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Journal of Photochemistry and Photobiology C: Photochemistry Reviews 14 (2013) 53– 71

Contents lists available at SciVerse ScienceDirect

Journal of Photochemistry and Photobiology C:Photochemistry Reviews

journa l h o me pa g e: www.elsev ier .com/ locate / jphotochemrev

eview

inding of serum albumins with bioactive substances – Nanoparticles to drugs

elvaraj Naveenraj, Sambandam Anandan ∗

anomaterials and Solar Energy Conversion Lab, Department of Chemistry, National Institute of Technology, Tiruchirappalli 620 015, India

r t i c l e i n f o

rticle history:eceived 11 May 2012eceived in revised form 5 September 2012ccepted 11 September 2012vailable online 19 September 2012

eywords:erum albumins

a b s t r a c t

The interactions of human and bovine serum albumins (HSA and BSA) with various drugs and nanomate-rials receive great attention in the recent years owing to their significant impact in the biomedical field.Although there are various techniques available for studying such interactions, fluorescence spectroscopyis the most appealing one due to its high sensitivity and straightforwardness. Detailed information aboutthe interactions of drugs and nanomaterials with serum can be deducted from a mass of informationaccumulated by the fluorescence quenching studies. The present review emphasizes the interaction ofvarious nanomaterials, antibiotics, anticancer drugs, anti-inflammatory agents, dyes, flavonoids, and cer-

luorescence quenchinganoparticlesuantum dotsnticancer agentsntibioticslavonoids

tain noxious materials with HSA and BSA. In particular, we focus on the interactions of serum albuminwith nanomaterials having different size and stabilizing agents with various receptors. This review helpsin understanding the structural features of drugs/nanomaterials crucial for not only their affinity forserum albumin but also their optimum pharmacological activities.

© 2012 Elsevier B.V. All rights reserved.

eceptors

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542. Serum albumins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543. Fluorescence quenching studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554. Binding capability of serum albumins with nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

4.1. Metal nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584.2. Semiconductor nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604.3. Dendrimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

5. Organic molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615.1. Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615.2. Anticancer agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635.3. Anti-inflammatory agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645.4. Organic dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655.5. Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665.6. Noxious materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

6. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

∗ Corresponding author. Tel.: +91 431 2503639; fax: +91 431 2500133.E-mail addresses: sanand@nitt.edu, sanand99@yahoo.com (S. Anandan).

389-5567/$20.00 © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jphotochemrev.2012.09.001

5 d Photobiology C: Photochemistry Reviews 14 (2013) 53– 71

T

cUrto

1

mfiTbchbaoi

lsftsatfloiitidtu

oosa(ib

Fig. 1. The crystal structure of HSA. The domains are color-coded as follows: red,domain I; green, domain II; blue, domain III. The A and B sub-domains within each

taining colloidal osmotic pressure in blood, serum albumins canplay a dominant role in the drug disposition and efficacy since

4 S. Naveenraj, S. Anandan / Journal of Photochemistry an

Selvaraj Naveenraj obtained his undergraduate degreein Chemistry from the American College, India (2004)and his post graduate degree in Chemistry from Madu-rai Kamaraj University, India (2006). He also obtained hisMaster of Philosophy degree in Inorganic Chemistry fromUniversity of Madras, India (2007), where he worked onthe synthesis of nanomaterials. Currently, he is a PhDstudent in the research group of Professor Anandan. Hewas awarded the P.S. Lakshminarayanan Prize for Pro-ficiency in Chemistry while doing his under graduation,and qualified in the Graduate Aptitude Test in Engineering.His research interests include synthesis of nanomaterialsand their applications in photocatalysis and biosensors.

o-date he has authored four research articles.

Sambandam Anandan obtained his doctoral degree inChemistry from the University of Madras, India under thesupervision of Prof. P. Maruthamuthu, where he workedon Dye-Sensitized Solar Cells. After two postdoctoralterms at Chungnam National University in South Koreaand Hong Kong University of Science & Technology, heworked as a visiting researcher at National Institute ofAdvanced Industrial Science and Technology (AIST) inJapan. Subsequently, he joined the Central Electrochemi-cal Research Institute in India and later National Instituteof Technology, Tiruchirappalli, where he is now an Asso-ciate Professor of Physical Chemistry, leading the researchgroup “Nanomaterials and Solar Energy Conversion Pro-

esses”. He had also spent short periods at University of Melbourne, Feng Chianiversity, University of Loughborough, and University of Alicante. His recent

esearch interests include hybrid semiconductor nanomaterials and their applica-ions in, solar cells, photocatalysis, electrocatalysis, and biosensors. He is the authorf ca 100 research articles.

. Introduction

Investigations on the interactions of drug molecules and nano-aterials with various proteins receive considerable interest in the

eld of chemistry, life science and clinical medicine for decades.he nature and the magnitude of these interactions influence theiosafety, delivery rate, pharmacological response, therapeutic effi-acy and the design of drugs. Hence studies on these interactionselp in understanding the structural features essential for theioaffinity of drugs and nanomaterials toward the pharmacologicalctivity [1–4]. Since serum albumin is essential in the drug deliveryf vertebrates, it is the ideal model for studying the drug–proteinnteractions in vitro.

Optical techniques such as absorption spectroscopy, circu-ar dichroism, ellipsometry, differential light scattering, Ramanpectroscopy, and fluorescence spectroscopy are powerful toolsor studying the drug–protein interactions in vitro due toheir exceptional sensitivity, speed, theoretical foundations, andtraightforwardness [4–7]. Among the various optical techniques,n incalculable amount of information is acquired about the struc-ural fluctuations and the microenvironment surrounding theuorescent labels of proteins from the measurements and analysesf fluorescence spectra, fluorescence lifetime, fluorescence polar-zation, etc. Hence, fluorescence spectroscopy plays a pivotal rolen the investigation of interactions between the drug molecule andhe receptor (serum albumins). In particular, fluorescence quench-ng studies are widely utilized for revealing the accessibility of arug/nanomaterial (quencher) to the fluorophore moiety in a pro-ein, which in turn helps us to understand the nature and thenderlying mechanism of drug–protein interactions [8].

Here we review the recent literature about the interactionsf human and bovine serum albumins (HSA and BSA) with vari-us drug molecules and nanomaterials studied using fluorescencepectroscopy. The review is organized as follows. Descriptionbout (i) HSA and BSA which are essential in the drug delivery;

ii) pivotal role of fluorescence quenching studies in determin-ng the interactions and binding of drugs with HSA/BSA; (iii) theiological applications of metal, semiconductor and metal-doped

domain are depicted in dark and light shades, respectively.

From Ref. [9].

nanoparticles, and their binding capability toward serum albumins;and (iv) the binding capability of various organic molecules suchas antibiotics, anticancer drugs, anti-inflammatory agents, dyes,flavonoids and noxious materials toward serum albumins.

This review does not seek to provide an absolute review of allarticles published on drug–serum albumin interactions, rather itprovides a snapshot of the assortment of fluorescence quench-ing studies involving the interactions and emphasizes on someof the key research directions and paradigms emerging in thisarea.

2. Serum albumins

Serum albumins, the most abundant soluble protein in thesystemic circulation comprising 52–60% in plasma, are synthe-sized by the parenchymal cells of the liver and exported asa non-glycosylated protein. They possess a half life in circula-tion of 19 days. Serum albumins consist of amino acid chainsforming a single polypeptide with well-known sequence, whichcontain three homologous �-helices domains (I–III) that assem-bled to form a heart shaped molecule whose dimensions are80 A × 80 A × 80 A × 30 A. Each domain contains 10 helices and isdivided into anti-parallel six-helix and four domains (A and B)extensively cross-linked by disulfide bridges. Fig. 1 shows the crys-tal structure of HSA illustrating I–III domains [9]. Serum albuminsare clearly an extraordinary globular protein molecule of manifoldbiological and pharmacokinetic functions. They are capable of bindreversibly with a large variety of relatively insoluble endogeneousand exogeneous ligands even though their principal function is totransport metabolites such as nutrients, hormones, fatty acids and avariety of pharmaceuticals. Apart from an important role in main-

it increases the apparent solubility of hydrophobic drugs in theplasma [9–14]. Serum albumins serve as the depot for the inter-acting bioactive substance and also it can be circulated through thesystem in the body. The binding affinity of any substance to serum

S. Naveenraj, S. Anandan / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 14 (2013) 53– 71 55

SA anF

anaainsdbtto

iarfBrs2aporFtbiIdaIiamaiaaf(oT

Fig. 2. Three dimensional structure of serum albumins [(a) Hrom Ref. [30].

lbumin is one of the major factors that determine the pharmacoki-etics i.e., time course of drug absorption, distribution, metabolism,nd excretion. In addition to the time course, the binding affinitylso determines the bioavailability of drugs [15]. If the binding affin-ty is low, the initial step of pharmacokinetics (drug absorption) isot feasible. In the case of moderate binding affinity of bioactiveubstances to serum albumins, the absorption and distribution ofrugs to various tissues are feasible. On the other hand, when theinding affinity is high, the absorption of drug is feasible but its dis-ribution to the required tissues is be limited due to the stability ofhe complex, which in turn adversely affects the pharmacokineticsf the drug.

Among serum albumins, HSA and BSA are extensively stud-ed due to their significance in the pharmacology field. Both HSAnd BSA display approximately 80% sequence homology and aepeating pattern of disulfides. The molecular weights are 66 kDaor BSA and 66.5 kDa for HSA. The tertiary structures of HSA andSA show 76% similarity [16]. Crystal structure analyses haveevealed that HSA contains 585 amino acid residues with 17 tyro-yl residues and only one tryptophan (Trp) located at position14 along the chain (subdomain IIA); whereas, BSA contains 582mino acid residues with 20 tyrosyl residues and two trypto-hans located at positions 134 and 212 and Trp-134 at the surfacef the molecule [11,12,16–20,10,21–29]. The chemical microenvi-onment of Trp-212 in BSA is similar to that of Trp-214 in HSA.ig. 2 shows the three dimensional structure of HSA and BSA withryptophan residues in green color [30]. There are two principalinding sites present in serum albumins (shown in Fig. 1) located

n the subdomain IIA (Sudlows site I: warfarin-binding site) andIIA (Sudlows site II: indole/benzodiazepine site). Although theyiffer in their affinities, they appear to be homologs in both BSAnd HSA [15,20]. Site I appears to be capacious; whereas, siteI appears to be smaller, or narrower. Site II shows less flexibil-ty than that of site I because binding at site II is often stronglyffected by stereoselectivity. The binding affinity offered by site I isainly through hydrophobic interactions; whereas, site II involves

combination of hydrophobic, hydrogen binding, and electrostaticnteractions [9,31]. Absorption peak maxima of serum albuminsre around 280 nm. The intrinsic fluorescence of serum albuminsppears at 340 nm when excited at 280 nm which is originating

rom the three aromatic l-amino acid (tryptophan (Trp), tyrosineTyr), and phenylalanine (Phe)) residues. Indeed, the intrinsic flu-rescence of serum albumins is mainly contributed by the Trp andyr residues because of the low fluorescence quantum efficiency of

d (b) BSA], with tryptophan residues shown in green color.

phenylalanine. The intrinsic fluorescence characteristics are verysensitive to the microenvironment of the fluorescent residues orchanges in the local surroundings of serum albumins, such asconformational transition, biomolecular binding and denaturation[32].

