A paclitaxel precursor nano-drug, and a preparation method and application thereof

Abstract

The application provides a paclitaxel precursor nanomedicine and a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The application comprises a paclitaxel dimer prodrug and 7-ethyl 10-hydroxy-camptothecin loaded by the paclitaxel dimer prodrug. The application takes the paclitaxel dimer prodrug as a self-assembly monomer to construct a synergistic treatment nanosystem of PTX-SN38, and has the advantages of water dispersibility, biocompatibility, tumor-specific response type and synergistic treatment, so that the limitations of paclitaxel and SN38 in antitumor treatment can be effectively solved.

Description

Technical Field

[0001] This application relates to a paclitaxel precursor nanomedicine, its preparation method, and its application, belonging to the field of medicinal chemistry technology. Background Technology

[0002] Paclitaxel (PTX) is a widely used natural chemotherapy drug for tumors, exhibiting high antitumor activity and low toxicity. However, its poor water solubility and biostability make it difficult to accumulate in tumor areas, significantly reducing its anticancer efficacy. The microenvironment of high ROS and GSH concentrations within tumor cells can serve as an ideal stimulus-response condition for prodrug design. Disulfide bonds can respond simultaneously to ROS and GSH, converting into sulfones or thiol groups, which then trigger the hydrolysis of the linker, releasing PTX. However, monotherapy with chemotherapy drugs easily leads to drug resistance in tumor cells, affecting treatment efficacy.

[0003] Camptothecin (CPT) possesses antitumor activity. The antitumor mechanism of camptothecin and its derivatives differs from that of paclitaxel. As a topoisomerase I (TOPO I) inhibitor, it binds to TOPO I to form a CPT-TOPO I-DNA ternary complex, inhibiting DNA synthesis and ultimately leading to cancer cell death. 7-Ethyl-10-hydroxycamptothecin (SN38) is the main active metabolite of the clinical anticancer drug irinotecan (CPT-11) and has been formally shown to be effective against various cancers such as rectal cancer, small cell lung cancer, and lymphoma. Its bioactivity is 100-1000 times that of CPT-11. However, SN38's hydrophobicity (almost insoluble in water and most medical oil solvents) and instability at physiological pH limit its clinical translation. Summary of the Invention

[0004] In view of this, this application provides a paclitaxel precursor nanomedicine that synergistically achieves therapeutic effects with the loading, which not only solves the problems of poor water solubility and difficulty in tumor delivery of the chemotherapy drugs PTX and SN38, but also has tumor targeting and achieves synergistic anti-tumor effects of the two drugs with different mechanisms of action.

[0005] Specifically, this application is implemented through the following scheme:

[0006] A paclitaxel precursor nanomedicine comprising a paclitaxel dimer prodrug and 7-ethyl-10-hydroxy-camptothecin (SN38) loaded thereon.

[0007] Furthermore, as a preferred option:

[0008] The paclitaxel dimer prodrug is PTX2-SS or PTX2-C8.

[0009] The mass ratio of the paclitaxel dimer prodrug to 7-ethyl-10-hydroxy-camptothecin is 100 to 4:1. More preferably, the mass ratio of the paclitaxel dimer prodrug to 7-ethyl-10-hydroxy-camptothecin is 100 to 10:1.

[0010] The nanomedicine provided by the above scheme uses paclitaxel dimer prodrug as a self-assembling monomer to construct a synergistic therapeutic nanosystem of PTX-SN38. It also has the advantages of water dispersibility, biocompatibility, tumor-specific response, and synergistic therapy, which can effectively solve the limitations of paclitaxel and SN38 in anti-tumor therapy.

[0011] The above-mentioned method for preparing paclitaxel precursor nanomedicines involves co-assembling paclitaxel dimer precursors with bovine serum albumin and directly doping them with 7-ethyl-10-hydroxy-camptothecin to obtain nanomedicines loaded with 7-ethyl-10-hydroxy-camptothecin.

[0012] Specifically, the above preparation process is described as follows: paclitaxel dimer prodrug is dissolved in tetrahydrofuran, 7-ethyl-10-hydroxy-camptothecin solution is added, and after mixing evenly, it is slowly added dropwise to bovine serum albumin aqueous solution, stirred continuously, and dialyzed to obtain nanomedicine loaded with 7-ethyl-10-hydroxy-camptothecin.

