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Insights into the Thermodynamics of Polymer Nanodot–Human Serum Albumin Association: A Spectroscopic and Calorimetric Approach Arpan Bhattacharya, Somnath Das, and Tushar Kanti Mukherjee Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02658 • Publication Date (Web): 30 Oct 2016 Downloaded from http://pubs.acs.org on October 31, 2016

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Langmuir

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Insights into the Thermodynamics of Polymer

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Nanodot–Human Serum Albumin Association: A

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Spectroscopic and Calorimetric Approach†

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Arpan Bhattacharya, Somnath Das, and Tushar Kanti Mukherjee*

6

Discipline of Chemistry, Indian Institute of Technology Indore, Simrol, Khandwa Road, Indore-

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453552, M.P., India.

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† *

Electronic supplementary information (ESI) available. Corresponding author. E-mail: [email protected]; Tel: +91-7312438779. 1 ACS Paragon Plus Environment

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Abstract. With the advent of newer luminescent nanoparticles (NPs) for bioimaging

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applications, their complex interactions with the individual biomolecules need to be understood

12

in great detail, prior to their direct application into the cellular environments. Here we have

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present a systematic and detailed study of the interaction between luminescent polymer nanodot

14

(PND) and human serum albumin (HSA) in its free and ligand-bound state with the help of

15

spectrophotometric and calorimetric techniques. At physiological pH (pH=7.4), PND quenches

16

the intrinsic fluorescence of HSA as a consequence of ground-state complex formation. The

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binding stoichiometry and various thermodynamic parameters have been evaluated by using

18

isothermal titration calorimetry (ITC) as  well  as  van’t  Hoff  equation.  It  has  been found that the

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association of PND with HSA is spontaneous (ΔG0 = -32.48 ± 1.24 kJ mol-1) and is driven by a

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favorable negative standard enthalpy change (ΔH0 = -52.86 ± 2.12 kJ mol-1) and unfavorable

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negative standard entropy change (ΔS0 = -68.38 ± 2.96 J mol-1 K-1). These results have been

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explained by considering hydrogen bonding interactions between amino and hydroxyl groups (–

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NH2 and –OH) of PND and carboxylate groups (-COO-) of glutamate (Glu) and aspartate (Asp)

24

residues of HSA. The binding constant of PND with HSA is estimated to be 4.90 ± 0.19 × 105 M-

25

1

. Moreover, it has been observed that warfarin bound HSA (war-HSA) shows a significantly

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lower binding affinity (Kb= 1.15 ± 0.19 × 105 M-1) towards PND, while ibuprofen bound HSA

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(ibu-HSA) shows a slightly lower affinity (Kb= 3.47 ± 0.13 × 105 M-1) compared to the free

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HSA. In addition, our results reveal that PND displaces warfarin from the site I (subdomain IIA)

29

of HSA due to the partial unfolding of the war-HSA. We hope that the present study will be

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helpful to understand the fundamental interactions of these biocompatible PNDs with various

31

biological macromolecules.

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1. Introduction

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In recent times, carbon-based nanomaterials, particularly carbon dots (CDs) have

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emerged as a most versatile candidates for biomedical applications owing to their excellent water

36

solubility, smaller size, low toxicity, chemical inertness, easy surface functionalization and large

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surface area with comparable photoluminescence (PL) properties compared to the inorganic

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core-shell quantum dots (QDs).1-6 Different types of CDs with distinct intrinsic structures such

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as carbon nanodots (CNDs)3,4, carbon quantum dots (CQDs)7, and polymer nanodots (PNDs)

40

have been reported in the literature.8,9

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Among these, polymer nanodots (PNDs) have gained enormous attention in recent

42

years.8-13 They are composed with crossed-linked polymer-like structure and show enhanced

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PL.9 Recently, Liu et al. have shown that N-doped PNDs contain no aromatic carbons (sp2

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hybridized) instead they are composed with C-N heterocycles, which is entirely different from

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the conventional aromatic carbon riched CDs.12 These PNDs show excellent optoelectronic

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properties as well as good biocompatibility and low cytotoxicity comparable to CDs.14 As a

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consequence of these advantages, they have been successfully used in several biomedical

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applications.10,11 For example, Zhu et al. have shown that non-conjugated PNDs prepared from

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branched PEI exhibit minimal cytotoxicity for rat adrenal pheochromocytoma (PC12) cells.9

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Although these recent studies have successfully demonstrated the role of PND as an optical

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marker in many bioimaging applications, there is a growing concern to understand the

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fundamental interaction mechanism of PND with individual biomolecules such as proteins.

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It has often been observed that the physicochemical properties of various NPs

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significantly alter in the presence of proteins.15-17 On the other hand, various proteins have been

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found to lose their native-like secondary structure and activity in contact with NPs surface.18,19 3 ACS Paragon Plus Environment

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The dynamic layer of proteins at  the  NP  surface  is  known  as  “protein  corona,” which determines

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the ultimate interaction of NP with living systems.20,21 Therefore it is utmost important to

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understand the mechanistic aspects of protein adsorption on the NP surface at the molecular-

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level prior to their direct introduction into the cellular environments. While several studies have

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thoroughly investigated the mechanistic aspects of various protein-NP complexes,22–25 very less

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is known about the influence of PND on the structure and stability of serum albumins which are

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the most abundant protein constituent in blood plasma.

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Serum albumin is the most abundant protein in the bloodstream and present at a

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concentration of ∼50 mg/mL.26 The majority of the secondary structure of HSA consist of α-

65

helix (~67%) with six turns and 17 disulfide bridges.27,28 The tertiary structure consists of three

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homologous domains (I, II, and III) and each domain contains two subdomains A & B.

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Human serum albumin (HSA) contains one tryptophan (Trp-214) residue in the subdomain IIA.

