Research Article

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Stable Self-Assembly of Bovine Lactalbumin Exhibits TargetSpecic Antiproliferative Activity in Multiple Cancer Cells
Sailendra Mahanta and Subhankar Paul*
Structural Biology and Nanomedicine Laboratory, Department of Biotechnology and Medical Engineering, National Institute of
Technology, Rourkela 769008, Odisha, India
S Supporting Information
*

ABSTRACT: Self-assembly of a protein is a natural

phenomenon; however, the process can be performed under
a suitable condition in vitro. Since proteins are nontoxic,
biodegradable, and biocompatible in nature, they are used in
various industrial applications such as biocatalyst, therapeutic
agent, and drug carriers. Moreover, their exible structural
state and specic activity are being used as sensors and
immensely attract many new applications. However, the
inherent potential of protein self-assembly for various
applications is yet to be explored in detail. In this study,
spherical self-assembly of bovine -lactalbumin (nsBLA) was
synthesized using an optimized ethanol-mediated desolvation
process with an average diameter of approximately 300 nm. The self-assembly was found to be highly stable against thermal, pH,
and proteases stress. When nsBLA was administered in various cancer cells, it demonstrated high cytotoxicity in three dierent
cancer cells via reactive oxygen species (ROS) generation, whereas it exhibited negligible toxicity in normal human and murine
cells. When nsBLA was conjugated with folic acid, it improved the cytotoxicity and perhaps mediated through enhanced cellular
uptake in cancer cells through binding with folate receptors. Further, experimental results conrmed that the cancer cell death
induced by nsBLA was not caused by apoptosis but a necrotic-like death mechanism. When compared with a well-known
protein-based anticancer agent BAMLET (bovine -lactalbumin made lethal against tumor cell), the self-assembled BLA clearly
exhibited higher cytotoxicity to cancer cells than BAMLET. While BAMLET exhibits poor biocompatibility, our nsBLA
demonstrated excellent biocompatibility to normal cells. Therefore, in this study, we prepared self-assembled -lactalbumin that
exhibits strong inherent antiproliferative potential in multiple cancer cells which can be used for ecient therapeutic approach in
cancer.
KEYWORDS: self-assembled nanostructured bovine -lactalbumin, BAMLET, self-assembly, cellular uptake, biocompatibility

1. INTRODUCTION
Biological systems that include proteins and DNAs are precisely
regulated and functioned by self-assembly. Modern nanobiotechnology is a rapidly growing area of research where the
natural biomolecules such as proteins and DNAs are being used
as fundamental units for assembling newly designed nanoassembly and nanobiomaterials for various applications.
Articially synthesized biomolecules-based nanostructures
constructed by self-assembly are in high demand for the
development of various kinds of novel biomaterials because of
their nonimmunogenic, nontoxic nature and biodegradability as
well as exible structure which can easily be modied based on
the application. Proteins-based self-assembly is complex, and
such nanostructures may achieve novel properties and functions
that otherwise perhaps would not be possible using any other
material. They have the potential to be used in commercial
applications such as biocatalysis,1 tissue engineering,2 biomineralization,3,4 cascade reactions,5,6 regenerative medicine,7
therapeutics, and drug delivery systems.8,9 They can also nd
nanotechnology application in designing eective scaold
2015 American Chemical Society

structures due to their strength and stiness such as with

other engineered polymers.10 Self-assembly of proteins made
articially have close resemblance with naturally occurring
protein clusters and can be used for the development of
functionalized supramolecular polymers.11
While self-assembly of a protein exhibits outstanding
applications, the inherent folding state of the molecule denes
the specic supramolecular structure and molecular function.
Moreover, change of protein conformation under stress can
also be useful to develop protein nanostructures or selfassembly having a novel function. For example, spherical hybrid
microassembly of both lysozyme and apo bovine -lactalbumin
(BLA) was reported with a size range of 14 m.12 In another
study, it was found that thermal stress could also induce
structural changes to form co-supramolecular assembly of
lysozyme and apo BLA with an average size of more than 5
Received: July 7, 2015
Accepted: October 6, 2015
Published: October 6, 2015
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m.13 However, in both cases, the protein assemblies were not
in nanosize range and since no cross-linker was used, no
stability analysis of the assembly was performed. Arroyo-Mayo
and co-workers, however, used 1.3% glutaraldehyde and 3 h of
cross-linking for the preparation of BLA nanoparticles which
show a size range of 100200 nm.14 Nanoparticles of bovine
and human serum albumin have also been reported.1519
Such nanostructures have already been administered in vitro
to nd many applications such as drug and gene delivery in
medicine.17,18,20 Protein-based self-assembly are usually nontoxic, less immunogenic, biocompatible, and biodegradable, and
thus they are ideally suited for drug conjugation, loading, and
release.21 Although they carry and release drug molecules in
target tumor sites, the side eects of the drugs cannot be
prevented. However, if the protein self-assembly itself acts as a
therapeutic agent through forming a deadly fold or can be made
lethal against diseases such as cancer without conjugating with
drug molecules, the postadministration side eects could be
grossly prevented.
In this study, we report a simple, ecient, and reproducible
desolvation technique for the synthesis of nano-self-assembly of
bovine -lactalbumin (nsBLA) using glutaraldehyde as a
chemical cross-linker. The synthesis process of nsBLA involves
well-orchestrated coordination of multiple steps. Detail
preparation, characterization, stability, and its antiproliferative
activity in multiple cancer cells are reported. We also examined
the cellular uptake of nsBLA and folate-based targeting of
cancer cells. Moreover, we compared the antiproliferative
activity and biocompatibility of nsBLA with BAMLET, a widely
studied anticancer agent.
Numerous evidence exists in the literature that supports the
inherent cytotoxic potential of BLA. BLA was identied to
inhibit mammary epithelial cells.22 BLA exists in monomeric,
dimeric, and multimeric forms. Multiple reports showed that in
three forms and in aggregate state BLA caused cytotoxicity in
cancer cells.2327 An altered folding state of BLA was already
reported to develop a lethal conformational state when the
protein conjugated with oleic acid and formed as BAMLET that
was shown to have strong antitumor activity.2830 However,
some reports hold oleic acid responsible for the selective
toxicity toward cancer cells.3133 Most striking was the
experimental results reporting the toxicity of BAMLET in
normal cells mainly contributed by oleic acid that perhaps
hinders its future use in cancer.32
Therefore, in this study we show that stable nsBLA can be
prepared eciently, and such stable protein self-assembly can
be used as a therapeutic agent against multiples cancers.

