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Safety Concerns towards the Biomedical Application of PbS
Nanoparticles: An approach through Protein-PbS
Interaction and Corona Formation
Amit Kumar Bhunia1, Pijus Kanti Samanta2, Satyajit Saha1, Tapanendu Kamilya3 *
1. Department of Physics & Technophysics, Vidyasagar University, Paschim Medinipur, 721102, India
2. Department of Physics, Ghatal R.S. Mahavidyalaya, Paschim Medinipur-721212, India
3. Department of Physics, Narajole Raj College, Paschim Medinipur-721211, India
1. Calculation of Fractal Dimension
We have analyzed the structure of ‘PbS NPs-BSA’ corona by fractalyse software to
calculate the fractal dimension. This software uses the Radial mass distribution method to
calculate the fractal dimension. The Hausdorff dimension, D, is related as S∝RD, where, S is the
area covered by each structure, R is the average distance from the center of mass of a structure to
its perimeter. The slope of linear fit of ln(S) vs ln(R) gives the value of D and is calculated to be
~ 1.7.
2. Fluorescence Quenching Measurement
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The PbS NPs-BSA binding kinetics and equilibrium as well as conformational change of
BSA is also analyzed by fluorescence quenching measurements.
𝐹
⁡ 𝐹0 = 𝐾𝑆𝑉 [𝑄] + 1
(2)
F0 and F represent the steady-state fluorescence intensities of flourephore in the absence and
presence of PbS NPs, respectively. KSV corresponds to the Stern-Volmer quenching constant and
[Q] represents the concentration of PbS NPs. The values of KSV for 20, 30 and 40oC are
summarised in Table-1. The increment of KSV with increasing temperature signifies that the
quenching mechanism of BSA is a dynamic quenching process in presence of PbS NPs and the
strength of interaction increases with temperature. The F0/F versus [Q] plots at different
temperature are shown in Fig. 8 (a).
The binding constant K along with the number of binding sites (n) between BSA and PbS NPs at
different temperatures are calculated using the following equation:
𝐹0 −𝐹
⁡log⁡[
𝐹0 −𝐹
The plot of ⁡𝑙𝑜𝑔⁡[
𝐹
𝐹
] = log 𝐾 + 𝑛⁡𝑙𝑜𝑔 [𝑄]
(3)
] versus 𝑙𝑜𝑔⁡[𝑄] gives a straight line and the slope determines the value of n.
The intercept of the straight line on Y-axis determines the value of⁡𝑙𝑜𝑔 𝐾⁡. The values of ⁡𝑙𝑜𝑔 𝐾⁡
at 20, 30 and 40oC are summarized in Table I, for comparison. In favour of positive cooperative
reaction, n›1, reveals that once one protein molecule is bound to the NPs, its affinity for the NPs
gradually increases in a super-linear fashion. However, in case of negative cooperative reaction,
n‹1, the binding strength of the protein with the NPs becomes weaker as further proteins adsorb.
As well as, for a non-cooperative reaction, n=1.
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3. Binding with Tryptophan
The interaction PbS NPs with TRY is studied to investigate the probable binding sites of
BSA with PbS NPs. The value of n in case of interaction of PbS NPs with TRY at 40oC is almost
matched with the PbS NPs-BSA complex.
4. van’t Hoff equation and thermodynamic parameter calculation
The thermodynamic parameters ∆H (change in enthalpy) as well as ∆S (change in
entropy) for BSA and PbS NPs interaction are studied to account for the main forces contributing
to the stability of BSA is determined by using the van’t Hoff equation
∆𝐻
ln 𝐾 = − 𝑅𝑇 +
∆𝑆
𝑅
(4)
K is analogous to the binding constant at corresponding temperature (T) in oC; R is the
universal gas constant. The free energy change is analyzed by following relationship
∆G =⁡ ∆𝐻 ⁡ −𝑇∆𝑆=−𝑅𝑇𝑙𝑛 𝐾
(5)
In general, mainly four types of interactions occur between bare NPs and biological
macromolecules: hydrogen bond, van der Waals interaction, electrostatic and hydrophobic
interaction etc. According to the value of ∆H and ∆S, the model of interaction can be predicted
as:
i) ∆H > 0 and ∆S > 0; represent hydrophobic interaction.
ii) ∆H < 0 and ∆S < 0; represent van der Waals and hydrogen bonding.
iii) ∆H < 0 and ∆S > 0; represent electrostatic bonding.
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5. CD Spectroscopy
CD spectroscopy is used to analyze the change of secondary structure of BSA in
interaction with PbS. PbS NPs binding associated conformational change of BSA is analyzed by
fitting the CD spectra with K2D3 software. The positive peak at 190 nm and two negative peaks
at 208 and 222 nm state that BSA is α-helix rich protein.