[0013] The method for preparing the paclitaxel dimer prodrug is as follows: Paclitaxel is dissolved in dichloromethane, and a linker, EDC·HCl and DMAP are added sequentially. After stirring and reacting, EDC·HCl and DMAP are added again, and the reaction is continued until complete. The dichloromethane is removed by rotary evaporation, the resulting solid is dissolved again, purified by column chromatography, dissolved again by rotary evaporation, and precipitated in ice water to obtain the paclitaxel dimer prodrug.

[0014] When the linker is 3,3-dithiodipropionic acid, the stirring reaction temperature is room temperature (20-30°C), and the resulting paclitaxel dimer prodrug is denoted as PTX2-SS.

[0015] When the linker is octanoic acid, the stirring reaction temperature is 30-40°C, and the resulting paclitaxel dimer prodrug is denoted as PTX2-C8.

[0016] The above preparation process is based on a strategy of co-assembling antitumor prodrugs with synergistic chemotherapeutic drugs, achieving synergistic delivery and treatment of the chemotherapeutic drug paclitaxel (PTX) and 7-ethyl-10-hydroxycamptothecin (SN38). First, paclitaxel dimer prodrugs (especially PTX2-SS) are synthesized using disulfide bond linkers (such as 3,3-dithiodipropionic acid) that exhibit dual responsiveness to high concentrations of glutathione (GSH) and reactive oxygen species (ROS) in the tumor microenvironment. This prodrug can self-assemble into nanoparticles (NPs) in the aqueous phase. Based on this, SN38 is directly incorporated during the co-assembly of PTX2-SS with bovine serum albumin (BSA), rapidly constructing nanomedicines co-loaded with PTX and SN38.

[0017] The above-mentioned paclitaxel precursor nanomedicines are used in the preparation of antitumor agents. This not only solves the problems of poor water solubility and difficulty in tumor delivery associated with the chemotherapy drugs PTX and SN38, but also demonstrates tumor targeting and achieves a synergistic antitumor effect through the different mechanisms of action of the two drugs. Attached Figure Description

[0018] Figure 1 The synthesis process of PTX2-SS.

[0019] Figure 2 For PTX2-SS mass spectrometry analysis.

[0020] Figure 3 PTX2-C8 is characterized, and part (a) in the figure is... 1 The H NMR spectrum, part (b) is the mass spectrum.

[0021] Figure 4 A schematic diagram of the co-assembly and intracellular degradation of PTX2-SS and SN38.

[0022] Figure 5 The figure shows a comparison of the particle sizes of PTX2-SS NPs and SN38@PTX2-SS NPs. Part (a) represents PTX2-SS NPs, part (b) represents PTX2-SS:SN38 = 100:1, part (c) represents PTX2-SS:SN38 = 50:1, and part (d) represents PTX2-SS:SN38 = 20:1.

[0023] Figure 6 The particle size comparison of small-ratio SN38@PTX2-SS NPs is shown in the figure. Part (a) is PTX2-SS:SN38=10:1, part (b) is PTX2-SS:SN38=5:1, and part (c) is PTX2-SS:SN38=4:1.

[0024] Figure 7To show the stability of different nanoparticles at room temperature, part (a) of the figure shows the DLS radius of the nanoparticles after standing at room temperature for 8 days, and part (b) shows the ratio of the second DLS analysis result to the first.

[0025] Figure 8 The figure shows the encapsulation efficiency of SN38 in nanoparticles with different proportions. Part (a) of the figure represents the concentration of SN38 in SN38@PTX2-SS NPs, and part (b) represents the encapsulation efficiency of SN38.

[0026] Figure 9 To analyze the anti-tumor cell proliferation ability of nanoparticles with different ratios using the MTT assay, part (a) of the figure shows the cell survival rate after 24 hours of B16F10 incubation, part (b) shows the cell survival rate after 24 hours of HeLa cell incubation, part (c) shows the cell survival rate after 48 hours of B16F10 incubation, and part (d) shows the cell survival rate after 48 hours of HeLa cell incubation.

[0027] Figure 10 To analyze the anti-tumor cell proliferation ability of different PTX dimer nanoparticles after 24 h using the MTT assay, part (a) of the figure represents B16F10 cells and part (b) represents HeLa cells.

[0028] Figure 11 Live and dead staining analysis of B16F10 cells after co-incubation of 10 μmol / L nanoparticles (based on PTX) for 12 h.

[0029] Figure 12 Fluorescence imaging of B16F10 cells after co-incubation with 10:1 SN38@PTX2-SS NPs, 5:1 SN38@PTX2-SS NPs, and 10:1 SN38@PTX2-C8 NPs, respectively. Part (a) of the figure shows the fluorescence imaging after 3 h of co-incubation, and part (b) shows the fluorescence imaging after 6 h of co-incubation.

