Carrier-free co-assembled nanoparticles, preparation method and application thereof

Abstract

The application discloses a carrier-free co-assembly nanoparticle and a preparation method and application thereof, and belongs to the field of nanomedicine.The carrier-free co-assembly nanoparticle is a co-assembly nanoparticle of a chemotherapeutic drug and gossypol, a co-assembly nanoparticle of a chemotherapeutic drug and gossypol modified by a polyethylene glycol modifier, or a co-assembly nanoparticle of a chemotherapeutic drug and gossypol loaded with a hydrophobic fluorescent substance; the chemotherapeutic drug includes one or more than two of cabazitaxel, docetaxel, paclitaxel, doxorubicin hydrochloride, epirubicin hydrochloride, 5-fluorouracil, cisplatin, oxaliplatin, irinotecan, SN38 and camptothecin.The application first constructs a carrier-free hybrid nano-assembly of gossypol and a chemotherapeutic drug, gossypol can significantly enhance the sensitivity of tumor cells to clinically common chemotherapeutic drugs under a non-cytotoxic dose condition, and the gossypol-driven chemosensitization has a good selectivity between normal cells and cancer cells, which is expected to create a favorable therapeutic window for safe and effective chemotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a carrier-free co-assembled nanoparticle, its preparation method, and its application. Background Technology

[0002] Prostate cancer (PCa) is one of the most common malignant tumors, ranking second among cancer-related deaths in men worldwide. Currently, clinical treatments for PCa include surgery, chemotherapy, endocrine therapy, cryotherapy, and radiotherapy, with chemotherapy being the most commonly used treatment. However, most chemotherapy drugs suffer from narrow therapeutic windows, poor pharmacokinetics, non-specific distribution in the body, and significant toxic side effects. In recent years, the development of nanomedicine delivery technology has helped address the inherent limitations of chemotherapy drugs, improving their adverse physicochemical properties to some extent and prolonging their circulation time in the body, potentially providing more new treatment options for prostate cancer patients. However, nanoformulations may have issues such as low drug loading, premature drug leakage, excipient-related toxicity, and insufficient accumulation at the tumor site, leading to poor efficacy. Therefore, the development of highly effective and safe novel chemotherapy strategies has attracted our attention.

[0003] Carrier-free nanomedicine delivery systems, constructed independently of carrier materials and assembled from pharmacologically active compounds, avoid carrier-related toxicity and significantly improve drug loading efficiency. Furthermore, their formulation and manufacturing processes are relatively simple, facilitating large-scale production. Simultaneously, carrier-free co-assembly nanotechnology can be used for combination therapy, enabling precise control of drug co-loading ratios when co-delivering multiple drugs, demonstrating promising application prospects.

[0004] Therefore, how to construct hybrid nanoassemblies containing chemosensitizers and chemotherapeutic drugs based on carrier-free nanomedicine delivery systems to achieve combined therapy is an important topic that urgently needs to be studied. Summary of the Invention

[0005] The purpose of this invention is to design and construct a co-assembled nanomedicine delivery system of chemotherapeutic drugs and gossypol, and its application in drug delivery, and to provide a carrier-free co-assembled nanoparticle, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A carrier-free co-assembled nanoparticle, wherein the carrier-free co-assembled nanoparticle is a co-assembled nanoparticle of a chemotherapeutic drug and gossypol, a co-assembled nanoparticle of a chemotherapeutic drug modified with a polyethylene glycol modifier and gossypol, or a co-assembled nanoparticle of a chemotherapeutic drug loaded with a hydrophobic fluorescent substance and gossypol; wherein the chemotherapeutic drug includes one or more of cabazitaxel, docetaxel, paclitaxel, doxorubicin hydrochloride, epirubicin hydrochloride, 5-fluorouracil, cisplatin, oxaliplatin, irinotecan, SN38, gemcitabine, and camptothecin.

[0008] This invention also discloses a method for preparing carrier-free co-assembled nanoparticles as described above, comprising the following steps: dissolving a chemotherapeutic drug and gossypol, or a chemotherapeutic drug, gossypol and a polyethylene glycol modifier, or a chemotherapeutic drug, gossypol, a polyethylene glycol modifier and a fluorescent substance in an organic solvent to obtain a mixed solution; adding the mixed solution dropwise to water to spontaneously form uniform nanoparticles; and then removing the organic solvent by rotary evaporation to obtain carrier-free co-assembled nanoparticles.

[0009] The present invention also discloses the application of carrier-free co-assembled nanoparticles as described above, or carrier-free co-assembled nanoparticles obtained by the preparation method described above, in a drug delivery system.

[0010] The present invention also discloses the application of carrier-free co-assembled nanoparticles as described above, or carrier-free co-assembled nanoparticles obtained by the preparation method described above, in the preparation of antitumor drugs.

[0011] The present invention also discloses the application of carrier-free co-assembled nanoparticles as described above, or carrier-free co-assembled nanoparticles obtained by the preparation method described above, in injection, oral or topical drug delivery systems.

[0012] This invention utilizes carrier-free self-assembly technology to design and construct a binary hybrid nanoassembly formed by co-assembling chemotherapeutic drugs and chemotherapeutic sensitizer gossypol (GSP) for synergistic sensitization chemotherapy regimens. Implementing embodiments of this invention will have the following beneficial effects: (1) This invention is the first to construct a carrier-free hybrid nanoassembly of chemotherapeutic sensitizer GSP and chemotherapeutic drugs. GSP can significantly enhance the sensitivity of tumor cells to commonly used clinical chemotherapeutic drugs under non-cytotoxic dosage conditions, and the GSP-driven chemotherapeutic effect has good selectivity between normal cells and cancer cells, which is expected to create a favorable therapeutic window for safe and effective chemotherapy. (2) Utilizing the molecular engineering nanoassembly characteristics of chemotherapeutic sensitizer GSP and chemotherapeutic drugs, the constructed carrier-free binary hybrid nanoassembly has ultra-high drug loading capacity and synchronous drug delivery characteristics. After accumulating in large quantities in tumor tissue through the EPR effect, it specifically releases chemotherapeutic drugs under the reduced microenvironment conditions at the tumor site, thereby exerting a synergistic sensitization chemotherapy effect. It has reduction-sensitive response drug release characteristics, which is conducive to achieving highly efficient and low-toxicity anti-tumor effects. (3) The carrier-free binary hybrid nanoassemblies constructed by the present invention through precise hybridization nanoassemblies can promote tumor cell apoptosis and inhibit tumor cell proliferation by downregulating the expression of the anti-apoptotic protein Bcl-2, thereby exerting a synergistic anti-tumor effect of sensitizing chemotherapy. (4) The preparation process is simple, safe, non-toxic and easy to industrialize by using a one-step nanoprecipitation method. Attached Figure Description

[0013] Figure 1 The graph shows the cytotoxicity results of GSP on RM-1, 4T1, 3T3 and L02 cells in Example 1.

