Supplementary Data
1. METHODS
1.1. Turbidity Test
Nanoparticles prepared under different conditions, as mentioned above, were
subjected to turbidity test. The turbidity of the samples was analyzed
spectrophotometrically at 600 nm as a function of time to study the possibility of
aggregation of the particles.
1.2.Reverse phase HPLC analysis
Isocratic reverse phase high performance liquid chromatography was performed using
Young Lin (RYL9100 HPLC, Young Lin, Korea) equipped with a vacuum degasser
(YL9101), high pressure dual plunger pumps (YL9111), rheodyne model injector
(7725i loop injector) and a dual wavelength detector (YL9120 UV/VIS). The sample
volume of 80 μL was injected and separation was carried out on C-18 reverse phase
analytical column (150 mm × 46 mm, Altech altima C18). The mobile phase,
water:acetonitrile (95:5) containing 0.1% TFA was eluted at a flow rate of 1 ml/min.
The temperature was maintained at 25 °C throughout the study. Absorbance of the
eluent was monitored at 220 nm (YL9120 UV/Vis detector) and the data was analysed
using Autochro-300 software.
1.3. Characterization of soy protein nanoparticles
The particle size and zeta potential of soy protein nanoparticles before and after
conjugation was determined using Brookhaven Zeta PALS instrument at Centre for
Nano Science and Engineering (CeNSE), Indian Institute of Science (IISc),
Bangalore, India. The morphology of the nanoparticles was evaluated using
Transmission Electron Microscope (TEM) at a protein concentration of 200µg/ml. A
drop of the sample was cast-dried on a copper grid and subsequently treated with infra
red rays in order to completely dry the nanoparticles. The nanoparticles on the copper
grid were examined using TEM (Philips CM200 TEM, SAIF, IIT-Bombay, India),
with operating voltage of 200 kV.
1.4. Structural stability of protein using DSC
Differential scanning calorimetry (DSC) was performed using a Mettler DSC (Mettler
Toledo, Greifensee, Switzerland) at SAIF, IISC, Bangalore. For DSC measurements,
15 mg of particles were loaded in aluminium pans. A scan rate of 10°C/min was
employed to heat the samples from 0 to 250 °C. An empty pan was used as reference.
Analysis was performed under nitrogen purge of 60 ml/min. Calorimetric parameters
were analyzed using the STARe Software.
2. RESULTS AND DISCUSSION
2.1. Stability of soy nanparticles by turbidity test
Fig 1A depicts the results of the turbidometry experiment, which could complement
the zeta potential measurements.
Higher zeta potential values will lead to greater
repulsion between the particles, and therefore the probability of aggregation becomes
least.1 As could be inferred, turbidity of the suspension was least in the presence of
5.4% NaOH, the concentration at which the zeta potential was the maximum. At all
other concentrations of NaOH, the turbidity was higher.
Based on these
experiments, it was clear that particles formed at 5.4% NaOH were smaller in size
and stable. Fig. 1B showed that the turbidity values of the sample were very low at
pH 6.2 and accordingly indicates the higher stability of particle. This highest stability
of the particle could be explained as a result of repulsion effect of the surface charge
of -30 mV, so preventing the aggregation of the particle.2 Thus pH 6.2 was found to
favour the formation of desired size of the particle with higher stability. Particles
were also found to be stable for longer duration at a glutaraldehyde concentration
above 28 µg/mg (Fig. 1C). It could be inferred that samples with either lower
concentration (<21 µg/mg) or absence of glutaraldehyde, the stability is very poor
which could be explained as a result of dissociation of the nanoparticle into free
polymer because of insufficent cross linker.1,3
2.2. Reverse phase HPLC analysis
The chromatogram profile of NP, AV and C-AV was performed to characterize the
particle based on its hydrophobic property Fig 2. NP showed a single peak at RT of
5.3 minute. On comparison of chromatogram pattern of AV with C-AV, only two
peaks with retention time (RT) of 5.3 and 6.2 was observed in AV while the C-AV
sample showed three peaks (5.3, 6.7 and 7.1 RT) with increase in RT. Separation of
molecule in RP-HPLC depends on the hydrophobic binding of the solute in the
mobile phase to the immobilized hydrophobic ligand on the stationary phase. This
increase in hydrophobicity of a molecule is characterized by increase in the RT of
solute in the eluent.4 Thus, increase in the RT of C-AV when compare to AV proves
that there is a change in the hydrophobicity of the antivenom on conjugation to
nanoparticles and thereby provides supporting information on confirmation of
conjugation of antivenom to the nanoparticles with structural modification.
