Preparation method of pH (potential of hydrogen) and GSH (glutathione) double-response cyclodextrin nanoparticles

Abstract

The invention relates to a preparation method of pH and GSH dual-response cyclodextrin nanoparticles. The preparation method comprises the following steps: S1, preparing a 10mg / mL lecithin absolute ethyl alcohol solution; s2, a DSPE-PEG 2000-UM206 solution with the concentration of 9 mg / mL is prepared; s3, preparing a water phase; s4, preparing an organic phase; and S5, nano-precipitate self-assembly: in a round-bottom flask, rapidly magnetically stirring the water phase at room temperature, dropwise adding the organic phase into the water phase at a speed of less than 1 mL per minute, and stirring at room temperature for 2 hours. And S6, purification of the nanoparticles: transferring the solution into a 30 kDa ultrafiltration tube, centrifuging for 20 minutes at 4000 * g, and cleaning with PBS for 3 times to obtain the acetalated disulfide bond beta-cyclodextrin dimer nanoparticles. When the pH and GSH double-response cyclodextrin nanoparticle prepared by the preparation method of the pH and GSH double-response cyclodextrin nanoparticle is used for delivering HHT, the pH and GSH double-response cyclodextrin nanoparticle has a sensitive pH / GSH double-response function and an efficient tumor killing effect, has a good treatment effect on OCSCs, can give full play to the tumor killing effect of the HHT, and also can effectively reduce the drug side effect of the HHT.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles. Background Technology

[0002] Osteosarcoma is the most common primary bone cancer in adolescents, with high recurrence and metastasis rates, making treatment difficult. Tumor stem cells play a crucial role in tumor formation, development, and recurrence, and their cell cycle quiescence and drug resistance further complicate treatment. Simultaneously, the high heterogeneity of osteosarcoma, partly due to asymmetric cell division and genetic and epigenetic variations, also presents challenges to treatment.

[0003] Delivering anti-tumor drugs via drug-loaded nanoparticles is a common approach to cancer treatment. PLGA nanoparticles are one such existing drug-loaded nanoparticle. The degradation of PLGA nanoparticles is influenced by the ratio of PLA to PGA; a higher PLA ratio leads to increased hydrophobicity, resulting in slower degradation kinetics. For example, the degradation time of PLGA (75:25) in vivo is approximately 4-5 months. Studies have specifically investigated the molecular degradation kinetics of PLGA at different pH levels, finding that the degradation rate did not accelerate under acidic conditions. This slower degradation rate may be advantageous in drug delivery, providing sustained drug release over extended periods. Although PLGA nanoparticles exhibit good stability, their non-responsive nature makes them unsuitable for treating osteosarcoma and osteosarcoma tumor stem cell-like cells (OCSCs). In the treatment of osteosarcoma and OCSCs, we hope that nanoparticles can rapidly and effectively release the tumor-killing drug HHT after precisely targeting the OCSC tissue. On the other hand, due to the high toxicity of HHT, even if the nanoparticles effectively encapsulate the drug and prevent it from entering the bloodstream, a lower dose of HHT is still necessary for safety reasons. Therefore, how to fully exert the tumor-killing effect of HHT while effectively reducing its side effects is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles, so as to solve the technical problem of how to fully exert the tumor-killing effect of HHT while effectively reducing the drug side effects of HHT.

[0005] This invention is achieved through the following technical solution: A method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles includes the following steps: S1. Prepare a 10 mg / mL anhydrous ethanol solution of lecithin; S2. Prepare a 9 mg / mL DSPE-PEG2000-UM206 solution; S3. Preparation of aqueous phase: Add 0.6 mL of 10 mg / mL anhydrous ethanol solution of lecithin and 1 mL of 9 mg / mL DSPE-PEG2000-UM206 solution to 15 mL of PBS solution, stir thoroughly in an oil bath at 65 °C for 1 hour, and then slowly cool to room temperature. At this point, the solution is in the form of a microemulsion. S4, Organic phase: Weigh 50 mg of acetalized disulfide bond β-cyclodextrin dimer and dissolve it in 1.9 mL of anhydrous DMSO, then vortex to mix. S5, Nanoprecipitate self-assembly: In a round-bottom flask, the aqueous phase is rapidly magnetically stirred at room temperature, and the organic phase is added dropwise to the aqueous phase at a rate of less than 1 mL per minute, and stirred at room temperature for 2 hours. S6. Purification of nanoparticles: The solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 4000×g for 20 minutes. After washing with PBS three times, acetalized disulfide bond β-cyclodextrin dimer nanoparticles were obtained.

[0006] Furthermore, the method for preparing anhydrous ethanol solution of lecithin is as follows: Weigh 100 mg of lecithin and dissolve it in 10 mL of anhydrous ethanol. Heat and vortex or use sonication to aid dissolution to form a 10 mg / mL anhydrous ethanol solution of lecithin.

[0007] Furthermore, the method for preparing the DSPE-PEG2000-UM206 solution is as follows: Weigh 5 mg of distearylphosphatidylacetamide-polyethylene glycol 2000 and 4 mg of DSPE-PEG2000-UM206 (total mass 9 mg), dissolve them in 1 mL of PBS buffer, sonicate and vortex to aid dissolution, forming a 9 mg / mL DSPE-PEG2000-UM206 PBS solution.

[0008] Furthermore, the preparation method of DSPE-PEG2000-UM206 in step S2 is as follows: S21. Activate the carboxyl group of DSPE-PEG2000-COOH: Quickly weigh 24 mg of DSPE-PEG2000-COOH into a round-bottom flask, add 5 mL of anhydrous DMSO, then weigh 23 mg of EDC·HCl and 6.9 mg of N-hydroxysuccinimide, add them into the round-bottom flask, and stir magnetically at room temperature (25℃) for 4 hours. S22, Dehydration condensation: Weigh 20 mg of UM206 lyophilized peptide, add it to a round-bottom flask and continue to stir magnetically for 28 hours. S23, Dialysis: Transfer the product to a 2kDa dialysis bag and dialyze for 48 hours to remove the catalyst and DMSO; S24. Ultrafiltration: Centrifuge the dialyzed liquid at 4000×g and 4℃ for 20 minutes using a 3 kDa ultrafiltration tube to remove unreacted distearylphosphatidylacetamide-polyethylene glycol 2000, UM206 and other byproducts. S25, freeze-dried, store at -20℃.

[0009] Furthermore, before step S21, the round-bottom flask, magnetic stir bar, bottle cap, etc. are cleaned and dried to minimize the influence of water.

[0010] Furthermore, the sequence of the polypeptide UM206 is: CNKTSEGMDGCEL.

[0011] Furthermore, the synthesis method of acetalized disulfide bond β-cyclodextrin dimer is as follows: 1.5g SCD was weighed and dissolved in 15mL anhydrous DMSO, and then 6mL 2-methoxypropylene was added. The mixture was magnetically stirred and dissolved. Then, 24mg pyridine p-toluenesulfonate was weighed and added to the above reaction solution. The reaction was carried out at room temperature for 3 hours. Finally, 0.45mL triethylamine was added to terminate the reaction. The product was precipitated with deionized water, washed three times by centrifugation, and freeze-dried to obtain the product as a white powder.