[0030] Figure 13 For in vivo anti-tumor treatment in mice, part (a) of the figure shows the tumor volume change over 10 days after administration, part (b) shows the body weight change curve over 10 days after administration, part (c) shows the tumors removed after euthanasia of mice in different experimental groups after treatment, and part (d) shows the HE staining of tumor tissues from different experimental groups after treatment. Detailed Implementation

[0031] Example 1

[0032] This embodiment describes the synthesis and purification of paclitaxel dimer PTX2-SS, as follows:

[0033] First, accurately weigh 200.0 mg (0.23 mmol) of PTX and dissolve it in 5 mL of dichloromethane. After complete dissolution by stirring, add 27.3 mg (0.13 mmol) of 3,3-dithiodipropionic acid, 90.0 mg (0.47 mmol) of EDC·HCl, and 2.9 mg (0.024 mmol) of DMAP sequentially, and stir at room temperature (approximately 20 °C) for 1 hour. Then, add 45.0 mg (0.23 mmol) of EDC·HCl and 2.9 mg (0.024 mmol) of DMAP again, and continue stirring for 24 hours. Monitor the reaction progress by HPLC. After the reaction is complete, remove the dichloromethane by rotary evaporation, and completely dissolve the resulting solid in a small amount of DMSO. Purify the product by silica gel column chromatography using ethyl acetate:n-hexane as the eluent in a ratio of 6:4 (v / v). After removing all eluent by rotary evaporation, dissolve the product again in a small amount of DMSO. The DMSO solution of the product was slowly added dropwise to ice water to allow the product to precipitate gradually. The product was collected by centrifugation and dried in an oven at 37°C. The purity of the obtained product was analyzed by HPLC, and NMR and mass spectrometry were performed.

[0034] The reaction of paclitaxel dimer PTX2-SS is as follows Figure 1 As shown, PTX2-SS was generated in a one-pot reaction at room temperature (RT) based on PTX and 3,3-dithiodipropionic acid under the catalysis of EDC·HCl and DMAP. After the reaction was completed, the product PTX2-SS was obtained by rotary evaporation-column chromatography-rotary evaporation-precipitation-centrifugation. The product PTX2-SS was then obtained. 1 The characteristic peak at 3.65 ppm attributed to 2'-OH was not observed in the ¹H NMR spectrum (deuterated reagent CDCl₃). Furthermore, due to the formation of an ester bond, the characteristic peak of 2'-CH at 4.83 ppm shifted to a lower field, to 5.57 ppm. Two methylene-CH₂- characteristic peaks of 3,3-dithiodipropionic acid appeared in the product, and the carboxyl-COOH characteristic peak of 3,3-dithiodipropionic acid disappeared. This indicates that PTX and 3,3-dithiodipropionic acid underwent a combination reaction, and the reaction site was 2'-OH.

[0035] PTX2-SS 13 The characteristic absorption peaks of the two methylene-CH2- groups of 3,3-dithiodipropionic acid can also be clearly observed in the C10 NMR spectrum. Furthermore, from... Figure 2 [PTX2-SS + Na] can be observed in the PTX2-SS mass spectrum. + (m / z: 1904.7) and 1 / 2 [PTX2-SS + 2Na] 2+The characteristic peak (m / 2z: 964.2) is consistent with the molecular weight of PTX2-SS (1882.07), further proving the successful synthesis of PTX2-SS.

[0036] Example 2

[0037] This embodiment describes the synthesis and purification of paclitaxel dimer PTX2-C8, as follows:

[0038] First, accurately weigh 200.0 mg (0.23 mmol) of PTX and dissolve it in 5 mL of dichloromethane. After complete dissolution by stirring, add 22.6 mg (0.13 mmol) of octanoic acid, 90.0 mg (0.47 mmol) of EDC·HCl, and 2.9 mg (0.024 mmol) of DMAP sequentially, and stir at approximately 30°C for 1 hour. Then, add 45.0 mg (0.23 mmol) of EDC·HCl and 2.9 mg (0.024 mmol) of DMAP again, and continue stirring for 30 hours. Monitor the reaction progress by HPLC. After the reaction is complete, remove the dichloromethane by rotary evaporation, and completely dissolve the resulting solid in a small amount of DMSO. Purify the product by silica gel column chromatography using ethyl acetate:n-hexane as the eluent in a ratio of 6:4 (v / v). After removing all eluent by rotary evaporation, dissolve the product again in a small amount of DMSO. The DMSO solution of the product was slowly added dropwise to ice water to allow the product to precipitate gradually. The product was collected by centrifugation and dried in an oven at 37°C. The purity of the obtained product was analyzed by HPLC, and NMR and mass spectrometry were performed.

