Synthesis and characterisations of AuNR, AuNRB and AuNRBR
AuNR was successfully synthesised with an average size of around 50 nm, as observed by TEM (Fig. 2a). The zeta potential of AuNR (Fig. 2c) was around + 35 mV, which was due to the cationic surfactant of CTAB. The AuNR was modified with BSA to increase biocompatibility, as demonstrated by the TEM (Fig. 2b). As shown in Fig. 2c, the zeta potential decreased to around − 18 mV compared with AuNR, thereby suggesting the successful conjugation of BSA to the surface of AuNR. To further identify the successful modification of BSA, UV was used to determine that the transverse and longitudinal peaks of AuNRB experienced redshift compared with AuNR (Fig. 2d), which was due to the plasmonic coupling of AuNR in this system. Moreover, AuNRB was conjugated with RGD by characterisation with the zeta potential of around − 17 mV (Fig. 2c). Besides, RGD labelled with BODIPY was conjugated to AuNRB along with the change of the solution colour from pink to blue after purification (Fig. 2c, insertion), thereby indicating that RGD was successfully conjugated to AuNRB. The photothermal property of AuNRBR was studied by using an 808 nm laser. Figure 2e shows that the temperature of the solutions rose was dependent on the concentration of AuNRBR. Interestingly, AuNRBR solutions (CAu=80 µg/mL) exhibited rather high temperature, which increased up to 50 °C within 5 min. In addition, the thermal images of AuNRBR had good photothermal conversion efficiency, thereby indicating the good photothermal property of AuNRBR due to the plasmonic coupling effect (Fig. 2f).
Neutrophils Loading AuNRBR and the release efficiency when irradiated with an 808 nm laser
Firstly, the neutrophils were isolated from the mice bone marrow. The purified neutrophils were doubly stained with FITC-CD11b and PE/Cy7 Ly-6G/Ly-6C and analysed by flow cytometry. The yield was about 2.5 × 106 cells/mouse, and the purity of neutrophils was approximately 95% (Fig. 3a, b). The morphology of the obtained neutrophils was observed directly under the microscope. They were polymorphonuclear and small, with sizes of approximately 8 µm (Fig. 3c).
Considering the short lifespan (only a few hours after isolation from blood) and quick phagocytosis ability of neutrophils, the incubation time was optimized to ensure the effective loading amount of AuNRBR. Figure 3d shows that neutrophils incubated with FITC-labelled AuNRBR showed more fluorescence signals with time going by according to flow cytometry analysis results. At the post-incubation time of 60 min, the fluorescence intensity was the highest, thereby suggesting that the ideal incubation time in which viability was not affected was 60 min. The neutrophils loading AuNRBR were also observed by CLSM. The same conclusion was drawn, i.e. neutrophils incubated with AuNRBR for 60 min showed stronger green fluorescence compared with those incubated for 10 min or 30 min, as observed by CLSM (Fig. 3e). Therefore, the incubation time of 60 min was particularly well suited to load AuNRBR into neutrophils.
To investigate AuNRBR release mediated by 808 nm laser irradiation, the content of AuNRBR was determined at each interval time. As shown in Fig. 3f, most AuNRBR remained in neutrophils, and only around 15% AuNRBR was detected in the medium at 12 h, thereby indicating that AuNRBR/N was stable in a normal physiological environment. However, a rather rapid release of AuNRBR from AuNRBR/N occurred when irradiated with an 808 nm laser. Approximately 80% AuNRBR was released from AuNRBR/N within 2 h. AuNRBR was capable of being released efficiently from AuNRBR/N under laser irradiation, thereby further exerting antitumour function.
Cellular Uptake And Cytotoxicity Of AuNRBR And AuNRBR/N
The cell uptake was estimated by using CLSM and flow cytometry to prove the targeting effect of AuNRBR. As shown in Fig. 4a, Lewis cells incubated with AuNRBR showed stronger fluorescence signals compared with AuNRB, as observed by CLSM. The cellular uptake was further measured quantitatively by flow cytometry. As shown in Fig. 4b, the mean fluorescence intensity of AuNRBR was much higher than that of AuNRB, which implied that the modification of RGD could increase the cellular uptake efficiently. The phenomenon above was due to the specific binding of RGD to integrin receptors expressed on Lewis cells [18].
Next, we detected the cytotoxicity of AuNRBR and AuNRBR/N to tumor cells with or without laser irradiation. As illustrated in Fig. 4c, AuNRBR/N exhibited little cytotoxicity to Lewis cells at 6 and 12 h post-incubation without laser irradiation, which demonstrated the excellent biosafety of AuNRBR/N without laser irradiation and selectivity for tumour ablation in PTT. In contrast, AuNRBR/N was able to inhibit the proliferation of Lewis cells effectively both at 6 and 12 h post-incubation with laser irradiation (Fig. 4d). Higher concentration of Au (40, 60 and 80 µg/mL) displayed stronger toxicity, in which Lewis cell viability was below 50% and close to around 10%. The potent cytotoxicity of AuNRBR/N was ascribed to the stronger photothermal effect of AuNRBR and extracellular trap net released from neutrophils [19]. Moreover, the cell viability of group AuNRBR/N at 12 h post-incubation decreased compared with that at 6 h post-incubation, indicating that more AuNRBR was delivered to Lewis cells with extended incubation time. Therefore, AuNRBR/N was capable of killing tumour cell in vitro with laser irradiation.
AuNRBR/N Promote Endothelial Permeability In Vitro and Tumour Targeting In Vivo.
