What is Nanotechnology?What are Nanoparticles?What are the properties of Nanoparticles they should possess for Agricultural applications?Methods of production of Nanoparticles?Types of Nanoparticles based on Origin and Composition? Applications of Nanotechnology in Agriculture?

1. WELCOM
E Presentation by
G Mallikarjuna,
BAM/19-13.

2. NANOTECHNOLOGY and
It's applications in crop improvement
programmes

3. Contents
• What is Nanotechnology?
• What are Nanoparticles?
• Properties of NP's for Agricultural applications
• Types of NP's
• Methods of production
• Applications in Agriculture
• Case study

4. What is Nanotechnology?
• Nanotechnology is the manipulation or self-assembly of individual atoms, or
molecules, or molecular clusters into structures to create materials and devices
with new or vastly different properties.
• It is related to materials,systems and processes which operate at a scale of 100
nanometers (nm) or less.
• The first ever concept of nanotechnology was presented in 1959 by the
famous professor of physics Dr. Richard P.Feynman.
• The term "Nano-technology" had been coined by Norio Taniguchi in 1974.
Shaima et.al,2015.

5. What are Nanoparticles ?
• Nanoparticle is defined based on the size at which fundamental characters
different from those of the corresponding bulk material .
• In this scale, physical, biological and chemical characteristics of materials are
fundamentally different from each other and often unexpected actions are seen
from them.
• Size of Nanoparticles is 1 to 100 mm.
• A nanometer is one billionth of a meter.
• For comparison, the wavelength of visible light is between 400 nm and 700
nm.A Leukocyte has the size of 10000 nm, a bacteria 1000-10000 nm, virus 75-
100 nm, protein 5-50 nm, glucose 1nm, deoxyribonucleic acid (DNA) ~2 nm
(width), and an atom ~0.1 nm.
Waleed Fouad
Abobatta,2018.

7. The properties of nanomaterial for agricultural
applications
1. Providing actual concentration .
2.Controlled release of fertilizers or pesticides in response to certain conditions.
3. Improved targeted activity.
4. Lower Eco harmful with safe and relaxed transport.
Waleed Fouad Abobatta,2018.

8. Types of Nanoparticles based on Composition
NPs types are divided in two main groups: organic and inorganic NPs.
1.Organic NP'S:The NP'S which are prepared with organic elements are
called Organic NP'S.
Eg:- Micelles, Dendrimers, Liposomes, Hybrid and compact polymeric NPs.
2.Inorganic NP'S:The NP'S which are prepared with inorganic elements are
called inorganic NP'S.
Eg:- Fullerenes, Quantum dots, Silica and Gold NPs.

9. Typesof
Nanoparticles

11. Types of Nanoparticles based on Origin
1.Natural Origin :
Nanoparticles are generated naturally by erosion, fires, volcanoes and marine
wave action.
2.Engineered NPs (ENPs) :
Nanomaterials that are intentionally produced and designed with specific
properties related to their shape, size, surface properties, and chemistry.
3.Man-made :Nanoparticles are also produced by human activities such as
coal combustion, vehicle exhaust and weathering rubber tires.

12. Methods of Nanoparticle Production
1.Top-down” systems:
• Where tiny manipulations of little number of atoms or molecules fashion
elegant patterns, through mechanical-physical methods like grinding, milling
and crushing for producing nanoparticles.
• This method use for producing Nano composites and Nano-grained bulk
materials like metallic and ceramic nanomaterials in extensive size distribution
(10 - 1000 nm) .
2.Bottom-up system:
• In ‘Bottom-up’ building up, numerous molecules self-assemble in parallel steps,
as a function of their molecular recognition characters, this processing produces
more complex structures from atoms or molecules.
• This method produce a uniform controlling sizes, shapes and size ranges of
nano materials.

13. • Waleed Fouad Abobatta,2018.
Top-down
System
Bottom-up
Systen

14. Applications of Nanotechnology in Agriculture
Nanotechnology
in
Agriculture
1.Crop improvement
2.Soil management
3.Water management
4.Post harvest
technology
5.Plant disease diagnosis
6.Bioprocessing
7.Precision farming
9.Monitering the identity
and quality of
Agricultural produce
8.Nanofertilizers
and
Nanoherbicides

