A triptolide-loaded nitroxide radical polymer nanoparticle, a preparation method thereof and application thereof in small cell lung cancer

Abstract

The application provides a triptolide-loaded nitroxide radical polymer nanoparticle and a preparation method and application thereof in small cell lung cancer, and belongs to the technical field of nano medicine. The nanoparticle provided by the application uses a nitroxide radical polymer as a carrier, the nitroxide radical structure of which not only gives the nanoparticle good stability and biocompatibility, but also enables triptolide (TP) to perform long circulation in the body and mainly accumulate in tumor sites, thereby actively reducing oxidative stress damage caused by TP. The nanoparticle can effectively inhibit the viability of human or murine small cell lung cancer cells in vitro, and shows better antitumor effect than free TP in a subcutaneous transplantation tumor and an orthotopic tumor model, and can significantly reduce systemic toxicity caused by TP, especially damage to testicular tissue and protection of male fertility.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to a nitrogen-oxygen free radical polymer nanoparticle loaded with triptolide, its preparation method, and its application in small cell lung cancer. Background Technology

[0002] Small cell lung cancer (SCLC) is a highly malignant neuroendocrine tumor, accounting for approximately 10-15% of all lung cancer cases. It is characterized by rapid growth, early metastasis, and an extremely poor prognosis. The 5-year survival rate for patients with extensive-stage SCLC is only 2-5%. Although initial chemotherapy (such as platinum-based drugs combined with etoposide) is effective, almost without exception, patients relapse and develop resistance within one year. In recent years, while immunotherapy has brought some survival benefits, its overall clinical efficacy remains limited. Therefore, the development of novel and highly effective treatment strategies is urgently needed.

[0003] Triptolide (TP) is a natural diterpenoid tricyclic oxide isolated from the traditional Chinese medicine Tripterygium wilfordii, possessing broad-spectrum and potent antitumor activity. Studies have shown that TP can effectively inhibit the progression of various malignant tumors, including gastric cancer, cervical cancer, pancreatic cancer, and non-small cell lung cancer. However, the clinical application of TP faces two major obstacles: firstly, its extremely poor water solubility leads to low bioavailability; secondly, it exhibits severe systemic toxicity, particularly to the kidneys and reproductive organs (such as the testes), especially reproductive toxicity, which limits its development and application as a systemic therapeutic agent.

[0004] To overcome the aforementioned shortcomings of TP, researchers have explored various strategies, including structural modification of the TP molecule to develop derivatives (such as Minnelide and LLDT-8), and encapsulation using delivery systems such as liposomes, polymer micelles, and albumin nanoparticles. However, existing technologies still have the following limitations: First, while most TP derivatives improve water solubility to some extent, their antitumor activity is often significantly reduced, and systemic toxicity remains unresolved; second, reported TP nanoformulations (such as TP liposomes and PLGA nanoparticles) mainly focus on improving pharmacokinetics and tumor targeting, but their effectiveness in improving reproductive toxicity is lacking or ineffective; third, current technologies have not yet been able to significantly reduce the specific damage to the male reproductive system (testes, spermatogenesis, and fertility) while maintaining or enhancing the anti-SCLC efficacy of TP.

[0005] Therefore, developing a novel nano-formulation that can maintain or even enhance the efficacy of TP in treating SCLC, while significantly reducing its systemic toxicity, and especially protecting male reproductive function, is of significant clinical value and urgently needed. Summary of the Invention

[0006] The purpose of this invention is to provide nitric oxide radical polymer nanoparticles loaded with triptolide, their preparation method, and their application in small cell lung cancer. The nanoparticles provided by this invention can maintain or even enhance the efficacy of triptolide in treating SCLC, while significantly reducing its systemic toxicity, particularly protecting male reproductive function.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a nitrogen-oxygen radical polymer nanoparticle loaded with triptolide, comprising a nitrogen-oxygen radical polymer carrier and triptolide encapsulated in the nitrogen-oxygen radical polymer carrier; The nitrile radical polymer support is obtained by RAFT polymerization and oxidation treatment of nitrile polymer monomers and octadecyl methacrylate.

[0008] Preferably, the nitrogen-oxygen polymer monomer includes one of (meth)acrylates, (meth)acrylamides, amino acids, esters, and ethyleneimines.

[0009] Preferably, the structural formula of the nitrile radical polymer support is shown in Formula I: Formula I; In Formula I, R1 and R2 are independently C1-C6 alkyl, C1-C6 substituted alkyl, aromatic or substituted aromatic groups, x is an integer from 10 to 1000, and y is an integer from 5 to 500.

