Self-assembling supramolecular dendrimer nanosystem for PET imaging of tumors

Amphiphilic Dendrimer 1 Bearing NOTA Terminals Is Able to Complex with Ga(III) and Self-Assemble into Small and Uniform Nanomicelles.

The NOTA-bearing dendrimer 1 devised for PET imaging in this work was synthesized starting with the amine-terminated amphiphilic dendrimer, which was conjugated with the reagent NODA-GA(tBu)₃, followed by subsequent deprotection. The dendrimer 1 was obtained with an excellent overall yield of 83% (Fig. 2A and SI Appendix, Scheme S1B), and its chemical structure was confirmed using ¹H, ¹³C, and 2D NMR and high-resolution mass spectrometry (HRMS), which revealed the characteristic signals corresponding to the chemically conjugated NOTA entities (SI Appendix, Figs. S1 and S2A). The characteristic signals for NOTA moieties in 1 could not be observed when simply mixing the amine-terminating dendrimer with the NOTA reagent in the absence of the coupling agent. This indicates the successful covalent conjugation of the NOTA functionality on the dendrimer terminals. Chelation of the isotope ⁶⁹Ga³⁺ by 1 using ⁶⁹GaCl₃ (Fig. 2A and SI Appendix, Scheme S1B), followed by dialysis to remove free ⁶⁹Ga³⁺, yielded the nonradioactive dendrimer [⁶⁹Ga]Ga-1. HRMS showed the isotopic pattern characteristic of the triply charged species [[⁶⁹Ga]Ga-1+3H]³⁺ in addition to the molecular weight peak (Fig. 2B and SI Appendix, Fig. S2B), confirming the successful complexation of four ⁶⁹Ga(III) ions within the dendrimer 1. Since ⁶⁹Ga³⁺ possesses a quadrupolar moment and very low NMR sensitivity (23), well-resolved NMR spectra for [⁶⁹Ga]Ga-1 could not be obtained. We therefore further studied [⁶⁹Ga]Ga-1 using isothermal titration calorimetry (ITC) (Fig. 2C), which demonstrated the spontaneous formation of the [⁶⁹Ga]Ga-1 complex (ΔG = −6.99 kcal/mol), resulting from the balanced and favorable contributions of both the enthalpic (ΔH = −4.05 kcal/mol) and the entropic component (−TΔS = −2.94 kcal/mol). Finally, the molar ratio identified by the ITC-derived number of occupied sites (n = 3.9) (Fig. 2C) confirmed the 4:1 stoichiometry for each [⁶⁹Ga]Ga-1 complex as derived from the HRMS analysis described above. All of these results substantiate the practical and reliable synthesis of amphiphilic dendrimers bearing either free NOTA terminals or Ga³⁺-chelated NOTA terminals.

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Fig. 2.

Synthesis of the amphiphilic dendrimer 1 and its chelation with the nonradioactive isotope [⁶⁹Ga]Ga³⁺ at the terminals. (A) Synthesis scheme: (i) (a) NODA-GA(tBu)₃, PyBOP, NMM, DMF, 30 °C, 72 h; (b)TFA, CH₂Cl₂, 30 °C, 16 h. (ii) [⁶⁹Ga]GaCl₃, 1.0 M HCl, 20 °C, 15 min. (B) High-resolution mass spectrum showing the isotopic pattern characteristic of the triply charged species [[⁶⁹Ga]Ga-1+3H]³⁺. The Inset shows the calculated isotopic pattern. (C) Isothermal titration calorimetry (ITC) curve for chelation of Ga³⁺ with dendrimer 1. The Inset shows measured heat power versus time elapsed during titration.

