A close-up on doxorubicin binding to c-cyclodextrin: an elucidatingspectroscopic, photophysical and conformational study{

Resmi Anand, Stefano Ottani,* Francesco Manoli, Ilse Manet and Sandra Monti*

Received 1st December 2011, Accepted 1st December 2011

DOI: 10.1039/c2ra01221a

The association of doxorubicin (DOX) with c-cyclodextrin (c-CyD) was studied in phosphate buffer

of pH 7.4, at 22 uC by performing titration experiments monitored with circular dichroism (CD), UV-

vis absorption, and fluorescence. Global analysis of multiwavelength spectroscopic data obtained at

different DOX concentrations was performed by taking into account the DOX monomer–dimer

equilibrium. Formation of c-CyD:DOX 1 : 1, 2 : 1, 1 : 2 and 2 : 2 complexes was evidenced. The

stability constants and the absolute CD, UV-vis absorption and fluorescence spectra of all the

complexes were determined. c-CyD did not prove to be able to disrupt the DOX dimer when the latter

is the predominant form in solution. The triplet state absorption and kinetic properties of DOX in the

presence of c-CyD and in ethanol were also determined by laser flash photolysis. The excited singlet

and the triplet features indicated the environment experienced by DOX in the CyD complexes is

ethanol-like. The structure of the c-CyD:DOX 1 : 1 complex was investigated by Molecular

Mechanics (MM) and Molecular Dynamics (MD) for both the neutral and the positively charged

(–NH3+ in the daunosamine moiety) DOX forms. The possibility of interaction of both the aglycone

and the daunosamine parts with the c-CyD cavity was evidenced.

Introduction

Anthracyclines represent an extremely important class of antic-

ancer drugs, ranking among the most potent ones ever developed.

They are usually employed in the treatment of leukaemia and

various solid tumors, in particular breast cancer and Kaposi’s

sarcoma. They exert their action by interfering with DNA cutting

and reclosing in a three component complex with DNA and

Topoisomerase, thereby providing a cytostatic effect.1,2 The first

anthracyclines isolated from the bacterium Streptomyces peucetius

in the 60s were doxorubicin (DOX, also known as Adriamycin,

Scheme 1) and daunorubicin (also known as daunomycin).

Because of their great efficacy these two derivatives have been

continuously applied in clinical practice, despite the development

of forms of resistance by cancer cells and serious side effects related

to low cardiac tolerability and necrotic action at the injection site.

Looking for a better compromise between activity and toxicity

of anthracyclines a lot of research efforts were invested in the

identification of drug structural modifications. On the other hand,

studies have focused on the development of improved delivery

techniques based on the employment of biocompatible and

biodegradable carriers, like micelles and liposomes,3 polymeric

architectures,4–6 and nanoparticles.7–10 Delivery systems were also

designed to contrast the tendency of these drugs to aggregate.

Self-aggregation represents a serious drawback in their clinical

use, because it may effectively compete with DNA binding thereby

limiting the pharmacological activity. When incorporated in

micelle-forming polymeric nanocarriers the DOX monomer

displayed a major antitumor activity whereas the DOX dimer

has no antitumor activity by itself.11

Cyclodextrins (CyDs) are biocompatible cyclic oligosacchar-

ides, made of a-D-glucopyranose units joined by a(1–4) linkages

(Scheme 1B), that form macrocycles with a hydrophilic exterior

surface and a hydrophobic cavity able to host lipophilic guests.

For a long time they have received considerable attention as

carriers able to improve solubility, stability and bioavailability of

drugs.12 Many derivatized CyDs and various CyD-based nanoas-

semblies, loading drugs via weak non covalent interactions13–19 or

via labile covalent bonds,6,20–22 have been synthesized and

proposed as delivery systems in preclinical studies. The binding

of DOX to CyDs has been addressed since the 90s. At that time it

was reported that methyl-b-CyD potentiates the activity of DOX

on both sensitive and multidrug-resistant cell lines.23–27 Since

then, the interest for this topic has been continuously growing.

Self-aggregation of DOX (evidenced in aqueous solution by UV-

vis absorption,28 circular dichroism (CD)29 and NMR spectro-

scopy30) is likely perturbed by inclusion of the drug in a CyD

cavity or a CyD nanoassembly. Therefore, gaining insights into

the binding modes of DOX to CyD systems is of direct relevance

to the optimization of the use of this drug.

As regards natural CyDs, DOX binds significantly to c-CyD,

whereas it possesses lower affinity for b-CyD and a-CyD.31–34

Istituto per la Sintesi Organica e la Fotoreattivita, ISOF-CNR,via P. Gobetti 101, 40129, Bologna, Italy. E-mail: monti@isof.cnr.it{ Electronic Supplementary Information (ESI) available. See DOI:10.1039/c2ra01221a/

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Formation of a c-CyD-DOX complex with 1 : 1 stoichiometry has

been reported.31,32,35 In this study we re-examined in detail the

complexation of DOX with c-CyD in aqueous medium, perform-

ing accurate titrations monitored with circular dichroism (CD),

UV-vis absorption and fluorescence. Thanks to the different sign

of the dichroic signal of monomer and dimer in the 500 nm

absorption region, the CD technique yielded valuable information

on the presence of the dimer and its disruption.36,37 By global

analysis of multiwavelength CD data taken at different DOX

concentrations we evidenced the formation of c-CyD:DOX

complexes of higher stoichiometries, i.e. 2 : 1, 1 : 2 and 2 : 2 in

addition to 1 : 1, not reported previously, and we determined the

stability constants and the individual CD features of all these

complexes. We obtained information on the environment

experienced by DOX in the CyD complexes from the excited

singlet state properties of the fluorescent DOX monomer and

from the triplet state properties of the non fluorescent DOX dimer

by laser flash photolysis. We also studied the association of DOX

with c-CyD in the 1 : 1 stoichiometry from the structural point of

view by Molecular Dynamics Simulations, with explicit solvent

and examined the interaction of either the aglycone or the

daunosamine moiety with the CyD cavity.

