Near-infrared Aza-BODIPY nanoparticles based on D-A-D structure for bioimaging

1. Introduction

Biological imaging techniques have a vital role in elucidating the complex chemical composition, biomolecular interactions, and structural functions of living organisms, as well as facilitating the diagnosis and treatment of related diseases.^1,2,3,4 However, the complex and highly organized structures of live cells and organisms present significant challenges in the field of biological imaging. Optical fluorescence imaging has become an important technique among various imaging modalities in biological research and biomedical applications, due to its noninvasive detection capabilities, high sensitivity, and excellent resolution.^5,6,7

Traditional fluorescence imaging methods encounter challenges such as intrinsic autofluorescence signals, significant cellular damage, and limited penetration depth of excitation light.^8,9 Consequently, fluorescent probes emitting at wavelengths below 550nm are often unsuitable for various biomedical applications. Increasing the wavelength from 550nm to 700nm can enhance penetration depth by up to fourfold.¹⁰ This phenomenon has sparked growing interest in long-wavelength probes, particularly near-infrared (NIR) fluorescent probes. These probes offer deeper photon penetration, minimize optical damage, and reduce interference from biological autofluorescence, resulting in higher signal-to-noise ratios. As a result, NIR fluorescence imaging is quickly emerging as a highly promising analytical technique in the field of bioimaging.^{11,12,13,14,15}

Organic dyes represent a significant category of novel fluorescent labeling materials.¹⁶ Among them, BODIPY dyes stand out due to their high fluorescence quantum yield, narrow absorption and emission spectra, excellent chemical and photostability, and versatile modification sites.^17,18,19,20 Aza-BODIPY, a structural analog of BODIPY, incorporates a nitrogen bridge in its core that connects two pyrrole rings, replacing the meso-carbon atom in the original BODIPY structure.^21,22,23 This modification enhances the electron density in the $\pi$-conjugated system, resulting in an approximate 80nm redshift in both absorption and emission wavelengths. Consequently, the operational range of aza-BODIPY falls primarily within the NIR region.^24,25,26

A typical method to obtain NIR emission for detection or imaging involves lengthening the conjugation of the molecular structure. Although extending the $\pi$-conjugated chain can yield greater redshifts, it may compromise solubility and complicate synthesis.²⁷ An alternative strategy involves constructing and modifying donor–acceptor (D-A) architectures on BODIPY, which enhances NIR emission and facilitates redshifts while avoiding the aforementioned limitations.^{19,28,29,30,31,32} Furthermore, addressing the inherent hydrophobicity of aza-BODIPY in biological systems is essential. One approach involves forming water-soluble NPs encapsulated with amphiphilic fragments, which can target tumor cells via the enhanced permeability and retention (EPR) effect.^{33,34,35,36,37}

In this work, we synthesized aza-BODIPY molecules exhibiting NIR emission using straightforward methods and cost-effective materials. To create a D-A-D framework, methoxy groups and triphenylamine were introduced into the aza-BODIPY framework, resulting in a redshift of the fluorescence emission band into the NIR region. For comparative analysis, we performed demethylation with boron tribromide to yield Aza–BDP-OH, and substituted a hydrogen atom on the pyrrole ring with an iodine atom to obtain Aza–BDP-I, which significantly enhanced the dye’s Stokes shift. The resulting NPs formed through the self-assembly of amphiphilic fragments, exhibit excellent water solubility and biocompatibility, enabling effective targeting of tumor cells via the EPR effect (Scheme 1). These characteristics position the synthesized NPs as promising candidates for NIR bioimaging applications.

2. Materials and Methods

2.1. Materials

4′-Bromoacetophenone, diphenylamine, and N-Iodosuccinimide (NIS) were sourced from Shanghai Aladdin Company. The Cell Counting Kit-8 (CCK-8) was sourced from Biyuntian Company. Chloroform was acquired from Sinopharm Group, phosphate-buffered saline (PBS), petroleum ether 50–$70^{\circ }$C (PE), poly(maleic anhydride-alt-1-octadecene) (PMHC${}_{18}$, $\mathrm{\text{MW}}=30–50$KDa) and poly(ethylene glycol) methyl ether (mPEG-NH₂, $\mathrm{\text{MW}}=5$KDa) were purchased from Adamas. Poly(maleic anhydride-alt-1-octadecene)-poly(ethylene glycol) (PMHC${}_{18}$-mPEG) were synthesized following established procedures (S2, S3, S23).³⁸ The solvents employed for synthesis underwent single distillation, while high-purity solvents were used for the research procedures.