A549) and normal cell lines such as HaCaT (human keratinocyte cell
line) and 3T3 (mouse broblast cells) were obtained from NCCS,
Pune, India.
2.2. Synthesis of nsBLA and BAMLET. nsBLA was prepared by
the standard desolvation technique14 followed by cross-linking with
glutaraldehyde (GTD). An amount of 10 mg of BLA was dissolved in
2 mL of Milli-Q water and was stirred for 15 min at 500 rpm. Ethanol
(8 mL) was added at the rate of 1 mL/min dropwise, during the
stirring process. GTD was added immediately to a nal concentration
of 0.1%, and the contents were allowed to stir for 8 h at 500 rpm. The
solution was centrifuged (25000g, 30 min, 4 C) and the pellet was
dispersed in 3 mL of Milli-Q water. Excess GTD was removed by 5
cycles of washing at 25000g for 30 min. As described by Mahanta et
al.,34 0.1% GTD was used as optimized concentration for cross-linking.
BLA of 1, 3, 5, 10, and 20 mg was used as starting material for
optimizing the self-assembly synthesis. The duration of cross-linking
was found to be 8 h.
BAMLET was prepared using a protocol described by Kamijima et
al.35 A mixture of protein and fatty acid (1:120 mol equiv) was
dissolved in 5 mL of PBS (pH 7.4) and incubated at 50 C. After
incubation for 10 min, the mixture was cooled to room temperature
and excess oleic acid was carefully removed by centrifugation.
2.3. Characterization. For FESEM, dried nsBLA sample was
placed in the sample chamber of the FESEM instrument and images
were captured using a voltage of 5 kV. For AFM analysis, nsBLA was
placed on a fresh mica sheet and dried under a continuous ow of N2
gas. The mica sheets were gently washed with Milli-Q water and dried
in a desiccator overnight. For imaging of the samples, standard tapping
mode was used, and 2080 N/m nominal spring constant of the
cantilever was used with a scan rate of 0.5 Hz. A sample volume of 10
L of nsBLA was diluted with 1990 L of deionized water and
analyzed for size analysis and -potential measurement in a Malvern
Zetasizer Nano-ZS dynamic light scattering (DLS) analyzer.
CD measurement was recorded in a wavelength range of 200 and
250 nm, with a resolution of 0.2 nm, a bandwidth of 1.0 nm, a scan
speed of 100 nm/min, and a standard sensitivity using a cuvette of
path length of 0.1 cm. Protein was dissolved in 20 mM sodium
cacodylate buer, pH 7.4. The CD spectrum was reported in
millidegrees. Percentage -helix, -sheet, and random coil were
calculated using the software provided along with the instruments.
For uorescence spectroscopy analysis, protein samples were
prepared in 20 mM sodium phosphate buer at pH 7.4. Trp
uorescence emission spectrum of BLA (60 g/mL) was recorded in
the range of 300500 nm at an excitation wavelength of 290 nm. The
excitation and emission slit widths were set at 10 nm each. Background
corrections were made with buer without protein in all cases.
The nsBLA-bound ANS uorescence was used for monitoring the
hydrophobic surface character of the protein assembly. The method of
measuring surface hydrophobicity of proteins using ANS (relative
measurement) has been widely used.3642 Optimized ANS concentration was added to nsBLA solution prepared in 20 mM phosphate
buer, pH 7.4. nsBLA-bound ANS uorescence emission spectra were
measured at an excitation wavelength of 350 nm. The slit widths of 5
and 10 nm were used for excitation and emission bandpasses,
respectively, with a scan rate of 240 nm/min. In all cases, protein
solutions were incubated for 30 min at 25 C.
For FTIR analysis, samples were analyzed at pH 7.0 in ATR mode.
The spectral range was used between 4000 and 500 cm1 with a
resolution of 2 cm1 and 25 scans per spectrum.
2.4. Stability of nsBLA. nsBLA solution (0.2 mg/mL) was heated
in a temperature range of 2080 C using the Peltier accessory. The
change in CD and Trp uorescence signal was recorded across the
entire temperature range. Similarly, the sample was evaluated for
stability at varying pH, i.e., 5.010 at 25 C. For measuring the relative
uorescence emission spectra of nsBLA, 60 g/mL of each sample was
excited at 290 nm, and the relative uorescence emission measurements were recorded between 300 and 600 nm within a temperature
range of 2080 C. The relative emission uorescence intensity of
both of the samples was also measured to evaluate for stability at
varying pH (510) at 25 C. For ANS-based uorescence study,

