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International Journal of Pharmaceutics: X logoLink to International Journal of Pharmaceutics: X
. 2024 Nov 14;8:100302. doi: 10.1016/j.ijpx.2024.100302

Trastuzumab-functionalized SK-BR-3 cell membrane-wrapped mesoporous silica nanoparticles loaded with pyrotinib for the targeted therapy of HER-2-positive breast cancer

Xing Liu 1, Wenwen Shen 1,⁎
PMCID: PMC11609682  PMID: 39624341

Abstract

In this study, the trastuzumab-functionalized SK-BR-3 cell membrane-wrapped mesoporous silica nanoparticles loaded with pyrotinib (Tra-CM-MSN-PYR) were prepared for targeted therapy of HER2-positive breast cancer. Transmission electron microscopy (TEM) characterization showed that MSN had a spherical morphology with mesoporous channels and that the structure of Tra-CM-MSN was a cell membrane (CM) layer successfully coated on the surface of MSN. A cellular uptake assay demonstrated that FITC-labeled Tra-CM-MSN were taken up by SK-BR-3 breast cancer cells, which illustrated that Tra-CM-MSN had good targeting ability compared with CM-MSN and MSN. In vivo imaging experiments demonstrated significant accumulation of FITC-labeled Tra-CM-MSN in tumor tissues, further proving that Tra-CM-MSN have superior targeting properties. Cell apoptosis experiments suggested that Tra-CM-MSN-PYR significantly inhibited the proliferation of SK-BR-3 breast cancer cells. The results of in vivo animal experiments also showed that Tra-CM-MSN-PYR significantly inhibited tumor growth. These results indicate that Tra-CM-MSN-PYR has potential application as a targeted therapy for HER2-positive breast cancer in the future.

Keywords: Pyrotinib, Mesoporous silica nanoparticles, SK-BR-3 cell membrane, Trastuzumab, HER2-positive breast cancer, Targeted therapy

Graphical abstract

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1. Introduction

Breast cancer is a common malignant tumor and one of the main causes of death affecting women's health worldwide (Harbeck et al., 2017). Therefore, effective treatment of breast cancer is essential to improve women's quality of life. HER2-positive breast cancer is a kind of highly malignant breast tumor with HER-2 overexpression on the surface of cancer cells (Brouwer et al., 2019). This type of tumor is highly invasive, has a poor prognosis and is more likely to metastasize to the viscus (Stanowicka-Grada et al., 2023; Pernas et al., 2022).

At present, chemotherapy is regarded as the main clinical treatment strategy for HER2-positive breast cancer in addition to surgery. Commonly used chemotherapy drugs include paclitaxel (Zhu et al., 2019), docetaxel (Ma et al., 2022), doxorubicin (Chen et al., 2018), etc. Compared with traditional chemotherapy drugs, pyrotinib (PYR) is a novel small molecule irreversible EGFR/HER2 double tyrosine kinase inhibitor that can covalently bind to adenosine triphosphate (ATP) binding sites in intracellular kinase regions of HER1, HER2 and HER4 (Xu et al., 2021). PYR can also prevent the formation of HER family homodimers/heterodimers, inhibit self-phosphorylation, and prevent the activation of downstream signaling pathways, thus inhibiting the growth of tumor cells (Xuhong et al., 2019; Wu et al., 2022). Therefore, pyrotinib has a significant advantage in the treatment of HER2-positive breast cancer and shows a strong antitumor effect in HER2-overexpressing xenograft tumor models. However, PYR can cause diarrhea, poorly absorbed, cleared too fast, and other problems, which seriously affect their therapeutic effect.

Therefore, it is necessary to develop a nanodrug targeted delivery system that can reduce the toxic side effects of drugs and improve their antitumor effects. In recent years, various nanodrug carriers, such as liposomes (Alavi et al., 2019; Fulton et al., 2023), polymer micelles (Ghosh et al., 2021; Panagi et al., 2022), manganese dioxide (Song et al., 2016), and mesoporous silicon dioxide (Tang et al., 2012a, Tang et al., 2012b; He et al., 2014), have been widely used in the delivery of chemotherapy drugs. Among them, mesoporous silica nanoparticles (MSN) have been proven to be highly promising candidate materials for drug delivery due to their advantages of easy synthesis, easy surface modification, fine drug loading performance and good biocompatibility. However, simple silica drug carriers lack targeting, cannot distinguish normal cells from tumor cells, and easily cause systemic toxicity. Therefore, to make mesoporous silica an ideal drug carrier, modification of its surface is needed to improve its aggregation ability at the tumor site (Lin et al., 2021; Kuthati et al., 2013). Cell membranes have provided a new idea for cancer treatment as novel biomimetic materials (Fang et al., 2023; Zou et al., 2019; Li et al., 2018). In existing research, the cell membrane has been used as the coating material, mainly including red cell membranes (Risinger et al., 2020; Han et al., 2019; Xia et al., 2019), platelet membranes (Hu et al., 2015; Jiang et al., 2020; Mei et al., 2020), bacterial cell membranes (Strahl et al., 2017; Grabowski et al., 2021), leukocyte membranes (Parodi et al., 2013), cancer cell membranes (Linh et al., 2022; Gao et al., 2020; Jin et al., 2019), etc. This kind of biomimetic nanoparticle facilitates a long cycle and has a slow-release effect. In addition, bionic nanomedicine carriers have good biocompatibility and good targeting because of the specific proteins on the surface of the cell membrane. Therefore, cell membrane-coated nanocarriers have good application potential.

