1.
Name of Institute:- NDRI Karnal
Project 1:Title:- Biosafety of Nanomaterials
Name of the PI:- Dr. Gautam Kaul
Main objectives: To test a representative set of manufactured nanomaterials (Carbon
nanotubes and nanoparticles) for safety analysis in vitro and in vivo system.
Report:
Nanotechnology policy worldwide aims to ensure that nanotechnology is developed in a
responsible way. The opportunities offered by nanotechnology research should be enabled,
while at the same time possible negative effects or negative perceptions of this new technology
should be appropriately managed. The biosafety of increasing use of nanofertilizers and
nanopesticides in Agriculture is at best obscure. In USA, Environmental Protection Agency
(EPA) along with Food and Drug Administration (FDA) regulates nanotechnology products
under existing statutory authorities. In Europe, REACH (Registration, Evaluation,
Authorisation and Restriction of Chemicals and Classification) controls nanosafety measures.
Asia-Pacific has its own agencies for controlling the safety of NMs. In Australia, the National
Industrial Chemicals Notification and Assessment Scheme (NICNAS) control NMs safety. In
India, there is as such no agency decidated towards nanosafety of NMs. The Organisation for
Economic Co-operation and Development (OECD) provides guidance through documents on
characterization, dosimetry & risk assessment issues of NMs. American Society for Testing
Materials (ASTM) Standards on NM characterization, environmental safety, informatics and
terminology. The European Union Reference Laboratory (EURL) for alternatives to animal
testing provides in vitro test recommendations. Considering all these, the nanosafety of
nanomaterials is mandatory for the responsible use of nanotechnology. Many mammals get
exposed to these nanomaterials, which can reach almost every cell of the mammalian body,
causing the cells to respond against nanoparticles (NPs) resulting in cytotoxicity and/or
genotoxicity. The important key to understand the toxicity of nanomaterials is that their minute
size, smaller than cellular organelles, allows them to penetrate the basic biological structures,
disrupting their normal function. There is a wealth of evidence for the noxious and harmful
effects of engineered NPs as well as other nanomaterials. The rapid commercialization of
nanotechnology field requires thoughtful, attentive environmental, animal and human health
safety research and should be an open discussion for broader societal impacts and urgent
toxicological oversight action.Several classes of nanomaterials have emerged today as
principal agents for targeted drug delivery, disease diagnostics and therapeutics. Plenty of
evidence exists for them addressing their beneficial and adverse effects. As a consequence,
confusion has perpetuated regarding their use in context of their size, shape, form and route.
Considering these issues, we designed a small study intended to clarify what is meant by toxic
nature with respect to class, size, shape, route of nanomaterial and how. We took two
nanomaterials namely Silica Nanoparticles(SN) and Multiwalled carbon Nanotubes, MCNTs
and compared their in vivo toxicity taking albino mice as an animal model. Presently,
conflicting data regarding behavior of these nanomaterials with macromolecules like protein
and lipid at cellular level in cell lines as well as in animal models generated interest within us
to appreciate them. The mice were treated orally with the single dose of 50 ppm MWCNTs and
intraperitonially with10 mg/kg, 25 mg/kg and 50 mg/kg of BW of MSNs and 1.5 mg/kg, 2.0
mg/kg and 2.5 mg/kg BW of MWCNTs. Pattern of liver enzyme markerslike AST, ALT, ALP
in serum along with total protein level were evaluated after 7 days post exposure. Changes in
organ coefficient of different organs were statistically insignificant at all doses. No significant
differences in enzyme levels were observed among different doses but with control. Of three
enzymes assayed, AST displayed a peculiar pattern especially in MWCNTs (IP) treated group.
Total protein level was high in orally treated MWCNTs group. The results showed that
MWCNTs even at much smaller doses than SNs displayed similar toxicity levels, based on
which
it
was
concluded
that
toxicity
of
MWCNTs
is
higher
than
SNs.
*
*
*
*
*
*
*
*
*
Fig : Liver injury marker enzyme levels in
serum after exposure to single dose of
MWCNTs at varying concentrations (1.5, 2.0
and 2.5 mg/kg) on 7th day after exposure. A.
Alanine aminotransferase (ALT). B. Aspartate
aminotransferase
(AST).
C.
Alkaline
phosphatase (ALP). Bars with different
superscripts indicate significant difference
(p<0.05).
Fig : Liver enzyme markers and total protein level in mice
serum following oral dosing of MWCNTs at 50 ppm. Bars with
different superscripts indicate significant difference (p<0.05).
This statement is well supported by data generated which shows that all liver enzymes
assessed in this work were raised even at lower doses of MWCNTs as compared to
significantly higher doses of MSNs. The fig shows values of different enzymes and
total protein assayed under different groups. MWCNTs when administered through IP
route and has been found to accumulate specifically inside Kupffer cells in hepatic
tissue. Liver is the primary organ responsible for metabolism and excretion of drugs
and molecules through its well established hepatic reticuloendotheilal system.An
overall result analysis of our present work also portrays MWCNTs being placed at a
higher toxicity level as compared to SNs. In the event of damage to hepatic cells there
is a consequent increase in hepatic enzymes which have been shown to be markers
for liver damage .This hepatic toxicity have been found to be a combined result of
recognition ability of Kupffer cells for MWCNTs as well as slow clearance rate which
further leads to persistence of these nanomaterials in liver for a longer period of time.
Continued persistence in liver leads to aggregation which has been observed to be
the primary reason for toxicity and at the same time explains about their slow
biodegradation. All liver enzymes assessed in the present work showed significant
increase when compared to no treatment group. Of these AST exhibited a particular
pattern. In case of ALT and ALP, although there was significant difference between
control and treatment values but the treatment values were more or less within normal
physiological range. But AST values in treatment groups were way above the normal
values. AST is found throughout the body at many sites where it acts as an indicator
for tissue or cellular damage. The greatest concentrations are found in heart muscle,
followed by liver, skeletal muscle, kidney, and brain in decreasing order. Following
damage to these tissues, increased amounts of AST are released and enter the
bloodstream. As per routineclinical diagnosis, AST elevations are most often seen in
heart and liver pathological states like acute myocardial infarction, congestive heart
failure, pulmonary infarction etc. AST is a sensitive marker for liver as well as smooth
muscle damage and is used in both diagnosis and monitoring of liver disease. Its
increase in nanomaterial exposed mice confirms its efficacy as a potent serum
biomarker for liver. These findings will surely put an impetus to the ever growing
interest in the current as well as future researchers to search for novel and concrete
means of addressing the antagonistic effects of nanomaterials but at the same time
enhancing their beneficial features.
Project 2;Title:- Nano encapsulation of Functional Ingredients for their Delivery.
Name of the PI:- Dr.(Mrs) Bimlesh Mann.
Main objectives:

