Metal clusters (e.g.
, f-c-c structure) of Yb were synthesized previously by the bio-reduction method to generate high metal content, particularly as blood pool agents.16
However, their large size (i.e.
, >6 nm, the renal clearance threshold and resultant poor biological elimination poses potential issues regarding long-term safety, similar to the barriers facing quantum dots or carbon nanotubes (SWNT). Very recently Lu et. al.
reported a Yb-based nanoparticle stabilized with oleic acid and modified with the biocompatible polymer DSPE-PEG 2000 for conventional CT imaging.17
However, to the best of our knowledge, the use of ytterbium as a nanoparticulate spectral “multi color” CT contrast agent has not been previously reported. The design of these particles involved concentrating a hydrophobic Yb-complex (ytterbium (III) 2,4-pentanedionate) within a phospholipids-entrapped, intravascular colloidal nanoparticle (>150 nm).
Self-assembled Yb nanocolloids were prepared from organically soluble trivalent ytterbium complex suspended in polysorbate. The core was encapsulated in a phospholipids monolayer through high-pressure homogenization. Briefly, the synthesis process involved suspending commercially available ytterbium (III) 2,4-pentanedionate in polyoxyethylene (80) sorbitan monooleate followed by microfluidization as a 20% (v/v) colloidal suspension with a 2% (v/v) phospholipids surfactant in nanopure water ().
Figure 1 Synthesis and physico-chemical characterization of self-assembled Ytterbium nanocolloids (YbNC). Schematic describing the preparation of Yb-enriched YbNC: (i) Suspension of Yb(III)-2,4-pentanedionate in Polyoxyethylene (20) sorbitan monooleate, vigorously (more ...)
The surfactant mixture was comprised of phosphatidylcholine (lecithin-egg PC), (99 mol%), dipalmitoyl phosphatidylethanolamine caproyl biotin (1 mol% w/v, PE-biotin) for in vitro avidin–biotin coupling of homing ligands. YbNC was purified by exhaustive dialysis through 10 kDa MWCO membrane against nanopure water (0.2μM) (). Multiple syntheses resulted in colloidal nanoparticles with mean particle size of 240 ± 30 nm using dynamic light scattering with zeta potentials in the range of −12mV to −18mV and low polydispersity (<0.2). In a typical preparation, ytterbium content was determined by ICP-OES, as 0.41 mg/ml of the 20% colloidal suspension, which corresponded to approximately 1,200K Yb atoms/nanoparticle with 1.1×1012 particles/ml. The Yb nanoparticles vialed and sealed under argon have exhibited significant shelf-life stability with <7% change in hydrodynamic diameter and polydispersities over two months at 4°C.
The nanoparticles were further characterized in the anhydrous state by transmission electron microscopy (TEM) and atomic force microscopy (AFM). For TEM, Yb nanoparticles were fixed with 2.5% glutaraldehyde and sequentially stained with osmium tetroxide and tannic acid before embedding in Polybed812. Post-stained in uranyl acetate and lead citrate, these particles were imaged on a Electron Microscope (Jeol 100CX) and observed to be spherical with a distinct dark lipid periphery (). AFM particle height was 110 ± 40 nm, reflective of their compressible nature.
A prototype spectral CT system (Philips Research, Hamburg, Germany) utilizing a single-slice, photon-counting detector featuring six energy bins (Gamma Medica Inc., Northridge, California, USA) was used to evaluate the Yb nanocolloids. In an initial experiment, YbNC in aqueous suspension was imaged. shows a representative cross section of the experimental setup rendered as a conventional CT image. It shows a pattern of X-ray lucent tubes filled with serially diluted YbNC suspension (YbNC:H2O - 1:2, 1:4, 1:8, 1:16, 1:32, 1:64) and two additional reference tubes filled with water and calcium chloride, respectively. Using spectral CT processing tools, the K-edge information was decomposed individually into its photoelectric absorption, Compton effect and k-edge components. The obtained ytterbium-selective image was then superimposed using color-coding (yellow: low concentration, red: high concentration) on the conventional CT image (). Absolute Yb concentrations derived from the K-edge image were compared with the concentrations obtained by inductively coupled plasma optical emission spectrometry (ICP-OES), revealing a good linear correlation between both results (; R2 = 0.987). This allows linear mapping of concentrations to the ytterbium image, which makes spectral CT a quantitative imaging technique.
