Synthesis, characterization and properties of the Nanocom-ICG
In a typical experiment, AuNRs were synthesized according to the seed-mediated template-assisted protocol with some modifications. As Fig. 1a and Fig. S1a show, AuNRs exhibit good uniformity with a length of 59 ± 3.6 nm and a width of 9.3 ± 1.2 nm. For the coat of silicon dioxide, the thickness of the silica layer can be tuned by adjusting the amount of TOES in the reaction. In our study, the thickness of the coated silica layer was 15 nm (Fig. 1b andFig. S1b). A polymer layer consisting mainly of poly (N-isopropylacrylamide, PNIPAM) was coated onto the surface of the [email protected]2 according to the seeded precipitation polymerization method with some modifications. The PNIPAM layer undergoes a reversible phase transition in aqueous solution from an extended hydrophilic chain to a condensed hydrophobic globule when the temperature is raised above 32 °C. As shown in Fig. 1c and d, the thickness of the PNIPAM layer was optimized to be~65 nm for efficient drug loading and tumour targeting, and it is also seen from the TEM image that the prepared nanoprobe has excellent uniformity. Dynamic light scattering (DLS) further revealed thatNanocom-ICGhas a narrow size distribution with an average diameter of 200.9 nm (Fig. 1e). The AuNR has two adsorption bands: a weak transverse surface plasmon resonance wavelength (TSPRW) at approximately 520 nm and a strong longitudinal surface plasmon resonance wavelength (LSPRW) at approximately 790 nm (Fig. 1f). After being coated with a silica layer, the LSPRW of the AuNRs exhibits a redshift of approximately 15 nm. After PNIPAM layer growth, the longitudinal band of gold nanorods showed a blueshift of approximately 20 nm. Obviously, the LSPRW after polymer coating and ICG loading remained in the NIR region, which permits the photos to penetrate biological tissues with relatively high transmissivity.
Drug loading and controlled release
In this work, ICG was loaded into the nanocomplex through electrostatic interactions, preparing Nanocom-ICG. The amount of ICG loaded into nanoplatform was determined by a UV/vis spectrophotometer at 780 nm, and the loading content of ICG was estimated to be 15 wt %. In aqueous solution, ICG tends to form more stable aggregates at high concentrations (>2 μg/mL), and this is associated with a decreasein fluorescence due to the formation of fluorescence quenching centres.A decrease in peak fluorescence intensity due to the quenching effects was observed (Fig. S2), which suggests that ICG forms aggregates upon adsorption into the nanocomplex. In addition to this PNIPAM layerwith a surprising amount of drug loading, Nanocom-ICG has a thermal response characteristic that shrinks after the temperature increases, so that controlled release of the drug can be achieved. As shown in Fig. 2a, after Nanocom-ICG was irradiated with an 808 nm laser for 5 min, the PNIPAM layer of Nanocom-ICG was significantly shrank, and this property would be beneficial forthe thermoresponsive release of the drug.In aqueous solution, ICG tends to form aggregates at high concentrations, and this is associated with a decline in monomer fluorescence. When these aggregated drugs are released from Nanocom-ICG, the fluorescence can be observeddue to the formation of the monomer. As expected, as the temperature of the solution increased, the fluorescence signal of ICG gradually increased (Fig. 2b). The release profile of the drug at different temperatures is shown in Fig. 2c. Nanocom-ICG shows rapid drug release when the solution temperature reaches 60 °C, and the release rate is significantly higher than in the 20 °C and 40 °C incubation groups.The drug release rate can be significantly enhanced by laser irradiation due to the heat generated by the photothermal effect of the gold nanorods, which induces shrinkage of the PNIPAM layer. These drug release results indicated that Nanocom-ICG possesses excellent temperature-triggered drug release behaviour. Furthermore, the Nanocom-ICG was suspended in saline or DMEM with 10% FBS for 2 weeks (Fig. S3), and no observable agglomeration as well as the color change occurred under two different pH envionments, indicating that Nanocom-ICG excellent colloidal stability.
