Journal of Molecular Liquids 282 (2019) 169–176

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Journal of Molecular Liquids

journal homepage: www.elsevier.com/locate/molliq

Biopolymer nanoparticle surface chemistry dictates the nature and

extent of protein hard corona
Aalok Basu a,b, Sonia Kundu a, Chitra Basu c, Sumanta Kumar Ghosh a, Runa Sur c, Arup Mukherjee a,d,⁎
a
Department of Chemical Technology, University of Calcutta, 92 A.P.C. Road, Kolkata 700009, West Bengal, India
b
Dr. B.C. Roy College of Pharmacy and Allied Health Sciences, Bidhannagar, Durgapur 713206, West Bengal, India
c
Department of Biophysics, University of Calcutta, 92 A.P.C. Road, Kolkata 700009, West Bengal, India
d
Department of Biotechnology, Maulana Abul Kalam Azad University of Technology, BF 142, Sector 1, Salt Lake City, Kolkata 700064, West Bengal, India

a r t i c l e i n f o a b s t r a c t

Article history: Biopolymer nanoparticles functionalized with targeting ligands serve as carriers for a wide range of therapeutic
Received 27 August 2018 payloads to desired sites of the body. Upon introduction into the blood stream, the nanoparticles tend to bind
Received in revised form 5 February 2019 with the plasma proteins in its immediate vicinity, forming a protein corona. In this work, we have investigated
Accepted 4 March 2019
the effects of differently functionalized PLGA nanoparticles on the formation of protein corona. It was observed
Available online 5 March 2019
that nanoparticles of uniform size range adsorbed protein in varying amounts and the final hard corona differed
Keywords:
structurally with respect to nanoparticle surface functionalization. A combination of fluorescence spectroscopy
PLGA nanoparticles and FTIR studies revealed that the association of plasma proteins with PLGA nanoparticles during corona forma-
Protein corona tion was intricately guided by the nanoparticle surface chemistry. Densitometric analysis through one dimen-
Nanoparticle-plasma protein interaction sional gel electrophoresis further showed that the plasma proteins present in the hard corona were different
for each case of surface functionalization. The aggregation of plasma protein during corona formation as studied
through Thioflavin T fluorescence and circular dichorism spectroscopy was also found to significantly differ with
each nanoparticle type. It is evident that surface chemistry of biopolymeric nanoparticles defines the final corona
form in the physiological environment, and this study on detailed understanding of nanoparticle-protein corona
would help to develop nanomedicines for clinical applications.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction
Advancements in the field of biopolymeric nanoparticle engineering
have rapidly promoted their utility in medical diagnostics and thera-
peutic interventions, such as pin pointed targeting of drug, enhanced
cellular uptake, reduced toxicity, and programmed release of their pay-
load [1]. Poly (lactic-co-glycolic acid) (PLGA) is one widely used bio-
polymer in designing of nano-scale materials, and can be differentially
tailored to serve specific needs. Functionalization of PLGA nanoparticles
with site-specific ligands to hit cellular targets, polyelectrolyte deposi-
tions to improve cellular uptake or PEGlyation to reduce elimination
by phagocytosis are of the common strategies to develop smarter drug
delivery devices [2–4].
Despite of advances in preclinical development of nanoparticle drug
delivery systems, there has always been a therapeutic shortfall upon ex-
posure of nanoparticles in the physiological environment. A survey con-
ducted by Warren C.W. Chan and co-workers revealed that only 0.7% of
administered dose of nanoparticles are effectively delivered to its
⁎ Corresponding author at: Department of Chemical Technology, University of Calcutta,
92 A.P.C. Road, Kolkata 700009, West Bengal, India.
E-mail address: arupm1234@gmail.com (A. Mukherjee).
intended target site [5]. Whenever a nanoparticle presents itself in a bi-
ological milieu, its surface is known to get quickly embedded in plasma
proteins, thus altering the biological identity of the nanoparticle. This
form, termed by K. A. Dawson as “protein corona”, affects the cellular
uptake and subsequent fate of the nanoparticle in vivo [6,7]. Nanoparti-
cle protein corona is currently conceived as dynamic physiological ac-
tivity which ultimately equilibrates in a plasma protein adsorption [8].
