Powerful inner/outer controlled multi-target magnetic nanoparticle drug carrier prepared by liquid photo-immobilization

Abstract

Nanomagnetic materials offer exciting avenues for advancing cancer therapies. Most researches have focused on efficient delivery of drugs in the body by incorporating various drug molecules onto the surface of nanomagnetic particles. The challenge is how to synthesize low toxic nanocarriers with multi-target drug loading. The cancer cell death mechanisms associated with those nanocarriers remain unclear either. Following the cell biology mechanisms, we develop a liquid photo-immobilization approach to attach doxorubicin, folic acid, tumor necrosis factor-α, and interferon-γ onto the oleic acid molecules coated Fe3O4 magnetic nanoparticles to prepare a kind of novel inner/outer controlled multi-target magnetic nanoparticle drug carrier. In this work, this approach is demonstrated by a variety of structural and biomedical characterizations, addressing the anti-cancer effects in vivo and in vitro on the HeLa, and it is highly efficient and powerful in treating cancer cells in a valuable programmed cell death mechanism for overcoming drug resistance.

Figure 1. The molecular structures of the four kinds of photoactive molecules and the chemical processes for preparing photoactive DOX, FOL, TNF-α, and IFN-γ (a), general procedure of photo-immobilization sequence for multi-target MNPs (b), the corresponding nano-particles and sketch of photo-immobilization apparatus in the solid (upper, OA-MNPs-a, OA-MNPs-b, and OA-MNPs-c) (c) and liquid (lower, OA-MNPs-d) photo-immobilizations (d).

Figure 2. SEM images (a), AFM micrographs (x, y-scale of 1 μm) (b), size distribution (c), FTIR spectra (d), Raman spectra (e), and Grafting ratios (f) for the four kinds of OA-MNPs: OA-MNPs-a, OA-MNPs-b, OA-MNPs-c, and OA-MNPs-d. The yellow bar in the SEM images stands for 150 nm.

Figure 3. Measured efficacy and toxicities data in vivo.

(a). Photos for tumor-bearing mice treated by the four kinds of OA-MNPs: (a) OA-MNPs-a, (b) OA-MNPs-b, (c) OA-MNPs-c, and (d) OA-MNPs-d. The yellow bar stands for 1 cm. (b). Tumor volume (left) and survival rate (right) of the nude mice as a function of time (day) upon the treatment by (a) OA-MNPs-a, (b) OA-MNPs-b, (c) OA-MNPs-c, and (d) OA-MNPs-d. The animal test is plotted with the significance p < 0.05 labeled by symbol *, 0.001 < p < 0.01 labeled by symbol **, and p < 0.001 labeled by symbol ***. The standard deviation for a and b is n = 6. (c). Prussian blue staining of tumor tissue upon the treatment by (a) OA-MNPs-a, (b) OA-MNPs-b, (c) OA-MNPs-c, and (d) OA-MNPs-d. The yellow bar stands for 15 μm. The white arrow means that the cells are treated by the OA-MNPs, and the black arrows mean that the tumor tissue and liver tissue. (d). Anti-ssDNA analysis of tumor tissue upon the treatment by (a) OA-MNPs-a, (b) OA-MNPs-b, (c) OA-MNPs-c, and (d) OA-MNPs-d. The yellow bar stands for 15 μm. The black arrows mean that the tumor tissue and liver tissue. (e). Magnetic hysteresis loops of tumor tissues upon the treatment by (a) OA-MNPs-a, (b) OA-MNPs-b, (c) OA-MNPs-c, and (d) OA-MNPs-d. (f). Serological and toxicology test of nude mice, including the density of platelet (left), the density of white blood cell (middle), and the density of red blood cell (right), upon the treatment by (a) OA-MNPs-a, (b) OA-MNPs-b, (c) OA-MNPs-c, and (d) OA-MNPs-d. The serological and toxicology test are plotted with the significance p < 0.05 labeled by symbol *, 0.001 < p < 0.01 labeled by symbol **. The bars stand for the standard deviations (n = 3).

Figure 4. Measured efficacy data in vitro.

HeLa cells were treated with (a) OA-MNPs-a, (b) OA-MNPs-b, (c) OA-MNPs-c, and (d) OA-MNPs-d for 48 hours. (a) and (b). Cells morphology as evaluated by light microscopy (Yellow bars, 15 μm) and transmission electron microscopy (Yellow bars, 5 μm). The white arrow means that the cells are uptaked by the OA-MNPs. Only one of three representative experiments is shown here. (c). Measured cell mortalities data by flow cytometry (n = 3). (d). Measured cell cycle data by flow cytometry (n = 3). (e). Measured mRNA expression of p53, Bax, Bcl-2 and caspase-3 by PCR and RTPCR analysis. In the PCR analysis, data are representative of three experiments. In the RTPCR anaylsis, the relative levels of p53, Bax, Bcl-2 and caspase-3 are plotted with the significance p < 0.05 labeled by symbol *, 0.001<p<0.01 labeled by symbol **, and p<0.001 labeled by symbol ***, in comparison with the OA-MNPs-a group. The bars stand for the standard deviations (n = 3). (f). Measured protein expression data of TNFR1, IFNR2, FOLR1, and caspase-3 (cleaved caspase-3) by western blotting. Blots were re-probed for β-actin and used as control for equal loading of proteins. Protein expression determined using BandScan software. The relative levels are plotted with the significance p < 0.05 labeled by symbol *, 0.001 < p < 0.01 labeled by symbol **, and p < 0.001 labeled by symbol ***, in comparison with the OA-MNPs-a group. The bars stand for the standard deviations (n = 3).