration and transfer of A375 cells to adipocytes. Similarly, in complete culture medium or under conditions of co-culture of A375 cells and adipocytes alone, the number of adipocytes migrating across the membrane is relatively small. However, when adipocytes are co-cultured with the A375-adipocyte system, the number of adipocytes migrating across the membrane increases. This may be because when A375 cells and adipocytes are present at the same time, the secretion of a certain factor promotes the migration of adipocytes to A375 cells, which is equivalent to the recruitment effect of A375 cells on adipocytes.

[0077] Example 6: Western blot assay of marker cytokines

[0078] A375 cells were planted at a density of 1.2 × 10⁶ cells per well. 6A375 cells were seeded at a density of [number] cells per well in 6-well plates and co-cultured with 3T3-L1 cells and mature adipocytes (Apo) in Transwell chambers (0.8 μm) for 72 h. Another group was co-incubated with sodium oleate for 72 h. A375 cells from each group were then collected, and total cellular protein was extracted. Western blotting was used to detect the expression of three proteins mediating fatty acid transport, binding, and uptake on the A375 cell membrane: FATP1, FABP4, and CD36. β-actin was used as an internal control to verify the mechanism by which co-culturing A375 cells with mature adipocytes (Apo) promotes fatty acid uptake in A375 cells.

[0079] Mature adipocytes (Apo) and 3T3-L1 preadipocytes were seeded into 6-well plates and co-cultured with A375 cells in Transwell chambers (0.8 μm) for 72 h. Apo and 3T3-L1 cells were then collected, and the expression of FABP4 on the cell membranes of the two cell types was detected by Western blotting.

[0080] Figure 6 (A) Western Blot analysis of proteins related to fatty acid uptake on the A375 cell membrane after co-culturing A375 cells with 3T3 cells, sodium oleate, and Apo, respectively, in Example 6; (B) Relative quantification of the expression of related proteins in each group. We used Western Blot experiments to verify whether co-culturing A375 cells with mature adipocytes (Apo) could induce A375 cells to express more of these related proteins. The A375 cells in the control group were untreated, and the expression levels of the three proteins were all low. The protein levels of the 3T3-A375 group co-cultured with 3T3 cells did not increase significantly (FATP1, FABP4, and CD36 were 1.17 times, 2.16 times, and 1.14 times that of the control group, respectively). Sodium oleate was used to simulate the lipid environment surrounding melanoma. The expression levels of the three proteins in A375 cells treated with sodium oleate were 0.68 times, 4.03 times, and 0.61 times that of the control group, respectively. The protein expression levels of the Apo-A375 group co-cultured with mature adipocytes were significantly increased, at 2.39 times, 4.47 times, and 2.34 times that of the control group, respectively. The above results indicate that co-culturing A375 cells with mature adipocytes promotes the expression of proteins on the A375 cell membrane that mediate fatty acid transport and uptake. When mature adipocytes are loaded with TP-SS-PA targeting palmitic acid, it is more easily taken up by A375 cells into the cytoplasm, thereby exerting an anti-tumor effect.

[0081] Figure 6(C) Western blot analysis of FABP4 on the cell membranes of 3T3 cells and Apo cells after co-culturing A375 cells with 3T3 cells and Apo cells, respectively, in Example 6; (D) Relative quantification of FABP4 expression in each group. The results showed that when detecting protein expression in adipocytes by Western blot, adipocytes lacked CD36 and the FATP protein family, while the expression level of FABP4 increased significantly after co-culture, as shown in Fig. 3-5. The FABP4 expression levels of 3T3-L1 cells and mature adipocytes (Apo cells) were 1.41 times and 2.92 times that of the control group, respectively. This result demonstrates that co-culturing melanoma cells with adipocytes also promotes an increase in the expression level of FABP4 in adipocytes.

[0082] Example 7: Laser confocal imaging of pTP-Apo uptake by A375 cells

[0083] This study investigated the uptake of pTP-Apo by A375 cells. BODIPY 505 / 515 is a lipophilic bright green fluorescent dye used to detect lipid droplets in adipocytes and lipids in various structures, exhibiting good selectivity and high fluorescence efficiency. In this experiment, pTP-Apo was stained with BODIPY fluorescence and co-cultured with A375 cells in Transwell chambers. The A375 cells were observed during the final detection to examine whether lipid droplets within adipocytes could be transported to and uptake by A375 cells.

[0084] Figure 7 This is a colocalization fluorescence imaging of A375 cells and their taken-up lipid droplets (LDs) in Example 7. Scale bar: 20 μm. The laser confocal colocalization results show that a small number of lipid droplets (green) are distributed around the A375 cell nucleus (blue), proving that A375 cells can take up lipid droplets from adipocytes and convert them for their own use.

