Indocyanine Green–Inspired Polymeric Chromophores With Intrinsic Photostability for Near‐Infrared Imaging

ABSTRACT

Indocyanine green (ICG), the only FDA‐approved near‐infrared (NIR) dye, is widely used in bioimaging but suffers from rapid photobleaching and nonspecific biodistribution. Here we introduce KR‐NIR‐P, a polymerized form of ICG that preserves its NIR fluorescence while markedly enhancing stability. Under continuous illumination, KR‐NIR‐P retained ∼66% of its initial fluorescence at 200 s, compared to ∼40% for free ICG within the first 50 s. The polymer was efficiently internalized into immortalized cancer cells (HeLa, HSC‐3) and primary human dermal fibroblasts (HDFs), exhibiting predominant cytosolic localization with >90% cell viability even at 20 µM, confirming excellent biocompatibility. The inclusion of both immortalized and primary cells underscores the polymer's broad compatibility across physiologically diverse models. This straightforward polymerization strategy provides a robust and durable NIR imaging platform, overcoming the key drawbacks of ICG and broadening its utility for advanced biomedical research and clinical translation.

Indocyanine green (ICG) is widely used for near‐infrared imaging but suffers from poor photostability. Here, an indocyanine green–inspired polymeric NIR chromophore is introduced, exhibiting intrinsically enhanced photostability and biocompatibility. The polymeric architecture enables stable fluorescence in cells, 3D spheroids, and in vivo imaging, offering a robust carrier‐free platform for NIR bioimaging.

1. Introduction

Near‐infrared (NIR) dyes play a crucial role in bioimaging due to their ability to penetrate biological tissues more effectively than visible‐light dyes. The NIR region (approximately 650–900 nm) is especially valuable for in vivo imaging because it minimizes absorption and scattering by key biological components such as water and hemoglobin [1, 2, 3]. Additionally, NIR light greatly reduces tissue auto‐fluorescence, which can obscure imaging clarity with visible‐spectrum dyes [4, 5]. his spectral range, often referred to as the “optical window,” enables deeper penetration and higher contrast by minimizing absorption and auto‐fluorescence [6, 7]. Therefore, the development of new NIR imaging materials is vital for advancing medical diagnostic and therapeutic techniques, enabling deeper and more precise imaging capabilities [8, 9, 10, 11, 12, 13].

Indocyanine green (ICG) is one of the most representative NIR dyes and has been widely used in medical imaging since its FDA approval in 1959. With advancements in clinical imaging technology, ICG has been applied in various fields, particularly in ophthalmic angiography, assessments of vascular integrity, and tumor margin visualization, playing a critical role in real‐time guidance during diagnostics and surgery [14, 15]. To date, ICG remains the only FDA‐approved NIR fluorescent dye, establishing a high benchmark for the safety and utility of NIR dyes in clinical settings. This also highlights the challenges in developing new NIR dyes that meet the rigorous safety and efficacy standards required for clinical approval. Despite decades of research, no other NIR dye has successfully navigated the complex regulatory pathway to FDA approval, either to replace ICG or surpass its performance. This underscores the difficulty of achieving optimal bioavailability, safety, and efficacy in new NIR dye candidates.

ICG does have significant limitations, including poor photostability and nonspecific biodistribution, which hinder its long‐term imaging performance and often necessitate repeated high‐dose administrations [16, 17]. These issues complicate clinical procedures and increase patient burden. To overcome ICG's shortcomings, numerous efforts have been made to improve its photostability. For example, Yeroslavsky et al. incorporated ICG into polymeric micelles, enhancing its chemical and thermal stability via energy transfer [18]. Kwon et al. developed nanostructured ICG dimers that demonstrated improved photothermal efficiency and photostability [19]. Furthermore, Yang et al. assembled ICG into nanobubbles, achieving enhanced fluorescence stability and monodispersity [20]. To mitigate these drawbacks, numerous strategies have been explored. Encapsulation in micelles or liposomes improves solubility and provides partial shielding, dimer‐based engineering modulates excited‐state interactions, and nanoformulations such as nanobubbles enable improved dispersion and short‐term stability. While these approaches offer incremental improvements, they rely primarily on physical encapsulation or supramolecular assembly, leaving the chromophore itself chemically unmodified and thus vulnerable to dye leakage, aggregation, and formulation‐dependent photophysical behavior. Consequently, three fundamental challenges remain: 1) the need to enhance the intrinsic stability of the chromophore itself rather than relying on external protection, 2) the additional synthetic or formulation complexity required by encapsulation‐based strategies, and 3) the limited reproducibility arising from carrier‐ and formulation‐dependent photophysical behavior.

