Biomimetic nanovesicle co-delivery system impairs energy metabolism for cancer treatment | Journal of Nanobiotechnology

Cell culture

Mouse mononuclear macrophage cell line RAW264.7 and mouse breast cancer cell line 4T1 were both purchased from the American Type Culture Collection (ATCC, USA). Both cell lines were grown in the complete DMEM (Dulbecco’s modified Eagle’s medium) medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified cell culture incubator at 37 °C with 5% CO2.

Preparation of drug-loaded liposomes (Met-3BP-Lip)

Liposomes are synthesized via thin-film hydration by a standard extrusion method. To obtain a liposome membrane, 100 mg of phospholipid and 5 mg of cholesterol were weighed, dissolved in chloroform, evaporated under negative pressure at 37 °C for 4–5 h, and dried in a vacuum oven at 37 °C overnight to completely remove the organic solvent. The required amounts of Met and 3BP were weighed and dissolved in PBS before being added as an aqueous phase to blank liposome membranes to form a homogeneous emulsion. Finally, the above-mentioned liquid was stirred at room temperature for 2 h to be hydrated, and the particle size was controlled by a liposome extruder or an ultrasonic cell crusher. The Met-3BP-Lip emulsion was obtained.

Extraction of M1 macrophage membranes

M1 macrophage membranes were isolated from the RAW 264.7 cells using previously reported extraction steps with minor modifications to the experimental procedure [15, 19]. Briefly, RAW 264.7 cells were stimulated with 100 ng/mL lipopolysaccharide (LPS, Sigma-Aldrich) for 24 h to induce M1 macrophage polarization [21] Then, M1 macrophages were harvested with a cell scraper at a concentration of 8 × 106 cells/mL and resuspended in 1× Tris-Mg buffer (Beyotime Biotechnology) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The collected cells were passed through a 400 nm liposome extruder without a polycarbonate membrane and extruded back and forth 20 times to rupture the membrane. The disrupted cell homogenate was mixed with 1 M sucrose to make the final sucrose concentration of 0.25 M. Then, the samples were centrifuged at 2000 rpm for 15 min at 4 °C to collect the supernatant. This process was repeated once more to remove the remaining organelles. Finally, the purified cell membrane pellet was obtained by centrifuging the supernatant at 10,000 rpm for 15 min at 4 °C. The extracted macrophage membranes will be used for preparation of RMEL NPs. The protein content in the purified macrophage cell membrane was determined by a BCA protein concentration assay kit (Beyotime Biotechnology).

Preparation of Met-3BP-Lip@M1

Met-3BP-Lip@M1 was prepared by a physical extrusion method. Briefly, the purified macrophage membranes were mixed with Met-3BP-Lip@M1 in a volume ratio of 1:1 and then passed through a liposome extruder equipped with a 200 nm polycarbonate membrane for 20 consecutive extruded times.

Nanoparticle characterizations

The particle size, polydispersity index (PDI), and zeta potential of the nanoparticles were measured using dynamic light scattering (DLS, Malvern Instruments, UK). The morphological features of liposomes were observed by transmission electron microscopy (TEM, JEM-1200EX, Japan). For TEM detection, the samples were diluted with PBS (pH 7.4), added to the copper grid surface and dried before negative staining using uranyl acetate solution (1%, w/v).

Drug loading and encapsulation efficiency

Drug loading and encapsulation efficiency were determined after ultracentrifugation at 10,000–20,000 rpm for 45 min and methanol-induced demulsification. The amount of Met was detected by a UV–Vis spectrophotometer (Agilent, Cary Eclipse, USA) at a wavelength of 204 nm. The calibration curve for Met was linear from 2 to 20 µg/mL with a correlation coefficient of R2 = 0.9997. The amount of 3BP was quantified using a high-performance liquid chromatography (HPLC, Agilent 1200, USA) equipped with C18 chromatographic column (150 mm × 4.6 mm, 5 μm). The mobile phase was trifluoroacetic acid aqueous solution and trifluoroacetic acid acetonitrile solution (9:1, V/V). The samples run at a flow rate of 1 mL/min. The detection wavelength was 233 nm. The calibration curve for 3BP was linear in the range 0.09375–3 mg/mL with a correlation coefficient of R2 = 0.9997.

The formulas for drug loading (DL) and encapsulation efficiency (EE) are as follows:

$$\text{DL}\;(\%)=\text{weight}\;\text{of}\;\text{encapsulated}\text{drug}/\text{total}\;\text{weight}\text{of}\;\text{liposome}\times 100\%;$$


In vitro drug release

In vitro drug release was determined by a dialysis method in PBS at pH value of 6.8 with constant stirring. Briefly, nanoparticle solutions were sealed into a dialysis bag (MWCO, 3500), immersed in a beaker with PBS as release media, and stirred at 100 rpm at 37 °C. The release medium was replaced at predetermined time intervals and the released drug content was measured using a UV–visible spectrophotometer or a HPLC as described previously.

