Preparation method of FZD1 active targeting nanoparticles
By preparing FZD1 active targeting nanoparticles, DSPE-PEG2000-UM206 was synthesized using DSPE-PEG2000-COOH and polypeptide UM206, and then combined with PLGA to form a three-layer structure of nanoparticles. This solved the problem of poor targeting of osteosarcoma cells and OCSCs, and achieved highly efficient targeted phagocytosis of osteosarcoma cells and OCSCs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE FIRST AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIVERSITY
- Filing Date
- 2026-02-11
- Publication Date
- 2026-04-17
AI Technical Summary
Existing technologies are insufficient to effectively promote the phagocytosis of nanoparticles by osteosarcoma cells and improve the targeting of OCSCs, especially tumor stem cells.
By preparing FZD1 actively targeted nanoparticles, DSPE-PEG2000-UM206 was synthesized using DSPE-PEG2000-COOH and polypeptide UM206. Combined with polylactic-co-glycolic acid copolymer (PLGA) and DSPE-PEG2000-UM206, a three-layer structure of nanoparticles was formed, including a nanoparticle core, a lipid stabilizing layer and a hydrophilic outer layer, thereby achieving active targeting of FZD1.
It improved the phagocytic efficiency of osteosarcoma cells on nanoparticles, especially the targeted phagocytic efficiency of OCSCs, and enhanced the uptake capacity of nanoparticles in OCSCs.
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Figure CN121868253A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing FZD1 actively targeted 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] Currently, small molecule drugs remain the primary treatment for osteosarcoma. An effective method for small molecule drug delivery is the use of nanomedicine delivery systems. These systems integrate small molecule compounds into nanomaterials through encapsulation or adsorption, forming drug nanoparticles and enabling highly efficient drug delivery. These drugs can be administered orally, by injection, nebulization, and other methods. Nanomedicine delivery systems hold immense potential in tumors. However, promoting the phagocytosis of nanoparticles by osteosarcoma cells and improving the targeting of nanoparticles to a range of FZD1-highly expressing tumor cells, such as osteosarcoma stem-like cells (OCSCs), remains a critical challenge. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide a method for preparing FZD1 active targeting nanoparticles to solve the technical problem of how to promote the phagocytosis of nanoparticles by osteosarcoma cells and improve the targeting of nanoparticles to OCSCs.
[0005] This invention is achieved through the following technical solution: A method for preparing FZD1 actively targeted nanoparticles, characterized by comprising the following steps: S1. Synthesize DSPE-PEG2000-UM206 using DSPE-PEG2000-COOH and polypeptide UM206; S2. FZD1 active targeting nanoparticles were synthesized using polylactic acid-glycolic acid copolymer and DSPE-PEG2000-UM206. The FZD1 active targeting nanoparticles comprise a three-layer structure, consisting of a polymer material encapsulating a hydrophobic compound in the nanoparticle core, a lipid-stabilizing layer formed by a middle monolayer of lecithin, and an outermost hydrophilic outer layer surrounded by DSPE-PEG2000-UM206.
[0006] Furthermore, the sequence of the polypeptide UM206 is: CNKTSEGMDGCEL.
[0007] Furthermore, the method for synthesizing DSPE-PEG2000-UM206 using DSPE-PEG2000-COOH and polypeptide UM206 is as follows: S11. Activate the carboxyl group of DSPE-PEG2000-COOH in a round-bottom flask; S12, Dehydration condensation: Weigh 20 mg of UM206 lyophilized peptide, add it to a round-bottom flask and stir magnetically for 28 hours. S13, Dialysis: Transfer the product to a 2 kDa dialysis bag and dialyze for 48 hours to remove the catalyst and DMSO; S14. Ultrafiltration: Centrifuge the dialyzed liquid at 4000xg and 4℃ for 20 minutes using a 3K Da ultrafiltration tube to remove unreacted DSPE-PEG2000, UM206 or other byproducts. S15. Store after freeze-drying.
[0008] Furthermore, the freeze-dried DSPE-PEG2000-UM206 was stored at -20℃.
[0009] Further, the method for activating the carboxyl group of DSPE-PEG2000-COOH is as follows: 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 NHS, add them to the round-bottom flask, and stir magnetically at room temperature (25℃) for 4 hours.