In this work, we designed KR‐NIR‐P by polymerizing a sulphonated indole dimer (compound 3), which was selected because its heptamethine‐like conjugated framework and sulphonated functionalities are structurally analogous to those of ICG (Figure 1). Building on this molecular scaffold, we developed KR‐NIR‐P, a polymerized NIR chromophore designed to achieve intrinsic stabilization without reliance on external carriers, enabling improved photostability and reproducible photophysical behavior. The polymer matrix greatly improves the photostability of the dye, allowing for longer imaging sessions without the rapid degradation that typically plagues ICG. The photostability tests in our study demonstrate that KR‐NIR‐P is far more resistant to photodegradation than ICG. Under identical laser irradiation conditions, KR‐NIR‐P shows a dramatically slower photobleaching rate than ICG. This suggests that using KR‐NIR‐P could significantly reduce the need for repeat dye administration in clinical imaging, thereby improving procedural efficiency and patient comfort [21, 22]. Moreover, we found that KR‐NIR‐P is readily taken up by various cell lines (including human breast and skin cells, as well as mouse tumor cells) without affecting cell viability or causing cytotoxic effects. Importantly, the polymer localizes in the cytosol and is excluded from the nucleus, suggesting a selective uptake mechanism that is critical for targeted imaging and therapy. This selectivity could open new avenues for the use of NIR dyes in precision medicine.

FIGURE 1.

(a) Chemical structure and (b) synthetic scheme of the NIR polymer (KR‐NIR‐P), illustrating the polymerization strategy used to enhance photostability.

In conclusion, the development of KR‐NIR‐P represents a significant advance in the field of NIR dyes. It maintains the favorable safety profile of FDA‐approved ICG while providing enhanced imaging capabilities that could enable wider applications in both research and clinical settings. This breakthrough marks the beginning of a new era for NIR bioimaging and photothermal therapy, with important implications for next‐generation NIR dyes that combine safety, efficacy, and advanced functionality.

2. Experimental

2.1. Materials and Methods

All chemicals and solvents were obtained from commercial sources and used as received unless otherwise specified. Reaction progress was monitored by thin‐layer chromatography (TLC) on silica gel plates and visualized under ultraviolet irradiation. ¹H NMR spectra were acquired using a Bruker 300 MHz spectrometer, and chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane as an internal standard. Mass spectrometric analysis was performed using electrospray ionization (ESI‐MS). Melting points were measured using a digital melting point apparatus and are reported without correction. UV‐Vis absorption spectra were collected using a V‐770 double‐beam UV‐Vis‐NIR spectrophotometer (Jasco), and photoluminescence measurements were carried out on an FLS1000 fluorescence spectrometer (Edinburgh Instruments).

2.2. Synthesis and Characterization of KR‐NIR‐P, and Optical Property Measurements

The synthetic route for KR‐NIR‐P is summarized in Figure S1. In brief, an aromatic diamine precursor was sequentially converted into a benzimidazole‐based intermediate through diazotization, reduction, and cyclization reactions. Subsequent functionalization steps, including sulphonation, were employed to enhance water solubility, followed by a condensation reaction to generate the cyanine‐based polymeric NIR fluorophore, KR‐NIR‐P.