3. Fluorescence quenching studies

Considering the interaction of various molecules with serumalbumins, changes in their aggregation state may be easily deducedas intrinsically fluorescent serum albumins are very sensitive tolocal changes in the polarity, conformation and/or exposure to thesolvent. The interaction will lead to modifications in fluorescenceintensity-decrease (‘quenching’) or increase (‘enhancement’). Flu-orescence quenching may result from variety of processes such asexcited state reactions, molecular rearrangements, energy transfer,ground-state complex formation (static quenching) or collisionalinteractions (dynamic quenching) [8]. The interacting moleculequenches the intrinsic fluorescence of serum albumin with or with-out any shift (red- or blue-shift) in the emission peak maxima. Ifthe interacting molecule quenches the fluorescence without affect-ing the spectral maximum, the hydrophobicity and polarity in themicroenvironment of the fluorophore (Trp or Tyr) is not altered.For example, gold nanoparticles quench the fluorescence of serumalbumins [33] without having any effect on the fluorescence spec-tral maximum (Fig. 3). If the interacted molecule quenches thefluorescence with a blue-shift in the spectral maximum, it indicatesa decrease in the polarity or an increase in the hydrophobicity ofthe microenvironment surrounding the fluorophore site; whereasa red-shift should be indicative of an increase in the polarity ordecrease in the hydrophobicity of the microenvironment [34]. Forexample, the fluorescence of BSA quenched by silver nanoparticles(Fig. 4) is accompanied by a blue shift in the emission maximum ofBSA [35].

The fluorescence quenching for interacted molecule (quencher(Q)) and protein (P) can be analyzed by the Stern–Volmer equation[36]:

F0

F= 1 + KSV [Q ] = 1 + kq�0[Q ] (1)

where F0 and F denotes the steady state fluorescence intensitiesin the absence and the presence of the quencher Q, respectively,kq is the bimolecular quenching rate constant, �0 is the averagelifetime of the protein, [Q] is the concentration of the quencher, KSV

56 S. Naveenraj, S. Anandan / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 14 (2013) 53– 71

300 350 400 450 5000

250

500

750

Flu

ore

sc

en

ce

in

ten

sit

y / a

.u.

Wavelength (nm)

A

G

300 350 400 450 5000

40

80

120

160

A

G

Flu

ore

sc

en

ce

in

ten

sit

y / a

.u.

Wavelength (nm)

a

b

Fig. 3. Fluorescence spectra of serum albumin (4 �M) [(a) BSA and (b) HSA]quenched by gold nanoparticles in the concentration range of 0–8 �M.

From Ref. [33].

Fig. 4. Fluorescence spectra (top to bottom) of BSA and BSA in presence of sil-ver nanoparticles (a–e) concentrations of 0.0903 × 10−9, 0.225 × 10−9, 0.451 × 10−9,0.677 × 10−9 and 0.8127 × 10−9 M.

From Ref. [35].

Fig. 5. Classical Stern–Volmer plot of F0/F versus [Au] for HSA in the presence of

different gold nanoparticles having sizes: (1) 8, (2) 10, (3) 16, (4) 25, (5) 34, (6) 41,(7) 47, (8) 55, and (9) 70 nm.

From Ref. [65].

is the Stern–Volmer quenching constant [37]. The above equation isapplied to determine KSV by linear regression of a plot of F0/F against[Q]. For example, the classical Stern–Volmer plots for HSA in thepresence of gold nanoparticles having different sizes are shown inFig. 5. The Stern–Volmer quenching constants for serum albuminsby various bioactive substances are summarized in Table 1.

The fluorescence quenching can either be dynamic or static.Dynamic quenching refers to a process where the fluorophoreand the quencher interact during the excited-state lifetime of thefluorophore; whereas, static quenching refers to the formationof the fluorophore–quencher complex in the ground state. Staticquenching can easily be distinguished from dynamic quenching byexamining their temperature dependence, or by the lifetime mea-surements. If the quenching is dynamic, the bimolecular quenchingconstant, which is diffusion-dependent, increases with raise intemperature. Whereas in the case of static quenching, the quench-ing constant decreases with raise in temperature because the

stability of the complex between the fluorophore and the quencheris lowered at higher temperatures. Similarly, if an increase in theconcentration of the quencher has no effect on the fluorescence life-time of serum albumins, then it reveals that the quenching follows

Table 1Stern–Volmer constants, KSV , for the quenching of serum albumins by various bioac-tive substances.

S. No. System Stern–Volmer constant, KSV

1 BSA–Au NP (8 nm) 5.95 × 10−8

2 BSA–Au NP (10 nm) 5.79 × 10−8

3 BSA–Au NP (16 nm) 5.13 × 10−8

4 BSA–Au NP (25 nm) 4.37 × 10−8

5 BSA–Au NP (34 nm) 3.60 × 10−8

6 BSA–Au NP (41 nm) 2.90 × 10−8

7 BSA–Au NP (47 nm) 2.63 × 10−8

8 BSA–Au NP (55 nm) 2.39 × 10−8

9 BSA–Au NP (70 nm) 2.09 × 10−8

10 BSA–MPA stabilized CdTe QDs 1.20 × 10−6

11 BSA–CYS stabilized CdTe QDs 1.79 × 10−6

12 BSA–TGA stabilized CdTe QDs 2.84 × 10−6

13 BSA–G4 PAMAM-OH dendrimers 2.87 × 10−3

14 BSA–G3.5 PAMAM dendrimers 3.83 × 10−3

15 BSA–G4 PAMAM dendrimers 8.38 × 10−3

16 BSA–G6.0 PAMAM-PC 48.52 × 10−3

17 BSA–G6.0 PAMAM-NH2 25.78 × 10−3

18 BSA–G5.5 PAMAM-OH 7.84 × 10−3

S. Naveenraj, S. Anandan / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 14 (2013) 53– 71 57

0 10 20 30 40 5010

100

1000

10000

100000C

ou

nts

Time (ns)

Fc

F

trqno(

sm

l

woctScbotb

Fo

F

-6.0 -5.8 -5.6 -5.4 -5.2 -5.0

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

HSA

BSA

log

[(F

0-F

)/F

]

log [Au]

Fig. 8. Modified Stern–Volmer plot for serum albumins in the presence of sono-

KSV = KA[Q ]n−1 (3)

Table 2The binding constant, KA , for the interaction of serum albumins with various bioac-tive substances.

S. No. System Binding constant,KA (M−1)

1 HSA–sonochemically synthesized Au NP 5.68 × 104

2 BSA–sonochemically synthesized Au NP 3.71 × 104

3 HSA–Au NP 1.12 × 107

4 BSA–Au NP 7.71 × 107

5 BSA–GSH stabilized CdTe QDs 8.89 × 105

6 BSA–CYS stabilized CdTe QDs 2.01 × 105

7 BSA–MPA stabilized CdTe QDs 1.11 × 105

5

ig. 6. Fluorescence decay curves of BSA in the presence of Au nanoparticles in theoncentration range of 0–8 �M.

rom Ref. [33].

he static mechanism; whereas, for dynamic quenching the fluo-escence lifetime of serum albumins decreases with increase in theuencher concentration [8,37,38]. For example, the addition of goldanoparticles does not show any effect on the fluorescence lifetimef BSA [33] (Fig. 6); whereas; the addition of benzo[a]phenazineBAP) lowers the fluorescence lifetime of BSA (Fig. 7) [39].

When drug molecules bind independently to a set of equivalentites on serum albumin, the equilibrium between free and boundolecules is given

og[

F0 − F

F

]= log KA + n log[Q ] (2)

here KA is the binding constant to a site and n is the numberf binding sites. Hence the binding parameters, KA and n, can bealculated using the values of intercept and slope obtained fromhe plot of log[(F0 − F)/F] versus log[Q]. For instance, modifiedtern–Volmer plot for serum albumins in the presence of sono-hemically synthesized gold nanoparticles is shown in Fig. 8. Theinding constant for the interaction of serum albumins with vari-

us bioactive substances is summarized in Table 2. If the value ofhe binding constant KA is in the range 1–15 × 104 M−1, then theinding affinity is moderate [31]. By considering Eqs. (1) and (2),

0 10 20 30 40 501

10

100

1000

10000

100000

Co

un

ts

Time (ns)

ig. 7. Fluorescence decay curve of BSA (a) and BSA (b) in the absence and presencef BAP. [BAP] ranges from 0, 0.1, 0.2 and 0.3 �M.

rom Ref. [39].

chemically synthesized gold nanoparticles.

From Ref. [33].

the relation between the Stern–Volmer quenching constant and thebinding constant is

8 HSA–CUR 3.12 × 109 HSA–DAC 6.36 × 102

10 BSA–CUR 3.42 × 106

11 BSA–DAC 2.92 × 103

12 HSA–EDAC 3.55 × 104

13 BSA–EDAC 7.41 × 103

14 HSA–DMACA 5.13 × 104

15 BSA–DMACA 4.17 × 104

16 HSA–tangeretin 3.52 × 104

17 HSA–nobiletin 3.66 × 106

18 HSA–naringenin 2.73 × 104

19 HSA–naringin 2.78 × 103

20 HSA–narirutin 4.68 × 104

21 BSA–galangin 6.43 × 105

22 BSA–kaempferol 2.58 × 106

23 BSA–quercetin 3.65 × 107

24 BSA–myricetin 4.54 × 108

25 HSA–flavone 1.95 × 104

26 HSA–7-hydroxyflavone 3.55 × 106

27 HSA–chrysin 1.07 × 106

28 HSA–baicalein 9.12 × 105

29 BSA–flavone 1.95 × 104

30 BSA–7-hydroxyflavone 1.48 × 107

31 BSA–chrysin 1.20 × 106

32 BSA–baicalein 4.68 × 105

33 BSA–myricetin 1.84 × 108

34 BSA–dihydromyricetin 1.36 × 104

35 BSA–quercetin 3.65 × 107

36 BSA–quercitrin 6.47 × 103

37 BSA–DDN 6.26 × 103

38 BSA–DSS 1.51 × 104

5 d Photobiology C: Photochemistry Reviews 14 (2013) 53– 71

weo

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madpotitdtotttcaci

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R

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wtnt

300 350 400 4500.00

0.04

0.08

0.12

Wavelength (nm)

Ab

so

rba

nc

e

0

50

100

150

200

Flu

ore

scen

ce in

ten

sit

y / a

.u.

