A self-assembling amphiphilic dendrimer nanotracer for SPECT imaging

Bioimaging has revolutionized modern medicine, and nanotechnology can oﬀer further specific and sensitive imaging. We report here an amphiphilic dendrimer able to self-assemble into supramolecular nanomicelles for eﬀective tumor detection using SPECT radioimaging. This highlights the promising potential of supramolecular dendrimer platforms for biomedical imaging.

Medical imaging plays an important role in modern medicine by providing accurate information relating to diagnosing, grading and staging diseases as well as monitoring treatment response and efficacy. 17][8] EPR results in nanoparticle specific tumor accumulation thanks to the leaky vasculature and dysfunctional lymphatic system characterizing the tumor microenvironment. 9 addition, nanosystems carrying and incorporating abundant imaging reporters can significantly amplify the contrast signal for better imaging and diagnosis.Consequently, a myriad of nanosystems have been explored for PET and SPECT imaging of tumors. 2,6,10e have recently used the self-assembling amphiphilic dendrimer Ga-1 to establish an innovative nanotracer for PET imaging (Fig. 1). 11This dendrimer is composed of a long hydrophobic alkyl chain and a hydrophilic poly(amidoamine) (PAMAM) dendron bearing the PET radionuclide 68 Ga(III) complexed within the macrocyclic chelator NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) at the peripheral units (Ga-1 in Fig. 1).It selfassembles into small and stable nanomicelles, which can effectively accumulate in tumors.These nanomicelles deliver excellent results in PET imaging of different tumors, including some which could not be detected with the standard clinical PET agent [ 18 F]FDG (2-fluorodeoxyglucose). 11 The performance of this dendrimer radiotracer is largely ascribed to the beneficial combination of its unique multivalent dendrimeric structure and the relevant EPR effect.
Motivated by the promising PET imaging results, 11 we aimed at further exploiting self-assembling nanotechnology for constructing Fig. 1 Self-assembling dendrimer nanosystems based on the amphiphilic dendrimers 1 and 2 bearing radionuclide, for positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging of tumors, respectively.
effective dendrimer-based radiotracers for SPECT imaging.2][3] In addition, SPECT is more readily accessible and less expensive than PET in the clinics, even if PET has higher sensitivity and resolution.Recent progress in advanced detection technology and the combination with computed tomography (CT) has considerably improved the resolution and sensitivity of SPECT, placing SPECT as a quantitative imaging modality similar to PET. 3,12 Also, SPECT radiotracers generally have longer half-lives, which allows the characterization of slow kinetic processes and long biological events that take hours or days.Common radionuclides used in SPECT imaging include technetium-99m ([ 99m Tc]Tc), indium-111 ([ 111 In]In) and iodine-123 ([ 123 I]I).Although [ 99m Tc]Tc is the most widely used, [ 111 In]In has a relatively longer half-life (2.8 days).Accordingly, in this study, we chose [ 111 In]In as the radionuclide to develop a dendrimer radiotracer for SPECT imaging with the aim of monitoring and measuring long and slow biological processes such as tumor development and treatment.
Chelation of 111 In 3+ to a thermodynamically stable and kinetically inert complex is a fundamental requirement for SPECT imaging in order to prevent the release of free radionuclide. 4,5he cyclic chelator DOTA (1,4,7,10-tetraaza-cyclododecane-1,4,7,10tetraacetic acid) and the acyclic chelator DTPA (diethylenetriaminepentaacetic acid) are the most frequently used in nuclear medicine. 13In general, DOTA forms more stable complexes with radionuclide ions than the acyclic chelator DTPA because of the entropically favorable pre-organized and rigid binding sites within the DOTA ring.Nevertheless, the process of metal-complex formation is often slow for DOTA, and requires high temperature and long reaction times.Yet, as DOTA is the ''gold standard'' chelator for 111 In 3+ , 13 and on the basis of our previous experience in developing the NOTA-conjugated dendrimer to complex with the radionuclide Ga 3+ (Ga-1) for PET imaging (Fig. 1), 11 we selected DOTA as the chelator to construct the amphiphilic dendrimer 2 which, in turn, was complexed with 111 In 3+ to generate In-2 for SPECT imaging (Fig. 1 and 2).Knowing that the DOTA ring is considerably larger than that of NOTA, we initially worried about the eventual synthetic difficulty stemming from steric hindrance of the DOTA terminals in 2. Gratifyingly, we could reliably synthesize the DOTA-conjugated dendrimer 2 with high yield (Fig. 2A).Also of note, we successfully prepared the stable dendrimer complex In-2 at 37 1C within 2 hours.Importantly, In-2 self-assembled into small and uniform nanomicelles for effective SPECT imaging of tumors.We present below the synthesis and characterization of the amphiphilic dendrimer 2 and its complex with In 3+ as well as the nanomicelles formed with the obtained dendrimer In-2 for SPECT imaging.
