An intelligent drug delivery system of cantharidin

Abstract

This invention belongs to the field of biomedical technology and relates to a smart drug delivery system for cantharidin. This invention utilizes a polymer hybrid carrier with precise pH response to deliver cantharidin, achieving efficient delivery and significantly improving the tissue distribution and drug release properties of cantharidin. In particular, this system exhibits excellent lysosomal escape capabilities, significantly enhancing the efficacy and safety of cantharidin in treating breast cancer.

Description

Technical Field

[0001] This invention relates to a cantharidin nanomedicine with precise pH response and its preparation method. Background Technology

[0002] Cantharidin (CTD) is a natural terpene toxin found in insects of the family Bryophyta in the order Coleoptera. It is a poorly soluble cytotoxic drug. Studies have shown that cantharidin has inhibitory effects on various malignant tumors, including breast cancer, liver cancer, and lung cancer. However, its toxic side effects have long limited its application in the anti-tumor field, hindering its therapeutic efficacy. Currently reported nanomedicines containing cantharidin mainly include liposomes, nanoparticles, nanoemulsions, and exosomes. However, these drugs often suffer from instability or leakage during delivery, resulting in generally high systemic toxicity. Furthermore, the reported cantharidin delivery systems lack pH-responsiveness, making them susceptible to lysosomal phagocytosis and degradation, hindering effective cytoplasmic drug release and leading to unsatisfactory efficacy. Therefore, constructing pH-responsive cantharidin nanomedicines is of great significance for the development and utilization of cantharidin. Summary of the Invention

[0003] We used the ionizable graft copolymer Soluplus and the biocompatible amphiphilic polymer mPEG-PLA to construct a pH-responsive delivery carrier. Through single-factor analysis combined with Box-Behnken design-response surface methodology, we prepared cantharidin nanomedicine with stable delivery and pH-responsive functions for the treatment of breast cancer.

[0004] This invention provides a cantharidin nanomedicine with precise pH response function, the nanomedicine comprising cantharidin and a composite carrier material; the composite carrier material comprises polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer and mPEG-PLA.

[0005] Preferably, the polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer is Soluplus. ® And / or the mPEG-PLA is selected from one or more of mPEG2000-PLA2000, mPEG2000-PLA3000, and mPEG2000-PLA4000.

[0006] In some implementations, the mPEG-PLA is selected from mPEG2000-PLA3000.

[0007] In some embodiments, the mass ratio of cantharidin to the composite carrier material is 1:1 to 15, preferably 1:15.

[0008] In some embodiments, the mass ratio of polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer to mPEG-PLA in the composite carrier material is 0.5~5:1, preferably 4.7~5:1.

[0009] In some embodiments, the mass ratio of cantharidin, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer and mPEG-PLA is 1:12.4~12.5:2.5~2.6, preferably 1:12.4:2.6.

[0010] In some embodiments, the nanomedicine is prepared as follows: Cantharidin, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and mPEG-PLA are weighed in the prescribed amounts, dissolved in an appropriate amount of organic solvent, and the mixed organic phase containing cantharidin, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and mPEG-PLA is uniformly injected into the aqueous phase under the condition of ultrasonication with an ice-water bath probe. The mixture is then ultrasonicated, ultrafiltered, and resuspended to obtain CTD-SP.

[0011] In some embodiments, the mPEG-PLA synthesis method is as follows: a certain proportion of mPEG-2000, lactide (D,L-LA) and Sn(Oct)2 are placed in a dry reaction flask, nitrogen is purged to remove oxygen, and the reaction is carried out under vacuum at 135~140 ℃ for 72~80 h. After cooling, an appropriate amount of dichloromethane is added to the product to fully dissolve it, and the product is precipitated in diethyl ether. The lower precipitate is filtered and dried under vacuum at room temperature to constant weight to obtain mPEG-PLA white powder. The mass ratio of mPEG to D,L-LA is 1:1, 2:3 or 1:2 to prepare copolymers mPEG2000-PLA2000, mPEG2000-PLA3000 or mPEG2000-PLA4000 with different hydrophilicity-hydrophobicity segment ratios.

[0012] In some embodiments, the dosage of the CTD-SP is 0.25~1.5 mg / mL, preferably 0.41~0.5 mg / mL.

[0013] In some embodiments, the ultrasonic power is 30~100W, preferably 50W.