Quencher (PTK)

absorption

Donor (HSA)

fluorescence

Overlap

I ntegral

J

8 S. Naveenraj, S. Anandan / Journal of Photochemistry an

hich suggests that the Stern–Volmer quenching constant becomequal to the binding constant when the number of binding sites isne.

The force of interaction between drugs and biomolecules maynclude electrostatic interactions, multiple hydrogen bonds, weakan der Waals interactions, and hydrophobic interactions, whichan be evaluated using the signs and magnitudes of thermodynamicarameters such as enthalpy change (�H), free energy change (�G)nd entropy change (�S). These parameters can be calculated usinghe following Van’t Hoff thermodynamic equations

nKA2

KA1= 1

R

(1T1

− 1T2

)�H (4)

G = −RT ln KA = �H − T�S (5)

here KA is the binding constant at the corresponding temperature, and R is the gas constant. The negative value of �G reveals that thenteraction proceeds spontaneous at the standard state. Accordingo the views of Ross and Subramanian [40], when �H < 0 or �H ≈ 0nd �S > 0, the main force is due to electrostatic interactions; whenH < 0 and �S < 0, the main force is due to van der Waals or hydro-

en bonding, and when �H > 0 and �S > 0, the main force is due toydrophobic interactions. The thermodynamic parameters such asH, �G, and �S for the interaction of serum albumins with various

ioactive substances are summarized in Table 3.Förster resonance energy transfer (FRET) [41] is a sensitive

ethod for the detection of interactions between drug moleculesnd serum albumin. FRET efficiency can be used to evaluate theistance between the bound drug molecule and the fluorophoreresent in serum albumins [8,42]. According to FRET, the transferf energy, which occurs through the direct electrodynamic interac-ion between the primarily excited molecules and their neighbors,s controlled by the following aspects: (1) the fluorescence quan-um yield of the donor, (2) the relative orientation of the transitionipoles of the donor and acceptor, (3) overlap integral betweenhe fluorescence spectrum of the donor and absorption spectrumf the acceptor, and (4) the distance (r0) between the donor andhe acceptor [30]. The Förster theory points out that the energyransfer efficiency ‘E’, in addition to its dependence on the dis-ance between the acceptor and the donor, depends upon theritical energy transfer distance, R0 (Förster distance; the distancet which the efficiency of energy transfer is 50%). Hence the effi-iency of energy transfer for a single donor–single acceptor systems expressed by the following equation:

= 1 − F

F0= R6

0

R60 + r6

0

(6)

he magnitude of R0 depends on the fluorescence spectrum of theonor (serum albumins) and absorption spectrum of the acceptorbound drug molecule). R0 is expressed as follows

60 = 8.8 × 10−25[�2n−4�D J] (7)

here �2 is the spatial orientation factor related to the geometry ofhe donor and acceptor dipoles, which is 2/3 for random orientations in fluid solution, n is the refractive index of the medium (1.36or BSA and 1.336 for HSA), �D is the fluorescence quantum yield ofhe donor (0.118 for BSA and 0.15 for HSA), J is the spectral overlapntegral, which is given by

=∫ ∞

0F(�)ε(�)�4 d�∫ ∞0

F(�)d�(8)

here F(�) is the corrected fluorescence intensity of the donor inhe wavelength range from � to (� + ��), with the total intensityormalized to unity and ε(�) is the molar extinction coefficient ofhe acceptor at �. Fig. 9 shows the overlap plots of fluorescence

Fig. 9. Overlap plots of fluorescence spectra of HSA with absorption spectra of PTK.

From Ref. [43].

spectra of HSA with the absorption spectra of perylene-3,4,9,10-tetracarboxylate tetrapotassium salt [43]. Using the above threeEqs. (6)–(8), the donor-to-acceptor distance, r0, can be calculated.If r0 < 7 nm [44,45] and 0.5R0 < r0 < 1.5R0 [46], the probability ofenergy transfer from serum albumins to bioactive substances ishigh.

Synchronous fluorescence spectroscopy introduced by Lloyd[47,48], which involves the simultaneous scanning of excitationand the fluorescence monochromators of a fluorimeter, whilemaintaining a fixed wavelength difference (��) between them, is asimple and effective means to measure the fluorescence quenchingand the possible shift of the maximum emission wavelength (�max)relative to the alteration of the polarity around the chromophoreat physiological conditions. When �� is stabilized at 15 nm or60 nm, synchronous fluorescence offers the characteristics of tyro-sine residues or tryptophan residues in the serum albumins [49].In the synchronous fluorescence spectra, the fluorescence intensitydecreases with or without any shift in the emission maximum. Adecrease in fluorescence intensity without any shift indicates thatthe microenvironment around that particular residue is not dis-turbed. Red-shift is indicative of an increase in the hydrophilicityaround the fluorophore in serum albumin. Blue-shift should be dueto an increase in the hydrophobicity around the fluorophore moi-ety [50,51]. Fig. 10 shows synchronous fluorescence spectrum ofHSA with �� = 15 nm in the absence and presence of (a) colloidalAgTiO2 and (b) colloidal TiO2 nanoparticles in the concentrationrange of 0–3 × 10−5 M [52,53].

4. Binding capability of serum albumins with nanoparticles

During the past decade, a tremendous attention has beenfocused on recognizing the interactions of nanomaterials withbiomolecules [54]. The size-dependent tunable optical propertiesof nanomaterials make them promising for various innova-tive biomedical applications – from diagnosis to therapy [55].Hence evaluation of the interactions between nanomaterials andbiomolecules such as serum albumins becomes noteworthy.

4.1. Metal nanoparticles

Gold (Au) nanoparticles, one of the noble metal plasmon-resonant nanoparticles where the collective coherent oscillation

of free electrons enables intense light absorption [56,57], hold agreat promise for biology and medicine due to their interestingsize-dependent tunable optical properties. Moreover the biocom-patibility and stability of Au nanoparticles make them excellent

S. Naveenraj, S. Anandan / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 14 (2013) 53– 71 59

Table 3The thermodynamic parameters �H, �G, and �S for the interaction of serum albumins with various bioactive substances.

S. No. System �H (kJ mol−1) �G (kJ mol−1) �S (J mol−1 K−1)

1 HSA–ketaconazole −30.25 −33.15 9.762 BSA–ketaconazole −17.96 −31.75 46.313 HSA–Jatrorrhizine −10.89 −27.55 56.274 HSA–PZFX −84.86 −28.98 −186.575 HSA–Farrerol −18.51 −32.43 47.526 HSA–INOD 9.66 −25.25 121.227 HSA–AHC −3.66 −27.65 82.458 BSA–SMZ −57.6 −24.0 −111.09 BSA–RF −5.93 −27.36 74.1610 HSA–GEM −13.96 −21.68 26.9211 BSA–GEM −15.76 −25.03 32.6812 HSA–EDAC 152.29 −22.93 59013 BSA–EDAC 81.42 −22.28 35014 HSA–DMACA 113.28 −23.21 46015 BSA–DMACA 190.90 −24.85 72016 HSA–EGCG −22.59 −27.45 16.2317 HSA–theasinesin −12.29 −24.09 −19.4718 HSA–ADNR −21.01 −28.18 24.7119 HSA–ODNR −17.97 −29.03 36.5220 HSA–genistein −22.24 −28.04 19.6021 HSA–trans-resveratrol −20.69 −27.42 23.3722 HSA–docetaxel −41.07 −27.00 −49.7223 HSA–HNF −31.10 −33.16 6.8724 BSA–TPP 70.96 −23.27 318.3525 BSA–TMEOPP −64.56 −19.7 167.9726 BSA–TClPP 30.02 −24.93 −133.8927 BSA–NCTPP 44.63 −32.92 264.6828 BSA–NCTMPP 61.01 −34.17 324.8629 BSA–NCTAPP 72.28 −34.48 364.3830 HSA–AR2 −4.512 −30.85 88.3831 HSA–AR73 253.4 −44.6 103032 HSA–DY9 −14.27 −28.03 47.6233 BSA–Sudan II −12.6 −22.9 −35.334 BSA–Sudan IV −1.79 −23.4 −73.835 BSA–MG −27.25 −23.96 −11.2336 BSA–RB −79.61 −38.38 −143.3737 BSA–Mordant Red −50.49 −35.36 −50.8838 BSA–DBSBL 227.2 −28.9 88639 BSA–AY −21.94 −30.65 30.0440 BSA–CGR −12.67 −29.78 58.6041 BSA–DDN −238.21 −17.54 −711.86

2

cdtacmhcov

sm[ssssnnbs1d

42 BSA–DSS 72.643 BSA–C3 −140.744 BSA–C1.3 −117.8

andidates for in vivo phototherapy of cancer [58], the sensitiveetection of HIV-1 in plasma [59], cell imaging due to the sensi-ive detection of Adenosine triphosphate (ATP) in live cells [60],nd distinguishing among various virus types [61]. Upon simpleonjugation of gold nanoparticles onto therapeutically inactiveonovalent small organic molecules, they can be converted into

ighly active drugs that effectively inhibit HIV-1 fusion to human Tells [62]. Due to the above vast biomedical applications, the studyn interaction of gold nanoparticles with serum albumins becomesital.

The binding of serum albumins with gold nanoparticles synthe-ized using NaBH4 reduction method and sonochemical reductionethod were investigated using optical techniques by Gao et al.

63] and Naveenraj et al. [33]. The effect of these gold nanoparticles,ynthesized by different methods, on the fluorescence spectra oferum albumins show gradual decrease in the fluorescence inten-ity of serum albumins without any changes to their fluorescencepectral shape and maximum, which indicates the formation ofon-fluorescent ground state complexes (static quenching mecha-ism). The same fluorescence quenching trend is observed for theinding of gold nanospheres and gold nanorods with BSA in the

tudy conducted by Iosin et al. [64]. The binding constant (Table 2,–2) of sonochemically synthesized gold nanoparticles with HSAeduced from modified Stern–Volmer plot (Fig. 8) is found to be

−20.86 301.58−33.5 −365.7−45.2 −248.0

about 1.5 times greater than that of BSA. This binding constant indi-cates that the affinity of HSA for Au nanoparticles synthesized bythe sonochemical method is more than that of BSA [33]; whereas,in the case of Au nanoparticles synthesized by the NaBH4 reduc-tion method (Table 2, 3–4), BSA shows more affinity than HSA [63].The difference in the binding affinity can be attributed to the factthat sonochemically synthesized nanoparticles have large surfacearea which induces a higher binding interaction with biomoleculesand causes the rapid transfer of drugs to the tissue. Also, the sono-chemically synthesized nanoparticles show uniform size and shape,which is evident from high resolution transmission electron micro-graphs. In the binding affinity study conducted by Pramanik et al.[65] using Au nanoparticles having diameters of 8, 10, 16, 25, 34,41, 47, 55 and 70 nm, which are synthesized by varying the [Au] tocitrate ratio, the Stern–Volmer constant, KSV (Table 1, 1–9) increaseswith decrease in the size of Au nanoparticles (Fig. 5). In other words,the fluorescence quenching is more efficient in the case of smallerAu nanoparticles, which suggests that smaller particles will havemore binding interaction due to the large surface area.