2. EXPERIMENTAL SECTION
2.1. Materials. Bovine -lactalbumin (BLA), glutaraldehyde, oleic
acid, n-acetylcysteine (NAC), N-hydroxysuccinimide (NHS), 8anilinonaphthalene-1-sulfonic acid (ANS), and gallic acid were
purchased from Sigma-Aldrich, Bangalore, India. MTT assay kit,
DMEM (Dulbeccos minimum essential medium), fetal bovine serum
(FBS), antibiotics, ethanol, sodium cacodylate, disodium EDTA,
protease inhibitor cocktail, DAPI, acridine orange, proteinase K, folic
acid, and sodium phosphate buer were purchased from HiMedia
India Pvt. Ltd., Mumbai. Hsp90, -actin, and Caspase 3 primary
antibodies and their respective secondary antibodies were purchased
from Abcam, Kolkata. Caspase 3 inhibitor (218750) and Triton-X
were purchased from Calbiochem, Mumbai, India. Plasticwares and
glasswares were purchased from Tarsons, Kolkata, India and Borosil,
Mumbai, India, respectively. All of the cell lines (breast cancer, MCF-7
and MDAMB-231; cervical cancer, HeLa; lung adeno-carcinoma,
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Figure 1. Characterization of nsBLA. (A) FESEM. (B) AFM image of nsBLA. (C) DLS particle size analysis of nsBLA. (D) CD spectra of native
BLA and nsBLA. The inset shows the secondary structural components of the samples. (E) Relative tryptophan uorescence spectra. (F) ANS
uorescence spectra. (G) FTIR spectra of both samples. All of the experiments were carried out at 25 C.
The stability of nsBLA against Proteinase K (PK) was evaluated by
analyzing their proteolytic susceptibilities. PK (3 M) was applied to
both the protein samples (200 g/mL) and incubated for 10 min. The
reaction was stopped by adding 1 mM PMSF (PK inhibitor). The
absorbance of both samples was recorded in a wavelength range of
250305 nm, and necessary baseline corrections were made. The
extent of degradation of the nsBLA sample was measured from the
drop in absorbance at 280 nm.

