Results & Discussion
The PARACEST agent, Eu-DOTA-Gly4, was coupled to the G5PAMAM dendrimer by using Eu-DOTA-Gly4 NHS ester, to perform an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/NHS coupling method to form the PARACEST dendrimer Eu-G5PAMAM with a moderate 17% yield (Figure 1). The mass spectrum showed that an average of 42 Eu-DOTA-Gly4 ligands were conjugated to the G5PAMAM dendrimer. Multiple carboxylates of the Eu-DOTA-Gly4 ligands were activated during synthesis, which may have led to the conjugation of one Eu-DOTA-Gly4 ligand to multiple amine end groups of the dendrimer. An average of 42 Eu-DOTA-Gly4 ligands per dendrimer suggests that each Eu-DOTA-Gly4 was conjugated to three amine end groups. This mass spectrum also showed a low, broad feature at approximately twice the mass-to-charge ratio of the main peak, which indicated a low level of dimerization of some dendrimers. This dimerization may have resulted from activation of multiple carboxylates of the Eu-DOTA-Gly4 ligands, although the low level of dimerization suggests that the dilute conditions during synthesis minimized this problem.
The fluorescent agent, DyLight 680, was coupled to Eu-G5PAMAM, to form DyLight 680-Eu-G5PAMAM, with a high 93% yield. The mass spectrum showed an average of one DyLight 680 ligand per dendrimer. The same procedure was used to couple DyLight 680 to a G5PAMAM dendrimer without the PARACEST agent, which also resulted in an average of one fluorophore per dendrimer.
In vitro CEST MRI
The dual-modality contrast agent, DyLight 680-Eu-G5PAMAM, showed a large CEST effect at +55 ppm (Figure 2A). The CEST spectrum of DyLight 680-Eu-G5PAMAM was identical to the CEST spectra of Eu-G5PAMAM [ALI ET AL., UNPUBLISHED DATA] and was comparable to Eu-DOTA-Gly4. Small 'bumps' at approximately -50, -30, 30 and 70 ppm in the CEST spectrum were attributed to pulse imperfections that the authors have observed in similar CEST MRI studies. CEST from the primary amines and amides within the dendrimer may have also created CEST at these large chemical shifts. However, these other chemical groups would have to possess very slow or very fast exchange rates to create small 'bumps' relative to the large CEST effect at +55 ppm, which seems unlikely.
The chemical exchange saturation transfer effect of DyLight® 680-Eu-generation 5 polyamidoamine. (A) The spectrum shows a CEST effect at +55 ppm. (B) The CEST magnetic resonance images of DyLight 680-Eu-G5PAMAM show a contrast dependence on concentration. (C) A Hanes-like linear analysis method shows that CEST of DyLight 680-Eu-G5PAMAM fits a two-pool model, Hanes-like fitting shown as a green line). For comparison, the Hanes-like analysis of Eu-G5PAMAM as reported in  is also shown. (D) The CEST dependence on concentration determined from the Hanes-like analysis for DyLight 680-Eu-G5PAMAM and Eu-G5PAMAM as reported in  are the same (the green line exactly overlays on the blue line).
CA: Contrast agent; CEST: Chemical exchange saturation transfer; G5PAMAM: Generation 5 polyamidoamine; M0: Selective saturation at -55 ppm; MS: Selective saturation at +55 ppm.
The concentration dependence of CEST for this agent (Figure 2B) showed an excellent fit to a two-pool model using a Hanes-like linear analysis method (Figure 2C & D). The PARACEST dendrimer, Eu-G5PAMAM, also showed CEST at +55 ppm and a similar fitting to the Hanes-like method (as determined from). These results demonstrated that the presence of the fluorescent agent on DyLight 680-Eu-G5PAMAM did not affect the agent's ability to generate CEST. In addition, Eu-G5PAMAM and DyLight 680-Eu-G5PAMAM each generate a 1% CEST effect with 14 µM on a PARACEST agent basis. For comparison, 530 µM of the small molecule PARACEST agent, Eu-DOTA-Gly4, is needed to generate a 1% CEST effect under identical conditions (as determined from). Therefore, coupling 42 PARACEST agents to the dendrimer resulted in a 38-fold improvement in sensitivity (on a per dendrimer basis), which shows that the CEST effect almost exactly scaled with the concentration of the agent on the dendrimer.
