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Superparamganetic iron oxide-based contrast agents in magnetic resonance imaging (MRI) have offered new possibility for early detection of lymph nodes and their metastases. According to important role of nanoparticle size in biodistribution, magnetite nanoparticles coated with different polyethylene glycol (PEG) concentrations up to 10/1 PEG/iron oxide weight ratio in an ex situ manner. To predict the PEG-coated nanoparticle behavior in biological media, such as blood stream or tissue, colloidal stability evaluation was performed to estimate the coating endurance in different conditions. Accordingly, optical absorbance measurements were conducted in solutions with different values of pH and NaCl concentrations. The results indicated that at neutral pH condition, nanoparticles treated by 3/1 ratio possessed better stability parameters. Investigating at high pH of 10 resulted in superior stability for bare magnetite nanoparticles due to its higher electrophoretic mobility. Coating material was attacked at acidic solutions which cause samples with higher PEG weight ratio to be settled slower. In various ionic strengths of 10−5 to 0.1 M, 3/1 ratio samples offered greater resistivity to sedimentation. The nanoparticles were further investigated by exposure to L929 cell and following up the iron uptake within cells. Finally, detection sensitivities in lymph nodes were evaluated. Particle uptake and the most signal reduction for in vivo MRI studies were also obtained by nanoparticles acquiring lower PEG contents that showed better colloidal stability.