2.3. Characterization of the nanoparticles with and without conjugation to
antivenom by TEM
TEM analysis showed that the particles were nearly spherical in shape with the inner
layer of protein forming hard cornea surrounded by a floppy structure of weakly
bound protein with rough surface area Fig. 3. Similar morphology of soy protein
nanoparticle with honeycomb inner structure was reported by Zhang et al 5 prepared
by using CaCl2 as an inducer. Antivenom conjugated nanoparticles were found to
have increased particle size (ranging from 200 – 600 nm). It can be observed that the
AV antibodies are attached on the core soy protein nanoparticles.
2.4.Thermal stability of the protein
Thermal denaturation of soy protein, soy protein nanoparticles (AV and C-AV are
presented in the main manuscript) was studied using differential scanning
calorimeter (DSC) (Fig. 4), in order to get further insights on the stability of the
conjugated nanoparticles with respect to its denaturation temperature and enthalpy
(∆H). The DSC thermogram of soy protein showed a single endothermic peak with
onset (To) of 92.74 and peak (Tp) denaturation temperature at 97.40°C representing
the abundance of glycinin rich fraction in the soy protein isolate.6,7 Nanoparticles
formulated from hydrolysate of soy protein (NP) showed a single exothermic peak in
DSC thermogram with To of 96.7°C and Tp of 100.02°C indicating the increased
stability of the protein which could probably because of the structural modification
in the protein during the process of nanoparticles formation.8
References
1. Z. Teng, Y. Luo, Q. Wang, Nanoparticles Synthesized from Soy Protein: Preparation,
Characterization, and Application for Nutraceutical Encapsulation. Journal of
Agricultural and Food Chemistry 60 (2012) 2712-2720.
2. I.D. Morrison, S. Ross, Colloidal dispersions: suspensions emulsions, and foams. John
Wiley & Sons, New York 2002, p. 177-190.
3. C. Weber, C. Coester, J. Kreuter, K. Langer. Desolvation process and surface
characterisation of protein nanoparticles. International Journal of Pharmaceutics 194
(2000) 91−102.
4. M.I. Aguilar, Reversed-Phase High-Performance Liquid Chromatography. Springer
Protocol (2003) 9-22. DOI: 10.1385/1-59259-742-4:9.
5. J. Zhang, L. Linag, T. Tian, L. Chen, M. Subirade, Preparation and in vitro evaluation
of calcium-induced soya protein isolate nanoparticles and their formation mechanism
study. Food Chemistry 133 (2012) 390-399
6. A.M. Hermansson, Physico-chemical aspects of soy protein structure formation.
Journal of Texture Studies 9 (1978) 33-58.
7. B. German, S. Damodaran, J.E. Kinsella, Thermal dissociation and association
behavior of soy proteins. Journal of Agricultural and Food Chemistry, 30 (1982) 807–
811.
8. J.S. Renkema, H. Gurppen, T.V Vliet, Influence of ph and ionic strength on heatinduced formation and rheological properties of soy protein gels in relation to
denaturation and their protein compositions.
Chemistry 50 (2002) 6064-6071.
Journal of Agricultural and Food
Figures
Figure 1: Stability of soy protein nanoparticles at different (A) NaOH concentrations (B)
pH (C) glutaraldehyde concentrations by turbidity analysis.
Figure 2: Reverse phase HPLC thermogram of NP, AV and C-AV using water:acetonitrile
(95:5) as a mobile phase.
Figure 3: The transmission electron microscopy image of the soy protein nanoparticles (A &
B) and soy protein nanoparticles conjugated to antivenom (C & D) .
Figure 4: Differential scanning thermogram of (A) soy protein, (B) soy protein nanoparticles