Structural confirmation of KR‐NIR‐P was achieved by ¹H NMR spectroscopy, and molecular weight analysis was conducted using gel permeation chromatography (GPC). The number‐average molecular weight (M_n ≈ 13,000 g mol^{− 1}) corresponds to an average degree of polymerization of approximately 15–16, based on the molecular weight of the sulphonated indole dimer unit. Photostability measurements were performed using a homemade optical setup (Figure S9). ICG and KR‐NIR‐P were dissolved in water at a concentration of 20 µM. A 785 nm continuous‐wave diode‐pumped solid‐state laser (PI‐ECL‐785‐900‐FC, Process Instruments) with an output power of 10 mW was continuously applied to the samples, and emission spectra were recorded at 1 s intervals. For KR‐NIR‐P, four independent measurements were conducted. The resulting spectra were normalized to the respective main emission peak intensities of ICG and KR‐NIR‐P.

2.3. Biocompatibility and Imaging

Cell viability was evaluated using a water‐soluble tetrazolium salt (WST) assay. HeLa, HSC‐3, and Detroit 573 cells were seeded in 96‐well plates and incubated with serial dilutions of KR‐NIR‐P (up to 20 µM). After incubation, absorbance at 450 nm was measured using a microplate reader, and cell viability was expressed as a percentage relative to untreated controls. For the WST assay, each concentration was measured using six technical replicates (six wells per condition in a 96‐well plate), and the data are presented as mean ± SD. For fluorescence imaging, cells were seeded on chamber slides and treated with KR‐NIR‐P (10 µM) for 6 h. Following fixation, samples were washed, mounted, and imaged using an inverted fluorescence microscope equipped with an EMCCD camera. Excitation was provided by a 785 nm continuous‐wave laser, and emission was collected using a long‐pass filter to suppress excitation light.

2.4. Cell Culture and 3D Spheroid Formation

HeLa, HSC‐3, and Detroit 573 cells were cultured under standard conditions using appropriate growth media supplemented with fetal bovine serum and antibiotics. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ and passaged upon reaching ∼80% confluency. Fibroblast experiments were conducted using cells between passages 3 and 6. 3D spheroids were generated using ultra‐low attachment plates. Cells were seeded at high density and cultured until compact spheroids formed, with periodic medium replacement. Fully formed spheroids were subsequently incubated with KR‐NIR‐P (10 µM) prior to imaging analysis. The Korea Basic Science Institute Institutional Animal Care and Use Committee approved all the experimental protocols (KBSI‐IACUC‐25‐29 and KBSI‐IACUC‐26‐27). All animal experiments were conducted according to the ARRIVE guidelines.

2.5. Biocompatibility Assays, qRT‐PCR amd Western Blot

All experiments were performed in triplicate unless otherwise stated. Data are presented as mean ± standard deviation (SD). Statistical differences between the two groups were assessed using the unpaired Student's t‐test. A p value < 0.05 was considered statistically significant. Data analysis was conducted using GraphPad Prism software (version 8.0).

2.5.1. MTT

The MTT reagent is reduced to insoluble purple formazan by mitochondrial reductase enzymes in metabolically active cells. After treatment with varying concentrations of ICG or KR‐NIR‐P (10 nM ∼ 100 µM) for 24 and 48 h, MTT solution was added directly to each well at a final concentration of 0.5 mg/mL and incubated at 37°C for 4 h in a humidified incubator with 5% CO₂. Following incubation, the medium was carefully aspirated and replaced with an equal volume of dimethyl sulfoxide (DMSO) to solubilize the formazan crystals. Absorbance was measured at 570 nm using a microplate reader (Tecan, Männedorf, Switzerland). Cell viability was calculated as a percentage relative to untreated control cultures [23].

2.5.2. qRT‐PCR

Following treatment with 1 µM of ICG or KR‐NIR‐P for 24 h, total RNA was extracted from HDF cells using TRI reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions. Reverse transcription was performed using SuperScript II reverse transcriptase and oligo(dT) primers (Invitrogen) to synthesize cDNA. Quantitative real‐time PCR (qRT‐PCR) was conducted using a QuantStudio 3 Real‐Time PCR System (Thermo Fisher Scientific, Waltham, MA). For each reaction, 20 ng of cDNA was used in duplicate according to the manufacturer's protocol [24]. All primer sequences were specific to human genes and are listed in Table 1. Target genes included markers related to apoptosis, oxidative stress, inflammation, and cell cycle regulation.

