Quantitative Biodistribution Research Articles

Effect of molar dose on the in vivo tissue biodistribution profile of FAP-targeted radioligand therapeutics.

177Lu-OncoFAP-23 is a novel FAP-targeted radioligand therapeutic (RLT) with high and prolonged tumor residence time and promising preclinical efficacy. In this work, we investigated the correlation between the injected molar dose and the in vivo tumor-to-organ ratios and tumor-targeting performance of 177Lu-OncoFAP-23. We evaluated the quantitative biodistribution profile of 177Lu-OncoFAP-23 at different molar doses (i.e., 3 to 2250nmol/kg) in tumor-bearing mice by means of ex vivo gamma counting, we included 177Lu-OncoFAP and 177Lu-BiOncoFAP as experimental controls. The biodistribution profile of 177Lu-OncoFAP-23 strongly depends on the molar dose injected. Molar doses below 30nmol/kg result in unwanted uptake of the compound in healthy organs, while doses higher than 725nmol/kg determined a reduced tumor uptake due to receptor saturation. We identified an optimal molar dose ranging from 90 to 250nmol/kg, characterized by elevated tumor uptake and adequate tumor-to-organ ratios. 177Lu-OncoFAP-23 presents a favorable in vivo biodistribution profile at molar doses ranging from 90 to 250nmol/kg in tumor-bearing mice. Our results guide the design of the first-in-human Phase I clinical trial with this novel FAP-targeted radioligand therapeutic.

Site-Specific Modification of Native IgGs with Flexible Drug-Load.

Homogeneous, site-specifically conjugated antibodies have shown to result in antibody-drug conjugates (ADCs) with improved efficacy and tolerability compared to stochastically conjugated ADCs. However, precisely controlling the drug load as well as attaching multiple payload moieties to the antibody remains challenging. Here, we demonstrate the simple and direct modification of native IgG-antibodies at the residue glutamine 295 (Q295) without the need for any protein engineering with flexible drug-to-antibody ratios of one or multiple payloads. The conjugation is enabled through short, positively charged lysine containing peptides and native, commercially available microbial transglutaminase. In proof-of-concept studies, HER2-targeting ADCs based on trastuzumab were generated with drug-to-antibody ratios (DARs) of 2 and 4 of the same or different payloads using orthogonal conjugation chemistries. Quantitative biodistribution studies performed with 111In-radiolabeled conjugates showed high tumour uptake and low accumulation of radioactivity in non-targeted tissues. A single dose study of trastuzumab conjugated to the highly potent payload α-Amanitin demonstrated complete and long-lasting tumour remission and was well-tolerated at all dose levels tested.

Quantitative Biodistribution of OMVs Using SPECT/CT Imaging with HYNIC-Duramycin Radiolabeling.

Introduction: Bacterial outer membrane vesicles (OMVs) are emerging as important players in the host-microbiome interaction, while also proving to be a promising platform for vaccine development and targeted drug delivery. The available methods for measuring their biodistribution, however, are limited. We aimed to establish a high-efficiency radiolabeling method for the treatment of OMVs. Methods: 99mTc-HYNIC-duramycin was incubated with OMVs isolated from E. coli BL21(DE3) ΔnlpI ΔlpxM. Radiolabeling efficiency (RLE) and radiochemical purity (RCP) were measured with size-exclusion high-performance liquid chromatography. The biodistribution was quantitatively measured in mice using SPECT/CT imaging. Results: RLE was 81.84 ± 2.03% for undiluted OMV suspension and 56.17 ± 2.29% for 100× dilution. Postlabeling purification with a spin-desalting column results in 100% radioactivity in the OMV fraction according to HPLC, indicating 100% RCP of the final product. The biodistribution was found to be in line with previous data reported in the literature using other OMV tracking attempts. Conclusions: Our findings illustrate that using HYNIC-duramycin for labeling of the OMVs enhances efficiency and is easily implementable for in vivo imaging studies, significantly improving upon earlier methods.