[0010] Further, before S11, the round-bottom flask, magnetic stirrer, and bottle cap are cleaned and dried.
[0011] Furthermore, the method for synthesizing FZD1 actively targeted nanoparticles using polylactic acid-glycolic acid copolymer and DSPE-PEG2000-UM206 is as follows: S21. Preparation of anhydrous ethanol solution of lecithin: Weigh 100 mg of lecithin, dissolve it in 10 mL of anhydrous ethanol, heat and vortex or use sonication to aid dissolution, to form an anhydrous ethanol solution of lecithin with a concentration of 10 mg / mL. Preparation of S22 and DSPE-PEG2000-UM206 solution: Weigh 5 mg of DSPE-PEG2000 and 4 mg of 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. S23. 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 time, the solution is in microemulsion state. S24. Preparation of organic phase: Weigh 50 mg PLGA and dissolve it in 1.9 mL acetonitrile, then vortex to mix. S25, 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. S26. The nanoparticles were purified to obtain the FZD1 active targeting nanoparticles.
[0012] Furthermore, the method for purifying the nanoparticles involves transferring the solution to a 30 kDa ultrafiltration tube, centrifuging at 4000×g for 20 minutes, and washing three times with PBS.
[0013] The beneficial effects of this invention are as follows: The FZD1 active targeting nanoparticles prepared by the method of the present invention can promote the phagocytosis of PLGA nanoparticles by osteosarcoma cells, and OCSCs have high phagocytosis efficiency of the FZD1 active targeting nanoparticles prepared by the method of the present invention.
[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 A schematic diagram illustrating the synthesis of the targeting unit DSPE-PEG2000-UM206; Figure 2Molecular dynamics simulations were used to evaluate the interaction and structural stability of UM206 or DSPE-PEG2000-UM206 with FZD1: (A) Molecular docking diagram of the extracellular region of UM206 and FZD1 protein; (B) RMSD curve of the complex formed by UM206 and FZD1; (C) Molecular docking diagram of the extracellular region of DSPE-PEG2000-UM206 and FZD1 protein; (D) RMSD curve of the complex formed by DSPE-PEG2000-UM206 and FZD1; (E) SASA curves of the FZD1 / UM206 system and the FZD1 / DSPE-PEG2000-UM206 system; (F) Protein gyration radius analysis diagram; (G) Hydrogen bond analysis diagram; (H) MM / PBSA analysis diagram. Figure 3 MALDI-TOF mass spectra of DSPE-PEG2000-COOH, UM206, and DSPE-PEG2000-UM206: (A) MALDI-TOF results for DSPE-PEG2000-COOH; (B) MALDI-TOF results for UM206; (C) MALDI-TOF results for DSPE-PEG2000-UM206. Figure 4 In the image, (A) is the particle size distribution of PLGA nanoparticles; (B) is the particle size distribution of PLGA@UM206 nanoparticles; (C) is the particle size distribution of Cy5 / PLGA nanoparticles; and (D) is the particle size distribution of Cy5 / PLGA@UM206 nanoparticles. Figure 5 In the image, (A) is a transmission electron microscope (TEM) image of the CP; (B) is a TEM image of the CPU. Figure 6 The graph shows the phagocytosis of CP and CPU nanoparticles by HOS cells at different time points using laser confocal microscopy. Figure 7 In the figure, (A) flow cytometry was used to detect the phagocytosis of CP nanoparticles by HOS cells at different time points; (B) flow cytometry was used to detect the phagocytosis of CPU nanoparticles by HOS cells at different time points; (C) quantitative statistical results of average fluorescence intensity. Figure 8 This is a diagram showing the phagocytosis of CP and CPU nanoparticles by OCSCs detected by laser confocal microscopy. Detailed Implementation
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] This invention provides a technical solution: a method for preparing FZD1 actively targeted nanoparticles, comprising the following steps: S1. Synthesize DSPE-PEG2000-UM206 using DSPE-PEG2000-COOH and polypeptide UM206; The sequence of the polypeptide UM206 is: CNKTSEGMDGCEL.
