Carboplatin-loaded nano-carrier drug delivery system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and provides a nano-carrier drug delivery system loaded with carboplatin as well as a preparation method and application of the nano-carrier drug delivery system. The preparation method comprises the following steps: (1) dissolving AZ-COOH, EDCI and HOBT in DMSO, and activating to obtain a first mixed solution; dSPE-PEG2000-NH2 and TEA are dissolved in DMSO (dimethyl sulfoxide), and a second mixed solution is obtained after stirring; dropwise adding the first mixed solution into the second mixed solution, reacting, dialyzing, and drying to obtain a nano-carrier DSPE-PEG-CAI; (2) dissolving the nano carrier DSPE-PEG-CAI and carboplatin in an organic solvent to obtain an organic phase; adding ultrapure water, continuously performing ultrasonic treatment, and volatilizing the organic solvent to obtain a water phase; and carrying out aqueous phase dialysis, and filtering to obtain a nano carboplatin DSPE-PEG-CAI-CBP aqueous solution. The carboplatin-loaded nano-carrier drug delivery system provided by the invention can improve the targeting property and intracellular enrichment degree of drugs, and passes through a biological barrier established by a tumor microenvironment, so that the targeting selectivity to triple negative breast cancer cells is enhanced, and the side effects of carboplatin treatment are reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a carboplatin-loaded nanocarrier drug delivery system, its preparation method, and its application. Background Technology

[0002] Although carboplatin (CBP) is widely used as a first-line chemotherapy drug in the treatment of breast cancer, it has significant side effects and is prone to causing drug resistance. Therefore, researchers at home and abroad have proposed developing effective nanocarriers to precisely deliver carboplatin to tumor cells, which could help improve treatment efficacy, reduce side effects, and decrease the occurrence of drug resistance.

[0003] Over the past decade, various technologies have been developed for drug targeting in cancer, as well as prodrug testing, drug structure modification, and carrier-mediated drug delivery. While these strategies can improve treatment outcomes, they also have drawbacks. Invasive techniques introduce unwanted substances into the body, increasing the risk of intracranial infection; prodrug testing methods are prone to loss of drug activity and stability after modification. In recent years, colloidal drug carrier systems, particularly nanoparticles, have emerged as an emerging field in drug delivery systems that target cancer cells via cell signaling pathways. Nanoparticles are essentially solid colloidal particles with an average size between 10 and 1000 nanometers (typically 50-300 nanometers), in which drug-loaded substances are loaded, dissolved, passively adsorbed, or covalently bound to the polymer surface. As an excellent drug delivery system, nanocarriers have the ability to improve drug stability, solubility, and targeting, while also modulating drug release rates and improving transmembrane transport efficiency. These advantages make nanocarriers a promising drug delivery platform, offering new solutions for treating various diseases.

[0004] While nanoparticles offer significant improvements in water solubility, biodistribution, bioavailability, and toxicity, their effectiveness in tumor accumulation remains limited due to the biological barriers established by the tumor microenvironment, even with passive targeting. Carbonic anhydrase IX (CA IX) is a key downstream gene product in a hypoxia-inducible factor (HIF)-mediated signaling cascade. CA IX is responsible for reversible carbon dioxide hydration and the production of carbonate and protons, regulating the pH balance between intracellular (pHi) and extracellular (pHe). These processes promote cancer cell survival, invasion, and migration. CA IX is known to be overexpressed on the surface of highly invasive triple-negative breast cancer cells. Therefore, CA IX inhibitors (CAIs), which have a high affinity for CA IX, can enhance targeting selectivity for triple-negative breast cancer cells.

[0005] 1,2-Distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol (DSPE-PEG) is a phospholipid-polymer conjugate widely used in drug delivery. It is a biocompatible, biodegradable, and amphiphilic material that can be functionally modified with various biomacromolecules to achieve specific functions.

[0006] Therefore, this invention develops an effective nanocarrier. By introducing the DSPE-PEG-CAI nanocarrier as a delivery method for carboplatin, a commonly used drug in the treatment of breast cancer, a new approach is provided. This allows for more precise and effective delivery of carboplatin to tumor cells, thereby improving the therapeutic effect of breast cancer, reducing side effects during treatment, and reducing the occurrence of chemotherapy resistance. Summary of the Invention

[0007] The purpose of this invention is to provide a carboplatin-loaded nanocarrier drug delivery system, its preparation method, and its application. By using the structurally stable and biocompatible DSPE-PEG-CAI nanocarrier, a new route is provided for the delivery of carboplatin, a commonly used drug in breast cancer treatment. This effectively encapsulates and protects carboplatin, thereby improving drug targeting and intracellular enrichment. Furthermore, it can cross the biological barrier established by the tumor microenvironment, enhancing the targeting selectivity for triple-negative breast cancer cells, thus enhancing efficacy and reducing the side effects of carboplatin treatment.

