Near-infrared induced phase-shifted ICG/Fe3O4 loaded PLGA nanoparticles for photothermal tumor ablation

Near-infrared (NIR) laser-induced photothermal therapy (PTT) uses a photothermal agent to convert optical energy into thermal energy and has great potential as an effective local, minimally invasive treatment modality for killing cancer cells. To improve the efficacy of PTT, we developed poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) encapsulating superparamagnetic iron oxide (Fe3O4), indocyanine green (ICG), and perfluoropentane (PFP) as synergistic agents for NIR laser-induced PTT. We fabricated a novel type of phase-shifting fluorescent magnetic NPs, Fe3O4/ICG@PLGA/PFP NPs, that effectively produce heat in response to NIR laser irradiation for an enhanced thermal ablation effect and a phase-shift thermoelastic expansion effect, and thus, can be used as a photothermal agent. After in vitro treatment of MCF-7 breast cancer cells with Fe3O4/ICG@PLGA/PFP NPs and NIR laser irradiation, histology and electron microscopy confirmed severe damage to the cells and the formation of many microbubbles with iron particles at the edge or outside of the microbubbles. In vivo experiments in mice with MCF-7 tumors demonstrated that Fe3O4/ICG@PLGA/PFP NPs could achieve tumor ablation upon NIR laser irradiation with minimal toxicity to non-irradiated tissues. Together, our results indicate that Fe3O4/ICG@PLGA/PFP NPs can be used as effective nanotheranostic agents for tumor ablation.

Previously, we developed PLGA microbubbles loaded with doxorubicin and Fe 3 O 4 NPs and demonstrated their suitability for dual MR/ultrasound (MR/US) imaging of sentinel lymph nodes and their anti-tumor efficacy for lymph node metastasis 47 .
Recently, many studies have focused on liquid perfluorocarbon (PFC) droplets, which can be vaporized into gas bubbles via active US sonication or laser irradiation 48,49 . This property of vaporization has been effectively employed for US imaging 50 , cancer therapy via vessel occlusion [51][52][53] , targeted drug delivery [54][55][56] , and thermal ablation of tumor cells 57,58 . However, this technology has yet to be combined with a dual NIR light-absorbing agent along with encapsulation in polymeric NPs toward the development of an effective PTT approach.
Here, we report the development of novel pefluoropentane (PFP)-based PLGA NPs loaded with Fe 3 O 4 NPs and ICG (Fe 3 O 4 /ICG@PLGA/PFP NPs) for photothermal tumor ablation (Fig. 1). Both Fe 3 O 4 NPs and ICG contribute to the ability of this NIR light-absorbing agent to efficiently convert absorbed light into heat. The liquid PFP core with a lower boiling point is easily converted into gas upon heating to physiological temperatures, and this thermoelastic expansion results in tissue extrusion deformation to enhance the thermal ablation effect in the local tumor area. Our results show that these dual NIR light-absorbing PFC-based polymeric NPs can be used as effective nanotheranostic agents in anti-tumor treatments.

Materials and Methods
Materials. ICG, poly vinyl alcohol (PVA, Mw = 30,000-70,000), and PLGA (lactide: glycolide = 50:50, Mw = 10,000) were obtained from Sigma-Aldrich (USA). Fe 3 O 4 NPs (diameter = 10 nm) treated with oleic acid were purchased from Ocean Nano Tech Inc. (USA). PFP was purchased from Alfa Aesar (UK). De-ionized (DI) water was purified using a Milli-Q Gradient System. Other regents of analytical grade were used without further purification. All experiments involving use of animals were performed in accordance with the relevant guidelines  Fluorescence images of Hoechst 33342/PI co-stained MCF-7 cells incubated with Fe 3 O 4 /ICG@PLGA/PFP NPs (0.5 mg/mL) and exposed to the 808 nm laser irradiation (1 W/cm 2 , 5 min). Live and dead cells were stained with Hoechst 33342 (blue color) and PI (red color), respectively. and regulations, approved by the Ethics Committee at the second Xiangya Hospital of Central South University in China.  47 . Briefly, 500 mg PLGA and 2 mL Fe 3 O 4 NPs suspension (31 mg Fe/ mL) were added to 10 mL of chloroform and stirred well, and then 2 mL ICG solution (1.25 mg/mL) and 4 mL liquid PFP were added and emulsified for 1 min in an ice bath using an ultrasonic processor. Then 50 mL cold PVA solution (5% w/v) was poured into the emulsion, which was homogenized for 5 min at 9500 rpm in an ice bath using a homogenizer. Subsequently, 100 mL DI water was added to the mixture and mixed for 2 h. Finally, the NPs were washed with DI water three times and kept at 4 °C until characterization. The same procedure was used to prepare Fe 3 O 4 /ICG@PLGA NPs without PFP, Fe 3 O 4 @PLGA/PFP NPs without ICG, and ICG@PLGA/ PFP NPs without Fe3O4 as controls. The cells in each experimental well were exposed to 808 nm laser irradiation (1 W/cm 2 ) for 5 min, and control samples were exposed to laser irradiation. Cell viability was determined using the CCK assay.