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Being one of the most abundant proteins in the blood plasma, serum albumin forms the first layer

69

of the long-lived hard corona on the NPs surface. To date, several studies have been performed

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to understand the interaction of various NP on the structure and stability of serum albumins. 17, 23,

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25,29-31

27,28

Earlier, Lesniak et al. have reported that silica NPs in the absence of serum show stronger

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adhesion to the cell membrane with higher internalization efficiency in comparison to medium

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containing serum.17 Earlier, Sharma et al. have studied the interaction of HSA with bimetallic

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Au/Ag alloy NP. They have observed that HSA retains its 76% esterase activity upon adsorption

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on the Au/Ag NP surface.30 While, most of these earlier studies with serum albumins reported

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moderate to significant alteration of the secondary structure of the native protein upon surface

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adsorption, very less is known about the subsequent physicochemical properties of

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conformationally

altered

serum

albumins.

Moreover,

the

structure,

stability,

and 4

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physicochemical properties of HSA in the presence of PND have not been explored earlier. In

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the present work, we have investigated the influence of PND on the structure and stability of

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human serum albumin in its free and ligand-bound state.

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2. Experimental Section.

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2.1. Materials. Human serum albumin (HSA, lyophilized powder, essentially fatty acid-free) and

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Warfarin were purchased from Sigma-Aldrich. D-Glucose was purchased from Fisher Scientific,

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Glycine and Ibuprofen were purchased from TCI chemicals. Milli-Q water was obtained from a

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Millipore water purifier system (Milli-Q Integral).

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2.2. Sample Preparation, Quantum Yield (QY) and Concentration Determination. All the

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solutions were prepared in 7.4 Tris-HCl buffer. Different concentration HSA was prepared by

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dissolving the required amount of HSA (MHSA= 66,437 g mol-1) in pH 7.4 Tris-HCl buffer. For

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Isothermal Titration Calorimetric (ITC) experiments, all solutions were degassed before loading

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the samples. PND solution (2 µμL for each injection) was injected from an injection syringe

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continuously rotating at 300 rpm to the sample chamber containing HSA solution. The interval

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time between the two injections was kept at 120 s. Subsequently, control experiments were

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performed by injecting the PNDs to the buffer in the absence of protein to correct the heat

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change due to the mixing and dilution. The QY of synthesized PNDs was estimated using

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quinine sulfate as reference according to the following equation

97

𝛷   =   𝛷  (𝐼 /𝐼 )  (𝜂 /𝜂 )  (𝐴 /𝐴 )

(1)

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where ϕ is the QY, I is the integrated PL intensity, η is the refractive index of the solvent, and A

99

is the optical density (absorbance value is below 0.10 at the excitation wavelength). The

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subscript  “st”  stands  for  standard  and  “u”  stands for the unknown sample. The molar extinction

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coefficient (εm) of the synthesized PNDs was determined from the radiative rate constant (kf) by

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following the previous literature.32 kf is determined from the very well-known equation,𝑘 =

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  𝛷 ⁄𝜏 , where ϕf is the quantum yield of the PNDs and τf is the lifetime of the PNDs. By using

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kf, εm is calculated to be ~3.28×103 M-1cm-1 from the equation mentioned elsewhere33 which is

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consistent with the earlier literature value for the other CDs.6 From the obtained εm, we have

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determined the concentration of synthesized PND, which matches well with that estimated from

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average mass obtained using mass spectrometry (Figure S1 of the supporting information)

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following the earlier literature.31 The fluorescence spectra for HSA in the presence of PNDs was

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corrected for the absorbance at 295 nm as the absorption spectrum of PND increases almost

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monotonically at the wavelength range of 200-400 nm.34

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2.3. Synthesis of PND. PND was synthesized according to the previously reported method with

112

little modification (Scheme 1).35 Briefly, 30 mmol of glucose and 30 mmol of glycine were

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dissolved in Milli-Q water (30 mL) and sonicated for 5 min. Then the solution was transferred to

114

a Teflon-coated stainless   steel   autoclave   (50   mL)   and   heated   at   150˚C   for   two hours.

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Subsequently, the reactor was cooled to ambient temperature, and a black-brown solution was

116

obtained suggesting the formation of PNDs. Then the solution was filtered through a 0.22 µμm

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syringe filter (Whatman) to remove the larger particles and then the solution was dialyzed

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(MWCO 3.5 kDa) for 24 h. Finally, the brown transparent aqueous PNDs solution was obtained.

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Scheme 1. Schematic Representation of the Synthesis of PND Using Glucose and Glycine.

130 131

2.4. Instrumentation. Absorption spectra were recorded in a quartz cuvette (10 × 10 mm) using

132

a  Varian  UV−Vis  spectrophotometer  (Carry  100  Bio).  The  fluorescence  spectra  were  recorded  in  

133

a quartz cuvette (10 × 10 mm), using Fluoromax-4 Spectrofluorimeter (HORIBA Jobin Yvon,

134

model FM-100) with excitation and emission slit width at 2 nm. Circular Dichroism (CD) spectra

135

were acquired by a JASCO J-815 CD spectropolarimeter using a quartz cell of 1 mm path length.

136

Scans were made with a slit width of 1 mm and speed of 20 nm/min. The α-helix content of HSA

137

has been estimated from the following equation36

138

𝛼 − 𝐻𝑒𝑙𝑖𝑥  (%) =

(

)

× 100

(2)

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where MRE is the mean residue ellipticity which is calculated from the observed ellipticity

140

values at 222 nm by using the following equation,36

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                                                                                     𝑀𝑅𝐸

142

where Cm is the molar concentration of the protein, n is the no. of amino acids residues present in

143

the protein and l is the path length (0.1 cm). The isothermal titration calorimetric (ITC)

144

experiments were performed using Nano ITC, TA instruments. The data were analyzed by

145

NanoAnalyze software v2.4.1 and fitted with the independent site binding model. FTIR spectra

146

were recorded for KBr pellets using a Bruker spectrometer (Tensor-27). The powder X-ray

147

diffraction spectrum (XRD) was recorded on a Rigaku SmartLab, Automated Multipurpose X-

148

ray Diffractometer with a Cu Kα source (the wavelength of X-rays was 0.154 nm). Transmission

149

electron microscopy (TEM) images were recorded on a Field Emission Gun-Transmission

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Electron Microscope (Model-Tecnai G2, F30) operating at an accelerating voltage of 300 kV.