Recently the interaction of silver (Ag) nanoparticles with BSAusing fluorescence spectroscopy was studied by Mariam et al. [35]

owing to the fact that Ag nanoparticles have potential antimicrobialand antiplatelet/antithrombolytic activities. The silver nanopar-ticles quench the fluorescence of BSA with a blue-shift in their

60 S. Naveenraj, S. Anandan / Journal of Photochemistry and Pho

Fig. 10. Synchronous fluorescence spectrum of serum albumin with �� = 15 nmic

F

epiScpt

aaaic

4

pbStauTpdppTameildn

n the absence and presence of colloidal Ag-TiO2 (a) and colloidal TiO2 (b) in theoncentration range of 0–3 × 10−5 M.

rom Ref. [52,53].

mission maximum (Fig. 4). The non-linearity of Stern–Volmerlots in Fig. 4 indicates that both static and dynamic quench-

ng are involved. Further, ‘n’ value obtained from the modifiedtern–Volmer plot and the blue-shift in the synchronous fluores-ence spectra (�� = 60 nm) indicate that Ag nanoparticles lower theolarity or increase the hydrophobicity of the microenvironment ofhe tryptophan residues in BSA.

In this section, fluorescence quenching studies on the inter-ction of metal nanoparticles such as gold and silver with serumlbumins is discussed. These studies illustrate the effect of the sizend stabilizing agent of metal nanoparticle on the hydrophobic-ty of the binding site. These studies will be valuable during theonstruction of nanomedicines based on Au and Ag nanoparticles.

.2. Semiconductor nanoparticles

Semiconductor nanocrystals (quantum dots) enthused over theast decade from electronic materials and physics to biological andiomedical areas [66] due to their unique photophysical properties.emiconductor TiO2 nanoparticles, a well-known photocatalyst inhe deactivation of microorganisms and viruses, of various sizesnd morphologies exhibit cytotoxicity toward some tumors underltraviolet light (UV) excitation. Also, interactions of nanosizediO2 complexed with antibodies make it a visible light-inducedhototoxic agent against human brain cancer [67]. Even in theark condition, TiO2 nanoparticles deform the tumor cell colonyattern by arresting their growth [68]. Owing to the bactericidalroperties either on their own or upon illuminated with UV light,iO2 nanoparticles are used in many products including fabrics [69]nd filters [70]. Further, it is used as a food colorant, additive inany cosmetic creams, and medicines [71]. It has been hypoth-

sized that the doping of transition metal nanoparticles to titania

mproves the light absorption capability of TiO2, increases its carrierifetime by scavenging electrons from the surface of the semicon-uctor, and improves the biological activities [72]. Pt–TiO2 hybridanoparticles show efficient cytotoxicity toward microbial cells in

tobiology C: Photochemistry Reviews 14 (2013) 53– 71

water when irradiated with near-UV light. Pt–TiO2 and Au–TiO2nanocrystals show effective cytotoxic effect toward cancer cellsunder near-UV light irradiation than that of undoped TiO2 nanopar-ticles [73].

Due to these fabulous applications of TiO2 nanoparticles, theinteraction of commercially available TiO2 nanoparticles (20 nm)and TiO2 nanoparticles synthesized by hydrolysis method (1.4 nm)with human serum albumin were investigated by Sun et al. [71]and Kathiravan et al. [74]. The effect of commercially available TiO2nanoparticles on the fluorescence spectra of HSA is to decrease thefluorescence intensity of HSA with a red shift in the emission max-imum; whereas, TiO2 nanoparticles synthesized by the hydrolysismethod show a decrease in fluorescence intensity without any shiftin the emission maximum. In both cases, the quenching follows thestatic mechanism i.e., the formation of ground state complexes.The synchronous fluorescence spectra indicate that the commer-cially available TiO2 nanoparticles disturb the environments of bothtyrosine and tryptophan residues present in HSA; whereas; TiO2nanoparticles synthesized by the hydrolysis method only affectsthe microenvironment of tyrosine residues in HSA.

The binding abilities of TiO2 nanoparticles and Ag doped TiO2nanoparticles with serum albumins were investigated using opti-cal techniques by Kathiravan et al. [52,53]. They inferred that bothTiO2 and Ag-TiO2 nanoparticles quench the fluorescence withoutany shift in the emission maxima. Lifetime measurements confirmthat both nanoparticles follow the static quenching mechanism.In the synchronous fluorescence spectra of serum albumins at�� = 60 nm, there is no shift in the emission wavelength, whichconfirms the absence of binding site near the tryptophan residue.In the synchronous fluorescence spectra (�� = 15 nm) of serumalbumins, Ag-TiO2 addition showed a red-shift (Fig. 10a), whichsuggests that the binding site is near the tyrosine region and theenvironment is more polar (or less hydrophobic) [75] and moreexposed to the solvent molecules [76]. On the other hand, pure TiO2nanoparticles show a blue-shift (Fig. 10b), which suggests that thebinding site is also near tyrosine region but it is in less polar (ormore hydrophobic) environment and less exposed to the solventmolecules.

Among various quantum dots (QDs), CdSe and CdSe/ZnScore/shell QDs are extensively applied in biological applicationssuch as cell labeling and bio-imaging due to their exceptionalstability and high photoluminescence quantum yield [77]. Theinteractions of CdSe and CdSe/ZnS core/shell QDs with BSA areinvestigated by Ju et al. [78] and Dzagli et al. [79]. Both CdSe andCdSe/ZnS QDs interact with BSA through static quenching (non-fluorescent ground state complex formation). CdSe QDs quench thefluorescence of BSA without any shift in the emission maximum;whereas, CdSe/ZnS quench the fluorescence of BSA with blue-shiftin the emission maximum. This observation suggests that the pres-ence of ZnS in the shell reduces the polarity around the tryptophanresidues, which is evident from the blue-shift in the emission maxi-mum [80]. van der Waals force and hydrogen bonds play major rolesin the interactions of CdSe QDs with BSA; whereas, electrostaticinteractions important for CdSe/ZnS QDs.

In continuation, due to the applications of CdTe QDs in the fieldof bio-imaging, immunofluorescence and phototherapy, the inter-action of CdTe QDs with BSA are investigated by Liang et al. [81],Idowu et al. [82] and Wang et al. [83]. In the size comparative bind-ing study conducted by Liang et al. [81], TGA stabilized CdTe QDsof size 1.8, 2.6 and 3.1 nm are synthesized just by varying the reac-tion time. As the size of TGA stabilized CdTe QDs is increased, theirbinding interaction with BSA increases. This behavior of CdTe QDs

is probably due to the changes in the relative size of BSA and CdTeQDs during the binding. In the study conducted by Idowu et al.[82], CdTe QDs of size 2.3, 3.0 and 3.2 nm were synthesized by vary-ing the stabilizers mercaptopropionic acid (MPA), l-cysteine (CYS),

d Photobiology C: Photochemistry Reviews 14 (2013) 53– 71 61

a(BwIdCB(poQob

bcGncctB

nteoi

4

hiltd[SdwhtsPs

PdsTGOaPbdStPa

aqT

S. Naveenraj, S. Anandan / Journal of Photochemistry an

nd thioglycolic acid (TGA). Stern–Volmer quenching constantsTable 1, 10–12) suggests that the binding ability of these QDs withSA follows the order: QD (TGA) > QD (CYS) > QD (MPA). In otherords, the binding ability of QDs increases with increase in size.

n the study conducted by Wang et al. [83], CdTe QDs of meaniameter 3.2 nm are synthesized by varying the stabilizers [MPA,YS and glutathione (GSH)]. The binding ability of these QDs withSA (Table 2, 5–7) follows the order: QD (GSH) > QD (CYS) > QDMPA), which suggests that the interaction depends on the cap-ing agent of QDs. Xiao et al. [84] investigated the interactionf 2.04 nm green-emitting QDs (G-QDs) and 3.79 nm red-emittingDs (R-QDs) with HSA. The binding constant suggests that the sizef CdTe QDs affected the affinity for HSA; larger QDs show strongerinding.

Owing to the applications of CdS nanoparticles in the field ofiology and medicine, the interaction studies of CdS nanoparti-les with BSA is vital and are investigated by Jhonsi et al. [85] andhali [86]. Starch-capped, thioglycerol-capped, or uncapped CdSanoparticles interact with BSA by the formation of ground stateomplex. Starch-capped CdS nanoparticles quench the fluores-ence of BSA with blue-shift in the emission maximum; whereas,hioglycerol capped CdS nanoparticles quench the fluorescence ofSA with red-shift.

In this section, studies on the interactions of semiconductoranoparticles such as TiO2, CdSe, CdSe/ZnS, CdTe and CdS nanopar-icles or QDs with serum albumins are discussed. Illustrated are theffect of nanoparticle size, doping agents, capping agents and shelln the hydrophobicity of the binding site. These studies are of greatmportance for nanomedical and in vivo bioimaging applications.

.3. Dendrimers

Polyamidoamine (PAMAM) dendrimers, a nanoscopic polymer,ave been applied as carrier molecules for magnetic resonance

maging (MRI) contrast agents, near-infrared (NIR) fluorescentabels and transfection vectors in gene therapy [87–89]. Due tohese applications, the interaction of different generation PAMAMendrimers with BSA has been investigated by Shcharbin et al.90], Klajnert et al. [91,92] and Yanming et al. [93]. In the study byhcharbin et al. [90], the interaction of generation 2 (G2) PAMAMendrimers and generation 6 (G6) PAMAM dendrimers with BSAas evaluated. G2 PAMAM dendrimer has a diameter of 2.4 nm withighly flexible structure, while G6 PAMAM dendrimer has a diame-er of 6.5 nm with rigid spherical structure. Stern–Volmer constantsuggested that G2 PAMAM dendrimers have more affinity than G6AMAM dendrimers toward BSA, which can be attributed to theirize and structure.