In addition, to further improve the targeting efficacy, ligands can be modified on the cell membrane surface to achieve a dual targeting effect. Commonly used ligands include hyaluronic acid (Soliman et al., 2022; Chandra et al., 2023), monoclonal antibodies (Roccatello et al., 2020; Faghfuri et al., 2015; Kimiz-Gebologlu et al., 2018), and RGD polypeptides (Cheng et al., 2019); Fang et al., 2012). Trastuzumab (Swain et al., 2023; Perez et al., 2021; Cameron et al., 2017) is a monoclonal antibody against HER-2-positive breast cancer; it works by binding to human epidermal growth factor receptor 2 (HER2) on the surface of breast cancer cells and blocking the formation of HER-2 homodimers and has been widely used as a targeted agent against HER2-positive breast cancer cells.

Inspired by the above, in this study, trastuzumab-modified cancer cell membrane-coated mesoporous silica nanoparticles loaded with pyrotinib were used as drug carriers for the targeted therapy of HER2-positive breast cancer. We used the mesoporous structure of MSN as a reservoir to absorb a large amount of PYR and then coated the surface of MSN with a Tra-modified SK-BR-3 cell membrane (CM) (Zhao et al., 2021). Because of the dual targeting ability of CM and Tra, trastuzumab-functionalized SK-BR-3 cell membrane-coated mesoporous silica nanoparticles loaded with pyrotinib (Tra-CM-MSN-PYR) were specifically guided to target homologous breast cancer cells with HER2 overexpression. This study verified the targeting and antitumor ability of Tra-CM-MSN-PYR through in vitro cell experiments and in vivo animal experiments, providing a new method for the treatment of HER2-positive breast cancer.

2. Materials and methods

2.1. Materials

Pyrotinib (SHR1258, PYR) was ordered from Jiangsu Hengrui Pharmaceutical Co., Ltd. (Jiangsu, China) with >99 % purity. Trastuzumab was purchased from Roche Pharmaceuticals Co., Ltd. (Shanghai, China). Cetyl trimethyl ammonium bromide (CTAB) and tetraethyl orthosilicate (TEOS) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), ethanol, methanol and paraformaldehyde were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). SK-BR-3 breast cancer cell lines were obtained from KeyGen Biotech (Jiangsu, China). Thiazolyl blue tetrazolium bromide (MTT), propidium iodide (PI), an Annexin V-FITC apoptosis detection kit, trypsin, Triton X-100, bovine serum albumin, McCoy's 5 A medium and fetal bovine serum (FBS) were purchased from Beijing Dingguo Changsheng Biotech Co., Ltd. (Beijing, China). Phenylmethanesulfonyl fluoride (PMSF) and a mitochondrial membrane potential assay kit (JC-1) were provided by Beyotime Biotechnology Co., Ltd. (Shanghai, China). Cleaved caspase-3 was obtained from Abcam (Cambridge, UK).

2.2. Preparation of Tra-CM-MSN-PYR

2.2.1. Cell culture

SK-BR-3 breast cancer cells were cultured in 90 % McCoy's 5 A medium containing 1 % antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL) and 10 % FBS. SK-BR-3 breast cancer cells were preserved with 90 % culture medium (90 % McCoy's 5 A medium containing 10 % FBS) and 10 % DMSO at −80 °C.

2.2.2. Extraction of CM

First, SK-BR-3 breast cancer cells were cultured in a cell culture flask. Then, SK-BR-3 cells were digested in trypsin and collected into centrifuge tubes after centrifugation. After treatment with 0.2 % PBS hypotonic solution for 24 h, the cell suspension was centrifuged for 10 min at 1250 rpm, and the supernatant was discarded. NaHCO3 (1 mM), EDTA (0.2 mM) and PMSF (100 mM) were added to the centrifuge tube. The cell suspension was transferred to a homogenizer and homogenized 30 times in an ice bath. Finally, the SK-BR-3 breast cancer cell membranes (CM) were obtained by gradient centrifugation at 4 °C and stored at 20 °C.

2.2.3. Preparation of Tra-CM

NHS and EDC were dissolved (5 mg/mL) and added to the CM suspension to activate carboxyl groups on the proteins embedded in the CM (Rezki et al., 2021). Next, trastuzumab was dissolved in PBS (1 mg/mL) and incubated with CM for 6 h at 4 °C (Tra-CM). The Tra-CM sample suspension was centrifuged at 12000 ×g for 30 min. The supernatant was collected to analyze the amount of bound protein using a BCA kit (Noh et al., 2016). The grafted amount of Tra was calculated using the following equation.

Pac=Pi−Pnc

Pac is the amount of connected Tra (mg), Pi is the initial amount of Tra (mg), and Pnc is the amount of nonconnected Tra (mg) in the supernatant.

Bradford protein concentration assay kit were used to further analyze the amount of binding proteins. The Bradford method is based on the association of Coommassie Brilliant Blue G-250 with the basic and aromatic amino acids of proteins, especially arginine. After binding (Arginine) in an acidic medium, the solution turns blue, the maximum absorption peak of the solution migrates from 465 nm to 595 nm, and the color change is proportional to the protein concentration. Therefore, the concentration of protein in solution can be determined by detecting the absorbance at 595 nm. Compared with other methods such as the BCA method, the Bradford method is not affected by the chemical substances in most samples, especially, the effect of the reducing agent.

2.2.4. Synthesis of MSN

Mesoporous silica nanoparticles (MSN) were prepared. CTAB (450 mg) was completely dissolved in 346 mL (13 %) of ethanol water solution. Ammonia water was added to the above solution to adjust the pH to 11.5. The mixture was heated to 75 °C, followed by the dropwise addition of 2.5 mL TEOS. The sample was stirred for 2 h at 75 °C and allowed to stand at room temperature for 24 h. The resulting mixture was centrifuged at 12000 rpm for 15 min and washed three times with anhydrous ethanol. Finally, the dried products were calcined in air at 550 °C for 5 h (Jiang et al., 2021).

