Synthesis and characterization of the bimodal AuMN-DTTC probe
The synthetic scheme of AuMN-DTTC is described in . Briefly, the parental MN was synthesized according to the reported procedure.6
The gold nanoparticles (AuNP) were doped on MN by reduction of HAuCl4
in the presence of sodium citrate. Following purification, the deposited gold seeds were further enlarged by reduction of HAuCl4
in the presence of hydroxylamine (). A Raman active dye molecule, DTTC, and the stabilizing polymer group, PEG, were later introduced onto the gold surface.36
The resulting material went through several purification steps to remove free MN and AuNP in the suspension. PEG not only protects the gold nanoparticles from degradation and aggregation and ensures a longer circulation half-life, but also keeps the Raman active dyes intact on the surface by forming a protective shell around them.36
Synthesis and characterization of the MRI-SERS active nanomaterial (AuMN-DTTC)
The absorbance spectrum of the probe was monitored by UV-Vis spectroscopy and compared with AuNP and MN to determine the existence of gold nanostructures in DTTC functionalized gold-doped superparamagnetic iron oxide particles (AuMN-DTTC). As seen in , there is a plasmon peak at 530 nm on the absorbance spectrum of AuMN-DTTC similar to the 520 nm plasmon peak of AuNP, which is a strong indication of the presence of gold nanostructures.9, 13
The absorbance spectrum of MN alone did not have such a feature, indicating that the plasmon peak associated with AuMN-DTTC was due to the presence of gold nanostructures. We also compared the color of the resulting probe, AuMN-DTTC, with AuNP and MN and we observed that the AuMN-DTTC (0.4 mM Fe, 1.5 mM Au) had a dark red color, whereas MN (0.4 mM Fe) had a light brown color10
and AuNP (1.5 mM Au) had a red color ( legend).9
The red color of the AuMN-DTTC and its absence in MN is a strong indication of the presence of gold in the composition of AuMN-DTTC.41
We also observed that the darker red color of AuMN-DTTC relative to AuNP is the result of hydroxylamine reduction and enlargement of the AuNP seeds on MN. These results demonstrated the incorporation of gold nanostructures in the resulting probe.
We performed relaxivity measurements on solutions of MN, AuMN-DTTC, and AuNP to determine whether the superparamagnetic feature of MN was preserved in AuMN-DTTC. The R1 and R2 values of AuMN-DTTC (40.3 ± 5.1 and 110.9 ± 8.4 mM−1sec−1 respectively) were comparable to those of the parental MN probe ().
The elemental analysis of AuMN-DTTC along with the component MN and AuNP was performed to fully quantify the gold and iron content of the final material. Our results indicated that the iron composition of AuMN-DTTC was comparable to the parental MN, whereas the gold content of AuMN-DTTC was similar to AuNP (). These results established that our probe effectively incorporated the iron and gold components.
Since the goal is to apply a given contrast agent in vivo
, it is important to control the size of the agent within a certain range given that nanoparticles that are too large are biologically incompatible and nanoparticles that are too small cannot generate sufficient signal for imaging.13, 42
We first analyzed our probes by dynamic light scattering and determined that the average particle sizes of AuMN-DTTC, AuNP, and MN were 94.13 + 1.6, 23.52 + 0.34 and 32.53 + 0.26 nm, respectively (Suppl. Fig. 1
). The zeta potential values for AuMN-DTTC, AuNP, and MN were −2.27 + 0.28, −37.3 + 2.58 and −6.62 + 0.94 mV, respectively.
Several electron microscopy experiments were carried out to obtain structural information about AuMN-DTTC. We first acquired low-resolution transmission electron microscopy (LWTEM) images to determine the overall structure of the probe. As seen in , the gold nanostructures, observed as the electron-dense dark-colored component of the overall probe structure, are complexed with the dextran coated superparamagnetic iron oxide nanoparticles, the less electron-dense light-colored component in the TEM image. As seen in the figure, there are several gold nanoparticles per AuMN-DTTC particle to serve as substrates for SERS.