151

Samples were placed on a carbon copper grid and air dried before imaging. The

152

spectrum was recorded on an AVANCE III 400 Ascend Bruker BioSpin International AG 400

153

MHz NMR spectrometer. Mass spectrum was recorded using electrospray ionization (ESI)

154

quadrupole time-of-flight liquid chromatography-mass spectrometer (Bruker Daltonik) in

155

methanol as a solvent by negative-mode ESI. Dynamic light scattering (DLS) and Zeta potential

156

measurements were performed on a Brookhaven particle size analyzer (Model 90 Plus). All the

157

samples for DLS measurements were filtered through a 0.22 mm syringe filter (Whatman).

158

Fluorescence decays were recorded on an HORIBA Jobin Yvon picosecond time-correlated

159

single photon counting (TCSPC) spectrometer (model Fluorocube-01-NL). The samples were

160

excited at 279 nm by a nanosecond diode laser (Deltadiode, Model: DD-280). The decays were

161

collected with the emission polarizer at a magic angle of 54.7° by a photomultiplier tube (TBX-

 (𝑑𝑒𝑔. 𝑐𝑚 . 𝑑𝑚𝑜𝑙

) =  

×

×

(3)

13

C NMR

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07C). The instrument response function (IRF, fwhm ~1.2 ns.) was recorded using a dilute

163

scattering solution. The fluorescence decays were analyzed using IBH DAS 6.0 software by the

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iterative reconvolution method, and the goodness of the fit was judged by reduced χ-square (χ2)

165

value. The decays were fitted with bi-exponential function

𝐹(t) =

𝑎 exp(−𝑡⁄𝜏 )                                                                  (4)

166 167

where F(t) denotes normalized PL decay and a1 and a2 were the normalized amplitude of decay

168

component τ1 and τ2, respectively. The average lifetime was obtained from the following

169

equation 〈𝜏〉 =

𝑎 𝜏                                                                                                            (5)

170 171 172 173 174 175 176 177

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3. Results and Discussion.

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3.1. Characterization of PND. The synthesized PND was characterized by FTIR spectroscopy,

180

powder XRD, elemental analysis, NMR spectroscopy, HRTEM, and DLS measurements.

181 182 183 184 185 186 187 188 189 190 191 192 193 194

Figure 1. (A) FTIR spectrum, (B) HRTEM image of synthesized PND; the inset shows the

195

magnified image of a single PND, (C) Size distribution histogram of PND from DLS

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measurements (D) powder XRD patterns of PND; the inset shows the elemental analysis of

197

synthesized PND. (E) 13C NMR spectrum of PND dissolved in D2O.

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Figure 1A shows the FTIR spectrum of synthesized PND. The broad peak at 3422 cm-1 is

199

assigned to the stretching vibration of the –OH and –NH moieties. The peak at 1408 cm-1 arises

200

due to –CH2 scissoring. Two peaks at 1631 and 2930 cm-1 are assigned to the characteristics

201

absorption band of –N–H and –C–H stretching, respectively. Figure 1B shows the HRTEM

202

image of synthesized PND. They are spherical in nature and show a mean diameter of 4.68 ±

203

0.06 nm. The inset shows the magnified image of a single PND. The mean hydrodynamic

204

diameter is estimated to be 6.24 ± 0.8 nm from DLS measurement (Figure 1C).

205

The zeta potential of PND is 1.52 ± 0.76 mV at pH 7.4. Figure 1D displays the powder

206

XRD patterns of PND. A broad peak at 2θ =28.9° (d = 0.31 nm) has been observed, which

207

signifies the amorphous nature of synthesized PND. Elemental analysis reveals 50.36% carbon,

208

36.66% oxygen, 7.76% nitrogen, and 5.22% hydrogen (Figure 1D, inset).

209

indicates that the signals in the range of 40-50 ppm are due to the presence of aliphatic (sp3)

210

carbon atoms (Figure 1E), which matches well with the earlier reported NMR spectrum of

211

PND.35 Moreover, no peak due to aromatic carbon has been observed in the range of 125-150

212

ppm, which clearly signifies that the synthesized PND is structurally different from those of

213

CDs.4 The PL QY is estimated to be 8.4% using quinine sulfate as a reference.24

13

C NMR spectrum

214

Figure 2A shows the absorption and normalized PL spectra (λex=375 nm) of synthesized

215

PND. The absorption spectrum of PND exhibits a prominent peak at 293 nm, which exactly

216

matches with the earlier reported value.35 A distinct PL band centered at 465 nm has been

217

observed upon excitation at 375 nm (Figure 2A). Moreover, the PL maxima strongly depend on

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excitation. The peak position shifts to lower energy (red shift) with the increase in excitation

220

wavelength (Figure 2B). This phenomenon could be either due to the presence of multiple

221

excited states in each PND or due to the quantum confinement effect similar to that has been

222

observed earlier for CDs.9,37 The PL decay trace of PND shows a tri-exponential decay kinetics

223

having lifetime components of 2.57 (29 %), 9.86 (16 %) and 3.79 ns (55 %) with an average

224

lifetime of 2.56 ± 0.07 ns (Figure S2 and Table S1 of the Supporting information).

(A)

Absorption Emission

0.4

0.2

0.0

300

400

500

600

Wavelength (nm)

700

PL. Intensity(a.u.)

the excitation wavelength. Maximum PL intensity has been observed at 460 nm upon 375 nm

PL. Intensity (Normalized)

218

Absorbance

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300 nm 350 nm 375 nm 400 nm 430 nm 460 nm 500 nm

(B)

300

400

500

600

700

Wavelength (nm)

225

Figure 2. (A) Absorption (red line) and normalized PL spectrum (blue line) of PND at λex = 375

226

nm. (B) Changes in PL spectra of PND at different excitation wavelengths.

227

3.2. Steady-State and Time-Resolved PL Measurements of PND-HSA System. The PL peak

228

position and quantum yield of PND remain unaltered in the presence of HSA (Figure S3 of the

229

supporting information). However, substantial changes have been observed in the intrinsic

230

fluorescence of HSA upon addition of different concentrations of PND. HSA shows an intrinsic 12 ACS Paragon Plus Environment

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fluorescence at 336 nm (λex= 295 nm) mainly due to the presence of Trp-214 in subdomain IIA.