[0085] Example 8: Evaluation of the in vitro anti-melanoma effect of the pTP-Apo Tripterygium wilfordii biomimetic drug delivery system

[0086] 1. Construction of an A375 melanoma cell xenograft model in nude mice

[0087] A375 cells were cultured continuously until the desired cell number was reached, at which point the cells were harvested and the cell density was adjusted to 2 × 10⁻⁶. 7 / mL. Expose the right axilla of the nude mouse, disinfect with alcohol, and inoculate the corresponding site with 0.2mL of cell suspension from an ice bath using a 1mL syringe. Record the long diameter (a) and short diameter (b) of the nude mouse tumor, and calculate V using the formula V = (a × b) / mL. 2 ) / 2 to calculate tumor volume.

[0088] 2. In vivo distribution of the pTP-Apo biomimetic drug delivery system

[0089] When the tumor grows to 200mm 3 Nude mice were treated by subcutaneous peritumoral injection of 100 μL of DiR-labeled Apo and pTP-Apo, respectively. The fluorescence distribution and fluorescence intensity (Ex / Em = 750 / 780 nm) of DiR-Apo in the nude mice were observed by IVIS system at 0, 1, 3, 6 and 12 h after administration.

[0090] 3. Evaluation of the in vivo antitumor efficacy of the pTP-Apo biomimetic drug delivery system

[0091] When the tumor grows to 100mm 3 Subsequently, nude mice were randomly divided into 5 groups using EXCEL. On days 0, 2, 4, 6, 8, 10, and 12, they were treated with saline, Apo, TP, pTP, and pTP-Apo, respectively. The saline, TP, and pTP groups received conventional tail vein injection. Considering the close connection between adipocytes and A375 melanoma cells, all experimental groups using Apo as a carrier received a 100 μL subcutaneous injection near the tumor. Regarding the selection of the subcutaneous injection site, considering that the drug solution would penetrate downwards due to gravity for better effect, we selected two points on the upper semicircle of the tumor site when the nude mouse was crawling, administering the drug twice to maximize the drug's contact area with the tumor. The dosage for each group is shown in Table 1. Tumor volume and mouse weight were measured every two days. After treatment, the nude mice were euthanized, the tumors were removed and weighed, and the tumor growth inhibition rate (%) was calculated as [(average tumor weight of control group - average tumor weight of treatment group) / average tumor weight of control group] × 100%.

[0092] Table 1. Administration methods and dosages for each group

[0093]

[0094] 4. H&E staining, TUNEL staining and immunohistochemistry of tumor tissue

[0095] Tumor tissues from each group of nude mice were fixed with 4% paraformaldehyde for 24 hours, then dehydrated using an alcohol gradient dehydrator to prepare paraffin sections for later use. The paraffin sections were then stained with H&E, TUNEL, and DAPI to counterstain cell nuclei, and examined under a light microscope. Immunohistochemical analysis was performed on the paraffin sections of the tumor tissues.

[0096] 5. In vivo safety evaluation of the pTP-Apo biomimetic drug delivery system

[0097] Two nude mice were dissected in each group to obtain heart, liver, spleen, lung, and kidney tissues. The tissues were fixed with 4% paraformaldehyde, dehydrated in an alcohol gradient dehydrator, and prepared into paraffin sections for later use. Then, H&E staining was performed on each section.

[0098] Figure 8(A) shows the A375 cell subcutaneous xenograft model in nude mice constructed in Example 8; (B) shows the fluorescence imaging of DiR-labeled Apo in tumor-bearing nude mice at 0, 1, 3, 6, and 12 hours after subcutaneous injection near the tumor. Following injection near the tumor, DiR-labeled Apo remained distributed near the tumor tissue, diffused into the tumor tissue between 3 and 6 hours, and was absorbed and shrank by the tumor tissue after 12 hours. We hypothesize that pTP-Ce6-Apo, after entering the nude mouse, remains relatively fixed at the tumor site, thereby achieving passive targeted drug release, and the drug is taken up by the tumor tissue to kill tumor cells.

[0099] Figure 8(C) shows the solid tumors in each group in Example 8; (D) shows the tumor growth inhibition rate in each group. The anti-tumor proliferative activity was evaluated by constructing a subcutaneous xenograft model of A375 melanoma cells. In the saline and Apo groups, tumor growth was rapid due to the absence of drug action, while tumor growth in the drug-treated groups was inhibited to varying degrees. The results indicate that adipocyte injection near the tumor does not promote tumor growth; the average tumor inhibition rates in the TP group, pTP group, and pTP-Apo group were 81.91%, 66.30%, and 77.22%, respectively. The adipocyte-based Tripterygium wilfordii biomimetic drug delivery system can effectively inhibit tumor growth.