Similar to our previous synthesis of the NOTA-dendrimer 1, 11 we conjugated the amine-terminated amphiphilic dendrimer with the reagent DOTA-GA(tBu) 4 , followed by deprotection for preparing 2 (Fig. 2A).Compared to the synthesis of 1, we halved the quantity of the reagent DOTA-GA(tBu) 4 from 4 to 2 equivalents, which considerably simplified the purification procedure while maintaining the high synthesis yield of 88% for 2. The chemical structure and integrity of 2 was analyzed and confirmed using 1 H, 13 C and 2D NMR and high-resolution mass spectrometry (HRMS), which exhibited the signals characteristic of the chemically conjugated DOTA groups (Fig. S1 and S2, ESI †).Chelation of the stable isotope 115 In 3+ by 2 was performed using 115 InCl 3 at 37 1C, pH 5.0 for 2 hours (Fig. 2A), and the final dendrimer In-2 was obtained in pure form as a white solid after dialysis to remove the free 115 In 3+ .The successful complexation of four 115 In 3+ by each molecule of the dendrimer 2 was confirmed using HRMS, which showed the isotopic pattern characteristic of the triply charged species [ 115 In-2 + 3H] 3+ in addition to the expected molecular weight peak (Fig. 2B and Fig. S3B, ESI †). 14 It is important to mention that the formation of DOTA complexes with metal ions usually requires high temperature at 95 1C and long reaction time of several hours because of the slow binding kinetics of DOTA.Remarkably, we successfully chelated In 3+ with the DOTA-conjugated dendrimer 2 at relatively low temperature (37 1C) within 2 h.This may be ascribed to the steric congestion created at the dendrimer terminals, 15 making the DOTA entities in 2 more reactive and hence favorably promoting their complexation with In 3+ rapidly and at lower temperature to form the stable complex In-2.
To corroborate the reliable synthesis of In-2, we studied the formation of the complex between dendrimer 2 and In 3+ using isothermal titration calorimetry (ITC).Specifically, a solution of 2 at 100 mM was titrated with the solution of InCl 3 at pH = 5.0 and 37 1C (see the ESI † for details).The left panel in Fig. 2C shows that the interaction between the DOTA cages of 2 and the In 3+ cations is characterized by a robust exothermic behavior, reflecting an enthalpy-driven binding process (DH = À5.25 kcal mol À1 ) led by strong coordination interactions between the In 3+ and the DOTA cages of 2. Notably, the entropic component (ÀTDS = À2.61kcal mol À1 ) also favors the stability of In-2.This is probably due to a synergistic effect of the hydrophobic interactions between the apolar dendrimer tails, which aggregate together with the concomitant release of water and ions from the charged surfaces when they form complexes with the cations.Accordingly, the spontaneous formation of the In-2 complex is highly thermodynamically favorable, with a Gibbs free energy (DG) value of À7.86 kcal mol À1 .Interestingly, ITC measurements show that the number of 115 In 3+ in In-2 is 4.02, which confirms the ideal stoichiometry of 4 : 1.Taken together, the ITC results provide evidence that the synthesis of In-2 was successful, and that In-2 is a stable complex.
We next studied the self-assembly of In-2 in solution.In-2 spontaneously self-assembled into small and spherical nanoparticles with average dimensions around 18 nm, as revealed by transmission electron microscopy (TEM) (Fig. 3A).Also, dynamic light scattering (DLS) analysis confirmed the formation of small nanoparticles with sizes around 19 nm, which is typical for nanomicelles (Fig. 3B).The formed nanoparticles were stable, with the critical micelle concentration (CMC) being 60 AE 10 mM (Fig. S4, ESI †).Further molecular dynamics (MD) simulations confirmed the spontaneous aggregation of In-2 into spherical micelles.Fig. 3C illustrates a representative configuration of the stable In-2 micelles obtained at the end of the computational process starting from a random distribution of In-2 in solution.The calculated average micelle diameter was around 15 nm, in close agreement with the values obtained using experimental techniques (TEM and DLS).Along the entire MD trajectories, all the In 3+ /DOTA terminal groups were nicely located at the micellar periphery without any back-folding observed, as evident from the relevant radial distribution function of the terminal groups shown in Fig. 3D.
Encouraged by the favorable self-assembly properties of In-2, we prepared the corresponding radioactive dendrimer complex [ 111 In]In-2 for SPECT imaging.We obtained the [ 111 In]In-2 complex with a satisfying radiochemical purity over 91 AE 2% along with a high molar activity of 1.09 AE 0.15 GBq mmol À1 .In addition, this radiochemical purity and integrity was maintained for up to 30 hours at 37 1C in human serum (Fig. 4A).On the basis of the high radiochemical purity and stability, we performed SPECT imaging using [ 111 In]In-2 in orthotopically xenografted mice bearing tumors derived from a human pancreatic adenocarcinoma tumor cell line, SOJ-6 (Fig. 4B).Co-registration with CT enabled precise, anatomical localization of SPECT signals for further quantification.The biodistribution of

Fig. 3
Fig. 3 Self-assembling of the amphiphilic dendrimer In-2 into small and uniform nanomicelles.(A) Transmission electron microscopy (TEM) image, (B) dynamic light scattering (DLS) measurement, and (C and D) computer modeling of the self-assembled nanoparticles formed by In-2.(C) Final image of the In-2 self-assembly process into spherical micelles as obtained from atomistic molecular dynamics (MD) simulations.The different parts of the In-2 molecules are represented as spheres (atom color: grey, hydrocarbon chain; yellow, DOTA cage; black, In 3+ ), while water molecules are shown as aqua transparent spheres.The first water shell surrounding each molecule/micelle is highlighted as a light cyan transparent contour.(D) Radial distribution function of the In(III)-bearing terminals as a function of the distance from the center of mass of the In-2 micelles.