Jiang Xindong from SYUCT & Sun Xiaohong from CMU et al.: Novel NIR-II 3,5-Julolidinyl aza-BODIPY for Photothermal Therapy of Gliomas Stem Cells by Brain Stereotactic Injection

This paper explores a novel NIR-II 3,5-julolidinyl aza-BODIPY, which is uesd for photothermal therapy of gliomas stem cells by brain stereotactic injection.

1.Abstract

Functional dyes with absorption in the second near-infrared window (NIR-II, 900-1700mm) demonstrate immense potential in photothermal therapy. The preparation of a novel NIR-II 3,5-julolidinyl aza-BODIPY (JLD) is first confirmed by X-ray crystallographic analysis. The molecular packing structure facilitates a J-type aggregation stacking mode. Self-assembled JLD-nanoparticles (JLDNPs) emit fluorescence (λ_em = 962 nm, Φ_f = 0.1%), covering a peak range in the NIR-II window from 900 to 1300 nm. The photothermal conversion efficiency is 79% high. With brain stereotactic injection, JLDNPs have a good performance in inhibition on tumors under NIR-II 915 nm laser irradiation by promoting the apoptosis in glioblastoma cells and inhibiting stem cell characteristics so as to influence malignant events, such as migration and invasion.

2.Research Purpose

This paper aims to develop a novel NIR-II (900-1700 nm) 3,5-julolidinyl aza-BODIPY (JLD) dye for photothermal therapy of glioma stem cells by brain stereotactic injection.

3.Research Contents

(1)Synthesis and Structural Confirmation: The molecular structure of 3,5-julolidinyl aza-BODIPY (JLD) is successfully synthesized and confirmed by X-ray crystallographic analysis for the first time.

(2)Optical Properties: JLD nanoparticles (JLDNPs), exhibiting a subspherical form in aqueous solution, can emit fluorescence at a wavelength of 962 nm with a fluorescence quantum yield of 0.1% and its absorption band covering a NIR-II window range of 900-1300 nm.

(3)Photothermal Conversion Efficiency: The photothermal conversion efficiency of JLDNPs is 79% high.

(4)Effect of Vitro Photothermal Therapeutic: In vitro experiments, JLDNPs effectively inhibit the proliferation of gliomas stem cells and induce apoptosis under 915 nm laser irradiation.

(5)Effect of Vivo Photothermal Therapy: In vivo experiments, JLDNPs manifest significant effect by brain stereotactic injection, such as inhibiting tumor growth and reducing tumor volume.

(6)Mechanism of Action: JLDNPs influence the malignant behavior of tumor cells by promoting apoptosis and inhibiting the stemness of glioblastoma cells, such as migration and invasion.

Research Schemes:

Figure 1. Oak Ridge Thermal Ellipsoid Plot (ORTEP) view of JLD (CCDC: 2351762) (ellipsoids displaced at the 30% probability level): (a) Front view and (b) side view of the molecular structure ; (c, d) Monoplast structure; (e) Normalized absorbance and (f) normalized emission spectra of JLD

Figure 2. (a) Self-assembled JLD-NPs; (b) Dynamic light scattering (DLS) of JLD-NPs in aqueous solution; (c) Transmission electron microscopy (TEM) image of JLD-NPs in aqueous solution with scale of 200 nm; (d) Absorbance of 20 μM JLD-NPs in water; (e) Fluorescence of 20 μM JLD-NPs in water; (f) Temperature changes of JLD-NPs at different concentrations (20−80 μM) by 915 nm laser irradiation (0.8 W cm−2); (g) Photothermal conversion of 80 μM JLD-NPs by 915 nm laser irradiation at different power densities (0.2−1.0 W cm−2); (h) Research on the photothermal stability during five heating-cooling articulations; (i) Temperature response curve of JLD-NPs in aqueous solution in irradiation and natural cooling conditions; (j) Linear fitting of −ln(θ) versus time

Figure 3. Photothermal effects of JLD-NPs on GSCs apoptosis and ROS levels under 915 nm laser irradiation: (a) CCK8 assay for cell viability of GSCs treated with different concentrations of JLD-NPs; (b) Western blot analysis of apoptosis- associated protein expression (BCL2, BAX, Cleaved-Caspase3) in GSCs treated with different concentrations of JLD-NPs; (c) Flow cytometer analysis of apoptosis in each group; (d-f) Detection of ROS release in each group using the DCFH probe via immunocytochemistry (figure d) and flow cytometer (figure e, f). The green fluorescence produced by DCFH represents the intracellular ROS level. Proportional scale = 100 μm. Legend: (*) p < 0.05, (**) p < 0.01, compared with the JLD-NPs = 20 μM group. For clarity, 20 μM JLD-NPs are abbreviated as NPs.

Figure 4. Effects of JLD-NPs on stem cell characteristics and invasiveness of GSCs under 915 nm laser irradiation: (a) Western blot to analysis small molecular stem cells markers levels in four groups (Nestin, SOX2, and CD133); (b) Limiting dilution analysis and (c) tumor sphere formation assay to evaluate changes in the tumor sphere formation and colonyforming ability in vitro of GSCs (magnification 100×). Proportional scale = 100 μm; (d) Transwell assay to detect the invasion ability of GSCs in each group (magnification 400×). Proportional scale = 100 μm. For clarity, 20 μM JLD-NPs are abbreviated as NPs.

Figure 5. Effects of JLD-NPs on an in-situ intracranial glioblastoma mouse model under 915 nm laser irradiation: (a) Schematic diagram of the mouse experimental procedure; (b) evaluation of tumor size in different groups using hematoxylin-eosin (H&E) staining. Proportional scale = 3 mm; (c) statistical graph showing tumor size; (d) immunohistochemical assessment of the proliferation marker ki-67. Proportional scale = 100 μm; (e)  representative immunofluorescence images showing ROS (magnification 200×). Proportional scale = 100 μm; (f) statistical analysis of ROS release expression in four groups of mice; (g) immunofluorescence analysis of TUNEL (cell apoptosis) and the stem cell marker Nestin expression levels (magnification 400×). Proportional scale = 100 μm. For clarity, 20 μM JLD-NPs are abbreviated as NPs.

4.Conclusion

We demonstrate that JLDNPs are an effective photothermal therapeutic agent capable of achieving high photothermal conversion efficiency in the NIR-II region. Furthermore, they exhibit significant therapeutic effects against glioma stem cells through intracranial stereotactic injection.

Corresponding Authors:

Xindong Jiang: Shenyang University of Chemical Technology, Liaoning and Shenyang Key Laboratory of Functional Dyes and Pigments, Shenyang, China, DC, 110142; ORCID: 0000-0002-9780-1124;

Xiaohong Sun: The Fourth Affiliated Hospital of China Medical University, Department of Neurology, Shenyang, China, DC, 110032; Scientific Experiment Center, China Medical University, Shenyang, China, 110032;

Rong Shang: Graduate School of Advanced Sciences of Matter, Hiroshima University, Hiroshima, Japan, DC, 739-8526;

DOI：https://doi.org/10.1021/acsmaterialslett.4c01447