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Figure from:Structure-property relationships in manganese oxide-mesoporous silica nanoparticles used for T< sub> 1</sub>-weighted MRI and simultaneous anti-cancer drug delivery

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Fig. 6. (a—c) CLSM images of tumor tissue after 24 h intravenous administration of DOX-Mn-MSNs into mice with different magnifications (Fig. b is the magnified image of pane area in a, and Fig. d is the magnified image of corresponding pane area in b); (d) Biodistributions of DOX in major organs of mice after 24 injection of free DOX and DOX-Mn-MSNs via intravenous injection (inset: magnified Figure of the DOX concentrations in tumor). To evaluate the potential of Mn-MSNs as the drug delivery system for cancer chemotherapy, a typical anti-cancer chemo- therapeutic agent doxorubicin (DOX) was chosen as the model drug to investigate the drug loading and intracellular release behavior. The drug loading amount on the support is 382 mg/g, as deter- mined by UV—vis characterization. The in vitro drug release prop- erties achieved at different pH values (pH = 7.4 , 6.0 and 5.0) demonstrated the sustained and pH-responsive DOX release profile from DOX-loaded Mn-MSNs (Fig. 5a), which is determined by the electrostatic interaction between positively charged DOX molecules and negatively charged mesopores. This pH-responsive character is very important because pH values in different tissues and cellular The in vivo therapeutic potential was assessed by intravenous injection of free DOX and DOX-loaded Mn-MSNs (DOX-Mn-MSNs) into tumor-bearing mice at a DOX dose of 5 mg/kg. Mice were scarified 24 h after the injection of drugs, and the major organs

Figure 6 (a—c) CLSM images of tumor tissue after 24 h intravenous administration of DOX-Mn-MSNs into mice with different magnifications (Fig. b is the magnified image of pane area in a, and Fig. d is the magnified image of corresponding pane area in b); (d) Biodistributions of DOX in major organs of mice after 24 injection of free DOX and DOX-Mn-MSNs via intravenous injection (inset: magnified Figure of the DOX concentrations in tumor). To evaluate the potential of Mn-MSNs as the drug delivery system for cancer chemotherapy, a typical anti-cancer chemo- therapeutic agent doxorubicin (DOX) was chosen as the model drug to investigate the drug loading and intracellular release behavior. The drug loading amount on the support is 382 mg/g, as deter- mined by UV—vis characterization. The in vitro drug release prop- erties achieved at different pH values (pH = 7.4 , 6.0 and 5.0) demonstrated the sustained and pH-responsive DOX release profile from DOX-loaded Mn-MSNs (Fig. 5a), which is determined by the electrostatic interaction between positively charged DOX molecules and negatively charged mesopores. This pH-responsive character is very important because pH values in different tissues and cellular The in vivo therapeutic potential was assessed by intravenous injection of free DOX and DOX-loaded Mn-MSNs (DOX-Mn-MSNs) into tumor-bearing mice at a DOX dose of 5 mg/kg. Mice were scarified 24 h after the injection of drugs, and the major organs

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Fig. 1. Schematic representation for the preparation of manganese oxide dispersed MSNs (Mn-MSNs) by the O/R-HT strategy. The “soft templates”, typically employed fc synthesizing mesoporous materials and evenly be restricted within mesopores, could react in-situ with potassium permanganate (KMnO,) by chemical oxidation-reduction (O/R by which manganese oxide nanoparticles are formed and retained within nanopores (Step A). The relaxivity of manganese oxide within mesopores could be enhanced by he< treatment under reducing atmosphere (Step B). Finally, water molecules could diffuse freely into mesopores, and interact with manganese paramagnetic centers.