Having the Ga³⁺-chelated NOTA-terminating dendrimer [⁶⁹Ga]Ga-1 at hand, we studied its self-assembling properties. Although [⁶⁹Ga]Ga-1 is highly soluble in water (≥10 mg/mL), it self-assembles in water with a critical micelle concentration (CMC) of 64 ± 1 μM. We also assessed the octanol–water partition coefficient of [⁶⁹Ga]Ga-1 (log P), with the P value being −140 ± 17. This indicates that the [⁶⁹Ga]Ga-1 nanoparticles are hydrophilic, an advantageous feature for bioimaging. Dynamic light scattering (DLS) analysis also revealed that [⁶⁹Ga]Ga-1 formed small nanoparticles with average dimensions around 14 nm (Fig. 3A), a typical size for nanomicelles. The formed nanoparticles were stable and maintained similar size up to 1 wk (SI Appendix, Fig. S3). The effective formation of [⁶⁹Ga]Ga-1 nanomicelles was further confirmed by transmission electron microscopy (TEM) imaging, which showed small and spherical nanoparticles (Fig. 3B). We also examined the formation of [⁶⁹Ga]Ga-1 nanomicelles using atomistic molecular-dynamics (MD) simulations (24, 25). We observed that, during the timescale of the MD course (1.0 μs), the randomly distributed [⁶⁹Ga]Ga-1 complexes spontaneously aggregate into spherical micelles (Fig. 3 C and D) with an effective average radius of 6.9 ± 0.2 nm. Accordingly, the corresponding average [⁶⁹Ga]Ga-1 micelle diameter of 13.8 nm is in excellent agreement with the experimental value obtained from DLS and TEM (Fig. 3 A and B). Further examination of the conformational structures of the formed nanomicelles (Fig. 3D) and the radial distribution of the terminal functions (Fig. 3E) revealed that the terminals bearing the Ga(III) functions are all located on the micellar periphery, and no backfolding was observed. Collectively, the structural and physicochemical properties exhibited by these self-assembled nanomicelles make them ideal candidates for nanotechnology-based imaging purposes, as presented below.

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Fig. 3.

Self-assembling of the amphiphilic dendrimer [⁶⁹Ga]Ga-1 into small and uniform nanomicelles. (A) Dynamic light scattering (DLS) measurement, (B) transmission electron microscopy (TEM) image, and (C–E) computer modeling of the self-assembled nanostructures formed by [⁶⁹Ga]Ga-1. (C) Final image of the [⁶⁹Ga]Ga-1 self-assembly process into spherical micelles as obtained from atomistic molecular-dynamics (MD) simulations. Different parts of the [⁶⁹Ga]Ga-1 molecules are represented as spheres (atom color: gray, hydrocarbon chain; lavender, NOTA cage; hot pink, Ga³⁺), while water molecules are shown as aqua transparent spheres. The first water shell surrounding each molecule/micelle is highlighted as a dark aqua transparent contour. (D) Zoomed image of a [⁶⁹Ga]Ga-1 micelle as extracted from the equilibrated portion of the MD trajectory. Colors as in C. Water molecules are not shown for clarity. (E) Radial distribution function of the Ga(III)-bearing terminals as a function of the distance from the center of mass of the [⁶⁹Ga]Ga-1 micelles.

Radionuclide ⁶⁸Ga-Labeled Dendrimer Facilitates Excellent PET Imaging of Tumors.

Motivated by the favorable self-assembling properties of [⁶⁹Ga]Ga-1, we prepared the corresponding radioactive dendrimer [⁶⁸Ga]Ga-1 for PET imaging. Using freshly generated [⁶⁸Ga]GaCl₃, the [⁶⁸Ga]Ga-1 complex was obtained with a radiochemical purity of 91.9 ± 2.3% soon after synthesis (SI Appendix, Fig. S4A). In addition, radiolabeling was stable up to 2 h after synthesis at room temperature and at 37 °C, both in 0.9% NaCl solution and in human serum (SI Appendix, Fig. S4B). The [⁶⁸Ga]Ga-1 complex therefore satisfies the two important prerequisites for PET imaging, namely, high radiochemical purity and stability.

We then carried out PET imaging using the self-assembled nanomicelles of [⁶⁸Ga]Ga-1 to quantify their tumor targeting and uptake in various xenograft mouse models of cancer, including human prostate carcinoma (22Rv1 cell line; Fig. 4A), human glioblastoma (U87 cell line; Fig. 4B), human colorectal adenocarcinoma (HT-29 cell line; Fig. 4C), and human pancreatic adenocarcinoma (SOJ-6 cell line; Fig. 4D). In almost all cases, the observed effective tumor uptake and tumor-to-background ratios with [⁶⁸Ga]Ga-1 were superior to those obtained with [¹⁸F]FDG, the clinical gold reference for PET imaging in oncology (Fig. 4). [¹⁸F]FDG PET is based on the high consumption of glucose needed by the fast proliferating and nutrient-greedy cancer cells, and the major drawback of [¹⁸F]FDG is its physiological accumulation in tissues with high glucose consumption, like myocardium and brain (26), which increases the background signal and has a negative impact on the image quality. It should also be mentioned that certain tumors have low [¹⁸F]FDG uptake, including tumors derived from human glioblastoma U87 cells and human pancreatic adenocarcinoma SOJ-6 cells. Remarkably, our [⁶⁸Ga]Ga-1 nanoparticles had no preferential accumulation in the brain and a low uptake in the heart in all cases. Also, [⁶⁸Ga]Ga-1 successfully imaged tumors with both high and low [¹⁸F]FDG uptake, and the PET signal ratios were up to 14-fold higher with [⁶⁸Ga]Ga-1 nanoparticles than with [¹⁸F]FDG (Fig. 4). Therefore, we reasoned that the difference in [¹⁸F]FDG and [⁶⁸Ga]Ga-1 nanoparticle-based imaging could be ascribed to different tumor uptake mechanisms, with EPR-based tumor accumulation being the most plausible one for the uptake of [⁶⁸Ga]Ga-1.

