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Spectroscopic analysis of Muga silk nanoparticles synthesized by microwave

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Article  in  Biotechnology and Applied Biochemistry · April 2020

DOI: 10.1002/bab.1932

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 Prithvi Asapur1
Spectroscopic analysis of Muga silk Indrani Banerjee1
∗

nanoparticles synthesized by microwave P. D. Sahare 2

Santosh Mahapatra3
method

1 School of Nano Sciences, Central University of Gujarat, Gandhinagar,

Gujarat, India
2 Department of Physics & Astrophysics, University of Delhi, Delhi, India
3 Department of Physical Sciences, School of Basic and Applied Sciences,
Central University of Punjab, Bathinda, Punjab, India

Abstract
Muga silk nanoparticles (MSNP) were synthesized using a ˜240 nm in size. The optical properties of these nanoparticles
microwave-assisted radiolysis method. The effect of were studied by UV–vis. spectroscopy and
microwave on the Muga protein secondary structures was photoluminescence. For studying thermal properties,
analyzed. The evolution of the secondary structure from differential scanning calorimetry was performed that revealed
random coils to the β-sheets was determined by using FTIR, early glass transition, which could be attributed to the
circular dichroism and X-ray diffraction techniques. The results presence of water and proteins. It also revealed that
showed that Muga silk fibroin protein contained the primary nanoparticles are thermally stable. Such studies are important
structure in silk-I state. When the protein was irradiated with for understanding more about the MSNP and would be
microwave, nanoparticle synthesis was possible having silk-II beneficial for their further wide applications. © 2020 International
state imparting crystallinity. The silk nanoparticles were Union of Biochemistry and Molecular Biology, Inc. Volume 00, Number 0,
characterized by a particle size analyzer and found to be of Pages 1–11, 2020

Keywords: Muga silk nanoparticles, microwave synthesis, UV–vis

spectroscopy, photoluminescence (PL), FTIR

1. Introduction nanoparticles possess outstanding properties that are advanced

and different from bulk polymer, with improved properties, such
Nanoparticles have achieved a breakthrough in optics, ther-
as, high surface to volume ratio, electrical, optical, mechanical,
apeutics, bioengineering, imaging, and diagnosis. The topic
biodegradable properties, and biocompatibility [1]. Among the
of interest in biomedical applications nowadays involves bio-
various natural polymers, such as, collagen, alginate, chitosan
compatible natural polymeric nanoparticles. These polymeric
silk, etc. Silk has been more attracted due to its exceptional
qualities like, biocompatibility, superior mechanical properties,
and biodegradability. Silk fibroin (SF) is histocompatible, non-
Abbreviations: MSNP, Muga silk nanoparticles; MSF, Muga silk fibroin; toxic, and also optically active, which makes it widely applicable
RC, random coil; RGD, Arg-Gly-Asp; PVDF, polyvinylidene fluoride; MWCO, to the biomedical and other fields [2,3].
molecular weight cut off; FTIR, Fourier transformed infrared; CD, circular
dichroism; XRD, X-ray diffraction; PSA, particle size analyser; UV, ultraviolet;
Silkworm silk can be classified into two major classes:
PL, photoluminescence; DSC, differential scanning calorimetry; FSD, mulberry and non-mulberry silk. The majority of the SF study
Fourier self-deconvolution; LB, Luria Broth; BSA, bovine serum albumin; is carried on domesticated Bombyx mori and Nephila clavipes
ROS, reactive oxygen species; MSN, mesoporous silica nanoparticles.. spider and a lot of literature is available for various biomedical
∗ Address for correspondence: Prof. P. D. Sahare, PhD, Department of
applications [2]. Mulberry silk, B. mori is widely exploited as
Physics & Astrophysics, University of Delhi, Delhi - 110007, India; compared to wild or non-mulberry silk and very limited study
Tel.: +91-11-27667793; Fax: +91-11-27667061; e-mail:
pdsahare@physics.du.ac.in.
is available in the literature on potential wild silk. Some of
Received 6 February 2020; accepted 23 April 2020
the recent studies on wild silk show various advantages over
DOI: 10.1002/bab.1932
its counterpart due to putative cell attachment and the RGD
Published online in Wiley Online Library
(Arg-Gly-Asp) sequence that increases the binding affinity on
(wileyonlinelibrary.com) receptor cell surface beneficial for stem cell therapy and other