[0014] Figure 2 This is a graph showing the results of GSP enhancing the chemosensitivity of RM-1 cells to CTX in Example 1.

[0015] Figure 3 This is a graph showing the results of GSP enhancing the chemosensitivity of RM-1 cells to DTX in Example 1.

[0016] Figure 4 This is a diagram showing the results of GSP enhancing the chemosensitivity of RM-1 cells to DOX in Example 1.

[0017] Figure 5 This is a diagram showing the results of GSP enhancing the chemosensitivity of RM-1 cells to OXP in Example 1.

[0018] Figure 6 This is a graph showing the results of GSP enhancing the chemosensitivity of RM-1 cells to DDP in Example 1.

[0019] Figure 7This is a diagram showing the results of GSP enhancing the chemosensitivity of RM-1 cells to GEM in Example 1.

[0020] Figure 8 This is a graph showing the results of GSP enhancing the chemosensitivity of 4T1 cells to CTX in Example 1.

[0021] Figure 9 This is a graph showing the results of GSP enhancing the chemosensitivity of 3T3 cells to CTX in Example 1.

[0022] Figure 10 This is a graph showing the results of GSP enhancing the chemosensitivity of L02 cells to CTX in Example 1.

[0023] Figure 11 IC of hybrid nanoassemblies with different molar ratios as described in Example 2 50 Value results (using RM-1 cells as a model).

[0024] Figure 12 IC of hybrid nanoassemblies with different molar ratios as described in Example 2 50 Value results (using 4T1 cells as a model).

[0025] Figure 13 This is an appearance diagram of the CTX / GSP hybrid nanoassembly of Example 3.

[0026] Figure 14 This is a transmission electron microscope image of the CTX / GSP hybrid nanoassembly of Example 3.

[0027] Figure 15 This is a molecular docking simulation diagram of the CTX / GSP hybrid nanoassembly in Example 4.

[0028] Figure 16 This is a diagram showing the disruption of molecular forces in the CTX / GSP hybrid nanoassembly of Example 4.

[0029] Figure 17 This is a physical stability diagram of the CTX / GSP hybrid nanoassembly in Example 5.

[0030] Figure 18 This is an in vitro drug release diagram of the CTX / GSP hybrid nanoassembly in Example 6.

[0031] Figure 19 These are confocal microscopy images of the CTX / GSP hybrid nanoassembly of Example 8 at 0.5h and 4h.

[0032] Figure 20 Flow cytometry quantification of cellular uptake at 0.5 h and 4 h for the CTX / GSP hybrid nanoassembly of Example 8.

[0033] Figure 21 The results show the cytotoxicity of the CTX / GSP hybrid nanoassemblies in Example 9 against RM-1 cells.

[0034] Figure 22 The results show the cytotoxicity of the CTX / GSP hybrid nanoassembly of Example 9 on 4T1 cells.

[0035] Figure 23 The results show the cytotoxicity of the CTX / GSP hybrid nanoassembly of Example 9 on 3T3 cells.

[0036] Figure 24 The results show the cytotoxicity of the CTX / GSP hybrid nanoassembly of Example 9 on L02 cells.

[0037] Figure 25 The blood drug concentration-time curve of the CTX / GSP hybrid nanoassembly in Example 10 is shown.

[0038] Figure 26 The tissue distribution of the CTX / GSP hybrid nanoassembly in Example 12 is shown in in vivo fluorescence imaging.

[0039] Figure 27 The tissue distribution of the CTX / GSP hybrid nanoassembly in Example 12 (in vitro fluorescence imaging).

[0040] Figure 28 The image shows a mouse tumor in an in vivo antitumor experiment of the CTX / GSP hybrid nanoassembly in Example 13.

[0041] Figure 29 This is a mouse tumor growth curve from the in vivo antitumor experiment of the CTX / GSP hybrid nanoassembly in Example 13.

[0042] Figure 30 This is a statistical graph showing the tumor bearing rate in mice during the in vivo antitumor experiment of the CTX / GSP hybrid nanoassemblies in Example 13.

[0043] Figure 31 The images show the TUNEL and H&E staining of tumor tissues in the in vivo antitumor experiment of the CTX / GSP hybrid nanoassemblies in Example 13.

[0044] Figure 32 This is a graph showing the weight changes of tumor-bearing mice in Example 14.

[0045] Figure 33 The results are the liver and kidney function analysis results of tumor-bearing mice in Example 14.

[0046] Figure 34These are pathological sections of other tissues and organs from the tumor-bearing mice in Example 14. Detailed Implementation

[0047] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0048] The inventors discovered that GSP can significantly enhance the sensitivity of tumor cells to commonly used clinical chemotherapeutic drugs under non-cytotoxic dosage conditions. To further explore the feasibility and related mechanisms of GSP-sensitized chemotherapy, the inventors selected chemotherapeutic drugs used in the clinical treatment of prostate cancer as model drugs and designed and constructed carrier-free hybrid nanoassemblies of GSP and chemotherapeutic drugs using small molecule nanoassembly technology. In this model, GSP acts as a chemotherapeutic agent. The inventors found that the GSP-driven chemotherapeutic effect exhibits good selectivity between normal cells and cancer cells, which holds promise for creating a favorable therapeutic window for safe and effective chemotherapy.