[0014] In some embodiments, the ultrasound duration is 30-180 s, preferably 80-90 s, and more preferably 82 s.

[0015] In some embodiments, the organic solvent is selected from one or more of acetone, methanol, ethyl acetate and dichloromethane, preferably acetone.

[0016] In some implementations, the response pH is 5 to 5.5.

[0017] The present invention also provides the application of the aforementioned cantharidin nanomedicine with precise pH response function in the preparation of drugs for treating breast cancer.

[0018] Beneficial effects

[0019] Delivery leakage and lysosomal escape are key challenges in antitumor drug delivery. Before reaching the tumor, drugs must first ensure encapsulation stability and long-term circulation capacity to enhance tumor targeting and reduce systemic toxicity. Furthermore, drugs taken up by tumor cells must overcome the lysosomal barrier to achieve true cytoplasmic drug release. Here, we employed polymer co-processing technology to construct a polymer hybrid carrier with precise pH response to deliver cantharidin. We optimized the formulation and process of cantharidin polymer hybrid nanoparticles (CTD-SP) using Box-Behnken design-response surface methodology, achieving efficient cantharidin delivery and significantly improving its tissue distribution and release performance. In particular, this system exhibits excellent lysosomal escape capabilities, significantly enhancing the efficacy and safety of cantharidin in treating breast cancer. Attached Figure Description

[0020] Figure 1 Box-Behnken 3D and 2D response plots obtained using the comprehensive index OD as the response value. (A) Response relationship between drug / polymer ratio, drug concentration and OD value; (B) Response relationship between ultrasound time, drug concentration and OD value; (C) Response relationship between ultrasound time, drug / polymer ratio and OD value; (D) Contour plot of drug concentration, drug / polymer ratio and OD value; (E) Contour plot of drug concentration, ultrasound time and OD value; (F) Contour plot of drug / polymer ratio, ultrasound time and OD value.

[0021] Figure 2 Characterization of CTD-SP. (A) Scanning electron microscope image of CTD-SP; (B) X-ray diffraction pattern of CTD-SP (the diffraction peaks of CTD are located within the red dashed box, and the diffraction peaks of the polymer are located at the positions indicated by the purple arrows); (C) Particle size distribution of CTD-SP; (D) Zeta potential of CTD-SP.

[0022] Figure 3 Release curves of CTD-SP and free cantharidin under different pH conditions (n=3 for each group).

[0023] Figure 4Comparison of cytotoxicity between CTD-SP and free cantharidin. (A) IC50 values ​​of CTD-SP and free cantharidin against 4T1 cells at 24 hours; (B) IC50 values ​​of CTD-SP and free cantharidin against 4T1 cells at 48 hours; (C) Comparison of cell viability 24 hours after administration of CTD-SP and free cantharidin; (D) Comparison of cell viability 48 hours after administration of CTD-SP and free cantharidin (ns p > 0.05, * p < 0.05, *** p < 0.001, n = 6); (E) Cell morphology 24 hours and 48 hours after administration of CTD-SP and free cantharidin.

[0024] Figure 5 4T1 cell uptake of Dir-SP and free Dir. (A) Fluorescence uptake images of Dir-SP at different time points; (B) Enlarged images of Dir-SP and free Dir (at 90 minutes); (C) Fluorescence uptake images of free Dir at different time points; (D) Statistical analysis of fluorescence intensity of Dir-SP and free Dir; (E) Flow cytometry analysis of DiR-SP uptake by 4T1 cells at different incubation times; (F) Statistical analysis of fluorescence intensity of DiR-SP and free Dir uptake by 4T1 cells using flow cytometry (** p < 0.01, *** p < 0.001, n = 3 / group).

[0025] Figure 6 Precise pH response of CTD-SP and colocalization of coumarin 6-SP with lysosomes. (A) Colocalization images of lysosomes with green fluorescent labeling observed by confocal microscopy; (B) Lysosomal fluorescence imaging at different time points; (C) Precise pH response of CTD-SP; (D) Comparison of Pearson colocalization coefficients at different time points (*** p < 0.001, n = 6).

[0026] Figure 7 In vivo distribution of Dir-SP. (A) In vivo fluorescence distribution within 24 hours after tail vein injection of Dir-SP and free Dir; (B) Fluorescence accumulation at the tumor site within 24 hours after tail vein injection of Dir-SP and free Dir; (C) Fluorescence imaging of ex vivo tissues; (D) Statistical graph of fluorescence intensity of ex vivo tissues (*** p < 0.001, n = 3).