Klajnert et al. [91,92] investigated the interaction of G3.5AMAM dendrimers, G4 PAMAM dendrimers and G4 PAMAM-OHendrimers with BSA. All these PAMAM dendrimers possess theame core molecule and have 64 end groups on their surface.heir diameters are similar and approximately equal to 4.0 nm.3.5 PAMAM dendrimers, G4 PAMAM dendrimers and G4 PAMAM-H dendrimers are terminated with COOH groups, NH2 groupsnd OH groups, respectively. G3.5 PAMAM dendrimers and G4AMAM dendrimers quench the intrinsic fluorescence of BSA withlue-shift in the emission maxima; whereas, G4 PAMAM-OH den-rimers quench the fluorescence without any spectral shift. Thetern–Volmer constant (Table 1, 13–15) suggests that the affinityoward BSA follows the order G4 PAMAM-OH dendrimers < G3.5AMAM dendrimers < G4 PAMAM dendrimers. This order in theffinity is related with their functional end groups.

In a similar interaction study done by Yanming et al. [93], G5.5nd G6.0 PAMAM dendrimers are compared. These dendrimersuenched the intrinsic fluorescence of BSA through statically.he quenching is accompanied by blue-shift in the case of G6.0

Fig. 11. Molecular structure of rifamycins. Rifamycin SV (RFSV), rifandin (RFD),rifampin (RFP) and rifapentine (RFPT).

PAMAM-NH2 and G5.5 PAMAM-OH; whereas, in the case ofG6.0 PAMAM-PC, red-shift is observed. Stern–Volmer quenchingconstants (Table 1, 16–18) suggest that the strength of interactionsof BSA with the dendrimers is in the order: G6.0 PAMAM-PC > G6.0PAMAM-NH2 > G5.5 PAMAM-OH. These results suggest that thestrength of the interactions strongly depend on the functionalgroups on the surface of the dendrimer.

In this section, the binding interactions of dendrimers withserum albumins are discussed. Studies illustrate that the gener-ation and the functional end groups of dendrimers are critical fortheir interactions with serum albumins. In summary, a critical needin the field of nanobiotechnology is the study of binding interac-tions of various nanoparticles with biomolecules such as serumalbumins. The interactions of biologically significant nanoparticlessuch as metal nanoparticles, semiconductor QDs and dendrimerswith serum albumin are discussed. The results reviewed here illus-trate the symbiotic relationship that nanotechnology shares withbiology.

5. Organic molecules

5.1. Antibiotics

The interaction of rifamycin antibiotics (Fig. 11), an antibacterialdrug often used in the treatment of tuberculosis, with SA has been

investigated by Yang et al. [94], which indicates that among therifamycin antibiotics rifamycin SV (RFSV), rifandin (RFD), rifampin(RFP) and rifapentine (RFPT), the fluorescence quenching occurs inthe order RFPT > RFP > RFD > RFSV. This observation suggests that

6 d Photobiology C: Photochemistry Reviews 14 (2013) 53– 71

iiq1ivoni

dMqcttctag

ttqaoTfipftbsbhstmza

D[7flflioemwPfitpeFiAaoaa

a

Fig. 12. Interaction modes between Farrerol and HSA. Residues around 8 A of theligand are displayed only. The residues of HSA and the ligand structure are rep-

BSA to decrease the fluorescence intensity and shift the emissionmaximum to the red. The thermodynamic parameters suggest thatthe key force of binding between SMZ and BSA is weak van derWaal’s interaction and hydrogen bonding; whereas, for RF, it is

2 S. Naveenraj, S. Anandan / Journal of Photochemistry an

ncrease in the bulkiness of the substituent (R2 moiety) in rifamycins responsible for its high quenching efficiency. RFP and RFPTuench the fluorescence of SA with a red-shift (15 nm for HSA and0 nm for BSA) in the emission maxima, which indicates increase

n the polarity or decrease in the hydrophobicity of the microen-ironment surrounding the fluorophore site; whereas, no shift isbserved in the case of RFSV. The interaction of RFD with HSA doesot involve any shift in the emission maximum; whereas, their

nteractions with BSA accompany a red-shift (10 nm).The interactions of antibacterial agents curcumine (CUR) and

iacetylcurcumine (DAC) with serum albumins are investigated byohammadi et al. [95]. Both CUR and DAC (lipophilic molecules)

uench the fluorescence of serum albumins statically. The bindingonstant (Table 2, 8–11) suggests that HSA is having more affinityoward curcumine than BSA. Even though DAC has more antibac-erial activity than CUR against multiresistant bacteria, the bindingonstants suggest that CUR strongly binds with serum albuminshan DAC, which indicates that the phenolic OH group of CUR playsn important role in the interaction; whereas, the phenolic acetylroup plays an important role in the antibacterial activity.

The antiviral and antifungal agent 1-benzoyl-4-p-chlorphenylhiosemicarbazide (BCPT) [96], the inhibition antibacterial agentetracyclines [97,98], and the antifungal agent ketoconazole [99]uench the fluorescence of serum albumins (HSA and BSA) with

blue shift in the emission maxima, which suggests increasef hydrophobicity in the region surrounding the tryptophan site.hese antibiotics interact with serum albumin by the complexormation which is evident from their Stern–Volmer quench-ng constant at different temperatures. Using the thermodynamicarameters, it is suggested that the force acting on the complexormation of serum albumin with BCPT is hydrophobic interac-ions; whereas, for tetracyclines, it is electrostatic. The acting forceetween ketaconazole and BSA (Table 3, 1–2) is mainly electro-tatic in addition to hydrophobic interactions; whereas, the forceetween Ketoconazole and HSA is synergy of electrostatic andydrophobic interactions. In the case of BCPT, the quenching con-tant of HSA is more than that of BSA; whereas, in the case ofetracycline and ketoconazole, the quenching constant of BSA is

ore than that of HSA. However, in the case of BCPT and ketocona-ole, FRET calculations indicate that the distance between BSA andcceptor is larger than that of HSA.

The interactions of antibiotics such as Jatrorrhizine [100],aunomycin [101], Pazufloxacin mesilate (PZFX) [102], Farrerol

103], indolone-N-oxide derivatives (INOD) [104], and 8-Acetyl--hydroxycoumarin (AHC) [105] with HSA are also studied byuorescence techniques. Jatrorrhizine and Daunomycin lower theuorescence emission intensity of HSA with a conspicuous change

n the emission spectrum. INOD lowers the fluorescence intensityf HSA without any change in the emission maxima. PZFX low-rs the fluorescence intensity of HSA with red-shift in emissionaxima; whereas, AHC lowers the fluorescence intensity of HSAith blue-shift in emission maxima. Jatrorrhizine, Daunomycin,

ZFX, Farrerol and INOD form complex with HSA which is con-rmed from the binding constants at different temperatures. Usinghe thermodynamic parameters (Table 3, 3–7), the force of com-lex formation between Jatrorrhizine and HSA is hydrophobic andlectrostatic interactions; whereas, the force of interaction of PZFX,arrerol and AHC with HSA are hydrogen bonding and hydrophobicnteractions. Such hydrogen bond formation of Farrerol (Fig. 12) andHC (Fig. 13) around the hydrophobic cavity near Try-214 in HSA islso apparent from molecular docking studies [103,105]. The forcef interaction between Daunomycin and HSA is hydrogen bonding

nd electrostatic interactions; whereas, hydrophobic interactionsre responsible for the complexation of INOD with HSA.

The interactions of antibacterial drug Sulfamethoxazole (SMZ)nd antimycobacterial drug Rifampicin (RF) with BSA are studied by

resented using ball and stick model. Hydrogen bonds between the ligand and theprotein are represented using yellow dashed lines.

From Ref. [103].

Naik et al. [106] and Kamat and Seetharamappa [107], respectively.SMZ interacts with BSA to decrease the fluorescence intensity andshift the emission maximum to the blue; whereas, RF interacts with

Fig. 13. Interaction mode between AHC and HSA, only residues around 6.5 A of theligand are displayed. The residues of HSA and the ligand structure are all repre-sented using stick model. The hydrogen bond between the ligand and the protein isrepresented using yellow dashed line.

From Ref. [105].

S. Naveenraj, S. Anandan / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 14 (2013) 53– 71 63

System KA 106 (M-1)

BSA-NCTPP 0.74

BSA-NCTMPP 1.24

BSA-NCTAPP 1.40

System (T= 293K) KA 106 (M-1)

BSA-TPP 1.37

BSA-TMEOPP 2.33

BSA-TClPP 3.51

F ate (Ep P, (ii) N

hsar

scas

5

i4(iGc

ig. 14. Molecular structure of anticancer agents (a) (i) (−)-epigallocatechin-3-gallorphyrins (i) TPP, (ii) TClPP, (iii) TMEOPP, and (d) N-confused porphyrins (i) NCTP

ydrophobic interactions. From the value of the number of bindingites, n, only one independent class of binding site (i.e., Trp-214) ispparent for the BSA–SMZ system; whereas, both the tryptophanesidues of BSA are exposed during the interaction of BSA with RF.

In this section, the binding interaction studies of antibiotics witherum albumins using fluorescence quenching technique are dis-ussed, which illustrate the effect of antibacterial, antiviral andntifungal agents on serum albumins. These studies are of greatignificance in the pharmacology of antibiotics.

.2. Anticancer agents

The interactions of the antineoplastic agent Gemc-tabine hydrochloride (GEM), and the antitumor agents-(dimethylamino)cinnamic acid (DMACA) and trans-ethyl-p-

dimethylamino)cinnamate (EDAC) with serum albumins arenvestigated by Kandagal et al. [108], and Singh and Mitra [109].EM lowers the intrinsic fluorescence of serum albumins withouthanging the emission maximum and the shape of the emission

GCG) and (ii) theasinesin, (b) anthracycline derivatives (i) ADNR and (ii) ODNR, (c)CTMPP, (iii) NCTAPP.

spectrum; whereas, EDAC and DMACA induce a slight blue-shiftand a red-shift in the emission maxima, respectively. The effect oftemperature on the Stern–Volmer constants confirms that GEM,EDAC and DMACA quench the intrinsic fluorescence through thestatic quenching mechanism. The binding constants (Table 2,12–15) suggest that DMACA interacts more strongly and pref-erentially with the albumins when compared with EDAC. Thethermodynamic parameters (Table 3, 10–15) suggest that bothhydrogen bonding and hydrophobic interactions play a role inthe binding of GEM to serum albumins; whereas, hydrophobicinteraction and less electrostatic interaction play a role in thebinding of both EDAC and DMACA to serum albumins.