Materials and methods

Materials

Doxorubicin (DOX, Adriamycin), was purchased from ALEXIS

Biochemicals and used without further purification; c-CyD

(Fluka) was used as received. Water was purified by passage

through a Millipore MilliQ system. 0.01 M phosphate buffer at

pH 7.4 was used. DOX was easily dissolved at a concentration of

2 6 1024 M in this medium.

Spectroscopic measurements

Ultraviolet absorption spectra were recorded on Perkin-Elmer

Lambda 9 or Lambda 950 spectrophotometers. The spectra of

the DOX-CyD mixtures were registered using CyD solutions as

the reference. Circular dichroism spectra were obtained with

a Jasco J-715 dichrograph. Fluorescence experiments were

carried out on y1.0 6 1025 M solutions in 1 cm cells having

an absorbance of ¡0.1 at the excitation wavelength. Steady state

fluorescence spectra were registered on a SPEX Fluorolog 111

spectrofluorimeter. Fluorescence lifetimes in air-saturated solu-

tions were measured with a time correlated single photon

counting system (IBH Consultants Ltd.). A nanosecond LED

at 465 nm was used as the excitation source and the emission was

detected at 590 nm. The software package for analysis of

emission decays was provided by IBH Consultants Ltd. DOX

fluorescence quantum yield was determined on excitation at

480 nm using Ru(bpy)3Cl2 as reference (W = 0.028, in air

equilibrated solution).38

CD and fluorescence titrations were recorded at constant drug

concentration on varying CyD host concentration. The best

complexation model and the association constants were deter-

mined by multivariate global analysis of multiwavelength data

from a series of 9–11 spectra corresponding to different mixtures,

using the program SPECFIT/32 (v.3.0.40, TgK Scientific). The

optimization procedure is based on singular value decomposition

and non linear regression modelling by the Levenberg–

Marquardt method. The calculation also afforded the individual

spectra of the associated species (see ESI{ SI-4 for further

details).

Nanosecond laser flash photolysis

The pulse of a Nd-YAG laser, operating at 532 or 266 nm (20 ns

FWHM, 2 Hz), was suitably shaped passing through a

rectangular, 3 mm high and 10 mm wide window, and providing

a fairly uniform energy density, incident onto the sample cell

(a pulse of 3.5 mJ at 266 nm corresponds to 12 mJ cm22). A front

portion of 2 mm of the excited solution was probed at a right

angle, the useful optical path for analyzing light being 10 mm.

A266 was y0.5–1 over 1 cm. Ar-saturated solutions were used.

The sample was renewed after few laser shots. The temperature

was 295 K.

Computational methods

Interactions between c-cyclodextrin (c-CyD) and doxorubicin

(DOX) was studied by Molecular Mechanics (MM) and

Molecular Dynamics (MD) methods. The initial geometry of c-

CyD was taken from its crystallographic structure.39 For DOX,

Scheme 1 (A) Doxorubicin (DOX), (B) schematic of a c-CyD glucopyranose unit.

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both the neutral and the positively charged molecules (–NH3+ in

the daunosamine moiety) were considered. The initial atomic

coordinates of these two species were obtained by ab initio

quantum-mechanical geometry optimizations, using the Gaussian

09 program suite40 at the B3LYP/6-31+G(d,p) level of theory.

Water solvent effects were simulated by the conductor-like

polarizable continuum model (CPCM).

According to the method proposed by Raffaini et al.,41

different initial molecular arrangements were prepared, as the

DOX is alternatively rotated by 180u, around an axis perpendi-

cular to the ring plane, to place the aglycone moiety facing the c-

CyD secondary (larger) rim (structure I) or far from it (structure

II). Two additional arrangements have been prepared with the

DOX molecule facing the c-CyD primary (narrower) rim with

the aglycone (structures Ib) and the daunosamine (structure IIb),

respectively. These four geometries are reported in Scheme 2

and have been used as the starting geometries for both the

neutral DOX and the positively charged DOX (–NH3+ in the

daunosamine moiety). Initial geometries with the charged DOX

molecules are labelled by adding the suffix plus to the previously

defined labels. However, the charged structures are not reported

in Scheme 2 since they differ only by a proton.

Initial geometries, topologies and force field parameter files

were prepared by the package Antechamber,42 using the General

Amber Force Field (GAFF).43 Antechamber was also used to

solvate the molecules in a water box of appropriate size using the

TIP3BOX water model. The periodic box used in MM and MD

enclosed a total of y2050 water molecules. Moreover, the excess

positive charge in the protonated systems was compensated by a

Cl2 ion. The MM and MD runs were performed by the package

NAMD,44 using the compatibility features with the GAFF.

Initial geometries were minimized and optimized and subse-

quently heated up to 300 K. The systems were allowed to relax

and equilibrate at this temperature and at a pressure of 1 atm for

a simulation time of 1 ns. MD production runs were carried on

at constant temperature and pressure by using Langevin

dynamics for an additional simulation time of 10 ns, at least.

MD runs were performed with completely unconstrained bonds

and a time step of 0.5 fs was used for compatibility with the

vibrational frequencies of bonds with hydrogen atoms. Frames

with the system geometries have been recorded at regular time

intervals along these trajectories. The molecular geometries

reported in this work were obtained by the graphical package

VMD.45 VMD plugins were used to perform some of the analysis

of the MD trajectories.