[0027] Figure 8Evaluation of the in vivo antitumor activity and safety of CTD. (A) Tumor volume statistics; (B) Mouse weight change statistics; (C) Survival analysis (median survival of the control group, free cantharidin group and CTD-SP group were 24 days, 25 days and 44 days, respectively); (D) H&E staining map of major organs (40×) (ns p > 0.05, ** p < 0.01, *** p <0.001, n = 5). Detailed Implementation

[0028] Materials and Methods

[0029] 1. Materials

[0030] 1.1 Cantharidin reference standard, 98.0% by mass, was purchased from Chengdu Mansite Biotechnology Co., Ltd.; cantharidin raw material was purchased from Sigma-Aldrich, Germany; Soluplus (polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer) was purchased from BASF, Germany (catalog number: 50539897); mPEG-PLA was synthesized in the laboratory; MTT was purchased from Beijing Coolaber Technology Co., Ltd.; fetal bovine serum (FBS) was purchased from Shanghai Nongsheng Biotechnology Co., Ltd.; 1640 culture medium, trypsin, and penicillin-streptomycin were purchased from Gibco, USA; PBS buffer was purchased from Wuhan Sewell Biotechnology Co., Ltd.; near-infrared fluorescent dye DiR was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 4',6-diamidinyl-2-phenylindole (DAPI) was purchased from Beijing Bio-Top Technology Co., Ltd.; coumarin 6 was purchased from Shanghai Maokang Biotechnology Co., Ltd.; lysosomal red fluorescent probe was purchased from Dalian Meilun Biotechnology Co., Ltd.

[0031] 1.2 BALB / c mice, female, 6–8 weeks old, weighing (18 ± 2) g, were purchased from Beijing SPAFE Biotechnology Co., Ltd. Mouse 4T1 mammary cancer cells were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. All animals used in the above experiments complied with the standards of the Animal Ethics Committee.

[0032] 2. Methods

[0033] 2.1. Preparation of mPEG-PLA

[0034] Synthesis method and molecular weight of mPEG-PLA: Stannous octoate (Sn(Oct)2) was used as a catalyst, and methoxy polyethylene glycol (mPEG-2000) was used as an initiator, via ring-opening polymerization. A certain proportion of mPEG-2000, lactide (D,L-LA), and Sn(Oct)2 were placed in a dry reaction flask, and nitrogen was purged to remove oxygen. The reaction was carried out under vacuum at 135 °C for 72 h. After cooling, an appropriate amount of dichloromethane was added to the product to fully dissolve it. The product was then precipitated in diethyl ether, and the lower precipitate was collected, filtered, and vacuum dried to constant weight at room temperature to obtain a white mPEG-PLA powder. By adjusting the mass ratio of mPEG to D,L-LA (1:1, 2:3, 1:2), copolymers with different hydrophilic / hydrophobic segment ratios (mPEG2000-PLA2000, mPEG2000-PLA3000, mPEG2000-PLA4000) were prepared.

[0035] 2.2. Preparation of CTD-SP

[0036] CTD-SP was prepared using a solvent injection method. Precise amounts of cantharidin raw material (CTD), Soluplus (S), and mPEG2000-PLA3000 (P) were weighed and dissolved in an appropriate amount of organic solvent. The mixed organic phase containing the dissolved cantharidin and polymer materials was uniformly injected into the aqueous phase under ultrasonic conditions with an ice-water bath probe. The ultrasonic power was 50 W, and the peristaltic pump flow rate was 0.6 mL / min. After ultrafiltration and resuspending, CTD-SP was obtained. Blank carrier SP was prepared using the same method.

[0037] 2.3. Determination of CTD-SP encapsulation efficiency and drug loading

[0038] The CTD in CTD-SP was quantified by high performance liquid chromatography, and the encapsulation efficiency (EE) and drug loading (DL) of CTD-SP were calculated according to formulas (1) and (2).

[0039] (1)

[0040] (2)

[0041] W1 represents the amount of encapsulated drug; W2 represents the amount of drug input; and W represents the total amount of drug and materials input.