The interactions of (−)-epigallocatechin-3-gallate (EGCG) andits polymer theasinesin with HSA are studied by Maiti et al.[110] and Ge et al. [111], respectively. Both EGCG and theasinesin

(Fig. 14a), which are present in green tea, are anticancer agentsor antioxidants. Both EGCG and theasinesin lower the intrinsicfluorescence of HSA with red-shift in the wavelength maximum,which indicates a more polar environment in the static complexes

64 S. Naveenraj, S. Anandan / Journal of Photochemistry and Pho

Fig. 15. Stereoview docking pose of HSA and EGCG. Residues of interest and EGCGhave been represented as sticks. The protein chain has been truncated at differentpoints for clarity. The binding pocket of site I is clearly visible with Trp-214 withinhr

F

brmcbhbad

3Oiomst1tHi

dflidoastmtthmai

spwpbd

ydrogen bonding distance of EGCG. The entrance to the pocket is lined with polaresidues.

rom Ref. [110].

etween the quencher and HSA. In both cases, the tryptophanesidue (Trp-214) of HSA is involved in the interaction. The ther-odynamic parameters (Table 3, 16–17) deduced using the binding

onstants suggests that van der Waals interactions and hydrogenonding are the forces for the binding of EGCG to HSA; whereas,ydrophobic and electrostatic interactions play major role in theinding of theasinesin with HSA. Molecular docking studies (Fig. 15)lso support the hydrogen bonding and changes in the polarityuring the interaction of EGCG with HSA [110].

The interactions of anthracycline derivatives (Fig. 14b) such as0-azido-30-deamino daunorubicin (ADNR) [112] and 4′-O-(�-l-leandrosyl)daunorubicin (ODNR) [113] with HSA are investigated

n detail. Both ADNR and ODNR quench the intrinsic fluorescencef HSA, which is accompanied by blue-shifts in the emission maxi-um through static quenching. Stern–Volmer quenching constants

uggest that ADNR quenches HSA more efficiently than ODNR dueo its steric constraint. The thermodynamic parameters (Table 3,8–19) suggests that both hydrophobic and electrostatic interac-ions are the major forces involved in the interaction of ADNR withSA; whereas, only hydrophobic interactions are involved in the

nteraction of ODNR with HSA.The interactions of anticancer agents such as genistein [7],

ocetaxel [114], chlorin derivatives [115], 2-hydroxy-3-nitro-9-uorenone (HNF) [116] and trans-resveratrol [117] with HSA are

nvestigated using fluorescence spectroscopy. Genistein, chlorinerivatives, and trans-resveratrol lower the intrinsic fluorescencef HSA with no other spectroscopic changes; whereas, docetaxelnd HNF lower the fluorescence intensity with slight blue-hift in the emission maximum. Genistein, docetaxel, HNF, andrans-resveratrol interact with HSA through the static quenching

echanism; whereas, chlorin derivatives interact dynamically. Thehermodynamic parameters (Table 3, 20–23) suggest that genis-ein, and trans-resveratrol bound to HSA are mainly based on theydrophobic and electrostatic interactions. It also suggests that theajor binding forces that act on docetaxel are hydrogen bonding

nd van der Waals interactions; whereas, for HNF, it is hydrophobicnteraction.

The interactions of cancer therapeutic and diagnostic agentsuch as tetraphenylporphyrin (TPP), tetraparachlorophenylpor-hyrin (TClPP), and tetraparamethoxyphenylporphyrin (TMEOPP)

ith BSA are studied by Tian et al. [118]. All these porphyrin com-ounds (Fig. 14c) lower the intrinsic fluorescence of BSA withlue-shift in the emission maximum, which suggests that all theerivatives can bind tightly to BSA. The binding constant, KA

tobiology C: Photochemistry Reviews 14 (2013) 53– 71

(table in Fig. 14c) suggests that the porphyrins bind with BSAby complex-formation and it also suggests that the binding ofTClPP to BSA is notably stronger than that of TMEOPP or TPP.The difference in the binding strength can be attributed to theeffect of electron-withdrawing or electron-donating substitutionsin the three porphyrin compounds. The thermodynamic parame-ters (Table 3, 24–26) confirm that hydrophobic interactions playthe major role in the binding of TPP and TClPP to BSA, while thebinding of TMEOPP is mainly based on van der Waals interactions.

In a similar study, the interactions of N-confused porphyrinssuch as N-confused tetraphenylporphyrin (NCTPP), N-confusedtetraparamethylphenylporphyrin (NCTMPP) and N-confused tetra-paraacetoxyphenylporphyrin (NCTAPP) with BSA are studied byYu et al. [119]. All these porphyrin compounds (Fig. 14d) quenchthe intrinsic fluorescence of BSA, which is accompanied by ablue-shift in the emission maximum. The binding constants sug-gest that the binding affinity (table in Fig. 14d) follows thetrend: NCTPP < NCTMPP < NCTAPP, which means NCTAPP has thestrongest ability to bind with BSA and NCTPP has the weakest. Thisdifference in the strength of binding can be attributed to the pres-ence of the longer branch chain (acetoxy group) which promotesstrong binding. The thermodynamic parameters (Table 3, 27–29)suggest that hydrophobic interactions play the major role in thespontaneous interaction of these porphyrin compounds with BSA.The results obtained from synchronous fluorescence spectra indi-cate that the binding site of all these porphyrins is next to thetryptophan residue.

The interactions of anticancer agents such as Gossypol, Vin-cristine sulfate (VS), and Berbamine are investigated by Yang et al.[120], Kamat and Seetharamappa [107], and Cheng et al. [121],respectively. Fluorescence quenching studies suggest that Gossy-pol, VS and Berbamine quench the fluorescence intensity of BSA bythe static quenching mechanism without any changes in the emis-sion maximum. The thermodynamic parameters suggest that thebinding of Gossypol to BSA might involve hydrophobic and electro-static interactions; whereas, for VS, it is hydrophobic interactions.The binding forces that act on Berbamine–BSA system are the weakvan der Waals interactions and hydrogen bonding. The number ofbinding sites (n), for Gossypol and Berbamine is found to be 1. Thesynchronous fluorescence spectrum confirms that Gossypol affectsthe conformation around the tyrosine residues in BSA. The upwardcurvature of Stern–Volmer plot for VS–BSA system indicates thatboth tryptophan residues of BSA are exposed to the drug, VS.

In this section, fluorescence quenching studies on the interac-tion of anticancer agents with serum albumins is discussed. Thesestudies illustrate the substituent effect on the hydrophobicity ofthe binding site. Generally, substitution of acetyl and methyl groupslowers the binding affinity but in the case of N-confused porphyrins,the substitution of acetyl and methyl group increases the bindingaffinity. These studies will be of considerable use in the field ofpharmacology and clinical medicine.

5.3. Anti-inflammatory agents

The interaction of Dexamethasone (DEX) with serum albuminshas been investigated by Naik et al. [122] (Fig. 16). DEX, a potentsynthetic member of the glucocorticoid class of steroid hormones,acts as an anti-inflammatory agent and immunosuppressant. DEXlowers the fluorescence intensity of serum albumins with a slightblue-shift in the emission maximum by the formation of a staticcomplex. The number of binding sites, n, for DEX–BSA and DEX–HSAindicate that there is one independent class of binding site on BSA

and HSA for DEX. The binding constant (table in Fig. 16) suggeststhat HSA and BSA have almost the same binding affinity towardDEX. The thermodynamic parameters suggest that the main inter-actions between DEX and HSA are hydrogen bonding and weak van

S. Naveenraj, S. Anandan / Journal of Photochemistry and Pho

System (T= 288 K) KA x 104 (M-1)

HSA-DEX 3.80

BSA-DEX 3.78

ds

caflmHcagtmfi

(aaopC

quenching mechanism. The thermodynamic parameters (Table 3,

FB

Fig. 16. Molecular structure of Dexamethasone (DEX).

er Waals interactions; whereas, for BSA, hydrophobic and electro-tatic interactions dominate.

The interactions of non-steroidal anti-inflammatory drugs Rofe-oxib and Ketoprofen with HSA are investigated by Qi et al. [123],nd Bi et al. [124], respectively. Rofecoxib quenches the intrinsicuorescence of HSA with a slight red-shift in the emission maxi-um; whereas, Ketoprofen quenches the intrinsic fluorescence ofSA without affecting the emission maximum. The Stern–Volmeronstants at different temperatures confirm that both Rofecoxibnd Ketoprofen interact with HSA through the formation of around state complex. The thermodynamic parameters suggesthat weak van der Waals and hydrogen bond interactions are the

ajor forces underlying the binding of Rofecoxib to HSA; whereas,or Ketoprofen, electrostatic interactions and hydrogen bonding arenvolved.

Anti-inflammatory agents such as Colchicine [125], Ellagic acidEA) [126], Cromolyn Sodium (CS) [127], and Shikonin [128] inter-ct and bind with HSA. The fluorescence intensity of HSA quencheslong with a blue-shift in the emission maximum on the addition

f Colchicine, EA, and Shikonin; whereas, the quenching is accom-anied by a red-shift on the addition of CS. The interactions ofolchicine, EA, CS and Shikonin with HSA result in the formation

ig. 17. Molecular structure of dyes. (a) Perylene-3,4,9,10-tetracarboxylate tetrapotassiuBS and (iii) BIS.

tobiology C: Photochemistry Reviews 14 (2013) 53– 71 65

of ground-state complexes. The thermodynamic parameters sug-gest that van der Waals interactions and hydrogen bonding are theinteraction forces for the binding of Colchicine and EA with HSA.Electrostatic interactions play major roles in the binding of CS withHSA; whereas, hydrophobic interactions and hydrogen bonding areinvolved in the case of Shikonin.

Fluorescence quenching studies on the interactions of steroidaland non-steroidal anti-inflammatory agents with serum albuminsare considered in this section. Even though serum albumins differ intheir affinities, the anti-inflammatory agent DEX shows equivalentbinding affinity with both HSA and BSA.

5.4. Organic dyes

As dyes (Fig. 17) are being increasingly used for clinical andmedicinal purposes, studies of their interactions with serum albu-min are vital. The interaction of thermoresponsive organic dyeperylene-3,4,9,10-tetracarboxylate tetrapotassium salt (PTK) withserum albumin is investigated by Naveenraj et al. [43]. Perylenederivatives for biochemical and pharmacological applications aresynthesized using PTK (Fig. 17a). PTK quenches the fluorescence ofserum albumins with the emergence of isoacitinic points (Fig. 18),which indicates that the quenching depends on the formation ofa complex between PTK and serum albumins. The binding con-stant (table in Fig. 17a) suggests that the affinity of HSA for PTKis more than that of BSA. The value of �G indicates the spontaneityin the binding of PTK to serum albumin. Synchronous fluorescencespectra indicate the proximity of the binding site to the tyrosinemoiety, and the changes in the microenvironment and molecularconformation of serum albumin.

Due to the toxicity, carcinogenicity and the mutagenic nature ofazo dyes, the interactions of azo dyes such as C.I. Acid Red 2 (AR2),C.I. Acid Red 73 (AR73) and C.I. Direct Yellow 9 (DY9) with HSA areinvestigated by Ding et al. [129], Guo et al. [130], and Yue et al. [131].All these azo dyes quench the fluorescence of HSA through the static

30–32) suggest that both hydrophobic and hydrogen bond inter-actions play major roles in the interactions of AR2 and DY9 withHSA; whereas, only hydrophobic interactions are involved in the

System (T= 298 K) KA 104 (M-1)

HSA-PTK 49.4

BSA-PTK 3.72

System (T= 293 K) KA 104 (M-1)

BSA–Sud an II 1.22

BSA–Sud an IV 1.48

m salt (PTK), (b) azo dyes (i) Sudan I, (ii) Sudan IV, (c) squaraine dyes (i) BHS, (ii)

66 S. Naveenraj, S. Anandan / Journal of Photochemistry and Pho

300 40 0 50 00

100

200

300

A

M

Flu

ore

sc

en

ce

in

ten

sit

y /

a.u

.

