Multicolor cyanine dendritic dots for real-time, high-resolution tracking of nanoparticle biodistribution

Abstract

Fluorescence imaging has become a powerful tool for visualizing nanoparticles in drug delivery systems, and multicolor fluorescence imaging offers an intuitive approach to exploring complex biological systems. Here, we present multicolor cyanine-based polylysine (PLL) dendritic dots (Cy3/Cy5/Cy7-PLL; up to eight generations) with a well-defined single-molecule structure and bright, photostable fluorescence. Using these probes, we achieved low-crosstalk three-channel tracking in live mice, enabling high-resolution, real-time visualization of nanoparticle biodistribution and within-subject comparisons that reduce inter-animal variability. By systematically modulating size and surface chemistry, we investigated how these parameters govern biodistribution, encompassing their efficacy in in vitro and in vivo tumor imaging, as well as nanoparticle tracking at both cellular and organ levels. We demonstrate that these multicolor dendritic dots serve as an advanced method for simultaneous, real-time tracking of nanoparticles with different traits, offering significant potential for future applications in various biomedical fields.

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Introduction

Fluorescence imaging is a powerful and versatile tool for visualizing biological processes with high contrast and quantitative capability, playing a pivotal role in fundamental biomedical research and clinical diagnostics.^1–4 Organic nanodots have become an attractive platform for biomedical imaging due to their exceptional biocompatibility, low cytotoxicity, and facile functionalization, which enable stable in vivo imaging.^5,6 These nanodots are formed by encapsulating organic fluorescent dyes within biocompatible nanomaterials like polymers,^7–11 dendrimers,^12–14 polymeric nanoparticles^15,16 and liposomes.^17–19

Multicolor fluorescence imaging is a powerful and intuitive approach for observing complex biological systems, as it allows for the simultaneous visualization of multiple targets or processes.^20–23 The core advantage of this technique is its ability to significantly increase research efficiency and information throughput in both in vitro and in vivo studies by combining two or more distinct fluorescent probes.²⁴ For instance, Sletten's group reported a multicolor imaging system utilizing near-infrared (NIR) fluorophores with different excitation lasers, enabling real-time imaging of different organs in mice.²⁵ Xu's group showed high-contrast imaging of mouse brain using multicolor three-photon fluorescence imaging.²⁶ Emerging approaches move beyond color visualization to sensing-with-imaging (FLIM or FRET, spectral or lifetime multiplexing),^27–31 and to complementary photoacoustic imaging, Raman/SERS,³² afterglow,³³ and NIR-II window^34,35 for deeper, low-background, quantitative readouts. Together, these trends motivate probe platforms that remain bright, exhibit minimal spectral/lifetime crosstalk, and possess well-defined structures for reliable co-administration.

The size and surface chemistry of nanoparticles are critical determinants of their biodistribution and therapeutic efficacy.^36–42 While multicolor imaging holds great promise for intuitively and conveniently addressing these complex relationships, a common limitation in current multicolor imaging research is the lack of detailed studies on how size and surface chemistry influence the distribution within diverse cellular compartments and living organisms.^43,44 Most studies predominantly focus on large nanoparticles (>100 nm), with minimal attention paid to elucidating the effects of subtle size variations (e.g., <10 nm differences) on their in vivo behavior.⁹ There is also a notable scarcity of reports specifically investigating the impact of subtle surface chemistry variations on nanoparticle biodistribution using multicolor fluorescent nanodots.⁴⁵ What's more, previous studies have primarily concentrated on macroscopic biodistribution patterns, overlooking the application of multicolor imaging to deeply elucidate tumor-targeting mechanisms and complex metabolic pathways.^46,47 Bridging these gaps requires multiplexable probes that (i) maintain low crosstalk under simultaneous administration, (ii) preserve brightness and photostability for real-time tracking, and (iii) provide controlled, isostructural series to decouple the roles of particle size and surface chemistry in vivo.

Building on our previous dye-cored polylysine (PLL) dendritic dots with customizable fluorescence emission, molecularly defined architectures and remarkable fluorescence brightness,^48–50 we here established a multicolor cyanine dendritic-dot platform (Cy3/5/7-Gn, n = 1–8) that enabled real-time tracking of multiple dots and study of structure–biodistribution relationships. The library encompassed cyanine 3, cyanine 5, and cyanine 7 as fluorescent cores separately, with generations ranging from G1 to G8. Through diverse surface modification of β-carboxylic amides anhydrides, we prepared Cy-G8-X with precisely controlled surface chemistry (X represents the surface moiety). In vitro study showed that these multicolor cyanine dendritic dots facilitate high-contrast, low-crosstalk three-channel multicolor imaging. To comprehensively investigate the interplay between dendritic dots’ traits and their in vivo behavior, these multi-color dots were co-injected into the same tumor-bearing mice, enabling real-time and concurrent tracking of distinct formulations within a single biological system (Fig. 1). This design reduced animal usage and inter-animal variability while remaining generalizable to diversified fluorescent probes, thereby providing a more robust and efficient tool for exploring structure-biodistribution relationships in complex biological environments.