2.2. Synthesis of Aza–BDP–OCH₃

B-3 (1.0g, 2.1mmol) and ammonium acetate (4.86g, 63mmol) were combined in a 30mL of n-butanol and heated to $115^{\circ }$C and stirred for 24h. Following this, the solvent was removed under reduced pressure, and the resulting mixture was filtered, washed with ethanol, and dried to produce a brownish-purple powder. This powder was then dissolved in an anhydrous DCM (40mL) in a N₂ atmosphere. DIPEA (6mL, 34.4mmol) was added, following 30min of stirring, BF${}_3\cdot {\mathrm{\text{Et}}}_2$O (10mL, 79.2mmol) was introduced gradually in a dropwise manner. The mixture was agitated at room temperature for 24h. Upon completion of the reaction, DCM was employed for extraction. The crude product was subsequently purified using column chromatography (DCM:$\mathrm{\text{PE}}=1$:1), resulting in a metallic purple-black solid (300mg, two-step yield: 20%). ¹H NMR (400MHz, Chloroform-$d)\delta 8.05$ (dd, $J=12.9$, 8.9Hz, 8H), 7.35 (dd, $J=8.5$, 7.3Hz, 8H), 7.25–7.19 (m, 8H), 7.15 (td, $J=7.3$, 1.3Hz, 4H), 7.08 (d, $J=9.0$Hz, 4H), 7.03–6.97 (m, 6H), 3.92 (s, 6H).${}^{13}$C NMR (101MHz, CDCl${}_3)\delta$149.95, 146.61, 130.96, 130.64, 129.53, 125.98, 124.43, 120.44, 114.04, 55.42, 31.52, 30.15, 29.71. HR-MS (ESI): m/z calcd for C${}_{58}$H${}_{44}$BF₂N₅O₂ [$\mathrm{\text{M}}+1$]${}^{+}$: 892.359, found: 892.371.

2.3. Synthesis of Aza–BDP–I

In a nitrogen atmosphere, Aza–BDP–OCH₃ (100mg, 0.11mmol) and N-iodobutyrylamide (63.1mg, 0.28mmol) were mixed in a 30mL of DCM. Following this, a 10mL of acetic acid was introduced, and the mixture was agitated at room temperature for 12h. The resulting product was extracted using DCM. The crude product was subsequently purified using column chromatography (DCM:$\mathrm{\text{PE}}=1$:2), resulting in a deep blue solid (51.4mg, yield: 49%). ¹H NMR (400MHz, Methylene Chloride-$d_2)\delta 7.82(\mathrm{\text{dt}},J=9.0$, 2.1 Hz, 4H), 7.70–7.62 (m, 8H), 7.41 (tt, $J=7.5$, 2.0Hz, 4H), 7.27 (t, $J=9.3$Hz, 6H), 7.13–7.06 (m, 4H), 7.05–6.96 (m, 10H), 3.90 (s, 6H). ${}^{13}$C NMR (101MHz, CD₂Cl${}_2)\delta 160.97$, 149.15, 145.33, 144.25, 142.00, 132.77, 132.34, 132.09, 127.49, 123.28, 122.81, 119.59, 117.51, 113.62, 55.41, 29.69, 13.89. HR-MS (ESI): m/z calculated for C${}_{58}$H${}_{42}$BF₂I₂N₅O₂ 1143.149, found: 1143.147.