Wavel eng th (nm)

A

M

300 40 0 50 00

100

200

300

A

M

Flu

ore

sc

en

ce

in

ten

sit

y /

a.u

.

Wavel eng th (nm)

A

M

a

b

Isoa citinic

point

Isoa citinic

point

Fig. 18. Fluorescence spectra (�ex = 280 nm) of serum albumin (3 �M) [(a) HSA and(b) BSA] quenched by PTK in the concentration range of 0–4 �M. From A to M curve,PTK concentrations are 0, 0.33, 0.67, 1, 1.33, 1.67, 2, 2.33, 2.67, 3, 3.33, 3.67, and4

F

btdio

a(aaiwtgtaHi

(l

Flavonoids are a large group of polyphenolic natural prod-

�M.

rom Ref. [43].

inding of AR73 to HSA. Synchronous fluorescence results indicatehat the hydrophobicity decreases around the tryptophan residuesuring the interaction of AR2 with HSA; whereas, the hydrophobic-

ty around the tryptophan residues increases during the interactionf DY9 with HSA.

The interactions of anthraquinone dye Alizarin Red S (ARS),nionic dye C. I. Acid Green 1 (AG1) and fluorescein dye Eosin BEB) with HSA are investigated by Ding et al. [132], Yue et al. [133]nd Yang et al. [134] owing to their biolabeling applications. ARSnd AG1 quench the intrinsic fluorescence of HSA without any shiftn the emission maximum; whereas, EB quenches the fluorescence

ith a blue-shift in the emission maximum. All these dyes quenchhe fluorescence of HSA through the formation of non-fluorescentround-state complexes. The thermodynamic parameters suggesthat hydrophobic interactions play major roles in the interaction ofll the dyes with HSA, but during the interactions of ARS and EB withSA, hydrogen bonding plays a role in addition to the hydrophobic

nteractions.

The interactions of azo dyes such as Sudan II and Sudan IV

Fig. 17b) with BSA are investigated by Lu et al. [135] for toxico-ogical importance. Both Sudan I and Sudan IV quench the intrinsic

tobiology C: Photochemistry Reviews 14 (2013) 53– 71

fluorescence of BSA through statically. The binding constant values(table in Fig. 17b) estimated for Sudan II and Sudan IV suggest thatSudan IV easily and stably binds with BSA when compared withSudan II, which may be attributed to the structural peculiaritiesof Sudan IV (methylphenyl and azo groups). The thermodynamicparameters (Table 3, 33–34) suggest that hydrogen bonding or vander Waals interactions plays major roles in the binding of Sudan IIand Sudan IV with BSA.

Jisha et al. [136] investigated the interactions of squarainedyes (Fig. 17c) such as bis(2,4,6-trihydroxyphenyl)squaraine(BHS), bis(3,5-dibromo-2,4,6-trihydroxyphenyl) squaraine (BBS),and bis(3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine (BIS) withserum albumins for their biological importance as NIR fluorescentlabels and photodynamic therapeutic agents. HSA shows higheraffinity toward all these squaraine dyes than BSA. BHS binds withserum albumins at the binding site I; whereas, BBS and BIS bind atsite II due to their steric constraints arising from the presence ofhalogen atoms. The uniqueness of these dyes is their substituentsize-dependent selectivity at site II.

The interactions of dyes such as Malachite Green (MG) andRose Bengal (RB) with BSA are investigated by Zhang et al. [137]and Shaikh et al. [138], respectively. MG, a triarylaminnethanedye, is used as food colorant, medical disinfectant and fungicide,but it is exhibiting carcinogenic, genotoxic, mutagenic and terato-genic properties. RB, the ophthalmic dye, is used as a biologicalstain, an antiviral agent, and a detection tool for organic anionspresent in the liver plasma. The dyes MG and RB lower the intrinsicfluorescence of serum albumins without any changes to the emis-sion wavelength or the shape of the emisson bands. MG quenchesthe intrinsic fluorescence through the static mechanism; whereas,RB quenches the intrinsic fluorescence dynamically. The thermo-dynamic parameters (Table 3, 35–36) suggest that van der Waalsinteractions and hydrogen bonding are the major forces of interac-tions of MG and RB with BSA.

The interactions of mutagenic and carcinogenic dyes such as C.I.Mordant Red dye 3, Disperse Blue SBL (DBSBL), Acid Yellow 11 (AY)and Congo Red (CGR) with BSA are studied by Ding et al. [139],Guo et al. [140], Pan et al. [141] and Zhang et al. [142], respec-tively for their toxicological importance. Both anthraquinone dyesC.I. Mordant Red 3 and DBSBL quench the fluorescence intensityof BSA, which is accompanied by blue-shift in the emission max-ima; whereas, AY and CGR quench the fluorescence intensity ofBSA without any shift in the emission maxima. The thermodynamicparameters (Table 3, 37–40) suggest that hydrophobic interactionsare responsible for the binding of DBSBL with BSA; whereas, elec-trostatic interactions are responsible for the AY. Hydrogen bondingand van der Waals interactions are major binding forces for C.I.Mordant Red; whereas, hydrophobic interactions and hydrogenbonding are the forces for the binding of CGR to BSA. C.I. MordantRed 3, AY and CGR show slight blue-shifts in the synchronous spec-trum of BSA for the tyrosine residue. In the synchronous spectra,C.I. Mordant Red shows a slight shift in the emission maximum oftryptophan residue; whereas, no shift is observed in the case of AYor CGR.

In this section, we discussed the fluorescence quenching stud-ies associated with the interactions of various dyes with serumalbumins and illustrated the effect of different substituents on thebinding interaction.

5.5. Flavonoids

ucts that are found ubiquitously in plants of higher generaand they possess novel nutritional and therapeutic properties ofhigh potency and low systemic toxicity. Hence their interaction

d Pho

wm

hQHtftie

baAaapwcicbetahb2cabccia

(Saaith3tnnrti

nNnqasSlf

(b[o

S. Naveenraj, S. Anandan / Journal of Photochemistry an

ith serum albumins is vital in understanding the role of theseolecules in biological process.The interactions of flavonoids such as quercetin, rutin, and

yperin (Fig. 19a) with HSA are investigated by Bi et al. [143].uercetin, rutin and hyperin quench the intrinsic fluorescence ofSA through the static mechanism. The binding constant suggests

hat the binding capacity of these three flavonoids with HSA is in theollowing order rutin < hyperin < quercetin, which can be attributedo the steric hindrance effect. The thermodynamic parametersndicate that the force of binding for these flavonoids is mainlylectrostatic.

The interactions of flavonoids chrysin [144], alpinetin [145],aicalein [146], and wogonin [147] (Fig. 19b) with BSA arelso investigated by the fluorescence quenching techniques.lpinetin, baicalein, chrysin and wogonin are having anti-tumornd anti-inflammatory properties. In addition, alpinetin showsnti-bacterial properties and chrysin shows antihypertensionroperties. Baicalein quenches the intrinsic fluorescence of BSAithout any shift in the emission maximum. The intrinsic fluores-

ence of BSA is quenched upon the addition of alpinetin, whichs accompanied by a red-shift in the emission maximum; whereas,hrysin and wogonin quench the intrinsic fluorescence with a slightlue-shift in the emission maximum. The thermodynamic param-ters suggest that hydrophobic interactions play a major role inhe binding of alpinetin, baicalein, chrysin, and wogonin to BSA. Inddition to the hydrophobic interactions, electrostatic force andydrogen bonding are involved in the binding of wogonin andaicalein to BSA. Alpinetin shows a slight red-shift (from 283.6 to86.8 nm) in the emission maximum in the synchronous fluores-ence spectrum of tryptophan residues; whereas, chrysin shows

slight blue-shift (from 284 to 281 nm). Alpinetin shows a slightlue shift (289.2–286.4 nm) in the emission maximum in the syn-hronous fluorescence spectrum of tyrosine residues; whereas,hrysin does not show any shift, which indicates that chrysin specif-cally binds with the tryptophan residues of BSA; whereas, alpinetinffects the microenvironment of both tryptophan and tyrosine.

The interactions of flavonones such as tangeretin and nobiletinFig. 19c) with HSA are investigated by Cao et al. [148]. Althoughtern–Volmer plots are non-linear, the interactions of tangeretinnd nobiletin with HSA follow the static quenching mechanisms their kq values are greater than the rate of collisional quench-ng. The binding constant of nobiletin (Table 2, 16–17) is greaterhan that of tangeretin, which suggests that the affinity for HSAas been improved by the methylation of tangeretin at position′. Hydrophobic interactions play the major role in their interac-ion with HSA. The interactions of flavonones such as naringin andarirutin (Fig. 19d) are investigated by Xiao et al. [149]. At 8.0 �M,aringin and narirutin quenched 21.80% and 14.54% of BSA’s fluo-escence through the static quenching mechanism, which indicateshat naringin has more affinity toward BSA than narirutin. The bind-ng constants also suggest that naringin has more affinity for BSA.

The interactions of flavonones such as naringenin, naringin andarirutin (Fig. 19d) with HSA are investigated by Cao et al. [148].aringenin quenches the fluorescence of HSA, which is accompa-ied by a red-shift in the emission maximum; whereas, naringinuenches the fluorescence without any spectral shift. The inter-ctions of naringenin, naringin and narirutin with HSA follow thetatic quenching mechanism, which is evident from the lineartern–Volmer relation. At 8.0 �M, the fluorescence quenching fol-ows the trend: naringin < naringenin < narirutin. The same trend isollowed in the binding affinity (Table 2, 18–20) toward HSA.

The interaction of flavonoids such as hyperoside, myricetin

Myr), tiliroside and troxerutin (Fig. 19e) with BSA are investigatedy Qin et al. [150], Tian et al. [151], Hu et al. [152] and Wang et al.153]. Hyperoside and troxerutin quench the intrinsic fluorescencef BSA with a blue-shift in the emission maximum; whereas, Myr

tobiology C: Photochemistry Reviews 14 (2013) 53– 71 67

and tiliroside quench with a slight red-shift. From the thermody-namic parameters, it is apparent that hydrophobic interactions arethe forces of interaction of hyperoside and troxerutin with BSA;whereas, both hydrophobic and electrostatic interactions play mainroles in the interaction of Myr with BSA. The synchronous fluores-cence spectra suggest that hyperoside and troxerutin affect onlythe microenvironment of typtophan moiety in BSA; whereas, Myrand tiliroside affect the microenvironments of both typtophan andtyrosine moieties in BSA.

