Self-sensitizing dihydroartemisinin nano-assembly, preparation and application thereof

Abstract

The application belongs to the technical field of new adjuvants and new dosage forms of pharmaceutical preparations combined therapy, and particularly relates to a self-sensitization type dihydroartemisinin nano-assembly and a preparation and application thereof. The application provides a nano-assembly co-assembled by DHA and Fc and a preparation method thereof. The application further provides a PEGylated self-sensitization type dihydroartemisinin nano-assembly and a preparation method and application thereof; the nano-assembly co-assembled by the above DHA and Fc and a PEG modification agent. The self-sensitization type dihydroartemisinin nano-assembly provided by the application has the characteristics of high drug loading and low toxic side effects. The application further provides new strategies and more choices for the development of drug delivery, and meets the urgent needs of the clinical sensitization chemotherapy drug strategy in preparations.

Description

Technical Field

[0001] This invention belongs to the technical field of new excipients and new dosage forms for combination therapy of pharmaceutical preparations, specifically relating to a self-sensitizing dihydroartemisinin nanoassembly and its preparation and application. Background Technology

[0002] Chemotherapy is one of the most commonly used strategies in cancer treatment, especially for tumors that cannot be surgically removed or have metastasized. However, most chemotherapeutic drugs are cytotoxic and have drawbacks such as low solubility, poor stability, narrow therapeutic window, and unfavorable pharmacokinetic properties. Existing formulation strategies also suffer from low delivery efficiency and poor tumor targeting, resulting in poor clinical efficacy and severe toxic side effects from chemotherapy.

[0003] The antitumor activity of dihydroartemisinin (DHA) is limited by several factors, including trace elements such as iron. The content of free iron ions in tumor cells is significantly higher than in normal cells, and DHA needs to bind with intracellular iron. 2+ This process forms an active intermediate that exerts a toxic effect on tumor cells. Therefore, changes in intracellular iron levels within tumor cells affect the anti-tumor efficacy of DHA; the higher the intracellular iron content, the greater the sensitivity of tumor cells to DHA. Thus, intervention targeting intracellular iron levels in tumor cells may help enhance the anti-tumor effect of DHA.

[0004] While DHA possesses potent anti-tumor effects, effective accumulation of DHA at tumor sites remains a crucial prerequisite for its therapeutic efficacy. With the rapid development of nanotechnology, various nanocarriers, including liposomes, polymers, and mesoporous silica, have been used to improve drug delivery efficiency. However, traditional nanocarriers often exhibit poor affinity for drugs, leading to carrier-related toxicity. In recent years, carrier-free nanomedicine delivery systems (CFNDS) have emerged as a promising cancer treatment strategy. In CFNDS, certain drug molecules have been found to self-assemble into stable nanoparticles driven by various intermolecular forces. Small-molecule self-assembled nanomedicines offer significant advantages over traditional carrier-based nanomedicines (such as liposomes and nanocapsules), including ease of preparation, good reproducibility, carrier-free nature, absence of excipient-related toxicity, high drug loading capacity, and high delivery efficiency. In addition to self-assembly, some drug molecules can co-assemble into binary or ternary self-sensitizing nanoparticles, although they cannot perform nanoassembly on their own. Beyond these advantages, hybrid nanosystems can also provide a universal drug co-delivery nanocarrier. Unlike drug co-encapsulation based on carrier materials, the development of pure drug-based co-assemblies enables highly synchronized drug delivery. Therefore, the emergence of CFNDS (Chemical Component-Based Drug Delivery Systems) offers a new strategy for co-delivery of drugs.

[0005] In recent years, carrier-free nanoparticles that self-assemble from pure drugs have shown great promise in drug delivery, especially for certain anticancer drugs that can self-assemble into stable nanoparticles without the need for carrier materials. Constructing self-sensitizing nanoassemblies with multiple drug molecules holds even greater potential in combination therapies. Researching and developing a self-sensitizing nanoassembly that enhances chemotherapy is an important and urgent research topic. Summary of the Invention

[0006] Based on the technical problems described in the background art, the present invention further aims to solve the problem of Fe in tumors. 2+ Insufficient DHA can lead to inadequate anti-tumor effects, as well as problems such as poor water solubility of DHA, low drug loading due to encapsulation in polymers, drug leakage, and excipient-related toxicity.