1
 Biotechnology and
Applied Biochemistry

targeted therapies [3–6]. Also, it has improved physicochemical, Research Laboratories (Mumbai, Maharashtra, India), and
mechanical and thermal properties [4]. absolute ethanol [99%], acetone [99%], cellulose dialysis tubing
Highly valued Muga silk from Antheraea assama is a wild (MWCO 12–14 kDa) from Sigma–Aldrich (St. Louis, Missouri,
silk that is largely unexplored. Muga silk has high mechanical USA).
property and durability compared with other silks and hence is
in demand in the international market. It has unique biophysical 2.2. Preparation of sericin free silk from the cocoon
properties like tenacity, golden luster and high absorbance of The Muga silk cocoon was cut into large pieces with simulta-
UV radiation [7,8]. neous removal of the worm, which was disposed of later. Then
There are several methods reported for the synthesis the cocoon pieces were degummed by boiling 0.021 M sodium
of Bombyx morisilk nanoparticles, for instance, desolvation, carbonate (Na2 CO3 ) for 45 Min and subsequently washed and
supercritical fluid technology, ion induction, microemulsion, cooled by rinsing with distilled water several times for the
electro-spraying, milling and so on. [3,6,9–13]. These methods removal of sericin protein and salt. The cooled silk was kept for
have various drawbacks like, a lengthy process, the use overnight drying at 50 °C.
of chemicals and less yield, which led to introduction of 2.3. Extraction of SF
other more resourceful methods, such as, radiolysis methods The extraction of Muga silk fibroin (MSF) was carried out by
[3,14]. Radiolysis methods include plasma etching, electron the previously reported method for other non-mulberry protein
irradiation, microwave synthesis and so on, of which microwave dissolution with minor changes [18,21,22]. One gram of small
synthesis method is found to be more rapid, environment- cut-pieces of degummed Muga silk was heated in a mixture
friendly, takes short reaction time, facile purification, improved of Ca(NO3 )2 :ethanol:distilled water at (1:4:1.5) at 2.5 w/v% at
production yield and undergoes controllable conditions [14–16]. 120 °C for 8–9 h. The solution was then cooled and filled in
Recently, radiolysis methods are used for the synthesis of silk a cellulose dialysis bag with MWCO 12–14 kDa followed by
nanoparticles, for example, methods reported by Gao et al. [15] dialysis using deionized water for 3 days to remove residual
using the electron-beam synthesis of silk nanoparticles and salts. The silk solution was then centrifuged at RCF 7,650g
Ko et al. [16] and Wongkongsak et al. [17] for the microwave- for 20 Min. The fibroin solution was then filtered through a
assisted synthesis of silk nanoparticles. 0.2 μm PVDF membrane filter and concentrated using 20%
The main interest of our present research work is to PEG 20,000. The extracted fibroin solution was stored at 4 °C
analyze the effect of microwave on the Muga silk protein for for further use. The concentration of protein was determined
its conversion to nanoparticles. The interaction of microwave gravimetrically by weighing the remaining dried protein.
radiation with the polymer would lead to chain aggregation,
chain scission, formation of double bonds, molecular, and 2.4. Synthesis of MNSP by microwave method
electronic transitions [14]. Such internal defects would lead Muga silk fibroin solution at a concentration of 10 mg/ml of
to the formation of induced centers that would make changes which 30 ml was taken into a flask and exposed to microwave
in the structure and optical bandgap in the silk protein irradiation at 200 °C for 25 Min as reported earlier for B. mori
solution. In the present work, the optical properties of MSNP with minute modifications [16,17]. The reaction was cooled by
were studied by UV–vis. spectroscopy and photoluminescence keeping the flask in ice for 5 Min. The resulting solution was
(PL), their structural defects by circular dichroism (CD), centrifuged at RCF 1,464g for 30 Min twice to remove large
Fourier transform infrared (FTIR) spectroscopy, and X-ray aggregates. The supernatant was filtered through a 0.2 μm
diffraction (XRD). Thermal analysis was done by differential PVDF syringe filter and lyophilized.
scanning calorimetry (DSC). Eventually, such studies on Muga
silk nanoparticles (MNSP) by different spectroscopic and 2.5. UV–vis and PL spectroscopy
other techniques would help us to extend the applicability of UV–vis. spectra were recorded on a Shimadzu UV-1800 using
these nanoparticles. These MNSP could find its application a 1 cm wide quartz cuvette from 200 to 500 nm. PL excitation
in drug delivery [19], optics and photonics, electronics and and emission spectra were recorded using a xenon lamp
optoelectronic applications [20], bioimaging [15], and so on. as an excitation source with an effective slit width of 1 nm
Also, the Muga silk protein has RGD sequence that helps in (Fluorescence spectrometer; Horiba Fluorolog 3–21) from
binding affinity for cell surface which find its application in 240–500 nm.
gene transfer [3–6]. 2.6. Dynamic light scattering
The average hydrodynamic radius and zeta potential measure-
ments were taken on a Zetasizer Nano ZS90 DLS system at
2. Materials and Methods 25 °C (Malvern Instruments, Malvern, Worcestershire, UK).
2.1. Materials
Cocoons of Muga silk was gesturally provided by Central Silk 2.7. FTIR spectroscopy
Board, Guwahati, Assam. Calcium nitrate (Ca(NO3 )2 , 99%), FTIR spectra were recorded on PerkinElmer (Waltham, Mas-
sodium carbonate (Na2 CO3 , 99%), 0.2 μm polyvinylidene sachusetts, USA) FTIR spectrometer (Spectrum 65 series) using
fluoride (PVDF) membrane filter were obtained from Sisco KBr pellet techniques. The transmittance (%) was recorded