[0042] 2.4. Single-factor experiment

[0043] Single-factor design and parameters: The effects of different solvents (acetone, methanol, ethyl acetate, dichloromethane), composite material ratios S:P (10:1, 5:1, 2:1), drug-to-carrier mass ratio (CTD:SP) (1:10, 1:15, 1:20), drug dosage (0.25, 0.5, 1 mg / mL), and sonication time (30, 60, 120 s) on the encapsulation efficiency and drug loading of CTD-SP were investigated. Drug dosage refers to the drug concentration after the drug and carrier material are mixed and dissolved in the organic solvent.

[0044] 2.5. Box-Behnken Design Optimization Prescription

[0045] 2.5.1. Box-Behnken Design Experiment Scheme

[0046] Based on the results of the single-factor experiment, the CTD-SP formulation was further optimized using BBD-RSM. Three factors that significantly affected the encapsulation efficiency and drug loading were selected as independent variables X1, X2, and X3, with encapsulation efficiency and drug loading as response values ​​Y1 and Y2, respectively. CTD-SP was prepared according to the obtained experimental design, and the data were normalized according to formula (3) to convert them into d (standard values ​​of 0 to 1). The overall desirability (OD) was calculated using the equal weighting method of formula (4) as a comprehensive index. The experimental results were fitted using Design Expert version 13 software to obtain a binary multiple regression equation and then analyzed.

[0047] (3)

[0048] (4)

[0049] Y i This is the actual measured value; Y min and Y max The minimum and maximum values ​​in the experiment

[0050] 2.5.2. Response Surface Optimization and Prediction

[0051] By fixing one of the independent variables X1, X2, and X3, we obtain a three-dimensional surface plot and a two-dimensional contour plot of the response value of the other two factors. Here, we use the comprehensive index OD as the response value to analyze the impact of changes in each factor within a certain range on the OD value and predict the optimal prescription.

[0052] 2.5.3. Process Validation and Characterization

[0053] Three batches of CTD-SP were prepared using optimized formulation conditions for process validation. The encapsulation efficiency and drug loading were measured, the deviation between the actual and predicted values ​​were calculated, the reliability of the response surface prediction was examined, and the particle size, PDI, zeta potential, XRD and appearance morphology were characterized.

[0054] 2.6. In vitro release rate assessment

[0055] Using free cantharidin as a control, the in vitro release characteristics of CTD-SP at pH=7.4, 6.5, and 5.5 were investigated using the dialysis bag method. After adjusting the drug concentration, 2 mL each of CTD-SP and free cantharidin were placed in dialysis bags and then in 20 mL of release medium (phosphate buffer containing 1% Tween 80, pH=7.4, 6.5, and 5.5). Samples were taken at (37 ± 1) ℃ and 100 r / min at 1, 2, 4, 8, 12, 24, 36, 48, and 60 h, and the same volume of release medium was added. The cantharidin content in the samples was determined by high-performance liquid chromatography, and the cumulative release rate was calculated (5).

[0056] (5)

[0057] Q i V1 represents the cumulative release rate; V2 represents the sample volume at each time point; c1, ..., c i CTD concentration at each time point; V0 is the volume of the drug solution in the dialysis bag; c0 is the concentration of the drug solution in the dialysis bag.

[0058] 2.7. In vitro anti-breast cancer activity study

[0059] 2.7.1. Investigation of Cytotoxicity and Morphological Changes

[0060] 4T1 cells were seeded at a density of 5000-8000 cells / well in 96-well plates and cultured in a cell culture incubator (37 ℃, 5% CO2) until adherent growth. CTD-SP was added at concentrations of 81.55, 40.77, 20.38, 10.19, 5.09, 2.54, 1.27, and 0.64 μM, respectively. A separate well was prepared with the same concentration of free cantharidin as a control. Cell morphology changes were observed after 24 h and 48 h. Cell viability was assessed using the MTT assay, and IC50 was calculated. 50 .

[0061] (6)

[0062] 2.7.2. Investigation of cellular uptake

[0063] Collect 4T1 cells in the logarithmic growth phase at a concentration of 1×10⁻⁶. 4Cells were seeded in confocal microscopy dishes at a density of 1 cell per dish and cultured until they adhered to the surface. Three groups were set up: a Control group, a DiR-SP (DiR-labeled vector SP) group, and a free DiR group. The final concentration of DiR in each group was 2 μg / mL. 1 mL of DiR was added to each dish and the cells were cultured in an incubator for 15 min, 30 min, 60 min, and 90 min, respectively. After reaching the time point, the cells were removed, washed three times with pre-cooled PBS, fixed with 4% paraformaldehyde for 15 min, and the cell nuclei were stained with the fluorescent dye DAPI. The cells were then observed under a confocal microscope and the corresponding fluorescence intensity was analyzed.