The microvascular endothelial cell penetrating ability of AuNRBR/N was studied by using a transwell technology (Fig. 5a) and by using the HUVEC as the cell monolayer. As shown in Fig. 5b, AuNRBR/N exhibited lower permeability (2%) through the monolayer in the absence of fMLP, and nearly all AuNRBR/N stayed in the upper chamber (86%), thereby suggesting that AuNRBR/N failed to pass through the endothelium layer efficiently. However, the amount of AuNRBR increased up to about 38% in the presence of fMLP, along with a decreased percentage of AuNRBR in the upper chamber (50%). The results suggested that AuNRBR/N could be recruited by inflammatory factor and then migrate across blood vessels into the tumour site efficiently.
Next, to evaluate the elevated tumour targeting efficiency and endothelial permeability of AuNRBR/N and AuNRBR in vivo, AuNRBR/N and AuNRBR were intravenously injected into Lewis tumour-bearing mice and real-time fluorescence imaging technique to detect the fluorescence intensity in tumor. Compared with AuNRBR, the area of tumour-bearing mouse administered with AuNRBR/N displayed significant fluorescence signal at 12 h post-injection. As time went on, the signal began to degrease, and the signal of both groups at 12 h post-injection was stronger than that at 24 and 48 h (Fig. 5c, left). These direct images of major organs and tumours at 24 h post-injection had a similar tendency with that presented above (Fig. 5c, right). Although AuNRBR group could target tumour to some degree due to the RGD peptide, AuNRBR/N showed enhanced tumour targeting efficiency, which originated from the optimal combination and tumour-homing ability of neutrophils recruited by chemotactic factor. This result suggested that AuNRBR/N has potent tumour targeting ability.
The specific targeting of AuNRBR/N to the tumour was further investigated by CLSM. As shown by Fig. 6a, the slice of tumour-bearing mice injected with AuNRBR/N exhibited stronger fluorescence intensity than that of AuNRBR, suggesting that AuNRBR/N could be efficiently delivered to the tumour tissue due to the inflammation-mediated neutrophil migration. The weak fluorescence signal of AuNRBR group still illustrated RGD peptide-mediated active targeting.
To obtain the dynamics of AuNRBR/N in tumour after intravenous injection, the tumours were taken out and digested, followed by staining with PE/Cy7-conjugated anti-mouse Ly-6G/Ly-6C to analyse by flow cytometry. As seen from Fig. 6b, AuNRBR/N could be significantly recruited into tumours, and around 30% of neutrophils in the tumour originated from AuNRBR/N at 12 h post-injection. The ratio of exogenously injected neutrophils decreased after 12 h and descended to around 5% at 48 h post-injection (Fig. 6c). The AuNRBR/N could be effectively recruited into the tumour and eliminated gradually.
AuNRBR/N induce potent antitumour therapy in vivo
The antitumour therapy experiment was designed as a schematic diagram shown in Fig. 7a. Firstly, the temperature of the Lewis tumour was detected with laser irradiation after administration of AuNRBR or AuNRBR/N. As shown in Fig. 7b, the temperature of the Lewis tumour increased up to 55 °C within 5 min when exposed to the 808 nm laser irradiation (0.5 W/cm2), thereby exhibiting a stronger photothermal therapeutic efficiency of AuNRBR/N to the tumour. Interestingly, AuNRBR/N showed a more significantly enhanced photothermal effect than AuNRBR, which induced the temperature increase to around 42 °C within 5 min (Fig. 7c). The elevated temperature confirmed the PTT efficiency of AuNRBR/N.
Then, the sizes of tumours were continuously monitored for 15 days. As shown in Fig. 8a, b, the tumour volume of mice injected with PBS quickly increased, which indicated that laser irradiation alone had no therapeutic effect on the tumour. However, the tumour of AuNR- treated mice was inhibited modestly compared with PTT alone due to limited EPR effect. Moreover, the tumour growth was obviously delayed when the mice were administrated with AuNRBR, thereby suggesting the active targeting ability of AuNRBR. Apparently, complete suppression of the growth of tumour treated with AuNR or AuNRBR was difficult, thereby indicating the limited targetability, which contributed to the undesired therapeutic effect. However, the tumour growth of mice was significantly inhibited when injected with AuNRBR/N. The enhanced therapeutic effect for AuNRBR/N was ascribed to the remarkable targeting efficiency of neutrophil, thereby leading to a higher amount of AuNRBR/N recruited into the tumour. AuNRBR/N would further release AuNRBR for penetration into deep tumour tissue, thereby leading to a robust therapeutic effect. The body weight was also stable during the therapy, which suggested the biosafety of AuNRBR/N (Fig. 8c). As seen in Fig. 8d, the median survival increased from 55 days for the control group to 60 days for the AuNR group. A more significant increase in the median survival (95 days) was observed in the AuNRBR group. For the AuNRBR/N-treated group, the median survival reached over 120 days. The administration of AuNRBR/N led to a significant increase in median survival of Lewis tumour-bearing mice. Thus, AuNRBR/N can significantly inhibit the growth of the tumour due to the neutrophil-based multistage delivery system and the improved active targeting of AuNRBR to the tumour. To study the potential toxicity, major organs (heart, liver, spleen, lung and kidney) were stained with hematoxylin and eosin (H&E). As shown in Fig. 9, no significant microscopic lesions were revealed in all three groups during 15 days compared with the control group, indicating that AuNRBR/N was biocompatible and safe.