[0010] Preferably, the method for preparing the nitroxide radical polymer support includes: A nitrogen oxypolymer monomer, octadecyl methacrylate, an initiator, a RAFT chain transfer agent, and a solvent are mixed and subjected to a polymerization reaction to obtain a polymerization precursor. The polymerization precursor and oxidant are mixed and oxidized to obtain a nitrogen oxide free radical polymer carrier.

[0011] Preferably, the particle size of the nitrogen-oxygen radical polymer nanoparticles loaded with triptolide is 150~200nm.

[0012] This invention also provides a method for preparing the nitric oxide radical polymer nanoparticles loaded with triptolide as described in the above technical solution, comprising: Tripterygium wilfordii, a nitroxide free radical polymer carrier, and an organic solvent were mixed to obtain a mixed solution; The mixed solution was mixed with water, and then dialyzed and dried sequentially to obtain nitrogen oxide free radical polymer nanoparticles loaded with triptolide.

[0013] Preferably, the mass ratio of triptolide to the nitric oxide free radical polymer carrier is 1:(8~12).

[0014] Preferably, the mass ratio of triptolide to the volume ratio of the organic solvent is 1 mg: (0.5~5) mL.

[0015] Preferably, the volume ratio of the organic solvent to water is 1:(5~15).

[0016] The present invention also provides the application of the triptolide-loaded nitric oxide radical polymer nanoparticles described in the above technical solution in the preparation of drugs for treating small cell lung cancer, which can enhance the anti-SCLC efficacy of triptolide and reduce its specific damage to the male reproductive system.

[0017] This invention provides nitric oxide radical polymer nanoparticles loaded with triptolide, comprising a nitric oxide radical polymer carrier and triptolide encapsulated within the nitric oxide radical polymer carrier; the nitric oxide radical polymer carrier is obtained by RAFT polymerization and oxidation treatment of a nitric oxide polymer monomer and octadecyl methacrylate. This invention uses a nitric oxide radical polymer as a carrier, whose nitric oxide radical structure not only endows the nanoparticles with good stability and biocompatibility, but also allows triptolide (TP) to circulate in vivo for a long time, mainly accumulating at the tumor site, thereby actively reducing oxidative stress damage caused by TP. While significantly enhancing the in vivo anti-SCLC efficacy, the nanoparticles can also significantly reduce the systemic toxicity of TP, especially its protective effect on testicular tissue, reaching the level of reversing TP reproductive toxicity. The results of the examples show that the nanoparticles provided by this invention can effectively inhibit the viability of human or mouse small cell lung cancer cells in vitro, exhibit superior anti-tumor effects compared to free TP in subcutaneous xenograft and orthotopic tumor models, and can significantly reduce the systemic toxicity caused by TP, especially the damage to testicular tissue, thus protecting male fertility. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the preparation process of the nanoparticles in Example 1; Figure 2 The infrared spectrum of the nitrogen-oxygen radical polymer support in Example 1; Figure 3 The 1H NMR spectrum of the nitrogen oxide radical polymer support in Example 1; Figure 4 The particle size distribution diagrams are of the nitrogen-oxygen polymer carrier and nanoparticles prepared in Example 1. Figure 5 The Zeta potential diagrams are for the nitrogen-oxygen polymer carrier and nanoparticles prepared in Example 1. Figure 6 TEM image of the nanoparticles prepared in Example 1; Figure 7The results show the in vitro anti-SCLC activity of the nanoparticles in Example 1; Figure 8 The results show the inhibitory effect of nanoparticles on subcutaneous SCLC xenografts in Example 1; Figure 9 The results show the inhibitory effect of nanoparticles on the in-situ SCLC model in Example 1; Figure 10 The results show the in vivo safety and reproductive toxicity evaluation of the nanoparticles in Example 1; Figure 11 The results show the effect of nanoparticles on male fertility in Example 1. Detailed Implementation

[0019] The present invention provides a nitrogen-oxygen radical polymer nanoparticle loaded with triptolide, comprising a nitrogen-oxygen radical polymer carrier and triptolide encapsulated in the nitrogen-oxygen radical polymer carrier.

[0020] The nanoparticles provided by this invention include nitrogen oxide free radical polymer carriers.

[0021] In this invention, the nitric oxide radical polymer carrier is obtained by RAFT polymerization and oxidation treatment of nitric oxide polymer monomer and octadecyl methacrylate.

[0022] In this invention, the nitrogen-oxygen polymer monomer preferably includes one of (meth)acrylates, (meth)acrylamides, amino acids, esters, and ethyleneimines. As one embodiment, the nitrogen-oxygen polymer monomer may specifically be dimethylaminoethyl methacrylate, with the structural formula [structure omitted]. .

