Berberine mediated fluorescent gold nanoclusters in biomimetic erythrocyte ghosts as a nanocarrier for enhanced photodynamic treatment

Abstract

Photodynamic therapy (PDT) is a well-established cancer treatment method that employs light to generate reactive oxygen species (ROS) causing oxidative damage to cancer cells. Nevertheless, PDT encounters challenges due to its oxygen-dependent nature, which makes it less effective in hypoxic tumor environments. To address this issue, we have developed a novel nanocomposite known as AuNC@BBR@Ghost. This nanocomposite combines the advantageous features of erythrocyte ghost membranes, the photoresponsive properties of gold nanoclusters (AuNC) and the anticancer characteristics of Berberine (BBR) for cancer treatment. Our synthesized AuNC efficiently produce ROS, with a 25% increase in efficiency when exposed to near-infrared (NIR) irradiation. By harnessing the oxygen-carrying capacity of erythrocyte ghost cells, AuNC@BBR@Ghost demonstrates a significant improvement in ROS generation, achieving an 80% efficiency. Furthermore, the AuNC exhibit tunable emission wavelengths due to their excellent fluorescent properties. In normoxic conditions, treatment of A549 lung carcinoma cells with AuNC@BBR@Ghost followed by exposure to 808 nm NIR irradiation results in a notable increase in intracellular ROS levels, accelerating cell death. In hypoxic conditions, when A549 cells were treated with AuNC@BBR@Ghost, the erythrocyte ghost acted as an oxygen supplement due to the residual hemoglobin, alleviating hypoxia and enhancing the nanocomposite's sensitivity to PDT treatment. Thus, the AuNC@BBR@Ghost nanocomposite achieves an improved effect by combining the advantageous properties of its individual components, resulting in enhanced ROS generation and adaptability to hypoxic conditions. This innovative approach successfully overcomes PDT's limitations, making AuNC@BBR@Ghost a promising nanotheranostic agent with significant potential for advanced cancer therapy.

Fig. 1. Synthesis and characterization of AuNC@BBR nanocomposite: (a) schematic depicting the preparation process of AuNC@BBR; (b) UV-visible absorption spectra of AuNC, BBR and the AuNC@BBR composite; (c) hydrodynamic particle sizes and (d) zeta potential of BBR, AuNC and AuNC@BBR in aqueous media; high-resolution transmission electron microscopy (HR-TEM) images of (e) AuNC, (f) a single AuNC, and (g) the AuNC@BBR composite. Scale bar represents 15 nm, 3 nm and 100 nm respectively.

Fig. 2. Characterization of hemoglobin-lysed erythrocyte blood fraction: (a) schematic illustrating the preparation process of ghost and AuNC@BBR-loaded ghost; (b) comparison of UV-vis absorbance spectra at different stages of ghost preparation (inset: color changes during sequential centrifugation and washes); (c) hydrodynamic particle size and (d) zeta potential of AuNC@BBR, ghost and AuNC@BBR@Ghost in aqueous solution; UV-vis absorbance spectra of (e) ghost and (f) AuNC@BBR@Ghost in oxygenated, deoxygenated and reoxygenated conditions; and (g) oxygen-dissociation curves of ghost and AuNC@BBR@Ghost.

Fig. 3. Fluorescence microscopic images: (a) A549 cells incorporated with AuNCs, emitting RGB fluorescence. With an increasing excitation wavelength, the emission band shifts to longer wavelength, confirming the distribution of nanoclusters sizes. (i) Optical image, (ii) AuNC (ex/em: 405/430–460 nm range, shown in blue), (iii) AuNC (ex/em: 488/510–560 nm range, shown in green), (iv) AuNC (ex/em: 514/610–660 nm range, shown in red) and (v) merged image of (i) to (iv); and (b) fluorescence microscopic images of A549 cells incorporated with BBR (20 μm). (i) Optical image, (ii) BBR (ex/em: 405/540–560 nm) and (iii) merged image of (i) and (ii). Scale bar represents 25 μm. Objective: 40× (oil immersion).

Fig. 4. Structural analysis of RBC, ghost and AuNC@BBR@Ghost: (a and b) optical microscopic images of RBC and ghost respectively. Objective: 40× and the embedded scale bar represents 20 μm; and (c) fluorescence microscopic images of ghost cells incorporated with AuNC@BBR complex at different focal planes. (i) Optical image, (ii) AuNC (ex/em: 514/610–660 nm, shown in red) and (iii) BBR (ex/em: 405/540–560 nm, shown in yellow) (iv) merged image of (i) to (iii). Scale bar represents 5 μm. Objective: 100× (oil immersion).

Fig. 5. Time-dependent measurement of ROS generation under NIR irradiation using DCF probe: (a) time-dependent DCF absorbance changes induced by control, BBR, AuNC, AuNC@BBR and AuNC@BBR@Ghost; (b) time-dependent DCF absorbance changes induced by AuNC. The inset shows the rate constants for the AuNC sample under NIR irradiation; (c) time-dependent DCF absorbance changes induced by AuNC@BBR@Ghost; and (d) the rate constants for the AuNC@BBR@Ghost sample under NIR irradiation.

Fig. 6. Cell viability study using MTT assay for A549 cells treated with various concentration of AuNC and BBR for incubation periods of 24 h and 48 h. (a and b) cells under normoxic conditions and (c and d) cells under hypoxic conditions (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Data are mean ± SD.

Fig. 7. Detection of intracellular ROS generation in A549 cell line under both normoxia and hypoxia conditions. The detection was performed using an oxidized DCFDA probe incorporated into samples with and without NIR irradiation (wavelength: 808 nm; power density: 0.75 w cm⁻²). (a) Fluorescence microscopic images (ex/em: 488/520–550 nm; objective: 20×) scale bar represents 100 μm; and (b) measurement of DCF fluorescence intensity (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant difference between untreated and treated samples. Data are mean ± SD.

Fig. 8. Comparison of the PDT efficacy of AuNC@BBR with and without ghost treatment under NIR irradiation (wavelength: 808 nm; power density: 0.75 w cm⁻²); (a) discrimination of live and dead A549 normoxia and hypoxia cell line using violet fluorescent and DiOC5(3) staining, observed through confocal fluorescence microscopy (objective: 20×). The scale bar represents 100 μm; (b) cell viability of A549 cells assessed through MTT assay. Cells were treated with AuNC, BBR, AuNC@BBR, and AuNC@BBR@Ghost associated PDT treatment (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant difference between untreated and treated samples. Data are mean ± SD.