Results and discussion

Selfassociation of DOX in buffer

The absorption spectrum of DOX in phosphate buffer at pH 7.4

displays bands at 288 and 480–500 nm relevant to the two

allowed 1A A 1La and 1A A 1Lb p,p* transitions, polarized

along the short and long axis, respectively.29 A shoulder around

320–380 nm is associated to n,p* transitions of the three CLO

groups in the molecule, partially forbidden by electric dipole.36

Self-aggregation of DOX affects the band shapes and the molar

absorption coefficients that tend to decrease at increasing

concentrations. The spectral profile in the visible region is

strongly influenced by the protonation state of the aglycone

moiety, but is practically insensitive to protonation of the

daunosamine moiety. At pH 7.4 the aglycone part is neutral,

whereas the daunosamine is protonated.29,36 Bands at 252 and

233 nm have been assigned to the aglycone moiety,46 with some

contribution of the daunosamine moiety.30

At concentrations below 5 6 1023 M a simple dimerization

model is sufficient to describe DOX aggregation.30 A set of

absorption spectra obtained upon DOX dilution in the range

5.0 6 1025 M—1.0 6 1027 M (see ESI,{ SI-1, Fig. S1) was

globally analysed adopting this model with the program

SPECFIT/32 (see Materials and Methods and ESI,{ SI-4). A

dimerization constant with log(Kd/M21) = 4.8 ¡ 0.1 was

extracted, in reasonable agreement with the literature data.28,47,48

The spectrum of the DOX dimer in solution was also determined

and is reported together with that of the monomer in the inset of

Fig. S1 of the ESI.{ It has been recently suggested by Agrawal

et al. on the basis of 2D NOESY spectra that the geometrical

arrangement of the two DOX units in the dimer consists of

stacking of the aglycone moieties in either parallel or antiparallel

orientation, with the methoxy substituent of the D ring pointing

towards the exterior or the interior of the interplanar space,

respectively (Scheme 3).30 The strong hypochromicity of the

Scheme 2 Initial geometries for c-CyD/DOX system assumed for

Molecular Mechanics (MM) and Molecular Dynamics (MD) calcula-

tions: I and II, aglycone and daunosamine units facing secondary c-CyD

rim, respectively; Ib and IIb, aglycone and daunosamine units facing the

primary c-CyD rim, respectively.

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absorption bands of the DOX unit in the dimeric state (see ESI,{Fig. S1) is consistent with both arrangements.

Anthracyclines are endowed with an intrinsic CD due to

several asymmetric carbon centers. The C7 and C9 configuration

are of particular importance in the explored wavelength region

(Scheme 1).29,36 The CD spectrum of DOX 1.6 6 1024 M is

characterized by negative bands at 202 nm, 293 nm, 516–547 nm

and positive bands at 233 nm, 252 nm, 352 nm and 453 nm. The

positive–negative split dichroic signal in the 420–580 nm region

is due to the presence of DOX in dimeric form. The presence of

the amino sugar affects the De for the band corresponding to

the p,p* transition polarized along the long axis, due to the

enhancement of the DOX molecular dissymmetry.29 At DOX

concentration ¡1.0 6 1025 M the CD spectrum does not exhibit

a prominent negative signal at 530–550 nm and suggests a

predominance of the monomer over the dimer.

Association of DOX to c-cyclodextrin

UV-Vis absorption and circular dichroism titration at ‘‘high’’

DOX concentration. A solution of DOX 1.6 6 1024 M in

phosphate buffer at pH 7.4 was prepared. In these conditions

DOX was largely dimeric. Increasing concentrations of c-CyD in

the range 2.0 6 1024 M–1.6 6 1022 M induced rather small

UV-Vis absorption variations: a 2–3 nm blue-shift of the visible

band, small increase of absorbance at 288 nm and 233 nm and

decrease at 252 nm (see Fig. S3 in the ESI,{ SI-2). Differently,

the presence of the chiral CyD host greatly increased the optical

asymmetry of the drug electronic transitions. In fact, the CD

changes were very large: (i) an overall increase of the signal

accompanied by a red shift of ca. 5–7 nm, (ii) formation of a new

intense negative band at 264 nm, concomitant with a blue shift of

the peak at 293 nm to 288 nm and appearance of a shoulder at

306 nm and (iii) a small shift of the positive peak from 352 to

362 nm, the only one not displaying an intensity increase

(Fig. 1A,B). The persistence of the positive–negative splitting in

the visible region indicated that c-CyD is not able to disrupt the

DOX dimer, the predominant form at this DOX concentration,

but associates with it as such.28,30,47

The sets of spectra of Fig. 1A and 1B were globally analyzed

with SPECFIT/32. Several complexation models were tried,

involving 1 : 1, 2 : 1, 1 : 2 and 2 : 2 c-CyD:DOX complexes in

various combinations. We included the DOX dimerization

equilibrium with a fixed constant of log(Kd/M21) = 4.8. The

CD spectrum of DOX alone at concentration 5 6 1026 M was

measured in a 10 cm cell and was assigned to the DOX monomer

and also fixed in the calculation. A good fit over the whole 200–

600 nm range was found for a model with a single 2 : 2 CyD:DOX

complex with association constant log(K22/M23) = 10.8 ¡ 0.2,

Durbin Watson (DW) factor of 1.5 (relative error of fit 3.1%) in

the 200–280 nm range and DW =1.9 (relative error of fit 6.2%) in

the 250–600 nm range. The individual spectra of all the species

involved in the equilibria are shown in Fig. S2 of the ESI,{ SI-2.