TABLE 1.

Primer sequences for quantitative RT‐PCR.

Genes	Forward	Reverse
CASP3	GGA AGC GAA TCA ATG GAC TCT GG	GCA TCG ACA TCT GTA CCA GAC C
CASP8	AGA AGA GGG TCA TCC TGG GAG A	TCA GGA CTT CCT TCA AGG CTG C
CASP9	GTT TGA GGA CCT TCG ACC AGC T	CAA CGT ACC AGG AGC CAC TCT T
BAX	TCA GGA TGC GTC CAC CAA GAA G	TGT GTC CAC GGC GGC AAT CAT C
BCL2	ATC GCC CTG TGG ATG ACT GAG T	GCC AGG AGA AAT CAA ACA GAG GC
SOD1	CTC ACT CTC AGG AGA CCA TTG C	CCA CAA GCC AAA CGA CTT CCA G
SOD2	CTG GAC AAA CCT CAG CCC TAA C	AAC CTG AGC CTT GGA CAC CAA C
SOD3	ACG CTG GCG AGG ACG ACC TG	GCT TCT TGC GCT CTG AGT GCT C
GPX4	ACA AGA ACG GCT GCG TGG TGA A	GCC ACA CAC TTG TGG AGC TAG A
IL1B	CCA CAG ACC TTC CAG GAG AAT G	GTG CAG TTC AGT GAT CGT ACA GG
IL6	AGA CAG CCA CTC ACC TCT TCA G	TTC TGC CAG TGC CTC TTT GCT G

2.5.3. Western Blot Analysis

HDF cells were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA) supplemented with protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland). Lysates were homogenized and centrifuged to collect the supernatant. Protein concentrations were determined using a BCA assay (Thermo Fisher Scientific). Equal amounts of protein were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto nitrocellulose membranes (Bio‐Rad, Hercules, CA). Membranes were blocked and incubated overnight at 4°C with primary antibodies against PARP, cleaved caspase‐3, cleaved caspase‐9, Bax, HO‐1, and LC3 (Cell Signaling Technology, Danvers, MA), with GAPDH (BD Biosciences, San Jose, CA) used as the endogenous loading control. After washing, membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL) [24].

2.6. Statistical Analysis

All quantitative data are presented as mean ± standard deviation (SD). For cell viability assays (WST and MTT), each experimental condition was measured using six technical replicates. qRT‐PCR and Western blot analyses were performed using three independent biological replicates. Data were evaluated for normality and outliers; no data points were excluded from the final analysis. Statistical comparisons between two groups were conducted using an unpaired two‐tailed Student's t‐test. A p value < 0.05 was considered statistically significant. All statistical analyses and data visualizations were performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA).

