| Objective: To prepare temperature and p H dual responsive core-shell-structured magnetic fluorescent mesoporous silica nanoparticles for magnetic targeting drug delivery and photodynamic therapy.Methods: 1. Synthesis and characterization of multifunctional magnetic mesoporous silica nanoparticles First, the core of nonporous silica doped with FITC coated Fe3O4 was fabricated through solvothermal reaction and reverse micelle method, then mesoporous silica doped with Zn Pc as the middle layer was further coated on the core by modified sol-gel process, finally multifunctional magnetic mesoporous silica nanoparticles(FSMP-NPs) were prepared with the polymer shell modified on the middle layer through seed precipitation polymerization. Transmission electron microscopy, X ray diffraction, Fourier Transform infrared spectroscopy and hydrodynamic diameter were respectively employed to characterize the particle size, ordered mesoporous structure, morphology and thermosensitivity. 2. Drug loading and releasing investigation of FSMP-NPs Using Doxorubicin hydrochloride as a model drug to investigate the drug loading behavior in p H 7.4 and controlled releasing behavior at different temperature(25 C and 37 C) and p H(p H 5.5 and p H 7.4) conditions. 3. Photodynamic investigation of FSMP-NPs By using 1,3-diphenylisobenzofuran as a singlet oxygen sensor, we divided the mixed solution of FSMP-NPs+DPBF into 9 tubes respectively irradiated with a 680 nm LED lamp for different time, then UV visible spectrophotometer was used to detect the absorbance change of DPBF in every tube at 417 nm, and the same concentration of DPBF was used as control to evaluate the photodynamic effect of FSMP-NPs. 4. Cytotoxicity and magnetic targeting of FSMP-NPs First of all, MTT assay was used to evaluate the cytotoxicity of FSMP-NPs at different concentrations. Then 25 μg/m L FSMP-NPs, 5 μg/m L DOX, 30 μg/m LDOX-FSMP-NPs were respectively irradiated with a 680 nm LED lamp for 10 min, 20 min, 30 min, 40 min and 50 min to value the cell viability. Finally, to value the synergistic effect of chemotherapy and photodynamic therapy, the cytotoxicity of FSMP-NPs+Light, DOX+Light and DOX-FSMP-NPs+Light were compared with FSMP-NPs, DOX and DOX-FSMP-NPs that without light. 30 μg/m L DOX-FSMP-NPs was added into a 35 mm cell culture dish, then a 2.5 cm small circular magnet was put under the central dish bottom, after DOX-FSMP-NPs were cultured with Hela cells for 10 min, 4 h and 8 h, the enrichment of nanoparticles in magnetic or non magnetic field condition and the cytotoxicity of DOX to Hela cells with the prolonged culturing time were observed under the inverted fluorescence microscope to evaluate the magnetic targeting of FSMP-NPs.Results: 1. Synthesis and characterization of multifunctional magnetic mesoporous silica nanoparticles The TEM results showed that black Fe3O4 nanocrystals were successfully coated in the mesoporous silica matrix; XRD results indicated that FSMP-NPs had two-dimensional hexagonal mesoporous phase structure, which similar to MCM-41, and NIPAM and AA were successfully polymerized on the surface of mesoporous silica; FTIR results showed that modified FSMP-NPs can still maintain the complete structure of mesoporous skeleton while the synthetic FSMP-NPs with thermosensitivity was demomstrated by DLS. 2. Drug loading and releasing investigation of FSMP-NPs Drug loading results showed that FSMP-NPs had a high drug loading content of(206.75±17.59)μg/mg and efficiency of(68.91±5.86)wt%. Drug releasing results showed that FSMP-NPs had a significant temperature and p H response. At the temperature of 37℃, in p H 5.5, the cumulative DOX release in 24 h was as high as(63.76±0.50)wt%, while in p H 7.4 was only(11.20±0.21)wt%; at the temperature of 25℃, in p H 5.5, the cumulative DOX release in 24 h was about(56.13±0.60)wt%, while in p H 7.4 was only(9.91±0.21)wt%.3. Photodynamic investigation of FSMP-NPs The results indicated that in the mixed solution of FSMP-NPs+DPBF, when the irradiated time prolonged, the absorbance of DPBF at 417 nm continued to decline; however in the solution of DPBF only, there was no apparent absorbance decrease. 4. Cytotoxicity and magnetic targeting of FSMP-NPs MTT tests indicated that, the average cell viability of FSMP-NPs in 25 μg/m L was(91.78±0.72)%, with no toxicity to cells, however FSMP-NPs in 30 μg/m L with an average cell viability of(80.19±8.69)% began to exhibit cytotoxicity(P<0.01). When irradiated by a 680 nm LED lamp for different time, the cell viability of DOX did not change with the time prolonged while DOX-FSMP-NPs and FSMP-NPs were significantly decreased, and the cytoxicity of DOX-FSMP-NPs with light was higher than that of DOX. The results of chemotherapy combined photodynamic therapy revealed that, the cell viability of DOX+Light almost was not changed when compared with DOX, that is chemotherapy. However, when compared with FSMP-NPs, the cell viability of FSMP-NPs+Light that is photodynamic therapy was significantly reduced from(91.78±0.72)% to(66.78±6.78)%; and compared with FSMP-NPs+Light, DOX and DOX-FSMP-NPs, the cell viability of DOX-FSMP-NPs+Light that is chemotherapy combined photodynamic therapy was only(11.33±1.91)%(P<0.01). Magnetic targeting results indicated that in the magnetic area, the enrichment of DOX-FSMP-NPs can be realized just in 10 min when cultured with Hela cells, while in non magnetic area, only a few enrichment of the particles. And when DOX-FSMP-NPs were cultured for 8 h, compared with non magnetic area, the magnetic area showed a higher cell death and DOX release.Conclusion: Temperature and p H dual responsive core-shell-structured magnetic fluorescent mesoporous silica nanoparticles were successfully prepared. DOX has been loaded into the nanoparticles with a high drug loading content and encapsulation efficiency and the release behavior has revealed obvious temperature and p H dependence. When irradiated with a 680 nm LED lamp, FSMP-NPs can produce singlet oxygen cause oxidative damage to tumor cells. With magnetic targeting, FSMP-NPs can not only beused as a targeting drug carrier for controlled release, but also be expected to achieve a synergistic effect by combining chemotherapy and photodynamic therapy. |
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