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Fig. 4.

Radiolabeled dendrimer [⁶⁸Ga]Ga-1 for PET imaging of various tumors. Tumor targeting and uptake assessment of [⁶⁸Ga]Ga-1 in mouse ectopic xenograft models of (A) prostate adenocarcinoma (22Rv1 cell line), (B) glioma (U87 cell line), (C) colorectal adenocarcinoma (HT-29 cell line), and (D) pancreatic adenocarcinoma (SOJ-6 cell line), in comparison with [¹⁸F]FDG, the clinical gold reference for PET imaging in oncology. (Top) Representative examples of PET images. The orange arrows indicate tumor positions. (Middle) Quantifications from PET images of tumor uptake expressed as mean ± SD percentage injected dose per gram (n = 3; except for 22Rv1, n = 4; *P ≤ 0.05, Kruskal–Wallis test). (Bottom) Quantifications from PET images of tumor-to-background ratios (right forelimb biceps muscle taken as background reference). All data were expressed as mean ± SD percentage (n = 3; *P ≤ 0.05, Kruskal–Wallis test).

EPR-Based Tumor Uptake of [⁶⁸Ga]Ga-1 for Effective PET Imaging.

To investigate the uptake mechanism and obtain evidence for the EPR-based tumor accumulation of [⁶⁸Ga]Ga-1, we carried out a comparative study of PET imaging for tumor detection using the dendrimer nanosystem [⁶⁸Ga]Ga-1 and the small molecular complex [⁶⁸Ga]Ga-NOTA (SI Appendix, Scheme S1A) in three pancreatic cancer models, namely, ectopic SOJ6- and LIPC-xenografted mice (Fig. 5 A and B) and orthotopic SOJ6-xenografted mice (Fig. 5C). [¹⁸F]FDG was used as the reference control in all studies. Coregistration with CT enabled precise, anatomical localization of PET signals for further quantification in tumors. The overall excellent tumor-to-background ratios highlighted the high quality of images and tumor targeting obtained with the [⁶⁸Ga]Ga-1 nanosystem. Indeed, the tumor uptake of the [⁶⁸Ga]Ga-1 nanoparticles was significantly higher than that of the simple [⁶⁸Ga]Ga-NOTA complex and the clinical reference [¹⁸F]FDG (up to 100-fold increase) not only in the two ectopic pancreatic cancer mouse models (Fig. 5 A and B) but also in the orthotopic pancreatic cancer model (Fig. 5C). Moreover, we observed a significant negative correlation between the tumor uptake of [⁶⁸Ga]Ga-1 and tumor blood flow as measured by 3D-Doppler (Fig. 5D). This suggests that [⁶⁸Ga]Ga-1 is more likely to accumulate in low-turbulent blood flow tumors, where the EPR effect contribution takes effect (27, 28). These results demonstrated that the significantly enhanced tumor uptake and the resulting superior imaging quality of [⁶⁸Ga]Ga-1 indeed stem from the nanoparticle-based EPR effect of the tumor microenvironment, which is distinctly different from the uptake mechanism of [¹⁸F]FDG. The different uptake mechanisms of [⁶⁸Ga]Ga-1 and [¹⁸F]FDG mean that these two agents can be used in a complementary manner for tumor imaging, with [⁶⁸Ga]Ga-1 offering superior imaging, especially for the tumors that are otherwise undetectable using the clinical reference [¹⁸F]FDG.

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Fig. 5.

