br mg of DOX MSN CaCO
10 mg of DOX/[email protected] were vigorously mixed with as-ex-tracted cancer cell membrane (1 mg) in 5 mL aqueous solutions. After continuous sonication for 5 min in ultrasonic bath, the final product of DOX/[email protected]@CM was collected and extruded from the poly-carbonate porous membrane.
2.7. Nanoparticle characterization
MSNs, DOX/[email protected], and DOX/[email protected]@CM were im-aged by transmission electron microscopy (TEM) (Tecnai G2F30, FEI, USA) at 80 kV. UV absorbance was recorded by a DNM-9602 microplate reader (Beijing Perlong New Technology Co., Ltd., Beijing, China). N2 adsorption/desorption assay was performed by using a Micromeritics ASAP 2020 M apparatus. The pore size was calculated from desorption branches of isotherms by the Barrrett-Joyner-Halenda (BJH) method, while the surface areas were calculated by the Brunauer-Emmett-Teller (BET) method. Fourier Transform Infrared (FT-IR) spectra were re-corded on a Fourier transform infrared spectrometer (Spectrum Two, Perkin-Elmer) over the spectral region of 500 cm−1 to 4000 cm−1. TGA was conducted from 25 °C to 800 °C by using a diamond DSC/TG/GTA instrument (TA Q600) with a heating rate of 10 °C/min under a ni-trogen purge of 40 mL/min.
DOX/[email protected] or DOX/[email protected]@CM with equivalent DOX content of 1 mg in a dialysis bag was placed in a tube containing 10 mL of pH 7.4 (PBS), pH 6.5 (PBS) or pH 5.0 (acetate buﬀer) and shaken at 37 °C. At predetermining time intervals, 5 mL of the solution was taken out to calculate the release amount of DOX by detecting UV–vis ab-sorption, while another 5 mL of fresh buﬀer was added into the original
Scheme 1. Schematic illustration of the synthesis and pH-triggered drug release of DOX/MSN/[email protected]
solutions for further drug release.
2.9. Cell uptake and intracellular drug release
To detect cell uptake of DOX/[email protected]@CM, LNCaP-AI Pam3CSK4 were planted into a 35 mm glass bottom dish and cultured for 24 h. Then the cells were incubated with DOX/[email protected]@CM for dif-ferent time intervals (2 h, 4 h and 8 h). After nuclear stained by Hoechst 33342, cells were washed with PBS for 3 times and imaged under a confocal laser scanning microscopy (CLSM, Zeiss LSM780). To de-monstrate the homologous targeting properties, the cellular uptake in MCF-7 cells for 8 h incubation was also evaluated though the same protocol. Meanwhile, the LNCaP-AI cells but pre-treated with NH4Cl (50 μM) for 45 min at 37 °C, before incubated with DOX/ [email protected]@CM was also used as a control to demonstrate the en-dosome/lysosome pH triggered intracellular drug release.
2.10. In vitro cytotoxic assay
LNCaP-AI cells were seeded in a 96-well plate at a density of 5000 cells per well. After incubated for 24 h, free DOX, DOX/ [email protected]@CM or [email protected]@CM with the equivalent DOX concentration ranged from μg/mL to 16 μg/mL were added to each well. After 24 h or 48 h of incubation, 10 μL of CCK8 solution was added to each well and further incubated for 2 h. At last, the absorbance of each well at 450 nm was measured with a microplate reader (SpectraMax M5e). Cell viability (%) = (Asample-Ablank)/(Acontrol-Ablank)
× 100%. Asample and Acontrol are the absorbance of the treated cells (as mentioned above) and the untreated cells, respectively. Ablank is the absorbance of CCK-8 reagent itself without staining any cells. All ex-periments were performed in quadruplicate. The results are presented as the mean ± standard deviation (SD). Meanwhile, Live/Dead cell assay was also used to qualitatively assess the cell viability. The cells
treated as indicated above were analyzed by using a Calcein-AM/PI Double Stain Kit incorporating with a fluorescence microscope (Zeiss Axio Vert.A1).
2.11. Cell apoptosis analysis
2 mL of cell suspension (1 × 106 cells/mL) was added into a 6-well plate. After incubation for 24 h, LNCaP-AI cells were treated with free DOX or MSN/[email protected]@CM for 48 h. For cell apoptosis analysis, the cells were collected, and stained by a Annexin V-FITC/PI apoptosis assay kit before detected by a flow cytometry (FCM, BD FACSVerse, USA).
2.12. In vivo antitumor therapy
The animal studies have been conducted strictly under the guide-lines of “National animal management regulations of China”, and ap-proved by the Animal Ethics Committee of Fujian Medical University. For cancer model preparation, six-week-old
BALB/c nude mice (obtained from China Wushi, Inc.) were injected subcutaneously with 106 LNCaP-AI cells. When the tumor volume reached ∼100 mm3, the mice were used in the following anti-tumor therapy studies.The LNCaP-AI tumor-bearing mice were randomly di-vided into four groups with five mice in each group as follows: PBS injection (group i), [email protected]@CM injection (group ii), DOX/ [email protected]@CM injection (group iii), and free DOX injection (group iv), respectively. The injection volume was 100 μL per mouse; the in-jection doses based on DOX were 5 mg/kg, the administration method was intravenous injection, and repeated at day 0, day 4, day 8. The tumor volume was recorded every 2 days for 14 days, and the tumor diameter at each time point was measured by using an electronic ca-liper. The tumor volume was calculated according to the following formula: Tumor volume (V) = length × width2/2. Relative volume V/