The interactions of flavonoids such as galangin, kaempferol,quercetin and myricetin (Fig. 19f) with BSA are investigated byXiao et al. [154] because of their potential antioxidant activities.All these flavonoids quench the intrinsic fluorescence of BSA withblue-shift in the emission maximum and follow the static quench-ing mechanism. The binding constants (Table 2, 21–24) follow thetrend: galangin < kaempferol < quercetin < myricetin, which sug-gests that the binding affinity of flavonoids toward BSA increaseswith increase in the number of hydroxyl groups on the B-ring.

The interactions of flavones such as flavone, 7-hydroxyflavone,chrysin and baicalein (Fig. 19g) with serum albumins are inves-tigated by Xiao et al. [155]. Flavone quenches the intrinsicfluorescence of serum albumins with red-shift in the emis-sion maximum; whereas, 7-hydroxyflavone, chrysin and baicaleinquench with blue-shift in the emission maximum, whichsuggests that the presence of hydroxyl group increases thehydrophilicity around the binding site. Stern–Volmer constantsand binding constants (Table 2, 25–32) suggest that the affin-ity of flavonoids toward serum albumins follows the trend:7-hydroxyflavone > chrysin > baicalein > flavone. This trend indi-cates that 7-hydroxyflavone containing only one hydroxyl grouphas the highest binding affinity. But hydroxyl groups introduced atpositions C-5, C-6, and/or C-7 of flavones weaken the affinities forserum albumins due to the formation of the intra-molecular hydro-gen bond. These results show that the optimal number of hydroxylgroups introduced to the ring A of flavones is one. The binding con-stant also suggests that BSA is having more affinity toward theseflavones than HSA.

The interactions of myricetin and dihydromyricetin (Fig. 19h)with BSA are investigated by Liu et al. [156]. Myricetin quenchesthe fluorescence of BSA with blue-shift in the emission maxi-mum; whereas, dihydromyricetin quenches with red-shift. Thebinding constants (Table 2, 33–34) suggest that myricetin has moreaffinity toward BSA than dihydromyricetin, which indicates thathydrogenation in ring C suppresses the binding interaction. Theinteractions of quercetin and quercitrin (Fig. 19i) with BSA are alsoinvestigated by Xiao et al. [154]. Quercitrin is the 3-O-�-glucosideof quercetin. Both quercetin and quercitrin quench the fluorescenceof BSA with blue-shift in the emission maxima, i.e. static quench-ing. The binding constant (Table 2, 35–36) of quercetin is greaterthan that of quercitrin, which indicates that glycoside substitutionat the C-ring position weakens the binding affinity due to steric hin-drance. The same trend is followed in the case of other flavonoidssuch as baicalein, genistein and daidzein [157].

The interactions of daidzein (DDN) and 3′-daidzein sulfonicsodium (DSS) are investigated (Fig. 19j) by Shang and Li [158].Both DDN and DSS quench the fluorescence of BSA through thenon-fluorescent ground-state complex formation (static quench-ing). The binding constants (Table 2, 37–38) suggest that DSS hasmore affinity for BSA than DDN, which is attributed to the presenceof SO3Na. The thermodynamic parameters (Table 3, 41–42) indicatethat the interaction of DDN with BSA is driven mainly by hydrogenbonding and van der Waals interactions; whereas, the binding of

DSS with BSA is driven mainly by hydrophobic forces.

Xiao et al. [159] reported some of the structural elements thatinfluence the affinities of flavonoids for HSA. One or more hydroxylgroups in the B ring of flavonoids enhance the binding affinity for

68 S. Naveenraj, S. Anandan / Journal of Photochemistry and Photobiology C: Photochemistry Reviews 14 (2013) 53– 71

Fig. 19. Molecular structure of flavonoids (a) (i) quercetin, (ii) rutin, and (iii) hyperin, (b) (i) chrysin, (ii) alpinetin, (iii) baicalein and (iv) wogonin, (c) (i) tangeretin and(ii) nobiletin, (d) (i) naringenin, (ii) naringin and (iii) narirutin, (e) (i) hyperoside, (ii) myricetin (Myr), (iii) tiliroside and (iv) troxerutin, (f) (i) galangin, (ii) kaempferol, (iii)quercetin and (iv) myricetin, (g) (i) flavone, (ii) 7-hydroxyflavone, (iii) chrysin and (iv) baicalein, (h) myricetin and dihydromyricetin, (i) quercetin and quercitrin, (j) daidzein(DDN) and 3′-daidzein sulfonic sodium (DSS).

S. Naveenraj, S. Anandan / Journal of Photochemistry and Pho

System (T= 293 K) KA 104 (M-1)

BSA–IDC 2.8

BSA–KDC 1.0

System (T= 293K) KA 105 (M-1)

BSA–C3 7.31

BSA– C1.3 1050

Fig. 20. Molecular structure of noxious substances (a) neonicotinoids insecti-c37

Hotw

wdgrd

5

ptoMoo

aitmq(Hn

pm[HaPspiBtit

ide (i) IDC, (ii) KDC and (b) hydroxychromone derivatives of coumarin (i)-hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-on (C3) (ii) 1,3-dihydroxy-,8,9,10-tetrahydro-6H-benzo[c]chromen-6-on (C1.3).

SA; whereas, the hydroxyl group in the C-ring weakens the affinityf flavonoids. Methylation of hydroxyl groups enhances the affini-ies for HSA; whereas, hydrogenation of the C2 C3 double bond asell as glycosylation lower the affinities for HSA.

In this section, reports on the interaction of various flavonoidsith serum albumins are summarized. In particular, the effects ofifferent substituents such as methyl group, methoxy group, andlycosyl group on the binding interactions are summarized. Fluo-escence quenching studies confirm that these substances interactissimilarly with BSA/HSA due to the steric hindrance.

.6. Noxious materials

The interaction of organic phosphorous pesticide methylarathion (MP) with SA is investigated by Silva et al. [160] for theiroxicological importance. MP quenches the intrinsic fluorescencef SA through the static mechanism. At 1:1 molar ratio of MP/SA,P quenches about 6.0% fluorescence of HSA and 4.3% fluorescence

f BSA, which suggest that MP quenches the intrinsic fluorescencef HSA more efficiently than that of BSA.

The interactions of neonicotinoids insecticide (Fig. 20a) suchs imidacloprid (IDC) and its keto analog (KDC) with HSA arenvestigated [161] for their toxicological importance. IDC quencheshe intrinsic fluorescence of HSA with red-shift in the emission

aximum; whereas, KDC quenches without any shift, but bothuenching follow the static mechanism. The binding constantstable in Fig. 20a) suggest that IDC has more affinity towardSA than KDC, which may be attributed to the presence of theitroimine group.

The interactions of the fungicide thiophanate methyl (MT), theesticide pentachlorophenol (PCP) and the herbicide bensulfuron-ethyl (BM) with HSA are studied by Li et al. [162], Wang et al.

163] and Ding et al. [164]. Due to the interaction of MT withSA, the intrinsic fluorescence intensity of HSA decreases, which isccompanied by a blue-shift in the emission maximum; whereas,CP and BM quench the intrinsic fluorescence of HSA without anypectral shift. MT, PCP and BM interact with HSA to form com-lexes. The thermodynamic parameters suggest that hydrophobic

nteractions are the major forces of the interaction of MT, PCP and

M with HSA. Also, hydrogen bonding plays a role in the interac-ion of MT with HSA; whereas, hydrogen bonding and electrostaticnteractions play key roles in the binding of BM with HSA. Inhe synchronous spectrum of HSA, both PCP and BM red-shift the

tobiology C: Photochemistry Reviews 14 (2013) 53– 71 69

emission maximum of the tryptophan residue. On the other hand,PCP blue-shifts the emission maximum of the synchronous spec-trum of tyrosine residue; whereas, BM does not produce any shift.

The interactions of Toxic Cr4+ ion compounds such as potassiumdichromate K2Cr2O7 and potassium chromate K2CrO4 with BSA arestudied by Zhang et al. [165]. Both the Cr4+ compounds quenchthe fluorescence of BSA with blue-shift in the emission maximum,which suggests an increase in the hydrophobicity around the bind-ing site and static quenching. The quenching constants suggestthat K2Cr2O7 has more affinity than K2CrO4. The thermodynamicparameters suggest that the electrostatic interactions play a majorrole in the binding reaction.

The interactions of two substituted hydroxychromonederivatives of coumarin (Fig. 20b) namely 3-hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-on (C3) and1,3-dihydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-on(C1.3) with BSA are investigated [166] for their hepatotoxic impor-tance. C3 quenches the fluorescence of BSA through both static anddynamic mechanisms; whereas, C1.3 quenches through the staticquenching mechanism. The binding affinity of C1.3 toward BSA ismore than that of C3, which is related to the number of hydroxylgroups present. The thermodynamic parameters (Table 3, 43–44)suggest that hydrogen bonding and van der Waals interactionsplay major roles in the binding of these two coumarin derivativesto BSA.

Fluorescence quenching studies on the interaction of noxiousmaterials including pesticides and insecticides with serum albu-mins are discussed in this section. Among these noxious materials,hepatotoxic coumarin derivatives C3 and C1.3 interact dissimilarlywith BSA/HSA due to the presence of hydroxyl group, which sug-gests the role of substituents in the binding interactions.

6. Conclusion

A critical need in the field of pharmacology is the study of the invitro binding interactions of various drugs with biomolecules suchas serum albumins. Fluorescence spectroscopy is a powerful toolto accomplish this need. In this review, the fluorescence quench-ing studies involving the interaction of bioactive substances withserum albumins are discussed. The results reviewed here exemplifythe dependence of the size and stabilizing agent of nanoparticleson their interactions with BSA/HSA. The organic molecules such asantibiotics, anticancer drugs, anti-inflammatory agents, flavonoidsand dyes having different substituents interact dissimilarly withserum albumins due to the steric hindrance which provides impor-tant clues for the design of effective drugs for the treatmentof various diseases. Such molecular interactions between serumalbumins and the drugs using fluorescence quenching are vitalpharmacokinetic processes such as drug distribution and trans-portation. In addition, the predicted concentrations of drugs inblood serum determine the pharmaceutical effects of the drug.Therefore, this review is of great significance in the fields of phar-macology and clinical medicine.

Acknowledgements

Author SA thanks DST, New Delhi (SR/S1/PC-49/2009) for thesanction of major research grant. Also author SA thank DST for sanc-tioning FIST (SR/FST/CSI-190/2008 dated 16th March 2009) andNanomission projects. Author SN thanks his institute for selectinghim in MHRD fellowship.

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