Global analysis of the set of absorption spectra of Fig. S3 in SI-2

with the same model afforded log(K22/M23) = 10.8 ¡ 0.1 (Durbin

Watson factor 2.0) in excellent agreement with the CD result. The

individual absolute absorption spectra of all the components are

reported in the inset of Fig. S3 in the ESI,{ SI-2. To confirm the

stoichiometry of complexation a continuous variation experi-

ment49 was performed at 1.0 6 1023 M total c-CyD + DOX

concentration, a compromise between the drug limited solubility/

aggregation tendency and the need to have distinctive signals from

complexes in the 1024–1023 M c-CyD concentration range. The

absolute value of the ellipticity at 264 nm associated to the

complexation progression (see Fig. 1B) was corrected subtracting

the DOX intrinsic signal, (|Dh264|) and was plotted vs. the DOX

molar fraction (Fig. 1E). The plot is characterized by a broad

asymmetric bell-shape profile with maximum at ca. 0.6 molar

fraction. This indicates a significant, but not exclusive, presence of

1 : 2 complexes in the equilibrium mixture (exclusive presence of

1 : 2 stoichiometry would give a maximum at 0.7 molar fraction).

Likely the contemporary presence of both the 1 : 2 and 2 : 2

stoichiometries is the best model to describe the c-CyD

complexation at ‘‘high’’ DOX concentrations. Indeed assuming

this model analysis of the data of Fig. 1A and B afforded

satisfactory fits with log(K12/M22) = 7.80 ¡ 0.04 and log

(K22/M23) = 10.48 ¡ 0.21 (DW = 1.8 and relative error of fit

1.04% in the UV and DW = 2.4 and error of fit 5.42% in the UV-

vis). The extracted spectra of the 1 : 2 and 2 : 2 complexes are

represented in Fig. 1C,D. In the UV range the spectra appear

almost identical to each other, both being characterized by nega-

tive peaks at 207 and 264 nm with De # 240 and 210 M21 cm21,

respectively, and an intense positive peak at 234 nm of De #70 M21 cm21. Also in the UV-vis range the CD spectra of the 1 : 2

and 2 : 2 complexes are similar to each other with positive/

negative splitting of the lowest energy band and negative peaks of

different relative size at 264, 288, 306 nm. In Fig. 1F the

comparison between experimental and calculated ellipticity values

is shown at 290 nm, a wavelength in a critical region, for the model

Scheme 3 Proposed doxorubicin dimer structures, (A) parallel, (B) antiparallel arrangement.30

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Fig. 1 Ellipticity changes of DOX 1.6 6 1024 M in 0.01 M phosphate buffer at pH 7.4 and 22 uC, titrated with c-CyD in the concentration range

2.0 6 1024 M - 1.6 6 1022 M: (A) cell path 0.2 cm; (B) cell path 0.5 cm. The signal of c-CyD alone was subtracted. (C), (D) Absolute CD spectra of

DOX dimer (green), 1 : 2 c-CyD:DOX complex (magenta) and 2 : 2 c-CyD:DOX complex (blue) for log Kd/M21 = 4.8 and log(K12/M22) = 7.80 and

log(K22/M23) = 10.48. The spectrum of free DOX monomer (red) was fixed in the calculations. (E) Modified Job plot of absolute value of ellipticity of

c-CyD–DOX mixtures at 264 nm, (|Dh264|), after subtraction of the signal of DOX alone, vs. DOX molar fraction, total concentration of DOX and

c-CyD = 1023 M. (F) Comparison of experimental (N) and calculated (line) ellipticity at 290 nm for the model with 2 : 2 complex only (red, log(K22/

M23) = 10.8) and 1 : 2 + 2 : 2 complexes (blue).

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with a 2 : 2 complex only and the model with both 1 : 2 and 2 : 2

complexes.

On the basis of the above analysis of the complexation

equilibrium we can safely conclude that c-CyD is not able to

disrupt the DOX dimer when the latter is the predominant DOX

form in solution, rather forming associates in 1 : 2 and 2 : 2

stoichiometry. Given the geometrical arrangement of the two

DOX units in the dimer (Scheme 3),30 it is relevant to gain

information about the possibility for either the aglycone or the

daunosamine moieties to interact with the c-CyD macrocycle. To

this goal, in addition to a MM and MD investigation (see

below), an acid–base titration monitoring the absorption of

DOX in the pH 6–11 interval was carried out. Deprotonation of

the daunosamine–NH3+ and of one of the phenolic OH groups

of the aglycone ring B in aqueous medium occurs with pK values

of ca. 8.15 and 10.16, respectively50,51 and produces large

changes in both the UV region (252 and 233 nm bands) and in

the visible region (Fig. 2A). Encapsulation of the sugar or the

aglycone moieties in the CyD cavity was expected to perturb the

relevant deprotonation process. Actually in the presence of c-

CyD 1.2 6 1022 M (ca. 99% DOX complexed in either 1 : 2 or

2 : 2 stoichiometry with a large predominance of the latter one)

the pH induced spectral modifications were much smaller over

the whole 230–600 nm spectral range. This indicated higher pKs

in the CyD environment for both the deprotonation steps and

suggested the interaction of both the aglycone and the

daunosamine moieties with the c-CyD cavity.

UV-Vis absorption, circular dichroism titration at ‘‘low’’ DOX

concentration. The complexation of the DOX monomer was

investigated at a drug concentration of 1.0 6 1025 M, with c-

CyD concentration varying from 5.0 6 1025 M to 1.2 6 1022 M

(Fig. 3A,B). In these conditions the monomer:dimer ratio is ca.