[0049] Therefore, the present invention obtained by the inventors based on these insights is as follows.

[0050] The inventors have designed and constructed a binary hybrid nanoassembly formed by co-assembling a chemotherapeutic drug and a chemotherapeutic sensitizer, gossypol (GSP), using carrier-free self-assembly technology for synergistic sensitization of chemotherapy regimens. Based on this, the present invention discloses a carrier-free co-assembled nanoparticle, which can be a chemotherapeutic drug and gossypol co-assembled nanoparticle, a chemotherapeutic drug and gossypol co-assembled nanoparticle modified with a polyethylene glycol modifier, or a chemotherapeutic drug and gossypol co-assembled nanoparticle loaded with a hydrophobic fluorescent substance.

[0051] Furthermore, the chemotherapy drugs include one or more of cabazitaxel (CTX), docetaxel (DTX), paclitaxel (CTX), doxorubicin hydrochloride (DOX), epirubicin hydrochloride (ADR), 5-fluorouracil (5-FU), cisplatin (DDP), oxaliplatin (OXP), irinotecan (IR), SN38, gemcitabine (GEM), and camptothecin (CPT). Preferably, the chemotherapy drug is CTX.

[0052] In one specific embodiment, the molar ratio of the chemotherapeutic drug to gossypol is 1:20 to 20:1. Preferably, the molar ratio of the chemotherapeutic drug to gossypol is 1:3.

[0053] In one specific embodiment, the molar ratio of the chemotherapy drug to the polyethylene glycol modifier is 60:40 to 90:10.

[0054] In one specific embodiment, the molar ratio of the chemotherapeutic drug to the fluorescent substance is 60:40 to 90:10.

[0055] In one specific embodiment, the polyethylene glycol modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG, and PE-PEG. Preferably, the polyethylene glycol modifier is DSPE-PEG. 2K and / or DSPE-SS-PEG 2K .

[0056] In one specific embodiment, the molecular weight of the polyethylene glycol modifier is 200 to 20,000.

[0057] In one specific embodiment, the fluorescent material includes one or more of coumarin-6, rhodamine, DiR, DiI, Cy5, and Cy7.

[0058] In one specific embodiment, the chemotherapy drug and gossypol are co-assembled through π-π stacking, hydrophobic interactions, and intermolecular hydrogen bonds.

[0059] This invention also discloses a method for preparing carrier-free co-assembled nanoparticles as described in any embodiment of this invention, comprising the following steps:

[0060] When the carrier-free co-assembled nanoparticles are co-assembled nanoparticles of chemotherapy drugs and gossypol, the preparation method includes the following steps: dissolving the chemotherapy drugs and gossypol in an organic solvent to obtain a mixed solution; adding the mixed solution dropwise to water to spontaneously form uniform nanoparticles; and then removing the organic solvent by rotary evaporation to obtain carrier-free co-assembled nanoparticles.

[0061] When the carrier-free co-assembled nanoparticles are chemotherapeutic drugs modified with polyethylene glycol and gossypol co-assembled nanoparticles, the preparation method includes the following steps: dissolving the chemotherapeutic drug, gossypol and polyethylene glycol modifier in an organic solvent to obtain a mixed solution; adding the mixed solution dropwise to water to spontaneously form uniform nanoparticles; and then removing the organic solvent by rotary evaporation to obtain carrier-free co-assembled nanoparticles.

[0062] When the carrier-free co-assembled nanoparticles are chemotherapeutic drugs and gossypol co-assembled nanoparticles loaded with hydrophobic fluorescent substances, the preparation method includes the following steps: dissolving chemotherapeutic drugs, gossypol, polyethylene glycol modifiers and fluorescent substances in an organic solvent to obtain a mixed solution; adding the mixed solution dropwise to water to spontaneously form uniform nanoparticles; and then removing the organic solvent by rotary evaporation to obtain carrier-free co-assembled nanoparticles.

[0063] In one specific embodiment, the organic solvent includes one or more of methanol, ethanol, tetrahydrofuran, and dimethyl sulfoxide. Preferably, the organic solvent is tetrahydrofuran / anhydrous ethanol (V / V) = 1:1.

[0064] The present invention also discloses the application of carrier-free co-assembled nanoparticles as described in any embodiment of the present invention, or carrier-free co-assembled nanoparticles obtained by the preparation method as described in any embodiment of the present invention, in drug delivery systems, preparation of antitumor drugs, injection administration, oral administration, or local administration systems.

[0065] Specifically, this invention utilizes the molecular engineering nanoassembly characteristics of the chemosensitizer GSP and chemotherapeutic drugs to construct, for the first time, a carrier-free hybrid nanoassembly of chemosensitizer GSP and chemotherapeutic drugs. This assembly possesses ultra-high drug loading capacity and simultaneous drug delivery, exerting a synergistic chemosensitizing effect and exhibiting reduction-sensitive response drug release characteristics, which is beneficial for achieving highly efficient and low-toxicity anti-tumor effects. Simultaneously, the preparation process is simple, safe, and free of toxic side effects, making it easy to industrialize. Furthermore, it can promote tumor cell apoptosis and inhibit tumor cell proliferation by downregulating the expression of the anti-apoptotic protein Bcl-2, thereby exerting a synergistic chemosensitizing anti-tumor effect. It shows promising application prospects in drug delivery systems, tumor treatment, injection administration, oral administration, or local administration systems.

[0066] The following are specific embodiments.

[0067] Example 1: GSP-sensitized chemotherapy and its selective sensitization verification

[0068] Using RM-1 cells (mouse prostate cancer cells), 4T1 cells (mouse breast cancer cells), 3T3 cells (mouse embryonic fibroblasts), and L02 cells (human normal hepatocytes) as cell models, the cytotoxicity of GSP solution to the four different cell lines was investigated using the MTT assay. The chemosensitizing effect of GSP at a non-cytotoxic dose (500 nM) on CTX was also investigated.