Current concepts indicates that the corona comprise of a tightly
bound, “hard” near-monolayer of proteins which is again shielded by
a “soft” layer of loosely associated proteins [9]. The proteins adsorbed
in the hard corona have longer residence time making it both physiolog-
ically relevant [10] and easier to isolate [11]. Proteins upon adsorption
on the nanosurface experience imbalance in entropy and change in
structural conformations [12]. Such changes can be either reversible or
irreversible [13] and is a function of nanoparticle surface curvature
[14–16]. A number of reports in past few years laid down the fact that
nanoparticles have a profound effect on protein fibrillogenesis, which
is characterized by formation of cross β sheets at the expense of native
α helical structure [17].
Most of the initial works are concentrated upon formation and pro-
teomics of the corona around different metal nanoparticles [18–20].
More recent studies depicted that surface charge and size of

https://doi.org/10.1016/j.molliq.2019.03.016
0167-7322/© 2019 Elsevier B.V. All rights reserved.
170 A. Basu et al. / Journal of Molecular Liquids 282 (2019) 169–176
nanoparticles play a significant role in corona formation and cellular in-
teractions thereafter [21,22]. Several workers have attempted to regu-
late the protein corona formations around the nanoparticle by tuning
their surface-bound ligands [23], introduction of zwitterionic function-
alities [24], or through synthesis of PEGylated copolymers [25]. How-
ever, works on nano-bio interface of biopolymeric nanoparticles and
their functionalized counterparts are fewer in number. Kreuter (2013)
and Langer (2015) have investigated the proteins eluted from the co-
rona around PLGA nanoparticles through SDS PAGE, followed by diges-
tion, purification and peptide identification with mass spectroscopy
[19,26]. Coronated nanoparticles are though the final form in physiolog-
ical environment, a proper insight in that domain is rather sparse. PLGA
is a Food and Drug Administration (FDA) approved biopolymer and is
garnered lot of attention for its biocompatibility and degradability
[27]. Our work has been intended to investigate the effect of surface
functionalization of PLGA nanoparticles on the formation of their hard
corona structures. Initially, bare pluronics stabilized and polyelectro-
lytes such as heparin and chitosan coated PLGA nanoparticles of uni-
form size range were synthesized. The structure of the hard corona
around these nanoparticles and their underlying chemical interactions
formed the basis of our study. Affinity constants of different nanoparti-
cle types derived from fluorescence spectroscopy were collectively used
with FTIR data to explore the molecular interactions occurring in
nanoparticle-protein interfaces. Further investigations to identify the
effects of these molecular interactions on protein secondary structures
were also carried out through Thioflavin T fluorescence and circular di-
chroism spectroscopy. It is expected that this work would shed some in-
sight into mechanism of protein corona on functionalized biopolymeric
nanoparticles commonly used in clinical settings.
2. Materials and methods
2.1. Materials
PLGA (50:50), 85% deacetylated chitosan (MW medium), heparin
sodium, pluronic F -127, Bradford reagent for protein assay, lyophilized
human plasma and human serum albumin (purified) were all procured
from Sigma Aldrich (St. Louis, US). All solvents, including water, were of
HPLC grade and obtained from Spectrochem Pvt. Ltd. (Mumbai, India).
Potassium bromide for IR analysis was purchased from Merck (Darm-
stadt, Germany). Thioflavin T dye for fibrillation experiments were pur-
chased from TCI Chemicals Pvt. Ltd. (Chennai, India).
2.2. Nanoparticle synthesis
PLGA nanoparticles (Pln) were prepared in solvent diffusion tech-
niques perfected earlier [28]. Briefly, 50 mg of the polymer was dis-
solved in 5 mL of acetone, and dropped into 10 mL aqueous pluronic
F-127 (1% w/v) solution under magnetic stirring. Stirring was continued
for 3 more hours and the nanoparticles formed were harvested by cen-
trifugation at 14,500 rpm. Pln were re-dispersed in 10 mL of water and
collected after centrifugation for subsequent polyelectrolyte coating.
