Over the years, there has been significant advancement in the field of nanotechnology, propelling plasmonic photothermal therapy (PPTT) as a viable method for removing tumor growths and other similar pathogenic tissues. However, in order to realize such PPTT applications, it is important to ensure that the structures of nanocomposites are rigorously regulated.
Nanocomposites should preferably have the following characteristics:
- An ability to be viewed easily in vivo
- A targeting component
- Efficient absorption and transformation of photon energy into heat
Gold nanorods or Au NRs can efficiently change photon energy into heat, which leads to hyperthermia and causes in vivo and in vitro suppression of the tumor growth. The light-to-heat conversion property of the gold nanostructures can be improved considerably under near-infrared [NIR (808nm)] irradiation by covering them in reduced graphene oxide (rGO).
Recently, Au NRs covered were developed that were further conjugated with Cy7-NHS NIR dye and TAT peptide to ensure in vivo visualization on nanocomposites and selective targeting of human astrocytoma U87MG tumor cells, respectively. Cy7-NHS NIR dye is a commercially available product. The Au [email protected]/Cy7 nanocomposites, thus obtained, were utilized for PPTT of Swiss nude mice with U87MG-originating tumor model.
In vivo Imaging
Under the guidance of Dr. P. Mariot, studies were carried out in which experimental procedures and animals were used as per the local animal ethical committee of the University of Sciences and Technologies of Lille.
For this analysis, male Swiss nude mice of six weeks old were used and these were kept in cages surrounded with air filters and placed in a temperature-regulated room with a 12 hour dark /12 hour light cycle. The animals were given a standard diet with drinking water available ad libitum. All experiments involving animals were carried out according to the institutional ethnical guidelines.
Mice with tumor growths were prepared so as to assess the PPTT efficiency of the Au [email protected]/Cy7 in vivo. This was done by inoculating a 5×106 U87MG cell suspension in 50% (v/v) Matrigel for each mouse. Once in two days, the growth of the tumor was tracked by determining the tumor size with calipers as well as by measuring the volume. As soon as the tumor reached to approximately 500mm3 in volume, Au [email protected]/Cy7 was intravenously administered.
In order to ensure considerable statistical data, each group was designated with three animals for tracking the growth of tumor. Approximately 20 hours after the intravenous injection of nanostructures, Fournier Medical Solution’s mobile continuous wave laser was used to irradiate the tumor tissue at power density of 0.5 to 2.0Wcm and1808nm for about 5 minutes. This was repeated two times with an interval of 2 minutes. Thermovision A40 infrared camera was then used to control the surface temperature of the irradiated skin areas.
Following 24 hours of irradiation, certain animals were anesthetized and subjected to cervical dislocation in accordance with institutional ethical guidelines. Excised tissues were used for successive fluorescent measurement and histological examination. Progression of tumor in both untreated and treated groups was assessed by determining the volume of the tumor for 15 successive days.
The Au [email protected]/Cy7 was tracked in vivo with the Bruker In-Vivo Xtreme, fitted with an interline front-illuminated 16MP CCD detector, 400W broad-band, high lumen flux xenon fluorescence illuminator as well as with excitation filters and emission filters spanning from 400 to 770nm and from 520 to 850nm, respectively. The animal´s welfare was maintained through a feedback-regulated, animal body temperature control unit. Bruker Molecular Imaging (MI) software was then used for data analysis.
Histological Validation and Analysis
Mice were anesthetized and subjected to cervical dislocation for histological analysis, and tissues like liver, heart, lung, kidneys, tumor and lymph node were extracted from the mice at day 1 following the intravenous injection of Au [email protected]/Cy7. The excised tissues were then fixed with neutral buffered formalin solution of 4% and preserved in paraffin as per the standard laboratory protocol.
These were later stained with eosin and hematoxylin (H&E). This was followed by capturing the images with Zeiss AxioImager A1 optical microscope utilizing 10x, 40x and 100x objectives with standard (halogen) light and utilizing color filter with 620 to 650nm transmission to view the Au [email protected]/Cy7 in vivo.