3. Results and Discussion

3.1. Design, Synthesis and Characterization of KR‐NIR‐P

KR‐NIR‐P was synthesized via a controlled polymerization of a sulphonated indole dimer (compound 3) under mild conditions (Figure S1). KR‐NIR‐P was synthesized from a sulphonated indole dimer, which was selected due to its structural similarity to ICG and its suitability for controlled polymerization [25, 26, 27, 28]. Compared with encapsulation‐based or dimeric ICG formulations, the polymerization strategy presented here provides intrinsic stabilization by covalently integrating the chromophore into a polymer backbone. This structure suppresses aggregation, prevents dye leakage, and eliminates carrier‐dependent variability, thereby overcoming the three major limitations of conventional ICG engineering approaches. The resulting KR‐NIR‐P exhibits consistent and carrier‐independent photo physical behavior, underscoring the mechanistic advantage of polymerization over formulation‐based methods. Notably, no metal catalysts are required in the process, making it both cost‐effective and environmentally friendly [29, 30]. As shown in Figure S1, the first step is the synthesis of (2,2′‐dimethyl‐[1,1′‐biphenyl]‐4,4′‐diyl)bis(hydrazine) (compound 1) from an aromatic diamine using sodium nitrite in the presence of HCl. The addition of acetic acid and stannous chloride then facilitates cyclization to form an indole derivative (compound 2), which provides the structural backbone for the polymer. Next, the indole intermediate is N‐alkylated and ionized with propane sultone to yield 3,3′‐(2,2′,3,3,3′,3′,6,6′‐octamethyl‐3H,3′H‐[5,5′‐biindole]‐1,1′‐diium‐1,1′‐diyl)bis(propane‐1‐sulfonate) (compound 3). Finally, this sulphonated indole dimer (compound 3) is condensed with glutaconaldehyde dianil hydrochloride (in the presence of trimethylamine and acetic anhydride in methanol) to produce the target cyanine‐based NIR polymer, KR‐NIR‐P. All synthetic intermediates were characterized by ¹H and ¹³CNMR (Figures S2–S5). UV–Vis spectroscopy was used to monitor the polymerization progress of KR‐NIR‐P (Figure S6a). Initially, the reaction mixture showed an absorption peak at 486 nm. As the reaction proceeded, the absorption maximum (λ_max) red‐shifted to 625 nm, which is shorter than the typical absorption wavelengths of cyanine dyes (generally 750–800 nm). To further drive the polymerization to completion, we added triethylamine (TEA) up to 6 equivalents (Figure S6b). This addition led to the appearance of a new absorption peak at 769 nm, while the intermediate peaks at 473 and 627 nm diminished, indicating successful progression of the polymerization. Under these optimized conditions, KR‐NIR‐P was obtained with the desired optical properties. Gel permeation chromatography analysis (Figure S7) revealed a number‐average molecular weight (M_n) of approximately 13000 g/mol and a weight‐average molecular weight (M_w) of ∼24900 g/mol, with a polydispersity index (PDI) of 1.88. Based on the GPC‐derived M_n of 13 000 g/mol and the molecular weight of the sulphonated indole dimer (∼ 850 g/mol), the average degree of polymerization (n) was estimated to be approximately 15∼16. This n‐value and the measured PDI are consistent with a step‐growth polymerization process and support the formation of a linear conjugated polymer backbone. These values indicate that the polymer is well‐defined with a relatively narrow molecular weight distribution, which helps ensure consistent physical and chemical properties. The absence of metal catalysts in the synthesis not only reduces environmental impact and production costs but also aligns with green chemistry principles.

3.2. Optical Properties and Photostability of KR‐NIR‐P and ICG

The optical properties of KR‐NIR‐P were evaluated in direct comparison to ICG, a conventional NIR dye widely used in biomedical imaging. The UV‐Vis absorption spectra (Figure 2a) show that KR‐NIR‐P has a maximum absorbance around 800 nm, very similar to that of ICG (∼780 nm). Likewise, the photoluminescence (PL) spectra (Figure 2b) display nearly identical emission maxima for KR‐NIR‐P and ICG, indicating that the polymer retains the beneficial optical characteristics of ICG for imaging applications. In methanol, the molar extinction coefficient (ε) of KR‐NIR‐P was determined to be 8.53 × 10⁴ M^{− 1} cm^{− 1} (Figure S8c), and the fluorescence quantum yield (PLQY) of KR‐NIR‐P was 0.64%, whereas that of ICG was 2.9% (Figure S8). The most striking advantage of KR‐NIR‐P is its dramatically enhanced photostability (Figure 2c). Under continuous illumination, KR‐NIR‐P retained approximately 66% of its initial fluorescence intensity after 200 s, whereas ICG's fluorescence dropped to about 40% within the first 50 s. This substantial improvement in photostability is attributed to the polymer matrix effectively stabilizing the ICG chromophore against photodegradation. Furthermore, the enhanced stability of ICG within the polymer matrix is closely associated with the stabilization of hydrophobic regions inherent to the ICG molecular structure. Previous studies have reported that adsorption of these hydrophobic domains onto other hydrophobic components can improve fluorescence stability. [31]. In the KR‐NIR‐P matrix, intramolecular hydrophobic interactions between ICG molecules within the polymer chains provide additional structural stabilization, which in turn contributes positively to maintaining the integrity of the polymethine structure responsible for its fluorescence characteristics. At the same time, the polymeric confinement partially shields the chromophore from molecular oxygen, thereby reducing oxygen accessibility to the excited state and suppressing singlet‐oxygen–mediated photodegradation. In addition, the polymer architecture restricts the uncontrolled aggregation that typically occurs with free ICG in solution an aggregation pathway known to accelerate photobleaching thereby further contributing to the improved photostability of KR‐NIR‐P [32, 33]. The greatly improved photostability of KR‐NIR‐P extends the usable imaging duration and reduces the need for repeated dye administration during both clinical and experimental procedures. In summary, KR‐NIR‐P exhibits optical properties comparable to ICG but with the critical added benefit of superior resistance to photobleaching. This combination positions KR‐NIR‐P as a highly promising candidate for advanced biomedical imaging applications, as it overcomes the key photobleaching limitation of conventional NIR dyes like ICG.