The chitosan or heparin polyelectrolyte (0.05% w/v) was dissolved in
water and the solution pH was maintained at 6. Pln nanosuspension in
water (3% w/v, 2 mL) was then added drop-wise into a 10 mL polyelec-
trolyte solution under magnetic stirring. Stirring was continued for
5 min more and the resultant chitosan or heparin surfaced nanoparti-
cles (PlnC or PlnH) were harvested by centrifugation at 14,500 rpm.
A solute free technique was perfected to obtain nanoparticles in nar-
row size window [29]. Gravity driven sedimentation cycle was carried
out to separate nanoparticles from solutions. Briefly, nanoparticle sus-
pension was first subjected to a mild centrifugation at 6000 rpm to sed-
iment any large particles. The supernatant was collected and
centrifuged again at 14,500 rpm. Sediment particles were re-dispersed
in particle free water and the stocks were maintained at 4 ± 0.5 °C for
further studies.
2.3. Nanoparticle characterization
Particle analysis in all cases were carried out in a Zetasizer (Malvern
Nano ZS, Malvern, UK) and measurements were completed in dispos-
able polystyrene cuvettes against 4 mW He-Ne laser using a back scat-
tering angle of 173°. Electrophoretic mobility was recorded in PBS
(50 mM, pH 7.4) and zeta potentials were recorded for comparative
analysis.
TEM micrographs of nanoparticles were obtained using JEOL JEM
2100 HR transmission electron microscope capable of point to point res-
olution. Highly diluted samples were deposited on carbon coated cop-
per grid (Ted Pella Inc., US) and micrographs were collected at a low
accelerating voltage of 120 kV.
Polyelectrolytes on nanoparticles were quantified using colorimetric
assays. Chitosan interaction with alizarin red dye (0.1% w/v) at 571 nm
was used to develop a concentration dependent standard graph, y =
0.002x + 0.084, R2 = 0.998. The chitosan deposit on PlnC was estimated
from the concentration difference between the initial solution and the
post harvested supernatants. Three separate batches of experiments
were run and the data were averaged for comparative purposes. Hepa-
rin was quantified using toluidine blue color reactions [30]. Different
concentrations of heparin standard solutions were first reacted with to-
luidine blue (0.025% w/v) and the absorbance at 562 nm was used to
generate a standard graph y = 0.038x + 0.127, R2 = 0.992. Heparin
content on nanoparticles was derived from the difference of polyelec-
trolyte initial solution concentration and the post harvesting accumu-
lated supernatant content. An average of three batch experiment was
recorded and the data were presented in percentage.
2.4. Nanoparticle protein corona
Lyophilized human plasma was initially dissolved in 1 mL of PBS
(50 mM, pH 7.4). The reconstituted solution was then diluted to 1% v/
v for use in subsequent experiments [31]. Nanoparticles dispersed in
PBS (500 μg mL−1) were incubated with the plasma solutions for 1 h
at 37 ± 0.5 °C. The solutions were then centrifuged at 14,500 rpm in
order to obtain hard corona nanoparticles. Individual pellet deposits
were dispersed in 1 mL PBS and washed thrice by centrifugation in
order to ensure complete removal of any loosely bound proteins. The
final hard corona nanoparticles were re-suspended in PBS for DLS mea-
surements prior to in-depth analysis.
2.5. Protein quantification
Bradford assay (Sigma Aldrich, US) was performed to estimate the
mass of protein on nanoparticles surface before and after the formation
of hard corona [32]. Briefly, 1 mL of 1% plasma solution was incubated
with or without 500 μg of nanoparticles for 1 h. 0.1 mL of the superna-
tant acquired after centrifugation was mixed with 3 mL of Bradford re-
agent, and analyzed at 595 nm using a UV–Visible spectrophotometer
(UV 1800 Shimadzu, Japan). An absorbance (y) vs. concentration
(x) graph, y = 0.690x − 0.071, R2 = 0.999, was initially developed
from standard HSA solutions in PBS and used for protein quantification
throughout.