[0069] Subsequently, using RM-1 cells as a tumor cell model, the sensitizing effect of GSP at a non-cytotoxic dose (500 nM) on various other commonly used chemotherapeutic drugs (DTX, DOX, OXP, DDP, GEM) was investigated. After digestion of healthy cells, an appropriate amount of freshly prepared culture medium was added to prepare a cell suspension of a certain concentration (approximately 10,000 cells / mL). The cell suspension was mixed thoroughly and added to 200 μL of cell suspension per well in a 96-well plate, and cultured in a cell culture incubator (37℃, 5% CO2) for 12–24 h. Then, drug-containing cell culture medium (200 μL / well) was added. Drug-free culture medium was added to the wells seeded with cells as control wells, and drug-free culture medium was added to the unseeded wells as zeroing wells (n=4), and culture continued for 48 h. Then add 20 μL of the prepared MTT solution and continue incubation in a cell culture incubator for 4 h. After incubation, discard the supernatant, add 200 μL of DMSO to each well, shake for 10 min, and measure the absorbance at 490 nm using a microplate reader.

[0070] Cytotoxicity results such as Figures 1-10 As shown, at micromolar concentrations, GSP alone exhibited almost no cytotoxicity against RM-1 mouse prostate cancer cells, 4T1 mouse breast cancer cells, 3T3 mouse embryonic fibroblasts, and L02 human hepatocytes. Figure 1 Interestingly, we found that GSP at an ineffective concentration of 0.5 μM significantly enhanced the sensitivity of RM-1 cells to CTX, DTX, DOX, OXP, DDP, and GEM. Figures 2-7 Among them, GSP has the most significant sensitizing effect on CTX chemotherapy, promoting the IC50 of CTX. 50 The value decreased by 100 times or more. Figure 2 and Figure 8 More importantly, under the same conditions, this chemosensitizing effect was not observed in the two normal cell lines, 3T3 and L02. Figure 9 and Figure 10 As shown, GSP had little effect on the cytotoxicity of CTX in these two normal cell lines. These results indicate that GSP-driven chemosensitization exhibits good selectivity between normal and cancer cells, potentially creating a favorable therapeutic window for safe and effective chemotherapy.

[0071] Example 2 GSP n / CTX n Screening for optimal dosage ratios of hybrid nanoassemblies

[0072] Preparation of CG NPs with different molar ratios: Hybrid nanoparticles with different molar ratios (GSP:CTX(n / n) = 20:1, 15:1, 10:1, 8:1, 6:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:6, 1:8, 1:10, 1:15, 1:20) were prepared by a one-step nanoprecipitation method. The specific preparation process is as follows: 2 mg of CTX powder and 2 mg of GSP powder were accurately weighed and dissolved in 400 μL of organic solvent (tetrahydrofuran / anhydrous ethanol (V / V) = 1:1) to prepare 5 mg / mL CTX stock solution and 5 mg / mL GSP stock solution. According to the required molar ratio, the corresponding amounts of GSP stock solution and CTX stock solution were taken, and then 100 μL of mixed organic solvent (tetrahydrofuran / anhydrous ethanol (V / V) = 1:1) was added. After vortex mixing, a mixed stock solution was obtained. Add 2 mL of deionized water and a stir bar to a 5 mL vial, place it on a magnetic stirrer, and add 300 μL of the mixed mother liquor dropwise while stirring vigorously. Continue stirring for 3 min. Then, transfer the formulation containing organic solvent to a 25 mL round-bottom flask, remove the organic solvent under reduced pressure using a rotary evaporator at 30 °C, and then add an appropriate amount of deionized water to make up to 2 mL. This yields hybrid nanoparticles (CG NPs) with different molar ratios of 0.5 mg / mL, which are then stored at 4 °C. The particle size and particle size distribution of the prepared nanoparticles are shown in Table 1.

[0073] Table 1 GSP with different GSP / CTX molar ratios n / CTX n Particle size and particle size distribution of hybrid nanoassemblies

[0074]

[0075] Table 1 shows that the nanoparticle size is concentrated between 90 nm and 150 nm. Further, the MTT assay was used to investigate the cytotoxicity of hybrid nanoparticles with different molar ratios of GSP and CTX on tumor cells to determine the optimal dosage ratio. Using RM-1 and 4T1 cells as cell models, the prepared hybrid nanoparticles with different molar ratios (GSP / CTX = 20:1, 15:1, 10:1, 8:1, 6:1, 5:1, 3:1, 1:1) were diluted with prepared culture media to create a series of drug-containing media with varying concentrations, and the IC50 of the hybrid nanoparticles with different molar ratios was determined using the MTT assay. 50 The result is as follows Figure 11 and Figure 12 As shown, when the molar ratio of GSP to CTX is 3:1, its IC 50The lowest value indicates the strongest tumor cell killing ability. Therefore, considering both the assembly ability and the synergistic effect of the hybrid nanoassemblies in enhancing chemosensitivity, a GSP to CTX molar ratio of 3:1 was ultimately determined to be the optimal dose ratio.

[0076] Example 3: Preparation of CTX / GSP hybrid nanoassemblies

[0077] Preparation of non-PEGylated hybrid nanoassemblies (np-GC NAs): 2 mg of CTX powder and 2 mg of GSP powder were accurately weighed and dissolved in 400 μL of organic solvent (tetrahydrofuran / anhydrous ethanol (V / V) = 1:1) to prepare 5 mg / mL CTX stock solutions and 5 mg / mL GSP stock solutions, respectively. 130 μL of the GSP stock solution and 70 μL of the CTX stock solution were then added, followed by the addition of 100 μL of mixed organic solvent (tetrahydrofuran / anhydrous ethanol (V / V) = 1:1). The mixture was vortexed until homogeneous, yielding 300 μL of mixed stock solution. Add 2 mL of deionized water and a stir bar to a 5 mL vial, place it on a magnetic stirrer, and add 300 μL of the mixed mother liquor dropwise while stirring vigorously. Continue stirring for 3 min. Then transfer the formulation containing organic solvent to a 25 mL round-bottom flask, remove the organic solvent under reduced pressure using a rotary evaporator at 30 °C, and then add an appropriate amount of deionized water to make up to 2 mL to obtain np-GCNAs.