Electron microscopy images of AuMN-DTTC using LWTEM and HRTEM/STEM/EDS
In order to resolve the structure further we performed high-resolution transmission electron microscopy (HRTEM), scanning transmission microscopy (STEM) and energy-dispersive X-Ray spectroscopy (EDS) on AuMN-DTTC. As seen in , the dark-colored component (gold nanostructures) of the probe has a metallic lattice that is distinct from the light-colored component (iron oxide nanoparticles). The arrows show the lattices of both gold and iron oxide nanoparticles. This result not only confirms that both components seen in TEM images are metallic nanoparticles, but also that they represent different metallic nanostructures (original HRTEM image is provided in Suppl. Fig. 2
We focused on the dark and light colored region of the probe using STEM/EDS in order to obtain spectroscopic information about the AuMN-DTTC (P1 and P2, map). As seen in the EDS spectra in , the dark-colored component in the structure is indeed composed of gold (P1), whereas the light-colored region is composed of iron (P2). The Cu peak in the spectum is due to the copper grid used for imaging.
After obtaining the spectroscopic information, we constructed an elemental map on the agent () to see if the Au, Fe and S elements are present in the structure and how they are organized spatially with respect to each other. As seen in , PEG is immobilized on gold via
thiol bond as confirmed by colocolization of gold and sulfur in the elemental maps. Also, the iron element co-localizes with the light-colored region of the map and is closely associated with the gold. By contrast an elemental map of sodium ion, a control map, did not detect a distinct spatial signature (Suppl. Fig. 3
). These results along with the results of elemental analysis not only prove that the AuMN-DTTC nanostructures are composed of gold and iron oxide nanoparticles that are complexed with each other, but they also demonstrate that the gold nanostructures are PEGylated through gold-thiol chemistry.
Stability of AuMN-DTTC in high-salt conditions and serum
After completing the characterization of our probe, we performed a series of experiments to validate the stability of AuMN-DTTC in serum and in high salt conditions relative to citrate stabilized AuNP. Citrate stabilized gold nanoparticles are stable in water but in the presence of high-salt conditions the nanoparticles irreversibly aggregate, resulting in a shift in plasmon resonance.43
As seen in , the gold nanoparticles aggregate after addition of 0.4 M NaCl and 1xPBS. The aggregation was monitored by changes in optical density at 520 nm and 610 nm as an indicator of red shift 9, 44
over the course of forty minutes. As seen in , the AuNP aggregate immediately after addition of 0.4 M NaCl and 1xPBS. This is evident from the decrease in the 520-nm peak and the increase of absorbance in the red shoulder due to the aggregation-induced plasmon resonance absorbance shift to longer wavelengths. A further decrease in both the 520 and 610-nm peaks is due to precipitation of the aggregated particles. After developing this approach to monitor the aggregation of AuNP, we performed similar stability measurements of AuMN-DTTC. We first obtained the absorbance spectrum of AuMN-DTTC and observed a 530-nm plasmon peak. After addition of 0.4 M NaCl and 1xPBS, we did not see a red shift away from the 530-nm peak. However, we observed a slight decrease in OD at 530 nm and 610 nm, which is due to dilution of the probe with the NaCl/PBS stock solution (). No aggregation was observed over the course of forty minutes (), indicating that the AuMN-DTTC preparation had long-term stability in high-salt conditions. In a more biologically relevant context, we measured the absorbance spectrum of our probe (AuMN-DTTC) in fetal bovine serum and did not observe any changes in the 530-nm and 610-nm peaks over forty minutes (), suggesting the suitability of AuMN-DTTC for in vivo
delivery through the circulation. The lack of aggregation in high-salt conditions was also confirmed by assessment of the spin-spin relaxation of the solution, as described below () and particle size measurements by dynamic light scattering ().
UV-Vis absorbance studies of probe stability in FBS or 0.4 M NaCl and 1xPBS
UV-Vis absorbance, relaxivity and particle size studies of probe stability in cysteamine and/or 0.4 M NaCl and 1xPBS
Association of the gold and iron nanostructure components in AuMN-DTTC
For the purposes of our work, it is important to determine that the MN and gold nanostructures are strongly associated with each other and do not dissociate. To investigate this, we induced aggregation of AuMN-DTTC with a concentrated thiol-containing molecule, cysteamine. At high concentrations cysteamine can replace the PEG molecules on the gold surface by exchanging the gold-thiol attachment of PEG with that of cysteamine. As the PEG molecules are displaced, the probe is no longer stable and tends to aggregate and finally to precipitate. To promote this, we added a 50 mM solution of cysteamine into the AuMN-DTTC solution. As seen in , the 530-nm peak started to decrease and shift to longer wavelengths after the addition of cysteamine, whereas the absorbance over 610 nm increased initially, after which it started to decrease as the aggregated probes precipitated to the bottom of the cuvette. Aggregation is followed by precipitation as the larger clusters of particles cannot stay in suspension, resulting in less absorbent material in the suspension and a corresponding decrease in the 530-nm peak and red shoulder.