[0023] In this invention, the structural formula of the octadecyl methacrylate is as follows: .

[0024] In this invention, the preferred structural formula of the nitrogen-oxygen radical polymer support is shown in Formula I: Formula I.

[0025] In Formula I, R1 and R2 are independently preferably C1-C6 alkyl, C1-C6 substituted alkyl, aromatic or substituted aromatic groups, x is preferably an integer from 10 to 1000, and y is preferably an integer from 5 to 500. As one embodiment, R1 and R2 may be independently specifically methyl, ethyl, propyl, pyrrolyl, piperidinyl, morpholinyl or pyridinyl.

[0026] In this invention, the molecular weight of the nitrogen-oxygen free radical polymer carrier is preferably 2000~50000; the molecular weight of the nitrogen-oxygen polymer (x part in Formula I) is preferably 1000~40000.

[0027] In this invention, the method for preparing the nitroxide radical polymer support preferably includes: A nitrogen oxypolymer monomer, octadecyl methacrylate, an initiator, a RAFT chain transfer agent, and a solvent are mixed and subjected to a polymerization reaction to obtain a polymerization precursor. The polymerization precursor and oxidant are mixed and oxidized to obtain a nitrogen oxide free radical polymer carrier.

[0028] In this invention, nitrogen-oxygen polymer monomers, octadecyl methacrylate, initiator, RAFT chain transfer agent and solvent are mixed and subjected to polymerization reaction to obtain polymerization precursor.

[0029] In this invention, the molar ratio of the nitrogen-oxygen polymer monomer to octadecyl methacrylate is preferably (25~35):6, more preferably 30:6.

[0030] This invention does not impose any particular limitation on the type of initiator; any initiator conventional in the art can be used. As one embodiment, the initiator may specifically be azobisisobutyronitrile (AIBN).

[0031] In this invention, the molar ratio of the initiator to the nitrogen-oxygen polymer monomer is preferably (0.001~0.002):1, more preferably 0.0017:1.

[0032] This invention does not specifically limit the type of RAFT chain transfer agent; any conventional RAFT chain transfer agent in the art can be used. As one embodiment, the RAFT chain transfer agent may specifically be 2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid.

[0033] In this invention, the molar ratio of the RAFT chain transfer agent to the nitrogen-oxygen polymer monomer is preferably (0.01~0.02):1, more preferably 0.017:1.

[0034] This invention does not specifically limit the type of solvent; it can be selected based on the solubility of the raw materials. As one embodiment, the solvent may specifically be anhydrous tetrahydrofuran.

[0035] In this invention, the preferred volume ratio of the amount of nitrogen-oxygen polymer monomer to the solvent is 30 mmol: (20~40) mL, more preferably 30 mmol: 30 mL.

[0036] The present invention does not impose any particular limitation on the mixing method of the nitrogen-oxygen polymer monomer, octadecyl methacrylate, initiator, RAFT chain transfer agent and solvent. Any mixing method known to those skilled in the art can be used to mix the components evenly.

[0037] After mixing, the present invention preferably deoxygenates the mixed system by bubbling with nitrogen for 20-40 minutes. The present invention does not have a specific limitation on the flow rate of the nitrogen gas; any nitrogen flow rate well known to those skilled in the art can be used.

[0038] In this invention, the polymerization reaction temperature is preferably 60-70°C; the polymerization reaction time is preferably 10-15 hours; and the polymerization reaction is preferably carried out under a nitrogen atmosphere. As one embodiment, the polymerization reaction temperature can specifically be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, or 70°C; and the polymerization reaction time can specifically be 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, or 15 hours.

[0039] After the polymerization reaction is completed, the product of the polymerization reaction is preferably subjected to vacuum concentration, precipitation, filtration and drying in sequence.

[0040] The present invention does not impose any special limitations on the operations of vacuum concentration, filtration and drying; any technical solution known to those skilled in the art can be used.

[0041] In this invention, the precipitant used for precipitation is preferably n-hexane; the temperature of the n-hexane is preferably -5 to 5°C. This invention does not have a specific limitation on the amount of n-hexane used, as long as sufficient precipitation is ensured. As one embodiment, the system concentrated under reduced pressure is added dropwise to n-hexane under stirring conditions.

[0042] After obtaining the polymerization precursor, the present invention preferably mixes the polymerization precursor with an oxidant and performs an oxidation treatment to obtain a nitrogen oxide free radical polymer carrier.

[0043] In one embodiment, the oxidant may specifically be hydrogen peroxide; the mass concentration of the hydrogen peroxide may specifically be 30%; the mass ratio of the polymerization precursor to the volume of hydrogen peroxide may be 0.5g:(1~10)mL, or may specifically be 0.5g:2mL, 0.5g:5mL or 0.5g:8mL.