[0008] In a first aspect, the present invention provides a method for preparing a carboplatin-loaded nanocarrier drug delivery system, comprising the following steps:

[0009] (1) Preparation of nanocarrier DSPE-PEG-CAI

[0010] AZ-COOH, EDCI, and HOBT were dissolved in DMSO and activated to obtain the first mixed solution; DSPE-PEG was then... 2000 -NH2 and TEA were dissolved in DMSO and stirred to obtain a second mixed solution; the first mixed solution was added dropwise to the second mixed solution, and after the reaction, it was dialyzed and dried to obtain the nanocarrier DSPE-PEG-CAI;

[0011] (2) Preparation of nanocarboplatin DSPE-PEG-CAI-CBP

[0012] The nanocarrier DSPE-PEG-CAI and carboplatin were dissolved in an organic solvent and sonicated to obtain an organic phase. The organic phase was then rapidly added to ultrapure water and sonicated continuously. The organic solvent was then evaporated or recovered to obtain an aqueous phase. The aqueous phase was dialyzed to obtain a filtered aqueous solution of nanocarboplatin DSPE-PEG-CAI-CBP.

[0013] Preferably, the mass ratio of AZ-COOH, EDCI, and HOBT is 31:27:19.

[0014] Preferably, the solid-liquid ratio of the first mixed solution is less than or equal to 77 mg: 1 mL.

[0015] Preferably, the DSPE-PEG 2000 The mass ratio of -NH2 to TEA is 100:11.

[0016] Preferably, the solid-liquid ratio of the second mixed solution is less than or equal to 111 mg: 1 mL.

[0017] Preferably, the activation temperature is 10-30℃ and the time is at least 2 hours; the stirring temperature is 10-30℃ and the time is at least 2 hours; and the reaction time is at least 24 hours.

[0018] Preferably, the mass ratio of DSPE-PEG-CAI to carboplatin is 2-15:2.

[0019] Preferably, the organic solvent is THF, the solid-liquid ratio in the organic phase is 4-17 mg:1 mL, and the solid-liquid ratio in the aqueous phase is 4-17 mg:5 mL.

[0020] Preferably, the ultrasonic treatment power is 100-150W and the ultrasonic time is 10-30min.

[0021] Preferably, the recovered organic solvent is recovered by using a rotary evaporator, wherein the temperature of the rotary evaporator is 40-60℃, the rotation speed is 100-200rpm, and the vacuum pressure is 0.65-0.8Mpa.

[0022] Preferably, the drying method is freeze drying or vacuum drying, the dialysis parameters are MWCO: 1000-3000 Da, and the filtration membrane is a 0.22 μm sterile membrane.

[0023] Secondly, the present invention also provides a carboplatin-loaded nanocarrier drug delivery system prepared by the above-described preparation method.

[0024] Thirdly, the present invention also provides the application of the above-mentioned carboplatin-loaded nanocarrier drug delivery system in the preparation of a drug system for treating breast cancer.

[0025] The beneficial effects of this invention are:

[0026] This invention introduces a novel delivery route for carboplatin by using a structurally stable, biocompatible polyethylene glycol liposome nanocarrier, DSPE-PEG-CAI, which carries a carbonic anhydrase inhibitor. This effectively encapsulates and protects carboplatin, improving drug targeting and intracellular accumulation. It can also cross the biological barrier established by the tumor microenvironment, thereby enabling more precise and effective delivery of carboplatin to tumor cells. This enhances the targeting selectivity for triple-negative breast cancer cells, thereby improving the therapeutic effect of breast cancer, reducing side effects during treatment, and reducing the occurrence of chemotherapy resistance. Attached Figure Description

[0027] Figure 1 The 1H NMR spectrum of the nanocarrier DSPE-PEG-CAI;

[0028] Figure 2 The morphology of DSPE-PEG-CAI-CBP nanoparticles under a transmission electron microscope (scale bar: A, B: 200 nm; C, D: 50 nm).

[0029] Figure 3 The release curve of nanocarboplatin DSPE-PEG-CAI-CBP with a drug carrier mass ratio of 2:15 after 48 hours in an environment of pH 7.4;

[0030] Figure 4 The parameters are: particle size, polymer dispersion coefficient, zeta potential, drug loading rate, and encapsulation efficiency of nanocarboplatin with different drug carrier mass ratios (A is particle size, B is polymer dispersion coefficient, C is zeta potential, D is drug loading rate, and E is encapsulation efficiency; where 2:2, 2:5, 2:10, and 2:15 refer to the mass ratio of carboplatin to nanocarrier self-assembly, respectively).