[0022] The method for synthesizing DSPE-PEG2000-UM206 using DSPE-PEG2000-COOH and polypeptide UM206 is as follows: S11, activates the carboxyl group of DSPE-PEG2000-COOH; The method for activating the carboxyl group of DSPE-PEG2000-COOH is as follows: 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 NHS into the round-bottom flask, and stir magnetically at room temperature (25℃) for 4 hours.
[0023] Before S11, clean and dry the round-bottom flask, magnetic stirrer, and bottle cap to minimize the impact of water.
[0024] S12, Dehydration condensation: Weigh 20 mg of UM206 lyophilized peptide, add it to a round-bottom flask and stir magnetically for 28 hours. S13, Dialysis: Transfer the product to a 2 kDa dialysis bag and dialyze for 48 hours to remove the catalyst and DMSO; S14. Ultrafiltration: Centrifuge the dialyzed liquid at 4000xg and 4℃ for 20 minutes using a 3K Da ultrafiltration tube to remove unreacted DSPE-PEG2000, UM206 or other byproducts. S15. Store after freeze-drying. The freeze-dried DSPE-PEG2000-UM206 should be stored at -20℃.
[0025] Please refer to Figure 1 As shown, the targeting unit DSPE-PEG2000-UM206 is obtained by dehydration condensation of the carboxyl group of DSPE-PEG2000-COOH and the amino group of UM206 in an EDC / NHS system.
[0026] S2. FZD1 active targeting nanoparticles were synthesized using polylactic acid-glycolic acid copolymer (hereinafter referred to as PLGA) and DSPE-PEG2000-UM206.
[0027] The sequence of the polypeptide UM206 is: CNKTSEGMDGCEL.
[0028] The method for synthesizing FZD1 actively targeted nanoparticles using polylactic acid-glycolic acid copolymer and DSPE-PEG2000-UM206 is as follows: S21. Preparation of anhydrous ethanol solution of lecithin: Weigh 100 mg of lecithin, dissolve it in 10 mL of anhydrous ethanol, heat and vortex or use sonication to aid dissolution, to form an anhydrous ethanol solution of lecithin with a concentration of 10 mg / mL.
[0029] Preparation of S22 and SPE-PEG2000-UM206 solutions: 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.
[0030] S23. 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. Stir thoroughly in an oil bath at 65°C for 1 hour, then slowly cool to room temperature. The solution is now in a microemulsion state.
[0031] S24. Preparation of organic phase: Weigh 50 mg PLGA and dissolve it in 1.9 mL acetonitrile, then vortex to mix.
[0032] S25, 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, and stirred at room temperature for 2 hours.
[0033] S26 nanoparticles were purified to obtain PLGA@UM206 nanoparticles, namely FZD1 active targeting nanoparticles.
[0034] The method for purifying the nanoparticles is as follows: the solution is transferred to a 30 kDa ultrafiltration tube, centrifuged at 4000×g for 20 minutes, and washed three times with PBS.
[0035] Molecular dynamics simulations of UM206 and DSPE-PEG2000-UM206 with FZD1: The binding of FZD1 to UM206 and DSPE-PEG2000-UM206 was detected using Gromacs (version 2022.3) molecular dynamics simulations to explore the dynamic processes of molecule-protein interactions.
[0036] (1) Preparation of structure files: A search for the 3D structure of FZD1 in UniProt (https: / / www.uniprot.org / uniprotkb / Q9UP38 / entry#structure) yielded a result with UniProt ID Q9UP38. As of December 5, 2023, no experimentally obtained 3D structure for Q9UP38 was available; therefore, we downloaded the FZD1 structure file predicted by AlphaFold (https: / / alphafold.ebi.ac.uk / entry / Q9UP38). Furthermore, structure files for UM206 and DSPE-PEG2000-UM206 were created in Pymol (version 2.5.2).
[0037] (2) Simulation conditions: The experiment was conducted at atmospheric pressure (1 Bar) and static temperature (300 K), using an Amber99sb-ildn force field, with water molecules as the solvent (Tip3p water model) and the addition of Na. + Ions are used to neutralize the total charge of the simulated system.
[0038] (3) Molecular dynamics simulation system: After minimizing energy using the steepest descent method, isothermal and isochoric ensemble (NVT) equilibration and isothermal and isobaric ensemble (NPT) equilibration are performed for 100,000 steps respectively, with a coupling constant of 0.1 picoseconds and a duration of 100 picoseconds.