232

Figure 3A displays the changes in the intrinsic fluorescence of HSA upon gradual addition of

233

PND. The fluorescence quantum yield decreases progressively upon addition of increasing

234

concentrations of PND. The fluorescence intensity of HSA is quenched by 66% with a red shift

235

of 9 nm in the presence of 4.50 µμM of PND. It is well known that any change in the polarity

236

around Trp residue of HSA results in alteration of the fluorescence quantum yield and peak

237

position.38 In general, quenching in fluorescence intensity with a red shift in the intrinsic

238

emission of HSA indicates partial unfolding of the native protein structure near Trp-214

239

residue.38 Hence, the observed 66% quenching with 9 nm red shifts in the intrinsic fluorescence

240

of HSA might be due to the partial unfolding of the native HSA structure in the presence of

241

PND. To support this argument, we have performed near and far-UV CD measurements of HSA

242

in the absence and presence of PND. The far-UV CD spectrum of HSA shows two minima at

243

208 and 222 nm indicating the α-helix rich structure (Figure 3A inset). The α-helix content of

244

HSA is estimated to be 66.43% (Table S2 of the supporting information). A noticeable change in

245

the CD spectrum of HSA has been observed in the presence of 4.50 µμM PND (Figure 3A inset).

246

The secondary structure analysis reveals that the α-helix content decreases to 60.11% (9.51%

247

loss of helicity) in the presence of 4.50 µμM PND (Table S2 of the supporting information). These

248

changes clearly signify the altered conformation of adsorbed HSA on the surface of PND. The

249

near-UV CD spectrum of HSA displays two negative peaks at 262 and 269 nm due to the

250

presence of phenylanaline residues (Figure S4 of the supporting information).39 Two week

251

shoulders at 277 and 284 nm have also been observed from the tyrosyl residues.39 Importantly,

252

no significant changes have been observed in the near-UV CD spectrum of HSA upon addition

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of PND, signifying that the overall tertiary structure of HSA remains unchanged (Figure S4 of

254

the supporting information).

255

The mechanism of the observed quenching could be either due to static quenching,

256

dynamic quenching or a combination of both. To know the extent and mechanism of quenching,

257

we have used Stern-Volmer equation which can be expressed as follows = 1 + 𝐾 [𝑄] = 1 + 𝑘 𝜏 [𝑄]

258

(6)

259

where F0 and F are the fluorescence intensities of the fluorophore in the absence and presence of

260

quencher, respectively. KSV is the Stern-Volmer constant. kq and τ0 are the quenching rate

261

constant and the lifetime of the fluorophore in the absence of any quencher, respectively. The

262

values of KSV and kq can be evaluated from a plot of F0/F against concentrations of the quencher.

263

Figure 3B shows the Stern-Volmer plot at 298 K. The plot is linear suggesting a single type of

264

quenching mechanism which could be either static or dynamic quenching. The estimated

265

quenching rate constant is 1.68 ×1014 M-1s-1 at 298 K. Notably; the estimated quenching rate

266

constant is ~ 4 to 5 order of magnitude higher than that for purely dynamic quenching

267

mechanism.40 These results indicate that the observed fluorescence quenching of HSA in the

268

presence of PND might be due to the formation of static ground-state complex formation.

269

Similar kind of static quenching mechanisms for other protein-NP complexes have been

270

observed earlier. 17, 23, 25,29-31

271

Table 1. Fluorescence Lifetime Components of HSA (λex = 279 nm) in the Absence and

272

Presence of Different Concentrations of PND at pH 7.4.

Systems

a1

τ1 (ns)

a2

τ2 (ns)

< τ> (ns)

χ2

2  μM  HSA

0.28

3.51

0.72

6.58

5.72 ± 0.06

1.09

2  μM  HSA+  4.50  μM  PND

0.29

3.37

0.71

6.64

5.69 ± 0.05

14 1.06

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Langmuir

273

Time-resolved decay measurements have been performed to establish the quenching

274

mechanism. Figure S5 of the supporting information shows the decay traces of HSA in the

275

absence and presence of PND. HSA shows a bi-exponential decay trace and has average

276

lifetimes of 5.72 ns with lifetime components of 3.51 (28%) and 6.58 ns (72%).32,33 The decay

277

curve remains unaltered in the presence of highest concentration (4.50 µμM) of PND. The values

278

of fitted parameters are summarized in Table 1. It is evident from Table 1 that the fluorescence

279

decay of HSA remains bi-exponential in the presence of 4.50 µμM of PND with an average

280

lifetime of 5.69 ns. The time-resolved Stern–Volmer plot is generated using the Stern–Volmer

281

equation which can be expressed as follows

282





〈 〉

= 1 + 𝐾 [𝑄]

(7)

283

where 〈τ0〉 and 〈τ〉 are the average lifetimes of the fluorophore in the absence and presence of

284

quencher, respectively. Figure 3B shows the time-resolved Stern–Volmer plot for PND-HSA

285

system. The plot is parallel to the X axis, signifying a lack of any lifetime quenching of HSA by

286

PND. These results clearly signify the static quenching mechanism between PND and HSA due

287

to the formation of ground-state complex formation.

15 ACS Paragon Plus Environment

(vi)

0 -6

Page 16 of 39

HSA +4.50  M PND

(C)

HSA

4.96 0.62 nm

-12 -18 -24 200

210

220

230

240

Wavelength (nm)

360

400

Wavelength (nm)

(B)

440

298 K

3.0

/

Intensity (a.u.)

3.0

(i)

CD (mdeg)

(A)

320

HSA+PND

2.5

2.5

2.0

2.0

1.5

1.5

1.0

/

0

F0/F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fl. Intensity. (a.u)

Langmuir

9.79 0.80 nm

1.0 0

1

2

3

[PND] (M)

4

5

1

10

Diameter (nm)

288

Figure 3. (A) Changes in fluorescence spectra (λex= 295 nm) of HSA with increasing

289

concentrations of PND (i) 0, (ii)  0.60,  (iii)  1.30,  (iv)  1.90,  (v)  3.20  and  (vi)  4.50  μM  at pH 7.4.