[0012] Further, the synthesis method of SCD is as follows: 483 mg of cystine hydrochloride and 2.3 g of triethylamine were weighed and added to 20 mL of anhydrous DMSO solution and dissolved completely. 702 mg of CDI was weighed and added dropwise to the aforementioned solution. The reaction was carried out under N2 protection at room temperature with magnetic stirring for 3 hours. Then, the above reaction solution was added dropwise to 20 mL of DMSO solution containing 4.98 g of amino-β-cyclodextrin, and the reaction was continued with stirring for 24 hours. Thin-layer chromatography was used for detection during the reaction. The mobile phase was n-butanol:methanol:water:30% ammonia water = 4:3:2:3, and basic potassium permanganate was used as the colorimetric reagent. After the reaction was completed, the solvent DMSO was removed by freeze-drying. 50 mL of pure water was added to dissolve the residue, and the insoluble matter was removed by filtration. The product was purified by Sephadex-CM chromatography with a mobile phase of 0.05 M to 0.5 M ammonium bicarbonate aqueous solution. The corresponding eluent was collected and freeze-dried to obtain the product, which was a white, fluffy solid.

[0013] The beneficial effects of this invention are as follows: When pH and GSH dual-responsive cyclodextrin nanoparticles prepared by the method described in this invention deliver HHT, they exhibit sensitive pH / GSH dual-responsiveness and highly efficient tumor-killing activity, showing good therapeutic effects on OCSCs. They can fully exert the tumor-killing effect of HHT while effectively reducing the drug side effects of HHT.

[0014] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the synthesis of the targeting unit DSPE-PEG2000-UM206; Figure 2 This is a schematic diagram of AcSCD synthesis; Figure 3 In the image, (A) Fourier transform infrared spectra of NH2-β-CD, cystamine, SCD, and AcSCD; (B) Hydrogen nuclear magnetic resonance spectra of NH2-β-CD, cystamine, SCD, and AcSCD; Figure 4 The MALDI-TOF mass spectrum of the SCD; Figure 5 The results show the particle size and zeta potential of four types of drug-loaded nanoparticles. Figure 6 In the image, (A) is a transmission electron microscope (TEM) image of HP; (B) is a TEM image of HPU; (C) is a TEM image of HA; and (D) is a TEM image of HAU. Figure 7 A standard curve for the determination of HHT by high performance liquid chromatography; Figure 8 The images show in vitro drug release curves of HPU and HAU nanoparticles at different pH and GSH conditions; (A) drug release curve of pH / GSH dual-response HHT-loaded nanoparticle CAU under different pH and GSH conditions; (B) drug release curve of control PLGA nanoparticles under different pH and GSH conditions; (C) drug release curve comparison of CPU and CAU at pH 7.4; (D) drug release curve comparison of CPU and CAU at pH 6.5 + 10mM GSH; (E) drug release curve comparison of CPU and CAU at pH 5.5 + 10mM GSH. Figure 9 A graph was created to evaluate the cytotoxic effects of several drug-loaded nanoparticles on HOS cells using CCK-8 assays. (A) A bar chart comparing the cytotoxic effects of HHT monotherapy, HP, HPU, HA, and HAU nanoparticles on HOS cells at different HHT concentrations. (B) A graph comparing the IC50 calculation results based on nonlinear fitting curves.

[0016] Figure 10 A schematic diagram demonstrating the therapeutic effect of drug-loaded nanoparticles on subcutaneous OCSCs in a nude mouse in vivo model; Figure 11 To evaluate the efficacy of drug-loaded nanoparticles in inhibiting the growth of subcutaneous OCSCs tumors; (A) photographs of subcutaneous OCSCs tumors in each group at the end of treatment; (B) bar chart of subcutaneous OCSCs tumor weight in each group at the end of treatment; (C) curve of OCSCs subcutaneous tumor volume change during treatment; (D) curve of nude mouse body weight change during treatment. Figure 12 Histological evaluation of subcutaneous tumor sections of OCSCs in each treatment group; immunohistochemistry of H&E staining (A), KI67 (B), PNCA (C) and Tunel staining (D) in each treatment group; Figure 13 A graph showing the changes in RNA expression of osteosarcoma stemness-related genes SOX2 (A), PROM1 (CD133, B), FZD1 (C), CTNNB1 (D), TCF7L1 (E), FBLIM1 (F), LAMC1 (G), MAGED2 (H) and SCL7A8 (I) after HHT, HP, HPU, HA and HAU nanoparticle treatment for subcutaneous OCSCs. Figure 14 The results show the complete blood count and liver and kidney function of nude mice with subcutaneous OCSCs tumors after treatment with drug-loaded nanoparticles; bar charts showing the results of (A) white blood cells, (B) red blood cells and (C) platelets after treatment in each group, and (D) alanine aminotransferase, (E) aspartate aminotransferase, (F) creatinine and (G) urea after treatment in each group.

[0017] Figure 15 H&E staining images of the major organs of nude mice in each treatment group. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0019] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0020] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0021] In the above description of the present invention, it should be noted that the terms "one side," "the other side," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of the invention is conventionally placed during use. These terms are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0022] Furthermore, terms such as "identical" do not imply that components must be absolutely identical; minor differences are permissible. The term "perpendicular" simply means that the positional relationship between components is more perpendicular than "parallel," not that the structure must be perfectly perpendicular; a slight tilt is acceptable.

[0023] This invention provides a technical solution: a method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles, characterized by comprising the following steps: S1. Prepare a 10 mg / mL anhydrous ethanol solution of lecithin; specifically, weigh 100 mg of lecithin, dissolve it in 10 mL of anhydrous ethanol, and heat and vortex or use sonication to aid dissolution to form a 10 mg / mL anhydrous ethanol solution of lecithin.

[0024] S2. Prepare a 9 mg / mL DSPE-PEG2000-UM206 solution; specifically, weigh 5 mg distearylphosphatidylacetamide-polyethylene glycol 2000 (hereinafter referred to as DSPE-PEG2000) and 4 mg DSPE-PEG2000-UM206 (total mass 9 mg), dissolve them in 1 mL of PBS buffer, sonicate and vortex to aid dissolution, forming a 9 mg / mL DSPE-PEG2000-UM206 PBS solution.

[0025] In step S2, DSPE-PEG2000-UM206 (please refer to...) Figure 1 The preparation method of ) is as follows: S21. Activation of the carboxyl group of DSPE-PEG2000-COOH: Quickly weigh 24 mg of DSPE-PEG2000-COOH into a round-bottom flask and add 5 mL of anhydrous DMSO. Then weigh 23 mg of EDC·HCl and 6.9 mg of N-hydroxysuccinimide (hereinafter referred to as NHS) and add them to the round-bottom flask. React at room temperature (25°C) with magnetic stirring for 4 hours. Before step S21, clean and dry the round-bottom flask, magnetic stir bar, cap, etc., to minimize the influence of water.

[0026] S22, Dehydration Condensation: Weigh 20 mg of the UM206 lyophilized peptide and add it to a round-bottom flask. Continue magnetic stirring for 28 hours. The sequence of the peptide UM206 is: CNKTSEGMDGCEL.

[0027] S23. Dialysis: Transfer the product to a 2 kDa dialysis bag and dialyze for 48 hours to remove the catalyst and DMSO.