[0044] In this invention, the oxidation treatment temperature is preferably room temperature; the oxidation treatment time is preferably 2-4 hours, more preferably 3-4 hours; and the oxidation treatment is preferably carried out under stirring conditions. This invention does not impose any special limitations on the stirring method and rate; stirring techniques well-known to those skilled in the art can be used.

[0045] After oxidation treatment, the product is preferably placed in a dialysis bag and dialyzed with deionized water for 24 hours, followed by freeze-drying. The molecular weight cutoff of the dialysis bag is preferably 3500 Da. The water is preferably changed every 4 hours during the dialysis process. The present invention does not impose specific limitations on the temperature and time of freeze-drying, as long as sufficient moisture is achieved.

[0046] This invention first prepares an amphiphilic block copolymer via RAFT polymerization, and then performs an oxidation treatment to oxidize the tertiary amine groups on the block copolymer into nitroxide radicals (the structure of oxidized tertiary amines), obtaining a nitroxide radical polymer carrier. This carrier can self-assemble in water to form stable nanomicelles. Its hydrophobic segments form the core for loading the hydrophobic drug triptolide, while the hydrophilic segments with nitroxide radicals form the shell, giving the nanoparticles good stability and biocompatibility.

[0047] In this invention, the particle size of the triptolide-loaded nitric oxide radical polymer nanoparticles is preferably 150-200 nm; the zeta potential of the triptolide-loaded nitric oxide radical polymer nanoparticles is preferably 0.5-0.7 mV; and the encapsulation efficiency of triptolide in the triptolide-loaded nitric oxide radical polymer nanoparticles is preferably 40-60%.

[0048] This invention first prepares a carrier through RAFT polymerization and oxidation treatment. Its nitrile radical structure not only endows nanoparticles with good stability and biocompatibility, but also allows TP to circulate in vivo for a long time, mainly accumulating in the tumor site, thereby actively reducing oxidative stress damage caused by TP. TP is encapsulated in the carrier to prepare nanomedicines, which are then applied to the treatment of SCLC. This fills the gap in the field of TP nano-formulations for the treatment of SCLC, a subtype with high malignancy, easy recurrence, and limited treatment options. While significantly enhancing the in vivo anti-SCLC efficacy (superior to free TP), the nanoparticles can greatly reduce the systemic toxicity of TP, especially the protective effect on testicular tissue, reaching the level of reversing TP reproductive toxicity. Male mice treated with nanoparticles showed intact seminiferous epithelial structure, normal sperm count, and complete recovery of fertility, while the free TP group showed severe testicular atrophy and infertility. This effect has not been achieved in any previously reported TP derivatives or nano-formulations.

[0049] Compared with the prior art, the present invention has the following significant advantages: 1. Significantly reduced systemic toxicity, especially reproductive toxicity: In acute toxicity models, the same dose of free TP caused severe liver damage and hemorrhage, while no significant abnormalities were observed in the nanoparticle (PNO-PSA@TP) group. In chronic treatment models, free TP caused severe testicular atrophy, a sharp decrease in sperm count, and loss of fertility in mice, while the testicular morphology, sperm count, and fertility of mice in the PNO-PSA@TP treatment group were not significantly different from those in the normal control group. This indicates that the PNO-PSA nanocarrier can effectively shield the systemic toxicity of TP, especially its specific protective effect on the male reproductive system, clearing a key obstacle for its clinical translation.

[0050] 2. Significant Anti-SCLC Synergistic Effect: The PNO-PSA@TP nanoparticles prepared in this invention maintain the highly efficient killing activity of TP against various SCLC cell lines (including human and mouse cells) in vitro, and exhibit a stronger tumor growth inhibitory effect than free TP in subcutaneous xenograft tumors and orthotopic SCLC mouse models in vivo. This is attributed to the nanocarrier improving the pharmacokinetic properties of TP and enhancing its accumulation in tumor tissues.

[0051] This invention also provides a method for preparing the nitric oxide radical polymer nanoparticles loaded with triptolide as described in the above technical solution, comprising: Tripterygium wilfordii, a nitroxide free radical polymer carrier, and an organic solvent were mixed to obtain a mixed solution; The mixed solution was mixed with water, and then dialyzed and dried sequentially to obtain nitrogen oxide free radical polymer nanoparticles loaded with triptolide.

[0052] This invention involves mixing triptolide, a nitroxide free radical polymer carrier, and an organic solvent to obtain a mixed solution.

[0053] In this invention, the mass ratio of triptolide to the nitric oxide free radical polymer carrier is preferably 1:(8~12), more preferably 1:10.

