Amine functionalized PEG-coated nanoparticles (NP-PEG-NH2
) were prepared by a process described in our previous work.
Nanoparticles synthesized by this method exhibited a core size of 10-15 nm by TEM and were stable in phosphate buffered saline (PBS), without flocculating, for several months.
Biocompatible PEG serves as a coating to reduce protein adsorption and non-specific macrophage uptake, ultimately prolonging serum half-life in vivo
Well-established for its non-fouling properties, PEG is FDA-approved and employed in a wide variety of biomedical applications.
Here PEG also serves as a linking agent providing terminal functional groups for the conjugation of ligands, such as CTX and potential therapeutic agents with iron oxide nanoparticles. The number of reactive amine functional groups available for conjugation of ligands was determined to be ~30 per nanoparticle by quantification of pyridine-2-thione following reaction with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP).
The conjugation of the targeting agent CTX, a recombinant peptide originally purified from the venom of the Leiurus quinquestriatus
scorpion, was performed through a three-step reaction illustrated in . Initially, a free sulfhydryl group was grafted to CTX through modification with 2-Iminothiolane-HCl (Traut's Reagent; ), as opposed to the heterobifunctional linker, N-succinimidyl-S-acetylthioacetate (SATA) which was previously utilized at this stage. NP-PEG-NH2
was then reacted with succinimidyl iodoacetate (SIA) to yield sulfhydryl reactive nanoparticles (NP-PEG-SIA; ). Upon combination of the thiol-modified CTX with the NP-PEG-SIA (), stable thioether linkages were formed between the nanoparticle and the peptide. The resulting NP-PEG-CTX nanoprobe was purified by GPC chromatography and stored in PBS for subsequent use. The improved synthetic procedure eliminates the SATA deprotection and purification processes and produces higher yield of products, substantially reducing overall processing time and potential contamination.
Figure 1 Illustration of CTX conjugation to PEG-amine coated nanoparticles. Chemical reaction scheme for (a) adding a free sulfhydryl reactive group to CTX via Traut's Reagent, (b) iodoacetate functionalization of NP-PEG-NH2, and (c) formation of a thioether linkage (more ...)
Nanoparticle surface modification and CTX conjugation were confirmed by Fourier transform infrared spectroscopy (FTIR). IR spectra of (a) bare iron oxide nanoparticles, (b) NP-PEG-NH2
, and (c) NP-PEG-CTX are shown in . The spectrum for the bare nanoparticles (a) exhibits peaks characteristic of iron oxide, most notably that of -OH found on the oxide surface at 3400 cm-1
. Following surface modification with the amine-terminal PEG silane (b), methylene peaks at 2916 and 2860 cm-1
were observed. Successful PEG attachment is further confirmed by the carbonyl bands at 1642 and 1546 cm-1
which correspond to the amide bonds within the structure of the bifunctional PEG silane.
The peak at 1105 cm-1
corresponding to the Si-O bond confirms the bonding between the silane and the nanoparticle. Following CTX conjugation (c), the methyl symmetric/asymmetric stretch located at 2960 cm-1
corresponding to the alanine residues of the CTX is observed. The broad peak at 1631 cm-1
indicates that both primary and secondary amines in residues such as lysine and arginine are present. Additionally, the peaks at 1422 cm-1
and 1380 cm-1
indicate carbonyl and C-C stretching, respectively, found in the peptide.
FTIR spectra of (a) bare nanoparticles, (b) NP-PEG-NH2, and (c) NP-PEG-CTX.
The targeting specificity of NP-PEG-CTX for glioma cells was assessed by the intracellular uptake of this nanoprobe in comparison to a non-targeting NP-PEG-SIA control. 9L cells were incubated with the nanoprobes at a concentration range of 0-150 μg Fe/ml for 2 hrs at 37°C. Nanoprobe uptake was quantified by intracellular iron content determined by a colorimetric ferrozine-based assay.
displays the intracellular iron content per cell after incubation. Both nanoprobes exhibited concentration dependent uptake that tended to saturate at high particle concentrations. Significantly higher intracellular iron content was observed in cells incubated with the NP-PEG-CTX than those incubated with NP-PEG-SIA at all nanoprobe concentrations indicating the preferential binding of the NP-PEG-CTX targeting probe to 9L cells. An approximate 10-fold increase in preferential uptake was observed at the highest nanoprobe concentration tested (150 μg Fe/mL). The minimal NP-PEG-SIA uptake by 9L cells at the highest nanoprobe concentration, is believed to be attributed to the PEG coating on nanoparticles, which has been shown to facilitate nanoparticle internalization into cancer cells.[22,27]
Uptake of nanoprobes by 9L cells as determined by ferrozine assays.