2 Spectroscopic Analysis of Muga Silk Nanoparticles

 FESEM micrograph of Muga silk nanoparticles. X-ray diffractogram of Muga silk fibroin and Muga
FIG. 1 FIG. 2 silk nanoparticles.

3.1.2. Morphology of Muga nanoparticles

in the wavenumber range of 400–4,000 cm−1 . The lyophilized The morphology of MNSP was observed by using a field
Muga silk sample of about 2 mg was added in anhydrous emission scanning electron microscope (FESEM). Figure 1 is
KBr (200 mg), mixed thoroughly, and pelleted using a die the micrograph that indicates the morphology and size of MNSP
and hierodulic press. The pellet was used to record the FTIR synthesized by the microwave method. The micrograph shows
spectrum. The spectrum of each sample was recorded at 64 the elongated morphology with average particle size ∼200 nm
scans (recorded 64 times and averaged out). that matches well with the measured one using PSA.

2.8. Circular dichroism 3.2. Secondary structure analysis

CD spectra were recorded on a JASCO CD spectrometer J- 3.2.1. XRD analysis
1500. Spectra for both far-UV and near-UV with a sample The conformational transitions, degree of crystallinity, size of
concentration of 0.1 mg/ml in D/W. solid crystals of silk protein and the silk nanoparticles were
obtained by XRD analysis. Silk protein is semi-crystalline
and has three types of crystalline structures along with
2.9. X-ray diffraction
the amorphous state in the form of random coils. Silk-I
Wide-angle XRD patterns of Muga silk protein and nanoparticle
corresponds to the glandular state before crystallization,
were measured by X-ray diffractogram on GNR APD 2000 using
silk-II represents β-sheet crystalline structure and silk-III
Cu-Kα radiation at 40 kV and 30 mA in the 2θ range 5°–40°
constitutes helical conformation [2,9,23]. Figure 2 shows the
with λ = 1.54 Å at the scanning rate 3° per Min.
X-ray diffractogram of MSF and its nanoparticles. Table 1
represents the consolidated peaks observed for both, that is,
2.10. Differential scanning calorimetry the protein (MSF) and its nanoparticle (MSNP) samples. The
DSC studies of Muga protein and nanoparticles were done d-spacing for the corresponding peaks are also mentioned.
in Perkin Elmer DSC 6000 in a nitrogen atmosphere with The table shows that before microwave irradiation the MSF
temperature range 30–300 °C with an increase of 10 °C/Min contains both random coils and a β-sheet crystalline structure
and 3 mg of sample was used each time. (as a result of intermolecular hydrogen bonding) with a
predominant constituent of random coil conformation (silk-
I). The MSF undergoes conformational changes mostly from
3. Result and Discussion random coils to β-sheets structures to form MSNP (silk-II). The
3.1. Nanoparticles characterization XRD results are in agreement with FTIR and CD data discussed
3.1.1. Particle size analysis later (Sections 3.2.3 and 3.2.3, respectively). Silk protein is
The hydrodynamic size of the nanoparticles synthesized by semi-crystalline, where the small crystallites are distributed in
the microwave method was analyzed by particle size analyzer the amorphous matrix. The crystallite content contributes to
(PSA) (Zetasizer Nano ZS90 DLS system). MNSP showed a the stability of protein [24]. The percentage of crystallinity to be
particle diameter of 240 nm with a 0.44 polydispersity index. determined by the area of the crystalline peak to the total peaks