[0054] As one embodiment, the organic solvent may specifically be dimethyl sulfoxide; the mass ratio of triptolide to the volume ratio of the organic solvent is preferably 1 mg: (0.5~5) mL, more preferably 1 mg: 1 mL.

[0055] The present invention does not have any special limitations on the operation of mixing the triptolide, nitroxide free radical polymer carrier and organic solvent. The raw materials can be fully mixed by means of technical solutions known to those skilled in the art.

[0056] After obtaining the mixed solution, the present invention mixes the mixed solution with water, and then performs dialysis and drying in sequence to obtain nitrogen oxide free radical polymer nanoparticles loaded with triptolide.

[0057] The present invention preferably involves adding the mixed solution dropwise to water under ultrasonic conditions, and after the addition is complete, continuing to sonicate for 5 to 10 minutes, followed by stirring for 1 to 3 hours.

[0058] In one embodiment, the water can be deionized water; the volume ratio of the organic solvent to water can be 1:(5~15), or more specifically 1:10; the ultrasonic power can be 200W; the ultrasonic working cycle can be 2s operation followed by 3s interval; the dropping rate of the mixed solution can be 0.5mL / min; and the stirring temperature can be room temperature. This invention does not impose any special limitations on the stirring method and rate; stirring techniques well-known to those skilled in the art can be used.

[0059] In this invention, the molecular weight cutoff of the dialysis bag used for dialysis is preferably 3500D; the dialysis time is preferably 7-9 hours, more preferably 8 hours; and the water is preferably changed twice during the dialysis process.

[0060] After dialysis, the present invention preferably filters the dialyzed system through a 0.22μm filter membrane and then dries it.

[0061] In this invention, the drying process is preferably freeze-drying. This invention does not impose any particular limitations on the temperature and time of the freeze-drying; any technical solution well-known to those skilled in the art can be used.

[0062] The present invention also provides the application of the triptolide-loaded nitric oxide radical polymer nanoparticles described in the above technical solution in the preparation of drugs for treating small cell lung cancer (SCLC).

[0063] In one embodiment, the present invention can mix the nitric oxide radical polymer nanoparticles loaded with triptolide with a pharmaceutically acceptable carrier and / or excipient to prepare a pharmaceutical composition; the pharmaceutical composition may be in the form of an injection or a lyophilized powder for injection.

[0064] In this invention, the SCLC includes multiple molecular subtypes represented by human NCI-H69, NCI-H82, NCI-H211 cell lines and mouse mSCLC1 and mSCLC2 cell lines; the treatment is achieved through the following mechanism: TP activates Caspase-3-dependent Gasdermin E (GSDME) lysis, induces tumor cell pyroptosis, thereby effectively inhibiting the growth of SCLC.

[0065] In the application described in this invention, the dose of TP used for SCLC cell lines is 0~100 nmol / L or the dose of TP used for SCLC mouse models is 0.4~0.8 mg / kg.

[0066] The nanoparticles provided by this invention can alleviate the reproductive toxicity caused by TP (specifically manifested as testicular tissue damage, shedding of seminiferous epithelial cells, atrophy of seminiferous tubules, significant reduction in sperm count, and decreased male fertility) when treating SCLC. They can significantly reduce the distribution and toxic effects of TP in non-target organs (especially the testes), protect testicular tissue structure and spermatogenesis, and maintain normal sperm count and male mouse fertility.

[0067] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0068] Example 1 A nitrogen-oxygen radical polymer nanoparticle loaded with triptolide, comprising a nitrogen-oxygen radical polymer carrier and triptolide encapsulated in the nitrogen-oxygen radical polymer carrier; The structural formula of the nitroxide radical polymer carrier is as follows: Its left-end group is a chain transfer agent fragment, x is 30, and y is 10; The preparation method of the nitrogen-oxygen free radical polymer carrier is as follows: (1) 30 mmol of dimethylaminoethyl methacrylate, 6 mmol of octadecyl methacrylate, 0.05 mmol of initiator (azobisisobutyronitrile), 0.5 mmol of RAFT chain transfer agent (2-[dodecylthio(thiocarbonyl)thio]-2-methylpropionic acid) and 30 mL of anhydrous tetrahydrofuran are mixed, nitrogen is bubbled to remove oxygen for 30 min, and the reaction is carried out at 65 °C under nitrogen atmosphere for 12 h. After the reaction is completed, the volume is reduced to 5~6 mL under reduced pressure, and hexane at 0 °C is added dropwise under stirring to precipitate. After filtration, the mixture is vacuum dried to obtain the polymerization precursor. (2) Mix 0.5g of the polymerization precursor obtained in step (1) with 5mL of 30wt% hydrogen peroxide and stir at room temperature for 4h. Transfer the reaction solution to a dialysis bag (3500Da) and dialyze with deionized water for 24h. Change the water every 4h during the dialysis process. Freeze dry to obtain the nitrogen oxide free radical polymer carrier, denoted as PNO-PSA. The method for preparing the triptolide-loaded nitric oxide radical polymer nanoparticles is as follows: 10 mg of nitric oxide radical polymer carrier, 1 mg of triptolide, and 1 mL of dimethyl sulfoxide are mixed to obtain a mixed solution; under 200 W ultrasonic conditions (2 s on, 3 s off), the mixed solution is added dropwise to 10 mL of deionized water at a rate of 0.5 mL / min. After the addition is complete, ultrasonication is continued for 10 min, and then the mixture is stirred at room temperature for 2 h. The mixture is then transferred to a dialysis bag (3500 Da) and dialyzed with deionized water for 8 h, with the water changed twice during the dialysis process. After filtration through a 0.22 μm filter membrane, the mixture is freeze-dried to obtain triptolide-loaded nitric oxide radical polymer nanoparticles, denoted as PNO-PSA@TP.