(Invited) Designing Quantum Dots for Multiplexed Deep-Tissue Imaging in the NIR-II

The combination of bright, tunable semiconductor quantum dots (QDs) emitting in the shortwave infrared or second near infrared window (SWIR/NIR-II, 1000 – 1600 nm) and InGaAs camera-equipped preclinical imagers opens the door to non-invasive, multiplexed, deep tissue imaging in live mice. While imaging in the NIR-I is like imaging through fog due to highly scattering tissue, imaging in the SWIR/NIR-II is more like imaging through pond water, enabling deeper imaging with much improved resolution. We synthesized multiple PbS/CdS core/shell QDs with emission wavelengths spanning 1000-1600 nm. These particles have been encapsulated in lipid-PEG micelles and up to three colors (so far) dosed into nude mice for imaging on a Photon Etc IR VIVO imager equipped with a hyperspectral Bragg grating and 6 bandpass filters, enabling demixing of the discrete colors. We have obtained exceptionally clear images of lymphatic drainage and the vasculature system, including visualization of lymphatic valve opening in videos. Interestingly, following intravascular injection, the liver and spleen are very visible and distinct, enabling the non-invasive visualization of a key nanomedicine drug delivery parameter (i.e., are the nanoparticles accumulating in the liver or the spleen?). Image processing investigations are expanding our ability to demix colors as well as to determine the average depth of the contrast agent, based on the wavelength-dependent attenuation of the photoluminescence based on water absorption. In addition to the photophysical advantages of using QDs for NIR-II, elemental analysis of blood and tissue samples enables quantitative biodistribution and pharmacokinetic studies. Ongoing work involves the application of this imaging approach to assess the impact nanomaterial surface properties and route of delivery on their pharmacokinetics and the use of deep tissue paired agent imaging for non-invasive “optical biopsy” of orthotopic tumors. Current data are already visually stunning, and ongoing imaging highlights new opportunities for research with every experiment! Figure 1

(Invited) Quantitative in Vitro/In Vivo Fluorescence Bioimaging with Graphene Quantum Dots

Graphene Quantum Dots (GQDs) are novel carbon nanomaterials consisting of few layers of partially functionalized graphitic carbon. Their nanometer-scale size together with functional groups render GQDs more water soluble and biocompatible than many other nanocarbon constructs. Like many nanocarbons, GQDs possess structure-dependent optical properties, exhibiting confinement-defined fluorescence in the visible and defect-originated emission in the near-infrared (NIR). These properties give GQDs a certain advantage for applications in therapeutic tracking, imaging, analyte detection and targeted drug/gene delivery. In addition to tracing the location of therapeutic or analyte, GOD fluorescence allows for several modes of quantitative assessment within these applications. In vitro it is utilized to evaluate relative amounts of therapeutics or analytes present within the cells at a certain time period. In vivo NIR emission of the GQDs-based sensors implanted under the skin can help detect trace quantities of analyte in the body, while their NIR fluorescence in animal organs can quantify relative therapeutic uptake over time.The present work introduces novel studies and analytical tools exploring and enhancing such imaging modalities. Although GQD in vitro fluorescence microscopy imaging is often used to quantitate their (and payload) uptake and excretion, this method suffers from multiple discrepancies including the analysis of dead cells, cellular debris and non-internalized nanomaterials, and can be also time and resource-consuming. Our work aiming to circumvent these issues utilizes Artificial Intelligence (AI) approach to provide more accurate quantification of the uptake/excretion of the GQDs and their payload. In this application the AI algorithms developed in house do not only optimize the imaging, but also provide automated image analysis streamlining the assessment of a variety of GQD biomedical applications including therapeutic delivery and targeting. In vivo quantitative animal imaging with several different types of GQDs, from pristine to doped with rare earth metals for NIR fluorescence enhancement, is performed to evaluate GQD organ accumulation over time. Defect or dopant-originated NIR GQD fluorescence in the range of 900 – 1060 nm due to its high penetration depth can be observed from the organs of sedated live mice and utilized for GQD uptake tracking. NIR imaging of the excised organs allows for quantitative biodistribution assessment with further microscopic confirmation of the GQD content in organ tissue slices. Quantitative optical imaging and analysis presented in this work for GQDs can aid the further advancement of these novel nanomaterials in therapeutic delivery and analyte detection applications in vitro and in vivo.

