| Objective: Iron oxide based core-mesoporous shell magnetic nanoparticles(MNPs)have been popularly used in various cancer theranostic applications due to their excellent properties,such as biocompatibility,magnetic resonance imaging,tumor targeting easily achieved by an external magnet as well as high capacity of drug loading.Conventional core-shell MNPS were usually synthesized from inside to outside.This method has strict requirements to the interface properties of magnetic cores and the precursors of coating shell.The shape and size of MNPs are significantly influenced by that of the pre-synthesized magnetic cores.Most of core-shell MNPs have only single T2 W MRI imaging ability.Herein,we propose a new “outside to inside” synthetic strategy for core-mesoporous shell structural MNPs,where hollow mesoporous Zr O2 nanospheres which exhibit an intrinsic property for both CT imaging and drug loading were used as the shell and the magnetic cores were produced in the cavity of the shell.Modified multiple cores-mesoporous shell MNPs,Fe3O4@Zr O2-PEG were obtained using this strategy.Then characterization,CT/MRI dual modal imaging,DOX loading,magnet and p H dual responsive drug delivery and magnetic targeting cancer chemotherapy were evaluated for liver cancer in vitro and in vivo.Methods: Firstly,mesoporous hollow Zr O2 NPs were prepared via a template method using Si O2 templates.Ferrous and ferric aqueous solution were sealed into the inner space of the hollow Zr O2 nanospheres with a simple physical forced penetration method.Afterwards,ultrasmall superparamagnetic iron(USPIO)nanoparticls were produced through coprecipitation in ethanol inside the cavity of mesoporous Zr O2 hollow nanospheres.Then the as-made Fe3O4@Zr O2 were then PEGylated and Fe3O4@Zr O2-PEG(magnetic mesoporous zirconia nanoparticls,M-MZNs)were obtained.The characterization,CT/MRI dual modal imaging,DOX loading and p H sensitive release were evaluated in vivo.Hep G2 cells were treated with a varying concentration M-MZNs-PEG with(0.78 to 100 ?g/m L)for 24 h and the cytotoxicity was detected with the CCK-8 analysis.We also investigated the cytotoxicity of M-MZNs-PEG on Hep G2 cells under an external magnetic field.Prussian blue staining were used to evaluate endocytosis behavior of M-MZNs-PEG by Hep G2 cells and the effect of magnetic fields on promoting internalization.Cellular internalization of M-MZNs-PEG after 24 h co-incubation in Hep G2 cells was further confirmed by Bio-TEM.LSCM and flow cytometry were employed to observe and quantify the fluorescence of intracellular DOX released from the nanocarrier of M-MZNs-DOX.The in vitro therapeutic effect of M-MZNs-DOX on cancer cells was evaluated with CCK-8 kit.Hep G2 cells were treated with free DOX and M-MZNs-DOX at various DOX concentrations for 24 h with or without an external magnetic field for the first 0.5 h of treatment.The CT/MRI dual modal imaging in vivo of M-MZNs-DOX was further verified in H22 liver cancer bearing mice via intratumorally injection.Magnetic targeting CT/MRI in vivo study were further conducted 3 hours post intravenous injection with a cylinder Nd Fe B permanent magnet fixed on the surface at the tumor site for 1 hour.Thereafter,mice were sacrificed and tumor and major organs were stained with Prussian blue to qualitatively analyze targeting and biodistribution of M-MZNs-DOX.Fifteen mice with mean tumor volume of 162 ± 20 mm3 were randomly separated into five groups:(1)a control group injected with saline(control);(2)M-MZNs-PEG treated group;(3)free DOX treated group;(4)M-MZNs-DOX treated group without external magnetic field(M-);(5)M-MZNs-DOX treated group with an external magnetic field(M+).Free DOX(2 mg/kg)or M-MZNs-DOX(equally containing 2 mg/kg of DOX)were injected via caudal vein every 4 days for 4 times.Tumors growth and body weight of the mice were monitored during the treatment.Magnetic targeting antitumor effect of M-MZNs-DOX treatment was further analyzed by staining apoptotic cells with TUNEL method.H&E staining of major organs was used to assess the safety.Results: Well-dispersed uniform nanoscale structures characterized with a round-shaped Zr O2 spheres enwraping multiple USPIO NPs were successfully synthesized.The mean size of Fe3O4@Zr O2 is 155.23 ± 7.10 nm.The zeta potential of M-MZNs was-17.9 m V and the hydrodynamic diameter was 231.4nm.The magnetization curve of M-MZNs-PEG shows its superparamagnetic behavior owing to absence of hysteresis loops,with a Ms of about 26.5 emu/g.The BET estimated specific surface area of M-MZNs were 307.066 m2/g.The pore size in M-MZNs-PEG is 3.803 nm and the pore volumes(VP)is 0.783 cm3/g.A a delivery