Fig. 1. Schematic representation for the preparation of manganese oxide dispersed MSNs (Mn-MSNs) by the O/R-HT strategy. The “soft templates”, typically employed fc synthesizing mesoporous materials and evenly be restricted within mesopores, could react in-situ with potassium permanganate (KMnO,) by chemical oxidation-reduction (O/R by which manganese oxide nanoparticles are formed and retained within nanopores (Step A). The relaxivity of manganese oxide within mesopores could be enhanced by he< treatment under reducing atmosphere (Step B). Finally, water molecules could diffuse freely into mesopores, and interact with manganese paramagnetic centers.
Fig. 2. Morphological, structural and in vitro MRI characterizations of Mn-MSNs after Hz reduction. TEM (a) and STEM (a,) images of Mn-MSNs, EDS elemental mappings of Mn (a2. Si (a3) and O (a4) elements on a Mn-MSNs. Plots of b) 7; and c) 5! versus Mn concentrations for Mn-MSNs before and after Hz reduction (inset: T,- and T2.-weighted MRI result obtained from aqueous suspensions of Mn-MSNSs before and after H reduction).
Fig. 2. Morphological, structural and in vitro MRI characterizations of Mn-MSNs after Hz reduction. TEM (a) and STEM (a,) images of Mn-MSNs, EDS elemental mappings of Mn (a2. Si (a3) and O (a4) elements on a Mn-MSNs. Plots of b) 7; and c) 5! versus Mn concentrations for Mn-MSNs before and after Hz reduction (inset: T,- and T2.-weighted MRI result obtained from aqueous suspensions of Mn-MSNSs before and after H reduction).
Fig. 3. FITC-Mn-MSNs are internalized by MCF-7 cells. The MCF-7 cells were incubated with the nanoparticles (FITC-Mn-MSNs, 50 g/mL) for 6 h. After incubation, the cell nucleus were stained by DAPI (blue), and the extracellular fluorescence was quenched by trypan blue and the endocytosed particles with FITC-label (green) inside cells were detected by CLSM (a,—a3); Three-dimensional fluorescence reconstructions of nanoparticle-endocytosed MCF-7 cells (b;—b3) to demonstrate the internalization of nanoparticles within cancer cells; Three-dimensional fluorescence reconstructions of nanoparticle-endocytosized MCF-7 cells after nucleus and lysosomes double-staining (c;—c4), to demonstrate the intra- cellular location of Mn-MSNs nanoparticles (nuclei (c;): blue fluorescence of DAPI staining; lysosomes (cz): red fluorescence of Lyso-tracker red staining; Mn-MSNs (c3): green fluorescence of FITC grafting). The yellow fluorescence signal in overlay image (c4), which comes from the superposition of the red and green, suggests that some Mn-MSNs have entered lysosomes while the rests are in the cytoplasm; selected bio-TEM images of MCF-7 cells after co-incubation with Mn-MSNs nanoparticles for 24 h showing (d,) Mn-MSNs uptaken by MCF-7 cells by endocytosed process, (d2) Mn-MSNs trapping in vesicles inside the cell and (d3) endosomal escape of nanoparticles into the cytoplasm. (For inter- pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) paramagnetic center within mesopores, EDS elemental mapping of Mn, Si and O elements in Mn-MSNs was conducted (Fig. 2a;—aq and Fig. S3). The result shows that the manganese element is homogeneously distributed in the whole mesoporous silica matrix without the accumulation at the particle surface, demonstrating the pore-confined formation and deposition of Manganese oxide nanoparticles within mesopores. Inductively coupled plasma atomic emission spectrometry (ICP-AES) results show that the manganese element content in Mn-MSNs is 5.94% (w/w), which could be facilely tuned by varying the initial synthetic parameters such as MnOq concentrations, reaction time and temperature. The Mn-MSNs nanoparticles exhibit small- angle X-ray diffraction (SAXRD) peaks at 2.5, 4.3 and 5.0° that are characteristic of (100), (110) and (200) planes of the typical Fig. 2 shows the morphology, structural and chemical char- acteristics of Mn-MSNs after Hz reduction. The Mn-MSNs exhibits spherical morphology, high dispersity and two-dimensional hexagonally ordered pore array (Fig. 2a and Figs. S1, S2 and S4). To further investigate the distribution of manganese