Characterization of Fe
For Hoechst 33342/propidium iodide (PI) staining, MCF-7 cells (10 5 cells/well) were incubated in 6-well plates at 37 °C with 0.5 mg/mL Fe 3 O 4 /ICG@PLGA/PFP NPs and then irradiated by the NIR laser (808 nm, 1 W/ cm 2 ) for 5 min. The cells were stained with a mixed solution of Hoechst 33342 and PI at 4 °C for 30 min and In groups I, II, and IV, the nude mice received an intratumor injection of 50 µL saline or Fe 3 O 4 /ICG@PLGA/PFP NPs (2 mg/mL). Two hours after injection, the tumors of mice in groups III (laser only) and IV (NPs + laser) were irradiated with the 808 nm laser at a power density of 1.0 W/cm 2 for 5 min. The mice in groups I (saline) and II (NPs only) received no laser irradiation as controls. The temperature changes in the tumors were monitored by an infrared thermal imaging camera (Ti27, Fluke, USA). The tumor volume was calculated according to the formula: (tumor width) 2 × (tumor length)/2. The tumor size in each mouse was recorded every 2 days for 14 days.
One day after the different treatments, tumor tissues were harvested and evaluated via hematoxylin and eosin (H&E) staining, TdT-mediated dUTP nick end labeling (TUNEL) staining, and TEM examination for assessment of therapeutic efficacy.
Biological toxicity assessment. For assessment of the biological toxicity of the developed NPs, 10 healthy female BALB/c nude mice were intravenously injected with 0.2 mL of 2 mg/mL Fe 3 O 4 /ICG@PLGA/PFP NPs. Five additional mice were injected with saline as controls. Blood samples were collected for serum biochemistry assays at 3 and 14 days after injection of Fe 3 O 4 /ICG@PLGA/PFP NPs. After sacrifice at the final time point, the major organs including the liver, spleen, kidney, heart, lung and brain were harvested, and sections were stained with H&E. Statistical analysis. All data are presented as means ± standard deviation (SD). Analysis of variance was used to analyze the data. Differences were considered significant if p < 0.05.  (Fig. 2a,b), average diameter of 289.6 ± 67.4 nm, and polydispersity index of 0.028 (Fig. 2d). In TEM (Fig. 2c), scattered black spots with diameter of 10 nm were clearly observed on the shells of Fe 3 O 4 /ICG@ PLGA/PFP NPs, confirming the encapsulation of Fe 3 O 4 NPs in the Fe 3 O 4 /ICG@PLGA/PFP NPs. Furthermore, the amount of Fe 3 O 4 NPs encapsulated in the Fe 3 O 4 /ICG@PLGA/PFP NPs was determined by atomic absorption spectrometry to be 113.55 ± 3.12 μg/mL. The encapsulation efficiency of ICG was 45.53% ± 1.61% as measured by steady state spectrophotometry. The UV-Vis-NIR absorption spectra of Fe 3 O 4 /ICG@PLGA/PFP NPs, ICG, and Fe 3 O 4 NPs displayed strong absorption in PBS in the range of 400-900 nm (Fig. 2e). The spectrum of Fe 3 O 4 NPs exhibited no obvious peak, whereas that of free ICG had a peak around 770~790 nm. The absorption spectrum of Fe 3 O 4 /ICG@PLGA/PFP NPs displayed a small peak around 790~810 nm with an obvious red shift, which is consistent with previously published results for ICG 33, 57-61 , confirming that ICG was successfully encapsulated in the NPs. The observed absorbance in the NIR region verified that the Fe 3 O 4 /ICG@PLGA/PFP NPs could serve as a good photo-absorbing agent for tumor PTT.