3 : 1 in the absence of c-CyD. Increasing c-CyD concentrations

induced intensity increase of both the positive band at 470 nm

and the negative one at 288 nm; the signals at 252 nm, 233 nm

and 202–205 nm also gained intensity but no new bands

appeared in the visible. The sets of CD spectra in Fig. 3A and

3B were analysed again with various complexation models with

1 : 1, 2 : 1, 1 : 2 and 2 : 2 c-CyD:DOX complexes in various

combinations, all including the DOX monomer–dimer equili-

brium. In no case the calculation attained convergence with

inclusion of DOX monomer–dimer equilibrium, so we neglected

it. The best model for the whole spectral window (200–600 nm)

was that involving a contemporary presence of 1 : 1 and 2 : 1 c-

CyD:DOX complexes. The relevant optimized binding constants

were log(K11/M21) = 2.7 ¡ 0.2 and log(K21/M22) = 4.4 ¡ 0.5,

with DW parameters in the 1.7–2.4 range. The individual spectra

of the various species are reported in Fig. 3C and 3D. The

agreement between calculated and experimental ellipticities at

representative wavelengths is shown in Fig. S6 of the ESI,{ SI-3.

We cannot exclude that omission of the dimerization equilibrium

may have led to more approximate association constants and

spectra for the complexes. However is worth noticing that the

calculated binding constant for the 1 : 1 complex is in good

agreement with values previously reported in literature34,35 and

the extracted spectrum for the free DOX monomer is very close

to that experimentally measured at 5 6 1026 M (see Fig. 1).

Global analysis of the UV-vis absorption titration data,

performed upon fixing the 1 : 1 association constant, confirmed

the magnitude of the 2 : 1 association constant (Table 1) and

afforded the absolute absorption spectra of the 1 : 1 and 2 : 1

complexes (see Fig. S5 in the ESI,{ SI-3).

Fluorescence. The emission of DOX was also used to

investigate the complexation process. The method which has to

be applied at low DOX concentrations, afforded information on

the monomer complexation, since the dimer is known to be not

emissive at all.30 A DOX 1.0 6 1025 M solution in phosphate

buffer at pH 7.4 exhibited a structured fluorescence spectrum

with lmax = 590 nm. In the presence of increasing concentrations

of c-CyD from 1.0 6 1024 to 1.6 6 1022 M, for lexc = 510 nm

(in a fairly good isosbestic region, see Fig. S5 in the ESI,{ SI-3),

both a progressive increase of the main peak at 590 nm and a

modification of the vibronic features of the spectrum were

observed (Fig. 4A). Global analysis of the whole set of emission

Fig. 2 (A) Absorption spectra of DOX 1.7 6 1024 M in 0.01 M phosphate buffer in the range of pH 6–11, reference water, 22 uC. (B) The same in the

presence of c-CyD 1.2 6 1022 M; reference c-CyD solutions. Cell 0.5 cm. Insets: detail of the 210–270 nm range.

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spectra confirmed the binding model of the CD and UV-Vis

analysis and afforded the stability constants of the 1 : 1 and 2 : 1

complexes, log(K11/M21) = 2.3 ¡ 0.3 and log(K21/M22) = 4.9 ¡

0.1 (Durbin Watson parameter 2.2). The individual fluorescence

contribution of each species in solution is reported in Fig. 4B.

The area under each of the spectral profiles of Fig. 4B is

proportional to the corresponding emission quantum yield

(Wf). Using the value of Wf = 0.039 for DOX in buffer (see

Materials and Methods), we obtained a value of Wf11=

0.032 for the 1 : 1 and Wf21= 0.048 for the 2 : 1 complex. The

550 nm/590 nm intensity ratio (I/II) in the emission spectrum of

DOX is a parameter probing the environment of the hydro-

xyanthraquinone-centered emitting state, because from a value

of ca. 0.80 for water, the ratio progressively diminishes in

protic solvents of decreasing polarity (e.g. 0.57 in ethanol, 0.51

in 1-heptanol).52 In the spectra of Fig. 4B I/II changes from

0.73 in pure buffer to 0.60 in the 1 : 1 complex and 0.51 in the

2 : 1 complex. Thus the I/II values in the complexes tend to

those of DOX in alcoholic solvents, indicating the excited state

of the drug experiences close proximity with the hydroxyl

Fig. 3 Ellipticity changes of DOX 1.0 6 1025 M in 0.01 M phosphate buffer at pH 7.4 and 22 uC, titrated with c-CyD in the concentration range

5.0 6 1025 M–1.2 6 1022 M: (A) cell path 2 cm; (B) cell path 4 cm. The signal of c-CyD alone was subtracted. (C), (D) Absolute spectra of DOX

monomer (red), 1 : 1 (cyano) and 2 : 1 (blue) c-CyD:DOX complexes, for log(K11/M21) = 2.7 ¡ 0.2 and log(K21/M22) = 4.4 ¡ 0.5.

Table 1 Association constants of Doxorubicin:CyD complexes in aqueous media at pH 7.4, determined with various spectroscopic techniques at 22 uCby global analysis of titration experiments with the Specfit/32 program

DOX complexes Binding constants Technique

DOX2 (DOX dimerization) log(Kd/M21) = 4.8 ¡ 0.1 UV-vis absorptionc-CyD:DOX 2 : 2 log(K22/M23) = 10.48 ¡ 0.21 CDc-CyD:DOX 1 : 2 log(K12/M22) = 7.80 ¡ 0.04 CDc-CyD:DOX 1 : 1 log(K11/M21) = 2.7 ¡ 0.2 CD

log(K11/M21) = 2.3 ¡ 0.3 Fluorescencec-CyD:DOX 2 : 1 log(K21/M22) = 4.4 ¡ 0.5 CD

log(K21/M22) = 4.9 ¡ 0.1 Fluorescencelog(K21/M22) = 4.9 ¡ 0.4 UV-vis absorption

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groups of the CyD rim and feels a decrease in the environ-

mental polarity.