[0028] S24. Ultrafiltration: Centrifuge the dialyzed liquid at 4000×g and 4℃ for 20 minutes using a 3 kDa ultrafiltration tube to remove unreacted DSPE-PEG2000, UM206 and other byproducts.

[0029] S25, freeze-dried, store at -20℃.

[0030] S3. Preparation of the aqueous phase: Add 0.6 mL of 10 mg / mL anhydrous ethanol solution of lecithin and 1 mL of 9 mg / mL DSPE-PEG2000-UM206 solution to 15 mL of PBS solution, and stir thoroughly in an oil bath at 65°C for 1 hour. Afterward, slowly cool to room temperature. The solution is now in a microemulsion state.

[0031] S4, Organic phase: Weigh 50 mg of acetalized disulfide bond β-cyclodextrin dimer (hereinafter referred to as AcSCD) and dissolve it in 1.9 mL of anhydrous DMSO, then vortex to mix.

[0032] The synthesis method of AcSCD is as follows: 1.5 g of disulfide-bonded β-cyclodextrin dimer (hereinafter referred to as SCD) was dissolved in 15 mL of anhydrous DMSO, and then 6 mL of 2-methoxypropylene was added. The mixture was magnetically stirred until dissolved and homogeneous. Then, 24 mg of pyridine p-toluenesulfonate was added to the above reaction solution, and the reaction was carried out at room temperature for 3 hours. Finally, 0.45 mL of triethylamine was added to terminate the reaction. The obtained product was precipitated with deionized water, washed three times by centrifugation, and freeze-dried to obtain a white powder.

[0033] The synthesis method of SCD is as follows: 483 mg of cystine hydrochloride and 2.3 g of triethylamine were weighed and added to 20 mL of anhydrous DMSO solution and dissolved completely. 702 mg of CDI was weighed and added dropwise to the aforementioned solution, and the reaction was carried out under N2 protection at room temperature with magnetic stirring for 3 hours. Then, the above reaction solution was added dropwise to 20 mL of DMSO solution containing 4.98 g of amino-β-cyclodextrin (hereinafter referred to as NH2-β-CD), and the reaction was continued with stirring for 24 hours. Thin-layer chromatography was used for detection during the reaction, with the mobile phase being n-butanol:methanol:water:30% ammonia water = 4:3:2:3, and basic potassium permanganate as the colorimetric reagent. After the reaction was completed, the solvent DMSO was removed by lyophilization, 50 mL of pure water was added to dissolve the residue, insoluble matter was removed by filtration, and purification was carried out by Sephadex-CM chromatography with the mobile phase being 0.05 M to 0.5 M ammonium bicarbonate aqueous solution. The corresponding eluent was collected, and the product was obtained by lyophilization, which was a white, fluffy solid. After characterization confirms successful synthesis, the next step is carried out.

[0034] like Figure 3 As shown, we first activated cystamine with CDI and triethylamine, and then reacted it with twice the amount of NH2-β-CD to form a disulfide-bonded CD-SS-CD (SCD) dimer that responds to glutathione (hereinafter referred to as GSH). Figure 2 A, B). Then, pH and GSH-responsive AcSCDs were synthesized via acetalization of SCD. Figure 2 C).

[0035] S5. Nanoprecipitate self-assembly: In a round-bottom flask, the aqueous phase is rapidly stirred magnetically at room temperature, and the organic phase is added dropwise to the aqueous phase at a rate of less than 1 mL per minute. The mixture is stirred at room temperature for 2 hours.

[0036] S6. Purification of nanoparticles: The solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 4000×g for 20 minutes. After washing with PBS three times, acetalized disulfide bond β-cyclodextrin dimers @UM206 nanoparticles were obtained, which are the pH and GSH dual-responsive cyclodextrin nanoparticles.

[0037] Infrared spectral and hydrogen nuclear magnetic resonance characterization of SCD and AcSCD materials: We performed acetalization on the SCD and characterized the AcSCD after acetalization using Fourier transform infrared spectroscopy and hydrogen nuclear magnetic resonance spectroscopy.

[0038] The infrared spectral characterization method for the material is as follows: a small amount of dry, anhydrous material powder is placed in the Fourier transform infrared spectroscopy detection stage and directly pressed into a pellet for detection.

[0039] The method for characterizing the material by 1H NMR is as follows: Weigh about 10 mg of the material powder and place it in a glass bottle. Use a glass pipette to draw 600 μL of deuterated DMSO as a solvent, dissolve it by sonication, and then transfer it to an NMR tube for 1H NMR identification.

[0040] Infrared spectroscopy results show ( Figure 3 A) SCD contains both the hydroxyl group of cyclodextrin and the amino group of cystamine. In AcSCD, due to 3010-3780cm -1 The absorption peak of the hydroxyl group at 2995 cm⁻¹ decreased significantly, while the absorption peak at 29 -1 A distinct new characteristic peak for the stretching vibration of the methyl group was observed.

[0041] In the 1H NMR spectra of NH2-β-CD, cystamine, SCD, and AcSCD ( Figure 3 (B) Comparing NH2-β-CD and SCD, we find that the absorption peak at approximately 1.2 in NH2-β-CD is a characteristic peak of amino hydrogen, while the corresponding position is not observed in SCD. This indicates that the amino group in NH2-β-CD participated in the chemical reaction, while no amino group is present in SCD. Simultaneously, new peaks with chemical shifts of 6.3 and 6.0 appear in SCD, which are characteristic peaks of the newly formed amide hydrogen from NH2-β-CD and cystamine, and the amide hydrogen itself in cystamine, respectively. Furthermore, in SCD, the two hydrogens originally located on the methylene group connecting the amino group in NH2-β-CD, with chemical shifts of 2.8 and 2.9, migrate to around 3.3, further indicating that the amino group in NH2-β-CD participated in the chemical reaction. A new peak appears at position 2.7, which is a characteristic peak on the methylene group connecting the disulfide bond in cystamine. All of the above evidence indicates that SCD was successfully synthesized.

[0042] Comparing SCD and AcSCD, we find that the absorption peaks near chemical shifts of 4.8 and 3.8 in SCD are characteristic peaks of the hydroxyl hydrogen and hydroxymethyl hydrogen on the six-membered ring, respectively, while these peaks do not appear at the corresponding positions in AcSCD. This indicates that both the hydroxyl and hydroxymethyl groups in SCD participated in the reaction. The characteristic peak at chemical shifts of approximately 1.2-1.3 in AcSCD is the characteristic peak of the two methyl groups on the carbon atom attached to the terminal methoxy group. These results indicate that AcSCD was successfully synthesized.

[0043] MALDI-TOF characterization of materials: Weigh an appropriate amount of lyophilized SCD material and perform MALDI-TOF mass spectrometry analysis. The solvent is methanol, and the matrix is ​​2,5-dihydroxybenzoic acid.

[0044] Figure 4 This is the MALDI-TOF mass spectrum of an SCD. The theoretical molecular weight of an SCD is 2472, and the quasi-molecular ion peak is [M+Na]. +The measured value was 2495 m / z, which is 2495.3350, perfectly matching the theoretical value. This result demonstrates the successful synthesis of the β-cyclodextrin dimer SCD linked by cystamine (-SS-) (hereinafter referred to as β-CD).