290

The inset shows the changes in far-UV CD spectra of HSA in the absence and presence of 4.50

291

μM  of  PND at pH = 7.4. (B) Overlap of time-resolved (triangle) and steady-state (circle) Stern-

292

Volmer plots at 298 K (C) Size distribution histograms from DLS measurements for HSA (blue

293

bars) and HSA-PND complex (magenta bars). The mean diameters with standard deviation are

294

mentioned in the figure.

295

Further evidence of their ground state association comes from DLS measurements. Figure

296

3C shows the size distribution histograms of HSA in the absence and presence of PND. The

297

mean hydrodynamic diameter of HSA is estimated to be 4.96 ± 0.62 nm (Figure 3C). However, a

298

clear shift of the histogram has been observed in the higher size region for the PND-HSA

299

system. The mean diameter increases to 9.79 ± 0.80 nm in the presence of 4.50 µμM of PND

16 ACS Paragon Plus Environment

Page 17 of 39

300

(Figure 3C). This increase in diameter with unimodal distribution strongly supports our proposed

301

mechanism of static ground state association between PND and HSA. Moreover, a noticeable

302

decrease in the zeta potential of HSA has been observed in the presence of PND, signifying the

303

association between PND and HSA (Figure S6 of the supporting information). Next, we have

304

performed ITC measurements to estimate various thermodynamic parameters and driving force

305

associated with the PND-HSA association process.

306

3.4. Isothermal Titration Calorimetry (ITC). ITC has been extensively used to investigate

307

various protein-NP interactions.41-44 The sign and magnitude of thermodynamic parameters can

308

provide a comprehensive understanding of the molecular-level interaction mechanism between

309

PND and HSA. Figure 4A displays the calorimetric profile for the titration of PND with HSA at

310

ambient temperature. Figure 4B shows the heat change per mole of injection of PND against the

311

molar ratio (PND: HSA) at each injection after correcting the heat change for dilution. The heat

312

change of the titration is fitted by using an independent site binding model for the determination

313

of the thermodynamic parameters. All the fitted parameters are listed in Table 2.

Time (sec) 0

315 316 317 318 319 320 321 322 323 324

Heat rate (J/s)

314 2.5

kJ/mol of Injection

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

0

500 1000 1500 2000 2500 3000

(A)

HSA+PND Buffer+ PND

2.0 1.5 1.0 0.5 0.0

(B)

-10 -20 -30 -40 -50 -60 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Molar Ratio 17 ACS Paragon Plus Environment

Langmuir

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Page 18 of 39

325

Figure 4. Isothermal titration calorimetric (ITC) profile for the titration of HSA in the presence

326

of PND at 298 K. (A) Shows the heat flow as a function of time per injection of the PND (red

327

profile) after correcting the heat change of dilution (black profile) at 298 K. (B) Shows the heat

328

evolved against the molar ratio of (PND: HSA) at 298 K (blue dots). The solid line is the fitted

329

curve.

330

The association constant (Kb) between PND and HSA is estimated to be 4.90 ± 0.19 ×105

331

M-1 at 298K, which is similar to the previously reported values for different NP–protein

332

complexes.37,41 This indicates that PND has high binding affinity towards HSA at physiological

333

conditions. The binding stoichiometry (n) is found to be 1.19 ± 0.10, which is reasonable by

334

considering their sizes. The overall association process between PND and HSA is spontaneous

335

with a negative standard free energy change (ΔG0 = -32.48 ± 1.24 kJ mol-1). Moreover, the

336

process is characterized by a negative standard enthalpy change (ΔH0 = -52.86 ± 2.12 kJ mol-1)

337

and a negative standard entropy change (ΔS0 = -68.38 ± 2.96 J mol-1 K-1). Earlier, based on the

338

magnitude and sign of the thermodynamic parameters Ross and Subramanian have proposed a

339

conceptual model for association mechanisms between various ligands with proteins.45 Later,

340

various groups have proposed different molecular forces behind the NP-protein association,

341

namely electrostatic, hydrophobic, hydrogen bonding or van der Waals interactions. 17, 23, 25,29-31

342

Generally, processes triggered by hydrophobic interactions between protein and ligand proceed

343

with a large positive entropy change along with positive enthalpy change while reactions

344

initiated by electrostatic interactions proceed with a positive entropy change with a small

345

positive or negative enthalpy change. However, for the present system neither hydrophobic nor

346

electrostatic interactions can account for the estimated thermodynamic parameters. A similar

347

kind of negative entropy change along with the negative enthalpy change was observed in many 18 ACS Paragon Plus Environment

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Langmuir

348

association processes and has been assigned due to the involvement of hydrogen bonding

349

interactions.25,45,46 Notably the unfavorable negative entropy change during the association

350

process might be due to the conformational restriction of surface exposed flexible amino acid

351

residues of HSA and surface functional groups of PND upon association.41,46 Here, it is

352

important to mention that the size and surface charge of the associating species strongly

353

influence the interaction mechanism. Hence, it is important to have a closer look at the size and

354

surface charge of PND and HSA to validate the hydrogen bonding interactions between them.

355

Table 2. Thermodynamic Parameters Obtained From the ITC Experiments for the

356

Interaction of PND with HSA.

357

Parameters 358 359 360

HSA-PND System

Kb ( ×105 M-1 )

4.90 ± 0.19

n

1.19 ± 0.10

ΔH0 (kJ mol-1)

-52.86 ± 2.12

ΔS0 (J mol-1 K-1)

-68.38 ± 2.96

ΔG0 (kJ mol-1)

-32.48 ± 1.24

361 362

The mean hydrodynamic diameter of PND is 6.24 ± 0.8 nm and contains –NH2 and –OH

363

groups on its surface. The estimated zeta potential of PND at pH 7.4 is ~1.521 ± 0.76 mV. This

364

relatively small value of zeta potential indicates that the surface of PND remains in neutral form

365

at pH 7.4. On the other hand, the isoelectric point (pI) of HSA is ~ 4.7 at 298 K and hence at

366

physiological pH (pH = 7.4) HSA has overall negative charge.27 Out of the three domains of

367

HSA, domain I and II contain the majority of the negatively charged aspartate (Asp) and

368

glutamate (Glu) residues, while domain III of HSA is neutral.27 Moreover, control experiment in

19 ACS Paragon Plus Environment

Langmuir

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Page 20 of 39

369

pH 7.4 PBS with 150 mM NaCl shows no effect of salt on the quenching process (Figure S7 of

370

the supporting information). This observation clearly ruled out the possibility of electrostatic

371

interactions between PND and HSA. Hence, by considering their sizes, surface potentials, and

372

estimated thermodynamic parameters, we propose that the most probable mechanism of their

373

association process involves hydrogen bonding interactions between surface exposed amino and

374

hydroxyl groups (–NH2 and -OH) of PND with carboxylate groups (-COO-) of glutamate (Glu)

375

and aspartate (Asp) residues of HSA.