[0007] This invention discloses a self-sensitizing dihydroartemisinin nanoassembly, wherein the self-sensitizing nanoassembly is composed of an organic peroxide and an iron donor;

[0008] Furthermore, the organic peroxide is selected from artemisinin, dihydroartemisinin, artesunate, and artemether;

[0009] Furthermore, the iron donor is one or more of ferrocene, hydroxymethylferrocene, or carboxymethylferrocene;

[0010] Furthermore, the organic peroxide is selected from dihydroartemisinin (DHA);

[0011] Furthermore, the iron donor is selected from ferrocene (Fc);

[0012] This invention discloses a method for preparing the above-mentioned self-sensitizing dihydroartemisinin nanoassemblies, specifically including the following steps:

[0013] Dihydroartemisinin (DHA) and ferrocene (Fc) were dissolved in organic solvents respectively. The mixture was then added dropwise to water while stirring. After the reaction was complete, the organic solvent was removed to obtain a self-sensitizing dihydroartemisinin nanoassembly.

[0014] Furthermore, the molar ratio of the organic peroxide to the iron donor is (7:1) to (1:1);

[0015] Furthermore, the organic solvent is selected from any one or more of ethanol, tetrahydrofuran, and dimethyl sulfoxide;

[0016] Furthermore, in the preparation method, the method for removing the organic solvent is vacuum rotary evaporation, ultrafiltration, or membrane permeation.

[0017] This invention discloses a PEGylated self-sensitizing dihydroartemisinin nanoassembly, wherein the PEGylated self-sensitizing dihydroartemisinin nanoassembly is composed of the above-mentioned self-sensitizing dihydroartemisinin nanoassembly and a modifier.

[0018] Furthermore, the modifier is selected from PEG modifiers with a molecular weight of 200-2000;

[0019] Furthermore, the PEG modifier is selected from any one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG, and PE-PEG;

[0020] Furthermore, the modifier is selected from DSPE-PEG. 2K .

[0021] This invention discloses a method for preparing the PEGylated self-sensitizing dihydroartemisinin nanoassemblies, specifically including the following steps:

[0022] PEG modifier, dihydroartemisinin and ferrocene were dissolved in organic reagents respectively. The mixture of the three was added dropwise to water under stirring. After the reaction was completed, the organic solvent was removed to obtain PEGylated self-sensitizing dihydroartemisinin nanoassemblies.

[0023] Furthermore, the molar ratio of the organic peroxide to the iron donor is (7:1) to (1:1), and the mass ratio of the organic peroxide to the PEG modifier is (10:90) to (90:10).

[0024] Furthermore, the organic solvent is selected from any one or more of ethanol, tetrahydrofuran, and dimethyl sulfoxide;

[0025] Furthermore, in the preparation method, the method for removing the organic solvent is vacuum rotary evaporation, ultrafiltration, or membrane permeation.

[0026] Furthermore, the solvent is preferably tetrahydrofuran.

[0027] Furthermore, the preferred molar ratio of DHA to Fc is 3:1.

[0028] This invention provides the application of PEGylated self-sensitizing dihydroartemisinin nanoassemblies in the preparation of drug delivery systems.

[0029] This invention provides the application of PEGylated self-sensitizing dihydroartemisinin nanoassemblies in the preparation of drugs for treating tumors.

[0030] This invention provides the application of PEGylated self-sensitizing dihydroartemisinin nanoassemblies in the preparation of injectable, oral, or topical drug delivery systems.

[0031] The present invention provides a pharmaceutical composition comprising the PEGylated self-sensitizing dihydroartemisinin nanoassemblies and pharmaceutically acceptable excipients.

[0032] This invention provides the use of the above-described pharmaceutical composition in the preparation of drugs for treating tumors.