The photothermal and photodynamic properties of Nanocom-ICG
Gold nanoparticles have excellent photothermal conversion efficiency, which can generate very high temperatures with a short laser irradiation time. In this experiment, we used infrared thermography to detect the heat-producing ability of the Nanocom-ICG under 808 nm laser irradiation. As shown in Fig. 2d and e, the temperature of Nanocom-ICG increasesthe fastest during the irradiation process, reaching 63.6 °C degrees in the 6th minute after irradiation, while the ICG and AuNRs groups reached temperatures of 43.9 and 57.1 °C, respectively. The results demonstrate that Nanocom-ICG benefits from the two heat sources (ICG and AuNRs) and therefore has excellent heat production capacity. Simultaneously, the singlet oxygen (1O2) generation capacity of Nanocom-ICG was quantitatively determined by measuring the fluorescence intensity changes of a specific singlet oxygen trapper (singlet oxygen sensor green, SOSG). The fluorescence spectra are shown in Fig. 2f, and a significant increase in singlet oxygen production was observed with increasing irradiation time, indicating that Nanocom-ICG has the ability to undergo photodynamic therapy. These results demonstrate that Nanocom-ICG possessed prospective photodynamic and photothermal qualities for further combination therapy. It is also worth noting that the absorption peak of this probe overlaps with the absorption peak of ICG, and such overlap greatly increases the singlet oxygen yield of the loaded ICG by maximizing the local field enhancement and protecting the drug against photo-degradation with the help of the high absorption cross-section of the AuNRs. As expected, after 5 min of laser irradiation, the amount of 1O2 produced by Nanocom-ICG was significantly higher than that of the free ICG at the same ICG concentration (Fig. S4 and S5).
To demonstrate the utility of the Nanocom-ICG for in vitro/in vivo applications, in the next experiment, the cytotoxicity of Nanocom-ICG was examined by the CCK-8 assay inA549 cells. As Fig. 3a shows, the CCK-8 data indicated no cytotoxicity of free ICG, nanocom or Nanocom-ICG in the concentration range of 0-24 μg/mL (ICG equivalent), and slight cytotoxicity occurred at a concentration of 48 μg/mL. Additionally, the cell index experiment results showed that nanocom and Nanocom-ICG did not affect the proliferation of cells.In addition, we used the growth index of the cells to evaluate the effect of the nanoprobe on the cells. As shown in Fig. 3b, after A549 cells were incubated with PBS, nanocom and Nanocom-ICG for 72 h, there was no significant difference in the cell proliferation curves between the three groups. Moreover, the flow analysis further proves that the nanoprobe has excellent biosafety. As shown in Fig 3c, after A549 cells were incubated with a series of Nanocom-ICG in different concentrations (0-48 μg), there were no obvious apoptosis and necrosis in the concentration range. These results indicated that Nanocom-ICG possessed excellent biocompatibility in A549 cells. The excellent biocompatibility of the probe facilitated the next in vivo and in vitro therapeutic applications.
Cellular uptake and localization
In the next experiment, the fluorescence of ICG was used to assess the cellular uptake of Nanocom-ICG. The confocal fluorescence microscope showed obvious fluorescence in the cytoplasm after treatment with ICG and Nanocom-ICG, suggesting efficient cellular uptake and intracellular distribution of Nanocom-ICG (Fig. 4a). Additionally, the fluorescence intensity in Nanocom-ICG-treated cells washigher than that in ICG-treated cells, which demonstrates the prominent delivery and protection of ICG from Nanocom-ICG. In addition, we measured the cellular uptake of ICG and Nanocom-ICG by flow cytometry. The flow cytometry results show that Nanocom-ICG exhibits more efficient drug delivery capabilities after incubation with A549 cells for 2 h or 8 h than thoseof ICG (Fig. 4b). To more precisely observe the intracellular localization of Nanocom-ICG, the ultrastructure of the A549 cells was observed by TEM after A549 cell treatment with Nanocom-ICG for 24 h. As shown in Fig. 4c, Nanocom-ICG is clearly visible and distinct in the cell due to its high electron density, and most of the Nanocom-ICG is located in cellular vesicles, mainly endosomes and lysosomes. These observations are consistent with the results of the flow cytometry assay, and these results suggest the efficient cellular uptake of Nanocom-ICG, which is the basis for the optimal in vitro therapeutic effect.
In vitro PTT/PDT therapeutic effects
In this study, the PTT/PDT combination therapy effects of Nanocom-ICG was first examined by a double-stain assay inA549 cells. A549 cells were incubated with free ICG (20 μg/mL), nanoplatform and Nanocom-ICG (20 μg/mL ICG equivalent) for 24 h and then irradiated with an 808 nm laser at a power density of 0.8 W/cm2 for 6 min. Finally, these cells were stained with calcein AM and PI staining solution. A series of fluorescence images are shown in Fig. 5a, and it can clearly be observed that Nanocom-ICG kills more cells than either free ICG or the nanocomplex alone after laser irradiation treatment. For the free ICG- or nanocomplex-treated groups, most of the cells were still alive after 6 min of laser irradiation. Additionally, the results of the flow cytometry apoptosis analysis were consistent with the double staining results, and as shown by the flow analysis files (Fig. 5b), as Nanocom-ICG induced the most cancer cell apoptosis among all treatment groups. Moreover, the cells in the PBS-treated group caused very little significant apoptosis and death of cells after irradiation, which also proved that the laser power and irradiation time we selected were safe for normal tissues. Combined with the results of cellular uptake, these results suggested that the PTT/PDT combination effect and enhanced cellular uptake are responsible for the improved therapeutic efficiency of Nanocom-ICG.
