Enhanced Hepatobiliary Excretion of Micelle encapsulated Upconverting Nanoparticles using in vivo PET and Optical Imaging

Abstract

Purpose: Nanoparticle (NP) accumulation and toxicity in variable organs have been an obstacle for biomedical application. Therefore, the characteristics of good excretion and biocompatibility of NPs are very important. One of the candidate biocompatible NPs, upconverting nanoparticle (UCNP) has emerged for biomedical application recently. Herein, I demonstrated that micelle encapsulated 64Cu-labeled upconverting nanoparticles (micelle encapsulated 64Cu-NOTA-UCNPs) showed substantial hepatobiliary excretion within 24 hours by in vivo positron emission tomography (PET) and also upconversion luminescence imaging (ULI). A long half-life (762 min) radiotracer 64Cu is a competent material to evaluate biodistribution and quantification for 72 hours. And I quantified the excretion of micelle encapsulated 64Cu-NOTA-UCNPs by PET and ex vivo method and revealed the excretory pathway by PET, ULI and transmission electron microscopy (TEM).

Methods: NaYF4:Yb3+, Er3+ NPs (UCNPs) were encapsulated and labeled with NOTA-C18 and 64Cu. After micelle encapsulated 64Cu-NOTA-UCNPs was intravenously injected in 6-week-old male BALB/c nude mice (weight = 24.1 ± 0.5 g, dose = 40 ± 0.6 μCi/50 μL, n = 7), whole body microPET was performed to obtain serial time point images (0.25, 1, 2, 4, 8, and 24 hours, n = 3 / 0.25, 1, 2, 4, 8, 24, 48 and 72 hours, n = 4) for in vivo biodistribution. To compare study of free 64Cu (n = 3), 64Cu-NOTA-C18 (n = 3) and 64Cu-NOTA-UCNPs (n = 7), microPET was performed as serial time points (0.25, 1, 2, 4, 8, and 24 hours). For ex vivo biodistribution, micelle encapsulated 64Cu-NOTA-UCNPs were injected into a male BALB/c mouse via the tail vein (weight = 20.1 ± 0.5 g, dose = 1 μCi/100 μL). The injected mice were sacrificed in serial time points (1, 4, 8, 24, and 72 hours, 3 mice for collection of feces and urine, n = 3, respectively, total n = 18) of post-injection. For ULI biodistribution study, UCNPs were injected intravenously in BALB/c nude mice (weight = 24.1 ± 0.5 g, dose = 0.158 mg/300 μL, total n = 7). In vivo, in situ and ex vivo ULIs were obtained as serial time points (1, 2, 4, 8, and 24 hours). Collected feces and urine were evaluated by microPET and ULI. Extracted liver was evaluated by TEM to reveal the hepatobiliary excretion as two time points (1 hour, n = 3, and 24 hours, n = 4).

Results: Using micelle encapsulation method, the final yield of micelle encapsulated 64Cu-NOTA-UCNPs was 40%. The radiochemical purity of micelle encapsulated 64Cu-NOTA-UCNPs was 99%. In vivo whole body microPET image revealed the remarkable hepatobiliary excretion according to the delayed time points. Hepatic uptake persisted until 8 hour delay image, but it was markedly decreased at 24 hour delay image and further decreased until 72 hours. Linearly increased uptake along the bowel was shown after 1 hour and the uptake was further extended along the intestine at 2, 4 and 8 hour delay images. Also, micelle encapsulated 64Cu-NOTA-UCNPs showed faster hepatic excretion than free 64Cu and the biodistribution of micelle encapsulated 64Cu-NOTA-UCNPs was different from that of 64Cu-NOTA-C18. Similarly, in our ex vivo biodistribution study, over 80% clearance from initial liver uptake within 72 hours and over 40% of total injected dose excretion by feces within 24 hours after injection of NPs have been revealed. The concentration of liver was increased till 8 hours and markedly decreased at 24 hours. Gradual increase of the concentration of intestine had been manifested till 8 hours and the concentration of intestine was decreased at 24 hours. Collected feces in ex vivo biodistribution consisted of 40.9%. Likewise, in vivo, in situ and ex vivo ULI revealed good hepatobiliary excretion. However, we could obtain the sufficient signal for in vivo ULI which was 13 times higher amount (158 μg/mouse) than that for the dose of PET imaging (12 μg/mouse). In 1 hour post-injection ex vivo image, liver, spleen and lung showed positive signals which were consistent with PET image. The intestinal uptake was not well-visualized in in vivo image, but after extraction and exposure of intestinal lumen, UCNP signal was clearly detected. Additionally, the evidences of hepatobiliary excretion of micelle encapsulated 64Cu-NOTA-UCNPs were obtained by ULI, PET and TEM. ULI revealed UCNP signal in all collected feces and urine. PET showed excretory 64Cu-NOTA-UCNPs in all collected feces. In TEM images, UCNPs were observed inside of sinusoid, space of Disse, hepatocyte, Kupffer cells and bile canaliculi at 1 hour after injection. However, NP was not found inside of hepatocyte, but remained in Kupffer cells at 24 hours after injection.

Conclusions: In conclusion, I demonstrated the feasibility of bimodal in vivo imaging characteristics of micelle encapsulated 64Cu-NOTA-UCNP and showed the substantial hepatobiliary excretion through in vivo microPET, ULI, and ex vivo biodistribution study. This bimodal imaging characteristic is ideal for the evaluation of excretion pattern of NP, because PET imaging is suitable for accurate quantification of biodistribution and ULI can be used for the confirmation of UCNP. Moreover, by well characterization of imaging property and excretion pattern of micelle encapsulated 64Cu-NOTA-UCNPs, NPs could be used for bimodal ULI and PET imaging agent for a variety of purposes by adding further functional moiety.