Biotechnology and Applied Biochemistry 3

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XRD data of Muga protein and its nanoparticles

TABLE 1

Sample 2θ (degree) d-Spacing (Å) Structure Crystal size (nm) Crystallinity (%)

MSF 9.93 4.46 silk-I 3.38 44.18%

23.26 1.94 silk-II 0.81

28.77 1.59 silk-I 0.27

MSNP 14.98 2.97 silk-II 30.44 53.21%

18.49 2.42 silk-II 0.56

19.16 2.34 silk-II 10.64

23.18 1.95 silk-II 1.31

29.62 1.55 silk-I 0.21

33.67 1.38 silk-II 10.96

in the XRD graph as shown in Eq. (1). Microwave synthesized position of the peaks along with assignments. In the case of
nanoparticle show higher crystallinity of 53.21% as compared MSF the Amide-I, -II, and -III were observed at 1,646, 1,530,
with the MSF protein being 44.18%. The interplanar spacing and 1,239 cm−1 , respectively, whereas, in MSNP peak shifts
(d) and crystal size (D) was calculated by the Braggs equation corresponding to Amide-I and -II were observed and the shifted
(question 2) and Scherrer’s equation (question 3) [25] as given positions are 1.673 and 1,534 cm−1 , respectively. Evolution of
below: new peaks at 1,109 and 953 cm−1 in MSNP was also observed,
  Area of crystalline peaks which could be attributed to v(C–C), v(C–O), and r(CH2 ), (A)n ,
Crystallinity % = × 100 (1) respectively.
Area of all peaks
The Amide-I of the proteins is widely studied due to its
Bragg’s equation: sensitivity for secondary structures that are thought to be
nλ due to the transition of dipole mechanism, bond coupling, and
d= (2) hydrogen bonding. Hence, the Amide-I peak was deconvoluted
2sinθ
to determine the fraction of secondary structures by the
Scherrer’s equation:
Fourier self-deconvolution (FSD) method. Figure 3C shows
kλ the deconvoluted Amide-I peak of MSF and MSNP. MSF shows
D= (3)
βcosθ a peak at 1,645 cm−1 that corresponds to the random coil
(RC), two peaks at 1,618 and 1630 cm−1 that correspond to
β-sheets and a peak at 1,662 cm−1 that corresponds to β-turns,
3.2.2. FTIR analysis whereas, MSNP shows a peak at 1,646 cm−1 corresponding
FTIR analysis is the rapid and reliable technique to study to RC, two peaks at 1,631 and 1,687 cm−1 corresponding to
the protein secondary structure based on the principle that β-sheets and two peaks at 1,674 and 1,662 cm−1 corresponding
H-bonding in secondary structures have characteristics IR ab- to β-turns. The results reveal that MSF is predominant with RC,
sorption frequencies and also the intensity and position of the and microwave-induced stress leads to the formation of more
amide bonds are sensitive to the molecular conformations. The of β-sheets and β-turns, which is more stable structures than
effect of microwave on Muga silk protein secondary structures RC in MSNP.
were analyzed by performing FTIR measurements. The ab- Table 3 shows the secondary structure content in the
sorption bands representing Amide-I (1,600–1,700 cm−1 ) and samples. The table also shows an increase in the β-sheets and
Amide-II (1,500–1,600 cm−1 ) show vibrational C=O stretching β-turns content in MSNP with a huge decrease in RC content
and N–H bending, respectively [25–27]. Figure 3A shows the as compared to MSF. Microwave has led to the conversion of
FTIR spectra of Muga fibroin protein and its nanoparticles. the random coil to β-sheets structures with two intermediate
All the samples showed three amide peaks at their respective structures of α-helix (the result of intramolecular hydrogen
regions, that is, Amide-I at 1,600–1,700 cm−1 that arises due bonding) and β-turns. The nanoparticles synthesized by
to C=O stretching vibration, Amide-II at 1,500–1,600 cm−1 , microwave method MSNP showed 0.76 times greater β-sheet
which is due to C–N stretching, C=O in-plane bending and and 3.35 times greater of β-turns content from its protein
N–H in-plane bending and Amide-III at 1,200–1,300 cm−1 is MSF that could be due to the formation of antiparallel β-sheet
due to C–N stretching and N–H bending. Table 2 shows the through inter-molecular non-covalent interactions [8]. The