The spin-spin relaxation time (T2) values of AuMN-DTTC before and after addition of cysteamine were monitored to demonstrate the stability of the association between the gold and magnetic components of the probe. It has been shown numerous times in the literature that the T2 value of a superparamagnetic nanoparticle solution decreases as the dispersed nanoparticles assemble into clusters. This event has been termed magnetic relaxation switching.10, 45–47
This feature of magnetic nanoparticles has been used to detect different analytes using MRI.5, 47, 48
Here, we used this feature of MN to test whether or not the T2 relaxation time of the AuMN-DTTC solution changes as the nanoparticles aggregate. A decrease in T2 relaxation time further confirms that the MN and gold nanoparticles are strongly associated. As seen in , after addition of cysteamine to an AuMN-DTTC (0.15 mM Fe) suspension, the T2 relaxation time immediately decreased from 144.6 to 72.6 ms. This is expected as the cysteamine aggregates AuMN-DTTC through the gold nanoparticle component. As observed by absorbance measurements, after a certain threshold, large aggregates cannot stay in suspension and tend to precipitate. This was observed as an increase in the T2 relaxation time because, as the AuMN-DTTC precipitated, fewer nanoparticles remained in solution to generate a T2 effect. Further validation of our hypothesis was obtained by cysteamine treatment of an MN (0.15 mM Fe). We did not observe an effect on the T2 relaxation time of the solution, indicating that the corresponding change in the T2 relaxation time of AuMN-DTTC is the result of aggregation of the component gold nanostructures.
Finally, to confirm the aggregation of our probe upon thiol replacement, we monitored the particle size change of AuMN-DTTC with and without the addition of cysteamine. When we added cysteamine into a solution of AuMN-DTTC in water, we observed an immediate increase in particle size, which became more dramatic with time. These data further confirmed that the changes in the absorbance spectra and T2 relaxation times of the agent are due to aggregation of our probe by cysteamine treatment. Consistent with our earlier results, cysteamine treatment had no effect on the particle size of MN ().
Overall the relaxivity, UV-Vis absorbance and particle size measurements suggest that AuMN-DTTC is stable in water, serum and high-salt conditions, which is important for its application in vivo. The cysteamine treatment experiments indicate that the MN and AuNP components of our nanomaterial are strongly associated with each other.
In silico MRI and SERS measurements on AuMN-DTTC
After characterizating our probe and thoroughly investigating issues related to its stability, we focused our attention on evaluating the suitability of AuMN-DTTC as an MRI-SERS contrast agent. To that end, we first performed MRI on in silico phantoms comprising solutions in water of AuMN-DTTC and the control probes AuMN, AuNP, MN and PBS. The iron content of MN, AuMN and AuMN-DTTC was normalized to 0.45 mM prior to the scan and the gold content of AuNP was normalized to AuMN-DTTC and AuMN (1.6 mM Au). As seen in the T2-weighted MR images (), the signal intensity of the AuMN-DTTC and AuMN solutions was comparable to MN and visibly lower than AuNP and PBS. This effect is consistent with the propensity for superparamagnetic iron oxides to shorten the transverse (T2) relaxation time of surrounding protons and reflects the capacity of our agent, like parental MN, to generate contrast on MR images. A quantitative assessment of T2 relaxation times was obtained using a multiecho MRI analysis. The T2 relaxation times of AuMN-DTTC, AuMN and MN were 29.23 ± 1.45, 32.13 ± 3.26 and 31.58 ± 1.7 msec, respectively, whereas PBS and AuNP had T2 relaxation times of 783 ± 102 and 811 ± 75 msec, respectively, indicating that the latter solutions were non-magnetic (). These data confirmed the validity of AuMN-DTTC as an MRI contrast agent.