The DOX fluorescence decay was little affected by the

presence of c-CyD. The decay kinetics was monoexponential

with tf = 1.02 ns in buffer and 1.13 ns in presence of c-CyD

5.0 6 1023 M (DOX ¢ 80% complexed in 1 : 1 or 2 : 1 c-

CyD:DOX stoichiometries).53 Considering the relation of Wf and

tf with the radiative (kr) and non radiative (knr) rate constants of

the emitting state (eqn (1)):

Wf = kr tf = kr /(kr + knr) (1)

we observe that kr and knr do not substantially change in the c-

CyD complexes compared to buffer (kr # 3–4 6 107 s21 and knr

# 9 6 108 s21). We collect in Table 1 the binding constants of

the various DOX complexes and in Table 2 their photophysical

parameters.

Triplet state of CyD:DOX complexes

The triplet state properties of the guest are generally useful to

gain information on drug environment in CyD complexes.54

Flash photolysis of a 1.6 6 1024 M DOX solution in Ar-

saturated phosphate buffer of pH 7.4 in the presence of 1.6 61022 M c-CyD was therefore carried out with 532 nm laser

excitation. In these conditions . 96% of DOX is associated in

1 : 2 or 2 : 2 stoichiometry. A weak differential absorption with

bands at 350, 420 and 480 nm was observed (Fig. 5A). Its decay

appeared as monoexponential with time constant t = 4.1 ¡

0.1 ms. It was assigned to the population of the DOX triplet

state.55 Given the adopted experimental conditions the actual

assignment was to the triplet of the DOX dimer in the c-CyD

complex(es). Unfortunately, detection of the triplet absorption

at DOX concentrations # 1 6 1025 M was not possible in our

laser flash photolysis apparatus. Thus we could not directly

reveal the triplet features of monomeric DOX in the c-CyD

complexes. However the spectral profile and the lifetime of the

transient in Fig. 5A are similar to those observed in EtOH (see

Fig. 5B), where the drug is monomeric. It can be inferred that

the triplet state absorption is not significantly affected by the

DOX pairing in the dimer (at least in its qualitative features)

and probes an alcoholic environment, as expected on the basis

of the excited singlet properties of the monomeric DOX

complexes. It is worth observing the triplet lifetime in water is

sensibly shorter (t = 1.7 ms) than that in EtOH and in c-CyD,

see also Table 2.55

Analysis of molecular dynamics trajectories for c-CyD:DOX 1 : 1

association

The interaction between c-CyD and DOX was studied by MM

and MD modelling (see all the details of the computational

methods in the Materials and Methods section). The main

purpose of this study was to investigate the ability of the DOX

molecule to interact with the c-CyD cavity with different

molecular portions, (A, B, C, D)-rings and the sugar moiety.

Fig. 4 (A) Fluorescence intensity changes of DOX 1.0 6 1025 M in 0.01 M phosphate buffer at pH 7.4 and 22 uC, upon titration with c-CyD in the

range 1.0 6 1024 M–1.6 6 1022 M. (B) Separated emission spectra of DOX monomer (red); 1 : 1 (cyano); 2 : 1 (blue) c-CyD:DOX complexes,

corresponding to log(K11/M21) = 2.3 ¡ 0.3 and log(K21/M22) = 4.9 ¡ 0.1.

Table 2 Photophysical parameters of doxorubicin in various media at pH 7.4, 22 uC

Photophysical parameters

tT/1026 s tf/1029 s Wf kr/107 s21 knr /108 s21

DOX in buffer 1.7 1.0 0.039 3.9 9.6DOX in EtOH 3.9c-CyD:DOX 1 : 2 or 2 : 2 complex 4.1 — — — —c-CyD:DOX 1 : 1 complex $1.1 0.032 $2.9 $8.8c-CyD:DOX 2 : 1 complex $1.1 0.048 $4.4 $8.7

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Some tentative MD runs showed that, in a water box of

appropriate size, the c-CyD-DOX complex forms rather rapidly

and, under constant pressure and temperature, the reciprocal

arrangements of the partners are quite persistent in their main

geometrical parameters, even for long MD runs. Table 3 reports

some relevant data obtained from the analysis of the MD

trajectories. All the averages in this table are computed by the

values obtained at fixed time intervals along the trajectories.

Average values of the total energies are all negative and their

standard deviations are less than 1%. The mass-weighted radius

of gyration, rgyr, has been obtained for the c-CyD, the DOX and

the complex by eqn (2):

rgyr ~

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

X

i~n

i~1

wi ri { rmeanð Þ2 !,

X

i~n

i~1

wi

!

v

u

u

t (2)

where wi is the mass of atom i, ri its position and rmean is the

position of the center of mass. Moreover, the visual inspection of

the results obtained from MD simulation shows that all the

initial geometries reported in Scheme 2 lead to stable complexes.

As reported in Table 3, after the equilibration stage, for the given

temperature, pressure and number of molecules of the MD runs,

values of energies and geometrical parameters display small

standard deviations from their average values. The same trend

applies to values of the Root Mean Square Deviations (RMSD)

of the complex conformation from a common reference frame,

after a simulation run-time . 2 ns. Thus, after the equilibration

stage, the c-CyD and DOX molecules reach rapidly a stable

reciprocal structural arrangement. The relative placements of the

DOX aglycone and daunosamine moieties with respect to the c-

CyD cavity are substantially preserved for MD runs of 10 ns,

while the c-CyD and DOX molecules undergo minor confor-

mational rearrangements (see SI-5 Fig. S8). The degree of

interpenetration of the two components can be estimated by the

values of rgyr of the complex, which is systematically lower than

the sum of the two components and quite similar to the

corresponding rgyr of the c-CyD molecule.