[0039] (4) Running the free molecular dynamics simulation: The process consists of 5,000,000 steps with a step size of 2 femtoseconds, lasting a total of 100 nanoseconds. After the simulation is completed, the trajectories are analyzed and the root mean square deviation (RMSD), solvent-accessible surface area (SASA), radius of gyration, hydrogen bond, and molecular mechanics Poisson-Boltzmann surface area (MM / PBSA) of each amino acid's motion trajectory are calculated.
[0040] After 100 nanoseconds of simulation, the interactions between UM206 and FZD1, as well as between DSPE-PEG2000-UM206 and FZD1, showed good performance, with considerable system stability and obvious interaction characteristics.
[0041] RMSD analysis: RMSD represents the distance between the same atom in different structures. A smaller RMSD value indicates higher protein stability, while a larger RMSD value indicates a change in the protein backbone's structural conformation during the simulation. Firstly, it can be seen that both UM206 and DSPE-PEG2000-UM206 can effectively dock with the extracellular region of the FZD1 protein (please refer to...). Figure 2 A- Figure 2 C). The RMSD curve shows that the system underwent a simulation of 7 × 10⁻⁶. 4 After picosecond treatment, the structure stabilized, with the RMSD of free UM206 around 0.5 nm and the RMSD of the FZD1 / UM206 complex around 1.25 nm. This indicates that the structures of the two free systems and the complex system are stable and in equilibrium. Figure 2 B). The mixture of DSPE-PEG2000-UM206 and FZD1 stabilized much earlier (approximately 1×10⁻⁶). 4 Picosecond laser). Initially, DSPE-PEG2000-UM206 showed a rapid increase due to its initial conformational change from long chains to clusters, exhibiting significant variations. However, it quickly stabilized, at which point the RMSD of the complex of FZD1 and DSPE-PEG2000-UM206 was around 1.5 nm. Figure 2 D).
[0042] SASA Analysis: SASA, or solvent-accessible biomolecular surface area, is used to assess the surface area of protein molecules exposed in solution. The results showed that the SASA values of both the FZD1 / UM206 and FZD1 / DSPE-PEG2000-UM206 systems were relatively high before binding, but decreased after binding and stabilized after 6 × 10⁴ picoseconds, indicating that the surface area of the protein decreased after binding. Figure 2 E).
[0043] Protein gyration radius analysis: Protein gyration radius is used to assess the overall compactness of proteins. The results showed that the protein gyration radius in the stabilized FZD1 / UM206 system was smaller than that in the FZD1 / DSPE-PEG2000-UM206 system, indicating that the FZD1 / UM206 structure was more compact. However, overall, the protein gyration radius in both systems decreased over time and then tended to stabilize. This demonstrates that the binding of both UM206 and DSPE-PEG2000-UM206 to FZD1 leads to a more compact protein molecule. Figure 2 F).
[0044] Hydrogen bond analysis: Hydrogen bonds and hydrophobic interactions play important roles in maintaining protein conformation. Furthermore, hydrogen bonds between amino acids can alter the internal geometry of proteins; therefore, we calculated the number of hydrogen bonds in the ligand-protein complex. In this simulation, the number of hydrogen bonds in both systems mostly remained between 5 and 15. FZD1 generated many hydrogen bonds in its interactions with UM206 or DSPE-PEG2000-UM206, indicating that the interaction between FZD1 protein and the peptide is strong and stable. Figure 2 G).
[0045] MM / PBSA analysis: We calculated the binding free energy and interactions using MM / PBSA to obtain the total free energy of the complex. The results showed that the combined total free energy of UM206 and FZD1 was -29.56 ± 5.62 kcal / mol, with an affinity of 2.03 × 10⁻⁶. - 22 The average combined total free energy of M, DSPE-PEG2000-UM206, and FZD1 is -33.80 ± 6.21, and the affinity at this point is 1.57 × 10⁻⁶. -25 M. Lower affinity indicates a stronger interaction between the target molecule and the ligand, meaning that both the UM206 peptide and DSPE-PEG2000-UM206 exhibit good interaction strength and stability with the protein. Figure 2 H).