376

3.3. Effect of Temperature on the Association Process. Figure 5A shows the effect of

377

temperature on the steady-state Stern-Volmer plots. It is evident that the slope of the plot

378

decreases with increase in temperature from 298 to 308K. This result clearly signifies the

379

destabilization of the PND-HSA complex with the increase in temperature as expected for the

380

static ground state quenching process. The estimated Stern-Volmer constants at different

381

temperature are listed in Table 3. From these Stern-Volmer plots we have estimated binding

382

constant (Kb) and binding sites (n) from the Scatchard equation, which can be expressed as

383

follows

384

𝑙𝑜𝑔[(𝐹 − 𝐹)/𝐹] = 𝑙𝑜𝑔𝐾 + 𝑛𝑙𝑜𝑔[𝑄]

(8)

385

where Kb is the binding constant of the PND-HSA complex and n is the number of binding sites.

386

The plot of log[(F0 - F) /F] against log[Q] should yield a straight line having an intercept of log

387

Kb and a slope equal to n. Figure 5B show the Scatchard plot at three different temperatures

388

which are linear in nature. The estimated binding constant (Kb) and no of binding sites (n) are

389

summarized in Table 3. Here it is important to note that the Kb value estimated from Scatchard

390

plot matches well with that estimated from ITC experiment. As expected the Kb value decreases

391

with increase in temperature which is in agreement with the static quenching mechanism. 20 ACS Paragon Plus Environment

Page 21 of 39

392

3.0 393

2.5

F0/F

394

(A)

298 K 308 K 318 K

2.0 1.5

395

1.0 0

396

0.4 397

log [(F0- F)/F]

398

0.2

(B)

0.0

1

2

3

4

5

-5.8

-5.6

-5.4

-5.2

[PND] (M) 298 K 308 K 318 K

-0.2

399

-0.4 -0.6

400

-0.8

-6.2

13.6 402

-6.0

log [PND]

401

(C)

13.2 12.8

403 404

ln Kb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

12.4 12.0

405

11.6

406

11.2 0.0031

407 408

ln Kb=6434/T-8.45 0.0032

0.0033

0.0034

1/T

409 410 411

Figure 5. (A) Steady-state Stern–Volmer plots of HSA at three different temperatures (298, 308,

412

and 318 K) as a function of PND concentrations. (B) Scatchard plots of the HSA-PND complex

21 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 39

413

at three different temperatures (298, 308, and 318 K). (C) van’t   Hoff   plot   for   the   HSA-PND

414

complex.

415 416

417

Next, we have estimated various thermodynamic parameters of the association processes of PND- HSA systems using van’t Hoff equation, which can be expressed as follows 𝑙𝑛𝐾 =







(9)

418

where R is the gas constant, ΔS0 is the standard entropy change and ΔH0 is the standard enthalpy

419

change during the association process between HSA and PND at temperature T. The plot of ln Kb

420

against 1/T for three different temperatures should yield a straight line, from which we have

421

evaluated the changes in enthalpy (ΔH0) and entropy (ΔS0) of the association processes between

422

HSA and PNDs (Figure 5E and F). The   Gibb’s   free   energy   change (ΔG0) during the binding

423

processes has been calculated by using the equation

424

∆𝐺 = ∆𝐻 − 𝑇∆𝑆

(10)

425

All the estimated thermodynamic parameters are summarized in Table 3. The association

426

process is found to proceed with a negative standard enthalpy change (ΔH0 = -53.49 ±1.52 kJ

427

mol-1) and negative standard entropy change (ΔS0 = -70.25 ± 4.90 J mol-1 K-1), giving rise to an

428

overall negative standard free energy change (ΔG0 = -32.55 ± 0.06 kJ mol-1) which is in good

429

agreement with ITC results. The thermodynamic parameters obtained from temperature variation

430

studies further support our proposed model of the ground state association between PND and

431

HSA through hydrogen bonding interactions.

432 433 22 ACS Paragon Plus Environment

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Langmuir

434

Table 3. Stern–Volmer Quenching Constants (KSV) for the HSA–PND Complex at Three

435

Different Temperatures.

436

Parameters 437 438 439 440 441

HSA-PNDs

Temperature

298 K

308 K

318 K

Ksv (×105 M-1)

4.20 ± 0.06

3.83 ± 0.07

3.49 ± 0.05

Kb (×105 M-1)

5.01 ± 0.04

2.57 ± 0.06

1.26 ± 0.06

∆G0 (kJ mol-1)

-32.55 ± 0.06

n

1.07 ± 0.01

0.99 ± 0.01

0.98 ± 0.01

R2

0.998

0.998

0.998

-31.85 ± 0.02 -31.15 ± 0.04

∆H0 (kJ mol-1)

-53.49 ±1.52

∆S0 (J mol-1 K-1)

-70.25 ± 4.90

442 443

3.6. Association between PND and Ligand-Bound HSA. HSA has three homologous domains

444

(I, II, and III) and each domain contains two subdomains (A and B). According to Sudlow et al.,

445

the principal ligand binding sites of HSA are located in the hydrophobic cavities of subdomains

446

IIA (site I) and IIIA (site II).47 Earlier, several studies have been performed to understand the

447

interaction of various drugs on the structure and stability of serum albumins.47-53 However, the

448

interaction of NP with serum albumin in the presence of drug has been less explored. To know

449

the influence of PND on the structure and stability of ligand-bound HSA, we have used warfarin

450

(site I marker) and ibuprofen (site II marker) for selective labeling of site I and site II of HSA,