2.6. Steady-state fluorescence quenching studies
Intrinsic fluorescence from 1% v/v human plasma in PBS was first re-
corded in a spectrofluorimeter (Perkin-Elmer LS-5, PerkinElmer Inc.,
US). Different concentrations (0 to 40 μg mL−1) of each nanoparticle
types were incubated with plasma at 37 °C for 1 h [33]. The quenching
of emission spectra were recorded over a range of 290–400 nm with ex-
citation wavelength set at 280 nm, and the data were plotted in a log-
log graph to derive plasma protein affinity constant in each case.
 A. Basu et al. / Journal of Molecular Liquids 282 (2019) 169–176
2.7. FT IR studies
FT IR spectral analysis was performed using Jasco-670 Plus FTIR in-
strument (Jasco, Japan). Spectral analysis was carried out at a resolution
of 4 cm−1 and an average of 128 scans were recorded over the mid IR
ranges of 4000–400 cm−1. Individual sample of nanoparticles were ly-
ophilized and then pressed in IR grade KBr to develop transparent pel-
lets [34]. Gathered data were stacked using Bio-Rad software (Bio-
Rad, KnowItAll, USA) for analysis of bare nanoparticles and protein co-
rona interactions.
2.8. Gel electrophoresis
Plasma protein binding onto nanoparticle surface was determined
by 1 D sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS PAGE) following a method explained elsewhere [21].
Nanoparticle-protein hard coronas obtained from the procedure as de-
scribed in Section 2.4 was re-suspended in 20 μL of 1× loading buffer.
Samples were incubated at 90 °C for 2 min, cooled to room temperature.
Nanoparticle surface bound proteins were separated on 10% polyacryl-
amide SDS PAGE in an electric field run under constant voltage on 100
V for 45 min. The resulting gels were fixed by Coomassie blue stain
and recorded using a gel documentation system (BioRad, Hercules),
and gel densitometry was performed using Bio-Rad Gel Doc using
Quantity One software. Plasma and purified human serum albumin
were also run along-side for comparison.
2.9. Thioflavin T (ThT) dye interactions studies
Nanoparticles (Pln, PlnC or PlnH) were dispersed in 1% v/v human
plasma in PBS and incubated at physiological temperature (37 ± 0.5
°C) for 1 h. Samples were then re-incubated with ThT (10 μM) and the
fluorescence emissions were measured at physiological temperature
(37 ± 0.5 °C) in each case [35]. The excitation and emission wavelength
were set at 450 nm and 482 nm, respectively. Amyloid-like plasma fi-
brillation was further induced in 1% v/v human plasma in PBS at 60 °C
for 1 h and that effect was assumed as the positive control.
2.10. Circular dichroism
Far-UV CD measurements of the samples (from Section 2.9) were
performed on a JASCO J-815 CD spectro-polarimeter with a thermostat-
ically controlled cell holder at 25 °C, in a cuvette of 0.1 cm path length.
The scan speed was set as 100 nm min−1 and ellipticity was recorded
between 190 and 250 nm from an average of three scans.
2.11. Statistical analysis
All the experiments were performed in triplicates and their results
are expressed as their mean ± S.D. Data from comparative studies
were interpreted using student t-test and the difference was considered
significant as p b 0.05.
3. Results & discussion
3.1. Nanoparticle characterization
Our studies were carried out on surface functionalized PLGA nano-
particles, which present great potential in various therapeutic applica-
tions [2]. Pluronics stabilized PLGA nanoparticles obtained through
solvent diffusion techniques were subsequently coated with heparin
or chitosan. Highly mono-dispersed nanoparticles (Fig. S1), either in
pristine form or coupled with chitosan or heparin, were obtained
through selective centrifugation and were used to explore the effect of
nanosurface functionalization on protein corona formation. Electron
microscopy confirmed that the particles were spherical in shape and
171
thus eliminated the possibility of affecting the corona structure due
morphological differences [15].
