Here, we describe the construction of a composite nanoparticle-based activatable probe that can be injected systemically into mice to sense matrix metalloproteinase (MMP) expression in vivo
. The choice of this experiment stemmed from our previous experience with building activatable probes using gold nanoparticles (GNPs) as templates.15
GNPs can efficiently absorb energy emitted from an adjacent fluorophore and induce a fluorescently quenched state to the overall nanostructure.16
The unique surface energy transfer (SET) properties of GNPs allow them to offer larger valid fluorescence quenching distances (~ 20 nm) than those of traditional fluorescence resonance energy transfer (FRET). For example, this feature has been harnessed to construct a protease-sensitive probe by using a protease substrate as the spacer to bridge the GNP and the fluorophores. Such probes are of potential clinical relevance in assessing diseases with up-regulation of proteases, such as MMPs, in cancer.17
The feasibility and superiority of such an approach has been demonstrated by us and others.15–16, 18
The success, however, was mainly achieved in vitro
, with in vivo
applications hampered by the unfavorable thiol-gold chemistry, which tends to be labile in a thiol-rich environment such as blood.19–20
When injected intravenously, gold-based activatable nanoparticles (GANPs) may encounter substantial off-target activation, which jeopardizes the role of GANPs as site-specific probes. There have been efforts to utilize multi-thiol anchors to improve the stability of the complexation, but with limited success.21–22
Our strategy is to replace GNP with a flower-shaped, Au-Fe3
composite nanoparticle. We expect that with the additional iron oxide surface, we can steer clear of gold-thiol chemistry, while still keeping fluorophores in close vicinity to GNPs, resulting in probes that can be activated in areas of interest after systemic administration. The nanoparticle design is illustrated schematically in . We have chosen GPLGVRG, a sequence with MMP selectivity (such as MMP-2, -9 and -13), as the bridging substrate to be coupled with Cy5.5, a widely used near-infrared dye molecule. On the other end of the peptide we covalently linked an anchoring unit, TDOPA (tri-dihydroxyphenylalanine, ). We and other researchers confirmed in previous studies that dopamine and its analogs can bind with high affinity to the surface of iron oxide nanoparticles (IONPs).23–25
TDOPA is essentially a modified version of such an anchor with improved affinity.26
In parallel, we applied a thiolated poly(ethylene glycol) (SH-PEG5000
) to passivate the gold surface. This measure aims to improve the physiological stability of the overall nanoconjugates, and protect the gold surface from cross-tethering by Cy5.5-GPLGVRG-TDOPA. Although first reported by us in 2005,9
nanoparticles have not yet been investigated in the context of nanomedicine. We chose a flower-shape, rather than dumbbell-shaped structure, because a flower-shape bears a multitude of iron oxide “petals” on each GNP core--an architecture that favors accommodation of more ligands in the vicinity of the GNPs, thereby maximizing the use of GNP as a quenching unit.
Figure 1 a) Schematic illustration of the formation and working mechanism of FANPs. b) High resolution TEM of the flower-like Au-Fe3O4 nanoparticles. Scale bar = 20 nm. c) Enlarged TEM image of a representative flower-like Au-Fe3O4 nanoparticle. The diameter of (more ...)
Synthesis of a) Cy5.5-GPLGVRG-TDOPA and b) Cy5.5-GPLGVRG-Cys. i) 2% DIPEA/DMF, ii) TFA/ethanedithiol/thioanisole/water (80/10/5/5/, v/v/v/v/).
nanoparticles were synthesized according to a previously published protocol9
with minor modification. We first prepared 8 nm GNPs by reducing HAuCl4
in tetralin with oleylamine. We then used the GNPs as seeds to grow Fe3
onto the GNP surface via
pyrolysis using iron pentacarbonyl (Fe(CO)5
) as the precursor. As shown in , the IONPs, with a diameter of 13.4 ± 3.5 nm, grew as “petals” around the 8 nm GNP cores, which have higher density and appear darker in TEM images. Each flower-like nanoparticle has an average of 2–4 such petals wrapping around the cores (). This observation was in accordance with the inductively coupled plasma (ICP) results, which found an estimated 3:1 ratio between the IONP moiety and the GNP moiety (based on the assumption that each GNP has a diameter of 8 nm and each IONP a diameter of 13 nm). It is noticeable that the longest distance between the IONP surface and the GNP surface (labeled as red arrows in ) is about 9.7 ± 1.4 nm, which is well within valid distance for effective quenching. Iron mapping by energy-filtered TEM () found almost no overlap between iron and gold, indicating that no alloy was formed during the pyrolysis, and the surface engineering can be processed by considering separate GNPs and IONPs.