[0064] 2.7.3. Investigation of Lysosomal Escape

[0065] 4T1 cells were collected and seeded in confocal microscopy dishes. After cell adhesion and growth, C6-SP (coumarin 6-labeled vector SP), 2 μg / dish, was added and incubated for 2 h and 4 h, respectively. Cells were then stained with 100 nM Lyso Tracker Red for 1 h, washed three times with PBS, and observed under a confocal microscope. 0.1 mol / L NaOH solution was added to the CTD-SP, 10 μL each time, titrating to pH 10. Then, 0.1 mol / L HCl solution was added, 10 μL each time, titrating to pH 4. The pH change was recorded to investigate the proton buffering capacity of CTD-SP.

[0066] 2.8. In vivo targeting evaluation

[0067] Collect 4T1 cells, prepare single-cell suspensions, and use 1×10⁻⁶ cells per cell. 6 Cells were inoculated into the mammary pads of BALB / c mice at a rate of 100 cells per mouse. The tumors were allowed to grow to 400 mm in size. 3 The mice were randomly divided into DiR-SP and free DiR groups, with three mice in each group. The fluorescence concentration was uniform. After being injected into the body via the tail vein (2 μg / mouse), in vivo imaging was performed at 1, 5, 12 and 24 h to examine the fluorescence distribution. After 24 h, the mice were euthanized by cervical dislocation, and their heart, liver, spleen, lung, kidney and tumor tissues were dissected and photographed in vitro to examine the fluorescence content of each tissue.

[0068] 2.9. Evaluation of in vivo anti-breast cancer activity and safety

[0069] 2.9.1. Pharmacodynamic Investigation

[0070] Two weeks after acclimatization, BALB / c mice were shaved from the abdomen. Mouse breast cancer cells (4T1) in the logarithmic growth phase were collected and formulated to a concentration of 1×10⁻⁶. 6 A single-cell suspension of 150 μL was inoculated into the mammary pads of BALB / c mice, and the tumor volume was allowed to progressively grow to 60-80 mm.3 A round, firm mass indicates successful tumor modeling. Tumor-bearing mice were randomly divided into Control (PBS injection), cantharidin-free, and CTD-SP groups, with five mice in each group. Mice in each group received the drug via tail vein injection at a dose of 0.25 mg / kg, 200 μL each time. Mice in the Control group received the same volume of saline as the drug group each time. The drug was administered four times, with 48-hour intervals between each administration. Other tumor-bearing mice were also randomly divided into Control (PBS injection), free cantharidin, and CTD-SP groups, and administered the drug in parallel with the three groups described above.

[0071] 2.9.2. Histopathological Analysis

[0072] A tumor-bearing mouse model was established and randomly divided into a Control group (injected with PBS), a free cantharidin group, and a CTD-SP group. The mice were administered the drug via tail vein injection every other day for four doses. Fifteen days after the first administration, the mice were euthanized by cervical dislocation. The heart, liver, spleen, lungs, kidneys, and tumor tissue were dissected and prepared into paraffin sections for H&E staining. The prepared sections were photographed under a microscope to observe their histological morphology.

[0073] 3. Results

[0074] 3.1. Single-factor analysis

[0075] Explanation of single-factor screening results:

[0076] (1) Using acetone as the reaction solvent, the EE of the prepared CTD-SP was (72.51 ± 4.24)% and the DL was (5.07 ± 0.34)%, both of which were significantly higher than those of other groups. Therefore, acetone was determined to be the best reaction solvent in the single-factor study.

[0077] (2) When the S:P mass ratio is 5:1, the EE of CTD-SP is (81.83 ± 2.86)% and the DL is (5.63 ± 0.35)%, both of which are higher than those of other groups. Therefore, the optimal composite material ratio S:P is determined to be 5:1 in the single-factor study.

[0078] (3) When the reaction solvent is fixed as acetone and the S:P ratio of the composite material is 5:1, different drug-carrier mass ratios are screened. The ratios of 1:10 and 1:20 have poor stability. 1:15, which has a higher EE than other groups and better stability, is selected as the best drug-carrier mass ratio in the single-factor study.