[0046] Based on the above results, it can be concluded that the binding of FZD1 protein to UM206 or DSPE-PEG2000-UM206 makes the protein molecule more compact, reduces its surface area, and also forms many hydrogen bonds. The results indicate that there is a stable interaction between the two.
[0047] MALDI-TOF characterization of DSPE-PEG2000-UM206: Figure 3 These are MALDI-TOF mass spectra of DSPE-PEG2000-COOH, UM206, and DSPE-PEG2000-UM206. The theoretical molecular weight of DSPE-PEG2000-COOH is around 2000, and the detection results show that it is mainly concentrated between 2000 and 2750. Figure 3 A). UM206 is a 13-peptide (CNKTSEGMDGCEL) with a theoretical molecular weight of 1386. The measured value is 1386.660, which perfectly matches the theoretical value. Figure 3 B). The DSPE-PEG2000-UM206 product after the reaction showed characteristic peaks of 3500-4500, proving the successful synthesis of the material. Figure 3C).
[0048] Preparation of PLGA nanoparticles loaded with Cy5 NHS ester: (1) Preparation of lecithin anhydrous ethanol solution: Weigh 100mg of lecithin, dissolve it in 10mL of anhydrous ethanol, heat and vortex or use sonication to aid dissolution, to form a 10mg / mL lecithin anhydrous ethanol solution.
[0049] (2) Preparation of DSPE-PEG2000 solution: Weigh 9 mg of DSPE-PEG2000, dissolve it in 1 mL of PBS buffer, sonicate and vortex to aid dissolution, and form a 9 mg / mL DSPE-PEG2000 PBS solution. (3) Preparation of aqueous phase: Add 0.6 mL of 10 mg / mL anhydrous lecithin ethanol solution and 1 mL of 9 mg / mL DSPE-PEG2000 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 time, the solution is in microemulsion state.
[0050] (4) Preparation of Cy5 methanol solution: Accurately weigh 5 mg of Cy5 NHS ester and dissolve it in 1 mL of anhydrous methanol, protected from light. Vortex to mix well and prepare a 5 mg / mL Cy5 methanol solution.
[0051] (5) Preparation of organic phase: Weigh 50 mg PLGA and dissolve it in 1.9 mL acetonitrile. After mixing, add 0.1 mL of 5 mg / mL Cy5 methanol solution and vortex to mix in the dark.
[0052] (6) Nanoprecipitate self-assembly: In a round-bottom flask, the aqueous phase is rapidly stirred magnetically at room temperature in the dark. 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.
[0053] (7) Purification of nanoparticles: The solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 4000×g for 20 minutes. The solution was washed three times with PBS to obtain PLGA nanoparticles loaded with Cy5 NHS ester, namely Cy5 / PLGA nanoparticles (hereinafter referred to as CP).
[0054] Preparation of the FZD1 actively targeted nanoparticles loaded with Cy5 NHS ester: (1) Preparation of lecithin anhydrous ethanol solution: Weigh 100mg of lecithin, dissolve it in 10mL of anhydrous ethanol, heat and vortex or use sonication to aid dissolution, to form a 10mg / mL lecithin anhydrous ethanol solution.
[0055] (2) Preparation of DSPE-PEG2000-UM206 solution: Weigh 5mg DSPE-PEG2000 and 4mg DSPE-PEG2000-UM206 (total mass 9mg), dissolve in 1mL PBS buffer, sonicate and vortex to aid dissolution, and form 9mg / mL DSPE-PEG2000-UM206 PBS solution.
[0056] (3) Preparation of aqueous phase: Add 0.6 mL of 10 mg / mL anhydrous lecithin ethanol solution 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 time, the solution is in microemulsion state.
[0057] (4) Preparation of Cy5 methanol solution: Accurately weigh 5 mg of Cy5 NHS ester and dissolve it in 1 mL of anhydrous methanol, protected from light. Vortex to mix well and prepare a 5 mg / mL Cy5 methanol solution.
[0058] (5) Preparation of organic phase: Weigh 50 mg PLGA and dissolve it in 1.9 mL acetonitrile. After mixing, add 0.1 mL of 5 mg / mL Cy5 methanol solution and vortex to mix in the dark.
[0059] (6) Nanoprecipitate self-assembly: In a round-bottom flask, the aqueous phase is rapidly stirred magnetically at room temperature in the dark. 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.