2.2.5. Synthesis of Tra-CM-MSN-PYR

PYR was loaded into the nanoscale mesoporous structure of MSN (MSN-PYR) by the adsorption method. In brief, PYR (20 mg) was dissolved in 5 mL distilled water, and 100 mg MSN was added to the PYR solution and allowed to stir for 6 h and stand for 30 min. The MSN-PYR was separated by centrifugation, washed with water and freeze dried. After that, Tra-CM (5 mg) was dissolved in 1 mL water, to which 100 mg MSN-PYR was added and stirred for 5 h. The mixtures were incubated for 30 min and completely adsorbed before being centrifuged for precipitation and freeze-dried to obtain Tra-CM-MSN-PYR.

2.3. Determination of drug loading

Tra-CM–MSN-PYR (5 mg) was dispersed in 10 mL of deionized water (pH = 7.4) and ultrasonicated for 30 min. The Tra-CM-MSN-PYR suspension was incubated for 1 h and then centrifuged. The supernatant was collected. The collected PYR solution was measured by UV (UV-2000, Unico, USA) at a wavelength of 260 nm. The drug load according to the following formula:

LCloading contnet=weight of loading drugtotal weight of manocomposites×100%

2.4. Characterization

2.4.1. Morphology and characterization of nanomaterials

The morphological structures of MSN and Tra-CM-MSN were observed using transmission electron microscopy (TEM) (JEM-1200EX; JEOL, Tokyo, Japan). The size and zeta potentials of MSN, CM-MSN and Tra-CM-MSN-PYR were obtained using a laser dispersion particle size analyzer (Nano-ZS90, Malvern, UK). XRD for both low and wide angles of the synthesized MSN was carried out by an X-ray refractometer with Cu—K radiation (Rigaku Geigerfex XRD, company, Japan, 30 kV and 30 mA Philips). The structure of MSN were determined and analyzed by BET test with a specific surface area and pore characteristics. (Beckman Coulter, USA).

2.4.2. Stability of MSN and Tra-CM-MSN

The targeting effects of Tra-CM-MSN-PYR were achieved by CM surface proteins. To characterize whether the encapsulation was successful, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used. Membrane proteins were extracted from CM, CM-MSN, and Tra-CM-MSN with RIPA lysis buffer and further measured with BCA analysis. Then, the protein samples were prepared. Then, 10 % separated rubber and 5 % concentrated rubber were selected and made. The protein samples and marker were added to the sample well for electrophoresis at 80–100 V. After staining with Coomassie Brilliant Blue, image development was performed under a microscope and analyzed by Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA).

2.4.3. Stability of MSN and Tra-CM-MSN

To verify the stability of MSN and Tra-CM-MSN, PBS solutions of MSN and Tra-CM-MSN were placed in a refrigerator at 4 °C for 7 days. The particle size and zeta potential of the PBS solution were measured every day, and the in vitro stability was determined by observing the changes in particle size and zeta potential.

2.4.4. Safety study

A hemolysis test was performed to verify the safety of MSN and Tra-CM-MSN in vitro. The whole blood of rats was taken and centrifuged at 1500 rpm/min for 15 min before being washed with PBS 3 times. The red blood cells obtained in the previous step were diluted to 2 % with normal saline. The MSN and Tra-CM-MSN were mixed with 2 % red blood cells and incubated for 2 h at 37 °C. The mixture was centrifuged at 1500 rpm/min for 15 min. Normal saline and deionized water were used as negative and positive controls, respectively. The absorbance was measured at 560 nm using an enzyme marker. The formula for the hemolysis rate was as follows:

Hemolysis rate=absorbance of Sample−absorbance of negative controlabsorbance of positive control−absorbance of negative control×100%

2.5. In vitro drug release study

In vitro drug release studies were conducted in a shaker (SHZ-82, Jintan Science Analysis Instrument Co., Ltd., Jiangsu, China) at 37 °C. The drug release medium was phosphate buffer solution (PBS, pH = 7.4). First, the sample was added to 20 mL of dissolved medium, the release medium (1 mL) was collected at predetermined time intervals, and the same volume of fresh buffer was added. Then, after the release medium was filtered through a microporous filter membrane, the PYR concentration in the filtrate was measured by UV (UV-2000, Unico, USA) at 260 nm. All experiments were repeated in triplicate using the above methods to determine drug release.

2.6. In vitro cell assay

2.6.1. Cell uptake assay

Fluorescence microscopy (Leica, Wetzlar, Germany) was used to observe cell uptake. SK-BR-3 and MCF-7 breast cancer cells were cultured in 6-well plates. Then, when the number of SK-BR-3 cells reached 80 %, the cells were incubated with FITC-labeled MSN, CM-MSN and Tra-CM-MSN for 3 h at 37 °C. Subsequently, the cells were fixed with 4 % paraformaldehyde solution for 10 min at room temperature and permeabilized with 0.1 % Triton for 10 min at 4 °C. Then, cells were blocked with 1 % bovine serum albumin (BSA) for 30 min at 37 °C. Next, the nuclei were stained with Hoechst33342 for 30 min, and the cytoskeletons were stained with phalloidin for 20 min at 37 °C. Finally, the cells were observed under a fluorescence microscope.

Furthermore, when the number of SK-BR-3 and MCF-7 cells reached 80 %, the cells were incubated with FITC-labeled CM-MSN for 3 h at 37 °C. Cells were treated in the same way as above. Finally, the cells were observed under a fluorescence microscope.