2.4. Synthesis of Aza–BDP–OH

In a nitrogen atmosphere, a 100mg (0.11mmol) of Aza–BDP–OCH₃ was dissolved in a 40mL of DCM. At $-70^{\circ }$C, a 160$\mu$L of BBr₃ (1 M solution in DCM, three equivalents) was introduced gradually, the resulting mixture was agitated for 1h, then allowed to room temperature, where it was stirred for 24h. To quench the BBr₃, a 10mL of MeOH was slowly added at $-70^{\circ }$C. The solvent was subsequently evaporated under reduced pressure, and water was incorporated, followed by a pH adjustment to 7–8 using Na₂CO₃. The resulting mixture was extracted with DCM. Ultimately, column chromatography (DCM:$\mathrm{\text{MeOH}}=1$:3) yielded a deep purple solid (25.7mg, yield: 24%). ¹H NMR (400MHz, DMSO-$d_6)\delta 10.02$ (s, 2 H), 8.05 (dd, $J=17.5$, 8.9Hz, 8 H), 7.46–7.33 (m, 10 H), 7.22–7.13 (m, 12 H), 6.95–6.82 (m, 8 H). ${}^{13}$C NMR (101MHz, DMSO-$d_6)\delta 159.53$, 155.76, 150.20, 146.23, 144.76, 141.66, 131.57, 131.19, 130.38, 126.42, 125.50, 123.77, 123.39, 119.68, 117.65, 116.12, 55.38. HR-MS (ESI): m/z calculated for C${}_{56}$H${}_{40}$BF₂N₅O₂ [M–1]${}^{+}$: 862.32, found: 862.31.

2.5. Characterization of Aza–BDPs

The analysis of ¹H NMR and ${}^{13}$C NMR was conducted using a Bruker Advance 400MHz NMR spectrometer, employing DMSO-d₆, chloroform-d and dichloromethane-d₂ as the solvents. High-resolution-mass (HR-MS) spectra were measured on an AB Sciex X-500B QTOF-mass spectrometer. For the assessment of UV-visible absorption and fluorescence emission, an HP-8453 UV spectrophotometer and a Fluoromax-4 spectrophotometer were utilized, respectively.

2.6. Preparation of Aza–BDPs NPs

The Aza–BDP–OCH₃ (1mg) and amphiphilic polymer PMHC${}_{18}$–mPEG (5mg) were dispersed in chloroform and ultrapure water ($V_{\mathrm{\text{chloroform}}}:V_{\mathrm{\text{water}}}=1$ : 5), followed by ultrasonic emulsification for 10min using an ultrasonic cell disruptor. After emulsification, the organic solvent was evaporated in a rotary evaporator to obtain amphiphilic PEG-encapsulated Aza–BDP–OCH₃ NPs. The preparation methods for Aza–BDP–OH NPs and Aza–BDP–I NPs were identical to that of Aza–BDP–OCH₃ NPs. For biological experiments, PBS was used in place of ultrapure water for the synthesis of Aza–BDPs NPs.

2.7. Characterization of Aza–BDPs NPs

The characterization of Aza–BDPs NPs was performed using transmission electron microscopy (TEM) with a Hitachi HT7800 model from Japan, operating in bright field mode, to evaluate their morphology. The particle size of the Aza–BDPs NPs and zeta potential were measured with the Zetasizer Lab dynamic light scattering instrument (DLS, Malvern, UK) at room temperature. A Shimadzu UV-1900 spectrophotometer (Japan) was used to record the absorption spectra. Fluorescence data were collected with an Edinburgh FS5 spectrometer (UK). Using a PerkinElmer IVIS Lumina III imaging system (USA), fluorescent micrographs of the Aza–BDP nanoparticles (NPs) were captured under 720nm laser excitation.

2.8. Hemolytic experiment of Aza–BDPs NPs

Blood (1mL) was collected from the eyes of mice and diluted with 2mL of PBS, followed by multiple washings (1200rpm, 5min) to separate the red blood cells (RBCs). The isolated RBCs were mixed in 4mL PBS. The RBC suspension was incubated with Aza–BDP–OCH₃ NPs at varying concentrations of 10, 25, 50, 75, and 100$\mu \mathrm{\text{g}}\cdot {\mathrm{\text{mL}}}^{-1}$, as well as with PBS and deionized water. After incubating for 5h, the samples were centrifuged at 13,000rpm for 15min to collect the supernatant. The absorbance of each solution was then measured at 570nm. The hemolysis experiments for Aza–BDP–OH NPs and Aza–BDP–I NPs were conducted using the same method as for Aza–BDP–OCH₃ NPs.