[0078] Preparation of PEG-modified hybrid nanoassemblies (p-GC NAs): The preparation method of p-GC NAs differs from that of np-GC NAs in that: 130 μL of GSP stock solution and 70 μL of CTX stock solution are taken, and then 57 μL of mixed organic solvent is added. After vortex mixing until homogeneous, 257 μL of mixed stock solution is obtained. 2 mg of DSPE-PEG is accurately weighed. 2K It was dissolved in 200 μL of mixed organic solvent to prepare 10 mg / mL DSPE-PEG. 2K Mother liquor. Add 257 μL of the mixed mother liquor dropwise while stirring, followed by 43 μL of DSPE-PEG. 2K For the mother liquor (addition method), continue stirring for 3 minutes, and the remaining operations are the same as for np-GC NAs.

[0079] Preparation of reduction-sensitive PEGylated hybrid nanoassemblies (sp-GC NAs): The preparation method of sp-GC NAs differs from that of p-GC NAs in that it uses DSPE-SS-PEG. 2K Replace DSPE-PEG 2K The rest of the operations are the same.

[0080] The particle size, particle size distribution, and zeta potential of the prepared np-GC NAs, p-GC NAs, and sp-GC NAs were detected, and the results are shown in Table 2.

[0081] Table 2. Characterization of np-GC NAs, p-GC NAs and sp-GC NAs

[0082] Hybrid nanoassemblies Particle size (nm) Particle size distribution Zeta potential (mV) np-GC NAs 103.60±0.47 0.11±0.08 -6.11±1.21 p-CGC NAs 109.70±1.41 0.11±0.06 -28.00±0.49 sp-GC NAs 105.90±1.89 0.15±0.08 -25.90±1.04

[0083] The results are as follows Figure 13 As shown in Table 2, the hybrid nanoparticles, including np-GC NAs, p-GC NAs, and sp-GC NAs, were a yellow, opalescent colloidal solution. All three types (np-GC NAs, p-GC NAs, and sp-GC NAs) assembled to form relatively uniform nanoparticles. The particle sizes of the prepared np-GC NAs, p-GC NAs, and sp-GC NAs were approximately 105 nm, with a PDI of approximately 0.11, which is beneficial for the accumulation of nanomedicines at the tumor site via the EPR effect. The potential of the prepared np-GC NAs was approximately -6 mV, while the potentials of the modified p-GC NAs and sp-GC NAs were approximately -25 mV to -28 mV. This indicates that the modified hybrid nanoparticles exhibited stronger charge repulsion, effectively preventing nanoparticle aggregation and precipitation, and prolonging their circulation time in the blood. Furthermore, the morphology of the hybrid nanoparticles was observed using transmission electron microscopy, and the results are shown below. Figure 14 As shown, the prepared np-GC NAs, p-GCNAs and sp-GC NAs hybrid nanoparticles are all spherical structures with uniform size.

[0084] Example 4 Assembly Mechanism of CTX / GSP Hybrid Nanoassemblies

[0085] The molecular interaction forces between CTX and GSP in hybrid nanoparticles were investigated using molecular docking simulation technology on the YinFu cloud computing platform to explore the assembly mechanism driving the hybrid nanoparticles. The chemical structures of CTX and GSP were plotted using Chemdraw 17.1 software, then converted to 3D structures, and energy minimization was performed under an MFF94 force field. Finally, semi-flexible docking was performed using the AutoDock Vina program to output the optimal conformation and corresponding assembly forces. Simultaneously, the particle size of the hybrid nanoparticles after incubation with sodium dodecyl sulfate (SDS, 50 mM), sodium chloride (NaCl, 50 mM), and urea (Urea, 50 mM) was measured using a Malvern particle size analyzer to further verify the intermolecular forces driving the assembly of the hybrid nanoparticles. np-GCNAs, p-GCNAs, and sp-GCNAs were incubated with 50 mM SDS, NaCl, and Urea at a concentration of 0.1 mg / mL for 3 h, and their particle size changes were measured at 0 min and 3 h, respectively.

[0086] The results are as follows Figure 15 As shown, the assembly process of the hybrid nanoparticles between CTX and GSP is mainly driven by multiple forces, including π-π stacking forces (green dashed lines), hydrophobic interactions (brown dashed lines), and hydrogen bonds (blue dashed lines). These forces both drive the assembly of the hybrid nanoparticles and ensure their stable existence. Subsequently, we further verified the molecular forces between CTX and GSP in the hybrid nanoparticles using three force-breaking agents: NaCl, Urea, and SDS. The results are as follows. Figure 16 As shown, the particle size of np-GC NAs increased after incubation with NaCl for 3 hours, while the particle sizes of p-GC NAs and sp-GC NAs showed no significant change. This demonstrates that PEGylation of hybrid nanoparticles can reduce the electrostatic interactions between nanoparticles. The particle sizes of np-GC NAs, p-GC NAs, and sp-GC NAs all increased significantly after incubation with Urea or SDS for 3 hours, respectively. These results demonstrate that electrostatic interactions, hydrogen bonds, and hydrophobic interactions jointly drive the assembly process of hybrid nanoparticles, with hydrophobic interactions being the primary driving force.

[0087] Example 5: Physical stability of CTX / GSP hybrid nanoassemblies

[0088] Stability study (simulating physiological conditions): 1 mL and 1 mg / mL of p-GC NAs and sp-GC NAs were placed in 19 mL of PBS (pH 7.4) containing 10% FBS (v / v) and incubated in a shaker (37℃, 100 rpm). Particle size changes were measured at preset time points (0 min, 1 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h). Results are as follows: Figure 17 As shown, the particle size variation of sp-CG NPs is not significant, indicating that the reduction-sensitive sp-CG NPs also have good stability in the simulated in vivo environment.