[0060] (7) 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 Cy5 / PLGA@UM206 nanoparticles, namely the FZD1 active targeting nanoparticles loaded with Cy5 NHS ester (hereinafter referred to as CPU).
[0061] Characterization of actively targeted nanoparticles: Particle size determination of various PLGA nanoparticles, namely PLGA nanoparticles, PLGA@UM206 nanoparticles, CP, and CPU: 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 of the nanoparticles.
[0062] Malvern particle size analysis showed that the average particle sizes of unloaded PLGA and PLGA@UM206 nanoparticles were 173.4 ± 3.868 nm, respectively. Figure 4 A) and 181.5±3.118nm ( Figure 4(B) indicates that the nanoparticle size slightly increased after attaching the active targeting unit DSPE-PEG2000-UM206. Furthermore, the CP and CPU particle sizes also increased after encapsulating Cy5 NHS ester, reaching 192.6 ± 4.05 nm, respectively. Figure 4 C) and 201±2.998nm ( Figure 4 D). These four groups of nanoparticles have a narrow distribution and uniform particle size, and can be used for subsequent experiments.
[0063] Transmission electron microscopy results of CP and CPU: CP and CPU 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.
[0064] Transmission electron microscopy revealed that the CP nanoparticles were regular spherical in shape, with full morphology and clear edges. The CPU nanoparticles were also regular spherical, with indistinct edges and a faint outer "coating layer." The particle sizes of both nanoparticles were consistent with Malvern's particle size results. Figure 5 ).
[0065] An experiment on the uptake of CP and CPU nanoparticles by osteosarcoma HOS cells: Cell lines: Human osteosarcoma cell lines HOS, 143B, and MNNG / HOS (in this invention, 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.
[0066] Cell treatment: (1) Preparation of nanoparticles: Take 50 μL of synthesized CP and CPU nanoparticles and add them to 2 mL of methanol. Vortex the nanoparticles to destroy them. Prepare standard curves with different concentrations of Cy5 methanol solutions and then use a fluorescence spectrophotometer to measure the Cy5 content in the nanoparticles.
[0067] (2) Cell preparation: HOS cells were seeded in confocal dishes at a rate of 1×10⁵ cells / dish in advance and cultured in a cell culture incubator for 12 hours.
[0068] (3) Drug administration: After preparing complete culture medium with nanoparticles of each group at a concentration of 2 μg / mL Cy5, the nanoparticles were added to a confocal dish. After incubation for 0, 0.5, 1, 2, 4 and 8 hours, the culture medium was discarded and the cells were cleared three times with PBS warmed to 37℃.
[0069] Laser confocal microscopy was used to investigate the uptake of nanoparticles by HOS: (1) Process the cells according to the cell processing procedure described above.
[0070] (2) Cell fixation and blocking: Fix with 4% paraformaldehyde for 10 minutes and wash 3 times with TBST, then block with QuickBlock™ immunostaining blocking solution for 10-30 minutes.
[0071] (3) Staining: After removing the blocking solution, add 200 μL of diluted phalloidin working solution and incubate at room temperature in the dark for 45 minutes. Then stain with DAPI at room temperature for 5 minutes, and wash the cells three times with TBST on a shaker for 5 minutes each time.
[0072] The anti-fluorescence quenching sealing solution was added to the confocal dish for laser confocal imaging and observation.
[0073] The uptake of CP and CPU nanoparticles by HOS cells at different time points was observed by laser confocal microscopy. Figure 6 No Cy5 red fluorescence was observed in cells without the addition of nanoparticles (0 hours). After co-incubation for 0.5 hours, Cy5 fluorescence from nanoparticles in both groups began to appear on the cell membranes of HOS cells, and the number of nanoparticles in HOS cells increased with prolonged incubation time, exhibiting a time-dependent change. We observed that at the same time point (e.g., 1 hour), HOS cells exhibited more cytotoxicity of CPU than CP cells. Furthermore, CPU cells appeared in the cytoplasm at 2 hours, and there was more Cy5 fluorescence signal in the cytoplasm at 4 and 8 hours. In contrast, the CP group's nanoparticles only showed significant cytoplasmic localization after 4 hours. The results suggest that the addition of the active targeting unit DSPE-PEG2000-UM206 promotes the endocytosis of nanoparticles by HOS cells. In addition, we found that CP and CPU nanoparticles aggregated in HOS cells, and this aggregation did not disappear even after 8 hours.