A thorough characterization of the nanomaterials was performed
through independent experimental procedures to assert polyelectrolyte
deposition on the particle surface. Polyelectrolyte mass deposition on
the nanoparticles was quantified using alizarin red and toluidine blue
based colorimetric reactions for chitosan and heparin, respectively
[36]. In case of chitosan coating, the entrapment efficiency was calcu-
lated to be 74.3%, while for heparin the value was 51.7%.
FT IR spectra also confirmed the deposition of chitosan and heparin
on the PLGA nanoparticle as in their individual cases. The pluronics sta-
bilized Pln spectrum exhibited a sharp characteristics peak at
1759 cm−1 due to presence of carbonyl group, while vibrations for
C\\O and C\\H were recorded at 1456 cm−1 and 1169 cm−1. Coating
of chitosan upon Pln could be easily identified from the presence of
\\OH peak at 3438 cm−1, and amide band stretch at wavenumbers
1636 cm−1, and 1533 cm−1. Heparin functionalized Pln exhibited char-
acteristics stretching of CH2SO3− at 959 cm−1 and vibrations of COO−
groups at 1632 cm−1. A wide band at around 3426 cm−1 was prominent
due to presence overly expressed hydroxyl moieties in heparin mole-
cules. The peak at 1759 cm−1 from Pln was preserved in both cases of
PlnC and PlnH.
Further, the zeta potential for Pln, PlnC and PlnH were found to be
−19.7, +22.3 and −38.4 mV, respectively. These recordings revealed
the changes in nano-surface chemistry in each type of particles and
well correlated with the FT IR observations. The negative charge of Pln
was prevalent due to the exposed carboxylic polymer chain groups of
PLGA. Chitosan coating with exposed amide groups provided a positive
charge to PlnC, whereas the presence of sulphonate and carboxylate
groups of heparin were responsible for a high charge density on PlnH
surface.
3.2. Protein corona formation
Protein corona formation was studied by incubating an equal
amount (500 μg) of differently functionalized nanoparticles for 1 h in
PBS containing 1% human plasma. Evolution of protein corona around
the nanoparticle is a dynamic phenomenon involving reversible and ir-
reversible attachment of protein entities on the surface [24]. Hard co-
ronas of the differently functionalized nanoparticles obtained through
centrifugation, were of sole interest for our work. Dynamic light scatter-
ing (DLS) analysis was performed under dilute conditions provided the
initial evidences of protein interactions on nanoparticle surface. The re-
sults showed an increase in the hydrodynamic diameter (Fig. 1A) of in-
dividual particles ranging from 52.1% (for Pln) to 171.2% (for PlnC), with
the adsorption of varying amounts of plasma proteins (Fig. 1B). DLS
measurements also exhibited a divergence from the original polydisper-
sity index (data not shown), suggesting particle aggregation and
changes in overall dispersion pattern (Fig. 1C).
Similar observations recorded from the zeta potential analysis
(Fig. S2) were used to interpret the stability of colloids in plasma envi-
ronment. Exposure of nanoparticles to plasma results in lowering of
charge density with instantaneous binding of oppositely charged pro-
teins [37]. However, the prevalence of a slight negative charge on the
nanomaterial surface at pH 7.4 was observed due to the subsequent ad-
sorption of serum albumin [38]. The corona of PlnC faced a maximum
electrostatic aggregation effect in biological media, therefore tends to
settle down more. The results further showed that minimum deviation
of zeta potential values occurred in case of the Pln- protein corona,
whose stability is mediated by steric repulsion offered by the amphi-
philic nature of the pluronics [39].
