PVP-coated iron oxide nanoparticles (PVP-IOs) were synthesized by the thermal decomposition of iron carbonyl (Fe(CO)5
) in N,N-Dimethylformamide (DMF).37
The particle size of the PVP-IOs was modulated by controlling the ratio of PVP to Fe(CO)5
. A decrease in particle size was observed by transmission electron microscopy (TEM) when the PVP concentration increased from 0.07 to 0.33 g/mL. Under other fixed reaction conditions, the highest PVP concentration (0.33 g/mL) resulted in particles with the smallest particle size, approximately 7.6 nm (). As the PVP concentration decreased to 0.27, 0.18 and 0.07 g/mL, the average particle size changed to 23.4 (), 36.8 () and 65.3 nm (). For convenience, we labeled the samples as PVP-IO-8, PVP-IO-23, PVP-IO-37 and PVP-IO-65, respectively. PVP was immobilized on the surface of the IO nanocrystals via coordination interaction through its carbonyl group,38
rendering the IO nanoparticles dispersible in water, as well as preventing them from having uncontrolled growth.
(Left) TEM images of as-synthesized IO nanoparticles: a) PVP-IO-8, b) PVP-IO-23, c) PVP-IO-37, d) PVP-IO-65. (Right) Corresponding particle size histograms obtained by statistical analysis over ~150 particles.
The hydrodynamic diameters of PVP-IO nanoparticles in aqueous solution were determined by dynamic laser scattering (DLS) analysis (). Compared to the particle sizes observed in TEM images, the corresponding hydrodynamic diameters of PVP-IO nanoparticles are much larger. The observed hydrodynamic diameter changed from 32.2 to 70.7, 102.4 and 118.3 nm as the core size of nanoparticles increased from 8 to 23, 37, and 65 nm, respectively. All the PVP-IOs are slightly positively charged while Feridex is negatively charged as indicated by the zeta potential measurement (see ). These results are attributed to the hydrophilic polymer coating present on the surface, similar to the other reported formulations.11, 39–40
This biocompatible PVP coating may also enhance the blood circulation time and stabilize colloidal solution.41
Hydrodynamic diameter distributions of PVP-IO samples determined by dynamic light scattering (DLS) measurements.
Average nanoparticle size determined by TEM, average hydrodynamic diameter obtained by DLS, zeta potential, and corresponding R2 relaxation values.
To evaluate the T2 enhancing capabilities of the four PVP-IOs, aqueous solutions of the PVP-IOs at different Fe concentrations were investigated by T2-weighted MRI on a 7.0 T small animal MR scanner (GE Healthcare). T2-weighted phantom images decreased significantly in signal intensity with increasing Fe concentration, due to the dipolar interaction of the magnetic moments of the particles and protons in the water (), making the images darker. This behavior indicates that PVP-IOs generate MR contrast on T2-weighted sequences, and are promising T2 MRI contrast agents.
a) T2-weighted MR images of PVP-IOs in aqueous solution with various concentrations at 7 T, b) Graphs of 1/T2 against the Fe concentration for PVP-IOs, compared with Feridex.
shows the relaxation rates, 1/T2
, of PVP-IOs as a function of Fe concentration. The plots were well-fit by linear functions within the analyzed range of Fe concentration. It has been observed previously that relaxation rates vary linearly with Fe concentration:42
is the observed relaxation rate in the presence of magnetic nanoparticles, 1/T02
is the relaxation rate of pure water, and the product [Fe]R239
is the relaxation by the field inhomogeneities, i.e., the susceptibility effect induced by the magnetic ion ([Fe] is iron concentration and R2
is the transverse relaxation rate, which represents the efficiency of the magnetic nanoparticles as a contrast agent). The specific relaxivity coefficients (R2
values), determined by the slope of 1/T2
against Fe concentration, increase with increasing particle size, hence the R2
value of PVP-IOs can be expressed as:43–44
is a constant, dNP
is the diameter of the nanoparticle, D
is the diffusion coefficient, μ
is the magnetic moment of the nanoparticles, γ
is the gyromagnetic ratio of the water proton, CNP
is the concentration of the nanoparticles, and J
) is the spectral density function. According to the above equation, R2
is proportional to the magnetic moment μ2
. Although magnetism is an intrinsic property of bulk materials, the magnetic properties of nanopartilces are strongly dependent on their size, shape and surface properties.18, 45–47
Cheon and coworkers have systematically studied the relationships among size, magnetism and relaxivity of uniform-sized IOs. It was found that larger IOs have a larger magnetization and higher R2
relaxivity.18, 45, 47
Thus, PVP-IO-65 nanoparticles, having the highest magnetic moments, will presumably distort the magnetic field most efficiently, and cause the greatest enhancement of the R2
relaxation value. Indeed, as the PVP-IO size increased, R2
steadily rose from approximately 173.37 to 203.86, 239.98 and 248.89 mM−1
for PVP-IO-8, PVP-IO-23, PVP-IO-37 and PVP-IO-65, respectively. Notably, the R2
values for all PVP-IOs were found to be higher than that of the well-known commercial MRI contrast agent Feridex (127.48 mM−1
). The magnetic moment in IO is due to the localized electron density and hence strongly depends on the degree of crystallographic order.48
As the synthetic procedure of PVP-IOs was performed at relatively high temperature compared to the alkaline coprecipitation method for Feridex, an improved crystallinity can be obtained.37
This, in conjunction with the smaller core size and higher polydispersity of Feridex, explains the relatively higher relaxivities of PVP-IOs (see ).
