Design, Synthesis, and Characterization of FBPD NPs
The multipurpose FBPD NPs were synthesized following the strategy as shown in Fig. 2a. The double-emulsion approach to synthesize FBPD NPs has been reported by our group before has been skillfully operated [35]. A series of characterizations have been conducted on the as-synthesized NPs. The TEM images (Fig. 2c) showed that the FBPD NPs had a well-defined spherical shape, and the SEM image (Fig. 2b) revealed that homogeneous size distribution of FBPD NPs. The mean hydrodynamic diameter of the FBPD NPs was 295.3 ± 40.3 nm (Fig. 2d). The size range indicated that it could readily transport in the blood vessel and accumulate into tumor tissue via the representative enhanced permeability and retention (EPR) effect [33]. In addition, the zeta potential of FBPD NPs was (-7.99 ± 6.66) mV. The EE and DL of the FBPD NPs were calculated by UV spectrophotometry. UV-spectra of Dox dispersed in saline at different concentrations (20, 25, 30, 35, 40, and 50 µg/mL) and the corresponding relationship between concentrations of Dox and absorbance (Fig. 2e, 2f). The EE was (87.48 ± 0.19%) for Bi2S3 and (25.21 ± 3.05%) for DOX, respectively. The DL was (3.38 ± 0.07%) for Bi2S3 and (4.07 ± 0.49%) for DOX, respectively. The result displayed that FBPD NPs could carry oil-solute and soluble drugs. While these FBPD NPs could act as photothermal conversion agents for therapeutic tumor ablation, due to its excellent imaging performance, these FBPD NPs could be potentially used as contrast agents for CT and PA simultaneously during the therapy, offering guidance for subsequent photothermal ablation and evaluating the potential of treatment results post entering the tumor site.
Different releasing behaviors were observed for Dox for FBPD NPs when in the presence and absence of laser irradiation (Fig. 2g). The percentage of DOX released almost reached 46.2% at 4 h after laser irradiation, while the percentage of Dox released reached 49.7% at 48 h in the control group. This is probably due to the addition of phase-changing PFP into the FBPD NPs. The laser irradiation triggered the liquid-gas phase conversion and subsequently induced a faster release of Dox.
Morphology and characterization. (a) Schematic diagram for the fabrication of FBPD NPs. (b) SEM and (c) TEM of FHMP NPs. (d) Size distribution of FBPD NPs. (e) UV-spectra of FBPD NPs dispersed in saline in different concentrations (20, 25, 30, 35, 40, 45, 50 µg/mL) and (f) corresponding relationship between concentration of FBPD NPs and absorbance. (g) In vitro release of DOX in FBPD NPs with or without laser irradiation.
In vitro cytotoxicity assay of FBPD NPs
The cytotoxicity against SKOV-3 cells was assessed by the CCK-8 protocol. As shown in Fig. 3, FBPD NPs with laser irradiation demonstrated more effective cell growth suppression than that of FBPD NPs group without laser irradiation. In addition, while both in the presence of laser irradiation, the group of FBP NPs resulted in low cell viability. Furthermore, when compared with FBPD NPs group and BPD NPs group, it was found that added FA resulted in more significant growth inhibition on SKOV-3 cells. The result showed that the chemotherapeutic approach, combined with photothermal techniques had an excellent tumor-suppression. After connected with FA, it could more effectively deliver antitumor drugs to the tumor region, which will greatly improve the anti-cancer effects.
Relative cell viability of SKOV-3 cells after different treatments (n = 3, *p < 0.01).
In vitro targeting ability of FBPD NPs
Confocal microscopy and flow cytometry were used to investigate the intracellular uptake of FBPD NPs. As shown in CLSM images, NPs contained Dox present red fluorescence, and DAPI-labeled SKOV-3 cells exhibit blue fluorescence (Fig. 4a). As expected, in the treatment group, numerous FBPD NPs with red fluorescence were internalized within the cells after 3 h of co-incubation. On the other hand, only a few BPD NPs assembled within the cells in the control group. Flow detection shows the same trend (Fig. 4b). The group treated with FBPD NPs was observed with much stronger fluorescence in the cells than the group treated with BPD NPs, especially at 3 h after incubation, which demonstrated that FA-labelled NPs had the strongest accumulation ability.
In vitro targeting ability. (a) Intracellular uptake of BPD NPs and FBPD NPs as observed by CLSM after various intervals of incubation. The scale bars are 100 µm. (b) Flow cytometry analysis of intracellular uptake of BPD NPs and FBPD NPs.
In vivo targeting ability of FBPD NPs
To further verify the targeting efficiency in vivo, the SKOV-3 tumor-bearing mice model was created, and fluorescence pictures were executed at certain time points after the intravenous administration with DiR-labeled FBPD NPs or BPD NPs. After injection of DiR-stained FBPD NPs, strong fluorescence signals were observed in the tumor area, while only weak signals were found in tumors in mice treated with BPD NPs (Fig. 5a). According to Fig. 5b, the fluorescence intensities with tumor regions reached a peak at 3 hours after FBPD NPs administration. Furthermore, in order to determine the distribution of NPs in the body, main organs and tumors were collected 24 hours post-injection for excised fluorescence imaging (Fig. 5c), and the relevant fluorescence intensities were quantified (Fig. 5d).