Photoacoustic imaging of a cyanine dye targeting bacterial infection

The development of a non-invasive infection-specific diagnostic probe holds the potential to vastly improve early-stage detection of infection, enabling precise therapeutic intervention and potentially reducing the incidence of antibiotic resistance. Towards this goal, a commercially available bacteria-targeting Zinc(II)-dipicolylamine (ZnDPA)-derived fluorophore, PSVue794, was assessed as a photoacoustic (PA) imaging probe (PIP). A radiolabeled version of the dye, [99mTc]Tc-PSVue794, was developed to facilitate quantitative biodistribution studies beyond optical imaging methods, which showed a target-to-non-target ratio of 10.1 ± 1.1, 12 h post-injection. The ability of the PIP to differentiate between bacterial infection, sterile inflammation, and healthy tissue in a mouse model, was then evaluated via PA imaging. The PA signal in sites of sterile inflammation (0.062 ± 0.012 a.u.) was not statistically different from that of the background (0.058 ± 0.006 a.u.). In contrast, high PA signal was detected at sites of bacterial infection (0.176 ± 0.011 a.u.) as compared to background (0.081 ± 0.04 a.u., where P ≤ 0.03). This work demonstrates the potential of utilizing established fluorophores towards PAI and utilizing PAI as a modality in the distinction of bacterial infection from sites of sterile inflammation.

Design of a Fibroblast Activation Protein-Targeted Radiopharmaceutical Therapy with High Tumor-to-Healthy-Tissue Ratios.

Because of upregulated expression on cancer-associated fibroblasts, fibroblast activation protein (FAP) has emerged as an attractive biomarker for the imaging and therapy of solid tumors. Although many FAP ligands have already been developed for radiopharmaceutical therapies (RPTs), most suffer from inadequate tumor uptake, insufficient tumor residence times, or off-target accumulation in healthy tissues, suggesting a need for further improvements. Methods: A new FAP-targeted RPT with a novel ligand (FAP8-PEG3-IP-DOTA) was designed by combining the desirable features of several previous ligand-targeted RPTs. Uptake and retention of [111In]In or [177Lu]Lu-FAP8-PEG3-IP-DOTA were assessed in KB, HT29, MDA-MB-231, and 4T1 murine tumor models by radioimaging or ex vivo biodistribution analyses. Radiotherapeutic potencies and gross toxicities were also investigated by monitoring tumor growth, body weight, and tissue damage in tumor-bearing mice. Results: FAP8-PEG3-IP-DOTA exhibited high affinity (half-maximal inhibitory concentration, 1.6 nM) and good selectivity for FAP relative to its closest homologs, prolyl oligopeptidase (half-maximal inhibitory concentration, ∼14.0 nM) and dipeptidyl peptidase-IV (half-maximal inhibitory concentration, ∼860 nM). SPECT/CT scans exhibited high retention in 2 different solid tumor models and minimal uptake in healthy tissues. Quantitative biodistribution analyses revealed tumor-to-healthy-tissue ratios of more than 5 times for all major organs, and live animal studies demonstrated 65%-93% suppression of tumor growth in all 4 models tested, with minimal or no evidence of systemic toxicity. Conclusion: We conclude that [177Lu]Lu-FAP8-PEG3-IP-DOTA constitutes a promising and safe RPT candidate for FAPα-targeted radionuclide therapy of solid tumors.

Quantitative pharmacokinetic and biodistribution studies for fluorescent imaging agents.

Pharmacokinetics and biodistribution studies are essential for characterizing fluorescent agents in vivo. However, few simple methods based on fluorescence imaging are available that account for tissue optical properties and sample volume differences. We describe a method for simultaneously quantifying mean fluorescence intensity of whole blood and homogenized tissues in glass capillary tubes for two fluorescent agents, ABY-029 and IRDye 680LT, using wide-field imaging and tissue-specific calibration curves. All calibration curves demonstrated a high degree of linearity with mean R2 = 0.99 ± 0.01 and RMSE = 0.12 ± 0.04. However, differences between linear regressions indicate that tissue-specific calibration curves are required for accurate concentration recovery. The lower limit of quantification (LLOQ) for all samples tested was determined to be < 0.3 nM for ABY-029 and < 0.4 nM for IRDye 680LT.

Performance of single-cell ICP-MS for quantitative biodistribution studies of silver interactions with bacteria

Single-cell inductively couple plasma mass spectrometry (SC-ICP-MS) method development for the study of silver interactions with bacteria to provide information about silver biodistribution in bacteria and spheroplasts.