3.3. Nanoparticle protein interaction studies
Elucidating the interaction of protein on nanoparticle surface is one
important step to gain detailed information on protein corona
172 A. Basu et al. / Journal of Molecular Liquids 282 (2019) 169–176
Fig. 1. (A) Particle size distribution for (i) Pln (ii) PlnC (iii) PlnH in absence and presence of human plasma (1%) (B) Protein adsorption quantification (C) Transmission electron
micrographs of (i) Pln (ii) PlnC (iii) PlnH in presence of human plasma (1%).
formation. Preliminary information about nanoparticle-protein interac-
tion was obtained from tryptophan fluorescence spectroscopy, as tryp-
tophan emission is highly specific to changes in local environment [40].
A gradual quenching of fluorescence intensity between 300 and 450 nm
at λex = 295 nm was observed for all three nanoparticle types (Fig. 2),
which can be attributed to the conformational changes of human
serum proteins around their tryptophan residues upon interaction
with the nanoparticle surface.
Nanoparticle-protein binding constants in 1% human plasma were
derived from log [(F0-F)/(F-Fs)] vs. log [nanoparticles] plots for all
three types of nanoparticles. It was observed that among the other
types of nanoparticles, PlnC had the maximum affinity towards plasma
proteins (Kb = 2.96), due to a typical guest-host complex formation
[41].The binding constant value was found to be at a lesser extent (Kb
= 2.37) in case of PlnH and least (Kb = 1.12) for the pluronics stabilized
Pln. The results clearly suggested that surface engineered biopolymeric
nanoparticles associated differently with plasma proteins under similar
conditions, despite of identical morphological characteristics.
The underlying interactions leading to the nanoparticle-protein
binding was further investigated through FT IR spectroscopy. Amide I
band at 1630 cm−1- 1658 cm−1 (due to C_O stretch) and amide II
band at 1525 cm−1- 1547 cm−1 (associated with C\\N stretch and
N\\H in-plane bending) for plasma protein were recorded and moni-
tored for subsequent nanoparticle-protein interaction studies. The in-
tensity of amide I band is related to secondary structure of protein
whereas amide II signal is attributed to protein adsorption on particle
surface [42]. Detailed analysis of protein interactions was done through
IR experiments, where 1650 cm−1- 1658 cm−1 region of amide I peak is
a representation of α-helical structures and 1620 cm−1- 1645 cm−1 re-
gion signifies presence of the β-sheets [13]. Therefore, alterations in
these bands characteristics would help explore how nanoparticle sur-
face chemistry affects the stability of native proteins which participate
in corona formation. Pln when exposed to 1% human plasma, exhibited
the dual protein peaks (for amide I and amide II) at their exact positions,
while retaining the characteristic peaks of pluronics stabilized nanopar-
ticles (Fig. 3). This indicated the absence of any chemical interactions
among the protein residues and Pln, and that the corona formation
around the Pln was merely due to surface deposition of plasma proteins.
In case of chitosan coated nanoparticles, the bands originally assigned to
the amide stretch for PlnC were not detected upon co-incubation with
1% human plasma. However, amide I band of plasma exhibited a signif-
icant shift from 1650 cm−1 to 1644 cm−1, along with a shift of amide II
peak from 1538 cm−1 to 1551 cm−1. These interactions were expressed
due to intermolecular hydrogen bond formation between carbohydrate
moieties and plasma protein during surface adsorption [43]. Similarly,
when PlnH were co-incubation with plasma, there has been a marked
up-shift (from 1539 cm−1 to 1543 cm−1) of amide II band of plasma
protein due to their adsorption on the nanosurface. While peaks
owing to different groups from pluronics and PLGA (C_O, C\\O,
C\\H) remained intact, a disappearance of peak at 959 cm−1 was ob-
served. The FTIR results suggested that certain degrees of electrostatic
interactions were pronounced between the amino groups of plasma
protein and negatively charged sulphonates and carboxylates of hepa-
rin. Such protein-glycosaminoglycans interactions often require expo-
sure of the binding site in protein through re-alignment of basic
amino acid residues [44] and may contribute to the alternations in pro-
tein tertiary structures.
