WATER SOLUBLE MAGNETITE NANOPARTICLES Fe3O4 – OLEIC ACID-TWEEN 80 : STABILITY, REDOX ACTIVITY AND TRANSPORT IN HEPATOPANCREATIC CELLS OF THE MANGROVE CRAB UCIDES CORDATUS Hector Aguilar Viorino1, Priscila Ortega2,Flavia P. Zanotto2, Breno Pannia Espósito1
[email protected] 1Institute of Chemistry. University of São Paulo. São Paulo – SP, 05508-000, Brazil. 2 Institute of Biosciences. Department of Physiology. University of São Paulo. São Paulo – SP, 05508-900, Brazil.
Introduction
λexc/λem=485/520 nm
Biomodels are used to assess the consequences of human activity on animals and their habitat. The edible crab Ucides cordatus, endemic to the mangrove, one of the most populated and degraded ecosystems in Brazil, is used as a bioindicator of toxic metal contamination. In this study, hepatopancreatic cells of U. cordatus were exposed to water-soluble nanoparticles (NP) with three different sizes (5, 8, 10 nm), coated with oleic acid-detergent, with the objective to evaluate the transport of this metal to hepatopancreatic cells of the animal model. The correlation of metal incorporation with hepatopancreatic cell type, NP stability and NP redox activity was tested.
Sample preparation Insertion of samples pH=7.8 in Ucides Buffer
The hepatopancreatic cells was then labeled with CAL-AM. The cellular transport was measured each 90 seconds in real time using a spectrofluorometer.
Experimental Nanoparticles Synthesis[1]
Table 1. Concentrations and percentage (as compared to total iron) of redox-active iron in 40 µM samples of Nps (average ± s.d. of 4 measurements)
Hexane
NP-OA/ Hexane
NP-OA /Tween 80
Nanoparticles of Magnetite
Redox Activity (µM)
%
5 nm
6.66 ± 0.23
16.7
8 nm
5.71 ± 0.48
14.3
Soluble NPs
5.63 ± 0.16
14.1
DHR
O
ROS
Ascorbate O
NH2Cl
FeIII
24 hours
+
FeIII
OCH3 C
fluorescent Probes
Iron–Fluoroscen Probes
Calcein / Fl-transferrin / DFO-Fl
0.8 0.6 0.4 0.2
0.2 0.5
1.0
1.5
2.0
DFO-Fl *
1.0
Rhodamine
Transport of iron in cell [3,4] The animals were collected at Itanhaém, São Paulo coast, and acclimatized in the vivarium of the University of São Paulo. For the experiment, the hepatopancreas were separated from crab. The organ ,was used for the dissociation of hepatopancreatic cells through mechanical method for the dissociation was used. Hepatopancreatic cells were further separated using the sucrose gradient method.
Blank ( 1.6%,Tween 80)
1.0
Fe(II) (a) 0.8 NP 12(b,c) (a,c) 0.6 NP 15 NP 18(b) 0.4 0.2
20
40
60
80
100
0.6 0.4 0.2
20
Time (s)
40
60
0.5
80
100
1.0
1.5
2.0
Iron concentration (M)
FlTf
C Fluorescence (r.u.)
1.0
*
*
0.5
1.0
Fe(II) 5 nm 8 nm 10 nm
*
0.8 0.6 0.4 0.2 0.0 0.0
1.5
2.0
Iron concentration (M)
Figure 1. Quenching of 2 µM (A) calcein (B) DFO-Fl (C) FlTf caused by the NPs with 2.0 0.0 µM of iron concentration.
B Cell
1.0 Blank ( 1.6%,Tween 80)
Fe(II)(a) 0.8 NP 12(a,c) (c) 0.6 NP 15 NP 18(b,c) 0.4 0.2
1.0 Blank( 1.6%,Tween 80)
Blank ( 1.6%,Tween 80)
(a) Fe(II) 0.8 NP 12(a,c) 0.6 NP 15(c) NP 18(b,c)
Fe(II)(a) NP 12(b) NP 15(c) NP 18(b)
0.4
0.2 0.0
0.0
0
Fe(II) 5 nm 8 nm 10 nm
*
0.8
F Cell
0.0
0
(quenched)
→ Acknowledgements
1.0
0.0
O
Iron Metallodrugs
R Cell Fluorescence (r.u.)
Determination of free
H2N
Fluorescence (r.u.)
E cell
Fluorescence (r.u.)
OCH3 C
[2,3] iron
0.4
B
Our results indicate that the NPs are very stable against different fluorescent chelators, probably because iron is not readily available from the mineral core and/or beneath the outer layer of oleic acid. Despite this stability, we found the equivalent to 5.63 -7.16 μM of redox-active iron, which could be a surface phenomenon.
NH2.HCl
O
H
Fe3+ +
0.6
0.0 0.0
HCl.H2N
1 hour
0.8
0.0 0.0
Thermal decomposition method
Pro-oxidant
* *
Fe(II) 5 nm 8 nm 10 nm
*
*
Iron concentration (M)
10 nm
[2] Activity
1.0
H2O
Tween 80
-Hexane
NP-OA
Fluorescence (r.u.)
Results and discussion
Fluorescence (r.u.)
265 °C
Calcein
A
Fluorescence (r.u.)
Fe(acac)3 ROH RCOOH RNH2 Ph2O
Fluorimeter
0
20
40
60
80
100
20
40
60
80
100
Time (s)
Time (s)
Time (s)
0
Figure 2. Iron transport in hepatopancreatic cells of Ucides cordatus represented as fluorescence (r.u.) in relation to time (s), during 90 s. The dashed line represents the addition of iron compound ~ 800 µM, final concentration (30s), where letters represents significant statistic difference (ANOVA), P