optimized ANS concentration was added into the nsBLA solution in

20 mM phosphate buer, pH 7.4. Solutions were incubated for 30 min
at 25 C. Protein-bound ANS uorescence was measured at 2080 C
and with varying pH (510) at an excitation wavelength of 350 nm
with a slit width of 5 and 10 nm for excitation and emission
bandpasses, respectively. In all of the experiments, native BLA was
used as a control.
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2.5. Cell Viability Assay. The cytotoxicity study was examined
using MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] assay.43 Cells were cultured in DMEM containing 10%
fetal bovine serum and 0.1 mg/mL penicillin and streptomycin. Cells
were seeded at a density of 1 104 cells/mL in a 96-well plate
containing 300 L of DMEM in each well and were allowed to
proliferate for 24 h. nsBLA and BAMLET were administered to the
cells, and the eect was monitored by MTT assay at the end of the
next 24 h. MTT (10 L of 5 mg/mL stock) was added 6 h before the
end point. At the end, the medium was removed, 100 L of DMSO
was added to dissolve the formazan formed, and the absorbance was
measured at 595 nm. The absorbance values were recorded, the
percentage cell viability was calculated from the absorbance values, and
the result was plotted.
2.6. Cellular Uptake of nsBLA. We performed the uorescence
imaging of MCF-7 cancer cells using a confocal laser scanning
microscope. Fluorescent dyes such as DAPI were used to stain the
nucleus, and acridine orange was used to observe the cellular uptake of
nsBLA. The cells were exposed to nsBLAacridine orange conjugate
(see Methods in the Supporting Information (SI)) for 12 h. At the end
of 12 h, we observed the cells for the presence of nsBLA particles
exhibiting orange uorescence inside the cytoplasm of MCF-7 cells.
2.7. Folate Receptor-Based Cancer Targeting. MDAMB-231 is
one such cell line that expresses folic acid (FA) receptors on its
surface. Hence, we conjugated nsBLA with NHS-FA using 0.1%
glutaraldehyde as a cross-linker leading to the formation of the
nsBLAfolic acid conjugate (nsBLA-FA). For the preparation of the
nsBLA-FA conjugate, the protocol reported by Li et al. was followed.19
The protocol for preparing N-hydroxysuccinimide ester of folate, i.e.,
NHS folate, is already reported.44
We also kept A549 cells that are FR (folic acid receptor) negative
and used as a control. Various concentrations (13.33333.33 g/mL)
of nsBLA-FA conjugate were administered to both MDAMB-231 cells
and A549 cells, and MTT assay was performed as per the protocol
mentioned earlier.
2.8. Evaluation of Reactive Oxygen Species Mediated Cell
Death. To ascertain reactive oxygen species (ROS) generation for
nsBLA-mediated cancer cell death, we applied nsBLA in both MCF-7
and MDAMB-231 cells and kept a corresponding group pretreated
with 2 mM acetylcysteine (NAC), a ROS inhibitor. The MTT assay
was performed as per the protocol mentioned in section 2.5.
2.9. Evaluation of Caspase 3 Mediated Apoptotic Cell Death.
Cell viability assay of MCF-7 induced by nsBLA and BAMLET was
performed in the presence of 100 g/mL gallic acid and Caspase 3
inhibitor. GA induces apoptosis in various cells mediated by Caspase 3.
We also performed the Caspase 3 as well as Hsp90 expression study to
support our cell viability results. We treated MCF-7 cancer cells with
nsBLA and BAMLET for 12 h. After treatment, cells were collected
and lysed in lysis buer in the presence of protease inhibitor cocktail.
The supernatant was obtained after centrifugation at 12500g for 10
min at 4 C. The concentration of protein in the supernatants was
measured by the bicinchoninic acid (BCA) assay. Then equal amounts
of protein (60 g) were separated by 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto
the nitrocellulose membrane (Millipore, Bengaluru, India). The blots
were incubated with respective primary antibodies overnight at 4 C
followed by incubation with secondary antibody for 4 h at 37 C.
Chemiluminescent detection was performed by the chemiluminescent
documentation system.
2.10. Hemocompatibility Assay. Freshly collected human blood
was gently resuspended and centrifuged at 1000g for 10 min. The
supernatant was removed, and the erythrocyte collected at the bottom
was washed three times by gentle suspension with 10 times the volume
of pyrogen free saline (0.9% NaCl). The erythrocyte pellet was gently
resuspended in normal saline and diluted to 0.8% (v/v). The solution
was placed in a sterile centrifuge tube, and various test samples were
administered to evaluate their hemolytic potential. Triton X (1%) was
used as a positive control, and erythrocyte suspension treated with
none was used as a negative control. The absorbance of the
hemoglobin released was measured at 405 nm. The percentage

hemolysis was calculated at 1 h, and the graph was plotted between

percent hemolysis and time (h).
2.11. Statistical Analysis. The data were represented as mean
SEM. Statistical analysis was carried out by one-way ANOVA followed
by Tukeys test using Graph Pad Prism 5.0. The p-value at <0.001
(***) was found to be statistically signicant vs the control group, by
one-way ANOVA and post-Tukeys test. All data are expressed as
mean SEM; n = 3.