Since we aimed at functionalizing both types of surfaces, it was important to know the accessibility of ligands to the gold core. From two dimensional TEM imaging it is impossible to determine the arrangement of the petals around the gold cores; therefore we used electron tomography to acquire many views of the specimen, which enabled us to generate its three dimensional structure. As shown in , a surface rendered tomogram, the gold core is accessible by the surroundings, even though it is surrounded by IONPs.
These as-synthesized Au-Fe3
nanoparticles were coated with a thick layer of oleic acid/oleylamine and, therefore, could not be dispersed in water.8
To render the particles water-soluble and to impart the functional motifs, we incubated the Au-Fe3
nanoparticles, which were in a CHCl3
/DMSO=2:1 mixture, with Cy5.5-GPLGVRG-TDOPA and SH-PEG5000
(see the Methods section) at room temperature (r.t.) overnight. As a comparison, we also incubated Au-Fe3
nanoparticles and IONPs (13 nm in diameter, oleic acid/oleylamine coated, from Ocean Nanotech) with Cy5.5-GPLGVRG-TDOPA (but without SH-PEG5000
); and, moreover, incubated Au-Fe3
nanoparticles with Cy5.5-GPLGVRG-Cys. The Cy5.5-GPLGVRG-Cys sequence was gold-philic and was used in our previous studies to prepare GANPs.15
As discussed above, we anticipated that by replacing cysteine with TDOPA, the peptide could become iron-philic and be immobilized on an IONP surface, instead of a gold surface. As a control, GANPs were also prepared based on the previously published protocol.15
After incubation, all the nanoparticles were washed and redispersed in water with sonication. The supernatant from each washing step was combined and stored for subsequent chemical analysis. The Cy5.5-GPLGVRG-TDOPA can be efficiently immobilized on the iron oxide surface of both IONPs and Au-Fe3O4 nanoparticles (as assessed by the Cy5.5 content in the supernatant). However, the Cy5.5-GPLGVRG-TDOPA, when used alone, failed to convey good water solubility to either the IONPs or Au-Fe3O4 nanoparticles. Cy5.5-GPLGVRG-Cys gave good solubility for IONPS, but not for Au-Fe3O4 nanoparticles. The Au-Fe3O4 nanoparticles incubated with both Cy5.5-GPLGVRG-TDOPA and SH-PEG5000, on the other hand, were readily water soluble after incubation. This suggests the successful immobilization of SH-PEG5000 on the gold surface and confirms its critical role as a stabilizing agent. By assessing the content of Cy5.5 in the supernatant, we were able to determine the amount of peptide immobilized onto the nanoparticles, hereafter referred to as flower-like activatable nanoparticles (FANPs). According to the inductively coupled plasma (ICP) results, and assuming a diameter of 8 nm for the GNP core in the FANPs, each FANP was estimated to have about 152 ± 12 copies of Cy5.5-GPLGVRG-TDOPA, compared to 85 ± 8 copies per GANP. Since each GANP has a core size of 20 nm, its surface is 6 times greater than that of GNP in the FANPs. Such an increase in peptide loading, despite a loss of gold surface, was attributed to the three petals extending from the gold surface and, once again, suggests that the peptide loading occurred on iron instead of on gold surface. The overall size of FANPs was 40.0 ± 4.3 nm, as analyzed by dynamic lighter scattering.
As shown in , FANPs have a broad absorption in the visible range (400–800 nm), contributed mainly by the iron oxide moiety. In addition, their spectra exhibit two shoulders at around 560 and 675 nm, which are characteristic absorption bands of GNPs and Cy5.5, respectively. One indicator of the agglomeration of GNPs is a red-shift of the 560 nm peak.7
We used such a feature to analyze the stability of FANPs under various conditions, including PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4), MMP reaction buffer (100 mM Tris, 200 mM NaCl, 5 mM CaCl, 0.1% Brij, pH 7.2) and a concentrated dithiothreitol (DTT) solution (10 mM, in water). Under all these conditions, we observed no aggregation, either visually or spectrometrically, indicating good stability of the FANPs.
Figure 3 a) Uv-vis spectra of FANPs under various conditions. b) Stability test of FANPs in PBS, FBS and DTT. Compared with GANPs, FANPs showed a much better stability than GANPs in a thiol-rich environment. c) Test of activation capacity by incubating FANPs with (more ...)