[0031] Figure 5 Images show cellular uptake of two different drugs, DSPE-PEG-CAI-CBP and DSPE-PEG-CBP, as well as the quantitative average fluorescence intensity values ​​of the two drugs within cells (A is the cellular uptake image of the two different drugs under a confocal microscope, B is the quantitative average fluorescence intensity value of the two drugs at different time periods, the image scale bar is 100 μm, ***p < 0.001, ****p < 0.0001, n indicates no significance).

[0032] Figure 6 The cell survival rate of MDA-MB-231 cells after 72 hours of treatment with carboplatin monotherapy (CBP) and nano-carboplatin (DSPE-PEG-CAI-CBP) was determined by different concentrations of carboplatin.

[0033] Figure 7 This is a comparison of tumor size after 30 days of sacrifice and dissection in mice from the nano-CBP group, CBP group, and PBS group in animal experiments.

[0034] Figure 8 These are the tumor volume growth curves of different groups in animal experiments (A is the growth curve of the nano-CBP group, CBP group and PBS group, B is the nano-CBP group, C is the CBP group, and D is the PBS group).

[0035] Figure 9 This is an analysis of Ki-67 in animal experiments in the nano-CBP group, CBP group, and PBS group (A is Ki-67 immunostained sections, B is quantitative analysis of Ki-67 expression levels).

[0036] Figure 10 These are HE-stained sections of different tissues from the nano-CBP group, CBP group, and PBS group. Detailed Implementation

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] Example 1: Preparation of Nanocarboplatin DSPE-PEG-CAI-CBP

[0041] I. Experimental Materials

[0042] Acetazolamide-carboxylic acid, abbreviated as AZ-COOH; 1-ethyl-3-(3-dimethylpropylamine)carbodiimide, abbreviated as EDCI; 1-hydroxybenzotriazole, abbreviated as HOBT; dimethyl sulfoxide, abbreviated as DMSO; distearylphosphatidylethanolamine-polyethylene glycol-amino, abbreviated as DSPE-PEG. 2000 -NH2; Triethanolamine, abbreviated as TEA; Tetrahydrofuran, abbreviated as THF.

[0043] II. Experimental Procedure

[0044] (1) Preparation of nanocarrier DSPE-PEG-CAI

[0045] 31 mg (0.11 mmol) AZ-COOH, 27 mg (0.14 mmol) EDCI, and 19 mg (0.140 mmol) HOBT were dissolved in 1 mL DMSO and activated at room temperature (10-30℃) for 2 hours to obtain activated AZ-COOH (first mixed solution). 100 mg (0.036 mmol) DSPE-PEG was added. 2000 -NH2 and 11 mg (0.110 mmol) TEA were dissolved in 1 mL DMSO and stirred at room temperature for 2 hours to obtain a second mixed solution. The first mixed solution was then added dropwise to the second mixed solution and reacted at room temperature for 24 hours. After the reaction was complete, the reaction solution was dialyzed against a dialysis bag (MWCO: 1000 Da) for 72 hours to remove unreacted raw materials and solvent, yielding a DSPE-PEG-CAI nanocarrier solution. The product was then freeze-dried to obtain the polyethylene glycol liposome nanocarrier DSPE-PEG-CAI carrying a carbonic anhydrase inhibitor.

[0046] (2) Preparation of nanocarboplatin DSPE-PEG-CAI-CBP

[0047] Carboplatin and the nanocarrier DSPE-PEG-CAI were weighed at mass ratios of 2:2, 2:5, 2:10, and 2:15 (see Table 1), dissolved in 1 mL of THF, and sonicated (100 W, 10 min) to ensure uniform drug dispersion in the organic phase. 5 mL of ultrapure water was preheated to 30°C (to reduce solvent interfacial tension), and the organic phase liquid was rapidly added to the ultrapure water under sonication. Sonication continued (100 W, 30 min) to promote self-assembly. After sonication, the mixture was stirred with a magnetic stirrer for 48 hours to allow THF to fully evaporate. The mixture was then transferred to a rotary evaporator (40°C, 200 rpm, 20 min) to remove residual THF. Next, the solution was placed in a dialysis bag (MWCO 3.5kDa) and dialyzed in deionized water for 24 hours (changing the water every 4 hours) to remove free carboplatin and obtain a suspension of DSPE-PEG-CAI-CBP nanoparticles. The suspension was then filtered through a 0.22μm sterile filter membrane to obtain a THF-free and well-encapsulated DSPE-PEG-CAI-CBP aqueous solution (5ml in total).