[0039] PTX2-C8 1 H NMR spectrum (see) Figure 3 Part (a) of the PTX2-SS mass spectrum is very similar to that of PTX2-SS. The disappearance of the 2'-OH side chain of paclitaxel in the 1H NMR spectrum of PTX2-C8, and the change in the 2'-CH position due to ester bond formation, are also observed. Unlike PTX2-SS, the carbon chain of octanoic acid contains three methylene-CH2- atoms in different chemical environments. Due to the absence of electron-withdrawing S atoms, the hydrogen atom absorption signals of methylene groups b and c should be at a higher field (b: 1.55 ppm, c: 1.26 ppm), while the hydrogen atom of methylene group a is at a lower field (2.40 ppm) due to electron-withdrawing by the carbonyl group. The PTX2-C8 mass spectrum (see...) Figure 3 Part (b) in the diagram also shows M / z: [PTX2-C8+Cl] - Signal peaks, mass spectrometry results and 1 The combined H NMR also indicates the successful synthesis of PTX2-C8.

[0040] Example 3

[0041] This embodiment describes the preparation of paclitaxel precursor nanomedicine, and the process is as follows:

[0042] Nanoparticles were prepared by co-assembling PTX2-SS with SN38 in BSA solution using the method described in Example 1. 2.0 mg of PTX2-SS was dissolved in 2 mL of tetrahydrofuran, and different volumes of SN38DMSO solution (2 mg / mL) were added. The mixture was then thoroughly stirred using a pipette. The solution was then slowly added dropwise to 10 mL of BSA aqueous solution (1 mg / mL) with thorough stirring. After the addition was complete, stirring was continued for 2 hours. The nanoparticle suspension was then placed in a dialysis bag with a molecular weight cutoff of 1000, the organic solvent was removed, and the water was changed every 4 hours. Dialysis was performed 3-4 times, and the solution was stored at room temperature. Nanoparticles of PTX2-SS and SN38 with mass ratios of 100:1, 50:1, 20:1, 10:1, 5:1, and 4:1 were prepared, and the resulting nanoparticles were denoted as X:1 SN38@PTX2-SS NPs (X=100, 50, 20, 10, 5, 4).

[0043] Combination Figure 4 In this embodiment, different proportions of SN38 solution were added during the self-assembly of PTX dimers to co-assemble them into nanoparticles. The addition of BSA improves the water solubility of the hydrophobic drugs PTX and SN38, while also enhancing the stability of the nanoparticles and controlling drug release.

[0044] Figure 5 , Figure 6 TEM images and DLS analysis of nanoparticle suspensions prepared by co-assembly with different proportions of SN38: The opacity of the nanoparticle suspension gradually increased with increasing SN38 proportion. When the mass ratio of SN38 to nanoparticles increased to 5:1, the opacity significantly improved, possibly due to the substantial increase in nanoparticle size. The morphology and size of nanoparticles with different proportions were characterized using TEM and a nanoparticle size analyzer. TEM images show that all nanoparticles exhibited regular spherical shapes, but the monodispersity of nanoparticle size varied considerably with different SN38 proportions, possibly due to variations in the amount of SN38 coated within individual nanoparticles, and this did not show a linear relationship with the SN38 proportion. It is also evident that when the mass ratio reached 5:1, the nanoparticle diameter rapidly increased to approximately 150 nm, leading to a significant increase in the opacity of the nanoparticle suspension, but the overall nanoparticles still maintained a uniform and regular spherical shape.

[0045] Compared to simple paclitaxel dimer self-assembled nanoparticles (see...) Figure 5In part (a) of the study, even for co-assembled nanoparticles with the lowest SN38 specific gravity, the hydrodynamic diameter surged from 139 nm to 186 nm, and the polydispersity index (PDI) increased from 0.18 to 0.29. Specifically, the hydrated particle size and PDI values ​​of the 100:1, 50:1, and 20:1 SN38@PTX2-SS NPs nanoparticles increased (see [reference needed]). Figure 5 (see parts (b), (c), and (d) in the text), but the hydrated particle size of the 10:1 nanoparticles decreased and the PDI value dropped significantly to 0.20 (see...). Figure 6 (see section (a)). Furthermore, the hydrated particle size and PDI of nanoparticles with a larger SN38 specific gravity increased significantly again (see section (a)). Figure 6 (parts (b) and (c) in the text).