In vitro Optical Imaging
Conjugation of Dylignt 680 to Eu-G5PAMAM shifted the absorption maximum (labs) and fluorescence emission maximum (lem) of the fluorescent agent by 10 nm, relative to the corresponding bands for the free dye (Figure 3). This red shift is consistent with conjugation of a fluorescent dye to a nanocarrier. Quenching was not observed from the fluorescence emission of DyLight 680 in DyLight 680-Eu-G5PAMAM, relative to the fluorescence emission of the free dye. This result confirmed that potential fluorescence quenching originating from Eu(III) was negligible, therefore, the presence of the PARACEST agent on DyLight 680-Eu-G5PAMAM did not affect the agent's ability to generate fluorescence.
Emission fluorescence spectra. Conjugation of DyLight® 680 to the G5PAMAM dendrimer caused a 10-nm red shift in the maximum emission wavelength. The dual-modality contrast agent DyLight 680-Eu-G5PAMAM also showed a 10-nm red shift, which demonstrated that the presence of the paramagnetic chemical exchange saturation transfer agent on the dendrimer did not effect the emission wavelength.
G5PAMAM: Generation 5 polyamidoamine.
In vivo CEST MRI
An anatomical MR image showed the location of the U87 glioma (Figure 4A). The CEST MR image contrast before and after the administration of the PARACEST agents was used to determine the dynamic change in % CEST during the study (Figure 4B). Based on image noise, a CEST effect of ≥5.2% had a 95% probability that the CEST effect was real. The MR images prior to injection showed no significant CEST effect, but this significance threshold was exceeded immediately after injection of the agent. A strong CEST effect was first visualized at the tumor rim 2.8 min after injection, which can be attributed to the hypervascular rim typically observed in malignant glioma tumors. The CEST effect persisted during the remainder of the MRI study, which was attributed to the enhanced permeability and retention effect that is typically observed with nano-sized agents in glioma tumors. For comparison, the contralateral brain tissue did not show a statistically significant CEST effect throughout the MRI scan session. The temporal results shown in Figure 4C demonstrate variability in the baseline measurement prior to injection, which was attributed to pulse imperfections. This variability was greater after injection of the agent, which was attributed to motion artifacts (despite the use of a stereotactic holder) and pulse imperfections. This 'noise' in Figure 4C has been observed in similar in vivo CEST MRI studies.[7,8,13,17] This result indicated that CEST MRI could detect the presence of the dual-modality contrast agent in the glioma, which detected the glioma with excellent specificity, relative to normal brain tissue.
In vivo chemical exchange saturation transfer MRI. (A) The left panel shows an anatomical image that identifies the location of the tumor prior to administration of the agent. The right panel highlights the location of the tumor. (B) Paramagnetic CEST maps detected the agent in the U87 tumor but not in the contralateral tissue. The maps are labeled with the time-point relative to injection. (C) The temporal change in CEST shows rapid accumulation and persistence of the agent in the U87 tumor. CEST was greater than the 95% probability threshold in the U87 tumor, but was less than the probability threshold for the contralateral tissue.
CEST: Chemical exchange saturation transfer.
This experimental approach only required the acquisition of CEST MR images at one saturation frequency, which simplified the acquisition method relative to approaches that require multiple saturation frequencies. This approach also accounted for static effects that influence the contrast of a MR image with selective saturation, including endogenous magnetization transfer, direct saturation of water, and B0 and B1 magnetic field inhomogeneities. However, the use of one saturation frequency cannot account for other dynamic changes caused by the agent. For example, macrocyclic Eu(III) chelates have recently been shown to have a significant T2ex rate that can cause dynamic darkening of the image. Although Eu(III) chelates have very low T1 relaxivities, the high ratio of Eu(III) chelates per dendrimer may compensate for low T1 relaxivity and may possibly cause a change in image contrast. Yet the statistically significant, dynamic changes in image contrast after injection still ensure that the MRI method detected the agent.
To elucidate the influence of T1, T2ex, magnetic susceptibilities and magnetization transfer on CEST MRI, a complimentary analysis method is needed that can accurately determine the concentration of the agent without also being influenced by these other physicochemical effects. Biosensor imaging of redundant deviation in shifts (BIRDS) has been used to detect lanthanide chelates using chemical shift imaging, and has outstanding potential to provide this complimentary analysis to further evaluate CEST. BIRDS has been employed to simultaneously measure temperature and pH during a single in vivo study, which provides additional information that may improve biomedical diagnoses.[35,36] BIRDS chemical-shift imaging and CEST MRI studies have not yet been demonstrated in the same in vivo system. As described below, including fluorescence imaging may compliment or validate future BIRDS chemical-shift imaging–CEST MRI studies.
