The boron-10 content in the polyglycerol dendrimer was investigated by quantitative neutroncapture therapy (QNCR). The
B-enriched PGLD samples were introduced into polyethylene bags. After evacuating the bags, the samples were exposed to a thermal neutron flux of 3.6
over a period of 35 min and Makrofol E as detector.
B-PGLD evaluation2.2.1. Citotoxicity
The cytotoxicity of
B-PGLD extracts was evaluated against Chinese hamster ovary (CHO)cells, ATCC CHO k1 (American Type Culture Collection, ATCC), according to ISOguidelines . Serial diluted PGLD-ChB extracts were added to a CHO cell culture. CHOcells were grown in Eagle minimum essential medium – fetal calf serum (MEM–FCS) and1% penicillin-streptomycin solution, in a plastic tissue culture at 37°C and atmosphere of 5%CO
. After monolayer propagation, the culture medium was removed and the cells werewashed with calcium and magnesium phosphate saline buffer. The culture was treated with0.25% trypsin solution to detach the cells from the culture tissue. After trypsinization, thecells were transferred to a screw-capped plastic tube, centrifuged, and washed twice withcalcium and magnesium free phosphate saline buffer. The cells were resuspended in MEM-FCS and adjusted to give 100 cells/mL. A volume of 2 mL of this cell suspension was seededto each 60 mm-diameter assay culture dish and incubated for about 5 h for adhesion of thecells. The culture medium was then replaced by 5mL of fresh MEM-FCS, in the control plates, and by undiluted (100%) and successively diluted extracts (50, 25, 12.5, and 6.25%),in culture dishes with the adhered cells. All concentrations were tested in triplicate. After incubation of the culture dishes for 7 days (37°C, 5% CO
) for the cell colonies formation,the cytotoxicity was evaluated quantitatively, based on the cell viability. After the incubationtime, the medium was removed from the dishes and after fixation with buffered salineformalin solution; the colonies were stained with Giemsa. The number of visible colonies oneach dish was counted and compared with the number of colonies in the CHO control dish.Phenol solution (0.02%) and polyethylene extract (60 cm
in 60 mL MEM-FCS) were used as positive and negative controls, respectively. The results were expressed as percent cellsurvival from control per volume of extract tested.
Sixteen female Wistar rats weighting 200 g were used in the
experiments. On the back of each rat, four incisions about 3 cm long each were made through the skin up to theunderlying muscular tissue layer under aseptic conditions. Boronated
B-PGLD was placedover the incisions, ensuring that the wounds were completely covered. After different periodof time, i.e: 3, 7, 15 and 30 days, the rats were sacrificed by using an excess of diethyl ether.Implants with surrounding tissues were carefully dissected from the dorsal muscle site at thecorresponding times. Histological evaluations using hematoxylin-eosin staining techniquewere performed to assess tissue response to membranes used and progression of healing. Thesamples were immersed-fixed in 10% (v/v) formalin in 0.1 M phosphate buffer (pH = 7.4). Inthe histopathological examination, the fixed samples were embedded in paraffin, sectionedinto slices of 5 µm thickness and then stained with hematoxylin and eosin. The preparedsections were examined using optical microscopy (Nikon, Eclipse E 400).
INAC 2005, Santos, SP, Brazil.