In vivo targeting ability. (a) In vivo fluorescence imaging and tumor targeting of a mouse at 0, 0.5, 1, 2, 3, 4, 6, and 24 h post-injection of SKOV-3 tumor-bearing mice. (b) Quantitative fluorescence intensity of tumor tissue at different times (n = 3). (c) Bio-distribution of DiR-labeled BPD NPs or FBPD NPs in major organs excised from mice at 24 h post-injection. (d) Quantitative bio-distribution of BPD NPs or FBPD NPs in mice as determined by the average FL intensities of organs and tumors. (Values are means ± sd., n = 3.)
In Vitro and In Vivo CT Imaging
Based on deep tissue penetration and 3D structure imaging advantages, CT imaging is one of the most helpful diagnostic methods [36]. The high atomic number of bismuth has strong X-ray absorption ability, which can significantly improve the contrast of CT imaging. Furthermore, Bi-nanoparticles has the advantages of low toxicity, small particle size, long blood circulation time, and surface modification [37]. Therefore, the experiment investigated the CT contrast imaging both in vitro and in vivo of FBPD NPs. When in vitro, it has been found that as the concentration of FBPD NPs increased, the CT signal value also increased and showed a good linear relationship (Fig. 6a). The CT imaging property in vivo was further evaluated on SKOV-3 tumor-bearing mice. As shown in Fig. 6c, after i.v. injection of FBPD NPs or BPD NPs, the group of FBPD NPs showed an distinct bright effect within the tumor area. The CT signal intensities of the tumor regions were measured with Pacs software, and it showed that the CT signal value peaked at 3 h (Fig. 6b). Furthermore, the pseudo-colored images were also recorded, it clearly indicated the increasement of CT imaging. This result demonstrated that FA has excellent targeting properties; therefore, could FBPD NPs could be delivered to the tumor region efficiently.
In Vitro and In Vivo CT Imaging. (a) In vitro CT contrast images and CT values of FBPD NPs at different concentrations. (b) Changes of CT-signal intensities within tumor regions at corresponding time points. (c) In vivo CT images of tumors on SKOV-3 tumor-bearing mice after i.v. injection of BPD NPs or FBPD NPs as recorded at different time points. The top row shows black and white images, and bottom row represents the pseudo-colored images.
In vitro and in vivo PA imaging
To evaluate the capability of FBPD NPs as a PA probe, PA images were obtained using FBPD NPs as the contrast agent both in vivo and in vitro. Compared with other imaging methods, PA imaging has very high sensitivity [20]. Bi2S3 has been used as a PA contrast agent because of its strong absorbance in the NIR region [31]. While applied in vitro, PA imaging performance of FBPD NPs dispersed in PBS at different concentrations were recorded (Fig. 7a). Furthermore, the PA signal intensities increased linearly with the concentration of NPs increased from 1–5 mg/mL, suggesting that FBPD NPs could effectively achieve PA imaging. During the in vivo assessment, PA imaging of SKOV-3 tumor-bearing mice was conducted at different time points after i.v. injection of BPD NPs or FBPD NPs (Fig. 7b), and the relevant quantitative analyses of PA intensities were executed (Fig. 7c). Before i.v. injection, the tumor showed only a weak PA signal. The PA signal reached the maximum approximately 3 h after injection with FBPD NPs. The reason could be the actively tumor-targeting induced by FA modification tumor cells at 3 h, and specifically bind to the folate-receptor on the tumor surface resulting in a high degree of accumulation of FBPD NPs in the tumor. In contrast to the FBPD NPs injection, few PA signal was discovered after BPD NPs injection. These results demonstrated that FBPD NPs could be served as a excellent contrast agent for PA imaging, which achieves diagnostic-imaging guidance and monitoring during tumor therapy.
In vitro and in vivo PA imaging. (a) In vitro PA contrast images and PA values of FBPD NPs at different concentrations. (b) PA images of tumor regions in SKOV-3 bearing-mice after i.v. injections of BPD NPs or FBPD NPs at varied time intervals (0, 0.5, 1, 2, 3, 4, 6, and 24 h). (c) PA signal at tumor regions in SKOV-3 tumor-bearing mice.
Photothermal and Photo-induced Phase Change performance of FBPD NPs
To assess the photothermal performance of FBPD NPs, the temperature changes of FBPD NPs aqueous solution were recorded by an infrared thermal imaging camera after the NIR irradiation (808 nm). After laser irradiation at 1.5 W/cm2 for 5 min, FBPD NPs showed a rapid temperature increase at all concentrations. Comparatively, the PBS did not show any obvious temperature rise as the given conditions (Fig. 8a). During the laser on-off cycles, the maximum temperature of FBPD NPs did not drop significantly after the cycle, indicated that it has good thermal stability (Fig. 8b).
