By:Nasreen Ahmad

12PGD538Adv. PG Diploma in Nanotechnology

Centre of Excellence in Materials Science (Nanomaterials)

Dept. of Applied Physics, AMU, Aligarh.

PtNH3

ClNH3

Cl

H

HHH H

H

Mitigation of Cisplatin induced toxicities by Nanocurcumin in

male wistar rats

Under Supervision of:Prof. Alim Hussain NaqviCo-ordinator,Centre of Excellence in Materials Science (Nanomaterials)Dept. of Applied Physics, AMU, Aligarh.

Under co-supervision of: Prof. M. Mobarak Hossain

Director, Interdisciplinary Brain Research Centre,

Faculty of Medicine,AMU, Aligarh.

Plan of Talk

What is Cisplatin?

What is Nanocurcumin?

How to synthesize nanocurcumin?

Is it really Nanocurcumin?

How did we do it?- Experimental design.

Is it really effective?-our findings.

Pt

NH3

ClNH3

Cl

H

HHH H

HCisplatin

It is a chemotherapeutic drug.

[molecular formula- H6Cl2N2Pt, molar mass-301.1g/mol]

It is used to treat various types of cancers.

It is accompanied by various toxicity including, Neurotoxicity, Genotoxicity, Ototoxicity, Nephrotoxicity and Hepatotoxicity

Image showing enlarged rat stomach on cisplatin exposure

Pt

NH3

ClNH3

Cl

H

HHH H

H Hepatocellular carcinoma in rats

H

HHH H

H

ATPADP

Pt

NH 3

ClNH3

Cl

H

HHH H

H

Adenine Guanine Cytosine Thymine

H

HH HHH

Pt

N N

H

H

H H

H

H

NN

H

H

HH H

H

2Cl

Adenine

Guanine

Cytosine Thymine

Pt

N NH

H

H H

H

H

NN

APOPTOSIS

 

 

H3CO

H3C

OH

OH

OH

O

Nanocurcumin (diferuloylmethane)

antioxidant, anti-inflammatory, antimicrobial

hepato and nephro-protective , myocardial infarction protection

A natural product extracted from curcuma longa

Soluble in water while curcumin was not soluble.

More bioavailability due to small sizes.

Synthesis of NanocurcuminDried Turmeric Ethanol:water::4:1

Shake vigorously for 10-15 min to obtain the shown image

Left on standing overnight.

{The upper clear liquid was collected and this process was repeated until a clear solution was obtained}

(dropwise in to water at 95 ºC)

(Nanocurcumin)

Dried &stored for further characterisation

Kept in liquid form for oral dosing to subject rats.

Experimental Design

Brain taken for Biochemical & Protein Estimations Blood Sample

collected for Hematological Estimations

Femur (Bone marrow) collected for Genotoxicity Estimations

Epididymis collected for the assessment of Sperm Abnormalities

Experimental Procedures

TISSUE IS HOMOGENIZED TO MAKE 10 % HOMOGENATE

(W/V)

POST MITOCHONDRIAL SUPERNATANT (PMS) IS PREPARED BY CENTRIFUGATION AT 10000rpm

FOR 20 MINUTES

CONTROL

Cisplatin GRP

Nanocurcumin GRP 1 (0.5mg/kg)

Treatment GRP 2(NC-1+cis)

Rat Brain

Nanocurcumin GRP 2 (1mg/kg)

Treatment GRP 1(NC-0.5+cis)

Assessment Parameters

Non-Enzymatic assays

• Non-Protein thiol• GSH Assay• TSH Assay

Biochemical Estimation

Lipid Peroxidation

Antioxidant Enzyme Assay Glytathion-S-

Transferase estimation

Reproductive toxcity

Hematological Estimations

Genotoxicity Estimations

Micronucleus Test

UV-Vis Spectroscopy

Absorbance maxima at 420nm

Fourier Transmission Infrared Microscopy

peaks at 3415 cm−1

corresponds to OH stretching vibrations

at 1,608 cm−1 were attributed to carboxylate OCO asymmetric stretching

at 1,588 cm−1 was assigned to C–OH deformation vibration with contribution of O–C–O symmetric stretching vibration of carboxylate group

(A)Figure shows the TGA of nanocurcumin and fig (B)shows the DSC of nanocurcumin

Differential Scanning Calorimetry & Thermogravimetric Analysis

first phase transition at 100.934ºC and the second phase transition at 440.699ºC

air dehydrated from 49 to 140 °C and the free curcumin in the product melted at ~ 250 °C

TEM images show sizes in the range of 10-20nm.

Transmission Electron Microscopy

GSH

Effect of different concentration of Nanocurcumin on cisplatin altered GSH level in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). It is measured as µmoles GSH/gm tissue. Significant difference are indicated by (*)when compared with control and (#) when compared with cisplatin group.

Effect of different concentration of Nanocurcumin on cisplatin induced depletion of non-protein thiols in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). It is measured as µmoles NP-SH/gmtissue.Significant difference are indicated by (*)when compared with control and (#) when compared with cisplatin group.

Non-Protein Thiol

Total ThiolEffect of different concentration of Nanocurcumin on cisplatin altered T-SH level in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). It is measured as mmoles TSH/gm tissue. Significant difference are indicated by (*)when compared with control and (#) when compared with cisplatin group

Lipid Peroxidation

Effect of different concentration of Nanocurcumin on cisplatin induced lipid peroxidation (LPO) in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). LPO is measured as µmoles TBARS formed/hr/gm tissue.

Glutathion-S-Transferase

Effect of different concentration of Nanocurcumin on cisplatin altered GST activity in brain of Male Wistarrats.Values are expressed as mean ± standard error (SE, n=6). It is measured as nmoles of CDNB conjugated/min/mg protein.

(E)

(D)(C)

(F)

(A) (B)

Micronucleus Test

(A) Represent normal bone marrow cells while (B) shows the micronucleated cells, (C&D) shows the normal cells,and (E&F)shows decline in MNs count with protection of nanocurcumin.

Dose Dependent Comparative representation of Micronucleus

Dose-dependent profile of Micronucleated Polychromatic Erythrocytes by Cisplatin and Nanocurcumin in Rattus norvegicus.

Total Sperm Abnormality

Fig (A) showing the normal sperm, (B,C,D) coiled tail, amorphous head, broken head, reversed head position, and coiled sperms showing the abnormality caused by the cisplatin.

(A) (B) (C)

(D)

Dose Dependent Comparative representation of Sperm Abnormalities

Frequency distribution of sperm abnormalities in cisplatin and Nanocurcumin treated R. norvegicus

Hematological Assessments

(A, C& D) showing the normal blood cells and (B,E&F) shows various kind of anomalies in blood cells of rat by the treatment of cisplatin.

(A) (B)

(C) (D)

(F)(E)

Inference From This Study?? Small sizes of Nanocurcumin enhanced its bioavailability.

Proved to be a potent candidate for the management of cisplatin

related side effects.

And Much More… Further we are also investigating liver, kidney and heart samples for

different analysis.

Also samples are being investigated for histopathological effects.

Comet assays are also being done.