2.6.2. Cytotoxicity analysis

To evaluate the cytotoxicity of PYR on SK-BR-3 breast cancer cells, an MTT assay was conducted. SK-BR-3 cells were counted and seeded into a 96-well plate at 5000 cells per well for 48 h. Then, PYR, MSN-PYR and Tra-CM-MSN-PYR at different drug concentrations (0.2 μg/mL, 0.4 μg/mL, 0.8 μg/mL, 1 μg/mL, 4 μg/mL, 8 μg/mL) were added to a 96-well plate and incubated for 48 h. In addition, to evaluate the cytotoxicity of MSN on SK-BR-3 cells, suspensions at different concentrations (500, 250, 120, 60, 30, 15 μg/mL) were added to the 96-well plate and cultured for 48 h. MTT solution (5 mg/mL) was added and incubated for 4 h under dark conditions. DMSO (150 μL) was added to 96 wells and placed in a shaker and shaken for 10 min under dark conditions. The absorbance value (OD) of formazan was measured at 492 nm with a microplate reader (VERSAmax, Molecular Devices, Sunnyvale, CA, USA). Cell viability was calculated using the following formula:

Cell viability=ODtODc×100%

ODt represents the absorbance of cells in the drug preparation treatment group, and ODc represents the absorbance of cells in the control group.

2.6.3. Flow cytometric detection of apoptosis

SK-BR-3 breast cancer cells were seeded in a 6-well plate and incubated for 24 h at 37 °C in a 5 % CO2 incubator. After dividing the cells into 4 groups (Control, PYR, MSN-PYR and Tra-CM-MSN-PYR groups) and treating them with PYR, MSN-PYR and Tra-CM-MSN-PYR (equivalent to 0.8 μg/mL PYR) for 48 h, the cells were digested with trypsin. Then, the collected cells were placed in another centrifuge tube, and 500 μL of binding buffer was added for resuspension. Annexin V-FITC and PI (1:1) were added and mixed gently in dark conditions. Finally, the cell samples were tested with a flow cytometer (Agilent Biosciences Inc., USA).

2.6.4. Measurement of mitochondrial membrane potential (MMP)

SK-BR-3 breast cancer cells were cultured in 12-well plates overnight. Cells were treated with PYR, MSN-PYR and Tra-CM-MSN-PYR (equivalent to 0.8 μg/mL PYR) for 24 h. JC-1 staining working solution was prepared at a ratio of JC-1 (200×):ultrapure water:JC-1 staining buffer (5×) = 1:160:40. Then, 500 μL of JC-1 staining working solution was added to each well and incubated at 37 °C for 30 min. During the incubation period, an appropriate amount of JC-1 staining buffer (1×) was prepared at a ratio of JC-1 staining buffer (5×): distilled water = 1: 4 and placed in an ice bath. After incubation at 37 °C, the working solution was aspirated, the cells were washed twice with JC-1 staining buffer (1×), and 2 mL of cell culture medium was added. Immediately, the cells were observed under a fluorescence microscope (Leica, Wetzlar, Germany).

2.6.5. Western blot assay on the expression of apoptosis protein

The Western blot assay were implementated to analyze the expression of Bcl-2, Bax and cleaved-caspase-3. The SK-BR-3 breast cancer cells were cultured in a 15 mL cell culture flask and treated with different preparations (equivalent to 10 μg/mL PYR) for 48 h. The adherent cells were scraped off on ice, which were treated with 300 μL of lysis buffer. After 0.5 h, the cell suspension centrifugated at 12,000 rpm for 30 min at 4 °C. The total protein concentration of the collected supernatant was determined by BCA analysis and the protein samples were prepared. Then, the polyamide gel electrophoresis was performed, included seven steps. The first step was to select 12 % separated rubber and 5 % concentrated rubber to make rubber. The second step was to polyacrylamide gel electrophoresis, which the protein samples were added to the sample well for electrophoresis at 90 V. The third step was to cut the appropriate size of 0.22 μM PVDF film, soak in methanol for activation and mold transfer foor for 60 min with a current of 300 mA. The fourth step was to use BSA to seal at room temperature for 2 h. The fifth step was to add the working concentration of first antibody and shake it overnight at 4 °C. The sixth step was to incubate the second antibody at room temperature for 2 h. The final step was analyzed by Quantity One Dimensional analysis software (Bio-Rad, Hercules, USA) after soaking PVDF films in ECL Plus ultra-sensitive luminescent solution.

2.6.6. Immunofluorescence assay on the expression of apoptosis protein

SK-BR-3 breast cancer cells were cultured in 24-well plates for 24 h and then treated with different pharmaceutical products (equivalent to 0.8 μg/mL PYR) for 24 h. The cells were fixed with 4 % paraformaldehyde solution for 10 min, permeabilized with Triton X-100 for 20 min, and blocked with BSA for 1 h to 2 h in turn. Then, cleaved caspase 3 was added to 24-well plates overnight at 4 °C. The cells were incubated for 2 h with the secondary antibody, and the nuclei were stained with Hoechst 33342. Fluorescence microscopy was used to observe cell immunofluorescence.

2.7. In vivo animal experiment

2.7.1. Establishment of a mouse tumor model

BALB/c nude mice (females, 6–8 weeks, 18–20 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). All BALB/c nude mice were fed in an SPF-free environment. The experiment was carried out under the Animal Management Regulations of Jinzhou Medical University (2022). SK-BR-3 breast cancer cells (5 × 106) were injected into the vicinity of the right front leg of nude mice. Two weeks later, there were noticeable tumor masses, which indicated that the tumor model was successfully established.

2.7.2. In vivo targeting assay

FITC-labeled MSN, CM-MSN and Tra-CM-MSN were injected into tumor-bearing mice via the tail vein. After 3 h, the mice were sacrificed, and the main organs (heart, liver, spleen, lung, kidney) and tumors were collected. All tissues were observed by an in vivo imaging system (IVIS Spectrum, PerkinElmer, Waltham, MA) at excitation wavelengths of 518 nm and 494 nm.