[0048] Table 1. Mass data for different ratios of carboplatin to nanocarriers

[0049]

[0050] Example 2: Determination of particle size, drug loading rate, and encapsulation efficiency of nano-carboplatin DSPE-PEG-CAI-CBP

[0051] I. Experimental Methods

[0052] 1.1 Morphological observation of nanocarboplatin and nanocarrier

[0053] Morphological observation was performed using transmission electron microscopy (TEM) after negative staining. 10 μL of the nanocarboplatin aqueous solution (drug carrier mass ratio 2:15) prepared in Example 1 was added to a copper grid and allowed to precipitate for 1 minute. The supernatant was then absorbed with filter paper. 10 μL of 2% phosphotungstic acid (pH 7.0) was added for negative staining, and the mixture was allowed to stand for 1 minute. The supernatant was then absorbed with filter paper. After drying at room temperature for several minutes, electron microscopy imaging was performed at 80 kV. The images were then acquired and analyzed under a TEM microscope. The morphology of the nanocarrier was observed using the same method.

[0054] In vitro release of 1.2 nm carboplatin DSPE-PEG-CAI-CBP

[0055] Cut the dialysis bag to a suitable length and soak it in deionized water for 12 hours, then equilibrate it with phosphate-buffered saline (PBS) for 1 hour. Take 1 mL of the prepared nanoparticle suspension (drug carrier mass ratio 2:15) and place it into the dialysis bag, sealing both ends with a sealing clip (to avoid air bubbles). Immerse the dialysis bag in a 50 mL beaker containing PBS buffer and place it in a 37°C constant-temperature shaking water bath (100 rpm). At 0.5, 1, 2, 4, 8, 12, 24, and 48 hours, remove 2 mL of solution from the release medium and immediately add 2 mL of fresh PBS to maintain a constant volume. Measure the platinum content of the collected liquid samples using ICP-OES to compare the platinum release under simulated normal tissue conditions.

[0056] 1.3 Particle size determination of nanocarboplatin DSPE-PEG-CAI-CBP

[0057] The particle size, polydispersity (PDI), and zeta potential of the nanocarboplatin nanoparticles (drug carrier mass ratios of 2:2, 2:5, 2:10, and 2:15) prepared in Example 1 were determined using a nanoparticle size and zeta potential analyzer (DLS) under suitable conditions (temperature: 25°C, detection angle: 173° backscatter mode). Each sample was tested in triplicate, and the average value was taken.

[0058] 1.4 Determination of drug loading and encapsulation efficiency of nano-carboplatin DSPE-PEG-CAI-CBP

[0059] Using inductively coupled plasma optical emission spectrometry (ICP-OES), the platinum content of small amounts of nanoparticle suspensions (drug-carrier mass ratios of 2:2, 2:5, 2:10, and 2:15, respectively) was determined. The mass of carboplatin in the nanocarboplatin DSPE-PEG-CAI-CBP drug was calculated based on the mass conversion, and the loading efficiency (LE%) and encapsulation efficiency (EE%) of the prepared drug were calculated. The calculation formulas are as follows:

[0060] LE%= ×100% (Formula 1)

[0061] EE%= ×100% (Formula 2).

[0062] II. Experimental Results

[0063] 2.1 Characterization of nanocarboplatin and nanocarboplatin

[0064] The characterization NMR spectrum of the nanocarrier DSPE-PEG-CAI prepared in Example 1 is as follows: Figure 1 As shown, the active hydrogen of the amine group is around 1.7 ppm. Comparing the reactions before and after, it can be seen that the active hydrogen of the amine group disappears after substitution. Simultaneously, two sets of peaks appear around 2.3 ppm, representing hydrogen on the adjacent carbon of the amide. This NMR data confirms the successful preparation of the DSPE-PEG-CAI amphiphilic polymer.

[0065] The nanocarboplatin DSPE-PEG-CAI-CBP (drug carrier mass ratio of 2:15) prepared in Example 1 was stored at 4°C for 15 days. The morphology of the DSPE-PEG-CAI-CBP nanoparticles was observed by transmission electron microscopy (TEM). The results showed that the nanoparticles exhibited a uniform spherical or near-spherical structure with a smooth surface, good dispersibility, and no obvious agglomeration (see [link to article]). Figure 2 This indicates that it has good short-term stability and is suitable for subsequent in vivo experiments.