[0033] This invention provides the application of the above-described pharmaceutical composition in the preparation of a drug delivery system.

[0034] This invention provides the use of the above-described pharmaceutical composition in the preparation of injection, oral, or topical drug delivery systems.

[0035] The advantages of this invention over the prior art are as follows:

[0036] This invention provides a self-sensitizing nanoassembly composed of DHA and Fc; wherein the presence of Fc can enhance the anti-tumor effect of DHA by providing Fe. 2+ This invention accelerates the DHA peroxide bridge cleavage reaction, thereby enhancing the antitumor effect of DHA. Simultaneously, the self-sensitizing nanoassembly provided by this invention not only possesses high drug loading capacity and good stability but also exhibits low toxicity, meeting the clinical demand for highly effective and low-toxicity formulations. It provides a new strategy for the application of sensitized DHA in the preparation of antitumor drugs. Furthermore, it provides an effective nanoplatform for the development of carrier-free self-sensitizing nanoassemblies and sensitized chemotherapy.

[0037] This invention provides a PEGylated self-sensitizing nanoassembly. In the 4T1 mammary tumor model of BALB / c mice, the DHA / Fc self-sensitizing nanoassembly exhibits strong anti-tumor activity, providing a new approach for the preparation of drugs to treat tumors. Attached Figure Description

[0038] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0039] Figure 1 This is a particle size distribution diagram of the PEGylated self-sensitized dihydroartemisinin nanoassembly of Example 8 of the present invention.

[0040] Figure 2 These are confocal microscopy images of the PEGylated self-sensitizing dihydroartemisinin nanoassemblies of Example 9 of the present invention at 2 hours and 4 hours.

[0041] Figure 3 The results of flow cytometry quantification of cellular uptake of the PEGylated self-sensitized dihydroartemisinin nanoassembly of Example 9 of the present invention at 2 hours and 4 hours are shown.

[0042] Figure 4 The results show the cytotoxicity of the PEGylated self-sensitized dihydroartemisinin nanoassembly of Example 10 of this invention.

[0043] Figure 5 This is a mouse tumor growth curve from the in vivo anti-tumor experiment of Example 11 of the invention.

[0044] Figure 6 This is an ex vivo photograph of a mouse tumor used in an in vivo anti-tumor experiment according to Example 11 of the invention.

[0045] Figure 7 This is a statistical chart of tumor weight in an in vivo anti-tumor experiment according to Example 11 of the invention.

[0046] Figure 8 This is a graph showing the changes in mouse body weight during the in vivo anti-tumor experiment of Example 11 of the invention. Detailed Implementation

[0047] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0048] Example 1: Proportional Screening of DHA / Fc Self-Sensitizing Dihydroartemisinin Nanoassemblies

[0049] DHA and Fc were dissolved in tetrahydrofuran to obtain 1 mg / mL DHA and Fc solutions. With a total mass of 0.2 mg DHA and Fc, and molar ratios of 7:1, 5:1, 3:1, 1:1, 1:3, 1:5, or 1:7, and a total volume of 200 μL for the mixed solutions, seven aliquots of the mixed solutions with different DHA and Fc molar ratios were prepared. Each of these seven mixed solutions was slowly added dropwise to 2 mL of deionized water, and stirred at 1300 rpm for 90 s to prepare DF NPs. Organic reagents were removed by vacuum rotary evaporation at 30 °C. The solution was then brought to a final volume of 2 mL with deionized water, and the particle size was measured using a Malvern particle size analyzer.

[0050] The results of the particle size and polydispersity index of the prepared nano-formulations are shown in Table 1.

[0051] Table 1. Particle size and polydispersity index of DHA / Fc self-sensitizing dihydroartemisinin nanoassemblies at different molar ratios

[0052]

[0053] As shown in Table 1, when the DHA / Fc molar ratio is between 1:1 and 1:7, neither DHA nor Fc can assemble well, resulting in large particle sizes or precipitation. However, when the DHA / Fc molar ratio is between 3:1 and 7:1, both DHA and Fc exhibit good assembly ability, and the prepared nanoassemblies are around 130 nm in size. A preliminary optimal DHA / Fc ratio of 3:1 is selected.