[0045] Preparation of high-harbinose base (hereinafter referred to as HHT) drug-loaded nanoparticles based on polylactic-co-glycolic acid copolymer (hereinafter referred to as PLGA): Preparation of anhydrous ethanol solution of lecithin: Weigh 100 mg of lecithin, dissolve it in 10 mL of anhydrous ethanol, heat and vortex or sonicate to aid dissolution, to form an anhydrous ethanol solution of lecithin with a concentration of 10 mg / mL.

[0046] Preparation of DSPE-PEG2000 solution: Weigh 9 mg of DSPE-PEG2000 and dissolve it in 1 mL of PBS buffer. Sonicate and vortex to aid dissolution, forming a 9 mg / mL DSPE-PEG2000 PBS solution. Preparation of DSPE-PEG2000-UM206 solution: Weigh 5 mg of DSPE-PEG2000 and 4 mg of DSPE-PEG2000-UM206 (total mass 9 mg), dissolve them in 1 mL of PBS buffer, sonicate and vortex to aid dissolution, forming a 9 mg / mL DSPE-PEG2000-UM206 PBS solution.

[0047] Preparation of the aqueous phase: Add 0.6 mL of 10 mg / mL anhydrous ethanol solution of lecithin and 1 mL of 9 mg / mL DSPE-PEG2000 solution or DSPE-PEG2000-UM206 solution to 15 mL of PBS solution, and stir thoroughly in an oil bath at 65°C for 1 hour. Afterward, slowly cool to room temperature. The solution is now in a microemulsion state.

[0048] Organic phase: Weigh 50 mg of PLGA and dissolve it in 1.5 mL of acetonitrile. Then weigh 20 mg of HHT, dissolve it in anhydrous DMSO, and add it to the PLGA-acetonitrile solution. Vortex to mix. Nanoprecipitate self-assembly: In a round-bottom flask, stir the aqueous phase rapidly with a magnetic stirrer at room temperature in the dark. Add the organic phase dropwise to the aqueous phase at a rate of less than 1 mL per minute and stir at room temperature for 2 hours.

[0049] Purification of nanoparticles: The solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 4000×g for 20 minutes. The nanoparticles were washed three times with PBS to obtain HHT / PLGA (hereinafter referred to as HP) and HHT / PLGA@UM206 (hereinafter referred to as HPU) nanoparticles. If necessary, the nanoparticles were freeze-dried and stored at -20°C.

[0050] Preparation of dual-responsive HHT drug-loaded nanoparticles based on pH and GSH dual-responsive cyclodextrin nanoparticles: Preparation of anhydrous ethanol solution of lecithin: Weigh 100 mg of lecithin, dissolve it in 10 mL of anhydrous ethanol, heat and vortex or sonicate to aid dissolution, to form an anhydrous ethanol solution of lecithin with a concentration of 10 mg / mL.

[0051] Preparation of DSPE-PEG2000 solution: Weigh 9 mg of DSPE-PEG2000 and dissolve it in 1 mL of PBS buffer. Sonicate and vortex to aid dissolution, forming a 9 mg / mL DSPE-PEG2000 PBS solution. Preparation of DSPE-PEG2000-UM206 solution: Weigh 5 mg of DSPE-PEG2000 and 4 mg of DSPE-PEG2000-UM206 (total mass 9 mg), dissolve them in 1 mL of PBS buffer, sonicate and vortex to aid dissolution, forming a 9 mg / mL DSPE-PEG2000-UM206 PBS solution.

[0052] Preparation of the aqueous phase: Add 0.6 mL of 10 mg / mL anhydrous ethanol solution of lecithin and 1 mL of 9 mg / mL DSPE-PEG2000 solution or DSPE-PEG2000-UM206 solution to 15 mL of PBS solution, and stir thoroughly in an oil bath at 65°C for 1 hour. Afterward, slowly cool to room temperature. The solution is now in a microemulsion state.

[0053] Organic phase: Weigh 50 mg AcSCD and HHT and dissolve them separately in 1 mL of anhydrous DMSO. After they are fully dissolved, mix them together and shake to mix.

[0054] Nanoprecipitate self-assembly: In a round-bottom flask, the aqueous phase was rapidly stirred magnetically at room temperature in the dark. The organic phase was added dropwise to the aqueous phase at a rate of less than 1 mL per minute, and the mixture was stirred at room temperature for 2 hours.

[0055] Purification of nanoparticles: The solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 4000×g for 20 minutes. The nanoparticles were washed three times with PBS. The resulting products were HHT / AcSCD (hereinafter referred to as HA) and HHT / AcSCD@UM206 (hereinafter referred to as HAU) nanoparticles. HHT / AcSCD@UM206 is a dual-response HHT drug-loaded nanoparticle constructed based on the pH and GSH dual-response cyclodextrin nanoparticles described in this invention. If necessary, the nanoparticles were freeze-dried and stored at -20°C.

[0056] Determination of nanoparticle size and zeta potential: Take an appropriate amount of the prepared nanoparticles, dilute them to 1 mL, add them to a special cuvette, and use a Malvern laser particle size analyzer to determine the particle size and surface zeta potential of the nanoparticles.

[0057] We constructed four drug-loaded nanoparticles based on PLGA and AcSCD materials, with or without the active targeting unit DSPE-PEG2000-UM206: HP, HPU, HA, and HAU. The results showed that the HP nanoparticle size was 176.5 ± 4.555 nm. Figure 5 A), HPU increased slightly, at 193.3±2.546nm ( Figure 5 B). HA particle size is 212±3.273 nm ( Figure 5 C), similarly, the particle size of HAU increased slightly after adding DSPE-PEG2000-UM206, to 221.3±9.079nm. Figure 5 D). Overall, the particle size distribution of the four types of nanoparticles is uniform.

[0058] like Figure 5 As shown in Figure E, we investigated the potentials of four nanoparticles. HP and HPU nanoparticles carried a certain amount of negative charge on their surfaces, while the AcSCD nanoparticles HA and HAU had very little negative charge, approaching a neutral state. The measured average potentials of HP, HPU, HA, and HAU were -9.23±1.12 mV, -7.65±0.636 mV, -1.45±0.176 mV, and -0.698±0.343 mV, respectively.

[0059] Transmission electron microscopy (TEM) analysis of nanoparticles: Nanoparticles were diluted with PBS and dropped onto a wax plate to form nanoparticle droplets. A copper mesh was then carefully inserted into the nanoparticle droplet at a 45-degree angle and placed at the bottom. After standing at room temperature for 10 minutes, the copper mesh was removed and placed on filter paper to air dry any excess liquid. The morphology of the nanoparticles was observed using a transmission electron microscope.

[0060] Transmission electron microscopy images of four types of drug-loaded nanoparticles: Transmission electron microscopy (TEM) results showed that all four drug-loaded nanoparticles were spherical in shape, with relatively uniform size, consistent with Malvern's particle size analysis. In the CPU and CAU actively targeted nanoparticles, the outermost layer was a "coating layer," caused by the presence of the UM206 hydrophilic peptide on the outermost layer. Figure 6 ).