An MTT assay using mouse macrophage cell line RAW264.7 was performed to evaluate the cytotoxicity of PVP-IOs (). PVP-IOs at six different concentrations, ranging from 0.4 to 250 μg Fe/mL, were incubated with macrophage cells for 24 h. The cell viability obtained by the MTT assay was expressed as a fraction of viable cells and normalized to that of cells without co-incubation with PVP-IOs (blank control). After incubation, the cell viability was maintained up to ~90% compared with the control. The MTT results indicate that the PVP-IOs showed little to no cytotoxicity even at the highest concentration (250 μg Fe/mL), which exceeds by one order of magnitude the concentrations of conventional iron-oxide-based-MRI contrast agents typically used in mice (1–20 mg/kg).49
Cell viability of macrophage cells treated with various concentrations of PVP-IOs and Feridex measured by the MTT assay.
To investigate the effect of size on cellular uptake, PVP-IOs solutions with various concentrations (0.4–250 μg Fe/mL) were co-incubated with macrophage cells for 1 h, and the quantitative amount of iron uptake was measured by ICP-AES. In , a plot of the amount of iron uptake by cells versus size of PVP-IOs showed that the cellular uptake was heavily dependent upon size, with the uptake of PVP-IO-37 > PVP-IO-65 > PVP-IO-8 > PVP-IO-23. The maximum uptake by cells occurred at a nanoparticle size of 37 nm, which was 1.3, 2.8 and 5.3 times the uptake of nanoparticles of size 65 nm, 23 nm, and 8 nm, respectively. Our result that 37 nm is the optimal size for cell uptake corresponds well to recent investigations of other particles. For instance, Chithrani et al.
found that 50 nm gold nanoparticles with surfaces modified by citric acid had higher cellular uptake rates than 14 and 74 nm nanoparticles.34
Lu et al.
also found that, for a range of 30–280 nm mesoporous silica nanoparticles, 50 nm particles had the maximum uptake by HeLa cells.35
Figure 5 (a) Cellular uptake of PVP-IOs and Feridex by macrophage cells at various concentrations with an incubation time of 1 h. (b) Cellular uptake of PVP-IOs and Feridex by macrophage cells as a function of incubation time at Fe concentration of 10 μg/mL. (more ...)
indicates that, in all the samples, the uptake of nanoparticles significantly increased in the first 4 h, but the uptake rate gradually slowed and reached a plateau at 4–8 h, depending on the size. This plateau effect is in agreement with a previous study by Chithrani et al.34
The average uptake rates during the first 4 h were 1.17, 1.36, 2.06, 1.56, 0.76 pg/cell per hour for PVP-IO-8, PVP-IO-23, PVP-IO-37, PVP-IO-65, and Feridex, respectively. Compared with other IOs, PVP-IO-37 exhibits the highest uptake rate. It has been well-documented that the cellular uptake of substances can be affected by a group of factors, such as size, shape, surface charge, roughness and functional groups on the surface.27–35
Size, in particular, was found to be a critical criterion, and can in large part decides the exact mechanisms by which nanomaterials get internalized. It is generally acknowledged that substances in the range of 10–30 nm can diffuse across plasma membrane freely or through membrane channels, while the larger particles are mainly carried into cell by pinocytosis or phagocytosis.30, 50
Even within the latter category, the internalization pathway can still be different. As pointed out by Conner and Schmid, there are at least three types of endocytosis for nanoparticles: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin- and caveolae-independent endocytosis.50
The detailed mechanism for the uptake of PVP-IOs is not clear at this stage. However, it is clear that the uptake is size-dependent, and among all the four tested formulas, PVP-IO-37 demonstrated the fastest and highest uptake. On the contrary, Feridex showed a much slower uptake rate, which we attributed to its high polydispersity and its slightly negatively charged coating (in comparison to the slightly positively charged PVP coating).
