HA-AuNPs/FDF for Highly Sensitive Detection of Hyaluronidase, Tumor-targeting Fluorescence Cell Imaging and Phototherapy

doi:10.6023/A23060283

Abstract

In this paper, a novel composite nano diagnostic and therapeutic agent HA-AuNPs/FDF was constructed for highly sensitive fluorescence detection of hyaluronidase (HAase), tumor-targeting fluorescence cell imaging and photodynamic/photothermal synergistic therapy. HA-AuNPs/FDF was formed by the electrostatic self-assembly of FDF, a diketopyrrolopyrrole-based conjugated small molecule, and HA-AuNPs, the gold nanoparticles functionalized with the tumor-targeting biomolecule hyaluronan (HA). FDF generated strong fluorescence under the excitation of near-infrared (NIR) light, and HA-AuNPs will quench the fluorescence of FDF through fluorescence resonance energy transfer (FRET). However, the overexpression of hyaluronidase (HAase) in tumor cells can gradually degrade HA, so FDF was released and the fluorescence of FDF was gradually recovered. Therefore, the fluorescence recovery of HA-AuNPs/FDF can be used for the rapid quantification of HAase. Experiments have shown that this method can achieve good linear response within the range of 0.25~2.25 U/mL with a detection limit of 0.04 U/mL. On this basis, after respective incubation with HA-AuNPs/FDF for 4 h, NIR fluorescence imaging was performed on human cervical cancer tumor cells (HeLa cells), HeLa cells pretreated with HA, and mouse embryonic fibroblasts (NIH-3T3 cells) to study the tumor-targeting capability of HA-AuNPs/FDF. In addition, after incubation with HA-AuNPs/FDF for 20 min, NIR fluorescence imaging was also performed on HeLa cells to detect the changes in fluorescence intensity over time and study the capability of HAase to activate fluorescence. All the results showed that HA-AuNPs/FDF was readily endocytosed by HeLa cells via the receptor CD44 and degraded by intracellular overexpressed HAase, and that it can be successfully used as a fluorescence probe activated by HAase for fluorescence cell imaging of HeLa cells. Furthermore, as FDF is a material with photothermal and photodynamic therapeutic potential, HA-AuNPs/FDF exhibited the single linear oxygen yield of 23.7%, with a photothermal conversion efficiency of 21.3% and good photothermal stability. The cytotoxicity of HA-AuNPs/FDF was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and live/dead cell staining experiments using calcein-acetoxymethyl ester (AM) and propidium iodide (PI) double staining kit. The results showed that the number of dead HeLa cells was significantly increased under laser irradiation as compared to that under dark conditions, confirming that HA-AuNPs/FDF can effectively inhibit the proliferation of HeLa cells by photodynamic/photothermal synergistic therapy. This system expands the ideas for achieving precise and efficient tumor diagnosis and therapy.

Key words: diketopyrrolopyrrole (DPP), nano diagnostic and therapeutic agent, hyaluronidase, fluorescence imaging, phototherapy

Cite this article

Yanqin Huang, Lijun Li, Shupei Yang, Rui Zhang, Xingfen Liu, Quli Fan, Wei Huang. HA-AuNPs/FDF for Highly Sensitive Detection of Hyaluronidase, Tumor-targeting Fluorescence Cell Imaging and Phototherapy[J]. Acta Chimica Sinica, 2023, 81(12): 1687-1694.

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Fig. & Tab. 10

Figure 1. (a) Structural formula of FDF and HA; (b) preparation of HA-AuNPs; (c) schematic diagram of the detection of HAase and tumor-targeting fluorescence imaging and phototherapy for tumor cells

Figure 2. (a) UV-Vis-NIR absorption spectra of HA-AuNPs/FDF, HA-AuNPs and FDF (the concentration of FDF is 1 mg/mL); (b) Zeta potential of HA-AuNPs/FDF, HA-AuNPs and FDF

Figure 3. (a) TEM image of HA-AuNPs; (b) TEM image of HA-AuNPs/FDF; (c) hydrodynamic sizes of HA-AuNPs; (d) hydrodynamic sizes of HA-AuNPs/FDF

Figure 4. (a) Fluorescence spectra of FDF (black solid line), fluorescence spectra of HA-AuNPs/FDF (red solid line), and fluorescence spectra of HA-AuNPs/FDF with the increase of HAase concentration (blue dash dot line); measurements were performed in phosphate buffer solution (PBS) (10 mmol/L, pH 5.6), [FDF]=0.025 mg/mL; (b) ΔI as a function of HAase concentration

检测方法	线性范围/(U•mL⁻¹)	检测限/(U•mL⁻¹)	文献
比色法	0~240	24	[7]
酶谱法	0.625~5	0.625	[12]
荧光法	0.01~10	0.004	[19]
荧光法	0.05~2	0.02	[18]
荧光法	0~1.3	0.075	[17]
荧光法	0.5~100	0.14	[9]
荧光法	1.25~50	0.63	[16]
荧光法	0.25~2.25	0.04	本文

Table 1. Comparison of different methods for HAase detection

Figure 5. Effects of various potential interferents on ΔI during the detection of HAase (HAase: 2.0 U/mL; EcoRI, Lysozyme, Trypsin and Thrombin: 10 U/mL)

Figure 6. (a) UV-Vis absorption spectra of DPBF in the aqueous solutions containing HA-AuNPs/FDF at different irradiation time (635 nm, 30 mW/cm2); (b) photodegradation rate of DPBF in the aqueous solutions containing HA-AuNPs/FDF or MB; (c) 1O2 detection in HeLa cells after incubation with HA-AuNPs/FDF, using DCFH-DA as a fluorescent probe

Figure 7. (a) Temperature elevation curve of HA-AuNPs/FDF at the same concentration (1 mg/mL) under laser irradiation with different powers; (b) photothermal response of HA-AuNPs/FDF (1 mg/mL) under laser irradiation (635 nm, 1 W/cm2) for 600 s and then the laser was shut off, and this operation was cycled five times; (c) single photothermal cycle of the process in (b); (d) linear time versus -ln θ obtained from the cooling period of (c)

Figure 8. (a) NIR fluorescence imaging of (1) HeLa cells, (2) HeLa cells pretreated with HA, and (3) NIH-3T3 cells after incubation with HA-AuNPs/FDF for 4 h; (b) changes in fluorescence intensity over time in HeLa cells incubated with HA-AuNPs/FDF for 20 min

Figure 9. (a) Cell viability of HeLa cells treated with HA-AuNPs/FDF under dark conditions; (b) cell viability of HeLa cells treated with HA-AuNPs/FDF under light irradiation (635 nm, 0.8 W/cm2) for 8 min; (c) live/dead staining of HeLa cells treated with HA-AuNPs/FDF without or with light irradiation (635 nm, 0.8 W/cm2) for 10 min

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HA-AuNPs/FDF用于透明质酸酶的高灵敏检测、肿瘤靶向细胞荧光成像和光疗