With recent developments in nanotechnology, plasmonic photothermal therapy (PPTT) has become a powerful tool for elimination of pathogenic tissues, e.g. tumor growths. To achieve such PPTT applications, however, the nanocomposite structures need to be strictly controlled. Ideally, nanocomposites should have the following traits: (1) efficient absorption and conversion of photon energy into heat, (2) a targeting component, and (3) a capacity to be visualized easily in vivo.

Gold nanorods (Au NRs) are known for their ability to convert photon energy into heat efficiently, resulting in hyperthermia, and thus a suppression of tumor growth in vitro and in vivo. Wrapping gold nanostructures in reduced graphene oxide (rGO) greatly enhances their light-to-heat conversion under NIR (808 nm) irradiation.1 We have recently reported2 the creation of Au NRs covered with rGO, which were additionally conjugated with TAT peptide to ensure selective targeting of human astrocytoma U87MG tumor cells,3 and with commercially available Cy7-NHS NIR dye to ensure visualization on nanocomposites in vivo. The resulting Au NRs@rGO-PEG-Tat/Cy7 nanocomposites were used for PPTT of Swiss nude mice with model U87MG-originating tumors.

In vivo and ex vivo Fluorescence Imaging of Functionalized Gold/Graphene Nanocomposites during Plasmonic Photothermal Therapy in MiceTetiana Dumych,1,2# Kostiantyn Turcheniuk,3# Pascal Mariot,4 Natasha Prevarskaya,4 Julie Bouckaert,2 Volodymyr Vovk,1 Valentyna Chopyak,1 Rabah Boukherroub,3 Sabine Szunerits3, Rostyslav Bilyy,1,2

Author Information: 1 - Danylo Halytsky Lviv National Medical University, 79010, Lviv, Ukraine; 2 - Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université Lille 1,

CNRS UMR 8576, 59655 Villeneuve d’Ascq, France; 3 - Institut d’Electronique, de Microélectronique et de Nanotechnologie (IEMN), UMR CNRS8520, Université Lille1, Avenue

Poincaré-BP 60069, 59652 Villeneuve d’Ascq, France; 4 - Laboratoire de Physiologie Cellulaire INSERM U1003, Equipe Labellisée par la Ligue Nationale, Contre le Cancer et

LABEX (Laboratoire d’excellence), Université Lille1, 59655 Villeneuve d’Ascq, France; # - equally contributed

Protocol

In vivo Imaging

Studies involving animals and experimental protocols were conducted in accordance with the local animal ethical committee (C59-00913; protocol CEEA 202012) of the University of Sciences and Technologies of Lille, under the supervision of Dr. P. Mariot (59-009243).

Six-week old male Swiss nude mice (Charles River, France) were used for this investigation. The mice were housed in cages covered with air filters in a temperature-controlled room with a 12-h light/12-h dark cycle and kept on a standard diet with drinking water available ad libitum. All animal experiments were performed in agreement with institutional ethnical guidelines.

To evaluate the in vivo PPTT efficiency of Au NRs@rGO-PEG-Tat/Cy7, tumor-bearing mice (subcutaneous U87MG xenografts) were prepared by inoculating a suspension of 5×106 U87MG cells per mouse (in the flank) in 50 % (v/v) Matrigel.4 Tumor growth was monitored every two days by measuring the dimensions with calipers and calculating the volume. When the tumor grew to about 500 mm3 in volume, Au NRs@rGO-PEG-Tat/Cy7 (150 µL for 1 mg/mL per mouse) was injected intravenously. To assure significant statistical data, three animals were used in each group for monitoring tumor growth. After 20 hours post-injection of the nanostructures, the tumor tissue was irradiated using a portable continuous wave laser (Gbox model, Fournier Medical Solution, France) at 808 nm and power density of 0.5 – 2.0 W cm-1 for up to 5 min, 2 times, with 2 min interval. The surface temperature of the irradiated areas of skin was controlled by Infrared Camera (Thermovision A40). 24 hours after irradiation, some animals were sacrificed with cervical dislocation under anesthesia in agreement with institutional ethical guidelines. Tissues were excised for subsequent fluorescent measurement and histological analysis. Tumor progression in treated and untreated groups was evaluated by measuring the tumor volume for the following 15 days.