Iron oxide nanoparticles have been extensively developed for liver MR imaging. The working mechanism is that the IOs, after i.v. administration, are rapidly taken up by the hepatic Kupffer cells, resulting in a decrease of MR signal intensity by shortening proton T2
relaxation times in the neighborhood. To first assess the in vivo
MRI effects of PVP-IOs, we performed T2
-weighted MRI in normal, healthy Balb/c mice before and after administration of PVP-IOs. The intravenous dose was 2.5 mg Fe (measured by ICP-AES) per kg of mouse body weight, and a 7.0 T MRI apparatus was used to collect data from the liver. To quantify the contrast enhancement, regions of interest (ROIs) (Figure S1
) were selected on the T2
-weighted MR images of the liver (). The signal-to-noise ratio (SNR) was calculated according to the equation: liver signal-to-noise ratio SNRliver
, (where SI stands for signal intensity and SD stands for standard deviation) and the average relative liver signal intensities of mice (SNRpost
) were plotted at different time points (). Relative contrast enhancement was defined as signal decrease ΔSNR = (SNRpre
. Compared with the pre-images, the images taken 10 min after administration showed some hypointensities in the liver, which maximized at 1 h time point. ΔSNR values of the PVP-IO-37, PVP-IO-65 and Feridex were significantly larger than those of PVP-IO-8 and PVP-IO-23. It has been previously reported that the relaxivity of magnetic nanoparticles is size-dependent, which grows as particle size increases.18, 45, 47
On the other hand, the cellular uptake of nanoparticles, also being size-dependent, maximizes at certain value, as reported by other groups34–35
and observed in the current study. Both factors are believed to play a role in determining the liver contrast enhancement. The relatively lower contrast enhancement of smaller nanoparticles (PVP-IO-8 and PVP-IO-23) is likely attributed to their rapid depletion from the blood stream, their lower uptake into Kupffer cells, and their naturally smaller relaxivities, while the outperformance of PVP-IO-37 is due to the close-to-maximum R2
and the best macrophage engulfment. The Prussian blue staining results confirmed that the “darkening” in the liver was caused by IO nanoparticle accumulation (Figure S2
In vivo mouse liver MR images at different time points after intravenous administration of PVP-IOs and Feridex at a dose of 2.5 mg/kg.
Quantification of relative SNRliver collected before and after administration of PVP-IOs in normal, healthy Balb/c mice (n = 3/group), compared with Feridex, at the dose of (a) 2.5 mg Fe/kg and (b) 1.0 mg Fe/kg.
We then looked at the in vivo
effects of Feridex on MRI. Prior research has shown that clearance by the RES cells in the liver and spleen of rats becomes saturated when more than 1015
particles are injected in a single bolus.51
To avoid saturation, we injected at a dose of 1.0 or 2.5 mg Fe/kg, a dose which is at least 100 times less concentrated (with the assumption that the organ weight of mice is approximately one-tenth that of rats). At 1 h after administration, the ΔSNR of PVP-IO-37 was slightly higher than those of PVP-IO-65 and Feridex at both doses (). However, the advantage is not as dramatic compared with their difference in uptake at the in vitro
level. Such a result was attributable to the complicated in vivo
environment. During circulation, the particles may tangle with serum proteins or form aggregates, both of which will lead to an increased overall particle size and defy them from the predicated behavior. Nonetheless, the significant signal intensity (SI) changes induced by PVP-IO-37 promise its use in identifying small focal hepatic lesions, including tumor metastases.
To evaluate the efficacy of the PVP-IOs in enhancing MRI contrast in hepatic lesions, in vivo
MR images were evaluated with nude mice bearing orthotopic Huh7 liver cancer before and after administration of PVP-IO-37 and Feridex, with a dose of 1.0 mg Fe/kg of body weight (). Since hepatic tumors either do not contain RES cells or the activity of their RES cells is reduced, they do not accumulate nanoparticles as efficiently as normal tissue.52–53
Thus the tumor cells appear as bright spots on a T2
- or T2
*-weighted image, against surrounding normal tissues which undergo particle accumulation and are manifested as hypointensities. Regions of interest were selected around the tumor and liver parenchyma for measurement of the signal intensity. Contrast-to-noise ratio (CNR) was defined as CNR = (SNRtumor
with SNR = SImean
, where SI denotes the tumor or liver intensity and SD is the standard deviation of the noise in the image. The increase in tumor-to-liver contrast is defined as ΔCNR = (CNRpost
. We observed, at the 1 h time point, a contrast change (ΔCNR) of 81 ± 8% with the commercial Feridex, which was lower than that of the PVP-IO-37 (94 ± 6%) (). This was consistent with the observation from normal mice. As discussed above, compared with the surrounding normal liver tissue, tumor tends to have a low IO nanoparticle uptake. Such a difference caused a less dramatic signal drop in the tumor area and was manifested as a contrast between tumor and normal tissue on T2/T2*-weighted MR images.
MR images of Huh7 orthotropic liver cancer model at different time points after intravenous administration of PVP-IO-37 and Feridex (1 mg Fe/kg). Arrows indicate the pseudo-positive contrast from the tumor.
Mean values of relative CNR in MR images on orthotropic liver cancer model before and after administration of PVP-IO-37 nanoparticles, compared with Feridex, at the dose of 1.0 mg Fe/kg (n = 3/group).