2.7.3. In vivo antitumor effect

When the tumor volume reached 300 mm3, nude mice were randomly divided into four groups (n = 3). Normal saline, PYR, MSN-PYR, or Tra-CM-MSN-PYR (equivalent to 10 mg/kg PYR) was injected into nude mice via the tail vein once every 3 days for a total of 7 times. Before each dose, we measured the body weight and the longest and shortest diameters of the tumor. The volume of the tumors was obtained according to the following formula:

Volume of tumor=lonest diamerter×shortest diameter22×100%

The tumor inhibition rate was obtained according to the following formula:

Tumor inhibition rate=1−WtWc×100%

Wc is the weight of the tumor in the saline group, and Wt is the average weight of the tumor in each drug treatment group.

The mice were sacrificed after the last dose, and then, the tumor tissue and major organs (hearts, livers, spleens, lungs, kidneys) were removed. After that, the tumors were fixed with paraformaldehyde and embedded in paraffin. The tumors samples were sliced and were stained using ki67 for immunohistochemistry study to assess necrosis of tumor cells. Hematoxylin and eosin (H&E) were used to stain the tumor tissue and major organs (hearts, livers, spleens, lungs, kidneys) to assess antitumor effects and biosafety in vivo. The sections were observed with a fluorescence microscope (Leica DMI 4000B, Germany).

2.7.4. In vivo pharmacokinetic study

Female SD rats were bought (180–220 g) from the Department of Laboratory Animal Science of Jinzhou Medical University. Twelve rats were randomly divided into 3 groups, fasted for 12 h, and injected PYR, MSN-PYR and Tra-CM-MSN-PYR (equivalent to PYR 5 mg/kg) through the tail vein. After administration, 1.5 mL of blood was collected from the fundus venous plexus at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24 h, and whole blood was placed in heparin sodium tube, centrifuged at 5000 r/min for 5 min, plasma was separated, and imatinib mesylate was used as the internal standard. The blood concentration was determined by LC/MS method.

2.8. Statistical analysis

The experimental results were analyzed using GraphPad Prism (version 8.0) and reported as the mean ± SD. In addition, all data were analyzed to determine the difference in means between groups. When p < 0.05, it was considered to be statistically significant.

3. Results and discussion

3.1. Characterization of Tra-C-HMSN-PYR

A schematic diagram of the Tra-CM-MSN-PYR preparation process and its antitumor effect is shown in Scheme 1. First, MSN were prepared, and PYR was loaded onto the MSN by the adsorption method. As a new type of mesoporous structure drug carrier, MSN are often used for preparing new nano-doses as an ingredient and have high drug loading capacity and good biocompatibility (Jiang et al., 2021). CM was extracted from SK-BR-3 breast cancer cells, and Tra was connected to the CM surface by amide bonds to obtain Tra-CM. After Tra-CM was coated on the MSN, Tra-CM-MSN as PYR carriers positively targeted the surface of SK-BR-3 breast cancer cells with high HER2 expression, thereby exerting a good therapeutic effect. CM has a phospholipid bilayer structure and unique protein components, which is beneficial for improving the stability of drug molecules and homotype aggregation targeting capabilities. In light of these advantages, the targeting ability, systemic circulation time and stability of CM-MSN can be enhanced. SK-BR-3 breast cancer cells have specific proteins. This kind of protein can specifically recognize the corresponding tumor cells in vivo, allowing the MSN coated in SK-BR-3 CMs to be more targeted to homotype tumor cells and tissues (Linh et al., 2022; Gao et al., 2020; Jin et al., 2019). It is well known that EGFR/HER-2 are highly expressed in SK-BR-3 cells. The trastuzumab-functionalized SK-BR-3 cancer cell membrane-coated MSN (Tra-CM-MSN) should further improve the active targeting of cancer cell membrane-coated nanoparticles and improve the antitumor effect of PYR (Swain et al., 2023; Perez et al., 2021; Cameron et al., 2017). Then, we characterized the resulting Tra-CM-MSN-PYR. In Fig. 1A, the structures of MSN and Tra-CM-MSN were observed by TEM. The TEM images showed that MSNs had a spherical structure with mesoporous pores and a diameter of 70 nm. The structure of Tra-CM-MSN was a cell membrane (CM) layer successfully coated on the surface of MSN. The DLS results in Fig. 1B indicated that the particle size of the MSNs was 75.5 ± 4.4 nm (PDI: 0.167). After CM modification, the size of the CM-MSN was 90.83 ± 7.7 nm (PDI: 0.237), which was 15 nm larger than that of MSN. This result illustrated that the SK-BR-3 cell membrane was successfully encapsulated on the MSN. The Tra-CM-MSN particle size was 102.1.3 ± 6.5 nm (PDI: 0.211) after modification of the CM-MSN surface with Tra, indicating that Tra was successfully grafted onto CM. In Fig. 1C, the zeta potential of the MSN was −17.13 ± 2.25 mV. In comparison, after MSN were coated with CM, the potential of CM-MSN became −20.43 ± 2.72 mV. After CM-MSN were modified with Tra, the zeta potential of the Tra-CM-MSN was −24.07 ± 1.67 mV. The changes in Tra-CM-MSN particle size and zeta potential before and after Tra-CM modification demonstrated the successful preparation of Tra-CM-MSN. The SDS–PAGE experiment in Fig. 1D shows that CM-MSN and Tra-CM-MSN had electrophoretic bands consistent with those of SK-BR-3 breast cancer cell vesicles, which further demonstrated the successful preparation of Tra-CM-MSN. The stability analysis of MSN and Tra-CM-MSN was conducted by particle size and zeta potential for 7 d, as shown in Fig. 1E and Fig.1F. The particle size and zeta potential of MSN and Tra-CM-MSN did not vary noticeably, which confirmed that the nanosystem was stable. Hemolysis experiments were used to study the biosafety of MSN and Tra-CM-MSN. As shown in Fig. 1G and Fig. 1H, 350 μg/mL MSN resulted in slight hemolysis, while Tra-CM-MSN did not produce hemolysis at the same concentration. This result showed that Tra-CM-MSN obviously had good biological safety. XRD were used to analyze the presence of MSN, which MSN had no evident characteristic diffraction peaks in Fig. 1I. The BET results in Fig. 1J show that the surface areas of MSN were 675 g/m2. The test evaluated the number of Tra attachments by Bradford protein detection, and its standard diagram was shown in Fig. 1K. BCA kit standard graph is shown in corresponding supplements in Fig. S1.The number of Tra attachments were 30 % ∼ 40 %.The drug loading in Tra-CM-MSN-PYR were 19.95 ± 3.21 %. The MSN and Tra-CM-MSN in vitro release of the drug was analyzed as shown in Fig. 1L. The PYR release in MSN-PYR was more than 70 % within 6 h. The results showed that MSN promoted the rapid release of PYR. After Tra-CM were coated on the MSN, PYR was released slowly from Tra-CM-MSN-PYR, and the release in MSN-PYR was more than 50 % within 48 h. The results of drug release in vitro showed that the stability of Tra-CM-modified MSN was improved. The absorption rate and bioavailability of Tra-CM-MSN-PYR were further accelerated. This means that Tra-CM-MSN-PYR prolongs the systemic circulation time of drugs, reduces drug loss, and is conducive to improving drug absorption. To further validate this idea, the pharmacokinetics of Tra-CM-MSN-PYR rats were evaluated in Fig. 1M. The plasma half-lives (t1/2) of Tra-CM-MSN-PYR, MSN-PYR, and PYR were 5.09 ± 0.55 h, 3.07 ± 0.75 h, and 2.08 ± 0.37 h, respectively. It is obvious that the results of pharmacokinetic experiments supported this view, which effectively avoided being captured by the reticuloendothelial system. The AUCs (0-∞) of Tra-CM-MSN-PYR, MSN-PYR, and PYR were 32.94 ± 2.870, 13.44 ± 2.14, and 6.46 ± 1.28, respectively. This results proved that Tra-CM-MSN-PYR effectively enhanced the bioavailability of PYR.

