| Purpsoe:FA-BSA modified polymeric magnetic micelles are investigated as a specific contrast agent for tumor targeting and magnetic resonance imaging in vitro and in vivo.Methods:1. reparation:(1) Mono-dispersed SPIONs were synthesized by chemical co-precipitation and modified with oleic acid.(2) The cationic amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers were synthesized by free radical polymerization.(3) Cationic polymeric magnetic micelles denoted as unmodified magnetic micelles were prepared by self-assembly.(4) FA-BSA modified magnetic micelles were synthesized by functionalizing the cationic magnetic micelles with FA-BSA by electrostatic complexation.2. Characterization:(1)1H NMR was conducted to investigate the chemical structure of the amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers. The structures of the SPIONs, amphiphilic poly(HFMA-co-MOTAC)-g-PEGMA copolymers, and FA-BSA modified magnetic micelles were assessed by FTIR.(2) The morphology of the magnetic micelles was examined by TEM at200kV. The hydrodynamic size and size distribution were measured using DLS.(3) The thermogravimetric analysis was performed on the Perkin Elmer TGA-7. The magnetic properties were studied on a VSM at298K under an applied magnetic field.(4) The transverse relaxivity (r2) of the magnetic micelles was determined using a3.0-T whole body MR scanner in combination with an8-channel wrist joint coil and tested by T2-weighted multi-echo spin echo sequence.3. In-vitro studies:(1) The cytotoxicity of the magnetic micelles was estimated by means of the MTT assay.(2) Cell uptaken studies:The human hepatoma cell lines Bel-7402(FR-positive) and Hep3B (FR-negative) were incubated with the FA-BSA modified magnetic micelles or unmodified magnetic micelles dissolved in DMEM at different iron concentrations. The unmodified magnetic micelles were used as controls. The uptake of both the FA-BSA modified and unmodified magnetic micelles is evaluated by different methods including MR imaging, intracellular iron content determination, and iron-specific Prussian blue staining. The uptaken results were carried out quantitative, qualitative and pathological analysises, respectively.4. In-vivo studies:(1) Establishment of human hepatoma xenografts in nude mice.(2) In vivo MRI:Different contrast agents including the T1contrast agent Gd-DTPA, T2contrast agents Feridex, unmodified magnetic micelles, and FA-BSA modified magnetic micelles were used via tail vein administration, in order to investigate the feasibility of the FA-BSA modified magnetic micelles in specific MR imaging of tumors. The first three were used as control groups.(3) Histochemistry analysis:the magnetic micelles in the liver and tumor tissues of the aforementioned tumor-bearing nude mice were evaluated by Iron-specific Prussian blue staining.(4) Tumor iron content determination:The uptake percentages of the unmodified and FA-BSA modified magnetic micelles in tumor tissue were determined by Spectr AA240FS at24h after tail vein administration.Results:1. The unmodified magnetic micelles and FA-BSA modified magnetic micelles were prepared successfully.2. Characterization:(1) The results of’H NMR and FTIR confirmed the chemical structure and construction of unmodified magnetic micelles and FA-BSA modified magnetic micelles.(2) Both of unmodified and FA-BSA modified magnetic micelles were spherical core-shell structures tested by TEM. The average hydrodynamic particle size of unmodified and FA-BSA modified magnetic micelles tested by DLS were about161.5nm (PDI=0.26),196.1nm (PDI=0.26), respectively.(3) The content in the unmodified and FA-BSA modified magnetic micelles were calculated to be27.5wt%and19.2wt%, respectively. The saturation magnetization values of the unmodified and FA-BSA modified magnetic micelles were13.9emu/g and5.5emu/g, respectively.(4) The transverse relaxivity (r2) of the unmodified and FA-BSA modified magnetic micelles tested by multi-echo T2-weighted MR imaging were243.9mM-1s-1and179.5mM-1s-1, respectively.3. In-vitro studies:(1) Cell viability of over80%was maintained from the human normal liver cell line HL-7702after incubation for24h and48h by increasing the iron concentration from25μg/mL to100μg/mL, indicating that both the FA-BSA modified and unmodified magnetic micelles have no obvious cytotoxicity and are suitable probes in biomedical applications.(2) Cell uptaken results:Both FR-positive Bel-7402cells and FR-negative Hep3B cells showed enhanced cellular uptake of FA-BSA modified magnetic micelles comparing with unmodified magnetic micelles. But the average uptake rate of FA-BSA modified magnetic micelles in Bel-7402cells was higher than Hep3B cells. The uptake of unmodified magnetic micelles in Bel-7402was similar to that in Hep3B cells. The average uptake percentages of unmodified magnetic micelles by Hep3B cells and Bel-7402cells were calculated to be (6.17±0.19)%,(6.43±0.45)%, respectively. The average uptake percentages of FA-BSA modified magnetic micelles by Hep3B cells and Bel-7402cells were (8.83±0.41)%,(12.07±1.29)%, respectively.4. In-vivo studies:(1) The T2signal intensity of tumor in the group treated with the FA-BSA modified magnetic micelles decreases most significantly between1h to24h. While the tumors with Feridex and unmodified magnetic micelles show minimal contrast change in the same period.(2) Prussian blue staining performed on the group with Feridex shows large accumulation of magnetic particles in the liver, whereas the group with unmodified magnetic micelles shows less blue spots. In contrast, the FA-BSA modified magnetic micelles do not accumulate much in the liver. Prussian blue staining of the tumor tissues was performed at24h post-injection of contrast agents. Accumulation of blue spots in the tumor tissues observed from the folate-targeted groups is obviously larger than that of untargeted groups. There are a small number of blue spots trapped in the tumor vascularity in the groups of Feridex and no visible blue spots in the groups of Gd-DTPA.(3) Tumor iron content determination: The uptake percentages of the unmodified and FA-BSA modified magnetic micelles in tumor tissue were calculated to be (1.54±0.041)%,(2.91±0.035)%, respectively.Conclusions:1. Polymer magnetic micelles and FA-BSA modified magnetic micelles are successfully synthetized.2. Both In vitro and in vivo studies demonstrate that FA-BSA modified magnetic micelles have excellent MR imaging effect and high selectivity and sensitivity to FR-positve hepatoma cells and tissue, indicating their potential use as a negative targeted MRI contrast agent for folate-receptor overexpressing cancer.3. The unmodified magnetic cells also maintain good tumor targeting and MR imaging ability. |
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