FIGURE 2.

(a) UV–Vis absorption spectra of KR‐NIR‐P and ICG in methanol; (b) photoluminescence spectra of KR‐NIR‐P and ICG in methanol, excited at 785 nm; (c) photostability under continuous 785 nm illumination in water (output power: 10 mW), where emission spectra were recorded at 1 s intervals. (KR‐NIR‐P in red, ICG in black).

3.3. Cellular Uptake and Biocompatibility of KR‐NIR‐P

To evaluate the biomedical applicability of KR‐NIR‐P, we conducted detailed studies of its cellular uptake and biocompatibility across multiple cell lines, including HeLa (human cervical carcinoma), HSC‐3 (human oral squamous carcinoma), and Detroit 573 (human skin fibroblast) cells. Representative bright‐field and fluorescence microscopy images (Figure 3a–c) show clear and efficient internalization of KR‐NIR‐P by these cells. The PL images reveal that KR‐NIR‐P is predominantly localized in the cytoplasm, with minimal nuclear staining, which is important for reducing potential genotoxic effects. Quantitative fluorescence analysis further confirmed significant uptake of KR‐NIR‐P in all tested cell lines. Merged bright‐field/PL images show a homogeneous cytoplasmic distribution of KR‐NIR‐P, suggesting that uptake likely occurs via endocytic pathways. To further validate cellular uptake at the population level, we performed wide‐field, low‐magnification fluorescence imaging at three distinct positions within the same culture area. As shown in Figure S10a, all positions displayed consistent KR‐NIR‐P fluorescence across the entire cell population, confirming uniform and widespread uptake. High‐magnification imaging (Figure S10b) additionally verified that the fluorescence originated from intracellular regions, with signals predominantly localized in the cytoplasm. Biocompatibility was assessed via a standard WST assay by exposing cells to increasing concentrations of KR‐NIR‐P (0 to 20 µM) for 6 h (Figure 3d). Cell viability remained high in HeLa, HSC‐3, and Detroit 573 cells at all tested concentrations, with no statistically significant reduction compared to untreated controls. Even at the highest concentration (20 µM), KR‐NIR‐P exhibited negligible cytotoxicity, indicating an excellent biocompatibility profile. These results collectively demonstrate the safety and efficacy of KR‐NIR‐P for cellular imaging applications. The robust cellular uptake, uniform intracellular localization, and minimal cytotoxicity observed reinforce KR‐NIR‐P's potential as an innovative NIR imaging agent. This thorough evaluation provides a strong foundation for further development and eventual clinical translation of KR‐NIR‐P as a safer, more stable, and highly effective alternative to conventional NIR dyes such as ICG.

FIGURE 3.

Cellular uptake and biocompatibility of KR‐NIR‐P in various cell lines. (a‐c) Bright‐field (BF), fluorescence (PL), and merged images of (a) HeLa, (b) HSC‐3, and (c) Detroit 573 cells after incubation with KR‐NIR‐P. (d) Cell viability (WST) assay results for HeLa (black), HSC‐3 (red), and Detroit 573 (blue) cells treated with increasing concentrations of KR‐NIR‐P (0‐20 µM). Data represent mean ± SD of six technical replicates.