Scheme 1.

Scheme 1

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Schematic diagram of the Tra-CM-MSN-PYR preparation process and its antitumor effect

Fig. 1.

Fig. 1

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(A) TEM images of MSN and Tra-CM-MSN. Scale bars: 100 nm. (B) The particle size distribution of MSN, CM-MSN and Tra-CM-MSN by DLS. (C) Zeta potential of MSN, CM-MSN and Tra-CM-MSN. (D) SDS-PAGE analysis of (a) SK-BR-3 cell membrane, (b) CM-MSN and (c)Tra-CM-MSN. (E) 7 d stability test of MSN and (F) Tra-CM-MSN. (G, H) Hemolysis experiments of MSN and Tra-CM-MSN. (I) The XRD for both low and wide angles of the synthesized MSN.(J) The textural properties (BET test) of the synthesized MSN. (K) The standard graph of Bradford protein assay. (L) The drug release curves in PBS (pH = 7.4) containing 0.05 % SDS (PYR). (M) The drug concentration time curve in vivo. All data represent the mean ± SD (n = 3).

3.2. Targeting study

The targeting of Tra-CM-MSN was analyzed by an in vitro cell uptake experiment and an in vivo imaging experiment. First, SK-BR-3 cellular uptake images were observed by an inverted fluorescence microscope (Fig. 2A and Fig. 2B). The nucleus was stained blue with Hoechst 33342, and the cytoskeleton was stained red with rhodamine phalloidin, while FITC-labeled MSN, CM-MSN and Tra-CM-MSN produced green fluorescence. The cellular uptake of Tra-CM-MSN was significantly higher than that of MSN and CM-MSN. This result indicated that modification of the MSN surface with Tra-CM improved the targeting ability of MSN. The targeting specific of CM-MSN was analyzed by an in vitro cell uptake experiment As a control, the MCF-7 HER-2 negative breast cancer cells did not exhibit specific uptake of CM-MSN Fig. 2C and Fig. 2D. This indicated that after MSN was coated with the SK-BR-3 cell membrane, the targeting ability was improved due to the homologous binding ability of the cell membrane with the same type. Furthermore, Tra introduced as a targeted ligand further enhanced the active targeting of MSN.

Fig. 2.

Fig. 2

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(A) Cellular uptake images of FITC-labeled MSN, CM-MSN and Tra-CM-MSN. Scale bars: 200 μm. (B) The fluorescence intensity of CLSM images in A. (C) Cellular uptake images of FITC-labeled MSN, CM-MSN and Tra-CM-MSN. Scale bars: 200 μm. (D) The fluorescence intensity of CLSM images in C. (E) Tissue fluorescence imaging of FITC-labeled MSN, CM-MSN and Tra-CM-MSN. (F) The fluorescence intensity of tissue in C. All data represent the mean ± SD (n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Then, to further prove the targeting of Tra-CM-MSN, tissue fluorescence imaging assays were performed by an in vivo imaging system. After FITC-labeled MSN, CM-MSN and Tra-CM-MSN were injected into tumor-bearing nude mice for 3 h, the fluorescence signals of the main organs are shown in Fig. 2E and Fig. 2F. The fluorescence intensities of the tumors in the FITC-labeled Tra-CM-MSN group were significantly higher than those of the FITC-labeled MSN and CM-MSN group. Moreover, the ratio of fluorescence intensity between the tumor and liver also explained the good tumor targeting of Tra-CM-MSN at the animal level. This result confirmed that after MSN were modified by Tra-CM, the targeting ability of MSNs was improved due to the binding capacity of Tra with EGFR/HER2 and the homotypic aggregation ability of SK-BE-3 breast cancer cells. Therefore, Tra-CM-MSN, as a targeted delivery system, has excellent application in targeted therapy of HER2-overexpressing breast cancer.

