- Cellular uptake of gold covered maghemite superparamagnetic nanoparticles and their effects on mouse melanoma B16 cells

Short description: Apoptosis is the process of programmed cell death strictly controlled by enzymes. When the critical temperature for triggering apoptosis is reached (around 42°C), the sustained thermal stress degenerates the regulation process. As some cancer cells are more susceptible to high temperatures than normal cells, such cancerous cells can be treated thermally. Therefore, by heating the tissue above 42oC, the cells could be destroyed selectively. There is a renewed interest in magnetic hyperthermia as a treatment method for cancer. To achieve this, a dose of superparamagnetic nanoparticles can be injected into a region of malignant tissue prior to exposure to an alternating magnetic field. If the field is strong enough and has an optimum frequency, the magnetic nanoparticles will absorb energy and will heat the surrounding tissue, affecting mostly the infected cells.

The purpose of this experimental study is to make a hystopathological investigation of the melanoma, after injecting gold coated maghemite nanoparticles, using UV fluorescence and after magnetic hyperthermia 60 minutes at 120 kHz and 8 mT using FTIR and Raman characterization.

The synthesised gold coated maghemite superparamagnetic nanoparticles were successfully used in  mid-range alternating magnetic fields for magnetic hyperthermia treatment of mice B16 melanoma and in hystopathological investigation of the melanoma, after injecting gold coated maghemite superparamagnetic nanoparticles, using UV fluorescence .

Results: The results certify the applicability of gold coated maghemite nanoparticles in the study of melanoma architecture and defining of melanoma edges.

The γ-Fe2O3 superparamagnetic nanoparticles, functionalized with gold or polyethylene glycol of 6000Da, are injected into the body intratumoral or intravenously and diffuse selectively into tumour tissues. Applying an alternating magnetic field of 120 kHz and 45 Oe will heat up the magnetic nanoparticles raising the temperature of the melanoma cells. The entire tumour volume is heated above 42oC for 30 minutes. We analyzed tumour surface temperature variation and body core temperature during alternating magnetic field exposure using a thermal digital camera HITACHI KP-M2RP. The KP-M2R is a monochrome camera using a 1/2" image size CCD with near infra red sensitivity and features high resolution and performance.

Histopathological aspect of B16 melanoma (Haematoxylin-Eosin stain, x60).

The apoptosis of B16 melanoma cells was studied by UV and FTIR spectrometry. This allows us to identify the superficial chemical bonds of the samples: healthy tissue, untreated melanoma, and magnetic hyperthermia treated melanoma. Four important spectral domains of interest were identified: 1) 3000-2800cm-1, this domain is characterized by spectral bands which can be attributed to the C-H vibration mode appearing both in tissue proteins and lipid cells found in the fresh tumour; for the magnetic hyperthermia treated tumours there was a drastic decrease of the intensity of these spectral bands, which can be explained by the occurring of an oxidation process during cell apoptosis. 2) 1800-1350cm-1, this domain is characterized by spectral bands which can be attributed to amidic groups from the protein skeleton; for the magnetic hyperthermia treated tumours there was a drastic shift of these spectral bands. At the same time another spectral band appeared, having a maximum centred on 1724cm-1, which indicates cellular apoptosis. 3) 1350-900cm-1, this domain is characterized by spectral bands which can be attributed to phosphodiester bonds in nucleic acids; this area offers the most relevant characterization of cellular apoptosis. For the magnetic hyperthermia the spectral band found at 1080-1030cm-1 shifts and increases in intensity (P=O), the spectral band found at 1240cm-1 disappears completely, while a new spectral band appears at 1283cm-1 as a consequence of an oxidation process which is characteristic to cellular apoptosis. 4) 900 – 500 cm-1, the intensity in this spectral bands increases  very much for tumour tissues and the intensity is low for skin and for the melanoma post-magnetic hyperthermia, so suggesting tissue recovery. The intensity in about 800 cm-1 spectral range decreases dramatically for tumours.

B16 melanoma after magnetic hyperthermia B16 melanoma, reference sample B16 melanoma with AuSPION healthy mouse skin with AuSPION healthy mouse skin without AuSPION About 600 cm-1 – spectral band which can be attributed to the vibration mode of the C-C bonds from the phenylalanine (622 cm-1) and tyrosine (640 cm-1). About 700 cm-1 – spectral band which can be attributed to the deformation mode of the C-H bonds from the lipids.   About 800 cm-1 – spectral band which can be attributed to the vibration mode of the P-O bonds within the lipids and nucleic acids. The intensity in this spectral bands increases  very much for tumour tissues and the intensity is low for skin and for the melanoma post-magnetic hyperthermia, so suggesting tissue recovery. The intensity in about 800 cm-1 spectral range decreases dramatically for tumours     B16 melanoma after magnetic hyperthermia B16 melanoma, reference sample B16 melanoma with AuSPION healthy mouse skin with AuSPION healthy mouse skin without AuSPION The spectral band present at about 1600 cm-1 observed in the specters of healthy skin without gold nanoparticles can be attributed to the vibration mode of the C=O bond in the amide I group from the protein structure, in the a-helix conformation, of normal tissue. The difference between normal and damaged tissue is associated with an increased peak intensity or slight shift of the spectral band maximum 1608 and 1702 cm-1).
2800-3000 cm-1 region. This band can be attributed to the vibration mode of the C-H bonds in protein (for healthy tissue) and in lipids (for tumours). This variation can be attributed to the modification of the secondary structure of the protein, as a result of the transformation from α phase to β phase. This confirms FTIR observation, the occurring of an oxidation process during cell apoptosis.

B16 melanoma after magnetic hyperthermia B16 melanoma, reference sample B16 melanoma with AuSPION healthy mouse skin with AuSPION healthy mouse skin without AuSPION 890 cm-1 – spectral band which can be attributed to the stretching mode of the C-C bond (n(C-C)). The intensity of this spectral band decreases for tumor tissues due to modifications in the protein structure. This band can also be attributed to the collagen present in normal skin. 970 cm-1 – appears only in tumours and can be attributed to the vibration mode of the asymmetric C-N bonds within lipids. After magnetic hyperthermia remains a small intensity band suggesting the existence of tumor cells. 1004 cm-1 – spectral band which can be attributed to the deformation mode of the aromatic ring in

phenylalanine (present in keratin). The keratin has the tendency to increase its concentration due to damages, leading to a higher peaks in the Raman spectra. 1030 cm-1 – spectral band characteristic to a normal tissue. It can be attributed to the vibration mode of the CH2 bonds (n(C-H)) in phospholipids. The higher peak in tumour. 1064 cm-1 – spectral band characteristic to the asymmetric vibration mode of the bonds in the PO2- group, attributed both to lipids (1055 cm-1) and nucleic acids (1064 cm-1). 1134 and 1173cm-1 – spectral bands which can be attributed to the stretching mode of the C-C bonds. The intensity of these bands increase as the tumor tissue develops, attributed to the increase of lipid concentration.