PREPARATION OF FERRIC OXIDE NANOPARTICLES AND EVALUATION OF ANTIOXIDANT PROPERTY IN CEREBRAL ISCHEMIA REPERFUSION MODEL
A-597
Shikha S, Neha K, Dimple C Department of Pharmaceutical Sciences and Research, Punjabi University, 147002, Patiala, India RESULTS
ABSTRACT The present study aims to prepare ferric oxide nanoparticles and to investigate its antioxidant property in reperfusion induced cerebral injury. The mean hydrodynamic diameter of obtained ferric oxide nanoparticles with change in molar ratios of Fe2+/Fe3+ from 0.5 to1 was found to be 174.4nm and 313.3nm respectively.The mean hydrodynamic diameter of PVP coated ferric oxide nanoparticles was found to be 469.3nm. Ferric oxide nanoparticles showed a negative zeta potential value of 10.8mV and PVP coated ferric oxide nanoparticles was -11.3mV. X-Ray diffraction showed average crystalline size of 17nm and 41nm for uncoated and PVP coated ferric oxide nanoparticles. There was a dose dependent increase in the percentage antioxidant activities as measured by modified DPPH method.Ferric oxide nanoparticles (10mg/kg and 20mg/kg, 1 ml i.p. doses of uncoated and PVP coated) were administered before cerebral ischemia. Global cerebral ischemia was induced by bilateral carotid artery occlusion for 15 minutes followed by reperfusion for 24 hrs. After 24 hrs animals were sacrificed and the antioxidant effect of uncoated and PVP coated ferric oxide nanoparticles was determined.Cerebral infarct size was estimated using triphenyltetrazolium chloride (TTC) staining, thiobarbituric acid reactive substances and glutathione levels. Administration of ferric oxide nanoparticles significantly reduced cerebral infaract size. Thus, ferric oxide nanoparticles have a great potential in reducing incidence of global cerebral ischemia.
Table 1. DPPH scavenging % (absorbance at 570nm) by the uncoated and coated Ferric oxide nanoparticles at different concentration
DPPH Scavenging % of uncoated ferric oxide nanoparticles
Iron oxide nanoparticles conc(mg)
No. of observation
DPPH Scavenging % coated ferric oxide nanoparticles
1
5
8.33 ± 0.98
6.21 ±0.78
2
10
13.89 ± 0.71
9.89 ± 0.98
3
15
15.2 ± 0.51
12.34 ± 0.19
4
20
23.2 ± 0.61
18.93 ± 0.38
5
50
35.23 ± 0.20
25.04 ± 0.28
Measurement of cerebral infract size
Treatment of cerebral ischemia/reperfusion has been limited due to poor cerebral cell penetration. Ferric oxide nanoparticles are an ideal candidates for this purpose because of their small size, improved permeability across cell membrane. Moreover, they have been shown to be endogenous scavenger of OH and ONOO-.
60 a
%Cerebral infract size
50
OBJECTIVE oxide
b
40 b 30
20
10
By weight
Sham
Magnetic properties of prepared ferric oxide nanoparticles was determined using Superconducting Quantum Interference Device (SQUID) magnetometer (Quantum Design MPMS-7, Company Quantum Design USA, at IIT, Delhi.
Control
Uncoated 10 mg/kg
1
Uncoated 20 mg/kg
Coated 10 mg/kg
Coated 20 mg/kg
Figure 2: Effect of ferric oxide nanoparticles (uncoated and PVP (coated) on cerebral infract size in mice subject to global cerebral ischemia followed by reperfusion. Each column represents the mean ± SEM, n = 3. a=P < .05 versus Sham; b=P < .05 versus control ; c=P < .05 versus coated 10 mg/kg (Ip)
Administration of ferric oxide nanoparticles at dose of 10mg/kg and 20mg/kg, i.p 24 hrs before cerebral ischemia and reperfusion was markedly reduced cerebral infarct. This clearly reveals neuroprotective property of ferric oxide nanopaticles in preventing cerebral ischemia infarct size and TBARS concentration.
CONCLUSION
TBARS 10 TBARS(nmol/mg of protein)
Measurement of particle size and polydispersity index was performed by photon correlation spectroscopy (PSC) using a Zetasizer 3000 (Malvern instruments).
DISCUSSION
b,c
0
Chemical Co-precipitation method was used to prepare uncoated ferric oxide nanoparticle using ferrous sulfate and ferric chloride and then ferric oxide nanoparticles were coated with PVP.
SEM reveals morphology of both ferric oxide nanoparticles and PVP coated nanoparticleas as having inverse spinel structure.
Global cerebral ischemia of 15minutes followed by reperfusion for 24hrs produced neuronal injury as indicated by cerebral infaract size by volume and weight method. Administration of ferric oxide nanoparticles (10mg/kg and 20mg/kg, 1ml I.p doses of uncoated and PVP coated) 60 minutes before inducing cerebral ischemia and reperfusion was found to be neuroprotective because it markedly reduced cerebral infaract size ; it helps in preventing global cerebral ischemia,it decrease theincreased TBARS concentration in brain mitochondria and supernatant fractions and thus it prevent global cerebral ischemia, it increased the reduced GSH level during oxidative stress and thus it show antioxidant effect and prevents global cerebral ischemia
Fig.1 (A) Sham group (B)Ischemic control (C)10mg/ml (1mlI.P)uncoated (D) 10mg/ml(1ml I.P)coated
RATIONALE
METHOD
X-ray diffraction , average crystalline size of ferric oxide nanoparticles as 17nm and 41nm for PVP coated ferric oxide nanoparticles; indication of inverse cubic spinel structure of nanoparticles
Antioxidant activity of ferric oxide uncoated and coated nanoparticles was measured by DPPH quenching activity. There was dose dependant increase in the percentage antioxidant activities for all the concentration tested.; indicate more antioxidant activity of uncoated ferric oxide than PVP coated ferric oxide nanoparticles
REVIEW OF LITERATURE
To prepare and evaluate antioxidant potential of ferric nanoparticles in cerebral ischemia reperfusion model in mice.