In vitro PTT effect of Fe 3 O 4 /ICG@PLGA/PFP NPs. To study the effectiveness of using Fe 3 O 4 /ICG@
PLGA/PFP NPs in PTT, aqueous suspensions of Fe 3 O 4 /ICG@PLGA/PFP NPs, mixture of ICG and Fe 3 O 4 NPs, free ICG, Fe 3 O 4 NPs, and PBS were exposed to 808 nm NIR laser irradiation with a power density of 1.0 W/ cm 2 for 10 min. The rapidly increasing temperature of Fe 3 O 4 /ICG@PLGA/PFP NPs allowed them to act as an effective photothermal nanoagent for PTT. As shown in Fig. 3a, no obvious temperature change was observed when PBS was exposed to NIR laser irradiation. In contrast, under the same concentration (0.2 mL solution with 5 μg/mL ICG or 113 μg/mL Fe) and laser irradiation conditions, the maximum temperatures achieved in the free ICG, Fe 3   In contrast, no temperature increases were observed in tumors after treatment with saline, NPs only, or laser irradiation only ( Fig. 5a and b).
The photographs in Fig. 6a show the black scars that appeared over the sites of tumors injected with Fe 3 O 4 / ICG@PLGA/PFP NPs upon laser irradiation. In these mice, the tumor size declined significantly, and the scar tissue gradually fell off within 14 days laser irradiation. However, in the three control groups treated with saline only, NPs only, or laser irradiation only, the volumes of tumors increased by more than 12-fold from day 0 to day 14 after treatment (Fig. 6b). Moreover, mice in the three control groups showed a survival range of 17-23 days after treatment, whereas all mice treated with Fe 3 O 4 /ICG@PLGA/PFP NP injection and laser irradiation remained alive at 48 days after treatment with complete tumor ablation achieved by PTT (Fig. 6c). These results demonstrate that Fe 3 O 4 /ICG@PLGA/PFP NPs could effectively inhibit tumor growth via their photothermal efficacy.
The therapeutic effectiveness of Fe 3 O 4 /ICG@PLGA/PFP NPs in PTT was also investigated by H&E staining, TUNEL staining, and TEM examination of tumor tissues harvested from the different groups of mice 1 day after treatment. H&E staining revealed severe damage with coagulative necrosis, leaving a mass of red-stained substance in tumor tissue injected with NPs and exposed to laser irradiation. Comparatively, no notable damage was found in the three control groups (Fig. 6d). TUNEL staining further verified the presence of many more necrotic cells in tumor tissues of mice treated with the NPs and laser irradiation than in tumor tissues of the other three groups, demonstrating the greater damage to tumor cells achieved via the photothermal effect of Fe 3 O 4 /ICG@ PLGA/PFP NPs (Fig. 6d). In addition, on TEM images, coagulative necrosis was more evident in the tumor tissues of mice treated with the NPs and laser irradiation than in that of the other three groups, which was consistent with our H&E staining results (Fig. 7). In the representative image showed in Fig. 7d, after treatment with NPs and laser irradiation, no clear cell structure could be distinguished, most cell membranes and nuclear membranes had ruptured or disintegrated (indicated by blue arrows), organelles had disappeared, and only remnants of the nuclear shadow remained. Moreover, the tumor blood vessels had ruptured, resulting in leakage of red blood cells. Comparatively, in the three control groups, the tumor tissue retained the original structure with no obvious damage (Fig. 7a-c). Finally, many microbubbles (red arrows) appeared in the tumor tissues treated with NPs and laser irradiation, with Fe particles expelled to the edge or outside of the microbubbles (Fig. 7d). This revealed that most Fe 3 O 4 /ICG@PLGA/PFP NPs absorbed the light energy and converted it into heat upon laser irradiation, and the PFP in the NPs underwent a phase shift to form microbubbles. In tumor tissues treated with NPs only without laser irradiation, only a small portion of NPs produced microbubbles (Fig. 7b). We think this may have occurred after harvesting of the tissue during the heated polymerization process of the TEM sample operation, when a temperature exceeding 70 °C is commonly used; at such temperatures, some of the NPs in the excised tissues could absorb the heat, resulting in microbubble formation.
In vivo toxicity of Fe3O4/ICG@PLGA/PFP NPs. To further assess the in vivo toxicity of Fe 3 O 4 /ICG@ PLGA/PFP NPs, major organs (heart, liver, spleen, lung, kidney, and brain) of healthy BABL/c mice injected with Fe 3 O 4 /ICG@PLGA/PFP NPs (0.2 mL, 2 mg/mL) via the tail vein were harvested,sectioned, and stained with H&E for histological analysis at 14 days post-injection. In addition, blood samples of these healthy BABL/c mice were collected and analyzed at 3 and 14 days post-injection. No noticeable organ damage or inflammatory lesion were observed in H&E stained sections of major organs from mice treated with Fe 3 O 4 /ICG@PLGA/PFP NPs, indicating that the Fe 3 O 4 /ICG@PLGA/PFP NPs did not induce appreciable toxic side effects in treated animals (Fig. 8a). Moreover, two indicators of hepatic function, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and two indicators of renal function, urea nitrogen (UREA) and creatinine (CREA) levels, were measured in blood samples from NP-treated mice. As shown in Fig. 8b, the levels of these four indicators were within normal ranges with no significant differences between the different time points (all *p < 0.05). Thus, the histological examination and serum biochemistry results indicate that Fe 3 O 4 /ICG@PLGA/PFP NPs at the tested dose have no cytotoxic effects in healthy mice in the absence of laser irradiation.

Conclusion
The multifunctional Fe 3 O 4 /ICG@PLGA/PFP NPs synthesized in this study showed promising results as a photothermal agent for cancer treatment. The photo-absorbance by Fe 3 O 4 NPs and ICG co-embedded in the Fe 3 O 4 / ICG@PLGA/PFP NP shell, along with the microbubbles generated upon the liquid-gas phase shift of PFP encapsulated in the NPs process make it possible to achieve photothermal tumor therapy. This successful demonstration of the use of nanobiotechnology for NIR laser-induced PTT provides an alternative modality for effective nanotheranostics.