[0054] Example 2: Organic reagent screening based on the preparation of DHA / Fc self-sensitizing dihydroartemisinin nanoassemblies

[0055] DHA and Fc were dissolved in different solvents (anhydrous ethanol, tetrahydrofuran, tetrahydrofuran / anhydrous ethanol = 1:1, methanol, acetonitrile, N,N-dimethylformamide) to obtain 1 mg / mL DHA and Fc solutions. From the above six DHA and Fc solutions dissolved in different organic reagents, 164 μL of DHA solution and 36 μL of Fc solution were taken and mixed thoroughly. The six mixed solutions were then slowly added dropwise to 2 mL of deionized water, and stirred at 1300 rpm for 90 s to prepare DF NPs. The organic reagents were removed by vacuum rotary evaporation at 30 °C. The volume was adjusted to 2 mL with deionized water, and the particle size was measured using a Malvern particle size analyzer.

[0056] Table 2. Particle size and polydispersity index of DHA / Fc self-sensitizing dihydroartemisinin nanoassemblies in different solvents

[0057]

[0058] As shown in Table 2, the self-sensitizing dihydroartemisinin nanoassemblies prepared with tetrahydrofuran had the smallest particle size and PDI. Tetrahydrofuran is preliminarily selected as the preferred organic reagent.

[0059] Example 3: Rotational speed screening of DHA / Fc self-sensitizing dihydroartemisinin nanoassemblies

[0060] DHA and Fc were dissolved in tetrahydrofuran to obtain 1 mg / mL DHA and Fc solutions. 164 μL of the DHA solution and 36 μL of the Fc solution dissolved in tetrahydrofuran were taken separately and mixed thoroughly, resulting in four aliquots. Each of the four aliquots was slowly added dropwise to 2 mL of deionized water, and the mixture was stirred at different speeds for 90 s to prepare DF NPs. Organic reagents were removed by vacuum rotary evaporation at 30 °C. The volume was adjusted to 2 mL with deionized water, and the particle size was measured using a Malvern particle size analyzer.

[0061] Table 3. Particle size and polydispersity index of DHA / Fc self-sensitized dihydroartemisinin nanoassemblies at different rotation speeds

[0062]

[0063] As shown in Table 3, the particle size and PDI of the prepared self-sensitized dihydroartemisinin nanoassemblies were optimal at a rotation speed of 1300 rpm. A rotation speed of 1300 rpm was initially selected as the optimal value.

[0064] Example 4: Screening of stirring time in the preparation of DHA / Fc self-sensitizing dihydroartemisinin nanoassemblies

[0065] DHA and Fc were dissolved in tetrahydrofuran to obtain 1 mg / mL DHA and Fc solutions. Four portions of each solution were prepared by mixing thoroughly: 164 μL of the DHA solution and 36 μL of the Fc solution dissolved in tetrahydrofuran. Each of these four mixed solutions was slowly added dropwise to 2 mL of deionized water at a stirring speed of 1300 rpm for 30, 60, 90, and 180 s, respectively. Organic reagents were removed by vacuum rotary evaporation at 30 °C. The solution was then brought to a final volume of 2 mL with deionized water, and the particle size was measured using a Malvern particle size analyzer.

[0066] Table 4. Particle size and polydispersity index of DHA / Fc self-sensitizing dihydroartemisinin nanoassemblies under different stirring times.

[0067]

[0068] As shown in Table 4, the particle size and PDI of the prepared self-sensitized dihydroartemisinin nanoassemblies were optimal when the stirring time was 60 s. The preliminary preferred stirring time is 60 s.