3. RESULTS AND DISCUSSION

3.1. Preparation and Characterization of Self-Assembled Nanostructured Bovine -Lactalbumin.
Although BLA of 0.5, 1.5, 2.5, 5, and 10 mg/mL were used
as the starting material, only 5 and 10 mg/mL protein produced
regular and spherical nanostructures. However, below 5 mg/mL
concentration produced smaller particles with heterogeneity in
size (see SI Figure S1). GTD concentration was optimized for
cross-linking by observing its eect on the potential (see SI
Figure S3) and the shape of the particles using FESEM imaging
(see SI Figure S2). Although we used 0.1, 0.3, 1, 3, and 8%
GTD for the self-assembly process, GTD of 0.1% only
produced nsBLA with a mean size of 300 nm (see SI Figure
S2) and a potential of +40 mV (see SI Figure S3). The results
signied the synthesis of the smallest spherical nsBLA with high
stability in solution. A GTD concentration of 0.3% and higher
produced larger particles (SI Figure S2). The optimum
duration of cross-linking was found to be 8 h as it produced
homogeneous nsBLA; however, below 8 h, the process
produced irregular particle size due to incomplete cross-linking
(see SI Figure S4).
FESEM and AFM images conrmed the spherical shape of
nsBLA and a mean size of 300 nm (Figure 1A,B). DLS particle
size analysis (Figure 1C) revealed the hydrodynamic diameter
of nsBLA ranging from 100 to 700 nm with a mean of 300 nm.
Arroyo-Mayo et al.,14 however, used 1.3% glutaraldehyde and
3 h of cross-linking for the preparation of BLA nanoparticles.
While they achieved an average size of nanoparticles of 246 nm
with a pI of 3.61, we reported a closely similar average size (300
nm) of nanoassembly with a potential of +40 mV. Since
glutaraldehyde was the key factor in contributing charge, the
concentration of the cross-linker therefore can make a large
dierence in net surface charge of the nanoparticle. Hence, the
overall structural state of BLA in nsBLA might be dierent than
the nanoparticle prepared by them.
The spherical shape of particles provides many advantages
including lower resistance against ow and higher penetration
capacity across cell membranes. CD analysis (Figure 1D)
clearly revealed the change of secondary structural components
of BLA in nsBLA that was associated with a little increase of
both -helix and -sheet amounts (Figure 1D). Moreover, it
also showed a substantial decrease of CD signal that indicated a
gross structural change of the protein. Trp uorescence
measurement of nsBLA recorded a substantial drop in
uorescence indicating the quenching of intrinsic Trp
uorescence whereas the emission maximum remained
unchanged, perhaps occurring due to glutaraldehyde crosslinking of the BLA (Figure 1E).
ANS uorescence result of nsBLA (see Figure 1F) clearly
revealed the drop in protein-bound ANS uorescence. Since
protein-bound ANS uorescence is a measure of surface
hydrophobicity,45 this fact conrmed the lower surface
hydrophobicity of nsBLA than native BLA. The uorescent
hydrophobic dye 8-anilinonaphthalenesulfonate (ANS) has
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Figure 2. Thermal stability of BLA and nsBLA in a temperature range of 2080 C. (A) Change in CD spectra at 222 nm. (B) Change in Trp
uorescence intensity at 350 nm. (C) Change in ANS uorescence intensity at 475 nm. Stability of BLA and nsBLA in a pH range of 510. (D)
Change in ANS uorescence intensity at 475 nm. (E) Change in Trp uorescence intensity at 350 nm. (F) UVvis spectroscopic measurement of
native BLA and nsBLA before and after treatment with 3 M Proteinase K (PK). All data were expressed as the mean of three readouts generated
from the instrument.

value drastically. While at a glutaraldehyde concentration of

0.1%, we observed a potential of +40 mV, at 0.3%
glutaraldehyde it showed a value of 30 mV. However, beyond
the concentration of 1% glutaraldehyde, the potential showed
positive values (see SI Figure S3). The reason perhaps is the
net surface charge of nsBLA, which was the combined
contribution of both BLA and the cross-linker, glutaraldehyde.
Moreover, the specic conformational state of BLA in nsBLA
might play a crucial role in contributing the surface charge.
Similarly, it might also be the glutaraldehyde that was a key
factor in controlling overall surface charge,and hence, the
concentration of the cross-linker therefore can make a large
dierence in the net surface charge of the nanoassembly.
3.2. Preparation and Characterization of BAMLET.
BAMLET was prepared and directly used in our experiments.
The CD spectra of BAMLET was also monitored (SI Figure
S5), and cytotoxicity in various cancer cells was investigated to
conrm its anticancer activity. BAMLET demonstrated
cytotoxicity in cancer cells such as MCF-7, MDAMB231, and
A549 cancer cells. The strong anticancer potential in multiple
cancer cells and the CD spectra of BAMLET closely resembling
the one synthesized by Kamijima et al.35 proved the successful
synthesis of BAMLET.
3.3. Stability Studies of nsBLA. 3.3.1. Thermal and pH
Stability. The thermal stability analysis of nsBLA across a
temperature range of 2080 C was performed by CD and Trp
uorescence measurement attached with a Peltier system. The
CD result clearly showed that nsBLA remained stable in a
temperature range of 2070 C, with a rapid change in signal

been used widely as a probe for the presence of hydrophobic

patches on the protein surfaces.36,41 ANS is a unique surfacespecic uorescent probe that gives characteristic uorescence
emission upon binding with hydrophobic residues present on
the protein surface. However, the free dye in polar medium
shows negligible emission of uorescence. It is usually
considered that if ANS uoresces intensely upon binding a
protein or protein surface, the binding sites must be
hydrophobic. Hence, our result also signies that nsBLA
gains higher dispersibility in water that helps in smooth ow in
the liquid medium.
FTIR analysis clearly revealed the presence of amide I and
amide II bonds for nsBLA. From Figure 1G, nsBLA showed a
clear change in both the shapes of the spectra and peak
positions, which indicates a change in the secondary structure
of the BLA in the conjugates. The native BLA shows the
distinct peak for the amide I and amide II bands; however, the
amide I band shows a slight right shift in the position in nsBLA,
indicating a structural change. Moreover, the amide I band in
nsBLA is split into two peaks at 1654.87 and 1695.66 cm1 that
also conrm cross-linking. The amide II band in nsBLA was
attened and broad compared to native BLA, which may be due
to the interaction with GTD.
While Arroyo-Mayo and co-workers achieved an average size
of nanoparticles of 246 nm with a pI of 3.61, we reported a
closely similar average size (300 nm) of nanoassembly with a
potential of +40 mV.14
From Figure S3, we observed that the use of various
concentrations of glutaraldehyde inuenced the potential
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Figure 3. Cell viability assay of nsBLA and BAMLET in four dierent cancer cell lines: (A) MCF-7, (B) MDAMB231, (C) HeLa, and (D) A549.
The control experiment was also performed with (E) HaCaT cell (human keratinocytes) and (F) 3T3 cell (mouse broblast).