[0069] The encapsulation efficiency of the nitric oxide radical polymer nanoparticles loaded with triptolide was determined to be 50% by high performance liquid chromatography.

[0070] The preparation process of nanoparticles in Example 1 is as follows: Figure 1 As shown.

[0071] The infrared spectrum of the nitrile radical polymer support in Example 1 is as follows: Figure 2 As shown, the 1H NMR spectrum is as follows Figure 3 As shown. From Figure 2 As can be seen, a new NO stretching vibration peak appears at 950 cm⁻¹. -1 Nearby. From Figure 3 As can be seen, when the tertiary amine is oxidized, the electron density of the protons on the N-methyl and N-methylene groups decreases, thus enhancing the deshielding effect and causing the resonance peak to shift to a lower field, with the shift changing from 3.2 to 3.6.

[0072] The particle size distribution of the nitrogen-oxygen polymer support and nanoparticles prepared in Example 1 is shown in the figure below. Figure 4 As shown, the Zeta potential is as follows Figure 5 As shown, the TEM image of the prepared nanoparticles is as follows. Figure 6 As shown. From Figures 4-6 As can be seen, the average particle size of the nanoparticles is 170 nm, the Zeta potential is 0.56 mV, the morphology is spherical, and the distribution is uniform.

[0073] Application Example 1 In vitro anti-SCLC activity validation of PNO-PSA@TP nanoparticles: Human SCLC cell lines NCI-H211 and NCI-H82, and mouse SCLC cell line mSCLC1 were seeded in 96-well plates (5000 cells / well). After overnight culture, different concentrations of free TP or PNO-PSA@TP (TP equivalent concentration: 0~100 nM) were added, while the control group was treated with an equal volume of PBS. After 48 h of treatment, cell viability was measured using the CCK-8 assay. The results are shown below. Figure 7As shown in Figure A. Figure 7 Results showed that the inhibitory activity of PNO-PSA@TP against the two SCLC cell lines was not significantly different from that of free TP, and the IC50 value was [not specified]. 50 The values ​​were all in the range of 10–20 nM. Western blot analysis was used to detect the protein expression levels of caspase-3, cleaved caspase-3, GSDME-F, and GSDME-N in NCI-H211, NCI-H82, and mSCLC1 cells after treatment with TP (20 nM) or PNO-PSA@TP (equivalent to 20 nM TP) for 24 h. The results are as follows: Figure 7 As shown in B. For Western blotting analysis, cells were lysed in 1×SDS sample buffer and denatured by heating at 100°C for 12 min. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis. Primary antibodies used were rabbit anti-Caspase3, Cleaved caspase3, GSDME-F, and GSDME-N; secondary antibody used was goat anti-rabbit HRP. Western blotting images were obtained using an SH-Magic 523 Mini system with enhanced chemiluminescence (see [link to image]). Figure 7 B). Figure 7 Western blot results for cell line B showed that after 24 h of treatment with TP (20 nM) or PNO-PSA@TP, the expression levels of cleaved caspase-3 and GSDME-N (the N-terminal active fragment of GSDME) proteins were significantly increased in NCI-H211, NCI-H82, and mSCLC1 cells, while the expression of pro-caspase-3 and GSDME-F (full-length GSDME) was correspondingly reduced, indicating that the drug can activate caspase-3 / GSDME pathway-mediated pyroptosis. Simultaneously, bright-field microscopy images showed morphological changes in NCI-H211, NCI-H82, and mSCLC1 cells after 24 h of treatment with TP (20 nM) or PNO-PSA@TP (equivalent to 20 nM TP), scale bar 100 µm. Results are as follows: Figure 7 As shown in C. Figure 7 Under a bright-field microscope, cells in the treatment group exhibited typical pyroptosis morphological features, including cell swelling and bubble-like protrusions, while cells in the control group maintained normal morphology, indicating that pyroptosis of SCLC cells was successfully induced.