[0069] Example 5: Dropping rate screening for the preparation of DHA / Fc self-sensitizing dihydroartemisinin nanoassemblies

[0070] DHA and Fc were dissolved in tetrahydrofuran to obtain 1 mg / mL DHA and Fc solutions. Three portions of each solution were prepared by mixing thoroughly: 164 μL of the DHA solution and 36 μL of the Fc solution dissolved in tetrahydrofuran. Each of these three mixed solutions was slowly added dropwise to 2 mL of deionized water at different rates: 1300 rpm, stirring time of 60 s, and dropwise rates of 0.5, 1, and 2 s / d. Organic reagents were removed by vacuum rotary evaporation at 30 °C. The volume was brought to 2 mL with deionized water, and the particle size was measured using a Malvern particle size analyzer.

[0071] Table 5. Particle size and polydispersity index of DHA / Fc self-sensitizing dihydroartemisinin nanoassemblies at different dropping rates

[0072]

[0073] As shown in Table 5, the particle size and PDI of the prepared self-sensitized dihydroartemisinin nanoassemblies were optimal when the dropping rate was 1 s / d. The preliminary preferred dropping rate was 1 s / d.

[0074] Example 6: Preparation of DSPE-PEG from PEGylated self-sensitized dihydroartemisinin nanoassemblies 2K Content screening

[0075] DHA and Fc were dissolved in tetrahydrofuran to obtain a 1 mg / mL DHA solution and an Fc solution. DSPE-PEG was then used. 2K Dissolved in anhydrous ethanol, 1 mg / mL DSPE-PEG was obtained. 2K Solutions. Take 164 μL of DHA solution, 36 μL of Fc solution, and DSPE-PEG solution respectively. 2K (22 μL, 50 μL, 86 μL) were mixed thoroughly and slowly added dropwise to 2 mL of deionized water at 1300 rpm for 60 s. Organic reagents were removed by vacuum rotary evaporation at 30 °C. The volume was brought to 2 mL with deionized water, and the particle size was measured using a Malvern particle size analyzer.

[0076] Table 6. Different DSPE-PEGs 2K Particle size and polydispersity index of PEGylated self-sensitized dihydroartemisinin nanoassemblies at different concentrations

[0077]

[0078] As shown in Table 6, DSPE-PEG 2K At a content of 20%, the prepared PEGylated self-sensitizing dihydroartemisinin nanoassemblies exhibited the best particle size and PDI. Preliminary selection of DSPE-PEG was made. 2K The content is 20%.

[0079] The DSPE-PEG 2K Content is DSPE-PEG 2K The proportion of quality to total quality.

[0080] Example 7: Preparation of DSPE-PEG from PEGylated self-sensitized dihydroartemisinin nanoassemblies 2K Joining Method Filtering

[0081] DHA and Fc were dissolved in tetrahydrofuran to obtain a 1 mg / mL DHA solution and an Fc solution. DSPE-PEG was then used. 2K Dissolved in anhydrous ethanol, 1 mg / mL DSPE-PEG was obtained. 2K Solution. Internal addition method: Take DHA solution (164 μL), Fc solution (36 μL), and DSPE-PEG solution respectively. 2K(50 μL) Mix well and slowly add it dropwise to 2 mL of deionized water at 1300 rpm for 60 s. External addition method: Mix 164 μL of DHA solution and 36 μL of Fc solution well and slowly add it dropwise to 2 mL of deionized water, then add 50 μL of DSPE-PEG. 2K The stirring speed was 1300 rpm, and the stirring time was 60 s. Organic reagents were removed by vacuum rotary evaporation at 30℃. The volume was brought to 2 mL with deionized water, and the particle size was measured using a Malvern particle size analyzer.

[0082] Table 7. Different DSPE-PEGs 2K Particle size and polydispersity index of PEGylated self-sensitized dihydroartemisinin nanoassemblies under different addition methods

[0083]

[0084] As shown in Table 7, DSPE-PEG 2K When added internally, the prepared self-sensitizing dihydroartemisinin nanoassemblies exhibited the best particle size and PDI. DSPE-PEG 2K The preferred method for adding it is internal addition.