In vivo & ex vivo Optical Imaging
Macroscopic fluorescence imaging validated that the CEST MRI results were generated by accumulation of the dual-modality contrast agent in the glioma. In vivo fluorescence imaging showed accumulation of the contrast agent in the brain (Figure 5A). The comparison of ex vivo fluorescence imaging (Figure 5B) and the anatomical MR image (Figure 5C) further confirmed that the accumulation occurred in the glioma tumor (Figure 5D). The ex vivo study may also have been accomplished by detecting the fluorescence from Eu(III) in the chelate. However, Eu(III) chelates typically have weak fluorescence, so the in vivo study would have been difficult to accomplish, based on Eu(III) fluorescence. Therefore, we elected to use the DyLight 680 fluorophore for the in vivo and ex vivo studies for consistency.
Macroscopic fluorescence imaging. (A) The in vivo fluorescent image of the rat head overlayed on an x-ray image shows the presence of the agent in the U87 tumor in the brain. (B) The ex vivo fluorescence image of the whole brain also detected the agent in the brain (fluorescent image was overlayed on x-ray image of the whole brain). (C) The coronal MRI shows the location of the U87 tumor. (D) The ex vivo fluorescence image was also overlayed on the MRI to show that the agent was located in the U87 glioma. The anatomy of the brain was correlated between MRI and x-ray images.
The specificity of in vivo CEST MRI for glioma detection was further validated with ex vivo fluorescence microscopy, which showed that the contrast agent accumulated in the glioma tumor, but not in the contralateral brain tissue (Figure 6A & B). Fluorescence microscopy at micrometer spatial resolution was conducted following lectin staining, which identified vessel lumen and DAPI staining that identified viable cellularity (Figure 6C–F). The multicolor microscopy images of the glioma showed that the contrast agent extravasated across the vessel lumen. Microscopy images of the contralateral tissue showed that the agent remained in the vessel lumen and did not extravasate into normal brain tissues. This result provides a plausible rationale for the persistence of the CEST effect in the glioma during the in vivo MRI study, which further validates the in vivo CEST MRI result. This result also demonstrates the advantages of using a nano-sized dendrimer when developing new imaging agents detecting gliomas.
Microscopic fluorescence imaging. (A) Strong, pervasive distribution of the agent was visualized in tumor tissue. (B) Weak, focal distribution was visualized in the contralateral tissue. (C) A high-resolution view showed that the agent extravasated across the endothlium in tumor tissue, (D) but not in contralateral tissue. (E & F) Overlays of fluorescence from the agent, lectin staining of endothelium and 4',6-diamidino-2-phenylindole staining for viable cellularity, validated the spatial distribution of the agents observed in (C & D). The exposure time for tumor area and contralateral brain was kept identical. The images of (D & F) were enhanced to show fluorescent activity in the vessels.
Merits of Dual-modality Imaging
The in vivo and ex vivo imaging of the dual-modality contrast agent showed excellent potential utility for identifying the location of gliomas. In vivo CEST MRI immediately following injection of the agent highlighted the hypervascular rim of the tumor (Figure 4B, at the 10.8-min time-point), which was similar but not identical to the location of the tumor rim identified by the anatomical MR image (Figure 4A). This result indicates that CEST MRI may provide a more sensitive diagnosis of the hypervascular rim of the tumor than the anatomical image, which demonstrates the merits of using an exogenous contrast agent for glioma detection. The persistence of the CEST MRI contrast throughout the glioma contributed to the identification of the tumor location at a millimeter scale.
In vivo and ex vivo fluorescence imaging also showed the location of the tumor, which demonstrated that similar intrasurgical fluorescence imaging with a wide-field view may improve sensitivity for detecting tumor locations at a millimeter scale. The high-resolution ex vivo fluorescence imaging demonstrated that similar intrasurgical fluorescence imaging may detect tumor features at a micrometer scale. Thus, a PARACEST-fluorescence imaging contrast agent offers potential to diagnose gliomas at multiple spatial scales, before and during surgical resection of gliomas and other pathological tissues.
Nanomedicine. 2012;7(12):1827-1837. © 2012 Future Medicine Ltd.