Fig. 1 Schematic of multicolor cyanine-cored dendritic-dot platform for in vivo tracking and biodistribution. The platform consists of multifunctional cyanine-cored polylysine dendritic dots with Cy3, Cy5, and Cy7 cores (Cy3: green, Cy5: red, Cy7: magenta). The 8th generation of cyanine dendritic dots was selected for surface modification with various anhydrides, enabling co-injection of different nanodot formulations into a single mouse for simultaneous, real-time tracking of their distinct biodistribution.

Results and discussion

Synthesis and characterization of high-generation cyanine-cored dendritic dots

In prior work, we have demonstrated the feasible preparation of three small-molecular-cyanine-dye-cored dendritic nanodots up to generation 5 (Cy3 or Cy7) or 8 (Cy5) through iterative lysine esterification and deprotection.⁴⁹ Here, we synthesized all three types of cyanine-cored PLL fluorescent dendritic dots up to generation 8 (Fig. 2A). Synthetic routes, chemical structures and yields for amine-terminated cyanine dyes and cyanine-cored dendritic dots are shown in Fig. S1–S4 and Table S1. All dendritic dots exhibited precise structures confirmed by ¹H-NMR (Fig. S5 and S6). High-performance liquid chromatography (HPLC) analysis revealed single peaks for Cy-Gn dendritic dots (n = 1–8, Fig. S7, S8), with elution times progressively decreasing as the nanodot generation increased, reflecting polarity changes associated with generation growth and demonstrating high purity. Similarly, gel permeation chromatography (GPC) traces (Fig. S9) showed unimodal and narrow peaks, confirming excellent monodispersity. The hydrodynamic diameters of dendritic dots measured by dynamic laser scattering (DLS) were 6.5 nm for G6, 7.5 nm for G7, and 8.5 nm for G8 (Table S2), confirming size dependence on generation other than core structure.

Fig. 2 Synthesis, photophysical properties and surface modification of multicolor cyanine dendritic dots. (A) Synthesis of polylysine fluorescent dendritic dots up to generation 8 (Cy-Gn, n = 1–8). (B) Normalized fluorescence of Cy3-, Cy5-, and Cy7-cored dendritic dots (2 µM in Milli-Q water). (C) Top: fluorescence imaging of a multicolor Cy-G8 mixture (2 µM in Milli-Q water). Bottom: UV-vis absorption and emission spectra of the same mixture, with excitation/emission at λ_ex/λ_em = 520/570 nm (Cy3), 620/670 nm (Cy5), and 720/790 nm (Cy7). (D) Table of photophysical properties. (E) Surface modification of β-carboxylic amide anhydrides and corresponding modified structures.

Generation-dependent photophysical properties

The photophysical property investigation showed that the fluorescence intensity of all three types of cyanine dendritic nanodots increased generation-dependently: G1 showed the lowest brightness, while G8 displayed the highest (Fig. 2B and Fig. S10), which is consistent with the previous observation of Cy5-cored dendritic dots.⁴⁹ The observed improvements in brightness and quantum yield may stem from a combination of factors, including suppressed aggregation-induced quenching, inhibited intermolecular rotation, and reduced non-radiative decay resulting from trans-cis photoisomerization. Cyanine dendritic dots maintained stable absorption/emission maxima (Cy3: 550–570 nm, Cy5: 645–665 nm, and Cy7: 743–760 nm) (Fig. 2C). Taking Cy7-cored nanodot as a representative, high generation nanodot Cy7-G8 maintained nearly identical absorption/emission maxima compared to free Cy7-NH₂. Under the same concentration, the molar absorption coefficient (ε) was sustained, while the fluorescence quantum yield (Φ_F) increased markedly with generation. Φ_F of Cy7-G8 achieved 7.2%, which is a 2.8-fold enhancement over Cy7-NH₂ (Fig. 2D and Table S3). This strategy is exceptionally effective for significantly enhancing the fluorescence quantum yield of fluorescent molecules. Instead of relying on a single modification, it works by fundamentally altering the molecule's physical properties to favor radiative emission over competing energy loss pathways. Given their aqueous miscibility, we further characterized Cy3, Cy5 and Cy7 dendritic dots in aqueous solution. Using selective excitation wavelengths (λ_ex), distinct emission wavelengths (λ_em) with negligible spectral overlap were observed (Fig. 2C and Fig. S11), enabling simultaneous multicolor imaging and quantitative spectral discrimination.