4 Spectroscopic Analysis of Muga Silk Nanoparticles

 (A) FTIR spectra of Muga silk fibroin protein (MSF) molecules [33,34]. For example, Desai et al. [35] have shown
FIG. 3 and Muga silk nanoparticles (MSNP). (B)
that the cellular uptake of 100-nm nanoparticles was 2.5 and
Secondary structural content (%). (C)
Deconvolution of Amide-I of MSF and MSNP. six times higher than 1 and 10 μm microparticles, respectively.
It was also reported in a similar study that cellular uptake of
100-nm nanoparticles was 15–250 times higher than 1- and
conversion of RC to β-sheet due to microwave would be due to 10-μm microparticles [36].
bond breakages and new bond formations during the process. Two important features of SF nanoparticles including
The β-sheet structure is related to the crystallinity of the protein pH-dependent drug release and lysosomal accumulation of
that imparts mechanical strength to the protein [8,29]. Hence, SF nanoparticles have confirmed the ability of drug-loaded
we can say that an increase in the β-sheet of MSNP makes it SF nanoparticles to serve as a lysosomotropic anticancer
more crystalline than the protein, which has also been verified nanomedicine. The main feature of silk among other polymers
by the XRD data. Moreover, the Muga protein is non-mulberry is its potential as a lysosomotropic drug delivery platform. A
silk which has polyalanine (A)6 residues in its sequence, which wide range of synthetic and natural polymers have been used
could have also contributed to the formation of β-nanocrystals as lysosomotropic delivery systems. However, as mentioned
that must have also helped for the increment of β-sheet. The earlier, silk as a natural polymer has an intrinsic capability
increase in the crystallinity increases the hydrophobicity that to response to pH changes in order to initiate drug release.
finds its application in drug delivery as slow degradable carrier Therefore, drug release is achieved in response to pH without
systems as it can sustain more in the aqueous medium [1]. any chemical modifications [37]. When using biomaterials for
Silk proteins are FDA-approved polymers that have been drug delivery applications, it is also necessary to consider their
used successfully as both drug delivery systems and sutures. clearance mechanism from the body. MSNP being very small in
These proteins have excellent mechanical properties, a flexible size would have advantage for this purpose than the SF.
preparation process and high biocompatibility [32]. It is also In the context of protein-based drug delivery systems, silk
known that particle size is the most important factor when is a potentially useful natural polymer, which has been used to
designing drug delivery systems and nanoparticles would deliver peptide and protein molecules also. Unfortunately, there
have added advantage due to its surface to volume ratio as are not many studies that discuss the role of silk nanostructure
compared with microparticles. Therefore, it is crucial to use in protein delivery systems. For example, Hofer et al. [38]
nanoparticles for the drug delivery and targeting therapeutic investigated the use of recombinant spider silk protein eADF4

Biotechnology and Applied Biochemistry 5

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Position assignments and conformation

TABLE 2

Sample Wavenumber (cm−1 ) Assignment band Conformation Ref. no.