The fluorescent activities were measured on an F-7000 Fluorescence Spectrophotometer (Hitachi). Despite the large amount of Cy5.5-GPLGVRG-TDOPA on the surface, the FANPs showed very low fluorescence activity (, excitation at 675 nm, emission at 690 nm), which was apparently due to the excellent quenching effect of Au-Fe3O4 nanoparticles. Incubating FANPs in PBS or serum (fetal bovine serum, Invitrogen) at 37°C did not increase the overall fluorescence activity substantially (), suggesting the reliability of the TDOPA-Fe bonding under these conditions. One important criterion of the activatable probes, as we addressed earlier, is their stability against non-specific activation in a thiol-rich environment. To study this, we incubated FANPs and GANPs, at the same Cy5.5 concentration (600 nM) with DTT (a final concentration of 10 mM) and monitored the change of fluorescence over time. We found that under such conditions DTT can almost immediately cleave all the Cy5.5-GPLGVRG-Cys attached to GANPs, but had a minimal impact on FANPs. This observation corresponded well with our expectation that Cy5.5-GPLGVRG was immobilized on the FANPs via DOPA-Fe binding, and therefore was much less liable to thiol cleavage.
After confirming the stability, we then moved forward to test the activation capacity of FANPs by MMP-13. We incubated FANPs with MMP-13 (3 µg/ml) at 37°C in MMP reaction buffer, and measured the fluorescence activities at pre-determined time points (). In a control group, we added an MMP-inhibitor (MMP inhibitor III, a broad-spectrum inhibitor of various MMPs, EMD Bioscience, 8 µM) in combination with MMP-13 to test whether the activation could be inhibited. Without MMP-13, the FANPs were very stable in the buffer solution, showing almost no signal change within the observation period (420 min). The addition of MMP-13, however, caused a dramatic signal increase, which was efficiently blocked in the control group by the addition of the MMP-inhibitor. After 420 min incubation, the activation in signal (between the +MMP-13 group and the −MMP-13 group) was determined to be 17-fold. Such activation was also found to be dependent on MMP-13 concentration. When incubated with MMP-13 at different concentrations (0.2, 0.5, 1, 2, and 4 µg/ml, at 37°C for 12 hrs), we observed a proportional increase of fluorescence (). This signal change was visualized better during a phantom study of the activated FANP solution (, on a Maestro 2 imaging system, Cri, Woburn, MA). In general, FANPs showed MMP-specific activation and a much improved stability compared to GANPs. These findings, in conjunction with non-toxicity (), make FANP a candidate imaging probe for in vivo detection of MMPs.
MTT assays with FANPs at different concentrations.
The in vivo
tests were performed on an SCC-7 (head and neck squamous cell carcinoma) tumor xenograft model, which is known to express high levels of MMP.15
Briefly, separate groups of mice received injections by tail vein of either FANPs or GANPs in 200 µl PBS (150 nmol Cy5.5/kg), and full-body optical images of the mice were acquired at selected time points (30, 60, 120 and 240 min) postinjection (p.i.) using an “orange” filter (640–820 nm). In the FANP group the signals were found to be almost exclusively in the tumor area as early as 30 min after the particle injection, and the intensity developed steadily throughout the observation period (). By drawing regions of interest (ROIs) around the tumor areas, we were able to assess the average signals in the tumor area, which were 414.6 ± 36.2, 855.4 ± 134.0, 1164.7 ± 233.0, 1523.26 ± 2.6 ×106
at 30, 60, 120 and 240 min p.i. For the GANP group, however, the signals were found mostly in the liver at early time points (30, 60 and 120 min), and only at the 240 min p.i. time point did we observe weak signals from the tumor (46.8 ± 13.5 ×106
at 240 min). To further confirm that the signals from tumors in the FANP group were indeed induced by MMP cleavage, in another control group we injected MMP inhibitor (1 mg/kg) intratumorally into the mice 30 min prior to the FANP injection. The injection of inhibitor induced an almost complete blocking of the FANP activation, as tumor uptake was found to be 26.4 ± 3.1, 31.4 ± 18.7, 27.8 ± 37.5, and 13.6 ± 1.4 ×106
at 30, 60, 120 and 240 min p.i., respectively.
In vivo NIRF imaging after the injection of FANPs, with and without the pre-injection of MMP inhibitor. In a control group, GANPs at the same Cy5.5 dose were injected.