Fig. 3. FITC-Mn-MSNs are internalized by MCF-7 cells. The MCF-7 cells were incubated with the nanoparticles (FITC-Mn-MSNs, 50 g/mL) for 6 h. After incubation, the cell nucleus were stained by DAPI (blue), and the extracellular fluorescence was quenched by trypan blue and the endocytosed particles with FITC-label (green) inside cells were detected by CLSM (a,—a3); Three-dimensional fluorescence reconstructions of nanoparticle-endocytosed MCF-7 cells (b;—b3) to demonstrate the internalization of nanoparticles within cancer cells; Three-dimensional fluorescence reconstructions of nanoparticle-endocytosized MCF-7 cells after nucleus and lysosomes double-staining (c;—c4), to demonstrate the intra- cellular location of Mn-MSNs nanoparticles (nuclei (c;): blue fluorescence of DAPI staining; lysosomes (cz): red fluorescence of Lyso-tracker red staining; Mn-MSNs (c3): green fluorescence of FITC grafting). The yellow fluorescence signal in overlay image (c4), which comes from the superposition of the red and green, suggests that some Mn-MSNs have entered lysosomes while the rests are in the cytoplasm; selected bio-TEM images of MCF-7 cells after co-incubation with Mn-MSNs nanoparticles for 24 h showing (d,) Mn-MSNs uptaken by MCF-7 cells by endocytosed process, (d2) Mn-MSNs trapping in vesicles inside the cell and (d3) endosomal escape of nanoparticles into the cytoplasm. (For inter- pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) paramagnetic center within mesopores, EDS elemental mapping of Mn, Si and O elements in Mn-MSNs was conducted (Fig. 2a;—aq and Fig. S3). The result shows that the manganese element is homogeneously distributed in the whole mesoporous silica matrix without the accumulation at the particle surface, demonstrating the pore-confined formation and deposition of Manganese oxide nanoparticles within mesopores. Inductively coupled plasma atomic emission spectrometry (ICP-AES) results show that the manganese element content in Mn-MSNs is 5.94% (w/w), which could be facilely tuned by varying the initial synthetic parameters such as MnOq concentrations, reaction time and temperature. The Mn-MSNs nanoparticles exhibit small- angle X-ray diffraction (SAXRD) peaks at 2.5, 4.3 and 5.0° that are characteristic of (100), (110) and (200) planes of the typical Fig. 2 shows the morphology, structural and chemical char- acteristics of Mn-MSNs after Hz reduction. The Mn-MSNs exhibits spherical morphology, high dispersity and two-dimensional hexagonally ordered pore array (Fig. 2a and Figs. S1, S2 and S4). To further investigate the distribution of manganese
Fig. 4. In vivo MR imaging capability of Mn-MSNs after H2 reduction. A time-course (0, 5, 30, 60 min) of the signal enhancement in T;-weighted MRI of tumor (a;—ag), liver (ci—c. and kidney (d\—d4) of a tumor-bearing mouse after intravenous injection of Mn-MSNs; b;—b,: magnified images of corresponding circled area in ay—a4. To assess the effectiveness of Mn-MSNs as MRI CAs, the magnetic resonance relaxivity of the nanoparticles was measured using a clinical 3.0 T human clinical scanner. The in vitro longitu- dinal relaxation rate (1/T,) and transverse relaxation rate (1/T2) as a function of the manganese ion concentrations of Mn-MSNs before and after Hz reduction were evaluated. As shown in Fig. 2b and c, Mn-MSNs have an 1; value of 0.45 mM~!s~! and rz value of 9. mM~'s~! before the heat treatment. However, the specific relaxivity of Mn-MSNs was found to significantly increase after Hz reduction. The r; and rz values of Mn-MSNs after Hz reduction reached 2.28 and 15.9 mM~'s~!,5.1 and 1.7 times higher than their original values before Hz reduction, respectively. It can be inferred that the increased relaxivities of the Mn-MSNs after Hz reduction MCM-41-type mesoporous materials, respectively (Fig. S5a). The SAXRD results indicate that Mn-MSNs possess ordered meso- porous arrays. The wide-angle XRD patterns (WAXRD, Fig. S5b) exhibit no apparent diffraction peaks for crystallized manganese oxide nanoparticles, demonstrating the absence of large manganese oxide particles formed on the material matrix during the synthetic process. Nitrogen adsorption-desorption technique was employed to characterize the pore structures of Mn-MSNs after the Hz reduction, which shows that the isotherms of Mn- MSNs exhibit the typical Langmuir IV type hysteresis loop, indi- cating the presence of well-defined mesopores (Fig. S6a). In addition, Mn-MSNs are highly porous with a surface area of 665 m?/g, pore volume of 0.99 cm?/g and pore size of 2.4 nm (Fig. S6b), indicating that the mesopore system of MSNs remain open and penetrable after the deposition of manganese oxide species.