Ferric oxide nanoparticles showed negative zeta potential 10.8mV and PVP coated ferric oxide nanoparticles -11.3mV; Indication of stability of particle suspension in water.
SQUID magnetometer reveals saturation magnetization of ferric oxide and PVP coated ferric oxide nanoparticles as 26emu-1 and 63emu-1;indicate magnetic properties
INTRODUCTION Nanoparticles have been applied for various purposes, including diagnostic and therapeutics in technology and medicine, owing to their unique physiochemical properties. Iron (II, III) oxide nanoparticles (IONPs) have attracted much attention because of their magnetic properties but also owing to their great potential in biomedical and invivo clinical applications. Particles of iron oxide have been administered intravenously for over 50 years initially for treatment of anemia. Emerging experimental and clinical applications in CNS capitalize on both the physical and magnetic properties of Ferric oxide nanoparticles. Ferric oxide nanoparticles show an antioxidant property according to the literature.
Ferric oxide nanoparticles showed mean hydrodynamic diameter with change in molar ratio of Fe2+/Fe3+ from 0.5 to 1 as 174.4nm and 313nm. The mean hydrodynamic diameter of PVP coated ferric oxide nanoparticles was 369.3nm, TEM photograph revealed presence of particles below 200nm and 500nm for both uncoated and coated ferric oxide nanoparticles ,indication of extended circulation time in the blood and decreased uptake by reticuloendothelial system.
9
a
8
b b
7
b,c
6 5 4 3 2
In the present study transient cerebral global ischemia and reperfusion model have been employed. This model mimics clinically observed cerebral ischemia due to cardiac arrest.Prolonged global ischemia results in neuronal death irrespective of post ischemic reperfusion.Therefore, global cerebral ischemic of hort duration followed by reperfusion has been employed in the present study of ferric oxide nanoparticles .
1
The morphological examination of nanoparticles was performed by transmission electron microscopy (TEM) (Tecnai 20 G2 S TWIN) set at 200 kV. We evaluated invitro antioxidant activity of both coated and uncoated ferric oxide nanoparticles was by DPPH method. We also evaluated in-vivo test for antioxidant property of both uncoated and coated ferric oxide nanoparticles on infarct size ischemic mice by TTC (triphenyltetrazolium chloride staining).
0 Sham Uncoated 20 mg/kg
1 Control Coated 10 mg/kg
Uncoated 10 mg/kg Coated 20 mg/kg
Figure 3: Effect of ferric oxide nanoparticles (uncoated and PVP coated) on TBARS formation in mice subject to global cerebral ischemia followed by reperfusion. Each column represents the mean ± SEM, n = 3. a=P < .05 versus Sham; b=P < .05 versus control; c=P < .05 versus coated 10 mg/kg (Ip)
We studied the biochemical parameters for antioxidant activity of ferric oxide uncoated and PVP coated nanoparticles by TBARS (Thiobarbituric acid reactive substance).
2
3 4 5 6
Name of group
Dose
Sham control
vehicle–pretreated
Uncoated nanoparticles Uncoated nanoparticles Coated nanoparticles Coated nanoparticles
10 mg/kg (1ml, i.p.) 20 mg/kg (1ml, i.p.) 10mg/kg (1ml, i.p.) 20mg/kg (1ml, i.p.)
3) Serpen, A., Capuano, E., Fogliano, V and Gokmen, V . (2007). A new procedure to measure the antioxidant activity of in soluble foot components. J. Agric. Food Chem., 55 (19) : 7676-7681
Procedure Surgical procedure Mice were orally administered vehicle 1 h prior to being subjected to 10 ml of global cerebral ischemia by bilateral carotid artery occlusion (BCAO)followed by reperfusion for 24 h. Administered nanoparticles 1h before subjecting them to global cerebral ischemia same same same
1) Arsalani, N., H. Fattahi, H. and Nazarpoor, M. (2010). Synthesis and characterization of PVP-functionalized superparamagnetic Fe3O4 nanoparticals as an MRI contrast agent. eXPRESS polymer letters, 4: 329-330. 2) Paul, S., Saikia, J. P., Samdarshi, S.K. and Kumar, B. (2009). Investigation of antioxidant property of iron oxide particles 1’-1’ diphenylpicryl-hydrazyle (DPPH) method. J. Magn. Magn. Mater., 321:3621-3623
EXPERIMENTAL PROTOCOL GP no 1
REFERENCES
ACKNOWLEDGEMENT I am really thankful to my mentor and guide Dr Dimple Chopra and Department of Pharmaceutical Sciences and Drug Resarch, Punjabi University, Patiala (A) SEM (B) SEM (C) TEM (D) TEM
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of ferric oxide nanoparticles of PVP coated ferric oxide nanoparticles of ferric oxide nanoparticles of PVP coated ferric oxide nanoparticles