[0079] (4) With other conditions unchanged, the results of the two groups with ultrasound time of 60 s and 120 s were similar. At 60 s, EE and DL were slightly higher. Therefore, the optimal ultrasound time was initially determined to be 60 s in the single-factor study, and further refined in the response surface design.

[0080] (5) When the drug concentration is 0.5 mg / mL, the EE and DL of CTD-SP are both high. As the drug concentration increases, the EE and DL of the prepared CTD-SP decrease significantly. Therefore, the optimal drug concentration is selected as 0.5 mg / mL in the single factor study.

[0081] After single-factor screening, the optimal solvent was determined to be acetone, the CTD:S:P mass ratio was 1:12.5:2.5, the sonication time was 60 s, and the drug dosage was 0.5 mg / mL. Among these factors, the drug-to-carrier mass ratio, sonication time, and drug dosage had a significant impact on the encapsulation efficiency and drug loading of the formulation. These three factors were selected for the next step of response surface methodology optimization.

[0082] 3.2. Box-Behnken Design Optimization Prescription

[0083] 3.2.1. Fitting the Box-Behnken Design Model

[0084] Based on the results of the single-factor experiment, the dosage, drug-to-carrier mass ratio, and ultrasonic time were used as independent variables X1, X2, and X3, respectively. Calculations were performed for formulation and injection, and the experimental results were fitted to obtain a multivariate binomial regression equation: OD = 0.9333 + 0.0567X1 - 0.1278X2 + 0.0531X3 + 0.1013X1X2 - 0.0707X1X3 - 0.0823X2X3 - 0.2982X1² - 0.3475X2² - 0.2168X3², R² = 0.9909, R... adj 2 =0.9793, R pre =0.9635, indicating that the regression model fits well and has a high degree of agreement with reality, making it suitable for prescription optimization studies. Analysis of variance using OD values ​​showed that all fitted models were statistically significant (P < 0.05), and the lack-of-fit terms were not significant (P > 0.05), indicating that unknown factors have little interference with the experimental results and that the model is highly reliable.

[0085] 3.2.2. Response Surface Optimization and Prediction

[0086] Box-Behnken 3D and 2D response plots obtained using the comprehensive index OD as the response value. Figure 1 As shown in AF), the denser the contour lines, the more significant the impact of changes in factor values ​​on the response value within this range. The red area represents high response values, and the blue area represents low values. The center point of the three sets of overlapping red areas is the predicted optimal formulation process. The predicted optimal CTD-SP formulation has a drug concentration of 0.41 mg / mL, CTD:S:P = 1:12.4:2.6, and an ultrasonic time of 82 s.

[0087] 3.2.3 Preparation of the optimal prescription CTD-SP

[0088] CTD-SP was prepared using a solvent injection method. Precise amounts of cantharidin raw material (CTD), Soluplus (S), and mPEG2000-PLA3000 (P) were weighed and dissolved in an appropriate amount of acetone. The mixed organic phase containing the dissolved cantharidin and polymer materials was uniformly injected into the aqueous phase under ultrasonic conditions with an ice-water bath probe. The ultrasonic power was 50 W, and the peristaltic pump flow rate was 0.6 mL / min. After ultrafiltration and resuspension, CTD-SP was obtained. The dosage concentration of CTD-SP was 0.41 mg / mL, CTD:S:P = 1:12.4:2.6, and the ultrasonic time was 82 s.

[0089] 3.2.4. Process Validation and Characterization

[0090] As shown in Table 1, the actual values ​​deviated from the predicted values ​​in terms of drug loading (0.99%) and encapsulation rate (1.06%), both of which were close to the predicted values, proving that the model fits well and that the BBD-RSM-optimized CTD-SP prescription has high reliability.

[0091] Table 1. Process Validation Indicators and Evaluation (n=3)

[0092] Evaluation indicators actual value Predicted value RD (%) EE (%) 81.91 ± 1.84 81.05 1.06 DL (%) 5.12 ± 0.12 5.07 0.99

[0093] The optimized CTD-SP formulation is clear and exhibits a pale blue opalescence. A small amount of diluted CTD-SP was dropped onto a silicon wafer and observed under a scanning electron microscope. The resulting CTD-SP particles were spherical with a uniform size distribution. Figure 2 A). Characterization by X-ray diffraction revealed distinct diffraction peaks of cantharidin at 15.69° and 32.02°. These peaks were also present in the physical mixture of the polymer material and cantharidin, while neither peak was observed in the CTD-SP. This indicates that cantharidin can be well encapsulated within the polymer carrier, existing in an amorphous form. Figure 2 B).