Localization of nanoprobes in the cells was visualized with TEM () which shows 9L cells incubated with either NP-PEG-SIA (a) or NP-PEG-CTX (b) at a concentration of 100 μg Fe/mL for 1 hr at 37°C prior to fixation for microscopy. Consistent with the nanoparticle uptake assay (), no appreciable NP-PEG-SIA nanoparticle accumulation was observed in the 9L cells (). In comparison, NP-PEG-CTX nanoprobes were clearly observed in the cytoplasm of the cells (). The presence of these nanoprobes in the lysozomes indicates the nanoprobes were internalized by the cells after binding to the membrane surface. This result is consistent with the receptor-mediated endocytosis of CTX upon preferential binding to MMP-2.
The high-magnification image reveals that the nanoprobes maintain their original morphology and size within the cytoplasm of the cells. The internalization of the nanoprobe suggests their ability to provide persistent MRI contrast enhancement as well as their potential to serve as a carrier for drug delivery.
TEM images of 9L cells incubated with (a) NP-PEG-SIA and (b) NP-PEG-CTX.
We then examined the efficacy of CTX-conjugated nanoprobes to target glioma cells and provide contrast enhancement for MRI. MRI phantom experiments were performed using 9L cells exposed to the nanoprobes under the same conditions described above. After cell culture, cells were suspended and encased in an agarose mold at a concentration of 1 × 10-7 cells/mL for MR imaging. A series of MR images were acquired using a conventional spin-echo pulse sequence on a 4.7T spectrometer, with varying echo times (TE) to construct an R2 (1/T2) relaxivity map for each sample. T2-weighted MR images and R2 color maps of samples containing 9L cells incubated with various concentrations of the nanoprobes are shown in . Compared to the tumor cells incubated with the non-targeting NP-PEG-SIA (), cells incubated with the NP-PEG-CTX () displayed a significant negative contrast enhancement (darkening) and higher R2 relaxivity at each of the nanoprobe concentrations tested. The signal enhancement observed in the cells incubated with the targeting nanoprobe is consistent with the uptake results describe above. The R2 relaxivity determined from the slope of the linear fit for cells cultured with the NP-PEG-CTX nanoprobe (5.20 ms/mmol) displayed a ~24-fold higher relaxation rate than that observed for the cells incubated with the non-targeting NP-PEG-SIA (0.22 ms/mmol) further signifying the targeted contrast enhancement of the targeting NP-PEG-CTX.
Figure 5 T2-weighted MR phantom images and corresponding R2 relaxivity maps of 9L cells incubated with (a) NP-PEG-SIA and (b) NP-PEG-CTX. R2 values plotted as a function of incubation concentration display a near linear correlation consistent with the nanoprobe (more ...)