[0074] Flow cytometry was used to investigate the uptake of nanoparticles by HOS: (1) Process the cells according to the above cell processing procedure, digest with trypsin, centrifuge and resuspend in PBS.
[0075] (2) The fluorescence intensity of Cy5 was directly detected by flow cytometry.
[0076] As co-incubation time increases, CP ( Figure 7 A) and CPU ( Figure 7 B) The proportion of positive HOS cells is increasing. Quantitative analysis of the average fluorescence intensity shows that the phagocytosis of both nanoparticles by HOS cells is time-dependent. Furthermore, from 0.5 hours onwards, the average fluorescence intensity of the CPU group is higher than that of the CP group (…). Figure 7 C). The above results suggest that the DSPE-PEG2000-UM206 targeting unit can promote the phagocytosis of PLGA nanoparticles by HOS cells.
[0077] Statistical methods: All data from this experiment were analyzed using SPSS 26.0 statistical software. Two-way ANOVA and Bonferroni tests were used for inter-group comparisons. A p-value less than 0.05 was considered statistically significant. The following symbols were used to represent the significance level: NS (no significance) indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.
[0078] Cellular uptake experiments of OCSCs on CP and CPU nanoparticles: OCSC tumor spheres cultured for 7 days were isolated using a 70 μm sterile filter and washed once with PBS. Two groups of nanoparticles were prepared in serum-free tumor stem cell culture medium at a concentration of 2 μg / mL Cy5, and OCSCs were co-incubated with the nanoparticles for 0, 0.5, 1, 2, 4, and 8 hours, with nuclei stained using Hoechst 33342 live cell staining solution during the last 10 minutes. The co-incubated OCSC spheres were then isolated again using a 70 μm sterile filter and washed 2-3 times with PBS. Subsequently, the tumor spheres were seeded into confocal dishes under a microscope, and 3D scanning was performed using laser confocal microscopy.
[0079] To observe the phagocytic effect of OCSCs on nanoparticles, we performed 3D scanning of OCSC spheres for nanoparticle uptake. Figure 8 It can be seen that after 0.5 hours of co-incubation, the actively targeted nanoparticles (CPU) already showed a significant Cy5 fluorescence signal, and the Cy5 fluorescence intensity in the OCSCs spheres increased with the extension of CPU co-incubation time. However, the Cy5 fluorescence signal of the non-targeted CP nanoparticles was very weak at 0.5 hours. Although the intensity of CP in OCSCs also increased with time, the Cy5 fluorescence signal was still not very obvious at 4 hours or 8 hours. The results show that both CP and CPU increased with the extension of co-incubation time, indicating that the OCSCs spheres have a time-dependent phagocytosis effect on both types of nanoparticles. However, there was a significant difference in phagocytosis efficiency between the two groups of nanoparticles, proving that DSPE-PEG2000-UM206 can effectively promote the uptake of nanoparticles by OCSCs.
[0080] In the above section, we performed molecular dynamics simulations of UM206 with FZD1 protein before and after coupling with DSPE-PEG2000. The results showed that DSPE-PEG2000-UM206 also had a good interaction with FZD1. Therefore, we chemically coupled the hydrophilic peptide UM206 to the carboxyl terminus of DSPE-PEG2000-COOH to target FZD1 in osteosarcoma. The polymer nanoparticles were prepared using a one-step modified nanoprecipitation self-assembly method. The polymer and hydrophobic compound were dissolved in an organic solvent (a water-miscible organic solvent, such as DMSO, methanol, acetonitrile, etc.). Then, the organic phase was dropwise injected into an aqueous phase containing lipid DSPE-PEG2000-UM206, vortexed, and then homogenized to reduce the particle size to the nanoscale. Finally, solid polymer nanoparticles with a "core-shell" structure were obtained. Based on this method, the obtained polyFZD1 actively targeted nanoparticles have a three-layer structure, including: 1. a polymer material encapsulating a hydrophobic compound in the nanoparticle core; 2. a lipid-stabilizing layer formed by a middle monolayer of lecithin; and 3. an outermost hydrophilic outer layer surrounded by DSPE-PEG2000-UM206. The active targeting unit DSPE-PEG2000-UM206 can effectively bind to FZD1. Adding the active targeting unit to the nanoparticles promotes the uptake of PLGA nanoparticles in osteosarcoma cells and increases their phagocytic efficiency in OCSCs. In other words, the FZD1 actively targeted nanoparticles prepared by the method of this invention can promote the phagocytosis of PLGA nanoparticles by osteosarcoma cells, and OCSCs exhibit high phagocytic efficiency of the FZD1 actively targeted nanoparticles prepared by the method of this invention.