[0094] The prepared CTD-SP was diluted appropriately, and the particle size and zeta potential were measured. The particle size was (58.1 ± 0.85) nm, the PDI was (0.128 ± 0.01), and the zeta potential was (-26.6 ± 1.04) mV. The particle size distribution was uniform and stable, the system carried a negative charge, and all conformed to a normal distribution. Figure 2 C, 2D).

[0095] 3.3. In vitro release rate study of the optimal formulation CTD-SP

[0096] Depend on Figure 3 It was found that CTD-SP remained stable at pH 7.4 within 60 h, with no cantharidin release detected in the medium; a slight release occurred at pH 6.5, with a cumulative release of approximately 10%; and a cumulative release of approximately 75% occurred at pH 5.5. Compared with the CTD-SP group, the release rate of free cantharidin was basically consistent in all three environments, with no significant difference, and it was almost completely released within 12 h. In contrast, CTD-SP, in the lysosomal simulated environment at pH 5.5, showed a cumulative release of approximately 50% within 12 h. These results indicate that CTD-SP possesses significant sustained-release properties and pH sensitivity.

[0097] 3.4. In vitro anti-breast cancer activity study of the optimal formulation CTD-SP

[0098] 3.4.1. Investigation of Cytotoxicity and Morphological Changes

[0099] The cytotoxic effects of CTD-SP and free cantharidin on 4T1 cells were concentration-dependent. The IC50 values ​​of CTD-SP and free cantharidin at 24 h were [not specified in the original text]. 50 The concentrations were 18.02 μM and 33.32 μM, respectively. Figure 4 A), during 48 hours of IC 50 The concentrations were 1.80 μM and 3.51 μM, respectively. Figure 4 B), Statistical analysis of the effects of drugs on cell viability, such as... Figure 4 C, 4D, it is evident that the cytotoxic effect of CTD-SP was significantly superior to that of free cantharidin (ns p > 0.05, * p < 0.05, *** p < 0.001, n = 6 / group). Observe the changes in cell morphology ( Figure 4 E) It can also be seen that the cells in the Control and Blank groups grew normally, while the free cantharidin and CTD-SP groups showed varying degrees of cell shedding and death, and the cell density in the CTD-SP group decreased more significantly.

[0100] 3.4.2. Investigation of cellular uptake

[0101] DiR-SP can be effectively taken up by cells. Within the observation time range, the fluorescence signal significantly increased with the extension of the cell uptake time of DiR-SP, and was significantly better than the fluorescence intensity of the free DiR group. Figure 5 AD), a magnified view of the local area shows that DiR-SP has a clearer advantage ( Figure 5 B), by flow cytometry ( Figure 5 E, 5F) further validated this conclusion. (**p < 0.01, ***p < 0.001, n = 3 / group).

[0102] 3.5. Evaluation of the Lysosomal Escape Capacity of the Optimal Prescription CTD-SP

[0103] like Figure 6 As shown in Figures A and B, the vector SP was labeled with coumarin-6, and lysosomes were labeled with the red fluorescent dye Lysotrcaker. The figures show that at 2 h, the vector was clearly endocytosed and remained within the endosomes (early lysosomes) in 4T1 cells. As the endosomes matured, they swelled, which may be due to the protonation of the hybrid vector designed to exert a proton sponge effect through the protonation of tertiary amine residues. Figure 6 (C, D) As time progressed, by 4 h, the intensity of red fluorescence in the visible region of lysosomes decreased, and the yellow fluorescence colocalization signal was significantly weakened. Simultaneously, the quantitative data showed Pearson colocalization coefficients (C, D). Figure 6 D) was also significantly reduced, consistent with the characteristics of lysosomal escape. (*** p < 0.001, n = 6 / group).