The hemolysis rate of the red blood cells was determined by the formula below :

The absorbance values at the corresponding wavelength are represented by A_sample, A_PBS, and A_water, which denote the sample, PBS, and deionized water, respectively.

2.9. Cell culture

The Shanghai Institute of Biochemistry and Cell Biology provided the 4T1 mouse breast cancer cells, which were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. These cells were kept in a humidified incubator at $37^{\circ }$C with 5% carbon dioxide and 95% air.

2.10. Cell viability assay

The 4T1 cell line was transferred to a 96-well plate, with each well containing 5000 cells, and incubated for 24h. The RPMI 1640 medium served as the growth environment, with Aza–BDP–OCH₃ NPs introduced at varying concentrations of 0, 1, 2, 5, 10, 25, 50, 75 and 100$\mu \mathrm{\text{g}}\cdot {\mathrm{\text{mL}}}^{-1}$. Following an incubation period of 48h, the assessment of cell viability was conducted using the CCK-8 assay kit,^39,40 adhering to the instructions provided by the manufacturer. Each well was supplied with 90$\mu$L of cell culture medium along with 10$\mu$L of CCK-8 solution and then incubated for an additional 1.5h. The absorbance was recorded at 450nm with the aid of a microplate reader. To reduce variability, each concentration was tested in five replicates ($n=5$). The cytotoxicity testing methods for Aza–BDP–OH NPs and Aza–BDP–I NPs were identical to those employed for Aza–BDP–OCH₃ NPs.

The rate of cell viability was determined using the following calculation :

$A_s$, $A_c$, and $A_b$ correspond to the absorbance values of the experimental group, the control group and the blank group, respectively.

2.11. Preparation of animal tumor model

Five-week-old female BALB/c-nu mice were sourced from the Shanghai Institute of Planned Parenthood Research to develop the 4T1 tumor model. This institute is accredited by the Shanghai Quality Supervision and Inspection Institute of Experimental Animals (SCXK 2018-0006). A suspension of 4T1 cells ($2\times 10^6$ cells mL${}^{-1}$, 100$\mu$L) was injected into the axillary region of the mice’s hind legs. Seven days post-injection, the 4T1 tumor model was employed for in vivo imaging studies.

2.12. Fluorescence imaging in vivo

The Aza–BDPs NPs (100$\mu$L, 2mM) were injected intravenously into BALB/c-nu mice. Under 720nm laser irradiation, Fluorescence images were obtained from the mice at 1h and 6h using a small animal imaging system (PerkinElmer IVIS Lumina III, USA). Subsequently, the mice were euthanized, and their primary organs and tumors were collected for fluorescence imaging of the various tissues.

3. Results

3.1. Synthesis and characterization

The synthetic route of Aza–BDPs is schematically illustrated in Scheme 2. A carbon–nitrogen coupling reaction was carried out using acetophenone and diphenylamine, facilitated by a palladium catalyst and a base, resulting in the formation of 4-(N,N-diphenylamino)acetophenone.⁴¹ This compound was subsequently reacted with 4-methoxybenzaldehyde to synthesize Aza–BDP–OCH₃ with a D-A-D structure, following established procedures in the literature.^42,43 The methoxy group in Aza–BDP–OCH₃ was further demethylated with BBr₃ to generate Aza–BDP–OH. Subsequently, Aza–BDP–I was produced by replacing a hydrogen atom on the pyrrole ring with an iodine atom. Finally, the synthesized compounds were analyzed using NMR and HRMS to determine their structures. Characterization results confirmed the successful synthesis of the target compounds (provided in SI).

3.2. Absorption and fluorescence

The UV–Vis absorption spectra of Aza–BDPs were recorded in CH₂Cl₂ at a concentration of $1\times 10^{-5}$ M. The optical data obtained are shown in Table 1, while the associated absorption spectra are displayed in Fig. 1(a). Aza–BDP–OCH₃ and Aza–BDP–OH displayed identical normalized absorption spectra, with a maximum absorption wavelength of 787nm, attributable to the similar electron-donating properties of their hydroxyl and methoxy groups. In contrast, Aza–BDP–I exhibited a maximum absorption wavelength of 763nm, with the observed blue shift resulting from changes in the molecular energy gap. All three compounds showed two distinct absorption bands in the 500–850nm range, which were ascribed to the ${}^1\pi$–${\pi }^{\ast }$ transitions occurring within the Aza-BODIPY aromatic framework.