451

respectively.54

452

The fluorescence spectrum of HSA shows 32 nm red shifts with marginal enhancement

453

(1.06 times) in the intensity upon addition of warfarin (Figure 6A). These spectral changes

454

signify the binding of warfarin in subdomain IIA of HSA.55 Earlier, it has been proposed that 23 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 39

455

mainly hydrophobic interactions of warfarin with the polar amino acid residues in subdomain

456

IIA of HSA contribute to its stability inside the binding pocket.56 The 32 nm red shifts in the

457

fluorescence peak of warfarin bound HSA (war-HSA) indicate a significant increase in the

458

polarity around Trp-214 residue of HSA. Moreover, the marginal enhancement in the

459

fluorescence intensity of HSA upon binding to warfarin might be due to the removal of

460

neighboring quencher residues near Trp-214.56 These results signify that the binding of warfarin

461

in the subdomain IIA of HSA results in significant alteration of the native conformation of HSA

462

around Trp-214. This argument gains support from near and far-UV CD measurements (Figure

463

6A inset and Figure S8 of the supporting information). Figure 6A inset shows the far-UV CD

464

spectrum of HSA in the absence and presence of warfarin. Although the spectral shape and peak

465

positions remain same in the presence of warfarin, the ellipticity becomes more negative. The

466

secondary structure analysis of HSA in the presence of warfarin reveals that the α-helix content

467

increases from 66.43 to 69.80% (Table S2 of the supporting information). These spectral changes

468

clearly indicate the altered conformation of HSA in the presence of warfarin. To monitor the

469

changes in the tertiary structure of HSA in the presence of warfarin, we have performed near-UV

470

CD measurements. As mentioned earlier, HSA shows characteristic near-UV CD spectrum

471

below 300 nm, with two negative peaks at 262 and 269 nm.39 On the other hand, warfarin shows

472

no noticeable near-UV CD signal (Figure S8 of the supporting information). However,

473

significant spectral changes have been observed upon addition of warfarin into HSA (Figure S8

474

of the supporting information). Although the positions of two negative peaks of HSA remain

475

unchanged, the intensity of the 269 nm peak increases. More significant changes have been

476

observed beyond 300 nm (Figure S8 of the supporting information). A new positive peak appears

477

in the wavelength rage of 305 to 340 nm. The origin behind this new peak is due to the induced

24 ACS Paragon Plus Environment

Page 25 of 39

478

ellipticity of HSA bound warfarin as a consequence of highly asymmetric binding site of HSA

479

(site I).39 Hence, near and far-UV CD measurements reveal the binding of warfarin in

480

hydrophobic binding site of HSA and results in significant alteration of the secondary as well as

481

tertiary structure of native HSA. In contrast, the fluorescence spectrum of HSA remains

482

unaltered upon addition of ibuprofen (Figure 6B). It is known that ibuprofen binds strongly at the

483

subdomain IIIA of HSA.47,58 The unchanged fluorescence spectrum of HSA in the presence of

484

ibuprofen is due to the fact that the Trp-214 residue is situated in the subdomain IIA of HSA,

485

which is farther away from the binding site of ibuprofen in subdomain IIIA. Moreover, far-UV

486

CD measurements reveal a marginal change in the secondary structure of HSA in the presence of

487

ibuprofen (Figure 6B inset). Secondary structure analysis reveals marginal loss of α-helix

488

content from 66.43% to 65.60% in the presence of ibuprofen. On the other hand, near-UV CD

489

measurements reveal unchanged tertiary structure of HSA in the presence of ibuprofen (Figure

490

S8 of the supporting information). Next, we have explored the interactions of PND with the

491

conformationally modified war-HSA and ibu-HSA.

(iv)

216

225

234

400

450

500

550

(B)

(i)

(ii)

(iii)

-10 -15 -20

207

225

234

Wavelength (nm)

350

400

450

500

550

600

Wavelength (nm)

(D)

-0.2 -0.4 HSA HSA+Warfarin HSA+Ibuprofen

0

CD (mdeg)

0.0

-0.8

216

(iv)

600

(C)

-0.6

HSA ibu-HSA ibu-HSA+ PND

-25

Wavelength (nm) 0.4 0.2

499

207

Wavelength (nm)

350

496

498

-16 -24

495

497

-8

-5

CD (mdeg)

(iii)

0

HSA war-HSA war-HSA+ PND

Fl. Intensity. (a.u.)

494

(i)

(ii)

Fl. Intensity. (a.u.)

493

Fl. Intensity. (a.u.)

492

CD (mdeg)

0

(A)

log [(F0-F)/F]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(ii)

-5

HSA HSA+Warfarin war-HSA+PND Warfarin

-10

-15

(iii)

270

300

330

360

390

Wavelength (nm)

(iv) (i)

-1.0

25 -6.2

-6.0

-5.8

-5.6

-5.4

-5.2

350

400

ACS Paragon Plus Environment

log [PND]

450

500

550

Wavelength (nm)

600

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 39

500

Figure 6. (A) Fluorescence spectra of (i) HSA, (ii) HSA-warfarin binary systems in the absence and

501

presence   of   (iii)   1.30   μM,   and   (iv)   4.50   μM concentrations of PND. The inset shows the far-UV CD

502

spectra of HSA, HSA-warfarin and HSA-warfarin-PND systems. (B) Fluorescence spectra of (i) HSA, (ii)

503

HSA-ibuprofen binary systems in the absence and presence of   (iii)   1.30   μM,   and   (iv)   4.50   μM

504

concentrations of PND. The inset shows the far-UV CD spectra of HSA, HSA-ibuprofen and HSA-

505

ibuprofen-PND systems. (C) Scatchard plot of the HSA-PND complex in the absence and presence of

506

warfarin and ibuprofen. (D) Fluorescence spectra (λex=330 nm) of (i) warfarin, (ii) HSA bound warfarin,

507

(iii) HSA bound warfarin with PND, and (iv) PND bound HSA with warfarin. The inset shows the

508

corresponding near-UV CD spectral changes.