Fig. 4. In vivo MR imaging capability of Mn-MSNs after H2 reduction. A time-course (0, 5, 30, 60 min) of the signal enhancement in T;-weighted MRI of tumor (a;—ag), liver (ci—c. and kidney (d\—d4) of a tumor-bearing mouse after intravenous injection of Mn-MSNs; b;—b,: magnified images of corresponding circled area in ay—a4. To assess the effectiveness of Mn-MSNs as MRI CAs, the magnetic resonance relaxivity of the nanoparticles was measured using a clinical 3.0 T human clinical scanner. The in vitro longitu- dinal relaxation rate (1/T,) and transverse relaxation rate (1/T2) as a function of the manganese ion concentrations of Mn-MSNs before and after Hz reduction were evaluated. As shown in Fig. 2b and c, Mn-MSNs have an 1; value of 0.45 mM~!s~! and rz value of 9. mM~'s~! before the heat treatment. However, the specific relaxivity of Mn-MSNs was found to significantly increase after Hz reduction. The r; and rz values of Mn-MSNs after Hz reduction reached 2.28 and 15.9 mM~'s~!,5.1 and 1.7 times higher than their original values before Hz reduction, respectively. It can be inferred that the increased relaxivities of the Mn-MSNs after Hz reduction MCM-41-type mesoporous materials, respectively (Fig. S5a). The SAXRD results indicate that Mn-MSNs possess ordered meso- porous arrays. The wide-angle XRD patterns (WAXRD, Fig. S5b) exhibit no apparent diffraction peaks for crystallized manganese oxide nanoparticles, demonstrating the absence of large manganese oxide particles formed on the material matrix during the synthetic process. Nitrogen adsorption-desorption technique was employed to characterize the pore structures of Mn-MSNs after the Hz reduction, which shows that the isotherms of Mn- MSNs exhibit the typical Langmuir IV type hysteresis loop, indi- cating the presence of well-defined mesopores (Fig. S6a). In addition, Mn-MSNs are highly porous with a surface area of 665 m?/g, pore volume of 0.99 cm?/g and pore size of 2.4 nm (Fig. S6b), indicating that the mesopore system of MSNs remain open and penetrable after the deposition of manganese oxide species.