Fluorescence spectra were recorded in the same solution (Fig. 1(b)), revealing emission peaks at 835nm for Aza–BDP–OCH₃ and 825nm for Aza–BDP–OH, respectively, with corresponding Stokes shifts of 48nm and 38nm. Notably, Aza–BDP–I exhibited a significant redshift in its emission spectrum, resulting 66nm Stokes shift. The solvatochromic behavior of Aza–BDPs was examined across a range of solvents (Fig. S1), revealing a positive solvent effect. This phenomenon can be ascribed to the intramolecular charge transfer (ICT) effect resulting from the D-A-D configuration established by triphenylamine, the Aza–BODIPY core, and the phenyl ring. Consequently, the fluorescence emission of the Aza–BDPs results from the combined contributions of ${}^1\pi$–${\pi }^{\ast }$ transitions and ICT effect. The measured absolute fluorescence quantum yields for Aza–BDP–OCH₃ and Aza–BDP–OH in CH₂Cl₂ were found to be 0.583% and 2.1%. In contrast, the fluorescence quantum yield of Aza–BDP–I dropped to 0.064%, likely due to the heavy atom effect of iodine, which enhances intersystem crossing (ISC) processes. These findings demonstrate that structural modifications significantly impact the photophysical properties of these Aza–BODIPY derivatives, highlighting their potential for NIR biological imaging applications.

3.3. Theoretical calculation

To enhance our understanding of the connection between molecules structure and photophysical properties, we utilized density functional theory to conduct quantum chemical calculations. The results of the structural optimization showed that the dihedral angles between the Aza–BODIPY core of Aza–BDPs and the $\gamma$-position phenyl ring are $17.9^{\circ },18.3^{\circ }$, and $37.6^{\circ }$ respectively, while those angles with the $\alpha$-position phenyl ring are $27.8^{\circ },27.8^{\circ }$, and $48.9^{\circ }$ respectively (Fig. 2(a)). The increased atomic volume at positions 2 and 6 in Aza–BDP–I leads to a stronger torsional conformation along the main chain. This suggests that the introduction of halogen substituents enhances the flexibility of the molecular geometry, potentially explaining the larger Stokes shift observed in Aza–BDP–I.⁴⁰ The electronic structure analysis (Fig. 2(b)) shows nearly identical highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) for Aza–BDP–OCH₃ and Aza–BDP–OH. This similarity explains the complete overlap of their normalized absorption spectra. The HOMO of Aza–BDPs is primarily localized in the core structure of Aza-BODIPY, suggesting a crossover of the $\pi$-conjugated orbitals. Additionally, the strong electron-donating triphenylamine group contributes to the HOMO distribution across the substituents. The HOMO of these three compounds exhibits extensive delocalization throughout the entire molecular framework, the LUMO is predominantly divided among the Aza-BODIPY backbone. This distribution suggests effective ICT within Aza–BDPs.

3.4. Preparation of nanoparticles and characterization

Water-soluble Aza–BDPs NPs were synthesized to address the strong hydrophobicity of Aza–BODIPY. These NPs were created through the self-assembly of Aza–BDPs with the amphiphilic polymer PMHC${}_{18}$–mPEG. They displayed similar absorption (Fig. 3(a)) and NIR emission spectra (Fig. 3(b)). The absorption characteristics of Aza–BDPs NPs remained unchanged (no redshift or blueshift), indicating that the distorted conformation of Aza–BDPs effectively inhibits $\pi$–$\pi$ stacking in the aggregated state. The absolute fluorescence quantum yields of Aza–BDP–OCH₃ NPs, Aza–BDP–OH NPs, and Aza–BDP–I NPs in water were measured at 0.074%, 0.068%, and 0.032%, respectively. Compared to Aza–BDPs, the NPs exhibited a reduction in fluorescence quantum yields, probably as a result of aggregation-induced quenching (ACQ) effects.