[0089] Example 6: In vitro drug release from CTX / GSP hybrid nanoassemblies

[0090] Using PBS (pH 7.4) containing 20% ​​tetrahydrofuran (v / v) as the release medium, the in vitro release behavior of p-GC NAs and sp-GC NAs prepared from Example 3 under reducing conditions was investigated by dialysis with CTX and GSP under reducing conditions, with or without (0 mM) DTT and with (5 mM) DTT. 0.5 mL of p-GC NAs and sp-GC NAs with a formulation concentration of 1 mg / mL were placed in dialysis bags (molecular weight cutoff: 8000-12000) and immersed in 30 mL of release medium, then placed in a shaker (37°C, 100 rpm). At predetermined time points (0 h, 0.05 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h), 200 μL of the liquid was collected in liquid chromatography vials, and 200 μL of the corresponding fresh release medium was immediately added.

[0091] The results are as follows Figure 18 As shown, the release of CTX and GSP from sp-GC NAs is consistent, with the cumulative release amount reaching over 80%. Figure 18 -A represents the cumulative release of CTX. Figure 18 -B represents the cumulative release of GSP. As shown in the figure, without the addition of DTT, the cumulative release of CTX and GSP in both p-GC NAs and sp-GC NAs was less than 20%. However, with the addition of 5 mM DTT, the cumulative release of CTX and GSP in both p-GC NAs and sp-GC NAs increased, and the release rate and cumulative release of CTX and GSP in sp-GC NAs were significantly enhanced. This result indicates that reducing conditions can significantly improve the rapid release of CTX and GSP from sp-GC NAs. Tumor sites have a high redox microenvironment, and PEG-modified hybrid nanoparticles bridged by disulfide bonds have been experimentally proven to have reduction-responsive properties, enabling selective and efficient drug release at tumor sites, while drug release in normal tissues is poor. This is beneficial for hybrid nanoparticle formulations to achieve highly effective and low-toxicity chemotherapy.

[0092] Example 7: Preparation of Cy7-labeled hybrid nanoassemblies

[0093] Preparation of Cy7-p-GC NAs: The difference between the preparation method of Cy7-p-GC NAs and the preparation method of p-GC NAs is that Cy7-p-GC NAs are prepared using Cy7-DSPE-PEG. 2K Replace DSPE-PEG 2K The rest of the operations are the same.

[0094] Preparation of Cy7-sp-GC NAs: The difference between the preparation methods of Cy7-sp-GC NAs and p-GC NAs lies in the use of Cy7-DSPE-SS-PEG. 2KReplace DSPE-SS-PEG 2K The rest of the operations are the same.

[0095] Example 8: Cellular uptake of CTX / GSP hybrid nanoassemblies

[0096] After normal digestion, RM-1 cells in good growth condition were centrifuged at low speed (1000 rpm / min, 3 min) to remove trypsin. A cell suspension of approximately 50,000 cells / mL was prepared by adding freshly prepared RPMI 1640 medium. The cell suspension was thoroughly mixed and added to 1 mL of cell suspension per well of a 24-well plate containing cell spreaders. The cell culture plates were then incubated in a cell culture incubator (37℃, 5% CO2) for 12-24 hours. Cy7Sol, Cy7-p-GC NAs, and Cy7-sp-GC NAs were diluted with fresh blank RPMI 1640 medium to achieve a Cy7 concentration of 200 ng / mL. Add 1 mL of fresh culture medium containing Cy7 Sol or Cy7-labeled hybrid nanoparticles to each well, and then incubate in a cell culture incubator for 0.5 h and 4 h. At the specified time points, remove the cell culture plate, discard the drug-containing culture medium, and immediately add cold PBS (pH 7.4) to stop cell uptake. Wash three times with cold PBS (pH 7.4), add 1 mL of tissue fixative, fix at room temperature for 15 min, wash three times with cold PBS (pH 7.4), add 500 μL of Hoechst dye, incubate at room temperature for 10 min, remove the dye, wash three times with cold PBS (pH 7.4), mount the slides, invert them onto a glass slide with anti-fluorescence attenuation mounting medium, observe the results using a confocal microscope and take pictures.

[0097] Cell uptake was then quantitatively observed using flow cytometry. Cells were plated as above in 12-well plates (200,000 cells / well). Drug administration was the same as above. After drug administration and incubation, the cell culture plate was removed, the drug-containing medium was discarded, and cold PBS was immediately added to stop cell uptake. The cells were washed three times with cold PBS (pH 7.4), the PBS (pH 7.4) was removed, 300 μL of trypsin was added to each well, and after digestion for 1 min, the cells were pipetted off, 1 mL of prepared RPMI 1640 medium was added to stop digestion, and the cell suspension was transferred to a labeled 1.5 mL EP tube. After centrifugation at low temperature (4℃, 1000 rpm) for 3 min, the supernatant was discarded, and the cells were resuspended in 500 μL of PBS (pH 7.4). After filtration through a 70 μm cell filter membrane, the cells were added to flow cytometry tubes, and cell uptake was measured using flow cytometry.

[0098] The results are as follows Figure 19 and Figure 20As shown, the hybrid nanoassemblies labeled with Cy7 (Cy7-p-GC NAs and Cy7-sp-GC NAs) exhibited higher intracellular fluorescence intensity than Cy7 Sol, but there was no significant difference in cellular uptake efficiency between Cy7-p-GC NAs and Cy7-sp-GC NAs, demonstrating that the hybrid nanoassemblies have higher cellular uptake efficiency than free Cy7, and the cellular uptake efficiency of the hybrid nanoparticles increased with time, exhibiting time-dependent characteristics.