16. Applications of Nanotechnology in Crop improvement
programmes
• Crop improvement refers to the genetic alteration of plants to satisfy human needs.
• Nanotechnology has the practical application in crop improvement through gene
transfer mediated by Np's/Nano devices.
• Nanoparticles which are involved in gene transfer are viz.,Gold NP's,Starch
NP's,Mesoporous silica NP's,Carbon Nanotubes(CNT).
• Types of Gene transfers using Nanoparticles :
1.Polymer based gene transfer
2.Liposome based gene transfer
3.Biobeads based gene transfer

17. 1.Polymer based gene transfer :
• Cationic polymers with physiological pH can be combined with DNA or
siRNA through electrostatic attractions and reduce the size of DNA and/or
RNA into nano-sized particles and also providing protection and promoting
cellular uptake.,thereby facilitating gene delivery.
• Currently, several important polymers are being used as vectors for gene
delivery.
• These include PEI(Polyethylene imine), chitosan, polyamidoamine, gelatin, and
cationic polysaccharides

19. 2.Liposome based gene transfer :
• Liposomes are artificial phospholipid vesicles used for the delivery.
• They can be preloaded with DNA by two common methods- membrane
membrane fusion and endocytosis thus forming DNA- liposome complex.
• This complex fuses with the cell membrane of target cell and to release the
contents into the cell.
• Animal cells. plant cells, bacteria, yeast protoplasts are susceptible to
lipofection method.

20. Liposome based gene
transfer

21. •3.Biobeads based gene transfer :
• Micrometer-sized calcium alginate beads referred to as "bio-beads" that
encapsulate plasmid DNA molecules carrying a reporter gene.
• Ca-alginate nanoparticles with an average size around 80 nm in diameter are
very efficient gene carriers, in comparison with widely-studied PEI/DNA
complexes.
• NIH 3T3 cells in the exponential growth phase were detached with 1X
EDTA-trypsin (Irvine Scientific, Santa Ana, CA) from a culture dish (Falcon,
Franklin Lakes, NJ).
• A totalof 5×104 NIH 3T3 cells were inoculated on a plastic petri dish(35 mm
in diameter) with 2 mL of high-glucose Dulbecco’s modified Eagle medium
(DMEM, Mediatech Inc., VA) supplemented with 10% fetal bovine serum
(FBS, Irvine Scientific).

22. • After 24-h incubation, Ca-alginate nanoparticles encapsulated with 2 µg
plasmid DNA encoding eGFP(plRES2-EGFP vector, Clontech, Palo Alto, CA)
were suspended in 2 mL of growth medium and layered on top of the NIH 3T3
cells which were attached on the bottom of the culture dish.
• NIH 3T3 cells exposed to pDNA encapsulated Ca-alginate nanoparticles were
incubated for transfection.
• All cellcultures were performed at 37°C and balanced with 5% CO2 in a 100%
humidified incubator.

23. Hazards :
• Nanoparticles as pesticides, fertilizers or in other formulations, when air-borne,
may deposit on above ground parts of plants.They may plug stomata and create
a fine physical and toxic barrier layer on stigma preventing pollen tube
penetration. They may also enter the vascular tissue and impair translocation of
water, minerals and photo synthates.
• Animals may inhale nanoparticles resulting into various ill effects and
disorders. The particles may enter the bloodstream.
• They may also create new kinds of contamination of soils and waterways due
to enhanced transport, longer persistence and higher toxicity.
• Air-borne nanoparticles present some specific hazards for human health; they
may enter the body through the respiratory system.
• Due to the entry of nanoparticles into lungs and blood stream, there is
possibility of inflammation, protein fibrillation and induction of genotoxicity.

24. Case study

26. Introduction :
• It has been demonstrated that the clinical risks of using viral vectors for gene
therapy are obstacles for their practical use even though virus-mediated gene
delivery offers high transfection efficiency.
• As a result, much attention has been focused on the design of synthetic
cationic vectors for nucleic acid condensation and delivery.
• Despite the promise of developing clinically useful nonviral vectors, most
nonviral gene carriers provide less efficient gene transfer, especially in escape
from endosomal vesicles.
• In this study, we used Caalginate nanoparticles as gene delivery vehicles due
to biocompatible and biodegradable features of Ca-alginate.
• Moreover, once ingested via endocytosis pathways, they can undergo quick
erosion and elicit osmotic swelling, hence facilitating endosomal escape of
gene to cytosol.