[0085] Example 8: Characterization of PEGylated self-sensitized dihydroartemisinin nanoassemblies

[0086] DHA and Fc were dissolved in tetrahydrofuran to obtain a 1 mg / mL DHA solution and an Fc solution. DSPE-PEG was then used. 2K Dissolved in anhydrous ethanol, 1 mg / mL DSPE-PEG was obtained. 2K Solutions. Take 164 μL of DHA solution, 36 μL of Fc solution, and DSPE-PEG solution respectively. 2K (50 μL) was mixed thoroughly and slowly added dropwise to 2 mL of deionized water at 1300 rpm for 60 s. Organic reagents were removed by vacuum rotary evaporation at 30 °C. The volume was brought to 2 mL with deionized water, and the particle size was measured using a Malvern particle size analyzer. The particle size, PDI, and Zeta potential of the prepared PEGylated self-sensitizing dihydroartemisinin nanoassemblies (DFP nanoparticles) were determined using a Malvern particle size analyzer in triplicate.

[0087] The results are as follows Figure 1The DFP nanoparticles shown in Table 8 have a particle size of 112.80 ± 1.44 nm, a polydispersity index of 0.27 ± 0.02, and a zeta potential of -12.1 ± 3.55 mV. Furthermore, the DFP nanoparticles exhibit high drug loading, with DHA loading at 65.6 wt% and Fc loading at 14.4 wt%. In summary, the prepared PEGylated self-sensitizing dihydroartemisinin nanoassemblies (DFP nanoparticles) have uniform particle size and high drug loading.

[0088] Table 8. Characterization of PEGylated self-sensitized dihydroartemisinin nanoassemblies

[0089]

[0090] Example 9: Cellular uptake of PEGylated self-sensitized dihydroartemisinin nanoassemblies

[0091] Qualitative examination of cell uptake using confocal fluorescence microscopy: 4T1 cells in good growth condition were digested and centrifuged to remove trypsin, resuspended in fresh RPMI-1640 medium, diluted, and prepared to a concentration of 5 × 10⁻⁶ cells / mL. 4 Cell suspension at a concentration of cells / mL. After thorough mixing of the cell suspension, [the solution is prepared] at 5 × 10 [units]. 4 Cells were seeded at a density of 1 / well into 24-well plates pre-placed with cell spreaders and incubated at 37°C in a 5% CO2 incubator for 12 h. Cy7 solution and Cy7-labeled DFP nanoparticles (DFP-Cy7 nanoparticles) were diluted separately using fresh RPMI-1640 medium to achieve a Cy7 concentration of 400 ng / mL. Specifically, the 24-well plates were removed, the old medium was discarded, and the diluted Cy7 solution and DFP-Cy7 nanoparticles were added. The plates were incubated at 37°C for 2 or 4 h, respectively. The drug-containing medium was discarded, and uptake was immediately stopped with cold PBS (pH 7.4). The plates were washed three times with cold PBS (pH 7.4), fixed with 1 mL of tissue fixative at room temperature for 10 min, washed three times with cold PBS (pH 7.4), stained with 500 μL of Hoechst 33342 staining solution for 10 min, and washed three times with cold PBS (pH 7.4). Remove the smear from the 24-well plate and place it upside down on a glass slide with a mounting medium containing anti-fluorescence attenuation agent. Observe the cell uptake behavior and take fluorescence images using a confocal fluorescence microscope.

[0092] The results are as follows Figure 2As shown, the Cy7-labeled PEGylated self-sensitized dihydroartemisinin nanoassemblies, DFP-Cy7 nanoparticles, exhibit higher intracellular fluorescence intensity than Cy7 solution, demonstrating that the PEGylated self-sensitized dihydroartemisinin nanoassemblies have higher cellular uptake efficiency than the free solution. Furthermore, the cellular uptake efficiency of the PEGylated self-sensitized dihydroartemisinin nanoassemblies increases with time, exhibiting time-dependent cellular uptake behavior.

