Targeted new peptide based nanoparticles toward high EGFR expressing cancer cells for MRI

In this study, the various core sizes of manganese ferrite nanoparticles(MnFe2O4) conjugated with D4 peptide (MnFe2O4-PEG-D4) were synthesized. The highrelaxivityMnFe2O4 nanoparticles were obtained by thermal decomposition of Iron acetylacetonate and manganese acetylacetonate in hydrophobic solution athigh boiling process. The surface ofMnFe2O4 nanoparticles were coated with polyethylene glycol (PEG) and EGFR peptide ligand (D4:Leu-Ala-Arg-Leu-Leu-Thr) to improve their dispersion and ability to target EGFR. The negative signal enhancement of EGFR expressing cancer cells(SKBR-3 and PC-3) were significantly higher than that of low EGFR expressing cells (HEK-293).IntroductionMolecular Imaging has recently been developed very rapidly and extensively in biotechnology [1]. Tumor-targeted drug delivery can enhance the effectivenessof therapy while decreasing the systemic toxicity of these drugs. We synthesizedMnFe2O4 nanoparticles for MR imaging are coated with polyethylene glycol,which has been frequently used as a drug carrier because of its biocompatibility, water solubility and their ability to escape capture by macrophages.MnFe2O4-PEG conjugate with D4 peptide substrate (Fig 1.) were synthesized to examine the binding affinity of EGFR using in vitro MR imaging.MethodsIron acetylacetonate(Fe(acac)3) and manganese acetylacetonate (Mn(acac)2) precursors were mixed in a molar ratio of 2:1, and reacted in benzyl ether solventcontaining oleic acid, oleylamine and, 1,2-hexadecanediol at 300°C for 1 hour to produce manganese ferrite nanoparticles (MnFe2O4). The MnFe2O4nanoparticles have various core sizes were synthesized through the same procedure while the amount of solvent was controlled. We tested two cancer cell lineswith different levels of EGFR expression: SKBR-3 and PC-3. In addition, we had chosen HEK-293 cells as negative cell for control which lacks of EGFRreceptors. All cells were incubated with MnFe2O4-PEG-D4 (0.5 mM Fe), washed by PBS buffer and scanned by 3.0 T MRI.Results and DiscussionThe manganese ferrite nanoparticles(MnFe2O4) was synthesized and characterized by TEM (Fig 2.), SQUID, and FT-IR. Moreover, the TEM results showedthat the well-dispersed nanoparticles which coated with PEG. The detection of saturation magnetization by SQUID magnetometry demonstrated thatMnFe2O4-PEG-D4 has high saturation magnetization (84 emu/g) and small coercivity (about 5 G). The internalization of MnFe2O4-PEG-D4 nanoparticles intopositive cells was confirmed by in vitro MR imaging study (Fig 3.). With theMnFe2O4-PEG-D4 conjugates, the detection of the SKBR-3 cell line occurredwith a noticeable negative enhancements(T2-weighted MR images). As the relative EGFR expression level increased, the MR contrast increased consistently.The signal intensity of positive cells in the present ofMnFe2O4-PEG-D4 is significantly lower than that of negative cells. No signal intensity change wasobserved for the negative cells in the presence and absence of MnFe2O4-PEG-D4.ConclusionWe have successfully synthesized and characterizedMnFe2O4-PEG-D4 nanoparticles, which was shown well-dispersed, high relaxivity and high saturationmagnetization. Moreover, we have demonstrated the D4 peptide substrate that was conjugated toMnFe2O4 nanoparticles and shown to enhance binding to andentry into EGFR expressing cells by MR imaging studies. References[1] Reynolds, F.; Loughlin, T.; Weissleder, R.; Josephson, L. Anal. Chem. 2005, 814-817.[2] onghai, L.; Ruijiao, Z.; Xianghua W.; FASEB J, 19, 2005, 1978-85.Figure 2. Structure formula of MnFe2O4-PEG-D4 Figure 1. The TEM images of MnFe2O4 nanoparticles (A),enlarged images ofMnFe2O4 nanoparticles 12.4 ± 0.9 nm (B).Figure 3. T2-weighted images of positive and negative cells after the treatment with or without o.5 mM MnFe2O4–PEG-D4. (a) SKBR-3 cells (b) PC-3 cells (c) HEK-293 cells. Upper: cells treatment without contrast agent. Lower: cells treatment with contrast agent. Proc. Intl. Soc. Mag. Reson. Med. 18 (2010)3751