To develop various types of nano delivery system encapsulating bioactive
components and micronutrients
 To study the interaction and compatibility of the nanostructured materials with
food matrix in the model food systems.
 To assess the bioactivity and safety aspects of the delivery system
Report:
Clove and thyme oils have been described as having useful pharmacological
properties. Their antimicrobial activity is well established and has been attributed to a
number of typicalsubstituted aromatic molecules, such as eugenol and thymol.
Micro/nano encapsulation of bioactive molecules helps easily incorporation into the
food systems, recovers their poor solubility, sensitivity to processing conditions and
adverse effects on the sensory attributes of foods. In the present investigation, clove
oil and thyme oil are encapsulated in the form ofoil in water (O/W) nanoemulsionsby
usingultrasonicator and high speed homogenizer.The clove oil and thyme oil were
taken as the inner oil phase (O) (1-10% w/v) and the outer aqueous phase (W) was
prepared by mixing different emulsifiers such as Whey Protein Concentrate (WPC),
maltodextrin inmillipore water. Nanoemulsion prepared by sonification using 1% clove
oil and 1% Tween-80 (particle size- 39.58 ± 5.25nm) was stable up to 8 months at
25℃.The clove oil nanoemulsions prepared by homogenization using whey protein
concentrate-maltodextrin conjugate showed particle size, zeta potential and poly
dispersity index were 199.2±2.88nm,
-36.46±0.48mV, 0.139±0.02 . The
nanoemulsions were stable to different food processing conditions like heat
treatments, different ionic strengths (0.1-1M) and pH ranging from 3.0-7.0.The
emulsion was also stable during freeze drying.Scanning electron micrographs and
transmission electron micrographs of the stable nanoemulsions confirmed the
spherical structure of emulsion droplets. The antimicrobial activity of the prepared
nanoemulsions were measured against E. coli, Bacillus subtilis, Candida lipolytica,
Aspergillusflavus,Salmonella typhiimurium, Listeria monocytogens, E. coli O157:H7,
and Shigella. It was observed that Minimum Inhibitory concentration (MIC) of stable
formulations were 1% (50µL) in case of all tested microorganisms and 0.6% (30µL) in
Salmonella typhiimurium, Candida lipolytica, andAspergillusflavus.Six log cycle
reductions was observed on addition of 0.8% and 1% concentration of nanoemulsion
after 4h and 8 h of incubation with Bacillus subtilis and E. coli, respectively. Most stable
formulation also showed similar minimum inhibitory concentration after freeze
drying.The paneer was dipped in antimicrobial nanoemulsions to assess the suitability
of this emulsion as a delivery medium.Four log cycle reductions in microbial count was
observed in treated paneer as compared to control duringstorageat 5℃and thereby
increased their shelf life from 6 days to 15 days.Hence, these nanoemulsions system
can be used as an effective delivery system for poorly soluble bioactive components.
Project 3:Title:- Structural and Functional Characterization of Exosome nanoparticle of
Buffalo Milk as a Model for Potential Application as Biomarkers and for
Developing New Cell Delivery System.
Name of the PI:- Dr. Dheer Singh
Main objectives:

Isolation and structural characterization of milk exosomes

Biochemical and molecular composition of exosomes from buffalo milk

Functional characterization of isolated exosomes in intracellular communication
using appropriate cell lines

Feasibility study to encapsulate molecules like siRNA as well for developing
nano formulation of traditional herbal medicines/therapeutics.
Report:
Progress: Milk is a natural nutraceutical produced by mammals. The nanovesicles of
milk play a role in horizontal gene transfer and confer health-benefits to milk
consumers. These nanovesicles contain miRNA, mRNA and proteins which mediate
the intercellular communication. During the period, we isolated and characterized the
buffalo milk-derived nanovesicles by dynamic light scattering (DLS), nanoparticle
tracking analysis (NTA), scanning electron microscopy (SEM), Western probing and
Fourier transform infrared (FTIR) spectroscopy. The DLS data suggested a bimodal
size distribution with one mode near 50 nm and the other around 200 nm for the
nanovesicles. The NTA and SEM data also supported the size of nanovesicles was
within a range of 50-200 nm.
Scanning electron microscopy of
fixed and dehydrated and gold
coated exosomes on glass
substrate from milk
CD81
Western probing of exosomes from milk using cd81 antibody
The buffalo milk-derived nanovesicles ranged from 50-200 nm comprising proteins, lipids,
polysaccharides and nucleic acids, including immune related miRNAs robust to household
milk storage methods.