observed only at 80 C. However, free BLA showed a rapid

change of CD signal even at 50 C, as reected from the plot of
CD signal at 222 nm vs temperature (Figure 2A and SI Figure
S6A,B), which implied that nsBLA is highly thermostable. The
Trp uorescence measurements of native BLA and nsBLA,
between 300 and 500 nm, were also recorded at an excitation of
290 nm, under varying temperature conditions. We observed a
drastic change in the uorescence intensity of native BLA
compared to nsBLA. Although nsBLA demonstrated excellent
thermal stability until 80 C, a large red shift in peak was
observed beyond 50 C indicating an increase of polarity of the
environment around the Trp (Figure 2B, SI Figure S6C,D).
The increase in hydrophobic surface character of a protein is
positively correlated with the stability of proteins against
thermal stress.46,47 Hence, estimation of the surface hydrophobicity of the protein surface under varying temperature
conditions will help in assessing the thermal stability of the
protein. ANS uorescence emission spectra of nsBLA were
recorded within a temperature range of 2080 C. The results

(Figure 2C, SI Figure S6E,F) showed the decrease of proteinbound ANS uorescence with the increase in temperature while
the reverse was observed for nsBLA. Both of these results
clearly indicate that the surface hydrophobicity of native BLA
decreased sharply with an increase in temperature. However,
under the same condition the surface hydrophobicity of nsBLA
was slightly increased which is a good indicator of thermal
stability. The CD, Trp uorescence, and ANS uorescence
measurements conrmed the thermal stability of nsBLA.
The stability study of nsBLA across a pH range of 510 was
performed in a spectrouorimeter. We observed a drastic drop
in the native BLA-bound ANS uorescence intensity of native
BLA (Figure 2D, SI Figure S7A). However, the nsBLA-bound
ANS uorescence remained unchanged (Figure 2D, SI Figure
S7B). The Trp uorescence measurement of the samples
within the pH range of 510 (Figure 3D,F) also showed the
higher stability of nsBLA than free BLA (Figure 2E, SI Figure
S7C,D). The results clearly indicate that, with the increase in
pH from 5 to 10, the Trp present on the surface of native BLA
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Figure 4. Fluorescence microscopic imaging of MCF-7 cells at 0, 3, 6, and 12 h postadministration of acridine-conjugated nsBLA: (A) 0, (B) 3, (C)
6, and (D) 12 h.

(Figure 3B) and A549 cell lines (see Figure 3D), BAMLET
demonstrated lower antiproliferative activity than nsBLA.
Moreover, BAMLET showed little antiproliferative activity in
HeLa cells (10%) (Figure 3C), while nsBLA demonstrated
90% cell death. HeLa cells are highly invasive compared to
MDAMB-231, A549, and MCF-7, and hence, we concluded
that nsBLA possessed higher antiproliferative potential
compared to BAMLET. Moreover, nsBLA showed negligible
toxicity compared to BAMLET when applied to HaCaT
(human keratinocyte cells) and 3T3 (murine broblast cells)
(Figure 3E,F). We have used 0.1% GTD as the nal
concentration for the preparation of nsBLA. To rule out the
possibility of cytotoxicity from GTD, we performed the
cytotoxicity study of GTD. We found that 0.1% GTD did
not show any cytotoxic eect in both normal (HaCaT) and
cancer cells (MCF-7) (see SI Figure S9).
3.5. Cellular Uptake Study. Fluorescent dyes such as
DAPI and acridine orange were used to observe the cellular
uptake of nsBLA. Cells stained with DAPI clearly exhibited blue
uorescence for the nucleus in live cells (Figure 4A). nsBLAconjugated acridine orange exhibited orange uorescence
(Figure 4BD) in MCF-7 cells.
At the end of 12 h postadministration, we observed the
appearance of nsBLA particles exhibiting orange uorescence
inside the cytoplasm of MCF-7 breast cancer cells (Figure 4D),
indicative of cellular uptake of nsBLA in MCF-7 cells.