[0081] 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 FZD1 actively targeted nanoparticles, characterized in that, Includes the following steps: S1. Synthesize DSPE-PEG2000-UM206 using DSPE-PEG2000-COOH and polypeptide UM206; S2. FZD1 active targeting nanoparticles were synthesized using polylactic acid-glycolic acid copolymer and DSPE-PEG2000-UM206. The FZD1 active targeting nanoparticles comprise a three-layer structure, consisting of a polymer material encapsulating a hydrophobic compound in the nanoparticle core, a lipid-stabilizing layer formed by a middle monolayer of lecithin, and an outermost hydrophilic outer layer surrounded by DSPE-PEG2000-UM206.
2. The method for preparing FZD1 actively targeted nanoparticles according to claim 1, characterized in that, The sequence of the polypeptide UM206 is: CNKTSEGMDGCEL.
3. The method for preparing FZD1 actively targeted nanoparticles according to claim 1, characterized in that, The method for synthesizing DSPE-PEG2000-UM206 using DSPE-PEG2000-COOH and polypeptide UM206 is as follows: S11. Activate the carboxyl group of DSPE-PEG2000-COOH in a round-bottom flask; S12, Dehydration condensation: Weigh 20 mg of UM206 lyophilized peptide, add it to a round-bottom flask and stir magnetically for 28 hours. S13, Dialysis: Transfer the product to a 2 kDa dialysis bag and dialyze for 48 hours to remove the catalyst and DMSO; S14. Ultrafiltration: Centrifuge the dialyzed liquid at 4000xg and 4℃ for 20 minutes using a 3K Da ultrafiltration tube to remove unreacted DSPE-PEG2000, UM206 or other byproducts. S15. Store after freeze-drying.
4. The method for preparing FZD1 actively targeted nanoparticles according to claim 3, characterized in that: The freeze-dried DSPE-PEG2000-UM206 should be stored at -20℃.
5. The method for preparing FZD1 actively targeted nanoparticles according to claim 3, characterized in that, The method for activating the carboxyl group of DSPE-PEG2000-COOH is as follows: 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 NHS into the round-bottom flask, and stir magnetically at room temperature for 4 hours.
6. The method for preparing FZD1 actively targeted nanoparticles according to claim 3, characterized in that: Before S11, clean and dry the round-bottom flask, magnetic stirrer, and bottle cap.
7. The method for preparing FZD1 actively targeted nanoparticles according to claim 1, characterized in that, The method for synthesizing FZD1 actively targeted nanoparticles using polylactic acid-glycolic acid copolymer and DSPE-PEG2000-UM206 is as follows: S21. Preparation of anhydrous ethanol solution of lecithin: Weigh 100 mg of lecithin, dissolve it in 10 mL of anhydrous ethanol, heat and vortex or use sonication to aid dissolution, to form an anhydrous ethanol solution of lecithin with a concentration of 10 mg / mL. Preparation of S22 and DSPE-PEG2000-UM206 solution: Weigh 5 mg of DSPE-PEG2000 and 4 mg of 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. S23. 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 time, the solution is in microemulsion form. S24. Preparation of organic phase: Weigh 50 mg PLGA and dissolve it in 1.9 mL acetonitrile, then vortex to mix. S25, 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. S26. The nanoparticles were purified to obtain the FZD1 active targeting nanoparticles.
8. The method for preparing FZD1 actively targeted nanoparticles according to claim 7, characterized in that: The method for purifying the nanoparticles is as follows: the solution is transferred to a 30 kDa ultrafiltration tube, centrifuged at 4000×g for 20 minutes, and washed three times with PBS.