3.4. In Vitro 3D Spheroid Uptake and In Vivo SLN Imaging of KR‐NIR‐P

To investigate the penetration and distribution characteristics of KR‐NIR‐P under more physiologically relevant conditions than conventional 2D cultures, we employed 3D multicellular spheroid models. Spheroids were generated from HeLa, HSC‐3, and Detroit 573 cells and subsequently treated with 10 µM KR‐NIR‐P. Representative bright‐field (BF), photoluminescence (PL), and merged images are shown in Figure 4a–c. The fluorescence images revealed robust and uniform signals across the spheroid cross‐sections, indicating that KR‐NIR‐P effectively penetrated into and accumulated within the deeper layers of the 3D structures. This substantial penetration highlights the polymer's ability to diffuse through densely packed cellular environments, an important feature for tumor imaging and therapeutic applications. The consistent uptake across spheroids derived from multiple cell lines further underscores the broad applicability of KR‐NIR‐P. These results establish 3D spheroid models as a valuable intermediate platform that provides insight into tissue‐level delivery behaviors prior to in vivo evaluation.

FIGURE 4.

In vitro 3D spheroid uptake and in vivo SLN imaging of KR‐NIR‐P. (a–c) BF, PL, and merged images of HeLa, HSC‐3, and Detroit 573 spheroids treated with KR‐NIR‐P, showing strong fluorescence throughout the 3D structures. (d) In vivo BF, PL, and merged images at 0, 2, and 24 h after subcutaneous footpad injection of KR‐NIR‐P, indicating time‐dependent transport to the draining SLN. (e) Ex vivo BF, PL, and merged images of excised SLNs showing selective accumulation in the injected‐side SLN. Scale bars: 100 µm (a–c) and 1 cm (d,e).

To assess the initial in vivo behavior of KR‐NIR‐P, we performed SLN imaging following subcutaneous injection into the mouse footpad. As shown in Figure 4d, fluorescence was initially restricted to the footpad immediately after injection, whereas at 2 h a clear signal emerged in the draining SLN. By 24 h, the SLN displayed a markedly stronger and accumulated fluorescent signal. Consistent with the in vivo findings, Figure 4e presents ex vivo imaging results, which further confirmed selective retention of KR‐NIR‐P in the lymph node on the injected side. These results indicate that KR‐NIR‐P maintains excellent optical and chemical stability in vivo and can effectively traffic through lymphatic pathways, supporting its potential utility for in vivo NIR imaging applications.

3.5. Toxicity of KR‐NIR‐P and ICG

We compared the cytotoxic profiles of KR‐NIR‐P and ICG. Cell viability was measured by MTT assay after 24 and 48 h of exposure to various concentrations of each compound (10, 100 nM, 1, 10, 100 µM; Figure 5a,b). Across all concentrations and time points, there were no significant differences in viability between KR‐NIR‐P–treated and ICG‐treated cells. At the highest concentration (100 µM), both KR‐NIR‐P and ICG caused a reduction in cell viability after 24 h, but the extent of this effect was similar for both compounds.

FIGURE 5.

Cytotoxicity and molecular responses to ICG and KR‐NIR‐P. (a,b) MTT viability assays after 24 h and 48 h treatment with ICG or KR‐NIR‐P (10 nM – 100 µM). (c) Western blot analysis of PARP, caspase‐3, caspase‐9, Bax, HO‐1, and LC3 following 1 µM treatment for 24 h. (d) Densitometry analysis of corresponding proteins. Data represent mean ± SD (n = 3). Significance was assessed using unpaired two‐tailed t‐tests (^* p < 0.05 versus control).

Importantly, in addition to immortalized cell lines (e.g., HeLa, HSC‐3, Detroit 573), we also examined human dermal fibroblast (HDF) cells, which are primary cells directly isolated from human tissue. Unlike immortalized cells that are genetically modified to proliferate indefinitely and often exhibit tumor‐like behavior, HDFs represent a non‐cancerous, physiologically relevant model. KR‐NIR‐P exhibited negligible cytotoxicity in HDFs, further supporting its excellent biocompatibility.