[0066] 2.2 nm Carboplatin DSPE-PEG-CAI-CBP in vitro release characteristics

[0067] Experimental results showed that, under simulated physiological conditions, the cumulative release rate of the nanoparticles was 22.73% within 24 hours and reached 23.37% within 48 hours, exhibiting typical sustained-release characteristics in the release curve (see...). Figure 3 The initial release rate is relatively fast (0–8 hours), then gradually slows down, which is consistent with the pattern of hydrophilic drugs being slowly released from nanocarriers through diffusion mechanisms.

[0068] 2.3 Results of particle size determination of nanocarboplatin

[0069] The results showed that, except for the carboplatin nanoparticles with a drug carrier mass ratio of 2:10, whose diameter range was greater than 100 nm, the diameter range of other carboplatin nanoparticles was 80–100 nm, consistent with the hydrodynamic particle size results determined by dynamic light scattering (DLS) (see...). Figure 4 (A) DLS analysis showed that the average particle size of the nanoparticles with a drug-carrier mass ratio of 2:15 was 89.11±6.04 nm, and the mean polydispersity index (PDI) was 0.30±0.05, indicating that the nanocarboplatin formulation has good monodispersity (PDI < 0.3) (see A). Figure 4 (B in the text). This narrow distribution characteristic is attributed to the optimization of the ultrasonic-assisted self-assembly process in the nanoprecipitation method, which effectively controls the uniformity of the particles. Furthermore, the average Zeta potential of these nanoparticles is -5.14 ± 0.67 mV (see [reference needed]). Figure 4 The negative surface charge of the C group indicates its excellent colloidal stability in aqueous solution, which can reduce particle aggregation through electrostatic repulsion, providing a basis for its long-term circulation characteristics in organisms. This also further verifies the hydrophilic modification effect of the polyethylene glycol (PEG) chains on the nanoparticle surface. PEGylation not only reduces the immunogenicity of the nanoparticles but also enhances their stability through steric hindrance.

[0070] Drug loading performance and encapsulation efficiency of 2.4 nm carboplatin DSPE-PEG-CAI-CBP

[0071] The platinum content in nanoparticles with a drug carrier mass ratio of 2:15 was quantitatively analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES). The results showed that the equivalent mass of carboplatin per milliliter of nanoparticle suspension was 0.19 ± 0.002 mg. Based on the initial dosage (2 mg carboplatin, 15 mg DSPE-PEG-CAI nanocarrier), the drug loading rate (LE%) and encapsulation efficiency (EE%) of DSPE-PEG-CAI-CBP were calculated to be 6.48% and 51.98%, respectively. The drug loading performance of the final formulation met the requirements of subsequent pharmacodynamic experiments.

[0072] Application Example 1: In vitro experiments

[0073] I. Experimental Methods

[0074] 1.1 Assessment of Tumor Cell Uptake and Intracellular Fluorescence Intensity Changes

[0075] (1) According to the instructions for Ce6 (dihydroporphyrin e6), the experimental dosage (100 μg / mL) was guided. Carboplatin, DSPE-PEG-CAI and Ce6 were weighed in a mass ratio of 4:30:1. Carboplatin, DSPE-PEG and Ce6 were prepared under light-protected conditions using the method in step (2) of Example 1. Ce6-loaded DSPE-PEG-CAI-CBP (CAI modified group, drug carrier mass ratio of 2:15) and Ce6-loaded DSPE-PEG-CBP (control group).

[0076] (2) MDA-MB-231 triple-negative breast cancer cells were cultured at 3 × 10⁻⁶ cells per dish. 5 The cells were seeded at a density of 1 cell / mL into confocal culture dishes and incubated overnight.

[0077] (3) Mix Ce6-loaded DSPE-PEG-CAI-CBP and DSPE-PEG-CBP with complete culture medium at a volume ratio of 3:7 and add them to different culture dishes. Divide MDA-MB-231 cells into two groups and incubate for 2 hours, 4 hours, 6 hours, 12 hours, 18 hours and 24 hours respectively.

[0078] (4) After incubation, remove the culture medium, wash the cells with PBS, add 1 mL of 4% paraformaldehyde to each dish of cells and fix at room temperature for 20-30 minutes, then remove excess formaldehyde, and wash each dish of cells with 1 mL of PBS 3 times, 5 minutes each time.

[0079] (5) Remove the washing solution, add 200 μL of DAPI staining solution, cover the sample, and incubate at room temperature for 20 minutes. Remove the DAPI staining solution, and wash each plate of cells three times with 1 mL of PBS for 5 minutes each time.

[0080] (6) Observe fluorescent cell images under a confocal laser scanning microscope.