moved inside the protein core, which resulted in the decrease in

Trp uorescence emission intensity. In the case of nsBLA, we
observed a fairly stable uorescence intensity indicating no
conformational change around Trp residue. The preceding
results clearly ensured the stable structural conformation of
BLA in nsBLA.
3.3.2. Stability of snLYZ against Proteinase K. We also
performed the PK-mediated digestion to nd out its resistance
against protease digestion. PK usually digests the non-native
proteins very fast rather than the native one. In this case, nsBLA
was partially denatured and unfolded but self-assembled in the
presence of 0.1% GTD. The spectroscopic measurement of
nsBLA after PK digestion exhibited almost no change in the
peak position of the spectra suggesting increased stability
against PK digestion compared to native BLA (Figure 2F). The
increased stability of nsBLA against PK may be due to the fact
that the digestion sites were inaccessible to PK due to the selfassembly of BLA.
3.4. Cytotoxicity Study. The cytotoxicity study of nsBLA
was examined in four dierent cell lines: breast cancer (MCF-7
and MDAMB-231), cervical cancer (HeLa), and lung
carcinoma (A549) using a range of concentrations (16.66
333.33 g/mL). Results in Figure 3AD clearly showed strong
cytotoxicity of nsBLA in all cancer cell lines. While nsBLA
showed more than 80% cell death in all cell lines after 24 h of
administration, BAMLET exhibited comparable toxicity only in
the MCF-7 cell line (see Figure 3A); however, in MDAMB-231
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3.6. Targeting Cancer Cells via Folate Receptor
Binding. We observed an increase in the inhibition of cell
proliferation of MDAMB-231 cells by nsBLA-FA than nsBLA
alone. We also found an increase in conjugate cytotoxicity in
MDAMB-231 cells compared to A549 cells (Figure 5). This

expression level was slightly increased after the treatment of

nsBLA, however, more pronounced with BAMLET treatment.
The result clearly indicates that the increased expression of
Hsp90 after treatment of nsBLA was due to the generation of
excessive stress or ROS formation in MDAMB-231cells.
3.9. Hemolysis Assay. While nsBLA produced negligible
hemolysis (SI Figure S8A,B), both BAMLET and oleic acid
demonstrated strong hemolytic activity (SI Figure S8C,D).
Further, we found that our prepared nsBLA showed strong
hemocompatibility until a concentration as high as 333.33 g/
mL; however, BAMLET showed profound hemolysis even
beyond 16.66 g/mL concentration where BAMLET killed
only 10% cancer cells (Figure 3). Therefore, comparing the
results, nsBLA may be considered to be a far better choice to
use in cancer therapy than BAMLET.
In Figure 7, we developed a mechanistic model based on our
experimental ndings on how our nsBLA caused the death of
various cancer cells.

4. CONCLUSION
In this study, we have successfully reported the preparation of
stable self-assembled nanostructured bovine -lactalbumin with
spherical shape having 300 nm mean diameter using chemical
cross-linking method. The amount of cross-linker, glutaraldehyde (GTD), was used as minimal as possible (0.1%) and its
nontoxicity was examined in normal cells. The whole synthesis
process was optimized with regard to average diameter, size
distribution of nsBLA, and amount of precursor protein,
glutaraldehyde concentration, and stability. The nanoassembly
was found to be highly stable against a wide range of pH,
temperature, and protease stress. In order to show its
applications, we demonstrated its strong antiproliferative
potential in three dierent cancer cells such as MCF-7 and
MDAMB-231 (breast), HeLa (cervical), and A549 (lung) via
ROS generation with negligible toxicity in normal cells such as
HaCaT (human keratinocytes) and 3T3 (mouse embryonic
broblast cells). We also demonstrated that folate-conjugated
nsBLA enhanced cytotoxicity through improved cellular uptake
of nsBLA in cancer cells expressing folate receptor and
cytotoxicity in MDAMB-231 cells (FR + ve).
To compare the anticancer potential of nsBLA, we
synthesized the widely studied protein-based anticancer agent
BAMLET using a well-known protocol and compared their
antiproliferative activity. We proved in the study that nsBLA
exhibited higher cytotoxicity than BAMLET. Moreover, while
BAMLET exhibited poor biocompatibility, nsBLA demonstrated excellent compatibility in normal cells as well as human
erythrocytes.
There were multiple reports which proved that the cytotoxicy
of BAMLET was due to the fatty acid, i.e., oleic acid, but not
due to the protein part.31,48,49 This fact has raised concern
about the anticancer potential of BLA protein in BAMLET.
However, Fontana et al.48 stressed the importance of the
protein part of HAMLET-like complexes and did not rule out
the possibility of its toxicity under certain experimental
conditions. In fact, Delgado et al.49 recently reported that, in
BAMLET, oleic acid was probably located on the surface of
BLA and thus protected BLA from proteolysis. Therefore, it
might be possible that, in BAMLET, BLA could not exhibit its
inherent cytotoxicity in cancer cells due to surface shielding.
However, in our present study, we proved categorically that
the nanostructured self-assembled BLA (nsBLA) caused the
sole toxicity in multiple cancer cells sparing normal cells

Figure 5. Cell viability assay of FA-conjugated nsBLA in MDAMB-231

cells and A549 cells. The inset shows the percentage of cell viability of
A549 and MDAMB-231 cells treated with nsBLA and nsBLA-FA
conjugates at a dose of 133.33 g/mL.