3.4. Nanoparticle-bound protein profile analysis
Nanoparticle-protein binding affinities and chemical interactions
are the key factors in determining the number and types of proteins
which constitutes the hard corona structure [18]. Proteins present in
the hard corona of functionalized nanoparticles were therefore sepa-
rated, using one dimensional gel electrophoresis (Fig. 4a), to perceive
the nature of protein corona surrounding the nanoparticles. Coomassise
stained gel for 1% v/v human plasma exhibited typical bands corre-
sponding to most abundant proteins such as transferrin (80 kDa) [45],
serum albumin (67 kDa), IgG (heavy chain, 50 kDa), apolipoprotein E-
1 (34 kDa) [38,46]. The protein patterns arising from the hard coronas
of the nanoparticles revealed the adsorption of all the major plasma pro-
teins with varying intensity for each nanoparticle type. The observations
were found to be very similar to other works on biopolymeric nanopar-
ticles [47,48].
The data were further verified from the band intensity profiles
(Fig. 4b), obtained from QuantityOne software, of gel image. Albumin
is the most abundant plasma protein (~44 g L−1), characterized by var-
ious binding sites, specific structural conformation and was found to be
 A. Basu et al. / Journal of Molecular Liquids 282 (2019) 169–176 173

Fig. 2. (A) Steady state fluorescence quenching of Trp214 signal upon interaction with increasing concentration (0 to 40 μg mL−1) of different nanoparticles at 37 °C (i) Pln (ii) PlnC (iii)
PlnH. (B) Binding constant derived from spectral data.

adsorbed on all the nanoparticles. Albumin attachment to PlnH was The relative density of these three proteins were slightly higher on the
maximum probably due to its high surface charge (−38.4 mV) at surface of chitosan coated PLGA nanoparticles. Chitosan is positively
pH 7.4. Apart from albumin, other proteins such as transferrin, IgG and charged polymer, and can induce innate immunological response in bi-
apolipoprotein E-1 were strongly adsorbed on the nanoparticle surface. ological system [49]. Thus, PlnC was found to be more associated with

Fig. 3. FTIR spectra of different nanoparticles and their hard corona.

174 A. Basu et al. / Journal of Molecular Liquids 282 (2019) 169–176

Fig. 4. Analysis of human plasma protein on nanoparticles (a) Resolved SDS-PAGE of adsorbed plasma proteins on nanoparticles (b) Histograms of band intensities of major adsorbed
proteins.

immunoglobulins (IgG) than other nanoparticles. This was significantly
lower in case of PlnH because heparin coating, unlike chitosan, ensures
reduced activation of complement system [50]. Protein adsorption upon
Pln surface was significantly lower in comparison to other cases, as pre-
viously quantified through the Bradford assay (Fig. 1b). These observa-
tions perfectly rationalize the outcomes obtained from the detailed
protein-nanoparticle interaction studies through IR and fluorescence
spectroscopy. The corona surrounding the nanoparticles was found to
composed of plasma proteins of different kinds and amount, the binding
of which rested upon the nature of the nanoparticle surface chemistry.
3.5. Effect of nano-surface chemistry on protein aggregation
Nanoparticles are potential candidates known to cause major con-
formational changes in protein structure [37,51] and the hard corona
is one site where nanoparticles come in close vicinity of different pro-
teins. FTIR studies had primarily revealed that adsorption of plasma pro-
teins on the surface engineered nanoparticles was due to chemical
interactions and could lead to the loss of protein tertiary structures. De-
stabilization of native protein structure through misfolding and expo-
sure of hydrophobic residues marks the initiation of protein
aggregation and amyloid formation [52]. We, therefore, investigated
the effects of nanoparticle surface chemistry on plasma protein
aggregation using a combination of Thioflavin T fluorescence and circu-
lar dichroism spectroscopy (CD). ThT is cross beta sheet specific dye
used to elucidate whether a certain testing agent modulate the assem-
bly or disassembly of protein aggregates or amyloids [53]. Freshly
reconstituted human plasma (1%) exhibited a low degree of ThT fluo-
rescence intensity due to presence of proteins (Fig. 5A), such as
microglobulins and glycoproteins which feature native beta sheet con-
figurations [54,55]. Plasma upon incubation at 60 °C showed a large in-
crease in ThT fluorescence response at 480 nm. Albumin, the major
protein plasma, undergoes conformation change to amyloid-like struc-
tures upon incubation at elevated temperatures [56], and was greatly
responsible for upsurge for ThT fluorescence intensity. This observation
was regarded as the positive control. Plasma samples co-incubated with
Plns beared a low degree of ThT intensity. This is due to the fact that
fewer number of proteins were adsorbed on the Pln surface and faced
structural alteration [57]. Among the different nanoparticles co-
incubated with plasma, ThT response was maximum in case of PlnC.