The stability of the complexes along the trajectories is

consistently confirmed by their negative interaction energies

and by the small standard deviations in the values of energy

and radius of gyration. Fig. 6 reports some relevant structures

extracted from the MD trajectories. The labels correspond to the

initial geometries of the complexes in Scheme 2. As far as

simulation conditions are concerned, these geometries should be

regarded as illustrative of stable structures, since temperature

fluctuations and collisions with solvent molecules are not able to

separate molecules and convert to different complexes.

Comparison of geometries in Fig. 6 with data in Table 3 shows

that the Ib+ initial setting leads to the more stable complex, as

confirmed by the lowest values of the Energy, Interaction Energy

and rgyr. This final geometry corresponds to the one reported in

the literature32 and it is achieved quite early (, 1 ns) in the MD

Fig. 5 Difference absorption spectra observed 300 ns after excitation with a 20 ns laser pulse, cell path 1 cm, at 22 uC: (A) 1.6 6 1024 M DOX in Ar-

saturated 0.01 M phosphate buffer at pH 7.4 in the presence of 1.6 6 1022 M c-CyD, laser pulse 532 nm, 3 mJ. (B) 7.0 6 1025 M DOX in EtOH, laser

pulse 266 nm, 2 mJ. Insets: decay profiles at 350 nm.

Table 3 Average values of energies and mass-weighted radius of gyration computed from the Molecular Dynamics trajectories for the startinggeometries in Scheme 2

Initial geometryEnergy average[kcal mol21]

Interaction energyAverage [kcal mol21]

rgyr (complex)Average [A]

rgyr (c-CyD)Average [A]

rgyr (DOX)Average [A]

I 216 347 ¡ 114 238 ¡ 5 6.17 ¡ 0.08 6.00 ¡ 0.08 4.75 ¡ 0.08II 215 129 ¡ 110 241 ¡ 6 6.26 ¡ 0.08 6.55 ¡ 0.22 4.36 ¡ 0.07I+ 217 365 ¡ 118 246 ¡ 7 6.12 ¡ 0.08 6.00 ¡ 0.07 4.77 ¡ 0.07II+ 215 623 ¡ 111 231 ¡ 5 6.44 ¡ 0.17 6.24 ¡ 0.13 4.56 ¡ 0.06Ib 217 586 ¡ 120 228 ¡ 3 6.32 ¡ 0.14 5.93 ¡ 0.20 4.78 ¡ 0.06IIb 217 671 ¡ 120 228 ¡ 3 6.37 ¡ 0.10 6.09 ¡ 0.09 4.78 ¡ 0.10Ib+ 219 962 ¡ 122 247 ¡ 3 6.14 ¡ 0.05 6.51 ¡ 0.09 4.82 ¡ 0.04IIb+ 217 787 ¡ 118 222 ¡ 4 6.80 ¡ 0.17 6.08 ¡ 0.07 4.77 ¡ 0.07

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trajectory, by insertion of the D-ring into the c-CyD cavity

through the primary (narrow) rim. Table 3 shows that

interaction of DOX with the c-CyD primary rim is energetically

more favored, probably by stabilization with the solvent

molecules since the interaction energies are not consistently

lower as compared to complexes interacting on the secondary

(large) rim. Actually, rgyr of the I and I+ complexes show that a

good packing can be achieved even by interaction on the

secondary rim and that especially the I+ structure can be of

significance in the complex formation. Inspection of Fig. 6

suggests that hydroxyl groups on the c-CyD rims interact

preferably with the conjugated ring system of the DOX,

particularly with rings B and C. This is confirmed by comparing

the distribution of distances between the c-CyD center of mass

and the B or D-ring, respectively (see SI-5 Fig. S7). The B-ring is

consistently closer to the c-CyD center of mass than the

peripheral D-ring, a trend which is even more pronounced in

the Ib+ complex.

Fig. 6 shows that insertion of the DOX daunosamine moiety

into the c-CyD cavity gives rise to stable complexes too. The –

NH2 group can cross from the secondary to the primary rim in

complex II, while the positively charged –NH3+ is confined

outside the secondary rim in complex II+. NOESY experiments

performed by Bekers et al.32 showed that the distance between

hydrogen atoms H2 of the c-CyD and H49 of the daunosamine is

about 3 A for a stable complex. However, the same investigators

couldn’t obtain optimized molecular models consistent with this

result. This point has been investigated in the present work by

computing the radial pair distribution function, g(r), for all

possible pairs of the DOX H4’ atom with the H2 atoms of the c-

CyD along the trajectories of the eight studied complexes. The

pair distribution function is defined as the probability of finding

a second particle as a function of distance from an initial particle

and its values for the investigated complexes are reported in

Fig. 7. Data in this figure show that only complex II+, with the

daunosamine moiety into the c-CyD cavity, comes close to

the NOESY experimental constraint. All other complexes, even

the most stable Ib+, display probabilities that become significant

only at larger distances. Results in Fig. 7 are also consistent with

the distance of 5.7 A for the neutral DOX-(S)-isomer obtained

Fig. 6 Typical geometric arrangements obtained in the MD simula-

tions. The labels correspond to the initial settings in Scheme 2.

Fig. 7 Radial pair distribution function, g(r), of the possible pairs between the DOX H4’ atom with the H2 atoms of the c-CyD. Values corresponding

to interactions with the secondary and the primary rim of the c-CyD are reported in the left and right plot, respectively.

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previously by molecular modeling.32 According to the geometries

in Fig. 6 and data in Fig. 7, it can be concluded that a distance of

3 A is not compatible with the daunosamine H4’ atom interacting

with the exterior of the c-CyD molecule,32 which would

correspond to larger distances. Instead, it should stem from

contributions of structures similar to complex II+, where the

daunosamine moiety is inside the c-CyD cavity. The structure of

these complexes suggests a possible rationale for the interaction

of the DOX molecule with two c-CyD units (2 : 1 complex) as

well as for the interaction of the DOX dimer with two c-CyD

units (2 : 2 complex). Higher order complexes will be the object

of further molecular modelling investigations.