may be due to the fact that the MDAMB-231 cells express

folate receptors on the membrane surface that facilitated the
entry of nsBLA-FA inside the cells. However, the A549 cells do
not express FR, hence the entry of nsBLA inside the cytoplasm
follows the normal route of entry into cells.
3.7. Cell Death by Reactive Oxygen Species. We
observed that the administration of NAC completely inhibited
the ROS-based cell death induced by nsBLA (Figure 6AD) in
both MCF-7 and MDAMB-231 cells, which indicates sucient
ROS production in cancer cells which subsequently triggers
rapid cell death of MCF-7 cells. However, in BAMLET, we
observed partial inhibition of cell death (Figure 6AD),
indicating a secondary death mechanism, besides the ROSbased mechanism. Our prepared BAMLET was already
reported to cause apoptosis-mediated cell death. However,
the lysosomal-mediated cell death was also observed when
prepared using other methods.28 Therefore, our result also
validates the fact of its multiple cell death mechanism.
However, nsBLA that has a higher potential of cancer cell
death than BAMLET triggers only an ROS pathway.
3.8. Cell Viability Assay Using Caspase 3 Inhibitor.
From the uptake study, we found that the cell and nucleus size
was increased after the treatment of nsBLA, which is contrary
to the normal apoptosis process where cell volume shrinkage is
observed. Hence, we performed an independent cell viability
assay in the presence of Caspase 3 inhibitor that prevents
Caspase-mediated apoptosis and gallic acid which induces
Caspase 3 mediated apoptosis in MDAMB-231 cells. From the
results (Figure 6E), we observed that the cell death induced by
nsBLA was Caspase 3 independent which clearly ruled out the
possibility of cell death due to apoptosis.
We also examined Caspase 3 expression to ascertain our
results obtained from Figure 6F, along with Hsp90 expression
that positively correlates with the measure of the stress. We
found that the basal expression level of Caspase 3 hardly
changed compared to control. However, the Caspase 3 level
slightly increased for BAMLET. Moreover, the Hsp90
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Figure 6. Reactive oxygen species (ROS) mediated cell death of cancer cells. (A and B) MCF-7 cells were treated with nsBLA and BAMLET in the
presence and absence of NAC at 24 and 48 h. (C and D) Experiments on MDAMB-231 cells. (E) MTT assay of MDAMB-231 cells administered
with nsBLA in the presence and absence of Caspase 3 inhibitor (written as C in Figure). (F) Western blot analysis of Caspase 3 and Hsp90
expression in MDAMB-231 cells treated with nsBLA and BAMLET.

unharmed. Here, we did not use any second component such as

oleic acid that was used in BAMLET/HAMLET; instead, we
prepared self-assembly of BLA protein by the cross-linking
using GTD at very low concentrations (0.1%). At such low

concentration of GTD, no cytotoxic eect was observed in

normal as well as cancer cells (see SI Figure S9). Hence, the
cytotoxicity caused in our studies must be from nsBLA alone.
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Figure 7. Model of cell killing induced by nsBLA. (I) Native BLA is converted to its self-assembled state by the simple process of desolvation
followed by cross-linking. (II) nsBLA enters cancer cells through the normal route of endocytosis. (III) nsBLA generates ROS within the cancer
cells. The cancer cells are already in a stressed state, and the increased stress in the form of ROS generated by nsBLA and reconstituted nsBLA add
to the stress of the cancer cells. The cancer cells are not able to cope with the additional amount of stress from ROS leading to cell death.

Thus, from our study, nsBLA emerged as a far better

performer than BAMLET in terms of its lethality against cancer
cells and biocompatibility in normal cells. Such self-assembly of
BLA not only helps in cancer therapy but proves a better
therapeutic option than existing nanoparticles-based protein
and peptide delivery mediated therapeutics in cancer.

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ASSOCIATED CONTENT

S Supporting Information
*

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acsami.5b06076.
Method of preparation of nsBLA-acridine orange
conjugates, results of optimization of the amount of
starting material, size, stability, duration of glutaraldehyde
cross-linking of bovine -lactalbumin for the preparation
of its self-assembled nanostructure, BAMLET characterization using by CD spectroscopy, thermal stability of
self-assembled nanostructured BLA in a temperature
range of 2080 C monitored by CD, tryptophan as well
as ANS uorescence spectroscopy, stability of nsBLA
under varying pH monitored by uorescence spectroscopy, hemolytic assay of self-assembled nanostructured
BLA and its comparison with BAMLET, and eect of
glutaraldehyde on normal and cancer cells (PDF)

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: spaul@nitrkl.ac.in. Tel.: +91-0661-2462284 (O); +910661-2463284 (R). Fax: +91-0661-2462022.

Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
We deeply thank the Department of Biotechnology (DBT),
Government of India for nancial support (Grant No. BT/
PR13853/NNT/28/481/2010) and the National Institute of
Technology Rourkela, Government of India for providing the
research facility for carrying out this work.
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