27. Materials and Methods :
Preparation of Ca-alginate Nanoparticle :
• First, 0.25 g of AOT (Sigma) was dissolved into a vial pre-filled with 13.11 g of
toluene.
• Then, 0.5% sodium alginate solution (medium viscosity; viscosity of 2% solution
is approximately 3,500 cps at 25°C, Sigma) was added dropwise into the vial.
• Various phase appearance (from transparency to turbidity) was obtained during
the process of adding alginate solution.
• For each distinct phase,the mixture was vortexed and waited for 10 minutes prior
to the addition of 2 mL of filtered calcium chloride solution (2% by weight) for
obtaining alginate-based nanodroplets by viture of crosslinking.
• After carefully washed with acetone and deionized water to remove all residual
AOT and toluene, the vial was centrifuged (33,000 ×g) for 30 min and a small
white pellet of material was obtained.

28. • Finally, the prepared nanoparticles were re-suspended with deionized water.
• The procedure of encapsulating plasmid DNA into Ca-alginate nanoparticles
was the same as the one just described above, except pDNA was pre-mixed
with 0.5% sodium alginate solution
Transfection of NIH 3T3 Cell :
• NIH 3T3 cells in the exponential growth phase were detached with 1X EDTA-
trypsin (Irvine Scientific, Santa Ana, CA) from a culture dish (Falcon, Franklin
Lakes, NJ).
• A total of 5×104 NIH 3T3 cells were inoculated on a plastic petri dish(35 mm
in diameter) with 2 mL of high-glucose Dulbecco’s modified Eagle medium
(DMEM, Mediatech Inc., VA) supplemented with 10% fetal bovine serum
(FBS, Irvine Scientific).

29. Contd...
• After 24-h incubation, Ca-alginate nanoparticles encapsulated with 2 µg
plasmid DNA encoding eGFP(plRES2-EGFP vector, Clontech, Palo Alto, CA)
were suspended in 2 mL of growth medium and layered on top of the NIH 3T3
cells which were attached on the bottom of the culture dish.
• NIH 3T3 cells exposed to pDNA encapsulated Caalginate nanoparticles were
incubated for transfection.
• All cell cultures were performed at 37°C and balanced with 5% CO2 in a 100%
humidified incubator.
• For comparison, transfection of NIH 3T3 cells was also performed using
PEI/DNA complexes (MW of PEI = 750 kDa; N/P ratio = 6).

30. Contd...
Statistical Analysis :
• All of the experimental data were obtained in triplicate and presented as mean
± standard deviation.
• Statistical comparison by the analysis of variance was done at a significance
level of P < 0.01 based on the Student’s t-test.

31. Result and discussion :
• To examine the efficacy of Ca-alginate nanoparticles in facilitating endosomal
escape of pDNA, NIH 3T3 cells were incubated with nanoparticles obtained
from the reverse microemulsion template (i.e., point A of Figure 1) for 24, 48
and 72 h,respectively.
• Percentages of eGFP-expressing NIH 3T3 cells were determined by both
bright-field and fluorescent microscopy (see Figure 3-I).
• For comparison,transfection of NIH 3T3 cells was also performed using
PEI/DNA complex (see Figure 3-II) which has been known for its capability of
enhancing pDNA release fromendosomes.
• As shown in Figure 3, the discrepancy of transfection rate between using
PEI/DNA complexes (53%)and pDNA encapsulation in Ca-alginate
nanoparticles (8.5%) was prominent after 24-h incubation.

32. • However, after 48-h incubation, the transfection rate using Ca-alginate
nanoparticles encapsulated with pDNA was greatly increased up to 48%, in
comparison with the one using PEI/DNA complexes (55%).
• The reason causing such dramatic enhancement of transfection rate is probably
because endosomal swelling, elicited by Ca-alginate nanoparticles, reaches its
peak after 48-h incubation,hence expediting the release of encapsulated pDNA
into cytosol.
• Transfection rates were also quantitatively determined by measuring the
intensity of eGFP expression in cells using a fluorimetric microplate reader
with an excitation wavelength of 472 nm and emission wavelength of 512
nm(shown inFigure 4).
• This result was consistent with the one given in Figure 3.

33. Conclusion :
• The size of Ca-alginate nanoparticles was significantly modulated by the ratio
of toluene, AOT, and sodiumalginate solution.
• Ca-alginate nanoparticles with an average size of 80 nm were able to
encapsulate plasmid DNA and ferry the gene into non-phagocytic cells via
endocytosis pathway.
• It seems that the alginate-based gene carriers can assist DNA escape within 1-2
day timeframe from the endosomal lumen into the cytoplasmic space, leading to
high transfection efficiency.

34. THANK YOU
"Next big thing is very Small"...