In vivo tracking of Au NRs@rGO-PEG-Tat/Cy7 was performed with the Bruker In-Vivo Xtreme, equipped with an interline front-illuminated (FI) 16 MP CCD detector, 400 Watt broad-band, high Lumen flux Xenon Fluorescence illuminator and by use of excitation filters ranging from 400 - 770 nm in combination with emission filters ranging from 520 - 850 nm. A feedback regulated animal body temperature control unit maintained the animal´s wellfare. The data was analyzed with Bruker Molecular Imaging (MI) software.

Histological Validation and Analysis

For histological analysis, mice were sacrificed with cervical dislocation under anesthesia and tissues (heart, liver, kidneys, lung, lymph node, tumor), were excised from the mice at day 1 post Au NRs@rGO-PEG-Tat/Cy7 injection, and then fixed with 4 % neutral buffered formalin solution and embedded in paraffin according to the standard laboratory protocol. The sliced tissues were stained with hematoxylin and eosin (H&E). Images were taken using an optical microscope (Zeiss AxioImager A1) using 10 x, 40 x and 100 x objectives with normal (halogen) light and using color filter with transmission of 620-650 nm to visualize Au NRs@rGO-PEG-Tat/Cy7.

Results and Conclusions

NIR Fluorescence Imaging and Biodistribution

In vivo biodistribution of the Au NRs@rGO-PEG-Tat/Cy7 composites in nude, U87MG tumor bearing mice (see Protocol) was evaluated by monitoring Cy7-NHS NIR dye fluorescence non-invasively. As seen in Figure 1, some accumulation of Au NRs@rGO-PEG-Tat/Cy7 appeared to have occurred in the tumor within 20-24 hours post injection. The strongest NIR fluorescence signals were detected from the tumor tissues due to the long blood circulation time and enhanced permeability and retention effect (EPR).5 Importantly, fluorescence was detected in the feces, indicating that Au NRs@rGO-PEG-Tat/Cy7 is cleared from the body through bladder and intestine.

Figure 1

In vivo biodistribution analysis of functionalized Au NRs@rGO-PEG-Tat/Cy7 nanocomposites using the Bruker In-Vivo Xtreme. 24 hours post injection of NRs@rGO-PEG-Tat/Cy7, mice were imaged using Ex/Em = 750/830 nm filter pair to visualize the Cy7 dye on targeted gold-graphene nanocomposites. The mouse was imaged in 3 positions - from below, left and right flank (only 2 positions are shown here, to demonstrate liver and tumor uptake). Nanocomposites have accumulated in liver, kidneys, tumor and lymph nodes. Cy7 intensity signal was evaluated in tumor (11.4 nW) and lymph node (2.4 nW).

In vivo Photothermal Treatment (PPTT) of Mice with Implanted U87MG Tumor Cells

PPTT treatment was assessed in nude mice bearing subcutaneous human astrocytoma U87MG tumor cells, 20 hours post intravenous injection of Au NRs@rGO-PEG-Tat/Cy7 nanocomposites (see Protocol). Mice were subjected to NIR laser irradiation using a portable continuous wave laser with 808 nm excitation, where an optical fiber was placed 6 cm above the tumor (Figure 2B). Several laser power intensities were tested2 to optimize the treatment and provide the tumor heating up to 50 - 52 °C, without burning the skin. A power of 0.7 W cm-2 satisfied those conditions. Figures 2C and 2D demonstrate the distribution of nanocomposites in the organs of a representative mouse, at 3 hours post irradiation. PPTT of tumor seems to destroy the

nanocomposite completely, since ex vivo analysis exhibited no NIR fluorescence. Furthermore, extensive tumor cellular damage was indicated by macroscopic internal hemorrhages in the tumor. Finally, the PPTT effect appeared to be restricted to the area of treatment, as other mouse organs (liver and kidney) continued to have NIR fluorescent signals and no visible hemorrhaging foci on ex vivo analysis (Figures 2C and 2D).