3.3. In vitro apoptosis analysis

Prior to drug-loaded formulation, the cytotoxic effects of MSN obtained by MTT assay are shown in Fig. 3A. After a 48 h incubation with MSN, cell viability remained above 90 %, which showed that MSN have excellent cell biocompatibility. The cell viability of PYR, MSN-PYR and Tra-CM-MSN-PYR was analyzed using the MTT assay. As shown in Fig. 3B, compared with the PYR and MSN-PYR groups, the Tra-CM-MSN-PYR group showed the most potent anticancer effect at all tested concentrations. The IC50 values of PYR, MSN-PYR and Tra-CM-MSN-PYR were 5.03 ± 0.16 μg/mL, 2.07 ± 0.02 μg/mL and 0.42 ± 0.06 μg/mL, respectively. The results showed that the encrustation of Tra-CM by MSN enhanced the accumulation of the drug in tumor cells. An Annexin V-FITC binding assay was carried out using Annexin V-FITC and propidium iodide (PI) to confirm cell apoptosis. As shown in Fig. 3C and Fig. 3D, Tra-MSN-PYR showed significantly higher apoptosis than pure PYR and MSN-PYR. The percentage of apoptotic cells treated with Tra-CM-MSN-PYR was 39.77 ± 13.10 %. In contrast, the corresponding values for pure PYR and MSN-PYR were 19.9 ± 10.69 % and 27.90 ± 10.91 %, respectively. The above results suggested that Tra-CM-MSN as carriers could improve the function of promoting cell apoptosis. JC-1 is an ideal fluorescent probe widely used to detect MMP, which is closely related to cell apoptosis. When MMP is high, JC-1 gathers in the matrix of the mitochondria to form a polymer that can show red fluorescence. When MMP is low, JC-1 cannot form a polymer in the matrix of mitochondria and shows green fluorescence. The decrease in mitochondrial membrane potential is a landmark event in the early stage of apoptosis. MMP can be easily detected by the transformation of JC-1 from red to green fluorescence. At the same time, the ratio change of JC-1 from red fluorescence to green fluorescence can be used as an indicator of early apoptosis. The decrease in the ratio of red fluorescence to green fluorescence represents cell apoptosis. According to Fig. 3E and Fig. 3F, the red/green fluorescence ratio of the Tra-CM-MSN-PYR group decreased significantly compared with that of the PYR and MSN-PYR groups. The JC-1 assay results further indicated that Tra-CM-MSN-PYR could significantly promote the apoptosis of SK-BR-3 breast cancer cells, which was also consistent with the flow cytometry results. All these results suggested that Tra modification of CM-coated MSN can significantly enhance the apoptosis-promoting effect of PYR in vitro.

Fig. 3.

Fig. 3

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(A) The cell viability of SK-BR-3 breast cancer cells after incubation with MSNs. (B) The cell viability of SK-BR-3 breast cancer cells after incubation with PYR, MSN-PYR and Tra-CM-MSN-PYR. (C, D) Flow cytometry analysis of the apoptosis ratio and statistical analysis. (E) Fluorescence image of SK-BR-3 breast cancer cells stained with JC-1 after different treatments. Scale bars: 100 μm. (F) The ratio changes of red fluorescence intensity to green fluorescence intensity in E represented the dissipation of MMP. All data represent the mean ± SD (n = 3) (p < 0.05; p < 0.01; p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Immunofluorescence analyses of apoptotic protein expression (cleaved caspase 3) were performed to more fully verify the proapoptotic effect. The immunofluorescence images and the intensity quantitative analysis of cleaved caspase 3 in SK-BR-3 breast cancer cells with different treatments are presented in Fig. 4 A and Fig. 4B. The green fluorescence intensity of cleaved caspase 3 in the MSN-PYR group was enhanced compared with that in the PYR group. This result proved that MSN-PYR reduced the toxic side effects and promoted the apoptosis of SK-BR-3 breast cancer cells. In addition, the green fluorescence intensity of cleaved caspase-3 in the Tra-CM-MSN-PYR group was significantly enhanced compared with that in the MSN-PYR groups, proving that Tra-CM-MSN-PYR greatly promoted apoptosis of SK-BR-3 breast cancer cells more than MSN-PYR.

Fig. 4.

Fig. 4

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(A, B) Cleaved caspase 3 immunofluorescence images and intensity quantitative analysis of SK-BR-3 breast cancer cells after different treatments. Cleaved-caspase3 apoptotic protein was stained green fluorescence. Cell nuclei were stained blue fluorescence with Hoechst-33,342. Scale bars: 50 μm. (C) Western blotting was used to detect the expression of cytokines (Bcl-2, Bax, Cleaved-Caspase 3) in different groups. (D-G) Quantitative analysis of the expression levels of Bcl-2, Bax, Cleaved-Caspase-3, and Bax/Bcl-2. All data represent the mean ± SD (n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Western blot was used to evaluate the expression of Bcl-2, Bax, Cleaved-Caspase 3 proteins in Fig. 4C. Quantitative analysis of the expression levels of Bcl-2, Bax, Cleaved-Caspase-3, and Bax/Bcl-2 in Fig. 4 (D-G), showed that the expression of the apoptotic proteins Bax and cleaved caspase-3 in the Tra-CM-MSN-PYR group was the most striking, and the expression of Bcl-2 was the weakest in all groups. Bax has a proapoptotic effect, and Bcl-2 can inhibit cancer cell apoptosis. Cleaved caspase-3 can destroy cell function and promote cell apoptosis. The combination of Bax/Bcl-2 can form an apoptotic dimer. When Bax/Bcl-2 increases, it will promote the release of apoptosis factors and cascade with Caspase protein to induce apoptosis. In all groups, the Bax/Bcl-2 ratio of the Tra-CM-MSN-PYR group was still the largest, indicating that Tra-CM-MSN-PYR could significantly promoted the apoptosis of SK-BR-3 breast cancer cells.