[0061] Drug loading and encapsulation efficiency of HHT drug-loaded nanoparticles: The HHT content in HP, HPU, HA, and HAU nanoparticles was determined by high-performance liquid chromatography (HPLC). First, HHT was weighed and dissolved in chromatographic-grade methanol, then diluted with water to prepare standard sample solutions of 0.1, 0.5, 1, 5, 10, 50, 100, 500, and 1000 μg / mL. These solutions were filtered through a 0.2 μm pore size filter and transferred to 2 mL HPLC vials. The HHT standard sample solutions were analyzed using HPLC, and a standard curve was established. The chromatographic conditions were as follows: C18 bonded silica column; mobile phase A was 0.01 mol / L KH₂PO₄ solution adjusted to pH 2.5 with H₃PO₄; mobile phase B was methanol; A:B = 60:40; flow rate 1 mL / min; injection volume 20 μL; λmax = 288 nm; column temperature 30 °C.

[0062] Weigh 5 mg of different lyophilized HHT drug-loaded nanoparticles and dissolve them in 5 mL of chromatographic grade methanol. After vortexing, centrifuge at 12000 rpm for 10 min. Collect the supernatant, filter through a 0.2 μm pore size filter, and transfer to a 2 mL liquid chromatography vial. Perform chromatographic analysis according to the above conditions. Calculate the peak area using high-performance liquid chromatography software, obtain a standard curve based on the concentration, and calculate the encapsulation efficiency and drug loading of the nanoparticles using the following formulas: Encapsulation efficiency % = (Mass of HHT in nanoparticles / Mass of HHT added to the system) × 100%; Drug loading % = mass of HHT in nanoparticles / mass of HHT-loaded nanoparticles × 100%.

[0063] We determined the liquid chromatography standard curve of HHT ( Figure 7 Within the concentration range of 0 μg / mL to 1000 μg / mL, the R² value was 0.999, indicating good linearity of the standard curve, which meets the requirements.

[0064] We then determined the drug loading and encapsulation efficiency of the four drug-loaded nanoparticles, and the results are shown in Table 1.

[0065] Table 1. HHT drug loading and encapsulation efficiency of different drug-loaded nanoparticles.

[0066] In vitro release of nanoparticles: Different masses of Na₂HPO₄ were weighed to prepare PBS buffer solutions with pH values ​​of 7.4, 6.5, and 5.5. Simultaneously, different masses of GSH were weighed and dissolved in pH 7.4 PBS solution to prepare 10 mM and 20 mM GSH solutions. A 10 mM GSH solution with pH 6.5 was then prepared. 5 mg of lyophilized CPU or CAU nanoparticles were dissolved in 2 mL of each of the different solutions, and the solutions were incubated at 37°C with shaking. Samples were released at 0, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 hours, centrifuged at 12000 rpm for 10 minutes, and the supernatant was used for high-performance liquid chromatography (HPLC) to determine the HHT concentration.

[0067] In vitro release behavior of CPU and CAU nanoparticles: like Figure 8 As shown in Figure A, HAU showed low HHT release at pH 7.4. HHT release gradually accelerated as the pH decreased, reaching approximately 80% release after 24 hours at pH 5.5. Similarly, HHT release increased with increasing GSH concentration; approximately 60% of HAU was released after 48 hours in a 20 mM GSH solution. At the same pH, increasing GSH concentration further enhanced the drug release capacity of the nanoparticles, particularly in a pH 5.5 + 10 mM GSH solution, where CAU nanoparticles achieved approximately 90% drug release after 12 hours and almost complete release after 36 hours.

[0068] The HHT release from HPU nanoparticles was low in pH 7.4 and pH 6.5 + 10mM GSH solutions. Although the HHT release increased slightly in pH 5.5 + 10mM GSH solution, the cumulative drug release rate of PLGA-loaded nanoparticles was less than 20%. Figure 8 B). It can be seen that at pH 7.4, drug release from both CPU and CAU nanoparticles is very slow. Figure 8 C). Under pH 6.5 + 10 mM GSH conditions, CAU's drug release far exceeds that of CPU nanoparticles ( Figure 8 D), under pH 5.5 + 10mM GSH conditions, the difference in drug release between CAU and CPU was more pronounced. Figure 8 E). Therefore, the results show that PLGA nanoparticles are not sensitive to pH and GSH, but AcSCD drug-loaded nanoparticles can exhibit ideal pH / GSH dual-responsive drug release ability. That is, when the pH and GSH dual-responsive cyclodextrin nanoparticles prepared by the preparation method of this invention deliver HHT, they can exhibit ideal pH / GSH dual-responsive drug release ability.

[0069] Cell lines: Human osteosarcoma cell lines HOS, 143B, and MNNG / HOS (in this paper, we use MN / H to represent MNNG / HOS cells) were all purchased from the American Type Culture Collection (ATCC) and all underwent STR cell line identification.

[0070] Laboratory animals: Female nude mice (BALB / c-nu) aged 4-6 weeks, weighing 18-20g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. After purchase, they were housed at the Experimental Animal Center of Chongqing Medical University, which has SPF (Special Purpose Animal) housing conditions. They were kept in a room temperature environment with 12-hour light-dark cycles, and had free access to water and food. An acclimatization feeding period of one week was implemented. The animal experimental protocol involved in this study was reviewed by the Experimental Animal Management and Use Committee of Chongqing Medical University (IACUC-CQMU) and complies with animal protection, welfare, and ethical principles, as well as relevant national regulations on experimental animal welfare and ethics. The approval number is: IACUC-CQMU-2023-0436.

[0071] CCK-8 assay for the cytotoxicity of drug-loaded nanoparticles against osteosarcoma cells (hereinafter referred to as HOS): The HHT mass in the nanoparticles was calculated using encapsulation efficiency and drug loading results. Different groups of drug-loaded nanoparticles were prepared with the same HHT concentration (0.1 μg / mL). Cells were first seeded in 96-well plates and cultured overnight at 37°C with 5% CO2. The next day, cell status was observed. When cell confluence was 50%-70%, the culture medium was aspirated, and a specific concentration of drug-containing medium was added. After culturing in a cell culture incubator for 24 hours, the 96-well plates were removed, the culture medium was carefully aspirated, and the cells were gently washed once with PBS warmed to 37°C. Then, 110 μL of thoroughly mixed CCK-8 working solution (10 mL CCK-8 solution added to 100 μL of complete culture medium) was added to each well. The plates were incubated at 37°C for 1-4 hours, and the absorbance of the samples at 450 nm was measured using a microplate reader. The IC50 value was estimated using a nonlinear regression model.