[0081] 1.2 In vitro tumor cell cytotoxicity assessment

[0082] (1) Take MDA-MB-231 cells in the logarithmic growth phase, digest them with 0.25% trypsin, count them, dilute them with complete culture medium, and adjust the density to about 5×10⁻⁶ cells. 4 cells / mL.

[0083] (2) Seed the diluted cells into 96-well plates, 100 μL per well, and fill the edge wells with PBS to reduce evaporation interference. Pre-culture at 37°C and 5% CO2 for 24 hours to achieve a cell adhesion rate of 60% to 70%.

[0084] (3) Remove the old culture medium, divide DSPE-PEG-CAI-CBP and carboplatin into two groups and serially dilute them (the half-lethal concentration needs to be determined in the preliminary experiment). Add 100 μL of drug-containing culture medium to each well, add 100 μL of cell-free culture medium to the blank control well, and add 100 μL of complete culture medium to the negative control well. Set up 6 replicates for each group and incubate in an incubator for 72 hours.

[0085] (4) Remove the old culture medium and add 100 μL of the pre-prepared 10% CCK-8 reagent to each well, avoiding the formation of air bubbles. Gently shake the 96-well plate to mix well and incubate at 37°C in the dark for 1 to 4 hours (the optimal color development time needs to be determined by preliminary experiments).

[0086] (5) Use an ELISA reader to measure absorbance (OD) at a wavelength of 450 nm, with a reference wavelength of 600 nm for background subtraction. Wipe the bottom of the well plate before reading the data to ensure there is no liquid residue or fingerprint interference.

[0087] (6) Perform data calculations to determine cell viability, use GraphPad Prism to plot dose-response curves, and calculate IC50. 50 Value. The formula for calculating cell viability is:

[0088] Survival rate (%) = ×100% (Formula 3)

[0089] II. Experimental Results

[0090] 2.1 Assessment of Cellular Uptake and Fluorescence Intensity Changes

[0091] The results showed that in the CAI-modified group, the intensity of red fluorescence in the cytoplasm significantly increased with prolonged incubation time (4h→12h), and the fluorescence signal distribution gradually diffused from punctate aggregation to the entire cytoplasm. At 12 hours, a clear spatial separation was observed between the CAI-modified group and the cell nucleus (DAPI blue fluorescence), suggesting that the DSPE-PEG-CAI-CBP nanoparticles had completed internalization and may have escaped into the cytoplasm. In contrast, the fluorescence intensity of the control group was generally weaker, with a signal intensity of approximately 45% of that of the CAI-modified group at 4 hours, and the fluorescence was mostly distributed at the cell edge, indicating lower internalization efficiency (see...). Figure 5 (A in the middle).

[0092] The mean fluorescence intensity (MFI) of the cytoplasmic region was quantified using ImageJ software. The results showed that the MFI of the CAI-modified group increased non-linearly with incubation time, with MFI values ​​at 2, 4, 6, and 12 hours being 18.56 ± 2.34, 31.31 ± 0.95, 39.14 ± 0.43, and 31.63 ± 4.40, respectively. The MFI of the control group increased slowly, reaching only 23.91 ± 5.91 at 6 hours, approximately 61% of that of the CAI-modified group (****p < 0.0001), indicating that its uptake mainly relied on passive diffusion (see...). Figure 5 (B in the text) further confirms that CAI in the CAI-modified group can accelerate the internalization of DSPE-PEG-CAI-CBP nanoparticles into cells.

[0093] 2.2 CCK-8 Cytotoxicity Assay

[0094] Experimental results showed that both formulations exhibited significant concentration-dependent cytotoxic effects, but the nanoparticles were significantly more toxic than the free drug (see [link to study]). Figure 6 Specifically, 72 hours after the drug was added, the cell survival rate in the DSPE-PEG-CAI-CBP group decreased from 72.14% (carpa concentration of 105 μM) to 1.19% (carpa concentration of 400 μM), while the survival rate in the free carboplatin group only decreased from 70.88% to 18.00%, indicating that DSPE-PEG-CAI-CBP nanoparticles have a significant killing advantage at high doses.

[0095] Application Example 2: In vivo experiments

[0096] I. Experimental Methods

[0097] 1.1 Constructing an animal model

[0098] Fifteen female BALB / c nude mice (4-6 weeks old, weighing 18-22g) were selected and housed in SPF-grade cages (temperature 25 ± 2°C, humidity 50 ± 10%, 12-hour light-dark cycle). During the experiment, the mice were confined in polypropylene cages with free access to food and water. Wooden bedding was used to line the cages, and the bedding was changed frequently to allow the animals to acclimatize to the environment for one week. Next, logarithmic growth phase MDA-MB-231 cells were collected and resuspended in PBS to a concentration of 5 × 10⁻⁶. 7 cells / mL, 100 μL of cell suspension (approximately 5 × 10⁶ cells / mL) was subcutaneously injected under the right axilla of each nude mouse. 6 The tumor cells were observed daily, and the experiment began when the tumor volume reached 100-150 mm³ (approximately 7-10 days).