In the experiment of photo-induced phase change, before laser irradiation, FBPD NPs did not change significantly under the optical microscope (Fig. 8c); After exposed to laser irradiation, FBPD NPs undergone a liquid-gas phase change, and the volume of FBPD NPs increases significantly (Fig. 8d). This result indicated that the NPs could easily transform into gas while heating to physiological temperature due to the lower boiling point of PFP [17]. The PFP had eventuated a phase change after being heated, then resulted in increase in the size of the FBPD NPs, which was subsequently more helpful for the release of drugs and the thermal ablation effect in the tumor area.
Photothermal and Photo-induced Phase Change performance. (a) Plot of temperature change (△T) of PBS and FBPD NPs at different concentrations (0, 1, 2, 3, 4, and 5 mg/mL) as a function of irradiation duration using an 808 nm laser (1.50 W/cm2). (b) Cycled heating profiles of FBPD NPs (concentration: 100 µL, 5 mg/mL) irradiated by an 808 nm laser (1.50 W/cm2) for five laser on/off cycles. (c) The imaging of FBPD NPs under the optical microscope before an 808 nm laser (1.50 W/cm2). (d) The imaging of FBPD NPs under the optical microscope after an 808 nm laser (1.50 W/cm2).
In vivo cytotoxicity assay of FBPD NPs.
The combination of PTT and chemotherapy, an efficient therapy for tumors, causes lesser damage to surrounding normal tissues. According to cytotoxicity assay in vitro, seven groups (control, laser only, FBP NPs + laser group, BPD NPs group, BPD NPs + laser group, FBPD NPs group, and FBPD NPs + laser group) were performed to evaluate the therapeutic efficacy of FBPD NPs. After conducting different treatments, the inhibitory effect on SKOV-3 tumor growth is shown in Fig. 9b. The volumes of tumor and the weights of mice were measured every two days (Fig. 9c, 9d), meanwhile, photographs of these mice were taken every two days for fourteen days after the therapy (Fig. 9a). The result displayed the the tumor volume in the FBPD NPs + laser group was much smaller than those of the other groups. It showed that chemotherapy combined photothermal therapy with FA-modification could inhibit tumor growth significantly. Compared to FBP NPs + laser group and laser only group, it was found that the tumor growth in FBP NPs + laser group was significantly inhibited, which indicated that laser irradiation with FBP NPs also showed a suppression effect on the tumor growth. The result demonstrated that FA-loaded NPs could deliver Dox and Bi2S3 to the tumor area and specifically kill the tumor. Furthermore, it had been reported that hyperthermia at 41℃-45℃ would incur a synergistic effect together with traditional therapeutic approaches es such as chemotherapy in tumor treatment [38]. Our experimental results presented consistency with reported mechanisms.
In vivo cytotoxicity. (a) Photographs of SKOV-3 tumor-bearing mice of seven groups taken during 14 d period after various treatments. (b) Photographs of tumors dissected from mice of seven groups after various treatments. (c) Time-dependent tumor volume curves of seven groups after various treatments (n = 5). (d) Time-dependent body weights of seven groups after various treatments (n = 5).
Hematoxylin-eosin (H&E) staining, proliferating cell nuclear antigen (PCNA) staining, and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining on tumor slices better affirmed the synergistic PTT/ chemotherapy effect (Fig. 10a). As shown in the H&E-staining tumor sections, there were much more deformed nucleus in PTT/ chemotherapy group than tumor sections in other groups, prompting serious necrosis of tumor cells. Furthermore, the apoptosis of cancer cells in the FBP NPs + laser group was more serious than that in laser only group. The rusult of TUNEL and PCNA staining followed the parallel tendency. Representative apoptosis cells were prompted by brown nucleus in the TUNEL staining. PCNA staining on tumor slices showed the in vivo proliferative abilities of seven groups where proliferative tumor cells were stained into brown. The TUNEL and PCNA staining indicated that synergistic PTT + chemotherapy possessed the highest efficacy than other groups. In addition, heart, liver, spleen, lung, and kidney of mice were conducted H&E staining after the treatments to survey the relevant pathological toxicity. Insignificant negative effect was discovered during the treatment course for all groups, indicating no obvious histological damage in the treatment group (Fig. 10b).
(a) H&E, TUNEL and PCNA staining on tumor sections from SKOV-3 tumor-bearing mice after various treatments. (b) H&E staining of the major organs (heart, liver, spleen, lung, and kidney) of SKOV-3 tumor-bearing mice after different treatments. The scale bars are 50 µm.
Biosafety assay of FBPD NPs
In order to explored the short/long-term potential toxicity of FBPD NPs, a blood biochemical assay and H&E staining of major organs were verified on Balb/c mice. The result of blood biochemical revealed that negligible changes after different treatments (Fig. 11A), indicating the satisfactory biocompatibility of FBPD NPs. In addition, no obvious damage to major tissues was discovered, suggesting the the high therapeutic biosafety of FBPD NPs (Fig. 11B).
Biosafety of FBPD NPs. (a) Assay of blood index after i.v. injection of FBPD NPs (n = 5). (b) Images of H&E stained slices of major organs (scale bar = 50 µm).