MSF and MSNP 1646, 1673 Amide I, C=O stretching, C–N stretching Random coil, [23,24,27–31]
β-sheet

1530, 1534 Amide II, C–N stretching, C=O in plane Random coil
bending, N–H in plane bending

1239, 1239 Amide III, C–N stretching and N–H Random coil
bending

1403 and 1333, 1436 CH3 group frequency of alanine and CH2 , –
CH3 (A)n /β-sheet
1056, 1046 r(CH2 ) Random coil

1109 v(C–C), v(C–O) –

953 r(CH2 ), (A)n β-sheet

As discussed earlier, MSF contains both random coils and

Conformation content from the deconvolution of a β-sheet crystalline structure (as a result of intermolecular
TABLE 3 amide I FTIR spectra hydrogen bonding) with a predominant constituent of random
coil conformation (silk-I). The MSF undergoes conformational
β-Sheet β-Turns α-Helix Random changes mostly from random coils to β-sheets structures to
Test Sample (%) (%) (%) coil (%)
form MSNP (silk-II). It should be mentioned that the β-sheet
Muga Protein 19.95 13.30 – 66.73 has an asymmetrical structure featuring hydrogen side chains
(MSF) from glycine on one side and methyl side chains from alanine
on the other, creating hydrophobic domains. Strong hydrogen
MSNP 35.23 57.93 7.3 6.82
bonds and van der Waals forces between the methyl and
hydrogen groups on opposing sides allow inter-sheet stacking
particles (MWCO ≈ 27 kDa) for carrying the high molecular in the crystals and thus, generate a thermodynamically stable
weight protein lysozyme. However, proteins with a molecular structure [41].
weight comparable or higher than that of 66 kDa (e.g., bovine Lammel et al. [42] reported that silk particles with silk II
serum albumin [BSA]) were not found to permeate into the structure showed that small molecules (Alcian blue, rhodamine
protein particle matrix and were found to bind only to the B, and crystal violet) had 95% loading efficiency because of
surface. By taking the advantage of the electrostatic interaction a charge–charge interaction with silk particles (The negative
between the negatively charged protein particles and positively surface charge of silk particles with positively charged small
charged groups lysozyme, they were loaded onto negatively molecules). They also have found that the release of these three
charged these protein particles (size ≈ 521 nm) with an molecules is highly related to their charge and the structure
efficiency of almost 100%. They have also reported that of silk. For instance, the structure of silk II and the pH of
substantial quantities of lysozyme diffused into the matrix of solution could induce the burst release of molecules at all-time
these particles instead of rather absorbing onto the surface. intervals [42], which is not possible using SF. In this regard, the
Later they observed that the release of lysozyme from such present study on synthesis of MSNP using a very simple method
protein particles was related to the pH and ionic strength of microwave irradiation and their systematic spectroscopic
of the medium used. The maximum release of lysozyme studies about the transformation from silk I into silk II could
was observed after 24 h in medium only with acidic pH pave ways to develop such drug delivery systems for their
(<7.0) and no significant release was observed in medium therapeutic, imaging, and biosensing applications.
with a neutral pH (∼7.0) even after 28 days. There are
only a few studies reported in the literature where it is 3.2.3. CD analysis
mentioned that SF and biological molecules could interact via The secondary structures in Muga silk protein and nanoparti-
electrostatic interaction, hydrophobic attraction, and Coulomb cles in solution form was determined by far- and near-UV CD
forces [32,39,40]. However, there is still less knowledge about spectroscopy. Far UV (190–250 nm) spectrum is indicative of the
the exact mechanism of these interactions and needs more protein secondary structure, since it comprises the absorption
investigations. of peptide bonds. Near-UV spectrum (250–300 nm) includes

6 Spectroscopic Analysis of Muga Silk Nanoparticles

 (A) and (B) Secondary structure content (%). (C) Far and near UV range CD analysis of Muga silk fibroin and Muga silk
FIG. 4 nanoparticles.