Surface modification on high-generation cyanine dendritic dots

We then developed a series of dendritic dots with surface modification of various β-carboxylic amides. The cyanine nanodot Cy-G8 carries primary amines from its lysine branches. While this ensures good solubility, the overall surface charge remains strongly positive which is not suitable for in vivo applications.⁵¹ We therefore reacted Cy-G8 with anhydrides to provide defined surface chemistries with mild charges (Fig. 2E and Fig. S12). The resulting conjugates are denoted as Cy-G8-X, where X corresponds to acetic (Ac), succinic (Sa), 4-fluorophthalic (Fp) or 3,3-dimethyl-glutaric (Dg) anhydride. ¹H-NMR confirmed successful substitution (Fig. S13–S14). After amidization, the chemical shift of the lysine methylene protons (–C̲H̲_2̲NH₂) moved from around 2.9 to 3.1 ppm (–C̲H̲_2̲NHC=O), indicating complete modification. All Cy-G8-X samples display single HPLC peaks, indicating good purity and monodispersity (Fig. S15). The surface charge of the dendritic dots changed from highly positive to mild negative charges (−10 to 0 mV) (Fig. S16). Meanwhile, DLS measurements gave comparable hydrodynamic diameters of ∼10 nm, indicating surface chemistry was the sole variable (Table S4). MTT assays confirmed negligible cytotoxicities of the β-carboxylic amides-terminated dendritic dots (Fig. S17). Both acetylated Cy-G8 and Cy-G8-X dendritic dots exhibited high biocompatibility (≥90% viability) across 10–100 µM, a trend consistently observed in both malignant 4T1 and non-malignant NIH-3T3 cells. In contrast, the amine-terminated Cy-G8 proved highly toxic, exhibiting less than 50% cell viability at a concentration as low as 10 µM. This confirms that the surface modification is key to conferring biocompatibility. Hemolysis assays corroborated the hemocompatibility of Cy-G8-X dendritic dots: at 1 and 5 mg mL⁻¹ (bracketing and exceeding the estimated maximal systemic concentration), hemolysis remained <5% over 4 h (Fig. S18). In addition, at 7 days post-injection, plasma biochemical assays and hematology parameters were within reference intervals and did not differ significantly from the PBS control, supporting the in vivo safety of Cy-G8-X (Fig. S19–S20).

Size-dependent in vivo biodistribution and tumor targeting

To assess feasibility based on dendritic dots’ size-dependent properties, we evaluated the performance of multicolor in vivo tumor imaging using three dendritic cyanine dots (Cy3-G6-Ac, Cy5-G7-Ac, and Cy7-G8-Ac) in the same mouse with an established 4T1 subcutaneous tumor model (Fig. 3A). Following intravenous administration, all three dendritic dots exhibited rapid systemic distribution followed by progressive tumor accumulation. Fluorescence signals were pseudo-colored as follows: Green for Cy3, Red for Cy5, and Magenta for Cy7. Fluorescence quantification in tumors and normal tissues across multiple time points revealed distinct pharmacokinetic behaviors (Fig. 3B). Cy3-G6-Ac showed fast tumor accumulation but rapid clearance within 4 h, indicating the shortest retention. In contrast, Cy7-G8-Ac achieved the highest tumor-to-normal tissue contrast, with TNR (tumor-to-normal ratio) of ∼6 at 24 h post-injection, reflecting prolonged tumor retention and superior imaging performance. Organ fluorescence further uncovered size-dependent biodistribution patterns. The smallest (Cy3-G6-Ac, 6.7 nm) peaked in the liver at 2 h post-injection, the intermediate size (Cy5-G7-Ac, 8.6 nm) at 4 h, and the largest (Cy7-G8-Ac, 10.9 nm) at 12 h, indicating progressively delayed hepatic clearance with increasing nanodot diameter. Correspondingly, liver signal-to-noise ratios (SNR-liver) rose with nanodot size, confirming both enhanced hepatic retention and prolonged systemic circulation (Fig. 3C). Real-time abdominal imaging 1–2 h after injection revealed distinct renal-excretion profiles: bladder fluorescence was evident for Cy3-G6-Ac and Cy5-G7-Ac but absent for Cy7-G8-Ac (Fig. S21). These observations point to an ∼8.6 nm threshold for renal clearance; particles below this limit were rapidly eliminated in urine, whereas those above ∼10 nm were preferentially cleared through the hepatobiliary route.^9,52,53 These results identified G8 as the optimal nanoparticle generation and size for subsequent imaging applications.