The sizes and morphologies of the three NPs were analyzed using DLS and TEM. The DLS analysis revealed the hydrated diameters of Aza–BDP NPs measured 250nm, 280nm, and 275nm, respectively. TEM images revealed that all NPs displayed a spherical shape, maintaining a mean diameter of 150nm, and they were stably dispersed in water. The smaller sizes noted in the TEM measurements were likely a result of the contraction of the hydration layer in the dehydrated samples (Figs. 3(c)–3(e)). Previous studies have demonstrated that NPs ranging from 10nm to 300nm can concentrate at tumor sites through the EPR effect.^44,45,46 Zeta potential measurements were conducted in PBS buffer solution at a pH value of 7.4, the results indicated the NPs exhibited good colloidal stability over a period of three days. Additionally, the comparison of different batches of Aza–BDPs NPs also demonstrated the good stability (Fig. S4). Furthermore, NPs coated with PEG-based polymers demonstrate excellent stability in blood circulation. Therefore, these NPs are well suited for in vivo tumor fluorescence imaging.

3.5. Biocompatibility assessments of Aza–BDPs NPs

Biocompatibility is essential for the biomedical application of nanomaterials. The cytotoxicity of Aza–BDPs NPs toward 4T1 cells using the Cell Counting Kit-8 (CCK-8) assay to assess biocompatibility. The cell viability of Aza–BDPs NPs exceeded 90%, with no significant cytotoxicity observed, even at concentrations up to 100$\mu \mathrm{\text{g}}\cdot {\mathrm{\text{mL}}}^{-1}$ (Figs. 4(a)–4(c), S5). Additionally, we conducted in vitro hemolysis tests to assess the interactions between blood components and Aza–BDPs NPs. At varying concentrations of Aza–BDPs NPs from 10$\mu \mathrm{\text{g}}\cdot {\mathrm{\text{mL}}}^{-1}$ to 100$\mu \mathrm{\text{g}}\cdot {\mathrm{\text{mL}}}^{-1}$, the hemolysis rate remained relatively unchanged (Figs. 4(d)–4(f)). These results indicate that these materials are suitable for biomedical imaging applications.

3.6. In vivo fluorescence imaging of mice tumors

Applying Aza–BDPs NPs to in vivo tumor imaging experiments. Tumor models were developed by administering 4T1 cells into the hind limb of female BALB/c-nu mice that were five weeks old. Optical imaging was performed seven days post-tumor inoculation, with Aza–BDPs NPs administered via tail vein injection. Imaging assessments were conducted at 1 and 6h post-injection (Fig. 5). We observed that the fluorescence intensity in the tumor tissue significantly increased 1h after the injection of Aza–BDP-I NPs, and then gradually decreased after 6h. In contrast, the fluorescence in the liver increased, indicating the metabolism of Aza–BDP-I NPs over time. Aza–BDP-I NPs effectively outlined the tumor tissue, demonstrating their potential as effective tumor imaging agents, while the other two types of NPs showed increased fluorescence in the abdominal cavity 1h after injection, which gradually decreased after 6h, indicating that these two NPs are not suitable as tumor imaging agents (S7–S10).

3.7. In vivo metabolism of Aza–BDP-I NPs

To investigate the metabolic routes of Aza–BDP-I NPs, two mice were dissected (Fig. 6). Dissection and analysis were conducted 1h after NPs post-injection. One hour after injection, Aza–BDP-I NPs exhibited low-fluorescence intensity in the upper region of the abdomen, attributed to limited accumulation of the material, whereas the stomach and lower abdominal cavity showed the brightest fluorescence. These observations suggest that Aza–BDP-I NPs, despite tail vein injection, follow the hepatobiliary metabolic pathway: Circulating in the bloodstream, entering the bile, and ultimately being metabolized via the digestive system. Six hours after injection, the fluorescence was mainly localized in the liver area, with a significant decrease in brightness in the stomach, further validating the proposed metabolic pathway of Aza–BDP-I NPs.

Examination of the organs indicated that the fluorescence level detected in the liver was higher at 6h post-injection compared to 1h, while the brightness in the tumor tissue decreased (Fig. 7). This observation corroborates the proposed metabolic pathway. Furthermore, a comparison of fluorescence intensity between tumor and liver tissues at 1h post-injection showed higher intensity in the tumor despite its smaller volume, indicating excellent passive targeting capabilities of Aza–BDP-I NPs. This indicates that Aza–BDP-I NPs can accumulate in tumor sites and facilitate tumor imaging through EPR effects. The NIR emission characteristics of these NPs play a significant role in tumor imaging.