The efficacy of the nanoprobe to specifically target gliomas and provide MRI contrast enhancement in vivo
were evaluated with nude mice bearing 9L xenograft tumors. The specificity of the nanoprobes was evaluated by comparing the contrast enhancement of the tumor regions of mice receiving NP-PEG-CTX and those receiving NP-PEG-SIA at the same concentration, as well as comparing the contrast enhancement between tumor and normal tissue regions. Initially, anatomical images were acquired in the coronal and sagittal planes to determine the tumor position along the flank of the animal, as shown in and , respectively. Axial cross-sectional images were then acquired through the tumor region using multi-spin echo pulse sequences to obtain a series of images over a range of TEs. Each mouse was imaged prior to, and at various time points after, intravenous injection of the nanoprobes at a dosage of 6 mg Fe/kg. R2 relaxivity maps were then generated to quantify contrast enhancement resulting from the accumulation of the nanoprobes. The preferential targeting of the glioma tumor in mice receiving the NP-PEG-CTX (), in comparison to NP-PEG-SIA (), was evident in MR images acquired 3 hrs post-injection. Tumor contrast enhancement in the superimposed R2 change map displays a significantly higher intensity and a more thorough enhancement of the overall tumor in the mouse injected with NP-PEG-CTX than in the mouse receiving the NP-PEG-SIA non-targeting probe. The inhomogeneous distribution of the nanoprobes observed in the tumor is due to heterogeneous nature of this type of tumor which is consistent with other reported studies.[28,29]
Slight accumulation of the NP-PEG-SIA in the tumor is believed due to the enhanced permeability and retention (EPR) effect common for nanoparticles[30,31]
and nonspecific uptake by glioma cells at the tumor margin.[32,33]
This result also suggests that although a non-targeting nanoprobe may accumulate in tumors through the passive uptake by cells, as demonstrated in other nanoparticle systems,[5-7,29,32,33]
targeting nanoprobes can significantly increase the particle accumulation and thus the contrast enhancement. Importantly, targeting nanoprobes can differentiate between tumor and muscle cells and thus preferentially accumulate in tumor over normal tissue regions as demonstrated below. This not only enhances the identification of tumor regions for more thorough surgical resection and reduces collateral damage to surrounding healthy tissue, but also allows for potential targeted drug delivery at low-dosage administration reducing toxic side effects.
Figure 6 Representative images from three independent experiments with similar results. MRI anatomical image of a mouse in the (a) coronal plane with the dotted line displaying the approximate location of the axial cross sections displayed in (c) and (d). Anatomical (more ...)
The accumulation and retention of the targeting nanoprobe in tumor and surrounding normal tissue were evaluated at intervals of 0, 0.3, 2, 12, and 24 hrs post-injection to quantify the nanoprobe accumulation and define the time window when animals can be imaged to achieve the maximum MRI contrast, as well as to gain an understanding of the pharmacokinetics of this nanoprobe. This was done by monitoring the change in R2 in tumor () and normal () tissues over time. Nanoprobe accumulation in the tumor increased sharply upon injection of the NP-PEG-CTX nanoprobe and up to a maximum at the 12 hr time point resulting in R2 change of ~16 ms-1. At 24 hrs, the change in R2 for the tumor region dropped to ~10 ms-1. The result indicates that the nanoprobe has a prolonged blood half-life sufficient to reach and accumulate in the target tumor prior to clearance by the liver or spleen and that the best imaging window is 2-25 hrs post-injection. The prolonged retention of the nanoprobe in tumors (in tens of hours) may prove to be particularly valuable in clinical settings. Compared to tumors, adjacent muscle showed only a maximum change of less than 5 ms-1 in R2 that occurred also at 12 hr imaging time point, which is significantly less than the R2 change in tumor. These results further support the specificity of these nanoprobes for glioma tumors and correlate well with the results obtained from the in vitro assays shown above.
Figure 7 Change in R2 relaxivity in (a) tumor and (b) normal tissue regions in mice injected with NP-PEG-CTX over a time course of 24 hrs post-injection. Mean values were obtained from three independent experiments with the error bars displaying the standard deviation (more ...)
Finally, we performed histological analysis on tissues obtained from various clearance organs (kidney, spleen, and liver), in which nanoprobe accumulation was expected to occur, to investigate for signs of acute toxicity. Tissues were harvested 2 days after injection of the nanoprobes and fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Tissue sections () were reviewed by a pathologist with expertise in veterinary pathology for evidence of tissue toxicity. No apparent toxicity was observed in the tissues from the animals receiving the nanoprobes in comparison to mice receiving no injection. Furthermore, mice injected with nanoprobes underwent physical and neurologic evaluations to detect any acute toxicity associated with the administration of the nanoprobes. No differences in eating, drinking, grooming, exploratory behavior, activity, physical features (e.g., coat quality), or neurologic status were observed between the mice injected with nanoprobes and the mice receiving no nanoprobes.
H &E stained tissue sections from mice receiving no injection (a,b,c) and mice injected with NP-PEG-SIA (d, e, f) or NP-PEG-CTX (g, h, i) 2 days post-injection. Tissues were harvested from kidney, spleen, and liver, respectively.