Fig. 5. (a) The release profiles of DOX from DOX-loaded Mn-MSNs at different pH values (pH = 7.4, 6.0 and 5.0; inset: digital pictures of the releasing media in 600 min of release). (b) CLSM images of MCF-7 cells incubated with DOX-loaded Mn-MSNs (DOX concentration: 10 pg/mL) for different time intervals (2 h, 4 h and 6 h; blue fluorescence (bj;, bz; and b3;): nuclei stained with DAPI; red fluorescence (b12, C22 and b32): DOX molecules; (b;3, bz3 and b33): merged images of blue and red fluorescence; (by4, bz4 and b34): fluorescence intensity of paned area in by, bz1 and b3,, respectively). (c) Viabilities of MCF-7 cells when incubated with free DOX and DOX-loaded Mn-MSNs at different concentrations as assessed by MTT protocol. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) It is known that excellent cell uptake of the CAs guarantees the efficient cell imaging by MRI for diagnosis and intracellular drug delivery for chemotherapy. The Mn-MSNs nanoparticles were covalently conjugated with the typical organic fluorescent dye- fluorescein isothiocyanate (FITC-Mn-MSNs) using simple silane conjugation chemistry, to make them visible by confocal laser scanning microscopy (CLSM) for the observations of interaction and intracellular location of nanoparticles. Breast cancer MCF-7 cells were chosen as the modal cells, which were incubated with fluo- rescent nanoparticles for 6 h at 37 °C at a dose of 50 ug/mL. In order to distinguish the nanoparticles only attached to the membrane : ‘The effectiveness of Mn-MSNs after H2 reduction as the MRI CAs in vivo was assessed employing a 3.0 T scanner. Fig. 4 shows the Mn-MSNs contrast-enhanced T,-weighted MRI of a tumor-bearing
Fig. 5. (a) The release profiles of DOX from DOX-loaded Mn-MSNs at different pH values (pH = 7.4, 6.0 and 5.0; inset: digital pictures of the releasing media in 600 min of release). (b) CLSM images of MCF-7 cells incubated with DOX-loaded Mn-MSNs (DOX concentration: 10 pg/mL) for different time intervals (2 h, 4 h and 6 h; blue fluorescence (bj;, bz; and b3;): nuclei stained with DAPI; red fluorescence (b12, C22 and b32): DOX molecules; (b;3, bz3 and b33): merged images of blue and red fluorescence; (by4, bz4 and b34): fluorescence intensity of paned area in by, bz1 and b3,, respectively). (c) Viabilities of MCF-7 cells when incubated with free DOX and DOX-loaded Mn-MSNs at different concentrations as assessed by MTT protocol. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) It is known that excellent cell uptake of the CAs guarantees the efficient cell imaging by MRI for diagnosis and intracellular drug delivery for chemotherapy. The Mn-MSNs nanoparticles were covalently conjugated with the typical organic fluorescent dye- fluorescein isothiocyanate (FITC-Mn-MSNs) using simple silane conjugation chemistry, to make them visible by confocal laser scanning microscopy (CLSM) for the observations of interaction and intracellular location of nanoparticles. Breast cancer MCF-7 cells were chosen as the modal cells, which were incubated with fluo- rescent nanoparticles for 6 h at 37 °C at a dose of 50 ug/mL. In order to distinguish the nanoparticles only attached to the membrane : ‘The effectiveness of Mn-MSNs after H2 reduction as the MRI CAs in vivo was assessed employing a 3.0 T scanner. Fig. 4 shows the Mn-MSNs contrast-enhanced T,-weighted MRI of a tumor-bearing
Fig. 7. T; 1(a) and 1? (b) versus Mn concentrations for Mn-SBA-15 before and after Hz reduction (inset: T;- and T2-weighted MRI results obtained from aqueous suspensions of Mn- SBA-15 before and after Hz reduction); T;- (c, d): and T2- (e, f) weighted MR images of a mouse tumor site before (c, e) and after (d, f) intratumoral injections for 60 min with Mn- SBA-15 (after Hz reduction).
Fig. 7. T; 1(a) and 1? (b) versus Mn concentrations for Mn-SBA-15 before and after Hz reduction (inset: T;- and T2-weighted MRI results obtained from aqueous suspensions of Mn- SBA-15 before and after Hz reduction); T;- (c, d): and T2- (e, f) weighted MR images of a mouse tumor site before (c, e) and after (d, f) intratumoral injections for 60 min with Mn- SBA-15 (after Hz reduction).