SYNTHESIS OF MNPs

(30/01/25)

• PRINCILPLE :
• The synthesis of magnetic nanoparticles (MNPs) is based on
the co-precipitation technique.
• The method relies on the simultaneous precipitation of ferrous
(Fe²⁺) and ferric (Fe³⁺) ions in an alkaline medium.
• When FeCl₃·6H₂O (ferric chloride) and FeSO₄·7H₂O (ferrous
sulfate) are dissolved, they dissociate into Fe³⁺ and Fe²⁺ ions in
aqueous solution.
• Upon adding NH₄OH (ammonium hydroxide) dropwise, the
solution becomes alkaline, and Fe²⁺ and Fe³⁺ co-precipitate to
form magnetite (Fe₃O₄) nanoparticles.
SYNTHESIS OF MNPs
(30/01/25)

• MATERIALS REQUIRED :
• Ferric chloride hexahydrate (FeCl₃·6H₂O) – 50mM
• Ferrous sulfate heptahydrate (FeSO₄·7H₂O) – 25mM
• Deionized water (dH₂O)
• Ammonium hydroxide (NH₄OH), 30% solution
SYNTHESIS OF MNPs
(30/01/25)
• PROCEEDURE :
• 1. Preparation of Iron Salt Solutions:
• Dissolve 50 mM of FeCl₃·6H₂O in 15 mL of dH2O.
• Dissolve 25mM of FeSO₄·7H₂O in 15 mL of dH2O.
• Stir both solutions separately for 30 minutes to ensure
complete dissolution.
• 2. Mixing of Iron Solutions:Stir the mixed solution for
an additional 30 minutes.
• 3. Precipitation of Magnetic Nanoparticles:Add 30%
NH₄OH solution dropwise to the iron solution while
continuously [Link] the pH to 10 by carefully
adding NH₄[Link] nanoparticles will form at pH
10.
SDS-PAGE & Silver staing Based Detection
of Proteins Adsorbed on Nanoparticles
from Conditioned Media (06/01/25 &
07/01/25)

• Sample : Conditioned medium containing NPs of different

concentration , which has been centrifuged and washed.
• Purpose : Identification of Proteins Adsorbed on Nanoparticles from Cell
grown medium.
• Result : After silver staing Dark brown colour is appeared throughout the
gel . Destaing is performed to remove background stains . But failed to
Retain bands while removing the background stains.
• Entire gel is turned into clear .
• Reason : 1. Oversensitization
• 2. Overdevelopment
• 3. Staining condition (4 °C)
• 4. Light induced background staining
SDS-PAGE & Silver staing Based
Detection of Proteins Adsorbed on
Nanoparticles from Conditioned Media
(08/01/25)

• Sample : Conditioned medium

containg NPs of different
concentration , which has been
centrifuged and washed.
• Purpose : Identification of
Proteins Adsorbed on
Nanoparticles from Cell grown
medium.
• Result : Out of 6 wells only the
last 2 well showed bands. High
background staining .
• Reason ...
SDS-PAGE & Silver staing Based
Detection of Proteins Adsorbed on
Nanoparticles from Conditioned Media
(09/01/25 & 10/01/25)

• Sample : Conditioned medium

containg NPs of different
concentrations.
• Result : No bands optained ,
brown dense protein containg
areas present in each well.
• Background was clear.
• Reason : Overloading protein
samples during SDS-PAGE can
lead to smearing and poor
resolution in silver-stained gels.
Excessive protein content mig
ht result in smearing and blurr
y bands . (Rabilloud, T. 2012)
SDS-PAGE & Silver staing Based
Detection of Proteins Adsorbed on
Nanoparticles from Conditioned Media
(13/01/25 & 14/01/25)

• Sample : Conditioned medium

containg NPs of different
concentration.
• Result : No bands optained ,
brown dense protein containg
areas present in each well.
• Background was clear.
• Reason : Overloading protein
samples during SDS-PAGE can
lead to smearing and poor
resolution in silver-stained gels.
Excessive protein content mig
ht result in smearing and blurr
y bands . (Rabilloud, T. 2012)
Investigation of Human Serum Albumin
(HSA) Interaction with Nanoparticles of
Varying Concentrations.
(20/01/25 & 21/01/25)

• Sample : 10 µl 0.1mg/ml HSA with

different concentration of NPs (25ml)
• In 24 hr incubation 2 methods
followed
• [Link] with SSB,centrifuge , and
supernatant added (1:1) BAND INTENSITY
• [Link] heating , pellets added. BAND INTENSITY
7445.836
• Result : Bands are obtained only the 6548.894
method 1 showed high intensity
bands.
• Note : In all well The NPs
Concentration is directly proportional 2683.752
1811.024
to band intensity except the heated
samples, which shows inverse 87.192 59.778
proportionality . 1hr (750) 1hr (250) 24hr (750) 24hr (250) 24hr Htd 24hr Htd
(750) (250)
Why Does HSA Produce Two Bands in SDS-
PAGE After Incubation with Nanoparticles?