[0099] 1.2 Group Dosing

[0100] Fifteen nude mice were randomly divided into three groups (n = 5). Based on the feasibility of in vivo experiments in nude mice and the safety of repeated administration, the experimental groups received intraperitoneal injections of DSPE-PEG-CAI-CBP and carboplatin monotherapy, respectively, while the control group received intraperitoneal injections of PBS buffer. The groups were named nano-carboplatin group (nano-CBP), carboplatin monotherapy group (CBP), and PBS control group (PBS). Carboplatin was administered at a concentration of 1.5 mg / kg, with injections given at 3-day intervals (days 0, 3, 6, 9, 12, and 15), for a total of 6 injections. Tumor volume was measured on days 0, 6, 12, 18, 24, and 30 after drug injection, and the tumor volume was calculated based on the major and minor axes. The mean body weight of each group was measured every 3 days for 30 consecutive days.

[0101] 1.3 Evaluation of therapeutic effect

[0102] 1.3.1 Changes in tumor volume

[0103] After the start of drug administration, the average body weight of each group of animals was measured every 3 days, and the long diameter (L) and short diameter (W) of the tumor were measured with vernier calipers. The tumor volume (V) was calculated based on the long and short diameters, and then the tumor inhibition rate (TSR) was calculated. The calculation formulas are as follows:

[0104] V(mm3)= (Formula 4)

[0105] TSR (%)= ×100% (Formula 5).

[0106] After the in vivo experiments were completed, curves showing changes in tumor volume and body weight were plotted for each group.

[0107] 1.3.2 Analysis of the cell proliferation marker Ki-67

[0108] All nude mice were euthanized on day 30 (fasting was allowed for 24 hours prior to euthanasia, but water was permitted). Tumors were removed after euthanasia, fixed in 4% paraformaldehyde for 24 hours, and embedded in paraffin (4 μm thickness) for sectioning. A portion of the sections was stained with hematoxylin and eosin (HE), dewaxed to water, stained with hematoxylin for 5 minutes, differentiated with hydrochloric acid alcohol, stained with eosin for 2 minutes, dehydrated, cleared, and mounted. Histopathological changes (such as tumor necrosis and fibrosis, lymphocyte infiltration, etc.) were observed under a microscope. Another portion of the sections was used for Ki-67 immunohistochemistry. First, antigen retrieval was performed by immersing the sections in sodium citrate buffer (pH 6.0), autoclaving for 2 minutes, and blocking with 3% BSA at room temperature for 30 minutes. Primary antibody incubation was performed using rabbit anti-human Ki-67 monoclonal antibody (1:200) overnight at 4°C, followed by secondary antibody incubation with HRP-labeled goat anti-rabbit IgG (1:500) for 1 hour at room temperature. Finally, staining was performed using DAB, followed by hematoxylin counterstaining and mounting with neutral resin. Under a microscope, cell nuclei appearing as brownish-yellow granules were identified as Ki-67 positive. Five high-power fields (×200) were randomly selected, and the percentage of positive cells was calculated for quantitative analysis.

[0109] 1.4 Safety Evaluation

[0110] After euthanizing all nude mice on day 30, livers and kidneys from each group of nude mice were collected, fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (method as in 1.3.2). Histopathological changes were observed under a microscope.

[0111] II. Experimental Results

[0112] 2.1 Inhibitory effect of DSPE-PEG-CAI-CBP on tumor growth

[0113] After a 30-day treatment cycle, the inhibitory effect of DSPE-PEG-CAI-CBP nanoparticles on MDA-MB-231 xenografts was significantly better than that of free carboplatin. The tumor volume in the nanocarboplatin group was visibly smaller than that in the other two groups (see...). Figure 7 On day 30, the tumor volume in the nanocarboplatin group was 456.16 ± 125.62 mm³ (initial volume 112.70 ± 40.72 mm³), significantly lower than that in the free carboplatin group (782.28 ± 413.08 mm³, initial volume 116.31 ± 33.55 mm³) and the PBS control group (888.97 ± 216.55 mm³, initial volume 131.00 ± 35.35 mm³). Tumor growth curves showed that the tumor growth rate slowed after day 18 of treatment in the nanocarboplatin group. Although the growth rate in the free carboplatin group was lower than that in the PBS group, there was no statistically significant difference, and the tumors continued to grow (see...). Figure 8 ).