[0074] Application Example 2 Evaluation of the in vivo anti-SCLC efficacy and reproductive toxicity of PNO-PSA@TP nanoparticles: 1. Subcutaneous xenograft tumor model To evaluate the in vivo antitumor efficacy of PNO-PSA@TP, subcutaneous xenograft tumor models of mSCLC1 and NCI-H82 were first established. Tumor-bearing mice were treated every two days with PBS (solvent control), TP (0.4 mg / kg), or PNO-PSA@TP (equivalent to 0.4 mg / kg TP), respectively. The drugs were administered intraperitoneally every two days for a total of five administrations. Tumor volume and body weight were measured every three days. Mice were sacrificed after treatment, and tumor weight was collected. The results are as follows: Figure 8 As shown. Figure 8 The results showed that, compared with the solvent control group, the tumor growth curve of mice in the PNO-PSA@TP group was significantly flatter (Figures A and D), the volume of the excised tumors was significantly smaller at the end of treatment (Figures B and E), and the tumor weight was also significantly reduced (Figures C and F), indicating that PNO-PSA@TP has superior or comparable antitumor activity to TP. Immunohistochemical staining results showed that the number of phosphorylated histone H3 (p-H3) positive cells in NCI-H82 tumor sections of the PNO-PSA@TP group was significantly reduced (Figure G), suggesting that tumor cell proliferation was inhibited.

[0075] 2. In-situ SCLC model validation An orthotopic SCLC model was established using CgrpCreER / + and TKO mice via tamoxifen induction. Mice were randomly assigned to groups and treated with the same regimen; results were as follows. Figure 9 As shown. Figure 9 The results showed that after PNO-PSA@TP treatment, the tumor area in the lung tissue of genotype mice was significantly smaller than that in the solvent control group. Figure 9 AB). Meanwhile, serum lactate dehydrogenase (LDH) level detection results showed that PNO-PSA@TP treatment significantly reduced serum LDH levels in orthotopic SCLC mice (AB). Figure 9 (C), further confirming the antitumor effect of the preparation.

[0076] 3. Reproductive toxicity evaluation C57BL / 6 mice were treated with a single high dose (0.8 mg / kg) of TP or PNO-PSA@TP (equivalent to 0.8 mg / kg TP) for 1 day (acute injury model), or with a low dose (0.4 mg / kg) every two days for 21 days (chronic injury model) to establish acute and chronic toxicity models. Representative H&E staining images of the liver, kidneys, spleen, and testes of mice after a single 1-day treatment with 0.8 mg / kg TP or PNO-PSA@TP (equivalent to 0.8 mg / kg TP) are shown below. Figure 10 As shown in Figure B, scale bar 50µm; serum AST, ALT (C), and creatinine (D) levels in mice are as follows: Figure 10As shown in C~D; after 21 days of treatment with low-dose TP or PNO-PSA@TP every two days, representative H&E staining images of the liver, kidney, and spleen of mice are shown. Figure 10 As shown in Figure E, scale bar 50µm; mouse serum AST, ALT and creatinine levels are as follows: Figure 10 As shown in F; after chronic treatment with 0.4 mg / kg TP or PNO-PSA@TP (equivalent to 0.4 mg / kg TP), the morphology of mouse testes was as follows. Figure 10 As shown in G, H&E staining images of mouse testicular sections after chronic treatment with 0.4 mg / kg TP or PNO-PSA@TP (equivalent to 0.4 mg / kg TP) in a chronic injury model are shown below. Figure 10 H is shown, with a scale bar of 200µm. In the chronic injury model, the quantitative results of testicular injury in mice after chronic treatment with 0.4 mg / kg TP or PNO-PSA@TP (equivalent to 0.4 mg / kg TP) are shown below. Figure 10 As shown in I~K. From Figure 10 As can be seen, in the acute toxicity model, high-dose (0.8 mg / kg) treatment with free TP caused severe liver damage (significantly elevated serum AST / ALT, liver tissue hemorrhage and necrosis) and kidney damage (elevated serum creatinine) in mice, while no obvious abnormalities were observed in the PNO-PSA@TP group. However, both caused severe testicular damage at this dose.