[0072] In vitro killing effects of several drug-loaded nanoparticles on parental HOS cells: We used CCK-8 to evaluate the cytotoxic effects of different drug-loaded nanoparticles on HOS cells over 24 hours. Figure 9As shown in Figure A, a statistically significant difference was observed between HAU and HP nanoparticles at 0.001 μg / mL HHT, but no statistically significant differences were found among the other groups. At 0.01 μg / mL HHT, HAU exhibited significantly stronger cytotoxicity against HOS than the other four groups. At HHT concentrations of 0.1 and 1 μg / mL, HAU remained the most cytotoxic, with HA also showing stronger cytotoxicity than HHT, HP, and HPU. At 10 μg / mL, although HAU and HA still exhibited the strongest cytotoxicity, cell viability was already very low in all groups. While HPU showed stronger cytotoxicity than HP at 0.1 μg / mL, at all concentrations, the drug-loaded PLGA nanoparticles HP and HPU showed no significant difference compared to HHT alone. Nonlinear regression curves were used to fit the IC50 values ​​of each group (…). Figure 9 B). The results showed that the IC50 of the HP group (0.126 μg / mL) was higher than that of the HHT monotherapy control group (0.106 μg / mL). The IC50 of HPU (0.103 μg / mL) was not significantly different from that of the HHT monotherapy group. However, the IC50 of the HA group (0.042 μg / mL) was more than half lower than that of the previous three groups, and the IC50 of HAU (0.014 μg / mL) was three times lower than that of the HA group. The results indicate that at 24 hours, PLGA-based nanoparticles could not enhance the cytotoxic effect of HHT at the same concentration, while nanoparticles self-assembled based on AcSCD material could enhance the in vitro toxicity of HHT to HOS at the same concentration, and the FZD1 actively targeted pH / GSH dual-responsive AcSCD nanoparticle HAU could further promote the cytotoxic effect of HHT.

[0073] Evaluation of the efficacy of drug-loaded nanoparticles in subcutaneous tumorigenesis of osteosarcoma tumor stem cells (hereinafter referred to as OCSCs): OCSCs subcutaneous tumorigenesis model: OCSCs cultured to the third generation from HOS were digested, centrifuged, and resuspended in sterile PBS. Cell counts were performed, and the cells were resuspended to a concentration of 1×10⁶ cells / 0.1 mL. Under aseptic conditions, 100 μL of PBS containing 1×10⁶ OCSCs was injected subcutaneously into nude mice. Animals were observed and tumor formation was assessed every two days post-inoculation.

[0074] Experimental Groups: Sixty nude mice with subcutaneous OCSC tumors were randomly divided into 6 groups of 10 mice each. The dosage for the nanoparticle group was calculated based on the concentration of HHT. (1) Blank control group: Nude mice with subcutaneous tumor formation were injected with sterile PBS only; (2) HHT monotherapy group: HHT was injected via tail vein at a dose of 0.5 mg / kg; (3) HP nanoparticle group: HP nanoparticles were injected into the tail vein at a dose of 0.5 mg / kg. (4) HPU nanoparticle group: HPU nanoparticles were injected via the tail vein at a dose of HHT 0.5 mg / kg; (5) HA nanoparticle group: HA nanoparticles were injected via the tail vein at a dose of 0.5 mg / kg of HHT; (6) HAU nanoparticle group: HAU nanoparticles were injected into the tail vein at a dose of HHT 0.5 mg / kg.

[0075] Dosage regimen: Subcutaneous tumor formation of OCSCs was established in nude mice on day -14. Starting from day 0, 0.1 mL was administered via tail vein every 2 days for a total of 7 administrations, and the weight and tumor growth of the nude mice were observed.

[0076] Tissue sample collection and processing: Blood: Whole blood was collected from nude mice after enucleation. 20 μL of whole blood was added to 180 μL of complete blood count (CBC) diluent and thoroughly mixed before CBC analysis. The remaining whole blood was allowed to stand for at least 1 hour until clear stratification occurred, then centrifuged at 3000 rpm at 4°C for 10 minutes. Serum was collected for liver and kidney function tests.

[0077] Tissue Samples: After euthanizing the animal, subcutaneous tumors, heart, liver, spleen, lungs, and kidneys were removed, weighed, and organ indices were calculated. Tumor tissue was photographed and its volume measured. Samples were labeled and paraffin-embedded or stored at -80°C as needed. H&E staining and immunohistochemistry: Tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and then subjected to routine H&E staining and immunohistochemical staining. Tunel staining: Tunel staining was performed according to the kit instructions, and images were taken under an upright fluorescence microscope. RT-qPCR: The primers were synthesized by Cyclopsidae and their sequences are as follows: Subcutaneous tumor tissues of OCSCs treated with various nanoparticle groups were collected on ice. Appropriate amounts of Buffer QLS lysis buffer were added for low-temperature tissue grinding, homogenization, and lysis. For tissues with insufficient mass, two samples from the same group were pooled before homogenization.

[0078] Subsequent RNA extraction, reverse transcription, and RT-qPCR steps can be performed according to the kit (Aikerui) instructions and the standard procedures.

[0079] Statistical methods All experimental data in this section were analyzed using SPSS 26.0 statistical software. One-way ANOVA was used for comparisons between multiple groups, followed by the Bonferroni test. For data with multiple concentrations or time points and different groupings, two-way ANOVA and the Bonferroni post-hoc test were used for comparisons between groups. A p-value less than 0.05 was considered statistically significant. The following symbols were used to represent the significance level: NS (nosignificance) indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0080] Evaluation of the therapeutic effect of drug-loaded nanoparticles on OCSCs in nude mice: We have already demonstrated that HA and HAU can promote the killing of HOS cells in vitro. Next, we will observe the in vivo efficacy of drug-loaded nanoparticles against OCSCs using a nude mouse subcutaneous tumorigenesis model. Figure 10 ).

[0081] The therapeutic effect of drug-loaded nanoparticles on subcutaneous OCSCs tumors: After each treatment group, the tumor images of the subcutaneous OCSCs are as follows: Figure 11 As shown in Figure A, the OCSCs tumors in the PBS control group were large and relatively uniform in size. Two nude mice died before the end of the 30-day treatment period (white circles represent samples from these two prematurely deceased mice). Free HHT had a certain inhibitory effect on the growth of subcutaneous OCSCs, but the tumor size varied among different nude mice within the group. Tumor morphologies of varying sizes also appeared in each nanoparticle treatment group, especially in the HP group, where the difference in tumor size was significant, indicating that HP treatment exhibits some heterogeneity. In contrast, the tumors in the HAU group were relatively uniform in size, demonstrating that HAU has good homogeneity in treating subcutaneous OCSCs. Statistical analysis of tumor weight results showed that HAU tumors had the lightest weight (…). Figure 11 B). In all other treatment groups compared to the control, tumor weight was reduced, but there were no statistically significant differences among the other treatment groups (HHT, HP, HPU, HA). This was observed in conjunction with the tumor growth volume curves ( Figure 11C), the subcutaneous tumors of OCSCs in the control group grew rapidly, with the tumor volume decreasing on days 24 and 27 due to the deaths of one nude mouse on days 22 and 26, respectively. Furthermore, the tumor volume in each treatment group was smaller than that in the control group. There was no significant difference in tumor growth curves between free HP and HHT monotherapy. The antitumor effect of the non-responsive, actively targeted nanoparticle HPU was superior to that of the non-targeted nanoparticle HP. It can be seen that the efficacy of the pH / GSH dual-responsive nanoparticle HA was better than that of the non-responsive HP nanoparticles, and the tumor inhibitory effect of HAU was also stronger than that of HPU. Among all groups, HAU nanoparticles showed the strongest inhibitory effect on the growth of OCSCs subcutaneous tumors, with tumor volume remaining almost stable at the initial treatment level for the first 12 days and showing a decreasing trend after 12 days. There were no statistically significant differences in the changes in body weight of nude mice among the groups during various treatment periods. Figure 11 D).