Similar responses of chitosan were recorded during its interaction
with serum albumin [41]. Chitosan functionalization disrupted alpha
helical conformations of adsorbed proteins, and led to acceleration of
protein aggregation on the nanoparticle surface. Therefore, it can be en-
visaged that the corona surrounding the PlnC comprised mostly of ag-
gregated protein. A low degree of aggregation was also observed

Fig. 5. (A) ThT fluorescence response of 1% (v/v) blood plasma co-incubated with and without nanoparticles. (B) Far UV CD spectra of 1% (v/v) blood plasma co-incubated with and without
nanoparticles.
 A. Basu et al. / Journal of Molecular Liquids 282 (2019) 169–176
during association of PlnH with plasma protein, possibly due to exis-
tence of weak electrostatic interactions.
CD was also used to measure the conformational changes occurring
in plasma protein upon nanoparticle interactions. Negative peaks at 208
and 222 nm arise from π → π* and n → π* transitions of carbonyl groups
present in the polypeptide chains of serum albumin. Plasma incubated
at 60 °C showed a disappearance of the negativity at 208 and 222 nm
due to breakdown of α-helical forms (Fig. 5B). Appearance of single
negative peak at 215 nm indicated an increase of β-sheet content
[58,59]. Co-incubation with Pln showed minimum change in the CD
spectra as compared to that of fresh plasma. However, more deviations
in terms of mdeg values observed in case of PlnC and PlnH revealed that
PlnC and PlnH affected the native conformations of protein in the vicin-
ity of the nanoparticles. ThT fluorescence and CD spectroscopy observa-
tions thus suggested that different nanosurface functionalization affects
the native protein conformations in the corona microenvironment. Fur-
ther studies under in vivo conditions would help understand how pro-
tein aggregation upon nanoparticle surface would interfere the
biodistribution of the nanoparticles and affect the efficiency of site-
specific delivery of payload.
4. Conclusion
In this work, we had intended to systematically explore and com-
pare the physico-chemical interactions occurring between some typi-
cally used PLGA nanoparticles and human plasma proteins, and
understand the effect of such interactions on protein corona formations.
It is now a known fact that nanoparticles exposed to biological media
triggers the formation of a protein rich layer surrounding the nanopar-
ticle surface, which re-defines the identity of the particles in terms of its
therapeutic capacities. Our findings suggested that composition and
thickness of the protein corona vary with surface functionalities of bio-
polymer nanoparticles. It was intriguing to observe that adsorption of
plasma protein altered the native protein conformations, and led to for-
mation of protein aggregates. These nanosurface-plasma protein inter-
actions will prove key players in determining the immunological
identity, systemic uptake and residence time, and toxicological aspects
of surface engineered nanoparticles. This kind of work is thus expected
to set the stage for further studies by highlighting the behaviour of
biopolymeric nanoparticles in biological environment, and address the
issues related to translational development of nanomedicines.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.molliq.2019.03.016.
Acknowledgement
The authors would like to thank TEQIP project in the University of
Calcutta for providing instrumental facilities on no cost basis. A research
fellowship sponsored to author CB from University Grant Commission,
India is gratefully acknowledged. The authors would also like to declare
that there is no conflict of interest in publication of this article.
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