carrier for dororubicin,drug loading and drug encapsulation efficiency were calculated to be 49.5% and 20.2%,respectively.The p H-sensitive release of DOX was explored in PBS solution with different p H(5.5 and 7.4).The cumulative released content of DOX was less than 12.5% in the condition of p H 7.4 in 24 h,while more than 25% of DOX was released in acidic condition(p H = 5.5)in 24 h.M-MZNs could be CT/MRI dual modal imaging agent and the relaxivity r2 value is calculated to be 79.88 m M-1S-1.M-MZNs-PEG has no obvious cytotoxicity,and the presence of magnetic stimuli didnot increase its cytotoxicity even under a high concentration of 100 ?g/m L.M-MZNs-PEG could be concentration dependently uptaken by tumor cells after 4 hours treatment and the endocytosis could be enhanced by the magnetic field.DOX was released from M-MZNs-DOX and accumulated in nuclei after 4 h of incubation.Cells in the control group showed nearly no fluorescence and the fluorescence intensity was only 1.26 ± 0.14(X104).However,obvious fluorescence was detected in the other three groups.The values of fluorescence intensity(X104)were 15.20 ± 0.15,20.59 ± 0.43 and 29.95 ± 1.10 for the group of free DOX,DOX-NPs M-and DOX NPs M+,respectively.M-MZNs-DOX have effective and magnetic-responsively targeting inhibition activity against Hep G2.The tumor site exhibits obvious enhancement on CT images and much lower SI on T2 W MRI after intratumorally injection of the nanocomposites.The turmor site was positively and negatively enhanced on CT and T2 WI images,respectively,3 h after injection.CT value of tumor increased from 44.35±4.35 HU before injection to 53.67±3.66 HU after injection(P<0.05),with an enhancement rate of 21.25±3.55% and SI decreased from 1116.63±197.27 before injection to 929.54±155.22 after injection(P<0.05),with a SI decrease rate of 16.64±1.56%.The contrast enhancement effect on both CT and MR imaging can be improved by the magnet placed on body surface near the tumor.CT value of tumor increased from 42.70±2.80 HU pre injection to 61.39±1.90 HU post injection(P<0.05),with an enhancement rate of 44.03±6.18%,and SI decreased from 1141.59±81.95 pre injection to 795.54±120.20 post injection(P<0.05),with a SI decrease rate of 30.59±5.74%.Blue dots staining Fe in the MNPs were clearly observed in tumor tissue after injection of M-MZNs-DOX and absent in the control group.Importantly,blue dots are significantly more in the tumor under magnetic field than that without the magnetic field.Besides,blue stains mainly existed in the liver,spleen,while they were absent in brain,heart,kidney and muscle tissues.M-MZNs-PEG showed no anti-cancer effect compared with the control group,while free DOX and M-MZNs-DOX exhibited tumor inhibition effect.Moreover,the therapeutic effect in the group of M-MZNs-DOX M+ was better than that in the previous groups.The final tumor volume in the control group was 4057.1 ± 166.4mm3,4028 ± 418.1mm3 in the M-MZNs group(P>0.05 versus control group),2833.7 ± 376.7mm3 in the DOX group(P<0.05 versus control group),2021.8 ± 265.7mm3 in the M-MZNs-DOX M-group(P<0.05 versus control group and DOX group),1286.9± 166.2mm3 in the M-MZNs-DOX M+ group(P<0.05 versus all the above groups).During the experiment span,body weights of the mice increase steadily with similar variation in mean among all the groups.As indicated by the result of TUNEL analysis,the relative apoptosis rate was 1.00 ± 0.24 in the control group,1.10 ± 0.27 in the NPs(P>0.05,versus control group),6.98 ±1.16 in the DOX group(P<0.05 versus control group),17.83 ±1.31 in the M-group(P<0.05 versus control group and DOX group),and;33.36499±3.20429 in the M+ group(P<0.05 versus all the above groups).No pathological damages is observed in all of the tissues from the major organs,including muscle,kidney,spleen,liver,lung,heart,and brain in all the groups,demonstrating good biosafety of M-MZNs-DOX.Conclusion: In this study,we developed a novel modified multiple cores-mesoporous shell MNP,M-MZNs through a facile “outside-to-inside” method.The as-made M-MZN possesses well nanoscale characterization.Such M-MZNs-PEG exhibits CT/MRI dual modal imaging,large drug payload capacity and could be used as a p H sensitive and magnetically responsive drug delivery for DOX.This novel nanodrug showed magnetic targeting CT/MRI dual modal imaging and anticancer effect in tumor bearing mice without obvious side effects.This nanoparticle delivery systems could enhance the efficacy of chemotherapy.Our work provides a new strategy for cancer theranostics using MNPs of dual modal imaging and magnetic targeting abilities. |
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