[0082] Histological evaluation of the therapeutic effect of drug-loaded nanoparticles on subcutaneous OCSCs tumors: To further evaluate the therapeutic effects of various drug-loaded nanoparticles on subcutaneous OCSC tumors, we performed histological observation of the tumor tissue at the end of treatment. The results showed that, for H&E staining, the subcutaneous OCSC tumor tissue in the blank control group was structurally intact, with densely packed tumor cells. Different degrees of tissue necrosis were observed in all treatment groups, with tumor cells exhibiting nuclear pyknosis and fragmentation, and loss of cell integrity. Particularly in the HAU group, a large number of cells died, with only a very small percentage of tumor cells maintaining normal structure. Figure 12 A). We also used KI67 and PCNA to assess the proliferation of subcutaneous OCSCs. For example... Figure 12 As shown in B and C, all treatment groups exhibited varying degrees of inhibitory effects on the proliferation of OCSC subcutaneous tumors, with the HAU group showing the strongest inhibitory effect, exhibiting almost no actively proliferating cells. Using TUNEL staining, we further observed the effects of each nanoparticle on apoptosis in OCSC subcutaneous tumors. Figure 12 (D). The results showed that all treatment groups induced apoptosis in OCSCs to varying degrees. Similarly, HAU showed the brightest and widest range of green fluorescence, indicating the strongest apoptosis signal. The results demonstrate that HAU has a strong inhibitory effect on the proliferation of OCSCs and a promoting effect on apoptosis.

[0083] Changes in the expression of osteosarcoma stemness-related genes in OCSCs after drug-loaded nanoparticle therapy: We extracted RNA from subcutaneous tumor tissue samples of OCSCs to observe the regulatory effects of different treatment groups on the expression of osteosarcoma stemness-related genes. We first evaluated the osteosarcoma tumor stemness marker SOX2 (… Figure 13 A) and CD133 (PROM1, Figure 13B), whose gene expression decreased in all treatment groups, indicating that HHT treatment can effectively inhibit the stemness of OCSCs. For SOX2, HPU was lower than HP, and HAU was lower than HA, indicating that DSPE-PEG2000-UM206 can enhance the inhibitory effect of nanoparticles on SOX2. In addition, the expression levels of SOX2 and CD133 were the lowest in the HAU group, indicating that HAU nanoparticles have the strongest tumor stemness inhibitory ability on subcutaneous OCSCs. Then, RNA was also measured for the Wnt gene, which is related to the tumor stemness of osteosarcoma. It can be seen that FZD1 ( Figure 13 C) Significant differences were observed among the groups. Free HHT showed no significant difference in its inhibitory effect on FZD1, while all nanoparticle groups reduced FZD1 expression. Compared to untargeted nanoparticles (HP or HA), targeted nanoparticles (HPU or HAU) had lower FZD1 levels, with HAU showing the most pronounced decrease in FZD1 expression. CTNNB1 ( Figure 13 D) was also expressed at the lowest level in the HAU group, and showed some degree of decrease in other treatment groups as well. TCF7L1 ( Figure 13 The RNA expression level of E) was highly consistent across the four nanoparticle groups. We then evaluated four other key genes related to osteosarcoma stemness. The results showed that FBLM1 (… Figure 13 F) and SLC7A8 ( Figure 13 The expression trends of I) were similar across groups; their expression levels decreased in all treatment groups, and the nanoparticle group further reduced their RNA expression. Regarding LAMC1 ( Figure 13 For G), HAU showed the most significant inhibition of its expression. However, untargeted HP and HA nanoparticles tended to upregulate LAMC1 RNA. Furthermore, MAGED2 expression levels did not differ significantly among the groups. Figure 13 H).

[0084] Preliminary safety evaluation of drug-loaded nanoparticles We examined the blood routine and liver and kidney function of nude mice after nanoparticle treatment. Figure 14 The results showed that HHT monotherapy in nude mice with subcutaneous OCSC tumors caused leukopenia, while the nanoparticle treatment group did not experience a decrease in leukocytes. Figure 14 (A) demonstrates that nanoparticles possess the potential protective function against the inhibitory side effects of HHT on leukocytes in nude mice. Free HHT monotherapy also caused a decrease in erythrocyte count in nude mice; the increase in erythrocyte count from untargeted nanoparticles HP and HA was not statistically different from the HHT group, while actively targeted nanoparticles HPU and HAU did not cause a decrease in erythrocyte count. Figure 14 B). No significant effect on platelet count was observed after treatment in any group. Figure 14C). Similarly, there was no change in alanine aminotransferase (ALT) levels in any of the treatment groups. Figure 14 D). Aspartate aminotransferase (AST) decreased in all treatment groups, but there was no statistically significant difference between the treatment groups. Figure 14 E). Renal function indicator creatinine ( Figure 14 F) and urea ( Figure 14 G) also showed no significant changes after treatment. The results indicate that 0.5 mg / kg HHT monotherapy may have some effect on the hematopoietic system of nude mice with OCSC subcutaneous tumors, but drug-loaded nanoparticles did not cause such changes, especially the active targeting nanoparticles, which had little effect on the hematopoietic system.

[0085] H&E staining was used to observe whether there was substantial organ damage to the major organs of nude mice in each treatment group. Figure 15 In the control group, the spleen tissue showed a clear capsule structure, a clear boundary between the red and white pulp, abundant white pulp, and no obvious abnormalities. However, in the free HHT group, the boundary between the red and white pulp in the spleen tissue was unclear. No obvious abnormalities were observed in the spleen of any of the nanoparticle treatment groups. No significant pathological structural changes were found in the other major organs (heart, liver, lung, and kidney). These results indicate that the pH / GSH dual-responsive FZD1 actively targeted HHT-loaded nanoparticles have good biocompatibility.

[0086] We successfully synthesized HHT-loaded PLGA and AcSCD nanoparticles using a modified nanoprecipitation self-assembly method, and formed FZD1 actively targeted nanoparticles by adding the active targeting unit DSPE-PEG2000-UM206. We first evaluated the drug release of HPU and HAU under different pH and GSH conditions in vitro. The results showed that the HAU material could release HHT responsively under low pH and high GSH conditions, and exhibited good stability at pH 7.4. However, in in vitro cytotoxicity experiments, the therapeutic effects of HP and HPU were not significantly different from those of free HHT. We only performed a 24-hour cytotoxicity assay, and combined with the drug release curve, the drug release of HPU under pH 5, 5+10mM GSH conditions was only 20% after 24 hours. This is because HHT exerts its protein synthesis inhibitory function and needs to act in the cytoplasm, while HHT loaded in PLGA nanoparticles cannot be rapidly released intracellularly. The IC50 of HP was even greater than that of HHT alone, which is also related to the low phagocytic efficiency of HP nanoparticles by cells. However, HPU significantly outperformed free HHT in inhibiting tumor volume in OCSCs in vivo. The 30-day in vivo treatment period provided ample time for HPU nanoparticles to release the drug. In vivo experiments showed that various nanoparticles inhibited the growth of subcutaneous tumors in OCSCs, and all treatment groups, except for HPU, showed no difference in efficacy between HPU and HHT monotherapy; all other nanoparticles enhanced the tumor-killing effect of HHT. At the end of treatment, except for HAU, there were no statistically significant differences in tumor quality among the other treatment groups. This is consistent with the heterogeneity of osteosarcoma stem cells, which leads to inconsistent sensitivity of OCSCs to drug efficacy.