3.4. In vivo antitumor effect and safety

The in vivo antitumor capability of Tra-CM-MSN-PYR was shown in Fig. 5A. The Raw PYR group and MSN-PYR group suppressed tumor growth to a lower level. In contrast, the antitumor efficacy in the Tra-CM-MSN-PYR group was much more significant than that in the other groups. The final tumor volumes in the Tra-CM-MSN-PYR group were approximately 95.20 ± 33.31 mm3. This result demonstrated that Tra-CM-MSN-PYR could significantly enhance the effect of PYR treatment. After 21 days of administration, the tumor tissue was removed, and photographs of tumors in different treatment groups were shown in Fig. 5C. The tumor volume of the Tra-CM-MSN-PYR group was significantly smaller than that of the other groups. This result was consistent with 5A, indicating that Tra-CM-MSN-PYR could significantly inhibit tumor growth.

Fig. 5.

Fig. 5

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(A) Tumor volume, (B) body weight and (C) tumor images of different treatment groups after 21 days of treatment in different groups. (D) Tumor H&E staining in different treatment groups. Scale bars: 100 μm. (E) Tumor Ki67 immunohistochemical imaging in different treatment groups. Scale bars:100 μm. (F) H&E staining of the main organs (heart, liver, spleen, lung and kidney) in different treatment groups. Scale bars: 100 μm. All data represent the mean ± SD (n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Tumor weights were determined, and the tumor inhibition rates of PYR, MSN-PYR and Tra-CM-MSN-PYR were 21.86.1 ± 6.40 %, 52.57 ± 10.50 %, and 62.50 ± 6.30 %, respectively. These results further demonstrated that Tra-CM-MSN-PYR could significantly inhibit tumor growth. H&E staining of tumor tissues is displayed in Fig. 5D. As predicted, in line with the above results, tumor tissue of mice treated with Tra-CM-MSN-PYR showed the most 785-significant apoptosis and the best antitumor outcome compared with the other treatment groups. In Fig. 5E Antigen Ki67 immunohistochemical staining showed that the brown nuclei of proliferative cells in the Tra-CM-MSN-PYR group were significantly less abundant than those of the other groups. This means that Tra-CM-MSN-PYR can significantly improve the anti-tumor activity of PYR in vivo. Antigen Ki67 immunohistochemical staining results were consistent with those of HE staining.The changes in mouse body weight are presented in Fig. 5B. The body weight of the PYR group increased during the first nine days and then gradually decreased with time, mainly due to the toxicity of PYR. In comparison, the body weights of the MSN-PYR group and Tra-CM-MSN-PYR group continued to increase, and the body weight gain of the Tra-CM-MSN-PYR group increased at the most significant level during 21 days of administration. According to relevant reports, PYR could stimulate the digestive tract and cause weight loss. These results suggested that Tra-CM-MSN-PYR could reduce the toxic side effects of the drug and increase the weight of the mice while playing an antitumor role. This result indicated that Tra-CM-MSN-PYR significantly increased drug antitumor efficacy and had excellent biological safety. H&E sections of the heart, liver, spleen, lung and kidney are shown in Fig. 5F and showed no significant pathological changes between the control group and each administration group, which proved the excellent biological safety of Tra-MSN-PYR. In summary, Tra-CM-MSN-PYR has considerable promise in the treatment of HER2-positive breast cancer.

4. Conclusion

Trastuzumab-functionalized SK-BR-3 cancer cell membrane-wrapped mesoporous silica nanoparticles (Tra-CM-MSN) were successfully prepared for the targeted delivery of pyrotinib. In vitro cell experiments and in vivo animal experiments showed that Tra-CM-MSN-PYR exhibits powerful antitumor effects, has significant targeting ability and can significantly promote the apoptosis of SK-BR-3 HER2-positive breast cancer cells. These results demonstrated that Tra-CM-MSN-PYR has potential application as a targeted therapy for the dosage regimen of HER2-positive breast cancer.

CRediT authorship contribution statement

Xing Liu: Writing – original draft, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Wenwen Shen: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation.

Declaration of competing interest

The authors declare no competing financial interest. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Acknowledgments

The authors acknowledge the financial support received from the Department of Education of Liaoning Province (Grant number JYTJCZR2020047, 2020), Wu Jieping Medical Foundation (Grant number 320.6750.2023-18-126), The funding of Scientific Research of The First Affiliated Hospital of Jinzhou Medical University (Grant number FYQKR-202202), The funding of Scientific Research of The First Affiliated Hospital of Jinzhou Medical University (Grant number KYTD-2022001), and Beijing Medical Award Foundation (Grant number YXJL-2020-0941-0744).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpx.2024.100302.

Contributor Information

Xing Liu, Email: liux2@jzmu.edu.cn.

Wenwen Shen, Email: shenwenwen20210719@163.com.

Appendix A. Supplementary data

Supplementary material
mmc1.docx (24.2KB, docx)

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material
mmc1.docx (24.2KB, docx)

Data Availability Statement

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

Articles from International Journal of Pharmaceutics: X are provided here courtesy of Elsevier

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