Biodistribution of Nanocom-ICG in vivo
To achieve targeted delivery and release of the loaded drug, the AuNRs were encapsulated by a PNIPAM layer. We expected that the heat generated by the photothermal effect of the gold nanorods would induce shrink age of the PNIPAM layer. It has been shown that PNIPAM can trigger loaded drug release through photothermal effectsupon irradiation with an NIR laser. In this study, Nanocom-ICG was intravenously injected into mice, and then we observed the fluorescence of ICG in the tumour-bearing mice by using a Maestro in vivo optical imaging system. As expected, a series of fluorescence images are shown in Fig. 6a. The Nanocom-ICG was mainly distributed in the liver 30 min post-injection. Over time, the fluorescence signal gradually appeared in the tumour site and reached a peak at 4 h post-injection. To further observe the distribution of photosensitive drugs in the main organs and tumoursites of experimental animals, we performed an in vivo imaging experiment. As shown in Fig. 6b and c, we can clearly observe that the fluorescence signals of the tumour site were the strongest, and these results fully indicated the ability of the nanoprobes to target the drug delivery towards the tumour site. In the following experiment, according to the photoacoustic signal of gold nanorods, we further observed aggregates of Nanocom-ICG at the tumour site by using a photoacoustic imaging system.The photoacoustic imaging images are shown in Fig. 6d. After the nanoprobe was injected intravenously into the experimental mice, the PA signals of the tumour site gradually increased and reached a peak at 12 h post-injection. These imaging results clearly demonstrated the prominent tumour-targeted aggregate and heat-induced release ability of Nanocom-ICG, which will facilitate subsequent in vivo antitumour therapy.
Antitumour efficacy of Nanocom-ICG In Vivo
To interpret the synergistic efficacy of PTT and PDT mediated by Nanocom-ICG, A549 tumour-bearing mice were randomly divided into 4 groups and injected with saline, ICG, Nanocom or Nanocom-ICG into a tail vein. At 4 h post-injection, the photothermal/photodynamic combination antitumour therapy was performed with 808 nm laser irradiation. In the whole process of laser treatment, the photothermal effect of Nanocom-ICG was monitored through an infrared thermal imaging camera. As shown in Fig. 7a, oncethe Nanocom-ICG-treated mice were exposed to the laser at a power density of 0.8 W/cm2, the temperature of the tumour region increased rapidly to 41.3 °C after 3 min of irradiation, and the surrounding healthy tissue showed a moderate increase in temperature. However, in the ICG- and AuNR-treated groups, the temperature of the tumour region reached 35.6°C or 38.6°C, respectively, after irradiation. We think that the reasons why the tumour region in the Nanocom-ICG treatment group reached the highest temperature are due to the high number of Nanocom-ICG aggregates at the tumour site and the dual heat production from the AuNRs and ICG. In addition, one minute after laser irradiation, the temperature of the tumour region in eachgroup quickly declined back to body temperature (Fig. S6), suggesting that the power density and irradiation time are likely to be safe.
Fourteen days after laser irradiation, as shown in Fig. 7b, in the free ICG and nanoplatform treatment groups, the tumour growth was relatively rapid. Moreover, the tumour volumes of the free ICG and nanoplatform groups were significantly larger than those of the Nanocom-ICG group, indicating that single thermal therapy or photodynamic therapy with mild laser treatment alone is ineffective in inhibiting tumour growth (Fig. 7d). In addition, consistent with the tumour growth results, the Nanocom-ICG group had the highest survival rate among all experimental groups (Fig. 7c). In addition, Western blot analysis of twomain HSP members, HSP90 and HSP70, in the tumour tissues revealed significantly increased expression after treatment with Nanocom-ICG and laser irradiation (Fig. 7e), proving the enhanced thermal therapy effects of Nanocom-ICG. Subsequently, we performed TUNEL and H&E experiment to observe the therapeutic effectsinthe tumourtissue in all experimental groups. The results are shown in Fig.8a and further confirmed that Nanocom-ICG achieved the best treatment effect. Compared with other treatment groups, many dead cells were clearly observed atthe tumour site of the Nanocom-ICG treated group. The excellent antitumour therapeutic benefits from the efficient aggregates of Nanocom-ICG in thetumour region and controlled drug release resulted in photodynamic/photothermal combination therapy. In addition, there were no obvious damage or inflammatory lesions observed in the main organs (liver, lung, heart, spleen, kidney) of the Nanocom-ICG treatment group 21 days after i.v. injection (Fig. 8b), demonstrating that Nanocom-ICG does not cause acute side effects and shows excellent biocompatibility.