• HSA is a single polypeptide chain (~66 kDa), so

under ideal conditions, it should give a single
band in SDS-PAGE.
• 1. Partial Denaturation of NP-Bound HSA :
• One band corresponding to free, fully denatured
HSA (~66 kDa)
• A second band for partially denatured or modified
HSA.
• Reference: Dobrovolskaia & McNeil (2007)
showed that NP-protein interactions can lead to
altered protein electrophoretic mobility in SDS-
PAGE.
• 2. HSA Fragmentation or Proteolysis:
• Heating at 95°C (or above) in the presence of reducing agents
(β-Mercaptoethanol) can destabilize HSA.
• This may weaken intramolecular interactions, causing: Cleavage
at structurally weak regions.
• 3. Protein Corona Structural Variants:
• When nanoparticles interact with proteins, they can induce
conformational changes due to hydrophobic interactions,
hydrogen bonding, or electrostatic forces.
• This results in different structural variants of HSA, which
migrate differently in SDS-PAGE.
• Reference: Monopoli et al. (2012) demonstrated that
nanoparticle-induced protein corona formation leads to different
protein conformations, affecting electrophoretic migration.
Investigation of HSA Interaction with
Nanoparticles of Varying Concentrations.
(27/01/25 & 28/01/25)

• Sample : 100 µl 0.1mg/ml HSA with

different concentration of NPs (500ml
final Volume)
• PBS used = EDTA based.
• Heat with SSB,centrifuge , and
supernatant added (44 µl sample
and 6 µl SSB )
• 6x loading dye used
• Well size increased
• Result : During comb removal the
wells are deformed because of it’s
increased size, making it difficult to
load sample properly. There is a
Concentration dependend increase in
the darkness of streaks.
 (03/01/2025)
• Control , 250 , 500 , 750 , 1000.
• 250 µg/ml, the band intensity is high
compared to the trend, meaning it does not
follow a smooth increase as seen in other
concentrations.
• Sample washed 3 times BAND INTESITY
BAND INTESITY

123169.566

107819.222
102648.122

91011.746

17937.848

control 250 500 750 1000

 (07/02/25)

• Here band intensity inversely proportional to

the concentration . ( Both MF and Normal
conditions )
• Sample centrifuged and washed 3 times
BANDINTENSITY
BANDINTENSITY
23630.111
22517.182
20117.271
19487.149

12402.04
10895.614

275.678

control MF 250 MF 750 250 500 750 1000

(19/02/15)

• 4x washing

• Band intensity
directly BAND INTENSITY
BAND INTENSITY

proportional to
concentration of
15844.472

NPs

6086.903

3302.146 3243.004
2510.033
1216.326
265.021

control MF 250 MF 750 250 500 750 1000

INDUCED HAEMOLYSIS USING
PHENYLHYDRAZINE
(03/03/2025)
• Phenylhydrazine (C₆H₅NHNH₂) is an aromatic hydrazine compound
known for its oxidative stress-inducing properties in biological
systems.
• Phenylhydrazine interacts with hemoglobin (Hb), oxidizing
ferrous iron (Fe²⁺) to ferric iron (Fe³⁺), forming
methemoglobin (MetHb) (Winterbourn, 1985).
• Phenylhydrazine undergoes autoxidation in vitro,
generating reactive oxygen species (ROS) like superoxide
(O₂⁻) and hydrogen peroxide (H₂O₂). These ROS oxidize
Fe²⁺ in hemoglobin to Fe³⁺, forming methemoglobin
(MetHb) (Gutteridge, 1982).
• Since MetHb cannot bind O₂, hemoglobin’s oxygen
transport function is impaired.
• Oxidative damage weakens RBC membranes, making
them more susceptible to hemolysis.
• ROS attack RBC membrane lipids, initiating lipid
peroxidation and disrupting the phospholipid bilayer.
• Lipid peroxidation products, such as
malondialdehyde (MDA), further degrade membrane
integrity (Siems et al., 2000).
• Phenylhydrazine modifies hemoglobin, leading to
denaturation and aggregation into Heinz bodies (Reilly et
al., 1991).
• Heinz bodies are insoluble protein aggregates that bind to
the inner membrane, impairing RBC deformability and
increasing splenic clearance (Jacob & Winterbourn, 1987).
 (07/03/25) W/O
(04/03/25) W/O MF MF