509

The fluorescence intensity of war-HSA decreases gradually upon addition of increasing

510

concentrations of PND (Figure 6A). The fluorescence intensity is quenched by 2-times with 9

511

nm red shifts in the presence of 4.50 µμM PND. Similar quenching in fluorescence intensity of

512

ibu-HSA has been observed upon addition of PND. The fluorescence intensity of ibu-HSA is

513

quenched by 2.74-times with 10 nm red shifts in the presence of 4.50 µμM PND. To know the

514

influence of bound warfarin and ibuprofen on the PND-HSA association process, we have

515

estimated the binding constants (Kb) from the Scatchard plot in the presence of these two site

516

markers (Figure 6C & Table 4).

517

Table 4. Estimated Binding Constants of PND with HSA, war-HSA, and ibu-HSA.

518

Systems 519 520

Binding constant Kb (105 M-1)

R2

HSA-PND

5.01 ± 0.04

0.99

War-HSA +PND

1.15 ± 0.19

0.99

Ibu-HSA +PND

3.47 ± 0.13

0.99

26 ACS Paragon Plus Environment

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Langmuir

521

It is evident from Table 4 that the binding constant of PND with war-HSA and ibu-HSA

522

decreases by 77% and 30%, respectively compared to that of free HSA. These results clearly

523

signify that in the presence of ligands, PND shows altered affinity towards HSA (Scheme 2).

524

This is particularly important for in vivo situation where various ligands can modulate their

525

association process. The lower affinity of PND towards war-HSA compared to ibu-HSA

526

indicates that the domain II of HSA actively participates in the association process through

527

hydrogen bonding interactions. However, we cannot rule out the involvement of domain I of

528

HSA as it also contain negatively charged Asp and Glu residues, which can also participate in

529

the hydrogen bonding interaction with PND. Moreover, binding of warfarin in subdomain IIA of

530

HSA can also trigger a local conformational change in domain I, which may result in the lower

531

binding affinity towards PND. Hence, our results indicate the possible involvement of domain I

532

and domain II of HSA in the association process with PND through hydrogen bonding

533

interactions. Next, to know the fate of bound warfarin in war-HSA, we have monitored the

534

intrinsic fluorescence of warfarin in the war-HSA complex in the absence and presence of PND.

535 536 537 538 539 540 541

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Scheme 2. Schematic Illustration of the Interactions of PND with war-HSA and ibu-HSA.

543

Warfarin shows a fluorescence peak at 390 nm upon excitation at 330 nm.59 The

544

fluorescence intensity of warfarin increases by 82% with a blue shift of ~8 nm in the presence of

545

HSA (Figure 6D). Similar enhancement in the fluorescence intensity of warfarin has been

546

observed earlier upon binding with HSA.59,60 However, the peak intensity of warfarin in war-

547

HSA decreases by 67% upon addition of 4.50 µμM of PNDs. This can be explained by

548

considering dissociation of bound warfarin in war-HSA as a consequence of PND-HSA

549

association (Scheme 2). Moreover, it has been observed that the fluorescence intensity of

550

warfarin increases by only 34% in the presence of pre-formed PND-HSA complex. This clearly

551

suggests that the presence of PND directly inhibits the binding of warfarin at the subdomain IIA

552

of HSA. These arguments gain support from near-UV CD measurements. Near-UV CD spectrum

553

of HSA bound warfarin shows a positive peak in the wavelength range of 305 to 340 nm due to

554

the induced ellipticity in warfarin (Figure 6D inset).39 Upon addition of PND, this positive peak

555

disappears completely signifying the dissociation of bound warfarin from site I of HSA as a

556

consequence of altered conformation of native HSA upon association with PND.

557

4. Conclusions

558

In summary, we have demonstrated the detailed mechanism and thermodynamics of

559

interaction between PND and human serum albumin in its free and ligand-bound state. At

560

physiological pH, PND quenches the intrinsic fluorescence of HSA due to the formation of

561

ground state complex. The estimated thermodynamic parameters from ITC and van’t   Hoff  

562

equation suggest the involvement of hydrogen bonding interactions between the surface amine

563

and hydroxyl groups of PND and carboxylate groups of HSA. Ligand-bound HSA shows a lower

564

binding affinity towards PND due to the altered conformation of native HSA. It has been 28 ACS Paragon Plus Environment

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565

observed that the binding constant of PND towards war-HSA significantly lower than that of ibu-

566

HSA possibly due to the involvement of domain II in the association process through hydrogen

567

bonding interactions. Moreover, it has been observed that the association of PND with HSA

568

inhibits the binding of warfarin at the subdomain IIA of HSA. Overall, our results reveal that the

569

physicochemical properties of HSA change significantly upon association with PND through

570

hydrogen bonding interactions.

571

Acknowledgements. The authors thank IIT Indore for providing the infrastructure, experimental

572

facilities, and financial support. This work is supported by Council of Scientific and Industrial

573

Research grant no. 01(2695)/12/EMR-II. The authors thank SIC, IIT Indore for instrumental

574

facilities. The authors thank Dr. Saptarshi Mukherjee and Miss Narayani Ghosh from the Indian

575

Institute of Science Education and Research, Bhopal for their help during isothermal titration

576

calorimetry experiments. The authors thank Professor Anindya Datta and Mr. Avinash Kumar

577

Singh from the Indian Institute of Technology Bombay for their help during dynamic light

578

scattering experiments. The authors also thank SAIF IIT Bombay for TEM measurements.

579

Supporting Information Available: Mass spectrum of as-synthesized PND, PL decay trace of

580

PND, changes in the PL spectra of PNDs in the absence and presence of HSA, near-UV CD

581

spectral changes of HSA in the presence of PND, fluorescence decay traces of HSA in the

582

absence and presence of PND, zeta potential of PND, HSA and PND-HSA mixture, effect of 150

583

mM NaCl on the fluorescence spectra of HSA-PND complex, near-UV CD spectral changes of

584

HSA in the presence of warfarin and ibuprofen, tables of PL lifetime components of PND and 𝛼-

585

helix contents of HSA in the absence and presence of PND, warfarin and ibuprofen. This

586

material is available free of charge via the Internet at http://pubs.acs.org.

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Author Information

589

Corresponding Author

590

* E-mail: [email protected]; Tel: +91-731-2438-779

591

Notes

592

The authors declare no competing financial interests.

Page 30 of 39

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