MRI and Raman spectroscopy in silico
After confirming that AuMN-DTTC has magnetic properties similar to MN, we analyzed the potential of our agent to also generate a surface enhanced Raman scattering (SERS) effect. To that end, we monitored the Raman spectra on AuMN-DTTC, AuMN, AuNP and DTTC. We observed amplification of the efficiency of Raman scattering associated with AuMN-DTTC but not with any other controls. As we hypothesized, AuMN, which was designed as a SERS inactive material due to the absence of the DTTC Raman reporter on the gold surface, does not generate the Raman signature observed with AuMN-DTTC. The observed peaks in the SERS spectrum of AuMN-DTTC are very sharp, narrow and clearly distinguished from the rest of the spectrum (). These findings demonstrate that AuMN-DTTC is not only useful as an MRI contrast agent but also has potential applications as a SERS-active imaging probe.
In vivo MRI and SERS measurements of AuMN-DTTC
The strong contrast-enhancing capabilities of AuMN-DTTC demonstrated by our in silico studies suggested the possibility that this probe can also represent a suitable in vivo MRI-SERS contrast agent. To test this hypothesis, we first investigated whether we could obtain T2 contrast with our bimodal contrast agent in a living subject. To do so we injected AuMN-DTTC and AuNP at equivalent gold concentrations in the right and left gluteal muscle, respectively, of female nude mice as demonstrated in . After injection, we imaged the animals using a standard MRI multiecho T2-weighted protocol. The AuNP-injected area appeared bright, due to edema, which has a higher T2 value than surrounding muscle. By contrast, we observed a notable area of signal loss associated with the AuMN-DTTC injected area, which is an indication of contrast agent accumulation (). Quantitative analysis of tissue relaxation times at the injection sites revealed that the T2 value of non-injected muscle tissue was 33.4 ± 2.5 msec, whereas muscle tissue injected with AuMN-DTTC or AuNP had T2 relaxation times of 20.3 ± 2.2 and 157.4 ± 38.8 msec, respectively ().
MRI and Raman spectroscopy in vivo
Having established that AuMN-DTTC can generate excellent MRI contrast in vivo, we performed Raman spectroscopy to determine whether this probe can generate SERS signal. Measurements were performed prior to and following intramuscular probe injection in the live mouse. The mouse was placed on Raman spectroscopy platform and imaged by placing the Raman probe directly over the injected muscle, as shown in . AuMN-DTTC was injected in the right gluteal muscle, whereas AuMN, which is a SERS-inactive derivative of our probe, was injected in the left gluteal muscle. After the scan, the animal was sacrificed and the muscle tissue excised. The final data were analyzed by comparing the in vivo AuMN-DTTC spectra to those of AuMN-DTTC in silico, AuMN in vivo, and noninjected skin tissue. The results demonstrated a clear surface enhanced Raman scattering observed both in vivo and ex vivo. It was also apparent that the SERS signatures obtained in vivo and ex vivo matched those gathered from AuMN-DTTC in silico. As seen in the , the marked SERS peaks in AuMN-DTTC in silico completely overlapped with the SERS peaks in vivo and in excised tissue. However the SERS effect was not observed in the skin nor the tissue injected with our MRI-active but SERS-inactive AuMN derivative. These findings assured us of the value of our probe, not only to monitor biological events by MRI, an established in vivo imaging modality, but also to apply SERS in order to gather complementary information from the same environment.
Developing materials that can be used for clinical or pre-clinical in vivo imaging using multiple modalities has been a very active area of research in the imaging field. Confirming the presence of a disease-related abnormality with multiple imaging methods by using only one type of material will result in the rapid acquisition of reliable data, especially when one considers the increasing acceptance of multi-modality instrumentation by the clinical and research communities. This type of data acquisition will also facilitate the collection of complementary information for a thorough characterization of biological phenomena. Here we report a novel nanoparticulate material with bimodal potential that can be imaged using magnetic resonance imaging and Raman spectroscopy. We demonstrate that the described probe is highly stable, biocompatible, and detectable using MRI and Raman spectroscopy by virtue of the SERS effect. This probe was fabricated without the use of bio-incompatible chemicals during the synthesis process. Importantly, the probe can be visualized both in silico and in vivo in deep tissue in live animal. Considering the paucity of examples describing in vivo SERS-active probes and the importance of developing multimodal imaging strategies, we feel that the development of our agent is both timely and valuable as a new molecular imaging tool.