To further elucidate the particle distribution and activation, we sacrificed the mice after imaging at the 4 h time point and collected tumor as well as other major organs for ex vivo
analysis. We first arranged the organs on a dark plate and subjected them to ex vivo
imaging on a Meastro 2 optical imaging system. The intensities were then analyzed by the software provided by the vendor (Maestro 2.10.0), and are illustrated as a histogram in . Signals from tumors were well correlated with those observed from the in vivo
imaging, and signals from the FANP group were 10 times more intense than those from the blocking group. The GANPs, on the other hand, showed only a marginal increase in tumor signals compared to the blocking group. Notably, while almost no signals were observed from blood in both FANP groups, we found a strong signal from the blood in the GANP group. This confirmed our previous concern that GANPs may be non-specifically activated in blood plasma, where glutathione (GSH) is present at high concentration. Besides tumor, the other major intensity-contributing organ was liver, which is not surprising considering the 40 nm diameter of the FANPs. Such a size, together with the antifouling effect afforded by the PEG, also explains the accumulation of FANPs in tumors, which was likely mediated by the enhanced permeability and retention (EPR) effect.3
Ex vivo imaging of tumor and major organs after the 4th hr of in vivo imaging. The intensities from the organs, quantified by the software provided with the Maestro 2 imaging system, are illustrated as histograms in the upper panel.
We then performed a series of immunostaining experiments to study FANP distribution and activation in the tissues, for both the MMP blocking and non-blocking groups. We found that the MMP inhibitor did not have a major impact on the tumor accumulation of particles, as manifested by similar Prussian blue staining patterns for tumor tissues in both groups (). As expected, we observed similarly high levels of MMP expression in both tissues, but found almost no Cy5.5 signals in the blocking group. This, together with the Prussian blue results, further confirms our assumption that without active MMPs (the MMP inhibitor does not regulate the MMP expression--rather, it antagonizes and blocks MMP from cleaving the GPLGVRG substrate), the FANPs stayed in an optically well-quenched state in the tumor. On the contrary, we observed strong Cy5.5 signals from the non-blocking group, and their distribution superimposed well with the MMP distribution. This again confirmed our postulation that the activation of FANP probes was specifically mediated by MMPs. Other than in the tumor, we also found significant levels of FANPs in the liver (). This is in accordance with the NIRF results, which showed that liver was a major contributor to the signal. Notably, because FANPs were distributed and activated in the intracellular space, we observed a substantial loss of FANPs by washing, as indicated by both Prussian blue staining and fluorescent immunostaining.
Figure 7 a) Prussian blue and fluorescent immuostaining results with tumor tissues. While particle distribution and MMP-13 expression were similar, only in the non-blocking group did we observe signals from FANPs. b) Prussian blue staining of tissues from liver, (more ...)
In the current study, by introducing a second iron oxide moiety, we were able to construct an activatable probe that can specifically sense MMP up-regulation in vivo
after systemic injection. Several factors considered in the design appear to benefit the role of the composite nanoconjugate as an efficient probe. First, both GNPs and IONPs are regarded as biologically safe materials, and have been separately studied as scaffolds for imaging/therapeutic applications.3–4, 19
Second, although not as effective as GNP, IONPs have been reported to possess an optical quenching effect,27
which alleviates the possible loss of energy-absorbing ability by moving fluorophores to the iron oxide moiety. Third, the unique flower-like architecture of FANPs allows more fluorophores to be loaded onto a single nanoparticle than with GANPs. This is also an appealing attribute in that each fluorophore on the FANP might in theory be quadruply quenched by: (1) its fluorophore neighbor, (2) the underlying IONP, (3) the other IONPs nearby, and (4) the GNP core. Fourth, although we removed substrates to the IONP surface, this does not mean that the gold surface is not useful. On the contrary, the immobilization of PEG on the gold surface was critical to the stability of the nanoconjugates in a physiological environment. Unlike for GANPs, where Cy5.5-GPLGVRG-Cys is working as the key substrate (and whose unintended detachment disables the particle’s ability to serve as an activatable probe), in the FANP system the PEG5000
-SH “merely” serves as a stabilizing agent, and such a function seems not to be affected by a thiol-rich environment.
Overall, we have demonstrated that an additional phase incorporated into a composite nanoparticle can be harnessed to optimize the strengths of each nanocomponent. GNPs provide an excellent quenching effect, but have labile surface chemistry in an in vivo environment; the IONPs, on the other hand, provide a robust surface conjugation technique, but do not have an appealing quenching effect. By combining the two, we have been able to engineer the surface and thereby integrate the strengths of each. The study has shown a novel way to tailor a material’s role in creating imaging/therapeutic agents. It will be interesting to tap further into the potential of such a transition from simple sphere-like GNPs to complex flower-like nanostructures. For example, it should be possible to optimize performance by adjusting the architecture to increase the number of petals in the flower-shaped nanoparticle, or to tune the size and shape of each individual component. Moreover, the same technique that was used to target MMP expression in the current study can be readily applied to detect other proteases that are implicated in cancer pathogenesis.