6. FTIR Spectroscopy
PbS NPs binding associated unfolding of BSA is analyzed by fitting the amide I band
(1600-1700 cm-1) of FTIR spectrum. The unfolding, intra and intermolecular associations of
protein were studied by monitoring the peak positions and width of amide bands within a fixed
range by FTIR analysis. The FTIR absorption spectra of amide-I band (1600-1700 cm-1) is due to
C=O stretching modes of peptide linkages of BSA. The vibrational energies of the carboxyl
group depend in reality on the different conformations of the protein, such as -helix, -sheet, turns and intra and intermolecular aggregates. The determination and the assignment of the
spectral components of the amide-I band can provide the information on the protein secondary
structure. A Gaussian multiple-peak-fitting procedure has been employed to study the amide I
band of FTIR spectra by using Microcal Origin 7.5 software after baseline correction. The quality
of the fitting was evaluated based on the χ2 values (on the order of 10-6) and the square of the
correlation coefficient (R2) values 0.999. The multiple peaks resulted from the deconvolution will
provide us the conformations of BSA in different condition and to identify its component and, in
particular, to determine the corresponding peak frequencies. The percentage area of the
deconvoluted peaks gives the relative area of the components. It is worth noting that, in all the
spectra considered in the present work, the maximum number N of the components which can be
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safely identified in the deconvoluted amide I band does not exceed N=5 to have a meaningful
fitting.
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Figure-S1:
(a) Absorption spectrum of PbS NPs, (b) PL spectrum of PbS NPs
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Figure-S2:
Absorption spectra: (a) pure BSA with CBSA=0.1 mg/mL, (b)- (e) represent the absorption of
BSA-PbS NPs complex with CBSA=0.1 mg/mL and CPbS= 0.01 (b), 0.03 (c), 0.05 and (d) 0.09
mg/mL (e), respectively.
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Figure-S3:
(a): Shift of absorption wavelength (∆λ) of BSA with CPbS. (b) Change of absorption intensity
(∆A) of BSA with CPbS.
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Figure-S4:
Schematic representation of interaction and corona formation of PbS NPs and BSA
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Figure-S5:
Fig. S5(i): (a) represents the fluorescence spectrum of pure BSA with CBSA=0.1 mg/mL. Fig.
S5(i) (b) – (e) represent the fluorescence spectra of BSA in BSA-PbS NPs complex with
CBSA=0.1 mg/mL and CPbS= 0.01, 0.03, 0.05, 0. 1 mg/mL. The spectra are taken at 20oC. Fig.
S5(ii): (a) represents the fluorescence spectrum of pure BSA with CBSA=0.01 mg/mL. Fig. S5(ii):
(b) – (e) represent the fluorescence spectra of BSA in BSA-PbS NPs complex with CBSA=0.1
mg/mL and CPbS= 0.01, 0.03, 0.05, 0. 1 mg/mL. The spectra are taken at 30oC. Fig. S5(iii): (a)
represents the fluorescence spectrum of pure BSA with CBSA=0.01 mg/mL. Fig. S5(iii): (b) –(e)
represent the fluorescence spectra of BSA in BSA-PbS NPs complex with CBSA=0.1 mg/mL and
CPbS= 0.01, 0.03, 0.05, 0. 1 mg/mL. The spectra are taken at 40oC.
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Figure-S6:
Fig. S6(a) represents the F0/F versus Q plot for PbS NPs-BSA complex. Fig. S6(b) represents the
log K versus 1/T plot for PbS NPs-BSA complex.
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Figure-S7:
Fig. S7(a) (a) represents the fluorescence spectrum of pure TRY with CTRY=0.1 mg/mL. Fig.
S7(a) (b) – (e) represent the fluorescence spectra of TRY in TRY-PbS NPs complex with
CTRY=0.1 mg/mL and CPbS= 0.01, 0.03, 0.05, 0. 1 mg/mL. The spectra are taken at 40oC. Fig.
S7(b) represents F0/F versus Q plot for TRY-PbS NPs complex.
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Figure-S8:
CD Spectra; (a) pure BSA (b) BSA-ZnO complex
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Figure-S9:
FTIR Spectra: (a) Amide bands of BSA-PbS NPs complex. (b) Amide-I band of BSA-PbS
NPs complex. Green lines represent the curves fitted by multiple peaks fitting by Microcal
Origin.
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TABLE I
BSA
BSA
BSA
TRY
T
(K)
293
303
313
313
KSV
mM-1
1.09
1.58
1.90
1.74
Log K
( µM)-1
2.348
2.564
3.708
2.950
ΔG°
(kJ/mol)
-5.717
-6.456
-9.646
-7.355
ΔH°
(kJ/mol)
ΔS°
(J/mol)
N
-51.39
15.574
14.823
13.311
0.8086
0.9006
1.1282
1.0718
Table I: Binding parameters and thermodynamic parameters of BSA–PbS NPs
TABLEII
Conformers
A1


T
A2
FTIR
Area (%)
Position (cm-1)
A
B
A
B
17.34 0.87 1605.2 1613.2
12.50 04.44 1626.3 1626.8
62.82 60.01 1650.7 1649.6
04.68 32.58 1674.2 1685.1
02.66 02.10 1688.1 1704.9
CD
 /
CD
FTIR
A
B
A
B
A
B
0.13
8.65 8.34
0.12
68.13 68.04
0.198
0.074
-
Table II: Fitting parameters of amide-I band for BSA
A: pure BSA, B: BSA-PbS NPs complex. Area (%) = 100 represents the total area under curve.