Cytotoxicity assay

Cytotoxicity of drugs was quantitatively analyzed using the standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 4T1 cells were seeded in 96-well cell culture plates at 8000 cells per well. When the cells reach 60–70% confluence, complete cell culture medium containing Met (0, 2.5, 5, 10, 20, 40, 60, and 80 mM) or 3BP (0, 5, 10, 20, 40, 60, 80 and 100 µM) were added and the cells were treated for 24 h. Furthermore, to examine the combination index (CI) of the two drugs, Met and 3BP, we treated 4T1 cells with different concentration ratios of Met:3BP (1000, 500, 250, 125, 83.333, and 62.5) and incubated them for 24 h. After 24 h, cytotoxicity was assessed using the MTT assay. After completing the above experiments, the optimal drug binding ratio was selected to prepare Met-3BP-Lip and Met-3BP-Lip@M1, and the cytotoxicity of the formulations was evaluated by MTT method. Briefly, 20 µL of MTT solution (5 mg/mL) was added into each well and the cells were incubated at 37 °C for 4 h. The medium was then replaced with 150 µL DMSO and mixed for 15 min on a shaker. Finally, the optical density (OD) value at the wavelength of 570 nm was measured by a microplate reader (Bio-Tek Instruments Inc., USA). Cytotoxicity calculation formula: cell viability (%) = (OD treatment group − OD blank group/OD control group − OD blank group) × 100%. CI values for drugs were calculated using Calcusyn software (version 1.0, CompuSyn, Inc., USA).

In vitro cellular uptake

We firstly seeded 4T1 cells at a density of 1 × 105 cells per well in 35 mm glass bottom dishes. When the cell density reached about 60% confluence, Cyanine5 (Cy5) fluorescently labeled NPs (Liposome or Lip@M1) were added and the cells were incubated for 2 h. After being washed 3 times with PBS (pH 7.4), the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then washed 2 times with PBS. Finally, the nuclei were stained with 2 µg/mL DAPI for 10 min and washed 3 times with PBS before adding anti-fluorescence quenching mounting solution. Fluorescence images were obtained using a confocal laser scanning microscope (CLSM, Leica, Germany).

To further quantitatively analyze cellular uptake, the treated 4T1 cells were collected and fixed with 4% paraformaldehyde for 10 min and washed 3 times with PBS. Intracellular fluorescence intensity was measured using a flow cytometer (FACS Calibur; BD Biosciences, UK) and analyzed with FlowJo V10 software.

Cell cycle analysis

Cell cycle assessment was performed using propidium iodide (PI) staining. Briefly, after being treated with PBS, Met, 3BP, Met&3BP, Met-3BP-Lip or Met-3BP-Lip@M1 for 24 h, the 4T1 cells were harvested, washed, resuspended in 100 µL of cold PBS, and fixed with 1 ml of pre-chilled 70% ethanol at 4 °C for 30 min. The cells were washed twice with PBS, treated with RNase A (100 µg/mL) for 30 min, and incubated with propidium iodide (50 µg/mL) for 30 min at room temperature in the dark before the analysis by flow cytometry.

Apoptosis assay

Apoptosis assay was carried out using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, US). Briefly, the treated 4T1 cells were collected, washed twice with PBS, resuspended in binding buffer, and incubated with FITC Annexin V solution and PI solution at room temperature for 15 min in the dark before the analysis by flow cytometry.

Glucose uptake and lactate production

Glucose and lactate concentrations were measured using a glucose concentration assay kit and a lactate assay kit (Solarbio Life Science, China) according to the manufacturer’s instructions. The treated 4T1 cells were analyzed by UV–Vis spectrophotometry.

3D tumor spheroid

4T1 tumor spheroids were formed in microwell devices using out established method [15, 22, 23]. When 4T1 tumor spheroids reached 200 μm in diameter, they were treated with Liposome, Lip@M1, Met, 3BP, Met&3BP, Met-3BP-Lip, or Met-3BP-Lip@M1 and new medium were added every two days. The growth of tumor spheroids was observed and recorded using a light microscope (OLYMPUS, Japan). ImageJ software was used for analysis, and the experimental results were expressed as volume change rate and circularity of tumor spheroids. Tumor spheroid volume V = (π × dmax × dmin)/6. Roundness (%) = 100 − (R − r)/R × 100 (R: the radius of the smallest circumscribed circle; r: the largest inscribed concentricity degree circle).

In vivo antitumor efficacy study

All animal experiments were approved by the Animal Ethics Committee of Nantong University. BALB/C female mice (6–8 weeks old) were purchased from the Experimental Animal Center of Nantong University. A 4T1 tumor-bearing BALB/C model was established by subcutaneously injecting 4T1 cells (1 × 106 cells/mL) into the flank regions of the mice. When the tumor volume grew to 100 mm [3], all mice were randomly divided into 8 groups (n = 5) and treated with PBS, Liposome, Lip@M1, Met, 3BP, Met & 3BP, Met-3BP-Lip, and Met-3BP-Lip@M1 every 2 days via tail vein injection. The body weight and tumor volume of the mice were recorded after the injection. The tumor volume was calculated by the following formula: V = 0.5a × b2, where a and b represent the long and short diameters of the tumor, respectively. At the 15th day post-injection, the mice were sacrificed, and their tumors and major organs (the heart, liver, spleen, lung and kidney) were harvested and fixed in 4% paraformaldehyde solution for histology analysis.

Histological analysis

The preserved tumors and major organs were embedded in paraffin, cut into 5 μm thick tissue sections, and stained with hematoxylin-eosin (H&E) staining for pathological evaluation. The tumor sections were also stained using Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) diaminobenzidine (DAB) apoptosis detection kit for the apoptosis analysis.

Statistical analysis

All experiments were repeated at least 3 times, and the results were expressed as mean ± SD. All statistical analyses were performed using GraphPad Prism 6 software. Quantitative data were analyzed using a t-test (for two groups) or one-way ANOVA (for multiple groups). A p-value < 0.05 was considered statistically significant. *p value < 0.05, **p value < 0.01, ***p value < 0.001, ****p value < 0.0001.