[0093] Flow cytometry was used to quantitatively assess cell uptake: 4T1 cells in good growth condition were digested and centrifuged to remove trypsin, resuspended in fresh RPMI-1640 medium, diluted, and prepared at 2×10⁶ cells / mL. 5 Cell suspension at a concentration of cells / mL. After thorough mixing of the cell suspension, [the solution is prepared] at 2 × 10 [units]. 5 Cells were seeded at a density of 1 / well into pre-filled 12-well plates and incubated at 37°C in a cell culture incubator containing 5% CO2 for 12 h. Cy7 solution and Cy7-labeled DFP nanoparticles (DFP-Cy7 nanoparticles) were diluted separately using fresh RPMI-1640 medium to achieve a Cy7 concentration of 400 ng / mL. Specifically, the 12-well plates were removed, the old medium was discarded, and the diluted Cy7 solution and DFP-Cy7 nanoparticles were added. The plates were incubated at 37°C for 2 or 4 h, respectively. The drug-containing medium was discarded, and the uptake was immediately stopped with cold PBS (pH 7.4). The cells were washed three times with cold PBS (pH 7.4), the PBS (pH 7.4) was removed, and 300 μL of trypsin was added to each well to digest the cells. Then, 1 mL of culture medium was added to stop the digestion. The digested cells were transferred to 1.5 mL EP tubes and centrifuged at low temperature for 3 min (4°C, 1000 rpm). Discard the supernatant, resuspend the cells in 500 μL of PBS (pH 7.4), filter through a 70 μm cell sieve, and add them to a flow cytometry tube. Quantitatively measure cell uptake using a flow cytometer.

[0094] The results are as follows Figure 3 As shown, the results are consistent with the qualitative results of confocal fluorescence microscopy. The Cy7-labeled self-sensitizing nanoassemblies DFP-Cy7 nanoparticles exhibit stronger cellular uptake capacity than Cy7 solution, and show a time-dependent effect.

[0095] Example 10: Cytotoxicity of PEGylated self-sensitized dihydroartemisinin nanoassemblies

[0096] Using 4T1 cells as a model, the cytotoxicity of PEGylated self-sensitized dihydroartemisinin nanoassemblies was investigated using the MTT assay. Specifically, 80% confluent 4T1 cells with good growth were transferred to a clean bench, the old culture medium was discarded, and 1 mL of trypsin was added for pre-digestion to wash away residual culture medium and dead cells. This was discarded, and 1 mL of trypsin was added again to digest the cells for approximately 1 minute until they could be dispersed in clumps. 4 mL of culture medium was added to stop the digestion. The cell suspension was completely transferred to a centrifuge and centrifuged (1000 rpm, 3 min). The culture medium containing trypsin was discarded, and 5 mL of fresh culture medium was added to resuspend the cells. The cells were then diluted to a concentration of 1 × 10⁴ cells / mL. 4T1 cells were centrifuged at 2 × 10⁴ cells / mL. 3 Cells were seeded at a density of 200 μL per well in 96-well plates, with 60 wells per plate. Sterile PBS (pH 7.4) was added to the outermost ring of the 96-well plates. The plates were then incubated at 37°C with 5% CO2 for 12 h until cells reached the desired morphology before drug administration. Cells were treated with different concentrations of DHA, Fc, DF, and DFP nanoparticles at the same concentration of DHA and / or Fc for 48 h. After 48 h of culture, the 96-well plates were removed and co-incubated at 37°C with 5 mg / mL MTT (25 μL / well) for 4 h. The culture medium was discarded, and DMSO (200 μL / well) was used to dissolve the generated formazan. Finally, the absorbance at 490 nm was measured using a multi-mode microplate reader.

[0097] Cytotoxicity results such as Figure 4 As shown, although Fc exhibits almost no cytotoxicity at this concentration, the combination therapy of Fc and DHA (DF solution) showed stronger cytotoxicity than the DHA solution, confirming that Fc can sensitize the antitumor effect of DHA. Notably, the PEGylated self-sensitizing dihydroartemisinin nanoassemblies (DFP nanoparticles) prepared in this invention exhibit stronger in vitro antitumor activity than the DF solution, which should be attributed to their highly efficient cellular uptake capacity.