Histological analysis of the tumor tissue confirmed that there was an uptake of Au NRs@rGO-PEG-Tat particles by tumor stroma cells in some areas (Figure 2E). The Au NRs@rGO-PEG-Tat particles were visualized as black particles by H&E staining and as strongly absorbing substance using illumination with 620 - 650 nm light (not shown). Additionally, tumor histology sections confirmed the presence of destructed blood vessels and hemorrhagic foci.

Lastly, measurement of tumor sizes in PPTT treated mice revealed a statistical decrease in tumor volume following treatment. 6 days after PPTT tumor size decreased by 39.0 ± 16.7 % (n = 3). In the untreated group tumor volume increased by 52.5 ± 10.1 %, (n = 3). The difference between treated and untreated groups was statistically significant, according to Mann-Whitney U-test. More details on animal survival can be found in [2].

Photothermal treatment (PPTT) of tumor-bearing mice by using Au NRs@rGO-PEG-Tat/Cy7 photothermal nanocomposites. A) Mice bearing tumor, 24 hours post injection of nanocomposites. B) PPTT of mice with 808 nm laser (not visible) and using Au NRs@rGO-PEG-Tat/Cy7. The red spot is a signal from 650 nm guidance laser. C/D) Organs of mice showing accumulation of nanocomposites, imaged at (D) Ex/Em 750 / 830 nm. PPTT completely destroyed nanocomposites in tumor tissue while non-irradiated tissues show still a fluorescence signal, e.g. lymphatic node, kidneys, liver. E) Histological analysis on tumor revealed massive accumulation of nanocomposites (black) in tumor cells (blue arrows) and in walls of vessels (not shown) resulting in massive vessel destruction (red arrows).

Figure 2

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References

[1] Lim D-K, Barhoumi A, Wylie RG, Reznor G, Langer RS, Kohane DS. Enhanced photothermal effect of plasmonic nanoparticles coated with reduced graphene oxide. Nano Lett. 2013; 13:4075-4079.

[2] Turcheniuk K, Dumych T, Bilyy R, Turcheniuk V, Bouckaert J, Vovk V, Chopyak V, Zaitsev V, Mariot P, Prevarskaya N, Boukherroub R, Szunerits S. Plasmonic photothermal cancer therapy with gold nanorods/reduced graphene oxide core/shell nanocomposites. RSC Advances 2016; 6:1600-1610.

[3] Lim SP, Garzino-Demo A. The human immunodeficiency virus type 1 Tat protein up-regulates the promoter activity of the beta-chemokine monocyte chemoattractant protein 1 in the human astrocytoma cell line U-87 MG: role of SP-1, AP-1, and NF-kappaB consensus sites. J Virol 2000; 74:1632-1640.

[4] Worthington P, Pochan DJ, Langhans SA. Peptide Hydrogels - Versatile Matrices for 3D Cell Culture in Cancer Medicine. Front Oncol 2015; 5:92.

[5] Chen R, Wang X, Yao X, Zheng X, Wang J, Jiang X. Biomaterials 2013; 34:8314-8322.

Acknowledgement

We acknowledge Horizon 2020 project 690836 “Pathogenes and Graphene” for financial support.

We thank Dr. Jens Waldeck (Bruker BioSpin MRI GmbH, Ettlingen, Germany) and Dr. Anna Yudina (Bruker BioSpin SAS, Wissembourg, France) for their support on Analysis Software and Application support as well as critical reviewing of research results presented here.

Conclusion

In summary, it could be demonstrated that the use of in vivo and ex vivo fluorescent imaging for monitoring the biodistribution and persistence of gold/graphene oxide nanorods in a PPTT mouse model is feasible. Deep tissue, in vivo visualization of nanocomposites was achieved by conjugation with commercially available cyanine dye Cy7. This nanocomposite construct will allow investigators in future studies to evaluate both the efficacy and completeness of PPTT in various mouse model systems. The study demonstrates that new functionalized nanomaterials also enable efficient monitoring and destruction of solid tumors. Thus they might serve as an excellent multi-functional theranostic agent in photothermal therapeutic applications, providing both efficient targeting, visualization and destruction of tumor tissue.