Fig. 3 Multicolor in vivo imaging for size-dependent biodistribution. (A) Longitudinal multicolor fluorescence of 4T1 tumor-bearing mice following intravenous co-injection of Cy3-G6-Ac, Cy5-G7-Ac, and Cy7-G8-Ac (10 nmol each). (B) and (C) Quantitative biodistribution analysis derived from (A), depicting time-dependent tumor-to-normal tissue ratio (TNR) and liver signal-to-normal tissue ratio (SNR-Liver). Data presented as mean ± SD (n = 3).

Core-independent biodistribution with identical size and surface chemistry

In both in vivo and in vitro studies, we investigated dendritic dots sharing identical sizes and surface chemistries to explore the influence of fluorescent core on bio-behavior. After surface modification through Sa conjugation, we co-administered three cyanine-cored G8 dendritic dots Cy3-G8-Sa, Cy5-G8-Sa, and Cy7-G8-Sa via intravenous injection. The biodistribution profiles of these three dendritic dots were strikingly similar, with accumulation observed in both tumor and liver tissues (Fig. 4A and Fig. S22). Peak accumulation in the target tissues occurred approximately 24 h post-injection (Fig. 4B and C). Additionally, Cy7-G8-Sa exhibited the highest TNR, a result attributed to the longer wavelength of Cy7, which minimizes interference from endogenous fluorescence in biological tissues. Mice were euthanized at 48 h post-injection, and liver and tumor tissue sections were analyzed using confocal microscopy (Fig. S23). Fluorescence from the three channels showed high colocalization, with Pearson's correlation coefficients (PCC) of 0.75, 0.71, and 0.72, confirming the consistent distribution of the cyanine dendritic dots with identical size and surface chemistry within the tissues. Cellular internalization also revealed analogous results for different cyanine-cored dendritic dots. A mixture of these dendritic dots was incubated with 4T1 cells for 3 h, each at the same concentration. The dendritic dots exhibited high colocalization coefficients with each other, demonstrating nearly identical cellular uptake profiles (Fig. 4D, E and Fig. S24). To further investigate the impact of fluorescent core, we performed a “color switch” experiment:⁴³ each cyanine core (Cy3-G8, Cy5-G8, Cy7-G8) was paired with each surface chemistry (Sa, Fp, Dg) in all possible pairings (Fig. 4F). Dendritic dots sharing the same surface chemistry exhibited similar uptake profiles regardless of core, whereas particles with identical cores but different surface chemistries differed markedly (Fig. 4G). Notably, Fp-modified dendritic dots showed a higher internalization efficiency in tumor cells than their Sa- or Dg-functionalized counterparts, supporting the fluorination-enhanced uptake effect. Collectively, these results suggest that dendritic dots’ core structure does not affect the biological behavior of the nanoparticles when particle size and surface chemistry are held constant.

Fig. 4 Comparative evaluation of multicolor dendritic dots with distinct fluorescent cores. (A) In vivo multicolor fluorescence of 4T1 tumor-bearing mice following intravenous co-injection of Cy-G8-Sa (10 nmol each). (B) and (C) Quantitative biodistribution profiles derived from (A). (D) Cellular uptake of Cy-G8-Sa (3 µM, 3 h; scale bars = 25 µm). (E) Colocalization analysis of G8-Sa dendritic dots in (D). (F) and (G) Cellular imaging of multicolor cyanine dendritic dots with varied surface modifications (5 µM, 3 h; scale bars = 25 µm). Data presented as mean ± SD (n = 3).

