br V V was the initial tumor volume before
V0 (V0 was the initial tumor volume before treatment) was used to evaluate the relative tumor growth ratio. After 14 days of treatment, the tumor of each nude mouse was excised and weighed. Meanwhile, the dissected tumor tissues were fixed in formalin, embedded in paraﬃn, sectioned into slices for H&E, Ki67 immunohistochemistry analyzed by optical microscope (Axio Lab.A1, Germany).
2.13. Statistical analysis
The data were analyzed by using GraphPad Prism 7 software. Student’s t-test or ANOVA were used for determining the significant diﬀerence. P < 0.05 represented statistical significance.
3. Results and discussion
3.1. Synthesis and characterization of MSNs and [email protected]
The DOX-loaded mesoporous silica nanoparticles with a CaCO3 layer as smart gatekeeper were prepared as illustrated in Scheme 1. Firstly, MSNs were synthesized according to a template-guided sol-gel method reported by Shi et al . In this approach, CTAC and TEOS were respectively used as a template and silica source, while TEA as a base to control the preparation process which will influence the final particle size. Afterwards, the obtained nanoparticles were extracted with NaCl/methanol mixture to remove CTAC to give mesoporous silica nanoparticles (MSNs). The channels of MSNs were then loaded with DOX via electrostatic interaction and hydrogen bonding role . Subsequently, the CaCO3 coated MSN (DOX/[email protected]) was pre-pared using a water-in-oil (w/o) micro-emulsion method . Finally, DOX/[email protected] was further cloaked with prostate cancer cell membrane fragments by extruding from the polycarbonate porous membrane.
The morphology of MSNs was characterized by TEM. As shown in Fig. 1A, MSNs presented a uniform size around 100 nm, a spherical shape, a well particle monodispersity, as well as an obvious mesoporous structure. For DOX/[email protected], a clear thin layer surrounded MSNs in Fig. 1B demonstrated the successful capping of CaCO3 as gate-keeper. Meanwhile, this figure revealed that DOX loading had no obvious
influence on the morphology of the MSNs, as nearly the same mor-phology, size distribution and dispersity in comparison with the bare MSNs. Meanwhile, DLS results (Fig. 1 C) also demonstrate the negli-gible change of size distribution after DOX loading. UV–vis spectra were used to confirm the DOX loading into MSNs. As shown in Fig. 1D, the characteristic 915759-45-4 peak located at 480 nm could be also observed in the absorption curves of DOX/MSN and DOX/[email protected], while MSN and [email protected] didn’t exhibit this absorption peak. In addition, the N2adsorption-desorption isotherms curves of MSN and DOX/MSN were also recorded to verify the loading of DOX into MSNs (Fig. 1E and 1 F), as the BET surface area decreased from 1542 m2/g to 1046 m2/g. To improve the stability of DOX/[email protected], the cell membrane fragments (CM) was adsorbed onto these nanoparticles. TEM revealed the successful coating of CM layer on the surface and the well-disper-sion of obtained DOX/[email protected]@CM (Fig. 2A). The intermediates at each step and the final product of DOX/[email protected]@CM were characterized by fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA). As shown in Figure S1, the FT-IR spectra of DOX/MSN showed both the typical peaks at 1578 cm−1 (assigned to C]O stretching vibration of DOX) and 1093 cm−1 (as-signed to the Si-O-Si bond stretching of MSN). Compared with DOX/ MSN, a new peak around 1420 cm−1 was found in the spectrum of DOX/[email protected], which is mainly resulted from carbonate vibration of CaCO3. After further coated with cancer cell membranes (CM), the PO43- (from phospholipid molecules in CM) characteristic absorption located at 1257 cm−1 appeared in the spectrum of DOX/[email protected]@CM. Furthermore, compared with other intermediates, the final product of DOX/[email protected]@CM showed most significant weight loss at high temperature (Figure S2). These results confirmed the suc-cessful preparation of DOX/[email protected]@CM. In order to further verify the coating layer is resulted from the LNCaP-AI cells, the protein ingredient of DOX/[email protected]@CM was analysed by using gel elec-trophoresis. As shown in Fig. 2B, DOX/[email protected]@CM showed the same electrophoresis patterns as cancer cell lysate and cancer cell membrane. Furthermore, western blot analysis also demonstrated the well retention of the membrane proteins on our nanoparticles, as the specific marker of claudin-1 belonging to the membrane proteins also appeared in the pattern of DOX/[email protected]@CM in comparison with
Fig. 2. Characterization of DOX/
[email protected]@CM. (A) TEM of DOX/ [email protected]@CM. (B) SDS-PAGE analysis of cell lysate (i), cell membrane (ii), and DOX/ [email protected]@CM (iii). Samples were stained with Coomassie blue. (C) Western blot analysis of membrane-specific protein (claudin-1) in cell lysate (i), cell membrane (ii), and DOX/ [email protected]@CM (iii). (D, E) Size distribu-tion of DOX/[email protected] (D) and DOX/ [email protected]@CM (E) in water or PBS with 10% FBS measured by DLS (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).