[0046] Using a nanoparticle size analyzer, we investigated the stability of different nanoparticles at room temperature. The results are as follows: Figure 7 As shown, after standing at room temperature for one week, the size of SN38 nanoparticles with specific gravities ranging from 0 to 10:1 did not change significantly, but the hydrodynamic diameters of both types of nanoparticles with higher specific gravities increased to over 300 nm. This indicates that high specific gravities of SN38 affect the stability of co-assembled nanoparticles.

[0047] The properties of the nanoparticles and paclitaxel dimers prepared in the above examples were further analyzed.

[0048] (1) Analysis of SN38 concentration and SN38 encapsulation efficiency in paclitaxel precursor nanomedicine

[0049] The concentration of SN38 in the paclitaxel nanoparticle prodrug was determined by HPLC. 500 μL of the nanoparticle suspension was centrifuged at 15000 rpm for 20 min to completely precipitate the nanoparticles. After removing the supernatant, 100 μL of DMSO was added to dissolve PTX2-SS and SN38, and the mixture was filtered through an organic filter membrane. The composition was then analyzed by HPLC. Next, 1.0 mg of SN38 was accurately weighed and completely dissolved in 1 mL of DMSO. Analysis was performed under the same HPLC conditions to determine the location of SN38 and the concentration of SN38 using the external standard single-point method. The encapsulation efficiency of SN38 was then calculated. The concentration C of SN38 was calculated using the following formula. SN38 and encapsulation rate ee%.

[0050] .

[0051] Where A 样 The peak area of ​​the sample in HPLC, A 标 C refers to the peak area of ​​the standard reference standard in HPLC. 标This refers to the concentration of the standard reference substance.

[0052] The results are as follows Figure 8 As shown: with the increase of the SN38 addition ratio, the concentration of SN38 in the nanoparticles also gradually increases (see...). Figure 8 (See part (a)). However, at lower SN38 ratios (PTX2-SS:SN38 = 100:1 or 50:1), the concentration and encapsulation efficiency of SN38 in the nanoparticles are low, with encapsulation efficiencies of only 42% and 44%, respectively; while when the ratio reaches 20:1, the encapsulation efficiency increases significantly to 71%; at a ratio of 10:1, the encapsulation efficiency reaches a maximum of 78%, and then decreases rapidly (see part (a)). Figure 8 (See part (b)). This is because a certain amount of organic solvent was used during the co-assembly process, causing some SN38 to dissolve in the THF-water mixture, which has a significant impact on the self-assembly system with a low initial concentration of SN38. In addition, BSA, which did not participate in the self-assembly to form nanoparticles, can also provide a hydrophobic structure, promoting the dissolution of free SN38.

[0053] (2) In vitro cytotoxicity

[0054] PTX and SN38 have different antitumor mechanisms. To evaluate the inhibitory effect of different nanoparticles on cell proliferation, the MTT assay was used to analyze the anti-cancer cell proliferation ability of paclitaxel nanoparticles containing different concentrations of SN38. Mouse melanoma B16F10 cells and cervical cancer cells HeLa cells were selected as cell models and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and DMEM medium, respectively. The cell culture incubator temperature was kept constant at 37 °C and the carbon dioxide concentration was 5%. The medium was changed regularly, and the cells were passaged 1:3 when the cell density reached 80%. In this process, the applicant compared the cytotoxicity of five nanoparticles—PTX2-SS NPs, 50: 1SN38@PTX2-SS NPs, 20: 1SN38@PTX2-SS NPs, 10: 1SN38@PTX2-SS NPs, and 5: 1SN38@PTX2-SS NPs—at different concentrations (0.01, 0.1, 1, 10, and 50 μmol / L, based on PTX).