[0099] Example 9: Cytotoxicity of CTX / GSP Hybrid Nanoassemblies

[0100] Using RM-1, 4T1, 3T3, and L02 cells as cell models, the antitumor cell proliferation activity of the solution (CTX), mixed solution (GC), and hybrid nanoparticles (p-GC NAs and sp-GC NAs) was evaluated by the MTT assay. Results are as follows: Figure 21 and Figure 22 As shown, sp-GC NAs exhibited the strongest cytotoxicity against both RM-1 and 4T1 cells compared to CTX Sol, GC Sol, and insensitive p-GC NAs. In contrast, the slow drug release of insensitive p-GC NAs, despite good stability and efficient cellular uptake, resulted in lower cytotoxicity, again highlighting the crucial importance of rapid drug release in tumor cells for the efficacy of nanomedicine therapy. Notably, the mixed solution (GC Sol) showed only a slight advantage compared to CTX Sol, which may be attributed to the different cellular uptake behaviors of CTX and GSP. Therefore, even simultaneous exposure of GC Sol to RM-1 and 4T1 cells does not guarantee an optimal synergistic dose ratio. These results suggest the necessity of simultaneous and co-administered drug delivery. It is noteworthy that, under the same conditions, both sp-GC NAs and p-GC NAs showed lower cytotoxicity against 3T3 and L02 cells compared to CTX Sol. Figure 23 and Figure 24 Insensitive p-GC NAs exhibit slow drug release rates in both tumor and normal cells. Unlike tumor cells, the lower redox levels in normal cells lead to inefficient drug release from sp-GC NAs, resulting in good therapeutic selectivity between tumor and normal cells. In summary, sp-CG NPs not only enhance antitumor effects through co-assembly with GSPs but also provide ideal therapeutic selectivity due to their tumor-specific drug release characteristics. This self-sensitizing nanomedicine has the potential to become a highly efficient and safe novel chemotherapy modality for cancer.

[0101] Example 10 Pharmacokinetic Study of CTX / GSP Hybrid Nanoassemblies

[0102] SD rats (200–220 g) were randomly divided into three groups to investigate the pharmacokinetic behavior of Cy7 Sol, Cy7-p-GCNAs, and Cy7-sp-GCNAs (n = 6). Cy7 Sol, Cy7-p-GCNAs, and Cy7-sp-GCNAs (at a Cy7 concentration of 2 mg / kg) were administered via tail vein injection. Following tail vein injection, blood was collected via ocular vein at predetermined time points (2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h), and plasma was collected by centrifugation (8000 rpm, 3 min). The blood concentration of Cy7 was determined using a multi-functional microplate reader under excitation wavelength of 744 nm and emission wavelength of 776 nm.

[0103] Table 3 Pharmacokinetic parameters of Cy7 sol and Cy7-labeled nanoformulations (n=6)

[0104] preparation Measurement <![CDATA[AUC 0-24h (μg / mL*h)]]> <![CDATA[t 1 / 2 (h)]]> <![CDATA[MRT 0-24h (h)]]> Cy7 Sol Cy7 2.6±0.4 1.6±0.7 0.8±0.2 Cy7-p-GC NAs Cy7 7.0±1.3 3.7±2.2 2.2±0.3 Cy7-sp-GC NAs Cy7 5.9±1.3 5.2±4.4 2.3±0.6

[0105] a) Area under the plasma concentration-time curve to infinity(μg / mL*h); b) Half-life(h); c) Mean residence time (h).

[0106] The results are as follows Figure 25 As shown, the area under the drug-time curve (AUC) of the Cy7 Sol, Cy7-p-GCNAs, and Cy7-sp-GCNAs groups was calculated using DAS software. 0-24h ), half-life (t) 1 / 2 ) and mean residence time (MRT) 0-24h Pharmacokinetic parameters (Table 3) were obtained. After Cy7 solution was administered to rats via tail vein injection, Cy7 was rapidly cleared from the bloodstream with an elimination half-life of 1.6 ± 0.7 h. This short blood circulation time is unfavorable for drug accumulation at tumor sites. However, nanoparticles modified with PEG and redox-sensitive PEG significantly increased the drug's time in the bloodstream, and the half-lives (t) of Cy7-p-GC NAs and Cy7-sp-GC NAs were significantly improved. 1 / 2The efficacy was increased by 2.3 times and 3.3 times, respectively. Simultaneously, after modification with PEG and redox-sensitive PEG, the nanoparticle surface was encapsulated by a hydrophilic shell, preventing the nanoparticles from being recognized and phagocytosed by the reticuloendothelial system, thus slowing the clearance rate and resulting in higher AUC. The area under the drug-time curve (AUC) of Cy7-p-GC NAs and Cy7-sp-GC NAs were also increased. 0-24h The mean retention time (MRT) of the nanoparticles relative to the solution was increased by 2.7 times and 2.3 times, respectively. 0-24h The duration of blood circulation was also significantly prolonged. It is noteworthy that the pharmacokinetic behavior of the hybrid nanoparticles modified with PEG and those modified with redox-sensitive PEG was not significantly different. This is likely because both PEG-modified and redox-sensitive PEG-modified hybrid nanoparticles possess good assembly ability and physical stability. Furthermore, since the rats were not inoculated with tumors and lacked a high redox microenvironment, the modified nanoparticles remained relatively stable during blood circulation, maintaining their intact nanostructure. Therefore, both methods significantly increased blood circulation time and exhibited good pharmacokinetic behavior.

[0107] Example 11: Establishment of the RM-1 tumor-bearing C57BL / 6 mouse model

[0108] RM-1 cells in good growth condition and in the logarithmic growth phase were digested normally with 0.05% trypsin, and then digestion was terminated with freshly prepared RPMI 1640. Cells were collected by low-speed centrifugation (1000 rpm, 3 min), and resuspended in sterile PBS (pH 7.4) to uniformly disperse them to a concentration of 5 × 10⁻⁶. 7 Cell suspensions of RM-1 cells / mL were prepared and placed in an ice box. Hair was removed from the right posterior flank of C57BL / 6 mice to reduce hair interference with imaging. 100 μL of well-mixed RM-1 cells (5 × 10⁶ cells / mL) were injected using an insulin syringe. 6 Cells were inoculated subcutaneously on the right posterior back of mice.

[0109] Example 12: In vivo distribution of CTX / GSP hybrid nanoassemblies

[0110] RM-1 cell suspension was inoculated into C57BL / 6 mice, and tumors were allowed to grow to approximately 300-400 mm². 3 At that time, a biological distribution experiment was conducted. For example... Figure 26 and Figure 27As shown, the Cy7 Sol, Cy7-p-GC NAs, and Cy7-sp-GC NAs groups all reached their maximum accumulation at the tumor site 4 hours after administration. The Cy7 solution was quickly cleared from the bloodstream, so its accumulation at the tumor site was less than that of the nanoparticle group, and it was basically cleared after 24 hours. The hybrid nanoparticles Cy7-p-GC NAs and Cy7-sp-GC NAs, due to their strong stability, longer blood circulation time, and ability to passively target tumor tissue through the EPR effect, had a higher accumulation in the tumor tissue.

