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Quantitative in vivo imaging of intratumorally injected 125I-labeled nanoparticles using a benchtop 2D multi-pinhole X-ray system

This study demonstrates that a benchtop 2D multi-pinhole X-ray imaging system using an energy-resolving detector can accurately quantify the intratumoral retention and biodistribution of 125I-labeled nanoparticles in mice, revealing high tumor selectivity and low off-target uptake to support preclinical optimization for nano-brachytherapy.

Original authors: Taeyun Kim, Taewan Kim, Hwijoon Jeong, Byeongchan Lee, Jun Young Lee, Jeong Hoon Park, Sung-Joon Ye

Published 2026-07-25

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CC BY 4.0

Original authors: Taeyun Kim, Taewan Kim, Hwijoon Jeong, Byeongchan Lee, Jun Young Lee, Jeong Hoon Park, Sung-Joon Ye

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

Imagine you are a tiny delivery driver trying to drop off a special package inside a specific house in a chaotic, crowded city. The problem is that the city's traffic police (your body's immune system) are very good at spotting delivery trucks and stopping them at the main checkpoints (the liver and spleen) before they ever reach the target house. Even if you get close, the package might leak out of the truck and get lost in the wrong neighborhoods. Scientists have been trying to figure out how to get these "nanoparticle" packages—tiny, engineered specks of matter—to stay exactly where they are needed, like inside a tumor, without getting lost or causing trouble elsewhere. To solve this, they need a way to watch these tiny packages move in real-time, like a security camera that can see through the walls of a mouse to track a glowing speck of gold. This is the world of preclinical imaging, where researchers use special cameras to see how medicine behaves inside living animals before it ever reaches a human patient.

Now, picture a team of scientists who decided to try a different strategy: instead of driving the package through the city streets (injecting it into the bloodstream), they decided to walk right up to the front door of the target house and hand-deliver it (injecting it directly into the tumor). But to make sure this "hand-delivery" actually worked, they needed a super-sensitive camera to watch the package over several days. They built a custom "benchtop" camera system—a device that sits on a table like a regular microscope but uses a special type of detector (a Cadmium Zinc Telluride sensor) that acts like a high-tech metal detector. This camera doesn't just see the package; it sees the unique "fingerprint" of light (characteristic X-rays) that the radioactive iodine inside the package emits.

In this study, the researchers used these special nanoparticles, which are like tiny golden marbles coated in a slippery, invisible shield (polyethylene glycol) to avoid the traffic police. They loaded these marbles with a radioactive tag, Iodine-125, and injected them directly into tumors in three mice. They then used their custom camera to take pictures of the mice at different times: right after the injection, and then again at 3, 6, 24, 96, and 120 hours later.

The results showed that the camera was incredibly good at its job. It could detect even the tiniest amount of the radioactive tag, down to 0.05 microcuries, and the pictures it took matched perfectly with the actual measurements taken after the experiment. When they looked at the data, they saw a clear story: right after the injection, a lot of the nanoparticles were still in the tumor, but some leaked out quickly. However, after the first 24 hours, the remaining nanoparticles stayed put. By the time 120 hours (five days) had passed, the tumor held onto about 26.41% of the total injected dose, which is a huge amount for a tiny spot. Meanwhile, the "traffic police" organs, like the liver and spleen, only held onto a tiny fraction (about 1.17% in the liver), and the thyroid (which often catches radioactive iodine) stayed relatively clean, holding about 1.92%.

The scientists found that the "direct hand-delivery" method worked much better than the usual "drive-through" method, where most of the medicine gets stuck in the liver before reaching the tumor. They also confirmed that the nanoparticles didn't break apart and release their radioactive tag too early, because the thyroid didn't light up like a beacon. This study suggests that injecting these special nanoparticles directly into a tumor, combined with this new, affordable camera system, is a promising way to keep the medicine exactly where it's needed for a long time, while keeping the rest of the body safe. It's a bit like successfully dropping a secret message into a specific mailbox and watching it stay there for days, without the post office stealing it or the neighbors finding it.

Technical Summary

Problem Statement
Radiolabeled nanoparticles (NPs) are critical for preclinical imaging and nano-brachytherapy, yet their utility is often hindered by challenges in accurate in vivo localization and quantification. Systemic administration frequently results in off-target accumulation in the reticuloendothelial system (RES), particularly the liver and spleen, which complicates quantitative evaluation and reduces effective tumor delivery. Furthermore, the potential for deiodination in radioiodinated tracers can lead to unwanted thyroid uptake, confounding biodistribution data. While micro-SPECT offers sensitive imaging, it is often resource-intensive and inaccessible for high-throughput screening. There is a need for practical, cost-effective preclinical workflows that support multi-timepoint imaging to capture retention kinetics and validate tumor selectivity without the complexity of full tomographic reconstruction.