[0055] First, we compared cell survival after incubation with the two cell types for 24 hours, such as... Figure 9As shown in sections (a) and (b), compared to PTX2-SS NPs, at lower concentrations (0.01–1 μmol / L), the three nanoparticles 50:1 SN38@PTX2-SS NPs, 20:1 SN38@PTX2-SS NPs, and 5:1 SN38@PTX2-SS NPs did not exhibit significant inhibitory activity against B16F10 cell proliferation. This is partly due to their larger size hindering cell entry, and partly because the in vitro cell culture system was conducted under physiological conditions, where the antitumor activity of SN38 is considerably affected. In experiments targeting the inhibition of HeLa cell proliferation, all nanoparticles except 50:1 SN38@PTX2-SS NPs showed higher cytotoxicity than PTX2-SS NPs, which is related to the characteristics of tumor cells. At high drug concentrations, PTX2-SS NPs and 50:1 SN38@PTX2-SS NPs, 20:1 SN38@PTX2-SS NPs, and 5:1 SN38@PTX2-SS NPs exhibited considerable inhibitory ability on cell proliferation. Among them, 10:1 SN38@PTX2-SS NPs showed the highest cytotoxicity in both cell lines, regardless of high or low drug concentrations, inhibiting tumor cell growth.

[0056] Subsequently, we extended the incubation time to 48 h to allow for a more complete release of PTX and SN38 after the nanoparticles entered the cells. The results were as follows... Figure 9 As shown in sections (c) and (d), after co-incubation with cells for 48 h, the cell proliferation inhibition abilities of 20:1 SN38@PTX2-SS NPs, 10:1 SN38@PTX2-SS NPs, and 5:1 SN38@PTX2-SS NPs were all superior to PTX2-SS NPs. When the PTX concentration was 50 μmol / L, the cell proliferation inhibition rate reached over 90%. The 5:1 SN38@PTX2-SS NPs even showed a better cell proliferation inhibition effect than the 10:1 SN38@PTX2-SS NPs. By comparing the MTT assay results at 24 h and 48 h, we can also see that the cytotoxicity after 48 h incubation was generally stronger than that after 24 h. This demonstrates the sustained-release capability of the PTX nanoprodrug for the active drug, which will be more beneficial in improving therapeutic efficacy.

[0057] To demonstrate the response of disulfide bonds to GSH and ROS species, leading to the release of active PTX and SN38, in Example 2, we synthesized PTX2-C8 using octanoic acid of the same carbon chain length as a linker. Following the same procedure as with PTX2-SS, we prepared the corresponding nanomedicines PTX2-C8 NPs and 10:1 SN38@PTX2-C8 NPs. Subsequently, we performed MTT assays using B16F10 and HeLa cells, respectively, and compared the results with those of PTX2-SS NPs and 10:1 SN38@PTX2-SS NPs to analyze their ability to inhibit tumor cell proliferation. The results are as follows: Figure 10 As shown, regardless of the cell line, at low drug concentrations, 10:1 SN38@PTX2-C8 NPs did not exhibit significantly superior cytotoxicity compared to PTX2-CC NPS. However, at high drug concentrations, the latter showed stronger cytotoxicity than PTX2-C8 NPs. Furthermore, the cell proliferation inhibition capacity of both PTX2-C8 NPs and 10:1 SN38@PTX2-C8 NPs was significantly lower than that of PTX2-SS NPs and 10:1 SN38@PTX2-SS NPs, especially at low drug concentrations where the difference in cytotoxicity was substantial.

[0058] Since PTX prodrugs linked to octanoic acid can only be hydrolyzed by intracellular carboxylesterases, the hydrolysis rate is relatively slow, which is detrimental to the release of PTX and SN38. Introducing disulfide bonds into the carbon chain not only benefits the PTX dimer's free rotation due to the longer bond length and more suitable bond angle, but also facilitates self-assembly in aqueous solution, promoting nanoparticle stability. Furthermore, the disulfide bonds respond to the highly expressed GSH and ROS species in tumor cells, cleaving under the influence of GSH to generate thiol groups and under the influence of ROS to generate sulfones or sulfoxides, thereby promoting PTX release. Therefore, compared to PTX2-C8 NPs and 10:1 SN38@PTX2-C8 NPs, PTX2-SS NPs and 10:1 SN38@PTX2-SS NPs exhibit superior cell proliferation inhibition capabilities.

