EXPERIMENTAL SET-UP

Succinylation of Soy protein isolate (SPI)

Synthesis of SSPI nanoparticles


Drug loading


Coating of chitosan onto bifenthrin loaded NPs


Characterization


In-vitro Release profile experiment


Box test (In-vivo termite detection)





Preparation and optimization of soy protein isolate nanoparticles
Synthesis of SPI nanoparticles: First the SPI will be dispersed in a pH 9 buffer, followed
by the addition of succinic anhydride. After surface modification with succinic anhydride, the
SPI solution will be passed through a dialysis membrane overnight to remove the excess of
succinic anhydride and samples will then be freeze dried. The freeze dried particles instantly
form nanoparticles (Fig. 2) when added to an aqueous solution such as water. By changing the
succinylation ratio, a variety of different sized particles will form and will then be analysed by
TEM, SEM, DLS and a zetasizer.

Aim 2. Loading and in-vitro release of bifenthrin using optimised
nanoformulations
Bifenthrin will be loaded into SPI nanoparticles using the adsorption method. 100mg of
Bifenthrin will be added to ethanol to form a solution. To the above solution, 100mg of SPI
nanoparticles will be added and then stirred for 24 hours. Particles will then be centrifuged and
the supernatant will be analysed for Bifenthrin content. The SPI loaded with Bifenthrin will then
be dried in a vacuum and stored at RT for further use.

Aim 3. Coating of Chitosan onto bifenthrin loaded nanoparticles to prepare
stable nanosuspension
As shown in Scheme 1, Bifenthrin loaded nanoparticles will be added to a positively charged CS
solution which will form instant chitosan coated nanoparticles. These nanoparticles will be
separated and tested for their bifenthrin content and the optimised formulation will be used in the
in-vivo box test.

Aim 4. I n-vivo termite detection using box test (designed by FMC) of
optimised formulation
FMC will carry out an in-vivo box test on the optimised nanoformulations using Coptotermes
acinaciformis, a termite widely spread throughout Australia. As depicted in Scheme 1, optimised
nanoformulations will be sprayed into a wooden box. The termites will then be given entry to the
box. If a termite passes from one end to the other without detecting the toxic bifenthrin it will
indicate the effectiveness of these nanoformulations. This test will also give us an idea as to
whether our formulation needs altering or not.



SEM ANALYSIS

Succinylated Soy protein Nanoparticles in the ratio of 1:1 which is freeze dried for
2 hours and dispersed in water for the SEM observation.


5% of Bifenthrin loaded in SSPI Nanoparticles in the ratio of 1:1, which is
dispersed in the ethanol as a solvent for the SEM analysis.




SOY PROTEIN

Synthesis of SPI nanoparticles: First the SPI will be dispersed in a pH 9 buffer, followed by
the addition of succinic anhydride. After surface modification with succinic anhydride, the SPI
solution will be passed through a dialysis membrane overnight to remove the excess of succinic
anhydride and samples will then be freeze dried. The freeze dried particles instantly form
nanoparticles (Fig. 2) when added to an aqueous solution such as water. By changing the
succinylation ratio, a variety of different sized particles will form and will then be analysed by
TEM, SEM, DLS and a zetasizer.

AIM : Obtain Succinylated Soy Protein (SSPI) in different ratios.
PROCEDURE:
Take 200mg of Soy Protein Isolates (SPI) dissolve in 20ml of PBS
Take 100mg, 200mg, 300mg and 400mg Succinic anhydride to obtain different ratios
1:0.5, 1:1, 1:1.5 and 1:2 respectively by dissolving in PBS.
Maintain the pH between 8-9 by adding 2M NaOH and stirring continuously
Stabilize pH to 8.5
Dialyse the solution in nanopure water for 24hr at 4C.
Freeze drying for 2 days.
S.No Soy Protein Isolates (mg) Succinic anhydride (mg) Ratio Obtained
1. 200 100 1:0.5
2. 200 200 1:1
3. 200 300 1:1.5
4. 200 400 1:2

AIM 2: Analyse the different sized particles and Zeta potential using DLS.
PROCEDURE:
Take 0.5 mg of SSPI of different ratios produced.
Mix the SSPI in 2ml of water and Bifenthrine in ethanol (As Bifenthrine is least soluble
in water) and analyse the particle size and Zeta potential using DLS.
Mix bifenthrin and SSPI in ethanol and process the sample using rotavapour .
Disperse the particles in water as well as ethanol to analyse the particle size and Zeta
potential using DLS.
RESULTS:
Table 1:
S.No SSPI (mg) Ratio of SSPI
used
Water (ml) PDI Z.
Average
Size(d.
Nm)
Zeta
Potential
(mV)
1. 0.5 1:0.5 2 0.241 328.4 -35.23
2. 0.5 1:1 2 0.240 352.8 -82.88
3. 0.5 1:1.5 2 0.330 391.6 -82.2
4. 0.5 1:2 2 0.245 294.3 -87.5
Table 2:
S.No Bifenthrin PDI Z.Average Size Zeta Potential
1. 2mg/ 2ml 1.002 1992.4 d.nm 0.6
DRUG: BIFENTHRIN









Here we use two methods:
1) We will add Chitosan dissolved in acetic acid to the SSPI nanoparticles dispersed in
water.
2) We will add Chitosan dissolved in acetic acid to the SSPI nanoparticles dispersed in
acetic acid.