| Objective: This study was aimed to prepare a multifunctional nano-drug delivery system (YSA-BHQ1/TAT-FITC/MSN@DOX) for anti-cancer drug delivery and imaging based on reversal of peptide charge, and to explore its in vivo and in vitro activities for cancer imaging and therapy.Methods: The shapes and sizes of YSA-BHQ1/TAT-FITC/MSN@DOX were observed with TEM and SEM. The infrared spectra were inspected with a FT-IR spectrometer. The zeta potentials of the nano-particles were measured with a Malvern zetasizer. The loading efficiency and amount of the DOX in the nano-particles were calculated according to the in vitro release behavior of the DOX from YSA-BHQ1/TAT-FITC/MSN@DOX. The fluorescence of YSA-BHQ1/TAT-FITC/MSN@DOX under different pH conditions was observed with a fluorescence microscope. The fluorescence intensities were measured with a Fluostar Optima fluorescence micro-plate reader. Nitrogen adsorption-desorption isotherms were acquired with a Quantachrome Nova Brunauer-Emmett-Teller specific surface area analyzer. The surface areas of the nano-particles were calculated by the Brunauer-Emmett-Teller means. The pore size distribution was determined from the nitrogen adsorption isotherms by the Barrett-Joynere-Halenda method. Thermogravimetric analysis was performed with a thermogravimetry analyzer. The in vitro cytotoxicities of nano－particles or DOX-loaded nanoparticles were assessed with the standard CCK-8 test.The relative percentage of apoptotic cells was detected by flow cytometry. The cellular uptake and intracellular distribution of nano-particles in the MCF-7 and HEK293 cells was observed with a CLSM. The anti-tumor efficacy and NIRF imaging were studied using MCF-7 cells bearing armpit tumor nude mice. The tumor volume and body weight were monitored. At the end of the experiment, 20 μL blood from each mouse was collected and the white blood cell (WBC) was counted to assess the blood toxicity of DOX formulations. Then mice were sacrificed and its organs were excised for Ihematoxylin and eosin (H&E) staining. Tumors were excised for in situ apoptosis analysis by TUNEL. The immuno-histochemistry of CD31 was performed to assess the anti-tumor effect of different DOX formulations. NIRF images were acquired before injection and after injection dynamically at 1 h, 5 h, 12 h,24 h, and 48 h. After 48 h, the mice were sacrificed and tumor, heart, lung, liver,spleen, and kidney were collected for the ex vivo fluorescent imaging.Results: YSA-BHQ1/TAT-FITC/MSN@DOX was obviously larger than MSN. The zeta potential of MSN, COOH-MSN, TAT-FITC/MSN, and YSA-BHQ1/TAT-FITC/MSN were -21.7 ± 7.5 mv,-44.3 ± 13.5mv, -28.2 ± 9.2 mv, and -5.1 ± 1.4 mv,respectively. The acidic atmosphere accelerated the release of DOX molecules. The loading efficiency and encapsulating efficiency of DOX were 8.6± 1.4% and 39.3±3.5%, respectively. The fluorescence of YSA-BHQ1/TAT-FITC/MSN@DOX was quenched under neutral or alkaline conditions and emitted in an acidic environment.The wt% of CPTS, TAT-FITC, Cit, YSA-BHQ1, and DOX in the YSA-BHQ1/TAT-FITC/MSN@DOX was 7.2 ± 2.4%.14.4 ± 4.1%.6.7 ± 1.9%、4.6 ±4.5%、8.2 ±2.7%, respectively. The specific surface area and pore volume of YSA-BHQ1/TAT-FITC/MSN@DOX were 157.4 ± 23.1 m2/g and 0.27 ± 0.07 mL/g, respectively. The average pore size of YSA-BHQl/TAT-FITC/MSN@DOX was 0.7± 0.15 nm. The viability of MCF-7 cells treated with YSA-BHQ1 /TAT-FITC/MSN@DOX was equivalent to that of cells treated with free DOX. YSA-BHQ1/TAT-FITC/MSN@DOX caused obvious apoptosis of MCF-7 cells and had a lesser effect on the apoptotic ratio of HEK293 cells. YSA-BHQ1/TAT-FITC/MSN had little influence on the apoptosis of MCF-7 and HEK293 cells. The YSA-BHQ1/TAT-FITC/MSN@DOX demonstrated a stronger anti-tumor efficacy and a lesser toxicity than free DOX on nude mice bearing MCF-7 cell tumor. The fluorescence of YSA-BHQ1/TAT-Cy5.5/MSN@DOX was concentreated on the tumor site and sustained until 48 h post injection.Conclusions: A novel multiffunctional nano-drug delivery system (YSA-BHQ1/TAT-FITC(Cy5.5)/MSN@DOX) based on reversal of peptide charge was successfully developed for anti-cancer drug delivery and imaging. It had good ability to target MCF-7 cells and could entered into MCF-7 cells via EphA2 receptor-mediated endocytosis. However, it had limited toxicity to HEK293 cells. The YSA-BHQ1/TAT-Cy5.5/MSN@DOX demonstrated a stronger anti-tumor efficacy and a lesser toxicity compared to free DOX on MCF-7 tumor bearing nude mice. The fluorescence of YSA-BHQ1/TAT-Cy5.5/MSN@DOX was concentreated on the tumor site and sustained until 48 h postinjection. Therefore, YSA-BHQ1/TAT-FITC(Cy5.5)/MSN@DOX could be a promising nano-drug delivery system for cancer imaging and therapy. |
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