[0059] Cell viability staining analysis is a commonly used method in biology for identifying cell viability. This application employs a live / dead cell dual staining kit (Calcein-AM / PI) for viability / dead cell staining analysis. Calcein-AM can easily cross the membrane of live cells, forming the membrane-impermeable polar molecule Calcein through enzymatic cleavage, which remains inside the cell and emits strong green fluorescence. Pyridine iodide (PI), on the other hand, can only cross the membrane of dead cells, reacting with double-stranded DNA and emitting red fluorescence. The combined use of Calcein-AM and PI allows for simultaneous fluorescence dual staining of both live and dead cells. We performed viability / dead cell staining analysis on six nanoparticles: PTX2-C8 NPs, 10:1 SN38@PTX2-C8 NPs, PTX2-SS NPs, and 20:1, 10:1, and 5:1 SN38@PTX2-SSNPs. The PTX concentration was 10 μmol / L, and the nanoparticles were incubated for 12 hours before staining. The staining was observed using a biofluorescence microscope, and the results are as follows: Figure 11 As shown in the figure. After co-incubation for 12 h, varying degrees of red fluorescence were observed in all experimental wells. The 5:1 SN38@PTX2-SS NPs and 10:1 SN38@PTX2-SS NPs nanoparticles showed the strongest red fluorescence signal, which is consistent with the MTT results. After incubation with 20:1 SN38@PTX2-SS NPs and PTX2-SS NPs, comparable red fluorescence signals were observed, but the green fluorescence intensity of live cells in the PTX2-SS NPs group was lower, and the cells were spherical. In contrast, the green fluorescence in the 20:1 SN38@PTX2-SS NPs group was brighter, and the cells were tightly adhered to the wells. This indicates that PTX2-SS NPs are more cytotoxic than 20:1 SN38@PTX2-SS NPs. The PTX2-C8 NPs and 10:1 SN38@PTX2-C8 NPs groups showed less red fluorescence signal and a large number of highly active tumor cells were observed. The results of the live / dead staining experiment were generally consistent with the MTT assay results. Compared with the control, the number of live cells was significantly reduced after co-incubation with different nanoparticles, which confirms that the nanomedicine of this application has a significant inhibitory effect on tumor cells.

[0060] (3) Cellular uptake behavior analysis

[0061] Cellular uptake behavior experiments were conducted on the co-assembled nanoparticles, selecting 10:1 SN38@PTX2-SS NPs, 5:1 SN38@PTX2-SS NPs, and 10:1 SN38@PTX2-C8 NPs as the research subjects. Since SN38 exhibits significant blue light under UV excitation, no other fluorescent substances were introduced to affect the stability and size of the nanoparticles. Due to the adhesive effect of PDA, HSA-PDA@PTX2-SS NPs were facilitated to enter cells, while PTX2-SS NPs were relatively slower; therefore, the incubation time was appropriately extended to 3 or 6 hours. Figure 12 As shown, after co-incubating nanoparticles with cells for 3 hours, obvious blue fluorescence was observed in the cytoplasm, indicating that after entering the cells via endocytosis, the nanoparticles mainly reside in the daughter cytoplasm. Due to structural similarity, the fluorescence intensity displayed in cells after incubation of 10:1 SN38@PTX2-SS NPs and 10:1 SN38@PTX2-C8 NPs for 3 hours was similar, indicating that their entry rates into cells were comparable. Compared to the two 10:1 nanoparticles, although 5:1 SN38@PTX2-SS NPs had a larger particle size, its higher SN38 content (…) Figure 12 The control group consisted of cells without nanoparticles, thus exhibiting a higher fluorescence intensity than the 10:1 SN38@PTX2-SS NPs. When the co-incubation time was extended to 6 h, the fluorescence intensity in the cytoplasm significantly increased, indicating that the uptake of nanoparticles by cells is time-dependent.

[0062] Through the characterization and analysis of the nanoparticles and the SN38 loading capacity analysis described above, it is evident that the nanoparticles formed when PTX2-SS:SN38 = 10:1 not only possess suitable nanoscale size and polydispersity but also exhibit the highest SN38 encapsulation efficiency, demonstrating the best tumor cell proliferation inhibition effect in in vitro cell experiments. Therefore, we further explored in vivo experiments to verify its antitumor effect.

[0063] (4) In vivo anti-tumor research and histological analysis

[0064] The same PBS group and three other experimental groups were set up, and the changes in tumor volume and health status of mice in each group were recorded over 10 days. Due to the excessively rapid growth of tumors in the PBS group mice, some mice died on the eighth day of treatment. In accordance with the principles of experimental ethics, it was necessary to sacrifice the mice and collect tumor tissue on the tenth day.

[0065] like Figure 13 As shown, during the experiment, the tumors in the PBS group mice grew rapidly, and by day 10, the average tumor volume had exceeded 1400 mm.3 In the remaining nanomedicine treatment groups, the growth rate of subcutaneous tumors in mice was significantly inhibited, while the PTX2-SS NPs group did not show a significantly better effect than PTX2-C8 NPs. Although both PTX2-SS NPs and PTX2-C8 NPs showed significant therapeutic effects, overall, the mouse tumors continued to grow. In the 10:1SN38@PTX2-SS NPs group, the volume of subcutaneous tumors in mice showed a decreasing trend in the early stages of treatment. During the 10-day treatment, the mouse tumors grew by less than 100 mm. 3 The experimental results demonstrated the advantages of synergistic treatment with SN38 and PTX, effectively inhibiting tumor growth. Throughout the treatment process, the mice in each experimental group maintained relatively good health.