[0104] 3.6. Optimal Prescription CTD-SP Targeting Evaluation

[0105] Depend on Figure 7 As shown in A and 7B, weak fluorescence signals were detected at the tumor site in mice with free DiR only at 24 h. In contrast, DiR-SP fluorescence signals reached the tumor site 1 h after in vivo and accumulated there over time, reaching a fluorescence value of 6 × 10⁻⁶ at 24 h. 7 AU. Mouse tissue was dissected 24 h later for in vitro fluorescence imaging and tissue fluorescence statistics were performed. Figure 7 (C, 7D) As can be seen, the DiR-SP group showed obvious fluorescence distribution in the tumor, while free DiR showed no fluorescence, consistent with the in vivo imaging results, indicating that DiR-SP has good targeting ability. (*** p < 0.001, n = 3 / group).

[0106] 3.7. Evaluation of the in vivo anti-breast cancer activity and safety of the optimal formulation CTD-SP

[0107] like Figure 8 As shown in Figure A, the tumor volume in the Control group mice grew rapidly, showing a linear growth trend; the free cantharidin group had a certain inhibitory effect on tumors in the early stage of administration, but the tumor tissue expanded rapidly after administration was stopped, with an overall trend similar to the Control group, and no significant difference in the middle and late stages; the CTD-SP group had a significant inhibitory effect on tumors during administration, and the tumor growth trend remained slow after administration was stopped, showing a significant difference in tumor volume compared with the Control group and the free cantharidin group (tumor volume: Conrtol group > free cantharidin group > CTD-SP group). Meanwhile, Figure 8As shown in Figure B, there was no significant difference in body weight changes among the tumor-bearing mice in each group. However, as the tumor volume and weight increased, the CTD-SP group mice maintained a stable relative body weight. Figure 8 As shown in the C-survival chart, the survival time of mice in the CTD-SP group was significantly longer than that of the other two groups, with a statistically significant difference. Mouse tissues were observed at the same magnification using the software "CaseViewer". Figure 8 (D) In ​​both the Control group and the free cantharidin group, alveolar septal thickening was observed in the lung tissue. No significant pathological changes were observed in the major organs of the CTD-SP group, but significant nuclear condensation and rupture were observed at the tumor site. Compared to the free cantharidin group, the necrotic area was larger. These results indicate that CTD-SP has stronger tumor inhibition and better biocompatibility than free cantharidin. (ns p > 0.05, ** p < 0.01, *** p < 0.001, n = 5 / group).

Claims

1. A cantharidin nanomedicine with precise pH response, characterized in that, The nanomedicine comprises cantharidin and a composite carrier material; the composite carrier material comprises a polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer and mPEG-PLA; the polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer is Soluplus®; the mPEG-PLA is selected from mPEG2000-PLA3000; the mass ratio of cantharidin, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer and mPEG-PLA is 1:12.4~12.5:2.5~2.6; the nanomedicine responds to a pH of 5~5.

5.

2. The cantharidin nanomedicine as described in claim 1, characterized in that, The preparation method of the nanomedicine is as follows: Cantharidin, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and mPEG-PLA are weighed in the prescribed amount, dissolved in an appropriate amount of organic solvent, and the mixed organic phase containing cantharidin, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, and mPEG-PLA is uniformly injected into the aqueous phase under the condition of ultrasonication with an ice-water bath probe. The mixture is then ultrasonicated, ultrafiltered, and resuspended to obtain cantharidin polymer hybrid nanoparticles (CTD-SP). The dosage of CTD-SP is 0.41~0.5 mg / mL; the ultrasonication time is 80~90 s; and the organic solvent is selected from acetone.

3. The cantharidin nanomedicine according to any one of claims 1-2, characterized in that, The method for synthesizing mPEG-PLA is as follows: mPEG-2000, lactide (D,L-LA), and Sn(Oct)2 are placed in a dry reaction flask, nitrogen is purged to remove oxygen, and the reaction is carried out under vacuum at 135~140 ℃ for 72~80 h. After cooling, an appropriate amount of dichloromethane is added to the product to fully dissolve it, and the product is precipitated in diethyl ether. The lower precipitate is filtered and dried under vacuum at room temperature to constant weight to obtain white mPEG-PLA powder. The mass ratio of mPEG to D,L-LA is 2:

3. mPEG2000-PLA3000 is thus prepared.

4. The cantharidin nanomedicine as described in claim 2, wherein the CTD-SP dosage is 0.41 mg / mL.

5. The cantharidin nanomedicine according to claim 2, wherein the ultrasound time is 82s.

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