NIR Fluorescence Imaging and Biodistribution
In vivo biodistribution of the Au [email protected]/Cy7 composites in nude, U87MG tumor bearing mice was evaluated by monitoring Cy7-NHS NIR dye fluorescence non-invasively. As seen in Figure 1, some accumulation of Au [email protected]/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). Importantly, fluorescence was detected in the feces, indicating that Au [email protected]/Cy7 is cleared from the body through bladder and intestine.
Figure 1. In vivo biodistribution analysis of functionalized Au [email protected]/Cy7 nanocomposites using the Bruker In-Vivo Xtreme. 24 hours post injection of [email protected]/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 (11.4nW) and lymph node (2.4nW).
In vivo Photothermal Treatment (PPTT) of Mice with Implanted U87MG Tumor Cells
In nude mice with subcutaneous human astrocytoma U87MG tumor cells, PPTT treatment was evaluated after 20 hours of injecting the Au [email protected]/Cy7 nanocomposites intravenously. Following this, the mice were irradiated with NIR laser using a mobile continuous wave laser with an excitation of 808nm. Here, an optical fiber was located 6cm over the tumor (Figure 2B).
Figure 2. Photothermal treatment (PPTT) of tumor-bearing mice by using Au [email protected]/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 [email protected]/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 / 830nm. 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).
A number of laser power intensities were analyzed for optimized treatment as well as to heat up the tumor to 50-52°C without causing any burn to the skin. These conditions were satisfied with a 0.7Wcm-2 power.
Figures 2C and 2D show how the nanocomposites are distributed in a specified mouse’s organs after 3 hours of irradiation. Tumor PPTT appears to damage the nanocomposite entirely, as ex vivo analysis did not show NIR fluorescence. Additionally, large internal hemorrhages in the tumor indicated widespread damage in the tumor cells.
The effect of PPTT appeared to be rather limited to the treatment area, because while other organs of the mouse such as kidney and liver had NIR fluorescent signals, they lacked noticeable hemorrhaging foci on ex vivo analysis, as shown in Figures 2C and 2D.
When the tumor tissue was histologically studied, it was observed that tumor stroma cells in certain areas absorbed the Au [email protected] particles (Figure 2E). The Au [email protected] nanoparticles were seen as powerfully absorbing substance using 620 to 650nm light illumination and as black particles with H&E staining. Further, histology sections of tumor validated the presence of hemorrhagic foci and damaged blood vessels.
In mice treated with PPTT, tumor size measurement showed a statistical reduction in the tumor volume subsequent to treatment. Six days following PPTT, the size of the tumor reduced by 39.0 ± 16.7 % (n = 3), while in the untreated group, the volume of the tumor increased by as much as 52.5 ± 10.1 %, (n = 3). Considerable difference was seen between the untreated and treated groups, in accordance with the Mann-Whitney U-test.
It has been shown that in vitro and in vivo fluorescent imaging can be effectively applied for tracking the biodistribution and perseverance of gold and graphene oxide nanocomposites in PPTT-based mouse models. Conjugation with cyanine dye Cy7 helped in achieving in vivo deep tissue visualization of nanocomposites.
In future studies, these nanocomposites will enable researchers to assess the completeness as well as the efficacy of PPTT in different systems of mouse models. The study shows that efficient tracking and damage of solid tumors can also be achieved with new functionalized nanomaterials. Therefore, such nanomaterials can act as good multi-functional theranostic agent in photothermal therapeutic purposes, offering highly efficient targeting, observation, and annihilation of tumor tissues.
Produced from material originally authored by Tetiana Dumych,1,2 Kostiantyn Turcheniuk,3 Pascal Mariot,4 Natasha Prevarskaya,4 Julie Boukaert,2 Volodymyr Vovk,1 Valentyna Chopyak,1 Rabah Boukherroub,3 Sabine Szunerits,3 and Rostyslav Bilyy,1,2
1Danylo Halytsky Lviv National Medical University, 79010, Lviv, Ukraine
2Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), Université Lille 1, CNRS UMR 8576, 59655 Villeneuve d’Ascq, France
3Institut d’Electronique, de Microélectronique et de Nanotechnologie (IEMN), UMR CNRS8520, Université Lille1, Avenue Poincaré-BP 60069, 59652 Villeneuve d’Ascq, France
4Laboratoire 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
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