[0111] Example 13: In vivo pharmacodynamic evaluation of CTX / GSP hybrid nanoassemblies

[0112] The tumor volume of the tumor-bearing C57BL / 6 mice was approximately 100 mm. 3 Mice were randomly divided into 6 groups of 5 mice each: Saline, CTX Sol, GSP Sol, GC Sol, p-GC NAs, and sp-GC NAs. Drugs were administered via tail vein every two days for a total of 3 doses, at a dose equivalent to 4 mg / kg CTX and / or 7.5 mg / kg GSP. Tumor volume was measured and recorded daily in the tumor-bearing C57BL / 6 mice. On day 8 after drug administration, all tumor-bearing mice were weighed and sacrificed. Heart, liver, spleen, lung, kidney, and tumor tissues were isolated. Tumor tissues were photographed and weighed, and the tumor bearing rate was calculated. Blood samples were collected for liver and kidney function analysis and to detect testosterone levels. TUNEL staining analysis was performed on the excised tumors.

[0113] The results are as follows Figures 28-31 As shown, the tumors in the saline group of tumor-bearing mice grew rapidly, reaching a volume of 1200 mm² on day 8. 3 The CTX Sol and GSP Sol treatment groups showed significant tumor inhibition, with tumor volume of approximately 600 mm. 3Furthermore, the differences in antitumor effects between the two were not significant. We also found that, compared to the CTX Sol and GSP Sol dosing groups, the mixed solution group (GC Sol) and the insensitive hybrid nanoparticle group (p-GC NAs) exhibited stronger antitumor activity. This indicates that the antitumor effect of relying on only one chemotherapeutic drug for antitumor treatment is limited. Notably, p-GC NAs showed better antitumor effects compared to the mixed solution group (GCSol). This is because the solution was taken up less by cells, and its pharmacokinetic behavior was poor. Simple mixing alone cannot simultaneously deliver the two drugs to the tumor site, making it difficult to achieve a good synergistic sensitization and antitumor effect. In contrast, the reduction-sensitive hybrid nanoparticles (sp-GC NAs) exhibited the best antitumor effect, with the smallest tumor volume and tumor burden, and the highest degree of tumor cell apoptosis and / or necrosis. This is because sp-GC NAs not only exhibited good cellular uptake, pharmacokinetic behavior, and tumor tissue accumulation capacity, but also efficiently, rapidly, and simultaneously released CTX and GSP under the high redox microenvironment of the tumor site, exerting a synergistic sensitization and antitumor effect.

[0114] Example 14 Preliminary safety evaluation of CTX / GSP hybrid nanoassemblies

[0115] Body weight changes in tumor-bearing C57BL / 6 mice: Starting from day 0 of the first administration, the body weight of tumor-bearing C57BL / 6 mice was measured daily, and a trend graph of body weight changes over time was plotted. The safety of the drug was preliminarily evaluated through changes in mouse body weight. The results showed that there were no significant changes in body weight in tumor-bearing C57BL / 6 mice in different formulation treatment groups. Figure 32 ).

[0116] Evaluation of Liver and Kidney Function Indicators: On day 8 after drug administration, before euthanizing mice for organ separation, blood was collected by enucleation of the eyeballs into coagulation tubes. The blood was then centrifuged at low speed (4000 rpm) for 5 min, and the supernatant was collected. Wuhan Saiweier Biotechnology Co., Ltd. was commissioned to evaluate liver function by measuring the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and kidney function by measuring the levels of creatinine (CR) and blood urea nitrogen (BUN). The results showed that serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) were all within the normal range. Figure 33 )

[0117] Hematoxylin-eosin (H&E) staining of organs and tissues: Mice were sacrificed on day 8 after drug administration, and the heart, liver, spleen, lungs, and kidneys were isolated. Residual blood was washed away with physiological saline, and the organs were then fixed in a tissue fixative containing 4% paraformaldehyde. Wuhan Saiwei Biotechnology Co., Ltd. was commissioned to section and stain the major organs to observe the lesions in each tissue. The results showed that no obvious tissue lesions were observed in the major organs (heart, liver, spleen, lungs, and kidneys) after treatment. Figure 34 ).

[0118] The in vivo safety of the nano-formulation was comprehensively evaluated by analyzing mouse body weight changes, liver and kidney function indicators, HE staining results of various organs and tissues, and hemolysis experiments. All results indicate that the reduction-sensitive hybrid nanoassemblies sp-CG NPs possess potent antitumor activity and good biocompatibility.

[0119] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A carrier-free co-assembled nanoparticle, characterized in that, The carrier-free co-assembled nanoparticles are co-assembled nanoparticles of a chemotherapy drug modified with a polyethylene glycol modifier and gossypol; the chemotherapy drug is cabazitaxel. The molar ratio of the chemotherapy drug to the gossypol is 1:3; The chemotherapy drug and gossypol are co-assembled through one or more of the following: π-π stacking, hydrophobic interaction, and intermolecular hydrogen bonding. The molar ratio of the chemotherapy drug to the polyethylene glycol modifier is 60:40 to 90:10; The polyethylene glycol modifier is DSPE-PEG. 2K and / or DSPE-SS-PEG 2K ; The method for preparing carrier-free co-assembled nanoparticles includes the following steps: Chemotherapy drugs, gossypol, and polyethylene glycol modifiers were dissolved in an organic solvent to obtain a mixed solution. The mixed solution was then added dropwise to water, where it spontaneously formed uniform nanoparticles. The organic solvent was then removed by rotary evaporation to obtain carrier-free co-assembled nanoparticles.

2. The carrier-free co-assembled nanoparticles according to claim 1, characterized in that, The organic solvent includes one or more of methanol, ethanol, tetrahydrofuran, and dimethyl sulfoxide.

3. The application of carrier-free co-assembled nanoparticles as described in any one of claims 1-2 in the preparation of antitumor drugs.

Images