Multicolor imaging for in vivo tracking and biodistribution

Nanoparticles with distinct surface modifications exhibited differential biodistribution patterns across various tissues and organs. However, the relationship between surface chemistry and biological behaviors is often assessed in different individuals, which can introduce variability due to inter-individual differences. To address this issue, we employed multicolor imaging to track the localization of dendritic dots in 4T1 subcutaneous tumor-bearing mice. The experimental group for tail administration consisted of a mixture of Cy3-G8-Sa, Cy5-G8-Fp, and Cy7-G8-Dg (Fig. 5A). The in vivo fluorescence monitoring was presented in Fig. S25. Fluorescence signals were captured and quantitatively analyzed (Fig. 5B). The TNR showed that dendritic dots with Sa surface modification exhibited higher fluorescence intensity compared to those with Fp and Dg modifications. Tumors and major organs of the tumor-bearing mice were harvested for ex vivo imaging 12 h post-injection, revealing the distribution of multicolor fluorescence intensities across the body (Fig. 5C and Fig. S26). Quantitative analysis of SNR in various organs and tumors showed distinct surface chemistry-dependent biodistribution patterns (Fig. 5D). Tumors exhibited the highest accumulation of Sa-modified dendritic dots, while the liver showed the greatest accumulation of Dg-modified dendritic dots. The spleen and lungs, by contrast, displayed dominant accumulation of Fp-modified nanoparticles. Histological analysis of tumor and organ sections confirmed these findings (Fig. 5E and Fig. S27). At the tissue level, liver and spleen sections exhibited significant colocalizations of three surface-modified cyanine dendritic dots, with PCC between pairs of channels ranging from 0.5 to 0.75 (Fig. 5F). In contrast, lung and tumor sections exhibited much lower PCC values, ranging from 0.25 ∼ 0.35, indicating tissue-specific localization of nanoparticles with different surface chemistries in both tumors and lungs.

Fig. 5 In vivo multicolor fluorescence imaging of surface-modified cyanine dendritic dots in 4T1 tumor-bearing mice. (A) Whole-body fluorescence at 12 h post-intravenous co-injection of Cy3-G8-Sa, Cy5-G8-Fp, Cy7-G8-Dg (10 nmol each). (B) Time-dependent TNR quantification. (C) Ex vivo fluorescence of tumors and major organs at 12 h post-injection. (D) Quantitative analysis of (C). (E) Confocal fluorescence imaging of tumor sections from (C): nuclei (DAPI, blue); Cy3-G8-Sa (green), Cy5-G8-Fp (red), Cy7-G8-Dg (magenta). Scale bar = 200 µm. (F) PCC for pairwise channel comparisons in organ slices. Data presented as mean ± SD (n = 3).

We hypothesize that the co-localization of various dendritic dots within immune organs is closely correlated with macrophages.⁵⁴ The liver and spleen, which are rich in macrophages, exhibited high PCC due to non-specific phagocytosis of nanoparticles by macrophages. In contrast, the lower macrophage density in lungs and tumors resulted in reduced non-specific phagocytosis, which accounted for the observed decrease in nanoparticle co-localization. To test this hypothesis, we co-incubated a mixture of Cy3-G8-Sa, Cy5-G8-Fp, and Cy7-G8-Dg dendritic dots with Raw264.7 cells. Confocal microscopy was then employed to visualize the cellular uptake of three surface-modified cyanine dendritic dots (Fig. S28). All dendritic dots were internalized greatly and each pair exhibited high co-localization, with PCC above 0.75, suggesting that the macrophages non-specifically phagocytose nanoparticles regardless of their surface modifications.

Conclusions

This study introduces a series of multicolor cyanine-cored dendritic dots with precise control over their size and surface chemistry for real-time nanoparticle tracking, achieving simultaneous, high-contrast in vivo visualization with minimal spectral crosstalk within a single biological system. We established a Cy3/Cy5/Cy7 cyanine-cored polylysine dendritic dot library (Cy-Gn, n = 1–8) with molecularly defined structures, high purity, and generation-dependent brightness, enabling low-crosstalk three-channel imaging. By diverse β-carboxylic anhydride amidization, we generated charge-mild Cy-G8-X with high biocompatibility and negligible hemolysis. “Color-switch” experiments demonstrated core-independent bio-behavior, whereas size and surface chemistry governed cellular uptake and organ distribution. We identified G8 as optimal for tumor imaging and an ∼8.6 nm renal-clearance threshold. Tissue-level co-localization further revealed surface-dependent distribution in tumors and lungs, while fluorescence in liver and spleen was largely surface-independent due to macrophage phagocytosis. These multicolor cyanine dendritic dots can be used for advanced fluorescence-based diagnostic and structure–activity relationship studies, with far-reaching implications for biomedical research and medicine development.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: experimental section, Fig. S1–S28 and Tables S1–S4. See DOI: https://doi.org/10.1039/d5tb01962a.

Acknowledgements

We thank the National Natural Science Foundation of China (52425311, 52495010, 52073249, 21875211), the National Key Research and Development Program of China (2021YFA1201201).

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Footnote

† These authors contributed equally to this work.

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