07-03-2025 W/O
04-03-2025 W/O MF MF
PC (PhNHNH2) 100 100
NC (PBS) 0 0
250
(PhNHNH2) 4.747 11.451
1000
(PhNHNH2) 7.937 30.645
250 control 0.828 1.595
1000 control 2.431 2.028
(04/03/25 )&
(07/03/35) Without
MF
PC NC
250

1000 07-03-2025 W/O

04-03-2025 W/O MF MF
PC (PhNHNH2) 100 100
NC (PBS) 0 0
250 (PhNHNH2) 4.747 11.451
1000
(PhNHNH2) 7.937 30.645
250 control 0.828 1.595
1000 control 2.431 2.028
PC ( DH20 ) 100 100
(07/03/35) With
MF

PC NC
250

1000 07/03/25 MF
PC (PhNHNH2) 100
NC (PBS) 0
250 (PhNHNH2) 10.96
1000
(PhNHNH2) 25.12
250 control 20.523
1000 control 27.346
PC ( DH20 ) 100
 INFERENCE :
• 250 µg/mL CCIONPs: Showed minor hemolysis.(Even in the
presence of PhNHNH2)
• 1000 µg/mL CCIONPs: Showed slightly higher hemolysis but
still much lower than the positive control .
• Curcumin is a strong antioxidant that scavenges ROS, which
could counteract the oxidative stress caused by
phenylhydrazine.
• Curcumin also prevents lipid peroxidation in RBC membranes.
• IONPs have been reported to possess antioxidant properties ,
They exists in Fe²⁺ and Fe³⁺ states, which can stabilize free
radicals under certain conditions.
• At higher concentrations (1000 µg/mL), the iron oxide may
enhance oxidative stress. Resulting heamolysis.
DPPH DEGRADATION ASSAY (Cur-
IONPs)
DPPH DEGRADATION ASSAY
(Cur-IONPs)-(11/02/25)

Degradation
11/02/25 %
250 25.699
500 40.31
750 48.49
1000 69.53

0
DPPH DEGRADATION ASSAY
(Cur-IONPs)-(13/02/25)

Degradation
13/02/25 %

250 53.030

500 82.191

750 91.582

1000 95.133
DPPH DEGRADATION ASSAY
(Cur-IONPs)-(14/02/25)

Degradation
14/02/225 %

250 56.467
500 76.949
750 82.339
1000 91.692
DPPH DEGRADATION ASSAY
(Cur-IONPs)-(14/02/25) &
(13/02/25)

13/02/2514/02/225

250 53.030 56.467

500 82.191 76.949

750 91.582 82.339

1000 95.133 91.692

 INFERENCE :
• DPPH degradation assay using curcumin-conjugated iron
oxide nanoparticles showed a proportional increase in
degradation percentage with increasing nanoparticle
concentration.
• Curcumin is a well-known antioxidant due to its phenolic
hydroxyl groups (-OH), which can directly scavenge free
radicals.
• Cur-IONPs should show higher DPPH degradation than
plain IONPs at the same concentration.
• References: (Ghosh et al., 2021)
DPPH DEGRADATION ASSAY
(MNPs)
DPPH DEGRADATION
ASSAY
(MNPs)-(25/02/25)

25/02/25. 25/02/25.
(1) (2)
250 4.900 26.770
500 20.986 28.590
750 25.789 16.115
1000 44.648 44.854
DPPH DEGRADATION
ASSAY
(MNPs)-(11/03/25)

11/03/25.
250 14.367
500 28.322
750 32.857
1000 51.887
 DPPH DEGRADATION
ASSAY
(MNPs)-
(25/02/25)&(11/03/25)

25/02/25.
(1) 11/03/25.
250 4.900 14.367
500 20.986 28.322
750 25.789 32.857
1000 44.648 51.887
 INFERENCE :
• MNPs showed a proportional increase in degradation
percentage with increasing nanoparticle concentration.
This trend suggests that the nanoparticles play a crucial
role in scavenging DPPH radicals.
• This NPs have intrinsic antioxidant properties due to
their ability to undergo redox cycling between Fe²⁺ and
Fe³⁺.
• Fe²⁺ donates an electron to the DPPH radical (DPPH˙) and
converts it into a non-radical (DPPH-H) form.
• Higher nanoparticle concentrations provide more Fe²⁺
sites for this redox reaction, leading to increased
degradation.
• References: (Zhang et al., 2019)