To further assess the biological effects of KR‐NIR‐P relative to ICG, we analyzed expression levels of key molecular markers after a 24‐h treatment at 1 µM. We examined apoptosis‐related genes (caspase‐3, ‐8, ‐9, and Bax, which promote apoptosis, and Bcl‐2, which opposes apoptosis). Typically, when apoptosis is triggered, pro‐apoptotic markers increase, and cells upregulate Bcl‐2 as a compensatory response. In our experiments, the KR‐NIR‐P–treated group showed a significant upregulation of caspase‐3, ‐8, ‐9, and Bax at the mRNA level (Figure S11a), whereas the ICG‐treated group did not. Apoptosis is often associated with elevated reactive oxygen species (ROS) that cause oxidative stress, which can be monitored via antioxidant response markers (Figure S11b). However, we observed no significant changes in the expression of antioxidant genes in either treatment group, suggesting that intracellular oxidative conditions remained stable. Similarly, we detected no notable changes in inflammation‐related markers, indicating that neither treatment elicited an inflammatory response (Figure S11c). In addition, cell‐cycle analysis revealed no significant alterations in cell cycle regulators, implying that KR‐NIR‐P does not perturb normal cell cycle progression (Figure S11d). Although KR‐NIR‐P triggered an increase in pro‐apoptotic mRNA markers, it is crucial to determine whether this leads to actual apoptotic protein activation, since cellular outcomes depend on protein‐level changes (Figure 5c,d). Notably, despite the transcript‐level differences, the protein levels of key apoptotic markers (PARP, caspase‐3, caspase‐9, and Bax) remained essentially unchanged in cells treated with KR‐NIR‐P compared to those treated with ICG or untreated controls. Likewise, protein levels of HO‐1 (an antioxidant enzyme) and LC3 (an autophagy marker) showed no significant differences between KR‐NIR‐P and ICG treatments. These results indicate that under our experimental conditions, KR‐NIR‐P does not induce significant cytotoxic or stress responses relative to ICG at both the cellular and molecular levels.

4. Conclusion

KR‐NIR‐P represents a significant advancement over traditional NIR dyes, primarily due to its dramatically enhanced photostability. While retaining optical properties comparable to indocyanine green (ICG), including similar absorption and emission profiles, KR‐NIR‐P exhibits far superior resistance to photobleaching, maintaining a high fluorescence intensity under prolonged illumination. This improvement, achieved through the strategic polymerization of ICG, greatly extends the feasible imaging duration and mitigates the challenges associated with ICG's rapid photodegradation. Extensive evaluations of KR‐NIR‐P's biological interactions further highlight its suitability for biomedical applications. The polymer shows robust and consistent cellular uptake across various human cell lines, with preferential cytoplasmic localization and no adverse nuclear accumulation, indicating effective and safe internalization pathways. Comprehensive testing confirmed the excellent biocompatibility of KR‐NIR‐P at clinically relevant concentrations, both in immortalized cell lines and in primary human dermal fibroblasts (HDFs), which more closely represent normal human tissue. Gene and protein expression analyses provided additional insight into cellular responses. A modest increase in apoptosis‐related transcripts was observed. However, these changes did not translate into activation of the corresponding proteins. This type of transcript–protein divergence is commonly reported as a mild and reversible cellular adjustment to new chemical structures rather than activation of functional apoptotic signaling. Representative antioxidant, cytokine, and cell cycle markers also showed no detectable changes, further supporting that major stress pathways were not engaged. Together with the viability data, these findings indicate that KR‐NIR‐P behaves similarly to ICG in terms of cellular safety. This underscores KR‐NIR‐P's safe profile and supports its translational potential for clinical applications. Additionally, 3D spheroid studies provided key insights into KR‐NIR‐P's excellent deep‐tissue penetration and uniform distribution within complex tumor‐like structures, ensuring thorough tumor coverage, which is essential for effective imaging and therapy. Collectively, these characteristics establish KR‐NIR‐P as a highly promising NIR imaging agent that combines superior photostability with excellent biocompatibility and tissue penetration capabilities. KR‐NIR‐P offers clear advantages over conventional ICG, significantly enhancing imaging efficiency and therapeutic potential, and it marks a valuable step forward in the development of advanced NIR biomedical imaging technologies.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll73356‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.