[0066] Hematologic and Escherichia coli (H&E) staining analysis is a commonly used analytical method in medical diagnosis. Therefore, after treatment, mice were euthanized and the tumors were removed for H&E staining analysis. Figure 13 As shown in section (d) of the diagram: In the PBS control group, melanoma grew rapidly, and a large number of tumor cells were visible in the stained sections. The nucleoli were clearly visible and densely distributed, indicating vigorous tumor cell proliferation. PBS had almost no effect on the growth of tumor cells and tissues. In contrast, the tumor tissue sections of the nanomedicine experimental group showed a significant decrease in the density of cell nuclei and partial nucleolysis, indicating that tumor cell proliferation was inhibited by the nanomedicine.

[0067] In summary, PTX2-SS and SN38 were co-assembled into nanoparticles in BSA protein solution, combining two chemotherapeutic drugs with antitumor activity to exert a synergistic antitumor effect. Characterization of the nanoparticles revealed that different proportions of SN38 affected the size and stability of the co-assembled nanoparticles. HPLC analysis of the SN38 encapsulation efficiency determined that a PTX2-SS to SN38 mass ratio of 10:1 resulted in 10:1 SN38@PTX2-SS NPs with suitable particle size, stability, and optimal encapsulation efficiency. Subsequent in vitro cell experiments and in vivo antitumor experiments in mice also demonstrated that 10:1 SN38@PTX2-SS NPs exhibited the best antitumor activity. Furthermore, we used octanoic acid of the same carbon chain length to replace 3,3'-dithiodipropionic acid to synthesize PTX dimers and conducted comparative studies. The paclitaxel dimer with dual-responsive disulfide bonds showed superior antitumor activity as a prodrug. The method of this invention is simple and easy to obtain the target nanoparticles.

Claims

1. A paclitaxel precursor nanomedicine, characterized in that: It includes a paclitaxel dimer prodrug and a 7-ethyl-10-hydroxy-camptothecin loaded thereon, wherein the paclitaxel dimer prodrug is PTX2-SS or PTX2-C8; The preparation method of paclitaxel precursor nanomedicine is as follows: Step 1: Dissolve paclitaxel in dichloromethane, add linker, EDC·HCl and DMAP in sequence, stir and react, add EDC·HCl and DMAP again, continue stirring and reacting until complete, remove dichloromethane by rotary evaporation, dissolve the obtained solid again, purify by column chromatography, rotary evaporation, dissolve again, precipitate in ice water to obtain paclitaxel dimer prodrug; The linker is 3,3-dithiodipropionic acid, and the stirring reaction is at room temperature. The resulting paclitaxel dimer prodrug is denoted as PTX2-SS. The linker is octanoic acid, and the reaction temperature is 30–40°C. The resulting paclitaxel dimer prodrug is designated PTX2-C8. Step 2: Paclitaxel dimer prodrug is co-assembled with bovine serum albumin and directly doped with 7-ethyl-10-hydroxy-camptothecin to obtain nanomedicine loaded with 7-ethyl-10-hydroxy-camptothecin.

2. The paclitaxel precursor nanomedicine according to claim 1, characterized in that: The mass ratio of paclitaxel dimer prodrug to 7-ethyl-10-hydroxy-camptothecin is 100 to 4:

1.

3. The paclitaxel precursor nanomedicine according to claim 1, characterized in that: The mass ratio of paclitaxel dimer prodrug to 7-ethyl-10-hydroxycamptothecin is 100 to 10:

1.

4. The paclitaxel precursor nanomedicine according to claim 1, characterized in that: Paclitaxel dimer prodrug was dissolved in tetrahydrofuran, and 7-ethyl-10-hydroxy-camptothecin solution was added. After mixing evenly, the mixture was slowly added dropwise to bovine serum albumin aqueous solution, and stirring was continued. After dialysis, nanomedicine loaded with 7-ethyl-10-hydroxy-camptothecin was obtained.

5. The application of the paclitaxel precursor nanomedicine of claim 1 in the preparation of anti-melanoma agents and anti-cervical cancer agents.

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