[0114] 2.2 Pathological section analysis of tumor tissue

[0115] In the nanocarboplatin group, tumor tissue showed extensive coagulative necrosis, with fibrosis and abundant lymphocyte infiltration surrounding the necrotic area; the free carboplatin group showed a smaller necrotic area and lower lymphocyte infiltration density compared to the nanocarboplatin group (see...). Figure 10 Immunohistochemical staining of Ki-67 cells and quantitative analysis of their proliferation activity showed that the proportion of Ki-67-positive cells in the nanocarboplatin group was 9.98%, a decrease of 40.20% compared to the PBS control group (16.69%) (**p < 0.01); the positive rate in the free carboplatin group was 14.26%, significantly higher than that in the nanoparticle group (*p < 0.05), indicating that the targeted nanosystem more effectively inhibits tumor cell proliferation (see...). Figure 9 ).

[0116] In summary, the nanocarboplatin DSPE-PEG-CAI-CBP prepared in this invention can cross the biological barrier established by the tumor microenvironment, deliver carboplatin to tumor cells more precisely and effectively, enhance the targeting selectivity for triple-negative breast cancer cells, and effectively inhibit the proliferation of triple-negative breast cancer cells.

[0117] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. 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 the present invention should be determined by the appended claims.

Claims

1. A method for preparing a carboplatin-loaded nanocarrier drug delivery system, characterized in that, Includes the following steps: (1) Preparation of nanocarrier DSPE-PEG-CAI AZ-COOH, EDCI, and HOBT were dissolved in DMSO and activated to obtain the first mixed solution; DSPE-PEG was then... 2000 -NH2 and TEA are dissolved in DMSO, and a second mixed solution is obtained after stirring. The first mixed solution was added dropwise to the second mixed solution, and after the reaction, it was dialyzed and dried to obtain the nanocarrier DSPE-PEG-CAI; (2) Preparation of nanocarboplatin DSPE-PEG-CAI-CBP The nanocarrier DSPE-PEG-CAI and carboplatin were dissolved in an organic solvent and sonicated to obtain an organic phase. The organic phase was then rapidly added to ultrapure water and sonicated continuously. The organic solvent was then evaporated or recovered to obtain an aqueous phase. The aqueous phase was dialyzed and filtered to obtain an aqueous solution of nanocarboplatin DSPE-PEG-CAI-CBP.

2. The preparation method according to claim 1, characterized in that, The mass ratio of AZ-COOH, EDCI, and HOBT is 31:27:

19.

3. The preparation method according to claim 2, characterized in that, The solid-liquid ratio of the first mixed solution is less than or equal to 77 mg: 1 mL.

4. The preparation method according to claim 1, characterized in that, The DSPE-PEG 2000 The mass ratio of -NH2 to TEA is 100:

11.

5. The preparation method according to claim 4, characterized in that, The solid-liquid ratio of the second mixed solution is less than or equal to 111 mg: 1 mL.

6. The preparation method according to claim 1, characterized in that, The activation temperature is 10-30℃ and the time is at least 2 hours; the stirring temperature is 10-30℃ and the time is at least 2 hours; the reaction time is at least 24 hours.

7. The preparation method according to claim 1, characterized in that, The mass ratio of DSPE-PEG-CAI to carboplatin is 2-15:

2.

8. The preparation method according to claim 1, characterized in that, The organic solvent is THF, the solid-liquid ratio in the organic phase is 4-17 mg:1 mL, and the solid-liquid ratio in the aqueous phase is 4-17 mg:5 mL.

9. The preparation method according to claim 1, characterized in that, The ultrasonic treatment power is 100-150W, and the ultrasonic time is 10-30min.

10. The preparation method according to claim 1, characterized in that, The recovered organic solvent is obtained by recovering organic reagents using a rotary evaporator. The rotary evaporator has a temperature of 40-60℃, a rotation speed of 100-200rpm, and a vacuum pressure of 0.65-0.8MPa.

11. The preparation method according to claim 1, characterized in that, The drying method is freeze drying or vacuum drying, the dialysis parameters are MWCO: 1000-3000 Da, and the filtration membrane is a 0.22 μm sterile membrane.

12. A carboplatin-loaded nanocarrier drug delivery system prepared by the preparation method according to any one of claims 1-11.

13. The application of the carboplatin-loaded nanocarrier drug delivery system as described in claim 12 in the preparation of a drug system for treating breast cancer.

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