Quantitative biodistribution of nanoparticles in plants with lanthanide complexes

The inefficient distribution of fertilizers, nutrients, and pesticides on crops is a major challenge in modern agriculture that leads to reduced productivity and environmental pollution. Nanoformulation of agrochemicals is an attractive approach to enable the selective delivery of agents into specific plant organs, their release in those tissues, and improve their efficiency. Already commercialized nanofertilizers utilize the physiochemical properties of metal nanoparticles such as size, charge, and the metal core to overcome biological barriers in plants to reach their target sites. Despite their wide application in human diseases, lipid nanoparticles are rarely used in agricultural applications and a systematic screening approach to identifying efficacious formulations has not been reported. Here, we developed a quantitative metal-encoded platform to determine the biodistribution of different lipid nanoparticles in plant tissues. In this platform lanthanide metal complexes were encapsulated into four types of lipid nanoparticles. Our approach was able to successfully quantify payload accumulation for all the lipid formulations across the roots, stem, and leaf of the plant. Lanthanide levels were 20- to 57-fold higher in the leaf and 100- to 10,000-fold higher in the stem for the nanoparticle encapsulated lanthanide complexes compared to the unencapsulated, free lanthanide complex. This system will facilitate the discovery of nanoparticles as delivery carriers for agrochemicals and plant tissue-targeting products.

Radiolabeling of Platelets with 99mTc-HYNIC-Duramycin for In Vivo Imaging Studies

Following the in vivo biodistribution of platelets can contribute to a better understanding of their physiological and pathological roles, and nuclear imaging methods, such as single photon emission tomography (SPECT), provide an excellent method for that. SPECT imaging needs stable labeling of the platelets with a radioisotope. In this study, we report a new method to label platelets with 99mTc, the most frequently used isotope for SPECT in clinical applications. The proposed radiolabeling procedure uses a membrane-binding peptide, duramycin. Our results show that duramycin does not cause significant platelet activation, and radiolabeling can be carried out with a procedure utilizing a simple labeling step followed by a size-exclusion chromatography-based purification step. The in vivo application of the radiolabeled human platelets in mice yielded quantitative biodistribution images of the spleen and liver and no accumulation in the lungs. The performed small-animal SPECT/CT in vivo imaging investigations revealed good in vivo stability of the labeling, which paves the way for further applications of 99mTc-labeled-Duramycin in platelet imaging.

Hybrid core-shell particles for mRNA systemic delivery.

mRNA based infectious disease vaccines have opened the venue for development of novel nucleic acids-based therapeutics. For all mRNA therapeutics dedicated delivery systems are required, where different functionalities and targeting abilities need to be optimized for the respective applications. One option for advanced formulations with tailored properties are lipid-polymer hybrid nanoparticles with complex nanostructure, which allow to combine features of several already well described nucleic acid delivery systems. Here, we explored hyaluronic acid (HA) as coating of liposome-mRNA complexes (LRCs) to investigate effects of the coating on surface charge, physicochemical characteristics and biological activity. HA was electrostatically attached to positively charged complexes, forming hybrid LRCs (HLRCs). At different N/P ratios, physico-chemical characterization of the two sets of particles showed similarity in size (around 200nm) and mRNA binding abilities, while the presence of the HA shell conferred a negative surface charge to otherwise positive complexes. High transfection efficiency of LRCs and HLRCs in vitro has been obtained in THP-1 and human monocytes derived from PBMC, an interesting target cell population for cancer and immune related pathologies. In mice, quantitative biodistribution of radiolabeled LRC and HLRC particles, coupled with bioluminescence studies to detect the protein translation sites, hinted towards both particles' accumulation in the hepatic reticuloendothelial system (RES). mRNA translated proteins though was found mainly in the spleen, a major source for immune cells, with preference for expression in macrophages. The results showed that surface modifications of liposome-mRNA complexes can be used to fine-tune nanoparticle physico-chemical characteristics. This provides a tool for assembly of stable and optimized nanoparticles, which are prerequisite for future therapeutic interventions using mRNA-based nanomedicines.

PD-L1/TLR7 dual-targeting nanobody-drug conjugate mediates potent tumor regression via elevating tumor immunogenicity in a host-expressed PD-L1 bias-dependent way

BackgroundVarious tumors are insensitive to immune checkpoint blockade (ICB) therapy. Toll-like receptors (TLRs) establish the link between innate and adaptive immunity, which can assist T-cell activation and serve as promising...

Precise quantitative evaluation of pharmacokinetics of cisplatin using a radio-platinum tracer in tumor-bearing mice.