[0098] Example 11: In vivo antitumor experiment of PEGylated self-sensitized dihydroartemisinin nanoassemblies

[0099] The in vivo antitumor activity of DHA / Fc nanoparticles was studied using 4T1 tumor-bearing mice (20-22g). First, 100 μL of 4T1 cells (5 × 10⁻⁶ g) were... 7 (Number of tumor cells / mL) was subcutaneously injected into the right back of mice. When the tumor volume reached 100 mm², the tumor was treated. 3Around 10:00 AM, tumor-bearing mice were randomly divided into 5 groups (n=5). They were then treated 5 times every other day with physiological saline, DHA solution, Fc solution, DF solution, and DFP nanoparticles, with a DHA concentration of 5 mg / kg (dose, DHA:Fc = 3:1 molar ratio). Mouse weight and tumor volume were measured and recorded daily during treatment. After the final treatment, the mice were sacrificed, and the removed tumors were weighed.

[0100] The results are as follows Figures 5-7 As shown, after treatment with saline solution, the tumor proliferated rapidly. Similarly, the Fc solution showed poor antitumor effect. In contrast, DHA and DF solutions effectively inhibited tumor growth; DFP nanoparticles showed the strongest antitumor effect in mice. During treatment, the mice's body weight did not change significantly. Figure 8 This indicates that the PEGylated self-sensitized dihydroartemisinin nanoassemblies exhibit good biological tolerance.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A self-sensitizing dihydroartemisinin nanoassembly, characterized in that, The self-sensitizing dihydroartemisinin nanoassemblies are composed of organic peroxides and Fe. 2+ The donor is co-assembled, with an organic peroxide to iron donor molar ratio of 3:1; wherein the organic peroxide is dihydroartemisinin, and the Fe... 2+ The donor was ferrocene.

2. A PEGylated, self-sensitizing dihydroartemisinin nanoassembly, characterized in that, The PEGylated self-sensitizing dihydroartemisinin nanoassembly is composed of the self-sensitizing dihydroartemisinin nanoassembly according to claim 1 and a PEG modifier; wherein the PEG modifier is DSPE-PEG. 2K DSPE-PEG 2K The content is 10%~30%.

3. The method for preparing the self-sensitizing dihydroartemisinin nanoassemblies according to claim 1, characterized in that, The process includes the following steps: dissolving dihydroartemisinin and ferrocene separately in an organic solvent, adding the mixed solution of the two solutions dropwise to water at a rate of 1 s / d while stirring, stirring at 1300 rpm for 60 s to spontaneously form a uniform hybrid nanoassembly, and finally removing the organic solvent by vacuum rotary evaporation to obtain a self-sensitizing dihydroartemisinin nanoassembly; the organic solvent is tetrahydrofuran.

4. The method for preparing the self-sensitizing dihydroartemisinin nanoassemblies as described in claim 3, characterized in that, The method for removing organic solvents is vacuum rotary evaporation, ultrafiltration, or membrane permeation.

5. The method for preparing the PEGylated self-sensitizing dihydroartemisinin nanoassemblies according to claim 2, characterized in that, The process includes the following steps: dissolving the PEG modifier, dihydroartemisinin, and ferrocene into an organic reagent, adding the mixture of the three solutions dropwise into water at a rate of 1 s / d while stirring, stirring at 1300 rpm for 60 s, removing the organic solvent after the reaction is complete, and obtaining the PEGylated self-sensitizing dihydroartemisinin nanoassemblies; the organic solvent is tetrahydrofuran.

6. The method for preparing the PEGylated self-sensitizing dihydroartemisinin nanoassemblies as described in claim 5, characterized in that, The method for removing organic solvents is vacuum rotary evaporation, ultrafiltration, or membrane permeation.

7. A pharmaceutical composition comprising the PEGylated, self-sensitizing dihydroartemisinin nanoassembly of claim 2 and pharmaceutically acceptable excipients.

8. The use of the PEGylated self-sensitizing dihydroartemisinin nanoassembly of claim 2 or the pharmaceutical composition of claim 7 in the preparation of antitumor drugs.

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