Figure 2 While conventional CT renders an image providing information about the overall attenuation, commonly represented in Hounsfield units (a), spectral CT is capable of separating the K-edge information and selectively image Ytterbium (b). The Yb k-edge signal (more ...)
The detection sensitivity of the prototype spectral CT scanner has been significantly improved through recent developments in image reconstruction technique from the usual algorithms of filtered back projection to statistical image reconstruction techniques.18
The applicability of this technique for Yb imaging was conceptually tested using a mouse blood pool study. In this preliminary study, the animal was briefly anesthetized with Isoflurane to effect and injected with YbNC (150 μl) into the tail vein. Two minutes later, the animal was euthanized and the blood pool was imaged for the metal with spectral CT. The scan parameters were set to: tube voltage 130 kV, tube current 50 μA, threshold energies 25-46-61-64-76-91 keV, views per turn 900, rotation time 72 s, slice increment 0.5 mm, reconstructed FOV 60 × 60 mm2
, pixel size 0.1 × 0.1 mm2
shows a representative cross section through the heart. While a change in contrast due to Yb is barely visible in the conventional CT image (), the K-edge image using spectral CT and iterative reconstruction presents the YbNC with a high signal to noise ratio (). To the best of our knowledge, this represents the first spectral CT imaging with Yb nanoparticles. Owing to the sensitivity limitations of the spectral CT system, only high concentrations in the heart were successfully separated from background noise. With on-going improvements of the spectral CT prototype, low concentrations as envisioned in the vascular system are expected to become visible in the future. The lack of spectral CT signal from other major organs can be explained due to the relatively short circulation time (two minutes after intravenous administration) of the YbNC. Although more in depth in vivo experiment is warranted in the future, this preliminary experiment demonstrates that YbNC can be successfully imaged with spectral CT at a concentration when the contrast was barely visible with conventional CT.
Figure 3 Blood pool imaging in mouse after bolus application of nontargeted Yb nanocolloids (6ml/kg). (a) Pseudo-conventional CT image composed from spectral measurements, slice through heart (dashed line). Statistical image reconstruction of Yb signal after 1 (more ...)
The pharmacokinetics and biodistribution of these particles were studied in a mouse model. Following intravenous injection (25μl), the concentration of ytterbium in serial blood samples as a function of time was determined by ICP-OES (). Pharmacokinetic profiles of YbNC, followed a two-compartment bi-exponential model (y = 40.9*e(−.028*x)+84.1*e(−.002*x). The half-lives were 25.2 and 303 min for the distribution and clearance phases, respectively. Bio-distribution of YbNC was determined by analyzing the major organs following intravenous administration of YbNC (25 μl) in mice at 2h, 24h and 7 days (n=3 / time point) using ICP-OES. These results were consistent with the expected clearance of the particle through reticuloendothelial system (RES) as evident by the high organ uptakes in liver, spleen and kidney. Interestingly, the relatively high accumulation of YbNC in liver has been noticed, which is not typically observed in this type of colloidal nanoparticles. Further detailed investigation is warranted to study the in vivo biodistributive properties of these particles. Finally, the whole body bioelimination of the YbNC (25μl i.v.) was tested in mice at 2h, 24h and 7days (n=3/time point). These data showed approximately 90% elimination of the metal over the 7 day study, with 11±3% remaining at that time.
Figure 4 In vivo pharmacokinetics, biodistribution and clearance of YbNC in mouse. (a) pharmacokinetic profile of nontargeted YbNC with a biexponential fit [y=0.0456*exp(−0.0391*x)+0.1022*exp(−0.0018*x)). (b) Organ distribution of YbNC based on (more ...)
Spectral CT holds great potential for a broad range of clinical applications, such as breast cancer screening and kidney stone characterization, but we anticipate that the initial major impact of the technology will be for molecular imaging beginning with the urgent diagnosis of acute coronary syndrome in EDs.