ObjectiveThe platinum-based antineoplastic drug cisplatin is commonly used for chemotherapy in clinics. This work aims to demonstrate a radio-platinum tracer is useful for precisely quantifying small amounts of platinum in pharmacokinetics studies.MethodsA cisplatin radiotracer (radio-cisplatin) was synthesized, and a comprehensive evaluation of cisplatin over 7 days after its intravenous injection into nude mice bearing a subcutaneous lung tumor (H460) was conducted.ResultsA biphasic retention curve in the whole body and blood was observed [T1/2(α) = 1.14 h, T1/2(β) = 5.33 days for the whole body, and T1/2(α) = 23.9 min, T1/2(β) = 4.72 days for blood]. The blood concentration decreased within 1 day after injection. Most of the intact cisplatin was excreted via the kidneys in the early time points, and a small part was distributed in tissues including tumors. The plasma protein binding rate of cisplatin increased rapidly after injection, and the protein-bound cisplatin remained in the blood longer than intact cisplatin. The peak uptake in H460 tumors was 4.7% injected dose per gram at 15 min after injection, and the area under the curve (AUC0–7 days) was approximately one-half to one-third of the AUC0–7 days in the kidneys, liver, and bone, where some toxicity is observed in humans.ConclusionThe radio-platinum tracer revealed the highly quantitative biodistribution of cisplatin, providing insights into the properties of cisplatin, including its adverse effects. The tracer enables a precise evaluation of pharmacokinetics for platinum-based drugs with high sensitivity.

Quantitative Biodistribution and Pharmacokinetics Study of GMP-Grade Exosomes Labeled with 89Zr Radioisotope in Mice and Rats.

For the successful clinical advancement of exosome therapeutics, the biodistribution and pharmacokinetic profile of exogenous exosomes in various animal models must be determined. Compared with fluorescence or bioluminescence imaging, radionuclide imaging confers multiple advantages for the in vivo tracking of biomolecular therapeutics because of its excellent sensitivity for deep tissue imaging and potential for quantitative measurement. Herein, we assessed the quantitative biodistribution and pharmacokinetics of good manufacturing practice-grade therapeutic exosomes labeled with zirconium-89 (89Zr) after systemic intravenous administration in mice and rats. Quantitative biodistribution analysis by positron emission tomography/computed tomography and gamma counting in mice and rats revealed that the total 89Zr signals in the organs were lower in rats than in mice, suggesting a higher excretion rate of exosomes in rats. A prolonged 89Zr signal for up to 7 days in most organs indicated that substantial amounts of exosomes were taken up by the parenchymal cells in those organs, highlighting the therapeutic potential of exosomes for the intracellular delivery of therapeutics. Exosomes were mainly distributed in the liver and to a lesser extent in the spleen, while a moderately distributed in the kidney, lung, stomach, intestine, urinary bladder, brain, and heart. Exosomes were rapidly cleared from the blood circulation, with a rate greater than that of free 89Zr, indicating that exosomes might be rapidly taken up by cells and tissues.

Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21.

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory activity of NK and CD8+ T-cells. Here we describe four formats of novel IL21-based antibody–cytokine fusion proteins, targeting the extra domain A (EDA) of fibronectin and explore their potential for cancer treatment. The fusion proteins were designed, expressed, and characterized. F8 in single-chain diabody (scDb) format fused to IL21 at its C-terminus exhibited a promising profile in size exclusion chromatography (SEC) and SDS-PAGE. The lead candidate was further characterized in vitro. A cell-based activity assay on murine cytotoxic T-cells showed that human IL21, compared to murine IL21 partially cross-reacted with the murine receptor. The prototype was able to recognize EDA as demonstrated by immunofluorescence analysis on tumor sections. In an in vivo quantitative biodistribution experiment, F8(scDb)-murine IL21 did not preferentially accumulate at the site of disease after intravenous injection, suggesting that additional protein engineering would be required to improve the tumor-homing properties of IL21-based product.