Methodology
The authors developed and utilized a benchtop 2D multi-pinhole X-ray imaging system to detect characteristic X-ray emissions from iodine-125 ( $^{125}$ I). The system integrates a High Energy X-ray Imaging Technology (HEXITEC) 2 × 2 cadmium zinc telluride (CZT) detector with a multi-pinhole collimator. The detector operates at room temperature with an energy range of 4–200 keV and features a 4 cm × 4 cm active area composed of four tiled CZT modules. The collimator consists of four tungsten pinholes (2 mm inner diameter, 4.88 mm outer diameter) within a lead structure.

The study employed polyethylene glycol (PEG)-coated gold-core/silver-shell NPs labeled with $^{125}$ I ( $^{125}$ I-NPs). The system was first calibrated using phantom solutions of varying activities (3.9 to 500 $\mu$ Ci) to establish a linear relationship between activity and K-shell characteristic X-ray photon counts (27.5 keV). Image processing involved background subtraction, nonuniformity correction using an $^{241}$ Am source, and reconstruction via the maximum likelihood expectation maximization (MLEM) algorithm.

In the animal study, three CT26 tumor-bearing BALB/c mice received intratumoral injections of 500 $\mu$ Ci $^{125}$ I-NPs. In vivo imaging was conducted at 0, 3, 6, 24, 96, and 120 hours post-injection with 10-minute scan times. Following the final in vivo scan, mice were euthanized, and excised tumors and major organs (liver, spleen, thyroid, kidneys) were imaged ex vivo using the same system for endpoint validation.

Key Contributions

System Implementation: This work presents the first in vivo demonstration of characteristic X-ray imaging of $^{125}$ I using a benchtop multi-pinhole X-ray system equipped with a HEXITEC 2 × 2 CZT detector.
Quantitative Workflow: The study establishes a quantitative workflow combining multi-timepoint in vivo imaging with endpoint ex vivo validation on the same platform, reducing inter-animal variability and eliminating the need for different modalities (e.g., gamma counters) for validation.
Intratumoral Kinetics: The research provides a time-resolved assessment of $^{125}$ I-NP retention following intratumoral injection, distinguishing between early clearance and long-term retention phases.

Results

System Performance: The imaging system demonstrated a strong linear relationship between activity and photon counts ( $R^2 = 0.9992$ ). The detection limit was estimated at 0.05 $\mu$ Ci based on a contrast-to-noise ratio (CNR) of 5.
Biodistribution Kinetics: In vivo imaging revealed a marked decline in tumor activity within the first 24 hours, followed by a slower clearance phase through 120 hours. Liver uptake was highest immediately post-injection but decreased over time. Thyroid uptake showed an early increase but remained relatively stable, indicating limited deiodination.
Endpoint Quantification: At 120 hours, ex vivo imaging confirmed high tumor selectivity. The tumor retained $26.41 \pm 2.36\%$ of the injected dose (%ID) and $172.65 \pm 15.45\%$ ID/g. In contrast, liver uptake was low ( $1.17 \pm 0.90\%$ ID; $0.77 \pm 0.60\%$ ID/g), and thyroid uptake was $1.92 \pm 0.17\%$ ID ( $94.22 \pm 8.15\%$ ID/g). Uptake in the spleen and kidneys was indistinguishable from background.
Validation: The difference between in vivo and ex vivo measurements ranged from 0.03 to 3.51 $\mu$ Ci across organs, supporting the quantitative reliability of the 2D imaging system.

Significance and Claims
The paper claims that the benchtop multi-pinhole X-ray imaging system enables rapid, repeated, and quantitative assessment of intratumoral retention and biodistribution of $^{125}$ I-NPs. By demonstrating high tumor retention ( $~26\%$ ID) and low off-target uptake at 120 hours following intratumoral injection, the study supports the strategy of direct intratumoral administration to maximize local delivery while minimizing systemic exposure and RES sequestration. The authors posit that this system serves as a practical alternative to resource-intensive tomographic systems for preclinical optimization, particularly when tumor residence time and normal tissue safety are primary design criteria. The study concludes that the observed biodistribution profile, validated by consistent in vivo and ex vivo data, supports the suitability of these NPs for applications where sustained intratumoral localization is required.

Technical Summary

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