[0087] In this invention, we employed a dual-response active targeted delivery strategy. Compared to non-responsive active targeted drug-loaded nanoparticles (HPU) and pH / GSH dual-responsive non-targeted drug-loaded nanoparticles (HA), HAU prepared based on pH and GSH dual-responsive cyclodextrin nanoparticles exhibited the best anti-osteosarcoma effects both in vitro and in vivo, enhancing OCSC apoptosis while inhibiting OCSC proliferation in vivo. This is attributed to its precise OCSC targeting, sensitive pH / GSH dual responsiveness, and highly efficient killing ability of the HHT payload.

[0088] In summary, HHT / AcSCD@UM206 (HAU), prepared based on pH and GSH dual-responsive cyclodextrin nanoparticles, demonstrated excellent targeting ability and sensitive osteosarcoma responsiveness, fully leveraging the tumor-killing effect of HHT. It inhibited the proliferation of subcutaneous OCSCs and promoted apoptosis, while reducing the expression of multiple osteosarcoma tumor stem cell markers such as FZD1, SOX2, and CD133. Simultaneously, it effectively reduced the drug side effects of HHT. Therefore, combining targeted and characteristically responsive nanomedicine delivery systems with highly efficient tumor-killing drugs holds great potential for the treatment of osteosarcoma and its tumor stem cells, providing a new approach for clinical treatment.

[0089] When pH and GSH dual-responsive cyclodextrin nanoparticles prepared by the method described in this invention deliver HHT, they exhibit sensitive pH / GSH dual-responsiveness and highly efficient tumor-killing activity, showing good therapeutic effects on OCSCs. They can fully exert the tumor-killing effect of HHT while effectively reducing the drug side effects of HHT.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles, characterized in that, Includes the following steps: S1. Prepare a 10 mg / mL anhydrous ethanol solution of lecithin; S2. Prepare a 9 mg / mL DSPE-PEG2000-UM206 solution; S3. Preparation of aqueous phase: Add 0.6 mL of 10 mg / mL anhydrous ethanol solution of lecithin and 1 mL of 9 mg / mL DSPE-PEG2000-UM206 solution to 15 mL of PBS solution, stir thoroughly in an oil bath at 65 °C for 1 hour, and then slowly cool to room temperature. At this point, the solution is in the form of a microemulsion. S4, Organic phase: Weigh 50 mg of acetalized disulfide bond β-cyclodextrin dimer and dissolve it in 1.9 mL of anhydrous DMSO, then vortex to mix. S5, Nanoprecipitate self-assembly: In a round-bottom flask, the aqueous phase is rapidly magnetically stirred at room temperature, and the organic phase is added dropwise to the aqueous phase at a rate of less than 1 mL per minute, and stirred at room temperature for 2 hours. S6. Purification of nanoparticles: The solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 4000×g for 20 minutes. After washing with PBS three times, acetalized disulfide bond β-cyclodextrin dimer nanoparticles were obtained.

2. The method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles according to claim 1, characterized in that... The method for preparing anhydrous ethanol solution of lecithin is as follows: Weigh 100 mg of lecithin and dissolve it in 10 mL of anhydrous ethanol. Heat and vortex or use sonication to aid dissolution to form a 10 mg / mL anhydrous ethanol solution of lecithin.

3. The method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles according to claim 1, characterized in that, The method for preparing DSPE-PEG2000-UM206 solution is as follows: Weigh 5 mg distearylphosphatidylacetamide-polyethylene glycol 2000 and 4 mg DSPE-PEG2000-UM206, dissolve them in 1 mL of PBS buffer, sonicate and vortex to aid dissolution, and form a 9 mg / mL DSPE-PEG2000-UM206 PBS solution.

4. The method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles according to claim 1, characterized in that, The preparation method of DSPE-PEG2000-UM206 in step S2 is as follows: S21. Activate the carboxyl group of DSPE-PEG2000-COOH: Quickly weigh 24 mg of DSPE-PEG2000-COOH into a round-bottom flask, add 5 mL of anhydrous DMSO, then weigh 23 mg of EDC·HCl and 6.9 mg of N-hydroxysuccinimide, add them to the round-bottom flask, and stir magnetically at room temperature for 4 hours. S22, Dehydration condensation: Weigh 20 mg of UM206 lyophilized peptide, add it to a round-bottom flask and continue to stir magnetically for 28 hours. S23, Dialysis: Transfer the product to a 2kDa dialysis bag and dialyze for 48 hours to remove the catalyst and DMSO; S24. Ultrafiltration: Centrifuge the dialyzed liquid at 4000×g and 4℃ for 20 minutes using a 3 kDa ultrafiltration tube to remove unreacted distearylphosphatidylacetamide-polyethylene glycol 2000, UM206 and other byproducts. S25, freeze-dried, store at -20℃.

5. The method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles according to claim 4, characterized in that: Before step S21, clean and dry the round-bottom flask, magnetic stir bar, bottle cap, etc., to minimize the influence of water.

6. The method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles according to claim 4, characterized in that, The sequence of the polypeptide UM206 is: CNKTSEGMDGCEL.

7. The method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles according to claim 1, characterized in that, The synthesis method of acetalized disulfide bond β-cyclodextrin dimer is as follows: 1.5 g SCD was weighed and dissolved in 15 mL anhydrous DMSO, and then 6 mL 2-methoxypropylene was added. The mixture was magnetically stirred and dissolved. Then 24 mg pyridine p-toluenesulfonate was weighed and added to the above reaction solution. The reaction was carried out at room temperature for 3 hours. Finally, 0.45 mL triethylamine was added to terminate the reaction. The product was precipitated with deionized water, washed three times by centrifugation, and freeze-dried to obtain the product as a white powder.

8. The method for preparing pH and GSH dual-responsive cyclodextrin nanoparticles according to claim 7, characterized in that, The synthesis method of SCD is as follows: 483 mg of cystine hydrochloride and 2.3 g of triethylamine were weighed and added to 20 mL of anhydrous DMSO solution and dissolved completely. 702 mg of CDI was weighed and added dropwise to the above solution. The reaction was carried out under N2 protection at room temperature with magnetic stirring for 3 hours. Then, the above reaction solution was added dropwise to 20 mL of DMSO solution containing 4.98 g of amino-β-cyclodextrin and the reaction was continued with stirring for 24 hours. Thin-layer chromatography was used for detection during the reaction. The mobile phase was n-butanol:methanol:water:30% ammonia water = 4:3:2:3, and alkaline potassium permanganate was used as the colorimetric reagent. After the reaction was completed, the solvent DMSO was removed by freeze drying. 50 mL of pure water was added to dissolve the residue. The insoluble matter was removed by filtration. The product was purified by Sephadex-CM chromatography. The mobile phase was 0.05 M to 0.5 M ammonium bicarbonate aqueous solution. The corresponding eluent was collected and freeze-dried to obtain the product, which was a white fluffy solid.

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