4. Discussion

NIR bioimaging technology plays a crucial role in the diagnosis of tumors.⁶ To achieve high-quality NIR fluorescence imaging, it is of great importance to obtain NIR fluorescence nanoprobes. In this study, we constructed a D-A-D structure by incorporating triphenylamine groups and phenyl ring-modified groups with the Aza–BODIPY core, which effectively extended the emission wavelength of the molecule into the NIR region.^29,31 The twisted conformation of the triphenylamine group effectively suppresses $\pi$–$\pi$ stacking in the aggregated state, while the incorporation of the heavy atom iodine further enhances the twisting conformation of the molecule.⁴³ These modifications account for the larger Stokes shift observed in Aza–BDP-I.

Nanoparticles are one of the commonly used approaches to address the hydrophobicity issue of Aza-BODIPY dyes, we encapsulated them within the amphiphilic polymer to create Aza–BDPs NPs. These NPs not only exhibit desirable NIR emission properties but also demonstrate excellent dispersion in aqueous solutions, maintaining a uniform particle size. Additionally, they also exhibit good stability. In subsequent biological evaluations, Aza–BDPs NPs showed remarkably low cytotoxicity even at a concentration of 100$\mu \mathrm{\text{g}}\cdot {\mathrm{\text{mL}}}^{-1}$, and the hemolysis rates remained stable across a concentration range of 10–100$\mu \mathrm{\text{g}}\cdot {\mathrm{\text{mL}}}^{-1}$. Collectively, these results indicate that these NPs possess significant potential for applications in biological systems.^36,37

We applied Aza–BDP NPs to in vivo tumor imaging experiments. Aza–BDP-I NPs demonstrated a longer half-life and enhanced targeting efficiency compared to Aza–BDP–OCH₃ NPs and Aza–BDP–OH NPs. Through anatomical studies of the mice, we confirmed that the metabolic pathway of Aza–BDP-I NPs follows the hepatobiliary metabolism route: they circulate in the bloodstream, enter the bile, and are ultimately metabolized and excreted through the digestive system.

However, our study has certain limitations and opportunities for further enhancement. NIR-II fluorescence provides deeper tissue penetration; therefore, additional molecular modifications may be necessary to achieve emission within this wavelength range.⁴⁷ Moreover, heavy atoms can improve the intersystem crossing efficiency, thereby augmenting the generation of reactive oxygen species and enhancing the efficacy of photodynamic therapy.⁴³ Future research should focus on the integration of tumor diagnosis and treatment.

5. Conclusions

We successfully synthesized three Aza-BODIPY molecules, which self-assembled into NPs with the amphiphilic segment PMHC${}_{18}$-mPEG. The synthesized aza-BODIPY molecules exhibit NIR emission, and DFT calculations indicate that their twisted conformation of Aza–BDPs successfully inhibits $\pi$–$\pi$ stacking in the aggregated form. Following NPs formation, these molecules maintain their excellent optical properties while exhibiting good biocompatibility and low biological toxicity. In vivo imaging results for Aza–BDP-I NPs demonstrate their efficacy in delineating tumor morphology and their subsequent elimination via hepatobiliary excretion. Therefore, we believe that these Aza-BODIPY NPs offer a promising, safe and effective NIR fluorescent platform for bioimaging applications.

Acknowledgments

This work was supported by the National Key R&D Program of China (2023YFA0913600), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX23-1480). H. Liao and Q. Meng contributed equally to this work.

Conflict 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.

Supplementary Material

The Supplementary Information are available at: https://www.worldscientific.com/doi/suppl/10.1142/S1793545825410032.

ORCID

Haitao Liao  https://orcid.org/0009-0008-4800-8120

Qingxuan Meng  https://orcid.org/0009-0001-8881-8477

Yuhao Li  https://orcid.org/0000-0001-7146-8645

Senqiang Zhu  https://orcid.org/0000-0002-8198-0703

Rui Liu  https://orcid.org/0000-0002-8227-6064

Hongjun Zhu  https://orcid.org/0000-0001-8447-6660