Abstract 1866: Pharmacokinetics and biodistribution of formatted Affimer® biotherapeutics targeting PD-L1

Abstract Programmed Cell Death-Ligand 1 (PD-L1) is part of the immune checkpoint system involved in preventing autoimmunity. PD-L1 is upregulated on tumors cells and binds to its receptor, PD-1, expressed by immune cells in the tumor microenvironment. Anti-PD-L1 immunotherapies have shown to be effective treatments both as monotherapies and in combinations with chemotherapies and radiotherapies, providing long term responses in a subset of patients. We have developed a PD-L1 Affimer antagonist that could differentiate itself from the current clinically approved mAbs due to its smaller size, improved tissue penetration and alternative routes of delivery such as a subcutaneous injection. It also opens up the possibility of generating additional PD-L1 antagonist therapeutics such as bispecifics and cytokine fusions. The Affimer biotherapeutic scaffold is a 14kDa monomeric protein based on the human protease inhibitor Stefin A, lacking post-translational modifications such as disulfide bonds and glycosylation. We have identified a range of high-affinity competitive binders to human PD-L1 confirmed by SPR using Fc formatted antigen, competition ELISA and blockade of PD-L1/PD-1 axis using various cell-based assays. We demonstrated efficacy in a humanized PD-L1 MC38 tumor growth inhibition model and compared it to approved PD-L1 antibody therapies. Pharmacokinetics (PK) and biodistribution are key parameters that influence the efficacy and safety of therapeutic proteins. Half-life extension of lead Affimer proteins was achieved by fusing to a human Fc region of an IgG (designated AVA004).Pharmacokinetic analysis of AVA004 Fc molecules were performed in C57Bl/6, humanized FcRn/HSA mice and cynomolgus after single intravenous administration. Tissue distribution following intravenous administration of AVA004 Fc to humanized NOG mice bearing orthotopic MDA-MB-231 tumor cells was evaluated using radiolabeled 125I- AVA004 Fc. Biodistribution imaging studies were conducted in mice bearing A375 tumor cells using IRDye800 conjugated to AVA004 Fc followed by ex vivo imaging. A more quantitative biodistribution study was conducted in the same model using SPECT imaging DOTAGA bioconjugated AVA004 Fc molecules radiolabeled with 111Indium. The imaging biodistribution allowed us to select the most favorable molecule based on its tumor/plasma and tumor/liver ratios. Citation Format: Estelle Adam, Emma Jenkins, Floriane Laurent, Jennifer Hillman, Maureen West, Emma Stanley, David Sanford, Agnieszka Kownacka, Céline Mothes, Alexandra Oudot, Vincent Matthew, David Gilfoyle, Barry Jones, William Bachovchin, Amrik Basran, Cyril Berthet. Pharmacokinetics and biodistribution of formatted Affimer® biotherapeutics targeting PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1866.

Innentitelbild: Implications of Quenching‐to‐Dequenching Switch in Quantitative Cell Uptake and Biodistribution of Dye‐Labeled Nanoparticles (Angew. Chem. 28/2021)

Innentitelbild: Implications of Quenching‐to‐Dequenching Switch in Quantitative Cell Uptake and Biodistribution of Dye‐Labeled Nanoparticles (Angew. Chem. 28/2021)

Inside Cover: Implications of Quenching‐to‐Dequenching Switch in Quantitative Cell Uptake and Biodistribution of Dye‐Labeled Nanoparticles (Angew. Chem. Int. Ed. 28/2021)

A quenching-to-dequenching switch of fluorescence-labeled nanoparticles in biological environments is unveiled by Chun-Xia Zhao and co-workers in their Research Article on page 15426. With dye molecules in a solid state, the release and/or degradation of the dye-labeled nanoparticles in biological environments led to a several-fold increase in fluorescence intensity due to the state switch, which has significant implications in quantitative cell uptake and biodistribution studies.

Implications of Quenching‐to‐Dequenching Switch in Quantitative Cell Uptake and Biodistribution of Dye‐Labeled Nanoparticles

AbstractA general strategy to carry out cell uptake and biodistribution studies is to label nanoparticles (NPs) with a fluorescent dye. However, the comparative study of different dye‐loaded NPs remains difficult owing to uncontrolled dye quenching and de‐quenching. Here we compared two types of dye‐labeled NPs and demonstrated their distinct properties. NPs with dye molecules at a solid state suffer from dye quenching, so the dye release and/or NP degradation in biological environments leads to a several‐fold increase of fluorescence intensity despite the same amount of NPs, owing to the state switch from quenching to de‐quenching. In contrast, NPs with dye molecules at a soluble state exhibit no quenching effect. To standardize the comparative study, we propose two possible solutions: using lower dye loading or using medium analysis for quantifying cell uptake of NPs. This work provides valuable insights into selecting valid quantification methods for bio‐nano studies.