EPR-based tumor uptake of [⁶⁸Ga]Ga-1 for effective PET imaging. Comparison of the tumor accumulation of the dendrimer nanosystem [⁶⁸Ga]Ga-1, the small molecular complex [⁶⁸Ga]Ga-NOTA, and the clinical reference [¹⁸F]FDG in ectopic (A) SOJ6- and (B) LIPC-xenografted mice as well as in (C) orthotopic SOJ6-xenograft mice. (Top) Representative examples of [¹⁸F]FDG (Left), [⁶⁸Ga]Ga-NOTA (Middle), and [⁶⁸Ga]Ga-1 (Right) PET images. The orange arrows indicate tumor positions. (Middle) Quantifications from PET images of tumor uptake, expressed as mean ± SD percentage injected dose per gram (n = 3; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, Kruskal–Wallis test). (Bottom) Quantifications from PET images of tumor-to-background ratios (right forelimb biceps muscle taken as background reference), expressed as mean ± SD percentage (n = 3; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Kruskal–Wallis test). (D) Correlation between [⁶⁸Ga]Ga-1 PET signal in tumors and tumor blood flow assessed by 3D-Doppler (percentage) in orthotopic SOJ6-xenografted mice (n = 3; two time points; Pearson correlation R² = 0.751; *P = 0.0255).

Advantageous Pharmacokinetics and Safety Profile of [⁶⁸Ga]Ga-1 for PET Imaging.

Encouraged by these promising PET imaging results of [⁶⁸Ga]Ga-1, we further studied the pharmacokinetics and biodistribution of [⁶⁸Ga]Ga-1 in the orthotopic xenograft mice using µPET dynamic acquisitions (Fig. 6). This allowed us to trace and quantify the distribution of [⁶⁸Ga]Ga-1 across body organs from 5 min up to 2 h postinjection. Unlike drug delivery procedures, which require a constant supply of drug to the target tissue, bioimaging demands a rather rapid and intense accumulation of the imaging agent within tumor lesions and a fast clearance of the nonspecific, nonfixed radiotracer. By virtue of its distinct nanostructure, [⁶⁸Ga]Ga-1 has a biological elimination half-life of 171 ± 42 min, as obtained from pharmacokinetic analysis of [⁶⁸Ga]Ga-1 in blood samples (SI Appendix, Fig. S5). This biological half-life of [⁶⁸Ga]Ga-1 is particularly advantageous for PET imaging, as it allows effective image capture shortly after its administration (29, 30). This feature, coupled with the short physical half-life [⁶⁸Ga]Ga (68 min), allows for both rapid and optimal imaging and fast elimination of radioactivity from the body. Further studies showed that the uptake of [⁶⁸Ga]Ga-1 in tumors was steadily increased, while that in the liver was significantly decreased within the 2-h imaging period (Fig. 6). This finding is also in line with the characteristic size and charge features of the [⁶⁸Ga]Ga-1 nanoparticles. Indeed, their nanosize allowed them to be readily trapped and enriched in the tumor via the EPR effect, and in the liver through the reticuloendothelial system, whereas their elimination was balanced between the kidneys and the intestines (Fig. 6). Collectively, the combination of high tumor signal stemming from the EPR effect, short biological half-life, and the absence of accumulation in most of the organs except the liver, all contribute to the favorable pharmacokinetic properties of the [⁶⁸Ga]Ga-1 nanoparticles for PET imaging of tumors.

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Fig. 6.

The biodistribution and pharmacokinetics of [⁶⁸Ga]Ga-1 in the orthotopic xenograft mice using µPET dynamic acquisitions. (A) Representative images from PET imaging of [⁶⁸Ga]Ga-1 biodistribution in an orthotopic SOJ6-xenograft mouse 10, 20, 30, 60, 90, and 120 min after i.v. injection (fused PET and contrast-enhanced CT maximum intensity projections images). The orange ovals indicate tumor position. (B) Quantifications in organs from PET imaging of [⁶⁸Ga]Ga-1 biodistribution in mice 5, 10, 20, 30, 60, 90, and 120 min after i.v. injection. Organ uptakes are represented as mean ± SD percentage injected dose for each organ and each time point (n = 3 mice).

It should be mentioned that, during the experimental period, the mice receiving [⁶⁸Ga]Ga-1 did not show any abnormal behavior or adverse effects. Healthy mice administered with [⁶⁸Ga]Ga-1 showed no cytokine inducement, neither blood biochemistry defects nor organ damage, even when the administered dose of [⁶⁸Ga]Ga-1 was 10 times higher than the PET imaging dose (SI Appendix, Figs. S6–S8). These results confirm that [⁶⁸Ga]Ga-1 is devoid of toxic effects while delivering superior PET imaging quality.