Schematic illustration of conversion of random

Illustration 1 coil to β-sheets. with the FTIR results which also shows the increase in the
β-sheets after the microwave stress effect on Muga protein.
The schematic illustration of these transformations is as shown
the absorption of the aromatic amino acid side chains and it in the following illustration (Illustration 1):
is indicative of the protein tertiary structure [43]. Figure 4A Figure 4B illustrates the near UV analysis of the same
illustrates the far UV analysis of MSF and MSNP. The figure samples. The region gives the absorption from aromatic amino
shows that the MSF and MSNP give a strong negative peak acid side chains and also represents the contribution of disulfide
at 197 and 195 nm, which corresponds to π0 → π ∗ transition bonds. This analysis involves several influencing factors like
indicating the molecular conformation of both being primarily polar groups, hydrogen bonding, and polarizability, the rigidity
composed of random coils. Figure 4B shows the percentage of proteins, highly mobile side chains having lower intensities,
content of secondary structure, the peaks analyzed in the and so on [43]. The figure shows the peaks between 254 and
CD software for secondary structures for both the samples. 270 nm for phenylalanine, along with peaks corresponding to
The secondary structure content (%) of β-sheets increases in tyrosine arising between 277 and 284 nm and tryptophan (Trp)
the MSNP as compared to MSF. This increase in the β-sheets peak observed between 281 and 295 nm.
from 45.3% to 52.6% is due to the effect of microwave on the
protein getting converted to nanoparticles. Inversely, there was 3.3. Study of optical behavior
a significant decrease in the random coil content from 43.3% 3.3.1. Analysis of UV-vis. spectra
to 32.8% in MSNP. The results show that the random coils The light absorption by the silk protein polymer and its
gradually get converted to the metastable β-sheets structure nanoparticle leads to the transition of electrons from the
through the formation and removal of intermediate structures, ground state to excited state [14,25]. The optical properties
such as, α-helix and β-turns. The CD results are in agreement of Muga silk protein fibroin and nanoparticles synthesized

Biotechnology and Applied Biochemistry 7

 Biotechnology and
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(A) UV–vis. spectra. (B) Bandgap and (C) Urbach localized states tailoring into the bandgap was estimated from
FIG. 5 energy of Muga silk fibroin (MSF) and Muga silk
the Urbach plot of ln(A) versus (hν) as shown in Fig. 5C. The
nanoparticle (MSNP).
estimated values were 0.69 and 0.84 eV for MSF and MSNP
samples, respectively. The refractive indices were calculated
by microwave methods were characterized by UV–vis. spec- from the Lorentz–Lorentz equation for electronic polarizability
troscopy. Figure 5 shows the UV–vis. spectra of the Muga with a modified form of:

protein and nanoparticles, respectively. MSF and MSNP show n2 − 1 Eg
a strong absorption peak at 274 and 275 nm, respectively, = 1−
n2 + 2 20
corresponding to the absorption of aromatic amino acids with
The refractive indices were found to be 5.02 and 4.79 for
π –π * transitions. No remarkable change in the absorption peak
MSF and MSNP samples.
was observed due to microwave. A minor shift in peak to higher
wavelength could be due to new crosslinks formed or due to 3.3.2. PL analysis
the oxidized form of aromatic amino acids that must have led The photoluminescent property of MNSP synthesized by the
to the formation of nanoparticles. microwave method was studied by using a fluorescence
The Tauc plots of (Ahν)2 (where A is the measured ab- spectrometer. Figure 6 shows the excitation spectrum and five
sorbance) against incident photon energy of hν were found to overlapping bands could be seen at around 255, 260, 275,
be linear over the range 3.45 to 3.49 eV. The bandgap for the 284, and 292 nm. The excitation bands around 255–260 nm
SF and the nanoparticle were determined from the extrap- could be attributed to phenylalanine, 275 nm band to tyrosine
olation of the linear portion to the intercept of the ordinate and 284–294 nm bands to Trp [46,47]. The emission spectra
corresponding to A = 0 [44,45]. The estimated bandgaps were excited by these wavelengths (in the range of 240–280 nm)
4.84 and 4.98 eV for MSF and MSNP, respectively. The width of are also shown in Fig. 7. It could also be seen that all the

8 Spectroscopic Analysis of Muga Silk Nanoparticles

 Differential scanning colorimetry of Muga silk
FIG. 8 fibroin and Muga silk nanoparticles.