[0100] Figure 8(E) shows TUNEL fluorescence staining in Example 8; (F) shows tumor sections stained with H&E and Ki67, scale bar 50 μm. TUNEL fluorescent reagent can stain fragmented nuclear DNA in the early stages of apoptosis. Since DNA fragmentation is rare in normal proliferating cells, the stronger the green fluorescence of TUNEL, the more tumor cells undergo apoptosis. Except for the Control and Apo groups, which showed almost no green fluorescence, the other groups all exhibited varying degrees of tumor cell apoptosis. In the Ki67 tumor sections, the Control and Apo groups had the most brown granules, indicating active tumor cell proliferation, while the pTP-Apo group had fewer brown granules, indicating that it significantly inhibited tumor cell proliferation.

[0101] Figure 8(G) shows the H&E staining of major organs in each group of nude mice in Example 8, scale bar 50 μm. The results showed that the liver tissue of the TP group exhibited sinusoidal dilation and congestion, and disordered hepatocyte arrangement. The kidney tissue of the TP group showed cortical and medullary interstitial congestion, indicating that TP has certain toxicity when administered via tail vein alone. The tissue sections of other groups showed no obvious abnormalities compared with the Control group, indicating that modifying TP helps to reduce the damage of TP to important organs and makes it more biosafetyable.

[0102] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A biomimetic drug delivery system based on the fat uptake pathway of Tripterygium wilfordii, characterized in that, The aforementioned Tripterygium wilfordii biomimetic drug delivery system uses mature adipocytes as drug carriers to encapsulate the triptolide derivative TP-SS-PA; the triptolide derivative TP-SS-PA is coupled to triptolide and palmitic acid using disulfide bonds as connecting arms.

2. The Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway according to claim 1, characterized in that, The aforementioned triptolide derivative TP-SS-PA has the chemical structural formula shown in Formula I:

3. The Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway according to claim 1, characterized in that, The aforementioned Tripterygium wilfordii biomimetic drug delivery system was prepared by co-incubating mature adipocytes with the Tripterygium wilfordii derivative TP-SS-PA.

4. The Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway according to claim 1, characterized in that, The mature adipocytes were obtained by inducing differentiation from 3T3-L1 preadipocytes.

5. The Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway according to claim 1, characterized in that, The drug loading capacity of the Tripterygium wilfordii biomimetic drug delivery system is 13.26±0.89μg / 10. 6 cell.

6. A method for preparing a Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway as described in any one of claims 1-5, characterized in that, The process includes the following steps: synthesizing the triptolide derivative TP-SS-PA, inducing 3T3-L1 preadipocytes to differentiate into mature adipocytes Apo, and then co-incubating the triptolide derivative TP-SS-PA with the mature adipocytes to obtain the aforementioned triptolide biomimetic drug delivery system.

7. The preparation method of the Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway according to claim 6, characterized in that, The method for co-incubating mature adipocytes with TP-SS-PA is as follows: 1×10 6 Mature adipocytes were added to 2 mL of PBS solution containing 40 μg / mL TP-SS-PA and incubated at 37°C and 5% CO2 for 1 h to construct the Tripterygium wilfordii biomimetic drug delivery system.

8. The preparation method of the Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway according to claim 6, characterized in that, The preparation method of the triptolide derivative TP-SS-PA is as follows: using palmitic acid as raw material, a disulfide bond is introduced by reacting 3,3-dithiodipropionic acid with acetic anhydride, and using 4-dimethylaminopyridine as catalyst, a palmitic acid derivative is synthesized, which is coupled with triptolide through a disulfide bond, and the triptolide derivative TP-SS-PA is prepared by one-step esterification.

9. The preparation method of the Tripterygium wilfordii biomimetic drug delivery system based on the fat uptake pathway according to claim 6, characterized in that, The method for preparing mature adipocytes is as follows: 3T3-L1 preadipocytes are seeded in 6-well plates. When the cells grow to the confluence state, 2.5 mL of induction medium I is added to the cell culture medium and cultured for 48 h. The induction medium II is replaced and cultured for another 48 h. Then, the cells are cultured continuously in complete culture medium for 11 days to obtain mature adipocytes. The induction medium I is a complete culture medium containing IBMX, dexamethasone, and insulin. The induction medium II is a complete culture medium containing insulin.

10. The use of the Tripterygium wilfordii biomimetic drug delivery system as described in any one of claims 1-5 in the preparation of a therapeutic drug for malignant melanoma.

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