[0077] In the chronic treatment model (0.4 mg / kg, consistent with the efficacy experiment dose), the results showed that mice in the free TP group had significant testicular atrophy, weight loss, damage to seminiferous tubule structure, reduction in seminiferous epithelial cells, and a significant decrease in epididymal sperm count; while the above indicators in mice in the PNO-PSA@TP group were not significantly different from those in the normal control group.

[0078] Quantitative analysis of testicular damage after long-term treatment with 0.4 mg / kg TP or PNO-PSA@TP (equivalent to 0.4 mg / kg TP) showed that TP significantly reduced the testicular index ( Figure 10 I) Sperm count (10J), seminiferous tubule diameter and epithelial height, and increased tunica albuginea thickness (10K); while the PNO-PSA@TP treatment group showed no significant difference compared with the control group.

[0079] Male mice were treated with 0.4 mg / kg TP or an equivalent amount of PNO-PSA@TP (based on TP) for 3 weeks, and then mated with untreated female mice at a female-to-male ratio of 2:1 for 1 week. Further fertility experiments showed that, compared to only 33% (2 / 6) of female mice in the free TP group who became pregnant and had significantly fewer litters, the PNO-PSA@TP group had a pregnancy rate of 83% (5 / 6), and the litter size was comparable to the control group. Figure 11 (AB). These results clearly demonstrate that PNO-PSA@TP nanoparticles successfully circumvented the male reproductive toxicity caused by TP.

[0080] In summary, this invention successfully constructed a nitric oxide radical polymer nanoparticle loaded with triptolide (PNO-PSA@TP). This nanoparticle significantly enhances the therapeutic effect against small cell lung cancer while unexpectedly and substantially reducing the inherent systemic toxicity of triptolide, particularly its toxicity to the male reproductive system, effectively protecting fertility. Therefore, this invention provides a safe, efficient, and clinically translational novel nanomedicine for the treatment of small cell lung cancer.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nitrogen-oxygen radical polymer nanoparticle loaded with triptolide, comprising a nitrogen-oxygen radical polymer carrier and triptolide encapsulated in the nitrogen-oxygen radical polymer carrier; The nitrile radical polymer support is obtained by RAFT polymerization and oxidation treatment of nitrile polymer monomers and octadecyl methacrylate.

2. The nitrogen-oxygen radical polymer nanoparticles loaded with triptolide according to claim 1, characterized in that, The nitrogen-oxygen polymer monomers include one of (meth)acrylates, (meth)acrylamides, amino acids, esters, and ethyleneimines.

3. The nitrogen-oxygen radical polymer nanoparticles loaded with triptolide according to claim 1 or 2, characterized in that, The structural formula of the nitrogen oxide radical polymer support is shown in Formula I: Formula I; In Formula I, R1 and R2 are independently C1-C6 alkyl, C1-C6 substituted alkyl, aromatic or substituted aromatic groups, x is an integer from 10 to 1000, and y is an integer from 5 to 500.

4. The nitrogen-oxygen radical polymer nanoparticles loaded with triptolide according to claim 1, characterized in that, The method for preparing the nitroxide radical polymer support includes: A nitrogen oxypolymer monomer, octadecyl methacrylate, an initiator, a RAFT chain transfer agent, and a solvent are mixed and subjected to a polymerization reaction to obtain a polymerization precursor. The polymerization precursor and oxidant are mixed and oxidized to obtain a nitrogen oxide free radical polymer carrier.

5. The nitrogen-oxygen radical polymer nanoparticles loaded with triptolide according to claim 1, characterized in that, The particle size of the nitrogen-oxygen radical polymer nanoparticles loaded with triptolide is 150~200nm.

6. A method for preparing the nitric oxide radical polymer nanoparticles loaded with triptolide according to any one of claims 1 to 5, comprising: Tripterygium wilfordii, a nitroxide free radical polymer carrier, and an organic solvent were mixed to obtain a mixed solution; The mixed solution was mixed with water, and then dialyzed and dried sequentially to obtain nitrogen oxide free radical polymer nanoparticles loaded with triptolide.

7. The preparation method according to claim 6, characterized in that, The mass ratio of triptolide to the nitric oxide free radical polymer carrier is 1:(8~12).

8. The preparation method according to claim 6, characterized in that, The mass ratio of triptolide to the volume ratio of the organic solvent is 1 mg: (0.5~5) mL.

9. The preparation method according to claim 6, characterized in that, The volume ratio of the organic solvent to water is 1:(5~15).

10. The application of the triptolide-loaded nitric oxide radical polymer nanoparticles according to any one of claims 1 to 5 in the preparation of drugs for treating small cell lung cancer can enhance the anti-SCLC efficacy of triptolide while reducing its specific damage to the male reproductive system.

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