Concluding remarks

The c-CyD-DOX complexation in aqueous buffer is a process

characterized by a great intrinsic complexity, mainly due to the

DOX self-aggregation. In the present study the complexation

equilibria were analysed taking into account the DOX mono-

mer–dimer equilibrium, but it was not considered that the DOX

dimer exists in two conformations, parallel and antiparallel, each

of them expected to have a peculiar affinity for c-CyD. So, our

analysis yields a single spectrum for a given stoichiometry even

though it may represent different conformations. In spite of this

approximation, UV-vis absorption, CD and fluorescence clearly

proved the formation of multiple c-CyD:DOX complexes. 1 : 1,

2 : 1, 1 : 2 and 2 : 2 association stoichiometries were evidenced

for c-CyD ¡ 1.6 6 1022 M. The accurate study of the CD

allowed to demonstrate that the complexes of monomeric

DOX with one (1 : 1) or two CyD units (2 : 1) prevail at DOX

concentrations ¡1025 M, whereas complexes of dimeric

DOX with one (1 : 2) or two c-CyD units (2 : 2) dominate at

DOX concentrations . 1024 M. The formation of higher order

c-CyD:DOX complexes was not reported in previous literature.32

The stability constants of all the complexes, determined by

global analysis of titration data from several spectroscopic

techniques, showed good self-consistency (Table 1), which

reinforces the reliability of the individual CD, fluorescence and

UV-Vis absorption spectra for a given stoichiometry (Fig. 1, 3, 4

and Fig. S1, S4 and S5 in the ESI{).

The CD spectra of the 1 : 1 and 2 : 1 complexes in the UV

region, when compared to each other, point to opposite dichroic

contributions for the association of the first and the second c-

CyD unit to monomeric DOX (Fig. 3C); the corresponding

profiles in the visible (Fig. 3D) indicate the CD is increased in

intensity and blue shifted in the 1 : 1 complex and somewhat

decreased in intensity and red-shifted in the 2 : 1 complex. These

features suggest the aglycone moiety interacts with c-CyD in the

first complexation step while the daunosamine unit interacts in

the second one.56

The MM and MD trajectories support the CD interpretation.

Geometries in Fig. 6 (Ib+) and data in Table 3 show that the

most favourable interaction is that with DOX aglycone part

approaching the c-CyD primary rim. The complex geometry is

characterized by the hydroxy-anthraquinone core (ring C and B)

embedded in the cavity with long axis parallel to the c-CyD axis

and ring D protruding out of the CyD secondary rim. A further

possible complex geometry is that represented in Fig. 6 (I+)

where the flexible c-CyD macrocycle folds to embrace the

aglycone part. The shape of the fluorescence spectra (Fig. 4B) is

consistent with both Ib+ and I+ structures. The quantum yields

and the lifetimes in both 1 : 1 and 2 : 1 complexes appear to be

little modified with respect to those of the free molecule in

agreement with the existence of structures with a large exposure

of the dihydroxy-anthraquinone part to solvent. MM and MD

simulations evidence the daunosamine moiety can also interact

favourably with the c-CyD macrocycle from the secondary rim

side. The sugar unit penetrates more deeply into the cavity when

the amino group is not protonated (compare structure II and II+

in Fig. 6). Thus the calculations support that two c-CyD units

can be associated to the aglycone and the daunosamine moieties

in the 2 : 1 complex.

The positive–negative split dichroic signal in the 420–580 nm

region, due to the presence of the p,p* transitions of the DOX

dimer, is maintained in the 1 : 2 and 2 : 2 complexes, indicating

c-CyD is not capable of disrupting the stacking interaction of the

aromatic chromophores of DOX in either parallel or antiparallel

arrangement. The pH effects on the UV-Vis absorption spectrum

(Fig. 2) demonstrate c-CyD strongly perturbs the acid–base

equilibria of DOX in the ‘‘dimer’’ concentration regime, both

those of the aglycone moiety, more clearly reflected in the

spectral changes at l . 500 nm, and likely also that of the

daunosamine. Considering the experimental conditions, where

the 2 : 2 complex largely predominates, and the geometries

proposed for the DOX dimer (Scheme 3)30 c-CyD might access

both the aglycone pair and the amino sugar tails. The possibility

for the latter complexation mode is supported by structures II

and II+ in Fig. 6.

We finally remark that CD spectra of the higher order c-CyD

complexes, molecular modeling results and pH effects on

electronic absorption consistently support the conclusion that

primary binding involves the aglycone part whereas secondary

binding involves the daunosamine moiety.

Acknowledgements

We thank the FP7 PEOPLE-ITN project n. 237962-CYCLON for

the funding of the research. We also thank Dr Daniele Cesini for

his help and continuous support on the grid computing system.

We also wish to acknowledge the Distributed Unified Computing

for Knowledge (DUCK) cooperation on porting MD applications

to the Comput-ER infrastructure (www.comput-er.it).

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54 S. Monti and S. Sortino, Chem. Soc. Rev., 2002, 31, 287–300.55 A. Andreoni, E. J. Land, V. Malatesta, A. J. McLean and T. G.

Truscott, Biochim. Biophys. Acta, Gen. Subj., 1989, 990, 190–197.56 The spectra of the complexes are characterized by positive bands in

the visible, as expected for monomeric DOX. However in the 1 : 1species a slightly negative CD at 550 nm is likely due to some 1 : 2complex, not considered in the analysis neglecting the DOX dimerfraction present in solution.

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