PL excitation spectrum of Muga silk fibroin and located in the hydrophobic protein domains, whereas the
FIG. 6 Muga silk nanoparticles.
emission at 640 nm was attributed to the intrinsic recombi-
nation in the semiconductor NCs. It is important to note here
even after the conjugation of the NC/micelles system with the
BSA, it seems that the conjugation process does not perturb
the optical properties of the NC when embedded in the mi-
celles and the micelles were effective in protecting the NC
surface.
In a similar studies by Meena and Sahare [50] studied the
interactions of protein corona in the Escherichia coli in Luria
Broth (LB) media with that of ZnO nanoparticles (ZnO NP) and
mesoporous silica nanoparticles (MSN). They studied emission
spectra (excited by 250 nm UV light) consisted of two bands,
one at around 430 nm and another weak band at 485 nm that
were attributed to the fluorescence spectra of Trp. Further, in
case of ZnO NP, it was observed that initially the fluorescence
intensity increased slightly but later it decreased with time. In
fact, ZnO nanoparticles interact with the biomolecules in the LB
media and the corona is formed due to proteins like Trp. The
PL emission spectra of Muga silk fibroin and Muga
increase in the fluorescence intensity could thus be attributed
FIG. 7 silk nanoparticles. to energy transfer from Zn NP to TrP in the beginning but
with time more reactive oxygen species (ROS) were formed
due to catalytic nature of ZnO NP that may be responsible for
forming new complexes or even disintegrate Trp resulting in
emission spectra (curves a–e) are similar except their relative decreasing the fluorescence activity (intensity). However, not
intensities. After deconvolution of one of the spectra (curve b), much decrease in fluorescence intensity was observed as the
it revealed that they consist of four overlapping bands peaking Trp protein present in the media did not interact with the
at around 290, 300, 315, and 340 nm. The bands 290 and 315 mesoporous nanoparticles. The nano-size pores present in
could be attributed to phenylalanine, whereas the bands at the MSN might be blocking the passage inside the cavity for
300 and 340 nm could be due to tyrosine and Trp, respectively biological macromolecules and which might have helped in
[47,48]. Thus, from the PL excitation/emission spectra it may adsorption of the biomolecules but did not disintegrate as no
be concluded that the MSNP consist of phenylalanine, tyrosine, ROS were formed. Such studies show that it is important to
and Trp proteins or their residues. study the interactions of nanoparticles with the biomolecules
The PL and absorption spectra of organically capped to see their biocompatibility and toxicity before they could be
CdSe@ZnS NCs incorporated in PEG modified phospholipids used in drug delivery and for other biomedical applications.
micelles and bioconjugated with BSA have been reported
by Depalo et al. [49] where the observed PL spectrum con- 3.4. Thermal analysis
sisted of two bands, that is, a broad band cantered at around 3.4.1. Differential scanning calorimetry
330 nm and a narrow band peaking at 640 nm, the first one Figure 8 shows the DSC thermogram of Muga silk (MSF) and
ascribed to BSA due to the presence of two Trp residues, Muga silk nanoparticles (MSNP). The thermogram exhibits a

Biotechnology and Applied Biochemistry 9

 Biotechnology and
Applied Biochemistry

MNSP’s applicability in the biomedical field and especially, in

Thermal decomposition of different biopolymers developing a drug delivery system for several lifesaving drugs.
TABLE 4

Sr. Biopolymeric Decomposition 5. Acknowledgements

no. nanoparticle temperature Reference
The research was funded by UGC for a Ph.D. fellowship. A
1. Chitosan nanoparticles 216 °C [55] special thanks to the Central silk board (CSB) for providing silk
cocoons.
2. BSA nanoparticles 230 °C [56]
3. Collagen nanoparticles 102 °C [57]
6. Conflict of Interest
4. Bombyx nanoparticles 288 °C [58] The authors declare no conflict of interest.

broad endothermic peak at 81.22 and 90.73 °C with enthalpy

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